Harmonizing Lipidomics

Vol. 18 / No. 5 / May 2019

HARMONIZING LIPIDOMICS

CONTENTS

NEWS FEATURES PERSPECTIVES

2 26 43 EDITOR’S NOTE HARMONIZING LIPIDOMICS PROFESSIONAL DEVELOPMENT Working knowledge Researchers strive to agree on what ASBMB webinar offers financial advice they’re measuring 3 44 NEWS FROM THE HILL 40 PUBLIC AFFAIRS Advocates visit Capitol Hill MEET KARIN MUSIER–FORSYTH What we learned from the ATP At Ohio State, the JBC associate editor and tRNA trailblazer uses ASBMB resources as 4 teaching tools for grad students 46 MEMBER UPDATE ESSAY 7 26 Of mice and scientists RETROSPECTIVE 48 Mary Jane Osborn (1927 – 2019) HILL DAY 10 IN REMEMBRANCE 43 11 LIPID NEWS Lipid regulation of mitochondria 12 A YEAR OF (BIO) CHEMICAL ELEMENTS For May, it’s in your bones: 46 calcium and phosphorus 13 40 JOURNAL NEWS 13 B acterial drug synergies hide in plain sight 15 The mystery of metformin 17 H ow to catch ovarian cancer when it’s curable 18 JLR celebrates diamond jubilee 48 with special reviews 20 From the journals 24 SCIENCE NEWS Circulating oxysterol levels

MAY 2019 ASBMB TODAY 1 EDITOR’S NOTE

THE MEMBER MAGAZINE OF THE AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY Working knowledge By Comfort Dorn OFFICERS COUNCIL MEMBERS Gerald Hart Squire J. Booker President Victoria J. DeRose ore than 30 years ago, I was chemistry and molecular biology. Blake Hill employed as the parish admin- As an undergrad, grad student Jennifer DuBois Audrey Lamb Secretary James M. Ntambi Mistrator of a church, a huge, and (maybe) postdoc, you’ve learned Toni M. Antalis Celia A. Shiffer crumbling building in an underserved a whole lot about science. But how Treasurer Takita Felder Sumter area of Washington, D.C. The parish much were you taught about having a Kelly Ten-Hagen JoAnn Trejo ethos was to make maximum use career as a scientist? More to the point, EX-OFFICIO MEMBERS of the building by opening it up to how much were you not taught? How Robert S. Haltiwanger ASBMB TODAY EDITORIAL neighborhood service providers. much knowledge did you have to pick Carla Koehler ADVISORY BOARD Co-chairs, 2020 Annual I was in my early 30s, a young up outside the lab — about choosing Rajini Rao Meeting Program Committee Chair mother with a degree in liberal arts a career path, finding a job, starting a Cheryl Bailey Floyd “Ski” Chilton and a vague interest in improving the lab, or managing a budget and Chair, Education and Henrik Dohlman world. My boss, the rector, was about personnel? Professional Development Peter J. Kennelly Committee Beronda Montgomery 10 years older, one of the first women You’ve probably learned a lot from Daniel Raben A. Maureen Rouhi ordained as a priest in the Episcopal your experience, which is, as they say, Chair, Meetings Committee Melissa Vaught Church. The parish staff was bare the best teacher. Would you be will- Sonia Flores Binks W. Wattenberg bones and the budget small. ing to share some of that hard-won Chair, Minority Affairs Committee ASBMB TODAY One dank winter afternoon, the knowledge? Susannna Greer Angela Hopp heat stopped working. Complaining The August issue of ASBMB Today Chair, Public Outreach Executive Editor phone calls came from the preschool traditionally is given over to the vast Committee [email protected] Comfort Dorn and free clinic upstairs. Our resident topic of careers. It’s an opportunity for Matthew S. Gentry Managing Editor custodian (referred to in Anglican- society members to pool their collec- Chair, Public Affairs [email protected] Advisory Committee speak as a sexton) was AWOL, so the tive wisdom and help each other with John Arnst rector and I made our way into the the stuff they really need to know. Sandra Weller Science Writer Chair, Publications [email protected] bowels of the church basement to the Every career has its McDonald & Committee Laurel Oldach furnace, an ancient behemoth squat- Miller valves. We’d like to hear about Lila M. Gierasch Science Writter ting in a dark corner. It required the yours. Maybe you want to write an Editor-in-chief, JBC [email protected] Ed Marklin regular draining of something called essay. Maybe you just have a few words A. L. Burlingame Web Editor a McDonald & Miller valve, a bit of of wisdom. Either way, drop me a line Editor, MCP [email protected] Nicholas O. Davidson Allison Frick maintenance that had gone neglected. at [email protected]. Deadline for the Editor-in-chief, JLR Media Specialist We wrestled the valve open and, as August issue is June 3. Kerry-Anne Rye [email protected] steaming rusty water gushed into a Barbara Gordon Editor-in-chief, JLR bucket (and onto our shoes), the rector Executive Director Comfort Dorn (cdorn@ [email protected] sighed and gave me a deadpan look. asbmb.org) is the managing “Another thing they didn’t teach me editor of ASBMB Today. Follow in seminary.” her on Twitter @cdorn56. Which brings me to careers in bio- For information on advertising, contact Pharmaceutical Media Inc. at 212-904-0374 or [email protected]. Corrections In an article about Angela Gronenborn on page 29 of the April issue the first sentence in the fifth paragraph should read, “In 1988, Gronenborn joined the National Institutes of Health where, alongside Marius Clore and Adriaan Bax, she started to work on HIV, supported by the HIV-targeted www.asbmb.org/asbmbtoday antiviral program.” PRINT ISSN 2372-0409 In an article about JLR junior associate editors on page 15 of the April is- Articles published in ASBMB Today reflect solely the authors’ views and not sue, Gissette Reyes–Soffer’s name was misspelled and Stephen Young should the official positions of the American Society for Biochemistry and Molecular Biology or the institutions with which the authors are affiliated. Mentions of have been listed as Brandon Davies’ mentor. products or services are not endorsements.

2 ASBMB TODAY MAY 2019 NEWS FROM THE HILL Advocates visit Capitol Hill By Benjamin Corb

wenty undergraduate, graduate and postdoctoral students joined Tmembers of the Public Affairs Ad- visory Committee and public affairs staff to participate in the American Society for Biochemistry and Mo- lecular Biology’s annual Hill Day on March 28, visiting their elected representatives to discuss Congress’ continued support for biomedical research. Although President Donald Trump’s budget request for fiscal year 2020 called for 5 percent cuts to all science funding agencies in the government, the advocates were heartened by the warm reception they received. Martha Cyert, associate chair of biology at Stanford University, said, “It was encouraging to hear staffers share their support for my science and their understanding of the importance of basic research in helping to discover treatments for diseases. They really COURTESY OF CALLAN FRYE seem to get it.” Callan Frye, a graduate student at the Medical University of South Carolina, shows his enthusiasm for The agenda for this year’s Hill Day advocacy at the ASBMB’s 2019 Hill Day. For more photos, turn to page 48. advocates focused largely on asking for increases in funding at the This year’s advocates came from Gentry has been a member of the National Institutes of Health, the 24 states and conducted 83 meet- PAAC for five years and is a Hill Day National Science Foundation and ings over the course of their day on veteran. For some, like Alex Black- the Department of Energy’s Office the Hill. The student participants, burn, a Ph.D. student at the Univer- of Science. But the discussions with selected from a pool of applicants by sity of Idaho, this Hill Day was their lawmakers and their staffs extended the ASBMB’s public affairs staff, ar- first taste of advocacy. to issues beyond funding, indicating a rived in Washington, D.C., the night “This was a very positive, very fun Congress that has a nuanced under- before Hill Day for a crash course in experience,” Blackburn said. “I got to standing of and curiosity about how being an advocate. PAAC members meet really great people on both sides science works. stayed through the following day for of the aisle. When I get back to Idaho, Kristine Deibler, a postdoc from meetings with NIH and NSF leaders. I look forward to telling my col- the University of Washington, was “This Hill Day experience is among leagues that they should consider get- surprised by some of the topics. “My the most rewarding opportunities that ting involved themselves. I definitely senator’s staff was very direct and ASBMB (offers) for its members,” would love to do this again.” interested in hearing about my per- said Matt Gentry from the University spectives related to the issue of sexual of Kentucky, the outgoing PAAC Benjamin Corb (bcorb@ harassment in science,” Deibler said. chair. “ASBMB isn’t the only scientific asbmb.org) is director of public “I was so encouraged to see Congress society that holds events like this, but affairs at the ASBMB. Follow him on Twitter @bwcorb. paying such close attention to this in my experience the organization and very serious issue.” staff put on the best show by far.”

MAY 2019 ASBMB TODAY 3 MEMBER UPDATE Member update By Erik Chaulk

Bruns named focuses on understanding protein In 2009, she joined the Program in modifications involved in cell signal- Systems Immunology and Infectious Mr. Homecoming ing as well as absolute quantification Disease Research, now the Laboratory Professor of chemistry Kerry Bruns of molecular representation and of Immune System Biology, at the has received the Southwestern Univer- interaction. NIAID. sity 2018 Mr. Homecoming Award. After obtain- Presented by the university’s ing her Ph.D. in Gottesman named alumni association, the Mr. Home- biochemistry at the coming Award is given to a faculty University of Basel visiting professor member who has garnered the respect in 2003, Nita– Susan Gottesman is among four of former students. Lazar completed NITA–LAZAR individuals announced in December Bruns earned his Ph.D. from New postdoctoral train- as new Vallee visiting professors. Mexico State University in 1987 ing at Stony Brook The visiting professor program and his B.A. from University and the Massachusetts sponsored by the Vallee Foundation Western New Institute of Technology. promotes intellectual exchange and Mexico University in 1981. In addition to teaching general chemistry In memoriam: Hugh Forrest BRUNS and biochemistry Former University of Texas professor Hugh Forrest died Nov. 16. He courses, he leads was 94. Southwestern University’s premedical The youngest of five sons, Forrest was born and raised in Glasgow, advisory committee, which provides Scotland. He remained in Scotland for his undergraduate studies, professional development program- graduating from the University of Glasgow in 1944. ming and handles the application He earned his first Ph.D. in 1947 studying therapeutic process for students interested in chemical compounds at the University attending medical school. of London and earned a second Ph.D. in He also organizes the health care 1951 from the University of Cambridge professionals’ breakfast at homecom- researching pteridine biosynthesis. ing each year, providing students the Forrest left for the United States in opportunity to connect with alumni. 1951 after receiving a U.S. Public Health Bruns is retiring at the end of the Service fellowship at the California Insti- year. Colleagues say he has had a tute of Technology, where he continued significant, long-lasting impact upon his research into pteridines. the Southwestern community. In 1955, Forrest joined the University FORREST of Texas at Austin as a postdoctoral fel- Nita–Lazar promoted low. He remained there for nearly five decades, becoming a professor in 1962 and a professor emeritus in 1993. to senior investigator Forrest served as a mentor to numerous postgraduate students during Aleksandra Nita–Lazar was pro- his tenure. moted in December to senior investi- The University of London awarded him a doctor of science degree in gator with the National Institutes of 1970, and he was elected as a fellow to the Royal Society of Edinburgh Health National Institute of Allergy in 1979. and Infectious Diseases. He is survived by his three children, Eleanor, Anne and Hugh, and Nita–Lazar leads the Functional his six grandchildren. Cellular Networks Section, which

4 ASBMB TODAY MAY 2019 scientific partnership by offering works to support sustainable pros- First conferred in 2005, this award senior scientists an opportunity to perity through research, education, focuses on a specific country or region work at premier biomedical research policy and diplomacy. each year, recognizing individuals for institutes. Basu is a J.C. Bose national fellow outstanding scientific mentorship. A National in the department of chemistry, Bose The award consists of two prizes of Institutes of Health Institute, Kolkata, India. She has $10,000, one awarded for midcareer distinguished contributed significant research in the mentorship and one for lifetime professor, Gottes- area of Mycobacterium tuberculosis achievement in mentorship. man is co-chief in pathogenesis. Corbett is a biochemist, and her GOTTESMAN the laboratory of Varshney is professor in the research primarily focuses on deter- molecular biology and head of the department of mi- mining the function of evolutionarily biochemical genetics section at the crobiology and cell conserved RNA binding proteins. National Cancer Institute. biology and chair Mentorship has played a significant Gottesman’s research focuses on of the division of role in Corbett’s career. Susan Wente, post-transcriptional mechanisms of biological sciences a Vanderbilt University professor and regulation in bacterial systems. at the Indian In- chair of the judging committee for VARSHNEY As a Vallee visiting professor, Got- stitute of Science, the award, stated in a Nature press tesman will spend a one-month sab- Bangalore, India. Varshney’s lab uses release that Corbett “reaches outside batical in another lab of her choice. E. coli to study mechanistic aspects of of her own laboratory and is a leader protein synthesis and DNA repair. at her university and in her field, with Basu, Varshney elected The election of this new class a passion for gender represents efforts to increase the ratio equity.” to World Academy of women in the organization as well Corbett is one Joyoti Basu and Umesh Varshney as scientists from countries that have of four scientists are among 46 new fellows elected to been underrepresented or unrepre- to receive the sented in the academy’s membership. 2018 awards and the World Academy of Sciences. CORBETT Founded in shares the midca- 1983, the World Corbett receives reer achievement award with Kjersti Aagaard, Baylor College of Medicine. Academy of Sci- mentoring award ences is a global Erik Chaulk (echaulk@ organization based Emory University biology profes- asbmb.org) is a peer-review in Trieste, Italy, that sor Anita Corbett has won the 2018 coordinator and digital publica- BASU seeks to promote Nature Award for Mentoring in tions web specialist at the ASBMB. science in developing countries and Science.

Upcoming ASBMB events and deadlines

National Stroke Awareness Month

Y 7 Serine Proteases in Pericellular Proteolysis and Signaling abstract deadline 23 Tips for Applying for ASBMB Accreditation webinar 28 Serine Proteases in Pericellular Proteolysis and Signaling early registration deadline MA 1 Marion B. Sewer Distinguished Scholarship for Undergraduates deadline

3–7 Art of Science Communications Course begins (first week) 4 Transforming Education in the Molecular Life Sciences abstract deadline 12 Transforming Education in the Molecular Life Sciences late registration deadline 13–15 IMAGE grant-writing workshop JUNE 27 National HIV Testing Day

Eye health, in support of UV Safety Month by the American Academy of Ophthalmology

7 Mass Spectrometry in the Health and Life Sciences: Molecular & Cellular Proteomics final abstract deadline. 25 Emerging Roles for the Nucleolus oral abstract submission deadline

JULY 25–28 Transforming Education in the Molecular Life Sciences 28 Serine Proteases in Pericellular Proteolysis and Signaling poster abstract submission deadline

MAY 2019 ASBMB TODAY 5 WELCOME, NEW ASBMB MEMBERS

Meshal Alfheed, Qassim University Callan Frye, Medical University of South Carolina Elizabeth Nolan, Massachusetts Institute of Technology Yousef Alharbi, Qassim University Ciara Golliher, University of North Florida Rachel Osborn, Otterbein University Aishah Al-Jarallah, Kuwait University Matthew Greseth, Medical University of South Carolina Michael Parsons, Marshall University Abdullah Alrashed, Qassim University Gerald Grunwald, Thomas Jefferson University Kathryn Pearce, Ohio Northern University Chenoa Arico, University of Texas at El Paso Jeisac Guzman Rivea, University of Puerto Rico, Oksana Penezina, Tokyo Chemical Industry Co. Akanksha Bansal, University of Calgary Río Piedras Campus Otto Phanstiel, University of Central Florida Ruthie Barbas, Spectrum Brands Inc. Lillian Hewitt, University of North Florida Hannah Richter, University of Pennsylvania Jessica Beckham, Middle Tennessee State Laszlo Homolya, Hungarian Academy of Sciences University Neishaly Rivera, Universidad Interamericana Nazar Hussein, Kent State University de Puerto Rico Manas Biswal, University of South Florida Joseph Hwang, MilliporeSigma Antonieta Salguero, Johns Hopkins University Ashis Biswas, New Mexico State University Houssine Ikhlef, University of Central Florida Fernanda Santos, Adventist University of Health Kaylee Brabham, St. Bonaventure University Sciences Camille Immanuel, University of Kentucky Martin Brenneman, Ohio Northern University Claire Schaef, St. Bonaventure University Mahnoor Izhar, University of North Florida Henry Brothers (no affiliation) Samantha Schwartz, Emory University Jodiene Johnson, Fisk University Alexandra Caguana, Montclair State University James Scott, Kennesaw State University Paul Johnson, University of North Georgia Philip Cammarata, St. Bonaventure University Sally Seder, Montclair State University Geoffrey Kapler, Texas A&M Health Science Mariangeles Campos, Montclair State University Center Vandana Sekhar, University of Central Florida Ciara Carpenter, Montclair State University College of Medicine Jack Kavanaugh, University of North Florida Kathy Castor, Montclair State University Savita Sharma, University of Kentucky Debra Kellogg–Yelder, BioCryst Pharmaceuticals Saiful Chowdhury, University of Texas at Arlington Inc. Yujiang Shi, Harvard Medical School David Cobrinik, University of Southern California Saran Kone, St. Bonaventure University Tala Shourbagitello, University of North Florida Tyler Cook, Loyola University Chicago Michael Larock, St. Bonaventure University Melanie Silva, Montclair State University Brock Couch, Middle Tennessee State University Katherine Leon Hernanzed, Montclair State Andrew Smith, Metropolitan State University Aimee Cruikshank, University of North Florida University of Denver Anjelica DaSilva, University of North Florida Yunan Li, University of Minnesota Patrick Sochor, Arizona State University Crsitiano Dias, New Jersey Institute of Technology Melissa Locke, University of California, Berkeley Kennon Stewart, Tulane University Emre Dikici, University of Miami Broderick Lu, University of Massachusetts Natalie Sumser, Otterbein University Boston Aiste Dobrovolskaite, University of Central Krissie Tellez, Stanford University Phillippe Ly, California State University, Long Florida Dorothea Tholl, Virginia Tech Beach Amalia Dolga, University of Groningen Claire Todd, Otterbein University Sonal Mahindroo, St. Bonaventure University Kristiann Dougherty, Palm Beach Atlantic Ashutosh Tripathi, Texas A&M University University Ramon Martinez, University of Maryland, Baltimore Trent Tucker, Otterbein University Garrett Dunlap, Harvard University Nathaniel McClure, St. Bonaventure University Takeshi Uemura, Amine Pharma Research Yaritza Escamilla, University of Texas Rio Grande Institute Valley Gillian Melikian, Providence College Abraham Villa Mundo, University of Texas Michael Merchant, University of Lousiville Serena Estevez, Hillsborough Community College at El Paso Carlen Merritt, Marshall University Brandon Eudy, University of Florida Sarah Waldron, Kennesaw State University Jyotsna Mishra, Medical College of Wisconsin Baggio Evangelista, University of North Carolina Adrianna White, University of North Florida at Chapel Hill Holly Moots, University of Central Florida Charles White, James Madison University Nathan Forney, Otterbein University Young–Jae Nam, Vanderbilt University Medical Jarrod French, Stony Brook University Center

6 ASBMB TODAY MAY 2019 RETROSPECTIVE Mary Jane Osborn (1927–2019) By Sandra Weller & Lawrence Rothfield

ary Jane Osborn, a prominent biomedical scientist, a trailblaz- Mer for women in science, and a former president of the American So- ciety for Biochemistry and Molecular Biology, died Jan. 17 in Farmington, Connecticut. She was 91. The cause was complications following emergency surgery. Osborn’s research career began in the late 1950s, a time when women were notoriously underrepresented in all fields of science and many major university biomedical research departments had no tenured female faculty. In the decades that followed, the number of female scientists in university departments increased significantly, although female faculty ALL PHOTOS COURTESY OF SANDRA WELLER were (and continue to be) significantly underrepresented at the higher Mary Jane Osborn poses with her former Ph.D. student Inger Damon, who now directs the Division of High- academic ranks. Although she always Consequence Pathogens and Pathology in the Centers for Disease Control and Prevention’s National Center for Emerging and Zoonotic Infectious Diseases and is a captain in the U.S. Public Health Service. said she personally never felt she was professionally discriminated against, Osborn was keenly aware of the to celebrate women in science. would have made her a remark- systemic problems faced by women After hearing of Osborn’s death, able role model of any gender, but in science. In recognition of this, the Lucy Shapiro, director of the Beck- she was a woman in a world where University of Connecticut Medi- man Center for Molecular and Ge- few women had a chance to shine. cal School, where she was a faculty netic Medicine at Stanford University, And shine she did. When she spoke, member for 42 years, established the said, “Mary’s breadth of knowledge, people listened. When she published, annual Osborn Lectureship in 2002 scientific rigor and acute intelligence her papers became the gold standard.” Osborn made her first important scientific contribution in 1957 as a research fellow at the University of Dr. Osborn was a remarkable woman, a fabulously Washington, when she discovered successful biochemist and microbiologist who was the mechanism of action of the drug methotrexate, one of the earliest ‘‘ always the smartest person in the room. She had cancer chemotherapeutic agents. The research was published in the Journal a profound influence on my career by serving as a of Biological Chemistry. Methotrexate role model and sounding board for ideas. also is widely used for patients with rheumatoid arthritis and psoriatic — Sandra Weller arthritis. Osborn’s first female faculty recruit As a faculty member at the New ’’ York University, Albert Einstein and University of Connecticut medical

MAY 2019 ASBMB TODAY 7

Remembrances These are excerpts from remembrances by scientists who worked with Mary Jane Osborn. Read the complete texts at asbmb.org/asbmbtoday.

In 1982, Mary Jane was selected to give a Harvey lecture. It’s a tremendous honor to be chosen to give this lecture, and the whole lab drove to New York to hear her present “Biogenesis of the outer membrane of Salmonella.” I knew MJO would give a wonderful talk, that was a given, but I honestly will never forget that night. Our group arrived and sat together. We watched as the Harvey Society members in their formal attire filed into the theaterlike hall, and then Mary Jane was introduced. From the moment she took the podium, she owned the room, expertly This photo of Mary Jane Osborn was taken for an presenting an amazing body of work. There could be absolutely no doubt Annual Review of Microbiology memoir she wrote she had truly advanced the field. I returned to the lab both tremendously recently. “It is very touching to me, as it shows frailty but also intense curiosity,” Sandra Weller said proud to be part of her group and inspired to continue my studies. of the photo.

— Pamela A. Marino schools, she became a leader in studies of bacterial endotoxin (also known as At first, my lab was right across the hall from Mary’s operation. I was lipopolysaccharide). This highly toxic a bit scientifically lonely, having moved from a large lab as a postdoc- molecule is located on the surfaces of toral fellow to a new department where it was just me and a technician. a large group of bacteria that include Fortunately, I quickly learned that I was always welcome to join Mary the causative agents of life-threatening at the blackboard outside her lab or in her office. She was never too busy diseases such as typhoid fever and to listen to my latest experiment or idea and then explore it with me. I meningococcal meningitis. This work always left the blackboard or her office with great advice and new ideas. prompted her to confront a puzzling My focus on DNA tumor viruses was unrelated to Mary’s interests, but paradox — many cells are capable of fabricating very large complex she generously shared her time to mentor a young colleague. molecules, such as endotoxin, that lie — Thomas Shenk outside the cell body despite the fact that all of the building blocks and the energy sources required for their Her influence is such that in my current research seminar I show slides synthesis are inside the cell. Begin- of two of her papers. Despite the fact that both of these were published in ning in 1972, she used a combination the 1970s, they remain very relevant. One of these describes the Osborn of bacterial genetics, biochemistry and electron microscopy to solve method for purifying the outer membrane. In this paper she notes that all of the components of the outer membrane are made inside the cell, and therefore mechanisms that transport these components across the cell ON THE WEB envelope to the cell surface must exist. I have spent the better part of my For an inspiring audio interview career trying to understand these mechanisms. and links to some of Mary Jane Osborn’s seminal papers in the — Thomas J. Silhavy Journal of Biological Chemistry and other journals, go to asbmb.org/asbmbtoday.

8 ASBMB TODAY MAY 2019 Mary Jane Osborn, Lawrence Rothfield and Sandra Weller pose together at a University of Connecticut departmental retreat in 2005.

She was the jewel in the crown of this medical school to New York City, often several times a month, to attend concerts and ballet and university. and opera performances. She was a ‘‘ special fan of the Paul Taylor Dance — Lawrence Rothfield Company, American Ballet Theater A longtime’’ colleague and sometime competitor and New York City Ballet. In 1968, she was one of the found- ing faculty who shaped the curricu- key aspects of this problem. In her Science Board, the governing body lum and character of the new medical seminal 1972 JBC papers, she de- of the National Science Foundation, school of the University of Connecti- scribed the technique that made these the country’s major funder of basic cut in Farmington. She served as chair experiments possible, now universally scientific research. of the medical school’s department of known as the Osborn method, which Osborn served on the ASBMB microbiology from1980 to 2002. She still is used in laboratories around the Council from 1974 to 1975, and remained there as a professor of mi- world. in 1981 she was elected ASBMB crobiology and of molecular biology Her research gained her interna- president, the second woman to hold and biophysics until her retirement tional renown, leading to her election that office. in 2014. to the American Academy of Arts and In the 1990s, she became inter- Sciences in 1977 and to the National ested in space science research. She Sandra Weller ([email protected]) is a Board of Academy of Sciences in 1978, 10 chaired NASA’s Committee on Space Trustees professor and chair of the department years after assuming her first faculty Biology and Medicine, which pro- of molecular biology and biophysics at UConn position. She was the president of duced a report that plotted the U.S. Health. several important biomedical societies space biology research program in the Lawrence Rothfield ([email protected]) and the recipient of many honors and first decade of the 21st century. is a professor emeritus in the department of molecular biology and biophysics at UConn awards. In 1980, President Jimmy Osborn had a keen interest in Health. Carter appointed her to the National poetry and the arts, and she traveled

MAY 2019 ASBMB TODAY 9 IN REMEMBRANCE d

The following ASBMB members died in 2018 and early 2019.

Alfred Alberts Julian Gomez-Cambronero Mary Jane Osborn 1931–2018 1960–2018 1927–2019 Thomas August Dwight Hall Woon Paik 1927–2019 1940–2018 1925–2019 Claude Baxter Geoffrey Hendy Joanne Ravel 1923–2018 1948–2018 1924–2018 Gunter Blobel Lowell Hokin Peter Reichard 1936–2018 1924–2018 1925–2018 Subir Bose Jerard Hurwitz Gordon Shore 1931–2018 1928–2019 1945–2018 Paul Boyer Jack Kirsch Donald Small 1918–2018 1934–2018 1931–2019 Ray Brown Laszlo Lorand Thomas Steitz 1924–2018 1923–2018 1940–2018 Peter Condliffe Barbara Low Frank Talamantes 1922–2018 1920–2019 1943–2018 Minor Coon Lewis Lukens Martha Vaughan 1921–2018 1927–2018 1926–2018 Richard Cowart Yves Marcel John Vournakis 1949–2018 1937–2018 1939–2018 Paul Englund Julian Marsh Tamio Yamakawa 1938–2019 1926–2018 1921–2018 Mark Fisher Andrei Medvedev Wu-Kuang Yeh 1954–2018 died 2018 1942–2018 Hugh Forrest Henry Metzger 1924–2018 1932–2018

We recently learned of the passing of these members.

William Claycomb Kenneth Kopple Deneys Van der Westhuyzen 1943–2016 1930–2013 1945–2017 Mahendra Jain Robert Lester William Wells 1938–2017 1929–2017 1927–2017

For news stories, remembrances and a video tribute, go to asbmb.org/asbmbtoday.

10 ASBMB TODAY MAY 2019 LIPID NEWS Lipid regulation of mitochondria By Yoshihiro Adachi, Miho Iijima & Hiromi Sesaki

itochondria are dynamic CL induces Drp1 origomerization organelles that grow, divide PA inhibits constriction of Drp1 oligomers and fuse in most of the human M Drp1 body’s cells; these dynamic membrane processes are critical for mitochondrial health. PA PA PA transport CL Our laboratory studies mitochon- PA drial division and fusion, focusing on ER three mechanochemical dynamin- CL PA PA stimulates related GTPases: Drp1, mitofusin MitoPLD mitofusin-mediated OM fusion and Opa1. Drp1 is a soluble GT- Pase that splits the mitochondrial membrane. Mitofusin and Opa1 are CL integral membrane GTPases that work together to fuse the mitochondrial Mitochondria Opa1-CL mediates IM fusion membranes. Mutations in each of HIROMI SESAKI ET AL. these enzymes lead to human diseases Cardiolipin, or CL, in the mitochondrial outer membrane promotes oligomerization of Drp1 to drive mitochon- that mainly affect central and periph- drial division; CL in the inner membrane mediates fusion through heterotypic interactions with Opa1. eral nervous systems. Mitochondrial division and fusion need to be balanced to main- from initiating the constriction of the chondria at these sites. Mitochondrial tain functional mitochondrial size, mitochondrial membranes, likely cre- PA levels also may be regulated by structure and distribution within cells. ating a priming step for mitochondrial this interorganellar interaction. This dynamic balance is controlled by division. Binding sites for CL and PA PA changes the mitochondrial several layers of mechanisms, includ- are different in Drp1; therefore, these membrane’s biophysical properties ing gene expression, post-translational phospholipids may create different and facilitates mitofusion-mediated modifications and protein degradation degrees of the regulation through a membrane fusion. Opa1, similar to of these GTPases and their binding combination of single or concurrent Drp1, binds CL, and this interaction partners. In addition, mitochondrial binding to Drp1. drives membrane fusion. Therefore, phospholipids play important roles in The production of CL and PA is lipid transport and synthesis coupled regulation of mitochondrial dynamics. a dynamic process in the OM. As to intramitochondrial contact site Drp1 interacts with two phospho- described above, CL is transported to dynamics and interorganellar interac- lipids, cardiolipin, or CL, and phos- the OM from the IM. This transport tions play key roles in controlling phatidic acid, or PA, in the mitochon- may be regulated through dynamic mitochondrial dynamics. drial outer membrane, or OM. CL interactions at the intramitochondrial is synthesized in the mitochondrial OM-IM contact sites. In the OM, Yoshihiro Adachi ([email protected]) is a inner membrane, or IM, and a frac- there is a phospholipase D, MitoPLD, postdoctoral fellow in Hiromi Sesaki’s lab in the tion of CL is transported to the OM. which converts CL to PA. Since Mito- department of cell biology at the Johns Hopkins Drp1 is recruited to the mitochondria PLD directly binds Drp1, conversion University School of Medicine. through its receptor proteins on the of stimulatory CL to inhibitory PA Miho Iijima ([email protected]) is an associ- surface of mitochondria; binding to may happen locally in the vicinity of ate professor in the department of cell biology CL stimulates Drp1 to assemble into the division machinery. PA at the Johns Hopkins University School of Medicine. high-order oligomers that function as also is produced in the endoplasmic a division machinery. The machinery reticulum, or ER, and imported into Hiromi Sesaki ([email protected]) is a profes- is regulated further by PA. Binding to the OM through ER-mitochondrial sor in the department of cell biology at the Johns Hopkins University School of Medicine. PA restrains the assembled machinery contact sites. Drp1 often divides mito-

MAY 2019 ASBMB TODAY 11 A YEAR OF (BIO) CHEMICAL ELEMENTS For May, it’s in your bones: calcium and phosphorus By Quira Zeidan

he International Year of the Periodic Table marks the 150th Tanniversary of Dimitri Men- deleev’s periodic system. The Ameri- can Society for Biochemistry and Molecular Biology is joining the celebration with a series of articles on biochemical elements. Since January, we have presented hydrogen; iron; sodium, potassium and chlorine; and copper. May is arthritis awareness month, so we selected calcium and phospho- rous, the two components of the mineral salt hydroxyapatite that makes up about 65 percent of the human adult bone mass. With chemical symbol Ca and atomic number 20, calcium is clas- sified in the periodic table as an alkaline earth metal. In chemical reactions, calcium easily loses the two valence electrons in its outermost orbital to form ionic compounds that CALCIUM & PHOSPHORUS contain dipositive Ca+2. At 3 percent of the Earth crust’s An electrical impulse traveling from a motor neuron results in the release of calcium from the muscle’s mass, calcium is the fifth most intracellular stores. Ca+2 binds to the inhibitory troponin-tropomyosin complex, allowing myosin and actin abundant element and the third most filaments to slide past one another causing muscle contraction. Adenosine triphosphate is hydrolyzed in the common metal after iron and alu- process. Relaxation follows when cytoplasmic calcium is removed. minum. Most of the Earth’s calcium is found as a carbonate mineral in transmitting the outside information which binds to its receptor on the limestone — sedimentary rock that to the interior of the cell. In bacteria, receiving cell, opening ion channels contains fossilized sea life. Calcium this calcium signaling system regulates and allowing the influx of extracel- carbonate makes corals, sea shells and chemotaxis — or movement toward lular Ca+2. At synapses, the inflow pearls when Ca+2 released by weather- a chemical stimulus — and flagellar of calcium propagates the electrical ing reacts with seawater bicarbonate. rotation. signal to the receiving neuron, and at Calcium is essential in biology. In mammals, cells respond to neuromuscular junctions, it triggers Both prokaryotes and eukaryotes hormones by activating the phos- muscle contraction in the receiving maintain low intracellular free Ca+2 phoinositide 3-kinase signaling path- fiber. via ion channels, transporters and way that leads to high intracellular Phosphorus — with chemical sym- calcium-sequestering proteins. In Ca+2 and expression of calcium-depen- bol P and atomic number 15 — response to environmental changes, dent genes. Excited neurons release intracellular Ca+2 rapidly rises, the neurotransmitter acetylcholine, CONTINUED ON PAGE 14

12 ASBMB TODAY MAY 2019 JOURNAL NEWS Bacterial drug synergies hide in plain sight Metagenomics may speed discovery of future therapeutic combinations By Laurel Oldach

hile on the hunt for a molecule with therapeutic Wpotential, Peter Mrak and his colleagues made a more sweep- ing discovery: Every known isolate of bacteria that produces the immu- nosuppressive drug rapamycin also can make a second compound that enhances rapamycin’s effect. “Put into simple words, this feels like finding an ancient treasure map in your grandfather’s attic,” Mrak said. Rapamycin was isolated in the 1970s from bacteria found on Easter Island. The molecule gives those bacteria a competitive edge by suppressing the growth of fungi in the soil. At first, researchers took a cue from nature and tried using rapamycin to treat fungal infections. Then they HULEROY0/PIXABAY found that it also suppresses growth and metabolism in human cells, nota- Rapamycin is named for Rapa Nui, or Easter Island, where the bacteria that produce it were initially isolated. bly those of the immune system. The drug is now used to prevent trans- called actinoplanic acids. This family After finding a few candidates, they plant rejection and stop tissues from of molecules had been isolated from showed by targeted mutagenesis that a growing into coronary stents. other microbes in the late 1990s, but cluster of closely spaced genes on the Mrak and an international team no one had worked out how they are Steptomyces genome can function as at the Swiss pharmaceutical com- synthesized. Mrak and colleagues no- a sort of biochemical assembly line to pany Novartis recently sought other ticed that actinoplanic acids contain generate actinoplanic acids, including beneficial compounds from the same a chemical group called a tricarbal- making the tricky tricarballylate. bacteria. Mining for natural products lylate that would be very difficult for When Mrak and colleagues looked with pharmaceutical potential in a synthetic chemist to produce in the for other bacteria that might carry this bacteria from diverse environments is lab and wondered whether they could gene cluster, they noticed something a standard approach with high success exploit bacterial means of synthesizing surprising. Every one of the fully rates. But in this case, the researchers that chemical group. sequenced bacterial isolates known found more than a single molecule. The researchers used a bioinformat- to make rapamycin also carried the In culture media that had been ics tool called antiSMASH to comb genes to make actinoplanic acid. This used to grow a rapamycin-producing through the Streptomyces genome, strain of Streptomyces, the scientists hunting for possible enzymes involved found a group of secondary products in actinoplanic acid production. CONTINUED ON PAGE 14

MAY 2019 ASBMB TODAY 13 JOURNAL NEWS

CONTINUED FROM PAGE 12

is a reactive nonmetal that combines for growth by oxidizing H2PO3– or a sugar, a nitrogenous base and three with other elements mainly by shar- phosphite to inorganic phosphate. phosphate groups. The addition of ing electrons via covalent bonds. Phosphate groups are major structural phosphoryl groups to proteins during Free phosphorus is rare; the element components of nucleotides, which are phosphorylation changes protein normally is found in compounds in the building blocks for nucleic acids activity and/or cellular localization, oxidation states of +3, +5 and -3. like DNA and RNA. Phospholipids regulating a plethora of cell-signaling Phosphorus is the 11th most com- — which contain a hydrophobic fatty events. mon element on Earth. About one acid “tail” and a hydrophilic phos- gram of phosphate is found for every phate “head” — form lipid bilayers kilogram of the Earth’s crust, mostly that constitute cellular membranes. Quira Zeidan (qzeidan@ in the form of oxidized inorganic Most cellular metabolic reactions asbmb.org) is the ASBMB’s rocks formed over millions of years. are driven by chemical energy har- education and public outreach coordinator. Follow her on Twitter Phosphorus is required for all nessed from the cleavage of adenosine @quirazeidan. life. Some bacteria derive energy triphosphate, a molecule that contains

CONTINUED FROM PAGE 13

suggested that microbes might benefit patients is amplified when the drug said. “Considering that some (natural from having both molecules. is combined with molecules that, like products) show their best only in When the researchers tested actinoplanic acid, inhibit an enzyme combination with another molecule, actinoplanic acid and rapamycin as called farnesyltransferase. That com- there may be a number of new me- fungal growth inhibitors, they found bination of drugs is in clinical tests dicinal compounds hiding among the that the two molecules’ combined for cancer now. The authors say their natural products already discovered.” effect was greater than you’d expect approach could be used to find other DOI: 10.1074/jbc.RA118.005314 from simple addition. In other words, co-occurring groups of biosynthetic the molecules work synergistically. genes more rapidly. Laurel Oldach (loldach@ The research appears in the Journal “We could potentially shorten the asbmb.org) is a science writer of Biological Chemistry. path toward new therapies by learning for the ASBMB. Follow her on Twitter @LaurelOld. Doctors have found by trial and from examples which have evolved in error that the effect of rapamycin in nature over millions of years,” Mrak

SUMMER SESSION BEGINS IN JUNE

Course begins first week of June

14 ASBMB TODAY MAY 2019 JOURNAL NEWS The mystery of metformin By Martin J. Spiering

ean Sterne discovered in 1959 that metformin, or dimethylbiguanide, Jlowers blood glucose levels in people with Type 2 diabetes. Re- searchers now are studying metformin as a therapeutic agent for managing such maladies as fatty liver disease, cardiovascular disorders and cancer. Yet while the mode of action for other drugs — including the anticancer agent Taxol, the analgesic aspirin and the antidepressant Prozac — is well understood, metformin’s exact molecular mechanism remains a mystery. This is not for lack of trying. The journey to discover how metformin works includes two seminal studies published in the Journal of Biologi- WIKIMEDIA COMMONS cal Chemistry, now recognized as Metformin is prescribed to control Type 2 diabetes, but the drug’s exact molecular mechanism remains JBC Classics. a mystery. Mohamad-Yehia El-Mir and colleagues reported in 2000 that metformin inhibits respiratory complex I I in isolated mitochondria, Fontaine an important advance, according to in the mitochondria of liver cells, or said, but only in state 3 of complex David Carling, senior author of the hepatocytes. This suggested that the I, when ATP is produced, a state that second JBC Classics paper, which authors had hit the bullseye of met- the El-Mir study did not examine. was published in 2002. However, “it formin’s molecular target. However, However, the metformin concentra- wasn’t clear how metformin was caus- there was a wrinkle. tions required to affect complex I ac- ing AMPK to be activated, what the “Metformin’s effects on complex tivity directly are consistently higher molecular mechanisms were,” he said. I were indirect,” said Eric Fontaine, than those yielding the indirect effect, This provided an opportunity for one of the researchers involved in this suggesting the two effects are not Carling’s lab, which had a long track work and now at Université Grenoble linked. Confusion about metformin’s record of studying AMPK. Alpes in France. This indirect effect effect on complex I persists in the Metformin’s effect on AMPK decreased cellular respiration and literature and likely will be resolved intrigued Carling because “AMPK was highly specific, solely affecting only after metformin’s molecular acts as a very important, perhaps the mitochondrial complex I — but only target is pinpointed. most important energy sensor, within in intact hepatocytes. Another clue to metformin’s cel- mammalian cells,” he said. “If we put metformin into isolated lular target came to light in a 2001 Having previously developed a mitochondria, we did not observe an paper reporting that metformin skeletal muscle cell line called H-2Kb inhibition of complex I,” Fontaine activates AMP-activated protein that provided a reliable model to said. The reason for this finding kinase, or AMPK. Gaochao Zhou study AMPK in muscle cells, Carling remains unclear, but metformin’s and colleagues observed that met- and his group were ideally positioned indirect effect has been reproduced by formin-induced AMPK activation to take the next steps to look at the authors of the El-Mir paper and suppresses glucose production in the AMPK’s role in mediating by other labs. liver through gluconeogenesis and metformin’s effects. Later studies have reported that also promotes glucose uptake into metformin directly inhibits complex skeletal muscle. The study represented CONTINUED ON PAGE 16

MAY 2019 ASBMB TODAY 15 JOURNAL NEWS

CONTINUED FROM PAGE 15 small changes in AMP,” Carling said. including complex I. So, although metformin is much Carling believes researchers are “The first thing was that we got less potent than rosiglitazone in in- much closer to finding out whether really good (AMPK) activation in this creasing the AMP–to–ATP ratio, both or how metformin interacts with cell line, so that was brilliant,” he said. appear to activate AMPK through the complex I or other cellular structures. “We were able to confirm and repro- same general mechanism. “It’s been “(Complex I) is an obvious one to duce that metformin really did give a an interesting journey for the AMPK do,” he said. very robust activation of AMPK.” field to realize that the levels of change Perhaps the long journey to solving This was no small feat; metfor- in the nucleotides that you need are the mystery of metformin’s direct min fails to get into cells that lack a incredibly subtle,” Carling said. target will soon come to an end. specific transporter, organic cation In an ironic twist, later studies have Three of the scientists who made transporter 1, or OCT1, a technical shown that AMPK is, in fact, activat- major contributions to these find- detail that wasn’t common knowl- ed via two routes: one through AMP- ings have since died: Mohamad-Yehia edge at the time and may still bedevil to-ATP ratio-stimulated phosphoryla- El-Mir and Xavier Leverve, first and research into metformin’s effects tion by the upstream kinase LKB1 senior authors, respectively, of the in cells. What’s more, the H-2Kb and another via calcium-induced El-Mir et al. paper, and Lee Fryer, first cells, unlike some commonly used phosphorylation by another enzyme, author of the Carling group study. cell lines, express high levels of an calcium/calmodulin-dependent kinase Fontaine worked closely with upstream kinase — liver kinase B1, kinase 2, or CAMKK2. El-Mir for about two years and or LKB1 — that phosphorylates and “It turns out there are two distinct remembers him as a great friend and activates AMPK. pathways, just not the ones that we colleague. And Leverve, he said, was “The hindsight is that (the cells) thought,” Carling said. an inspiring mentor. “He was a fan- were able to take up metformin,” Both Carling’s work on AMPK tastic man — very, very charismatic. Carling said, “and they expressed the and that of El-Mir and colleagues Everybody who met him wanted to upstream kinase that is required for on complex I show that metformin work with him.” activation of AMPK in response to profoundly alters cellular energetics. Leverve’s training and experience metformin.” “I think there are a lot of knock-on as a physician in an intensive care With all these then-unknown fac- effects that interfere with complex I,” unit may have spurred his interest in tors in alignment, Carling’s research Carling said. bioenergetics, leading him to study team set out to test whether metfor- This might explain why it’s been so complex I and the compounds acting min and another antidiabetic drug, hard to pin down metformin’s direct on it. “He had a very open mind rosiglitazone, activate AMPK by target, but metformin’s elusive activity from the clinic, especially in the ICU, increasing the AMP-to-ATP ratio, in the lab, its relatively weak effects where there is a kind of an energy fail- a well-known AMPK inducer. The on the AMP-to-ATP ratio and its ure during a septic shock,” Fontaine authors first demonstrated that rosi- impaired uptake into some cells might said. glitazone activates AMPK, a finding be why it continues to be an efficient Carling said he appreciates that his not previously reported, and that it and well-tolerated antidiabetic drug. lab’s work has been nominated as a does so by dramatically increasing “The fact that it is a fairly poor JBC Classic, but he also noted, “It’s the AMP-to-ATP ratio. However, drug turns out to be its strength — it’s a great shame that Lee is not around metformin apparently did not mea- very unusual,” Carling said. “I think to see this recognition. Lee was a very surably change the ratio. This finding metformin is a great example of why productive scientist and had a big prompted the authors to conclude you shouldn’t get too hung up with impact on my lab.” that rosiglitazone and metformin mechanism (when you start) to test activate AMPK via distinct routes. whether a drug is effective.” This article has been edited for But there was a hitch to that This hasn’t stopped scientists’ ASBMB Today. Read more JBC Classics conclusion — with refinements in search for metformin’s direct target in at jbc.org. analytical methods, additional studies hopes of unraveling how it exerts its have shown that AMPK is sensitive effects and improving on those effects even to very small changes in the to more efficiently manage diabetes or AMP-to-ATP ratio. So small, in fact, cancers that have emerged as potential Martin J. Spiering (mspiering@ that Carling and his team had been targets of metformin-based therapies. asbmb.org) is the technical edi- unable to see them. Recent advances such as cryo-EM tor at the Journal of Biological Chemistry. Follow him on Twitter “We didn’t have sensitive enough have enabled detailed structural @spieringmj. methods available for detecting very studies of larger protein complexes,

16 ASBMB TODAY MAY 2019 JOURNAL NEWS How to catch ovarian cancer when it’s curable Researchers use proteomics to develop diagnostic tool from liquid biopsy By Laurel Oldach

ewer than half of ovarian cancer patients survive for five years Fafter their diagnosis. According to the American Cancer Society, this is because only about one-fifth of ovarian cancer cases are detected early, when the chances of successful treatment and recovery are highest. “If we could change this reality by detecting (ovarian cancer) at a curable stage, we could save many lives,” said Keren Levanon, a physician-researcher at Chaim Sheba Medical Center in VIRGINIA COMMONWEALTH UNIVERSITY Israel. A section of an ovary stained with hematoxylin and eosin, showing a roughly star-shaped tumor. This tumor In the journal Molecular & Cel- can be identified as aggressive because of its micropapillae, or outgrowths. lular Proteomics, researchers led by Levanon and Tamar Geiger of Tel cargo that may vary between normal out of every four healthy volunteers Aviv University report a new test for and malignant tissues. as healthy. It outperformed previous ovarian cancer that outperforms pre- Using proteomics, the researchers proteomics-based tests, which had less vious tests. They hope it can be used compared thousands of proteins in than 60 percent sensitivity. to screen women who are genetically uterine microvesicles from 12 healthy Though the study used fluids col- predisposed to the disease. volunteers and 12 cancer patients. lected during surgery, the proteomic The researchers searched for signa- Then they used machine-learning test can also be run on samples tures of cancer in uterine fluid sam- algorithms to search for patterns collected in a minimally invasive pro- pled by liquid biopsy during surgery. of protein abundance that differed cedure similar to intrauterine device They compared samples from women among the samples. insertion. The authors propose that it with ovarian cancer who had surgical “We developed a diagnostic set may help young women whose risk of treatment and from volunteers who of nine proteins that distinguishes developing ovarian cancer is known to had gynecological surgery for reasons women with ovarian cancer from be high in deciding whether to have unrelated to cancer, such as uterine healthy women with greater sensi- their ovaries removed as a preventative fibroids or benign ovarian cysts. tivity and specificity than reported measure. The method of isolating mi- Bodily fluids contain many pro- before,” Levanon said. crovesicles from bodily fluids to detect teins. Strong signals from the most The researchers then tested the set’s faint cancer signals also may have common proteins can mask signals accuracy in 152 women, 37 of whom promise for other difficult-to-detect from smaller amounts of cancer-linked were known to have ovarian cancer. types of cancer. proteins that also might be present. To The test had 70 percent diagnostic DOI: 10.1074/mcp.RA119.001362 overcome that difficulty, researchers sensitivity, meaning that it correctly isolated microvesicles from the uterine detected cancer in 25 of the 37 study Laurel Oldach (loldach@ fluid. Because microvesicles are shed participants who truly had cancer, asbmb.org) is a science writer from cells, they contain almost none including all early-stage cases; it also for the ASBMB. Follow her on Twitter @LaurelOld. of the signal-masking blood plasma had 76 percent specificity, meaning proteins. Instead, they contain protein that it correctly identified about three

MAY 2019 ASBMB TODAY 17 JOURNAL NEWS JLR celebrates diamond jubilee with special reviews By Laurel Oldach

diamond traditionally symbol- izes a 60th anniversary, and Athis month, the Journal of Lipid Research marks 60 years with a sparkling collection of special review articles. The JLR started as a quarterly journal in October 1959 in response to an explosion in methods and research in lipid biochemistry. It began publishing monthly in 1983, and in 2003 it joined the American Society for Biochemistry and Molecular Biology journals family. Since its 50th anniversary, the JLR K. PRENTICE ET AL./JLR 2019 has sponsored lectures at meetings in Each JLR perspective article features a graphical abstract that can be downloaded as a PowerPoint slide the lipid biochemistry field. For the for teaching. This one is from a review on fatty acid binding protein 4 by Kacey Prentice and colleagues at journal’s 60th anniversary, the editors Harvard University and Harvard School of Public Health. invited each of the more than 50 membrane. What’s more, hydropho- regulator of T and B cell activity, from researchers who have been recognized bic molecules such as cholesterol can within the cell where it’s made to with these invited lectures to write an be recognized and promote inflam- outside the cell, where it is sensed by update on his or her area of research. mation. G protein-coupled receptors. While not all the scientists could • Leukotrienes are lipid signaling participate, the 60th anniversary party mediators that contribute to ana- is off to an exciting start with the first Lipids in metabolism phylaxis and white blood cell traffic. set of reviews. The synthesis of a leukotriene from a and metabolic disorders These articles touch on several membrane lipid sometimes requires Energy storage in the form of broad themes in lipid science. They several cells to chip in. Robert Mur- triglycerides in adipocytes in lipid explain recent advances in well-known phy and Giancarlo Folco discuss how droplets is a basic building block areas, such as the role of lipid storage leukotriene synthesis is regulated in of obesity — and it also plays into in metabolic disorders, and also areas concert with other lipid metabolism insulin resistance and other metabolic that may not be so familiar, such as pathways. problems. Several articles discuss what transcellular biosynthesis of signal- • Macrophages contribute to we’ve learned in recent years about ing molecules. Whatever your focus atherosclerosis, or blood vessel plaque coordinating triglyceride formation. in lipid research, you’re sure to find formation, through inflammation. • Rosalind Coleman describes how something interesting. Here’s a quick Alan Tall and Marit Westerterp the enzymes that build triglycerides overview of the articles published review the hypotheses for how excess interact with other proteins to get so far. cholesterol can contribute to inflam- these energy-storing lipids made — matory signaling in macrophages and and used — at appropriate times. Lipids in immune signaling neutrophils. • Phosphatidic acid phosphatases Many diffusible signaling mol- • Sarah Spiegel and colleagues clip a phosphate group from phospha- ecules that affect immune activity describe a transporter that moves tidic acid to make a simpler backbone originally were components of the cell sphingosine-1-phosphate, a lipid that can be built into triglycerides.

18 ASBMB TODAY MAY 2019 JOURNAL NEWS

Companion reviews from Karen Reue standing for an old and familiar and Huan Wang and from George drug; other times it’s about targeting Carman and Gil-Soo Han discuss the new signaling pathways. human and yeast versions of these • Niacin was the earliest drug for enzymes. dyslipidemia. Johan Auwerx and • Without communication between colleagues review recent research tissues, whole-body lipid metabolism suggesting a new mechanism of ac- never could be coordinated. Marcus tion to explain why it lowers blood Seldin and Aldons Lusis describe their triglycerides. work using systems genetics to inves- • Lysophosphatidic acid is a tigate variability in how adiponectin G. TIGYI ET AL./JLR 2019 powerful antiapoptotic signal, but from fat tissues is received in the liver. This graphical abstract is from a review by Gabor like many such signals, it also can Tigyi and colleagues on the therapeutic potential of promote tumor growth. Gabor Tigyi Lipids in the development lysophosphatic acid type 2 receptor agonists. and colleagues describe their effort to develop an LPA mimic with less of rare diseases • The latest genetic disorder of dramatic negative side effects. Two reviews highlight genetic sphingolipids was discovered in • Finally, a review on inhibitors disorders of sphingolipid metabolism. 2017. Julie Saba describes the 50-year of the PI3 kinase pathway, a new Sphingolipids are found in mem- journey from the first description of class of drugs for cancer, highlights branes, where they can act as receptors a sphingolipid breakdown enzyme to the importance of signaling that or regulate membrane protein activity; the identification of a disease caused happens downstream of lipids. they also can work as signaling mole- when it is disrupted. Lewis Cantley and coauthors write cules after a little metabolic tweaking. about how cancer cells evade PI3K • In the past, many genetic diseases Lipids in pharmaceutical inhibition and how to reduce that resistance. seemed unrelated, but as our under- science standing of sphingolipid synthesis and turnover improves, it has become clear Along with our emerging under- Laurel Oldach (loldach@ that several are linked by disruptions standing of lipids’ roles in physiology asbmb.org) is a science writer to sphingolipid processing, Richard have come advances in how scientists for the ASBMB. Follow her on Twitter @LaurelOld. Proia and co-authors explain in their can modulate lipid-related signaling. review. Sometimes this means a new under-

MAY 2019 ASBMB TODAY 19 JOURNAL NEWS

From the journals By Gelareh Abulwerdi, Jonathan Griffin & Kerri Beth Slaughter

We offer a selection of recent early-onset Alzheimer’s disease. The study was published in the Journal of papers on a variety of topics from mechanism connecting this muta- Biological Chemistry. the American Society for Biochem- tion to the protein accumulation DOI: 10.1074/jbc.RA118.004511 istry and Molecular Biology’s three is unknown, however. Meng Lu journals, the Journal of Biological and colleagues at the University of Chemistry, the Journal of Lipid Cambridge revealed details of this Advancing phage Research, and Molecular & Cellular process by establishing stable cell lines proteomics Proteomics. expressing the mutation and observing them with fluorescence-lifetime Bacteriophages, or phages, are vi- and super-resolution imaging. They ruses that infect bacteria, and they are Peering into Alzheimer’s found that Arctic mutant Abeta42 ubiquitous across the planet. Phage protein aggregation formed five unique types of aggre- studies have provided fundamental gates that degraded more slowly than knowledge for the development of The hereditary Arctic mutation of those in wild-type cells. The authors molecular biology and important 42-amino-acid beta-amyloid peptide, suggest their model lays groundwork biotechnologies such as the CRISPR- known as Abeta42, causes aggres- for studying how intracellular amyloid Cas system. sive aggregation of the peptide in affects cellular compartments. The Researchers are working to

Signal transduction in sperm development Spermatogenesis is a highly regulated cell differentiation process that gives rise to the male gamete known as the sperm cell. This process takes place within the seminiferous tubules of the testis. During the last step of the cell differentiation process, the developing gametes undergo a number of structural changes in which they elongate, lose most of their cy- toplasm and gain important functional features such as the flagellum. The gametes then are released to continue their maturation process in the epididymis. Fertility deficiencies and offspring disorders can occur ENVER KEREM DIRICAN/WIKIMEDIA COMMONS if any of the steps in spermatogenesis are disrupted. This scanning electron micrograph shows human spermatozoa at 2,500 Previous studies of spermatogenesis have uncov- times magnification. ered the hormone pathways that drive the differentiation process. However, other areas of regulation, such tant signaling events during spermatogenesis. They as signal transduction during sperm development, coupled metal oxide affinity chromatography with require further exploration. For instance, protein mass spectrometry to generate a profile of the human phosphorylation is a signaling event that plays a role testis phosphoproteome that included 174 phos- in cell cycle regulation, cell death, cell differentiation phorylated kinases. They used their data to identify and other critical biological pathways. Protein phos- the most active kinases in the human testis, such as phorylation has been identified as a crucial player in cyclin-dependent kinase 12 and p21-activated kinase regulation of testis-specific events. 4, which function in splicing regulation and cell In a paper published in Molecular & Cellular survival, respectively. In the future, their findings may Proteomics, Judit Castillo and colleagues from Lead be used to validate targets for drug development for Pharma and the OncoProteomics Laboratory in the male fertility and testicular tumors. Netherlands performed phosphoproteomics studies DOI: 10.1074/mcp.RA118.001278 using human testicular tissue to identify impor- — Kerri Beth Slaughter

20 ASBMB TODAY MAY 2019 JOURNAL NEWS

Role of sphingosine in mitochondrial dysfunction and TBI

Traumatic brain injury, or TBI, is a public health concern in the developed world among young people who play contact sports and the elderly, who are prone to falling. Events after TBI can be separated into primary and secondary injuries. Primary injury occurs nearly instantly after TBI, while secondary injury begins usually within minutes or hours and includes events such as immune cell infiltration and activation, cytokine release and apoptosis. One secondary injury event is mitochondrial dysfunction. In a paper published in the Journal of Lipid Research, Sergei Novgorodov and colleagues at the University of South Carolina write about some mechanisms by which enzymes involved in sphingolipid metabolism lead to mitochondrial dysfunction and exacerbation of secondary injury after TBI. Sphingolipids play important roles in cells. Some, IAN MACDONALD such as sphingomyelins, are major building blocks Mitochondria, such as the one in this drawing, are the powerhouses of of cellular membranes, while others, ceramides and the cell. In this study, researchers show that sphingosine can disrupt sphingosine, are essential in cell proliferation, dif- mitochondrial function. ferentiation and programmed cell death. More than 40 enzymes in the brain metabolize sphingolipids. histological and pathological changes associated with The researchers showed that events after TBI cause human TBI. The NCDase-deficient mice had 37 stimulation of one of these enzymes, called neutral percent lower levels of mitochondrial sphingosine ceramidase, or NCDase, leading to accumulation of in their injured brains than the wild-type mice. The mitochondrial sphingosine and eventually mitochon- finding that sphingolipids can cause mitochondrial drial dysfunction exacerbating secondary injury. dysfunction, which in turn can exacerbate neuro- To investigate the role of sphingosine in secondary inflammation, could open new avenues for drug brain injury, Novgorodov’s team examined NCDase- discovery. deficient mice and wild-type mice that underwent DOI: 10.1194/jlr.M091132 controlled cortical impact injury to reproduce the — Gelareh Abulwerdi unravel crucial aspects of phage biol- tion. They identified several bacterial are limited by low resolution and ogy, including the phage and bacterial proteins that were expressed only the requirement for highly purified proteome during infection. Tran- during infection. Their results also samples. Xiunan Yi and colleagues at scriptomic and metabolomic studies generated a more comprehensive view the University of Texas present a new have been conducted to analyze gene of the phage proteome that will be technique that combines single-cell expression during phage infection, important for future studies of phage lysis with electron microscopy. In but proteomic analysis is required to biology. their report in the Journal of understand how gene expression is DOI: 10.1074/mcp.RA118.001135 Biological Chemistry, they write regulated post-transcription. that they used this method to image In a paper published in Molecular and count ribosomes of single Cae- & Cellular Proteomics, Marie- Up close and personal norhabditis elegans embryos through- Laurence Lemay and colleagues with ribosomes out several stages of development. The from Laval University in Quebec authors conclude that this method City describe how they used high- The ability to visualize macromo- holds significant promise for applica- throughput proteomics techniques lecular structures of single cells could tions in developmental, evolutionary to characterize the viral and bacterial aid understanding of a host of cellular and disease-related studies. proteomes at different stages of infec- activities. However, current methods DOI: 10.1074/jbc.RA118.006686

MAY 2019 ASBMB TODAY 21 High fructose diet and or EVs, but their role in infection is the development of a new approach not completely understood. Caroline to malaria vaccine development. metabolic syndrome Coelho at Johns Hopkins University DOI: 10.1074/mcp.RA118.000997 Have you looked at the sugar and colleagues report in the Journal content on your favorite soft drink or of Biological Chemistry that these Lipid profiles and chronic juice? Soft drinks and juices are high EVs contain several virulence factors in fructose. A high-fructose diet can including the pore-forming toxin lis- kidney disease lead to dyslipidemia and is a major teriolysin O. High-resolution fluores- Data on the association between risk factor for development of cardio- cence imaging captured real-time EV dyslipidemia and end-stage renal dis- vascular disease, or CVD, and insulin release from bacteria inside infected ease, the final stage of chronic kidney resistance. Andrew Butler at Saint mammalian cells. The authors suggest disease, are rare, and the results are Louis University, in collaboration that EVs are used for toxin release, not conclusive. Such data would be with researchers at the University of which adds to the current understand- valuable for a medical practitioner California, Davis, studied the role of ing of L. monocytogenes’ pathogenic putting a CKD patient on a drug reg- fructose-fed diet in developing CVD. strategy. imen. Ching-Wei Tsai and colleagues In a paper published in the Journal of DOI: 10.1074/jbc.RA118.006472 at China Medical University per- Lipid Research, they write that they formed a 13-year prospective study of fed rhesus monkeys beverages sweet- Immunomic screening of more than 4,500 patients with CKD ened with fructose-containing sugar between the ages of 20 and 90 to find and measured plasma lipid profiles as the Plasmodium proteome the association between lipid trajecto- well as lipidomic changes. They were Malaria is caused by a single-celled ries and risk of developing ESRD. In the first researchers to show a posi- eukaryotic parasite in the Plasmodium a paper published in the Journal of tive correlation between high fructose genus. This parasite is spread from Lipid Research, Tsai and colleagues consumption and an increase in the mosquitoes to humans, particularly in report that higher baseline levels of quantity of large lipoprotein particles tropical and subtropical regions of the lipoproteins such as LDL cholesterol such as very-low density lipoproteins, world, leading to severe disease and and total cholesterol correlated with known as VLDLs, that are associated sometimes death. No vaccine exists to higher risk of developing ESRD in with CVD. Furthermore, they showed prevent malaria, and drug resistance patients with CKD. Overall, the that administration of fish oil reversed poses a major threat to fighting the incidence of ESRD in CKD patients the increased shift in the quantity infection. with high baseline triglyceride was 49 of large lipoprotein particles. They Developing a malaria vaccine has percent higher. showed a strong correlation between proved challenging, and additional DOI: 10.1194/jlr.P084590 concentration of plasma ApoC3, a studies are needed to identify new protein component of VLDLs, and parasite proteins associated with A protein that propelled elevation in the number of VLDLs. protection in humans. A limited life toward multicellularity ApoC3 affects the metabolism of li- number of malaria proteins have been poproteins, and its expression is a risk investigated, and a more comprehen- The basement membrane, or BM, factor for CVD. Next time, check the sive screening of the malaria proteome is an extracellular matrix component sugar content and think twice before using antibodies able to provide that is essential to tissue functions. you enjoy your favorite sweet drink. protection against infection could BM played a key role in the evolu- DOI: 10.1194/jlr.M089508 advance vaccine development. tionary leap of unicellular organ- In a paper published in Molecular isms to multicellularity, but its exact Bacterial extracellular & Cellular Proteomics, Anthony structure and function during this vesicles as virulent bombs Siau and colleagues from the Nanyang ancient transition are unknown. Carl Technological University in Singapore Darris and colleagues at Vanderbilt The bacterium Listeria mono- screening of the malaria proteome us- University now report that the BM cytogenes is the cause of listeriosis, ing antibodies able to provide protec- protein Goodpasture antigen-binding an illness that can present a major tion against infection. After the initial protein, or GPBP, likely played a key threat to pregnant women, newborns screen, the researchers used predictive role in this transition. By comparing and others with weakened immune criteria to calculate the likelihood of the genomes of some metazoans and systems. This organism has been protection for each antigen. Their unicellular organisms, they found found to secrete tiny lipid-bilayered proteomic techniques for studying that GPBP-2 is the oldest isoform of spheres called extracellular vesicles, protection provided a framework for GPBP and possibly functioned both

22 ASBMB TODAY MAY 2019 NAGASHIMA ET AL. A reporter gene assay shows the response of tobacco BY-2 cells to the caryophyllene structural analog caryophyllene oxide. How plants sniff out danger of VOCs and noticed that caryophyllene encouraged Plants cannot hear or see impending threats, but the expression of stress-responsive genes. To find they can smell them. Volatile organic compounds, which plant molecule was binding this VOC, they or VOCs, released by herbivore-infested plants can used a pulldown assay in which proteins extracted be sensed by other plants, which then, in response to from tobacco leaves were incubated with caryoph- the danger nearby, upregulate defense-related genes. yllene-linked beads. After separating the beads from While animals sense odors through membrane recep- the extract with magnets and washing them, they tors in their nervous systems, the same receptors are found that TPLs remained bound to caryophyllene. rarer and have different jobs in plants. It seems likely The role of TPLs in plant olfaction was probed that plants have their own unique way of sensing further by overexpressing the protein in tobacco VOCs, but molecular mechanisms that could explain cells. After exposing these cells to caryophyllene, they these olfactory processes have evaded researchers. found that the upregulation of defense-related genes In a study published in the Journal of Biologi- was reduced, which could implicate TPLs in the cal Chemistry, Ayumi Nagashima and a team of response of plant gene expression to caryophyllene. researchers from across Japan have made a significant This study provides evidence for VOC sensing step toward understanding plant olfaction by show- via a nuclear protein. Future studies could aim at ing that gene-suppressing TOPLESS-like proteins, illuminating the long-hidden mechanism underlying or TPLs, are critical for tobacco plants’ sensing and plant olfaction. responding to VOCs. DOI: 10.1074/jbc.RA118.005843 The researchers exposed tobacco cells to an array — Jonathan Griffin intracellularly and extracellularly been associated with elevated levels homeostasis and metabolism. in early metazoans. The GPBP-1 of intracellular glucosylceramide, DOI: 10.1074/jbc.RA118.005876 isoform emerged later in chordates as known as GlcCer, but how these an extracellular component, which lipids traverse cellular membranes is Gelareh Abulwerdi (gelareab@ the authors suggest likely allowed for not well understood. In a study in the umaryland.edu) is a Ph.D. candi- the development of epithelial tissues. Journal of Biological Chemistry, date at the University of Maryland, Baltimore. Follow her on Twitter Their results were published in the Bartholomew Roland and colleagues @gelareh_science. Journal of Biological Chemistry. from Vanderbilt University and Kyoto DOI: 10.1074/jbc.RA118.006225 University confirmed that certain P4- Jonathan Griffin (jgriffin@ asbmb.org) is a science communi- ATPases play a direct role in GlcCer cator for all ASBMB journals. Fol- Linking lipid transport to transport by observing uptake of low him on Twitter @spelledjon. metabolic disease fluorescently tagged GlcCer into yeast and human cells. They also found that Kerri Beth Slaughter Mutations in lipid transporting a central glutamine of the TM4 trans- ([email protected]) is a phospholipid flippases, or P4-ATPas- membrane segment is required for graduate student in the biochem- es, can lead to a variety of metabolic GlcCer transport. The authors suggest istry department at the University of Kentucky. Follow her on Twitter diseases, including insulin resistance that certain GlcCer-transporting @KB_Slaughter. and obesity. These mutants also have P4-ATPases may be critical for lipid

MAY 2019 ASBMB TODAY 23 SCIENCE NEWS Circulating oxysterol levels A new tool in detecting breast cancer By Kian Kamgar-Parsi

inding cancer before it has progressed or spread is crucial Fwhen trying to fight the disease. Despite this, early cancer detection remains a challenge. Cancers often can lie undetected without symptoms until it’s too late for effective treatment. Recent research, however, may present a new path to overcoming this obstacle. In an article in the journal Clinical Chemistry and Laboratory Medicine, F. Peter Guengerich of the Vanderbilt School of Medicine and an international team showed a correlation between certain circulating molecules and the presence of cancerous cells. Oxygenated metabolites of cholesterol, called oxysterols, are molecules tumors grow and are removed. levels change after tumor removal formed naturally through metabolic Particularly surprising to Gueng- and how those changes correlate with processes and play important roles erich and his team was that one oxy- survival rates. Researchers also want in mediating cholesterol and lipid sterol, 7-ketocholesterol, or 7-keto, to determine if changes in oxysterols metabolism. By measuring the blood actually decreased in concentration indicate cancer metastasis. levels of oxysterols in breast cancer pa- when tumors grew and increased This research project was limited; tients before and after tumor removal, in concentration after tumors were only 24 patients were involved and Guengerich and colleagues discovered removed. Previously, many scientists the study lacked healthy control that certain concentrations changed, believed that more cancer would subjects for comparison. “Cancer is indicating a potential role for them in mean more oxysterols. a heterogeneous type of disease, and cancer biology. “Even in this field, people have the we need more people and time to be “In general, some people think idea that all oxysterols are bad, but clinically useful,” Guengerich said. that most cancer is caused by things our work indicates that that may not Oxysterols such as 7-keto may not in the environment, like smoking or be true,” said Guengerich, who is a yet be useful biomarkers, but they the things they eat,” Guengerich said. deputy editor of the Journal of Bio- present a promising avenue of research “Turns out there’s a lot of stuff going logical Chemistry. “Levels of individ- and raise new questions: What causes on in our own bodies, like metabo- ual oxysterols are going up and down the change in oxysterol levels? Are lism, that’s driving cancer.” in relation to a cancer, so they’re not these changes a cause of breast cancer Oxysterols have been implicated necessarily all good or all bad, but all or a side effect? When answers are in breast cancer in the past, but this have their own type of biology.” found, they could lead to new tools in new research indicates that their role This richer picture of oxysterol the fight against breast cancer. may be more complex than previ- behavior gives rise to the hope that ously thought. While researchers the monitoring of oxysterols could Kian Kamgar-Parsi (kkamgar@ have known for years that there are become an early warning system for umich.edu) studied biophysics at multiple types of oxysterols, it turns cancer, although Guengerich warns the University of Michigan and is that we’re not there yet. Studies are a consultant for the pharmaceuti- out that these oxysterols can behave cal industry. very differently from each other when needed to determine how oxysterol

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MAY 2019 ASBMB TODAY 25 HARMONIZING LIPIDOMICS

26 ASBMB TODAY MAY 2019 FEATURE Harmonizing lipidomics Researchers strive to agree on what they’re measuring By Laurel Oldach

hatting at a conference a few people with a given disease have twice years ago, lipidomics research- as much of a certain lipid as healthy Cers Markus Wenk and Andrej individuals. If later studies back up Shevchenko noticed a problem. the finding, the molecule in question “We were having coffee over a begins to look like a promising analyte break during the sessions, and we were to screen for the disease. thinking, ‘There are a lot of papers For a lipid to be useful in the clinic, flying around now on plasma lipido- however, researchers must agree on the mics,’” Wenk, identity of the molecule in question of the National and on how much of it to expect in a University of healthy person. Lipidomics researchers Singapore, said. are working toward such agreements; Many of their field-wide project is mostly col- those studies legial but sometimes contentious and were looking provides a case study of how scientists WENK for biomarkers, create systems of measurement. molecules that change reliably during the course of Lipidomic beginnings a disease and might someday be used for diagnosis. To detect potential lipid The most common lipid biomarker biomarkers, researchers compare the is cholesterol. Clinical laboratories level of many lipid species in blood use an assay with colorimetric readout plasma from patients to those of to measure cholesterol in a patient’s healthy volunteers — often without blood plasma. A lab’s confidence in determining an absolute molar quan- its measurement comes from running tity of either. control samples with each assay and “The situation is very odd,” said from external certification. Shevchenko, of the Max Planck Insti- Lipid researchers have found nu- tute of Molecu- merous molecules that might someday lar Cell Biology predict diseases such as Alzheimer’s, and Genetics. cancer and metabolic disorders as “We cannot effectively as cholesterol predicts the compare data … risk of heart attack and stroke. But You iden- cholesterol is by far the most abun- tify significantly dant lipid in the blood. (See box on changing lipids, SHEVCHENKO page 28: What is a lipid?) To measure and do maybe molecules with much lower concen- thousands of analyses, but I cannot trations, scientists must use methods compare my results with yours.” with higher sensitivity, such as mass Not all biological studies must be spectrometry, or MS. strictly quantitative. Western blots, Forty years ago, using a mass fluorescence imaging and many spectrometer to measure lipids seemed Opposite: These colorful stripes are adapted from a proteomics approaches rely on semi- impossible. The technique starts by figure showing the wide variety of molar quanti- quantitative measurement. Likewise, ionizing molecules to measure their ties of various lipids in human plasma. For more a lipidomics study might show that mass-to-charge ratio; lipids, many of information, see page 37.

MAY 2019 ASBMB TODAY 27 FEATURE

QUEHENBERGER ET AL. JLR 2010

The LIPID MAPS consortium established a classification system for lipid molecules. This figure, from the consortium’s study of the lipidome of a pooled plasma sample, shows how the six major lipid classes (fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterols and prenols) are synthesized and interconverted as well as how they relate to other lipid metabolites.

What is a lipid?

Lipids are partly or completely hydrophobic lipids fall into six classes. small molecules. The simplest is a fatty acid, a Lipids famously form the plasma membranes carboxylic acid with a hydrocarbon tail. By adding that contain cells and eukaryotic organelles, and fatty acids to other molecules via esterification, triglycerides are stored for energy in lipid droplets. or by elaborating on a few other building-block Lipids also can function as coenzymes (for example, molecules like acetyl coenzyme A, cells can make ubiquinone in the mitochondrial electron transport a tremendous variety of molecules that fit into the chain, or vitamin K) and as diffusible signaling lipid class; LIPID MAPS’ database includes some molecules (a well-known example is S1P). 40,000 molecular species. In animal cells, these

28 ASBMB TODAY MAY 2019 which are nonpolar, can be difficult Three review articles in quick suc- to ionize. (See box on page 30: How cession pointed to a trickle of papers does mass spectrometry work?) demonstrating robust mass spectro- When he started measuring lipids metric measurement of lipids and in the 1980s, Richard Gross, a mass argued that, like the growing field of spectrometrist at Washington Univer- proteomics, high-throughput lipid sity in St. Louis, would homogenize measurement was poised to take off. buckets of samples to harvest enough of certain subcellular membranes to detect. Though the fundamentals The legacy of LIPID MAPS of mass spectrometry are the same, Before lipidomics even entered instrumentation has advanced a great the lexicon, Edward Dennis of the deal since then. University of California, San Diego, “Nothing has changed in the last says he made a case to the National 40 years,” Gross joked, “except the Institutes of Health that the field ion sources have improved by five or- would be a great investment. The ders of magnitude, the ion traps have NIH budget had doubled in the pre- improved, and so has detector resolu- vious five years. In 2000, the National tion. We’ve gone from working with Institute for General Medical Sciences garbage cans and introduced a new funding mechanism WIKIPEDIA/THETWEAKER canoe paddles called the glue grant intended to Many lipidomics studies use electrospray ionization. to working in propel interdisciplinary science for- This photo shows the ionization source in a mass spectrometer. The sample is ionized while it’s Eppendorfs.” ward with up to 10 years of funding sprayed from the needle, and gaseous ions then Gross’ lab was for projects with multiple principal enter the mass detector to the right. among the first investigators. Dennis thought he had to measure lipids just the project. extracted from GROSS “Developing an integrated metabo- tissue by MS, lomics system capable of character- publishing its first paper on the work izing the global changes in lipid me- in 1984. “From my perspective, that tabolites is a daunting task, but one was the beginning of lipidomics,” he that is important said, “although lipidomics wasn’t go- to undertake,” ing to be a word for 20 years.” Dennis wrote What took the technique so long in his initial ap- to catch on? Kai Simons, former di- plication. rector of the Max Planck Institute of The Lipid Molecular Cell Biology and Genetics, Metabolites and blames the molecular revolution in Pathways Strat- DENNIS biology. egy, or LIPID “It was DNA, RNA, proteins,” MAPS for short, got the grant. Its 12 Simons said. “There was so much PIs set out to develop rigorous labora- to do. Many of the most creative tory and bioinformatics techniques young researchers wanted to do what for lipidomics with approximately they saw before $73 million from the NIGMS over them — and 10 years. lipids were not By current estimates, human blood included.” contains millimoles of hydropho- Nonetheless, bic cholesterol but only tenths of a analytical chem- nanomole of the hydrophilic signaling ists continued to lipids called eicosanoids: a 10 million- make technical SIMONS fold difference in molar abundance. progress. In With such chemical diversity and 2003, the word “lipidomics” made its varied abundance, lipid measure- debut in the peer-reviewed literature. ment requires multiple approaches.

MAY 2019 ASBMB TODAY 29 FEATURE

ADAPTED FROM WORK BY PHILIPE HUPE/WIKIMEDIA Lipidomics experiments often begin by separating a sample through liquid chromatography (top). After ionization, the sample is passed through one or more mass analyzers (middle). The spectrum from the first detector (bottom left) represents ionized whole molecules. In tandem mass spectrometry, some parent ions are broken down and further examined by a second mass analyzer, which produces the second spectrum (bottom right). For more on how product ions are formed, see page 33.

How does mass spectrometry work? Mass spectrometry is the process of ionizing a sample, the same composition but different structures) and separating the ions by mass and charge, detecting the ionization suppression, which happens when two ana- ions, and recording a spectrum. lytes affect each other’s chances of picking up a charge. The output can be viewed as a series of mass-to-charge Because differences in technical approaches affect ioniza- ratios (also called features or m/z peaks) whose height tion and other variables, instrumentation and workflow represents their abundance. By determining the elemen- may introduce biases into different labs’ estimates of lipid tal composition of a peak, researchers can determine ratios. which molecule it might represent — a process called To estimate the amount of a lipid in a sample ac- annotation. curately, a researcher must compare its abundance to a Many experimental approaches elaborate on the core known concentration of a standard — usually a lipid ionization-separation-detection workflow. For example, synthesized either with a heavy isotope of carbon or using tandem mass spectrometry, a researcher can choose hydrogen or with an unnatural odd-numbered acyl a peak from the first spectrum and break it down further chain. The standard needs to have chemical properties as into constituent ions. It’s also possible to separate a close as possible to the lipid of interest so it will behave sample into fractions before ionizing it. roughly the same way in the mass spectrometer. Accord- Whether to fractionate samples is a long-running ing to Al Merrill of Georgia Tech, choosing standards can debate in the field. In shotgun lipidomics, complex lipid be difficult for researchers interested in a lot of lipids at extracts are injected straight into the mass spectrometer. once. By contrast, liquid chromatography separates lipids “There are so many lipids that they occupy a very large according to their size or chemical properties before ion- fraction of the possible masses that would be detected by izing them. mass spectrometry,” Merrill said. “Once you add these Analysis of a spectrum can be complicated by factors new materials, there’s a potential that they occupy the such as the existence of lipid isomers (compounds with space of something else that you might need to analyze.”

30 ASBMB TODAY MAY 2019 The LIPID MAPS consortium chose in 2006 the field was in its infancy,” core labs specializing in different lipid he said. “Just being able to see lipids classes to lead methods development and quantify (them) … was a big for each class. Al Merrill of Georgia step forward. People would look at Tech led the sphingolipid core. this with big respect. But that doesn’t “From the very beginning, the mean the right thing to do was to hope was that the field was going to standardize the approaches. Because explode,” Merrill said. “Our goal was there were no approaches to standard- to do anything ize. Right? There’s no way around we could to this — just to wait and let the field accelerate that develop.” process, and “When do you standardize? I think seeing it happen he makes a good point,” Dennis said would be the in reply. “(But) I don’t think LIPID biggest reward MAPS attempted to set the standard MERRILL for our efforts.” protocol or ever said it did. LIPID The consor- MAPS developed the best damn pro- tium developed a widely used system tocol it could, used that approach to for classifying lipid types, referring to measure plasma, and published it.” them by standard names and drawing them consistently. The American labs worked with leading Japanese Quantifying a lipidome and European lipid biochemists to A LIPID MAPS capstone proj- reach consensus on the system before ect was to quantitate as many lipid publishing it. According to Dennis, species as possible in human plasma, this collaboration helped to clarify using the technologies the consortium NATIONAL INSTITUTE FOR STANDARDS AND TECHNOLOGY later conversations. had developed. They chose a plasma Standard Reference Material 1950, a pooled sample “Everyone in the world has ac- sample supplied by the National In- of plasma from 100 volunteers, has been the subject of numerous lipidomics studies. cepted our nomenclature, our clas- stitute of Standards and Technology, sification and our structure drawing or NIST, pooled from 100 volunteers, program,” Dennis said. “There is no middle-aged men and women whose conference that argues about nomen- ethnicity matched the average U.S. clature … it’s just accepted by the population. worldwide community.” NIST develops many such refer- LIPID MAPS labs used identical ence materials for calibration of instruments to determine structures clinical assays. This particular sample, and quantities of thousands of lipid called Standard Reference Material species and built a website where 1950, came with certified measure- researchers could search for features ments of cholesterol, triglycerides, in mass spectra and match them to free fatty acids, and several steroid known structures. hormones and lipid vitamins. Oth- After the project’s funding expired erwise, the lipid content was largely in 2013, its large-scale collaborative unknown. projects came to a halt, but most Each LIPID MAPS core labora- of its researchers kept working in tory used the quantitative techniques lipidomics. that it had optimized for its class of Shevchenko, who was trained in interest to extract lipids from a vial of proteomics and has worked with SRM 1950, measure them and report lipids for about 20 years, believes back. The consortium measured just LIPID MAPS settled on instruments shy of 600 lipid species in six classes and approaches a little too early in the and published its findings in the process of global methods develop- Journal of Lipid Research. ment. In 2011, the year after the LIPID “With all my respect to colleagues, MAPS study was published, analytical

MAY 2019 ASBMB TODAY 31 FEATURE

chemist John Bowden took a and independent of the instrumenta- job at NIST. With the field of lipido- tion … and the type of data process- mics expanding, he saw the post as ing tools you use.” an opportunity The disagreement among labs was to contribute to considerable. “The problem was that standardization. (NIST) wanted to be neutral,” said “I tried to Kai Simons, the former Max Planck spread the mes- Institute director who now runs Li- sage at NIST potype, a company that participated that we needed in the study. “They didn’t want to BOWDEN to help stan- provoke a critical discussion, which dardize and har- would have been quite devastating.” monize the community in this type of Reception of the study, published measurement,” said Bowden, now a in the Journal of Lipid Research, was professor at the University of Florida. as mixed as the laboratories’ estima- “At the time, it was not a community- tions of lipid concentration. wide priority.” Oliver Fiehn, a metabolomics Bowden and his team launched a expert at the University of California, comparison exercise in 2014, using Davis, who participated, said that SRM 1950 to determine the vari- when measurements are technically ability in lipid identification and challenging and protocols differ, quantitation among 30 labs. Bowden expecting perfect concordance is told ASBMB Today in 2017, “We unrealistic. “People always think that (asked): ‘How much variability exists we should all get the same results … in the community right now, with (but) every single ring trial shows all the different methodologies and that there’s always a distribution of philosophies values.” for measuring Fiehn called the study a success. lipids?’” “Is the glass half full or half empty? If Each lab (others) say it’s half empty, I’d say its measured lipids half full.” in triplicate Xianlin Han, who wrote one of the according to its 2003 lipidomics review articles, called ULMER usual proto- the study a failure. “It turned out the cols, reporting result could be a threefold differ- concentration estimates for anywhere ence,” said Han, of the University of from 100 to 1,500 species. Candice Texas Health Sciences Center at San Ulmer, a postdoc on Bowden’s team, Antonio. “So what do the data mean? said the guidelines were intentionally Later on, if I want to study lipidomics vague. “We gave them free rein (in in plasma, I can order) to see, if there’s no guidance report any data, provided, how do they go about as- refer to this signing concentrations?” paper, and say, After anonymizing individual labs’ ‘Hey, my data measurements, Bowden’s team calcu- are within this lated a consensus mean, a median of range.’ That’s the average concentration measured a disaster. The HAN at multiple labs, for each lipid spe- outcome would cies. The median of means was more be very different if the organizers had robust than a weighted average to spiked a few standards into the NIST outlier values, of which there were plasma sample as controls.” several. Ulmer said, “Our idea with For Bowden, establishing the lack the consensus mean values was to of agreement on how to conduct and provide concentrations that are robust quantify lipidomics experiments was

32 ASBMB TODAY MAY 2019 LAUREL OLDACH

This schematic, based on information from LIPID MAPS, shows one way structural isomers can introduce challenges in mass spectrometry–based lipidomics. The phosphatidylcholine (top) and phosphatidylethanolamine (middle) molecules shown have the same chemical formula. After soft ionization, both have the same mass-to-charge ratio and appear as one peak in an MS1 spectrum (lower left), even though they may behave differently as part of a membrane. More destructive ionization enables researchers to differentiate the two by their fragmentation in MS2 (lower right).

Levels of lipid identification According to analytical chemist John to be analyzed with tandem mass spectrometry. Bowden, who led a NIST lipidomics study, Moreover, because distinct molecular spe- “Every level of identification of a lipid requires cies can have the same mass-to-charge ratio, some specific piece of data to support it.” researchers must reckon with overlapping peaks. When analyzing a mass spectrometry experi- For example, the two phospholipids pictured ment, researchers interpret a feature, usually are structural isomers. Though they consist of a spectrum peak, to annotate it or label its different head groups with four distinct acyl identity. There is a limit to how much structural chains and have distinct chemical properties, information a researcher can determine from a they have the same elemental makeup. To tell parent ion: the lipid’s class or head group, the them apart, researchers would need to break combined length of its fatty acyls, and how down a parent ion into smaller parts. many carbon-carbon double bonds or rings are LIPID MAPS and the Lipidomics Standards in the structure. It’s impossible to tell how fatty Initiative seek to promote annotation that acyl groups are oriented on the head group, and specifies the degree of certainty about a lipid’s where in a fatty acyl chain double bonds occur, exact molecular structure according to how an without breaking the molecule into smaller ions experiment was carried out.

MAY 2019 ASBMB TODAY 33 FEATURE

precisely the point. “At the time, we into the field,” O’Donnell said, “there really did not know what the exact is- are emerging issues around how you sues were in lipid measurement across identify lipids.” the community,” he said. The major challenge is that any The study helped participating one feature in an MS spectrum could laboratories spot problems in their represent a large number of mo- workflows and got the community lecular species. To be sure of a lipid’s talking about measurement quality, structure, a researcher needs several Bowden said. “There was a thinking dimensions of information, far more that everybody was doing the best we than a single MS run provides. (See could, given the resources … but if box, page 33: Levels of lipid identifi- we put enough energy into certain ar- cation.) eas, then maybe we can actually make Bowden and better lipidomics measurements.” his NIST colleagues wrote Identifying features a primer on reporting one’s What would it take to improve degree of certain- lipidomics measurement? ty in identifying Valerie O’Donnell, a professor a peak in a spe- EKROOS at Cardiff University, specializes in cial issue of the analysis of phospholipids and their journal Biochimica et Biophysica Acta signaling byproducts. “Up until 12 in 2017. In the same issue, another or 15 years ago, lipid research was paper explicitly called for standardiza- a relatively small field,” O’Donnell tion of lipidomics data. Its authors said. “Everyone knew each other; it’s include two scientists Bowden met always been an during the NIST study: Kim Ekroos, extremely col- an independent lipidomics consultant laborative but in Finland, and Gerhard Liebisch relatively small of the University of Regensburg in field of special- Germany. ists who worked Liebisch and Ekroos had come on individual across a troubling case of peak mis- lipid classes.” O’DONNELL identification in a 2015 paper in the Everything journal Clinical Chemistry. Using changed around 2005, O’Donnell standard metabolomics, the authors said, when new benchtop mass spec- reported finding six lipids that were trometers made the technology ac- higher in healthy volunteers’ serum cessible to more labs. “From then on, than in samples you didn’t have to be a physicist and from people with understand how to take apart and put Type 2 diabetes. back together a mass spectrometer to “We question use it to measure lipids.” the identities Instrument resolution also of the reported improved, making it possible to biomarkers and distinguish lipid species more accu- the lack of vali- LIEBISCH rately. According to Bowden, “We’re dation thereof,” uncovering all kinds of new lipids, Liebisch, Ekroos and colleagues wrote and we’re able to separate out lipids in a reply to the editors. They pointed you couldn’t separate out before.” out that the authors had reported These factors have drawn more more information about acyl chain researchers to look into the lipidome. length and position for some lipid But that growth brings challenges. species than their data could reason- “With lots of new people coming ably provide. Moreover, some species

34 ASBMB TODAY MAY 2019 were at unreasonable masses, suggesting they might be wrongly identified. The authors of the original study protested that they’d given adequate information under guidelines from the Metabolomics Society’s standardization initiative. There was, they conceded, an error in one of their annotations: a lipid species with one double bond was described incor- rectly as having none, accounting for the wrong mass. Liebisch said the exchange demonstrated that “lipids should be con- sidered a special case of metabolites, where simple matching of (spectral) features is not sufficient.” It also motivated the pair to think about problems in the field. Ekroos said, “We are starting to see quite a few errors in published data already … that we want to somehow try to clear up.” The LIPID MAPS website, redesigned in 2018, curates MS data on lipids and collects information about Michael Wakelam, director of the methods and tools. Babraham Institute associated with Cambridge University, has been do- headed the continuation of LIPID ing lipidomics MAPS. When NIH funding ended in studies for more 2013, lead bioinformatician Shankar than 25 years Subramaniam, a University of Cali- and agrees with fornia, San Diego, professor, landed a Ekroos. “We are small bridge grant to keep the website seeing more and online. more of this,” “They were at the point where WAKELAM Wakelam said of they were thinking that it may have error-prone to get shut down,” O’Donnell said. papers. “I think it’s actually a byprod- “I felt that was really bad, because we uct of the success of the field.” use this (tool) all the time, and we couldn’t do our work without it.” LIPID MAPS returns Wakelam, Dennis, Subramaniam and O’Donnell applied to the U.K.- By 2016, it was clear to many based Wellcome Trust to revitalize the experts that lipidomics researchers database. The project migrated to the needed help with standardization U.K., and curation of lipid species and reproducibility. Today, three and MS data resumed on a redesigned overlapping groups are working on website. the problem. One has begun work to “LIPID MAPS wants to play a big revitalize LIPID MAPS as a resource role in signposting our lipid biochem- for researchers in the field, a second istry colleagues to what’s out there for has focused on technical problems in big data analysis,” O’Donnell said. plasma lipidomics, and a third wants “Previously, it was a research proj- to reform publishing and reporting ect … but at the moment it’s really standards for all lipidomics data. (about) the database and having it The first team, led by O’Donnell available as a global online free open and Wakelam in the U.K., has spear- access resource.”

MAY 2019 ASBMB TODAY 35 FEATURE

MATEJ ORESIC This group photo was taken at a 2017 forum on harmonizing plasma lipidomics held in Singapore. After they noticed confusion in the field, Markus enkW (third from right) and Andrej Shevchenko (fifth from left) invited fellow lipidomics researchers to meet and hash out a path toward reference values for lipids in the blood.

The Singapore workshop teach you how to do lipidomics,’ the field will not respond,” Shevchenko At the 2016 Lipidomics Forum, said. “We have to be united.” where Wenk and Shevchenko shared Wenk agreed. “I don’t think you impressions over that cup of coffee, can tell scientists what they have to the two also gave the opening and do. You need consensus. We’ve been closing keynotes; they discussed find- very careful about this.” ing, respectively, quantitative varia- Shevchenko and Wenk reached tions within a healthy individual’s out to established PIs working on plasma lipidome and further vari- plasma lipidomics. Dennis, Ekroos, ability among individuals according Han, Liebisch, Wakelam and 13 to age, ethnicity, sex and prescription others responded, and in April 2017, medications. the two hosted a two-day meeting Both thought groundwork was in Singapore. Members of the group needed before biomarker discovery specialized in various lipid chemis- studies went forward. “Rather than tries using a variety of instruments study disease or complications, one and platforms. Their mission: figure needs to first actually get an idea of out how to harmonize studies of the healthy baselines,” Wenk said. plasma lipidome. That means finding out which “Of course, we all do it a little lipids are stable in healthy adults and differently,” Dennis said. “Nobody which ones vary by the hour — and said, ‘Oh, you do it right, Mr. X, we’ll determining how labs might reliably all just use your protocol,’ because measure each of those. Wenk and everybody’s invested in different ap- Shevchenko knew that if the goal proaches.” was consensus on these points, they The workshop hadn’t been going needed other scientists to buy in. on long when Tze Ping Loh spoke “If you publish a small article and up. Loh, a consulting physician in say, ‘Hey guys, we are in a position to the laboratory medicine department

36 ASBMB TODAY MAY 2019 at Singapore’s National University agree to measure Hospital, was a rare bird among the in the same way. panelists, a clinician in a room full of To define a basic scientists. reference interval “We’re all doing analytical chemis- is to come to a try,” Shevchenko said. “To get grants, consensus on we used to call (our work) biomarker what an analyte LOH or diagnostic discovery. But we don’t is, how to mea- do diagnosis. And then Tze Ping, the sure it accurately and reproducibly, in guy who actually does diagnosis … which populations it should be mea- explained to us what a diagnostic sured, and the statistical procedures is actually and how far we are from to calculate it. (See box on page 39: (that). He really hit us in the head.” Talking about reference intervals.) Loh explained the practice in The cholesterol test is a good laboratory medicine of establishing example. In its report, a clinical lab reference intervals, the range of con- notes how the patient’s total choles- centrations of a molecule measured terol levels compare to recommended in a chosen population when all labs targets. That gives a robust health

BURLA ET AL. / JLR 2019 A schematic diagram shows estimated molar quantities of various lipid classes in human plasma, measured by mass spectrometry. Each vertical line represents an individual lipid species, and horizontal bands of color represent range estimates. Lipidomics researchers are trying to narrow down these estimates and make sure they match ranges known from clinical chemistry.

MAY 2019 ASBMB TODAY 37 FEATURE

BURLA ET AL./JLR 2019 A lipidomics workflow schematic developed by participants at the Singapore lipidomics forum shows many points where a researcher’s decisions can introduce variability in a lipidomics experiment — even before a sample is loaded into the mass spectrometer. For example, many labs store and handle blood samples on ice to slow biochemical reactions and delay lipid oxidation. But that chilling may activate platelets, which then release bioactive lipids. Research groups interested in measuring those particular lipids reproducibly may handle samples at room temperature, perplexing colleagues from other labs.

indicator that any doctor or nurse can and working toward evidence-based interpret. agreements on other technicalities. Loh’s interjection helped move the At a follow-up meeting in Novem- conversation from debate over the ber, researchers were invited to help merits of different technical approach- select the lipid species most amenable es to comparing measurements among to clinical development. “When you these platforms, Shevchenko said. He come to all this granularity — they’re attributes the productive conversation all scientists, right?” Wenk said. “By in part to Wenk. paring it to a single task, my hope “This is Markus’ talent. He’s a guy is that it will be easier to come to a who has a vision to organize people general best solution to that particular without insulting them. So he got us problem.” together, with (our) different plat- The group settled on ceramides forms, different approaches, different and bile acids because these two backgrounds,” Shevchenko said. classes of molecule have well-defined Galvanized by that first day, the roles in disease and are thought to be workshop’s attendees started to lay out abundant, durable and easy to isolate. a plan for harmonization, beginning The research community can now with broad-strokes guidelines for work toward defining a method for lipidomics research. measuring these specific lipids and Balancing biases and adjudicat- determining their range in healthy ing conflicts among research camps individuals, Wenk said. can derail attempts to standardize. The article in the Journal of Lipid The standards initiative Research that came out of the Singa- pore working group took no stance Ekroos and Liebisch, the research- on most technical questions. Instead, ers who took biomarker hunters to the authors stressed the importance of task in Clinical Chemistry, partici- using internal standards, determining pated in the Singapore meeting but molar concentrations of each analyte found it too narrow in scope. In early

38 ASBMB TODAY MAY 2019 2018, they launched the Lipidomics helpful, could be an added burden for that are spearheading this,” Bowden Standards Initiative, or LSI. The two researchers. said. “But I think the ultimate goal is said they want to focus on getting “One needs to leverage what’s that everybody gets involved at some standards for reporting methods there,” he said. “One of those things point, and it’s not just a couple of and identifying lipids accepted by is a roster, or a carousel as I call it, of people.” researchers in the field and demanded small to medium-sized conferences.” However the field organizes its next by journals. Wenk envisions a decentralized steps, Shevchenko said, he sees enthu- “That’s an initiative that is more approach to standardizing the field, siastic support from the community. overarching than ours, that aims at where researchers come together at “I love the words ‘critical mass,’” he standardization in principle,” Wenk conferences, hash out clearly defined said. “We’ve reached a critical mass said. “It’s a much more heroic effort.” issues as they did in Singapore and of knowledge, of experience and of Modeling their project on efforts then send a report on to the next interest in transparent and confident in metabolomics and proteomics, conference. lipidomics.” Liebisch and Ekroos started a website Conference organizers including Scientists who contributed expertise with guidelines for the design and Keystone, Gordon and the American but were not quoted in this story include analysis of quantitative lipidomics Society for Mass Spectrometry have Shankar Subramaniam, Eoin Fahy, experiments, which Liebisch de- added lipidomics meetings to their Erin Baker, Bo Burla and Tze Ping scribed as a summary of current best programming for this year and 2020. Loh. practices. They also provided space for These come in addition to existing discussion about the protocols. periodic meetings in Europe, Aus- Laurel Oldach (loldach@asbmb. “We can’t really write these rules in tralia and Japan. The LSI and LIPID org) is a science writer for the stone today,” Ekroos said. “They have MAPS plan to host workshops at the ASBMB. Follow her on Twitter @ to be pretty open. But they should at Keystone meeting. LaurelOld. least guide people to do more what is “I think there’s a pocket of people correct.” Perceiving a common mission, the LSI quickly partnered with LIPID Talking about reference intervals MAPS. The groups held a joint workshop at the European Lipido- A conversation between Andrej Shevchenko and his mother, a mics Meeting in October to introduce physician, illuminates the gap between discovery lipidomics and what both projects. might be useful in the clinic. This is Shevchenko’s account of their “I haven’t seen so much debate and exchange, lightly edited for clarity: argument, constructive discussion, in a long time at a meeting,” O’Donnell My mom is a medical doctor. She’s had an M.D. for like 45 years as a practicing said. “People got really animated physician in a humble neighborhood, no science. She knows that I’ve been doing about different methods and different this plasma analysis in a clinical context, and she once said to me, “You know what, approaches.” Andrej? I think you do rubbish.” Participants debated “everything I was like, “Why?” from the start of the acquisition process right through to the data She said, “When a clinical analysis comes to my desk — talk about cholesterol, analysis … and reporting guidelines,” for example, or blood pressure — I have a table. I know the norm and I know the O’Donnell said. variation … Do you have these sort of ranges for normal values in patients?” I said, “No. We are kind of in the process.” A carousel of meetings She said, “You guys are cheating people. You’re saying you are aiming at So what happens next in lipido- diagnostics, but how can you talk about diagnosis if you don’t have normal mics? values?” Other fields of analytical chemistry, such as proteomics and metabolo- I was stumped. I said, “Yeah, but we are finding biomarkers …” mics, have formed standing com- “Forget about this, it is all crap. Before you arrive at a reference value, what the mittees, and the LSI seems poised normal parameter is, it’s all noise.” to follow that model. But Wenk believes forming a society for the And she is right. That’s what I think. harmonization of lipidomics, while

MAY 2019 ASBMB TODAY 39 FEATURE Meet Karin Musier–Forsyth At Ohio State, the JBC associate editor and tRNA trailblazer uses ASBMB resources as teaching tools for grad students By John Arnst

he pseudolife of a retrovirus is tRNA synthetases. We’re also inter- complicated. ested in single-domain trans-editing T Once it has infiltrated a host proteins. cell, the virus — here, HIV — must A long time ago, my interest in make use of a reverse transcriptase tRNA got me interested in retrovi- primer, lysine tRNA, to help tran- ruses, because retroviruses use tRNAs scribe its viral RNA into DNA that to prime reverse transcription. For can be sent to a host cell’s nucleus. example, HIV-1 specifically packages There, the new viral DNA will be a host cell tRNA group, that’s lysine- integrated into host DNA and hijack specific tRNAs, into the virus particle the mechanisms of DNA transcrip- for that purpose. We’re interested in tion to pump out fresh copies of the mechanisms of how the tRNAs viral mRNA. These will either be are selectively packaged and also how used as new genomes for viral cells or the genomic RNA is selectively pack- translated at host ribosomes to create aged into the virus particle. viral proteins — the collective raw A big question in the field is how ingredients, plus some stolen shield- and why exactly two copies of the ing from the host cell’s surface, for genomic RNA are packaged into new viral particles. every retroviral particle, when Gag as- In her lab at Ohio State University, COURTESY OF KARIN MUSIER–FORSYTH sembly of an immature virus particle Karin Musier–Forsyth probes the A proud alumna of Eckerd College, Musier–Forsyth can happen even in the absence of editing mechanisms of aminoacyl- is a member of the liberal arts school’s National genomic RNA. (Author’s note: The Advisory Council, and visits the St. Petersburg, tRNA synthetases, enzymes that at- Florida campus at least once a year. Gag polyprotein coordinates the as- tach amino acids onto transfer RNAs sembly, budding and maturation of during synthesis of HIV proteins at HIV virions.) But if genomic RNA is ribosomes. expressed in the cell, it gets packaged, Musier–Forsyth joined Ohio State Originally a Floridian, Musier–For- and exactly two copies get packaged. University as an Ohio Eminent Schol- syth grew up in St. Petersburg, where So how does that selective packaging ar in 2007. She has been a member of she attended Eckerd College and re- work? the editorial board of the Journal of ceived a bachelor’s degree in chemistry We’ve recently probed the confor- Biological Chemistry since 2012 and in 1984. She did her graduate work mational dynamics of the 5’ untrans- became an associate editor in January at Cornell University, earning a Ph.D. lated region of the genome, which is 2018. She spoke with John Arnst, in 1989. After an American Cancer a critical region for packaging of the ASBMB Today’s science writer, about Society postdoctoral fellowship at the RNA. We can see different factors, her work. The interview has been Massachusetts Institute of Technology both cellular and viral factors, that edited for clarity and length. from 1989 to 1992, she started as an shift the conformational dynamics, assistant professor at the University What is your group focused on? and we think these conformational of Minnesota, where she was named shifts are important for the packag- Merck professor of chemistry in 2003 We’re interested in two broad areas, ing process. I should mention our and distinguished McKnight univer- one being fidelity mechanisms in collaboration with James Munro at sity professor in 2006 and received protein translation. Specifically, we’re Tufts University. We’re using single- the Camille-Dreyfus Teacher Scholar focused there on quality control (or molecule methods to look at the Award in 1996. editing) mechanisms by aminoacyl- dynamics of these conformational

40 ASBMB TODAY MAY 2019 COURTESY OF KARIN MUSIER–FORSYTH Musier–Forsyth’s lab, currently more than 20 members strong, boasts research associates, postdoctoral fellows, lab technicians and undergraduate and graduate students from a variety of backgrounds. This group photo of the lab was taken in 2017. changes. We’ve been collaborating for aminoacyl-tRNA synthetases and Georgia Tech and got a real flavor for about two years, and we’re just about HIV — they’re much more than just what graduate school was like. I had to publish our first paper on this. housekeeping proteins, and their two co-authored papers as a result of All these interactions and packag- other activities are a big, emerging that, and I realized at that point that I ing processes involve protein-RNA area. I just edited a thematic series in was much more excited about research interactions, so that’s another way I JBC that covers some of these nonca- than medical school. sometimes describe my lab: we’re in- nonical functions of aminoacyl-tRNA As a postdoc, you have to decide terested in protein-RNA interactions. synthetases and their role in human if you’re going to go into industry or disease. academics or some alternative career. What was your academic I thought I’d give academics a shot, background and training? Did anything occur in a milestone because I really like working with sort of way that made you choose students and teaching and training When I was an undergraduate science as a career? students in the lab. studying chemistry, I became really interested in biochemistry, so I did I was interested in math and sci- How have your students my Ph.D. at Cornell in biophysical ence and was deciding between medi- responded to using ASBMB chemistry, and then I did a postdoc cal school and graduate school — I resources and JBC reviews at MIT with Paul Schimmel study- think that’s a common decision that in the classroom? ing aminoacyl-tRNA synthetases students that are interested in those and tRNA recognition. That’s where areas make. Some of my undergradu- For the first time, together with I really got interested in this family ate research experiences pushed me in two colleagues at OSU, I’m teaching a of enzymes that specifically charge the direction of graduate school and writing class for graduate students. It’s tRNAS, and I’ve been working in that a career in research. Specifically, one a small class, and I found the ASBMB area ever since. summer I was in a Research Experi- and JBC resources really useful, We also have a link between ence for Undergraduates program at especially Kaoru Sakabe’s Due

MAY 2019 ASBMB TODAY 41 the years, so now I’m excited to serve as an AE and help shape the future of scientific publishing at a journal that’s so well-respected and has been around for so long.

What do you do outside the lab? Any advice for balancing life in the lab with life outside of it? I think it’s really important to have life outside of work. I enjoy jogging and swimming in the summer. I have a 15-year-old son; and my husband, my son and I go on vacations a few times a year. We like hiking, camp- ing, skiing, things that get us out- doors and active. We just came back

NICHOLAS FORSYTH from spring break skiing in Utah. It was great — while we were there, it Musier–Forsyth and her husband, Craig Forsyth, also a biochemist at Ohio State University, have been skiing together for 34 years. Their son took this photo. snowed three feet. We also have a Siberian sled dog, a Samoyed named Lola. We didn’t take Diligence columns. My class is a mix also I think it’s useful to have them her skiing, but she would have loved of first-year through fifth-year gradu- experience that process. The JBC it. She definitely loves when it’s cold ate students, so they have different minireviews were perfect for that, outside here. I also walk/jog her every levels of what they know and what because they’re short, and the students day, so that also keeps me active. they’ve experienced, but I found that could pick one that interests them. these resources are very useful, espe- They’ve done a great job with that. Do you have any advice cially for the early students, if they I also picked some JBC reviews; for young scientists? haven’t published yet. we went through parts of them and I teach my students how to write a talked about how to write a good in- You should make sure that your review, because faculty often ask their troduction. We looked at the figures, work is something you’re really pas- students to help them write one, and because now we have a wonderful sionate about. Then you’ll never be figure consultant/artist at JBC. The bored or unhappy. It’s important reviews are perfect for second-year to have hobbies and do things with students, because they’ve had a little family and friends, but a significant experience but they’re just getting into portion of your day is going to be the research. spent at work. So if you love and are passionate about what you do, then How is your new role with JBC you’re going to be happier overall. so far? I find the work that I do at the university and at JBC to be really I really like being an associate edi- satisfying. It’s great to be able to teach tor of a society-based journal that’s and train graduate students and then run by scientists from the editor- see my graduate students go on and in-chief down to the AEs and the get postdocs and jobs and start train- editorial board members. Because it’s ing students of their own. run by scientists, I think the review process is really fair; the people that John Arnst (jarnst@ are reviewing the papers are also going asbmb.org) is an ASBMB KARIN MUSIER–FORSYTH to be wanting to publish someday. Today science writer. Follow him on Twitter @ arnstjohn. Musier–Forsyth’s dog is a four-year-old Samoyed I’ve had a great experience both named Lola. as a JBC author and as a reviewer over

42 ASBMB TODAY MAY 2019 PROFESSIONAL DEVELOPMENT ASBMB webinar offers financial advice By Jeff Pines

oth IRA. Credit cards. Student loans. Car loans. Checking Raccount. Personal finance can be confusing and a source of stress. Getting into good habits when you’re in your 20s or 30s can reduce the likelihood of long-term problems. To help instill those habits, the American Society for Biochemistry and Molecu- lar Biology recently offered a financial advice webinar for its members. Eric Goodbar, managing director at BNY Mellon Wealth Management, delivered the talk. Here are some highlights. “You can collapse the hype and jargon to a few clear concepts you can A key component of a financial the amount when you get pay raises. use throughout your career,” Goodbar plan is putting aside money for an This percentage is for people who said. emergency fund. Goodbar warned plan to live modestly, he said. Retirees The starting point is the checking that anyone might be out of work who plan to travel extensively will account. “Your checking account is for a time and it’s essential to have need to save more. the root of the financial tree of life. It’s enough money for three to six Goodbar made a couple of reading like an organic footprint. It’s essential months of living expenses in a savings recommendations: to balance your checkbook monthly,” account. Consider how much you • “The Infographic Guide to Goodbar said. He explained that spend on housing, food, utilities and Personal Finance: A Visual Reference balancing your checkbook will enable other monthly expenses. “Plan for the for Everything You Need to Know” you to track what you earn; what worst. Hope for the best,” he said. by Michele Cagan and Elisabeth you pay out for rent, bills and other Once you have a budget plan and Lariviere expenses; and what you keep. an emergency fund, consider invest- • “The Infographic Guide to Per- “Data capture of your income and ing for retirement. For investors in sonal Finance: A Visual Reference for spending pattern leads to a viable their 20s or 30s, the goal should be Everything You Need to Know” by budget plan,” he said. compounding interest, not maximiz- Tycho Press ing performance, Goodbar said. “Investing for the future is a bit Jeff Pines (jpines5720@ Join us on Twitter like forecasting the outcome of meta- gmail.com) has worked as a The ASBMB will host a Twitter bolic pathways,” he said. journalist since 1994, covering chat on finances for scientists There is a plethora of rules out corporations and investors for Bloomberg News, Bridge News in May. Follow us on Twitter there on how much to invest. Good- and Dow Jones and writing @asbmb for date and time in- bar suggests the 4 percent rule. Take about a variety of topics for formation, mark your calendar, your salary and multiply it by 4 per- service and housing providers and plan to join us with your cent. For example, let’s say you earn for the elderly. He served in the U.S. Air Force as a public affairs questions and comments. $50,000 a year. Multiply by 4 percent specialist. and you get $2,000 a year. Readjust

MAY 2019 ASBMB TODAY 43 PUBLIC AFFAIRS What we learned Graduates talk about the ASBMB’s Advocacy Training Program By Daniel Pham

goal of the American Society for Biochemistry and Molecular Biology is to provide our members with opportunities and tools to become lifetime advocates for science. Our Advocacy Training Program, implemented last June, A is doing just that. More than 20 ASBMB members have gone through this six-month externship, which provides hands-on training and advocacy experience, becoming community leaders in the process.

We invited participants in the first ATP cohort to share their experience and what they have learned through the program. Read their responses in the paragraphs below.

The greatest lesson I learned from being an ATP delegate was how to refine my advocacy strategy. I started the program well aware of the numerous challenges scientists face, but the ATP gave me an opportunity to critically think about how to realistically address those challenges. I learned that one of the most important aspects of advocacy is perfecting “the ask,” a specific policy request made to a legislator regarding a topic. It must be realistic, strategic and ambitious. A good “ask” also requires the advocate to possess a certain level of prudence when considering who they are advocating to. These invaluable lessons have carried over into my personal life, where I have taken initiatives to advocate for graduate students at large and evoke institutional change. Aria Byrd Graduate student University of Kentucky

During the ATP I co-founded the Health Sciences Student Advocacy Association at my university (@oneWSUHS). Our advocacy group focuses on innovating ways to amplify student voices on campus and beyond. STEM culture needs to change, and the fastest way to achieve change is to train scientists to advocate for change from within and teach them how to talk with legislators about change (and funding). Shannon Kozlovich Graduate student Washington State University

The ASBMB ATP made me dedicate time to connect with my congressional representatives and work on issues I find important. In the three months since the ATP program ended, I have started multiple policy projects including a pathway analysis of recycling and analyzing the scientific backgrounds of members of Congress. Sage Arbor Faculty member Marian University

44 ASBMB TODAY MAY 2019 Participating in the ATP took me to a new level in my involvement in science communication and advocacy. Through listening to speakers such as state representatives and science advocacy experts, engaging in hands-on advocacy, and learning about the fundamentals of policy, I was able to strengthen my skills to become an effective advocate. With the support and knowledge I gained from the ATP, I founded an ASBMB Student Chapter at my institution. Currently, I’m the president of the chapter I started, a member of ASBMB’s engagement working group, and vice chair of the ASBMB ATP alumni working group. Kelly McAleer Undergraduate College of New Jersey

Through the ATP, I was able to learn about effective advocacy strategies for all of science. We heard from experienced advocates as well as local representatives, which allowed us to construct effective advocacy messages. We were also able to hear from other scientists and cohort members with similar goals, which gave us fresh ideas and provided a support network for questions. I was able to sit down in offices and talk face-to-face with representatives at the local and federal levels to discuss topics such as STEM education and science funding. The ATP furthered empow- ered me to explore this passion for science communication and advocacy, and I am so grateful to have had the opportunity. Bailey Weatherbee Undergraduate student University of Delaware

Being a part of the ATP opened up the world of science policy to me, since I had very limited experience with the field during graduate school. Through working with the ATP, I’ve had mean- ingful interactions with elected officials as well as campaigns. I focused my efforts on working with my local congressional representatives to support legislation that combats sexual harassment in academic science — this is something I continue to work on. I also utilized the skills I obtained from the ATP to create a 2018 midterm science policy voter guide for my community, which is something I would have never thought to tackle before. Christa Trexler Postdoctoral fellow University of California, San Diego

The ATP has granted me the tools I need to be a science advocate. Before this program, I knew little about how laws that fund scientific research actually work or how my actions could influence them. Now I understand how to set up and have effective meetings with my representatives and organize advocacy events in my own community. In the most recent government shutdown, for example, I was able to guide peers in my department in writing letters to our state representatives to make our voices heard. I would not have had the courage or knowledge to do any of this without the ATP. Daniel Wilson Graduate student Carnegie Mellon University

Be an advocate Daniel Pham (dpham@ asbmb.org) is the ASBMB’s The third ATP training session begins in June. We are accepting applications public affairs manager. Follow through May 17. Learn more about the program and apply today at him on Twitter @dpham20. asbmb.org/advocacy/atp/.

MAY 2019 ASBMB TODAY 45 ESSAY Of mice and scientists Following instructions and creativity in research training By Daniel Bolon

hen I was a 22-year-old graduate student, I was instructed Wto add dry ice to a cage containing a white lab mouse and to wait until the carbon dioxide released from the dry ice purged the oxygen from the cage air and the mouse died. I had been taught to follow instructions, and I did as I had been taught. I asphyxiated two mice that day. I used an oven mitt to protect my hand from the cold of the dry ice pellets. I first weighed the pellets to ensure they would provide enough carbon dioxide to kill each mouse. Then I lifted the plastic lid on the top of the cage and poured the dry ice into a metal container with warm water in it. The warm water made the dry ice bubble rapidly and hastened the release of carbon dioxide gas from the pellets. I COURTESY OF DAN BOLON quickly closed the lid. Then I waited This photo of Daniel Bolon was taken when he was a graduate student, around the time he was first anxiously and uncomfortably. Within instructed to euthanize mice in the lab. minutes, each mouse lay down and stopped moving. After a further minute, my mentor checked that they not consider the possibility of asking were dead. Later that day, I harvested if what I was doing was okay or, even organs and analyzed the levels of a further, if it was sensible. I did not few proteins, though the purpose and value my own concerns and ideas. details of those experiments have long Why didn’t I value my own ideas? since departed from my memory. I do It was a habit from my childhood, vividly remember how I felt as I sacri- formed for good reason — it helped ficed the mice. I felt a tightness in my me to feel worthwhile. Following stomach, as if I were about to vomit, instructions made my parents happy, and a frightening sense of power — and it also made my teachers happy, imagine Voldemort with severe food which in turn gave me a sense of poisoning. accomplishment and meaning and I also felt something that I did not was a large factor in my decision to appreciate at the time — comfort and study science. Following instructions trust in following instructions. Expe- felt like eating ice cream on a warm rienced scientists had developed these day — it was satisfying and filled me instructions, so it was okay to follow with joy. them. This trust and comfort were Some 20 years later, I am a profes- so strong that they overwhelmed my sor who teaches and mentors stu- uncertainty about my actions. I did dents. I wonder about what and how

46 ASBMB TODAY MAY 2019 I should be teaching them. I wonder or clearly. Asking such questions as provide opportunities to practice free about the value of following instruc- a professor also drove me nuts. If a thinking and open communication tions and how this can be both a student struggled to answer, I would while learning to value our own ideas critical part of science and at the same wonder if they felt like a failure. My and wisdom. time an impediment to free thought, attempts to foster free thinking felt Is there a tradeoff between fol- creativity and ethics. I am convinced inefficient or counterproductive. lowing instructions and creativity in that both instruction following and In my search for more effective research training? Maybe there is, and free thought have an important role ways to foster confidence and creative encouraging free thinking will come in education. As a scientific commu- thinking, I stumbled on ethics. Dur- with a cost. My experience indicates nity, we already are adept at teaching ing casual conversations with students that the cost is minor compared to the the value of following instructions. and postdocs, I often would search benefits. I feel an urgent need to encourage for topics of mutual interest. What So, when is it appropriate to sacri- free thinking in the early stages of topics bring out broad interest from fice mice for science? science education. Devaluing personal scientists independent of their status I cannot answer that question for concerns and ideas is common among from student to leader? Ethical ques- you, but I hope my thoughts may graduate students in the sciences. As tions are the best answer I’ve found, help you to consider and value your an educator, I seek opportunities for and the conversations they engender own perspective and encourage others students to share their ideas and gain can stimulate creativity and confi- to do the same. confidence and a sense of value in dence. Discussions of ethical ques- their perspectives. tions provide a proverbial roundtable I have tried many unsuccessful where the perspectives of all scientists Daniel Bolon (dan.bolon@ approaches. For example, in the midst can carry equal weight: When is it umassmed.edu) is a professor in the department of biochemistry of explaining a 20-step experiment, I appropriate to sacrifice animals in and molecular pharmacology at would ask young scientists to consider research? When is it acceptable to the University of Massachusetts why a step was important. This type genetically engineer plants, animals Medical School. His lab studies of questioning drove me nuts when I and humans? Is it sensible for humans molecular evolution and seeks to understand how genotype was a student because it took all my to design robots to kill? What human contributes to phenotype. Follow energy to take in new instructions, population control measures, if any, him on Twitter @BolonDan. leaving me unable to think freely are appropriate? These discussions

MAY 2019 ASBMB TODAY 47 HILLDAY!

COURTESY OF ELIZABETH HUNSAKER Above: Elizabeth Hunsaker (left) and Callan Frye, grad students at Duke University and the Medical University of South Carolina, respectively, board the subway under the U.S. Capitol building with Michael Schaller, a professor at the University of West Virginia, during the ASBMB’s Hill Day in March. COURTESY OF DANIEL BASTARDO BLANCO

Upper right: Dorothy Shippen (left), a professor at Texas A&M University; Katherine Friedman, an associate professor and director of graduate studies at Vanderbilt University; and Daniel Bastardo Blanco, a graduate student at St. Jude Children’s Research Hospital pose for a selfie in front of the U.S. Capitol dome.

Below: Jazmine Benjamin (left) and Alanna Condren, grad students at the University of Alabama at Birmingham and the University of Illinois at Chicago, respectively, ride the subway under the U.S. Capitol with Anita Corbett, a professor at Emory University.

COURTESY OF DAN WILSON

COURTESY OF ALEXANDRA CHIRAKOS Above, center photo: Dan Wilson (left), a graduate student at the Univer- sity of Pittsburgh, meets with U.S. Rep. Michael Doyle, D-Pa., during the ASBMB’s Hill Day in March. At right is Jeff Brodsky, professor of biological sciences at the University of Pittsburgh. Above: Alexandra Chirakos, a graduate student at the University of Notre Dame, poses by the door of a congressman’s office. For more on Hill Day, see page 3.

COURTESY OF JAZMINE BENJAMIN

48 ASBMB TODAY MAY 2019 Washington University School UC Davis Health, School of Medicine of Medicine Assistant Project Scientist Cryo-EM Research Specialist

The Washington University Center for Cellular Imaging (WUCCI) is The Department of Physiology & Membrane Biology at the Univer- seeking an Cryo-Electron Microscopy Research Specialist to aug- sity of California, Davis, School of Medicine, seeks to hire a full-time ment current Cryo-EM staff capacity in the operation and support of employee as an Assistant Project Scientist to perform research func- the center’s current Titan Krios G3 and future Talos Arctica Cryo-EM tions in development and validation of recombinant antibodies and in protein engineering. platforms. Successful applicants will work in conjunction with existing staff under the general supervision of the Center Director. They will Although the Project Scientist is expected to work independently serve as one of the point people responsible for the oversight and under the general guidance of an academic member with an inde- maintenance of the Titan Krios and Talos Arctica platforms under the pendent research program (i.e., Professor, Professional Researcher, supervision of the Director as well as training and assisting users. Specialist in Cooperative Extension, etc.), he/she is not required to develop an independent research program or reputation. He/she They will also have the opportunity to work on correlative microscopy will carry out research or creative programs with supervision by an technology development projects currently underway in the research individual in an academic title that carries with it automatic Principal arm of the center. status. The Project Scientist does not usually serve as a Principal http://www.asbmb.org/Careers/Jobs/80002/ Investigator but may do so by exception. http://www.asbmb.org/Careers/Jobs/80000/

Wisconsin Institute for Discovery College of the Holy Cross University of Wisconsin–Madison Continuing Non-Tenure Track Position Biology and Health Professions Advising Professor in Metabolic Flux

We are seeking an established Principal Investigator, but will consider The Department of Biology and Health Professions Advising at the applicants at the Assistant Professor level, with expertise in metabo- College of the Holy Cross invites applications for a full-time Continu- lism and flux analysis as it relates to human health. Ph.D in biochemis- ing Non-Tenure-Track faculty position to begin in August 2019. We try or related field required. Candidates for associate or full professor are seeking a candidate who could contribute to high demand biol- rank must meet criteria for appointment at rank per UW School of ogy courses in areas of Cell and Molecular Biology such as Micro- Medicine and Public Health guidelines for appointment and promo- biology, Biochemistry, Genetics, Cell Biology, or Nutrition as well as tion on the tenure track. We seek applicants with a track record of biology courses for non-majors with a preference for courses related outstanding research accomplishments and exhibiting a strong commit- to health and human biology. In addition, the successful candidate ment to mentoring and teaching in a highly collegial and collaborative will join the Health Professions Advising Committee (HPAC) and also academic environment. serve as an Associate Health Professions Advisor at the College. http://www.asbmb.org/Careers/Jobs/79941/ http://www.asbmb.org/Careers/Jobs/79729/ JBC REVIEWS.pdf 1 2/22/19 4:12 PM

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