6Th Meeting of the International Bioiron Society September 6-10, 2015 Zhejiang University Hangzhou, China

6th Meeting of the International BioIron Society September 6-10, 2015 Zhejiang University Hangzhou, China

Featuring Special Events: Educational Introductory Course September 6, 2015 “Essentials of BioIron for Clinicians and Scientists”

“Meet the Expert” Sessions for Trainees September 8 – 10, 2015

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Silver Level

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Educational Grant Provider Welcome to Hangzhou and BioIron 2015 IBIS

Dear friends and colleagues,

Welcome to China for the Sixth Congress of the International Bioiron Society (IBIS)(Bioiron 2015) at Zhejiang University in Hangzhou. This is the first time the biennial meeting of IBIS has been held in China, and it provides the opportunity for many Chinese scientists and clinicians working in the iron field to attend Bioiron for the first time. It also enables those from other parts of the world to experience the fascinating culture of China.

The bioiron area continues to expand and diversify, with important advances and discoveries both in fundamental aspects of iron homeostasis and iron-related disorders. Bioiron 2015 will highlight the best of these advances, and will provide a stimulating environment for scientific discussion and exchange. Whether you are an experienced iron worker or just starting out in this fascinating field, there will be something to hold your attention.

The scientific program was planned by the IBIS Board in collaboration with the Local Organizing Committee (LOC). Local arrangements and logistics were organized by the LOC with the support of dedicated staff at WJ Weiser and Associates. The educational activities that were introduced at the last Bioiron meeting: the Introductory Course and the Meet-the-Expert breakfasts, were a great success and will be held again at Bioiron 2015. These activities are specifically aimed and students and early career investigators, but all delegates are welcome to attend the Introductory Course.

Hangzhou is recognized as one of China’s most beautiful cities, and Zhejiang University provides a very pleasant environment for our sixth congress. We hope you will take advantage of your time in China to interact with the local people and enjoy some of the fine attractions and wonderful cuisine that Hangzhou and China in general are able to offer.

We wish to warmly thank all our sponsors for their generous contributions to this event, and especially their support of the educational activities and bursaries. Without their support it would be much more difficult to organize an event such as this.

Thank you again for joining us in Hangzhou.

Greg Anderson, PhD, IBIS President Fudi Wang, MD, PhD, Chair of the Local Organizing Committee Robert Fleming MD, PhD, IBIS President-Elect

Sixth Congress of the International BioIRon Society Page 3 Table of Contents IBIS

IBIS Board of Directors & Committees...... 5 General Meeting Information ...... 6 Social Events ...... 11 Hangzhou Sightseeing / Optional Tours ...... 12 Program Schedule...... 15 Keynote Speakers ...... 51 Speaker Abstracts ...... 54 Podium Abstracts ...... 82 Poster Abstracts...... 148 Alphabetical Author Index ...... 324

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OFFICERS LOCAL ORGANIZING COMMITTEE Greg Anderson, BSc, MSc, PhD, President Fudi Wang, Chair Robert E. Fleming, MD, President-Elect Yan-Zhong Chang Suzy Torti, PhD, Secretary Huijun Chen Dorine W. Swinkels, MD, PhD, Treasurer Sijin Liu Clara Camaschella, MD, Past-President Guangjun Nie Xiaoyun Shen DIRECTORS Xia Yin Gaetano Cairo, PhD Lu Zhao Caroline Enns, PhD Robert W. Evans, BA, PhD BURSARY COMMITTEE Elizabeta Nemeth, PhD Gaetano Cairo, Chair Caroline Philpott, MD Robert Evans Stefano Rivella, PhD Caroline Philpott Suzy Torti

EXECUTIVE OFFICE Heather Swanson, Executive Director

Abstract Reviewers

Greg Anderson, BSc, MSc, PhD Robert Fleming, MD Stefano Rivella, PhD Jodie Babbitt, MD David Frazer, PhD Paul A. Sharp, BSc, PhD Pierre Brissot, MD Tomas Ganz, MD, PhD Kaila S. Srai, BSc, MSc, PhD Ioav Cabantchik, MD, PhD Yutaka Kohgo, MD, PhD V. Nathan Subramaniam, MSc, PhD Gaetano Cairo, PhD Sijin Liu, PhD Dorine W. Swinkels, MD, PhD Clara Camaschella, MD Gawain MColl, PhD Igor Theurl, MD James Connor, PhD Gordon McLaren, MD Suzy Torti, PhD James Duce, PhD Martina Muckenthaler, PhD Shinya Toyokuni, MD, PhD Richard Eisenstein, PhD Sant-Rayn Pasricha, PhD, FRSCP, Vip Viprakasit, MD Caroline Enns, PhD FRCPA Chris Vulpe, MD, PhD Robert W. Evans, BA, PhD Caroline Philpott, MD Fudi Wang, PhD

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Dates of the Conference

September 6 – 10, 2015

On Sunday, September 6, registration will open at 11:00 and the Welcome Reception will begin at 18:30.

Venue

The Zijingang Campus of Zhejiang University Yuhangtang Rd. 866 Xihu District Hangzhou, China, 310058 Phone: +86 571 8517 2244

Language

The official language of the Bioiron 2015 Conference will be English.

The native residents of Hangzhou, like those of Zhejiang and southern Jiangsu, speak a Wu dialect. However, the Wu dialect varies throughout the area where it is spoken; therefore, Hangzhou's dialect differs from regions in southern Zhejiang and southern Jiangsu. As the official language defined by China's central government, Mandarin is the dominant spoken language.

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Map of Zhejiang University, Hangzhou

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Weather

Hangzhou weather is generally warm and mild year round, with abundant sunshine and rainfall. There are four clear-cut seasons – a short and rainy spring, a hot and humid summer, a cool and clear autumn, and a dry and cold winter.

There are two rainy seasons in the region – one is the Plum Rain Season beginning in late June through early July; the other comes with heavy rains and potential typhoons in August and September. High tourist season occurs in spring (Mar. to May) and autumn (Sep. to Nov.) when the average temperature is 17 C (63 F).

Money

The official currency of China is the Chinese Yuan.

Chinese money is based on the decimal system; there are ten Fen in one Jiao and ten Jiao to each Yuan. Notes: 1 Jiao, 2 Jiao, 5 Jiao, 1 Yuan, 2 Yuan, 5 Yuan, 10 Yuan, 20 Yuan, 50 Yuan, 100 Yuan Coinage: 1 Fen, 2 Fen, 5 Fen, 1 Jiao, 2 Jiao, 5 Jiao, 1 Yuan

Cards: Visa, Master Card, a.m.erican Express, Discover Card, Diners Club, Federal Card, Million Card, and JCB credit cards are accepted at most hotels and state run shops in major cities. Travelers should be prepared to pay in Yuan when shopping in smaller shops, at restaurants, and in smaller hotels.

Local Time

All times listed observe the 24-hour clock in GMT+8:00.

Electricity

Supply: 220V AC with a variety of plug types. Most common are the two prong straight plug (North a.m.erican style) and three prong (Australian style). If you have a three prong North a.m.erican style plug you will need an adapter.

Emergency Number

For police, dial 110. For fire, dial 119. For a.m.bulance, dial 120.

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Registration / Information Desk

The registration/information desk hours are as follows: Location: Zi jin’ gang Campus, Zhejiang University Sunday, September 6 11:00 – 20:00h Monday, September 7 08:00 – 18:30h Tuesday, September 8 07:00 – 14:30h Wednesday, September 9 07:00 – 18:15h Thursday, September 10 07:00 – 18:15h

Registration fee for participant includes: • Admission to Opening Ceremony, all scientific sessions and the Introductory Course • Congress materials (notebook, pen, badge, certificate of attendance) • Complete Online Program which includes full abstracts • Coffee breaks, lunch and afternoon refreshments • Admission to Welcome Reception on Sunday, September 6, 2015

Registration fee for accompanying person includes: • Coffee breaks, lunch, and afternoon refreshments • Admission to Welcome Reception on Sunday, September 6, 2015

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Name Badges

Attendees are required to wear their name badges at all times while at the meeting.

Poster Sessions

Poster Presentations can be set-up on Monday, September 7 from 07:00 and must be set up prior to 10:30. Posters should remain on display until at least the end of Poster Session 2, but may remain on display until 10:00 on Thursday September 10.

• Poster Walk Session I: Monday, September 7 (16:00 - 19:00) • Presenters with ODD numbered posters should stand in front of their poster • Poster Walk Session II: Wednesday, September 9 (16:00 – 19:00) • Presenters with EVEN numbered posters should stand in front of their poster *Any posters left behind will NOT be held by staff. Presenters/authors are 100% responsible for their posters, and IBIS does not hold any liability.

Tourist Information

Tourist information is available from your hotel concierge.

Lunch

Lunch will be served Sunday, Monday, Wednesday and Thursday in QiZhen Hotel (Haoyue Hall 1st Floor & Oufang Hall 2nd Floor).

Dress

The meeting dress is business casual throughout the conference, except for the Gala Dinner, which is formal dress.

Special Needs

If you have special needs (e.g. due to disability) or special dietary needs, please call +1-(847) 517-7225 or e-mail [email protected].

Liability

BioIron will not be liable for illness, accidents or thefts suffered by participants or accompanying persons during the conference or their stay in China.

We would like to remind IBIS members, especially senior members, that they may support the "Travel awards program" of the Society. Even small donations will help young scientists and physicians to attend the BioIron 2017 Meeting.

Please visit our web site at http://www.bioiron.org and click over “Travel fund donations.“ Thank you very much.

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Welcome Reception

Date: Sunday, September 6, 2015 Time: 18:30 – 20:00h Location: Zi jin’ gang Campus, Zhejiang University Attire: Casual Cost: One ticket included in Attendee & Accompanying Person registration fees. Additional tickets can be purchased for $25 USD.

Gala Dinner & Awards Ceremony

Date: Thursday, September 10, 2015 Time: 19:30 – 24:00h Location: Sofitel Hangzhou Westlake Transportation to and from location will be provided. Attire: Formal Dress Cost: $120 USD Registration for the Gala Dinner is closed.

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West Lake ( Xī西湖 Hú ) Hangzhou's most famous scenic sight. Technically, there are 10 Scenes of the West Lake and 10 New Scenes given by Emperor Kangxi of the Qing dynasty, but they are often seasonal (Snowfall Over Broken Bridge, etc). Rather than make a checklist and walking back and forth looking for them, simply spend a clear day wandering the circumference of the lake and the causeways, take a ferry to the islands, and you will probably cover most of the sites anyway. The "West Lake" itself can be divided into countless smaller sites, from Mr. Guo's villa to "Orioles Singing in the Willows".

The "West Lake Scenic Area" itself is very large. This section only covers areas in the immediate vicinity of the lake. Other spots are covered in later sections.

Boat Ride: There are numerous providers of boat rides on the lake. The official tour operator sells tickets from official ticket stalls for boat trips covered by insurance, with clear pricing and which covers all parts of the lake. There are lots of private gondola rides with varying insurance coverage, range and prices.

Sitting on a bench, overlooking West Lake

The Broken Bridge—(桥) is the most elegant and romantic site in Hangzhou West Lake Scenic Area. The classic view of West Lake seen from the Broken Bridge is quite fascinating, and the legend of Lady White Snake also brings this ancient bridge much more charm of culture and history.

Lesser Yingzhou Isle (Three Pools Mirroring the Moon)— Built in the early 1600s, this is the largest island on the lake. When there is a full moon, candles inside the pagodas are lit, and in the candle light it appears as though you see the moonlight (if you are romantic enough to see it), hence the name.

Mid-Lake Pavilion— Built in 1552, it is the oldest island in Hangzhou. There is a Chinese inscription on the Qing Dynasty-era stone arch in which the Qing Emperor wrote "Chong Er" or "Endless Love".

Lord Ruan's Mound— This is a mound they made from piling up dirt after dredging the lake 200 years ago. However, it is not just a dirt mound. At night (summer), entertainment activities are occuring in the garden on the island.

Hubin #X Park— Hubin Parks 1, 3, 6 and probably the numbers in between are the parks between the West Lake and Hubin Road to the east. Relatively newly-designed as the West Lake Tunnel that goes underneath was being built in early 2004, these parks are good to sit for a bit, buy ice cream or a newspaper, and most importantly hire a boat from the cluster of boat docks at each park.

Su Causeway— Almost 3km long, this causeway dates from the year 1189 and has a bunch of willows and peach trees. It is long north-south causeway that starts by the Shangri-La on Beishan Road and goes all the way down to Nanshan Road.

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Bai Causeway— Starting at the eastern end of Beishan Road, this cause way leads to Solitary Hill and cuts off the distances between, say, Hubin Road and the Shangri La.

Solidary Hill and Zhongshan Park— Where Loud Wai Lou restaurant is located, this is the only natural island on the lake. At least three emperors constructed palaces are here. Besides an expensive restaurant, the popular area is the home of the Xiling Seal-Engravers' Society, and the seals, calligraphy, engraving-masters, and relics that go along with it.

Yang Causeway— This is more than 3km long and one road west of the Su Causeway. It starts at the intersection of Beishan and Shuguang Road (which becomes Yang Causeway once you are south of this intersection); the causeway runs north-south. Yang Causeway includes Quyuan Garden (aka Qu Garden aka Qu Courtyard), which is the most popular spot to see tons of lotus blossoms (late spring > summer). The water area to the west of the top of Yang Causeway is Maojiabu Scenic area, with orchids blended into the water scenery. Another tourist spot on Yang Causeway is Mr. Guo's Villa, which was built in 1907 and is considered one of the most "classical" gardens in Hangzhou. At the southern end of the causeway, just before Nanshan Road, is a fish-viewing pond.

King Qian's Memorial, (Qian Wang Ci)– Five kings of the Wuyue Kingdom are buried here in this memorial on the south end of the lake off Nanshan Road.

Zhejiang Museum[www.zhejiangmuseum.com/en/index.jsp], ()— is the largest comprehensive museum in Zhejiang province and collects many rarely-seen treasures founded in Zhejiang, especially the celadon porcelains. It shows the elite culture of traditional China in Jiangnan region. The headquarters is located on 25 GuShan Road, on the north side of West Lake Scenic Area.

Zhejiang West Lake Gallery, ()— is a must-see tourist attraction near to the classic site of Ping Hu Qiu Yue. Zhejiang West Lake Gallery is a famous art education and exhibition center in Zhejiang province.

Wushan Square, ( Wu Shan Guang Chang)— Wushan Square and Wushan Hill are a major town center in Hangzhou. The view from the top is excellent on a clear day, and there are also trails around the hills from behind the pagoda. The pagoda itself has been modernized with an elevator and nice open-air teahouse at the top, but the original bell is still intact and in use. This area also features easy access to Hefang Jie shopping street at the base of the hill, full of small pedestrian streets and shopping stalls. It is also extremely close to the West Lake itself.

Huqingyutang Traditional Chinese Medicine Museum,()—, is the only themed museum with traditional Chinese medicine in China. Huqingyutang is also a traditionally famous medicine store keeping the traditional medicine culture of ancient China. It was created by Hu Xueyan, a representative of Huizhou Merchants.

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Other nearby places of interest: • The world's largest tidal bore races up the Qiantang River through Hangzhou reaching up to 12 m (39 ft) in height. • The Residence of Hu Xueyan () located on Yuanbao Street was built in 1872 by Hu Xueyan, a native of Anhui, a very successful businessman. It was restored and opened to the public in 2001. • Xixi National Wetland Park. Established with the aim of preserving the wetland ecological system, it covers an area of about 10km2. Fish ponds and reed beds have been restored and it is home to many types of birds. It holds a temple and several historic rural houses. • Hangzhou Botanical Garden • Hangzhou Zoo • Old China Street on He Fang Street (He Fang Jie), which offers various souvenirs and renowned Longjing tea. • Jade Springs (Yu Quan) • West Lake Cultural Square is the tallest building in the city and houses the Zhejiang Natural History Museum and Zhejiang Museum of Science and Technology. • Qiandao Lake is a man-made lake with the largest number of islands. These islands are different in size and shape, and have distinctive scene. • Grand Canal • Tianducheng, a 31-km² housing development outside Hangzhou. It contains a subscale replica of the French Eiffel Tower and hundreds of French-architecture-influenced buildings

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Sixth Congress of the International BioIron Society

SUNDAY, SEPTEMBER 06, 2015

OVERVIEW

11:00 - 20:00 Registration/Information Desk Open Location: QiZhen Lobby (1st Floor)

18:30 - 20:00 Welcome Reception Location: QiZhen Hotel (Haoyue Hall, 1st Floor & Oufang Hall, 2nd Floor)

GENERAL SESSION

Introductory Course: Essentials of BioIron for Clinicians and Scientists

12:00 - 13:00 Open Buffet Lunch Location: QiZhen Hotel (Haoyue Hall, 1st Floor & Oufang Hall, 2nd Floor)

13:00 - 13:15 Introductory Remarks Location: QiZhen Hall (3rd Floor)

13:15 - 15:45 Session I: Chairs: Robert E. Fleming, MD Prem Ponka, MD, PhD, FCMA

The Chemistry of Iron in Biological Systems Speaker: Ioav Cabantchik, MD, PhD

Cellular Iron Homestasis Speaker: Caroline Enns, PhD

Systemic Iron Homestasis Speaker: Elizabeta Nemeth, PhD

Model Systems for Studying Iron Biology Speaker: Surjit K. Srai, BSc, MSc, PhD

Nutritional Anemia and Iron Supplementation Speaker: James Collins, PhD

15:45 - 16:15 Coffee Break Location: QiZhen Hall Corridor

16:15 - 18:15 Session II: Chairs: James Connor, PhD Sonia Levi, PhD

Anemia of Inflammation and Cancer Speaker: Robert E. Fleming, MD

Primary Iron Overload Speaker: Clara Camaschella, MD

Hemoglobin Disorders and Secondary Iron Overload Speaker: Suthat Fucharoen, MD

Diagnosing and Treating Iron Disorders Speaker: Yutaka Kohgo, MD, PhD

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18:30 - 20:00 Welcome Reception Location: QiZhen Hotel (Haoyue Hall, 1st Floor & Oufang Hall, 2nd Floor)

MONDAY, SEPTEMBER 07, 2015

OVERVIEW

8:00 - 18:30 Registration/Information Desk Open Location: QiZhen Lobby (1st Floor)

8:00 - 15:00 Exhibit Hours Location: QiZhen Hall Corridor (3rd Floor)

GENERAL SESSION

8:30 - 9:00 Opening Ceremony/Welcome Address Location: QiZhen Hall (3rd Floor)

9:00 - 10:30 Plenary Session I: Systemic Iron Regulation Chairs: Caroline Enns, PhD Yin Xia, PhD

9:00 - 9:25 Erythroid Regulation of Systemic Iron Metabolism Speaker: Tomas Ganz, PhD, MD

9:25 - 9:50 TMPRSS6 and BMP6 In Iron Homeostasis: A Matter of Balance Speaker: Laura Silvestri, PhD

9:50 - 10:15 BMP Signaling in Systemic Iron Regulation Speaker: Jodie Babitt, MD

10:15 #1 ADMINISTRATION OF A SMALL MOLECULE HIF-2 INHIBITOR REDUCES IRON OVERLOAD IN A MOUSE MODEL OF HEMOCHROMATOSIS Yaomin Chen, PhD, Julio Ruiz, PhD and Richard Bruick, PhD UT Southwestern Medical Center Presented By: Richard Bruick

10:30 - 11:00 Coffee Break Location: QiZhen Hall Corridor

11:00 - 12:00 Keynote Lecture I Location: QiZhen Hall (3rd Floor) Chair: Greg Anderson, PhD

Iron and Global Health Speaker: Andrew Prentice, MD

12:00 - 13:00 Lunch Location: QiZhen Hotel (Haoyue Hall 1st Floor & Oufang Hall 2nd Floor)

13:00 - 14:30 Concurrent Session I and Session II

Concurrent Session I: Iron Metabolism in the Kidney Location: QiZhen Hall (3rd Floor) Chairs: Jonathan Barasch, MD, PhD Dorine W. Swinkels, MD, PhD

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13:00 - 13:30 Iron Handling of the Kidney SPECIALIZED KIDNEY INTERCALATED CELLS DEFEND THE URINE SYSTEM BY CHELATING SIDEROPHORES WITH THE BIOMARKER OF KIDNEY INFLAMMATION AND INJURY KNOWN AS NGAL-SIDERCALIN- LCN2 Speaker: Jonathan Barasch, MD, PhD

13:30 #2 DEFICIENCY OF TRANSFERRIN AND NON-TRANSFERRIN IRON RESULTS IN CHRONIC KIDNEY DISEASE Andong Qiu, PhD , Melanie Viltard, PhD , Rong Deng, Bsc , Xueqiao Wang, Bsc , Max Werth, PhD , Yue Yu, Bsc , Neal Paragas, PhD , Abby Sakar, Bsc , Shaun Darrah, Msc , Roger Boles, Bsc , Efrat Bruck, Bsc , Alexander Rittenberg, MD , Danielle Recanati, Bsc , Kristen McNierney, Bsc , Rosemary Sampogna, MD and Jonathan Barasch, MD, PhD Tongji University; Institute for European Expertise in Physiology, Paris, France; Columbia University Presented By: Andong Qiu

13:45 #3 HEPCIDIN: A NOVEL TREATMENT FOR HEME-MEDIATED KIDNEY INJURY? Rachel van Swelm, Jack Wetzels, Vivienne Verweij, Coby Laarakkers, Jeanne Pertijs, Rosalinde Masereeuw and Dorine Swinkels Radboudumc Presented By: Dorine Swinkels

14:00 #4 HEPCIDIN AS A MAJOR COMPONENT OF RENAL ANTIBACTERIAL DEFENSES AGAINST UROPATHOGENIC ESCHERICHIA COLI Dounia Houamel, Nicolas Ducrot, Thibaud Lefebvre, Boualem Moulouel, Marie-Agnes Sari, Sarah Millot, Sophie Vaulont, Erick Denamur, Herv Puy, Carole Beaumont, Laurent Gouya and Zoubida Karim INSERM U1149, Centre de Recherche sur l inflammation, Universit Paris Diderot, Laboratory of Excellence, GREx, 16 rue Henri Huchard, 75018 Paris, France. Presented By: Zoubida Karim

14:15 #5 IMPORTANT ROLES OF HEPHAESTIN AND CERULOPLASMIN IN IRON TRANSPORT OF MOUSE KIDNEY Guohao Liu, Bo Jiang, Undergraduate, Jiashuo Zheng, Undergraduate, Mengxia Chen, Undergraduate and Huijun Chen, Professor Medical School of Nanjing University Presented By: Guohao Liu

Concurrent Session II: Iron Homeostasis in Non-Mammalian Systems Location: QiuShi Hall (3rd Floor) Chairs: Gawain McColl, PhD Bing Zhou, PhD

13:00 #6 STUDYING IRON HOMOESTASIS IN DROSOPHILA Bing Zhou Tsinghua University Presented By: Bing Zhou

13:18 #7 FERRITIN ASSEMBLY IN THE INTESTINES OF DROSOPHILA MELANOGASTER Fanis Missirlis, PhD and Abraham Rosas-Arellano, PhD Centro de Investigaci n y de Estudios Avanzados del Instituto Polit cnico Nacional Presented By: Fanis Missirlis

13:36 #9 THE ACTIVE ROLE OF MULTICOPPER OXIDASE-1 IN IRON METABOLISM IN DROSOPHILA: A FERROXIDASE AND /OR AN ASCORBATE OXIDASE? Minglin Lang, PhD¹, Maureen J. Gorman, PhD² and Michael R. Kanost, PhD² ¹ College of Life Science University，University of Chinese Academy of Sciences，Beijing 100049, China; ²Department of Biochemistry, Kansas State University，Manhattan, Kansas 66506 USA Presented By: Minglin Lang

13:54 #10 IRON HOMEOSTASIS IN C. ELEGANS: A MODEL OF AGEING Gawain McColl, PhD The Florey Institute of Neuroscience and Mental Health Presented By: Gawain McColl Sixth Congress of the International BioIRon Society Page 17 2015 IBIS Program Schedule IBIS

14:12 #11 CHARACTERIZATION OF NON-VERTEBRATE FERROPORTIN HOMOLOGUES Maria Carmela Bonaccorsi di Patti , Fabio Polticelli , Valentina Tortosa , Pier Antonio Furbetta , Tiziana Persichini and Giovanni Musci Sapienza University of Rome; University Roma Tre; University of Molise Presented By: Maria Carmela Bonaccorsi di Patti

14:30 - 16:00 Concurrent Session III and Session IV

Concurrent Session III: Heme Biology Location: QiuShi Hall (3rd Floor) Chairs: Janis L. Abkowitz, MD Iqbal Hamza, PhD

14:30 - 15:00 Heme Coordination with Globin During Red Cell Maturation Speaker: Janis L. Abkowitz, MD

15:00 #12 MACROPHAGE-HEPCIDIN EXPRESSION IN RESPONSE TO HEMIN IS MEDIATED VIA ERK-SIGNALLING PATHWAY Naveen Kumar Tangudu, MSc, Bet l Alan, MSc, Dilay Lai, Sabine H bner, PhD and Maja Vujic Spasic, Prof Dr Institute for Comparative Molecular Endocrinology, University of Ulm Presented By: Naveen Tangudu

15:15 #13 GENERATION AND CHARACTERIZATION OF Hmox1 LysM-Cre MICE: A SMALL SUBSET OF RESIDUAL Hmox1-POSITIVE MACROPHAGES ACCOUNTS FOR EFFICIENT IRON RECYCLING Carine Fillebeen, PhD , Daniel Garcia-Santos, PhD, Konstantinos Gkouvatsos, MD, PhD, Marzell Buffler, Christiane Becker, PhD, Klaus Sch mann, MD, Prem Ponka, MD and Kostas Pantopoulos Lady Davis Institute for Medical Research and McGill University; Lady Davis Institute for Medical Research and Department of Medicine, McGill University, Montreal, Quebec, Canada Presented By: Kostas Pantopoulos

15:30 #14 ELUCIDATION OF THE MECHANISM OF IRON TRANSPORT FORM PLASMA TRANSFERRIN TO MITOCHONDRIAL FERROCHELATASE: FURTHER EVIDENCE FOR THE KISS AND RUN HYPOTHESIS Amel Hamdi, PhD, Tariq Roshan, MD, Alex D. Sheftel, PhD and Prem Ponka, MD,PhD,FCMA McGill University, Lady Davis Institute for Medical Research Presented By: Amel Hamdi

15:45 #15 CRUCIAL ROLE OF FLVCR1A IN THE MAINTENANCE OF INTESTINAL HEME HOMEOSTASIS Veronica Fiorito, PhD , Marco Forni, MD , Lorenzo Silengo, Prof , Fiorella Altruda, Prof and Emanuela Tolosano, Prof Molecular Biotechnology Centre, University of Torino, Italy; EuroClone S.p.A Research Laboratory, Molecular Biotechnology Centre, University of Torino, Italy Presented By: Veronica Fiorito

Concurrent Session IV: Iron and Cancer Location: QiZhen Hall (3rd Floor) Chairs: Sijin Liu, PhD Shinya Toyokuni, MD PhD

14:30 #16 CANCER-ASSOCIATED BUT EPO-RESISTANT ANEMIA IS CAUSED BY IRON DEFICIENCY IN A SPONTANEOUSLY TUMOR-DEVELOPING MOUSE MODEL Markus Thiersch, PhD , Maja Ruetten, DVM , Milena Costa da Silva, PhD , Martina Muckenthaler, PhD and Max Gassmann, DVM University of Zurich; University of Z rich; University of Heidelberg Presented By: Max Gassmann

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14:45 #17 NOVEL FLUORESCENT PROBE DETECTING CATALYTIC FERROUS IRON Shinya Toyokuni, MD, PhD , Takahiro Mukaide, PhD , Yuka Hattori, MD, PhD , Fumiya Ito , Masahiko Mori, MD , Yuki Ohara, MD , Tasuku Hirayama, PhD and Hideko Nagasawa, PhD Nagoya University Graduate School of Medicine; Nagoya University; Gifu Pharmaceutical University Presented By: Shinya Toyokuni

15:00 #18 EXPRESSION OF THE IRON EXPORTER FERROPORTIN IS CONTROLLED BY MIR-20A IN LUNG CANCER Kamesh Rajendra Babu, MSc, MPhil and Martina Muckenthaler, PhD Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, University of Heidelberg, Germany Presented By: Kamesh Babu

15:15 #19 IRON LEVELS DEFINE TWO POPULATIONS OF TUMOR ASSOCIATED MACROPHAGES Milene Costa da Silva , Francesca Vinchi, PhD , Adelheid Cerwenka, PhD and Martina Muckenthaler, PhD University of Heidelberg, Germany; DKFZ, Heidelberg; University of Heidelberg Presented By: Milene Costa Da Silva

15:30 #20 IRON EXCESS DELAYS DISEASE DEVELOPMENT IN MURINE MODELS OF MULTIPLE MYELOMA Alessandro Campanella, PhD, Silvia Galvan, Jessica Bordini, Maurilio Ponzoni, MD, Maria Teresa Sabrina Bertilaccio, PhD and Clara Camaschella, MD San Raffaele Scientific Institute, Milan, Italy Presented By: Alessandro Campanella

15:45 #21 MYELOID ZINC FINGER 1 (MZF-1) SUPPRESSES PROSTATE TUMOR GROWTH THROUGH ENHANCING FERROPORTIN-CONDUCTED IRON EGRESS Yue Chen , Zhihong Zhang , Kuo Yang , Jin Du , Yong Xu and Sijin Liu State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco- Environmental Sciences, Chinese Academy of Sciences; Department of Urology, The Second Hospital of Tianjin Medical University; School of Life Science, Tsinghua University Presented By: Yue Chen

16:00 - 19:00 Poster Walk I Location: ShunShui Hall, Lizhou Hall and Yangming Hall (4th Floor) Refreshments available. Beer and Wine service to begin at 17:00

Poster# 1 IRON CHELATION BY BIOPOLYMERS FOR MAINTAINING COLONIC HEALTH Richard Horniblow¹, Yemisi Latunde-Dada, PhD², Tariq Iqbal, MBBChir³, Zoe Pikramenou, PhD¹ and Chris Tselepis, PhD¹ ¹University of Birmingham; ²Kings College London; ³Queen Elizabeth Hospital Birmingham (Presented By: Richard Horniblow)

Poster# 3 SELF-ASSEMBLY OF FERRITIN NANOCAGES INTO ONE- AND TWO- DIMENSIONAL ARRAYS INDUCED BY POLY (?,L-LYSINE) Guanghua Zhao, PhD and Rui Yang, PhD China Agricultural University (Presented By: Guanghua Zhao)

Poster# 5 EFFECTS OF PARENTERAL IRON LOADING AND/OR IRON CHELATION ON THE EXPRESSION OF DUODENAL IRON TRANSPORT MACHINERIES IN THALASSEMIA MICE Patarabutr Masaratana, MD, PhD¹, Chanita Sanyear, MSc², Wiraya Eamsaard, BSc³, Saovaros Svasti, PhD4 and Suthat Fucharoen, MD, PhD4 ¹Department of Biochemistry, Faculty of Medicine Siriraj Hospital; ²Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Thailand; ³Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand; 4Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Thailand (Presented By: Patarabutr Masaratana)

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Poster# 7 MACROPHAGE IRON METABOLISM PROFILE IN PRO-ATHEROGENIC CONDITIONS François Canonne-Hergaux, PhD¹,³,4, Liliana Marques, Anne Negre-Salvayre and Luciana Costa² ¹INSERM UMR 1043; ²Departamento da Promoção da Saúde e Prevenção de Doenças Não Transmissíveis, Instituto Nacional de Saúde Doutor Ricardo Jorge, Lisboa, Portugal; ³CNRS UMR 5282; 4Université de Toulouse, UPS, Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse, France. (Presented By: François Canonne-Hergaux)

Poster# 9 IV IRON SUPPLEMENTATION ASSOCIATED WITH RETINAL DEGENERATION IN MICE AND MAN Joshua Dunaief, MD,PhD¹, Delu Song, MD, PhD¹, Yafeng Li, BA¹, Ying Song, MD¹, Liangliang Zhao, MD², Chenguang Wang, MD, PhD² and Levi Kanu, BA¹ ¹University of Pennsylvania; ²Second Hospital of Jilin University (Presented By: Joshua Dunaief)

Poster# 11 CROSSTALK BETWEEN OBESITY AND NEURODEGENERATION; ADIPONECTIN-MEDIATED MODULATION OF IRON FLUX VIA DEXRAS1 IN THE BRAIN Yong Chen, PhD, Lauren Mathias, BS, Rexford Ahima, PhD and Sangwon Kim, PhD University of Pennsylvania (Presented By: Sangwon Kim)

Poster# 13 INTERLEUKIN 6 REGULATES IRON RELATED PROTEINS THROUGH C-JUN N-TERMINAL KINASE ACTIVATION IN BV2 MICROGLIAL CELL LINES Jun Wang, PhD & MD Department of Physiology, Shandong Provincial Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology (Presented By: Jun Wang)

Poster# 15 THE EFFECT OF ESTROGEN ON IRON METABOLSIM IN ASTROCYTES AND NEURONS Manman Xu, bachelor Department of Physiology, Shandong Provincial Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology, Medical College of Qingdao University (Presented By: Manman Xu)

Poster# 19 DESIGN OF NOVEL FLUORESCENT MITOCHONDRIA-TARGETED PEPTIDES WITH IRON SELECTIVE SENSING ACTIVITY Robert Hider¹, Vincenzo Abbate, PhD¹, Olivier Reelfs, PhD² and Charareh Pourzand, PhD² ¹King's College London; ²Bath University (Presented By: Robert Hider)

Poster# 23 GALLIUM NANOPARTICLE: A SINGLE DRUG TARGETING IRON METABOLISM TO TREAT HIV-TB CO-INFECTION IN HUMAN MACROPHAGES Seoung Choi, PhD, Bradley Britigan, MD and Prabagaran Narayanasamy, PhD University of Nebraska Medical Center (Presented By: Bradley Britigan, MD)

Poster# 25 HFE POLYMORPHISMS AFFECT SURVIVAL OF BRAIN TUMOR PATIENTS Sang Lee, PhD, Becky Slagle-Webb, BS, Jonas Sheehan, MD, Junjia Zhu, PhD, Joshua Muscat,PhD, Michael Glantz, MD and James Connor, PhD Pennsylvania State University (Presented By: Sang Lee)

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Poster# 27 ALTERATIONS IN THE IRON REGULATORY GENE SIGNATURE ARE ASSOCIATED WITH DECREASED SURVIVAL IN LOW GRADE GLIOMA Cody Weston, PhD¹, Joseph Klobusicky, PhD², Jennifer Weston, MLIS³, James Connor, PhD³, Steven Toms, MD² and Nicholas Marko, MD² ¹Penn State Hershey College of Medicine; ²Geisinger Medical Center, Danville, PA; ³Penn State College of Medicine, Hershey, PA (Presented By: Cody Weston)

Poster# 29 EFFECT OF IRON DEPLETION AND REPLETION ON THE RELATIONSHIPS OF EXPRESSION OF CARDIAC IRON-SULFUR PROTEINS AND CARDIAC HYPERTROPY OF RATS YihFong Liew, PhD¹, Chia-Shu Lee, MS¹, Ya-Yun Cheng, MS¹, Ming-Chen Yu, MS² and Ning-Sing Shaw, PhD² ¹Fu-Jen Catholic University; ²National Taiwan University (Presented By: YihFong Liew)

Poster# 31 EFFECTS OF IRON CHELATOR DEFEROXAMINE ON NUCLEOLAR MORPHOLOGIC AND FUNCTIONAL CHANGE AND CELL GROWTH IN HUMAN BREAST CANCER MCF-7 CELLS YihFong Liew, PhD, Yu-Shan Lin, MS, Wei-Chih Chen, MS and Ya-Yun Cheng, MS Fu-Jen Catholic University (Presented By: YihFong Liew)

Poster# 33 TRANSCRIPTOMIC ANALYSIS OF IRON METABOLISM GENES IN THP-1 DERIVED MACROPHAGES IDENTIFIES HEPHL-1 AS POTENTIAL REGULATOR OF MACROPHAGE IRON EXPORT Amin Sobh, Zouhair Attieh, PhD, Huijun Chen, PhD, Alex Loguivov, PhD and Chris Vulpe, MD, PhD University of California, Berkeley (Presented By: Amin Sobh)

Poster# 35 THE MONOCLONAL ANTI-TRANSFERRIN ANTIBODY SYNDROME: A WAY FOR A BETTER UNDERSTANDING OF IRON METABOLISM Pierre Brissot, Martine Ropert, Lénaïck Detivaud, Béatrice Fimbel D’hauthuille, Mathilde Gautier, Patricia Leroyer, Anne-Marie Jouanolle and Olivier Loreal National center of reference for rare genetic iron overload diseases, Inserm-UMR 991, Laboratory of biochemistry, Laboratory of molecular genetics and genomics, Hematology department Trousseau Hospital (Tours). Pontchaillou University hospital, Rennes, Fr (Presented By: Pierre Brissot)

Poster# 37 CANCER RELATED ANEMIA HAS A HIGH PREVALENCE AND ADVERSELY IMPACTS QUALITY OF LIFE Giridhar Kanuri¹, Ritica Sawhney, MSc², Girish Raju, MD³, Jeeva Varghese, MSc4, Madonna Britto4 and Arun Shet, MD, AB (Haem/onco)³ ¹Wellcome Trust- DBT Hematology Research Division; ²Wellcome Trust- DBT Hematology Research Division, St. Johns Research Institute, Bangalore, Karnataka 560064 India; ³Department of Medical Oncology, St. John’s Medical College Hospital, Bangalore, Karnataka, India; 4College of Nursing, St. John’s National Academy of Health Sciences, Bangalore, Karnataka, India (Presented By: Giridhar Kanuri)

Poster# 39 IRON IMPORT TO UNUSUAL TRYPANOSOMA BRUCEI MITOCHONDRIA - THE ROLE OF MITOFERRIN HOMOLOG Jan Mach, Dominik Arbon, Ivo F. Scheiber, Jan Tachezy and Robert Sutak Charles University in Prague (Presented By: Jan Mach)

Poster# 41 INFLAMMATION-INDUCED IRF-1 FACTOR DOWNREGULATES FERROPORTIN IRON EXPORTER Catherine Mura, PhD¹, Rafiou Agoro, MSc², Francois Erard, PhD³ and Valerie Quesniaux, PhD³ ¹UMR7355 CNRS, University Orleans; ²UMR 7355 CNRS; ³UMR7355 CNRS (Presented By: Catherine Mura)

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Poster# 43 INVESTIGATION OF LINKS BETWEEN HEPCIDIN TRANSCRIPTIONAL CONTROL AND CELLULAR ENERGY STATUS Ana Rita da Silva¹,³,4, Katarzyna Mleczko-Sanecka¹,4, Amol Tandon², Sven Sauer², Matthias W. Hentze³ and Martina U. Muckenthaler¹,4 ¹Molecular Medicine Partnership Unit, Heidelberg, Germany; ²Metabolism Center, University of Heidelberg, Heidelberg, Germany; ³European Molecular Biology Laboratory, Heidelberg, Germany; 4University of Heidelberg, Heidelberg, Germany (Presented By: Ana Rita da Silva)

Poster# 45 FERRITIN INHIBIT DOXORUBICIN-INDUCED BREAST CANCER CELL DEATH Benjaporn Buranrat¹ and James R. Connor² ¹Faculty of Medicine, Mahasarakham University, 44000, Thailand; ²Department of Neurosurgery, The Pennsylvania State University Hershey Medical Center, Hershey, PA, USA (Presented By: Benjaporn Buranrat)

Poster# 47 REGULATION OF HEPCIDIN BY INFLAMMATION IN IMMUNE CELLS Rafiou Agoro, MSC¹ and Catherine Mura, PhD² ¹UMR-7355 CNRS Orleans; ²CNRS - Orleans (Presented By: Rafiou Agoro)

Poster# 49 DEFERIPRONE AND DEFERASIROX AS PROMISSING CANDIDATE LIGANDS FOR SKIN PHOTOPROTECTION BY LIGHT-ACTIVATED CAGED-IRON CHELATORS Sharareh Houshmandyar, MSc, Olivier Reelfs, PhD, DSc, Tina Radka, PhD, Ian M. Eggleston, PhD and Charareh Pourzand, PhD, DSc University of Bath (Presented By: Sharareh Houshmandyar)

Poster# 51 PREVALENCE OF HYPOFERRITINEMIA IN BLOOD DONORS Marta Clavero Olmos, Nadia Matskiv, Ines Valdes Gross, Ana López Aparicio, Alejandro García- Espona Pancorbo, Rosa Peraita and Alejandro del Castillo Rueda Hospital General Universitario Gregorio Marañón (Presented By: Marta Clavero Olmos)

Poster# 53 FUNCTIONAL PROPERTIES OF DIVALENT METAL TRANSPORTER 1 (DMT1) ON THE OUTER MITOCHONDRIAL MEMBRANE (OMM) Michael Garrick, PhD¹, Andrew Ghio, MD², Lin Zhao, MD¹, Stephen T. Koury, PhD¹, Natascha A. Wolff, PhD³, Frank Thévenod, MD, PhD³, Sultan Chowdhury, PhD4, Alison B. Cutts, PhD4, Jay A. Cadieux, PhD, PMP4, Y. Paul Goldberg, MD, PhD4, Charles Cohen, MD, PhD4, James F. Collins, PhD5 and Laura M. Garrick, PhD¹ ¹University at Buffalo; ²USA EPA; ³Witten/Herdecke University; 4Xenon Pharmaceuticals; 5University of Florida (Presented By: Michael Garrick)

Poster# 55 DCYTB EXPRESSION DURING ERYTHROPOIESIS Heewon Choi, MSc and G.O. Latunde-Dada, PhD King's College London (Presented By: G.O. Latunde-Dada)

Poster# 57 MUTATION SCREENING IN CHINESE PATIENTS AFFECTED BY HEREDITARY HEMOCHROMATOSIS Yongwei Wang¹, Gang Liu, PhD², Bing Han, PhD, MD³, Yangzhong Chang, PhD4 and Guangjun Nie, PhD² ¹CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology; ²National Center for Nanoscience and Technology; ³Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100730, China; 4Laboratory of Molecular Iron Metabolism, College of Life Science, Hebei Normal University, Shijiazhuang, 050024, Hebei Province, China (Presented By: Yongwei Wang)

Poster# 59 UNRAVELLING THE ROLE OF OXIDATIVE DAMAGE AND REPLICATION STRESS IN THE INDUCTION OF DNA DAMAGE RESPONSE SIGNALING IN DMT1-DEFICIENT HEMATOPOIESIS Monika Horvathova, Zuzana Zidova, Katarina Kapralova, Leona Raskova-Kafkova, Zuzana Somikova, Jana Kucerova and Vladimir Divoky

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Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic (Presented By: Monika Horvathova)

Poster# 61 HEPCIDIN REPRESSION BY PROMOTER DNA HYPERMETHYLATION IN NON- VIRALHEPATOCELLULAR CARCINOMA Natascia Campostrini, Silvia Udali, Michela Corbella, Patrizia Guarini, Annalisa Castagna, Patrizia Pattini, Andrea Ruzzenente, Alfredo Guglielmi, Sara Moruzzi, Alberto Ferrarini, Massimo Delledonne, Luigi Perbellini, Antonia Franceschi, Sang-Woon Choi, Domenico Girelli and Simonetta Friso Department of Medicine, University of Verona, Italy (Presented By: Domenico Girelli)

Poster# 63 GENOTYPE-PHENOTYPE STUDIES IN 70 IRIDA PATIENTS Luigia De Falco², Mariasole Bruno¹,³, Laura Silvestri, Caroline Kannengiesser, Erica Moran, Claire Oudin, Marco Rausa, Jessica Aranda, Bienvenida Argiles, Idil Yenicesu, Maria Falcon-Rodriguez, Ebru Yilmaz-Keskin, Ulker Kocak, Carole Beaumont, Clara Camaschella, Bernard Grandchamp, Mayka Sanchez and Achille Iolascon ¹CEINGE - Biotecnologie Avanzate; ²CEINGE, Biotecnologie Avanzate; ³2Department of Medicine, Section of Internal Medicine, University of Verona (Presented By: Mariasole Bruno)

Poster# 65 CANCER STEM CELLS: IS THERE A ROLE FOR IRON? Zuzana Rychtarcikova², Sandra Lettlova², Jiri Neuzil²,³ and Jaroslav Truksa, PhD¹ ¹Academy of Sciences of the Czech Republic; ²Academy of Sciences of the Czech Republic, Institute of Biotechnology, Prague, Czech Republic; ³School of Medical Science, Griffith University, Southport, Qld, Australia (Presented By: Jaroslav Truksa)

Poster# 67 HYPERGLYCEMIA DYSREGULATES RENAL IRON HOMEOSTASIS Rajiv Kumar, PhD Jawaharlal Nehru University (Presented By: Rajiv Kumar)

Poster# 69 DECREASED HEPCIDIN LEVELS CORRELATE WITH ELEVATED PLASMA AND TISSUE IRON PARAMETERS IN PATIENTS AND MURINE DISEASE MODELS OF DIABETES Sandro Altamura¹,³, Stefan Kopf², Julia Glockenmeier¹,³, Peter Nawroth² and Martina U. Muckenthaler¹,³ ¹Dep. of Pediatric Oncology, Hematology and Immunology - University of Heidelberg, Germany; ²Department of Internal Medicine I and Clinical Chemistry, University of Heidelberg, Heidelberg, Germany; ³MMPU Molecular Medicine Partnership Unit (Presented By: Sandro Altamura)

Poster# 71 FENUGREEK PLANT AS AN ANTI-CANCER REMEDY INDUCES APOPTOTIC GENES Kholoud Khoja and Yemisi Latunde-Dada, Lecturer, Diabetes and Nutritional Sciences Division Kings College London (Presented By: Kholoud Khoja)

Poster# 73 BIOLOGIC CHARACTERIZATION OF IRON CHELATOR SIH, OXIDATIVE STRESS-ACTIVATED PROCHELATOR BSIH AND THEIR METABOLITES Hana Jansova, Jan Bures, Petra Kovarikova and Tomas Simunek Charles University in Prague, Faculty of Pharmacy in Hradec Kralove (Presented By: Hana Jansova)

Poster# 75 PROTECTIVE EFFECTS OF ROSMARINIC ACID AGAINST IRON-INDUCED NEUROTOXICITY IN SK-N-SH CELLS Le Qu, Huamin Xu, Hong Jiang and Jun-Xia Xie Department of Physiology, Shandong Provincial Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology, Medical College of Qingdao University, Qingdao, 266071, China (Presented By: Le Qu)

Poster# 77 LIVER HFE PROTEIN CONTENT IS REGULATED BY IRON LEVELS IN VIVO Jan Krijt, Jana Frydlova, Petr Prikryl, Iuliia Gurieva and Martin Vokurka Charles University in Prague, First Faculty of Medicine (Presented By: Jan Krijt)

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Poster# 81 BMP6 REGULATES HEPCIDIN PRODUCTION INDEPENDENTLY OF THE HEMOCHROMATOSIS-ASSOCIATED MOLECULES HFE, TFR2, AND HJV Chloe Latour², Celine Besson-Fournier², Delphine Meynard², Laura Silvestri³, Ophelie Gourbeyre², Patricia Aguilar-Martinez4, Paul J. Smith5, Mark D. Fleming5, Marie-Paule Roth² and Helene Coppin, PhD¹ ¹Inserm-U1043; ²Inserm; ³San Raffaele Scientific Institut; 4Hopital Saint-Eloi; 5Hardward Medical School (Presented By: Helene Coppin)

Poster# 83 MULTI-COPPER FERROXIDASES PLAY AN IMPORTANT ROLE IN BRAIN IRON METABOLISM Ruiwei Jiang, Postgraduate, Mengxia Chen, Undergraduate, Jiashuo Zheng, Undergraduate and Huijun Chen, Professor Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University (Presented By: Jiashuo Zheng)

Poster# 85 INSULIN SIGNALLING IS IMPAIRED IN IRON-LOADED PRIMARY HEPATOCYTES Jithu James, MSc¹, Joe Varghese, MD², Sophie Vaulont, PhD³, Andrew Mckie, PhD4 and Molly Jacob, MD, PhD² ¹Christian Medical College, Vellore; ²Christian Medical College, Vellore, India; ³INSERM, U1016, Institut Cochin, Paris, France; 4Division of Diabetes and Nutritional Sciences, King’s College, London, UK (Presented By: Jithu James)

Poster# 87 IRON DEFICIENT RBCS FROM GAMBIAN CHILDREN AND PREGNANT WOMEN ARE RESISTANT TO FIELD ISOLATES OF FALCIPARUM MALARIA Morgan Goheen, BS¹, Bakary Darboe, DMLS², Ebrima Danso, BSc², Mamadou Bah, Foundation Degree², Muna Affara, PhD², Amat Bah, MsC², Rita Wegmuller, PhD², Andrew Prentice, PhD³ and Carla Cerami, MD,PhD¹ ¹University of North Carolina; ²MRC-Gambia; ³London School of Hygiene & Tropical Medicine (Presented By: Carla Cerami)

Poster# 89 MACROPHAGES UNDER STRESS ERYTHROPOIESIS ARE ASSOCIATED WITH A CHARACTERISTIC GENE EXPRESSION PROFILE Ritama Gupta, Piali Mukherjee, Igor Theurl, Tara Arvedson and Stefano Rivella, PhD Children's Hospital of Philadelphia (Presented By: Stefano Rivella)

Poster# 91 IDENTIFICATION OF CYS LIGANDS FOR THE [2FE-2S] AND [4FE-4S] CLUSTERS IN DRE2: BOTH CLUSTERS ARE ABSOLUTELY ESSENTIAL FOR THE FUNCTION Yan Zhang, PhD¹, Andrew Dancis, MD² and Eiko Nakamaru-Ogiso, PhD² ¹School of Pharmaceutical Science and Technology, Tianjin University; ²University of Pennsylvania (Presented By: Yan Zhang)

Poster# 93 A NOVEL NANO-IRON SUPPLEMENT (IHAT) TO SAFELY COMBAT IRON DEFICIENCY AND ANAEMIA Dora Pereira, PhD, MEng¹, David Frazer, PhD², Mohamad Aslam, BSc², Greg Anderson² and Jonathan Powell, PhD² ¹Medical Research Council Human Nutrition Research; ²MRC Human Nutrition Research (Presented By: David Frazer)

Poster# 95 TARGETING STORAGE LESION IN RED BLOOD CELL TRANSFUSION Xi Huang, PhD¹, Kanika Brown, BS² and Alan Perlstein, MS² ¹Ex Vivo Dynamics, Inc.; ²Ex Vivo Dynamics, Inc. 423 127th Street, New York, NY 10017, USA (Presented By: Xi Huang)

Poster# 97 THE REGULATION OF HIF-1 ON IRON METABOLISM IN COLON CANCER CELL LINES UNDER HYPOXIA Li Zhu, PhD, Mengwan Zhang, Master, Qianqian Luo, Master and Dan Wang, Master Institute for Nautical Medicine (Presented By: Li Zhu)

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Poster# 99 NOVEL BETA-CASEIN NANOCARRIERS FOR ORAL IRON DELIVERY Mohammed Gulrez Zariwala, PhD², Soma Somavarapu, PhD³, Robert Evans, PhD¹, Sebastien Farnaud, PhD and Derek Renshaw, PhD ¹Centre for Electronic Systems Research, Brunel University; ²Faculty of Science and Technology, University of Westminster; ³School of Pharmacy London (Presented By: Robert Evans)

Poster# 101 CHANGES OF MOTOR COORDINATION ABILITY AND SPATIAL LEARNING ABILITY IN CHRONIC IRON-INTOXICATED MICE Fengjv Jia, Ning Song, Junxia Xie and Hong Jiang Department of Physiology, Shandong Provincial Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology, Medical College of Qingdao University, Qingdao, 266071, ChinaShandong Provincial Collaborative Inn (Presented By: Fengjv Jia)

Poster# 103 FUNCTIONAL ANALYSIS OF GLRX5 MUTANTS REVEALS DISTINCT FUNCTIONALITIES OF GLRX5 PROTEIN Gang Liu, PhD¹, Yongwei Wang, BS¹, Greg Anderson, PhD², Clara Camaschella, PhD, MD³, Yanzhong Chang, PhD4 and Guangjun Nie¹ ¹CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, China, Beijing, 100190; ²Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia; ³Vita-Salute University and IRCCS San Raffaele Scientific Institute, Milan, Italy; 4Laboratory of Molecular Iron Metabolism, College of Life Science, Hebei Normal University, Shijiazhuang, 050024, Hebei Province, China (Presented By: Guangjun Nie)

Poster# 105 LACK OF MACROPHAGE FERROPORTIN AFFECTS WOUND HEALING Stefania Recalcati, MD, PhD, Elena Gammella, PhD, Paolo Buratti, BSc, Massimo Locati, MD, PhD and Gaetano Cairo, PhD University of Milan (Presented By: Stefania Recalcati)

Poster# 107 EFFECT OF PHLEBOTOMY ON LEARNING AND MEMORY FUNCTION IN CERULOPLASMIN MICE M.R. Liu, Master, X.F. Zhang, BS, P.P. Yu, BS, Z.H. Shi, PhD, Y.Z. Chang*, PhD and Peng Yu Key Laboratory of Animal Physiology, Biochemistry & Molecular Biology of Hebei Province, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Laboratory of Molecular Iron Metabolism, College of Life Science, Hebei Normal University (Presented By: Peng Yu)

Poster# 109 BONE IRON CONTENT IN POSTMENOPAUSAL WOMEN WITH HIP FRAGILITY FRACTURES: CORRESPONDENCE TO BONE STATUS AND IMPLICATIONS FOR POSTMENOPAUSAL OSTEOPOROSIS Li Guangfei¹ and Xu Youjia, PhD² ¹The Second Affiliated Hospital of Soochow University; ²The Second Affiliated Hospital of Soochow University (Presented By: Li Guangfei)

Poster# 111 THE ETIOLOGY AND PATHOGENESIS OF THE DYSMETABOLIC IRON OVERLOAD SYNDROME Heyang Wang¹, Chen Ling², Lusha Wu, Xin Jiang, Fang He, Xuan Zhen, Yuxiao Tang and Min Li, MD¹ ¹Second Military Medical University; ²University of Florida (Presented By: Min Li)

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Poster# 113 PROLACTIN SUPPRESSING DRUG BROMOCRIPTINE REGULATES HEPCIDIN THROUGH IRON HOMEOSTASIS IN MICE Shang-Yuan Liu, MD¹, Jing Wang, PhD², Peng Yu, PhD¹, Yu-Mei Fan, PhD¹, Guofen Gao, PhD¹, Xiang-Lin Duan, Bsc¹, Yaru Zhou, MD³, Shu-E Zhao, MD³, Guanjun Nie, PhD4 and Yan-Zhong Chang¹ ¹Hebei Normal University; ²Chinese Academy of Sciences Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology of China; ³The Third Hospital of Hebei Medical University; 4National Center for Nanoscience and Technology of China (Presented By: Yan-Zhong Chang)

Poster# 117 HEPCIDIN INVOLVED IN THE REGULATION OF IRON HOMEOSTASIS DURING PREGNANCY AND LACTATION IN RATS Guofen Gao, Shang-Yuan Liu, Hui-Jie Wang, Tian-Wei Zhang, Sai-Shou Zhang, Xiang-Lin Duan, Shu-E Zhao and Yan-Zhong Chang Hebei Normal University (Presented By: Guofen Gao)

Poster# 119 FXN III IS A NOVEL FUNCTIONAL FRATAXIN ISOFORM Shuangying Hao Nanjing University (Presented By: Shuangying Hao)

Poster# 123 IPSC-DERIVED HUMAN NEURONS AS DISEASE MODELS TO STUDY NEURODEGENERATION WITH BRAIN IRON ACCUMULATION DISORDERS Sonia Levi, PhD¹, Daniel Orellana, PhD², Paolo Santambrogio, PhD², Alicia Rubio-Garrido, PhD², Vania Broccoli, PhD³, Paola Venco, PhD4 and Valeria Tiranti, PhD4 ¹Vita-Salute San Raffaele University; ²San Raffaele Scientific Institute; ³CNR-Institute of Neuroscience; 4IRCCS-C. Besta. (Presented By: Sonia Levi)

Poster# 125 MICE ARE POOR HEME ABSORBERS AND DO NOT REQUIRE INTESTINAL HMOX1 FOR DIETARY HEME IRON ASSIMILATION Carine Fillebeen, PhD, Konstantinos Gkouvatsos, MD, PhD, Gabriela Fragoso, Annie Calvé, Daniel Garcia-Santos, PhD, Marzell Buffler, Christiane Becker, PhD, Klaus Schümann, MD, Prem Ponka, MD, Manuela Santos, PhD and Kostas Pantopoulos Lady Davis Institute for Medical Research and McGill University (Presented By: Kostas Pantopoulos)

Poster# 127 A HIGH FAT DIET MODULATES IRON METABOLISM BUT DOES NOT PROMOTE LIVER FIBROSIS IN HEMOCHROMATOTIC HJV-/- MICE Ranjit Padda, MSc, Konstantinos Gkouvatsos, MD, PhD, Maria Guido, MD, Jeannie Mui, Hojatollah Vali, PhD and Kostas Pantopoulos Lady Davis Institute for Medical Research and McGill University (Presented By: Kostas Pantopoulos)

Poster# 129 NEUROPROTECTIVE EFFECTS OF GINKGETIN AGAINST NEURO-INJURY IN PARKINSON’S DISEASE MODEL INDUCED BY MPTP VIA CHELATING IRON Y.Q. Wanf, BS, M.Y. Wang, BS, X.R. Fu, BS, Peng Yu, G.F. Gao, PhD, Y.M. Fan, PhD, X.L. Duan, BS, B.L. Zhao, Phd, Y.Z. Chang, PhD and Zhenhua Shi*, PhD Key Laboratory of Animal Physiology, Biochemistry & Molecular Biology of Hebei Province, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Laboratory of Molecular Iron Metabolism, College of Life Science, Hebei Normal University (Presented By: Peng Yu)

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Poster# 131 MITOCHONDRIAL FERRITIN DELETION AGGRAVATE A? INDUCED DAMAGE IN THE MICE BRAIN Pei-Na Wang, Qiong Wu and Yan-Zhong Chang Hebei Normal University (Presented By: Pei-Na Wang)

Poster# 133 THE EFFECTS OF SEX ON BRAIN IRON STATUS IN RATS Qian Hao, Qiong Wu and Yan-Zhong Chang Hebei Normal University (Presented By: Qian Hao)

Poster# 135 INVESTIGATING THE ROLE OF NO IN ANEMIA OF INFLAMMATION Marc Mikhael, BSc, PhD¹, Costantine Daher, PhD¹ and Prem Ponka, MD, PhD² ¹Lebanese American University; ²McGill University (Presented By: Marc Mikhael)

Poster# 137 ALTERATIONS IN CARDIAC IRON STATUS, ELECTROPHYSIOLOGICAL RESPONSES AND TRANSCRIPT LEVELS IN MOUSE MODELS OF DIETARY AND GENETIC IRON LOADING Kristy Martin, Bachelor², Daniel Johnstone, PhD³, Ross Graham, PhD4, Dirk van Helden, PhD², Karen Kerr, PhD², Sharon Hollins, Bachelor², Derek Laver, PhD², John Olynyk, PhD5, Debbie Trinder, PhD6 and Liz Milward, PhD¹ ¹University of Newcastle; ²School of Biomedical Sciences and Pharmacy and the Hunter Medical Research Institute, the University of Newcastle, Australia; ³Bosch Institute and Discipline of Physiology, University of Sydney, Australia; 4School of Biomedical Sciences and 5 Curtin Health Innovation Research Institute, Curtin University of Technology, Australia; 5Department of Gastroenterology, Fremantle Hospital, Australia; 6School of Medicine and Pharmacology, Fremantle Hospital and Harry Perkins Institute of Medical Research, University of Western Australia, Australia (Presented By: Liz Milward)

Poster# 139 SYSTEMIC AND LOCAL IRON ACCUMULATION LEADS TO OXIDATIVE STRESS IN TYPE 2 DIABETES MELLITUS MICE Min Chen, Bo Jiang, Bachelor and Huijun Chen, Doctor Medical School of Nanjing University (Presented By: Min Chen)

Poster# 141 THE ROLE OF THE MITOCHONDRIAL FERRITIN IN THE HEART AND TESTES Federica Maccarinelli, Maria Regoni, Maura Poli, PhD, Michela Asperti, Paola Ruzzenenti, Magdalena Gryzik and Paolo Arosio, PhD University of Brescia (Presented By: Federica Maccarinelli)

Poster# 143 VALPROIC ACID INDUCES HEPCIDIN EXPRESSION Marie-Laure Island, PhD¹, Thibault Cavey², Patricia Leroyer³, Martine Ropert², Pierre Brissot³ and Olivier Loréal³ ¹INSERM-UMR 991 and National Reference Center for Rare Genetic Iron Overload Diseases, CHU Pontchaillou, Rennes, France; ²INSERM UMR 991 and CHU Pontchaillou, Department of Biochemistry, Rennes France; ³INSERM UMR 991 and University of Rennes 1, France (Presented By: Marie-Laure Island)

Poster# 145 EXOME SEQUENCING IN HFE C282Y HOMOZYGOTES WITH EXTREME HEPATIC IRON OVERLOAD REVEALS MODIFYING GENES FOR THE DEVELOPMENT OF CIRRHOSIS Mary Emond, PhD², Christine McLaren, PhD³, Tin Louie, PhD², Jie Wu, PhD³, Lawrie Powell, MD, PhD¹, Pradyumna Phatak, MD4, James Barton, MD5, Paul Adams, MD6, Lyle Gurrin, PhD7, John Phillips, PhD8, Charles Parker, MD8, Katrina Allen, FRACP, PhD, Deborah Nickerson, PhD², Gregory Anderson, PhD¹, Nathan Subramaniam, PhD¹, Gordon McLaren, MD¹° and Grant Ramm, PhD¹ ¹QIMR Berghofer MRI; ²University of Washington; ³University of California, Irvine; 4Rochester General Hospital; 5Southern Iron Disorders Center; 6London Health Sciences Centre; 7University of Melbourne; 8University of Utah; Murdoch Children’s Research Institute; ¹°VA Long Beach Healthcare System (Presented By: Grant Ramm)

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Poster# 147 A CANDIDATE MECHANISM FOR THE ASSOCIATION OF HFE C282Y WITH REDUCED LDL- CHOLESTEROL Dan Yin, BE, PhD¹, Ibrahim AY Hamad, MSc², Jutta Palmen, MSc³, Anastasia Kalea, PhD³, Andrew Smith, PhD³, Philippa Talmud, DSc³, Steve Humphries, PhD³ and Ann Walker, PhD³ ¹BGI-Wuhan, Wuhan, China, BGI-Shenzhen, Shenzhen 518083, China & UCL, London WC1E 6JF, UK; ²UCL, London WC1E 6JF, UK & Alazhar University, Damietta 34511, Egypt; ³UCL, London WC1E 6JF, UK (Presented By: Dan Yin)

Poster# 149 THE METAL-ION TRANSPORTER ZIP8 (SLC39A8) AND IRON TRANSPORT ACROSS THE PLACENTA Wei Zhang, PhD, Supak Jenkitkasemwong, PhD, Alan Chan, BS and Mitchell Knutson, PhD University of Florida (Presented By: Wei Zhang)

Poster# 151 IRON OVERLOAD PERTURBS COPPER DISTRIBUTION IN MICE Jung-Heun Ha, Caglar Doguer, Xiaoyu Wang, Shireen R. Flores and James F. Collins Food Science and Human Nutrition Dept., University of Florida (Presented By: James F. Collins)

Poster# 153 EFFECTS OF DIFFERENTIATION ON REGULATION OF INTESTINAL IRON ABSORPTION: CELL AND MOLECULAR MECHANISMS Gordon D. McLaren¹, Khushin Patel², Hyder Said², Gregory J. Anderson³ and Hamid M. Said¹ ¹Department of Veterans Affairs Long Beach Healthcare System, Long Beach, CA, and Department of Medicine, University of California, Irvine, CA; ²Department of Veterans Affairs Long Beach Healthcare System, Long Beach, CA; ³QIMR Berghofer Medical Research Institute, Brisbane, Australia, and Faculty of Medicine and Biomedical Sciences, The University of Queensland, Brisbane, Australia (Presented By: Gordon D. McLaren)

Poster# 155 WHAT COULD INSULIN TEACH US ABOUT HEPCIDIN? A COMPARATIVE STUDY BETWEEN THE IRON HORMONE HEPCIDIN AND INSULIN Kosha Metha, PhD², Mohammed Gulrez Zariwala, PhD, Robert Evans, PhD¹, Derek Renshaw, PhD and Sebastien Farnaud, PhD ¹Brunel University; ²University of Berdfordshire (Presented By: Robert Evans)

Poster# 157 CLINICAL FACTORS AFFECTING NTBI LEVELS: INSIGHTS FROM DIFFERENCES BETWEEN A NOVEL FLUORESCENT BEAD METHOD AND THE ESTABLISHED NTA METHOD Maciej Garbowski, MD, Yongmin Ma, PhD, Patricia Evans, PhD, Somdet Srichairatanakool, PhD, Suthat Fucharoen, MD, Robert Hider, PhD and John Porter, MD UCL (Presented By: Maciej Garbowski)

Poster# 159 SECOND INTERNATIONAL ROUND ROBIN FOR THE QUANTIFICATION OF SERUM NON-TRANSFERRIN-BOUND IRON AND LABILE PLASMA IRON IN PATIENTS WITH IRON- OVERLOAD DISORDERS Louise de Swart², Jan Hendriks³, Lisa van der Vorm4, Ioav Cabantchik5, Patricia Evans6, Eldad Hod7, Gary Brittenham8, Yael Furman, Boguslaw Wojczyk¹°, Mirian Janssen¹¹, John Porter6, Vera Mattijssen¹², Bart Biemond¹³, Marius MacKenzie², Raffaella Origa¹4, Renzo Galanello¹4, Robert Hider¹5 and Dorine Swinkels, MD, PhD¹ ¹Radboud University Medical Centre Nijmegen; ²Department of Hematology, Radboud University Medical Center, Nijmegen, the Netherlands; ³Department of Health Evidence, Radboud University Medical Center, Nijmegen, the Netherlands; 4Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, the Netherlands; 5Department of Biochemical Chemistry, Hebrew University of Jerusalem, Israel; 6Department of Haematology, University College London, UK; 7 Department of Pathology and Cell Biology, Columbia University Medical Center, New York, USA; 8 Department of Pediatrics, Columbia University Medical Center, New York, USA; Aferrix Ltd., Tel-Aviv, Israel; ¹°Department of Pathology and Cell Biology, Columbia University Medical Center, New York; ¹¹Department of Internal Medicine, Radboud University Medical Center, Nijmegen, the Netherlands; ¹²Department of Hematology, Rijnstate Hospital, Arnhem, The Netherlands; ¹³Department of Hematology, Academic Medical Center, Amsterdam, The Netherlands; ¹4Regional Microcythemia Hospital, University of Cagliari, department of Biomedical Science and Biotechnology, Cagliari, Italy; ¹5Institute of Pharmaceutical Science, King’s College London, UK (Presented By: Dorine Swinkels)

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Poster# 161 IRON, CHOLESTEROL AND MIR122: DEMONSTRATION OF THE PRESENCE OF THE “DELETED” MIR122 GENE IN HEPG2 CELLS Ibrahim Hamad, MSc¹, Yue Fei, MSc¹, Anastasia Kalea, PhD¹, Dan Yin, PhD², Andrew Smith, PhD¹, Jutta Palmen, MSc¹, Steve Humphries, PhD¹, Philippa Talmud, DSc¹ and Ann Walker, PhD¹ ¹UCL, London; ²Wuhan, China/UCL,London (Presented By: Ann Walker)

Poster# 163 MANGANESE NEUROTOXICITY DERIVES IN PART FROM PERTURBED FE/ IRE/ IRP REGULATION IN RATS AND MODELS OF HUMAN NEURONS Vivek Venkataramani, MD², Yanyan Liu, PhD³, Catherine Cahill, PhD³, Katharina Fernsebner, PhD4, Bernhard Michalke, PhD4, Hong Jiang, MD5, Scott Ayton, PhD6, Ashley Bush, MD6, Xudong Huang, PhD³ and Jack Rogers, PhD¹ ¹MGH/Harvard; ²University of Goettingen; ³MGH/ Harvard; 4Munich, Germany; 5Qingdao, China; 6Melbourne, Australia (Presented By: Jack Rogers)

Poster# 165 WHAT MAKES A PRESUMED ZINC TRANSPORTER, SLC39A13, EXPORT IRON? Guiran Xiao, PhD, Mengran Zhao, PhD and Bing Zhou, Prof Tsinghua University (Presented By: Guiran Xiao)

Poster# 167 ALR PROTEIN, A CRITICAL PROTEIN IN CARDIAC DEVELOPMENT, REGULATES CELLULAR IRON HOMEOSTASIS AND MATURATION OF CYTOSOLIC FE/S PROTEINS BY PARTICIPATING IN MITOCHONDRIAL TRANSPORT OF ATP-BINDING CASSETTE (ABC)-B8 Jimmy Chnag, PhD and Hossein Ardehali, MD, PhD Northwestern University (Presented By: Hossein Ardehali)

Poster# 169 IRON-DEPENDENT EPIGENETIC REGULATION BY THE HISTONE DEMETHYLASE KDM4A IN THE HEART Yu Jin Chung, Magda Wolna, Peter Robbins, DPhil and Samira Lakhal-Littleton, DPhil University of Oxford (Presented By: Yu Jin Chung)

Poster# 171 SECONDARY IRON OVERLOAD INTERFERES WITH ERYTHROFERRONE SIGNALING Martin Vokurka, MD, PhD, Luliia Gurieva, MD, Jana Frydlova, PhD, Petr Prikryl, PhD and Jan Krijt, PhD 1st Medical Faculty, Charles University, Prague, Czech Republic (Presented By: Martin Vokurka)

Poster# 173 INTERROGATING HEME LEVELS USING SUBCELLULAR TARGETING OF HEMOPROTEIN PROBES Xiaojing Yuan¹, Ana Beatriz Walter Nuno Da Silva, MS² and Iqbal Hamza, PhD³ ¹University of Maryland; ²Universidade Federal do Rio de Janeiro, Brazil; ³University of Maryland, USA (Presented By: Xiaojing Yuan)

Poster# 175 MITOFERRIN DEFICIENCY PROVOKES AN ECDYSONE SYNTHESIS IMPAIRMENT AND AN IMMUNE SYSTEM OVERREACTION Jose Llorens, PhD¹, Christoph Metzendorf, PhD², Fanis Missirlis, PhD³ and Maria Lind, PhD4 ¹Uppsala University; ²Department of Comparative Physiology, Uppsala University. Uppsala, Sweden. Biochemistry Center, Heidelberg University, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany; ³Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados. Mexico City, Mexico.; 4Department of Comparative Physiology, Uppsala University. Uppsala, Sweden. (Presented By: Jose Llorens)

TUESDAY, SEPTEMBER 08, 2015

OVERVIEW

7:00 - 14:30 Registration/Information Desk Open Location: QiZhen Lobby (1st Floor)

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7:30 - 12:00 Exhibit Hours Location: QiZhen Hall Corridor (3rd Floor)

GENERAL SESSION

7:15 - 8:30 Meet the Experts I: Novel Molecular Technologies for Investigation Iron Homeostasis Location: Haoyue Hall (1st Floor) Moderators: Iqbal Hamza, PhD Martina Muckenthaler, PhD Students/Trainees ONLY - Space is limited.

8:30 - 10:00 Plenary Session II: Structure and Function of Iron-Related Proteins Location: QiZhen Hall (3rd Floor) Chairs: Huijun Chen, PhD Robert W. Evans, BA, PhD

8:30 - 8:55 The NTBI Transporter SLC39A14: From Function to Structure Speaker: Mitchell Knutson, PhD

8:55 - 9:20 The Ferritins: Complex, Multifunctional Protein Nanocages with Self-Synthesized, Internal Iron Minerals Speaker: Takehiko Tosha, PhD

9:20 - 9:45 Structural and Functional Studies of a Divalent Metal Transporter Speaker: Mika Jormakka, PhD

9:45 #22 NEW INSIGHTS INTO FERRITIN TRAFFICKING, POLARIZATION AND SECRETION Marianna Truman-Rosentsvit, Adi Bukris and Esther G. Meyron-Holtz Technion Israel Institute of Technology Presented By: Esther Meyron-Holtz

10:00 - 10:30 Coffee Break Location: QiXhen Hall Corridor (3rd Floor)

10:30 - 12:00 Plenary Session III: Special Symposium - Cardiac Iron Homeostasis Location: QiZhen Hall (3rd Floor) Chairs: Greg Anderson, PhD Clara Camaschella, MD

10:30 - 11:00 Iron in the Heart: A Paradigm of Organ Susceptibility to Siderosis and Sideropenia. Etiopathology and Treatment Speaker: Ioav Cabantchik, MD, PhD

11:00 - 11:30 Cardiomyopathies and Iron Status: A Clinical Outlook Speaker: Antonio Piga, MD

11:30 - 12:00 The Cardiac Hepcidin/Ferroportin Axis is Important for Cardiac Iron Homestasis and Function Speaker: Samira Lakhal-Littleton, BSc DPhil

12:00 - 12:30 Business Meeting (IBIS Members)

WEDNESDAY, SEPTEMBER 09, 2015

OVERVIEW

7:00 - 18:15 Registration/Information Desk Open Location: QiZhen Lobby (1st Floor)

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7:30 - 15:00 Exhibit Hours Location: QiZhen Hall Corridor (3rd Floor)

GENERAL SESSION

7:15 - 8:30 Meet the Experts II: Genetics of Human Iron Disorders Location: Haoyue Hall (1st Floor) Moderators: V. Nathan Subramaniam, MSc, PhD Christopher Vulpe, MD, PhD Students/Trainees ONLY - Space is limited.

8:30 - 10:00 Plenary Session IV: Iron Loading Hemoglobinopathies Location: QiZhen Hall (3rd Floor) Chairs: Suthat Fucharoen, MD Domenico Girelli, MD, PhD

8:30 - 8:55 Regulatory Role of Iron Transport in Beta-Thalassemia Speaker: Yelena Ginzburg, MD

8:55 - 9:20 Iron Overload in the Hemoglobinopathies & Transfusion-Dependent Anemias A Clinical Perspective Speaker: Joy Ho, MBBS,DPhil,FRACP

9:20 - 9:45 What is the Unmet Need In Iron Chelation Practice in 2015: Experiences From Hemoglobinopathies Speaker: Vip Viprakasit, MD

9:45 #23 LACK OF BETA-1 INTEGRIN LIMITS STRESS EYTHOPOIESIS AND SPLENOMEGALY IN BETA- THALASSEMIA Roberta Chessa, PhD , Bart J. Crielaard, MD, PhD , Ritama Gupta , Carla Casu, PhD , Rick Feldman, PhD and Stefano Rivella, PhD CHOP; Bayer Healthcare Presented By: Stefano Rivella

10:00 - 10:30 Coffee Break

10:30 - 12:00 Plenary Session V: Iron, Infection and Inflammation Location: QiZhen Hall (3rd Floor) Chairs: Francois Canonne-Hergaux, PhD Martina Muckenthaler, PhD

10:30 - 10:55 Iron Deficient RBCS From Gambian Children and Pregnant Women are Resistant to Field Isolates of Falciparum Malaria Speaker: Carla Cerami, MD, PhD

10:55 - 11:20 Inflammation-Driven Heme/Iron Cytotoxicity in Parkinson's Disease Speaker: Raffaella Gozzelino, PhD

11:20 - 11:45 Responses of the Gut Microbiota to Supplementary Iron: A Survey at the Host- Microbial Interface Speaker: Guus Kortman, PhD

11:45 #24 LIPOSOME-ENCAPSULATED H-FERRITIN IMPROVES SURVIVAL IN A SOD1 MUTANT MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS James Connor, PhD, Amanda Snyder, PhD, Achuthamangalam Madhankumar, PhD, Elizabeth Neely, BS, Elias Rizk, MD, Olivia Hess and Zachary Simmons, MD Penn State University College of Medicine Presented By: James Connor

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12:00 - 13:30 Lunch Location: QiZhen Hotel (Haoyue Hall - 1st Floor & Oufang Hall - 2nd Floor)

13:30 - 14:30 Keynote Lecture II Location: QiZhen Hall (3rd Floor) Chair: Fudi Wang, PhD

Vitamin C and Fe Based Enzymes in Somatic Cell Reprogramming Speaker: Duanqing Pei, PhD

14:30 - 16:00 Concurrent Session V and Session VI

Concurrent Session V: Cell Death and Autophagy with an Iron Twist Location: QiuShi Hall (3rd Floor) Chairs: Paolo Arosio, PhD Gaetano Cairo, PhD

14:30 - 14:55 Ferroptosis: A New Name for an Old Way to Die Speaker: Marcus Conrad, PhD

14:55 - 15:20 Role of NCOA4 Protein in DNA Replication Control and Ferritinphagy Speaker: Francesca Carlomagno, PhD

15:20 #25 EFFECT OF DIETARY IRON INTAKE ON MITOPHAGY, MITOCHONDRIAL BIOGENESIS AND MITOCHONDRIAL DYNAMIC IN SKELETAL MUSCLE OF RATS YihFong Liew, PhD, Hsuan-Lin Chen, MS and Dong-Lin Tsai, MS Fu-Jen Catholic University Presented By: YihFong Liew

15:33 #26 CHARACTERIZATION AND CLONING OF HUMAN NUCLEAR RECEPTOR COACTIVATOR 4 (NCOA4), THE CARGO RECEPTOR MEDIATING FERRITINOPHAGY Magdalena Gryzik, PhD Student, Maura Poli, PhD, Margherita Di Somma, PhD Student, Claudia Saraceno, PhD, Paola Ruzzenenti, PhD Student, Michela Asperti, PhD Student, Maria Regoni, PhD Student and Paolo Arosio, PhD University of Brescia Presented By: Magdalena Gryzik

15:46 #27 INTRACELLULAR IRON DEREGULATION INDUCES SENESCENCE PHENOTYPE IN FIBROBLASTS FROM A NEUROFERRITINOPATHY PATIENT Sonia Levi, PhD , Anna Cozzi, PhD , Paolo Santambrogio, PhD and Chiara Fiorillo, PhD Vita-Salute San Raffaele University; San Raffaele Scientific Institute; IRCCS Stella Maris, Pisa Presented By: Sonia Levi

Concurrent Session VI: Novel Therapies for Iron Disorders Location: QiZhen Hall (3rd Floor) Chairs: Elizabeta Nemeth, PhD Stefano Rivella, PhD

14:30 - 15:00 Exploiting Hepcidin and Its Pathway to Limit Iron Absorption and Erythroid Iron Intake Speaker: Stefano Rivella, PhD

15:00 #28 HEPARINS AS INHIBITORS OF HEPCIDIN: MECHANISM OF ACTION Michela Asperti, PhD Student , Maura Poli, PhD , Paola Ruzzenenti, PhD Student , Annamaria Naggi, PhD , Natascia Campostrini, PhD , Magdalena Gryzik, PhD Student , Federica Maccarinelli , Domenico Girelli, MD and Paolo Arosio, PhD University of Brescia; Ronzoni Institute of Milano; University of Verona Presented By: Michela Asperti

15:15 #29 MONOCLONAL ANTIBODIES NEUTRALIZING HEMOJUVELIN DOWNREGULATE HEPCIDIN: CANDIDATE DRUGS FOR THE TREATMENT OF IRIDA AND ANEMIA OF CHRONIC DISEASE

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Andreas Popp, MD , Preethne Boeser , Suzana Kovac, MD , Daniela Casarrubea, MD , Dunja Ferring- Appel , Matthias W. Hentze, Prof, MD and Bernhard K. Mueller, PhD, MD AbbVie Deutschland GmbH & Co. KG; EMBL, Heidelberg, Germany Presented By: Andreas Popp

15:30 #30 IRON CHELATORS IN NANOFORMULATIONS FOR SPECIFIC TARGETING AND EFFECTIVE REMOVAL OF MACROPHAGE IRON FOR TREATMENT OF IRON OVERLOAD DISEASES Guangjun Nie , Shanshan Guo, BS , Gang Liu, PhD and Greg Anderson, PhD CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, China, Beijing, 100190; Iron Metabolism Laboratory, Queensland Institute of Medical Research, Brisbane Queensland, Australia, 4029 Presented By: Guangjun Nie

15:45 #31 TARGETING TMPRSS6 USING ANTISENSE TECHNOLOGY FOR THE TREATMENT OF β- THALASSEMIA Shuling Guo, PhD , Mariam Aghajan, PhD , Carla Casu, PhD , Sara Gardenghi, PhD , Sheri Booten , Stefano Rivella, PhD and Brett Monia, PhD Isis Pharmaceuticals; Weill Cornell Medical College Presented By: Shuling Guo

16:00 - 19:00 Poster Walk II Location: Shunshui Hall, Lizhou Hall and Yangming Hall (4th Floor) Refreshments available. Beer and Wine service to begin at 17:00

Poster# 2 NOVEL 3-HYDROXYPYRIDIN-4-ONE HEXADENTATE LIGAND-BASED POLYMERIC IRON CHELATOR: SYNTHESIS, CHARACTERIZATION AND ANTIMICROBIAL EVALUATION Tao Zhou, Yingjun Zhou, MSc, Xiaole Kong, PhD, Junpei Li, MSc, Yongmin Ma, PhD and Robert Hider, PhD Zhejiang Gongshang University (Presented By: Tao Zhou)

Poster# 4 SILVER NANOPARTICLES INDUCED RNA POLYMERASE-SILVER BINDING AND RNA TRANSCRIPTION INHIBITION IN ERYTHROID PROGENITOR CELLS Sijin Liu Chinese Academy of Sciences (Presented By: Sijin Liu)

Poster# 6 IRON BIOAVAILABILITY OF SWEET POTATO AND MORINGA LEAVES IN COMPARISION WITH LEAFY GREEN VEGETABLES COMMONLY CONSUMED IN GHANA Francis Amagloh, PhD², Richard McBride, BSc¹ and Tatiana Christides¹ ¹University of Greenwich, Faculty of Engineering and Science, United Kingdom; ²University for Development Studies, Ghana (Presented By: Tatiana Christides)

Poster# 8 GLP-1 AND FE CHELATION INDEPENDENTLY RESCUE NAF-1 DEFICIENT INSULINOMA B CELLS (WOLFRAM SYNDROME 2) BY REDUCING MITOCHONDRIAL LABILE IRON AND ROS FORMATION Rachel Nechushtai, DSc¹, Erol Cerasi, MD², Gil Leibowitz, MD² and Ioav Cabantchik, MD, PhD¹ ¹Institute of Life Sciences, Hebrew University of Jerusalem; ²Hadassah Medical Center, Hebrew University of Jerusalem (Presented By: Rachel Nechushtai)

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Poster# 10 ATRANSFERRINEMIA: AN ULTRA-RARE IRON-LOADING ANAEMIA - REPORT OF 6 CASES FROM 4 FAMILIES Anna Barqué², Francisco Fuster², Cristina Díaz de Heredia, PhD, MD³, Eunice S. Edison, PhD, MD4, Katja Moser, MD5, Erica Morán, PhD², Rekha Athiyarath4, Jessica Aranda², Ana M. Rojas, PhD6, Ilona Kleine, PhD7 and Mayka Sanchez, PhD¹ ¹Josep Carreras Leukaemia Research Institute (IJC), Diagnostics in Iron Metabolism Disease (D IRON) and Iron Metabolism: Regulation and Diseases Group. and Institute of Predictive and Personalized Medicine of Cancer (IMPPC). Badalona, Barcelona.; ²Josep Carreras Leukaemia Research Institute (IJC), Diagnostics in Iron Metabolism Disease (D IRON). Badalona, Barcelona and Institute of Predictive and Personalized Medicine of Cancer (IMPPC). Badalona, Barcelona.; ³Vall d'Hebron University Hospital, Pediatric Haematology-Oncology Service. Barcelona; 4Christian Medical College Hospital, Haematology Department. Vellore, India; 5Klinik für Kinder- und Jugendmedizin Hospital, Pediatrics and Neonatology Department. Aschaffenburg, Germany; 6Campus University Hospital Virgen del Rocío. Institute of Biomedicine of Seville. Sevilla, Spain; 7Sanquin Blood Supply Foundation, Medical Department Plasma Products. Amsterdam, the Netherlands (Presented By: Mayka Sanchez)

Poster# 12 EFFECTS OF ACUTE EXERCISE ON IRON METABOLISM IN RATS Yu Wang², Ning Zheng³, Jia Chen³, Peng Yu, PhD4, Yanzhong Chang, PhD4 and Yuqian Liu, PhD¹ ¹College of Physical Education, Hebei Normal University; ²College of Physical Education, Hebei Normal University; ³College of Physical Education, Hebei Normal University. Laboratory of Molecular Iron Metabolism, College of Life science, Hebei Normal University,Shijiazhuang,050024; 4Laboratory of Molecular Iron Metabolism, College of Life science, Hebei Normal University,Shijiazhuang,050024 (Presented By: Yuqian Liu)

Poster# 14 ERR GAMMA CONTROLS SALMONELLA INFECTION BY MODULATING HOST IRON HOMEOSTASIS Don-Kyu Kim, PhD¹, Jae-Ho Jeong, PhD², Ki-Sun Kim, MS³, Yoon Seok Jung, BS³, Hyon Choy, PhD² and Hueng-Sik Choi, PhD³ ¹Chonnam University/Department of Biotechnology; ²Chonnam National University Medical School/Department of Microbiology; ³Chonnam University/National Creative Research Initiatives Center for Nuclear Receptor Signals and Hormone Research Center, School of Biological Sciences and Technology (Presented By: Don-Kyu Kim) Poster# 16 LOW DIETARY IRON INTAKE RESTRAINS THE INTESTINAL INFLAMMATORY RESPONSE AND PATHOLOGY OF ENTERIC INFECTION BY FOOD-BORNE BACTERIAL PATHOGENS Guus Kortman, PhD¹, Michelle Mulder¹, Thijs Richters¹, Nanda Shanmugam, PhD², Estela Trebicka, BSc², Jos Boekhorst, PhD³, Harro Timmerman, PhD³, Rian Roelofs, BSc¹, Erwin Wiegerinck, BSc¹, Coby Laarakkers, BSc¹, Dorine Swinkels¹, Albert Bolhuis, PhD4, Bobby Cherayil, PhD² and Harold Tjalsma, PhD¹ ¹Radboud University Medical Center; ²Massachusetts General Hospital; ³NIZO Food Research BV; 4University of Bath (Presented By: Guus Kortman, PhD)

Poster# 20 SPECIATION OF NON-TRANSFERRIN-BOUND IRON Yongmin Ma¹ and Robert Hider, PhD² ¹Zhejiang Chinese Medical University; ²King's College London (Presented By: Yongmin Ma)

Poster# 22 PROFILIN-2, A NEW PLAYER IN IRON METABOLISM Sara Luscieti², Pietro Pilo Boyl, PhD³, Bruno Galy, PhD4, Lucía Gutiérrez, PhD5, Maya Shvartsman, PhD², Jorge Couso², Alejandro Negro², Maria Puerto Morales, PhD5, Matthias W. Hentze, PhD, MD4, Walter Witke, PhD³ and Mayka Sanchez, PhD¹ ¹Josep Carreras Leukaemia Research Institute (IJC), Diagnostics in Iron Metabolism Disease (D IRON) and Iron Metabolism: Regulation and Diseases Group. and Institute of Predictive and Personalized Medicine of Cancer (IMPPC). Badalona, Barcelona.; ²Josep Carreras Leukaemia Research Institute (IJC), Iron Metabolism: Regulation and Diseases Group. and Institute of Predictive and Personalized Medicine of Cancer (IMPPC). Badalona, Barcelona.; ³University of Bonn, Institute of Genetics, Bonn, Germany; 4European Molecular Biology Laboratory (EMBL), Heidelberg, Germany; 5Instituto de Ciencia de Materiales de Madrid (ICMM-CSIC), Madrid, Spain (Presented By: Mayka Sanchez)

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Poster# 24 HFE GENOTYPE AND A FORMULATED DIET CONTROLLING FOR IRON STATUS ATTENUATE EXPERIMENTAL CEREBRAL MALARIA IN MICE Dominique Leitner, PhD², Jose Stoute, MD¹, Mary Landmesser, BS¹, Elizabeth Neely, BS¹ and James Connor, PhD¹ ¹Penn State University; ²Mount Sinai Icahn School of Medicine (Presented By: James Connor)

Poster# 26 DEVELOPMENT OF NOVEL THERAPEUTIC AGENTS FOR NEUROBLASTOMA THROUGH IRON AND CHOLESTEROL METABOLISMS STUDY Sang Lee, PhD, Becky Slagle-Webb, BS, Cara-Lynne Schengrund, PhD and James Connor, PhD Pennsylvania State University (Presented By: Sang Lee)

Poster# 28 TOWARDS THE IDENTIFICATION OF THE MAMMALIAN ASCORBATE-STIMULATED PLASMA MEMBRANE FERRICYANIDE REDUCTASE Alfons Lawen, Dr rer nat¹, Anurag A. Atnerkar, BSc¹ and Darius J. R. Lane, p² ¹Monash University; ²Sydney University (Presented By: Alfons Lawen)

Poster# 30 CHARACTERIZATION OF FERROPORTIN DISEASE-RELATED RESIDUES PREDICTED TO CONFER HEPCIDIN RESISTANCE S. Aschemeyer, B. Qiao, A. Sek, M. Jormakka, T. Ganz and E. Nemeth University of California, Los Angeles (Presented By: S. Aschemeyer)

Poster# 32 NEXT-GENERATION SEQUENCING: APPLICATION OF A NOVEL PLATFORM TO THE ANALYSIS OF ATYPICAL IRON DISORDERS Cameron McDonald, PhD, Lesa Ostini, Daniel Wallace, Alyson Lyons, Darrell Crawford and V. Nathan Subramaniam QIMR Berghofer (Presented By: Cameron McDonald)

Poster# 34 IRON-DEFICIENT MILK OF HEPHAESTIN KNOCKOUT MICE UNDERLIES A HAIR LOSS PHENOTYPE IN PUPS ASSOCIATED WITH INCREASED ANDROGEN RECEPTOR EXPRESSION IN THE SKIN Amin Sobh, Kathryn Page, PhD, Erica Lachenauer, Julie Luong, Chenchen Han, Hiroko Irimagawa, Yuanchi He, David Killilea, PhD, David Frazer, Gregory Anderson, PhD, Brie Fuqua, PhD and Chris Vulpe, MD, PhD University of California, Berkeley (Presented By: Amin Sobh)

Poster# 36 FUNCTIONAL CHARACTERIZATION AND EVALUATION OF A THIRD CASE OF SIDEROBLASTIC ANEMIA WITH BIALLELIC MUTATIONS OF GLRX5 Raêd Daher, Abdellah Mansouri, Sophie Bayart, Alain Martelli, Isabelle Callebaut, Ly Sunnaram, Julien Goustille, Claire Oudin, Anne Marie Jouanolle, Carole Beaumont, Hervé Puy, Zoubida Karim and Caroline Kannengiesser INSERM U1149, Université Paris Diderot, AP-HP, Département de Génétique, Hôpital Bichat, Laboratory of Excellence GR-Ex, 16 rue Henri Huchard, 75018 Paris, France (Presented By: Raêd Daher)

Poster# 38 IRON IS A RISK FACTOR FOR ATHEROSCLEROSIS: CONFIRMATION OF THE IRON HYPOTHESIS IN A MOUSE MODEL OF HEMOCHROMATOSIS Francesca Vinchi, PhD¹, Sandro Altamura, PhD¹, Milene Costa da Silva, MSc¹, Bruno Galy, PhD², Matthias W. Hentze, MD, PhD¹ and Martina U. Muckenthaler, PhD¹ ¹Molecular Medicine Partnership Unit (MMPU), University of Heidelberg & EMBL; ²German Cancer Research Center (DKFZ) (Presented By: Francesca Vinchi)

Poster# 40 ROLE OF OXIDATIVE STRESS ON REGULATION OF IRON HOMEOSTASIS GENES IN NEURONAL CELL: IMPLICATION IN IRON ACCUMULATION Som Dev, MSc¹, Sanju Kumari, MSc¹, Neena Singh, MD, PhD² and Chinmay K. Mukhopadhyay, PhD¹ ¹SCMM, JNU, New Delhi; ²Case Western Reserve University, Cleveland (Presented By: Som Dev)

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Poster# 42 DISRUPTION OF THE HEPCIDIN/FERROPORTIN REGULATORY CIRCUITRY CAUSES INCREASED PULMONARY IRON CONTENT AND RESTRICTIVE LUNG DISEASE Joana Neves¹,²,4, Milene Costa da Silva¹,²,4, Dominik Leitz²,5, Raman Agrawal²,5, Bruno Galy³, Matthias W. Hentze³, Marcus A. Mall²,5, Sandro Altamura¹,²,6 and Martina U. Muckenthaler¹,²,6 ¹University of Heidelberg, Heidelberg, Germany; ²Molecular Medicine Partnership Unit (MMPU), Heidelberg, Germany; ³European Molecular Biology Laboratory (EMBL), Heidelberg, Germany; 4Graduate Program in Areas of Basic and Applied Biology, University of Porto, Portugal; 5Department of Translational Pulmonology, Translational Lung Research Center, Member of the German Center for Lung Research, University of Heidelberg, Heidelberg, Germany; 6equal contribution (Presented By: Joana Neves)

Poster# 44 FERROPORTIN AND HEPCIDIN IN RETINAL IRON HOMEOSTASIS Delu Song, MD, PhD¹, Milan Theurl², Esther Clark³, Jacob Sterling³, Sandro Altamura4, Bruno Galy5, Matthias Hentze6, Martina Muckenthaler4, Steven Grieco7 and Joshua Dunaief³ ¹University of Pennsylvania; ²Department of Ophthalmology and Optometry, Innsbruck Medical University, Innsbruck, Austria; ³F. M. Kirby Center for Molecular Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania, United States; 4Department of Pediatric Hematology, Oncology and Immunology, University of Heidelberg, Germany; 5European Molecular Biology Laboratory, Meyerhofstrasse, Heidelberg, Germany; 6Molecular Medicine Partnership Unit, Heidelberg, Germany; 7Department of Neuroscience, University of Florida, Miami, Florida, United States (Presented By: Delu Song)

Poster# 46 THE EFFECT OF OKRA (ABELMOSCHUS ESCULENTUS LINN) AND QUERCETIN ON OXIDATIVE STRESS IN STABLY EXPRESSED HFE NEUROBLASTOMA SH-SY5Y CELL LINE Nootchanat Mairuae, PhD¹, Sang Lee, PhD², Poonlarp Cheepsunthorn, PhD³, Walaiporn Tongjaroenbuangam, PhD4 and James R. Connor, PhD5 ¹Faculty of Medicine, Mahasarakham University; ²College of Medicine/Milton S. Hershey Medical Center, Hershey, PA, USA; ³Faculty of Medicine, Chulalongkorn University, Thailand; 4Faculty of Medicine, Mahasarakham University, Thailand; 5College of Medicine/Milton S. Hershey Medical Center, Hershey, PA, USA (Presented By: Nootchanat Mairuae)

Poster# 48 EVALUATION OF THE PHOTOPROTECTIVE POTENTIAL OF NOVEL HEXADENTATE IRON CHELATORS AGAINST SUNLIGHT-INDUCED DAMAGE TO SKIN CELLS Olivier Reelfs, PhD, DSc², Vincenzo Abbate, PhD³, Robert C. Hider, PhD³ and Charareh Pourzand, PhD, DSC¹ ¹University of Bah; ²University of Bath; ³King's College London (Presented By: Charareh Pourzand)

Poster# 50 IRON DEFICIENCY AND RESTLESS LEGS SYNDROME. A PROSPECTIVE STUDY IN BLOOD DONORS Marta Clavero Olmos, Nadia Matskiv, Ines Valdes Gross, Ana López Aparicio, Alejandro García- Espona Pancorbo, Rosa Peraita and Alejandro del Castillo Rueda Hospital General Universitario Gregorio Marañón (Presented By: Marta Clavero Olmos)

Poster# 52 REVISITING THE ROLE OF THE HFE IN ENTEROCYTES Betül Alan, MSc and Maja Vujic Spasic, Prof Dr Institute for Comparative Molecular Endocrinology, University of Ulm (Presented By: Betül Alan)

Poster# 54 COMPARATIVE EVALUATION OF MANAGEMENT BY MACROPHAGES OF INTRAVENOUS PHARMACEUTICAL IRON FORMULATIONS James Connor, PhD¹, Xuesheng Zhang, PhD¹, Anne Nixon, BS¹, Becky Webb, BS¹ and Joseph Perno, MD, PhD² ¹Penn State University College of Medicine; ²Luitpold Pharmaceuticals, Inc. (Presented By: James Connor)

Poster# 56 THE PREDICTION OF FLUORINATED 3-HYDROXYPYRIDIN-4-ONE LOG P VALUES Yu-Lin Chen, Dr¹, Dave Barlow, Dr², Yongmin Ma¹ and Robert Hider, Dr³ ¹Zhejiang Chinese Medical University; ²King's College London; ³King's College London (Presented By: Yongmin Ma)

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Poster# 58 DEVELOPMENT OF PEPTIDE THERAPEUTICS FOR IRON DISORDERS Daniela Goncalves, MSc¹, Gautam Rishi, PhD², Randy Aliyanto, BSc¹, Johan Rosengren, PhD¹, Nathan Subramaniam, Prof², David Frazer, PhD² and Richard Clark, PhD¹ ¹School of Biomedical Sciences, University of Queensland; ²QIMR Berghofer Research Institute, Brisbane (Presented By: Daniela Goncalves)

Poster# 60 NICOTIANAMINE IN TOMATO PLANT FLUIDS AS AFFECTED BY IRON DEFICIENCY AND IRON RESUPPLY Pablo Diaz-Benito, MSc, Anunciación Abadía, PhD, Javier Abadia, PhD and Ana Álvarez- Fernández, PhD Spanish Council for Scientific Research (CSIC) (Presented By: Javier Abadia)

Poster# 62 HEMOPEXIN PRESERVES CARDIAC FUNCTION BY LIMITING HEME-DRIVEN TOXICITY ON CARDIOMYOCYTES Giada Ingoglia, PhD student¹, Alessandra Ghigo, PhD¹, Francesca Vinchi, PhD², James Cimino, Dr¹, Lorenzo Silengo, PhD¹, Emilio Hirsch, PhD¹, Fiorella Altruda, PhD¹ and Emanuela Tolosano, PhD¹ ¹University of Torino (Italy); ²University of Heidelberg (Germany) (Presented By: Giada Ingoglia)

Poster# 64 TOWARD BETTER PATHOPHYSIOLOGICAL CHARACTERIZATION AND THERAPEUTIC SOLUTION IN ?-THALASSEMIA TRAIT PATIENTS WITH IRON OVERLOAD. PRELIMINARY REPORTS FROM AN ONGOING STUDY Natascia Campostrini, Fabiana Busti, Sadaf Badar, Alberto Ferrarini, Luciano Xumerle, AnnaChiara Giuffrida, Giovanna De Matteis, Riccardo Manfredi, Paola Capelli, Annalisa Castagna, Massimo Delledonne, Oliviero Olivieri and Domenico Girelli Department of Medicine, University of Verona, Italy (Presented By: Domenico Girelli)

Poster# 66 EXPRESSION PROFILING OF IRON METABOLISM–RELATED GENES IN TAMOXIFEN RESISTANT BREAST CANCER CELLS Veronika Tomkova², Zuzana Rychtarcikova¹, Sandra Lettlova² and Jaroslav Truksa² ¹Academy of Sciences of the Czech Republic, Institute of Biotechnology; ²Academy of Sciences of the Czech Republic, Institute of Biotechnology, Prague, Czech Republic (Presented By: Zuzana Rychtarcikova)

Poster# 68 TOWARDS HARMONIZATION OF WORLDWIDE HEPCIDIN ASSAYS: IDENTIFICATION OF A COMMUTABLE REFERENCE MATERIAL Lisa van der Vorm, Coby Laarakkers, Siem Klaver, Cas Weykamp and Dorine Swinkels, MD, PhD Radboud University Medical Centre Nijmegen (Presented By: Dorine Swinkels)

Poster# 70 ATOH8 MRNA LEVELS DO NOT REGULATE HEPCIDIN EXPRESSION IN HUMAN HEPATOCYTE CULTURES Marie-Laure Island, PhD¹, Nadia Fatih², Patricia Leroyer², Lenaick Detivaud¹, Marie-Paule Roth³, Helene Coppin³, Pierre Brissot¹ and Olivier Loreal¹ ¹INSERM-UMR 991 and National Reference Center for Rare Genetic Iron Overload Diseases, CHU Pontchaillou, Rennes, France; ²INSERM UMR 991; ³INSERM-UMR 1043, and University of Toulouse, France (Presented By: Marie-Laure Island)

Poster# 72 MOUSE GENETIC BACKGROUND IMPACTS BOTH ON IRON AND NON-IRON METALS METABOLISM PARAMETERS AND ON THEIR RELATIONSHIPS Thibault Cavey², Martine Ropert², Marie De Tayrac³, Edouard Bardou-Jacquet4, Marie-Laure Island4, Patricia Leroyer5, Claude Bendavid², Pierre Brissot4 and Olivier Loreal¹ ¹INSERM UMR 991 and University of Rennes 1, Rennes, France; ²INSERM-UMR 991 and CHU Pontchaillou, Department of Biochemistry, Rennes, France; ³CNRS-UMR 6290, and CHU Pontchaillou, Molecular Genetics and Genomics Department Rennes, France; 4INSERM-UMR 991 and National Reference Center for Rare Genetic Iron Overload Diseases, CHU Pontchaillou, Rennes, France; 5INSERM-UMR 991 and University of Rennes 1, Rennes, France (Presented By: Olivier Loreal)

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Poster# 74 GLUTATHIONE-HEME, THE MAIN COMPONENT OF THE CYTOSOLIC HEME POOL? Robert Hider¹, Ann Smith, PhD² and Xiaole Kong, PhD¹ ¹King's College London; ²University of Missouri (Presented By: Robert Hider)

Poster# 76 THE CHEMOKINE CCL2 IS A NOVEL MODIFIER OF TISSUE IRON LEVELS AND A PREDICTOR OF DISEASE SEVERITY IN HEREDITARY HEMOCHROMATOSIS Katarzyna Mleczko-Sanecka¹,¹°,¹¹, Sandro Altamura¹,¹°,¹¹, Mingang Zhu¹,¹°,¹¹, Milene Costa da Silva¹,¹°, Maja Vujic Spasic¹,¹°, Claudia Guida²,¹°, Oriana Marques³, Matthew Lawless4, Dorine Swinkels5, Regina Maus6, Jorge P. Pinto³, Wen-Pin Chen7, Nikolas Gunkel8, Margarida Lima, Rainer Pepperkok²,¹°, Christine E. McLaren7, Ulrich Maus6, Heiko Runz¹,¹°, Graca Porto³,¹¹ and Martina U. Muckenthaler¹,¹°,¹¹ ¹University of Heidelberg, Germany; ²European Molecular Biology Laboratory (EMBL), Heidelberg, Germany; ³University of Porto, Portugal; 4University College Dublin, Mater Misericordiae University Hospital, Dublin, Ireland; 5Radboud University Medical Center, The Netherlands; 6Hannover School of Medicine, Germany; 7University of California, Irvine, CA, United States; 8German Cancer Research Center (DKFZ), Heidelberg, Germany; CHP- Hospital Santo Antonio, Porto, Portugal; ¹°Molecular Medicine Partnership Unit (MMPU), Heidelberg, Germany; ¹¹Equal contribution (Presented By: Katarzyna Mleczko-Sanecka)

Poster# 78 IRON DEFICIENCY IN ALCOHOLIC LIVER DISEASE: A CONSEQUENCE OF CHANGES IN PROTEINS INVOLVED IN DUODENAL IRON ABSORPTION? Molly Jacob, MBBS, MD, PhD, Jithu James, MSc, Joe Varghese, MBBS, MD, DNB, Kavita Rasalkar, MBBS, MD, Ramya Raghavan, MBBS, MD and CE Eapen, MBBS,MD, DM Christian Medical College, Vellore, India (Presented By: Molly Jacob)

Poster# 80 BRAIN IRON LOCALIZES TO MYELIN AND AFFECTS EXPRESSION OF GENES RELATED TO MYELIN AND NEURODEGENERATION WITH BRAIN IRON ACCUMULATION DISORDERS IN MICE WITH DISRUPTION OF HFE AND TFR2 Moones Heidari, MSc¹, Dan Johnstone, PhD², Brianna Bassett, B Biomed Hons¹, Ryan Horn¹, Kristy Martin, B Biomed Sci¹, Dylan Huff¹, James Montgomery, B Biomed Hons¹, Conceicao Bettencourt, PhD³,Anita Chua, PhD4, Ross Graham, PhD5, John Olynyk, MD, PhD4, Debbie Trinder, PhD6 and Liz Milward, PhD¹ ¹School of Biomedical Sciences and Pharmacy/University of Newcastle; ²Bosch Institute and Discipline of Physiology, University of Sydney; ³Department of Molecular Neuroscience, UCL Institute of Neurology; 4Institute for Immunology and Infectious Diseases, Murdoch University; 5School of Biomedical Sciences, Curtin University of Technology; 6School of Medicine and Pharmacology, The University of Western Australia (Presented By: Moones Heidari)

Poster# 82 THE ROLE OF MULTI-COPPER FERROXIDASES IN VIVO IRON METABOLISM Shunli Liu, Jiashuo Zheng, Undergraduate, Mengxia Chen, Undergraduate and Huijun Chen, Professor Medical School of Nanjing University (Presented By: Shunli Liu)

Poster# 84 INSULIN RESISTANCE IN DIET-INDUCED OBESE MICE IS ASSOCIATED WITH DYSREGULATION OF IRON HOMEOSTASIS Joe Varghese, MD¹, Jithu Varghese James, MSc¹, Andrew McKie, PhD² and Molly Jacob, MD, PhD¹ ¹Christian Medical College, Vellore; ²King's College, London (Presented By: Joe Varghese)

Poster# 86 A PRELIMINARY STUDY OF THE LINK BETWEEN IRON INCREASE IN DOPAMINERGIC CELLS AND PARKINSON’S DISEASE Kosha Metha, PhD², Bushra Ahmed, PhD, Mohammed Gulrez Zariwala, PhD, Robert Evans, PhD¹ and Sebastien Farnaud, PhD ¹Brunel University; ²University of Bedfodshire (Presented By: Robert Evans)

Poster# 88 CYTOPROTECTIVE EFFECT OF FERRITIN H IN RENAL ISCHEMIA REPERFUSION INJURY Suzy Torti, PhD, Heather Hatcher, PhD, Lia Tesfay, MS and Frank Torti, MD University of Connecticut Health Center (Presented By: Suzy Torti)

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Poster# 90 DIFFERENTIAL EXPRESSION OF CERULOPLASMIN AND ZYKLOPEN IN HUMAN DUCTAL CARCINOMA TISSUE AND MODEL CELL LINES Rania Darwish, MS², Rasha Mismar, MS², Pamela Nabhan, MS², Amin Sobh, MS³, Mhenia Haidar, PhD4, Julnar Usta, PhD5, Chris Vulpe, MD PhD6 and Zouhair Attieh, PhD¹ ¹American University of Science and Technology (AUST); ²American University of Science and Technology; ³University of California, Berkeley; 4Lebanese University; 5American University of Beirut; 6University of Florida (Presented By: Zouhair Attieh)

Poster# 92 WATER SOLUBLE MAGNETITE NANOPARTICLES FE3O4 – OLEIC ACID-TWEEN 80 : STABILITY, REDOX ACTIVITY AND TRANSPORT IN HEPATOPANCREATIC CELLS OF THE MANGROVE CRAB UCIDES CORDATUS Hector Aguilar Vitorino, PhD¹, Priscila Ortega, PhD², Flavia Pinheiro Zanotto, PhD² and Breno Pannia Espósito, PhD¹ ¹University of São Paulo - Institute of Chemistry; ²University of São Paulo - Biosciences Institute (Presented By: Hector Aguilar Vitorino)

Poster# 94 DISTINCT METABOLOMIC PROFILES IN INBRED MOUSE STRAINS AND CORRELATION WITH IRON Rola Zeidan, BSc, MSc, Daniel Medina-Cleghorn, BS, Kathryn Page, PhD, Stela McLachlan, PhD, Eleazar Eskin, PhD, Daniel Nomura, PhD and Chris Vulpe, MD, PhD University of Florida (Presented By: Rola Zeidan)

Poster# 96 SIRT2 MEDIATED-DEACETYLATION OF NUCLEAR FACTOR (ERYTHROID-DERIVED 2)-LIKE 2 (NRF2) REGULATES CELLULAR IRON HOMEOSTASIS Xiaoyan Yang, MD, PhD, Athanasios Vassilopoulos, PhD, Seong-Hoon Park, PhD, David Gius, MD, PhD and Hossein Ardehali, MD, PhD Northwestern University (Presented By: Hossein Ardehali)

Poster# 98 GENETICALLY CONTROLLED UPTAKE OF FERRITIN AS AN MRI CONTRAST AGENT Christoph Massner, Dipl Biol (to)¹, Giorgio Pariani, Dr², Hannes Rolbieski², Felix Sigmund, MSc² and Gil Gregor Westmeyer, Prof Dr²,³ ¹Nuklearmedizinische Klinik, Klinikum rechts der Isar, Technische Universität München, München/ Institute for Biological and Medical Imaging, Institute for Developmental Genetics, Helmholtz Zentrum München, München; ²Institute for Biological and Medical Imaging, Institute for Developmental Genetics, Helmholtz Zentrum München, München; ³Nuklearmedizinische Klinik, Klinikum rechts der Isar, Technische Universität München, München (Presented By: Christoph Massner)

Poster# 100 DEVELOPMENT OF MITOCHONDRIA-TARGETED IRON CHELATORS FOR THE TREATMENT OF FRIEDRICH ATAXIA Roxana Yesenia Pastrana Alta, PhD¹, Maria Têresa Machini, PhD², Shana O. Kelley, PhD³ and Breno Pannia Espósito, PhD¹ ¹Institute of Chemistry/Laboratory of Bioinorganic Chemistry and Metallodrugs, São Paulo, Brazil; ²Institute of Chemistry/Laboratory of Peptide Chemistry, Department of Biochemistry, São Paulo, Brazil; ³Department of Biochemistry/Faculty of Medicine, University of Toronto, Toronto, Canada (Presented By: Roxana Yesenia Pastrana Alta)

Poster# 102 NOVEL HEPCIDIN MIMETICS Greg Bourne, PhD¹, Mark Smythe, PhD², Brian Frederick, PhD³, Sonya Scott, PhD4, Simone Vink, PhD4, Jacob Ulrik Fog, PhD5, Pernille Tofteng-Shelton, PhD5 and Praveen Madala, PhD4 ¹Protagonist; ²Protagonist Therapeutics Inc. 306 Carmody Road PO Box 6421, Brisbane Qld. 4067 and Institute for Molecular Bioscience University of Qld., St Lucia Qld 4067; ³Protagonist Therapeutics Inc. 521 Cottonwood Dr, Milpitas, CA, USA 95035; 4Protagonist Therapeutics Inc. 306 Carmody Road PO Box 6421, Brisbane Qld. 4067 and Institute for Molecular Bioscience University of Qld., St Lucia Qld 4067; 5Zealand Pharma A/S, Smedeland 36, 2600 Glostrup, Copenhagen, Denmark (Presented By: Greg Bourne)

Poster# 120 DIFFERENTIATED FUNCTIONS OF IRP1 AND IRP2 Huihui Li Nanjing University (Presented By: Huihui Li)

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Poster# 122 REPROGRAMMING OF IRON METABOLISM IN CANCER Junxia Min, MD, PhD Institute of Translational Medicine/Zhejiang University (Presented By: Junxia Min)

Poster# 124 SERUM HEPCIDIN AND PROHEPCIDIN IN VEGAN POPULATION Kamila Balusikova, Jan Gojda, Jana Patockova, Jan Kovar and Michal Andel 3rd Faculty of Medicine, Charles University in Prague (Presented By: Kamila Balusikova)

Poster# 126 HFE AND HJV EXHIBIT OVERLAPPING FUNCTIONS FOR IRON SIGNALING TO HEPCIDIN: GENETIC EVIDENCE FROM SINGLE AND DOUBLE KNOCKOUT MICE Patricia Kent, MSc, Nicole Wilkinson, PhD, Marco Constante, PhD, Carine Fillebeen, PhD, Konstantinos Gkouvatsos, MD, PhD, John Wagner, MSc, Marzell Buffler, Christiane Becker, PhD, Klaus Schümann, MD, Manuela Santos, PhD and Kostas Pantopoulos Lady Davis Institute for Medical Research and McGill University (Presented By: Kostas Pantopoulos)

Poster# 128 SERUM FERRITIN AND FIBROBLAST GROWTH FACTOR 23 ARE ASSOCIATED WITH LIVER FIBROSIS DIAGNOSED BY TRANSIENT ELASTOGRAPHY IN HIV/HCV CO-INFECTED PATIENTS Giada Sebastiani, MD², John Wagner, MSc, Kathleen Rollet, Marina Klein, MD and Kostas Pantopoulos¹ ¹Lady Davis Institute for Medical Research and McGill University; ²McGill University Health Center (Presented By: Kostas Pantopoulos)

Poster# 130 THE INFLUENCE OF INTERMITTENT HYPOBARIC HYPOXIA ON THE BRAIN IRON METABOLISM IN ADULT SPRAGUE DAWLEY RATS Qiong Wu¹, Yaru Li² and Yan-Zhong Chang² ¹Hebei Normal Unversity; ²Hebei Normal University (Presented By: Qiong Wu)

Poster# 132 MITOCHONDRIAL FERRITIN ATTENUATES THE ROTENONE-INDUCED TOXICITY IN DROSOPHILA MELANOGASTER Yu-Jing Gou, Guofen Gao, Peng Yu, Zhen-Hua Shi and Yan-Zhong Chang Hebei Normal University (Presented By: Yu-Jing Gou)

Poster# 134 IRON DEFICIENCY IN OVERWEIGHT AND OBESE POPULATION Lu Zhao, Xiangyi Zhang, Ye Shen, Xuexian Fang, Youfa Wang and Fudi Wang Zhejiang University (Presented By: Lu Zhao)

Poster# 136 EFFECTS OF PERSISTENT C19ORF12 DOWN-REGULATION UPON CELLULAR IRON HOMEOSTASIS Claudia Saraceno, PhD, Paola Ruzzenenti, Michela Asperti, PhD, Federica Maccarinelli and Dario Finazzi, MD University of Brescia (Presented By: Federica Maccarinelli)

Poster# 138 CATECHOLAMINE STRESS HORMONES REGULATE CELLULAR IRON HOMEOSTASIS BY A POSTTRANSCRIPTIONAL MECHANISM MEDIATED BY IRON REGULATORY PROTEIN Nisha Tapryal, PhD, Vishnu Vivek, MSc, Som Dev, MSc and Chinmay K. Mukhopadhyay, PhD SCMM, JNU, New Delhi (Presented By: Som Dev)

Poster# 140 HEPARAN SULFATE PROTEOGLYCANS IN THE BMP6/SMAD SIGNALING Paola Ruzzneneti, PhD Student, Maura Poli, PhD, Michela Asperti, PhD Student, Magdalena Gryzik, PhD Student and Paolo Arosio, PhD University Of Brescia (Presented By: Paola Ruzznenti)

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Poster# 142 THE EFFECT OF NEUROINFLAMMATION ON IRON REGULATION IN CELLS OF THE CENTRAL NERVOUS SYSTEM Surjit Kaila Srai, PhD¹, Veronika Kallo, MRes¹, Roberta Ward, PhD² and David Dexter, PhD² ¹University College London; ²Imperial College (Presented By: Surjit Kaila Srai)

Poster# 144 DEFEROXAMINE ATTENUATES LIPOPOLYSACCHARIDE-INDUCED INFLAMMATORY RESPONSES AND PROTECTS AGAINST ENDOTOXIC SHOCK IN MICE Yumei Fan¹, Shengnan Wang² and Xianglin Duan² ¹College of Life Science, Hebei Normal University; ²Laboratory of Molecular Iron Metabolism, College of Life Science, Hebei Normal University (Presented By: Yumei Fan)

Poster# 146 A DOUBLE KNOCK OUT OF HEPCIDIN AND IL6 DEMONSTRATES INDEPENDENT ROLES OF THE TWO GENES IN ANEMIA OF INFLAMMATION Sara Gardenghi², Ritama Gupta, Lori Bystrom, Roberta Chessa and Stefano Rivella, PhD¹ ¹Children's Hospital of Philadelphia; ²Weill Cornell Medical College (Presented By: Stefano Rivella)

Poster# 148 SERUM FROM INDIVIDUALS GIVEN A SINGLE ORAL DOSE OF IRON SUPPLEMENTATION SUPPORTS INCREASED BACTERIAL GROWTH James Cross, BSc², Richard Bradbury, PhD², Anthony Fulford, PhD³, Amadou Jallow, Foundation Degree², Rita Wegmuller, PhD², Andrew Prentice, PhD³ and Carla Cerami, MD,PhD¹ ¹University of North Carolina; ²MRC-Gambia; ³London School of Hygiene and Tropical Medicine (Presented By: Carla Cerami)

Poster# 150 DIFFERENCES IN CLINICAL MANIFESTATIONS OF HEMOCHROMATOSIS IN HFE C282Y HOMOZYGOTES WITH EXTREME HIGH AND LOW IRON PHENOTYPES GD McLaren¹, LW Powell², PD Phatak³, JC Barton4, PC Adams5, VN Subramaniam², LC Gurrin6, JD Phillips7, C Parker7, KJ Allen8, W-P Chen, MJ Emond¹°, DA Nickerson¹°, GA Ramm², GJ Anderson² and CE McLaren ¹VA Long Beach Healthcare System and Department of Medicine, University of California, Irvine; ²QIMR Berghofer Medical Research Institute and University of Queensland, Brisbane, Australia; ³Rochester General Hospital, Rochester, NY; 4Southern Iron Disorders Center, Birmingham, AL; 5London Health Sciences Centre, London, ON, Canada; 6University of Melbourne, Melbourne, Australia; 7University of Utah, Salt Lake City, UT; 8Murdoch Childrens Research Institute, Melbourne, Australia; University of California, Irvine, CA; ¹°University of Washington, Seattle, WA (Presented By: GD McLaren)

Poster# 152 PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ALPHA (PPAR?) CONTRIBUTES TO HEPCIDIN UP-REGULATION IN MICE DURING FASTING Yihang Li, PhD, Princy Prasad, MS, Ian Miller, BSc, Cedric Langhi, PhD, Ángel Baldán, PhD and Robert Fleming, MD Saint Louis University (Presented By: Yihang Li)

Poster# 154 COMPARATIVE STUDY OF THE BIOAVAILABILITY OF DIFFERENT IRON SUPPLEMENTS TO BACTERIA Darlene Dagos, MSc², Priya Selvam, MSc, Patrick Kelly, BSc, Robert Evans, PhD¹, Mohammed Gulrez Zariwala, PhD, Derek Renshaw, PhD and Sebastien Farnaud, PhD ¹Brunel University; ²Life Sciences Dept. University of Bedfordshire (Presented By: Robert Evans)

Poster# 156 LEAD NEUROTOXICITY DERIVES IN PART FROM PERTURBED FE/ IRE/ IRP REGULATION IN MODELS OF HUMAN NEURONS Catherine Cahill, PhD², Hong Jiang, MD, PhD³, Xudong Huang, PhD², Jack Rogers, PhD¹ and Ann Smith, PhD4 ¹MGH. Harvard; ²MGH/Harvard; ³Univ. of Qingdao; 4Univ. Missouri, Kansas City (Presented By: Jack Rogers)

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Poster# 158 DIETARY IRON AND GENETIC VARIATION ALTER METALLOSTASIS IN MICE Kathryn E. Page, PhD², Brie K. Fuqua, PhD¹, Stela McLachlan, PhD³, Hiro Irimagawa², Yuanchi He², David W. Killilea, PhD4, Brian W. Parks, PhD5, Simon T. Hui, PhD5, Eleazar Eskin, PhD6, Aldons J. Lusis, PhD5 and Chris D. Vulpe, MD, PhD7 ¹Department of Physiological Sciences, University of Florida, Gainesville, FL and Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA; ²Department of Nutritional Sciences & Toxicology, University of California, Berkeley, CA; ³Centre for Population Health Sciences, The University of Edinburgh, UK; 4Nutrition and Metabolism Center, Children's Hospital Oakland Research Institute, Oakland, CA; 5Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA; 6Department of Computer Science & Human Genetics, University of California, Los Angeles, CA; 7Department of Nutritional Sciences & Toxicology, University of California, Berkeley, CA and Department of Physiological Sciences, University of Florida, Gainesville, FL (Presented By: Brie K. Fuqua)

Poster# 160 IRON REFRACTORY IRON DEFICIENCY ANEMIA (IRIDA) CASE SERIES IN THE NETHERLANDS: A HETEROGENEOUS DISEASE Albertine Donker, MD², Paul Brons, MD, PhD², Dirk Bakkeren, PhD³, Michiel van Gelder, MD, PhD4, Bernd Granzen, MD, PhD4, Mirian Janssen, MD, PhD², Alexander Rennings, MD, PhD², Anita Rijneveld, MD, PhD5, Charlotte Schaap, MD², Frank van de Veerdonk, MD, PhD², Andre Vlot, MD, PhD6, Marten Nijziel, MD, PhD³, Thom Vlasveld, MD, PhD7, Vera Novotny, MD, PhD² and Dorine Swinkels, MD, PhD¹ ¹RadboudUMC; ²RadboudUMC, Nijmegen, Netherlands; ³Maxima Medical Centre, Veldhoven, Netherlands; 4Academic Medical Centre Maastricht, Maastricht, Netherlands; 5ErasmusMC, Rotterdam, Netherlands; 6Alysis Hospital, Arnhem, Netherlands; 7Bronovo Hospital, The Hague, Netherlands (Presented By: Dorine Swinkels)

Poster# 162 A NOVEL PROTEIN INVOLVED IN IRON TRANSPORT IN DROSOPHILA MELANOGASTER Xudong Wang¹ and Bing Zhou² ¹School of life sciences, Tsinghua University; ²School of life sciences,Tsinghua University (Presented By: Xudong Wang)

Poster# 164 SCREENING FOR TRANSITION METAL TRANSPORTING FUNCTIONS OF DROSOPHILA ZIPS AND ZNTS Sai Yin, PhD and Bing Zhou, Prof Tsinghua University (Presented By: Sai Yin)

Poster# 166 REGULATION OF HEPCIDIN AND GDF15 IN ANEMIC PATIENTS WITH TYPE 2 DIABETES WITHOUT OVERT RENAL IMPAIRMENT Jun Hwa Hong, PhD², Yeon-Kyung Choi, PhD², Byong-Keol Min¹, Kang Seo Park, PhD³, Kayeon Seong, PhD4, Byoungjun Choi¹, In Kyu Lee, PhD² and Jung Guk Kim, PhD² ¹Department of Biomedical Science, Graduate School, Kyungpook National University, Daegu, South Korea; ²Department of Internal Medicine, Kyungpook National University School of Medicine, Daegu, South Korea; ³Department of Internal Medicine, Eulji University School of Medicine, Daejeon, South Korea; 4College of Nursing, Taegu Science University, Daegu, South Korea (Presented By: Byoungjun Choi)

Poster# 168 EFFECT OF IRON ON DROSOPHILA HEMOLYMPH PROTEOME Mengran Zhao¹, Guiran Xiao, PhD² and Bing Zhou, PI ¹tsinghua university; ²School of Life Sciences, Tsinghua University (Presented By: Mengran Zhao)

Poster# 170 THE IRON METABOLISM PROTEIN IRP2 IS OVER EXPRESSED IN COLORECTAL ADENOCARCINOMA Sarah Evans, Matthew Bedford, Richard Horniblow, Neeraj Lal, Andrew Beggs, Tariq Iqbal, Olga Tucker and Chris Tselepis University of Birmingham (Presented By: Richard Horniblow)

Poster# 172 STRESS SIGNALING OF HEME-REGULATED EIF2? KINASE IN ERYTHROBLASTS AND MACROPHAGES DURING IRON-RESTRICTIVE ERYTHROPOIESIS Jane-Jane Chen, PhD MIT (Presented By: Jane-Jane Chen) Sixth Congress of the International BioIRon Society Page 42 2015 IBIS Program Schedule IBIS

Poster# 174 DIFFERENTIAL REGULATION OF HEPCIDIN EXPRESSION IN LIVER AND BRAIN OF NEWBORN MICE IN RESPONSE TO SYSTEMIC INFLAMMATION Feng Qi, MD¹, Mary Migas, MS² and Robert Fleming, MD² ¹Peking University First Hospital; ²Saint Louis University (Presented By: Feng Qi)

Poster# 176 POTENTIAL THERAPEUTIC APPLICATIONS OF COMBINING THE USE OF JAK2 INHIBITORS AND MINIHEPCIDIN PEPTIDES IN MICE AFFECTED BY BETA-THALASSEMIA C. Casu¹, R. Chessa¹, R. Oikonomidou¹, B. MacDonald² and S. Rivella¹ ¹Children’s Hospital of Philadelphia (CHOP); ²Merganser Biotech LLC (Presented By: C. Casu)

THURSDAY, SEPTEMBER 10, 2015

OVERVIEW

7:00 - 18:15 Registration/Information Desk Open Location: QiZhen Lobby (1st Floor)

GENERAL SESSION

7:15 - 8:30 Meet the Experts III: The Mouse as a Model to Study Iron Metabolism Location: Haoyue Hall (1st Floor) Moderators: David Frazer, PhD Francesca Vinchi, PhD Students/Trainees ONLY - Space is limited

8:30 - 10:00 Concurrent Session VII and VIII

Concurrent Session VII: Iron Speciation and Its Modification by the Use of Therapeutics Location: QiZhen Hall (3rd Floor) Chairs: Robert Hider, BSc, PhD John Porter, MD, FRCP

8:30 - 8:40 Introduction to Session: Speciation of Iron in Normal Tissue Speaker: Robert Hider, BSc, PhD

8:40 - 9:05 Distribution of Iron in Systemic Iron Overload Speaker: John Porter, MD, FRCP

9:05 - 9:30 NTBI and LPI Assays as Clinical Indicators of Iron Overload and Treatment Efficacy Speaker: Dorine W. Swinkels, MD, PhD

9:30 #32 SYSTEMIC INFLAMMATION INFLUENCES THE ABILITY OF DEFERIPRONE TO CHELATE IRON FROM SPECIFIC BRAIN REGIONS IN PARKINSON S DISEASE PATIENTS Antonio Bastida, MD , Roberta Ward, Mphil, PhD , Paula Piccini, MD and David Dexter, PhD⁴ Imperial College; Imperial College, London UK; Imperial College. London, UK; ⁴Imperial College, London, UK Presented By: Roberta Ward

9:45 #33 ORAL AND INTRAVENOUS IRON: THERAPEUTIC OPTIONS AND DIFFERENCES AMONG THE PREPARATIONS Susanna Burckhardt Vifor Pharma Presented By: Susanna Burckhardt

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Concurrent Session VIII: Iron and Tissue Injury Location: QiuShi Hall (3rd Floor) Chairs: Esther Meyron-Holtz, PhD Grant A. Ramm, PhD

8:30 - 9:00 Mechanisms of Iron-Induced Hepatic Pathology in Haemochromatosis: The Role of the Hepatic Stellate Cell-Liver Progenitor Cell Niche Speaker: Grant A. Ramm, PhD

9:00 #34 DEXRAS1/DMT1 PATHWAY PLAYS A MAJOR ROLE IN OXIDATIVE STRESS-MEDIATED NEURONAL CELL DEATH Yong Chen, PhD, Reas Khan, PhD, Alyssa Cwanger, BS, Ying Song, PhD, Joshua Dunaief, MD, PhD, Kenneth Shindler, MD, PhD and Sangwon Kim, PhD University of Pennsylvania Presented By: Sangwon Kim

9:15 #35 THE EFFECT OF ANTI-INFLAMMATORY PROPERTIES OF FERRITIN LIGHT CHAIN ON LIPOPOLYSACCHARIDE-INDUCED INFLAMMATORY RESPONSE IN MURINE MACROPHAGES Yumei Fan, Yanzhong Chang, PhD and Xianglin Duan College of Life Science, Hebei Normal University (Presented By: Yumei Fan)

9:30 #36 IRON AND AGING: A NOVEL APPROACH AGAINST SKIN NATURAL AGING AND PHOTO-AGING Xi Huang, PhD , Jun Ou, BS and Guo Ting Wang, BS Marivan Skincare Inc.; Marivan Cosmetics Inc.; Marivan Cosmetics, Inc. (Presented By: Xi Huang)

9:45 #37 TRANSVERSE RELAXATION AND VOLUMETRIC NEURAL CHANGES IN THE H67D HFE MOUSE MODEL AND COGNITIVELY NORMAL HEALTHY H63D-HFE HUMAN GENOTYPE CARRIERS Mark Meadowcroft, PhD , Douglas Peters, MS , Carson Purnell, BS , Jian-Li Wang, MD, PhD , Paul Eslinger, PhD , Megha Vasavada, PhD , Qing Yang, PhD and James Connor, PhD The Pennsylvania State University - College of Medicine; The Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania, USA Presented By: Mark Meadowcroft

10:00 - 10:30 Coffee Break Location: QiZhen Hall Corridor (3rd Floor)

10:30 - 12:00 Concurrent Session IX and Session X

Concurrent Session IX: The Regulation of Iron Metabolism Location: QiZhen Hall (3rd Floor) Chairs: Delphine Meynard, PhD Kostas Pantopoulos, PhD

10:30 #38 LOW INTRACELLULAR IRON INCREASES THE STABILITY OF MATRIPTASE-2 Ningning Zhao , Christopher Nizzi , Sheila Anderson , Jiaohong Wang , Akiko Ueno , Hidekazu Tsukamoto , Richard Eisenstein , Caroline Enns and An-Sheng Zhang Oregon Health & Science University; University of Wisconsin-Madison; University of Southern California Presented By: An-Sheng Zhang

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10:45 #39 A MUTATION IN THE IRON-RESPONSIVE ELEMENT OF ALAS2 IS A MODIFIER OF CLINICAL SEVERITY IN ERYTHROPOIETIC PROTOPORPHYRIA Sara Luscieti , Sarah Ducamp, PhD , Hanna Manceau, PhD⁴, Caroline Kannengiesser, PhD⁵, Marguerite Hureaux, PhD⁶, Gael Nicolas, PhD , Zoubida Karim, PhD , Jean Charles Deybach Deybach, PhD, MD⁷, Laurent Gouya Gouya, PhD, MD⁸, Herve Puy Puy, PhD, MD⁸ and Mayka Sanchez, PhD Josep Carreras Leukaemia Research Institute , Diagnostics in Iron Metabolism Disease (DIRON) and Iron Metabolism: Regulation and Diseases Group. and Institute of Predictive and Personalized Medicine of Cancer (IMPPC). Badalona, Barcelona.; Institute of Predictive and Personalized Medicine of Cancer (IMPPC). Badalona, Barcelona.; INSERM U1149 CNRS ERL 8252, Centre de Recherche sur l inflammation, Paris. and Laboratory of excellence, GR-Ex, Paris, France; ⁴INSERM U1149 CNRS ERL 8252, Centre de Recherche sur l inflammation, Paris, France. and AP-HP, Centre Fran ais des Porphyries, H pital Louis Mourier, Colombes.; ⁵INSERM U1149 CNRS ERL 8252, Centre de Recherche sur l inflammation, Paris. and Universit Paris Diderot, site Bichat, Sorbonne Paris Cit , France; ⁶INSERM U1149 CNRS ERL 8252, Centre de Recherche sur l inflammation; ⁷AP-HP, Centre Fran ais des Porphyries, H pital Louis Mourier, Colombes.; ⁸INSERM U1149 CNRS ERL 8252, Centre de Recherche sur l inflammation, Paris. and Laboratory of excellence, GR-Ex, Paris, France. and Universit Paris Diderot, site Bichat, Sorbonne Paris Cit , France (Presented By: Mayka Sanchez) Presented By: Mayka Sanchez Fernandez

11:00 #40 HEPCIDIN AND ATOH8 EXPRESSION ARE DECREASED IN MURINE MODELS OF HEMOCHROMATOSIS HOWEVER HEPCIDIN EXPRESSION IS UNAFFECTED BY LOSS OF ATOH8 Yihang Li, PhD , Princy Prasad, MS , Ian Miller, BSc , Sally Nijim, Andrew McKie, PhD and Robert Fleming, MD Saint Louis University; King's College London Presented By: Yihang Li

11:15 #41 HEPCIDIN DIFFERENTIALLY REGULATES FERROPORTIN EXPRESSION IN SUCKLING MICE David Frazer, Sarah Wilkins, Deepak Darshan, Cornel Mirciov and Greg Anderson QIMR Berghofer Medical Research Institute, Brisbane, Australia Presented By: David Frazer

11:30 #42 HEME AND IRON CONTROL OF MACROPHAGE PLASTICITY IS PREVENTED BY THE HEME SCAVENGER HEMOPEXIN AND THE IRON CHELATOR DFO Francesca Vinchi, PhD , Milene Costa da Silva, MSc , Giada Ingoglia, MSc , Sara Petrillo, MSc , Nathan Brinkman , Adrian Zuercher, PhD⁴, Emanuela Tolosano, PhD and Martina U. Muckenthaler, PhD Molecular Medicine Partnership Unit , University of Heidelberg & EMBL; Molecular Biotechnology Center & Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy; CSL Behring, Research & Development, Kankakee, IL, USA; ⁴CSL Behring, Research & Development, Bern, Switzerland (Presented By: Francesca Vinchi)

11:45 #43 A NOVEL IRON-MEDIATED MECHANISM FOR DEVELOPMENT OF INFLAMMATORY BOWEL DISEASE Shirly Belizowski, Abraham Nyska, Avi Zuckerman, Fabio Cominelli, Orly Savion and Esther Meyron-Holtz Technion Israel Institute of Technology, Haifa, Israel Presented By: Esther Meyron-Holtz

Concurrent Session X: Iron Nutrition and Supplementation in Populations Location: QiuShi Hall (3rd Floor) Chairs: Sant-Rayn Pasricha, PhD, FRSCP, FRCPA Fudi Wang, PhD

10:30 - 11:00 How Do We Solve a Problem Like Anaemia? From Biology to Policy and Back Again Speaker: Sant-Rayn Pasricha, PhD, FRSCP, FRCPA

11:00 - 11:30 Clinical Studies in Iron and Infection Speaker: Kamija Phiri, PhD

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11:30 #44 COMMON VARIANTS AND HAPLOTYPES IN THE TF, TNF AND TMPRSS6 GENES ARE ASSOCIATED WITH IRON STATUS IN A FEMALE BLACK SOUTH AFRICAN POPULATION Wanjiku Gichohi, PhD , Alida Melse-Boonstra, PhD , Dorine Swinkels, PhD , Michael Zimmermann, PhD⁴, Edith Feskens, PhD and Gordon Towers, PhD⁵ Jomo Kenyatta University of Agriculture and Technology; Division of Human Nutrition, Wageningen University, Wageningen, the Netherlands; The Laboratory of Genetic, Endocrine and Metabolic Diseases, Department of Laboratory Medicine, Radboud University Medical Centre, Nijmegen, the Netherlands; ⁴Laboratory of Human Nutrition, Institute of Food Nutrition and Health, Department of Health Sciences and Technology, Swiss Federal Institute of Technology, Zurich, Switzerland; ⁵Centre of Excellence for Nutrition, North West University , South Africa (Presented By: Wanjiku Gichohi)

11:45 #45 IMPROVED IRON ABSORPTION BY CONSUMPTION OF PROBIOTICS Michael Hoppe , Elisabeth Gramatkovski , Gunilla nning, Malin Bj rklund, Niclas Larsson and Lena Hulth n Institute of Medicine, University of Gothenburg; Inst of Medicine Presented By: Lena Hulthen

12:00 - 13:30 Lunch Location: QiZhen Hotel (Haoyue Hall - 1st Floor & Oufang Hall - 2nd Floor)

13:30 - 15:00 Concurrent Session XI and Session XII

Concurrent Session XI: Inflammation, Microorganisms and Iron Location: QiZhen Hall (3rd Floor) Chairs: Xiangming Fang, MD Maja Vujic Spasic, PhD

13:30 #46 THE ROLE OF IRON AND HEPCIDIN IN YERSINIA ENTEROCOLITICA INFECTION Debora Stefanova, Joao Arezes, MS, Victoria Gabayan, BS, Tomas Ganz, MD, PhD, Yonca Bulut, MD and Elizabeta Nemeth, PhD University of California, Los Angeles Presented By: Debora Stefanova

13:45 #47 ANAEMIA ASSOCIATED WITH INTESTINAL INFLAMMATION Debbie Trinder, PhD , Anita Chua, PhD , Roheeth Delima, PhD , Desiree Ho, PhD , Borut Klopcic, PhD , John Olynyk, MD and Ian Lawrance, MD, PhD University of Western Australia; University of Western Australia and Murdoch University; Murdoch University and Curtin University Presented By: Deborah Trinder

14:00 #48 PARADOXICAL TISSUE IRON DEPLETION AND HEPCIDIN INDEPENDENT DOWNREGULATION OF THE IRON EXPORTER FERROPORTIN1 DURING SALMONELLA INFECTION Fran ois Canonne-Hergaux, PhD , , , Alexandra Willemetz, Sean Beatty, Etienne Richer, Aude Rubio, Anne Auriac, Ruth J. Milkereit, Olivier Thibaudeau, Sophie Vaulont and Danielle Malo INSERM UMR 1043; CNRS UMR 5282; Universit de Toulouse, UPS, Centre de Physiopathologie de Toulouse Purpan , Toulouse, France. (Presented By: Fran ois Canonne-Hergaux)

14:15 #49 EFFICACY OF EARLY VERSUS DELAYED TREATMENT WITH IRON AND ERYTHROPOIETIN IN A MOUSE MODEL OF ACUTE AND SEVERE ANEMIA OF INFLAMMATION Airie Kim, MD, PhD, Eileen Fung, PhD, Eeman Khorramian, Victoria Gabayan, BS, Elizabeta Nemeth, PhD and Tomas Ganz, PhD, MD University of California, Los Angeles Presented By: Airie Kim

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14:30 #50 NON-TRANSFERRIN-BOUND IRON (NTBI) IS ESSENTIAL FOR RAPID GROWTH AND VIRULENCE OF VIBRIO VULNIFICUS Jo o Arezes, Deborah Stefanova, Victoria Gabayan, Yonca Bulut, Elizabeta Nemeth and Tomas Ganz University of California, Los Angeles / University of Porto Presented By: Jo o Arezes

14:45 #51 ACTIVIN B UTILIZES ACTIVIN TYPE II RECEPTORS, BMP TYPE I RECEPTORS, HEMOJUVELIN AND SMAD1/5/8 SELECTIVELY IN HEPATOCYTES TO PROMOTE HEPCIDIN INDUCTION BY INFLAMMATION IN MICE Susanna Canali, PhD , Amanda B. Core, PhD , Kimberly B. Zumbrennen-Bullough, PhD , Maria Merkulova, PhD , Alan Schneyer, PhD , Antonello Pietrangelo, MD, PhD and Jodie L. Babitt, MD Program in Anemia Signaling Research, Division of Nephrology, Program in Membrane Biology, Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School; Department of Veterinary and Animal Science, UMass-Amherst, Amherst, MA; Center for Hemochromatosis, University Hospital of Modena and Reggio Emilia, Modena Italy Presented By: Susanna Canali

Concurrent Session XII: Hemochromatosis and Other Forms of Primary Iron Overload Location: QiuShi Hall (3rd Floor) Chairs: Pierre Brissot, MD Christine E. McLaren, BS, MS, PhD

13:30 - 13:55 The Search for Modifier Genes in HFE-Related Hemochromatosis: Exome Sequencing in C282Y Homozygous Men with Extreme Phenotypes Identifies a GNPAT Polymorphism Associated with Severe Iron Overload Speaker: Gordon McLaren, MD

13:55 #52 THE USE OF NEXT-GENERATION SEQUENCING TO SCREEN THE BRITISH POPULATION FOR DISORDERS OF IRON REGULATION USING A 16 GENE PANEL Kathryn Robson, PhD , Patricia Bignell, BSc Hons , Wale Atoyebi, FRCPath , Joanne Mason, PhD , Pavlos Antoniou, PhD and Anna Schuh, PhD MRC Institute of Molecular Medicine, University of Oxford; Molecular Haematology Department, John Radcliffe Hospital; BRC/NHS Oxford Molecular Diagnostics Centre, Oxford Presented By: Kathryn Robson

14:10 #53 DISSECTING THE CONTRIBUTION OF UNREGULATED MACROPHAGE IRON RECYCLING AND DIETARY IRON UPTAKE IN GENERATING SYSTEMIC IRON OVERLOAD IN HEMOCHROMATOSIS Regina Kessler , , Matthias W. Hentze , Martina U. Muckenthaler , and Sandro Altamura , Dep. of Pediatric Oncology, Hematology and Immunology - University of Heidelberg, Germany; EMBL - European Molecular Biology Laboratory, Heidelberg, Germany; MMPU Molecular Medicine Partnership Unit Presented By: Sandro Altamura

14:25 #54 HETEROZYGOUS MUTATIONS IN BMP6 PRO-PEPTIDE LEAD TO INAPPROPRIATE HEPCIDIN SYNTHESIS AND MODERATE IRON OVERLOAD IN HUMANS Raed Daher, Caroline Kannengiesser, Dounia Houamel, Thibaud Lefebvre, Edouard Bardou-Jacquet, Nicolas Ducrot, Caroline de Kerguenec, Anne-Marie Jouanolle, Pierre Bedossa, Dominique Valla, Laurent Gouya, Carole Beaumont, Pierre Brissot, Herv Puy, Dimitri Tchernitchko and Zoubida Karim INSERM U1149, Centre de Recherche sur l inflammation, Universit Paris Diderot, Laboratory of Excellence, GREx, 16 rue Henri Huchard, 75018 Paris, France. Presented By: Zoubida Karim

14:40 #55 NON-HFE HAEMOCHROMATOSIS: ESTIMATION OF THE GLOBAL PREVALENCE FROM ANALYSIS OF NEXT-GENERATION SEQUENCING DATA Daniel Wallace, BSc , PhD and V. Nathan Subramaniam, PhD QIMR Berghofer Medical Research Institute (Presented By: Daniel Wallace)

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15:00 - 15:30 Coffee Break Location: QiZhen Hall Corridor (3rd Floor)

15:30 - 17:00 Concurrent Session XIII and Session XIV

Concurrent Session XIII: Oxygen, Iron and Erythropoiesis Location: QiuShi Hall (3rd Floor) Chairs: Cameron McDonald, PhD Guangjun Nie, PhD

15:30 #56 SUPPRESSED LIVER HEPCIDIN EXPRESSION DESPITE EXOGENOUS FERRI- TRANSFERRIN IN MICE WITH IRON DEFICIENCY ANEMIA IS ASSOCIATED WITH UPREGULATION OF MARROW ERYTHROFERRONE Yihang Li, PhD , Princy Prasad, MS , Ian Miller, BSc , Yelena Ginzburg, MD , Stefano Rivella, PhD and Robert Fleming, MD Saint Louis University; New York Blood Center; Children's Hospital of Philadelphia Presented By: Yihang Li

15:45 #57 ROLE OF MATRIPTASE-2 ON HEPCIDIN SUPPRESSION IN RESPONSE TO ERYTHROPOIESIS NEEDS Aude Rubio, Engineer , Oph lie Gourbeyre, Engineer , Herbert Lin, MD, PhD , H l ne Coppin, PhD , Marie-Paule Roth, MD, PhD and Delphine Meynard, PhD Inserm U1043-CPTP-Paul Sabatier University; Program in Anemia Signaling Research - Program in Membrane Biology/Center for Systems Biology-Massachusetts General Hospital Presented By: Delphine Meynard

16:00 #58 INCREASED HEPCIDIN EXPRESSION IN Β-THALASSEMIC MICE TREATED WITH APO- TRANSFERRIN IS ASSOCIATED WITH DECREASED ERK1/2 PATHWAY ACTIVATION AND ENHANCED P-SMAD1/5/8 NUCLEAR TRANSLOCATION Huiyong Chen , , Tenzin Choesang , Petra Pham , Weili Bao , Maria Feola ,⁴, Huihui Li , , Mark Westerman , Guiyuan Li , Antonia Follenzi⁴, Lionel Blanc⁵, Stefano Rivella⁶, Robert E. Fleming⁷ and Yelena Ginzburg New York Blood Center, New York, NY; Central South University, Changsha, PR China; Intrinsic Lifesciences, LLC, La Jolla, CA; ⁴University of Piemonte Orientale, Amedeo Avogadro, Novara, Italy; ⁵The Feinstein Institute for Medical Research, Manhasset, NY; ⁶Weill Cornell Medical College, New York, NY; ⁷Saint Louis University, St Louis, MO Presented By: Huiyong Chen

16:15 #59 UNCOVERING THE ROLE OR HEME OXYGENASE 1 IN THE PATHOPHYSIOLOGY OF β- THALASSEMIA Daniel Garcia dos Santos, PhD , Zuzana Zidova, Msc , Marc Mikhael, PhD , Stefano Rivella, PhD⁴, Monika Harvathova, PhD⁵ and Prem Ponka, MD, PhD Lady Davis Institute/McGill University; Palacky University, Czech Republic; Lebanese American University, Lebanon; ⁴Weill Cornell Medical College New York; ⁵University, Czech Republic Presented By: Prem Ponka

16:30 #60 IRON AND ERYTHROPOIESIS: A NOVEL ROLE FOR TRANSFERRIN RECEPTOR 2 IN STRESS ERYTHROPOIESIS Gautam Rishi, MSc, Daniel Wallace, PhD, Eriza Secondes, BSc Hons and Nathan Subramaniam, PhD QIMR Berghofer Medical Research Institute, Brisbane, Australia Presented By: V. Nathan Subramaniam

16:45 #61 EXPRESSION OF ERYTHROFERRONE IN MURINE MODELS OF ANAEMIA Cornel Mirciov, Sarah Wilkins, Greg Anderson and David Frazer QIMR Berghofer Medical Research Institute, Brisbane, Australia Presented By: Cornel Mirciov

Concurrent Session XIV: Iron and the Central Nervous System Location: QiZhen Hall (3rd Floor) Chairs: Yan-Zhong Chang, PhD James Duce, PhD

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15:30 - 15:50 The Influence of Amyloid-B Precursor Protein Proteolytic Processing on Neuronal Iron Homeostasis Speaker: James Duce, PhD

15:50 #62 DIFFERENTIAL MRI RELAXATION IN ALZHEIMER S PATIENTS WITH MUTANT HFE AND TRANSFERRIN GENOTYPES Mark Meadowcroft, PhD , Douglas Peters, MS , Carson Purnell, BS , Jian-Li Wang, MD, PhD , Paul Eslinger, PhD , Megha Vasavada, PhD , Qing Yang, PhD and James Connor, PhD The Pennsylvania State University - College of Medicine; The Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania, USA.; The Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania, USA Presented By: Mark Meadowcroft

16:04 #63 LYSOSOMAL IRON MODULATES SYNAPTIC EXCITABILITY VIA DEXRAS1/DMT1 PATHWAY IN HIPPOCAMPUS Rachel White, PhD, Anup Bhattacharya, BS, Yong Chen, PhD, Madeleine Byrd, BS, Gregory Carlson, PhD and Sangwon Kim, PhD University of Pennsylvania Presented By: Sangwon Kim

16:18 #64 RETINAL IRON LOADING AND ABNORMAL VISUAL FUNCTION IN A MOUSE MODEL OF HEMOCHROMATOSIS Ali Shahandeh , Dan Johnston, PhD , Alice Brandli, B Med Chem and Liz Milward, Associate Prof University of Newcastle; The Bosch Institute and Discipline of Physiology, University of Sydney, Australia; School of Biomedical Sciences and Pharmacy, The University of Newcastle, Australia Presented By: Ali Shahandeh

16:32 #65 ASTROCYTE HEPCIDIN REGULATE IRON TRAFFIC ACROSS THE BRAIN ENDOTHELIUM Lin-Hao You , Bing-Jie Zheng , Shu-Min Wang , Yun-Zhe Ci , Peng Yu , Zhen-Hua Shi , Yu-Mei Fan , Li- Peng Wang , Shi-Yang Chang , Tracey A. Rouault , Gregory J. Anderson , Fudi Wang⁴, Xiang-Lin Duan and Yan-Zhong Chang Hebei Normal University; Eunice Kennedy Shriver National Institute of Child Health and Human Development; QIMR Berghofer Medical Research Institute; ⁴Zhejiang University Presented By: Yan-Zhong Chang

16:46 #66 MUTATIONS IN THE FLVCR1 GENE CAUSE HEREDITARY SENSORY AND AUTONOMIC NEUROPATHY TYPE 2 Deborah Chiabrando , Giulio Valperga , Fiorella Altruda , Lorenzo Silengo , Maja Di Rocco , Marco Castori , Ingo Kurth⁴ and Emanuela Tolosano Department of Molecular Biotechnology and Health Sciences, University of Torino, Italy; Second Unit of Pediatrics, G. Gaslini Institute, Genova, Italy., Genova, Italy; Department of Medical Genetics, San Camillo-Forlanini Hospital, Sapienza University of Rome and San Camillo - Forlanin, Rome, Italy.; ⁴Institute of Human Genetics, Jena University Hospital, Jena, Germany Presented By: Deborah Chiabrando

17:00 - 18:20 Plenary Session VI: Best of Posters Location: QiZhen Hall (3rd Floor)

17:00 #67 IDENTIFICATION OF CYS LIGANDS FOR THE [2FE-2S] AND [4FE-4S] CLUSTERS IN DRE2: BOTH CLUSTERS ARE ABSOLUTELY ESSENTIAL FOR THE FUNCTION Yan Zhang, PhD , Andrew Dancis, MD and Eiko Nakamaru-Ogiso, PhD School of Pharmaceutical Science and Technology, Tianjin University; University of Pennsylvania Presented By: Yan Zhang

17:08 #68 A DOUBLE KNOCK OUT OF HEPCIDIN AND IL6 DEMONSTRATES INDEPENDENT ROLES OF THE TWO GENES IN ANEMIA OF INFLAMMATION Sara Gardenghi , Ritama Gupta, Lori Bystrom, Roberta Chessa and Stefano Rivella, PhD Children's Hospital of Philadelphia; Weill Cornell Medical College Presented By: Stefano Rivella

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17:16 #69 THE METAL-ION TRANSPORTER ZIP8 (SLC39A8) AND IRON TRANSPORT ACROSS THE PLACENTA Wei Zhang, PhD, Supak Jenkitkasemwong, PhD, Alan Chan, BS and Mitchell Knutson, PhD University of Florida Presented By: Wei Zhang

17:24 #70 DISCOVERY OF A SECONDARY BMP-SMAD-HEPCIDIN REGULATORY CIRCUIT IN HEPATIC SMAD7-DEFICIENT MICE Peng An, PhD , Hao Wang, PhD , Qian Wu, PhD , Zhuzhen Zhang, PhD , Zhidan Xia, PhD , Xinhui Wang, PhD , Guoli Li, PhD , Yan Chen, PhD , Junxia Min, MD, PhD and Fudi Wang, MD, PhD School of Medicine, Zhejiang University; Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences Presented By: Peng An

17:32 #71 EXOME SEQUENCING IN HFE C282Y HOMOZYGOTES WITH EXTREME HEPATIC IRON OVERLOAD REVEALS MODIFYING GENES FOR THE DEVELOPMENT OF CIRRHOSIS Mary Emond, PhD , Christine McLaren, PhD , Tin Louie, PhD , Jie Wu, PhD , Lawrie Powell, MD, PhD , Pradyumna Phatak, MD⁴, James Barton, MD⁵, Paul Adams, MD⁶, Lyle Gurrin, PhD⁷, John Phillips, PhD⁸, Charles Parker, MD⁸, Katrina Allen, FRACP, PhD⁹, Deborah Nickerson, PhD , Gregory Anderson, PhD , Nathan Subramaniam, PhD , Gordon McLaren, MD¹⁰ and Grant Ramm, PhD QIMR Berghofer MRI; University of Washington; University of California, Irvine; ⁴Rochester General Hospital; ⁵Southern Iron Disorders Center; ⁶London Health Sciences Centre; ⁷University of Melbourne; ⁸University of Utah; ⁹Murdoch Children s Research Institute; ¹⁰VA Long Beach Healthcare System Presented By: Grant Ramm

17:40 #72 CROSSTALK BETWEEN OBESITY AND NEURODEGENERATION; ADIPONECTIN-MEDIATED MODULATION OF IRON FLUX VIA DEXRAS1 IN THE BRAIN Yong Chen, PhD, Lauren Mathias, BS, Rexford Ahima, PhD and Sangwon Kim, PhD University of Pennsylvania Presented By: Sangwon Kim

17:48 #73 CD81 PROMOTES BOTH THE DEGRADATION OF TRANSFERRIN RECEPTOR 2 (TFR2) AND THE TFR2-MEDIATED MAINTENANCE OF HEPCIDIN EXPRESSION Juxing Chen, PhD and Caroline Enns, PhD Oregon Health & Science University Presented By: Caroline Enns

17:56 #74 MULTI-COPPER FERROXIDASES PLAY AN IMPORTANT ROLE IN BRAIN IRON METABOLISM Ruiwei Jiang, Postgraduate, Mengxia Chen, Undergraduate, Jiashuo Zheng, Undergraduate and Huijun Chen, Professor Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University Presented By: Jiashuo Zheng

18:04 #75 BONE IRON CONTENT IN POSTMENOPAUSAL WOMEN WITH HIP FRAGILITY FRACTURES: CORRESPONDENCE TO BONE STATUS AND IMPLICATIONS FOR POSTMENOPAUSAL OSTEOPOROSIS Li Guangfei and Xu Youjia, PhD The second affilicated hospital of Soochow University; The second affilicated hospital of Soochow University Presented By: Li Guangfei

18:12 #76 IRON OVERLOAD PERTURBS COPPER DISTRIBUTION IN MICE Jung-Heun Ha, Caglar Doguer, Xiaoyu Wang, Shireen R. Flores and James F. Collins Food Science and Human Nutrition Dept., University of Florida Presented By: James Collins

18:15 Concluding Remarks

19:30 23:45 Gala Dinner/Award Ceremony Location: Sofitel Hangzhou Westlake * Buses will depart promptly at 19:30, please check with the Registration Desk for bus departure location.

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Monday, September 7, 2015 11:00 – 12:00 Keynote Lecture I Chair: Greg Anderson, PhD Iron and Global Health Invited Speaker: Andrew M. Prentice, MRC Unit, The Gambia & London School of Hyg & Trop Med, UK

Estimating the global prevalence of nutrient deficiencies is an imprecise science easily manipulated by the choice of diagnostic methods and cut-offs employed. Nonetheless iron deficiency is probably the most prevalent micronutrient deficiency worldwide and, in various forms, iron is the most widely administered of all therapeutic agents. This creates a challenge in low-income settings where infectious diseases are still highly prevalent because there is little doubt that iron is the most critical nutrient in terms of host-pathogen competition for nutritional resource. The evidence for this claim is derived from numerous sources ranging from theoretical (inferences from genomic investment in iron regulatory pathways in host and pathogens), experimental (effect of iron administration on the course of experimental infections) and clinical (adverse outcomes from large-scale supplementation trials with iron). This has created a policy impasse in need of urgent resolution. There would appear to be three possible routes forward: better control of infectious diseases in poor populations (a long-term goal requiring economic transition); design of safer ways to administer iron; or acceptance of the fact that some children will suffer serious adverse consequences in the course of programmes aimed at eliminating iron deficiency (the ‘greater good’ argument). Fortunately, recent rapid progress in describing the molecular regulation of iron metabolism is yielding critical insights that should help us navigate towards methods for safe iron therapies. In recent decades nutritionists had the view that human infants were poorly designed to absorb iron and hence required large unphysiological bolus doses of readily absorbable iron. The discovery of hepcidin and elucidation of its interaction with enterocyte ferroportin have revealed that, far from being poor iron absorbers, children under threat of infection are working hard to exclude iron and large bolus doses cause iatrogenic disease by over-whelming their iron chaperone systems. It is also now clear that unabsorbed iron from such large doses alters the gut microbiome with a displacement of benign symbionts (lactobacilli and bifidobacteria) by potential pathogens and the creation of an inflammatory dysbiosis. These insights have reset the parameters for the development of next-generation supplements that will be both more effective and safer in low-income settings. New insights into the mechanisms by which iron supplementation increases the risk of P falciparum malaria also point a way forward. Here the latest evidence suggests that reticulocytes and young RBCs are much more susceptible to invasion and hence children recovering from a state of iron-deficiency anemia will inevitably pass through a period of transiently elevated risk of malaria. The practical implication is that iron supplements should (initially at least) be given under the cover of malaria prophylaxis. The role of hecipin-mediated iron redistribution (blood stream hypoferremia with elevated macrophage iron) likely has an important bearing on the etiology of intracellular infections and points the need for a holistic view of infectious pathology as opposed to the usual ‘silo’ approach. Finally we believe that optimal iron regulation is critically involved in the safe transition from fetal to extrauterine life and that this is a key area for future research investment.

Sixth Congress of the International BioIRon Society Page 51 Keynote Speakers IBIS

Andrew Prentice founded the MRC International Nutrition Group at LSHTM in 1999. Born in Uganda, he studied in East Africa and the UK obtaining a BSc in Biochemistry followed by a PhD in Nutrition from Darwin College, Cambridge. He worked in the MRC Dunn Nutrition Unit’s rural field station in Keneba, The Gambia from 1978-83. In 1983 he returned to the MRC Dunn Clinical Nutrition Centre in Cambridge to become Head of Human Energy Metabolism where he specialised in studying the regulation of energy balance with a particular focus on obesity. In 1998 he became scientific director of the MRC Keneba fieldstation and of the Nutrition Programme for MRC The Gambia Unit, a role he still maintains.

His current research spans the 4 research areas of ING with special interest in early life programming of immune function, nutrient-gene interactions (especially in relation to iron and infectious diseases) and reproductive nutrition, with research collaborations in Gambia, Chile, Bangladesh, Kenya, and Tanzania. He has been a member of numerous national and international advisory committees, and held senior posts in several academic associations. He is a former member of The Bill & Melinda Gates Foundation’s Global Health Discovery Expert Group, and currently chairs the Wellcome Trust’s Expert Review Group for Physiology in Health and Disease, and the NIH/NICHD/Gates Research Review Group in Iron and Malaria. His work has attracted several international awards, most recently the EV McCollum International Lecturer Award from the American Society of Nutrition 2010/11, and the 5th George G Graham Lectureship 2011 from Johns Hopkins University. Prentice heads (with Hal Drakesmith) the Gates Foundation Funded HIGH Consortium (Hepcidin and Iron in Global Health).

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Wednesday, September 9, 2015 13:30 – 14:30 Keynote Lecture II Chair: Fudi Wang, PhD Vitamin C and Fe Based Enzymes in Somatic Cell Reprogramming Invited Speaker: Duanqing Pei, PhD

DUANQING PEI PhD is Professor of stem cell biology and also serves as the Director General (President) at the Guangzhou Institutes of Biomedicine and Health (GIBH), Chinese Academy of Sciences, in Guangzhou, China. He obtained his PhD from the University of Pennsylvania in 1991 and trained as a postdoctoral fellow at the University of Michigan before becoming a faculty member at the University of Minnesota School of Medicine in 1996. He joined the Medical Faculty at Tsinghua University in Beijing China in 2002 and moved to the newly formed GIBH in 2004.

Upon returning to China, he once again changed his field of study and started working on pluripotency first and then reprogramming. The Pei lab in Tsinghua began to publish in the stem cell field on the structure and function of Oct4, Sox2, FoxD3, Essrb, and Nanog, and their interdependent relationship towards pluripotency. Based the understanding of these factors, the Pei lab was the first in China to create mouse iPSCs using a non-selective system, and then improved the iPS process systematically. The Pei lab subsequently disseminated the iPS technology in China by providing not only resources, but also training workshops. Recent publications from the Pei lab includes the discovery of vitamin C as a potent booster for iPSC generation and the histone demethylases Jhdm1a/1b are key effectors of somatic cell reprogramming downstream of vitamin C, as well as a mesenchymal to epithelial transition initiates the reprogramming process of mouse fibroblasts. Now, his lab continues to explore new ways to improve iPS technology, dissect the reprogramming mechanisms driven by Oct4/Sox2/Klf4 or fewer factors, and employ iPSCs to model human diseases in vitro.

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Monday, September 7, 2015 9:00 – 9:25 Plenary Session I: Systemic Iron Regulation Chairs: Caroline Enns and PhD Yin Xia, PhD Erythroid Regulation of Systemic Iron Metabolism Speaker: Tomas Ganz, PhD, MD; University of California Los Angeles

The production of erythrocytes in the marrow consumes most of the circulating plasma iron. It has been known for more than fifty years that increased erythropoietic activity after hemorrhage, hypoxia or other erythropoietic stimuli causes a physiologic response of increased iron absorption and release of iron from stores. Pathological interaction of erythropoiesis with iron metabolism is manifested in anemias with ineffective erythropoiesis, i.e. diseases characterized by expansion of erythropoietic precursors that fails to generate a proportional increase in mature erythrocyte production. In ineffective erythropoiesis, intestinal iron absorption is increased and may cause lethal iron overload. Accumulated evidence indicates that these physiologic responses and the pathological effects are partly mediated by hepcidin suppression but also by direct effects of hypoxia on the duodenum. Hepcidin suppression is attributed to “erythroid regulators”, factors secreted by erythroid precursors. The first reported regulator is GDF15, a member of the bone morphogenetic protein family, whose involvement in hepcidin suppression is best documented in human anemias with ineffective erythropoiesis. It does not appear to mediate physiologic responses to hemorrhage in mice or humans, or in ose odels o -thalassemia. Because of the inapplicability of mouse models, definitive evidence for its role in the pathogenesis of iron overload in ineffective erythropoiesis may require studies of GDF15 antagonists in appropriate patients. Another erythroid regulator is erythroferrone (ERFE, Fam132b, CTRP15), a member of the α speraly proded ha ad re erythrolasts ad reatly ded y erythropoet Treatment of hepatocytes with ERFE suppresses hepcidin expression with an EC50 in the picomolar range. In mice, plasma protein measurements document that bioactive concentrations of ERFE are induced by hemorrhage or erythropoietin administration. Plasma ERFE concentrations are also increased in the Th3/+ re -thalassemia model. Based on mouse ERFE KO studies, ERFE mediates early suppression of hepcidin after hemorrhage or erythropoietin administration, and is partially responsible for iron overload in a ose odel o -thalassemia. However, despite normalization of hepcidin in Erfe-/-Th3/+ mice at an early age, the thalassemic animals continue to accumulate iron faster than WT mice pointing to partially hepcidin- resistant hyperabsorption of iron. Moreover, in Erfe-/-Th3/+ mice the slope of hepcidin increase with liver iron content is intermediate between the slopes in Th3/+ and WT mice. These observations suggest the existence o addtoal ators that rease ro asorpto ad otrte to ro oerload -thalassemia, likely including duodenal hypoxia caused by anemia, and at least one additional suppressor of hepcidin. Ongoing studies will test the applicability of these findings to human physiology and the pathogenesis of iron-loading anemias.

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Monday, September 7, 2015 9:25 – 9:50 Plenary Session I: Systemic Iron Regulation Chairs: Caroline Enns and PhD Yin Xia, PhD TMPRSS6 and BMP6 In Iron Homeostasis: A Matter of Balance Speaker: Laura Silvestri, PhD; Regulation of Iron Metabolism Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy and Vita-Salute San Raffaele University, Milan, Italy

L. Silvestri1,2, A. Nai1,2, A. Pagani1,2 and C. Camaschella1,2

1 Regulation of Iron Metabolism Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy 2 Vita-Salute San Raffaele University, Milan, Italy

Hepcidin, the soluble peptide hormone produced by the liver, is regulated by multiple stimuli as iron, inflammation/infection, hypoxia, erythropoiesis, gluconeogenesis and testosterone. Hepcidin deregulation causes two genetic disorders with opposite phenotype: hemochromatosis (iron overload due to low hepcidin) and IRIDA (iron deficiency anemia due to high hepcidin). Hepcidin expression is mainly regulated by the BMP-SMAD pathway, which is activated by iron through bone morphogenetic protein 6 (BMP6). To gain further insights into the mechanisms regulating hepcidin, we have analyzed the transcriptional regulation of Bmp6 in the different liver cells population in physiologic and disease models of iron overload and deficiency. We confirmed that hepcidin is expressed almost exclusively by hepatocytes (HCs) and Bmp6 is produced also by non-parenchymal cells (NPCs), mainly sinusoidal endothelial cells (LSECs). We propose that NPCs, sensing the iron flux, not only induce hepcidin increase through Bmp6 with a paracrine mechanism to control systemic iron homeostasis but, controlling hepcidin, they also regulate their own ferroportin, inducing iron retention or release and further modulating Bmp6 production in an autocrine manner. This mechanism, that contributes to protect HCs from iron loading or deficiency, is lost in disease models of hepcidin production (Rausa et al., 2015). Hepcidin is reduced in conditions of iron deficiency, hypoxia and increased erythropoiesis. TMPRSS6 is the main physiologic inhibitor of hepcidin that in vitro inactivates the liver BMP-SMAD pathway through the cleavage of the BMP coreceptor hemojuvelin (HJV) (Silvestri et al., 2008) at position R121 and R326 (Rausa et al., 2015). The relationship between the newly identified hepcidin inhibitor erythroferrone (Erfe) and TMPRSS6 remains unclear. We have demonstrated that, inactivation of Tmprss6 in the Hbbth3/+ thalassemia mice ameliorates iron overload and anemia, irrespective of erythropoietin levels that remain high. We found that Erfe expression is increased in the thalassemia mice model and in the double mutant animals, suggesting that Tmprss6 is indispensable for erythropoiesis-mediated hepcidin inhibition (Nai et al., 2012). To explore the potential role of Tmprss6 in Erfe mediated hepcidin downregulation, we treated Tmprss6 KO mice and iron deficient, iron replete and iron loaded littermates with EPO to increase Erfe production and showed that Erfe efficiently inhibits hepcidin in iron replete and iron deficient conditions but this was not the case in Tmprss6 KO mice. We found that Erfe is unable to inhibit hepcidin when the BMP-SMAD signaling is hyperactive, as in the absence of Tmprss6 or in iron overload, demonstrating a signal hierarchy in the regulation of iron homeostasis.

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Monday, September 7, 2015 9:50 – 10:15 Plenary Session I: Systemic Iron Regulation Chairs: Caroline Enns and PhD Yin Xia, PhD BMP Signaling in Systemic Iron Regulation Speaker: Jodie Babitt, MD; Massachusetts General Hospital, Harvard Medical School

Mutations in hemojuvelin are the leading cause of juvenile-onset hereditary hemochromatosis, a severe iron overload disorder that presents in the first to third decades of life and is characterized by hypogonadotropic hypogonadism, cardiomyopathy, arthropathy, and cirrhosis. The discovery that hemojuvelin is a bone morphogenetic protein (BMP) co-receptor helped to identify this signal transduction pathway as a key mediator of systemic iron balance. BMP signaling is a central transcriptional regulator of the iron hormone hepcidin, which downregulates expression of the iron exporter ferroportin to inhibit iron entry of iron into the circulation from dietary sources, iron recycling macrophages, and hepatocyte stores. The BMP signaling pathway is critical for hepcidin regulation by iron, and also intersects with most other known hepcidin regulators. This plenary lecture focuses on the molecular mechanisms of hepcidin regulation by the BMP pathway in response to iron.

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Monday, September 7, 2015 13:00 – 13:30 Concurrent Session I: Iron Metabolism in the Kidney Chairs: Jonathan Barasch, MD and PhD Dorine W. Swinkels, MD, PhD Specialized Kidney Intercalated Cells Defend the Urine System by Chelating Siderophores with the Biomarker of Kidney Inflammation and Injury Known as NGAL-Sidercalin-LCN2 Speaker: Jonathan Barasch, MD, PhD; University of Washington

A. Qiu, T. Shen, K. Xu, N. Paragas, J. Barasch University of Washington, Tongji and Columbia University.

The nephron is a long tubule of epithelial cells differentiated into domains with unique protein, electrolyte and water transport systems, but iron trafficking to the kidney and the recycling of iron from the nephron has not received much attention. We have identified many surprises such as the specialized locations of TfR1, Fpn, and DMT1 suggesting a complex path for iron. In addition, special cases or iron trafficking have been detected in kidney damage, urinary infection, and iron overloaded states. Mapping the kidney response to ferric and heme-iron is now just beginning.

The NGAL-Siderocalin-Lcn2 is a small beta barrel protein that binds the catechol residues of Enterochelin (Ent) with 3 positively charged amino acids (R. Strong, Fred Hutchinson). Serum NGAL is captured by megalin: deletion of megalin resulted in the appearance of urinary NGAL and when we mutated surface residues to bypass recognition by megalin, NGAL appeared in the urine as well. Loading with Ent, permitted the chelation of serum NTBI, and its disposal in the urine. Hence NGAL binds siderophore:iron and is competent to traffic and dispose of iron.

Urine NGAL is normally present at 20ng/ml, but upon tissue injury by ischemia, sepsis, obstruction, NGAL RNA rises (in 3-6 hours) 10-1000 fold, in dose response with the stimulus. NGAL-Luc2 reporter mice demonstrate intense luminescence from the medulla of the ischemic kidney and cross-transplantation between knockout and wildtype mice demonstrated kidney dependent uNGAL. In >2500 human patients, we show that the amount of NGAL predicted true kidney injury, mortality and dialysis rather than simple, reversible changes in creatinine typical of hemodynamic variations which cause pre-renal azotemia. By in situ hybridization, we located the source of NGAL to the kidney intercalated cell, a cell specializing in the secretion of H+, IL-18, RNase-7, all of which are involved in antimicrobial activity.

Deletion of TLR4 resulted in little if any NGAL expression and bacterial overgrowth of a urinary tract infection with UTI. NGAL knockout resulted in prolonged UTI as well. To delete intercalated cells, we identified a critical transcription factor called Cp2L1 which controls the specification of the intercalated cell and its differentiation from prinicipal cells; upon deletion of CP2L1 we identified uniform collecting ducts missing intercalated cells. These mice demonstrated bacterial overgrowth. To determine whether intercalated cells expressed specialized genes relevant to UTI, we developed a method of in situ RNA labeling and found that these cells were the primary responders against UTI, expressing a number of iron sulfur genes critical in controlling the inflammatory response and iron capture.

In sum, NGAL which is best known as a marker of acute kidney injury, is a rapid response gene targeting Enterochelin and a few other siderophores in a pattern which is necessary and sufficient for bacteriostasis in E. Coli UTI. NGAL can traffic long distances with iron, and serve also to chelate or to deliver iron to the urinary system. NGAL is a special case of extracellular chelation of iron with a cofactor, and is one component of a complex iron chelation and recycling mechanism in the kidney.

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Tuesday, September 8, 2015 8:30 – 8:55 Plenary Session II: Structure and Function of Iron-Related Proteins Chairs: Huijun Chen, PhD and Robert W. Evans, BA, PhD The NTBI Transporter SLC39A14: From Function to Structure Speaker: Mitchell Knutson, PhD; University of Florida

SLC39A14 (commonly known as ZIP14) is a cell-surface transmembrane protein that can mediate the cellular uptake of non-transferrin-bound iron (NTBI), which appears in the plasma during iron overload. NTBI is widely believed to be a major contributor to iron loading of the liver, pancreas, and heart, the three tissues that commonly display iron-related pathologies in iron overload disorders such as hereditary hemochromatosis and thalassemia major. We have investigated the role of SLC39A14 in iron metabolism by using Slc39a14-/- mice (Jenkitkasemwong et al., Cell Metab, 2015). We found that Slc39a14-/- mice display markedly reduced uptake of plasma NTBI by the liver and pancreas, indicating that SLC39A14 is the main route of NTBI uptake by these organs. To test the role of SLC39A14 in tissue iron loading, we crossed Slc39a14-/- mice with Hfe-/- and Hfe2-/- mice, animal models of type 1 and type 2 (juvenile) hemochromatosis, respectively. Slc39a14 deficiency in hemochromatotic mice greatly diminished iron loading of the liver and prevented iron deposition in hepatocytes and pancreatic acinar cells. The role of SLC39A14 in tissue iron loading was additionally examined in the context of dietary iron overload (1% carbonyl iron for 4 weeks). Similar to hemochromatotic mice deficient in Slc39a14, dietary iron-loaded Slc39a14-/- mice failed to load iron in hepatocytes and pancreatic acinar cells. Collectively, these studies in mice suggest that SLC39A14-mediated NTBI uptake is the main route of iron loading of the liver and pancreas in iron overload disorders.

In addition to transporting NTBI (as Fe2+), SLC39A14 can transport Zn2+, Mn2+, Cd2+, and Co2+ into cells. Yet, the membrane topology of SLC39A14 and the amino acid residues that confer metal-ion selectivity are unknown. We have used epitope tagging and immunofluorescence as a first step to delineate the topology of SLC39A14 and site-directed mutagenesis to identify residues critical for iron and zinc transport. We conclude that SLC39A14 contains 8 transmembrane (TM) domains with extracellular N- and C-termini. Amphipathic TM domains 4 and 5 are involved in metal transport, with a number of negatively charged amino acids being required for Fe2+/Zn2+ transport. Amino acid residues conferring metal-ion selectivity have also been identified. These structure-function studies offer insight into how a broad-scope metal-ion transporter transports iron.

Supported by NIH grant R01 DK080706.

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Tuesday, September 8, 2015 8:55 – 9:20 Plenary Session II: Structure and Function of Iron-Related Proteins Chairs: Huijun Chen, PhD and Robert W. Evans, BA, PhD The Ferritins: Complex, Multifunctional Protein Nanocages with Self-Synthesized, Internal Iron Minerals Speaker: Takehiko Tosha, PhD

Takehiko Tosha1,2 & Elizabeth C. Theil1

1Children’s Hospital Oakland Research Institute, UCSF Benioff Children's Hospital Oakland, Oakland 94609, USA, 2RIKEN SPring-8 Center, Hyogo 679-5148, Japan

Ferritin is the family name of a group of nanocage proteins that reversibly concentrate intracellular iron for living cells; iron is central to many biological processes including energy metabolism, respiration, and cell division; iron mineralization in ferritin consumes oxygen, conferring antioxidant activity and transcriptional regulation (MARE/ARE promoter) with antioxidant proteins like heme oxygenase. Ferritin protein structure of ferritin is a protein nanocage (see figure), self-assembled from multiple subunits, usually 24, and studded with a multiple, functional sites (iron ion channel, transport, and oxidation and mineral nucleation). Ferritin enzyme sites oxidize ferrous to ferric similarly to other diiron iron oxygenases (ribonucleotide reductase or methane monooxygeanse, e.g.) but uniquely use both iron and oxygen as enzyme substrates; the iron oxy products of ferritin enzyme action are the ferritin biomineral precursors : 2+ 3+ 3+ + 2 Fe + O2 + 2 H2O → [Fe -O-O-Fe ] → → 2 H + (Fe2O3 • H2O) protein -caged solid, The nanocage structure of ferritin (see figure) is distinctive, known and admired for over 40 years. Particularly remarkable is the amazing symmetry of protein cage. Ferritin protein subunits (folded polypeptides align (spontaneously in vitro) with two-fold, three- fold and four-fold symmetry. Now, ferritin protein cage symmetry is understood to be part of ferritin function. The functions of ferritin structural symmetry axes are: 1. Two-fold axes (subunit dimers) stabilize the protein cage; 2. Three-fold axes (subunit trimers) form ion channels where cytoplasmic Fe2+ ions enter and dissolved/reduced Fe2+ ions dissolved/reduced from iron mineral exit ferritin protein cages; 3. Four-fold axes (subunit tetramers): organize ferric mineral nucleation of ferritin diferric enzyme products. (Part support: USPS:NIH-DK20251 and The CHORI

Loop-loop Interactions in ferritin protein cages. Left: 24 Foundation). subunit ferritin protein cage with two, proximal, protein subunits (yellow & green) aligned along the 2-fold (dimer) symmetry axes Right: close up of subunit dimers showing amino acid interactions in amino acids in subunit loops: red (positive charge); blue (negative charge). (Bernnachioni et al., Biochim Biophys Acta. 2015 (in press: pii: S1570- 9639(15)00047-3.doi: 10.1016/j.bbapap.2015.02.011

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Tuesday, September 8, 2015 9:20 – 9:45 Plenary Session II: Structure and Function of Iron-Related Proteins Chairs: Huijun Chen, PhD and Robert W. Evans, BA, PhD Structural and Functional Studies of a Divalent Metal Transporter Speaker: Mika Jormakka, PhD; Structural Biology Program, Centenary Institute, Sydney, Australia; Faculty of Medicine, University of Sydney, Sydney, Australia

R. Taniguchi, H. E. Kato, J. Font, C. N. Deshpande, M. Wada, K. Ito, R. Ishitani, O. Nureki

In vertebrates, the iron exporter ferroportin releases Fe2+ from cells into plasma, thereby maintaining iron homeostasis. The transport activity of ferroportin is suppressed by the peptide hormone hepcidin, which exhibits upregulated expression in chronic inflammation, causing iron-restrictive anemia. However, due to the lack of structural information about ferroportin, the mechanisms of its iron transport and hepcidin-mediated regulation remain largely elusive. Here we report the crystal structures of a putative bacterial homolog of ferroportin, BbFPN, in both the outward- and inward-facing states. A comparison of the two structures revealed that BbFPN undergoes an intra-domain conformational rearrangement during the transport cycle. We identified a substrate-metal binding site, based on structural and mutational analyses. This and additional findings will be discussed.

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Tuesday, September 8, 2015 10:30 - 11:00 Plenary Session III: Special Symposium - Cardiac Iron Homeostasis Chairs: Greg Anderson, PhD and Clara Camaschella, MD Iron in the Heart: A Paradigm of Organ Susceptibility to Siderosis and Sideropenia. Etiopathology and Treatment Speaker: Ioav Cabantchik, MD, PhD; DellaPergola Chair in Life Sciences, The Hebrew University, Jerusalem, Israel

Cardiac iron has been the focus of studies aimed at understanding its role in acute, chronic and often fatal cardiomyopathies (CM). Those features have been associated with systemic and regional siderosis, but also with iron deficiency (ID) unrelated to anemia. In thalassemia major (TM) systolic or diastolic dysfunctions have been causatively associated with cardiosiderosis, independent of other concomitant processes. That has largely relied on the recovery of siderotic hearts following chelation regimens that demonstrably reduce siderotic foci. However, mechanistically, the labile components of plasma NTBI are those that in systemic siderosis infiltrate the heart via resident channels/transporters raising the pool of labile cell iron and thereby leading to ROS and oxidative damage. Being labile iron the primary target of chelators, it raises the question if iron detoxification that is apparently sufficient for reversing/preventing systemic siderosis, can also be attained in regional siderosis, which demands sparing systemic iron or chelating it but redeploying it subsequently to minimize losses. Reversal of hypertrophic cardiomyopathy by chelation regimens that spare systemic iron has been recently shown in patients with Friedreich's ataxia, a paradigm of locoregional siderosis caused by dysfunctional iron utilization due to faulty ISC synthesis. On the opposite side of THE CM spectrum are iron disorders associated with ID. In mild-to-moderate cases, ID induces hemodynamic adaptations (increased cardiac output and O2 delivery) that permit adequate CV compensation via a combined increase in heart rate and stroke volume accompanied by diastolic and systolic LV chamber sizes. In severe ID, LV function deteriorates concomitant with myocardial contractility. Attempts to explain how IDA and/or ID per se might cause CM have yet to provide a verifiable explanation as to the rapid clinical reversibility evoked by iron replacement, mostly via parenteral routes. Biochemically, mitochondria, as the major factory of cell iron processing, have been implicated in cardiosiderotic disorders of either iatrogenic or genetic origin. With limited abilities of cardiomyocytes to prevent sustained infiltration of NTBI forms present in hemosiderosis or sequester excess "imported" metal into ferritin shells or eliminate labile cell iron via egress pathways (into an NTBI-rich medium), those are the mitochondria that absorb labile forms of metal that, on the one hand, provoke organelle damage but also deplete other compartments from metabolically essential iron. A similar chemical scenario ensues when labile iron ingress into mitochondria is not met by a commensurate utilization due to a faulty metabolic machinery and/or an apparently limited ability to extrude excess metal. It is also curious that most mutations that adversely impact mitochondrial functions lead in fact to neurological disorders earlier and/or in more cases than to overt heart diseases, as in FA. It has been speculated that the myocardium might be endowed with metabolic plasticity and sufficient robustness to evoke compensatory measures for coping with mitochondrial dysfunctions or quality control mechanims that can spare them from degenerative pathways described in neuronal degeneration. Hitherto, our knowledge about the molecular mechanisms underlying cardiac iron handling is rather limited. However, the subject is gradually gaining attention by both basic and clinical investigators, particularly following the clinical success attained by pharmacological interventions aimed at bringing aberrant systemic and cellular iron to their respective homeostatic levels.

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Tuesday, September 8, 2015 11:00 - 11:30 Plenary Session III: Special Symposium - Cardiac Iron Homeostasis Chairs: Greg Anderson, PhD and Clara Camaschella, MD Cardiomyopathies and Iron Status: A Clinical Outlook Speaker: Antonio Piga, MD; Department of Clinical and Biological Sciences, University of Torino, Italy

Both iron deficiency and excess may worsen an existing cardiomyopathy regardless the etiology, but only the latter is causative of a specific cardiomyopathy. Cardiac iron overload, by means of chronic oxidative damage, leads to progressive diastolic and systolic dysfunction, up to congestive heart failure. Impaired endothelial function and arrhythmias up to cardiac arrest are often associated. The natural history and clinical course in untreated patients is one of clinically silent cardiomyopathy for many years, followed by malignant arrhythmias and acutely impaired myocardial function. Many and disparate conditions may include an iron-related cardiopathy, starting with primary iron overload due to genetic disorders of iron metabolism as hereditary hemochromatosis and other non HFE-related disorders. Secondary iron overload may descend from iatrogenic (oral or intravenous) iron excess, from repeated transfusions, and from iron-loading anemias, where ineffective erythropoiesis suppresses the hepcidin production, causing hyper-absorption of dietary iron and systemic iron overload even in the absence of transfusions. In each of the above conditions the prevalence of cardiac involvement, onset modalities and severity will depend on specific pathophysiological patterns and the coexistence of worsening/mitigating factors. Regardless the underlying condition, what is important from the clinical point of view is the possibility of reverting an existing cardiopathy or preventing it when the risk is high. This is seldom possible in most of the cardiomyopathies, but not with the iron-related one, due to its toxic nature, directly related to myocardial iron accumulation, with the important prospect of complete resolution with treatments directed at iron removal rather than at myocardial performance. In the absence of anemia, a systemic iron overload may be efficiently removed by a phlebotomy program, otherwise iron chelation therapy is indicated. Three chelators are approved for human use, with deferiprone appearing the most effective at removing cardiac iron and the fastest in improving cardiac function. An intriguing hypothesis regards the possibility of preventing and reversing cardiac iron loading by modulation of calcium channels activity, as shown in cellular and animal models. A first translational study of this type using a calcium channels blocker as amlodipine in thalassemic patients showed a significant strengthening of the efficacy of standard iron chelation. This result has been recently demonstrated in a randomized controlled trial. A difficult challenge are conditions where the total body iron is normal but a part is “misplaced” for genetic or acquired causes. Often the consequences affect only one (or a few) organ(s)/system(s) (regional siderosis). A paradigmatic example is Friedreich’s ataxia where the frataxin deficit leads to mitochondrial iron load, but the consequences are severe for the nervous system and the myocardium. The cardiac iron content is normal but the cardiomyopathy share many features, including being the leading cause of death, with conditions of systemic iron overload like thalassemias. The ability of iron chelation with deferiprone to improve the cardiac status in both conditions, regardless of iron status, may have important implications for other conditions where a regional siderosis is relevant. Another important step for a wider application of this treatment has been the long-term safety profile with very low prevalence of neutropenia/agranulocytosis or “iron-deficient” erythropoiesis.

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Wednesday, September 9, 2015 7:15 - 8:30 Meet the Experts II: Genetics of Human Iron Disorders Genetics of Human Iron Metabolism: Non-HFE Genes and Genetic Modifiers Iron Overload Speaker: V. Nathan Subramaniam, MSc, PhD ; QIMR Berghofer Medical Research Institute, Brisbane, Australia.

Many disorders of primary iron overload and iron deficiency have a genetic origin. Hereditary haemochromatosis (HH) due to mutations in the HFE gene accounts for about 80-90% of HH in European populations with the remainder ascribed to mutations in other genes (termed non-HFE). While mutations in the HFE gene are not so common, mutations in the non-HFE genes are being increasingly identified in non- European populations including the Asia-Pacific region. A combination of low awareness, high cost and non- standardised methodology for definitive diagnosis is likely leading to under-recognition of non-HFE HH. As many countries achieve improved nutrition, access to healthcare and advanced diagnostic capabilities, it is possible that hitherto unrecognised hereditary iron overload conditions will be unmasked. Genes which have been associated with haemochromatosis, besides HFE, include: Hemojuvelin, mutated in type 2A or juvenile HH; Hepcidin, mutated in type 2B HH; Transferrin Receptor 2, mutated in type 3 HH, and Ferroportin, mutations of which are associated with type 4 HH or ferroportin disease. A number of potential genetic modifiers of iron disorders have also been recently identified including the GNPAT gene. With the advent of next-generation based sequencing it is expected that additional genes and genetic modifiers of iron metabolism will be identified. This session will focus on the genetics of human iron overload.

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Wednesday, September 9, 2015 7:15 - 8:30 Meet the Experts II: Genetics of Human Iron Disorders Genetics of Human Iron Disorders: Hematological and Iron Metabolism Traits Speaker: Christopher Vulpe, MD, PhD; Center for Environmental and Human Toxicology, Veterinary Medicine, University of Florida, Gainesville

Stela McLachlan1 and Chris Vulpe2 1Centre for Population Health Sciences, The Usher Institute of Population Health Sciences and Informatics, The University of Edinburgh, Edinburgh, UK; 2Center for Environmental and Human Toxicology, Veterinary Medicine, University of Florida, Gainesville

Both hematological parameters and iron status are routinely measured in clinical practice. Commonly measured red blood cell (RBC) endpoints include hemoglobin (Hb), hematocrit (Hct), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV) and red blood cell count (RBCC). Similarly, serum iron, transferrin, transferrin saturation and serum ferritin are commonly used to assess iron status. Ongoing work has uncovered significant unique and common genetic contributions to the phenotypic expression of both hematological endpoints and iron status. Large scale genetic association studies on RBC traits, including up to ~71,800 people by means of meta-analysis, identified more than 100 genes potentially influencing one or more of these traits with many of them consistently identified across studies and even different ethnic groups [1-12]. Iron-related genes feature prominently among these associations. Most notably, HFE (associated with Hb, MCH and MCV) and TMPRSS6 (associated with Hb, Ht, MCH, MCHC and MCV) show strong association with RBC traits across different populations. Recent large scale genetic study of iron status on ~48,000 Europeans [13] showed that an unexpectedly high proportion of loci known to affect erythrocyte phenotypes also affect iron, transferrin and ferritin. The study identified ABO as associated with ferritin, which was previously shown to affect most of the RBC traits [1, 5, 8, 10, 14, 15], however, it is not clear if “ABO variation primarily affects iron stores and therefore erythrocyte count, or vice versa.” Several QTLs identified in mouse studies of RBC traits [16] contain genes identified in human studies [1]. For example, Atp2b4 is a peripheral membrane calcium ATPase with no obvious connection to hemoglobin synthesis or structure but is found to be associated with MCHC in both studies. Equally so, Trim58 (tripartite motif-containing 58) is strongly expressed in bone marrow but has no established function but significantly associated with RBCC in both studies. Trim10, encoding Tripartite motif- containing 10, a protein required for terminal differentiation of erythroid cells in vitro, located in Hb and MCH mice QTLs [17] has shown to be associated with MCHC in humans [1]. Similar studies to identify still undetected contributors to the underlying genetic basis of both RBC traits (reviewed in [18, 19]) and iron status traits are needed in both mice and humans, especially given that some of the identified loci play roles in cardiovascular risk and lipid metabolism. References 1. van der Harst, P., et al., Seventy-five genetic loci influencing the human red blood cell. Nature, 2012. 492(7429): p. 369-75. 2. Benyamin, B., et al., Common variants in TMPRSS6 are associated with iron status and erythrocyte volume. Nat Genet, 2009. 41(11): p. 1173-5. 3. Chen, Z., et al., Genome-wide association analysis of red blood cell traits in African Americans: the COGENT Network. Hum Mol Genet, 2013. 22(12): p. 2529-38. 4. Ganesh, S.K., et al., Multiple loci influence erythrocyte phenotypes in the CHARGE Consortium. Nat Genet, 2009. 41(11): p. 1191-8. 5. Kamatani, Y., et al., Genome-wide association study of hematological and biochemical traits in a Japanese population. Nat Genet, 2010. 42(3): p. 210-5. 6. Kullo, I.J., et al., A genome-wide association study of red blood cell traits using the electronic medical record. PLoS One, 2010. 5(9). 7. Soranzo, N., et al., A genome-wide meta-analysis identifies 22 loci associated with eight hematological parameters in the HaemGen consortium. Nat Genet, 2009. 41(11): p. 1182-90.

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8. Lo, K.S., et al., Genetic association analysis highlights new loci that modulate hematological trait variation in Caucasians and African Americans. Hum Genet, 2011. 129(3): p. 307-17. 9. Chambers, J.C., et al., Genome-wide association study identifies variants in TMPRSS6 associated with hemoglobin levels. Nat Genet, 2009. 41(11): p. 1170-2. 10. Li, J., et al., GWAS of blood cell traits identifies novel associated loci and epistatic interactions in Caucasian and African-American children. Hum Mol Genet, 2013. 22(7): p. 1457-64. 11. Traglia, M., et al., Association of HFE and TMPRSS6 genetic variants with iron and erythrocyte parameters is only in part dependent on serum hepcidin concentrations. J Med Genet, 2011. 48(9): p. 629-34. 12. Ding, K., et al., Genetic Variants That Confer Resistance to Malaria Are Associated with Red Blood Cell Traits in African-Americans: An Electronic Medical Record-based Genome-Wide Association Study. G3 (Bethesda), 2013. 3(7): p. 1061-8. 13. Benyamin, B., et al., Novel loci affecting iron homeostasis and their effects in individuals at risk for hemochromatosis. Nat Commun, 2014. 5: p. 4926. 14. Ferreira, M.A., et al., Sequence variants in three loci influence monocyte counts and erythrocyte volume. Am J Hum Genet, 2009. 85(5): p. 745-9. 15. Hong, K.W., et al., Association between the ABO locus and hematological traits in Korean. BMC Genet, 2012. 13: p. 78. 16. Davis, R.C. , et al., Genome-wide association mapping of blood cell traits in mice. Mamm Genome. 2013 Apr;24(3-4):105-18. 17. Bartnikas, T.B., et al., QTLs for murine red blood cell parameters in LG/J and SM/J F(2) and advanced intercross lines. Mamm Genome. 2012 Jun;23(5-6):356-66. 18. Okada, Y. and Y. Kamatani, Common genetic factors for hematological traits in humans. J Hum Genet, 2012. 57(3): p. 161-9. 19. Chami, N. and G. Lettre, Lessons and Implications from Genome-Wide Association Studies (GWAS) Findings of Blood Cell Phenotypes. Genes (Basel), 2014. 5(1): p. 51-64.

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Wednesday, September 9, 2015 8:30 – 8:55 Plenary Session IV: Iron Loading Hemoglobinopathies Chairs: Suthat Fucharoen and MD Domenico Girelli, MD, PhD Regulatory Role of Iron Transport in Beta-Thalassemia Speaker: Yelena Ginzburg, MD; New York Blood Center

Iron is an essential component of oxygen-carrying proteins (e.g. Hb). Since most body iron is destined for Hb synthesis, erythropoiesis and iron metabolism are inextricably linked. Dietary absorption, storage, and recycling of iron maintain its stable concentration in circulation, enabling the production of 6 gm of Hb daily. Hepcidin, secreted primarily by hepatocytes, is the principal regulator of iron homeostasis, including dietary iron absorption, iron recycling by macrophages, and the release of iron from hepatic stores. Hepcidin expression is induced by iron loading and inflammatory cytokines and suppressed by hypoxia, iron deficiency, ad eete erythropoess -thalasseas are a rop o dseases ased y tatos the -globin gene resulti reded or aset -globin synthesis, anemia, expanded and ineffective erythropoiesis, and ro oerload espte ro oerload patets ad e th -thalassemia, hepcidin levels are not increased. Low hepcidin causes increased iron absorption ad s plated ro oerload -thalassemia. The effect of anemia and/or ineffective erythropoiesis is dominant over iron in regulating hepcidin. The mechanisms of this suppressive effect have not been fully elucidated but likely include factor(s) secreted by erythroid prersors e roth deretato ator ad tsted astrlato s deted - thalassemic humans and mice, respectively, and Erfe identified as a physiologic erythroid regulator of hepcidin, increased in oe arro ro -thalassemic and phlebotomized wild type mice, and suppressed hepcidin expression in vitro). We have previously shown that exogenous apo-transferrin (apoTf) results in increased Hb, reduced reticulocytosis, decreased Epo, reversed splenoealy, ad reased hepd epresso - thalassemic mice and more recently demonstrate that apoTf normalizes Erfe expression in sorted erythroid prersors ad reerses syste ro oerload -thalassemic mice, strongly suggesting that iron depletion improves ineffective erythropoiesis and thus increases hepcidin expression despite relative iron deficiency. Our current data reveals that apoTf, despite relative iron restricted erythropoiesis and decreased MCH, results in a decrease of TfR1 expression on erythroid precursors. Specifically, our preliminary data suggests that apoTf functions via an effect on TfR1 trafficking, increasing enucleation to improve erythropoiesis in vitro. Furthermore, we previously demonstrate decreased soluble TfR1 in apoTf-treated -thalassemic mice. In addition, TfR1+/- mice, despite characteristics of ineffective erythropoiesis (i.e. increased serum Epo, erythroid expansion (without changes in Hb), and higher Erfe expression), exhibit no change in hepcidin expression, suggesting that cellular and/or soluble TfR1 is involved in erythroid hepcidin regulation. We thus analyzed TfR1 haplo-sey -thalassemic mice and demonstrate a reversal of ineffective erythropoiesis similar to apoTf-treated -thalassemic mice. TfR1 haplo-set -thalassemic mice exhibit normalized enucleation in vivo relate to -thalassemic mice. Despite a relative decrease in liver and serum iron concentrations relative to -thalassemic mice and increased Erfe expression relative to wild type mice, hepcidin expression is increased in TfR1 haplo-set -thalassemic mice, suggesting that TfR1 provides an Erfe independent regulation of hepcidin. We hypothesize that hepcidin expression changes in response to soluble TfR1 and demonstrate that in vitro administration of holoTf and soluble TfR1 results in hepcidin suppression relative to holo aloe prary hepatoytes ro ld type or -thalassemic mice. Taken together, these data reveal the complexity and importance of the nuances of iron trafficking and their impact on the effectiveness of erythropoess ad relato o ro etaols, dysrelated -thalassemia.

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Wednesday, September 9, 2015 8:55 - 9:20 Plenary Session IV: Iron Loading Hemoglobinopathies Chairs: Suthat Fucharoen and MD Domenico Girelli, MD, PhD Iron Overload in the Hemoglobinopathies & Transfusion-Dependent Anemias – A Clinical Perspective Speaker: Joy Ho, MBBS,DPhil,FRACP; Royal Prince Alfred Hospital & University of Sydney; Massachusetts General Hospital, Harvard Medical School

Significant advances have been made in the management of iron overload in the hemoglobinopathies, to prevent and reduce iron-related organ damage. For many years iron-overload cardiomyopathy has been the most important cause of mortality in thalassemia major (TM). The development of new oral iron chelators has widened treatment options and improved non-adherence. Magnetic resonance imaging (MRI) for the measurement of liver and cardiac iron load are increasingly used to individualise therapy. Techniques such as transient elastography are used to evaluate liver fibrosis, while assays for labile plasma iron are being assessed for their clinical utility. The efficacy of oral deferiprone and deferasirox has been evaluated in phase III clinical trials, but studies on their efficacy in specific organs are continuing, particularly on cardiac iron. Whether the same protocols and targets can be applied to other transfusion-dependent anemias is also important to establish. A multi-centre Australian study (MILE) was conducted to evaluate change in cardiac and liver iron load by MRI over one year of chelation by deferasirox, including predominantly TM patients but also patients with sickle cell disease (SCD) and myelodysplastic syndrome (MDS). A significant reduction in cardiac iron load was demonstrated in the study, particularly in patients with normal and moderate baseline cardiac load. Significant reductions in liver iron concentration (LIC) were also observed, especially in those with baseline LIC above target range. Despite these advances a recent, large multi-centre Australian epidemiological study (TIMES) (n=243) has shown a high prevalence of iron overload in transfusion-dependent (TM, MDS, other anemias) and non- transfusion dependent thalassemia (NTDT) patients. Despite the widespread availability of iron chelation, many susceptible patients still had cardiac (T2* <20 ms) and hepatic (liver iron concentration (LIC) >5 mg/g dw for NTDT and >7 mg/g dw for other groups) siderosis - 10% and 47% of patients respectively. Hepatic siderosis was found in 32% of TM patients (who all received ICT) and >50% in the other disease groups, while a high prevalence (22%) of cardiac siderosis was found in TM patients. In 220 patients analyzed, few (3.4%) patets th , or other aea types th ser errt had e d, confirming the utility of this SF chelation target in clinical practice. The mechanisms of iron load in TM likely differ from MDS and may account for the increased predisposition to cardiac iron loading. Studies have shown that cardiac loading and unloading often lag behind hepatic iron, suggesting that de-ironing of the liver is important in reducing cardiac iron, supported by results such as the Australian MILE study demonstrating that patients with a lower baseline LIC had a greater reduction in cardiac iron than those with a higher LIC. Possible causes for the high prevalence of iron overload in chelated patients include suboptimal use of or adherence to chelators or inefficacy; further investigation to define such causes will be important, possibly suggesting a need for improvements in the modes of administration and/or the development of newer agents. These include hepcidin agonists and activin receptor ligand traps, the latter confiming the impact of ineffective erythropoiesis on iron regulatory pathways and highlights the exciting prospect of controlling iron through modulating hepcidin while improving erythropoiesis.

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Wednesday, September 9, 2015 9:20 - 9:40 Plenary Session IV: Iron Loading Hemoglobinopathies Chairs: Francois Canonne-Hergaux, PhD and Martina Muckenthaler, PhD What is the Unmet Need In Iron Chelation Practice in 2015: Experiences From Hemoglobinopathies Vip Viprakasit, MD, DPhil (Oxon), FRCPT Division of Hematology and Oncology, Department of Pediatrics & Thalassemia Center, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

Iron overload is an unavoidable consequence of regular blood transfusion in patients with transfusion dependent thalassemia (TDT). This leads to several complications secondary to iron toxicity including cardiac dysfunction, liver cirrhosis endocrinopathies and growth disturbances. Due to human have no mechanism to excrete this etra ro ot the oly easre s y the se o ro helato therapy oeer, lal eay o ro helato therapy (ICT) in transfusion dependent thalassemia (TDT) can be variable due to several factors. These include; asele est ro stats, otos trasso ro , type, dose ad ode o adstrato o ro chelators, compliance and putative genetic background that might underlie biological response to iron overload and its treatment.

Previously, we have shown that up to 40% of our Thai TDT patients who received monotherapy of deferasirox (DFX) >35 mg/kg/day did not respond well to once daily dosing but could get improved after their administration have been adjusted to twice daily. In addition, up to 55% of our TDT patients do not respond to deferiprone (DFP) monotherapy. In a follow up study, the majority of this population showed a clinical response after we increased the DFP dose up to 94 mg/kg/day in a combination with deferoxamine (DFO). However a number of TDT patients in whom their compliance were not compromised, they did not show any clinical response as there was no decline in their baseline serum ferritin (SF) and liver iron concentration (LIC) by MRI-T2* and they represent a population who seems to have ‘clinical resistance’ to current standard iron chelation therapy. This is an important unmet clinical need in term of iron chelator choices and requires a further basic research to search for a better chelator with an improved chelation properties with acceptable and tolerable adverse events.

Finally, a modern, direct organ targeting iron monitoring by means of magnetic resonance imaging (MRI) to determine liver iron concentration (LIC) and myocardial iron concentration through the measurement of either R2 or T2* parameters have become a standard practice in developed world. This modality has been shown to proe satly o the patets ortalty ad ordty seeral ohorts ro the , taly, reee ad yprs r reet aalyss halad has also sho that or patets sral as satly etter in our center than the others in our country, owing to the fact that we have incorporated such monitoring into our practice since 2009. However, the accessibility of thalassemia patient to this monitoring remains obscure due to the cost of MRI and its subsequent analysis. We proposed a model based on an MRI network that is operate saa otres sa a ro ssesset y hroh ths etor, e can successfully perform over 750 scans in over 500 patients over the last three years. This ‘poor’ man approach remains to be an alternative to provide an access to this standard monitoring for people who desperately need this measurement.

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Wednesday, September 9, 2015 10:30 - 10:55 Plenary Session V: Iron, Infection and Inflammation Chairs: Francois Canonne-Hergaux, PhD and Martina Muckenthaler, PhD Iron Deficient RBCS From Gambian Children and Pregnant Women are Resistant to Field Isolates of Falciparum Malaria Speaker: Carla Cerami, MD, PhD

M. M. Goheen1, B. Darboe2, E. Danso2, M. Bah2, M. Affara3, A. Bah2, R. Wegmuller2, A. M. Prentice4, C. Cerami1 ; University of North Carolina

Clinical studies show that iron deficiency protects against malaria, and that administration of iron to iron- deficient individuals increases the risk of malaria. However, the precise mechanisms by which iron deficiency confers this protection from malaria remain poorly understood and elucidation of these mechanisms may enable new antimalarial strategies. We have previously reported that parasite invasion of and growth in iron deficient RBCs is reduced, however all of this work was done using RBCs from donors living in a non-malaria endemic area (the United States) and with standard laboratory strains of P. falciparum. In an effort to further evaluate the pathogenesis of P. falciparum infection in people with iron deficiency, we have now quantified parasite cellular phenotypes from several clinical strains of P. falciparum isolated from The Gambia in vitro with erythrocytes from Gambian individuals with iron deficiency. Specifically, we have collected RBCs from Gambian children (ages 6 months to 2 years) and pregnant Gambian women (2nd and 3rd trimester) with hemoglobin levels less than 11 g/dL. Then, using flow cytometry-based assays, we separately examined the effect of iron deficiency on overall parasite growth and, merozoite invasion of, RBCs. We demonstrate that P. falciparum erythrocytic stage growth in vitro increases directly in relation to mean corpuscular hemoglobin concentration in RBCs. We also describe the development of a field ready RBC barcoding assay that allows for direct comparison of parasite invasion into two cell populations labeled with the same fluorophore at differing concentrations. Using this assay, we demonstrate that P. falciparum invasion is reduced in iron deficient RBCs from Gambian children. Additionally, we show that Gambian field isolates of P. falciparum exhibit the same phenotype of reduced invasion and growth in iron deficient RBCs. These investigations leverage novel tools and clinical samples from a malaria endemic country to refine our understanding of an ancient relationship between parasite and host. Further investigations of this relationship can improve our fundamental understanding of P. falciparum pathogenesis and enable the development of strategies to treat and prevent both iron deficiency and malaria.

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Wednesday, September 9, 2015 10:55 – 11:20 Plenary Session V: Iron, Infection and Inflammation Chairs: Francois Canonne-Hergaux, PhD and Martina Muckenthaler, PhD Inflammation-Driven Heme/Iron Cytotoxicity in Parkinson's Disease Speaker: Raffaella Gozzelino, PhD; Chronic Diseases Research Center (CEDOC), Faculty of Medical Sciences (FCM), NOVA University of Lisbon

Heme is a metallo-compound, essential for the survival of most organisms. However, the ability of the central iron (Fe) atom, contained within its protoporphyrin ring, to participate in the Fenton chemistry and generate highly reactive hydroxyl radicals renders heme potentially toxic. Under inflammatory conditions, the release of heme from hemoproteins leads to unfettered oxidative stress, a deleterious effect associated with tissue damage and disease severity that dictates the outcome of infectious diseases. As these are often associated with a certain degree of hemolysis and vascular leakage, it is possible that under these conditions the cytotoxicity of heme/Fe may affect also less accessible organs, such as the brain, thus increasing the risk and severity of neurodegenerative diseases. This hypothesis has been investigated in mice, in which the exogenous administration of heme or the release of this molecule upon infection was shown to enhance the severity of Parkinson’s disease. While the cytotoxicity of heme relies on the ability of Fe to participate in redox reactions, the disruption of Fe homeostasis in peripheral immune cells increases the susceptibility to PD and critically contributes to neurodegeneration.

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Wednesday, September 9, 2015 11:20 - 11:45 Plenary Session V: Iron, Infection and Inflammation Chairs: Francois Canonne-Hergaux, PhD and Martina Muckenthaler, PhD Responses of the Gut Microbiota to Supplementary Iron: A Survey at the Host-Microbial Interface Speaker: Guus Kortman, PhD; Radboud University Medical Center, Nijmegen, The Netherlands

Iron is a highly abundant metal on earth and is vital for virtually all organisms, including most bacterial species. Nonetheless, iron deficiency is the most prevalent human nutrition disorder worldwide and is generally treated by oral iron administration. In particular infants and young children in tropical underdeveloped countries with high infection pressure are vulnerable to iron deficiency. Ironically, iron supplementation has been associated with an increased burden of infectious diseases and accumulating evidence suggests that unabsorbed iron can stimulate growth and virulence of bacterial pathogens in the intestinal environment. Simultaneously, host iron status influences the intestinal defense against pathogens. The human gut is the natural habitat for a unique large and dynamic bacterial community. Major functions include important trophic effects on intestinal epithelia, on immune structure and function, and protection of the colonized host against invasion by pathogenic microbes via the production of trophic metabolites and immunostimulatory molecules. The healthy gut microbiota is a balanced community that is shaped by host immunity and metabolism and by environmental factors. Perturbations of the balanced gut microbiota have been associated with gastrointestinal disorders such as diarrhea, gastroenteritis and chronic inflammatory bowel disease. Importantly, oral iron administration has been associated with a potentially more pathogenic gut microbiota profile and an increase in diarrhea and gut inflammation. Our work covered the multifaceted aspects of nutritional iron with respect to growth, composition, metabolism and pathogenicity of the gut microbiota in relation to human health. In a randomized controlled trial with Kenyan infants that received iron fortification, we have shown that after 4 months the iron-fortified groups had a lower relative abundance of beneficial Bifidobacteriaceae and a higher relative abundance of potentially pathogenic Enterobacteriaceae, while also the ratio of Enterobacteriaceae to Lactobacillaceae was increased. Importantly, iron fortification increased the abundance of pathogenic E. coli strains and caused elevated fecal calprotectin levels, indicating increased intestinal inflammation. With in vitro models we have shown that iron can increase growth and the adhesion to intestinal epithelial cells of pathogenic gut microbiota members such as Salmonella Typhimurium. Furthermore, supplementary iron also increased S. Typhimurium pathogenicity in a simple in vivo C. elegans intestinal model. In a kinetic model for the human large intestine (TIM-2) we evaluated the effects of supplementary iron on gut microbial metabolism. Metabolome profiling showed that gut microbial activity markedly shifted from a saccharolytic to a proteolytic profile in response to iron, in particular with ferrous sulfate. Importantly, cell viability tests showed increased cytotoxicity of metabolite-containing effluent from iron-rich conditions. Next, metagenomic analyses showed a general enrichment of bacterial motility-chemotaxis systems under iron-rich conditions. These data indicate that iron preparations induce a more hostile intestinal environment characterized by i) reduction of beneficial microbes, ii) increased levels of bacterial metabolites that can impair the barrier function of the gut wall, and iii) increase of virulence-associated pathways of enteric pathogens. Together, these findings implicate that more safe iron preparations are warranted. An ideal iron preparation is well absorbed by the host, but does not have adverse effects on the gut microbiota composition and activity. Some potential beneficial approaches of oral iron administration will be discussed.

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Wednesday, September 9, 2015 14:30 – 14:55 Concurrent Session V: Cell Death and Autophagy with an Iron Twist Chairs: Paolo Arosio, PhD and Gaetano Cairo, PhD Ferroptosis: A New Name for an Old Way to Die Speaker: Marcus Conrad, PhD; Helmholtz Zentrum München, Institute of Developmental Genetics

Ferroptosis was initially described as a novel form of non-apoptotic cell death that can be induced by a set of small molecules based on their ability to selectively kill engineered human fibroblasts overexpressing oncogenic H-RAS. Being clearly distinct from apoptosis and other reported forms of regulated necrotic cell death, iron-dependent lipid peroxidation has been recognized as one of the hallmarks of this cell death paradigm. Cysteine availability either through direct uptake of cystine via the cystine-glutamate antiporter (System xc-) or via the transsulfuration pathway, glutathione biosynthesis and maintenance of glutathione peroxidase 4 (GPX4) activity emerge to be important upstream events to prevent ferroptosis. Thereby, GPX4 stands out for its unique activity to efficiently detoxify oxidized esterified fatty acids in membranes. Recently, direct genetic evidence was provided that ferroptosis is not only limited to specific tumors treated with ferroptosis inducing agents, but that ferroptosis is a pathologically relevant form of cell death in vivo causing kidney tubule cell death and acute renal failure in GPX4 knockout mice and in wildtype mice subjected to ischemia/reperfusion injury in kidney and hepatic tissue. Cardiolipin oxidation in GPX4 knockout kidneys appears to be an early biochemical event preceding oxidative modification of other phospholipid species and outer mitochondrial membrane rupture. Given the prominent role of oxidized lipids in ferroptotic signaling, deciphering the oxi-lipidome language will be instrumental for a better understanding of how this impacts on ferroptotic cell death. Additional studies are required to better illuminate the in vivo importance of iron in ferroptosis and to unveil yet-unrecognized players in the ferroptotic pathway. Nonetheless, the finding that oxidative stress is translated via the GPX4/lipid oxidizing system into a specific cell death cascade amenable to pharmacological intervention opens new possibilities for redox-targeted therapies.

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Wednesday, September 9, 2015 14:55 – 15:20 Concurrent Session V: Cell Death and Autophagy with an Iron Twist Chairs: Paolo Arosio, PhD and Gaetano Cairo, PhD Role of NCOA4 Protein in DNA Replication Control and Ferritinophagy Speaker, Francesca Carlomagno, PhD; Universita’ degli Studi di Napoli Federico II

Roberto Bellelli, Giorgia Federico, Maria Domenica Castellone, Alessandro Matte, David Colecchia, Achille Iolascon, Mario Chiariello, Lucia De Franceschi, Massimo Santoro, Francesca Carlomagno

Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita’ degli Studi di Napoli Federico II, 80131, Napoli, Italy; Istituto di Endocrinologia ed Oncologia Sperimentale del CNR, 80131, Napoli, Italy

NCOA4 is a transcriptional coactivator of nuclear hormone receptors that undergoes gene rearrangement in human cancer. We recently showed that NCOA4 is able to control DNA replication by interacting with and regulating CMG complex, the processive helicase of the replication fork. NCOA4 null mouse embryonic fibroblasts display unscheduled DNA replication origin activation, which results in replication stress and premature senescence. In addition, NCOA4 has been recently identified as a cargo receptor mediating autophagic ferritin degradation. By analysing NCOA4 null mice iron metabolism, we observed that NCOA4 deficiency leads to iron accumulation in liver and spleen. Although NCOA4 null mice display iron overload signs, they also suffer from a mild microcytic hypochromic anaemia that was exacerbated under an iron- deprived diet regimen. On the contrary, when fed with an iron-enriched diet, NCOA4 null mice die prematurely, featuring signs of liver damage. Thus, NCOA4 paly a dual role; it acts as a regulator of DNA replication origins that helps preventing inappropriate DNA synthesis and replication stress but also as a controller of ferritinophagy and iron homeostasis. If and how these distinct functions might crosstalk is currently unknown, although one can speculate that this dual activity of NCOA4 may be co-evoluted in order to coordinate iron availability with cell cycle progression.

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Wednesday, September 9, 2015 14:30 – 15:00 Concurrent Session VI: Novel Therapies for Iron Disorders Chairs: Elizabeta Nemeth and PhD; Stefano Rivella, PhD Exploiting Hepcidin and Its Pathway to Limit Iron Absorption and Erythroid Iron Intake Speaker: Stefano Rivella, PhD; Department of Pediatrics, Division of Hematology Children's Hospital of Philadelphia

Department of Pediatrics, Division of Hematology Children's Hospital of Philadelphia

ess ro deposto tal oras s the a ase o ordty ad ortalty patets aeted y - thalassemia. Inappropriately low levels of the liver hormone hepcidin are responsible for the increased iron absorption, leading to toxic iron accumulation in many organs. Interestingly, modulation of iron metabolism can also have significant positive effects on the aberrant erythropoiesis seen in ß-thalassemia. Several studies have shown that targeting iron absorption could be beneficial in reducing or preventing iron overload as well ameliorating the anemia in ß-thalassemia, showing promising preclinical data. Some of the new approaches include targeting Tmprss6, the main suppressor of hepcidin expression; the use minihepcidins, small peptide hepcidin agonists that target ferroportin; administration of transferrin, which reduce erythroid iron intake; and new approaches that target iron regulatory molecules in the intestine, limiting intestinal iron absorption. Additionally, direct modulation of erythropoiesis might lead to beneficial effects on iron metabolism. In fact, due to the suppressive nature of the erythropoiesis on hepcidin expression, approaches that limit erythroid expansion and ineffective erythropoiesis (such as targeting the Smad2/3 signaling) are also showing beneficial effects on iron metabolism. In conclusion, understanding the mechanisms that control iron metabolism and erythropoiesis is paving the way to novel therapeutic approaches to reduce or prevent iron overload and aelorate aea -thalassemia.

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Thursday, September 10, 2015 8:30 – 8:40 Concurrent Session VII: Iron Speciation and Its Modification by the Use of Therapeutics Chairs: David Frazer, PhD and Francesca Vinchi, PhD Speciation of Iron in Normal Tissue Speaker: R.C. Hider; King’s College London

This overview describes the major transit and storage forms of iron that exist in normal serum and in tissues. Extracellular Iron In normal mammals, extracellular iron is transported in the serum bound to ferritin, transferrin, hemopexin and albumin. There is a vanishing small amount of iron bound to low molecular weight ligands (M.Wt < 500) due to the high iron affinity of apotransferrin. Iron bound to ferritin and transferrin is present in the iron(III) redox slate. Hemopexin and albumin bind hematin (iron(III) coordinated to protophoryrin) and heme. Hemopexin binds heme more tightly than any other protein (K > 109 M-1), and rapidily removes heme and hematin from albumin when not fully saturated. Both transferrin and hemopexin have specific receptors that are capable of releasing bound iron in a controlled fashion. Intracellular Iron Cells require a kinetically labile form of iron which can donate iron to cytosolic iron-dependant apoenzymes, of which there are over 400. Iron(II)glutathione has recently been suggested to adopt this role, it being kinetically labile and protected from autoxidation by the presence of excess glutathione, typically > 2 mM.1 The concentration of the labile iron pool is in the rego o 2 It has been further proposed that iron(II)glutathione is the mitochondrial labile iron pool and so is responsible for the supply of iron for heme and iron sulfur cluster synthesis.2 Heme is a prosthetic group for numerous hemoproteins and plays an important role in cell differentiation and yet, like inorganic iron, heme can redox cycle causing cell damage. Mammalian cells contain a cytosolic heme pool hh seres oth prersor ad relatory tos, reah oetratos o soe cell types. As with the cytosolic labile iron pool, glutathione forms a conjugate with hematin which possesses a markedly improved water solubility and a much lower tendency to redox cycle, both properties rendering it a suitable candidate for the cytosolic heme pool.3,4 Unlike iron(II)glutathione, the stable redox state of iron in hematin-glutathione is iron(III). The major storage form of intracellular iron is ferritin, which is being continually broken down in lysosomes5 to a range of low molecular weight iron containing species, including iron-citrate, iron-cysteine and iron- glutathione. The iron associated with this mixture of iron complexes is effluxed from the lysosome via the DMT1 transporter to supplement the cytosolic labile iron pool. 1. Hider RC and Kong XL (2011) Biometals 24, 1179-1187. 2. Hider RC and Kong XL (2011) Dalton. Trans. 42, 3220-3229. 3. Shviro Y and Shaklai (1987) Biochem. Pharmacol. 36, 3801-3807. 4. Hider RC, Smith A and Kong XL (2015) 6th IBIS meeting, Poster 74. 5. Kidane TZ, Sauble E and Linder MC (2006) Am. J. Physiol. Cell Physiol. 291, C445-C455.

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Thursday, September 10, 2015 9:05 – 9:30 Concurrent Session VII: Iron Speciation and Its Modification by the Use of Therapeutics Chairs: Robert Hider, BSc, PhD and John Porter, MD, FRCP NTBI and LPI Assays as Clinical Indicators of Iron Overload and Treatment Efficacy Speaker: Dorine W. Swinkels, MD, PhD; Radboudumc, Nijmegen, the Netherlands

The predominant forms of iron present in living entities are associated with proteins such as transferrin in the major circulating fluid and haem and ferritin in cells. However, the iron binding capacity of the iron transport protein transferrin can be surpassed when a substantial amount of iron enters the circulation due to excessive iron absorption from the diet or an outpour of iron from cell stores. In these conditions non-transferrin-bound- iron (NTBI; circulating iron not bound to transferrin, ferritin or haem) appears in the plasma. Plasma NTBI is apparently comprised of several subspecies, which may be classified either by their chemical composition, chemical reactivity and susceptibility to chelation. As for the chemical composition NTBI is heterogeneous and thought to consist of Fe(III) bound to albumin and citrate and potentially to acetate, malate and phosphate. Of these, citrate has the highest affinity for Fe(III), and under physiological conditions two isoforms dominate, i.e. monomeric and oligomeric Fe(III) complexes. The fraction of plasma NTBI that is redox-active and chelatable is designated labile plasma iron (LPI). Although not much is known about the in vivo behavior of the iron complexes assumed to represent NTBI, these complexes have been shown experimentally to be taken up by susceptible cell types, including hepatocytes, cardiomyocytes and pancreatic islet cells, with consequent oxidant injury. NTBI and LPI are thought to play a major role in various disorders that are dominated by accumulation of excess iron in the circulation and tissues, among which are hereditary hemochromatosis (HH), iron-loading anemias (-thalassemia major and intermedia), myelodysplastic syndromes and sickle-cell disease after transfusion. Studies in patients with various iron-loading disorders have shown a reduction in NTBI and LPI upon phlebotomy and chelation therapy that is associated with an improved prognosis. Therefore, NTBI and LPI assays are promising as therapeutic targets and for the evaluation of iron overload and the efficacy and compliance of iron-lowering therapies. Due to the complexity and potential clinical importance of NTBI, several assays have been developed for its detection. In a recent Round Robin study comprising samples from patients with various iron loading disorders that were send out to laboratories performing 10 worldwide leading assays, we found that absolute levels differed considerably between assays and were lower for LPI than for NTBI. Some assays also reported negative values. Assays were reproducible with high between-sample and low within-sample variation. Assays correlated and correlations were highest within the same group of NTBI and LPI assays. Increased transferrin saturation, but not ferritin, was a good indicator for the presence of circulating NTBI iron forms. We conclude that before NTBI and LPI assays can be introduced into clinical practice rigorous validation and standardization of assays, consensus on how to report the results, and clinical outcome studies to determine clinically relevant assay formats and their toxic thresholds are needed.

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Thursday, September 10, 2015 8:30 – 9:00 Concurrent Session VIII: Iron and Tissue Injury Chairs: Esther Meyron-Holtz, PhD and Grant A. Ramm, PhD Mechanisms of Iron-Induced Hepatic Pathology in Haemochromatosis: The Role of the Hepatic Stellate Cell-Liver Progenitor Cell Niche Speaker: Grant A. Ramm, PhD; Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, Brisbane, Australia.

The development and progression of hepatic fibrosis in HFE-associated haemochromatosis is highly variable and not solely due to the severity of iron overload. Clinical cofactors are known to influence progression and genetic factors may also play a role. A variant of GNPAT (glyceronephosphate O-acyltransferase) was recently identified as a modifying gene for extreme iron loading in exosome sequencing studies of patients with Hemochromatosis (1), with similar such studies identifying potential modifiers of cirrhosis predisposition (2). The precise mechanisms responsible for increased hepatocyte iron stores progressing to portal fibrosis, cirrhosis and ultimately hepatocellular carcinoma in hemochromatosis remain unclear. Excess hepatic iron leads to oxidative stress-induced liver cell damage which is proposed to play a role in Kupffer cell activation and subsequent transformation of liver fibroblast precursors, hepatic stellate cells, into collagen-producing myofibroblasts responsible for hepatic fibrosis in Haemochromatosis. However, these hepatic stellate cells do not work in isolation and are influenced by their hepatic paracrine environment, mediated by the local production of soluble mediators as well as by direct cell-cell contact with resident and infiltrating cell populations. Continuous iron loading of hepatocytes has been shown to lead to impaired hepatocyte replication, which has recently been linked to the proliferation of a population of stem-like bipotential liver progenitor cells (LPCs) and their differentiation into reactive biliary cells in a histological event termed the ductular reaction. Both the presence of the ductular reaction and portal inflammation are strongly associated with hepatic fibrosis progression. It has been proposed that LPC proliferation and the ductular reaction are crucial for liver fibrogenesis and that cross-talk between LPCs, hepatic stellate cells and macrophages may drive this fibrogenic pathway. Our studies have implicated a role for tissue-derived ferritin as well as putative ferritin receptors, key cytokines including the tumour necrosis factor (TNF) family members TNF-like weak inducer of apoptosis (TWEAK) and lymphotoxin-beta (LT -associated), growth factor molecules such as jagged1/notch, RANTES and ICAM-1, as well as specific microRNAs, in regulating various different aspects of hepatic stellate cell/LPC interaction, the ductular reaction and hepatic fibrogenesis.

(1) McLaren et al., Hepatology, 2015;62:429-439. (2) Emond et al., BioIron 2015 Proceedings, 2015; Abstract #219.

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Thursday, September 10, 2015 10:30 – 11:00 Concurrent Session X: Iron Nutrition and Supplementation in Populations Chairs: Sant-Rayn Pasricha, PhD, FRSCP, FRCPA and Fudi Wang, PhD How Do We Solve a Problem Like Anaemia? From Biology to Policy and Back Again Speaker: Sant-Rayn Pasricha, PhD, FRSCP, FRCPA; University of Oxford

Anaemia affects 800 million children and women worldwide according to latest estimates. The World Health Organization has developed the ambitious target of reducing the prevalence of anaemia in women of reproductive age by 50% by 2025, even though progress on anaemia control has been disappointing. Several critical gaps remain in how we biologically conceive of the problem of anaemia, how we define it, how we measure it, and how we intervene to address it, especially among young children. While anaemia in children has been linked to serious health and economic outcomes including impairments in growth and short and long term cognitive, motor and behavioral development, evidence that iron interventions alleviate these outcomes is inconclusive. Among children, iron interventions can be delivered through iron supplementation, home- fortification using multiple micronutrients, and central food fortification. These strategies reduce anaemia prevalence, but evidence of benefit on functional health outcomes has not been obtained. In contrast, convincing evidence of harm from iron interventions has been observed in several randomized controlled trials. These concerns raise the possibility that iron interventions may not provide an overall benefit to children in low- income countries at high risk of both anaema and infection. Mathematical-modeling-based risk-benefit analysis may provide insights into the net benefit on health which can be achieved by iron interventions, and the cost at which these are obtained. The contribution to the overall burden of anaemia from iron deficiency is uncertain and difficult to define. Meta-analyses suggest iron supplementation can alleviate about 30% of the burden of anaemia in malaria endemic countries and 70% in non-endemic countries. Epidemiologic data support the hypothesis that the contribution to anaemia of iron deficiency varies greatly between countries. However, measurement of iron status (and hence iron deficiency) is difficult in settings where infection is highly prevalent, as all iron indices are affected by inflammation. The World Health Organization is presently undertaking a major effort to define the utility of ferritin as an index of iron status (both deficiency and overload) and to devise solutions for adjusting ferritin in the context of inflammation during nutrition surveys. However, it may also be worth redefining the problem from ‘iron deficient’ to ‘in need of and ready to accept iron’; in this context, biomarkers that predict iron absorption may be appropriate. Hepcidin can predict iron absorption and may be useful in defining which children should be given iron in malaria-endemic settings. Hepcidin has also been shown to be a useful index of iron status among adolescents living in malaria non-endemic settings, and among adult women in developed settings. Hepcidin appears to reflect elevated iron needs during pregnancy, supporting the rationale for universal iron supplementation in this particular population. More broadly, the concept of ‘anaemia’ may need revisiting. Thresholds of Hb used to define anaemia have not been examined for decades, and the role of present cutoffs in defining the problem among the young, the elderly, during pregnancy and among different populations is uncertain.

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Thursday, September 10, 2015 11:00 – 11:30 Concurrent Session X: Iron Nutrition and Supplementation in Populations Chairs: Sant-Rayn Pasricha, PhD, FRSCP, FRCPA and Fudi Wang, PhD Clinical Studies in Iron and Infection Speaker: Kamija Phiri; PhD; University of Malawi, Malawi

Anaemia which affects xx people worldwide has largely been attributed to iron deficiency. Iron has paradoxically been shown to be both beneficial and also detrimental as its believed to exacerbate infection in the human body. Although there have been numerous studies, the exact interaction between iron and infection remains unclear. Findings of a large case control study carried out in southern Malawi will be discussed that showed that iron deficiency was associated with less risk of infection i.e. iron was protective in a group of children with severe anaemia. Further the contribution of iron deficiency versus other aetiologies in development of severe anaemia will be presented. Lastly anaemia pathogenesis studies will be discussed with regard to iron and infection.

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Thursday, September 10, 2015 13:30 – 13:55 Concurrent Session XII: Hemochromatosis and Other Forms of Primary Iron Overload Chairs: Pierre Brissot, MD and Christine E. McLaren, BS, MS, PhD The Search for Modifier Genes in HFE-Related Hemochromatosis: Exome Sequencing in C282Y Homozygous Men with Extreme Phenotypes Identifies a GNPAT Polymorphism Associated with Severe Iron Overload Speaker: Gordon McLaren, MD

GD McLaren1,2, MJ Emond3, VN Subramaniam4,5, PD Phatak6, JCBarton7, PC Adams8, JB Goh4,5, CJ McDonald4, LW Powell4,5, LC Gurrin9, KJ Allen10, DA Nickerson3, Tin Louie3, GA Ramm4,5, GJ Anderson4,5, and CE McLaren2 1VA Long Beach Healthcare System, Long Beach, CA; 2University of California, Irvine, CA; 3University of Washington, Seattle, WA; 4QIMR Berghofer Medical Research Institute; 5University of Queensland, Brisbane, Australia; 6Rochester General Hospital, Rochester, NY; 7Southern Iron Disorders Center, Birmingham, AL; 8London Health Sciences Centre, London, ON, Canada; 9University of Melbourne, Melbourne, Australia; 10Murdoch Childrens Research Institute, Melbourne, Australia

Phenotypic variability among homozygotes for the HFE C282Y polymorphism has long been recognized. Although recent screening studies have suggested that HFE-hemochromatosis is often relatively mild, some patients develop severe iron overload and organ damage. Such variability in the severity of iron overload suggests the possibility of genetic modifiers. To address this question, we conducted exome sequencing of DNA from 35 HFE C282Y male homozygotes with either markedly increased iron stores (n=22; cases) or normal to mildly increased iron stores (n=13; controls) in order to identify rare and common causal variants associated with variability of disease expression in HH. The 35 participants, residents of the U.S., Canada, and Australia, reported little or no alcohol consumption. After quality control filtering, sequencing data included 82,068 single nucleotide variants and 1,403 insertions/deletions (indels); 10,337 genes were tested for a difference between cases and controls. We identified the polymorphism GNPAT p.D519G (1556A>G; exon 11; chromosome 1q42; rs11558492) as the most significantly different variant between cases and controls (p=0.033 by the likelihood ratio test after correction for multiple comparisons). Sixteen of 22 case participants had the GNPAT p.D519G polymorphism (15 heterozygotes, 1 homozygote); no control participant had this polymorphism. GNPATp.D519G is common among people of European descent (allele frequency 20.6%) and might interact with aberrant HFE to increase the risk of hepatic iron overload. We compared the allele frequencies of GNPAT p.D519G in the present 22 cases and 13 controls with that of 4300 European Americans in the NHLBI Exome Sequencing Project Exome Variant Server. The allele frequency in cases was greater than that of European Americans (38.6% vs. 20.6%, respectively; p = 0.0076). The allele frequency in controls was significantly lower (0% vs. 20.6%, p = 0.0054).

To examine functional consequences of GNPAT deficiency, we performed siRNA-based knockdown of GNPAT in the human liver cell line HepG2/C3A. This resulted in a >17-fold decrease in expression of the messenger RNA encoding the iron-regulatory hormone, hepcidin. Our data indicate that GNPAT p.D519G is associated with a high-iron phenotype in male HFE C282Y homozygotes and may participate in hepcidin regulation, thereby modifying severity of iron overload. The fact that 6 of 22 cases in our studies did not have the GNPAT p.D519G allele suggests that there may be other genetic modifiers of phenotypic variability in these C282Y homozygotes. It is important to examine the prevalence of the GNPAT p.D519G allele among C282Y homozygotes in other populations. Our results identify GNPAT as a candidate gene for expanded studies to examine its function in regulating iron absorption and metabolism and to identify newly-diagnosed C282Y homozygotes whose risk for development of severe iron overload is great.

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Thursday, September 10, 2015 15:30 – 15:50 Concurrent Session XIV: Iron and the Central Nervous System Chairs: Yan-Zhong Chang and PhD James Duce, PhD The Influence of Amyloid-β Precursor Protein Proteolytic Processing on Neuronal Iron Homeostasis Speaker: James Duce, PhD

Bruce X. Wong1, Andrew Tsatsanis2, Linh Q. Lam1,3, James Dachtler2, Paul A. Adlard1,4, Ashley I. Bush1,4 and James A. Duce1,2,4. 1The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Victoria, Australia. 2School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, West Yorkshire, United Kingdom. 3Department of Pharmacology and Therapeutics, The University of Melbourne, Parkville, Victoria, Australia. 4Department of Pathology, The University of Melbourne, Parkville, Victoria, Australia.

Introduction: Iron is an integral cofactor in many metabolic processes involved in transcriptional signalling, synapse formation and neuroplasticity, in all of which a function for amyloid-β precursor protein (APP) has also been heavily implicated but remains unclear. Intraneuronal iron imbalance is a predominant catalyst for reactive oxygen species production, particularly within iron accumulating neurodegenerative diseases such as Alzheimer’s disease. While APP has historically been associated with AD due to the prevalence of the amyloidogenic processing of APP to produce amyloid- peptde, e reetly dsoered that has a role in neuronal iron homeostasis by, in part, promoting iron efflux through cell surface stabilization of the iron pore ferroportin. Methods: The requirement for surface presented APP to stabilise ferroportin and facilitate neuronal iron efflux was established with cell surface biotinylation assays, flow cytometry and confocal microscopy. Manipulation of the proteolytic processing of APP to alter the cellular location of APP was carried out by suppression of required secretase expression or activity, or stable transfection of familial mutations in APP known to alter its processing pathway. Neuronal iron homeostasis was monitored indirectly by iron responsive protein expression (e.g. Ferritin) or directly by the Calcein-AM assay. Results: Detailed cell surface characterization confirms that the location of ferroportin on the neuron surface is increased upon iron incubation and is dependent upon APP. By altering the proteolytic processing of APP at the cell surface, consequential changes in neuronal iron homeostasis arise. Enhancing the amyloidogenic pathway of APP processing leads to intracellular iron accumulation. Conclusion: With increased amyloidogenic processing of APP being a major contributor to sporadic AD, these studies increase our understanding as to why iron accumulation and increased susceptibility to reactive oxygen species neurotoxicity are prevalent with this disease.

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Podium #1 ADMINISTRATION OF A SMALL MOLECULE HIF-2 INHIBITOR REDUCES IRON OVERLOAD IN A MOUSE MODEL OF HEMOCHROMATOSIS Yaomin Chen, PhD, Julio Ruiz, PhD and Richard Bruick, PhD UT Southwestern Medical Center

Members of the Hypoxia Inducible Factor (HIF) family have been implicated in the progression of several diseases using a variety of genetic models. Previous work demonstrated that the HIF-2 transcription factor could be selectively inhibited with small molecules that bind within an internal cavity located in the HIF-α -B domain. These antagonists are now suitable for in vivo studies. Here we demonstrate that a HIF-2 antagonist is efficacious in the treatment of hemochromatotic mice lacking either the HFE or HJV genes, models of iron overload disease. These mice fail to express adequate levels of hepcidin, a hormone secreted from the liver that promotes degradation of the iron export protein ferroportin. Consequently, these mice absorb iron in an unregulated manner with the excess iron accumulating in tissues such as the liver. Administration of the HIF-2 inhibitor reduces the expression of key proteins required for iron absorption in the duodenum including Divalent metal transporter 1 (DMT-1) and Duodenal cytochrome B (DcytB), required for iron uptake by enterocytes, in addition to ferroportin. As a result, dietary iron absorption is decreased in the treated animals, lowering serum iron levels and reducing the iron burden within the livers of hemochromatotic mice. These studies demonstrate the therapeutic potential for HIF-2 inhibitors for the treatment of iron overload diseases. This work was supported by Peloton Therapeutics.

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Podium #2 DEFICIENCY OF TRANSFERRIN AND NON-TRANSFERRIN IRON RESULTS IN CHRONIC KIDNEY DISEASE Andong Qiu, PhD1, Melanie Viltard, PhD2, Rong Deng, Bsc3, Xueqiao Wang, Bsc1, Max Werth, PhD3, Yue Yu, Bsc3, Neal Paragas, PhD3, Abby Sakar, Bsc3, Shaun Darrah, Msc3, Roger Boles, Bsc3, Efrat Bruck, Bsc3, Alexander Rittenberg, MD3, Danielle Recanati, Bsc3, Kristen McNierney, Bsc3, Rosemary Sampogna, MD3 and Jonathan Barasch, MD, PhD3 1Tongji University; 2Institute for European Expertise in Physiology, Paris, France; 3Columbia University

Iron deficiency in pregnancy is the most common single nutrient deficiency in the world, and it results in prematurity and low birth weights. However, neither the species of iron required for organogenesis, nor the specific defects induced by deficiency of different iron species have been identified. Here, we investigated the response of the embryonic kidney to periconceptual maternal iron deficiency (PM-ID) and to cell lineage autonomous transferrin iron deficiency (ATf-ID) by generating a conditional ablation of the transferrin receptor 1 (TfR1) gene. Our studies demonstrated that the iron requirement was an effect of two different pools of iron, transferrin (Tf) and non-transferrin-bound iron (NTBI), which were both required for optimal kidney development in a temporal and spatial sequence. PM-ID depleted both Tf iron and NTBI in embryos, which resulted in hypoplastic kidneys and even anephria, characterized by markedly fewer glomeruli and proximal tubules, and defective patterning of ureteric bud (UB) branching. Transcript analysis revealed that the development of proximal tubules was highly sensitive to iron deficiency and to widespread hypoxia induced by PM-ID. Studies in TfR1 knockout mice found that kidney development was initiated in ATf-ID embryos, but ATf-ID specifically in metanephric mesenchyme retarded the development of the initiated kidney and resulted in anephria at birth, demonstrating that NTBI supported the initial stages of kidney development whereas Tf iron was required during maturation towards birth. ATf-ID in the nephron progenitor cells, condense mesenchyme, resulted in only mild hypoplasia early in the development, but severely cystic kidneys after birth, indicating the increasing requirement of Tf iron for postnatal kidney development, growth and function. In particular, ATf-ID in stroma demonstrated normal kidneys and in UB only led to occasional hypoplastic and hydronephrotic postnatal kidneys, indicating that NTBI could fully support the development of stroma, and possibly the UB as well.

Remarkably, maternal iron supplementation at mid-gestation (E11) achieved nearly full rescue of nephron number with PM-ID, but not with ATf-ID, further highlighting the necessity of Tf iron for nephrogenesis. Hence, our results demonstrate that both Tf iron and NTBI were combinatorially required for metanephric kidney development and a switch occurs in late gestation from NTBI to Tf iron reflecting compartment-specific iron sensitivity, and that iron depletion resulted in hypoplastic chronic kidney diseases the timing and specificity of which was the result of the sequence of NTBI and Tf-iron utilization.

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Podium #3 HEPCIDIN: A NOVEL TREATMENT FOR HEME-MEDIATED KIDNEY INJURY? Rachel van Swelm, Jack Wetzels, Vivienne Verweij, Coby Laarakkers, Jeanne Pertijs, Rosalinde Masereeuw and Dorine Swinkels Radboudumc

In multiple clinical observational studies it has been demonstrated that increased urinary hepcidin levels are associated with reduced risk of developing acute kidney injury (AKI) due to hemolysis and hypoxia in cardiac surgery patients. This study aimed to get more insight in renal hepcidin handling and its potential protective effects against heme-mediated AKI. C57Bl/6 mice were treated with i) a single i.p. dose of 10 µg human hepcidin-25 (hhep25) to study renal handling of systemic hepcidin, ii) a single i.v. dose of 5 mg hemoglobin (Hb) to induce AKI, and iii) Hb combined with hhep25 to evaluate the protective effects of hhep25 on Hb-mediated kidney injury. Hhep25 levels in plasma and urine were measured and distinguished from murine endogenous hepcidin-1 levels using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Systemically administered hhep25 was cleared rapidly from plasma and excreted in the urine. Hhep25 was biologically active in mice as observed by a negative feedback on mouse endogenous hepcidin1 plasma levels (60% reduction, p<0.05) and hepatic mRNA expression (10-fold decrease, p<0.05). In addition to hhep25, we also detected the smaller isoforms hhep22 and hhep20 in urine, but not in plasma, showing that hhep25 is degraded in the tubular lumen. Urine hhep25 was 20-fold increased in megalin deficient mice compared to control (p<0.05) and immunofluorescence staining showed that hepcidin was present in tubules expressing megalin, but NOT in megalin-deficient tubules, demonstrating that megalin is responsible for hhep25 uptake in the proximal tubules.. Administration of hhep25 simultaneously or 4h after Hb injection in wildtype mice significantly attenuated the Hb-induced rise in urinary NGAL (p<0.05) and KIM1 (p<0.05) levels, and renal IL6 (p<0.05) and NGAL (p<0.05) mRNA expression. Interestingly, simultaneous administration of Hb and hhep25, but not Hb or hhep25 alone, resulted in an increase in renal Hamp1 mRNA expression (15 fold, p<0.05), suggesting that besides systemic filtered hepcidin, also locally produced hepcidin may be involved in renal protection. Administration of hhep25 to Hb-treated mice reduced renal mRNA expression of HO-1, DMT1, H-ferritin and L- ferritin (all p<0.05), possibly reflecting the mechanisms by which hepcidin exerts its protective effects. In conclusion, systemic hepcidin is filtered to the urine, partly reabsorbed via megalin in the proximal tubules and degraded in the tubular lumen. Moreover, our data suggest that both systemically delivered hepcidin and locally produced hepcidin are involved in renal protection against heme-induced AKI.

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Podium #4 HEPCIDIN AS A MAJOR COMPONENT OF RENAL ANTIBACTERIAL DEFENSES AGAINST UROPATHOGENIC ESCHERICHIA COLI Dounia Houamel, Nicolas Ducrot, Thibaud Lefebvre, Boualem Moulouel, Marie-Agnes Sari, Sarah Millot, Sophie Vaulont, Erick Denamur, Hervé Puy, Carole Beaumont, Laurent Gouya and Zoubida Karim, PhD INSERM U1149, Centre de Recherche sur l’inflammation, Université Paris Diderot, Laboratory of Excellence, GREx, 16 rue Henri Huchard, 75018 Paris, France.

Introduction: Urinary tract infection (UTI) is most often caused by uropathogenic Escherichia coli (UPEC), which have the ability to adapt to iron limitation and the strong host defenses of the urinary tract. Hepcidin is The iron-regulatory peptide that was also shown to exhibit antimicrobial activity. We recently demonstrated that hepcidin controls iron excretion by regulating its reabsorption in the distal nephron. Here we were interested in addressing its role in UTI, which remains largely unknown. Methods: Experimental UTI was induced in wild-type (WT) and hepcidin knockout mice (Hepc-/-) using the uropathogenic CFT073 strain. We measured renal cytokine levels by multiplex ELISA and measured bacterial growth with a Tecan Infinite reader. Hepcidin expression was estimated level in both liver and kidney by RT-qPCR and in the serum by LCMSMS. BMP6 and IL-6 pathways were investigated in mice and in mIMCD-3-infected renal cells. Results: Using microdissected renal tubules, we showed that hepcidin is preferentially expressed in distal nephron tubules. In Hepc-/- mouse kidneys, iron accumulated in the medulla, the Atp4a pump was significantly repressed, and urine was alkalinized. Compared with infected WT mice, Hepc-/- mice showed a dramatic increase in renal bacterial load. However, bacterial infection was significantly dampened by the pretreatment of WT mice with hepcidin. In addition, infected Hepc-/- mice exhibited decreased iron accumulation in the renal medulla and a significant attenuation of the renal inflammatory response. Importantly, we were able to demonstrate in vitro the bacteriostatic activity of hepcidin against CFT073. Altogether, these results strongly support a protective role of renal hepcidin in UTI, its deficiency aggravating the course setting of the disease. Interestingly, CFT073 was found to repress renal hepcidin, both in vivo and in cultured mIMCD-3 renal cells, via a reduced SMAD-pathway, suggesting a bacterial strategy to escape the antimicrobial activities of hepcidin. Conclusion: We provide new mechanisms by which hepcidin contributes to renal host defense and suggest that targeting renal hepcidin offers a promising strategy to prevent bacterial infection. Further experiments are in progress including UTI in kidney-specific knockout host mice and In vitro studies to highlight the molecular mechanism by which UPEC affect hepcidin expression.

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Podium #5 IMPORTANT ROLES OF HEPHAESTIN AND CERULOPLASMIN IN IRON TRANSPORT OF MOUSE KIDNEY. Guohao Liu, Bo Jiang, Undergraduate, Jiashuo Zheng, Undergraduate, Mengxia Chen, Undergraduate and Huijun Chen, Professer Medical School of Nanjing University

Many researches about the role of multi-copper ferroxidases (MCFs) in iron homeostasis were focused on the tissues classically associated with iron absorption and metabolism, which is namely the duodenum, the liver and the reticuloendothelial system. However, the role of MCFs in kidney iron transport remains poorly understood. The proteins, which is related with iron metabolism, were found in the kidney of rats, mice and human bodies and they play very important roles. We used Hephaestin (HEPH) and Ceruloplamin (CP) single (Heph KO, Cp KO) or double (Heph/Cp KO) knockout mouse model to study the roles of MCFs in the kidney. We found there are both gene and protein of Heph or Cp expression in the cortex of the all phenotype mice at 6 months of age, while the gene expressions of Heph and Cp were significant up regulated in cortex of the Heph/Cp KO mice compared to other phenotype mice. This data suggests that Heph RNA and protein still remained in the cortex of kidney of Heph KO mice, Cp RNA and protein also remained in the cortex of kidney of Cp KO mice, and both of Heph and Cp genes and protein remained in the cortex of kindey of Heph/Cp KO mice. We found that Heph gene and protein deleted in the kidney medulla of the Heph KO mouse, Cp gene and protein deleted in the medulla of the Cp KO mouse, and both gene and protein of Heph and Cp deleted in the kidney medulla of Heph/Cp KO mice. Furthermore, we did peal's staining for mouse kidney, and found that iron is accumulated in medulla of Heph/Cp KO mice, however neither in the kidney medulla of Heph KO and Cp KO mice nor in the kidney cortex of all phenotype mice. These results indicate that the double knockout of Heph and Cp genes could lead to the iron deposition in mouse kidney, which reminds us the important roles of MCFs in mouse kidney iron transport and the subsequent impact on the whole body iron homeostasis.

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Podium #6 STUDYING IRON HOMOESTASIS IN DROSOPHILA Bing Zhou Tsinghua University

Drosophila melanogaster, or the fruit fly, is a genetically tractable organism that has been widely used to help unravel a number of fundamental biological questions. We have been trying to use this model organism platform to study how iron is metabolized, aiming to complement existing bioiron research. We found Drosophila ferritin, which is secreted, is responsible for absorbing iron from the diet and delivering to other tissues of the body. Likewise, ferritin also participates in effluxing iron for detoxification purpose. In a serendipitous finding, the enormous amount of ferritin iron in the Drosophila gut is surprisingly provided by a previously suspected zinc importer, ZIP13. In addition to ferritin, iron proteins residing in the secretion pathway such as collagen modifying enzymes lysyl hydroxylase and prolyl hydroxylases also needs iron from dZIP13. Deficiency of dZIP13 leads to collagen defects, iron shortage in Drosophila ferritin and as a result, a significant reduction of iron level in the body. These findings not only greatly enhanced our understanding of Drosophila iron metabolism, they also helped explain the etiology of one kind of human EDS (Ehlers–Danlos syndrome) disease associated with hZIP13 mutation. References 1. Xiao G, Wan Z, Fan Q, Tang X, Zhou B. The metal transporter ZIP13 supplies iron into the secretory pathway in Drosophila melanogaster. Elife. 3:e03191. doi: 10.7554/eLife.03191. 2014 2. Tang X, Zhou B. Ferritin is the key to dietary iron absorption and tissue iron detoxification in Drosophila melanogaster. FASEB J. 27(1):288-98. 2013

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Podium #7 FERRITIN ASSEMBLY IN THE INTESTINES OF DROSOPHILA MELANOGASTER Fanis Missirlis, PhD and Abraham Rosas-Arellano, PhD Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional

Fruit flies store iron into ferritin, a protein complex of 12 H and 12 L subunits that assembles within the secretory system of specialized intestinal cells and is then released into the hemolymph. In the brief period (of approximately one hour) that follows dietary iron intake, but precedes the assembly of the ferritin complex and its iron loading, a distinct subcellular localization of the H and L subunits has been detected using green fluorescent protein (GFP) tagged H subunits and specific antibodies against L subunits (Missirlis et al. 2007). This observation suggested that the assembly process of the ferritin complex might be regulated by iron, yet post-translational regulation of ferritin biosynthesis has not been studied in any organism. Here, we characterize new genetic tools based on monomeric cherry fluorescent protein (mCherry) tagging of the ferritin L subunit. We have generated flies expressing simultaneously mCherry-tagged L subunits and GFP-tagged H subunits and we have imaged the ferritin assembly process in vivo. Our observations suggest that the H and L subunits are present in intestinal tissue even under iron-limiting conditions; however, they are largely kept in separate vesicular pools when iron is scarce. Separation of the ferritin subunits by two-dimensional protein gel electrophoresis suggests that post-translational modifications can differentiate the individual ferritin subunits when assembled or when present in separate pools under iron sufficiency and limitation, respectively. Reference Missirlis F, Kosmidis S, Brody T, Mavrakis M, Holmberg S, Odenwald WF, Skoulakis EM, Rouault TA. Homeostatic mechanisms for iron storage revealed by genetic manipulations and live imaging of Drosophila ferritin. Genetics (2007) 177:89-100.

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Podium #8 – Switched to Poster #175

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Podium #9 THE ACTIVE ROLE OF MULTICOPPER OXIDASE-1 IN IRON METABOLISM IN DROSOPHILA: A FERROXIDASE AND /OR AN ASCORBATE OXIDASE? Minglin Lang, PhD1, Maureen J. Gorman, PhD2 and Michael R. Kanost, PhD2 1College of Life Science, University of Chinese Academy of Sciences, Beijing 100049, China; 2Department of Biochemistry, Kansas State University，Manhattan, Kansas 66506, USA

Iron is an essential metal involved in many fundamental biochemical processes within a living cell or organism. In mammals, ceruloplasmin and hephaestin, two enzymes known as multicopper oxidases, have been well characterized as having iron oxidase (ferroxidase) activity and playing critical roles in iron metabolism. Surprisingly, we know little of multicopper ferroxidases from invertebrates, and the mechanisms of iron metabolism in invertebrates (including model organisms such as Drosophila melanogaster) are not well understood. Our previous work has identified a multicopper oxidase-1 (DmMCO1) function as a ferroxidase, and it’s essential in iron metabolism in D. melanogaster. This is based on the following several lines of evidences: Knockdown of DmMCO1 markedly reduced iron accumulation in the iron cells of both wandering larvae and adults, as well as in whole body of wandering larvae and adults. Moreover, knockdown of DmMCO1 increased the longevity of flies cultured on food supplemented with a toxic concentration of iron, and strong knockdown of DmMCO1 resulted in pupal lethality, indicating that DmMCO1 is an essential gene. qRT-PCR results showed that knockdown of DmMCO1 resulted in a significant reduction of DMT1 mRNA; because DMT1 transports iron from the food into the midgut epithelial cells, this reduction in DMT1 could explain the reduction in iron in knockdown insects. Furthermore, we purified recombinant DmMCO1 and verified that it has ferroxidase activity. Recent in vitro study showed that the purified recombinant DmMCO1 has excellent ascorbate oxidase activity, and the previous three predicted iron binding residues in DmMCO1 are not required for ferroxidase or laccase activity, but two of the residues (His374 and Asp380) influence oxidation of ascorbate. As iron homeostasis and ascorbate homeostasis are linked via redox homeostasis, the result suggests that DmMCO1 is also possible influence iron homeostasis through its ascorbate oxidase activity. However, whether DmMCO1 does function as a ferroxidase and/or an ascorbate oxidase in Drosophila need further experiments to determine. Taken together, as iron has numerous implications in human neurological diseases and Drosophila is used to model a number of human syndromes including Parkinson’s disease and Alzheimer’s disease, our understanding of how iron is handled in this organism is of great interest and significance.

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Podium #10 IRON HOMEOSTASIS IN C. ELEGANS: A MODEL OF AGEING Gawain McColl, PhD The Florey Institute of Neuroscience and Mental Health

Iron is essential for eukaryotic biochemistry. However, systematic trafficking and storage is required to maintain supply of iron while preventing it from catalysing unwanted reactions, particularly the generation of oxidising reactive species. Iron dyshomeostasis has been implicated in major age-associated diseases including cancers, neurodegeneration and heart disease. Although oxidative stress is widely studied, there has been comparatively less focus on cellular iron in ageing models. Here I will explore iron homeostasis in an ageing context in the nematode, Caenorhabditis elegans. Our understanding of which genes and signaling pathways modulate longevity and ageing rate has enormously benefited from this simple multicellular animal. Taking advantage of this basis I will discuss X-ray fluorescence imaging, native- metalloproteomic analysis and transgenic approaches to better understand the cause and consequence of altered iron coordination and distribution in ageing. I will explore how and where iron homeostasis is lost during C. elegans ageing, and its relationship to the age-related elevation of damaging reactive oxygen species. In addition, I will discuss methods to examine iron metabolism across different scales of biological organisation, from the cellular level to populations of animals. My laboratory recently reported that wild type C. elegans utilise the iron storage protein, ferritin, to sustain longevity, buffering against exogenous iron. As post-reproductive C. elegans senesce iron escapes from safe-storage in ferritin, increasing cellular Fe2+ load, and elevating the generation of reactive species. These findings and further investigations are consistent with the hypothesis that iron-mediated processes contribute to biological ageing. I propose that loss of iron homeostasis may be a fundamental and inescapable consequence of senescence that may represent a critical target for therapeutic strategies to improve health outcomes in ageing.

Sixth Congress of the International BioIRon Society Page 91 Podium Abstracts IBIS

Podium #11 CHARACTERIZATION OF NON-VERTEBRATE FERROPORTIN HOMOLOGUES Maria Carmela Bonaccorsi di Patti1, Fabio Polticelli2, Valentina Tortosa2, Pier Antonio Furbetta1, Tiziana Persichini2 and Giovanni Musci3 1Sapienza University of Rome; 2University Roma Tre; 3University of Molise

Introduction. Ferroportin (Fpn), a membrane protein belonging to the Major Facilitator Superfamily (MFS) of transporters, is the only vertebrate iron exporter known so far. Mutations of Fpn lead to the so-called ‘ferroportin disease’ or type 4 haemochromatosis, characterized by two distinct iron overload phenotypes depending on whether the mutations affect the protein’s activity or its degradation pathway. Structural models of Fpn have allowed to identify a potential iron binding site and other residues important for the function of the protein. However, many questions on how Fpn works still require an answer and an experimentally determined structure of the protein is lacking. Objective. In this work a search for non-vertebrate homologues of Fpn has been carried out with the objective of identifying a potentially simpler protein that might be amenable for structural and functional studies. Structural models of some of these Fpn homologues have been obtained and heterologous expression and partial characterization of the proteins has been performed. Methods. A BLAST search was performed to identify non-vertebrate homologues of human Fpn. Selected non-vertebrate Fpn structural models were obtained in the inward-open, occluded and outward-open conformations based on the threading/ab initio modeling approach implemented in I-TASSER. Recombinant Fpn was constitutively expressed in the methylotrophic yeast Pichia pastoris strain GS115. A GFP fusion was expressed in HEK293 cells and subcellular localization was determined by confocal microscopy. Results and discussion. Few non-vertebrate Fpn homologues were identified in the NCBI Non Redundant protein sequence database. Non-vertebrate Fpns were mostly found to be shorter than human Fpn. All residues belonging to the iron binding site were conserved; however, Cys326 which is required for binding of hepcidin was not conserved. C. elegans Fpn 1.1 (CeFpn) was chosen as a first target for structural and functional characterization. Molecular modeling of CeFpn showed that the protein conforms to the MFS general organization with 12 TM helices, with extra- and intracellular loops substantially shorter compared to human Fpn. Heterologous expression of CeFpn in P. pastoris demonstrated low expression levels but a single homogeneous band was evident in Western blots of membrane extracts. Confocal microscopy analysis of a GFP fusion expressed in HEK293 cells demonstrated that the protein was present mostly in intracellular vesicles. Conclusion. Non-vertebrate homologues of Fpn can represent good alternatives to the human protein for structural and functional studies.

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Podium #12 MACROPHAGE-HEPCIDIN EXPRESSION IN RESPONSE TO HEMIN IS MEDIATED VIA ERK-SIGNALLING PATHWAY Naveen Kumar Tangudu, MSc, Betül Alan, MSc, Dilay Lai, Sabine Hübner, PhD and Maja Vujic Spasic, Prof Dr Institute for Comparative Molecular Endocrinology, University of Ulm

Macrophages are the key immune cells actively involved in iron metabolism. Inflammatory and infection stimuli shape the macrophages towards a proinflammatory M1-phenotype, hallmarked by iron retention and increased expression of iron- hormone hepcidin. As professional phagocytotic cells, macrophages are also capable of acquiring iron from senescent erythrocytes or scavenge haemoglobin and home which are then degraded within the phagolysozome. In addition, macrophages uptake iron in the form of transferrin-bound iron through TfR1-mediated endocytosis and by the transmembrane transport of iron by DMT1. However, the consequences of iron uptake on macrophage intracellular iron levels and on hepcidin expression have not been fully clarified. We therefore set to investigate whether non-transferrin and transferrin-bound iron (NTBI and TBI, respectively) modulate macrophage-hepcidin expression and intracellular macrophage iron levels. Treatment of murine bone marrow derived macrophages (BMDM) with ferric ammonium citrate (200µM; 16h) or haemoglobin (25µM; 16h) significantly increased intracellular iron pool (2.2-fold and 1.8-fold, respectively) while no changes in macrophage-hepcidin mRNA expression were measured. In contrast, treatment with hemin (25µM; for 16h) or TBI (3.5%) resulted in a significant hepcidin mRNA induction (4.3-fold; p<0.0003 and 4.8-fold; p<0.006, respectively) without affecting intracellular iron levels. For the hemin-mediated hepcidin induction, the presence of the Erk/MEK signalling, but not Nf- b or p38-MAPK, as examined using respective inhibitors, is obligatory. Similarly to hemin, LPS treatment (100ng/ml; 2h) of BMDM resulted in significant induction of hepcidin expression; however, Erk/MEK, Nf- b or p38-MAPK pathways were dispensable for LPS-mediated hepcidin induction. The relevance of our in vitro findings was further investigated in mice upon short-term hemin (35µmol/kg for 16h) and LPS injections (0.25mg/kg for 6h). An almost 20-fold increase of hepcidin mRNA expression was observed in the spleen of hemin-injected mice which correlated with the hemopexin and Il-6 mRNA induction. In line with our in vitro observations, increased levels of pErk in the spleen of hemin-injected mice were measured implicating for a possible contribution of the Erk/MEK signalling pathway in the regulation of hepcidin expression in vivo. Surprisingly none of these acute-phase mediators was altered in the liver of hemin-injected mice. The outcome of heme-injections in mice on hepcidin expression are in contrast to LPS effects since the latter caused a prominent induction of a series of acute phase proteins (including hepcidin, hemopexin, Il-6) in the liver but not in the spleen. Interestingly, LPS injections in mice caused a rapid activation of Jak/Stat3 signalling pathway in the liver without affecting the levels of pErk proteins. Notably, no marked induction of the Jak/Stat3 signalling pathway has been observed neither in the spleen of hemin-injected mice nor in BMDM upon hemin treatment. Despite the fact that both hemin and LPS signal through a similar Tlr4-receptor mechanisms, thereby affecting the innate immune responses, the regulation of hepcidin expression by hemin and LPS occurs in a tissue-specific manner and is controlled by distinct signalling pathways. Jointly our data demonstrate that hepcidin expression is triggered in macrophages in response to hemin treatment and requires, at least in part, the presence of Erk/MEK signalling pathway.

Sixth Congress of the International BioIRon Society Page 93 Podium Abstracts IBIS

Podium #13 GENERATION AND CHARACTERIZATION OF Hmox1LysM-Cre MICE: A SMALL SUBSET OF RESIDUAL Hmox1- POSITIVE MACROPHAGES ACCOUNTS FOR EFFICIENT IRON RECYCLING Carine Fillebeen, PhD2, Daniel Garcia-Santos, PhD, Konstantinos Gkouvatsos, MD, PhD, Marzell Buffler, Christiane Becker, PhD, Klaus Schümann, MD, Prem Ponka, MD and Kostas Pantopoulos1 1Lady Davis Institute for Medical Research and McGill University; 2Lady Davis Institute for Medical Research and Department of Medicine, McGill University, Montreal, Quebec, Canada

Tissue macrophages play a central role in systemic iron balance by recycling iron from senescent red blood cells (RBCs) during erythrophagocytosis. The pathway involves enzymatic degradation of heme via heme oxygenase 1 (HO-1), and export of liberated inorganic Fe2+ to the bloodstream via ferroportin. Macrophages from Hmox1-/- mice exhibit defective recycling of heme iron and sensitivity to heme accumulation, which leads to splenic atrophy and iron redistribution to the kidneys. We generated Hmox1LysM-Cre mice bearing myeloid-specific disruption of the Hmox1 gene to explore how the lack of HO-1 in macrophages affects body iron metabolism. Effective ablation of Hmox1 was validated in thioglycollate-elicited peritoneal macrophages (TEPMs). As expected, Hmox1LysM-Cre TEMPs showed decreased viability in response to in vitro erythrophagocytosis. Nevertheless, Hmox1LysM-Cre mice did not develop any signs of anemia and their hematological and serum iron parameters did not significantly differ from those of Hmox1fl/fl controls up to the age of 18 months. The lack of significant pathology persisted even under stress conditions of acute or chronic hemolytic anemia triggered by phenylhydrazine. The Hmox1LysM-Cre mice did not manifest increased intestinal iron absorption or altered tissue iron distribution compared to controls. However, they developed progressive splenomegaly. Kinetic experiments revealed an early delay in clearance of injected 59Fe-labeled RBCs from the blood. Importantly, 59Fe-heme metabolism was shifted to the spleen of Hmox1LysM-Cre mice, while it predominated in the liver of Hmox1fl/fl controls. Consistently, HO-1 expression was readily detectable in red pulp splenic macrophages and completely absent from Kupffer cells of Hmox1LysM-Cre mice. The lack of HO-1 did not compromise Kupffer cell density in Hmox1LysM-Cre livers. Hmox1-positive macrophages were recruited to both Hmox1LysM-Cre liver and spleen following phenylhydrazine-induced hemolysis. Considering that the LysM- Cre system promotes disruption of target genes in 83-98% of tissue macrophages, our data suggest that a small subset of Hmox1-positive myeloid cells in Hmox1LysM-Cre mice is expanded and differentiated to iron-recycling macrophages. We are currently characterizing this cell population, which maintains almost physiological body iron homeostasis. Our data warrant consideration in light of other studies using the LysM-Cre technology for macrophage-specific disruption of iron metabolism genes.

Sixth Congress of the International BioIRon Society Page 94 Podium Abstracts IBIS

Podium #14 ELUCIDATION OF THE MECHANISM OF IRON TRANSPORT FORM PLASMA TRANSFERRIN TO MITOCHONDRIAL FERROCHELATASE: FURTHER EVIDENCE FOR THE “KISS AND RUN” HYPOTHESIS Amel Hamdi, PhD, Tariq Roshan, MD, Alex D. Sheftel, PhD and Prem Ponka, MD,PhD,FCMA McGill University, Lady Davis Institute for Medical Research

In developing red blood cells (RBC), more than 90% of iron (Fe) has to enter mitochondria where ferrochelatase, the final enzyme in the heme-synthesis pathway that inserts Fe2+ into protoporphyrin IX, resides. Strong evidence exists for specific targeting of Fe toward mitochondria in immature RBC, in which Fe acquired from transferrin (Tf) continues to flow into mitochondria even when the synthesis of protoporphyrin IX is suppressed [Borová et al. (1973) Biochim Biophys Acta 320: 143; Richardson et al. (1996) Blood 87: 3477]. It has been hypothesized [Ponka P (1997) Blood 89: 1] that, in hemoglobin-producing cells, there is a direct relaying of Fe from endosomes to mitochondria. This hypothesis has been supported by our findings that: 1) iron, delivered to mitochondria via the Tf pathway, is unavailable to cytoplasmic chelators [Sheftel et al. (2007) Blood 110: 125]; 2) endosome mobility is essential for the efficient incorporation of 59Fe from 59Fe-Tf-labeled endosomes into heme [Zhang et al. (2005) Blood 105: 368]; 3) in reticulocytes, endosomes continuously traverse the cytosol and touch mitochondria as documented by confocal laser microscopy; and 4) the endosome-mitochondrion interaction increases chelatable mitochondrial iron [Sheftel et al. (2007) Blood 110: 125]. Using 3D live confocal imaging, we have confirmed that the delivery of Fe to its final destination in developing RBC involves a transient interaction of endosomes with mitochondria. To further demonstrate the junction between these organelles, we used a novel method based on flow cytometry analyses of lysates (“flow sub-cytometry”) obtained from reticulocytes with fluorescently labeled endosomes (Alexa Green Transferrin) and mitochondria (MitoTracker Deep Red). Using this approach, we have identified three distinct populations: endosomes, mitochondria, and a population of particles labelled with both fluorescent markers representing endosomes interacting with mitochondria. This strategy has been used in studies on reticulocytes and erythroblasts subjected to various experimental conditions. In this study, we also attempted to identify molecular partners involved in the endosome-mitochondria interaction. Using co-immunoprecipitation and pull-down strategies, we aimed to recognize proteins interacting with the extra-endosomal (intracellular) loops of DMT1, which may be involved in interactions with mitochondria. The co-immunoprecipitated proteins were separated based on their molecular weights, stained using Coomassie and/or Silver gels and identified by mass spectrometry and western blotting. Using these strategies, we co-immunoprecipitated (from MEL cells and reticulocytes lysates) proteins that were pulled down with DMT1. We have identified the voltage-dependent anion channel (VDAC), which is located at the outer membrane of the mitochondria [Graham et al. (2004) Curr Top Dev Biol. 59: 87], as one of DMT1 interacting partners using specific antibodies against VDAC and western blotting. These results indicate the physical contact between endosomes and mitochondria. In addition, to define the possible role of DMT1-VDAC interactions in mediating iron uptake, we used a siRNA to silence VDAC expression in MEL cells and then measured 59Fe incorporation into heme. These studies revealed decreased 59Fe incorporation into MEL cells with silenced VDAC. Hence, our findings provide a strong support for the hypothesis that this outer-membrane mitochondrial protein is involved in the interaction with endosomes.

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Podium #15 CRUCIAL ROLE OF FLVCR1A IN THE MAINTENANCE OF INTESTINAL HEME HOMEOSTASIS Veronica Fiorito, PhD1, Marco Forni, Dr2, Lorenzo Silengo, Prof1, Fiorella Altruda, Prof1 and Emanuela Tolosano, Prof1 1Molecular Biotechnology Centre, University of Torino, Italy; 2EuroClone S.p.A Research Laboratory, Molecular Biotechnology Centre, University of Torino, Italy

Introduction. The maintenance of heme homeostasis, mucosa cell renewal and redox environment in the intestine is essential to permit digestion, absorption, cell proliferation, cell apoptosis, and immune response and to avoid the development of gut disorders. The Feline Leukemia Virus, subgroup C, Receptor 1a (FLVCR1a) is a heme exporter with an established role in erythroid cells, macrophages and hepatocytes. Flvcr1a is expressed in almost all cell types including intestinal cells, suggesting a role for this protein also in this tissue. This work investigates the role of FLVCR1a in the intestine. Methods. To analyse the role of Flvcr1a in vivo, an intestine specific conditional Flvcr1a-knockout mouse was generated. Moreover, Flvcr1a- depleted Caco2 cells were used to test Flvcr1a function in vitro. Results. The data show that FLVCR1a does not participate in the absorption of dietary heme, whereas it is involved in the export of de novo synthesized heme from intestinal cells. The loss of Flvcr1a is associated to a decrease of intestinal cell proliferation and to alterations in the peculiar homeostasis of proliferating cells, including the maintenance of their redox status. The involvement of Flvcr1a in these processes renders this exporter crucial for the survival of mice in a model of ulcerative colitis. Discussion. The reported data indicate that FLVCR1a-mediated heme export does not contribute to dietary heme absorption processes. By exporting the excess of de novo synthesized heme from intestinal cells, FLVCR1a participates in intestinal cell proliferation and in the maintenance of the redox status and metabolic activity of intestinal proliferating cells. Thanks to these functions, FLVCR1a collaborates to protect mice from ulcerative colitis. Conclusions. The present work represents the first description of the function of the heme exporter FLVCR1a in the intestine and an advance in the understanding of heme metabolism in this tissue. The findings shed light on the role of heme export in heme absorption processes and unravel a new role for heme export in the control of mucosal renewal and in proliferating cell redox status and metabolic activity, demonstrating for the first time a crucial role for FLVCR1a in maintaining intestinal homeostasis in both physiologic and pathologic conditions.

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Podium #16 CANCER-ASSOCIATED BUT EPO-RESISTANT ANEMIA IS CAUSED BY IRON DEFICIENCY IN A SPONTANEOUSLY TUMOR-DEVELOPING MOUSE MODEL Markus Thiersch, PhD1, Maja Ruetten, DVM2, Milena Costa da Silva, PhD3, Martina Muckenthaler, PhD3 and Max Gassmann, DVM1 1University of Zurich; 2University of Zürich; 3University of Heidelberg

1Institute of Veterinary Physiology, Vetsuisse Faculty and Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich; 2Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich; 3Department of Paedriatic Oncology, Hematology and Immunology, University of Heidelberg

Depending on the cancer type and the severity of hemorrhage and inflammation, tumor patients frequently suffer from the so-called anemia of cancer (AC) that compromises radio- and chemotherapy as well as their quality of life. Erythropoietin (Epo) is the first choice for AC treatment but reduced iron availability may render this medication ineffective. Even more critical are some recently published studies indicating that some cancer cells express the receptor for Epo and that Epo treatment may enhance tumor growth. To develop new strategies to treat AC we analyzed mice that spontaneously develop mammary carcinoma (Trp53floxWapCre; Jonkers et al., 2001) for iron-related parameters. With growth progression of suppuratively inflammed and necrotic carcinosarcomas, the animals developed AC. In more detail, mice display severe splenomegaly and excessive myelopoiesis in both, bone marrow and spleen and showed a pronounced reduction in hemoglobin and hematocrit levels as well as diminished erythrocyte numbers. With progression of the disease, free plasma iron was reduced, transferrin levels increased and mRNA expression of the hepatic iron hormone hepcidin was diminished. These observations strongly suggest that the tumor-bearing mice suffer from iron deficiency. Epo treatment of these mice was unable to restore hemoglobin levels likely due to reduced iron availability. Experiments are in progress to identify the cause of reduced plasma iron level as well as the impact of reduced iron availability on tumor growth and progression. In this disease model for AC we will test novel therapeutic strategies to ameliorate anemia in cancer patients.

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Podium #17 NOVEL FLUORESCENT PROBE DETECTING CATALYTIC FERROUS IRON Ohara, MD2, Tasuku Hirayama, PhD3 and Hideko Nagasawa, PhD3 1Nagoya University Graduate School of Medicine; 2Nagoya University; 3Gifu Pharmaceutical University

Iron overload of a chronic nature has been associated with various human diseases, including infection, carcinogenesis, and atherosclerosis. Recently, a highly specific turn-on fluorescent probe (RhoNox-1; RhoNox-M) specific to labile ferrous iron Fe(II), but not to labile ferric iron Fe(III), was developed. The evaluation of Fe(II) is more important than Fe(III) in vivo in that Fe(II) is an initiating component of the Fenton reaction. Here we applied this probe to frozen sections of an established Fenton reaction-based rat renal carcinogenesis model with an iron chelate, ferric nitrilotriacetate (Fe-NTA). Catalytic iron induces the Fenton reaction specifically in the renal proximal tubules that finally leads to a high incidence of renal carcinogenesis. Notably, this probe reacted with Fe(II) but with neither Fe(II)-NTA, Fe(III) nor Fe(III)-NTA in vitro. Prominent red fluorescent color was explicitly observed in and around the lumina of renal proximal tubules 1 h after an intraperitoneal injection of 10-35 mg iron/kg Fe-NTA, which was dose-dependent, according to semiquantitative analysis. The RhoNox-1 signal colocalized with the generation of hydroxyl radicals, as detected by hydroxyphenyl fluorescein (HPF). The results demonstrate the transformation of Fe(III)-NTA to Fe(II) in vivo in the Fe-NTA-induced renal carcinogenesis model. Therefore, this probe would be useful for localizing catalytic Fe(II) in a variety of studies. Diverse applications of this novel probe to biological samples such as tissues, human amniotic fluids and endometriosis would be presented and discussed.

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Podium #18 EXPRESSION OF THE IRON EXPORTER FERROPORTIN IS CONTROLLED BY MIR-20A IN LUNG CANCER Kamesh Rajendra Babu, MSc, MPhil and Martina Muckenthaler, PhD Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, University of Heidelberg, Germany

The hepcidin/ferroportin regulatory system adjusts systemic iron levels in response to altered iron availability, erythropoietic activity and inflammatory cues. Ferroportin (FPN) is the only known mammalian iron exporter. It binds to the iron hormone hepcidin, which mediates its internalization and degradation. Hepcidin synthesis is controlled by miR-122, a microRNA highly expressed in hepatocytes (Castoldi et al., JCI. 2011). We now show that FPN expression is controlled by miR-20a, which is involved in the regulation of fundamental cellular processes such as cell proliferation and apoptosis. RNA sequencing data obtained from ‘The Cancer Genome Atlas (TCGA)’ revealed significant inverse correlation between expression of FPN and miR-20a in lung cancer samples. A bioinformatic search identified 3 highly conserved target sites for miR-20a within the FPN-3’UTR. The functionality of this interaction was validated by dual-luciferase reporter assays in the hepatocytic cell line Huh7. Transfection of cultured cells with miR-20a significantly suppressed luciferase activity of a reporter containing the wild-type FPN-3’UTR, whereas luciferase activity was rescued in constructs containing a FPN- 3’UTR with mutated binding sites. miR-20 plays a critical role in cancer development. We therefore hypothesized that modulation of the iron export activity of cancer cells by miR-20 may control cellular iron availability, which is a contributing factor to enhance cell proliferation. We analyzed expression of endogenous FPN and miR-20a in cancer-derived cell lines and show a significant inverse correlation of FPN mRNA levels in two non-small cell lung cancer cell lines (H1299 and H1650). Importantly, transfection of a miR-20a mimic or inhibitor significantly altered FPN mRNA levels in the expected manner. Moreover overexpression or knockdown of miR-20a and FPN affects cancer cell proliferation and colony formation. Our results suggest that increased expression of miR-20 in lung cancer may down regulates FPN levels, which causes intracellular iron retention that in turn favors DNA synthesis and increased cell division.

Sixth Congress of the International BioIRon Society Page 99 Podium Abstracts IBIS

Podium #19 IRON LEVELS DEFINE TWO POPULATIONS OF TUMOR ASSOCIATED MACROPHAGES Milene Costa da Silva1, Francesca Vinchi, PhD1, Adelheid Cerwenka, PhD2 and Martina Muckenthaler, PhD3 1University of Heidelberg, Germany; 2DKFZ, Heidelberg; 3University of Heidelberg

Tumor-associated macrophages (TAMs) play an important role in the tumor microenvironment. TAMs acquire a M2-like macrophage phenotype and contribute to tissue remodeling, tumor growth and angiogenesis, hallmarks for cancer invasion. Likely, they also recycle iron from erythrocytes released as a consequence of angiogenesis in the tumor or retain iron upon tumor-related inflammation. The functional consequences of iron accumulation in TAMS are not well established. Here, we investigated whether iron levels affect TAM polarization and function and/or influence tumor growth. Macrophage subpopulations were studied in a mouse model of Lewis Lung Carcinoma (LLC) and macrophage infiltration and iron-related parameters were analyzed by FACS, iron measurements, histology, immunohistochemistry, RT-qPCR and co-culture of macrophages and cancer cells. In addition, iron distribution was studied in human histological slides from lung cancer patients. Both, in lung cancer patients and in a mouse model for LLC we detected two populations of TAMs that differed in their iron content. Interestingly, the iron levels in the TAMs correlates with the differentiation state of the macrophages: iron-loaded macrophages are of the inflammatory (M1-like) phenotype, whereas iron-spared macrophages polarize towards the M2- like phenotype. Co-culturing of LLC cells and macrophages induce the polarization of the macrophages towards an M2- like phenotype. Interestingly, this can be shifted to a M1-like inflammatory phenotype by applying different sources of iron. We show that iron can influence macrophage differentiation in the tumor microenvironment. We described two different populations of iron-related TAMs that might have relevant impact for cancer cell proliferation and the metastatic potential of the tumor.

Sixth Congress of the International BioIRon Society Page 100 Podium Abstracts IBIS

Podium #20 IRON EXCESS DELAYS DISEASE DEVELOPMENT IN MURINE MODELS OF MULTIPLE MYELOMA Alessandro Campanella, PhD, Silvia Galvan, Jessica Bordini, Maurilio Ponzoni, MD, Maria Teresa Sabrina Bertilaccio, PhD and Clara Camaschella, MD San Raffaele Scientific Institute, Milan, Italy

Background Multiple myeloma (MM) is a malignant proliferation of bone marrow (BM) plasma cells (PC) characterized by the release of monoclonal component (M-spike) in serum. Recently it has been demonstrated that a small increase of iron favors MM cell growth in both allogeneic and syngeneic transplanted mouse models. However, since redox homeostasis is fundamental for PC biology we hypothesized that iron limitation may favor long-term survival of malignant cells. In agreement studies on transgenic Vk*MYC mouse model suggested that iron sequestration by BM macrophages causes microenvironment iron restriction (Bordini et al, 2015). We aimed at investigating the effect of iron excess in MM to exploit whether iron toxicity may limit the disease progression. Materials and Methods We used the Vk*MYC model and a xenograft model generated by injecting U266 MM cells in immunodeficient Rag2-/-gc- /- mice. Mice were treated with iron-dextran (250 mg/Kg) or saline. In Vk*MYC the disease development was assessed by the M-spike evaluation; in xenograft mice the disease progression was assessed by flow cytometry, histology and measuring the overall survival. We analyzed erythropoietic markers by flow cytometry and gene expression by qRT-PCR. Results Vk*MYC mice, characterized by slow disease progression, were treated with a single dose of iron-dextran before disease development (at 20 weeks) and we followed the disease onset and progression at 35, 50 and 80 weeks. Iron supplementation reduced from 40% to 25% the number of affected mice at 35 weeks. Then the iron effect was lost in males, while in females it persisted up to 50 weeks. In female mice iron supplementation reduced also disease expansion since M-spike levels remained stable up to 80 weeks. We observed that the erythroid precursors, reduced in Vk*MYC mice, were normalized in both gender treated with iron. In the xenograft mice, characterized by rapidly progressive disease, iron rich BM macrophages and strong reduction of BM erythroid precursors, a single dose of iron-dextran after disease engraftment reduced BM U266 cells expansion, increased median survival compared to control saline treated mice (128 vs 105 days), reduced inflammation and restored bone marrow erythropoiesis. In both models iron supplementation induced a transient increase of transferrin saturation and liver iron storage and strongly promoted liver hepcidin expression. Histological examination did not reveal evident signs of liver toxicity. Conclusion Microenvironment iron restriction is induced by BM macrophages in both models of MM studied. We hypothesize that iron toxicity caused by supplemented iron decreased MM cell survival favoring erythroid maturation. Since disease develops very slowly in Vk*MYC and iron is rapidly stored to avoid toxicity the effect on disease development is transient in this model. Iron supplementation to 50-week-old Vk*MYC mice already affected by the disease is in progress to asses the effect of iron on disease control and on the response to pharmacological treatments.

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Podium #21 MYELOID ZINC FINGER 1 (MZF-1) SUPPRESSES PROSTATE TUMOR GROWTH THROUGH ENHANCING FERROPORTIN-CONDUCTED IRON EGRESS Yue Chen1, Zhihong Zhang2, Kuo Yang2, Jin Du3, Yong Xu2 and Sijin Liu1 1State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences; 2Department of Urology, The Second Hospital of Tianjin Medical University; 3School of Life Science, Tsinghua University

Increasing evidence suggests that disorders in iron metabolism affect tumorigenesis and tumor behaviors, and that excess tumor iron stimulates tumor progression through various mechanisms such as enhancing DNA replication and energy metabolism. Ferroportin (FPN) is the only known iron exporter in mammalian cells, and it determines global iron egress out of cells. FPN reduction leads to decreased iron efflux and increased intracellular iron that consequentially aggravates the oncogenic effects of iron. Although previous studies suggest that myeloid zinc finger 1 (MZF-1) is a multifaceted transcription factor that may function as either an oncogene or a tumor suppressor. MZF-1 was recently identified as a transcription factor that regulates FPN expression. Thus far, however, the molecular mechanisms underlying MZF-1 – FPN signaling in cancers are largely unknown. Here, we found a significant reduction of FPN levels in prostate tumors relative to adjacent tissues, and demonstrated a crucial role of FPN in tumor growth through controlling tumor iron concentration. Inhibition of MZF-1 expression led to reduced FPN concentration, coupled with resultant intracellular iron retention, increased iron-related cellular activities and enhanced tumor cell growth. In contrast, increase of MZF-1 expression restrained tumor cell growth through promoting FPN-conducted iron egress. Importantly, we demonstrated that AP4 and c-Myb jointly modulated MZF-1 transcription, and that miR-492 was also directly involved in regulating MZF-1 concentration through binding to the 3’ untranslated regions of its mRNA. These results correlate with reduced AP4 and c-Myb expression and elevated miR-492 expression found in prostate tumors as compared with adjacent tissues that resulted in diminished MZF-1 and FPN. Moreover, we demonstrated that alterations of AP4, c-Myb and miR-492 levels significantly affected tumor cell growth. Targeting molecules within the MZF-1 – FPN signaling thus appears to be a promising approach to restrain prostate cancer.

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Podium #22 NEW INSIGHTS INTO FERRITIN TRAFFICKING, POLARIZATION AND SECRETION Marianna Truman-Rosentsvit, Adi Bukris and Esther G. Meyron-Holtz Technion – Israel Institute of Technology

INTRODUCTION: Ferritin in mammalians is mainly considered an intracellular cytosolic iron storage protein, however, in numerous immunofluorescence images published in the literature ferritin appears to be in a punctate uneven distribution which could be due to ferritin enrichment in lysosomes. Moreover, possible functions of a secreted form of ferritin have been demonstrated in recent years. Thus, we aimed to elucidate pathways of intracellular ferritin-trafficking, polarization and distribution between different subcellular compartments and possible routes for ferritin secretion. METHODS: Subcellular fractionation by centrifugational methods and glycerol gradient followed by immunoblotting were employed in murine macrophage primary cultures and cell-lines. Immunofluorescence was used to further analyse ferritin subcellular distribution in macrophages and its polarization in epithelial cells. The classical ER-Golgi secretion pathway was inhibited by adding Brefeldin A (BFA), followed by pulse chase analysis. Autophagy, as an example of an entry-point into non-classical pathways, was manipulated by RNAi and chemically by both, activating and inhibitory agents. Lysosomal function inhibitors were also applied. Effects of all these substances on intracellular ferritin distribution and its secretion from macrophages were evaluated. RESULTS and DISCUSSION: Subcellular fractionations and immunofluorescence demonstrated ferritin both in the cytosol and the lysosome, with significant enrichment of the lysosomally processed short L-subunit, the S-subunit, in the lysosomal fraction. This suggested both, controlled processing and stability of ferritin in the lysosome. We also found evidence that in epithelial cells ferritin distribution is polarized. Interestingly, ferritin in enterocytes appeared in a punctate pattern and was enriched near the apical pole while in the Sertoli cell line MSC1 ferritin was enriched near the basal pole, in concert with our earlier findings in vivo. BFA did not inhibit ferritin secretion from macrophages even when the Golgi was dissociated. Also, ferritin did not co-localize with the ER-Golgi machinery and BFA did not cause intracellular ferritin accumulation, while inhibitors of lysosomal function caused a significant accumulation. Further, inhibition and activation of the autophagy pathway inhibited and activated ferritin secretion respectively. Moreover, we confirmed that ferritin colocalized with the autophagy receptor NCOA4 in these cells. Taking these and additional results together we have evidence that cytosolic ferritin enters the endo-lysosomal machinery and utilizes two non-classical secretion routes involving lysosomes and lysosome-related organelles. CONCLUSION: The aforementioned findings show that lysosomes play important roles in cellular trafficking and secretion of ferritin, shedding new light on possible functions for ferritin in tissue-iron distribution and cellular and systemic iron homeostasis. Specifically, ferritin secretion may play a central role in iron distribution in the brain and thus constitute an important link in the elucidation of the many neurodegenerative diseases accompanied by impaired iron-distribution in the brain, such as Parkinson's and Alzheimer's diseases.

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Podium #23 LACK OF BETA-1 INTEGRIN LIMITS STRESS EYTHOPOIESIS AND SPLENOMEGALY IN BETA-THALASSEMIA Roberta Chessa, PhD1, Bart J. Crielaard, MD, PhD1, Ritama Gupta1, Carla Casu, PhD1, Rick Feldman, PhD2 and Stefano Rivella, PhD1 1CHOP; 2Bayer Healthcare

After blood loss, the production of red cells must be increased by stress erythropoiesis. This phenomenon is associated with increased proliferation and reduced differentiation of the erythroblasts, leading to a net increase in the number of progenitor erythroid cells and red cells (erythron). In normal conditions, after expansion of the pool of erythroblasts, these ells eetally deretate to erythroytes ad the aea resoles oeer, dseases sh as -thalassemia, production of healthy mature erythrocytes is impaired, resulting in anemia. Over time, the expansion, rather than the differentiation, of the erythron further exacerbates the ineffective erythropoiesis (IE), reducing the ability of the erythroid progenitors to generate erythrocytes. Interrupting the interaction between macrophages and erythroblasts (MEI) in thalassemia models is efficacious in reducing IE and alleviating the disease phenotype. We speculate that these molecules are also responsible for the homing of erythroid progenitor cells to extramedullary organs, such as the spleen and liver. Our studies in erythroblasts indicate that integrin beta- t1) and also intracellular molecules such as focal adhesion kinase (Fak1), Talin-1 and Sharpin might play a role in stress erythropoiesis. Furthermore, there is increased interaction between Itgb1 and Fak1 in erythroblasts co-cultured with macrophages as demonstrated by immunocytochemistry and in vitro proty lato assays addto, taret ether t1 or Fak1 prevents expansion of erythroid cells when cultured in the presence of macrophages. Strikingly, using t1 together with Ter119 as selection parameters in flow cytometry, a distinct subset of erythroblasts, not discernable using CD44 or CD71, was observable, which we found to be part of the mixed orthochromatic erythroblast/reticulocyte population as determined with CD44 expression. Enucleation of erythroblasts was accompanied by a marked loss of 1 epresso, dat that t1 may th3/+ be involved in erythroblast enucleation and differentiation. We crossed Hbb mice with animals in which 1 or Fak1 were floxed and carrying an inducible Cre-recombinase (Mx1-Cre). From these animals, we investigated three different th3/+ fl/fl th3/+ fl/fl models; two obtained from breeding (Hbb -1 -Mx1-Cre and Hbb -Fak1 -Mx1-Cre) and one by bone marrow th3/+ fl/fl transplant (BMT) of hematopoietic stem cells (HSCs) of Hbb -1 -Mx1-Cre animals into wt mice to generate thalassemic animals that expressed the floxed 1 only in hematopoietic cells. After serial administration of Poly(I)- Poly(C) [poly(I:C)] the animals were analyzed for their erythropoiesis in the bone marrow and spleen. All the animals treated th poly shoed poplatos o t1 or Fak1 negative cells in the bone marrow and spleen. This indicated that all the HSCs were successfully depleted of the I1 or Fak1 gene. Interestingly, the spleen weight of all the treated animals was reduced, on average, 50% compared to untreated thalassemic mice. Similar results were seen also in th3/+ fl/fl Hbb -1 -Mx1-re aals eerated throh hereore, t1 and Fak1 might contribute to the pathophysiology of thalassemia and their removal might result in reduced stress erythropoiesis, erythroid proliferation and, as a consequence, amelioration of splenomegaly. Iron analysis and quantification of Erythroferrone (ERFE) are in proress to ealate the pat o deplet t ad a o these ehass e are o the proess o identifying compounds that target MEI and, in particular, 1. Such molecules might be utilized for development of new treatments for thalassemia or additional disorders of aberrant erythropoiesis.

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Podium #24 LIPOSOME-ENCAPSULATED H-FERRITIN IMPROVES SURVIVAL IN A SOD1 MUTANT MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS James Connor, PhD, Amanda Snyder, PhD, Achuthamangalam Madhankumar, PhD, Elizabeth Neely, BS, Elias Rizk, MD, Olivia Hess and Zachary Simmons, MD Penn State University College of Medicine

The misregulation of iron and subsequent oxidative stress are consistent features shared between humans with Amyotrophic Lateral Sclerosis (ALS) and in animal models of the disease. H-ferritin has ferroxidase activity and limits the toxic potential of iron, making it an attractive therapy to pursue in ALS. One of the disadvantages of most systemically- delivered treatments for neurological diseases is that they exert their biological effects not only at their target sites but also at peripheral tissue and cells. This often results in dilution of the agent below therapeutic levels to the target tissue; a way to increase efficacy and reduce the amount of agent administered is to utilize liposomal drug carriers. The objective of this work is to determine if infusion of liposome-encapsulated iron-poor H-ferritin has neurorescue properties in a murine model of ALS. Two types of liposomes were used: those that were not directed to any specific cell type and those that were directed to microglia by the presence of lipopolysaccharide (LPS). At 90 days of age, mice with the SOD1G93A mutation underwent surgery allow for continuous infusion of liposome-encapsulated H-ferritin into the lateral ventricle. Disease onset was assessed by performance on the rotarod apparatus, and endpoint was determined by the inability of the animal to right itself. A significant impact on lifespan occurred in mice treated with H-ferritin encapsulated by non-targeted liposomes, resulting in a 10.5 day extension of survival as compared to untreated SOD1G93A mice (median values). Specific delivery of H- ferritin to microglia was of limited benefit in extending lifespan. A plausible explanation for this outcome is over-stimulation of microglia by accessing them through the TL4 receptor. Indeed, histological examination of lumbar spinal cord sections indicated less extensive microglial activation at end-stage in the group of mice treated with non-targeted liposomes carrying H-ferritin as compared to the group of animals treated with the LPS-directed liposomes. Furthermore, the motor neurons that remained at end-state in the non-targeted liposomal group had thick, extensively branched projections: features not seen in the LPS-targeted group. Our intervention in the animal model is of particular relevance to the clinical population because our intervention occurs at the peri-symptomatic stage of the disease; this would correlate to the time in which human patients would begin to notice symptoms and seek treatment in the clinic. Therefore, our therapy may be of greater clinical benefit than those compounds tested while animals are asymptomatic.

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Podium #25 EFFECT OF DIETARY IRON INTAKE ON MITOPHAGY, MITOCHONDRIAL BIOGENESIS AND MITOCHONDRIAL DYNAMIC IN SKELETAL MUSCLE OF RATS YihFong Liew, PhD, Hsuan-Lin Chen, MS and Dong-Lin Tsai, MS Fu-Jen Catholic University

Introduction: Prolonged iron deficiency results in more severely impaired mitochondrial function in skeletal muscle than other tissues. However, how the removal of damaged mitochondria in iron-deficient skeletal muscle is still not well understood. Mitophagy is a cellular metabolic process that mediates the selective degradation of dysfunctional or damaged mitochondria via lysosomal machinery. In addition, mitophagy are opposite, but coordinate with mitochondrial biogenesis to determine the mitochondrial content, structure and function. Therefore, the aim of this study is to investigate the effect of dietary iron intake on mitochondrial dynamics and biogenesis in skeletal muscle. Methods: Thirty-two weaning male Wistar rats were randomly assigned to four groups, including adequate-iron control group (AI, 35 mg Fe/kg diet), moderate iron-deficient group (MID, 10 mg Fe/kg diet), severe iron-deficient group (ID, < 5 mg Fe/kg diet), and severe iron-deficient pair-fed group (IPF, 35 mg Fe/kg diet). After four weeks, the rats were sacrificed and corrected gastrocnemius muscle for determination of mitochondrial ultrastructure by electron microscopy, isolation of cytosolic and mitochondrial fraction, measurement of mitochondrial copy number, and detection of molecular marker, including mitophagy, mitochondrial dynamic, mitochondrial biogenesis by immunoblotting. Results: Electron microscopy revealed the mitochondria became small and oval, and had extensive loss of internal cristae, and induced the formation of autolysosomes in severe iron-deficient skeletal muscle. Moreover, molecular marker for mitophagy, including LC3 (microtubule-associated protein 1 light chain 3), PINK1 (PTEN-induced putative kinase 1), Parkin, polyubiquitin, p62, and p-ULK-1 (phospho-UNC-51-like kinase 1), BNIP3 (Bcl-2/adenovirus E1B 19-kDa interacting protein 3), and mitochondrial Beclin-1 protein levels were increased in iron-deficient skeletal muscle. The molecular marker of mitochondrial dynamic, Mfn (mitofusin) 1 and Mfn2, Drp1 (dynamin related protein 1) protein levels were increased, but mitochondrial Opa1 (optic atrophy 1) protein levels was decreased in iron-deficient skeletal muscle. Iron deficiency also increased mitochondrial copy number and regulator of mitochondrial biogenesis Tfam (mitochondrial transcription factor A) expression. The protein expression of mTOR (mammalian target of rapamycin) and phospho-mTOR were decreased, but the p-AMPK/AMPK (AMP activated protein kinase) ratio were increased in iron deficient skeletal muscle. Conclusion: Our study demonstrated iron deficiency induces mitophagy via the PINK1/Parkin, BNIP3/Beclin-1 and AMPK/mTOR pathways in skeletal muscle. To compensate extensive loss of mitochondria, iron deficiency also activates mitochondrial biogenesis in skeletal muscle.

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Podium #26 CHARACTERIZATION AND CLONING OF HUMAN NUCLEAR RECEPTOR COACTIVATOR 4 (NCOA4), THE CARGO RECEPTOR MEDIATING FERRITINOPHAGY Magdalena Gryzik, PhD student, Maura Poli, PhD, Margherita Di Somma, PhD student, Claudia Saraceno, PhD, Paola Ruzzeneti, PhD student, Michela Asperti, PhD student, Maria Regoni, PhD student and Paolo Arosio, PhD University of Brescia

Ferritin stores and detoxifies cellular iron by sequestering up to 4000 Fe atoms in its large cavity. The recycling of this iron is crucial and it seems to involve the nuclear receptor coactivator 4 (NCOA4) that acts as a selective cargo-receptor mediating ferritin autophagy (ferritinophagy). It is a protein of 614 amino acids, and about 70 kDa, predominantly localized in the cytoplasm, containing a coiled-coil domain promoting protein-protein interactions and two motifs, LXXLL and FXXLF, involved in interactions with nuclear receptors. NCOA4 has been identified as a transcriptional coactivator of several nuclear hormone receptors, although it may be also involved in carcinogenesis, inflammation, erythropoiesis, microtubule activity during mitosis, DNA replication origins activation. The aim of the study was to analyze its expression and relation to iron metabolism regulation and produce biologically active NCOA4 for its biochemical characterization. We set up qRT-PCR assays for human and mouse NCOA4. We found NCOA4 mRNA to be expressed at various levels in all analyzed material - mouse tissues (liver, kidney, heart, brain, spleen, lung, thymus, testis), the human cell lines (neuroblastoma SH-SY5Y cells, hepatocellular carcinoma HepG2 cells, HeLa cells, primary fibroblasts, embryonic kidney HEK293 cells) and the human placenta. The expression of NCOA4 mRNA was observed to be low in HepG2 cells, mouse lung and spleen, whereas in primary fibroblasts as well as in HeLa and neuronal SH-SY5Y cells, and in mouse testis and brain, the NCOA4 transcript level was high. Within mouse brain, all parts have shown similar expression level. Thus, there is no evident relationship between NCOA4 expression and organs of iron storage, and its highest levels were found in mouse testis and human fibroblasts. Iron deprivation and supplementation by incubation with the chelator desferoxamine and ferric ammonium citrate respectively, did not affect NCOA4 mRNA level in hepatic and neuronal cells indicating it not to be iron-regulated. The down-regulation of NCOA4 in these cells modified the expression of the iron-regulated proteins, such as ferritin and transferrin receptor 1 (TfR1), even under conditions of different iron availability. Moreover, HepG2 cells were treated with different concentration of H2O2 and their viability was analyzed using MTT assay. We found that NCOA4 down-regulation protected the cells from the toxic effect of hydrogen peroxide, perhaps because it reduces iron availability. The full-length human NCOA4 was cloned into pET-12a vector with a 6-His tag at C-terminus and expressed in E. coli. The denaturation, refolding and purification of the recombinant NCOA4 is ongoing. The produced and purified recombinant human NCOA4 will be analyzed for biochemical properties, used for antibody production and to characterize the interaction between NCOA4 and ferritin. Moreover, the identification of NCOA4 binding site for ferritin is in progress. In conclusion, our preliminary results support the hypothesis that NCOA4 can carry ferritin to the lysosomes, control its degradation and has a role in cellular iron homeostasis. The study of NCOA4 involvement in the targeting of ferritin to autophagosomes may clarify one mechanism of regulation of cellular iron homeostasis.

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Podium #27 INTRACELLULAR IRON DEREGULATION INDUCES SENESCENCE PHENOTYPE IN FIBROBLASTS FROM A NEUROFERRITINOPATHY PATIENT Sonia Levi, PhD1, Anna Cozzi, PhD2, Paolo Santambrogio, PhD2 and Chiara Fiorillo, PhD3 1Vita-Salute San Raffaele University; 2San Raffaele Scientific Institute; 3IRCCS Stella Maris, Pisa

Neuroferritinopathy (NF) is a rare dominant inherited movement disease belonging to the group of NBIA disorders, caused by alterations of the L-ferritin gene. The hallmark of this disease is the presence in the patient’s brain of abundant spherical inclusions positive for iron, ferritin and ubiquitin stains. We characterized fibroblasts from a 42-years old Italian patient affected by parkinsonism and cognitive impairment. The gene mutation is the already described 469-484Ins16nt causing a sequence alteration of the L-peptide at C-terminus. The patient fibroblasts were analyzed in comparison with three healthy control subjects for iron metabolism, cell oxidative status and senescence. The NF fibroblasts showed the hallmark of the disorder: ferritin/iron aggregates detected by immunofluorescence and ESI, respectively. Experiments of 55Fe-ferritin incorporation resulted in reduced protein functionality (~1.5 fold). Iron homeostasis analysis indicated decreased (~10 fold) TfR1 expression, increased amount of oxidized proteins and ROS production (~1.4 fold), reduced viability in basal condition (~ 2 fold) and after iron treatment (~ 3 fold) in NF fibroblasts respect to the control cells. Reduced proliferation and ATP production, other then induction of SA-ß-GAL activity in NF fibroblasts suggested a senescence phenotype. The SA-ß-GAL activity was increased after addition of iron and hydrogen peroxide and it was reversed after treatment with an antioxidant agent. Furthermore, the NF fibroblasts showed morphology alteration, the presence of cytoplasmic vacuolation and increased autophagosome formation, confirming the senescence cells status. For the first time we reported in a model of neurodegeneration the evidence that the lack of intracellular iron buffering, due to ferritin defect, triggers cells senescence via oxidative stress. The financial support of Telethon–Italy (Grant n°GGP10099) is gratefully acknowledged

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Podium #28 HEPARINS AS INHIBITORS OF HEPCIDIN: MECHANISM OF ACTION Michela Asperti, PhD Student1, Maura Poli, PhD1, Paola Ruzzenenti, PhD Student1, Annamaria Naggi, PhD2, Natascia Campostrini,PhD3, Magdalena Gryzik, PhD Student1, Federica Maccarinelli1, Domenico Girelli, MD3 and Paolo Arosio, PhD1 1University of Brescia; 2Ronzoni Institute of Milano; 3University of Verona

Hepcidin, is the master regulator of systemic iron homeostasis. This hormone stimulates the internalization and degradation of ferroportin (the only iron exporter) and controls the absorption of the dietary iron and the recycle of hemoglobin iron. Hepcidin is controlled by iron, inflammation, hypoxia and anemia. Its deregulation is the cause of different diseases, such as hemochromatosis, Iron-refractory Iron deficiency anemia (IRIDA) and Anemia of Chronic diseases. For this reason hepcidin and its regulation pathways are interesting targets for new therapies to cure iron disorders. Different hepcidin modulators are in study and among them are heparins, anticoagulant drugs used in clinics for more than seven decades. We demonstrated that heparin has a strong anti-hepcidin activity, acting mainly by interfering with the BMP6/SMAD signalling, essential for hepcidin expression. Since the anticoagulant activity of heparins limits their use as anti-hepcidin agents, different approaches have been used to modify the pentasaccharide domain responsible of high affinity binding to antithrombin. It can be modified by periodate oxidation and borohydride reduction (glycol-split heparins) or by introducing extra sulfates obtaining super sulfated (SS) heparins. We demonstrated that both gs and SS heparins are effective inhibitors of hepcidin in vitro and in mice and they modify systemic iron homeostasis without evident side effects. Thus heparins are promising drugs for reducing hepcidin but the structure can be optimized to obtain a compound with the strongest activity. For this reason we are analysing the anti-hepcidin activity of heparins with different chemical modifications. We found that 2-O de-sulfation, N-acetylation, and a molecular weight <7-8 kD reduce the anti- hepcidin potency, suggesting that it requires many binding sites. Heparins are structurally similar to the Heparan Sulfates (HS) exposed on hepatic cells and the finding that they strongly modify hepcidin expression indicates an important role of local HS in hepcidin expression. To verify this hypothesis we chemically or genetically modify the HS biosynthesis. Our data show that: - Treatment of HepG2 cells with 25-100mM sodium chlorate (an inhibitor of HS synthesis) strongly reduced pSMAD and hepcidin level (50-90%); - Silencing the major enzymes involved in HS biosynthesis Ext1 and Ext2 (that catalyse the addition of GlcA and GlcNAc to the nascent chain) and Ndst1 (that removes acetyl group and adds sulphate groups) in hepatic cells, caused a reduction of hepcidin expression (about 60%) and of pSMAD; - The HepG2 cells overexpressing heparanase, (an enzyme for HS modification/maturation) showed a low level of hepcidin expression (50-60% of controls) and also transgenic mice for heparanase that have altered HS structure in liver and other organs, showed a low level of liver hepcidin expression (about 50%) and iron overload. In conclusion, we have identified non-anticoagulant heparins with high anti-hepcidin activity in vivo that are potential drugs for disorders with hepcidin excess. In addition our data show that cellular/liver heparan sulfates participate in the regulation of hepcidin expression and could be a new therapeutic target.

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Podium #29 MONOCLONAL ANTIBODIES NEUTRALIZING HEMOJUVELIN DOWNREGULATE HEPCIDIN: CANDIDATE DRUGS FOR THE TREATMENT OF IRIDA AND ANEMIA OF CHRONIC DISEASE Andreas Popp, Dr1, Preethne Boeser1, Suzana Kovac, Dr2, Daniela Casarrubea, Dr2, Dunja Ferring-Appel2, Matthias W. Hentze, Prof Dr2 and Bernhard K. Mueller, PD Dr1 1AbbVie Deutschland GmbH & Co. KG; 2EMBL, Heidelberg, Germany

No breakthrough has been achieved in the development of therapies for anemia of chronic disease (ACD) in the past. Here we describe humanized monoclonal antibodies (Ab1 and 2) targeting hemojuvelin (RGMc) to decrease hepcidin by neutralizing hemojuvelin. In healthy animals (mice, rats, cynomolgus monkeys) Ab1 and 2 increase serum iron and decrease the serum iron binding capacity. Histopathologically, iron is released from spleen macrophages (reduced iron in the spleen) and accumulates in periportal hepatocytes. The effects are dose-dependent and negatively correlate with hepcidin expression. After treating female rats with a single dose of 20 mg/kg, the more potent antibody Ab2 significantly reduced serum hepcidin compared to control levels for 6 weeks. In toxicology studies performed in rats and cynomolgus monkeys for up to 13 weeks with Ab1, no adverse effects were observed, in particular no degeneration or inflammation due to the iron accumulation in the liver. The effects showed complete or partial recovery after a 12 week treatment free period. These effects in healthy animals motivated us to test the antibodies in preclinical models of Anemia of Chronic Disease (ACD). In peptidoglycan polysaccharide-treated rats, chronic arthritis with normocytic, normochromic anemia and low serum iron manifest three weeks after a single ip injection. We treated rats starting at this time point once per week for four weeks with either of the two antibodies, or the isotype control antibody. Weekly hemoglobin (Hb) measurements demonstrate significant improvement of Hb levels already after the second application, and this effect lasted for 2 weeks. As expected, Ab2 showed higher potency. In a murine inflammatory model, hepcidin mRNA increases with an expression peak at 6 hours following heat-killed Brucella abortus (HKBA) injection. The Ab2 was administered 30 minutes after HKBA injection to determine suppression of inflammation-induced hepcidin mRNA. Our results demonstrate that the antibody significantly decreases hepcidin mRNA compared to IgG-treated mice. These data further support the conclusion that the anti-RGMc antibodies decrease

Finally we tested Ab2 in a genetic anemia mouse model (Tmprss6-/-), which reflects the rare human genetic disorder iron refractory iron deficiency anemia (IRIDA), in a single dose experiment and in a second study with a single dose cross-over design. After a single dose of 20 mg/kg of Ab2, anemia in the Tmprss6-/- mice was ameliorated for 2 weeks. The cross over experiment confirmed these results. Histopathology performed at the end of the cross over study revealed no abnormalities in spleen and liver. The bone marrow showed that treatment with Ab2 rescued erythropoiesis in homozygous Tmprss6-/- mice, while mice treated with hIgG displayed a moderate to marked decrease of erythropoiesis in the bone marrow.

These data show in multiple pre-clinical models that anti-RGMc antibodies effectively rescue anemia by suppressing hepcidin, opening new horizons to use anti-RGM antibodies as a new therapeutic approach for patients suffering from ACD and IRIDA.

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Podium #30 IRON CHELATORS IN NANOFORMULATIONS FOR SPECIFIC TARGETING AND EFFECTIVE REMOVAL OF MACROPHAGE IRON FOR TREATMENT OF IRON OVERLOAD DISEASES Guangjun Nie1, Shanshan Guo, BS1, Gang Liu, PhD1 and Greg Anderson, PhD2 1CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, China, Beijing, 100190; 2Iron Metabolism Laboratory, Queensland Institute of Medical Research, Brisbane Queensland, Australia, 4029

Iron overload disorders represent an important class of human diseases. Some, such as hereditary hemochromatosis, result from increased dietary iron absorption, while others can result from frequent blood transfusions. Organs commonly affected by iron overload are the liver, spleen, heart and endocrine glands. For the patients affected by the secondary iron overload diseases (i.e., through multiple transfusions), iron initially localized within Kupffer cells and portal macrophages. Chemicals known as iron chelators are used to remove iron from the body, particularly in secondary iron overload diseases. Deferoxamine (DFO) is the most commonly used iron chelator in clinical practice and is generally administered by subcutaneous infusion over a long period of time because of the short half-life time in the blood, representing a major treatment burden for the patients. With the aim of developing more effective methods for delivering iron chelators, we have examined whether amphiphilic copolymer nanoparticles (NPs), which can specifically target to liver, spleen and other organs, can be used to deliver DFO efficiently. Mice were loaded with iron dextran administration, then treated with free DFO and DFO-NPs. Both free DFO and DFO-NPs were able to significantly reduce serum iron (157.34, 110.60 and dl or ro oerload, ad -NPs groups), transferrin saturation (98.77%, 64.38% and 41.73% for iron overload, DFO and DFO-NPs groups), tissue ferritin expression and tissue iron levels, but DFO-NPs consistently proved more effective. Much of this effect is assumed due to the specific targeted delivery of DFO to the macrophages in the liver and spleen by the NPs, which have been widely perceived as a natural defense mechanism of body for foreign particulates. Histological study and in vitro study confirmed that indeed tissue macrophages are the target of DFO-NPs and the encapsulated in DFO was more effective at depleting iron than free DFO. Systemic evaluation of DFO toxicity revealed that this preparation was even less toxic than DFO alone. Taken together, our current results suggest a new powerful strategy for removal of iron specific from tissue macrophages using nanoformulation of DFO and such a strategy lays a foundation for treatment of diseases with specific iron overload in macrophages.

Sixth Congress of the International BioIRon Society Page 111 Podium Abstracts IBIS

Podium #31 TARGETING TMPRSS USING ANTISENSE TECHNOLOGY FOR THE TREATMENT OF -THALASSEMIA Shuling Guo, PhD1, Mariam Aghajan, PhD1, Carla Casu, PhD2, Sara Gardenghi, PhD2, Sheri Booten1, Stefano Rivella, PhD2 and Brett Monia, PhD1 1Isis Pharmaceuticals; 2Weill Cornell Medical College

Antisense technology is a powerful drug discovery approach for identifying oligonucleotide analogs that can specifically modify RNA expression through multiple mechanisms including RNase H–mediated degradation of RNA and modulation of RNA splicing. We have successfully applied this technology towards targeting a number of transcripts in a wide-range of therapeutic areas. Beta-thalassemia, one of the most common genetic disorders worldwide, is characterized by reductions in beta-globin and ineffective erythropoiesis. This in turn leads to suppression of hepcidin, a peptide hormone that serves as the master regulator of iron homeostasis. Inappropriately low levels of hepcidin trigger increased dietary iron absorption resulting in iron overload, which is the major cause of morbidity and mortality in beta-thalassemia patients. TMPRSS6 is a transmembrane serine protease mainly produced by hepatocytes that negatively regulates hepcidin expression. Previous mouse and human genetic data from multiple groups suggest that lowering TMPRSS6 expression could up-regulate hepcidin and ameliorate many of the disease symptoms associated with beta-thalassemia. We identified potent antisense oligonucleotides (ASOs) against mouse TMPRSS6. Downregulation of TMPRSS6 with ASO treatment resulted in dose-dependent hepcidin upregulation and reduction in serum iron and transferrin saturation in normal mice. In a mouse model of beta-thalassemia (th3/+ mice), which effectively recapitulates beta-thalassemia intermedia in humans, TMPRSS6 reduction resulted in induction of hepcidin and dramatic reductions of serum transferrin saturation (from 55-63% in control group down to 20-26% in treatment group). Liver iron concentration (LIC) was also greatly reduced (40-50%). Moreover, anemia endpoints were significantly improved with ASO treatment, including increases in red blood cells (~30-40%), hemoglobin (~2 g/dl), and hematocrit (~20%); reduction of splenomegaly; decreases in serum erythropoietin levels; improved erythroid maturation as indicated by a strong reduction in reticulocyte number and a normalized proportion between the pool of erythroblasts and enucleated erythroid cells. Encouraged by the strong pharmacology of TMPRSS6 suppression in animal models, we initiated an effort to identify a human TMPRSS6 clinical candidate with a liver-targeted delivery strategy. Over 2000 ASOs were screened in cell lines and the most active compounds were evaluated in rodent tolerability studies. A human TMPRSS6 transgenic mouse model was established enabling evaluation of ASO activity toward human TMPRSS6 transcript in vivo. Furthermore, lead compounds were tested in a 3-month study in normal monkeys. With repeated dosing, TMPRSS6 mRNA levels in monkey liver were reduced by >90%, accompanied by time-dependent reductions of serum iron (from ~100-120ug/dl to <40ug/dl), transferrin saturation (from ~30-35% to <10%), and hemoglobin. These compounds were well tolerated in rodents and in monkeys. Collectively, our data demonstrate that TMPRSS6 ASO could be an effective therapeutic for patients with beta- thalassemia and related disorders. A Phase 1 clinical trial is planned to initiate in 2016.

Sixth Congress of the International BioIRon Society Page 112 Podium Abstracts IBIS

Podium #32 SYSTEMIC INFLAMMATION INFLUENCES THE ABILITY OF DEFERIPRONE TO CHELATE IRON FROM SPECIFIC BRAIN REGIONS IN PARKINSON’S DISEASE PATIENTS Antonio Bastida, MD2, Roberta Ward, MPhi,l PhD1, Paula Piccini, MD3 and David Dexter, PhD4 1Imperial College; 2Imperial College, London UK; 3Imperial College. London, UK; 4Imperial College, London, UK

The use of chelation therapy to remove iron from specific brain regions in various neurodegenerative diseases and improve clinical symptoms has been shown to be of benefit in clinical trials. However not all patients respond positively to such treatment. We therefore investigated whether efficacy of brain iron chelation was related to systemic inflammation. Twenty three early stage PD patients were recruited to the clinical trial and administered 20mg/kg or 30 mg/kg or placebo daily for 24 weeks. MRI T2* semi-quantitated the iron content in caudate, dentate nuclei, red nucleus, putamen, globus pallidus and substantia nigra at 0, 12 and 24 weeks. Both ferritin and the pro-inflammatory cytokine IL-6 were assayed in the plasma every 4 weeks. A positive relationship was evident between IL-6 and ferritin concentrations at time 0, P=0.005. . In deferiprone-treated PD patients where the initial ferritin value was <100ng/ml, it was noteworthy that there was a greater decrease in brain iron in the dentate and caudate nucleus, as well as the SNc, as assessed by T2* MRI, than in those PD patients where the plasma ferritin level commenced at values >100ng/ml. The changes in the T2* measurements in the dentate nuclei of PD patients administered 30 mg/kg inversely correlated with the levels of IL-6, i.e. the higher the levels of the pro-inflammatory the smaller the change in T2* measurement. No such correlations were evident in this brain region of PD patients administered 20 mg/kg Inflammation clearly influences the ability of the chelators to remove iron from specific brain regions. This could relate to the fact that IL6 will induce the synthesis of hepcidin which decreases ferroportin levels thereby increasing cellular iron content particularly within the neurons and microglial cells.

Sixth Congress of the International BioIRon Society Page 113 Podium Abstracts IBIS

Podium #33 ORAL AND INTRAVENOUS IRON: THERAPEUTIC OPTIONS AND DIFFERENCES AMONG THE PREPARATIONS Susanna Burckhardt Vifor Pharma ORAL AND INTRAVENOUS IRON: THERAPEUTIC OPTIONS AND DIFFERENCES AMONG THE PREPARATIONS S. Burckhardt, Vifor Pharma, St. Gallen, Switzerland

Different therapeutic options are available to treat iron deficiency anemia. The administration of oral iron is a convenient, inexpensive, and effective treatment. However, long-term use of widely prescribed ferrous salts is limited by frequent side effects, such as nausea, vomiting, constipation, and metallic taste. When these adverse effects are not tolerated and result in non-compliance, oral iron is ineffective, the iron need is high, or a fast correction of iron deficiency is required, intravenous iron is then the treatment of choice. For both forms, oral and intravenous iron, different classes of drugs exist. The pathways by which the drugs are metabolized are specific for each product. Ferrous salts and iron polymaltose complex, a polynuclear iron(III)- oxyhydroxide-based complex, are two diverse examples of commonly used oral iron drugs, which display distinct uptake mechanisms. Despite comparable extent of utilization, administration of a therapeutic dose of the two drugs leads to significant serum iron increase along with formation of non-transferrin-bound iron only in the case of ferrous salts. Thus, serum iron cannot be used to assess the bioavailability of oral iron preparations. All iron compounds used for intravenous iron therapy consist of a polynuclear Fe(III)-oxyhydroxide/oxide core surrounded by different types of carbohydrates which stabilize the core and protect it against further polymerization. The nature of the carbohydrate determines the stability of the complex as well as the metabolic pathway followed upon administration. Currently available preparations can be divided in two classes with distinct types of correlations between physicochemical parameters: non-dextran-containing and dextran-based complexes. Non-dextran-containing complexes exhibit a good correlation between molecular weight distribution and complex stability. Low molecular weight complexes such as sodium ferric gluconate (weht aerae olelar eht a ad ro srose a are ea ad moderately strong, respectively, whereas the higher molecular weight complex ferric carboxymaltose (Mw a s very stable. Dextran-based complexes such as iron isomaltoside 1’000 (Mw a, ro detras kDa), and ferumoxytol (Mw a eht hh stalty depedetly o ther olelar eht ased o the different stability of the complexes, together with the pH and the osmolarity of the solutions, a wide range of maximal single iron doses (from 62.5 mg up to more than 1’000 mg), administration modes (injection and infusion), and administration times (15 min up to several hours) characterize the various preparations, which evidently are not interchangeable. The impact of the different physico-chemical properties on the metabolism of the complexes is particularly evident when comparing the pharmacokinetics parameters. For instance, the half-life of the weak complex sodium ferric gluconate is less than 2 hours, whereas those of the stable dextran-containing complexes is more than 20 hours. Thus, a clear distinction has to be made among the various preparations, which cannot be considered equivalent.

Sixth Congress of the International BioIRon Society Page 114 Podium Abstracts IBIS

Podium #34 DEXRAS1/DMT1 PATHWAY PLAYS A MAJOR ROLE IN OXIDATIVE STRESS-MEDIATED NEURONAL CELL DEATH Yong Chen, PhD, Reas Khan, PhD, Alyssa Cwanger, BS, Ying Song, PhD, Joshua Dunaief, MD, PhD, Kenneth Shindler, MD, PhD and Sangwon Kim, PhD University of Pennsylvania

Iron is the most abundant metal in our body participating in a wide variety of metabolic processes (oxygen transport, electron transfer, DNA synthesis, etc.) but also having the potential for deleterious effects. Especially, unless appropriately chelated, iron plays a key role in the formation of harmful oxygen radicals that ultimately cause peroxidative damage to vital cell structures. Thus, evolving organisms were compelled to solve one of the many paradoxes of life, i.e., to keep “free iron” at the lowest possible level and yet in concentrations allowing its adequate supply for the synthesis of heme proteins and other iron-containing molecules. Due to iron's unique chemical nature and the brain heterogeneity, it has been very difficult to study iron in the central nerve system (CNS). Recently, we have recently identified a novel signaling cascade in neurons whereby stimulation of glutamate-NMDA receptors activates neuronal Nitric Oxide Synthase (nNOS), leading to S-nitrosylation and activation of small GTPase Dexras1 which, via Acyl-CoA Binding Domain 3 (ACBD3) and Divalent Metal Transporter (DMT1), physiologically induces iron uptake. This is the first demonstration of a link between neurotransmission and active regulation of iron trafficking in the brain. Moreover, a membrane permeable iron chelator substantially reduces NMDA-excitotoxicity suggesting that Dexras1-mediated iron influx plays a crucial role in NMDA/NO-mediated cell death. We here report that iron influx is elicited by nitric oxide but not by other pro-apoptotic stimuli such as H2O2, staurosporine. Genetic deletion of Dexras1 abolishes NMDA-induced cell death in vitro and in vivo. Furthermore, we have confirmed that Dexras1/DMT1 pathway is involved in various neurodegenerative diseases associated with iron imbalance. Thus our studies suggest that Dexras1 plays a critical role in iron-elicited neurotoxicity and may be potential therapeutic targets for various neurodegenerative diseases.

Sixth Congress of the International BioIRon Society Page 115 Podium Abstracts IBIS

Podium #35 THE EFFECT OF ANTI-INFLAMMATORY PROPERTIES OF FERRITIN LIGHT CHAIN ON LIPOPOLYSACCHARIDE- INDUCED INFLAMMATORY RESPONSE IN MURINE MACROPHAGES Yumei Fan, Yanzhong Chang, PhD and Xianglin Duan College of Life Science, Hebei Normal University

Ferritin light chain (FTL) reduces the free iron concentration by forming ferritin complexes with ferritin heavy chain (FTH). Thus, FTL competes with the Fenton reaction by acting as an antioxidant. In the present study, we determined that FTL influences the lipopolysaccharide (LPS)-induced inflammatory response. FTL protein expression was regulated by LPS stimulation in RAW264.7 cells. To investigate the role of FTL in LPS-activated murine macrophages, we established stable FTL-expressing cells and used shRNA to silence FTL expression in RAW264.7 cells. Overexpression of FTL significantly decreased the LPS-induced production of tumor necrosis factor alpha (TNF-α, terle -, tr oxide (NO) and prostaglandin E2 (PGE2). Additionally, overexpression of FTL decreased the LPS-induced increase of the intracellular labile iron pool (LIP) and reactive oxygen species (ROS). Moreover, FTL overexpression suppressed the LPS-induced activation of MAPKs and nuclear factor- - otrast, odo o y sh shoed the reverse effects. Therefore, our results indicate that FTL plays an anti-inflammatory role in response to LPS in murine macrophages and may have therapeutic potential for treating inflammatory diseases.

Sixth Congress of the International BioIRon Society Page 116 Podium Abstracts IBIS

Podium #36 IRON AND AGING: A NOVEL APPROACH AGAINST SKIN NATURAL AGING AND PHOTO-AGING Xi Huang, PhD1, Jun Ou, BS2 and Guo Ting Wang, BS3 1Marivan Skincare Inc.; 2Marivan (Hunan) Cosmetics Inc.; 3Marivan (Hunan) Cosmetics, Inc.

Iron is one of the most abundant transition metals in the human body. Although the human body has developed sophisticated systems of iron absorption, menstruation and skin desquamation seem to be the few important routes to excrete iron. Yet, skin as an important target of iron and iron-mediated skin aging and photo-aging have not drawn considerable attention, particularly in older women when menstruation ceases. In the present study, we tested a hypothesis that removal of iron from ferritin in the skin reduces oxidative stress and prevents skin natural aging and photo-aging. We have shown that iron levels in skin in the form of ferritin, an iron storage protein, increase significantly from premenopause to postmenopause over the 10-year transition period from 42 to 51 years old. We have further shown that iron from ferritin not only enhances oxidant formation, but also upregulates UVA- mediated matrix metalloproteinase-1 activity in dermal fibroblasts by directly activating ERK pathway or indirectly through keratinocytes releasing TNF-alpha. Since iron in ferritin is nonessential and is a causative factor of skin damage, we have developed a technology targeting ferritin to prevent iron-mediated skin natural aging and photo-aging. This de-ironizing inducer (D.I.I.) technology is based on: 1) ferritin has a strong binding affinity for ferric ion (Fe3+) but not for ferrous ion (Fe2+) and 2) “free” released Fe2+ is unstable in aqueous solution at pH>4.5. Application of D.I.I. reduces Fe3+ to Fe2+ and precipitates it immediately after its release from ferritin, effectively making iron unavailable for skin damage. A clinical trial has shown that D.I.I. significantly improved skin appearance by reducing 30% wrinkles over a period of 4 weeks. Taken together, our results indicate that iron is an important risk factor of skin natural aging and photo-aging. This study is especially relevant in older postmenopausal women because, in addition to estrogen deficiency, our study shows that increased iron in skin contributes to its aging. Due to the side effects of hormone replacement therapy, iron removal through D.I.I. technology presents a novel approach to improve skin appearance.

Sixth Congress of the International BioIRon Society Page 117 Podium Abstracts IBIS

Podium #37 TRANSVERSE RELAXATION AND VOLUMETRIC NEURAL CHANGES IN THE H67D HFE MOUSE MODEL AND COGNITIVELY NORMAL HEALTHY H63D-HFE HUMAN GENOTYPE CARRIERS Mark Meadowcroft, PhD1, Douglas Peters, MS2, Carson Purnell, BS2, Jian-Li Wang, MD, PhD2, Paul Eslinger, PhD2, Megha Vasavada, PhD2, Qing Yang, PhD2 and James Connor, PhD2 1The Pennsylvania State University - College of Medicine; 2The Pennsylvania State University – College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania, USA

Introduction: A common mutation that leads to systemic iron overload within the hereditary hemochromatosis (HHC) protein (H63D-HFE) has been associated with neurodegenerative diseases such as ALS, Parkinson’s disease, and Alzheimer’s disease. H63D mutations result in iron dyshomeostasis, increased oxidative stress, glutamate release, tau phosphorylation, and alterations in inflammatory response. The H63D-HFE mutation is prevalent within the general population; approximately 29.7%. A mouse model has been generated that contains the mouse homolog for the human H63D gene, the H67D knock-in. H67D mice exhibit alterations in their iron management proteins, increased neuronal oxidative stress, aberrant gliosis, and disrupted cholesterol dynamics; all which leads to an increase in neurodegeneration and memory deficits. The overarching goal of this research was to evaluate the transverse relaxation profiles and volumetric measures in the mice and contrast these to human H63D carrier metrics. Methods: Human Work: Thirty-two cognitively normal healthy Caucasian subjects (16F, 16M) were selected for inclusion in this study design. All subjects were administered a battery of cognitive tests and determined to be cognitively normal. A total of 15 subjects (9F, 6M) were heterozygous for the minor G allele (H63D/+). A 3DT1-weighted scan and a multi-echo T2- weighted spin-echo protocol were obtained. R2 relaxation maps were normalized to the template brain and voxel based analysis was performed lster se ad p Mouse work: Twenty mice, 10 HFE-H67D mice and 10 C57BL/6 (WT), were anesthetized with 1.5% isoflurane and placed within a 7.0 T MRI system and 35 mm birdcage volume coil (Bruker BioSpin). Animals were imaged at baseline (9 months) and twelve months later with the same imaging protocol. A four-echo 3D-T2-weighted dataset was acquired and parametric relaxation R2 maps were generated. R2 relaxation maps were normalized to the template brain and voxel based analysis was perored lster se ad p Results: Voxel-wise comparison of H63D and wild-type cognitively normal whole human brain R2 relaxation rates demonstrate alterations in the neuronal R2 relaxation rate within H63D carriers compared to the wild-type patients (Fig. 1). H63D patients exhibit a decrease in R2 relaxation rate within white matter throughout the brain. The most prominent decrease in R2 rate is seen in the frontal white matter, extending throughout the entirety of this white matter region. Parametric relaxation maps of H67D mice show a group based decrease in R2 relaxation rate compared to controls. These regions are exclusively white matter in origin, similar to the human data. Discussion: The MRI data presented here demonstrate that cognitively normal H63D HFE carriers have relaxometry alterations in their brains. The data demonstrate that there is an HFE genome related alteration in the transverse R2 relaxation rate in cognitively normal H63D human and H67D mouse carriers compared to controls. The widespread decrease in R2 relaxation rate in both species is limited to white matter tracks for which the cause is speculated to be multifaceted.

Sixth Congress of the International BioIRon Society Page 118 Podium Abstracts IBIS

Podium #38 LOW INTRACELLULAR IRON INCREASES THE STABILITY OF MATRIPTASE-2 Ningning Zhao1, Christopher Nizzi2, Sheila Anderson2, Jiaohong Wang3, Akiko Ueno3, Hidekazu Tsukamoto3, Richard Eisenstein2, Caroline Enns1 and An-Sheng Zhang1 1Oregon Health & Science University; 2University of Wisconsin-Madison; 3University of Southern California

Matriptase-2 (MT2) is a type II transmembrane serine protease that is predominantly expressed in hepatocytes. It suppresses the expression of hepatic hepcidin, an iron regulatory hormone, by cleaving membrane hemojuvelin (HJV) into an inactive form. HJV is a bone morphogenetic protein (BMP) co-receptor. Here we report that MT2 is upregulated under iron deprivation. In HepG2 cells stably expressing the coding sequence of MT2 gene, TMPRSS6, incubation with apo-transferrin or membrane impermeable iron chelator, desferroxamine, was able to increase MT2 levels. This increase did not result from the inhibition of MT2 shedding from the cells. Rather, studies using a membrane permeable iron chelator, SIH, revealed that depletion of cellular iron was able to decrease the degradation of MT2 independently of internalization. We found that lack of the putative endocytosis motif in its cytoplasmic domain largely abolished the sensitivity of MT2 to iron-depletion. Neither acute nor chronic iron deficiency was able to alter the association of Tmprss6 mRNA with polyribosomes in the liver of rats indicating a lack of translational regulation by low iron levels. Studies in mice showed that Tmprss6 mRNA was not regulated by iron or the BMP-mediated signaling with no evident correlation with either Bmp6 mRNA or Id1 mRNA, a target of BMP signaling. These results suggest that regulation of MT2 occurs at the level of protein degradation rather than by changes in the rate of internalization and translational or transcriptional mechanisms and that the cytoplasmic domain of MT2 is necessary for its regulation.

Sixth Congress of the International BioIRon Society Page 119 Podium Abstracts IBIS

Podium #39 A MUTATION IN THE IRON-RESPONSIVE ELEMENT OF ALAS2 IS A MODIFIER OF CLINICAL SEVERITY IN ERYTHROPOIETIC PROTOPORPHYRIA Sara Luscieti2, Sarah Ducamp, PhD3, Hanna Manceau, PhD4, Caroline Kannengiesser, PhD5, Marguerite Hureaux, PhD6, Gael Nicolas, PhD3, Zoubida Karim, PhD3, Jean Charles Deybach Deybach, PhD, MD7, Laurent Gouya Gouya, PhD, MD8, Herve Puy Puy, PhD, MD8 and Mayka Sanchez, PhD1 1Josep Carreras Leukaemia Research Institute (IJC), Diagnostics in Iron Metabolism Disease (D IRON) and Iron Metabolism: Regulation and Diseases Group. and Institute of Predictive and Personalized Medicine of Cancer (IMPPC). Badalona, Barcelona.; 2Institute of Predictive and Personalized Medicine of Cancer (IMPPC). Badalona, Barcelona.; 3INSERM U1149 CNRS ERL 8252, Centre de Recherche sur l’inflammation, Paris. and Laboratory of excellence, GR-Ex, Paris, France; 4INSERM U1149 CNRS ERL 8252, Centre de Recherche sur l’inflammation, Paris, France. and AP-HP, Centre Français des Porphyries, Hôpital Louis Mourier, Colombes.; 5INSERM U1149 CNRS ERL 8252, Centre de Recherche sur l’inflammation, Paris. and Université Paris Diderot, site Bichat, Sorbonne Paris Cité, France; 6INSERM U1149 CNRS ERL 8252, Centre de Recherche sur l’inflammation; 7AP-HP, Centre Français des Porphyries, Hôpital Louis Mourier, Colombes.; 8INSERM U1149 CNRS ERL 8252, Centre de Recherche sur l’inflammation, Paris. and Laboratory of excellence, GR-Ex, Paris, France. and Université Paris Diderot, site Bichat, Sorbonne Paris Cité, France

Erythropoieticprotoporphyria (EPP) is a rare constitutive haematological disorder, characterized by protoporphyrin (PP) accumulation resulting in acute photosensitivity and rarely in severe liver disease. Most of the patients presented with partial ferrochelatase (FECH) deficiency causing free-PP accumulation. Few patients harboured gain-of-function mutations of the erythroid isoform of delta-aminolevulinic synthase (ALAS2) and show a distinct biochemical feature with a high level of zinc-PP. Nevertheless about 5% of protoporphyric families are FECH and ALAS2 mutation-negatives. The proband is an 18 year old Caucasian girl referred to the French Center of Porphyria because of acute photosensitivity suggesting EPP. High level of free and Zn PP in erythrocytes confirms the diagnosis. Iron metabolism parameters were compatible with iron deficiency. FECH enzyme activity was normal and no punctual or large FECH gene mutation was identified. Moreover, the proband did not harbour the FECH low-expressed allele IVS3-48C. The father and one uncle of the proband presented with zinc- and free PP accumulation in erythrocytes associated with mild or absent photosentivity. ALAS2 gene was sequenced and the c.-38T>C (NM_000032.4) variation located in the IRE of ALAS2 was found in heterozygous state. This change is absent of the 6500 exome sequencing in the Exome Variant Server database. In vitro competitive EMSA experiments demonstrate that the mutation reduce Iron Regulatory Protein 1 (IRP1) binding affinity what will de-repress ALAS2 mRNA translation, producing more protein. A linkage study allowed us to exclude ALAS2 gene as the causative gene for EPP in this family.The ALAS2IRE mutation was inherited from the healthy mother (normal upper levels of ZnPP) and did not co-segregrate with the PP accumulation in the family. Linkage analysis in the family combined with exome sequencing in the proband revealed a new causative gene for EPP. Functional analyses are ongoing to confirm the role of this new gene in EPP. The ALAS2 IRE mutation will act as a modifier, increasing the severity of the disease in the proband. We identified the first-ever described mutation in the IRE of ALAS2. This variation abrogates in vitro the binding of recombinant IRP1 and confers clinical severity for EPP. Mutations in this regulatory element may play an important role in other rare cases of EPP. Funding: Work supported by grant SAF2012-40106 from Spanish Secretary of Research, Development and Innovation (MINECO) and grant CIVP16A1857 “Ayudas a proyectos de InvestigaciónenCiéncias de la Vida - Fundación Ramón Areces”, 2014 SGR225 (GRE) Generalitat de Catalunya and economical support from FundacióInternacionalJosep Carreras i de la Obra Social “la Caixa” Spain to M.S. M.S. held a research contract under the Ramón y Cajal program from the Spanish Ministry of Science and Innovation (RYC-2008-02352).

Podium #40 Sixth Congress of the International BioIRon Society Page 120 Podium Abstracts IBIS

HEPCIDIN AND ATOH8 EXPRESSION ARE DECREASED IN MURINE MODELS OF HEMOCHROMATOSIS HOWEVER HEPCIDIN EXPRESSION IS UNAFFECTED BY LOSS OF ATOH8 Yihang Li, PhD1, Princy Prasad, MS1, Ian Miller, BSc1, Sally Nijim, Andrew McKie, PhD2 and Robert Fleming, MD1 1Saint Louis University; 2King's College London

Atoh8 is a member of the bHLH transcription factor family, and hepatic expression of atoh8 mRNA is up-regulated by dietary iron and down-regulated by stimulation of erythropoiesis in mice, in parallel with changes in hepcidin mRNA (hamp1). It is a candidate transcription factor in the regulation of Hamp1 by iron status. Here we performed studies evaluating the contribution of genes regulating hepcidin to the regulation of Atoh8, as well as the role of Atoh8 in the regulation of hepcidin. In the former studies, wild type FVB and three hereditary hemochromatosis (HH) mouse models, Hfe-/-, TfR2Y245X/Y245X and Hfe-/-TfR2Y245X/Y245X mice, were placed on diets containing 60 ppm or 25,000 ppm iron at weaning for two weeks before euthanasia and tissue collection. Liver expression of Hamp1 and Atoh8 were evaluated. Additionally, the WT and HH mice were intraperitoneally injected with 30 mg/kg phenylhydrazine (PHZ) on days 49 and 50, and then euthanized on day 53. Liver and spleen non-heme iron concentrations, and serum hematologic and iron parameters were measured. The liver mRNAs for hamp1, atoh8, and bmp6 were measured by quantitative RT-PCR. Hfe-/- and Tfr2Y245X/Y245X mice each demonstrated lower Hamp1 and Atoh8 expression than did WT mice, despite hepatic iron loading. WT mice demonstrated the expected increases in Hamp1 and Atoh8 expression in response to dietary iron loading. Hamp1 increased in the HH mice with iron loading; however Atoh8 expression was not statistically different. The addition of Hfe knockout to the TfR2Y245X/Y245X mutation suppressed Hamp1 expression further, but had no additive effect on Atoh8. PHZ treatment at 30 mg/kg mouse significantly decreased red blood cells and hematocrit, caused severe splenomegaly, and down-regulated Hamp1 and Atoh8 in WT mice, as well as in HH mice. These results are consistent with a role for Hfe and TfR2 in normal signaling to Hamp1 and Atoh8, but also suggest some regulation of Hamp1 in response to dietary iron independent of a change in Atoh8 expression. To evaluate the role of Atoh8 in the regulation of hepd, toh eo hooyos, heteroyos ad e ere plaed o dets ota stadard pp and euthanized at 5- ees o ae he toh eo hooyos e ere satly saller se tha the or heterozygous littermates, but did not differ in any measured iron parameter (serum iron, hematocrit, liver iron, liver hamp1 and bmp6 he toh eo hooyos e also dd ot der ro heteroyos e response. These observations indicate that HFE and TFR2 are necessary for normal Atoh8 as well as hepcidin expression; however they do not support an essential role for Atoh8 in hepcidin regulation by iron demand.

Sixth Congress of the International BioIRon Society Page 121 Podium Abstracts IBIS

Podium #41 HEPCIDIN DIFFERENTIALLY REGULATES FERROPORTIN EXPRESSION IN SUCKLING MICE David Frazer, Sarah Wilkins, Deepak Darshan, Cornel Mirciov and Greg Anderson QIMR Berghofer Medical Research Institute, Brisbane, Australia

Introduction: Intestinal iron absorption in adults is regulated by the liver-derived peptide hepcidin, which binds to the iron export protein ferroportin, causing its internalization and degradation. Our previous studies in rats suggest that iron absorption during the suckling period is refractory to stimuli that would normally increase hepcidin production in weaned animals. To better understand the regulation of iron absorption during the suckling period, we used mice to extend our previous studies and characterised the relationship between hepcidin, ferroportin and iron absorption during this crucial stage of life. Materials and Methods: We treated 15 and 25 day old wild-type C57BL6/ARC mice with 300mg/kg iron dextran and studied them 4 days later. In addition, the effect of hepcidin overexpression on intestinal absorption was investigated in matriptase2 knockout mice at the same ages. Blood was collected for serum iron analysis, and liver and spleen tissue and duodenal enterocytes were collected for gene and protein expression profiles. Intestinal iron absorption was measured by assessing the whole body retention of oral 59Fe. To determine whether ferroportin was involved in iron absorption during the suckling period, absorption was measured in intestine-specific ferroportin knockout mice. The effect of hepcidin protein on ferroportin was examined by administering synthetic hepcidin (1mg/g body weight) intraperitoneally to 15 and 25 day old mice. The animals were sacrificed 24 hours later and duodenal ferroportin expression was determined by western blotting and immunohistochemistry. Results: We found that intestinal iron absorption was exceptionally high during suckling in mice and dropped rapidly to adult levels upon weaning. Consistent with our studies in rats, there was very little decrease in absorption in suckling mice injected with iron dextran, with the absorption of a dose of 59Fe remaining above 82%. This was despite a large increase in the expression of hepatic hepcidin. Similar results were seen in suckling matriptase2 knockout mice, confirming that high hepcidin did not affect absorption. However, absorption was decreased significantly (to less than 20% of littermate control levels) in 15 day old ferroportin knockout mice, indicating that ferroportin is responsible for the majority of the iron absorbed at this time. The injection of synthetic hepcidin did not decrease the expression of ferroportin protein in 15 day old mice but there was a significant decrease in 25 day old animals. Immunofluorescent localisation of ferroportin showed that the protein localised to the basolateral membrane of duodenal enterocytes in both 15 and 25 day old mice. Basolateral expression of ferroportin decreased in response to hepcidin administration in 25 day old animals, but not in 15 day old mice. Decreases in splenic ferroportin levels were seen in both age groups. Discussion and Conclusions: These data demonstrate that the high iron absorption that occurs during the suckling period is mediated by ferroportin. However, ferroportin is not responsive to hepcidin mediated degradation at this time, despite being expressed on the basolateral membrane. In contrast, hepcidin is able to regulate the levels of ferroportin in other body tissues such as the spleen. This suggests that enterocyte-specific alterations to ferroportin that prevent hepcidin binding and/or changes to the ferroportin degradation pathway during suckling allow iron absorption to remain high regardless of hepcidin expression levels.

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Podium #42 HEME AND IRON CONTROL OF MACROPHAGE PLASTICITY IS PREVENTED BY THE HEME SCAVENGER HEMOPEXIN AND THE IRON CHELATOR DFO Francesca Vinchi, PhD1, Milene Costa da Silva, MSc1, Giada Ingoglia, MSc2, Sara Petrillo, MSc2, Nathan Brinkman3, Adrian Zuercher, PhD4, Emanuela Tolosano, PhD2 and Martina U. Muckenthaler, PhD1 1Molecular Medicine Partnership Unit (MMPU), University of Heidelberg & EMBL; 2Molecular Biotechnology Center & Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy; 3CSL Behring, Research & Development, Kankakee, IL, USA; 4CSL Behring, Research & Development, Bern, Switzerland

A broad spectrum of diseases, such as sickle cell anemia, hemorrhages, atherosclerosis, malaria or sepsis are hallmarked by hemoglobin and heme release as well as elevated levels of circulating and/or localized pro-inflammatory cytokines. Heme drives sterile inflammation mediated by TLR4 activation, indicating that products of erythrocyte hemolysis can be considered as damage-associated molecular pattern molecules (DAMPs). In particular, heme acts as a pro-inflammatory molecule that activates endothelial and immune system cells by inducing cytokine production. We now show that the heme scavenger hemopexin (Hx) protects RES macrophages from heme overload in heme-challenged Hx- null mice, reducing cell activation as well as cytokine and ROS (reactive oxygen species) production. Importantly, treatment of bone-marrow derived macrophages (BMDM) with aged RBCs, heme or iron (Fe-NTA) polarizes macrophages towards the M1 pro-inflammatory phenotype, independent of the initial differentiation state of the macrophages. This effect can be prevented by co-treatment with the heme scavenger Hx or the iron chelator desferrioxamine (DFO). Consistently, tissue macrophages derived from mice injected with heme or from mice with sickle cell disease show iron accumulation and a M1-like pro-inflammatory phenotype. Interestingly, both ROS accumulation and activation of the TLR4 signaling pathway trigger the pro-inflammatory phenotypic switching of macrophages induced by heme. The administration of exogenous Hx to heme-challanged wild-type mice or sickle mice rescues heme-induced M1 macrophage polarization, thanks to its ability to scavenge free heme. The finding that heme and iron drive polarization of macrophages towards the M1 pro-inflammatory phenotype is of pathophysiological relevance for those conditions in which free heme levels are increased and cytokine production is observed. Based on our data we propose that the therapeutical administration of Hemopexin will be beneficial to counteract not only heme-driven endothelial activation, as previously reported, but also heme-driven macrophage activation.

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Podium #43 A NOVEL IRON-MEDIATED MECHANISM FOR DEVELOPMENT OF INFLAMMATORY BOWEL DISEASE Shirly Belizowski, Abraham Nyska, Avi Zuckerman, Fabio Cominelli, Orly Savion and Esther Meyron-Holtz Technion– Israel Institute of Technology, Haifa, Israel

Background: Iron accumulation in inflammatory lesions has been described in many settings but a molecular mechanism for this accumulation was not completely elucidated. We hypothesized that an iron independent activation of RNA-binding activity of the Iron Regulatory Protein (IRP)1, via reactive oxygen and nitrogen species is the driving force for this iron accumulation. Methods: To test this hypothesis, we examined a mouse-model of inflammatory bowel disease, the α overexpressing mouse (TNF) and an epithelial cell model for changes in iron homeostasis during inflammation and analyzed the effect of IRP1 and 2 deletions on the course of the inflammation. Differential analysis of the different cell-types within the inflamed tissue was performed. Results: We found that the terminal ileitis, which is developed in the TNF mouse, was accompanied not only by a local iron accumulation but also by profound iron redistribution within the inflamed tissue. While the local and infiltrating macrophages accumulated iron, the inflamed epithelial cells showed a state of significant iron deficiency and elevated iron flux, which was mimicked in the epithelial cell model of inflamed Caco-2 cells. The significant iron redistribution was accompanied by elevated levels of the transcription factor Hypoxia inducible factor (α, y dstte altered - binding activity of both IRP1 and IRP2 in the different local cell types, and by altered levels of the proteins involved in the cellular iron homeostasis. These new dynamics of iron homeostasis were completely reversed by the deletion of IRP1 in the TNF mouse and the Crohn’s like picture of inflammation of the TNF mouse was not detectable in the TNF x IRP1-/- mice. In contrast, TNF x IRP2-/- mice showed severe transmural ileitis. Discussion: The non-iron mediated activation of IRP1 during inflammation triggered an impaired iron homeostasis within the inflammatory lesion that led to the accumulation of iron in the local immune cells. The iron accumulation in these cells and the iron depletion of the epithelial cells both support the recruitment of a systemic immune response and the propagation of an initially local and close to physiologic inflammation of the gut to a severe inflammation with massive infiltration of systemic immune cells. Conclusion: The finding that targeted deletion of IRP1 but not IRP2 completely abolished the inflammation in the TNF mouse suggests that IRP1 may be a target for specific inhibition to treat inflammatory bowel diseases.

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Podium #44 COMMON VARIANTS AND HAPLOTYPES IN THE TF TNF α AND TMPRSS GENES ARE ASSOCIATED ITH IRON STATUS IN A FEMALE BLACK SOUTH AFRICAN POPULATION Wanjiku Gichohi, PhD1, Alida Melse-Boonstra, PhD2, Dorine Swinkels, PhD3, Michael Zimmermann, PhD4, Edith Feskens, PhD2 and Gordon Towers, PhD5 1Jomo Kenyatta University of Agriculture and Technology; 2Division of Human Nutrition, Wageningen University, Wageningen, the Netherlands; 3The Laboratory of Genetic, Endocrine and Metabolic Diseases, Department of Laboratory Medicine, Radboud University Medical Centre, Nijmegen, the Netherlands; 4Laboratory of Human Nutrition, Institute of Food Nutrition and Health, Department of Health Sciences and Technology, Swiss Federal Institute of Technology, Zurich, Switzerland; 5Centre of Excellence for Nutrition, North West University (Potchefstroom campus), South Africa

Introduction: It is unknown whether single nucleotide polymorphisms (SNP) associated with iron status in European and Asian populations have the same relationship within the African population. We aimed to investigate associations of reported SNPs with iron status in a female black South African cohort. Additionally, we aimed to investigate gene-gene interactions that influence iron status. Methods: Blood samples were obtained from women (n=686; range 32–86 years) which were part of the Prospective Urban and Rural Epidemiology (PURE) study. Hemoglobin, serum ferritin, serum transferrin receptor and body iron stores concentrations were determined. Thirty two single nucleotide polymorphisms in 10 genes were genotyped. Results: In the TF gene, serum ferritin and body iron were significantly lower in the heterozygote genotype (AG) of rs1799852 (p=0.01 and 0.03 respectively) and serum transferrin receptor (sTfR) concentrations significantly higher (P=0.004) compared to the homozygote minor allele genotype (AA), while transferrin receptor and body iron concentrations were significantly lower in the heterozygote genotype (AG) of rs3811647 (both P=0.03) compared to the homozygote wild type (AA) and minor allele groups (GG). The chromosome 6 allele combination (AAA) consisting of rs ad rs oth α ad rs as assoated th hher odds or lo ser ferritin concentrations (serum ferritin<15µg/L; OR:1.86 (95%-CI, 1.23-2.79)) compared to the allele combinations AGA, GGT and AGT. The chromosome 22 allele combination (GG) consisting of rs228918 and rs228921 in the TMPRSS6 gene was associated with lower odds for increased sTfR concentrations (sTfR>8.3mg/L; OR:0.79 (95%-CI,0.63-0.98) compared to the allele combination AA. ssso ad olso aros s ad allele oatos the , α ad ees are assoated with iron status in female black south Africans; however, these association patterns are different compared to European ancestry populations. This stresses the need for population specific genomic data.

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Podium #45 IMPROVED IRON ABSORPTION BY CONSUMPTION OF PROBIOTICS Michael Hoppe2, Elisabeth Gramatkovski2, Gunilla Önning, Malin Björklund, Niclas Larsson and Lena Hulthén1 1Institute of Medicine, University of Gothenburg; 2Inst of Medicine

Worldwide, iron deficiency and low iron status are common. An imbalance to meet high iron requirements with inadequate iron intake and low iron bioavailability is the predominant reason for iron deficiency in populations subsisting on plant based diets, independent of gender. Strategies to increase the intake of foods rich in iron, as well as dietary factors with enhancing effect on iron absorption are therefore important. Intake of ascorbic acid and meat stimulates absorption. Lactic acid fermented foods may also improve the non-heme iron absorption. This study comprise four cross-over double radio iron isotope single-blinded trials in young healthy women to investigate if Lactobacillus plantarum can increase the iron absorption. In the first trial the iron absorption was increased with 100% when L. plantarum 299v was present in a beverage based on oats and with a high content of phytates. The absorption of iron from fruit drinks high in ascorbic acid and supplemented with iron was studied in two trials. Here the iron absorption was increased with about 50% in the Lactobacillus plantarum 299v drink in comparison with a fruit drink without bacteria. Thus, the enhancing effect has been observed both in foods with a low bioavailability but also in foods with a high bioavailability of iron. In two recently finished trials, capsules with freeze dried Lactobacillus plantarum 299v was given together with a breakfast bun meal. The capsules also contained iron, folic acid and ascorbic acid. The formula gave a significant increase of the iron absorption in comparison with a control capsule without the bacteria. The iron enhancing effect of Lactobacillus plantarum 299v is specific and beyond the effect of ascorbic acid and organic acids. The mechanism behind the effect could be related to the colonization of Lactobacillus plantarum 299v in the first part of the small intestine where the iron absorption takes place. Studies have been started to further study the mechanisms.

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Podium #46 THE ROLE OF IRON AND HEPCIDIN IN YERSINIA ENTEROCOLITICA INFECTION Debora Stefanova, Joao Arezes, MS, Victoria Gabayan, BS, Tomas Ganz, MD, PhD, Yonca Bulut, MD and Elizabeta Nemeth, PhD University of California, Los Angeles

Introduction Hepcidin, the sole known iron regulatory hormone, controls iron distribution between plasma and tissues. Its interaction with the iron exporter ferroportin causes iron retention within the target cells and consequent decrease in extracellular iron concentration. Hepcidin production is potently induced by inflammatory signals. The resulting decrease in plasma iron is proposed to be an innate immune mechanism to inhibit bacterial replication and spread. Yersinia enterocolitica is an extracellular food-borne pathogen, which typically affects the digestive tract. However, it is known to cause severe systemic infection (yersinosis) in iron-overloaded or immune-compromised patients causing high mortality. Furthermore, it is one of the most common pathogens associated with blood transfusions due to its ability to grow at ambient temperatures much lower than 37C. As epidemiological and clinical data suggest a correlation between iron overload and yersinosis, we used a mouse model to evaluate the role of iron and hepcidin in Yersinia infection, and investigate the therapeutic potential of hepcidin agonists in yersinosis. Materials and Methods Wild type (WT) and hepcidin knockout (HKO) mice were iron-loaded or iron-depleted by dietary manipulations and were infected with Yersinia enterocolitica through oral gavage. Mortality rate was correlated to serum and liver iron concentration, bacterial spread (tissue CFU assay and histological analysis) and inflammatory parameters. Synthetic hepcidin analogues (minihepcidins) were administered to iron-loaded HKO or WT mice for several days, starting either pre- or post-infection. Results Iron-loaded HKO mice were highly susceptible to Yersinia enterocolitica infection with mortality rate reaching 100% at the highest dose (108 CFUs per mouse), in contrast to 0% mortality in WT mice. Iron-depletion of the HKO mice was sufficient to prevent yersinosis-associated lethality. Improved survival correlated with low serum and liver iron levels and diminished bacterial spread to the liver and spleen. Pre- and post-infection treatments of HKOs with minihepcidins efficiently lowered serum iron without affecting liver iron stores, and significantly prevented mortality (70% to 100% survival rate). Even in iron dextran-injected WT mice, which do not die of yersinosis, minihepcidin treatment had beneficial effects and decreased bacterial dissemination to organs. Discussion HKO mice are highly susceptible to yersinosis-associated mortality as a result of their high plasma iron concentrations and inability to lower plasma iron in the face of infection. Protective effect of minihepcidin treatment that lowered plasma iron levels but not the stores, confirmed that it is extracellular iron availability rather than total iron concentration that promotes yersinosis. It remains to be determined which forms of extracellular iron are able to promote Yersinia enterocolitica growth and dissemination. The ability of minihepcidins to prevent yersinosis-associated lethality suggest they may be useful therapeutic option for yersinosis.

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Podium #47 ANAEMIA ASSOCIATED WITH INTESTINAL INFLAMMATION Debbie Trinder, PhD1, Anita Chua, PhD2, Roheeth Delima, PhD1, Desiree Ho, PhD1, Borut Klopcic, PhD1, John Olynyk, MD3 and Ian Lawrance, MD, PhD1 1University of Western Australia; 2University of Western Australia and Murdoch University; 3Murdoch University and Curtin University

We have previously demonstrated that increased dietary iron intake enhanced intestinal inflammato ad signalling promoting colonic tumour development1. Systemic inflammation is also known to regulate the synthesis of the ler ro relatory horoe hepd ad odlate ro etaols a a depedet pathay he a o this study was to investigate the effects of intestinal inflammation on systemic inflammation, erythropoiesis and the regulation of iron metabolism. Mice were treated with a single dose of azoxymethane (7.4 mg/kg ip) and dextran sodium sulphate (DSS; 2% w/v) for 3 or 7 days (3d or 7d) to induce intestinal inflammation. Colonic inflammation was assessed by histology. Haematology was easred y stadard ethods ee epresso as detered y realte , plasa ad erythropoet levels by ELISA and phosphorylation of Stat3 and protein expression by immunoblot. Intestinal inflammation was enhanced with increasing duration of DSS treatment. After 3d of DSS treatment, there was a moderate increase in inflammatory scores and this was further enhanced by approately old ater d o treatment (p<0.01) with evidence of severe inflammatory infiltration, mucosal erosion and crypt damage. Systemic laato as osered at d t ot d th plasa leels reased p ad ler tat phosphorylation ehaed ater d p hle plasa as ot deteted at d er epresso o ate phase protes Omm and Saa were also elevated at 7d (p<0.01) while expression was negligible at 3d. Early signs of iron deficiency anaemia were detected with reduced mean cell haemoglobin (MCH) (p<0.05) at 3d and the degree of anaemia increased with the exacerbation of intestinal inflammation at 7d with significantly decreased haemoglobin, haematocrit, MCH and mean cell volume and increased reticulocyte count (p<0.01). Furthermore, plasma erythropoietin levels (p<0.01) and splenic gene expression of the erythroid signalling molecule, erythroferrone (p<0.05) were elevated at both 3d and 7d consistent with the presence of stress erythropoiesis. er hepd ee epresso as dorelated p ad the iron exporter, ferroportin protein levels were increased (p<0.05) at 3d in response to erythropoiesis. Hepcidin expression was also decreased (p<0.01) at 7d in response to anaemia, despite reased plasa epresso ad liver Stat3 phosphorylation. However, ferroportin gene expression was negligible and protein levels remained low consistent with the presence of systemic inflammation. These findings suggest moderate intestinal inflammation caused anaemia and the erythropoietic signal stimulated the hepderroport pathay to rease the spply o ro or erythropoess eere testal laato ased a additional systemic inflammatory response that blunted ferroportin expression and is likely to contribute to tissue iron retention and enhanced anaemia. These results suggest both anaemia of inflammation and iron deficiency anaemia were associated with intestinal inflammation. 1Chua et al. PlosOne 2013;8[11]e78850.

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Podium #48 PARADOXICAL TISSUE IRON DEPLETION AND HEPCIDIN INDEPENDENT DOWNREGULATION OF THE IRON EXPORTER FERROPORTIN1 DURING SALMONELLA INFECTION François Canonne-Hergaux, PhD1,2,3, Alexandra Willemetz, Sean Beatty, Etienne Richer, Aude Rubio, Anne Auriac, Ruther J. Milkereit, Olivier Thibaudeau, Sophie Vaulont and Danielle Malo 1INSERM UMR 1043; 2CNRS UMR 5282; 3Université de Toulouse, UPS, Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse, France.

Introduction: Retention of iron in tissues macrophages through upregulation of hepatic hepcidin (Hepc) and consequent downregulation of the iron exporter ferroportin1 (Fpn1) is thought to participate in the establishment of anemia of inflammation occurring after infection. However, some recent works proposed that, in the context of intracellular pathogens infection, a loss of iron from macrophages due to an upregulation of Fpn1 occur in order to limit intracellular iron access for pathogen proliferation. To better understand the regulation of Fpn1 in the setting of intracellular bacterial infection, we investigated in mice (in vivo) as well as in mouse macrophages (in vitro), the regulation of Fpn1 after infection with Salmonella, an intracellular pathogen presenting a strong cellular tropism for macrophages.

Methods: Mice presenting tissue macrophages with basal iron (A/J, 129S6 and C57BL/6 mice), high iron (recombinant congenic AcB61 mice with hemolytic anemia) or low iron (Hepc-deficient mice) were infected intravenously with Salmonella enterica serovar Typhimurium or serovar Enteriditis. Liver and spleen iron levels and localization were assessed by Perl’s staining or by acid digestion of tissue samples. Bacterial load was followed by measuring the colony- forming unit (CFU) in liver and spleen. Liver and spleen were collected for mRNA (RT-PCRq) and protein (western blot and immunohistofluorescence) expression analysis of Fpn1, Hepc and HO1 (marker of infection and inflammation). Bone marrow derived macrophages (BMDM) isolated from CD1 and C57BL/6 mice were infected with Salmonella Typhimurium and expression of Fpn1 and HO1 were followed by RT-PCRq.

Results & discussion: During the course of in vivo Salmonella Typhimurium infection, upregulation of HO1 and downregulation of Fpn1 protein in macrophages of the spleen (A/J and AcB61 mice) and the liver (AcB61 mice) were observed despite no significant changes of the level of hepatic Hepc in both strains of mice. In AcB61 liver and spleen, decrease of Fpn1 protein occurred without a significant change at the level of Fpn1 mRNA suggesting a negative translational regulation. In addition, Fpn1 expression at both mRNA and protein levels was strongly decreased during Salmonella Typhimurium or Enteriditis infection in Hepc-deficient mice. Interestingly, such negative regulation of Fpn1 in Hepc-deficient mice was observed in both hepatocytes and tissues macrophages. The expression of Fpn1 mRNA was also strongly decreased during the course of in vitro infection of both CD1 and C57BL/6 derived BMDM with Salmonella Typhimurium. Importantly, in A/J, 129S6 and C57BL/6 mice, the downregulation of Fpn1 was associated with decrease iron stores in both liver and spleen. In infected Hepc-deficient mice, we also observed a strong decrease of the hepatic iron content with some iron redistribution in cells lining the centrolobular zones and around inflammatory foci.

Conclusion: Contrasting with recent works, in our mouse models of Salmonella infection, the expression Fpn1 is strongly repressed in a Hepc independent manner at both mRNA and protein levels. Effort need to be made to understand at the molecular level these regulations as well as the concomitant tissue iron depletion observed in Salmonella infection.

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Podium #49 EFFICACY OF EARLY VERSUS DELAYED TREATMENT WITH IRON AND ERYTHROPOIETIN IN A MOUSE MODEL OF ACUTE AND SEVERE ANEMIA OF INFLAMMATION Airie Kim, MD, PhD, Eileen Fung, PhD, Eeman Khorramian, Victoria Gabayan, BS, Elizabeta Nemeth, PhD and Tomas Ganz, PhD, MD University of California, Los Angeles

INTRODUCTION: Acute anemia of inflammation (AI) is a pathology that accompanies fulminant inflammatory conditions, including sepsis, trauma, and burns. This subset of AI presents similarly to chronic AI, with normocytic anemia, shortened erythrocyte lifespan, impaired erythropoiesis, and hypoferremia despite intact iron stores. Despite its prevalence and significance, acute AI is undertreated because of the unclear efficacy of traditional therapies. In this study, we have used a mouse model of acute and severe inflammatory anemia induced by heat-killed Brucella abortus (HKBA) to study the effectiveness of iron supplementation and/or exogenous erythropoietin (EPO) in treating acute AI. METHODS: After two weeks on an iron-adequate diet, mice were injected with HKBA or saline, then analyzed at 14 days for iron and hematologic parameters. To study the effects of different treatments and their timing, groups of these mice were treated early (days 1,2 after HKBA) or late (days 7,8) with subcutaneous iron only, EPO only, or the combination of iron and EPO. These experiments were repeated in hepcidin-1 knockout (HKO) mice to understand the role of hepcidin in response to different treatments. RESULTS: The early treatment group showed no significant difference in hemoglobin changes among the treatment groups (EPO/Fe -7.3 g/dL vs. EPO -8.4 g/dL vs. Fe -7.8 g/dL vs. Saline -7.3 g/dL). In the late treatment group, the EPO/Fe combination was most effective as these mice had the smallest drop in hemoglobin (EPO/Fe -5.5 g/dL vs. Saline -9.2 g/dL; p<0.001), with an increased reticulocyte product index (EPO/Fe 15.9 vs. Saline 10.8; p<0.05). In HKO mice, anemia was much milder than in WT mice (by 3.9 g/dL), but neither early nor late treatment caused further attenuation of anemia. CONCLUSIONS: In our HKBA mouse model of acute and severe inflammation, late treatment with iron supplementation and exogenous EPO was able to partially reverse the development of AI, with a concurrent increase in reticulocytosis. HKO mice had a much smaller hemoglobin drop than wild-type mice with no further improvement in AI despite identical treatments. Thus, hepcidin ablation alone is protective against AI and may supersede any potential benefit from EPO and/or iron supplementation.

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Podium #50 NON-TRANSFERRIN-BOUND IRON (NTBI) IS ESSENTIAL FOR RAPID GROWTH AND VIRULENCE OF VIBRIO VULNIFICUS João Arezes, Deborah Stefanova, Victoria Gabayan, Yonca Bulut, Elizabeta Nemeth and Tomas Ganz University of California, Los Angeles / University of Porto

Iron overload disorders are associated with increased risk of severe infections by siderophilic bacteria. We have recently shown that hepcidin-induced hypoferremia is a critical host defense mechanism to protect mice from Vibrio vulnificus infection. The effect of hypoferremia on bacteria is unlikely to be due to decreased transferrin-bound iron, as this form of iron is inaccessible at the early stage of infection. NTBI, a form of iron usually present in the serum of patients with iron overload, has previously been associated with increased V. vulnificus growth, suggesting that the removal of NTBI from circulation early in the infection may be critical for host resistance. To address this issue, we measured V. vulnificus growth in standard broth and in mouse sera, supplemented with various concentrations of Ferric Ammonium Citrate (FAC), a form of NTBI. We observed that V. vulnificus growth was enhanced by FAC in broth, and that FAC was absolutely required for bacterial growth in serum. Furthermore, bacteria only grew when the amount of FAC added was sufficient to saturate transferrin, coinciding with the appearance of NTBI in serum. In addition, bacterial growth only occurred when Fe(III), and not Fe(II), was added to the serum. We hypothesized that Ferric Uptake Regulator [FUR] - induced changes in bacterial gene expression might explain the rapid bacterial growth in the presence of NTBI. However, tat stra as eally lethal ro-overloaded hepcidin knockout mice as the wild type strain of V. vulnificus, suggesting that another mechanism is responsible for the iron-induced bacterial growth and virulence in mice. To explore other potential mechanisms by which ferric iron could induce V. vulnificus pathogenesis, we performed RNA sequencing of the V. vulnificus transcriptome, comparing bacteria grown with or without FAC. We found 127 differentially expressed genes that are currently being analyzed to clarify the genetic response triggered by NTBI. Altogether, our data demonstrate that NTBI is required for rapid V. vulnificus growth in serum and may be the factor causing lethal infections in iron-overloaded hosts.

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Podium #51 ACTIVIN B UTILIZES ACTIVIN TYPE II RECEPTORS, BMP TYPE I RECEPTORS, HEMOJUVELIN AND SMAD1/5/8 SELECTIVELY IN HEPATOCYTES TO PROMOTE HEPCIDIN INDUCTION BY INFLAMMATION IN MICE Susanna Canali, PhD1, Amanda B. Core, PhD1, Kimberly B. Zumbrennen-Bullough, PhD1, Maria Merkulova, PhD1, Alan Schneyer, PhD2, Antonello Pietrangelo, MD, PhD3 and Jodie L. Babitt, MD1 1Program in Anemia Signaling Research, Division of Nephrology, Program in Membrane Biology, Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School; 2Department of Veterinary and Animal Science, UMass-Amherst, Amherst, MA; 3Center for Hemochromatosis, University Hospital of Modena and Reggio Emilia, Modena Italy

Induction of the iron regulatory hormone hepcidin contributes to the anemia of inflammation by restricting iron availability. Bone morphogenetic protein 6 (BMP6) signaling is a central transcriptional regulator of hepcidin. Recently, the BMP/TGF- speraly eer t as sho to e ded y lpopolysaharde lthoh t typally sals a a different subset of SMAD proteins (SMAD2/3) compared with BMP6 (SMAD1/5/8), Activin B was reported to stimulate noncanonical SMAD1/5/8 signaling and hepcidin expression in hepatocytes. Here, we investigated the detailed molecular mechanism of Activin B-mediated hepcidin regulation and its functional significance in vivo. We showed that low concentrations of Activin B, but not Activin A, stimulate prolonged SMAD1/5/8 signaling and hepcidin expression in liver cells to a similar degree as canonical SMAD2/3 signaling, and with similar potency compared with BMP6. Activin B stimulates hepcidin via classical Activin type II receptors ACVR2A and ACVR2B, noncanonical BMP type I receptors ALK2 and ALK3, and SMAD5. The co-receptor hemojuvelin binds directly to Activin B and facilitates Activin B-SMAD1/5/8 signaling. Activin B-SMAD1/5/8 signaling occurs selectively in hepatocyte-derived cells and is not enabled by hemojuvelin in other cell types. Finally, the Activin inhibitor follistatin-315 has no effect on basal hepcidin expression, but blunts hepcidin induction by lipopolysaccharide or heat-killed Brucella abortus in mice. Our data elucidate a novel mechanism for noncanonical SMAD activation by BMP/TGF- speraly eers, ad support a functional role for Activin B in hepcidin induction by inflammation in vivo. Targeting the Activin B-hepcidin pathway may lead to the development new therapies for anemia of inflammation.

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Podium #52 THE USE OF NEXT-GENERATION SEQUENCING TO SCREEN THE BRITISH POPULATION FOR DISORDERS OF IRON REGULATION USING A 16 GENE PANEL Kathryn Robson, PhD1, Patricia Bignell, BSc Hons2, Wale Atoyebi, FRCPath2, Joanne Mason, PhD3, Pavlos Antoniou, PhD3 and Anna Schuh, PhD3 1MRC Institute of Molecular Medicine, University of Oxford; 2Molecular Haematology Department, John Radcliffe Hospital; 3BRC/NHS Oxford Molecular Diagnostics Centre, Oxford

Background: Next-generation sequencing (NGS) is a valuable tool. Recent improvements in accuracy, reductions in the cost of NGS and exon capturing platforms, have extended the applicability of targeted exon sequencing to UK National Health Services (NHS) diagnostic laboratories. This has enabled the identification of a wider range of variants, thus contributing to our understanding of the heritability of complex diseases, including disorders of iron metabolism. Aims: We sought to investigate the clinical utility of NGS to understanding iron disorders by sequencing 16 genes involved in iron regulation by designing a TruSeq Custom Amplicon (TSCA) panel, the Oxford Iron Panel. Methods: Samples were run on the Illumina MiSeq platform. The TSCA panel was designed with 340 amplicons covering 16 genes: HFE, HFE2, HAMP, TFR2, SLC40A1, TF, ALAS2, CP, TMPRSS6, SLC11A2, HEPH, FTL, FTH, BMP4, BMP6 and SMAD4. These genes were selected as many are known to have a role in iron metabolism. A number of these genes are mutated in diseases such as haemochromatosis, hyperferritinaemia and iron deficiency disorders (IRIDA). Patient samples are routinely referred to the BRC/NHS Oxford Molecular Diagnostics Centre, Oxford, United Kingdom for molecular investigation of disorders of iron metabolism. They have ferritin/transferrin saturation/serum iron parameters lying outside the normal range. The vast majority of these patients have an iron overload phenotype. The first step in the pipeline is C282Y mutation analysis. Those patients who lack two copies of the C282Y mutation were investigated further using the Oxford Iron Panel. Multiplex ligation-dependent probe amplification (MLPA) is required to identify large deletions as NGS only identifies mutations within a sequencing read. Results: Three hundred and eighty two diagnostic samples have been screened by NGS using the Oxford Iron Panel. We identified mutations in 13/16 genes. Two hundred and eighty three patients were Caucasian in origin, of whom 91(32%) had causative mutations detected by NGS. Ninety-nine of the samples were from other ethnic groups, mainly Asian, of which 31(31%) were found to have causative mutations. Overall, likely pathogenic mutations were found in 121 samples (32%). Twenty-six were novel mutations; 14 were missense mutations, 9 were small deletions, one was a small insertion and another a nonsense mutation. Three patients had novel copy number variation mutations (deletions). Digenic inheritance was more common than expected; 18 (15%) of the patients had mutations in more than one gene. In general the phenotype was iron overload. Conclusion: The 16 gene Oxford Iron Panel is an effective method for the clinical diagnosis of multi-gene and single large gene disorders. It is now in routine diagnostic use. We found mutations in 32% of our patient group; the additional diagnostic value of this panel is screening ethnic groups not previously suitably screened using the 2 common HFE mutations, 31% were found to be positive for a causative mutation. The Oxford Iron Panel turn around time is comparable to conventional screening and is cost effective. This preliminary study demonstrates the potential advantages of NGS in a clinical setting.

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Podium #53 DISSECTING THE CONTRIBUTION OF UNREGULATED MACROPHAGE IRON RECYCLING AND DIETARY IRON UPTAKE IN GENERATING SYSTEMIC IRON OVERLOAD IN HEMOCHROMATOSIS Regina Kessler1,3, Matthias W. Hentze2, Martina U. Muckenthaler1,3 and Sandro Altamura1,3 1Dep. of Pediatric Oncology, Hematology and Immunology - University of Heidelberg, Germany; 2EMBL - European Molecular Biology Laboratory, Heidelberg, Germany; 3MMPU Molecular Medicine Partnership Unit

Systemic iron levels are balanced by the hepatic iron hormone hepcidin and its “receptor” ferroportin (Fpn) to prevent the pathological consequences of iron overload or iron deficiency. Hepcidin binding to the iron exporter FPN reduces dietary iron export from duodenal enterocytes and iron recycling from aging erythrocytes in reticuloendothelial macrophages. Mutations as the Fpn(C326S), that disrupt the hepcidin/FPN regulatory loop, cause an uncontrolled iron export from spleen and duodenum resulting in systemic iron overload (Altamura et al, 2014). By applying cre/lox technology, we generated mouse lines expressing the Fpn(C326S) mutation only in duodenal enterocytes (Villin-Cre/FpnC326S) or in macrophages (Lyz-Cre/FpnC326S) to dissect the single contribution of these two iron exporting cell types in generating iron overload. 10-week old C57BL6/J congenic male mice have been analyzed in this study. Mice carrying the Fpn(C326S) mutation exclusively in the duodenum show identical hematological alterations as found both in hemochromatotic patients and in constitutive Fpn(C326S) mice: increased hemoglobin (Hb), hematocrit (HCT) and mean corpuscular volume (MCV). Serum iron content and transferrin saturation are strongly increased further consistent with the hemochromatotic phenotype. Hepatic iron measurement revealed severe iron deposition that correlates with increased hepcidin expression. Elevated hepcidin levels cause iron retention in reticuloendothelial macrophages of the spleen generating splenic iron overload. Interestingly, Lyz-Cre/FpnC326S mice that show hepcidin independent macrophage iron export failed to show alterations in hematological parameters and serum iron levels. However the spleen is iron depleted, in line with a constitutively expressed iron exporter. By contrast hepatic iron content and hepcidin expression are similar compared to control mice. Our results show for the first time that increased duodenal iron export is the major if not the only contributor in the generation of systemic iron overload in hemochromatosis. This finding opens new insights in developing pharmacological strategies aimed to specifically limit dietary iron import for the treatment of primary and secondary iron overload diseases.

Sixth Congress of the International BioIRon Society Page 134 Podium Abstracts IBIS

Podium #54 HETEROZYGOUS MUTATIONS IN BMP6 PRO-PEPTIDE LEAD TO INAPPROPRIATE HEPCIDIN SYNTHESIS AND MODERATE IRON OVERLOAD IN HUMANS Raed Daher, Caroline Kannengiesser, Dounia Houamel, Thibaud Lefebvre, Edouard Bardou-Jacquet, Nicolas Ducrot, Caroline de Kerguenec, Anne-Marie Jouanolle, Pierre Bedossa, Dominique Valla, Laurent Gouya, Carole Beaumont, Pierre Brissot, Hervé Puy, Dimitri Tchernitchko and Zoubida Karim, PHD INSERM U1149, Centre de Recherche sur l’inflammation, Université Paris Diderot, Laboratory of Excellence, GREx, 16 rue Henri Huchard, 75018 Paris, France.

Introduction: Hereditary hemochromatosis is a heterogeneous group of genetic disorders characterized by parenchymal iron overload reflecting the defective expression of liver hepcidin, the main regulator of iron homeostasis. Results from animal models and from patients have shown that HAMP gene mutations as well as mutations in genes implicated in the regulatory pathways of hepcidin expression (e.g. HFE, TFR2 and HJV) are responsible for hereditary hemochromatosis where the severity of iron overload is correlated with the degree of hepcidin deficiency. The bone morphogenetic protein BMP6 is the key endogenous regulatory cytokine that regulates hepcidin expression via SMAD signaling. Indeed, BMP6 null mice exhibit severe phenotype of iron overload and very low level of hepcidin. Because no BMP6 mutations have been reported in humans so far, we postulated that these mutations might be responsible for a mild late-onset rather than a severe phenotype of iron overload. Therefore, we analyzed the BMP6 gene in cohort of patients with primary mild to moderate iron overload and in whom mutations in known hereditary hemochromatosis genes had been excluded.

Methods: BMP6 gene was analyzed by exons and exon-intron boundary sequencing. Family studies for five patients and a replication cohort analysis were performed. Serum levels of hepcidin was measured by liquid chromatography-tandem mass spectrometry and BMP6 expression was explored in liver biopsies from patients by immunohistochemistry. The functions of mutant and normal BMP6 were assessed in transfected cells using immunofluorescence, real-time quantitative PCR, and immunoblot analyses.

Results: We identified 3 heterozygous missense mutations in BMP6 (p.Pro95Ser, p.Leu96Pro, and p.Gln113Glu) in 6 unrelated patients with unexplained iron overload (9% of our cohort). These mutations were detected in less than 1% of controls. Family studies indicated dominant transmission. Serum levels of hepcidin were inappropriately low in patients. A low level of BMP6, compared with controls, was found in a biopsy from 1 patient. In cell lines, the mutated residues in the BMP6 propeptide resulted in defective secretion of BMP6; reduced signaling via SMAD1, SMAD5, and SMAD8; and significant defect of hepcidin production. Conclusions: We identified 3 heterozygous missense mutations in BMP6 in patients with unexplained iron overload. These mutations lead to loss of signaling to SMAD proteins and reduced hepcidin production. These mutations might increase susceptibility to mild-to-moderate late onset iron overload. We therefore propose that BMP6 is a new susceptibility gene for iron overload in Caucasians and speculate that it could be the principal susceptibility gene in other ethnic groups where HFE mutations are rarely present.

Sixth Congress of the International BioIRon Society Page 135 Podium Abstracts IBIS

Podium #55 NON-HFE HAEMOCHROMATOSIS: ESTIMATION OF THE GLOBAL PREVALENCE FROM ANALYSIS OF NEXT- GENERATION SEQUENCING DATA Daniel Wallace, BSc (Hons), PhD and V. Nathan Subramaniam, PhD QIMR Berghofer Medical Research Institute

Introduction: The majority of hereditary haemochromatosis (HH) cases are caused by homozygosity for the p.C282Y mutation in the HFE gene. The prevalence of this HFE-related form of HH has been well-studied and is most prevalent in northern European populations. Other forms of iron overload can be caused by mutations in HFE2, HAMP, TFR2 or SLC40A1. These non-HFE forms of iron overload occur world-wide but to our knowledge there is no data on their population prevalence. The aims of this study were to estimate the population prevalence of non-HFE forms of HH. Methods: To estimate the population prevalence of the various forms of non-HFE HH we analysed the allele frequencies of all known disease-causing variants in the HFE, HFE2, HAMP, TFR2 and SLC40A1 genes among publically available databases containing next-generation sequence data from whole genome and whole exome sequencing projects using wANNOVAR. Results: A literature search identified 151 variants that have been previously associated with iron overload and were considered pathogenic. Of these 44 were represented among either the 1000 Genomes (1000G), Exome Sequencing Project (ESP6500) or Exome Aggregation Consortium (ExAC) sequence databases. The frequencies of these variants were combined with those of 41 additional, previously unreported, major structural variants (including frameshift, premature stop codon, initiator codon, splice donor or splice acceptor mutations) to determine the combined pathogenic allele frequencies for each gene and each sequence database (Table 1). This data was then used to determine the predicted HH genotype carrier rates (Table 1). The predicted prevalence of HFE-related HH closely matched previous estimates from similar populations, with the p.C282Y mutation being most prevalent. Of the non-HFE forms of iron overload, TFR2-, HFE2- and HAMP-related forms are predicted to be rare (Table 1); with a maximum prevalence for TFR2-HH of 1 in 62,500 among East Asians. Significantly, 7 SLC40A1 mutations that have been previously associated with the autosomal dominant ferroportin disease were identified in several populations, being most prevalent among the African and African-American populations. Conclusions: For the first time we have been able to provide an insight into the population prevalence of non-HFE forms of iron overload. The prevalence of TFR2-, HFE2- and HAMP-related forms of HH appear rare relative to the HFE form. Genotypes associated with ferroportin disease appear to be significantly more prevalent and may contribute to iron overload, especially in people of African origin.

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Podium #56 SUPPRESSED LIVER HEPCIDIN EXPRESSION DESPITE EXOGENOUS FERRI-TRANSFERRIN IN MICE WITH IRON DEFICIENCY ANEMIA IS ASSOCIATED WITH UPREGULATION OF MARROW ERYTHROFERRONE Yihang Li, PhD1, Princy Prasad, MS1, Ian Miller, BSc1, Yelena Ginzburg, MD2, Stefano Rivella, PhD3 and Robert Fleming, MD1 1Saint Louis University; 2New York Blood Center; 3Children's Hospital of Philadelphia

Ferri-transferrin regulates hepcidin expression directly by hepatocellular receptors as well as indirectly by erythropoietic signals. We examined the effects of ferri-transferrin on hepcidin expression in iron-deficient mice with or without anemia. Dietary iron content was manipulated to result in iron sufficiency (control), iron deficiency without anemia (ID), or iron deficiency anemia (IDA). Control mice were maintained on a 250 ppm iron diet. ID mice were generated in colonies maintained on 60 ppm Fe diet and then changed to 2 ppm Fe diet at 4 weeks until sacrifice at 6 weeks. IDA mice were born to colonies on 2 ppm Fe diet and maintained on 2 ppm Fe diet. Mice from each group were administered 10 mg recombinant human ferri-transferrin (Fe2-hTf) or vehicle intraperitoneally, and euthanized 6 hours later. Serum iron parameters and tissue iron distribution were analyzed. Liver hamp1 and bmp6, and marrow erfe mRNA levels were measured by quantitative RT-PCR. The ID and IDA mice had significantly lower serum Fe, transferrin saturation, hepatic and splenic Fe, with even lower values in the IDA mice (serum Fe: 377, 265 and 121 µg/dL; transferrin saturation: 82%, 66% and 21%; hepatic Fe: 894, 326 and 132 µg/g dry; splenic Fe: 920, 502, 244 µg/g dry, for the control, ID and IDA mice respectively; P < 0.05 for each). As expected, liver hamp1 and bmp6 mRNA expression were decreased in the ID group compared with control, and further decreased in the IDA group. IDA mice demonstrated significantly lower body weight, hematocrit, and elevated serum EPO as well as increased bone marrow erfe mRNA levels, whereas the ID mice did not differ from the control mice in these measurements levels (weight: 22g, 22g and 18g; HCT: 43%, 42% and 25%; EPO: 154, 221 and 5561 pg/ml; relative marrow erfe mRNA level: 1, 1.6, 5.9, for control, ID and IDA mice respectively;). In response to the Fe2-hTf injection, the ID mice demonstrated elevated serum Fe (265 to 656 µg/dL), transferrin saturation (66% to 89%) and liver hamp1 mRNA levels (6.8-fold), but no significant change was observed in tissue Fe, liver bmp6, or marrow erfe expression. In contrast, after the Fe2-hTf injection in the IDA mice, serum Fe, serum EPO, tissue Fe, transferrin saturation, liver hamp1 and bmp6 mRNA level did not significantly change; however, marrow erfe expression was increased even further (3.4-fold, P = 0.02). These findings demonstrate that ferri-transferrin up-regulates hepcidin in the setting of iron deficiency, but only without concomitant anemia. The observation that marrow erfe expression is further up-regulated by ferri-transferrin in the setting of iron-deficiency anemia suggests a mechanism to ensure ongoing hepcidin suppression during iron replenishment until erythroid needs have been met.

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Podium #57 ROLE OF MATRIPTASE-2 ON HEPCIDIN SUPPRESSION IN RESPONSE TO ERYTHROPOIESIS NEEDS Aude Rubio, Engineer1, Ophélie Gourbeyre, Engineer1, Herbert Lin, MD, PhD2, Hélène Coppin, PhD1, Marie-Paule Roth, MD, PhD1 and Delphine Meynard, PhD1 1Inserm U1043-CPTP-Paul Sabatier University; 2Program in Anemia Signaling Research - Program in Membrane Biology/Center for Systems Biology-Massachusetts General Hospital

Hepcidin, main regulator of iron homeostasis, is repressed by stimulated erythropoiesis in order to increase iron availability for red blood cells production. Although several candidates have been proposed, it has been recently demonstrated that erythroferrone (erfe) is the circulating erythroid factor responsible for the hepcidin suppression. Iron refractory iron deficiency anemia (IRIDA) is a hereditary recessive anemia due to a defect in the TMPRSS6 gene encoding for matriptase-2 protein. In IRIDA patients and Tmprss6-/- mice, the severe anemia is associated with activated Bmp-Smad pathway and increased hepcidin levels. However, IRIDA patients and Tmprss6-/- mice are unable to respond to erythropoietin (EPO) injection suggesting that matriptase-2 could play a role in hepcidin repression in response to erythropoiesis needs. To understand the role of matriptase-2, we first evaluated the erythroid signaling in Tmprss6-/- mice through the measurement of EPO level in the serum and erfe mRNA expression in the bone marrow and spleen. In Tmprss6-/- mice, EPO and Erfe are adequately induced in regard to their degree of anemia suggesting that the erythroid signaling is maintained in these mice. We hypothesized that lack of hepcidin inhibition in response to EPO in Tmprss6-/- mice could be explained by the high EPO and Erfe levels already present. To address this question, we resolved the anemia of Tmprss6-/- mice through iron injections, thus normalizing EPO and erfe levels, and injected these mice with EPO. As expected, EPO injection leads to an induction of Erfe mRNA expression but still did not suppress hepcidin mRNA expression. Lack of matriptase-2 induces an increase of hepcidin expression through the stimulation of the Bmp-Smad pathway. Interestingly we have observed that the Bmp-Smad pathway activation plays a key role on hepcidin suppression in response to EPO injection. Indeed, in response to EPO injection, hepcidin expression is strongly inhibited in Bmp6-/- mice, a mouse model of disrupted Bmp-Smad pathway with an iron overload, whereas it is not inhibited anymore in WT mice with a similar iron overload where the Bmp-Smad pathway is strongly activated. In Tmprss6-/- mice injected with iron, the anemia is corrected but the Bmp-Smad pathway is still activated. To determine if the blunted suppression of hepcidin expression is due to the activated Bmp-Smad pathway and not directly dependent on matriptase-2, we are currently generating Bmp6-/--Tmprss6-/- double knock-out mice. In this mouse model, the double KO mice present a similar phenotype to Bmp6-/- mice: no anemia and no activated Bmp-Smad signaling. If matriptase-2 has a role in hepcidin repression in response to EPO, double KO mice should not present a hepcidin suppression as observed in Bmp6-/- mice injected with EPO. In conclusion, we have demonstrated that Tmprss6-/- mice present an appropriate level of EPO and Erfe expression for their degree of anemia. Our data also show that the correction of the anemia did not restore the correct the erythroid suppression of hepcidin expression suggesting a role for matriptase-2 in this regulation. However, because of the influence the Bmp-Smad pathway activity on EPO response, we are currently evaluating the role of matriptase-2 in double KO mice Bmp6-/--Tmprss6-/- injected with EPO.

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Podium #58 INCREASED HEPCIDIN EPRESSION IN -THALASSEMIC MICE TREATED WITH APO-TRANSFERRIN IS ASSOCIATED WITH DECREASED ERK1/2 PATHWAY ACTIVATION AND ENHANCED P-SMAD1/5/8 NUCLEAR TRANSLOCATION Huiyong Chen1,2, Tenzin Choesang1, Petra Pham1, Weili Bao1, Maria Feola1,4, Huihui Li1,2, Mark Westerman3, Guiyuan Li2, Antonia Follenzi4, Lionel Blanc5, Stefano Rivella6, Robert.E Fleming7 and Yelena Ginzburg1 1New York Blood Center, New York, NY; 2Central South University, Changsha, PR China; 3Intrinsic Lifesciences, LLC, La Jolla, CA; 4University of Piemonte Orientale, Amedeo Avogadro, Novara, Italy; 5The Feinstein Institute for Medical Research, Manhasset, NY; 6Weill Cornell Medical College, New York, NY; 7Saint Louis University, St Louis, MO

Iron overload is the principal cause of morbidity and mortality in patients th -thalassemia. Inappropriately low hepcidin s plated the ro oerload patets th -thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin injections result in relatively iron restricted erythropoess, aelorate eete erythropoess, ad rease hepd epresso thth -thalassemia intermedia (thalassemic) mice. We now explore the effect of exogenous apo-transferrin on signaling pathways (i.e. Smad and Erk) and circulating parameters thought to participate in hepcidin regulation in vivo and ex vivo in wild type (WT) and thalassemic mice. Our results demonstrate that apo-transferrin increases both serum hepcidin concentration (603 vs. 306 ng/ml, P<0.0001) and liver mRNA expression (P=0.003) despite decreased circulating serum iron and parenchymal liver iron concentrations. Increased hepcidin in apo-transferrin treated thalassemic mice is unrelated to changes in liver Bmp6 mRNA expression (1.1-fold increase, n=8 per group, P=0.49) but correlates well with serum BMP2 concentration (1.3-fold increase, n=6-9 per group, P=0.03). Hepatocytes from thalassemic mice exhibit more pErk1/2 and a higher ratio of pErk1/2:Erk1/2 relative to WT mice, and apo-transferrin treated thalassemic mice exhibit an obvious suppression of the Erk1/2 pathway and enhanced nuclear translocation of pSmad1/5/8 (left figure). These findings suggest an inhibitory effect of the Erk pathway on hepcidin expression. We thus evaluate the effect of increasing doses of Erk inhibitor U0126 on freshly isolated mouse hepatocytes and demonstrate a dose-dependent increase in hepcidin expression (3.7-fold increase at 50µM U0126, P=0.0003), no change in cellular pSmad1/5/8, and induction in nuclear pSmad1/5/8 (right figure). In addition, combination of BMP2 and U0126 ex vivo demonstrate a synergistic effect on hepcidin expression. To further evaluate the role of circulating factors on the regulation of hepcidin expression with apo-transferrin treatment, we found a significant increase in hepcidin expression is observed in hepatocytes exposed to mouse serum relative to untreated cells, and hepatocytes treated with serum from thalassemic mice demonstrate suppressed hepcidin expression relative to treatment of WT mice serum (P=0.007). Changes in Smad phosphorylation mimick results in hepcidin expression. Furthermore, primary hepatocytes concurrently treated with serum and neutralizing anti-BMP2/4 antibodies have relatively suppressed hepcidin expression in each condition relative to cells treated with serum alone. Lastly, erythroferrone expression is increased in sorted orthochromatophilic bone marrow erythroblasts in thalassemic relative to WT mice and normalized by apo-transferrin injection in thalassemic mice. No differences are observed either in GDF11, GDF15 or TWSG1 in sorted bone marrow orthochromatophilic erythroblasts. These findings support the importance of erythroferrone as an erythroid regulator in thalassemic mice, suggest that the effect in Hbbth1/th1 mice and Hbbth3/+ mice are comparable, and provides further evidence that treatment with apo-transferrin reverses ineffective erythropoiesis in thalassemic mice. In total, our findings support a model in which treatment of Hbbth1/th1 mice with apo-transferrin decreases bone marrow erythroferrone expression, decreases hepatocellular Erk activation, and increases nuclear Smad activation to increase liver hepcidin expression.

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Podium #59 UNCOVERING THE ROLE OR HEME OXYGENASE 1 IN THE PATHOPHYSIOLOGY OF -THALASSEMIA Daniel Garcia dos Santos, PhD1, Zuzana Zidova, Msc2, Marc Mikhael, PhD3, Stefano Rivella, PhD4, Monika Harvathova, PhD5 and Prem Ponka, MD, PhD1 1Lady Davis Institute/McGill University; 2Palacky University, Czech Republic; 3Lebanese American University, Lebanon; 4Weill Cornell Medical College New York; 5University, Czech Republic

Thalassemias are a heterogeneous group of red blood cell disorders ranging from a clinically severe phenotype requiring life-saving transfusions (thalassemia major) to a relatively moderate symptomatic disorder, sometimes requiring transfusions (thalassemia intermedia). Thalassemia minor, the least severe form of the disorder, is characterized by minimal to mild symptoms. Though considered a major cause of morbidity and mortality worldwide, there is still no universally available cure for thalassemia major. The reason for this is, at least in part, due to the lack of full understanding of pathophysiology of thalassemia. The underlying basis of thalassemia pathology is the premature apoptotic destruction of erythroblasts causing ineffective erythropoeisis. Normally, the assembly of adult hemoglobin osst o a tetraer o to α- ad to -lo has eatres a ery tht oordato o α- ad -globin chain sythess oeer, -thalassea, -lo sythess s dshed as α-globin accumlato hle α- thalassemia the opposite scenario occurs. Unpaired globin chains that accumulate in thalassemic erythroblasts are bound to hee addto, -thalassea a erythrod spe protease destroys eess α-globin chains, likely leading to the generation of a pool of “free” heme in erythroblasts. “Unshielded” heme is toxic, but this toxicity will likely be augmented, if heme oxygenase 1 (HO-1) can release iron from heme. So far, virtually no information about the expression of HO-1 in erythroblasts has been produced. However, we have recently provided unequivocal evidence that this enzyme is present in several model erythroid cells1. Based on this novel and important fd, e hypothese that -thalassemic erythroblasts HO-1-mediated release of iron from heme is the aor lprt resposle or elllar daae o test ths hypothess, e eploted the ose odel o -thalassemia known as th3/th3. Our data indicates that HO- epresso s reased the ler, splee ad dey o -thalassemic mice compared to wild-type mice. Importantly, we observed that erythropoietin-mediated erythroid differentiation of fetal ler ells solated ro -thalassemic fetuses have increased levels of HO-1 at mRNA and protein levels as well as a decrease in phosphorylated eIF2-α leels errt leels ere reased -thalassemic FL cells suggesting increased heme catabolism and iron release. To investigate the contribution of HO- to the patholoy assoated th -thalassemia, wild-type and thalassemic (th3/+) mice were injected with 40 µmoles/kg/d of tin-protoporphyrin IX (SnPP, HO-1 inhibitor) during a 4-ee perod, tes a ee r reslts sho that -thalassemic mice injected with SnPP display a decrease in the spleen index, hemoglobin levels, red blood cell counts, reticulocyte counts and liver iron content when compared to eted -thalassemic mice. Additionally, HO-1 inhibition reduced ineffective erytropoess -thalassemia mice. r reslts date that -thalassemic erythroblasts have inappropriately high levels of “free” heme that is continuously degraded by HO-1. Further research is needed to determine whether iron liberated from heme by HO-1 is directly resposle or the daae o -thalassemic erythroblasts. 1Garcia-Santos D, et al. Heme oxygenase 1 is expressed in murine erythroid cells where it controls the level of regulatory heme. Blood 123 (14): 2269-77, 2014.

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Podium #60 IRON AND ERYTHROPOIESIS: A NOVEL ROLE FOR TRANSFERRIN RECEPTOR 2 IN STRESS ERYTHROPOIESIS Gautam Rishi, MSc, Daniel Wallace, PhD, Eriza Secondes, BSc Hons and Nathan Subramaniam, PhD QIMR Berghofer Medical Research Institute, Brisbane, Australia

Introduction: Iron metabolism and erythropoiesis are interlinked. A constant supply of iron is required for red blood cell production; erythropoiesis itself regulates iron metabolism by modulating the expression of hepcidin (Hamp), the liver- expressed iron-regulatory hormone. Transferrin receptor 2 (TFR2) plays an important role in the regulation of hepcidin. Mutations in TFR2 lead to the iron overload disorder Hereditary Haemochromatosis Type 3 which is associated with an inappropriate hepcidin response to body iron levels. TFR2 is expressed at high levels in the liver, with some expression observed in the bone marrow (BM). Using a hepatocyte-specific TfR2 mouse knockout model we have shown that hepatic expression of TfR2 is required to maintain systemic iron homeostasis. Recent studies published by our group and others have suggested a novel role for TfR2 in erythropoeisis. We examined the role of TfR2 in stress erythropoeisis by generating transgenic mice lacking TfR2 in haematopoietic tissue. ethods a-Cre+/- male e ere red th eale e to eerate a-Cre-/- (control) and a-Cre+/- (KO) mice. Control and KO mice were fed either a control (68 mg/Kg iron), an iron-deficient (2-10 mg/Kg iron) or an iron-rich diet (20 g/Kg carbonyl iron) for 2 weeks. Haematological parameters were measured from whole blood samples. Total serum iron levels, transferrin saturation, hepatic and splenic iron concentrations were measured. The expression of genes involved in iron metabolism and erythropoiesis was determined using qRT-PCR in the BM, liver and spleen. Flow cytometry was used to determine erythroid development in the BM and spleen. Results: Mice fed an iron-deficient diet exhibited extramedullary haematopoiesis (EMH) in the spleen. The EMH phenotype was more severe in the spleens of KO mice as indicated by increased expression of haematopoietic markers. Haematopoietic markers and Epo (erythropoietin) mRNA expression was increased in the livers of KO mice fed an iron- deficient diet. In addition, H&E staining of the liver sections of KO mice fed an iron-deficient diet showed increased number of erythroid cells, suggesting increased EMH in the liver. Flow cytometric analysis of the BM and splenocytes revealed an increase in the number of polychromatic erythroblasts in the KO mice fed an iron-deficient diet. Discussion: We demonstrate that TFR2 is essential for stress erythropoeisis caused by dietary iron deficiency. Erythropoeisis is a known negative regulator of Hamp; KO mice fed an iron-deficient diet showed increased erythropoiesis and a downregulation of liver Hamp mRNA levels as compared to control mice. The decrease in Hamp could be due to either increased Epo expression in the livers or increased erythropoietic activity in the BM, spleens and livers of KO mice as compared to the control mice fed an iron-deficient diet. We did not observe any significant changes in mRNA expression levels of Gdf15 and Fam132b in the BM of control and KO mice suggesting that these proposed erythroid regulators may not be involved in the down regulation of Hamp observed here.

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Podium #61 EXPRESSION OF ERYTHROFERRONE IN MURINE MODELS OF ANAEMIA Cornel Mirciov, Sarah Wilkins, Greg Anderson and David Frazer QIMR Berghofer Medical Research Institute, Brisbane, Australia

Iron is crucial to many biological functions, but quantitatively the most important sink for iron is for haemoglobin production in new red blood cells. The amount of iron in the plasma, and hence its availability for erythrocyte synthesis, is determined by the liver-derived iron regulatory hormone hepcidin. When erythropoiesis is stimulated, hepcidin levels decline and iron enters the plasma through the enhanced release of iron stored within macrophages and through increased intestinal iron absorption. Although the full details of how stimulated erythropoiesis suppresses hepcidin have yet to be revealed, erythroferrone has emerged as a strong candidate in this regulatory role. In this study, we used mouse models of anaemia to examine the relationship between erythropoiesis and erythroferrone. or ose odels o aaea ere stded, to odels o -thalassemia (Hbbth3/+ [n=9] and Rbc14 [n=6]), the haemoglobin deficit mouse (Hbd [n=6]) and dietary iron deficient mice (IDA [n=6]). Mice with genetic defects were studied at 3.5 (younger) and 8 weeks (older) of age to take into account the effects of iron loading. For the IDA model, wild-type mice were placed on an iron deficient diet for six weeks from two weeks of age. Anaemia status was assessed using full blood counts, and iron status was examined by the measurement of non-haem iron in the liver and serum. Liver, bone marrow and spleen were taken for RNA isolation and subsequent cDNA synthesis. Quantitative real time PCR was used to measure the expression of hepcidin in the liver and erythroferrone in the bone marrow and spleen. Haemoglobin and haematocrit levels indicated that all models were anaemic compared to their controls. Erythroferrone was increased in both thalassaemia strains, but much higher levels were observed in the more severely affected Hbbth3/+ mice. Hepcidin expression was decreased only in younger Hbbth3/+ animals. In Rbc14 mice, erythroferrone may not have reached a sufficient threshold to trigger a hepcidin reduction, while in older Hbbth3/+ mice, increased liver iron stores may have been sufficient to counteract any erythroferrone-mediated hepcidin reduction. Severely iron deficient IDA mice had blunted erythropoiesis and very low hepcidin, but surprisingly erthroferrone was increased in the bone marrow and spleen, suggesting it may respond to alterations in body iron levels, independently of erythropoiesis. Hbd mice represent another situation where there is iron restricted blunting of erythropoiesis. Despite this, erythroferrone was increased in this mouse strain, particularly in younger animals. Even though erythroferrone increased, hepcidin did not decline in Hbd mice, suggesting again that it is the balance between competing factors, in this case possibly the increases in hepatic iron and/or transferrin saturation, which influences hepcidin expression. Decreases in hepcidin expression were associated with elevated erythroferrone. Although there were changes in erythroferrone without accompanying changes in hepcidin, some instances can be accounted for by liver iron levels. Surprisingly, erythroferrone expression was decreased in the Hbd and IDA mice even though erythropoiesis is blunted under these conditions. These results suggest that erythroferrone is responsive to both the erythropoietic rate and the level of iron supply to the bone marrow. Further research is required to clarify the role of erythroferrone in hepcidin regulation.

Sixth Congress of the International BioIRon Society Page 142 Podium Abstracts IBIS

Podium #62 DIFFERENTIAL MRI RELAXATION IN ALZHEIMER’S PATIENTS WITH MUTANT HFE AND TRANSFERRIN GENOTYPES Mark Meadowcroft, PhD1, Douglas Peters, MS2, Carson Purnell, BS2, Jian-Li Wang, MD, PhD2, Paul Eslinger, PhD2, Megha Vasavada, PhD2, Qing Yang, PhD2 and James Connor, PhD3 1The Pennsylvania State University - College of Medicine; 2The Pennsylvania State University – College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania, USA.; 3The Pennsylvania State University – College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania, USA

Introduction: Iron accumulation in the brain and oxidative stress are observed in a number of neurodegenerative disorders such as Alzheimer’s disease (AD). Common mutations that lead to high iron overload has been associated with two gene variants within the HFE gene, C282Y and H63D, and within the transferrin (Tf) gene, C21. Within Alzheimer’s disease these mutations are found with increased frequency in patients (estimates are between 20-50%). These mutations result in iron dyshomeostasis, increased oxidative stress, glutamate release, tau phosphorylation, and alteration in inflammatory response2. The goal of this work was to understand how HFE and C2 mutations effect transverse relaxation in the AD brain. Methods: Thirty-eight mild Alzheimer’s disease patients (13M, 25F) were enrolled. Of these, 7 subjects (1M, 6F) were heterozygous and 1 subject homozygous (1F) for the H63D mutation, 3 subjects (2M, 1F) were heterozygous C282Y mutation, and 4 subjects were heterozygous (2M, 2F) for the TfC2 mutation. An anatomical 3DT1-weighted and a multi- echo T2-weighted spin-echo (9 echoes, 11 – 99 ms) protocol were obtained and parametric relaxation R2 rate maps generated. AD patients were stratified into two groups: those with high iron mutations (IRON +; H63D, C282Y, or C2) and those with all wild-type genes (IRON –). For parametric map analysis, the relaxation maps were normalized to the teplate ra ad oel ased aalyss as perored lster se ad p Patient age and gender were used as covariates in the analysis. Results: The group based R2 parametric analysis demonstrates that AD patients with high iron mutations (IRON+) have increased R2 rates specifically within white matter regions of interest (Fig. 1). R2 measures were not found to correlate to decreased cognitive score in the IRON+ AD patients, as IRON + and – patients did not differ in their cognitive measures. Discussion: Increased iron load has been proposed as a putative risk factor and potential causative factor in the development of Alzheimer’s disease. MRI measures have shown differences in relaxation measures between AD patients and controls. The stratification of AD patients based on IRON + genetics and determination that there are relaxation differences specifically in white matter is a highly novel finding. The cause for the white matter relaxation metrics are believed to be multi-faceted and not only related to the high iron status of AD IRON + carriers. As the relaxation rate alterations are found exclusively in white matter, we hypothesize that there are a white matter modifications in AD IRON + patients. Considering that R2 relaxation rate is a factor of iron content and tissue structure, this pattern could be indicative of white matter alterations in Alzheimer’s disease3,4. Future longitudinal analysis on the relation of IRON + genetics and AD progression is planned. References: 1 – Feder et al. Nat Genet 1996, 13, 399-408, 2 – Nandar et al., The Journal of nutrition 2011, 141, 729S- 739S, 3 – Lu. et al., JAD, 2014;39(2):261-269, 4 – Bartzokis et al., Neurobiol Aging 2009;32(8):1341-1371.

Sixth Congress of the International BioIRon Society Page 143 Podium Abstracts IBIS

Podium #63 LYSOSOMAL IRON MODULATES SYNAPTIC EXCITABILITY VIA DEXRAS1/DMT1 PATHWAY IN HIPPOCAMPUS Rachel White, PhD, Anup Bhattacharya, BS, Yong Chen, PhD, Madeleine Byrd, BS, Gregory Carlson, PhD and Sangwon Kim, PhD University of Pennsylvania

Iron metabolism in the brain is similar to that of the rest of the body in that all of the major proteins associated with regulating iron in systemic circulation are also expressed in the central nervous system (CNS). However, iron acquisition by the CNS is problematic as the CNS is functionally separate from systemic circulation by the blood-brain barrier (BBB) and the blood-cerebrospinal barrier. Due to iron's unique chemical nature and the brain heterogeneity, it has been very difficult to study iron in the CNS and most of the studies of iron functionality in the brain have centered around its pathophysiological properties and participation in various neurodegenerative diseases as a catalyst leading to the production of reactive oxygen species. Previously, we identified a novel signaling cascade in neurons whereby stimulation of glutamate-NMDA receptors activates small GTPase, Dexras1, inducing iron movement into the neurons via an iron channel, DMT1. We demonstrated that Dexras1-mediated iron influx plays a crucial role in NMDA excitotoxicity. This association between iron flux and one of the most abundant excitatory neurotransmitter also made us hypothesize that Dexras1-mediated iron influx may have a role in normal neurophysiological processes. Hence, we investigated whether disruption of NMDA-Dexras1-DMT1 cascades in neurons affects hippocampal activity and excitability. We first confirmed that a pool of chelatable iron was rapidly generated at higher concentration in hippocampus CA1 area than in the dentate gyrus or CA3, and this change is dependent on NMDA receptor. We found that rapid disruption of Dexras1 pathway via chelating iron with a membrane permeable iron specific chelator, pyridoxal isonicotinoyl hydrazone (PIH), induced an increase in the frequency of spontaneous events and produced a 47% increase in evoked synaptic excitability. Also, iron chelation-mediated synaptic changes were abolished by DMT1 channel blocker or tissues prepared from Dexras1 knockout mice. To our surprise, we further identified that iron is released via Dexras1/DMT1 pathway from lysosome. Consequently, when we disrupts lysosomal function, iron-mediated modulation of synaptic excitability is abolished. These data reveal a novel mechanism that demonstrates an active role of intracellular iron released from a lysosome in modulating synaptic excitability.

Sixth Congress of the International BioIRon Society Page 144 Podium Abstracts IBIS

Podium #64 RETINAL IRON LOADING AND ABNORMAL VISUAL FUNCTION IN A MOUSE MODEL OF HEMOCHROMATOSIS Ali Shahandeh1, Dan Johnston, PhD2, Alice Brandli, B Med Chem2 and Liz Milward, Associate Prof3 1University of Newcastle; 2The Bosch Institute and Discipline of Physiology, University of Sydney, Australia; 3School of Biomedical Sciences and Pharmacy, The University of Newcastle, Australia

Iron has been linked to various serious eye diseases including age-related macular degeneration (AMD), however little is known about how iron affects the eye. Recent research in mouse models of hemochromatosis or brain iron loading1,2 provides evidence that iron accumulation in the retina can cause tissue pathology and molecular changes however effects on vision have not been assessed. We studied retinal iron accumulation in mice with defects in the hemochromatosis Hfe and transferrin receptor 2 genes on an AKR background (Hfe-/-xTfr2mut).3 Diaminobenzidine-enhanced Perls’ stain revealed substantial iron accumulation in the retinal pigment epithelium, photoreceptors, ciliary body and other structures in Hfe-/-xTfr2mut mice compared to wild- type mice. Immunofluorescent labelling for glial fibrillary acidic protein (GFAP), a marker of retinal astrocytes (Müller cells), provided evidence for penetration of radial glial fibres into the nuclear layer of the Hfe-/-xTfr2mut mice, a well- recognized sign of retinal stress and potentially also early retinal degeneration.4

Retinal function of 9-month-old mice was assessed by electroretinography, which measures the electrical activity generated by the light-sensitive cells in the retina in response to a light stimulus. Electroretinography was performed on dark-adapted anesthetized Hfe-/-xTfr2mut and wildtype AKR mice (n=6/group) between 10 am and 3 pm to reduce circadian fluctuation. We analysed the electroretinogram a-wave and b-wave, measures of photoreceptor and bipolar cell function. After normalization, there was no significant difference in amplitude or latency of the a-wave (p>0.05) but a reduction of 41% in b-wave amplitude (p=0.03) with 19% delay in the latency (p=0.003). The b-wave is a measure of outer nuclear bipolar cell function and reflects both photoreceptor and synaptic function in the outer plexiform layer of the retina. The observed changes are consistent with abnormalities involving Müller cells and bipolar cells.

We next considered the possibility that these effects were due solely to Hfe deficiency since unusual retinopathy with retinal pigment epithelium changes and altered visual function has been reported in association with the human C282Y HFE mutation5 and retinal pathology is seen in single mutant Hfe-/- mice in the absence of known Tfr2 mutations.2 To examine whether the observed effects were due to Hfe deficiency alone or whether the Tfr2 mutation may also contribute, we investigated single mutant Tfr2mut mice on the AKR background with normal Hfe. Electroretinogram parameters showed trends towards alterations in latency and amplitude of approximately half the magnitude of effects seen in double mutant Hfe-/-xTfr2mut but failed to reach significance (p>0.05), suggesting optimal visual function may require normal functioning of both Hfe and Tfr2. The results demonstrate for the first time that abnormal retinal iron loading in association with mutations in the Hfe and Tfr2 genes can lead to impaired visual function, in conjunction with glial cell responses reflecting retinal stress and degeneration. Ongoing studies in these models and in single mutant Hfe-/- mice aim to extend understanding of the roles of iron homeostasis in retinal function and provide further insights into the mechanisms underlying observations of unusual retinopathy in human hemochromatosis. 1Hadziahmetovic et al. Invest Ophthalmol Vis Sci, 2011;52:109-118. 2Gnana-Prakasam et al. Biochem J, 2009;424:243-252. 3Delima et al. Hepatology, 2012;56:585-593. 4Eisenfeld et al. Invest Ophthalmol Vis Sci, 1984;25:1321-1328. 5Zerbib et al. Retin Cases Brief Rep, 2015;9:190-194.

Sixth Congress of the International BioIRon Society Page 145 Podium Abstracts IBIS

Podium #65 ASTROCYTE HEPCIDIN REGULATE IRON TRAFFIC ACROSS THE BRAIN ENDOTHELIUM Lin-Hao You1, Bing-Jie Zheng1, Shu-Min Wang1, Yun-Zhe Ci1, Peng Yu1, Zhen-Hua Shi1, Yu-Mei Fan1, Li-Peng Wang1, Shi-Yang Chang1, Tracey A. Rouault2, Gregory J. Anderson3, Fudi Wang4, Xiang-Lin Duan1 and Yan-Zhong Chang1 1Hebei Normal University; 2Eunice Kennedy Shriver National Institute of Child Health and Human Development; 3QIMR Berghofer Medical Research Institute; 4Zhejiang University

The regulation of iron transport into brain through the blood brain barrier (BBB) plays an important role in maintaining brain iron homeostasis. Little is known about the mechanisms and regulation by which iron crosses the basal membrane of the BBB and then enters the brain. In our study, we confirmed directly for the first time that FPN1 played an exceedingly important role in the process of iron transport into the brain by using mice with FPN1 that was preferentially inactivated in the brain microvascular endothelial cells (BMVEC). Hepcidin secreted by astrocytes directly influenced FPN1 expression in BMVEC where the astrocytes and BMVECs contacted, and then regulated the iron passage into the brain. We provided the theoretical basis for further research on the disorders of brain iron metabolism and the related diseases.

Sixth Congress of the International BioIRon Society Page 146 Podium Abstracts IBIS

Podium #66 MUTATIONS IN THE FLVCR1 GENE CAUSE HEREDITARY SENSORY AND AUTONOMIC NEUROPATHY TYPE 2 Deborah Chiabrando1, Giulio Valperga1, Fiorella Altruda1, Lorenzo Silengo1, Maja Di Rocco2, Marco Castori3, Ingo Kurth4 and Emanuela Tolosano1 1Department of Molecular Biotechnology and Health Sciences, University of Torino, Italy; 2Second Unit of Pediatrics, G. Gaslini Institute, Genova, Italy., Genova, Italy; 3Department of Medical Genetics, San Camillo-Forlanini Hospital, Sapienza University of Rome and San Camillo - Forlanin, Rome, Italy.; 4Institute of Human Genetics, Jena University Hospital, Jena, Germany

FLVCR1 (Feline Leukemia Virus subgroup C Receptor 1) is a heme exporter that controls intracellular heme levels. Two different isoforms, FLVCR1a and FLVCR1b, export heme from the plasma membrane and mitochondria respectively. FLVCR1 is ubiquitously expressed and studies using mouse models reported a critical role for FLVCR1 in erythropoiesis, endothelial cell maintenance and regulation of cytochromes activity. Mutations in the FLVCR1 gene have been associated to Posterior Column Ataxia and Retinitis Pigmentosa (PCARP), an autosomal recessive disorder characterized by severe sensory ataxia and progressive pigmentary retinopathy. Here, we report the identification of FLVCR1 mutations in some individuals affected by a distinct neurodegenerative disorder: Hereditary Sensory and Autonomic Neuropathy type 2 (HSAN2). HSAN2 is an autosomal-recessive disorder characterized by impaired nociception and autonomic dysfunction. We found three individuals affected by HSAN2 without mutations in the classical genes responsible for this disorder. Two of these individuals also showed some features of PCARP. We performed exome-sequencing and identified novel pathogenetic mutations in the FLVCR1 gene. Both homozygous and compound heterozygous mutations in the FLVCR1 gene were identified. This work is aimed at understanding the functional consequences of FLVCR1 mutations in HSAN2. To address this issue, we compared fibroblasts and immortalized lymphocytes derived from HSAN2 patients with FLVCR1 mutations (patients cells) to those derived from healthy donors (controls cells). Heme content was comparable between controls and patients cells. However, heme oxygenase 1 (HO1) was induced in patets ells opared to otrols ellsollo the stlato o the edoeos hee sythess th - aminolevulinic acid (ALA), heme overload was observed in patients cells compared to controls cells. Patients cells also showed increased oxidative stress species (ROS), deregulation of antioxidant genes and increased apoptosis compared to control cells. We propose that different kinds of FLVCR1 mutations lead to a wide spectrum of clinical outcomes: PCARP, HSAN2 or a combination of the two disorders. Our data indicate that FLVCR1 mutations impair heme export activity leading to a transient accumulation of heme that is rapidly catabolized by HO1. Moreover, mutations in the FLVCR1 gene induce oxidative stress and apoptosis in patients-derived fibroblasts and lymphocytes. The phenotype of neurons derived from patients fibroblasts is currently under investigation. The nervous systems is highly susceptible to oxidative stress injury and ROS have been reported to contribute to the pathogenesis of other forms of neuropathies. We hypothesize that heme-induced oxidative stress could lead to the degeneration of sensory neurons. Further work is needed to understand the reason why mutations in an ubiquitously expressed heme exporter specifically affect nociception in HSAN2, vision and proprioception in PCARP.

Sixth Congress of the International BioIRon Society Page 147 Poster Abstracts IBIS

Poster #1 IRON CHELATION BY BIOPOLYMERS FOR MAINTAINING COLONIC HEALTHRichard Horniblow1, Yemisi Latunde- Dada, PhD2, Tariq Iqbal, MBBChir3, Zoe Pikramenou, PhD1 and Chris Tselepis, PhD11University of Birmingham; 2Kings College London; 3Queen Elizabeth Hospital Birmingham

Background Iron is central to the aetiology of gastrointestinal disease. Specifically, the toxic effects of excess, unabsorbed ‘luminal’ iron ingested from the diet has been shown to be important in the development of inflammatory bowel disease and intestinal carcinogenesis. A platform for therapeutic intervention is likely to involve chelation of this ‘luminal’ pool of iron specifically without interfering with systemic iron. Therefore, a range of iron-chelating polymers, known as alginates, have been tested for their iron binding capacity. Alginates are natural, non-absorbable and non-fermentable bio-polymers which can therefore be employed to bind residual dietary colonic iron without becoming absorbed during gastro-intestinal transit. Materials methods The structural and physicochemical properties of alginate-iron binding were assessed using high resolution scanning transmission electron spectroscopy (STEM) and circular dichroism spectroscopy. Cell lines were stimulated with iron in the presence and absence of sodium alginates (ManDH, ManLD, LFR, RF, GHB, PROT and KEL at 0.3% w/v) and effects on cellular iron levels were assessed using the radioactive (59Fe) uptake assays and ferritin analysis. CACOII models of intestinal absorption were performed to examine the dynamics of iron trafficking through the cell monolayer when treated with alginates. Furthermore, In vivo studies were performed to 1) assess the potential of alginate solutions (8% w/v) to bind iron within the gastrointestinal tract by utilising radioactive iron and to 2) assess its potential anti-cancer activity. Results A range of alginates were utilised and their iron binding potential was assessed. The chemical interaction between alginate and iron was studied using STEM, which revealed alginate-supported iron oxide nanoparticle formation. In vitro studies confirmed that alginate mediated cellular iron chelation as exemplified by decreased cellular iron levels and ferritin expression. The effect was most profound for one alginate, namely ManLD. Ferritin expression reduced by 60% (p = 0.03) in cells challenged with ManLD and iron compared to cells challenged with iron alone. Cellular iron also decreased by 70% (p=0.001) when treated similarly. Furthermore, ManLD was shown to decrease intestinal iron absorption by 83% (p= 0.002) compared to an iron only control and was also able to suppress tumorigenesis in a murine intestinal cancer model, increasing median survival by 26 days (p = 0.007) compared to mice treated with vehicle alone. Conclusions Sodium alginate is an excellent candidate for sequestering luminal iron present in the gastrointestinal tract and in vivo shows promising anti-neoplastic activity. These results underpin the rationale in utilising these types of natural and safe bio-polymers for the prevention and treatment of gastrointestinal disease.

Sixth Congress of the International BioIRon Society Page 148 Poster Abstracts IBIS

Poster #2 NOVEL 3-HYDROXYPYRIDIN-4-ONE HEXADENTATE LIGAND-BASED POLYMERIC IRON CHELATOR: SYNTHESIS, CHARACTERIZATION AND ANTIMICROBIAL EVALUATION Tao Zhou, Yingjun Zhou, MSc, Xiaole Kong, PhD, Junpei Li, MSc, Yongmin Ma, PhD and Robert Hider, PhDZhejiang Gongshang University

Wound infections are probably the most commonly treated condition after urinary tract infections. Iron is an essential cofactor of many biochemical pathways in both prokaryotic and eukaryotic species. Hence, limiting the amount of available iron should, in principle, inhibit microbial growth. Many microorganisms have evolved strategies to scavenge and absorb iron from the environment by the production and secretion of siderophores, which possess a high affinity and selectivity for iron(III). Such uptake can be interrupted by the introduction of high affinity iron-selective chelating agents. In our previous work, hexadentate 3-hydroxypyridin-4-ones have been demonstrated to possess marked antimicrobial activity. Generally, in comparison with low molecular weight chelators, polymeric chelators with high-molecular-weights possess low toxicity due to the fact that they are nonabsorbed by the gastrointestinal tract. Most of reported iron chelating polymers are based on bidentate structures, which possess a markedly lower affinity for iron(III). As it is difficult for each bidentate ligand to form part of an ideal octahedral iron(III) coordination site, thus the complexation of three bidentate ligands with iron will not be consistently strong. In contrast, hexadentate-based polymeric chelators possess much higher iron(III) affinity, and thus are expected to have stronger antimicrobial activity. In this study, a novel 3-hydroxypyridin-4-one hexadentate monomeric chelator has been synthesized using maltol as a starting material. This monomer was incorporated into polymers by copolymerization with 2-hydroxyethyl acrylate using azobisisobutyronitrile as an initiator. The physicochemical characterization of the monomeric hexadentate ligand has been undertaken by spectrophotometric titration. The monomeric chelator was found to possess very high affinity for iron(III), with a log stability constant of iron complex (logK)=33.6 and pFe3+=30.4. To compare the iron(III) affinity of the polymeric chelator and monomeric chelator, a study associated with competition between a fluorescent chelator and monomeric chelator or polymeric chelator was performed. The pFe values of monomeric and polymeric chelators were calculated to be 29.7 and 29.8 respectively, which indicates that the hexadentate moieties on the soluble polymer possess a similar affinity for iron(III) as isolated hexadentate unit. The molecular weight of polymer was determined to be about 180 KDa by gel permeation chromatography. In vitro antimicrobial activity of the monomeric and polymeric chelators against both Gram-positive bacteria (Staphyloccocus aureus, Bacillus subtilis) and Gram-negative bacteria (Escherichia coli, Salmonella spp., Pseudomonas aeruginosa) was evaluated by inhibition zone, MIC and MBC assays. The polymeric chelator exhibited similar inhibitory activity to monomeric chelator in most cases, both exerting superior inhibitory activity on all the five tested strains, when compared to EDTA. Thus the polymeric chelators possess potential application in the treatment of wound infection.

Sixth Congress of the International BioIRon Society Page 149 Poster Abstracts IBIS

Poster #3 SELF-ASSEMBLY OF FERRITIN NANOCAGES INTO ONE- AND TWO- DIMENSIONAL ARRAYS INDUCED BY POLY αL-LYSINE) Guanghua Zhao, PhD and Rui Yang, PhDChina Agricultural University

Electrostatic interactions play an important role in hierarchical protein assemblies. However, how to control the electrostatic interactions is a challenge. The wide occurrence of protein channels in nature offers a good opportunity to control the electrostatic interaction for higher-order hierarchical assemblies. Shell-like protein, ferritin has six 4-fold channels which are distributed on the protein with an octahedral symmetry, but the size of the channels are too small to allo lare olele sh as polyα, -Lysine) with polymerization degree of 15 (PLL15) to enter into protein cage. Unlike animal ferritin, the pore size of the 4-fold channels of mature soybean seed ferritin (mSSF) appears to be larger because all E-helices of 12 H-1 subunits are deleted after assembly with H-2 analogues into a protein shell. mSSF is a heteropolymer consisting of H-1 and H-2 in a 1:1 ratio, and contains 6 expanded 4-fold channels. To enable PLL15 enter into the 4-fold channels as a linker to bridge ferritin nanocages together, first, we built an efficient method for biomimetic synthesis of mSSF (rmSSF) via genetic engineering and chemical approaches, the structure of which is characterized by its expended 4-fold channels. Second, polycationic PLL15 with the size in length as ~4-5 nm can bind with rmSSF through the 4-fold channel-directed electrostatic attraction in the absence and presence of urea. Third, we found that ferritin nanocages can self-assemble into 1D array of linear chains without urea intervention and into 2D square arrays in the presee o rea ded y polyα, -lysine) at pH 7.0 as suggested by TEM, dynamic light scattering, and fluorescence titration analyses. In contrast, PLL analogues with less or larger size in length hardly facilitated rmSSF self-assembly into the above 1D and 2D arrays. Thus, the length of PLL is pre-requisite for these self-assembly. More importantly, the size of these arrays is able to be controlled by tuning the PLL15/protein ratio or reaction time. Taken together, the ferritin channels can be used to manipulate electrostatic interactions that direct protein self-assembly into higher ordered arrays. These findings provide guidelines for fabricating higher ordered assembly of a number of known shell-like proteins that own protein channels.

Sixth Congress of the International BioIRon Society Page 150 Poster Abstracts IBIS

Poster #4 SILVER NANOPARTICLES INDUCED RNA POLYMERASE-SILVER BINDING AND RNA TRANSCRIPTION INHIBITION IN ERYTHROID PROGENITOR CELLS Sijin LiuChinese Academy of Sciences

Due to its antimicrobial activity, nanosilver (nAg) has become the most widely used nanomaterial. Thus far, the mechanisms responsible for nAg-induced antimicrobial properties and nAg-mediated toxicity to organisms have not been clearly recognized. Silver (Ag) ions certainly play a crucial role, and the form of nanoparticles can change the dissolution rate, bioavailability, biodistribution, and cellular uptake of Ag. However, whether nAg exerts direct “particle-specific” effects has been under debate. Here, we demonstrated that nAg exhibited a robust inhibition on RNA polymerase activity and overall RNA transcription through direct Ag binding to RNA polymerase, which is separated from the cytotoxicity pathway induced by Ag ions. nAg treatment in vitro resulted in reduced hemoglobin concentration in erythroid cells; in vivo administration of nAg in mice caused profound reduction of hemoglobin content in embryonic erythrocytes, associated with anemia in the embryos. Embryonic anemia and general proliferation deficit due to the significant inhibition on RNA synthesis, at least partially, accounted for embryonic developmental retardation upon nAg administration. To date, there is no conclusive answer to the sources of nAg-mediated toxicity: Ag ions or “particle-specific” effects, or both. We here demonstrated that both Ag ions and nAg particles simultaneously existed inside cells, demonstrating the “Trojan horse” effects of nAg particles in posing biological impacts on erythroid cells. Moreover, our results suggested that “particle- specific” effects could be the predominant mediator in eliciting biological influences on erythroid cells under relatively low concentrations of nAg exposure. The combined data highlighted the inhibitory effect of nAg on RNA polymerase activity through a direct reciprocal interaction.

Sixth Congress of the International BioIRon Society Page 151 Poster Abstracts IBIS

Poster #5 EFFECTS OF PARENTERAL IRON LOADING AND/OR IRON CHELATION ON THE EXPRESSION OF DUODENAL IRON TRANSPORT MACHINERIES IN THALASSEMIA MICE Patarabutr Masaratana, MD, PhD1, Chanita Sanyear, MSc2, Wiraya Eamsaard, BSc3, Saovaros Svasti, PhD4 and Suthat Fucharoen, MD, PhD41Department of Biochemistry, Faculty of Medicine Siriraj Hospital; 2Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Thailand; 3Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand; 4Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Thailand

Background: Thalassemia, one of the global health problems, is caused by mutations of genes encoding adult globin has reslt reded lo sythess ad alae etee α ad o-α lo has erythrod ells e o the major life-threatening complications of thalassemia is iron overload caused by increased iron absorption and/or blood transfusion. Iron chelators have therefore been used to reduce tissue iron deposition. In thalassemia, the expression of iron regulatory peptide, hepcidin, is affected by several regulators including iron loading and ineffective erythropoiesis. However, the expression of iron transport machineries in the duodenum is not only regulated by systemic signal through hepcidin but also by cellular iron status. It is noteworthy that iron status of enterocytes as well as the response of duodenal iron transport machineries to iron chelation and iron loading have not been fully elucidated. Materials and methods: Male wild type C57BL/6J and th3/+ (BKO; thalassemia mouse model) mice aged 7 weeks old were given intramuscular injection of 5 mg iron dextran for 2 consecutive days. After 2-week equilibration, the mice were administered deferoxamine (DFO) intraperitoneally or deferiprone (DFP; L1) orally for 14 days. Saline injection was used as control. The mice were sacrificed 24 hours after the last dose of the iron chelator. Liver non-heme iron levels were spectrophotometrically measured by ferrozine-based assay. The expression of hepcidin and iron transport machineries was studied by real-time PCR. Results and discussion: Tissue non-heme iron measurement and Prussian blue staining suggested that iron dextran injection resulted in dramatic iron accumulation in the liver of both wild type and BKO mice. However, iron chelator administration had no effect on liver non-heme iron levels. Complete blood count revealed that none of erythrocyte parameters in BKO mice was affected by iron dextran or iron chelator administration. Notably, liver hepcidin mRNA expression was relatively higher in BKO compared to wild type mice. In wild type mice, liver hepcidin expression was increased by iron dextran administration whereas a trend toward increased expression was noted in BKO mice. In both phenotypes, hepcidin mRNA expression was not statistically affected by iron chelator treatment. The expression of duodenal iron transport machineries along with their response to iron loading and/or iron chelation will be presented.

Sixth Congress of the International BioIRon Society Page 152 Poster Abstracts IBIS

Poster #6 IRON BIOAVAILABILITY OF SWEET POTATO AND MORINGA LEAVES IN COMPARISION WITH LEAFY GREEN VEGETABLES COMMONLY CONSUMED IN GHANA Francis Amagloh, PhD2, Richard McBride, BSc1 and Tatiana Christides11University of Greenwich, Faculty of Engineering and Science, United Kingdom; 2University for Development Studies, Ghana

Introduction: Iron deficiency anaemia (IDA) is a significant public health problem in Northern Ghana especially amongst women and children. Leafy green vegetables are major contributors to iron intake in this part of the world; poor iron bioavailability from these food sources may be part of the reason for the high prevalence of IDA. Evidence suggests that sweet potato and Moringa leaves might be better sources of bioavailable iron, compared with other leafy green vegetables, as both have high levels of iron, and also beta-carotene - a dietary factor that has been suggested to improve iron bioavailability. Aims/Hypothesis: Our research aims were to evaluate iron bioavailability of sweet potato and Moringa leaves in comparison with other leafy green vegetables commonly consumed in Ghana. We hypothesized that iron uptake from sweet potato and Moringa leaves would be higher compared with the other tested vegetables. Methods: We used the Caco-2 cell/in vitro digestion system; Caco-2 cell ferritin formation was used as a surrogate marker of iron bioavailability. In addition, we also measured levels of other nutrients and dietary factors known to affect iron bioavailability: beta-carotene, iron, calcium, zinc, ascorbate, phytates and polyphenols. Results: Iron bioavailability from all tested vegetables was poor despite relatively high absolute levels of iron in the leaf samples (14.5 - 24.6 mg/100 grams dry weight); there was no statistically significant difference in iron uptake between any of the tested varieties or the control sample with no added iron. Levels of phytates and polyphenols, known inhibitors of iron uptake, were high and probably accounted for the low iron bioavailability of tested leaves. As expected, beta-carotene levels were highest in the sweet potato and Moringa leaves (ranging from 47-98 micrograms retinol activity equivalent)/gram freeze dried leaf) - approximately 100% more compared with the other leafy green vegetables, with the exception of the purple leafed sweet potato variety tested that had approximately the same amount of beta-carotene as the commonly consumed vegetables. Conclusion: In our in vitro model neither sweet potato nor Moringa leaves demonstrated good iron bioavailability suggesting that increased consumption of these vegetables would not lead to improved iron status. However, both leaves were good sources of beta-carotene, and further testing in vivo to evaluate whether they could impact on vitamin A status may be warranted.

Sixth Congress of the International BioIRon Society Page 153 Poster Abstracts IBIS

Poster #7 MACROPHAGE IRON METABOLISM PROFILE IN PRO-ATHEROGENIC CONDITIONS François Canonne-Hergaux, PhD1,3,4, Liliana Marques, Anne Negre-Salvayre and Luciana Costa21INSERM UMR 1043; 2Departamento da Promoção da Saúde e Prevenção de Doenças Não Transmissíveis, Instituto Nacional de Saúde Doutor Ricardo Jorge, Lisboa, Portugal; 3CNRS UMR 5282; 4Université de Toulouse, UPS, Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse, France.

Introduction: Atherosclerosis is currently considered as a chronic inflammatory disease, in which the subendothelial accumulation of macrophages and their differentiation in lipid-laden foam cells through the uptake of oxidized LDL (oxLDL) constitute key events in atherogenesis. In such process, iron could constitute a modifiable factor and characterization of the iron metabolism profile of the different macrophages phenotypes present in atherosclerotic lesions may provide insight on their potential protective or pro-atherogenic role. A new macrophage phenotype (Mox) driven by exposure to oxidized phospholipids was recently described in murine models of atherosclerosis. Herein, we investigated the effect of oxLDL as well as inflammatory stimuli on macrophage polarization and iron metabolism profile. Methods: Murine bone marrow derived macrophages (BMDM) were treated with native LDL, acetylated LDL, oxLDL (Mox atator, ro ador ativator). Expression of ferroportin-1 (Fpn1, iron exporter), heme oxygenase-1 (HO1, Mox marker), ferritin (Ft, iron storage protein), hepcidin (Hepc, iron regulator), ceruloplasmin (Cp, oxidase) and interleukine-6 (IL6, inflammation and M1 marker) were followed by quantitative PCR. Protein expression of Fpn1 and HO1 was analyzed by immunofluorescence and In-cell Western Blotting. To study the implication of Nrf2 (the master regulator of antioxidant response) in our cellular model, experiments were further conducted with BMDM prepared from Nrf2 null mice. Results and discussion: oxLDL treated macrophages (Mox) presented increased HO1 and Ft expression with basal Fpn1 protein level at cell surface despite the significant increase of Fpn1 mRNA. Upregulation of HO1 and Fpn1 mRNA was specific to LDL oxidative modification and was mediated by Nrf2 in murine macrophages. Interestingly, the downregulation of both Cp isoforms and the upregulation of Hepc expression observed in Mox macrophages suggest that Fpn1 stabilization at the cell surface could be compromised as well as its iron transport activity. Simultaneous exposure to o ad dated that o polarato s ot doat oer , reslt a ed o pheotype closer to M1 than Mox showing increased expression of IL6, HO1 and Ft combined with Fpn1 downregulation. The soluble and secreted Cp isoform (sCp) and not the glycosylphosphatidylinositol-membrane anchored Cp (GPI-Cp) was upregulated by pro-inflammatory conditions either combined or not with oxLDL which suggest a major role for sCp in inflammation. Cp has the capacity to oxidize LDL and a possible consequence of the significant sCp upregulation in M1 and Mox/M1 macrophages could be increased local LDL oxidation. Our observations suggest potential mechanisms for macrophage iron retention, LDL oxidation and production of pro-inflammatory cytokines in atherosclerotic lesions, which are in agreement with the iron hypothesis proposed by Sullivan that macrophage iron accumulation could contribute to plaque destabilization and disease progression. Conclusion: Altogether, our results suggest that a microenvironment rich in oxLDL and pro-inflammatory cytokines likely promotes macrophage iron retention and lipid accumulation while macrophage secretion of pro-inflammatory cytokines and sCp could further enhance local inflammation and LDL oxidation.

Sixth Congress of the International BioIRon Society Page 154 Poster Abstracts IBIS

Poster #8 GLP-1 AND FE CHELATION INDEPENDENTLY RESCUE NAF-1 DEFICIENT INSULINOMA B CELLS (WOLFRAM SYNDROME 2) BY REDUCING MITOCHONDRIAL LABILE IRON AND ROS FORMATION Rachel Nechushtai, DSc1, Erol Cerasi, MD2, Gil Leibowitz, MD2 and Ioav Cabantchik, MD, PhD11Institute of Life Sciences, Hebrew University of Jerusalem; 2Hadassah Medical Center, Hebrew University of Jerusalem

Wolfram syndrome 2 (WFS-2) is a neurodegenerative disorder that is also characterized by diabetes associated with insulin deficiency due to ß-cell dysfunction. WFS-2 is caused by a deficiency in NAF-1, a 2Fe-2S protein that resides both in the ER and mitochondrial outer membranes (1). We have recently shown that repressed expression of the NAF-1 protein causes cell structure-function abnormalities, comprising maldistribution of iron (depleted in cytosol and accumulating in mitochondria), leading to oxidative damage with ensuing autophagy and apoptosis (1). As these abnormalities are partly reversed by chelation, we hypothesized that iron may have a pathophysiological role in WFS-2 and hence constitute a potential pharmacological target. The present study was prompted by a dramatic response observed in a WFS2 patient treated with the GLP-1 receptor agonist exendin-4 (Exenatide). The treatment resulted in a 70% reduction in insulin requirement so that after 2-month Exanitide the insulin secretion stimulated with IV glucose along with glucagon and arginine was raised by 7-fold (!). This led us to explore whether the amelioration of ß-cell properties in NAF-1 deficient pancreatic cells attained by exendin-4 treatment is also associated with changes in cell iron parameters affected by NAF-1 deficiency and amenable to correction by iron chelation. Results. WFS-2 cell models were obtained by knocking down NAF-1 expression in rat insulinoma ß-cells (INS-1E) using inhibitory RNAs (si oligos or sh transfection). The NAF-1 knocked-downed (kd) cells showed: a. increased levels of mitochondrial labile iron (detected by RPA fluorescence), b. a rise in reactive oxygen species formation (DHE fluorescence), c. mitochondrial membrane depolarization (TMRE fluorescence) and d. decreased maximal O2 consumption (Seahorse). The mitochondrial properties affected by NAF-1 repression of expression were largely reversed by pre-treatment of cells with the chelator deferiprone (DFP 50 ùM o/n) or with 100 nM exendin-4 for 48 hrs. Exendin-4 also reduced the susceptibility of NAF-1- deficient cells to apoptosis when triggered by the chemical ER stressor thapsigargin. Discussion. The fact that GLP-1-based treatment, known to amplify insulin secretion and ameliorate ß-cell survival under stress conditions, can reverse ß-cell dysfunctions elicited by NAF-1 deficiency, and that moderate iron chelators can mimic these protective effects, raises important questions of both biochemical and clinical relevance: 1. By which mechanisms does NAF-1 repression compromise ß-cell survival? 2. How do iron chelators and GLP-1 stimulators reverse these mechanisms? 3. Which factors modulated by GLP-1 are regulated by NAF-1, such that in their absence (as in WFS- 2) they enhance oxidative stress in an iron-dependent manner? These and other questions, explored by work in progress, may pave the road for gaining insights about the pathophysiogical role of NAF-1 in WFS-2 diabetes, and specifically about the mechanisms by which GLP-1 corrects the iron dysfunctions in ß-cells in WFS-2 and possibly also in multiple iron overload disorders. (1) Tamir et al (2015) Structure–function of NEET proteins uncovers their role as key regulators of iron and ROS in health and disease, BBA Rev. Biomembr. 1853:1294–315

Sixth Congress of the International BioIRon Society Page 155 Poster Abstracts IBIS

Poster #9 IV IRON SUPPLEMENTATION ASSOCIATED WITH RETINAL DEGENERATION IN MICE AND MAN Joshua Dunaief, MD,PhD1, Delu Song, MD, PhD1, Yafeng Li, BA1, Ying Song, MD1, Liangliang Zhao, MD2, Chenguang Wang, MD, PhD2 and Levi Kanu, BA11University of Pennsylvania; 2Second Hospital of Jilin University

Introductio ro dysrelato otrtes to retal deeerato seeral dseases, ld aerelated alar degeneration (AMD), aceruloplasminemia, Friedreich's Ataxia, and Pantothenate Kinase Associated Neurodegeneration. This is not surprising, as the retina has high metabolic activity, oxygen concentration, and easily oxidized polyunsaturated fatty acid. A recent clinical case suggested that IV iron administration may cause increased retinal iron and retinal degeneration. Methods: Early onset AMD was detected in a patient 11mo after IV Venofer treatment (300mg X3 weekly doses). Retinal imaging was performed, including retinal photography, optical coherence tomography and autofluorescence. Mice were given IV Venofer (1.2mg X 12 weekly doses) through the tail vein, then aged to 9 months. Retinal iron accumulation was measured directly by VIP-enhanced Perls’ stain and indirectly using immunolabeling for ferritin and quantitative PCR for transferrin receptor and ferritin. Results: The 43 year old patient with macular degeneration was found 11 months after Venofer infusion to have many drusen, the extracellular subretinal deposits that define AMD. Drusen were not seen in exams over the previous five years by the same ophthalmologist. She had been given Venofer to treat iron deficiency anemia. She is a vegetarian with heavy menses and an otherwise unremarkable medical history. She had a maternal uncle who developed AMD in his 70s. In the mice, retinal photos showed white dots resembling drusen and histology showed extracellular subretinal deposits. Perls’ stain showed iron accumulation in the retinal pigment epithelium and diminished transferrin receptor mRNA levels consistent with the increased iron. Discussion: These results suggest that IV Venofer may increase iron levels in the retinal pigment epithelium and increase the risk of AMD in certain genetically susceptible individuals. The patient described herein is in the highest risk category for AMD based on genotyping of complement alleles. It is possible that she would have developed macular degeneration by this age, even without the IV Venofer. However, causality is suggested by the exceptionally early age of onset and the temporal sequence of Venofer treatment 11mo prior to diagnosis. Mouse data support this hypothesis, although the dose used in mice was about 25X higher than the patient’s dose on a mg/kg basis. Conclusions: The case presented herein, along with the mouse data, suggest that further studies should be conducted on the risk of exacerbating retinal degeneration with iron supplementation.

Sixth Congress of the International BioIRon Society Page 156 Poster Abstracts IBIS

Poster #10 ATRANSFERRINEMIA: AN ULTRA-RARE IRON-LOADING ANAEMIA. REPORT OF 6 CASES FROM 4 FAMILIES Anna Barqué2, Francisco Fuster2, Cristina Díaz de Heredia, PhD, MD3, Eunice S. Edison, PhD, MD4, Katja Moser, MD5, Erica Morán, PhD2, Rekha Athiyarath4, Jessica Aranda2, Ana M. Rojas, PhD6, Ilona Kleine, PhD7 and Mayka Sanchez, PhD1

1Josep Carreras Leukaemia Research Institute (IJC), Diagnostics in Iron Metabolism Disease (D IRON) and Iron Metabolism: Regulation and Diseases Group. and Institute of Predictive and Personalized Medicine of Cancer (IMPPC). Badalona, Barcelona.; 2Josep Carreras Leukaemia Research Institute (IJC), Diagnostics in Iron Metabolism Disease (D IRON). Badalona, Barcelona and Institute of Predictive and Personalized Medicine of Cancer (IMPPC). Badalona, Barcelona.; 3Vall d'Hebron University Hospital, Pediatric Haematology-Oncology Service. Barcelona; 4Christian Medical College Hospital, Haematology Department. Vellore, India; 5Klinik für Kinder- und Jugendmedizin Hospital, Pediatrics and Neonatology Department. Aschaffenburg, Germany; 6Campus University Hospital Virgen del Rocío. Institute of Biomedicine of Seville. Sevilla, Spain; 7Sanquin Blood Supply Foundation, Medical Department Plasma Products. Amsterdam, the Netherlands Transferrin (TF) is a glycoprotein that facilitates the transport of ferric iron in blood. It is the ligand for transferrin receptor1 (TFR1), which enables cellular iron uptake in various tissues. Transferrin synthesis is increased in conditions of iron deficiency anaemia. Atransferrinemia or hypotransferrinemia (OMIM#209300, OPRHA1195) is an ultra-rare autosomal recessive disease characterized by a severe deficiency of serum transferrin which causes inefficient erythropoiesis, severe hypochromic microcytic anemia and iron overload in different non-hematopoietic tissues (liver and heart). In the literature there are described only 14 families with 16 cases, 8 of which have been characterized at the molecular level. Our Diagnostics unit in Iron Metabolism Disease (D·IRON) is involved in the clinical, genetic, molecular and computational characterization of 6 new worldwide cases of atransferrinemia: 2 Spanish, 2 Turkish and 2 Indian. The existence of these cases underlines the fact that atransferrinemia should be considered, even if it is an ultra-rare disease, as the cause of a previously unexplainable hypochromic microcytic anaemia with iron overload. An appropriate diagnosis would lead to the correct implementation of an adequate treatment to minimize the severe health complications related to iron overload. Therefore, it is critical to distinguish atransferrinemia from other hereditary anaemias (thalassemia, non-sideroblastic anaemia), acquired anaemias (celiac disease, myelodisplastic syndrome) or nutritional iron deficiency anaemia to improve patients’ quality of life. Funding: Work supported by grant SAF2012-40106 from Spanish Secretary of Research, Development and Innovation (MINECO) and grant CIVP16A1857 “Ayudas a proyectos de Investigación en Ciéncias de la Vida - Fundación Ramón Areces” , 2014 SGR225 (GRE) Generalitat de Catalunya and economical support from Fundació Internacional Josep Carreras i de la Obra Social “la Caixa” Spain to M.S. M.S. held a research contract under the Ramón y Cajal program from the Spanish Ministry of Science and Innovation (RYC-2008-02352).

Sixth Congress of the International BioIRon Society Page 157 Poster Abstracts IBIS

Poster #11 CROSSTALK BETWEEN OBESITY AND NEURODEGENERATION; ADIPONECTIN-MEDIATED MODULATION OF IRON FLUX VIA DEXRAS1 IN THE BRAIN Yong Chen, PhD, Lauren Mathias, BS, Rexford Ahima, PhD and Sangwon Kim, PhDUniversity of Pennsylvania

Obesity and metabolic syndrome are associated with changes in adipokines, insulin resistance and inflammatory cytokines which have deleterious effects on brain structure and function. Adiponectin is secreted by adipocytes and has major insulin-sensitizing, anti-inflammatory and anti-oxidant properties. Circulating adiponectin levels are decreased in obesity and type 2 diabetes. In addition to targeting neurons in the hypothalamus and brainstem, studies including ours have also shown that adiponectin has neuroprotective effects in various types of brain injury. Dexras1 is a small GTPase, highly expressed in the neurons and plays a central role in neuronal iron trafficking. We have shown previously that stimulation of glutamate-NMDA receptors activates neuronal nitric oxide synthase (nNOS), leading to S-nitrosylation of Dexras1 and a physiological increase in iron uptake through DMT1. Here we report that adiponectin suppresses Dexras1 GTPase activity via PKA dependent manner. Our in vitro phosphorylation studies utilizing various recombinant kinases revealed that PKA is the only kinase which regulates iron flux via Dexras1. Moreover, our mutagenesis analysis showed that Dexras1 is phosphorylated at a single residue, Serine253. We further confirmed that phosphodead form (serine 253 to alanine) displayed a dramatic increase in iron uptake while phosphomimetic form (serine 253 to glutamic acid) decreases it. Interestingly, phosphomimetic form of Dexras1 did not yield S-nitrosylation of this protein nor an increase in iron influx upon NO treatment. Moreover, activation of PKA pathway reduces S-nitrosylation of Dexras1 as well as iron uptake. Hence these findings suggest that a potential mechanism by which adiponectin protects neurons from iron- catalyzed neurotoxicity as well as a functional cross talk between S-nitrosylation and phosphorylation. Moreover, our studies will provide novel therapeutic strategies to neurodegenerative disorders associated with metabolic syndrome and other diseases.

Sixth Congress of the International BioIRon Society Page 158 Poster Abstracts IBIS

Poster #12 EFFECTS OF ACUTE EXERCISE ON IRON METABOLISM IN RATS Yu Wang2, Ning Zheng3, Jia Chen3, Peng Yu, PhD4, Yanzhong Chang, PhD4 and Yuqian Liu, PhD11College of Physical Education, Hebei Normal University; 2College of Physical Education, Hebei Normal University; 3College of Physical Education, Hebei Normal University. Laboratory of Molecular Iron Metabolism, College of Life science, Hebei Normal University,Shijiazhuang,050024; 4Laboratory of Molecular Iron Metabolism, College of Life science, Hebei Normal University,Shijiazhuang,050024

Iron plays an important role in athletes’ aerobic ability. Some athletes has Iron deficiency anemia (IDA) after longtime strenuously exercise, which is harmful to health and performance. To reveal effects of acute exercise on iron metabolism and provide some methods for preventing exercise induced anemia, 30 male SD rats (8-week old, 220g±10g) were randomly divided into two groups, the control group (CG) and the exercise group (subjected to exercise on a treadmill for 2 consecutive days, running at the speed of 30m/min until exhaustion). Rats were sacrificed immediately (E0h), 3h (E3h), 6h (E6h), 24h (E24h) after exercise. The hepatic hepcidin andPGC-αs ere eaed y -PCR. The expression of hepatic ferroportin l (FPN1) and iron absorption proteins in duodenum were examined by Western blot. The results were as follows,1) Compared with CG, the content of serum iron in E0h and E3h were significantly decreased (P<0.01) and increased sharply in E6h then decreased at E24h(P<0.01), while the serum iron in E24h was increased than that of E0h and E3h(P<0.01). These results suggested that serum iron levels was reduced with the consumption of acute exhaustive exercise. The significantly increased iron in E6h might because of the hemolysis caused by strenuously running. 2) The expression of hepatic PGC-α reased ater ate eerse t ea to rede ater h he t reached the highest level. The content of IL-6 in serum was increased in E3h (P<0.01), and decreased in E24h. IL-6 and PGC-α are de o laato ad they de the reased hepd he laato has dya haes ater acute exercise, the peak inflammation was E3h to E6h. 3) Compared with CG, the expression of hepatic hepcidin mRNA in E3h and E6h were significantly increased (P<0.05), while hepcidin mRNA was decreased in E24h (P<0.01). The previous research showed FPN1 was negatively regulated by hepatic hepcidin. The decreased hepcidin resulted in the high level of FPN1, as a result, more iron was released into the circulation from iron absorption and iron storage. The iron content of liver and spleen of E24h were significantly decreased. 4) What was the mechanism which resumed the serum iron in E24h? The Western blot results showed the iron absorption proteins in duodenum including divalent metal transporter 1 (DMT1-IRE) and FPN1 were increased in E24h compared with those in E3h and E6h (P<0.01). The increased DMT 1 imported more iron into the absorption cells, and the FPN1 exported the iron into circulation. The expression of hepatic FPN1 in E24h was significantly higher than that of E3h and E6h(P<0.01). Liver and spleen are the main iron storage, and iron is exported from these tissues into plasma through FPN1, the sole known cellular iron exporter. The results indicated that iron absorption and iron release were both increased to meet the consumption of iron during recovery. The inflammation was the main factor influencing the iron metabolism. We concluded that inflammation- Hepcidin-FPN1 pathway might play an important role in the regulation of iron metabolism during acute exercise in rats.

Sixth Congress of the International BioIRon Society Page 159 Poster Abstracts IBIS

Poster #13 INTERLEUKIN 6 REGULATES IRON RELATED PROTEINS THROUGH C-JUN N-TERMINAL KINASE ACTIVATION IN BV2 MICROGLIAL CELL LINES Jun Wang, PhD, MDDepartment of Physiology, Shandong Provincial Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology

Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by the loss of dopaminergic (DA) neurons in the substantia nigra (SN) and the subsequent DA depletion in the striatum. Extensive studies have demonstrated that microglia activation and nigral iron accumulation play a key role in the pathogenesis of PD. The activated microglia showed amplified levels of iron deposit, however, the relationship between microglia activation and iron accumulation was not fully elucidated. In the present study, we aimed to investigate how the iron level affects interleukin-6 (IL-6) synthesis in BV2 microglia, as well as the effect of IL-6 on cellular iron metabolism in BV2 microglia. The results were as follows: IL-6 mRNA was up-regulated after FAC treatment for 12 h in BV2 cells. Iron regulatory protein 1 (IRP1) was up-regulated and iron exporter ferroportin1 (FPN1) was down-regulated after IL-6 was treated for 24 h in BV2 cells. Phosphorylated c-Jun N-terminal kinase (JNK) increased significantly after IL-6 was treated in BV2 cells for 1 h compared with the control. Pretreatment with JNK inhibitor SP600125 attenuated the up-regulation of IRP1 and downregulation of FPN1 compared with IL-6 treated group in BV2 cells. The results suggest that iron load can increase IL-6 mRNA expression in BV2 cells. IL-6 up-regulates IRP1 expression and down-regulates FPN1 expression in BV2 microglial cells through JNK signaling pathways. Key words: microgliainterleukin 6iron regulatory protein 1ferroportin 1JNK

Sixth Congress of the International BioIRon Society Page 160 Poster Abstracts IBIS

Poster #14 ERR GAMMA CONTROLS SALMONELLA INFECTION BY MODULATING HOST IRON HOMEOSTASIS Don-Kyu Kim, PhD1, Jae-Ho Jeong, PhD2, Ki-Sun Kim, MS3, Yoon Seok Jung, BS3, Hyon Choy, PhD2 and Hueng-Sik Choi, PhD31Chonnam University/Department of Biotechnology; 2Chonnam National University Medical School/Department of Microbiology; 3Chonnam University/National Creative Research Initiatives Center for Nuclear Receptor Signals and Hormone Research Center, School of Biological Sciences and Technology

Hepcidin is a major regulator of iron metabolism and intracellular bacterial pathogens depend on iron acquisition for their survival within host cells. Here, we report that nuclear receptor ERRgamma is a novel transcriptional mediator of Salmonella-mediated regulation of hepcidin and iron metabolism. Hepatic ERRgamma gene expression was induced by Salmonella-stimulated interleuklin-6 (IL-6) signaling, and led to induction of hepcidin and hypoferremia in mice. Conversely, liver-specific ablation of ERRgamma gene expression blocked Salmonella-mediated induction of hepcidin, and normalized the hypoferremia by Salmonella infection. An inverse agonist of ERRgamma ameliorated Salmonella- mediated hypoferremia through reduction of ERRgamma-mediated hepcidin gene expression, and performed a potent antimicrobial function for the intracellular growth of Salmonella. Control of iron metabolism by an ERRgamma-specific inverse agonist could be a novel therapeutic approach for protection against intracellular bacteria.

Sixth Congress of the International BioIRon Society Page 161 Poster Abstracts IBIS

Poster #15THE EFFECT OF ESTROGEN ON IRON METABOLSIM IN ASTROCYTES AND NEURONS Manman Xu, Bachelor Department of Physiology, Shandong Provincial Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology, Medical College of Qingdao University

Precious studies have demonstrated that estrogen affects iron metabolism in peripheral tissues. Then what is the role of estrogen on the brain iron metabolism? In this study, we investigated the effect of estrogen on the iron transport proteins as well as its mechanisms. The results were as follows: Iron exporter ferroportin1 (FPN1) and iron importer divalent metal trasporter as prelated ater estroe as treated or h prary ltred astroytes ro relatory prote ad hypoa dle atoralpha α as pregulated, but hypoxia inducible factor 2 alpha α reaed haed ater estroe as treated or h prary ltred astroytes eros, as prelated hle reaed haed ater estroe as treated or h prary ltured neurons. IRP1 was dorelated hle α ad α reaed haed after estrogen was treated in primary cultured neurons. he reslts sest that estroe a rease ad epressos y eleat α ad epressos in astrocytes. However, the decreased expression of IRP1 may account for the increased FPN1 in neurons.

Key words: Astrocyte, Neuron, estrogen, Iron

Sixth Congress of the International BioIRon Society Page 162 Poster Abstracts IBIS

Poster #16 LOW DIETARY IRON INTAKE RESTRAINS THE INTESTINAL INFLAMMATORY RESPONSE AND PATHOLOGY OF ENTERIC INFECTION BY FOOD-BORNE BACTERIAL PATHOGENS Guus Kortman, PhD1, Michelle Mulder1, Thijs Richters1, Nanda Shanmugam, PhD2, Estela Trebicka, BSc2, Jos Boekhorst, PhD3, Harro Timmerman, PhD3, Rian Roelofs, BSc1, Erwin Wiegerinck, BSc1, Coby Laarakkers, BSc1, Dorine Swinkels1, Albert Bolhuis, PhD4, Bobby Cherayil, PhD2 and Harold Tjalsma, PhD11Radboud University Medical Center; 2Massachusetts General Hospital; 3NIZO Food Research BV; 4University of Bath

Introduction and objectives Orally administrated iron is suspected to increase susceptibility to enteric infections among children in infection endemic regions. We investigated the effect of dietary iron depletion and supplementation on the pathology and local immune responses in intestinal infection models. Materials and Methods 6 Weeks old wild-type C57BL/6 mice were put on an iron-restricted (2-6 mg Fe/Kg), normal-iron diet (45 mg Fe/Kg), or high-iron diet (225 mg Fe/Kg) (n=5 per group). After 2 weeks the mice were orally challenged with Citrobacter rodentium. After another 2 weeks the gut microbiome was determined and inflammatory markers were assessed in plasma, tissue and faeces. Survival of Caenorhabditis elegans infected by Salmonella Typhimurium, pre-incubated with increasing iron concentrations, was tested (a simple gut infection model). Results Dietary intervention significantly altered tissue iron stores and microbiome analysis revealed profound iron- and infection- induced shifts. After iron deprivation Parabacteroides became dominant, while animals on the medium/high-iron containing diets had an Allobaculum dominated microbiota. Remarkably, Enterorhabdus abundance correlated with fecal lipocalin-2 and calprotectin. Levels of these innate defensive molecules and markers of inflammation were lower in iron- deprived mice. These mice also tended to have a lower grade of colon pathology and to gain more weight. Surprisingly, similar to animals on the iron-deficient diet, mice on the high-iron diet also showed decreased intestinal immune responses compared to the normal-iron diet. Notably, iron-deprivation was associated with prolonged survival of the nematode Caenorhabditis elegans after infection with S. Typhimurium. From this data it follows that iron increases the pathogenicity of this pathogen. Conclusion Together, these data show that iron limitation restricts disease pathology upon bacterial infection in two different animal models. Our data also showed decreased intestinal inflammatory responses of mice fed on high-iron diets. It suggests that iron influences many processes at the intestinal host-pathogen interface and that the clinical outcome of oral iron administration is difficult to predict as this may highly depend on host iron status, immune status and the gut microbiota composition.

Sixth Congress of the International BioIRon Society Page 163 Poster Abstracts IBIS

Poster #17 - WITHDRAWN

Sixth Congress of the International BioIRon Society Page 164 Poster Abstracts IBIS

Poster #18 - WITHDRAWN

Sixth Congress of the International BioIRon Society Page 165 Poster Abstracts IBIS

Poster #19 DESIGN OF NOVEL FLUORESCENT MITOCHONDRIA-TARGETED PEPTIDES WITH IRON SELECTIVE SENSING ACTIVITY Robert Hider1, Vincenzo Abbate, PhD1, Olivier Reelfs, PhD2 and Charareh Pourzand, PhD21 King's College London; 2Bath University

Mitochondrial labile iron (LI) plays a crucial role in oxidative injuries and pathologies. It can catalyze the overproduction of reactive oxygen species via Fenton chemistry, ultimately causing cellular damage and death [1]. It is thus important to be able to accurately and selectively monitor levels of mitochondrial LI under both physiological and pathological conditions. At present, there is no organelle-specific sensitive iron sensor that specifically accumulates in the mitochondria and reliably monitors levels of LI in this organelle. In this work, we report the design, synthesis, physico-chemical and biological evaluation of novel fluorescent and highly specific mitochondria iron sensors, using the family of mitochondrial- homing ‘SS-peptides’ (short cell-permeable signal peptides mimicking mitochondrial import sequence [2]) as carriers of highly specific iron chelators for sensitive evaluation of the mitochondrial LI. All the peptides were prepared using orthogonal solid-phase peptide synthesis (SPPS), fluorescently labeled at the C-terminus, cleaved from the solid support and purified via semi-preparative reverse phase-high performance liquid chromatograph (RP-HPLC). The purity of the peptides was confirmed via analytical RP-HPLC and the compounds were further characterized using high-resolution mass spectrometry (HRES-MS). Epifluorescence and confocal microscopies using specific trackers for mitochondria, lysosomes and endoplasmic reticulum (ER) were employed to assess subcellular localization of the compounds in primary foreskin fibroblast FEK4 cells. Microscopy analysis of subcellular localization of a small library of fluorescently labelled SS-like tetra- and penta-peptides identified dansyl (DNS) as the lead fluorophore for the subsequent synthesis of chimeric iron chelator-peptides of either catechol (compounds 10-11) or hydroxypyridinone (HPO) (compounds 13-14) type. Such compounds were demonstrated to preferentially accumulate in the mitochondria as judged by fluorescence microscopy studies using organelle-specific markers. The iron-sensing ability of these chimeric compounds was confirmed by fluorescent quenching and de- quenching studies both in solution and in cells, with the HPO-based compound 13 exhibiting the highest sensitivity towards iron modulation. The intramolecular fluorophore-chelator distance and the iron affinity both influenced probe sensitivity towards iron. Importantly, compound 11, which lacks iron chelating ability due to methylation of one phenolic oxygen, was not iron-sensitive. This strongly suggests that the iron-dependent quenching effect occurred solely via ligand- metal complex formation. These compounds represent the first example of highly sensitive mitochondria-targeted iron chelating probes with significant potential to monitor mitochondrial LI level.

References: [1] Cabantchik, Z. I. (2014) Labile iron in cells and body fluids. Physiology, pathology and pharmacology. Front. Pharmacol. 5, 1-10. [2] Szeto, H. H. and Schiller, P. W. (2011) Novel therapies targeting inner mitochondrial membrane-from discovery to clinical development . Pharm. Res. 28, 2669-2679.

Sixth Congress of the International BioIRon Society Page 166 Poster Abstracts IBIS

Poster #20 SPECIATION OF NON-TRANSFERRIN-BOUND IRON Yongmin Ma1 and Robert Hider, PhD21 Zhejiang Chinese Medical University; 2King's College London

Non-transferrin-bound iron (NTBI) appears in the serum of patients and has been associated with multiple organ malfunctions. Although the nature of NTBI is uncertain, the majority of species are various complexes of low-molecular- mass ligands such as citrate and a low affinity iron binding protein such as albumin, but definitive evidence is still lacking. Gel filtration associated with inductively coupled plasma mass spectrometry (GF-ICP-MS) has been employed to assess the iron speciation containing ferritin, albumin and citrate. Since the gel filtration column is incapable of separating protein with similar molecular weight, such as apo-transferrin from albumin, an additional method for this separation was employed. Ion exchange column is one selection for such separation. In addition, the combined complex of hepatoglobin and haemoglobin on gel filtration column flows out at same time as that of ferritin, making a difficult assessment of ferritin- bound iron level. This can be resolved by employing gel filtration on HPLC, as the haemoglobin has strong absorption wavelength at 410 nm, whereas ferritin has no absorption at this wavelength. The same method was employed to separate the combined complex of albumin-hemin from albumin-bound iron. Based on the above combined methods, the concentrations of NTBI speciation in different component are able to be calculated and the NTBI level of sera from different iron overloaded patient type is investigated and compared with the fluorescent bead-based assay1. Reference Ma Y, Podinovskaia M, Evans PJ, Emma G, Schaible UE, Porter J, Hider RC. A novel method for non-transferrin-bound iron quantification by chelatable fluorescent beads based on flow cytometry. Biochem J. 2014, 463, 351-362

Sixth Congress of the International BioIRon Society Page 167 Poster Abstracts IBIS

Poster #21 CD81 PROMOTES BOTH THE DEGRADATION OF TRANSFERRIN RECEPTOR 2 (TFR2) AND THE TFR2-MEDIATED MAINTENANCE OF HEPCIDIN EXPRESSION Juxing Chen, PhD and Caroline Enns, PhD Oregon Health & Science University Mutations in transferrin receptor 2 (TfR2) cause a rare form of the hereditary hemochromatosis (HH), resulting in iron overload predominantly in the liver. TfR2 is primarily expressed in hepatocytes and is hypothesized to sense iron levels in the blood to positively regulate the expression of hepcidin through activation of the BMP-signaling pathway. Hepcidin is a peptide hormone that negatively regulates iron egress from cells and thus limits intestinal iron uptake. In this study, a yeast two-hybrid approach using the cytoplasmic domain of TfR2 identified CD81 as an interacting protein. CD81 is an abundant tetraspanin in the liver. Co-precipitations of CD81 with different TfR2 constructs demonstrated that both the cytoplasmic and ecto-transmembrane domains of TfR2 interact with CD81. Knockdown of CD81 using siRNA significantly increased TfR2 levels by increasing the half-life of TfR2, indicating that CD81 promotes degradation of TfR2. Previous studies showed that GRAIL, an ubiquitin E3 ligase, targets CD81 for degradation. Knockdown of GRAIL in Hep3B-TfR2 cells increased TfR2 levels, consistent with inhibition of CD81 ubiquitination. These results suggest that downregulation of CD81 by GRAIL targets TfR2 for degradation. Surprisingly, knockdown of CD81 decreased hepcidin expression, implying that the TfR2/CD81 complex is involved in the maintenance of hepcidin mRNA. Moreover, knockdown of CD81 did not affect the stimulation of hepcidin expression by BMP6, but increased both the expression of ID1 and SMAD7, direct targets of BMP-signaling pathway, and the phosphorylation of ERK1/2, indicating that the CD81 regulates hepcidin expression differently from the BMP and ERK1/2-signaling pathways.

Sixth Congress of the International BioIRon Society Page 168 Poster Abstracts IBIS

Poster #22 PROFILIN-2, A NEW PLAYER IN IRON METABOLISM Sara Luscieti2, Pietro Pilo Boyl, PhD3, Bruno Galy, PhD4, Lucía Gutiérrez, PhD5, Maya Shvartsman, PhD2, Jorge Couso2, Alejandro Negro2, Maria Puerto Morales, PhD5, Matthias W. Hentze, PhD, MD4, Walter Witke, PhD3 and Mayka Sanchez, PhD1

1Josep Carreras Leukaemia Research Institute (IJC), Diagnostics in Iron Metabolism Disease (D IRON) and Iron Metabolism: Regulation and Diseases Group. and Institute of Predictive and Personalized Medicine of Cancer (IMPPC). Badalona, Barcelona.; 2Josep Carreras Leukaemia Research Institute (IJC), Iron Metabolism: Regulation and Diseases Group. and Institute of Predictive and Personalized Medicine of Cancer (IMPPC). Badalona, Barcelona.; 3University of Bonn, Institute of Genetics, Bonn, Germany; 4European Molecular Biology Laboratory (EMBL), Heidelberg, Germany; 5Instituto de Ciencia de Materiales de Madrid (ICMM-CSIC), Madrid, Spain The IRPs/IRE regulatory network plays a central role in the control of cellular iron homeostasis. Using a high throughput approach, we have previously identified novel IRP1 and IRP2 interacting mRNAs. Among the identified mRNAs, we studied more in depth Profilin2 (Pfn2), a protein involved in endocytosis and neurotransmitters release. The aim of this work is to characterize Pfn2 as a novel IRPs target mRNA and study its role in iron homeostasis. Mouse and human Pfn2 mRNAs were tested by non-radioactive competitive electrophoretic mobility shift assays (EMSA) for the binding to IRP1 and IRP2. To test the responsiveness of Pfn2 to IRP activity, Pfn2 mRNA levels were analyzed in mice with intestinal IRP1 and IRP2 deficiency. The labile iron pool (LIP) was measured in HeLa and Hepa1-6 cell lines with transient or stable overexpression of Pfn2. Tissues derived from Pfn2 knock-out mice were analyzed for iron content, measured by atomic absorption or colorimetric assay, and for mRNA and protein levels of iron-related genes. Combination of EMSA experiments and bioinformatic analyses allowed the identification of a novel and conserved 3’UTR iron responsive element in Pfn2 mRNA with an atypical hexanucleotide apical loop (AAGUGG). Pfn2 mRNA levels were significantly reduced (~20-25%) in duodenal samples from mice with IRP1 and IRP2 intestinal specific ablation, suggesting that IRPs exert a positive effect on Pfn2 mRNA expression in vivo. Overexpression of Pfn2 cDNA in HeLa and Hepa1-6 cells reduces LIP levels compared to control cells. Finally, analysis of Pfn2 KO mice showed iron accumulation in discrete areas of the brain (olfactory bulb, hippocampus and midbrain) together with an hepatic iron deficiency with ferritin reduction. Our results indicate that Pfn2 is controlled by the IRP regulatory system in vivo and that Pfn2 is a new iron gene that modulates iron homeostasis in cell lines and mice. Funding: Work supported by grant SAF2012-40106 from Spanish Secretary of Research, Development and Innovation (MINECO) and grant CIVP16A1857 “Ayudas a proyectos de Investigación en Ciéncias de la Vida - Fundación Ramón Areces” , 2014 SGR225 (GRE) Generalitat de Catalunya and economical support from Fundació Internacional Josep Carreras i de la Obra Social “la Caixa” Spain to M.S. M.S. held a research contract under the Ramón y Cajal program from the Spanish Ministry of Science and Innovation (RYC-2008-02352).

Sixth Congress of the International BioIRon Society Page 169 Poster Abstracts IBIS

Poster #23 GALLIUM NANOPARTICLE: A SINGLE DRUG TARGETING IRON METABOLISM TO TREAT HIV-TB CO-INFECTION IN HUMAN MACROPHAGES Seoung Choi, PhD, Bradley Britigan, MD and Prabagaran Narayanasamy, PhD University of Nebraska Medical Center

Human Immunodeficiency Virus (HIV) and Mycobacterium tuberculosis (TB) are two major infectious causes of morbidity and mortality worldwide. Individuals are often simultaneously infected with both agents, with the outcome and severity worse under such circumstances. Treatment of both HIV and TB is challenging due to the need for prolonged treatment regimens that have significant toxicity and emerging drug resistance. Thus, an urgent need exists for simplified, long acting and effective co-treatment regimens. An agent that inhibits both HIV and TB would be ideal. We believe that it can be achieved through targeting the mononuclear phagocytes (MP), which include monocytes, macrophages, monocyte- derived macrophages (MDMs), and dendritic cells that are natural reservoir for both HIV and TB. We have previously demonstrated that gallium (Ga(III)) disrupts TB and HIV iron-dependent metabolism. Therefore, we developed MP- targeted Ga(III) nanoparticles to treat both HIV and TB simultaneously. MDM-targeted long-acting nanoformulations to treat HIV-TB co-infection and corresponding sustained drug releasing property were determined. Ga (III) nanoparticles reduced the growth of both HIV (Figure 1A) and TB (Figure 1B) and were able to provide sustained drug release for 15 days and controlled the HIV-TB growth considerably. These data provide the basis for a much-needed new approach for effectively treating HIV-TB co-infections and eliciting improved clinical outcomes. Figure 1. Comparison of gallium (III) free drug (GaT) with gallium (III) nanoparticle (NP-GaT) and non-Ga treated MDMs co-infected with HIV and M. tuberculosis. The data are from day 5, day 10 and day 15 post single dose treatment with Ga and 10 days post-infection with HIV/TB. Data shows significant growth reduction of HIV (reverse transcriptase activity, panel B) and TB (CFU, panel A) by GaT and NP-GaT for up to 15 days.

Sixth Congress of the International BioIRon Society Page 170 Poster Abstracts IBIS

Poster #24 HFE GENOTYPE AND A FORMULATED DIET CONTROLLING FOR IRON STATUS ATTENUATE EXPERIMENTAL CEREBRAL MALARIA IN MICE Dominique Leitner, PhD2, Jose Stoute, MD1, Mary Landmesser, BS1, Elizabeth Neely, BS1 and James Connor, PhD11 Penn State University; 2Mount Sinai Icahn School of Medicine

Plasmodium falciparum infects about 500 million individuals each year. A small but significant number of infections lead to complications such as cerebral malaria (CM). CM is associated with myelin damage and neurological deficits in survivors, and iron status is thought to impact the outcome of infection. We evaluated whether a mouse model of CM (experimental CM; ECM) with Plasmodium berghei ANKA was altered by dietary iron deficiency (ID) or genetic iron overload (H67D HFE). We found that H67D mice had increased survival over H67H (wildtype) mice. Moreover, a specifically designed formulation diet increased survival regardless of whether the diet was iron deficient or iron adequate. To determine potential mechanisms underlying demyelination in ECM, we measured Semaphorin4A (Sema4A) levels in the brain because we found it is cytotoxic to oligodendrocytes. Sema4A was increased in wildtype mice that developed ECM while consuming standard rodent chow, consistent with a decrease in myelin basic protein (MBP); an indicator of myelin integrity. The brains of ID and H67D mice had lower levels of Sema4A. MBP was decreased in brains of mice fed the ID diet as previously reported. We also examined erythropoietin (Epo), which is under consideration for treatment of CM, and IL-6, which is known to increase during infection. We found that plasma Epo was elevated and IL-6 was low in H67D mice and in the mice fed the formulation diets. These data reveal a paradigm-shifting concept that maintaining iron status may not increase the mortality associated with malaria as long as there is a strategy to modulate the immune response, which should include decreased Sema4A in brain, elevated Epo, and lower IL-6 in plasma.

Sixth Congress of the International BioIRon Society Page 171 Poster Abstracts IBIS

Poster #25 HFE POLYMORPHISMS AFFECT SURVIVAL OF BRAIN TUMOR PATIENTS Sang Lee, PhD, Becky Slagle-Webb, BS, Jonas Sheehan, MD, Junjia Zhu, PhD, Joshua Muscat, PhD, Michael Glantz, MD and James Connor, PhD Pennsylvania State University

The HFE (high iron) protein plays a key role in the regulation of body iron. HFE polymorphisms (H63D and C282Y) are the common genetic variants in Caucasians. Based on frequency data, both HFE polymorphisms have been associated with increased risk in a number of cancers. The prevalence of the two major HFE polymorphisms in a human brain tumor patient populations and the impact of HFE polymorphisms on survival have not been studied. In the present study, there is no overall difference in survival by HFE genotype. However, male GBM patients with H63D HFE (H63D) have poorer overall survival than wild type HFE (WT) male GBM (p=0.03). In GBM patients with the C282Y HFE polymorphism (C282Y), female patients have poorer survival than male patients (p=0.05). In addition, female metastatic brain tumor patients with C282Y have shorter survival times post diagnosis than WT patients (p=0.02) or male metastatic brain tumor patients with C282Y (p=0.02). There is a tendency toward a lower proportion of H63D genotype in GBM patients than a non-tumor control group (p=0.09) or other subtypes of brain tumors. At present, we are analyzing the association between HFE polymorphisms and GBM patient’s survival of The Cancer Genome Atlas (TCGA) data. In conclusion, our study suggests that HFE genotype impacts survival of brain tumor patients in a gender specific manner. We previously reported that glioma and neuroblastoma cell lines with HFE polymorphisms show greater resistance to chemo and radiotherapy. Taken together, these data suggest HFE genotype is an important consideration for evaluating and planning therapeutic strategies in brain tumor patients. [This work was supported in part by the Tara Leah Witmer Endowment and Pennsylvania Department of Health.]

Sixth Congress of the International BioIRon Society Page 172 Poster Abstracts IBIS

Poster #26 DEVELOPMENT OF NOVEL THERAPEUTIC AGENTS FOR NEUROBLASTOMA THROUGH IRON AND CHOLESTEROL METABOLISMS STUDY Sang Lee, PhD, Becky Slagle-Webb, BS, Cara-Lynne Schengrund, PhD and James Connor, PhD Pennsylvania State University

Neuroblastoma (NB) is the most common childhood extracranial solid malignancy. A significant problem is that at the time of diagnosis about 50% are metastatic and despite aggressive multimodality therapy advanced disease remains difficult to treat successfully. Previously, we report that C282Y HFE (mutant form of the iron metabolism protein HFE, known to be a risk factor for certain cancers) stably transfected human NB cells have significantly elevated levels of iron and cholesterol and changes in expression of genes encoding proteins involved in cholesterol metabolism relative to wild type (WT) HFE cells. The C282Y NB cells were also more sensitive to U18666A (an inhibitor of the last step in cholesterol synthesis) than WT HFE cells. Based on these observations, we studied the relationship between HFE polymorphism, iron and cholesterol metabolisms in human NB cell lines to identify a novel treatment strategy for this cancer. Genotyping of nine human neuroblastoma cell lines indicated that only the SK-N-AS cells expressed the C282Y HFE mutation. None of the cells tested expressed the H63D mutation of HFE. In general, iron levels in human NB cells increased during the time they were cultured (~ 3 days) while cholesterol levels of cells maintained in medium containing 1% fetal bovine serum (FBS) or 10% lipoprotein deficient serum decreased. The drug resistant human NB CHLA-171 cells had higher levels of total iron than human NB CHLA-136 (drug resistant) and SH-SY5Y (drug sensitive) cells as well as significantly more total cholesterol than the other cells during the first 48 hours in culture. The drug resistant NB cells (eg., SK-N-AS & CHLA-171 cells, CHLA-136 cells have not been tested) were less susceptible to the iron chelator, deferoxamine (DFO) than drug sensitive NB cells (eg., SH-SY5Y). The drug resistant SK-N-AS cells are also more resistant to sphingosine kinase inhibitor (SKI) than drug sensitive SH-SY5Y cells. However, the drug resistant NB cells are more sensitive to simvastatin than drug sensitive NB cells. In addition, the drug resistant CHLA-171 cells are more vulnerable to serum deprivation than the SH-SY5Y and SK-N-AS NB cells. This result implies that a NB with high iron/cholesterol might respond well to anti-angiogenesis therapy. In summary, the current data suggest that the need for iron is enhanced during cell proliferation, while cellular cholesterol is used for building new cells or synthesized more slowly during cell growth. The present data also indicates that cholesterol is essential for the growth of drug resistant NB cells, and the effect of inhibition of its synthesis has a significant impact on cell viability when its availability to the cells is low. [This work was supported by Children’s Miracle Network Fund.]

Sixth Congress of the International BioIRon Society Page 173 Poster Abstracts IBIS

Poster #27 ALTERATIONS IN THE IRON REGULATORY GENE SIGNATURE ARE ASSOCIATED WITH DECREASED SURVIVAL IN LOW GRADE GLIOMA Cody Weston, PhD1, Joseph Klobusicky, PhD2, Jennifer Weston, MLIS3, James Connor, PhD3, Steven Toms, MD2 and Nicholas Marko, MD21 Penn State Hershey College of Medicine; 2Geisinger Medical Center, Danville, PA; 3Penn State College of Medicine, Hershey, PA

Background: Iron is a tightly regulated micronutrient that is necessary for cell division in normal tissue. Previous studies have established that a signature of iron regulatory genes can predict survival in breast tumors. Recent evidence suggests that dysregulation of iron regulatory proteins may play a role in brain cancer pathophysiology. Methods: We use public data from The Cancer Genome Atlas (TCGA) to study the association between survival and expression levels of 61 genes coding for iron regulatory proteins in patients with low grade brain gliomas (LGG). Specifically, we utilized the Coxnet package to performe feature selection using elastic net regularization and compared this model to the 1-Nearest Neighbor algorithm and random selection. To prevent overfitting in our Coxnet model, 10-fold cross-validation was implemented. Results: Using feature reduction strategies we identified a novel, optimized subset of 10 iron regulatory genes (STEAP3, HFE, TMPRSS6, SFXN1, TFRC, SLC25A37, ALAD, SLC25A28, TFR2, and HIF1AN) whose differential expression defines two phenotypic groups with differential survival of 62.9 months (94.5 vs 31.6 months, p< 10-6). The correlation between predicted relative risks and survival on the Coxnet model was -0.27, compared to a 0.30 correlation for the 1NN algorithm performed on all 61 iron regulatory genes. Therefore, feature selection eliminated 51 of the candidate genes with only a slight reduction in accuracy. Conclusions: This work indicates that iron metabolism affects tumor progression in multiple types, promising broader applicability for therapeutic and predictive strategies focusing on iron metabolism. The genes identified in this study are risk factors in other cancers, suggesting that targeting these genes could provide therapeutic and prognostic tools with utility beyond low grade glioma.

Sixth Congress of the International BioIRon Society Page 174 Poster Abstracts IBIS

Poster #28 TOWARDS THE IDENTIFICATION OF THE MAMMALIAN ASCORBATE-STIMULATED PLASMA MEMBRANE FERRICYANIDE REDUCTASE Alfons Lawen, Dr rer nat1, Anurag A. Atnerkar, BSc1 and Darius J. R. Lane, p21 Monash University; 2Sydney University

Introduction: Eukaryotic cells possess transplasma membrane electron transfer (tPMET) systems capable of reducing extracellular electron acceptors at the cost of cytosolic electron donors. tPMET has been suggested to be involved in otraserrod ro redto ad ptae e hae, hoeer, sho, that at least the ase o the ascorbate-stimulated tPMET activity, this is not the case and that transplasma membrane ascorbate/dehydroascorbate (DHA) cycling can instead promote NTBI reduction and uptake by human erythroleukemia (K562) cells and rodent astrocytes [2,3]. Ascorbate-stimulated ferricyanide reduction, in contrast to ferric citrate reduction, is dependent on intracellular ascorbate [4].We have previously reported on an ascorbate-stimulated plasma membrane ferricyanide reductase in human erythroleukemia (K562) cells and rodent primary astrocytes [1]. We hypothesised the enzyme responsible to be a member of the cytochrome b561 family [1,5]. Aims: In this study we aimed to establish whether members of the cytochrome b561 family of proteins are expressed at the plasma membrane of K562 cells. Methods: We employed Western blot analyses in whole cell extracts from K562 and PC12 cells to establish the expression of the cytochrome b561 proteins: duodenal cytochrome b561 (Dcytb), lysosomal cytochrome b561 (Lcytb), chromaffin granule cytochrome b561 (CGcytb), gene product 101F6, ferric chelate reductase 1 (FRRS1, SDR-2). We then employed indirect immunofluorescence labelling and confocal microscopy to analyse the subcellular localisation of the cytochrome b561 isozymes expressed in these cells. Results: PC12 cells expressed all isoforms and K562 expressed all isoforms except Lcytb. Confocal microscopy of K562 cells showed all isoforms in intracellular compartments, but only Dcytb was expressed at the plasma membrane. Discussion: Our data suggest Dcytb to be the identity of the long-sought ascorbate-stimulated plasma membrane ferricyanide reductase. We plan to employ siRNA to knock down this enzyme in order to establish the molecular identity of the reductase.

[1] Lawen, A. and Lane, D. J. R. (2013): Mammalian iron homeostasis in health and disease: uptake, storage, transport and molecular mechanisms of action. Antiox. Redox Signal. 18, 2473-2507. doi: 10.1089/ars.2011.4271. [2] Lane, D. J. R. and Lawen, A. (2008): Non-transferrin iron reduction and uptake are regulated by transmembrane ascorbate cycling in K562 cells. J. Biol. Chem. 283, 12701-12708. doi: 10.1074/jbc.M800713200. [3] Lane, D. J. R., Robinson, S. R., Czerwinska, H., Bishop, G. M. and Lawen, A. (2010): Two routes of iron accumulation in astrocytes: ascorbate-dependent ferrous iron uptake via the divalent metal transporter (DMT1) plus an independent route for ferric iron. Biochem. J. 432, 123-132. doi: 10.1042/BJ20101317. [4] Lane, D. J. R., Robinson, S. R., Czerwinska, H. and Lawen, A. (2010): A role for Na+/H+ exchangers and intracellular pH in regulating vitamin C-driven electron transport across the plasma membrane. Biochem. J. 428, 191-200. doi: 10.1042/BJ20100064.4. [5] Lane, D. J. R. and Lawen, A. (2009): Ascorbate and plasma membrane electron transport – enzymes vs. efflux. Free Rad. Biol. Med. 47, 485-495. doi: 10.1016/j.freeradbiomed.2009.06.003.

Sixth Congress of the International BioIRon Society Page 175 Poster Abstracts IBIS

Poster #29 EFFECT OF IRON DEPLETION AND REPLETION ON THE RELATIONSHIPS OF EXPRESSION OF CARDIAC IRON- SULFUR PROTEINS AND CARDIAC HYPERTROPY OF RATS YihFong Liew, PhD1, Chia-Shu Lee, MS1, Ya-Yun Cheng, MS1, Ming-Chen Yu, MS2 and Ning-Sing Shaw, PhD21 Fu-Jen Catholic University; 2National Taiwan University

Introduction: Impaired of energy metabolism appear to play an important role in cardiac dysfunction and progression to cardiac hypertrophy and heart failure. Iron-sulfur proteins in mitochondria plays a pivotal role in electron transport in respiratory complexes, and generates ATP. Severe iron deficiency has been shown leads to anemia, cardiac hypertrophy, impaired function of iron-sulfur proteins and energy metabolism. Whether iron deficiency result in cardiac hypertrophy associates with dysfunction of regulating in the iron-sulfur protein is still unrevealed. Therefore, the aim of this study is to investigate dietary iron depletion and repletion on the expression of iron-sulfur proteins in heart of rat. Methods: Weaning Wistar male rats were fed an iron-deficient diet for 3 week, and then replete with 6, 12, 18, 24 and 35 Fe mg.kg diet-1 for 2 weeks. In addition, one group of rat received an iron sufficient diet for all 5 weeks. Blood was collected for measurement of hemoglobin. The heart was rapidly excised for preparation of cytosolic and mitochondrial fraction, and measured the enzyme activities of succinate dehydrogenase (SDH), cytosolic aconitase (c-aconitase) and mitochondrial aconitase (m-aconitase). The expression of IscS, IscU, frataxin, m-aconitase and SDH subunit B protein levels were detected by Western blot analysis using with commercial specific antibody. Results: Iron deficiency caused a significant reduced hemoglobin concentration by 63%, and both heart weight and the heart to body weight ratio were increased by 37 and 57% after the depletion period, respectively. The enzyme activities of c-aconitase, m-aconitase and SDH were decreased by 32%, 24% and 33% in the heart of iron-deficient group, respectively. Both of cardiac SDH subunit B and IscU protein levels were decreased in iron-deficient group, however, cardiac m-aconitase, mitochondrial IscS and frataxin protein levels remained unchanged. At the end of the repletion period, hemoglobin concentration displayed that gradually restored to normal. The heart weight, cytosolic aconitase and SDH activities were also restored to normal after repletion with 12 mg Fe kg diet-1. In contrast, the enzyme activity of mitochondrial aconitase did not restore to normal levels after repletion with 35 mg Fe kg diet-1. In addition, the cardiac mitochondrial IscU protein levels were still increased after repletion with any iron contents. Conclusion: Iron deficiency results in the decline in enzyme activity of iron-sulfur containing protein due to limitation of iron sulfur cluster formation from lack of iron, suggesting that the dysfunction of iron-sulfur protein leads to impaired energy metabolism contributing to progression of heart hypertrophy.

Sixth Congress of the International BioIRon Society Page 176 Poster Abstracts IBIS

Poster #30 CHARACTERIZATION OF FERROPORTIN DISEASE-RELATED RESIDUES PREDICTED TO CONFER HEPCIDIN RESISTANCE S.Aschemeyer, B. Qiao, A. Sek, M. Jormakka, T. Ganz and E. Nemeth University of California, Los Angeles

Ferroportin (Fpn), the only known cellular iron exporter, transports iron into plasma from enterocytes absorbing dietary iron, macrophages recycling aged erythrocytes, and hepatocytes which store iron. The hormone hepcidin binds to ferroportin, causing its ubiquitination, endocytosis, and degradation, thus inhibiting the entry of iron into plasma. However, mutations in ferroportin can lead to hepcidin resistance by interference with hepcidin binding, with binding-induced conformational change, or with the generation of covalent modifications or secondary binding events that trigger endocytosis. Regardless of the mechanism, hepcidin resistance results in increased duodenal iron absorption and iron overload, clinically characterized by elevation in both serum transferrin saturation and serum ferritin. To understand the contribution of different ferroportin residues to the binding or other effects of hepcidin, we generated all known human ferroportin mutations that conferred hepcidin resistance based on the patient iron parameters: Y64N, S71F, V72F, N144D, N185D, G204S, D270V, C326S, S338R, Y501C, D504N, and H507R. We established stably-expressing inducible Fpn mutant cell lines, as transient transfection is not reliable in assessing hepcidin resistance. All the mutants demonstrated membrane localization by microscopy. Using intracellular ferritin as a proxy for cellular iron export, we confirmed that nearly all of these mutants were partially or completely resistant to the effect of hepcidin on iron export. For each mutant, we will next analyze hepcidin binding, ubiquitination of Fpn after hepcidin addition, and endogenous turnover of Fpn, and integrate it with the most recent structural data on Fpn. Obtaining a structural understanding of the effect of hepcidin on Fpn based on these Fpn substitutions will aid in the development of improved therapeutics for iron overload disorders and iron-restricted anemias.

Sixth Congress of the International BioIRon Society Page 177 Poster Abstracts IBIS

Poster #31 EFFECTS OF IRON CHELATOR DEFEROXAMINE ON NUCLEOLAR MORPHOLOGIC AND FUNCTIONAL CHANGE AND CELL GROWTH IN HUMAN BREAST CANCER MCF-7 CELLS YihFong Liew, PhD, Yu-Shan Lin, MS, Wei-Chih Chen, MS and Ya-Yun Cheng, MS Fu-Jen Catholic University

Introduction: The nucleolus is the organelle of the nucleus where the place for the ribosome biogenesis, including of ribosomal RNA (rRNA) transcription, pre- rRNA processing and ribosomal assembly. Nucleolar morphological changes and ribosome biogenesis have been shown to coordinate with regulation of cell proliferation and cell death. Elevated cellular iron indicates demand for growth of cancer cells, therefore decreasing iron level by iron chelator results in inhibiting of cancer cell growth. Whether the iron chelator inhibits cell proliferation associate with the morphological and functional changes in nucleolus is still unrevealed. Methods: Deferoxamine was used to investigate the anti-proliferative mechanism of iron chelation in MCF-7 cells. Cell cycle analysis performed by flow cytometry with propidium iodine staining. Cell proliferation was investigated by BrdU incorporation. Nucleolar morphological analysis was detected by AgNOR (silver staining for nucleolus organizer regions) staining. The expression of protein level was measured by using Western blotting. Results: A total of 3430 proteins from MCF-7 human breast cancer cells were identified by using of stable isotope dimethyl label coupled with LC/MS/MS. Two hundred fifty eight proteins were up-regulated, and 164 proteins were down- regulated in iron chelator deferoxamine (DFO)-treated cells for 48hr, respectively. Most of these up-regulated proteins are involved in pre-rRNA processing, ribosomal assembly, and apoptosis/cell death. In contrast, most of the down-regulated proteins are involved in energy metabolism, protein metabolic process, cell cycle, and cell proliferation. DFO treatment led to the accumulation of cells arrested in G0/G1 phase, with concomitant decreased cell populations in S and G2/M phases and BrdU incorporation. In addition, DFO treatment also caused nucleolar morphologic changes, with concomitant reduced the number of nucleoli by 30%. The rRNA processing relative ribonucleoprotein Nop58 and an essential role in oxidative protein folding Ero1L-α prote leels ere reased y ad treatet, respetely contrast, the protein levels of ribosome export adaptor NMD3 was reduced by 55% in DFO-treated cells. Conclusion: In summary, our data indicates that iron chelator induces the morphologic and functional changes in nucleolus, and suggesting this associates with regulation of ribosome biogenesis and function, and involves in regulation of cell growth in human breast cancer MCF-7 cells.

Sixth Congress of the International BioIRon Society Page 178 Poster Abstracts IBIS

Poster #32 NEXT-GENERATION SEQUENCING: APPLICATION OF A NOVEL PLATFORM TO THE ANALYSIS OF ATYPICAL IRON DISORDERS Cameron McDonald, PhD, Lesa Ostini, Daniel Wallace, Alyson Lyons, Darrell Crawford and V. Nathan Subramaniam QIMR Berghofer

Introduction: Atypical iron disorders represent a spectrum of iron dysregulation ranging from juvenile onset iron overload through to iron-refractory iron-deficiency anaemia. The most common form of primary iron overload disease, HFE-related hereditary hemochromatosis, accounts for anywhere between 60-95% of cases of iron overload in European populations. However, a significant and growing number of atypical iron disorders are being identified, particularly within non-European populations. The development of targeted next-generation sequencing applications now promises a clinically viable option for the diagnosis of such rare disorders, with this approach proving to have significant utility where standardized testing has failed to identify the underlying molecular basis of disease. Methods: The whole transcripts of 39 genes either currently associated with genetic iron overload or anaemia, or for which evidence from animal models or in vitro experiments demonstrates a contribution to iron homeostasis, and promoter regions from 11 of these, where targeted using a custom Ampliseq panel. This panel was then used for next- generation sequencing of gDNA from a female subject with atypical iron overload. Following identification of the potentially causative mutation, expression constructs were generated of wild-type (WT) Transferrin Receptor 2 (TFR2), mutagenesis performed, and constructs expressed in HepG2/C3A cells. Results: Sequencing identified a potentially causative mutation in the TFR2 gene which was predicted to be deleterious by several algorithms. Functional characterization of the novel nucleotide substitution (NM_003227.3: c.T1627A) which results in an I529N substitution was performed alongside the previously reported, but not cellularly characterized M172K mutation. Cellular analysis of the mutant proteins indicates that these amino acid substitutions affect the localization of the protein resulting in its retention in the endoplasmic reticulum and thus failure to function at the cell surface (Fig 1). Conclusion: We have successfully applied our next-generation sequencing approach, utilizing a novel targeted gene panel, to identify the underlying genetic cause in a case of atypical iron overload where standard testing had been unable to provide a genetic diagnosis. We report the identification of a novel p.I529N mutation in TFR2 which results in a failure in trafficking of the protein to the cell surface, similar to that seen for the p.M172K mutant. In this case study we have demonstrated the utility and viability of targeted next-generation sequencing for identifying causative mutations. We believe that our gene panel and this novel approach will provide an important resource to the scientific and clinical community for the identification the genetic basis of atypical iron disorders.

Sixth Congress of the International BioIRon Society Page 179 Poster Abstracts IBIS

Poster #33 TRANSCRIPTOMIC ANALYSIS OF IRON METABOLISM GENES IN THP-1 DERIVED MACROPHAGES IDENTIFIES HEPHL-1 AS POTENTIAL REGULATOR OF MACROPHAGE IRON EXPORT Amin Sobh, Zouhair Attieh, PhD, Huijun Chen, PhD, Alex Loguivov, PhD and Chris Vulpe, MD, PhD University of California, Berkeley

Macrophage iron metabolism affects a variety of key biological functions. Iron recycling by macrophages comprises the primary source of iron for erythropoiesis. In addition, iron handling by different macrophage populations contribute to their distinctive functions. Pro-inflammatory (M1) macrophages use iron sequestration as a bacteriostatic strategy whereas anti-inflammatory (M2) macrophages spontaneously release iron to fulfill their role in tissue repair and tumor growth. The difference in iron metabolism between M1 an M2 cells requires differential regulation of certain cellular components. Although many of these components were previously identified, a transcriptomic approach could further provide insights into the novel molecular mechanisms of iron metabolism in M1 and M2 macrophages. We used RNAseq analysis to identify transcriptomic changes during the differentiation of THP-1, the human monocytic cell line, into M1 and M2 macrophages. Our approach confirmed the differential expression of iron-related genes between M1 and M2 cells observed in other studies and revealed a differential regulation of HephL-1, a member of the multicopper ferroxidase family. HephL-1 exhibited high expression in M2 macrophages and this expression correlated with ferroxidase activity in these cells. Our results suggest that HephL-1 is the ferroxidase involved in iron export from macrophages.

Sixth Congress of the International BioIRon Society Page 180 Poster Abstracts IBIS

Poster #34 IRON-DEFICIENT MILK OF HEPHAESTIN KNOCKOUT MICE UNDERLIES A HAIR LOSS PHENOTYPE IN PUPS ASSOCIATED WITH INCREASED ANDROGEN RECEPTOR EXPRESSION IN THE SKIN Amin Sobh, Kathryn Page, PhD, Erica Lachenauer, Julie Luong, Chenchen Han, Hiroko Irimagawa, Yuanchi He, David Killilea, PhD, David Frazer, Gregory Anderson, PhD, Brie Fuqua, PhD and Chris Vulpe, MD, PhD University of California, Berkeley

The link between hair loss and iron deficiency has been established in many studies. Androgenetic alopecia (AA), one of the most common causes of hair loss in women is associated with decreased serum ferritin levels. We previously generated knockout mice that lack Hephaesetin (Heph), a ferroxidase involved in intestinal iron efflux. Heph null mice (Heph-/-) exhibited an iron-deficient phenotype. Neonates born to Heph-/- dams developed a truncal hair loss phenotype that resolved after weaning. The onset of hair loss (~day 14) is associated with a decrease in tissue iron levels. The same phenotype was observed in wild-type pups fed by Heph-/- dams that exhibited low milk iron levels. These data indicated that iron-deficient milk underlies the hair loss. We sought to determine whether this hair loss phenotype was similar to that of AA by studying the expression levels of androgen receptor (AR) in the skin. The hair-less back skin showed elevated AR transcript and protein levels indicating an androgen-mediated disorder. The hair loss phenotype was not associated with elevated serum dihydrotestosterone (DHT) levels. We aim to determine whether elevated AR levels in the skin result from systemic iron deficiency or local iron insufficiency in the hair-less skin tissue. We will further investigate the relationship between iron deficiency and AR up-regulation using comparative genomic and transcriptomic approaches. Our study can identify iron-regulated pathways that are essential for normal hair development.

Sixth Congress of the International BioIRon Society Page 181 Poster Abstracts IBIS

Poster #35 THE MONOCLONAL ANTI-TRANSFERRIN ANTIBODY SYNDROME: A WAY FOR A BETTER UNDERSTANDING OF IRON METABOLISM Pierre Brissot, Martine Ropert, Lénaïck Detivaud, Béatrice Fimbel d’Hauthuille, Mathilde Gautier, Patricia Leroyer, Anne- Marie Jouanolle and Olivier Loreal National center of reference for rare genetic iron overload diseases, Inserm-UMR 991, Laboratory of biochemistry, Laboratory of molecular genetics and genomics, Hematology department Trousseau Hospital (Tours). Pontchaillou University hospital, Rennes, Fr

Background and aim. Monoclonal anti-transferrin antibody is a rare situation observed in monoclonal gammopathy of undetermined significance (MGUS). Our aim was to explore iron metabolism in one case of this intriguing syndrome. Methods. Besides the usual blood iron parameters and MRI liver iron assessment, serum hepcidin, non-transferrin bound iron (NTBI), labile plasma iron (LPI), HFE and non-HFE mutations, and serum transferrin sialylation were studied. In addition, protein A affinity chromatography and western blotting using human anti-transferrin antibody were performed to analyse the interaction between immunoglobulin and transferrin. Results. Serum iron and transferrin (Tf) concentrations were extremely high (96.9 µmol/L and 5,0 g/L, respectively). Serum ferritin was moderately increased (608 µg/L ; N<300). There was no MRI liver iron excess. HFE and non-HFE gene mutations were absent. Hepcidin/ferritin ratio was strongly decreased (<0.1 – N: 4-30). Tf was observed in serum and in the IgG eluate fraction after affinity precipitation, confirming the presence of IgG directed against transferrin. Purified transferrin addition to the serum, prior to immunoglobulin affinity purification, increased the amount of purified immunoglobulin transferrin complexes, suggesting the presence, in the serum, of unbound anti-transferrin IgG. High LPI concentration (0.92µmol/L ; N<0.5µmol/L) was noticed, contrasting with normal NTBI level (< 0.5 µmol/L). The basic electrophoretic profile did not provide interpretable information whereas, after immunoprecipitation of transferrin-IgG complexes, the remaining free transferrin glycoforms were clearly found. Discussion. The very high levels of serum iron and transferrin concentration suggest that the transferrin-IgG interaction could modify the transferrin ability to link iron. The increase of transferrin concentration could be a compensatory mechanism aiming to maintain normal availability of the immunoglobulin-free transferrin for iron. The IgG-transferrin complex could generate a form of acquired functional hypotransferrinemia triggering, in turn, transferrin oversynthesis. Another mechanism could be an iron-deficiency-like situation. Indeed, a disturbed interaction between transferrin iron and transferrin receptor 1 could impede iron delivery to cells, thus creating cellular iron deprivation, a condition leading an increase of transferrin synthesis. The contrast between high LPI and normal NTBI concentrations strongly supports that these two iron forms correspond to different biochemical species. Conclusion. The abnormalities of iron metabolism associated with monoclonal transferrin antibody raise important questions on the way this type of monoclonal immunoglobulin impacts iron status, especially in favoring the production of reactive plasma iron and in altering transferrin-iron delivery to the cells. References 1. Alyanakian MA, et al. (2007) Blood 109, 359-361. 2. Forni GL, et al. (2013) Am J Hematol 88, 1045-1049. 3. Ropert M, et al. (2015) Am J Hematol (in press).

Sixth Congress of the International BioIRon Society Page 182 Poster Abstracts IBIS

Poster #36 FUNCTIONAL CHARACTERIZATION AND EVALUATION OF A THIRD CASE OF SIDEROBLASTIC ANEMIA WITH BIALLELIC MUTATIONS OF GLRX5 Raêd Daher, Abdellah Mansouri, Sophie Bayart, Alain Martelli, Isabelle Callebaut, Ly Sunnaram, Julien Goustille, Claire Oudin, Anne Marie Jouanolle, Carole Beaumont, Hervé Puy, Zoubida Karim and Caroline Kannengiesser INSERM U1149, Université Paris Diderot, AP-HP, Département de Génétique, Hôpital Bichat, Laboratory of Excellence GR-Ex, 16 rue Henri Huchard, 75018 Paris, France

Introduction: Congenital sideroblastic anemia (cSA) is associated with the presence of ring sideroblasts in the bone marrow, resulting from dysfunctional heme synthesis or processing, and consequently an iron overload in mitochondria. cSA is most commonly caused by defect in the gene encoding the first enzyme of heme biosynthetic pathway ALAS2, giving rise to X-linked sideroblastic anemia (XLSA). However, few cases with severe phenotypes have been reported showing a recessive cSA, caused by mutations in SLC25A38 and GLRX5 (2 cases) genes. GLRX5 (Glutaredoxin 5), a mitochondrial protein of 156 amino acids, is involved in the biogenesis of the Fe-S clusters. In this study, we describe a third case of cSA with mutations in GLRX5. Methods: Functional study was carried out on lymphoblastoid cell line derived either from patient or control. The quality of the mitochondrial DNA was investigated by slot blot and electrophoresis. Protein expressions of iron carriers, iron regulatory sensors IRP1/2, and that of markers of oxidative stress were studied by Western blot. The activities of the total aconitase, complexes I /IV, ferrochelatase, as well as the synthesis of ATP were measured. Cell proliferation was assessed by an MTT test (tetrazolium salt). Results: Our patient is 17 years-old and was diagnosed with sideroblastic anemia at the age of 14. She was treated with iron chelator (Exjade) because of a moderate iron overload (confirmed by MRI). Anemia was treated by blood transfusions (twice). The search for ALAS2 and SLC25A38 mutations by fluorescent sequencing was negative. We identified compound heterozygous mutations in GLRX5 gene: p.Cys67Tyr and p.Met128Lys. Molecular modeling of GLRX5 protein confirmed the involvement of the p.cys67 in Fe-S clusters binding and predicted an involvement of p.Met128Lys in protein-protein interaction. The ex vivo studies on lymphoblastoid cells showed altered quality and content of mitochondrial DNA. This was associated with decreased protein expression of COX-2, which is encoded by a mitochondrial gene. Both the activity of the respiratory chain (complexes I and IV) and the ATP synthesis were significantly reduced. The expression and activity of the ferrochelatase enzyme, which catalyses the terminal step in heme biosynthesis pathway was highly reduced. Total aconitase activity was highly decreased. The expression of IRP2 (Iron Regulatory Protein 2) but not IRP1 was increased. The expression of cytosolic L-ferritin was reduced but transferrin receptor 1 (TFR1) remained unchanged. In addition, the expression of MnSOD (anti-oxidative mitochondrial enzyme) was enhanced and reduced glutathione (GSH) was decreased indicating the presence of an oxidative stress. Finally, cell proliferation of these lymphoblastoid cells was significantly lower compared to control. Conclusion: In this study, we demonstrate that biallelic mutations p.Cys67Tyr and p.Met128Lys of GLRX5 are responsible for congenital sideroblastic anemia. We also show that GLRX5 deficiency leads to global mitochondrial dysfunction and loss of Fe-S assembly, and alters heme biosynthesis pathway and cell viability.

Sixth Congress of the International BioIRon Society Page 183 Poster Abstracts IBIS

Poster #37 CANCER RELATED ANEMIA HAS A HIGH PREVALENCE AND ADVERSELY IMPACTS QUALITY OF LIFE Giridhar Kanuri1, Ritica Sawhney, MSc2, Girish Raju, MD3, Jeeva Varghese, MSc4, Madonna Britto4 and Arun Shet, MD AB (Haem/onco)31 Wellcome Trust- DBT Hematology Research Division; 2Wellcome Trust- DBT Hematology Research Division, St. Johns Research Institute, Bangalore, Karnataka 560064 India; 3Department of Medical Oncology, St. John’s Medical College Hospital, Bangalore, Karnataka, India; 4College of Nursing, St. John’s National Academy of Health Sciences, Bangalore, Karnataka, India

Background: Cancer related anemia (CRA) adversely affects Quality of Life (QoL) and overall survival in patients with cancer. We studied the prevalence and etiology of anemia in cancer patients and evaluated the relationship between haemoglobin (Hb) and QoL. Methods: Using a cross sectional study design, 218 cancer patients attending a tertiary medical center oncology clinic were prospectively enrolled after obtaining informed consent. Standard laboratory assays and the Functional Assessment of Cancer Therapy-Anemia (FACT-An) tool, standardized for language, were used. Sex adjusted WHO cut-off values deed aea ad the solle traserr reeptor slo errt o deed ro deiency anemia (IDA) superimposed on anemia of inflammation (AI). Results: Mean age was 51+13 years and 60% were female. Most patients had breast cancer (37%), followed by gastro intestinal (17%), lymphoma (15%) and head and neck cancers (10%). The spectrum of clinical stages of cancer was: stage I (7.7%), stage II (19%), stage III (24%), and stage IV (26%). Mean Hb in cancer patients without anemia was 13.3±1 and in cancer patients with anemia was 10.2±1.5 (p <0.001). Among the cancer patients that were anemic (n=139), the majority had IDA (n=83; 60%) suggesting a high prevalence of nutritional anemia. There was a significant lear relatoshp etee ad o peara p On multivariate analysis, every gram rise in Hb was accompanied by a 3.1 unit rise in the QoL score (p=0.003; CI=0.19-5.33). However, subgroup analysis in the patients with CRA showed a 3.9 rise in QoL units in patients with IDA+AI (n=83, p=0.1) whereas in cancer patients with AI alone the QoL rise is only 2.6 units (n=37, p=0.2). Breast cancer patients without CRA (n=33) had significantly higher QoL scores than breast cancer patients with CRA (n=52) (Mean QoL score 141±34 vs. 129±31; p=0.02). Conclusion: CRA is associated with a poor quality of life. CRA is accompanied with a high prevalence of IDA in this patient population. These data suggest a rationale for iron therapy in CRA patients with documented IDA, considering the beneficial effects of IV iron in other chronic disease settings (Anker et al NEJM 2009). However, the negative consequences of increasing metabolically available iron in cancer patients (Pinnix et al SciTranslMed 2010) warrant further study.

Sixth Congress of the International BioIRon Society Page 184 Poster Abstracts IBIS

Poster #38 IRON IS A RISK FACTOR FOR ATHEROSCLEROSIS: CONFIRMATION OF THE IRON HYPOTHESIS IN A MOUSE MODEL OF HEMOCHROMATOSIS Francesca Vinchi, PhD1, Sandro Altamura, PhD1, Milene Costa da Silva, MSc1, Bruno Galy, PhD2, Matthias W Hentze, MD, PhD1 and Martina U. Muckenthaler, PhD11Molecular Medicine Partnership Unit (MMPU), University of Heidelberg & EMBL; 2German Cancer Research Center (DKFZ)

Iron accumulates in atherosclerotic lesions but its role in atherogenesis is still debated. In the “iron hypothesis” (1981), Sullivan proposed that iron is detrimental for the cardiovascular system promoting atherosclerosis progression. Epidemiological data and studies in animal models provided conflicting evidence regarding the role of iron in atherosclerosis. In this study we aim to dissect the role of iron in atherosclerosis and to discriminate how different iron sources contribute to its pathogenesis. We generated a novel mouse model of type IV Hereditary Hemochromatosis (HH) (Altamura et al., 2014), on a ApoE-null genetic background (HH ApoE-null), to study the susceptibility to atherosclerosis. The point mutation within the hepcidin binding site of ferroportin, causes systemic iron overload and macrophage-iron deficiency. This provides a unique model to dissect the contribution of (1) systemic iron overload (high transferrin saturation and NTBI levels) and (2) iron deficiency in macrophages to atherogenesis. Mice were analyzed at 3, 6 and 12 months of age. We show that in 3 month-old HH ApoE-null mice the innominate artery lesion area is significantly smaller compared to age-matched ApoE-null controls. Interestingly, at 6 and 12 months of age the phenotype is reversed and an increased lesion size and number is observed. HH ApoE-null mice show increased levels of circulating oxLDL and iron deposition in the artery media layer, which is associated with increased aortic lipid peroxidation and increased expression of anti-oxidant genes. Moreover, these mice show signs of endothelial activation and dysfunction, including higher aortic expression of adhesion molecules and reduced nitric oxide availability. It is of note that aortic macrophages from HH ApoE-null mice are iron deficient compared to ApoE-null mice and show higher expression of M2-polarization markers and lower expression of the M1 markers. This suggests that iron depletion of macrophages is beneficial in atherosclerosis, preventing polarization towards a M1-like pro-inflammatory phenotype and foam cell formation. Collectively, these data suggest that macrophage-iron depletion is protective for early atherosclerosis, while severe systemic iron overload promotes atherosclerosis progression, thus supporting the iron hypothesis. However, in this model, the potentially protective effect of macrophage-iron depletion is not sufficient to counteract the strongly pro-atherogenic effect of high circulating iron. Rescue experiments on aged HH ApoE-null mice are ongoing by maintaining mice on an iron-deficient diet and by treatment with iron chelators.

Sixth Congress of the International BioIRon Society Page 185 Poster Abstracts IBIS

Poster #39 IRON IMPORT TO UNUSUAL TRYPANOSOMA BRUCEI MITOCHONDRIA - THE ROLE OF MITOFERRIN HOMOLOG Jan Mach, Dominik Arbon, Ivo F. Scheiber, Jan Tachezy and Robert Sutak Charles University in Prague

Parasitic microorganisms acquire all the iron from its host, thus this essential element is considered to be one of the most crucial virulence factors. There is virtually no free extracellular iron present in the host and parasites had to evolve different pathways to obtain host iron and to utilize it. Trypanosoma brucei is a tse-tse fly-transmitted causative agent of African sleeping sickness in humans and nagana in domestic animals. Trypanosomes undergo a complex life cycle, with two main forms, the procyclic form inhabiting the gut of a tse-tse fly and the bloodstream form being pathogenic in vertebrate hosts. The cyclic changes are accompanied by dramatic alterations in the parasite’s metabolism. Particularly its peculiar mitochondrion undergoes remarkable changes between the two principal life stages. It was shown that trypanosomal iron demands are satisfied by specific import of iron-binding proteins from the vertebrate host. In contrast, the insect form takes up iron efficiently from ferric complexes via a reductive mechanism. However mechanism of iron import to the mitochondria, where it is mostly required, is still unknown. In yeast, Mrs3 and Mrs4 proteins (orthologous to mitoferrins in metazoans) have been shown to function in iron transport to this organelle. We found a distinct homolog of Saccharomyces cerevisiae Mrs3/4 genes in the T. brucei genome database – mitochondrial carrier protein 17 (MCP17). This protein contains predicted mitochondrial targeting sequence and multiple transmembrane domains. We raised polyclonal antibody against MCP17 peptide, showing mitochondrial localization of MCP17. To test its possible role in iron transport to mitochondria, we prepared tetracycline-inducible MCP17 knock-down cell line of procyclic T. brucei cells. Growth of this line is severely impaired and the activities of several iron-containing enzymes (fumarase, aconitase and succinate dehydrogenase) are significantly decreased in knock-down line, suggesting an indispensable role of MCP17 in trypanosomal iron metabolism.

Sixth Congress of the International BioIRon Society Page 186 Poster Abstracts IBIS

Poster #40 ROLE OF OXIDATIVE STRESS ON REGULATION OF IRON HOMEOSTASIS GENES IN NEURONAL CELL: IMPLICATION IN IRON ACCUMULATION Som Dev, MSc1, Sanju Kumari, MSc1, Neena Singh, MD, PhD2 and Chinmay K. Mukhopadhyay, PhD11SCMM, JNU, New Delhi; 2 Case Western Reserve University, Cleveland

Introduction: Neuronal iron accumulation and oxidative stress are intimately associated with several neurodegenerative diseases. While, iron is known to catalyze oxidative stress induced neuronal damages but role of oxidative stress on neuronal iron accumulation is not much revealed. Cells including neurons have evolved highly regulated mechanisms for controlling cellular iron levels. In neurons iron uptake is controlled by transferrin receptor-1 (TfR1) and/or by divalent metal transporter-1 (DMT1); whereas taken up iron is stored in ferritin to avoid iron-mediated damage and for the need of other cellular activities whenever necessary. Ferritin-H (Ft-H) is the predominant component of neuronal ferritin content. Cells also control iron level by releasing it through unique iron exporter ferroportin (Fpn). In brain, Fpn stability is regulated by ferroxidase ceruloplasmin (Cp) in astroglia and by Amyloid Precursor Protein (APP) in neurons. Dysregulation of uptake, storage or release components of iron homeostasis in response to oxidative stress may result into cellular iron accumulation. Objective: The current study was undertaken to understand the regulation of iron homeostasis genes in response to oxidative stress and possible implication in increased intracellular iron level detected in neurodegenerative diseases. Observations: The neuroblastoma SH-SY5Y cells were subjected to oxidative stress by addition of hydrogen peroxide (H2O2) and as a result the expression of iron exporter ferroportin was decreased significantly at protein level but not at transcript level. We detected a significant increase in degradation of Fpn and simultaneous decrease in APP, while there was no detectable change in TfR1 and DMT1. Decrease in Fpn should increase labile iron pool (LIP) and subsequent expression of Ft-H for iron storage by affecting iron regulatory protein-iron responsive element (IRP-IRE) interaction. Surprisingly, we detected decreased Ft-H translation due to conversion of cytosolic aconitase to iron regulatory protein (IRP1) and Ft-H mRNA due to decrease in its enhancer activity. Glutathione biosynthesis inhibitor L-buthionine sulfoximine (BSO) treatment also led to Fpn degradation and decreased Ft-H expression. All these events resulted in increased intracellular iron pool in a time dependent manner. Conclusions: This study reveals a complex regulation of neuronal iron release and iron storage components in response to H2O2 that may explain iron overload condition detected in neurodegenerative diseases associated with oxidative stress. To best of our knowledge this is the first report of ferroportin degradation in response to oxidative stress in any cell type including neuron.

Sixth Congress of the International BioIRon Society Page 187 Poster Abstracts IBIS

Poster #41 INFLAMMATION-INDUCED IRF-1 FACTOR DOWNREGULATES FERROPORTIN IRON EXPORTER Catherine Mura, PhD1, Rafiou Agoro, MSc2, Francois Erard, PhD3 and Valerie Quesniaux, PhD31 UMR7355 CNRS, University Orleans; 2UMR 7355 CNRS; 3UMR7355 CNRS

In mammals systemic iron homeostasis is tightly controlled through the hepcidin-ferroportin axis. Ferroportin (Fpn1) is the only transmembrane exporter of iron to blood. Hepcidin once secreted in blood acts as a hormone, binding to the Fpn1, inducing its internalization and lysosomal degradation, and resulting in a decrease in iron export to the circulating blood. Hepcidin is transcriptionally regulated by iron status and inflammation which in turn negatively controlled Fpn1 expression. Fpn1 mRNA levels are also up-regulated by iron levels and hypoxia, and down-regulated by inflammation. Hypoferremia observed upon inflammation result from hepcidin-ferroportin interaction but also from the downregulation of Fpn1 mRNA Here, the molecular mechanism of Fpn1 mRNA downregulation upon inflammation has been studied. We used wild type and knock out mice, as well as RAW264.7 macorphage-like cells to study the molecular pathway of Fpn1 regulation upon inflammation. We constructed a series of clones into the luciferase reporter vector containing either the upstream or downstream sequences of the mouse Fpn1 coding region, and assessed reporter activity after transient transfection. We also performed electrophoretic mobility shift assay and siRNA silencing studies to determine the regulatory factor involved in the Fpn1 downregulation. In mice, hepatic Fpn1 mRNA levels correlated with hepatic iron content, and Fpn1 is downregulated in liver and macrophages after systemic LPS treatment. In the liver, inflammation-induced Fpn1 downregulation can be antagonized in vivo by iron loading, conversely, the combination of iron deficiency and LPS treatment led to a more drastic decrease of the Fpn1 mRNA level in liver. Fpn1 mRNA was efficiently prevented in the absence of Tlr4 or of both Myd88 and Trif; but only moderately reduced in Myd88-/- mice as well as in Trif-/-. In RAW264.7 monocytic cells Fpn1 mRNA was rapidly downregulated after LPS stimulation. Cells incubated with ferric ammonium citrate (FAC) exhibited an increase in Fpn1 mRNA levels while incubation with holo-transferrin (holo-TF) did not significantly affect Fpn1 mRNA levels. However, LPS combined with FAC or holo-TF led to a partial reversal of LPS- induced Fpn1 mRNA down-regulation. In addition to LPS agonist of Tlr4, Fpn1 downregulation can be induced by different TLR agonists, through MyD88-or TRIF-adaptors. Transient transfection of reporter vector analysis indicated the upstream proximal coding region as containing the negative regulatory elements required for LPS downregulation. In silico analysis of the DNA sequence revealed a high score motif matching for consensus Interferon Response Element 1/2 (IRF-1/2) binding site is located within the highly conserved 5’UTR sequence of Fpn1 mRNA of mouse and human species. Electrophoretic mobility shift assays revealed an interaction between a nuclear factor from LPS treated mice and Fpn1 sequence. Silencing Irf1 gene in RAW264.7 cells using selective siRNA significantly abrogated the ability of LPS to downregulate Fpn1 expression in RAW264.7 cells. The binding of Interferon Regulatory Factor 1 (IRF-1) transcription factor to the interferon response element (IFR-E) of the 5’UTR- region of Fpn1 appears to be responsible of Fpn1 promoter repression upon inflammation.

Sixth Congress of the International BioIRon Society Page 188 Poster Abstracts IBIS

Poster #42 DISRUPTION OF THE HEPCIDIN/FERROPORTIN REGULATORY CIRCUITRY CAUSES INCREASED PULMONARY IRON CONTENT AND RESTRICTIVE LUNG DISEASE Joana Neves1,2,4, Milene Costa da Silva1,2,4, Dominik Leitz2,5, Raman Agrawal2,5, Bruno Galy3, Matthias W. Hentze3, Marcus A. Mall2,5, Sandro Altamura1,2,6 and Martina U. Muckenthaler1,2,61 University of Heidelberg, Heidelberg, Germany; 2Molecular Medicine Partnership Unit (MMPU), Heidelberg, Germany; 3European Molecular Biology Laboratory (EMBL), Heidelberg, Germany; 4Graduate Program in Areas of Basic and Applied Biology, University of Porto, Portugal; 5Department of Translational Pulmonology, Translational Lung Research Center, Member of the German Center for Lung Research, University of Heidelberg, Heidelberg, Germany; 6equal contribution

The lung is an organ with unique characteristics being specialized in the gas exchange between the atmosphere and the blood stream. In addition, it exerts a critical role in host defense at the interface between the environment and the body. As all other organs, the lung needs to prevent excess of iron, which causes cellular damage due to the generation of reactive oxygen species (ROS). The risk for oxidative damage in the lung is exacerbated by its continuous exposure to high oxygen levels making iron detoxification within airway epithelial cells and alveolar macrophages an essential mechanism to prevent lung failure. The aim of our study is to determine the consequences of unbalanced iron homeostasis in the lung. We analyzed a mouse model of hereditary hemochromatosis type 4 (FPN C326S) hallmarked by a disrupted hepcidin/ferroportin regulatory circuitry and severe systemic iron overload (Altamura et al., 2014). Biochemical and histological analysis of the lung demonstrates that FPN C326S mice exhibit an age dependent increase in pulmonary iron content localized in ciliated airway epithelial cells, alveolar macrophages, alveolar type II cells and smooth muscle cells surrounding pulmonary arteries. Increased pulmonary iron levels correlate with an increase in ROS-mediated lipid peroxidation, suggesting that iron-mediated oxidative stress could contribute to the pathogenesis of lung diseases. To investigate this hypothesis, we performed measurements of lung function and blood oxygen saturation. These analyses revealed signs of restrictive lung disease in FPN C326S mice, with a decrease in total lung capacity and lung compliance. Interestingly, when compared with wild-type littermates, FPN C326S mice present a significant reduction in blood oxygen saturation. Experiments are ongoing to better understand the development of restrictive lung disease and decreased blood oxygen saturation.

Sixth Congress of the International BioIRon Society Page 189 Poster Abstracts IBIS

Poster #43 INVESTIGATION OF LINKS BETWEEN HEPCIDIN TRANSCRIPTIONAL CONTROL AND CELLULAR ENERGY STATUS Ana Rita da Silva1,3,4, Katarzyna Mleczko-Sanecka1,4, Amol Tandon2, Sven Sauer2, Matthias W. Hentze3 and Martina U. Muckenthaler1,4 1Molecular Medicine Partnership Unit, Heidelberg, Germany; 2Metabolism Center, University of Heidelberg, Heidelberg, Germany; 3European Molecular Biology Laboratory, Heidelberg, Germany; 4University of Heidelberg, Heidelberg, Germany

Metabolic diseases, such as obesity, metabolic syndrome and diabetes are commonly associated with alterations in iron metabolism. The master regulator of systemic iron homeostasis is hepcidin, which controls systemic iron fluxes. Hepcidin transcriptional control was recently linked to gluconeogenic responses during starvation, as well as to the nutrient-sensing mTOR signaling pathway commonly associated with metabolic alterations. The overall aim of this study is to understand links between iron homeostasis and cellular metabolism. The Krebs cycle is a major pathway for energy production that integrates both anabolism and catabolism of biomolecules. Under metabolic conditions when elevated levels of ATP and citrate are observed, citrate controls a negative feedback loop that inhibits both glycolysis and the Krebs cycle. Since citrate communicates the cellular energy status and regulates energy production, we tested whether it controls endogenous hepcidin mRNA expression. In primary hepatocytes, inhibition of an enzyme that consumes citrate, the ATP citrate lyase, either by RNAi or a small molecule inhibitor, induces hepcidin expression. Moreover, hepatocytes supplemented with citric acid potently induce hepcidin mRNA levels in a time and dose-dependent manner. Importantly, this response is abolished upon RNAi of the plasma membrane citrate transporters corroborating the need for citrate to act intracellularly. As citrate communicates the energy status of the cell, time-resolved experiments are ongoing to analyze the rate of glycolysis and oxidative phosphorylation in response to citrate to link the rate of cellular energy production with hepcidin transcriptional control. We further demonstrate that citric acid supplementation of hepatocytes increases Id1 mRNA levels suggesting activation of the BMP/SMAD signaling pathway. Consistently, RNAi against Bmpr1A and Smad4 mRNA significantly attenuates the hepcidin response to citrate. Taken together, our work suggests that increased citrate levels activate hepcidin expression via the BMP/SMAD signaling pathway. We expect that this work will link the metabolic status of cells with the hepcidin response and may shed light on the question why iron levels are often altered in metabolic disorders.

Sixth Congress of the International BioIRon Society Page 190 Poster Abstracts IBIS

Poster #44 FERROPORTIN AND HEPCIDIN IN RETINAL IRON HOMEOSTASIS Delu Song, MD, PhD1, Milan Theurl2, Esther Clark3, Jacob Sterling3, Sandro Altamura4, Bruno Galy5, Matthias Hentze6, Martina Muckenthaler4, Steven Grieco7 and Joshua Dunaief3 1University of Pennsylvania; 2Department of Ophthalmology and Optometry, Innsbruck Medical University, Innsbruck, Austria; 3F. M. Kirby Center for Molecular Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania, United States; 4Department of Pediatric Hematology, Oncology and Immunology, University of Heidelberg, Germany; 5European Molecular Biology Laboratory, Meyerhofstrasse, Heidelberg, Germany; 6Molecular Medicine Partnership Unit, Heidelberg, Germany; 7Department of Neuroscience, University of Florida, Miami, Florida, United States

Purpose: The retinal localization of Ferroportin (Fpn), the only known cellular iron exporter, is critical for understanding retinal iron flux. Hepcidin produced locally in the retina may regulate retinal Fpn. Methods: Immunofluorescence was used to label Fpn in the retinas of from mice with four different genotypes (WT; Fpn C326S, in which Fpn is resistant to hepcidin regulation; hepcidin knockout; and ceruloplasmin/hephaestin double knockout). AAV2/8- CMV-hepcidin was co-injected with AAV2/8-CMV-GFP into the left eye of C57BL/6J male hepcidin knockout mice (N=10). The right eye received only AAV-GFP as a control (N=10). The retinas were immunostained to assess the levels and localization of iron, ferritin, hepcidin, Fpn, and transferrin receptor. Results: Fpn is on the abluminal side of the outer plexiform layer vascular endothelial cells, on Müller glial cells, and on the basolateral side of the retinal pigment epithelium (RPE). No difference in Fpn levels was observed in the retinal pigment epithelial cells (RPE) or Muller glail cells from above mentioned genotypes. Fpn C326S mice had systemic iron overload and the fastest retinal iron accumulation of any hereditary model studied to date. They also had hypertrophy and autofluorescence of the RPE. Ferritin immunoreactivity in blood vessels, Müller cells and RPE was increased in areas that received co-injection of AAV-GFP and AAV-hepcidin when compared to control retinas that only received AAV-GFP. Moreover, AAV-hepcidin caused a decrease in RPE Fpn and transferrin receptor levels when compared to control retinas. Conclusions: Our results suggest a model in which iron is imported through the retinal vasculature and exported from the basal side of the RPE. Because Fpn immunolabeling was not altered in RPE or Muller glia from mice with hereditary iron overload or those carrying the Fpn C326S mutation, Fpn in these cells may not normally be regulated by endogenous hepcidin. When hepcidin was augmented by gene transfer, it caused elevated ferritin levels in vascular endothelial cells. Retinal hepcidin may normally inhibit retinal iron influx through the retinal vasculature, trapping iron in the vascular endothelial cells.

Sixth Congress of the International BioIRon Society Page 191 Poster Abstracts IBIS

Poster #45 FERRITIN INHIBIT DOXORUBICIN-INDUCED BREAST CANCER CELL DEATH Benjaporn Buranrat1 and James R. Connor21 Faculty of Medicine, Mahasarakham University, 44000, Thailand; 2Department of Neurosurgery, The Pennsylvania State University Hershey Medical Center, Hershey, PA, USA

Ferritin has a wide range of functions in the body including iron storage, iron homeostasis, iron delivery, immunosuppression, angiogenesis, and cell proliferation. Ferritin is overexpressed in many cancer cells, but the functionis still uncertain. In this study, we determined the effects of ferritin in protecting the breast cancer cell line MCF-7 against anticancer drug doxorubicin. The results showed that ferritin treatment activated MCF-7 cell proliferation in a dose- and time-dependent. Inhibition of ferritin heavy chain (FTH) and ferritin light chain (FTL) by siRNA sensitized cells to doxorubicin. Moreover, treatment with doxorubicin alone significantly induced reactive oxygen species (ROS) formation and the cell cycle dependent kinase inhibitor protein p21, whereas ferritin reduced ROS and p21 expression. Conclusion, ferritin plays a critical role in protecting breast cancer cell against the anticancer drug doxorubicin. Targeted reduction of ferritinfrom cancer cell may be a useful strategy for reducing chemoresistance in breast cancer. Keywords: ferritin, doxorubicin, breast cancer, reactive oxygen species

Sixth Congress of the International BioIRon Society Page 192 Poster Abstracts IBIS

Poster #46 THE EFFECT OF OKRA (ABELMOSCHUS ESCULENTUS LINN) AND QUERCETIN ON OXIDATIVE STRESS IN STABLY EXPRESSED HFE NEUROBLASTOMA SH-SY5Y CELL LINE Nootchanat Mairuae, PhD1, Sang Lee, PhD2, Poonlarp Cheepsunthorn, PhD3, Walaiporn Tongjaroenbuangam, PhD4 and James R. Connor, PhD5 1Faculty of Medicine, Mahasarakham University; 2College of Medicine/Milton S. Hershey Medical Center, Hershey, PA, USA; 3Faculty of Medicine, Chulalongkorn University, Thailand; 4Faculty of Medicine, Mahasarakham University, Thailand; 5College of Medicine/Milton S. Hershey Medical Center, Hershey,PA, USA

Iron accumulation in the brain and increased oxidative stress are consistent observations in many neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS). Currently, the evidence has shown that oxidative stress related neurodegeneration is not only caused by disturbed iron metabolism. One mechanism for disrupting iron homeostasis is through expression of the HFE gene variant. Increased incidence of HFE H63D variant has been reported in AD and ALS. In this study, we investigated the possibility that the anti- inflammatory folk medicine okra (Abelmoschus esculentus) and quercetin could mitigate oxidative stress in HFE stably expressing SH-SY5Y cells. The results showed that treatment of H63D HFE cells either with okra or quercetin significantly decreased reactive oxygen species (ROS), hydrogen peroxide (H2O2) and protein oxidation compared to untreated cells. We also established that okra treatment dramatically decreases intracellular iron levels in H63D cells compared to untreated cells. These results suggest okra may be beneficial in people expressing the H63D variant to reduce the risk of neurodegenerative diseases related to oxidative stress. Further in vivo studies would help confirm this.

Sixth Congress of the International BioIRon Society Page 193 Poster Abstracts IBIS

Poster #47 REGULATION OF HEPCIDIN BY INFLAMMATION IN IMMUNE CELLS Rafiou Agoro, MSC1 and Catherine Mura, PhD21 UMR-7355 CNRS Orleans; 2CNRS – Orleans

Hepcidin has been isolated on the basis of its antibacterial peptide structure. Hepcidin is a 25 amino acid peptide sharing a characteristic structure with other antibacterial peptides capable of disrupting bacterial membranes. Hepcidin also acts as an iron regulatory hormone binding to the transmembrane ferroportin iron exporter resulting in a decrease in iron export from enterocytes and macrophages to the circulating blood. Hepcidin expression is regulated mainly at the transcriptional level by iron and inflammation. BMP receptor type I /smad1/5/8/4 pathway can be activated by BMP6 released factor correlated to iron loading and by iron-saturated transferrin interacting with TfR2/HFE. Inflammation upregulates hepcidin expression via cytokines, like IL-6, and also via activin b which activates BMP receptor. Here, the molecular mechanism of hepcidin mRNA regulation in response to inflammation has been studied. We used C57BL/6 wild type mice as well as TLR4, MyD88, TRIF deficient and double deficient MyD88/TRIF mice treated with LPS agonist for in vivo study. We analyzed hepcidin mRNA levels in liver, peritoneal macrophages. We also used bone marrow derived macrophages (BMDM), dentritic cells (BMDC), and RAW264.7 macorphage-like cells to study the molecular pathway of hepcidin regulation upon inflammation. RAW264.7 cells were treated with various PAMPs and DAMPs (LPS, MPLA, Pam3CSK4, poly(I:C), R848, Imiquimod, CpG, ATP, ADP) to analyze the transduction signal pathway and expression of hepcidin. In mice, hepatic hepcidin mRNA levels correlated with hepatic iron content, and hepcidin is upregulated in liver and peritoneal macrophages after systemic LPS treatment. In the liver, inflammation-induced hepcidin upregulation can be exacerbated in vivo by iron loading and antagonized by iron deficiency. In peritoneal macrophages, BMDM, and BMDC the hepcidin mRNA basal levels are much lower than in the liver, but the inflammation-induced hepcidin upregulation is much drastic. Inflammation-induced hepcidin mRNA upregulation was efficiently prevented in the absence of Tlr4, double deficiency MyD88/TRIF or Myd88, but not in TRIF deficient mice. In RAW264.7 monocytic cells incubated with ferric ammonium citrate (FAC) or holo-transferrin (holo-TF) did not significantly affect hepcidin mRNA levels. However, LPS combined with FAC or holo-TF led to an enhanced of upregulation hepcidin mRNA compared to LPS alone, as observed in vivo. In addition to LPS agonist of Tlr4, hepcidin can be upregulated by different TLR agonists (MPLA, Pam3CSK4, R848, Imiquimod, CpG) through MyD88-adaptor, but not TRIF or other pathways of signal transduction. Due to the dual role of hepcidin, hormone for systemic iron regulation and antimicrobial peptide, the hepcidin response to inflammation stimulators in liver and antigen presenting cells (APCs) can play a differential role on invading bacteria. The APCs may provide high local hepcidin release to neutralize bacteria.

Sixth Congress of the International BioIRon Society Page 194 Poster Abstracts IBIS

Poster #48 EVALUATION OF THE PHOTOPROTECTIVE POTENTIAL OF NOVEL HEXADENTATE IRON CHELATORS AGAINST SUNLIGHT-INDUCED DAMAGE TO SKIN CELLS Olivier Reelfs, PhD, DSc2, Vincenzo Abbate, PhD3, Robert C. Hider, PhD3 and Charareh Pourzand, PhD, DSC1 1University of Bah; 2University of Bath; 3King's College London

We have previously demonstrated that exposure of human skin cells to physiologically relevant doses of Ultraviolet A (UVA, 320-400 nm), the oxidising component of sunlight, leads to immediate and dose-dependent increases in labile iron (LI) [1]. We further showed that at the cellular level, UVA-mediated release of reactive LI plays a key role in the increased susceptibility of skin cells to UVA-mediated oxidative membrane damage and necrotic cell death and is likely to exacerbate damage caused by further exposure [2, 3]. The strong hexadentate iron chelator desferrioxamine (DFO) has been shown to suppress cell damage and necrotic cell death in skin cells by moderating the amount of intracellular labile iron pool [2-4], but because of its hydrophilic nature and high molecular weight, this compound has not been pursued further for skin photoprotection via topical application. Bidentate and tridentate chelators with appropriate lipohilicity and molecular weight may be better candidate photoprotectants but at low iron concentrations, ligands such as catechols and hydroxypyridinones are not powerful iron scavengers [5]. We hypothesized that hexadentate analogues, designed in a similar fashion to siderophore structures [6] are likely to be more effective in this role, although inevitably they possess a higher molecular weight. In the present study, we designed and synthesized a series of novel hexadentate tri-catechol- and tri-hydroxypyridinone-based iron chelators. We then evaluated their photoprotective potential against UVA-induced oxidative damage and cell death, using cultured primary skin fibroblasts FEK4 as a cell model. Time-course and dose- response experiments using the spectrophometric MTT and flow cytometric Annexin V/propidium iodide uptake assays revealed that these compounds are not significantly toxic in skin fibroblasts up to the tested concentration of 50 uM. Following UVA irradiation, only tri-catechol-based chelators provided significant protection against oxidative damage and necrotic cell death. The protection afforded was markedly higher than that observed with DFO and the photoprotective effect of the tri-catechol-based chelators was related to their iron-chelating properties, as saturation of the compounds with iron abolished their protective action against UVA-induced oxidative damage and cell death. In conclusion, the novel family of tri-catechol-based iron chelators described herein shows promising properties giving them potential for use in protecting skin against the deleterious effects of sunlight. [1] Pourzand C, Watkin RD, Brown JE and Tyrrell RM (1999). Proc Natl Acad Sci. USA 96(12), 6751-6756. [2] Zhong JL, Yiakouvaki A, Holley P, Tyrrell RM and Pourzand C (2004). J. Invest. Dermatol. 123(4), 771-780. [3] Aroun A, Zhong JL, Tyrrell RM and Pourzand C (2012). Photochem. Photobiol. Sci. 11(1):118-34. [4] Reelfs O, Eggleston IM and Pourzand C (2010) Curr. Drug Metab. 11(3):242-249. [5] Liu ZD and Hider RC (2002). Med Res Rev 22(1):26-64. [6] Hider RC and Zhou T (2005). Ann N Y Acad Sci 1054:141-54.

Sixth Congress of the International BioIRon Society Page 195 Poster Abstracts IBIS

Poster #49 DEFERIPRONE AND DEFERASIROX AS PROMISSING CANDIDATE LIGANDS FOR SKIN PHOTOPROTECTION BY LIGHT-ACTIVATED CAGED-IRON CHELATORS Sharareh Houshmandyar, MSc, Olivier Reelfs, PhD, DSc, Tina Radka, PhD, Ian M. Eggleston, PhD and Charareh Pourzand, PhD, DSc University of Bath

In order to protect cells from the skin against iron-catalysed damage caused by ultraviolet A (UVA, 320-400nm), the oxidising component of sunlight [1,2], we have recently developed a series of light-activated caged iron chelators (CIC). CIC strategy was achieved by blocking the critical iron-binding function of two chosen parental iron chelators of the isonicotinoyl hydrazone family PIH and SIH with a photo-labile caging group. CIC molecules, whose activity is released on demand in situ in response to UVA [3,4], allow safe delivery of iron chelators that may otherwise cause toxicity to skin cells by starving them of the essential iron required for various cellular functions, notably cell division. The success of the CIC strategy depends crucially on the choice of the parental chelator selected for caging as its inherent toxicity/antiproliferative activity may still become an issue upon release by UVA. As a first step towards identifying the best ligand(s) for caging purposes, we evaluated the cytotoxicity and photoprotective potential of two well-known clinical iron chelators deferiprone (DFP) and deferasirox (DFX) in primary skin fibroblasts in culture. The high selectivity of these ligands for iron as well as their inherent lipophilic nature make them suitable for skin photoprotection via topical delivery. The caging of such chelators has the advantage of further increasing their lipophilicity and therefore improving their delivery to the skin. Skin fibroblasts were incubated with a range of concentrations of the ligands for 24-48 hours and the level of cytotoxicity was assessed using the MTT assay. Neither of the ligands exhibited significant cytotoxicity up to the tested concentration of 100 uM. The evaluation of photoprotective potential of the ligands was performed 24 and 48 hours after UVA irradiation using the spectrophotometric MTT and flow cytometric Annexin V/propidium iodide uptake assays. The results revealed that both ligands provide substantial protection against UVA-induced oxidative damage and cell death at the highest concentration tested of 100 uM. These results suggest that both DFP and DFX are good candidate ligands for caging and the CIC approach in skin photoprotection. [1] Pourzand C, Watkin RD, Brown JE and Tyrrell RM (1999). Proc Natl Acad Sci. USA 96(12), 6751-6756. [2] Zhong JL, Yiakouvaki A, Holley P, Tyrrell RM and Pourzand C (2004). J. Invest. Dermatol. 123(4), 771-780. aoa , ao , l-Qenaei A, Dowden J, Pourzand C (2006). J Invest Dermatol 126(10):2287-95. [4] Reelfs O, Eggleston IM and Pourzand C (2010) Curr. Drug Metab. 11(3):242-249.

Sixth Congress of the International BioIRon Society Page 196 Poster Abstracts IBIS

Poster #50 IRON DEFICIENCY AND RESTLESS LEGS SYNDROME. A PROSPECTIVE STUDY IN BLOOD DONORS. Marta Clavero Olmos, Nadia Matskiv, Ines Valdes Gross, Ana López Aparicio, Alejandro García-Espona Pancorbo, Rosa Peraita and Alejandro del Castillo Rueda Hospital General Universitario Gregorio Marañón

INTRODUCTION Restless legs syndroe s stroly led to ro deey elar lood doato ay lead to ro deey Contradictory results in previous studies in the scientific literature are found; some studies suggest an increased risk of RLS in blood donors, and others, fail in finding any relation. This kind of study has not been replicated in Spain, where blood donation is altruistic and tightly regulated. METHODS We designed a prospective study in two Blood Donation Units of the Public Health-Care System in Madrid (Spain), to study iron metabolism and the prevalence of RLS symptoms in blood donors. All blood donors underwent a physical examination including blood pressure, body mass index (BMI) and capillary haemoglobin (HB) test (discarding for donation those with HB<12 g/dl). The study was approved by the hospital Ethic’s Committee. Sample recruitment, after signing the Informed Consent, underwent a four issues questionnaire about RLS symptoms and an additional blood test to analyze serum ferritin levels. RESULTS 179 blood donors were included in the study, 101 women (56%), and 78 men (44%). Mean age was 43.18 years IC 95% (41.46-44.90) and mean BMI was 25.86 kg/m2 IC 95% (25.20-26.52). Blood donors were divided in two groups: first time donors (n=44, 24.58%) and regular blood donors (n=135, 75.42%); the average in donating time (in years) was 10.88 IC 95% (9.29-12.47) and the average of donations/year 2.059 IC 95% (1.89-2.23). Mean serum ferritin levels were 48.69µg/L, IC 95% (40.05-57.33) in women, and 131.81 µg/L, IC 95% (110.06-153.5) in men. 79 donors (44.4%) had serum ferritin levels < 50 µg /L, with predominance of women (n=50, 64.9%) and regular donors (n=66, 49.3%). 7 donors (4%) (all of them regular donors), (5 F/ 2M) had serum ferritin levels < 12 µg /L. Mean serum ferritin levels in first time donors were 136.47 µg/L, IC 95% (101.89-171.05) and 79.72 µg/L in regular donors, IC 95% (67.60-91.83) P=0.007. Twenty-eight blood donors (15.4%, 18 women) answered ‘yes’ to at least one of the questionnaire issues.15 donors answered ‘yes’ to 3-4 questions (53.6%). Women had a non-significant higher risk to have at least one RLS symptom when adjusting for all variables (age, ferritin, BMI, first or regular donors), p=0,053. We did not find any association neither between low serum ferritin levels and RLS symptoms (p=0.97), nor between serum ferritin < 50 µg /L and RLS symptoms (p=0.88). DISCUSSION It is not usual to assess iron metabolism in Spain blood donation units, where the only item which can discard donation is HB. Normal HB values can coexist with iron deficiency and unleash RLS. Nevertheless we could not demonstrate this second item. In our study there is a high prevalence of low serum ferritin levels, but this does not increase RLS prevalence. CONCLUSIONS Regular blood donation, when regulated in similar conditions as in Spain, reduces iron stores to ferritin concentration < 50 µg/L but this fact does not increase the risk of RLS symptoms. Women donors have lower ferritin values, and a higher risk to have, at least, one RLS symptoms.

Sixth Congress of the International BioIRon Society Page 197 Poster Abstracts IBIS

Poster #51 PREVALENCE OF HYPOFERRITINEMIA IN BLOOD DONORS Marta Clavero Olmos, Nadia Matskiv, Ines Valdes Gross, Ana López Aparicio, Alejandro García-Espona Pancorbo, Rosa Peraita and Alejandro del Castillo Rueda Hospital General Universitario Gregorio Marañón

INTRODUCTION: It is well known that continuous blood extractions decrease significantly iron deposits. Despite this fact, it is not usual to assess iron status in Spain blood donation units, where the only item in iron metabolism which can discard donation is the presence of anemia, defined as hemoglobin (HB) levels under 12 g/dL. METHODS: We designed a prospective study in two Blood Donation Units of the Public Health-Care System, in Madrid (Spain), to study iron status. We also analyzed if the iron deficiency could have discarded the donation. The study was approved by the hospital’s Ethic Committee. All blood donors underwent a physical examination including blood pressure, body mass index (BMI) and capillary haemoglobin test. All donors with HB under 12 g/dL were excluded. Sample recruitment, after signing the Informed Consent, underwent an additional blood test to determine serum ferritin levels, transferring saturation (TfS) and soluble transferrin receptors. Donors with hypoferritinemia were followed-up and received treatment with oral or parenteral iron supplements. RESULTS: 179 blood donors were included in the study, 101 women (56%), and 78 men (44%). Mean age was 43.18 years IC 95% (41.46-44.90) and mean BMI was 25.86 kg/m2 IC 95% (25.20-26.52). Blood donors were divided into: first time donors (n=44, 24.58%) and regular blood donors (n=135, 75.42%), where the mean years donating was 10.88 IC 95% (9.29- 12.47) and the average of donations/year was 2.059 IC 95% (1.89-2.23). Mean serum ferritin levels were 48.69µg/L, IC 95% (40.05-57.33) in women, and 131.81 µg/L, IC 95% (110.06-153.5) in men. 79 donors (44.4%) had serum ferritin levels < 50 µg /L, with predominance of women (n=50, 64.9%) and regular donors (n=66, 49.3%). 7 donors (4%), (5 F/ 2M) had serum ferritin levels < 12 µg /L, all of them regular donors. Mean serum ferritin levels in first time donors were 136.47 µg/L, IC 95% (101.89-171.05) and 79.72 µg/L in regular donors, IC 95% (67.60-91.83) P=0.007.An association between serum ferritin levels and high BMI was found (Rho Sperman 0.298, p< 0.001). DISCUSSION: Hypoferritinemia is expected to be present in usual blood donors. This could unleash anemia and eliminate potential blood donors from the donating system. Some authors promote a systematic use of iron supplements in donors. This strategy may also be related to side effects, since long-term consequences are not well known. It may be indicated to assess iron metabolism values in high-risk donors, in which rapid and appropriate supplementation could avoid altruist blood donors looses. CONCLUSIONS: Regular blood donation, when regulated in similar conditions as in Spain, reduces iron stores to ferritin concentration < 50 µg/L. Women donors have lower serum ferritin values, in contrast to high BMI, which have higher serum ferritin levels. We recommend to investigate iron status in high-risk donors, in order to initiate prompt supplementation which could help to maintain blood donation habits.

Sixth Congress of the International BioIRon Society Page 198 Poster Abstracts IBIS

Poster #52 REVISITING THE ROLE OF THE HFE IN ENTEROCYTES Betül Alan, MSc and Maja Vujic Spasic, Prof Dr Institute for Comparative Molecular Endocrinology, University of Ulm

Hfe-Hereditary Hemochromatosis (Hfe-HH) is a prevalent iron overload disorder characterised by low expression of iron- hormone hepcidin which signals for increased intestinal iron absorption and progressive iron accumulation in numerous tissues. While hepatic-Hfe is required to control hepcidin expression and thereof the proper distribution of iron in the body, little is known on the role of Hfe in the enterocytes. We previously demonstrated that the lack of Hfe in the duodenum of young mice does not impair hepcidin expression (Vujic Spasic et al., 2007) thus excluding the function of duodenal-Hfe in hepcidin control. Yet, whether duodenal-Hfe may play a role in regulating iron metabolism independently of hepcidin has not been addressed. Based on the data that the onset of Hfe-HH in patients occurs in late adulthood, we postulated that the effects of duodenal-Hfe on modulating iron homeostasis may correlate with aging. We therefore set to investigate the impact of the Hfe in the duodenum using mice deficient for Hfe exclusively in enterocytes (HfeVillinCre mice) at 60 weeks of age by using quantitative real-time PCR and Western Blot analysis. In contrast to our previous analyses which demonstrated that duodenal-Hfe is not required to maintain physiological iron homeostasis in young mice (Vujic Spasic et al., 2007), we observed profound changes in systemic iron homeostasis in aged HfeVillinCre mice. More precisely, deletion of Hfe in the enterocytes led to increased expression of major intestinal iron transporter genes (Dcytb, TfR1, DMT1, Slc40a1) suggesting an increased iron uptake, the finding also observed in Hfe-/- mice. Consequently, increased iron uptake resulted in significant iron accumulation in the liver, kidney, lung, heart and spleen of HfeVillinCre mice, reflecting the systemic iron overload condition. In line with this, 3-fold (p < 0.02) increase in hepatic hepcidin expression was measured suggesting that i) the lack of duodenal-Hfe does not impair hepcidin expression, and more importantly, that ii) elevated levels of hepatic hepcidin are not sufficient to prevent uncontrolled iron- uptake in HfeVillinCre mice. Intriguingly, iron overload in the spleen of HfeVillinCre mice is likely to be caused by increased uptake of non-transferrin-bound iron (i.e. via heme-scavenging pathway) as increased expression of the heme-scavenger hemopexin and its receptor CD91 was measured. By contrast, TfR1-mediated iron uptake pathway was unaffected in the spleen of HfeVillinCre mice (nc in TfR1, Steap3, Nramp1). Similar to our observation in the spleen, we observe elevated expression of heme transporter genes, such as HCP-1, Abcg2 and Flvcr1, in the duodenum of HfeVillinCre mice suggesting for the elevated heme-iron uptake by duodenal cells as well. Our data implicate for a previously unrecognised role for Hfe in the duodenum required to maintain systemic iron homeostasis independent of hepcidin. We speculate that Hfe may impair iron metabolism by interacting with possible heme transporters thereby regulating the heme-iron uptake and this occurs in hepcidin-independent manner.

Sixth Congress of the International BioIRon Society Page 199 Poster Abstracts IBIS

Poster #53 FUNCTIONAL PROPERTIES OF DIVALENT METAL TRANSPORTER 1 (DMT1) ON THE OUTER MITOCHONDRIAL MEMBRANE (OMM) Michael Garrick, PhD1, Andrew Ghio, MD2, Lin Zhao, MD1, Stephen T. Koury, PhD1, Natascha A. Wolff, PhD3, Frank Thévenod, MD, PhD3, Sultan Chowdhury, PhD4, Alison B. Cutts, PhD4, Jay A. Cadieux, PhD, PMP4, Y. Paul Goldberg, MD, PhD4, Charles Cohen, MD, PhD4, James F. Collins, PhD5 and Laura M. Garrick, PhD1 1University at Buffalo; 2USA EPA; 3Witten/Herdecke University; 4Xenon Pharmaceuticals; 5University of Florida

DMT1 is an importer of ferrous iron and other metals on plasma membranes and an exporter of the same metals on endosomal membranes. We have previously shown that DMT1 is also present on the OMM by multiple approaches [1,2]. Mitochondria are the site of most intracellular iron flux; yet the mechanism for iron entry through the OMM is uncertain even though the redox activity of mitochondria makes careful management of iron important there. We proposed that iron and other metals enter this organelle via DMT1. We present here evidence that OMM DMT1 carries out such a function. DMT1 has four major isoforms differing at the 5’ and 3’ ends of the mRNA where the options are exons 1A or 1B and 16 or 17, respectively. Exon 16 in the extended version contains an iron responsive element (IRE) while it does not when truncated and followed by exon 17 so 1A/+IRE, 1A/-IRE, 1B/+IRE and 1B/-IRE designate the options. We relied on human embryonic kidney cells engineered to overexpress the 1A/+IRE and 1B/-IRE isoforms in response to doxycycline induction for many of the experiments. Mitochondria from these cells import very little 54Mn unless the cells were induced to express DMT1. Incorporation is time dependent and more rapid at pH 6 than 7.4. It is readily blocked by Xen602, a specific DMT1 inhibitor. The inhibitor is more effective when mitochondria express 1A/+IRE DMT1 than when they express 1B/-IRE DMT1, a result that also occurs with intact cells. Mitochondrial import is also much more sensitive to an uncoupler than cellular uptake. We predict that OMM DMT1 levels will be particularly elevated when the mitochondria originate from a tissue where iron flux is high due to energy demands or high levels of heme synthesis in mitochondria. Testing that prediction, we found high levels in kidney derived mitochondria [1,2] and now similarly find high levels in murine erythroleukemia cells after induction to erythroid differentiation and in enterocytes after isolation from iron-deficient rats. When the mitochondria come from the kidneys of rats homozygous for the Belgrade mutation (G185R in DMT1), they have undetectable uptake of Mn; but when from the kidneys of wild type (+/+) rats, mitochondria have substantial Mn import activity. Recently we have begun to test for mitochondrial iron import by multiple assays. The results support the argument that OMM DMT1 is not only capable of importing Mn into mitochondria, but it also allows Fe to pass into that organelle. 1. Wolff et al. (2014) FASEB J 28:2134-45. 2. Wolff et al. (2014) Channels 8:458-66.

Sixth Congress of the International BioIRon Society Page 200 Poster Abstracts IBIS

Poster #54 COMPARATIVE EVALUATION OF MANAGEMENT BY MACROPHAGES OF INTRAVENOUS PHARMACEUTICAL IRON FORMULATIONS James Connor, PhD1, Xuesheng Zhang, PhD1, Anne Nixon, BS1, Becky Webb, BS1 and Joseph Perno, MD, PhD2 1Penn State University College of Medicine; 2Luitpold Pharmaceuticals, Inc.

There is a significant clinical need for an effective treatment of iron deficiency in a variety of diseases. Iron absorption at the level of the gut is tightly regulated; attempts to treat iron deficiency through oral compounds have not been routinely successful, and the treatment itself results in unpleasant side effects. As a result, a number of iron compounds that can be administered intravenously have been developed. There are little data, however, on how intravenous delivery of iron, which may provoke oxidative stress in both cells and tissue, is handled in the body. The mechanism by which the body handles intravenous iron injections is thought to begin in the reticuloendothelial system. Macrophages are known to acquire iron under multiple physiological and clinical conditions through phagocytosis, but the release of that iron to participate in iron replenishment is poorly if at all understood. Therefore we examined the ability of the macrophages to acquire iron from various iron formulations [iron dextran, iron sucrose, sodium ferric gluconate, ferumoxytol, iron isomaltoside and ferric carboxymaltose] that are in use clinically and how they impact cytokine release and oxidative stress in macrophages. Analyses were performed for indices of oxidative stress within macrophage mitochondria and cytoplasm. Compromise to cell membrane integrity was measured by lactate dehydrogenase (LDH) release and cell viability monitored by MTT assay. Ferroportin and H-ferritin expression and secretion, cytokine secretion, and iron uptake and release by macrophages were also determined. In the macrophages, loss of cell viability was limited to iron sucrose although LDH release occurred at low concentrations for sodium ferric gluconate and eventually for all compounds except ferric carboxymaltose. Iron uptake was compound dependent; best for ferumoxytol and iron isomaltoside and least for iron sucrose and sodium ferric gluconate. Iron was released from macrophages as secretion of ferritin or as ferrous iron via ferroportin. However, the release of ferrous iron from macrophages could be blocked by hepcidin. Exposure of arophaes to err aroyaltose ad ro detra reslted a reased release o tor eross ator α Finally, direct exposure of iron compounds to macrophages resulted in no significant changes in their oxidative stress. The results of this study have shown that the compounds can differ in their cellular uptake, cytotoxicity profile and release of inflammatory cytokines, but not oxidative stress. The data clearly demonstrates that iron released from macrophages is possible in two modalities following uptake of iron from the various compounds. Macrophages are likely to be critical for managing the iron effectively in vivo and there was a clear difference in the iron uptake and release in the compounds. The ability of hepcidin to block iron release from macrophages is relevant to the potential efficacy of the use of these iron compounds under inflammatory conditions and may work to diminish some of the effect of intravenous iron.

Sixth Congress of the International BioIRon Society Page 201 Poster Abstracts IBIS

Poster #55 DCYTB EXPRESSION DURING ERYTHROPOIESIS eewon Choi, MSc and G.O. Latunde-Dada, PhD King's College London

DCYTB a ferric reductase, plays an important role in the process of iron absorption. While STEAP3 has been implicated in erythropoeisis, that of DCYTB has not been defined. The aim of this project was to study the effect of cell differentiating compounds on DCYTB and STEAP3 mRNA expression and protein function in erythropoeitic cell lines. Three different human erythropoeitic lines namely K562, U937 and HL-60, were used in this study. HT-29 cancer cells and HuTu-80 cells were used as a duodenal cell for comparison. The cells were treated with butytate, and hemin, succinyl acetone and DMSO. DCYTB and STEAP3 mRNA expression was determined by quantitative real-time polymerase chain reaction (qPCR). Ferric reductase assay was carried out to confirm protein function with ferrous-ferrozine complex formation using the spectrophotometer. Erythropoeitic cell lines treated with butyrate exhibited a decrease in both DCYTB and STEAP3 mRNA expression. Furthermore, HuTu-80 cells treated with butyrate also showed a decrease in DCYTB mRNA expression, although STEAP3 mRNA expression was increased in these cells. The effect of hemin alone and in combination with succinyl acetone showed an increased trend in up-regulation of DCYTB and STEAP3 mRNA levels in HL-60 cells, while the results in other cells were not consistent. There were gene specific differences in DCYTB and STEAP3 mRNA levels. Ferric reductase activity in erythropoeitic cell lines was not significantly influenced by butyrate. In conclusion, both DCYTB and STEAP3 mRNA expression was down regulated by butyrate in erythropoeitic cell lines. DCYTB mRNA expression was also down regulated by butyrate in HuTu-80 cells. Hemin seems to enhance DCYTB and STEAP3 mRNA levels in erythropoeitic cells. DMSO shows a time-course down regulation of STEAP3 mRNA levels in K562 cells. DCYTB could also exert ferric reductase activities in humans during erythropoiesis. References Choi J, Masaratana P, Latunde-Dada GO, Arno M, Simpson RJ, McKie AT. (2012). Duodenal reductase activity and spleen iron stores are reduced and erythropoiesis is abnormal in Dcytb knockout mice exposed to hypoxic conditions.J Nutr. 142(11):1929-34. Su D, May JM, Koury MJ, Asard H.(2006). Human erythrocyte membranes contain a cytochrome b561 that may be involved in extracellular ascorbate recycling. J Biol Chem. 281(52):39852-9. Lambe T, Simpson RJ, Dawson S, Bouriez-Jones T, Crockford TL, Lepherd M, Latunde-Dada GO, Robinson H, Raja KB, Campagna DR, Villarreal G Jr, Ellory JC, Goodnow CC, Fleming MD, McKie AT, Cornall RJ.(2009). Identification of a Steap3 endosomal targeting motif essential for normal iron metabolism. Blood. 113(8):1805-8.

Sixth Congress of the International BioIRon Society Page 202 Poster Abstracts IBIS

Poster #56 THE PREDICTION OF FLUORINATED 3-HYDROXYPYRIDIN-4-ONE LOG P VALUES Yu-Lin Chen, Dr1, Dave Barlow, Dr2, Yongmin Ma1 and Robert Hider, Dr31 Zhejiang Chinese Medical University; 2King's College London; 3King's College London

There are many putative therapeutic applications for iron-chelating agents. The fluorinated 3-hydroxypyridin-4-ones (HPOs) achieve the improvement of pharmacokinetic profile and blood brain barrier permeability. The further optimisation of HPO structures for drug candidates is still required. The partition coefficient, P, is one of fundamental properties for drug discovery. In this study, the log P values of 38 HPOs were predicted using nine existing methods, CLogP, miLogP, ALOGPs, AC_logP, ALOGP, MLOGP, KOWWIN, XLOGP2 and XLOGP3. The insufficient prediction result for all 38 HPOs was obtained by the methods. The HPOs were further divided into two groups, 24 non-fluorinated (NF) and 14 fluorinated (F) HPOs because the fluorine atom possesses the highest electronegativity, which significantly affects the electron distribution on the HPO ring. The adequate prediction result for 24 NFHPOs were obtained by the existing methods, the best of which was miLogP. In contrast, the result for 14 FHPOs remained poor. Subsequently, a novel property-based log P prediction method using stepwise multiple linear regression for 14 FHPOs was developed with sufficient accuracy (r2: 0.98 and mean of absolute deviations: 0.06 log units). The predictive power of this model was further tested using leave- one-out cross validation and the result was acceptable. The success of this model can be attributed to two main aspects. Firstly, the targeted structures are relatively small and rigid. Secondly, the chosen molecular descriptors can distinguish the structures. Interestingly, the log P value of FHPOs can be described as a linear combination of radial basis functions. In the future, FHPO candidates can be screened using this model before chemical synthesis.

Sixth Congress of the International BioIRon Society Page 203 Poster Abstracts IBIS

Poster #57 MUTATION SCREENING IN CHINESE PATIENTS AFFECTED BY HEREDITARY HEMOCHROMATOSIS Yongwei Wang1, Gang Liu, PhD2, Bing Han, PhD, MD3, Yangzhong Chang, PhD4 and Guangjun Nie, PhD2 1CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology; 2National Center for Nanoscience and Technology; 3Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100730, China; 4Laboratory of Molecular Iron Metabolism, College of Life Science, Hebei Normal University, Shijiazhuang, 050024, Hebei Province, China

Hereditary hemochromatosis is a group of inherited iron overload disorders associated with pathogenic defects in the genes of hemochromatosis (HFE), hemojuvelin (HJV/ HFE2), transferrin receptor 2 (TfR2), ferroportin (FPN1), ferritin, heavy polypeptide 1 (FTH1) or hepcidin (HAMP). HJV is a bone morphogenetic protein (BMP) co-receptor which could form a membrane-associated protein complex with HFE and TfR2 that positively regulates the transcription of hepcidin via the BMP-SMAD signaling pathway. FTH1 is the subunit of ferritin, which is responsible for the iron storage. Ferroportin is the only identified iron efflux protein and is very important for iron release into the circulation and is regulated by hepcidin to maintain body iron homeostasis. Pathogenic mutations in HJV and HAMP genes cause a juvenile form of hereditary hemochromatosis with loss of function or low level of hepcidin in liver, while the mutations identified in the HFE and TfR2 genes lead to iron deposition in the liver and other organs in adults. Herein, we identified two putative pathogenic mutations (c. 187 C>G, c. 325 C>T) in HFE gene, a putative pathogenic mutation (c. 714 C>G) in TfR2 gene, a putative pathogenic mutation (c. 626 C>T) in FPN1 gene and five putative pathogenic mutations (c. 9 G>C, c.18 G>C, c.860 T>G, c. 962 G>A, c.963 C>A) in HJV gene in a cohort of 12 Chinese patients affected by hereditary hemochromatosis and depicted two family pedigrees with unique features. Furthermore, our data showed that there is a 400 kb deletion in the HJV gene from one of the patients. We also investigated the possible molecular mechanism of the deletion for the severe iron overload phenotype via whole genome sequencing. Taken together, our study provides new insights about hereditary hemochromatosis in Chinese population.

Sixth Congress of the International BioIRon Society Page 204 Poster Abstracts IBIS

Poster #58 DEVELOPMENT OF PEPTIDE THERAPEUTICS FOR IRON DISORDERS Daniela Goncalves, MSc1, Gautam Rishi, PhD2, Randy Aliyanto, BSc1, Johan Rosengren, PhD1, Nathan Subramaniam, Prof2, David Frazer, PhD2 and Richard Clark, PhD1 1School of Biomedical Sciences, University of Queensland; 2QIMR Berghofer Research Institute, Brisbane

Inadequate modulation of the ferroportin-hepcidin axis brings about two phenotypically contrasting groups of disorders, one in which increased hepcidin levels cause or contribute to iron-restrictive anaemias, and another characterised by hepcidin deficiency, which underlies the iron overload observed in nearly all forms of hereditary haemochromatosis. This research concerns the development of peptide therapeutics based on two distinct methodologies. The first focuses on HFE, the most frequently mutated protein in haemochromatosis, and the molecular function of which is still largely unclear. Briefly, peptide entities able to disrupt the interaction between HFE and transferrin receptor 1 (TfR1) will be explored with the intent of increasing hepcidin transcription. This would be of use not only to investigate HFE- mediated signalling, but also as a potential therapy for iron loading disorders. On the basis of the crystal structure of the HFE/TfR1 complex (Bennet, 2000), a series of peptides was designed and synthesised by automated Fmoc-based solid-phase peptide synthesis. Testing is being carried out using an in situ proximity ligation assay (Duolink®) and Hepa1-6 cells stably expressing FLAG-tagged mouse HFE and endogenous TfR1. The results to date are encouraging with one of the peptides showing a significant decrease in HFE/TfR1 complex formation, relative to both untreated and non-specific peptide-treated Hepa1-6 cells. The second approach entails hepcidin replacement. Mini-hepcidins are small, rationally designed, hepcidin agonists amenable to optimization of activity and drug-like properties, and thus are potentially applicable to the treatment or prevention of iron overload. In this strand, end-to-end cyclisation and N-methylation of mini-hepcidins is being examined for the introduction of favourable conformational restrictions and optimisation of biostability, whilst retaining target affinity and specificity. A series of mini-hepcidins has been synthesised via Fmoc-based solid-phase peptide synthesis, and tested for their activity in a cell-based assay in which the decrease in GFP-tagged ferroportin is measured by flow cytometry. The results regarding the introduction of end-to-end macrocyclisation are promising, with cyclic mini-hepcidin 9 (DTHFPICIF) showing the same level of activity of its linear counterpart. Regrettably, cyclic doubly N-methylated analogues appear to be approximately 10-fold less active than cyclic mini-hepcidin 9, but some loss in activity would be acceptable if the peptide series is shown to be pharmacologically superior. In the immediate future, a second set of inhibitors for HFE/TfR1 complex formation will be examined. In addition, the remaining analogues in the mini-hepcidin series will be synthesised and tested, not only for bioactivity, but also for stability and membrane permeability. NMR analysis will then be used to correlate the structure and conformational restrictions of the peptides with their physiological properties.

Sixth Congress of the International BioIRon Society Page 205 Poster Abstracts IBIS

Poster #59 UNRAVELLING THE ROLE OF OXIDATIVE DAMAGE AND REPLICATION STRESS IN THE INDUCTION OF DNA DAMAGE RESPONSE SIGNALING IN DMT1-DEFICIENT HEMATOPOIESIS Monika Horvathova, Zuzana Zidova, Katarina Kapralova, Leona Raskova-Kafkova, Zuzana Somikova, Jana Kucerova and Vladimir Divoky Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic

Divalent metal transporter 1 (DMT1) is essential for intestinal iron absorption and erythroid iron utilization. We have previously shown that DMT1 deficiency impairs erythroid differentiation, induces apoptosis of erythroid precursors and causes suppression of colony-forming capacity of erythroid progenitors. Decreased hemoglobinization of DMT1-mutant burst forming unit-erythroid (BFU-E) colonies was associated with the activation of heme-regulated inhibitor (HRI) kinase, which specifically phosphorylates the alpha st o the earyot tato ator eα ad atteates lo translation. In addition, we observed that DMT1-mutant red blood cells (RBCs) have higher levels of reactive oxygen species (ROS) than wild-type (wt) RBCs despite significantly increased activity of anti-oxidative defense enzymes. This indicates that exaggerated anti-oxidative defense in DMT1-mutant RBCs is not sufficient to eliminate ROS effectively. The contributing factor, causing ROS generation that exceeds anti-oxidant capacity of the DMT1-mutant erythrocyte defense system, is likely tissue hypoxia, as documented by increased expression of hypoxia target genes in DMT1-mutant reticulocytes. In this study, we sought to determine whether and how DMT1-mutant proliferating hematopoietic cells cope with iron deficiency and oxidative damage. For these analyses we used fetal liver (FL) erythroblasts or nucleated bone marrow (BM) cells derived from DMT1-tat e e rst ored reased phosphorylato o eα DMT1-mutant FL cultures, both undifferentiated and those differentiated for 48 hours. In DMT1-mutant mice the attenuated globin translation was associated with BM hypoxia as documented by increased expression of hypoxia-target genes. Previous studies have deostrated that statos o hro stress, ostttely phosphorylated eα leads to oerepresso o eα phosphatase yd yd s a arer o tedey to reoer ells ro edoplasat reticulum stress and to resume translation. Indeed, the expression of Myd116 was significantly upregulated in DMT1- mutant BM when compared to wt BM. We also observed increased expression of the main ROS scavenging enzymes, superoxide dismutase and catalase, in DMT1-mutant BM. Consistently, elevated ROS formation was detected in DMT1- mutant FL cultures. We hypothesized that hypoxia and increased ROS could result in oxidative stress-induced DNA damage signaling and therefore evaluated markers of ATR- and ATM-dependent DNA-damage response (DDR). We observed increased phosphorylation of an ATR target, Chk1(Ser345), in DMT1-mutant BM in comparison with wt BM. On the other hand, no differences in the levels of phospho-H2AX(Ser139) between DMT1-mutant mice and wt littermates suggested that the extent of DNA damage in DMT1-mutant cells is below the threshold that activates more robust ATM- dependent DDR signaling. Since it is known that several proteins involved in nucleotide metabolism, DNA replication and DNA repair utilize iron as a cofactor, ATR/Chk1 activation could also indicate ongoing replication stress in DMT1-mutant proliferating cells. Consistently, we observed accumulation of DMT1-mutant BM erythroblasts in the S-phase of cell cycle. Currently, we study in more details to what extent it is oxidative or replication stress that fuels ATR-dependent DNA damage signaling in iron-restricted hematopoiesis caused by DMT1-deficiency. Acknowledgement: We thank prof. Ponka and D. Garcia-Santos (McGill University, LDI, Montreal, Canada) for their help with FL cultures. Grant support: Czech Science Foundation, 15-13732S; Ministry of Health Czech Republic, NT13587; Grant of Palacky University, LF_2015_015.

Sixth Congress of the International BioIRon Society Page 206 Poster Abstracts IBIS

Poster #60 NICOTIANAMINE IN TOMATO PLANT FLUIDS AS AFFECTED BY IRON DEFICIENCY AND IRON RESUPPLY Pablo Diaz-Benito, MSc, Anunciación Abadía, PhD, Javier Abadia, PhD and Ana Álvarez-Fernández, PhD Spanish Council for Scientific Research (CSIC)

Iron deficiency is one of the most limiting factors for biomass production in plants and affects 30% of crops worldwide. Plants are the major source for Fe in human and animal nutrition, and therefore Fe deficiency is one of the most prevalent nutritional problems worldwide. The mechanisms plants use to acquire and transport metals from the soil to edible parts are not completely understood. It has long been proposed that a significant fraction of metals would be present in plant fluids not as free ions but in less reactive chemical forms, e.g., non-covalently bound to organic ligands, to prevent uncontrolled binding, and also because free metals often exert some degree of toxicity. Among the possible ligand candidates is the non-proteinogenic amino acid nicotianamine (NA) that is known to chelate many metals, including Fe(II) and Fe(III). There is still scarce information on the levels of NA in plant fluids, due to sampling and analytical difficulties. Here, we study the effect of the Fe plant status on NA concentrations in different plant fluids, including apoplastic and intracellular fluids of roots and developed leaves, as well as in xylem sap. Tomato (Solanum lycopersicum) plants were grown for 11 days in half-strength Hoagland solution including either 0 (Fe-deet plats or e–EDTA (Fe- sufficient plants). Then, some Fe-deet plats ere treated th e(III)–EDTA, and fluids of all plants were sampled at different times in the following 48 hours. Plant fluids were assessed for micronutrients and NA concentrations using ICP-MS and HPLC-ESI/(TOF-MS), respectively. Twelve hours after Fe-resupply, Fe concentrations in root apoplastic and intracellular fluids, xylem sap and leaf intracellular fluid increased markedly, whereas the Fe concentration in leaf apoplastic fluid was not changed. Nicotianamine concentrations also increased in root fluids 12 hours after Fe- resupply, whereas no change was observed in xylem sap and a significant decrease occurred in leaf fluids. These data support that NA is involved in Fe long-distance transport mechanisms. ACKNOWLEDGEMENTS: Supported by the Spanish Ministry of Economy and Competitiveness (projects AGL2012- 31988 and AGL2013-42175-R, co-financed with FEDER) and the Aragón Government (groups A03).

Sixth Congress of the International BioIRon Society Page 207 Poster Abstracts IBIS

Poster #61 HEPCIDIN REPRESSION BY PROMOTER DNA HYPERMETHYLATION IN NON-VIRAL HEPATOCELLULAR CARCINOMA Natascia Campostrini, Silvia Udali, Michela Corbella, Patrizia Guarini, Annalisa Castagna, Patrizia Pattini, Andrea Ruzzenente, Alfredo Guglielmi, Sara Moruzzi, Alberto Ferrarini, Massimo Delledonne, Luigi Perbellini, Antonia Franceschi, Sang-Woon Choi, Domenico Girelli and Simonetta Friso Department of Medicine, University of Verona, Italy

Introduction. DNA methylation, the major epigenetic feature of mammalian DNA, consists in the covalent binding of a methyl group to the 5’ carbon of cytosines in CpG dinucleotide sequences and acts for gene expression regulation. DNA methylation is involved in development of cancer but the epigenetic signatures implied in non-viral hepatocellular carcinoma (HCC) are still poorly known. Hepcidin, the main regulator of iron homeostasis, has been previously described as being repressed in HCC, nevertheless a direct effect of DNA methylation on hepcidin gene (HAMP) transcriptional regulation has not been investigated, so far. By using an array-based, genome-wide DNA methylation study combined with gene expression profiling, we recently observed a link between the transcriptional repression of HAMP and the hypermethylation of its promoter region, in a sample set of liver tissues from non-viral HCC-affected patients. Aims of the present study were to investigate the functional effect of methylation at HAMP promoter site by an in vitro cell culture model and the downstream effect of HAMP regulation by measuring tissue iron content in neoplastic liver samples of HCC affected patients. Methods. Twenty-seven patients undergoing curative surgery for HCC were enrolled. Exclusion criteria were: coexisting hepatitis B (HBV) or C virus (HCV) infections, chronic and/or acute inflammatory diseases and haematological disorders (including autoimmune liver diseases and hereditary hemochromatosis) and the presence of decompensate liver cirrhosis (Child-Pugh B, C). Venous blood samples were drawn from each subject for routine laboratory tests for iron parameters (serum iron content, ferritin, etc.). Histological confirmed HCC and homologous non-neoplastic liver tissues were obtained from each patient. The tissues were analyzed according to HAMP expression, evaluated by RealTime RT-PCR using TaqMan assays, and to metal iron content analyzed by ICP-MS (Inductively Coupled Plasma Mass Spectrometry). Human liver carcinoma cells, HepG2, were treated with different concentrations of 5-Aza--deoxycytidine, a DNA methyltransferases inhibitor, and the effects on HAMP expression were analyzed in treated and non-treated cells. Results&Discussion. Gene expression analysis showed HAMP down-regulation in HCC tissues as compared to non- neoplastic tissue. The experiments with human liver carcinoma cells evidenced a marked increased in hepcidin transcription when DNA methylation was inhibited, thus revealing the role of DNA hypermethylation at promoter site on HAMP transcriptional repression. The tissue iron content measurement highlighted significantly lower levels of the metal in HCC as compared to non-neoplastic liver tissues. Conclusion. The transcriptional modulation of HAMP, as firstly shown in our previous report, was validated by RealTime RT-PCR in a larger cohort of patients. The in vitro experiments, on HepG2 cells, after treatment with 5-Aza-- deoxycytidine, confirmed the involvement of DNA methylation mechanisms in the repression of HAMP. The transcriptional repression of HAMP by promoter DNA methylation in non-viral HCC opens up to intriguing speculations on the role of iron metabolism regulatory mechanisms in hepatocellular carcinogenesis. Further epigenetic and biomolecular analyses in our sample set are ongoing with the purpose of improving the knowledge on the relationship between cancer, iron homeostasis and the ferroportin-hepcidin axis regulatory processes.

Sixth Congress of the International BioIRon Society Page 208 Poster Abstracts IBIS

Poster #62 HEMOPEXIN PRESERVES CARDIAC FUNCTION BY LIMITING HEME-DRIVEN TOXICITY ON CARDIOMYOCYTES Giada Ingoglia, PhD student1, Alessandra Ghigo, PhD1, Francesca Vinchi, PhD2, James Cimino, Dr1, Lorenzo Silengo, PhD1, Emilio Hirsch, PhD1, Fiorella Altruda, PhD1 and Emanuela Tolosano, PhD1 1University of Torino (Italy); 2University of Heidelberg (Germany)

Hemopexin is the plasma protein with the highest binding affinity for heme. Recently, it was demonstrated that hemopexin may be used as a therapeutic tool to prevent endothelial and cardiovascular damage in animal models of hemolytic diseases 1-4.

To investigate the mechanism of hemopexin (Hx)-mediated protection, we analysed Hx-/- and Hx+/+ mice under basal conditions and after treatment with the hemolytic agent phenylhydrazine. Moreover, we performed experiments on primary cardiomyocytes and cardiomyoblast-like cell line H9C2 treated with heme in the presence or absence of hemopexin.

We found that the heart of Hx-/- mice accumulated heme. Heme accumulation resulted in the up-regulation of Heme Oxygenase (HO)-1 and Ferroportin expression and in the down-regulation of Transferrin Receptor (TfR1) and Divalent Metal Transporter 1 (DMT1), and it was associated with increased oxidative stress. These data were confirmed on neonatal cardiomyocytes. Heme loading in the heart of Hx-/- mice led to oxidation of Calcium/calmodulin-dependent protein kinase II (CaMKII) and phosphorylation of Ryanodine receptor 2 (RYR2) at the serine 2814 suggesting that alteration of redox status due to heme accumulation might alter Ca2+ homeostasis within the cell. In agreement with this conclusion, functional analysis of the heart demonstrated reduced fractional shortening not associated to cardiac remodelling in Hx-/- mice. Finally, treatment of wild-type mice with phenylhydrazine reduced fractional shortening and this reduction was rescued by the concomitant administration of exogenous hemopexin demonstrating that heme excess resulting from hemolysis is responsible for the impairment of cardiac function.

These data demonstrate an important homeostatic role of circulating hemopexin in the control of heme accumulation within cardiomyocytes and in the maintenance of cardiac function. They may have important clinical implications for the use of hemopexin as a drug to preserve heart function in hemolytic disorders, especially in those patients that require a hro trasso ree as ors -thalassemia.

References 1. Vinchi F, De Franceschi L, Ghigo A, et al. Hemopexin therapy improves cardiovascular function by preventing heme- induced endothelial toxicity in mouse models of hemolytic diseases. Circulation. 2013;127(12):1317-1329. 2. Belcher JD, Chen C, Nguyen J, et al. Heme triggers TLR4 signaling leading to endothelial cell activation and vaso- occlusion in murine sickle cell disease. Blood. 2014;123(3):377-390. 3. Ghosh S, Adisa OA, Chappa P, et al. Extracellular hemin crisis triggers acute chest syndrome in sickle mice. J Clin Invest. 2013;123(11):4809-4820. 4. Schaer DJ, Buehler PW, Alayash AI, Belcher JD, Vercellotti GM. Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins. Blood. 2013;121(8):1276-1284.

Sixth Congress of the International BioIRon Society Page 209 Poster Abstracts IBIS

Poster #63 GENOTYPE-PHENOTYPE STUDIES IN 70 IRIDA PATIENTS Luigia De Falco2, Mariasole Bruno1,3, Laura Silvestri, Caroline Kannengiesser, Erica Moran, Claire Oudin, Marco Rausa, Jessica Aranda, Bienvenida Argiles, Idil Yenicesu, Maria Falcon-Rodriguez, Ebru Yilmaz-Keskin, Ulker Kocak, Carole Beaumont, Clara Camaschella, Bernard Grandchamp, Mayka Sanchez and Achille Iolascon 1CEINGE - Biotecnologie Avanzate; 2CEINGE, Biotecnologie Avanzate; 3Department of Medicine, Section of Internal Medicine, University of Verona

Iron-refractory iron-deficiency anemia or IRIDA (Online Mendelian Inheritance in Man number #206200, ORPHA209981) is an inherited recessive anemia unresponsive to oral iron treatment but with a slow persistent response to intravenous iron. IRIDA is characterized by microcytic hypochromic anemia with low serum iron, low transferrin saturation, and normal/high ferritin levels. Serum hepcidin is inappropriately normal/high for the low iron status and accounts for the absent/delayed response to treatment. Up to now 41 different mutations in the TMPRSS6 gene have been reported. We performed a genotype-phenotype correlation analysis in 70 pediatric IRIDA patients (39 from the literature and 31 new recruited patients) divided into two groups: Group A (n=44), patients with two missense mutations and patients with one missense mutation in combination with another type of mutation (nonsense, frameshift or splicing mutation); group B (n=26), patients with two nonsense mutations, two frameshift mutations, two splicing mutations, one nonsense and one frameshift mutation and one frameshift and one splicing mutation. Therefore, we attempted to assess whether genotype- phenotype correlation studies may help the classification of IRIDA patients to predict their clinical severity and response to iron treatment. We first evaluated the age at diagnosis and we found that this tends to be lower in group A (4.20±3.85) than in group B (3.09±4.12) (P=0.29). Hemoglobin levels are statistically significant lower in group B (7.78±1.24g/dL) than in group A (8.68±1.34g/dL) (P=0.01). MCV is also significantly lower in group B (54.46±5.21fl) than in group A (59.46±6.17fl) (P=0.0018). Transferrin saturation and serum ferritin showed a trend towards reduction in patient group B, however the differences with group A were not statistically significant in either case. The same was true for serum iron with lower but not statistical values in group B than in group A. Finally, we also analysed the hepcidin levels with regards to the genetic alterations. Patients bearing more severe mutations (group B) present statistically significant higher serum hepcidin levels (n=15 185.60±131.97) than patients in group A (n=18 92.04±51.11) (P=0.05). This data is in agreement with our above observation regarding group B as the IRIDA patients with more severe anemia (lower hemoglobin and lower MCV). Patients with severe mutations (group B) have statistically significant lower hemoglobin and MCV values and higher serum hepcidin levels than patients with two missense mutations or one missense mutation in combination with other type of mutations. We also have attempted to correlate the different genotype of the two groups (A and B) in regards to response to oral iron treatment. We found that patients in group A present a better response to oral iron treatment than those in group B (32.3% versus 20%), however, this difference is not statistically significant (P=0.36) (ratio of good responders to oral iron therapy is 11 in group A versus 5 in group B). In conclusion, we hypothesized that the IRIDA genotype not only determine the severity of the anemia, including the hepcidin levels, but also it may help in predicting the responsiveness to oral iron treatment.

Sixth Congress of the International BioIRon Society Page 210 Poster Abstracts IBIS

Poster #64 TOWARD BETTER PATHOPHYSIOLOGICAL CHARACTERIATION AND THERAPEUTIC SOLUTION IN - THALASSEMIA TRAIT PATIENTS WITH IRON OVERLOAD. PRELIMINARY REPORTS FROM AN ONGOING STUDY Natascia Campostrini, Fabiana Busti, Sadaf Badar, Alberto Ferrarini, Luciano Xumerle, AnnaChiara Giuffrida, Giovanna De Matteis, Riccardo Manfredi, Paola Capelli, Annalisa Castagna, Massimo Delledonne, Oliviero Olivieri and Domenico Girelli Department of Medicine, University of Verona, Italy

Background: A certain degree of iron overload (IO) is sometimes seen in subjects wth -thalassea trat , a ld form of non-transfusion-depedet -thalassemia. The pathogenesis is unclear, but recently a population study in Sri aa hldre has sho that s haratered y ld hepd sppresso de to reased erythropoietic activity (Jones E, Blood 2015). In individual patients, this may be further aggravated by genetic (i.e. mutations in hemochromatosis genes) or acquired factors (e.g. alcohol abuse or non-alohol ler dseases reatet o is problematic, since “standard” large-volume phlebotomies are not feasible in mildly anemic subjects, as well as the lack of approval of oral iron chelators and of specific guidelines. Deferoxamine (DFO) is the only approved therapy, but it is poorly applicable because of inconvenient parenteral administration and side effects. Sporadic case reports have suggested the use of “mini-phleotoes patets th , t easlty ad eay o sh approah has ot been evaluated in patients’ series. Aims: hs stdy has ee desed to etter haratere ators oled the deelopet o releat patients, and to evaluate feasibility and efficacy of mini-phlebotomies to remove IO in this condition. Patients and methods: Up to now we have erolled osete patets th ales ad eales, ea age 58 years) addressed to our tertiary referral center for Iron Disorders in Verona (northern Italy). IO was documented through increased ferritin levels (in most cases >1000 ng/ml, in repeated assays), and either MRI or liver biopsy when indicated. All subjects gave written informed consent. The study plan includes collection of detailed information about clinical history through a structured questionnaire, measurement of serum hepcidin by in-house mass spectrometry-based method, and a comprehensive genetic analysis of the five hemochromatosis genes (HFE, HFE2, HAMP, TFR2, SLC40A1) through next-generation-sequencing (NGS). Preliminary results: Mean Hb values were not uniform (9.5-13.5 g/dl), in agreement with the known phenotypic variability o the he aerae leels o hepd ere hher tha those osered the eeral poplato ad the hepcidin/ferritin ratio was quite low. A substantial alcohol consumption (> 100 g/day) appears a common acquired cofactor patets th , hle p to o perored patets dd ot reeal potetally pathoe arats out of the known H63D in HFE. Nine patients started treatment with “mini-phlebotomies” and four of them have reached the iron depletion. None of the patients experienced a worsening of anemia during the treatment. Conclusions: In these patients, the high levels of hepcidin suggested that hepcidin response to IO was conserved. Moreover, the low hepcidin/ferritin ratio, used to correct for hepcidin changes according to iron stores, was in agreement th a relate hepd deet or reo, the orree o lally sat ro oerload s ot rare subjects, and is likely multifactorial in most cases, with high alcohol intake and the H63D variant appearing as the most frequent cofactors contributing to further hepcidin suppression. Preliminary results suggest mini-phlebotomies as a valuable approach for this peculiar category of patients.

Sixth Congress of the International BioIRon Society Page 211 Poster Abstracts IBIS

Poster #65 CANCER STEM CELLS: IS THERE A ROLE FOR IRON? Zuzana Rychtarcikova2, Sandra Lettlova2, Jiri Neuzil2,3 and Jaroslav Truksa, PhD1 1Academy of Sciences of the Czech Republic; 2Academy of Sciences of the Czech Republic, Institute of Biotechnology, Prague, Czech Republic; 3School of Medical Science, Griffith University, Southport, Qld, Australia

Cancer stem cells represent a distinct subpopulation of tumour cells which probably participate in developing cancer resistance and therapy failure and exhibit major alterations in signalling pathways regulating cellular proliferation, invasivity and ability to undergo apoptosis. Our current research focuses on the biology of these cells and mainly on their iron metabolism that has not been described as yet. We have analysed tumour spheres generated in vitro that exhibit properties of cancer stem cells and we detected alterations in the amount of labile iron pool within these cells and also in their sensitivity to iron chelation suggesting alterations in their iron metabolism. Furthermore, by using the Fluidigm nano qPCR technology, we have analysed the expression of genes related to iron metabolism and detected major significant alterations in the expression of ACO1, ABCB10, CYBRD1, FXN, GLRX5, QSOX1, TFRC and other iron metabolism-related genes. The role of most of these genes in carcinogenesis is only beginning to appear and their role in the biology of cancer stem cell has not been studied at all. In conclusion, we propose that iron and its metabolism plays a role in the biology of cancer stem cells. Their iron metabolism shows major alterations compared to cancer cells lacking the stem cell properties and it appears that especially mitochondrial iron metabolism is significantly altered. We propose that studying and possibly targeting the iron metabolism of these cells should be considered as an option to affect their biology and could be eventually utilized in designing novel therapies that target specifically cancer stem cells.

Sixth Congress of the International BioIRon Society Page 212 Poster Abstracts IBIS

Poster #66 EXPRESSION PROFILING OF IRON METABOLISM–RELATED GENES IN TAMOXIFEN RESISTANT BREAST CANCER CELLS Veronika Tomkova2, Zuzana Rychtarcikova1, Sandra Lettlova2 and Jaroslav Truksa2 1Academy of Sciences of the Czech Republic, Institute of Biotechnology; 2Academy of Sciences of the Czech Republic, Institute of Biotechnology, Prague, Czech Republic

Tamoxifen is a widely used anti-cancer drug used to treat estrogen positive breast cancers. Despite an undisputed benefits of this drug in cancer therapy, a significant portion of patient eventually develops resistance, metastases and secondary tumours which are the actual case of death. The important role of cancer stem cells in the development of tamoxifen resistance has been proposed, suggesting that acquiring resistance is connected with gaining the stem cells properties and expression of the epithelio-mesenchymal transition (EMT) markers which are connected with poor response to therapy and eventual failure of the treatment. Our study focuses on the biology of tamoxifen resistant cells with particular interest in their iron metabolism that has not been studied so far. We have generated the tamoxifen resistant cells by the long term in vitro cultivation with increasing concentrations of tamoxifen. The generated cells exhibit properties of cancer stem cells and show significant inhibition of the ER signalling pathway. We have analysed the expression profile of the iron metabolism-related genes by using the Fluidigm nano qPCR approach and we have observed significant alterations in the expression of ACO1, ABCB10, GLRX5, HEPH, HFE, QSOX1, TMPRSS6 and also other genes. The role of most of these genes in the tamoxifen resistance has not been documented so far and is completely novel. In summary, the expression profile of iron metabolism–related genes is significantly altered in the tamoxifen resistant breast cancer cells we propose that iron metabolism may be affected and regulated by estrogen signalling. Further research could shed light on previously undescribed role of iron in tamoxifen resistance.

Sixth Congress of the International BioIRon Society Page 213 Poster Abstracts IBIS

Poster #67 HYPERGLYCEMIA DYSREGULATES RENAL IRON HOMEOSTASIS Rajiv Kumar, PhD Jawaharlal Nehru University

Introduction: Hyperglycemia causes major renal complication during diabetes due to increase in oxidative stress. Oxidative stress induces deleterious effects in conjunction with redox active iron. However, there is little understanding so far regarding the kidney iron homeostasis in hyperglycemic condition. Cellular iron balance is maintained by coordinated regulation of iron uptake by transferrin receptor 1 (TfR1), iron storage by ferritin (Ft) and iron release by ferroportin 1 (Fpn). Any change in the expression of these key regulators of iron homeostasis may lead to alteration of cellular iron pool. Objective: The main objective of the current study was to investigate the effect of hyperglycemia on the iron homeostasis regulators such as TfR1, Ft and Fpn in kidney tissue and cell line. Methods: Wistar rats were rendered diabetic using streptozotocin (60mg/kg of body weight) for 28 weeks and then immunohistochemistry for TfR1 and Fpn1 was performed in kidney tissues. To examine iron accumulation Perl’s staining was performed. To understand mechanism of cellular regulation of iron homeostasis, COS7 cells were treated with equivalent concentrations of glucose reported in diabetic condition. Expressions of TfR1, Ft, hepcidin and Fpn were studied using Western blot and/or qRT-PCR analyses. Met-[S35] metabolic labeling, iron uptake using 55Fe-transferrin and other standard biochemical techniques were also used in this study. Results: Immunohistochemistry revealed a strong induction of TfR1 in the proximal tubular cells of diabetic rat kidney, while Fpn expression was also reduced. Perl’s staining showed iron deposition in kidney of diabetic rats. The expression of hepatic hepcidin was elevated in 28-week diabetic rats. Treatment of COS7 cells by increasing concentration of glucose resulted in induction of TfR1. Increase in cellular iron uptake was confirmed by radiolabeled iron and increased Ft level, while Fpn expression was unaltered. There was no change in TfR1 mRNA in both COS7 cells and diabetic rat kidney. Flag-tagged TfR1 expression was unaltered by higher glucose treatment in COS7 cells, while metabolic labeling experiment suggested TfR1 regulation was controlled by translational mechanism. Discussion and conclusion: Regulation of kidney iron homeostasis is less explored than most of the other tissues or cell types; particularly, in a pathogenic condition like hyperglycemia. Here we revealed that hyperglycemic condition could alter iron homeostasis resulting into renal iron overload. High glucose exposure to COS7 cell increased intracellular iron by translational regulation of TfR1. In streptozotocin treated diabetic rats renal iron overload might be contributed by increasing TfR1 expression and decreased Fpn expression probably due to increase in hepatic hepcidin expression. This finding may imply a role of iron in pathogenesis of progressive diabetic nephropathy.

Sixth Congress of the International BioIRon Society Page 214 Poster Abstracts IBIS

Poster #68 TOWARDS HARMONIZATION OF WORLDWIDE HEPCIDIN ASSAYS: IDENTIFICATION OF A COMMUTABLE REFERENCE MATERIAL Lisa van der Vorm, Coby Laarakkers, Siem Klaver, Cas Weykamp and Dorine Swinkels, MD, PhD Radboud University Medical Centre Nijmegen

Background: Bioactive hepcidin is a 25-amino-acid hepatic peptide hormone controlling physiological iron homeostasis. As a key player in iron metabolism hepcidin holds great promise as a potential target of therapy and biomarker for diagnosis and monitoring of iron disorders. Therefore, many methods to quantify hepcidin in body fluids have been developed, which can be roughly divided based on their analytical principle into mass spectrometry (MS) and immunochemical (IC) assays. The two previous Round Robin studies organized by our lab found that absolute values of hepcidin levels as measured by various methodologies differ substantially (Kroot et al. 2009, 2012). Consequently, reference intervals and decision limits are method dependent, thereby complicating the interpretation of results. Thus, harmonization of hepcidin assays is essential to facilitate future implementation of plasma hepcidin in research and clinical practice. Methods: We applied a toolbox developed by the Consortium for Harmonization of Clinical Laboratory Results of the American Association for Clinical Chemistry (AACC) containing technical procedures to achieve harmonization for measurands such as hepcidin, for which no higher-order reference measurement procedures and materials are available (Weykamp et al. 2012, http://www.harmonization.net/oversight/Pages/DevelopingTaskForce.html). Following the steps described in this toolbox, 11 participating laboratories, housing 14 worldwide leading hepcidin assays (7 MS and 8 IC), agreed to measure (in triplicate) 64 blinded and randomized samples, which were collected and prepared in March and April and will be send out in May 2015. Of these latter samples, 24 are candidate reference material (cRM) samples of 4 different types that will be tested for their suitability as an international calibrator for hepcidin assays. These potential calibrators comprise human native plasma samples and value-assigned human blank plasma samples spiked with synthetic hepcidin-25, which are either frozen or lyophilized. Furthermore, we will assess whether addition of a cryolyoprotectant could improve the stability of hepcidin in the cRMs when subjected to freezing and lyophilization. The remaining 40 samples are native plasma samples representing a wide range of hepcidin levels, allowing assessment of several important characteristics of the participating methods (e.g. linearity, reproducibility) and cRMs, most importantly their commutability i.e. whether they behave analogous to patient samples in the various assays. (Potential) results and promises: Laboratories are requested to report results before the end of June 2015, allowing us to promptly analyze data according to the pre-determined approach of the toolbox and to present results at Bio-Iron in September 2015. If we succeed to identify a suitable calibrator for hepcidin assays, periodic calibration of an internal/external standard at the reference material will ensure that results between different methods are equivalent and thus comparable. Eventually, worldwide harmonization of hepcidin methods will allow i) to develop and apply universal reference intervals and consistent clinical decision limits for medical care and best practice guidelines ii) pooling and comparison of data from various studies to facilitate medical research and its translation to the clinic. *Hepcidin harmonization team: H. Drakesmith, A. Armitage, SR Pasricha (Bachem kit); S. Bansal; D. Girelli, N. Campostrini, F. Tagliaro; M. Fillet; O. Itkonen; N. Tomosugi; A. Bamberg, K. Pitts (Corgenix Inc.); M. Herkert (DRG); R. Konrad, J.H. Sloan (Eli Lilly); M. Westerman, G. Olbina (Intrinsic LifeSciences).

Sixth Congress of the International BioIRon Society Page 215 Poster Abstracts IBIS

Poster #69 DECREASED HEPCIDIN LEVELS CORRELATE WITH ELEVATED PLASMA AND TISSUE IRON PARAMETERS IN PATIENTS AND MURINE DISEASE MODELS OF DIABETES Sandro Altamura1, 3, Stefan Kopf2, Julia Glockenmeier1, 3, Peter Nawroth2 and Martina U. Muckenthaler1, 3 1Dep. of Pediatric Oncology, Hematology and Immunology - University of Heidelberg, Germany; 2Department of Internal Medicine I and Clinical Chemistry, University of Heidelberg, Heidelberg, Germany; 3MMPU Molecular Medicine Partnership Unit

Hereditary hemochromatosis (HH) was initially referred to as «bronze diabetes», suggesting a link between iron metabolism and diabetes. More recently, studies demonstrated elevated hepatic iron levels in patients with diabetes. In addition, elevated plasma levels of iron and/or ferritin were shown to be prognostic factors for the development of diabetes. The aim of this study is to understand the molecular mechanisms that cause diabetic iron overload and to elucidate the role of increased iron levels in the generation of reactive metabolites and diabetic late complications. Patients with diabetes mellitus type 2 and healthy controls were registered in the Diabetes and Stress Study (HEIDIS- Study) in Heidelberg. Written consent was acquired according to the Declaration of Helsinki. Type 1 diabetes was induced in C57BL6 mice by repeated STZ injections (60mg/kg body weight, for 5 days) and blood glucose levels were controlled by insulin administration. db/db mice were analyzed as a model for type 2 diabetes. Animal experimentation was approved by the “Regierungspräsidium Karlsruhe”, Germany. 115 type 2 diabetes patients and 26 healthy controls were included in this study. CRP>2 was used as exclusion criteria to avoid an inflammatory contribution to changes in iron-related parameters. The diabetic status of the patients was biochemically reconfirmed via HbA1c measurement. Interestingly, diabetic patients show a significant increase in serum iron content that is reflected by an increase in transferrin saturation and by augmented ferritin levels. Serum hepcidin levels show a marked decrease in the diabetic cohort compared to controls explaining the increased systemic iron content. Similar findings were obtained in type 1 and type 2 mouse models of diabetes. Hepcidin levels are decreased both in diabetic patients and mouse models of type 1 and type 2 diabetes causing an increase in systemic iron content. Experiments are ongoing to identify the molecular mechanisms responsible for the hepcidin dysregulation.

Sixth Congress of the International BioIRon Society Page 216 Poster Abstracts IBIS

Poster #70 ATOH8 MRNA LEVELS DO NOT REGULATE HEPCIDIN EXPRESSION IN HUMAN HEPATOCYTE CULTURES Marie-Laure Island, PhD1, Nadia Fatih2, Patricia Leroyer2, Lenaick Detivaud1, Marie-Paule Roth3, Helene Coppin3, Pierre Brissot1 and Olivier Loreal1 1INSERM-UMR 991 and National Reference Center for Rare Genetic Iron Overload Diseases, CHU Pontchaillou, Rennes, France; 2INSERM UMR 991; 3INSERM-UMR 1043, and University of Toulouse, France

Background. Atoh8 is a basic helix loop helix transcription factor involved in neuronal fate and development which has been described as a modulator of tissue differentiation. It has also been reported that, in mice, iron burden co-regulated the mRNA levels of both Atoh8 and hepcidin, the systemic regulator of iron metabolism (1) Moreover, data obtained in vitro in human kidney HEK293 cell line and in vivo in mice suggested that ATOH8 could be a positive regulator of hepcidin mRNA expression (2), and thus take place in the control of iron metabolism. Aim. Our goal was to obtain a better understanding of the relationship between ATOH8 expression and hepatic hepcidin expression. Material and Methods. We analysed mouse culture hepatocytes maintained in differentiated state in a co-culture system using rat liver epithelial cells (RLEC), and pure culture of freshly human isolated hepatocytes, which are known to longer maintain their differentiated functions. ATOH8 mRNA levels were, or not, modulated using siRNA strategy. ATOH8 and hepcidin 1 mRNA levels were analysed by quantitative RT-PCR. Cell cultures were, or not, exposed to BMP6, IL6 or iron. Results. In mouse liver, Atoh8 was mainly expressed in freshly isolated hepatocytes compared to enriched non parenchymal hepatic cells. In the coculture model associating mouse hepatocytes and RLEC, which permits maintenance of hepatocyte differentiation and high level of hepcidin expression, Atoh8 mRNA expression levels in hepatocytes were higher than in pure primary culture. Inhibition of Atoh8 mRNA levels in hepatocytes by specific siRNAs (70% drop) was associated to a 200% increase of hepcidin1 mRNA levels. BMP6 and IL6 exposure of cell cultures induced an increase of both Atoh8 and hepcidin mRNA levels. In human hepatocytes, ATOH8 specific siRNAs decreased significantly ATOH8 mRNA levels (30% remaining) but did not impact significantly hepcidin mRNA expression levels. As expected, BMP6 and IL6 significantly increased hepcidin mRNA levels. In addition, ATOH8 mRNA levels were also significantly increased by BMP6 but, conversely, decreased by IL6. Finally, ferric ammonium citrate (10µM) did not modulate significantly ATOH8 mRNA expression. Discussion. Our data indicates that: i) Atoh8 expression could significantly impact hepcidin mRNA expression in mouse but not in human hepatocytes; ii) BMP6 induced both Atoh8 and hepcidin mRNA levels in human hepatic cells and in mouse hepatocytes, as reported in vivo in mouse, likely through the BMP/SMAD pathway; iii) IL6 may differentially regulate ATOH8 in mouse and in human cells; iv) FAC exposure does not seem to exert a direct impact on ATOH8 expression levels in human hepatocytes. Conclusion. The present data supports the view that, in human hepatocytes, ATOH8 may have a lower impact on hepcidin expression than previously suggested in mouse. References : (1) Kautz L, et al. Blood. 2008 ;15;112:1503-9. (2) Patel N, et al. Br J Haematol. 2014;164:586-96.

Sixth Congress of the International BioIRon Society Page 217 Poster Abstracts IBIS

Poster #71 FENUGREEK PLANT AS AN ANTI-CANCER REMEDY INDUCES APOPTOTIC GENES Kholoud Khoja and Yemisi Latunde-Dada, Lecturer, Diabetes and Nutritional Sciences Division Kings College London

Iron overload has been implicated in the incidence of carcinogenesis in tissues such as those of the breast, intestine and liver [1;2]. This is potentiated by oxidative stress and inflammatory cytokines that characterize both iron overload conditions. Naturally-occurring antioxidant plant products are employed in the management of cancer and these could have implications on iron metabolism. There is evidence that that fenugreek (Trigonella foenum graecum) could be used to treat cancer because of its ability to enhance the expression of apoptotic genes [3]. The mechanisms and the signally pathways that are induced are however not yet known. Moreover, active ingredients from Fenugreek could have antioxidant effects [4] and these will be assessed in cell culture for their impact on the expression and regulation of antioxidants and iron metabolism genes. Consequently, the potential of these products to inhibit cell proliferation and enhance apoptosis will be assessed. The findings will enhance the management of cancer in iron-loaded disorders. Flavonoids from Fenugreek (Trogonella foenum-graecum) will be investigated for their efficacy in prevention of iron-induced tumorigenesis in cultured cells (MCF7, MCF10). The objective of this study is to explore the impact of Fenugreek in the regulation of cancer progression using MCF7 as a breast cancer cell line. Fenugreek extract was obtained from Sigma and its effect on the viability of MCF7 cells was studied using the Sulforhodamine B (SRB) assay. Gene expression was done by qPCR. Iron uptake using the ferrozine assay was studied in MCF7 cells and this was compared with non-cancerous control cells, MCF10. In general, 50-200 ug/µl of Fenugreek significantly inhibited the growth of MCF7 cells. These results were confirmed further by increased mRNA expression of p53 and Bax genes in MCF7 cells that were exposed to Fenugreek. However, cells exposed to Fenugreek and iron in combination showed decreased expression of those genes. Iron uptake by Ferrozine assay tends to be higher in MCF7 cells when compared with MCF10 ( non-cancerous cells). Following to this, the mechanisms or the signal pathway involved in responses observed will be investigated. According to the result attempts will be made to obtain sufficient information about the active ingredients in Fenugreek. Finally, the outcomes of cell culture studies will be tested in animal models of cancer and iron overload.

References 1. Mascitelli L, Goldstein MR. Inhibition of iron absorption by polyphenols as an anticancer mechanism. QJM 2011;104:459-61. 2. Marques O, da Silva BM, Porto G, Lopes C. (2014). Iron homeostasis in breast cancer. Cancer Lett. 347(1):1-14. 3. Khoja KK, Shaf G, Hasan TN, Syed NA, Al-Khalifa AS, Al-Assaf AH, Alshatwi AA. (2011). Fenugreek, a naturally occurring edible spice, kills MCF-7 human breast cancer cells via an apoptotic pathway. Asian Pac J Cancer Prev.;12(12):3299-304 4. Amin A, Alkaabi A, Al-Falasi S, Daoud SA. Chemopreventive activities of Trigonella foenum graecum (Fenugreek) against breast cancer. Cell Biol Int 2005;29:687-94.

Sixth Congress of the International BioIRon Society Page 218 Poster Abstracts IBIS

Poster #72 MOUSE GENETIC BACKGROUND IMPACTS BOTH ON IRON AND NON-IRON METALS METABOLISM PARAMETERS AND ON THEIR RELATIONSHIPS Thibault Cavey2, Martine Ropert2, Marie De Tayrac3, Edouard Bardou-Jacquet4, Marie-Laure Island4, Patricia Leroyer5, Claude Bendavid Bendavid2, Pierre Brissot4 and Olivier Loreal1 1INSERM UMR 991 and University of Rennes 1, Rennes, France; 2INSERM-UMR 991 and CHU Pontchaillou, Department of Biochemistry, Rennes, France; 3CNRS-UMR 6290, and CHU Pontchaillou, Molecular Genetics and Genomics Department Rennes, France; 4INSERM-UMR 991 and National Reference Center for Rare Genetic Iron Overload Diseases, CHU Pontchaillou, Rennes, France; 5INSERM-UMR 991 and University of Rennes 1, Rennes, France

Background. The phenotypic variability of iron-related diseases, including genetic HFE-related iron overload, suggests that yet undetermined factors modulate thedisease clinical expression. Iron is reported to potentially interact in vitro, in animals and in clinical studies, with other metals. These elements raise the question of the impact of non-iron metals on the control of normal and abnormal iron metabolism. In addition, it has been shown that mouse genetic background may modulate iron metabolism. Aim. Our objectives were, in mice of three genetic backgrounds, to characterize: i) the impact of genetic background on the parameters of iron and non-iron metals, and ii) the links between iron and several non-iron metals. Material and Methods. 30 normal male mice (C57BL/6, Balb/c and DBA/2; n=10 for each group), fed with the same diet, were studied. Quantification of iron (Fe), zinc (Zn), cobalt (Co), copper (Cu), manganese (Mn), magnesium (Mg) and rubidium (Rb) was performed by Inductively Coupled Plasma - Mass Spectrometry (ICP/MS) in plasma, erythrocytes, liver and spleen. Plasma transferrin saturation was determined. Hepatic hepcidin1 mRNA level was evaluated by quantitative RT-PCR. Results. As previously reported, iron parameters were modulated by genetic background with significantly higher values for plasma iron parameters and liver iron concentration in DBA/2 and Balb/c strains. Hepatic hepcidin1 mRNA level was lower in DBA/2 mice. None of the iron parameters was correlated with hepcidin1 mRNA levels. Principal component analysis of the data obtained for non-iron metals indicated that metals parameters stratified the mice according to their genetic background. Plasma and tissue metal parameters that are dependent or independent of genetic background were identified. Moreover, links were found between plasma and tissue content of iron and some other metals in the four studied matrices: in plasma, mainly between transferrin saturation and Cu; in erythrocytes, primarily between hepatic hepcidin 1 mRNA and haemoglobin levels; in liver and spleen, mainly between Fe, Co and Mn on the one hand, between hepatic hepcidin 1 mRNA and Rb one the other hand. Conclusions. Our data : i) confirms the impact of the genetic background on iron parameters, ii) shows that genetic background may also play a role in the metabolism of non-iron metals, iii) identifies links between iron and other metals which may have implications in the understanding and, potentially, the modulation of iron metabolism.

Sixth Congress of the International BioIRon Society Page 219 Poster Abstracts IBIS

Poster #73 BIOLOGIC CHARACTERIZATION OF IRON CHELATOR SIH, OXIDATIVE STRESS-ACTIVATED PROCHELATOR BSIH AND THEIR METABOLITES Hana Jansova, Jan Bures, Petra Kovarikova and Tomas Simunek Charles University in Prague, Faculty of Pharmacy in Hradec Kralove

Iron chelators are commonly used for treatment of iron overload, but they have promising applications also in oxidative stress-induced pathological states without systemic iron overload. Recent evidence suggest that oxidative stress is a common denominator of numerous cardiovascular diseases. Salicylaldehyde isonicotinoyl hydrazone (SIH) is an experimental aroylhydrazone iron chelator. Because of its small size and lipophilicity, SIH can be administered orally and enters cells to chelate the intracellular pool of redox-active iron and is therefore able to efficiently block the iron-dependent production of hydroxyl radicals and thereby protect cardiac cells against oxidative damage. However, there is a risk of toxicity caused by iron deficiency. Another disadvantage of SIH is also its rapid hydrolysis due to the labile hydrazone bond. To avoid affecting iron homeostasis under physiological conditions and to increase the stability, boronate-masked prochelator (BSIH) has been designed [1]. It does not bind ions until mask is removed in the disease-specific conditions of oxidative stress. In this study, we aimed to examine potential cardioprotective effects, own toxicities and iron chelating properties of SIH, BSIH and their metabolites and/or degradation products. H9c2 rat embryonal cardiomyoblast-derived cell line was used. Neutral red uptake assay was used for assessment of cellular viability. Calcein assay and calcein-AM assay were used to determine the chelating activity and efficiency of BSIH activation by hydrogen peroxide (H2O2) to effective chelator in solution and inside the cells. Epifluorescence microscopy was used for photodocumentation of mitochondrial inner membrane potential by JC-1 probe. HPLC-UV method was used for determination of SIH and BSIH stability and for identification of their metabolites/degradation products. Our in vitro experiments have demonstrated conversion of BSIH in the presence of H2O2 to effective chelator SIH in solution as well as in cells where its activity reached up to 70 % of SIH chelating activity. We confirmed higher stability of BSIH in medium as well as in cells compared to SIH. Whereas SIH showed dose-dependent decrease of cellular viability, BSIH did not display any sign of viability reduction. SIH significantly protected cells against injury caused by H2O2 (200 at oetratos , hereas at oetrato µM and at its higher concentrations its protective effect was even more pronounced than the parent chelator. Importantly, we observed that SIH degradation product, salicylaldehyde, was ale satly presere elllar alty at oetratos µM. This protective property correlated with its ability to chelate iron. Its activity reached almost 50 % of SIH chelating activity. SIH has been shown to protect guinea pig or rat isolated cardiomyocytes and other cell types against damage induced by H2O2 or by other pro-oxidants. We observed that SIH could be toxic in physiological conditions due to the iron deficiency. Whereas, prochelator BSIH shows higher stability and does not display almost any inherent toxicity while retaining the protective properties. Its protective properties have been also previously described on ARPE-19 cells. We observed that one of the degradation products, salicylaldehyde, preserves some protective properties of SIH. This study was supported by the Czech Science Foundation (grant 13-15008S). [1] L.K. Charkoudian, et al., J. Am. Chem. Soc., 128:12424–12425; 2006.

Sixth Congress of the International BioIRon Society Page 220 Poster Abstracts IBIS

Poster #74 GLUTATHIONE-HEME, THE MAIN COMPONENT OF THE CYTOSOLIC HEME POOL? Robert Hider1, Ann Smith, PhD2 and Xiaole Kong, PhD1 1King's College London; 2University of Missouri

Recently, glutathione (GSH) has been proposed as a key component of the cytosolic and mitochondrial labile iron pool [1, 2]. GSH possesses a buffering role, via it’s cysteine residue for cytosolic iron(II) and offers a means for the selection of iron in its incorporation into a wide range of iron-dependent enzymes and electron transfer proteins. GSH, when in excess, is able to protect iron(II) from autoxidation [1]. Heme is a prosthetic group for numerous hemoproteins and plays an important role in controlling protein synthesis and cell differentiation [3] and yet non-protein-bound heme can cause cell damage. Heme can exist as an iron(II) complex (heme) or an iron(III) complex (hemin) and will redox cycle between these two oxidation states generating free radicals. Mammalian cells contain a cytosolic heme pool which serves both precursor and regulatory functions [4-6]. The concentration of this pool has been estimated to reach 10 µM in rat reticulocytes [7]. The chemical nature of this heme pool is unknown. In parallel with the ability of GSH to bind iron(II), we wondered if a similar interaction might occur with heme. The binding of the extremely hydrophobic heme moiety to the charged tripeptide would undoubtedly result in increased water solubility and might be expected to reduce the rate of heme partitioning into membranes. Indeed, GSH does bind hemin forming a 1:1 complex with an affinity constant of 2.3 x 103 M, which although relatively weak, dominates hemin under typical cytosolic conditions, where [GSH] deed, ah ad oorers deostrated that prevents erythrocyte damage by hemin. In an earlier publication, Shviro and Shaklai [9] also demonstrated that GSH is a scavenger of free hemin and calculated that at least 97% of cytosolic hemin is complexed with glutathione. These data led to their proposal that GSH may serve physiologically as the cytosolic chelator of free hemin [9]. We have spectroscopic evidence for the formation of a GSH/heme conjugate when hemin is treated with dithionite in the presence of excess GSH. Furthermore, with high concentrations of GSH (2mM) this conjugate is stable in the presence of oxygen. In the light of these preliminary studies we propose that GSH is the major cytosolic ligand for both hexaquo- iron(II) and heme and is involved in intracellular trafficking of both species.

[1] Hider RC and Kong XL (2011) Biometals 24, 1179-1187. [2] Hider RC and Kong X (2013) Dalton Trans. 42, 3220-3229. [3] Ponka P (1997) Blood 89, 1-25. [4] Garcia-Santos D, Schranzhofer M, Horvathova M, et al, (2014) Blood, 123, 2269-2277. [5] Vinchi F, Ingoglia G, Chiabrando D, et al, (2014) Gastroenterology, 146, 1325-1338. [6] Correia MA, Sinclair PR and De Matteis F (2011) Drug Metabolism Reviews 43, 1-26. [7] Garrick MD, Scott D, Kulju D, et al (1999) Biochim. Biophys. Acta 1449, 125-136. [8] Sahini VE, Dumitrescu M, Volanschi E, et al (1996) Biophysical Chemistry 58, 245-253. [9] Shviro Y and Shaklai N (1987) Biochem. Pharmacol. 36, 3801-3807.

Sixth Congress of the International BioIRon Society Page 221 Poster Abstracts IBIS

Poster #75 PROTECTIVE EFFECTS OF ROSMARINIC ACID AGAINST IRON-INDUCED NEUROTOXICITY IN SK-N-SH CELLS Le Qu, Huamin Xu, Hong Jiang and Jun-Xia Xie Department of Physiology, Shandong Provincial Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology, Medical College of Qingdao University, Qingdao, 266071, China

Rosmarinic acid (RA) is a naturally occurring polyphenolic compound, and is composed of caffeic acid and danshensu. Our previous studies have confirmed RA could protect against 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl pyridine cation (MPP+) induced cell injury. Improving evidence showed iron-ded α-synuclein aggregation played important roles in the etiology of Parkinson's disease (PD). However, whether RA could protect dopaminergic neurons throh ht the areato o α-synuclein in PD is unclear. And the regulation mechanisms underlying this inhibition were not elucidated. Therefore, the experiment proposed to explore the effects of rosmarinic acid against iron- ided α-synuclein aggregation in dopaminergic cells and elucidate the possible mechanisms in the SK-N-SH cells. Results showed that iron could reduce the mitochondrial transmembrane potential ( ad de α-synuclein aggregation in the SK-N-SH cells. In accordance with iron responsive element/iron regulatory protein (IRE/IRP) system, ro old rease the leels o α-syle d reslts also shoed that pretreatet△ old restore the reduction induced by iron and alleviate iron induced α-synuclein aggregation. Further results showed RA pretreatment old ht ro ded α-synuclein aggregation by up-regulating hemeoxygenase-1 (HO-1). In addition, RA pretreatet old derease the leels o α-synuclein via decreasing the protein levels of IRP1. These results provide new findings and new strategies for the prevention and treatment of PD. ey ords arsos dsease ro α-synuclein; rosmarinic acid Acknowledgements This work was supported by the grants from the National Program of Basic Research sponsored by the Ministry of Science and Technology of China (2011CB504102), the National Foundation of Natural Science of China (81430024, 31371081, and 31271131), Excellent Innovative Team of Shandong Province, Taishan Scholars Construction Project, Shandong. *E-mail address: [email protected] (J. Xie).

Sixth Congress of the International BioIRon Society Page 222 Poster Abstracts IBIS

Poster #76 THE CHEMOKINE CCL2 IS A NOVEL MODIFIER OF TISSUE IRON LEVELS AND A PREDICTOR OF DISEASE SEVERITY IN HEREDITARY HEMOCHROMATOSIS Katarzyna Mleczko-Sanecka1,10,11, Sandro Altamura1,10,11, Mingang Zhu1,10,11, Milene Costa da Silva1,10, Maja Vujic Spasic1,10, Claudia Guida2,10, Oriana Marques3, Matthew Lawless4, Dorine Swinkels5, Regina Maus6, Jorge P. Pinto3, Wen-Pin Chen7, Nikolas Gunkel8, Margarida Lima9, Rainer Pepperkok2,10, Christine E. McLaren7, Ulrich Maus6, Heiko Runz1,10, Graca Porto3,9,11 and Martina U. Muckenthaler1,10,11 1University of Heidelberg, Germany; 2European Molecular Biology Laboratory (EMBL), Heidelberg, Germany; 3University of Porto, Portugal; 4University College Dublin, Mater Misericordiae University Hospital, Dublin, Ireland; 5Radboud University Medical Center, The Netherlands; 6Hannover School of Medicine, Germany; 7University of California, Irvine, CA, United States; 8German Cancer Research Center (DKFZ), Heidelberg, Germany; 9CHP- Hospital Santo Antonio, Porto, Portugal; 10Molecular Medicine Partnership Unit (MMPU), Heidelberg, Germany; 11Equal contribution

Most patients with hereditary hemochromatosis (HH) are homozygous for the C282Y mutation in the HFE gene, but only a small percentage develops overt clinical symptoms. Environmental or genetic factors are thought to modify tissue iron overload and disease severity in these patients. To uncover genetic modifiers of iron levels we performed a targeted RNAi screen for genes that affect transferrin uptake. The screen identified the chemokine (C_C motif) ligand (CCL2) as a suppressor of cellular transferrin uptake. Analysis of iron-related parameters in Ccl2 knock-out mice reveals decreased serum iron levels and transferrin saturation, iron-overload in the spleen and mild iron accumulation in the liver. This phenotype seems not to be is not explained by an apparent deregulation of the hepcidin-ferroportin regulatory axis or the presence of proinflammatory conditions. A combination of magnetic splenic cell fractionation and FACS analysis demonstrates that iron-loaded leukocytes express aberrantly high TFR1 levels. Furthermore, the spleen contains proportionally more iron-rich B cells that show substantially higher TFR1 expression compared to iron-recycling F4/80- bright macrophages. These data suggest that at least two factors contribute to high splenic iron levels in Ccl2 knock-out mice: (1) alteration of the cellular composition of iron-rich splenic cell types and (2) inappropriately high TFR1-mediated iron uptake from the plasma. Finally, we show that CCL2 levels may serve as a marker for disease severity in patients with HH. Among individuals homozygous for the C282Y HFE mutation, CCL2 levels negatively correlate with liver iron overload and clinical manifestations of HH at the time of diagnosis. Collectively, these data suggest that appropriate CCL2 expression is required to prevent excessive tissue iron accumulation and that the CCL2 profile may be used as a novel biomarker predicting symptomatic disease in HH. These findings broaden the growing spectrum of homeostatic roles of CCL2 that reach beyond its originally identified functions in immune cells recruitment under inflammatory conditions.

Sixth Congress of the International BioIRon Society Page 223 Poster Abstracts IBIS

Poster #77 LIVER HFE PROTEIN CONTENT IS REGULATED BY IRON LEVELS IN VIVO Jan Krijt, Jana Frydlova, Petr Prikryl, Iuliia Gurieva and Martin Vokurka Charles University in Prague, First Faculty of Medicine

Introduction: Mutations in the HFE gene were identified as a cause of hereditary hemochromatosis already in 1996; however, the exact function of the HFE protein as well as its possible regulation by iron status are still not known. So far, no in vivo data on the response of liver Hfe protein to iron overload or iron deficiency have been published. Experimental: The aim of this study was to determine the Hfe protein content in plasma membrane-enriched fractions of mouse and rat liver. Male C57BL/6 mice were fed an iron deficient diet since weaning, or were injected with iron dextran (400 mg/kg) at 8 weeks of age. Weaned Wistar rats were fed an iron deficient diet for four weeks. Erythropoietin was administered to mice and rats for four days. Hfe in liver plasma membrane-enriched fraction was determined by immunoblotting using a commercial antibody, band specificity was confirmed by analyzing livers from Hfe-/- mice. Results: Feeding of iron deficient diet to mice for two weeks decreased Hfe protein content to 60%. Liver Hamp mRNA content was decreased to about 10%. Expression of erythroferrone in the spleen was unchanged. Feeding of iron deficient diet for 8 weeks resulted, as expected, in a dramatic decrease of Hamp mRNA (to less than 1%). Liver Hfe protein content decreased to 30%, and Tmprss6 protein was increased to 200%. Hematocrit was decreased and erythroferrone expression in the spleen was increased. In rats fed an iron deficient diet for four weeks, Hfe protein content was decreased to 50%, while Tmprss6 protein content increased to 350%. Liver Hamp mRNA was decreased, and splenic erythroferrone mRNA was increased. Administration of iron dextran to mice increased Hfe protein content to 250 %. In both mice and rats, administration of erythropoietin increased liver Tmprss6 protein content (to 150 and 200 % respectively). Hfe protein content was not significantly changed by erythropoietin treatment. Discussion: Hfe protein participates in the regulation of hepcidin expression; however, its exact function is not yet known. Results from this study demonstrate that, in liver plasma membrane-enriched fraction, Hfe protein content is regulated by iron status. In mice fed an iron deficient diet, the decrease in Hfe protein apparently preceded the development of iron deficiency anemia and the activation of the erythroferrone pathway. Based on these results, we speculate that Hamp expression could be controlled by changes in hepatic Hfe protein content. The iron deficiency-induced decrease in Hamp expression apparently occurs in two phases. The relatively low decrease of Hamp mRNA, which is seen at two weeks, could be mediated by downregulation of the Hfe protein. With the subsequent development of iron deficiency anemia the erythroferrone pathway is activated, resulting in further profound downregulation of Hamp expression. The increase of the Tmprss6 protein, which is observed at this phase, suggests that Tmprss6-mediated proteolytic activity could play a role in the activation of erythroferrone signaling. It has been proposed that the Hfe protein cooperates with transferrin receptor 2 protein. It has also been proposed that Hfe participates in the hemojuvelin signaling pathway. The presented method for Hfe immunodetection will provide new possibilities to address these issues.

Sixth Congress of the International BioIRon Society Page 224 Poster Abstracts IBIS

Poster #78 IRON DEFICIENCY IN ALCOHOLIC LIVER DISEASE: A CONSEQUENCE OF CHANGES IN PROTEINS INVOLVED IN DUODENAL IRON ABSORPTION? Molly Jacob, MBBS, MD, PhD, Jithu James, MSc, Joe Varghese, MBBS, MD, DNB, Kavita Rasalkar, MBBS, MD, Ramya Raghavan, MBBS, MD and CE Eapen, MBBS,MD, DM Christian Medical College, Vellore, India

Alcoholic liver disease (ALD) has been reported to be associated with iron overload. Increased absorption of dietary iron in such patients is a possible mechanism that may underlie this. It is not known whether duodenal proteins involved in iron absorption are altered in those with ALD.

Patients with ALD, who were required to undergo a medically-indicated upper gastrointestinal endoscopy, were recruited into the study after informed consent. Patients who underwent an endoscopy for investigation of dyspepsia and were found to have no endoscopic abnormalities served as control subjects. Duodenal mucosal tissue obtained was used to determine expression levels of divalent metal transporter 1 (DMT1), ferroportin, duodenal cytochrome b (dcytb) and hypoxia-inducible factor 2-α -α, all o hh are oled o-heme iron absorption. Blood obtained was used for estimation of hemoglobin, C-reactive protein (CRP), ferritin, iron, hepcidin (the central regulator of iron homeostasis) and tests of liver function. Data were analyzed by student’s t-test or Mann-Whitney test and by Pearson’s or Spearman’s correlational coefficients, as appropriate. A p value of less than 0.05 was considered statistically significant.

Parameters of liver function were found to be deranged in patients with ALD (n = 24). They had significantly lower levels of hemoglobin and serum iron and higher levels of serum CRP than control subjects (n = 30). Expression levels of duodenal DMT1, ferroportin and dcytb, and serum levels of hepcidin were significantly lower in these patients, when compared with controls. Correlational analysis showed positive correlations between hemoglobin and serum markers of iron status. Expression levels of many of the duodenal proteins involved in iron absorption also correlated positively with one another. The most interesting of these was a significant positive correlation between mRNA expression levels of HIF- α ad dyt ales or prothro te those th dd ot orrelate th those o heolo or ser ro or ferritin.

This study showed no evidence of iron overload in patients with ALD; on the contrary, they appeared to be iron-deficient, as evidenced by lowered hemoglobin and serum iron levels. These lowered levels did not correlate with a history or evidence of bleeding. In view of this, we postulate that the lowered iron status in these patients may be due to decreased absorption from the duodenum. This is supported by finding lowered expression levels of duodenal DMT1, dcytb and ferroportin in these patients. These findings were, however, associated with decreased serum hepcidin levels. This appears to be contradictory, as a fall in hepcidin levels is known to up-regulate these proteins. A possible explanation for this apparent discrepancy may be the involvement of HIF-α, hh s o to relate T1 and dcytb expression. The positive correlation seen between HIF-α ad dyt leels ths stdy spports ths tre or s arrated to investigate this aspect further.

In conclusion, patients with ALD showed evidence of iron deficiency, not overload. This may be a result of decreased absorption of dietary non-heme iron, possibly due to decreased expression of duodenal DMT1, dcytb and ferroportin. The possible role of HIF-α edat sh reded epresso reres orato

Sixth Congress of the International BioIRon Society Page 225 Poster Abstracts IBIS

Poster #79 - WITHDRAWN

Sixth Congress of the International BioIRon Society Page 226 Poster Abstracts IBIS

Poster #80 BRAIN IRON LOCALIZES TO MYELIN AND AFFECTS EXPRESSION OF GENES RELATED TO MYELIN AND NEURODEGENERATION WITH BRAIN IRON ACCUMULATION DISORDERS IN MICE WITH DISRUPTION OF HFE AND TFR2 Moones Heidari, MSc1, Dan Johnstone, PhD2, Brianna Bassett, B Biomed Hons1, Ryan Horn1, Kristy Martin, B Biomed Sci1, Dylan Huff1, James Montgomery, B Biomed Hons1, Conceicao Bettencourt, PhD3, Anita Chua, PhD4, Ross Graham, PhD5, John Olynyk, MD PhD4, Debbie Trinder, PhD6 and Liz Milward, PhD1 1School of Biomedical Sciences and Pharmacy/University of Newcastle; 2Bosch Institute and Discipline of Physiology, University of Sydney; 3Department of Molecular Neuroscience, UCL Institute of Neurology; 4Institute for Immunology and Infectious Diseases, Murdoch University; 5School of Biomedical Sciences, Curtin University of Technology; 6School of Medicine and Pharmacology, The University of Western Australia

The rare inherited diseases categorized as Neurodegeneration with Brain Iron Accumulation (NBIA) are often fatal disorders characterized by iron accumulation in the basal ganglia. We investigated brain iron distribution and compared transcriptome changes in mice with disruption of HFE and TFR2 on an AKR background (Hfe-/-xTfr2Y245X) to NBIA-related transcript profiles from human basal ganglia. Diaminobenzidine-enhanced Perls' stain shows iron labelling throughout the brain in wildtype and mutant mice, particularly in myelinated fiber tracts in the basal ganglia. Iron increases with age (3, 6, 9 and 12 months) and is greater in Hfe-/-xTfr2mut tha ldtype e at all aes rop o-localization of iron and myelin was confirmed by Luxol fast blue and proteolipid protein 1 immunolabeling of myelinated fibers. Although iron co-localizes with a few Nissl-positive cells, neuronal localization could not be confirmed by NeuN labeling. Instead iron primarily co-localizes to myelin and myelin-associated or perivascular cells, which triple in density in Hfe-/-xTfr2mut compared to wildtype cerebrum by 3 months of age (p=0.01, n=4/group). Some of these are Olig2-positive putative oligodendrocytes however many remain unidentified, failing to stain with Olig2 or other oligodendrocyte markers, with Iba1 (microglia), GFAP (astrocytes) or NG2 (pericytes). To maximize iron status, Hfe-/-xTfr2mut mice were switched to 2% carbonyl iron-supplemented diet for three weeks before sacrifice at 10 weeks of age. This does not increase brain iron in wildtype mice or alter expression of transcripts described below (p>0.05). Brain iron, assessed by inductively coupled plasma-atomic emission spectroscopy, non-heme iron and ferritin immunoblotting, was over 1.4-fold greater in Hfe-/-xTfr2mut mice (p, rop Microarray revealed significantly decreased brain transcripts for transferrin, transferrin receptor 1 and hepcidin in the Hfe-/-xTfr2mut brain (p<0.05). Transcripts of five NBIA-linked genes were significantly decreased (including ceruloplasmin and the myelin-related genes phospholipase A2 group 6, fatty acid 2-hydroxylase, chromosome 19 open reading frame 12 and ATPase 13A2). Real time RT-PCR confirmed these findings (p<0.05). A further 14 myelin-related genes were also significantly down-regulated by microarray including the oligodendrocyte marker 2’,3’-cyclic nucleotide 3’ phosphodiesterase, myelin oligodendrocyte glycoprotein and myelin-associated oligodendrocytic basic protein. There was significant overlap (p<1x10-4) between genes differentially expressed in Hfe-/-xTfr2mut brain and two basal ganglia gene co-expression modules enriched for NBIA genes. These modules were identified by unsupervised weighted gene co-expression network analysis of the whole-transcriptome profiles from ten brain regions in neuropathologically normal adult human brain (n=101), and validated on pediatric and adult basal ganglia expression datasets. In conclusion, these findings demonstrate higher brain iron content in Hfe-/-xTfr2mut mice, primarily localized in myelin, some oligodendrocytes and other still unidentified cells. The altered expression of several NBIA genes and numerous other myelin-related genes, together with the correspondence between the mouse transcriptome changes and human NBIA gene networks, provides compelling evidence that myelin-associated iron accumulation drives alterations in molecular systems involved in NBIA neuropathogenesis. This research provides new insights into NBIA pathogenesis and into possible brain abnormalities that may occur in other iron disorders and links to myelin.

Sixth Congress of the International BioIRon Society Page 227 Poster Abstracts IBIS

Poster #81 BMP6 REGULATES HEPCIDIN PRODUCTION INDEPENDENTLY OF THE HEMOCHROMATOSIS-ASSOCIATED MOLECULES HFE, TFR2, AND HJV Chloe Latour2, Celine Besson-Fournier2, Delphine Meynard2, Laura Silvestri3, Ophelie Gourbeyre2, Patricia Aguilar- Martinez4, Paul J. Smith5, Mark D. Fleming5, Marie-Paule Roth2 and Helene Coppin, PhD1 1Inserm-U1043; 2Inserm; 3San Raffaele Scientific Institut; 4Hopital Saint-Eloi; 5Hardward Medical School

Background & Aims: Hereditary hemochromatosis, which is characterized by inappropriately low levels of hepcidin, increased dietary iron uptake, and systemic iron accumulation, has been associated to mutations in the HFE, TfR2, or HJV genes. However, it is still not clear whether these molecules intersect in vivo with BMP6/SMAD signaling, the main pathway upregulating hepcidin expression in response to elevated hepatic iron. To determine whether the hemochromatosis-associated proteins HFE and TfR2 and the signaling pathway transduced by BMP6 really intersect, we took advantage of our observation that hepcidin gene expression is only partially repressed in Bmp6-deficient mice, particularly in females. We thought that there was a possibility that Hfe and/or Tfr2 could operate in a molecular pathway independent of Bmp6 to regulate hepcidin expression. If it were, then the loss of Hfe and/or Tfr2 would further repress hepcidin expression in Bmp6-deficient mice and this greater repression would be easily detectable in females. To answer this question, we used Bmp6- and Hjv-deficient mice which share a similar phenotype and assessed what was the impact of loss of Hfe or Tfr2 on these mice. Methods: e proded e dole oot or p ad -microglobulin (a surrogate for the loss of Hfe) and for Bmp6 and Tfr2, and we compared their phenotype (hepcidin expression, Bmp/Smad signaling, hepatic and extrahepatic tissue iron accumulation) with that of single Bmp6-deficient mice and that of mice deficient for Hjv alone or in combination with Hfe or Tfr2. Results: Whereas the phenotype of Hjv-deficient females was not affected by loss of Hfe or Tfr2, that of Bmp6-deficient females was considerably worsened, with decreased Smad5 phosphorylation, compared with single Bmp6-deficient mice, further repression of hepcidin gene expression, undetectable serum hepcidin, and massive iron accumulation not only in the liver but also in the pancreas, the heart and the kidneys. Conclusions: These results suggest that there are two signaling pathways regulating hepcidin. One, transduced by BMP6, does not require HJV. The other, activated by another BMP, requires HJV as a coreceptor and the participation of HFE and/or TfR2. These results, which suggest that BMP6 regulates hepcidin by an HFE/TfR2/HJV-independent pathway, improve our understanding of the regulation of iron homeostasis and have significant implications for the treatment of forms of hereditary hemochromatosis associated with mutations in HFE, TfR2, or HJV.

Sixth Congress of the International BioIRon Society Page 228 Poster Abstracts IBIS

Poster #82 THE ROLE OF MULTI-COPPER FERROXIDASES IN VIVO IRON METABOLISM Shunli Liu, Jiashuo Zheng, Undergraduate, Mengxia Chen, Undergraduate and Huijun Chen, Professor Medical School of Nanjing University

There are two known vertebrate multi-copper ferroxidases (MCFs) proteins: hephaestin (HEPH) and ceruloplasmin (Cp), which play a central role in body iron metabolism facilitating iron transport by oxidizing ferrous iron to the ferric form. In this study, we seek to clarify the role of MCFs in vivo iron metabolism by analyzing and comparing the effects of Heph (Heph KO), Cp (Cp KO) and double (Heph/Cp KO) knockout on systemic iron homeostasis. We focus two MCFs gene knockout on Heph and Cp KO mice to study their roles in the whole body iron metabolism. We collected mouse liver samples and extracted total RNA for RT-qPCR analysis, and purified protein for western blot assay from all of phenotype mice. Our results shown that both gene expression of Ferritin and Haem oxygenase-1HO-1were significantly higher in Heph/Cp KO mouse liver than Heph KO, Cp KO and WT control mouse, respectively. The ferritin protein expression was up-regulated in Heph/Cp KO mouse liver than the other phenotype mice, it suggested that there is an oxidative stress caused by iron overload in Heph/Cp KO mouse liver. In additional, Ferroportin1(Fpn1) and Transferring Receptor1(TfR1) mRNA expression were no significantly different in the single or double KO mouse liver, while TfR1 protein expression was significantly down-regulated in the Cp KO and Heph/Cp KO mouse, and Fpn1 protein only significantly higher in the Heph/Cp KO mouse liver compared to other phonotype mice. This result supported that the protein of TfR1 was mainly affected by local iron condition, and Fpn1 protein was regulated by systemic iron requirements. Our work suggested that Heph and Cp interact in liver iron transport and a secondary oxidative stress injury exists when iron accumulated in the liver.

Sixth Congress of the International BioIRon Society Page 229 Poster Abstracts IBIS

Poster #83 MULTI-COPPER FERROXIDASES PLAY AN IMPORTANT ROLE IN BRAIN IRON METABOLISM Ruiwei Jiang, Postgraduate, Mengxia Chen, Undergraduate, Jiashuo Zheng, Undergraduate and Huijun Chen, Professor Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University

That iron plays important functions in the brain and iron accumulation in the central nervous system (CNS) is a common feature of many neurodegenerative diseases. Multi-copper ferroxidases (MCFs) play an important role in cellular iron metabolism. They partner with the iron export protein ferroportin (FPN1) and rapidly oxidize iron, facilitating cellular iron release. However, the role of MCFs in the CNS in health and disease remains poorly characterized. We used knockout mouse models for two MCFs, Hephaestin (Heph) and Ceruloplasmin (Cp), to study their roles in the brain. We measured the iron contents and ferritin protein levels in different brain regions and found increased iron and ferritin in Heph KO mouse brain cortex, hippocampus, cerebellum and brainstem regions compared to WT and Cp KO mouse brain regions at 6-7 month of age. Furthermore, we generated Heph/Cp double knockout mice, which appeared a deeply iron accumulation in the mouse brain regions, the iron concentration is 5-10 times higher than the wild type control mice, especially in the cerebellum. For understanding that whether excessive iron is related with oxidative stress, we have determined SOD and MDA contents in mouse brain regions. We found a significant decrease of brain SOD contents in Heph/Cp double knockout mice compared to WT control, and a significant increase of brain MDA contents in Heph/Cp double knockout mice compared to WT mice. Several brain cells, including oligodendrocytes and astrocytes, likely play the important roles in the brain iron metabolism. Mutant of MCFs could interrupt intracellular iron transport. We have purified primary brain cells from Heph KO, Cp KO and WT mouse, and grown them into ologodendrocytes and astrocytes by the special growth media. We identified that Cp gene expression in astrocytes and Cp KO astrocytes have a severely iron efflux impaired with ROS injury; similarly Heph gene expression in natural oligodendrocytes and Heph KO oligodendrocytes have a iron efflux impaired with ROS injury. Our results suggest that ablation of Heph or Cp results in disordered brain iron homeostasis and brain cell oxidative stress.

Sixth Congress of the International BioIRon Society Page 230 Poster Abstracts IBIS

Poster #84 INSULIN RESISTANCE IN DIET-INDUCED OBESE MICE IS ASSOCIATED WITH DYSREGULATION OF IRON HOMEOSTASIS Joe Varghese, MD1, Jithu Varghese James, MSc1, Andrew McKie, PhD2 and Molly Jacob, MD, PhD1 1Christian Medical College, Vellore; 2King's College, London

Hepcidin is the central regulator of systemic iron homeostasis. Iron overload has been reported to be associated with insulin resistance. The interactions between hepcidin and insulin resistance are not well understood. In order to study this, male C57Bl/6 mice aged 6-8 weeks were fed a high-fat diet (HFD) (60% calories derived from fat) for 4, 8, 12, 16, 20 or 24 weeks to render them insulin-resistant. Glucose tolerance test (GTT) and insulin tolerance test (ITT) were done before initiation and after completion of feeding. Fasting plasma insulin and glucose levels were measured and the homeostatic model assessment – insulin resistance (HOMA-IR) index was calculated. Serum levels of hepcidin and ferritin were estimated, using commercially available ELISA kits. Iron content of liver was estimated by the bathophenanthrolene method. Expression of genes of interest in the liver was determined by quantitative real-time PCR (qPCR). HFD induced glucose intolerance from 4 weeks of feeding onwards; this persisted up to 24 weeks. Insulin resistance, as assessed by insulin tolerance test (ITT), increased significantly after 8 weeks of feeding and progressively worsened after 20 and 24 weeks of HFD feeding. This was associated with increased fasting plasma insulin levels and HOMA-IR index. Serum hepcidin levels were significantly lower after 20 and 24 weeks of HFD feeding and serum ferritin levels were increased at 12, 20 and 24 weeks. Liver iron content (µg of iron per gram dry liver weight) decreased progressively with HFD feeding for 12 weeks up to 24 weeks. However, the total liver iron contents (measured as µg of iron per gram wet liver weight multiplied by liver weight) in HFD-fed mice were not significantly different from those in mice fed the control diet, as the HFD-fed mice were found to have hepatomegaly. Liver hepcidin (HAMP1) mRNA expression in HFD-fed mice tended to be down-regulated at 16, 20 and 24 weeks. Transferrin receptor 1 (TfR1), ferritin and heme oxygnease-1 (HO-1) mRNA levels were elevated at the same time points. Expression of other genes associated with hepatic iron metabolism (matriptase 2 (TMPRSS6), transferrin receptor 2 and ferroportin) were not significantly affected by the HFD. Expression of key regulators of gluconeogenesis, peroxisome proliferator-activated receptor c coactivator 1 (PGC1) and cAMP-regulatory element binding protein H (CREBH), also remained unchanged. These results show that high-fat feeding resulted in peripheral insulin resistance and hyperinsulinemia. This was associated with increased hepatic mRNA expression of TfR1 (involved in cellular iron uptake) and ferritin (involved in iron storage) and decreased hepatic HAMP1 expression and blood hepcidin levels. It is possible that these changes predispose to development of hepatic iron overload over time. Such a situation may underlie the association of insulin resistance and iron overload. In summary, mice fed a high-fat diet developed insulin resistance that was associated with dysregulation of several hepatic proteins involved in iron homeostasis. Work is ongoing to further elucidate the interactions involved in these changes.

Sixth Congress of the International BioIRon Society Page 231 Poster Abstracts IBIS

Poster #85 INSULIN SIGNALLING IS IMPAIRED IN IRON-LOADED PRIMARY HEPATOCYTES Jithu James, MSc1, Joe Varghese, MD2, Sophie Vaulont, PhD3, Andrew Mckie, PhD4 and Molly Jacob, MD, PhD2 1Christian Medical College, Vellore; 2Christian Medical College, Vellore, India; 3INSERM, U1016, Institut Cochin, Paris, France; 4Division of Diabetes and Nutritional Sciences, King’s College, London, UK

Mild-to-moderate iron overload has been reported to be associated with insulin resistance. However, the interaction between intracellular iron and insulin signaling is poorly understood. In order to study possible interactions between these two, primary mouse hepatocytes were isolated from wild-type or hepcidin knockout (hepc KO) mice. Hepatocytes from hepKO mice are known to be iron-overloaded. After an overnight incubation in microplates (16 hrs), these cells were treated with insulin (10 nM concentration for 5 minutes). Cells were then lysed in RIPA buffer. The lysates were used for western blotting analyses to determine the levels of phosphorylated Akt (Ser 473), insulin receptor substrate-1 (IRS-1), phosphorylated lyoe sythase ase p-G er ad phosphorylated -activated kinase (p-AMPK [Thr172]). Total RNA was also isolated from the cells, reverse-transcribed and used for quantitative PCR (qPCR) assays to determine expression levels of various genes of interest. Hepc KO hepatocytes were also treated with an iron chelator, desferroxamine (DFO), at varying concentrations for 16 hours, before insulin treatment (as described above). Hepc KO hepatocytes were found to have significantly higher intracellular iron levels than wild-type hepatocytes. Baseline p-Aktlevels (before insulin stimulation) were found to be higher and insulin-induced pAkt levels were lower in hepc KO hepatocytes than in wild-type hepatocytes. Pre-treatment with DFO before the insulin treatment was found to attenuate these effects in hepc KO hepatocytes. Levels of IRS-1 and p-, hh are p-stream and down-stream respectively of Akt in the insulin signaling pathway, were not different in hepc KO hepatocytes. Levels of p-AMPK, a key sensor of intracellular energy levels, were also not affected in hepc KO hepatocytes, when compared to wild-type cells. Gene expression analyses by qPCR showed elevated levels of mRNA for phosphoenol pyruvate carboxykinase (PEPCK) in the hepc KO hepatocytes, suggestive of increased gluconeogenesis. These cells also showed elevated levels of heme oxygenase-1 (HO-1) mRNA, an antioxidant enzyme, suggesting presence of oxidative stress. Expression of transferrin receptor 1 (TfR1) was significantly reduced,a probable consequence of elevated intracellular iron levels in hepc KO hepatocytes. These changes were also found to be attenuatedby pre-treatment of hepc KO hepatocytes with DFO. In conclusion, insulin-induced phosphorylation of Akt was impaired in hepc KO hepatocytes, which are iron-overloaded. This effect was significantly attenuated after iron chelation of these cells. These results suggest that increased intracellular iron levels impaired insulin signaling. This may contribute to insulin resistance seen in conditions characterized by iron overload, such as thalassemia and hemochromatosis.

Sixth Congress of the International BioIRon Society Page 232 Poster Abstracts IBIS

Poster #86 A PRELIMINARY STUDY OF THE LINK BETWEEN IRON INCREASE IN DOPAMINERGIC CELLS AND PARKINSON’S DISEASE Kosha Metha, PhD2, Bushra Ahmed, PhD, Mohammed Gulrez Zariwala, PhD, Robert Evans, PhD1 and Sebastien Farnaud, PhD 1Brunel University; 2University of Bedfodshire

Although Parkinson’s disease (PD) was described nearly 200 years ago by James Parkinson, its causes and mechanisms are still very poorly understood. PD is simply defined as a degenerative disorder of the central nervous system with motor symptoms resulting from the death of dopamine-generating cells in the substantia nigra. An increase in iron deposits in the substantia nigra pars compacta has been proposed to be a hallmark of the disease, and is believed to be responsible for oxidative damage linked to progressive dopaminergic neuro-degeneration. Whereas in haemochromatotic patients a 10-20 fold increase in iron stores seems to be necessary before clinical manifestations are observed, for PD patients only a 2-fold increase has been proposed to be sufficient to mediate disease progression. This suggests that comparatively to other cell types such as hepatocytes and macrophages, brain cells and in particular dopaminergic neurones might be particularly sensitive to iron increase with a distinct regulation. Because most of published studies, which try to understand the mechanism of PD, relate mainly to the animal model, progression of the disease in human cells still remains to be fully characterised. This preliminary study is using a human-relevant model to investigate the progression of PD state particularly in relation to iron metabolism. In this human cellular model, neural progenitor cells ReNcells were differentiated into dopaminergic cells, and treated with 6-OHD. Following 6-OHD treatment, the dopamine and iron levels were monitored over 48h. A decrease in intracellular dopamine after 2h treatment confirmed a PD phenotype but the rise that followed suggests a recovering phenomenon that is atypical of PD. Intracellular iron level indicates a decrease at 4h-time point, which correlates with mRNA expression of TFRC and SLC40a1 measured at time 0h, followed by a major increase at 48h-timepoint which exceeds by 3-fold the level observed in untreated cells. In conclusion, it is proposed that the iron excess measured in response to 6OH-DOPA treatment supports the iron excess observed in the brain of PD patients, and suggests further study to elucidate its mechanism.

Sixth Congress of the International BioIRon Society Page 233 Poster Abstracts IBIS

Poster #87 IRON DEFICIENT RBCS FROM GAMBIAN CHILDREN AND PREGNANT WOMEN ARE RESISTANT TO FIELD ISOLATES OF FALCIPARUM MALARIA Morgan Goheen, BS1, Bakary Darboe, DMLS2, Ebrima Danso, BSc2, Mamadou Bah, Foundation Degree2, Muna Affara, PhD2, Amat Bah, MsC2, Rita Wegmuller, PhD2, Andrew Prentice, PhD3 and Carla Cerami, MD,PhD1 1University of North Carolina; 2MRC-Gambia; 3London School of Hygiene & Tropical Medicine

Clinical studies show that iron deficiency protects against malaria, and that administration of iron to iron-deficient individuals increases the risk of malaria. However, the precise mechanisms by which iron deficiency confers this protection from malaria remain poorly understood and elucidation of these mechanisms may enable new antimalarial strategies. We have previously reported that parasite invasion of and growth in iron deficient RBCs is reduced, however all of this work was done using RBCs from donors living in a non-malaria endemic area (the United States) and with standard laboratory strains of P. falciparum. In an effort to further evaluate the pathogenesis of P. falciparum infection in people with iron deficiency, we have now quantified parasite cellular phenotypes from several clinical strains of P. falciparum isolated from The Gambia in vitro with erythrocytes from Gambian individuals with iron deficiency. Specifically, we have collected RBCs from Gambian children (ages 6 months to 2 years) and pregnant Gambian women (2nd and 3rd trimester) with hemoglobin levels less than 11 g/dL. Then, using flow cytometry-based assays, we separately examined the effect of iron deficiency on overall parasite growth and, merozoite invasion of, RBCs. We demonstrate that P. falciparum erythrocytic stage growth in vitro increases directly in relation to mean corpuscular hemoglobin concentration in RBCs. We also describe the development of a field ready RBC barcoding assay that allows for direct comparison of parasite invasion into two cell populations labeled with the same fluorophore at differing concentrations. Using this assay, we demonstrate that P. falciparum invasion is reduced in iron deficient RBCs from Gambian children. Additionally, we show that Gambian field isolates of P. falciparum exhibit the same phenotype of reduced invasion and growth in iron deficient RBCs. These investigations leverage novel tools and clinical samples from a malaria endemic country to refine our understanding of an ancient relationship between parasite and host. Further investigations of this relationship can improve our fundamental understanding of falciparum pathogenesis and enable the development of strategies to treat and prevent both iron deficiency and malaria.

Sixth Congress of the International BioIRon Society Page 234 Poster Abstracts IBIS

Poster #88 CYTOPROTECTIVE EFFECT OF FERRITIN H IN RENAL ISCHEMIA REPERFUSION INJURY Suzy Torti, PhD, Heather Hatcher, PhD, Lia Tesfay, MS and Frank Torti, MD University of Connecticut Health Center

Oxidative stress is a major contributor to kidney injury following ischemia reperfusion. Ferritin, a highly conserved iron- binding protein, is a key protein in the maintenance of cellular iron homeostasis and protection from oxidative stress. Ferritin mitigates oxidant stress by sequestering iron and preventing its participation in reactions that generate reactive oxygen species. Ferritin is composed of two subunit types, ferritin H and ferritin L. Using an in vivo model that enables conditional tissue-specific doxycycline-inducible expression of ferritin H in the mouse kidney, we tested the hypothesis that an increased level of H-rich ferritin is renoprotective in ischemic acute renal failure. Prior to induction of ischemia, doxycycline increased ferritin H in the kidneys of the transgenic mice nearly 6.5-fold. Following reperfusion for 24 hours, induction of neutrophil gelatinous-associated lipocalin (NGAL, a urine marker of renal dysfunction) was reduced in the ferritin H overexpressers compared to controls. Histopathologic examination following ischemia reperfusion revealed that ferritin H overexpression increased intact nuclei in renal tubules, reduced the frequency of tubular profiles with luminal cast materials, and reduced activated caspase-3 in the kidney. In addition, generation of 4-hydroxy 2- nonenal protein adducts, a measurement of oxidant stress, was decreased in ischemia-reperfused kidneys of ferritin H overexpressers. These studies demonstrate that ferritin H can inhibit apoptotic cell death, enhance tubular epithelial viability, and preserve renal function by limiting oxidative stress following ischemia reperfusion injury. Supported in part by NIH R01 CA 171101.

Sixth Congress of the International BioIRon Society Page 235 Poster Abstracts IBIS

Poster #89 MACROPHAGES UNDER STRESS ERYTHROPOIESIS ARE ASSOCIATED WITH A CHARACTERISTIC GENE EXPRESSION PROFILE Ritama Gupta, Piali Mukherjee, Igor Theurl, Tara Arvedson and Stefano Rivella, PhD Children's Hospital of Philadelphia

Macrophages have been implicated in iron recycling during erythropoiesis and maintaining the structure of erythroblastic islands consisting of a central macrophage surrounded by erythroid cells in different stages of differentiation. Others and we have previously shown that macrophages contribute to stress erythropoiesis. Using clodronate-encapsulated liposomes to deplete macrophages, we showed that in a model of acute stress, phlebotomy, there was impaired recovery from anemia. In a chronic model of erythroid stress, Polycythemia Vera, we showed that clodronate treated animals had reduced erythrocytosis, reticulocytosis and splenomegaly, hallmarks of the disease. We used iron loaded and hepcidin knock out .mice to show that this effect of macrophages is not solely related to their ability to recycle iron. These findings necessitate study of processes within the macrophage itself that might facilitate stress erythropoiesis and characterization of macrophage transcriptome signatures associated with the same. Bone marrow macrophages have been identified previously as CD115Low GR1Low F4/80High cells. Extending this to the spleen, we sorted and sequenced singlet macrophages in the bone marrow and spleen of two mouse models of stress erythropoiesis: acute stress induced by phlebotomy and chronic stress by bone marrow transplanted Polycythemia Vera. We found that splenic macrophages show more significant changes upon induction of stress (both acute and chronic) in comparison to bone marrow macrophages. The mean of normalized count of each gene when plotted against fold change (Figure) show that the acute- and chronically-stressed splenic macrophages differ significantly over non-stressed macrophages but not over each other. We further found that stressed splenic macrophages have a characteristic gene expression profile associated with erythropoietic stress, common to both models of induced and chronic stress. We used Ingenuity Pathway Analysis (IPA) to look at biological processes encompassing our significantly upregulated candidates [(False discovery rate) FDR 10%], upstream analysis of our candidates and network analysis. Some biological processes of highest significance were cellular growth and proliferation, hematological system development and function, inflammatory response, response to infection, cellular adhesion and cellular movement. We validated some of our key candidates by flow cytometry, such as the receptor for the anti inflammatory cytokine, IL-10 and the cellular adhesion molecule ICAM-1 (Intracellular cell adhesion molecule-1). Upstream analysis using IPA predicted activation of transcription factors such as Notch1, Jun, RelA etc. The iron exporter ferroportin was significantly upregulated in the transcriptome signature of our stressed macrophages and on the cell surface of the same. Given that our stressed “pro erythroid” macrophages upregulate ferroportin we are looking to investigate transcriptome signatures of macrophages on the opposite end of the spectrum, i.e. those downregulating ferroportin. Previous work by the group of Dr. Muckenthaler has shown that injection of the TLR (Toll like receptor) ligand FSL-1 downregulates ferroportin (reference 1) in a hepcidin-independent fashion. We reproduced this data and are currently investigating transcriptome signatures of macrophages downregulating ferroportin as well as from macrophages from a different model of chronic stress, beta thalassemia. 1) Guida C, Altamura S, Klein FA, Galy B, Boutros M, Ulmer AJ, Hentze MW, Muckenthaler MU. A novel inflammatory pathway mediating rapid hepcidin-independent hypoferremia. Blood. 2015;125(14):2265-75.

Sixth Congress of the International BioIRon Society Page 236 Poster Abstracts IBIS

Poster #90 DIFFERENTIAL EXPRESSION OF CERULOPLASMIN AND ZYKLOPEN IN HUMAN DUCTAL CARCINOMA TISSUE AND MODEL CELL LINES Rania Darwish, MS2, Rasha Mismar, MS2, Pamela Nabhan, MS2, Amin Sobh, MS3, Mhenia Haidar, PhD4, Julnar Usta, PhD5, Chris Vulpe, MD PhD6 and Zouhair Attieh, PhD1 1American University of Science and Technology (AUST); 2American University of Science and Technology; 3University of California, Berkeley; 4Lebanese University; 5American University of Beirut; 6University of Florida

Besides being an essential element for many cellular functions, iron (Fe) has lately emerged as a key molecule in carcinogenesis. This is due to the fact that Fe can lead to the formation of reactive oxygen species (ROS) due to its loosely bound electrons on its outer shell. ROS are molecules containing oxygen that can lead, when produced in excess, to DNA damage via oxidative stress, an event implicated in cellular neoplastic transformation. Studies have shown higher serum Fe levels in breast cancer patients compared with normal subjects, even though their dietary Fe intake was lower. Studies have also showed increased Fe staining in colon carcinoma tissue as well as the overexpression of Fe import proteins and down regulation of Fe export proteins including hephaestin (Heph), a member of the multicopper ferroxidase (MCF) family that includes ceruloplasmin (Cp) and zyklopen (Zp) as well. MCF proteins mediate cellular Fe release by the Fe transporter ferroportin (FPN). Pinnix et al. reported a marked decreased in FPN expression in ductal carcinoma (DC) of the breast compared to normal tissue. The expression decrease was further noted as DC tissue became more invasive. To test the hypothesis that DC cells down-regulate Fe export proteins to sequester more Fe needed for cell proliferation, we investigated the expression of MCF transcripts and proteins in normal and DC tissue and model cell lines. Immunostaining using MCF-specific antisera in in situ mammary DC showed a decrease in Cp and Zp expression compared to normal mammary tissue while no Heph expression was observed. Similarly, and employing quantitative PCR using MCF-specific primers on RNA extracted from DC and Normal tissue revealed marked decrease in Cp and Zp transcripts with no detectable Heph transcript. Ion-coupled plasma mass spectrometry (ICP-MS) revealed higher Fe levels in DC tissue than normal one. Quantitative PCR on RNA extracted from MCF10A, MCF7 and MDA-MB231 model cell lines showed a lower expression of Cp and Zp in MCF7 and MDA-MB231 cells compared to MCF10A cells with no detectable Heph expression. Estrogen treated MCF7 and MDA-MB231 cells had a significant decrease in Cp and Zp expression compared to that of non-treated cells, while no significant difference was observed in treated versus non- treated MCF10A cells. These results suggest that DC tissue and DC model cells lines down-regulate the expression of MCF Cp and Zp transcripts and proteins, similar to FPN, to reduce cells’ ability of release Fe. Future experiments will probe the expression of Cp and Zp proteins using immunoblotting as well as the assessment of cellular Fe levels using ICP-MS in treated and non-treated MCF10A, MCF7 and MDA-MB231 cells. The role of miRNA will be then evaluated in the observed Cp and Zp reduction.

Sixth Congress of the International BioIRon Society Page 237 Poster Abstracts IBIS

Poster #91 IDENTIFICATION OF CYS LIGANDS FOR THE [2FE-2S] AND [4FE-4S] CLUSTERS IN DRE2: BOTH CLUSTERS ARE ABSOLUTELY ESSENTIAL FOR THE FUNCTION Yan Zhang, PhD1, Andrew Dancis, MD2 and Eiko Nakamaru-Ogiso, PhD2 1School of Pharmaceutical Science and Technology, Tianjin University; 2University of Pennsylvania

Yeast Dre2 (human anamorsin/CIAPIN1 homologue) was identified as a component essential for cytosolic Fe-S cluster biosynthesis. Dre2 itself contains redox active Fe-S clusters and forms a complex with the diflavin protein Tah18, which transfers electrons from NADPH to the cytosolic Fe-S cluster biosynthesis machinery. Furthermore, Tah18/Dre2 may act as a ferric reductase involved in cofactor assembly of the di-iron enzyme ribonucleotide reductase. Dre2 contains a nonessential N-terminal methyltransferase domain that does not bind SAM and has no methyltransferase activity. The C- terminal domain however contains eight evolutionarily conserved cysteine residues and is absolutely essential. The nature of the prosthetic groups in Dre2 has been controversial. We were the first to demonstrate that Dre2 contains both [2Fe- 2S] and [4Fe-4S] clusters, while others have reported that Dre2 has a single [2Fe-2S] or two [2Fe-2S] clusters. A more recent study confirmed our finding and showed that the [2Fe-2S] in Dre2 could be reduced by Tah18 using the electrons from NADPH. However, the [4Fe-4S] in Dre2 with a lower redox potential was incapable of receiving electrons from Tah18, leaving the function of this prosthetic group mysterious. We constructed individual substitution mutants of total 9 Dre2 cysteines, 8 conserved C-terminal cysteine residues and a non-conserved C116. We heterologously expressed all 9 mutants and the wt Dre2 protein in E. coli and purified them for Electron Paramagnetic Resonance (EPR) characterization. EPR results allowed us to clearly distinguish the [2Fe-2S] cluster ligands from the [4Fe-4S] cluster ones. The C252, C263, C266, and C268 mutants lost the EPR signals assigned for the [2Fe-2S] cluster, while the C311, C314, C322 and C324 mutants lost the [4Fe-4S] cluster signals. We also observed spin interaction between the two clusters, which along with the oxygen sensitivity of the [4Fe-4S] may account for the failure to recognize this cluster in some of the previous studies. With the mutants carrying a single Fe-S cluster, we were able to perform spin simulation and obtain the g values of each cluster. Using a yeast shuffle strain we further demonstrated for the first time that both clusters are essential for cell viability. Finally, our EPR spectra of the human wt anamorsin exhibited temperature-dependent patterns, similar to those of the yeast counterpart, suggesting the coexistence of both [2Fe-2S] and [4Fe-4S] clusters in the human protein. This is consistent with our previous finding that human anamorsin can complement the yeast dre2 null mutant.

Sixth Congress of the International BioIRon Society Page 238 Poster Abstracts IBIS

Poster #92 WATER SOLUBLE MAGNETITE NANOPARTICLES FE3O4 – OLEIC ACID-TWEEN 80 : STABILITY, REDOX ACTIVITY AND TRANSPORT IN HEPATOPANCREATIC CELLS OF THE MANGROVE CRAB UCIDES CORDATUS Hector Aguilar Vitorino, PhD1, Priscila Ortega, PhD2, Flavia Pinheiro Zanotto, PhD2 and Breno Pannia Espósito, PhD1 1University of São Paulo - Institute of Chemistry; 2University of São Paulo - Biosciences Institute

Introduction Biomodels are used to assess the consequences of human activity on animals and their habitat. The edible crab Ucides cordatus, endemic to the mangrove, one of the most populated and degraded ecosystems in Brazil, is used as a bioindicator of toxic metal contamination. In this study, hepatopancreatic cells of U. cordatus were exposed to water- soluble nanoparticles (NP) with three different sizes (5, 8, 10 nm), coated with oleic acid-detergent, with the objective to evaluate the transport of this metal to hepatopancreatic cells of the animal model. The correlation of metal incorporation with hepatopancreatic cell type, NP stability and NP redox activity was tested. Method and materials Monodisperse magnetic NP of magnetite (Fe3O4) coated with oleic acid (OA) have been synthesized by high-temperature reaction of Fe(acac)3 in phenyl ether with alcohol, oleic acid and oleylamine, followed by suspension in tween 80/water (1:9) [1]. NP stability in buffered medium against fluorescent probes was tested with calcein, 5-DTAF apo-transferrin and FTIC desferrioxamine [2,3]. The redox activity was quantified by measuring the rate of oxidation of the probe dihydrorhodamine 123 [3]. The different types of hepatopancreatic cells (embryonic, resorptive, fibrillar and blister; respectively E, R, F and B) were separated using a discontinuous sucrose gradient at concentrations of 10-40% [4]. Intracellular (cytosolic) iron was quantified by loading the cells with acetomethoxy-calcein followed by a titration curve with calcein [2]. Results and Discussion Our results indicate that the NPs are very stable against different fluorescent chelators, probably because iron is not readily available from the mineral core and/or beneath the outer layer of oleic acid. Despite this stability, we found the equivalent to 5.63 - o redo-active iron, which could be a surface phenomenon. The transport of NPs to the interior of cells will depend on the size, nature and charge of its surface [5]. Iron loading to E (0.67 - ad - ells ere slar ease ther a to s asorpto o trets the other hand F (0.43 - ad - ells, resposle or desto ad storae o trets, dsplayed lower load and therefore was not detected by the fluorescent probe in large amounts [6]. Conclusions In this work three small magnetic NPs were synthesized and displayed similar stability in physiological buffered medium. Iron loading to hepatopancreatic cells of U. cordatus depends on the type of cell, and is related to their function and developmental stage. Acknowledgments We would like to thank CAPES and FAPESP for financial support References [1] A.D. Arelaro. Journal of Magnetism and Magnetic Materials 320 (2008) 335–338 [2]Esposito BP, Epsztejn S, Breuer W, Cabantchik ZI. A review of fluorescence methods for assessing labile iron in cells and biological fluids. Anal Biochem. (2002) 304: 1–18. [3] Vitorino HA, Mantovanelli L, Zanotto FP, Espósito BP .Iron Metallodrugs: Stability, Redox Activity and Toxicity against Artemia salina. PLoS ONE(2015) ; 10(4) [4] P. Ortega ,F.Zanotto. Aquatic Toxicology 157 (2014) 21–29. [5] L.Shang , G. Nienhaus.Journal of Nanobiotechnology (2014), 12:5 [6] P. Ortega ,F. Zanotto. Iheringia, Série Zoologia. (2014) 104(3):347-354

Sixth Congress of the International BioIRon Society Page 239 Poster Abstracts IBIS

Poster #93 A NOVEL NANO-IRON SUPPLEMENT (IHAT) TO SAFELY COMBAT IRON DEFICIENCY AND ANAEMIA Dora Pereira, PhD, MEng1, David Frazer2, Mohamad Aslam, BSc2, Greg Anderson2 and Jonathan Powell, PhD2 1Medical Research Council Human Nutrition Research; 2MRC Human Nutrition Research

Ferritin is the biological storage form of iron. It is composed of up to eight nanoparticles of iron oxo-hydroxide stored in a protein shell that renders the iron particles both nano-disperse and sufficiently unstable to be utilised biologically. When ingested in either plant or animal based foods, it is well absorbed. The exact mechanism of absorption is not resolved- either the iron is gradually broken down in the acidic, gastric lumen at a rate that matches later intestinal absorption or the particle resists total gastric degradation and is taken up whole by the small bowel enterocytes and gradually broken down intra-lysosomally to join the common iron pool. Either way, undigested nanoparticulate iron oxo-hydroxide should not be reactive in the same way that soluble iron is and thus should be poorly available to colonic bacteria or to participate in redox reactions at the epithelial surface. At the Medical Research Council Human Nutrition Research Unit in Cambridge, we have been developing a cheap, synthetic ferritin core mimetic for safe and effective iron supplementation. Iron hydroxide adipate tartrate (IHAT) is a tartrate-modified, nano-disperse Fe(III) oxo-hydroxide, formed in an adipate buffer, with similar functional properties and small primary particle size (2-5 nm) as the iron form found in ferritin. My presentation will provide an overview of the preclinical data we have collected for in the last 10 years: Efficacy: A study with iron deficient women in the UK showed that (i) bioavailability (i.e. red blood cell incorporation) of iron from IHAT was nearly 80% that of iron from ferrous sulphate, the gold standard of iron supplementation and (ii) IHAT had slower kinetics of serum iron absorption, raising serum iron levels and transferrin saturation slowly and less aggressively (at 4 hours) than the same dose of iron from ferrous sulphate. Four independent rodent studies have shown that IHAT is equivalent to ferrous sulphate at correcting haemoglobin levels in anaemia. Pharmacokinetics: Absorption of IHAT was significantly increased in iron-deficient mice compared to iron-sufficient mice, showing that systemic absorption of IHAT is normally regulated by body iron levels. Basolateral export of iron derived from IHAT from the intestinal enterocyte into the blood circulation, was via ferroportin (as with ferrous sulphate). Expression of duodenal DMT1, expression of liver hepcidin, liver iron, splenic iron and duodenal iron levels were all similar between the 2 groups. Safety proof-of-concept in vitro assays: IHAT had no adverse effect on gut cell viability, even at doses 10-20 fold in excess of those possible in the gut lumen, whereas soluble ferrous iron was markedly toxic at 14-fold lower doses than IHAT. IHAT had no adverse effects on epithelial cell monolayer integrity. Safety proof-of-concept in vivo studies: In an ‘enteric infection’ rodent model, IHAT was much less available to potential enteropathogens than ferrous sulphate and led to a more beneficial gut microbiome, with increasing Lactobacilli after 14 days supplementation. In a ‘healthy colon’ rodent model, IHAT did not negatively impact the gut microbiome after 28 days of supplementation. Mechanism of absorption in vitro and in vivo studies: In cellular and animal studies, IHAT was shown to be taken up apically as whole nanoparticles, without the need for luminal or mucosal iron redox activity, in a separate mechanism to the DMT1-driven uptake of conventional soluble iron supplements.

Sixth Congress of the International BioIRon Society Page 240 Poster Abstracts IBIS

Poster #94 DISTINCT METABOLOMIC PROFILES IN INBRED MOUSE STRAINS AND CORRELATION WITH IRON Rola Zeidan, BSc, MSc, Daniel Medina-Cleghorn, BS, Kathryn Page, PhD, Stela McLachlan, PhD, Eleazar Eskin, PhD, Daniel Nomura, PhD and Chris Vulpe, MD, PhD University of Florida

Introduction: Differences between the strains in their metabolic profiles and the variation in metabolic profiles in response to environmental influences are partly genetically determined and this difference could be used to map genes underlying it. Methods: Six strains of inbred mice were studied: A/J, AKR/J, BALB/cJ, C57BL/6J, C3H/HeJ, DBA/2J. Eight replicates from each strain, all males, were on iron defined diet (35ppm iron) and were sacrificed at 10 weeks of age. Liver samples were obtained, lipid extraction was performed and the metabolome was analyzed using Triple Quadrupole LC/MS run (LCMSMS) to obtain the metabolomics profile (141 lipid metabolites). Results were normalized according to the dry weight of the liver sample and the internal standard. Normalized data was checked for outliers (2 samples removed), log transformed and analyzed by ANOVA. Bonferroni correction has been applied to account for multiple testing (p=0.00035). Pearson’s r of mean strain values of each metabolite was used to calculate correlation of metabolites between the strains and between metabolites and iron. Results: Around 60% of the metabolites show statistically significant differences between the strains by nominal p-value (p<0.05). When we applied Bonferroni correction, there are 21 metabolites significantly different between the strains, which accounts to around 15% (Table 1). 13 metabolites showed correlation above +/-0.8 with iron (Table 1), with additional 8 showing correlation above +/-0.7. Conclusions: Our finding of reproducible differences in metabolite profiles between inbred strains suggests that mapping of the underlying genetic variation responsible for the differences could be possible using a larger inbred and recombinant inbred mouse QTL mapping panel.

Sixth Congress of the International BioIRon Society Page 241 Poster Abstracts IBIS

Poster #95 TARGETING STORAGE LESION IN RED BLOOD CELL TRANSFUSION Xi Huang, PhD1, Kanika Brown, BS2 and Alan Perlstein, MS2 1Ex Vivo Dyncamics, Inc.; 2Ex Vivo Dynamics, Inc. 423 127th Street, New York, NY 10017, USA

Red blood cell (RBC) transfusion is a critical, life-saving treatment for severe anemia caused by disease or chemotherapy, or by blood loss due to trauma or major surgery. Yet, RBC transfusion has been under scrutiny as a causative element in worse clinical outcomes, longer hospital stays, higher readmission rate, and increased risk of morbidity and mortality. Routine blood storage is up to 42 days for RBC units. During storage, a fraction of RBCs becomes damaged, releasing hemoglobin (Hb) and its metabolites such as heme and iron. Infusion of these hazardous components, collectively called “storage lesion”, overwhelms a patient’s normal ability to clear these transfusion byproducts and results in enhanced complications and adverse outcomes. Until now, there is no clinical solution to mitigate the risk from RBC storage lesion. Hb is a potent scavenger of nitric oxide (NO), which modulates vascular contractility. Free Hb binds NO ~1,000-times faster than RBC-bound Hb. Thus, Hb released from lysed RBCs disrupts NO homeostasis causing blood vessel constriction, which leads to further hemolysis within 2 h transfusion, hypertension, and acute lung injury. Based on the property of fast reaction of free Hb with NO, we tested a hypothesis that NO group could be used to sequester and retain free Hb, heme, and iron. To provide a proof of the concept, we used nitrocellulose membrane and regular paper filter to compare their ability to retain Hb and iron complex. We found that nitro compounds have strong affinity for Hb and iron compounds but not for calcium compounds. Since nitrocellulose is highly inflammable and is made with nitric acid, it is not suitable for RBC separation. We then used nitrosobenzene, a NO mimetic, and carried out a surface plasma resonance (SPR)-based study. We identified two binding sites, one very strong (0.46 µM) and one weak (990 µM) binding site to Hb. The affinity of nitrosobenzene to Hb appears to be stronger than those of retinal and retinoic acids to retinoid binding proteins (1 µM range). In conclusion, certain NO materials can be used to selectively remove storage lesion from packed RBC units immediately before transfusion. Success in this technology development will reduce blood transfusion-associated complications thereby lowering healthcare costs and providing anemic patients with a higher quality of life (Patent pending).

Sixth Congress of the International BioIRon Society Page 242 Poster Abstracts IBIS

Poster #96 SIRT2 MEDIATED-DEACETYLATION OF NUCLEAR FACTOR (ERYTHROID-DERIVED 2)-LIKE 2 (NRF2) REGULATES CELLULAR IRON HOMEOSTASIS Xiaoyan Yang, MD, PhD, Athanasios Vassilopoulos, PhD, Seong-Hoon Park, PhD, David Gius, MD, PhD and Hossein Ardehali, MD, PhD Northwestern University

Background: Sirtuins (SIRTs) are NAD+-dependent deacetylases and critical regulators of energy metabolism and response to oxidative stress. Metabolic processes and antioxidant activity also need iron, however, too much iron can also be toxic when in excess. Thus, we hypothesized that SIRTs regulate cellular iron levels to ensure adequate supply of this element for their biological functions, while keeping its levels below toxic levels. We specifically focused on SIRT2, one of the least characterized sirtuin that is mainly present in the cytoplasm.

Methods and Results: Iron content was significantly lower in SIRT2-/- mouse embryonic fibroblasts (MEFs) compared to SIRT2+/+ MEFs (non-hee ro s ol prote, p, ad ee epresso o erroport-1 (FPN1), the major cellular iron exporter, was also significantly increased in SIRT2-/- MEFs. Similarly, silencing SIRT2 in HepG2 cells decreased cellular iron levels and increased FPN1 expression, indicating that enhanced FPN1 in SIRT2 knockout or knockdown condition increases iron export and reduces cellular iron. SIRT2 levels were also increased with iron chelation, while iron supplementation had the opposite effect. To investigate the underlying mechanism, we focused our studies on nuclear factor (erythroid-derived 2)-like 2 (NRF2), a known regulator of FPN1. Our results demonstrated that NRF2 is upregulated and translocates into the nucleus in SIRT2-/- MEFs and knocking down NRF2 in SIRT2-/- MEFs reverses iron deficiency and FPN1 expression. Furthermore, NRF2 is acetylated by P300/CBP and can be deacetylated by SIRT2. Finally, to confirm the role of SIRT2 in iron regulation, cellular heme and non-heme iron in the heart (major iron- consuming organ) and liver (major iron-storage organ) were measured in wild type (WT) and SIRT2-/- mice. Heme and non-heme iron content were significantly decreased in SIRT2-/- mouse livers compared to WT livers (heme: 2.25 vs. 1.65 nmol/mg protein, p=0.002; non-hee ro s ol prote, p Furthermore, heme levels were also significant decreased in the heart, while non-heme iron was not significantly altered.

Conclusions: Our results indicate that SIRT2 regulates cellular iron homeostasis by deacetylating NRF2 and altering iron export through FPN1. Thus, SIRt2 is a novel regulator of cellular iron, which works independent of the iron regulatory protein (IRP) system.

Sixth Congress of the International BioIRon Society Page 243 Poster Abstracts IBIS

Poster #97 THE REGULATION OF HIF-1 ON IRON METABOLISM IN COLON CANCER CELL LINES UNDER HYPOXIA Li Zhu, PhD, Mengwan Zhang, Master, Qianqian Luo, Master and Dan Wang, Master Institute for Nautical Medicine

When exposed to hypoxia, hypoxia-inducible factor-1 (HIF-1) remains stable and regulates varieties of physiological activities by regulating the expression of many kinds of proteins, including some iron metabolism-related proteins. This regulation often happens during cancer development. Colorectal cancer (CRC) is the third most common cause of cancer- related deaths in industrialized countries. Iron overload caused by excess dietary iron uptake or hereditary mutations has been identified as a risk factor for CRC. To understand the regulation of HIF-1 on iron metabolism under hypoxia, three colon cancer cell lines Caco-2, HT-29, and HCT116 were used. These cells were exposed to hypoxia (1% O2) for different time periods (0h, 4h, 8h, 12h, 24h) and cell viabilities, as well as cell morphology were observed. The expression of HIF-1 and iron metabolism-related proteins, including iron regulatory protein 1 and 2 (IRP1, 2), divalent metal transporter 1 (DMT1), transferrin receptor 1 (TfR1), ferroportin 1 (Fpn1) were detected by Western blot. The uptake of ferrous iron and the total iron level were measured using fluorescent probe and spectrophotometric method. Digoxin, has been reported to inhibit HIF-1 transcriptional activity by preventing HIF-α prote traslato o as sed to ht HIF-1 level under hypoxia and estimate the effect of HIF-1 on iron metabolism. The data indicated that: (1)The cell viability and viable cell number of three colon cancer cells were affected differently when exposed to hypoxia (1%O2) for different time periods: the viability of Caco-2 and HCT116 cells were reduced for 4-24 h under hypoxia, the viability of HT-29 increased for 4-8 h while reduced for 12-24 h; the viable cell of Caco-2 and HT-29 increased while HCT116 decreased; (2) The protein level of HIF-1 markedly increased for all three cell lines under hypoxia. The expression of both IRPs and DMT1 increased when exposed to hypoxia; The expression of TfR1 under hypoxia was various in three cell lines, which was up-regulated in HT-29, down-regulated in HCT116 while remained almost invariant in Caco-2; (3) Hypoxia promoted the uptake of ferrous iron in a time-independent manner but reduced the total iron level in three cell lines with the time of hypoxia; (4) Digoxin treatment inhibited obviously the expression of HIF-1 and also affected the expression of some iron metabolism-related proteins under hypoxia. When HIF-1 was inhibited under hypoxia, IRP1 and TfR1 decreased in three cell lines, while IRP2 increased in HT-29 but reduced in HCT116 and Caco-2. Fpn1 decreased in Caco-2 and HT-29 cell lines but unchanged in HCT116. There was no obvious effect of HIF-1 inhibition on DMT1 expression under hypoxia; (5) Inhibition of HIF-1 by digoxin inversed the effect of hypoxia on the uptake of ferrous iron and total iron level in three cell lines. Taken together, HIF-1 involved in the regulation of iron metabolism in colon cancer cell lines under hypoxia through alternation of the expression of iron metabolism-related proteins and affected the uptake of ferrous iron and total iron level.

Sixth Congress of the International BioIRon Society Page 244 Poster Abstracts IBIS

Poster #98 GENETICALLY CONTROLLED UPTAKE OF FERRITIN AS AN MRI CONTRAST AGENT Christoph Massner, Dipl Biol (to)1, Giorgio Pariani, Dr2, Hannes Rolbieski2, Felix Sigmund, MSc2 and Gil Gregor Westmeyer, Prof Dr2,3 1Nuklearmedizinische Klinik, Klinikum rechts der Isar, Technische Universität München, München/ Institute for Biological and Medical Imaging, Institute for Developmental Genetics, Helmholtz Zentrum München, München; 2Institute for Biological and Medical Imaging, Institute for Developmental Genetics, Helmholtz Zentrum München, München; 3Nuklearmedizinische Klinik, Klinikum rechts der Isar, Technische Universität München, München

Throughout recent years our understanding of iron trafficking and storage on a cellular level has been growing immensely. This knowledge can be utilized for developing of genetically controlled MRI contrast agents for applications in non- invasive preclinical gene reporter imaging of entire organisms. We measured the T2 relaxation rates of HEK293T cells loaded with ferritin during overexpression of transferrin receptor 1 (TfR1) and yellow fluorescent protein (YFP) as a control. The cell viability was determined by trypan blue staining and cell counting. The intracellular iron content was calculated based on a ferrozine assay1 and cell count. The elevated uptake of horse spleen ferritin (HSFt) in HEK293T overexpressing TfR1 resulted in 60% increased T2 relaxation rate after one or two days expression of the respective proteins without observable toxic effects. Our results show that genetic control over ferritin uptake through the overexpression of TfR1 results in elevated MRI contrast and cellular iron loading. As reported, this probably occurs via ferritin binding on the cell surface receptor and subsequent internalization2. Since ferrihydrite can be exchanged by more magnetizable iron species in the ferritin cavity3 and ferritin can cross the blood brain barrier4, this system has the potential to outperform superparamagnetic iron-oxide (SPIO) based imaging in brain research with respect to delivery and cell specific addressability.

1. Riemer, J., Hoepken, H. H., Czerwinska, H., Robinson, S. R. & Dringen, R. Colorimetric ferrozine-based assay for the quantitation of iron in cultured cells. Anal. Biochem. 331, 370–5 (2004). 2. Li, L. et al. Binding and uptake of H-ferritin are mediated by human transferrin receptor-1. Proc. Natl. Acad. Sci. U. S. A. 107, 3505–10 (2010). 3. Cao, C. et al. Magnetic characterization of noninteracting, randomly oriented, nanometer-scale ferrimagnetic particles. J. Geophys. Res. 115, B07103 (2010). 4. McCarthy, R. C. & Kosman, D. J. Iron transport across the blood-brain barrier: development, neurovascular regulation and cerebral amyloid angiopathy. Cell. Mol. Life Sci. (2014). doi:10.1007/s00018-014-1771-4

Figure 1: TfR-dependent MRI contrast enhancement. Signal change after loading of YFP overexpressing HEK293T cells as a control and TfR overexpressing HEK293T cells with HSFt (black bars) shown for one and two days protein expression respectively. Control cells overexpressing YFP and TfR1 without incubation of HSFt (grey bars).The middle and bottom rows show the respective MR images (TR: 6000 ms, TE: 21.6 ms, Matrix: 256) and color photographs of the cell pellets.

Sixth Congress of the International BioIRon Society Page 245 Poster Abstracts IBIS

Poster #99 NOVEL BETA-CASEIN NANOCARRIERS FOR ORAL IRON DELIVERY Mohammed Gulrez Zariwala, PhD2, Soma Somavarapu, PhD3, Robert Evans, PhD1, Sebastien Farnaud, PhD and Derek Renshaw, PhD 1Centre for Electronic Systems Research, Brunel University; 2Faculty of Science and Technology, University of Westminster; 3School of Pharmacy London

Introduction: Iron deficiency is the most prevalent nutritional disorders worldwide (WHO). Ferrous sulphate (FeSO4) is the most common iron supplement/fortificant, however, it causes gastrointestinal (GI) side effects and has a poor sensory profile. Encapsulation approaches are used to overcome this, but poor absorption is a limitation. Milk proteins, being non- toxic and readily available, are increasingly being utilised as encapsulation shell material for bioactives. Beta-casein is one of the six main milk proteins found in mammalian milk, and is known to self-assemble into natural micelles that transport nutrients to neonates. Previous research has used modified beta casein micelles for delivery of hydrophobic drugs and nutrients. We aimed to formulate and characterise novel beta-casein nanocarriers for iron delivery. Methodology: Blank (BC-Blank) and ferrous iron (FeSO4) loaded beta-casein nanocarriers (BC-NC) were prepared using cold homogenisation method. Nanocarrier physiochemical characteristics were assessed by particle size, zeta potential and morphological analysis (scanning electron microscopy; SEM). Iron uptake from test formulations was compared by Caco-2 cell uptake experiments using simulated GI fluid, with intracellular ferritin protein as a measure of iron absorption. Cellular cytotoxity in the presence of increasing concentrations of blank and iron loaded nanocarriers (BC-Blank and BC- NC) was determined by alamarBlue® cell viability assay (48 hour and 72 hour time-points). Iron absorption from BC-NC was compared against pure FeSO4 as reference as well as unformulated mixture of beta-casein plus ferrous sulphate (BC-Free). Results: Beta-casein nanocarrier formulations were found to be within a submicron size range (BC-NC: 93.98 ± 1.77 nm, BC-NC: 127.47 ± 6.7 nm) favourable towards intestinal permeability. All nanocarrier formulations were found to be non- toxic under test conditions. Poor caco-2 iron absorption was observed from BC-free (105.44 ± 14.91 ng ferritin/mg cell protein). However, significantly enhanced iron absorption was observed from the nanocarrier formulation BC-NC (454 ± errt ell prote hher tha ree erros slphate otrol .28 ng ferritin/mg cell protein). Summary: Free beta-casein plus unentrapped iron mixture demonstrated poor cellular iron absorption. Iron loaded beta- casein nanocarriers fabricated under specific physiochemical conditions were able to deliver high levels of iron to intestinal cells. Our results suggest that formulation of ferrous iron loaded nanocarriers with beta-casein may impart enhanced membrane permeability properties that results in high cellular delivery. We thus demonstrate for the first time the formulation and characterisation of novel beta-casein nanocarriers as vehicles for intestinal delivery of ferrous iron for nutritional applications.

Sixth Congress of the International BioIRon Society Page 246 Poster Abstracts IBIS

Poster #100 DEVELOPMENT OF MITOCHONDRIA-TARGETED IRON CHELATORS FOR THE TREATMENT OF FRIEDRICH ATAXIA Roxana Yesenia Pastrana Alta, PhD1, Maria Têresa Machini, PhD2, Shana O. Kelley, PhD3 and Breno Pannia Espósito, PhD1 1Institute of Chemistry/Laboratory of Bioinorganic Chemistry and Metallodrugs, São Paulo, Brazil; 2Institute of Chemistry/Laboratory of Peptide Chemistry, Department of Biochemistry, São Paulo, Brazil; 3Department of Biochemistry/Faculty of Medicine, University of Toronto, Toronto, Canada

Introduction Friedreich ataxia (FA) is the most common recessive ataxia and is characterized by a mixed spinocerebellar and sensory ataxia frequently associated to cardiomyopathy [1]. Early biochemical studies revealed that dysregulation of iron metabolism is a key feature of the disease, mainly characterized by mitochondrial iron accumulation and by decreased activity of iron-sulfur cluster enzymes [2]. However, the role of frataxin deficiency on this essential biosynthetic pathway is still poorly understood. Targeting excess iron in this organelle is proposed as a means to ameliorate FA symptoms. Desferrioxamine (DFO) is a bacterial siderophore with high affinity for iron, but low cell penetration. Mitochondria-penetrating peptides (MPP) are short peptide sequences comprising alternating lipophilic and cationic residues. MPP: TAT49-57 (H-Arg-Lys-Lys-Arg-Arg-Gln- Arg-Arg-Arg-OH)[3], 1A (H-Cha-Arg-Cha-Lys-Cha-Arg-Cha-Lys-NH2)[3], SS-02 (H-Dmt-Arg-Phe-Lys-NH2)[4] and SS-20 (H-Phe-Arg-Phe-Lys-NH2) [4] were synthesized and appended to the DFO unit. Those peptides were selected for conjugation because they permeate cytosolic and mitochondrial membranes, exhibit no toxicity toward human cells, and are composed of artificial amino acids, therefore not degraded by proteases [3]. Material and methods Peptides were prepared as reported [5]. Conjugation of the –NH2 group of DFO with –COOH of succinic anhydride linked with the terminal –NH2 of the resin-bound peptides was achieved using 2.5 molar excess (relative to the resin capacity) of a solution of DFO, four equivalents of HBTU and HOBt and eight equivalents DIPEA in DMF for 1h.The peptide/DFO conjugates are referred to as mtDFO. A fluorophore-labeled analogues, mtDFO-carboxy-tetramethylrhodamine(TAMRA), were also prepared [5]. The peptides were then cleaved from the resin with trifluoroacetic acid and purified by reverse- phase HPLC. The purified DFO-peptide conjugates were characterized by MALDI-MS. Quantification of mtDFO-TAMRA was done using an extinction coefficient of 92,000M-1cm-1at560nm, in methanol. A bicinchoninic acid (BCA) assay (Thermo Scientific) was used to quantify unlabeled peptides, mtDFO. In order to assess the intracellular localization of mtDFO, fluorescence imaging studies were carried out with the TAMRA-labeled analog. Toxicity was in HeLa cells using CCK-8 viability dye was examined (Dojindo, Rockville). Results All peptides and conjugates were obtained at 90% purity, with an average yield of 4%. Localization of mtDFO was first assessed in HeLa cells, for they have a well-defined mitochondrial network that can be readily imaged. Representative images revealing colocalization of the TAMRA conjugate with MitoTracker Deep Red (Invitrogen) for the mtDFO:DFO-1A- TAMRA (Figure1). None of the compounds displayed significant toxicity. Fig. 1: Structure and mitochondrial localization of DFO-1A-TAMRA. Conclusions We have prepared and characterized four mitochondria-traceable conjugates of DFO that may find use in the treatment of FA. References: [1] Koeppen et al., Acta Neuropathol. Commun, 2013, 1:26; [2] HélènePuccio et al.,Frontiers in Pharmacology, 2013, 5,130:1-11; [3] Shana O. Kelley. et al. Chem. Bio. Chem., 2009,10, 2081–2088; [4] Hazel H. Szeto. et al. The Journal of Biological Chemistry, 2004, 279(33) 34682–34690. [5] Simon P. Wisnovsky, 2013 Chemistry & Biology 20, 1323–1328. Acknowledgments CAPES and FAPESP for the financial support.

Sixth Congress of the International BioIRon Society Page 247 Poster Abstracts IBIS

Poster #101 CHANGES OF MOTOR COORDINATION ABILITY AND SPATIAL LEARNING ABILITY IN CHRONIC IRON- INTOXICATED MICE Fengjv Jia, Ning Song, Junxia Xie and Hong Jiang Department of Physiology, Shandong Provincial Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology, Medical College of Qingdao University, Qingdao, 266071, ChinaShandong Provincial Collaborative Inn

Abstract: Environmental factors contribute to the development of Parkinson's disease (PD). High iron intake might be associated with an increased risk of PD. In the present study, we observed the motor symptom and non-motor symptom (spatial learning and memory abilities) in one-year-old C57/BL6 mice with dietary supplements of 3% carbony iron for 1 and 3 months, respectively. The residence time on rotarod treadmills was shorter in 3-month-treated female mice in rotarod test, whereas there was no change in 1-month-treated mice. However, there were no changes of the residence time in the male mice treated for both 1 and 3 months. The contents of dopamine (DA), dihydroxy-phenylaceticacid (DOPAC) and homovanillic acid (HVA) were measured by HPLC. The contents of DA and HVA were decreased by 55.6% and 29.6% respectively, in 3-month-treated female mice. The DA turnover rate was decreased by 38.1%. Interestingly, in 1-month-treated male group, the content of HVA and the level of DA turnover rate were increased by 36.7% and 61.1%. For 3-month-treated male group, the contents of DA, DOPAC and HVA were decreased by 46.3%, 40.6% and 37.3%, respectively. The DA turnover rate was decreased by 27.2% in this group. Spatial learning and memory ability was measured using Morris water maze. The latency to escape on the hidden platform was reduced in iron-intoxicated male mice for 1 month in the training session. Yet, no changes were detected in all the groups in any of the parameters of the probe session (average speed, time to destination, average proximity, platform crossings, relative time in the training and opposite quadrant). In conclusion, the results showed that dietary iron supplement for 1 month and 3 months could alter both motor symptom and spatial learning ability. Keywords: Iron; Motor symptom; Spatial learning and memory abilities; Parkinson's disease Acknowledgement: This work was supported by grants from the National Program of Basic Research sponsored by the Ministry of Science and Technology of China (2011CB504102) and the National Foundation of Natural Science of China (81171207, 81430024).

Sixth Congress of the International BioIRon Society Page 248 Poster Abstracts IBIS

Poster #102 NOVEL HEPCIDIN MIMETICS Greg Bourne, PhD1, Mark Smythe, PhD2, Brian Frederick, PhD3, Sonya Scott, PhD4, Simone Vink, PhD4, Jacob Ulrik Fog, PhD5, Pernille Tofteng-Shelton, PhD5 and Praveen Madala, PhD4 1Protagonist; 2Protagonist Therapeutics Inc. 306 Carmody Road PO Box 6421, Brisbane Qld. 4067 and Institute for Molecular Bioscience University of Qld., St Lucia Qld 4067; 3Protagonist Therapeutics Inc. 521 Cottonwood Dr, Milpitas, CA, USA 95035; 4Protagonist Therapeutics Inc. 306 Carmody Road PO Box 6421, Brisbane Qld. 4067 and Institute for Molecular Bioscience University of Qld., St Lucia Qld 4067; 5Zealand Pharma A/S, Smedeland 36, 2600 Glostrup, Copenhagen, Denmark

Hepcidin is a 25–amino acid peptide and is critical for iron homeostasis. The known molecular target of hepcidin is the protein ferroportin, which functions as a trans-membrane channel for the export of cellular iron. Hepcidin thus induces the endocytosis and proteolysis of ferroportin, to decrease the delivery of iron to plasma. Hepcidin is structurally complex (4- disulfide-cross linked beta-hairpin), difficult to synthesize, and prone to aggregation. A hepcidin mimetic with drug-like properties is thus highly desirable. Protagonist has utilized its proprietary VectrixTM technology platform and ‘scaffold hopping’ to identify novel hepcidin mimetics. The ability to identify disulphide-rich peptides (DRP) that allow the functional residues to be engineered within the DRP is referred to as scaffold hopping. Figure 1 represents the overall strategy. VectrixTM determines the functional epitopes within the query in 3-D space and appends these residues upon a new scaffold that represents a similar structural shape. The scaffolds are filtered based on selection criteria including the ability to manipulate medicinal chemistry to optimize potency and overall PK stability. A series of queries were initiated to find the correct scaffold. Scaffold Selection included the N-termini (AA1-AA9), the Antibody Binding surface, the C-terminal AA10-AA25, and combinations thereof. Ranking of each search was based upon a number of parameters including number of residues and disulphide bond overlay (solvent exposure), synthesis, RMS value and finally steric invasion of the query in relation to hepcidin. Utilizing this approach, Protagonist discovered potent hepcidin mimetics. Two potent chemical classes were developed with good physiochemical characteristics that improved solubility with enhanced stability compared to hepcidin. Several potent analogues possess >24 h half-life in human serum. One peptide was shown to possess a similar PK/PD profile to hepcidin in adult mice.

Sixth Congress of the International BioIRon Society Page 249 Poster Abstracts IBIS

Poster #103 FUNCTIONAL ANALYSIS OF GLRX5 MUTANTS REVEALS DISTINCT FUNCTIONALITIES OF GLRX5 PROTEIN Gang Liu, PhD1, Yongwei Wang, BS1, Greg Anderson, PhD2, Clara Camaschella, PhD, MD3, Yanzhong Chang, PhD4 and Guangjun Nie1 1CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, China, Beijing, 100190; 2Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia; 3Vita-Salute University and IRCCS San Raffaele Scientific Institute, Milan, Italy; 4Laboratory of Molecular Iron Metabolism, College of Life Science, Hebei Normal University, Shijiazhuang, 050024, Hebei Province, China

GLRX5 is a 156 amino acid mitochondrial protein that plays an essential role in mitochondrial iron-sulfur cluster transfer. Mutations in this protein were reported to result in sideroblastic anemia and variant nonketotic hyperglycinemia in human. Recently, we have characterized a Chinese congenital sideroblastic anemia patient who has two compound heterozygous missense mutations (c. 301 A>C and c. 443 T>C) in his GLRX5 gene. Herein, we developed a GLRX5 knockout K562 cell line and studied the biochemical functions of the identified pathogenic mutations and other conserved amino acids with predicted essential functions. We observed that the K101Q mutation (due to c. 301 A>C mutation) may prevent the binding of [Fe-S] to GLRX5 protein, while L148S (due to c. 443 T>C mutation) may interfere with [Fe-S] transfer from GLRX5 to iron regulatory protein 1 (IRP1), mitochondrial aconitase (m-aconitase) and ferrochelatase. We also demonstrated that L148S is functionally complementary to the K51del mutant with respect to Fe/S-ferrochelatase, Fe/S- IRP1, Fe/S-succinate dehydrogenase and Fe/S-m-aconitase biosynthesis and lipoylation of pyruvate dehydrogenase ople ad α-ketoglutarate dehydrogenase complex. Furthermore, we demonstrated that the mutations of highly conserved amino acid residues in GLRX5 protein can have different effects on downstream Fe/S proteins. Collectively, our current work demonstrates that GLRX5 protein is multifunctional in [Fe-S] protein synthesis and maturation and defects of the different motifs of the protein will lead to distinct clinical presentation.

Sixth Congress of the International BioIRon Society Page 250 Poster Abstracts IBIS

Poster #104 DELETION OF THE MULTICOPPER FERROXIDASE ZYKLOPEN IN MICE LEADS TO HAIR ABNORMALITIES BUT DOES NOT AFFECT SYSTEMIC IRON HOMEOSTASIS IN ADULTS Yan Lu, PhD1, Brie K. Fuqua, PhD2, Kathryn Page, PhD3, Christopher D. Vulpe, PhD4 and Gregory J. Anderson, PhD5 1QIMR Berghofer Medical Research Institute; 2Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, QLD Australia & Department of Nutritional Science and Toxicology, University of California at Berkeley, Berkeley, CA, USA; 3Department of Nutritional Science and Toxicology, University of California at Berkeley, Berkeley, CA, USA; 4Department of Nutritional Science and Toxicology, University of California at Berkeley, Berkeley, CA, USA. Current address: Department of Physiology, School of Veterinary Medicine, University of Florida, Gainesville, FL, USA; 5Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, QLD Australia

Zyklopen (Zp), the third member of the multicopper ferroxidase (MCF) family, shares high sequence homology with Hephaestin (Hp) and Ceruloplasmin (Cp). Like Hp and Cp, Zp possesses a ferroxidase activity that is decreased upon copper deprivation. Zp is expressed in multiple tissues, including the kidney, retina, placenta and mammary gland [1]. Here we report the results from the global deletion of Zp in C57BL/6J mice. Exon 2 of the gene encoding Zp, Hephl1, was targeted using the cre-lox system. Exon 2 is located near the start of the protein-coding region and encodes residues that make up the Type II Cu site required for ferroxidase activity. At birth, Zp-/- pups were of normal size and the genotype ratios of the pups were normal (male pups: Zp+/+ 25%, Zp+/- 50%, Zp-/- 25%; female pups: Zp+/+ 30%, Zp+/- 43%, Zp-/- 27%; approximately 100 pups/gender). Homozygous breeding pairs produce litters of reasonable size (average 6 pups per litter) and with normal gender ratios (male:female=1:1). Zp-/- mice reach adulthood and do not die prematurely. At 8-10 weeks of age, the haematological parameters (Hb, Hct, MCV and MCH) of male and female Zp-/- mice were normal, as was their blood iron status (serum iron, TIBC and Tf saturation). Zp-/- mice, but not Zp+/- mice, are easily distinguished by their truncated, curly whiskers and a subtle loss of pigmentation in the fur on their ventral side. Because hair abnormalities are associated with copper deficiency, we examined serum copper levels, as well as serum Cp protein expression and serum p-phenylenediamine oxidase activity at 8-10 weeks of age, but all parameters were normal. We also measured the levels of multiple metals in the liver, kidney and spleen by ICP-OES, but no changes were found. As Zp is one of the MCFs, and deletion of Hp and Cp results in severe anaemia, we investigated whether mice lacking Zp, Cp and Hp (Hp-/yCp-/-Zp-/-, TKO) had a more severe phenotype than those lacking both Hp and Cp (Hp-/yCp-/-, DKO). Other than abnormal whiskers and hair, TKO mice had a phenotype grossly similar (in terms of body weight and haematological parameters) to that of DKO mice. The current studies indicate that Zp is not essential in mice and we have found no evidence that it plays a systemically important role in the transport of iron or other metals in adults. However, Zp may play a localized role, as suggested by the abnormalities in whisker formation. Reference: 1. Chen H, et al. Identification of zyklopen, a new member of the vertebrate multicopper ferroxidase family, and characterization in rodents and human cells. J Nutr. 2010 Oct;140(10):1728-35.

Sixth Congress of the International BioIRon Society Page 251 Poster Abstracts IBIS

Poster #105 LACK OF MACROPHAGE FERROPORTIN AFFECTS WOUND HEALING Stefania Recalcati, MD, PhD, Elena Gammella, PhD, Paolo Buratti, BSc, Massimo Locati, MD, PhD and Gaetano Cairo, PhD University of Milan

OBJECTIVE Macrophages play an important role in the wound healing process, which consists of overlapping phases of inflammation, tissue formation and remodeling. In particular, alternatively activated (M2) macrophages, that are associated with inflammation resolution and tissue repair, are major players in wound healing. Since we showed that ferroportin (Fpn) expression is elevated in M2 macrophages, we investigated the role of macrophage iron in wound repair using a mouse model with macrophage-specific Fpn inactivation. MATERIALS AND METHODS Mice carrying a floxed Fpn allele (Fpnflox/flox, generously provided by Nancy Andrews) were bred to mice expressing Cre under the control of the macrophage-specific promoter LysM in the C57BL/6 background (FpnLys/Lys), and backcrossed for more than 10 generations. Fpn expression was evaluated by quantitative RT-PCR and immunoblotting. Full-thickness excisional skin wounds on the back of FpnLys/Lys and Fpnflox/flox mice were evaluated by macroscopical, histological and biochemical analyses at 2, 7, and 12 days after excision. Wound cellular profile was assessed by FACS analysis for different leukocytes populations and stromal cells. Cytokine production was evaluated by RT-PCR analysis of mRNAs in FACS-sorted macrophages, and at the protein level by ELISA in wound tissue. RESULTS Characterization of our FpnLys/Lys mice showed effective Fpn inactivation in macrophages with >90% reduction of Fpn mRNA levels in bone marrow derived macrophages. This result was confirmed by immunoblot and histochemical analyses of Fpn. Functional evidence for efficient deletion of Fpn was provided by the increased iron accumulation revealed by Perl's staining in spleen macrophages of FpnLys/Lys adult mice. Iron sequestration in reticuloendothelial cells results in early life anemia, that disappears after weaning. Macroscopical analysis showed that closure of full thickness excisional skin wounds was delayed in FpnLys/Lys mice, as compared to control Fpnflox/flox littermates, at all time points under observation. Histological analysis supported this data, as it showed that FpnLys/Lys mice have reduced re-epithelization, predominance of immature granulation tissue, less fibroblasts and higher inflammation characterized by granulocytes infiltrate. Moreover, in FpnLys/Lys mice, increased iron content was evident in the macrophages localized in healing wounds. CONCLUSION These results indicate that lack of Fpn, with consequent disruption of iron export from macrophages, affects wound healing, possibly by impairing the phenotypic switch from inflammatory macrophages to macrophages governing tissue repair

Sixth Congress of the International BioIRon Society Page 252 Poster Abstracts IBIS

Poster #106 THE EFFECT OF DIETARY HABITS ON IRON STATUS AMONG REPEATED BLOOD DONORS IN HONG KONG Tak Hei Stephanie Liang, MPH, BSc1, Cheuk Kwong Lee, MBBS, MRCP, FHKCP, FHKAM2 and Simin Liu, MD, MS, ScD, MPH1 1Brown University; 2Hong Kong Blood Transfusion Service

Background: Iron deficiency is a common nutritional disorder among regular blood donors. The high donor deferral rate due to iron deficiency has alarmed the Hong Kong Red Cross Blood Transfusion Service (HKBTS). As dietary iron plays an important role in preventing iron deficiency, understanding the dietary habits of blood donors may help us to identify potential interventions that can reduce risk of iron deficiency and improve donor safety. Objective: To examine whether there is an association between repeated blood donors’ dietary habits (including vegetarianism, mealtime stability, consumption of coffee or tea and consumption of iron-rich food) and their iron status. Method: In 2014, we administered a dietary questionnaire to 512 repeated whole blood donors, randomly selected from all repeated blood donors by the HKBTS. Responses were analyzed with chi-square test and multivariable logistic regression. Results: Iron deficiency was present in 25.4% of the participants, and 77.7% of them were female donors. No significant increased odds of iron deficiency were observed with vegetarian status or consumption of tea and coffee in the multivariable logistic regression. Participants with irregular mealtime were about 2 times more likely to be iron deficient. Donors who reported the lowest consumption of iron-rich food had 2.34 [95% CI 1.08, 5.08, p<0.05] higher odds in becoming iron deficient. Conclusion: The results indicate that female donors have a higher chance in becoming iron deficient. High mealtime stability and iron-rich food consumption were associated with lower risk of iron deficiency among repeated donors. These data indicate that regular blood donors should be encouraged to increase their consumption of iron-rich food and have regular meals.

Sixth Congress of the International BioIRon Society Page 253 Poster Abstracts IBIS

Poster #107 EFFECT OF PHLEBOTOMY ON LEARNING AND MEMORY FUNCTION IN CERULOPLASMIN MICE M.R. Liu, Master, X.F. Zhang, BS, P.P. Yu, BS, Z.H. Shi, PhD, Y.Z. Chang*, PhD and Peng Yu Key Laboratory of Animal Physiology, Biochemistry & Molecular Biology of Hebei Province, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Laboratory of Molecular Iron Metabolism, College of Life Science, Hebei Normal University

Introduction: Alzheimer's disease (AD) is one of the most common types of neurodegenerative diseases in the world, and it is characterized by the gradual reduction of memory function, senior cognitive function degradation. It is pathologically sho as the deposto o solle aylod eta , traelllar erorllary tales, phosphorylato o a prote and the loss of neurons. It is found that ro deposts the loato o plae patets, ad the ro a proote the progression of the AD, thus, it is predicted that reducing iron levels might be an effective prevention strategy. At present, the therapy of iron chelating agent was limited in its application in clinic due to its side effects, which implies that it is profound to find a simpler and less side effects method of decreasing the iron levels for the prevention and delay of AD. Phlebotomy has been reported as a treatment method for hereditary hemochromatosis to reduce iron contents. However, it is not yet known if phlebotomy can reduce brain iron levels and improve the cognitive function of the brain. Methods: Ceruloplasmin knockout (CP-/-) mice were treated with phlebotomy at a regular interval for two months. After that, the learning and memory function was detected by Morris water maze, the iron levels in the brain were detected by Atomic Absorption Spectrum (AAS), and iron levels in serum and other organs were detected by biochemical methods. Moreover, the side effects of phlebotomy were evaluated in mice. Results and discussions: Data showed that CP-/- mice treated with Phl (CP-/- Phl) need less time to search and reach the platform, and the tracks were simpler compared with that of CP-/- mice without phlebotomy (CP-/- Con). Data from AAS showed that the iron contents were decreased in CP-/- Phl mice. It is also found that phlebotomy decreased the iron contents in liver, but has no effects on spleen iron contents, serum iron contents, blood parameters and IL-6 expression in CP-/- mice. These suggest that phlebotomy can decrease brain iron contents, elevate the cognitive function and has low side effects in CP-/- mice. ConclusionsPhlebotomy might be a promising strategy to improve the function of learning and memory in CP-/- mice. Key words: ceruloplasmin, iron, learning, memory, Morris water maze

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Poster #108 DIVALENT METAL TRANSPORTER 1 IS INVOLVED IN THE REGULATION OF IRON METABOLISM BY GABA Peng Yu1, M. Zhang, Master2, H. Ding, Master, S.M. Wang, PhD, Y.Q. Liu, PhD, X.L. Duan*, B and Y.Z. Chang*, PhD 1Key Laboratory of Animal Physiology, Biochemistry & Molecular Biology of Hebei Province, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Laboratory of Molecular Iron Metabolism, College of Life Science, Hebei Normal University; 2Key Laboratory of Animal Physiology, Biochemistry & Molecular Biology of Hebei Province, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Laboratory of Molecular Iron Metabolism, College of Life Science, Hebei Normal University,

Introduction: Gamma-aminobutyric acid (GABA) can change the iron contents in rat brain, but the underlying molecular mechanism is not clear. Our previous data showed that divalent metal transporter 1 (DMT1) might be associated with the changes of brain iron metabolism induced by L-Dopa, but it is unknown if DMT1 is involved in the regulation of iron metabolism by GABA. Methods: To explore the effect of GABA on iron metabolism and its regulation mechanism, the cell viability, total iron contents, non-transferrin-bound iron (NTBI) uptake and the expression of iron metabolism related proteins were examined with MTT assay, inductively coupled plasma mass spectrometry (ICP-MS), radioactive 55Fe liquid scintillation counting analysis, immunofluorescence technique and western blot methods in PC12 cells. Results and discussions: Data showed that GABA induced the increase of total iron contents and NTBI uptake, and that the increased expression of DMT1 protein was correlated with the changes of iron metabolism in PC12 cells. Further investigations revealed that the translocation of NF- to les ht e oled the reased epression of DMT1 in PC12 cells treated with GABA. ConclusionsThese findings suggest that DMT1 may be the key molecule in the regulation of iron metabolism and that NF- as oled the relato o ells ater treatet Key words: DMT1, NF-, -aminobutyric acid, iron, PC12

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Poster #109 BONE IRON CONTENT IN POSTMENOPAUSAL WOMEN WITH HIP FRAGILITY FRACTURES: CORRESPONDENCE TO BONE STATUS AND IMPLICATIONS FOR POSTMENOPAUSAL OSTEOPOROSIS Li Guangfei1 and Xu Youjia, PhD2

1The Second Affiliated Hospital of Soochow University; 2The Second Affiliated Hospital of Soochow University Summary: Data gathered from 202 postmenopausal women with hip fragility fractures in a hospital demonstrate that the association between high bone iron content and low bone mineral density is more prominent than that between high serum ferritin and low bone mineral density.

Introduction: Despite extensive in vivo and clinical studies showing that higher serum ferritin is associated with acceleratedbone loss and low bone mineral density, the association between bone iron content and bone mineral density (BMD) among human beings has not been clarified. In the present study, we investigated and compared the association between femoral head bone iron content and bone mineral density, based on the data from 202 postmenopausal women with hip fragility fractures. Methods: This is a retrospective clinical study from the date of Second Affiliated Hospital of Soochow University, including 202 postmenopausal women with hip fragility fractures aged 56-93 years. BMD was measured using dual X-ray absorptiometry, and osteoporosis was diagnosed by the World Health Organization definition. The bone tissue iron content was measured by the inductively coupled plasma mass spectrometry (ICP-MS). The iron of bone tissue was stained by prussian blue. Results: Initially, we divided the subjects into eight age groups with the interval of 5 years old. During aging , BMD values at all measured sites decreased and serum ferritin concentrations increased meanwhile. Simultaneously, concurrent but inverse changes occurred between femoral head bone iron content and BMD values. Multiple regression analysis showed that serum ferritin was inversely associated with BMD values at only one site of five measured sites, while femoral head bone iron content was inversely associated with BMD values at four sites of five measured sites. Furthermore, when we divided these women into bone iron content quartiles, the odds for prevalent osteoporosis were 5.798–fold (95% CI=2.328-14.438) higher in subjects in the highest quartile compared with those in the lowest quartile. In addition, bone iron prussian blue staining confirmed the bone iron content in the corresponding quartile. Conclusions: These results provide the first clinical evidence that the bone iron content is associated with BMD, and the association maybe more prominent than that between serum ferritin and BMD.

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Poster #110 IRON OVERLOAD INCREASES OSTEOCLASTOGENESIS AND AGGRAVATES THE EFFECTS OF OVARIECTOMY ON BONE MASS Wang Xiao, PhD2 and Xu Youjia1 1The Second Affiliated Hospital of Soochow University; 2The Second Affiliated Hospital of Soochow University

Postmenopausal osteoporosis is a metabolic disease associated with estrogen deficiency. Numerous studies have shown the positive correlation between iron accumulation and postmenopausal osteoporotic status. Although previous studies have demonstrated that estrogen or iron alone has an effect on bone metabolism, their combined effects are not well defined. In an in vivo mouse model, we found that bone mass was minimally affected by an excess of iron in the presence of estrogen. Once the source of estrogen was removed (ovariectomy), iron accumulation significantly decreased bone mass. These effects were accompanied by fluctuations in the level of oxidative stress. To determine whether these effects were related to bone formation or bone resorption, primary osteoblasts, RAW264.7 cells, and BMMs were used for in vitro experiments. We found that iron accumulation did inhibit the activity of osteoblasts; however, estrogen had little effect on this inhibition. In contrast, iron promoted osteoclast differentiation through the production of ROS. Estrogen, a powerful reactive oxygen scavenger, suppressed this effect in osteoclasts. Our data provided a direct evidence that iron affected the bone mass only in the absence of estrogen. The inhibition of estrogen on iron-induced osteopenia was particularly relevant to bone resorption rather than boneformation.

Sixth Congress of the International BioIRon Society Page 257 Poster Abstracts IBIS

Poster #111 THE ETIOLOGY AND PATHOGENESIS OF THE DYSMETABOLIC IRON OVERLOAD SYNDROME Heyang Wang1, Chen Ling2, Lusha Wu, Xin Jiang, Fang He, Xuan Zhen, Yuxiao Tang and Min Li, MD1 1Second Military Medical University; 2University of Florida

Dysmetabolic iron overload syndrome (DIOS) is now frequently found in patients with chronic metabolic diseases such as nonalcoholic fatty liver disease and metabolic syndrome. DIOS has been confirmed to play an important role in the onset and development of chronic metabolic diseases by causing oxidative stress injury in the liver, increasing cellular lipid burden through enhancing liver cholesterol synthesis and participating in liver fibrosis. Iron reduction therapy, such as phlebotomy or an iron restricted diet, could markedly decrease metabolic alterations and liver enzymes. However, the etiology and pathogenesis of DIOS remains unclear. In the present studies, we observed that: 1. A mild iron overload in patients with prediabetes and T2D by using a magnetic resonance imaging (MRI) T2* gradient-recalled-echo technique; 2.The hyperinsulnemia could induce hepatic iron overload by up-regulating liver TFR1 via the PI3K/AKT/mTOR/IRP2 pathway, which may be one of the main reasons for the occurrence of DIOS; 3. The hepcidin can be directly regulated by insulin, and the suppressed liver hepcidin synthesis may be an important reason for the iron overload in DM2; 4. The hepatocyte nuclear factor-α -α has a sppresse eet o hepd epresso y atat the pathay, specifically via BMPR1A5.The IL-6-hepcidin axis is up-regulated by psychological stress in rats, resulting in hypoferremia and increase of hepatic iron storage; 6.The glucocorticoid receptor and STAT5 in stress-induced upregulation of IRP-1, which subsequently enhances transferrin receptor-1expression and down-regulates ferritin, causing iron accumulation in the liver. Our research also found that iron overload could cause liver tissue inflammation by HNF4a – mir-122 – NF- pathays

Sixth Congress of the International BioIRon Society Page 258 Poster Abstracts IBIS

Poster #112 MOLECULAR AND GENETIC MECHANISM OF HEPCIDIN IN REGULATION OF BONE METABOLISM IN ZEBRAFISH Jiang Yu1 and Xu Youjia, PhD2 1The Second Affiliated Hospital of Soochow University; 2The Second Affiliaed Hospital of Soochow University

Hepcidin is a important factor in transcriptionly up-regulated in response to iron overload which is a risk factor of osteoporosis. The phenomenon of bone microarchitecture defects has been described in hepcidin knockout mouse but molecular and genetic mechanisms leading to bone loss are lacking. In the present study, we show that in zebrafish knock-down of hepcidin leading to iron overload results in mineralization loss in early intramembranous bones while cartilage formation is mostly unaffected,but coinjection of hepcidin cRNA partially restored these defects. Quantitative real-time PCR analyses showed down-regulation of expression of osteoblast-specific genes (runx2a, runx2b, ALP, and sp7).In vivo analyses using whole mount in situ hybridization show that osteoblast genes (runx2a ,sp7)maturation and activity delayed was observed after knock-down of hepcidin. Furthermore, we analyzed expression of bone defects in transgenosis GFP of osteoblast genes (runx2a ,sp7). Furthermore, we provide the first functional evidence for a role of hepcidn in regulation of metabolism of osteoblast genes.Luciferase reporter assays demonstrated that bmp2a enhances runx2a expression.We find that iron overload represses its expression through bmp2a. Cell transfection and co-IP assays revealed that bmp2a enhances runx2a expression through binding to nuclear receptor hjv,but iron overload repress its process. High throughput transcriptome sequencing in the zebrafish of hepcidin knock-down have confirmed its pathways in osteoblast metabolism.Taken together, we show that iron overload decreased bone formation probably by pathway of bmp and hjv affecting runx2 in zebrefish.

Sixth Congress of the International BioIRon Society Page 259 Poster Abstracts IBIS

Poster #113 PROLACTIN SUPPRESSING DRUG BROMOCRIPTINE REGULATES HEPCIDIN THROUGH IRON HOMEOSTASIS IN MICE Shang-Yuan Liu, MD1, Jing Wang, PhD2, Peng Yu, PhD1, Yu-Mei Fan, PhD1, Guofen Gao, PhD1, Xiang-Lin Duan, Bsc1, Yaru Zhou, MD3, Shu-E Zhao, MD3, Guanjun Nie, PhD4 and Yan-Zhong Chang1 1Hebei Normal University; 2Chinese Academy of Sciences Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology of China; 3The Third Hospital of Hebei Medical University; 4National Center for Nanoscience and Technology of China

Hepcidin plays a central role in iron homeostasis regulation. However, the regulation of hepcidin production and the connection of hepcidin regulatory factors with iron homeostasis are still to be fully explored. Herein we report a study on the effect of bromocriptine, a drug commonly used against hyperprolactinemia, on hepcidin expression and iron homeostasis in mice. Results indicated that intragastical bromocriptine gavage significantly up-regulated hepcidin expression in mouse liver, and increased the hepatic and splenic iron storage, without inducing significant change in serum iron and transferrin saturation. Analysis of iron-related protein levels of bromocriptine-treated mice showed upregulation of transferrin in serum, as well as TfR2, Fpn1, DMT1+IRE and p-ERK in iron-storing organs. Moreover, bromocriptine-treated mice also showed increased DMT1+IRE and Fpn1 in duodenum, suggesting that bromocriptine may enhance hepcidin expression by increasing duodenal iron uptake and the transportation of iron via transferrin into tissues. These results may add to the current knowledge of hepcidin and iron homeostasis regulation.Hepcidin plays a central role in iron homeostasis regulation. However, the regulation of hepcidin production and the connection of hepcidin regulatory factors with iron homeostasis are still to be fully explored. Herein we report a study on the effect of bromocriptine, a drug commonly used against hyperprolactinemia, on hepcidin expression and iron homeostasis in mice. Results indicated that intragastical bromocriptine gavage significantly up-regulated hepcidin expression in mouse liver, and increased the hepatic and splenic iron storage, without inducing significant change in serum iron and transferrin saturation. Analysis of iron-related protein levels of bromocriptine-treated mice showed up-regulation of transferrin in serum, as well as TfR2, Fpn1, DMT1+IRE and p-ERK in iron-storing organs. Moreover, bromocriptine-treated mice also showed increased DMT1+IRE and Fpn1 in duodenum, suggesting that bromocriptine may enhance hepcidin expression by increasing duodenal iron uptake and the transportation of iron via transferrin into tissues. These results may add to the current knowledge of hepcidin and iron homeostasis regulation.

Sixth Congress of the International BioIRon Society Page 260 Poster Abstracts IBIS

Poster #114 DISCOVERY OF A SECONDARY BMP-SMAD-HEPCIDIN REGULATORY CIRCUIT IN HEPATIC SMAD7-DEFICIENT MICE Peng An, PhD1, Hao Wang, PhD1, Qian Wu, PhD1, Zhuzhen Zhang, PhD1, Zhidan Xia, PhD1, Xinhui Wang, PhD1, Guoli Li, PhD1, Yan Chen, PhD2, Junxia Min, MD, PhD1 and Fudi Wang, MD, PhD11 School of Medicine, Zhejiang University; 2Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

Iron homeostasis is tightly regulated by the liver peptide hepcidin, thus preventing pathological changes in iron levels. Dysregulated hepcidin expression causes several iron disorders in patients. Therefore, understanding how hepcidin is regulated in vivo has high clinical relevance. The Bmp-Smads are the major hepcidin regulatory signal pathways. However, the in vivo physiological role of inhibitory Smad7 in hepcidin regulation has not been fully investigated. We generated hepatocyte-specific Smad7-knockout mouse (Smad7Alb/Alb) . We observed an upregulation of hepcidin expression and increased phosphorylation of Smad1/5/8 in both isolated primary hepatocytes and liver tissues of Smad7Alb/Alb mice. Moreover, Smad7Alb/Alb mice developed mild symptoms of iron-deficient anemia. Surprisingly, when challenged with an iron-rich diet, hepcidin expression was unchanged. To identify other possible regulators of hepcidin expression in Smad7Alb/Alb mice during iron overload, we analyzed the liver transcriptome sequencing results of these mice. Interestingly, the expression of TGF- htors ld a, ollstat, ad ad ere reased y -fold in iron-challenged Smad7Alb/Alb mice compared with control-treated Smad7Alb/Alb mice. These results were confirmed by qRT- PCR and supported by additional functional characterization. Notably, overexpressing either Bambi or follistatin significantly reduced both hepcidin expression and Smad1/5/8 phosphorylation. Our data indicate that hepatic Smad7 functions as a master regulator of iron homeostasis by negatively controlling the expression of hepcidin. Under basal conditions, Smad6 is not sufficient to fully replace Smad7. While upon high iron challenging, we discovered Smad6, Bambi, and/or follistatin reconstitutes a compensatory circuit —at least partially—for the function of Smad7. This newly defined regulatory mechanism thereby coordinates hepcidin expression during iron overload in the absence of Smad7. Our findings suggest the Smad7 together with its compensatory Smad6, Bambi, and/or follistatin might be promising therapeutic targets/pathways for treating iron disorders caused by dysregulated hepcidin expression.

Sixth Congress of the International BioIRon Society Page 261 Poster Abstracts IBIS

Poster #115 BACTERIAL SEPSIS IS A MAJOR CAUSE OF MORTALITY WORLDWIDE. INDIVIDUAL GENE MUTATIONS CAN COMPROMISE THE IMMUNE SYSTEM, FURTHER INCREASING SUSCEPTIBILITY TO BACTERIAL INFECTION. WHETHER HEREDITARY HEMOCHROMATOSIS, A GENETIC DISEASE OF IRON OVERLOAD, AFFECTS THE HOST’S IMMUNE RESPONSE TO BACTERIAL INFECTIONS IS CURRENTLY UNKNOWN. THEREFORE, WE PERFORMED IN VIVO BACTERIAL SCREENING IN SEVERAL GENETIC MOUSE MODELS OF HEMOCHROMATOSIS, INCLUDING HFE (HFE-/-), HEMOJUVELIN (HJV-/-), AND FERROPORTIN-1 (FPN1LYSM/LYSM) KNOCKOUT MICE. WE FOUND THAT HJV-/- MICE, BUT NOT HFE-/- OR FPN1LYSM/LYSM MICE, ARE HIGHLY SUSCEPTIBLE TO BOTH GRAM-NEGATIVE AND GRAM-POSITIVE BACTERIAL INFECTIONS IN PERITONEAL CAVITY. INTERESTINGLY, WE FOUND THAT PHAGOCYTIC CELLS IN THE PERITONEUM OF HJV-/- MICE PRESENTED REDUCED BACTERIAL CLEARANCE, IFN- SECRETION AND NITRIC OIDE PRODUCTION; IN CONTRAST, BOTH MIGRATION AND PHAGOCYTOSIS ARE NORMAL. RECONSTITUTING HJV EXPRESSION INCREASED PSTAT1 LEVELS AND NITRIC OXIDE PRODUCTION IN RAW264.7 MACROPHAGES. NOTABLY, MICE THAT LACKED HJV EXPRESSION SELECTIVELY IN MACROPHAGES HAD INCREASED SENSITIVITY TO BACTERIAL INFECTION, SIMILAR TO THE PHENOTYPE OBSERVED IN HJV-/- MICE. MECHANISTICALLY, WE FOUND THAT HJV FACILITATES IFN- SECRETION IA THE IL-12/JAK2/STAT4 PATHWAY. THESE FINDINGS REVEAL A NOVEL PROTECTIVE ROLE FOR HJV IN THE EARLY STAGES OF ANTIMICROBIAL DEFENSE, WHICH CAN POTENTIALLY BE APPLICABLE TO HEMOCHROMATOSIS PATIENTS WHO ARE AT RISK FOR INFECTIOUS DISEASE. Qian Wu1, Yuanyuan Shen, PhD2, Yunlong Tao, PhD2 and Fudi Wang, PhD,MD31 School of Public Health Zhejiang Universty; 2Institutes of Nutritional Sciences, Chinese Academy of Sciences; 3School of Public Health, Zhejiang University

Bacterial sepsis is a major cause of mortality worldwide. Individual gene mutations can compromise the immune system, further increasing susceptibility to bacterial infection. Whether hereditary hemochromatosis, a genetic disease of iron overload, affects the host’s immune response to bacterial infections is currently unknown. Therefore, we performed in vivo bacterial screening in several genetic mouse models of hemochromatosis, including Hfe (Hfe-/-), hemojuvelin (Hjv-/-), and ferroportin-1 (Fpn1LysM/LysM) knockout mice. We found that Hjv-/- mice, but not Hfe-/- or Fpn1LysM/LysM mice, are highly susceptible to both Gram-negative and Gram-positive bacterial infections in peritoneal cavity. Interestingly, we found that phagocytic cells in the peritoneum of Hjv-/- mice presented reduced bacterial clearance, IFN- sereto, ad nitric oxide production; in contrast, both migration and phagocytosis are normal. Reconstituting Hjv expression increased pStat1 levels and nitric oxide production in RAW264.7 macrophages. Notably, mice that lacked Hjv expression selectively in macrophages had increased sensitivity to bacterial infection, similar to the phenotype observed in Hjv-/- mice. Mechanistically, we found that Hjv facilitates IFN- sereto a the -12/Jak2/Stat4 pathway. These findings reveal a novel protective role for Hjv in the early stages of antimicrobial defense, which can potentially be applicable to hemochromatosis patients who are at risk for infectious disease.

Sixth Congress of the International BioIRon Society Page 262 Poster Abstracts IBIS

Poster #116 MECHANISMS OF IRON MISREGULATION IN HYPERTENSION INDUCED BY EPINEPHRINE AND NORADRENALIN Heng-Rui Chang1, Yun-Zhe Ci2, Hai-Lin Song2, Wen-Yue Wu2, Yaru Li2, Yan-Zhong Chang2 and Shu-E Zhao1 1The Third Hospital of Hebei Medical University; 2Hebei Normal University

Hypertension is a very common disease especially in the elderly population. Hypertension can be divided into primary hypertension and secondary hypertension, of which more than 90% is primary hypertension. The pathogenesis of primary hypertension contains increasing sympathetic nerve activity, activating Renin-Angiotensin-Aldosterone System (RAAS), renal retention of excess sodium, insulin resistance and so on. The latest study found that increased level of oxidative stress due to iron deposition around blood vessels is an important cause of angiotensin II induced hypertension. Is the iron misregulation involved in hypertension induced by activating sympathetic nerve system? To explore the question is an important study. This research starts from the clinical investigation to explore the relevance of serum iron levels and the change of serum epinephrine and norepinephrine in patients with hypertension, and further detects the regulatory mechanisims of epinephrine and norepinephrine on perivascular iron deposition using animal models. 1. The study screened out 38 male patients with first identified as hypertension without any treatment from the health examination population, the 38 healthy male as control. Their serum were collected, measuring norepinephrine, epinephrine, hepcidin, serum ferritin, serum transferritin receptor, serum iron, serum transferrin saturation, total iron binding capacity and other indicators. The results of the study are as follows: (1) Compared with healthy control, serum epinephrine, norepinephrine, serum ferritin, serum transferritin receptor, serum transferritin saturation and total iron binding capacity have a significant differences in the patients, but serum iron and hepcidin is not. The results indicated that epinephrine and norepinephrine have a significant role on the body iron levels. (2) Whether patients with hypertension or healthy controls, there is a strong correlation between epinephrine, norepinephrine and serum ferritin, serum transferritin receptor. This result indicated that epinephrine and norepinephrine may be significantly regulating the body's iron homeostasis. (3) The study also found that the weight，heart rate, serum total protein content of patients with hypertension was significantly higher, verifying that the weight and metabolic disorders were closely associated with the incidence of hypertension. 2. Based on the findings of clinical studies, we studied the effects of adrenaline and noradrenaline on body iron metabolism and iron levels around the ascending aorta. The molecular mechanisms causing the iron change by intravenously injecting epinephrine and norepinephrine 7 days and 14 days in male mice were also explored. The study found that: (1) injection of epinephrine and norepinephrine for 14 days significantly increased serum iron levels，decreased serum total iron binding capacity in mice. This result verified the results of clinical studies. (2) after injecting epinephrine and norepinephrine for 14 days, the L-ferritin and total iron levels of ascending aorta was significantly increased. (3) the study further revealed the molecular mechanisms of increased iron levels caused by adrenaline and norepinephrine in the ascending aorta which the expression of iron uptaking protein (TfR, DMT1 + IRE) increased to increase iron uptake and the expression of proteins FPN1 decreased to reduced iron release. This study based on finding the correlation between epinephrine, norepinephrine and body iron metabolism by clinical observations, and further revealed the molecular mechanisms of adrenaline and noradrenaline regulating the ascending aorta iron metabolism by increasing TfR, DMT1+IRE and decreasing FPN1 expression. Our findings not only deepen the understanding of hypertension pathogenesis, but will also provide new ideas for the prevention and treatment of hypertension.

Sixth Congress of the International BioIRon Society Page 263 Poster Abstracts IBIS

Poster #117 HEPCIDIN INVOLVED IN THE REGULATION OF IRON HOMEOSTASIS DURING PREGNANCY AND LACTATION IN RATS Guofen Gao, Shang-Yuan Liu, Hui-Jie Wang, Tian-Wei Zhang, Sai-Shou Zhang, Xiang-Lin Duan, Shu-E Zhao and Yan- Zhong Chang Hebei Normal University

Maternal iron homeostasis is especially important in pregnancy and lactation. However, the molecular iron mechanism and its regulation are least well understood in how iron uptake to fit the maternal requirement. We conducted this study by using female Sprague Dawley (SD) rats at different stages of gestation and lactation. The levels of non-heme iron, transferrin receptors 1 and 2 (TfR1 and TfR2), phosphorylated ERK protein (P-ErK), L-ferritin, divalentmetal transporter 1 (DMT1), ferroportin 1 (FPN1) and ceruloplasmin (Cp) in liver and spleen, hepatic hepcidin and erythropoietin receptor (EPOR) mRNA levels and FPN1 in small intestine, were examined. The results showed that the iron level increased slightly at the 9th day of pregnancy and declined significantly at the late stages of pregnancy and lactation. This iron level decline was regulated by the dramatically decreased hepcidin level, whose regulation was associated with the increased EPO level, and involved the TfR2 signaling as well. We also found that Cp may participate in the iron release in liver and spleen during pregnancy and lactation, and hepatic DMT1(+IRE) played a role in iron uptake during lactation through the classic pathway, Tf-TfR. This is the first time that the alterations of key molecules in iron metabolism during pregnancy and lactation were comprehensive studied, and the functional role of hepcidin and its possible regulatory pathways were also investigated. These results are fundamental to the understanding of iron homeostasis during pregnancy and lactation as inadequate iron intake is a highly prevalent problem in the pregnant women.

Sixth Congress of the International BioIRon Society Page 264 Poster Abstracts IBIS

Poster #118 THE EFFECT AND MECHANISM OF DEFEROXAMINE ON IRON ACCUMULATION-INDUCED BONE LOSS IN OVARIECTOMIZED C57BL/6J MICE Wang Sheng1 and Xu Youjia, PhD21 The Second Affiliated Hospital of Soochow University; 2The Second Affiliated Hospital of Soochow University

Iron has recently been recognized as an independent risk factor for postmenopausal osteoporosis, but the mechanisms leading to Iron-related bone loss are not fully determined. Reportedly, female menstrual discharge 36 mg iron a year, several years after postmenopausal can form the accumulation of iron. But the mice don’t have the phenomenon of menstruation, after ovariectomy the level of iron in the body is not to rise. So this research adopts the ovariectomized mice model of ferric ammonium citrate (FAC) intervention to simulate postmenopausal osteoporosis patients with iron accumulation. We evaluated the effect of iron in ovariectomized C57BL/6J mice and further investigated the potential benefits and the mechanism of deferoxamine (DFO), a powerful iron chelator, in this model. The model of iron accumulation described in this study demonstrated an apparent loss of bone mass, accompanied by the results of the Micro-CT of the femurs. Most importantly, we revealed that DFO not only was capable of reducing the iron accumulation of the serum and bone, but also attenuating the loss of bone mass. We also found that the DFO treatment significantly increased the OPG mRNA levels, and suppressed the RANKL mRNA levels, and the RANKL/OPG mRNA ratio. Therefore, we conclude DFO decreases bone mass loss induced by iron accumulation, possibly through the regulation of OPG/RANKL/ RANK pathway.

Sixth Congress of the International BioIRon Society Page 265 Poster Abstracts IBIS

Poster #119 FXN III IS A NOVEL FUNCTIONAL FRATAXIN ISOFORM Shuangying Hao Nanjing University

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by reduced expression levels of the frataxin gene (FXN) due to expansion of triplet nucleotide GAA repeats in the first intron of FXN. FXN is a mitochondrial protein which plays an important role in the regulation of intracellular iron trafficking, biogenesis of iron- sulfur cluster and heme, and removal of reactive oxygen species. We found a novel FXN isoform (FXN III) in our previous work, which is specifically expressed in heart tissue without mitochondrial targeting sequence. To investigate the functional role of FXN III, we transfected mammalian cells with the recombinant plasmid pcDNA3.1(-)-FXN III-myc and found that FXN III was localized more in nucleus than in cytosol. Overexpression of FXN III in mouse neuroblastoma cell line (N2a) remarkably increased the activity of aconitase and mitochondrial respiratory chain complex II and level of ATP content. To further assess whether the isoform may replace the canonical one, we knocked down the endogenous transcript with the specific siRNA and overexpressed FXN III. Strikingly, the activities of Fe-S containing enzyme (aconitase) and respiratory chain complex I/II and level of ATP content were all rescued by overexpression of FXN III. If gene therapy is considered for treatment of FRDA, FXN III could be a better candidate for this isoform may increase the activities of Fe-S containing enzymes more efficient than the canonical isoform. Our results indicated a fully functional isoform of FXN in vitro. The in vivo function needs to be determined and the mechanism of how a non-mitochondria targeting FXN isoform rescued FXN deficiency-induced mitochondrial dysfunction is under investigation. Keyword: Friedreich ataxia, frataxin, FXN isoform III, Fe–S cluster

Sixth Congress of the International BioIRon Society Page 266 Poster Abstracts IBIS

Poster #120 DIFFERENTIATED FUNCTIONS OF IRP1 AND IRP2 Huihui Li Nanjing University

Cellular iron homeostasis is mainly controlled by the iron-regulatory protein/iron-responsive element machinery in mammalian cells. Iron regulatory proteins (IRP1 and IRP2) posttranscriptionally control the expression of transcripts that contain iron-responsive element sequences. In consistence with others, we found that IRP1 or IRP2 deficiency reduced the iron uptake of mouse embryo fibroblasts. Moreover, IRP1 or IRP2 deficiency reduced the protein levels of FXN and ISCU, both of which are essential proteins for the biogenesis of iron-sulfur cluster. To determine the effect of IRP1 or IRP2 on iron-sulfur cluster biogenesis, we measured the activities of some iron-sulfur cluster-containing enzymes. Unexpectedly, the activities of mitochondrial aconitase and cytosolic xanthine oxidase were not diminished in IRP1-/- or IRP2-/- cells. However, complex I, II, and III activities of mitochondrial respiratory chain were markedly decreased in IRP2- /- cells. The activities of complex I and II were also decreased other than that of complex III in IRP1-/- cells. After normalization to protein levels of the subunits of complexes, IRP2-/- cells exhibited the significantly reduced activity of complex III. Furthermore, we found that IRP1-/- and IRP2-/- cells were sensitive to iron and H2O2 treatment compared to wild type. In particular, IRP1-/- cells were more sensitive to iron and H2O2 treatment than IRP2-/- cells. These results suggest that IRP1 is more important for defense against oxidative stress and IRP2 for mitochondrial respiration of electron transfer chain, which might give an explanation for neurodegenerative symptoms of IRP2-/- mice and for polycythemia and pulmonary hypertension symptoms of IRP1-/- mice. Key words: Iron regulatory proteins (IRP1 and IRP2), iron homeostasis, oxidative stress, mitochondrial respiration

Sixth Congress of the International BioIRon Society Page 267 Poster Abstracts IBIS

Poster #121 IMPROVED IRON ABSORPTION BY CONSUMPTION OF PROBIOTICS Michael Hoppe2, Elisabeth Gramatkovski2, Gunilla Önning, Malin Björklund, Niclas Larsson and Lena Hulthén1 1Institute of Medicine, University of Gothenburg; 2Inst of Medicine

Sixth Congress of the International BioIRon Society Page 268 Poster Abstracts IBIS

Poster #122 REPROGRAMMING OF IRON METABOLISM IN CANCER Junxia Min, MD, PhD Institute of Translational Medicine/Zhejiang University

Iron hemostasis is essential for maintenance of human health. Results from epidemiological studies implied iron imbalance plays roles in many cancer types. To date, the causal relationship has not been firmly established. To explore the role of iron metabolic reprogramming in cancer, we investigated potential roles of iron overload and iron deficiency in cancer initiation and progression. Furthermore, we carried out additional experiments to study the reprogrammed iron metabolic pathways in cancer. Our preliminary findings indicates that both iron overload and iron deficiency could lead to tumor cell proliferation. During iron metabolic reprogramming process in cancer, we found many pathways of iron storage, transport, and regulations are all disturbed. In addition, we found reprogrammed iron metabolic pathways are highly interconnected with those cancer-type dependent oncogenic signaling pathways. Together, our findings suggest that reprogramming of iron metabolism is a critical aspect of tumor cell survival. Further studies are warranted to assesse whether targeting iron reprogrammed oncogenic pathways might open a new avenue for cancer prevention and treatment.

Sixth Congress of the International BioIRon Society Page 269 Poster Abstracts IBIS

Poster #123 IPSC-DERIVED HUMAN NEURONS AS DISEASE MODELS TO STUDY NEURODEGENERATION WITH BRAIN IRON ACCUMULATION DISORDERS Sonia Levi, PhD1, Daniel Orellana, PhD2, Paolo Santambrogio, PhD2, Alicia Rubio-Garrido, PhD2, Vania Broccoli, PhD3, Paola Venco, PhD4 and Valeria Tiranti, PhD41Vita-Salute San Raffaele University; 2San Raffaele Scientific Institute; 3 CNR-Institute of Neuroscience; 4IRCCS-C. Besta.

Neurodegeneration with Brain Iron Accumulation (NBIA) is a group of rare genetic disorders characterized by radiological evidence of focal accumulation of iron in the brain. The main symptoms are associated with problems in movement, spasticity and cognitive impairment. Despite that the causative genes encode proteins not only involved in iron metabolism but also in other functions, such as fatty acids metabolism, mitochondria functioning, lysosomal and autophagosome activity, they share the alteration of brain iron metabolism and the lack of knowledge of the pathogenetic mechanisms controlling this phenotype. With the aim to clarify the molecular alteration leading to iron dysregulation, we have employed iPS cell technology for generating the in vitro disease neuronal models. We succeeded to obtain iPS clones from patients affected by alteration in PANK2, PLA2G6, C19orf12, COASY, WDR45. Here we described the results obtained on Pank-2 associated neurodegeneration (PKAN), caused by the deficiency of Pank-2, the mitochondrial enzyme involved in CoA synthesis. PKAN and matched healthy donors fibroblasts were reprogrammed by using the Sendai reprogramming vectors. The iPS clones were differentiated to generate neurons and characterized for mitochondrial functionality, oxidative status and iron metabolism. The PKAN iPSC-derived neurons display a reduced antioxidant defense (about 15% lower content of reduced glutathione), increased level of ROS development (about 20% higher) and abnormal electrophysiological properties respect to the control. Furthermore, PKAN iPSC-derived neurons present electron dense aggregates inside the mitochondria of a still unknown nature. In conclusion, we succeed in obtaining human neuronal models to study NBIA disorders and the preliminary results on PKAN derived neurons suggested altered oxidative status, abnormal mitochondrial functionality and morphology. The financial support of Telethon (Grant n°: GGP11088) and AISNAF is gratefully acknowledged.

Sixth Congress of the International BioIRon Society Page 270 Poster Abstracts IBIS

Poster #124 SERUM HEPCIDIN AND PROHEPCIDIN IN VEGAN POPULATION Kamila Balusikova, Jan Gojda, Jana Patockova, Jan Kovar and Michal Andel 3rd Faculty of Medicine, Charles University in Prague

People consuming food without any limitation have higher body iron stores when compared with people not consuming animal food (vegans). Since body iron stores positively correlates with the intake of especially red meat together with the fact that higher iron stores positively correlate with insulin resistance it can be assumed that the lower iron uptake in vegans is one of the factors increasing insulin sensitivity. A key hormone involved in iron homeostasis in the organism is hepcidin that functions as a negative regulator of iron absorption by enterocytes and is responsible for release of iron from the monocyte-macrophage system circulation. However, to this time, the hepcidin levels in a population of vegans were not studied. Our study included 31 vegans and 15 corresponding controls. Exclusion criteria were age under 18 years and the presence of any chronic disease with an impact on the intermediary metabolism. Each subject underwent a collection of peripheral blood under basal conditions. Following parameters of iron metabolism and glucose homeostasis were determined in serum: hepcidin, prohepcidin, ferritin, iron binding capacity, transferrin, serum iron level, blood glucose, insulin and C-peptide. A significant difference was found between the groups in the levels of hepcidin (vegans 6.43 ng.ml-1 vs. control 9.20 ng.ml-1, p = 0.016), prohepcidin (vegans 252.85 ng.ml-1 vs. control 456.47 ng.ml-1, p = 0.005) and glucose (4.80 mM vegans 1 vs. control 5.47 mM-1, p = 0.004). Other iron metabolism and glucose homeostasis parameters differences were off significance (ferritin, p = 0.12; iron binding capacity, p = 0.10; transferrin, p = 0.09; insulin, p = 0.07; C- peptide, p = 0.15). Multiple correlations found significant or borderline significant following parameters: hepcidin and prohepcidin (R 0.27, p = 0.07), prohepcidin and insulin (R -0.31, p <0, 05), hepcidin and ferritin (R 0.28, p = 0.068), hepcidin and iron binding capacity (R -0.26, p = 0.079), prohepcidin and transferrin (R -0.26, p = 0.08). These results showed that vegans compared with the control group present lower serum hepcidin and prohepcidin. This corresponds to an effort of the organism to increase the intake of iron through enterocytes, but also increased release of iron into circulation at the level of individual cells. These facts, together with the known negative association between vegan diet and insulin resistance may indicate that lower dietary intake of iron assists to the reduction of insulin resistance development. This study was funded within the scientific framework of research programs of Charles University in Prague, PRVOUK- P31 and UNCE 204015, and was supported by a Grant of the Ministry of Health of the Czech Republic, number NT/14416.

Sixth Congress of the International BioIRon Society Page 271 Poster Abstracts IBIS

Poster #125 MICE ARE POOR HEME ABSORBERS AND DO NOT REQUIRE INTESTINAL Hmox1 FOR DIETARY HEME IRON ASSIMILATION Carine Fillebeen, PhD, Konstantinos Gkouvatsos, MD, PhD, Gabriela Fragoso, Annie Calvé, Daniel Garcia-Santos, PhD, Marzell Buffler, Christiane Becker, PhD, Klaus Schümann, MD, Prem Ponka, MD, Manuela Santos, PhD and Kostas Pantopoulos Lady Davis Institute for Medical Research and McGill University

We generated Hmox1Vil-Cre mice with intestinal-specific ablation of heme oxygenase 1 (HO-1), the major heme catabolic enzyme, to dissect its role in heme iron assimilation. A high-hemin diet triggered partial induction of intestinal ferritin in Hmox1Vil-Cre mice, indicating compensatory heme catabolism by heme oxygenase 2 (HO-2). Intestinal Flvcr1a was also induced but hepatic HO-1 and hemopexin remained unaffected, excluding substantial export of intact heme to the bloodstream. 59Fe-hemoglobin absorption was miniscule and did not differ significantly between Hmox1Vil-Cre and control fl/fl Hmox1 mice. Contrary to dietary FeSO4, hemin only partially corrected iron deficiency anemia in wild type mice due to limited transport across the gut epithelium. We conclude that intestinal heme catabolism is an obligatory step for heme iron assimilation even in the absence of HO-1. Housekeeping HO-2 suffices for basal heme iron acquisition, while inducible HO-1 may be required for processing of higher heme loads in carnivores. Murine enterocytes cannot efficiently internalize heme from the intestinal lumen; thus, heme is a poor nutritional iron source for mice.

Sixth Congress of the International BioIRon Society Page 272 Poster Abstracts IBIS

Poster #126 Hfe AND Hjv EXHIBIT OVERLAPPING FUNCTIONS FOR IRON SIGNALING TO HEPCIDIN: GENETIC EVIDENCE FROM SINGLE AND DOUBLE KNOCKOUT MICE Patricia Kent, MSc, Nicole Wilkinson, PhD, Marco Constante, PhD, Carine Fillebeen, PhD, Konstantinos Gkouvatsos, MD, PhD, John Wagner, MSc, Marzell Buffler, Christiane Becker, PhD, Klaus Schümann, MD, Manuela Santos, PD and Kostas Pantopoulos Lady Davis Institute for Medical Research and McGill University

Functional inactivation of HFE or hemojuvelin (HJV) is causatively linked to adult or juvenile hereditary hemochromatosis, respectively. Systemic iron overload results from inadequate expression of hepcidin, the iron regulatory hormone. While HJV regulates hepcidin by amplifying bone morphogenetic protein (BMP) signaling, the role of HFE in the hepcidin pathway remains incompletely understood. We investigated the pathophysiological implications of combined Hfe and Hjv ablation in mice. Isogenic Hfe-/- and Hjv-/- mice were crossed to generate double Hfe-/-Hjv-/- progeny. Wild type control and mutant mice of all genotypes were analyzed for serum, hepatic and splenic iron content, expression of iron metabolism proteins, and expression of hepcidin and Smad signaling in the liver, in response to a normal or an iron- enriched diet. As expected, Hfe-/- and Hjv-/- mice developed relatively mild or severe iron overload, respectively, which corresponded to the degree of hepcidin inhibition. The double Hfe-/-Hjv-/- mice exhibited an indistinguishable phenotype to single Hjv-/- counterparts with regard to suppression of hepcidin, serum and hepatic iron overload, splenic iron deficiency, tissue iron metabolism, and Smad signaling, under both dietary regimens. We conclude that the hemochromatotic phenotype caused by disruption of Hjv is not further aggravated by combined Hfe/Hjv deficiency. Our results provide genetic evidence that Hfe and Hjv operate in the same pathway for the regulation of hepcidin expression and iron metabolism.

Sixth Congress of the International BioIRon Society Page 273 Poster Abstracts IBIS

Poster #127 A HIGH FAT DIET MODULATES IRON METABOLISM BUT DOES NOT PROMOTE LIVER FIBROSIS IN HEMOCHROMATOTIC Hjv-/- MICE Ranjit Padda, MSc, Konstantinos Gkouvatsos, MD, PhD, Maria Guido, MD, Jeannie Mui, Hojatollah Vali, PhD and Kostas Pantopoulos Lady Davis Institute for Medical Research and McGill University

Hemojuvelin (Hjv) is a membrane protein that controls body iron metabolism by enhancing signalling to hepcidin. Hjv mutations cause juvenile hemochromatosis, a disease of systemic iron overload. Excessive iron accumulation in the liver progressively leads to inflammation and disease, such as fibrosis, cirrhosis or hepatocellular cancer. Fatty liver (steatosis) may also progress to inflammation (steatohepatitis) and liver disease, and iron is considered as pathogenic cofactor. The aim of this study was to investigate the pathological implications of parenchymal iron overload due to Hjv ablation in the fatty liver. Wild-type (wt) and Hjv-/- mice on C57BL/6 background were fed a standard chow, a high fat diet (HFD) or a HFD supplemented with 2% carbonyl iron (HFD+Fe) for 12 weeks. The animals were analyzed for iron and lipid metabolism. As expected, all Hjv-/- mice manifested higher serum and hepatic iron, and diminished hepcidin levels compared to wt controls. The HFD reduced iron indices and promoted liver steatosis in both wt and Hjv-/- mice. Notably, steatosis was attenuated in Hjv-/- mice on the HFD+Fe regimen. Hjv-/- animals gained less body weight and exhibited reduced serum glucose and cholesterol levels. Histological and ultrastructural analysis revealed absence of iron-induced laato or ler ross, despte early ss o ler ry epresso o α sooth sle at e olde that parenchymal hepatic iron overload does not suffice to trigger progression of liver steatosis to steatohepatitis or fibrosis in C57BL/6 mice.

Sixth Congress of the International BioIRon Society Page 274 Poster Abstracts IBIS

Poster #128 SERUM FERRITIN AND FIBROBLAST GROWTH FACTOR 23 ARE ASSOCIATED WITH LIVER FIBROSIS DIAGNOSED BY TRANSIENT ELASTOGRAPHY IN HIV/HCV CO-INFECTED PATIENTS Giada Sebastiani, MD2, John Wagner, MSc, Kathleen Rollet, Marina Klein, MD and Kostas Pantopoulos1 1Lady Davis Institute for Medical Research and McGill University; 2McGill University Health Center

Background: Liver fibrosis is the most important factor affecting prognosis and management of patients with chronic liver diseases. HIV/HCV co-infection is characterized by a particularly fast progression of liver fibrosis towards cirrhosis and its end-stage liver complications. As such, identification of biomarkers associated with fibrosis progression is of paramount importance. Aims: We studied correlation of iron and inflammatory serum biomarkers with liver stiffness measured by transient elastography, a validated non-invasive tool to diagnose liver fibrosis. We selected these biomarkers because the iron and inflammatory status are known to affect liver disease progression. Methods: This pilot study included consecutive HIV/HCV co-infected patients who underwent transient elastography examination. All patients were on antiretroviral therapy and had HIV viral load <50 cp/mL. Following serum parameters were measured within a period of 6 months: hepcidin, iron, ferritin, transferrin saturation, fibroblast growth factor 23 (FGF23), interleukin 6 (IL- ad tor eross ator α α arse orrelato ad ltarate lear regression analysis were used to explore association with liver stiffness. Results: 45 patients (mean age 47 years, 78% male) were included. Median liver stiffness measurement was 7 kPa (interquartile range 5.2-14). 64% of the patients were HCV treatment naïve. Liver stiffness was significantly correlated with ferritin (r=0.51, p=0.001), transferrin saturation (r=0.36, p=0.003), FGF23 (r=0.36, p=0.02) and IL-6 (r=0.42, p=0.007). The results of multivariate analysis and relative regression coefficients are depicted in the Table. After adjustment, ferritin and FGF23 were independently associated with liver fibrosis. Conclusions: In HIV/HCV co-infected patients, serum ferritin and FGF23 are positive independent predictors of liver fibrosis. These data are consistent with recent findings identifying serum ferritin as independent predictor of advanced fibrosis and mortality in chronic liver disease patients of other etiologies. They also suggest that serum FGF23 has a potential as biomarker for liver fibrosis in HIV/HCV co-infected patients. Larger scale studies are required to validate the clinical significance of our data.

Sixth Congress of the International BioIRon Society Page 275 Poster Abstracts IBIS

Poster #129 NEUROPROTECTIVE EFFECTS OF GINKGETIN AGAINST NEURO-INJURY IN PARKINSON’S DISEASE MODEL INDUCED BY MPTP VIA CHELATING IRON Y.Q. Wang, BS, M.Y. Wang, BS, X.R. Fu, BS, Peng Yu, G.F. Gao, PhD, Y.M. Fan, PhD, X.L. Duan, BS, B.L. Zhao, PhD, Y.Z. Chang*, PhD and Zhenhua Shi*, PhD Key Laboratory of Animal Physiology, Biochemistry & Molecular Biology of Hebei Province, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Laboratory of Molecular Iron Metabolism, College of Life Science, Hebei Normal University

Abstract Disruption of neuronal iron homeostasis and oxidative stress are closely related to the pathogenesis of Parkinson's disease (PD). Ginkgetin, a natural biflavonoid isolated from leaves of Ginkgo biloba L, has many known effects, including anti-inflammatory, anti-influenza virus and anti-fungal activities, but its underlying mechanism of the neuroprotective effects in PD remains unclear. The present study utilized PD models induced by 1-methyl-4-phenylpyridinium (MPP+) and 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to explore the neuroprotective ability of ginkgetin in vivo and in vitro. Our results showed that ginkgetin could provide significant protection from MPP+-induced cell damage in vitro by decreasing the levels of intracellular reactive oxygen species and maintaining mitochondrial membrane potential. Meanwhile, ginkgetin dramatically inhibited cell apoptosis induced by MPP+ through the caspase-3 and Bcl2/Bax pathway. Moreover, ginkgetin significantly improved sensorimotor coordination in a mouse PD model induced by MPTP by dramatically inhibiting the decrease of tyrosine hydroxylase expression in the substantia nigra and superoxide dismutase activity in the striatum. Interestingly, ginkgetin could strongly chelate ferrous ion and thereby inhibit the increase of the intracellular labile iron pool through downregulating L-ferritin and upregulating transferrin receptor 1. These results indicate that the neuroprotective mechanism of ginkgetin against neurological injury induced by MPTP occurs via regulating iron homeostasis. Therefore, ginkgetin may provide neuroprotective therapy for PD and iron metabolism disorder related diseases. Keywords: Ginkgetin, oxidative stress, chelation, Parkinson’s disease, iron homeostasis.

Sixth Congress of the International BioIRon Society Page 276 Poster Abstracts IBIS

Poster #130 THE INFLUENCE OF INTERMITTENT HYPOBARIC HYPOXIA ON THE BRAIN IRON METABOLISM IN ADULT SPRAGUE DAWLEY RATS Qiong Wu1, Yaru Li2 and Yan-Zhong Chang2 1Hebei Normal Unversity; 2Hebei Normal University

Objective: Iron is an essential element in all living organisms and is required as a cofactor for oxygen-binding proteins. Iron metabolism, oxygen homeostasis and erythropoiesis are consequently strongly interconnected. In mammalian cells, exposure to a low-oxygen environment triggers a hypoxic response pathway centered on the regulated expression of the hypoxia-inducible transcription factor (HIF). Hypoxia has been shown to increase the expression of a variety of proteins involved in iron homeostasis. However, little is known about brain iron metabolism after intermittent hypobaric hypoxia (IHH) treatment. In this study, adult Sprague dawley (SD) rats were treated with IHH for 28 days, 8h per day and then we detected iron homeostasis in different brain areas of SD rats. Results: The protein level of hippocampus transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1) with IRE, DMT1 (-IRE), ferritin-H, iron regulatory protein (IRP) 2 and ceruloplasmin (CP) is elevated significantly while ferritin-L decreased. We have also found the down regulation of IRP1. We observe the same results in the cerebral cortex in the brain. Conclusion: We first discover that IHH has an influence on the brain iron homeostasis and the decreased ferritin-L corresponds to the down regulation of IRP1 indicating hypoxia can affect the expression of ferritin-L through IRE/IRP system. Although there is a marked increase in TfR1 expression that would lead to the raised level of LIP in cells. It can finally result in the higher ROS which can damage the cells. The concerned mechanisms involved in it remain to be deliberated.

Sixth Congress of the International BioIRon Society Page 277 Poster Abstracts IBIS

Poster #131 MITOCHONDRIAL FERRITIN DELETION AGGRAATE A INDUCED DAMAGE IN THE MICE BRAIN Pei-Na Wang, Qiong Wu and Yan-Zhong Chang Hebei Normal University

Objective: Mitochondrial ferritin (FtMt) is a novel iron homeostasis related protein located in the mitochondria. Over expression of FtMt participates in the regulation of iron distribution between cytosol and mitochondria. Data have shown that FtMt plays an important function in preventing neuronal damage in Parkinsonism. As a common neurodegenerative disorder, Alzheimer’s disease is closely related to the disruption of iron metabolism in brain. To study the role of FtMt in the orato o , e proded e odel throh et -35 into the lateral cerebral ventricle of FtMt-/- mice and FtMt+/+mice and then explored the levels of proteins involved in iron metabolism and oxidative stress. Results: orrs ater ae tests ad proe tests shoed that o ot o tt satly ehaed -35 induced neurotoxicity and led to memory failure. The levels of oxidative stress and cell apoptosis are enriched in FtMt-/- mice. The Ferritin-L protein are increased both in cerebral cortex and hippocampus; the expression level of FPN1 increased and TfR1 levels decreased in cerebral cortex but DMT1(-IRE)levels increased in FtMt-/- e hh eted -35. Conclusion: Our results show that knock out of FtMt can affect iron metabolism of mice and these influences are different deret ra reo tt plays a portat protete role preet eroal daae ded y -35.

Sixth Congress of the International BioIRon Society Page 278 Poster Abstracts IBIS

Poster #132 MITOCHONDRIAL FERRITIN ATTENUATES THE ROTENONE-INDUCED TOXICITY IN DROSOPHILA MELANOGASTER Yu-Jing Gou, Guofen Gao, Peng Yu, Zhen-Hua Shi and Yan-Zhong Chang Hebei Normal University

Neuronal iron homeostasis disruption and oxidative stress are closely related to the pathogenesis of Parkinson’s disease (PD). Mitochondrial ferritin (FtMt)，which is located in mitochondria， can store iron to reduce the content of reactive oxygen species and protect mitochondria from oxidative stress damage. In this study, we investigated the potential protective effects of mitochondrial ferritin against the toxicity induced by rotenone in Drosophila melanogaster(D. melanogaster).Newly born mitochondrial ferritin over expressed(Fer3HCH OE) drosophila adults, and wild-type drosophila adults were concomitantly exposed to rotenone (500 mM) and normal culture medium during the 0-30 days after eclosion. Fer3HCH OE adults had a better performance in the negative climbing capability， and a lower incidence of mortality when compared to wild-type flies，as well as a higher level of cephalic tyrosine hydroxylase( TH). It is suggested that FtMt may offset the detrimental effects of rotenone. So we can infer that the regulation of FtMt expression in neuronal system may provide a new therapeutic strategy to PD.

Sixth Congress of the International BioIRon Society Page 279 Poster Abstracts IBIS

Poster #133 THE EFFECTS OF SEX ON BRAIN IRON STATUS IN RATS Qian Hao, Qiong Wu and Yan-Zhong Chang Hebei Normal University

Iron plays essential roles in the human body. Studies have shown that iron is distributed differently in male and female Rats in liver, spleen, bone marrow, kidney, heart. However, the effects of sex on iron distribution in central nervous system are not well established. In this study, female and male Sprague Dawley rats were used at 4 months of age. The ferritin light chain (FTL), transferrin receptor1 (TfR1), ferroportin 1 (FPN1), divalent metal transporter 1 (DMT1) in the cortex, hippocampus, striatum, cerebellum, and olfactory bulb was determined by Western blot analysis. The results showed that the levels of FTL protein in the cortex, hippocampus, striatum, cerebellum, and olfactory bulb were higher in female rats than in male rats, but the levels of TfR1 protein were lower in female rats than in male rats. These data suggest that sex have effects on brain iron status. iron is distributed differently in central nervous system in male and female rats. However, the precise mechanisms need further study.

Sixth Congress of the International BioIRon Society Page 280 Poster Abstracts IBIS

Poster #134 IRON DEFICIENCY IN OVERWEIGHT AND OBESE POPULATION Lu Zhao, Xiangyi Zhang, Ye Shen, Xuexian Fang, Youfa Wang and Fudi Wang Zhejiang University

Our previous work has concluded a positive association between iron status/intake with Alzheimer’s disease, cardiovascular diseases and metabolic syndrome through meta-analysis. In this work, we systematically evaluate the association between iron deficiency and obesity. We critically assessed levels of iron hematological markers in overweight and obese patients, as well as the risks of iron deficiency and anemia in obesity. A total of 26 studies were included with 40, 014 participants and 13, 393 overweight or obese patients. Marked reductions in serum iron and TS% were observed in overweight and obese cases, suggesting the existence of iron deficiency in obese population. Consistently, overweight and obesity were associated significantly with increased risks of iron deficiency. In light of these findings, we concluded significant association between obesity and iron deficiency, and suggested the importance of early monitoring and treatment of iron deficiency in overweight and obese people.

Sixth Congress of the International BioIRon Society Page 281 Poster Abstracts IBIS

Poster #135 INVESTIGATING THE ROLE OF NO IN ANEMIA OF INFLAMMATION Marc Mikhael, BSc, PhD1, Costantine Daher, PhD1 and Prem Ponka, MD, PhD2 1Lebanese American University; 2McGill University

AI (anaemia of inflammation) often manifests in patients with chronic immune activation due to cancer, chronic infections, autoimmune disorders, rheumatoid arthritis and other diseases. The pathogenesis of AI is complex and involves cytokine- mediated inhibition of erythropoiesis, insufficient erythropoietin production and diminished sensitivity of erythroid progenitors to this hormone, and retention of iron in haemoglobin-processing macrophages. NO (nitric oxide) is a gaseous molecule produced by activated macrophages that has been identified as having numerous effects on iron metabolism. We have previously explored the possibility that NO affects iron metabolism in reticulocytes and our results suggested that NO may also contribute to AI. Here we report on additional experiments in mice treated with a NO donor and anemia in mice models of cancer. These investigations test the possibility that NO is a significant player in the etiology of anemia of inflammation, specifically during cancer.

Sixth Congress of the International BioIRon Society Page 282 Poster Abstracts IBIS

Poster #136 EFFECTS OF PERSISTENT C19ORF12 DOWN-REGULATION UPON CELLULAR IRON HOMEOSTASIS Claudia Saraceno, PhD, Paola Ruzzenenti, Michela Asperti, PhD, Federica Maccarinelli and Dario Finazzi, MD University of Brescia

Mutations in the gene C19orf12 gene have been associated to a specific subtype of neurodegeneration with brain iron accumulation (NBIA), called mitochondrial membrane protein-associated neurodegeneration, MPAN. The gene codes for two protein isoforms of 141 and 152 aminoacids, that carry a transmembrane domain and localize to the ER and mitochondria. The gene is particularly expressed in neurons, adipocytes and white blood cells and co-regulated with genes involved in fatty acids metabolism. Its down-regulation in drosophila led to bang sensitivity and neurodegeneration. The link between C19orf12 defects, neurodegeneration and iron accumulation in the brain is completely unknown. We investigated this connection creating models of the disease by transient or stable silencing of the gene in SH-SY5Y neuroblastoma cells and studying the effects upon iron related parameters. SH-SY5Y cells treated with 10nM of a specific s or h, shoed a ared redto o or trasrpt, that as assoated th reded leels o errt %) and increased expression of transferrin receptor and ferroportin proteins. To better characterize these observations, we generated SH-SY5Y cells with stable expression of a C19orf12 specific pre-miRNA and selected three clones with the strongest silencing (80–90%). As control we isolated clones with stable expression of a pre-miRNA predicted not to target any known gene. The first evident effect we observed was a reduction of the proliferation rate in the three silenced clones, without any increase of cell mortality. Western blot analyses of the main proteins involved in iron homeostasis confirmed the results obtained in the transient model and showed a significant increase of ferroportin (2-2,5 fold) and transferrin receptor-1 levels (2-3 fold) and a significant reduction of ferritin levels. Then we measured the aconitase activity, an iron-dependent enzyme. We observed that down-regulation of C19orf12 caused a significant reduction of both the cytosolic and mitochondrial enzymatic activity. We analyzed the cellular labile iron pool (LIP) by a fluorescent permeable metal sensor, calcein-AM. Cells were treated or not with ferric ammonium citrate (FAC) and loaded th ale-AM for 15 min at 37 °C. Fluorescence was monitored before and after the addition of the permeable iron chelator deferiprone. After iron supplementation, we observed a significant increase in LIP levels in the C19orf12 clones compared with the control ones; on the contrary the basal LIP levels didn’t show relevant differences between the two cell types. To investigate whether these perturbations in iron handling were associated with altered oxidative stress, we measured the amount of carbonylated proteins and observed an increase of oxidized proteins in the C19orf12 silenced clones. This was associated with a significant reduction of both cytosolic (SOD1) and mitochondrial (SOD2) activity with a correspondent decrease of SOD2 protein. Furtermore western blot analysis of two markers of autophagy, LC3B and Beclin1, showed increased levels of these proteins in the C19orf12 clones compared with the controls. Altogether our results show that reduced C19orf12 functioning is associated with altered expression of ferroportin, TFR-1 and ferritin proteins and defects in iron handling, that probably leads to increased oxidative stress and induction of autophagy. Understanding the molecular mechanisms underpinning these phenotype could be of relevance for the comprehension of MPAN pathogenesis.

Sixth Congress of the International BioIRon Society Page 283 Poster Abstracts IBIS

Poster #137 ALTERATIONS IN CARDIAC IRON STATUS, ELECTROPHYSIOLOGICAL RESPONSES AND TRANSCRIPT LEVELS IN MOUSE MODELS OF DIETARY AND GENETIC IRON LOADING Kristy Martin, Bachelor2, Daniel Johnstone, PhD3, Ross Graham, PhD4, Dirk van Helden, PhD2, Karen Kerr, PhD2, Sharon Hollins, Bachelor2, Derek Laver, PhD2, John Olynyk, PhD5, Debbie Trinder, PhD6 and Liz Milward, PhD1 1University of Newcastle; 2School of Biomedical Sciences and Pharmacy and the Hunter Medical Research Institute, the University of Newcastle, Australia; 3Bosch Institute and Discipline of Physiology, University of Sydney, Australia; 4School of Biomedical Sciences and 5 Curtin Health Innovation Research Institute, Curtin University of Technology, Australia; 5Department of Gastroenterology, Fremantle Hospital, Australia; 6School of Medicine and Pharmacology, Fremantle Hospital and Harry Perkins Institute of Medical Research, University of Western Australia, Australia

Background: Hemochromatosis causes cardiomyopathies and arrhythmia and myocardial infarction strongly contribute to premature mortality in severe secondary iron overload.1,2 We are using dietary (secondary) and genetic (primary) models of iron loading to study effects of cardiac iron status on heart gene expression and electrophysiological function. Methods: We used 10 week old wildtype AKR mice fed normal chow (control) or an iron-supplemented diet (2% carbonyl iron, three weeks) and two models of genetic hemochromatosis, Hfe knockout mice (Hfe-/-) and transferrin receptor 2 mutant mice (Tfr2mut). Hearts were studied using non-heme iron assays, 3,3’-diaminobenzidine (DAB)-enhanced Perls’ stain, ferritin immunoblotting, microarray, real time reverse transcription PCR (RT-PCR) and functional assessment by in vitro sinoatrial node isolation studies for detection of changes in contractile frequency in response to the sympathetic neurotransmitter noradrenaline and other agents. Results: Cardiac iron and ferritin levels did not differ significantly in Hfe-/- mice compared to controls whereas heart from the other models had significantly increased non-heme iron and ferritin (all p<0.05, control

Sixth Congress of the International BioIRon Society Page 284 Poster Abstracts IBIS

Poster #138 CATECHOLAMINE STRESS HORMONES REGULATE CELLULAR IRON HOMEOSTASIS BY A POSTTRANSCRIPTIONAL MECHANISM MEDIATED BY IRON REGULATORY PROTEIN Nisha Tapryal, PhD, Vishnu Vivek, MSc, Som Dev, MSc and Chinmay K. Mukhopadhyay, PhD SCMM, JNU, New Delhi

Introduction: In mammals, various psychosocial and physical stresses activate the peripheral sympatho- adrenomedullary and central catecholaminergic systems to elevate secretion of epinephrine (EPI) and norepinephrine (NE) in the circulation. These stress hormones, more commonly known as catecholamines, trigger a series of biological reactions including increased rate of energy metabolism, in order to prepare the organism to overcome or to adapt to the stressful events. Catecholamines enhance glycogenolysis in liver to increase glucose release in bloodstream to compensate the extra glucose consumption by various organs, including brain, heart and muscles for enhanced ATP production. During stress response, 40% increase in energy expenditure was reported in skeletal muscles’ alone. Since rate limiting enzymes of energy generating pathways such as TCA cycle and mitochondrial electron transport chain are dependent on iron; therefore, there should be an alteration in cellular iron requirement during stress induced catecholamine release..But so far, not much has been explored to understand cellular iron homeostasis during catecholamine exposure. Objective: This study was undertaken to explore the effect of catecholamines like epinephrine (EPI) and norepinephrine (NE) on iron homeostasis of hepatic and skeletal muscle cells. Observation: Here we show that epinephrine/norepinephrine regulates iron homeostasis components like transferrin receptor-1 and ferritin-H in hepatic and skeletal muscle cells by promoting the binding of iron regulatory proteins (IRPs) to iron responsive elements (IREs) present in the untranslated regions (UTRs) of transferrin receptor-1 and ferritin-H transcripts. Increased transferrin receptor-1, decreased ferritin-H and increased iron responsive element-iron regulatory protein interaction are also observed in liver and muscle tissues of epinephrine/norepinephrine injected mice. We demonstrate the role of epinephrine/ norepinephrine induced generation of reactive oxygen species on converting cytosolic aconitase (ACO1) into iron regulatory protein-1 to bind iron responsive elements present in UTRs of transferrin receptor-1 and ferritin-H. Our study further reveals that mitochondrial iron content and mitochondrial aconitase (ACO2) activity are elevated by epinephrine/ norepinephrine that are blocked by the antioxidant N-acetyl cysteine and iron regulatory protein-1 siRNA suggesting involvement of reactive oxygen species and iron regulatory protein-1 in this mechanism. Discussion and Conclusion: Little is known regarding regulation of iron homeostasis in response to stress hormones like EPI and NE. In this study, we found that EPI and NE both could increase IRE-IRP interaction to regulate iron homeostasis in hepatic and skeletal muscle cells. This study also revealed that EPI/NE could promote mitochondrial iron supply through IRE-IRP interaction. We speculate this alteration of cellular iron homeostasis is necessary to adapt to changes associated with these catecholamines induced metabolism and to restore the energy balance.

Sixth Congress of the International BioIRon Society Page 285 Poster Abstracts IBIS

Poster #139 SYSTEMIC AND LOCAL IRON ACCUMULATION LEADS TO OXIDATIVE STRESS IN TYPE 2 DIABETES MELLITUS MICE Min Chen, Bo Jiang, bachelor and Huijun Chen, Doctor Medical School of Nanjing University

Introduction: Increasing evidence suggests that iron accumulation is associated with the risk of diabetes mellitus (DM), h owever, the iron metabolism and mechanism in T2DM patients remains unclear. We have set up a T2DM mice model by i ntraperitoneal injection of low-dose streptozotocin (STZ) to study this issue. Methods: Three month old C57BL/6J wild-type (WT) mice were fed a high- fat diet (60% calories) or a standard chow diet for 1 month, and then injected intraperitoneally with either STZ at 40 mg/kg body weight for T2DM group or citrate buffer for control group for five days, and then both groups were kept on the standa rd chow diet for the additional one month (T2DM-5m) or three months (T2DM-7m). Results: We found that the protein expression of serum ceruloplasmin (CP) and liver heme oxygenase (HO- 1) were significantly increased in T2DM- 5m group compared to WT controls. Also we found a significant decrease of mRNA level of liver hepcidin and a remarkabl e increase of iron concentration in serum, brain, heart, liver and enterocytes in T2DM- 5m group compared to WT controls. But the mRNA level of liver hepcidin was inversely increased as time passed in the T 2DB- 7m group. Then, we checked the relative genes of oxidative stress and inflammation, and found that mRNA levels of the n uclear factor E2-related factor 2 (Nrf-2) and heme oxygenase (HO- 1) of mice liver were significantly increased in the T2DM-5m group, but no change in the T2DM- 7m group compared to WT controls, respectively. The mRNA levels of interleukin 6 (IL-6) and interleukin 1 beta (IL- of mice liver were significantly increased in the T2DM-7m group, but no change in the T2DM- 5m group compared to WT controls, respectively. Furthermore, we checked the superoxide dismutase (SOD) activity and l ipid peroxidation level by malondialdehyde (MDA) assay. We found that SOD activity was significantly decreased of seru m and liver in T2DM-5m group, but not in T2DM- 7m group compared to the same age of WT controls, and MDA content was significantly increased of serum and liver in T 2DM-5m group, but not in T2DM-7m group compared to the same age of WT controls. Conclusion: Those results suggest that the decreased expression of hepcidin at early of T2DM can lead to the local and systemic iron accumulation in T2DM mice, which contribute to oxidative stress reaction and subsequently inflammatory re sponse.

Sixth Congress of the International BioIRon Society Page 286 Poster Abstracts IBIS

Poster #140 HEPARAN SULFATE PROTEOGLYCANS IN THE BMP6/SMAD SIGNALING Paola Ruzzenenti, PhD Student, Maura Poli, PhD, Michela Asperti, PhD Student, Magdalena Gryzik, PhD Student and Paolo Arosio, PhD University of Brescia

Hepcidin is a master regulator of systemic iron availability controlling the functionality of ferroportin. Hepcidin expression occurs mainly in the liver where is regulated mostly by the BMP6/SMAD pathway that leads to the phosphorylation of SMAD1/5/8, the recruitment of SMAD4 and the translocation of the complex into the nucleus where it binds hepcidin promoter and activates its transcription. In addition the signaling merges with other pathways, the most important of which is the IL6/STAT3 pathway that is a central link connecting iron homeostasis and inflammation. The activation of BMP/SMAD pathway involves a large number of proteins: dimers of type I and of type II receptors, HJV (a GPI-anchor protein that acts as key BMP-co-receptor), TMPRSS6 (a membrane protease that negatively regulates HJV), Neogenin, HFE and TfR2. Although the crystallographic structure of some of these proteins is known, how they interact to form the functional complexes is not so clear. Our recent observation that heparins strongly repress BMP/SMAD pathway and hepcidin expression, suggests that the endogenous heparan sulfates (HSs) participate in offering multiple binding sites that would facilitate the formation of the signaling complexes. Our preliminary data seem to support the hypothesis that heparin antagonizes the role of endogenous heparan sulfate. First of all the three hepatic cells characterized by different panels of Heparan Sulfate Proteoglycans (HSPGs) (HepG2 with high level of glypican3; HuH7 and Hep3B with low glypican3; primary hepatocytes with high glypican4), differently regulate hepcidin expression in response to the treatment of Sodium chlorate (an inhibitor of HSs production) and after the silencing of key enzymes of HSs biosynthesis (Ext1, Ext2 and Ndst1). Our main goal is to identify the binding sites with HS of the proteins involved in the BMP/SMAD pathway that leads to the expression of hepcidin through their cloning and their mutagenesis in eukaryotic and bacterial vectors. We started with BMP6 that has a putative heparin binding domain (HBD) at the unstructured N-terminus domain and we found that a biotin-labelled synthetic peptide of this region showed high affinity binding to heparin observed using two approaches: Heparin/GAG Binding Plates and heparin coated Microcantilever biosensors. BMP6 also presents other basic regions in structured portion and the studies will be extended to these portions. To obtain a complete picture of the heparin binding capacity of the signalling complex, we need to produce different proteins that compose this signalling complex and to verify if they have HS (or heparin) binding sites. In silico analysis of the available crystallographic structures suggest the presence of putative heparin binding sites (HBS) also in type I and II receptors. To check the presence and localization of these HBS we started cloning and expressing in mammalian cells tagged forms of the ectodomains of BMP receptor type I and type II and their binding to cell bound HS analyzed by cell-cell contact assay between cells expressing normal HSs and mutant ones. Our preliminary data seem to confirm an important role of HSPGs in the BMP6/SMAD signaling and the identification of the actors and interaction sites will be defined.

Sixth Congress of the International BioIRon Society Page 287 Poster Abstracts IBIS

Poster #141 THE ROLE OF THE MITOCHONDRIAL FERRITIN IN THE HEART AND TESTES Federica Maccarinelli, Maria Regoni, Maura Poli, PhD, Michela Asperti, Paola Ruzzenenti, Magdalena Gryzik and Paolo Arosio, PhD University of Brescia

Mitochondrial ferritin (FtMt) precursor is encoded by an intronless gene and targeted to mitochondria, where it is processed in the mature form. The 22KDa protein has high homology to ferritin Hha or seee, strtre ad the presence of a ferroxidase center and readily assembles in a shell of 24 subunits that accumulates iron efficiently. The over-expression of FtMt in cultured cells showed that it competes with the cytosolic ferritins for cellular iron sequestration and protects the cells against oxidative damage, suggesting that it has an important role in iron homeostasis. FtMt expression appears not to be governed by cellular iron levels, and in mice and humans it is highly expressed in the testes, particularly in spermatocytes, in the heart and brain, all tissues with high metabolic activity and oxygen consumption and therefore rich in mitochondria. In human, FtMt protein was also found to accumulate in iron-loaded mitochondria of erythroblasts from patients with sideroblastic anemia (SA), but its biological role has not been established yet. To this aim we generated a mouse strain lacking FtMt by standard homologous recombination technology and followed it for more than two years. FtMt-KO mice are viable and do not show any evident pathological phenotype. Given the high level of FtMt expression in the heart, we challenged the mice by the cardiotoxic agent Doxorubicin (DOX) (15mg/Kg IP), and we found that FtMt-KO mice are more sensitive to DOX toxicity with enhanced mortality, altered heart morphology and signs of oxidative damage (Maccarinelli et al., 2014). We observed also that DOX treatment altered the testis morphology much more severely in FtMt-KO than in wild-type (WT) mice. This addressed our attention on the role of FtMt in the testis. The FtMt-KO mice are fertile, but we observed that male KO crossed with WT female gave raise to smaller litters than male WT mice. No difference was instead found between female KO and WT crossed with male WT mice, pointing to male fertility problems. To study this aspect, we initially performed histological analysis of testis paraffin sections of 12-week-old mice: FtMt-KO mice showed evident signs of oligozoospermia absent in the controls. Then we started analyzing sperm parameters: sperm samples were collected from the cauda epididymis, counted and analyzed at the light microscope. KO sperm samples showed a significant reduction of the sperm density and motility, and we observed some morphological alterations of the head. To study mitochondria functionality biochemical analysis for ATP levels and oxidative damage are ongoing. Our data suggested that FtMt has a significant protective role in the heart and testis, the two tissues where it is most highly expressed. It protects the cells against the DOX cardiotoxicity and has an important role in ensuring male fertility.

Sixth Congress of the International BioIRon Society Page 288 Poster Abstracts IBIS

Poster #142 THE EFFECT OF NEUROINFLAMMATION ON IRON REGULATION IN CELLS OF THE CENTRAL NERVOUS SYSTEM Surjit Kaila Srai, PhD1, Veronika Kallo, MRes1, Roberta Ward, PhD2 and David Dexter, PhD2 1University College London; 2Imperial College

Introduction: One of the key pathological features of sporadic neurodegenerative disorders is the accumulation of iron in areas of comprehensive neuronal cell death. The origin of this iron is unclear, but its accumulation may be related to inflammation observed in all neurodegenerative disorders. Our previous studies have demonstrated that activated glial cells are principally responsible in mediating inflammation, namely microglia, which can alter their own regulation of iron and that of neighbouring neurons and astrocytes (1). The purpose of this study is to assess the impact of the innate inflammatory response induced by lipopolysaccharide (LPS) on iron metabolism in microglial cells in vitro, and further investigate the effect of activated microglial cells on neurons in a co–culture system. Materials and Methods: N9 microglia cells were treated with LPS directly or LPS-treated microglia were co-cultured with N27 neurons for various time periods, up to 24h. The expression of the iron importers DMT1, Zip14, TfR1 and ferroportin (iron exporter) were measured with real-time PCR. Transferrin bound iron (TBI) and non-transferrin bound iron (NTBI) influx was assessed using radioactive isotope of iron (55Fe). Results: LPS treated N9 microglia displayed a significant up-regulation in DMT1 mRNA levels at 2h (2.5x), 4h (2.3x), 6h (1.6x), 12h (1.6x), and 24h (1.4x). Zip14 also showed up-regulatory responses at 2h (1.24x), 4h (2.11x), 12h (1.69x), and a decline in expression at 6h and 24h. TfR1 mRNA levels remained unchanged over the 24h period. This was mirrored by a significant increase of NTBI uptake (p<0.0001) at 24h but no change observed in TBI uptake. Ferroportin transcripts were highly up-regulated at 24h (6.3x). N27 neurons co-cultured with activated N9 cells displayed significant up-regulation of DMT1 at 6h (3.6x) and 24h (2.9x). Zip14 mRNA levels showed a very steep up-regulation profile at 2h (63.9x) and 4h (16.4x). NTBI uptake studies support the involvement of these genes in importing iron under inflammatory conditions at 6h (p<0.01), 12h (p<0.0001) and 24h (p<0.0001). Interestingly, TBI uptake did not show significant differences with or without inflammation present. However, TfR1 mRNA levels were significantly elevated at 4h (2.7x), 6h (3.2x), and at 24h (1.6x), and ferroportin mRNA levels were drastically down-regulated by 24h (p<0.0001) in response to inflammatory stimuli. Conclusion: LPS not only induces an inflammatory response in microglia but also changes the gene expression profile of iron importer and exporter proteins. Neuronal and microglial co-culture studies further support the effect of inflammation on iron regulating proteins that show increased influx of iron in both neurons and microglia. These results highlight that the inflammatory process can influence the iron content of brain cells, and provide further grounds for investigating the role of iron in neurodegeneration. 1. Dexter, D. Ward, R. Crichton, R. Kallo, V. Srai, S., K. 2013, Altered brain iron homeostasis in Parkinson’s disease and potential for iron chelation therapy. American Journal of Hematology, 88(5):E163 – E163.

Sixth Congress of the International BioIRon Society Page 289 Poster Abstracts IBIS

Poster #143 VALPROIC ACID INDUCES HEPCIDIN EXPRESSION Marie-Laure Island, PhD1, Thibault Cavey2, Patricia Leroyer3, Martine Ropert2, Pierre Brissot3 and Olivier Loréal3 1INSERM-UMR 991 and National Reference Center for Rare Genetic Iron Overload Diseases, CHU Pontchaillou, Rennes, France; 2INSERM UMR 991 and CHU Pontchaillou, Department of Biochemistry, Rennes France; 3INSERM UMR 991 and University of Rennes 1, France

Background. In the various forms of genetic hemochromatoses related to mutations in genes controlling hepcidin expression (HFE, TFR2, HJV), an abnormally low level of serum hepcidin is involved in the development of systemic iron overload. Venesections are used to remove iron excess but do not treat the underlying mechanism, and may exert a further lowering effect on hepcidin levels. During Beta-thalassemia, both anemia and the erythropoietic process decrease hepatic hepcidin expression, thus contributing to increase the iron overload burden. Venesections cannot be performed due to anemia, and iron chelators are also a symptomatic treatment of iron overload. Therefore, there is a need for an etiological therapeutic approach based on an increase of serum hepcidin levels. Aim. We investigated whether valproic acid, an antiepileptic drug and mood stabilizer with multiple biological functions, could be an inducer of hepcidin expression. Material and Methods. We analysed HepG2 cells and pure cultures of freshly isolated human hepatocytes. Cells were exposed to valproic acid from 125µM to 1mM. In vivo, wild type C57BM/6 mice were acutely exposed to VPA (50 or 300 mg/Kg intraperitoneally). Hepcidin mRNA levels were quantified by RT-PCR in cell cultures and in the mouse liver. In vitro, the impact of VPA on the transcription of hepcidin gene was analyzed through gene reporter strategy and luciferase activity. BMP/SMAD pathway was explored by performing SMAD1/5/8 western-blot and by analyzing the impact of SMAD4 siRNAs. Results. Exposure of HepG2 cells and cultured human hepatocytes to VPA induced a dose dependent increase of hepcidin mRNA levels. In vivo, we analyzed the VPA impact 24 hours after injection and found a significant decrease of serum iron coupled with an increase of unbound iron capacity. In HepG2 cells, the gene reporter strategy identified the BMP-Responsive element as a mediator of VPA activity on hepcidin transcription activity. However, phosphorylation of SMAD1/5/8 proteins was not significantly modulated by VPA exposure. In addition, inhibition of SMAD4 expression by SMAD4 siRNAs did not alter the inducer impact of VPA. Discussion. Our data indicates that: i) VPA induces hepcidin expression in human hepatocytes; ii) VPA induces, in mice, a decrease of serum iron thus showing its potential impact on iron metabolism; iii) The mechanism involves BPM- Responsive elements in the hepcidin promoter, the precise mechanism remaining to be dissected. Conclusion. The present data shows that hepatocyte hepcidin expression can be induced by valproic acid, therefore suggesting that this track could be followed to design therapeutic actions in the field of hepcidin deficiency related diseases.

Sixth Congress of the International BioIRon Society Page 290 Poster Abstracts IBIS

Poster #144 DEFEROXAMINE ATTENUATES LIPOPOLYSACCHARIDE-INDUCED INFLAMMATORY RESPONSES AND PROTECTS AGAINST ENDOTOXIC SHOCK IN MICE Yumei Fan1, Shengnan Wang2 and Xianglin Duan2 1College of Life Science, Hebei Normal University; 2Laboratory of Molecular Iron Metabolism, College of Life Science, Hebei Normal University

To examine the role of intracellular labile iron pool (LIP) in the induction of inflammatory responses, we investigated anti- inflammatory effects of the iron chelator deferoxamine (DFO) on lipopolysaccharide (LPS)-induced inflammatory responses in RAW264.7 macrophage cells and endotoxic shock in mice in present study. DFO significantly decreased LPS-induced production of tumor necrosis factor-α -α, terle- -, tr ode ad prostalad (PGE2). Our data also showed that DFO significantly attenuated expression of iNOS and COX-2 in LPS-stimulated RAW264.7 macrophages. Furthermore, administration of DFO significantly decreased the mortality and improved survival of septic mice with lethal endotoxemia in LPS-injected mice. We then found that DFO inhibited phosphorylation of ases sh as ad p, also hted deradato o α ded y hese reslts deostrate that iron plays a pivotal role in the induction of inflammatory response and against septic shock. DFO has effective inhibitory effects on production of inflammatory mediators via suppressing activation of NF- ad sal pathays t also has a protective effect on LPS-induced endotoxic shock in mice. Our findings open doors to further studies directed at exploring a new class of drug against septic shock or other inflammatory diseases by modulating cellular chelatable iron.

Sixth Congress of the International BioIRon Society Page 291 Poster Abstracts IBIS

Poster #145 EXOME SEQUENCING IN HFE C282Y HOMOZYGOTES WITH EXTREME HEPATIC IRON OVERLOAD REVEALS MODIFYING GENES FOR THE DEVELOPMENT OF CIRRHOSIS Mary Emond, PhD2, Christine McLaren, PhD3, Tin Louie, PhD2, Jie Wu, PhD3, Lawrie Powell, MD, PhD1, Pradyumna Phatak, MD4, James Barton, MD5, Paul Adams, MD6, Lyle Gurrin, PhD7, John Phillips, PhD8, Charles Parker, MD8, Katrina Allen, FRACP, PhD9, Deborah Nickerson, PhD2, Gregory Anderson, PhD1, Nathan Subramaniam, PhD1, Gordon McLaren, MD10 and Grant Ramm, PhD1 1QIMR Berghofer MRI; 2University of Washington; 3University of California, Irvine; 4Rochester General Hospital; 5Southern Iron Disorders Center; 6London Health Sciences Centre; 7University of Melbourne; 8University of Utah; 9Murdoch Children’s Research Institute; 10VA Long Beach Healthcare System

Introduction: Variability in the severity of both iron overload and clinical manifestations among HFE C282Y homozygotes are current unsolved problems in our understanding of hereditary hemochromatosis (HH). While hepatic iron accumulation likely has a direct effect on cirrhosis risk, genetic factors probably modify this risk. We previously conducted exome sequencing of DNA from C282Y homozygotes with markedly increased iron stores vs. those with normal/mildly increased iron and identified a GNPAT polymorphism, GNPAT p.D519G (1556A>G; exon 11; chromosome 1q42; rs11558492) associated with extreme hepatic iron overload in HH in 16 of 22 high iron cases, which was absent in all 13 subjects with no/mild iron loading1. In this study we used this ‘black swan’ approach to investigate potential modifying genes associated with cirrhosis in HH using exome sequencing of subjects with extreme hepatic iron overload, who either have cirrhosis (cases), or alternatively have no histological evidence of any fibrosis/cirrhosis (controls). Methods: Criteria for HFE C282Y homozygotes with extreme iron stores included serum ferritin >1000 µg/L at diagnosis and either (a) hepatic iron concentration >236 µmol/g dry weight, or (b) mobilized body iron >10g by quantitative phlebotomy. Hepatic fibrosis was assessed histologically, staged for fibrosis and classified as having cirrhosis (fibrosis stage, F4), any fibrosis (F1-F4), or no evidence of fibrosis (F0). Cases included 12 subjects (11 male) with cirrhosis and controls included 6 subjects (4 male) with no fibrosis. Exome sequencing was performed at the University of Washington Northwest Genomics Center and processed using the GATK Unified Genotyper. Ten candidate genes were examined: MYO, PNPLA3, PCSK7, TGF-beta, TNFa, AGT, TLR4, MPO, GSTP1 and SOD2. Association analysis using remaining exome data was performed using the burden test of Morris and Zeggini2 restricted to variants with >99% probability of functional consequences according to Combined Annotation Dependent Depletion (CADD) scores3. Results: There was no difference in prevalence of steatosis between cases (3/11, one unknown) and controls (2/6). Among the candidate genes, the rs738409 missense variant in PNPLA3 was found to be significantly more frequent among cases (7 of 12) than among controls (0 of 6) (p=0.005). In the whole exome association study, two genes were found with a false discovery rate of <5%, the most significant having seven mutations among cases and none among controls. Conclusions: PNPLA3 rs738409 contributes to the risk of cirrhosis in HFE C282Y homozygous individuals with extreme iron phenotype. This variant has previously been associated with steatosis and fibrosis in NAFLD and was weakly associated with fibrosis progression in HH. Exome sequencing on a small, carefully selected sample can identify plausible novel candidates for validation. 1Hepatology, 2015; doi:10.1002/hep.27711. 2Genetic Epidemiology, 2010;34(2):188-93. 3Nature Genetics, 2014;46(3):310-5.

Sixth Congress of the International BioIRon Society Page 292 Poster Abstracts IBIS

Poster #146 A DOUBLE KNOCK OUT OF HEPCIDIN AND IL6 DEMONSTRATES INDEPENDENT ROLES OF THE TWO GENES IN ANEMIA OF INFLAMMATION Sara Gardenghi2, Ritama Gupta, Lori Bystrom, Roberta Chessa and Stefano Rivella, PhD1 1Children's Hospital of Philadelphia; 2Weill Cornell Medical College

Inflammatory states seen in infection and other chronic disorders are often characterized by a condition called anemia of inflammation (AI). The iron deficiency in AI is predominantly due to an altered balance of the cytokine-interleukin-6 (IL6) and the hormone hepcidin (Hamp). IL6 has been implicated in inducing expression of hepcidin, which degrades the iron exporter ferroportin. We have previously shown that lack of IL6 or hepcidin in knockout mouse models (IL6-KO and Hamp-KO) injected with the heat-killed pathogen Brucella abortus (BA) results in improved recovery from anemia. This recovery was different in IL6-KO and Hamp-KO mice, suggesting that the two proteins contribute independently to AI. Here, we formally validated the independent role of IL6 and Hamp in AI by generating a double-knockout (DKO) mouse model lacking the expression of both. The DKO mice showed the most ameliorated phenotype following BA administration. BA-treated-DKO mice showed an increased number of erythroblasts in the bone marrow (BM) and spleen as seen by flow cytometry, in comparison to IL6-KO and Hamp-KO. Concurrently, compared to WT, Hamp-KO and IL6- KO animals, in DKO mice the reticulocyte count was already increased by week-2.. The anemia induced by the pathogen by week-1 was less severe in DKO mice. Moreover, both hemoglobin and RBC values measured at week-2 were the highest in DKO, followed by Hamp-KO and then IL6-KO. We also investigated RBC lifespan in these animals by measuring the turnover of biotinylated RBC over time. The turnover of the biotinylated RBC occurred in two phases. In the initial phase the percentage of biotinylated and non-biotinylated RBCs in the BA-treated animals remained the same, while in the second phase it decreased, indicating production of new RBCs. In Hamp-KO and the IL6-KO mice the first phase lasted for 7 days and 4 days respectively, while in DKO animals the percentage of biotinylated RBC had already started to decrease by day 4, indicating an accelerated production of new RBCs compared to single KO mice. Additionally, we used the RodentMAP®-v.3.1 (MyriadRBM) to quantify 51 serum inflammatory biomarkers, and Ingenuity Pathway Analysis to identify pathways activated in single KO as well as DKO mice. Of the top 10 pathways activated in all three models, 4 present in the IL6-KO were also activated in the DKO model, and the remaining 6 were unique. Five of the pathways that came up in Hamp-KO were also activated in DKO mice and the remaining 5 were unique. For instance, both DKO and Hamp-KO mice showed activation of Hmgb1 signaling, suggesting a response to limit inflammation and reduce tissue damage. Moreover, both the DKO and IL6-KO models showed activation of granulocyte adhesion and diapedesis, the former suggesting an inflammatory response associated with the infection while the latter possibly indicating mobilization of cells in response to the infection. A unique pathway activated in DKO mice was that associated with increased production of pluripotent stem cells, likely triggered by the damage observed to the BM and anemia and potentially responsible for the accelerated recovery observed in these animals. In conclusion, these results suggest that the absence of both IL6 and Hamp not only is associated with activation of pathways in common with the single KO, but also with unique features triggered by the concurrent depletion of the two genes. Potential clinical implications will be discussed.

Sixth Congress of the International BioIRon Society Page 293 Poster Abstracts IBIS

Poster #147 A CANDIDATE MECHANISM FOR THE ASSOCIATION OF HFE C282Y WITH REDUCED LDL-CHOLESTEROL Dan Yin, BE, PhD1, Ibrahim AY Hamad, MSc2, Jutta Palmen, MSc3, Anastasia Kalea, PhD3, Andrew Smith, PhD3, Philippa Talmud, DSc3, Steve Humphries, PhD3 and Ann Walker, PhD3 1BGI-Wuhan, Wuhan, China, BGI-Shenzhen, Shenzhen 518083, China & UCL, London WC1E 6JF, UK; 2UCL, London WC1E 6JF, UK & Alazhar University, Damietta 34511, Egypt; 3UCL, London WC1E 6JF, UK

HFE C282Y (rs18005 s reprodly assoated th loer leels o lo desty lpoproteholesterol eoede assoato stdes We hypothesised that this association was mediated by iron, possibly via iron responsive elements (IREs), influencing mRNA leels o other ees assoated th leels IREs were predicted or assoated ees o hepatoyt ell les, ep ad h , ere treated th err ammonium citrate (FAC), 30µM haemin, 10µM desferrioxamine or 100µM deerproe or h oloal replates assayed in triplicate). Relative RNA expression was determined using TaqMan assays.

Expression of positive control TFRC in treated versus untreated HepG2 cells was reduced by FAC (mean relative expression ± SEM, 0.58 ± 0.057, p=0.0034) and showed a trend towards lower levels after haemin treatment (0.70 ± 0.12, p=0.075). h ells, TFRC expression after FAC (1.07 ± 0.13, p=0.63) or haemin (1.05 ± 0.20, p=0.84) treatment was not significantly altered, possibly due to the “haemochromatotic” genotype of this cell line. TFRC expression was reased y deerproe oth ep , p ad h , p, ad lese as increased by desferrioxamine in both HepG2 (1.76 ± 0.20, p ad h , p

lee o assoated ees eoded predted s PCSK9, which binds to and promotes degradation of the LDL receptor (LDLR) and is a therapeutic target for hypercholesterolaemia, contained a predicted IRE, but it was in a coding exon. When compared to untreated cells, FAC caused a trend towards lower PCSK9 expression in HepG2 (0.62 ± , p ad loer epresso h , p Consistently, deferiprone caused a trend towards higher PCSK9 epresso ep , p ad reased epresso h , p=0.0035), while desferrioxamine caused trends towards increased PCSK9 expression in HepG2 (2.17 ± 0.48, p=0.059) ad h , p072). LDLR contained no predicted IREs and its expression was not significantly altered by FAC or haemin treatment in either cell line. Deferiprone increased LDLR expression in both HepG2 (1.53 ± 0.072, p ad h , p Similarly, desferrioxamine increased LDLR expression in HepG2 (1.72 ± , p ad ased a tred toards reased epresso h , p, erss treated cells. Both SORT1 and LDLRAP1 were predicted to contain IREs classically located ther tralated reos, t the only result tending towards significance after the four treatments in the two cell lines was for SORT1 expression after FAC treatment of HepG2 cells (0.71 ± 0.11, p=0.07).

hle le ots ere predted seeral assoated ees, they dd ot appear to edate a eeral lee o ro po assoated ee epresso However, repression of PCSK9 expression by iron could provide a potential mechanism underlying the association of HFE C282 th loer

Sixth Congress of the International BioIRon Society Page 294 Poster Abstracts IBIS

Poster #148 SERUM FROM INDIVIDUALS GIVEN A SINGLE ORAL DOSE OF IRON SUPPLEMENTATION SUPPORTS INCREASED BACTERIAL GROWTH James Cross, BsC2, Richard Bradbury, PhD2, Anthony Fulford, PhD3, Amadou Jallow, Foundation Degree2, Rita Wegmuller, PhD2, Andrew Prentice, PhD3 and Carla Cerami, MD,PhD1 1University of North Carolina; 2MRC-Gambia; 3London School of Hygiene and Tropical Medicine

Iron supplementation is standard policy for pregnant mothers and children in most low-income countries. However, the wisdom of universal iron supplementation campaigns has come under serious scrutiny starting in 2006 with the premature termination of a large trial in Tanzania after significant increases in the number of serious adverse outcomes and deaths in young children receiving iron supplements were observed. Initially, emphasis was focused on malaria, but it is clear that bacterial infections are also an important factor. Subsequent clinical trials of iron administration in African and Asian children have reported increased rates of malaria, respiratory infections, severe diarrhea, hospitalizations and death, but the mechanism(s) are unclear. Recent work has also shown that oral iron supplementation alters the gut microbiota and increases the virulence of many common bacterial pathogens. To investigate the possibility that oral doses of supplemental iron could promote bacterial growth in serum, we have used a series of ex vivo bacterial growth assays with sentinel organisms that were selected on the basis of their varying modes of pathogenesis and abilities to scavenge iron from the host. We will present data on our investigation of the ex vivo growth characteristics of five exemplar bacteria in adult sera collected before and 4h after oral supplementation with ferrous sulfate. Three gram negative bacteria, Escherichia coli, Yersinia enterocolitica and Salmonella enterica serovar Typhimurium and one gram positive bacteria, Staphylococcus epidermidis showed markedly elevated growth in serum collected after iron supplementation. Growth of Staphylococcus aureus, which preferentially scavenges heme iron, was unaffected. Growth rates were very strongly correlated with transferrin saturation (p<0.0001 in all cases). These data suggest that even modest oral supplementation in iron replete men could promote bacteremia by accelerating early phase bacterial growth prior to the induction of immune defenses.

Sixth Congress of the International BioIRon Society Page 295 Poster Abstracts IBIS

Poster #149 THE METAL-ION TRANSPORTER ZIP8 (SLC39A8) AND IRON TRANSPORT ACROSS THE PLACENTA Wei Zhang, PhD, Supak Jenkitkasemwong, PhD, Alan Chan, BS and Mitchell Knutson, PhD University of Florida

Introduction: The molecular mechanisms of iron transport across the placenta are not well understood. Studies using knockout mice demonstrate that the transmembrane iron-import proteins DMT1 and ZIP14 are dispensable for maternofetal iron transfer. We recently reported that ZIP8, the closest paralogue to ZIP14, transports iron, is regulated by iron, and is abundantly expressed in the placenta (Wang et al., J Biol Chem, 2012). Moreover, suppression of ZIP8 expression in BeWo cells, a placental cell line, decreased cellular uptake of iron. Collectively, these observations raise the possibility that ZIP8 plays a role in placental iron transport. The aims of the present study were to: (1) determine the cellular and subcellular localization of ZIP8 in placenta (2) assess the effect of global loss of Zip8 (Zip8-/-) on iron status of mouse embryos and placentas, (3) assess the effect of conditional loss of Zip8 on iron status by studying Meox2- Cre;Zip8flox/flox mice, which lack Zip8 everywhere except for placenta and extraembryonic visceral endoderm. Methods and Materials: Cellular and subcellular localizations of ZIP8 in human term placenta sections were studied by immunostaining. Iron status was assessed by measuring total and non-heme iron concentrations of embryos and placentas and Perls’ Prussian blue staining of tissue sections. Results: We found that placental ZIP8 was predominantly localized in syncytiotrophoblasts, cells that form the placental barrier. In syncytiotrophoblasts, ZIP8 colocalized with both transferrin receptor 1 (TfR1) and EEA1 (early endosome antigen 1). Zip8-/- mice did not survive beyond E 14.5. At E 14.5, Zip8-/- embryos were iron-deficient and markedly pale, consistent with iron-deficiency anemia. Total and non-heme iron concentrations were 54% and 43% lower in Zip8-/- embryos compared with those in wild-type (WT) controls. By contrast, total and non-heme iron concentrations in the placentas were 95% and 240% higher in Zip8-/- placentas compared with WT controls. In the placenta of Zip8-/- mice, iron was found to accumulate in syncytiotrophoblasts. Meox2-Cre;Zip8flox/flox mice survived longer than did Zip8-/- mice, but did not survive past E 17.5. At E 14.5, Meox2-Cre;Zip8flox/flox mice were less pale and displayed a less-severe iron deficiency than did Zip8-/- mice at the same gestational age. Total and non-heme iron concentrations were 27% and 23% lower in Meox2-Cre;Zip8flox/flox embryos compared with those in WT controls. Total and non-heme iron concentrations in the placentas were 15% and 113% higher in Meox2-Cre;Zip8flox/flox embryos compared with those in WT controls. In contrast to Zip8-/- placenta, Meox2-Cre;Zip8flox/flox placenta showed little iron accumulation in syncytiotrophoblasts. Conclusion: Colocalization of ZIP8 with TfR1 and EEA1 in placental syncytiotrophoblasts suggests that ZIP8 may serve as an endosomal iron transporter. The observation that Zip8-/- placenta accumulates iron in syncytiotrophoblasts suggests that without ZIP8 iron is retained in the placental barrier without being transported to the fetus efficiently. That Meox2- Cre;Zip8flox/flox embryos have a less severe iron-deficient phenotype than do Zip8-/- embryos is also consistent with a role for ZIP8 in placental iron transport. Supported by NIH grant R01 DK080706.

Sixth Congress of the International BioIRon Society Page 296 Poster Abstracts IBIS

Poster #150 DIFFERENCES IN CLINICAL MANIFESTATIONS OF HEMOCHROMATOSIS IN HFE C282Y HOMOZYGOTES WITH EXTREME HIGH AND LOW IRON PHENOTYPES GD McLaren1, LW Powell2, PD Phatak3, JC Barton4, PC Adams5, VN Subramaniam2, LC Gurrin6, JD Phillips7, C Parker7, KJ Allen8, W-P Chen9, MJ Emond10, DA Nickerson10, GA Ramm2, GJ Anderson2 and CE McLaren9 1VA Long Beach Healthcare System and Department of Medicine, University of California, Irvine; 2QIMR Berghofer Medical Research Institute and University of Queensland, Brisbane, Australia; 3Rochester General Hospital, Rochester, NY; 4Southern Iron Disorders Center, Birmingham, AL; 5London Health Sciences Centre, London, ON, Canada; 6University of Melbourne, Melbourne, Australia; 7University of Utah, Salt Lake City, UT; 8Murdoch Childrens Research Institute, Melbourne, Australia; 9University of California, Irvine, CA; 10University of Washington, Seattle, WA

Introduction: In a study to identify genetic modifiers of iron overload in C282Y homozygotes, we divided participants into groups of extreme low and high iron phenotypes.1 Here, we report the prevalences of selected clinical manifestations in an expanded sample of participants meeting these criteria. Methods: Participants were enrolled in the USA, Canada, and Australia.1 Increased iron stores ("cases") included serum ferritin (SF) >1000 µg/L at diagnosis and either (a) hepatic iron concentration >236 µmol/g dry weight or (b) iron >10 g removed by phlebotomy. Normal or mildly elevated iron stores (“controls”) included SF <200 µg/L for women and <300 or e, or ether a ae y th ro reoed y phleotoy to ahee ser errt , or age y th ro reoed y phleotoy e elded partpats ho osed ethaol d Bonferroni correction for multiple comparisons was applied to control the type I error. Results: There were 42 cases and 65 controls (69% male), including 98 Caucasians, 1 African American, and 8 unspecified. Mean (SD) ages at diagnosis were 51 (11.1) y (cases) and 55 (14.9) y (controls). Median SF was 2363 µg/L ases ad otrols ease atty ler ay rease rrhoss rs y ehasms other than iron stores, we evaluated a subgroup of 46 subjects with liver biosies showing no evidence of fatty liver. In this subgroup, mean ± SD serum alanine aminotransferase (ALT) was higher in cases (62 ± 52.8 U/L) than controls (26 ± 11.6 U/L), P=0.0072. Fibrosis was diagnosed in 10 of 24 (42%) cases and in none of 22 (0%) controls (P=0.0006). Cirrhosis was diagnosed in 9 of 24 (38%) cases and in none of 22 (0%) controls (P=0.0016). Among cases, mean iron removed was significantly greater in those with cirrhosis (26.0 ± 11.7 g) than in those without (12.4 ± 4.9), P=0.0130. Some cases who had large amounts of iron removed (up to 24 g) did not have cirrhosis. The prevalence of swollen or tender MP joints was higher in cases (13 of 22; 59%) than controls (1 of 20; 5%), P=0.0002. The prevalence of fatigue was greater in cases (16 of 24; 67%) than controls (3 of 21; 14%), P=0.0007. Conclusions: Prevalences of important clinical manifestations such as elevated ALT, hepatic fibrosis or cirrhosis, swollen or tender MP joints, and fatigue were greater in C282Y homozygotes with severe iron overload alone, without fatty liver, than in participants with little or no iron overload. Further research is needed to identify factors in addition to iron that influence the likelihood of cirrhosis in homozygotes with severe iron overload. 1Hepatology, 2015; doi:10.1002/hep.27711.

Sixth Congress of the International BioIRon Society Page 297 Podium Abstracts IBIS

Poster #151 IRON OVERLOAD PERTURBS COPPER DISTRIBUTION IN MICE Jung-Heun Ha, Caglar Doguer, Xiaoyu Wang, Shireen R. Flores and James F. Collins Food Science and Human Nutrition Dept., University of Florida

A common method to cause iron loading in rodents is feeding a high-iron diet, which presumably bypasses normal protective mechanisms (e.g. a block to absorption or storage in ferritin) and leads to increased body iron levels. We utilized this dietary iron-overload model in a mouse feeding study in which we also altered the dietary copper content. This study was intended to test the hypothesis that dietary copper would influence intestinal iron absorption during iron overload. Weanling C57Bl6 mice housed in overhanging, wire mesh-bottomed cages were fed one of 6 different AIN-93G- based diets for 4 weeks. Diets were identical except for iron and copper content and contained: low (11 ppm), normal (79 ppm) or high (8820 ppm) iron in combination with low (0.9 ppm), normal (8 ppm) or high (183 ppm) copper. All data reported below were from 4-8 mice and all reached statistical significance (p<0.05; 2-factor ANOVA). To our surprise, mice fed the high-iron diets with low and normal copper levels grew slower and were severely anemic, but the higher copper content normalized growth rate and corrected the anemia. Transferrin saturation, non-heme iron content in the liver, and hepatic hepcidin mRNA expression were all uniformly elevated in all groups of mice fed the high-iron diets (regardless of copper content). We also noted decreased heart copper content and cardiac hypertrophy in the mice fed the high-iron diet with low and normal copper, but again, the higher copper intake corrected this defect. Moreover, hepatic copper content was lower and serum ceruloplasmin activity was depressed in the high-iron fed mice with the low and normal copper content, both of these parameters were, however, restored to control levels in the high iron, high copper group. In sum, these findings implied to us that the high iron content in the diets with low and normal copper caused severe copper-deficiency anemia, but the higher copper content corrected the copper deficiency. We further postulated that the high iron content blocked intestinal copper absorption, causing systemic copper deficiency. We thus tested this hypothesis by performing in vivo intestinal copper absorption studies. Weanling mice were again fed the same diets for 4 weeks, fasted overnight and 64Cu was then administered to the mice by oral gavage. Mice were immediately given ad libitum access to the same diets and then sacrificed ~8 hours later. This time point ensured that no copper was excreted in the feces since intestinal transit time in mice is ~11 hours. This is important since copper can be excreted in the bile via the feces. Copper absorption was calculated as (cpms in the carcass [minus the GI tract]/ total cpms administered by gavage) x 100. Again, to our surprise, all mice accumulated the same amount of copper, demonstrating that copper absorption was NOT impaired. However, 64Cu accumulation in liver and blood was extremely low in all mice fed the high- iron diets. These data then suggest that high systemic iron levels alter copper distribution, thus potentially contributing to the pathology. Future experiments will examine this issue in genetic models of iron overload.

Sixth Congress of the International BioIRon Society Page 298 Podium Abstracts IBIS

Poster #152 PEROXISOME PROLIFERATOR-ACTIATED RECEPTOR ALPHA PPARα CONTRIBUTES TO HEPCIDIN UP- REGULATION IN MICE DURING FASTING Yihang Li, PhD, Princy Prasad, MS, Ian Miller, BSc, Cedric Langhi, PhD, Ángel Baldán, PhD and Robert Fleming, MD Saint Louis University

Nutritional deprivation has been reported to be associated with iron deficiency; however iron parameters of nutritional deprivation differ from that of dietary iron deficiency alone. In this study, 6 week old male AKR mice (N = 6) were fed diet with 60 ppm Fe ad libitum and fasted for overnight (18 hours). Tissue and blood samples were collected to evaluate iron parameters and gene expression in comparison to fed controls. All mice were provided water ad libitum. In the fasted mice, serum iron (321 and 199 µg/dL, respectively for fed and fasted mice, P = 0.001) as well as transferrin saturation (80% and 64% respectively for fed and fasted mice, P = 0.04) decreased significantly. Fasted mice also had elevated hematocrit (43% and 47%, respectively, for the fed and fasted mice, P = 0.001) in association with weight loss (22g and 18g, respectively, for the fed and fasted mice, P < 0.01), consistent with hemoconcentration. In addition, 18h fasting induced a significant increase in hepatic (735 and 953 µg/g dry, respectively for the fed and fasted mice, P < 0.05) and splenic non-heme iron content (414 and 488 µg/g dry, respectively for the fed and fasted mice, P < 0.001), and elevated hepatic hamp1 mRNA levels (4-fold increase from the fed group, P < 0.001). To elucidate the mechanism of the Hamp1 up-regulation, we measured liver creb-h and pparα levels, and observed elevated creb-h (by 4-fold, P < 0.001) and ppara mRNA levels (by 3-fold, P < 0.001) in the fasted mice. To further elucidate the roles of CREB-, ad α the regulation of Hamp1 during fasting, we performed fasting-refeeding studies in 10-12 week old male C57BL/6 mice. These mice (N = 4-6) were fed with chow ad libitum, fasted overnight, or fasted overnight then re-fed for 4 hours. Fasting at this age also induced an increase in liver pparα, creb-h, and hamp1 mRNA levels (by 2.5-, 3- and 6-fold, respectively), and refeeding over this timeframe was associated with decreases in pparα, creb-h, and hamp1 to levels that are similar to fed mice, even though the serum and tissue iron levels were not restored. To determine if Pα otrtes to the relato o hepd, e eaed the ast respose o α-/- mice. The fasting-associated increases in expression of hamp1 and creb-h ere dapeed the α-/- mice. In the WT mice, fasting induced a hamp1 increase of 2.6-fold (P , hereas the α-/- mice, the increase was only 0.8-fold (P = 0.11). Similarly, fasting induced a creb-h increase of 2.8-fold in the WT mice (P = 0.0003) but a 1.1-old rease the α-/- mice (P = 0.004). The differences in magnitude of change in hamp1 (P < 0.05) and creb-h (P epresso etee ad α-/- mice were statstally sat hese ds date that α otrtes to re-h and Hamp1 up-regulation during fasting- induced changes in iron metabolism.

Sixth Congress of the International BioIRon Society Page 299 Podium Abstracts IBIS

Poster #153 EFFECTS OF DIFFERENTIATION ON REGULATION OF INTESTINAL IRON ABSORPTION: CELL AND MOLECULAR MECHANISMS Gordon D. McLaren1, Khushin Patel2, Hyder Said2, Gregory J. Anderson3 and Hamid M. Said1 1Department of Veterans Affairs Long Beach Healthcare System, Long Beach, CA, and Department of Medicine, University of California, Irvine, CA; 2Department of Veterans Affairs Long Beach Healthcare System, Long Beach, CA; 3QIMR Berghofer Medical Research Institute, Brisbane, Australia, and Faculty of Medicine and Biomedical Sciences, The University of Queensland, Brisbane, Australia

Introduction: Differentiation of intestinal epithelial cells is associated with up- and down-regulation of the expression of a variety of genes, but little is known about the differentiation-dependent regulation of genes encoding proteins involved in intestinal iron absorption. We have addressed this issue using human-derived intestinal epithelial Caco-2 cells and crypt/villus epithelial cells isolated from wild-type mice as models. Methods: We examined expression of divalent metal-ion transporter-1 (DMT-1; brush border iron importer), ferroportin (FPN; basolateral iron exporter) and hephaestin (HEPH; basolateral iron oxidase). Caco-2 cells were grown in EMEM medium and harvested at pre-confluent, confluent and post-confluent (5 days after confluence) stages. mRNA expression was measured by real-te ad oraled th -actin. Protein expression was examined by Western Blot analysis using specific polyclonal antibodes, oraled th -actin. Promoter activity was investigated by Dual-Glow Luciferase assay using Renilla and Luciferase activity. Results: Differentiation of Caco-2 cells was associated with a significant up-regulation of iron uptake. This up-regulation was associated with a significant increase in the expression of DMT-1 and HEPH mRNA and protein, as well as increased expression of FPN mRNA, but not FPN protein. Similarly, iron uptake and the expression of DMT-1, HEPH, and FPN mRNAs was higher in mouse villus cells than crypt cells. Promoter-reporter constructs were used to show that the activity of the promoters of DMT-1 and FPN increased with differentiation of Caco-2 cells. Conclusions: These studies demonstrate that differentiation of intestinal epithelial cells is associated with up-regulation of the iron uptake process and increased expression of DMT-1, HEPH, and FPN. The expression of all these proteins of iron transport can be mediated at the post-transcriptional level according to intracellular iron availability, but our results indicate that up-regulation of DMT-1 and FPN during differentiation appears to be mediated at least in part via transcriptional regulatory mechanisms.

Sixth Congress of the International BioIRon Society Page 300 Podium Abstracts IBIS

Poster #154 COMPARATIVE STUDY OF THE BIOAVAILABILITY OF DIFFERENT IRON SUPPLEMENTS TO BACTERIA Darlene Dagos, MSc2, Priya Selvam, MSc, Patrick Kelly, BSc, Robert Evans, PhD1, Mohammed Gulrez Zariwala, PhD, Derek Renshaw, PhD and Sebastien Farnaud, PhD 1Brunel University; 2Life Sciences Dept. University of Bedfordshire

Iron deficiency is a major health issue worldwide. It is thought that 1.6 billion people, or approximately 25% of the population, are suffering from anaemia, 50% of which in developing countries is thought to be due to iron-deficiency. Over the last 50 years, the search for an effective iron supplement has intensified, but the problem of efficient uptake remains a major issue. Unfortunately, efficiency of iron uptake is not the only issue; a major problem faced by the scientific community is the fact that infectious agents do take advantage of iron supplement. Iron depravation is one of the first lines of defence for many organisms; for example, following infection by microorganisms, mammalian organisms, including human, respond by sequestering iron to reduce availability to the invading agents and therefore their growth, providing opportunity for other defence mechanisms to act. Unfortunately, in such instance iron availability is also reduced for the host often leading to iron deficiency and even anaemia if iron stores are insufficient. Preventing iron uptake from microorganisms is therefore to be considered when designing an iron supplement. This project is a comparative study of the bioavailability to bacteria of several iron supplements. Several models to assess bioavailability were compared by monitoring bacterial growth in different media, under different conditions, using different iron supplements. Bacterial growth and biofilms formations were monitored and compared, and the effects of different iron sources were determined, to potentially define the most suitable iron supplement.

Sixth Congress of the International BioIRon Society Page 301 Podium Abstracts IBIS

Poster #155 WHAT COULD INSULIN TEACH US ABOUT HEPCIDIN? A COMPARATIVE STUDY BETWEEN THE IRON HORMONE HEPCIDIN AND INSULIN Kosha Metha, PhD2, Mohammed Gulrez Zariwala, PhD, Robert Evans, PhD1, Derek Renshaw, PhD and Sebastien Farnaud, PhD 1Brunel University; 2University of Berdfordshire

Hepcidin was originally isolated as a 25-amino acid antimicrobial peptide in human plasma and urine, but was later on localised mainly in the liver where it was named the iron-hormone, although the endocrinological aspect of hepcidin is often omitted. Animal and cell culture studies have demonstrated that hepcidin could act as a negative regulator of iron absorption. Several studies have shown the link between iron and glucose metabolism, which in turn affects iron metabolic pathways. In addition, body iron stores have been confirmed to be positively correlated with serum insulin and blood glucose concentrations. Other studies have shown that hepcidin was expressed in the pancreas of rat and man, where histo-heal stdes loalsed the peptde elsely -cells of the islets of Langerhans. The localisation of ths peptde sested a l etee pareat -cells and iron metabolism in addition to their main function in blood glucose regulation. To further investigate the parallels between glucose and iron metabolisms, the present study provides a comparison between both hormones, Insulin and hepcidin, which strengthens the endocrinological identity of Hepcidin.

Sixth Congress of the International BioIRon Society Page 302 Poster Abstracts IBIS

Poster #156 LEAD NEUROTOXICITY DERIVES IN PART FROM PERTURBED FE/ IRE/ IRP REGULATION IN MODELS OF HUMAN NEURONS Catherine Cahill, PhD2, Hong Jiang, MD, PhD3, Xudong Huang, PhD2, Jack Rogers, PhD1 and Ann Smith, PhD4 1MGH. Harvard; 2MGH/Harvard; 3Univ. of Qingdao; 4Univ. Missouri, Kansas City

Iron supplementation is known to ameliorate the toxicity of Lead (Pb), a common environmental contaminant but the mechanism is unknown and remains to be defined. Although an essential nutrient, excessively high levels of iron can be toxic due to the catalytic generation of destructive hydroxyl radicals. We hypothesized that Pb disrupts iron homeostasis by competing for iron binding sites. We report that Pb disrupts the translational regulation of proteins via Fe/ IRE/ IRP that control intracellular iron homeostasis, including the iron storage protein, ferritin, and the Alzheimer’s amyloid precursor protein (APP) that stabilizes the iron exporter, ferroportin. Our data support that both Pb-II and Pb-IV prevent the protective induction of H-ferritin and APP protein in human neuroblastoma cells thus leading to elevated levels of cytosolic iron causing toxicity. Pb increased IRP1 binding to the cognate APP and H-ferritin IREs. Iron did not rescue cells from this inhibition of translation by lead. In conclusion, our data support that Pb generated a neurotoxicity that coincided with enhanced IRP/IRE-mediated repression of APP and ferritin translation in models of human neurons.

Sixth Congress of the International BioIRon Society Page 303 Poster Abstracts IBIS

Poster #157 CLINICAL FACTORS AFFECTING NTBI LEVELS: INSIGHTS FROM DIFFERENCES BETWEEN A NOVEL FLUORESCENT BEAD METHOD AND THE ESTABLISHED NTA METHOD Maciej Garbowski, MD, Yongmin Ma, PhD, Patricia Evans, PhD, Somdet Srichairatanakool, PhD, Suthat Fucharoen, MD, Robert Hider, PhD and John Porter, MD UCL

Plasma NTBI, a heterogeneously speciated iron pool, detectable across iron overload diagnoses typically with transferrin saturation (TfSat)>75%, is implicated in haemosiderotic organ complications. NTBI levels were measured in a diverse group of iron-overloaded patients (n=133: 85 thalassaemia, 7 SCD, 9 haemochromatosis, 3 MDS, 18 rare anaemias, 11 normal) and compared using the well-established nitrilotriacetate (NTA) assay, and a novel flow-cytometric assay based on bead-linked fluorescent chelator (CP851). In the NTA method, 80mM NTA scavenges NTBI species before ultrafiltration and subsequently donates it to a stronger detecting chelator. This leads to artefacts, either from NTA-bound iron being donated to apotransferrin, or from ferrotransferrin iron being removed by NTA. These problems are in principle circumvented using the CP851 assay, where direct iron scavenging by the fluorescent chelator CP851 causes fluorescence quenching which is detected by flow- cytometry. Thus the potential of iron-to-apotransferrin shuttling that occurs with the NTA method is here abrogated. This method may also in principle be able to access NTBI species unavailable to the NTA method. We have compared which factors affect NTBI levels in different patient subgroups between the two methods in order to draw insights about NTBI speciation and detection in different conditions. We have compared univariate regression slopes of both methods in patient subgroups and modelled NTA-NTBI, CP851-NTBI, their bias, and transferrin saturation (by urea gel electrophoresis), using multiple regression to compare predictors across models. Whilst the methods show poor agreement by Bland-Altman analysis both irrespective of diagnosis and within diagnoses, subgroup analyses provide some insight into factors affecting agreement. In transfused patients, the CP851 method detects 3-5 times more NTBI than the NTA method, in non-transfused patients it detects NTBI where that is absent by the NTA method. Clinical variables associated with disparity between the two methods include: splenectomy status, degree of erythropoiesis (based on levels of soluble transferrin receptors sTfR>ULN), and high levels of iron overload (serum ferritin>1500ug/L). There were significant slope differences between groups defined by the above four parameters. These differences can at least partially be explained by differences in TfSat between the respective groups (see figure). On multiple regression analysis, NTBI levels using the NTA method are most positively predicted by TfSat and splenectomy, and negatively predicted by sTfR. By contrast, with the CP851 method, NTBI levels are predicted much less so by TfSat, but also by splenectomy and chelation status. Bias between both measures is predicted negatively by TfSat, positively by splenectomy, chelator status and normal status (lack of iron overload diagnosis), while transferrin saturation by ferritin level and presence of iron overload diagnosis. Increased NTBI values with the CP851 method were used to identify paired NTA values as either false-positive or false-negative (according to iron removal from or donation to transferrin by NTA, respectively) for which clinical evidence of matching TfSat is provided in the figure. The two methods appear to be identifying different NTBI pools in different patient groups. We suggest that these methods initially could be used together to help identify which forms of raised NTBI are most closely associated with iron mal-distribution under a range of clinical diagnoses.

Sixth Congress of the International BioIRon Society Page 304 Poster Abstracts IBIS

Poster #158 DIETARY IRON AND GENETIC VARIATION ALTER METALLOSTASIS IN MICE Kathryn E. Page, PhD2, Brie K. Fuqua, PhD1, Stela McLachlan, PhD3, Hiro Irimagawa2, Yuanchi He2, David W. Killilea, PhD4, Brian W. Parks, PhD5, Simon T. Hui, PhD5, Eleazar Eskin, PhD6, Aldons J. Lusis, PhD5 and Chris D. Vulpe, MD, PhD7 1Department of Physiological Sciences, University of Florida, Gainesville, FL and Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA; 2Department of Nutritional Sciences & Toxicology, University of California, Berkeley, CA; 3Centre for Population Health Sciences, The University of Edinburgh, UK; 4Nutrition and Metabolism Center, Children's Hospital Oakland Research Institute, Oakland, CA; 5Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA; 6Department of Computer Science & Human Genetics, University of California, Los Angeles, CA; 7Department of Nutritional Sciences & Toxicology, University of California, Berkeley, CA and Department of Physiological Sciences, University of Florida, Gainesville, FL

Introduction: The great diversity in genetic disorders of iron metabolism in man, rodents, and other vertebrates suggests that multiple genes can contribute to the susceptibility to iron deficiency and to the severity of iron overload. Several studies have shown differences in iron homeostasis between inbred strains of mice. However, the variation in genetically attributed iron phenotypes cannot be fully explained by the set of genes currently known to be involved in iron metabolism. Association studies in humans and mice have identified new genomic loci associated with iron metabolism; however, causal genes in these loci are not known. To achieve increased resolution of the associated loci, we are collecting iron- related phenotype data from 110 different mouse strains that make up the Hybrid Mouse Diversity Panel (HMDP). We will associate genetic variants with iron phenotypes to identify novel genetic loci and identify causal genes involved in iron metabolism. Here we report preliminary phenotype data from a subset of strains from the HMDP panel. Methods: Male mice from six inbred mouse strains (A/J, AKR/J, BALB/cJ, C57BL/6J, C3H/HeJ, DBA/2J) were fed low (5 ppm), high (20000ppm), and sufficient (50ppm) iron diets for six weeks starting at 4 weeks of age. We assessed the levels of iron, calcium, copper, magnesium, manganese, and zinc in the heart, kidney, liver, pancreas, and spleen from each mouse using inductively coupled plasma atomic emission spectroscopy. The metal content was expressed as micrograms per gram dry weight of tissue. Means values for all metal-tissue-diet combinations for each mouse strain were compared by one-way ANOVA. Where a significant departure from homogeneity of variance was present we report Welch’s F. Results: In all six strains, most tissue iron levels, especially in the liver, increased with increasing dietary iron content, as expected. On the high iron diet, significant variation in tissue iron levels was observed between at least two mouse strains for all tissues examined. On the low and iron sufficient diets, variation between strains reached significance in the heart, liver, and spleen. The differences in the degree of change in iron levels between different tissues from mice on the different iron diets show how tissues respond differently to changes in dietary iron. The variation in other elemental profiles indicates multiple metal homeostasis differences between the strains. Conclusions: This study highlights the population diversity in the ability to handle iron stress (deficiency or overload), emphasizes the role of iron regulation in metallostasis, and indicates that genetic variation in this population plays an important role in maintaining metal homeostasis. The significant phenotype variation between these strains shows promise for the genetic mapping of these traits.

Sixth Congress of the International BioIRon Society Page 305 Poster Abstracts IBIS

Poster #159 SECOND INTERNATIONAL ROUND ROBIN FOR THE QUANTIFICATION OF SERUM NON-TRANSFERRIN-BOUND IRON AND LABILE PLASMA IRON IN PATIENTS WITH IRON-OVERLOAD DISORDERS Louise de Swart2, Jan Hendriks3, Lisa van der Vorm4, Ioav Cabantchik5, Patricia Evans6, Eldad Hod7, Gary Brittenham8, Yael Furman9, Boguslaw Wojczyk10, Mirian Janssen11, John Porter6, Vera Mattijssen12, Bart Biemond13, Marius MacKenzie2, Raffaella Origa14, Renzo Galanello14, Robert Hider15 and Dorine Swinkels, MD, PhD1 1Radboud University Medical Centre Nijmegen; 2Department of Hematology, Radboud University Medical Center, Nijmegen, the Netherlands; 3Department of Health Evidence, Radboud University Medical Center, Nijmegen, the Netherlands; 4Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, the Netherlands; 5Department of Biochemical Chemistry, Hebrew University of Jerusalem, Israel; 6Department of Haematology, University College London, UK; 7Department of Pathology and Cell Biology, Columbia University Medical Center, New York, USA; 8Department of Pediatrics, Columbia University Medical Center, New York, USA; 9Aferrix Ltd., Tel-Aviv, Israel; 10Department of Pathology and Cell Biology, Columbia University Medical Center, New York; 11Department of Internal Medicine, Radboud University Medical Center, Nijmegen, the Netherlands; 12Department of Hematology, Rijnstate Hospital, Arnhem, The Netherlands; 13Department of Hematology, Academic Medical Center, Amsterdam, The Netherlands; 14Regional Microcythemia Hospital, University of Cagliari, department of Biomedical Science and Biotechnology, Cagliari, Italy; 15Institute of Pharmaceutical Science, King’s College London, UK

Background: Non-transferrin bound iron (NTBI) and its labile (redox active) plasma iron component (LPI) are thought to be potentially toxic forms of iron originally identified in the serum of patients with iron overload. Methods: We compared 10 worldwide leading assays (6 NTBI and 4 LPI) as part of an international inter-laboratory study. Serum samples from 60 patients with 4 different iron-overload disorders in various treatment phases were coded and sent in duplicate for analysis to 5 different laboratories worldwide. Some laboratories provided multiple assays. Results: Overall, hhest assay leels ere osered or patets th treated heredtary heohroatoss ad - thalassemia intermedia, patients with transfusion-dependent (TD) myelodysplastic syndromes and patients with TD and helated -thalassemia major. Absolute levels differed considerably between assays and were lower for LPI than for NTBI. Four assays also reported negative values. Assays were reproducible with high between-sample and low within- sample variation. Assays correlate and correlations were highest within the same group of NTBI or LPI assays. Increased transferrin saturation, but not ferritin, was a good indicator for the presence of circulating NTBI iron forms. Conclusions: Although NTBI and LPI values of various assays are well correlated, the absolute values differed considerably between assays. However, the possibility of using NTBI and LPI measures as clinical indicators of overt iron overload and/or of treatment efficacy would largely depend on the rigorous validation and standardization of each assay vis-a-vis clinical outcomes, of both short and long term nature.

*This work is dedicated to the memory and in honor of Renzo Galanello, who was instrumental for the study until the end of his career.

Sixth Congress of the International BioIRon Society Page 306 Poster Abstracts IBIS

Poster #160 IRON REFRACTORY IRON DEFICIENCY ANEMIA (IRIDA) CASE SERIES IN THE NETHERLANDS: A HETEROGENEOUS DISEASE Albertine Donker, MD2, Paul Brons, MD, PhD2, Dirk Bakkeren, PhD3, Michiel van Gelder, MD, PhD4, Bernd Granzen, MD, PhD4, Mirian Janssen, MD, PhD2, Alexander Rennings, MD, PhD2, Anita Rijneveld, MD, PhD5, Charlotte Schaap, MD2, Frank van de Veerdonk, MD, PhD2, Andre Vlot, MD, PhD6, Marten Nijziel, MD, PhD3, Thom Vlasveld, MD, PhD7, Vera Novotny, MD, PhD2 and Dorine Swinkels, MD, PhD1 1RadboudUMC; 2RadboudUMC, Nijmegen, Netherlands; 3Maxima Medical Centre, Veldhoven, Netherlands; 4Academic Medical Centre Maastricht, Maastricht, Netherlands; 5ErasmusMC, Rotterdam, Netherlands; 6Alysis Hospital, Arnhem, Netherlands; 7Bronovo Hospital, The Hague, Netherlands

Pathogenic TMPRSS6 mutations result in impaired matriptase-2 function and consequently uninhibited hepcidin production, leading to Iron Refractory Iron Deficiency Anemia (IRIDA). This disease is characterized by microcytic, hypochromic anemia and serum hepcidin values that are inappropriately high for body iron levels. We describe a series of 21 phenotypically affected IRIDA patients in 20 families living in the Netherlands of which six were from Turkish and Moroccan descent. Although IRIDA is considered an autosomal recessive disease, in 8 out of these 21 cases only a heterozygous TMPRSS6 defect was demonstrated. Multiplex Ligation dependent Probe Amplification confirmed heterozygosity. Fifteen different pathogenic TMPRSS6 defects were found of which 8 are novel. Age of presentation, disease severity and response to iron supplementation were highly variable even for patients with similar TMPRSS6 genotypes. IRIDA patients diagnosed with a heterozygous TMRPSS6 mutation had a milder phenotype with respect to hemoglobin and MCV and presented significantly later in life with anemia. TSAT/hepcidin was significantly lower for bi-allelic affected IRIDA patients than for heterozygous IRIDA patients (median 0.50 %/nM, range 0.13-1.0 %/nM vs median 1.1, range 0.30-2.7%/nM, unpaired t-test p=0.013). In addition, heterozygous IRIDA patients had lower ratios than their heterozygous and wildtype relatives without an IRIDA phenotype (median 11%/nM, range 3.1-38%/nM, unpaired t-test p= 0.006). We conclude that IRIDA is a genotypically and phenotypically heterogeneous disease. We suggest a co-dominant rather than an autosomal recessive mode of inheritance. In case of absence of inflammation the TSAT/hepcidin ratio is a sensitive and specific diagnostic tool, even after iron supplementation has been given. The relatively high proportion of subjects descending from thalassemia endemic regions suggests that TMPRSS6 defects might give a survival advantage in these populations.

Sixth Congress of the International BioIRon Society Page 307 Poster Abstracts IBIS

Poster #161 IRON, CHOLESTEROL AND MIR122: DEMONSTRATION OF THE PRESENCE OF THE “DELETED” MIR122 GENE IN HEPG2 CELLS Ibrahim Hamad, MSc1, Yue Fei, MSc1, Anastasia Kalea, PhD1, Dan Yin, PhD2, Andrew Smith, PhD1, Jutta Palmen, MSc1, Steve Humphries, PhD1, Philippa Talmud, DSc1 and Ann Walker, PhD1 1UCL, London; 2Wuhan, China/UCL,London

MicroRNA 122 (miR-122) is highly expressed in the liver where it influences diverse biological processes and pathways, including hepatitis C virus replication and metabolism of iron and cholesterol. It is processed from a long non-coding primary transcript (~7.5 kb) and the gene has two evolutionarily-conserved regions containing the pri-mir-122 promoter and premir-122 hairpin region. Several groups reported that the widely-used hepatocytic cell line HepG2 had deficient expression of miR-122, previously ascribed to deletion of the pre-mir- 122 stem-loop region. We aimed to characterise this deletion by direct sequencing of 6078 bp containing the pri-mir-122 promoter and pre-mir-122 stem-loop region in HepG2 and Huh-7, a control hepatocytic cell line reported to express miR-122, supported by sequence analysis of cloned genomic DNA. In contrast to previous findings, the entire sequence was present in both cell lines. Ten SNPs were heterozygous in HepG2 indicating that DNA was present in two copies. Three validation isolates of HepG2 were sequenced, showing identical genotype to the original in two, whereas the third was different. The promoter chromatin status was investigated by Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE). Three biological replicates were assayed, each with 4 technical replicates). This showed that Huh-7 cells had (mean ± SEM) 6.2 ± 0.19- and 2.7 ± 0.01- old ore aessle hroat at the proal α-binding) and distal DR1 transcription factor sites, compared to HepG2 cells (p=0.03 and 0.001, respectively). This was substantiated by ENCODE genome annotations, which showed a DNAse I hypersensitive site in the pri-mir-122 promoter in Huh-7 that was absent in HepG2 cells. While the origin of the reported deletion is unclear, cell lines should be obtained from a reputable source and used at low passage number to avoid discrepant results. Deficiency of miR-122 expression in HepG2 cells may be related to relative deficiency of accessible promoter chromatin in HepG2 versus Huh-7 cells.

Sixth Congress of the International BioIRon Society Page 308 Poster Abstracts IBIS

Poster #162 A NOVEL PROTEIN INVOLVED IN IRON TRANSPORT IN DROSOPHILA MELANOGASTER Xudong Wang1 and Bing Zhou2 1School of Life Sciences, Tsinghua University; 2School of Life Cciences,Tsinghua University

Iron is an indispensible micronutrient critical for the development of almost all organisms. Dysfunction of iron homeostasis can cause severe anemia or hemochromatosis in human. In recent years, the mechanism of iron homeostasis in mammals and yeast have been intensively studied, but as a powerful model organism, the fruit fly Drosophila melanogaster is not sufficiently used in the iron study and how iron is utilized in the fly is poorly understood. In mammals, Ferroportin is the only known transmembrane protein transporting ferrous iron out of the duodenum enterocyte, in collaboration with Hepheastin. However, whether there exists a fly transmembrane protein involved in a similar function in intestinal iron absorption remains unknown. In the fruit fly, it is known that a major part of iron is exported via ferritin through the secretory pathway. Here, using various approaches including yeast two hybrid, bioinformatics and genetics, we identified a novel protein in the gut of the fly that can mediate iron efflux.

Sixth Congress of the International BioIRon Society Page 309 Poster Abstracts IBIS

Poster #163 MANGANESE NEUROTOXICITY DERIVES IN PART FROM PERTURBED FE/ IRE/ IRP REGULATION IN RATS AND MODELS OF HUMAN NEURONS Vivek Venkataramani, MD2, Yanyan Liu, PhD3, Catherine Cahill, PhD3, Katharina Fernsebner, PhD4, Bernhard Michalke, PhD4, Hong Jiang, MD5, Scott Ayton, PhD6, Ashley Bush, MD6, Xudong Huang, PhD3 and Jack Rogers, PhD1 1MGH/Harvard; 2University of Goettingen; 3MGH/ Harvard; 4Munich, Germany; 5Qingdao, China; 6Melbourne, Australia

Iron supplementation is known to ameliorate the toxiciy of mangansee (Mn), a common environmental contaminant but the mechanism is unknown and remains to be defined. Although an essential nutrient, excessively high levels of iron can be toxic due to the catalytic generation of destructive hydroxyl radicals. We hypothesized that Mn disrupts iron homeostasis by competing for iron binding sites. We report that Mn disrupts the translational regulation of proteins via Fe/ IRE/ IRP that control intracellular iron homeostasis, including the iron storage protein, ferritin, and the Alzheimer’s amyloid precursor protein (APP) that stabilizes the iron exporter, ferroportin. Our data support that Mn prevents the protective induction of H-ferritin and APP protein in human neuroblastoma cells thus leading to elevated levels of cytosolic iron causing toxicity. Mn increased IRP1 binding to the cognate APP and H-ferritin IREs. Iron did not rescue cells from this inhibition of translation by Mn. In manganese treated rats we observed reduction of APP levels while the Fe2+/ Fe3+ ratio shifted towards increased neurotoxicity. REDOX active oxidative intermediates were reduced with a concomitant increase in cell's viability after Mn treatement of cell lines when APP was over-expressed. In conclusion, our data support that Mn generated a neurotoxicity that coincided with enhanced IRP/IRE-mediated repression of APP and ferritin translation in models of human neurons.

Sixth Congress of the International BioIRon Society Page 310 Poster Abstracts IBIS

Poster #164 SCREENING FOR TRANSITION METAL TRANSPORTING FUNCTIONS OF DROSOPHILA ZIPS AND ZNTS Sai Yin, PhD and Bing Zhou, Prof Tsinghua University

The way in which Drosophila melanogaster absorbs iron from the diet remains poorly understood despite the significance of iron for larval growth. As previously reported, Drosophila Zip13, a presumed zinc importer, participates in intracellular iron transfer process as an iron transporter. We wonder whether there are any other Zips or ZnTs having transporting activity for metals other than zinc, in particular iron. We performed a quick screen for metal transporting functions of Zips or ZnTs, using gut-specific gal4 to overexpress these zinc transporters in the fly to test their metal sensitivity to zinc, iron, copper and manganese. We have already tested several plasma-membrane-localized Zips and ZnTs, and will update our progress in this work.

Sixth Congress of the International BioIRon Society Page 311 Poster Abstracts IBIS

Poster #165 WHAT MAKES A PRESUMED ZINC TRANSPORTER, SLC39A13, EXPORT IRON? Guiran Xiao, PhD, Mengran Zhao, PhD and Bing Zhou, Prof Tsinghua University

Our previous work report that Drosophila ZIP13 (Slc39a13), which is a presumed zinc importer, fulfills the iron effluxing role: it mediates iron export to the secretory pathway. This is the first time to report that a member of the ZIP transporter family functions as an iron exporter. Almost all other members of the family (SLC39A) so far identified appear to be importers, it would be interesting to know what sequences or structures endow dZIP13 with this unique property. Amino acid sequence comparisons of ZIP13 with other ZIP family members reveal some distinct differences. The most notable difference is in transmembrane domain TM4. The TM4 amino acid sequence for ZIP13s from different organisms are all DNFTHG whereas other closely related ZIPs are HNFTDG, i.e., the highly conserved region in TM4 has a D and H residue swap between ZIP13s and other ZIP members. In this study, we explore this question and found that this variation of ZIP13 might be a key factor for the iron transporting property of ZIP13.

Sixth Congress of the International BioIRon Society Page 312 Poster Abstracts IBIS

Poster #166 REGULATION OF HEPCIDIN AND GDF15 IN ANEMIC PATIENTS WITH TYPE 2 DIABETES WITHOUT OVERT RENAL IMPAIRMENT Jun Hwa Hong, PhD2, Yeon-Kyung Choi, PhD2, Byong-Keol Min1, Kang Seo Park, PhD3, Kayeon Seong, PhD4, Byoungjun Choi1, In Kyu Lee, PhD2 and Jung Guk Kim, PhD2 1Department of Biomedical Science, Graduate School, Kyungpook National University, Daegu, South Korea; 2Department of Internal Medicine, Kyungpook National University School of Medicine, Daegu, South Korea; 3Department of Internal Medicine, Eulji University School of Medicine, Daejeon, South Korea; 4College of Nursing, Taegu Science University, Daegu, South Korea

Aims. Patients are usually anemic in spite of decreased levels of erythropoietin (EPO) without overt renal impairment. Hepcidin, which is induced by inflammatory stimuli, is a critical factor in anemia. The levels of Growth differentiation factor 15(GDF 15), a putative anti-inflammatory cytokine, are elevated in type 2 diabetes (T2DM). Therefore, we studied the relationship between hepcidin and GDF15 in anemic T2DM patients without overt renal impairment. Methods. Among 1150 patients who were diagnosed their illness having T2DM from june 2006 to june 2014 in Kyungpook National University Hospital, we selected 55 anemic patients without overt renal impairment (serum creatinine < 1.5 mg/dL or estimated glomerular filtration rate > 60mL/min/1.73m2) and other severe disease, including malignancy, thyroid disease, rheumatic arthritis, liver disease, iron-deficiency anemia and other endocrine disease. We measured anthropometric and metabolic parameters. The serum iron, ferritin, interleukin-6(IL-6), erythropoietin, hepcidin-25 and GDF15 levels were measured as well. Results. Anemic T2DM patients without overt renal impairment showed a greater inflammatory state compared with non-anemic T2DM patients with increased serum hsCRP, ESR and IL-6 levels. Both hepcidin and GDF15 levels were increased that indicates a positive correlation in anemic T2DM patients. Conclusion. Without overt renal impairment, anemia in T2DM is associated with chronic inflammation, inducing elevated levels of hepcidin and GDF 15 regardless of the erythropoietin level.

Sixth Congress of the International BioIRon Society Page 313 Poster Abstracts IBIS

Poster #167 ALR PROTEIN, A CRITICAL PROTEIN IN CARDIAC DEVELOPMENT, REGULATES CELLULAR IRON HOMEOSTASIS AND MATURATION OF CYTOSOLIC FE/S PROTEINS BY PARTICIPATING IN MITOCHONDRIAL TRANSPORT OF ATP-BINDING CASSETTE (ABC)-B8 Jimmy Chnag, PhD and Hossein Ardehali, MD, PhD Northwestern University

Introduction: Disruption of Fe/S cluster maturation can lead to cellular iron accumulation and oxidative stress, as seen in the cardiomyopathy associated with Friedreich’s ataxia. Mutation of Augmenter of Liver Regeneration (ALR), a mitochondrial inter-membrane-space protein involved in mitochondrial protein import, is linked to increased oxidative stress. Yeast with deletion of Erv1, the yeast homolog of ALR, have cytosolic Fe/S cluster maturation defects and iron accumulation, but the role of ALR in iron homeostasis and Fe/S cluster maturation is unclear. ABCB8 is one of only two mitochondrial membrane proteins known to regulate cytosolic Fe/S cluster maturation. We hypothesized that ALR is critical for cytosolic Fe/S cluster maturation and iron homeostasis by regulating mitochondrial import of ABCB8. Results: Downregulation of ALR in vitro resulted in reduced cytosolic Fe/S cluster-containing enzyme activities and increased cellular iron uptake. Since ALR has mitochondrial- and cytosolic-specific isoforms, we performed knockdown- rescue studies of each isoform, and demonstrated that only the mitochondrial ALR is needed for the maturation of cytosolic Fe/S clusters. Because Fe/S clusters are synthesized in the mitochondria, we then assessed whether ALR can alter the levels or activity of ABCB7 and ABCB8, the two mitochondrial membrane proteins known to regulate the maturation of cytosolic Fe/S cluster proteins. Downregulation of ALR reduced the mitochondrial levels of ABCB8, while ABCB7 levels were not affected. We also identified defects in mitochondrial transport of ABCB8 as the mechanism for reduced mitochondrial ABCB8 levels with ALR knockdown. As ALR is involved in mitochondrial protein transport through its interaction with Mia40, we also demonstrated that knockdown of Mia40 recapitulated the ABCB8 mitochondrial transport defect seen in ALR downregulation and showed that Mia40 phsycially interacts with ABCB8. Conclusion: Our results indicate that ALR and its interaction partner Mia40 are involved in the transport of ABCB8 into the mitochondria, which in turn regulates cytoplasmic Fe/S cluster maturation. These findings provide insights into cellular iron regulation, with implications in cardiovascular disease.

Sixth Congress of the International BioIRon Society Page 314 Poster Abstracts IBIS

Poster #168 EFFECT OF IRON ON DROSOPHILA HEMOLYMPH PROTEOME Mengran Zhao1, Guiran Xiao, PhD2 and Bing Zhou, PI 1Tsinghua university; 2School of Life Sciences, Tsinghua University

Iron is an essential micronutrient involved in numerous biological processes. Cellular and systemic iron homeostasis are kept under tightly control to ensure nutritional availability while limiting the toxicity of excessive iron. But the mechanism of systemic iron regulation and transportation in Drosophila has not been elucidated. In this study, hemolymph was isolated from Drosophila larvae reared on standard cornmeal-agar medium or iron supplied/deficient food and performed a proteomic comparison by LC-MS/MS. Our SDS-PAGE analysis of protein map of Drosophila hemolymph presented some visual differences between the three batch. In addition, Proteomics analysis using LC-MS/MS allows the identification and isolation of novel candidate responding to systemic iron level. The latest processing will be updated in my poster.

Sixth Congress of the International BioIRon Society Page 315 Poster Abstracts IBIS

Poster #169 IRON-DEPENDENT EPIGENETIC REGULATION BY THE HISTONE DEMETHYLASE KDM4A IN THE HEART Yu Jin Chung, Magda Wolna, Peter Robbins, DPhil and Samira Lakhal-Littleton, DPhil University of Oxford

Epigenetic control of gene expression is an important mechanism of cellular response to physiological stimuli. In the context of the heart, epigenetic changes mediated by histone demethylases such as lysine demethylase 4A (KDM4A) have been implicated in cardiac hypertrophy in both humans and mice. KDM4A is a member of a novel class of epigenetic regulators that require iron for activity, but it remains unknown whether iron affects gene expression by modulating KDM4A enzymatic function. The aim of our study is to determine the changes in KDM4A activity and downstream epigenetic effects in response to changes in cardiac iron levels. To achieve this, we probed for changes in KDM4A- dependent histone marks in the hearts of iron-loaded and iron-deficient mice. We also utilized the cardiac cell-line HL-1 to gain further mechanistic insight of iron-mediated changes in histone methylation pattern. We report that the levels of the histone mark H3K9me3 are responsive to cardiac iron content, following dietary iron manipulation as well as in the context of genetic hemochromatosis. Preliminary results also indicate that the effect of iron on this histone mark likely involves multiple mechanisms, including changes in KDM4A protein levels. Taken together, our findings describe a novel pathway through which iron affects gene expression. Next, it would be important to determine, via chromatin immunoprecipitation, the global changes in gene expression downstream of H3K9me3 in response to changes in physiological iron levels. Further dissection of the epigenetic targets of iron in the heart should help uncover genes involved in the hypertrophic disease process.

Sixth Congress of the International BioIRon Society Page 316 Poster Abstracts IBIS

Poster #170 THE IRON METABOLISM PROTEIN IRP2 IS OVER EXPRESSED IN COLORECTAL ADENOCARCINOMA Sarah Evans, Matthew Bedford, Richard Horniblow, Neeraj Lal, Andrew Beggs, Tariq Iqbal, Olga Tucker and Chris Tselepis University of Birmingham

Introduction Colorectal adenocarcinoma remains a significant cause of morbidity and mortality on a worldwide basis. Of note, there is now an emerging body of evidence implicating iron in the malignant progression of this disease. Specifically, colorectal cancer cells have adapted to further acquire an excess of iron despite pre-existing increased intracellular iron levels. The mechanism that underpins this process is unknown, though likely to involve iron regulatory protein 2 (IRP2); the master regulator of intracellular iron. This study aimed to characterise the expression of IRP2 in colorectal adenocarcinoma and to determine the effect of its perturbation on cellular phenotype. Methods IRP2 mRNA expression was determined in 41 samples of colorectal adenocarcinoma and matched normal mucosa by qRT-PCR. Protein expression was quantified using immunohistochemistry performed on a separate cohort of 32 samples. IRP2 siRNA was then utilised to determine effect of IRP2 ‘knockdown’ on colorectal adenocarcinoma cell (RKO) phenotype, including iron loading (through ferrozine assay) and cell cycle progression (via FACS). Full ethical approval was granted for all studies. Results IRP2 was significantly over expressed at both the mRNA (median fold change 8.80, p<0.05) and protein (median fold change 1.67, p<0.05) level in adenocarcinomas. IRP2 mRNA expression was strongly and positively correlated to that of TfR1, being greatest in locally advanced (T3 and T4) and proximal colonic lesions. IRP2 ‘knockdown’ significantly decreased TfR1 expression at both the mRNA (by 33.7%, p<0.05) and protein (by 60.8%, p<0.05) level, leading to a decrease in colonocyte iron loading (47.2% vs. control, p<0.05) and subsequent accumulation of cells in G1 of the cell cycle. Discussion and Conclusions IRP2 is over expressed in colorectal adenocarcinoma relative to normal mucosa, facilitating increased cellular iron acquisition and perturbation of proliferation. Tumour IRP2 expression may therefore serve as a surrogate marker for increased tumour iron content and subsequent efficacy to therapy with iron chelation.

Sixth Congress of the International BioIRon Society Page 317 Poster Abstracts IBIS

Poster #171 SECONDARY IRON OVERLOAD INTERFERES WITH ERYTHROFERRONE SIGNALING Martin Vokurka, MD, PhD, Iuliia Gurieva, MD, Jana Frydlova, PhD, Petr Prikryl, PhD and Jan Krijt, PhD 1st Medical Faculty, Charles University, Prague, Czech Republic

Introduction: It has been known since 2002 that administration of erythropoietin (EPO) dramatically decreases hepatic hepcidin (Hamp) expression. In 2006, we demonstrated that this downregulation depends on accelerated erythropoiesis. In 2014, it was shown that the effect of erythropoiesis on Hamp expression is mediated by erythroferrone (ERFE, Fam132b), a protein secreted by erythroblasts. Since some patients who display elevated EPO levels also have transfusion-induced secondary iron overload, it was of interest to determine how secondary iron overload affects ERFE signaling. Experimental: Male C57BL/6 mice and female Wistar rats were treated with EPO (50 and 500 iU/animal/day respectively) for four days. Iron was administered as iron dextran at 400 mg/kg body weight one week before EPO treatment. Hamp expression was determined by real-time PCR, liver concentration of matriptase-2, hemojuvelin (Hjv) and Hfe proteins were determined by immunoblotting with commercial antibodies. Results: Administration of EPO decreased Hamp expression by two orders of magnitude in both mice and rats. However, if the animals were pretreated with iron dextran, the EPO-induced decrease of Hamp expression was completely prevented. These results suggest that secondary iron overload interferes with ERFE signaling. As expected, EPO administration increased Fam132b expression in the spleen; however, iron pretreatment did not influence the EPO- mediated increase of Fam132b mRNA. This indicates that the effect of secondary iron overload on ERFE signaling occurs posttranscriptionally. Since it is possibile that the ERFE signaling pathway utilizes known hepatocyte membrane proteins, which are known to control Hamp expression, we examined the effect of EPO and iron on liver Hjv, matriptase-2 and Hfe. EPO administration did not affect the liver protein content of Hjv and Hfe; however, it significantly increased matriptase-2 protein content. In contrast, iron administration decreased matriptase-2 protein content. We therefore speculate that the observed changes in matriptase-2 could affect the ERFE signaling pathway. Discussion: Administration of high doses of EPO to experimental animals is known to decrease Hamp expression by several orders of magnitude. It has been postulated that this decrease could be mediated by EPO-induced decrease of plasma iron content or transferrin saturation; however, the main candidates are specific proteins such as Gdf15. Recently, a new erythroblast-secreted factor, ERFE, has been demonstrated to control the EPO-induced Hamp regulation. Interestingly, administration of iron dextran is able to completely prevent the dramatic decrease of Hamp expression induced by EPO. Since iron dextran did not prevent the substantial EPO-induced increase of splenic Fam132b mRNA, it is possible to speculate that secondary iron overload interferes with some components of the ERFE-mediated signaling pathway. Interestingly, data from the literature suggest that EPO-mediated decrease of Hamp expression does not occur in mice lacking functional matriptase-2 protein. This suggest that matriptase-2 could be an important mediator of ERFE signaling. In agreement with this concept, we found increased matriptase-2 protein content in animals treated with EPO, and decreased matriptase-2 content in animals pretreated with iron. Overall, our results suggest that secondary iron overload interferes with ERFE-mediated hepcidin regulation, and point to an important role of matriptase-2 in ERFE signaling.

Sixth Congress of the International BioIRon Society Page 318 Poster Abstracts IBIS

Poster #172 STRESS SIGNALING OF HEME-REGULATED eIF2α KINASE IN ERYTHROBLASTS AND MACROPHAGES DURING IRON-RESTRICTIVE ERYTHROPOIESIS Jane-Jane Chen, PhD, A. Macias, S. Zhang, E. Paltrinieri, M. Cappellini, Institute for Medical Engineering and Science MIT, Institute for Medical Engineering and Science

Iron deficiency anemia is a very prevalent disease, yet molecular mechanisms by which iron and heme regulate erythropoiesis are not well understood. Heme-regulated eIF2a kinase (HRI) is highly expressed in erythroid precursors and senses intracellular heme concentrations to coordinate heme and globin synthesis for production of hemoglobin. In heme deficiency, HRI is activated, phosphorylates the α-subunit of eIF2 (eIF2a) and inhibits the translation of globin mRNAs. In addition, phosphorylation of eIF2a (eIF2aP) by HRI specifically enhances the translation of ATF4 mRNA for transcription activation of stress response genes. We have shown previously that HRI is necessary to promote erythroid differentiation in iron deficiency. To better understand the role of HRI-eIF2aΠ-ATF4 signaling in erythropoiesis, we generated a knockin mouse model that lacks eIF2aP specifically in the erythroid lineage by substituting the serine 51 with alanine (eAA mice). During normal adult steady-state erythropoiesis, eAA, Hri-/- and Atf4-/- mice did not exhibit significant erythroid phenotypes. After two months of iron deficiency, eAA mice developed stress erythropoiesis with splenomegaly. Additionally, the anemia of iron deficient eAA mice was not microcytic and hypochromic as seen in wild type (Wt) mice. Rather, the anemia was macrocytic and hyperchromic; the same as that of Hri-/- mice. Iron deficient Atf4-/- mice developed a more severe anemia than eAA or Hri-/- mice, and was microcytic and hypochromic. Thus, regulation of RBC cell size and hemoglobin content by HRI is mediated through eIF2aP, not ATF4. Consistent with the regulation of globin translation by eIF2aP, there were globin inclusions in reticulocytes of eAA and Hri-/- mice, but not Atf4-/- mice. There were inhibitions of erythroid differentiation starting at basophilic erythroblasts both in the bone marrow and spleen of eAA and Atf4-/- mice. The ROS levels in RBCs and reticulocytes were also elevated, indicating that HRI-eIF2aP-ATF4 signaling plays a more important role in mitigating oxidative stress in these late stage erythroid cells. While HRI was activated, no activation of ATF4 targets (CHOP, ATF5 and TRIB3) was observed in eAA and Atf4-/- erythroid precursors in contrast to Wt cells. These results demonstrate for the first time that both eIF2aP and ATF4 are necessary for iron restrictive erythropoiesis. Furthermore, iron and heme regulate erythropoiesis starting mostly from the basophilic erythroblast stage in which hemoglobin begins to be actively synthesized. Macrophages play a critical role in erythropoiesis especially under stress. We have shown earlier that macrophages from Hri-/- mice exhibited impaired maturation and erythrophagocytosis. Recently, we investigated the role of HRI signaling in erythroblastic island (EI) formation. Using methylcellulose colony culture assays, we found that there were increases in CFU-M (monocytic colony) from spleens of Hri-/-, Atf4-/- and eAA mice in iron deficiency. There was no significance difference in CFU-M colonies from the bone marrow of these mutant mice. Importantly, there was a decrease in numbers of native EI from the bone marrow and spleen of iron deficient Hri-/- mice by immunofluorescent microscopy. These observations further support the defect of maturation of Hri-/- monocytes to macrophages. EI work of Atf4-/- and eAA mice are in progress. In summary, HRI signaling is important in both erythroid and macrophage lineages during iron restrictive erythropoiesis.

Sixth Congress of the International BioIRon Society Page 319 Poster Abstracts IBIS

Poster #173 INTERROGATING HEME LEVELS USING SUBCELLULAR TARGETING OF HEMOPROTEIN PROBES Xiaojing Yuan1, Ana Beatriz Walter Nuno Da Silva, MS2 and Iqbal Hamza, PhD3 1University of Maryland; 2Universidade Federal do Rio de Janeiro, Brazil; 3University of Maryland, USA

Genetic studies in cell lines, zebrafish, mice, and humans have provided plausible evidence that intact heme must cross membranes, and embryonic development in zebrafish and mice occurs even in heme synthesis mutants. However, no studies have addressed if heme partitioning occurs and if so, how, where, when, and why since there are no molecular or imaging tools currently available to dynamically evaluate heme distribution in living cells hampering our understanding of heme trafficking. Here, we exploit the robust activity of two genetically-encoded hemoprotein reporters, horseradish peroxidase (HRP) and ascorbate peroxidase (APX), and show that heme levels can be monitored dynamically at the organellar and tissue level in single cells and whole animals. By systematically targeting HRP and APX to different subcellular compartments, we measure heme levels as a function of enzyme activity in the cytoplasm, nucleus, mitochondria, and various secretory compartments in mammalian cells. We show that these probes respond differentially to heme synthesized in the mitochondria from heme imported from exogenous sources. We extend these cell biological studies to in vivo models by engineering the HRP/APX reporters for tissue-specific expression in C. elegans. Using HRP/APX transgenic worms in an RNAi screening assay, we confirm the biochemical utility of the hemoprotein reporters, and adapt the assay to a high throughput format. Our subcellular HRP/APX probes will be a powerful tool for investigating the dynamic trafficking of heme in whole animal models.

Sixth Congress of the International BioIRon Society Page 320 Poster Abstracts IBIS

Poster #174 DIFFERENTIAL REGULATION OF HEPCIDIN EXPRESSION IN LIVER AND BRAIN OF NEWBORN MICE IN RESPONSE TO SYSTEMIC INFLAMMATION Feng Qi, MD1, Mary Migas, MS2 and Robert Fleming, MD2 1Peking University First Hospital; 2Saint Louis University

BACKGROUND: Hepcidin is an iron regulatory hormone produced primarily in the liver which negatively regulates dietary iron absorption and release of iron from tissue stores. Iron deficiency leads to a decrease in liver hepcidin expression and consequent increased serum iron; in contrast infections and other inflammatory conditions lead to an increase in liver hepcidin expression and consequent hypoferremia. Hepcidin has recently been demonstrated to be expressed in brain, where it is speculated to regulate local iron metabolism. The consequences of inflammation on liver and brain hepcidin expression and body iron distribution during the newborn timeframe have not been investigated. OBJECTIVE: We hypothesized that systemic inflammation during the newborn timeframe would result in high expression of liver hepcidin with consequent decreased availability of iron to certain tissues, including the developing brain. DESIGN/METHODS: Newborn mice were injected beginning at 4 days of age with endotoxin (LPS, n=3) or saline (n=5) at a dose of 500 mcg/kg daily for 7 days and sacrificed 6 hours after the last injection. Hepcidin expression (Hamp1 mRNA) in liver and brain was measured by real-time RT-PCR and normalized to ß-actin. Parameters of brain iron status including total non-heme brain iron concentrations, and transferrin receptor1 protein levels were measured. RESULTS: Mice exposed to endotoxin demonstrated markedly increased liver hepcidin mRNA expression (20-fold, P=0.003), Hematocrit levels were significantly lower in the treated group (30.0 ± 0.8%) compared to the control group (36.5 ± 1.4%, P=0.006). In contrast to the liver, hepcidin mRNA expression in the brain was decreased in the mice exposed to endotoxin (>25 fold, P<0.001). There was no change in total brain iron or in TfR1 protein levels. CONCLUSIONS: We conclude that chronic endotoxin exposure upregulates liver hepcidin expression in liver of newborn mice. We speculate that the consequent decreased circulating iron availability to the erythron led to the observed fall in hematocrit and contributed to the decreased hepcidin expression in the brain. These observations suggest that different relative effects of hypoferremia and inflammation on hepcidin expression in the liver and brain.

Sixth Congress of the International BioIRon Society Page 321 Poster Abstracts IBIS

Poster #175 MITOFERRIN DEFICIENCY PROVOKES AN ECDYSONE SYNTHESIS IMPAIRMENT AND AN IMMUNE SYSTEM OVERREACTION Jose Llorens, PhD1, Christoph Metzendorf, PhD2, Fanis Missirlis, PhD3 and Maria Lind, PhD4 1Uppsala University; 2Department of Comparative Physiology, Uppsala University. Uppsala, Sweden. Biochemistry Center, Heidelberg University, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany; 3Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados. Mexico City, Mexico.; 4Department of Comparative Physiology, Uppsala University. Uppsala, Sweden.

Introduction: Besides serving as energy power plants, mitochondria also participate in the biosynthesis of Fe/S clusters and heme. In addition to numerous other iron-dependent proteins, these mitochondrial-derived cofactors are present in cytochromes and ferredoxins required for steroidogenesis. In Drosophila melanogaster, ecdysone is the key steroid hormone that signals the termination of larval growth and the initiation of metamorphosis. Hemocytes also respond to circulating ecdysone by turning into a more active state. Fly mutants in genes encoding for steroidogenic enzymes, collectively known as the halloween genes, show developmental arrest as larvae. Flies lacking the mitochondrial iron carrier mitoferrin were also found to arrest developmentally under mild iron deficiency. We therefore asked whether the developmental arrest of mitoferrin mutants is due to impaired ecdysone synthesis. Results: We found that mitoferrin mutants grown on low iron food developed slower than controls and rarely initiated metamorphosis. Expression of some halloween genes was increased in mutant animals while the expression of the ecdysone inducible gene E74A was reduced, suggesting an impairment of ecdysone biosynthesis. The developmental defect of the mitoferrin mutant flies grown on low iron food was partially recovered upon dietary supplementation with 20- hydroxyecdysone, the active form of ecdysone. No rescue was observed when the mitoferrin mutants were grown on food supplemented with ecdysone-synthesis precursors (cholesterol or 7-dehydrocholesterol). In addition, in the third instar mitoferrin mutant larvae we observed a higher number of lamellocytes and melanotic masses produced by activated crystal cells, suggesting an activated state of the larval innate immune response. Discussion: Our results suggest that lack of the mitochondrial iron carrier mitoferrin leads to reduced ecdysone levels, likely due to a decreased Fe/S and heme biosynthesis and a consequent inactivation of steroidogenic enzymes and to a developmental arrest. Activation of the immune system in mitoferrin mutants is not explainable by low ecdysone, but may arise due to oxidative stress caused by dysbalances in cellular iron homeostasis.

Sixth Congress of the International BioIRon Society Page 322 Poster Abstracts IBIS

Poster #176 POTENTIAL THERAPEUTIC APPLICATIONS OF COMBINING THE USE OF JAK2 INHIBITORS AND MINIHEPCIDIN PEPTIDES IN MICE AFFECTED BY BETA-THALASSEMIA C. Casu1, R. Chessa1, R. Oikonomidou1, B. MacDonald2 and S. Rivella1 1Children’s Hospital of Philadelphia (CHOP); 2Merganser Biotech LLC;

Beta-thalassemia is a genetic disorder characterized by ineffective erythropoiesis (IE), extramedullary hematopoiesis (EMH), splenomegaly and iron overload mediated by low levels of hepcidin. EMH is associated with elevated Epo levels and hyperactivation of the protein kinase Jak2, and the use of JAK2 inhibitors can reverse splenomegaly in Hbbth3/+ mice, affected by Non-Transfusion Dependent Thalassemia (NTDT). However, in these animals we also observed exacerbation of the anemia. These last observations indicate that the use of JAK2 inhibitors can rapidly revert the splenomegaly, but at the expense of RBC production. Moreover, in animals affected by beta-thalassemia major (TDT), we observed that use of JAK2 inhibitors (JAK2i) further decreased splenomegaly compared to blood transfusion alone. Using a different approach, we recently showed that administration of minihepcidins (MH) to Hbbth3/+ can significantly reduced iron absorption, erythroid iron intake and cell damage, ameliorating the IE, anemia and iron overload. Based on these observations, we hypothesized that in NTDT mice acute administration of a JAK2i (for 10 ten days) will rapidly reduce the splenomegaly, while the MH will prevent the recurrence of splenomegaly and improve anemia and iron overload. We also hypothesized that, in animals affected by TDT, combining JAK2i inhibitors with MH will further decrease the splenomegaly, decrease the requirement of blood transfused and, potentially, the requirement of iron chelation. Looking at NTDT mice, a JAK2i was administered to 3 month old Hbbth3/+ animals by oral gavage twice at day for 10 days, followed by 3 weeks of subcutaneous injection with MH (M009) (2.625 mg/kg every other day). Compared to Hbbth3/+ untreated mice, flow cytometry studies demonstrated a large increase in the proportion of mature rather than immature erythroid cells. A 26% increase in RBC was observed together with increased hemoblobin level (1.9 g/dL) and reduction in reticulocyte count (45%) and in spleen weight (65%). Compared to animals treated with JAK2i for ten days, splenomegaly was further reduced (~20%) and anemia markedly improved. Analysis of iron parameters and generation of animals treated with MH alone for 3 weeks is in progress. Presently, adult mice affected by TDT are generated by infusion of Hbbth3/th3 fetal liver cells (FLC) into normal mice after irradiation. These recipient animals do not produce adult or fetal Hb and RBCs, as both the b1 and b2 beta-globin genes were deleted (th3 mutation) and mice do not harbor any fetal-globin gene. Therefore, this model is not useful to test drugs that have the potential to modify erythropoiesis and RBC production. Additional thalassemic models are available: Hbbth2/+ mice were generated with an insertion the neomycin gene into the b1 gene (th2 mutation), but do not show severe anemia, while Hbbth2/th2 die perinatally. Hbbth1/+ mice harbor a deletion of the b1 gene, showing little anemia, while Hbbth1/th1 are affected by NTDT. To establish a mouse model with a phenotype similar to that observed in patients who generate some RBCs but require blood transfusion, we isolated FLCs from different breedings. Hbbth1/th3 and Hbbth1/th2 FLCs were transplanted into normal mice. Our preliminary data indicate that these mice show more severe features that Hbbth3/+ (Fig.1) animals, but are still able to generate RBC. In conclusion, these are severe models of thalassemia that will be utilized to test

Sixth Congress of the International BioIRon Society Page 323 Alphabetical Index of Authors IBIS

ABADIA, JAVIER BARASCH, JONATHAN Poster #60 9/7/2015 13:00

ABKOWITZ, JANIS BONACCORSI DI PATTI, MARIA CAR 9/7/2015 14:30 9/7/2015 14:12 AB #11

AGORO, RAFIOU BOURNE, GREGORY Poster #47 Poster #102

AGUILAR VITORINO, HECTOR BRISSOT, PIERRE Poster #92 9/10/2015 13:30

ALAN, BETUL BRUNO, MARIASOLE Poster #52 Poster #63

ALTAMURA, SANDRO BURANRAT, BENJAPORN 9/10/2015 14:10 AB #53 Poster #45 Poster #69 BURCKHARDT, SUSANNA AN, PENG 9/10/2015 9:45 AB #33 9/10/2015 17:24 AB #70 Poster #114 CABANTCHIK, IOAV 9/6/2015 13:15 ANDERSON, GREG 9/8/2015 10:30 9/7/2015 11:00 9/8/2015 10:30 CAIRO, GAETANO 9/9/2015 14:30 ARDEHALI, HOSSEIN Poster #96 CAMASCHELLA, CLARA Poster #167 9/6/2015 16:15 9/8/2015 10:30 AREZES, JOO 9/10/2015 14:30 AB #50 CAMPANELLA, ALESSANDRO 9/7/2015 15:30 AB #20 AROSIO, PAOLO 9/9/2015 14:30 CANALI, SUSANNA 9/10/2015 14:45 AB #51 ASCHEMEYER, S. Poster #30 CANONNE-HERGAUX, FRANCOIS 9/9/2015 10:30 ASPERTI, MICHELA 9/9/2015 15:00 AB #28 CARLOMAGNO, FRANCESCA 9/9/2015 14:55 ATTIEH, ZOUHAIR Poster #90 CASU, CARLA Poster #176 BABITT, JODIE 9/7/2015 9:50 CERAMI, CARLA 9/9/2015 10:30 BALUSIKOVA, KAMILA Poster #124 CHANG, YAN-ZHONG 9/10/2015 15:30

Sixth Congress of the International BioIRon Society Page 324 Alphabetical Index of Authors IBIS

CHEN, HUIYONG DAHER, RAED 9/10/2015 16:00 AB #58 Poster #36

CHEN, MIN DEV, SOM Poster #139 Poster #40 Poster #138 CHEN, HUIJUN 9/8/2015 8:30 DUCE, JAMES 9/10/2015 15:30 CHEN, JANE-JANE Poster #172 DUNAIEF, JOSHUA Poster #9 CHEN, YUE 9/7/2015 15:45 AB #21 ENNS, CAROLINE 9/6/2015 13:15 CHIABRANDO, DEBORAH 9/7/2015 9:00 9/10/2015 16:46 AB #66 EVANS, ROBERT CHOI, BYOUNGJUN 9/8/2015 8:30 Poster #166 FAN, YUMEI CHRISTIDES, TATIANA 9/10/2015 9:15 AB #35 Poster #6 Poster #144

CHUNG, YU JIN FANG, XIANGMING Poster #169 9/10/2015 13:30

CLAVERO OLMOS, MARTA FIORITO, VERONICA Poster #50 9/7/2015 15:45 AB #15 Poster #51 FLEMING, ROBERT COLLINS, JAMES 9/6/2015 13:15 9/6/2015 13:15 9/6/2015 16:15

CONNOR, JAMES FRAZER, DAVID 9/6/2015 16:15 9/10/2015 7:15 9/9/2015 11:45 AB #24 Poster #24 FUCHAROEN, SUTHAT Poster #54 9/6/2015 16:15 9/9/2015 8:30 CONRAD, MARCUS 9/9/2015 14:30 FUQUA, BRIE Poster #158 COPPIN, HELENE Poster #81 GANZ, TOMAS 9/7/2015 9:00 COSTA DA SILVA, MILENE 9/7/2015 15:15 AB #19 GAO, GUOFEN Poster #117 DA SILVA, ANA RITA Poster #43 GARBOWSKI, MACIEJ Poster #157

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GARRICK, MICHAEL HO, JOY Poster #53 9/9/2015 8:55

GASSMANN, MAX HORNIBLOW, RICHARD 9/7/2015 14:30 AB #16 Poster #1 Poster #170 GICHOHI, WANJIKU 9/10/2015 11:30 AB #44 HORVATHOVA, MONIKA Poster #59 GINZBURG, YELENA 9/9/2015 8:30 HOUSHMANDYAR, SHARAREH Poster #49 GIRELLI, DOMENICO 9/9/2015 8:30 HUANG, XI 9/10/2015 9:30 AB #36 GONCALVES, DANIELA Poster #58 HULTHEN, LENA 9/10/2015 11:45 AB #45 GOU, YU-JING Poster #132 ISLAND, MARIE-LAURE Poster #70 GOZZELINO, RAFFAELLA Poster #143 9/9/2015 10:55 JACOB, MOLLY GRYZIK, MAGDALENA Poster #78 9/9/2015 15:33 AB #26 JAMES, JITHU GUIRAN, XIAO Poster #85 Poster #165 JANSOVA, HANA GUO, SHULING Poster #73 9/9/2015 15:45 AB #31 JIA, FENGJV HAMDI, AMEL Poster #75 9/7/2015 15:30 AB #14 JORMAKKA, MIKA HAMZA, IQBAL 9/8/2015 9:20 9/7/2015 14:30 9/8/2015 7:15 JUNXIA, MIN Poster #122 HAO, SHUANGYING Poster #119 KANURI, GIRIDHAR Poster #37 HAO, QIAN Poster #133 KARIM, ZOUBIDA 9/7/2015 14:00 AB #4 HEIDARI, MOONES 9/10/2015 14:25 AB #54 Poster #80 KHOJA, KHOLOUD HIDER, ROBERT Poster #71 9/10/2015 8:30

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KIM, AIRIE LI, YIHANG 9/10/2015 14:15 AB #49 9/10/2015 11:00 AB #40 9/10/2015 15:30 AB #56 KIM, DON-KYU Poster #152 Poster #14 LIANG, TAK HEI STEPHANIE KIM, SANGWON Poster #106 9/10/2015 9:00 AB #34 9/10/2015 16:04 AB #63 LIU, GUOHAO 9/10/2015 17:40 AB #72 9/7/2015 14:15 AB #5 Poster #11 LIU, SIJIN KNUTSON, MITCHELL 9/7/2015 14:30 9/8/2015 8:30 LIU, SHUNLI KOHGO, YUTAKA Poster #82 9/6/2015 16:15 LIU, YUQIAN KORTMAN, GUUS Poster #12 Poster #16 9/9/2015 11:20 LLORENS, JOSE Poster #175 KRIJT, JAN Poster #77 LOREAL, OLIVIER Poster #72 KUMAR, RAJIV Poster #67 LU, YAN Poster #104 LAKHAL-LITTLETON, SAMIRA 9/8/2015 11:30 MA, YONGMIN Poster #20 LATUNDE-DADA, YEMISI Poster #56 Poster #55 MACCARINELLI, FEDERICA LAWEN, ALFONS Poster #136 Poster #28 Poster #141

LEVI, SONIA MACH, JAN 9/6/2015 16:15 Poster #39

LI, GUANGFEI MAIRUAE, NOOTCHANAT 9/10/2015 18:04 AB #75 Poster #46 Poster #109 MASARATANA, PATARABUTR LI, HUIHUI Poster #5 Poster #120 MASSNER, CHRISTOPH LI, MIN Poster #98 Poster #111 MCCOLL, GAWAIN 9/7/2015 13:00

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MCDONALD, CAMERON NEVES, JOANA Poster #32 Poster #42 9/10/2015 15:30 NIE, GUANGJUN MCLAREN, CHRISTINE 9/10/2015 15:30 9/10/2015 13:30 PANTOPOULOS, KOSTAS MCLAREN, GD 9/10/2015 10:30 Poster #150 PASRICHA, SANT-RAYN MCLAREN, GORDON 9/10/2015 10:30 9/10/2015 13:30 PASTRANA ALTA, ROXANA MEADOWCROFT, MARK Poster #100 9/10/2015 15:50 AB #62 9/10/2015 9:45 AB #37 PEI, DUANQING 9/9/2015 13:30 MEYNARD, DELPHINE 9/10/2015 10:30 PHIRI, KAMIJA 9/10/2015 11:00 MEYRON-HOLTZ, ESTHER 9/10/2015 8:30 PIGA, ANTONIO 9/8/2015 11:00 MIKHAEL, MARC Poster #135 PONKA, PREM 9/6/2015 13:15 MILWARD, ADRIENNE Poster #137 POPP, ANDREAS 9/9/2015 15:15 AB #29 MIRCIOV, CORNEL 9/10/2015 16:45 AB #61 PORTER, JOHN 9/10/2015 8:30 MISSIRLIS, FANIS 9/10/2015 8:40 9/7/2015 13:18 AB #7 POURZAND, CHARAREH MLECZKO-SANECKA, KATARZYNA Poster #48 Poster #76 PRENTICE, ANDREW MUCKENTHALER, MARTINA 9/7/2015 11:00 9/8/2015 7:15 9/9/2015 10:30 QI, FENG Poster #174 MURA, CATHERINE Poster #41 QIU, ANDONG 9/7/2015 13:30 AB #2 NECHUSHTAI, RACHEL Poster #8 RAJENDRA BABU, KAMESH 9/7/2015 15:00 AB #18 NEMETH, ELIZABETA 9/6/2015 13:15 RAMM, GRANT 9/9/2015 14:30 9/10/2015 8:30 9/10/2015 17:32 AB #71 Poster #145

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RECALCATI, STEFANIA SWINKELS, DORINE Poster #105 9/7/2015 13:00 9/10/2015 9:05 RIVELLA, STEFANO 9/9/2015 14:30 TANGUDU, NAVEEN KUMAR 9/7/2015 15:00 AB #12 ROBSON, KATHRYN 9/10/2015 13:55 AB #52 TORTI, SUZY Poster #88 ROGERS, JACK Poster #156 TOSHA, TAKEHIKO Poster #163 9/8/2015 8:55

RUZZENENTI, PAOLA TOYOKUNI, SHINYA Poster #140 9/7/2015 14:30

RYCHTARCIKOVA, ZUZANA TRINDER, DEBORAH Poster #66 9/10/2015 13:45 AB #47

SANCHEZ FERNANDEZ, MAYKA TRUKSA, JAROSLAV 9/10/2015 10:45 AB #39 Poster #65 Poster #10 Poster #22 VARGHESE, JOE Poster #84 SHAHANDEH, ALI 9/10/2015 16:18 AB #64 VINCHI, FRANCESCA 9/10/2015 11:30 AB #42 SHENG, WANG Poster #38 Poster #118 9/10/2015 7:15

SILVESTRI, LAURA VIPRAKASIT, VIP 9/7/2015 9:25 9/9/2015 9:20

SOBH, AMIN VOKURKA, MARTIN Poster #33 Poster #171 Poster #34 VUJIC SPASIC, MAJA SONG, DELU 9/10/2015 13:30 Poster #44 VULPE, CHRISTOPHER SRAI, SURJIT 9/9/2015 7:15 9/6/2015 13:15 WALKER, ANN STEFANOVA, DEBORA Poster #161 9/10/2015 13:30 AB #46 WALLACE, DANIEL SUBRAMANIAM, V. NATHAN 9/10/2015 14:40 AB #55 9/9/2015 7:15 WANG, JUN SUBRAMANIAM, NATHAN Poster #13 9/10/2015 16:30 AB #60

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WANG, PEI-NA YUAN, XIAOJING Poster #131 Poster #173

WANG, FUDI ZEIDAN, ROLA 9/9/2015 13:30 Poster #94 9/10/2015 10:30 ZHANG, AN-SHENG WANG, XUDONG 9/10/2015 10:30 AB #38 Poster #162 ZHANG, YAN WANG, YONGWEI 9/10/2015 17:00 AB #67 Poster #57 Poster #91

WARD, ROBERTA ZHANG, WEI 9/10/2015 9:30 AB #32 9/10/2015 17:16 AB #69 Poster #149 WESTON, CODY Poster #27 ZHAO, LU Poster #134 WU, QIAN Poster #115 ZHAO, MENGRAN Poster #168 WU, QIONG Poster #130 ZHENG, JIASHUO 9/10/2015 17:56 AB #74 XIA, YIN Poster #83 9/7/2015 9:00 ZHOU, BING XU, MANMAN 9/7/2015 13:00 Poster #15 ZHOU, GUANGHUA YIH-FONG, LIEW Poster #3 9/9/2015 15:20 AB #25 Poster #29 ZHOU, TAO Poster #31 Poster #2

YIN, DAN ZHU, LI Poster #147 Poster #97 YIN, SAI Poster #164

YOUJIA, XU Poster #110

YU, JIANG Poster #112

YU, PENG Poster #107 Poster #108 Poster #129

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