Discovering the mechanism of action of

nutritional therapy and evolving an

innovative formula for the treatment of

Crohn’s disease

A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy

Moftah Hussin Alhagamhmad

School of Women’s and Children’s health Faculty of Medicine The University of New South Wales August 2015

Table of contents

List of abbreviations …………………………………………………………………………………………..V

List of Figures……………………………………………………………………………………………...... IX

List of Tables……………………………………………………………………………………………...... XII

Publications………………………………………………………………………………………………....XIII

Acknowledgements...... XIV

Chapter 1: Literature review and aims

1.1 Background and disease discovery………………………………………………………………………..1

1.2 Epidemiology…………………………………………………………………………………………..….2

1.3 Pathophysiology of Crohn’s disease

1.3.1 Alteration in the immune response…………………………………………………………….….6

1.3.2 Role of bacteria…………………………………………………………………………………..13

1.3.3 Role of genetics………………………………………………………………………………….26

1.3.4 Role of environmental factors…………………………………………………………………...28

1.4 Diagnosis of Crohn’s disease

1.4.1 History and clinical examination………………………………………………………………..33

1.4.2 Laboratory findings……………………………………………………………………………..33

1.4.3 Imaging studies& endoscopic evaluation……………………………………………………….34

1.4.4 Crohn’s disease activity index…………………………………………………………………...34

1.5 Symptomatology of Crohn’s disease 1.5.1 Intestinal Manifestations…………………………………………………………………………36

1.5.2 Extra-intestinal manifestations…………………………………………………………………..36

1.6 Management of Crohn’s disease

1.6.1 Corticosteroids Medications……………………………………………………………………...42

1.6.2 Immunomodulators……………………………………………………………………………….44

1.6.3 AminoSalcylic Acid drugs………………………………………………………….…………….46 1.6.4 Biological agents…………………………………………………………………….…………...48

1.6.5 Antibiotics……………………………………………………………………………….……….51

1.6.6 Probiotics and prebiotics………………………………………………………….……………...53

1.6.7 Surgery…………………………………………………………………………………………...55

1.7 Role of nutritional therapy in management of Crohn’s disease

1.7.1 Overview………………………………………………………………………………………….57

1.7.2Mechanisms of action of exclusive enteral nutrition (EEN).……...………………………………61

1.7.3 Nutritional benefits of diet therapy………………………………………………………………..74

1.7.4 EEN as a treatment option………………………………………………………………………...77

1.8 Novel nutritional treatments

1.8.1 Glutamine……………………………………………………………………………………...….93 1.8.2 Arginine……………………………………………………………………………………….…..96 1.8.3 Curcumin………………………………………………………………………………………….98 1.8.4 Omega-3 fatty acids……………………………………………………………………………...102 1.8.5 Vitamin D3……………………………………………………………………………………....104 1.9 Project aims and hypotheses ……………………………………………………………………………105

Chapter 2: Methodology 2.1 In Vitro model of IBD

2.1.1 Cell culture and induction of inflammation………………………………………………………107

2.1.2 Cell viability assessment………………………………………………………….……..……….115

2.1.3 Enzyme Linked Immunosorbent assay…………………………………………………………...119

2.1.4 Western blot…………………………………………………………………….……………...…122

2.1.5 RNA extraction and amplification by Real-Time PCR…………………………………………..128

2.1.6 Single- Labelling Immunofluorescence…………………………………………………………...132

2.1.7 Kinase Assay…………………………….………………………………………………………..134

2.1.8 Affymetrix assay………………………………………………………………………………….136

2.2 In vivo models of IBD

2.2.1 Background…………………………………….…………………………………………………..138

2.2.2 Mice strain and acclimatisation…………………………………………………………………....141

2.2.3 Experimental protocols and induction of colitis…………………………………………………...142 2.2.4 Treatment composition and preparation…………………………………………………..……….145

2.2.5 Evaluation of colitis and response to treatment…………………………………………………....147

2.3 Ex-Vivo human cultured colonic biopsies

2.3.1 Introduction…………………………………………………………………………………….….152

2.3.2 Participants and inclusion/exclusion criteria………………………………………………………153

2.3.3 Endoscopy and histology examination…………………………………………………………….154

2.3.4 Biopsy culture and treatment………………………………………………………………………155

2.3.5 Cytokines immunoassay and LDH activity………………………………………………………..156

Chapter 3: Investigating the anti-inflammatory properties of PF and elucidating the mechanisms in vitro.

3.1 Introduction……….……….…………………..……………………………………………………158

3.2 Hypothesis...... …...160

3.3 Aims………………………………………………………………………………………………....160

3.4 Results…………………………………………………………………………………………...….161

3.5 Discussion………………………………………………………………………...…………………214

3.6 Conclusion………………………………………………………………..…………………………232

Chapter 4: Developing a novel nutritional therapy with enhanced anti-inflammatory properties for CD in vitro.

4.1 Introduction………………………………………………………………………………………....233

4.2 Hypothesis …………………………………………………………………………………….…... 234

4.3 Aims...... …....234

4.4 Results………………………………………………………………………………………………236

4.5 Discussion…………………………………………………………………………………….…..…255

4.6 Conclusion…………………………………………………………………………………………..262

Chapter 5: Exploring efficacy of the novel nutritional therapy in murine model of colitis

5.1 Introduction ……………………………………………….……………………………………….263

5.2 Hypothesis...... …...263 5.3 Aims…….……….………………………………………………………………………………….263

5.4 Results……………………………………………………..……………………………………….265

5.5 Discussion…………………………………………………………………………………………..284

5.6 Conclusion………………………………………………………………………………………….292

Chapter 6: Using ex-vivo cultured colonic biopsies from Crohn’s patient to investigate effect of the novel nutritional therapy

6.1 Introduction………………………………….………………………………………………….…..293

6.2 Hypothesis…….……………………………………….…………………………………………....294

6.3 Aims…..….………………………………………………………………………………………….294

6.4 Results.……………………………………………………………………………………………...295

6.5 Discussion……….……………………………………………………………………………….….302

6.6 Conclusion…….…………………………………………………………………………………….305

Chapter 7: Final discussion and conclusions……………………………………………………………..306

Bibliography ………………………………………………………………………………………………..315 List of Abbreviations

ALA: Alpha-linolenic acid

ANOVA: Analysis of variance

5-ASA: 5-Aminosalicylates

ASCA: Anti-Saccharomyces cerevisiae antibodies

ATG16L1: autophagy-related 16-like 1

AZA: Azathioprine

BCA: Bicinchoninic acid

BME: Basal Medium Eagle

BMI: Body mass index

BSA: Bovine serum albumin

CARD15: Caspase activation recruitment domainfamily-15

CBC: Complete blood count

CD: Crohn’s disease

CDED: Crohn’s disease exclusion diet

CDAI: Crohn's disease activity index cDNA: complementary DNA

CE: Capsule endoscopy

CEACAM: Carcinoembryonic antigen-related cell adhesion molecule

COX-2: cyclooxgenase-2

CRP: C-reactive protein

CT: Threshold cycle

DAPI: 4', 6-diamidino-2-phenylindole

DEG: Differentially expressed gene

DGGE: Denaturing gradient gel electrophoresis

DHA: Docosahexaenoic acid

DIA: Disease activity index

DMSO: Dimethyl sulfoxide

DSS: Dextran sodium sulfate

ECCO: European Crohn's and Colitis Organization E: Enterography

ESPGHAN: European Society of Pediatric Gastroenterology, Hepatology and Nutrition

EEN: Exclusive enteral nutrition

EF: Elemental formula

EMSA: Electro-mobility shift assay technique

EPA: Epicosapentanenoic acid

ESR: Erythrocyte sedimentation rate

F: Faecalibacterium

Ȧ -3 FAs: Omega-3 fatty acids

FBS: Fetal bovine serum

FDR: False discovery rate adjusted P value

FL: Fecal Lactoferrin

GH: Growth hormone

GIT: Gastro-intestinal tract

HLH: Helix-loop-helix

H2SO4: Sulphuric acid

HRP: Horseradish peroxidase

IBD: Inflammatory bowel disease

IBDU: IBD unclassified

IFN: Interferon

IG: Immunoglobulin

IGF: Insulin like growth factor

Iț%: Inhibitor of kappa B

,țț,ț%NLQDVH

IL: Interleukin

IRAK: IL-1 receptor associated kinase

LCT: Long chain triglycerides

LDH: Lactate dehydrogenase

LPS: Lipopolysaccharide

LZ: Leucine zipper MAP: Mycobacterium Paratuberculosis

MAPK: Mitogen activated protein kinases

MCP: Monocyte chemoattractant protein

MDP: Muramyl dipeptide

MEKK1: Mitogen-activated protein kinase kinase kinase-1

MEM: Minimum Essential Medium mM: Millimole

MLCK: Myosin II regulatory light chain kinase

6-MP: 6-Mercaptopurine

MPO: Myloperoxidase

MTT: (3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide; Thiazolyl blue)

MTX: Methotrexate

NEAA: Non-Essential Amino Acids Solution

NDS: Normal donkey serum

NF-ț%1XFOHDUIDFWRU 1) -ț%

NO: Nitric Oxide

NOD2: Oligomerization domain-2

OTU: Operational taxonomic unit

PBMS: Peripheral mononuclear cells

PBS: Phosphate Buffered Saline

PCDAI: Paediatric Crohn’s disease activity Index

PF: Polymeric formula

PGA: Physician global assessment tool

PSC: Primary sclerosing cholangitis

PUFA: Polyunsaturated fatty acid

PVDF: Polyvinylidene difluoride

RAGE: receptor for advanced glycation end products

RMA: Multi-array average

RT-PCR: Real time polymerase chain reaction

SCFA: Short chain fatty acid TBN: Total parenteral nutrition

TGF-ß: Transforming growth factor beta

TGGE: Temperature gradient gel electrophoresis

TIRAP: TLR associated protein

TLR: Toll like receptors

TMB: 3, 3’, 5, 5’-Tetramethylbenzidine

TNBS: Trinitrobenzene sulfonic acid

TNF-Į7XPRXUQHFURVLVIDFWRU-alpha

TRAF: TNF-receptor associated factor

UC: Ulcerative colitis

WR: Working reagent List of Figures

1.1 Aetiology of malnutrition in patients with CD………………………………………………………...….41

2.1 TNBS and DSS colitis model protocols…………………………………………………………….…...144

2.2 A summary for the protocol of ex-vivo colonic biopsy study………………………….………………..157

3.1 Effect of glutamine, arginine, vitamin D3 and ALA on IL-8 production from HT29 cells in response to TNF-Į«««««««««««««««««««««……………………………………………... 163

3.2 Effect of combined glutamine, arginine, vitamin D3 and ALA versus standard PF on IL-8 production from HT29 cells in response to TNF-Į««««««««««««««««««««««««««166

3.3 Viability assay of HT29 cells treated with glutamine, arginine, vitamin D3 and ALA or PF………..…167

3.4 Effect of increasing glutamine concentration on IL-8 production from HT29 in response to TNF-Į«171

3.5 Viability assay of HT29 cells treated with increasing glutamine concentrations……….……………….172

3.6 Effect of increasing arginine concentration on IL-8 production from HT29 in response to TNF-Į««173

3.7 Viability assay of HT29 cells treated with increasing arginine concentrations………………………….174

3.8 Effect of increasing Vitamin D3 concentration on IL-8 production from HT29 in response to TNF-Į175

3.9 Viability assay of HT29 cells treated with increasing Vitamin D3 concentrations. …………………….176

3.10 Effect of increasing ALA concentration on IL-8 production from HT29 in response to TNF-Į«««177

3.11 Viability assay of HT29 cells treated with increasing ALA concentrations………….………………..178

3.12 Viability assay of Caco2 cell line treated with increasing concentrations of glutamine or arginine….180

3.13 MTT cellular activity assay of HT29 cell treated with increasing concentrations of glutamine or arginine………………………………………………………………………………………………………181

3.14 Effect of glutamine and arginine, individually and in combination, on IL-8 production from TNF-Į exposed HT29 cells………………………………………………………………………………………….184

3.15 Effect of glutamine and arginine, individually and in combination, on IL-8 production from TNF-Į exposed Caco2 cells ……………………………………………………………………………………...….185

3.16 Outcome of combined glutamine and arginine on mRNA expression of IL-8 in response to TNF-ĮLQ intestinal epithelial cells ……………………………………………………………………………….…....186 3.17 ,țț DQG SK-,țț responses in TNF-Į VWLPXODWHG +7 FHOOV LQFXEDWHG ZLWK RU ZLWKRXW JOXWDPLQH RU arginine ……………………………………………………………………………….……………………..189

3.18 7RWDODQGSKRVSKRU\ODWHG,ț%LQ71)-ĮVWLPXODWHG+7FHOOVLQFXEDWHGZLWKRUZLWKRXWJOXWDPLQHRU arginine ……………………………………………………………………………….……………………..192

3.19 Cytosolic and nuclear portions of P65 in TNF-Į VWLPXODWHG +7 FHOOV LQFXEDWHG ZLWK RU ZLWKRXW glutamine or arginine …………………………………………………………………………...... 195

3.20 Immunohistochemical expression and nuclear migration of P65 subunit of NF-ț%in TNF-ĮVWLPXODWHG HT29 cells incubated with or without glutamine or arginine……………………...... …...... 198

3.21 *OXWDPLQHLQKLELWV,țțHQ]\PHDFWLYLW\LQDGRVHGHSHQGHQWPDQQHU…………………. ………….…..201

3.22 $UJLQLQHVXSSUHVVHV,țțHQ]\PHDFWLYLW\LQDGRVHGHSHQGHQWIDVKLRQ ……………………………….202

3.23 Total and phosphorylated P38 MAPK in TNF-Į VWLPXODWHG +7 FHOOV incubated with or without glutamine or arginine ………………..……………………….……………………………………………...204

3.24 Targeting of NF-ț% DW PXOWLSOH UHJXODWRU\ OHYHOV E\ FR-supplementation of glutamine and arginine…………………………………………………………………………………………………...... 210

3.25 Targeting the Apoptosis and survival Role of PKR in stress-induced apoptosis pathway by glutamine and arginine co-supplementation………………………………………………… …...... 211

3.26 HSP60 and HSP70/TLR signaling pathway targeted by glutamine and arginine co- supplementation...... 212

3.27 Targeting NF-ț%WUDQVFULSWLRQIDFWRULQWHUmediating IL-18 signaling pathway by glutamine and arginine co-supplementation …………………………………………………….………………………………...….213

4.1 Viability assays of curcumin treated HT29 and INT407 cell lines………………….…………………..237

4.2 MTT cellular activity assay of HT29 cell treated with increasing concentrations of curcumin………....238

4.3 Effect of curcumin on IL-8 production from HT29 cell line in response to TNF-ĮVWLPXODWLRQ«241

4.4 Effect of curcumin on IL-8 production from INT407 cell line in response to TNF-ĮVWLPXODWLRQ««242

4.5 Effect of curcumin pre-LQFXEDWLRQRQ,ț%UHVSRQVHVLQ71)-ĮH[SRVHG+7FHOOV«««««««244

4.6 Effect of standard PF versus PFs enriched with increasing glutamine and arginine concentration on IL-8 production from TNF-ĮH[SRVHG+7FHOOV««««««««««««««««««««««««247 4.7 Effect of standard PF versus novel formula on IL-8 production from TNF-ĮH[SRVHG+7FHOOV««248

4.8 Viability assays of HT29 cells incubated with different treatments including the novel formula……....250

4.9 MTT cellular activity assay of HT29 cell treated with various treatments including the novel formula...... 251

4.10 7KUHHDFWLYHFRPSRQHQWVRIWKHQRYHOIRUPXODFRPSOHWHO\VXSSUHVV,țțDFWLYLW\««««««««253

5.1 Survival rates of Balb/C mice treated with or without standard PF or novel formula following TNBS………………………………………………………………………………………………………...266

5.2 Body weight changes in Balb/C mice treated with or without standard PF or novel formula following TNBS…………………………………………….……………………………………………………..……268

5.3 Colonic cytokine expression of Balb/C mice treated with or without standard PF or novel formula following TNBS……………………………………………………………………………………………..271

5.4 Average daily intake of standard PF and novel formula in C57BL/6 following DSS…………………..273

5.5 Body weight changes of DSS exposed C57BL/6 mice treated with or without standard PF or novel formula…………..…………………………………………………………………………………….….…275

5.6 Colon weight and length score of C57BL/6 mice treated with or without standard PF or novel formula following DSS course………………………………………………………………………………………..277

5.7 Colonic cytokine expression of C57BL/6 mice treated with or without standard PF or novel formula following DSS exposure…………………………………………………………………………………..…279

5.8 MPO activity assay in colonic tissue collected from DSS exposed C57BL/6 mice treated with or without standard PF or novel formula……………………………………………………………………………..…282

5.9 Histological assessment of local colon injury of C57BL/6 mice following DSS exposure treated with or without PF or novel formula………………………………………………...……………………………….283

6.1 Consort diagram shows flow of participants and biopsy collection……….…………………………….296

6.2 LDH activity in cultured colonic biopsies……………………………………………………………….297

6.3 TNF, IL-8 and IL-6 release from colonic biopsies in organ culture…………………………………..…301 List of Tables

1.1 Five randomized studies comparing EN to corticosteroids as induction treatment in paediatric CD patients…………………………………………………………………………………………………..…….60

1.2 Shows clinical and histological remission of 47 children with CD...... …...69

1.3 Body weight, BMI and blood markers of 28 adult CD patients before and after EF treatment…….....….76

2.1 Sequences of IL-8 and Ʌ2M primers used for RT-PCR analysis of TNF-ĮH[SRVHG+7FHOOOLQH«.129

2.2 Concentrations of glutamine, arginine and curcumin components per 1ml standard PF to create the Novel formula……………………………………………………………………………….……………………....146 2.3 Mice primers sense and anti-sense sequences……………………….…………………………………..151

3.1. Summary of DEG profile and related immune pathways in TNF-exposed HT29 and incubated with co- supplementation of glutamine and arginine…………………………….……………………………..208 Publications

A portion of this thesis is included in a patent application (below).

Applicants: NewSouth Innovations Pty Limited and the Sydney Children's Hospital Network; Title: Pharmaconutrient Composition; Application No: 2013904917

This thesis is also a product of the following publications:

1. Alhagamhmad, Moftah, Day Andrew, Lemberg Daniel and Leach Steven. "An update of the role of nutritional therapy in the management of Crohn’s disease." Journal of gastroenterology 2012 47(8): 872-882. Full paper

2. ALHAGAMHMAD, Moftah. LEMBERG, Daniel. DAY, Andrew and LEACH, Steven (2013). "Defining the anti-inflammatory benefits of curcumin in vitro." Journal of Gastroenterology and Hepatology 28(2): 134-139. Conference abstract

3. Alhagamhmad MH, Lemberg DA, Day AS, Leach ST. Therapeutic and preventative anti- inflammatory benefits of curcumin in vitro. OA Inflammation 2013 Oct 01; 1(2):11. Full paper

4. Alhagamhmad, Moftah, Steven Leach, Andrew S. Day, and Daniel Lemberg. "Mo1232 Development of an Enhanced Nutritional Therapy for the Treatment of Crohn's Disease." Gastroenterology 148, no. 4 (2015): S-644.Conference abstract

5. Alhagamhmad, Moftah, et al. "Mo1231 Attenuation of Intestinal Inflammation by Co- Supplementation of Glutamine and Arginine." Gastroenterology 148.4 (2015): S- 644. Conference abstract

6. Nahidi, Lily, Susan M. Corley, Marc R. Wilkins, Jerry Wei, Moftah Alhagamhmad, Andrew S. Day, Daniel A. Lemberg, and Steven T. Leach. "The major pathway by which polymeric formula reduces inflammation in intestinal epithelial cells: a microarray- based analysis." Genes & nutrition 10, no. 5 (2015): 1-9. Full paper

7. Alhagamhmad, Moftah, Steven Leach, Daniel Lemberg and Andrew S. Day. Changing patterns in the epidemiology of Crohn disease." Journal of Gastroenterology and Hepatology Research 2015 [in press]. Full paper

Acknowledgements

The one who is most deserving of thanks and praise from people is the God. I sincerely thank Allah for the wisdom, great favours and blessings he has been bestowed upon me during this research project, and indeed, throughout my life. I bear witness that none deserve to be worshiped except Allah and Prophet Muhammad peace up on him he is his slave and the last messenger. The lord has commanded us to be grateful for all of his blessings and we should fulfil all the necessary conditions, which are gratitude of the heart, gratitude of the tongue and gratitude every single part of our body to him.

The lord has also commanded us to be thankful for all people who helped us and made a positive impact in our life. Foremost, I would like to express my sincere gratitude to my supervisor Dr Steven Leach for his continuous support and for his precious time dedicated to my PhD study. I highly appreciate his patience, willingness to help, motivation, enthusiasm, and encouragement. Indeed, his guidance helped me a lot in all the time of research and thesis writing. He was so close to me throughout the three and half year study period sharing the ideas and working hard together. I should make it clear that the most valuable contributions were attributed to him. Certainly, I greatly EHQH¿WHG IURP KLV ZLGH UHVHDUFK EDVHG NQRZOHGJH DQG KLV DELOLW\ RI GHYHORSLQJ QHZ VFKHPHV Steven is one of greatest people that I had the chance to work with I wish him all success in his life. Besides my supervisor, I would like to thank my co-supervisor Professor Andrew Day, for his efforts in proof reading the drafts that was fast and very efficient; and for engaging me in new ideas and thinking broadly; and demanding a high quality of work. Of course, this project would not have been possible without his contribution and his genuine continuous advice.

I also would like to thank my father and my mother and all other family member including my wife and my two lovely daughters Noerhann and Ghazala for supporting me spiritually, emotionally and financially throughout my study. I should mention here my home country Libya for giving me the opportunity to finish my postgraduate study in such lovely place of the world Australia.

I should also thank various professionals with whom I had the opportunity to work. Firstly, I should knowledge Professor Avi Lemberg, head of Gastroenterology department for his profitable advice and helping in putting together research plans. He is also contributed largely in collecting colonic biopsies for the ex-vivo project that was part of this work. In the same regard I should mention all patients and their parents who accepted to be enrolled in my project and agreed to donate part of their tissue for the study. I pray for them to be cured and be in a better health condition. Moreover, I should thank Dr Nitin Gupta, Dr Ooei Keith, Dr Usha Krishan and Dr Leisza had been involved in collecting the colonic biopsies. Indeed, without their willingness and friendly cooperation that part of work would not be possible at all. Further I should made it clear the main source of funding of my project was from Gastroenterology department budget, as such I should again express my gratitude to Prof Lemberg who have not rejected any financial request related to my project. Secondly, I would like to thank Professor Anne Cunningham, head of Westfield research laboratory, for offering me such highly scientific environment to conduct most of my work and for the continuous financial support for my project. I should also acknowledge other staffs in the Westfield research Lab Dr Lily for helping in understanding basic cell culture techniques and Post graduate student Yuelia for helping in some immunofluorescent work. Thirdly, I should knowledge the staff in virology research lab Prince Of Wales Hospital for allowing me to carry out part of my experiments in their lab and use some of their equipment’s. Fourthly, my gratitude for all staff working in the animal house facility of UNSW including Professor Stephen Danon, Technician Sandy Spathose, Technician Robin and all other members for giving me the chance to conduct my animal work in their facility and helping in mice sourcing and the monitoring during the experiment. Additionally, I should be thankful to Professor Marc Wilkins and DR Susan Corley for conducting microarray analysis. Finally, I should thank Professor John Munday, Messay University New Zealand who helped in reading histology slides.

I shall offer my sincere appreciation for the learning opportunities provided by my annual review panel members Professor John Ziegler and Professor Adam Jaffle. Also I should recognise the genuine and friendly support from Professor Richard Lock, PhD coordinator in GRS, UNSW. I should remember him as a brother standing firm in my side whenever I feel weak and cannot do much by myself.

Last but not least the reviewers who accepted the request to review my thesis and dedicated part of their valuable time to assess my work. Chapter 1: literature review and aims

1.1 Background and disease discovery

Crohn's disease (also known as regional enteritis) is one form of inflammatory bowel disease (IBD). Crohn’s Disease (CD) can affect any part of the gastro-intestinal tract (GIT)

[1] and it is a chronic, episodic condition characterized by transmural inflammation and skip lesions [2]. In 1932, the American gastroenterologist Burrill Bernard Crohn and his two colleagues Ginzberg and Oppenheimer, were the first to describe the disease in many patients with inflammation of terminal ileum, and since then the disease was known as CD

[3]. CD is an emerging global health problem affecting a large number of populations throughout the world [4]. It has been reported that there are at least 70,000 Australian living with IBD, taking in consideration higher incidence of CD it is estimated there are around 41,570 cases of CD across the country [5]. Although CD is not a fatal disease, it significantly impairs the health, life quality, and psychosocial well-being of a substantial portion of affected patients [6-8]. Further, it is a disease with a significant cost burden related to health care utilization and productivity loss [9, 10]. 1.2 Epidemiology

CD is a worldwide illness and all age groups are vulnerable to the disease [11, 12], although, it is more prevalent in certain geographical areas and mostly affects adults [13].

The incidence of disease is generally more common in industrialized countries compared to developing countries [14]. Nevertheless, recent reports show that the incidence of disease is rising in both developed and less developed countries including China, South

Korea, India, Lebanon and Thailand [15]. Internationally, the incidence of CD ranges from

0.1 to 16 per 100,000 of the populations with higher rates recorded in Europe, the United

Kingdom and North America whilst lower rates reported from Africa, South America and

Asia [16]. In North America the incidence rates range from 3.1 to 14.6 per 100,000 of the population [17], and in Europe the incidence is reported to be 9.8 cases per 100,000 of the population [18]. As compared to Saudi Arabia where incidence was 0.94 per 100.000 of the population between 1983-2002 [16]. However, the highest records of incidence rate worldwide in the literature are from Geelong, Australia in 2008 where the incidence of disease was 17. 4 per 100,000, Canada in 2000 with 16.3 and in Canterbury, New Zealand in 2004 at 16.5 per 100,000 of the population [14]. More recently reports came from

Nelson, New Zealand of another population-based study where in 2012 the incidence was reported as 15.2 per 100,000 [19] and from North America where the annual incidence was approaching 20.2 per 100,000 [20].

Prevalence rate of CD is around 150 per 100,000 individuals worldwide [21]. The prevalence rates follow the incidence rates where the highest epidemiological data have been recorded from developed countries. In North America the prevalence varies from 44 to 201 per 100,000 of the population [11]. For instance, in the USA the prevalence of CD was 244 per 100,000 of the population in 2004 which considered one of the high prevalence rates worldwide [22]. However, the highest record was reported recently in

Europe with prevalence of 322 per 100,000 population [23]. In contrast, in less developed areas, despite that fact that the prevalence of disease is rapidly increasing, the overall prevalence rate remains much lower compared to Western countries [24].

There is an increasingly growing concern over the trend of the rapid increase in incidence and prevalence of the disease worldwide, and this is especially worrying with the sharp rise of the disease among the Asian population [24-26]. In one district of South Korea, based on a 20 year study, the incidence rate of CD rose significantly from 0.5 to 1.34 per

100,000 of the population [27]. In China the disease is no longer uncommon, according to a meta-analysis study reviewing descriptive data of CD (1950 to 2007) in one mainland city, the incidence is increasing annually by 0.848 [24]. Authors also found that the prevalence is rising by 2.29 per 100,000 of the population [24]. In Hong Kong, a hospital based cohort reported the incidence increased from 0.4 to 1 between 1990 and 2001[28].

Likewise, data from Singapore showed that the prevalence of CD has risen from 1.3 cases to 7.2 cases per 100,000 inhabitants [29]. Further, areas that have previously reported incidence rates amongst the highest in the literature are still experiencing a continuous growth in the number of people diagnosed with CD [30]. Denmark and Sweden have shown a rapid increase in the incidence rates of CD, rising from 4.1 per 100,000 in 1980 to

8.6 per 100.000 in 2005 [16]. In Wales (Cardiff) the incidence jumped from 5.5 in between

1986-1990 to 6.6 per 100.000 in between 1996-2005 [11]. In the USA the incidence rate of CD has almost doubled in the period in between 1991 to 2002 [31]. A Similar trend seen in

France when the incidence among paediatric patients increased from 3.5/100,000 in 1990 to 5.2/100,000 in 2005 [11]. Moreover, recent reports from Australasian studies also indicated there is an increase in rates of CD, especially in Australia and most notably among paediatric populations [19]. Phavichitr et al. [32] in a retrospective study of CD amongst Australian children in the state of Victoria, reported that incidence of CD increased almost 10 fold over 3 decades [32]. Israel, where CD rates are already considered high, has also experienced a rapid rise in rates of the disease [33]. The prevalence has increased by more than 4-fold in a 20- year period between 1987-2007 in kibbutz settlements in Israel [34]. A similar trend was also reported in another study of adolescent

Jewish Israelites, where the prevalence doubled over the 13 year period of study [35].

In terms of ethnic and racial distribution of CD the disease was considered to be a disease of Caucasians [4] and Jewish ethnic groups [36]. A study conducted in California, the rates of hospitalizations were equal among Caucasian and African Americans, however, the prevalence of CD among African American was two thirds of Caucasian American [37].

The authors also found that Hispanics and Asians were less affected by CD [37]. However, recent reports indicated that the disease is rapidly increasing among Asians [28], and more in migrants from Asia to West [38].

CD is a disease of adults where most of the cases of CD are diagnosed between the ages of

15 to 35 years [21]. However, there is a second peak for the disease which happens around age of 60 [4]. Of interest, there are also many cases of the disease in children 5 years of age and younger [39]. Overall, the disease remains common among the most productive section of community where 80 % of cases are below age of 40 [40]. Further, it is a slightly more common in females than males as the ratio of girls to boys is around 1.3 : 1 [41].

Nevertheless, in younger age groups the ratio is a slightly lower than in adults with a ratio of 0.97 to 1 girls to boys [41]. This was also shown in a 12 year prospective population based study, where investigators found that the incidence of CD was more common among adult female than their counterpart males, whereas boys were predominant among paediatric cases [39].

In summary, CD is a worldwide disease mostly affecting adults and is more common in people with Caucasia and Jewish ethnic backgrounds. Originally CD was considered a disease of western societies. However, recent reports show that it is an emerging disease throughout the world including less developed countries, particularly in Asia. Changing diet habit is a major environmental factor suggested to contribute to the recent trend toward the rapid rise, although, the effect of perinatal and early risk factors as well as improvements in living standards remain questionable. 1.3 Pathophysiology of Crohn’s disease

To date, CD remains a highly complex disorder with poorly understood pathogenesis [42].

Pathogenesis of CD is considered multi-factorial with genetics, microbial, immunologic, environmental, and other adjuvant factors contributing to the disease [43]. Histologically,

CD is characterized by patchy areas of inflammation with various degrees of oedema, erythema, ulceration and friability, resulting (in some individuals) in strictures [44] and/or sinus tract and fistula formation [45]. The primary lesion starts as a focal inflammatory infiltrate around the crypts that followed by ulceration of superficial mucosa and inflammatory cells invasion resulting in formation of non-caseating granulomas in the intestine [2, 46].

1.3.1 Alterations in the immune response

An altered immune regulation is one of the proposed underlying mechanisms of CD [47].

Although the exact mechanisms of initiation of the ongoing inflammatory processes are still poorly understood, the inappropriate activation of the immune system cells remains the hallmark of disease pathogenesis [48]. In CD, both innate (macrophage, neutrophil) and acquired (T and B cell) immune responses are activated with loss of tolerance to enteric commensal bacteria [49]. Immune (T) cells are activated via an interaction of the membrane bound Toll like Receptors (TLR), nucleotide binding oligomerization domain

(NOD) and bacterial components including lipopolysaccharide (LPS), peptidoglycan and bacterial lipoprotein [50, 51]. TLRs, key regulators of innate immune response, are characterised by an extracellular leucine-rich repeat domain and an interleukin-1 receptor type 1-like intracellular signalling domain [52]. There are at least 10 human TLR identified [53]. In the healthy intestine,

TLR are expressed in low numbers and are negatively regulated to maintain basal activation [54]; however, during inflammation their expression is up regulated [55].

Nuclear factor (NF)-ț%LVWKHPDMRUVLJQDOOLQJWDUJHWRI7/5DFWLYDWLRQ[56].

Of the NOD family of proteins, NOD2 works as an intracellular sensor for bacterial cell wall components [57]. NOD2 recognizes muramyl dipeptide (MDP), a component of the peptidoglycan present in the bacterial cell wall [58] and plays a role in the pathogenesis of

CD through selective binding with MDP leading to inappropriate activation of intracellular signaling pathways such as the NF-ț% SDWKZD\ [59]. NOD2 gene codes for CARD15

(caspase activation recruitment domainfamily-15), an intracellular protein expressed in a group of lamina propria cells (macrophages, neutrophils, dendritic cells, and Paneth cells) and plays a key role in intestinal innate immunity [51]. NOD2 expression is induced by inflammatory cytokines including Tumour necrosis factor (TNF)-ĮDQG interferon (IFN); however, its expression under normal conditions is low and is only elevated under inflammatory conditions [60]. NOD2 can associate through its CARD domain, with a group of protein kinases resulting in degradation of inhibitor of kappa B (Iț% SURWHLQVRI the NF-ț%SDWKZD\and thereby activation the IțB NLQDVH ,țț DQGVXEVHTXHQWSURGXFWLRQ of proinflammatory cytokines [61]. Following activation of the innate immune response, T helper cells (CD4+) also become activated and capable of triggering a series of pathological steps including synthesis and release of TNF-Į LQWHUOHXNLQ ,/ -2, and IFN-Ȗ ZKLFK IXUWKHU DFWLYDWH DQG DWWUact more inflammatory cells to release more pro-inflammatory cytokines [21]. Activated immune cells also start secreting prostaglandin E2 and leukotriene B4 that contribute to the vasodilatation and enhanced vascular permeability characteristic of CD [21]. Subsequently additional cytokines such as, IL-21 and IL-18, are also released from activated T cells contributing to ongoing mucosal inflammation [62, 63]. Thus, an imbalance between pro- inflammatory and anti-inflammatory cytokines is evident leading to an initiation and perpetuation of intestinal inflammation [64].

Increased levels of different cytokines in patients with active CD is evident [65, 66]. Pro- inflammatory cytokines levels that are altered in CD include IL-1 TNF, IL-6, IL-8 [67]; and anti-inflammatory cytokines levels that are altered include IL-4, IL-10 and

WUDQVIRUPLQJJURZWKIDFWRUȕ 7*)ȕ [67]. The imbalance in these key cytokines drives the dysregulated immune response seen in CD. Gene expression of many pro-inflammatory cytokines is driven through an activation of complex pathways, most notably the NF-ț% pathway, which plays a key role in CD pathogenesis [68, 69].

NF-ț%LVDPHPEHURIWKH5HOIDPLO\RILQGXFLEOHWUDQVFULSWLRQIDFWRUVRIZKLFKWKHUHDUH presently five known proteins: c-Rel (Rel), NF-ț% 3DQGLWVSUHFXUVRU3 3 5HO

A), NF-ț% 3 DQG LWV SUHFXUVRU 3  DQG 5HO % [70]. Functionally active NF-ț% encompasses homo- or hetero-dimers of these transcription factor proteins, which mostly include P65 as one of the molecules [71]. The transcription factor (NF-ț% LVDPDMRU regulator of the host immune and inflammatory response as well as it is involved in cell survival and protection from undergoing apoptosis [72, 73].

NF-ț%LVFRPSOH[HGLQWKHF\WRSODVPZKHUHLWLVLQDFWLYHWKURXJKDQLQWHUDFWLRQRIWKH

P65 or c-5HOVXEXQLWZLWKDJURXSRILQKLELWRU\SURWHLQV,ț%[74]. After receptor activation, a group of upstream kinases are activated, which then phosphorylate and activate NF-ț% inducing kinase (NIK) [75]. Activated NIK phosphorylate SURWHLQNLQDVHFRPSOH[,țțD key step in NF-ț% DFWLYDWLRQ WKDW SKRVSKRU\ODWHV ,ț% PHGLDWLQJ LWV XELTXLWLQDWLRQ DQG degradation [76]. 7KH,țțFRPSOH[LVFRPSRVHGRIWZRFDWDO\WLFVXEXQLWV,țțĮDQG,țțȕ

DQG DQ HVVHQWLDO UHJXODWRU\ VXEXQLW 1(02 ,țțȖ  [77] ,țțĮ DQG ,țțȕ NLQDVHV KDYH D catalytic domain at their N-terminal half and an activation loop (a leucine zipper [LZ] and a helix-loop-helix [HLH]) at their C-terminal half [78] $FWLYDWLRQ RI ,țț GHSHQGV RQ phosphorylation of its subunit [76]. The non-phosphorylated form of the activation loop overlaps over the kinase domain, preventing entry of ATP and peptide substrates and thereby interfering with activation of the catalytic domain [79]. Phosphorylation moves the activation loop away from the catalytic centre allowing its interaction with the substrates, thus its activation [80]. Later when the C-terminal is phosphorylated the enzyme reaches a low activity state and attached by phosphatases facilitating its deactivation [79] WKDWLVZK\,țțFRPSOH[DFWLYDWLRQRFFXUVLQDYHU\WUDQVLHQWIDVKLRQ[79,

81]. Once the ,ț%VXEXQLWLVSKRVSKRU\ODWHGE\,țțFRPSOH[LWLVUDSLGO\SRO\XELTXLWLQDWHGDQG degraded allowing translocation of P65 into the nucleus and initiating gene expression [76,

82, 83]. Migrated P65 binds to DNA response elements in gene promoter regions and initiates cytokine gene transcription [84]. In IBD, NF-ț%PHGLDWHVincreased production of pro-inflammatory cytokines in the gut mucosa: this is considered an essential element in the pathophysiology of intestinal inflammation [85]. Therefore, this regulatory pathway provides an attractive target to centrally block the inflammatory response [86].

NF-ț%FDQEHWDUJHWHGDWGLIIHUHQWUHJXODWRU\OHYHOVVWDUWLQJIURPWKHOHYHORIWKHXSVWUHDP signalling proteins including receptors and the other signalling molecules (receptor associated factors, adaptor proteins and the upstream kinases) to the level of nuclear gene promoter regions [87, 88]. However, targeting the pathway at the main regulatory step, the

,țț FDWDO\WLF DFWLYLW\ KDV JDLQHG PRVW RI WKH LQWHUHVW IRU WKH UHVHDUFKHUV LQ WKH GUXJ discovery area [89].

Additional pathways that also involved in CD pathogenesis include mitogen protein kinase cascades (MAPK) [90]. MAPKs family is a heterogeneous group of enzymes responsible for phosphorylating serine and threonine amino acids of numerous functional proteins of the intracellular signalling pathways [91]. There are several families of MAPKs including, extracellular regulated kinase 1/2 (ERK1/2), extracellular regulated kinase 3/4 (ERK3/4), extracellular regulated kinase 5 (ERK5), extracellular regulated kinase 7/8 (ERK7/8), P38 kinase, Nemo-like kinase (NLK) and the c-Jun N-terminal kinase (JNK) group [92]. In addition to their critical roles in several key cellular activities, including cell proliferation, differentiation, and survival or death, the MAPK signalling pathways have been implicated in the pathogenesis of human diseases[93].

Phosphorylation of specific amino acids in the motif region is a prerequisite and essential step for activation of each particular kinase [94]. Up on activation, MAPK can then stimulate other intermediary signalling regulatory pathways [95], or translocate to the nucleus to initiate gene expression of various cellular molecules including the inflammatory mediators [96]. In the context of IBD, involvement of MAPK in initiation of intestinal inflammation has been mainly centered about the role of P38 family [97, 98].

7KHUHDUHIRXU30$3NLQDVHVLVRIRUPVLQPDPPDOVĮȕȖDQGį$PRQJVWWKHVH3

0$3.LVRIRUPV3ĮLVWKHPRVWVWXGLHGDQGLVH[SUHVVHGLQPRVW FHOO W\SHV [99].

$FWLYDWHG3ĮKDVVKRZQWRSUHVHQWLQERWKF\WRSODVPDQGWKHQXFOHXVPHGLDWLQJVHYHUDO functions through targeting numerous cytoplasmic and nuclear targets [100]. During

LQIODPPDWLRQ 3Į 0$3. LV ODUJHO\ LPSOLFDWHG LQ UHJXODWLQJ WKH ELRV\QWKHVLV RI SUR- inflammatory cytokines (IL-8, IL-1, IL-6, anG 71)Į  [101], and other inflammatory mediators (COX2) [102]. The role of P38 in regulating these inflammatory mediators, which can arise at different levels, has been shown to be related to post-transcriptional regulation [103].That involves phosphorylation-activation of several substrate proteins including AU-rich element (ARE) binding proteins tristetraprolin (TTP) and poly (A)- binding protein PABP [104]. In addition to post-transcriptional regulations, P38 can affect gene transcription by phosphorylating and activating various transcription factors, most notably NF-ț%DQGperoxisome proliferator-activated receptor alpha [105, 106]. Inflammatory P38 MAPK is a critically involved in the pathogenesis of CD and its inhibition provides a novel therapeutic strategy [107]. Interestingly, in preclinical studies a number of P38 kinase inhibitors have been identified as potential treatments for IBD [108].

Further, several of these inhibitors have also progressed to clinical trial [97, 109].

Collectively, NF-ț% DQG 3 DUH DPRQJ WKH PDMRU UHJXODWRU\ SDWKZD\V LQYROYHG LQ &' pathogenesis and development. As a proof of concept, potential IBD treatments therefore should be investigated against these signaling pathways. 1.3.2 Role of bacteria

1.3.2.1 Overview

Several reports have ascertained a strong correlation between microbiota composition and various aspects of host health including immune response regulation [110]. Indeed, early development of the immune system is largely influenced by microbial colonization [111].

There is a substantial number of microbiota populating the mammalian host [112]. That necessities a critical role for the human host to recognise the commensal species involved in different physiological and immunological functions from pathogenic groups [113].

Disturbance in such microbe-host interactions predisposes to disruption in host immune tolerance, so that chronic inflammatory diseases may consequently develop [114].

In CD it is well recognised that an inappropriate mucosal immune response to luminal microbes plays a crucial role in the pathogenesis and development of disease [115]. That evidence is historically provided by the successful role of antibiotics in treating CD [116].

Further evidence comes from studies that have described a successful role for faecal diversion in preventing disease relapse [117]. With advances in the genome sequencing technology, the role of bacteria in the development of CD has become more focused on specific bacterial footprints (bacterial protein signals) associated with the disease [118].

However, the precise role that bacteria play in the pathogenesis of IBD remains elusive.

Nevertheless, there is a consensus that a shift in microbiota composition is the leading cause for high loads of bacterial antigens, which influence the host immune system to respond in an exaggerated perpetuating inflammatory response [119]. In a subset of patients (those with ileal CD) a group of the commensal bacteria invade and replicate in host cells and directly trigger the mucosal immune system [120]. Interestingly, it has been shown at the surface of microbial cells of CD patients there is a reduction in glycoprotein-

P2, facilitating bacterial adhesion to the mucosa [118]. As a result, bacteria can then bind to specific host adhesion receptors like carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), a highly expressed protein in the ileal mucosa, with the bacteria replicating and activating immune cells [121]. In addition, different mechanisms for the roles of various putative bacteria in the pathogenesis of CD have also been proposed.

However all share similar concepts in that loss of the normal host bacterial tolerance and a subsequent inappropriate triggering of the mucosal immune system, are the main driving forces for the tissue destruction and disease development [122, 123]. 1.3.2.2 Dysbiosis as an aetiopathogenesis of CD

Numerous studies have documented that there is a significant difference in the faecal microflora of healthy individuals and those with IBD [124, 125]. Dysbiosis, or an alteration in the diversity of microbiota, has been demonstrated in CD [126, 127]. It has been reported that more than 30% of the dominant microflora in CD belongs to new phylogenetic groups [127]. The shift from predominant “symbiont” microbes to potential harmful “pathobiont” microbes with a breakdown of host-microbial cross-talk is a potential driving force in the development of IBD [128].

The most defined changes in the gut microbiota noted in CD patients encompass depletion in Firmicutes, a major phylum present in the intestinal microbiota, in conjunction with an increase in Proteobacteria phylum (E. coli and Bacteroides) [129]. Amongst the Firmicutes, a reduction in the abundance of the commensal Faecalibacterium (F) prausnitzii in CD patients has been well documented [130, 131]. F. prausnitzii inhabits the gut mucosa and mediates anti-inflammatory activities through production of a protein with immunomodulating properties [132]. There is a strong association between depletion of this bacteria (thereby loss of its anti-inflammatory functions), and the disturbed immune tolerance to the intestinal microbial community, which may precede or perpetuate inflammation [133].

Early reports also show there is a reduction in faecal lactobacilli and bifidobacteria in CD patients [134]. With more progress made in this area, other species such as Dialister invisus and Clostridium clusters were also found fewer than in matched healthy controls [135]. Clostridia, particularly IV and XIV, are the main butyrate-producing bacteria in the gut [136]. Butyrate is a major fuel source for colonic epithelial cells [137], and exerts anti- inflammatory activity in the intestinal mucosa, through suppression of the NF-ț% signalling [138]. Butyrate is also required for maintaining integrity of epithelial barrier

[139]. Thus, loss of Clostridial clusters likely results in loss of these vital functions including loss of key anti-inflammatory metabolites and the cell-associated immunomodulatory ligands [140].

In addition to depletion of beneficial members of the microbiome, a relative increase in the abundance of potentially pathogenic species in the gut microbiota community has also been reported [141, 142]. While their role in disease pathogenesis has not been confirmed,

Bacteroidetes, Enterobacteria and Ruminococcus gnavus were observed more frequently in CD patients [127, 135, 143]. Enteroccoci, Clostridium perfringens and Bacteroides fragilis were specifically isolated more often from the mesenteric lymph nodes of surgically treated CD patients [144]. In a study that compared the abundance of bacteria in mesenteric lymph nodes in CD and healthy controls, it was noted that these species were identified in approximately 40 % of CD patients compared to only 10% of a healthy control group [145]. 1.3.2.3 Dysbiosis and risk of relapse

A newly emerged topic of particular interest is that dysbiosis is a leading factor in predicting disease relapse [146]. Microbial patterns in patients with recurrent disease has been determined to have reduced abundance of Firmicutes, in particular F. prausnitzii

[133], as well as increased Bacteroides and AIEC species [147, 148]. A pilot study explored changes in the microbiota profile of 12 CD patients at the time of surgical intervention and at 6 months follow-up [149]. In that study, biodiversity was lower in CD patients at the time of surgery and increased after surgery. However, biodiversity remained different to healthy subjects [149]. The study also found that CD patients with recurrent disease had a predominance of Enterococcus; whereas those who maintained remission showed predominant butyrate-producing Firmicutes [149]. Similar observations were also reported by Rajca et al. [150] in a recent cohort study that explored microbiota changes in

33 CD patients. Low counts of Firmicutes (Clostridium coccoides and F. prausnitzii ) correlated with higher rates of disease relapse [150]. More interestingly, the study showed that F. prausnitzii species in particular predicted the occurrence of relapse independent of raised inflammatory markers [150]. Thus, it appears that monitoring patients’ microbiota might provide a new prognostic tool for assessing risk of relapse and/or the recurrence following surgery in CD patients. However, further investigations are required to determine the sensitivity and specificity of using the identified species as a marker for disease relapse. 1.3.2.4 Antibiotic induced dysbiosis and the increased risk for CD

Antibiotics have the potential to alter gut microbial ecology [151]. Traditional culture- based techniques ascertained that exposure to antibiotics resulted in a reduction of the number of bacteria in human fecal samples [152]. With advancing technology in bacterial detection, such as 16S rRNA sequencing, major changes in the gut microbiota composition are observed following antibiotic administration [153]. A short course of ciprofloxacin, one of the commonly prescribed antibiotic, induced profound and rapid changes with a loss of diversity in the microbiota composition and incomplete resolution of composition one week after the completion of the antibiotic course [154]. Similarly, a 10 day course of amoxicillin led to a major shift in the main components of the fecal microbiota

(Bacteroides, Clostridium cluster IV, XIVa and Bifidobacterium), together with overgrowth of Enterobacteriaceae [155].

Antibiotic exposure can also affect microbial metabolism and function [156]. A multi-omic approach was undertaken to explore the abundance and function of gut microbiota in fecal

VDPSOHVFROOHFWHGIURPLQGLYLGXDOVZKRUHFHLYHGȕ-lactam antibiotics for 2 weeks [151].

Major dynamic changes were observed in the microbial community such as an early drop in the abundance of gram negative bacteria, a reduction in the diversity and new species colonization and late re-growth in the gram positive species [151]. Metabolic changes were also noted, with attenuation in the bacterial energetic status and impairment in metabolic functions [151]. Perturbation in the host microbial community can lead to various immunological effects and numerous clinical consequences [157]. It has been reported that antibiotic exposure in early life predisposes to an increased risk for the subsequent development of CD [158,

159]. In a meta-analysis that included 11 observational studies, antibiotic use was associated with the potential development of CD [160]. Case-control analysis of a population-based study involving IBD diagnosed patients also found that use of antibiotics

2-5 years before diagnosis, was significantly associated with higher chance of diagnosis

[161]. More interestingly, the study also showed that there is a dose dependent association between drug prescriptions and increased risk for development of CD [161].

There is debate whether dysbiosis precedes or follows IBD pathology [128]. With limited available knowledge, however, the association of antibiotic exposure with the increased incidence of CD suggests that microbial perturbation (dysbiosis) is likely be the leading cause for the initiation of inflammation and subsequent disease development, although host genetic susceptibility prerequisite might be required. Thus, limiting exposure and over prescription of antimicrobial agents, especially to those with a family history of IBD, might be of value in reducing the rising incidence of CD. 1.3.2.5 Diet-microbe interactions and the increased risk of dysbiosis

There is evidence that diet has profound effects on the gut microbiome, which influences the host–microbe cross-talk which is critical for intestinal and immune homeostasis [162].

Several reports indicate that the input of diet is critical for the microbial growth and shapes its architecture [163, 164]. In fact, influence of dietary components on the composition of the microbiota is observed during the initial colonization and is dependent on the type of feeding [165]. Breast fed infants has been found to have higher levels of Bifidobacteria with less abundance of Bacteroids, Clostridium Coccoides compared to the formula fed infants [166]. Beyond the neonatal period, gut microbiota undergoes various changes until it becomes more stable and resembles adult community around 3 years of age [167]. Furthermore the influence of diet is not restricted on the initial bacterial settlements and the effect continues throughout the life [168].

Microbial alterations accompanying diet include a change in the diversity (dysbiosis)

[169]. Under normal physiological condition there is pathobiont bacterial species coexisting with the commensal beneficial bacteria, but typically in very low abundance

[170]. Certainly, Ferimictus and Bacteroidetes account for 90% of the bacterial gut community [171], whereas the Actinobacter and Proteobacteria represent only less than

5% [172]. Interestingly, dietary manipulation, for example prebiotics, has been shown to modify the intestinal microbiome by fostering the growth and metabolic activity of the commensal bacterial species [173]. This approach is of significant importance in treating and/or preventing various chronic inflammatory diseases including, obesity and IBD [174,

175]. Contrarily, several dietary components have been found to negatively influence the compositional development of the microbial community and induce dysbiosis [176,

177]. Numerous human studies indicate that both over nutrition seen in the Western societies and malnutrition predominant in the underdeveloped countries, are leading factors for dysbiosis [178].

Western diets rich in sugar and fat has been found to correlate with overgrowth of

Firmicutes (Clostridium innocuum, Eubacterium dolichum, Catenibacterium mitsuokai), and reduction in the abundance of Bacteroids species [179]. Refined sugars containing diets has also been shown to increase overgrowth of the opportunistic Clostridial species (C. difficile and C. perfringens) [177]. In contrast, diets rich in complex carbohydrates promote less pathogenic bacteria (Enterobacteriaceae) and increases levels of beneficial Bifidobacteria [143, 180, 181]. In addition to the over nutrition states, malnutrition disorders have also been shown to have profound and sustained effects on the gut microbial composition [182]. It has been suggested that malnutrition delays the normal assemblage of the gut microbiota in early childhood and obligates toward lack of diversity

[183]. In a study involving sixty four Bangladeshi children to explore the relationships between nutritional disorders and the gut microbiota, persistent immaturity of the gut microbiota was a consistent finding among patients with severe acute malnutrition [184].

How precisely diet-microbe interaction contributes to intestinal inflammation remains elusive. However, it has been ascertained that diet is a leading factor in the perturbed host- microbe interplay and triggers initial nonspecific immune responses [185]. Exaggerated immune reactions may become chronic because of the constant influx of the microbial antigens in hosts with genetic defects in the mucosal barrier function and/or the innate immune system [186]. 1.3.2.6 Single causative bacteria for CD

An individual pathogenic organism as a single cause for IBD has also been proposed and a number of candidate bacteria have been identified [187, 188]. Nevertheless, conclusive evidence that a single bacterium contributes to disease pathogenesis is still lacking [144].

Infection with Mycobacterial species is commonly postulated in the aetiology of CD: an assumption based on the observed similarity between the disease and the bovine condition caused by Mycobacterium avium paratuberculosis (MAP) infection [189]. A germ-free human gut model established by subcutaneous transplantation of fetal intestine into immundeficient mice has shown MAP organisms are able to invade human gut epithelium and induce tissue damage and inflammation [190]. MAP is also frequently cultured from patients with CD, although the accuracy of such findings are questionable as environmental contamination has not always been excluded [191]. Moreover, a specific immune response to MAP with enhanced TNF-ĮSURGXFWLRQKDVEHHQGRFXPHQWHGLQ&' patients and macrophages infected with viable MAP, indicating MAP pathogenicity and ability to elicit inflammation [192, 193]. Bacterial DNA from MAP was found within granulomas of CD patients, although other types of bacteria DNA, including E. coli, were also found [194]. Dalton et al. [195] recently investigated associations between MAP and single nucleotide polymorphisms (SNPs) linked with CD, in a cohort of 84 MAP positive

CD patients . However, no associations were identified. Further, no correlation was found between NOD2 polymorphism and MAP serology in CD [196].

A role for Helicobacter (H) species in IBD has also been proposed [197, 198]. However, findings are varied and to-date has failed to support a definitive association for H species in IBD. In particular, H. pylori showed a negative correlation among IBD patients [199].

Zhang et al. [200] investigated the prevalence of H. pylori infection among 208 Chinese

IBD patients and found it was significantly lower in these patients compared to 416 matched controls. Furthermore, a recent meta-analysis involving 33 studies that included

4400 IBD patients, showed a significant negative association between H. pylori and IBD indicating a possible protective benefit for H. pylori infection against the development of

IBD [201].

E. coli and non-jejuni Campylobacter and other members of Proteobacteria, have also been linked with IBD pathogenesis [202]. Zhang et al.[203] isolated Campylobacter (C.) concisus from intestinal biopsies and found a higher prevalence of bacterial DNA with raised IgG levels in newly diagnosed pediatric CD patients [203]. Similar findings were found in another study involving faecal samples collected from newly diagnosed CD patients [204]. This study also showed higher prevalence of non-jejuni Campylobacter species including C. hominis , C. ureolyticus, C. showae, C. gracilis, C. rectus in CD samples than in matched healthy controls [204]. Importantly, immune reactive antigenic protein of C. concisus flagellin B, ATP synthase F1 alpha subunit, and outer membrane protein 18 were recently identified in CD patients [205]. However, the precise role of C. concisus in the development of IBD remains to be fully described. Nevertheless new evidence that strains from oral resident C. concisus are able to acquire zonula occludens toxin, known to increase intestinal permeability, may be a clue to its contribution to IBD pathogenicity [206]. Adherent-invasive E.coli (AIEC) is another important potential player in IBD pathogenesis

[207]. AIEC has been identified in the intestinal mucosa of CD patients, specifically associated with the ileal mucosa [148, 208]. Interestingly, antibody positivity to AIEC organisms was also more prevalent in CD patients and is associated with disease severity and fast progression, and the increased need for surgical intervention [199].

Additionally, other putative bacterial pathogen such as Staphylococcaceae,

Strepotococcaceae, Pseudomonas maltophilia, Mycobacterium kansasii and B2D+ strains of E. coli have also been isolated from patients with CD and are considered potential causative agents [144]. However, these organisms were also identified in other conditions

[209]. Disseminated Mycobacterium kansasii is also reported in patients with DiGeorge syndrome and in renal transplant recipients [210, 211] and is currently considered the second most common mycobacterial respiratory infection [212, 213].Additional associations have been also found with Klebsiella, Salmonella, Fusobacterium varium, and

Yersinia infections and it is thought these organisms might have a role in the disease relapse [199].

Collectively, many key questions remain in regards to single bacteria agents and/or major drifts in the microbiota composition (dysbiosis), and their role in CD pathogenesis. Better understanding of the microbiota-host interactions would be crucial for new insights into the unrevealed mechanisms of pathogenesis of microbial imbalances in diseases. 1.3.3 Role of genetics

There is evidence of a strong genetic component in the pathogenesis of CD [214]. It has been found that prevalence of the disease is higher in families with a history of CD compared to the general population, and is especially high among twins [42]. Siblings of affected patients have a 20-40 fold increased risk of developing the disease compared to normal population [145].

There are more than 163 genetic loci identified as risk factors increasing susceptibility for

IBD [215], with 71 specifically associated with CD [216, 217]. Some of these loci include

(NOD2/CARD15, TLR4, CARD9), (IL-23R, JAK2, STAT3, CCR6, ICOSLG),

(ATG16L1, IRGM, LRRK2), (IBD5, DLG5, PTGER4, ITLN1, DMBT1, XBP1), (HLA- region, TNFSF15/TL1A, IRF5, PTPN2, PTPN22, NKX2-3, IL-12B, IL-18RAP, MST1) and (TNFRSF6B) [218]. Of these genes, the NOD2/CARD15 gene has gained the most attention. Studies have shown that mutated NOD2 can trigger an inappropriate immune response to different pathogens, which contributes to development of CD [219]. NOD2 gene mutations enhance sensitivity of macrophages to NOD protein and MDP [21].

Further, NOD2 mutations result in a reduction in production of alpha-defensin, a small anti-bacterial proteins predominantly expressed in Paneth cells of the ileum acting as mucosal antibacterial barrier [220]. Therefore, individuals with NOD2 mutations are expose to developing CD through loss of mucosal barrier function [220]. Specific mutations within the NOD2/CARD15 gene have been identified as having strong association with CD [221]. NOD2 mutations (Arg702Trp, Gly908Arg and Leu1007 fsins C) are associated with 2-4 fold increase in heterozygous individuals and to 20-40 fold in homozygous individuals [222].

Mutations in the IL-23R and autophagy-related 16-like 1 (ATG16L1) genes are also associated with additional risk for developing CD [223, 224].The IL-23 R gene is located on chromosome 1p31 and its corresponding ligand IL-23, is a principal component of the immunoregulatory pathway and plays a role in the survival and differentiation of T helper cells into T helper-17 cells [225]. Therefore mutations in the IL-23 R gene will lead to disturbance of the immune system and an increased risk for developing CD [226].

However, the rare allele R381Q variant of IL-23R is protective against developing the disease [227]. This protection is mediated through weakening IL-23 induced T helper-17 cells function [228].

The ATG16L1 gene is located on chromosome 2 and is another susceptibility gene for development of CD [229]. One mutation involving a threonine to alanine substitution

(T300A) occurs at amino acid position 300 [230], and is responsible for decreased ability to remove intestinal micro flora, auto antigen presentation on major histocompatibility complex type 2 and altered process of intestinal apoptosis [231].

The TNF-ĮJHQHLVDQRWKHU&'VXVFHSWLELOLW\JHQH[232, 233]. In two CD cohorts it was found that a polymorphism in the TNF-Į JHQH SURPRWHU ZDV FRUUHODWHG ZLWK SDHGLDWULF onset of disease and colonic involvement [233]. 1.3.4 Role of environmental factors

Although the precise aetiology of CD, and the forces behind the changing epidemiology, remains unknown, there is a significant body of evidence supporting the relation between environmental factors including diet and IBD [234]. Recent epidemiological changes in

CD, rises in the incidence rates, have been attributed in part to the dietary preferences [11,

235, 236]. Altered diet habit in Asia, with increased westernisation of diet, is thought to be a leading factor responsible for the recent increases in the incidence of CD among Asian populations [25, 237]. Certainly, a multicentre case-control study in Japan, found a higher consumption of refined sugars and a high fat diet to be highly correlated with an increase in newly diagnosed cases of the disease [238]. On the other hand, diet therapy is increasingly becoming a preferred modality option in the treatment of IBD, as seen with enteral nutrition and its successful role in inducing remission in CD patients [239].

The double sword effect of diet indicates complexity in the interactions between diet and the host. Indeed, the precise reasons that diet contributes to the pathogenesis of CD remains poorly understood. Nevertheless, there is accumulating evidence that the contribution of dietary factors, at least in part, is attributed to inappropriate diet-microbe interactions [240]. Such perturbations of the flora may then lead to disturbances in host- immune cross-talk resulting in subsequent inflammation and tissue destruction [241].

Certainly, gut microbiota along with intestinal epithelial cells and the mucous layer, are required for proper functioning of the mucosal immune system [242]. Alterations of the composition of the intestinal microbiota community is a one potential connection linking diet to IBD development [243]. It has been ascertained that the constituents and function of the gut microbiome is largely determined by diet [244, 245].

Indeed, newly emerging modellings for studying human’s gut microbiota changes in response to diet, showed that 60% of microbial variability is defined by the host diet

[246]. For example, short chain fatty acids (SCFAs) produced by the colonic anaerobic bacteria following consumption of the dietary fibres of edible plants [247], have been found to alter the gut microbiome [248]. Greater diversity, higher predominance of beneficial bacteria Prevotella over Bacteroides and greater density in SCFA-producing bacteria were observed in faecal samples collected from children living in a rural African area consuming a diet rich in dietary fibre (high fermentable fibres) compared with matched children living in Europe eating a western style diet (with low fermentable fibres)

[249]. The latter group also showed increased levels of mucosa-associated enteric pathogens, including AIEC, which are commonly observed species in IBD patients [249].

Similar findings were reported from other studies involving carbohydrate deficient-diet

(low fibre contents) given to obese individuals that resulted in significant alterations in the large bowel bacterial inhabitants [250, 251]. All of these findings refer to the benefits of diet rich in fibres as opposed to the western diets deficient in SCFA sources. More importantly, these observations provide a clue about how diet induces intestinal pathology and possible links to modulating gut microbes.

High fat milk induced colitis and over growth of pathobiont species is also another relevant association that may explain the relationships between diet, microbes and IBD development [252]. Milk rich in fat has been shown to alter bile acid composition favouring expansion of sulphite-induced pathobionts, known bacteria capable of inducing colitis in mice [252].

Increasing bacterial adherence and migration across the mucosal barrier is also a potential dietary factor in the pathogenesis of disease [243]. Using an M-cell model (co-culture of epithelial cell line with human Peyer’s patches) Polysorbate-80, an emulsifier food product, increased translocation of mucosa–associated E.coli isolated from CD patients across M-cells [253]with translocation reduced by the addition of plant dietary fibres

[253].

Increasing intestinal permeability is another potential contributing factor of diet in perpetuation of inflammation and disease development [243]. It is well accepted that

‘leaky gut’ is a characteristic feature of CD [254]. Sodium caprate, a medium-chain fatty acid found in dairy products, increases intestinal permeability in the ileal samples of CD patients [255]. Gliadin, the toxic component of gluten, was also shown to enhance intestinal permeability and zonulin release, an immunomodulator involved in tight junction dysfunction, in human intestinal mucosa and epithelial cell lines [256, 257]. Damage of the protective mucous layer of the gut by various synthetic food additives (mono- and diglycerides of fatty acids) can also lead to increased intestinal permeability [258].

However, there remain many gaps in our understanding of the precise interrelationship between IBD and diet. Nevertheless, current available knowledge indicates that an inappropriate interplay of diet with the microbiota, together with the collateral effects on intestinal epithelial barrier, might be the triggers for disease development.

In addition to diet, perinatal and postnatal factors including type of feeding and early life exposure to drugs and vaccines has also been claimed to be environmental factors associated with the increase in various immunological diseases including IBD [259]. These factors are largely involved in mucosal homeostasis through influencing the initial colonization of the gut and the microbial balance in early life [260]. Further, the hygiene hypothesis is another environmental contributor suggested to contribute to the burgeoning prevalence of many inflammatory disorders including CD [145]. This theory supports the idea that different environmental changes such as better housing, better nutrition, improved hygiene, better living standards and sanitation have led to a rapid decline in infectious disease and therefore a weakening of the immune response to different pathogens in the early stages of life [20, 261]. The weakened immune response and an altered intestinal microbial community pose a risk and therefore the individual is more vulnerable to develop immunological diseases including CD [259].

Additionally, there is a correlation between CD and other environmental triggers including smoking and appendectomy [262]. Smoking has been identified as an important environmental factor in IBD [263]. It has been found there is a relationship between smokers and developing severe clinical disease [264]. The interaction between nicotine, macrophages and intestinal microvasculature leads to differing expressions of RNF138,

MT2A, and STEAP3 genes in smokers is the leading cause of disease severity [265]. Of interest, there is evidence that the effects of smoking disappears after cessation of smoking

[266, 267]. Cosens et al. [268] in a retrospective cohort study of cessation of smoking and course of CD revealed that flare-up rates, therapeutic needs, and disease severity are similar in both patients who stopped smoking and those who never smoked. In addition, the authors also showed that smokers who quit had a 65% lower risk of disease flare up during remission [268]. More interestingly is the association of smoking with ulcerative colitis (UC). Reports have shown that while smoking is a risk factor for developing CD, smoking is protective against UC [20]. Similarly, appendectomy appeared a strong risk factor for CD [269], but it is protective in UC [270].

In summary, to-date there is no a single causative agent responsible for developing CD; however the disease is a multi-factorial illness and different underlying complex mechanisms may be involved. Current evidence points to genetically susceptible individual developing inappropriate immune responses to different triggering factors including bacteria, nicotine and certain diets. Although mode(s) of how these risk factors contribute to the disease pathogenesis remains unknown, trafficking antigenic agents seems to be the key in initiating an uncontrolled compensatory immune response that results in over expression and release of immunoregulatory cytokines. 1.4 Diagnosis of Crohn’s disease

There is no single diagnostic test for CD, rather the diagnosis depends on a set of clinical, radiological, endoscopic and laboratory findings [271, 272].

1.4.1 History and Clinical examination

Detailed history and physical examination of suspected cases is the first step for diagnosis of CD [273]. History includes detailed questioning about the onset of symptoms, recent travel, food intolerances, contact with infectious enteric disease, medication (including antibiotics and non-steroidal anti-inflammatory drugs), smoking and family history of IBD

[272, 274]. General examination comprises general well-being, pulse rate, blood pressure, temperature, palpable masses, perineal and oral inspection, rectal digital examination, measurement of body weight, calculation of body mass index and pubertal status assessment for young groups [275].

1.4.2 Laboratory Findings

Complete blood count (CBC), erythrocyte sedimentation rate (ESR) and C-reactive protein

(CRP) can be routinely requested in patients with CD to support the diagnosis in suspicious cases and may indicate the activity of disease as well as nutritional consequences of the disease [276]. Further, there are various serological markers which are suggestive of CD

[277]. These include anti-saccharomyces cerevisiae antibodies (ASCA) which correlates with the disease activity and its oral manifestations [278], Escherichia coli anti-outer membrane porin c [277], anti-laminaribioside carbohydrate antibodies [279] and antibodies to CBir1 (anti-CBir1), a novel flagellin-like bacterial antigen [277]. Stool is often examined to exclude the presence of certain pathogens that may cause colitis and mimic the disease such as Clostridium difficile [272]. Further, stool may also be inspected for measuring specific inflammatory makers that have been found to correlate with gut inflammation [280]. Faecal lactoferrin (FL), faecal S100A12 and faecal calprotectin are some of the markers used as indicators of intestinal inflammation [281-

283]. Of interest, these new faecal markers, S100A12 and calprotectin, were found to be more sensitive and specific than standard tests (ESR, CRP, platelet count, and albumin) in detecting GIT inflammation [280]. This was shown in a study of 61 children with suspected IBD [280]. The authors identified that faecal S100A12 was highly specific in distinguishing IBD from other intestinal conditions. Therefore, it seems that the stool examination is becoming more essential to IBD screening and is a preferable tool compared to other invasive techniques.

1.4.3 Imaging studies & endoscopic evaluation

If IBD is suspected, endoscopic examination with histological analysis of intestinal biopsies is used to establish the diagnosis [46]. Single contrast upper GI tract radiologic series, capsule endoscopy, MR Enterography (MRE), CT Enterography (CTE) and abdominal ultrasonography are additional imaging tools usually used to support the diagnosis [2, 284, 285].

1.4.4 Crohn’s disease activity index

The Crohn's disease activity index (CDAI) is a research tool established in 1970 for assessment of illness activity in patients with CD [98]. It involves seven clinical and laboratory variables, which include the number of liquid or very soft stools per day, abdominal pain rating, general well-being rating, presence of complications, abdominal mass, Haematocrit, and % decrease from expected body weight. Generally, patients with a score <150 are considered to be in remission whilst those with a score >150 are considered to have active disease; and those with a score >450 are classified as having very severe disease [286].

In children, the Paediatric Crohn’s Disease Activity Index (PCDAI) was established to measure disease activity [287]. It is considered an accurate tool and is well correlated with the physician global assessment tool (PGA) [288]. The PCDAI score is calculated based on subjective descriptions of patient’s symptoms and objective parameters such as physical examination, growth measurements, and laboratory tests [289]. Modified PCDAI (Mod

PCDAI) is a new tool developed for assessing CD activity and is used mainly for research purposes [290]. It consists of laboratory measures of the PCDAI plus CRP. Authors reported that the Mod PCDAI correlates with PCDAI, PGA and faecal Calprotectin. 1.5 Symptomatology of Crohn’s disease

1.5.1 Intestinal Manifestations

The clinical manifestations of CD are vague and variable depending on the location, extent, and severity of involvement [46]. In general, chronic diarrhea is the most common presenting symptom followed by abdominal pain in 70% of patients, weight loss in 60% with blood and/or mucus in the stool in 40% to 50% of patients with CD [272].

1.5.2 Extra-intestinal manifestations

Extra-intestinal manifestations are common in CD patients occurring in 25% of cases

[291]. They include arthropathy, fever, growth failure, sexual maturation delay, oral lesions, cutaneous lesions, ophthalmological complications, hepatobiliary disorders and renal manifestations [292-294].

1.5.2.1 Fever

Fevers are seen in up to 40% of patients with IBD at the time of presentation [295]. Fevers are usually low grade and unrecognized but high spikes can occur [46].

1.5.2.2 Arthropathy

Joint involvement is the most common extra-intestinal manifestations of CD [296].

Arthropathy is a common complication of disease, occurring in approximately 25% of IBD cases, however in CD alone it occurs in 2-16% of patients [297]. Inflammatory arthropathy usually correlates with disease activity and response to treatment [46]. The clinical spectrum of spondylarthropathies includes axial symptoms, peripheral arthritis, dactylitis and enthesopathy [296]. Radiographic sacroiliitis is the most common type occurring in 12% of total patients, however it is usually asymptomatic and may not progress to ankylosing spondylitis [46]. Peripheral arthritis is the second commonest and is usually asymmetrical having a migratory nature [297].

1.5.2.3 Nutritional consequences of Crohn’s disease

Growth failure and malnutrition are often problematic in CD, especially in children as a large proportion of cases have weight loss at the time of diagnosis [298], and a significant number of them are stunted [299]. In a cohort of 431 children with CD, 60% of patients had weight loss at time of diagnosis, of those 27% had a weight that was less than the 3rd centile. Furthermore 30% were stunted (below the 10th centile of height for age), of those

13% having a height below 3rd centile [300]. Macronutrient (protein-losing enteropathy), blood loss (contributing to iron-deficient anaemia) and micronutrient deficiencies

(generally calcium, folic acid, iron, zinc, vitamin D, vitamin K and vitamin B12) were also detected [301]. The aetiology of this poor nutritional status is multi-factorial and may relate to poor intake, malabsorption, increased energy demands and increased micro- nutrients loss through stool [302-307] (Figure 1.1). The most common pattern of growth failure is linear growth retardation that is typically associated with delayed skeletal maturation [308], which is seen more often in males, and associated with pubertal delay

[307]. Ongoing inflammation is the most likely cause of growth failure consequent to high levels of pro-inflammatory cytokines such as TNF-Į DQG,/-6 leading to suppression of insulin like growth factor 1(IGF-1) activity [309-311]. The growth hormone (GH)/(IGF-1) axis is altered in IBD patients resulting in different biochemical changes ranging from GH deficiency to resistance [312]. However, these markers rapidly return to normal once the inflammation is resolved [313]. More recently it has been found that CD patients have a lower level of IGF-1 compared with healthy controls [314]. Exposure to corticosteroids also increases the risk of linear growth failure in children [315]. In the past, 20-30% of children with CD were shown to not reach their expected final adult height [316].

Therefore, taking these issues into consideration and making a thorough nutritional assessment in CD patients is of clinical importance during the management of disease, especially in the paediatric population [317, 318] .

1.5.2.4 Oral lesions

CD has different buccal clinical manifestations that range from small oral ulcers to cobble- stoning of the labial and buccal mucosa [319]. Oral apthous stomatitis lesions are the commonest oral lesions in CD and occur in approximately 21% of cases [320]. These lesions are not a specific feature for CD as they also occur in patients with ulcerative colitis

[321] and individuals who do not have IBD [322]. Lip swelling with or without fissures, angular cheilitis, gingival hypertrophy, linear ulcerations and tissue tags are also considered oral manifestations of CD, although they are less common than apthous ulcers that are more specific for CD [323]. 1.5.2.5 Cutaneous lesions

Dermatological involvement in patients with CD is not uncommon and can occur in 14 to

44 % of CD patients [324]. CD patients with cutaneous lesions fall into two groups, cutaneous direct and metastatic lesions [324]. Direct manifestations are the most common and include pyoderma gangrenosum, Sweet's syndrome, erythema nodosum, granulomatous dermatitis, necrobiosis lipoidica, epidermolysis bullosa acquisita and erythema multiforme [324]. Erythema nodosum and pyoderma gangrenosum are considered common among the direct group [325, 326]. In contrast, metastatic skin lesions are rare and less common than direct manifestations [327].

1.5.2.6 Ophthalmological manifestations

Eye involvement occurs in 3 to 6.3% of CD patients and is commonly associated with the colonic subtype [328]. Episcleritis is considered the most common eye manifestations in patients with CD [326]. Blurred vision, teary itchy eyes, ocular pain, photophobia, conjunctival or scleral hyperaemia, loss of visual acuity and possible blindness all are the ocular clinical manifestations of disease that patients may present with [329]. Ocular involvement can be due to the disease itself or as result of chronic use of steroids [330].

According to Felekis et al [331], eye manifestations in IBD patients are categorized into three broad groups: eye involvement that correlates with disease activity and tend to remit with the use of corticosteroids or surgical resection; involvement that is related to chronic usage of corticosteroids; and a third group called coincidental complications [331]. 1.5.2.7 Genitourinary complications

Urological manifestations can be seen in up to 23 % of adult patients with IBD (4-23%) and include ureteral calculi, enterovesical fistula, peri-vesicle infection, peri-nephric abscess, obstructive uropathy and amyloidosis [328]. Nephrolithiasis is more common in

IBD patients (8-19%) than in general population (0.1%) [332]. Calcium oxalate stones often occur in IBD patients [333].

1.5.2.8 Hepato-biliary manifestations

Hepato-biliary complications of CD include primary sclerosing cholangitis (PSC), small duct PSC, autoimmune hepatitis / PSC overlap, IGg4 cholangitis, gall bladder stones, portal vein thrombosis, hepatic abscess, fatty liver, supportive pylephlebitis, hepatic amyloidosis, granulomatous hepatitis, primary biliary cirrhosis, drug induced hepatitis and cholangiocarcinoma [334]. Choliathiasis can be seen in up to 25% of patients with CD as result of bile salt malabsorption resulting from inflammation in the terminal ileum [335].

Prevalence of PSC in CD patients is only 1% [336] however these patients have higher risk of cholangiocarcinoma, gall bladder carcinoma and hepatocellular carcinoma [337].

Further, PSC also increases the risk for developing colitis-associated colorectal cancer and dysplasia [338]. Malnutrition Under weight Micronutrient deficiency Altered body composition

C- A- B- Increased GIT nutrient Altered energy /nutrient Decreased dietary loss/requirements. metabolism intake

Figure 1.1 illustrates the aetiology of malnutrition in patients with CD. (A): Drug therapy and inflammatory response. (B): GIT inflammation, pain and decreased appetite. (C): Leaky gut, surgical resection decreased absorptive area, increased mucosal turn over and bleeding loss. 1.6 Management of Crohn’s disease

Due to the chronic and incurable nature of CD, patients usually suffer from repeated disease flares and frequent relapses [339]. The purpose of current therapies are therefore to relieve symptoms, afford better quality of life, minimize drug side-effects and support growth in the paediatric age group [340, 341]. Generally, the appropriateness of therapeutic interventions in CD depends on disease activity, site, behaviour and course of disease, response to previous medications and extra-intestinal manifestations [342]. The therapeutic options for CD treatment include corticosteroids, 5-aminosalicylates (5-ASA), antibiotics, immunosuppressive including azathioprine (AZA) and methotrexate (MTX), biological agents, endoscopic and surgical options, as well as nutritional therapy [273,

343].

1.6.1 Corticosteroids

One of the most reliable and effective medications in treating patients with CD is steroids, which can induce clinical remission in up to 80% of cases presenting with active disease

[344, 345]. Steroids exert their therapeutic benefits by regulating expression of several pro- inflammatory proteins including IL-1, IL-2, IL-6, IL-8, TNF, adhesion molecules and leukotrienes [346].This action is mediated through inhibition of the NF-ț% ZKLFK regulates the release of a large number of mediators involved in the immunological and inflammatory response in CD [347]. Oral prednisolone, a commonly prescribed steroid, is used for induction of remission in the active stage of disease, and is then tapered once improvement begins and inflammatory markers start normalising [343]. In a review of randomized controlled trials investigating the efficacy of corticosteroids in treating patients with CD, steroids were more effective than placebo in inducing remissions (RR 1.99; 95% CI 1.51 to 2.64; P < 0.00001) [348].

Further, in a population based cohort that assessed the efficacy of corticosteroids in treating IBD patients, 84% of CD patients had either complete or partial remission after one month of receiving prednisolone [349].

However steroids are also associated with low rates of sustained remission and mucosal healing, linear growth impairment, steroid dependency and increased risk of infection

[350]. In addition side-effects of steroids are often problematic, particularly in younger patients owing to their deleterious effects on growth and development [351-353].

However, new generations of steroid have been introduced, which potentially limit toxicity

[354]. Numerous studies have investigated the efficacy of budesonide in treating CD and found it is less likely to cause adverse effects compared to conventional steroids, although it is 13% less effective for the induction of remission in active disease [355].Similar findings were seen in that budesonide is superior to placebo in inducing remission in patients with CD, but less effective than old generation steroids [356, 357]. Therefore, budesonide is mainly indicated for treating patients with mild to moderate disease severity

[106, 358]. 1.6.2 Immunomodulators

Immunomodulators are used in conjunction with steroids in the treatment of CD [359].

They are given for patients with severe relapse, or require two or more corticosteroid courses within one year as well as patients who relapsed within short period of stopping steroids [360]. Further, they are also frequently prescribed for managing complications of the disease [361]. Thiopurines are a subclass of immunomodulators, which include AZA and its metabolite 6-Mercaptopurine (6-MP). The thiopurines are effective in inducing and maintaining remission in both adults [362-365] and children with CD [366].

In a review of 622 IBD patients (272 CD and 346 UC), azathioprine treatment induced remission in 45% of CD and 58% in UC patients [367]. However, in a recent systemic review and meta-analysis of randomized controlled trials of the efficacy of immunomodulators in CD, there is very little data supporting the use of AZA and 6-MP for induction of remission, although the drugs may be used as steroid sparing agent or as an adjunct to biological therapy in preventing relapse [368]. AZA and 6-MP are now widely recommended to reduce the risk of post-operative recurrence of CD [369] and preventing surgery in patients with non-penetrating and non-stricturing CD [370]. Further, in patients with moderate to severe CD, a combination of AZA with infliximab has been shown to induce a longer corticosteroid-free clinical remission compared to AZA as monotherapy

[371, 372].

Although thiopurines offer many therapeutic benefits for patients with CD, their side effects limit their clinical utilization [373]. The adverse effects of thiopurines can be classified into dose dependent and non-dependent effects [373]. The non-dose related or

“allergic” effects include fever, malaise, nausea, abdominal pain, diarrhoea, arthralgia and pancreatitis, which usually occur within days or weeks of thiopurines treatment [374]. The dose related or “ non-allergic” effects include bone marrow suppression and liver toxicity, which develop later (months or years) but may also occur very early following treatment

[374]. Generally, nausea, pancreatitis and bone marrow suppression are the most common side-effects of AZA and are more likely to occur in patients with thiopurine methyl transferase deficiency [374].

MTX is a kind of immunomodulator and has a limited role in inducing remission in patients with CD [368, 375]; however it is useful in maintaining remission. In a double blind study involving 76 patients with CD who were randomly assigned to receive either

MTX at dose of 15 mg once weekly for 40 weeks or placebo, steroid-free remission was maintained over 40 weeks in 65% of patients received MTX, as compared to only 39% in the placebo group [376]. 1.6.3 Aminosalcylic Acid drugs

5-ASA drugs are a group of compounds that have a long established use in IBD patients

[344, 377]. They have been used for a long time as the first line drug in treating mild to moderate active CD, however recently their role in managing CD has been questioned

[378]. A systemic review and meta-analysis of 6 randomized-controlled trials investigating the efficacy of 5-ASA in patients with active CD revealed that there was no benefit of mesalamine usage over placebo in induction of remission [379]. The authors also report that in 4 of the 6 trials, sulphasalazine was superior to placebo to achieve remission [379].

In a further systemic review that evaluated the efficacy of sulphasalazine or mesalamine in the treatment of mildly to moderately active CD compared to placebo and corticosteroids, the authors similarly concluded that 5-ASA drugs have a limited role in treating CD [380].

In that study, sulphasalazine had modest efficacy compared to placebo and was inferior to corticosteroids for treating mild to moderately active CD, and other preparations like olsalazine and mesalamine were not superior to placebo [380].

5-ASA drugs also appear to be not effective agents as maintenance therapy in CD [381]. In a systemic review of randomised controlled trials that investigated the effect of oral 5-ASA used for at least 6 months, found there was no evidence supporting the use of 5-ASA preparations as maintenance therapy for medically induced remission in patients with CD

[377]. However, 5-ASA drugs may have a role as maintenance therapy in patients with surgically induced remission [382]. In a meta-analysis of 11 randomised clinical trials involving 1282 CD patients who underwent surgical resection followed either by 5-ASA or placebo or no therapy, sulphasalazine was found to be of no benefit in preventing relapse

(RR=0.97; 95% CI=0.72–1.31) but mesalamine was more effective than placebo or no therapy in preventing relapse (RR=0.80; 95% CI=0.70–0.92) [383]. 1.6.4 Biological Agents

Biological therapies include infliximab, natalizumab, anti–IL-12 and anti–IFN-ȖDQWLERGLHV

[384]. The introduction of biological agents has resulted in numerous changes to the course of disease including rapidly achieved and sustaining clinical remission, complete and sustained mucosal healing, reduced dependence on steroids, reduction in hospital stay, reduced surgeries, fewer complications and improvement of patients’ overall quality of life

[385]. In a meta-analysis of clinical trials of adult patients with active and quiescent CD who were treated with biological therapies or placebo, biological therapy were shown to be superior to placebo in inducing remission in patients with active CD and in preventing relapse of quiescent CD [386].

Of the biological agents used to treat CD, infliximab, an anti-TNFĮ chimeric monoclonal antibody, has become a viable therapeutic option for inducing and maintaining remission of CD [387]. Infliximab is predominantly indicated in steroid-refractory, steroid-dependent and/or immunomodulators-refractory luminal CD, in patients who fail to tolerate conventional medications and in conjunction with surgical drainage in cases with complex fistulas [378, 388]. In refractory disease and in disease with fistula, the response rates for infliximab treatment is 79% for initial response and 54% for sustained response after 12 weeks [389, 390]. Further, infliximab is highly effective in treating extra-intestinal manifestations of the disease [391, 392]. Infliximab also improves anthropometric measures in children [393]. In a retrospective case series reviewing children with CD treated with infliximab, 83% of patients went in remission and mean body mass index Z

VFRUHV LPSURYHG VLJQLILFDQWO\ IURP í WR í P< 0.01) following one year of infliximab therapy [393]. Further, infliximab is effective in maintaining remission in CD patients, where prolonged steroid withdrawal can be achieved for up to 3 years while receiving infliximab [394]. Infliximab is also beneficial for preventing disease recurrence in patients who underwent surgical induction of remission [395]. Infliximab is also very effective in managing peri-anal conditions in patients with CD [396]. In a prospective study involving 52 CD patients with peri-anal fistulae treated with infliximab, 22 patients

(42.3%) had a complete response and 23 (44.2%) had a partial response to treatment [397].

Recent reports of the management of CD indicate that response to biological therapies can be further enhanced when combined as early as possible with immunosuppressive drugs

(AZA or MTX) [350]. A randomized-controlled trial involving 508 patients with active

CD who received 6 months of either AZA alone or infliximab alone or in combination found significantly more patients were in remission in the combination group compared to both the infliximab monotherapy group (56.8% vs 44.4%, P<0.02) or AZA monotherapy group (30.0%, P<0.001) [371]. Further, 43.9% of patients in the combination group had mucosal healing whereas only 30.1% in the infliximab group and 16.5% in the AZA group

[371]. Of interest, serious infections rates were reported less in the combination group

(3.9%) compared to the infliximab group (4.9%) or AZA group (5.6%) [371]. D’Haens et al. [398] undertook a study of 133 patients with active CD who randomly received either a combination of AZA (or MTX if AZA wasn’t tolerated) and infliximab or corticosteroids.

Following 6 months of treatment, about 60% of the combined therapy group were in remission and did not require corticosteroids or surgical resection, whereas only 35·9% of the corticosteroids group was in remission; with corresponding rates after 1 year at 61·5% and 42·2%, respectively [398]. The mechanism of Infliximab is that it directly neutralizes soluble TNF-Į DQG LQWHUDFWV with membrane bound TNF to eliminate or promote clearance of active inflammatory cells

[399]. Infliximab also influences TNF-Į LQGXFHG JUDQXORF\WH-macrophage colony stimulating factor expression [400], which promotes apoptosis of activated T-lymphocytes involved in the pro-inflammatory cytokines release [390]. Further, infliximab can down regulate mucosal angiogenesis by reducing production of vascular endothelial growth factor-A from mucosal fibroblasts [401] and may influence the CD40/ CD40 L pathway

[402].

Despite the many therapeutic advantages of biological therapy in CD there are concerns about long-term safety and the adverse effects of these agents [403]. Infusion and injection site reactions, psoriasiform eruptions, lupus-like disorders, vasculitis, granulomatous reactions, cutaneous infections and cutaneous neoplasm are some of the adverse effects reported in association with anti-TNF-ĮGUXJVXVH[404]. Loss of efficacy, especially after extended period of time, is another problem of biological therapies [391]. In a observational multicentre study of 152 CD patients receiving infliximab maintenance treatment, approximately 50% of patients lost the therapeutic response to infliximab following 5 years of treatment [405].

Thus, biological therapy has resulted in a revolution in treating steroid-resistant and complicated CD. Nevertheless, adverse effects, possible loss of efficacy and scarcity of available safety data of long term use may limit full the clinical utilization of these medications. 1.6.5 Antibiotics

Bacterial pathogens have been isolated from the gut lumen of CD patients [406, 407], and this evidence forms the argument for antimicrobial treatment in CD [408]. Antibiotics reduce the number of mucosa associated bacteria and prevent bacterial translocation [144].

In addition to bactericidal activity, metronidazole and ciprofloxacin antibiotics may act as immune-suppressants that may influence and ameliorate the inflammatory activity accompanying the disease [409].

Clinical trials of antimicrobial therapy in CD have shown inconsistent results [410]. In a double-blind placebo controlled trial involving CD patients treated with metronidazole, metronidazole was shown to be superior to placebo in reducing disease activity, although it was not effective in inducing remission [411]. The authors also showed that this benefit was confined to patient with colonic and ileocolonic involvement and not in patients with isolated ileal involvement [411]. The limited efficacy of metronidazole for cases with CD with colonic involvement may be attributed to presence of anaerobic bacteria including

Bacteroides in the colon [344]. Ciprofloxacin, from the quinolone antibiotic group, was shown to be as effective as 5-ASA drugs, and more effective than placebo, in treating 40

CD patients with mild to moderate disease [412]. Further, in a small preliminary study, CD patients with active disease were randomly assigned to receive either 500 mg ciprofloxacin or placebo for 6 months [413]. The outcome of this study showed a significant reduction in

PCDAI scores in the ciprofloxacin group compared to the placebo group [413]. Rifaximin is a semi-synthetic antibiotic and its efficacy was investigated in 68 patients with CD who received either rifaximin alone or in combination with steroid treatment [414]. In this study, clinical remission was achieved in 67% of patients receiving rifaximin as mono- therapy versus 58% in patients who received concurrent steroids [414]. In contrast, other clinical trials have shown that antibiotics offer no benefit in the management of CD patients [415]. A meta-analysis of 11 randomized placebo-controlled clinical trials involving 668 patients with CD receiving either antimicrobial therapy or placebo revealed no significant difference in clinical responses between antimicrobial treatment and placebo

[416].

Despite the controversy, antibiotic combinations have shown more favourable outcomes

[417]. In a retrospective study of 32 paediatric patients with CD treated with 8 weeks of azithromycin and metronidazole, clinical remission was achieved in 66% of patients [418].

In a randomized controlled trial involving 41 patients with CD who receive either a combination of ciprofloxacin and metronidazole or steroids, remission rates were comparable between the antibiotic group (45.5%) and the steroid group (63%) following a

12 week treatment course [419]. Further, antibiotics have been shown to have a role as maintenance therapy for CD patients who underwent surgical intervention [420]. In a randomized, placebo-controlled trial of metronidazole, a significant decrease in the incidence of severe endoscopic recurrence was observed after ileal resection [421]. In a further trial of CD patient who underwent surgical intervention, metronidazole when given orally with AZA was effective in decreasing the number of patients with postoperative recurrence of disease [422]. A similar conclusion was also made by Doherty et al. [423] in a meta-analysis reviewing two randomized controlled trials of postoperative CD patients. 1.6.6 Probiotics and prebiotics

Probiotics are viable microorganisms that have beneficial properties for the host when given orally at adequate dose [424]. Proposed probiotics include Lactic acid bacilli

Lactobacillus, Bifidobacterium, E.coli Nissle1917, Clostridium butyricium, Streptococcus salivirus thermophilus and Saccharomyces boulardii [425]. Prebiotics are non-digestible food ingredients that beneficially affect the host by selectively stimulating and fostering growth and/or activity of a limited number of bacterial species resident in the colon [426].

Because bacteria have been implicated in the pathogenesis of CD, it has been suggested that probiotics may be used as a therapy to modulate gut intestinal microflora and may ameliorate the altered immune response in CD [427]. Current evidence indicates that varying probiotic strains mediate their effects by receptor competition with microbial pathogens thereby preventing bacterial adhesion and translocation, enhancing epithelial barrier integrity, enhancing digestive and absorptive function of intestinal epithelium, bactericidal effect, immunomodulating effect and enhancing immune response of intestinal epithelium and lymphoid tissues [428-430].

Probiotics have been used as maintenance therapy for patients with CD. However, evidence of substantial clinical efficacy of probiotics is lacking [423, 431]. A systemic review of 6 randomized-controlled trials of patients with CD who commenced probiotics immediately after either surgical intervention or pre-treatment with steroids, indicated that probiotics had no effect in reducing postoperative recurrence or sustained clinical and endoscopic remission [432]. Similar conclusions were made by Rahimi et al [433], in a meta-analysis of eight randomized-controlled trials investigating probiotic efficacy in CD. The authors found that probiotics failed to maintain remission or to prevent clinical and endoscopic recurrence in patients [433]. Further, in a randomized prospective controlled double-blind trial of 63 patients with CD who underwent elective ileo-caecal resection, it was shown that administration of probiotics offered no benefit over placebo in reducing postoperative recurrence rates or lowering serum inflammatory parameters and clinical relapse rates [434].

It appears that probiotics might have limited roles in managing CD. However there is evidence that synbiotic therapy, a mixture of probiotics and prebiotics, is more beneficial for IBD patients [435]. In a clinical trial CD patients who had failed to achieve remission with prednisolone, 5-ASA or nutritional therapy, were then treated with synbiotic therapy

[436]. After a period of 12 months of synbiotic therapy, 6 of 10 patients had a complete response and 1 patient had a partial response. Further, of those who achieved remission, two patients were able to discontinue their prednisolone and four patients decreased their prednisolone. Although this was a small study, it suggested symbiotic therapy could produce good outcomes in difficult to manage patients. 1.6.7 Surgery

Due to chronicity and incurable nature of CD, a number of affected patients may need a surgical intervention at some stage of their disease [437]. Population based cohort studies of the natural history of patients with CD have shown that around one third of patients required surgical intervention early after initiation of medical treatment and one half of patients had surgery within 10 years of diagnosis [438]. A retrospective study of 907 patients with ileocecal CD revealed that resection rates were 61% and 83%, 1 and 10 years respectively, following diagnosis [439]. In addition, in that study approximately one third of cases relapsed after 5 years subsequent to their first resection.

Failure of medical therapy is considered the most common indication for surgery in patients with CD [440]. However, in a retrospective analysis from a single centre, intestinal obstruction was found to be the most common indication for CD patients who underwent surgery [441]. Generally, indications for surgery are variable and depend on location of disease, however poor response to medical treatment, intestinal obstruction, fistula complications, inflammatory mass, intra-abdominal or pelvic abscess, gut bleeding and persistent growth retardation are indications for surgery [442, 443]. Surgery remains one option for treating CD, especially in complicated and resistant disease. However, following the introduction of biological drugs (e.g. infliximab), rates of surgery in patients with CD are reducing [444].

In summary, there are several available therapeutic choices for the treatment of CD.

However, major adverse effects, drug resistance and loss of activity as well as incomplete efficacy all contribute to the limited and incomplete utilization of current medications.

Further, none of the previously discussed therapeutic options have addressed the nutritional complications of CD. The therapeutic options discussed so far aim to reduce inflammation, prolong the periods of remission and to minimise disease flares. Short of a cure, the rationale for therapeutic development is to produce more safe and effective therapies that can address all the disease aspects. 1.7 Role of nutritional therapy in management of Crohn’s disease

1.7.1 Overview

Nutritional treatment, utilizing exclusive enteral nutrition (EEN), has become another new viable therapeutic option for the treatment of CD [445-447]. EEN is the use of enteral nutrition as the sole nutritional source, which is given either as either elemental (EF), semi- elemental or polymeric Formula (PF) together with exclusion of a normal diet [448].

Beneficial effects on growth, nutritional values, safety and enhanced efficacy in inducing remission have been reported for EEN therapy in CD patients [449, 450]. Primarily, benefits of enteral nutrition in CD were first observed more than three decades ago [451].

The authors reported some patients whilst awaiting surgical treatment for management of their medically-resistant CD, went into remission during delivery of an EF as preoperative nutritional support [451].

Role of EEN in achieving clinical remission has been shown to be equivalent to steroids in children [452]. In the paediatric age group, EEN is considered a first-line therapy for the induction of clinical remission in active CD [453-456]. It has become a feasible and effective approach for managing children with active disease in many countries, such as

Europe [457, 458], Japan [459] and Australia [460]. Firstly, EEN can induce high remission rates of up to 85% in newly diagnosed patients which is comparable or superior to corticosteroids [461]. According to Heuschkel et al. [462] who reviewed five randomized controlled trials with a combined total of 147 paediatric patients with CD, the efficacy of EEN was comparable to corticosteroids in treating active CD in children where both the EEN and corticosteroids groups showed equal rates of clinical remission. The authors conclude that EEN is more attractive for practical use compared to corticosteroids, especially in children, as corticosteroids have many undesirable side effects [462] (Table

1.1). In a second meta-analysis in children with CD [298] and based on the results of four randomised control trials (n = 144), no significant differences in the remission rates between EEN and steroids were found (RR 0.97, 95% CI: 0.7-1.4) [298].

Secondly, EEN can improve weight/body mass index (BMI) Z-scores (Z-score is equivalent to standard deviations from the mean, for examples a Z-score of 1 represents 1 standard deviation greater than the mean) in malnourished patients [448]. Buchanan et al

[463] in one study of 114 children with CD who were treated with an 8 week course of

EEN, revealed that 88 children (80%) achieved remission with consequent reductions in

ESR and CRP (P< 0.001). Patients in remission had comparative improvements in weight and BMI Z-scores by the end of treatment. Weight median Z-score increased from -1.04 at start of treatment to -0.47 after 8 weeks of receiving EEN formula. Likewise, Z-score of

BMI jumped from -0.98 at 0 week to -0.03 at the 8th week of therapy.

Thirdly, EEN is highly effective in healing the inflamed bowel mucosa compared to corticosteroids [464]. In a prospective, 10-week open-label trial of children with active

CD, 19 children were randomised to receive PF and 18 to receive corticosteroids [465]. At week 10, the proportion of patients achieving clinical remission was comparable between the two groups (PF: 15/19 [79%]; corticosteroid group: 12/18 [67%]; P = 0.4). However, the proportion of children showing mucosal healing was significantly higher in the EEN group (14/19; 74%; 95% CI, 51%–89%) than in the corticosteroid group (6/18 [33%; 95%

CI, 16%–57%]; P < 0.05). At week 10 both endoscopic and histologic scores significantly decreased only in the EEN group (P < 0.001). In addition, EEN when used as the preferred therapy in this younger age group may postpone corticosteroid use [461, 466, 467], and maintain longer duration of remission than if corticosteroids are used as primary therapy

[461]. Authors References Duration Clinical remission Relative risk

(Weeks) EN steroids (95% CI)

Sanderson et al. [468] 6 8/9 7/8 1.02 (0.37-2.80) Seidman et al. [469] 3 8/10 5/9 1.20 (0.42-3.46)

Seidman et al. [470] 4 26/34 31/34 0.84 (0.50-1.41)

Thomas et al. [471] 4 12/12 12/12 1.0 (0.45-2.22) Ruuska et al. [472] 8 9/10 8/9 1.01(0.39 2.62)

Table 1.1 shows five randomized studies comparing EN to corticosteroids as induction treatment in paediatric CD patients. 1.7.2 Mechanisms of action of exclusive enteral nutrition

The precise mechanism through which EEN induces remission in CD is poorly understood.

However, there is accumulating evidence suggesting that EEN exerts its effect through modulating the exaggerate immune response, influencing bacterial flora, enhancing healing of the disrupted gut mucosa and reducing the exposure to microbial and/or dietary antigens

[473, 474].

1.7.2.1 Direct anti-inflammatory effect

Several clinical trials have shown that the EEN is capable of suppressing pro-inflammatory mediators in the inflamed intestine of CD patients [475-477]. Bannerjee et al [313], investigated the direct anti-inflammatory and nutritional effects of EEN. In their study conducted on 12 children with active CD, the participants were treated exclusively with a polymeric/elemental formula for 6 weeks with regular assessment of disease activity

(PCDAI), nutritional indices and inflammatory markers. They noted changes in inflammatory parameters (ESR and IL-6) by day 3 and PCDAI, CRP, and IGF-1 by day 7 with improvements in the nutritional parameters by day 14 and 21. The authors suggest that an early increase in IGF-1 during treatment is responsible for the anti-inflammatory effect of the EN, rather than nutritional restitution. Therefore the authors conclude the primary mode of action of EEN in induction of clinical remission in patients with CD is by direct anti-inflammatory effect, which usually precedes the nutritional benefits.

Further evidence of the direct anti-inflammatory effect of EEN was provided in another study in 2005, which was conducted by Yamamoto et al [464]. This trial consisted of a total of 28 adult patients with CD treated with an elemental diet for 4 weeks, with biopsies obtained from the terminal ileum and large bowel of all patients before and after treatment.

Additional mucosal biopsies were taken from a control group of 20 patients without inflammation when admitted for endoscopic examination. The authors observed that there was a difference in the cytokine levels between the CD group and the control group before treatment, but after receiving the EF there was no longer a difference in cytokine levels between the two groups. Concentrations of several inflammatory mediators, such as IL-ȕ

IL-6, IL-8, and TNF-ĮZHUHUHGXFHGZKLOHNH\DQWL-inflammatory cytokines such as IL-1 receptor antagonist were increased.

The direct anti-inflammatory effect of EEN has also been shown utilising in vitro models

[478]. De Jong et al. [479] were able to show, using three different epithelial cell lines, a significant reduction in the cellular IL-8 response when PF was added to counter the effects of an inflammatory stimulus. Further, they used a two compartment model that separated the PF from the inflammatory stimuli. The authors concluded that the anti- inflammatory effects of EEN on enterocytes are more likely to be the result of intracellular actions of the PF. However, the constituents of the PF responsible for these immunomodulatory effects in CD were not described in this study and remain unknown.

Additional experiments utilizing a similar in vitro model of IBD identified that PF can further alter immune responses through reduction of TNF-Į induced expression of adhesion molecules to suppress production of the pro-inflammatory cytokines including

IL-8 and TNF-Į [480]. Moreover, PF has also been shown to delay degradation of Iț% subunit, a prerequisite step required for activation of NF-ț%, though strong evidence that PF exerts its effect through influencing NF-ț% is yet to be identified [479]. Furthermore,

PF can also correct pro-inflammatory induced intestinal defects though modulation of

ROCK, MPRIP, and MYLK2 genes [480]. These observations indicate that PF may also directly alter the response of epithelial cells to pro-inflammatory stimuli.

Further, it has been suggested that amelioration of mesenteric fat following EEN treatment, is an additional potential mechanism for enteral diet in inducing remission in patients with

CD [481]. Hypertrophy of the mesenteric adipose tissue is a hallmark of CD and is implicated in the production of various adipokines [482]. It has been shown that EEN treatment is capable of restoring adipocyte morphology and decreasing the production of inflammatory mediator through a direct anti-inflammatory activity exerted on the inflamed mesenteric fat tissue in CD patients [483]. A recent study of sixteen CD patients who underwent ileal resection, in which half received EEN for four weeks before the resection, and the other patients had no nutritional intervention. Mesenteric fat samples collected during surgery were assessed for adipocyte size, adipokine production and adiponectin level. The adipocyte size from patients treated with EEN was much larger than that from patients with no nutritional therapy. Furthermore, protein levels of pro-inflammatory adipokines including TNF-alpha and leptin were lower, whereas adiponectin, a protein with anti-inflammatory property, was up-regulated in the EEN group [483].

Despite proven activity of PF in suppressing inflammation, the origin of that effect remains elusive. Nevertheless, fat content of the formula has been proposed as potential source [466]. Bamba et al. [484] in a randomized-controlled trial involving 28 patients with CD showed that excess n-6 long chain triglycerides (LCT) attenuates the effect of EN in treating active CD. A similar conclusion was made by Gassull et al [485], who reported lower remission rates when formulae with high content of polyunsaturated fatty acids

(PUVA) were used. In contrast, Zachos et al. [486] who reviewed seven clinical trials including 209 CD patients treated with EN formulas of differing fat content (low fat: <20 g/1000 kCal versus high fat: >20 g/1000 kCal), demonstrated no statistically significant difference in efficacy of formulas with the differing fat content, in inducing clinical remission. However, some argue that it is the quality, and to a lesser extent the quantity, of fat that will determine the efficacy of the EEN therapy [487]. In an in vitro study on IL-10 deficient mice, partial replacement of dietary (n-6) fatty acids with medium chain triglycerides reduces the incidence of spontaneous colitis in these animals [488].

Furthermore, it is of significant interest that Shoda et al [489], in a study carried out in

Japan examining the correlation between the epidemiology of CD and dietary habit changes, demonstrated that increased dietary intake oI DQLPDO SURWHLQ DQG Ȧ-6 polyunsaturated fatty acids with reduced intake of n-3 polyunsaturated fatty acids, may contribute to the development of CD.

It has been proposed that a PF containing bovine TGF-ß may have immunomodulatory effects based on the known anti-inflammatory properties of this cytokine [477]. Hartman et al [490], using a TGF-ß-enriched formula undertook studies where the formula was given to children with active CD for 8 weeks as their sole nutrition, followed by a 4 week period of controlled reintroduction of solid food. The TGF-ß-enriched formula was effective in inducing remission, mucosal healing, normalising inflammatory markers, and reducing pro-inflammatory cytokine mRNA levels. However, these studies have not been able to distinguish whether it is the TGF-ß or other constituents of the formula that primarily contribute to the beneficial properties of EEN. 1.7.2.2 Mucosal healing

Mucosal healing of the inflamed bowel leads to restoration of intestinal epithelium integrity, improved absorption of nutrients and resolution of protein loss [491].

Histological healing, and not clinical remission, is correlated with reductions in levels of pro-inflammatory cytokines [464]. Further, mucosal healing has become a goal in the treatment of patients with CD not only because persistence of inflammation is correlated with frequency of relapses [492], but also because poor mucosal healing contributes to interruption of normal growth patterns in children, consequent to high levels of circulating inflammatory cytokines [448]. Fell et al. [477] in a prospective cohort study of 29 children with active CD treated exclusively with PF, reported a remission rate of 75% with evidence of mucosal healing on endoscopy, together with a fall of inflammatory mediators.

In a further study on 47 children with CD, mucosal healing was observed in 64.8% of the children receiving EEN compared to 40% of the children who received corticosteroids

[461]. In addition, complete mucosal healing was achieved in seven patients on nutritional therapy whereas no patients receiving corticosteroids had complete mucosal healing [461]

(Table 1.2). The evidence indicates the EEN promotes mucosal healing in CD to a greater extent than corticosteroids, which may in part explain its role in inducing remission in CD patients. However, the question remains as to what extent mucosal healing is required to maintain remission, and whether it is necessary to follow-up and monitor mucosal healing.

Afzal et al. [493] in a prospective cohort study which assessed changes in quality of life and histological improvement in CD patients after EEN treatment, concluded that there was a poor correlation between mucosal healing and quality of life, although most patients did receive a high score of quality of life. Further studies are required to investigate the role of mucosal healing in disease outcomes and overall patient well-being.

Although EEN can enhance mucosal healing in treated IBD patients, the exact mechanisms remain elusive and poorly investigated. A few studies have investigated the effect of PF upon intestinal permeability [480]. Altered intestinal epithelial barrier function is one of features of CD [494] that is at least in part, attributable to pro-inflammatory cytokine production, such as TNF-Į [495]. TNF-Į-induced increase in intestinal epithelial TJ permeability is regulated by modulation of myosin II regulatory light-chain kinase

(MLCK) protein expression [496]. Nahidi et al [497], investigated the effects of therapies upon intestinal barrier function in the presence of TNF-Į LQ DQ in vitro model utilizing

Caco-2 epithelial monolayers. Activated cells were then treated with either PF or corticosteroids. Monolayer permeability was studied and MLCK gene expression was evaluated. TNF-Į exposure increased monolayer permeability, upregulated MLCK expression and reduced tight junction integrity. Interestingly, these changes were reversed completely with PF, but only partially with steroids. This restoration of gut barrier morphology and function by PF was noted to be principally associated with inhibition of

MLCK [497]. Thus, these findings provide some evidence about the molecular mechanisms of PF-induced mucosal healing and lack of mucosal healing with steroids.

These observations were further supported by studies utilizing a mouse model of colitis characterized by increased gut inflammatory markers and reduced gut barrier function

[498]. Interestingly, EEN treatment given as PF maintained gut barrier integrity and function, and reversed the inflammatory changes with amelioration of colitis. The authors also found these changes were correlated with down-regulation of gene expression of pro- inflammatory cytokines involved in disruption of tight junction proteins [498]. Duration of therapy Treatment P-value (8 weeks) Cs (n=10) EN (n=37) Clinical remission 9/10(90%) not significant 32/37(86.5%) Histological remission Partial 4/10(40%) 26/37(70%) <0.001 Complete 0/10(0%) 7/37(20%) <0.005

Table 1.2 Shows clinical and histological remission of 47 children with CD [461]. 1.7.2.3 Modification of the intestinal flora

An alteration in the host bacterial microflora is one of the distinctive features of CD and has significant implications upon the pathogenesis of disease [127]. Interestingly, there is evidence that treatment with EEN leads to profound modifications in the fecal microflora

[499]. Lionetti et al. [500] using Temperature Gradient Gel Electrophoresis (TGGE), examined the microflora in stool samples of CD patients who received EEN for 8 weeks along with samples from a comparative healthy control group. The authors observed a major modification of the faecal microflora in all patients treated with EEN, expressed as variable distributed bands on the TGGE profile. In contrast, all healthy controls had a stable profile [500].

Leach et al. [501] made additional observations when they examined changes in the bacterial diversity of paediatric CD patients treated with EEN. Stool samples were collected from six children diagnosed with CD at time of diagnosis, during the 6-8 weeks of EEN treatment, and then for up to 4 months following the end of this treatment. Stools were also collected from seven healthy control children. A significant modulation in stool bacterial composition was shown in EEN-treated patients but not in samples from the healthy group, using another molecular technique (Denaturing gradient gel electrophoresis;

DGGE) that included a broader selection of primers. This change in composition was still evident four months after the completion of therapy. Further, it was noted that changes in

Bacteroides species correlated positively with reduced disease activity [501]. A recent study by Kaakoush et al. [502] looked at the changes in the fecal microbiota of

CD patients using high throughout sequencing techniques. Stools were collected from five

CD children, before, during, and after EEN therapy and compared with samples from five healthy controls .The microbial diversity in CD patients was significantly lower than that in matched controls. Importantly, the authors noted that the number of operational taxonomic units (OTU), reflecting microbial diversity, decreased substantially upon starting EEN treatment and correlated with a drop in disease activity. It has been noted that disease remission was associated with a decrease in OTUs whereas an increase in OTUs corresponded with disease relapse. The authors were also able to identify a number of bacterial species within the Firimictus family that correlated with disease activity during and after EEN therapy [502].

In addition to the effects on fecal microflora, EEN treatment has also shown to alter mucosal flora in a study involved intestinal biopsies collected from six pediatric CD patients treated with PF [503]. Marked changes in the mucosal bacterial community were noted with restoration of the bacterial variability in the disease biopsies to that seen in the control samples [503].

How precisely treatment with EEN modulates changes in faecal or mucosa-associated flora remains elusive with several potential mechanisms proposed. The first is that formulas utilized for EEN have both low residue and prebiotic properties, and that these properties have the capacity to modify the gut microflora [500]. Starving bacteria by limiting the access to energy and/or nutrients during EEN might be an additional factor of how EEN affects the gut flora [480]. Further, it has been claimed that EEN might induce changes in the micro-environment of the colon as a result of alterations in pH, short-chain fatty acids or changes in specific bacterial growth factors [448]. However, further research is warranted to either support or refute these assumptions.

In addition to modification of the gut microbiota, it has been suggested that EEN can induce remission by excluding dietary factors responsible for inflammation, as recently termed “bacterial penetration cycle” [243]. It is well accepted that gut microbiota has a major role in triggering the mucosal immune system, a hallmark of CD pathogenesis [504] and specific diets and food additives affect and compromise the integrity of the intestinal epithelial barrier function [505, 506]. In the “bacterial penetration cycle” hypothesis, it has been claimed that the impairment in the intestinal epithelial barrier function, secondary to consumed dietary factors, allows adherence and trafficking of bacteria that then start replicating in epithelial cells and other mucosal immune cells. Penetration of bacteria might be made worse in susceptible individuals where failure of intracellular bacterial detection and clearance [507], results in triggering of the adaptive immune system and subsequent tissue destruction with further loss in the mucosal barrier, thereby a continuous cycle of diet-induced compromised mucosal barrier-bacterial penetration-inflammation

[243]. Therefore, how patients benefit from EEN while receiving the therapy might be, at least in part, relate to modulating this cycle of diet-facilitated bacterial penetration. 1.7.2.4 EEN and bowel rest

Resting the inflamed bowel has been suggested to be a further possible mechanism of

EEN. Greenberg et al. [508] investigated this hypothesis in a study where 51 patients with active CD were randomised into three treatment groups: total enteral nutrition group, total parenteral nutrition group or mixed partial parenteral nutrition and solid food. They found that each group achieved remission rates of approximately 60% and there was no significant different in the remission rates between the three groups. Therefore the authors conclude that bowel rest does not have a role in inducing remission. To date, it has not been clearly verified that EEN exerts clinical effects in patients with CD through decreasing an antigenic load and resting the injured mucosa. 1.7.3 Nutritional benefits of diet therapy

Nutritional therapy is capable of improving poor nutritional status, correcting negative nitrogen balance [305] and supporting growth of CD patients [445, 509]. EEN results in increased serum albumin level, weight gains and linear growth recovery shortly after commencing the therapy [467]. Knight et al. [467] in a study involving 44 children with newly diagnosed CD whom received nutritional therapy as sole therapy, observed that the mean serum albumin levels increased from 32 to 44 g/l over an 8 week period. Further, there was a significant rise in standardized Z-scores for weights from presentation to follow up after 1 year, consequent to EEN. A similar conclusion was made by Day et al

[460], in a study of twenty-seven children with CD, 15 of whom were newly diagnosed.

The 15 children with newly diagnosed disease gained an average of 4.7+/- 3.5 kg after eight weeks of EEN along with positive changes in body mass index and Z-scores for weight and increased serum albumin levels. Another advantage of nutritional therapy is that young patients with CD in pre- or early pubertal stages will likely catch up their height if they commence treatment before bone maturation occurs [308, 510]. Berni Canani et al. [461] in a study involving 47 children with CD where 37 of them received nutritional therapy (and 10 were given steroids), observed that nutritional therapy is more effective in enhancing linear growth recovery than steroids, as the ratio of corrected linear growth was much higher in the EEN group than corticosteroid group. Moreover, restoring already depleted levels of circulating micronutrients in patients with CD is another nutritional value attributed to EEN [511]. In a study involved 17 children with active CD who presented with suboptimal concentrations of several trace elements and vitamins had significant changes in micronutrients plasma levels along with improvements in body weight at the end of a 6 week EEN course [67].

Several clinical trials involving adults have shown promising results with weight gained, improved nutritional status and body composition within a short period of commencing nutritional treatment [512, 513]. Royal et al. [514] assessed the nutrition of 30 adult patients with newly diagnosed CD who received enteral therapy for 3 weeks. All patients gained weight (mean weight gain of 2 kg), which was accompanied by an increase in total body protein, fat and plasma transferrin levels. A similar observation was noticed by

Yamamoto et al. [464] in a study of 28 adult patients with CD who were treated with EF for 4 weeks. The median body weight, BMI, and serum albumin levels of all patients significantly increased, indicating improved nutritional status. Further, the blood inflammatory markers, ESR and CRP fell along with raised counts of lymphocytes and platelets [464] (Table 1.3).

Given the various nutritional consequences of CD, which may be worsened by corticosteroids, recommending nutritional therapy as the first line therapy for induction of remission in children and young adults with active disease is appropriate and well justified.

This approach should lead to nutritional improvements, including linear growth and assisting these children to achieve their height potential (particularly when treated before bone maturation). Marker Before Treatment (n = 28) After Treatment (n = 28) P-value Median Value (i.q.r.)

Body weight (kg) 50 (45–52) 52.5 (48–54) 0.0001

BMI (kg/m2) 19 (17–22) 19.5 (19–23) 0.0001

Albumin (g/dL) 3.0 (2.5–3.4) 3.4 (3.2–3.5) 0.0001

WBC count (x100/mm) 104 (89–121) 80 (68–88) 0.0001

Platelet count(x10000/mm) 52 (42–58) 31 (25–37) 0.0001

ESR (mm/h) 48 (42–60) 23 (12–32) 0.0001

CRP (mg/dL) 4.7 (3.0–6.0) 1.0 (0.1–2.9) 0.0001

Table 1.3 Represents body weight, BMI and blood markers of 28 adult CD patients before and after EF treatment [464]. 1.7.4 EEN as a treatment option

1.7.4.1 EEN as induction therapy in Children

The potential benefits of EN as a therapeutic option in CD were first documented almost 4 decades ago [515]. One of the earliest observations was reported by Ricour et al. [516] in

1976 on 5 children presented with severe CD who were prescribed an exclusive regimen encompassing EF and parenteral feeding given together. The authors noted that all children showed a dramatic improvement in their general condition and were capable to overcome the acute stage of disease [516]. Eight years later, the first controlled trial was undertaken involving 21 CD patients who were randomized to receive either corticosteroids or EN

[517]. The authors reported that both groups had equal remission rates represented by reductions in disease activity indices at 4 and 12 weeks assessments.

Subsequently, numerous randomized and non-randomized trials that involved larger number of patients have been undertaken to investigate the efficacy of EEN as an induction treatment in children with CD [448]. Thomas et al.[518], in a randomised controlled trial, recruited 24 children to receive either EEN utilizing EF or high dose steroids. Both groups had a similar improvement in disease activity and achieved equal remission rates.

Elemental diet was well tolerated by the majority of treated patients. Moreover, growth parameters such as growth velocity was significantly better in the EF treated group than in the group treated with steroids [518]. Similar findings were reported by Seidman et al.

[469, 519] in two randomized controlled trials involving 44 children given 4 weeks of

EEN. Matched controls treated with corticosteroids for induction of remission were also included. Remission rate among the forty-four EEN treated patients (a mean of 78%) was statistically incomparable to that of the steroid treated patients.

In 2000, the first meta-analysis was carried out by Heuschkel et al.[520] which included 5 randomised controlled paediatric trials with a total 147 children treated with EEN. This study ascertained that EEN and steroids were of equal efficacy in the induction of remission in children with active CD. However, with observed supported growth, enhanced development and avoidance of adverse effects of steroids indicated EEN was superior to conventional medications as a first line therapy in children with active CD. Seven years later, a similar conclusion was made by Dziechciarz et al. [298] in a meta-analysis that included 7 randomised controlled trials with a total of 204 paediatric CD patients. The authors also noted that there was no significant difference in induction of remission between EEN and corticosteroids. Subsequently numerous studies have been published and the majority support EEN as the treatment of choice, being superior to corticosteroids for induction of remission in a paediatric population with CD [463, 521, 522].

The most recent meta-analysis were gathered in the last edition of published consensus guidelines of of the European Society of Pediatric Gastroenterology, Hepatology and

Nutrition (ESPGHAN), and the European Crohn's and Colitis Organization (ECCO) on the medical management of paediatric CD 2014 [523]: seven randomized controlled trials were included with a total of 204 patients (100 in corticosteroid group, 104 in EEN group) comparing activity of different formula preparations with corticosteroid therapy. There was a considerable variability in regard to treatment duration (varying from 3 to 10 weeks), disease location and duration (new onset or relapsing disease), or associated treatment.

However, the overall conclusion was that induction of remission with EEN was equivalent to corticosteroids for paediatric CD and therefore it should be recommended as a treatment of choice in children with active disease [523]. 1.7.4.2 EEN as induction therapy in adults

In contrast to the conclusions regarding the use of EEN in children, EEN is considered to be less effective in adults [466, 524]. Early reports compared efficacy of EEN using EF to steroids in treating CD adult patients showed favorable outcomes of use of the enteral diet

[448, 525]. In the initial controlled trial almost 3 decades ago, in which adult patients were randomized to receive either prednisolone or an elemental diet, it was shown that the remission rates were equal in the two groups [517].

Subsequently several studies including meta-analyses revealed that corticosteroid is more effective than EF in inducing remissions in adult CD patients [526]. Griffiths et al. [527] in 1995 published a meta-analysis comprising eight trials with a total of 413 CD patients

(primarily adults). Enteral nutrition was inferior to corticosteroids in inducing clinical remissions in CD (pooled OR, 0.35; 95% confidence interval, 0.23-0.53). Later, Zachos et al. [486] published a Cochrane review that included 212 patients treated with enteral nutrition and 179 treated with steroids. The meta-analysis yielded a pooled OR of 0.36 favouring steroid therapy over EN (95% CI 0.23 to 0.56) [486]. However, more favorable outcomes were noted in a recent systemic review undertaken in 2013 by Wall et al.[528] where 11 studies, comparing the efficacy of EEN versus corticosteroids in adults. The authors came to the following preliminary conclusions: EEN as induction therapy, apart from Japan, it is not routinely utilized as first line treatment in adult CD; EEN therapy remission rates varied considerably: 3 out of 11 studies reported remission rates equal to

80% or above [517, 529, 530], whereas in the other 8 trials remission rates were achieved in half or less of the recruited patients [531]; The remission rate generally was higher in newly diagnosed patients. Moreover, the adherence with the treatment due to unpalatable formula (EF) was an issue in half of the studies. The authors also noted there was no strong evidence to ascertain if disease location, duration of disease or age of diagnosis affected

EEN therapy outcomes [171]. However, there is a tendency to support the use of EEN as a treatment option for those motivated to adhere to an EEN regimen and possibly those newly diagnosed with CD, especially if a more palatable formula like PF is used [528].

Guo et al.[532] investigated efficacy of EEN utilizing PF, known to have better palatability than other preparations [533], in adult patients with active CD and assessed the effect on the health-related quality of life. Remission rate was 85% of treated patients following a 4 week PF regimen. Further, there was a significant improvement quality of life score, social function and emotional status after treatment. In addition, around 61% of the recruited subjects expressed their willingness to receive the treatment again to induce remission if the disease relapsed [532].

Therefore, it appears that EEN can be effective in inducing remission in adult patients and may be a favourable choice if compliance can be improved. 1.7.4.3 EEN as maintenance therapy

Numerous paediatric and adult reports support a role for EN in the maintenance of remission and prevention of relapse in CD [448, 452]. There is a significant body of evidence indicating that ongoing low volume maintenance with EN can maintain medically induced remission for a prolonged time [534-536]. One of the early trials that assessed the effect of EN continuation on the disease activity in pediatric patients was undertaken by

Wilschanski et al.[537]. This study investigated the effect of EN when given as a nocturnal supplement with intake of normal diet during the day time. Sixty five children and adolescents with CD who were initially treated with EEN were reviewed retrospectively based on they were continued supplementary EN. The authors noted that patients who continued nasogastric EN feeding together with normal diet remained well longer than those who discontinued EN. Relapse rates were higher in the non-EN group at 6 and 12 months than the group that continued EN. In addition the nocturnally EN fed group experienced an improvement in their linear growth parameters [537].

Day et al. [460] also revealed that remission can be prolonged in CD by supplementary

EN. They carried out a study on a cohort of children with CD. Twenty-seven children received EEN in which fifteen children had newly diagnosed CD and 12 had long-standing disease. Twelve of 15 (80%) newly diagnosed CD and seven of 12 (58%) with long- standing disease entered remission. Further, four children after initial EEN continued on supplementary PF (without other medical therapies) and all subsequently maintained remission for an average follow-up period of 15.2 months [460]. Additionally, Duncan et al. [538] in a recent retrospective study, assessed the efficacy of

EN as a maintenance treatment option for paediatric CD alongside the use of azathioprine

(a commonly prescribed maintenance drug). Fifty nine pediatric patients were recruited for initial induction of remission by EEN. Forty eight patients who completed 8 weeks EEN and achieved clinical remission were randomized into 3 groups: one group continued on ongoing EN for a mean of 10 months, the second group was commenced on azathioprine whereas the third group had no treatment. Remission rates at 1 year in patients with ongoing EN were 60% (9/15) compared to 15% (2/13) in patients taking no treatment

(Pௗ ௗ  DQG    LQ SDWients taking azathioprine (Pௗ ௗ  [538]. These data indicate, taking into account the side effects of the immunosuppressive drug, that EEN is superior to the commonly used maintenance treatments in CD.

In adult patients, Yamamoto et al. [539] reported a prospective study of 40 adults with CD with medically achieved remission. In this study, twenty of this group continued receiving nutritional therapy with restriction of normal diet for one year, whereas the other twenty returned to normal diet, with no supplementary EN. One quarter of the maintenance group had clinical relapse over one year of observation, contrasting to a relapse rate of 60% in the normal diet group. Further, there was a significant elevation of mucosal levels of the inflammatory cytokines IL-ȕ ,/-6, and TNF-Į LQ WKH QRUPDO GLHW JURXS RQO\ 7KH authors concluded that nutritional therapy would maintain clinical and endoscopic remission in CD patients who continue receiving nutritional therapy in conjunction with restricted diet. A similar conclusion also was made by Takagi et al. [534] in a randomised study involving

51 adults with CD in remission. Twenty-six patients were randomly allocated to receive half of their daily caloric requirements with EF whilst the other 25 patients were allocated to receive a normal unrestricted diet. Lower relapse rates were recorded among the EF group compared to the free diet group, (34% vs 64%) after 12 months follow up.

Along with benefitting patients with medically induced remission, there is evidence that maintenance enteral supplements can also be successful in preventing relapse in patients after surgical induction of remission [536, 540, 541]. Yamamoto et al. [542] carried out a trial involving 40 patients with CD who underwent surgical resection and investigated whether patients can be offered benefit from EN after the surgery. Patients were allocated to receive nutritional therapy in conjunction with a restricted diet for 12 months or to receive normal unrestricted diet only. The group that received nutritional therapy with a restricted diet had a 5% clinical relapse compared to 35% for the normal diet group. The endoscopic recurrence was also higher in the normal diet group (40% after six months and

70% after one year) than in the restricted group (25% after six months and 30% after one year).

Thus, EEN appears to have a significant role in managing patients with chronic CD. It is capable of preventing clinical and endoscopic recurrence of the disease for an extended period of time, particularly if given with a restricted diet. Further studies assessing the optimal duration of such therapy are warranted. 1.7.4.4 EEN vs. PEN

In contrast to EEN, a partial enteral nutrition (PEN) regimen involves enteral diets given in conjunction with food intake, in which a significant portion of caloric source is provided from food [521]. In terms of effectiveness, it has been shown that PEN with a free diet is less effective in inducing complete remission or reducing acute phase reactants in active

CD compared to EEN [543]. In a trial involving pediatric CD patients (n = 50) who were randomized to receive either 100% EEN or 50% PEN as EF with free access to diet for 6 weeks. The remission rate with PEN (15%) was significantly lower than that with EEN

(42%). The authors also noted that only the EEN group had an increase in hemoglobin and albumin, and a fall in platelets and ESR [543]. Correspondingly, the recent consensus guidelines of the ECCO/ESPGHAN on the medical management of CD published in 2014, did not recommend the use of PEN as induction therapy for the treatment of CD [523].

Efficacy of EEN might depend, at least in part, on exclusion of free diet elements hypothesized to affect the microbiome or intestinal permeability. In line with these concepts, Sigall-Boneh et al. [544] investigated the effect of PEN as induction therapy, but with exclusion of particular dietary components. In their study 47 children and young adults with CD with mild to moderate disease severity were given a 6-week structured CD exclusion diet (CDED) that allowed access to specific foods and restricted exposure to all other foods, and up to 50% of dietary calories from PF. Up to 70% of recruited individuals went into remission by the end of the study together with substantial reduction in specific inflammatory markers [544]. This indicates that enteral diet might have utility as an induction therapy even if provided as PEN instead the conventional regimen when prescribed as a solely nutritional source (EEN). However, exclusion of any identified triggering dietary factors seems essential for better outcomes in this setting.

It should be noted that PEN as induction treatment is not yet established in clinical practice. Indeed, use of PEN in CD is currently restricted to patients with chronic disease as a maintenance therapy [521]. Remission could also be maintained by just prescribing nutritional therapy solely (EEN) as periodic extensive regimen [448]. Polk et al. [545] investigated the effect of intermittent EEN courses on growth and disease activity in children with CD. EEN was given as the exclusive nutrient source every 1 out of 4 months during the 1-year period. Growth parameters including height and weight velocity significantly increased. Steroid intake significantly decreased and significant improvement was seen in disease activity [545]. While early studies showed promising outcomes, this regimen is rarely prescribed. 1.7.4.5 EEN efficacy and disease location

It has been proposed that the efficacy of EEN in inducing remission in CD patients is linked with disease location [486]. In several studies it has been suggested that the most favorable outcomes of EEN in CD are achieved in patients who have small bowel disease, and those with isolated colonic disease are least likely to have benefit [546]. A prospective study by Afzal et al. [547] included 65 pediatric CD patients who were grouped into 3 categories based on their disease location: ileal, ileocolonic and colonic. All patients were treated with EEN, with clinical and histologic remission assessed. The isolated colonic disease group showed the least reduction in PCDAI scores, and the lowest clinical and endoscopic remission rates compared to the other two groups. Patients with colonic disease responded better if there was also ileal disease present [547]. Similar observations were seen in a study undertaken by Wilschanski et al. [537] it was noted that the individuals with isolated colonic CD were significantly less responsive to treatment with EEN than the patients with CD at other locations.

In contrast, a trial carried out by Buchanan et al.[463] involving CD patients who received

EEN for 8 weeks, demonstrated that disease phenotype including location did not influence efficacy of EEN. Patients only with isolated ileal disease had the lowest rate of remission, but no significant differences in the remission rates were observed among other disease locations [463]. Similar findings were reported by Day et al. [460] when they showed no difference in the remission rates of children with ileal disease and those with isolated colonic disease after receiving EEN utilizing PF for 8 weeks, however this involved a small patient group. Moreover, “The second European evidence-based Consensus on the diagnosis and management of CD 2010 and 2014” suggested that EEN is effective as induction treatment in pediatric CD irrespective of the disease location, although there is some evidence indicating children with colonic disease respond better if there is ileal involvement as well

[523, 548]. Indeed, looking more deeply to the data from Buchanan et al. [463] although it was not significantly different, patient with isolated colonic involvement had the lowest remission rate compared with other disease location groups. In this study 78.9% of the colonic disease group went into remission, whereas 86.2% of patients with ileocolonic disease involvement went into remission. Further trials to assess outcomes in different time points are warranted to ascertain whether disease location has an effect on EEN therapy outcome in CD. 1.7.4.6 PF vs. EF

Although elemental diet (amino acids as nitrogen source) and polymeric diet (complex protein as nitrogen source) differ considerably in nutritive composition, many clinical trials have shown they have similar efficacy in treating patients with CD [549-551]. The first- double blind randomized trial in children designed to compare the therapeutic efficacy of

PF with EF was undertaken by Verma et al.[551] in which 21 pediatric patients with active

CD were randomized to receive either PF or EF. The two formulas were identical except for the nitrogen source. The remission rates and inflammatory indices were equal in the two groups [551]. Similar observations were seen in another randomized, non-blinded, multicenter, controlled trial that assessed efficacy and safety of elemental and polymeric diet as the primary therapy in 16 children with active CD [552]. There was no significant difference between the formulas in remission rates at 6 wks. Further, patients treated with

PF gained significantly more weight than patients treated with EF suggesting polymeric diet may be superior to elemental diet where the primary aim is to increase the patient's weight [552]. Moreover, a recent trial by Grogan et al. [553] who compared the efficacy between the two formulas over 2 year follow-up. Over the study period remission rates were similar between the two groups. One third of patients also maintained remission for 2 years with no significant difference between the formulas. The mean time to relapse was

162 days in PF group and 183 days for the EF treated group (P > 0.05) [553].

Additionally, meta-analyses of dietary source of protein (PF vs. EF) does not appear to influence efficacy of enteral nutrition therapy for induction of remission in CD patients

[527]. A large review by Zachos et al. [486] included 14 trials comparing different formulations of EN for the treatment of CD. Eleven studies compared elemental formula to a non-elemental formula; three compared enteral diets of similar protein composition but different fat composition. Meta-analysis of ten trials with 334 patients demonstrated no difference in the efficacy of elemental versus non-elemental formulas (OR 1.10; 95% CI

0.69 to 1.75). Subgroup analysis of seven trials including 209 patients treated with EN formulas of differing fat content demonstrated no statistically significant difference in efficacy (OR 1.13; 95% CI 0.63 to 2.01). Similarly, effects of very low fat content or type of fat were investigated and did not show any a difference in efficacy in the treatment of active CD. Furthermore, analyses performed to evaluate the different types of elemental and non-elemental diets (elemental, semi-elemental and polymeric) showed no statistically significant differences. However, in terms of adherence to treatment, studies showed that polymeric diet is better tolerated with naso-gastric feeding required less frequently [523,

554]. Overall there appears to be no clinical difference in the choice of formula; however the lower cost and high palatability of PF make it more attractive for practical use. 1.7.4.7 EN and duration of treatment

For induction of remission via EEN therapy, a slow introduction of formula over a period of 2 to 3 days is generally recommended to ensure tolerance and decrease risk of diarrhea

[555]. Once the therapy is initiated, formulas would be given solely with exclusion of all additional diet for several weeks [556]. Although duration of EEN therapy varies between

IBD centers, it is usually given for up to 8 weeks [557]. In two recent surveys with regard to the duration of EEN therapy, participants most commonly reported ranges of 6 to 8 weeks [556, 558]. Consistently, as per the 2014 ESPGHAN and ECCO’s guidelines, 6 to 8 weeks of EEN was recommended if given as induction therapy in patients with active CD

[523].

Enteral diet (PEN) for maintenance of remission in CD is usually prescribed in conjunction with a restricted normal diet, in which up to half of the daily calorie requirement is provided by an elemental diet and the remaining half by a restricted diet [534, 542]. The other option is to supplement with an unrestricted normal diet throughout the day [460,

466]. However, the former regimen in which EN is given with restriction in diet appears to show more favourable outcomes than if EN is given with an unrestricted diet.

Although there is strong evidence of the efficacy of EN as a maintenance therapy in CD patients, the optimal duration of therapy remains ambiguous [559]. Different trials had used EN courses that in part, varied by patients’ compliance [560]. Wilschanski et al.

[537] reported that EN was given as a nocturnal supplement for 6 months in some patients and 12 months in others. In another trial by Duncan et al. [538] patients received ongoing

EN for a mean of 10 months. In contrast, trials that involved surgically induced remission, the follow up duration was of up to a mean of 2.6 years [541]. Further research is required to ascertain the optimal length of EN therapy when given to patients with chronic disease.

In summary, nutritional therapy (EEN) is an increasingly attractive option for CD. In many specialist centres in Europe and Japan, EEN is the standard treatment for active CD in children. This is due to its equivalent efficacy to corticosteroids in remission induction, enhanced mucosal healing, steroid-sparing effects, safety even with long-term use and the many nutritional benefits. Because many children with CD are under weight and a large proportion of them are stunted at presentation, it is clinically advantageous to use nutritional therapy as induction therapy. This will help recovery of impaired linear growth, promote weight gain, and correct other nutritional manifestations whilst permitting avoidance of adverse effects associated with conventional medications. The mechanism(s) of action of nutritional therapy are not yet completely understood. Nevertheless, current evidence shows that direct anti-inflammatory effects, mucosal healing and alteration of the intestinal microflora, contribute to the therapeutic properties of EEN. Finally, although much is known about the roles and mechanisms of EEN, there remain many gaps in our understanding. 1.8 Novel nutritional treatments

1.8.1 Glutamine

During the process of inflammation, nutrient utilization, including utilization of amino acids, is altered [561]. Protein loss and increased energy expenditure has been observed in various inflammatory conditions [562]. In such situations, amino acid metabolism is modified so that amino acids become a source for hepatic acute-phase protein synthesis and immunoglobulin production by immune cells [563]. These changes suggest there are specific amino acid requirements during catabolic states [564]. In such states, glutamine and arginine become conditionally essential amino acids required for cell survival and responses to injury [565, 566].

Glutamine is the most abundant amino acid in the human body [567]. It is an important nutritional source for enterocytes [568] and is required by T- lymphocytes for activation

[569]. Glutamine is utilized at a very high rate by intestinal epithelial cells and immunocytes [570]. Further, systemic and mucosal glutamine is markedly depleted in inflammatory conditions including CD [571]. Mucosal atrophy, disrupted intestinal barrier function, increased bacterial translocation and reduced glutathione synthesis are consequences of glutamine deprivation [564, 572]. Interestingly, there is evidence that glutamine supplementation can reverse these changes [573]. Glutamine supplementation can improve intestinal integrity and barrier function [574], stimulate intestinal epithelial cell proliferation [575] and reduce apoptosis of enterocytes [576]. Glutamine supplementation also prevents bacterial translocation, which is implicated in triggering inappropriate mucosal immune responses [577]. Numerous clinical trials of glutamine supplementation to treat critically ill and surgical patients have reported beneficial outcomes, including reduced infections rate, reduced morbidity and improved survival [578-582]. Further, glutamine has been shown to be an effective anti-inflammatory agent in many animal models [583-585]. Based on this evidence it has been proposed that glutamine supplementation may also be beneficial in

IBD. In one double-blind randomised controlled trial [586], 18 children with active CD received a 4-week course of either standard polymeric diet with low glutamine or a glutamine-enriched polymeric formula. However, there was no measurable advantage of the glutamine-enriched formula compared to the standard formula in induction of remission. Further, the glutamine enriched formula was also less effective in reducing the

PCDAI. Additional clinical trials of patients with CD have also shown that glutamine supplementation has little effect on intestinal inflammation, with the supplementation groups showing no differences in intestinal permeability, CRP, CDAI and length of hospital stay [587-588].Therefore, there remains some controversy regarding glutamine supplementation in the treatment of patients with IBD.

The benefits of glutamine have been proposed to be through the activation of the heat shock pathway and over expression of heat shock protein (HSP) 70, an important protein for cellular protection during stress [578, 581, 583]. In addition, the effect of glutamine on enterocytes is important for maintaining and improving intestinal integrity through increasing cell proliferation, reducing intestinal epithelial apoptosis and modulating the inflammatory response [589]. The anti-inflammatory activity of glutamine has previously been investigated in vivo and in vitro. One study using cultured intestinal epithelial cells showed that glutamine modulated the NF-ț%VLJQDOLQJSDWKZD\DQGLVWKHPDLQSURFHVVRI inhibiting inflammation [590]. However, it was not specifically elucidated where or how, glutamine was interacting with the NF-ț%SDWKZD\Moreover, glutamine can also down regulate TLR4 expression [591], which may reduce the inflammatory response to intestinal bacteria. Considering all the available data, the beneficial effects of glutamine appear substantial; however these benefits may also be dose dependent. Therefore, further research is needed to determine whether, and under what conditions, glutamine supplementation has a role in IBD therapy. 1.8.2 Arginine

Arginine is an amino acid with multiple metabolic and immunological functions [592].

Diet, endogenous synthesis and turnover of body proteins are the three main sources of free arginine in the body [593]. Approximately 40% of dietary arginine is catabolized by the intestine before entering the circulation [594]. The gut is also implicated in endogenous arginine synthesis that involves the intestinal-renal axis, in which the citrulline synthesized from glutamine in the small intestine is converted into arginine in the kidney [595]. During catabolic conditions, de novo synthesis of arginine fails to meet the increased demand resulting in disrupted body arginine homeostasis [596], thus arginine becomes an essential amino acid [597]. It has been reported that arginine deficiency in preterm babies results in severe metabolic derangements and multiple organ failure [596].

Several benefits have been observed for arginine supplementation during inflammation and catabolic states [598]. Arginine supplementation reduced the incidence of necrotizing enterocolitis in preterm infants [599]. In animal studies, arginine supplementation is also advantageous for the injured gut. Arginine enhanced peritoneal macrophage phagocytic activity in rats with gut-derived sepsis [600]. Following radiation enteritis, dietary arginine accelerated intestinal mucosal regeneration and enhanced bacterial clearance in treated mice [592]. Moreover, when rats were subjected to mesenteric ischemia, arginine administration improved the disrupted intestinal epithelial barrier [601] and accelerated repair of the damaged intestinal mucosa [602]. Arginine exerts these effects by altering several biochemical pathways [603]. One postulated target of arginine metabolism is the arginase pathway, through which polyamines are produced [604]. Polyamines play a key role in cell division, DNA replication and regulation of the cell cycle [605]. A further target is NO synthetase that is involved with NO production [606]. Arginine availability is one of the rate-limiting factors governing NO production [607]. Expression of inducible

NO synthetase is controlled predominantly by the NF-ț% VLJQDOLQJ SDWKZD\ [608].

However, the relationship between arginine and NF-ț%DFWLYLW\LVQRWZHOOGHILQHG 1.8.3 Curcumin

Turmeric (the common name for Curcuma Longa) is an Indian spice that belongs to the ginger family [609]. In ancient time, turmeric powder was utilized as a traditional and natural remedy for various health conditions such as joint pain, ulcers, liver disease, wounds and skin diseases [610]. The active ingredient of turmeric is curcumin with the chemical name of Diferuloylmethane [611]. Curcumin exhibits anti-microbial, anti- inflammatory, anti-oxidant and anti-neoplastic properties [612]. Because of these various activities, curcumin has been extensively investigated for benefits in managing chronic inflammatory conditions [613].

In IBD, curcumin is proposed as an attractive alternative therapy [614, 615] due to the undesirable side effects [616], expense [617] and refractoriness of conventional medicines

[618, 619]. In contrast, curcumin is considered as a safe and inexpensive supplement

[620]. Certainly, it has been ascertained that a dose up to 12 g a day of curcumin can be safely consumed by human [621]. Further, there is a significant body of evidence emerging from pre-clinical studies indicating potential benefits for curcumin supplementation in IBD

[622-625]. In several experiments involving various murine models of colitis, therapy with curcumin revealed numerous favourable benefits including body weight recovery, improved survival rates, substantial drop in the inflammatory indices, and enhanced healing of the local injured intestinal tissue [623, 624, 626-630]. Clinical trials of curcumin have included a pilot study involving five patients with chronic ulcerative proctitis and five patients with CD [631]. Curcumin was administered for 2 months to the patients with proctitis and for 3 months to the patients with CD. All patients with proctitis experienced symptom relief, which was consistent with significant reduction in inflammatory indices; in addition 4 of the 5 subjects were able to reduce concomitant medications. Four of the CD patients who completed the study showed a significant drop in disease activity and erythrocyte sedimentation rate. Moreover, in a randomized, double- blind, multicentre trial involving 89 patients with quiescent UC, 45 patients received curcumin plus sulphasalazine or mesalamine, and 44 patients received placebo plus sulphasalazine or mesalamine for 6 months [632], at the completion of the study, relapse rates were 4.65% in the curcumin-treated group compared to 20.51% in the placebo group.

These preliminary studies appear promising and suggest that curcumin may be a viable maintenance therapy for IBD patients.

Additionally, there is a newly evidence indicated that curcumin may be utilized as induction therapy in IBD. A recent double-blind study involved 50 patients with mild to moderate UC who did not respond to the maximum dose of mesalamine, were randomized to receive either curcumin capsules or placebo with continued mesalamine [633]. In this study, addition of curcumin was superior to placebo in inducing clinical and endoscopic remissions.

The anti-inflammatory properties of curcumin are attributed in part to its interference with activation of the intracellular signaling pathways, particularly NF-țB, P38 MAPK, Activated protein-1 (AP-1), signal transducer and activator of transcription (STAT) proteins and peroxisome proliferator-activated receptor-gamma (PPAR-Ȗ [634-636].

Modulation of NF-țB is of particular importance and is considered a putative target for intervention in IBD [637]. Curcumin inhibited activation of NF-ț% LQGXFLQJ NLQDVH DQG

WKHUHE\SUHYHQWHG,ț%GHJUDGDWLRQLQ+7-29 and Caco-2 intestinal epithelial cells cultured in vitro [638]. Moreover, in chemically induced colitis in mice, a curcumin-containing diet ameliorated colonic inflammation and prevented inflammatory cell tissue infiltration; NF-

ț%DFWLYDWLRQDQGH[SUHVVLRQRIpro-inflammatory cytokine messenger RNA in the colonic mucosa were inhibited [639]. Consistent with these observations, NF-ț%DFWLYDWLRQLQWKH inflamed colonic mucosa of rats is suppressed by curcumin [640].

In addition to NF-ț% [641], therapeutic benefits of curcumin have been determined to be mediated through suppression of the P38 MAPK pathway [642]. In a study involving rats subjected to experimental colitis, curcumin treatment substantially attenuated local colonic tissue damage and caused substantial reductions in the raised inflammatory indices in conjunction with attenuation in the up-regulation of P38 MAPK [634]. Further, in cultured intestinal mucosa from children and adults with IBD, curcumin inhibited the activity of

MAPK with subsequent reduction in proinflammatory cytokine production [641].

Collectively, curcumin has wide anti-inflammatory activities by mediating signalling transduction pathways and is shown to have the potential as a therapeutic choice for treating IBD patients. However, it remained underutilized in clinical practice that is at least in part due to its poor pharmacokinetics properties [642]. Nevertheless, based on the observations seen in the initial clinical studies that utilised curcumin in combination with current IBD treatments, it appears that co-administration of curcumin might be one viable option for better utilization.

1.8.4 Omega-3 fatty acids

Omega- IDWW\ DFLGV Ȧ-3 FAs) are anti-inflammatory substances found abundantly in marine fish that have several health benefits [643]. The beneficial effects of fish oil are related to LWVKLJKFRQWHQWRIȦ-3 PUFA, particularly epicosapentanenoic acid (EPA) and docosahexaenoic acid (DHA) [644]. These compounds have been used to treat various diseases such as Rheumatoid arthritis [645] and immunoglobulin (IG) A nephropathy [646] and are imperative in reducing mortality rates in cardiac diseases [647]. There are a

QXPEHU RI VWXGLHV LQ DQLPDO PRGHOV RI ,%' VKRZLQJ SURPLVLQJ UHVXOWV ZLWK Ȧ-3 FAs supplementation. Whiting et al. [648] LQPLFHPRGHORIFROLWLVKDVVKRZQWKDWȦ-3 FAs are capable of reducing colitis and colonic immunopathology in the mucosa of rodent.

Likewise, in different experimental model of colitis, it has been shown that dietary fish oil

ULFK LQ Ȧ-3 FAs extenuates the progression of inflammation and shortens the course of disease [649].

,Q ,%' WKH FOLQLFDO XVHIXOQHVV RI Ȧ-3 FAs supplements is still controversial. Although there is evidence supporting the use of these supplements as maintenance therapy for patients with CD [650]. In a double-blind, randomised, placebo-controlled clinical trial where 38 patients with CD in remission, receive either 5-ASA with enteric coated capsules

RIȦ-3 FAs or 5-ASA with placebo capsules [651]. After 1 year of follow up, the relapse

UDWHV ZHUH PXFK OHVV LQ WKH Ȧ-3 FAs coated capsules group than in the placebo capsule group [651]. However, several other clinical trials have shown conflicting results [652,

653]. Maclean et al [654] in a systemic review of 13 clinical trials that assessed the effects

RI Ȧ-3 FAs on clinical, sigmoidoscopic, or histologic scores, rates of induced remission and relapse, requirements for steroids and other immunosuppressive agents concluded that

WKHUHZDVQRWVXIILFLHQWGDWDWRUHDFKDFOHDUFRQFOXVLRQDERXWȦ-3 FAs in the treatment of

IBD. More recently Cabre et al. [655] reviewed 19 clinical trials of Ȧ-3 PUFA as therapeutic agents in IBD and reached the same conclusion there was no clear evidence

EHQHILWWLQJ,%'SDWLHQWVWUHDWHGZLWKȦ-3 FAs. 1.8.5 Vitamin D3

Vitamin D3 is a steroid hormone that plays a role in bone homeostasis and has a potent immuno-regulatory function [656]. It regulates differentiation, proliferation and function of the immune system cells [657, 658]. Several in vitro studies involving different human cell types have shown that vitamin D3 supplementation is capable of reducing the production of pro-inflammatory cytokines and enhancing release of various anti-inflammatory mediators

[659-662]. Jorgensen et al. [663] in one double-blind placebo-controlled study to assess the effect of vitamin D3 treatment to CD patients, showed that oral supplementation with 1200

IU vitamin D3 reduced the risk of relapse from 29% to 13%. Of interest, studies have also shown a correlation between low serum levels of Vitamin D3 and higher incidence rates of

CD [18, 664, 665]. Parallel to this, there are observational reports that indicate relapse rates of CD are influenced by seasonal changes, in some area the cases of relapse was higher in winter than other seasons [666]. Thus, there is a potential link between CD and vitamin D3 status, and vitamin D3 supplementation might be effective in reducing risk of developing as well as treating the disease. 1.9 Hypotheses and aims of this Project

1.9.1 Hypotheses

While PF has immunomodulating activity, the determinants of that property and the underlying mechanisms have not yet been identified. I hypothesize that the PF components including glutamine, arginine, vitamin D3 and fatty acids contribute to the anti- inflammatory properties of PF and that these effects are likely mediated through targeting intracellular major signaling pathways, especially the NF-ț% DQG P38 MAPK signaling cascades. I also hypothesize that manipulation of the concentrations of the active constituents of PF may be a method to enhance the anti-inflammatory properties of PF.

1.9.2 Research questions

1. How precisely does PF reduce inflammation in vitro?

2. Will manipulating the main active ingredients of PF enhance its anti-inflammatory properties? 3. Will enhancing the anti-inflammatory activity of PF make a better therapy?

1.9.3 Aims

1. Investigating the active anti-inflammatory ingredients of Polymeric Formula and

elucidating the mechanisms of action using in vitro model of IBD.

2. Developing a novel nutritional therapeutic formula with enhanced anti-

inflammatory properties in vitro.

3. Exploring the efficacy of a novel nutritional therapy in ameliorating gut

inflammation using murine model of colitis. 4. Examining the effectiveness of the novel therapy on human intestinal inflammation

utilizing ex-vivo cultured colonic biopsies. Chapter 2: Methodology

2.1 In vitro model of IBD

2. 1.1 Cell culture and induction of inflammation:

2.1.1.1 Background

The luminal surface of the human gut is lined with a highly polarized and self-renewal epithelium [667]. The primary function of these cells is absorbing nutrients [668, 669].

However, because they are in direct contact with gut lumen through their apical surface, intestinal epithelial cells form a protective barrier against different invasive infectious agents, toxins and antigenic factors [670]. The epithelial cells are also implicated in homeostasis of the complex mucosal immune response [670]. The mucosal immune responses play an imperative role in epithelial defensive mechanism against invasive pathogens [671, 672]. However, in intestinal inflammation, the mucosal immune response is altered and intestinal epithelial cells start secreting excessively number of cytokines

[673-675]. It is well documented that pro-inflammatory cytokines TNF-Į,/-Į,/-ȕDQG

IL-8 are produced largely in the inflamed intestinal mucosa of IBD patients [676-679]. The released cytokines are responsible for extensive histological damage in the intestinal epithelium of IBD patients [680-682]. These facts would largely support the notion that intestinal epithelial cells are to large extent involved in, and play an important role in tissue injury in IBD [57]. In line with this, a number of intestinal epithelial cell lines including

HT29, Caco2 and INT407 are being successfully replicated in lab as a modelling for intestinal inflammation [683, 684]. Therefore, in the current work these three cell lines were utilized as an in vitro model of IBD. Limitations of using these cell lines include mimicking just one single type of the intestinal mucosa cells, the epithelial mono-layer, which does not precisely reflect the overall complexity of the intestinal mucosa [685]. Moreover, Caco2 and HT29 cells are tumor cells derived from human colonic adenocarcinoma: as such these cell lines have unique properties different to the normal cells (e.g. genetic makeup, metabolic profile and other cellular physiological properties) [686, 687]. Further, there is a suggestion that the INT407 cell line has been misidentified and actually originated from Hela cells (glandular cell of the cervix) rather than, as previously thought, derived from embryonic intestinal cells

[688]. Additionally, cell culture conditions in which these cells are maintained may not represent the local environment in situ [689].

Remarkable progress has been made in the isolation and maintenance of intestinal cells derived from mammals in cell culture [690]. Among these advances are in vitro culture of intestinal stem cells and progenitors [691]. Certainly, generation of human proximal

(jejunal organoids) and regional (colon organoids) intestinal tissue in vitro from pluripotent stem cells has been established [691-693]. The cell-derived organoids should allow for better in vitro modelling for IBD. Nevertheless, these newly emerged methods, in addition to their complexity, are still at an early stage of development.

The currently utilized cell lines reproducibly display a number of properties characteristic of normal differentiated intestinal cells [687, 694]. Intestinal epithelial cells play a critical role in the homeostasis of intestinal mucosal immunity [695]. Further, the intestinal epithelium is largely involved in intestinal tissue injury and damage following mucosal homeostasis disruption through involvement in pro inflammatory cytokine production

[673, 675]. Therefore observations made using these cell lines replicate key in situ events occurring in IBD, although, the limitations of this model must be considered.

Basic cell culture methods have remained one of the most widely used models for studying intestinal structures and physiological function, and assisted in understanding the underlying pathology and investigating potential treatments [689]. De Jong et al. [479] examined the anti-inflammatory properties of PF using HT29, Caco2 and INT407 cell lines cultured in vitro, as a model of intestinal inflammation. Nahidi et al. [497] also utilized the caco2 cell line grown in culture plates to study effects of EEN using PF and other treatments including steroids and biological therapy on the intestinal epithelial barrier integrity and function. Moreover, in many studies, activity of several amino acids including glutamine and arginine, and their potential benefits in treating IBD, have been explored in vitro by utilizing cultured intestinal epithelial cell lines (HT29 and Caco2)

[576, 606, 696-699]. Further, a similar model was used for investigating various physiological functions including elucidating the mechanisms of actions, and the therapeutic implications of other nutritional supplements such as, vitamin D3, curcumin and omega-3 fatty acids [638, 700-704]. Overall, basic culture models have their limitations they are still are able to provide physiological and clinically relevant information. 2.1.1.2 Materials and reagents

x Cell lines

- HT-29 (ATCC HTB-38)

- Caco2 (ATCC HTB-37)

- INT407 (ATCC CCL6)

Cell lines were obtained from American Tissue Culture Type and stored at -180 °C. In all experimentations the cells were used between passages 40 to 50.

x Culture media

- McCoy’s 5A media-1X (Gibco-Invitrogen, Victoria, Australia)

- Minimum Essential Medium (MEM)-1X, Gibco-Invitrogen)

- Basal Medium Eagle (BME; Gibco-Invitrogen)

- 10% Fetal bovine serum (FBS, Gibco-Invitrogen)

- 1% Penicillin/Streptomycin (Gibco-Invitrogen)

- 1% Na pyruvate ( Gibco-Invitrogen)

- 1% Na bicarbonate ( Gibco-Invitrogen)

- 1% MEM Non-Essential Amino Acids Solution (MEM NEAA)-100X ( Gibco-

Invitrogen)

x Cell culture flask tubes and plates

- Cell culture flask tubes (T25 and T75 flask tubes, Greiner Bio-One, Victoria,

Australia)

- 24-well and 6-well plates (Becton Dickinson, NSW, Australia)

x Recombinant human cytokines -TNF-Į *LEFR-Invitrogen)

-IL-ȕ 6LJPD-Aldrich, NSW, Australia)

-INF-Ȗ 6LJPD-Aldrich)

- LPS (Sigma-Aldrich)

x Nutritional supplements (Treatments)

- Osmolite (PF, Abbott Nutrition, NSW, Australia)

- Ȧ-3 FAs (Canola oil, generic brand used for FRRNLQJULFKLQĮ-linolenic acid)

- L-Glutamine {(S)-2, 5-Diamino-5 oxopentanoic acid L-Glutamic acid 5-amide,

146.14 g/mol, Sigma-Aldrich}

- L-Arginine {(S)-2-Amino-5-guanidinopentanoic acid, 174.2 g/mol, Sigma-

Aldrich}

- Vitamin D3 {Į-Dihydroxycholecalciferol, Calcitriol, Sigma-Aldrich}

- Curcumin {(E,E)-1,7-bis(4-Hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5dione,

Diferuloylmethane, Sigma-Aldrich } 2.1.1.3 Methods

The protocols for the current work concerning cell culture model (cells growth, inflammation induction, time course etc.) had previously been verified and established by our research group, as reported in previously published works [705-708]. Further, these standardised protocols are consistent with work from other authors. Boonkaewwan et al.

[709] investigated proinflammatory cytokine (IL-8) release from HT29 cells in response to

TNF-Į DQG GHWHUPLQHG WKDW ,/-8 production is time and dose dependent. IL-8 levels plateaued after a 6 hour exposure time with 50 ng/ml TNF-Į.Van De Walle et al. [710] also explored the effect of several experimental parameters on inflammatory responses in the Caco2 cell line, and showed that a mixture of 50 ng/ml of TNF-ĮQJPORI,1)-Ȗ

25ng/ml IL-ȕDQGXJPO/36IRUKRXUVLQFXEDWLRQLQGXFHGDPD[LPDOF\WRNLQH ,/-8) production.

x Thawing down and establishing cell lines (HT29, Caco2 and INT407)

All work was performed in a class II lamina flow hood as follow: A vial of frozen cells was retrieved from liquid nitrogen storage and warmed in water bath at 37°C until the cell suspension was almost completely liquid. Cells were then taken to the lamina flow hood and mixed 1 ml of warm FBS and then transferred to 15 ml falcon tube filled with 10 ml of warm media. Cells with media were centrifuged at 1200 g for 5 minutes. The supernatant was discarded and cells were resuspended in another 10 ml of warm fresh media. Cells were then transferred to a T25 flask and placed in incubator at 37°C with 5% CO2 supply.

Culture media was replaced with fresh media every 2 to 3 days. x Passage of cells from T25 flask

Cells were passaged when 80% confluence was reached. Briefly, once cells were at 80% confluence, media was removed and the cells washed with 5ml of warm PBS for 30 seconds. PBS was removed and 5 ml of warm 0.25% trypsin was added to cells. The flask with trypsin was incubated at 37°C for 10 minutes before 5 ml of warm FBS was added to inhibit further trypsin action. The suspension containing detached cells was then centrifuged at 1500g for 5 minutes. The supernatant was discarded and cells were resuspended in fresh media and seeded onto plates for experimentation.

x Cell preparation for experimentation

Cells were seeded in 24 or 6-well plates and maintained in culture media. Cells were incubated at 37°C with 5% CO2 with media changed every alternative day. HT29 cells were cultured in McCoy’s 5A medium containing 10% FBS and 100U/mL penicillin/streptomycin. INT407 cells were grown in BME containing 10% FBS and

100U/ml penicillin/streptomycin whilst Caco2 cells were maintained in MEM supplemented with 20% FBS, 1% Penicillin/Streptomycin, 1% Na pyruvate, 1% Na bicarbonate and 1% MEM EAA. Experiments were conducted after 5 days incubation

(approximately 90% confluence reached) for HT29 and INT407 cells; or following 2 weeks incubation for Caco2 cells. x Induction of inflammation

For induction of inflammation, confluent cells were exposed to 50ng/ml of TNF-Į DQG incubated for 6 hours (HT29 cell line) [709], or 24 hours (INT407) [479].To induce an inflammatory response in Caco2, cells were exposed to a mixture of 50 ng/ml of TNF-Į

50 ng/ml of INF-Ȗ ng/ml IL-ȕDQGXJPO/36IRUKRXUV [710].

x Treatment protocol

Glutamine, arginine, alpha-linolenic acid (ALA) and PF were added directly in the media.

Vitamin D3 was solubilised in 100% ethanol and then added to cells to give a final ethanol concentration of 0.1% v/v in the media. Curcumin was solubilised in dimethyl sulphoxide

(DMSO) and added to the culture medium with a final DMSO concentration of 0.1% v/v in media. In the first series of experiments glutamine, arginine, vitamin D3 and fatty acids were tested at their concentrations present in PF (Appendix 1). These concentrations were obtained directly from the product information (Osmolite 1 Cal, Abbott- Australia) available on the manufacturer’s website, and were further validated after correspondence with representatives from Abbott.

(http://abbottnutrition.com.au/portals/0/img/all%20pages%20R1.pdf).

Because PF is given at a concentration of 1:5 to the media [711], glutamine, arginine, vitamin D3 and ALA when investigated as a part of PF, were added at 1/5 of their respective concentration in PF, as previously utilised [479]. 2. 1.2 Cell viability assessments:

2.1.2.1 Haemocytometer & Trypan blue

x Introduction

The trypan blue method was used to determine the number of viable cells presents in a cell suspension [712]. The principle is that live cells possess intact cell membranes and can exclude certain dyes, such as the trypan blue whereas dead cells absorb the dye and therefore appear blue under the microscope [713]. The dye exclusion method is a reliable indicator of cell viability in cell culture based research [714].

x Materials and reagents

- Haemocytometer ( BLAU BRAND, Germany)

- Microscope (Nikon Eclipse E600 POL Microscope, USA)

- Trypan blue (Sigma- Aldrich)

- Phosphate Buffered Saline (PBS), Gibco Invitrogen)

- 0.25% Trypsin (Gibco-Invitrogen)

x Method

Following experimentation, cells were washed twice with warm PBS then incubated with trypsin 0.5 mL per well for 15 minutes. Warm FBS (0.5 mL per well) was then added to inhibit trypsin. A total volume of 1mL (FBS and trypsin) was then transferred to a separate tube with tubes centrifuged at 1200g for 5 minutes. Supernatants were discarded and the pellet containing the cells gently resuspended in warm media. Equal volumes of cells

(25μl) and trypan blue (25μl) were mixed and added to a haemocytometer and viewed under a light microscope. Cell viability counts were conducted and the viability expressed as the percentage of unstained cells (viable) among the total cells. 2.1.2.2 MTT colorimetric assay kit

x Introduction

MTT (3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide; Thiazolyl blue)

reduction assay is a non-radioactive colorimetric assay utilizing MTT substrate to

assess the cellular activity as an indicator of viability [715]. MTT is a water soluble

tetrazolium salt yielding a yellowish solution when prepared in media lacking phenol

red [716]. Dissolved MTT is converted to an insoluble purple formazan by cleavage of

the tetrazolium ring by mitochondrial dehydrogenase enzymes of metabolically active

cells [717]. Water insoluble formazan can be solubilised and then dissolved material

can be measured spectrophotometrically yielding absorbance as a function of

concentration of converted dye [718]. The MTT based assay has been used to assess

cell viability, cell proliferation and drug cytotoxic effects in various studies involved

human cells grown in vitro [719-721].

x Materials and reagents

- MTT substrate (Sigma-Aldrich)

- McCoy´s 5A, phenol red-free media (Banksia Scientific Company, QLD,

Australia)

- DMSO (Sigma-Aldrich)

- Micro-plate reader (Bio-Rad, NSW, Australia)

- 96-well microtiter plates (Maxisorp Nunc, Victoria, Australia)

x Method

The assay was conducted according to the manufactures instructions. In brief, following experimentation, used culture media was replaced with 1ml/well phenol red-free

McCoy´s 5A media. One hundred microliters of MTT solution (5 mg/ml) was added to each well of cell culture and incubated further for 4 hours at 37°C with 5% CO2.

Following the incubation, dye was solubilized in 100μl DMSO and then the total 200μl of mixture was transferred to wells of a 96-well plate with absorbance read at a wavelength of 570 nM. The amount of converted dye, which represented metabolic activity, was determined by comparison to a standard curve.

2.1.3 Enzyme-linked immunosorbent essay

2.1.3.1 Background

Enzyme-linked immunosorbent essay (ELISA) technique was used to measure levels of

IL-8 of experiments involved intestinal epithelial cells cultured in vitro. IL-8 is a member of chemokine family which is a group of proteins produced during the inflammation by many different cell types including epithelial cells [722-727]. Resting cells usually secrete very low levels of chemokines, however, upon inflammation the secretion of IL-8 is up- regulated [728]. The up-regulation is mediated through activation of NF-ț% SDWKZD\ RI infiltrating immunocytes and tissue cells in response to pro-inflammatory cytokines TNF-

Į,1)-ȖDQG,/-ȕ[729, 730]. Chemokines are responsible for recruitment and activation of immunocytes, a character of chronic inflammation as in IBD [731]. Specifically, IL-8 plays role as a powerful neutrophil chemoattractant and activator that is accounting for perpetuation of inflammation in CD [66, 680, 682]. In a study involving intestinal mucosal samples collected from normal subjects and from patients with CD, IL-8 was among of the pro-inflammatory cytokines that were significantly high in CD patients compared to controls [732]. Additionally, in vitro settings, researchers were able to enhance IL-8 production from colonic epithelial cells replicated in culture media and treated with recombinant human cytokines [733-736]; and in numerous of these studies that involved cultured epithelial monolayer, IL-8 measured as a principle inflammatory marker for the model [737-739]. In this work, the three cultured cell lines (HT29, Caco2 and INT407) also exhibited a strong inflammatory response, presented with high level of measured IL-8, to the recombinant human cytokines. Therefore IL-8 levels were used as a proxy inflammatory marker for in vitro experiments. In addition to IL-8 measurements, TNF-ĮDQG,/-6 levels were also assessed, as additional inflammatory markers, for the experiments involved cultured colonic biopsies. TNF-ĮDQG

IL-6 play a pivotal role in the induction and amplification of the inflammatory cascade characterising gut inflammation [29, 358]. In CD, they are part of key cytokines contributing to the disease pathogenesis [740]; and their secretion is markedly enhanced

[741]. Further, they are among of spontaneously released inflammatory mediators from organ culture involving IBD gut mucosa [339].

2.1.3.2 Materials

- IL-8, IL-6 and TNF-Į +XPDQ $QWLERG\ Pair, ELISA Kits (Novex®-Invitrogen,

Victoria, Australia).

- Nunc-Immuno™ Micro Well™ 96 well solid plates (Maxisorp Nunc, Victoria,

Australia)

- Coating buffer A (8g NaCL, 1.13g Na2HPO4, 0.2g KCL to 1 litre distilled water,

PH to 7.4)

- Assay buffer (8g NaCL, 1.13g Na2HPO4, 0.2g KCL, 5g bovine serum albumin

(BSA), 1ml Tween 20 to 1 litre distilled water, PH to 7.4)

- Washing buffer {250ul Tween 20 (Promega, NSW, Australia) to 500ml PBS}

- Soluble 3,3',5,5'-tetramethylbenzidine (TMB) substrate (TMB: Thermo-Fischer

Scientific, Victoria, Australia)

- Stop solution 1.8N H2SO4 (Sigma-Aldrich)

- Micro-plate reader (Bio-Rad) 2.1.3.3 Method

Following experiments, cell and /or organ culture supernatants were collected and assayed in duplicate as follow: Coating solution was prepared by diluting the coating antibodies to

1μg/ml with coating buffer A. One hundred microliters of prepared coating solution was pipetted to each well of the 96-well plate and incubated further for 18 hours at 4°C.

Following the incubation, wells were aspirated and washed once with 200μl washing buffer before blocking the pate with 300 μl assay buffer per well for 1 hour at room temperature. Consequently, standards (1000, 500, 250, 125, 62.5, 31.25 and 15.62 pg/ml) and samples (1 in 2 and 1 in 20 dilutions) were added at 100 μl per wells together with 50

μl of detection antibody per well. After continual shaking at 250rpm for 2 hours, reagents were aspirated and the plate washed. Subsequently, 100μl of streptavidin-horseradish peroxidase (HRP) solution was added to each well and the plate was then incubated further for 30 minutes at room temperature. Following the incubation, plates were washed and loaded with 100μl per well of TMB substrate and gently shaken until colour development.

Once the colour reaction had progressed sufficiently, the reaction was stopped with addition of 1.8 N sulphuric acid (H2SO4) and the absorbance was read at 450 nM. The optical density readings were then converted to picograms per milliliter based on the standard curve obtained with the recombinant cytokine where 15.62 pg/ml was the lower detection limit of the assay. 2.1.4 Western blot

2.1.4.1 Materials

x Lysis buffers For western blots, a number of lysis buffers were used. For preparing whole cell extract,

Radio Immuno Precipitation Assay (RIPA) buffer was used. RIPA buffer is composed of

150 mM NaCl, 0.1% Triton X-100 (Astral Scientific Pty Ltd, NSW, Australia), 0.5% sodium deoxycholate (Sigma–Aldrich), 0.1%SDS (sodium dodecyl sulphate, Sigma-

Aldrich), 50 mM Tris-HCl pH 8.0 (Sigma-Aldrich), 1mM Na orthovanadate (Sigma-

Aldrich) 10 mM Na fluoride (Sigma-Aldrich) and a mixture of protease inhibitors{2

μg/ml Aprotinin, 10μg/ml Leupeptin and 1μg/ml Pepstatin A (Abcam, Cambridge, UK)}.

Tris-triton buffer was used for preparing cytosolic extract. It consists of 100 mM NaCl,

1% Triton X-100, 0.5%sodium deoxycholate ,0.1% SDS, 10 mM Tris-HCl, 1mM Na orthovanadate, 10% glycerol, 1mM ethylenediaminetetraacetic acid ( EDTA) , 1 mM

Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA, Sigma-Aldrich)

10mM Na fluoride and a mixture of protease inhibitors.

Nuclear extracts were prepared using 2 buffers; Hypotonic buffer for nuclear separation from whole cellular extract:10mM HEPES/KOH (Sigma-Aldrich), 10 mM KCl (Sigma-

Aldrich), 2 mM MgCl2 (Sigma-Aldrich), 0.1 mM EDTA and 1 mM Dithiothreitol (DTT,

Sigma-Aldrich); and saline buffer to destruct the nuclear envelop {50 mM HEPES/KOH,

50 mM KCl, 1 mM DTT, 300 mM NaCl, 0.1 mM EDTA, 10% glycerol (Sigma-Aldrich),

1mM Na orthovanadate, 10mM Na fluoride and a mixture of protease inhibitors}. x Micro BCA protein assay reagent kit

Whole protein concentrations were measured using bicinchoninic acid (BCA) {Pierce

Biotechnology- Thermo-Fischer Scientific}.

x Running buffer

25mM Tris base (Sigma-Aldrich), 190 mM glycine (Sigma-Aldrich) and 0.1% SDS

(Sigma -Aldrich)

x Loading buffer

Laemmli 2X buffer was the loading buffer for all the experiment. It is composed of 4% sodium dodecyl sulphate (SDS, Sigma-Aldrich), 10% 2-mercaptoethanol (Sigma -Aldrich),

20% glycerol (Sigma -Aldrich), 0.004% bromophenol blue (Sigma -Aldrich) and 0.125 M

Tris-HCl (Sigma -Aldrich).

x Washing buffer

TBST was the washing buffer and was prepared by making 10 times dilutions of TBS 10x in ultra-pure water with 1ml Tween20 (polysorbate, Sigma-Aldrich) added to the solution.

TBS 10x (concentrated TBS) is a mixture of 24.23 g Trizma HCl (Sigma-Aldrich), 80.06 g

NaCl and 800 ml ultra-pure water.

x Blocking buffer

Membranes were blocked using 5% BSA (Bovine serum albumin, Invitrogen) solution.

BSA is dissolved in TBST buffer and would be filtered before used. x Running gel, membranes, standards and Transfer apparatus

Tris-Glycine NB 10% gel (NuSep Ltd, NSW, Australia); PVDF membranes, Precision

Protein™ StrepTactin-HRP Conjugate, Precision Plus Protein™ WesternC™ Standards and Trans-Blot Turbo Transfer System were sourced from (Bio- Rad).

x Primary and secondary antibodies

For studying NF-ț%SRO\FORQDOUDEELWDQWL-IKK, anti-phosphorylated IKK, anti-Iț%DQWL-

SKRVSKRU\ODWHG,ț%DQGDQWL-P65 were sourced from Abcam. Polyclonal rabbit anti human- total and phosphorylated P38 of MAPK were also sourced from Abcam. Polyclonal rabbit anti-ȕ-actin antibody (Abcam) was the loading control. Secondary goat anti-rabbit antibody was sourced from (Bio- Rad).

x Immune-Star HRP chemiluminescent kit- for detection

Bands were detected by chemiluminescent detection method using Immun-Star HRP

Chemiluminescent Substrate Kit (Bio-Rad) and visualized by GelDoc (Bio-Rad). 2.1.4.2 Methods

x Cell culture, sample lysis and protein measurement

For western blot experiments, HT29 cells were seeded in 6-well plate at a concentration

106 cells/well, and grown until confluence. Supplements (glutamine, arginine or curcumin) were added to the confluent cells before being exposed to TNF-ĮRI ng/ml for 5, 15, 30 or 60 minutes. Following experimentation, cell culture plates were placed on ice and washed with cold PBS. PBS was then aspirated and 250 μl of cold Lysis buffer was added to each well of the 6-well plate. RIPA buffer was the selection for experiments involved whole cellular extract, whereas Tris-Triton was used in preparing cytosolic fraction and hypotonic buffer for nuclear extraction. After 15 minutes incubation with lysis buffer, adherent cells were scrapped off using a cold plastic cell scraper and then the cell suspension was transferred into a pre-cooled microcentrifuge tube. The tubes were then agitated for 30 minutes at 4°C followed by centrifugation at 16,000g for 20 minutes at 4°C.

Supernatant was then transferred to a fresh tube and the pellet was discarded. For preparing nuclear extracts, the supernatant was discarded and the pellet containing the nucleus was incubated further with 100 ȝORIFROGVDOLQHEXIIHUIRUPLQXWHVEHIore being centrifuged at 16,000g for 20 minutes at 4°C. Supernatants, reflecting the nuclear portion of cellular proteins, were collected and the remaining debris discarded.

x Total protein measure of cell lysates

For measuring protein concentrations, diluted albumin standards were prepared to give a set of protein standards from 2000 to 25 μg/ml. Working reagent (WR) was prepared according to the manufacturer protocol by mixing 49ml of BCA Reagent A with 1ml of

BCA Reagent B (50:1, Reagent A:B). Samples were diluted with PBS to give serial dilutions of 1:1, 1:10 and 1:100. Twenty five microliters of each standard and sample was added to each well of a microplate. Two hundred microliters of WR was then added to each well and the plate is incubated at 37°C for 30 minutes. After cooling the plate, the absorbance was read at 562nm using microplate reader.

x SDS PAGE and transfer to membrane

Equal volumes of cell lysate and 2X Laemmli loading buffer were mixed to give a 40μg of protein in loading buffer. The mixture of lysate and the loading buffer was heated at 100°C for 5 minutes to denature the proteins. Samples and 5μl of Precision Plus Protein™

WesternC™ were then loaded into wells of the SDS-PAGE gel. The gel was exposed to15 minutes at 100 V which then increased to 200V for 45 minutes. Subsequently, protein was transferred from the gel to a polyvinylidene difluoride (PDVF) membrane by the Trans-

Blot Turbo electrophoretic transfer apparatus.

x Blocking and probing the membrane & blot development and imaging

The membrane was blocked with blocking buffer for 1 hour at room temperature.

Membranes were probed with primary antibodies diluted in TBST buffer overnight at 4°C.

Then membranes were washed in TBST solution for 10 minutes and repeated for a further four washes. Subsequently, membranes were incubated with secondary antibodies conjugated with HRP for 1 hour at room temperature. Membranes were then incubated in a mixture of luminol and peroxide buffer in a 1:1 ratio for 3 to 5 minutes (chemiluminescent

Substrate) and exposed to Bio-Rad Versa Doc and bands visualized by GelDoc imager. 2.1.5 RNA Extraction and Amplification by Real-Time PCR (RT-PCR)

2.1.5.1 Materials

x RNA Extraction reagents TRIzol Total RNA isolation reagent (Invitrogen), Chloroform (Sigma-Aldrich), and

Isopropyl alcohol (Sigma-Aldrich)

x RNA quality and quantity assessment NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA)

x DNA removal kit

Turbo DNA- free kit (Ambion, Austin, TX, USA) containing 10X Dnase 1 buffer, Turbo-

Dnase 1 and Dnase inactivation reagent

x Complementary (c) DNA synthesis kit SuperScript® VILO™ cDNA Synthesis Kit (Life Technologies, USA), which is supplied with 10X SuperScript Enzyme Mix and 5X VILO™ Reaction Mix.

x DNA Amplification RT-PCRs were performed using Realplex Mastercycler (Eppendorf, Barkhausenwig,

Hamburg, Germany) and SYBR-Green fluorescence detection system: (iQ™ SYBR®

Green Supermix; Bio-Rad) and (Fast SYBR® Green Master Mix, Life Technologies)

x Primers & Nuclease free water

Sense and anti-sense primers of IL- ,QYLWURJHQ  DQG KRXVHNHHSLQJ JHQH ȕ2-

0LFURJOREXOLQȕ2M; Invitrogen) (Table 2.1) & nuclear- free water (Ambion) Primer* Orientation Sequence (5’-3’)

IL-8 Sense CCGGAAGGAACCATCTCACT

Anti-sense AACTTCTCCACAACCCTCTGC

ȕ0 Sense AGGCTATCCAGCGTACTCCAAAGA

Anti-sense AACGAGATCAACATCATGAACCA

Table 2.1 Shows sequences of IL- DQG ȕ0 SULPHUV XVHG IRU 57-PCR analysis of TNF-Į exposed HT29 cell line.

*[742]

2.1.5.2 Methods

x RNA Extraction and DNA removal For RNA extraction from cells in vitro, 1 ml of TRIzol was add per well, and then incubated room temperature for 5 minutes. For RNA extraction from mouse tissue, a piece of colon (50 mg) immersed immediately in 200μl RNA later solution (prepared in lab; sodium citrate, ammonium sulfate and EDTA, Sigma-Aldrich). Subsequently, 200 μl of chloroform was added to each 1 ml of TRIzol suspension and incubated further at room temperature for 3 minutes. The mixture was then centrifuged at 12,000g for 15 minutes at

4°C and the upper aqueous phase containing the RNA was separated and placed into

RNase free tube. To the approximate 500μl of separated aqueous phase, 0.5ml of isopropyl alcohol was added, and incubated together at room temperature for 10 minutes. Following incubation, the composite was centrifuged at 12,000g for 15 minutes at 4°C. Later the supernatants were discarded and 1 ml of 75% ethanol added to the pellet before being centrifuged at 7500 g for 5 minutes at 4°C. The last step was repeated 3 times before the pellet left to dry at room temperature for 10 minutes. Finally, air-dry pellets were re- suspended in 25μl nuclease free water. For DNA removal, 2.5μl of 10X Dnase 1 buffer and

1μl of Turbo-Dnase 1 were added to the 25μl of RNA and then incubated together at 37 °C for 20 minutes. Following incubation, 2.5μl of Dnase inactivation reagent added to the

RNA tube and incubated together at room temperature for 5 minutes. The mixture was centrifuged at 10,000g at room temperature for 1 minute to pellet the reagents and separate the pure RNA. The RNA was transferred to a new RNase free tube for storage at – 80 °C until use. One aliquot (1μl) of RNA was used to measure the absorbance using NanoDrop, through which the concentration and quality of RNA were assessed. x Complementary DNA synthesis kit and RNA amplification

Extracted RNA of high quality (OD 260/280> 1.8 and 260/230> 2) was reverse- transcribed to complementary DNA (cDNA). The reaction containing the RNA was kept on ice and the following added: 2μlof 10X SuperScript Enzyme Mix, 4μl 5X VILO™ Reaction Mix,

RNA (1μg) and nuclease free water to 20μl for each reaction. Subsequently, tube contents were vortexed and incubated at 25°C for 10 minutes, then for 60 minutes at 42°C before the reaction was terminated by heating the tube at 85°C for 5 minutes. Produced cDNA was amplified using the Realplex master cycler and SYPR-Green flourcent dye. For the in vitro study, the PCR reaction volume was 25μl, including 12.5μl of SYBR® Green

Supermix, 5μl of produced cDNA (10 ng), 3μl forward primer (300 nM), 3μl reverse primer (300 nM) and 1.5μl nuclear-free water. The reaction was initiated by heating the tubes at 95°C for 2 minutes, which followed by 40 cycles of 95 °C for 15 seconds, 60 °C for 15 seconds and 72 °C for 20 seconds. For the cDNA produced from mice tissue, The

PCR reaction volume was 20μl, including 10μl of Fast SYBR® Green Master Mix, 3 μl of produced cDNA (10 ng), 3μl forward primer (300 nM), 3μl reverse primer (300 nM) and 1

μl nuclear-free water. Tubes heated at 95°C for 20 seconds and followed by 40 cycles of

95 °C for 3 seconds and 60 °C for 30 seconds. Melting curve step for both reactions was included for checking homogeneity of PCR products as follow: 95°C for 15 seconds, 60°C for 15 seconds and 95°C for 15 seconds. Average threshold cycle (CT) was measured and gene expression quantified. Fold induction was calculated by using comparative CT method [449]. The expression is calculated as follow: 2- delta CT of control – delta CT of experiment. CT is the cycle at which arbitrary detection threshold is crossed. 2.1.6 Single-labelling Immunofluorescence

Fluorescence immunohistochemistry was performed on HT29 cells for investigating effect of supplements (glutamine and arginine) on the expression and nuclear translocation of

P65 subunit, as an indicator of the NF-ț% pathway activity, after TNF-ĮH[SRVXUH

2.1.6.1 Materials

x Glass coverslips {Thermo Scientific, ™ Nunc™ Lab-Tek™ II Chamber Slide™ System} x 100% methanol, {normal donkey serum (NDS), Sigma-Aldrich} x PBS (Invitrogen), humidified chamber x Primary ploy-rabbit anti-P65 antibodies (Abcam) x 488 Alex secondary goat anti-rabbit antibodies (molecular probes, Invitrogen) x 4',6-diamidino-2-phenylindole (DAPI), fluorescent stain (Vectashield Hard+SetMounting media with DAPI, Vector Labs, Fluoroshiel with DAPI, Sigma- Aldrich) x Zeiss Axioplan 2 imaging microscope and Axiovision software (Zeiss, Germany)

2.1.6.2 Method

Cells were grown on chamber slides at concentration 103. Once 70% of confluence reached, cells were incubated further with either glutamine and arginine or no treatment for

24 hours before being exposed to 100ng/ml TNF-Į IRU  KRXU &HOOV ZHUH IL[HG E\ emerging them in 100% ice-cold methanol solution and incubated further for 20 minutes at

-20°C. Later cells were rinsed with PBS 3 times of 5 minutes each. Nonspecific staining was blocked with 10% NDS for 30 minutes at room temperature. Samples were incubated overnight with anti-P65 primary antibodies at 1:400 dilutions. Fluorescence was detected by incubating with 488 Alex secondary goat anti-rabbit antibodies at for 30 minutes at room temperature (1:500 dilutions). Nuclei were counter stained with DAPI fluorescence mounting media (~200μl). Samples were then cover slipped and edges sealed with nail polish. Cells were viewed by Axioplan 2 microscope and images analysed using

Axiovision software. 2.1.7 Kinase assay

A new kit that can provide a non-LVRWRSLF VHQVLWLYH PHWKRG WR GLUHFWO\ PHDVXUH WKH ,țț activity was used. The kit replaces the outdate radioactive in vitro kinase assays by utilizing a peroxidase coupled anti-phospho- ,ț%Į6PRQRFORQDODQWLERG\DVDUHSRUWHU molecule. The assay is performed in 96-well ELISA plate pre-coated with recombinant

,ț%Į ,Q SUHVHQFH RI 0J DQG $73 PROHFXOHV ,ț%Į LV SKRVSKRU\ODWHG at two serine

UHVLGXHVE\IUDFWLRQDWHG,țțȕVXEXQLW7KHDPRXQWRISKRVSKRU\ODWHGVXEVWUDWHUHIOHFWLQJ

,țțDFWLYLW\FDQEHGLUHFWO\PHDVXUHGE\FDSWXULQJLWZLWKWKHVSHFLILFSHUR[LGDVHFRXSOHG anti-phospho- ,ț%Į6DQWLERG\ZKLFKFDWDO\VHWKH70%VXEVtrate to develop a colour reaction. The colour then can be quantified by micro-plate reader.

(http://www.cyclex.co.jp/main_eproduct_kinase.html#serine/threonine_kinase_kit).

2.1.7.1 Materials

- CycLex Ițț ĮDQGȕ$VVD\/Inhibitor Screening Kit (CycLex Co., Ltd, Japan)

The kit has the following materials: Microplate (ZHOOVDUHFRDWHGZLWKUHFRPELQDQW,ț%ĮDV substrate of IKK), 10X wash buffer, kinase buffer, 20X ATP, anti-Phospho-,ț%Į 6 monoclonal antibody, HRP conjugated anti-mouse IgG, substrate reagent (TMB), and Stop solution (sulphric acid).

-Ițț positive control ( CY-E1178-2, CycLex)

-10X K252a (Sigma-Aldrich) 2.1.7.2 Methods

The assay was conducted to study effect of glutamine and arginine (test samples) on Ițț activity. No enzyme control and two inhibitors, curcumin the natural inhibitor and K252a, the synthetic inhibitor of Ițț, were included. Glutamine and arginine were dissolved directly in kinase buffer. Curcumin and K252a were dissolved first in DMSO then added to the kinase buffer to give 0.5% v/v concentration of DMSO in the reaction buffer.

Additional solvent control (0.5% DMSO) was also included. The reaction started by pipetting 2μl of Ʌ- subunit of Ițț (10μunits) to DOO,ț%ĮSUH-coated wells, except the no enzyme control. This was followed by addition of 98μl of kinase reaction buffer (kinase buffer and 20x ATP at ratio of 1:20). Experimental wells contained glutamine, arginine, curcumin or K252a dissolved in the reaction buffer. Reagents were mixed well together and plate incubated for 30 minutes at 30°C. Wells then were aspirated and washed 4 times with washing buffer. Anti-Phospho-,ț%Į 6 DQWLERG\ —O  ZDV SLSHWWHG into each well, and the plate incubated further at room temperature for 30 minutes. Plate then washed and 100μl of HRP-conjugated anti-mouse IgG added into each well. Following 30 minute

LQFXEDWLRQDWURRPWHPSHUDWXUHDQGSODWHZDVKLQJȝOof the substrate was loaded into each well and incubated further at room temperature for 10 minutes. The colour reaction stopped by adding 100 ȝO of stop solution and the absorbance, reflecting amount of generated phosphorylated ,ț%Į read at 450 nM using the spectrophotometer. 2.1.8 Affymetrix microarray

Gene expression microarray analysis was conducted to comprehensively investigate the effect of co-supplementation of glutamine and arginine on the global gene expression profile of TNF-Į H[SRVHG +7 7KH DLP ZDV WR ILQG RXW DQ\ UHODWLRQ ZLWK WKHLU DQWL- inflammatory properties and the immune response related signaling pathways.

2.1.8.1 Microarray Experiments and Analysis

The pure extracted RNAs (500 ng; form un-treated cells, cells exposed to TNF-ĮDQGFHOOV exposed to TNF-Į DQG FR-incubated with glutamine and arginine) with the 260:280 and

260:230 rations between1.8 to 2.3 were used for microarray analysis. Each sample was additionally run on the Agilent Bioanalyzer RNA Nano 6000 chip to further assess the quality and integrity of the total extracted RNA. After scanning, array images were

DVVHVVHG E\ H\H WR FRQ¿UP VFDQQHU DOLJQPHQW DQG WKH DEVHQFH RI VLJQL¿FDQW EXEEOHV RU scratches on the chip surface. When the quality of the samples was acceptable, they were subjected to Affymetrix microarray analysis with an Affymetrix Human PrimeView™

(U219) GeneChip® according to the manufacturer’s guidance (Santa Clara, CA, USA) performed at Ramaciotti Centre for Gene Function Analysis. The protocols used for RNA microarray analysis were those available from Ramaciotti Centre for Genomics

(http://www.ramaciotti.unsw.edu.au). 2.1.8.2 Statistical Analysis

Analysis of the microarray data was undertaken using the Partek Genomic Solutions platform (Partek Inc.). Following import of the Affymetrix CEL files, the Robust Multi- array Average (RMA) method was used for background correction, quantile normalization across all chips, log2 transformation and median polish summarization. Differential expression was tested using one-way analysis of variance (ANOVA) with linear contrasts between groups. Gene lists were created using a false discovery rate adjusted P value

(FDR) of 0.05. We used MetaCoreTM from Thomson Reuters to conduct functional analysis of the differentially expressed genes including pathway analysis. 2.2 In vivo model of IBD: Experimental colitis

2. 2.1Background:

Animal experiments for human diseases are an integral and essential part in the pre-clinical research area [743]. Data generated from pre-clinical studies offer a meticulous guidance and potential relevance when considering clinical trial [744, 745]. It has been widely accepted that replicated experimental models would largely reflect human organ pathology in several aspects, including cause and course of the disease as well as accompanied physiological and pathological changes [746]. Taking the ethical issues and limitations into considerations, animal models remain an important resource to gain scientific knowledge

[747, 748]. Animal models offer flexibility for studying and exploring the underlying mechanism of various health conditions and have been used to gain significant progress in the understanding of human diseases, including IBD [749].

Amongst more than 20 available animal models for IBD [749], the trinitrobenzene sulfonic acid (TNBS) and dextran sodium sulfate (DSS) induced murine colitis models are often used (Wirtz, Neufert et al. 2007). TNBS and DSS murine colitis represents a robust, easily reproducible and suitable representative form of intestinal inflammation; that is why they are often employed animal models for studying IBD [749, 750]. TNBS induces colitis by haptenation of colonic proteins [750] resulting in a delayed-type hypersensitivity reaction mediated by CD4+ T helper 1 (TH1) cells [751]. This reaction results in severe transmural

DQG JUDQXORPDWRXV LQÀDPPDWLRQ DVVRFLDWHGZLWKLQ¿OWUDWLRQRI7FHOOVDQGPDFURSKDJHV

[752]. In addition, release of Th1 cytokines, such as TNF-Į DQG LQWHUIHURQ-Ȗ DQG RWKHU inflammatory mediators including, IL-12, IL-6 and IL-ȕ[753]. Macroscopically, injected TNBS causes transmural lesions, ulcerations, necrosis, loss of lining epithelium, oedematous thickened bowel, and loss of colon length [754, 755]. Clinically, in susceptible strain of mice once TNBS is given, the mice develop diarrhea, weight loss, anorexia and per rectal bleeding [756-758]. However, different mice strains respond differently to

TNBS. The SJL mice strain is considered the most susceptible and BALB/c strain is susceptible; whereas C57BL6 strain is resistant to TNBS [759].

In contrast, the DSS molecule has direct toxic effect on colonic epithelium [760]. It causes disruption and breakdown in integrity of the intestinal epithelial barrier, thereby allowing entry of luminal foreign antigens including microorganism, into mucosa and thereby perpetuation an inflammatory response [761]. DSS-induced colitis is associated with increased expression of different inflammatory mediators, Th1 (TNF-Į,/-ȕ,/-12, INF-

Ȗ  DQG 7K PHGLDWHG F\WRNLQHV OLNH ,/-6, IL-4, INF-Ȗ DQG ,/-10 [762-764]. Course and severity of the induced colitis varies with several factors. DSS is a molecule of 40 KD molecular weight produces a sever colitis in mice [761]. However, DSS dose and mice strain also alters the course of disease: DSS of 5% v/v concentration results in acute colitis after several days, whereas giving repeated or continuous cycles of 1-3% concentration leads to developing chronic disease [449, 765, 766]. C57BL/6 mice can develop either acute and/or chronic disease after receiving DSS, whist Balb/C strains can only develop acute colitis, as they completely recover once the acute stage has passed [764]. Although the validity of animal experiments for predicting the effectiveness of potential therapies in humans is subject to criticism [767], animal models still play a fundamental role in biological and medical research that to-date, cannot be replicated in vitro [768,

769]. In the context of IBD, the TNBS and DSS induced colitis models are considered two of the most commonly utilized protocols to study potential treatments, including enteral diets and the other nutritional supplements [770]. The anti-inflammatory activity of elemental diets with different fat compositions was explored in a recent study utilizing the

TNBS- experimental colitis model [771]. This model was also employed in several other studies investigating the immunomodulatory properties of numerous nutritional diets, such as ALA-enriched enteral formulas, glutamine and arginine amino acids supplementation, and curcumin supplements [639, 772-775]. Similarly, the DSS model has been used in several relevant published studies. The DSS mouse colitis model was utilized to explore the intestinal effects of a glutamine–rich diet [776]. In the same model, enteral diet enriched with glutamine, fiber, and oligosaccharide was shown to attenuate inflammation

[777], and dietary curcumin exerted chemopreventive effects on inflammation-induced colorectal carcinogenesis [778]. Thus, in the current work, both TNBS and DSS models were used to investigate the efficacy of nutrient supplements in the setting of intestinal inflammation. 2.2.2 Mice strain and acclimatization

Female Balb/C and C57BL/6 mice, 6-8 weeks old were obtained from the Animal

Resource Centre (Perth, Australia) and housed with no more than 6 mice per cage in the animal facility of the UNSW (Wallace Wurth Building, Randwick, Australia). Mice were acclimatized prior to experimentation on a standard pelleted diet with free water access for

1 week at controlled humidity and temperature condition with a 12hrs light- 12 hrs dark cycle. The Animal Research Ethics of UNSW approved all the experiments and related procedures (approval ID; 12/149B). 2.2.3 Experimental protocols and induction of colitis

2.2.3.1 TNBS colitis model

Following acclimatization, mice were randomly assigned into 4 experimental groups with

12 mice per group (Figure 2.1). First group, designated as a negative control, continued on standard chow with free water access for the experiment duration. In the second group, referred as a positive control, mice were injected with TNBS and continued on normal mouse diet and water for the experiment duration. The third and fourth group included mice that had TNBS injected rectally and then continued on either standard or novel formula for 1 week. For induction of colitis, mice were fasted first for 24 hrs with free access to drinking water. Once lightly anaesthetized in the induction chamber using 2-3% isoflurane vapor, mice were received 100μl of 45% ethanol solution containing 2.5mg of

TNBS (Sigma) by intra-rectal administration. A 20 gauge cannula fitted to a 1ml syringe was inserted 1cm proximal to the anal verge. The ethanol/TNBS solution was instilled slowly into the colon. Following instillation mice were held in the head down position for

30 sec to ensure distribution within the bowel and to prevent leakage. Mice were observed until recovered from anaesthetic then returned to their cages. Mice in the negative control group received only a 45% solution of ethanol administered intra-rectally. The day of intra- rectal administration was denoted as day 1. 2.2.3.2 DSS colitis model

Following acclimatisation, mice were divided into four experimental groups with 12 mice in each group (Figure 2.1). The negative control group had a free excess to standard diet and water for the duration of the experiment. In the second group, designated as a positive control, mice received 3% DSS (w/v) (M.W 40KD; Sigma-Aldrich) dissolved in drinking water for 5 days with access to mouse pellets. DSS was then stopped and mice continued on normal drinking water for another 2 weeks with free access to a mouse pellets . In the other two groups, mice were given 3% DSS in drinking water with free access to mouse pellets for 5 days. DSS was then stopped, and mice were put on either standard or novel formula diet for 2 weeks during night time (almost 18 hrs a day) with free water access for

6 hrs of day time. (A)

Groups Day 1 Day 7

TNBS injection Collection

Negative - Positive + continued on normal mice bullet + water

PF + continued on PF + water Novel formula + continued on Novel formula + water

(B)

Groups Day -5 -1 1 Day 14

DSS treatment Collection

Negative - Positive + continued on normal mice bullet + water

PF + continued on PF + water Novel formula + continued on Novel formula + water

Figure 2.1 Illustrates TNBS (A) and DSS (B) colitis model protocols. 2.2.4 Treatment composition and preparation

Following induction of colitis, mice in the third and fourth experimental groups were put on either standard PF (Osmolite, Abbott); or Novel formula prepared in the facility fresh on daily basis. Novel formula is comprised of standard PF with glutamine and arginine concentrations of 250 and 100 mM, respectively, plus curcumin added to a final concentration of 250 μM (Table 2.2). These concentrations were used because in vitro experiments established 50 mM glutamine, 20 mM arginine and 50 μM curcumin as optimal conventions (Chapter 4). However, we propose there is a 1 in 5 dilution factor of standard PF within the GI tract due to secretions, therefore formula concentrations need to be 5 times higher to have similar efficacy at the intestinal epithelium as compared to the in vitro epithelium. All the treatment regimens were given in liquid form in the drinking bottle ad libitum. The liquid was replaced every 24 hours to ensure there was no degradation in the activity of treatments. Mice that received formulas did not have access to mouse pellets, as the balanced nutritional formula supplied all necessary calories, protein, fat and vitamins required. Supplement Concentration (mM) Concentration per 1 ml (g)

Glutamine 250 0.03645

Arginine 100 0.0166

Curcumin 0.250 0.00009

Table 2.2 Concentrations of glutamine, arginine and curcumin components per 1ml standard PF to create the Novel formula. 2.2.5 Evaluation of colitis and response to treatment

Assessing severity colitis and efficacy of treatments in ameliorating inflammation in TNBS colitis was based on three tools: body weight of mice, colonic mRNA expression of TNF-

Į,/-6, IL-12 and monocyte chemoattractant protein (MCP)-1 and microscopic histology examination of colon. In DSS colitis, the same three tools were used but were expanded further to include histology evaluation, myeloperoxidase (MPO) assay and colon over weight score.

2.2.5.1 Body weight scale

Disease activity index (DIA) is a validated clinically based grading system used to quantitate and monitor the disease severity in mice [779]. In present work mice were observed twice a day throughout duration of experiment (1 week in TNBS experiment; and

19 days in DSS model) for any sign of distress, formula consumption, weight loss or death.

Body weight was recorded at the initiation of the experiment and then daily until end of the experiment. Along with changes in the body weight, mice were observed for stool consistency and presence of rectal bleeding. However, the DIA was calculated on body weight loss as diarrhea and bleeding scores were considered subjective and prone to variations. 2.2.5.2 PCR analysis of colonic cytokines mRNA expression

At completion of the experiment mice were euthanized using CO2 inhalation before the entire colon was removed and gently cleared of feces. Colons were divided into 3 portions, proximally to distally as follow: first portion for MPO assay; the second part for RNA isolation and the third and last rectal region for histology examination. Gene expression of

4 inflammatory mediators, TNF-Į ,/-6, IL-12, and MCP-1 were quantified relative to expression of housekeeping gene GAPDH as previously detailed (2.3.1) using specific mice primers sense and anti-sense sequences (Invitrogen) (Table 2.3).

2.2.5.3 Histology examination

To evaluate histological damage of colitis, colonic tissue (0.5 cm long) was fixed in 10% formalin (Sigma-Aldrich), embedded in paraffin and cut into cross sections. The sections were stained with hematoxylin and eoxin, and examined by light microscope. Histology was assessed by an experienced histologist who was blind to treatments, using an established histology score system as a follow: Colonic tissue sections were scored by a blinded observer using a score modified from previously published system [498], as follow: crypt damage (normal, 0 - severe crypt distortion 4), extent of inflammation

(normal, 0 - dense inflammatory infiltrate extends to serosa, 4), severity of inflammation

(normal, 0 - marked inflammation 4), and mucosal edema (absent, 0- extensive, 3). The histological damage score is the sum of each individual score and ranges from 0 (no injury) to 15 (maximal injury). 2.2.5.4 Myeloperoxidase assay

MPO is a heme protein abundantly present in neutrophil and other cells including monocytes [780]. Neutrophils play a vital role in the mucosal homeostasis by rapidly eliminating invading organisms, however they can also prominently contribute to intestinal inflammation induced pathology [781]. Neutrophils mediate their effects through different mechanisms involving numerous agents including MPO [782].

In this work MPO activity was quantified, as a marker of neutrophil infiltration, with the

O-dianisidine Method [783]. Briefly, Colonic tissue (50 mg) homogenized with 0.5% hexadecyltrimethylammonium bromide in 50 mM PBS, pH 6.0 (Sigma-Aldrich). Buffer ratio of 50 mg/mL was used. Tissue homogenates (7 μl) added in duplicate into a 96-well plate. ȝO RI  PJPO RI GLDQLVLGLQH GLK\GURFKORULde (Sigma-Aldrich) containing

0.00005% H2O2 (Sigma-Aldrich) was added to each of the wells. The absorbance was then read at 450nM and MPO activity measured. One unit of MPO is defined as the

DPRXQWQHHGHGWRGHJUDGHȝPRI+2O2 per minute at room temperature.

2.2.5.2 Colon weight over length score

Colon weight/ length ratio is a macroscopic assessment tool utilized to assess degree of colonic inflammation in experimental colitis models [784]. In several studies involving

DSS colitis model, along with other parameters, colon weight over length score was used for assessing degree of the local colonic inflammation and the response to the potential treatments [785, 786]. Indeed, it has been ascertained that this score (colon weight over length ) correlates with the local pathologic and histological changes, and considered one of the reliable markers for the assessment of colonic inflammation [447]. Basically, the score reflects local colon morphological changes manifesting as a marked reduction in the colon length together with increased weight due to the local tissue oedema [787]. In the current work, large bowels of the experimental mice were removed and the scores (length and weight) measured, as previously utilized [614]. In Brief, colons from the anus to the ileocolonic junction were cut along the anti-mesenteric border and gently cleared of faecal materials. Wet tissue weight and length were then calculated by tap measure and the score assessed.

Sequence (5’-3’)

Primer* Sense Anti-sense

GAPDH TGAAGCAGGCATCTGAGGG CGAAGGTGGAAGAGTGGGAG

TNF-ĮCCTCCCTCTCATCAGTTCTA ACTTGGTGGTTTGCTACGAC

IL-6 TAGTCCTTCCTACCCCAATTTCC TTGGTCCTTAGCCACTCCTTC

MCP-1 CTTCTGGGCCTGCTGTTCA CCAGCCTACTCATTGGGATCA

IL-12 CAGCCTTGCAGAAAAGAGAGC CCAGTAAGGCCAGGCAACAT

Table 2.3 shows forward and reverse sequences of mice primers.

*[788, 789] 2.3 Ex-vivo human cultured colonic biopsy

2. 3.1Introduction:

Several decades ago early trials to preserve intestinal tissue in vitro were attempted [790].

Early reports have shown that the integrity of ex-vivo cultured intestinal tissue can only be maintained for few hours [791]. However, enhancements in the organ culture techniques has allowed ex-vivo tissue survival for 2-3 weeks [792]. Organ culture technique largely contributed to understanding the intestinal disease pathology and analysis of numerous intestinal physiological functions [694]. Importantly, it has shown that the mucosal biopsies collected from IBD patients continue to produce several inflammatory mediators including cytokines [793]. That spontaneous release can be deployed to explore therapeutic benefits of potential therapies of IBD [794]. In order to test the efficacy of the novel formula in mitigating intestinal inflammation, similar approach was followed in the current work. Colonic biopsies were collected from patients with active CD patients were cultured with the novel formula and the response to the treatment assessed by measuring cytokines release. 2. 3.2 Participants, inclusion/exclusion criteria and sample size calculation:

Participants were children aged between 5 and 17 years who attended the Sydney

Children's Hospital IBD clinic with gastrointestinal symptoms and a suspicion of IBD requiring diagnostic endoscopy (groups 1). Patients were selected on the likelihood of CD, based upon history, clinical assessment, and screening blood tests. Children who had been on anti-inflammatory drugs 4 weeks prior or antibiotics 2 weeks prior to the endoscopy were excluded from the study. A control group (group 2) of subjects who underwent endoscopy and were confirmed not to have any inflammatory condition were included in the study.The aim was to establish whether the novel formulation can abrogate the inflammatory response in inflamed biopsies. The primary outcome measure was protein

IL-8 expression in the culture supernatants. Based on data of a study carried out by

McCormack et al.[732] who investigated the levels of chemokines and pro-inflammatory cytokines in colonic mucosal biopsies of normal subjects and patients with CD, supernatant IL-8 level were significantly higher in CD (319 ± 61 pg/mg) (P < 0.05) compared to controls (120 ± 32 pg/mg). Using power analysiV ZLWK Į DW  DQG 95% study power, a sample size of at 6 was sufficient to establish the difference in IL-8 expression between inflamed CD and control. Complete abrogation of the inflammatory response should result in IL-8 supernatant levels equivalent to control. Therefore a minimum sample size of 6 is sufficient to establish whether the novel formulation can abrogate the inflammatory response. Here, a total of 20 subjects were recruited who fulfilled all criteria for groups 1 and the criteria for group 2 (Figure 2.2). The Human

Research Ethics of UNSW approved the project and the related procedures (approval ID;

12/SCHN/449). 2. 3.3 Endoscopy and histology examination:

For suspected CD patients, biopsies were obtained from the colon, along with multiple biopsies from the upper gut for microscopic evidence of inflammation. The bowel was inspected for confluent deep linear ulcers, aphthoid ulcers, deep fissures, fistulas, skip lesions (segmental disease), cobblestoning and strictures which were considered macroscopic features suggestive for CD. When these features were observed, the patient was assigned to group 1 and three (3) additional colonic biopsies were collected for experimentation (Figure 2.2). The biopsies were collected from tissue immediately adjacent to the endoscopically affected area. Patients where no inflammatory features were observed, were assigned to group 2 and had one (1) additional colonic biopsy collected

(Figure 2.2). Subsequent to the endoscopy, biopsies collected for microscopic examination were reviewed by a histologist. If histological analysis did not confirm a diagnosis of CD for group 1 patients, then data collected from these patients were excluded from final analysis of the results (Figure 2.2). For group 2 if the histology report did not confirm that the collected tissues were normal, and then also the data obtained from these specimens were excluded (Figure 2.2). 2. 3.4 Biopsy culture and treatment:

Following collection at endoscopy, the biopsies were transferred in pre-weighed tubes containing culture medium (DMEM, Eurobio, Invitrogen) supplemented with {penicillin

(0.1U/L), streptomycin (1024U/L) and 10%FBS, Invitrogen}. After washing with PBS

(Invitrogen), biopsies were placed into a well of a plastic 24-well culture plate. The three biopsies from group 1 patients were cultured as follow: first biopsy was cultured with media alone; second biopsy was cultured with media and standard PF at ratio of 1 to 5; third biopsy was cultured with the novel formula at ratio of 1 to 5, as previously described

(2.1.1.3). The single biopsy collected from group 2 patients was cultured with media alone.

Culture plates were then placed in a chamber equilibrated with 5% CO2 and incubated for

18 h at 37°C. Following incubation, supernatants were collected and transferred into vials for storage at -80°C until analysis. 2.3.5 Cytokines immunoassay and lactate dehydrogenase enzyme activity measurement:

Culture media supernatants were assessed for concentrations of IL-6, IL- DQG 71)Į

(Novex®-Invitrogen) to establish the cytokine inflammatory response of the biopsies and effect of treatments. Levels of IL-6, IL- DQG 71)Į ZHUH PHDVXUHG XVLQJ (/,6$ technique, as previously detailed (2.1.3.3).

Tissue viability was assessed based on release of lactate dehydrogenase (LDH) enzyme per mg of tissue weight. LDH activity was measured in culture supernatants using the LDH activity assay kit (Sigma). In brief, according to manufacturer’s instruction, standards were prepared for colorimetric reaction and samples (25μl) were added to the wells in duplicates. After adjusting final volumes of standards and samples (supernatants) to a final volume 50μl with assay buffer, 50μl of reaction master mix was then added to each well.

Plates were incubated at 37°C with colour development in the well measured on a microplate reader (450nm) every 5 minutes for a total of 3 readings. Based on the calculated consumed NAD, LDH activity was determined as the amount of enzyme that catalyzes the conversion of lactate into pyruvate to generate 1.0mM of NADH per minute at 37 °C. Recruitment1- Recruitment

Children aged 5 to 17 years attending IBD clinic to undergo diagnostic endoscopy for suspected IBD

2-Diagnostic endoscopy

Visible inflammation at endoscopy No visible inflammation at endoscopy

Assinged to Group 1 (n=10) Assinged to Group 2 (n=10)

(3 biopsies collected) (1 biopsy collected)

3-Tissue culture

Biopsy 1 (no treament – Positive Control) Biopsy 1 (no treament – Negative Control)

Biopsy 2 (treated with existing Polymeric Formula)

Biopsy 3 (treated with the novel Formula)

A definitive diagnosis of CD or normal non-inflammaed bowel can only be made post endoscopy by histological examination. Therefore Biopsies may be excluded from the study if there is not a definitive diagnosis of CD (group 1) or a definitive normal bowel (group 2)

4-Microscopic Histological examination

Confirmed CD Not confirmed CD Normal bowel Other pathology excluding IBD

Group 1 Excluded from study Group 2 Excluded from study

Figure 2.2 a summary for the protocol of ex-vivo colonic biopsy study. Chapter 3: Investigating the active anti-inflammatory ingredients of

Polymeric Formula and elucidating the mechanisms of actions in vitro.

3.1 Introduction

Enteral nutrition utilizing PF or EF given as the sole nutritional source is now the preferred option for the management of young patients with CD [448]. While there is ample evidence supporting the benefits of PF, its mechanism of actions remains elusive [795].

Several clinical trials involving CD patients show that enteral nutrition using PF results in a fall of pro-inflammatory cytokines and correction of inflammatory indices in patients, indicating that PF has a strong immuno-modulatory effect [463, 464, 477, 796]. This suggests that the primary mode of action of PF may be due to its anti-inflammatory properties.

However, it remains unknown which constituent(s) of PF contribute to suppressing inflammation. In various studies glutamine, arginine, vitamin D and Ȧ-3 FAs supplements, which are present in PF, are shown to possess immuno-modulating effects [584, 649, 660,

797], and may be candidates that contribute to the anti-inflammatory effects of PF. This provides the first clue in explaining the primary therapeutic effect of PF in CD. However, these nutrients were not investigated as part of PF.

NF-ț%and P38 MAPK transcription factors play vital roles in the immune response and are determined as major regulators of gene expression of the inflammatory mediators including proinflammatory cytokines [798, 799]. Importantly, it has been proposed that glutamine and arginine can modulate their effects through the NF-ț% and P38 MAPK pathways [590, 800, 801]. However, strong evidence is lacking and the precise mechanistic interactions are not defined and require further investigations.

3.2 Hypotheses

The central hypothesis is that glutamine, arginine, vitamin D3 and ALA (all key ingredients of standard PF) contribute to PFs immuno-modulating effects. A further hypothesis is that the combination of glutamine and arginine ingredients of PF has a synergistic immunomodulatory effect on activated intestinal epithelial cells, which is mediated through modulation of the major intracellular signalling pathways.

3.3 Aims

1-([DPLQLQJHIIHFWVRIJOXWDPLQHDUJLQLQHYLWDPLQ'DQGȦ-3 Fas (as ALA) individually or in combination, and PF upon IL-8 production.

2- Exploring whether co-supplementation of glutamine and arginine is more effective in abrogating IL-8 production than if given individually.

3- Investigating the mechanisms of action of glutamine and arginine in attenuating NF-ț% signal transduction and stress-activated protein kinase P38 MAPK cascade. 4-Evaluating the effect of glutamine and arginine on the global pattern of gene expression of major components of the immune response related pathways. 3.4 Results

3.4.1 Glutamine, arginine and vitamin D3, but not ALA at their concentrations of PF significantly reduced IL-8 production from HT29 cell line in response to TNF-ĮDQG that effect was magnified with their combination.

HT29 cells were cultured until confluence as previously indicated (2.1.1.3). For experimentation, four replicates were included for each individual group. In the first group

(Negative control), confluent cells were maintained in media with no additional treatment.

In the other groups (positive control and treatments), confluent cells were exposed to TNF-

ĮIRULQGXFWLRQRILQIODPPDWLRQDVLQVHFWLRQ  ,QWKHWUHDWPHQWJURXSVWKH71)-Į exposed cells were then in addition treated with 12.7 mM glutamine, 1.8 mM arginine, 3.8 nM vitamin D3 and 0.72 mM ALA individually or in combination. Following 6 hours incubation supernatants were collected for subsequent IL-8 assays using the ELISA technique (2.1.3.3).

The production of IL-8 was greater following the addition of TNF-Į QJPO  LQ WKH positive control group compared with the negative control group. IL-8 level was increased to around 90 times compared to un-stimulated cells (P < 0.0001; Figure 3.1). Moreover, in the presence of 12.7 mM glutamine, IL-8 production was significantly decreased from

(16658 ± 1693) pg/ml to (11791 ± 1191) pg/ml (P < 0.05; Figure 1). Arginine (at 1.8mM) also considerably suppressed IL-8 production from TNF-Į VWLPXODWHG +7 FHOOV ,/-8 level was substantially reduced by arginine treatment from (16658 ± 1693) pg/ml to 9472 ±

1022 pg/ml (P = 0.001; Figure 3.1). Further, 3.8 nM vitamin D3 attenuated IL-8 levels from (16658 ± 1693) pg/ml to (9456 ± 740) pg/ml (P = 0.001; Figure 3.1). Because vitamin D3 was dissolved first in ethanol and then added to the media to give a final concentration of ethanol of 0.1% v/v in the media, effect of 0.1% ethanol on IL-8 production was tested in a separate experiment. IL-8 levels were not significantly different to control levels in the ethanol exposed cells (data not shown).

In contrast to the significant effect of previous supplements on reducing IL-8 level, ALA of 0.72mM had no effect on IL-8 production from TNF-ĮVWLPXODWHGFHOO7KH,/-8 level in

ALA treated cells was 13838 ± 1340pg/ml, showing no significant difference from the positive control (P = 0.12; Figure 3.1). Interestingly, combination of glutamine of 12.7 mM, arginine of 1.8 mM, vitamin D3 of 3.8 nM and ALA of 0.72mM resulted in further and more significant reduction in IL-8 production from HT29 cells in response to TNF-Į stimulation that dropped from16658 ± 1693 pg/ml to 6764 ± 1046 pg/ml ( P < 0.0001;

Figure 3.1). Figure 3.1 Effect of glutamine, arginine, vitamin D3 and ALA on IL-8 production from HT29 cells in response to TNF-Į. Cells were grown until confluence, and then cultured with glutamine, arginine, vitamin D3 and Ȧ-3 FAs individually or in combination at concentrations equivalent to their concentrations in PF. Cells were then exposed to TNF-ĮIRUKRXUVWKHQVXSHUQDWDQWVZHUH collected and assayed for IL-8 by ELISA. Glutamine, arginine, and vitamin D3, but not ALA, significantly attenuated IL-8 production, which further attenuated when these nutrients were added together (P (*) < 0.01, (**) < 0.001, (***) < 0.0001 and ns not significant); IL-8 levels were compared in treated groups to its level in the positive control group using one-way ANOVA test followed by Fischer least significance post hoc test. Negative: Negative control group. Positive: Positive control group (only TNF-Į Glu: Glutamine treated group. Arg: Arginine treated group. Vit D: Vitamin D3 treated group. ALA: Alpha-linolenic acid treated group. Combination: combination treated group (glutamine of 12.7 mM, arginine of 1.8 mM, vitamin D3 of 3.8 nM and ALA of 0.72 mM). Values represent mean and SEM of 4 replicates for each group 3.4.2 Standard PF was superior to combination of glutamine, arginine, vitamin D3, and ALA at their concentrations of PF in attenuating IL-8 level.

Culturing of HT29 cells was performed as previously described (2.1.1.3). Cells then were treated with either the combination (12.7 mM glutamine, 1.8 mM arginine, 3.8 nM vitamin

D3 and 0.72 mM ALA) or with standard PF at concentration of 1:5 to media. An inflammatory response was stimulated by exposure to 50ng/ml TNF-Į DQG FHOOV ZHUH incubated for 6 hours. Additionally, positive and negative control groups were included as in previous experiment. At the end of the incubation, supernatants were collected and IL-8 levels were measured as previously explained (2.1.3.3). PF treatment prompted a substantial reduction in IL-8 response to TNF-ĮH[SRVXUH([SUHVVLRQRI,/-8 was reduced from 20237±1914pg/ml in TNF-ĮVWLPXODWHGFHOOVWR±730pg/ml after PF treatment (P

< 0.0001; Figure 3.2). Similar results to the previous experiment were seen with the combination of glutamine, arginine, vitamin D3 and ALA treated group, where IL-8 levels were attenuated from 20237 ± 1914 pg/ml in TNF-ĮVWLPXODWHGFHOOVWR± 1960pg/ml

(P<0.0001; Figure 3.2). PF, with its whole list of ingredients, was more effective in blocking IL-8 release from TNF-Į VWLPXODWHG +7 FHOOV WKDQ WKH FRPELQDWLRQ WKH IRXU individual constituents (glutamine of 12.7 mM, arginine of 1.8 mM, vitamin D3 of 3.8 nM and ALA of 0.72 mM (P = 0.005; Figure 3.2). However, IL-8 levels remained statistically higher in both the combination and PF groups compared to the negative control group (P

<0.05; Figure 3.2). To assess cell viability, confluent HT29 Cells were treated with the four supplements individually or in combination; and with PF at 1:5 to media and incubated for 24 hours.

Subsequently, cell viability assay was conducted using trypan blue exclusion method

(2.1.2.1). Cell viability was maintained above 95% in all experimental groups and there was no significant difference in cell viability between the groups (Figure 3.3). Figure 3.2 Effect of combined glutamine, arginine, vitamin D3 and ALA versus standard PF on IL-8 production from HT29 cells in response to TNF-ĮCells were cultured with either a combination of glutamine, arginine, vitamin D3 and ALA at their concentrations in PF or with standard PF. Cells were then exposed to TNF-ĮIRUKRXUVZLWKVXSHUQDWDQWVFROOHFWHGDQGDVVD\HG for IL-8 by ELISA. Both the combination and standard PF considerably reduced IL-8 level compared to positive control group (P < 0.05 for both groups). Further, in both groups IL-8 levels remained statistically higher than in unstimulated cells (P < 0.05 vs. negative control group). However, PF was superior to the combination in reducing the IL-8 response (P = 0.005). Multiple comparisons were carried out between all groups for comparing IL-8 levels using one-way ANOVA test followed by Fischer least significance post hoc test; P<0.05 considered significant. Negative: Negative control group. Positive: Positive control group (only TNF-Į Combination: combination treated group (glutamine of 12.7 mM, arginine of 1.8 mM, vitamin D3 of 3.8 nM and ALA of 0.72 mM). Standard PF: Polymeric formula treated group. Data account for mean and SEM of 6 replicates for each group. Figure 3.3 Viability assay of HT29 cells treated with glutamine, arginine, vitamin D3 and ALA or PF. Cells were grown until confluence then were cultured with glutamine, arginine, vitamin D3 and alpha-linolenic acid individually or in combination; or with PF. After 24 hours incubation cells were stained with trypan blue and cell viability counts were conducted using a haemocytometer and a light microscope. In all groups cell viability was maintained above 95% and remained incomparable to the untreated cells group (P > 0.05 for treated groups vs. control group using one-way ANOVA test followed by Fischer least significance post hoc test) Control: untreated cells group. Glu: Glutamine treated group. Arg: Arginine treated group. VitD: Vitamin D3 treated group. ALA: Alpha-linolenic acid treated group. Combination: combination treated group (glutamine of 12.7 mM, arginine of 1.8 mM, vitamin D3 of 3.8 nM and ALA of 0.72 mM). Standard PF: Polymeric formula treated group. Dots represent the mean and SEM of 4 replicates for each group. 3.4.3 Glutamine, arginine and vitamin D3, but not ALA attenuated IL-8 release from

HT29 in response to TNF-Į LQ D GRVH GHSHQGHQW PDQQHU ZLWKRXW DQ\ GHWULPHQWDO effect on the cell viability.

HT29 cells were cultured in four replicates as previously described (2.1.1.3). Two controls were included as in previous experiment; negative control group, and positive control group (cells exposed to 50ng/ml TNF-Į  ,Q RWKHU JURXSV FHOOV ZHUH JURZQ XQWLO confluence was reached but in addition to TNF-ĮH[SRVXUHFHOOVZHUHWUHDWHGZLWKDUDQJH of concentrations of glutamine (0.5, 1, 2.5, 5, 7.5, 10, 15, 50, 120 and 240mM), arginine

(0.5, 2, 5, 10 and 50 mM), vitamin D3 (cells were pre-incubated with vitamin D3 at 1, 10,

30 and 100nM for 1 hour before being exposed to TNF-Į DQG$/$ FHOOVZHUHSUH- incubated with ALA for 2 days at concentrations 0.3, 0.72, 1.44, 3.6, 7.2 mM before TNF-

ĮZDVDGGHG 6XEVHTXHQWO\QXWULHQWWUHDWHGFHOOVZLWKDGGHG71)- ĮZHUHWKHQLQFXEDWed further for 6 hours. Glutamine, arginine and ALA were dissolved directly in culture media to the above concentrations whilst vitamin D3 was dissolved in ethanol and then added to cells with the final ethanol concentration kept constant 0.1% v/v in the culture media. Cell viability was assessed by the trypan blue method (2.1.2.1).

Glutamine has a significant inhibitory effect on IL-8 release from TNF-ĮVWLPXODWHG+7 cells. The inhibitory effect starts at a concentration of 10 mM when IL-8 levels are reduced from 22344 ± 1769 pg/ml to 17615 ± 2922 pg/ml with glutamine treatment, however, this decrease was not statistically significant compared to positive control group (P > 0.05).

Statistical significance is achieved at a concentration of 15mM when IL-8 dropped to 12968 ± 1862 pg/ml (P < 0.01; Figure 3.4). Further inhibition of IL-8 production was evident with increasing glutamine concentration as seen with 50 and 120mM glutamine

(P<0.001) and 240mM glutamine where IL-8 reduced by 5 fold compared to the positive control group to reach 4618 ± 319 pg/ml (P < 0.0001; Figure 3.4). This indicates that glutamine does have a dose dependent anti-inflammatory effect on TNF stimulated HT29 cells. Importantly, the cell viability of glutamine treated group remained above 95% even with high concentrations of glutamine (P > 0.05 glutamine treated groups vs untreated group; Figure 3.5). This refers to the fact that the reduction in the IL-8 level is likely due to the immuno-modulating effects of glutamine and nota a cytotoxic effect. Arginine also had a dose dependent effect on IL-8 production. The activity started at 2.5 mM arginine when

IL-8 levels were attenuated from 16465 ± 1820 pg/ml after TNF-ĮH[SRVXUHWR±

420 pg/ml (P = 0.04 versus positive control group; Figure 3.6). Increasing arginine concentration resulted in greater reduction in IL-8 level (P = 0.002 for 5mM, 0.001 for

10mM and < 0.0001 for 20 and 40mM arginine versus positive control group; Figure 3.6).

HT29 cells treated with arginine at 50 mM produced six-fold less IL-8 compared to the positive control cells (from 16465 ± 1820 to 2459 ± 223 pg/ml, P < 0.0001), however, it remained significantly lower than in 20mM arginine treated group (P = 0.04; Figure 3.6).

Of importance, cell viability remained high at all arginine concentrations (P > 0.05 arginine treated groups vs. untreated group; Figure 3.7).

This HT29 model of intestinal inflammation showed that vitamin D3 exhibited a strong anti-inflammatory effect. In presence of 1nM vitamin D3 treatment, IL-8 levels were significantly attenuated compared to the positive control group (from 1543 ± 706 to 10061 ± 841pg/ml) (P = 0.002; Figure 3.8). Interestingly, increasing vitamin D3 concentrations at

10 and 30 nM resulted in further reduction in the IL-8 response to TNF-Į P= 0.001 and

0.0005, respectively; Figure 3.8). The most significant suppression of IL-8 was observed with the highest tested concentration of vitamin D3 (100 nM) where IL-8 was reduced 2- fold compared the positive control group (from 15431 ± 706 to 7160 ± 474 pg/ml) (P <

0.0001; Figure 3.8). This indicates that in addition to glutamine and arginine, vitamin D3 also possesses a concentration dependent anti-inflammatory effect on intestinal inflammation. Importantly, vitamin D3 at all concentrations, had no cytotoxic effect, as cell viability remained above 90% (P > 0.05 vitamin D3 treated groups vs. untreated group;

Figure 3.9). In comparison ALA appeared to have no anti-inflammatory effect even at the highest concentration tested, as IL-8 levels were not significantly different to the positive control for any of the tested ALA concentrations (Figure 3.10). No effect on cell viability observed with ALA treatments (P > 0.05 ALA treated groups vs. untreated group; Figure

3.11). Figure 3.4 Effect of increasing glutamine concentration on IL-8 production from HT29 cells in response to TNF-Į. Cells were grown until confluence, and then cultured with increasing glutamine concentrations with 4 replicates for each group. Simultaneously, glutamine treated cells were exposed to TNF-Į  ng/ml) and incubated for 6 hours. Supernatants were collected and assayed for IL-8 by ELISA. Cells treated with glutamine of 15 mM and above showed a significant reduction in IL-8 level as compared with positive control group. (P (**) <0.01, (***) <0.001, (****) <0.0001, and (ns) >0.05 using one-way ANOVA test followed by Fischer least significance post hoc test). (-): Negative control group. (+): Positive control group (only TNF). (0.5 to 240): glutamine treated groups; cells were treated with differing ranges of glutamine concentrations and exposed to 50ng/ml TNF-ĮData represent mean and SEM mean of 4 replicates for each group. Figure 3.5 Viability assay of HT29 cells treated with increasing glutamine concentrations. Cells were grown until confluence was reached then cultured with increasing glutamine concentrations for 24 hours with 4 replicates for each group. Following the incubation cells were stained with trypan blue and cell viability counts were conducted using a haemocytometer and a light microscope. Glutamine treated cells showed no significant drop in the cell viability compared to control group (P > 0.05; groups were compared to control group using one-way ANOVA test followed by Fischer least significance test); Points represent mean and SEM of replicates. (0): 0 mM glutamine concentration (control group). (0.5 to 240): glutamine treated groups; cells were incubated with differing ranges of glutamine concentrations for 24 hours. Figure 3.6 Effect of increasing arginine concentration on IL-8 production from HT29 cells in response to TNF-Į. Cells were grown until confluence then cultured with increasing arginine concentrations of 4 replicates for each group. Simultaneously, arginine treated cells were exposed to TNF-Į ng/ml) and incubated for 6 hours. Supernatants were collected and assayed for IL-8 by ELISA. In presence of arginine IL-8 reduced in a dose dependent fashion (P (*) < 0.05, (**) < 0.001, (***) < 0.0001, arginine treatment groups were compared to positive control group) reaching best significance at 50mM (P = 0.04 vs. 20 mM arginine treated group). Statistical analysis of data conducted using one-way ANOVA test followed by Fischer least significance post hoc test. (-): Negative control group. (+): Positive control group (only TNF-Į). (0.5 to 50): arginine treated groups; cells were treated with differing ranges of arginine concentrations and exposed to 50ng/ml TNF-ĮValues presented with mean and SEM of replicates. Figure 3.7 Viability assay of HT29 cells treated with increasing arginine concentrations. Cells were grown until confluence was reached then cultured with increasing arginine concentrations (mM) for 24 hours. Following incubation cells were stained with trypan blue and cell viability counts conducted using a haemocytometer and a light microscope. Arginine showed no toxic effect even at high concentrations (P > 0.05; groups were compared to the control group using one-way ANOVA test followed by Fischer least significance test). (0): mM arginine concentration (control group). (0.5, 2.5, 5, 10, 20, 40 and 50): arginine treated groups; cells were incubated with differing ranges of arginine concentrations for 24 hours. Dots indicate mean and SEM of 4 replicates for each group. Figure 3.8 Effect of increasing vitamin D3 concentration on IL-8 production from HT29 cells in response to TNF-Į Cells were grown until confluence then cultured with increasing vitamin D3 concentrations. Simultaneously, cells were exposed to TNF-Į ng/ml) and incubated for 6 hours. Supernatants were collected and assayed for IL-8 by ELISA. Vitamin D3 treated cells showed a dose dependent reduction in IL-8 production in response to TNF-ĮVWLPXODWLRQ P, (**) < 0.01, (***) < 0.001, and (****) < 0.0001 using one-way ANOVA test followed by Fischer least significance post hoc test). (-): Negative control group. (+): Positive control group (only TNF). (1, 10, 30 and 100): vitamin D3 treated groups; cells were treated with differing ranges of vitamin D3 concentrations and exposed to 50ng/ml TNF-Į Values presented with mean and SEM of 4 replicates for each group. Figure 3.9 Viability assay of HT29 cells treated with increasing vitamin D3 concentrations. Cells were grown until confluence was reached then cultured with increasing vitamin D3 concentrations for 24 hours. Following incubations cells were stained with trypan blue and cell viability counts were conducted using a haemocytometer and a light microscope. Vitamin D3 treatment showed no significant drop in the cell viability compared to control group (P >0.05; groups were compared to control group using one-way ANOVA test followed by Fischer least significance test). (0): 0 nM of vitamin D3concentration (control group). (1, 10, 30 and 100): vitamin D3 treated groups. Dots presented with mean and SEM of 4 replicates for each group. Figure 3.10 Effect of increasing alpha-linolenic acid (ALA) concentration on IL-8 production from HT29 cells in response to TNF-Į Cells were grown until confluence then pre-incubated with ALA at the given concentrations for 48 hour before were exposed to 50ng/ml TNF-Į DQG incubated further for 6 hours. After incubation, supernatants were collected and assayed for IL-8 by ELISA. ALA had a negligible anti-inflammatory effect in all range of tested concentrations (P > 0.05; groups were compared to the positive control group using one-way ANOVA test followed by Fischer least significance test). (-): Negative control group. (+): Positive control group (only TNF- Į). (0.3, 0.7, 1.4, 3.5 and 7): ALA treated groups. Points presented with mean and SEM of 4 replicates.

Figure 3.11 Viability assay of HT29 cells treated with increasing ALA concentrations. HT29 cells were grown until confluence was reached then cultured with increasing ALA concentrations and incubated for 24 hours. Following incubations cells were stained with trypan blue and cell viability counts were conducted using a haemocytometer and a light microscope. No significant drop in the cell viability observed (P >0.05; groups were compared to control group using one-way ANOVA test followed by Fischer least significance test). (0): 0 mM of ALA (control group). (0.3, 0.7, 1.4, 3.5 and 7): vitamin D3 treated groups. Values presented with mean and SEM of 4 replicates. 3.4.4 Caco2 epithelial cell line tolerates high glutamine and arginine concentrations

Caco2 cells were grown under similar conditions with increasing glutamine (1, 10, 50, 100,

200 and 240 mM) and arginine (0.5, 1, 2.5, 5, 10, 20 and 50 mM) concentrations for 24 hours with cell viability assessed (2.1.2.1). Based on trypan blue staining, there was no loss of viability of Caco2 cells with increasing concentrations of either glutamine or arginine compared to the control (P > 0.05 for all treated groups; Figure 3.12). Comparable results were also obtained with the MTT assay. The assay assesses mitochondrial dehydrogenase enzyme activity and provides an indicator of the metabolic activity of cells. For these experiments, HT29 cells were incubated with glutamine at concentrations of 1, 10, 50, 100 and 240mM; or arginine at 1, 5, 10, 20 and 50 mM for 24 hours, and then the MTT activity measured as previously detailed (2.1.2.2). There was no drop in the MTT activity in all treated groups. However, there was a significant rise in treated cells with all tested glutamine concentrations compared to 0 mM control group (P<0.05; Figure 3.13).

Arginine of 5, 10 and 20 mM also showed a substantial rise in the MTT activity (P < 0.05 vs.0 mM arginine tread group; Figure 3.13). A

B

Figure 3.12 Viability assay of Caco2 cell line treated with increasing concentrations of glutamine or arginine. Cells were grown to confluence and then treated with increasing concentrations of glutamine (A) or arginine (B) for 24 hours then cell viability assessed by trypan blue exclusion assay. Mean and SEM is presented for 4 replicates. Cell viability in all treated groups was equivalent to the control (0 mM; P > 0.05) for all concentrations using one-way ANOVA test. A

B

Figure 3.13 MTT cellular activity assay of HT29 cell line treated with increasing concentrations of glutamine or arginine. Cells were grown until confluence and then treated with glutamine at concentrations of 0, 1, 10, 50, 100 and 240 mM (A) or arginine at concentrations of 0, 1, 5, 10, 20 and 50mM (B) for 24 hours. Media was replaced with a mixture of phenol red free media and MTT substrate and incubated further. Absorbance was read at 450nM using micro-plate reader. Four replicates are presented as well as mean and SEM .The activity in both glutamine and arginine treated groups at the tested concentrations was above the baseline of control group (untreated cells, only media), which was significantly high with all tested glutamine concentrations; and arginine of 5, 10 and 20 mM (P < 0.05 vs. 0 mM control group). 3.4.5 Combined glutamine and arginine attenuates proinflammatory cytokine production and m-RNA expression in intestinal epithelial cells stimulated with TNF-Į

HT29 and Caco2 cells were seeded in 24-well plates and grown until confluent (2.1.1.3).

Inflammation was induced with TNF-ĮDQGWKHQFHOOVZHUHLQFXEDWHGIXUWKHUZLWKHLWKHU

240 mM glutamine or 50 mM arginine individually or in combination. At the end of the incubation, supernatants were collected and IL-8 levels were measured as described earlier

(2.1.3.3). Glutamine treated HT29 cells produced 3-fold less IL-8 compared to the positive control cells (3847 ± 550 pg/ml compared to 13,659 ± 1700 pg/ml, P < 0.0001; Figure

3.14). Arginine treatment also exhibited strong anti-inflammatory activity that resulted in a significant decline in IL-8 production from 13,659 ± 1700 pg/ml to 3000 ± 299 pg/ml, (P <

0.0001; Figure 3.14). Moreover, when glutamine and arginine were combined there was a further significant reduction in IL-8 compared to the positive control group from 13659 to

151 ± 41pg/ml (P < 0.0001; Figure 3.14). The IL-8 in the supernatant was significantly lower when glutamine and arginine were combined compared to either glutamine (P =

0.005) or arginine (P = 0.025) alone. The combination of glutamine and arginine, but not glutamine nor arginine alone, completely abrogated the IL-8 response from HT29 cells stimulated with TNF-ĮDVWKHUHZDVQRVLJQLILFDQWGLIIHUHQFHLQWKHVXSHUQDWDQW,/-8 level compared to the negative control (P = 0.95; Figure 3.14).

Similar results were seen with Caco2 cells. A mixture of TNF-Į/36,/-ȕDQG,1)-Ȗ was used to induce Caco2 cells to produce 124.4 ± 14.8 pg/ml of IL-8 compared to an undetectable level of IL-8 in unstimulated cells. In response to the inflammatory stimuli, both glutamine and arginine amino acids significantly reduced the IL-8 production from

Caco2 cells (Figure 3.15). Supernatant IL-8 levels declined from 124.4 ± 14.8 pg/ml to

71.9 ± 4.4 pg/ml in the presence of glutamine (P = 0.001; Figure 3.15) and to 69.8 ± 9 pg/ml in the presence of arginine (P = 0.0007; Figure 3.15). Further, combined glutamine and arginine reduced IL-8 levels to 47.6 ± 12pg/ml (P < 0.0001), although IL-8 levels remained significantly higher than un-stimulated cells (P = 0.002; Figure 3.15).

To further support these findings IL-8 mRNA expression was also investigated. HT29 cells were grown in 6-well plates for five days (2.1.1.3). Experiments were conducted with 3 replicates in each group. Control groups were confluent cells with no inflammatory stimulus. Experimental groups were exposed to 100ng/ml of TNF-Į ZLWK RU ZLWKRXW glutamine (240 mM) and arginine (50 mM). Following 5 hrs incubation, RNA was extracted and IL-8 mRNA was quantified as previously detailed in (2.1.5). Raw Ct values were generated and folds of IL-8 expression were calculated relative to expression of the house keeping gene (Ʌ0 71)ĮH[SRVXUHSURPRWHGPRUHWKDQ-fold increase in the

IL-8 mRNA (P < 0.0001) compared to the control (Figure 3.16). With glutamine and arginine exposure, IL-8 mRNA expression dropped 4-fold compared to the positive control but remained 25-fold higher than the negative control (Figure 3.16). Figure 3.14 Effect of glutamine and arginine, individually and in combination, on IL-8 production from TNF-ĮH[SRVHG+7FHOOVCells were grown to confluence and then treated with either glutamine (240 mM) or arginine (50 mM); or a combination. TNF-Į  ng/ml) was added and cells incubated for a further 6 hours. Supernatants were collected and assayed for IL-8 by ELISA. Five replicates for each group as well as mean and SEM. (Neg): negative control (neither treatment nor TNF-Į  Pos): positive control (only TNF- Į). (Glu): glutamine at 240 mM + TNF- Į(Arg): arginine at 50 mM + TNF- Į (Glu/Arg): a combination of 240mM glutamine and 50mM arginine + TNF-Į(DFKRIJOXWDPLQHDQGDUJLQLQHLQGLYLGXDOO\VLJQLILFDQWO\DWWHQXDWHG IL-8 levels (P <0.0001 vs. positive control group). Combined glutamine and arginine resulted in further reduction in IL-8 level (P = 0.005 for glutamine treated group and P=0.02 for arginine treated group vs combined glutamine and arginine treated group). There was complete abrogation of IL-8 response in the combination treated group (P = 0.95 vs. negative control group). Analysis of data was conducted using one-way ANOVA followed by Fischer least significance test. Figure 3.15 Effect of glutamine and arginine, individually or in combination, on IL-8 production from TNF-ĮH[SRVHG&DFRFHOOVCells were cultured until confluent and then treated with either glutamine (240 mM) or arginine (50 mM) individually; or in combination. Treated cells were exposed to an inflammatory stimulus of 50 ng/ml TNF-Į ng/ml INF-Ȗ ng/ml IL-ȕ and 1μg/ml LPS for 24 hours. Supernatants were collected and assayed for IL-8 by ELISA. Points represent 5 replicates with the mean and SEM also presented. (Neg): negative control (neither treatment nor inflammatory stimulus). (Pos): positive control (only inflammatory stimulus). (Glu): glutamine at 240 mM + inflammatory stimulus (Arg): arginine at 50Mm + inflammatory stimulus (Glu/Arg): a combination of 240 mM glutamine and 50 mM arginine + inflammatory stimulus. Each of glutamine and arginine significantly attenuated IL-8 level in response to the inflammatory stimuli. Combined glutamine and arginine resulted in further significant reduction in IL-8 (P (*) < 0.01, (**) < 0.001 and (***) < 0.0001 vs. positive control group). Analysis of data was conducted using one-way ANOVA followed by Fischer least significance test. Figure 3.16 Outcome of combined glutamine and arginine on mRNA expression of IL-8 in response to TNF-ĮLQLQWHVWLQDOHSLWKHOLDOFHOOVHT29 cells were incubated with 100ng/ml TNF-Į in the presence or absence of glutamine (240 mM) and arginine (50 mM). After 5 hours incubation RNA was extracted from cells using the TRIzol method and cDNAs produced. RT-PCRs were performed and the IL- P51$ H[SUHVVLRQ PHDVXUHG UHODWLYH WR ȕ0 KRXVHNHHSLQJ JHQH XVLQJ SYBR-Green fluorescence detection method. (Control): (confluent cells neither treatment nor TNF-Į  TNF-Į : cells were exposed to TNF-Į (TNF-Į*OX/Arg): cells were exposed to TNF-Į and treated with combination glutamine and arginine. Cells exposed to TNF-Į KDG P-RNA expression raised 100-fold relative to the control group. However, in presence of combined glutamine and arginine treatment, the expression was reduced to 25-fold above the control. The columns represent the mean and SEM of 3 replicates of each group. Analysis of data was conducted using unpaired t Test (P (*) < 0.01, (**) < 0.0001 versus. control group). 3.4.6 Glutamine and arginine reduce TNF-ĮLQGXFHG,țțSKRVSKRU\ODWLRQ

To investigate the effects of glutamine and arginine on NF-ț% activity, the effects on the

,țț FRPSOH[ ZDV LQYHVWLJDWHG ,PPXQREORWWLQJ ZDV HPSOR\HG DV SUHYLRXVO\ GHWDLOHG

(2.1.4). HT29 cells were grown to confluence in 6-well plates. Negative controls (only media), positive controls (cells exposed to TNF-ĮQJPOIRUDQGPLQXWHV DV well as cells pre-incubated for 1 hour with either glutamine (240 mM) or arginine (50 mM) then stimXODWHG ZLWK 71)Į ZHUH LQFOXGHG 7ZR DGGLWLRQDO FRQWURO JURXSV ZHUH DOVR included where glutamine and arginine were added to cells without the addition of TNF-Į

Cell lysates were collected and total protein measured. Cell lysates were run on SDS-

PAGE with WKH DPRXQW DGGHG QRUPDOLVHG WR WRWDO SURWHLQ FRQFHQWUDWLRQV ,țț DQG phosphorylated-,țț3URWHLQVZHUHGHWHFWHGZLWKDQWL-,țțSULPDU\UDEELWDQWLERGLHV 

GLOXWLRQV  RU DQWL SKRVSKRU\ODWHG ,țț SULPDU\ UDEELW DQWLERGLHV  GLOXWLRQV  *RDW anti-rabbit IgG antibodies were utilized at 1:25,000 dilution to detect primary antibody binding. Bound antibodies were then visualised with chemi-luminescence and images captured and analysed with a GelDoc system.

TNF-Į LPPHGLDWHO\ DFWLYDWHG ,țț DV HYLGHQW E\ the appearance of a strong band of

SKRVSKRU\ODWHG ,țț DV HDUO\ DV  PLQXWHV DQG UHPDLQHG HYLGHQW DW  DQG  PLQXWHV following exposure to TNF-Į )LJXUH 3.17A). In the presence of glutamine, the phosphorylated-,țțDSSHDUHGUHGXFHGDWDQGPLQXWHV Figure 3.17B). Similarly, in

WKHSUHVHQFHRIDUJLQLQHWKHSKRVSKRU\ODWHG,țțDSSHDUHGUHGXFHGDWDQGPLQXWHV (Figure 3.17C). There was no visible change in the un-SKRVSKRU\ODWHG,țțEDQGLQDQ\RI the groups at any time point (Figure 3.17). A

5 15 30 (min)

- + + + TNF

IKK

Ph-IKK

Ǻ-actin

B

5 15 30 (min)

- + + + TNF

IKK

Ph-IKK

Ǻ-actin + + + + Glu

C

5 15 30 (min)

- + + + TNF

IKK

Ph-IKK

Ǻ-actin + + + + Arg Figure 3.17 ,țț and Ph-,țț responses in TNF-Į VWLPXODWHG +7 FHOOV LQFXEDWHG ZLWK RU without glutamine or arginine. Confluent cells were exposed to 100ng/ml TNF-Į A) or TNF-Į and 240 mM glutamine (B) or TNF-Į and 50 mM arginine (C). Cells were then incubated for 5, 15 and 30 minutes then cell lysates were collected. ,țț and phosphorylated ,țț were detected in the lysates by western blot using anti-,țț antibodies or anti-ph ,țț DQWLERGLHVǺ-actin was included as a loading control. Bands of phosphorylated ,țț were less visualised in cells treated with either glutamine or arginine without altering in total ,țț 3.4.7 Glutamine and arginine prevent TNF-Į GHSHQGHQW ,ț% SKRVSKRU\ODWLRQ DQG degradation.

To further investigate the activity of glutamine and arginineWKHHIIHFWRQ,ț%ZDVDOVR investigated using the procedure already described (4.4.3); however membranes were probed with the primary antibodies rabbit anti-,ț%DQGDQWL-SKRVSKRU\ODWHG ,ț%  dilution). In response to TNF-Į,ț% levels were lower at 5 and 15 minutes which were consistent with the earO\DSSHDUDQFHRISKRVSKRU\ODWHG,ț% )LJXUH3.18$ 2ILQWHUHVW,ț%

OHYHOVDSSHDUHGWRLQFUHDVHDWPLQXWHVDOWKRXJKSKRVSKRU\ODWHG,ț%ZHUHDOVRLQFUHDVHG

(Figure 3.18$ ,QJOXWDPLQHWUHDWHGFHOOVWKH,ț%EDQGDSSHDUHGVLPLODUDWDOO-time points,

(Figure 3.18%  )XUWKHU IRU SKRVSKRU\ODWHG ,ț% WKH SUHVHQFH RI JOXWDPLQH DSSHDUHG WR diminish the bands at each time point compared to the TNF-Į controls (Figure 3.18B).

Similarly, in the presence of arginine, cells showed an accumulation rather than a fall in

WKH OHYHO RI ,ț% ZLWK FRQWLQXRXV LQFUHPHQW LQ SURWHLQ H[SUHVVLRQ WKURXJKRXW WKH  minutes of TNF-Į H[SRVXUH )LJXUH 3.18&  &RUUHVSRQGLQJO\ SKRVSKRU\ODWHG ,ț% EORWV appeared less pronounced at all-time points following TNF-Į H[SRVXUH )LJXUH 3.18C).

Bands of the loading control Ʌ-actin were consistent in all groups indicating equal amounts of loaded protein (Figure 3.18). A

5 15 30 (min)

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,ț%

Ph-,ț%

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B

5 15 30 (min)

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Ǻ-actin + + + + Arg Figure 3.18 7RWDODQGSKRVSKRU\ODWHG,ț%LQ71)-ĮVWLPXODWHG+7FHOOVLQFXEDWHGZLWKRU without glutamine or arginine. Confluent cells were exposed to 100ng/ml TNF-Į A) or TNF-Į and 240 mM glutamine (B); or TNF and 50 mM arginine. Cells were then incubated for 5, 15 and PLQXWHVZLWKFHOOO\VDWHVFROOHFWHG,ț%DQGSKRVSKRU\ODWHG,ț%OHYHOVLQO\VDWHVZHUHDQDO\VHG by western blot using anti-,ț%DQWLERGLHVRUDQWL-SKR,ț%DQWLERGLHVRUȕ-actin anti-bodies as a ORDGLQJ FRQWURO %DQGV RI SKRVSKRU\ODWHG ,ț% ZHUH OHss evident in cells treated with either JOXWDPLQH RU DUJLQLQH &RUUHVSRQGLQJO\ FHOOV VKRZHG DFFXPXODWLRQ UDWKHU D GHJUDGDWLRQ RI ,ț% following TNF-ĮVWLPXODWLRQ 3.4.8 Glutamine and arginine inhibit TNF-ĮLQGXFHG nuclear migration of P65.

As a final measure of NF-ț% pathway activation, migration of the P65 subunit from the cytoplasm into the nucleus was investigated. Cells were treated as previously described

(4.4.3); however cell extracts were collected at 15, 30 and 60 minutes post TNF-ĮH[posure and both nuclear and cytosolic protein extracts were collected. For protein detection, membranes were probed with primary anti-P65 rabbit anti-human antibodies (1:1000 dilutions). TNF-Į LQGXFHG QXFOHDU WUDQVORFDWLRQ RI 3 IROORZLQJ  PLQXWHV RI 71F-Į exposure as there was an increase in the nuclear P65 band intensity (Figure 3.19A). At 30 minutes, P65 appeared to accumulate in the cytosol corresponding with decreased nuclear

P65 band intensity (Figure 3.19A). Interestingly, at 60 minutes the majority of P65 appears to have migrated into the nucleus again leaving a reduced proportion of P65 in the cytosol

(Figure 3.19A). In the presence of glutamine there was complete abolition of nuclear P65 at 30 minutes, together with diminished amount in the cytosol (Figure 3.19B). After 60 minutes the level of P65 in the cytosol fell further although there was a reappearance of nuclear P65 (Figure 3.19B). Similarly, in the presence of arginine, P65 nuclear bands were reduced at 15 minutes with complete loss of bands at 30 and 60 minutes (Figure 3.19C).

Simultaneously, there was also a drop in the cytosolic pool of P65 at 15, 30 and 60 minutes

(Figure 3.19C).

To further confirm the western blot findings, immunohistochemistry technique was utilized

(2.1.6). HT29 cells were seeded on glass slides and treated as follows: Negative control

(comprising unstimulated cells); TNF group (cells incubated with TNF-Į QJPO IRU hour); glutamine and arginine treatment groups in which cells were pre-incubated with either glutamine (240 mM) or arginine (50 mM) for 1 hour, then exposed to TNF-Į

(50ng/ml) for another 1 hour. Slides were then incubated with rabbit polyclonal antihuman

P65 antibody (dilution, 1:400). Primary antibody binding was detected by 488 Alexa secondary goat anti-rabbit antibodies. Nuclei were counter stained with DAPI fluorescence. Slides were visualized by Axioplan 2 immunofluorescent microscopy. In the control monolayers, NF-ț%p65 subunits were present abundantly in the cytoplasm (green colour), with minimal localization in the nucleus (blue colour) (Figure 3.20). Upon TNF-Į stimulation, a significant number of P65 subunits translocated into the nucleus (yellowish white colour; Figure 3.20). Interestingly, P65 trafficking was partially prevented by glutamine and almost completely prevented by arginine (Figure 3.20). More interestingly, the inhibited nuclear migration in both treatment groups was accompanied by a corresponding significant reduction in the expression of in the cytoplasm after 60 minutes of TNF-ĮH[SRVXUH )LJXUH3.20). A

15 30 60 (min)

- + + + TNF

Cytosolic P65

Nuclear P65

Ǻ-actin

B

15 30 60 (min)

- + + + TNF

Cytosolic P65

Nuclear P65

Ǻ-actin + + + + Glu

C

15 30 60 (min)

- + + + TNF

Cytosolic P65

Nuclear P65

Ǻ-actin + + + + Arg Figure 3.19 Cytosolic and nuclear portions of P65 in TNF-ĮVWLPXODWHG+7FHOOVLQFXEDWHG with or without glutamine or arginine. Confluent cells were exposed to 100ng/ml TNF-Į A) or with TNF-Į and 240 mM glutamine (B); or with TNF and 50 mM arginine and then incubated for 15, 30 and 60 minutes with both cytosolic and nuclear cell lysates collected. Cytosolic and nuclear P65 levels in the lysates were analysed by western blot using anti-P65 antibodies. Ǻ-actin was included as a loading control. Glutamine or arginine treatment was accompanied with inhibited nuclear migration and a corresponding significant reduction in the expression of P65.

1 2 3

A

B

C

D

Figure 3.20 Immunohistochemical expression and nuclear migration of P65 subunit of NF-ț% in TNF-ĮVWLPXODWHG+7FHOOVLQFXEDWHGZLWKRUZLWKRXWJOXWDPLQHRUDUJLQLQH Cells were grown on glass slide for 3 days as a control negative group (A). In the other groups, cells were exposed to TNF-ĮRIQJPOIRUKUSRVLWLYHFRQWURO(B). In the treatment groups, cells were pre- incubated with either 240 mM glutamine (C) or 50 mM arginine (D) for 1 hour, and then exposed to the TNF-Į IRU DQRWKHU  KRXU 6OLGHV ZHUH LQFXEDWHd with rabbit polyclonal antihuman P65 antibody (green colour) and fluorescence detected by 488 Alex secondary goat anti-rabbit antibodies. Nuclei were counter stained with DAPI fluorescence (blue colour). Cells exposed to TNF-ĮVKRZHGHQKDQFHGQXFOHDUWUDnslocation of P65 which is largely absent with glutamine or arginine treatments. The slides were visualized by Axioplan 2 immunofluorescent (40x magnification) illustrating epithelial monolayer histology (1), P65 expression (2) and the expression when nuclei were counterstained (3). 3.4.9 Glutamine and arginine directO\LQKLELW,țțDFWLYLW\

In the previous experiments, immunoblotting analysis indicated that in presence of

JOXWDPLQHRUDUJLQLQHWKHUHZDVDUHGXFWLRQLQSKRVSKRU\ODWHG,țțLQresponse to TNF-Į stimulation. To investigate whether glutamine and arginine directly and/or indirectly

VXSSUHVVHG,țțDFWLYLW\DNLQDVHDVVD\ZDVFRQGXFWHGXVLQJ a non-isotopic colorimetric kit as per manufacturer protocol (2.1.7). Briefly, the assay was performed in 96-well ELISA plates pre-FRDWHGZLWKUHFRPELQDQW,ț%Į*OXWDPLQHDQGDUJLQLQHZHUHGLVVROYHGGLUHFWO\ in kinase buffer to give final concentrations of 10, 50, 100 and 240mM for glutamine; and

2, 10, 20 and 50mM for arginine. K252a, synthetic kinase inhibitor, was dissolved in

DMSO then added to the kinase buffer to give a final concentration of 10mM with 0.5% v/v concentration of DMSO in the reaction buffer. Additional solvent control (0.5%

DMSO) and a no enzyme control were also included in the experiment. The reaction was initiated by adding ATP molecules to the fractionated ,țțȕ VXEXQLW 7KH DPRXQW RI phosphorylated substrate, reflecting ,țț activity, was measured by capturing the phosphorylated molecule with a peroxidase coupled anti-phospho-,ț%Į6DQWLERG\$ substrate was added that was converted to a colour product by the peroxidate and the generated colour was quantified by a spectrophotometer. In agreement with the manufacturer’s guidelines the fractionated ,țțȕ VXEXQLW GHYHORSHG D FRlour reaction of approximately 2.4 optical density (OD) units whereas, the no enzyme control did not exceed 0.06 O.D (Figure 3.21 7KHV\QWKHWLFLQKLELWRU.DFRPSOHWHO\LQKLELWHG,țț activity as the O.D was not different to the no enzyme control (P=0.19; Figure 3.21). The solvent control, DMSO alone, showed no significant reduction in the ,țț activity (P = 0.12 vs. ,țț control; Figure 3.21). Interestingly, when glutamine was added to the reaction, there was significant inhibition of ,țț activity beginning with the lowest glutamine concentration tested (10mM; Figure 3.21 ,QGHHGWKHLQKLELWLRQRI,țțDFWLYLW\LQFUHDVHG with higher glutamine concentrations (50 and 100 mM; Figure 3.21). Further, as with

K252a, the enzyme activity was completely abolished by glutamine at 240 mM concentration (P = 0.8 vs. K252a; Figure 3.21). Similarly, arginine showed a significant dose dependent attenuation in the ,țț enzyme activity (Figure 3.22). The maximal inhibition was reached at 50 mM when the ,țț activity was equivalent to K252a’s inhibition (P = 0.31; Figure 3.22). Figure 3.21 Glutamine inhibits ,țț enzyme activity in a dose dependent manner. Data are presented as raw O.D values (A) and enzyme activity as percentage activity compared to positive control (B).The kinase assay was conducted in 96-well ELISA plate pre-coated with recombinant ,ț%Į The reaction was initiated by adding ATP to the fractionated ,țț ȕVXEXQLW(,țț). No enzyme control (no ,țț) and synthetic inhibitor control at 10mM concentration (K252a 10) were included. Additional K252a’s solvent control (DMSO 0.5%) was included. Glutamine (Glu) dissolved directly in the kinase buffer to give final concentrations of 10, 50, 20, 100 and 240 mM. Optical density (O.D) was read by spectrophotometer and the activity assessed. Data represent mean and SEM of 5 replicates in each group. Analysis of data was conducted using one-way ANOVA test followed by Fischer least significance post hoc test (P (*) <0.01, (**) < 0.0001.vs ,țț control). Figure 3.22 Arginine suppresses ,țț enzyme activity in a dose dependent fashion. Data is presented as raw O.D values (A) and enzyme activity as percentage activity compared to positive control (B).The kinase assay was conducted in 96-well ELISA plate pre-coated with recombinant ,ț%Į The reaction was initiated by adding ATP to the fractionated ,țțȕVXEXQLW(,țț). No enzyme control (no ,țț) and synthetic inhibitor control at 10 mM concentration (K252a 10) were included. Additional K252a’s solvent control (DMSO 0.5%) was included. Arginine (Arg) dissolved directly in the kinase buffer to give final concentrations of 2, 10, 20 and 50 mM. Optical density (O.D) was read by spectrophotometer and the activity assessed. Data represent mean and SEM of 5 replicates in each group. Analysis of data was conducted using one-way ANOVA test followed by Fischer least significance post hoc test (P (*) < 0.01, (**) < 0.0001 vs. ,țț control). 3.5.0 Glutamine and arginine block TNF-ĮLQGXFHG30$3.SKRVSKRU\ODWLRQ

The effect of glutamine and arginine on P38 kinase signalling was next investigated. HT29 cells were cultured in 6-well plates as previously described (2.1.1.3). Negative control group (only media), Positive control (TNF-Į ng/ml), two treatment group’s glutamine

240mM or arginine 50mM, and two additional controls (glutamine or arginine without stimulation) were included. Cell lysates were collected and immune-blotting was conducted, as previously detailed (2.1.4). For protein detection, membranes were probed with primary anti-P38 rabbit anti-human antibodies (1:1000 dilutions) or anti– phosphorylated P38 primary rabbit antibodies (1:500 dilutions). Secondary goat anti-rabbit

IgG antibodies were utilized at 1:20,000 dilutions. Upon TNF-ĮVWLPXODWLRQ3DFWLYDWLRQ occurred after 15 minutes of the exposure, indicated by a strong band of phosphorylated

P38 (Figure 3.23A). The phosphorylated band remained at 30 minutes (Figure 3.23A). In contrast, the bands of phosphorylated P38 were less pronounced in cells pre-incubated with either glutamine (Figure 3.23B) or arginine treatments (Figure 3.23C). Correspondingly, blots of total P38 remained constant throughout the 30 minute observation period and did not differ between the groups (Figure 3.23B and C). A

5 15 30 (min)

- + + + TNF

P38

Ph-P38

Ǻ-actin

B

5 15 30 (min)

- + + + TNF

P38

Ph-P38

Ǻ-actin + + + + Glu

C

5 15 30 (min)

- + + + TNF

P38

Ph-P38

Ǻ-actin + + + + Arg

Figure 3.23 Total and phosphorylated P38 MAPK in TNF-ĮVWLPXODWHG+7FHOOVLQFXEDWHG with or without glutamine or arginine. Confluent cells were exposed to 100ng/ml TNF-Į A) or with TNF and 240 mM glutamine (B); or TNF-Į with 50 mM arginine (C). Cells were then incubated for 5, 15 and 30 minutes. Cell lysates were collected and protein concentrations were measured. Total and phosphorylated P38 in lysates were analysed by immunoblotting utilizing anti- P38 antibodies or anti-pho P38 antibodies; or Ʌ-actin anti-bodies as a loading control. Bands of phosphorylated P38 were less pronounced in cells treated with either glutamine or arginine. 3.5.1 Glutamine and arginine co-supplementation targets the NF-ț% signaling pathway in a microarray based analysis.

To investigate the effect of glutamine and arginine on a more general level, gene expression profiles of activated colonic epithelial cells using microarray analysis techniques were used (2.1.8). Briefly, HT29 cells grown in media (negative control

‘NCon’), as well as cells exposed to 100ng/ml TNF for 5 hrs (positive control ‘PCon’) and cells exposed to 100ng/ml TNF-Į ZKLOH FR-incubated with glutamine and arginine

(240/50mM; ‘GA’). Following incubation, RNA was extracted using the TRIzol method

(2.1.5.2). RNAs (500ng) from each group with high quality were subjected to Affymetrix microarray analysis (2.1.8.1). Analysis of the microarray data was undertaken using the

Partek Genomic Solutions platform (2.1.8.2).

TNF-Į HOLFLWHG DQ LPPXQH UHVSRQVH LQ WKH +7-29 cells which resulted in substantial changes in the expression of genes involved in immune processes, including NF-ț%UHODWHG genes (fold change > 2, FDR < 0.05; supplementary 2). The differentially expressed genes

(DEGs) comprised 50 significant immune related pathways (supplementary 2).

Interestingly, in a group stimulated with TNF-ĮEXWWUHDWHGZLWKFR-supplementation of glutamine and arginine, there were approximately 3305 DEGs compared to positive control group (fold change > 2, FDR < 0.05; supplementary 1). The DEGs were predominantly involved in 10 immune response related pathways (Table 3.1). In relation to

NF-ț% WKH PRVW LPSRUWDQW '(*V ZHUH FDWHJRUL]HG LQWR  FOXVWHUV Figure 3.24). Five groups were primarily involved in regulating the NF-ț%SDWKZD\DFWLYLW\DQGRQHFOXVWHU represented its final products (Figure 3.24). The first 5 groups were sub-classified based on the level of regulation: at the level of receptors such as TLR and TNFR; at the level of adaptor molecules such as TNF-receptor associated factor (TRAF; 2, 3 and 6), IL-1 receptor associated kinase (IRAK; 1and 2) and TLR associated protein (TIRAP); at the level of upstream kinases such as mitogen-activated protein kinase kinase kinase-

 0(..  DW WKH OHYHO RI ,țț LQFOXGLQJ ,țțĮ DQG ,țțȕ H[SUHVVLRQ DQG DW WKH OHYHO RI

DFWLYHGLPHUVDQGLQKLELWRU\,ț%LQFOXGLQJ1)-ț% 3 1)ț% 3 DQG,ț%Į )LJXUH

3.24). These signaling molecules which positively control NF-ț%ZHUHDOOGRZQUHJXODWHG by the glutamine and arginine treatment simultaneously with up regulating gene expression

RIWKHLQKLELWRU\,ț% 7DEOH3.1). The overall effect was inhibition of the NF-ț%SDWKZD\ resulting in down-regulated gene expression of the key pathway’s products including IL-8,

TNF, ICAM1 and HMGB1 (Figure 3.24). A detailed map of NF-ț% UHODWHG LPPXQH pathways, with illustration of DEGs by glutamine and arginine treatment, includes: Protein kinase R (PKR) pathway (Figure 3.25), HSP70/TLR signaling (Figure 3.26) and IL-18 signaling pathway (Figure 3.27). Table 3.1.Summary of DEG profile and related immune pathways in TNF-exposed HT29 and incubated with co-supplementation of glutamine and arginine.

Up regulated with Down regulated with Pathway Glu/Arg Glu/Arg FDR

1 Apoptosis and survival 2.318E- Role of PKR in stress- 11 TRAF3, TNF, IKK(cat), induced apoptosis TRAF6, eIF4E, TRAF2, eIF2S1, NFKBIB, NF-B, Caspase-8, IKK-alpha, PP2A regulatory p21, FasR(CD95), ATF-3, IFN- C/EBP z, I-kB, TAB2, gamma receptor, TLR4, PPP2R5A, NFKBIA, TNF-R1, IKK-beta, PP2A TRAM, ATF-3 catalytic, MSK2, IRF3

2 Immune response Role of STAT1, TRAF3, BAFF, 1.26E- PKR in stress-induced TNA, IKK, TRAF6, 10 antiviral cell response TIRAP, TRAF2, NFKBIB, NF-kB, Caspase-8, IKK- AFT-2/c, MEK6, IL-1RI, I- alpha, IFN-gamma receptor, kB, TAB2, c-Jun, TLR4, TNF-R1, IKK-beta, NFKBIA, ATF-2 IL8, MSK2, IRF3

3 Immune response Gastrin G-protein alpha-q, MEF2D, 6.65E- in inflammatory response AR-2/c-Jun, MEK6, IP3 10 receptor, JNK, I-kB, LARG, GRO-2, c-Jun, SOS, ATF-2, c-Fos, MEK1, IKK(cat), H-Ras, TRAF6, HB-EG, G-protein alpha- AKT, MEKK1, IKK-alpha, q/11, MEF2, PDK, PKC- CREB1, PTGS2 (COX-2), epsilon PI3K, IKK-beta, IL8

4 Immune response_HSP60 TNF, IKK(cat), TRAF6, 7.99E- and HSP70/ TLR TIRAP, NF-kB, NF- 10 signaling pathway AP-1, c-Jun/c-Fos, MEK6, kB1(p105), IKK-alpha, JNK, TAB3, I-kB, TAB2, IRAK1/2, Ubiquitin, E2N, HSP70, c-Jun, MEK1//2 , TLR4, IKK-beta, IL8, TPL2, CD14 ICAM1

5 Immune SP1, TNF, IL1RN, PI3K, 2.94E- response_HMGB1/RAGE MEK6, Tissue factor, JNK, NF-kB, NFKBIB, AKT, 09 signaling pathway I-kB, c-Jun, PAI1, CDC42, CREB1, TLR4, NFKBIA, MEK1//2, PLAT, PI3K, IL8, K-RAS, MIP-1beta ICAM1, HMGB1 6 Reproduction_GnRH AP-1, MEF2D, c-Jun/c- 6.47E- signaling Fos, MEK6, IP3 receptor, 09 JNK, MKP-1, FosB, NUR77, c-Jun, PKA-cat, SOS, ATF-2, c-Fos, MEK1, G-protein alpha-q/11, PLC- beta, FosB/JunB, ATF-3, PKC-epsilon, G-protein alpha-s, EGR1, JunB, H-Ras, Dynamin-1, Calmodulin CDC42, MEKK1, CREG1

7 Immune response_IL-18 SP!, TNF, IKK (cat), H- 7.87E- signaling AP-1, c-Jun/c-Fos, MEK6, Ras, TRAF6, Bcl-XS, 09 JNK, I-kB, PI3K (p85- PI3K, NF-kB, AKT, IKK- alpha), TAB2, c-Jun, alpha, PTGS2(COX-2), TRAM, MEK1/2, PDK IKK-beta, IL8, ICAM1

8 Immune response_IL-1 STAT1, TNF, IKK(cat), 5.11E- signaling pathway TRAF6, MEKK1, IKK- 08 AP-1, c-Jun, MEK6, IL- alpah, E2N, Ubiquitin, 1RI, Tissue factor, JNK, I- PTGS2(COX-2), IKK-beta, kB, TAB2, c-Jun, PAI1, IL8

9 Immune response_MIF- AP-1, CD44, c-Jun/c-Fos, 1.16E- induced cell adhesion, ROCK, JNK, MKP-1, H-Ras, NF-kB, AKT, 07 migration and VEFG-A, c-Jun, c-Fos, MEKK1, MMP-13, PI3K, angiogenesis PDK, MEK1/2, CXCR4 MMP-1, IL8, ICAM1

10 Development_HGF GAB1, CrkL, JNK, 1.74E- signaling pathway TCF7L2, PI3K, c-Jun, H-Ras, HGF receptor, AKT, 07 SOS, c-Fos, MEK1, E- MEKK1, FasR, cadherin, SNAIL1, PDK, PTGS2(COX-2), Beta- EGR1 catenin, PLAU

Figure 3.24 Targeting of NF-ț% DW PXOWLSOH UHJXODWRU\ OHYHOV E\ FR-supplementation of glutamine and arginine. Cells were exposed to 100ng/ml TNF for 5 hrs. In the other group, cells in addition to 100ng/ml TNF-ĮZHUHVLPXOWDQHRXVO\FR-supplemented with glutamine and arginine (240/50mM; ‘GA’). RNA was extracted and subjected to Affymetrix microarray analysis. Microarray analysis showed that glutamine and arginine targeted the gene expression of a number of key molecules involved in regulation of NF-ț% *HQH H[SUHVVLRQ RI ,ț%Į ZDV XS UHJXODWHG although the following genes were down regulated: Level of receptor, TLR and TNFR (1); level of adaptors TRAF, IRAK and TIRAP (2); at the level of upstream kinases MEKK1 (3); at the level of ,țț ,țțĮ DQG ,țțȕ H[SUHVVLRQ (4) DQG DW WKH OHYHO RI DFWLYH GLPHUV DQG LQKLELWRU\ ,ț% 1)-ț% 3 1)ț% 3 (5). As a result the NF-ț%WUDQVFULSWLRQIDFWRULQIOXHQFHGQHJDWLYHO\UHVXOWLQJ in down regulation some of its target genes including IL-8, COX2, TNF, ICAM1 and HMGB1. Figure 3.25 Targeting the Apoptosis and survival Role of PKR in stress-induced apoptosis pathway by glutamine and arginine co-supplementation. Cells were exposed to 100ng/ml TNF for 5 hrs. In the other group, cells in addition to 100ng/ml TNF-Į ZHUH VLPXOWDQHRXVO\ FR- supplemented with glutamine and arginine (240/50mM; ‘GA’). RNA was extracted and subjected to Affymetrix microarray analysis.This map included information from sets of differentially expressed genes of cells co-supplemented with Glutamine and arginine vs PCon. DEG is colored red if the gene is up regulated compared to PCon and blue if the gene is down regulated compared to PCon. Gene expressions of a number of key signaling molecules were down regulated in response to glutamine and arginine co-supplementation that negatively influenced NF-ț% transcription factor.

Figure 3.26 HSP60 and HSP70/TLR signaling pathway targeted by glutamine and arginine co-supplementation. Cells were exposed to 100ng/ml TNF for 5 hrs. In the other group, cells in addition to 100ng/ml TNF-Į ZHUH VLPXOWDQHRXVO\ FR-supplemented with glutamine and arginine (240/50mM; ‘GA’). RNA was extracted and subjected to Affymetrix microarray analysis. This map included information from sets of differentially expressed genes of cells co-supplemented with glutamine and arginine vs. PCon. DEG is colored red if the gene is up regulated compared to PCon and blue if the gene is down regulated compared to PCon. Gene expressions of a number of key signaling molecules were down regulated in response to glutamine and arginine co- supplementation that negatively influenced NF-ț%WUDQVFULSWLRQIDFWRU

Figure 3.27 Targeting NF-ț%WUDQVFULSWLRQIDFWRULQWHUPHGLDWLQJ,/-18 signaling pathway by glutamine and arginine co-supplementation. Cells were exposed to 100ng/ml TNF for 5 hrs. In the other group, cells in addition to 100ng/ml TNF-ĮZHUHVLPXOWDQHRXVO\FR-supplemented with glutamine and arginine (240/50mM; ‘GA’). RNA was extracted and subjected to Affymetrix microarray analysis. This map included information from sets of differentially expressed genes of cells co-supplemented with Glutamine and arginine vs. PCon. DEG is colored red if the gene is up regulated compared to PCon and blue if the gene is down regulated compared to PCon. Gene expressions of a number of key components of NF-ț% ZHUH down regulated in response to glutamine and arginine co-supplementation. 3.5 Discussion

These in vitro investigations have demonstrated that glutamine, arginine and vitamin D3, but not ALA, at concentrations equivalent to their concentrations in standard PF, supress the TNF-ĮPHGLDWHGSURGXFWLRQRIWKHSUR-inflammatory cytokine IL-8 in human intestinal epithelial cells. Further, glutamine, arginine and vitamin D3 all had strong dose-dependent anti-inflammatory effects, and combining glutamine and arginine further amplified the inhibitory effect on IL-8 production through influencing NF-ț%and P38 MAPK pathways.

Additionally, microarray analysis showed that there are 10 additional signaling transduction pathways that could be also involved with glutamine and arginine-mediated down-regulation of IL-8.

The results indicate that glutamine reduces IL-8 production under inflammatory conditions in human intestinal epithelium. This finding is consistent with the long standing concept that glutamine has immuno-modulating effects and is capable of suppressing cytokine production in inflammatory conditions [802]. An in vitro study involving intestinal gut epithelial cells showed that glutamine pre-treatment significantly decreased production of proinflammatory cytokines including IL-8 from cultured mucosal cells [803, 804]. Further, it has been shown that glutamine deprivation enhances IL-8 production from Caco2 cells exposed to LPS [805]. The present study is the first to examine the effect of high concentrations of glutamine on IL-8 production using an in vitro model of intestinal inflammation. Previously 10mM glutamine was the highest concentration given to cultured intestinal epithelial cells [801, 806, 807], although 15 mM glutamine treatment was used on human peripheral blood monocytes [808]. Of particular interest, treatment with 10 mM glutamine showed inconsistent results on IL-8 production from cytokine exposed intestinal cells. Marion et al. [807] reported that 10 mM glutamine treatment did not influence IL-8 production from intestinal epithelial cells under inflammatory conditions. In contrast,

Hubert-Buron et al. [589] demonstrated that 10 mM glutamine pre-treatment resulted in a considerable reduction in IL-8 production. This discrepancy was explained by presence of variable experimental conditions, including the timing of supplementation. In the current study, 12.7 mM glutamine was given simultaneously together with TNF resulting in significantly reduced IL-8 production. This might infer that both concentration and timing of supplementation would likely influence the immune-modulating effect of glutamine.

Indeed, in one experiment involving the two human intestinal cell lines, Caco2 and HCT-

8, increasing glutamine concentration from 2 to 10 mM significantly modulated chemokine secretion in a dose dependent fashion, in response to given inflammatory cytokines [807].

In a different model of peripheral blood cells exposed to LPS, increasing glutamine concentration from 1 to 15 mM also led to a gradual reduction in the expression of levels of numerous cytokines including IL-8 as well as TNF-Į,/-ȕDQG,/-6 [808]. Further, in another in vitro model involving murine intestinal cells, glutamine supplementation showed a dose dependent influence on the NF-ț%VLJQDOWUDQVGXFWLRQSDWKZD\[809]. All of these observations indicate that glutamine has dose dependent anti-inflammatory properties. Importantly, the current study has assessed a wider range of concentrations and demonstrated that higher concentrations of glutamine are more efficient than lower concentrations in reducing the chemokine response. It is also important to note that epithelial cells can tolerate a concentration of approximately 20 times higher than previously tested. This may be of clinical importance if higher concentrations of glutamine are used for the treatment of IBD.

The work presented here also indicates that arginine influences the inflammatory response in intestinal epithelial cells exposed to a pro-inflammatory cytokine. Arginine at 1.8 mM

(the concentrations in PF) significantly decreased IL-8 production from TNF-Į H[SRVHG

HT29 cells. However, the effect of arginine supplementation in intestinal inflammation has not been widely investigated. The first in vitro study which investigated the effect of arginine supplementation showed that 2 mM arginine treatment led to a significant reduction in IL-8 production from cytokine-exposed HCT-8 intestinal epithelial cells

(Marion, Coëffier et al. 2005). However, animal experiments of arginine supplementation have received more attention. Arginine supplementation was shown to enhance immune function and prolong the survival rates in severely injured and critically ill animals [810,

811]. In a study investigating burns and healing in rats, a high arginine diet reduced inflammatory cytokines production and improved the survival of animals that received burns [812]. Likewise, in rodents with endotoxin-induced lung injury, arginine supplementation ameliorated inflammatory responses in the injured lung, consistent with reduction in inflammatory cytokine production by alveolar macrophages [813]. In this work, arginine also exhibited a strong dose-dependent effect in blocking the pro- inflammatory IL-8. Based on the literature it is known that arginine has a strong anti- inflammatory effect under both basal and inflammatory conditions, which can be amplified with increasing concentrations. In an in vitro model involving peritoneal macrophages from healthy rats, increasing arginine concentration up to 2 mM down-regulated basal production of IL-6 and TNF-Į IURP FXOWXUHG FHOOV LQ D GRVH GHSHQGHQW IDVKLRQ [814].

Similar findings were seen in cultured peritoneal macrophages in vitro using cells collected from endotoxemic rats [815]. In the current study, the IL-8 suppressing effects of arginine can be enhanced further with increasing concentration of up to 50 mM, without detrimental effects on cell viability. Although the anti-inflammatory effect of arginine on inflammation involving the intestinal epithelium has not been widely investigated, the results of the current work utilising an in vitro model appear promising and necessitate further exploration.

Vitamin D3 at 3.8 nM was also shown to have a substantial anti-inflammatory effect in this model of intestinal inflammation. This observation is consistent with previous studies utilising different cell lines. In one study involving T lymphocytes, vitamin D3 supplementation inhibited inflammatory cytokine production [816]. Although it has not been widely investigated with in vitro models of intestinal inflammation, vitamin D3 also showed promising results in murine models of colitis [817, 818]. Vitamin D3 analogue ameliorated mucosal injury and decreased inflammatory cytokine expression in a dextran sodium sulphate induced colitis model [819]. In another study, vitamin D3 deficient mice developed an accelerated form of colitis, which was ameliorated by vitamin D3 supplementation [820]. Interestingly, it has been reported that patients with IBD have lower serum levels of vitamin D3 than healthy controls [821]. All of these findings indicate that vitamin D3 supplementation may be of clinical importance in treating and/or preventing conditions involving intestinal inflammation. Certainly, in a double-blind randomized controlled trial of 108 patients with CD, daily oral supplementation with vitamin D3 restored low levels of vitamin D3 to the normal range as well as diminishing the relapse rates over a one year of follow-up period [822]. Similar to glutamine and arginine, the anti-inflammatory effect of vitamin D3 was influenced by concentration. The dose response of vitamin D3 on intestinal inflammation in vitro has not been shown before.

However, in another in vitro study involving LPS-exposed human peritoneal macrophages, vitamin D3 pre-treatment resulted in significant reduction in TNF-ĮSURGXFWLRQ[823]. In that study cells were pre-incubated for 16 hours with various concentrations (0.1 to 100 nM) of vitamin D3 prior to LPS exposure. Similar to the current study, there was a significant reduction in the level of released mediators in response to inflammatory stimuli when cells were pre-incubated with vitamin D3, although more interestingly, there were further decreases in the inflammatory response with higher concentration of vitamin D3.

Panichi et al. [824] in a study involving peripheral mononuclear cells (PBMS) from normal subjects and from patients with chronic renal failure, were pre-incubated with a range of vitamin D3 concentrations (0.1 to 4 nM), also showed that vitamin D3 induced a significant dose dependent inhibition of TNF-Į and IL-ȕ SURGXFWLRQ IURP WKH 3%06 cells. Further, in another in vitro experiment involving blood leukocytes from cattle exposed to mitogen, A similar response to vitamin D3 treatment was shown with increasing concentrations (0.1 to 10 nM) of vitamin D3 resulting in a further reduction in

INF-Ȗ UHOHDVH IURP OHXNRF\WHV [825]. Therefore when considered together, all of these observations indicate that the anti-inflammatory properties of vitamin D3 are concentration dependent, and thereby can be enhanced with increasing concentrations. However, it should be noted that these preliminary observations have used very high levels of vitamin

D3. In the current study approximately 10 times the concentration of vitamin D3 (as compared to vitamin D3 concentrations in PF) was required for further augmenting the anti-inflammatory properties. If such high concentrations were utilized for therapy, there would be significant safety concerns. This therefore necessitates further investigations into immunomodulating property of vitamin D3 and consideration as to what role vitamin D3 supplementation has in IBD therapy.

In contrast to the other nutrients investigated, ALA showed no immunomodulating activity in this model of intestinal inflammation. This is contradictory to previous observations reported in the literature. Previously, it has been reported that ALA significantly inhibited

IL-6, IL-ȕDQG71)-ĮSURGXFWLRQIURPSHULSKHUDOEORRGPRQRQXFOHDUFHOOVFXOWXUHGIURP hypercholesterolemic individuals exposed to a diet high in ALA [826]. Further, in a trial with patients who received a diet rich in ALA for 3 months, there was a considerable reduction in systemic inflammation measured by low plasma levels of IL-6 from patients with dyslipidaemia [827]. In another study using an in vitro model of intestinal inflammation of Caco2 cells exposed to LPS and IL-ȕ$/$VLJQLILFDQWO\UHGXFHG,/-8 production, but not IL-6 [828] $OWKRXJK LQ WKH VDPH VWXG\ RWKHU SURGXFWV RI Ȧ-3 FAs significantly suppressed both IL-6 and IL-8 levels from the cells in response to inflammatory stimuli [828]. In the current study, ALA exhibited no anti-inflammatory effect even with high concentrations used. One explanation may be related to product

VHOHFWLRQ&DQRODRLOULFKLQ$/$ZDVWKHVRXUFHRIȦ-3 Fas used in the current study,

ZKLFK LVHTXLYDOHQWWR WKH Ȧ-3 Fas present in PF. However, other studies used the most

SRWHQW SXULILHG SURGXFWV RI Ȧ-3 Fas, such as EPA and DHA. Certainly, Chong-Jeh et al

[829] reported that fish oil rich in EPA and DHA can significantly inhibit the production of IL-ȕDQG71)-ĮLQFXOWXUHGPRQRF\WHVH[SRVHGWR/366LPLODUILQGLQJVZHUHUHSRUWHGLn another two in vitro experiments involving LPS-exposed human macrophages when given omegaven (rich in EPA and DHA) which significantly ameliorated TNF-ĮSURGXFWLRQIURP the cultured cells [830, 831]. Also, in an ex vivo model using murine macrophages exposed to inflammatory cytokines, fish oil led to a significant reduction in IL-ȕ,/-6 and TNF-Į

[832]. ,QDPRGHORIH[SHULPHQWDOFROLWLVLWZDVVKRZQWKDWGLHWDU\ILVKRLOULFKLQȦ-3 Fas extenuates the progression of inflammation and shortens the course of disease [649].

Moreover, the daily consumption of EPA and DHA for a period of 4 weeks resulted in inhibition of TNF-ĮDQG,/-6 from PBMC of healthy subjects [833]. In the present work there was no evidence supporting that Ȧ-3 Fas are strong anti-inflammatory agents in the setting of PF. However, canola oil rich in ALA was the only tested product and the pro- inflammatory cytokine IL-8 was the only inflammatory marker measured in this study.

7KHUHIRUHIXUWKHULQYHVWLJDWLQJȦ-3 Fas anti-inflammatory properties in vitro utilizing EPA

DQG'+$DVȦ-3 Fas, and measuring numerous inflammatory markers, may be required to establish the anti-inflammatory properties of Ȧ-3 Fas.

In this model of intestinal inflammation, PF also exhibited pronounced immunomodulating action. Several studies have supported this primary effect of enteral nutrition using PF. de

Jong et al. [707] demonstrated that PF has direct anti-inflammatory effects with reduction in IL-8 production from three different epithelial cell lines utilized as in vitro models of intestinal inflammation. In a further study involving culturing intestinal biopsies collected from patients with IBD, similar results were also obtained [834]. Despite these findings the precise mechanism by which PF reduces intestinal inflammation is unknown. The present study is the first to test in vitro, the anti-inflammatory effect of combining glutamine, arginine, vitamin D3 DQGȦ-3 FAs. Here, each of the 3 supplements glutamine, arginine and vitamin D3 EXW QRW Ȧ-3 Fas, at equivalent concentrations to their concentrations in PF, showed an effect on suppressing IL-8 production from TNF exposed intestinal epithelial cells. Importantly, when these nutrients were combined together, the overall effect was amplified indicating the combination may lead to synergistic or additive actions. However,

PF with its whole ingredients remained more effective in reducing IL-8 production than the combination of glutamine, arginine, and vitamin D3 DQGȦ-3 FAs. This suggests that there may be other unknown constituents or factors of PF with immuno-modulating effect. This may include other vitamins and minerals. In one trial involving diabetic patients with impaired glucose tolerance it has been shown that a combination of vitamin C and E improved insulin sensitivity due to inhibition of inflammation and oxidative stress in recruited patients [835]. In another study involving immunodeficient patients vitamin A supplementation resulted in enhanced immunoglobulin production and reduction in inflammatory response [836]. Further, zinc has also been shown to modulate immune response and its deficiency has deleterious effects on immune system [837]. In a mouse model of allergic inflammation, zinc was effective in ameliorating the inflammatory response in inflamed airways [838]. Thus, it seems that the anti-inflammatory properties of

PF may be the result of the combination of various molecules with immunomodulating effects, in addition to glutamine, arginine and vitamin D3 DQG OHVV OLNHO\ Ȧ-3 FAs.

Therefore, for better understanding of the precise mechanism of action of the PF, each single constituent has to be investigated individually. However, these investigations were beyond the scope of this thesis and were not attempted. Further, for first time these experiments demonstrated that glutamine and arginine can act synergistically in activated intestinal epithelial cell line, resulting in additive immunomodulatory effects. Lecleire et al. [801] in work that involved ex vivo biopsies taken from patients with CD and cultured with glutamine and arginine also showed that the combination of glutamine and arginine has additive and/or synergistic effects on decreasing production of proinflammatory cytokines. However, the authors found that arginine alone was unable to promote any anti-inflammatory activity in the cultured biopsies. Similar results of synergistic effects of combined glutamine and arginine have also emerged from animal work [839]. A model involving endotoxemic mice showed that a combination of glutamine and arginine is superior in ameliorating the inflammatory response in the intestinal mucosa to either agent given individually [840]. Indeed, arginine only significantly decreased TNF-ĮH[SUHVVLRQLQWKHLOHXPZKLOVWJOXWDPLQHVLJQLILFDQWO\ decreased both TNF-ĮDQG,/-10 expression, but only in the ileum. On the other hand, the combination of arginine and glutamine significantly decreased expression of both TNF-Į and IL-ȕLQWKHLOHXPDVZHOODVLQWKHMHMXQXPRIWUHDWHGPLFH[840]. All these data, along with concurrent experimental data, suggest that glutamine and arginine can synergize with each other and amplify their immunomodulating effects during intestinal inflammation.

This therefore further supports use of amino acid supplementation in IBD.

Mechanistically, immunoblotting findings ascertained that activity of glutamine and arginine is attributed to targeting the major intracellular signaling pathways. The results presented here show that immediately after adding TNF-Į1)-țB was activated as evident by phosphorylation of Ițț, which in turn led to phosphorylation and early partial drop in WKHOHYHORI,ț%6XEVHTXHQWO\WKH3VXEXQLWRI1)-țB was liberated and migrated into nucleus after 15 minutes of TNF stimulation. It is well documented that in HT-29 cells, the response to various stimuli including TNF-Į WKH F\WRSODVPLF ,ț% SURWHLQ LV QRW WRWDOO\ depleted [841]. Interestingly, glutamine blocked these sequential events and the net results were blocking downstream signal transduction and therefore down-regulation of IL-8 gene expression and cytokine production.

Previously, it has been shown in an in vitro study utilizing LPS treated Caco-2 cells, that

JOXWDPLQH GHSULYDWLRQ GHFUHDVHG WKH OHYHO RI WKH LQKLELWRU\ SURWHLQ ,ț% DQG HQKDQFHG nuclear P65 content, thereby resulting in higher IL-8 production [805]. Further, in the same experiment, the authors reported that adding glutamine at a low concentration (0.5 mM) partially prevented the LPS-induced IL-8 production, but without any reported effect on

LPS-LQGXFHGSKRVSKRU\ODWLRQRI,ț%[805]. Similar findings were also reported by Huang et al. [842] when they showed using Caco2 cells, the addition of glutamine (0.5 to 5 mM) reduced mRNA expression and IL-8 production without any reported changes in NF-ț% activity. There is no clear explanation for this discrepancy between our findings and previous work; however, the inconsistency may be due to the lower concentrations of

JOXWDPLQH KDYLQJ VXEWOH HIIHFWV RI SUHYHQWLQJ ,ț% GHJUDGDWLRQ DQGZLGHUUDQJHRI concentrations allows a greater ability to demonstrate that effect. Indeed, in a different experiment involving HTC-8 cells, glutamine at 10 P0ZDVHQRXJKWRLQFUHDVHWKH,ț% content and reduce IL-8 production [589]. Recently, in a model of disrupted epithelial barrier using Caco2 cells cultured in vitro, JOXWDPLQH SUHYHQWHG WKH GHFUHDVH LQ WKH ,ț% expression induced by methotrexate treatment [843]. On a different cell line based on lung epithelial cells, glutamine supplementation also prevented LPS induced NF-ț% QXFOHDU translocation [844]. In contrast, in a IHZVWXGLHVJOXWDPLQHKDGDGLIIHUHQWHIIHFWRQWKH,ț% state. For instance, in a recent study involving Caco2 cells cultured in the presence or

DEVHQFH RI LQIODPPDWRU\ F\WRNLQHV JOXWDPLQH VXSSOHPHQWDWLRQ HQKDQFHG ,țț phosphorylation followed by phosphor\ODWLRQ DQG GHJUDGDWLRQ RI ,ț% [845]. Also, in peritoneal macrophages, glutamine enhanced LPS-induced activation of NF-ț%

FRQVLVWHQWO\ZLWKDGHFUHDVHLQWKH,ț%SURWHLQH[SUHVVLRQLQWKH FXOWXUHG PDFURSKDJHV

[846]. Despite this controversy about the relationship between glutamine and the NF-ț% pathway, especially in in vitro studies, more consistent findings were reported from animal work [564]. In a rat model of colitis, glutamine promoted strong anti-inflammatory activity that prevented colonic mucosal damage of treated mice that was consistent with decreased

NF-ț%H[SUHVVLRQ[847]*OXWDPLQHDOVRLQKLELWHG,ț%ĮSKRVSKRU\ODWLRQGHJUDGDWLRQDQG

NF-ț% QXFOHDU WUDQVORFDWLRQ LQ D GLIIHUHQW PLFH PRGHO RI FROLWLV [848]. Fillmann et al.

[584] UHSRUWHG WKDW HQKDQFHG ,ț% H[SUHVVLRQ LQ WKH JOXWDPLQH-treated mice is associated

ZLWK FKDQJHV LQ ,țț FRPSOH[ DV D UHVXOW RI JOXWDPLQH VXSSlementation. Additionally,

Lecleire et al. [801] in their ex vivo project that involved intestinal biopsies collected from patients with CD, reported that glutamine is able to modulate the NF-ț% DFWLYLW\ LQ WKe cultured tissue. Collectively, previous research as well as the results presented here, points to the fact that glutamine can negatively regulate NF-ț% WUDQVFULSWLRQ IDFWRU DFWLYLW\ through targeting phosphorylation of its kinase subunits, in cultured colonic epithelial cell lines. Arginine also showed similar findings to glutamine in attenuating NF-ț% DFWLYLW\

$UJLQLQH EORFNHG ,țț SKRVSKRU\ODWLRQ DQG KHQFH VXSSUHVVHG ,ț% SKRVSKRU\ODWLRQ DQG degradation. Subsequently, this led to inhibition of P65 liberation and nuclear migration.

Arginine, a substrate of NO synthetase, is capable of ameliorating inflammatory responses in the cultured epithelial cells that involves the NO pathway [849]. However, the activity of arginine on NF-ț% KDV QRW EHHQ ZLGHO\ H[SORUHG ,QDVWXG\RISRUFLQHFXOWXUHG enterocytes, the authors reported that arginine inhibited LPS induced phosphorylation of the NF-ț% DFWLYH GRPDLQ EXW GLG QRW SURYLGH D GHWDLOHG PHFKDQLVWLF GHVFULSWLRQ [850].

However, there are claims that the inhibitory effect of arginine-induced NO on IL-8 production is likely related to the inhibition of the NF-ț%SDWKZD\,QDQDQLPDOPRGHORI

Duchene Dystrophy involving dystrophin-deficient mice, arginine decreased the level of inflammatory cytokines and enhanced muscle regeneration through down-regulation of

NF-ț%DFWLYLW\ZLWKFRQFRPLWDQWLQKLELWLRQRI,ț%GHJUDGDWLRQ[851]. Also, in a rat model of LPS-LQGXFHGLQMXU\DUJLQLQHSUHYHQWHGWKHGHFUHDVHLQ,ț%OHYHOVDQGLQKLELWHG1)-ț%

DNA binding [852]. The results presented here show that arginine’s immunomodulatory action is mediated, at least in part, through NF-ț%EORFNDGH

It is well documented that targeting of the NF-ț%SDWKZD\LVRIJUHDWFOLQLFDOVLJQLILFDQFH and therapeutic importance for treating IBD [853]. It is widely accepted that corticosteroids and sulphasalazine, due to their anti-inflammatory activity which are attributed to modulating NF-ț%KDYHEHQHILWWHGSDWLHQWVZLWK,%'[854, 855]. Targeting the NF-ț%SDWKZD\FDQEHDFKLHYHGDWGLIIHUHQWVWHSVLQFOXGLQJDWWKHOHYHORIWKHXSVWUHDP

DGDSWRUPROHFXOHVDQGDYDULHW\RIRWKHUVLJQDOOLQJFRPSRQHQWVOLNH,țțUHODWHGNLQDVHV [856-858]7KH,țțFRPSOH[ZKLFKOLHVDWDFHQWUDOMXQFWLRQLQWKHSDWKZD\KDVJDLQHG most of the interest and the focus from researchers in the drug discovery area [89, 859,

860]. Several common anti-inflammatory agents including sodium salicylate (aspirin) and

ASA related products have been reported to inhibit NF-ț%E\WDUJHWLQJ,țț[861-863]. The results presented here show that suppression of NF-ț%DFWLYLW\E\JOXWDPLQHDQGDUJLQLQH

LV PHGLDWHG E\ GLUHFW LQKLELWLRQ RI WKH ,țț enzyme, and thereby impairing the

SKRVSKRU\ODWLRQ RI ,ț% DQG VXEVHTXHQW DFWLYDWLRQ RI 1)-ț% 7KH UHGXFWLRQ LQ WKH ,țț complex activity by the two amino acids was dose dependent. This may provide a clue about the mechanistic basis for the synergistic interaction between these two agents.

Indeed, at lower concentrations, JOXWDPLQHDQGDUJLQLQHRQO\SDUWLDOO\VXSSUHVVHGWKH,țț complex activity. The mechanism by which glutDPLQH DQG DUJLQLQH EORFN ,țț

SKRVSKRU\ODWLRQPD\UHODWHWRLQWHUIHULQJZLWK$73PROHFXOHELQGLQJWRWKH,țț Ʌ subunit,

VLPLODUWRWKHV\QWKHWLF,țț inhibitor K252a. It has been reported that K252a, exhibits its inhibitory activity against the kinases through competing with the ATP molecule [864,

865]. Here, glutamine and arginine had similar activity to the K252a when added directly to the reaction buffer together with ATP molecules, suggesting the mechanism of action could be related to the ATP binding. Of interest, aspirin and its related ASA compounds

DUHNQRZQWRDOVRFRPSHWLWLYHLQKLELW$73ELQGLQJWR,țțȕ[862].

Importantly the immunoblotting analysis also revealed that glutamine and arginine prevented the TNF-Į LQGXFHG SKRVSKRU\ODWLRQ RI 3 3UHYLRXVO\ JOXWDPLQH KDV EHHQ shown to protect mice from induced dermatitis and lethal endotoxin shock, in part through deactivation of the P38 pathway [866, 867]. However, in a study involved Caco2 cell line have shown that down-regulation of LPS-stimulated IL-8 production with glutamine was not accompanied with reduction in P38 MAPK kinase activity [868]. Further, there is no previous specific evidence of the action of arginine on the P38 pathway. Nevertheless it has previously been shown in activated mast cell that the combination of glutamine and arginine can alter the phosphorylation of P38 [869]. Further, in a study involving colonic biopsies collected from patients with CD, this combination (glutamine and arginine) also altered expression level of P38 MAPK and this was responsible for the observed drop in the level of the inflammatory mediators measured [801]. The results presented here revealed that there were no noticeable changes in the expression level of total P38 MAPK but there was a clear decrease in phosphorylated P38 in the presence of glutamine and arginine. Thus, it appears that in addition to the inhibition of NF-ț%DFWLYDWLRQWKHDQWL- inflammatory property of glutamine and arginine is also mediated through altering MAPK

P38 activity. Of interest, signaling in MAPK P38 is suggested to be involved in pathogenesis of numerous inflammatory conditions including IBD [98, 107]. Further, targeting this kinase has gained much interest for researchers as a therapeutic strategy for various chronic inflammatory diseases [107, 870]. Thus, through blocking the signals transduced by NF-ț%DQG0$3K P38 pathways; glutamine and arginine supplementation might be of great therapeutic value for the treatment of IBD.

Microarray analysis of activated colonic epithelial cells showed there is clear evidence that co-supplementation of glutamine and arginine alters gene expression of several key signaling molecules involved in 10 different immune pathways. The effect was at different regulatory levels from the receptors to the target genes. Overall, this resulted in a net negative influence on NF-ț% WUDQVFULStion factors, resulting in down regulation of a number of its key end products. This suggests that glutamine and arginine may also control

NF-ț% LQGLUHFWO\ E\ PHGLDWLQJ H[SUHVVLRQ RI XSVWUHDP PROHFXOHV &HUWDLQO\ there is a cross talk with these cascades and NF-ț%WUDQVFULSWLRQIDFWRUDQG1)-ț%OLHVLQWKHFHQWUH of this network, mediating downstream signaling transduction. Indeed, NF-ț% LV often considered a key transcription factor in the immune response and is an intensely studied factor in the immune system [871]. Therefore, the activity of glutamine and arginine appears to be central in the regulation of this essential signaling pathway.

Signaling pathways and their components including transmembrane receptors, adaptor proteins and other subcellular signaling molecules, are often considered a double-edged sword [872-874]. Cells respond to various stimuli through activation of a number of stress pathways to control and limit the spread the invasive agent [875]. However, occasionally the cellular stress response is over exaggerated and progresses to a pathological condition

[876]. PKR is an example. PKR is an interferon-induced serine/threonine protein kinase that is activated by double-stranded RNA (dsRNA) [877]. PKR function is primarily to control cell growth and cell differentiation [878]. Its effect is mediated through sequential activation of numerous signaling pathways and transcription factors [879], most notably eIF-2a and NF-ț% [880]. Interestingly, a recent report revealed that PKR through a endoplasmic reticulum (ER) unfolded protein responses (UBRS), contributes to pathogenesis of intestinal inflammation [881]. Further, targeting PKR might be a potential approach for novel IBD therapies [881]. The results of this chapter indicate that TNF-Į induced a PKR exaggerated response is suppressed with glutamine and arginine co- supplementation. HSP-70 and its signaling pathway is an example of stress proteins that are induced in order to provide a protection against stressors [882]. HSP-70 has protective roles against numerous GIT diseases including providing a beneficial effects in IBD [883].

Nevertheless, there is evidence that HSP70/TLR4 signaling can induce proinflammatory cytokine production and contribute to the cellular immune response, providing an example of the dual nature of these pathway [884]. HSP70 utilizes both TLR2 and TLR4 to transduce its proinflammatory signal and activate NF-ț% transcription factors [885].

Interestingly, in the current work we observed both an enhancement in expression of protective HSP-70 by glutamine and arginine treatment, together with repression of

HSP70/TLR4 signaling.

Further glutamine and arginine amino acids were shown to also modulate the gastrin signaling pathway. Gastrin, a regulator of gastric acid secretion [886], also modulates the function of immune cells following cellular stress and injury [887]. There is evidence that

Gastrin and its receptor are up-regulated during inflammation including in GIT conditions

[887]. Gastrin induces expression of IL-8 and other pro-inflammatory mediators including cyclooxgenase-2 (COX-2) in part through activation of NF-ț%DQG30$3.[888, 889].

Moreover, the inflammatory cascade of the IL-1 and its related family member IL-18, which typically induces sequential activation of the NF-ț% DQG 0$3. pathways [890], was shown in the current work to also be inhibited with glutamine and arginine co- supplementation. IL-1 and IL-18 are primarily pro-inflammatory cytokines, able to stimulate expression of different genes associated with inflammation and autoimmune diseases [890] and have been shown to contribute to IBD pathogenesis [891]. Additionally, the microarray analysis also revealed down regulation of HMGB1 proinflammatory mediator, the end product of HMGB1/RAGE signaling. The High mobility group box 1 (HMGB1) is an intracellular protein with extracellular functions that promote inflammatory responses [892, 893]. The receptor for advanced glycation end products (RAGE) is the major receptor for the protein HMGB1 [894], although TLR2 and

TLR4 may also be receptors for the protein [895]. HMGB1/RAGE axis elicits responses by activating multiple signaling pathways including NF-ț% DQG 30$3. [896, 897].

Interestingly, HMGB1 is highly expressed in the inflamed intestinal tissue of IBD and is found abundantly in the stool samples [898]. Therefore HMGB1 has been proposed as a novel non-invasive fecal marker of intestinal inflammation in IBD patients [898]. A recent report also suggested that HMGB1may be a suitable biomarker for mucosal healing [899].

Finally, genome analysis also revealed glutamine and arginine co-supplementation can influence the migration inhibitory factor (MIF) induced cell adhesion and angiogenesis pathway. MIF is a pleotropic cytokine which plays a pivotal role in inflammatory and immune-mediated diseases [900, 901]. In addition, it mediates cellular adhesion, migration and vascular angiogenesis [902]. In IBD, this pathway mediates and amplifies the inflammatory response by allowing trafficking of immune cells into diseased tissues as well as altering the microvascular architecture [903, 904]. MIF also functions via activation of numerous signaling pathways including NF-ț%DQG0$3.[905, 906]. Thus

MIF and its signaling intermediates may be potential therapeutic targets for autoimmune diseases characterized by active immune cell recruitment and over expression of adhesion molecules. Indeed, targeting the intracellular adhesion molecule (ICAM) has gained much interest recently as a therapeutic target in IBD [907]. ICAM 1 is an example of MIF mediated adhesion molecules that are down-regulated by glutamine and arginine.

Collectively, the findings of this chapter further support that glutamine and arginine contribute to the therapeutic benefits of EEN therapy. 3.6 Conclusions

What is unique for this study is that glutamine, arginine and vitamin D3 were shown to be among the active constituents of PF responsible for suppressing inflammation. Moreover, this data also indicated that, in addition to those supplements, there are likely other ingredients of PF with an immunomodulating effect that may additionally contribute to the overall activity of PF. More interestingly, this work has shown that glutamine, arginine and vitamin D3 supplements all have dose dependent anti-inflammatory effects indicating there is potential to use these nutrients at higher concentrations. Further, the current work adds to the literature in that glutamine and arginine in combination, offers synergistic anti- inflammatory activity. That activity is modulated through influencing the NF-ț%DQG3

MAPK signaling pathways as well as a network of 10 intracellular immune response related pathways. At the molecular level, glutamine and arginine interfered with phosphorylation of the key components of the signaling pathways. Collectively, integration of those two amino acids glutamine and arginine could result in a synergy that is beneficial during intestinal inflammation. Accordingly, a high glutamine and arginine fortified enteral nutrition product should be evaluated in patients with IBD. Therefore, the aim of the next chapter will be to determine whether manipulation of the glutamine and arginine components of PF leads to enhanced anti-inflammatory properties in an in vitro model of

IBD. Chapter 4: Developing a novel nutritional therapy with enhanced anti- inflammatory properties for CD in an in vitro model of intestinal inflammation.

4.1 Introduction

Nutritional therapy is increasingly seen as an attractive therapeutic option in treating various medical diseases [908, 909]. In CD, as per the 2014 consensus guidelines of

ECCO/ESPGHAN on the medical management of pediatric CD, enteral diet given as EEN is now the preferred induction therapy for children with active disease [523]. However, this therapy remains underutilized [910] because of the long duration of induction treatment

(up to 10 weeks) [557] and even longer duration of maintenance therapy (several months)

[911]. Further, the role of EEN in achieving clinical remission in adults is considered to be less effective than standard drugs [466]. Developing a novel nutritional therapy with enhanced activity compared to the currently used formulas, is desirable. Therefore, there is a scope that nutritional therapy could be more widely utilised if the efficiency and/or efficacy of the formula is improved.

Increasing the concentrations of glutamine and arginine, two of the essential elements of standard PF as shown in the previous chapter (3), could be a viable option for enhancing the anti-inflammatory activity of PF. Those two conditionally essential amino acids with multiple immunological functions offer numerous benefits when given solely or part of immunomodulating nutritional formulas [565, 592]. Nevertheless, these nutrients showed limited and incomplete activity in various contexts [588, 912, 913]. This may be in part is due to these nutrients were given in suboptimal concentrations. A further investigation into using high concentrations of these nutrients in PF is warranted. Additionally, curcumin is another nutritional supplement that has been shown to have immunomodulating activities in pre-clinical and clinical studies [642]. In IBD, curcumin possess numerous potential therapeutic benefits, particularly when given in combination with other conventional drugs

[914]. Therefore, it has been proposed that curcumin may be an additional nutritional supplement that could be used to enhance the anti-inflammatory properties of PF.

However, it is not yet clear how best to utilise this nutritional supplement. Previously, using three human intestinal cell lines, HCT 116, HT29 and Caco2 cells, it was shown that curcumin pre-treatment inhibited and modulated gene expression of IL-8 in response to inflammatory cytokine exposure through influencing NF-ț%DFWLYLW\[638, 915]. However, it was not investigated whether curcumin had similar effects if given simultaneously with inducers of inflammation.

4.2 Hypotheses

The primary hypothesis is that immunomodulatory activity of curcumin in vitro culture is determined by its concentration and not timing of exposure to an inflammatory stimulus. A further hypothesis is that manipulating the concentrations of the two active components of

PF (glutamine and arginine) could improve its anti-inflammatory properties, which can be then further amplified with the addition of curcumin.

4.3 Aims Using intestinal epithelial cells cultured in vitro to:

1. Further define the preventative and therapeutic anti-inflammatory properties of curcumin.

2. Investigate the anti-inflammatory properties of glutamine and arginine-enriched PF together with addition of curcumin.

3. Further explore effects of glutamine, arginine and curcumin supplements individually and in combination on the Ițț activity of NF-ț%

4.4 Results

4.4.1 At high concentrations, curcumin reduces epithelial cell viability and activity

HT29 and INT407 cells were cultured until confluence (2.1.1.3). Cells were then exposed to increasing concentrations of curcumin (0, 10, 25, 50, 75 or 100 μM) solubilised in

DMSO and added to culture media, for 24 hours with cell viability measured (2.1.2.1). The final concentration of DMSO for all experiments remained constant at 0.1% v/v. At this concentration DMSO alone did not affect cell viability (Figure 4.1). Increasing concentrations of curcumin, (up to 50 μM), had no significant effect on cell viability, with the viability remaining above 90% for both cell lines (P > 0.05; Figure 4.1). However, curcumin concentrations of 75 μM and above, significantly decreased cell viability

(P<0.001; Figure 4.1). These observations were consistent with the cell activity assay as measured by the MTT assay kit (2.1.2.2). Curcumin, up to 50 μM concentration, showed an increase in the mitochondrial dehydrogenase enzyme activity, whilst cells exposed to 60

μM or higher concentrations of curcumin exhibited a significant drop in the activity as compared to controls (P < 0.001; Figure 4.2). Therefore, for further experimentation, only curcumin concentrations of up to 50 μM were used. A

B

Figure 4.1 Viability assays of curcumin treated HT29 and INT407 cell lines. HT29 (A) and INT407 (B) cells were exposed to curcumin at 10, 25, 50, 75 and 100 μM solubilised in DMSO and incubated for 24 hours. DMSO concentrations were kept constant in all treated cells with final DMSO concentration of 0.1% v/v to the media. Cell viability was measured by a haemocytometer and a light microscope using trypan blue. Dots represent the mean and SEM of 4 replicates for each group. Curcumin of 10, 25 and 50 μM in both cell lines maintained the cell viability curve above 90% and incomparable to the control group (P > 0.05). In contrast, cells exposed to 75 and 100μM in the both cell lines showed a significant drop in the cell viability (P < 0.0001 of 75 and 100 μM as compared with 0 μM curcumin group (DMSO only) using one way ANOVA test followed by Fischer least significance). Figure 4.2 MTT cellular activity assay of HT29 cell treated with increasing concentrations of curcumin. Cells were grown until confluence then exposed to curcumin at 0, 20, 40, 50, 60, 75 and 100 μM solubilised in DMSO and incubated for 24 hours. DMSO concentrations were kept constant in all curcumin exposed cells with final DMSO concentration of 0.1% v/v in the media. Following incubation the media was replaced with phenol red free media and MTT substrate. Dye was then dissolved and the absorbance read at 570 nM using micro-plate reader. Data presented with mean and SEM of 4 replicates for each group. Curcumin at 60 μM and above significantly reduced the cellular activity compared to the control (0μM curcumin) P (*) = 0.001, (**) = 0.0002, analysis by one way ANOVA test followed by Fischer least significance}. 4.4.2 Curcumin blocks IL-8 production from TNF-ĮH[SRVHGLQWHVWLQDOHSLWKHOLDOFHOOV

To investigate the dose-response of curcumin, HT29 and INT407 cells were seeded in 24- well plates and grown to confluence (2.1.1.3) then exposed to 10, 25 and 50 μM curcumin and TNF-Į DQG LQFXEDWHG IRU  RU  KRXUV ,Q DGGLWLRQ WR LQYHVWLJDWH ZKHWKHU SUH- incubation with curcumin alters the response to TNF-Į   DQG  —0 RI FXUFXPLQ were administered to cells for 24, 6 and 1 hour, then cells were exposed to TNF-Į DQG incubated for a further 6 hours. For all experiments, negative controls (cells with media only and no TNF-Į  DQG SRVLWLYH controls (cells exposed to TNF-Į RI QJPO  ZHUH included. In addition DMSO controls (cells exposed to 50ng/ml TNF-Į DQG  YY

DMSO) were also included. Following incubation cell supernatants were analysed for IL-8 by ELISA (2.1.3.3). Curcumin at the lowest concentration (10 μM) did not reduce the IL-8 response to TNF-Į P > 0.05 treated groups vs. positive control group; Figure 4.3A).

Further, pre-incubation did not change the cellular response to 10 μM curcumin (Figure

4.3A). However, at higher concentrations (25 and 50μM), curcumin did significantly reduce the amount of IL-8 released from HT29 cells in response to TNF-Į 3 < 0.01;

Figure 4.3B and C). Similarly, pre-incubation did not change the cellular response to curcumin (Figure 4.3B and C), but the concentration did alter IL-8 levels (Figure 4.4D).

In INT407cells, low concentrations of curcumin (10 and 25μM) had no suppressant effect on IL-8 production; conversely there was a rise in the IL-8 levels (Figure 4.4A and 4.4B).

However, curcumin at 50μM significantly reduced IL-8 production in response to TNF-Į

(P <0.001 vs. positive control; Figure 4.4C). Similar to HT29 cells, pre-incubation of cells with curcumin did not influence the cellular response to curcumin (P > 0.05 for all comparisons; Figure 4.4C); however, the curcumin concentration did alter IL-8 levels

(Figure 4.4D). DMSO alone at 0.1% v/v had no measurable effect on IL-8 production in either of the two cell lines (Figure 4.3 and 4.4). (A) (B)

(C) (D)

Figure 4.3 Effect of curcumin on IL-8 production from HT29 cells in response to TNF-Į HT29 cells were grown to confluence and pre-treated with varying concentrations of curcumin (10 [A], 25 [B] and 50 [C] μM) for varying times (24, 6, 1 and 0 [D] hrs). TNF-Į ng/ml) was added at time 0 and incubated for a further 6 hours. Supernatants were collected and assayed for IL-8 by ELISA. The reduction in IL-8 levels was dependent on the given concentration of curcumin, but not on timing of pre-incubation.{Analysis of data was conducted using one way ANOVA test followed by Fischer least significance P(*) < 0.01; (**) < 0.001; (ns) > 0.05 vs. positive control group)}. (A) (B)

(c) (D)

Figure 4.4 Effect of curcumin on IL-8 production from INT407 cell line in response to TNF-Į INT407 cells were grown to confluence and pre-treated with varying concentrations of curcumin 10 [A], 25 [B] and 50 [C] μM) for varying times (24, 6, 1 and 0 [D] hrs). TNF-Į ng/ml) was added at time 0 and incubated for a further 6 hours. Supernatants were collected and assayed for IL-8 by ELISA. Lower concentrations of curcumin had no effect in suppressing IL-8 production, rather may enhance the production; however, at 50 μM concentration regardless duration of pre- incubation, curcumin prompted significant reduction in the IL-8 level.{Analysis of data was conducted using one way ANOVA test followed by Fischer least significance (P (*) < 0.001; (ns) > 0.05, (**) < 0.01 vs. positive control group)}. 4.4.3 &XUFXPLQVXSSUHVVHV,ț%GHJUDGDWLRQLQLQWHVWLQDOHSLWKHOLDOFHOOV

To continue the investigations of curcumin and to determine whether curcumin was suppressing IL-8 through interaction with the NF-ț%SDWKZD\+7ZHUHVHHGHGLQD- wells plate. Cells were grown for 5 days before treated with 50 μM curcumin for 1 hour before or at the same time as administration of 50ng/ml TNF-Į &XUFXPLQ WUHDWHG FHOOV were then incubated with TNF-Į IRU   DQG  PLQXWHV )ROORZLQJ LQFXEDWLRQ FHOO lysates were collected and western blotting was performed on the lysates as detailed previously (2.1.4). Membranes were probed with the primary antibodies rabbit anti-,ț%RU anti-SKRVSKRU\ODWHG,ț% 1:1000 dilution) for overnight incubation at 4°C. Subsequently, blots were incubated with the secondary antibody goat anti-rabbit IgG (1: 25,000 dilution) and visualised by chemiluminescence.

The addition of TNF-ĮWR+7-FHOOVFDXVHGUDSLGGHJUDGDWLRQRI,ț%VHHQDWDQG

PLQXWHV ZLWK  ,ț% OHYHOV LQFUHDVLQJ DW  PLQXWHV Figure 4.5). This is consistent with

DSSHDUDQFH RI SKRVSKRU\ODWHG ,ț% DW  PLQXWHV DOWKRXJK SKRVSKRU\ODWHG ,ț% LV EDUHO\ detectable at 15 and 30 minutes (Figure 4.5). For curcumin treated cells (1 hour or no pre-

LQFXEDWLRQ WKHUHZDVPLQLPDOHYLGHQFHRI,ț%ORVVRUDSSHDUDQFHRISKRVSKRU\ODWHG,ț%

WKHUHLVHYLGHQFHRIDFFXPXODWLRQRI,ț% )LJXUH4.5). Figure 4.5 Effect of curcumin pre-LQFXEDWLRQRQ,ț%UHVSRQVHVLQ71)-ĮH[SRVHG+7FHOOV Confluent cells were treated with DMSO dissolved curcumin either for a 1 hour pre-incubation or at the same time as TNF-Į exposure. Following TNF-Į ng/ml) exposure cells were incubated IRUDQGPLQXWHVWKHQFHOOO\VDWHVZHUHFROOHFWHG,ț%OHYels in lysates was analysed by Western blot using anti-,ț%  .'D  DQG DQWL-SKRVSKRU\ODWHG ,ț%  .'D  DQG WRWDO SURWHLQ levels assessed with anti-ȕ-actin antibodies (40KDa). Curcumin treatments with or without pre- LQFXEDWLRQEORFN,ț%SKRVSKRU\ODWLRQDnd thereby its degradation in TNF-ĮVWLPXODWHG+7FHOOV 4.4.4 Novel formula completely blocks IL-8 production in response to TNF-Į stimulation

In the previous chapter, enrichment of an existing PF resulted in a dose-response reaction that progressively inhibited the IL-8 response in TNF-ĮWUHDWHGHSLWKHOLDOFHOO+RZHYHUWKH highest level of glutamine/arginine enrichment is not a physiologically viable in vivo option as the PF becomes overloaded with protein. Therefore, to determine an optimal

‘physiologically viable’ enriched PF, lower glutamine and arginine concentrations, that could be used to physiologically enrich PF, were tested. For this investigation HT29 cells were plated in a 24 well plate and grown until confluence (2.1.1.3). Cells were exposed to

50ng/ml TNF-ĮDQGJOXWDPLQHDQGDUJLQLQHDWWKHIROORZLQJFRQFHQWUDWLRQV

50/15, 50/20, 50/25, 50/30 and 240/50 mM. Parallel negative control cells (cultured with media only) and positive control cells (exposed to 50ng/ml TNF-Į LQ PHGLD  ZHUH included. After 6 hours incubation, supernatants were collected and analysed for IL-8 by

ELISA as detailed earlier (2.1.3.3). As previously shown standard PF considerably reduced

IL-8 production from HT29 cells in response to TNF-Į VWLPXODWLRQ 3 < 0.0001; Figure

4.6). Glutamine concentrations were kept constant at 50 mM but arginine concentrations were increased from 2 to 50 mM. There was a progressive reduction in IL-8 levels with increasing arginine concentrations that plateaued at 20 mM (Figure 4.6).This represented the optimal physiologically viable glutamine/arginine concentrations as further suppression of IL-8 could only be achieved by increasing the glutamine and arginine concentrations to levels that compromise safety of the therapy (Figure 4.6). In the next experiment, we wanted to determine if the addition of curcumin to physiologically viable enriched PF further suppressed the IL-8 response to TNF-Į)RUWKLV experiment HT29 cells were exposed to TNF-Į DQG WUHDWHG ZLWK HLWKHU VWDQGDUG 3) curcumin (50 μM) alone, glutamine (50 mM) and arginine (20 mM) enriched PF or glutamine (50 mM), arginine (20 mM) enriched PF with curcumin (50 μM) (Novel PF).

When curcumin was added to the enriched-PF, IL-8 levels were completely abrogated such that there was no significant difference in IL-8 compared to the negative control cells (P =

0.19; Figure 4.7). Figure 4.6 Effect of standard PF versus PFs enriched with increasing glutamine and arginine concentration on IL-8 production from TNF-Į H[SRVHG +7 FHOOV Cells were grown to confluence and then treated with either standard PF or glutamine and arginine-enriched PFs (glutamine to arginine: 50/2, 50/10, 50/20, 50/25, 50/30 and 240/50 mM). Simultaneously, TNF-Į (50ng/ml) was added at time 0 and incubated for 6 hours with IL-8 measured in the supernatants. With glutamine concentrations constant at 50 mM, increasing the arginine concentration of PF resulted in further reduction in IL-8 with the optimal ‘physiological enrichment of 50 mM glutamine, 20 mM arginine. Further reduction in IL-8 could only be achieve with the non- physiological concentrations of 240mM glutamine and 50mM arginine{P (ns) > 0.05, (*) < 0.05, (**) < 0.01 and (***) <0.001 vs. standard PF}. Analysis conducted using one-way ANOVA followed by Fischer least significance test. (Neg): negative control (neither treatment nor TNF-Į  (Pos): positive control (only TNF- Į  (PF): standard PF. (50/2, 50/10, 50/20, 50/25, 50/30 and 240/50) glutamine to arginine in mM concentrations of new PFs.

Figure 4.7 Effect of standard PF versus novel formula on IL-8 production from TNF-Į exposed HT29 cells. Cells were grown to confluence and then treated with either standard PF or glutamine and arginine-enriched PF with or without curcumin; or curcumin alone. Simultaneously, TNF-Į ng/ml) was added at time 0 and incubated for a further 6 hours. Supernatants were collected and assayed for IL-8 by ELISA. Data presented with mean and SEM of 4 replicates for each group. All treatments significantly reduced IL-8 level in response to TNF-Į 3 0.0001 treatments vs. positive control group). However, only the novel formula treatment (glutamine/arginine enriched PF with curcumin), but not enriched PF nor curcumin treatment groups completely blocked IL-8 level (P (ns)> 0.05, (*) < 0.01 and (**) < 0.001, respectively versus negative control group. Analysis of data was conducted using one-way ANOVA followed by Fischer least significance test. (Neg): negative control (neither treatment nor TNF-Į  Pos): positive control (only TNF- Į  (Standard PF): standard PF (12.7/1.8 glutamine to arginine concentrations in mM). (Curcumin): curcumin (50μM) was dissolved first in PF and then was added to media to give 0.1% v/v PF final concentration in media. (Enriched- PF): glutamine and arginine-enriched PF (50/20 glutamine to arginine concentrations in mM). (Novel formula): combination of enriched-PF and curcumin. 5.4.5 Novel formula maintains the viability and enhances the activity of intestinal epithelial cells

To exclude the possibility that diminished IL-8 levels was a result of cell death; cell viability experiments of cells exposed to the novel formula were conducted. HT29 cells were treated with either standard PF in a 1 to 5 dilution in media as previously described to give final concentrations of 12.7 mM glutamine and 1.8 mM arginine; or treated with PF enriched with increasing concentrations of glutamine and arginine as follows: (50/1.8,

50/10, 50/15, 50/20, 50/25, 50/30 and 240/50 mM), curcumin (50 μM) alone or novel formula for 24 hours. Following incubation cell viability was determined by trypan dye exclusion test (2.1.2.1). None of the treatments tested had any significant effect on cell viability following 24 hours of cell culture (P > 0.05; Figure 4.8).

In the next experiment was determined whether standard PF, enriched- PF, novel formula

(combination of curcumin with glutamine and arginine-enriched PF) or curcumin had an effect on cell activity. HT29 cells were exposed to treatments for 24 hours and cellular activity measured by the mitochondrial dehydrogenase enzyme activity assay (2.1.2.2).

Interestingly, the activity was significantly lower in the standard PF treated group compared to control group (P = 0.002), however, there was no statistical significant variation in cellular activity for any other treatments compared to the control group (P >

0.05; Figure 4.9). A

B

Figure 4.8 Viability assays of HT29 cells incubated with different treatments including the novel formula. Cells were incubated for 24 hours with either standard PF or PF with increasing glutamine and arginine concentrations, or curcumin alone (50 μM) alone or novel formula for 24 hours. Cell viability was assessed using Trypan blue exclusion. Data presented as mean and SEM of four replicates for each group. There was no significant difference in the cell viability for any of the treatments shown (P >0.05 using one-way ANOVA (–): negative control (no treatment). (Standard PF): standard PF (12.7/1.8 glutamine to arginine concentrations in mM). (50/2, 50/10, 50/20, 50/25, 50/30 and 240/50) glutamine to arginine in mM concentrations of new PFs. (Curcumin): curcumin (50μM) was dissolved first in PF and then was added to media to give 0.1% v/v PF final concentration in media. (Enriched- PF): Enriched PF (50/20 glutamine to arginine concentrations in mM). (Novel formula): combination of enriched-PF and curcumin. Figure 4.9 MTT cellular activity assay of HT29 cell treated with various treatments including the novel formula. HT29 cells were grown until confluence and then treated with standard PF, enriched PF, curcumin and novel formula for 24 hours. Old media were replaced with a mixture of phenol red free media and MTT substrate. Dye was dissolved in DMSO and the absorbance read at 450nM using micro-plate reader. Data presented as the mean and SEM of 4 replicates for each group. Enriched-PF, curcumin and novel formula treated groups showed no significant drop in the cell activity compared to control group (P > 0.05 in the treated groups vs. untreated control group) whereas standard PF had significantly lower activity than control group (P (*) < 0.01). Statistical analysis of data carried out using one way ANOVA test followed by Fischer least significance). (Control): negative control (no treatment). (Standard PF): standard PF (12.7/1.8 glutamine to arginine concentrations in mM). (Curcumin): curcumin (50 μM) was dissolved first in PF and then was added to media to give 0.1% v/v PF final concentration in media. (Enriched- PF): glutamine and arginine-enriched PF (50/20 glutamine to arginine concentrations in mM). (Novel formula): combination of enriched-PF and curcumin (50 μM). 41RYHOIRUPXODFRPSOHWHO\DEROLVKHV,țțDFWLYLW\

To determine if inhibition of IL-8 by the novel formula was a direct result of suppressing the NF-ț%SDWKZD\WKHFRPELQDWLRQRIJOXWDPLQHDUJLQLQHDQGFXUFXPLQZDVWHVWHGZLWK the IțțDVVD\The Ițțassay was employed as previously detailed (2.1.7.2). Glutamine and arginine were dissolved directly in kinase buffer and then added to the reaction buffer to give 12 mM (Glu 1) and 50 mM (Glu 2) concentrations for glutamine; and 2 mM (Arg 1) and 20 mM (Arg 2) concentrations for arginine. K252a and curcumin, at the concentration in the novel formula, were dissolved first in DMSO then added to the kinase buffer 10 mM and 50 μM respectively. Glutamine and arginine, in the all tested concentrations significantly reduced IțțDFWLYLW\ )LJXUH4.10). However, curcumin at 50 μM suppressed

,țț activity the most compared to all of the individual supplements. Importantly, the combination of glutamine 50 mM, arginine 20 mM and curcumin 50 μM showed a complete suppression in the IțțFRPSOH[DFWLYLW\ 3 = 0.66 vs. K252a; P = 0.10 vs. no Ițț control; Figure 4.10).

Figure 47KUHHDFWLYHFRPSRQHQWVRI1RYHOIRUPXODFRPSOHWHO\VXSSUHVV,țțDFWLYLW\The ,țț assay was conducted in 96-well ELISA plate pre-FRDWHGZLWKUHFRPELQDQW,ț%Į Reaction was initiated by adding ATP to the IUDFWLRQDWHG,..ȕVXEXQLW(IKK). No enzyme control (no IKK) and synthetic inhibitor control at 10 mM concentration (K252a) were included. Glutamine and arginine were dissolved directly in the kinase buffer. Final concentrations of glutamine were 12 mM reflecting its concentration in PF (Glu 1) and 50 mM representing novel formula concentration (Glu 2). Likewise, for arginine 2 mM (Arg 1) and 20 mM (Arg 2), respectively. Curcumin was dissolved first in DMSO and then added to the reaction buffer to give final concentration of 50μM (cur), representing novel formula concentration. Optical density measured by microplate reader and the activity assessed. Results presented as raw data showing generated SKRVSKRU\ODWHG ,ț% $  DQG ,țț DFWLYLW\ UHODWLYH WR SRVLWLYH FRQWURO %  (DFK RI JOXWDPLQH DQG arginine at their concentrations of PF considerably reduced kinase activity; however they exhibited further significant reduction in the activity when tested at their concentrations equal to novel formula. Curcumin also showed significant effect against kinase complex activity. More interesting, the combination of glutamine, arginine and curcumin (Glu 2/Arg 2+Cur) resulted in completely abolishing the kinase activity (P=0.66 vs. K252). Analysis of data was conducted using one-way ANOVA test followed by Fischer least significance post hoc test (P (*) < 0.01, (**) YV,țțFRQWURO 4.5 Discussion

These in vitro investigations demonstrate that curcumin can prevent the TNF-ĮPHGLDWHG production of IL-8 in human intestinal epithelial cells. Further, this inhibition of IL-8 is similar whether curcumin was introduced prior to or at the same time as TNF-Į stimulation. These results indicated that curcumin exerts an anti-inflammatory activity by

LPSDLULQJ WKH GHJUDGDWLRQ RI ,ț% WKHUHE\ PRGXODWLQJ WKH 1)-ț% VLJQDO WUDQVGXFWLRQ pathway. This work has also shown that altering glutamine and arginine concentrations in standard PF enhances the anti-inflammatory activity of the PF in vitro. This increased anti- inflammatory activity was achieved without any detrimental effects on the activity and/or viability of the intestinal epithelial cells. Moreover, addition of curcumin to glutamine and arginine-enriched PF further enhanced the anti-inflammatory activity and resulted in complete inhibition of IL-8 production in vitro. This anti-inflammatory activity can be attributed, to a certain extent, to glutamine and arginine suppressing ,țț complex activity and this suppression can be further amplified in combination with curcumin. Therefore by combining all these elements together, a novel formula can completely abrogate the IL-8 response to TNF-Į in vitro, and can be constructed that is potentially physiologically viable in vivo.

These investigations indicate that increased amino acid concentration in formulas may be beneficial. However, one limitation is that the high protein content of formulas may affect nutritional value. Based on the Schofield equation [916], which estimates basal metabolic rate from weight, the requirements of standard PF are translated to 40 ml (1.76 g protein) per kg body weight per day to give 35-40 kcal/kg body weight a day. Therefore protein intake can be increased up to 4-5 g per kg body weight per day in individuals receiving

2300 Kcal daily [917]. This is equivalent to the 40 ml of PF received per kg body weight per day, and therefore the formula can be further fortified with glutamine and arginine to an upper limit of 5 g protein (equivalent to 350 mM of amino acid). We estimate there is an effective 1 to 5 dilution of formula in the intestine [707]. Therefore 350 mM amino acid in formula would be diluted to 70 mM amino acid in the intestinal lumen; therefore the combination of 50 mM glutamine and 20 mM arginine seems the highest practice amino acids concentrations that would be considered for designing a new therapy. Interestingly, preliminary results shown in the previous chapters indicate these lower concentrations are less efficient in reducing the inflammatory response and blocking the NF-ț% pathway.

Thus, for development of a novel formulation, the addition of glutamine and arginine at these sub optimal concentrations would result in a formulation that does not completely inhibit the inflammatory response. To ensure a formula with maximal efficacy and optimum safety, the addition of further pharmaconutrients may be required. One proposed nutrient was curcumin, which has potential therapeutic implications in IBD [918].

The results in this chapter also indicated there is a minimal benefit with curcumin pre- incubation. However, it was clear that there was greater effect with increasing curcumin concentration regardless of pre-incubation time. Curcumin either given before or at the same time as an inflammatory stimulus was shown to attenuate pro-inflammatory cytokine production and modulate NF-ț% DFWLYLW\ UHSUHVHQWHG E\ LQKLELWLRQ RI ,ț% degradation.

This suggests that curcumin may be effective for both preventative and concurrent therapy. More interestingly, the addition of curcumin to glutamine and arginine-enriched PF augmented the anti-inflammatory properties of the standard PF resulting in complete abrogation in the expression of IL-8 production from intestinal epithelial cells in vitro.

This combination of nutrients may therefore have the potential to improve efficacy of the nutritional therapy.

The potential mechanism by which these nutrients suppress IL-8 production was also investigated. The NF-ț%VLJQDOWUDQVGXFWLRQSDWKZD\LVDQLPSRUWDQWUHJXODWRURIF\WRNLQH transcription in intestinal epithelial cells [68] and IL-8 production is tightly regulated by this pathway [919]. An important step in this signal transduction pathway is

SKRVSKRU\ODWLRQ RI ,ț% E\ WKH SURWHLQ NLQDVH FRPSOH[ ,țț [76]. De Jong et al [707] investigated a link between PF treatment and NF-ț%LQKLELWLRQLQFRORQLFHSLWKHOLDOFHOOV

DQGGHVFULEHGDGHOD\LQWKHGHJUDGDWLRQRIWKH,ț%VXEXQLWLQWKHSUHVHQFHRI3) In the current study, a kinase assay was employed to isolate one essential step of the NF-ț% signal transduction pathway and investigate the effect of the nutrients on this step. The results show that the nutrients have direct anti-inflammatory activity through inhibition of an essential step of the NF-ț%SDWKZD\ Both glutamine and arginine at the concentrations seHQ LQ VWDQGDUG 3) SDUWLDOO\ DWWHQXDWHG ,țț FRPSOH[ DFWLYLW\ 7KLV ILQGLQJ VXSSRUWV WKH hypothesis that both amino acids contribute to the anti-inflammatory properties of PF.

Moreover, when higher concentrations of glutamine and arginine were added together with

FXUFXPLQWKHVHWKUHHFRPSRQHQWVFRPSOHWHO\DEURJDWHGSKRVSKRU\ODWLRQRIWKHȕ-subunit

RIWKH,țțFRPSOH[UHVXOWLQJLQFRPSOHWHLQKLELWLRQRIWKHNLQDVHDFWLYLW\ Standard PF is considered a safe therapy and is currently utilized in many IBD clinics with no major adverse effects [460]. Although the in vitro model utilised in the current study is unable to ascertain tolerance in vivo, these results indicate no direct toxicity upon epithelial cells. De Jong et al. [707] in their previous work showed that standard PF has no measurable effect on the intestinal cell viability when cells were incubated for 18 hours with PF at different dilutions, including the current used dilution (1 in 5 to the media).

Here, using trypan blue exclusion, both standard PF and enriched PF together with added curcumin showed no significant drop in the viability compared to the control group.

Moreover, and more interestingly, the standard PF prompted a significant drop in cellular activity whereas the cellular activity of cells treated with enriched PF in presence of curcumin remained equivalent to untreated cells. Thus, the current preliminary observations of viability and activity indicate that the new combination of nutrients has no adverse effects on cells.

However, it is important to note that these experiments suggest that curcumin may have a narrow safety window. Cells treated with curcumin concentrations greater than 50 μM had a significant drop in cell viability that was in parallel with the major decrease in the mitochondrial dehydrogenase enzymes activity of treated cells. In contrast, one clinical trial has reported patients were able to tolerate up to 8 grams of curcumin daily for 3 months with no reported toxicity [920]. This apparent discrepancy is likely due to the poor pharmacokinetic properties of curcumin including very low bioavailability, poor absorption and rapid metabolic elimination [921] when curcumin is delivered in the absence of a vehicle. In one study it was found that the highest achieved peak serum concentration of curcumin in the peripheral blood was only 139 nM, measured 4 hours after a single oral dose of 12g curcumin [922]. Further, limited transepithelial flux and rapid metabolism in the gut epithelium are likely to impair curcumin exerting its beneficial effect locally on gut epithelium [923]. In our in vitro experiments curcumin was delivered in DMSO in the initial experiment and in PF (containing oils that increase the solubility of curcumin) in the later experiments. Both of these delivery methods would enhance delivery of curcumin to the cell cytoplasm compared to curcumin added in the absence of a vehicle.

These may explain why curcumin has shown better outcomes in both in vitro and ex-vivo settings than in clinical trials and may also explain the lack of toxicity despite high doses.

Therefore, to improve clinical outcomes and to fully utilize the properties of this agent, future clinical application of curcumin may benefit from further investigation of curcumin solubility and targeted-delivery of curcumin to the colon. However, improving delivery in vivo must also be done cautiously and with concurrent investigation of cell toxicity.

Collectively, the novel formula can be considered as a potentially safe nutritional therapy and possesses enhanced activity over the standard PF. However, further investigations warranted to validate these preliminary observations in vivo setting. There are a limited number of studies that compare standard formulas versus enriched diet in IBD patients and did not show any evidence that supported the use of an enriched nutritional diet over a standard diet [912, 924]. Akobeng et al. [912] performed a clinical trial in which the efficacy of a glutamine-enriched polymeric diet was compared with a standard low- glutamine polymeric diet in treating active CD patients. The results showed no difference between the two groups in the proportion of patients achieving remission. The authors also showed that glutamine-enriched formula is less effective in reducing disease activity [912].

Further, in a trial involving 24 patients with IBD who were scheduled to receive either standard total parenteral nutrition (TPN) or glutamine-enriched TPN, glutamine supplementation did not make any significant difference in the inflammatory or nutritional parameters between the two groups [588]. It is important to note that in these trials only the glutamine component was enhanced and in lower concentration than that utilized in the present work. Here, both glutamine and arginine components of PF were fortified at levels much higher than previously utilized together with addition of curcumin.

Although clinical trials of glutamine-enriched formula have not supported glutamine supplementation in CD, in experimental colitis models administration of glutamine- enriched enteral diet has shown promising results [925, 926]. In a chemically induced colitis model, rodents received either control diet or control diet fortified with low or high glutamine. Severity scores of colonic damage and the level of inflammatory cytokines

(TNF-Į DQG ,/-8) were lower in the glutamine groups than control group [773]. These measures were lower in the high glutamine group compared to low glutamine diet group

[773]. Similarly, in a mouse model of colitis, glutamine-enriched diet significantly reduced gene expression of pro-inflammatory cytokines and chemokines compared to the control diet [927]. Glutamine-supplemented enteral diet also improved nutritional status, ameliorated intestinal injury, reduced bacterial translocation, and enhanced survival rates of rats subjected to methotrexate-induced enterocolitis [928]. Several studies utilizing an arginine-enriched nutritional diet have also supported dietary arginine supplementation [929, 930]. Arginine-fortified diet improved survival rates in mice subjected to gut derived sepsis [931], and was more efficient in inducing mucosal damage recovery and enhancing bacterial clearance than a low arginine control diet in a rodent model of radiation enteritis [592]. Further, arginine-enriched TPN reduced TNF-Į

IL-ȕ DQG ,/-6 and enhanced cellular immunity after celiac ligation induced injury in septic rats [810]. Similar effects were seen also in LPS exposed macrophages cultured from diabetic rats treated with increasing arginine concentrations when high arginine diet showed better outcomes in suppressing inflammation compared to low arginine [815].

Additionally, advantages of curcumin-fortified diets were also documented in preclinical studies involving IBD models [932-934]. Nones et al. [932] using a spontaneously IBD model in multi-drug resistant deficient mice, showed that a curcumin-fortified diet significantly reduced the histological signs of colonic inflammation that was consistent with down-regulating proinflammatory pathways and up regulating protective pathways.

Villegas et al. [778] in a chemically induced mouse model of colitis, also showed that curcumin fed animals, but not control diet fed animals, experienced a significant decrease in the production of TNF-Į DQG ,1)-Ȗ LQIODPPDWRU\ F\WRNLQHV DQG D GHFUHDVH LQ expression of other inflammatory proteins.

Thus, along with the current findings, it appears that optimising and combining anti- inflammatory components in nutritional formulations is a viable option to enhance efficacy of nutritional therapy and requires further investigations. 4.6 Conclusions

Manipulating the glutamine and arginine components of standard PF enhanced the anti- inflammatory properties of currently used polymeric diet for CD in an in vitro model of

IBD. Enriched-PF was superior to standard PF in ameliorating TNF-Į LQGXFHG inflammatory response in the intestinal epithelial cells. This study also adds support that curcumin has the potential to induce and maintain remission of intestinal inflammation.

Curcumin whether given before or at the same time of inflammatory stimuli was shown to attenuate pro-inflammatory cytokine production and modulate ,ț% degradation of NF-ț%

Further, adding curcumin to the enriched-PF, to create a ‘novel formula', completely inhibited pro-inflammatory cytokine production in response to inflammatory stimuli without having a negative impact on both cell viability and activity. That effect in part is mediated through interfering with phosphorylation of IțțɅ subunit of NF-ț% E\ integration of the three active candidates of the novel formula (glutamine, arginine and curcumin). Similar mechanism can be attributed to the standard PF when glutamine and arginine candidates partially inhibited activity of the IțțFRPSOH[ZKLFKIRUWKHILUVWWLPH provides indirect evidence proofs the linkage between NF-ț%DQGWKHIRUPXla’s immune effects. Definitive preclinical studies involving murine colitis models are now required to establish the potential therapeutic effects of the novel therapy in ameliorating intestinal inflammation in vivo setting. Chapter 5: Exploring effectiveness of the novel nutritional therapy in two murine models of colitis.

5.1 Introduction

In the previous chapter (4) a novel PF was developed as a potential treatment for CD. This novel formula is composed of standard PF with altered glutamine and arginine concentrations together with addition of curcumin to the formula. In an in vitro cell culture model with TNF-ĮVWLPXODWHGFRORQLFHSLWKHOLDOFHOOVWKHQRYHO3)H[KLELWHGVXSHULRUDQWL- inflammatory activity compared to standard PF, as it completely suppressed pro- inflammatory cytokine production from the colonic epithelial cells without influencing cell viability or activity. In order to validate and support these preliminary observations, further investigation of the novel PF in pre-clinical studies using murine colitis models is required.

5.2 Hypotheses

That the novel formula will attenuate the induced inflammatory response, improve the local histological intestinal injury of experimental mice and will enhance recovery. The second hypothesis is that the novel formula is potentially more effective than standard PF in reversing inflammatory changes in murine models of colitis.

6.3 Aims 1. To investigate potentiality of the novel formula in ameliorating chemically induced intestinal inflammation in murine models of intestinal inflammation.

2. To explore whether the novel formula has superior effectiveness over the standard PF in normalizing the inflammatory profile and healing damaged tissue colon in murine models of intestinal inflammation. 5.4 Results

5.4.1 Effect of novel formula in the murine TNBS induced colitis model

In order to investigate the therapeutic potential of the novel formula, colitis was induced in

Balb/C mice using TNBS, as previously detailed (2.2.3.1). In brief, mice were received

TNBS dissolved in ethanol per rectum, and then were immediately fed either normal chow

(positive control); or standard PF or novel formula for 1 week. Novel formula is comprised of standard PF with glutamine and arginine at concentrations of 250 and 100 mM respectively, plus curcumin added to a final concentration of 250 μM (2.2.4). Parallel negative control group was also included where mice just had per rectal injection of ethanol without TNBS and put on a diet of normal mouse chow. Body weight was recorded and colon inflammation quantitated using RT-PCR (2.2.5).

5.4.1.1 Balb/C mice showed no significant drop in survival curve after TNBS injection.

Two out of 12 mice in the positive control group died within the first 48 hrs due to TNBS toxicity resulting in 16.6% mortality rate versus 0% in negative control group at the end of the experiment (Figure 5.1). Survival curve analysis using Kaplan Meier log rank test showed that this drop in survival rate was not significant (P > 0.05). Similarly, the two groups that received TNBS, but treated with either standard PF or novel formula, had also

2 deaths in each experimental group. Mortality rate again among treatments remained insignificant compared to control groups (P > 0.05; Figure 5.1). Figure 5.1 Survival rates of Balb/C mice treated with or without standard PF or novel formula following TNBS. Mice were randomized into 4 groups with 12 each. Control group (negative) in which mice had only ethanol injection without TNBS, whereas other groups had ethanol dissolved TNBS enema. Of TNBS groups, one group of mice continued on a normal mouse pellet diet (positive) with the other two groups treated with standard polymeric formula (standard PF) or the novel formula (novel formula) for 1 week. TNBS injection was designated as day 0. No significant drop in the survival curve was noted between groups by Kaplan Meier log rank test; P#>0.05 vs. negative control. 5.4.1.2 Novel formula prevented TNBS- induced weight loss in Balb/C mice.

In the positive control group following TNBS administration, mice lost weight to a mean of 83% of original body weight within the first 24 hours (P < 0.05 vs. negative control group) (Figure 5.2). The TNBS-induced weight loss continued for 3 days before weight began to recover, however weight was still below initial weight by day 7 (Figure 5.2). In contrast, PF fed mice exhibited only partial weight loss in response to TNBS, with a fall to a mean of 93% of original body weight at day 1 (Figure 5.2). However, weight loss was statistically insignificant compared to negative control group (P > 0.05). Weight in the standard PF group started rising by day 2 and remained not statistically different to negative control group (Figure 5.2). Novel formula treated mice continued to lose weight for the first 2 days that dropped significantly to a mean of 87 % of original body weight (P

< 0.05 vs. control group; Figure 5.2). Weight began to rise by day 3 with mice showing substantial weight gain such that weight was not statistically different to negative control at the end of the experiment (P > 0.05 vs. control group; Figure 5.2).

Figure 5.2 Body weight changes in Balb/C mice treated with or without standard PF or novel formula following TNBS. Mice were randomized into 4 groups. Control group (negative) were mice that had ethanol injection without TNBS, whereas other groups had ethanol dissolved TNBS enema. Mice that received TNBS and continued on normal mouse pellet diet were the positive control group (positive) and mice that received TNBS and treated with either standard PF diet (standard formula) or novel formula diet (novel formula) were the two treatment groups. Body weight was recorded daily starting from day 0 (TNBS given) till collection at day 7. Standard PF showed a considerable effect in preventing TNBS induced weight changes. By day 3 there was no significant difference in weight between noel formula group and negative control. By day 4 all TNBS injected mice including positive control gained weight and were not significantly different compared to control mice. Data represent body weight changes compared to original weight. Analysis done using one way Anova test followed by post hoc test; Ps (*) < 0.05 vs. negative control; (#) > 0.05 vs. negative control. 5.4.1.3 Balb/C mice did not develop colitis following TNBS.

Colonic tissue was collected at day 7 of the experiment. Tissue was subject to RT-PCR analysis of cytokine mRNA expression including IL-6, IL-12, TNF-ĮDQG0&3-1. There were no significant differences in IL-12, TNF-ĮRU0&3-1 expression in any of the TNBS groups compared to the negative control group (P > 0.05; Figure 5.3). IL-6 expression in the positive group was significantly higher than the negative control group (P < 0.05;

Figure 5.3). In addition, IL-6 in the treatment groups was significantly higher compared to the negative and was not significantly different to the positive control group (P > 0.05;

Figure 5.3). Figure 5.3 Colonic cytokine expressions of Balb/C mice treated with or without standard PF or novel formula following TNBS. Four groups were included, control group (negative) in which mice had ethanol injection without TNBS, whereas other groups had ethanol dissolved TNBS enema. Of the mice that received TNBS, one group continued on a normal mouse pellet diet (positive) one group was treated with a standard PF diet (standard formula) and one group was treated with a novel formula diet (novel formula) for 1 week starting from day 0 (TNBS given). Colonic tissues was collected at the end of the experimental period (7th day of TNBS administration) and stored in RNA later for RT-PCR analysis. Expression of TNF, IL-12 and MCP- 1 were not significantly different in positive control compared to negative control. IL-6 was significantly higher in all TNBS received groups including treatments compared to negative control group. Analysis done using one way Anova test followed by post hoc test; P(*) < 0.05 vs. negative control; (#) > 0.05 vs. negative control;(^) > 0.05 vs. positive control. 5.4.2 Effect of novel formula on DSS induced colitis

The efficacy of the novel formula was also assessed in the DSS mouse model of colitis.

Female C57BL/6 mice were provided with 3% DSS solution in the drinking water for 5 days to induce colitis (2.2.3.2). Following the induction of colitis, mice were exposed to either a regimen of water and normal mouse pellet diet (positive control); or standard PF diet (standard PF) or novel formula diet (novel formula) for 2 weeks. An additional group

(negative control) was included, in which animals were not exposed to DSS and continued on normal water and mouse pellet diet for the duration of the experiment. Consumption of formulas and body weight were recorded on a daily basis. At the completion of the experiment, colon weight and length measurements were taken. Colonic tissue was collected for MPO activity, cytokine expression and histological damage assessment

(2.2.5).

5.4.2.1 Novel formula fed mice had lower intake than standard PF given mice

Average daily consumption per mice was significantly higher in standard PF (10.8 ± 0.1 ml) than in novel formula (9.4 ± 0.2 ml) (P < 0.05; Figure 5.4A). Overall, the intake in novel formula group was 86% of total intake of standard PF fed group (Figure 5.4B).

Nevertheless, there was no significant difference in daily calorie ingestion between the two groups, with 10.9 ± 0.1 calories/mouse/day in standard PF group vs. 10.4 ± 0.2 calories/mouse/day in novel formula group (P > 0.05; Figure 5.4C). Further, all mice remained active and healthy with no reported deaths while on formulas for the 2 week treatment period. A

B

C

Figure 5.4 Average daily intake of standard PF and novel formula in C57BL/6 following DSS. Mice were given 3% DSS in drinking water for 5 days. Mice then received standard PF or novel formula. The intake in novel formula group was significantly lower (86% of total intake of standard PF) in novel formula fed group. However, both groups had similar calorie consumption. Data represent mean with SEM of each group; analysis was done by T test. 5.4.2.2 Novel formula prevented DSS- induced weight loss in C57BL/6 mice

In positive control mice, DSS induced a marked time-dependent weight loss starting from the completion of the 5 days of exposure to DSS, with weight loss peaking on the 6th day following the completion of DSS exposure (P < 0.05 compared to the negative control group; Figure 5.5). Body weight then recovered, however it remained significantly low compared to the negative control mice throughout the experiment (P < 0.05 vs. negative control; Figure 5.5). In contrast, mice that received DSS but were treated with the novel formula, exhibited less change in body weight (Figure 5.5). Weight in this group was significantly higher compared to the positive group from day 3 of treatment and remained higher for the duration of the experiment (Figure 5.5). Mice that received DSS and treated with standard PF showed numerical, but not significant weight loss compared to the negative control group (Figure 5.5). Comparing the two formula groups, there was no significant difference in weight between the two groups throughout the experiment (Figure

5.5). Figure 5.5 Body weight changes of DSS exposed C57BL/6 mice treated with or without standard PF or novel formula. Mice were given 3% DSS in drinking water for 5 days and then either continued on normal mouse pellet diet (positive control); or treated with standard PF or novel formula. Negative control mice were not exposed to DSS and continued on normal mouse pellet diet throughout the experiment. DSS induced a time dependent weight loss. Treatment with standard or novel formula substantially prevented DSS induced weight loss. There was no difference in body weight between the two treatments at any of the time points. Data represent mean of body weight changes for each group on each single day. Ps (*) < 0.05, (**) < 0.01, (***) < 0.001, (****) 0.0001 vs. negative control; (#) < 0.05 novel formula or standard PF vs. positive control; (^) > 0.05 novel formula vs. standard PF. Data were analyzed by one way Anova test followed by post hoc test. 5.4.2.3 Novel formula reversed changes to colon weight over length score in DSS exposed C57BL/6 mice

At completion of the experiment, the colons were collected with weight and length measured. DSS exposure caused colonic length shortening, which decreased from a mean of 6.3 ± 0.1 cm in negative control mice to a mean of 5.4 ± 0.1 cm in positive control mice

(P < 0.05; Figure 5.6A). In standard PF or novel formula groups, colon length also remained significantly lower compared to negative control mice with means of 5.1 ± 0.1 and 5.4 ± 0.1 cm respectively (P < 0.05; Figure 5.6A). Colon weight was increased significantly in positive control mice compared to negative control mice (237 ± 14 mg vs.

157.5 ± 6.5 mg respectively) (P < 0.05; Figure 5.6B). In treatment groups, both standard

PF and novel formula prevented increased colon weight as weight was not significantly different to the negative control group (147.5 ± 9.2 and 152.5 ± 9.2 mg respectively) (P >

0.05; Figure 5.6B). Further, colon weight/length scores increased significantly in positive control mice (P < 0.05 vs. negative control; Figure 5.6), but substantially reduced with standard PF or novel formula treatment (P <0.05 vs. positive control; Figure 5.6C). There was no significant difference in any of the scores between the two treatments (P > 0.05;

Figure 5.6). A B

(C)

Figure 5.6 Colon weight and length score of C57BL/6 mice treated with or without standard PF or novel formula following DSS course. Mice were given 3% DSS in drinking water for 5 days and then either continued on normal mouse pellet diet (positive control); or treated with standard PF or novel formula. Negative control mice were not exposed to DSS and fed only mouse pellet diet. At completion of the experiment colon length (A) and weight (B) measurements were recorded and colon over weight length score (C) calculated. Colon/ weight length score significantly increased following DSS treatment. However, in presence of novel formula or standard PF, score substantially reduced and were similar to negative control mice. There was no difference in any of the scores between treatment groups. Ps (*) < 0.05 vs. Negative control; (#) < 0.05 novel formula or standard PF vs. positive control; (&) > 0.05 novel formula vs. standard PF). Data analyzed by one way Anova test followed by post hoc test. 5.4.2.4 Novel formula inhibited DSS induced colonic proinflammatory cytokine expression in C57BL/6 mice

At completion of the experiment, colon was collected with mRNA extracted and used for pro-inflammatory cytokine gene expression by RT-PCR. DSS-induced colitis was

DFFRPSDQLHGE\DVLJQL¿FDQWXSUHJXODWLRQRIJHQHH[SUHVVLRQRI71)-Į,/-12, IL-6 and

MCP-1 in the colon. There was a 12-fold increase in the expression of TNF-ĮDIWHU'66 exposure (P < 0.05; Figure 5.7). When mice were treated with novel formula or standard

PF, expression of TNF-Į ZDV WZRIROG OHVV WKDQ SRVLWLYH FRQWURO JURXS 3 < 0.05 vs. positive control; Figure 5.7). Additionally, IL-12 expression was increased 14-fold following DSS exposure compared to negative control mice (P < 0.05; Figure 5.7).

However, expression was again considerably reduced (to 5.4 and 4.3 fold respectively) with standard PF or novel formula treatment (P < 0.05 vs. positive control; Figure 5.7).

Exposure to DSS also enhanced IL-6 mRNA expression to 69 times higher than in the negative control mice (P < 0.05; Figure 5.7). With PF or novel formula treatment, there was a marked reduction in the cytokine expression such that IL-6 expression was only 21 and 19 fold higher, respectively, than negative control mice (P < 0.05 vs. positive control;

Figure 5.7). Moreover, DSS also increased the expression of the chemokine MCP-1 to 35 times higher than in the negative control group (P < 0.05; Figure 5.7). Although there was a numerical drop in MCP-1 expression with formula treatment, the level was not significantly different to control levels (P > 0.05 vs. positive control; Figure 5.7).

Importantly, the expression of TNF-Į ,/-12, IL-6 and MCP-1 was not significantly different between the two treatment groups (Figure 5.7). Figure 5.7 Colonic cytokine expression of C57BL/6 mice treated with or without standard PF or novel formula following DSS exposure. Mice were given 3% DSS in drinking water for 5 days and then either continued on normal mouse pellet diet (positive control); or treated with standard PF or novel formula. Negative control mice were not exposed to DSS and continued on normal mouse pellet diet throughout the experiment. At completion of the experiment, colonic tissue was collected for cytokine expression by RT-PCR. Expression of TNF-Į,/-12, IL-6 and MCP-1 was up regulated in response to DSS. Apart of chemokine MCP-1 expression, other measured mediators were significantly reduced with both novel formula and standard PF treatment, but there was no significant difference between the two treatment groups.P (*) < 0.05vs. Negative control; (#) < 0.05 novel formula or standard PF vs. positive control; (^) > 0.05 novel formula or standard PF vs. positive control. Data analyzed by one way Anova test followed by post hoc test. 5.4.2.5 Novel formula attenuated colonic MPO activity of C57BL/6 mice following

DSS exposure

At completion of the experiment, colon was collected to assess MPO activity. MPO activity in the colon of the negative control mice was 368 ± 105 mU/mg of tissue. In response to DSS, the activity significantly increased to 1400 ± 174 mU/mg (P < 0.05;

Figure 5.8). Interestingly, in mice treated with novel formula the MPO activity considerably dropped to 379 ± 103 mU/mg tissue and to 716 ± 194 mU/mg with standard

PF treatment (Ps < 0.05 vs. positive control; Figure 5.8). However, the difference in MPO activity between the two formula treatments was not significantly different (P = 0.12;

Figure 5.8).

5.4.2.6 Novel formula partial ameliorated colonic local injury in C57BL/6 mice following DSS exposure

Colonic tissue was also assessed for histological damage. DSS substantially induced local histological changes in the colon of mice. Histological assessment that included crypt damage score, extent of inflammation score, severity of inflammation and local edema was significantly increased in the positive control group (P < 0.05; Figure 5.9). Formula treatment resulted in only a numerical drop in the total damage scores compared with that

RI PLFH LQ WKH SRVLWLYH FRQWURO JURXS ZKLFK ZDV QRW VWDWLVWLFDOO\ VLJQL¿FDQW (P > 0.05;

Figure 5.9). However, the crypt damage score was significantly lower in the novel formula group and standard PF group fed compared to the positive control group (P < 0.05 treatments vs. positive control; Figure 5.9). 7KHUH ZHUH QR VLJQL¿FDQW GLIIHUHQFHV in histological scores, between the treatment groups (Figure 5.9). Figure 5.8 MPO activity assay in colonic tissue collected from DSS exposed C57BL/6 mice treated with or without standard PF or novel formula. Mice were given 3% DSS in drinking water for 5 days and then either continued on normal mouse pellet diet (positive control); or treated with standard PF or novel formula. Negative control mice were not exposed to DSS and continued on normal mouse pellet diet throughout the experiment. At completion of the experiment colons were collected for MPO assay. MPO activity significantly increased following DSS exposure. However, with novel formula or standard PF treatment, the activity substantially reduced to a level in equivalent to normal control mice. No difference was in between treatment groups were observed. P (*) < 0.05 vs. Negative control; (#) < 0.05 novel formula or standard PF vs. positive control). Data analyzed by one way Anova test followed by post hoc test. Figure 5.9 Histological assessment of local colon injury of C57BL/6 mice following DSS exposure treated with or without PF or novel formula. Mice were given 3% DSS in drinking water for 5 days and then either continued on normal mouse pellet diet (positive control); or treated with standard PF or novel formula. Negative control mice were not exposed to DSS and continued on normal mouse pellet diet throughout the experiment. Colonic tissue was collected at completion of the experiment and embedded in paraffin. Slides were then stained with hematoxylin and eosin and viewed by light microscope. Histology scores included crypt damage score, mucosal inflammation, extent of inflammation and mucosal edema. DSS induced significant damage in mice colon as indicated by raised total histology score. Both treatments with standard or novel formula showed only numerical but not statistical reduction in the total score. However treatment with either standard PF or novel formula resulted in significantly attenuated crypt damage. P (*) < 0.05 vs. Negative control; (#) < 0.05 novel formula or standard PF vs. positive control; (&) > 0.05 novel formula or standard PF vs. positive control). Data analyzed by one way Anova test followed by post hoc test. 5.5 Discussion

An important finding of this study is that the novel formula effectively attenuated intestinal inflammation in vivo, in a mouse model of colitis. In the DSS model of colitis, exposure to

'66FDXVHGVXEVWDQWLDOLQÀDPPDWRU\FKDQJHVDQGWLVVXHLQMXU\LQWKHPRXVHFRORQZKLFK manifested as decreased body weight, enhanced MPO activity and up-regulation of colonic cytokine expression. Interestingly, these changes were substantially reversed by treatment with the novel formula. However, treatment with the novel formula did not show superiority over treatment with the standard PF. Nevertheless both treatments significantly prevented DSS induced weight loss, reduced MPO activity, suppressed pro-inflammatory cytokines mRNA expression and partially ameliorated local colonic tissue injury. It is worth noting that the novel formula had similar activity to standard PF but with15% less total volume. Moreover, novel formula showed no signs of toxicity or any observed undesirable adverse effects in the mice.

In agreement with previous published papers, TNBS injected per rectal induced weight loss in mice [447, 935]. Here Blab/c mice presented with features of TNBS toxicity within

48 hours of TNBS injection. TNBS toxicity manifested as a significant weight decrease and mortality. Weight remained significantly low throughout the first 3 days following

TNBS injection, and then began to increase before gradually approaching the original body weight. However, weights remained lower in the TNBS group compared to the negative control group and did not reach the pre-TNBS weight by day 7. Interestingly, PF treatment exhibited positive effects on the changes in weight in response to TNBS as early as the first 24 hrs. PF treatment prevented the initial early weight drop and mice steadily increased weight throughout the experimental duration. Similar effects were also seen with the novel formula treatment in that it there was substantially enhanced weight gain in treated mice; however, these effects were delayed compared to the standard PF treated mice. This suggests that standard PF may be superior to novel formula in preventing

TNBS–induced weight loss. However, weight change is just one tool for assessing the efficacy and may not precisely reflect overall therapy efficacy. Unfortunately daily intake was not recorded for the TNBS experiments therefore no interpretation can be made whether novel formula intake was different that standard PF intake.

Mortality rate in the Balb/C mice following TNBS challenge was less than previously reported in the literature [639, 936]. Because of low mortality rates among experimental groups, the survival curve was not significantly different to the negative control group.

However, it is not uncommon among studies utilizing this model that TNBS administration can result in statistically undistinguishable drop in the survival rate [935].

Further RT-PCR analysis of colonic tissue collected from TNBS treated Balb/C mice of

TNF-Į,/-12, and one chemokine (MCP-1) showed no significant up-regulation in gene expression of these inflammatory mediators. However, only one cytokine (IL-6) showed a slight but significant up-regulation with TNBS exposure. This indicates a minor local inflammatory response in this setting. However, these findings are consistent with the published literature. It has been reported that 1 week following TNBS challenge, only one third of mice enter the chronic disease phase of colitis [937]. Taking into consideration this ratio, two-thirds of the experimental mice would have normal intestinal inflammation. In similar studies using the TNBS murine model, PCR analysis of colonic tissue demonstrated that mRNA expression of TNF-Į,/-2, IL-12, and IL-18 peaked on day 2 and 3 after TNBS administration and all returned to baseline between days 5 and 7 [938].

In another study, relative expression of the inflammatory mediators including TNF-ĮDQG

IL-12 were significantly higher when mice were sacrificed 3 days after TNBS challenge

[939]. In another study when mice were euthanized on day 3, 5, 10, or 14 after TNBS challenges, the authors observed that colitis was most severe by day 3 where there was the highest colonic mRNA expression [940]. Here, the duration of the experimental protocol was extended to 7 days. The decision to leave the model for 7 days was to allow the formulas sufficient time to have an effect. In a previously published work with a different murine colitis model, standard PF was administrated for 2 and 4 weeks, with formula treatment showing favourable outcomes [941]. Even in clinical practice, standard PF given as EEN therapy is provided for several weeks [942]. In the current experiment it appears as though the mice were recovered after 7 days, as indicated by weight gain and low colonic cytokine expression. Therefore this model provided no opportunity to investigate the efficacy of the novel formula. Thus, the acute TNBS model appears to be the wrong model to explore potential treatments that may require a longer window to exert its effects.

Ultimately it would have been more favourable to have a model of chronic colitis, which may have been achieved through repeated TNBS injections. However, because of limited experience of this protocol [943], it was decided to proceeded to a DSS model of colitis as it was the most widely utilized model of chronic colitis. In the DSS mouse model of colitis, previous studies indicate that the disease usually starts

3 to 7 days following DSS administration, with disease progression monitored primarily by body weight loss. Therefore, mice may even be observed to gain weight in the first days of

DSS initiation [354, 447]. Consistently, here C57BL/6 mice did not show any significant weight changes over the 5 days of DSS exposure and the majority of mice gained weight.

Body weight loss began once DSS was ceased which continued to fall considerably and remained significantly low for the duration of experiment. Interestingly, both treatment groups showed favorable effects on body weight, although novel formula fed mice showed early a partial drop. However, it remained significantly high compared to the positive control and not significantly different to the standard PF group. Towards the end of the experiment, novel formula treatment group showed a dramatic weight gain with weight remaining higher than the negative control mice by the end of experiment. Novel treatment did not show any noticeable superior benefit in weight gaining over standard PF in the 2 weeks’ time. Nevertheless, less volume of novel formula was required for mice to gain the same weight as PF fed group, which may indicate superior therapeutic benefits.

In IBD models, due to the ongoing local inflammatory response, bowel of the experimental mice becomes shorter and heavier, and thereby colon weight over length score, a marker of tissue oedema, is increased [941]. Consistently, in the current model, the weight/length score significantly increased following DSS treatment, with complete reversal by both the novel and standard formulas; however, there was no difference between these formulas. It is well documented that DSS exposure induces expression of numerous inflammatory mediators, including proinflammatory cytokines, in the colon of experimental animals

[944]. Increase in the productiRQRIWKHVHSURLQÀDPPDWRU\PHGLDWRUVLVDWWULEXWHGWRWKH recruitment of circulating leukocytes into the colon [945]. In this study, the expression of

TNF-Į,/-6, IL-12 and the chemokine MCP-1 were significantly up-regulated with DSS treatment. Similar to our findings were demonstrated in a study involved DSS exposed

C57BL/6 mice [680]. There was an increase in the production of TNF-Į,/-6 and IL-12 proinflammatory cytokines which peaked on the 5th day following DSS and remained high until day 14. In the current study, the novel formula showed a substantial reduction in the up regulation of inflammatory cytokines, indicating strong anti-inflammatory effects of the novel treatment in the vivo setting.

Neutrophil secreted MPO is involved in lipid peroxidation required for initiation of the inflammatory process at the site of inflammation [782]. In setting of the inflammatory induced tissue damage, MPO activity becomes a definitive and essential marker of inflammation and is as sensitive as cytokine measurement and more accurate than histological examination [946]. Correspondingly, in murine experimental colitis studies including the DSS model, MPO enzyme activity measurement has become a reliable indicator of inflammation and is used as an indirect assessment tool of neutrophil infiltration into colonic tissue [770, 947, 948]. Along with up-regulated colonic cytokine expression, increased colon weight/length score and marked body weight decrease, MPO activity was also significantly higher following DSS administration. The increased enzyme activity indicates colonic neutrophil infiltrations that are involved in ongoing inflammation and tissue destruction. Importantly both standard and novel treatments substantially decreased MPO activity to a level statistically undistinguishable to negative control mice.

Of worth note, the reduction was more significant in the presence of the novel formula.

In addition to inflammatory cell infiltration, inflammation secondary to DSS exposure is characterized by edema, mucosal ulcerations and crypt destruction [449]. However, the histology appearance score showed only a numerical reduction with treatments, although there was a significant drop in crypt damage score along with reduced MPO activity.

Therefore it seems both formulas induced only partial histological remission in the 2 week time.

Initially, the findings in the mouse models appear unexpected and contradictory to observations seen in vitro. The novel formula had similar activity to standard PF and did not result in complete resolution of colitis. However, in the DSS model the mice had a lower intake of novel formula compared to the standard PF intake. This was considered to be primarily due to the higher caloric content of the novel formula where each 1 ml of novel formula has 1.13 calorie compared to only 1 calorie for each ml of standard PF.

Nevertheless, both formulas had similar activity in mitigating DSS colitis. It can be considered that less volume producing the same anti-inflammatory activity compared to standard PF may represent superior efficacy. Further, the novel formula seems to be, at least in mice, a safe potential therapy for CD. None of mice were unusually distressed while they were on the formula. Several drawbacks of mouse models were present in this chapter and it remains unknown whether different outcomes would be seen with different models and/or different length of treatments. The ideal approach would be monitoring experimental mice more frequently and treatment response being assessed over a wider period of time. However, limited time and resources prevented the inclusion of additional treatment groups. Indeed, investigating the therapeutic benefits of nutritional products usually requires giving these therapies for a period of several weeks to be able to show noticeable effects. In a study conducted to explore effect of a glucosamine rich diet on intestinal inflammation in a DSS model of colitis, the dietary supplements were given for 4 weeks [474]. Further, in an IL-10 knockout (IL-íí PRXVHPRGHORI,%'XVHGWRDVVHVVWKHHIILFDF\RID7*)ULFKHQWHUDO diet in mitigating colitis, the mice received the diet for 8 weeks [450]. Similarly, in another study, a fatty acid enriched diet attenuated intestinal inflammation when given for 8 weeks

[67]. Here, longer duration was not attempted to avoid the risk of spontaneous colitis recovery. A previous report had shown a new approach to initiating experimental colitis in mice with one cycle of a short 5 days course of DSS which was sufficient to induce progressive chronic colitis in C57BL/6 mice [274]. However, it was also considered that mice have a tendency to recover if DSS is stopped [511]. Therefore, mice were sacrificed 2 weeks after DSS treatment and this resulted only in mild colitis.

As this report is the first to examine the anti-inflammatory properties of this novel formula in vivo, it would now be relevant to undertake further investigations in other models.

Repeated cycles of DSS administration may be a viable option to induce a more severe chronic colitis. However, due to time and cost considerations these experiments were not included as part of this thesis, but might be attempted for future investigations. 5.6 Conclusion

Clear evidence that novel formula has superior efficacy over standard PF in ameliorating colitis is lacking at this stage. Certainly, in order to progress to translation of this therapy, further evidence needs to be collected. Nevertheless manipulating PF components may still have promise for better therapies. The novel formula exhibited several favourable outcomes in the DSS model of colitis and there were no reported adverse effects. Ex-vivo cultured colonic biopsy from CD patients is an alternative viable option to models of colitis at this stage, and will be the aim of the next chapter. Chapter 6: Using ex vivo cultured colonic biopsies from patients with Crohn’s disease to investigate the effect of the novel nutritional formula in attenuating intestinal inflammation

6.1 Introduction

Human gut mucosa comprises of intestinal epithelial cells (composed mainly of enterocytes) and supporting lamina propria hosting numerous inflammatory cells [949].

There is cross talk and a complex interaction between the gut microbiota and cells of the gut mucosa, which requires tight regulation to control the mucosal immune response and maintain homeostasis [695]. Disruption of intestinal homeostasis is a hallmark of IBD and results in increased production of proinflammatory cytokines [274]. Immune cells from the lamina propria, including monocytes, neutrophils, macrophages and lymphocytes, and to lesser extent epithelial cells, are the source of these cytokines [474, 941]. Due to this intricacy and multiplicity of gut responses that becomes even more complex in the setting of pathology, it is evident that an in vitro single cell type model of intestinal inflammation provides a limited picture of gut inflammation. Nevertheless, organ culture of gut mucosa can offer a more realistic model of intestinal inflammation in vitro [950].

It is well documented that human colonic biopsies can be successfully cultured in vitro for up to 48 hrs without detrimental effects on differentiation or metabolic activity [450].

Further, it is well established that intestinal biopsies collected from patients with CD spontaneously release proinflammatory cytokines including TNF-Į DQG ,/-6 as well as other mediators [678, 793]. Therefore, in this chapter the anti-inflammatory properties of the novel formula were further examined by utilizing ex vivo cultured colonic biopsies collected from paediatric patients with active CD.

6.2 Hypothesis

That novel formula will attenuate inflammation to greater extent than standard PF, in an organ culture model that utilizes colonic biopsies collected from patients with active CD

6.3 Aims

1- To examine the extent to which novel formula, in comparison to standard PF, reduces

cytokine release from ex vivo cultured biopsies collected from inflamed regions of the

gut mucosa of CD patients.

2- To assess the effect of the novel formula has on tissue viability of ex vivo cultured

intestinal mucosa. 6.4 Results

6.4.1 Novel formula has no detrimental effect on organ culture of colonic biopsies from CD patients

A total of 37 organ culture experiments were included in this study. Biopsies were collected from 10 normal subjects and 9 patients with confirmed CD (Figure 6.1). Tissue specimens of normal individuals were cultured with media only and represent the negative control. Colonic tissues of CD were cultured with either media only (positive control); standard PF or novel formula at ratio of 1 to 5 to the media as previously explained (2.2.4).

LDH release into culture media was assessed by measuring enzyme activity in the collected supernatants as a measure of tissue viability (2.3.5). LDH activity was 0.5 ± 0.1

U/mg of tissue for all biopsies collected (Figure 6.2A) LDH release was not significantly different between controls or any of the treatment groups (P > 0.05; Figure 6.2B). Assessed for eligibility (n =20) Excluded n = 0 Enrolment Endoscopy

Allocated to group 1, IBD (n = Allocated to group 2, controls (n = 10); have visible inflammation 10); no visible inflammation collection y s

p Three biopsies were collected One biopsy was collected from Allocation-

bio from each individual each individual

Confirmed Crohn’s disease Confirmed normal controls (n = (CD) (n = 9) 10)

Not confirmed CD (n = 1) Histology examination

Analyzed (n = 9)

Excluded from analysis (n = 1) Analyzed (n = 10)

Analysis diagnosed unclassified IBD (IBDU)

Figure 6.1 CONSORT diagram showing the flow of participants and biopsy collection. Children aged between 5 and 17 years with suspicion of IBD requiring diagnostic endoscopy were recruited. Based on the endoscopy findings, subjects were allocated to either IBD group (group 1) or control group (group 2). Three colonic biopsies were collected from group 1 and one biopsy from group 2 for experimentation. Collected biopsies were then utilized as an ex-vivo IBD modelling to assessing effectiveness of the novel formula in suppressing inflammation.

Figure 6.2 LDH activity in cultured colonic biopsies. Organ culture of intestinal biopsies collected from normal subjects with un-inflamed bowel (negative control) and from patients with active CD incubated and with media alone (positive control), standard PF or novel formula. Using colorimetric assay kit, LDH activity (A) was measured in the culture media and translated onto % enzyme release (B). LDH release, as an indicator of tissue viability, was not significantly different between groups. Data present mean and SEM. Analysis done by One way Anova test with least Fischer sufficient test; P#>0.05 vs. negative control. 6.4.2 Novel formula completely inhibited pro-inflammatory cytokine and chemokine production from the cultured intestinal mucosa of active CD patients

Following organ culture, concentrations of TNF-Į,/-6, and IL-8 were measured in the culture media using ELISA cytoset kits (2.3.5). The level of TNF-Į ZDV KLJKHU LQ supernatant collected from CD biopsies of median 24.25 and interquartile ranges of

(31.40-12.40) pg/mg tissue compared to normal control 0.87 (1.3 ± 0.70) pg/mg tissue (P <

0.05; Figure 6.3A). Interestingly, TNF-Į FRQFHQWUDWLRQ VXEVWDQWLDOO\ GURSSHG E\ approximately 0.70 (9.3 - 0.683) pg/mg tissue in CD biopsies incubated with novel formula (P < 0.05 vs. positive control; Figure 6.3A), resulting in a TNF-Į VXSHUQDWDQW concentration identical to that of negative control (P > 0.05; Figure 6.3A). Despite a trend toward lower TNF-ĮSURGXFHGE\VWDQdard PF treated CD biopsies 8.6 (15.3 - 2.6) pg/mg tissue, TNF-ĮVXSHUQDWDQWOHYHOZDV not different to positive control levels (P = 0.09 vs. positive control; Figure 6.3A).

IL-8 and IL-6 also showed a similar trend. IL-8 released into the supernatant in negative control biopsies was approximately 170.5 (372 - 28) pg/mg tissue but was significantly higher at 1193 (3563 - 306) pg/mg tissue in the culture media of CD biopsies (P < 0.05;

Figure 6.3B). IL-8 levels were equal to negative control 104 (277 -15) pg/mg tissue with the novel formula treatment (P > 0.05 vs. negative control; Figure 6.3B); and approximately 268 (608 -126) pg/mg tissue with standard PF treatment, which was not significant different to positive control (P = 0.72 vs. positive control; Figure 6.3B). Moreover, IL-6 production was approximately 7 times higher in CD biopsies compared to the negative control biopsies {680 (927- 370) vs. 40 (162- 15) pg/mg, P < 0.05; Figure

6.3C}. However, IL-6 levels were considerably decreased with novel formula treatment to

19.4 (91.3 -14) pg/mg tissue (P < 0.05; Figure 6.3C). Standard PF treatment also resulted in a reduction in IL-6 production 183 (214 - 50) pg/mg, although it did not reach significance compared to positive control levels (P = 0.07; Figure 6.3C)

Figure 6.3 TNF, IL-8 and IL-6 release from colonic biopsies in organ culture. Colonic biopsies were collected from normal subjects with un-inflamed bowel (negative control) and from patients with active CD incubated with media alone (positive control), standard PF or novel formula. Concentrations of TNF-Į(A), IL-8 (B) and IL-6 (C) were measured in the supernatants using the ELISA technique. Levels of the measured mediators were significantly higher in the positive control compared to the negative control. However, in presence of novel formula, concentration of measured cytokine/chemokine was substantially decreased to negative control levels. Standard PF showed only numerical reduction for the three measured mediators. Data present median and interquartile ranges and analysed by Kruskal-Wallis test; (*) P < 0.05 vs. negative control; (&) P > 0.05, (#) P < 0.05 vs. positive control. 6.5 Discussion

In the present study we report that the spontaneous The main finding of this chapter is that the novel formula was capable of ameliorating gut inflammation in biopsies collected from children with active CD. The effect of the formula on ex vivo cultured colonic biopsies was manifested by a decrease in the release of the key proinflammatory cytokines TNF-ĮDQG,/-6 and the chemokine IL-8. More interesting, the novel formula was superior to standard PF in attenuating released inflammatory mediators, resulting in levels equivalent to that of normal non-inflamed cultured mucosa. Further, suppression of cytokines and chemokines was achieved without altering LDH tissue release, indicating that the biopsies remained viable.

Consistent with previous reports in the literature, concentrations of released proinflammatory mediators is higher in organ cultures of biopsies from the inflamed mucosa compared to biopsies from the non-inflamed bowel [793]. What is unique in the current study is that novel formula prompted a strong anti-inflammatory response in the inflamed gut mucosa from CD patients. Following incubation with the novel formula, concentrations of cytokines in the supernatant was equivalent to that of the normal non- inflamed mucosa. Although levels were lower in standard PF treated biopsies compared to un-treated biopsies form CD patients, this difference was not significant. Two limitations are the small number of samples (10 biopsies for each group) and the observed variability in the measured cytokine levels between samples. These factors could explain the lack of difference between standard PF treatment and the positive controls. Studies that examine spontaneous cytokine release in cultured intestinal mucosa of patients with CD, commonly report variability with abnormal distribution in the concentrations of the measured mediators [678, 951]. Nevertheless, even with this limited data set, the novel formula was consistently superior to standard PF in mitigating mucosal inflammation.

It is well documented that standard PF possesses a down-regulatory effect on mucosal pro- inflammatory cytokine production in vivo and can induce remission in CD patients [464,

477]. However, it is less clear in ex vivo model system. Of the few studies to have investigated the immune effects of standard formulas on intestinal biopsies collected from

CD patients, have shown that standard formula have no or limited down-regulatory effects

[952]. In a study that involved intestinal biopsies of CD cultured with enteral diet (EF) the ratio of anti- to pro-inflammatory cytokine (IL-1ra to IL-ȕ LQFUHDVHGKRZHYHUWKHHIIHFW on other protective mediators (IL-10 to IL-ȕUDWLR ZDVQRWVLJQLILFDQW[834]. In another study, enteral diet utilizing EF showed no any anti-inflammatory activity on the cytokine production from cultured biopsies of active CD patients [952].

However, of note it has been demonstrated that combined glutamine and arginine have a substantial immune effect on the inflamed gut mucosa collected from CD patients [801] and those effects were largely dependent on the given concentrations of these two amino acids. Throughout this thesis it has been consistently shown that a novel formula possessing higher concentrations of glutamine and arginine was more effective than standard PF in reducing inflammation.

LDH, an indirect viability measurement in organ explant experiments [953], was detected in the culture supernatant. Interestingly, none of the treatment groups, including novel formula treatment, showed significant changes in LDH activity compared to that of biopsies from the un-inflamed bowel. Based on this finding, as well as finding in the previous chapters, indicates that novel formula is not toxic to cells and appears to be a potentially safe therapy similar to standard PF therapy.

Apart of the clinical studies that have shown a decrease in the cytokine level in response to enteral diet therapy in CD [477], there is some evidence to support the notion that the primary mode of action of the enteral diet is through direct anti-inflammatory effect [707].

However, this evidence is derived from experiments on intestinal epithelial cells only

[707]. Furthermore, studies that did involve organ culture of colonic biopsies from CD patients, did not include key pro-inflammatory mediators including TNF-Į DQG ,/-6 in their investigations [834]. TNF-Į,/-8 and IL-6 are considered master pro-inflammatory mediators serving vital functions in IBD pathogenesis [680, 944, 945], and are derived from mononuclear cells of the lamina propria, most notably from macrophages [511, 793].

The results of this chapter indicate that the novel formula has a strong direct anti- inflammatory effect that likely targets all the intestinal cells, including epithelial and non- epithelial types, involved in the inflammatory process. Further, this effect is manifested by the reduction of key inflammatory cytokines involved in the ongoing inflammatory response, and provides additional support that a novel formula may have superior therapeutic benefits in the treatment of CD. 6.6 Conclusion

Novel formula, but not standard PF, supressed inflammation in biopsies from patients with active CD when the former resulted in a complete abrogation in the cytokine production by cultured colonic biopsies. Further, no adverse effects on tissue viability were observed with treatment with the novel formula. These findings therefore strongly support the argument that manipulating conventional nutritional treatment of CD can result in a more effective safe therapy with enhanced immune effects. Chapter 7: Final discussion and conclusions

The initial aim of this thesis was to further define the anti-inflammatory properties of PF.

Four ingredients of PF were proposed as potential active components, and their immunomodulating effects were investigated in an in vitro model of intestinal inflammation. Glutamine, arginine and vitamin D3 were shown to be are among the key active ingredients of PF and responsible at least in part for the alleviated immune responses seen with enteral diet therapy. The results of this thesis also revealed novel active concentrations for these nutrients in suppressing inflammation suggesting there may be additional potential benefits if given at higher concentrations than currently utilized in conventional formulas. Further experiments were undertaken to elucidate the mechanisms of actions of PF. the two active components of PF glutamine and arginine, exhibited a synergistic anti-inflammatory effect on the activated intestinal epithelial cells. That effect was shown to be mediated through deactivation of the NF-ț%DQG30$3.SDWKZD\VDV well as targeting additional several immune response related pathways.

Although many gaps remain in our understanding, these initial in vitro experiments have revealed novel observations about the anti-inflammatory activity of the polymeric diet and their related mechanisms. de Jong et al. [479] previously had established that PF has a strong immunomodulating activity on intestinal epithelial cell lines cultured in vitro, but the origin of the observed activity was not explored and remained elusive. Further, in the clinical research setting and despite proven efficacy of EEN in treating CD patients, investigations were limited to assessing the effectiveness of PF in delineating inflammation and did not explore the underlying mechanisms [521, 560, 954]. Subsequently, a hypothesis was developed that a high glutamine and arginine fortified PF would be superior to a standard PF in attenuating an inflammatory response. Indeed, glutamine and arginine enriched PF exhibited broader spectrum immunomodulating activity over the standard PF in the cultured intestinal epithelial monolayers. More interestingly, introduction of curcumin to the amino acids fortified PF, which was subsequently called the “novel formula”, revealed further enhancements in the activity with complete abrogation of the inflammatory response in the in vitro model.

However, these novel observations were from immortalized colonic epithelial cells, which may not precisely represent the anti-inflammatory activity seen in the clinical setting.

Nevertheless, the contribution of this work to clinical outcomes should not be discounted as: intestinal epithelial cells play a critical role in intestinal immune homeostasis and considered a key component of the mucosal barrier [955], and disruption in the regulatory function of the epithelial barrier contributes to the etiology of CD [956]. Another limitation is that it has not been investigated what happens to PF when it passes through the stomach and small intestine before being delivered to the colon. Importantly, this work has established that the combination of glutamine, arginine and curcumin, if at sufficient concentrations at the epithelial surface, will likely block the inflammatory response by a mechanism that directly interferes with NF-ț%DFWLYLW\. Therefore, we postulate that this activity will likely be replicated in vivo, dependent on sufficient nutrient concentration being delivered to the epithelial surface. In part to overcome the limitations of the in vitro model, the effectiveness of the novel therapy was tested further and compared to standard PF in vivo utilizing TNBS and DSS murine models of colitis. In TNBS colitis, BALB/c mice recovered to their original body weight and colitis was attenuated 1 week following disease induction, indicating the model does not accurately represent the human situation where EEN treatment would be employed. An additional colitis model was attempted (DSS). Interestingly, administration of the novel formula reproducibly ameliorated colitis in the DSS treated mice accounting for down-regulation of cytokine/chemokine expression and prevention of immune cell infiltration, which led to local healing in the colonic mucosa and body weight recovery.

However, despite the therapeutic benefits and observed safety of the novel formula in the

DSS exposed mice, there was no clear evidence that the novel formula was superior compared to standard PF in mitigating colitis in these experimental mouse models.

Nevertheless, less given volume of the novel formula (85% of standard PF) produced similar effects that may indicate superiority of the novel formulation.

There were limitations of the animal models. An ideal approach would be to assess the response to treatments over a longer period of time, in order to not just identify how effective the therapy was at inducing disease remission but also how fast the response to treatment occurs. In the models used, mice tend to recover once the chemical inducers are removed [957]. Additionally, delivering the formula to the colon and achieving the desirable concentrations of glutamine and arginine into the colon is an unknown variable.

It has been reported that nearly all of dietary glutamine and up to 40 % of dietary arginine, are catabolised in the small intestine and do not reach the large bowel [958]. Given the large uptake of glutamine and arginine by the small intestine, it would be expected that only a small amount of these amino acids reach the large bowel. Therefore, this may account for the negligible effect of the novel formula on the colonic mucosa in the experimental colitis models. From a clinical perspective this may explain why diet therapy involving EEN is considered less effective in isolated colonic CD compared to CD with small bowel involvement [547].

Interestingly, Buchanan et al.[463] in a trial involved CD patients treated with EEN demonstrated that the disease phenotype including location dose not influence efficacy of

EEN. On contrary, Wilschanski et al.[537], noted that individuals with isolated colonic CD were significantly less responsive to treatment with EEN than the patients with other disease locations. Although the literature is still controversial regarding the relationship between disease location and efficacy of enteral therapy in CD, the observed discrepancy in the activity of the novel formula between the cell culture model and the experimental colitis models may suggest that disease location may influence EEN outcomes. Thus, it becomes clear that creating a suitable delivery system for the novel formula is vital to show more favourable outcomes.

Finally, in order to ascertain whether manipulating concentrations of the standard polymeric diet leads to a better therapy, as the mouse model results were equivocal, effectiveness of the novel formula was tested further on cultured colonic biopsies obtained from CD patients. The results showed the novel formulation prompted a strong anti- inflammatory activity that was superior to standard PF in reducing the release of key inflammatory mediators. This further supports the in vitro results from the colonic intestinal epithelial cells that the novel formula can completely suppress inflammation in mucosal tissue if suitable concentrations of the active ingredients are achieved. These results also emphasize the need for a suitable delivery method to ensure that the key components of the nutritional formula are delivered to the mucosa at their most active concentrations. Developing an optimal delivery method is out of the scope of the current work, but it would appear to be a logical further step prior to considering the future application of these findings to the clinical setting.

Collectively, the thesis findings provide strong evidence that current nutritional therapy utilizing traditional formula has a limited efficacy in knocking down the inflammatory response in intestinal inflammation; and that modifying concentration of candidate nutrients in the formula is one way of enhancing the therapeutic benefits of EEN therapy.

Testing the efficacy of the novel formula in a clinical trial on CD patients is one of the future directions and implications of this work. However, further investigation into influence of the route of administration on the formulas utility may be required. Indeed, achieving the active concentrations of the novel formula’s active components inside the body tissues and target cells seems the most challenging task of translating these findings into therapy. In healthy volunteers, the plasma level of glutamine was significantly raised following infusion of glutamine solution at a concentration 200 mm (0.57g/kg), however it plateaued at the level of 25% above the normal value [959]. Although it seems a safe approach, giving these nutrients at high concentrations may not be solely capable of achieving the desirable active levels inside the body. Nevertheless, influencing nutrients’ metabolism might be a potential avenue. Although intraluminal glutamine was not measured, targeting the intestinal glutaminase, as a potential treatment for hepatic encephalopathy, was successful in reducing intestinal ammonia and blocking glutamate production from glutamine in mice [960]. A similar concept might lead to favorable outcomes if implemented together with enteral diets rich in amino acids and requires further research.

Secondly, given the poor palatability of current enteral diet therapy (EEN) is proposed as one of the factors behind therapy underutilization [910], rational therapy design may be a fruitful approach for further enhancing the therapeutic benefits of EEN. Because this thesis established a link between nutrients and activity of NF-ț% transcription factors, this may allow a direct targeting of the NF-ț% signalling pathway and the potential synthesis of new therapies. For example, this could be applied in the setting of adult patients with CD where nutritional consequences are less of an issue than in children [961, 962] and therefore the therapy may not need to be delivered in a formula. The identified active components of the novel formula (glutamine, arginine and curcumin) might be given together at their desirable concentrations with exclusion of other ingredients, as a new potential pharmaceutical design for EN therapy to improving acceptability. Third future direction of the thesis is that novel formula in CD might be utilized in conjunction with other interventions. Standard polymeric diet is commonly prescribed solely as a primary therapy for CD patients [528]. However, there is a newly emerging tendency for the EEN therapy to be utilized in combination with pharmacological treatments or given as PEN [558]. Sigall-Boneh et al. [544] investigated the effect of PEN utilizing standard PF as an induction therapy with a structured CDED and noted promising findings. Up to 70% of recruited patients achieved remission after 6 weeks of receiving just 50% of their dietary calories as PF [544]. Further, there is a potential avenue for the novel therapy to be administrated along with biological therapies. Indeed, there is an accumulating evidence of benefits of the concomitant use of EN with infliximab to sustain the efficacy of the later, and to prevent loss of response with time [963]. Based on the observations seen in the current work of enhanced activity of the novel formula compared to standard PF, it is intriguing to speculate that these new protocols of using enteral diet in

CD would result in a more favorable outcome if the novel formulation was utilized.

Further, there is also a potential application for using novel formula as a treatment option in patients with UC. Unlike CD, the effect of EN therapy on UC patients has not been as yet determined [964]. However, the initial trials showed that EEN is t a safe and nutritionally adequate intervention in UC patients [965]. Importantly, the common features of CD and UC are that pathology is a result of inappropriate activation of the two major signal transduction pathways (NF-ț% DQG 30$3.  which govern altered cytokine production [966]. The results of the thesis determined that the active components of the novel formula can synergise with each other and abolish activity of those two regulatory pathways. Therefore the novel formula might also have therapeutic utility in UC.

The novel therapy may have potential in treating other chronic disorders. Recent reports indicate that EEN could become a possible anti-inflammatory treatment for juvenile idiopathic arthritis (JIA) as it has shown remarkable benefits in a 7 year old girl with arthritis resistant to biological therapy [967]. Although the pathophysiological role of EEN in JIA has not been determined, it is well documented that the disrupted immune response as in other autoimmune diseases contributes to the disease pathogenesis [968]. NF-ț% implicated in the pathogenesis of CD, also contributes to the development of various rheumatic disorders including JIA and is thereby considered a potential therapeutic target for these disorders [969]. Targeting the NF-ț%E\WKHFRPSRQHQWVRI3)DVLQGLFDWHGLQ this thesis would explain the therapeutic benefits of EEN in treating these chronic conditions. Not surprisingly, the biological therapies such as infliximab are now being utilised for treating both IBD and JIA [970, 971]. Of note, the results presented here indicate inhibition of NF-ț%ZRXOGEHPRUHHYLGHQWZLWK((1WKHUDS\LIWKHQRYHOIRUPXOD was utilized and thereby more therapeutic advantages would be expected. Certainly, higher concentrations of the active components have shown superior suppressing effects against

,ț% NLQDVH DFWLYLW\ RI 1)-ț% FRPSDUHG WR WKDW RI VWDQGDUG 3) ,Q DGGLWLRQ FXUFXPLQ D known natural inhibitor of NF-ț%[972], is not part of the standard polymeric diets, but is in the novel formula. In the same context of targeting NF-ț%WKHQRYHOIRUPXODFDQEH used as a cancer therapy. Indeed, it has been reported that NF-ț%SDWKZD\LVXSUHJXODWHG in leukemic patients [973], and its targeting considered a potential target for future therapies [974]. Further, similar perception could also be applied for other conditions, such as asthma, muscular disorders and various neurological conditions where the pivotal role of NF-ț%LQSDWKRJHQHVLVDQGWKHQHZO\HPHUJLQJHYLGHQFHVDVDWKHUDSHXWLFWDUJHW[975-

977]. Collectively, it seems there are numerous future directions for this work with several potential therapeutic implications as a result. However, future research should be directed toward, and be more focused within the context of IBD where the most visible applications of the novel therapy appear.

In conclusion, this thesis has contributed significant knowledge about the enteral diet and its mechanism of action to induce disease remission. This work has also contributed to designing a better nutritional therapy that should hopefully contribute to improving disease outcomes in near feature.

Bibliography

1. Bouma, G. and W. Strober, The immunological and genetic basis of inflammatory bowel disease. Nature Reviews Immunology, 2003. 3(7): p. 521-533. 2. Parray, F., et al., Crohn's disease: A surgeon's perspective. Saudi Journal of Gastroenterology, 2011. 17(1): p. 6. 3. Blumberg, R.S., Crohn disease. JAMA, 2008. 300(4): p. 439-40. 4. Karlinger, K., et al., The epidemiology and the pathogenesis of inflammatory bowel disease. European journal of radiology, 2000. 35(3): p. 154-167. 5. Wilson, J., et al., High incidence of inflammatory bowel disease in Australia: A prospective population-based Australian incidence study. Inflammatory bowel diseases, 2010. 16(9): p. 1550-1556. 6. Mittermaier, C., et al., Impact of depressive mood on relapse in patients with inflammatory bowel disease: a prospective 18-month follow-up study. Psychosomatic Medicine, 2004. 66(1): p. 79-84. 7. Loftus, E., P. Schoenfeld, and W. Sandborn, The epidemiology and natural history of Crohn’s disease in population-based patient cohorts from North America: a systematic review. Alimentary pharmacology & therapeutics, 2002. 16(1): p. 51-60. 8. Blondel-Kucharski, F., et al., Health-related quality of life in Crohn's disease: a prospective longitudinal study in 231 patients. The American journal of gastroenterology, 2001. 96(10): p. 2915-2920. 9. Cohen, R.D., et al., The cost of hospitalization in Crohn's disease. The American journal of gastroenterology, 2000. 95(2): p. 524-530. 10. Feagan, B.G., et al., Annual cost of care for Crohn's disease: a payor perspective. The American journal of gastroenterology, 2000. 95(8): p. 1955-1960. 11. Cosnes, J., et al., Epidemiology and natural history of inflammatory bowel diseases. Gastroenterology, 2011. 140(6): p. 1785-1794. e4. 12. Vestergaard, P. and L. Mosekilde, Fracture risk in patients with celiac disease, Crohn’s disease, and ulcerative colitis: a nationwide follow-up study of 16,416 patients in Denmark. American journal of epidemiology, 2002. 156(1): p. 1-10. 13. Siew, C., Changing Epidemiology and Future Challenges of Inflammatory Bowel Disease in Asia. Intestinal Research, 2010. 8(1): p. 1-8. 14. Wilson, J., et al., High incidence of inflammatory bowel disease in Australia: A prospective population based Australian incidence study. Inflammatory bowel diseases, 2010. 16(9): p. 1550-1556. 15. Ng, S.C., et al., Geographical variability and environmental risk factors in inflammatory bowel disease. Gut, 2013. 62(4): p. 630-649. 16. Lakatos, P.L., Recent trends in the epidemiology of inflammatory bowel diseases: up or down? World Journal of Gastroenterology, 2006. 12(38): p. 6102. 17. Loftus Jr, E., P. Schoenfeld, and W. Sandborn, The epidemiology and natural history of Crohn’s disease in population based patient cohorts from North America: a systematic review. Alimentary pharmacology & therapeutics, 2002. 16(1): p. 51-60. 18. Loftus, E.V., Clinical epidemiology of inflammatory bowel disease: incidence, prevalence, and environmental influences. Gastroenterology, 2004. 126(6): p. 1504-1517. 19. Day, A., D. Lemberg, and R. Gearry, Inflammatory Bowel Disease in Australasian Children and Adolescents. Gastroenterology research and practice, 2014. 2014. 20. Ponder, A. and M.D. Long, A clinical review of recent findings in the epidemiology of inflammatory bowel disease. Clinical epidemiology, 2013. 5: p. 237. 21. Quaglietta, L., et al., Functional consequences of NOD2/CARD15 mutations in Crohn disease. Journal of pediatric gastroenterology and nutrition, 2007. 44(5): p. 529. 22. Kappelman, M.D., et al., The prevalence and geographic distribution of Crohn's disease and ulcerative colitis in the United States. Clinical Gastroenterology and Hepatology, 2007. 5(12): p. 1424-1429. 23. Molodecky, N.A., et al., Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review. Gastroenterology, 2012. 142(1): p. 46- 54. e42. 24. Zheng, J.J., et al., Prevalence and incidence rates of Crohn's disease in mainland China: A meta analysis of 55 years of research. Journal of digestive diseases, 2010. 11(3): p. 161- 166. 25. Goh, K. and S.D. XIAO, Inflammatory bowel disease: a survey of the epidemiology in Asia. Journal of digestive diseases, 2009. 10(1): p. 1-6. 26. Leong, R.W.L., J.Y. Lau, and J.J.Y. Sung, The epidemiology and phenotype of Crohn's disease in the Chinese population. Inflammatory bowel diseases, 2004. 10(5): p. 646- 651. 27. Yang, S.K., et al., Epidemiology of inflammatory bowel disease in the Songpa Kangdong district, Seoul, Korea, 1986–2005: A KASID study. Inflammatory bowel diseases, 2008. 14(4): p. 542-549. 28. Ng, S.C., Epidemiology of inflammatory bowel disease: Focus on Asia. Best Practice & Research Clinical Gastroenterology, 2014. 28(3): p. 363-372. 29. Tong, B.C. and A. Barbul, Cellular and physiological effects of arginine. Mini reviews in medicinal chemistry, 2004. 4(8): p. 823-832. 30. Henderson, P., et al., Rising incidence of pediatric inflammatory bowel disease in Scotland. Inflammatory bowel diseases, 2012. 18(6): p. 999-1005. 31. Malaty, H.M., et al., Rising incidence of inflammatory bowel disease among children: a 12-year study. Journal of pediatric gastroenterology and nutrition, 2010. 50(1): p. 27. 32. Phavichitr, N., D.J. Cameron, and A. Catto-smith, Increasing incidence of Crohn's disease in Victorian children. Journal of gastroenterology and hepatology, 2003. 18(3): p. 329- 332. 33. El-Tawil, A.M., Jews and Inflammatory Bowel Diseases. J Gastrointestin Liver Dis, 2009. 18(2): p. 137-138. 34. Zvidi, I., et al., The prevalence of Crohn’s disease in Israel: a 20-year survey. Digestive diseases and sciences, 2009. 54(4): p. 848-852. 35. Levi, Z., et al., The increasing prevalence of inflammatory bowel diseases among jewish adolescents and the sociodemographic factors associated with diagnosis. Inflammatory bowel diseases, 2013. 19(9): p. 1867-1871. 36. Baumgart, D.C. and S.R. Carding, Inflammatory bowel disease: cause and immunobiology. The Lancet, 2007. 369(9573): p. 1627-1640. 37. Kurata, J., et al., Crohn's disease among ethnic groups in a large health maintenance organization. Gastroenterology, 1992. 102(6): p. 1940. 38. Foster, A. and K. Jacobson, Changing incidence of inflammatory bowel disease: environmental influences and lessons learnt from the South Asian population. Frontiers in pediatrics, 2013. 1. 39. Auvin, S., et al., Incidence, clinical presentation and location at diagnosis of pediatric inflammatory bowel disease: a prospective population-based study in northern France (1988-1999). Journal of pediatric gastroenterology and nutrition, 2005. 41(1): p. 49. 40. Freeman, H.J., Long-term natural history of Crohn’s disease. World journal of gastroenterology: WJG, 2009. 15(11): p. 1315. 41. Bernstein, C.N., et al., Epidemiology of Crohn's disease and ulcerative colitis in a central Canadian province: a population-based study. American journal of epidemiology, 1999. 149(10): p. 916. 42. Sartor, B., Bacteria in Crohn's disease: mechanisms of inflammation and therapeutic implications. Journal of clinical gastroenterology, 2007. 41: p. S37. 43. Cashman, K.D. and F. Shanahan, Is nutrition an aetiological factor for inflammatory bowel disease? European journal of gastroenterology & hepatology, 2003. 15(6): p. 607- 613. 44. Magro, F., et al., European consensus on the histopathology of inflammatory bowel disease. Journal of Crohn's and Colitis, 2013. 7(10): p. 827-851. 45. Geboes, K., Histopathology of Crohn’s disease and ulcerative colitis. Inflammatory bowel disease. Edinburgh, London, Melbourne: Churchill Livingstone Elsevier, 2003: p. 255– 276. 46. Hendrickson, B.A., R. Gokhale, and J.H. Cho, Clinical aspects and pathophysiology of inflammatory bowel disease. Clinical microbiology reviews, 2002. 15(1): p. 79. 47. Hume, G. and G.L. Radford-Smith, The pathogenesis of Crohn s disease in the 21st century. Pathology, 2002. 34(6): p. 561-567. 48. Balfour Sartor, R., Bacteria in Crohn's disease: mechanisms of inflammation and therapeutic implications. Journal of clinical gastroenterology, 2007. 41: p. S37. 49. Young, B., P. Woodford, and G. O'Dowd, Wheater's functional histology: a text and colour atlas. 2013: Elsevier Health Sciences. 50. Hume, G. and G.L. Radford-Smith, The pathogenesis of Crohn's disease in the 21st century. PATHOLOGY-SYDNEY-, 2002. 34(6): p. 561-567. 51. Di Sabatino, A., et al., Recent advances in understanding Crohn’s disease. Internal and Emergency Medicine, 2011: p. 1-13. 52. Randall, K.J., J. Turton, and J.R. Foster, Explant culture of gastrointestinal tissue: a review of methods and applications. Cell biology and toxicology, 2011. 27(4): p. 267-284. 53. Yoon, J.S., et al., Anti-inflammatory effect of quercetin in a whole orbital tissue culture of Graves' orbitopathy. British Journal of Ophthalmology, 2012. 96(8): p. 1117-1121. 54. Borruel, N., et al., Increased mucosal tumour neĐƌŽƐŝƐ ĨĂĐƚŽƌ ɲ ƉƌŽĚƵĐƚŝŽŶ ŝŶ ƌŽŚŶ͛Ɛ disease can be downregulated ex vivo by probiotic bacteria. Gut, 2002. 51(5): p. 659- 664. 55. Zhang, J.-G., et al., Characterization of an in vitro model for the study of the short and prolonged effects of myocardial ischaemia and reperfusion in man. Clinical Science, 2000. 99(5): p. 443-453. 56. Lordan, S., J.E. Kennedy, and C.L. Higginbotham, Cytotoxic effects induced by unmodified and organically modified nanoclays in the human hepatic HepG2 cell line. Journal of Applied Toxicology, 2011. 31(1): p. 27-35. 57. Maloy, K.J. and F. Powrie, Intestinal homeostasis and its breakdown in inflammatory bowel disease. Nature, 2011. 474(7351): p. 298-306. 58. MacDonald, T.T., et al., Regulation of homeostasis and inflammation in the intestine. Gastroenterology, 2011. 140(6): p. 1768-1775. 59. Hugot, J.P., et al., Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease. Nature, 2001. 411(6837): p. 599-603. 60. Rosenstiel, P., et al., TNF-ɲ ĂŶĚ /&E-ɶ ƌegulate the expression of the NOD2 (CARD15) gene in human intestinal epithelial cells. Gastroenterology, 2003. 124(4): p. 1001-1009. 61. Beutler, B., Microbe sensing, positive feedback loops, and the pathogenesis of inflammatory diseases. Immunological reviews, 2009. 227(1): p. 248-263. 62. De Nitto, D., et al., Interleukin-21 triggers effector cell responses in the gut. World Journal of Gastroenterology: WJG, 2010. 16(29): p. 3638. 63. Pizarro, T.T., et al., IL-18, a novel immunoregulatory cytokine, is up-regulated in Crohn’s disease: expression and localization in intestinal mucosal cells. The Journal of Immunology, 1999. 162(11): p. 6829. 64. Isaacs, K., R. Sartor, and S. Haskill, Cytokine messenger RNA profiles in inflammatory bowel disease mucosa detected by polymerase chain reaction amplification. Gastroenterology, 1992. 103(5): p. 1587. 65. Akazawa, A., et al., Increased expression of tumor necrosis factor-ɲŵĞƐƐĞŶŐĞƌZEŝŶ the intestinal mucosa of inflammatory bowel disease, particularly in patients with disease in the inactive phase. Journal of gastroenterology, 2002. 37(5): p. 345-353. 66. Daig, R., et al., Increased interleukin 8 expression in the colon mucosa of patients with inflammatory bowel disease. Gut, 1996. 38(2): p. 216-222. 67. Rogler, G. and T. Andus, Cytokines in inflammatory bowel disease. World journal of surgery, 1998. 22(4): p. 382-389. 68. Schottelius, A. and A. Baldwin Jr, A role for transcription factor NF-kB in intestinal inflammation. International journal of colorectal disease, 1999. 14(1): p. 18-28. 69. Fiocchi, C., Inflammatory bowel disease: etiology and pathogenesis. Gastroenterology, 1998. 115(1): p. 182-205. 70. Baldwin, A.S., Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappaB. Journal of Clinical Investigation, 2001. 107(3): p. 241-246. 71. ^ĐŚƌĞŝďĞƌ͕^͕͘^͘EŝŬŽůĂƵƐ͕ĂŶĚ:͘,ĂŵƉĞ͕ĐƚŝǀĂƚŝŽŶŽĨŶƵĐůĞĂƌĨĂĐƚŽƌʃŝŶŝŶĨůĂŵŵĂƚŽƌLJ bowel disease. Gut, 1998. 42(4): p. 477-484. 72. Yamamoto, Y. and R.B. Gaynor, Therapeutic potential of inhibition of the NF-ʃƉĂƚŚǁĂLJ in the treatment of inflammation and cancer. Journal of Clinical investigation, 2001. 107(2): p. 135. 73. Karin, M. and A. Lin, NF-ʃ Ăƚ ƚŚĞ ĐƌŽƐƐƌŽĂĚƐ ŽĨ ůŝĨĞ ĂŶĚ ĚĞĂƚŚ͘ EĂƚƵƌĞ ŝŵŵƵŶŽůŽŐLJ͕ 2002. 3(3): p. 221-227. 74. WhitesidĞ͕^͘d͕͘ĞƚĂů͕͘/ŬĂƉƉĂĞƉƐŝůŽŶ͕ĂŶŽǀĞůŵĞŵďĞƌŽĨƚŚĞ/ʃĨĂŵŝůLJ͕ĐŽŶƚƌŽůƐZĞů and cRel NF-ʃĂĐƚŝǀŝƚLJ͘dŚĞDKũŽƵƌŶĂů͕ϭϵϵϳ͘ϭϲ;ϲͿ͗Ɖ͘ϭϰϭϯ-1426. 75. Chen, Z.J., Ubiquitin signalling in the NF-ʃƉĂƚŚǁĂLJ͘EĂƚƵƌĞĐĞůůďŝŽůŽŐLJ͕ϮϬϬϱ͘ϳ;ϴͿ͗Ɖ͘ 758-765. 76. Karin, M. and Y. Ben-Neriah, Phosphorylation meets ubiquitination: the control of NF-ʃ activity. Annual review of immunology, 2000. 18(1): p. 621-663. 77. Rahman, M.M. and G. McFadden, Modulation of NF-ʃ ƐŝŐŶĂůůŝŶŐ ďLJ ŵŝĐƌŽďŝĂů pathogens. Nature Reviews Microbiology, 2011. 9(4): p. 291-306. 78. ĂŶĚŝ͕ ͕͘ Ğƚ Ăů͕͘ dŚĞ /ʃ ŬŝŶĂƐĞ ĐŽŵƉůĞdž ;/<<Ϳ ĐŽŶƚĂŝŶƐ ƚǁŽ ŬŝŶĂƐĞ ƐƵďƵŶŝƚƐ͕ /<<ɲ ĂŶĚ /<<ɴ͕ŶĞĐĞƐƐĂƌLJĨŽƌ/ʃƉŚŽƐƉŚŽƌLJůĂƚŝŽŶĂŶĚE&-ʃĂĐƚŝǀĂƚŝŽŶ͘Ğůů͕ϭϵϵϳ͘ϵϭ;ϮͿ͗Ɖ͘Ϯϰϯ- 252. 79. Karin, M., The beŐŝŶŶŝŶŐ ŽĨ ƚŚĞ ĞŶĚ͗ /ʃ ŬŝŶĂƐĞ ;/<<Ϳ ĂŶĚ E&-ʃ ĂĐƚŝǀĂƚŝŽŶ͘ :ŽƵƌŶĂů ŽĨ Biological Chemistry, 1999. 274(39): p. 27339-27342. 80. Holger, W., X. Shou-Hua, and S.W. Young, High throughput screening for protein kinase inhibitors. Combinatorial Chemistry & High Throughput Screening, 2005. 8(2): p. 181- 195. 81. Ravid, T. and M. Hochstrasser, NF-ʃ ƐŝŐŶĂůŝŶŐ͗ ĨůŝƉƉŝŶŐ ƚŚĞ ƐǁŝƚĐŚ ǁŝƚŚ ƉŽůLJƵďŝƋƵŝƚŝŶ chains. Current biology, 2004. 14(20): p. R898-R900. 82. Tak, P.P. and G.S. Firestein, NF-kappaB: a key role in inflammatory diseases. Journal of Clinical Investigation, 2001. 107(1): p. 7-12. 83. DiDonato, J., et al., Mapping of the inducible IkappaB phosphorylation sites that signal its ubiquitination and degradation. Molecular and Cellular Biology, 1996. 16(4): p. 1295- 1304. 84. EĞƵƌĂƚŚ͕D͘&͕͘ĞƚĂů͕͘LJƚŽŬŝŶĞ'ĞŶĞdƌĂŶƐĐƌŝƉƚŝŽŶLJE&Ͳʃ&ĂŵŝůLJDĞŵďĞƌƐŝŶWĂƚŝĞŶƚƐ with Inflammatory Bowel Disease. Annals of the New York Academy of Sciences, 1998. 859(1): p. 149-159. 85. Wullaert, A., M.C. Bonnet, and M. Pasparakis, NF-ʃ in the regulation of epithelial homeostasis and inflammation. Cell research, 2011. 21(1): p. 146-158. 86. Hollenbach, E., et al., Inhibition of RICK/nuclear factor- B and p38 signaling attenuates the inflammatory response in a murine model of Crohn disease. Journal of Biological Chemistry, 2005. 280(15): p. 14981. 87. Oeckinghaus, A. and S. Ghosh, The NF-ʃ ĨĂŵŝůLJ ŽĨ ƚƌĂŶƐĐƌŝƉƚŝŽŶ ĨĂĐƚŽƌƐ ĂŶĚ ŝƚƐ regulation. Cold Spring Harbor perspectives in biology, 2009. 1(4): p. a000034. 88. Aggarwal, B.B., Tumour necrosis factors receptor associated signalling molecules and their role in activation of apoptosis, JNK and NF-ʃ͘ŶŶĂůƐŽĨƚŚĞƌŚĞƵŵĂƚŝĐĚŝƐĞĂƐĞƐ͕ 2000. 59(suppl 1): p. i6-i16. 89. Karin, M., Y. Yamamoto, and Q.M. Wang, The IKK NF-ʃƐLJƐƚĞŵ͗ĂƚƌĞĂƐƵƌĞƚƌŽǀĞ for drug development. Nature reviews Drug discovery, 2004. 3(1): p. 17-26. 90. Waetzig, G.H., et al., p38 mitogen-activated protein kinase is activated and linked to TNF- signaling in inflammatory bowel disease. The Journal of Immunology, 2002. 168(10): p. 5342. 91. Broom, O., et al., Mitogen activated protein kinases: a role in inflammatory bowel disease? Clinical & Experimental Immunology, 2009. 158(3): p. 272-280. 92. Chen, Z., et al., MAP kinases. Chemical reviews, 2001. 101(8): p. 2449-2476. 93. Kyriakis, J.M. and J. Avruch, Mammalian MAPK signal transduction pathways activated by stress and inflammation: a 10-year update. Physiological reviews, 2012. 92(2): p. 689- 737. 94. Huang, C.-Y. and J.E. Ferrell, Ultrasensitivity in the mitogen-activated protein kinase cascade. Proceedings of the National Academy of Sciences, 1996. 93(19): p. 10078- 10083. 95. Seger, R. and E.G. Krebs, The MAPK signaling cascade. The FASEB journal, 1995. 9(9): p. 726-735. 96. Brunet, A., et al., Nuclear translocation of p42/p44 mitogen-activated protein kinase is required for growth factor-induced gene expression and cell cycle entry. The EMBO journal, 1999. 18(3): p. 664-674. 97. Schreiber, S., et al., Oral p38 mitogen-activated protein kinase inhibition with BIRB 796 for active Crohn’s disease: a randomized, double-blind, placebo-controlled trial. Clinical Gastroenterology and Hepatology, 2006. 4(3): p. 325-334. 98. Waetzig, G.H., et al., p38 mitogen-activated protein kinase is activated and linked to TNF-ɲ ƐŝŐŶĂůŝŶŐ ŝŶ ŝŶĨůĂŵŵĂtory bowel disease. The Journal of Immunology, 2002. 168(10): p. 5342-5351. 99. Jiang, Y., et al., Characterization of the structure and function of a new mitogen- ĂĐƚŝǀĂƚĞĚ ƉƌŽƚĞŝŶ ŬŝŶĂƐĞ ;ƉϯϴɴͿ͘ :ŽƵƌŶĂů ŽĨ ŝŽůŽŐŝĐĂů ŚĞŵŝƐƚƌLJ͕ ϭϵϵϲ͘ Ϯϳϭ;ϯϬͿ͗ Ɖ͘ 17920-17926. 100. Ben-Levy, R., et al., Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2. Current Biology, 1998. 8(19): p. 1049-1057. 101. Schieven, G.L., The biology of p38 kinase: a central role in inflammation. Current topics in medicinal chemistry, 2005. 5(10): p. 921-928. 102. Guan, Z., et al., Interleukin-ϭɴ-induced cyclooxygenase-2 expression requires activation of both c-Jun NH2-terminal kinase and p38 MAPK signal pathways in rat renal mesangial cells. Journal of Biological Chemistry, 1998. 273(44): p. 28670-28676. 103. Cuenda, A. and S. Rousseau, p38 MAP-kinases pathway regulation, function and role in human diseases. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 2007. 1773(8): p. 1358-1375. 104. Chrestensen, C.A., et al., MAPKAP kinase 2 phosphorylates tristetraprolin on in vivo sites including Ser178, a site required for 14-3-3 binding. Journal of Biological Chemistry, 2004. 279(11): p. 10176-10184. 105. Schindler, J., J. Monahan, and W. Smith, p38 pathway kinases as anti-inflammatory drug targets. Journal of dental research, 2007. 86(9): p. 800-811. 106. Hollenbach, E., et al., Inhibition of p38 MAP kinase-and RICK/NF-ʃ-signaling suppresses inflammatory bowel disease. The FASEB Journal, 2004. 18(13): p. 1550-1552. 107. Kumar, S., J. Boehm, and J.C. Lee, p38 MAP kinases: key signalling molecules as therapeutic targets for inflammatory diseases. Nature reviews Drug discovery, 2003. 2(9): p. 717-726. 108. Arulampalam, V. and S. Pettersson, Uncoupling the p38 MAPK kinase in IBD: a double edged sword? Gut, 2002. 50(4): p. 446-447. 109. Hommes, D., et al., Inhibition of stress-activated MAP kinases induces clinical improvement in moderate to severe Crohn's disease. Gastroenterology, 2002. 122(1): p. 7-14. 110. Hooper, L.V., D.R. Littman, and A.J. Macpherson, Interactions between the microbiota and the immune system. Science, 2012. 336(6086): p. 1268-1273. 111. Wesemann, D.R., et al., Microbial colonization influences early B-lineage development in the gut lamina propria. Nature, 2013. 501(7465): p. 112-115. 112. Qin, J., et al., A human gut microbial gene catalogue established by metagenomic sequencing. nature, 2010. 464(7285): p. 59-65. 113. Rakoff-Nahoum, S., et al., Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell, 2004. 118(2): p. 229-241. 114. French, N. and S. Pettersson, Microbe-host interactions in the alimentary tract: the gateway to understanding inflammatory bowel disease. Gut, 2000. 47(2): p. 162-163. 115. Pagliari, D., et al., The Interactions between Innate Immunity and Microbiota in Gastrointestinal Diseases. Journal of Immunology Research, 2015. 2015. 116. Greenbloom, S.L., A.H. Steinhart, and G.R. Greenberg, Combination ciprofloxacin and metronidazole for active Crohn's disease. Canadian journal of gastroenterology= Journal canadien de gastroenterologie, 1997. 12(1): p. 53-56. 117. Rutgeerts, P., et al., Effect of faecal stream diversion on recurrence of Crohn's disease in the neoterminal ileum. The Lancet, 1991. 338(8770): p. 771-774. 118. Juste, C., et al., Bacterial protein signals are associated with Crohn’s disease. Gut, 2014. 63(10): p. 1566-1577. 119. Abraham, C. and R. Medzhitov, Interactions between the host innate immune system and microbes in inflammatory bowel disease. Gastroenterology, 2011. 140(6): p. 1729- 1737. 120. Baumgart, M., et al., Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn's disease involving the ileum. The ISME journal, 2007. 1(5): p. 403- 418. 121. Barnich, N., et al., CEACAM6 acts as a receptor for adherent-invasive E. coli, supporting ileal mucosa colonization in Crohn disease. Journal of Clinical Investigation, 2007. 117(6): p. 1566. 122. Kelsen, J. and G.D. Wu, The Gut Microbiota and IBD, in Pediatric Inflammatory Bowel Disease. 2013, Springer. p. 35-42. 123. Magrone, T. and E. Jirillo, The interplay between the gut immune system and microbiota in health and disease: nutraceutical intervention for restoring intestinal homeostasis. Current pharmaceutical design, 2013. 19(7): p. 1329-1342. 124. Marchesi, J.R., et al., Rapid and noninvasive metabonomic characterization of inflammatory bowel disease. Journal of proteome research, 2007. 6(2): p. 546-551. 125. Zhang, M., et al., Structural shifts of mucosa-associated lactobacilli and Clostridium leptum subgroup in patients with ulcerative colitis. Journal of clinical microbiology, 2007. 45(2): p. 496-500. 126. Manichanh, C., et al., Reduced diversity of faecal microbiota in Crohn’s disease revealed by a metagenomic approach. Gut, 2006. 55(2): p. 205-211. 127. Seksik, P., et al., Alterations of the dominant faecal bacterial groups in patients with Crohn's disease of the colon. Gut, 2003. 52(2): p. 237-242. 128. Kaur, N., et al., Intestinal dysbiosis in inflammatory bowel disease. Gut microbes, 2011. 2(4): p. 211-216. 129. Loh, G. and M. Blaut, Role of commensal gut bacteria in inflammatory bowel diseases. Gut Microbes, 2012. 3(6): p. 544-555. 130. Sokol, H., et al., Low counts of Faecalibacterium prausnitzii in colitis microbiota. Inflammatory bowel diseases, 2009. 15(8): p. 1183-1189. 131. Fujimoto, T., et al., Decreased abundance of Faecalibacterium prausnitzii in the gut microbiota of Crohn's disease. Journal of gastroenterology and hepatology, 2013. 28(4): p. 613-619. 132. Quévrain, E., et al., Identification of an anti-inflammatory protein from Faecalibacterium prausnitzii, a commensal bacterium deficient in Crohn’s disease. Gut, 2015: p. gutjnl- 2014-307649. 133. Sokol, H., et al., Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients. Proceedings of the National Academy of Sciences, 2008. 105(43): p. 16731-16736. 134. &ĂǀŝĞƌ͕͕͘ĞƚĂů͕͘&ĞĐĂůɴ-d-galactosidase production and bifidobacteria are decreased in Crohn's disease. Digestive diseases and sciences, 1997. 42(4): p. 817-822. 135. Joossens, M., et al., Dysbiosis of the faecal microbiota in patients with Crohn's disease and their unaffected relatives. Gut, 2011. 60(5): p. 631-637. 136. Fava, F. and S. Danese, Intestinal microbiota in inflammatory bowel disease: friend of foe? World journal of gastroenterology: WJG, 2011. 17(5): p. 557. 137. Fleming, L. and M. Floch, Digestion and absorption of fiber carbohydrate in the colon. The American journal of gastroenterology, 1986. 81(7): p. 507-511. 138. Segain, J., et al., Butyrate inhibits inflammatory rĞƐƉŽŶƐĞƐ ƚŚƌŽƵŐŚ E&ʃ ŝŶŚŝďŝƚŝŽŶ͗ implications for Crohn's disease. Gut, 2000. 47(3): p. 397-403. 139. Vanhoutvin, S., et al., Butyrate-induced transcriptional changes in human colonic mucosa. PloS one, 2009. 4(8): p. e6759. 140. Nagalingam, N.A. and S.V. Lynch, Role of the microbiota in inflammatory bowel diseases. Inflammatory bowel diseases, 2012. 18(5): p. 968-984. 141. Frank, D.N., et al., Disease phenotype and genotype are associated with shifts in intestinal-associated microbiota in inflammatory bowel diseases. Inflammatory bowel diseases, 2011. 17(1): p. 179-184. 142. Lupp, C., et al., Host-mediated inflammation disrupts the intestinal microbiota and promotes the overgrowth of Enterobacteriaceae. Cell host & microbe, 2007. 2(2): p. 119-129. 143. Walker, A.W., et al., High-throughput clone library analysis of the mucosa-associated microbiota reveals dysbiosis and differences between inflamed and non-inflamed regions of the intestine in inflammatory bowel disease. BMC microbiology, 2011. 11(1): p. 7. 144. De Hertogh, G., et al., Evidence for the involvement of infectious agents in the pathogenesis of Crohn’s disease. World journal of gastroenterology: WJG, 2008. 14(6): p. 845. 145. Matricon, J., N. Barnich, and D. Ardid, Immunopathogenesis of inflammatory bowel disease. Self Nonself, 2010. 1(4): p. 299. 146. Tamboli, C., et al., Dysbiosis in inflammatory bowel disease. Gut, 2004. 53(1): p. 1-4. 147. Lepage, P., S. Mondot, and N. Vasquez, Bacterial Recolonization after Gut Resection for Crohn’s DIsease: Uniformity as an Essential Factor Toward Remission. Gut, 2009. 58. 148. Darfeuille-Michaud, A., et al., High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn’s disease. Gastroenterology, 2004. 127(2): p. 412- 421. 149. De Cruz, P., et al., Association between specific mucosa-associated microbiota in Crohn's disease at the time of resection and subsequent disease recurrence: A pilot study. Journal of gastroenterology and hepatology, 2015. 30(2): p. 268-278. 150. Rajca, S., et al., Alterations in the intestinal microbiome (dysbiosis) as a predictor of relapse after infliximab withdrawal in Crohn's disease. Inflammatory bowel diseases, 2014. 20(6): p. 978-986. 151. Pérez-Cobas, A.E., et al., Gut microbiota disturbance during antibiotic therapy: a multi- omic approach. Gut, 2013. 62(11): p. 1591-1601. 152. Nord, C., The effect of antimicrobial agents on the ecology of the human intestinal microflora. Veterinary microbiology, 1993. 35(3): p. 193-197. 153. Dethlefsen, L., et al., The pervasive effects of an antibiotic on the human gut microbiota, as revealed by deep 16S rRNA sequencing. PLoS biol, 2008. 6(11): p. e280. 154. Dethlefsen, L. and D.A. Relman, Incomplete recovery and individualized responses of the human distal gut microbiota to repeated antibiotic perturbation. Proceedings of the National Academy of Sciences, 2011. 108(Supplement 1): p. 4554-4561. 155. Young, V.B. and T.M. Schmidt, Antibiotic-associated diarrhea accompanied by large- scale alterations in the composition of the fecal microbiota. Journal of clinical microbiology, 2004. 42(3): p. 1203-1206. 156. Zeissig, S. and R.S. Blumberg, Life at the beginning: perturbation of the microbiota by antibiotics in early life and its role in health and disease. Nature immunology, 2014. 15(4): p. 307-310. 157. Russell, S.L., et al., Early life antibiotic-driven changes in microbiota enhance susceptibility to allergic asthma. EMBO reports, 2012. 13(5): p. 440-447. 158. Shaw, S.Y., J.F. Blanchard, and C.N. Bernstein, Association between the use of antibiotics in the first year of life and pediatric inflammatory bowel disease. American Journal of Gastroenterology, 2010. 105(12): p. 2687. 159. Hildebrand, H., et al., Early-life exposures associated with antibiotic use and risk of subsequent Crohn's disease. Scandinavian journal of gastroenterology, 2008. 43(8): p. 961-966. 160. Ungaro, R., et al., Antibiotics associated with increased risk of new-onset Crohn’s disease but not ulcerative colitis: a meta-analysis. The American journal of gastroenterology, 2014. 109(11): p. 1728-1738. 161. Shaw, S.Y., J.F. Blanchard, and C.N. Bernstein, Association between the use of antibiotics and new diagnoses of Crohn's disease and ulcerative colitis. The American journal of gastroenterology, 2011. 106(12): p. 2133-2142. 162. Sekirov, I., et al., Gut microbiota in health and disease. Physiological reviews, 2010. 90(3): p. 859-904. 163. Koropatkin, N.M., E.A. Cameron, and E.C. Martens, How glycan metabolism shapes the human gut microbiota. Nature Reviews Microbiology, 2012. 10(5): p. 323-335. 164. Carbonero, F., et al. Traditional African and Western Diets Select Distinct Phylogenetic and Functional Colonic Microbiota Among Different Populations. in Gatroenterology. 2012. Wb Saunders CO-Elsevier INC 1600 John F Kennedy Boulevard, STE 1800, PA 19103-2899 USA. 165. Harmsen, H.J., et al., Analysis of intestinal flora development in breast-fed and formula- fed infants by using molecular identification and detection methods. Journal of pediatric gastroenterology and nutrition, 2000. 30(1): p. 61-67. 166. Fallani, M., et al., Intestinal microbiota of 6-week-old infants across Europe: geographic influence beyond delivery mode, breast-feeding, and antibiotics. Journal of pediatric gastroenterology and nutrition, 2010. 51(1): p. 77-84. 167. Mackie, R.I., A. Sghir, and H.R. Gaskins, Developmental microbial ecology of the neonatal gastrointestinal tract. The American journal of clinical nutrition, 1999. 69(5): p. 1035s-1045s. 168. Claesson, M.J., et al., Gut microbiota composition correlates with diet and health in the elderly. Nature, 2012. 488(7410): p. 178-184. 169. Scott, K.P., et al., The influence of diet on the gut microbiota. Pharmacological research, 2013. 69(1): p. 52-60. 170. Chow, J. and S.K. Mazmanian, A pathobiont of the microbiota balances host colonization and intestinal inflammation. Cell host & microbe, 2010. 7(4): p. 265-276. 171. Ley, R.E., et al., Evolution of mammals and their gut microbes. Science, 2008. 320(5883): p. 1647-1651. 172. Eckburg, P.B., et al., Diversity of the human intestinal microbial flora. science, 2005. 308(5728): p. 1635-1638. 173. Macfarlane, G.T. and S. Macfarlane, Manipulating the Indigenous Microbiota in Humans: Prebiotics, Probiotics, and Synbiotics. The Human Microbiota: How Microbial Communities Affect Health and Disease, 2013: p. 315-338. 174. Delzenne, N.M., et al., Targeting gut microbiota in obesity: effects of prebiotics and probiotics. Nature Reviews Endocrinology, 2011. 7(11): p. 639-646. 175. Shanahan, F. and E.M. Quigley, Manipulation of the Microbiota for Treatment of IBS and IBD—Challenges and Controversies. Gastroenterology, 2014. 146(6): p. 1554-1563. 176. Dos Santos, V.M., M. Müller, and W.M. De Vos, Systems biology of the gut: the interplay of food, microbiota and host at the mucosal interface. Current opinion in biotechnology, 2010. 21(4): p. 539-550. 177. Brown, K., et al., Diet-induced dysbiosis of the intestinal microbiota and the effects on immunity and disease. Nutrients, 2012. 4(8): p. 1095-1119. 178. Devkota, S. and E.B. Chang, Nutrition, microbiomes, and intestinal inflammation. Current opinion in gastroenterology, 2013. 29(6): p. 603-607. 179. Turnbaugh, P.J., et al., The effect of diet on the human gut microbiome: a metagenomic analysis in humanized gnotobiotic mice. Science translational medicine, 2009. 1(6): p. 6ra14-6ra14. 180. Cummings, J., et al., The effect of meat protein and dietary fiber on colonic function and metabolism. II. Bacterial metabolites in feces and urine. The American Journal of Clinical Nutrition, 1979. 32(10): p. 2094-2101. 181. Pokusaeva, K., G.F. Fitzgerald, and D. van Sinderen, Carbohydrate metabolism in Bifidobacteria. Genes & nutrition, 2011. 6(3): p. 285-306. 182. Hashimoto, T., et al., ACE2 links amino acid malnutrition to microbial ecology and intestinal inflammation. Nature, 2012. 487(7408): p. 477-481. 183. Kane, A.V., D.M. Dinh, and H.D. Ward, Childhood malnutrition and the intestinal microbiome. Pediatric research, 2014. 184. Subramanian, S., et al., Persistent gut microbiota immaturity in malnourished Bangladeshi children. Nature, 2014. 185. de Medina, F.S., et al., Host–microbe interactions: the difficult yet peaceful coexistence of the microbiota and the intestinal mucosa. British Journal of Nutrition, 2013. 109(S2): p. S12-S20. 186. Sartor, R., Key questions to guide a better understanding of host–commensal microbiota interactions in intestinal inflammation. Mucosal immunology, 2011. 4(2): p. 127-132. 187. Chamberlin, W., et al., Mycobacterium avium subsp. paratuberculosis as one cause of Crohn’s disease. Alimentary pharmacology & therapeutics, 2001. 15(3): p. 337-346. 188. Chen, W., et al., Detection of Listeria monocytogenes by polymerase chain reaction in intestinal mucosal biopsies from patients with inflammatory bowel disease and controls. Journal of gastroenterology and hepatology, 2000. 15(10): p. 1145-1150. 189. Greenstein, R.J., Is Crohn's disease caused by a mycobacterium? Comparisons with leprosy, tuberculosis, and Johne's disease. The Lancet infectious diseases, 2003. 3(8): p. 507-514. 190. Golan, L., et al., Mycobacterium avium paratuberculosis invades human small-intestinal goblet cells and elicits inflammation. Journal of Infectious Diseases, 2009. 199(3): p. 350- 354. 191. Seksik, P., et al., Review article: the role of bacteria in onset and perpetuation of inflammatory bowel disease. Alimentary pharmacology & therapeutics, 2006. 24(s3): p. 11-18. 192. Olsen, I., et al., Isolation of Mycobacterium avium subspecies paratuberculosis reactive CD4 T cells from intestinal biopsies of Crohn’s disease patients. PLoS One, 2009. 4(5): p. e5641. 193. Nakase, H., et al., Involvement of mycobacterium avium subspecies paratuberculosis in dE&ͲɲƉƌŽĚƵĐƚŝŽŶĨƌŽŵŵĂĐƌŽƉŚĂŐĞ͗WŽƐƐŝďůĞůŝŶŬďĞƚǁĞĞŶDWĂŶĚŝŵŵƵŶĞƌĞsponse in Crohn's disease. Inflammatory bowel diseases, 2011. 17(11): p. E140-E142. 194. Ryan, P., et al., Bacterial DNA within granulomas of patients with Crohn's disease— detection by laser capture microdissection and PCR. The American journal of gastroenterology, 2004. 99(8): p. 1539-1543. 195. Dalton, J.P., et al., Detection of Mycobacterium avium subspecies paratuberculosis in patients with Crohn’s disease is unrelated to the presence of single nucleotide polymorphisms rs2241880 (ATG16L1) and rs10045431 (IL12B). Medical microbiology and immunology, 2014. 203(3): p. 195-205. 196. Bernstein, C.N., et al., Testing the interaction between NOD-2 status and serological response to Mycobacterium paratuberculosis in cases of inflammatory bowel disease. Journal of clinical microbiology, 2007. 45(3): p. 968-971. 197. Papamichael, K., P. Konstantopoulos, and G.J. Mantzaris, Helicobacter pylori infection and inflammatory bowel disease: Is there a link? World journal of gastroenterology: WJG, 2014. 20(21): p. 6374. 198. Bohr, U.R., et al., Identification of enterohepatic Helicobacter species in patients suffering from inflammatory bowel disease. Journal of clinical microbiology, 2004. 42(6): p. 2766-2768. 199. Hold, G.L., et al., Role of the gut microbiota in inflammatory bowel disease pathogenesis: what have we learnt in the past 10 years? World journal of gastroenterology: WJG, 2014. 20(5): p. 1192. 200. Zhang, S., et al., Role of Helicobacter species in Chinese patients with inflammatory bowel disease. Journal of clinical microbiology, 2011. 49(5): p. 1987-1989. 201. Rokkas, T., et al., The association between Helicobacter pylori infection and inflammatory bowel disease based on meta-analysis. United European Gastroenterology Journal, 2015: 60(2): p. 20-50. 202. Mukhopadhya, I., et al., IBD—what role do Proteobacteria play? Nature Reviews Gastroenterology and Hepatology, 2012. 9(4): p. 219-230. 203. Zhang, L., et al., Detection and isolation of Campylobacter species other than C. jejuni from children with Crohn's disease. Journal of clinical microbiology, 2009. 47(2): p. 453- 455. 204. Man, S.M., et al., Campylobacter concisus and other Campylobacter species in children with newly diagnosed Crohn's disease. Inflammatory bowel diseases, 2010. 16(6): p. 1008-1016. 205. Kovach, Z., et al., Immunoreactive proteins of Campylobacter concisus, an emergent intestinal pathogen. FEMS Immunology & Medical Microbiology, 2011. 63(3): p. 387- 396. 206. Zhang, L., et al., Campylobacter concisus and inflammatory bowel disease. World journal of gastroenterology: WJG, 2014. 20(5): p. 1259. 207. Chassaing, B., et al., Crohn disease–associated adherent-invasive E. coli bacteria target mouse and human Peyer’s patches via long polar fimbriae. The Journal of clinical investigation, 2011. 121(3): p. 966. 208. Martin, H.M., et al., Enhanced Escherichia coli adherence and invasion in Crohn’s disease and colon cancer. Gastroenterology, 2004. 127(1): p. 80-93. 209. Ehsani, L., et al., Fatal aortic pseudoaneurysm from disseminated Mycobacterium kansasii infection: case report. Human pathology, 2015. 46(3): p. 467-470. 210. Yin, S.M., et al., Disseminated Mycobacterium kansasii Disease in Complete DiGeorge Syndrome. Journal of Clinical Immunology, 2015: p. 1-4. 211. Kaur, P., et al., Disseminated Mycobacterium kansasii infection with hepatic abscesses in a renal transplant recipient. Transplant Infectious Disease, 2011. 13(5): p. 531-535. 212. Srivastava, S., et al., Rapid Drug Tolerance and Dramatic Sterilizing Effect of Moxifloxacin Monotherapy in a Novel Hollow-Fiber Model of Intracellular Mycobacterium kansasii Disease. Antimicrobial agents and chemotherapy, 2015. 59(4): p. 2273-2279. 213. Park, H., et al., Clinical Significance Of Mycobacterium Kansasii Isolates From Respiratory Specimens. Am J Respir Crit Care Med, 2015. 191: p. A6290. 214. Cadwell, K., et al., Virus-Plus-Susceptibility Gene Interaction Determines Crohn's Disease Gene< i> Atg16L1 Phenotypes in Intestine. Cell, 2010. 141(7): p. 1135-1145. 215. Brant, S.R., Promises, delivery, and challenges of inflammatory bowel disease risk gene discovery. Clinical gastroenterology and hepatology: the official clinical practice journal of the American Gastroenterological Association, 2013. 11(1): p. 22-26. 216. Amre, D.K., et al., NELL1, NCF4, and FAM92B genes are not major susceptibility genes for Crohn's disease in canadian children and young adults. Inflammatory Bowel Diseases, 2012. 18(3): p. 529-535. 217. Wang, M.-H., et al., A novel approach to detect cumulative genetic effects and genetic interactions in Crohn's Disease. Inflammatory bowel diseases, 2013. 19(9): p. 1799- 1808. 218. Van Limbergen, J., D.C. Wilson, and J. Satsangi, The genetics of Crohn's disease. Annual review of genomics and human genetics, 2009. 10: p. 89-116. 219. Philpott, D.J. and J. Viala, Towards an understanding of the role of NOD2/CARD15 in the pathogenesis of Crohn's disease. Best practice & research. Clinical gastroenterology, 2004. 18(3): p. 555. 220. Wehkamp, J., et al., NOD2 (CARD15) mutations in Crohn’s disease are associated with ĚŝŵŝŶŝƐŚĞĚŵƵĐŽƐĂůɲ-defensin expression. Gut, 2004. 53(11): p. 1658. 221. Rigoli, L., et al., Clinical significance of NOD2/CARD15 and Toll-like receptor 4 gene single nucleotide polymorphisms in inflammatory bowel disease. World Journal of Gastroenterology: WJG, 2008. 14(28): p. 4454. 222. Li, J., et al., Regulation of IL-8 and IL-ϭɴ ĞdžƉƌĞƐƐŝŽŶ ŝŶ ƌŽŚŶΖƐ ĚŝƐĞĂƐĞ ĂƐƐŽĐŝĂƚĞĚ NOD2/CARD15 mutations. Human molecular genetics, 2004. 13(16): p. 1715. 223. Massey, D.C.O. and M. Parkes, Genome-wide association scanning highlights two autophagy genes, ATG16L1 and IRGM, as being significantly associated with Crohn's disease. Autophagy, 2007. 3(6): p. 649. 224. Roberts, R.L., et al., IL23R R381Q and ATG16L1 T300A are strongly associated with Crohn's disease in a study of New Zealand Caucasians with inflammatory bowel disease. The American journal of gastroenterology, 2007. 102(12): p. 2754-2761. 225. McGovern, D. and F. Powrie, The IL23 axis plays a key role in the pathogenesis of IBD. Gut, 2007. 56(10): p. 1333. 226. Taylor, K.D., et al., IL23R haplotypes provide a large population attributable risk for Crohn's disease. Inflammatory bowel diseases, 2008. 14(9): p. 1185-1191. 227. Dubinsky, M.C., et al., IL-23 receptor (IL-23R) gene protects against pediatric Crohn's disease. Inflammatory bowel diseases, 2007. 13(5): p. 511-515. 228. Di Meglio, P., et al., The IL23R R381Q gene variant protects against immune-mediated diseases by impairing IL-23-induced Th17 effector response in humans. PLoS One, 2011. 6(2): p. e17160. 229. Glas, J., et al., The ATG16L1 gene variants rs2241879 and rs2241880 (T300A) are strongly associated with susceptibility to Crohn's disease in the German population. The American journal of gastroenterology, 2008. 103(3): p. 682-691. 230. Hampe, J., et al., A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1. Nature genetics, 2006. 39(2): p. 207- 211. 231. Cheng, J.F., et al., T300A polymorphism of ATG16L1 and susceptibility to inflammatory bowel diseases: a meta-analysis. World Journal of Gastroenterology: WJG, 2010. 16(10): p. 1258. 232. Segundo González, M., et al., TNF-alpha-308A promoter polymorphism is associated with enhanced TNF-alpha production and inflammatory activity in Crohn's patients with fistulizing disease. The American journal of gastroenterology, 2003. 98: p. 1101-1106. 233. Levine, A., et al., A polymorphism in the TNF-ɲ ƉƌŽŵŽƚĞƌ ŐĞŶĞ ŝƐ ĂƐƐŽĐŝĂƚĞĚ ǁŝƚŚ pediatric onset and colonic location of Crohn's disease. The American journal of gastroenterology, 2005. 100(2): p. 407-413. 234. Apostolopoulos, P., Environmental Factors in IBD. Annals of Gastroenterology, 2006. 19(2). 235. Jakobsen, C., et al., Environmental factors and risk of developing paediatric inflammatory bowel disease—A population based study 2007–2009. Journal of Crohn's and Colitis, 2013. 7(1): p. 79-88. 236. Neuman, M.G. and R.M. Nanau, Inflammatory bowel disease: role of diet, microbiota, life style. Translational Research, 2012. 160(1): p. 29-44. 237. Hanauer, S.B., Inflammatory bowel disease: epidemiology, pathogenesis, and therapeutic opportunities. Inflammatory bowel diseases, 2006. 12(5): p. S3-S9. 238. Sakamoto, N., et al., Dietary risk factors for inflammatory bowel disease A Multicenter Case Control Study in Japan. Inflammatory bowel diseases, 2005. 11(2): p. 154-163. 239. Stewart, M., A.S. Day, and A. Otley, Physician attitudes and practices of enteral nutrition as primary treatment of paediatric Crohn disease in North America. Journal of pediatric gastroenterology and nutrition, 2011. 52(1): p. 38-42. 240. Loh, G., Interactions between bacteria, host and diet in the intestine. 2013, Freie Universität Berlin, Germany. 241. Kau, A.L., et al., Human nutrition, the gut microbiome and the immune system. Nature, 2011. 474(7351): p. 327-336. 242. Tlaskalová-Hogenová, H., et al., The role of gut microbiota (commensal bacteria) and the mucosal barrier in the pathogenesis of inflammatory and autoimmune diseases and cancer: contribution of germ-free and gnotobiotic animal models of human diseases. Cellular & molecular immunology, 2011. 8(2): p. 110-120. 243. Levine, A. and E. Wine, Effects of enteral nutrition on Crohn’s disease: clues to the impact of diet on disease pathogenesis. Inflammatory bowel diseases, 2013. 19(6): p. 1322-1329. 244. Flint, H.J., Obesity and the gut microbiota. Journal of clinical gastroenterology, 2011. 45: p. S128-S132. 245. Gibson, G.R., et al., Dietary modulation of the human colonic microbiota: updating the concept of prebiotics. Nutr Res Rev, 2004. 17(2): p. 259-275. 246. Faith, J.J., et al., Predicting a human gut microbiota’s response to diet in gnotobiotic mice. Science, 2011. 333(6038): p. 101-104. 247. Hamer, H.M., et al., Review article: the role of butyrate on colonic function. Alimentary pharmacology & therapeutics, 2008. 27(2): p. 104-119. 248. Simpson, H. and B. Campbell, Review article: dietary fibre–microbiota interactions. Alimentary pharmacology & therapeutics, 2015. 249. De Filippo, C., et al., Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proceedings of the National Academy of Sciences, 2010. 107(33): p. 14691-14696. 250. Duncan, S.H., et al., Reduced dietary intake of carbohydrates by obese subjects results in decreased concentrations of butyrate and butyrate-producing bacteria in feces. Applied and environmental microbiology, 2007. 73(4): p. 1073-1078. 251. Russell, W.R., et al., High-protein, reduced-carbohydrate weight-loss diets promote metabolite profiles likely to be detrimental to colonic health. The American journal of clinical nutrition, 2011. 93(5): p. 1062-1072. 252. Devkota, S., et al., Dietary-fat-induced taurocholic acid promotes pathobiont expansion and colitis in Il10-/-mice. Nature, 2012. 487(7405): p. 104-108. 253. Roberts, C.L., et al., Translocation of Crohn's disease Escherichia coli across M-cells: contrasting effects of soluble plant fibres and emulsifiers. Gut, 2010. 59(10): p. 1331- 1339. 254. El-Tawil, A., Zinc supplementation tightens leaky gut in Crohn's disease. Inflammatory bowel diseases, 2012. 18(2): p. E399-E399. 255. Söderholm, J.D., et al., Augmented increase in tight junction permeability by luminal stimuli in the non-inflamed ileum of Crohn's disease. Gut, 2002. 50(3): p. 307-313. 256. Lammers, K.M., et al., Gliadin induces an increase in intestinal permeability and zonulin release by binding to the chemokine receptor CXCR3. Gastroenterology, 2008. 135(1): p. 194-204. e3. 257. Drago, S., et al., Gliadin, zonulin and gut permeability: Effects on celiac and non-celiac intestinal mucosa and intestinal cell lines. Scandinavian journal of gastroenterology, 2006. 41(4): p. 408-419. 258. Csáki, K.F., Synthetic surfactant food additives can cause intestinal barrier dysfunction. Medical hypotheses, 2011. 76(5): p. 676-681. 259. Aujnarain, A., D.R. Mack, and E.I. Benchimol, The role of the environment in the development of pediatric inflammatory bowel disease. Current gastroenterology reports, 2013. 15(6): p. 1-11. 260. Renz, H., P. Brandtzaeg, and M. Hornef, The impact of perinatal immune development on mucosal homeostasis and chronic inflammation. Nature Reviews Immunology, 2011. 12(1): p. 9-23. 261. Danese, S. and C. Fiocchi, Etiopathogenesis of inflammatory bowel diseases. World Journal of Gastroenterology, 2006. 12(30): p. 4807. 262. Molodecky, N.A. and G.G. Kaplan, Environmental risk factors for inflammatory bowel disease. Gastroenterology & hepatology, 2010. 6(5): p. 339. 263. Lakatos, P.L., et al., Is current smoking still an important environmental factor in inflammatory bowel diseases? Results from a population-based incident cohort. Inflammatory bowel diseases, 2013. 19(5): p. 1010-1017. 264. Johnson, G., J. Cosnes, and J. Mansfield, Review article: smoking cessation as primary therapy to modify the course of Crohn's disease. Alimentary pharmacology & therapeutics, 2005. 21(8): p. 921-931. 265. Nielsen, O.H., et al., Influence of smoking on colonic gene expression profile in Crohn's disease. PLoS One, 2009. 4(7): p. e6210. 266. Cottone, M., et al., Smoking habits and recurrence in Crohn's disease. Gastroenterology, 1994. 106: p. 643-643. 267. Timmer, A., L.R. Sutherland, and F. Martin, Oral contraceptive use and smoking are risk factors for relapse in Crohn's disease. Gastroenterology, 1998. 114(6): p. 1143-1150. 268. Cosnes, J., et al., Smoking cessation and the course of Crohn's disease: an intervention study. Gastroenterology, 2001. 120(5): p. 1093-1099. 269. Kaplan, G.G., et al., The risk of developing Crohn's disease after an appendectomy: a meta-analysis. The American journal of gastroenterology, 2008. 103(11): p. 2925-2931. 270. Koutroubakis, I. and I. Vlachonikolis, Appendectomy and the development of ulcerative colitis: results of a metaanalysis of published case-control studies. The American journal of gastroenterology, 2000. 95(1): p. 171-176. 271. Leighton, J.A., et al., Capsule endoscopy in suspected small bowel Crohn’s disease: economic impact of disease diagnosis and treatment. World Journal of Gastroenterology: WJG, 2009. 15(45): p. 5685. 272. Stange, E., et al., European evidence based consensus on the diagnosis and management of Crohn’s disease: definitions and diagnosis. Gut, 2006. 55(suppl 1): p. i1. 273. Lemberg, D.A. and A.S. Day, Crohn disease and ulcerative colitis in children: An update for 2014. Journal of paediatrics and child health, 2014. 274. Neurath, M.F., Cytokines in inflammatory bowel disease. Nature Reviews Immunology, 2014. 14(5): p. 329-342. 275. Fell, J., Update of the management of inflammatory bowel disease. Archives of Disease in Childhood, 2011. 276. Khan, K., et al., Role of serology and routine laboratory tests in childhood inflammatory bowel disease. Inflammatory bowel diseases, 2002. 8(5): p. 325-329. 277. Markowitz, J., et al., Age of diagnosis influences serologic responses in children with Crohn's disease: A possible clue to etiology? Inflammatory bowel diseases, 2009. 15(5): p. 714-719. 278. Russell, R., et al., Anti-Saccharomyces cerevisiae antibodies status is associated with oral involvement and disease severity in Crohn disease. Journal of pediatric gastroenterology and nutrition, 2009. 48(2): p. 161. 279. Dotan, I., et al., Antibodies against laminaribioside and chitobioside are novel serologic markers in Crohn's disease. Gastroenterology, 2006. 131(2): p. 366-378. 280. Sidler, M.A., S.T. Leach, and A.S. Day, Fecal S100A12 and fecal calprotectin as noninvasive markers for inflammatory bowel disease in children. Inflammatory bowel diseases, 2008. 14(3): p. 359-366. 281. de Jong, N.S., S.T. Leach, and A.S. Day, Fecal S100A12: a novel noninvasive marker in children with Crohn's disease. Inflammatory bowel diseases, 2006. 12(7): p. 566-572. 282. Pfefferkorn, M.D., et al., Utility of fecal lactoferrin in identifying Crohn disease activity in children. Journal of pediatric gastroenterology and nutrition, 2010. 51(4): p. 425-428. 283. Bunn, S.K., et al., Fecal calprotectin: validation as a noninvasive measure of bowel inflammation in childhood inflammatory bowel disease. Journal of pediatric gastroenterology and nutrition, 2001. 33(1): p. 14-22. 284. Leighton, J.A., The role of endoscopic imaging of the small bowel in clinical practice. The American journal of gastroenterology, 2010. 106(1): p. 27-36. 285. Triester, S.L., et al., A meta-analysis of the yield of capsule endoscopy compared to other diagnostic modalities in patients with non-stricturing small bowel Crohn's disease. The American journal of gastroenterology, 2006. 101(5): p. 954-964. 286. Best, W.R., et al., Development of a Crohn's disease activity index. National Cooperative Crohn's Disease Study. Gastroenterology, 1976. 70(3): p. 439. 287. Hyams, J.S., et al., Development and validation of a pediatric Crohn's disease activity index. Journal of pediatric gastroenterology and nutrition, 1991. 12(4): p. 449. 288. Hyams, J., et al., Evaluation of the pediatric Crohn disease activity index: a prospective multicenter experience. Journal of pediatric gastroenterology and nutrition, 2005. 41(4): p. 416-421. 289. Otley, A., et al., Assessing activity of pediatric Crohn's disease: Which index to use? Gastroenterology, 1999. 116(3): p. 527-531. 290. Leach, S.T., et al., Development and assessment of a modified pediatric Crohn disease activity index. Journal of pediatric gastroenterology and nutrition, 2010. 51(2): p. 232- 236. 291. Ephgrave, K., Extra-intestinal manifestations of Crohn's disease. Surgical Clinics of North America, 2007. 87(3): p. 673-680. 292. Ahmad, T., et al., The molecular classification of the clinical manifestations of Crohn's disease. Gastroenterology, 2002. 122(4): p. 854-866. 293. Repiso, A., et al., Extraintestinal manifestations of Crohn's disease: prevalence and related factors. Revista Espanola de Enfermedades Digestivas, 2006. 98(7): p. 510. 294. Mendoza, J., et al., Extraintestinal manifestations in inflammatory bowel disease: differences between Crohn's disease and ulcerative colitis]. Medicina clínica, 2005. 125(8): p. 297. 295. Jiang, L., et al., Retrospective survey of 452 patients with inflammatory bowel disease in Wuhan city, central China. Inflammatory bowel diseases, 2006. 12(3): p. 212-217. 296. Salvarani, C. and W. Fries, Clinical features and epidemiology of spondyloarthritides associated with inflammatory bowel disease. World Journal of Gastroenterology: WJG, 2009. 15(20): p. 2449. 297. Tomasello, G., M. D'arienzo, and A. Sanflippo, Arthritis and Crohn's disease: Our Experience. Journal Of Orthopedics, 2009. 298. Dziechciarz, P., et al., Meta-analysis: enteral nutrition in active Crohn’s disease in children. Alimentary pharmacology & therapeutics, 2007. 26(6): p. 795-806. 299. Newby, E., et al., Interventions for growth failure in childhood Crohn’s disease. Cochrane Database Syst Rev, 2005. 3. 300. Sawczenko, A. and B. Sandhu, Presenting features of inflammatory bowel disease in Great Britain and Ireland. Archives of Disease in Childhood, 2003. 88(11): p. 995-1000. 301. Yamamoto, T., M. Nakahigashi, and A. Saniabadi, Review article: diet and inflammatory bowel disease–epidemiology and treatment. Alimentary pharmacology & therapeutics, 2009. 30(2): p. 99-112. 302. Wiskin, A.E., S.A. Wootton, and R.M. Beattie, Nutrition issues in pediatric Crohn's disease. Nutrition in Clinical Practice, 2007. 22(2): p. 214-222. 303. Moorthy, D., K.L. Cappellano, and I.H. Rosenberg, Nutrition and Crohn's disease: an update of print and Web-based guidance. Nutrition reviews, 2008. 66(7): p. 387-397. 304. Goh, J. and C. O'Morain, Nutrition and adult inflammatory bowel disease. Alimentary pharmacology & therapeutics, 2003. 17(3): p. 307-320. 305. Shamir, R., Nutritional aspects in inflammatory bowel disease. Journal of pediatric gastroenterology and nutrition, 2009. 48: p. S86. 306. Hébuterne, X., et al., Nutritional consequences and nutrition therapy in Crohn's disease. Gastroenterologie Clinique et Biologique, 2009. 33: p. S235-S244. 307. Gerasimidis, K., P. McGrogan, and C. Edwards, The aetiology and impact of malnutrition in paediatric inflammatory bowel disease. Journal of Human Nutrition and Dietetics, 2011. 308. Savage, M., et al., Growth in Crohn's disease. Acta Paediatrica, 1999. 88: p. 89-92. 309. De Benedetti, F., et al., Interleukin 6 causes growth impairment in transgenic mice through a decrease in insulin-like growth factor-I. A model for stunted growth in children with chronic inflammation. Journal of Clinical Investigation, 1997. 99(4): p. 643. 310. Ballinger, A., et al., Growth failure occurs through a decrease in insulin-like growth factor 1 which is independent of undernutrition in a rat model of colitis. Gut, 2000. 46(5): p. 695. 311. MacRae, V., C. Farquharson, and S. Ahmed, The restricted potential for recovery of growth plate chondrogenesis and longitudinal bone growth following exposure to pro- inflammatory cytokines. Journal of endocrinology, 2006. 189(2): p. 319. 312. Wong, S., et al., The growth hormone insulin-like growth factor 1 axis in children and adolescents with inflammatory bowel disease and growth retardation. Clinical Endocrinology, 2010. 73(2): p. 220-228. 313. Bannerjee, K., et al., Anti-inflammatory and growth-stimulating effects precede nutritional restitution during enteral feeding in Crohn disease. Journal of pediatric gastroenterology and nutrition, 2004. 38(3): p. 270. 314. Street, M.E., et al., Relationships between serum IGF-1, IGFBP-2, interleukin-1beta and interleukin-6 in inflammatory bowel disease. Hormone Research in Paediatrics, 2004. 61(4): p. 159-164. 315. Shamir, R., M. Phillip, and A. Levine, Growth retardation in pediatric Crohn's disease: pathogenesis and interventions. Inflammatory bowel diseases, 2007. 13(5): p. 620-628. 316. Seidman, E., Nutritional therapy for Crohn's disease: Lessons from the Ste.-Justine hospital experience. Inflammatory bowel diseases, 1997. 3(1): p. 49-53. 317. Otley, A.R., R.K. Russell, and A.S. Day, Nutritional therapy for the treatment of pediatric Crohns disease. Expert Review of Clinical Immunology, 2010. 6(4): p. 667-676. 318. Wiskin, A.E., et al., Body composition in childhood inflammatory bowel disease. Clinical nutrition, 2011. 30(1): p. 112-5. 319. Harty, S., et al., A prospective study of the oral manifestations of Crohn's disease. Clinical Gastroenterology and Hepatology, 2005. 3(9): p. 886-891. 320. Jose, F.A., et al., Development of extraintestinal manifestations in pediatric patients with inflammatory bowel disease. Inflammatory bowel diseases, 2009. 15(1): p. 63-68. 321. Lisciandrano, D., et al., Prevalence of oral lesions in inflammatory bowel disease. The American journal of gastroenterology, 1996. 91(1): p. 7. 322. Natah, S., et al., Recurrent aphthous ulcers today: a review of the growing knowledge. International journal of oral and maxillofacial surgery, 2004. 33(3): p. 221-234. 323. Pittock, S., et al., The oral cavity in Crohn's disease. The Journal of pediatrics, 2001. 138(5): p. 767-771. 324. Farhi, D., et al., Misleading pustular plaques of the lower limbs during Crohn's disease: two case reports. Journal of Medical Case Reports, 2007. 1(1): p. 1-4. 325. Tavarela Veloso, F., Skin complications associated with inflammatory bowel disease. Alimentary pharmacology & therapeutics, 2004. 20: p. 50-53. 326. Levine, J.S. and R. Burakoff, Extraintestinal manifestations of inflammatory bowel disease. Gastroenterology & hepatology, 2011. 7(4): p. 235. 327. Palamaras, I., et al., Metastatic Crohn's disease: a review. Journal of the European Academy of Dermatology and Venereology, 2008. 22(9): p. 1033-1043. 328. Jose, F.A. and M.B. Heyman, Extraintestinal manifestations of inflammatory bowel disease. Journal of pediatric gastroenterology and nutrition, 2008. 46(2): p. 124. 329. Mintz, R., et al., Ocular manifestations of inflammatory bowel disease. Inflammatory bowel diseases, 2004. 10(2): p. 135-139. 330. Cury, D.B. and A.C. Moss, Ocular manifestations in a community-based cohort of patients with inflammatory bowel disease. Inflammatory bowel diseases, 2010. 16(8): p. 1393-1396. 331. Felekis, T., et al., Spectrum and frequency of ophthalmologic manifestations in patients with inflammatory bowel disease: A prospective single-center study. Inflammatory bowel diseases, 2009. 15(1): p. 29-34. 332. Larsen, S., K. Bendtzen, and O.H. Nielsen, Extraintestinal manifestations of inflammatory bowel disease: epidemiology, diagnosis, and management. Annals of medicine, 2010. 42(2): p. 97-114. 333. Pardi, D.S., et al., Renal and urologic complications of inflammatory bowel disease. The American journal of gastroenterology, 1998. 93(4): p. 504-514. 334. Navaneethan, U. and B. Shen, Hepatopancreatobiliary manifestations and complications associated with inflammatory bowel disease. Inflammatory bowel diseases, 2010. 16(9): p. 1598-1619. 335. Rothfuss, K.S., E.F. Stange, and K.R. Herrlinger, Extraintestinal manifestations and complications in inflammatory bowel diseases. World Journal of Gastroenterology, 2006. 12(30): p. 4819. 336. Chapman, R.W., Primary sclerosing cholangitis. Medicine, 2011. 39(10): p. 588-591. 337. Boonstra, K., et al., Primary sclerosing cholangitis]. Nederlands tijdschrift voor geneeskunde, 2010. 154: p. A1476. 338. Lindström, L., et al., Increased Risk of Colorectal Cancer and Dysplasia in Patients With Crohn's Colitis and Primary Sclerosing Cholangitis. Diseases of the Colon & Rectum, 2011. 54(11): p. 1392. 339. Barbul, A., et al., Arginine enhances wound healing and lymphocyte immune responses in humans. Surgery, 1990. 108(2): p. 331-6; discussion 336-7. 340. Lichtenstein, G.R., Emerging prognostic markers to determine Crohn's disease natural history and improve management strategies: a review of recent literature. Gastroenterology & Hepatology, 2010. 6(2): p. 99. 341. Cummings, J.R.F., S. Keshav, and S.P.L. Travis, Medical management of Crohn’s disease. BMJ: British Medical Journal, 2008. 336(7652): p. 1062. 342. Travis, S., et al., European evidence based consensus on the diagnosis and management of Crohn’s disease: current management. Gut, 2006. 55(suppl 1): p. i16. 343. Rampton, D.S., Management of Crohn's disease. BMJ, 1999. 319(7223): p. 1480. 344. Scribano, M. and C. Prantera, Medical treatment of active Crohn's disease. Alimentary pharmacology & therapeutics, 2002. 16: p. 35-39. 345. Gionchetti, P., et al., Role of conventional therapies in the era of biological treatment in Crohn’s disease. World Journal of Gastroenterology: WJG, 2011. 17(14): p. 1797. 346. Yang, Y.X. and G.R. Lichtenstein, Corticosteroids in Crohn's disease. The American journal of gastroenterology, 2002. 97(4): p. 803-823. 347. Irving, P., et al., Review article: appropriate use of corticosteroids in Crohn’s disease. Alimentary pharmacology & therapeutics, 2007. 26(3): p. 313-329. 348. Benchimol, E., et al., Traditional corticosteroids for induction of remission in Crohn's disease. Cochrane Database of Systematic Reviews, 2008. 2. 349. Faubion Jr, W.A., et al., Alimentary Tract. Gastroenterology, 2001. 121(2): p. 255-260. 350. Ordás, I., B.G. Feagan, and W.J. Sandborn, Early use of immunosuppressives or TNF antagonists for the treatment of Crohn's disease: time for a change. Gut, 2011. 60(12): p. 1754-1763. 351. Rutgeerts, P., et al., A comparison of budesonide with prednisolone for active Crohn's disease. New England Journal of Medicine, 1994. 331(13): p. 842-845. 352. Lichtenstein, G.R., et al., American Gastroenterological Association Institute technical review on corticosteroids, immunomodulators, and infliximab in inflammatory bowel disease. Gastroenterology, 2006. 130(3): p. 940. 353. Smith, P.A., Nutritional therapy for active Crohn’s disease. World journal of gastroenterology: WJG, 2008. 14(27): p. 4420. 354. Chassaing, B., et al., Dextran Sulfate Sodium (DSS)-Induced Colitis in Mice. Current Protocols in Immunology, 2014: p. 15.25. 1-15.25. 14. 355. Kane, S., et al., Systematic review: the effectiveness of budesonide therapy for Crohn's disease. Alimentary pharmacology & therapeutics, 2002. 16(8): p. 1509-1517. 356. Seow, C., et al., Budesonide for induction of remission in Crohn's disease. Cochrane Database of Systematic Reviews, 2008. 3(3). 357. Ford, A.C., et al., Glucocorticosteroid therapy in inflammatory bowel disease: systematic review and meta-analysis. The American journal of gastroenterology, 2011. 106(4): p. 590-599. 358. Costa, K.A., et al., l-Arginine Supplementation Prevents Increases in Intestinal Permeability and Bacterial Translocation in Male Swiss Mice Subjected to Physical Exercise under Environmental Heat Stress. The Journal of nutrition, 2014. 144(2): p. 218- 223. 359. Kozuch, P.L. and S.B. Hanauer, Treatment of inflammatory bowel disease: a review of medical therapy. World journal of gastroenterology: WJG, 2008. 14(3): p. 354. 360. Ardizzone, S., et al., Review: Immunomodulators for all patients with inflammatory bowel disease? Therapeutic Advances in Gastroenterology, 2010. 3(1): p. 31. 361. Biancone, L., et al., Maintenance treatment of Crohn's disease. Alimentary pharmacology & therapeutics, 2003. 17: p. 31-37. 362. Mantzaris, G.J., et al., Azathioprine is superior to budesonide in achieving and maintaining mucosal healing and histologic remission in steroid-dependent Crohn's disease. Inflammatory bowel diseases, 2009. 15(3): p. 375-382. 363. Reinisch, W., et al., A multicenter, randomized, double-blind trial of everolimus versus azathioprine and placebo to maintain steroid-induced remission in patients with moderate-to-severe active Crohn's disease. The American journal of gastroenterology, 2008. 103(9): p. 2284-2292. 364. Derijks, L., et al., Review article: thiopurines in inflammatory bowel disease. Alimentary pharmacology & therapeutics, 2006. 24(5): p. 715-729. 365. Das, K.M. and S.A. Farag, Current medical therapy of inflammatory bowel disease. World Journal of Gastroenterology, 2000. 6(4): p. 483-489. 366. Riello, L., et al., Tolerance and efficacy of azathioprine in pediatric Crohn's disease. Inflammatory bowel diseases, 2011. 367. Fraser, A., T. Orchard, and D. Jewell, The efficacy of azathioprine for the treatment of inflammatory bowel disease: a 30 year review. Gut, 2002. 50(4): p. 485. 368. Khan, K.J., et al., Efficacy of immunosuppressive therapy for inflammatory bowel disease: a systematic review and meta-analysis. American Journal of Gastroenterology, 2011. 106(4): p. 630-42. 369. Van Assche, G., et al., The second European evidence-based Consensus on the diagnosis and management of Crohn's disease: Special situations. J Crohns Colitis, 2010. 4(1): p. 63-101. 370. Picco, M.F., et al., Immunomodulators are associated with a lower risk of first surgery among patients with non-penetrating non-stricturing Crohn's disease. The American journal of gastroenterology, 2009. 104(11): p. 2754-2759. 371. Colombel, J.F., et al., Infliximab, azathioprine, or combination therapy for Crohn's disease. New England Journal of Medicine, 2010. 362(15): p. 1383-1395. 372. Waters, O.R. and I.C. Lawrance, Understanding the use of immunosuppressive agents in the clinical management of IBD. Current Drug Targets, 2011. 12(9): p. 1364-71. 373. Costantino, G., et al., Thiopurine treatment in inflammatory bowel disease: Response predictors, safety, and withdrawal in follow-up. Journal of Crohn's and Colitis, 2011. 374. Ansari, A., et al., Thiopurine methyltransferase activity and the use of azathioprine in inflammatory bowel disease. Alimentary pharmacology & therapeutics, 2002. 16(10): p. 1743-1750. 375. Willot, S., A. Noble, and C. Deslandres, Methotrexate in the treatment of inflammatory bowel disease: An 8-year retrospective study in a Canadian pediatric IBD center. Inflammatory bowel diseases, 2011. 376. Feagan, B.G., et al., A comparison of methotrexate with placebo for the maintenance of remission in Crohn's disease. New England Journal of Medicine, 2000. 342(22): p. 1627- 1632. 377. Akobeng, A. and E. Gardener, Oral 5-aminosalicylic acid for maintenance of medically- induced remission in Crohn’s disease. Cochrane Database of Systematic Reviews, 2005. 1. 378. Lichtenstein, G.R., S.B. Hanauer, and W.J. Sandborn, Management of Crohn's disease in adults. The American journal of gastroenterology, 2009. 104(2): p. 465-483. 379. Ford, A.C., et al., Efficacy of 5-aminosalicylates in Crohn's disease: systematic review and meta-analysis. The American journal of gastroenterology, 2011. 106(4): p. 617-629. 380. Lim, W.C. and S. Hanauer, Aminosalicylates for induction of remission or response in Crohn’s disease. Cochrane Database of Systematic Reviews, 2010. 12. 381. Levesque, B.G. and S.V. Kane, Searching for the Delta: 5-Aminosalicylic Acid Therapy for Crohn's Disease. Gastroenterology & Hepatology, 2011. 7(5): p. 295. 382. Gordon, M., et al., Oral 5-aminosalicylic acid for maintenance of surgically-in-duced remission in Crohn’s disease. status and date: Edited (no change to conclusions), published in, 2011(7). 383. Ford, A.C., et al., 5-aminosalicylates prevent relapse of Crohn's disease after surgically induced remission: systematic review and meta-analysis. The American journal of gastroenterology, 2010. 384. Rutgeerts, P., S. Vermeire, and G. Van Assche, Biological therapies for inflammatory bowel diseases. Gastroenterology, 2009. 136(4): p. 1182-1197. 385. Ricart, E., et al., Are we giving biologics too late? The case for early versus late use. World journal of gastroenterology: WJG, 2008. 14(36): p. 5523. 386. Ford, A.C., et al., Efficacy of biological therapies in inflammatory bowel disease: systematic review and meta-analysis. The American journal of gastroenterology, 2011. 106(4): p. 644-659. 387. Allez, M., et al., The efficacy and safety of a third anti-TNF monoclonal antibody in Crohn’s disease after failure of two other anti-TNF antibodies. Alimentary pharmacology & therapeutics, 2010. 31(1): p. 92-101. 388. Danese, S., et al., Review article: infliximab for Crohn’s disease treatment–shifting therapeutic strategies after 10 years of clinical experience. Alimentary pharmacology & therapeutics, 2011. 389. Arnott, I.D.R., et al., Clinical use of infliximab in Crohn’s disease: the Edinburgh experience. Alimentary pharmacology & therapeutics, 2001. 15(10): p. 1639-1646. 390. Ten Hove, T., et al., Infliximab treatment induces apoptosis of lamina propria T lymphocytes in Crohn's disease. Gut, 2002. 50(2): p. 206. 391. Rutgeerts, P., G. Assche, and S. Vermeire, Review article: infliximab therapy for inflammatory bowel disease–seven years on. Alimentary pharmacology & therapeutics, 2006. 23(4): p. 451-463. 392. Nielsen, O.H., et al., Use of biological molecules in the treatment of inflammatory bowel disease. Journal of internal medicine, 2011. 393. Sinitsky, D.M., et al., Infliximab improves inflammation and anthropometric measures in pediatric Crohn's disease. Journal of gastroenterology and hepatology, 2010. 25(4): p. 810-816. 394. Hyams, J.S., et al., Long-term outcome of maintenance infliximab therapy in children with Crohn's disease. Inflammatory bowel diseases, 2009. 15(6): p. 816-822. 395. Sorrentino, D., et al., Low-dose maintenance therapy with infliximab prevents postsurgical recurrence of Crohn's disease. Clinical Gastroenterology and Hepatology, 2010. 8(7): p. 591-599. e1. 396. Panes, J., et al., Crohns disease: a review of current treatment with a focus on biologics. Drugs, 2007. 67(17): p. 2511-2537. 397. Duff, S., et al., Infliximab and surgical treatment of complex anal Crohn’s disease. Colorectal Disease, 2011. 398. D'Haens, G., et al., Early combined immunosuppression or conventional management in patients with newly diagnosed Crohn's disease: an open randomised trial. The Lancet, 2008. 371(9613): p. 660-667. 399. Chowers, Y. and M. Allez, Efficacy of Anti-TNF in Crohns Disease: How Does it Work? Current Drug Targets, 2010. 11(2): p. 138-142. 400. Kirman, I., R.L. Whelan, and O.H. Nielsen, Infliximab: mechanism of action beyond TNF- [alpha] neutralization in inflammatory bowel disease. European journal of gastroenterology & hepatology, 2004. 16(7): p. 639. 401. Rutella, S., et al., Infliximab Therapy Inhibits Inflammation-Induced Angiogenesis in the Mucosa of Patients With Crohn's Disease. The American journal of gastroenterology, 2011. 106(4): p. 762-770. 402. Danese, S., et al., TNF-ɲ ďůŽĐŬĂĚĞ ĚŽǁŶ-regulates the CD40/CD40L pathway in the mucosal microcirculation: a novel anti-inflammatory mechanism of infliximab in Crohn’s disease. The Journal of Immunology, 2006. 176(4): p. 2617. 403. Baert, F., et al., Influence of immunogenicity on the long-term efficacy of infliximab in Crohn's disease. New England Journal of Medicine, 2003. 348(7): p. 601-608. 404. Moustou, A.E., et al., Cutaneous side effects of anti-tumor necrosis factor biologic therapy: a clinical review. Journal of the American Academy of Dermatology, 2009. 61(3): p. 486-504. 405. De Bie, C.I., et al., The duration of effect of infliximab maintenance treatment in paediatric Crohn’s disease is limited. Alimentary pharmacology & therapeutics, 2011. 406. Linskens, R., et al., The bacterial flora in inflammatory bowel disease: current insights in pathogenesis and the influence of antibiotics and probiotics. Scandinavian Journal of Gastroenterology, 2001. 36(234): p. 29-40. 407. Chacon, O., L.E. Bermudez, and R.G. Barletta, Johne's disease, inflammatory bowel disease, and Mycobacterium paratuberculosis. Annu. Rev. Microbiol., 2004. 58: p. 329- 363. 408. Erkan, P., et al., A retrospective trial comparing 5-ASA plus ciprofloxacin and metronidazole with 5-ASA and corticosteroids in the treatment of active Crohn's disease. Turkish Journal of Gastroenterology, 2000. 11(3): p. 227-230. 409. ISHIKAWA, T., et al., Metronidazole plus ciprofloxacin therapy for active Crohn's disease. Internal medicine, 2003. 42(4): p. 318-321. 410. Guslandi, M., Antibiotics for inflammatory bowel disease: do they work? European journal of gastroenterology & hepatology, 2005. 17(2): p. 145-147. 411. Sutherland, L., et al., Double blind, placebo controlled trial of metronidazole in Crohn's disease. Gut, 1991. 32(9): p. 1071. 412. Colombel, J.F., et al., A controlled trial comparing ciprofloxacin with mesalazine for the treatment of active Crohn's disease. The American journal of gastroenterology, 1999. 94(3): p. 674-678. 413. Arnold, G.L., et al., Preliminary study of ciprofloxacin in active Crohn's disease. Inflammatory bowel diseases, 2002. 8(1): p. 10-15. 414. Shafran, I. and P. Burgunder, Adjunctive antibiotic therapy with rifaximin may help reduce Crohn’s disease activity. Digestive diseases and sciences, 2010. 55(4): p. 1079- 1084. 415. Kale-Pradhan, P.B., et al., The role of antimicrobials in Crohn's disease. Expert Review of Gastroenterology & Hepatology, 2013. 7(3): p. 281-288. 416. Huang, J., et al., A meta-analysis of randomized controlled trials comparing antibacterial therapy with placebo in Crohn’s disease. Colorectal Disease, 2011. 13(6): p. 617-626. 417. Greenbloom, S.L., A.H. Steinhart, and G.R. Greenberg, Combination ciprofloxacin and metronidazole for active Crohn's disease. Canadian journal of gastroenterology= Journal canadien de gastroenterologie, 1998. 12(1): p. 53. 418. Levine, A. and D. Turner, Combined azithromycin and metronidazole therapy is effective in inducing remission in pediatric Crohn's disease. Journal of Crohn's and Colitis, 2011. 419. Prantera, C., et al., An antibiotic regimen for the treatment of active Crohn's disease: a randomized, controlled clinical trial of metronidazole plus ciprofloxacin. The American journal of gastroenterology, 1996. 91(2): p. 328. 420. Rutgeerts, P., et al., Ornidazole for prophylaxis of postoperative Crohn’s disease recurrence: a randomized, double-blind, placebo-controlled trial. Gastroenterology, 2005. 128(4): p. 856-861. 421. Rutgeerts, P., et al., Controlled trial of metronidazole treatment for prevention of Crohn's recurrence after ileal resection. Gastroenterology, 1995. 108(6): p. 1617-1621. 422. D'Haens, G.R., et al., Therapy of metronidazole with azathioprine to prevent postoperative recurrence of Crohn's disease: a controlled randomized trial. Gastroenterology, 2008. 135(4): p. 1123-1129. 423. Doherty, G., et al., Meta-analysis: targeting the intestinal microbiota in prophylaxis for post-operative Crohn’s disease. Alimentary pharmacology & therapeutics, 2010. 31(8): p. 802-809. 424. Weese, J.S., Evaluation of deficiencies in labeling of commercial probiotics. The Canadian Veterinary Journal, 2003. 44(12): p. 982. 425. Jones, J.L. and A.E. Foxx-Orenstein, The role of probiotics in inflammatory bowel disease. Digestive diseases and sciences, 2007. 52(3): p. 607-611. 426. Schrezenmeir, J. and M. de Vrese, Probiotics, prebiotics, and synbiotics—approaching a ĚĞĨŝŶŝƚŝŽŶϭ഻Ϯ഻ϯ͘ŵĞƌŝĐĂŶũŽƵƌŶĂůŽĨĐůŝŶŝĐĂůŶƵƚrition, 2001. 73(2): p. 361S-364S. 427. Jonkers, D. and R. Stockbrügger, Probiotics and inflammatory bowel disease. JRSM, 2003. 96(4): p. 167-171. 428. Fedorak, R.N. and K.L. Madsen, Probiotics and the management of inflammatory bowel disease. Inflammatory bowel diseases, 2004. 10(3): p. 286-299. 429. Ng, S., et al., Mechanisms of action of probiotics: recent advances. Inflammatory bowel diseases, 2009. 15(2): p. 300-310. 430. Yan, F. and D.B. Polk, Probiotics: progress toward novel therapies for intestinal diseases. Current Opinion in Gastroenterology, 2010. 26(2): p. 95. 431. Rolfe, V., et al., Probiotics for maintenance of remission in Crohn's disease. Cochrane Database of Systematic Reviews, 2006. 4. 432. Haller, D., et al., Guidance for substantiating the evidence for beneficial effects of probiotics: probiotics in chronic inflammatory bowel disease and the functional disorder irritable bowel syndrome. The Journal of nutrition, 2010. 140(3): p. 690S. 433. Rahimi, R., et al., A meta-analysis on the efficacy of probiotics for maintenance of remission and prevention of clinical and endoscopic relapse in Crohn’s disease. Digestive diseases and sciences, 2008. 53(9): p. 2524-2531. 434. Van Gossum, A., et al., Multicenter randomized-controlled clinical trial of probiotics (Lactobacillus johnsonii, LA1) on early endoscopic recurrence of Crohn's disease after ileo-caecal resection. Inflammatory bowel diseases, 2007. 13(2): p. 135-142. 435. Ocón, B., et al., Active hexose-correlated compound and Bifidobacterium longum BB536 exert symbiotic effects in experimental colitis. European journal of nutrition, 2013. 52(2): p. 457-466. 436. Fujimori, S., et al., High dose probiotic and prebiotic cotherapy for remission induction of active Crohn’s disease. Journal of gastroenterology and hepatology, 2007. 22(8): p. 1199-1204. 437. ĐŚŝƌƵƌŐŝĐnjŶĞ ĐŚŽƌŽďLJ >ĞƑŶŝŽǁƐŬŝĞŐŽ-Crohna, L., Surgical treatment of Crohn’s disease. Gastroenterologia Polska, 2010. 17(4): p. 261-266. 438. Peyrin-Biroulet, L., et al., The natural history of adult Crohn's disease in population- based cohorts. The American journal of gastroenterology, 2009. 105(2): p. 289-297. 439. Bernell, O., A. Lapidus, and G. Hellers, Risk factors for surgery and recurrence in 907 patients with primary ileocaecal Crohn's disease. British journal of surgery, 2002. 87(12): p. 1697-1701. 440. Fichera, A. and F. Michelassi, Surgical treatment of Crohn’s disease. Journal of Gastrointestinal Surgery, 2007. 11(6): p. 791-803. 441. Aarnio, M., J. Mecklin, and M. Voutilainen, The role of surgery in Crohn's disease: a retrospective analysis from a single hospital. Scandinavian journal of surgery: SJS: official organ for the Finnish Surgical Society and the Scandinavian Surgical Society, 2010. 99(4): p. 208. 442. Hurst, R.D., et al., Prospective study of the features, indications, and surgical treatment in 513 consecutive patients affected by Crohn's disease. Surgery, 1997. 122(4): p. 661- 668. 443. Farmer, R., W. Hawk, and R. Turnbull Jr, Indications for surgery in Crohn's disease: analysis of 500 cases. Gastroenterology, 1976. 71(2): p. 245. 444. Peyrin-Biroulet, L., et al., Impact of azathioprine and tumour necrosis factor antagonists on the need for surgery in newly diagnosed Crohn's disease. Gut, 2011. 60(7): p. 930- 936. 445. Rubio, A., et al., The efficacy of exclusive nutritional therapy in paediatric Crohn’s disease, comparing fractionated oral vs. continuous enteral feeding. Alimentary pharmacology & therapeutics, 2011. 446. Hiwatashi, N., Enteral nutrition for Crohn's disease in Japan. Diseases of the Colon & Rectum, 1997. 40: p. 48-53. 447. Wirtz, S., et al., Chemically induced mouse models of intestinal inflammation. Nature protocols, 2007. 2(3): p. 541-546. 448. Day, A., et al., Systematic review: nutritional therapy in paediatric Crohn’s disease. Alimentary pharmacology & therapeutics, 2008. 27(4): p. 293-307. 449. Solomon, L., et al., The dextran sulphate sodium (DSS) model of colitis: an overview. Comparative clinical pathology, 2010. 19(3): p. 235-239. 450. Coëffier, M.s., et al., Influence of glutamine on cytokine production by human gut in vitro. Cytokine, 2001. 13(3): p. 148-154. 451. Voitk, A.J., et al., Experience With Elemental Diet in the Treatment of Inflammatory Bowel Disease: Is This Primary Therapy? Archives of Surgery, 1973. 107(2): p. 329. 452. Heuschkel, R., Enteral nutrition in crohn disease: more than just calories. Journal of pediatric gastroenterology and nutrition, 2004. 38(3): p. 239. 453. Beattie, R.M., Enteral nutrition as primary therapy in childhood Crohn's disease: control of intestinal inflammation and anabolic response. Journal of Parenteral and Enteral Nutrition, 2005. 29(4 suppl): p. S151-S159. 454. Ruemmele, F.M., et al., Nutrition as primary therapy in pediatric Crohn's disease: fact or fantasy? The Journal of pediatrics, 2000. 136(3): p. 285. 455. Escher, J.C., et al., Treatment of inflammatory bowel disease in childhood: best available evidence. Inflammatory bowel diseases, 2003. 9(1): p. 34-58. 456. Heuschkel, R., Enteral nutrition should be used to induce remission in childhood Crohn’s disease. Dig Dis, 2009. 27(3): p. 297-305. 457. Levine, A., et al., Consensus and controversy in the management of pediatric Crohn disease: an international survey. Journal of pediatric gastroenterology and nutrition, 2003. 36(4): p. 464. 458. Lochs, H., et al., ESPEN guidelines on enteral nutrition: gastroenterology. Clinical nutrition, 2006. 25(2): p. 260-274. 459. Matstji, T., T. Sakurai, and T. Yao, Nutritional therapy for Crohn’s disease in Japan. Journal of gastroenterology, 2005. 40: p. 25-31. 460. Day, A.S., et al., Exclusive enteral feeding as primary therapy for Crohn’s disease in Australian children and adolescents: a feasible and effective approach. Journal of gastroenterology and hepatology, 2006. 21(10): p. 1609-1614. 461. Berni Canani, R., et al., Short-and long-term therapeutic efficacy of nutritional therapy and corticosteroids in paediatric Crohn's disease. Digestive and liver disease, 2006. 38(6): p. 381-387. 462. Heuschkel, R.B., et al., Enteral nutrition and corticosteroids in the treatment of acute Crohn's disease in children. Journal of pediatric gastroenterology and nutrition, 2000. 31(1): p. 8. 463. Buchanan, E., et al., The use of exclusive enteral nutrition for induction of remission in children with Crohn’s disease demonstrates that disease phenotype does not influence clinical remission. Alimentary pharmacology & therapeutics, 2009. 30(5): p. 501-507. 464. Yamamoto, T., et al., Impact of elemental diet on mucosal inflammation in patients with active Crohn's disease: cytokine production and endoscopic and histological findings. Inflammatory bowel diseases, 2005. 11(6): p. 580-588. 465. Borrelli, O., et al., Polymeric diet alone versus corticosteroids in the treatment of active pediatric Crohn's disease: a randomized controlled open-label trial. Clinical Gastroenterology and Hepatology, 2006. 4(6): p. 744-753. 466. Hartman, C., R. Eliakim, and R. Shamir, Nutritional status and nutritional therapy in inflammatory bowel diseases. World Journal of Gastroenterology: WJG, 2009. 15(21): p. 2570. 467. Knight, C., et al., Long-term outcome of nutritional therapy in paediatric Crohn's disease. Clinical nutrition, 2005. 24(5): p. 775-779. 468. Sanderson, I., et al., Remission induced by an elemental diet in small bowel Crohn's disease. Archives of disease in Childhood, 1987. 62(2): p. 123-127. 469. Seidman, E., et al., Elemental diet versus prednisone as initial therapy in Crohn’s disease: early and long term results. Gastroenterology, 1991. 100(5 Part 2): p. A250. 470. Seidman, E., et al., Semi-elemental (SE) diet versus prednisone in pediatric Crohn’s disease. Gastroenterology, 1993. 104: p. A778. 471. Thomas, A., F. Taylor, and V. Miller, Dietary intake and nutritional treatment in childhood Crohn's disease. Journal of pediatric gastroenterology and nutrition, 1993. 17(1): p. 75. 472. Ruuska, T., et al., Exclusive whole protein enteral diet versus prednisolone in the treatment of acute Crohn's disease in children. Journal of pediatric gastroenterology and nutrition, 1994. 19(2): p. 175. 473. Lucendo, A.J. and L.C. De Rezende, Importance of nutrition in inflammatory bowel disease. World journal of gastroenterology: WJG, 2009. 15(17): p. 2081. 474. Fiocchi, C., Production of inflammatory cytokines in the intestinal lamina propria. Immunologic research, 1991. 10(3): p. 239-246. 475. Beattie, R., et al., Polgmeric nutrition as the primary therapy in children with small bowel Crohn's disease. Alimentary pharmacology & therapeutics, 1994. 8(6): p. 609-615. 476. Breese, E., et al., The effect of treatment on lymphokine-secreting cells in the intestinal mucosa of children with Crohn's disease. Alimentary pharmacology & therapeutics, 1995. 9(5): p. 547-552. 477. Fell, J., et al., Mucosal healing and a fall in mucosal pro-inflammatory cytokine mRNA induced by a specific oral polymeric diet in paediatric Crohn's disease. Alimentary Pharmacology and Therapeutics, 2000. 14(3): p. 281-290. 478. Meister, D., et al., Anti-inflammatory effects of enteral diet components on Crohn's disease-affected tissues in vitro* 1. Digestive and liver disease, 2002. 34(6): p. 430-438. 479. de Jong, N.S.H., S.T. Leach, and A.S. Day, Polymeric Formula Has Direct Anti- Inflammatory Effects on Enterocytes in an in VitroModel of Intestinal Inflammation. Digestive diseases and sciences, 2007. 52(9): p. 2029-2036. 480. Nahidi, L., et al., Paediatric inflammatory bowel disease: A mechanistic approach to investigate exclusive enteral nutrition treatment. Scientifica, 2014. 2014. 481. Li, Y., et al., Influence of exclusive enteral nutrition therapy on visceral fat in patients with Crohn's disease. Inflammatory bowel diseases, 2014. 20(9): p. 1568-1574. 482. Desreumaux, P., et al., Inflammatory alterations in mesenteric adipose tissue in Crohn's disease. Gastroenterology, 1999. 117(1): p. 73-81. 483. Feng, Y., et al., Exclusive enteral nutrition ameliorates mesenteric adipose tissue alterations in patients with active Crohn's disease. Clinical Nutrition, 2014. 33(5): p. 850- 858. 484. Bamba, T., et al., Dietary fat attenuates the benefits of an elemental diet in active Crohn's disease: a randomized, controlled trial. European journal of gastroenterology & hepatology, 2003. 15(2): p. 151. 485. Gassull, M., et al., Fat composition may be a clue to explain the primary therapeutic effect of enteral nutrition in Crohn's disease: results of a double blind randomised multicentre European trial. Gut, 2002. 51(2): p. 164-168. 486. Zachos, M., M. Tondeur, and A.M. Griffiths, Enteral nutritional therapy for induction of remission in Crohn's disease. The Cochrane Library, 2007. 487. Gorard, D.A., Enteral nutrition in Crohn's disease: fat in the formula. European journal of gastroenterology & hepatology, 2003. 15(2): p. 115. 488. Mañé, J., et al., Partial Replacement of Dietary (n-6) Fatty Acids with Medium-Chain Triglycerides Decreases the Incidence of Spontaneous Colitis in Interleukin-10–Deficient Mice. The Journal of nutrition, 2009. 139(3): p. 603-610. 489. Shoda, R., et al., Epidemiologic analysis of Crohn disease in Japan: increased dietary intake of n-6 polyunsaturated fatty acids and animal protein relates to the increased incidence of Crohn disease in Japan. The American journal of clinical nutrition, 1996. 63(5): p. 741-745. 490. Hartman, C., et al., Nutritional supplementation with polymeric diet enriched with transforming growth factor-beta 2 for children with Crohn’s disease. Isr Med Assoc J, 2008. 10(7): p. 503-507. 491. D'Haens, G., Mucosal healing in pediatric Crohn's disease. The goal of medical treatment. Inflammatory Bowel Diseases, 2004. 10(4): p. 479-480. 492. Van Kemseke, C., J. Belaiche, and E. Louis, Frequently relapsing Crohn's disease is characterized by persistent elevation in interleukin-6 and soluble interleukin-2 receptor serum levels during remission. International journal of colorectal disease, 2000. 15(4): p. 206-210. 493. Afzal, N., et al., Improvement in quality of life of children with acute Crohn's disease does not parallel mucosal healing after treatment with exclusive enteral nutrition. Alimentary pharmacology & therapeutics, 2004. 20(2): p. 167-172. 494. McGuckin, M.A., et al., Intestinal barrier dysfunction in inflammatory bowel diseases. Inflammatory bowel diseases, 2009. 15(1): p. 100-113. 495. Wang, F., et al., Interferon-ɶĂŶĚtumor necrosis factor-ɲƐLJŶĞƌŐŝnjĞƚŽŝŶĚƵĐĞŝŶƚĞƐƚŝŶĂů epithelial barrier dysfunction by up-regulating myosin light chain kinase expression. The American journal of pathology, 2005. 166(2): p. 409-419. 496. Ma, T.Y., et al., Mechanism of TNF-ɲ ŵŽĚƵůĂƚŝŽŶ Žf Caco-2 intestinal epithelial tight junction barrier: role of myosin light-chain kinase protein expression. American Journal of Physiology-Gastrointestinal and Liver Physiology, 2005. 288(3): p. G422-G430. 497. Nahidi, L., et al., Differential effects of nutritional and non-nutritional therapies on intestinal barrier function in an in vitro model. Journal of gastroenterology, 2012. 47(2): p. 107-117. 498. Nahidi, L., et al., Inflammatory bowel disease therapies and gut function in a colitis mouse model. BioMed research international, 2013. 2013. 499. Lionetti, P., et al., P0127 PP Enteral Nutrtion-Induced Remission is asscociated with Profound modifications of intestinal microflora in Crohn's disease. Journal of Pediatric Gastroenterology and Nutrition, 2004. 39: p. S106. 500. Lionetti, P., et al., Enteral nutrition and microflora in pediatric Crohn's disease. Journal of Parenteral and Enteral Nutrition, 2005. 29(4 suppl): p. S173. 501. Leach, S., et al., Sustained modulation of intestinal bacteria by exclusive enteral nutrition used to treat children with Crohn’s disease. Alimentary pharmacology & therapeutics, 2008. 28(6): p. 724-733. 502. Kaakoush, N.O., et al., Effect of Exclusive Enteral Nutrition on the Microbiota of Children With Newly Diagnosed Crohn’s Disease. Clinical and translational gastroenterology, 2015. 6(1): p. e71. 503. Pryce-Millar, E., et al., P0610 Enteral nutrition therapy in Crohn’s disease changes the mucosal flora. Journal of Pediatric Gastroenterology and Nutrition, 2004. 39: p. S289. 504. Koboziev, I., et al., Role of the enteric microbiota in intestinal homeostasis and inflammation. Free Radical Biology and Medicine, 2014. 68: p. 122-133. 505. Shen, W., H.R. Gaskins, and M.K. McIntosh, Influence of dietary fat on intestinal microbes, inflammation, barrier function and metabolic outcomes. The Journal of nutritional biochemistry, 2014. 25(3): p. 270-280. 506. Lerner, A. and T. Matthias, Changes in intestinal tight junction permeability associated with industrial food additives explain the rising incidence of autoimmune disease. Autoimmunity reviews, 2015. 14(6): p. 479-489. 507. Barnich, N., J. Denizot, and A. Darfeuille-Michaud, E. coli-mediated gut inflammation in genetically predisposed Crohn's disease patients. Pathologie Biologie, 2013. 61(5): p. e65-e69. 508. Greenberg, G., et al., Controlled trial of bowel rest and nutritional support in the management of Crohn's disease. Gut, 1988. 29(10): p. 1309. 509. Lee, K.M., [Nutrition in inflammatory bowel disease]. Korean Journal of Gastroenterology/Taehan Sohwagi Hakhoe Chi, 2008. 52(1): p. 1-8. 510. Seidman, E., Nutritional management of inflammatory bowel disease. Gastroenterology Clinics of North America, 1989. 18(1): p. 129. 511. Grimm, M., et al., Interleukin 8: cells of origin in inflammatory bowel disease. Gut, 1996. 38(1): p. 90-98. 512. O'Morain, C., A. Segal, and A. Levi, Elemental diets in treatment of acute Crohn's disease. British medical journal, 1980. 281(6249): p. 1173-1175. 513. Capristo, E., et al., Effect of a vegetable-protein-rich polymeric diet treatment on body composition and energy metabolism in inactive Crohn's disease. European journal of gastroenterology & hepatology, 2000. 12(1): p. 5. 514. Royall, D., et al., Total enteral nutrition support improves body composition of patients with active Crohn's disease. Journal of Parenteral and Enteral Nutrition, 1995. 19(2): p. 95-99. 515. Tim, L., et al., The use of an elemental diet in gastro-intestinal diseases. South African medical journal, 1976. 50(43): p. 1752-1756. 516. Ricour, C., J. Duhamel, and C. Nihoul-Fekete, [Use of parenteral and elementary enteral nutrition in the treatment of Crohn's disease and ulcerative colitis in children]. Archives francaises de pediatrie, 1976. 34(6): p. 505-513. 517. O'moráin, C., A. Segal, and A. Levi, Elemental diet as primary treatment of acute Crohn's disease: a controlled trial. BMJ, 1984. 288(6434): p. 1859-1862. 518. Thomas, A., F. Taylor, and V. Miller, Dietary intake and nutritional treatment in childhood Crohn's disease. Journal of pediatric gastroenterology and nutrition, 1993. 17(1): p. 75-81. 519. Seidman, E., et al. Semielemental (SE) diet vs prednisone in pediatric Crohn disease, 1993. PA 19106-3399. 520. Heuschkel, R.B., et al., Enteral nutrition and corticosteroids in the treatment of acute Crohn's disease in children. Journal of pediatric gastroenterology and nutrition, 2000. 31(1): p. 8-15. 521. Critch, J., et al., Use of enteral nutrition for the control of intestinal inflammation in pediatric Crohn disease. Journal of pediatric gastroenterology and nutrition, 2012. 54(2): p. 298-305. 522. Grover, Z., R. Muir, and P. Lewindon, Exclusive enteral nutrition induces early clinical, mucosal and transmural remission in paediatric Crohn’s disease. Journal of gastroenterology, 2014. 49(4): p. 638-645. 523. Ruemmele, F., et al., Consensus guidelines of ECCO/ESPGHAN on the medical management of pediatric Crohn's disease. Journal of Crohn's and Colitis, 2014. 8(10): p. 1179-1207. 524. Zachos, M., M. Tondeur, and A. Griffiths, Enteral nutritional therapy for induction of remission in Crohn's disease. Cochrane Database Syst Rev, 2007. 1. 525. Gorard, D., et al., Initial response and subsequent course of Crohn's disease treated with elemental diet or prednisolone. Gut, 1993. 34(9): p. 1198-1202. 526. Fernández-Bañares, F., et al., How effective is enteral nutrition in inducing clinical remission in active Crohn's disease? A meta-analysis of the randomized clinical trials. Journal of Parenteral and Enteral Nutrition, 1995. 19(5): p. 356-364. 527. Griffiths, A.M., et al., Meta-analysis of enteral nutrition as a primary treatment of active Crohn's disease. Gastroenterology, 1995. 108(4): p. 1056-1067. 528. Wall, C.L., A.S. Day, and R.B. Gearry, Use of exclusive enteral nutrition in adults with Crohn’s disease: A review. World journal of gastroenterology: WJG, 2013. 19(43): p. 7652. 529. Engelman, J., et al. Comparsion of a semi elemental diet (Peptamen) with prednisolone in the primary treatment of active ileal Crohn disease. Gastroenterology. 1993, PA 19106-3399. 530. Okada, M., et al., Controlled trial comparing an elemental diet with prednisolone in the treatment of active Crohn's disease. Hepato-gastroenterology, 1990. 37(1): p. 72-80. 531. Gonzalez-Huix, F., et al., Polymeric enteral diets as primary treatment of active Crohn's disease: a prospective steroid controlled trial. Gut, 1993. 34(6): p. 778-782. 532. Guo, Z., et al., Effect of exclusive enteral nutrition on health-related quality of life for adults with active Crohn’s disease. Nutrition in Clinical Practice, 2013. 28(4): p. 499-505. 533. Assa, A. and R. Shamir, Impact of Therapeutic Strategies on Growth in Pediatric Inflammatory Bowel Disease. J Clin Cell Immunol, 2014. 5(235): p. 2. 534. Takagi, S., et al., Effectiveness of an ‘half elemental diet’as maintenance therapy for Crohn's disease: a randomized-controlled trial. Alimentary pharmacology & therapeutics, 2006. 24(9): p. 1333-1340. 535. Esaki, M., et al., Factors affecting recurrence in patients with Crohn’s disease under nutritional therapy. Diseases of the colon & rectum, 2006. 49: p. 68-74. 536. Yamamoto, T., et al., Enteral nutrition for the maintenance of remission in Crohn's disease: a systematic review. European journal of gastroenterology & hepatology, 2010. 22(1): p. 1. 537. Wilschanski, M., et al., Supplementary enteral nutrition maintains remission in paediatric Crohn's disease. Gut, 1996. 38(4): p. 543-548. 538. Duncan, H., et al., A retrospective study showing maintenance treatment options for paediatric CD in the first year following diagnosis after induction of remission with EEN: supplemental enteral nutrition is better than nothing! BMC gastroenterology, 2014. 14(1): p. 50. 539. Yamamoto, T., et al., Impacts of long-term enteral nutrition on clinical and endoscopic disease activities and mucosal cytokines during remission in patients with Crohn's disease: A prospective study. Inflammatory bowel diseases, 2007. 13(12): p. 1493-1501. 540. Ikeuchi, H., et al., Efficacy of nutritional therapy for perforating and non-perforating Crohn's disease. Hepato-gastroenterology, 2004. 51(58): p. 1050-1052. 541. Esaki, M., et al., Preventive effect of nutritional therapy against postoperative recurrence of Crohn disease, with reference to findings determined by intra-operative enteroscopy. Scandinavian journal of gastroenterology, 2005. 40(12): p. 1431-1437. 542. Yamamoto, T., et al., Impact of long-term enteral nutrition on clinical and endoscopic recurrence after resection for Crohn's disease: a prospective, non-randomized, parallel, controlled study. Alimentary pharmacology & therapeutics, 2007. 25(1): p. 67-72. 543. Johnson, T., et al., Treatment of active Crohn’s disease in children using partial enteral nutrition with liquid formula: a randomised controlled trial. Gut, 2006. 55(3): p. 356- 361. 544. Sigall-Boneh, R., et al., Partial enteral nutrition with a Crohn's disease exclusion diet is effective for induction of remission in children and young adults with Crohn's disease. Inflammatory bowel diseases, 2014. 20(8): p. 1353-1360. 545. Polk, D.B., J.A.T. Hattner, and J.A. Kerner, Improved growth and disease activity after intermittent administration of a defined formula diet in children with Crohn's disease. Journal of Parenteral and Enteral Nutrition, 1992. 16(6): p. 499-504. 546. de Bie, C., A. Kindermann, and J. Escher, Use of exclusive enteral nutrition in paediatric Crohn's disease in The Netherlands. Journal of Crohn's and Colitis, 2013. 7(4): p. 263- 270. 547. Afzal, N.A., et al., Colonic Crohn’s disease in children does not respond well to treatment with enteral nutrition if the ileum is not involved. Digestive diseases and sciences, 2005. 50(8): p. 1471-1475. 548. Van Assche, G., et al., The second European evidence-based Consensus on the diagnosis and management of Crohn's disease: Special situations. Journal of Crohn's and Colitis, 2010. 4(1): p. 63-101. 549. Grogan, J.L., et al., Enteral feeding therapy for newly diagnosed pediatric crohn's disease: A double-blind randomized controlled trial with two years follow-up. Inflammatory bowel diseases, 2012. 550. Rajendran, N. and D. Kumar, Role of diet in the management of inflammatory bowel disease. World journal of gastroenterology: WJG, 2010. 16(12): p. 1442. 551. Verma, S., et al., Polymeric versus elemental diet as primary treatment in active Crohn's disease: a randomized, double-blind trial. The American journal of gastroenterology, 2000. 95(3): p. 735-739. 552. Ludvigsson, J., et al., Elemental versus polymeric enteral nutrition in paediatric Crohn's disease: a multicentre randomized controlled trial. Acta Paediatrica, 2004. 93(3): p. 327- 335. 553. Grogan, J.L., et al., Enteral feeding therapy for newly diagnosed pediatric crohn's disease: A double-blind randomized controlled trial with two years follow-up. Inflammatory bowel diseases, 2012. 18(2): p. 246-253. 554. Rodrigues, A.F., et al., Does polymeric formula improve adherence to liquid diet therapy in children with active Crohn’s disease? Archives of disease in childhood, 2007. 92(9): p. 767-770. 555. Scholz, D., The Role of Nutrition in the Etiology of Inflammatory Bowel Disease. Current Problems in Pediatric and Adolescent Health Care, 2011. 41(9): p. 248-253. 556. Whitten, K.E., et al., International survey of enteral nutrition protocols used in children with Crohn's disease. Journal of Digestive Diseases, 2012. 13(2): p. 107-112. 557. Fell, J., Update of the management of inflammatory bowel disease. Archives of disease in Childhood, 2012. 97(1): p. 78-83. 558. Gråfors, J.M. and T.H. Casswall, Exclusive enteral nutrition in the treatment of children with Crohn’s disease in Sweden: a questionnaire survey. Acta Paediatrica, 2011. 100(7): p. 1018-1022. 559. Yamamoto, T., et al., Enteral nutrition for the maintenance of remission in Crohn's disease: a systematic review. European journal of gastroenterology & hepatology, 2010. 22(1): p. 1-8. 560. Akobeng, A.K. and A.G. Thomas, Enteral nutrition for maintenance of remission in Crohn's disease. The Cochrane Library, 2007. 561. Grimble, R.F., Interaction between nutrients, pro-inflammatory cytokines and inflammation. Clinical Science, 1996. 91(2): p. 121-130. 562. Utaka, S., et al., Inflammation is associated with increased energy expenditure in patients with chronic kidney disease. The American journal of clinical nutrition, 2005. 82(4): p. 801-805. 563. Le Floc'h, N., D. Melchior, and C. Obled, Modifications of protein and amino acid metabolism during inflammation and immune system activation. Livestock Production Science, 2004. 87(1): p. 37-45. 564. Chen, G., et al., Glutamine decreases intestinal nuclear factor kappa B activity and pro- inflammatory cytokine expression after traumatic brain injury in rats. Inflammation Research, 2008. 57(2): p. 57-64. 565. Kelly, D. and P.E. Wischmeyer, Role of L-glutamine in critical illness: new insights. Current Opinion in Clinical Nutrition & Metabolic Care, 2003. 6(2): p. 217-222. 566. Luiking, Y.C., et al., Sepsis: an arginine deficiency state? Critical care medicine, 2004. 32(10): p. 2135-2145. 567. Roth, E., et al., Regulative potential of glutamine—relation to glutathione metabolism. Nutrition, 2002. 18(3): p. 217-221. 568. Scaglia, F., New insights in nutritional management and amino acid supplementation in urea cycle disorders. Molecular genetics and metabolism, 2010. 100: p. S72-S76. 569. Carr, E.L., et al., Glutamine uptake and metabolism are coordinately regulated by ERK/MAPK during T lymphocyte activation. The Journal of Immunology, 2010. 185(2): p. 1037-1044. 570. Szeliga, M. and M. Obara-Michlewska, Glutamine in neoplastic cells: focus on the expression and roles of glutaminases. Neurochemistry international, 2009. 55(1): p. 71- 75. 571. Sido, B., et al., Low intestinal glutamine level and low glutaminase activity in Crohn’s disease: a rational for glutamine supplementation? Digestive diseases and sciences, 2006. 51(12): p. 2170-2179. 572. Smith, R.J. and D.W. Wilmore, Glutamine nutrition and requirements. Journal of Parenteral and Enteral Nutrition, 1990. 14(4 suppl): p. 94S-99S. 573. Rao, R.K. and G. Samak, Role of glutamine in protection of intestinal epithelial tight junctions. Journal of epithelial biology and pharmacology, 2012. 5(supplement 1): p. M7. 574. Benjamin, J., et al., Glutamine and whey protein improve intestinal permeability and morphology in patients with Crohn’s disease: a randomized controlled trial. Digestive diseases and sciences, 2012. 57(4): p. 1000-1012. 575. Yao, K., et al., Alpha-ketoglutarate inhibits glutamine degradation and enhances protein synthesis in intestinal porcine epithelial cells. Amino acids, 2012. 42(6): p. 2491-2500. 576. Evans, M.E., D.P. Jones, and T.R. Ziegler, Glutamine prevents cytokine-induced apoptosis in human colonic epithelial cells. The Journal of nutrition, 2003. 133(10): p. 3065-3071. 577. Sozen, S., et al., Prevention of bacterial translocation using Glutamine and Melatonin in small bowel ischemia and reperfusion in rats. Ann hal Chit, 2012. 83(2): p. 143-148. 578. Morrison, A.L., et al., Glutamine's protection against cellular injury is dependent on heat shock factor-1. American Journal of Physiology-Cell Physiology, 2006. 290(6): p. C1625. 579. Garrel, D., et al., Decreased mortality and infectious morbidity in adult burn patients given enteral glutamine supplements: A prospective, controlled, randomized clinical trial*. Critical care medicine, 2003. 31(10): p. 2444. 580. Boelens, P.G., et al., Glutamine-enriched enteral nutrition increases HLA-DR expression on monocytes of trauma patients. The Journal of nutrition, 2002. 132(9): p. 2580-2586. 581. Wischmeyer, P.E., et al., Glutamine administration reduces Gram-negative bacteremia in severely burned patients: a prospective, randomized, double-blind trial versus isonitrogenous control. Critical care medicine, 2001. 29(11): p. 2075. 582. Goeters, C., et al., Parenteral l-alanyl-l-glutamine improves 6-month outcome in critically ill patients*. Critical care medicine, 2002. 30(9): p. 2032. 583. Singleton, K.D. and P.E. Wischmeyer, Glutamine's protection against sepsis and lung injury is dependent on heat shock protein 70 expression. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 2007. 292(5): p. R1839. 584. Fillmann, H., et al., Glutamine inhibits over-expression of pro-inflammatory genes and down-regulates the nuclear factor kappaB pathway in an experimental model of colitis in the rat. Toxicology, 2007. 236(3): p. 217-226. 585. Li, N., et al., Glutamine decreases lipopolysaccharide-induced intestinal inflammation in infant rats. American Journal of Physiology-Gastrointestinal and Liver Physiology, 2004. 286(6): p. G914-G921. 586. Akobeng, A.K., et al., Double-blind randomized controlled trial of glutamine-enriched polymeric diet in the treatment of active Crohn's disease. Journal of pediatric gastroenterology and nutrition, 2000. 30(1): p. 78. 587. Hond, E.D., et al., Effect of long-term oral glutamine supplements on small intestinal permeability in patients with Crohn's disease. Journal of Parenteral and Enteral Nutrition, 1999. 23(1): p. 7. 588. Ockenga, J., et al., Glutamine-enriched total parenteral nutrition in patients with inflammatory bowel disease. European journal of clinical nutrition, 2005. 59(11): p. 1302-1309. 589. Hubert-Buron, A., et al., Glutamine pretreatment reduces IL-8 production in human intestinal epithelial cells by limitŝŶŐ/ʃɲƵďŝƋƵŝƚŝŶĂƚŝŽŶ͘dŚĞ:ŽƵƌŶĂůŽĨŶƵƚƌŝƚŝŽŶ͕ϮϬϬϲ͘ 136(6): p. 1461-1465. 590. Brasse-Lagnel, C.G., A.M. Lavoinne, and A.S. Husson, Amino acid regulation of mammalian gene expression in the intestine. Biochimie, 2010. 92(7): p. 729-735. 591. Kessel, A., et al., Treatment with glutamine is associated with down regulation of Toll like receptor 4 and myeloid differentiation factor 88 expression and decrease in intestinal mucosal injury caused by lipopolysaccharide endotoxaemia in a rat. Clinical & Experimental Immunology, 2008. 151(2): p. 341-347. 592. Gurbuz, A.T., J. Kunzelman, and E.E. Ratzer, Supplemental dietary arginine accelerates intestinal mucosal regeneration and enhances bacterial clearance following radiation enteritis in rats. Journal of surgical research, 1998. 74(2): p. 149-154. 593. Morris, S.M., Arginine metabolism: boundaries of our knowledge. The Journal of nutrition, 2007. 137(6): p. 1602S-1609S. 594. Wu, G. and J.S. MORRIS, Arginine metabolism: nitric oxide and beyond. Biochem. J, 1998. 336: p. 1-17. 595. Buentello, J.A. and D.M. Gatlin III, The dietary arginine requirement of channel catfish (< i> Ictalurus punctatus) is influenced by endogenous synthesis of arginine from glutamic acid. Aquaculture, 2000. 188(3): p. 311-321. 596. Wu, G., et al., Arginine deficiency in preterm infants: biochemical mechanisms and nutritional implications. The Journal of nutritional biochemistry, 2004. 15(8): p. 442-451. 597. Flynn, N., et al., The metabolic basis of arginine nutrition and pharmacotherapy. Biomedicine & pharmacotherapy, 2002. 56(9): p. 427-438. 598. Poeze, M., et al., Effects of l-arginine pretreatment on nitric oxide metabolism and hepatosplanchnic perfusion during porcine endotoxemia. The American journal of clinical nutrition, 2011. 93(6): p. 1237-1247. 599. Amin, H.J., et al., Arginine supplementation prevents necrotizing enterocolitis in the premature infant. The Journal of pediatrics, 2002. 140(4): p. 425-431. 600. Wang, Y.-Y., et al., Arginine supplementation enhances peritoneal macrophage phagocytic activity in rats with gut-derived sepsis. Journal of Parenteral and Enteral Nutrition, 2003. 27(4): p. 235-240. 601. Schleiffer, R. and F. Raul, Prophylactic administration of L-arginine improves the intestinal barrier function after mesenteric ischaemia. Gut, 1996. 39(2): p. 194-198. 602. Sukhotnik, I., et al., Oral arginine improves intestinal recovery following ischemia- reperfusion injury in rat. Pediatric surgery international, 2005. 21(3): p. 191-196. 603. Morris Jr, S.M., Arginine: master and commander in innate immune responses. Science signaling, 2010. 3(135): p. pe27. 604. Satriano, J., Arginine pathways and the inflammatory response: interregulation of nitric oxide and polyamines: review article. Amino acids, 2004. 26(4): p. 321-329. 605. Evoy, D., et al., Immunonutrition: the role of arginine. Nutrition, 1998. 14(7): p. 611-617. 606. Coeffier, M., R. Marion-Letellier, and P. Déchelotte, Potential for amino acids supplementation during inflammatory bowel diseases. Inflammatory bowel diseases, 2010. 16(3): p. 518-524. 607. Mori, M. and T. Gotoh, Regulation of nitric oxide production by arginine metabolic enzymes. Biochemical and biophysical research communications, 2000. 275(3): p. 715- 719. 608. Connelly, L., et al., Biphasic regulation of NF-ʃ ĂĐƚŝǀŝƚLJ ƵŶĚĞƌůŝĞƐ ƚŚĞ ƉƌŽ-and anti- inflammatory actions of nitric oxide. The Journal of Immunology, 2001. 166(6): p. 3873- 3881. 609. Mishra, P., Isolation, spectroscopic characterization and molecular modeling studies of mixture of curcuma longa, ginger and seeds of fenugreek. International Journal of PharmTech Research, 2009. 1(1): p. 79-95. 610. Zhou, H., C.S. Beevers, and S. Huang, Targets of curcumin. Current drug targets, 2011. 12(3): p. 332. 611. Bisht, S., et al., Polymeric nanoparticle-encapsulated curcumin (“nanocurcumin”): a novel strategy for human cancer therapy. J Nanobiotechnology, 2007. 5(3): p. 1-18. 612. Lantz, R., et al., The effect of turmeric extracts on inflammatory mediator production. Phytomedicine, 2005. 12(6): p. 445-452. 613. Yadav, V.R., et al., Novel formulation of solid lipid microparticles of curcumin for anti angiogenic and anti inflammatory activity for optimization of therapy of inflammatory bowel disease. Journal of Pharmacy and Pharmacology, 2009. 61(3): p. 311-321. 614. Yadav, V.R., et al., Novel formulation of solid lipid microparticles of curcumin for anti- angiogenic and anti-inflammatory activity for optimization of therapy of inflammatory bowel disease. Journal of Pharmacy and Pharmacology, 2009. 61(3): p. 311-321. 615. Hanai, H. and K. Sugimoto, Curcumin has bright prospects for the treatment of inflammatory bowel disease. Current pharmaceutical design, 2009. 15(18): p. 2087- 2094. 616. Stallmach, A., S. Hagel, and T. Bruns, Adverse effects of biologics used for treating IBD. Best Practice & Research Clinical Gastroenterology, 2010. 24(2): p. 167-182. 617. Rocchi, A., et al., Inflammatory bowel disease: a Canadian burden of illness review. Canadian journal of gastroenterology= Journal canadien de gastroenterologie, 2012. 26(11): p. 811-817. 618. Bantel, H., et al., Abnormal activation of transcription factor NF-ʃŝŶǀŽůǀĞĚŝŶƐƚĞƌŽŝĚ resistance in chronic inflammatory bowel disease. The American journal of gastroenterology, 2000. 95(7): p. 1845-1846. 619. Farrell, R. and D. Kelleher, Glucocorticoid resistance in inflammatory bowel disease. Journal of endocrinology, 2003. 178(3): p. 339-346. 620. Singh, U.P., et al., Alternative medicines as emerging therapies for inflammatory bowel diseases. International Reviews of Immunology, 2012. 31(1): p. 66-84. 621. Irving, G.R., et al., Prolonged biologically active colonic tissue levels of curcumin achieved after oral administration—a clinical pilot study including assessment of patient acceptability. Cancer prevention research, 2013. 6(2): p. 119-128. 622. Jian, Y.-T., et al., Preventive and therapeutic effects of NF-kappaB inhibitor curcumin in rats colitis induced by trinitrobenzene sulfonic acid. World J Gastroenterol, 2005. 11(12): p. 1747-1752. 623. Deguchi, Y., et al., Curcumin prevents the development of dextran sulfate sodium (DSS)- induced experimental colitis. Digestive diseases and sciences, 2007. 52(11): p. 2993- 2998. 624. Jiang, H., et al., Curcumin-attenuated trinitrobenzene sulphonic acid induces chronic colitis by inhibiting expression of cyclooxygenase-2. World Journal of Gastroenterology, 2006. 12(24): p. 3848. 625. Venkataranganna, M., et al., NCB-02 (standardized Curcumin preparation) protects dinitrochlorobenzene-induced colitis through down-regulation of NFkappa-B and iNOS. World Journal of Gastroenterology, 2007. 13(7): p. 1103. 626. Ung, V.Y., et al., Oral administration of curcumin emulsified in carboxymethyl cellulose has a potent anti-inflammatory effect in the IL-10 gene-deficient mouse model of IBD. Digestive diseases and sciences, 2010. 55(5): p. 1272-1277. 627. Salh, B., et al., Curcumin attenuates DNB-induced murine colitis. American Journal of Physiology-Gastrointestinal and Liver Physiology, 2003. 285(1): p. G235-G243. 628. Camacho-Barquero, L., et al., Curcumin, a< i> Curcuma longa constituent, acts on MAPK p38 pathway modulating COX-2 and iNOS expression in chronic experimental colitis. International immunopharmacology, 2007. 7(3): p. 333-342. 629. Billerey-Larmonier, C., et al., Protective effects of dietary curcumin in mouse model of chemically induced colitis are strain dependent. Inflammatory bowel diseases, 2008. 14(6): p. 780-793. 630. Lubbad, A., M. Oriowo, and I. Khan, Curcumin attenuates inflammation through inhibition of TLR-4 receptor in experimental colitis. Molecular and cellular biochemistry, 2009. 322(1-2): p. 127-135. 631. Holt, P.R., S. Katz, and R. Kirshoff, Curcumin therapy in inflammatory bowel disease: a pilot study. Digestive diseases and sciences, 2005. 50(11): p. 2191-2193. 632. Hanai, H., et al., Curcumin maintenance therapy for ulcerative colitis: randomized, multicenter, double-blind, placebo-controlled trial. Clinical gastroenterology and hepatology: the official clinical practice journal of the American Gastroenterological Association, 2006. 4(12): p. 1502. 633. Lang, A., et al., Curcumin in Combination With Mesalamine Induces Remission in Patients With Mild-to-Moderate Ulcerative Colitis in a Randomized Controlled Trial. Clinical Gastroenterology and Hepatology, 2015. 634. Camacho-Barquero, L., et al., Curcumin, a Curcuma longa constituent, acts on MAPK p38 pathway modulating COX-2 and iNOS expression in chronic experimental colitis. International immunopharmacology, 2007. 7(3): p. 333-342. 635. Singh, S. and A. Khar, Biological effects of curcumin and its role in cancer chemoprevention and therapy. Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Cancer Agents), 2006. 6(3): p. 259-270. 636. Taylor, R.A. and M.C. Leonard, Curcumin for inflammatory bowel disease: a review of human studies. Altern Med Rev, 2011. 16(2): p. 152-156. 637. :ŽďŝŶ͕ ͘ ĂŶĚ Z͘͘ ^ĂƌƚŽƌ͕ E&Ͳʃ ƐŝŐŶĂůŝŶŐ ƉƌŽƚĞŝŶƐ ĂƐ ƚŚĞƌĂƉĞƵƚŝĐ ƚĂƌŐĞƚƐ ĨŽƌ inflammatory bowel diseases. Inflammatory bowel diseases, 2000. 6(3): p. 206-213. 638. Jobin, C., et al., Curcumin blocks cytokine-mediated NF-ʃ ĂĐƚŝǀĂƚŝŽŶ ĂŶĚ proinflammatory gene expression by inhibiting inhibitory factor I-ʃŬŝŶĂƐĞĂĐƚŝǀŝƚLJ͘dŚĞ Journal of Immunology, 1999. 163(6): p. 3474-3483. 639. Sugimoto, K., et al., Curcumin prevents and ameliorates trinitrobenzene sulfonic acid– induced colitis in mice. Gastroenterology, 2002. 123(6): p. 1912-1922. 640. Motawi, T.K., S.M. Rizk, and A.H. Shehata, Effects of curcumin and Ginkgo biloba on matrix metalloproteinases gene expression and other biomarkers of inflammatory bowel disease. Journal of physiology and biochemistry, 2012. 68(4): p. 529-539. 641. Epstein, J., et al., Curcumin suppresses p38 mitogen-activated protein kinase activation, reduces IL-ϭɴ ĂŶĚ ŵĂƚƌŝx metalloproteinase-3 and enhances IL-10 in the mucosa of children and adults with inflammatory bowel disease. British journal of nutrition, 2010. 103(06): p. 824-832. 642. Epstein, J., I.R. Sanderson, and T.T. MacDonald, Curcumin as a therapeutic agent: the evidence from in vitro, animal and human studies. British journal of nutrition, 2010. 103(11): p. 1545-1557. 643. Fetterman Jr, J.W. and M.M. Zdanowicz, Therapeutic potential of n-3 polyunsaturated fatty acids in disease. Am J Health Syst Pharm, 2009. 66(13): p. 1169-79. 644. Lo, C.J., et al., Fish oil decreases macrophage tumor necrosis factor gene transcription by ĂůƚĞƌŝŶŐƚŚĞE&ʃĂĐƚŝǀŝƚLJ͘:ŽƵƌŶĂůŽĨ^ƵƌŐŝĐĂůZĞƐĞĂƌĐŚ͕ϭϵϵϵ͘ϴϮ;ϮͿ͗Ɖ͘Ϯϭϲ-221. 645. Goldberg, R.J. and J. Katz, A meta-analysis of the analgesic effects of omega-3 polyunsaturated fatty acid supplementation for inflammatory joint pain. Pain, 2007. 129(1): p. 210-223. 646. Donadio, J.V., et al., A randomized trial of high-dose compared with low-dose omega-3 fatty acids in severe IgA nephropathy. Journal of the American Society of Nephrology, 2001. 12(4): p. 791-799. 647. Bucher, H.C., et al., N-3 polyunsaturated fatty acids in coronary heart disease: a meta- analysis of randomized controlled trials. American Journal of Medicine, 2002. 112(4): p. 298-304. 648. Whiting, C.V., P.W. Bland, and J.F. Tarlton, Dietary n-3 polyunsaturated fatty acids reduce disease and colonic proinflammatory cytokines in a mouse model of colitis. Inflammatory bowel diseases, 2006. 11(4): p. 340-349. 649. Vilaseca, J., et al., Dietary fish oil reduces progression of chronic inflammatory lesions in a rat model of granulomatous colitis. Gut, 1990. 31(5): p. 539-544. 650. Calder, P.C., The use of n-3 polyunsaturated fatty acids as therapeutic agents for inflammatory diseases. Immunology, Endocrine & Metabolic Agents in Medicinal Chemistry, 2009. 9(1): p. 45-54. 651. Romano, C., et al., Usefulness of omega-3 fatty acid supplementation in addition to mesalazine in maintaining remission in pediatric Crohn's disease: A double-blind, randomized, placebo-controlled study. World Journal of Gastroenterology, 2005. 11(45): p. 7118. 652. Belluzzi, A., et al., Polyunsaturated fatty acids and inflammatory bowel disease. The American journal of clinical nutrition, 2000. 71(1): p. 339s-342s. 653. Turner, D., et al., Omega 3 fatty acids (fish oil) for maintenance of remission in Crohn's disease. The Cochrane Library, 2008. 654. DĂĐ>ĞĂŶ͕͘,͕͘ĞƚĂů͕͘^LJƐƚĞŵĂƚŝĐƌĞǀŝĞǁŽĨƚŚĞĞĨĨĞĐƚƐŽĨŶоϯĨĂƚƚLJĂĐŝĚƐŝŶŝŶĨůĂŵŵĂƚŽƌLJ bowel disease. The American journal of clinical nutrition, 2005. 82(3): p. 611-619. 655. Cabré, E., M. Mañosa, and M.A. Gassull, Omega-3 fatty acids and inflammatory bowel diseases–a systematic review. British Journal of Nutrition, 2012. 107(S2): p. S240-S252. 656. Stio, M., et al., The Vitamin D analogue TX 527 blocks NF-ʃ ĂĐƚŝǀĂƚŝŽŶ ŝŶ ƉĞƌŝƉŚĞƌĂů blood mononuclear cells of patients with Crohn's disease. The Journal of steroid biochemistry and molecular biology, 2007. 103(1): p. 51-60. 657. Deluca, H.F. and M.T. Cantorna, Vitamin D: its role and uses in immunology. The FASEB Journal, 2001. 15(14): p. 2579-2585. 658. Manolagas, S., et al. Vitamin D and the hematolymphopoietic tissue: a 1994 update. in Seminars in nephrology. 1994. 659. yƵĞ͕ D͘>͕͘ Ğƚ Ăů͕͘ ϭɲ͕ Ϯϱ-Dihydroxyvitamin D3 inhibits pro-inflammatory cytokine and chemokine expression in human corneal epithelial cells colonized with Pseudomonas aeruginosa. Immunology and cell biology, 2002. 80(4): p. 340-345. 660. ůŵĞƌŝŐŚŝ͕ ͕͘ Ğƚ Ăů͕͘ ϭɲ͕ Ϯϱ-Dihydroxyvitamin D3 inhibits CD40L-induced pro- inflammatory and immunomodulatory activity in Human Monocytes. Cytokine, 2009. 45(3): p. 190-197. 661. Barrat, F.J., et al., In vitro generation of interleukin 10–producing regulatory CD4+ T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)–and Th2- inducing cytokines. The Journal of experimental medicine, 2002. 195(5): p. 603-616. 662. Cantorna, M.T., et al., 1, 25-Dihydroxyvitamin D3 is a positive regulator for the two anti- encephalitogenic cytokines TGF-ß1 and IL-4. The Journal of Immunology, 1998. 160(11): p. 5314-5319. 663. Jørgensen, S.P., et al., Clinical trial: vitamin D3 treatment in Crohn’s disease–a randomized double-blind placebo-controlled study. Alimentary pharmacology & therapeutics, 2010. 32(3): p. 377-383. 664. Loftus Jr, E.V. and W.J. Sandborn, Epidemiology of inflammatory bowel disease. Gastroenterology Clinics of North America, 2002. 31(1): p. 1. 665. Harries, A., et al., Vitamin D status in Crohn's disease: association with nutrition and disease activity. Gut, 1985. 26(11): p. 1197-1203. 666. Zeng, L. and F. Anderson, Seasonal change in the exacerbations of Crohn's disease. Scandinavian journal of gastroenterology, 1996. 31(1): p. 79-82. 667. van der Flier, L.G. and H. Clevers, Stem cells, self-renewal, and differentiation in the intestinal epithelium. Annual review of physiology, 2009. 71: p. 241-260. 668. DeWitt, R.C. and K.A. Kudsk, The gut's role in metabolism, mucosal barrier function, and gut immunology. Infectious disease clinics of North America, 1999. 13(2): p. 465-481. 669. Luedtke-Heckenkamp, K. and H. Reinecker, Role of epithelial cells in mucosal immunobiology. IMMUNOLOGY AND MEDICINE SERIES, 2001. 31: p. 55-74. 670. Fusunyan, R.D., et al., Butyrate Enhances Interleukin (IL)-8 Secretion by Intestinal Epithelial Cells in Response to IL-ϭɴĂŶĚ>ŝƉŽƉŽůLJƐĂĐĐŚĂƌŝĚĞϭ͘WĞĚŝĂƚƌŝĐƌĞƐĞĂƌĐŚ͕ϭϵϵϴ͘ 43(1): p. 84-90. 671. Phalipon, A., et al., Secretory component: a new role in secretory IgA-mediated immune exclusion in vivo. Immunity, 2002. 17(1): p. 107-115. 672. McNabb, P.C. and T. Tomasi, Host defense mechanisms at mucosal surfaces. Annual Reviews in Microbiology, 1981. 35(1): p. 477-496. 673. Stadnyk, A., Cytokine production by epithelial cells. The FASEB Journal, 1994. 8(13): p. 1041-1047. 674. Kim, J.M., et al., Apoptosis of human intestinal epithelial cells after bacterial invasion. Journal of Clinical Investigation, 1998. 102(10): p. 1815. 675. Eckmann, L., M. Kagnoff, and J. Fierer, Epithelial cells secrete the chemokine interleukin- 8 in response to bacterial entry. Infection and immunity, 1993. 61(11): p. 4569-4574. 676. Sartor, R.B., Cytokines in intestinal inflammation: pathophysiological and clinical considerations. Gastroenterology, 1994. 106(2): p. 533. 677. Casini-Raggi, V., et al., Mucosal imbalance of IL-1 and IL-1 receptor antagonist in inflammatory bowel disease. A novel mechanism of chronic intestinal inflammation. The Journal of Immunology, 1995. 154(5): p. 2434-2440. 678. Reimund, J., et al., Increased production of tumour necrosis factor-alpha interleukin-1 beta, and interleukin-6 by morphologically normal intestinal biopsies from patients with Crohn's disease. Gut, 1996. 39(5): p. 684-689. 679. Jung, H.C., et al., A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion. Journal of Clinical Investigation, 1995. 95(1): p. 55. 680. Mitsuyama, K., et al., IL-8 as an important chemoattractant for neutrophils in ulcerative colitis and Crohn's disease. Clinical & Experimental Immunology, 1994. 96(3): p. 432- 436. 681. Keshavarzian, A., et al., Increased interleukin-8 (IL-8) in rectal dialysate from patients with ulcerative colitis: evidence for a biological role for IL-8 in inflammation of the colon. The American journal of gastroenterology, 1999. 94(3): p. 704-712. 682. Izzo, R., et al., Neutrophil-activating peptide (interleukin-8) in colonic mucosa from patients with Crohn's disease. Scandinavian journal of gastroenterology, 1993. 28(4): p. 296-300. 683. Asmuth, D.M., S.M. Hammer, and C.A. Wanke, Physiological effects of HIV infection on human intestinal epithelial cells: an in vitro model for HIV enteropathy. Aids, 1994. 8(2): p. 205. 684. Peterson, M.D., W.M. Bement, and M.S. Mooseker, An in vitro model for the analysis of intestinal brush border assembly. II. Changes in expression and localization of brush border proteins during cell contact-induced brush border assembly in Caco-2BBe cells. Journal of cell science, 1993. 105(2): p. 461-472. 685. Zweibaum, A., et al., Use of cultured cell lines in studies of intestinal cell differentiation and function. Comprehensive Physiology, 2011. 686. Remy, L., et al., Origin of intracellular lumina in HT 29 colonic adenocarcinoma cell line. Virchows Archiv B, 1985. 48(1): p. 145-153. 687. Cell, B., Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol. Cell, 1983. 47: p. 323-330. 688. Lacroix, M., Persistent use of “false” cell lines. International journal of cancer, 2008. 122(1): p. 1-4. 689. McKay, D., D. Philpott, and M. Perdue, Review article: In vitro models in inflammatory bowel disease research—a critical review. Alimentary pharmacology & therapeutics, 1997. 11(s3): p. 70-80. 690. Atreya, R. and M.F. Neurath, IBD pathogenesis in 2014: Molecular pathways controlling barrier function in IBD. Nature Reviews Gastroenterology & Hepatology, 2015. 12(2): p. 67-68. 691. Stelzner, M., et al., A nomenclature for intestinal in vitro cultures. American Journal of Physiology-Gastrointestinal and Liver Physiology, 2012. 302(12): p. G1359-G1363. 692. Jorge, M., et al., P-169 Generation of Human Regional Intestinal Tissue with Human Pluripotent Cell Derived Organoids. Inflammatory Bowel Diseases, 2014. 20: p. S96-S97. 693. Spence, J.R., et al., Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature, 2011. 470(7332): p. 105-109. 694. Kedinger, M., K. Haffen, and P. Simon-Assmann, Intestinal tissue and cell cultures. Differentiation, 1987. 36(1): p. 71-85. 695. Rimoldi, M., et al., Intestinal immune homeostasis is regulated by the crosstalk between epithelial cells and dendritic cells. Nature immunology, 2005. 6(5): p. 507-514. 696. Viallard, V., et al., Effect of glutamine deprivation and glutamate or ammonium chloride addition on growth rate, metabolism and differentiation of human colon cancer cell-line HT29. International Journal of Biochemistry, 1986. 18(3): p. 263-269. 697. Murnin, M., et al., Effects of glutamine isomers on human (Caco-2) intestinal epithelial proliferation, strain-responsiveness, and differentiation. Journal of Gastrointestinal Surgery, 2000. 4(4): p. 435-442. 698. Eckmann, L., et al., Nitric oxide production by human intestinal epithelial cells and competition for arginine as potential determinants of host defense against the lumen- dwelling pathogen Giardia lamblia. The Journal of Immunology, 2000. 164(3): p. 1478- 1487. 699. Brandsch, M., et al., Regulation of taurine transport in human colon carcinoma cell lines (HT-29 and Caco-2) by protein kinase C. American Journal of Physiology-Gastrointestinal and Liver Physiology, 1993. 264(5): p. G939-G946. 700. Vandewalle, B., N. Wattez, and J. Lefebvre, Effects of vitamin D 3 derivatives on growth, differentiation and apoptosis in tumoral colonic HT 29 cells: possible implication of intracellular calcium. Cancer letters, 1995. 97(1): p. 99-106. 701. Murillo, G., et al., Actions of vitamin D are mediated by the TLR4 pathway in inflammation-induced colon cancer. The Journal of steroid biochemistry and molecular biology, 2010. 121(1): p. 403-407. 702. Moon, D.-O., et al., Curcumin Decreases Binding of Shiga-Like Toxin-1B on Human Intestinal Epithelial Cell Line HT29 Stimulated with TNF-. ALPHA. and IL-1. BETA.: Suppression of p38, JNK and NF-. KAPPA. B p65 as Potential Targets. Biological and Pharmaceutical Bulletin, 2006. 29(7): p. 1470-1475. 703. ůůƌĞĚ͕ ͕͘͘ Ğƚ Ăů͕͘ WWZɶϭ ĂƐ Ă ŵŽůĞĐƵůĂƌ ƚĂƌŐĞƚ ŽĨ ĞŝĐŽƐĂƉĞŶƚĂĞŶŽŝĐ ĂĐŝĚ ŝŶ ŚƵŵĂŶ colon cancer (HT-29) cells. The Journal of nutrition, 2008. 138(2): p. 250-256. 704. Ăŝ͕&͕͘ĞƚĂů͕͘/ŶƚĞƌĂĐƚŝŽŶŽĨʘ-3 polyunsaturated fatty acids with radiation therapy in two different colorectal cancer cell lines. Clinical Nutrition, 2014. 33(1): p. 164-170. 705. Nahidi, L., et al., Osteoprotegerin in pediatric Crohn's disease and the effects of exclusive enteral nutrition. Inflammatory bowel diseases, 2011. 17(2): p. 516-523. 706. Nahidi, L., et al., P-081: The major pathway by which polymeric formula reduces intestinal inflammation in Crohn’s disease patients – a microarray-based analysis. 707. de Jong, N.S., S.T. Leach, and A.S. Day, Polymeric Formula Has Direct Anti-Inflammatory Effects on Enterocytes in an in VitroModel of Intestinal Inflammation. Digestive diseases and sciences, 2007. 52(9): p. 2029-2036. 708. ALHAGAMHMAD, M., et al., Defining the anti-inflammatory benefits of curcumin in vitro. Journal of Gastroenterology and Hepatology, 2013. 28(2): p. 134-139. 709. Boonkaewwan, C., et al., Specific immunomodulatory and secretory activities of stevioside and steviol in intestinal cells. Journal of agricultural and food chemistry, 2008. 56(10): p. 3777-3784. 710. Van De Walle, J., et al., Inflammatory parameters in Caco-2 cells: effect of stimuli nature, concentration, combination and cell differentiation. Toxicology in Vitro, 2010. 24(5): p. 1441-1449. 711. Chang, E.B., M.D. Sitrin, and D.D. Black, Gastrointestinal, hepatobiliary, and nutritional physiology. 1996: JB Lippincott Company. 712. Hoskins, J., G. Meynell, and F. Sanders, A comparison of methods for estimating the viable count of a suspension of tumour cells. Experimental cell research, 1956. 11(2): p. 297-305. 713. Hortigón-Vinagre, M.P. and F. Henao, Apoptotic Cell Death in Cultured Cardiomyocytes Following Exposure to Low Concentrations of 4-Hydroxy-2-nonenal. Cardiovascular toxicology, 2014: p. 1-13. 714. McMahon, T.A. and J.R. Rohr, Trypan Blue Dye is an Effective and Inexpensive Way to Determine the Viability of Batrachochytrium dendrobatidis Zoospores. EcoHealth, 2014: p. 1-4. 715. Hördegen, P., et al., In vitro screening of six anthelmintic plant products against larval Haemonchus contortus with a modified methyl-thiazolyl-tetrazolium reduction assay. Journal of ethnopharmacology, 2006. 108(1): p. 85-89. 716. Bahtiyar, M.O., et al., Follicular fluid of women with endometriosis stimulates the proliferation of endometrial stromal cells. Human Reproduction, 1998. 13(12): p. 3492- 3495. 717. Moon, E.-Y., et al., Clitocybins, novel isoindolinone free radical scavengers, from mushroom Clitocybe aurantiaca inhibit apoptotic cell death and cellular senescence. Biological and Pharmaceutical Bulletin, 2009. 32(10): p. 1689-1694. 718. Salvatori, D., et al., Maedi-visna virus, a model for in vitro testing of potential anti-HIV drugs. Comparative immunology, Microbiology and infectious diseases, 2001. 24(2): p. 113-122. 719. Damiano, J.S., et al., Cell adhesion mediated drug resistance (CAM-DR): role of integrins and resistance to apoptosis in human myeloma cell lines. Blood, 1999. 93(5): p. 1658- 1667. 720. Mickisch, G., et al., Chemosensitivity testing of primary human renal cell carcinoma by a tetrazolium based microculture assay (MTT). Urological research, 1990. 18(2): p. 131- 136. 721. Fischer, D., et al., In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis. Biomaterials, 2003. 24(7): p. 1121-1131. 722. Moser, B. and K. Willimann, Chemokines: role in inflammation and immune surveillance. Annals of the rheumatic diseases, 2004. 63(suppl 2): p. ii84-ii89. 723. Baggiolini, M., A. Walz, and S. Kunkel, Neutrophil-activating peptide-1/interleukin 8, a novel cytokine that activates neutrophils. Journal of Clinical Investigation, 1989. 84(4): p. 1045. 724. Baggiolini, M., P. Loetscher, and B. Moser, Interleukin-8 and the chemokine family. International journal of immunopharmacology, 1995. 17(2): p. 103-108. 725. Steiner, T.S., et al., Enteroaggregative Escherichia coli expresses a novel flagellin that causes IL-8 release from intestinal epithelial cells. Journal of Clinical Investigation, 2000. 105(12): p. 1769-1778. 726. Steiner, T., et al., Enteroaggregative Escherichia coli produce intestinal inflammation and growth impairment and cause interleukin-8 release from intestinal epithelial cells. Journal of Infectious Diseases, 1998. 177(1): p. 88-96. 727. Schuerer-Maly, C., et al., Colonic epithelial cell lines as a source of interleukin-8: stimulation by inflammatory cytokines and bacterial lipopolysaccharide. Immunology, 1994. 81(1): p. 85. 728. Epstein, F.H. and A.D. Luster, Chemokines—chemotactic cytokines that mediate inflammation. New England Journal of Medicine, 1998. 338(7): p. 436-445. 729. Täuber, M.G. and B. Moser, Cytokines and chemokines in meningeal inflammation: biology and clinical implications. Clinical infectious diseases, 1999: p. 1-11. 730. Elewaut, D., et al., NF-ʃ ŝƐ Ă ĐĞŶƚƌĂů ƌĞŐƵůĂƚŽƌ ŽĨ ƚŚĞ ŝŶƚĞƐƚŝŶĂů ĞƉŝƚŚĞůŝĂů ĐĞůů ŝŶŶĂƚĞ immune response induced by infection with enteroinvasive bacteria. The Journal of Immunology, 1999. 163(3): p. 1457-1466. 731. Papadakis, K.A. and S.R. Targan, The role of chemokines and chemokine receptors in mucosal inflammation. Inflammatory bowel diseases, 2006. 6(4): p. 303-313. 732. McCormack, G., et al., Tissue cytokine and chemokine expression in inflammatory bowel disease. Inflammation Research, 2001. 50(10): p. 491-495. 733. Gross, V., et al., Regulation of interleukin-8 production in a human colon epithelial cell line (HT-29). Gastroenterology, 1995. 108(3): p. 653-661. 734. Son, D.O., H. Satsu, and M. Shimizu, Histidine inhibits oxidative stress-and TNF-ɲ- induced interleukin-8 secretion in intestinal epithelial cells. FEBS letters, 2005. 579(21): p. 4671-4677. 735. tĂƌŚƵƌƐƚ͕͕͘^͘,ŽƉŬŝŶƐ͕ĂŶĚ'͘tĂƌŚƵƌƐƚ͕/ŶƚĞƌĨĞƌŽŶɶŝŶĚƵĐĞƐĚŝĨĨĞƌĞŶƚŝĂůupregulation ŽĨɲĂŶĚɴĐŚĞŵŽŬŝŶĞƐĞĐƌĞƚŝŽŶŝŶĐŽůŽŶŝĐĞƉŝƚŚĞůŝĂůĐĞůůůŝŶĞƐ͘'Ƶƚ͕ϭϵϵϴ͘ϰϮ;ϮͿ͗Ɖ͘ϮϬϴ- 213. 736. Sonnier, D.I., et al., TNF-ɲ/ŶĚƵĐĞƐsĞĐƚŽƌŝĂů^ĞĐƌĞƚŝŽŶŽĨ/>-8 in Caco-2 Cells. Journal of Gastrointestinal Surgery, 2010. 14(10): p. 1592-1599. 737. Hollebeeck, S., et al., Dimethyl sulfoxide (DMSO) attenuates the inflammatory response in vitro intestinal Caco-2 cell model. Toxicology letters, 2011. 206(3): p. 268-275. 738. Sergent, T., et al., Anti-inflammatory effects of dietary phenolic compounds in an in vitro model of inflamed human intestinal epithelium. Chemico-biological interactions, 2010. 188(3): p. 659-667. 739. Lammers, K., et al., Effect of probiotic strains on interleukin 8 production by HT29/19A cells. The American journal of gastroenterology, 2002. 97(5): p. 1182-1186. 740. Jarrett, E.E. and H.R. Miller, Production and Activities of IgE in Helminth Infection (Part 1 of 3). Vol. 31. 1982: Karger Publishers. 741. Tjellström, B., et al., Effect of exclusive enteral nutrition on gut microflora function in children with Crohn's disease. Scandinavian journal of gastroenterology, 2012. 47(12): p. 1454-1459. 742. Zhang, N., et al., The antiangiogenic effects of tyroservatide on animal models of hepatocellular carcinoma. Journal of cancer research and clinical oncology, 2009. 135(10): p. 1447-1453. 743. Webb, D.R., Animal models of human disease: inflammation. Biochemical pharmacology, 2014. 87(1): p. 121-130. 744. Collins, J.M., C.K. Grieshaber, and B.A. Chabner, Pharmacologically guided phase I clinical trials based upon preclinical drug development. Journal of the National Cancer Institute, 1990. 82(16): p. 1321-1326. 745. Glaze, E.R., et al., Preclinical toxicity of a geldanamycin analog, 17- (dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), in rats and dogs: potential clinical relevance. Cancer chemotherapy and pharmacology, 2005. 56(6): p. 637-647. 746. Hau, J., Animal models for human diseases, in Sourcebook of Models for Biomedical Research. 2008, Springer. p. 3-8. 747. Hanna, Z. and P. Jolicoeur. Mice as animal models for human disease. in Qatar Foundation Annual Research Forum. 2012. 748. Duty, S. and P. Jenner, Animal models of Parkinson's disease: a source of novel treatments and clues to the cause of the disease. British journal of pharmacology, 2011. 164(4): p. 1357-1391. 749. Jurjus, A.R., N.N. Khoury, and J.-M. Reimund, Animal models of inflammatory bowel disease. Journal of pharmacological and toxicological methods, 2004. 50(2): p. 81-92. 750. Maxwell, J.R., et al., Methods of inducing inflammatory bowel disease in mice. Current Protocols in Pharmacology, 2009: p. 5.58. 1-5.58. 37. 751. Hoffmann, J., et al., Role of T lymphocytes in rat 2, 4, 6-trinitrobenzene sulphonic acid ;dE^ͿŝŶĚƵĐĞĚĐŽůŝƚŝƐ͗ŝŶĐƌĞĂƐĞĚŵŽƌƚĂůŝƚLJĂĨƚĞƌɶɷdĐĞůůĚĞƉůĞƚŝŽŶĂŶĚŶŽĞĨĨĞĐƚŽĨɲɴd cell depletion. Gut, 2001. 48(4): p. 489-495. 752. Neurath, M.F., et al., Antibodies to interleukin 12 abrogate established experimental colitis in mice. The Journal of experimental medicine, 1995. 182(5): p. 1281-1290. 753. <ŝŶŽƐŚŝƚĂ͕<͕͘ĞƚĂů͕͘ZŽůĞŽĨdE&ͲɲŝŶŵƵƐĐƵůĂƌŝƐŝŶĨůĂŵŵĂƚŝŽŶĂŶĚŵŽƚŝůŝƚLJĚŝƐŽƌĚĞƌŝŶĂ dE^ͲŝŶĚƵĐĞĚĐŽůŝƚŝƐŵŽĚĞů͗ĐůƵĞƐĨƌŽŵdE&ͲɲͲĚĞĨŝĐŝĞŶƚŵŝĐĞ͘EĞƵƌŽŐĂƐƚƌŽĞŶƚĞƌŽůŽŐLJΘ Motility, 2006. 18(7): p. 578-588. 754. Gardiner, K., et al., Colitis and colonic mucosal barrier dysfunction. Gut, 1995. 37(4): p. 530-535. 755. Elson, C.O., et al., Hapten-induced model of murine inflammatory bowel disease: mucosa immune responses and protection by tolerance. The Journal of Immunology, 1996. 157(5): p. 2174-2185. 756. Khan, I. and M. Ali, Altered expression of the Na+/H+ exchanger isoform-3 in experimental colitis: effect of garlic. Molecular and cellular biochemistry, 1999. 200(1- 2): p. 77-84. 757. Dohi, T., et al., Hapten-induced colitis is associated with colonic patch hypertrophy and T helper cell 2–type responses. The Journal of experimental medicine, 1999. 189(8): p. 1169-1180. 758. Zhao, J., et al., R-spondin1, a novel intestinotrophic mitogen, ameliorates experimental colitis in mice. Gastroenterology, 2007. 132(4): p. 1331-1343. 759. Kawada, M., A. Arihiro, and E. Mizoguchi, Insights from advances in research of chemically induced experimental models of human inflammatory bowel disease. World Journal of Gastroenterology, 2007. 13(42): p. 5581. 760. Johansson, M.E., et al., Bacteria penetrate the inner mucus layer before inflammation in the dextran sulfate colitis model. PloS one, 2010. 5(8): p. e12238. 761. Perše, M. and A. Cerar, Dextran sodium sulphate colitis mouse model: traps and tricks. BioMed Research International, 2012. 2012. 762. Yan, Y., et al., Temporal and spatial analysis of clinical and molecular parameters in dextran sodium sulfate induced colitis. PloS one, 2009. 4(6): p. e6073. 763. Rees, V., Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines. Clinical & Experimental Immunology, 1998. 114(3): p. 385-391. 764. Melgar, S., A. Karlsson, and E. Michaëlsson, Acute colitis induced by dextran sulfate sodium progresses to chronicity in C57BL/6 but not in BALB/c mice: correlation between symptoms and inflammation. American Journal of Physiology-Gastrointestinal and Liver Physiology, 2005. 288(6): p. G1328-G1338. 765. Ten Hove, T., et al., Differential susceptibility of multidrug resistance protein-1 deficient mice to DSS and TNBS-induced colitis. Digestive diseases and sciences, 2002. 47(9): p. 2056-2063. 766. Kojouharoff, G., et al., Neutralization of tumour necrosis factor (TNF) but not of IL-1 reduces inflammation in chronic dextran sulphate sodium-induced colitis in mice. Clinical & Experimental Immunology, 1997. 107(2): p. 353-358. 767. McGonigle, P. and B. Ruggeri, Animal Models of Human Disease: Challenges in Enabling Translation. Biochemical pharmacology, 2013. 768. Löscher, W., Critical review of current animal models of seizures and epilepsy used in the discovery and development of new antiepileptic drugs. Seizure, 2011. 20(5): p. 359- 368. 769. Hansen, K. and C. Khanna, Spontaneous and genetically engineered animal models: use in preclinical cancer drug development. European Journal of Cancer, 2004. 40(6): p. 858- 880. 770. Morampudi, V., et al., DNBS/TNBS colitis models: providing insights into inflammatory bowel disease and effects of dietary fat. JoVE (Journal of Visualized Experiments), 2014(84): p. e51297-e51297. 771. Papada, E., et al., Anti-inflammatory effect of elemental diets with different fat composition in experimental colitis. British Journal of Nutrition, 2014. 111(07): p. 1213- 1220. 772. ,ĂƐƐĂŶ͕ ͕͘ Ğƚ Ăů͕͘ Ŷ ɲ-linolenic acid-rich formula reduces oxidative stress and inflammation by regulating NF-ʃ ŝŶ ƌĂƚƐ ǁŝƚŚ dE^-induced colitis. The Journal of nutrition, 2010. 140(10): p. 1714-1721. 773. Ameho, C., et al., Prophylactic effect of dietary glutamine supplementation on ŝŶƚĞƌůĞƵŬŝŶϴĂŶĚƚƵŵŽƵƌŶĞĐƌŽƐŝƐĨĂĐƚŽƌɲƉƌŽĚƵĐƚŝŽŶŝŶƚƌŝŶŝƚƌŽďĞŶnjĞŶĞƐƵůƉŚŽŶŝĐĂĐŝĚ induced colitis. Gut, 1997. 41(4): p. 487-493. 774. Kono, H., et al., Enteral diets enriched with medium-chain triglycerides and N-3 fatty acids prevent chemically induced experimental colitis in rats. Translational Research, 2010. 156(5): p. 282-291. 775. Mane, J., et al., Effect of L-arginine on the course of experimental colitis. Clinical Nutrition, 2001. 20(5): p. 415-422. 776. Benton, S., et al., Intestinal Effects of Alanyl-Glutamine Dipeptide-Supplemented Diet in a Mouse Model of DSS-Induced Colitis. The FASEB Journal, 2012. 26(1_MeetingAbstracts): p. 375.4. 777. Joo, E., et al., Enteral supplement enriched with glutamine, fiber, and oligosaccharide attenuates experimental colitis in mice. Nutrition, 2013. 29(3): p. 549-555. 778. Villegas, I., S. Sánchez-Fidalgo, and C.A. de la Lastra, Chemopreventive effect of dietary curcumin on inflammation-induced colorectal carcinogenesis in mice. Molecular nutrition & food research, 2011. 55(2): p. 259-267. 779. Murthy, S., et al., Combination therapy of pentoxifylline and TNF monoclonal antibody in dextran sulphate-induced mouse colitis. Alimentary Pharmacology and Therapeutics, 1999. 13: p. 251-260. 780. Nauseef, W.M., S.J. McCormick, and R.A. Clark, Calreticulin functions as a molecular chaperone in the biosynthesis of myeloperoxidase. Journal of Biological Chemistry, 1995. 270(9): p. 4741-4747. 781. Fournier, B. and C. Parkos, The role of neutrophils during intestinal inflammation. Mucosal immunology, 2012. 5(4): p. 354-366. 782. Zhang, R., et al., Myeloperoxidase functions as a major enzymatic catalyst for initiation of lipid peroxidation at sites of inflammation. Journal of Biological Chemistry, 2002. 277(48): p. 46116-46122. 783. Krawisz, J., P. Sharon, and W. Stenson, Quantitative assay for acute intestinal inflammation based on myeloperoxidase activity. Gastroenterology, 1984. 87(6): p. 1344-1350. 784. Togawa, J.-i., et al., Lactoferrin reduces colitis in rats via modulation of the immune system and correction of cytokine imbalance. American Journal of Physiology- Gastrointestinal and Liver Physiology, 2002. 283(1): p. G187-G195. 785. Aomatsu, T., et al., Neutralization of complement component C5 ameliorates the development of dextran sulfate sodium (DSS)-colitis in mice. Journal of clinical biochemistry and nutrition, 2013. 52(1): p. 72. 786. Laroui, H., et al., Dextran sodium sulfate (DSS) induces colitis in mice by forming nano- lipocomplexes with medium-chain-length fatty acids in the colon. PloS one, 2012. 7(3): p. e32084. 787. Camuesco, D., et al., The intestinal anti-inflammatory effect of quercitrin is associated with an inhibition in iNOS expression. British journal of pharmacology, 2004. 143(7): p. 908-918. 788. Kawanishi, N., et al., Exercise training attenuates hepatic inflammation, fibrosis and macrophage infiltration during diet induced-obesity in mice. Brain, behavior, and immunity, 2012. 26(6): p. 931-941. 789. Elsaid, F.G., Modification by Aqueous Extracts of Allium kurrat L. and Ricinus communis L. of Cyanide Nephrotoxicity on Balb/C Mice. 790. Wilson, T.H. and G. Wiseman, The use of sacs of everted small intestine for the study of the transference of substances from the mucosal to the serosal surface. The Journal of physiology, 1954. 123(1): p. 116-125. 791. Browning, T.H. and J.S. Trier, Organ culture of mucosal biopsies of human small intestine. Journal of Clinical Investigation, 1969. 48(8): p. 1423. 792. Autrup, H., Explant culture of human colon. Methods Cell Biol B, 1980. 21: p. 385-401. 793. Reimund, J.-M., et al., Mucosal inflammatory cytokine production by intestinal biopsies in patients with ulcerative colitis and Crohn's disease. Journal of clinical immunology, 1996. 16(3): p. 144-150. 794. Onken, J.E., et al., Bromelain treatment decreases secretion of pro-inflammatory cytokines and chemokines by colon biopsies< i> in vitro. Clinical Immunology, 2008. 126(3): p. 345-352. 795. Goulet, O., Nutrition in paediatric Crohn’s disease. South African Journal of Clinical Nutrition, 2010. 23(1). 796. Berni Canani, R., et al., Short-and long-term therapeutic efficacy of nutritional therapy and corticosteroids in paediatric Crohn's disease. Digestive and liver disease: official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver, 2006. 38(6): p. 381. 797. Suchner, U., D. Heyland, and K. Peter, Immune-modulatory actions of arginine in the critically ill. British Journal of Nutrition, 2002. 87(S1): p. S121-S132. 798. Tak, P.P. and G.S. Firestein, NF-ʃ͗ĂŬĞLJƌŽůĞŝŶŝŶĨůĂŵŵĂƚŽƌLJĚŝƐĞĂƐĞƐ͘:ŽƵƌŶĂůŽĨ Clinical Investigation, 2001. 107(1): p. 7. 799. Herlaar, E. and Z. Brown, p38 MAPK signalling cascades in inflammatory disease. Molecular medicine today, 1999. 5(10): p. 439-447. 800. Meng, Q., M. Pan, and R. Cooney, L-arginine attenuates interleukin-1 induced NF-KB activation in Caco-2 cells: P-0146. Inflammatory Bowel Diseases, 2009. 15: p. S51. 801. Lecleire, S., et al., Combined Glutamine and Arginine Decrease Proinflammatory Cytokine Production by Biopsies from Crohn's Patients in Association with Changes in Nuclear Factor-ʃĂŶĚƉϯϴDŝƚŽŐĞŶ-Activated Protein Kinase Pathways. The Journal of nutrition, 2008. 138(12): p. 2481-2486. 802. Wilmore, D.W. and J.K. Shabert, Role of glutamine in immunologic responses. Nutrition, 1998. 14(7): p. 618-626. 803. Coëffier, M., et al., Influence of glutamine on cytokine production by human gut in vitro. Cytokine, 2001. 13(3): p. 148. 804. Coëffier, M.s., et al., Glutamine decreases Interluekin-8 and Interluekin-6 but not nitric oxide and prostaglandins production by human gut in vitro. Cytokine, 2002. 18(2): p. 92- 97. 805. Liboni, K.C., et al., Glutamine modulates LPS-induced IL-ϴƉƌŽĚƵĐƚŝŽŶƚŚƌŽƵŐŚ/ʃͬE&-ʃ in human fetal and adult intestinal epithelium. The Journal of nutrition, 2005. 135(2): p. 245-251. 806. Wischmeyer, P.E., et al., Glutamine protects intestinal epithelial cells: role of inducible HSP70. American Journal of Physiology-Gastrointestinal and Liver Physiology, 1997. 272(4): p. G879-G884. 807. Marion, R., et al., Glutamine and CXC chemokines IL-8, Mig, IP-10 and I-TAC in human intestinal epithelial cells. Clinical Nutrition, 2004. 23(4): p. 579-585. 808. Yang, C., et al., Effects of glutamine on the cytokine expression of peripheral blood mononuclear cells]. Zhonghua yi xue za zhi, 2008. 88(7): p. 449. 809. Xue, H., A.J. Sufit, and P.E. Wischmeyer, Glutamine therapy improves outcome of in vitro and in vivo experimental colitis models. Journal of Parenteral and Enteral Nutrition, 2011. 35(2): p. 188-197. 810. Yeh, C.-L., et al., Effects of arginine-enriched total parenteral nutrition on inflammatory- related mediator and T-cell population in septic rats. Nutrition, 2002. 18(7): p. 631-635. 811. Tsai, H.-J., et al., Effects of arginine supplementation on antioxidant enzyme activity and macrophage response in burned mice. Burns, 2002. 28(3): p. 258-263. 812. Cui, X.-L., et al., Arginine-supplemented diet decreases expression of inflammatory cytokines and improves survival in burned rats. Journal of Parenteral and Enteral Nutrition, 2000. 24(2): p. 89-96. 813. Meldrum, D.R., et al., L-arginine decreases alveolar macrophage proinflammatory monokine production during acute lung injury by a nitric oxide synthase-dependent mechanism. The Journal of Trauma and Acute Care Surgery, 1997. 43(6): p. 888-893. 814. Breuillard, C., et al., Charlotte Breuillard, Linda Belabed, Sandra Bonhomme, Marie- Céline Blanc-Quintin, Nathalie Neveux, Rémy Couderc, Jean-Pascal De Bandt, Luc Cynober, Sylviane Darquy. Clinical Nutrition, 2012. 31(3): p. 415-421. 815. Bonhomme, S., et al., Arginine-supplemented enteral nutrition in critically ill diabetic and obese rats: A dose-ranging study evaluating nutritional status and macrophage function. Nutrition, 2012. 816. Jeffery, L.E., et al., 1, 25-Dihydroxyvitamin D3 and IL-2 combine to inhibit T cell production of inflammatory cytokines and promote development of regulatory T cells expressing CTLA-4 and FoxP3. The Journal of Immunology, 2009. 183(9): p. 5458-5467. 817. Lagishetty, V., et al., Vitamin D deficiency in mice impairs colonic antibacterial activity and predisposes to colitis. Endocrinology, 2010. 151(6): p. 2423-2432. 818. Daniel, C., et al., The new low calcemic vitamin D analog 22-ene-25-oxa-vitamin D prominently ameliorates T helper cell type 1-mediated colitis in mice. Journal of Pharmacology and Experimental Therapeutics, 2006. 319(2): p. 622-631. 819. Strauch, U.G., et al., Calcitriol analog ZK191784 ameliorates acute and chronic dextran sodium sulfate-induced colitis by modulation of intestinal dendritic cell numbers and phenotype. World Journal of Gastroenterology, 2007. 13(48): p. 6529. 820. Zhu, Y., et al., Calcium and ϭɲ͕ ϮϱͲĚŝŚLJĚƌŽdžLJǀŝƚĂŵŝŶ ϯ ƚĂƌŐĞƚ ƚŚĞ dE&Ͳɲ ƉĂƚŚǁĂLJ ƚŽ suppress experimental inflammatory bowel disease. European journal of immunology, 2005. 35(1): p. 217-224. 821. Pappa, H.M., et al., Vitamin D status in children and young adults with inflammatory bowel disease. Pediatrics, 2006. 118(5): p. 1950-1961. 822. Guillot, X., et al., Vitamin D and inflammation. Joint Bone Spine, 2010. 77(6): p. 552-557. 823. Cohen, M.L., et al., Regulation of TNF-&agr; by 1&agr;, 25-dihydroxyvitamin D3 in human macrophages from CAPD patients. Kidney international, 2001. 59(1): p. 69-75. 824. Panichi, V., et al., Calcitriol modulates in vivo and in vitro cytokine production: a role for intracellular calcium. Kidney international, 1998. 54(5): p. 1463-1469. 825. Nonnecke, B.J., et al., In vitro effects of 1, 25-dihydroxyvitamin D3 on interferon-gamma and tumor necrosis factor-alpha secretion by blood leukocytes from young and adult cattle vaccinated with Mycobacterium bovis BCG. International journal for vitamin and nutrition research. Internationale Zeitschrift für Vitamin-und Ernährungsforschung. Journal international de vitaminologie et de nutrition, 2003. 73(4): p. 235. 826. ŚĂŽ͕'͕͘ĞƚĂů͕͘ŝĞƚĂƌLJɲ-linolenic acid inhibits proinflammatory cytokine production by peripheral blood mononuclear cells in hypercholesterolemic subjects. The American journal of clinical nutrition, 2007. 85(2): p. 385-391. 827. ZĂůůŝĚŝƐ͕>͘^͕͘ĞƚĂů͕͘ŝĞƚĂƌLJɲ-linolenic acid decreases C-reactive protein, serum amyloid A and interleukin-6 in dyslipidaemic patients. Atherosclerosis, 2003. 167(2): p. 237-242. 828. Marion-Letellier, R., et al., Comparison of cytokine modulation by natural peroxisome proliferator–ĂĐƚŝǀĂƚĞĚƌĞĐĞƉƚŽƌɶůŝŐĂŶĚƐǁŝƚŚƐLJŶƚŚĞƚŝĐůŝŐĂŶĚƐŝŶŝŶƚĞƐƚŝŶĂů-like Caco-2 cells and human dendritic cells—potential for dietary modulation of peroxisome proliferator–ĂĐƚŝǀĂƚĞĚ ƌĞĐĞƉƚŽƌ ɶ ŝŶ ŝŶƚĞƐƚŝŶĂů ŝŶĨůĂŵŵĂƚŝŽŶ͘ dŚĞ ŵĞƌŝĐĂŶ ũŽƵƌŶĂů ŽĨ clinical nutrition, 2008. 87(4): p. 939-948. 829. Lo, C.-J., et al., Fish oil decreases macrophage tumor necrosis factor gene transcription ďLJĂůƚĞƌŝŶŐƚŚĞE&ʃĂĐƚŝǀŝƚLJ͘:ŽƵƌŶĂůŽĨ^ƵƌŐŝĐĂůZĞƐĞĂƌĐŚ͕ϭϵϵϵ͘ϴϮ;ϮͿ͗Ɖ͘Ϯϭϲ-221. 830. Babcock, T.A., et al., Modulation of lipopolysaccharide-stimulated macrophage tumor necrosis factor-ɲ ƉƌŽĚƵĐƚŝŽŶ ďLJ ʘ-3 fatty acid is associated with differential cyclooxygenase-2 protein expression and is independent of interleukin-10. Journal of Surgical Research, 2002. 107(1): p. 135-139. 831. Novak, T.E., et al., NF-ʃ ŝŶŚŝďŝƚŝŽŶ ďLJ ʘ-3 fatty acids modulates LPS-stimulated macrophage TNF-ɲ ƚƌĂŶƐĐƌŝƉƚŝŽŶ͘ ŵĞƌŝĐĂŶ :ŽƵƌŶĂů ŽĨ WŚLJƐŝŽůŽŐLJ-Lung Cellular and Molecular Physiology, 2003. 284(1): p. L84-L89. 832. Yaqoob, P. and P. Calder, Effects of dietary lipid manipulation upon inflammatory mediator production by murine macrophages. Cellular immunology, 1995. 163(1): p. 120. 833. Trebble, T., et al., Inhibition of tumour necrosis factor-ɲĂŶĚŝŶƚĞƌůĞƵŬŝŶϲƉƌŽĚƵĐƚŝŽŶďLJ mononuclear cells following dietary fish-oil supplementation in healthy men and response to antioxidant co-supplementation. British Journal of Nutrition, 2003. 90(02): p. 405-412. 834. Meister, D., et al., Anti-inflammatory effects of enteral diet components on Crohn's disease-affected tissues in vitro. Digestive and Liver Disease, 2002. 34(6): p. 430-438. 835. Rizzo, M.R., et al., Evidence for anti-inflammatory effects of combined administration of vitamin E and C in older persons with impaired fasting glucose: impact on insulin action. Journal of the American College of Nutrition, 2008. 27(4): p. 505-511. 836. Aukrust, P., et al., Decreased vitamin A levels in common variable immunodeficiency: vitamin A supplementation in vivo enhances immunoglobulin production and down regulates inflammatory responses. European journal of clinical investigation, 2000. 30(3): p. 252-259. 837. Prasad, A.S., et al., Antioxidant effect of zinc in humans. Free Radical Biology and Medicine, 2004. 37(8): p. 1182-1190. 838. Lang, C., et al., Anti-inflammatory effects of zinc and alterations in zinc transporter mRNA in mouse models of allergic inflammation. American Journal of Physiology-Lung Cellular and Molecular Physiology, 2007. 292(2): p. L577-L584. 839. Pohlenz, C., et al., Synergies between vaccination and dietary arginine and glutamine supplementation improve the immune response of channel catfish against< i> Edwardsiella ictaluri. Fish & Shellfish Immunology, 2012. 33(3): p. 543-551. 840. Zhou, X., et al., Preventive oral supplementation with glutamine and arginine has beneficial effects on the intestinal mucosa and inflammatory cytokines in endotoxemic rats. Amino acids, 2012. 43(2): p. 813-821. 841. Jobin, C., et al., Evidence for altered regulation of I kappa B alpha degradation in human colonic epithelial cells. The Journal of Immunology, 1997. 158(1): p. 226-234. 842. Huang, Y., et al., Glutamine decreases lipopolysaccharide-induced IL-8 production in Caco-2 cells through a non-NF-ʃƉϱϬŵĞĐŚĂŶŝƐŵ͘LJƚŽŬŝŶĞ͕ϮϬϬϯ͘ϮϮ;ϯͿ͗Ɖ͘ϳϳ-83. 843. Beutheu, S., et al., Glutamine and arginine improve permeability and tight junction protein expression in methotrexate-treated Caco-2 cells. Clinical Nutrition, 2013. 844. Hou, Y.-C., et al., Glutamine modulates lipopolysaccharide-induced activation of NF-ʃ via the Akt/mTOR pathway in lung epithelial cells. American Journal of Physiology-Lung Cellular and Molecular Physiology, 2012. 302(1): p. L174-L183. 845. Lesueur, C., et al., Glutamine induces nuclear degradation of the NF-ʃƉϲϱƐƵďƵŶŝƚŝŶ Caco-2/TC7 cells. Biochimie, 2012. 94(3): p. 806-815. 846. Rogero, M.M., et al., Effects of glutamine on the nuclear factor-kappaB signaling pathway of murine peritoneal macrophages. Amino acids, 2010. 39(2): p. 435-441. 847. ĞŒĞƌ͕͕͘ĞƚĂů͕͘dŚĞĞĨĨĞĐƚŽĨŐůƵƚĂŵŝŶĞŽŶƉĂŶĐƌĞĂƚŝĐĚĂŵĂŐĞŝŶdE^-induced colitis. Digestive diseases and sciences, 2006. 51(10): p. 1841-1846. 848. Kretzmann, N.A., et al., Effects of glutamine on proinflammatory gene expression and activation of nuclear factor kappa B and signal transducers and activators of transcription in TNBS-induced colitis. Inflammatory bowel diseases, 2008. 14(11): p. 1504-1513. 849. Marion, R., et al., L-Arginine modulates CXC chemokines in the human intestinal epithelial cell line HCT-8 by the NO pathway. Biochimie, 2005. 87(12): p. 1048-1055. 850. Tan, B., et al., L-Arginine stimulates proliferation and prevents endotoxin-induced death of intestinal cells. Amino acids, 2010. 38(4): p. 1227-1235. 851. Hnia, K., et al., L-arginine decreases inflammation and modulates the nuclear factor- ʃͬŵĂƚƌŝdž ŵĞƚĂůůŽƉƌŽƚĞŝŶĂƐĞ ĐĂƐĐĂĚĞ ŝŶ ŵĚdž ŵƵƐĐůĞ ĨŝďĞƌƐ͘ dŚĞ ŵĞƌŝĐĂŶ ũŽƵƌŶĂů of pathology, 2008. 172(6): p. 1509-1519. 852. Calkins, C.M., et al., L-arginine attenuates lipopolysaccharide-induced lung chemokine production. American Journal of Physiology-Lung Cellular and Molecular Physiology, 2001. 280(3): p. L400-L408. 853. Atreya͕/͕͘Z͘ƚƌĞLJĂ͕ĂŶĚD͘EĞƵƌĂƚŚ͕E&ͲʃŝŶŝŶĨůĂŵŵĂƚŽƌLJďŽǁĞůĚŝƐĞĂƐĞ͘:ŽƵƌŶĂůŽĨ internal medicine, 2008. 263(6): p. 591-596. 854. D'Acquisto, F., M.J. May, and S. Ghosh, Inhibition of nuclear factor kappa B (NF-B). Molecular interventions, 2002. 2(1): p. 22. 855. ƌĚŝƚĞ͕͕͘ĞƚĂů͕͘ĨĨĞĐƚƐŽĨƐƚĞƌŽŝĚƚƌĞĂƚŵĞŶƚŽŶĂĐƚŝǀĂƚŝŽŶŽĨŶƵĐůĞĂƌĨĂĐƚŽƌʃŝŶ patients with inflammatory bowel disease. British journal of pharmacology, 1998. 124(3): p. 431-433. 856. WŽŵĞƌĂŶƚnj͕ :͘>͘ ĂŶĚ ͘ ĂůƚŝŵŽƌĞ͕ E&Ͳʃ ĂĐƚŝǀĂƚŝŽŶ ďLJ a signaling complex containing TRAF2, TANK and TBK1, a novel IKK-related kinase. The EMBO journal, 1999. 18(23): p. 6694-6704. 857. Baud, V., et al., Signaling by proinflammatory cytokines: oligomerization of TRAF2 and TRAF6 is sufficient for JNK and IKK activation and target gene induction via an amino- terminal effector domain. Genes & development, 1999. 13(10): p. 1297-1308. 858. Li, Q. and I.M. Verma, NF-ʃƌĞŐƵůĂƚŝŽŶŝŶƚŚĞŝŵŵƵŶĞƐLJƐƚĞŵ͘EĂƚƵƌĞZĞǀŝĞǁƐ Immunology, 2002. 2(10): p. 725-734. 859. Greten, F.R. and M. Karin, The IKK/NF-ʃĂĐƚŝǀĂƚŝŽŶƉĂƚŚǁĂLJ—a target for prevention and treatment of cancer. Cancer letters, 2004. 206(2): p. 193-199. 860. Hehner, S.P., et al., The antiinflammatory sesquiterpene lactone parthenolide inhibits NF-ʃďLJƚĂƌŐĞƚŝŶŐƚŚĞ/ʃŬŝŶĂƐĞĐŽŵƉůĞdž͘dŚĞ:ŽƵƌŶĂůŽĨ/ŵŵƵŶŽůŽŐLJ͕ϭϵϵϵ͘ϭϲϯ;ϭϬͿ͗ p. 5617-5623. 861. Kopp, E. and S. Ghosh, Inhibition of NF-kappa B by sodium salicylate and aspirin. Science, 1994. 265(5174): p. 956-959. 862. Yin, M.-J., Y. Yamamoto, and R.B. Gaynor, The anti-inflammatory agents aspirin and ƐĂůŝĐLJůĂƚĞŝŶŚŝďŝƚƚŚĞĂĐƚŝǀŝƚLJŽĨ/ʃŬŝŶĂƐĞ-ɴ͘EĂƚƵƌĞ͕ϭϵϵϴ͘ϯϵϲ;ϲϳϬϲͿ͗Ɖ͘ϳϳ-80. 863. ĂƐƚƌŽ͕͕͘͘ĞƚĂů͕͘EŽǀĞů/<<ŝŶŚŝďŝƚŽƌƐ͗ɴ-carbolines. Bioorganic & medicinal chemistry letters, 2003. 13(14): p. 2419-2422. 864. Mizuno, K., et al., Staurosporine-related compounds, K252a and UCN-01, inhibit both cPKC and nPKC. FEBS letters, 1993. 330(2): p. 114-116. 865. Duran, C., L.-T. Chien, and H.C. Hartzell, Drosophila bestrophin-1 currents are regulated by phosphorylation via a CaMKII dependent mechanism. PloS one, 2013. 8(3): p. e58875. 866. Ayush, O., et al., Glutamine Suppresses DNFB-Induced Contact Dermatitis by Deactivating p38 Mitogen–Activated Protein Kinase via Induction of MAPK Phosphatase- 1. Journal of Investigative Dermatology, 2013. 133(3): p. 723-731. 867. Ko, H.-M., et al., Glutamine protects mice from lethal endotoxic shock via a rapid induction of MAPK phosphatase-1. The Journal of Immunology, 2009. 182(12): p. 7957- 7962. 868. Liboni, K., N. Li, and J. Neu, Mechanism of glutamine-mediated amelioration of lipopolysaccharide-induced IL-8 production in Caco-2 cells. Cytokine, 2004. 26(2): p. 57- 65. 869. Lechowski, S., et al., Combined arginine and glutamine decrease release of de novo synthesized leukotrienes and expression of proinflammatory cytokines in activated human intestinal mast cells. European journal of nutrition, 2013. 52(2): p. 505-512. 870. Mayer, R.J. and J.F. Callahan, p38 MAP kinase inhibitors: a future therapy for inflammatory diseases. Drug Discovery Today: Therapeutic Strategies, 2006. 3(1): p. 49- 54. 871. Tripathi, P. and A. Aggarwal, NF-kB transcription factor: a key player in the generation of immune response. Current Science, 2006. 90(4): p. 519-531. 872. Smith, J.A., Neutrophils, host defense, and inflammation: a double-edged sword. Journal of Leukocyte Biology, 1994. 56(6): p. 672-686. 873. Aggarwal, B.B., Signalling pathways of the TNF superfamily: a double-edged sword. Nature Reviews Immunology, 2003. 3(9): p. 745-756. 874. Huang, B., et al., TLR signaling by tumor and immune cells: a double-edged sword. Oncogene, 2008. 27(2): p. 218-224. 875. Matsuzawa, A. and H. Ichijo, Molecular mechanisms of the decision between life and death: regulation of apoptosis by apoptosis signal-regulating kinase 1. Journal of biochemistry, 2001. 130(1): p. 1-8. 876. Werner, T. and D. Haller, Intestinal epithelial cell signalling and chronic inflammation: From the proteome to specific molecular mechanisms. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2007. 622(1): p. 42-57. 877. Clemens, M.J., PKR—a protein kinase regulated by double-stranded RNA. The international journal of biochemistry & cell biology, 1997. 29(7): p. 945-949. 878. Garcia, M., E. Meurs, and M. Esteban, The dsRNA protein kinase PKR: virus and cell control. Biochimie, 2007. 89(6): p. 799-811. 879. Balachandran, S., et al., Activation of the dsRNA-dependent protein kinase, PKR, induces apoptosis through FADD-mediated death signaling. The EMBO journal, 1998. 17(23): p. 6888-6902. 880. Donze, O., et al., The protein kinase PKR: a molecular clock that sequentially activates survival and death programs. The EMBO journal, 2004. 23(3): p. 564-571. 881. Rath, E., et al., Induction of dsRNA-activated protein kinase links mitochondrial unfolded protein response to the pathogenesis of intestinal inflammation. Gut, 2012. 61(9): p. 1269-1278. 882. Santoro, M.G., Heat shock factors and the control of the stress response. Biochemical pharmacology, 2000. 59(1): p. 55-63. 883. Mizushima, T., Protective role of HSP70 against various gastrointestinal diseases. Current opinion in pharmacology, 2014. 19: p. 1-5. 884. Kaisho, T. and S. Akira, Critical roles of Toll-like receptors in host defense. Critical Reviews™ in Immunology, 2000. 20(5). 885. Asea, A., et al., Novel signal transduction pathway utilized by extracellular HSP70 role of Toll-like receptor (TLR) 2 and TLR4. Journal of Biological Chemistry, 2002. 277(17): p. 15028-15034. 886. Dockray, G., R. Dimaline, and A. Varro, Gastrin: old hormone, new functions. Pflügers Archiv, 2005. 449(4): p. 344-355. 887. Chao, C. and M.R. Hellmich, Gastrin, inflammation, and carcinogenesis. Current opinion in endocrinology, diabetes, and obesity, 2010. 17(1): p. 33. 888. Subramaniam, D., et al., Gastrin-mediated interleukin-8 and cyclooxygenase-2 gene expression: differential transcriptional and posttranscriptional mechanisms. Gastroenterology, 2008. 134(4): p. 1070-1082. 889. Hiraoka, S., et al., Gastrin induces CXC chemokine expression in gastric epithelial cells through activation of NF-ʃ͘ŵĞƌŝĐĂŶ:ŽƵƌŶĂůŽĨWŚLJƐŝŽůŽŐLJ-Gastrointestinal and Liver Physiology, 2001. 281(3): p. G735-G742. 890. Sims, J.E. and D.E. Smith, The IL-1 family: regulators of immunity. Nature Reviews Immunology, 2010. 10(2): p. 89-102. 891. Leach, S.T., et al., Local and systemic interleukin-18 and interleukin-18-binding protein in children with inflammatory bowel disease. Inflammatory bowel diseases, 2008. 14(1): p. 68-74. 892. Erlandsson Harris, H. and U. Andersson, Mini-review: The nuclear protein HMGB1 as a proinflammatory mediator. European journal of immunology, 2004. 34(6): p. 1503-1512. 893. Ulloa, L. and D. Messmer, High-mobility group box 1 (HMGB1) protein: friend and foe. Cytokine & growth factor reviews, 2006. 17(3): p. 189-201. 894. Kokkola, R., et al., RAGE is the major receptor for the proinflammatory activity of HMGB1 in rodent macrophages. Scandinavian journal of immunology, 2005. 61(1): p. 1- 9. 895. Yu, M., et al., HMGB1 signals through toll-like receptor (TLR) 4 and TLR2. Shock, 2006. 26(2): p. 174-179. 896. Salminen, A., et al., Activation of innate immunity system during aging: NF-kB signaling is the molecular culprit of inflamm-aging. Ageing research reviews, 2008. 7(2): p. 83- 105. 897. Qin, Y.-H., et al., HMGB1 enhances the proinflammatory activity of lipopolysaccharide by promoting the phosphorylation of MAPK p38 through receptor for advanced glycation end products. The Journal of Immunology, 2009. 183(10): p. 6244-6250. 898. Vitali, R., et al., Fecal HMGB1 is a novel marker of intestinal mucosal inflammation in pediatric inflammatory bowel disease. The American journal of gastroenterology, 2011. 106(11): p. 2029-2040. 899. Palone, F., et al., Role of HMGB1 as a Suitable Biomarker of Subclinical Intestinal Inflammation and Mucosal Healing in Patients with Inflammatory Bowel Disease. Inflammatory bowel diseases, 2014. 20(8): p. 1448-1457. 900. Nishihira, J., Macrophage migration inhibitory factor (MIF): its essential role in the immune system and cell growth. Journal of Interferon & Cytokine Research, 2000. 20(9): p. 751-762. 901. Nishihira, J. and K. Mitsuyama, Overview of the role of macrophage migration inhibitory factor (MIF) in inflammatory bowel disease. Current pharmaceutical design, 2009. 15(18): p. 2104-2109. 902. Lue, H., et al., Macrophage migration inhibitory factor (MIF): mechanisms of action and role in disease. Microbes and Infection, 2002. 4(4): p. 449-460. 903. Koutroubakis, I.E., et al., Role of angiogenesis in inflammatory bowel disease. Inflammatory bowel diseases, 2006. 12(6): p. 515-523. 904. Danese, S., et al., Adhesion molecules in inflammatory bowel disease: therapeutic implications for gut inflammation. Digestive and liver disease, 2005. 37(11): p. 811-818. 905. Amin, M.A., et al., Migration inhibitory factor up-regulates vascular cell adhesion molecule-1 and intercellular adhesion molecule-ϭǀŝĂ^ƌĐ͕W/ϯŬŝŶĂƐĞ͕ĂŶĚE&ʃ͘ůŽŽĚ͕ 2006. 107(6): p. 2252-2261. 906. Amin, M.A., et al., Migration inhibitory factor mediates angiogenesis via mitogen- activated protein kinase and phosphatidylinositol kinase. Circulation research, 2003. 93(4): p. 321-329. 907. Marafini, I., et al., Targeting Integrins and Adhesion Molecules to Combat Inflammatory Bowel Disease. Inflammatory Bowel Diseases, 2014. 908. Stickel, F., et al., Nutritional therapy in alcoholic liver disease. Alimentary pharmacology & therapeutics, 2003. 18(4): p. 357-373. 909. Evert, A.B., et al., Nutrition therapy recommendations for the management of adults with diabetes. Diabetes care, 2013. 36(11): p. 3821-3842. 910. Kansal, S., et al., Enteral Nutrition in Crohn’s Disease: An Underused Therapy. Gastroenterology research and practice, 2013. 2013. 911. Konno, M., et al., Long-term therapeutic effectiveness of maintenance enteral nutrition for Crohn's disease. Pediatrics International, 2015. 57(2): p. 276-280. 912. Akobeng, A.K., et al., Double-blind randomized controlled trial of glutamine-enriched polymeric diet in the treatment of active Crohn's disease. Journal of pediatric gastroenterology and nutrition, 2000. 30(1): p. 78-84. 913. Marik, P.E. and G.P. Zaloga, Immunonutrition in critically ill patients: a systematic review and analysis of the literature. Intensive care medicine, 2008. 34(11): p. 1980-1990. 914. Baliga, M.S., et al., Curcumin, an active component of turmeric in the prevention and treatment of ulcerative colitis: preclinical and clinical observations. Food & function, 2012. 3(11): p. 1109-1117. 915. Wang, X., et al., Curcumin inhibits neurotensin-mediated interleukin-8 production and migration of HCT116 human colon cancer cells. Clinical cancer research, 2006. 12(18): p. 5346-5355. 916. Schofield, W., Predicting basal metabolic rate, new standards and review of previous work. Human nutrition. Clinical nutrition, 1984. 39: p. 5-41. 917. Bilsborough, S. and N. Mann, A review of issues of dietary protein intake in humans. International journal of sport nutrition and exercise metabolism, 2006. 16(2): p. 129. 918. Larmonier, C., et al., Modulation of neutrophil motility by curcumin: implications for inflammatory bowel disease. Inflammatory bowel diseases, 2011. 17(2): p. 503-515. 919. Hoffmann, E., et al., Multiple control of interleukin-8 gene expression. Journal of leukocyte biology, 2002. 72(5): p. 847-855. 920. Cheng, A.-L., et al., Phase I clinical trial of curcumin, a chemopreventive agent, in patients with high-risk or pre-malignant lesions. Anticancer research, 2001. 21(4B): p. 2895. 921. Anand, P., et al., Bioavailability of curcumin: problems and promises. Molecular pharmaceutics, 2007. 4(6): p. 807-818. 922. Lao, C.D., et al., Dose escalation of a curcuminoid formulation. BMC complementary and alternative medicine, 2006. 6(1): p. 10. 923. Midura-Kiela, M.T., et al., Curcumin inhibits interferon-ɶ ƐŝŐŶĂůŝŶŐ ŝŶ ĐŽůŽŶŝĐ Ğpithelial cells. American Journal of Physiology-Gastrointestinal and Liver Physiology, 2012. 302(1): p. G85-G96. 924. Den Hond, E., et al., Effect of long-term oral glutamine supplements on small intestinal permeability in patients with Crohn's disease. Journal of Parenteral and Enteral Nutrition, 1999. 23(1): p. 7-11. 925. Fujita, T. and K. Sakurai, Efficacy of glutamine-enriched enteral nutrition in an experimental model of mucosal ulcerative colitis. British journal of surgery, 1995. 82(6): p. 749-751. 926. Vicario, M., et al., Dietary glutamine affects mucosal functions in rats with mild DSS- induced colitis. The Journal of nutrition, 2007. 137(8): p. 1931-1937. 927. Tian, Y., et al., Chemopreventive effect of dietary glutamine on colitis-associated colon tumorigenesis in mice. Carcinogenesis, 2013. 928. Fox, A.D., et al., Effect of a glutamine-supplemented enteral diet on methotrexate- induced enterocolitis. Journal of Parenteral and Enteral Nutrition, 1988. 12(4): p. 325- 331. 929. Shang, H.-F., et al., Effects of arginine supplementation on mucosal immunity in rats with septic peritonitis. Clinical Nutrition, 2004. 23(4): p. 561-569. 930. Caparrós, T., J. Lopez, and T. Grau, Early Enteral Nutrition in Critically III Patients With a High-Protein Diet Enriched with Arginine, Fiber, and Antioxidants Compared With a Standard High-Protein Diet. The Effect on Nosocomial Infections and Outcome. Journal of Parenteral and Enteral Nutrition, 2001. 25(6): p. 299-309. 931. Gianotti, L., et al., Arginine-supplemented diets improve survival in gut-derived sepsis and peritonitis by modulating bacterial clearance. The role of nitric oxide. Annals of surgery, 1993. 217(6): p. 644. 932. Nones, K., et al., The effects of dietary curcumin and rutin on colonic inflammation and gene expression in multidrug resistance gene-ĚĞĨŝĐŝĞŶƚ ;ŵĚƌϭĂоͬоͿ ŵŝĐĞ͕ Ă ŵŽĚĞů ŽĨ inflammatory bowel diseases. British Journal of Nutrition, 2009. 101(02): p. 169-181. 933. Murphy, E.A., et al., Curcumin's effect on intestinal inflammation and tumorigenesis in the Apc Min/+ mouse. Journal of Interferon & Cytokine Research, 2011. 31(2): p. 219- 226. 934. Murakami, A., et al., Curcumin combined with turmerones, essential oil components of turmeric, abolishes inflammation-associated mouse colon carcinogenesis. BioFactors, 2012. 935. Juhás, S., et al., Effects of borneol and thymoquinone on TNBS-induced colitis in mice. FOLIA BIOLOGICA-PRAHA-, 2008. 54(1): p. 1. 936. Arita, M., et al., Resolvin E1, an endogenous lipid mediator derived from omega-3 eicosapentaenoic acid, protects against 2, 4, 6-trinitrobenzene sulfonic acid-induced colitis. Proceedings of the National Academy of Sciences of the United States of America, 2005. 102(21): p. 7671-7676. 937. Scheiffele, F. and I.J. Fuss, Induction of TNBS colitis in mice. Current Protocols in Immunology, 2002: p. 15.19. 1-15.19. 14. 938. Hollenbach, E., et al., Inhibition of RICK/nuclear factor-ʃĂŶĚƉϯϴƐŝŐŶĂůŝŶŐĂƚƚĞŶƵĂƚĞƐ the inflammatory response in a murine model of Crohn disease. Journal of Biological Chemistry, 2005. 280(15): p. 14981-14988. 939. Bai, A., et al., AMPK agonist downregulates innate and adaptive immune responses in TNBS-induced murine acute and relapsing colitis. Biochemical pharmacology, 2010. 80(11): p. 1708-1717. 940. Elliott, D.E., et al., Exposure to schistosome eggs protects mice from TNBS-induced colitis. American Journal of Physiology-Gastrointestinal and Liver Physiology, 2003. 284(3): p. G385-G391. 941. Kusugami, K., et al., Elevation of interleukin-6 in inflammatory bowel disease is macrophage-and epithelial cell-dependent. Digestive diseases and sciences, 1995. 40(5): p. 949-959. 942. Alhagamhmad, M.H., et al., An update of the role of nutritional therapy in the management of Crohn’s disease. Journal of gastroenterology, 2012. 47(8): p. 872-882. 943. te Velde, A.A., M.I. Verstege, and D.W. Hommes, Critical appraisal of the current practice in murine TNBS-induced colitis. Inflammatory bowel diseases, 2006. 12(10): p. 995-999. 944. Mudter, J. and M.F. Neurath, Il-6 signaling in inflammatory bowel disease: Pathophysiological role and clinical relevance. Inflammatory bowel diseases, 2007. 13(8): p. 1016-1023. 945. Papadakis, K.A. and S.R. Targan, Role of cytokines in the pathogenesis of inflammatory bowel disease. Annual review of medicine, 2000. 51(1): p. 289-298. 946. Faith, M., et al., How reliable an indicator of inflammation is myeloperoxidase activity? Clinica Chimica Acta, 2008. 396(1): p. 23-25. 947. Vijay-Kumar, M., et al., Activation of toll-like receptor 3 protects against DSS-induced acute colitis. Inflammatory bowel diseases, 2007. 13(7): p. 856-864. 948. Southey, A., et al., Pathophysiological role of nitric oxide in rat experimental colitis. International journal of immunopharmacology, 1998. 19(11-12): p. 669-676. 949. Singh, I., Textbook of human histology:(with colour atlas & practical guide). 2011: Jaypee Brothers Publishers. 950. Seidl, K. and A.-F. Holstein, Organ culture of human seminiferous tubules: a useful tool to study the role of nerve growth factor in the testis. Cell and tissue research, 1990. 261(3): p. 539-547. 951. Pinna, G.F., et al., Celastrol inhibits pro-inflammatory cytokine secretion in Crohn’s disease biopsies. Biochemical and biophysical research communications, 2004. 322(3): p. 778-786. 952. Meister, D. and S. Ghosh, Effect of fish oil enriched enteral diet on inflammatory bowel disease tissues in organ culture: differential effects on ulcerative colitis and Crohn's disease. World journal of gastroenterology: WJG, 2005. 11(47): p. 7466-7472. 953. Borges, Á.M., G.D. Healey, and I.M. Sheldon, Explants of intact endometrium to model bovine innate immunity and inflammation ex vivo. American Journal of Reproductive Immunology, 2012. 67(6): p. 526-539. 954. Borrelli, O., et al., Polymeric diet alone versus corticosteroids in the treatment of active pediatric Crohn’s disease: a randomized controlled open-label trial. Clinical Gastroenterology and Hepatology, 2006. 4(6): p. 744-753. 955. Peterson, L.W. and D. Artis, Intestinal epithelial cells: regulators of barrier function and immune homeostasis. Nature Reviews Immunology, 2014. 14(3): p. 141-153. 956. Garrett, W.S., J.I. Gordon, and L.H. Glimcher, Homeostasis and inflammation in the intestine. Cell, 2010. 140(6): p. 859-870. 957. Alex, P., et al., Distinct cytokine patterns identified from multiplex profiles of murine DSS and TNBS-induced colitis. Inflammatory bowel diseases, 2009. 15(3): p. 341-352. 958. Rhoads, J.M. and G. Wu, Glutamine, arginine, and leucine signaling in the intestine. Amino acids, 2009. 37(1): p. 111-122. 959. Lowe, D.K., et al., Safety of glutamine-enriched parenteral nutrient solutions in humans. The American journal of clinical nutrition, 1990. 52(6): p. 1101-1106. 960. Díaz-Herrero, M.M., et al., THDP17 Decreases Ammonia Production through Glutaminase Inhibition. A New Drug for Hepatic Encephalopathy Therapy. PloS one, 2014. 9(10): p. e109787. 961. Goodhand, J.R., et al., Prevalence and management of anemia in children, adolescents, and adults with inflammatory bowel disease. Inflammatory bowel diseases, 2012. 18(3): p. 513-519. 962. Nic Suibhne, T., et al., High prevalence of overweight and obesity in adults with Crohn's disease: associations with disease and lifestyle factors. Journal of Crohn's and Colitis, 2013. 7(7): p. e241-e248. 963. Sazuka, S., et al., Concomitant use of enteral nutrition therapy is associated with sustained response to infliximab in patients with Crohn’s disease. European journal of clinical nutrition, 2012. 66(11): p. 1219-1223. 964. Triantafillidis, J.K., C. Vagianos, and A.E. Papalois, The Role of Enteral Nutrition in Patients with Inflammatory Bowel Disease: Current Aspects. BioMed research international, 2015. 2015. 965. Klaassen, J., et al., [Enteral nutrition in severe ulcerative colitis. Digestive tolerance and nutritional efficiency]. Revista medica de Chile, 1998. 126(8): p. 899-904. 966. Wei, J. and J. Feng, Signaling pathways associated with inflammatory bowel disease. Recent patents on inflammation & allergy drug discovery, 2010. 4(2): p. 105-117. 967. Berntson, L., Anti-inflammatory effect by exclusive enteral nutrition (EEN) in a patient with juvenile idiopathic arthritis (JIA): brief report. Clinical rheumatology, 2014: p. 1-3. 968. Sakaguchi, S., et al., Regulatory T cells and immune tolerance. Cell, 2008. 133(5): p. 775- 787. 969. Roman-Blas, J. and S. Jimenez, NF-ʃĂƐĂƉŽƚĞŶƚŝĂůƚŚĞƌĂƉĞƵƚŝĐƚĂƌŐĞƚŝŶŽƐƚĞŽĂƌƚŚƌŝƚŝƐ and rheumatoid arthritis. Osteoarthritis and cartilage, 2006. 14(9): p. 839-848. 970. Lahdenne, P., P. Vähäsalo, and V. Honkanen, Infliximab or etanercept in the treatment of children with refractory juvenile idiopathic arthritis: an open label study. Annals of the rheumatic diseases, 2003. 62(3): p. 245-247. 971. Ljung, T., et al., Infliximab in inflammatory bowel disease: clinical outcome in a population based cohort from Stockholm County. Gut, 2004. 53(6): p. 849-853. 972. Reyes-Gordillo, K., et al., Curcumin protects against acute liver damage in the rat by inhibiting NF-ʃ͕ƉƌŽŝŶĨůĂŵŵĂƚŽƌLJĐLJƚŽŬŝŶĞƐƉƌŽĚƵĐƚŝŽŶĂŶĚŽdžŝĚĂƚŝǀĞƐƚƌĞƐs. Biochimica et Biophysica Acta (BBA)-General Subjects, 2007. 1770(6): p. 989-996. 973. Baumgartner, B., et al., Increased IkB kinase activity is associated with activated NF-kB in acute myeloid blasts. Leukemia, 2002. 16(10): p. 2062-2071. 974. Vilimas, T., et al., Targeting the NF-ʃ ƐŝŐŶĂůŝŶŐ ƉĂƚŚǁĂLJ ŝŶ EŽƚĐŚϭ-induced T-cell leukemia. Nature medicine, 2007. 13(1): p. 70-77. 975. Barnes, P.J. and I.M. Adcock, NF-kB: a pivotal role in asthma and a new target for therapy. Trends in pharmacological sciences, 1997. 18(2): p. 46-50. 976. Messina, S., et al., Activation of NF-KB pathway in Duchenne muscular dystrophy: relation to age. Acta Myologica, 2011. 30(1): p. 16. 977. Venna, V.R., et al., NF-ʃĐŽŶƚƌŝďƵƚĞƐƚŽƚŚĞĚĞƚƌŝŵĞŶƚĂůĞĨĨĞĐƚƐŽĨƐŽĐŝĂůŝƐŽůĂƚŝŽŶĂfter experimental stroke. Acta neuropathologica, 2012. 124(3): p. 425-438. $SSHQGL[ ® Standard Energy OSMOLITE 1.0 – 1.2kcal/mL

Osmolite® is a 1.0kcal/mL complete and balanced isotonic fibre free tube feed.

INDICATIONS FEATURES UÊÊ ˆÃi>ÃiÊÀi>Ìi`ʓ>˜ÕÌÀˆÌˆœ˜ U Low residue, isotonic promotes U >ˆ˜Ìi˜>˜ViʘÕÌÀˆÌˆœ˜ÊÃÕ««œÀÌ tolerance Uʘ̜iÀ>˜ViÊÌœÊ Þ«iÀœÃ“œ>ÀÊi˜ÌiÀ˜> U Fat blend includes MCT oil feeding to facilitate fat absorption Uʘ̜iÀ>˜ViÊ̜ÊwLÀiÊVœ˜Ì>ˆ˜ˆ˜}Êi˜ÌiÀ> U Variety of presentation sizes feeds to reduce wastage

FACTS NUTRIENT INFORMATION Nutrient density (Cal/mL) 1.0 Avg Qty Avg Qty NUTRIENT Unit (kJ/mL) 4.2 Per 100mL Per 1000mL Protein (% Cal) 15.94 Energy kJ 422 4223 Carbohydrate (% Cal) 54.03 kcal 100 1004 Protein g 4.00 40 Fat (% Cal) 30.03 Carbohydrate g 13.56 136 kcal/N ratio 160:1 Sugars g 0.63 6.3 Non-protein kcal/N ratio 134:1 Fat g 3.40 34 Kosher Yes Saturated fat g 0.84 8.4 Gluten-free Yes Monounsaturated fat g 1.93 19.3 Lactose 0.01g/100mL Polyunsaturated fat g 0.48 4.8 Renal Solute Load (mOsm/L) 342 Dietary fibre, total g 0 0 Water g 84.9 849 Osmolality (mOsm/kgH 0) 288 2 VITAMINS N-6:N-3 Ratio 4.1:1 Vitamin A mcg RE 108 1080 RDI/AI volume 1550mL# Vitamin D3 mcg 0.73 7.3 # Recommended Dietary Intake (RDI) and Adequate Intake (AI) volumes Vitamin E mg | TE 2.1 21 based on ‘Nutrient Reference Values for Australia and New Zealand 2005’ Vitamin K1 mcg 5.2 52 Male 31-50y, specified volume meets 100% RDI/AI for micronutrients Vitamin C mg 10 100 excluding electrolytes with the exception of Folate (89% RDI), Magnesium (74%RDI). Folic acid mcg 23 230 Thiamin (Vitamin B1) mg 0.16 1.6

Riboflavin (Vitamin B2) mg 0.18 1.8 Vitamin B mg 0.22 2.2 INGREDIENTS 6 Vitamin B12 mcg 0.34 3.4 Water, maltodextrin, sodium and calcium caseinates, high Niacin mg NE1.7 17 oleic sunflower oil, canola oil, MINERALS (potassium citrate, Choline mg 56 560 calcium phosphate tribasic, magnesium chloride, potassium Biotin mcg 4.6 46 chloride, sodium citrate, potassium phosphate dibasic, Pantothenic acid mg 0.78 7.8 magnesium sulphate, ferrous sulphate, zinc sulphate, MINERALS manganese sulphate, cupric sulphate, sodium molybdate, Sodium mg 88 880 chromium chloride, sodium selenate, potassium iodide), mmol 3.83 38.3 soy protein isolate, MCT oil, soy lecithin, VITAMINS (choline Potassium mg 148 1480 chloride, ascorbic acid, dl-alpha tocopheryl acetate, mmol 3.79 37.9 niacinamide, calcium pantothenate, pyridoxine hydrochloride, Chloride mg 136 1360 thiamin hydrochloride, riboflavin, vitamin A palmitate, mmol 3.84 38.4 Calcium mg 68 680 folic acid, biotin, phylloquinone, vitamin D , cyanocobalamin), 3 Phosphorus mg 68 680 carrageenan. Magnesium mg 20 200 May contain: sodium chloride. Iodine mcg 11 110 Manganese mg 0.38 3.8 Copper mcg 170 1700 ORDERING INFORMATION Zinc mg 1.3 13 Units Abbott Nutrition Iron mg 1.4 14 Flavour Presentation per case Product Code Selenium mcg 6.0 60 Unflavoured 250mL Can 24 E711.12501 Chromium mcg 6.5 65 Unflavoured 500mL RTH 15 E711.15003 Molybdenum mcg 12 120 Unflavoured 1000mL RTH 8 E711.154

www.abbottnutrition.com.au

Abbott Australasia Pty Ltd. ABN 95 000 180 389. 32-34 Lord Street, Botany NSW 2019 Australia. Customer Service 1800 225 311. Date of Preparation: December 2010. Every effort is made to ensure all information is correct at time of publication, however is subject to change. Please refer to product labels for the most current information. This page has been downloaded from the Abbott Nutrition Australia website. This information is provided for use by a Healthcare Professional. ANPI-001-1210-1AU-B1 17 $SSHQGL[ Gene Symbol p-value(NConFold-Change( IL8 1.67E-10 -45.6516 IL8 1.26E-06 -34.344 CXCL1 7.53E-09 -32.8438 CCL20 1.94E-08 -32.8033 IL8 2.05E-06 -27.8025 PI3 1.04E-08 -21.898 TNFAIP2 1.40E-09 -20.9724 PHLDB2 0.00049552 -17.0663 CXCL3 1.31E-08 -14.8492 BIRC3 7.50E-09 -14.1478 BIRC3 1.95E-08 -13.9428 CXCL2 1.43E-07 -13.2228 PTGS2 9.06E-06 -13.0202 BIRC3 4.85E-09 -9.54519 CXCL10 9.93E-05 -8.99013 CXCL3 2.29E-07 -7.68866 TNFAIP3 4.56E-08 -7.55665 PLAU 8.56E-06 -7.08371 PHLDB2 0.00388046 -6.99807 SDC4 7.44E-07 -6.73687 SDC4 8.91E-07 -6.43613 LTB 2.84E-06 -6.07953 SPRR1B 1.33E-09 -5.98315 PTGS2 0.00021638 -5.54356 TRIM29 1.80E-10 -5.48774 ETS1 4.96E-05 -5.47884 LAMC2 5.66E-05 -5.44798 TNF 9.68E-09 -5.26621 SPRR1A 7.65E-07 -5.18267 TRIM29 2.24E-08 -5.06331 FAM43A 6.24E-08 -5.04552 S100A3 2.97E-06 -5.02244 LAMC2 6.50E-05 -4.97075 TRIM29 2.05E-06 -4.97035 SEMG1 7.09E-07 -4.81164 TAP1 6.98E-06 -4.79925 ETS1 9.76E-06 -4.60109 C15orf52 2.69E-05 -4.48959 RELB 2.09E-08 -4.42513 CD83 0.00015241 -4.39861 CXCL2 6.93E-08 -4.3939 ICAM1 1.26E-06 -4.33092 CXCL11 0.00146907 -4.04049 KLK10 9.16E-08 -3.97184 TNFAIP3 1.84E-06 -3.96563 ICAM1 1.51E-07 -3.89487 NFKBIA 3.80E-05 -3.8736 FOSL1 0.00484762 -3.87199 IFIH1 1.81E-05 -3.80373 NCEH1 2.97E-08 -3.80072 NFKBIE 3.04E-07 -3.80008 IFNAR2 2.26E-07 -3.62123 TNFSF10 6.42E-07 -3.61595 SPC25 0.00013611 -3.61458 IRF1 0.00579037 -3.54567 NFKBIA 6.80E-05 -3.54346 IRF1 0.00377138 -3.53651 PARP14 8.50E-05 -3.52345 TRIM31 4.35E-08 -3.44705 NFKBIA 6.06E-05 -3.36788 SERPINB8 1.07E-06 -3.34555 TNFSF10 9.90E-07 -3.29649 PHLDB2 0.00573575 -3.27837 PSMB9 4.92E-05 -3.27337 MKI67 3.37E-06 -3.25603 GABBR1 0.00256839 -3.235 IER3 0.00016626 -3.21831 TRIM31 6.54E-10 -3.18159 TAP2 2.14E-05 -3.17961 CDC25A 0.00134327 -3.16705 PARP14 3.58E-06 -3.15427 SPP1 0.00368724 -3.1381 IL32 4.86E-07 -3.13727 CSF2 1.40E-06 -3.13302 MKI67 6.33E-06 -3.13283 CXCL11 0.00261303 -3.10405 PIF1 0.00013578 -3.07694 LAMC2 4.90E-07 -3.05488 NMUR2 0.00043939 -3.05282 PARP14 5.81E-05 -3.02445 SPRR1A 4.09E-06 -3.02122 IFNGR1 3.77E-05 -2.97037 SLC6A14 0.00060196 -2.96872 SDC4 2.75E-08 -2.96351 RDM1 0.00098755 -2.95444 CXCL1 5.47E-06 -2.92927 SAMD9 0.00189338 -2.90572 LMNB1 1.61E-06 -2.87278 LMNB1 1.61E-06 -2.87278 NCAPG 1.85E-05 -2.8634 POLQ 4.35E-05 -2.80022 RDM1 2.21E-05 -2.79792 NFKB1 5.89E-07 -2.78811 OBSCN 0.00023207 -2.78666 C4BPB 0.00672533 -2.78295 --- 3.28E-05 -2.78172 AURKB 2.18E-07 -2.76275 IL32 2.59E-05 -2.7576 CDKN1A 0.00142469 -2.75415 DRAM1 2.32E-05 -2.74843 CDC20 1.40E-05 -2.73333 DDX58 0.00013798 -2.71133 EREG 6.68E-08 -2.71099 SLC28A3 0.00021396 -2.70615 POLA2 0.00016675 -2.70162 ATF5 1.74E-06 -2.68888 KLK10 2.05E-05 -2.67414 CXCL1 1.95E-05 -2.6638 FOXO1 9.77E-05 -2.64461 IRAK2 7.43E-05 -2.64458 CFLAR 0.00036186 -2.63844 APOBEC3B 1.70E-05 -2.63445 OAS3 5.10E-05 -2.59899 PSMB10 5.97E-07 -2.58304 KIF14 0.00025265 -2.56863 CYLD 1.25E-06 -2.56042 APOBEC3A 2.63E-05 -2.55924 HIST2H2AA3 8.26E-09 -2.55801 HIST2H2AA3 1.43E-07 -2.55448 BIRC5 4.58E-07 -2.55291 CCNF 5.23E-06 -2.55151 SERPINB8 4.65E-05 -2.54667 CDC25A 1.13E-07 -2.5455 POP1 8.96E-06 -2.53541 BTG3 5.42E-06 -2.52827 TNFRSF10B 4.96E-05 -2.52699 CDCA2 0.00025351 -2.52157 FOXM1 0.00022325 -2.5169 ZNF121 1.54E-05 -2.51145 CFLAR 1.21E-05 -2.51022 NEB 0.0002554 -2.50575 CDC45 0.00011928 -2.50409 MTAP 0.001706 -2.50277 ITGB8 0.00116568 -2.50101 PSMB10 1.05E-06 -2.49352 DLGAP5 0.00043216 -2.49328 CCNB1 0.00044245 -2.49284 FOXO1 2.63E-05 -2.4889 PSME2 4.08E-05 -2.4796 SEMA3C 0.00147606 -2.47871 ZNF121 3.72E-05 -2.45658 IKBKE 6.29E-05 -2.44989 MKI67 9.05E-05 -2.43672 PARP12 9.38E-05 -2.43208 PSMB10 4.49E-05 -2.41999 SEMA3A 0.00433784 -2.41643 DLGAP5 0.00047639 -2.4143 APOL6 5.52E-05 -2.41429 EMG1 3.11E-05 -2.40993 SQSTM1 0.0001529 -2.40329 SFR1 5.00E-07 -2.40137 TNS4 0.0002909 -2.40058 --- 0.00047281 -2.39481 HJURP 1.06E-05 -2.38841 MYO10 0.00281619 -2.38522 ITGAM 0.00357227 -2.38351 PHLDB2 0.00122278 -2.37967 PLK2 4.56E-07 -2.37492 TAP1 4.41E-05 -2.37051 NFKBIB 0.00130703 -2.36729 BLMH 0.00010914 -2.3485 SLC25A22 3.17E-09 -2.34602 XKRX 0.00099498 -2.34368 IL17RB 2.17E-06 -2.34034 OAS3 0.00634067 -2.33854 CDC45 2.88E-08 -2.33662 LOC10012788 2.19E-07 -2.33197 CDCA5 5.80E-05 -2.32004 MMD 1.95E-05 -2.31236 CDKN1A 0.00236192 -2.31011 AURKA 1.06E-05 -2.30651 RAET1L 0.0002105 -2.30334 RARRES1 0.00119561 -2.30154 KIF2C 4.00E-05 -2.29489 LOXL4 0.00103561 -2.28766 AURKA 0.0001434 -2.2708 TACC3 4.77E-06 -2.27071 MT2A 7.64E-05 -2.2604 SPAG5 0.00010038 -2.2505 ATP6V1G2-DD 3.09E-08 -2.24461 CHAC2 0.00116654 -2.24369 DENND1A 3.89E-07 -2.24065 NASP 4.11E-07 -2.2339 TPX2 4.12E-05 -2.23284 BIRC5 0.00033677 -2.23066 SPC24 0.00417729 -2.22805 SGK1 0.00391116 -2.22204 FAM40B 4.58E-06 -2.22084 CX3CL1 6.70E-06 -2.21899 DLEU2 0.00328653 -2.21114 FAS 0.00179251 -2.21054 SH3RF2 1.41E-07 -2.204 TROAP 9.72E-05 -2.1994 NFKBIB 0.0005494 -2.19241 NINJ1 9.94E-09 -2.19135 FKBP1A 6.85E-06 -2.19118 CENPF 0.00056957 -2.18958 STAP2 1.23E-05 -2.18597 ARHGAP11A 0.00078688 -2.17839 ESCO2 0.00210564 -2.1759 ARHGAP11A 0.00096575 -2.17406 ERAP2 0.00014715 -2.17136 LETM1 6.21E-05 -2.1698 KIF22 8.92E-05 -2.16591 RNF207 0.00010684 -2.16038 ANP32E 7.89E-06 -2.15895 UBE2C 0.00032459 -2.15789 FEN1 1.88E-05 -2.15721 CENPE 0.0002472 -2.1553 LRR1 0.00174642 -2.14855 LAMC2 0.00141974 -2.14581 SDF2L1 1.98E-06 -2.14568 TXNL4B 2.92E-05 -2.14292 SERPINA3 5.47E-05 -2.14242 CITED4 5.79E-06 -2.14129 NCAPG2 8.11E-08 -2.13947 KIF2C 0.00111775 -2.13621 HAUS7 5.27E-06 -2.13401 BIRC5 3.20E-05 -2.13189 SGK1 0.00456065 -2.12938 ITGAV 0.00069718 -2.12709 SSSCA1 2.24E-06 -2.12521 ATG4B 0.00157587 -2.12392 LAMC2 0.00090727 -2.123 CDKN3 0.00444416 -2.12056 NFE2L3 0.00026117 -2.1194 PHKG2 1.79E-05 -2.11891 PVRL2 4.41E-06 -2.11455 PDXK 5.39E-06 -2.11415 TAP2 0.0002201 -2.11282 DDX51 0.00082011 -2.11098 CXCL11 0.00489795 -2.10635 H2AFX 1.43E-05 -2.10634 CDKN3 0.00053378 -2.1049 HIST1H2BC 0.00036344 -2.10412 HMGA2 0.00033157 -2.09944 EYA3 6.66E-06 -2.09819 CHEK1 6.16E-05 -2.09644 C11orf82 0.00464614 -2.09404 NEB 0.00157148 -2.09026 FANCA 2.54E-05 -2.08906 C1orf112 0.00414059 -2.08817 ISG15 2.14E-06 -2.08771 PSMC3 9.69E-06 -2.08754 COMMD4 9.37E-06 -2.08575 ARPC3 3.49E-06 -2.08446 CDKN3 0.00081031 -2.08373 PHLDB2 0.00354153 -2.08247 GPATCH4 2.02E-05 -2.08212 LMNB1 0.00083712 -2.08169 SERPINA3 0.00074793 -2.08085 AP5S1 4.52E-06 -2.07645 TAP2 0.00084814 -2.07174 PSMB9 0.00099897 -2.0712 DDX56 1.11E-06 -2.06709 NCAPG 0.00117674 -2.06654 CCDC150 0.0001427 -2.06479 ARHGAP11A 0.00476908 -2.06372 HIST2H2AC 0.00078643 -2.06325 KIF23 0.00010372 -2.06314 RRP7A 6.19E-06 -2.06242 DTX3L 8.97E-05 -2.06075 CLSPN 9.46E-05 -2.06057 POLA2 7.40E-05 -2.05931 ANXA1 0.00041084 -2.05755 FAM54A 0.00058302 -2.05641 EP400NL 2.84E-05 -2.05621 CENPN 0.00047638 -2.05576 GJB2 7.29E-05 -2.05501 NCAPG2 6.39E-07 -2.0545 DDX23 5.39E-06 -2.05407 EZH2 0.00017665 -2.05368 EP400NL 2.07E-05 -2.05355 ESCO2 2.02E-06 -2.05052 CHMP6 8.23E-05 -2.04919 C10orf10 3.20E-05 -2.04857 LAMC2 0.00016782 -2.04819 GJB6 0.00586558 -2.04367 ANXA1 0.00033892 -2.04334 TRAF3 0.00130026 -2.04211 CCDC64 7.55E-08 -2.04174 RACGAP1 8.51E-05 -2.04116 CDCA3 0.00021881 -2.03917 NAA50 2.79E-05 -2.03849 AKIP1 5.30E-05 -2.03822 HDAC9 2.81E-05 -2.03725 SGK1 0.00529042 -2.03444 NUSAP1 0.0001796 -2.03371 MUC12 1.97E-05 -2.03359 ZNF593 3.24E-06 -2.03089 ANXA1 0.00084668 -2.03086 ABCF1 2.14E-05 -2.0305 FTSJ3 2.07E-06 -2.03024 TUBB 2.34E-05 -2.02859 ABHD10 5.35E-06 -2.02738 KIAA1524 0.00282608 -2.02654 NMU 0.00015747 -2.0257 HSPA5 1.71E-05 -2.02508 NEK2 0.00015297 -2.0238 ZNF697 0.00025918 -2.02297 MNF1 1.62E-05 -2.02083 IL32 5.23E-05 -2.0205 EYA3 1.86E-06 -2.01977 KIAA0754 1.99E-06 -2.01969 TUBB 6.36E-08 -2.01698 DTX3L 0.00019207 -2.01344 RRP7A 2.68E-06 -2.01229 METTL1 0.00060732 -2.00983 LAMC2 0.00128932 -2.00656 IKBKE 1.12E-05 -2.0049 NEDD9 0.00286003 -2.00456 EIF1AD 6.90E-05 -2.00426 EXOC4 1.61E-05 -2.00378 MTMR12 0.00016681 -2.00179 PTTG1 6.32E-05 -2.00161 HAUS7 0.00241134 -2.00037 TRIB1 0.00153993 2.00025 CXXC5 0.00361731 2.00048 CA12 1.31E-06 2.00252 PDPK1 1.82E-05 2.0027 ROCK1 4.87E-05 2.00284 CRTAP 0.00015616 2.00372 PKM 5.67E-06 2.00418 TSPAN12 0.00023166 2.00487 EPB41L4B 1.03E-05 2.00519 C20orf112 7.99E-05 2.00598 PHF3 0.00704887 2.00625 TNFSF15 0.00052715 2.00698 FAM47E 0.00029458 2.00924 RAD21 0.00151184 2.00966 SPRY2 0.0021831 2.00994 ZNF558 0.0014392 2.01052 APP 9.18E-06 2.01119 MPZL2 1.42E-07 2.01163 PTPRE 1.43E-05 2.01271 JMJD1C 0.00242671 2.01287 ABCD3 0.00257308 2.01346 MLL3 0.00544842 2.01358 TMTC4 0.0011171 2.01417 MTDH 0.00172776 2.0148 ENOPH1 0.00338843 2.01482 STAG2 0.00199773 2.01648 PHF2 0.00015498 2.01688 CAPZA1 0.00453937 2.01921 CCNY 6.45E-07 2.01941 AFF1 1.69E-06 2.0209 SEC24D 0.00074233 2.0209 RNASE4 2.30E-05 2.02178 LOC10050747 1.76E-05 2.02196 SYBU 2.81E-07 2.02204 HOXB3 0.000576 2.02207 TCEAL1 7.59E-06 2.02216 CAMK2D 2.34E-05 2.02227 HPGD 5.66E-05 2.02353 CDC42BPA 0.00097151 2.02376 EEF2 8.26E-06 2.02394 SMARCE1 0.00114992 2.02409 PTCH1 2.70E-05 2.02446 WSB1 0.00015458 2.02456 KLHL2 0.00270857 2.0253 CDX2 0.00107453 2.02532 APBB2 0.00278625 2.02567 TEP1 6.94E-06 2.02575 AJUBA 3.08E-06 2.0265 MAP2K1 0.00072572 2.02751 MUC20 1.05E-05 2.02786 CEACAM5 6.36E-07 2.02802 IGF2R 5.54E-07 2.03065 BRI3BP 0.00271821 2.03078 UBE2Q2 0.00167811 2.03148 ITM2C 3.12E-06 2.03164 EIF3F 0.0017817 2.03166 ZNF280D 0.00030773 2.03187 PTTG1IP 0.00325435 2.03255 BMP2 2.17E-05 2.03284 ATP6V1A 0.00097794 2.03371 PLXNA2 5.61E-08 2.03627 TIA1 0.00022788 2.03661 TMED4 3.30E-05 2.03675 TXNIP 0.00059138 2.03691 INSR 0.00018746 2.03864 KCNH2 0.00145248 2.03912 TCF12 0.00059877 2.04009 PBX1 0.00330128 2.04215 PPARA 3.81E-05 2.04312 TSC22D2 0.00024103 2.04333 WDR45L 0.0004218 2.04335 LOC10028749 0.00297267 2.0443 CTGF 0.00299243 2.04443 PIGP 8.18E-07 2.04471 BMPR2 0.00447917 2.04596 BIVM 2.52E-06 2.04693 CREB3L1 0.00067931 2.0471 EIF2AK3 0.00195058 2.04836 RPL15 1.12E-05 2.04901 TOMM20 0.00151716 2.0491 PKDCC 0.00217417 2.04962 PTPRR 0.00019064 2.05079 ITPR1 0.00152298 2.05141 ERRFI1 8.52E-05 2.05294 LOC10050654 2.07E-07 2.05331 ZDHHC9 2.96E-05 2.05384 KIAA1244 0.00168476 2.05438 UPRT 0.00024492 2.0544 LMAN2L 0.00399047 2.05573 RALGDS 7.91E-05 2.05614 PIM1 1.70E-06 2.0566 RPS6KA3 0.00245299 2.05678 RHPN2 0.00682356 2.057 FBXO16 0.00293563 2.05818 CAPN5 0.00063121 2.05948 SMARCA2 0.00279932 2.05995 SLC26A2 0.0002624 2.06095 CXXC5 0.00132717 2.06101 IL1RAP 5.69E-06 2.06153 SYBU 2.36E-06 2.06165 CDC73 0.0017051 2.06341 RBM47 0.00039168 2.06466 ZMIZ1 0.00038938 2.06587 ASAH1 0.00013133 2.0659 GNB1 0.0014235 2.0664 SPRED1 0.00018082 2.06641 RREB1 0.00286334 2.06679 RICTOR 0.00707175 2.06713 ITGB1 0.00195884 2.06717 KIAA1199 6.29E-06 2.06765 CLK1 0.00010635 2.06773 EIF4A2 3.56E-05 2.06831 TRIM4 1.03E-05 2.06857 NDFIP2 0.00241784 2.07109 PTCH1 4.25E-07 2.07335 SPRED2 0.00021635 2.0736 NPTN 0.00024987 2.07467 ZNF468 0.00308677 2.07474 NFAT5 5.01E-05 2.07485 APOBEC1 0.00014991 2.07586 POLI 0.0016491 2.07613 HEPH 0.00012378 2.07765 IPO7 0.00105121 2.07824 C17orf103 0.00020376 2.07914 DAB2 0.00026224 2.08065 KCNE3 3.52E-05 2.08141 GPR160 5.39E-08 2.08148 SPIN1 0.00380464 2.08417 SH3BGRL 0.00089552 2.08493 KLHDC10 0.00661472 2.08493 C3orf58 0.00695611 2.08632 ABCC4 0.00517554 2.08657 SLC40A1 2.65E-06 2.08826 OBFC1 0.00048478 2.08859 PDXDC1 0.00612004 2.08963 PPM1H 0.00019942 2.09255 STAU1 0.0023321 2.0931 LIMCH1 0.00024909 2.094 ZNF791 0.0021008 2.09457 ADNP 5.71E-07 2.0952 CA12 1.27E-06 2.09551 GNAQ 0.00062436 2.0967 PLCB1 0.00023647 2.0978 SLC17A5 0.00359253 2.09837 SEC23A 0.00090937 2.09839 CD164 0.00037121 2.09852 MED13L 0.00242535 2.09934 IQGAP1 0.00449428 2.09949 BPTF 2.13E-05 2.10047 ADD3 0.00025833 2.10078 ACVR1 2.02E-06 2.10088 PLS1 0.00290389 2.10142 NIPSNAP3A 0.00064729 2.10174 C15orf48 0.00011565 2.10296 IGFBP7 0.00472514 2.10384 PLOD2 0.00557754 2.10387 ABCG1 0.00031134 2.10513 ADNP 2.46E-07 2.10541 TMEM135 9.13E-05 2.10573 CLDND1 0.00217434 2.10619 CRBN 0.00415027 2.10695 MYLIP 0.00680775 2.11069 PIP5K1B 0.00091009 2.1116 TMCC1 5.38E-06 2.11171 SIAH1 0.00129181 2.11282 FAM108C1 0.00111093 2.11336 BMPR2 1.87E-05 2.11583 PIK3C2A 0.00181968 2.11651 SH3YL1 0.00018277 2.11701 TRIM4 0.00235072 2.11785 NPTN 0.00014199 2.12001 APP 8.57E-06 2.12456 RAB6B 0.00093166 2.12486 LETMD1 0.00033354 2.12531 MAN1A1 1.87E-06 2.12598 SOS2 0.00029812 2.12607 MTUS1 0.00020353 2.12678 PAM 4.60E-05 2.12701 HIPK2 0.00030886 2.12711 BPTF 1.66E-05 2.1322 KIAA0895 7.08E-06 2.13495 XRN2 0.0065624 2.13495 MEAF6 0.00024817 2.1358 RMI1 0.00063518 2.13814 AHCYL1 0.00013217 2.13827 GCOM1 6.17E-07 2.13937 FBXO11 0.00238111 2.13969 KIAA1211 0.00224768 2.14179 FAM172A 0.00014733 2.14253 DCAF7 0.00283803 2.14368 ADM 4.83E-05 2.14515 SERPINE2 0.00077425 2.14708 CD46 0.00287119 2.1477 --- 0.00050139 2.14789 TCF7L2 0.00054723 2.14802 PPP2R5C 2.02E-05 2.14966 SYT17 0.0038522 2.15013 FOXP1 0.00279064 2.1522 DACH1 0.00075256 2.15269 PDZK1 0.00078269 2.15274 HBP1 0.00025068 2.15351 ARID4B 0.00244249 2.15422 ZFP62 0.00703295 2.15601 VEGFA 4.39E-06 2.15713 OXR1 0.0003236 2.15716 ERGIC1 0.00081123 2.15721 CHRAC1 7.84E-05 2.15801 LETMD1 8.99E-05 2.15918 YPEL2 3.52E-06 2.16256 PIGP 1.57E-07 2.16267 FNBP1L 7.97E-06 2.16298 RALGDS 3.90E-05 2.16305 FAM177B 0.00147791 2.16393 CDYL 0.00064309 2.16461 MANEA 0.0017543 2.1653 TOX3 0.00014463 2.16631 NAP1L5 0.00029999 2.16708 APP 3.74E-06 2.16739 IRS1 0.00073412 2.1674 CD44 0.00185945 2.16848 TFF3 0.00166939 2.17094 MBNL2 0.00298026 2.1713 FXYD3 4.12E-07 2.17133 APP 2.15E-05 2.17232 PAPSS1 3.28E-06 2.17234 CPOX 2.68E-05 2.17392 CDKN1C 0.00261196 2.17412 PDZK1 0.00077075 2.17436 --- 3.16E-06 2.17442 PDE10A 0.00144628 2.17814 MOB1A 0.00627127 2.17843 ASAH1 4.71E-05 2.17871 Mar-07 0.00099468 2.17949 SEC23A 0.00263373 2.18002 PJA2 0.00041662 2.18198 STX3 1.68E-05 2.18295 RAB7L1 5.70E-05 2.1846 F5 9.30E-05 2.18475 SPTBN1 0.00321474 2.18493 AHNAK2 0.00020169 2.18516 GEM 6.26E-05 2.1854 CDC14B 3.87E-08 2.18658 SLC30A1 8.86E-07 2.1868 PICALM 0.00648867 2.18928 KLHL9 0.00030326 2.19069 MAP4K5 0.00090534 2.19139 TRERF1 0.00513554 2.19172 BCOR 0.00350721 2.19174 SLC2A1 9.14E-06 2.19256 ADAM10 0.00331953 2.1929 GCC2 0.00635325 2.19389 SCP2 0.00047513 2.19569 SMARCC1 0.00375729 2.197 LOC10028789 8.40E-05 2.19752 FOSL2 0.00135181 2.19861 CD46 0.00019489 2.19941 DNMT3A 0.00192606 2.20034 FAM63B 0.00612533 2.20053 SOX4 0.00023319 2.20126 HNRNPF 0.00027383 2.20196 ASNS 0.00130257 2.20333 SRP9 0.00158823 2.20385 ADH5 6.68E-07 2.20389 ZKSCAN1 0.00151878 2.20523 C5orf54 0.00042975 2.20536 CARD8 0.00225126 2.20723 VEGFA 0.00045883 2.21011 PROS1 0.0002455 2.21051 CXADR 0.00070885 2.21277 SLC2A13 2.28E-05 2.21376 PNMA1 8.28E-06 2.2138 NCOR1 0.00038641 2.21397 IGF2R 0.00067529 2.21715 ZNF292 0.0034763 2.21753 NPTN 0.00103553 2.2177 B3GALT5 2.00E-05 2.21805 PAPSS2 0.00204259 2.21924 KBTBD7 9.82E-05 2.21994 PPFIBP2 0.00010038 2.22354 SLITRK6 0.00139228 2.22372 FAM108B1 0.00549758 2.22534 CDH17 7.93E-06 2.22561 DHX9 4.21E-05 2.22602 VEGFA 1.45E-06 2.22645 FKBP9 7.16E-05 2.22676 ASNS 0.00166542 2.22806 ERBB3 2.31E-07 2.22908 GOLPH3 0.00471776 2.22921 ZNF395 0.00040319 2.23165 UPRT 0.00464115 2.23414 SUV420H1 0.00122168 2.23451 TSHZ1 0.00032107 2.2377 CHN2 0.00105375 2.23796 HKDC1 5.46E-07 2.23799 CHRAC1 0.00061227 2.2393 SLC16A4 0.00110536 2.24241 UFL1 0.00221395 2.24244 PRSS23 0.00012536 2.24246 USP14 0.00021912 2.24711 IQGAP2 0.00077589 2.24732 ARHGAP12 0.00219219 2.24741 APP 4.69E-06 2.25008 SLC39A6 0.00481765 2.25037 MCTP1 6.10E-05 2.25048 SLCO2B1 0.00107698 2.25072 SEPP1 3.25E-06 2.25079 FNDC3B 1.04E-06 2.25258 HILPDA 0.00017721 2.25286 FZD7 0.00013053 2.25286 SLC3A1 1.59E-06 2.25624 CEACAM6 5.18E-05 2.25666 TRAPPC8 0.0010133 2.25696 ATP7B 0.0003978 2.25736 LRP11 0.00532103 2.25991 RIT1 2.51E-06 2.26122 PPP2R5E 0.00059492 2.26137 SNHG7 6.93E-09 2.26151 LOC10050674 1.62E-06 2.26156 CD46 0.00155152 2.26376 LIMCH1 0.0017251 2.26536 SCIN 0.00025634 2.26559 SCP2 0.00033402 2.26568 EIF4A2 9.95E-09 2.26671 CCNG1 0.00213554 2.26718 APP 6.16E-06 2.26811 TFF2 0.00075488 2.26891 RALGPS1 0.0024966 2.26992 HSPH1 0.00287333 2.27063 APPL2 0.002988 2.27095 RABGAP1 0.00125457 2.27118 SLC2A1 0.00030474 2.2724 RAB5A 0.00020642 2.27397 WASL 0.00595827 2.27573 ABLIM1 0.00665221 2.28026 PKIB 0.00047718 2.28062 PTPRF 0.00045514 2.28243 FAM149B1 0.0007559 2.28297 PBX1 0.00478929 2.28304 KHDRBS1 0.00042709 2.28373 FAM73A 1.19E-05 2.28474 PRSS23 0.00034062 2.28542 MAP3K5 2.93E-05 2.2859 SNAP23 0.00186037 2.28741 SMAD7 0.00045436 2.28855 Sep-10 0.00288808 2.28858 EID1 0.00021702 2.28952 KHDRBS1 0.00547911 2.28992 RPL31 3.19E-05 2.29028 KIAA1984 0.00582419 2.29075 CEACAM6 0.00015696 2.29115 TPD52 7.43E-05 2.29298 GPA33 1.27E-06 2.29403 TGDS 0.00250498 2.29483 EIF4H 0.00044977 2.29487 FZD6 0.00379441 2.29711 SORL1 4.92E-06 2.29829 SLC26A2 0.00168658 2.30059 HIPK2 0.00048053 2.30077 LURAP1L 0.00624739 2.30272 TMEM181 0.00021488 2.30452 DENND4C 0.00093897 2.30565 SLC22A3 0.0003517 2.30779 LYZ 4.11E-05 2.30799 LYPLA1 0.00022619 2.30962 GULP1 0.0002982 2.31191 HPGD 8.94E-05 2.31332 CCPG1 0.00072655 2.31333 ENPP5 0.00206587 2.31457 CHMP4C 1.50E-05 2.3147 PTPRF 2.90E-05 2.31669 ALAD 3.72E-05 2.31737 HNMT 0.00031281 2.32231 TIMP2 1.77E-06 2.3237 ANXA13 0.00060294 2.327 CHN2 0.00053942 2.3272 SLC26A2 0.00316741 2.32829 PPP1R3B 0.00020303 2.32875 HKDC1 2.39E-06 2.32913 CCDC91 0.00425864 2.33234 SPAG9 0.00400363 2.33317 PABPC1 2.17E-07 2.33577 SERP1 0.00032436 2.33634 SNX25 4.17E-05 2.33804 PAG1 5.66E-07 2.33832 STYK1 0.00062217 2.33872 AHCYL1 6.72E-05 2.33886 FNDC3B 0.00010519 2.33896 DEPTOR 0.00586964 2.34191 SLC16A4 0.00019219 2.34297 CLINT1 5.44E-05 2.34402 LPCAT1 0.00369504 2.34417 ATP6V1B1 2.17E-05 2.34742 HSD17B11 4.30E-05 2.34775 GANAB 1.35E-05 2.34976 KLHL24 1.71E-06 2.35024 TMED2 0.00121715 2.35494 ADH5 2.38E-05 2.35681 GALNT7 0.00263621 2.35798 FOS 0.00032804 2.35825 ACVR1 2.47E-05 2.35966 EXOC5 0.00313937 2.3639 SLC44A5 5.36E-05 2.36437 STAU2 0.00443474 2.365 ANKRA2 6.58E-05 2.36566 SPTBN1 0.00386599 2.36722 FAM105A 1.47E-06 2.36848 PKIB 0.00158402 2.36906 EP300 0.00419007 2.36917 EPB41L4A-AS 5.29E-08 2.36945 PPP1R3D 0.00504675 2.36985 C5orf15 0.00293962 2.37213 TBC1D9 2.34E-06 2.37252 TSC22D3 0.00145031 2.3763 PPP2R5E 0.00079493 2.38091 SLC44A5 6.88E-05 2.38424 CPNE3 0.00168281 2.38428 ATP1B1 0.00654314 2.38531 RAB5A 0.00308161 2.38612 SNTB1 9.60E-06 2.38659 ASNS 0.0025207 2.38748 LIMCH1 0.00259169 2.38911 EIF2C4 5.64E-06 2.38961 BCL6 0.00026605 2.39174 PRKACB 0.00211515 2.39202 LOC642852 0.00013639 2.3927 KRCC1 4.91E-05 2.39497 LIMCH1 0.00197001 2.39711 PPFIBP2 0.00010961 2.39968 ARSJ 0.00036862 2.4002 KRT12 8.00E-05 2.40095 KCTD12 0.00077231 2.40363 SWAP70 0.0046767 2.40424 CD46 0.00115429 2.40517 SLC6A20 8.86E-06 2.40651 CDK19 0.00073181 2.40694 APP 1.28E-05 2.40779 APP 2.24E-06 2.4082 CD164 0.0036629 2.40845 FOS 0.00015843 2.40886 SLC7A2 0.00314145 2.40925 FXYD3 0.00064886 2.40931 SDHD 8.19E-05 2.41054 CCNG1 0.00215619 2.41077 SLC11A2 0.00355183 2.41617 RIOK3 0.00058003 2.41725 RIOK3 3.07E-05 2.42113 PRSS23 0.00021622 2.42214 IQGAP2 0.00627238 2.4227 SH3YL1 0.00051826 2.42363 SPIN1 0.00390908 2.42527 C9orf64 0.0001202 2.42538 DIP2C 0.00106542 2.42563 SBF2 4.58E-06 2.4268 LYPLA1 0.00073533 2.42782 --- 1.66E-08 2.42986 LMAN1 0.00094127 2.43226 ARL6IP5 1.32E-05 2.4324 ZFR 0.00066616 2.43993 IRF2BP2 5.29E-07 2.44054 BCAS1 0.00030658 2.44153 PIP5K1B 0.00010237 2.44257 RBM47 8.58E-06 2.44719 APPL2 0.00065971 2.45002 RAB6A 0.00124197 2.4541 THAP6 0.00439487 2.45424 TMCC1 3.13E-08 2.45444 VLDLR 0.00127889 2.45467 TRIB3 5.46E-06 2.45518 KLHL7 0.00090068 2.45527 AHCYL1 0.00037561 2.46158 GAL3ST4 0.00055818 2.46611 MAT2A 0.00027245 2.46635 CREBRF 0.00042108 2.46851 SLC4A4 5.40E-06 2.46978 LIMCH1 0.00126326 2.47129 ABCD3 0.0012792 2.48199 FAM134B 0.00704573 2.48325 SPRED2 9.51E-07 2.48696 EXTL2 0.00465466 2.49077 NPTN 0.00038777 2.49175 MLL5 0.00025366 2.49372 CDH17 3.33E-06 2.49687 F2RL1 0.00030335 2.49783 BNIP3L 8.44E-05 2.49795 ERGIC1 2.02E-05 2.49818 CEP57 0.00096482 2.49822 DYRK2 0.00423447 2.50183 APPL1 0.00394289 2.50206 KCTD12 0.00097873 2.506 C1orf63 0.00323488 2.50763 FHL2 6.68E-05 2.51113 WAC 0.00194628 2.51118 CERS2 2.16E-05 2.51139 RBM22 0.00684953 2.51202 RNF183 0.00080999 2.51234 RAB6A 0.00545408 2.51235 LEMD3 0.00389618 2.51246 MFSD6 0.00045879 2.51876 PCYOX1 0.00099011 2.51933 HSD17B12 0.00089871 2.52017 SH3PXD2A 0.00010954 2.52119 ITM2C 1.91E-05 2.52125 LETMD1 4.25E-05 2.52878 TMEM106B 0.00179012 2.52922 NFIL3 5.87E-06 2.53375 LIMCH1 0.0013753 2.53808 ATXN7L3B 0.00516343 2.53996 SLC11A2 0.00100111 2.54092 FOSL2 9.47E-05 2.54519 RNF219 4.06E-06 2.54595 GDPD1 0.00096867 2.5467 EEF2 8.04E-08 2.54678 NUPL1 0.00454137 2.55679 GCNT1 0.00116293 2.55682 ATP6V1A 0.00052783 2.55761 ASNS 0.00120148 2.56271 KDM4B 0.00172194 2.5633 KIAA1984 0.00321567 2.5651 EGLN1 0.00236456 2.56516 NIPAL1 0.00067202 2.56702 ANKH 3.38E-08 2.56786 RASA4 0.00018477 2.56903 STX3 0.00110435 2.57173 SMC3 0.00381872 2.57196 FOSL2 4.19E-06 2.57431 AK4 0.00035042 2.5757 ANKH 0.00013812 2.5772 XRN2 0.00579904 2.57732 GADD45B 6.83E-07 2.57826 TMCO3 4.69E-08 2.57986 ZNF277 0.00043689 2.58147 USP3 0.00031241 2.58155 EPRS 0.00165645 2.58273 PTPRB 1.34E-05 2.58309 AKAP7 0.00045384 2.5846 KHDRBS1 0.00040005 2.58579 NAP1L5 0.0013853 2.59538 ZNF793 0.00196259 2.59629 TCF7L2 0.00251567 2.59755 STX12 0.00292832 2.60162 ANXA13 0.00031306 2.60261 AMIGO2 9.39E-08 2.60426 FUCA1 0.0014553 2.60621 TMEM45B 4.40E-05 2.60789 GANAB 2.53E-06 2.61054 SLC7A1 8.04E-05 2.6139 MST4 0.00440763 2.61403 WSB1 2.04E-07 2.61516 GALNT3 0.00048431 2.61585 SMARCA2 0.0001641 2.61629 NDUFS1 4.30E-05 2.61652 CREBRF 4.08E-05 2.62046 PTPRB 0.00020983 2.62191 GPR64 7.33E-05 2.62217 KALRN 0.00014061 2.62262 SNX3 6.96E-05 2.62352 PIP5K1B 0.00015795 2.62389 BNIP3L 0.00016944 2.62704 DIP2C 0.00040371 2.62912 SORBS2 0.000598 2.63224 AKAP13 0.00400794 2.63529 PAPSS1 3.50E-05 2.63996 PFKFB3 1.48E-05 2.64014 RNF19A 4.01E-05 2.64146 CCNY 3.49E-07 2.64694 MXI1 4.45E-06 2.6471 FBXO11 0.00043204 2.65281 KDM3A 1.62E-08 2.65592 KCNH2 0.00130606 2.66889 IHH 3.31E-07 2.66957 SRI 4.03E-05 2.67008 PPARG 2.55E-10 2.6806 CD46 0.00112824 2.68303 ACVR1 2.64E-05 2.68469 PPFIBP2 0.00074461 2.69065 IRF2BP2 1.53E-06 2.69417 SLC31A1 0.00115939 2.69427 TCF12 0.00113474 2.69756 TTC3 0.0004029 2.70317 KLF5 0.00029153 2.70356 ANKRA2 0.00023495 2.70793 SLC40A1 9.01E-06 2.71965 PAPSS2 0.000113 2.73423 AKAP12 0.00447842 2.73729 FAM107B 8.16E-05 2.73794 PTPRN2 9.83E-05 2.73984 PSAT1 0.00475557 2.74126 PPP1CB 0.00219675 2.74206 SLC2A10 7.30E-05 2.74356 KIAA0895 5.12E-06 2.74888 SDHD 6.95E-05 2.75282 SDHD 6.95E-05 2.75282 CD164 0.00074143 2.76217 NTN4 2.14E-05 2.76771 SGPP1 0.00279644 2.77091 SERPINE2 0.00359196 2.77411 BTAF1 0.00097868 2.77416 BCAS1 4.97E-05 2.77491 FOS 1.41E-05 2.77545 UBXN4 0.0035077 2.77806 BTAF1 0.00036197 2.78367 ZNF569 9.27E-06 2.78455 CCNG1 0.00313857 2.79028 BMI1 0.00270831 2.79144 GAL3ST1 0.00073845 2.79373 CCNG2 0.00366682 2.79502 TCF12 0.00305049 2.79981 SNTB1 4.45E-06 2.80382 PLOD2 0.00092046 2.80549 PICALM 0.00431799 2.80779 AMIGO2 2.90E-06 2.81382 CD46 0.00164424 2.81557 CRIM1 3.43E-06 2.81858 MAP4K3 0.00153476 2.82113 JAG1 0.00399949 2.82184 ABCA1 5.89E-06 2.82998 --- 1.99E-07 2.83738 SIAH1 5.23E-05 2.83922 TSHZ1 6.14E-05 2.8409 CD46 0.00081823 2.84396 PRR5L 7.20E-07 2.84524 BHLHE40 9.36E-05 2.85128 BCL6 1.07E-07 2.85296 SLC7A1 8.18E-06 2.85444 PLA2G16 0.00125644 2.85615 EGLN1 0.00059448 2.85787 FHL2 7.85E-05 2.86744 SCARB2 0.0005337 2.87516 PPM1B 0.00097885 2.87618 MLL3 7.60E-06 2.87765 CDC14B 0.0010012 2.87767 EGLN3 3.66E-07 2.881 MAN1A1 2.05E-06 2.88355 RBM47 0.000108 2.89293 ZNF793 0.00019953 2.90663 AMIGO2 0.00010215 2.90801 SCARB2 0.00035555 2.91662 STYK1 0.00037477 2.92223 ZNF615 0.00021102 2.92383 HIPK2 7.67E-05 2.92787 MAP3K2 0.00172227 2.93515 PSPH 0.00038553 2.93562 LURAP1L 0.0019277 2.94296 TBX3 7.12E-08 2.95337 VEGFA 2.03E-09 2.95861 BBS10 0.0016528 2.95876 ERBB3 7.19E-06 2.96524 PPARG 4.11E-10 2.97081 SRSF11 0.00437481 2.97382 RPL31 1.23E-09 2.97517 CPOX 6.76E-05 2.97544 BCL6 2.03E-05 2.97715 NDRG3 9.05E-06 2.97973 NCKAP1 0.00012557 2.97985 KDM3A 2.50E-08 2.98029 SLC38A11 5.14E-06 2.98197 ZSWIM6 0.00070469 3.00106 MBNL1 0.0035917 3.00275 ANKRD37 3.08E-08 3.00439 CD46 0.00153057 3.00559 UACA 0.0018739 3.01759 SH3BP2 2.07E-05 3.02463 BNIP3L 2.28E-05 3.02658 PTPLB 0.00180823 3.02721 NDRG1 0.00023111 3.02774 DSP 2.59E-06 3.03028 ZNF615 0.00025309 3.03215 SORL1 1.57E-05 3.05379 FNBP1L 0.00050784 3.05704 TMCC1 7.13E-06 3.06062 ZNF655 0.00188243 3.06239 FUT11 3.73E-05 3.06679 OSBPL8 0.00032443 3.06871 KDM3A 1.18E-08 3.06933 PAPSS2 0.00090543 3.07243 FAM214A 3.70E-07 3.0799 KLHL24 0.00011337 3.07997 AKAP7 0.00186312 3.0836 SIPA1L2 1.57E-06 3.09126 FBXL17 0.00138195 3.09547 SHANK2 4.86E-05 3.10282 PAG1 1.52E-07 3.11378 FOXP1 0.00033345 3.11838 BCAS1 0.00052252 3.1315 SNTB1 0.00011547 3.13664 TDP2 5.49E-06 3.137 FCHO2 0.00486249 3.14189 RNASE4 5.64E-08 3.14411 PBX1 0.0008153 3.16213 FNDC3A 0.00223733 3.16324 SEMA6A 0.00025734 3.17456 TCEA1 0.00239051 3.18733 PJA2 0.00285521 3.18796 MSMO1 5.25E-05 3.19397 FAM175A 0.00035338 3.19539 CLC 1.42E-05 3.20375 AKAP9 0.00348513 3.20992 ZNF569 4.07E-05 3.21221 SLC25A24 0.00698828 3.21644 NIPSNAP1 9.55E-05 3.21646 PTPRB 1.02E-05 3.21734 SLC3A1 1.24E-05 3.21758 MMP28 0.00013783 3.23008 PSAT1 0.00099356 3.2406 MBNL2 0.00463912 3.26462 CTH 0.0009112 3.28178 FAM13A 0.00018208 3.28342 FAM134B 0.00093338 3.2899 C10orf118 0.00476483 3.29268 FBXL16 1.86E-05 3.30308 MAP4K3 0.00104355 3.31062 SLC16A4 0.00101124 3.32008 ZKSCAN1 0.0002385 3.33247 FHL2 3.17E-05 3.33323 SGK223 8.87E-09 3.34293 IRS2 4.60E-06 3.34603 BCAS1 0.00013767 3.35259 PDK4 0.00013454 3.35489 CEACAM5 2.44E-05 3.35868 PTPRB 4.17E-05 3.36383 REG4 5.89E-08 3.37425 ZNF571 2.45E-05 3.3888 KIAA0319 0.00086019 3.39113 TBX3 6.94E-08 3.39872 DSG2 4.76E-06 3.40605 ZKSCAN1 1.33E-05 3.40734 PLSCR4 6.00E-06 3.41386 MXI1 0.00011041 3.42366 ZKSCAN1 0.00207619 3.4337 NFAT5 0.00169574 3.44348 BNIP3L 1.15E-05 3.45748 BNIP3L 1.15E-05 3.45748 TCF7L2 0.00069482 3.46021 KLF5 0.00040713 3.47987 AMIGO2 1.65E-05 3.4906 HAS3 7.00E-07 3.50596 MXI1 6.97E-05 3.51721 KLF4 9.58E-08 3.52158 RBPMS2 2.44E-06 3.55221 IRF2BP2 1.24E-05 3.55562 ZNF571 2.29E-05 3.5577 PTPRR 0.00408284 3.57449 PCID2 0.00148517 3.58332 USO1 0.00023 3.5852 C4orf3 1.38E-05 3.59389 NDRG1 0.00052374 3.61966 GPX3 1.60E-05 3.62264 KLF4 1.30E-08 3.64856 C4orf3 7.98E-07 3.6574 AKAP7 0.00027955 3.72483 CDH17 2.19E-06 3.73279 MXI1 0.0001966 3.7368 ZNF292 0.00278959 3.74882 REG4 5.68E-05 3.76519 CDH17 4.09E-07 3.80789 PIM1 2.09E-08 3.81069 WSB1 1.80E-05 3.82744 FXYD3 0.00023156 3.83345 AKAP7 0.00016165 3.83811 SIPA1L2 4.68E-07 3.84207 ABHD3 2.07E-05 3.86251 ELF3 0.00516584 3.89139 TBX3 1.07E-07 3.89152 SNTB1 7.28E-08 3.89321 MANEAL 2.13E-05 3.9206 ELF3 0.00542911 3.93011 HAVCR1 0.00036282 3.93863 EIF4A2 2.16E-05 3.97119 PTPRR 0.00018521 3.98121 WASF2 0.0003484 4.0845 LINC00273 1.01E-08 4.08625 LETMD1 4.11E-05 4.10657 CA12 6.91E-07 4.26392 SLC2A1 7.30E-08 4.31608 ZNF571 8.64E-06 4.33825 SIPA1L2 3.12E-06 4.35112 CYP1B1 4.94E-08 4.41328 JAG1 0.00257822 4.41864 NDRG1 0.00019469 4.4908 CYP1B1 3.96E-08 4.50343 GPR64 0.00010481 4.5169 NDRG1 0.00014757 4.53681 ADM 7.75E-10 4.58611 DDIT4 2.09E-05 4.62652 FAM13A 7.22E-05 4.68833 PTPLB 0.00032322 4.72326 CYP1B1 1.78E-08 4.78707 ZNF540 7.42E-06 4.81533 ADM 3.07E-10 4.84499 KRCC1 5.41E-06 4.92111 ZNF28 8.81E-05 5.02174 NDRG1 4.46E-05 5.03528 AKAP7 5.39E-05 5.10936 PTPRR 3.57E-05 5.17739 GDF15 2.86E-06 5.36094 FAM13A 1.91E-05 5.50987 CYP1B1 4.17E-07 5.54481 EGLN3 0.0001528 5.60274 FNDC3B 1.98E-05 5.70685 SLC2A1 2.50E-08 5.85633 NDRG1 0.00043908 6.06471 FAM13A 0.00011604 6.21324 EGLN3 5.67E-05 6.5849 ZNF540 5.83E-06 6.74386 GDF15 4.43E-06 6.93436 ZNF540 1.34E-06 7.13352 ZNF540 2.95E-07 7.2152 GPR64 0.00011296 7.33337 GDF15 2.58E-06 10.2579 Gene Symbol p-value(NConFC GAvsNCon ZNF540 2.17E-09 -41.4421 MARS2 3.59E-09 -32.5038 ZNF540 2.24E-08 -31.8562 ZNF540 1.39E-08 -24.0735 ZNF540 4.19E-09 -21.7169 ZNF226 2.80E-12 -20.7055 SLC7A11 5.12E-05 -18.3189 TTC14 3.21E-09 -14.3987 MBD4 1.53E-06 -14.2491 LCMT2 1.42E-09 -14.1528 MIR17HG 1.42E-09 -14.1177 SLC7A11 6.75E-05 -14.0711 HNRNPA2B1 6.20E-11 -13.7482 FUT4 4.13E-09 -13.2899 CREB5 8.38E-09 -13.1439 TRMT13 2.47E-06 -12.9549 KIAA1984 8.53E-07 -12.6929 ADAT2 1.93E-10 -12.5531 ANKRD49 1.84E-09 -12.5493 PVRL4 1.51E-08 -12.3565 ZNF772 4.20E-08 -11.4411 TRMT13 4.79E-06 -10.887 AKAP12 6.63E-06 -10.5591 PPP1R3D 2.34E-06 -10.0996 PNISR 6.06E-09 -9.86199 ZNF564 8.88E-08 -9.65522 SFT2D3 7.86E-11 -9.62003 GPATCH2 2.55E-09 -9.27946 AKAP12 1.42E-05 -9.20991 MIR17HG 6.30E-09 -9.04803 EXOC8 2.59E-07 -8.74764 KBTBD6 4.64E-06 -8.70654 SASS6 4.43E-08 -8.57519 LUC7L3 2.48E-10 -8.50228 WDR5B 6.97E-10 -8.37767 KLHL9 3.08E-10 -8.29673 TTC14 3.01E-07 -8.2909 NSUN5P1 2.65E-08 -8.28689 NDC80 3.01E-08 -7.88219 TRIM32 7.32E-10 -7.84792 CAPN8 1.31E-09 -7.7713 PNISR 1.55E-09 -7.6762 KLHL9 4.07E-09 -7.61121 KLHL9 7.02E-08 -7.59332 ZNF321P 3.07E-08 -7.53999 ZNF572 1.24E-07 -7.34261 ZFP112 3.83E-06 -7.31182 HPS6 1.43E-11 -7.30503 JRKL 2.32E-08 -7.0737 AGPAT5 8.16E-07 -7.01356 PSAT1 0.00015076 -6.94776 AMIGO2 3.30E-07 -6.84885 FANCF 3.00E-07 -6.64287 COIL 8.03E-11 -6.63323 ANKRD20A5P 5.51E-08 -6.61404 IREB2 2.95E-11 -6.59247 KIAA1984 1.70E-05 -6.53316 ZNF571 7.31E-07 -6.44162 ZNF302 2.63E-06 -6.39259 HMGB1 1.59E-06 -6.34387 C15orf39 2.09E-10 -6.32016 KTI12 3.37E-08 -6.28281 NSUN5P2 5.96E-08 -6.24658 CSTF3 6.30E-10 -6.19484 SPATA6L 2.92E-06 -6.15184 EXOC8 8.16E-10 -6.14883 RAB7B 8.25E-10 -6.12758 AMIGO2 1.04E-06 -6.08058 INADL 9.29E-07 -5.85573 CASP8 8.72E-07 -5.84705 BCDIN3D 2.12E-08 -5.83468 PPP1R8 1.18E-05 -5.80733 ZNF552 3.06E-09 -5.79478 KCTD20 1.25E-06 -5.78806 AMIGO2 2.65E-10 -5.76106 ZNF607 6.31E-09 -5.75154 NR2C1 7.72E-07 -5.74774 HMGXB4 5.55E-08 -5.74263 HILPDA 2.11E-07 -5.7248 MAT2A 1.00E-06 -5.66904 ARL4A 3.90E-06 -5.6145 TGOLN2 7.55E-06 -5.61332 WDR5B 3.50E-07 -5.61279 ZNF239 5.50E-07 -5.61047 ZNF181 4.69E-06 -5.60796 PSMA1 3.23E-06 -5.59975 HILPDA 1.39E-09 -5.59355 CD59 4.53E-06 -5.58222 CSTF3 9.79E-08 -5.56714 CASP4 4.58E-07 -5.55457 CTTNBP2NL 3.56E-09 -5.54166 ZNF552 9.99E-09 -5.53079 NSUN5P1 2.45E-07 -5.50806 UTP15 1.01E-05 -5.45771 ZNF180 1.21E-07 -5.45638 NSD1 1.04E-07 -5.45189 ZNF485 2.85E-06 -5.42333 ZFC3H1 3.77E-09 -5.40795 MAK16 9.87E-08 -5.40059 THUMPD3 1.57E-05 -5.3971 RBM33 1.84E-11 -5.39427 DICER1 3.47E-06 -5.37367 TIGD1 6.55E-07 -5.35025 PTPRK 2.28E-05 -5.33433 ZNF235 6.01E-07 -5.2552 INADL 3.87E-08 -5.24106 ZNF780A 5.92E-08 -5.23845 FAM178A 3.19E-07 -5.23198 UTP3 3.34E-08 -5.21859 ZCCHC3 6.82E-09 -5.21781 ZNF345 3.87E-07 -5.20997 RANBP6 1.23E-10 -5.18836 TMEM177 1.54E-07 -5.18774 METTL18 6.35E-07 -5.17948 Mar-07 4.01E-09 -5.16243 ZNF571 1.77E-06 -5.16118 UTP3 1.57E-09 -5.13523 DFFB 7.00E-07 -5.11868 SDR42E1 5.74E-07 -5.10966 UMPS 1.61E-11 -5.10923 SLC25A27 9.77E-06 -5.08544 KIAA1586 9.90E-08 -5.07492 FOXQ1 4.67E-07 -5.02582 CSTF3 1.64E-08 -5.024 NSD1 1.32E-07 -5.024 SH3BP2 7.30E-07 -4.98228 SLC7A5 2.05E-05 -4.96847 ZNF12 1.49E-08 -4.96549 ZNF223 4.04E-05 -4.96057 ZNF502 2.74E-07 -4.95777 ARL17A 2.32E-07 -4.94368 PSAT1 0.00029197 -4.94296 PTAR1 3.42E-06 -4.94142 TGOLN2 3.74E-05 -4.91561 MMP1 0.0062869 -4.90868 MTERFD1 9.70E-06 -4.90621 UBN2 1.10E-07 -4.90112 PRR5L 3.71E-10 -4.89612 ZNF181 4.51E-06 -4.89144 ZFC3H1 5.01E-09 -4.88977 SLC25A37 1.71E-08 -4.88583 BHLHE41 5.68E-07 -4.87809 ZNF551 3.32E-07 -4.87316 ANKZF1 1.03E-07 -4.8729 ZSCAN16 1.05E-05 -4.87135 NDUFS2 6.33E-07 -4.8696 SLC25A37 1.37E-09 -4.86862 RPL4 8.01E-06 -4.86392 CDK12 3.35E-05 -4.83815 AGGF1 8.00E-09 -4.82466 CD58 9.89E-07 -4.80297 MOCS3 3.58E-05 -4.80103 ZNF551 5.55E-06 -4.77917 ZBTB6 1.63E-09 -4.76385 SLITRK6 2.11E-06 -4.7526 PSAT1 1.57E-05 -4.74035 LCMT2 7.05E-09 -4.73575 ZNF267 3.79E-05 -4.73454 PPP1R8 4.56E-10 -4.72953 MBLAC2 3.71E-05 -4.7291 GEN1 2.96E-06 -4.72799 CDC7 3.09E-08 -4.72473 MARS2 4.36E-06 -4.72033 MED7 3.31E-08 -4.71646 UTP15 4.65E-06 -4.70634 PTAR1 1.04E-07 -4.69638 NAPEPLD 2.66E-05 -4.69251 ZNF485 2.23E-05 -4.68909 ZNF594 4.00E-05 -4.68885 ARGLU1 6.58E-10 -4.68231 NSUN5P1 3.03E-07 -4.67713 DIDO1 3.80E-09 -4.66506 TRMT5 3.02E-08 -4.65911 C5orf54 1.59E-06 -4.64289 ZNF321P 1.51E-08 -4.63847 PIK3R4 1.45E-10 -4.62025 IREB2 6.54E-09 -4.62014 PNISR 5.81E-06 -4.62005 RPL37A 7.12E-07 -4.60765 THAP6 9.90E-05 -4.59928 MYSM1 5.05E-06 -4.58529 CUTC 2.46E-07 -4.57131 DHRS9 0.00066648 -4.57033 FOXQ1 0.00026443 -4.5435 ZNF180 2.70E-07 -4.53617 FIGNL1 5.02E-08 -4.52741 PSAT1 0.00015303 -4.51993 PSAT1 0.00014907 -4.51757 ZNF480 1.08E-07 -4.51165 Mar-07 5.94E-07 -4.51067 ZNF223 3.47E-05 -4.50151 TTC14 6.76E-07 -4.48415 PSAT1 0.00031359 -4.48162 FBXL5 2.16E-06 -4.47736 METTL2B 3.86E-05 -4.46505 ZNF232 4.21E-06 -4.4625 ASF1A 4.36E-06 -4.45838 MARS 3.13E-05 -4.43302 CD58 1.08E-06 -4.42682 RBM12 6.53E-06 -4.42102 C11orf73 9.25E-06 -4.4074 POLR1B 9.79E-06 -4.38954 SERPINB5 2.88E-05 -4.38213 --- 0.00027586 -4.37038 TRMT13 0.00055851 -4.3681 FXYD3 9.79E-06 -4.36809 SRSF1 1.61E-05 -4.35495 TRNT1 0.00023272 -4.34988 CD58 3.05E-06 -4.34449 TADA1 5.37E-06 -4.3291 ZNF302 3.92E-07 -4.32007 PDIK1L 9.53E-05 -4.31137 ZBTB3 8.75E-05 -4.30734 ITGA2 0.00041613 -4.30564 NAPEPLD 1.30E-05 -4.30107 MTERF 5.41E-12 -4.29983 TEFM 8.38E-07 -4.29567 ADAM19 0.00019586 -4.28541 ZNFX1-AS1 3.08E-08 -4.28268 WDR36 8.03E-07 -4.28043 MKRN3 4.71E-07 -4.27969 CCNL1 3.99E-05 -4.27644 ZNF571 9.90E-06 -4.2417 ZNF267 2.87E-08 -4.2387 NAPEPLD 2.52E-05 -4.23855 ZBTB24 1.39E-06 -4.23562 DEM1 2.68E-08 -4.23391 ANKRD22 2.70E-06 -4.22969 ZNF280C 1.34E-07 -4.22662 ZNF267 2.93E-05 -4.22295 ZNF232 4.88E-09 -4.21364 TSEN2 2.11E-09 -4.20416 MARS2 6.85E-05 -4.20142 CSTF2T 4.79E-08 -4.18801 OGT 1.02E-07 -4.18428 AMIGO2 1.42E-07 -4.18215 PAK1IP1 1.55E-08 -4.18128 METTL2B 3.53E-05 -4.15809 LCMT2 3.37E-06 -4.13123 PNISR 0.00099184 -4.12472 RNF43 0.00025201 -4.12262 CCNJ 1.30E-05 -4.1124 TFB1M 9.57E-06 -4.10167 SPRY1 0.00015547 -4.09972 SPRY1 0.00015547 -4.09972 CD58 2.56E-06 -4.09339 RRS1 2.43E-07 -4.0884 DCUN1D4 1.40E-08 -4.07773 TMEM186 2.99E-07 -4.07025 ZRANB2 1.75E-05 -4.06991 UHRF1BP1 5.48E-06 -4.06869 ERCC4 6.11E-06 -4.05679 ZNF398 2.30E-07 -4.05294 RBM12 1.59E-07 -4.0462 SMG8 6.19E-12 -4.04583 ZNF232 5.52E-09 -4.04272 UTP23 5.39E-08 -4.03832 MAK16 1.98E-05 -4.0377 C2orf69 3.40E-07 -4.03533 ASTE1 9.91E-06 -4.02825 ZNF195 7.14E-08 -4.02086 FUT4 4.67E-08 -4.01062 PIGW 2.40E-05 -4.00965 ZNF449 5.61E-05 -4.0031 TMEM177 1.68E-06 -3.99857 KCNE3 1.09E-07 -3.9984 ZNF614 4.98E-09 -3.99771 IMP3 1.32E-06 -3.99359 CKAP2L 2.36E-07 -3.99128 DDX28 4.55E-07 -3.98551 ZNF548 3.84E-05 -3.97974 ZNF169 4.72E-08 -3.97744 CTTNBP2NL 6.16E-06 -3.97619 TRNT1 8.68E-05 -3.97188 DCUN1D4 1.08E-09 -3.96779 ZNF215 1.79E-07 -3.96529 RIOK2 2.10E-05 -3.9603 NOL8 1.98E-06 -3.9598 KIAA0040 1.31E-06 -3.95976 TRMT13 0.00081049 -3.95535 FOXP1 2.45E-05 -3.95262 PRMT6 6.95E-06 -3.94109 CBLL1 1.12E-05 -3.9356 ZNF790 0.00012642 -3.92773 KLHL20 1.27E-06 -3.92507 ZNF557 1.67E-07 -3.92398 GTF2H2 0.00062961 -3.91801 NANP 0.00015425 -3.91387 LUC7L3 1.17E-06 -3.91075 ZNF33A 1.23E-07 -3.9097 C2orf49 4.79E-09 -3.90491 KIAA1731 7.60E-07 -3.90123 CREB5 8.33E-06 -3.89727 PRRC1 7.12E-09 -3.89489 ARGLU1 4.48E-10 -3.88782 PDCD7 0.00011331 -3.8845 RFC4 2.86E-07 -3.88353 CNEP1R1 2.13E-05 -3.85837 ARAP2 3.20E-05 -3.85544 GORAB 6.74E-06 -3.85195 ZNF193 6.96E-05 -3.85035 MTPAP 0.00032675 -3.84923 MTF2 4.14E-06 -3.84736 ZNF557 5.56E-08 -3.84721 PIK3R1 2.78E-05 -3.83893 KRAS 3.16E-09 -3.83613 ABLIM3 0.00027353 -3.83254 PKDCC 1.43E-09 -3.82824 TXNIP 1.22E-11 -3.82595 NAPEPLD 1.49E-05 -3.82171 ZNF169 1.26E-06 -3.80117 SETD6 4.77E-05 -3.79893 NUMB 4.26E-06 -3.79375 ZNF33A 2.02E-08 -3.79018 C15orf52 7.50E-05 -3.77657 ZNF350 1.20E-08 -3.77343 PIGM 5.34E-06 -3.77008 ZNF565 2.17E-07 -3.76246 ZNF514 1.32E-06 -3.76163 CCDC14 6.47E-09 -3.7573 BCL11B 1.04E-07 -3.75508 ZNF141 2.18E-06 -3.75371 IFIT5 4.46E-06 -3.7534 FAM217B 0.00041105 -3.7474 CTTNBP2NL 5.98E-09 -3.74726 HNRNPD 6.34E-05 -3.74542 SLC35G1 0.00037934 -3.74299 ZNF398 1.34E-05 -3.73686 UBXN1 4.52E-05 -3.72629 SRSF1 1.08E-05 -3.72624 SPIN4 4.36E-05 -3.72385 --- 1.63E-05 -3.72308 TMEM87A 0.00078169 -3.72058 DCUN1D4 3.24E-05 -3.71928 TAMM41 2.47E-06 -3.71653 CLDN2 0.00083011 -3.71327 ZNF354B 8.10E-06 -3.7132 GRPEL2 3.13E-07 -3.71227 IKBKB 7.55E-05 -3.70783 HMGA2 0.00023883 -3.69907 SRSF1 2.50E-07 -3.69688 AIM1L 5.58E-05 -3.69578 COG8 1.14E-07 -3.69502 PRR5L 8.85E-08 -3.69267 CLDN1 0.00080842 -3.68274 ALG2 5.34E-05 -3.68212 ZNF30 0.00017281 -3.6801 SETMAR 1.41E-05 -3.67898 POLI 1.85E-05 -3.67335 BTF3L4 8.01E-06 -3.67232 MTF2 2.00E-06 -3.6695 C1orf116 4.17E-06 -3.66155 NHLRC1 1.64E-05 -3.66044 PUS7L 4.91E-05 -3.65642 ARL14EP 2.03E-06 -3.65571 ZKSCAN4 0.00020385 -3.65069 ZNF227 7.30E-06 -3.63982 HYLS1 1.30E-05 -3.63785 PUS1 8.57E-07 -3.63725 CXorf26 4.58E-05 -3.63042 C1orf131 3.35E-05 -3.63024 EZH1 2.61E-07 -3.62989 FBXO3 4.70E-06 -3.62851 ANAPC7 1.83E-06 -3.62208 GTPBP4 1.84E-11 -3.61732 UMPS 9.49E-09 -3.61574 ZNF587 7.42E-08 -3.60324 TNRC6B 2.13E-05 -3.59846 PAPOLG 4.84E-06 -3.59649 GORAB 8.19E-05 -3.58185 CENPJ 2.19E-06 -3.57887 ZNF195 5.02E-07 -3.57601 FJX1 9.05E-05 -3.5699 MSH6 1.67E-05 -3.56729 MCM9 8.04E-11 -3.54757 ADAT2 1.52E-05 -3.54526 SRFBP1 6.49E-06 -3.54404 N4BP2L2 6.17E-06 -3.53375 CXorf26 1.61E-07 -3.53295 FOSL2 3.57E-05 -3.53128 NANP 4.34E-08 -3.53068 CD58 3.40E-07 -3.52327 RBM19 1.91E-06 -3.52211 FANCL 9.67E-06 -3.52165 KIAA1586 3.11E-08 -3.5216 MMAA 5.72E-08 -3.52034 GPR39 9.69E-09 -3.50455 KIAA2026 3.27E-05 -3.49899 PRPF38B 0.00029163 -3.4988 RBMXL1 4.64E-08 -3.49755 ZNF187 3.57E-07 -3.49376 CSTF3 4.17E-08 -3.49132 OPA3 7.91E-07 -3.4886 ZNF562 1.23E-05 -3.48855 HAUS3 1.12E-05 -3.4794 NOL8 1.72E-06 -3.47478 IMP3 2.42E-08 -3.47322 ZNF451 2.78E-05 -3.47176 UBASH3B 0.00336693 -3.46664 ASNS 4.01E-05 -3.45959 EXOSC3 7.69E-07 -3.45418 TRIM27 3.41E-08 -3.44893 MTPAP 1.72E-05 -3.44878 KIAA0020 4.51E-07 -3.44797 XPO4 1.34E-06 -3.44698 SPRY1 4.73E-05 -3.44401 ZNF292 4.29E-09 -3.44263 FOXP1 8.01E-07 -3.44233 ATXN1 1.21E-07 -3.44179 ASNS 6.33E-05 -3.43975 TBRG1 8.00E-07 -3.43005 DNTTIP2 4.22E-07 -3.4242 ZNF33A 2.43E-05 -3.41247 ASNS 0.00021559 -3.41127 ZNF187 6.81E-05 -3.40761 ZNF283 4.17E-05 -3.40624 RBMXL1 1.82E-05 -3.3914 CBWD1 0.00012566 -3.38465 ALS2 1.66E-05 -3.38199 ZNF181 0.00100996 -3.37892 NSUN5P1 6.39E-07 -3.37839 HMGXB4 7.47E-05 -3.37839 NSUN5P1 4.83E-05 -3.37802 GEMIN6 1.52E-06 -3.37467 N4BP2L2 2.35E-06 -3.37402 ENPP5 0.00013686 -3.37293 LARP4 2.78E-05 -3.37006 FANCF 5.22E-07 -3.36626 ALG2 1.01E-09 -3.36278 RBMXL1 1.91E-09 -3.35085 TNFAIP8 1.97E-05 -3.34954 LURAP1L 0.0008914 -3.34589 CLDN2 3.73E-08 -3.33845 ZNF195 1.38E-06 -3.33472 TMEM68 3.35E-06 -3.32595 FOXP1 0.00010528 -3.32582 GARS 1.08E-07 -3.32501 CPSF6 1.58E-08 -3.3232 C14orf169 2.94E-05 -3.32087 KBTBD6 6.88E-06 -3.3191 NGDN 1.44E-08 -3.31827 LARP4 0.00012673 -3.31306 ZNF195 4.66E-06 -3.30552 ZNF136 6.61E-06 -3.30472 SPRED1 1.68E-08 -3.30415 POLR1B 1.64E-05 -3.30377 ZRANB2 4.23E-11 -3.30349 TXNIP 6.01E-07 -3.30192 MPHOSPH10 2.69E-09 -3.30121 DMTF1 1.76E-08 -3.2995 ZNHIT6 3.72E-05 -3.29541 ZNF195 9.58E-07 -3.29401 POLR1B 2.11E-05 -3.29199 MINA 0.00094155 -3.29126 C9orf72 5.42E-06 -3.29014 ZNF791 5.89E-05 -3.2899 FAM200A 6.54E-05 -3.2861 PIK3R1 1.64E-08 -3.28405 THUMPD2 2.31E-07 -3.28144 MANEAL 6.99E-05 -3.27589 LRRFIP1 1.86E-07 -3.27415 MALL 2.55E-06 -3.27057 C1orf74 9.64E-06 -3.26806 --- 6.90E-06 -3.26482 --- 6.90E-06 -3.26482 NT5C1B-RDH 4.64E-09 -3.26417 C16orf91 1.91E-07 -3.26374 NAPEPLD 0.00106915 -3.26343 FADD 8.66E-07 -3.26014 NOL8 7.59E-06 -3.2561 MGEA5 2.67E-08 -3.25487 RIOK2 2.69E-05 -3.2538 ZNF708 0.0001459 -3.25238 ZNF195 1.27E-06 -3.25009 CLK2 9.86E-08 -3.24803 POLR1B 0.00012543 -3.24739 TAMM41 8.67E-08 -3.246 Mar-07 1.25E-05 -3.24562 MTERFD2 9.94E-06 -3.24494 RPP38 2.45E-08 -3.24463 ZNF248 4.77E-07 -3.24293 FEM1C 2.14E-07 -3.24205 FAM178A 0.00011197 -3.23653 WDR75 1.14E-05 -3.23588 ZNF789 0.00039005 -3.23473 AASDHPPT 0.00011655 -3.22883 CLPX 0.00151246 -3.22559 NAA15 3.87E-06 -3.22172 AHSA2 2.45E-07 -3.22129 NTSR1 4.29E-07 -3.22056 PNN 4.58E-09 -3.22049 RNF169 1.16E-05 -3.21885 HDHD3 7.05E-09 -3.21374 RWDD2A 1.16E-05 -3.21129 BRD8 1.27E-05 -3.20882 SH3RF1 6.25E-07 -3.20634 ANKRD10 2.62E-08 -3.19497 SPRYD4 2.52E-07 -3.1948 ZNF283 1.17E-05 -3.19235 OGT 2.69E-07 -3.18922 LRRC8B 9.82E-06 -3.18532 RUNDC1 7.28E-06 -3.18196 MRPL46 9.04E-09 -3.18031 ZNF30 0.00041135 -3.17213 ZNF16 1.86E-08 -3.17146 KIAA0754 1.85E-08 -3.17144 ZBTB33 1.05E-05 -3.17024 LUC7L 1.50E-05 -3.16728 FER1L4 7.85E-06 -3.16564 ZNF420 0.00085374 -3.16355 CLK2 2.24E-05 -3.16165 CDCA4 8.07E-05 -3.15972 MARS 3.29E-06 -3.15955 FBXL5 0.00013408 -3.15851 METTL2B 0.00029096 -3.15799 INPP4B 3.88E-05 -3.15619 RAD18 0.00014662 -3.15449 IRS1 3.25E-05 -3.15349 ZNF780A 6.26E-05 -3.15275 FAM222B 3.98E-08 -3.15267 ENPP5 1.28E-06 -3.14986 ZNF200 0.000255 -3.14963 POLR1B 1.15E-05 -3.1479 ZNF514 1.80E-06 -3.14732 KLHL31 0.00087657 -3.14451 GTPBP4 3.18E-07 -3.14335 FDXACB1 0.00124232 -3.13256 ZNF214 2.86E-05 -3.13133 MCM10 1.40E-06 -3.12956 RBM12 5.35E-06 -3.12836 EIF2S1 1.34E-06 -3.12746 BLZF1 0.00053232 -3.1259 ZNF17 4.97E-05 -3.1205 IPO7 8.16E-08 -3.11996 ZKSCAN1 2.11E-06 -3.11843 ZNF451 4.49E-05 -3.1139 STAG3 5.26E-05 -3.11381 USP36 3.86E-05 -3.11323 ZNF397 1.06E-06 -3.1129 FTSJD1 2.82E-07 -3.11194 DCAF4 1.51E-05 -3.10807 MALL 8.10E-06 -3.10786 FTSJD1 1.78E-06 -3.10736 ZC3HAV1L 5.40E-06 -3.10504 ADRB1 7.31E-06 -3.10496 RBPMS2 6.75E-06 -3.10156 ITGA2 0.00068011 -3.10145 LGR5 0.00035461 -3.09794 FADD 4.72E-10 -3.09563 STK4 7.39E-09 -3.09192 ZNF573 1.40E-08 -3.08815 ZNF627 3.97E-05 -3.08785 PNISR 0.0010842 -3.08775 GPATCH4 6.73E-05 -3.08685 CFL2 1.36E-05 -3.08638 RSBN1 5.74E-07 -3.08607 ZNF28 0.00132898 -3.0844 UTP23 0.00043951 -3.0838 RFXAP 0.00036773 -3.08263 DEM1 1.54E-07 -3.08018 ARL14EP 4.82E-08 -3.07928 GARS 1.98E-07 -3.07706 ZNF302 3.70E-05 -3.07623 PKDCC 7.81E-05 -3.07395 CCNL2 1.88E-06 -3.07277 PUS3 1.20E-05 -3.07221 TJP2 1.33E-05 -3.07108 MCTP1 3.68E-06 -3.06807 LARP4 3.74E-07 -3.06663 NAA25 1.14E-05 -3.06378 C1orf212 6.03E-08 -3.0627 GTPBP4 5.29E-11 -3.06242 UNKL 4.87E-05 -3.06224 ZNF295 1.35E-06 -3.06048 NSUN5P1 4.98E-08 -3.06031 LYSMD4 2.16E-05 -3.05968 WDR36 1.58E-05 -3.05919 EFNA4 2.87E-08 -3.05842 ZNF561 5.32E-06 -3.05784 UNC50 2.80E-06 -3.05615 PHF20L1 0.00081052 -3.05442 CCDC14 8.48E-07 -3.05411 ZNF805 2.03E-05 -3.05082 ATP2B1 0.00022077 -3.0501 ZNF791 6.67E-05 -3.04623 ZNHIT6 3.27E-05 -3.04274 ING3 2.99E-05 -3.04174 SAMD5 6.66E-06 -3.03927 ZNF529 1.24E-06 -3.03843 REG4 0.00023541 -3.03735 MRPS25 0.00021509 -3.03567 ZNF507 1.80E-07 -3.03281 ZNF561 7.61E-07 -3.03184 TARDBP 7.14E-07 -3.02994 WDR75 1.17E-06 -3.0286 GEMIN2 3.26E-05 -3.02837 DDX20 1.12E-07 -3.02746 RPP14 1.70E-06 -3.01719 CHAC2 0.00011108 -3.01686 SLC38A1 0.00011209 -3.01317 N6AMT1 4.56E-07 -3.01297 TADA1 8.12E-08 -3.00733 AJUBA 9.31E-06 -3.0063 ZNF175 4.37E-05 -3.00557 PILRB 2.45E-09 -3.00426 ZNF302 5.76E-05 -3.00178 PPM1B 4.76E-09 -3.00076 ZNF148 0.00011975 -3.00056 KIAA0930 8.36E-05 -3.00009 BLOC1S3 8.20E-08 -2.99861 ANKRD22 1.54E-05 -2.99685 MSANTD4 4.96E-05 -2.99353 ZFP112 1.76E-05 -2.99299 ETAA1 4.02E-05 -2.99169 ZNF337 9.87E-07 -2.99139 SMAD5 2.60E-08 -2.98837 C5orf34 3.68E-07 -2.98761 ZFP62 0.00071309 -2.98642 SNRPA1 1.50E-07 -2.98399 SMAD5 1.65E-05 -2.98385 ZNF791 0.00025395 -2.98252 PEX13 2.06E-06 -2.98198 MMAA 6.38E-08 -2.98123 BTAF1 0.00060089 -2.98048 ARHGAP29 0.00014431 -2.97861 C22orf29 1.53E-09 -2.97704 LTV1 0.0031824 -2.97525 MLKL 1.91E-06 -2.9752 GEMIN5 0.00029199 -2.97373 SNAPC5 4.55E-08 -2.97305 UBIAD1 3.43E-05 -2.97282 KBTBD3 1.05E-05 -2.972 ARL17A 0.00066282 -2.96992 BAG5 3.64E-05 -2.96974 RBM12 0.00169824 -2.96891 ANKRD50 1.32E-06 -2.96748 AGGF1 1.80E-06 -2.96566 ACTR5 3.93E-08 -2.96454 TRIM27 6.61E-08 -2.96439 ZNF195 4.19E-06 -2.9596 TTC30A 5.65E-07 -2.95912 ZNF544 4.28E-05 -2.95631 CLDN1 0.0057524 -2.95458 ZNF320 2.79E-06 -2.95447 OTUD6B 0.00026429 -2.95409 WSB1 6.57E-08 -2.95374 ELF2 3.71E-07 -2.95053 STK4 6.70E-10 -2.94981 ZNF526 2.24E-06 -2.94884 C5orf43 0.00050624 -2.94734 ZNF577 5.60E-05 -2.94311 ATG14 0.00016174 -2.94228 PTPRB 4.30E-06 -2.94161 TRNT1 0.00279646 -2.94119 MDM4 1.91E-07 -2.93985 RBM12B 5.01E-05 -2.93824 THAP6 2.47E-08 -2.93801 FNDC3B 0.00089178 -2.93797 MSANTD4 0.00021611 -2.93478 SPIN3 9.02E-07 -2.93471 VCPIP1 2.05E-06 -2.93391 SLC25A27 1.21E-05 -2.93139 THUMPD2 3.55E-05 -2.93121 LTV1 0.00014962 -2.92457 EIF3M 0.00027127 -2.92295 ZNF230 4.39E-05 -2.92193 ZNF121 4.02E-06 -2.92008 GEMIN2 0.00036888 -2.9178 PIGM 2.07E-05 -2.91777 TNFRSF21 0.00014725 -2.91724 KIAA0232 1.08E-06 -2.91647 FBXO3 2.14E-05 -2.91546 MCTP1 0.00171157 -2.91299 LTV1 0.00177128 -2.90943 C5orf30 2.21E-05 -2.90896 SH3KBP1 0.00010502 -2.9034 SNRNP35 9.82E-07 -2.90134 FANCL 1.10E-06 -2.89981 TTK 1.14E-05 -2.89963 BACH1 3.54E-10 -2.89891 MED4 2.35E-08 -2.89797 WSB1 2.42E-06 -2.89511 VHL 2.21E-07 -2.89254 DOLK 2.38E-05 -2.8902 KLF12 1.57E-07 -2.88936 C22orf29 0.00010487 -2.88838 VEZT 0.0029516 -2.88773 METTL3 0.00025336 -2.88293 TWISTNB 6.43E-06 -2.88213 SLC25A27 1.21E-05 -2.88005 PAQR3 0.00050683 -2.88002 ZMYM6 0.0003257 -2.87716 AHNAK2 1.66E-05 -2.87708 TMEM185B 7.56E-06 -2.87544 TWISTNB 5.17E-05 -2.87244 ARID5B 8.52E-06 -2.87217 KLHL23 1.27E-06 -2.87214 NOL11 5.79E-09 -2.87204 THUMPD1 3.69E-07 -2.87131 EGLN3 0.00480069 -2.87118 ZNF564 1.80E-07 -2.87059 DYRK2 4.01E-07 -2.8702 CCNT2 5.35E-05 -2.86724 NUB1 0.00026555 -2.86616 GIN1 0.00014651 -2.86453 ASB8 2.81E-05 -2.86352 RBM48 7.04E-05 -2.86326 MDM4 4.27E-07 -2.86255 FAM156A 3.67E-08 -2.8606 FASTKD2 8.65E-05 -2.85953 TRAF6 6.76E-06 -2.85946 ZNF24 8.06E-08 -2.85902 RIF1 3.84E-06 -2.85837 IFNGR1 0.00964615 -2.85763 ZNF224 1.40E-06 -2.85256 GOLGA8A 1.58E-06 -2.84989 CFL2 1.96E-05 -2.84911 PNISR 0.00037242 -2.84745 C19orf12 2.19E-06 -2.84689 SRSF5 1.75E-05 -2.84579 BEND3 6.45E-05 -2.84568 HOXA13 5.06E-05 -2.84408 KCTD15 1.00E-05 -2.84305 GORAB 1.15E-05 -2.84116 SLITRK6 0.00019581 -2.83872 TRIM27 1.67E-08 -2.83482 NSUN4 1.85E-05 -2.83191 GOLGA8A 3.48E-06 -2.83169 BLZF1 0.00023518 -2.83089 PNN 7.66E-11 -2.82888 ZEB1-AS1 1.09E-05 -2.82721 C16orf88 0.00074447 -2.82384 BRWD1 2.70E-05 -2.82376 NUMA1 7.34E-06 -2.82241 ZNF595 3.89E-06 -2.81949 CREBZF 2.87E-08 -2.81934 METAP2 3.76E-05 -2.81917 LYST 0.00027816 -2.81668 LTV1 3.49E-05 -2.81593 FOXP1 1.94E-06 -2.81537 C22orf29 8.20E-08 -2.81474 PLRG1 0.00055684 -2.81413 RNF219 7.99E-10 -2.81166 PNO1 4.43E-06 -2.81121 LIN37 1.88E-06 -2.81105 CYP1B1 1.52E-06 -2.80458 DHX57 0.00030501 -2.80309 DHX57 0.00030501 -2.80309 TRMT13 3.54E-05 -2.80179 FASTKD3 0.00012891 -2.80111 ZNF615 1.41E-07 -2.7989 SPATA5 5.10E-05 -2.79635 ANXA2R 4.18E-07 -2.79481 RNF146 4.77E-07 -2.79303 TRMT61B 1.06E-05 -2.79277 VCPIP1 0.00018538 -2.79167 KDM5A 1.27E-06 -2.79087 SWSAP1 0.00071015 -2.79003 DCBLD2 0.00196547 -2.78889 UBN2 5.80E-06 -2.78499 VCPIP1 2.46E-06 -2.78446 KIAA1199 2.69E-07 -2.78372 ZNF800 0.00027863 -2.78301 TGS1 0.00035312 -2.7828 DDX19A 2.58E-05 -2.78064 KIAA1432 1.11E-07 -2.78027 LRIG3 3.26E-05 -2.78024 LIPT1 0.00011077 -2.77779 DYRK1A 7.94E-09 -2.77525 FASTKD2 0.00036797 -2.77494 NEMF 3.35E-06 -2.7726 C5orf28 0.00513949 -2.7721 IFIT5 7.02E-06 -2.77193 BAG5 1.41E-07 -2.77107 POLQ 4.74E-05 -2.77068 GLMN 3.57E-05 -2.77048 MKLN1 1.44E-06 -2.76912 PNN 1.18E-08 -2.76646 DUSP11 1.24E-05 -2.76634 CENPC1 5.20E-05 -2.76507 ASPM 0.00010175 -2.76407 ZNHIT2 6.58E-07 -2.76389 LRRC58 1.45E-06 -2.76371 XKRX 0.00027378 -2.75834 MNT 5.61E-07 -2.75738 ZNF323 5.01E-05 -2.75699 PGBD4 1.82E-06 -2.75682 REV1 1.27E-07 -2.75536 ZNF146 4.01E-08 -2.75273 NPAT 9.06E-07 -2.75222 LRRC49 0.00119855 -2.7516 ZNRD1-AS1 4.51E-05 -2.75014 TSEN2 2.77E-05 -2.74827 CTPS1 7.53E-06 -2.7476 ZNF284 9.71E-07 -2.74465 TAF1A 0.00850695 -2.7433 EXOSC3 1.38E-06 -2.74244 CLDN12 1.46E-06 -2.74014 CTPS1 9.49E-05 -2.74007 ZNF766 5.74E-07 -2.73983 ZNF493 2.15E-06 -2.7398 ABCE1 0.00377105 -2.7389 NOL8 0.0110288 -2.73757 PNO1 1.57E-05 -2.7367 AKR1B10 1.48E-06 -2.73584 REV1 4.72E-06 -2.73219 PDE10A 0.00022328 -2.73182 RNF43 3.44E-07 -2.73149 C2orf69 5.54E-08 -2.73148 SETD6 0.00011604 -2.73026 TP53RK 8.93E-06 -2.72907 C12orf4 7.68E-05 -2.72903 RNF34 1.10E-05 -2.72892 ZNF84 3.28E-05 -2.72867 JRKL 7.32E-05 -2.72783 LOC10050764 0.00102597 -2.72744 NOL11 3.49E-07 -2.72707 DOPEY1 9.89E-06 -2.72581 USP6NL 3.14E-06 -2.72563 B3GALT6 1.78E-07 -2.72472 GPR110 0.00871053 -2.72441 CLN8 8.65E-07 -2.72437 PPFIBP2 0.00068299 -2.72289 RCL1 3.19E-06 -2.72212 ASNS 0.0007761 -2.72167 C6orf203 1.77E-05 -2.72161 ZNF544 2.03E-08 -2.72117 DPY19L1 5.05E-06 -2.71471 YARS2 1.98E-05 -2.71408 MCTP1 0.00136801 -2.71346 TXNDC15 0.00371141 -2.71236 MAPK6 0.00183957 -2.71204 CREBZF 5.25E-05 -2.71078 FBXO3 0.00111623 -2.71074 ARHGAP32 1.23E-06 -2.71022 NSUN4 0.00025158 -2.7094 PAQR3 0.00186541 -2.70909 SERPINB8 2.71E-05 -2.70876 STK4 4.56E-05 -2.70757 TAMM41 1.59E-05 -2.7075 ZNF12 6.10E-05 -2.70742 TNFAIP8 2.29E-07 -2.70265 ASF1A 0.00011653 -2.70239 UTP15 0.00013743 -2.70027 KLHL9 5.22E-06 -2.69988 TAF1A 0.0012325 -2.69539 NAPEPLD 0.00283546 -2.69492 ZNF681 3.19E-05 -2.69473 GINS3 5.45E-05 -2.69419 ZNF507 1.66E-06 -2.69368 TSEN15 2.14E-05 -2.69334 EPM2AIP1 0.00017836 -2.69154 ZNF543 6.32E-05 -2.69152 PIF1 0.00036446 -2.69121 POLR3F 3.86E-05 -2.69036 FANCF 2.63E-05 -2.68967 HMGXB4 0.0004627 -2.6889 TMCC1 4.16E-07 -2.6872 SEMA3A 0.0111246 -2.68677 RRP8 1.09E-06 -2.6866 CCNL2 8.60E-06 -2.6865 ANP32A 8.27E-05 -2.68648 ZNF562 0.00086731 -2.68628 ZNF860 0.00071363 -2.68592 TIGD2 1.43E-05 -2.68562 RNF43 1.99E-08 -2.68547 DDX52 3.30E-05 -2.68348 ENPP5 2.05E-05 -2.68195 FOXP1 0.0009473 -2.6818 NFXL1 0.00014859 -2.68173 FUBP1 2.72E-05 -2.68162 LRP8 0.0001573 -2.67866 ATP2B1 0.0040388 -2.67742 CHAMP1 1.09E-06 -2.67501 USP36 0.0007375 -2.6717 TGFBR2 1.31E-06 -2.6712 RIF1 9.24E-05 -2.67093 ZNF12 5.05E-05 -2.66824 TMEM138 3.53E-05 -2.66542 AGPS 0.00026833 -2.66404 SLC25A27 1.33E-06 -2.66221 MRFAP1L1 4.68E-09 -2.66088 PHF17 5.83E-06 -2.66059 ZNF681 1.95E-05 -2.65787 NKRF 1.36E-07 -2.65562 ZCCHC7 1.45E-05 -2.64946 ABCA1 1.05E-05 -2.64925 C3orf52 8.51E-07 -2.64892 POLR2D 2.63E-06 -2.64576 BCAR3 3.18E-06 -2.64571 MRPS10 0.00016212 -2.64569 MCPH1 4.53E-06 -2.64561 IFNGR1 9.59E-05 -2.64516 ATXN7L3B 0.00400541 -2.64395 EXOSC6 1.78E-05 -2.64268 ZNF12 1.49E-05 -2.64238 ZNF561 2.85E-05 -2.64233 KLHDC4 3.72E-08 -2.64123 CCDC12 1.02E-06 -2.64112 ASXL2 1.25E-05 -2.64098 TXNIP 2.30E-05 -2.64054 FARSB 2.56E-06 -2.63913 COX4I1 0.00022908 -2.63732 C12orf43 4.85E-05 -2.63623 LRRFIP1 1.80E-08 -2.63552 RNGTT 0.00252807 -2.63504 ZMYM1 0.0008472 -2.63364 SOWAHC 8.90E-08 -2.62923 LOC729678 1.60E-07 -2.62891 TSEN15 7.60E-05 -2.628 POP1 1.27E-07 -2.62796 BCLAF1 7.08E-09 -2.62734 C3orf62 3.75E-05 -2.62685 C18orf21 3.09E-07 -2.62662 RLIM 4.56E-08 -2.62604 PNN 1.31E-06 -2.62586 B3GALTL 3.98E-05 -2.62553 TROVE2 4.21E-05 -2.6252 CCDC84 9.09E-07 -2.62423 THUMPD3 0.00021447 -2.62398 PPARGC1A 1.13E-05 -2.62287 SPRR1B 5.18E-07 -2.61664 ZNF587 0.0033808 -2.6164 ALDH1B1 6.96E-05 -2.61565 ZC3HC1 6.63E-06 -2.61506 AKT2 8.88E-05 -2.61412 ZBTB8A 1.12E-05 -2.61306 NR2C2 1.15E-06 -2.61161 DDX21 4.05E-06 -2.61142 ZNF292 9.83E-08 -2.60983 PSD3 0.00031062 -2.60821 ZNF502 1.69E-07 -2.60733 GTPBP2 6.79E-09 -2.60619 SLC25A16 9.16E-05 -2.60472 BRWD1 0.00012718 -2.60452 SLC25A37 0.00011227 -2.60444 BTNL9 2.17E-07 -2.60363 TMCC1 1.70E-08 -2.60285 N6AMT1 0.00116587 -2.60199 CD59 0.00013601 -2.60177 CSTF2T 0.00017751 -2.6002 TRPS1 0.00086119 -2.59911 PLAGL2 3.80E-05 -2.59622 NHS 4.16E-05 -2.59293 B3GNT2 1.05E-06 -2.59215 ASPM 0.00073524 -2.59163 SETD4 0.00017406 -2.59163 OTUD6B 0.00616504 -2.5908 PPHLN1 0.00017589 -2.58983 TMEM43 4.89E-05 -2.58933 C1orf131 2.04E-06 -2.58921 DNAJC2 4.11E-08 -2.58911 EIF4E 0.00048066 -2.58734 SMAD2 8.31E-06 -2.58647 RNF111 6.50E-05 -2.58574 FOXP1 2.00E-09 -2.5856 LRRC58 2.18E-07 -2.58511 ABCE1 0.00092528 -2.58511 CLPX 0.0002248 -2.58424 UBIAD1 0.00010098 -2.58364 PILRB 2.32E-06 -2.58345 CCNJ 4.07E-05 -2.58342 RTN4IP1 1.38E-05 -2.58307 GTF2H2 0.00020233 -2.58235 TSR1 0.0001296 -2.58083 ZNF613 2.10E-07 -2.57974 JMJD7 8.50E-07 -2.57682 MTERFD3 3.45E-05 -2.57605 ZBTB39 1.54E-05 -2.57559 PPFIBP2 5.81E-05 -2.57532 CNO 3.15E-07 -2.57482 RIF1 3.34E-05 -2.57468 DEPDC7 0.00087128 -2.57468 LRP5L 1.96E-06 -2.57447 ZNF767 7.32E-05 -2.57427 LOC10065300 0.00043373 -2.57107 DEPDC7 0.00027644 -2.56909 PUM2 1.22E-09 -2.56825 PPWD1 1.77E-05 -2.56784 GPR158 0.00082642 -2.56693 ZNF615 0.00056679 -2.56454 MTERFD2 2.35E-07 -2.56237 STX17 0.00135163 -2.56093 PPWD1 0.00113966 -2.56021 MSS51 3.48E-06 -2.55953 ANKS4B 2.71E-05 -2.55939 ZNHIT6 0.00212988 -2.55934 FMN1 0.00225343 -2.55927 PELO 7.23E-05 -2.5548 KLHDC5 0.00262322 -2.55339 CEBPG 1.79E-07 -2.55332 SRSF5 3.82E-07 -2.55327 FAM98A 6.80E-06 -2.55295 MPHOSPH6 0.00011345 -2.5525 JAG1 0.00729104 -2.55184 ECE2 6.45E-07 -2.55152 DEPDC1 0.00266785 -2.55151 FUBP1 5.32E-05 -2.55086 SLC25A32 3.81E-07 -2.55031 RHOBTB1 2.44E-05 -2.54986 ZC2HC1A 0.00061344 -2.54935 VANGL1 5.76E-06 -2.54848 OPA3 6.58E-06 -2.54758 DCAF16 0.00012234 -2.54665 ZNF566 2.74E-05 -2.54486 RPS27A 1.51E-05 -2.54437 ENGASE 6.93E-07 -2.54186 SRSF5 1.84E-06 -2.54154 POLD3 2.83E-06 -2.54152 SMARCAD1 0.00032903 -2.53905 CD59 1.99E-07 -2.53895 TMOD1 8.91E-06 -2.5381 LOC10050599 1.15E-08 -2.53807 E2F8 0.00191886 -2.53765 CHCHD4 0.00021556 -2.53756 TRMT61B 1.16E-06 -2.53624 NR2C1 0.00172748 -2.53612 MFSD5 9.05E-07 -2.53485 TTC30B 5.37E-05 -2.53285 KIAA0040 5.85E-06 -2.53226 DNHD1 4.91E-07 -2.53071 ONECUT2 4.31E-08 -2.53034 STYX 0.00043467 -2.52997 C12orf66 2.64E-05 -2.52992 YAE1D1 7.95E-06 -2.52977 C3orf17 5.09E-05 -2.52832 POLR1B 6.92E-05 -2.52718 ZNF615 0.0009592 -2.52573 RUFY3 0.00011358 -2.52559 ATMIN 6.05E-05 -2.52422 C20orf112 6.99E-06 -2.52286 Mar-07 0.00028043 -2.52231 KLHDC5 2.86E-05 -2.5223 PAQR3 0.00039994 -2.52021 U2SURP 2.46E-05 -2.51983 PABPC1L 2.57E-07 -2.51952 SMARCAD1 0.00010303 -2.51711 C21orf91 1.73E-05 -2.51706 XIST 1.57E-08 -2.51596 PNN 5.95E-08 -2.51562 HNRNPU-AS1 8.92E-08 -2.51538 POLR3G 0.00873018 -2.51288 SLC2A1 1.06E-05 -2.51268 AMMECR1 8.01E-05 -2.51266 LOC10050947 6.37E-06 -2.51265 TRIM32 4.94E-08 -2.51215 SH3RF1 0.00012842 -2.50927 LOC10050947 0.00012973 -2.50923 DDX52 3.26E-05 -2.50771 BTAF1 1.86E-06 -2.50752 PUS1 1.45E-06 -2.50621 FOXP1 9.78E-10 -2.50511 EMG1 2.15E-05 -2.50484 CATSPER2 1.50E-06 -2.50458 ZNF91 0.00021838 -2.50353 FOXP1 5.64E-07 -2.50324 NKTR 5.06E-05 -2.50199 WDR43 2.44E-05 -2.50189 LYRM7 5.69E-06 -2.50162 AHSA2 3.83E-07 -2.50156 TYW5 4.15E-05 -2.50129 PSAT1 0.00077999 -2.50125 ATAD3B 4.68E-06 -2.50099 PPAP2B 2.92E-06 -2.49976 SYNCRIP 1.16E-05 -2.49962 COMMD5 1.70E-06 -2.49899 RPS3A 1.50E-06 -2.49839 TRIM13 0.00287547 -2.49769 RICTOR 9.05E-09 -2.49758 TRMT13 1.92E-05 -2.49616 PAFAH1B1 3.41E-07 -2.49585 CEP44 3.81E-05 -2.49514 ZNF330 1.71E-05 -2.49438 PLK3 0.00136198 -2.49321 ZNF416 2.04E-05 -2.49314 ZFP14 2.49E-07 -2.49276 TTI2 1.27E-07 -2.49084 BLM 1.44E-05 -2.48945 KANK1 1.65E-06 -2.48915 THADA 1.31E-05 -2.48837 BAG5 5.26E-06 -2.48709 ZNF234 0.00031977 -2.48704 MACC1 0.000166 -2.48699 CREB1 1.10E-05 -2.48677 BRI3BP 0.00047737 -2.4857 LOC10028751 4.93E-07 -2.48559 SCO2 4.12E-09 -2.48517 FAM13A 0.00130986 -2.48516 PPHLN1 0.00010904 -2.48497 CPSF6 3.78E-07 -2.4842 SGK494 0.00112853 -2.48391 TMEM60 2.28E-05 -2.48233 TIMM23 2.35E-05 -2.48125 RBM12 6.39E-05 -2.48104 ZNF827 0.00016945 -2.48062 SCAF11 3.58E-05 -2.47631 TXNIP 0.00029194 -2.47565 UNC50 4.91E-06 -2.47248 MAPK1IP1L 1.50E-05 -2.4723 TRIM32 8.65E-05 -2.47078 ZNF786 7.57E-05 -2.47023 XPO4 2.02E-05 -2.46884 CEP76 0.00199066 -2.46872 GEMIN4 1.02E-06 -2.46814 PDE10A 0.00120076 -2.46588 LYPLAL1 0.00221458 -2.46504 ZNF280D 0.00030429 -2.46288 ATAD2B 0.00309849 -2.46184 NOC3L 0.00129751 -2.45983 AKR7L 0.00235008 -2.4596 PNO1 0.00021167 -2.45927 BBS10 0.00539739 -2.45825 METTL16 4.88E-05 -2.45736 SETDB1 5.22E-08 -2.45732 PRMT6 0.00447422 -2.45564 PPFIBP2 3.82E-05 -2.45496 RAB18 0.00206568 -2.45452 TRIM35 0.00024904 -2.45222 GTF2H2 0.00157693 -2.45163 FMN1 0.00122586 -2.45026 ZNF766 2.35E-05 -2.44985 ZNF680 6.05E-08 -2.44817 SLC35B4 0.00046196 -2.44717 ZNF441 0.00035668 -2.44606 MIA3 6.06E-05 -2.44564 ENOSF1 0.00031617 -2.44405 CSRP2BP 0.00141989 -2.44365 SRSF8 1.93E-07 -2.44264 SLC2A1 0.00015874 -2.4413 SELRC1 5.02E-06 -2.44122 MTHFD1L 0.00040541 -2.44097 GPATCH2 0.00492674 -2.43844 MAP4K3 0.00407683 -2.43825 GNPNAT1 0.00058159 -2.43777 MCM3AP-AS1 7.35E-05 -2.43757 ABHD13 0.00070437 -2.43654 ANKS4B 0.00010685 -2.43598 EXOSC4 2.02E-06 -2.43531 CREBZF 3.12E-06 -2.43338 FAM208B 0.00792639 -2.43337 CCNA2 2.59E-05 -2.43279 BAG2 0.00539712 -2.43218 TNFRSF21 0.0001277 -2.43203 BRIX1 0.00017462 -2.43141 LRRFIP1 0.00888347 -2.43135 VKORC1L1 1.07E-05 -2.42991 FBXO38 0.00017683 -2.42987 HPDL 1.19E-06 -2.42981 SKP2 2.35E-06 -2.4292 BTAF1 4.02E-06 -2.42901 SLC2A1 7.43E-06 -2.4285 METTL14 0.00065067 -2.42601 RPL7L1 3.80E-08 -2.42594 MTF1 2.63E-06 -2.42523 DTL 0.00074329 -2.42294 DNAJC24 0.00038608 -2.42102 GK5 3.96E-05 -2.421 AHNAK2 1.33E-06 -2.41948 ZNF83 5.05E-05 -2.41749 GTF2E1 2.85E-05 -2.41731 DMTF1 0.0001399 -2.41717 MDM4 1.38E-07 -2.41661 KALRN 0.00028166 -2.41651 VWA2 5.87E-07 -2.41536 CUTC 4.67E-05 -2.41516 FASTKD2 0.00036036 -2.414 USP45 0.00664587 -2.41384 PCID2 0.0135696 -2.41328 GNL3L 9.62E-06 -2.41239 EXOC4 1.95E-06 -2.41238 TADA2A 3.39E-07 -2.41216 SLC38A1 9.42E-07 -2.41152 ZNF28 0.00051735 -2.41134 FLJ39632 0.00019653 -2.41103 ZNF765 6.56E-05 -2.40912 VANGL1 5.40E-05 -2.4079 BAG5 3.43E-06 -2.40568 ABCE1 5.24E-07 -2.40424 BLZF1 0.00110618 -2.40414 TNRC6B 0.00019093 -2.40396 BCLAF1 0.00214677 -2.40258 CCDC82 0.00275637 -2.4025 PHAX 2.26E-05 -2.40203 COX19 3.99E-06 -2.40015 PUS7 1.27E-05 -2.39955 CEP85 1.56E-07 -2.39792 AHCTF1 7.40E-06 -2.39781 PNN 4.99E-06 -2.39712 PLAA 0.00045791 -2.39599 PHC3 2.41E-08 -2.39506 CDKN2AIPNL 1.95E-05 -2.39458 LIN9 0.0123089 -2.39338 DHFRL1 4.05E-08 -2.39296 SYNCRIP 0.00032144 -2.3927 BORA 0.00402525 -2.39153 WDR35 0.00034028 -2.39129 RARS 3.37E-07 -2.39082 ZNF234 3.78E-05 -2.39022 PRPF38B 5.41E-09 -2.38914 NEMF 4.89E-05 -2.38849 TMEM43 0.00073734 -2.38819 SNRPD1 4.72E-05 -2.38711 CA13 2.09E-05 -2.38682 IREB2 3.67E-07 -2.38679 HSPBAP1 0.00502501 -2.38671 SRSF5 2.51E-06 -2.38531 AQP11 3.05E-05 -2.38531 KIAA0232 0.00016583 -2.38432 GLS 0.00093757 -2.38229 ALKBH2 3.29E-05 -2.38187 PMS1 2.36E-07 -2.38107 SLC38A1 3.74E-06 -2.37792 C10orf118 3.72E-06 -2.37736 SMC5 5.79E-07 -2.3764 MTF2 2.42E-05 -2.37595 TIMM8A 0.00295499 -2.37565 CKLF 0.00471297 -2.37502 FAM98A 0.00227146 -2.37473 RPGRIP1L 0.00067629 -2.37449 SRM 9.58E-05 -2.37364 PGAM5 2.60E-05 -2.37348 ZDHHC17 0.000487 -2.37294 DCUN1D4 1.02E-05 -2.37209 USP31 0.00094063 -2.37176 CDCA4 0.00125535 -2.3715 RPS6KB1 0.00010659 -2.37122 POLE2 0.00357035 -2.37078 COBL 0.00013708 -2.36997 FOXP1 3.05E-09 -2.36933 SEC24B 3.97E-06 -2.36911 DTWD1 0.00361652 -2.3685 C9orf41 7.97E-06 -2.36791 FEN1 6.95E-06 -2.36678 ZNF792 0.00138035 -2.3667 MRPL50 0.00015202 -2.36657 NIF3L1 0.00165716 -2.36557 PARP9 0.00197379 -2.36505 ZNF330 6.39E-06 -2.36488 RAB12 9.89E-05 -2.36472 DPY19L1 6.37E-05 -2.36443 PGAM5 0.00015852 -2.36323 THNSL1 2.49E-05 -2.36257 ZNF341 2.24E-05 -2.36229 KLF12 0.00010036 -2.36013 UBIAD1 9.95E-05 -2.3599 GCFC2 0.00070883 -2.35821 ZNF544 4.02E-05 -2.35805 KRT13 0.00052313 -2.35784 ZNF200 6.63E-05 -2.35563 FAM160B1 0.00061681 -2.35324 EXTL2 0.00679158 -2.35281 ZNF614 0.0020237 -2.35256 AASDHPPT 0.0104216 -2.35124 ZBTB8A 6.24E-05 -2.35006 SEC24B 0.00054541 -2.35005 TXNIP 0.00014565 -2.35001 RABL3 0.00209768 -2.34861 CDC37L1 0.0008297 -2.34775 CAPRIN2 6.48E-05 -2.34767 ARMC10 2.22E-07 -2.34705 TMEM161B 0.00067643 -2.34555 NSUN5P1 1.00E-06 -2.34549 GPR87 2.20E-08 -2.34546 TRMT44 3.59E-06 -2.34408 SOCS6 2.39E-06 -2.34386 PHAX 0.00055207 -2.34203 COMMD2 3.33E-06 -2.34123 AEBP2 0.00273088 -2.34078 PMS1 9.89E-05 -2.34032 METTL1 0.00013407 -2.34008 C5orf43 1.03E-05 -2.33895 BMP4 2.72E-06 -2.33822 ZADH2 0.0001697 -2.33787 RNF19A 6.20E-05 -2.33766 FANCL 0.00022917 -2.33731 DKC1 0.00019368 -2.3373 SACS 0.00041724 -2.33543 RBM48 2.70E-06 -2.33453 ZNF193 8.58E-05 -2.33421 ZNF148 0.00042314 -2.33392 ACTG1P4 3.09E-05 -2.3333 ZNF234 0.00039713 -2.33305 FNIP1 8.22E-06 -2.33303 CKAP2 0.00132893 -2.33296 BRWD1 0.00033091 -2.33251 OXSM 1.28E-05 -2.33168 PLAG1 9.98E-06 -2.33088 TRNT1 0.00015432 -2.33004 DTWD1 0.00251935 -2.3299 NUDCD1 0.00020008 -2.32946 RICTOR 1.04E-08 -2.32943 NDUFAF4 6.01E-06 -2.3276 FEN1 9.21E-05 -2.32759 PPAPDC1B 9.89E-05 -2.32506 RNF25 1.98E-05 -2.325 HS3ST1 4.85E-06 -2.32405 AGGF1 0.00027455 -2.32251 SPATA5L1 0.00025997 -2.32185 ZNF691 3.20E-05 -2.32165 DFFA 2.86E-06 -2.32022 C11orf82 0.00205852 -2.31874 METTL2B 6.68E-05 -2.31859 ABCE1 0.00269813 -2.31843 CSAD 0.00399569 -2.31828 ZNF527 0.00016918 -2.31581 CMTM3 0.00011072 -2.31457 DCAF16 0.00552974 -2.31425 C9orf72 0.00103913 -2.31251 FAM111A 3.56E-07 -2.3121 TLR4 1.55E-06 -2.31194 MOCS3 2.12E-05 -2.3114 ZNF786 0.00112976 -2.31011 NOC3L 0.00259706 -2.30909 MSL3P1 0.00020743 -2.30899 HJURP 1.50E-05 -2.30897 TMEM168 1.86E-06 -2.30875 ZNF473 4.30E-05 -2.3086 TRMT10C 3.41E-07 -2.30834 PALB2 0.00017392 -2.30826 ZNF595 2.16E-05 -2.30819 MUM1 4.11E-07 -2.30721 GEMIN2 0.0038124 -2.30694 CCRL2 4.49E-05 -2.30668 TMEM203 1.05E-05 -2.30622 MSTO1 3.48E-05 -2.30524 KIAA1432 0.00723556 -2.30511 RWDD3 7.36E-05 -2.30404 MAGEA2 0.00016471 -2.30342 OTUD4 9.90E-06 -2.30125 CEBPZ 1.37E-07 -2.30104 GIGYF2 0.00027622 -2.30087 MTHFD1L 0.00028348 -2.30079 ZCCHC7 7.32E-05 -2.29922 AURKAPS1 2.11E-06 -2.29759 NUPL1 0.00914181 -2.29591 DCLRE1C 0.0005295 -2.29479 MAP4K3 0.0101224 -2.29403 DCP1B 1.18E-08 -2.29394 INF2 0.00053423 -2.29234 GLRX2 9.56E-06 -2.29231 ENTPD7 0.00173743 -2.29101 ZNF346 1.13E-06 -2.2909 KIAA1217 0.00026654 -2.29089 TNS4 0.00044111 -2.29067 PLEKHA8 0.00010388 -2.28983 METTL13 4.57E-06 -2.28969 ZNF506 0.00022697 -2.28959 RIBC2 3.21E-05 -2.28937 SRM 6.21E-07 -2.28912 METTL3 0.00043553 -2.28867 SLC5A6 1.68E-05 -2.28819 AHNAK2 0.00022083 -2.28506 ZNF160 0.00034782 -2.28456 CLPX 1.86E-05 -2.28299 C14orf28 0.00049443 -2.28297 RBM48 0.00011028 -2.28244 SUV39H2 1.17E-05 -2.28124 FNDC3B 9.04E-07 -2.28072 AKAP2 0.00112079 -2.27921 PPAPDC2 0.00487136 -2.27762 UBLCP1 0.0002702 -2.27585 ACTG1P4 0.00544517 -2.27585 DYRK2 1.45E-07 -2.27551 MOB1B 1.46E-07 -2.27542 ZNF793 0.00500529 -2.27531 NOP16 9.04E-05 -2.27507 C2orf68 0.00304452 -2.27504 RPRD2 0.00171896 -2.27449 TSPAN14 5.01E-05 -2.27395 KIAA1598 0.00087066 -2.27372 NAA25 1.04E-05 -2.27332 FUBP1 2.06E-05 -2.2724 C5orf28 7.93E-05 -2.27151 TTI1 3.05E-08 -2.27108 AQP3 1.69E-05 -2.27095 SPIN3 0.00029751 -2.26984 ARHGAP29 0.0018138 -2.26975 RAP2B 0.00136162 -2.26888 PIBF1 0.00993242 -2.26881 HAUS8 0.00029978 -2.26804 PPHLN1 3.76E-06 -2.26779 EXOSC9 1.05E-05 -2.26696 ZBTB1 2.67E-06 -2.2669 PPWD1 0.00083619 -2.26687 DYRK2 0.00821275 -2.26671 DKC1 3.42E-06 -2.26628 HIPK2 0.00063646 -2.26598 LIF 0.00911302 -2.26574 HNRNPF 0.0002087 -2.2648 NFYA 0.00022956 -2.26462 SRSF10 0.00193625 -2.26446 ZCCHC2 0.00031831 -2.26433 UNC50 1.18E-06 -2.26428 --- 6.67E-05 -2.26427 CDK12 0.00993354 -2.26395 SMAD2 0.00180559 -2.2633 ZNF512 0.00030791 -2.26309 FKBP15 4.13E-05 -2.26287 BBS10 0.00212949 -2.26228 ANKRD12 7.44E-05 -2.26183 ATP5S 9.97E-05 -2.25763 BHLHE41 1.28E-05 -2.25729 SLTM 6.64E-05 -2.2569 MCM4 0.00078304 -2.25639 C12orf29 7.94E-05 -2.25499 OSTM1 3.14E-05 -2.25483 ABHD15 5.41E-05 -2.25467 SRSF5 7.00E-05 -2.25267 ZNF766 0.00031529 -2.25263 DDX11 1.43E-07 -2.25206 RAD1 0.00543578 -2.25193 VEZT 0.00018172 -2.25078 C6orf120 0.00042761 -2.25062 C1orf131 0.00130252 -2.24933 ZNF562 0.00016658 -2.24809 DIMT1 0.00010561 -2.24806 ZSCAN2 5.89E-05 -2.24666 CNPY2 3.72E-05 -2.24648 VKORC1L1 0.00038336 -2.24645 ODF2 0.00091728 -2.24643 SLC7A6 0.00043474 -2.24459 FAM98B 1.85E-06 -2.24376 PHIP 3.05E-06 -2.2431 DNM1 3.91E-05 -2.24196 HNRNPA3 3.96E-06 -2.24168 CLPX 5.23E-06 -2.24163 PLRG1 0.00285539 -2.24089 SLC25A27 0.00134905 -2.24072 AAK1 1.70E-06 -2.24061 MSTO1 4.13E-05 -2.2406 EEF1E1 0.00070332 -2.23952 ZNF638 4.30E-06 -2.23914 CWC15 1.39E-09 -2.23804 LYPLAL1 0.00032042 -2.23794 ESF1 0.00201906 -2.23775 ZNF449 0.00032477 -2.23717 MAP3K1 0.00014454 -2.23577 ZNF100 1.25E-07 -2.23515 ZNF121 9.39E-05 -2.23428 ZNF845 0.00024458 -2.2338 ZNF567 0.00135932 -2.2331 DDX52 0.0051653 -2.23296 AKAP2 0.00073763 -2.23252 ZMAT3 0.00177985 -2.23145 VKORC1L1 0.00119129 -2.23001 B3GALT5 1.89E-05 -2.22931 GART 8.65E-05 -2.2289 TMEM170B 0.00083495 -2.22692 CCNA2 0.00147427 -2.22673 FAM108C1 0.00016311 -2.22664 UTP6 5.77E-07 -2.22661 CPSF6 3.95E-08 -2.22601 MPHOSPH8 2.96E-07 -2.22588 ZNF764 9.69E-07 -2.22579 HEATR8 1.79E-06 -2.22461 SLC38A1 0.0010852 -2.22419 TNFRSF1A 1.36E-07 -2.22384 EXOC6 0.00037957 -2.22381 THNSL1 8.45E-05 -2.22352 CASC5 1.55E-05 -2.22351 NDUFAF4 2.45E-06 -2.2235 CATSPER2 2.67E-06 -2.22268 IKBKB 0.0002143 -2.22249 PRPF4B 0.00099362 -2.22147 INO80D 2.34E-08 -2.22126 LRRC8B 1.39E-05 -2.22083 ZNF784 0.00067103 -2.2195 KCNE3 0.000383 -2.21736 TMEM41A 4.03E-06 -2.217 RPF2 6.16E-08 -2.2169 C3orf17 0.00026303 -2.21683 CKAP2 0.00080994 -2.21508 C12orf76 0.00010316 -2.21457 LRP6 1.99E-06 -2.21455 TMEM133 0.0019724 -2.21455 RNGTT 0.00016272 -2.21365 PDCD2 4.99E-05 -2.21273 C12orf5 0.00454005 -2.20924 KBTBD3 2.45E-05 -2.20916 MST1R 5.95E-05 -2.20873 CCDC50 3.26E-06 -2.20869 RBM15 3.61E-07 -2.20864 ECD 3.52E-06 -2.20862 LYRM2 3.67E-06 -2.2078 POGK 1.99E-06 -2.20771 COIL 0.00319166 -2.20754 PROSER1 0.00010941 -2.20745 MINA 0.00013796 -2.20651 TSPYL4 3.31E-05 -2.20575 C12orf26 0.00014778 -2.20575 MRM1 0.00053251 -2.20556 PRPF4 0.00177293 -2.20307 SUPT7L 0.00058396 -2.20206 ZADH2 0.00035641 -2.20163 KIAA1598 0.0007628 -2.20153 DCLRE1C 8.46E-05 -2.20088 STAG3L2 4.28E-05 -2.20073 ZNF552 0.00333981 -2.20013 MARS 8.42E-06 -2.19998 MAP4K3 4.65E-05 -2.19799 C3orf17 1.46E-05 -2.1976 CEBPG 2.94E-07 -2.19711 TOMM5 1.04E-06 -2.19645 FTSJ2 1.26E-06 -2.19593 XIAP 6.35E-07 -2.19533 NIP7 2.49E-05 -2.19517 ZUFSP 5.09E-05 -2.19496 GINS1 2.99E-07 -2.19429 ZNF264 5.93E-07 -2.19378 C1orf21 2.09E-07 -2.19332 GLS 0.00118597 -2.19327 ZNF419 0.00047439 -2.19207 SFT2D2 0.00080829 -2.19182 NAPB 2.38E-05 -2.19168 SLC17A5 0.00027432 -2.19145 UCHL3 6.97E-05 -2.19124 CPSF6 5.25E-07 -2.19095 ZBTB1 0.00333166 -2.19091 TMTC3 0.00463598 -2.19079 PRSS23 0.00015879 -2.1902 LRCH1 0.00099519 -2.18898 RANBP6 2.40E-05 -2.18854 YTHDC2 4.33E-05 -2.1868 TSEN15 0.00729887 -2.18564 RIN2 2.53E-07 -2.18559 DLEU2 0.00360417 -2.18551 TSPAN14 3.50E-06 -2.18477 MTAP 0.00094892 -2.18452 FAM156A 0.00243493 -2.18418 DNAAF2 0.00246598 -2.18406 PILRB 7.19E-06 -2.1837 ZNF765 2.05E-05 -2.18309 ZKSCAN3 0.0002472 -2.1824 RAD18 4.30E-05 -2.18151 E2F8 0.0114369 -2.18143 PDE7A 7.45E-06 -2.18104 ZNF22 0.00159489 -2.18098 KIAA1704 0.00026727 -2.18095 TAF1A 0.00626683 -2.18001 YAP1 0.00045849 -2.17933 TCEB3 0.00027779 -2.17885 ZNF212 2.98E-05 -2.17871 ANO1 1.60E-07 -2.17846 RRN3 0.00025844 -2.17834 EPHA1 0.0002987 -2.17829 EIF3J 2.55E-06 -2.17816 CSTF1 1.27E-07 -2.17763 LRP8 0.00096807 -2.17731 TPM4 0.00548599 -2.17649 ZBED5 2.61E-07 -2.17648 OPA3 0.0005865 -2.17631 POLR3F 0.00273158 -2.17616 NR0B2 0.0004172 -2.17606 RPGRIP1L 5.04E-05 -2.17479 PUS1 4.27E-06 -2.17443 SRSF11 8.31E-07 -2.17434 ZNF193 5.46E-05 -2.17391 KIFC2 1.79E-05 -2.17391 KIAA1429 0.00015121 -2.17364 CSTF3 5.80E-06 -2.1729 GALNT4 6.64E-05 -2.17265 TIGD5 1.25E-05 -2.17234 LYRM7 9.60E-05 -2.17233 PHF19 0.00021987 -2.17155 CCDC120 1.59E-06 -2.17103 PHOSPHO2 6.33E-05 -2.17013 VCPIP1 0.00039512 -2.16901 NUP62CL 6.31E-05 -2.16824 PPFIBP1 0.00895429 -2.16745 ZNF419 3.19E-05 -2.16721 REXO2 8.39E-05 -2.16721 KLK10 0.0001516 -2.16597 CDCA4 9.25E-05 -2.1657 MARS 0.00099046 -2.16544 SERPINB5 0.00217107 -2.16541 TTLL4 1.38E-05 -2.16525 SAMD9 0.0128169 -2.16393 CCNF 2.85E-05 -2.16331 DNA2 3.75E-05 -2.16312 C2orf68 0.00057859 -2.1627 SRSF10 3.97E-05 -2.16143 C5orf28 0.0020775 -2.16114 C5orf28 0.0020775 -2.16114 MMAA 0.00020021 -2.15971 TTPAL 0.00027016 -2.1593 SRSF5 4.05E-05 -2.15909 ZFP62 1.51E-05 -2.159 DCAF16 6.14E-05 -2.15861 JRKL 0.00082878 -2.15844 MTF2 0.00045231 -2.15787 FRAS1 0.00379572 -2.1578 CHIC1 4.30E-06 -2.15774 ALKBH2 4.55E-05 -2.15768 SPRED1 3.83E-09 -2.15763 UBLCP1 0.00049839 -2.15749 MTHFD1L 0.00103576 -2.15643 ZDHHC23 4.09E-05 -2.15628 CPSF6 2.66E-07 -2.15553 SLC15A4 4.09E-05 -2.15491 C6orf228 0.00026536 -2.15486 EBNA1BP2 2.54E-05 -2.15474 TUBGCP3 0.00277401 -2.15444 FAM118A 7.89E-05 -2.15404 SDAD1 6.08E-05 -2.15275 RNF219 2.32E-05 -2.15248 CXorf23 0.00135893 -2.15242 MBD4 4.87E-05 -2.15178 BTAF1 0.00276499 -2.15026 MTERFD3 0.0027299 -2.14938 DDX31 0.00467369 -2.14912 TRMT10B 0.00081311 -2.14888 FAN1 0.00012104 -2.14887 BRIX1 8.62E-06 -2.14748 NSRP1 8.19E-06 -2.14746 CCNL2 8.97E-07 -2.14657 AIMP2 7.78E-06 -2.14548 PAG1 5.96E-06 -2.14547 CSTF1 4.89E-06 -2.14531 PRPF3 4.08E-05 -2.14458 SULF2 0.00015529 -2.14298 DDX17 8.06E-10 -2.14295 SKA3 3.53E-06 -2.14272 C10orf137 0.00132068 -2.14133 PHF20L1 0.00027342 -2.14002 RFC3 0.0027774 -2.13973 STX19 1.42E-06 -2.13971 MRPS21 5.04E-06 -2.13912 RNASEH2C 9.14E-05 -2.13876 CREBZF 2.85E-05 -2.13852 TPBG 0.00017448 -2.13809 PPP1R35 1.49E-05 -2.13801 ASF1A 0.00166405 -2.13764 GIGYF2 0.00024263 -2.13693 SLC6A20 0.0020977 -2.13648 HKDC1 6.33E-06 -2.13583 SLC35G2 0.00154679 -2.13567 ZNF225 0.00305895 -2.13527 MTERFD2 6.70E-05 -2.13503 GNL3 1.79E-06 -2.13449 DPH2 0.00028382 -2.13386 SOS1 1.21E-06 -2.13254 APOLD1 0.00464287 -2.13254 RALGAPA1 6.33E-07 -2.13202 ZNF577 0.00063029 -2.13182 ZNF706 8.54E-06 -2.13148 ZNF330 7.17E-05 -2.13116 MDM4 0.00028782 -2.13111 TBP 0.00015122 -2.13086 FNDC3B 9.41E-07 -2.12997 SMCR7L 9.50E-06 -2.12985 RNF26 0.0001383 -2.12958 PLEKHA5 0.0002118 -2.12892 NSD1 0.00016854 -2.12885 SERBP1 8.48E-08 -2.12849 MIS12 1.96E-05 -2.12834 PGBD3 9.69E-05 -2.12799 FAM208B 0.00645487 -2.12743 ITPRIPL2 4.82E-05 -2.12717 SLC7A1 0.00052876 -2.127 ZADH2 0.00201455 -2.12684 CSTF2 0.00016692 -2.12664 DIDO1 3.41E-05 -2.12662 DIDO1 3.41E-05 -2.12662 PARP2 0.00013009 -2.1266 TXNDC9 0.00016721 -2.1265 SLC38A1 0.00963417 -2.12636 EXPH5 2.69E-05 -2.12566 PIK3R1 0.00141401 -2.12543 LOC10028896 6.12E-06 -2.12507 DNAJC16 0.00073778 -2.12447 PRPF39 0.00089572 -2.12446 ZNF117 0.00275727 -2.12429 MAGI2 0.00042579 -2.1241 USP25 0.0038534 -2.12216 RALGPS2 1.33E-05 -2.12152 RPP40 0.00245796 -2.12142 USP15 0.00026609 -2.12129 ZBTB34 0.00021164 -2.12022 CDK12 0.0003856 -2.12017 MRS2 0.00359377 -2.11985 CASP8 0.00221396 -2.1195 DNAJC17 3.64E-06 -2.1185 GSDMB 5.50E-06 -2.11849 ERCC6L 0.00053984 -2.11843 TXLNA 1.93E-05 -2.11821 RICTOR 1.42E-05 -2.11754 JRK 0.00021034 -2.11685 ZKSCAN1 2.65E-05 -2.11665 NAA50 0.00024149 -2.11661 EGLN3 8.52E-06 -2.11625 IGIP 5.69E-05 -2.11593 TMEM60 2.55E-07 -2.11566 FAM126B 1.23E-05 -2.11564 EIF4A2 0.00231323 -2.11523 VSIG1 0.00690213 -2.11479 C3orf17 0.0135327 -2.11432 TAF9B 0.00190697 -2.11232 PIK3C3 2.97E-05 -2.11203 MSTO1 2.16E-05 -2.11174 KBTBD7 0.00016551 -2.11112 TTF1 0.00042158 -2.11094 RPS27A 5.85E-05 -2.11029 MIR17HG 0.00618219 -2.11022 ZNF638 1.31E-05 -2.10961 RPL7L1 8.33E-06 -2.1096 MORC4 0.00130197 -2.10896 MYNN 0.0003834 -2.10871 PIBF1 0.00459309 -2.10819 ATG14 9.59E-07 -2.10513 JRK 0.00036368 -2.10507 DDX20 6.85E-06 -2.10397 RBM26 0.00013678 -2.10375 TBX3 2.39E-06 -2.10351 GPATCH4 1.80E-05 -2.10284 LYRM2 4.41E-06 -2.10253 DACH1 0.00094777 -2.10077 GTF2H3 0.0109395 -2.10038 PTPN2 0.0128946 -2.1003 FRAT2 0.00053983 -2.10002 ZBED4 1.29E-05 -2.09994 C1orf116 0.00265481 -2.09982 C15orf23 0.0006076 -2.09979 FAM221A 0.00110459 -2.09952 SLC5A3 5.08E-05 -2.09951 CCDC66 0.00355498 -2.09915 MRP63 0.00038633 -2.09833 PPP2R1B 0.00368159 -2.09819 GEMIN7 3.20E-05 -2.09815 PWP1 0.00074393 -2.09681 C2orf63 0.00036662 -2.09651 ZNF28 0.00019502 -2.09581 RMI2 0.0003183 -2.09561 ZNF434 9.22E-06 -2.09528 TBRG1 0.00066067 -2.09466 ZSCAN20 8.50E-05 -2.0939 C5orf51 1.96E-06 -2.09378 ZNF609 0.0131599 -2.09335 USP6NL 0.00121188 -2.09334 LENG8 0.00183372 -2.0933 PPAT 8.64E-05 -2.09306 MIER3 1.19E-05 -2.09252 ZNF326 3.47E-06 -2.09235 ZNF816 0.00038551 -2.09192 DIEXF 0.00016318 -2.09189 ZNF643 0.00026055 -2.09169 FAM111B 0.00285845 -2.09152 CASP8AP2 0.00095462 -2.09106 RAD23B 0.00010932 -2.09086 PLEKHA5 0.00043261 -2.09048 WBP4 4.00E-05 -2.09045 E2F7 0.00277192 -2.08874 NDNL2 5.44E-06 -2.08794 RIF1 0.00018275 -2.08702 NOL6 0.00069275 -2.08687 EXOSC2 2.74E-07 -2.08653 CDC23 0.00291266 -2.08565 LMO7 0.00019555 -2.08429 MTERFD2 0.00027916 -2.08399 SYNCRIP 0.00110333 -2.08364 TMEM165 2.90E-05 -2.08314 ZNF567 0.00095472 -2.08276 PMM2 7.00E-05 -2.08228 MED9 0.0001332 -2.08217 PPARG 4.65E-09 -2.08192 PDDC1 3.13E-06 -2.08173 NT5C2 8.94E-07 -2.08142 RRP15 0.00067804 -2.08104 SMARCAD1 0.00020538 -2.0809 SMNDC1 0.00440629 -2.0802 SNAP23 0.00405651 -2.07991 ABI2 0.00019905 -2.07899 WDR20 0.00097868 -2.07858 SLC25A15 0.00018164 -2.07851 FAM65B 0.00839392 -2.07845 FAIM 0.00286961 -2.07835 FAIM 0.00286961 -2.07835 KRAS 0.00012394 -2.07669 CARS 3.81E-05 -2.07634 TMPO 0.00047915 -2.07625 MRPL15 6.00E-06 -2.07622 SLC7A1 0.0001548 -2.07622 NUPL2 0.00317006 -2.07618 CALB2 8.73E-06 -2.07599 AKAP2 0.00352178 -2.07585 GRWD1 0.00023174 -2.07574 NANOS1 6.41E-05 -2.07513 PANK2 1.60E-06 -2.07488 PELO 6.47E-05 -2.07482 PMEPA1 4.46E-08 -2.07442 MAP4K4 0.00146455 -2.07431 RPL7L1 6.21E-06 -2.07429 TIFA 0.00514406 -2.07423 RAB3IP 0.00250884 -2.07358 TAF1C 0.00018106 -2.07338 SLC25A15 0.00015095 -2.07299 UBN2 0.00037299 -2.07258 SPATA5L1 0.00386367 -2.07225 KPNA3 5.91E-05 -2.07194 RCHY1 0.00813916 -2.07142 ARL17A 0.00357506 -2.07141 DPH3 0.0001031 -2.07137 FAM175B 1.54E-08 -2.07099 MKI67IP 2.42E-07 -2.07094 WHSC1 3.84E-08 -2.0706 MYNN 1.80E-06 -2.07031 SLC35B4 1.23E-05 -2.06986 UCHL5 0.00351814 -2.06882 SDAD1 0.00204201 -2.06826 NUP85 2.46E-05 -2.06824 TIMM8B 3.53E-05 -2.06802 BDP1 4.69E-05 -2.06788 FAM222B 3.22E-07 -2.06727 OGT 8.61E-06 -2.06672 VANGL1 0.00134574 -2.06639 ARPC5L 0.00015899 -2.06605 UBE2F 0.00301323 -2.06574 LUC7L 1.40E-05 -2.06558 KBTBD3 1.03E-06 -2.06538 FAM135A 0.00030527 -2.06486 UBXN8 0.00025799 -2.06457 SERPINB8 9.31E-05 -2.06376 DNAJC30 0.00015749 -2.06324 NUPL2 0.00440407 -2.06298 SSX2IP 0.00087622 -2.06256 NCOA5 7.35E-05 -2.06245 CAST 0.00160811 -2.06234 SRRD 1.97E-07 -2.06211 O3FAR1 1.89E-05 -2.06193 UBE2F 0.00222309 -2.06175 MOCOS 0.00046664 -2.0615 USP37 4.92E-06 -2.061 C1orf174 2.31E-05 -2.06036 MET 0.000524 -2.06021 SLC25A28 4.92E-06 -2.05906 KIN 7.95E-07 -2.05757 C1orf135 1.98E-05 -2.05691 SRSF10 0.00010131 -2.05681 ADO 5.12E-06 -2.05672 FAM108C1 4.64E-08 -2.05658 BCLAF1 0.00383649 -2.0558 AARS 0.0001456 -2.05564 NVL 5.36E-06 -2.05544 BRIX1 2.13E-07 -2.05544 CRBN 9.29E-07 -2.05519 LIPT2 0.00023104 -2.05493 VEZT 0.00013959 -2.0544 QTRTD1 0.00019922 -2.05358 TRIM44 1.46E-06 -2.05319 HIPK2 0.00137206 -2.05318 MPHOSPH8 1.14E-05 -2.05316 MRTO4 8.28E-05 -2.05295 POGK 0.00166854 -2.05243 ATXN7L1 4.37E-07 -2.05235 RPL7L1 0.00020655 -2.05234 PPTC7 6.99E-05 -2.05226 PLEKHA8P1 0.00012041 -2.05225 MYNN 2.83E-05 -2.05147 ADORA2B 1.01E-05 -2.05143 G2E3 0.00021211 -2.05083 ATAD2B 0.00017391 -2.05079 ASCC3 1.61E-05 -2.04996 LRRC14 2.74E-05 -2.04995 UTP14A 3.53E-06 -2.04919 ARHGAP29 0.00166003 -2.04898 RBM41 0.00196578 -2.04863 GTPBP10 0.00011285 -2.04796 CLDN23 0.0048382 -2.04678 RBM26 0.00021337 -2.04668 FAM199X 6.92E-05 -2.04465 ERGIC2 0.00481713 -2.04458 JPH1 0.00756783 -2.04439 PAPD4 6.12E-05 -2.0442 MICAL2 7.75E-06 -2.04272 MGAT1 0.00195297 -2.04262 RNF14 0.00045633 -2.04255 DCAF16 0.00111191 -2.04228 KCTD14 0.00048158 -2.04088 EIF2B1 1.20E-05 -2.04067 BCLAF1 0.0125254 -2.04036 USP15 7.21E-05 -2.04007 FLJ39639 0.00192153 -2.03994 HAUS6 0.00190349 -2.0399 MTHFSD 1.47E-05 -2.03934 DTX3L 0.0001669 -2.03895 RICTOR 0.00787656 -2.03888 CELF1 1.31E-06 -2.03758 TNFAIP1 0.00044979 -2.03697 HSF2 0.00064049 -2.03672 MBOAT7 0.00015649 -2.03626 RWDD4 0.00224284 -2.03611 TRMT10C 0.00514479 -2.03587 EIF2S1 0.0003615 -2.03586 CLK2 1.15E-06 -2.0354 MRPS17 0.00059352 -2.03524 TNRC6B 4.92E-06 -2.03491 MRS2 0.00303304 -2.03438 MPHOSPH8 9.52E-06 -2.03427 PDS5A 0.00805105 -2.03421 PRPF4 4.64E-06 -2.03391 ZNF83 4.22E-08 -2.03389 RPAP2 0.00079592 -2.03373 MORC4 0.00166825 -2.03371 ZNF330 2.37E-06 -2.03273 TRERF1 0.00912868 -2.03266 RAB18 0.00223017 -2.03244 MTRF1 0.00046572 -2.03165 PON2 4.00E-06 -2.03158 PMS1 0.00071192 -2.03156 TRIM23 0.00015845 -2.03147 LRRFIP1 0.00101765 -2.03138 RABIF 0.00022254 -2.0312 PPM1B 0.0115402 -2.03076 PHF20 0.00055194 -2.03053 ATPIF1 0.00052546 -2.02927 FPGT 0.00050062 -2.0288 CYP1B1 2.70E-05 -2.0284 NETO2 0.00013657 -2.02815 IKZF5 0.00453857 -2.02797 MORC4 0.00060819 -2.02789 SKA1 0.00086882 -2.02786 LLPH 6.67E-05 -2.02774 ZNF805 1.80E-07 -2.02773 MREG 0.00589433 -2.02753 WARS 7.62E-05 -2.02734 SYNCRIP 7.17E-07 -2.02726 ARHGAP29 0.0132032 -2.02673 SPIN2A 0.00012713 -2.02644 BCLAF1 0.00854162 -2.02643 GALNT3 0.00043589 -2.02631 IL22RA1 2.22E-05 -2.02606 PNPT1 0.00055721 -2.02597 FNIP1 1.80E-05 -2.02584 FGD6 0.00415627 -2.02575 NUFIP2 2.91E-06 -2.02565 RAC1 1.47E-07 -2.02511 NKTR 8.87E-05 -2.02475 SLC17A5 0.0001493 -2.02442 LEPREL4 0.00053353 -2.02422 CLPX 5.65E-05 -2.02354 ZNF638 2.69E-07 -2.02204 ZNF2 0.00685856 -2.02146 RMI1 0.00109906 -2.02121 UCHL5 0.00521195 -2.02041 PAIP1 0.00036473 -2.02038 RARS 6.87E-06 -2.02019 FBXO3 0.00085101 -2.0201 MAPK1IP1L 0.00062326 -2.01964 BARD1 0.00043279 -2.01886 FAM175B 0.00173898 -2.01886 CDK5RAP2 0.0117815 -2.01885 C11orf92 0.00064976 -2.01883 DAB2 0.00139776 -2.01881 E2F8 0.00171592 -2.01877 AQP11 1.35E-05 -2.01865 RAB20 0.00011252 -2.01838 PRR15 1.63E-06 -2.01809 SCYL2 2.45E-06 -2.01765 IPO9 7.22E-05 -2.01652 LRRC49 0.00073976 -2.01629 ALG11 0.00020101 -2.01584 GTPBP10 0.002881 -2.01447 CDK6 3.10E-06 -2.01443 ZNF577 0.00021329 -2.01436 CTPS1 0.00890472 -2.01401 TP53RK 0.00067524 -2.01338 EFNA5 0.00352414 -2.01322 CCRL2 4.47E-05 -2.013 ZNF175 0.00069015 -2.01243 PEX11A 0.00017021 -2.01231 ZNF22 1.87E-05 -2.01215 ZSCAN21 0.00035458 -2.01196 CDC25A 1.70E-06 -2.01124 MARCKS 6.72E-05 -2.01079 TRMT13 0.00016915 -2.01067 MRPS10 0.00221178 -2.01065 TP53I3 4.11E-05 -2.0106 TFCP2 0.00412496 -2.00918 LRRC14 0.00029899 -2.00897 ZNF805 0.00033673 -2.00887 FBXO38 0.000787 -2.00851 KDM5A 0.00037249 -2.0084 HNRNPD 1.17E-06 -2.00804 SLC3A2 5.94E-06 -2.00767 COX19 0.00017344 -2.00748 WARS 0.00102979 -2.00745 EARS2 2.05E-05 -2.00742 RPRD1B 8.47E-06 -2.00727 NRF1 0.00249017 -2.00677 ARMC8 8.36E-06 -2.00566 HIF1AN 3.91E-06 -2.00557 SCEL 0.0116283 -2.00455 MAP7 0.00013271 -2.00342 CMTM3 0.00915248 -2.00297 MRPL39 0.00039618 -2.00255 ZNF252P 5.05E-05 -2.00251 CAPRIN1 0.00170829 -2.00198 SETX 3.68E-05 -2.00186 SMNDC1 8.36E-07 -2.0017 ZDHHC3 2.98E-05 -2.00167 ZNF133 0.00016322 -2.00166 DDX18 2.62E-08 -2.00154 EXOSC2 0.00057324 -2.00151 ADPRHL2 2.26E-06 -2.00141 ZC3H15 0.00062834 -2.00131 BTBD1 0.010084 -2.00128 SGK223 1.69E-06 -2.00099 APOL6 0.00037164 -2.00065 PPWD1 0.00153874 -2.00049 TARDBP 1.38E-09 -2.00047 SLC22A3 0.001362 -2.0004 C7orf29 2.56E-06 -2.00039 GPR37 0.00246619 2.00031 SEMA6A 0.0078994 2.00039 C1orf61 0.00025532 2.00045 LPP 0.00056209 2.00071 HIST1H2BC 0.00310626 2.00088 MYPN 9.90E-05 2.00108 PHF17 1.06E-06 2.00111 GOLGA1 0.00022874 2.00183 TSPAN12 1.79E-05 2.00258 SOS1 0.00128237 2.00288 IPMK 0.00099034 2.00391 OAT 9.32E-07 2.00445 EHD1 0.00802902 2.00486 C12orf36 6.01E-05 2.00505 MBP 1.43E-05 2.00569 IVNS1ABP 3.97E-06 2.00711 AOC3 0.00049433 2.00732 C5orf48 0.00157173 2.00872 SCUBE2 0.00058011 2.00909 PROX1 0.00070669 2.00911 DPY19L4 0.0070225 2.0106 EPHA4 3.96E-05 2.01197 PTRF 0.00013737 2.01219 NCAN 7.02E-07 2.01283 EIF4A2 3.54E-07 2.01371 DIP2C 0.00369745 2.01431 SUSD4 0.0008676 2.01462 ALAS1 9.79E-06 2.01474 AMPD1 0.00013185 2.01509 EPB41L5 8.21E-05 2.01516 CDK19 0.00569536 2.01563 MSL1 0.00090963 2.01672 FBXO36 0.0004108 2.01704 NABP1 8.06E-05 2.01815 KBTBD2 0.00012453 2.01837 SLC3A1 0.00074127 2.01927 PPARA 3.14E-05 2.02064 MLL5 0.00097885 2.02111 KCNH2 0.00157346 2.02216 ZFAND5 1.62E-05 2.02249 C1orf64 0.00044677 2.02277 TICAM2 0.00043952 2.02334 PPP1R15B 8.91E-05 2.02377 GAGE1 0.00069159 2.02439 GABBR2 3.57E-05 2.02562 THRSP 0.00032787 2.02592 CD55 0.00025276 2.02638 TLE4 0.00265507 2.02836 PTP4A1 7.56E-07 2.02947 NRXN2 0.00030163 2.0296 MCL1 5.82E-08 2.02968 RNF103 5.78E-07 2.02992 HSPH1 1.63E-08 2.03027 THSD4 0.00052068 2.03059 MBNL2 7.07E-05 2.03121 HTRA1 4.94E-05 2.03169 PER2 0.00246703 2.03335 ARHGAP19 0.00011148 2.03351 ERICH1 1.58E-05 2.03354 PHLPP1 0.00010391 2.03455 SFN 8.33E-05 2.03554 GRAMD1C 0.0016844 2.03725 PECAM1 8.95E-07 2.0378 YPEL2 7.19E-06 2.03835 LEKR1 0.0004516 2.03877 PTGS2 0.00145367 2.03907 SOX9 0.00163346 2.03909 RPS24 1.29E-05 2.04035 GPR37 0.00095636 2.0406 COBL 5.58E-09 2.04157 AFF4 0.00193191 2.0423 CDKN1C 0.00225879 2.04243 THRB 9.01E-06 2.04377 KRT78 7.64E-06 2.0442 EAF2 0.00041711 2.04506 SNX16 0.0005655 2.04567 TMEM41B 0.00073315 2.04588 DNAJB6 0.00361603 2.04608 PHF17 0.00048036 2.04666 N4BP2L1 0.00880026 2.04708 STMN1 3.20E-06 2.04753 RPS28 1.60E-05 2.049 CALB1 0.00029691 2.05012 TNFSF4 0.00016581 2.05078 TNFSF13B 0.00095106 2.05112 ANKRD10 5.28E-07 2.05275 SCML1 0.00321219 2.05542 EIF5 8.77E-07 2.0559 ZFP3 0.00060504 2.0583 AGPAT9 0.00025141 2.05896 ATF2 8.10E-07 2.05928 TSPYL2 6.74E-06 2.05947 CD1D 6.58E-06 2.06092 KCNMB4 0.0001714 2.06188 HOMER1 3.57E-07 2.06226 HOXB13 0.00192413 2.06257 DUSP14 1.66E-05 2.06273 NUAK1 0.00149931 2.06299 BMP2 0.00150895 2.06422 LIN54 0.00024772 2.06438 CREM 6.20E-05 2.06693 MEF2D 0.0015179 2.06797 CD55 0.0001435 2.06888 TRIM24 0.0004199 2.06922 PLSCR4 0.00045263 2.0703 EIF4A2 4.84E-05 2.07041 CRABP2 1.60E-05 2.07065 PRR19 0.00011732 2.07213 PTP4A1 1.52E-05 2.07309 HIVEP1 0.00510312 2.07363 BCL2L11 0.00133362 2.0758 ATP1B3 4.88E-06 2.07637 BFSP1 1.21E-06 2.07652 LOC388948 3.94E-06 2.07662 FLJ45340 7.42E-07 2.07835 S100A7A 0.00021884 2.0786 C18orf25 0.00175014 2.08031 GJC1 1.66E-05 2.08183 TNFRSF9 3.25E-05 2.08323 CCDC81 0.00016287 2.08565 NCOA3 0.00619342 2.08623 CRABP2 1.45E-06 2.08727 HOXA1 4.37E-05 2.08791 TLE3 0.00270275 2.08882 LRRC19 0.00047836 2.09105 IL19 0.0001814 2.09286 N4BP2L2 0.00016467 2.09361 HIST2H2BF 1.57E-05 2.094 OPRL1 0.00178667 2.09458 TMEM41B 0.00116413 2.09459 USP44 3.21E-06 2.09481 SCML1 5.37E-05 2.09483 DLL1 0.00019799 2.09486 PIP5K1A 7.21E-08 2.09539 FOXO4 1.88E-06 2.09632 IRX2 2.64E-06 2.09686 NPS 5.88E-06 2.09696 GPBP1 0.00116074 2.1008 CEP19 0.00343593 2.10149 KIAA0182 0.00326564 2.10161 TXLNG 0.00027097 2.10168 CHD3 9.73E-05 2.10462 CSRP2 0.00016643 2.10484 MSX1 1.96E-05 2.10643 KLHDC10 2.20E-06 2.10721 CPEB4 0.00793442 2.10968 KANSL1L 0.00073713 2.10981 PLGLB1 0.00471544 2.11038 KITLG 0.00028996 2.11059 NFAT5 3.34E-08 2.11076 ZNF77 1.03E-05 2.11158 DDX5 2.67E-07 2.1128 PARD6G-AS1 0.00019546 2.11394 KCTD10 1.14E-05 2.11462 RSRC2 2.11E-07 2.11507 C21orf91 0.00317147 2.11552 PITX2 0.0002449 2.11578 OSR2 0.00244182 2.11618 IVNS1ABP 9.42E-06 2.11659 TMEM45A 2.42E-05 2.11817 EGFL8 5.93E-06 2.12036 HNRPDL 1.40E-07 2.12051 MBNL2 0.00104378 2.12219 ZNF711 0.0128911 2.12223 BCL9L 0.00334544 2.12285 HSPH1 1.31E-07 2.12398 TIA1 0.00054885 2.12586 ZNF217 0.0007139 2.12593 PAPD7 0.00052124 2.12617 HOXB2 0.00015723 2.12617 MMP20 9.26E-05 2.12622 ZRANB1 3.65E-05 2.12743 TRIM36 0.00176314 2.128 DIP2C 0.00027085 2.1292 ATP1B3 2.86E-08 2.12938 XIST 2.36E-05 2.1307 SLC5A9 0.00605064 2.13331 MAPK9 1.42E-05 2.1354 ANKHD1 5.65E-07 2.13569 LGALSL 0.00085796 2.1383 IRX5 0.00073541 2.13867 BRIP1 0.00092841 2.1389 DNAJB9 0.00092111 2.13922 FAM124A 9.81E-07 2.14065 CPEB4 0.00373393 2.14443 GLUL 8.86E-06 2.14509 ZNF597 0.00018831 2.1452 VAMP1 0.00131568 2.14655 TRIM33 4.96E-06 2.14674 C6orf141 0.00027068 2.14706 HSPB3 0.00043542 2.14709 ACTA1 8.03E-05 2.14833 REL 0.0124658 2.15235 CDC42SE2 0.00248444 2.15573 --- 0.00020834 2.15721 TRIM24 4.32E-05 2.16049 DNAJB4 5.16E-05 2.16239 GSTTP1 0.00024776 2.16255 FAM53A 0.00648102 2.16312 EPB41L5 0.00157217 2.16406 SLC6A13 0.001221 2.16533 RPS2 1.73E-05 2.16535 EIF4A2 2.11E-05 2.16537 GSPT1 4.41E-05 2.17124 H2AFX 9.75E-06 2.17753 TRIM52 0.012705 2.1789 NR1H4 2.85E-06 2.17899 HIST1H2AB 1.48E-05 2.17988 OR2B6 3.23E-06 2.18078 CREBBP 1.88E-06 2.18144 MLL5 1.43E-05 2.18148 DBIL5P 0.00011442 2.18264 KCNJ3 0.00133882 2.18294 NFAT5 0.00707001 2.18298 KLF7 2.46E-05 2.18321 STRN 0.00138396 2.1838 SMURF2 7.49E-06 2.18893 SLC41A1 1.18E-05 2.18918 PELI1 2.72E-08 2.18943 EXT1 9.74E-06 2.18992 CHD4 1.64E-07 2.19491 DCDC2 4.88E-05 2.19515 KISS1 8.66E-07 2.19521 TUBB1 0.00012996 2.19608 DEPTOR 0.00910724 2.19741 CPEB3 1.02E-05 2.19762 TAF4B 0.00208178 2.19966 KIF1B 0.000148 2.19973 LOC10013309 1.40E-06 2.20224 ING1 1.80E-05 2.20391 FAM117B 0.00012728 2.20644 SOAT2 0.00021324 2.20821 YTHDF3 6.93E-06 2.20868 HDAC9 1.09E-05 2.21286 SOX4 4.68E-09 2.21599 ANK3 0.0136605 2.21913 LOC647859 2.72E-05 2.21942 EIF4A3 8.97E-11 2.22083 SHANK2 7.11E-05 2.22382 SOX4 0.00083529 2.22389 SMCHD1 3.94E-06 2.22552 PDK2 8.56E-05 2.22577 SGMS2 0.00185421 2.22752 TFAMP1 0.00012936 2.22755 HIST1H3A 2.06E-06 2.22839 FGFBP2 8.73E-05 2.22875 UBTD2 0.00017567 2.22938 CLOCK 0.00023821 2.2314 TMPRSS9 6.69E-05 2.2316 NR5A2 0.00010895 2.23183 ZKSCAN5 0.00029056 2.23325 ERMAP 6.42E-06 2.23408 ZNF295 1.54E-07 2.23434 C7orf61 4.06E-06 2.23602 ISL1 0.00089457 2.23702 GSK3B 7.12E-06 2.23859 PGAM5 4.33E-05 2.23879 SPATS2L 0.0002001 2.23935 SLC20A1 2.04E-06 2.24175 SPTY2D1 2.47E-08 2.24255 FAM179B 9.39E-05 2.24317 METTL12 2.79E-06 2.24525 KCNQ2 4.48E-05 2.24626 SESN2 0.00036847 2.24784 NR2F2 1.91E-07 2.25152 SORBS1 1.02E-05 2.25359 DST 0.0045593 2.25496 EPB41L5 1.51E-05 2.25814 TAB3 6.62E-05 2.25965 DEPTOR 0.00033866 2.26071 TOB2 0.00463228 2.26094 NFATC2 2.84E-06 2.26176 EHF 1.31E-05 2.26351 ARHGAP5 0.00054047 2.26534 GNRH1 0.00010107 2.2682 TNF 7.25E-06 2.26964 DHDH 0.00013787 2.27031 SLC3A1 1.47E-06 2.27108 SUSD4 2.68E-05 2.27114 PPM1K 2.97E-05 2.27227 CCP110 6.96E-05 2.27437 RSRC2 2.67E-08 2.2772 --- 1.75E-05 2.27944 CNNM4 7.46E-05 2.2817 FAM161A 0.00024502 2.28257 SAP18 2.99E-08 2.28426 RASSF9 0.00226983 2.28434 YPEL2 0.00627612 2.28555 EIF4A1 0.00010034 2.28605 UBL3 0.00293966 2.28688 SRSF6 0.00021864 2.28845 TUBA1A 0.00127164 2.28927 ZFX 0.00754588 2.28933 ZFX 0.00754588 2.28933 TIPARP 0.00017754 2.2897 LOC10012951 0.00012345 2.29109 DDX3X 8.08E-09 2.29616 TUBB2A 2.11E-05 2.29703 C16orf80 4.07E-08 2.29841 ANK3 0.00246671 2.2991 DCAF4L1 0.00028342 2.30103 KLRC1 0.00015021 2.30438 UBL3 3.45E-07 2.30663 YTHDF3 0.00023998 2.30796 ZNF184 0.00037571 2.30817 Mar-03 0.00034773 2.31077 SEC24A 1.26E-05 2.31213 YAP1 2.98E-08 2.31214 CXCL13 4.37E-06 2.31229 PIGR 2.24E-06 2.31351 KISS1 4.63E-08 2.3149 FOSL1 0.00105595 2.32134 CCDC74B 2.90E-05 2.32596 AMD1 0.00045724 2.32619 HSPH1 4.17E-06 2.32772 ITGB1BP1 1.84E-05 2.32792 HSPH1 2.12E-07 2.33031 FAM108B1 1.24E-07 2.33186 EIF4A2 9.71E-05 2.33193 ANGPTL4 1.03E-05 2.33196 DDX3X 2.30E-10 2.33388 OSR2 7.49E-05 2.33405 INTS2 0.00143301 2.33428 LIPG 9.86E-05 2.33468 OSR2 0.0041565 2.33583 SOX4 2.16E-08 2.33708 SESN2 1.50E-05 2.33762 UCP3 2.57E-06 2.33797 USP43 9.07E-05 2.34256 TXLNB 2.19E-05 2.34355 DCUN1D3 0.00096154 2.34469 TP53AIP1 3.32E-05 2.34572 ZSWIM3 1.87E-05 2.34581 HBG1 6.06E-05 2.34744 FRMD4B 3.60E-05 2.34832 PSPC1 7.29E-06 2.34979 FAM46B 4.63E-06 2.34982 SLC25A25 1.36E-05 2.35051 SPAST 0.00389974 2.35155 LOC219347 1.11E-05 2.35338 ANKRD34C 1.01E-06 2.35452 ACBD7 0.00010923 2.35496 THBS1 3.44E-05 2.35693 FAM54A 0.00015461 2.35778 ANP32E 9.79E-06 2.36089 LMLN 1.00E-07 2.36671 RABGEF1 0.00011528 2.37048 CSRP2 0.00043878 2.37056 GTF2IRD2 0.00044682 2.3712 ALB 2.72E-06 2.373 ARHGEF6 3.35E-06 2.37559 TSC22D3 0.00145252 2.37584 TRIM36 0.0010285 2.37611 LINC00597 0.00021091 2.37651 ATP8B2 2.11E-06 2.37869 ING1 4.36E-05 2.37877 ACTC1 9.73E-07 2.3821 NFAT5 1.12E-05 2.38525 HIST2H2AA3 2.92E-07 2.38572 TMEM217 4.00E-05 2.38672 APPBP2 1.64E-07 2.39006 CALB1 0.00167008 2.39019 C3orf58 1.43E-05 2.39099 KCNH2 0.00285231 2.39277 EFNB2 8.45E-08 2.395 RSRC2 1.03E-08 2.39526 LMO4 6.98E-07 2.3957 ZFX 0.00010483 2.39647 NCEH1 1.60E-06 2.39728 ZNF114 6.92E-05 2.39801 HMGCS1 9.26E-06 2.40647 PCDHA3 0.00017535 2.40673 ENPP2 0.00142897 2.40719 PYGM 0.00052323 2.4074 TUFT1 3.81E-05 2.40767 HCLS1 1.34E-05 2.41081 NABP1 0.00013247 2.41082 EZH2 3.38E-05 2.41222 PTMA 5.60E-06 2.41399 ITGB1BP1 7.70E-06 2.4145 EMP1 5.12E-09 2.41749 KDM5B 6.12E-07 2.41861 PIP5K1A 2.57E-08 2.41936 DIAPH2 8.45E-05 2.4194 PTP4A1 1.40E-06 2.42048 SOCS5 0.00026695 2.42283 VEGFA 0.00019821 2.42313 RGL1 1.54E-06 2.42641 HIST1H2BJ 0.00107863 2.42657 FERMT2 0.00018339 2.42895 PARD3 5.67E-05 2.42948 EFNB2 0.00709207 2.43161 NCEH1 0.00324715 2.43632 FOXO1 3.22E-05 2.43648 PELI1 0.00211969 2.43738 LAG3 1.20E-05 2.43848 HIST2H2AA3 1.37E-08 2.43917 EIF4A1 1.04E-06 2.44241 SERPINI1 6.31E-06 2.44432 HIST1H4A 0.00024372 2.44485 CDKL2 0.00015639 2.44488 CHKA 8.56E-05 2.44504 C6orf141 1.85E-08 2.44704 NEDD4L 0.00017344 2.44881 ARNTL 2.56E-05 2.45021 NPC1L1 5.56E-06 2.45348 LRRC8C 9.17E-08 2.45855 ABHD5 3.31E-07 2.46006 PDE2A 1.41E-06 2.46157 SDE2 0.00029807 2.4618 TRIB1 0.00022743 2.46243 GRAMD1B 0.00074653 2.46251 DSTNP2 2.47E-06 2.46551 DSTNP2 2.47E-06 2.46551 TUBB2A 1.07E-05 2.46666 TTC33 0.00010346 2.47068 TPST1 0.00014481 2.47074 FN1 0.00107947 2.47126 CA2 0.00044242 2.47687 ALLC 8.19E-05 2.477 PTP4A1 1.21E-08 2.48661 NEDD4L 0.00040252 2.48945 MRC2 1.15E-06 2.4903 METTL20 0.00031025 2.4903 RAB33A 2.65E-08 2.49559 VEGFA 1.46E-05 2.49629 TSC22D3 4.41E-07 2.49858 EMP1 0.00086125 2.50405 GPR75 0.00010463 2.50476 CHM 0.00077016 2.50522 NEK8 1.05E-08 2.50831 KDM6A 1.08E-05 2.5142 LGALSL 8.61E-08 2.51482 TFAMP1 2.07E-05 2.51958 SLC17A8 0.0001913 2.52062 TUFT1 0.00028908 2.52322 PTGS2 0.0122761 2.52525 CHD4 0.00464198 2.52593 CCNL1 0.00259443 2.52625 PRR20A 2.16E-05 2.52738 TFAP2C 3.05E-06 2.531 C16orf80 4.59E-06 2.53784 STARD4 1.24E-06 2.53949 REM2 3.64E-06 2.54098 TESK2 2.01E-05 2.54176 ZNF750 6.58E-05 2.54487 H3F3A 1.89E-05 2.54603 PTRF 1.78E-07 2.54679 CD55 4.24E-06 2.54798 ASXL1 8.75E-08 2.54906 INHBB 5.65E-05 2.55195 APLNR 3.53E-06 2.55488 DDX26B 0.0001364 2.55491 PCDHA1 4.67E-05 2.55496 CYP2B7P1 9.25E-05 2.55672 ANKRD34C 2.10E-05 2.56602 ZNF10 1.50E-06 2.56843 CDKN1C 0.00068405 2.57171 GPLD1 3.08E-05 2.57244 POLG2 0.000596 2.57317 PTGER3 5.45E-06 2.57386 RASL12 4.07E-05 2.57404 --- 0.0127856 2.57466 SRGAP2 2.61E-06 2.57504 NEDD4L 2.84E-06 2.57762 ANP32E 5.27E-05 2.57774 TRPC1 0.00031008 2.58009 RDH10 6.79E-05 2.58208 HIST1H2BD 0.00024851 2.58322 DNAJB6 1.22E-07 2.58562 DNAJB6 1.22E-07 2.58562 METTL20 1.61E-05 2.58868 HIST1H2AC 8.90E-05 2.58906 CCSAP 9.89E-05 2.58951 NR5A2 2.53E-05 2.58996 SEPP1 4.53E-06 2.59076 SERTAD2 0.00175861 2.59211 ELK4 2.45E-05 2.59391 STK17B 1.56E-05 2.59501 SAP18 4.64E-07 2.59513 C20orf111 4.09E-08 2.59567 CWC25 2.65E-06 2.60441 HSPA8 3.65E-05 2.60489 ACER2 4.77E-05 2.61001 SOCS5 1.78E-06 2.612 REL 0.00491721 2.61526 CCNG2 2.02E-06 2.61647 THAP2 4.77E-05 2.61978 MTSS1 1.34E-05 2.62438 SSBP3 0.00233 2.62549 SNX5 2.46E-06 2.62804 GDF11 0.00050451 2.62973 RPL27A 5.35E-05 2.63291 --- 2.76E-06 2.63553 HEG1 4.97E-06 2.6469 KIF3A 0.00150732 2.64708 CLK1 0.00025856 2.65197 SEMA4A 2.59E-06 2.65554 IER5 1.48E-06 2.65622 CHRFAM7A 1.48E-05 2.65747 LOC392288 4.85E-07 2.66418 CDKN2B 0.00037418 2.66418 UBXN7 1.78E-07 2.66428 CDRT1 6.17E-08 2.66832 NEDD4L 5.55E-07 2.67246 KLF10 0.00112563 2.67388 GPR68 5.16E-07 2.67738 FILIP1L 0.00123747 2.67989 ENPP2 0.00025472 2.68107 DDX3X 1.68E-07 2.68174 CMYA5 0.00014115 2.68197 HHEX 0.00020588 2.68333 RYBP 1.38E-05 2.68389 ELL2 4.87E-07 2.68575 GAB1 0.0020552 2.68802 IKZF4 0.00025812 2.69454 FPR2 2.21E-05 2.69456 IGF2BP2 0.00189659 2.70084 FRMD4B 1.23E-05 2.70344 SGK1 0.00028012 2.70421 ITGA10 2.56E-06 2.70843 GTF2IRD2 0.00018575 2.70982 NADKD1 5.74E-06 2.71281 BTG1 4.00E-07 2.71433 POU6F1 3.83E-05 2.71745 HIST1H2AB 6.72E-07 2.71847 SEPP1 4.71E-07 2.72018 ANKRD12 1.49E-07 2.72033 TUBB3 1.82E-06 2.72055 CRKL 1.99E-05 2.72126 PELI1 5.38E-05 2.72139 DUSP10 0.00034485 2.72318 BRI3 5.16E-06 2.72386 ANP32E 4.37E-06 2.72431 YPEL5 4.18E-10 2.72458 METTL7A 0.00012053 2.72577 C1orf114 6.67E-05 2.72629 EFNB2 0.00518327 2.72943 MCL1 8.23E-06 2.73315 IL20 2.17E-07 2.73838 TMC5 4.95E-05 2.73969 RBM33 5.96E-05 2.74783 TMPRSS2 0.0002555 2.76235 TSC22D2 0.00120207 2.76254 PELI1 0.00391503 2.76289 PLK2 0.00026466 2.76319 POU2F2 7.05E-07 2.76397 SMAD7 9.14E-05 2.76397 SLC45A4 0.00010435 2.76403 LOC10013425 0.00013027 2.76808 RSRC2 0.00011176 2.77211 PCF11 1.91E-07 2.77944 ID2 9.98E-11 2.78024 SIRT1 0.00218786 2.78538 DNAJB4 2.02E-06 2.78561 YPEL5 1.38E-09 2.78591 IRS2 1.94E-05 2.78975 GLCCI1 6.67E-07 2.79005 EIF4A2 2.62E-07 2.79061 EREG 3.98E-05 2.79932 DDX3X 1.08E-06 2.8049 HSPH1 0.00012239 2.80836 TOB1 0.00022371 2.8091 AREG 4.07E-08 2.81389 MAP2K6 0.00068915 2.81416 KIF27 1.32E-06 2.82077 HNRNPL 7.18E-06 2.82569 NADKD1 4.82E-05 2.82606 ZNF75A 1.68E-06 2.82784 RASL11A 0.00963792 2.82795 FRZB 8.16E-06 2.82809 NSUN7 1.25E-05 2.83426 STMN1 1.74E-08 2.83438 LHX4 1.63E-06 2.83503 ATF7IP2 2.02E-05 2.83565 CELF2 7.98E-08 2.841 PDE4C 1.92E-05 2.84375 KIF1B 2.40E-07 2.84972 RSRC2 0.00023081 2.84982 HIVEP1 7.25E-06 2.85134 INTS6 8.11E-05 2.85903 TNFRSF10D 1.43E-05 2.86009 HIPK3 1.07E-05 2.86178 ZNF34 3.60E-05 2.87028 COL21A1 7.01E-05 2.87191 ZNF711 7.98E-09 2.87243 HMP19 0.00011583 2.87318 KLF17 5.46E-05 2.87402 CSRNP1 3.03E-06 2.87585 PELI2 9.21E-06 2.87835 LOC10050640 2.41E-05 2.87918 MEF2D 0.00016264 2.88081 FAM108B1 0.0008891 2.88207 AMD1 3.28E-06 2.88463 SERTAD2 0.00015213 2.88669 RSRC2 1.66E-06 2.89182 --- 9.32E-06 2.89208 IDI2-AS1 3.82E-07 2.9069 FOXC1 1.94E-06 2.90701 KCNJ12 6.56E-08 2.91086 CPEB4 0.00268267 2.91361 NR4A3 0.00153013 2.91896 RYBP 4.81E-06 2.92333 TIPARP 0.0112692 2.92386 PNRC1 1.70E-09 2.93044 NKX3-1 1.47E-05 2.93065 ENDOU 3.97E-06 2.93207 CCDC62 7.56E-08 2.93773 PLK2 5.52E-08 2.94803 BTG1 4.57E-08 2.94864 ADM 2.18E-08 2.94888 PATL1 1.14E-06 2.9519 LOC10028778 8.38E-06 2.95309 USP38 1.74E-09 2.9584 CCNG2 0.00259038 2.9625 DAPK1 0.00054708 2.96304 DNAH12 5.77E-07 2.97043 HIST1H2BD 7.71E-08 2.97185 EREG 0.0006991 2.97335 BCL6 4.79E-05 2.97837 IRS2 1.36E-07 2.97903 IDI1 1.44E-06 2.98168 EGR2 2.56E-07 2.98333 KDM6A 6.20E-08 2.98452 BPGM 9.79E-06 2.98971 WDR26 2.54E-08 2.99156 PTGER3 0.00034762 2.99322 LONRF3 1.12E-08 3.0022 NADKD1 5.72E-05 3.00664 MAP1LC3B 1.06E-08 3.00691 WDR47 2.48E-06 3.00877 ZFP36 2.95E-06 3.01202 KLF2 0.00071567 3.01348 RBM15 6.81E-05 3.01688 PDE5A 4.53E-05 3.01949 MAP3K8 0.00013306 3.02045 WSB1 9.13E-05 3.02344 ANP32E 2.83E-07 3.02528 RHEBL1 0.00023685 3.02606 DNAJA1 1.63E-09 3.02738 TRA2B 3.07E-05 3.02808 ZKSCAN5 7.47E-05 3.03415 ETS1 6.74E-07 3.03448 SIRT1 4.79E-09 3.0399 LMO4 2.36E-05 3.0415 EPHA2 0.00034892 3.05039 KLF10 0.0006954 3.05193 SLC25A25 1.66E-05 3.05367 CCNE1 2.15E-07 3.06427 SPRY4 4.74E-06 3.06953 KL 1.69E-06 3.0743 FOSL1 4.16E-05 3.07737 PPP1R36 7.59E-06 3.07785 RERE 1.92E-05 3.079 AMD1 1.37E-05 3.09817 HIST1H2BD 8.30E-06 3.1002 BCL6 5.15E-08 3.10035 RICTOR 2.26E-06 3.10147 ABHD5 0.00012457 3.10291 FBXO48 0.00014438 3.10691 RYBP 1.67E-06 3.11158 ZNF75A 4.11E-06 3.11839 GAB1 9.32E-06 3.11945 LNP1 1.12E-06 3.12502 IL1R1 3.42E-06 3.1262 FRMD4B 0.00023248 3.13515 SERPINE1 0.00012107 3.13567 --- 5.57E-10 3.14762 GKAP1 0.00022654 3.14775 CYP2U1 8.78E-08 3.15398 TM2D2 4.78E-07 3.17048 FOSL1 0.0118678 3.17069 TYW1B 3.21E-07 3.17514 HSPH1 0.00025389 3.17594 IKZF5 1.36E-05 3.1776 MMP25 0.00028337 3.17889 PARD6B 4.16E-05 3.18747 SLC19A3 0.00292897 3.18756 PCF11 0.00021956 3.19187 TUBB8 0.00104537 3.19556 DDX3X 4.58E-05 3.20056 FOXD4 1.07E-08 3.20885 LRRC17 9.20E-06 3.20998 TAF7L 3.22E-05 3.21076 SPTY2D1 1.78E-05 3.22075 CCNE2 8.48E-05 3.22209 JMJD6 7.32E-06 3.22508 TRIM31 5.83E-10 3.22531 HES1 1.33E-07 3.22553 TSPAN12 2.66E-06 3.24252 IDI1 9.30E-06 3.25214 PAPD5 9.28E-06 3.25937 BPGM 9.16E-06 3.26297 SDC4 1.20E-08 3.26775 CCDC108 4.00E-08 3.26918 AMD1 1.71E-05 3.27684 ADM 9.68E-07 3.27761 FOXC1 0.00041141 3.27947 GAB1 0.00089822 3.28108 DNAJA1 4.63E-10 3.28733 NR4A1 0.00055973 3.28903 DAND5 1.33E-05 3.30229 ISL1 0.00129914 3.30436 HIVEP2 2.95E-09 3.3058 TRIM31 6.03E-08 3.30699 PNRC1 3.98E-06 3.30738 FAM101B 9.94E-07 3.31182 NFAT5 0.00209621 3.31232 GRAMD2 3.71E-08 3.32067 HSPA1L 6.54E-07 3.32107 HNRPDL 4.85E-07 3.32463 KCNJ1 7.66E-05 3.33349 CD14 0.00046604 3.33627 NR5A2 1.08E-05 3.33754 CDO1 4.83E-06 3.33824 MCL1 7.30E-06 3.33948 RBM47 3.79E-05 3.33952 AMD1 1.10E-05 3.34167 C16orf72 2.50E-05 3.34223 CLK4 0.00026631 3.34484 PCDHA1 2.12E-05 3.34812 ASXL1 7.90E-06 3.34887 EMP1 2.62E-06 3.35338 INTS6 1.53E-06 3.3548 ITPR1 7.94E-06 3.35581 CSPG5 7.99E-06 3.35657 CXCL1 2.98E-06 3.35753 POU2F2 2.76E-07 3.3629 RDH10 3.44E-06 3.36684 PCDHA1 1.96E-05 3.36962 ANKRD45 1.80E-10 3.37638 NOP2 1.12E-06 3.38013 ARIH1 9.53E-09 3.39032 TRA2B 0.00013468 3.39229 RGL1 0.00012614 3.39394 LARP6 2.46E-06 3.3952 ERO1LB 4.09E-05 3.39784 SERTAD3 6.07E-05 3.41568 LARP6 3.18E-05 3.4177 INTS2 1.37E-09 3.42374 CHN1 7.13E-06 3.42466 CHD1 3.11E-09 3.43256 SIRT4 3.57E-08 3.43763 FGFBP1 4.68E-07 3.44475 PTGS2 0.00256951 3.46126 PLK2 0.00019424 3.46184 ZBTB10 9.02E-05 3.46274 BCL6 1.43E-05 3.46798 AMD1 6.94E-06 3.4742 ZFAND2A 9.46E-11 3.47676 PCDHA1 3.15E-06 3.48131 GLB1L 1.25E-05 3.48375 BTK 1.02E-05 3.49167 NOP2 1.36E-07 3.506 RMND5A 7.60E-09 3.51226 HIST1H2BC 4.44E-06 3.52871 FBXO11 4.67E-05 3.53923 CYP2C9 9.31E-05 3.54426 KLHL24 4.28E-05 3.54672 ARC 1.31E-05 3.55815 ZNF503 4.00E-05 3.55987 OVOL1 1.56E-05 3.56409 ELL2 5.61E-06 3.57155 NEDD9 3.00E-05 3.57192 RYBP 8.00E-05 3.57489 TDRD6 3.08E-07 3.58588 SOS1 7.85E-10 3.58716 ZSWIM6 0.00022661 3.59018 PLGLA 0.00026532 3.59356 GADD45B 3.85E-06 3.59551 FAM46C 5.73E-05 3.59706 ZNF425 6.39E-05 3.60934 SLC5A5 0.00029351 3.61181 --- 6.73E-05 3.61599 ANKRD34C 2.99E-06 3.61799 MAP7D2 5.55E-06 3.62941 RPL13P5 0.00022623 3.64782 HIST1H2BC 1.43E-06 3.6495 USP12 1.27E-05 3.65687 HIST1H2BC 4.41E-05 3.66016 TNFSF15 2.96E-06 3.66181 GALNT5 1.58E-08 3.6639 CYP2U1 2.91E-08 3.67468 MEX3B 1.88E-05 3.68629 ZSWIM6 4.57E-08 3.68796 NOP2 6.53E-07 3.70869 KIF1B 1.66E-09 3.73078 DNAJA1 1.98E-10 3.73849 ADM 1.68E-09 3.77001 PTRF 0.00020493 3.78086 RGS4 2.74E-07 3.7875 DNAJA1 2.45E-09 3.79787 CENPL 2.17E-06 3.79897 PPP1R10 9.51E-05 3.80293 CXCL1 7.52E-07 3.80424 DDX3X 1.93E-06 3.82413 CD200 6.74E-06 3.83252 AKAP9 1.35E-06 3.853 EFNB2 8.81E-05 3.86091 C1orf114 6.14E-06 3.8611 C16orf72 6.24E-08 3.8701 ABHD5 1.03E-05 3.8754 SOCS3 2.19E-07 3.89079 FOSL1 0.00097264 3.90052 ZFAND5 2.12E-09 3.90364 --- 7.47E-07 3.9089 LOC10050944 3.16E-05 3.91573 SLA 2.92E-10 3.91719 RDH10 1.55E-07 3.9206 TP53AIP1 7.14E-07 3.9218 ABHD5 5.43E-06 3.92588 SLC25A44 1.33E-07 3.93941 CCP110 6.91E-08 3.94945 SGK1 4.40E-05 3.94968 CDKN2AIP 8.25E-07 3.95415 HIST1H3A 9.04E-07 3.9598 HNRPDL 1.39E-07 3.96333 TUBB8 3.54E-05 3.98343 ARL5B 0.00031938 3.98606 LOC10050551 3.35E-07 3.98809 LAG3 3.27E-10 3.98924 SGK1 5.60E-05 3.996 ATF3 3.36E-05 4.00285 MYLIP 5.19E-09 4.02328 ID2 5.74E-09 4.0259 TWSG1 2.12E-07 4.02619 CPED1 7.19E-05 4.03425 MMP10 0.0015616 4.037 DDIT3 1.25E-08 4.04345 C1orf63 2.69E-09 4.04962 DNAJB1 2.17E-05 4.05923 ADRB2 4.91E-05 4.06046 SNORA14B 2.14E-05 4.08591 PIGR 1.36E-07 4.08718 PTP4A1 3.69E-07 4.08933 LDHC 2.30E-05 4.09736 ACVR1C 4.22E-06 4.0978 CCNE2 1.22E-05 4.09991 HIST1H2BJ 1.12E-06 4.10058 SUCNR1 6.89E-06 4.11878 ZBTB1 2.43E-05 4.12337 EPHA2 0.00019963 4.13804 POU2F2 3.44E-06 4.15489 CPEB2 1.45E-06 4.15524 ITPR1 6.48E-06 4.15788 DNAJB9 0.00105917 4.16054 ID3 5.77E-08 4.18524 MICB 1.01E-07 4.19322 ATF3 2.52E-07 4.19527 ZNF75A 2.56E-06 4.19892 KCNN3 6.44E-10 4.20402 MYLIP 6.62E-05 4.20571 CCP110 1.86E-05 4.22068 SNX31 1.18E-09 4.23966 SERTAD3 9.73E-06 4.25052 DDX3X 0.00014607 4.26351 BAG3 1.59E-07 4.2661 IDI2 1.59E-08 4.26812 MAP3K8 2.54E-07 4.27857 AHR 5.00E-08 4.28813 PPP1R10 1.92E-06 4.29286 CCNYL1 1.62E-05 4.29572 DEFB135 8.27E-05 4.29806 SGK1 4.47E-05 4.33087 ITGB8 0.00011527 4.3405 AVIL 7.54E-05 4.35763 DNAJA1 7.55E-10 4.35989 DDIT3 1.32E-08 4.36297 SPDYA 5.64E-10 4.36716 TMPRSS12 3.48E-06 4.38073 HIVEP2 8.42E-09 4.38861 FILIP1L 5.33E-06 4.39191 BCL6 1.32E-06 4.40026 CCL4 8.08E-06 4.4011 BRD2 0.00011583 4.41242 DUSP5 0.00025772 4.41337 CDKN1C 6.92E-05 4.44952 MXRA7 3.39E-09 4.46718 MYLIP 1.36E-08 4.4846 LINC00341 1.08E-07 4.48548 TSC22D2 1.06E-07 4.51535 ABHD5 4.22E-05 4.51789 NUFIP2 0.00095881 4.54744 C1orf63 3.91E-09 4.54981 USP12 2.01E-07 4.55819 BRD2 5.69E-08 4.56563 CENPL 1.87E-07 4.58041 GAL 3.12E-08 4.58658 FOS 1.36E-06 4.59443 PCDHA2 5.59E-07 4.61112 CXCR4 2.61E-06 4.61896 PPP1R1C 0.00041442 4.63011 FOXD4L2 4.47E-09 4.63092 JUNB 2.38E-07 4.63614 PTGER4 0.00017873 4.65404 ELL2 8.27E-07 4.65435 INTS6 1.36E-07 4.66014 FOXA3 0.00010242 4.66082 LOC728741 1.47E-05 4.66841 SORBS1 2.38E-06 4.68225 NR4A1 0.00075632 4.70405 ING1 8.57E-10 4.71732 EML6 1.28E-05 4.71952 CYLD 1.07E-08 4.72655 CCNE1 1.60E-10 4.72726 CITED2 0.00087563 4.73609 SLC38A2 1.24E-06 4.7465 ARG2 3.43E-06 4.7624 LEP 1.91E-06 4.77708 --- 2.87E-07 4.77936 KRT12 4.71E-07 4.78245 CYP2U1 1.12E-06 4.81461 DUSP1 1.70E-08 4.81482 KCNQ1OT1 5.83E-06 4.86585 EGR1 1.11E-07 4.86751 POU2F2 2.45E-08 4.886 ZNF304 6.69E-05 4.89362 TSC22D2 7.51E-08 4.90216 DDX3X 0.00198344 4.92161 ARL5B 7.96E-05 4.95742 RND1 7.89E-06 4.97554 DIRAS3 3.10E-06 4.98563 PMAIP1 1.17E-07 4.99792 ERO1LB 4.39E-06 5.01646 LINC00312 6.51E-08 5.01748 TRNAU1AP 1.18E-07 5.02204 ZNF295 1.96E-06 5.02493 FOXD4L1 8.74E-09 5.08752 FKBP14 1.25E-07 5.116 TSC22D2 1.62E-07 5.16767 RGS16 2.97E-08 5.17871 DNAJB1 1.98E-06 5.1962 CD14 6.78E-06 5.19854 GADD45B 4.99E-07 5.20986 PTP4A1 1.08E-06 5.21374 INTS6 4.86E-08 5.23294 FKBP14 1.54E-06 5.25458 BCL2L11 6.79E-10 5.27528 FKBP14 2.32E-07 5.27985 RND3 2.74E-10 5.28762 IER2 3.42E-06 5.29109 ID3 1.53E-07 5.30064 RGS16 9.32E-08 5.31746 ELOVL3 5.07E-09 5.32898 HEG1 1.50E-07 5.35782 SLC6A13 4.82E-06 5.3879 USP38 1.58E-07 5.39395 KLF4 9.73E-10 5.40935 HIST1H2BK 4.11E-10 5.41029 SLC30A1 8.48E-11 5.41096 KRT4 1.86E-08 5.42644 SLC38A2 3.40E-06 5.47778 ZFAND5 1.47E-09 5.49015 HIST1H2BN 1.36E-05 5.49776 GADD45A 1.90E-06 5.5013 CYP2U1 2.28E-06 5.50875 JUN 2.69E-06 5.54473 LINC00281 2.29E-08 5.59626 SLC7A9 3.07E-08 5.64506 DUSP10 2.32E-08 5.67908 DDX26B 1.27E-06 5.67969 ARL5B 8.63E-06 5.68068 HSPA2 2.05E-07 5.69054 FOS 2.19E-07 5.73563 MED26 1.77E-07 5.73623 CXCR4 4.34E-06 5.75108 PMAIP1 1.74E-07 5.76175 IQCJ-SCHIP1 2.28E-06 5.77008 CCNYL1 2.32E-09 5.81471 CDKN2B 1.44E-06 5.84588 SPDYE6 2.16E-07 5.8689 NUFIP2 5.81E-09 5.87513 PDK4 5.28E-06 5.87587 GIMAP8 1.26E-05 5.88874 ARG2 3.45E-06 5.89484 ETS1 2.50E-06 5.91516 PIK3R3 5.98E-08 5.9226 NUFIP2 2.88E-08 5.93773 FAM22D 9.32E-06 5.94748 CXCL13 2.20E-08 5.98744 FLNC 6.17E-07 5.98901 PDE6C 5.23E-05 5.99969 RGS16 1.49E-06 6.0824 CXCR4 1.79E-06 6.08872 CATSPERG 5.27E-09 6.10185 RBBP6 1.44E-06 6.12056 TSC22D2 1.60E-09 6.12707 C3orf58 8.08E-06 6.13754 NR4A1 0.00207226 6.19017 ZC3H12A 1.66E-08 6.25166 KLF4 2.50E-09 6.27205 SLC38A2 7.76E-06 6.28443 FOS 4.56E-07 6.28745 AADAC 5.69E-06 6.29557 CXCR4 3.17E-06 6.29789 EIF4E 4.50E-06 6.31415 NR4A1 0.0036032 6.34879 CDKN1A 4.48E-06 6.35354 TNFSF11 4.65E-06 6.37761 RBBP6 3.75E-07 6.38147 FOS 5.74E-08 6.39402 GATA2 5.03E-07 6.43321 RND3 1.21E-08 6.46792 ZNF34 1.58E-06 6.46809 BIRC3 3.02E-08 6.47247 GPR83 8.94E-08 6.4848 TSC22D2 5.35E-10 6.58311 C1orf114 2.94E-06 6.59272 C4orf47 3.81E-08 6.59602 FLJ20518 2.55E-10 6.63464 ELL2 5.02E-08 6.68399 RBBP6 4.15E-09 6.69479 JUN 6.65E-07 6.7393 RASD1 6.90E-08 6.74689 DUSP1 4.67E-07 6.75781 ZNF439 2.08E-05 6.79799 OVOL1 1.82E-05 6.79962 LDHAL6B 5.89E-09 6.80785 SERTAD1 8.18E-05 6.83342 SPDYE1 5.07E-05 6.83908 RND1 1.94E-06 6.89365 SORBS1 1.66E-07 6.90043 DUSP10 6.74E-07 6.92868 RTN4 4.15E-05 6.94405 EREG 4.72E-05 6.96493 BAMBI 1.31E-08 6.97869 NUFIP2 1.39E-06 7.02403 DDR2 4.41E-08 7.06745 KBTBD8 9.53E-06 7.07328 BCL2L11 4.44E-05 7.09389 OVOL1 0.00022931 7.14189 ATF3 2.67E-05 7.14618 EIF5 1.81E-07 7.21552 ABHD5 1.53E-07 7.2335 ELL2 1.43E-06 7.25722 CDKN1C 2.51E-07 7.26254 DNAJB1 7.41E-08 7.27927 CTGF 2.24E-07 7.31184 JUN 5.97E-09 7.32383 IL11 1.74E-11 7.36829 FOXD4 1.54E-08 7.40699 KRT4 1.58E-10 7.42905 CYP2U1 1.32E-05 7.45423 GATA2 1.22E-09 7.51396 CDK5R1 2.33E-06 7.5148 FAM117A 4.29E-09 7.60863 ITGAM 5.14E-07 7.63482 RAB30 8.81E-07 7.78384 DDR2 1.22E-08 7.78993 ING1 7.72E-11 7.79071 CBX4 1.75E-08 8.00938 THBS1 1.42E-05 8.10222 CD83 8.16E-06 8.13676 BIRC3 1.70E-07 8.17865 JUN 1.04E-07 8.19457 HEXIM1 4.67E-08 8.28081 ARL14 6.44E-08 8.33626 CITED2 2.65E-08 8.3403 NXF1 1.18E-12 8.34477 S100A7 4.60E-10 8.39374 TAF7L 2.69E-08 8.41115 EREG 3.95E-11 8.45092 C1orf63 1.25E-07 8.613 RND3 2.25E-07 8.62292 SLA 3.87E-07 8.67789 DDR2 6.90E-09 8.69464 SNX31 5.15E-08 8.71693 KLHL15 1.92E-08 8.77879 DUSP10 1.96E-08 8.83505 FILIP1L 9.09E-07 8.87299 HIST1H2AG 1.01E-05 8.95799 CXCL2 1.54E-09 8.96023 TUBB7P 9.79E-09 9.00049 BIRC3 4.48E-08 9.04034 MGARP 4.26E-09 9.31254 CBX4 5.46E-11 9.32961 TIGD3 2.05E-08 9.41225 CDKN1A 2.17E-06 9.45328 ASXL1 2.19E-08 9.48245 EGR1 1.31E-05 9.49892 IER3 5.37E-07 9.53314 NXF1 7.24E-07 9.63628 THBS1 6.28E-08 9.84035 PPM1D 3.26E-07 9.86416 ARL4D 3.28E-06 10.0151 HEY1 9.17E-11 10.019 PPM1D 5.10E-11 10.2451 PCDHA1 2.68E-06 10.3765 BTG1 2.31E-07 10.5433 HEXIM1 1.70E-07 10.6045 CPEB2 3.99E-09 10.6127 CCL20 7.86E-07 10.6149 TNFAIP3 1.12E-08 10.6321 PPM1D 7.20E-09 10.6641 CRTAM 1.23E-07 10.6777 RAB30 1.70E-07 10.7082 TRIM69 2.21E-09 10.7109 SIX4 4.16E-07 10.8361 EID3 2.83E-07 10.8676 SLC16A10 5.44E-08 10.8828 NUFIP2 1.64E-10 10.9224 NR4A3 3.07E-07 11.0912 ID3 8.88E-11 11.178 LOC729974 9.08E-09 11.1787 CTGF 1.94E-09 11.1792 HIST1H2AG 1.69E-06 11.285 FGF18 2.66E-10 11.4018 KCNE4 4.42E-08 11.4918 HEXIM1 9.75E-08 11.8412 MYBL1 2.08E-07 11.9419 MEX3B 3.38E-09 11.9838 GDF9 9.75E-08 12.0759 MIR22 1.06E-09 12.1108 NR4A2 4.05E-08 12.2219 GADD45G 2.05E-09 12.5177 MCL1 8.66E-07 12.7442 SKIL 1.13E-07 13.4267 TNFAIP3 3.99E-09 13.4811 SKIL 5.39E-08 13.5043 CTGF 5.68E-08 13.7229 NFKBIA 5.55E-08 13.8585 PPM1D 4.00E-10 13.8794 MAFB 1.10E-08 14.0078 CYR61 2.95E-10 14.6192 S100A7 4.96E-10 14.647 HEXIM1 3.29E-11 14.6862 PPP1R15A 1.68E-11 14.8486 NFKBIA 7.21E-08 14.8488 ARL4D 3.55E-10 14.9152 HEXIM1 8.23E-11 15.3948 NFKBIA 5.98E-08 15.7358 CSRNP1 0.00043559 16.0455 SKIL 6.40E-08 16.1509 MAFB 4.16E-08 16.4181 HBEGF 9.64E-09 16.6126 IL8 7.65E-06 17.6712 NR4A1 0.00088644 17.6873 SNAI1 1.42E-11 18.1529 IL8 2.27E-09 18.7092 IL20 5.84E-09 18.7227 NR4A2 3.58E-09 18.7743 ETS1 3.79E-07 18.8318 FPR2 4.97E-08 18.8332 DDR2 8.47E-10 19.165 EGR3 5.55E-05 19.8129 C7orf53 9.03E-11 20.1712 MAFB 4.67E-09 20.727 IL8 4.34E-06 21.8904 ITGAM 9.53E-08 22.1896 GADD45B 3.91E-10 22.2414 HBEGF 3.64E-10 22.43 CD55 1.23E-08 23.2795 DDR2 1.57E-10 23.4443 ATF3 7.70E-05 23.4614 CXCL2 2.09E-08 23.4928 C1S 6.06E-10 23.8958 NR4A2 5.18E-10 23.9588 GADD45B 5.08E-12 24.2757 CXCL1 1.62E-08 25.2081 CYR61 4.93E-11 27.5999 THBS1 1.75E-08 28.6798 EGR1 1.71E-07 30.042 EGR1 5.11E-08 32.024 MIR22 5.35E-10 32.078 MIR22 2.83E-10 34.5238 FOSB 6.22E-07 34.9274 MCL1 4.53E-08 37.0463 MIR22 2.12E-10 44.191 DLX2 6.46E-11 46.7135 EGR1 3.06E-08 46.9231 FAM171B 1.85E-11 51.4024 EGR1 1.34E-08 53.982 MIR22 3.22E-09 56.7149 CXCL3 3.07E-10 56.7986 GEM 1.04E-11 59.2649 EGR2 7.99E-07 62.5398 FAM46C 7.20E-12 77.2194 ERVK3-2 2.08E-11 83.2066 CXCL3 8.12E-11 93.0077 FAM46C 2.14E-10 112.507 Gene Symbol p-value(PCon FC GAvsPCon MARS2 3.45E-09 -32.9629 PI3 5.41E-09 -27.1536 ZNF226 1.40E-12 -25.7905 MIR17HG 3.02E-10 -22.1972 C15orf52 8.30E-08 -16.9552 SLC7A11 7.86E-05 -15.8187 ADAT2 8.35E-11 -15.7057 SPRR1B 1.91E-11 -15.6558 NDC80 2.19E-09 -14.9527 FUT4 2.93E-09 -14.5861 LCMT2 1.36E-09 -14.3122 SLC7A11 7.16E-05 -13.8079 HNRNPA2B1 6.53E-11 -13.5647 MBD4 2.16E-06 -12.9178 MIR17HG 2.21E-09 -11.6089 ZNF772 4.23E-08 -11.4241 TNFAIP2 1.23E-08 -11.4084 TTC14 8.12E-09 -11.2931 SASS6 1.42E-08 -11.2317 PNISR 3.73E-09 -11.0888 NSUN5P1 9.06E-09 -10.6218 PHLDB2 0.0018877 -10.4433 ANKRD49 3.87E-09 -10.4227 EXOC8 1.48E-07 -9.98962 MMP1 0.00055974 -9.94326 PVRL4 3.84E-08 -9.79162 SFT2D3 8.50E-11 -9.44921 UTP15 1.59E-07 -9.31861 UMPS 7.28E-13 -9.277 GEN1 1.21E-07 -8.94402 NSUN5P2 1.15E-08 -8.79371 UTP15 1.12E-06 -8.71336 KBTBD6 4.69E-06 -8.68425 ZNF540 4.20E-07 -8.60629 TRIM29 1.83E-11 -8.55268 RAB7B 1.63E-10 -8.47711 CAPN8 8.71E-10 -8.47446 SPP1 0.00074065 -8.4682 TRIM29 1.55E-09 -8.45884 CREB5 5.33E-08 -8.38334 PIF1 5.36E-07 -8.28069 PTPRK 3.51E-06 -7.92678 HPS6 9.73E-12 -7.90401 UTP3 3.85E-09 -7.88773 IFNGR1 1.16E-07 -7.85711 AGPAT5 4.92E-07 -7.81883 ZNF564 2.30E-07 -7.77503 POLQ 8.50E-08 -7.75853 CCNJ 4.53E-07 -7.7474 TRMT13 1.85E-05 -7.70358 SERPINB5 1.65E-06 -7.68535 KIAA0040 3.29E-08 -7.64444 TMEM177 2.14E-08 -7.56815 CDC7 2.45E-09 -7.5126 SEMA3A 8.51E-05 -7.50415 UBASH3B 0.00010815 -7.39821 NSUN5P1 2.59E-08 -7.37015 DEM1 1.14E-09 -7.35921 NR2C1 2.25E-07 -7.34961 ZNF121 1.18E-08 -7.33364 NSD1 1.90E-08 -7.22227 GPATCH2 8.22E-09 -7.21179 COIL 5.27E-11 -7.20227 MAK16 2.19E-08 -7.19722 PPP1R8 4.54E-06 -7.09084 NSUN5P1 6.94E-08 -7.02049 CKAP2L 9.18E-09 -6.97401 INADL 3.92E-07 -6.95346 TRMT13 3.05E-05 -6.90542 SERPINB8 1.23E-08 -6.90443 SERPINB8 6.41E-08 -6.89833 CHAC2 9.15E-07 -6.76891 TRIM32 1.54E-09 -6.75561 CSTF3 3.99E-10 -6.75414 SRSF1 1.57E-06 -6.74809 IFIH1 7.49E-07 -6.70846 LTB 1.82E-06 -6.65016 UTP3 3.80E-10 -6.62172 CSTF3 3.93E-08 -6.6121 SPC25 5.15E-06 -6.60716 NSD1 3.94E-08 -6.54096 RBM33 6.47E-12 -6.50565 C15orf39 1.80E-10 -6.50101 METTL18 1.91E-07 -6.48293 XKRX 1.51E-06 -6.46466 FIGNL1 6.76E-09 -6.41706 ZNF232 3.96E-10 -6.41631 ZNF232 3.38E-10 -6.4121 KIAA0754 1.77E-10 -6.40532 ANKRD20A5P 6.53E-08 -6.39753 ZNF572 2.46E-07 -6.38285 KTI12 3.13E-08 -6.37394 HMGA2 1.38E-05 -6.34167 FAM178A 1.13E-07 -6.33945 SPRR1A 2.60E-07 -6.33877 SAMD9 2.92E-05 -6.28777 CASP4 2.43E-07 -6.26438 ZNF239 3.11E-07 -6.25336 EXOC8 7.57E-10 -6.23556 ZNF267 2.88E-09 -6.23497 CXorf26 4.61E-09 -6.21144 UBN2 3.10E-08 -6.13653 DICER1 1.88E-06 -6.04332 EMG1 4.50E-08 -6.03648 CLDN1 6.79E-05 -6.02725 SLC25A37 4.06E-10 -6.00065 TRMT5 6.98E-09 -5.99157 ZFP112 1.01E-05 -5.95557 ZNF180 7.52E-08 -5.95242 COG8 5.73E-09 -5.94893 C14orf169 8.79E-07 -5.92296 CDC25A 4.86E-05 -5.91775 PLAU 2.03E-05 -5.89103 INADL 2.05E-08 -5.86508 HYLS1 7.63E-07 -5.86192 ZNF480 2.34E-08 -5.85206 CUTC 5.90E-08 -5.84794 ZNF502 1.09E-07 -5.83759 PHLDB2 0.00699756 -5.81878 SDR42E1 2.83E-07 -5.80699 ZNF33A 1.06E-08 -5.80495 KLK10 1.03E-07 -5.79212 ZNF33A 1.38E-09 -5.79101 CSTF3 7.28E-09 -5.78706 ZNF321P 1.19E-07 -5.77606 CASP8 9.31E-07 -5.77421 KIAA1586 4.87E-08 -5.75161 DNTTIP2 1.49E-08 -5.73987 JRKL 7.03E-08 -5.71518 ZNF780A 3.68E-08 -5.69986 UBXN1 4.03E-06 -5.67428 IREB2 6.90E-11 -5.64401 ZNF232 1.12E-06 -5.63775 FANCF 7.07E-07 -5.62661 HMGXB4 6.29E-08 -5.61313 ZNF181 4.69E-06 -5.609 PAK1IP1 2.53E-09 -5.6087 CXCL10 0.00064507 -5.60035 ZNF551 2.36E-06 -5.58493 UMPS 5.60E-10 -5.56791 PIGW 3.72E-06 -5.56244 ZBTB6 6.50E-10 -5.55305 KIAA1984 2.90E-05 -5.54092 CCNF 1.89E-08 -5.51971 NSUN5P1 2.50E-06 -5.51572 HJURP 2.02E-08 -5.51476 GPR110 0.00025008 -5.50602 TNS4 9.63E-07 -5.49893 ARL4A 4.39E-06 -5.48884 ZNF121 1.14E-07 -5.48868 ZNFX1-AS1 6.74E-09 -5.4855 DDX28 6.44E-08 -5.47296 ARL17A 1.30E-07 -5.47108 NSUN5P1 2.74E-08 -5.45719 LUC7L3 2.43E-09 -5.45468 ZNF195 1.10E-08 -5.4152 MTERFD1 5.80E-06 -5.3941 ZNF302 6.24E-06 -5.39281 CDK12 1.90E-05 -5.3913 PTAR1 4.64E-08 -5.38032 GTPBP4 1.27E-12 -5.3799 TTK 1.78E-07 -5.34396 KIAA0020 2.55E-08 -5.33535 ZSCAN16 6.41E-06 -5.33135 PNISR 1.08E-08 -5.31393 NCAPG 2.78E-07 -5.31327 ZNF485 3.21E-06 -5.30644 MTERF 1.44E-12 -5.29026 IFNGR1 0.0004955 -5.27855 GINS3 5.50E-07 -5.27088 TMEM177 3.13E-07 -5.26397 IRAK2 6.67E-07 -5.25171 CD59 6.48E-06 -5.21849 ZNF557 7.95E-09 -5.1984 TTC14 3.03E-06 -5.19258 WDR5B 8.62E-09 -5.17006 ZNF267 2.41E-05 -5.15675 ASPM 1.59E-06 -5.15585 EXOSC3 5.59E-08 -5.13801 CDC25A 5.00E-10 -5.1196 KLHL9 3.96E-09 -5.10803 FEN1 1.80E-08 -5.10563 MMAA 4.73E-09 -5.09978 NSUN5P1 1.32E-09 -5.08715 ZNF552 6.49E-09 -5.08652 DCBLD2 6.22E-05 -5.07673 CENPJ 2.36E-07 -5.05786 CDCA4 4.22E-06 -5.05405 BCDIN3D 4.84E-08 -5.0476 ZNF283 3.69E-06 -5.04246 ZBTB24 4.90E-07 -5.02931 TRIM29 1.91E-06 -5.02899 S100A3 2.95E-06 -5.02841 DFFB 7.95E-07 -5.00676 IMP3 3.30E-07 -4.98972 CTTNBP2NL 1.57E-06 -4.98085 ABLIM3 6.68E-05 -4.96799 DEPDC1 4.85E-05 -4.96527 SRSF1 1.86E-06 -4.95878 INPP4B 2.11E-06 -4.95515 CXorf26 7.13E-06 -4.94782 GPR158 1.20E-05 -4.93838 TMEM186 8.90E-08 -4.93115 MCM10 6.06E-08 -4.92375 SETMAR 2.33E-06 -4.92133 RBM12 3.51E-06 -4.91933 USP36 1.97E-06 -4.9144 ZCCHC3 1.00E-08 -4.89152 RFC4 6.48E-08 -4.8867 NUB1 8.89E-06 -4.87588 SLC35G1 9.08E-05 -4.87226 RRP8 1.25E-08 -4.87095 RIOK2 6.15E-06 -4.869 BLM 9.15E-08 -4.86865 BORA 5.16E-05 -4.86022 PIK3R4 1.05E-10 -4.85935 MYSM1 3.61E-06 -4.85886 C11orf82 1.55E-05 -4.85554 ZNF280C 5.62E-08 -4.84956 MTF2 9.76E-07 -4.84734 TRNT1 0.00013291 -4.84721 ZNF451 3.47E-06 -4.84226 EXOC4 7.89E-09 -4.83387 DLEU2 1.81E-05 -4.83246 GTPBP4 1.52E-08 -4.83068 APOL6 3.09E-07 -4.83014 ANKRD22 1.22E-06 -4.82487 ZFC3H1 7.53E-09 -4.81718 DCUN1D4 4.84E-09 -4.79767 RRS1 8.85E-08 -4.79054 ZNF215 5.45E-08 -4.76437 SRSF1 4.64E-08 -4.76193 CCNA2 1.58E-07 -4.75493 ZNF180 2.04E-07 -4.74801 POP1 1.26E-09 -4.74385 PPP1R8 4.53E-10 -4.73491 ZNF540 3.52E-05 -4.72373 POP1 7.99E-08 -4.71657 NOL8 6.57E-07 -4.71511 GORAB 1.91E-06 -4.71439 PUS1 1.55E-07 -4.70949 PARP14 1.49E-05 -4.70331 METTL1 7.14E-07 -4.70316 C2orf69 1.31E-07 -4.67715 IKBKB 1.92E-05 -4.67382 POLR1B 6.85E-06 -4.66579 PNO1 1.11E-07 -4.65805 IMP3 3.12E-09 -4.65436 IFIT5 1.13E-06 -4.6515 DLGAP5 5.85E-06 -4.64787 SPP1 0.00092419 -4.64662 SPP1 0.00073352 -4.64482 CLDN1 0.00057507 -4.63954 SNRNP35 3.13E-08 -4.63672 ZNF223 5.79E-05 -4.63661 ANKRD22 7.58E-07 -4.63097 DEM1 7.91E-09 -4.62986 FAM111A 1.09E-09 -4.60975 WDR75 1.06E-06 -4.60838 ZNF321P 1.57E-08 -4.60633 FOXQ1 7.85E-07 -4.60393 MAK16 9.11E-06 -4.59639 POLR2D 3.93E-08 -4.58933 Mar-07 8.38E-09 -4.58004 STK4 4.00E-10 -4.57566 ZNF398 1.06E-07 -4.56637 CSTF2T 2.74E-08 -4.56415 PNO1 3.85E-07 -4.55508 PSMA1 1.01E-05 -4.55457 PARP14 2.79E-07 -4.54925 ZNF235 1.43E-06 -4.53231 ZNF790 5.65E-05 -4.52629 TEFM 6.12E-07 -4.51667 ATF5 3.30E-08 -4.51588 DOLK 1.05E-06 -4.50542 GRPEL2 8.66E-08 -4.49308 CTTNBP2NL 1.27E-08 -4.4902 ZNF557 6.82E-08 -4.4893 IFNAR2 5.20E-08 -4.48904 TBRG1 1.25E-07 -4.48794 SLC25A37 2.91E-08 -4.48767 EXOSC6 3.47E-07 -4.48462 PILRB 1.16E-10 -4.48325 RAD18 1.60E-05 -4.47565 CTPS1 2.02E-07 -4.47515 SKP2 1.73E-08 -4.4616 IREB2 8.21E-09 -4.45923 METAP2 1.54E-06 -4.42698 FAM98A 9.60E-08 -4.41931 ZNF302 3.40E-07 -4.41878 GPATCH4 6.29E-06 -4.41537 ZNF485 3.16E-05 -4.40846 MMAA 3.34E-09 -4.40187 RNF169 1.40E-06 -4.38902 GPATCH4 3.20E-08 -4.37836 C3orf52 1.64E-08 -4.3778 SPRYD4 2.57E-08 -4.37492 LOC10050764 5.25E-05 -4.3701 TAP2 2.50E-06 -4.36857 CKAP2 1.71E-05 -4.36777 VHL 9.63E-09 -4.3627 RNF25 1.23E-07 -4.36078 ZNF552 4.26E-08 -4.35609 ZNHIT2 1.96E-08 -4.35582 ZNF283 1.40E-06 -4.35163 RANBP6 3.79E-10 -4.34406 CSTF3 8.85E-09 -4.33987 CD59 2.51E-09 -4.33825 SEMA3A 0.00011639 -4.33425 ITGA2 0.00040453 -4.32943 MRPL46 9.00E-10 -4.32938 ZNHIT6 6.30E-06 -4.31966 HAUS8 2.20E-06 -4.31578 DLGAP5 7.42E-06 -4.31483 WDR75 8.72E-08 -4.31019 SH3KBP1 7.23E-06 -4.30529 BLMH 1.01E-06 -4.30262 AASDHPPT 1.89E-05 -4.29206 ING3 2.79E-06 -4.28851 POLR1B 3.61E-06 -4.28827 EXOSC4 1.97E-08 -4.28065 MAP4K4 0.00013016 -4.27375 DIDO1 6.76E-09 -4.26886 ZFC3H1 1.20E-08 -4.26725 OTUD6B 2.43E-05 -4.26674 RBMXL1 1.12E-08 -4.26612 KIAA1586 7.92E-09 -4.26545 TAMM41 9.95E-07 -4.26265 PPP1R3D 0.00013073 -4.26169 ZC3HC1 1.51E-07 -4.25934 ZNF551 7.75E-07 -4.24714 TADA1 6.04E-06 -4.24675 RTN4IP1 3.03E-07 -4.24074 DCUN1D4 6.86E-10 -4.23592 NTSR1 5.85E-08 -4.2357 GEMIN2 3.27E-06 -4.22092 GEMIN6 3.24E-07 -4.20514 NDUFS2 1.56E-06 -4.20397 ELF2 2.52E-08 -4.20264 CCNJ 1.07E-06 -4.18263 WDR5B 1.98E-06 -4.181 ALS2 4.04E-06 -4.17643 AGGF1 2.05E-08 -4.17126 ZBTB3 0.00010495 -4.17038 FUT4 3.62E-08 -4.16321 ANAPC7 7.10E-07 -4.163 SPP1 0.00084895 -4.16033 AKAP12 0.00051739 -4.15739 ZNF193 8.98E-07 -4.15736 GTPBP4 4.85E-12 -4.15403 NOL8 5.12E-07 -4.13816 ARL4C 0.00053635 -4.13543 MKRN3 5.93E-07 -4.13051 RNF43 0.00025095 -4.12582 ZNF195 3.04E-07 -4.12575 NFE2L3 1.14E-06 -4.11862 RPL4 2.12E-05 -4.11665 CCNB1 1.24E-05 -4.11567 FAM111B 2.05E-05 -4.10616 DTX3L 4.18E-07 -4.10529 CCDC14 3.49E-09 -4.09741 PIK3R1 3.17E-09 -4.08815 EIF2S1 1.87E-07 -4.08696 PRMT6 5.58E-06 -4.07826 SMG8 5.89E-12 -4.07426 TIGD1 3.37E-06 -4.06653 FADD 5.62E-11 -4.06254 C6orf203 8.63E-07 -4.06187 XPO4 4.22E-07 -4.06155 TRNT1 7.62E-05 -4.0609 NANP 1.58E-08 -4.06064 OPA3 2.71E-07 -4.05922 ZNF594 9.03E-05 -4.05693 TAF1A 9.07E-05 -4.05603 CTPS1 5.76E-06 -4.04973 FAM43A 2.51E-07 -4.04681 MYO10 8.74E-05 -4.04061 POLD3 6.61E-08 -4.03407 PILRB 6.35E-08 -4.03238 FASTKD2 7.81E-06 -4.02653 RCL1 1.49E-07 -4.02622 LRRC8B 1.89E-06 -4.02361 SPIN4 2.70E-05 -4.02152 MKI67 0.00050465 -4.01996 DDX11 7.91E-10 -4.01171 TMEM138 1.63E-06 -4.00664 LCMT2 2.12E-08 -4.00611 TGOLN2 4.94E-05 -4.00486 ARGLU1 1.89E-09 -4.00012 C1orf112 3.95E-05 -3.99362 NOL8 1.81E-06 -3.99061 LTV1 2.73E-06 -3.98756 C12orf43 2.15E-06 -3.98519 C5orf34 4.04E-08 -3.98025 AURKB 1.16E-08 -3.97695 GLMN 2.48E-06 -3.97547 ANKZF1 3.82E-07 -3.96853 UNKL 8.09E-06 -3.96805 MKI67 1.16E-06 -3.96054 KIAA1731 6.86E-07 -3.96047 BHLHE41 2.12E-06 -3.9506 ZCCHC7 6.50E-07 -3.95059 TMEM87A 0.0005684 -3.94663 CDC45 3.82E-06 -3.94116 ZNF193 6.07E-05 -3.93736 KLK10 9.74E-08 -3.93658 KIAA0040 1.66E-07 -3.92441 PNN 9.98E-10 -3.92287 POLR3F 2.26E-06 -3.9223 POLR1B 3.69E-05 -3.92208 ZNF544 9.85E-10 -3.91854 CCNA2 2.33E-05 -3.91593 RBM12 1.99E-07 -3.91577 NAA15 9.49E-07 -3.91419 CBLL1 1.17E-05 -3.91193 PARP14 9.70E-06 -3.91174 TWISTNB 6.41E-07 -3.9083 CALB2 2.65E-08 -3.90621 DHX57 3.19E-05 -3.9054 DHX57 3.19E-05 -3.9054 FEN1 1.43E-06 -3.90456 EXOSC9 9.70E-08 -3.89908 DDX52 1.94E-06 -3.89846 MKI67 9.22E-07 -3.89748 CLDN12 8.83E-08 -3.89623 GPR39 4.41E-09 -3.89505 POLE2 0.00013912 -3.89395 DGCR8 1.92E-07 -3.89344 NT5C1B-RDH 1.20E-09 -3.89071 ZNF398 1.04E-05 -3.88752 ZNF451 9.70E-06 -3.88611 DCLRE1C 7.12E-07 -3.88597 ERCC6L 4.01E-06 -3.88412 SPP1 0.00339128 -3.87333 STK4 7.33E-11 -3.87259 ATAD3B 1.28E-07 -3.86496 POLR1B 2.61E-06 -3.86382 KLHL9 2.03E-07 -3.86376 MINA 0.00037408 -3.86342 C2orf69 3.15E-09 -3.86339 LUC7L 3.68E-06 -3.8603 FER1L4 1.88E-06 -3.85899 CLDN2 1.25E-08 -3.85887 KCTD20 1.32E-05 -3.85827 AKAP12 0.00063195 -3.8575 TSEN2 3.86E-09 -3.85699 LARP4 4.78E-05 -3.85473 TFB1M 1.42E-05 -3.85279 MTPAP 0.00032807 -3.84649 IFIT5 5.60E-07 -3.83906 STAG3L2 3.63E-07 -3.83316 TAF9B 2.08E-05 -3.82841 CCDC82 0.00012516 -3.82827 ZNF223 8.95E-05 -3.82827 ARL14EP 8.78E-09 -3.82668 SHCBP1 0.000873 -3.8254 POLR1B 5.98E-06 -3.81829 LRP8 1.21E-05 -3.81175 ZNF195 6.21E-07 -3.8059 MAP3K1 1.82E-06 -3.80344 THUMPD3 0.00011107 -3.80297 SUV39H2 1.40E-07 -3.80275 GEMIN5 5.74E-05 -3.80252 ADAT2 9.52E-06 -3.80159 NHLRC1 1.28E-05 -3.7983 COMMD5 5.03E-08 -3.79186 ASPM 5.39E-05 -3.78651 DFFA 3.90E-08 -3.786 ZNF565 2.08E-07 -3.78508 CTTNBP2NL 5.58E-09 -3.78291 CCDC12 5.28E-08 -3.78274 MALL 1.94E-06 -3.77895 GORAB 5.85E-05 -3.77731 SNAPC5 6.69E-09 -3.77728 SRM 2.33E-06 -3.77266 TGOLN2 0.00016663 -3.76856 NUDT16 1.17E-05 -3.76807 PTGS2 0.00167658 -3.7617 ZNF502 7.61E-09 -3.75835 KLHDC4 1.84E-09 -3.75823 BLOC1S3 1.37E-08 -3.75285 RBM19 1.22E-06 -3.75178 GINS1 2.02E-09 -3.74894 UTP15 1.26E-05 -3.74755 MALL 9.42E-07 -3.74737 C1orf212 1.22E-08 -3.74678 UBLCP1 5.14E-06 -3.73943 ZNF449 8.53E-05 -3.73636 PIGM 5.70E-06 -3.73394 ERCC4 1.06E-05 -3.72927 EMG1 5.02E-08 -3.71894 SKA1 5.68E-06 -3.71629 CFL2 3.52E-06 -3.71547 LTV1 2.85E-05 -3.71416 PTPN2 0.0003315 -3.7078 TIPIN 1.68E-07 -3.70569 ZNF789 0.000169 -3.69986 COX4I1 1.98E-05 -3.69076 ZNF691 6.41E-07 -3.68603 WDR43 1.05E-06 -3.68547 TCEB3 3.60E-06 -3.68445 CDKN3 8.52E-05 -3.68246 FDXACB1 0.00048278 -3.67949 ZNF587 6.40E-08 -3.67638 PUS7L 4.76E-05 -3.67349 BAG5 1.36E-08 -3.67335 UTP23 1.06E-07 -3.67184 MCM10 5.39E-07 -3.66742 TWISTNB 3.86E-06 -3.66307 UBLCP1 6.73E-06 -3.66043 PUS1 5.67E-08 -3.65832 FARSB 1.73E-07 -3.65829 NSUN5P1 1.88E-08 -3.65766 DNAJC2 2.04E-09 -3.65611 ASF1A 1.55E-05 -3.64988 CDK5RAP2 0.00020908 -3.64859 SLC7A5 0.00012349 -3.64175 TRMT10C 5.30E-09 -3.64046 ZNF473 9.11E-07 -3.63963 FAM111B 2.23E-05 -3.63931 MRPS17 6.93E-08 -3.63502 DCAF16 7.98E-06 -3.634 TNS4 1.93E-05 -3.63344 C15orf23 6.55E-06 -3.63277 POLR3G 0.00106098 -3.63204 ATAD3A 2.60E-09 -3.62951 ZNF193 6.07E-07 -3.62935 LLPH 3.18E-07 -3.62589 TTC14 2.75E-06 -3.62537 FEN1 5.01E-07 -3.62446 RELB 8.37E-08 -3.62217 AMMECR1 4.50E-06 -3.62201 BRD8 5.37E-06 -3.61669 MTF2 2.22E-06 -3.61582 PPP2R1B 6.62E-05 -3.61563 FAM217B 0.00050503 -3.6149 LCMT2 8.18E-06 -3.61375 ZNF345 3.88E-06 -3.61319 ZNF195 4.67E-07 -3.61181 FAM118A 8.54E-07 -3.61014 RPS27A 8.64E-07 -3.60999 TPX2 6.32E-07 -3.60983 KIF2C 8.36E-07 -3.60959 SRRD 7.80E-10 -3.60759 MTAP 0.00014483 -3.60447 USP36 9.58E-05 -3.60252 ZNF12 1.27E-07 -3.60235 PDIK1L 0.00026924 -3.60231 WDR36 2.56E-06 -3.60146 NAPEPLD 0.00012697 -3.59829 RPP14 4.31E-07 -3.59824 THUMPD2 7.84E-06 -3.59721 MRPS25 6.92E-05 -3.59305 PUS3 3.79E-06 -3.58758 ZNF224 2.22E-07 -3.58652 MOCS3 0.00019489 -3.57761 PABPC1L 1.19E-08 -3.57753 ANP32A 9.53E-06 -3.57487 UBIAD1 8.29E-06 -3.57462 ZNF544 1.07E-05 -3.57445 MSTO1 7.09E-07 -3.57438 ASTE1 2.18E-05 -3.57139 UBIAD1 3.38E-06 -3.56664 OPA3 3.99E-07 -3.56201 ACTR5 8.70E-09 -3.5619 FOXQ1 0.00097166 -3.55805 INF2 1.73E-05 -3.55713 NSUN4 3.23E-06 -3.55176 SKA3 3.04E-08 -3.54817 MMP1 0.0104468 -3.54525 TAF1A 0.00216809 -3.54408 C16orf91 1.01E-07 -3.54376 ZMYM6 7.90E-05 -3.53974 PAPOLG 5.41E-06 -3.53962 LRRC14 1.62E-07 -3.53911 C15orf42 9.99E-07 -3.5354 SDC4 2.99E-05 -3.531 MDM4 4.43E-09 -3.52998 KIF14 2.37E-05 -3.52528 CHCHD4 1.79E-05 -3.52294 ZNF354B 1.16E-05 -3.52235 HMGXB4 5.68E-05 -3.5192 MPHOSPH10 1.62E-09 -3.51907 ZNF195 6.98E-07 -3.51826 MTAP 2.22E-05 -3.51667 NOL11 1.04E-09 -3.51569 ZNF267 9.12E-05 -3.51528 ZNF561 2.36E-07 -3.51488 BAG5 1.05E-05 -3.51471 UHRF1BP1 1.47E-05 -3.50857 FASTKD2 7.18E-05 -3.50763 SACS 1.73E-05 -3.50738 TRIM27 3.00E-08 -3.50698 CDCA4 7.35E-05 -3.50637 SRSF5 2.39E-08 -3.50499 ECE2 4.08E-08 -3.50452 BCLAF1 5.55E-10 -3.50184 CDC45 6.53E-10 -3.49796 BTBD7 5.30E-07 -3.49724 NGDN 9.61E-09 -3.49105 ZNF562 1.23E-05 -3.48581 GEMIN4 4.84E-08 -3.48416 LRR1 4.20E-05 -3.48215 PRR5L 3.97E-09 -3.48106 MARS2 2.96E-05 -3.47913 HMGXB4 7.60E-05 -3.47386 C1orf74 6.19E-06 -3.4721 FBXL5 1.14E-05 -3.47191 KLHL9 6.53E-06 -3.46617 EIF4E 5.83E-05 -3.46387 CFLAR 5.09E-05 -3.45841 Mar-07 3.53E-06 -3.45769 PNO1 1.49E-05 -3.45333 TAMM41 5.32E-08 -3.45244 PILRB 1.02E-07 -3.44869 UBIAD1 1.14E-05 -3.44843 NUMB 8.26E-06 -3.44825 TAP1 5.37E-05 -3.44825 HSPBAP1 0.0004701 -3.44751 MTPAP 1.72E-05 -3.44722 DBF4 6.60E-05 -3.44468 DDX20 3.91E-08 -3.44427 RBMXL1 1.54E-09 -3.44185 ZNF200 0.00014293 -3.43872 ARGLU1 1.15E-09 -3.43361 ZNF607 1.81E-07 -3.43328 PPHLN1 8.30E-06 -3.43208 RPL37A 5.05E-06 -3.43027 ZNF24 1.74E-08 -3.42934 UBN2 1.09E-06 -3.42655 TROAP 2.03E-06 -3.42417 MFSD5 6.47E-08 -3.42376 SRM 1.44E-08 -3.42076 POLR1B 6.24E-06 -3.41818 NIP7 4.48E-07 -3.41668 MARS 0.00015348 -3.41267 ANKRD10 1.54E-08 -3.41263 PIF1 1.52E-06 -3.40867 METTL16 3.28E-06 -3.4057 SFT2D2 2.37E-05 -3.40507 RIOK2 1.95E-05 -3.40404 NMUR2 0.00021704 -3.40095 MKI67 6.00E-06 -3.39718 PRPF38B 0.0003507 -3.3956 GART 2.22E-06 -3.39492 C12orf4 1.46E-05 -3.3923 EZH1 4.36E-07 -3.38928 ZNF284 1.65E-07 -3.38815 TIMM8A 0.00026199 -3.3863 CENPF 1.61E-05 -3.38526 TMEM68 2.96E-06 -3.38105 TUBB 5.04E-08 -3.38028 SLITRK6 1.90E-05 -3.37889 ZNF540 8.80E-05 -3.3747 CASC5 3.42E-07 -3.3735 ZNF766 9.82E-08 -3.37212 BARD1 4.61E-06 -3.36552 GEN1 9.93E-07 -3.36536 MCPH1 6.01E-07 -3.36413 AKT2 1.24E-05 -3.36401 C21orf91 1.51E-06 -3.36392 ZNF780A 3.97E-05 -3.36214 FANCL 3.28E-07 -3.36121 ARL17A 0.00029963 -3.35611 DDX56 8.68E-09 -3.35475 CDK12 7.50E-06 -3.35462 MRTO4 8.56E-07 -3.35421 SRFBP1 9.67E-06 -3.35144 LAMC2 2.33E-07 -3.35036 DDX11 6.60E-09 -3.35016 MED7 3.54E-07 -3.35004 MSTO1 1.32E-06 -3.34842 CD59 1.95E-05 -3.34537 THADA 1.05E-06 -3.34239 SETD6 2.52E-05 -3.34231 DCAF4 8.80E-06 -3.34167 WARS 6.82E-07 -3.34133 ZNF227 1.34E-05 -3.33816 GNPNAT1 5.43E-05 -3.33769 VNN1 0.0022165 -3.33761 KCTD14 6.39E-06 -3.3374 WHSC1 4.46E-07 -3.33654 LRRC14 2.98E-06 -3.33557 ZNF697 2.75E-06 -3.33515 TSR1 2.69E-07 -3.33124 NANP 0.00040761 -3.3311 RNF26 2.55E-06 -3.33011 TTLL4 2.45E-07 -3.32853 ZNF562 0.0002001 -3.32712 RRP1 5.70E-05 -3.32624 PRPF4 7.69E-05 -3.32246 RPP38 2.02E-08 -3.32199 TRAF6 2.03E-06 -3.32151 ADAM19 0.00083096 -3.32059 HMGB1 6.96E-05 -3.31988 TP53RK 1.80E-06 -3.3185 DTL 7.07E-05 -3.31779 SH3RF2 1.14E-07 -3.31687 PGAM5 1.42E-06 -3.31607 LMNB1 4.96E-07 -3.31549 LMNB1 4.96E-07 -3.31549 PHF17 9.23E-07 -3.3152 CD58 5.43E-07 -3.31366 ZNF146 7.90E-09 -3.3133 C22orf29 2.02E-08 -3.31214 HILPDA 4.70E-08 -3.31178 ZNF507 8.78E-08 -3.30767 SDC4 4.52E-05 -3.30608 LARP4 2.03E-07 -3.30605 MTERFD2 8.68E-06 -3.30461 ZNF195 4.67E-06 -3.30444 GRWD1 3.59E-06 -3.30378 PRPF3 7.56E-07 -3.30226 ARAP2 8.83E-05 -3.30214 ICAM1 8.02E-06 -3.30092 CCNT2 1.85E-05 -3.2998 ZNF593 2.47E-08 -3.2922 ZNF35 5.10E-08 -3.2905 ZNF195 9.79E-07 -3.28493 C16orf88 0.00027033 -3.28451 ZNF526 9.41E-07 -3.28247 ARHGAP11A 3.79E-06 -3.27931 FAM208B 0.00130288 -3.27918 NUP85 2.90E-07 -3.27877 SPC24 0.00027359 -3.27822 C10orf10 3.43E-07 -3.27773 RRP15 1.41E-05 -3.27392 FJX1 0.00016078 -3.26996 DCUN1D4 7.72E-05 -3.26642 THUMPD1 1.25E-07 -3.26526 ST3GAL1 1.46E-05 -3.26504 DDX52 3.57E-06 -3.26481 TAF1C 2.92E-06 -3.26403 ASCC3 1.69E-07 -3.26384 EXOSC2 2.86E-09 -3.26177 BAG5 2.19E-07 -3.26027 MAGOHB 4.14E-06 -3.25933 LYAR 2.06E-07 -3.25912 SERPINB5 9.43E-05 -3.25816 BLZF1 8.70E-05 -3.25115 SLC5A6 6.94E-07 -3.25011 SERTAD4 4.02E-07 -3.24826 ZNF225 0.00191679 -3.2472 TNFRSF21 6.87E-05 -3.24225 CFL2 7.24E-06 -3.23338 EIF2S1 5.62E-06 -3.23056 ANAPC7 6.15E-10 -3.22894 ZNF562 7.42E-06 -3.22789 CCDC137 1.23E-06 -3.2277 CTPS1 0.00029453 -3.22603 B3GALT6 4.03E-08 -3.22549 HDHD3 6.85E-09 -3.22466 HELLS 2.86E-06 -3.21888 TRIM27 3.28E-08 -3.21833 ZNF548 0.00015188 -3.21567 RARS 2.08E-08 -3.21553 ZNF136 8.13E-06 -3.2151 MSTO1 3.85E-07 -3.21432 TWISTNB 2.20E-05 -3.21206 CKAP2 2.24E-06 -3.20729 SLC6A14 0.00036189 -3.20646 SDF2L1 3.74E-08 -3.20629 LIF 0.00099315 -3.20525 SDR16C5 6.19E-08 -3.20403 LOC10028751 4.80E-08 -3.20399 CFLAR 1.47E-06 -3.20226 PPHLN1 3.40E-05 -3.20029 MLKL 1.06E-06 -3.19901 RBM12 4.50E-06 -3.19886 PTAR1 5.43E-05 -3.19876 ZNF587 0.00095643 -3.19828 ATG14 9.00E-05 -3.19265 CPSF6 3.78E-08 -3.18796 CCNL2 1.72E-08 -3.18757 PHAX 4.84E-05 -3.18575 NRF1 5.67E-05 -3.18046 MGEA5 3.23E-08 -3.18011 ZNF17 4.35E-05 -3.17812 SLC25A27 0.00017189 -3.17494 TCN1 0.0115126 -3.17443 ANO9 1.59E-07 -3.17354 LAMC2 0.00108512 -3.17274 SETD6 0.00015588 -3.17192 CNEP1R1 7.98E-05 -3.1701 ZNF181 7.11E-05 -3.16836 TXNIP 5.50E-11 -3.16757 ZNF609 0.00084929 -3.16705 SCO2 4.12E-10 -3.16304 PHF20L1 0.00065022 -3.16212 TRMT44 2.23E-07 -3.16047 ZNF337 6.28E-07 -3.15989 BEND3 2.90E-05 -3.15942 PPP1R8 0.00018523 -3.1586 KIF23 6.95E-06 -3.15749 CXCL11 0.00508314 -3.15634 CCNL2 1.52E-06 -3.15455 IPO7 7.48E-08 -3.15235 TARDBP 5.16E-07 -3.15141 DCLRE1C 4.08E-05 -3.1513 SPATA6L 0.00014934 -3.15057 CCDC50 1.04E-07 -3.14951 PPAN 3.85E-07 -3.14925 ANKRD13C 2.39E-05 -3.14485 ARL4C 0.00019071 -3.14435 CD58 1.68E-05 -3.14224 SLC25A32 5.66E-08 -3.14209 CKAP2L 1.46E-05 -3.14085 ZNF248 6.18E-07 -3.14027 SRSF5 2.80E-07 -3.1353 THUMPD2 3.34E-07 -3.13525 JMJD7 1.45E-07 -3.13502 RRP36 2.54E-06 -3.13133 PGP 4.21E-09 -3.12969 STAG3L3 2.09E-07 -3.12875 PHLDB2 0.00723292 -3.12811 UCHL5 0.000141 -3.12748 C1orf131 9.40E-05 -3.12306 ZNF767 1.52E-05 -3.12242 TARDBP 1.04E-11 -3.12144 C2orf49 2.74E-08 -3.11944 GEMIN2 0.00023242 -3.11856 LOC10013154 2.33E-05 -3.1143 MBLAC2 0.00045219 -3.11392 FGD6 0.0001431 -3.11279 MCM4 8.01E-07 -3.10752 AHSA2 3.32E-07 -3.10308 POLA2 5.79E-05 -3.10252 PGAM5 1.61E-05 -3.10171 RNF207 3.93E-06 -3.10145 HNRNPU-AS1 1.25E-08 -3.10111 TRIM27 7.53E-09 -3.09966 ICAM1 8.52E-07 -3.0986 TNFRSF21 1.71E-05 -3.09827 RAB12 9.77E-06 -3.09817 CKAP2 0.00136317 -3.09645 CDCA3 4.46E-06 -3.09544 PHAX 2.40E-06 -3.09318 CCL20 0.00042794 -3.09032 GCFC2 8.60E-05 -3.0886 ING5 9.96E-07 -3.08675 TTI1 1.39E-09 -3.08649 RBMXL1 3.63E-05 -3.086 VEZT 0.00199243 -3.08583 SETD4 4.43E-05 -3.0849 CENPE 1.03E-05 -3.08211 BACH1 2.04E-10 -3.08126 TACC3 2.60E-07 -3.07995 EXOSC3 5.02E-07 -3.07962 CSTF1 3.65E-09 -3.07828 TMEM70 2.10E-06 -3.07321 PMM2 1.69E-06 -3.07318 ZRANB2 7.85E-11 -3.07278 CATSPER2 9.98E-08 -3.0718 GK5 4.98E-06 -3.07085 MCM4 8.28E-07 -3.06826 SLC25A19 3.35E-08 -3.06814 ZNF30 0.00054461 -3.06809 OGT 1.02E-06 -3.06566 STK4 1.69E-05 -3.06109 MED4 1.45E-08 -3.06038 BORA 1.89E-05 -3.06016 OBSCN 0.00011628 -3.05992 C2orf18 0.00012673 -3.05656 ZNF609 0.00121809 -3.05601 ZAK 1.17E-05 -3.05555 TBP 5.55E-06 -3.05538 FTSJ2 4.65E-08 -3.05469 LRP8 6.07E-05 -3.04809 SNAPC4 2.12E-07 -3.04797 MRPS17 3.62E-06 -3.04587 ZNF16 2.63E-08 -3.04425 PINX1 1.68E-06 -3.04414 LUC7L 4.53E-09 -3.04246 RIF1 3.29E-05 -3.04168 C1orf135 3.94E-07 -3.04165 KRI1 1.73E-08 -3.04145 FAM156A 2.13E-08 -3.04016 RWDD2A 1.77E-05 -3.03879 ZNF595 2.07E-06 -3.03711 GABPB1 0.00022727 -3.0361 UBE2C 1.61E-05 -3.03544 RPS27A 1.82E-06 -3.03336 CLSPN 2.32E-06 -3.03171 TP53I3 6.68E-07 -3.03164 EXO1 1.21E-07 -3.03094 EIF5B 1.99E-10 -3.03058 ARHGAP11A 0.00025594 -3.02556 GEMIN7 8.96E-07 -3.02544 NCAPG 4.56E-05 -3.02431 ARL14EP 8.41E-06 -3.02376 CBWD1 0.00026979 -3.02334 RRM2 2.77E-07 -3.01987 NMI 0.00014822 -3.01906 MRFAP1L1 1.43E-09 -3.01874 RPS6KB1 1.34E-05 -3.01767 ZNF805 2.22E-05 -3.01572 LOC10050656 1.62E-07 -3.01485 METTL2B 0.00027968 -3.01399 PDLIM5 9.47E-05 -3.01381 ZCCHC10 3.27E-06 -3.01205 GJB6 0.00031193 -3.01121 ZNF507 6.20E-07 -3.0109 ZNF540 5.47E-05 -3.00988 GARS 2.45E-07 -3.00901 CEBPZ 9.50E-09 -3.00635 PARP9 0.00034303 -3.00628 LYSMD4 2.48E-05 -3.00515 MKI67 8.45E-05 -3.00447 GOLGA8A 2.11E-06 -3.00379 ZNF784 5.42E-05 -3.00375 FANCI 1.01E-05 -3.00374 VDR 1.14E-07 -3.00121 NOL11 1.50E-07 -2.9964 ACSL5 0.00023575 -2.99602 C5orf43 0.00045296 -2.99579 ZRANB2 0.00013691 -2.99528 PITPNB 1.10E-07 -2.99485 WHSC1 7.13E-10 -2.99366 AIMP2 2.83E-07 -2.99245 AIM1L 0.00023266 -2.98818 VANGL1 1.39E-06 -2.98661 ABCE1 6.54E-08 -2.98395 FASTKD3 8.01E-05 -2.98137 FADD 1.79E-06 -2.98011 OAS3 0.00129445 -2.97957 CITED4 2.10E-07 -2.97929 E2F4 6.92E-09 -2.97831 TIFA 0.00033284 -2.97742 U2SURP 5.82E-06 -2.97294 FRMD5 2.71E-05 -2.97216 MKLN1 7.79E-07 -2.96894 MUM1 3.40E-08 -2.96522 RAD1 0.00080649 -2.96518 KIF2C 7.22E-05 -2.96491 NAPEPLD 0.00015406 -2.96199 PINX1 4.81E-06 -2.95956 HEATR8 1.05E-07 -2.95439 CKAP2 7.55E-05 -2.95048 GSDMB 1.89E-07 -2.94976 N6AMT1 5.47E-07 -2.94942 C1orf212 8.93E-08 -2.94917 CEP78 2.15E-05 -2.94826 ETAA1 4.52E-05 -2.94732 ASB8 2.23E-05 -2.94645 EDEM3 8.88E-05 -2.94467 NAA15 1.50E-06 -2.94461 ALG2 0.00024304 -2.94398 AKAP2 7.49E-05 -2.9434 LIN37 1.27E-06 -2.94197 CMTM3 1.34E-05 -2.94096 SLC7A6 4.39E-05 -2.94036 ABHD13 0.0001655 -2.93908 ZNF302 6.77E-05 -2.93868 SYNCRIP 1.30E-08 -2.93824 SLC25A15 6.99E-06 -2.937 FAM199X 1.94E-06 -2.93674 FARSB 3.72E-11 -2.93432 TUBB 1.00E-09 -2.93419 ZNF564 1.50E-07 -2.92973 CHAMP1 4.82E-07 -2.92838 SPRR1A 5.31E-06 -2.92633 ST3GAL1 5.76E-05 -2.92584 DDX31 0.00047885 -2.92323 AGGF1 3.91E-05 -2.9229 GLRX2 9.40E-07 -2.92127 CENPN 1.93E-05 -2.92057 C9orf114 1.65E-05 -2.91887 KRAS 2.86E-08 -2.91813 KIFC1 1.54E-05 -2.91809 RRP1 1.95E-08 -2.91785 ZMYM1 0.00040964 -2.91378 E2F8 0.00074344 -2.90954 LENG8 0.00012098 -2.9079 RNF34 6.38E-06 -2.90734 MARS 6.47E-06 -2.90627 PARP2 6.92E-06 -2.90526 KIF15 0.00028219 -2.90367 MCTP1 0.00174787 -2.90326 CDKN3 3.04E-05 -2.90319 GARS 3.26E-07 -2.90315 MIR4723 1.18E-06 -2.90261 E2F7 0.00019856 -2.90151 BTF3L4 4.51E-05 -2.9001 HAUS7 0.00010387 -2.89903 ANO1 7.98E-09 -2.89795 ALG2 3.63E-09 -2.89748 PHAX 1.19E-05 -2.89507 PSME3 9.56E-06 -2.89384 LACC1 0.00025639 -2.89115 PRRC1 7.75E-08 -2.89022 FOXM1 7.32E-05 -2.88924 TSEN15 3.49E-05 -2.889 CD58 3.13E-05 -2.8888 PRPF4 1.13E-07 -2.88766 PRPF38B 7.92E-10 -2.88662 UBE2C 3.45E-06 -2.8853 TOE1 2.69E-07 -2.88351 C4BPB 0.00552668 -2.8828 PAQR3 0.00124239 -2.88117 RIF1 1.27E-05 -2.87975 CCDC14 1.40E-06 -2.87894 TIPIN 9.48E-07 -2.87496 CCDC84 3.92E-07 -2.87492 MND1 1.04E-05 -2.87413 ZNF614 6.90E-08 -2.87317 METTL2B 0.00060993 -2.87251 FTSJD1 5.62E-07 -2.8715 VNN1 0.00513301 -2.86935 YAF2 3.47E-05 -2.86869 PARP12 2.26E-05 -2.86759 ZNF187 1.76E-06 -2.86678 RIBC2 3.93E-06 -2.86525 PSME2 1.15E-05 -2.86242 CD58 2.40E-05 -2.86196 GTF2E1 6.19E-06 -2.86136 ENOSF1 8.81E-05 -2.85924 MDM4 2.45E-07 -2.859 CPSF6 2.83E-09 -2.85891 NPAT 6.46E-07 -2.85793 KRI1 4.88E-08 -2.85633 ZNF175 6.49E-05 -2.85606 ZNF589 5.41E-06 -2.8558 ATXN7 5.87E-05 -2.85432 ZNF295 2.47E-06 -2.84929 BTNL9 9.28E-08 -2.84843 SLC25A28 1.60E-07 -2.8471 RAB3IP 0.00018344 -2.84632 MRM1 5.94E-05 -2.84529 DKC1 3.57E-07 -2.84461 NMI 2.90E-05 -2.84044 TIMM23 1.15E-09 -2.8401 NFKBIE 2.97E-06 -2.83839 CAPRIN2 1.17E-05 -2.83738 KIAA1524 0.00017342 -2.83602 MACC1 5.58E-05 -2.83517 CPSF6 3.56E-08 -2.83427 TADA1 1.39E-07 -2.83177 HAUS3 5.29E-05 -2.82868 FGFR1OP 0.00068855 -2.82865 GXYLT1 4.74E-05 -2.82837 CREB5 8.26E-05 -2.82766 WDR4 6.78E-07 -2.82678 ZBTB8A 1.18E-05 -2.8253 DDX39A 8.08E-06 -2.82455 RACGAP1 3.33E-06 -2.82399 NR2C1 6.56E-05 -2.82333 CPSF6 6.46E-08 -2.82227 ZNF28 0.00014653 -2.82145 C1orf174 9.12E-07 -2.82106 SNAPC4 8.85E-09 -2.82058 MSS51 1.43E-06 -2.81917 CENPC1 4.45E-05 -2.81842 WDR6 3.39E-07 -2.8158 ITGA2 0.00128289 -2.81417 FAM136A 1.81E-07 -2.8134 VEZT 2.46E-05 -2.81337 KIF20B 3.80E-05 -2.81236 LARP4 0.0001062 -2.81189 CSRP2BP 0.00050251 -2.81187 CPSF6 1.58E-08 -2.80675 DUS2L 1.46E-07 -2.80442 UTP6 5.27E-08 -2.80402 TUBB 8.01E-07 -2.8036 TMEM201 2.28E-07 -2.80352 AHCTF1 1.68E-06 -2.80317 ZNHIT6 6.23E-05 -2.80233 CFLAR 0.00010316 -2.80229 SLC25A16 5.03E-05 -2.79975 ZNF346 1.49E-07 -2.79912 PTPN2 8.16E-05 -2.79885 TMEM185B 9.51E-06 -2.79868 EIF3M 0.00037186 -2.79864 MRTO4 2.08E-08 -2.79725 STAG3 0.00011891 -2.79628 ZNF230 6.25E-05 -2.79502 IFNGR2 1.43E-06 -2.79429 ENTPD7 0.00037698 -2.79275 CDKN3 6.01E-05 -2.79225 C3orf62 2.23E-05 -2.79129 VANGL1 1.45E-05 -2.78839 DNAJC17 2.03E-07 -2.788 LRRC8B 1.47E-06 -2.78779 TRIM11 7.36E-08 -2.78641 MDM4 5.50E-07 -2.78324 TUBB 2.00E-13 -2.78282 ZNF595 3.64E-06 -2.78233 REV1 4.02E-06 -2.78221 SLC35B4 0.000165 -2.78218 CDC23 0.00028389 -2.7819 TNFAIP8 1.75E-07 -2.78083 CCNL2 6.35E-06 -2.78061 RARS 2.20E-07 -2.78058 FASTKD2 0.00011335 -2.77807 DCAF16 5.31E-06 -2.77805 ZNF350 1.50E-07 -2.77606 UBE2N 3.39E-07 -2.77439 C7orf49 4.65E-07 -2.7728 FTSJD1 4.65E-06 -2.7728 MRPL20 1.29E-08 -2.77266 MSANTD4 9.04E-05 -2.77199 TYW5 1.68E-05 -2.77171 ICMT 3.64E-07 -2.77009 BUB1B 2.96E-05 -2.76963 RAB3IP 0.00023098 -2.76906 SPP1 0.00750129 -2.76889 ZUFSP 5.48E-06 -2.7652 ZNF323 4.89E-05 -2.76508 RRP7A 2.73E-07 -2.76441 ODF2 0.00016617 -2.76234 GORAB 1.47E-05 -2.76202 CEP55 7.13E-05 -2.76173 DTL 0.0012108 -2.76151 ALKBH2 8.42E-06 -2.76122 SPCS3 6.72E-08 -2.76008 NCAPH 1.67E-06 -2.75865 CDCA4 9.16E-06 -2.75779 LRIG3 3.49E-05 -2.7575 MRPL50 1.67E-06 -2.75734 DSN1 1.73E-05 -2.75676 PUS7 3.46E-06 -2.75553 SUV39H1 1.92E-06 -2.75426 JUP 3.81E-06 -2.75407 GOSR2 5.13E-06 -2.75221 UTP14A 1.45E-07 -2.75146 LRCH1 0.00014553 -2.75042 NAPEPLD 0.00044366 -2.7494 RNASEH2C 8.06E-06 -2.74836 TXLNA 1.35E-06 -2.7481 N4BP2L2 1.24E-05 -2.74505 GABPB1 2.81E-06 -2.74397 MAGEA12 8.70E-08 -2.74383 ANO1 3.66E-06 -2.74236 LUC7L 7.25E-07 -2.73837 MSANTD4 0.00036078 -2.73783 GPX2 3.12E-09 -2.73764 TADA2A 9.55E-08 -2.73676 NOP16 1.20E-06 -2.736 RABL3 0.00067275 -2.73555 JPH1 0.00085272 -2.73534 UBE2C 6.20E-06 -2.73533 TMEM41A 4.71E-07 -2.73486 NUMA1 9.63E-06 -2.73484 PVR 2.21E-07 -2.73459 LRRFIP1 2.79E-05 -2.73392 GRWD1 8.75E-08 -2.73336 KIAA2026 0.00019663 -2.73334 ZNF416 8.86E-06 -2.73289 C5orf30 3.70E-05 -2.73277 SNRPD1 1.38E-05 -2.73248 ZNF181 0.00359512 -2.73217 CEP85 4.15E-08 -2.73204 PWP2 3.63E-06 -2.72961 PAQR3 0.00074145 -2.72941 MTF1 8.39E-07 -2.72823 KLHL20 2.02E-05 -2.72786 RRP12 5.56E-08 -2.7269 PRC1 3.03E-07 -2.72507 LYRM2 4.24E-07 -2.72232 SRSF5 6.83E-07 -2.72211 USP45 0.00308861 -2.72051 EBNA1BP2 2.43E-06 -2.72028 HAUS7 4.16E-07 -2.71841 MCFD2 2.73E-05 -2.71802 IL1RN 0.00025496 -2.71752 MIR1292 3.67E-08 -2.71733 DPH3 6.97E-06 -2.71607 PSAT1 0.00056158 -2.71588 SLC25A22 6.72E-10 -2.71555 TOR1A 6.89E-05 -2.71537 MRPL39 2.09E-05 -2.71434 PSME4 2.84E-07 -2.71399 DIMT1 1.85E-05 -2.71087 SEMG1 4.06E-05 -2.71057 PPM1F 3.80E-06 -2.71017 LIPT1 0.00013484 -2.71 WDR73 2.67E-06 -2.70986 PSMD11 7.68E-09 -2.7097 MARS2 0.00111587 -2.70856 MKI67IP 1.20E-08 -2.70786 PSME4 4.26E-06 -2.70767 GXYLT1 0.00117254 -2.70696 ECD 4.31E-07 -2.70642 TLR4 3.15E-07 -2.70547 TAF1A 0.00133563 -2.70469 ZNF708 0.0005391 -2.70386 STYX 0.00025704 -2.70373 LRP5L 1.24E-06 -2.70266 DPH2 3.15E-05 -2.70163 MSL3P1 5.17E-05 -2.70096 NRGN 0.0001354 -2.70073 XPO4 8.87E-06 -2.69954 RPF2 7.32E-09 -2.699 MESDC1 1.28E-07 -2.69883 UCHL5 0.00052156 -2.6987 SMAD5 3.78E-05 -2.69858 FBXO3 0.00115259 -2.69835 HEATR1 4.50E-05 -2.69818 BIN3 1.34E-05 -2.69305 FXC1 4.88E-06 -2.6929 NAA25 3.29E-05 -2.6924 PIK3R1 0.00033183 -2.69217 METTL13 8.98E-07 -2.69204 ABCE1 0.00068731 -2.69134 LMNB1 8.34E-05 -2.69133 EFNA4 9.22E-08 -2.69073 BRD9 1.93E-09 -2.68926 WARS 7.06E-05 -2.68826 MSH5 9.75E-07 -2.68767 THOC1 3.19E-10 -2.68614 S100A16 1.79E-08 -2.68536 ZNF101 2.40E-06 -2.68493 C2orf68 0.00088789 -2.68454 VRK1 2.71E-05 -2.68242 ZNF212 3.74E-06 -2.68212 TTPAL 3.63E-05 -2.68165 PDCD2 7.71E-06 -2.68135 ZADH2 6.08E-05 -2.68034 AGPS 0.00025609 -2.67993 PARL 6.32E-10 -2.67801 RRP12 5.15E-06 -2.67573 SNRPA1 5.10E-07 -2.67504 DNAJC17 4.92E-05 -2.67402 ZNF419 3.90E-06 -2.67211 PUS1 4.95E-07 -2.67115 CCNL1 0.00085264 -2.67036 CLK2 5.49E-07 -2.66941 UCHL3 1.03E-05 -2.66923 ZNF544 1.26E-05 -2.66885 C9orf41 2.50E-06 -2.66707 SPATA5 7.55E-05 -2.66642 DLEU2 0.00082958 -2.66537 JRK 2.34E-05 -2.66415 MIR1292 1.22E-07 -2.66319 LTV1 0.00312288 -2.66092 METTL13 0.00030376 -2.66013 GRWD1 1.12E-10 -2.65911 EIF3B 2.55E-09 -2.65839 MRPS10 0.00015597 -2.65831 CSTF2 1.95E-05 -2.65782 ZNF845 5.06E-05 -2.65718 SELRC1 2.22E-06 -2.65667 MSH6 0.00015037 -2.65636 RBM6 1.75E-05 -2.65618 ZNF30 0.00136904 -2.65499 PPTC7 5.02E-06 -2.65363 TXNDC9 1.99E-05 -2.65189 ALKBH2 5.82E-06 -2.65158 DBF4 0.00049612 -2.65135 RUFY3 7.50E-05 -2.6511 RNF43 2.26E-08 -2.65045 TAF5L 2.66E-06 -2.64984 CDKN3 0.00011895 -2.64802 TEX10 3.97E-08 -2.64691 EBNA1BP2 6.17E-07 -2.64673 CCDC66 0.00056054 -2.64635 APOBEC3B 1.64E-05 -2.64529 PNISR 0.0002592 -2.64508 MRPL14 2.47E-07 -2.64388 RPP40 0.00040775 -2.64317 HIF1AN 1.78E-07 -2.64282 NAPB 3.62E-06 -2.64276 SGK494 0.00071196 -2.64028 LRRFIP1 2.52E-06 -2.63913 STX17 0.00109422 -2.63672 GGA2 4.41E-05 -2.63664 THRAP3 1.90E-07 -2.63637 FLJ39639 0.00020828 -2.63445 NVL 3.54E-07 -2.63433 NKTR 3.20E-05 -2.63367 ARMC10 6.71E-08 -2.63216 GJB4 1.16E-07 -2.63183 MPHOSPH8 4.87E-08 -2.63182 DDX24 2.06E-05 -2.63127 FANCD2 1.66E-06 -2.63004 CD58 0.00011159 -2.6297 UTP20 6.71E-05 -2.62876 RAB7B 8.15E-06 -2.62818 GNL2 2.17E-08 -2.62786 ZNF584 1.26E-05 -2.62724 CAPRIN2 1.60E-06 -2.62627 MAPK1IP1L 8.54E-06 -2.62612 FAM105B 4.10E-06 -2.62537 MALSU1 7.18E-10 -2.62514 RBM12 0.00370586 -2.62258 MMD 5.72E-06 -2.62197 SLTM 1.58E-05 -2.62193 ZNF441 0.00020149 -2.6204 CTPS1 0.0003546 -2.62018 GALNT4 1.05E-05 -2.61953 PPIL1 2.01E-05 -2.61934 CNPY2 8.33E-06 -2.61758 FAM135A 3.09E-05 -2.61736 XIST 1.06E-08 -2.61502 C2orf18 0.00213489 -2.61185 SWSAP1 0.00113842 -2.61166 TRIP13 3.49E-06 -2.61108 MRPL27 3.47E-08 -2.61098 RNF43 5.29E-07 -2.61088 TIMM23 1.47E-05 -2.61037 VEZT 1.26E-05 -2.60964 CDC6 2.69E-07 -2.60899 WDR85 3.23E-09 -2.60712 AKR1C1 2.01E-05 -2.60645 PHLDA1 0.00073983 -2.60619 MCM9 1.26E-09 -2.60503 DDX19A 4.51E-05 -2.60468 PNN 2.20E-06 -2.60421 WDR35 0.00016529 -2.60361 NOX1 0.00017806 -2.60346 ZNF187 0.0004787 -2.6027 LGR5 0.00119504 -2.6025 METTL14 0.00036976 -2.60243 LAMC2 0.00294563 -2.60151 TIMM10 1.58E-07 -2.60131 ADPRHL2 1.15E-07 -2.59905 RPUSD2 1.37E-05 -2.59879 ATF5 0.00223329 -2.5981 KIAA0232 7.76E-05 -2.59801 GIGYF2 3.81E-05 -2.59622 PGGT1B 3.18E-05 -2.59517 NDUFAF4 4.81E-07 -2.59432 ALG11 1.58E-05 -2.59408 DDX51 0.00013016 -2.59122 XPO4 0.00040561 -2.59092 GIN1 0.00032042 -2.59055 MIS18BP1 7.32E-06 -2.58923 FHOD1 4.96E-07 -2.58877 MTF2 8.63E-05 -2.58716 N6AMT1 0.00122491 -2.58437 PDE7A 1.26E-06 -2.58412 DDX31 2.19E-06 -2.58343 C19orf12 5.32E-06 -2.58083 AIFM2 2.03E-05 -2.58046 BRD8 6.14E-07 -2.58037 POLR3B 0.00016168 -2.57816 MED9 1.57E-05 -2.57775 ZNF573 7.55E-08 -2.57568 ABCF2 4.23E-07 -2.57458 ZNF561 3.60E-05 -2.57422 BRD8 2.74E-06 -2.57402 NUDCD1 8.22E-05 -2.5729 TRMT61B 1.01E-06 -2.57175 MMP7 6.77E-05 -2.5702 CLDN2 0.00690681 -2.57006 GPAM 0.00495844 -2.56932 CEP55 0.00035669 -2.5692 PSMG4 6.78E-07 -2.56733 GTF2H2 0.00704571 -2.56587 KLHDC5 2.45E-05 -2.56553 RAB3IP 0.00234421 -2.56483 YARS2 3.26E-05 -2.56466 RNF111 6.98E-05 -2.5645 TAF15 1.72E-07 -2.56445 MIR4723 3.94E-07 -2.56307 ABCF1 1.71E-06 -2.56243 IREB2 1.76E-07 -2.56177 C10orf10 4.68E-06 -2.56147 RFXAP 0.00134809 -2.56069 PSMB10 6.51E-07 -2.56058 ORC2 9.20E-06 -2.55994 LSG1 2.78E-05 -2.55951 SSSCA1 2.91E-07 -2.55919 PKD1P1 2.45E-06 -2.55899 MBD4 8.42E-06 -2.55874 CKAP2 7.24E-05 -2.55864 KLHL23 3.65E-06 -2.55803 FAM98B 4.61E-07 -2.5576 TNFRSF10B 4.48E-05 -2.55617 FAM222B 2.71E-07 -2.55605 TMPO 6.73E-05 -2.55443 ZNF320 1.01E-05 -2.55418 UGGT1 8.84E-06 -2.5528 ABCF2 1.84E-06 -2.55278 CMTM3 0.00145805 -2.55129 CENPN 6.47E-05 -2.55067 ZNF566 2.68E-05 -2.55067 SNRPA1 9.58E-10 -2.55046 TRNT1 6.78E-05 -2.55044 UBE2K 7.66E-09 -2.55041 UTP23 0.00164112 -2.54899 PPHLN1 1.11E-06 -2.54891 VPS33A 2.00E-06 -2.54869 DTWD1 0.00128609 -2.54859 CWC15 3.20E-10 -2.54826 ZNF397 6.21E-06 -2.54705 KIAA1984 0.0033718 -2.54694 PKDCC 4.81E-08 -2.54477 UBASH3B 0.00776531 -2.54241 ZNF766 0.00010584 -2.5422 ZNF45 1.71E-05 -2.54193 AK2 1.36E-06 -2.54125 HILPDA 5.79E-05 -2.54113 RARRES1 0.00053573 -2.54109 ZNF800 0.00056415 -2.53923 DPH3 1.78E-05 -2.53864 RIF1 1.12E-05 -2.53704 CUTC 2.95E-05 -2.53698 HPDL 7.66E-07 -2.53672 HELLS 7.87E-05 -2.5357 METTL2B 2.86E-05 -2.53525 NOM1 9.51E-05 -2.53503 DEPDC1 0.00090059 -2.53488 ZBTB33 6.59E-05 -2.53456 IDH3A 5.72E-05 -2.53363 CDKN3 0.00067845 -2.53283 SMC5 2.98E-07 -2.53259 TMEM165 3.65E-06 -2.53238 CRNKL1 5.94E-07 -2.53076 CSTF1 1.95E-05 -2.53022 C17orf56 2.58E-06 -2.52953 ZNF514 3.12E-05 -2.52879 TIMM23 2.13E-07 -2.52866 MCAT 4.78E-06 -2.52858 THEM4 7.52E-06 -2.52812 TARDBP 8.40E-09 -2.52708 WDR74 3.75E-07 -2.52663 SNAPC4 3.03E-10 -2.52623 C2orf18 0.00249658 -2.52596 RRP7A 1.97E-07 -2.52549 KLHL18 0.00019761 -2.52521 PNN 1.92E-06 -2.52404 PIGM 7.05E-05 -2.52392 BUB1 5.10E-05 -2.52202 FBXO3 7.31E-05 -2.52157 CIAPIN1 5.81E-06 -2.52082 RNGTT 4.74E-05 -2.52077 NAPEPLD 0.00031359 -2.52074 NUP85 1.64E-05 -2.52042 KIF22 1.99E-05 -2.51963 C11orf73 0.00044968 -2.5191 BCL11B 2.96E-06 -2.51864 RUNDC1 5.14E-05 -2.51627 SRPRB 5.98E-07 -2.51562 CA13 1.26E-05 -2.51555 KIF23 1.35E-05 -2.51478 TRIP13 1.60E-05 -2.51446 EBNA1BP2 0.00061608 -2.51441 SYNCRIP 1.10E-05 -2.51402 ALDH1B1 9.83E-05 -2.514 FAM178A 0.00071663 -2.51355 PWP1 2.47E-05 -2.51212 EYA3 1.50E-07 -2.51142 OAS3 6.91E-05 -2.51129 C12orf5 0.00175305 -2.51057 RPL7L1 5.82E-05 -2.51048 NFE2L3 8.29E-05 -2.50977 CSTF1 9.03E-07 -2.5059 FBXO3 8.66E-05 -2.50466 PDE4D 4.54E-05 -2.50454 NDUFAF4 2.85E-06 -2.50438 SNAPC4 2.58E-06 -2.5041 CLP1 1.07E-08 -2.50395 ALS2CL 6.83E-06 -2.5038 TBRG1 9.86E-06 -2.50345 ZNF83 3.67E-05 -2.50196 PSMG4 1.64E-06 -2.5012 CLK2 0.00014802 -2.50109 SH3RF1 5.59E-06 -2.50043 OGFOD1 5.82E-06 -2.50014 ARHGAP29 0.00055259 -2.49972 TAP2 4.25E-05 -2.4996 RLIM 7.54E-08 -2.49906 ABCA10 1.93E-05 -2.49818 LETM1 1.51E-05 -2.4964 PARP14 0.00677783 -2.49602 JRK 7.15E-05 -2.49512 ZC3HAV1 6.00E-07 -2.49416 GPR114 1.70E-06 -2.49396 CCDC50 0.00025486 -2.49336 LOC10028896 1.07E-06 -2.49249 ZNF234 2.54E-05 -2.49239 RBM41 0.00035251 -2.49227 GRIPAP1 1.70E-05 -2.49093 CXCL11 0.0094255 -2.49067 ABCF1 6.27E-07 -2.49008 U2AF1 2.30E-07 -2.48993 FANCF 5.22E-05 -2.48988 SFXN1 7.11E-07 -2.48975 ANKRD50 6.50E-06 -2.48933 SMAD5 1.49E-07 -2.48909 NAA50 7.10E-07 -2.48865 TMEM203 4.86E-06 -2.48843 ATMIN 6.89E-05 -2.48814 E2F8 0.00475971 -2.4878 MMP7 4.15E-05 -2.48756 ZNF493 5.34E-06 -2.48738 GNL3L 7.14E-06 -2.48643 NUP93 3.82E-07 -2.48625 DDX31 0.00024139 -2.48388 NOL6 0.00013729 -2.48348 KIAA0232 4.78E-06 -2.48207 CNOT2 4.43E-09 -2.4796 MCM8 9.67E-09 -2.47896 PPAT 5.79E-08 -2.47803 DGKE 6.29E-06 -2.47726 ZNF785 4.09E-05 -2.47726 TTF1 9.24E-05 -2.4771 HECTD2 0.00027492 -2.47538 NKRF 2.76E-07 -2.47511 ATP6V1G2-DD 2.23E-05 -2.4739 FAM118A 6.01E-07 -2.47341 SRP19 1.31E-10 -2.47332 ATP2B1 0.0010339 -2.47318 MTERFD2 1.50E-05 -2.47178 FANCL 0.00015519 -2.47142 PPP2R1B 5.33E-05 -2.47067 KIAA1432 3.55E-07 -2.46923 DDX24 2.36E-08 -2.46917 BAG2 0.00489673 -2.4682 MRS2 0.00133784 -2.46744 REV1 3.77E-07 -2.46709 SERBP1 1.53E-08 -2.46692 WBP4 6.92E-06 -2.4669 DNAJC9 8.91E-08 -2.46621 ASF1A 0.00025019 -2.46559 SUV39H2 9.57E-05 -2.46529 WDR77 0.00013009 -2.46501 SYNCRIP 0.00024852 -2.46488 SPIN3 4.53E-06 -2.46433 BCCIP 4.26E-06 -2.46428 ZNF165 0.00044515 -2.46348 THAP6 1.37E-07 -2.46293 SRSF5 6.19E-05 -2.4616 SLC38A1 0.00053758 -2.46083 C1orf144 0.0001761 -2.46082 RAD1 3.24E-06 -2.46009 UBE2F 0.0004845 -2.45897 PGBD3 2.25E-05 -2.45761 PGBD4 5.42E-06 -2.4572 ZNF2 0.00148276 -2.45705 RAB18 0.0020517 -2.45679 MTF2 1.74E-05 -2.45654 MELK 2.25E-06 -2.45605 E4F1 1.40E-07 -2.45581 TMOD1 1.23E-05 -2.45481 COX19 3.17E-06 -2.45469 SSPN 0.00944245 -2.4542 FAM193B 9.51E-08 -2.45387 DDX24 7.59E-05 -2.45264 ZBED5 6.75E-08 -2.45169 TXNDC9 5.68E-05 -2.45134 GOLT1B 8.30E-05 -2.45104 BID 2.58E-05 -2.45048 CEBPG 2.73E-07 -2.45046 C9orf72 6.30E-05 -2.45021 ZNF148 0.00057836 -2.4486 PTRH2 2.61E-07 -2.44857 NUP88 3.52E-06 -2.44763 EIF3B 8.78E-08 -2.44722 OSTM1 1.38E-05 -2.44608 ATG14 1.72E-07 -2.44588 DCUN1D5 3.48E-08 -2.44455 PNN 4.11E-08 -2.44425 PSMD1 5.75E-09 -2.44409 CCRL2 2.55E-05 -2.44356 TUBB 6.20E-06 -2.44342 ANXA2R 1.54E-06 -2.44214 ZCCHC7 0.000199 -2.44209 SULF2 4.28E-05 -2.44094 SEMA3C 0.00166022 -2.44023 IL8 0.00012257 -2.44005 RECQL5 8.52E-06 -2.43952 MRPL15 9.83E-07 -2.43862 PPP1CA 2.25E-07 -2.43845 FOXP1 0.00076139 -2.43819 MYBBP1A 1.05E-05 -2.43798 PRPF3 0.00022348 -2.43605 ANAPC4 1.54E-05 -2.43365 AKAP2 0.00095116 -2.43359 NUP35 0.00164172 -2.43258 PHF23 7.89E-08 -2.43243 C22orf29 1.13E-08 -2.43205 CREBZF 0.00013475 -2.43149 NCOA5 1.29E-05 -2.43057 CDC37L1 8.68E-06 -2.43048 C9orf156 1.50E-05 -2.43015 THUMPD3 0.00025908 -2.42916 HARS 1.71E-06 -2.42844 MMAA 6.39E-05 -2.42826 ZNF529 9.46E-06 -2.42767 METTL3 0.0002589 -2.42693 TIMM8B 9.64E-08 -2.42666 LRRC49 0.002893 -2.42655 NOC3L 0.00144082 -2.42643 TMEM60 5.21E-08 -2.42461 NKTR 1.30E-05 -2.42443 ZKSCAN4 0.00303699 -2.42436 NOP16 4.91E-05 -2.42358 LTV1 0.0107522 -2.42093 TUBB 6.12E-12 -2.41969 SVIL 0.00251929 -2.41955 COMMD5 2.72E-05 -2.41791 PPAT 1.93E-05 -2.41781 DDX55 4.80E-05 -2.41765 TCF7 3.63E-07 -2.41727 TMTC3 0.00220782 -2.41725 SAMD5 4.90E-05 -2.4167 GCLM 0.00079721 -2.4148 ERVMER34-1 0.00395543 -2.41439 TROAP 2.61E-05 -2.41353 RPAP2 0.00015544 -2.41334 ACAD9 0.00068783 -2.41182 ACAD9 0.00068783 -2.41182 NFKBIB 0.00113127 -2.41072 DYRK1A 3.35E-08 -2.40949 ZNF638 1.95E-06 -2.40949 RRP12 9.69E-06 -2.40941 ZNF202 8.80E-06 -2.40933 CSPG4 2.59E-05 -2.40931 SEH1L 2.48E-06 -2.40922 SMAD2 1.64E-05 -2.40914 RICTOR 1.34E-08 -2.40871 EFNB1 0.0001099 -2.40819 FANCI 1.14E-06 -2.40736 SERPINB8 8.48E-05 -2.40701 GABBR1 0.0137715 -2.40555 SSX2IP 0.00020896 -2.40389 PPP2R1B 2.96E-05 -2.40293 TRMT1 5.36E-05 -2.40261 DCP1B 6.98E-09 -2.40236 ZNF234 0.00030712 -2.40221 MIS12 5.34E-06 -2.40101 RBM6 4.76E-06 -2.40075 POLR3H 7.21E-06 -2.40013 RAB11FIP2 0.00237538 -2.40006 TROVE2 1.04E-05 -2.39977 SCO1 3.81E-05 -2.39974 ARPC5L 1.62E-08 -2.39967 PRPF4B 0.00011137 -2.39935 WDR36 0.00012287 -2.39878 EPHA1 0.0001196 -2.39841 GNPNAT1 1.84E-08 -2.39819 GNL3 4.80E-07 -2.39742 CATSPER2 1.17E-06 -2.39691 PPP1R35 4.34E-06 -2.39583 BBS12 0.0131355 -2.39554 PSMD7 9.08E-06 -2.39403 PANK2 3.05E-07 -2.39317 GTPBP2 1.68E-08 -2.39307 GLI2 0.00049239 -2.39243 PSRC1 2.97E-05 -2.39234 TRAF3 0.00028751 -2.3919 GOLGA8A 8.22E-06 -2.39152 FAM208B 0.00268715 -2.39148 LRRFIP1 4.99E-08 -2.391 PRPS1 1.71E-05 -2.39085 LRRFIP1 0.00021926 -2.39079 VCPIP1 1.05E-05 -2.38967 BCL2L12 3.34E-08 -2.38964 MAX 0.00072444 -2.38838 AKR1B10 5.51E-06 -2.38825 ARHGAP8 1.90E-05 -2.38775 GPSM2 4.05E-05 -2.3877 PMS1 2.29E-07 -2.38754 MET 0.00012522 -2.38744 TOMM40 2.16E-07 -2.38644 UBE2C 5.46E-05 -2.38638 TIMM17A 0.00017489 -2.38562 KDM2B 0.00015909 -2.38555 CCNH 2.10E-05 -2.38522 ZNF561 4.71E-05 -2.38488 TTL 0.00244299 -2.38409 BYSL 1.21E-09 -2.38362 FAM222B 0.00010383 -2.38347 WDR85 1.46E-08 -2.38193 BIRC5 9.36E-07 -2.38164 XPO4 0.000125 -2.38156 RFK 4.13E-09 -2.38152 TSN 1.09E-05 -2.38142 RPS3A 2.45E-06 -2.38124 SLC25A27 4.01E-06 -2.38072 EXOSC9 4.80E-06 -2.38038 SYNCRIP 0.0003271 -2.38007 MARS 3.61E-06 -2.37963 PDCD11 1.78E-06 -2.37953 EXOSC7 5.40E-08 -2.37922 GBP3 0.00676663 -2.3787 SRP19 3.44E-05 -2.37774 KLHDC5 0.00430386 -2.37737 NVL 3.76E-06 -2.37732 ZNF200 6.08E-05 -2.377 SAP30BP 7.67E-06 -2.37574 ARHGAP29 0.00126135 -2.37444 THAP7 8.94E-07 -2.37423 SLC25A19 4.02E-05 -2.37421 FAM40B 2.22E-06 -2.37356 SCNM1 2.52E-05 -2.37234 FLJ39639 4.81E-07 -2.3723 SLC35E4 1.89E-07 -2.3719 FANCA 6.27E-05 -2.37168 PISD 9.30E-07 -2.37113 EXOSC9 1.06E-09 -2.37093 NOL9 0.00013464 -2.37083 FANCI 3.10E-05 -2.37033 TYW5 0.00017939 -2.37025 ZNF805 2.68E-08 -2.37021 AKAP2 0.00080813 -2.37001 ZFAND2B 0.00029421 -2.36933 RAD18 1.83E-05 -2.36899 PSMC3 2.35E-06 -2.36817 C12orf4 0.00117755 -2.36811 BAG5 8.64E-06 -2.36698 MRPS17 0.00013568 -2.36666 DDX58 0.00043213 -2.36463 TAMM41 5.53E-05 -2.36435 LENG8 3.00E-08 -2.36408 CLPX 1.30E-05 -2.36378 ERAP2 6.36E-05 -2.36264 TSN 1.08E-05 -2.36259 HELLS 6.01E-05 -2.36213 RDM1 9.95E-05 -2.36144 FLAD1 2.03E-07 -2.36132 NTMT1 5.83E-08 -2.36047 KIFC2 7.40E-06 -2.36024 POGK 9.45E-07 -2.35999 GTPBP10 2.52E-05 -2.35963 IDH3A 2.07E-07 -2.35907 TJP2 0.00012611 -2.35899 EIF3B 7.15E-09 -2.35762 RDM1 0.00455844 -2.35719 TAF15 1.41E-08 -2.3568 GALNT3 9.60E-05 -2.35662 TCF19 0.00038189 -2.35651 SLX1A-SULT1 7.68E-08 -2.35597 POLR1A 8.57E-06 -2.35577 FBXO22 3.55E-06 -2.35558 OASL 0.00049343 -2.35557 FARSB 1.34E-07 -2.35545 ZNF169 6.23E-05 -2.35522 ZNF100 6.85E-08 -2.35493 DBF4 0.0004619 -2.35429 GAR1 0.00037163 -2.35417 TCF7 1.81E-05 -2.35328 TRNT1 0.0108491 -2.35301 GOSR2 1.66E-05 -2.35195 ATF7 0.00079221 -2.35185 SARNP 6.23E-07 -2.35172 NFKB1 3.15E-06 -2.34947 DDI2 0.00456772 -2.34932 FEM1C 4.11E-06 -2.34891 NEK3 3.14E-05 -2.34875 KIAA1429 7.02E-05 -2.34817 NOL12 1.55E-08 -2.34763 COQ2 2.52E-05 -2.34743 ATXN3 6.56E-05 -2.34684 ANXA1 0.00021135 -2.34657 NAP1L4 1.12E-08 -2.34588 CDK5RAP3 2.59E-07 -2.34557 GTF2H2 0.00046015 -2.34427 TTF1 0.00280459 -2.34369 YAE1D1 1.69E-05 -2.34353 PPARGC1A 3.30E-05 -2.34336 FAM200A 0.00086516 -2.34315 TIGD2 5.10E-05 -2.34289 GAR1 1.43E-05 -2.34233 RBM15 0.00025955 -2.34232 RPS6KA4 2.39E-06 -2.34232 CD47 1.17E-05 -2.34183 TRMT6 0.00026715 -2.3413 C1orf116 0.00015232 -2.34104 KPNA6 0.00012929 -2.34008 ZC3HAV1L 6.43E-05 -2.33984 ZNF20 2.75E-05 -2.33944 PDDC1 8.11E-07 -2.33872 HMOX2 1.18E-06 -2.33698 EIF6 5.21E-06 -2.33637 PSMC5 1.28E-08 -2.3363 BCL2L1 6.72E-09 -2.33624 TNFAIP1 0.00011457 -2.33601 MRPL16 1.39E-06 -2.33429 SRRD 0.00056148 -2.33377 SAP30BP 0.00046932 -2.33284 HMGB3 1.77E-05 -2.33282 MITD1 1.65E-05 -2.33234 PSMD6 1.10E-08 -2.33158 TOE1 1.32E-06 -2.33118 ZNF638 4.31E-06 -2.33052 NXT1 2.83E-07 -2.33051 DCBLD2 0.00827594 -2.33042 DUSP11 6.01E-05 -2.32994 MIR1292 2.05E-06 -2.32884 CENPI 9.00E-05 -2.32812 MTHFD1L 5.39E-06 -2.32797 KIAA0922 9.91E-06 -2.32785 MUC12 4.23E-06 -2.32781 EED 2.61E-08 -2.32665 PLEC 2.07E-05 -2.32571 LOC10050599 2.98E-08 -2.3249 TXNDC9 9.60E-05 -2.32464 TSEN54 9.74E-05 -2.32355 ZNF641 0.00036006 -2.32345 BLZF1 0.0040872 -2.32342 SP140L 2.60E-05 -2.32327 CTSC 1.95E-05 -2.32272 XIAP 3.34E-07 -2.32157 C17orf79 1.50E-06 -2.32114 PLK3 0.0023488 -2.3209 RNASEH1 7.07E-06 -2.32086 TNF 5.72E-06 -2.32028 PA2G4 8.33E-06 -2.31999 LMNA 1.89E-06 -2.31989 ZNF197 0.00033891 -2.31848 GGA3 1.52E-06 -2.31833 YKT6 0.00215625 -2.31663 RIF1 6.29E-05 -2.31551 EXOSC2 0.00013283 -2.31514 DBF4 5.49E-05 -2.31478 SGPP2 0.00049759 -2.31459 PDCD7 0.00359466 -2.31404 MRPS31P3 0.00056571 -2.31394 GNL2 1.58E-05 -2.31349 SPATA5L1 0.0002693 -2.31295 B3GNT2 3.35E-06 -2.31292 THADA 1.03E-05 -2.31277 MOCOS 0.00015103 -2.31072 NSRP1 0.00016615 -2.30966 ZCCHC7 7.01E-05 -2.30951 BCLAF1 0.00312916 -2.30944 GPR87 2.65E-08 -2.30784 JMJD4 8.79E-06 -2.30729 ABCD3 4.94E-06 -2.30721 BCLAF1 0.00025584 -2.30706 MYNN 5.00E-07 -2.30675 STAT1 0.00021016 -2.30671 SMARCAD1 0.00022691 -2.3065 FAM192A 2.35E-06 -2.30625 TOMM34 1.46E-05 -2.30511 BCLAF1 0.00147673 -2.30495 KPNA1 8.09E-07 -2.30492 ZNF845 7.09E-05 -2.30475 TFB2M 0.00010049 -2.30473 KPNA3 1.86E-05 -2.30446 GPCPD1 1.73E-05 -2.30233 C17orf85 8.90E-05 -2.30126 GON4L 5.13E-05 -2.3012 KPNA1 0.0001163 -2.30042 TBRG1 0.00027104 -2.29991 ODF2L 3.31E-05 -2.29983 PSME3 5.64E-06 -2.29915 CDS2 2.03E-06 -2.29876 MAT2A 0.00050329 -2.29855 SUGP2 4.86E-06 -2.29749 GCLC 0.00093766 -2.29741 LOC150776 9.66E-07 -2.29684 TRIM59 0.00076603 -2.29621 SMARCE1 9.03E-08 -2.29619 NR2C1 0.00358255 -2.29605 E2F6 0.00015496 -2.29538 DDX18 4.59E-09 -2.2951 TP53RK 0.0001846 -2.29483 ABCE1 0.0114293 -2.29326 MRPL55 4.91E-06 -2.29077 TUSC2 3.54E-08 -2.29061 PRPF38A 7.56E-07 -2.28924 PLAGL2 0.00012181 -2.28914 TMEM41A 1.19E-07 -2.2891 PSMB10 2.61E-06 -2.28756 ZNF764 7.12E-07 -2.2868 MTMR9 9.07E-06 -2.28617 UTP11L 2.31E-07 -2.28554 C9orf100 0.00014018 -2.28532 AJUBA 0.00010651 -2.28307 HSF2 0.00020843 -2.28265 CSE1L 5.15E-09 -2.28261 CSE1L 7.51E-09 -2.2825 PSMD7 7.54E-06 -2.28243 MAP3K1 0.00020223 -2.28188 ATF5 0.00112944 -2.28159 OTUD4 1.09E-05 -2.28111 ABHD10 1.32E-06 -2.28089 SNRPF 2.60E-07 -2.28085 CDC25B 8.15E-05 -2.28068 FASTKD5 5.65E-07 -2.28014 PLSCR1 0.00020802 -2.28013 TXNDC9 0.00017048 -2.27996 SLC7A6 0.00029654 -2.27912 NEMF 2.21E-05 -2.27794 CLCF1 0.00166181 -2.27789 MINA 0.00010059 -2.27758 FAIM 0.00131699 -2.27717 FAIM 0.00131699 -2.27717 CSNK1G2 6.04E-08 -2.27675 MCM4 1.69E-07 -2.27636 USP37 1.54E-06 -2.27559 DMTF1 6.08E-07 -2.27541 XPO5 2.00E-07 -2.27539 CENPA 0.00098926 -2.27511 PSPC1 8.82E-07 -2.27426 PSTK 0.00114317 -2.27411 C12orf45 1.04E-08 -2.27323 MPHOSPH6 0.00031963 -2.2732 PPWD1 0.00280872 -2.27318 EIF2B1 3.42E-06 -2.27308 ZNF84 0.00017052 -2.27274 NECAP2 4.82E-06 -2.27242 TNFRSF1A 1.05E-07 -2.27241 METTL2B 0.00310113 -2.27201 MBOAT7 4.90E-05 -2.27035 CHMP6 2.71E-05 -2.26932 TGS1 0.00013544 -2.26865 EP400NL 6.67E-06 -2.26841 ZNF559 0.00014372 -2.26757 FAM118B 2.95E-07 -2.26737 PHTF1 2.35E-05 -2.26726 CHEK1 2.64E-05 -2.26717 SGOL1 6.69E-06 -2.26716 RQCD1 0.00907505 -2.26657 ZNF692 9.19E-07 -2.26654 TOMM34 3.10E-08 -2.26636 SNRPA1 2.23E-06 -2.26606 ARFGAP1 1.50E-06 -2.26541 RPP25 0.00080844 -2.26442 SETDB1 1.30E-07 -2.26401 ETF1 4.08E-07 -2.2638 ZNF420 0.00764975 -2.26376 CCDC18 0.00146965 -2.26335 IKBKE 0.00013365 -2.26323 CDK5RAP3 8.74E-08 -2.26318 DNA2 2.31E-05 -2.26304 TMEM168 2.33E-06 -2.26245 RRP9 1.19E-05 -2.26202 SVIL 0.00098877 -2.26139 TRIM15 0.00113815 -2.26062 MAP4K4 0.00067061 -2.25996 ZNF234 0.00072961 -2.25967 EXOC6 0.00032692 -2.25947 EIF3J 1.68E-06 -2.25922 ESF1 0.00186764 -2.25918 MAP4K4 4.04E-05 -2.25878 MECOM 3.95E-08 -2.25867 FUBP1 0.00016483 -2.25848 LRRFIP1 5.95E-06 -2.25772 SOCS4 7.96E-07 -2.25685 SLC28A3 0.00095781 -2.2567 ZNF214 0.00043395 -2.2564 TOMM5 7.68E-07 -2.25563 ZBTB45 2.61E-05 -2.2556 CNDP2 2.49E-05 -2.25553 TIMM50 3.72E-06 -2.25433 EIF5B 0.00012201 -2.25427 PABPN1 0.0007775 -2.25379 MTERFD3 0.00185721 -2.25174 WDR43 3.66E-07 -2.25126 FAM160B1 0.00090462 -2.25113 SRRT 2.93E-07 -2.25075 DIEXF 7.65E-05 -2.24976 WDR89 3.10E-06 -2.24976 RPL7L1 1.29E-08 -2.2489 SMARCE1 1.07E-06 -2.24796 FAM20B 2.24E-06 -2.24747 ORC5 1.34E-05 -2.24667 CDC20 8.77E-05 -2.24654 CREB1 3.06E-05 -2.24547 NCAPG2 4.50E-08 -2.24547 HRAS 2.82E-06 -2.24479 CDC42EP2 2.02E-05 -2.24456 UBA5 2.87E-05 -2.24422 SVIL 0.00749945 -2.24419 C18orf21 1.61E-06 -2.24378 CELF1 0.00010255 -2.24348 FAM123B 4.42E-06 -2.24312 CFLAR 8.91E-07 -2.24295 PSMB9 0.00096277 -2.24289 CDC45 2.72E-07 -2.2412 PSMG3 4.19E-07 -2.24107 RBM48 0.00055866 -2.24105 FAS 0.00160039 -2.24061 CD59 3.01E-05 -2.24038 URB2 4.77E-05 -2.23959 BCLAF1 0.00639861 -2.23952 FBXO9 1.71E-05 -2.23925 C14orf159 1.98E-05 -2.23906 ITGA6 3.23E-05 -2.2389 TNRC6A 3.24E-06 -2.23876 KPNA1 0.00015492 -2.23861 POLE3 4.94E-07 -2.2383 TSR1 0.00045717 -2.23799 BTG3 1.87E-05 -2.23768 ZNF225 0.00208078 -2.23736 CCDC71 1.53E-05 -2.23682 FARSB 1.64E-07 -2.23679 MFAP3 7.91E-06 -2.23666 DDX27 0.00035297 -2.23616 PSMB10 9.74E-05 -2.23599 TRAF2 2.19E-09 -2.23579 FSBP 0.00024436 -2.23476 SNRPD1 1.05E-07 -2.23455 ZNF141 0.00015672 -2.23432 TSEN15 0.00012247 -2.2336 MRPS10 7.43E-07 -2.23353 FBXL5 0.00187519 -2.2334 TRA2A 2.99E-05 -2.23325 BAG2 0.00138245 -2.23321 ZNF562 2.79E-05 -2.23239 UBE2F 1.03E-05 -2.23231 TYW5 0.00022517 -2.23217 METTL2A 2.49E-05 -2.23206 WHSC1 0.00012228 -2.23168 LRRC58 1.05E-06 -2.23137 RPL7L1 8.61E-05 -2.23027 DNAJC24 0.0007928 -2.22943 C11orf83 1.17E-05 -2.22898 LOC10012788 3.66E-07 -2.22879 AAGAB 4.71E-06 -2.22868 PARPBP 0.00724327 -2.22782 INCENP 8.11E-05 -2.2273 LTB4R 4.11E-08 -2.22606 ANXA1 0.00014041 -2.22591 DNAJC9 1.69E-05 -2.2259 RAB11FIP2 7.58E-05 -2.22531 GEN1 2.01E-05 -2.22515 ITGA6 3.21E-07 -2.22494 SMG5 9.49E-07 -2.22443 QTRT1 4.03E-06 -2.22412 EBLN2 0.00020544 -2.22363 IK 9.92E-08 -2.22362 COBL 0.00025222 -2.22215 VEZT 4.81E-05 -2.22213 PPHLN1 1.09E-05 -2.2214 MTHFSD 5.45E-06 -2.22047 ZNF330 5.47E-05 -2.21967 TRMT61B 9.13E-05 -2.21914 AGAP4 6.21E-07 -2.21822 SRPK1 2.33E-06 -2.21744 ELOF1 1.37E-05 -2.21671 PHLDB2 0.00217396 -2.21656 FAM176C 0.00218543 -2.21625 CDK6 9.63E-07 -2.21601 BRWD1 0.00023744 -2.21559 BRWD1 0.00053046 -2.21514 FCHSD1 8.01E-06 -2.21461 CEP44 0.0001207 -2.21441 CNOT1 1.46E-05 -2.21438 USP31 0.00166842 -2.21393 CLPX 6.03E-06 -2.21327 DDX24 5.83E-06 -2.21317 C17orf53 0.00015191 -2.21241 AMIGO2 5.47E-07 -2.21217 TNRC6B 0.00093253 -2.21194 CLK2 4.14E-07 -2.21078 ATP11C 4.46E-05 -2.21021 DDX42 2.39E-07 -2.20967 PSAT1 0.0111468 -2.20868 MRPS12 8.41E-08 -2.20835 APOBEC3A 0.00010884 -2.20831 CHTOP 2.31E-09 -2.20765 PSME4 7.08E-08 -2.20517 KIN 3.39E-07 -2.2048 FAM110A 0.00010099 -2.20471 FGFR1OP 6.42E-08 -2.20464 EIF5B 0.00016354 -2.20411 EIF4E2 4.16E-06 -2.2041 WDR53 0.00025642 -2.20307 ZNF101 5.51E-06 -2.20292 CENPN 0.0017132 -2.20286 ZSCAN20 4.93E-05 -2.20217 RPL7L1 1.15E-07 -2.20209 CDC123 1.04E-08 -2.20199 PSMC1 2.91E-07 -2.20143 SS18 1.64E-06 -2.19997 RPL7L1 3.10E-06 -2.19972 C10orf137 0.00103847 -2.19958 ZNF2 0.00056442 -2.19939 OGT 9.21E-06 -2.19928 RICTOR 9.21E-06 -2.19925 NASP 1.67E-08 -2.19907 NMI 0.00083481 -2.19878 RPUSD4 2.42E-08 -2.19851 MIR17HG 0.00451365 -2.19816 NFATC2IP 0.00018663 -2.19809 PPP1CA 1.72E-06 -2.19773 EIF2S1 3.62E-06 -2.19771 TRIM32 2.21E-07 -2.19697 DGCR14 0.00331988 -2.19664 CATSPER2 6.07E-06 -2.19658 CFLAR 2.64E-05 -2.19425 VTI1A 9.07E-05 -2.19422 LRRC58 1.45E-05 -2.19395 PANK3 5.16E-05 -2.19315 WDR85 2.96E-06 -2.19275 C3orf17 1.50E-05 -2.1923 ABCE1 0.00418142 -2.1901 MTBP 0.00024376 -2.19002 C3orf17 0.00019905 -2.18992 LMO7 0.00011689 -2.18926 TXNL4B 2.31E-05 -2.18916 TMA16 0.00064334 -2.18883 FANCF 2.67E-05 -2.18859 C15orf63 1.29E-06 -2.18828 ZMAT3 0.00209742 -2.18805 SEC22A 1.17E-05 -2.18791 NUP210 0.00835121 -2.1879 ZNF169 9.06E-06 -2.18742 TIMM21 5.27E-10 -2.18661 ZNF585A 7.56E-07 -2.18628 AP3M2 0.00394953 -2.18622 NFATC2IP 3.27E-05 -2.18605 CDK1 0.00223172 -2.18502 CCDC64 3.16E-08 -2.18488 SLC25A15 8.62E-05 -2.18478 FANCA 2.78E-05 -2.18443 PPAPDC2 0.00661662 -2.18403 GCFC2 0.0159394 -2.18375 NSUN4 3.50E-07 -2.18325 PPTC7 1.25E-07 -2.18291 LRRFIP1 0.00012304 -2.18273 HRAS 3.41E-07 -2.18257 FBXO38 0.00047493 -2.18244 PPM1B 1.29E-07 -2.18236 SLC9A8 0.00019531 -2.1814 CHIC1 3.79E-06 -2.18115 C1orf131 1.21E-05 -2.18078 CDKN3 0.00158742 -2.18072 NUP155 4.90E-05 -2.18068 HNRNPD 0.003078 -2.18033 RPUSD4 4.20E-10 -2.18027 U2AF1 4.01E-08 -2.18012 EPM2AIP1 0.0010665 -2.17908 FOXRED1 1.32E-07 -2.17862 PNN 2.97E-07 -2.17852 HARS 3.49E-06 -2.17777 TRPS1 0.00338961 -2.17767 PARP2 1.39E-05 -2.17751 TXNIP 2.80E-05 -2.17717 UBE2V2 1.07E-07 -2.17658 SETX 1.39E-05 -2.17645 FANCA 1.60E-05 -2.17629 MRPL4 4.01E-05 -2.17587 BCLAF1 0.00457147 -2.17568 RUFY3 7.93E-05 -2.17559 SMG9 4.99E-05 -2.17534 MARS 0.00066526 -2.1753 STYX 4.05E-06 -2.17502 THAP5 0.00014752 -2.17447 WDR46 5.37E-05 -2.17435 SKA2 5.65E-05 -2.17399 MBD3 4.10E-05 -2.17362 MAP6D1 1.54E-05 -2.17344 LOC10028863 0.00013168 -2.17199 PHF19 0.00022001 -2.17141 CHRAC1 3.51E-06 -2.17121 IMP4 7.67E-07 -2.17069 ZNF343 1.88E-05 -2.16956 CDKL3 5.48E-05 -2.16922 SLC35B4 7.03E-06 -2.16899 ZNF202 1.37E-06 -2.16885 RNMTL1 4.52E-07 -2.16874 WDR91 0.00119878 -2.16815 SLC38A1 1.02E-05 -2.16803 CCNB2 3.95E-05 -2.16788 DNAJC10 0.00100103 -2.16784 DYRK2 2.58E-07 -2.16783 PDS5B 0.00191671 -2.16764 SNAPC4 4.31E-07 -2.16746 GTPBP5 4.12E-07 -2.16726 PWP1 0.00054219 -2.16689 TSN 3.55E-06 -2.1654 PNPT1 0.00020956 -2.165 BRIX1 7.87E-06 -2.16451 ZEB1-AS1 0.00013247 -2.16444 LYAR 0.00172309 -2.16346 PPIL2 1.27E-05 -2.16333 NUBP1 9.43E-05 -2.1631 FAM35A 1.75E-11 -2.16306 TRAPPC10 3.14E-05 -2.16254 CHUK 0.00016766 -2.16208 SDAD1 9.28E-06 -2.16149 LAS1L 5.47E-06 -2.16143 RUFY3 5.17E-05 -2.15933 POP5 1.01E-06 -2.15851 PELO 0.00034728 -2.15835 DENR 0.0022869 -2.15762 ACAD9 2.39E-06 -2.15738 MED20 4.50E-05 -2.15692 CWC22 1.49E-07 -2.1562 SDHAF2 3.23E-06 -2.15619 COMMD1 0.00010374 -2.15574 NMU 7.97E-05 -2.15572 ZNF512 0.00048949 -2.15538 RAD54L 2.18E-05 -2.15513 NEK3 3.17E-06 -2.15507 ZNF252P 2.16E-05 -2.15472 DPY19L1 4.95E-05 -2.15465 G2E3 0.00043634 -2.15426 FAM175B 9.25E-09 -2.15419 FXC1 9.09E-05 -2.15417 BEND3 1.32E-05 -2.15385 ZNF37A 6.79E-05 -2.15342 UBE2L3 0.00023632 -2.15314 MPHOSPH6 0.00018597 -2.15217 RNGTT 0.0101201 -2.15208 RMI1 6.74E-06 -2.15187 B3GALTL 0.00025775 -2.15141 DCAF13 2.00E-05 -2.15074 RUNX2 0.00049915 -2.15043 ENGASE 4.22E-06 -2.15032 SPRY1 0.00968016 -2.14951 SPRY1 0.00968016 -2.14951 FAM204A 9.21E-05 -2.14891 HIRA 4.50E-06 -2.14882 DNAAF2 0.00282507 -2.1486 RQCD1 1.15E-06 -2.14843 NUP43 2.54E-06 -2.1482 BMS1 8.17E-08 -2.14813 DSCC1 2.17E-05 -2.14738 SLC25A37 0.00085213 -2.14704 G6PD 1.98E-07 -2.14699 PDE12 2.69E-05 -2.14699 C5orf28 0.00014094 -2.14698 AKR1C1 0.00221839 -2.14688 FXN 9.14E-07 -2.14658 ZMYND19 6.17E-07 -2.14521 DNAJC16 0.00067405 -2.1447 SRSF10 0.00302646 -2.14443 TRIM35 0.00083241 -2.14417 ACER3 5.30E-06 -2.14404 RARRES3 0.00014259 -2.14399 SLC35B4 2.66E-05 -2.1439 THOC2 5.23E-08 -2.1439 MAP7D3 2.67E-05 -2.14372 TPM3 0.00064321 -2.14354 ZNF638 1.27E-07 -2.14312 CLIC4 0.00502618 -2.14291 DNM1 6.33E-05 -2.14279 PPP1R14B 1.65E-07 -2.14219 SAP30BP 1.26E-06 -2.14185 MRPL39 0.00028995 -2.14127 CXCL11 0.00430007 -2.14094 SLC52A3 0.00254676 -2.14045 RNF126 5.36E-05 -2.14039 ZNF84 0.00010183 -2.13986 OFD1 9.89E-06 -2.13969 WHSC1 9.55E-05 -2.13963 PSMC1 4.18E-07 -2.1395 ATXN7L1 2.59E-07 -2.13877 ZNF302 0.00079365 -2.13877 EIF6 3.92E-09 -2.13847 CELF1 7.14E-07 -2.13843 BMP4 7.38E-06 -2.13819 ARPC5L 0.0001243 -2.13772 ANKFY1 0.00032522 -2.1377 FAM156A 0.002915 -2.13747 FAM126B 1.09E-05 -2.13653 SAC3D1 6.36E-06 -2.13644 EPT1 1.37E-08 -2.13621 CDK1 1.59E-05 -2.13604 LOC10050947 3.44E-05 -2.13594 RMI2 0.0002625 -2.13544 ARHGAP11A 0.00027683 -2.1354 ETF1 1.61E-07 -2.13515 ST7 0.0003424 -2.13504 CSTF2 0.00349947 -2.13454 MAOB 2.63E-06 -2.13374 EHD4 8.84E-09 -2.1336 YWHAH 4.31E-06 -2.13349 PNN 1.73E-09 -2.13336 NOL12 2.27E-06 -2.13323 C1orf43 0.00027909 -2.1329 FAM83H 4.06E-07 -2.13248 PSMD12 3.85E-07 -2.132 PSMD12 4.90E-06 -2.13183 STAG3 1.43E-06 -2.13178 AMMECR1 0.00037147 -2.13122 ZNF506 0.00045251 -2.13113 ZDHHC17 0.00126225 -2.13087 LAMC2 0.00151237 -2.13071 RAET1L 0.00018413 -2.13012 TRAPPC2 2.13E-09 -2.12986 MICALL2 0.00029231 -2.12975 PSMD12 1.25E-07 -2.12958 ZNF613 1.73E-06 -2.12902 SRSF5 2.36E-06 -2.12856 RNF146 7.88E-06 -2.12823 S100A16 0.0003308 -2.12793 HNRNPD 5.60E-07 -2.12776 SNRPB2 7.70E-05 -2.12769 BCAS2 8.13E-05 -2.12747 MIS18BP1 0.00064168 -2.12689 ZNHIT6 0.00808924 -2.12616 CENPK 0.0001373 -2.12594 WDR67 0.00029394 -2.12572 GOT2 1.38E-07 -2.12531 PLD6 0.00014675 -2.12449 PHC3 9.92E-08 -2.12435 MYBBP1A 5.78E-07 -2.12413 CDC7 1.36E-05 -2.12378 IFI35 1.64E-06 -2.12376 TIMM21 2.91E-05 -2.12373 PPP6R3 3.84E-08 -2.12353 PHF5A 0.00023151 -2.12326 ELAC1 1.22E-06 -2.12297 ZNF292 4.95E-07 -2.12275 RALGPS2 1.33E-05 -2.12202 FAM129A 0.00164249 -2.12171 QTRTD1 0.00014032 -2.12167 KRI1 8.44E-06 -2.12147 EMILIN2 1.56E-05 -2.12085 HERC2P4 1.55E-05 -2.12051 TRDMT1 0.0133812 -2.12007 BIRC5 0.00055014 -2.11984 LEO1 5.28E-06 -2.11979 DKC1 0.00049395 -2.11912 FAM76B 6.84E-06 -2.11796 ZDHHC3 1.52E-05 -2.11796 DDX24 1.16E-06 -2.11774 TTI2 7.99E-07 -2.11744 ZBTB39 0.00010791 -2.1174 TOE1 6.67E-08 -2.11585 SAPCD2 2.21E-05 -2.11561 HCCS 0.00073374 -2.11548 RAD1 0.00137541 -2.11534 NFKBIB 0.00077133 -2.1153 SPRED1 1.39E-06 -2.11489 PGBD1 0.00322491 -2.11483 NAT10 1.29E-07 -2.11419 NOL6 5.27E-05 -2.11412 TIMM8A 4.22E-05 -2.1141 PDSS1 0.00055539 -2.11366 NEMF 0.0001675 -2.11358 TRIM47 2.92E-07 -2.11356 ATP6V0A2 0.0001425 -2.11288 RBM8A 2.07E-06 -2.11281 NSRP1 9.93E-06 -2.11228 GCSH 0.00042182 -2.11102 MZF1 3.46E-06 -2.11094 ZWINT 6.34E-07 -2.10947 SAPCD2 5.70E-06 -2.10936 ZNF317 6.57E-05 -2.10934 ZNF419 0.00068797 -2.1087 WDR20 0.00085308 -2.10857 VANGL1 0.00054713 -2.10823 POLA2 5.68E-05 -2.10809 AP5S1 3.76E-06 -2.10802 CFLAR 1.18E-06 -2.108 LOC10050975 5.79E-06 -2.10797 EIF2B1 0.00136178 -2.1077 PIK3C3 3.05E-05 -2.10725 LOC128322 0.00010868 -2.107 TTF2 0.00090942 -2.10699 VHL 0.000144 -2.10657 KIF11 3.14E-05 -2.10651 AP2S1 1.23E-06 -2.10644 NT5C2 0.00112439 -2.10643 RANGAP1 4.18E-06 -2.1063 PRPF39 0.00097174 -2.1062 SRSF8 1.06E-06 -2.1053 C5orf54 0.00067126 -2.10527 ASXL2 0.0001172 -2.10477 RAB23 0.00119366 -2.10449 GTPBP3 1.78E-06 -2.10444 ATXN3 0.00533732 -2.10429 NUP35 0.00079814 -2.10426 ADCK5 4.31E-05 -2.1042 QTRT1 4.44E-08 -2.10378 MPP3 0.00012309 -2.1036 DRAM1 0.00028478 -2.1035 ALKBH8 0.0042024 -2.10329 MRE11A 0.0138347 -2.10307 MORC4 6.23E-05 -2.1027 SMAD3 0.00031888 -2.10203 LRFN4 2.23E-06 -2.10178 RPF2 7.47E-08 -2.10152 FAM102B 0.00318393 -2.10128 CSAD 0.00825862 -2.10115 KDM5A 2.29E-05 -2.10087 GSG2 1.06E-06 -2.10084 ZNF35 5.66E-05 -2.10079 SYMPK 0.00028247 -2.10065 PHIP 6.54E-06 -2.10035 CARS2 0.0006523 -2.10015 RNF4 5.37E-06 -2.09989 LUC7L3 0.00022111 -2.09952 EXPH5 3.11E-05 -2.09892 DDX55 0.00014427 -2.09863 MAGEA2 0.00041173 -2.0979 MON1A 0.00016977 -2.0976 CD3EAP 6.77E-06 -2.09757 ABCF3 1.63E-05 -2.09716 IFI30 5.81E-05 -2.0963 C10orf2 1.42E-06 -2.09629 SND1-IT1 0.00012831 -2.09611 GNPTAB 0.00026176 -2.0957 XRCC6 1.18E-05 -2.09569 RPP30 3.82E-06 -2.09525 TRIM23 0.00011263 -2.09523 CACYBP 2.89E-06 -2.09417 C1orf216 0.00051696 -2.0941 PPWD1 0.00013516 -2.09407 MTF2 0.00091493 -2.09385 CREBZF 7.08E-07 -2.09379 CYB5B 0.00102044 -2.09358 SRSF5 0.00015073 -2.09334 PLEK2 2.65E-09 -2.09331 AGGF1 5.33E-05 -2.09323 ZNF280D 0.00129392 -2.09318 ANXA11 5.48E-05 -2.09311 ETF1 9.13E-08 -2.09241 CSTF3 9.03E-06 -2.09237 ANKS4B 0.0001965 -2.09228 TRIM21 0.0003914 -2.0917 NAT10 1.51E-05 -2.09129 AMIGO2 0.00160979 -2.09098 EIF3B 2.51E-08 -2.09082 RIOK1 0.00152567 -2.09017 N4BP2L2 0.0005164 -2.09014 GCSH 7.35E-07 -2.09003 DND1 2.49E-05 -2.08988 RBM14 2.47E-06 -2.08973 SETD8 1.11E-08 -2.08922 TSTA3 5.15E-08 -2.08871 SNHG9 1.52E-07 -2.08859 NETO2 9.84E-05 -2.08858 SSH1 2.30E-06 -2.08733 VWA2 3.10E-06 -2.08728 DNTTIP1 1.78E-06 -2.08713 MRPS23 1.67E-05 -2.08703 KIAA1598 0.00185994 -2.08688 BLMH 0.00019175 -2.08678 HP1BP3 1.16E-06 -2.08663 IKBKE 6.87E-06 -2.08656 KIAA1432 0.0142993 -2.08647 TP53RK 5.82E-05 -2.08636 ZNF133 0.00010258 -2.08634 UMPS 4.13E-05 -2.08582 TXLNA 1.60E-07 -2.08532 NOX1 0.00023436 -2.08526 OGFOD1 0.00112905 -2.08494 FBXO28 4.14E-08 -2.08489 GINS1 0.00078369 -2.0847 ARHGAP11A 0.0014201 -2.08468 ZNF121 0.00215404 -2.0844 EWSR1 1.58E-06 -2.08404 RRP15 6.66E-05 -2.08384 DEF8 0.00016782 -2.08324 CDKN2AIPNL 8.39E-05 -2.08321 EXOSC8 1.24E-06 -2.08277 POLR1C 1.13E-05 -2.08241 FAM108C1 3.94E-08 -2.08236 CDC42 0.00088934 -2.08215 UBE2F 0.00281295 -2.08212 C12orf66 0.0001832 -2.08174 ICT1 8.73E-05 -2.08174 MTAP 0.00533997 -2.08147 PNISR 0.0145818 -2.0814 C17orf70 3.60E-07 -2.08122 TMEM214 3.74E-06 -2.08081 MCTP1 0.00920478 -2.08067 IDH3A 0.00016844 -2.07939 ZNF643 0.00027893 -2.07813 MSH6 4.90E-05 -2.07798 SPIN2A 9.60E-05 -2.07779 SENP5 3.18E-05 -2.07769 PDS5A 0.00682534 -2.07761 TAP2 0.00082563 -2.07745 RTKN 3.02E-05 -2.07719 HEATR7A 1.78E-06 -2.07685 ZNF680 4.14E-07 -2.07665 RUVBL1 9.52E-08 -2.07601 PWWP2A 8.38E-08 -2.07582 RICTOR 4.27E-08 -2.0756 DCBLD2 0.0160214 -2.0755 CDK5RAP1 2.58E-06 -2.0749 RFC3 1.28E-07 -2.07485 GCSH 1.89E-07 -2.07427 SUB1 2.97E-06 -2.07422 EDARADD 0.00011451 -2.0733 FAM64A 0.00135937 -2.07326 GTF2H4 1.51E-06 -2.07325 PARP9 0.00102024 -2.07252 KCTD10 3.27E-06 -2.07249 RBM5 1.48E-05 -2.07185 RBM48 0.00029498 -2.07148 SUPV3L1 6.63E-05 -2.07143 GPATCH2 0.0068567 -2.07134 HNRNPA3 1.39E-07 -2.0711 ZKSCAN1 0.00010068 -2.0711 TMC7 0.00037624 -2.07109 DPH2 0.00285535 -2.07096 RPS6KB1 0.00674431 -2.07062 HNRNPA3 3.64E-07 -2.06939 KIAA0232 0.0006334 -2.06935 ARAP2 0.00342688 -2.06932 C5orf43 3.94E-05 -2.06917 MTF1 2.03E-05 -2.06907 ANXA1 0.00038784 -2.069 PSMC4 4.05E-06 -2.06882 FLJ39639 1.42E-06 -2.06862 ACP6 0.00011244 -2.06835 PDGFB 0.00479928 -2.06833 SLC25A27 0.00030726 -2.06823 GLS 0.00309403 -2.06739 CYP2R1 0.00070061 -2.06652 GTF3C3 0.00040356 -2.06628 MEF2BNB 0.00959195 -2.06614 RPL7L1 1.07E-05 -2.06543 RWDD1 1.04E-05 -2.06515 EIF2S1 6.76E-06 -2.06479 RAP2B 0.00305887 -2.06379 UBA6 4.52E-06 -2.06364 CCDC120 2.95E-06 -2.06354 SAE1 9.20E-06 -2.06296 MTAP 0.0061365 -2.06251 MPZL1 8.04E-05 -2.06179 ZNF33A 0.00148216 -2.06032 MMACHC 0.00011373 -2.0598 ZNF202 0.00015671 -2.05979 CXorf23 0.0020223 -2.05976 PUM2 1.64E-08 -2.05961 PEX13 7.57E-05 -2.0592 NUDT19 1.81E-05 -2.05859 CTAGE5 0.00021448 -2.05846 LANCL2 0.00010702 -2.05822 POLR3A 1.24E-06 -2.05795 HAUS2 5.58E-06 -2.05783 TUBGCP6 3.61E-05 -2.05751 UBE2F 0.00054662 -2.0573 CATSPER2 0.00042855 -2.05724 NSUN5 2.43E-05 -2.05724 TDG 5.13E-09 -2.05715 TRIM44 1.43E-06 -2.05697 ESCO2 0.00340786 -2.05682 TFCP2 0.00336651 -2.05663 KANK1 1.36E-05 -2.05631 UBXN8 0.00027007 -2.05574 POLR3C 0.00465652 -2.05541 METTL3 0.00373315 -2.05532 ADAR 2.75E-07 -2.05382 TBC1D1 0.00172823 -2.05375 ERGIC2 0.00464736 -2.05337 GTF2H3 0.0128673 -2.0533 BTN3A3 8.31E-05 -2.05225 NF1 0.0008352 -2.05213 UBE2L3 6.41E-07 -2.05154 MPP3 2.81E-05 -2.05107 IRF3 1.58E-07 -2.0504 MST1R 0.00013281 -2.0504 KIF4A 4.00E-05 -2.05038 PPP2R3C 0.00042063 -2.04962 WDR90 1.90E-08 -2.0489 LYPLAL1 0.00075915 -2.04831 KDM1B 1.27E-05 -2.04814 TMEM19 0.0006161 -2.04727 CDC37L1 0.0027239 -2.04644 BRCA1 0.00037022 -2.04621 ZNF561 0.00034787 -2.04583 DGKE 0.00173106 -2.04553 LINC00514 0.00066237 -2.04499 HS3ST1 2.06E-05 -2.04471 PAFAH1B1 3.30E-06 -2.04468 TIRAP 6.87E-06 -2.0446 HKR1 8.30E-06 -2.04459 NOP9 9.76E-05 -2.04342 FEM1A 0.00015869 -2.04341 SCEL 0.010062 -2.04339 PLRG1 0.00064916 -2.04324 CDC37L1 0.00841245 -2.04306 LRRC8E 0.00027944 -2.04244 RER1 4.39E-06 -2.04212 GNPNAT1 0.00373139 -2.04207 PALB2 0.00058195 -2.0418 ERAP2 0.00027669 -2.04129 ACTR1A 9.62E-05 -2.04125 TNPO2 6.98E-07 -2.04095 WDR3 2.04E-05 -2.04069 MCM3 2.03E-05 -2.04041 SDF2 4.43E-07 -2.0404 ZWILCH 0.00038951 -2.04029 SENP1 0.00271793 -2.03967 PDCD6 0.00341052 -2.03911 ZGPAT 1.46E-06 -2.0391 CAPN2 0.00045394 -2.03873 C16orf53 4.19E-06 -2.03816 MTO1 0.00441537 -2.037 RRM1 4.50E-07 -2.03693 ARPC5L 0.00018586 -2.03689 RABIF 0.0002159 -2.03681 PITPNA 0.00017359 -2.03669 GOSR2 6.80E-05 -2.03653 TRIM38 0.0002403 -2.03623 MCM3 2.48E-05 -2.03585 EYA3 9.64E-06 -2.03553 BFAR 8.68E-06 -2.03476 ATAD3A 7.14E-08 -2.03447 SMAD2 0.00439832 -2.0344 FKBP15 0.00013017 -2.03421 SLC38A1 6.65E-06 -2.03356 ABCF3 1.25E-05 -2.03351 ABHD10 0.00934195 -2.03345 SP1 9.07E-06 -2.03345 C12orf29 0.00023541 -2.03335 CFLAR 1.56E-05 -2.03334 EIF3B 1.87E-06 -2.03311 LIPT2 0.00025954 -2.03299 BRMS1 5.47E-08 -2.03267 KIF9 5.10E-06 -2.03267 MNF1 1.51E-05 -2.03263 CHMP1A 1.79E-05 -2.03207 UBASH3B 0.0145191 -2.03199 WDR77 1.24E-05 -2.03168 RAB14 0.00050334 -2.03147 AKR1C2 0.0007285 -2.03117 PTHLH 0.00132331 -2.03067 NUF2 0.00291945 -2.02963 BFAR 8.68E-06 -2.02947 CNOT2 2.95E-06 -2.02933 DDX60L 0.00291397 -2.02929 LRRC59 2.24E-06 -2.02917 CRYGS 0.00032521 -2.0291 ELK4 1.24E-05 -2.02908 GEMIN2 0.00998152 -2.02907 SNRPA 2.94E-05 -2.02851 SURF2 4.54E-05 -2.02845 DPY19L1 0.00030476 -2.02807 CXorf56 0.00043863 -2.0277 ACSL5 5.96E-05 -2.02764 MRPL47 4.51E-07 -2.02764 RBM12 0.00046201 -2.02753 IKBKB 0.00054185 -2.02735 CDC37 6.21E-06 -2.02644 ERGIC2 2.83E-05 -2.02619 AKR1C1 0.012038 -2.026 NUP35 0.00018843 -2.02574 ENOSF1 5.83E-06 -2.02566 CCDC150 0.0001765 -2.02555 C4orf27 1.01E-05 -2.02554 NDUFV3 0.00199945 -2.02493 ZHX1-C8ORF7 0.00030583 -2.02468 TMEM43 0.00051972 -2.02453 ZNF37A 0.00261767 -2.02448 SLC7A6 0.0001077 -2.02445 RAD51AP1 0.00103639 -2.02442 TIGD5 2.84E-05 -2.02437 AURKAIP1 6.34E-06 -2.02424 EXOSC10 5.18E-05 -2.02398 IRGQ 0.00116758 -2.02392 SLC3A2 5.39E-06 -2.02299 TATDN2 4.12E-07 -2.02274 KLF12 6.82E-06 -2.02267 TP53BP1 0.00025044 -2.02232 TRIM5 0.00049298 -2.02217 FUBP1 7.48E-05 -2.022 DTYMK 0.0001158 -2.02193 AAGAB 2.09E-07 -2.02139 CTNNB1 1.40E-05 -2.02134 DHFRL1 3.13E-07 -2.02111 KARS 2.24E-08 -2.02077 ATMIN 6.18E-06 -2.0207 ZNF83 4.61E-08 -2.02058 MED6 0.00113293 -2.02016 KLHL9 0.00010032 -2.02007 HSP90AA1 5.88E-06 -2.01953 SNRPB2 9.12E-07 -2.01924 MAGOH 0.00172907 -2.01918 TRIM41 4.16E-06 -2.01909 CMTM7 3.52E-08 -2.01891 MAGEA3 2.23E-06 -2.01874 RHOT2 2.29E-07 -2.01862 PSMD13 3.02E-07 -2.01721 TAF9 1.60E-05 -2.0172 CDC6 0.00063522 -2.01664 RPGRIP1L 0.00282198 -2.01653 KPNA4 6.74E-06 -2.01579 MTERFD2 0.0003975 -2.01564 EP400NL 3.62E-05 -2.015 DTX3L 0.00011589 -2.01497 DMAP1 3.58E-05 -2.01453 DYRK2 1.63E-05 -2.01439 SGOL2 0.0142634 -2.01387 A4GALT 0.00209347 -2.01331 MIER3 1.90E-05 -2.01328 RALGAPA1 7.47E-05 -2.01273 CDK5RAP1 3.72E-05 -2.01229 ANKRD40 3.15E-05 -2.0121 TNFRSF1A 9.85E-05 -2.01204 G6PD 1.64E-05 -2.01171 RNF216P1 3.68E-09 -2.01152 CENPP 5.95E-05 -2.0115 ARID5B 0.0002626 -2.01145 GMPPA 1.79E-05 -2.01125 NUSAP1 0.0002038 -2.01079 LIPG 2.16E-05 -2.01072 LIF 0.00551461 -2.01044 CCNF 0.00035692 -2.00969 MIR3661 9.42E-08 -2.00955 ZC3H10 4.21E-05 -2.00945 LRRC8C 2.71E-05 -2.00892 DOPEY1 0.00020503 -2.00865 RFT1 1.75E-05 -2.00839 POLR3H 0.00010032 -2.00826 HMOX2 2.27E-06 -2.00821 TSTA3 3.59E-07 -2.00694 ZNF586 0.00098014 -2.00676 TNFAIP8 0.00146609 -2.00529 EEF1E1 0.00195802 -2.00473 LOC10028751 0.00010488 -2.00421 ZNF260 1.81E-08 -2.00345 CYTH2 2.00E-06 -2.00328 RPIA 5.08E-05 -2.00306 SHFM1 0.00235945 -2.003 GMNN 2.62E-07 -2.0028 NMI 0.00491208 -2.0025 ATG16L1 0.0006085 -2.00249 EIF5B 8.14E-05 -2.00224 ATF5 0.00027246 -2.00158 GJB2 9.92E-05 -2.00097 PSMG4 5.69E-05 -2.00064 RDH13 0.00275415 -2.00054 SCEL 0.00712986 -2.0005 PLRG1 0.00755919 -2.00046 SLK 2.40E-06 -2.00041 TRMT10C 0.0059645 -2.00021 TAF15 3.22E-05 -2.00018 LOC729020 3.46E-05 -2.00015 SUMF2 5.95E-07 2.00049 MUC5B 1.33E-05 2.0008 FMNL2 0.00416206 2.00082 ZNF137P 1.07E-06 2.00091 COL21A1 0.00399514 2.00127 ATP8A1 1.13E-07 2.00148 AGAP1 0.00016621 2.00179 ITGB1BP1 6.00E-05 2.00198 HBP1 2.20E-09 2.00216 DMXL2 0.00070535 2.00224 PLS3 0.00269789 2.00318 RPL31 0.00013649 2.00332 LRRC8C 1.03E-06 2.00408 APOE 1.57E-06 2.00449 UBE2H 9.35E-06 2.00477 SLC5A8 2.66E-05 2.00498 SLC12A6 6.63E-06 2.00506 ABI1 0.00020672 2.00512 MAML3 8.62E-07 2.00519 NPC1L1 5.04E-05 2.00534 PDE6D 0.005813 2.00572 TRIM4 1.50E-05 2.00602 ARID1B 3.09E-06 2.00625 LDLR 0.00021033 2.00659 PTPRF 3.08E-07 2.00672 FAM210B 0.00011438 2.00767 CAPN10 0.00010319 2.00817 PLAT 0.00020042 2.00878 CREBBP 0.00011907 2.00899 ACVR1 0.00043286 2.00908 BCORL1 0.00078191 2.00968 TFF1 0.00076375 2.01025 TCF12 0.00012219 2.01036 BDH2 1.18E-07 2.01046 HERC4 2.41E-06 2.01062 BAGE2 0.00499657 2.01078 FGFR3 0.00023712 2.01116 CEACAM5 0.00171174 2.01129 CCDC92 1.13E-05 2.01237 WRB 1.42E-05 2.01257 MAOA 4.57E-09 2.01335 AGAP1 0.00085872 2.01349 GFPT1 6.54E-06 2.01352 CEP57 2.54E-07 2.01364 SF3B1 0.00857258 2.01373 RAB3A 4.03E-05 2.01478 UPRT 0.0101509 2.01496 DYNC2LI1 5.10E-07 2.01499 INSIG1 1.21E-05 2.01507 SEZ6L2 0.00018441 2.01513 NMRK1 1.91E-05 2.01518 C16orf80 2.09E-07 2.0153 LIMCH1 0.00037338 2.01568 NABP1 0.0016052 2.01682 PNRC2 0.00110942 2.01688 SPRED2 0.00029101 2.01749 PKIB 8.68E-05 2.0185 LIMCH1 0.00067583 2.01882 TMEM117 0.00020136 2.01958 ASH1L 0.00116707 2.02066 EPHX2 4.65E-05 2.0212 TIA1 0.00090227 2.02191 CREM 7.95E-05 2.02269 TFAMP1 0.00019335 2.02347 THAP9 2.58E-05 2.02391 DCAF6 8.61E-05 2.02431 MSMO1 3.17E-08 2.02436 IGF1R 0.0001431 2.02459 SDHD 0.00110142 2.02483 SDHD 0.00110142 2.02483 SPATA13 0.00035071 2.02573 ZMYM5 0.00574781 2.02576 TMEM55A 8.44E-05 2.02585 SUMF2 1.17E-07 2.02702 SLC24A1 0.0001347 2.02793 SGMS2 0.00410775 2.02797 NEU1 3.41E-07 2.02824 PBX3 0.00221257 2.02832 ZNF750 9.84E-05 2.02912 PPP1CC 0.00056751 2.02987 CAMTA1 0.00557493 2.03046 DNM2 0.00062744 2.03096 HSD17B11 1.11E-09 2.03133 IL19 0.00025003 2.03152 PELI1 7.11E-08 2.03187 PTK7 0.00029131 2.03257 XRN2 0.00948463 2.03406 PROC 1.21E-05 2.03443 MYB 3.76E-05 2.03447 RALGDS 7.74E-05 2.03491 PILRA 1.19E-05 2.0353 C1orf27 0.00116803 2.03534 NR2F1 0.0011512 2.0358 WLS 4.58E-07 2.03621 AK4 0.00260267 2.0364 TAPT1 3.76E-05 2.03641 MPPE1 0.00026099 2.03651 1.52E-06 2.0366 PTMA 3.59E-05 2.0377 C5orf15 0.0092117 2.03775 AOC3 0.00042117 2.03794 SEMA6A 0.00100742 2.0381 PAPD7 0.00079173 2.03848 EIF2C2 8.09E-05 2.03852 PTP4A1 9.77E-06 2.03911 CXCL2 5.21E-05 2.03924 LNX1 0.00109709 2.04035 BRIP1 0.00144761 2.04047 TCF12 0.00048198 2.0408 MYOF 1.95E-05 2.04084 FLJ23867 2.23E-05 2.0411 FZD7 0.0003582 2.04116 GANAB 6.24E-05 2.04158 ITFG1 0.00029515 2.04212 N4BP2L2 0.00021516 2.04247 ACSM3 2.94E-08 2.04282 PHF3 0.00607996 2.04304 CCSAP 0.00087933 2.04326 ZFAND1 7.52E-05 2.0437 EGFL8 0.00066347 2.0438 PSMD10 1.70E-05 2.04436 SYBU 2.62E-06 2.04442 RAP2A 0.00014883 2.04456 POC1B 0.00073026 2.04506 HIST1H2BJ 0.00435 2.0464 FHDC1 4.18E-05 2.04674 ATP2A2 0.00014925 2.04682 PLK2 0.00309043 2.048 EPB41L5 4.50E-05 2.04816 BRI3 9.09E-05 2.04847 C11orf75 1.73E-05 2.0485 LOC648771 0.0121787 2.05002 SCAMP1 0.00075362 2.0506 PPP2R5E 0.00146482 2.05087 SEC24A 9.94E-06 2.05115 LOC10050654 3.27E-06 2.05137 CTSL2 1.02E-05 2.05183 CTBS 0.00076027 2.05195 ACSF2 0.00013579 2.05223 MBD5 0.00772931 2.05251 CEP120 0.00041648 2.05259 SUSD4 0.00071837 2.05264 RC3H1 0.00148163 2.05347 FGFR2 0.00096165 2.05432 SH3BGRL 1.95E-05 2.0544 BAZ1A 0.00145655 2.05446 ALDH3B1 0.0004664 2.05453 C6orf106 0.00374613 2.05466 ING1 0.00019633 2.05477 ARHGEF12 0.00013905 2.05494 LOC10028778 0.00025047 2.05505 PPP3R1 0.00565493 2.05524 RBFOX2 0.00074472 2.05575 CUL4B 4.98E-08 2.05615 BCOR 0.00010873 2.05664 ZBTB20 0.00129078 2.05665 SPATA13 0.00368242 2.05694 EXT1 2.01E-05 2.05702 GPR75 0.00066699 2.05784 RBM39 3.32E-05 2.0579 AHCYL1 0.00024763 2.05819 HOXB13 0.0002176 2.0584 TRIQK 3.20E-07 2.05992 OXR1 0.0005147 2.05999 MAPK9 2.16E-05 2.06022 BCL2L11 0.00240919 2.06105 TMEM217 0.00017954 2.0619 WDFY1 0.00467312 2.06239 EZR 0.0158997 2.06326 ZMYM6 1.33E-07 2.06367 USP43 0.00028796 2.06386 SLC6A13 0.00188052 2.06453 PARD3 0.00028658 2.06481 ACADM 0.00047115 2.0649 KRCC1 0.00021992 2.06569 CASK 0.00125409 2.06593 PSPH 0.00550542 2.0665 CLDN11 0.00329934 2.06686 HNRPDL 1.95E-07 2.06729 WDR26 1.61E-05 2.06741 METTL12 7.31E-06 2.06761 PICALM 0.00038285 2.06774 PHLPP2 2.24E-05 2.06829 GPRC5C 0.0001268 2.06847 SCAMP1 0.00071821 2.06864 RGL1 9.17E-06 2.06875 PBX1 0.00294382 2.06908 HPGD 2.25E-05 2.06941 FKBP10 1.90E-06 2.07044 HNMT 1.31E-06 2.07103 SENP7 4.11E-05 2.0712 AUH 0.00010097 2.07197 TMEM135 1.08E-05 2.07257 FGF7 0.00324994 2.07288 TBC1D23 0.00668998 2.07296 GULP1 2.66E-05 2.07297 CPNE8 0.00016621 2.07341 RAB5A 0.00032096 2.07372 SATB2 0.00127283 2.07427 ATP6V1A 0.00080352 2.07462 EPC2 0.00015994 2.07485 TRIM4 0.00280704 2.07509 PLAGL1 0.00286112 2.07529 PABPC4 7.75E-05 2.07636 SAT1 1.98E-05 2.07653 ALLC 0.00044412 2.07725 RAP1GAP 6.51E-05 2.07784 BBS1 7.77E-09 2.078 SLC23A1 0.00038023 2.07931 SNX16 0.00012481 2.07976 FOLR1 7.60E-06 2.07991 PRDM2 0.00016498 2.08019 KBTBD2 5.44E-07 2.08023 ZNF277 0.00263009 2.08103 EIF4B 4.97E-07 2.08158 EIF3F 0.00141713 2.08193 SPIRE2 0.00126209 2.08236 CAT 0.00012896 2.08274 SGK1 0.00242575 2.08366 ARL6IP1 0.00151062 2.08387 TXLNG 0.00029602 2.08389 KCTD10 1.35E-05 2.08412 IGFBP2 5.01E-05 2.08438 CAMLG 2.20E-06 2.08441 RASSF9 0.00472994 2.08481 AKAP7 4.05E-06 2.08556 SH3BGRL 0.00022143 2.08593 HIVEP1 0.00014467 2.08636 MCL1 0.00043071 2.08645 NOP2 0.00010399 2.08698 HIST1H3A 4.46E-06 2.08744 MGAT4A 0.00091036 2.08831 LNX1 0.00203864 2.08844 POT1 0.00165354 2.08847 RAPGEFL1 0.0022367 2.08927 MSL1 9.40E-06 2.08942 IGF2R 0.00116873 2.08966 UGDH 0.00023488 2.0898 KIAA0564 9.83E-07 2.08982 IRX2 2.74E-06 2.09061 SLCO2B1 5.64E-05 2.09176 COG6 0.00436137 2.09242 MYOF 3.26E-05 2.09266 FABP1 9.20E-07 2.09299 FAM46A 0.00016598 2.0932 ZBTB43 0.0122395 2.09352 CDH1 9.06E-07 2.09379 RAG1 0.00469782 2.09392 KAT6B 2.45E-05 2.09416 SOAT2 0.00036196 2.09485 ZC3H6 0.00374207 2.09495 TRIM24 0.00036974 2.09504 OSBPL11 0.00392419 2.09522 SIAH1 0.00139177 2.09581 ADH5 8.16E-05 2.09591 EEF1A1 5.41E-09 2.09602 DNAJB6 5.64E-05 2.09646 LMLN 4.19E-07 2.09682 EMP1 2.80E-08 2.09719 NCEH1 0.00954908 2.09719 TBC1D8B 0.00093741 2.09734 CSRP2 0.00017269 2.09754 RPL15 8.45E-06 2.09763 ALDH2 2.36E-06 2.09764 RLIM 1.38E-06 2.09809 ATP5G2 6.64E-10 2.0996 BCL2L15 0.00047595 2.09979 TP53AIP1 0.00010526 2.09985 WRB 0.00016869 2.10147 IGSF1 5.67E-07 2.10168 PAPSS1 0.00030338 2.10204 LGMN 3.72E-06 2.10266 UVRAG 1.07E-05 2.10315 SRSF5 0.0141631 2.10317 CDKL2 0.00063455 2.10405 CYP2B7P1 0.00056117 2.10438 KLHL24 8.41E-06 2.10439 IER5 1.22E-05 2.10443 BAZ1A 0.00139844 2.10483 ABCG1 0.00058893 2.10574 IQGAP1 0.00437538 2.10643 SMARCA2 0.00016577 2.10685 CCDC126 0.00672312 2.10798 PVRL3 0.00206075 2.10805 MYO5A 5.33E-05 2.10863 DDR1 3.28E-07 2.109 POU3F1 8.17E-05 2.10922 PREPL 0.00187229 2.10955 EPRS 0.00732291 2.10957 NRP1 0.00051253 2.11 PBX1 0.00043053 2.11007 LOC10028778 0.00684427 2.11013 NKX3-1 0.00029413 2.11018 SERPINI1 3.01E-05 2.11049 TMPRSS2 0.00011608 2.11208 MAP2K1 7.12E-06 2.11233 KIAA0319 0.00049345 2.11261 USP43 0.00025295 2.11292 BAZ1A 0.0017995 2.11338 ENO2 0.00078503 2.11372 LIPE 0.00027183 2.11478 TRIM33 5.91E-06 2.11505 GCC2 0.0137419 2.1152 CCDC115 7.67E-08 2.11543 NADKD1 0.00062802 2.11552 CERS2 0.00012042 2.11555 VEGFA 4.77E-08 2.11619 JDP2 0.00010033 2.11656 CAPS 2.16E-08 2.11661 AHCYL1 0.00014658 2.11744 STXBP5 0.00225834 2.11768 ARSJ 0.00112532 2.11887 FAM179B 7.28E-05 2.11974 CALB1 0.00430673 2.12009 CD46 0.00028185 2.1204 CHKA 8.91E-05 2.12079 FAM46A 6.96E-07 2.12107 CXCL13 1.14E-05 2.12164 NBPF1 8.56E-07 2.12229 CALM1 9.39E-08 2.1224 PRLR 0.0016955 2.12274 FAM84B 0.00036839 2.123 HACE1 0.00045204 2.12374 MACROD2 0.00125762 2.12374 EMP1 1.61E-05 2.1254 CCP110 0.00262045 2.12545 AKIRIN2 7.91E-08 2.12627 RIOK3 4.23E-07 2.12802 PLOD2 0.00097256 2.12819 GLRX 3.19E-06 2.12952 TNIK 0.00332667 2.12976 IGF2R 3.00E-07 2.13029 FAM46A 9.46E-05 2.13165 TRAK1 1.93E-05 2.13226 SNX16 0.00201503 2.13366 RHOT1 7.11E-08 2.13373 ABI1 6.02E-05 2.13445 SLC16A3 0.0129576 2.13519 AGMO 2.90E-06 2.13569 JDP2 6.17E-05 2.13612 HOXA2 0.0001736 2.13764 KLRC1 0.00031232 2.13824 PAPSS1 1.06E-06 2.13843 ETS1 2.03E-05 2.13846 SLC3A1 0.00157393 2.13918 SLC4A7 0.00693428 2.14038 HIVEP2 0.0007733 2.14111 CDC14B 0.00028917 2.14173 KIAA0182 0.00276255 2.14384 TRIB1 0.00030225 2.14428 UVRAG 0.00378767 2.14526 TMED4 1.80E-05 2.14592 ABI1 2.83E-05 2.14612 PQLC3 0.00244018 2.14631 FOSL2 2.72E-05 2.14693 Mar-03 0.00068662 2.14716 SLC2A10 1.03E-05 2.14797 CD44 0.00201423 2.14845 RIT1 5.05E-08 2.14875 SPAG9 1.05E-05 2.14896 LYPLA1 0.00044855 2.14937 OSR2 0.00017055 2.14969 CAPN10 0.00029435 2.14994 STAU2 0.00870898 2.15081 TMEM245 4.60E-07 2.15081 TFAP2C 1.68E-05 2.15142 LIMCH1 0.00374536 2.15153 GLUD1 0.00092979 2.15169 ITM2C 1.53E-06 2.1522 STK17B 9.79E-05 2.15227 PDZK1IP1 2.92E-07 2.15274 BCL6 0.00028157 2.15302 RAB27B 0.0001936 2.15309 FKBP9 0.00108501 2.1531 ITPR2 0.00115465 2.15347 ANKRD34C 2.77E-06 2.15348 SCGB2A1 2.06E-05 2.15487 ADD3 3.24E-06 2.15494 PJA2 0.00046891 2.15541 ELK4 0.00014641 2.15564 RPS15A 0.00026225 2.15573 MBIP 6.67E-05 2.15604 ZNF295 2.35E-07 2.15688 ADD3 1.52E-06 2.1574 DCAF4L1 0.00051875 2.15785 INSIG1 9.92E-06 2.15793 LMAN1 0.00452359 2.15826 MLLT10 0.00074574 2.15888 LOC10050764 0.0005385 2.15891 CA9 7.58E-05 2.15902 MPP5 0.00653617 2.15961 SPATA13 1.42E-05 2.16046 WLS 0.00040841 2.16126 S100A7A 0.00014493 2.16188 FLRT3 8.44E-05 2.1619 TMEM41B 0.00086651 2.16209 HIST1H4A 0.00073634 2.16226 CLRN3 2.99E-05 2.16232 TAB2 0.00015308 2.1634 REM2 1.93E-05 2.16386 CTCFL 5.91E-05 2.16408 PDE8A 1.32E-05 2.16492 PHF17 6.54E-05 2.1653 UNC119 0.00114677 2.16533 PRMT2 1.39E-05 2.16536 RABGAP1 0.0018716 2.16684 SYNJ1 0.00022635 2.16734 MMP20 7.54E-05 2.16738 CPEB4 2.53E-05 2.16761 DPYSL2 0.00012128 2.16763 TBC1D8B 8.71E-05 2.16764 FREM2 0.00018609 2.16782 ABI1 0.00038062 2.16824 C1orf64 0.00021781 2.16844 SIDT2 1.90E-06 2.17305 NR1D2 0.00026411 2.17368 ANKRA2 0.00144885 2.17393 RIOK3 0.00033187 2.17402 PTPLB 0.0145014 2.17499 MAN1A1 6.25E-06 2.1752 BBX 0.00528704 2.1753 ZFAND5 6.81E-06 2.17548 TPP1 4.65E-06 2.17554 PRPS2 0.00134578 2.17567 HIST3H2A 4.73E-06 2.17617 SPEN 3.24E-06 2.17629 SNX16 1.33E-06 2.17636 EEF1A1 3.24E-09 2.17652 SLC41A1 1.25E-05 2.17777 PPP2R5E 0.00168559 2.17812 AFF4 0.001072 2.17835 RINL 0.00261766 2.17839 RPS15A 3.47E-09 2.17881 Mar-02 2.41E-05 2.17927 ITGB4 0.00044033 2.17928 MXD1 6.65E-08 2.17982 RGS10 2.85E-07 2.18039 LIMS1 0.006899 2.18204 ZNF655 0.00168498 2.18258 DYNC1I2 1.57E-08 2.18266 MKRN1 0.0002508 2.18305 MBNL1 0.00766948 2.18347 VPS13B 3.75E-05 2.18373 PIP5K1A 8.38E-08 2.18408 PRMT2 1.46E-05 2.18551 SKAP2 0.00139441 2.18586 CD46 0.00250082 2.18594 FAM46B 1.02E-05 2.18598 EAF2 0.00021073 2.18621 CAMSAP2 0.00299649 2.18634 ZNF10 8.10E-06 2.18734 CPD 4.17E-08 2.18843 SLC2A2 0.00019172 2.18926 LIMCH1 0.00419721 2.18995 TRIM2 0.00025939 2.18998 CWC25 1.56E-05 2.19103 C6orf106 0.00277525 2.19139 EIF1B 1.66E-07 2.19229 EIF3L 6.33E-05 2.19231 FLRT3 2.79E-05 2.19259 DSG2 0.00020487 2.19265 HDGF 6.42E-05 2.19396 ITM2C 1.82E-07 2.19439 CDNF 0.00087437 2.19481 TRIM36 0.00133734 2.196 PIK3C2B 5.44E-06 2.19611 PPP4R2 0.00741291 2.19835 PPIL6 0.00185522 2.20009 FAM107B 0.00054897 2.20023 RAB6A 0.00293157 2.20047 TMTC2 0.00487515 2.2011 GALNT12 1.74E-05 2.20143 LRP5 2.81E-05 2.20189 ULK1 6.43E-05 2.20193 FBXO48 0.00203378 2.20263 ALB 6.11E-06 2.20301 MYCL1 5.68E-07 2.20409 SYBU 9.37E-08 2.20484 ABI1 0.00047676 2.20637 USP44 1.73E-06 2.20652 KAT2B 0.00027895 2.20706 ACTA1 6.03E-05 2.20735 WDR47 4.58E-05 2.20798 SEMA6A 0.00073155 2.20862 TSC1 2.48E-07 2.20905 CPT1A 1.42E-06 2.20916 PDK2 9.19E-05 2.21031 HADHA 0.00501956 2.21249 CST1 0.00020918 2.21325 ACADM 1.54E-05 2.21341 KIF3A 0.0054653 2.21341 RSF1 0.00015328 2.21463 FOXO4 9.74E-07 2.21471 SMARCA2 0.00392291 2.21572 YY1 0.00430502 2.21582 AK7 0.0017639 2.21585 SPRED2 3.26E-06 2.21748 FAM124A 6.43E-07 2.21794 HDAC11 3.67E-05 2.2184 LOC646214 0.00093798 2.21884 EEF2 4.09E-08 2.21899 KLF10 0.00441373 2.21983 RAPGEF2 1.29E-09 2.21996 C11orf54 0.00014416 2.22107 TTBK2 0.00544319 2.22113 CLIC3 0.00133383 2.22236 FBXO11 0.00178583 2.22249 CST1 0.00056767 2.22262 MUC5B 8.24E-05 2.22273 WDFY3 7.91E-05 2.22324 HTRA1 1.77E-05 2.22508 OSBPL8 0.00179532 2.22666 PGM1 7.39E-05 2.22674 CSRP2 0.00076104 2.22701 ITM2C 5.27E-06 2.22709 SLC26A2 0.00017612 2.2272 OAT 2.49E-07 2.22736 ZNF75A 1.72E-05 2.22777 ING1 1.60E-05 2.2283 HSPH1 7.25E-08 2.2287 SC5DL 1.25E-06 2.22911 MYO9A 2.70E-07 2.22972 SNAPC3 0.00245508 2.23011 AHNAK 7.97E-08 2.2309 DHX9 4.10E-05 2.23133 LOC728802 0.00313713 2.23133 SUSD4 3.23E-05 2.2315 CAPN7 0.00086183 2.2317 EPHX2 0.00125768 2.23233 CCNG1 0.00161676 2.23305 TIMP2 2.74E-06 2.2336 CRTAM 9.49E-06 2.23392 PRR20A 7.17E-05 2.2346 GNAS 5.05E-07 2.23473 SHROOM3 1.14E-08 2.23475 TPD52 3.49E-05 2.23547 ACBD5 2.94E-07 2.23663 LAPTM4A 6.75E-05 2.23669 ETV5 0.00311158 2.23722 LPP 0.00017914 2.2385 FREM2 0.00023163 2.23871 SPIN1 0.00684144 2.23906 IL6ST 0.013075 2.23921 SLC25A25 2.26E-05 2.23965 ZNF114 0.00013383 2.23969 ARMCX3 0.00110266 2.24008 LRRC31 0.00032675 2.24119 RSL1D1 3.98E-07 2.24168 NPTN 7.95E-05 2.24175 KLF5 0.00133027 2.24182 STAG2 0.00118392 2.24183 ATF2 2.84E-07 2.24391 BRWD3 5.85E-05 2.24416 PDGFC 0.0001207 2.24429 HOPX 6.13E-06 2.24488 BPTF 1.26E-05 2.24542 PTP4A1 2.16E-07 2.24564 FOXO4 0.00018655 2.24567 PARP16 4.59E-05 2.24574 RABGAP1L 4.17E-06 2.2465 CAMK2D 0.00037877 2.24657 DNAJA1 3.40E-08 2.24754 SRD5A1 0.00967494 2.24755 RAB11FIP4 2.76E-05 2.24772 LOC219347 1.79E-05 2.24779 ABLIM1 0.00734931 2.24784 TXK 0.0012164 2.24869 DNAJA1 1.96E-08 2.24905 MAP2K1 0.0002603 2.24972 EIF1B 2.69E-07 2.24983 HES1 6.04E-06 2.25018 HOPX 1.39E-07 2.25095 PDE8A 6.89E-07 2.25232 PLXND1 4.80E-05 2.25233 C6orf48 3.69E-09 2.25236 TULP3 2.01E-05 2.25545 NPTN 0.00089252 2.25562 ZFX 0.00018578 2.2558 LIMS1 0.00502846 2.25601 TMEM41B 0.00028256 2.25601 CD1D 2.28E-06 2.25611 ARL6IP6 1.74E-07 2.25763 KDM4C 0.00214219 2.25802 GRAMD1B 0.00151118 2.25857 FRMD4B 0.00053611 2.25866 TCF12 0.00411281 2.25902 RAPGEFL1 0.00081649 2.2594 TAF4B 0.0016658 2.25955 SLC40A1 5.23E-05 2.25975 HCLS1 2.58E-05 2.26045 EIF2C4 1.01E-05 2.26117 RAB6B 0.00051883 2.26427 SNIP1 0.00015403 2.26455 GNA14 0.00121619 2.2665 CREG1 0.00080047 2.26797 TMEM106B 0.00398294 2.26819 INSR 0.00069619 2.27046 TRAK1 7.12E-06 2.27069 SH3BGRL 0.00011072 2.27085 ARSK 1.49E-05 2.27186 TOB1 9.64E-06 2.27212 DNMT3A 0.0014706 2.27262 FOSL1 0.00050277 2.27276 SLC16A4 0.00098432 2.27283 LGALS14 1.25E-06 2.27303 RHOBTB1 0.00684376 2.27312 C11orf1 1.40E-09 2.27342 PPP1R15B 2.47E-05 2.27454 C11orf54 3.23E-06 2.27569 RPS18 0.00096775 2.27677 MLL3 0.00817112 2.27737 MANBA 2.21E-06 2.27795 PGM2L1 0.0004102 2.27834 DYNC1H1 0.00259744 2.27951 LIMCH1 0.00367649 2.27952 RFX7 0.00074618 2.28036 ACADM 2.51E-05 2.28099 RPS28 4.62E-06 2.28381 FOLR1 1.87E-06 2.28544 PRKCE 5.98E-05 2.28552 RBFOX2 0.00032131 2.28606 SEMA4A 1.14E-05 2.2864 DUSP6 6.19E-05 2.28671 INSR 0.00108048 2.28701 CD46 0.00171138 2.28731 SC5DL 1.12E-05 2.28788 FABP1 6.86E-07 2.28843 PAM 2.08E-05 2.2906 NDUFS1 0.00014526 2.29106 PCK1 0.00071016 2.29124 SCD5 8.57E-05 2.29255 LRP11 0.0047948 2.29271 ANKRD34C 6.14E-05 2.29373 PDPK1 0.00012208 2.29389 PHF2 6.78E-05 2.2941 EID1 0.00021215 2.29506 MPP5 7.78E-05 2.2968 DUSP6 3.61E-05 2.29796 DBR1 0.0002784 2.29813 CBLB 1.27E-05 2.29826 DPYSL2 0.00015246 2.2988 LIMCH1 0.00270736 2.29926 RAB5A 0.00018538 2.2999 ZNF655 5.42E-07 2.30053 C11orf70 1.01E-07 2.30054 ENPP2 0.00203549 2.30097 MLL5 7.78E-07 2.30114 PKM 1.09E-06 2.30163 BDH2 3.98E-07 2.30237 RAPGEFL1 0.0155216 2.30252 RAB6A 0.00956805 2.30264 LMCD1 0.00033155 2.30296 IPMK 0.00025698 2.30326 DUSP10 0.00129455 2.30381 SOS1 0.00033844 2.30409 MANBA 1.74E-05 2.30415 CCDC91 0.00463932 2.30471 PDE2A 2.81E-06 2.30479 ZKSCAN1 0.00314882 2.30517 PCSK9 0.00013126 2.30604 ARHGEF37 0.00016916 2.30638 TCF12 0.0103193 2.3064 ARHGAP12 0.00177736 2.30683 C12orf23 0.0014974 2.30828 ABCG1 0.0002699 2.30844 ZMYM2 0.00020292 2.31057 OPRL1 0.00074678 2.31257 CHST13 0.00013505 2.31366 ITSN1 0.00453426 2.31374 RFX7 0.00441348 2.31453 GLUL 2.78E-08 2.3147 PNMA1 5.07E-06 2.31584 CTBS 0.00604731 2.31606 INSR 0.00029677 2.3161 TRAK1 1.47E-07 2.31612 ANKHD1 0.00014921 2.3163 METTL20 1.79E-05 2.31658 SLC4A4 3.28E-06 2.31727 KBTBD2 2.81E-05 2.31745 BMI1 0.0088807 2.31817 LYPLA1 0.00107381 2.31849 SPATS2 0.00224527 2.31874 TMEM45A 8.99E-06 2.31921 NR3C2 7.44E-05 2.32103 ISL1 0.0110066 2.32159 HPGD 0.00010906 2.32174 KLHDC10 0.00289532 2.32176 PLD1 0.00536919 2.32182 SERP1 0.00034227 2.32242 ITGB8 0.00568424 2.32326 TMEM2 0.00108001 2.3238 RTN3 0.00014049 2.32497 BCL9L 0.0114268 2.3258 ARL6IP5 2.28E-06 2.32594 SENP7 0.00050758 2.32657 GANAB 7.94E-06 2.32751 CALB1 8.11E-05 2.32925 C5orf48 0.00039654 2.32948 ZNF362 4.68E-05 2.32952 RABGEF1 0.00013552 2.32998 SLC2A1 2.22E-05 2.33071 PIK3C2A 0.00036311 2.33104 LNX1 0.00449963 2.33162 GYG2 0.00352741 2.33164 PABPC1 2.21E-07 2.33165 AMD1 2.37E-05 2.33226 CSNK2A2 5.30E-07 2.33378 NADKD1 2.40E-05 2.33419 ACER2 0.00013136 2.33454 FUCA1 0.00323534 2.33458 ARHGEF6 4.01E-06 2.33531 C12orf65 0.00288268 2.3356 SC5DL 2.91E-05 2.3369 MYB 5.74E-05 2.33818 PPL 0.0003167 2.33835 BCAS1 0.00053141 2.33891 HOXB13 0.00062822 2.33999 HOXA1 1.27E-05 2.3402 IGF1R 0.00362019 2.3407 FAM47E 0.00042859 2.34171 TNS3 0.00015376 2.34283 ST13 0.00062366 2.34297 ITM2C 0.00020225 2.34304 C11orf75 4.35E-06 2.34355 LGALS2 6.51E-05 2.34403 SOX9 0.00045882 2.34437 FREM2 0.00014225 2.34524 PCMTD1 1.23E-05 2.34602 TCF7L2 0.00777008 2.34744 TMEM159 0.0005049 2.34812 STK3 0.00208691 2.34905 TMEM135 0.00091167 2.34922 ERGIC1 0.00037097 2.35164 FBXL20 9.88E-06 2.35187 SH3BGRL 0.00029099 2.35254 KITLG 9.93E-05 2.35262 STX3 4.01E-05 2.3528 OSR2 0.00394403 2.35297 MTSS1 3.73E-05 2.35491 FRMD3 9.88E-06 2.35585 ZKSCAN5 0.00056189 2.35596 CPOX 0.00044883 2.35689 AHCYL1 0.00054299 2.35693 RB1CC1 0.00021101 2.35704 HSPH1 2.54E-09 2.35752 FNDC3A 0.0125916 2.35774 NFE2L2 0.00015109 2.35776 SRSF11 0.01633 2.35776 SLC7A2 0.00366012 2.35857 TRIO 0.00150945 2.35893 LRRC31 1.70E-07 2.35928 CYP1B1 4.95E-06 2.36002 KLHL7 6.67E-05 2.36018 PHF16 0.0037183 2.36155 NFIL3 1.17E-05 2.36305 HPN 7.25E-08 2.36317 CCP110 4.76E-05 2.36364 KISS1 3.63E-08 2.36537 ZNF655 0.00050834 2.36559 ZKSCAN1 0.00083702 2.36676 ARMCX3 0.00013452 2.36996 PLGLB1 0.00385964 2.37032 CCNY 1.07E-06 2.37037 MLL3 0.0003619 2.37046 MUC20 9.18E-05 2.37061 LMAN1 0.00297277 2.37147 EIF2AK3 3.92E-09 2.37183 TSHZ1 0.0002796 2.3722 UCP3 2.19E-06 2.37386 ZSWIM3 1.65E-05 2.3751 ZNF184 0.00029034 2.37568 KISS1 3.54E-07 2.37618 MUC5B 5.37E-05 2.37655 CBL 0.00019397 2.37698 ZNF467 0.00012443 2.37828 FAM179B 0.00013363 2.38103 ATF7IP2 9.39E-05 2.38115 KIAA2026 0.00016525 2.38258 PERP 7.63E-06 2.38322 HIST1H2BD 0.0004845 2.38463 FAM172A 5.04E-05 2.38493 RALBP1 0.0100035 2.38497 GRAMD1C 9.16E-07 2.38565 APP 2.37E-06 2.38603 APP 2.49E-06 2.38632 APLNR 6.93E-06 2.38709 DENND5A 1.51E-06 2.38868 CD164 0.00010471 2.38995 SLC11A2 0.00383218 2.39028 F3 0.00121029 2.3909 C6orf48 2.03E-10 2.39112 OVGP1 0.00016912 2.39147 CDRT1 1.88E-07 2.39413 HPGD 6.41E-05 2.39489 SCML1 0.00130554 2.39604 PMM1 1.57E-05 2.39732 LIMCH1 0.00108861 2.39802 TMEM159 3.42E-05 2.39914 PHLPP1 1.80E-05 2.39936 SWAP70 0.00473785 2.39969 YTHDF3 2.83E-06 2.39983 NEDD4L 2.90E-05 2.39995 TXLNB 1.72E-05 2.40092 SH3BGRL 1.92E-05 2.40228 EGLN1 0.00208625 2.40288 PHF2 2.45E-05 2.40353 SAMD13 2.77E-05 2.40606 LOC10013425 0.00041084 2.40633 DNAJA1 1.39E-07 2.40636 GOLGA4 0.0139302 2.40732 POLG2 0.00100628 2.4075 MEF2D 0.00066345 2.40933 FLRT3 3.46E-05 2.41044 ARNTL 0.00020569 2.41072 NUAK1 0.00036991 2.41078 ATP6V1A 0.00084182 2.41224 ESRP1 1.38E-06 2.41355 SLC11A2 0.00147919 2.41414 PIGP 1.15E-07 2.41417 ZNF750 0.00010561 2.41482 GLUL 2.43E-06 2.41497 MSL2 0.00047735 2.41529 CDH17 3.30E-06 2.41653 RSRC2 1.36E-08 2.41703 IKZF4 0.00061763 2.41706 CDC42SE2 0.00095382 2.42276 TAB3 3.32E-05 2.42378 ZNF644 0.0008496 2.42484 PLGLB1 0.00158621 2.42519 EEF2 1.82E-07 2.42705 CAMK2D 3.05E-06 2.42769 PLS1 0.00087625 2.42821 EZR 0.008227 2.42868 CCDC62 4.74E-07 2.42869 PCF11 0.00155423 2.42937 EIF4A2 4.50E-09 2.43022 NPTN 0.00047684 2.4305 MSL1 6.21E-08 2.43078 HSPA1A 6.83E-10 2.43113 EPB41L4B 2.60E-05 2.43154 SRD5A1 0.00155652 2.43209 STX12 0.00459873 2.43228 CEP57 9.80E-05 2.43251 C7orf61 1.64E-06 2.43331 PLAGL1 0.00132186 2.434 RALGAPA2 0.00112521 2.43448 NT5E 0.00569732 2.43502 PBX1 0.00036569 2.43586 PDPK1 1.98E-06 2.43786 VASH2 0.00499924 2.44053 HEPH 2.37E-05 2.44055 LMCD1 4.69E-06 2.4407 NRN1 0.00136422 2.44102 LPCAT1 0.00274199 2.44323 PLD1 0.00579352 2.44335 KCNMB4 3.01E-05 2.44522 PLK2 0.00208641 2.44636 TRIM36 0.0008113 2.44719 EEF2 9.51E-07 2.44761 FGFBP1 8.84E-06 2.44923 NEDD4L 0.00026368 2.44956 SMARCA2 0.00064413 2.44957 RPS15A 6.71E-05 2.45217 RHEBL1 0.00111765 2.4527 MED13L 0.00065149 2.45398 RAB33A 3.17E-08 2.45406 SH3BGRL 0.00019851 2.45431 PTPRR 0.0038436 2.45658 EGFL8 1.17E-06 2.45681 HBG1 3.91E-05 2.45786 TLE1 2.02E-05 2.45813 ZNF143 1.50E-05 2.45894 CD55 5.96E-06 2.46068 ABCG1 0.00088204 2.46161 ID1 0.0166914 2.46192 TXNDC16 5.55E-06 2.46201 FNBP1 1.74E-05 2.46284 KLF17 0.00019567 2.4635 ASAH1 2.15E-05 2.46389 C2orf55 0.00015483 2.46556 SORL1 2.37E-06 2.46571 MLL5 4.17E-05 2.4659 FKBP9 2.62E-05 2.46597 SLCO2B1 2.32E-05 2.46669 AMD1 0.00010696 2.46717 DUSP6 7.23E-05 2.46772 ISL1 0.00038504 2.46805 MST4 0.00634439 2.46809 NCOA3 0.00170647 2.46873 UBTD2 6.67E-05 2.46915 AKR7A3 6.20E-06 2.47093 SNX3 0.00011713 2.47159 FILIP1L 0.00220053 2.47175 SLC11A2 0.00082588 2.47215 GLRX 3.19E-05 2.47232 KIAA0513 0.00044913 2.4736 LOC10050654 2.30E-08 2.4737 MSL1 2.63E-06 2.47373 DDX3X 3.53E-06 2.47529 TAOK1 3.67E-05 2.4759 TICAM2 5.99E-05 2.47684 EIF2AK3 1.34E-05 2.47768 FXYD3 1.62E-08 2.47833 NCOA4 0.00232619 2.47988 HPGD 4.03E-05 2.48027 ZRANB1 7.27E-06 2.48299 ZKSCAN1 0.00015957 2.4852 SAP18 1.16E-08 2.48697 DPY19L4 0.00131853 2.48759 CREBBP 3.84E-06 2.48803 ATL2 0.0024862 2.49085 APP 1.59E-06 2.49088 BRD4 0.00078803 2.49105 NRG4 0.00017997 2.49257 IDH1 2.36E-05 2.49268 ARID4A 0.00015331 2.49269 CCNE1 1.43E-06 2.49339 LOC647859 5.68E-09 2.49402 THSD4 7.08E-05 2.4941 ROCK1P1 1.55E-05 2.49461 IRF2BP2 0.00018882 2.49613 REEP5 0.00776142 2.49686 PTP4A1 1.16E-08 2.49797 SIPA1L2 0.00017685 2.49845 PCDHA3 0.00012563 2.49894 RCOR3 6.16E-05 2.4992 BCL2L11 0.00025473 2.49934 APP 2.23E-06 2.50085 FBXL17 0.00010916 2.50384 POU2F2 1.81E-06 2.50449 CAPN5 9.94E-05 2.50453 SMCHD1 1.12E-06 2.50474 ST6GAL1 1.69E-06 2.50548 SEC24A 5.58E-06 2.50625 LIMCH1 0.0015507 2.50661 CREBRF 0.0001062 2.50742 IFRD1 0.00010173 2.50903 TAF4B 0.00115322 2.50958 PERP 3.66E-06 2.51025 C20orf111 5.76E-08 2.51043 RTN3 0.00029364 2.51476 CDKN1B 0.0031528 2.51582 PITHD1 7.50E-05 2.51649 COBL 4.50E-10 2.51693 CCP110 2.81E-06 2.51866 PDGFA 0.00211556 2.51868 EIF5 0.00030654 2.51924 MMP25 0.00143617 2.52155 ZIC2 0.0024719 2.52177 SCOC 0.00799291 2.522 PTPRB 0.00036468 2.52238 LGALSL 0.00019655 2.52254 MAP4K5 4.04E-05 2.52271 MTL5 7.87E-07 2.5228 LHX4 4.74E-06 2.52589 POU6F1 7.20E-05 2.52676 BMPR2 0.00046554 2.52736 CDC14B 7.48E-09 2.52797 SH3YL1 3.21E-05 2.52831 TES 0.0002078 2.52942 SCARB2 0.0013326 2.5314 PELI2 2.83E-05 2.53198 CYP1B1 3.59E-06 2.53293 NADKD1 0.00022365 2.53298 FAM149B1 0.00031355 2.53323 C10orf118 1.00E-06 2.53423 MBNL2 0.00100353 2.53484 WDFY3 3.25E-05 2.53555 ERBB3 5.60E-08 2.53572 RSF1 0.0159151 2.53748 RAB40B 2.32E-06 2.53749 CDC42BPA 0.00596792 2.53837 MCTP2 0.000798 2.54041 PRKACB 1.39E-05 2.54061 SYNC 2.17E-05 2.54163 EPB41L5 7.10E-06 2.54183 ZNF654 0.00137653 2.54603 GPLD1 3.36E-05 2.54766 ITM2C 1.55E-08 2.548 ARID4B 0.00066037 2.54983 PRDM2 2.42E-05 2.5505 WDR26 3.73E-07 2.55183 HNMT 0.00013637 2.55233 MAP3K2 0.00416374 2.55498 CREBRF 2.94E-07 2.55517 BBX 0.00137348 2.55546 HIST1H2BC 2.51E-05 2.55608 RFX7 0.00780093 2.55634 EGR2 1.06E-06 2.55711 ROCK1 3.52E-06 2.55738 N4BP2L1 0.00386381 2.55757 PICALM 0.00212123 2.55861 TTC33 2.85E-05 2.55871 RBM15 0.00024786 2.55897 RAC2 4.85E-07 2.56085 ZNF569 0.00023389 2.56105 PAPSS2 3.12E-05 2.56194 HSPA1A 8.50E-11 2.56323 DAPK1 2.43E-05 2.56382 RALGPS1 0.00097666 2.56489 C11orf54 1.29E-07 2.56534 SLC17A8 0.00016486 2.56542 FRAS1 2.92E-05 2.56559 SEC24A 2.23E-05 2.56572 PARD6B 0.0002222 2.56717 PIGP 2.30E-08 2.56793 MLL5 6.55E-07 2.56803 LIMCH1 0.00225759 2.56875 DEPTOR 0.00032105 2.56937 FAM161A 6.53E-05 2.57047 RNF32 0.00023905 2.57131 STEAP2 4.87E-06 2.57199 FPR2 3.32E-05 2.57291 DDX3X 2.48E-07 2.57763 HLA-DMA 0.00021243 2.57795 SNHG7 7.34E-10 2.57878 CPEB4 0.00088439 2.57911 PGRMC1 0.0001161 2.57943 PPP1R9A 0.00837434 2.57971 PPP3R1 0.00040519 2.57974 TNFSF15 4.56E-06 2.57992 HHEX 0.00028107 2.58107 GRAMD1A 0.00030656 2.58267 TPST1 9.84E-05 2.5831 ANK3 1.88E-06 2.58457 STARD4 2.68E-05 2.58464 ABI1 0.00023823 2.58504 TPD52 2.89E-05 2.58695 CD164 0.00222674 2.58702 KCTD12 0.00042794 2.58704 RSRC2 4.54E-09 2.5871 HIPK3 2.56E-05 2.58933 NR1H4 4.48E-07 2.59064 TDP2 1.03E-05 2.59141 CD46 0.00052422 2.59172 BPTF 5.16E-06 2.59179 DSEL 0.00052898 2.59246 MAP4K5 0.00021152 2.59449 BTK 0.0001011 2.59504 AMD1 8.13E-05 2.59551 GPX3 0.00013689 2.59587 CLK1 1.05E-05 2.59816 CCDC115 7.80E-08 2.59842 AK3 0.00083466 2.59935 TMEM2 2.22E-05 2.59954 C5orf4 3.38E-05 2.60045 INSR 1.63E-05 2.60111 SLC44A3 1.04E-09 2.60239 GPR160 4.12E-09 2.60265 HOPX 1.83E-06 2.60369 PTPRB 5.62E-05 2.60386 MBNL2 0.00071391 2.60391 ASXL1 7.04E-08 2.60458 ACTC1 3.91E-07 2.6046 ID2 1.91E-10 2.60482 CCPG1 0.00027059 2.60497 HIST1H2BD 3.50E-05 2.6067 KIF1B 5.52E-07 2.60699 CUL4B 3.94E-06 2.60787 CMYA5 0.00017725 2.60809 HSPH1 6.47E-08 2.60852 TPD52 2.20E-05 2.60897 DAND5 8.51E-05 2.6107 TCEA1 0.00750677 2.61114 C17orf103 2.19E-05 2.61155 UBE2G1 1.88E-08 2.61211 IL20 3.39E-07 2.61348 C10orf99 1.96E-06 2.61803 PCDHA1 0.00013548 2.61928 ANK3 0.00236522 2.62067 CRKL 2.71E-05 2.62656 IKZF5 6.26E-05 2.62771 ZNF654 5.13E-06 2.62816 CHRFAM7A 1.63E-05 2.62817 RCOR3 1.44E-07 2.62821 KLF5 0.00242752 2.62886 HNRPDL 3.53E-06 2.63265 FAM117B 2.39E-05 2.63269 INHBB 4.30E-05 2.63285 HIVEP2 2.29E-08 2.63309 ZFX 0.00296238 2.63353 ZFX 0.00296238 2.63353 C1orf114 8.90E-05 2.63355 FAM63B 0.00169266 2.6342 HBP1 3.77E-05 2.63543 B4GALT4 3.67E-06 2.63583 NDRG3 2.54E-05 2.63692 KIAA0319 0.00052289 2.63816 APP 4.62E-07 2.63866 ANKRD11 0.00757311 2.64124 FAM53A 0.00152611 2.64227 HIST1H2BC 0.00043658 2.6451 ENPP2 0.00028157 2.64711 HPGD 2.36E-07 2.64781 CLSTN1 0.00188339 2.64816 TBX3 5.98E-07 2.64822 BHLHE40 0.00016814 2.64854 DNAH12 1.61E-06 2.65009 TESK2 1.37E-05 2.6512 BCOR 0.00081928 2.65159 PITX2 2.84E-05 2.6536 PIK3C2A 0.00027254 2.65383 APP 4.40E-07 2.65497 ERGIC1 1.15E-05 2.65769 STAG2 0.00017694 2.66098 CSRNP1 6.01E-06 2.6619 ANGPTL4 2.80E-06 2.66346 RHPN2 0.00096233 2.66357 ARID2 0.00031972 2.66381 CELF2 1.46E-07 2.66456 PTTG1IP 0.00034292 2.66494 APP 4.84E-06 2.66584 SOX4 3.99E-05 2.66689 ABCG1 3.34E-05 2.66757 PPM1K 6.17E-06 2.67234 UNC93A 0.00025847 2.67438 RAB5A 0.00135626 2.67811 RRM2B 0.00049709 2.67823 HPGD 2.51E-05 2.68014 SERTAD2 0.001393 2.68014 PPP1R15B 0.00121383 2.68067 TNFAIP3 3.52E-05 2.68106 FAM214A 0.00054184 2.682 STX3 1.27E-07 2.68319 ANKRD45 1.41E-09 2.68351 SPAG9 1.92E-05 2.68451 PCDHA1 3.02E-05 2.68524 HOXA5 3.28E-06 2.6862 TXNDC16 0.00209136 2.68629 EEF2 4.73E-08 2.68638 SQLE 0.00194803 2.68697 SPATS2L 3.80E-05 2.68729 RYBP 1.36E-05 2.68816 SOX4 5.53E-10 2.69063 PPP2R5A 7.96E-05 2.69112 UFL1 0.00333324 2.69192 SERTAD2 0.0002593 2.69217 FCHO2 0.0108911 2.69766 ZBTB43 0.00118023 2.69843 TLE4 0.00023675 2.70008 CYP2U1 3.45E-07 2.70214 RTN3 6.88E-05 2.70281 ZFAND5 4.60E-07 2.70326 SLC26A2 0.00107177 2.70409 C4orf34 6.85E-08 2.70613 PAPSS1 3.29E-07 2.7062 NR2F2 9.65E-07 2.70652 FERMT2 7.25E-05 2.707 OPN3 2.38E-06 2.70873 SCOC 0.00978416 2.71305 PELI1 5.50E-05 2.71424 BNIP3L 4.11E-05 2.71444 STARD4 1.35E-05 2.71507 EMP1 1.44E-05 2.71588 IGF2BP2 0.00182587 2.71625 GLCCI1 8.47E-07 2.71854 EPB41L4B 3.73E-07 2.72095 LIPA 0.00163478 2.72151 TAF7L 0.00011401 2.72186 ARNTL 2.18E-08 2.72432 HOXA6 1.80E-05 2.72504 RASL12 2.47E-05 2.72557 NCKAP1 0.00024581 2.72561 HNMT 5.12E-07 2.72612 WASL 0.00170861 2.7282 XIST 1.91E-06 2.72958 CENPL 8.46E-06 2.73068 CLIP1 6.58E-07 2.73339 HEG1 3.71E-06 2.73375 COL21A1 0.00010326 2.73482 MLL5 6.22E-05 2.73633 C16orf80 2.27E-06 2.73643 F2RL1 0.00014326 2.73932 KCNJ12 1.14E-07 2.74008 SERTAD3 0.00029335 2.74142 EPB41L4A-AS 1.17E-08 2.74206 APP 2.08E-06 2.74366 MBNL2 3.27E-06 2.74634 DNAJA1 2.70E-09 2.7473 NXT2 0.00486594 2.74792 SSBP3 0.0017137 2.74895 CARD8 0.00040199 2.74945 CDKN1A 0.00064064 2.75032 CRABP2 7.85E-08 2.7515 SPRY4 1.17E-05 2.75382 GALNT3 0.00032707 2.75421 PRLR 5.81E-05 2.75485 PICALM 0.00112335 2.75494 BDH1 6.71E-05 2.75918 GULP1 6.48E-05 2.75923 PER2 0.00018785 2.75955 HERPUD1 9.77E-09 2.75995 BPTF 5.99E-07 2.76011 KIF27 1.60E-06 2.76076 ITM2C 0.00036812 2.76359 NDUFA4L2 1.37E-05 2.7637 GPR68 3.79E-07 2.76724 FAM108B1 2.10E-08 2.7698 PLOD2 0.0007066 2.76992 APPBP2 3.67E-08 2.77134 PTPRF 5.36E-06 2.77135 STRADB 6.64E-05 2.77197 CD46 0.00089473 2.77333 KLF10 0.0013054 2.7746 EFNB2 1.88E-08 2.77663 TMCO3 3.28E-09 2.77868 SRI 2.84E-05 2.78241 SOCS5 8.36E-05 2.78469 NEDD4L 1.38E-06 2.78609 PTPRR 0.0138048 2.78691 CHD4 0.00205692 2.78773 PPM1H 1.30E-05 2.78864 TM2D2 1.42E-06 2.78917 PIWIL4 4.75E-07 2.78931 KL 3.85E-06 2.7894 PIP5K1B 7.99E-05 2.79147 RASA4 9.40E-05 2.79167 PTPRF 8.20E-05 2.79299 CLK4 2.25E-05 2.79509 SLC16A4 4.14E-05 2.7958 NR5A2 1.35E-05 2.79686 SPAG9 7.52E-07 2.79883 GNA14 0.00058947 2.80276 EREG 0.00103914 2.80301 EFNB2 0.00289538 2.80438 NTN4 1.91E-05 2.8058 IQGAP2 0.00244308 2.80695 UBE2H 3.00E-05 2.8073 MUC20 3.32E-07 2.80763 HSD17B11 8.31E-06 2.80828 TTC3 0.00030158 2.80991 CSPG5 3.17E-05 2.81007 SCP2 5.46E-05 2.81312 SCP2 5.10E-05 2.81433 FRY 0.00165003 2.81553 ST8SIA6 0.00078185 2.81577 TSPAN12 4.85E-07 2.8171 USO1 0.00107784 2.81805 SIPA1L2 5.14E-06 2.81838 CSRNP2 3.03E-08 2.81848 RTN3 0.00127249 2.81854 GRAMD1C 8.38E-07 2.81855 GAB1 4.01E-08 2.81889 CDX2 5.56E-05 2.82092 PRKACB 0.00063413 2.82249 GPC4 6.41E-06 2.82468 SNTB1 4.14E-06 2.82688 FOXD4 3.25E-08 2.82696 RSBN1 1.31E-06 2.82715 CCNE1 8.16E-09 2.82906 CREBRF 2.13E-05 2.82979 CRABP2 6.31E-07 2.83323 PTGER3 2.27E-06 2.83361 JMJD6 2.03E-05 2.83541 KCNJ3 0.00015689 2.8356 ASAP3 9.05E-05 2.83604 USP3 0.00014921 2.83683 HOXB2 1.04E-05 2.83753 PPP1CB 0.00176147 2.83756 BPTF 1.39E-05 2.83795 LMAN1 0.00028804 2.83942 ZNF75A 8.83E-06 2.84 FHL2 2.32E-05 2.8412 ERBB3 1.02E-05 2.8417 LTBP1 1.65E-05 2.84963 AKAP9 0.00657545 2.85425 TRIQK 2.43E-07 2.85656 STYK1 0.0001224 2.85783 KIAA0895 3.59E-07 2.85939 IL1R1 7.01E-06 2.86363 ZFAND5 1.36E-08 2.86484 ANG 2.94E-06 2.8658 SAP18 1.82E-07 2.86691 MLL5 9.51E-07 2.86907 NTN4 2.01E-05 2.86981 LRRC17 2.21E-05 2.87081 EGLN1 0.00055584 2.87134 AMD1 2.50E-05 2.87164 PDE4C 1.77E-05 2.87215 RNF103 1.29E-08 2.87459 RIPK4 6.57E-05 2.87489 PIGH 1.74E-05 2.8756 C6orf141 3.68E-09 2.87686 OSR2 0.00020736 2.87791 CREBRF 0.00012427 2.87882 KDM3A 2.08E-08 2.87935 SHROOM3 8.02E-06 2.88084 FAM115A 0.00291311 2.88342 TFF3 0.00016876 2.88428 ARL6IP5 2.74E-06 2.88632 TBC1D9 3.52E-07 2.88682 SLC26A2 1.10E-05 2.88794 LONRF3 1.58E-08 2.8885 DNAJB9 7.13E-05 2.88894 PLEKHF2 6.23E-06 2.88959 STAU1 0.00016934 2.88995 JMJD1C 5.53E-07 2.8912 ITM2C 5.67E-06 2.89189 DNAJA1 1.82E-08 2.89227 CD46 0.0013802 2.89277 NR4A3 0.00159243 2.90062 HERPUD1 2.41E-08 2.90119 PMAIP1 5.30E-06 2.90343 ITGA10 1.36E-06 2.90935 DIDO1 1.92E-07 2.91091 LMO4 1.04E-07 2.91539 ROCK1 0.00012406 2.9163 ORAI3 2.39E-05 2.91668 SCAPER 3.73E-05 2.91924 GPA33 1.16E-07 2.92065 HERPUD1 2.74E-09 2.92119 PPARA 1.04E-06 2.92158 DST 0.00072824 2.9218 CDO1 1.36E-05 2.9225 CYP1B1 2.81E-05 2.92279 TOB2 0.00075175 2.92644 CRABP2 4.09E-08 2.93014 C4orf3 4.41E-06 2.93135 PHF17 1.79E-05 2.93215 SH3PXD2A 3.11E-05 2.93434 SESN2 3.75E-05 2.93578 CEP19 0.00025284 2.93657 CA12 1.02E-05 2.93901 INTS6 6.49E-05 2.94257 MLL3 0.00028155 2.9456 HAS3 2.76E-06 2.94662 PTPRM 7.63E-06 2.94731 IDI2-AS1 3.34E-07 2.95229 RNASE4 4.74E-07 2.95382 FAM161A 2.66E-05 2.95452 RYBP 4.40E-06 2.95456 KANSL1L 3.95E-05 2.9553 CDKN2B 0.00017183 2.95574 HSD17B12 0.00027675 2.95604 HNRPDL 1.28E-06 2.96129 SUCNR1 6.71E-05 2.96206 IER3 0.0002949 2.96215 CHD4 0.00168708 2.96315 FAM46A 9.89E-06 2.96454 SERTAD3 0.00010913 2.96703 CPEB4 0.00070477 2.96808 MLL3 0.00418547 2.96833 EIF4A2 4.97E-09 2.98169 Sep-10 0.00041856 2.98448 TDRD6 1.30E-06 2.98586 TMEM135 3.30E-06 2.98636 MBNL2 5.81E-05 2.99017 WNK1 7.39E-05 2.99067 FRMD4B 4.07E-06 2.99127 SCIN 2.34E-05 2.99254 NAP1L5 1.70E-05 2.99641 HMP19 8.43E-05 2.99693 GAL3ST4 0.00012358 2.99783 NR4A1 0.00099055 3.00352 LETMD1 4.17E-06 3.00411 CD164 0.00041174 3.00606 ANKRD12 5.97E-08 3.00897 WAPAL 0.00302666 3.00944 NEDD4L 9.15E-05 3.00946 FAM13A 0.00063138 3.01274 GLIS3 0.00119835 3.01326 CDH17 5.99E-07 3.01359 REG4 1.49E-07 3.0139 ZNF34 2.43E-05 3.01736 TRIM52 0.00156789 3.01938 PXK 0.00015191 3.01958 THBS1 3.71E-06 3.02036 PPP1R36 8.81E-06 3.0204 MXI1 1.39E-06 3.02228 KDM3A 2.20E-08 3.02411 METTL7A 5.38E-05 3.02419 PROS1 1.57E-05 3.02442 FAM46C 0.00018576 3.02463 JMJD1C 3.08E-07 3.02668 JMJD1C 8.11E-05 3.02908 BPTF 3.87E-06 3.03093 HIST1H2BD 6.47E-08 3.03291 SLC25A44 9.65E-07 3.03306 HIST1H2AC 2.43E-05 3.03667 LATS2 1.21E-05 3.04023 MAP1LC3B 5.70E-05 3.04057 TMEM135 0.00149235 3.04391 DDX3X 1.00E-05 3.05257 SLC2A13 1.10E-06 3.05286 AKAP13 0.00160918 3.0544 FOXC1 1.28E-06 3.05475 HSPH1 3.19E-07 3.05607 CYP2C9 0.00024934 3.05758 TMCO3 9.57E-09 3.05759 NR2F2 8.89E-09 3.05767 SMPD3 2.20E-05 3.06239 FNBP1 0.00072838 3.06278 FXYD3 1.24E-08 3.06352 PDE5A 4.05E-05 3.06496 KCNJ1 0.00013733 3.06545 STARD4 2.21E-07 3.06822 ZNF292 0.00746049 3.07079 BPGM 7.91E-06 3.07081 AKAP7 0.00189646 3.07436 ADD3 8.20E-06 3.07457 HIST1H3A 5.66E-06 3.08003 ARC 3.70E-05 3.08015 NEDD4L 2.65E-05 3.08019 MAP1LC3B 1.28E-07 3.08222 DEFB1 3.18E-06 3.08274 ZBTB11 0.00092907 3.08354 IRX5 3.30E-05 3.08532 CD46 0.00130079 3.08641 RGL1 0.00023683 3.09017 NAP1L5 6.78E-07 3.09076 ARL6IP1 0.00049169 3.09105 N4BP2L1 0.00045657 3.09114 BBS4 2.87E-05 3.09605 BPGM 1.36E-05 3.09791 ANKRD34C 9.40E-06 3.09969 CBL 0.00036489 3.10532 C6orf141 1.03E-05 3.10551 KDM3A 3.80E-09 3.10915 RBM47 9.86E-06 3.10958 TUFT1 4.08E-06 3.10987 TYW1B 3.76E-07 3.11445 ANK3 0.00027053 3.11489 EREG 1.90E-08 3.11728 LMO4 1.96E-05 3.11797 ZNF217 2.75E-05 3.11904 FAM101B 1.59E-06 3.11972 NEDD4L 1.38E-07 3.12532 TNFRSF10D 7.01E-06 3.12624 GDF11 0.00014175 3.1277 SLC4A4 0.00171962 3.12798 SLC19A2 0.00122334 3.13014 CHP1 8.17E-07 3.13376 EIF2AK3 5.81E-05 3.13935 CCNG2 0.00010787 3.14071 IER5 3.40E-07 3.14112 RGS4 1.12E-06 3.14163 PTGER3 0.00024939 3.1422 OVOL1 3.80E-05 3.14407 MSMO1 8.59E-06 3.14523 FAM13A 0.00072334 3.15019 CCNG1 0.00018466 3.15066 MAP3K8 9.76E-05 3.1543 SYT17 0.00022406 3.15469 NABP1 1.42E-05 3.15549 RGS16 3.04E-06 3.15646 SNTB1 7.67E-07 3.15718 SLC5A9 0.00036687 3.15814 ZKSCAN1 0.0032492 3.16014 NR1D2 0.00033694 3.16408 BNIP3L 1.62E-05 3.1668 INTS2 0.00014665 3.16707 ARID4B 0.0001196 3.16931 DIAPH2 8.70E-06 3.16957 EPB41L5 7.62E-05 3.1712 SNHG7 2.31E-10 3.17303 SH3YL1 6.43E-05 3.17389 RMND5A 1.70E-08 3.17687 ERO1LB 6.52E-05 3.17917 CNGA1 1.41E-06 3.17985 DDX26B 2.38E-05 3.18237 GNAQ 1.71E-05 3.18432 GRAMD2 5.16E-08 3.1885 NFAT5 4.60E-10 3.18944 C4orf3 3.20E-05 3.18951 ABHD5 3.93E-05 3.1909 VEGFA 1.05E-09 3.19194 CXCR4 0.00016195 3.19331 KIAA0895 1.45E-06 3.19518 MLL3 3.23E-06 3.1993 LETMD1 9.25E-06 3.20062 TSC22D2 0.0004521 3.20296 IQGAP2 4.52E-05 3.20592 MEIS2 0.00018684 3.20886 SESN2 8.62E-07 3.20894 SGMS1 5.33E-05 3.21136 PAPSS2 0.00012273 3.21769 KDM5B 4.32E-08 3.21841 SNTB1 9.59E-05 3.22088 BBX 0.00313489 3.22223 TUFT1 4.37E-05 3.2238 AKAP7 0.0006655 3.22508 TTC33 1.16E-05 3.228 ABHD3 6.86E-05 3.22912 RGS16 7.32E-07 3.23008 CCDC108 4.39E-08 3.232 CD14 0.00056644 3.2332 SERPINE1 9.72E-05 3.23616 DSTNP2 2.14E-07 3.2363 DSTNP2 2.14E-07 3.2363 CDK19 0.00015723 3.23663 NR5A2 3.95E-06 3.23916 CCNE2 5.81E-05 3.2397 SLC25A25 1.05E-05 3.24485 HOMER1 0.00027266 3.24763 RBM33 1.61E-05 3.25181 LINC00273 5.50E-08 3.25636 GTF2IRD2 3.67E-05 3.25704 ZFAND2A 1.61E-10 3.25765 ELL2 8.98E-08 3.26029 SLC16A4 0.00112243 3.26163 MAN1A1 2.72E-08 3.26191 GPBP1 3.10E-05 3.26195 GDPD1 0.00016554 3.26494 EFNB2 0.00184207 3.26541 TFF2 4.22E-05 3.26732 IDI1 6.84E-07 3.26802 LOC10050640 8.97E-06 3.26894 LOC10050944 0.00010238 3.26995 PFKFB3 2.49E-06 3.27245 GPR37 4.38E-05 3.27351 PAPSS2 2.88E-05 3.27386 SORL1 9.22E-06 3.27516 CCNE2 7.55E-05 3.27667 PCDHA1 2.47E-05 3.27784 SNX16 8.91E-06 3.28809 FAM177B 5.46E-05 3.28912 AKAP7 7.69E-05 3.28931 CNNM4 3.10E-06 3.28949 CYP2U1 6.81E-08 3.29053 ATF3 0.00011682 3.2963 ERMAP 1.69E-07 3.29738 CREBBP 3.45E-08 3.29796 SNTB1 2.50E-07 3.2983 CREB3L1 1.09E-05 3.30226 AMD1 1.62E-05 3.30314 TMC5 1.14E-05 3.30704 FBXO32 6.94E-09 3.30849 SLC5A5 0.00049754 3.31137 SCAPER 0.00021492 3.31228 ZNF711 0.00071152 3.3158 HOXC6 8.87E-10 3.31721 KLHL7 9.93E-05 3.31837 OVGP1 5.21E-06 3.31978 ABHD5 7.69E-05 3.32545 PJA2 0.00226467 3.32618 LIMA1 0.00593099 3.32731 FRZB 2.19E-06 3.33181 SIRT4 4.54E-08 3.33386 DEFB1 2.55E-06 3.33463 DNAJB6 8.29E-05 3.3351 HPGD 4.82E-07 3.33564 OSBPL8 0.00018324 3.34376 HOXC6 1.53E-09 3.34381 KIAA1211 7.33E-05 3.34387 PICALM 0.00158273 3.34425 ATG2B 7.16E-05 3.34706 MAP3K8 1.40E-06 3.35314 SLC26A2 0.00010437 3.35646 FGD4 4.43E-05 3.35978 SSTR1 0.00289926 3.36621 BNIP3L 1.39E-05 3.36677 BNIP3L 1.39E-05 3.36677 FHL2 2.36E-05 3.37311 SPAST 0.00034352 3.37472 BNIP3L 2.48E-05 3.38463 MICB 4.61E-07 3.38718 SCARB2 0.00012781 3.38881 DNAJB1 6.93E-05 3.3896 CDKN1C 3.89E-05 3.39485 PPARA 2.10E-07 3.40048 PATL1 3.62E-07 3.40091 THAP2 5.94E-06 3.4054 POU2F2 2.49E-07 3.40689 C4orf34 1.26E-07 3.41007 TRA2B 1.27E-05 3.41204 FOXC1 0.00032036 3.4131 ANXA13 4.19E-05 3.41591 SOCS5 1.79E-07 3.41683 ANK3 0.00097261 3.41852 WSB1 1.25E-06 3.42367 ARHGAP5 2.01E-05 3.42477 APOBEC1 1.66E-06 3.42482 RSRC2 4.23E-07 3.42531 LOC647859 5.44E-07 3.43076 CDKN1A 0.00034483 3.43238 RERE 8.58E-06 3.43307 ETS1 0.00061472 3.43718 PLXNA2 2.59E-10 3.44085 KIF1B 3.06E-09 3.44473 MAN1A1 4.87E-07 3.44597 PLCB1 3.06E-06 3.44625 PFKFB4 0.00364715 3.44659 INSIG1 4.72E-06 3.44663 SORBS1 1.69E-05 3.4486 CXCR4 1.69E-05 3.45381 MBNL2 0.00348938 3.45399 CCNG2 1.90E-07 3.45675 SLC38A11 1.61E-06 3.46198 TUBB3 2.44E-07 3.46445 SPAG9 0.00027831 3.46999 DIP2C 4.07E-06 3.47144 ANXA13 2.72E-05 3.47708 CCNG1 0.00017243 3.47748 DDX3X 2.55E-05 3.47755 HNRNPL 1.38E-06 3.47861 GLB1L 1.26E-05 3.48025 MAP2K6 0.0001669 3.48135 WDR26 7.19E-09 3.48503 RBM22 0.0009687 3.49304 EIF4A2 3.83E-07 3.49344 DNAJB6 8.64E-09 3.49533 DNAJB6 8.64E-09 3.49533 TIPARP 5.36E-06 3.50093 FBXL16 1.23E-05 3.50348 BCAS1 8.42E-06 3.50657 FRMD4B 1.52E-06 3.508 PAPSS2 0.00039905 3.51015 EIF4A2 3.22E-06 3.51489 YPEL5 4.46E-11 3.51615 GRAMD1C 2.02E-05 3.51819 LRRC19 5.81E-06 3.52055 IQCH 9.93E-05 3.52628 CRIM1 5.66E-07 3.52778 SOX4 2.24E-05 3.53222 RYBP 6.18E-07 3.53865 CPED1 0.00015903 3.54028 ALAD 9.66E-07 3.55267 SHANK2 1.22E-06 3.56119 USP38 3.69E-10 3.56167 THRB 3.63E-10 3.56517 PCF11 2.33E-08 3.57622 CHD1 2.26E-09 3.57707 FAM179B 1.75E-06 3.58655 CEP57 7.48E-05 3.59027 GPR37 8.63E-06 3.5988 ANKRD10 2.08E-09 3.60088 NIPSNAP1 4.37E-05 3.61115 KBTBD8 0.00030938 3.61339 GAB1 0.00031767 3.61488 ATP6V1B1 5.38E-07 3.62069 ZNF711 1.13E-09 3.62897 PIGR 3.13E-07 3.63072 SOS1 6.84E-10 3.65241 SLC45A4 1.35E-05 3.65269 SLC4A4 1.96E-07 3.65333 PLOD2 0.00016571 3.65528 BCAS1 7.76E-05 3.66376 CD200 9.07E-06 3.66608 SIRT1 1.04E-09 3.66702 BCAS1 1.39E-05 3.67062 ELF3 0.00666065 3.67216 SPTY2D1 6.97E-06 3.67396 ZBTB10 6.14E-05 3.67542 PAPD5 3.86E-06 3.68336 BMP2 1.50E-05 3.68547 JUNB 1.07E-06 3.68997 METTL20 9.34E-07 3.69017 HIST1H2BJ 2.28E-06 3.69171 CENPL 2.64E-06 3.6925 PIP5K1B 1.23E-05 3.70107 CDC14B 0.00020323 3.70467 WAC 1.34E-07 3.70579 SIRT1 0.00038221 3.71231 PLGLA 0.00021826 3.71273 SOX4 3.08E-10 3.71448 CPEB3 9.21E-08 3.71708 MAP7D2 4.71E-06 3.71733 FMO5 1.24E-07 3.72117 DSP 5.40E-07 3.72166 DEPTOR 6.37E-06 3.72639 ZFAND5 2.97E-09 3.72701 PTPRR 0.00021426 3.74119 CHN1 3.82E-06 3.7432 POU2F2 6.76E-06 3.74602 DDIT4 7.26E-05 3.74738 TMPRSS12 9.40E-06 3.74824 ELF3 0.00665685 3.74889 DNAJB4 4.23E-07 3.74962 CHKA 2.89E-06 3.75328 KIAA0319 0.00046506 3.7764 FZD7 1.45E-07 3.77754 FHL2 1.35E-05 3.78085 ATP7B 6.85E-06 3.78222 GALNT5 1.21E-08 3.79844 BPTF 9.20E-08 3.79855 DBIL5P 1.06E-06 3.80082 YPEL2 0.00023871 3.80167 PIK3R3 8.90E-07 3.80237 TOP1 0.00274912 3.80874 UBL3 3.79E-09 3.80987 VEGFA 1.12E-08 3.81099 AKAP7 0.00016831 3.81192 LOC10050551 4.57E-07 3.812 DIP2C 4.28E-05 3.82545 HIST1H2BN 0.0001017 3.82724 GPR64 4.20E-06 3.82789 NAP1L5 0.00010648 3.83095 KCNN3 1.24E-09 3.83107 NFKBIA 4.06E-05 3.83334 BPTF 9.34E-08 3.83503 PDZK1 9.10E-06 3.8374 SLC11A2 0.00693029 3.84043 RSRC2 1.06E-05 3.84963 THRB 2.47E-08 3.84982 ZNF75A 4.46E-06 3.85364 PBX1 0.00014877 3.86812 SECISBP2 0.00020125 3.87157 ZC3H12A 2.95E-07 3.87851 CLINT1 8.92E-07 3.88038 GADD45B 2.26E-06 3.88432 YPEL5 8.66E-11 3.88643 KDM4B 0.00011357 3.88836 LEMD3 0.00026038 3.89065 ARID4B 3.20E-05 3.8983 FAM134B 0.00049686 3.90134 UBXN7 7.50E-09 3.90778 SLCO2B1 1.78E-05 3.90916 TWSG1 2.59E-07 3.9105 FAM13A 0.00104811 3.91536 DAPK1 9.20E-05 3.92151 EGR1 4.44E-07 3.92235 FBXO11 2.40E-05 3.92293 BMP2 5.24E-08 3.92937 ERRFI1 2.78E-07 3.93355 SHANK2 9.47E-06 3.9342 C15orf48 5.38E-07 3.93574 ZNF655 0.00044811 3.94069 TUBB8 0.00030964 3.94143 KRCC1 2.01E-05 3.94531 ID2 6.62E-09 3.94556 CCNG1 0.00041903 3.94686 LDHC 2.85E-05 3.95731 MXRA7 7.70E-09 3.95822 AKAP9 1.12E-06 3.96154 PTP4A1 4.54E-07 3.96508 PDZK1 6.60E-06 3.97297 TBX3 9.22E-08 3.97616 ARIH1 2.81E-09 3.99241 SOCS3 1.83E-07 3.99385 ERO1LB 1.68E-05 3.99743 NR4A1 0.00163795 3.99806 EGLN1 0.00014131 4.00522 LAG3 3.13E-10 4.01427 METTL20 9.26E-06 4.03282 FKBP14 1.19E-06 4.03848 C10orf118 0.00173841 4.04247 FAM134B 0.00028707 4.0438 SCML1 2.12E-05 4.05172 ARNTL 4.63E-07 4.05594 ID3 7.01E-08 4.06614 EMP1 3.07E-05 4.06648 CXCR4 1.70E-05 4.07028 NR5A2 2.76E-06 4.07214 ITPR1 2.06E-06 4.08079 TRA2B 4.24E-05 4.08141 UBL3 5.77E-05 4.08341 TRPC1 1.19E-05 4.08475 LETMD1 1.06E-06 4.0855 EGLN3 0.0005532 4.09803 HIVEP2 1.31E-08 4.10875 NFKBIA 1.66E-05 4.11491 NFAT5 0.00011351 4.11509 KCNH2 5.96E-06 4.12344 ADRB2 4.46E-05 4.12693 SORBS1 2.92E-06 4.12973 GAL 6.17E-08 4.13138 ELL2 2.11E-06 4.13236 DUSP10 1.60E-07 4.13317 TP53AIP1 5.02E-07 4.13442 MXI1 3.37E-05 4.13808 RND1 2.28E-05 4.14061 STX3 4.67E-05 4.14869 CYP2U1 2.79E-06 4.1497 RPL31 8.91E-11 4.15261 LOC728741 2.90E-05 4.15526 CXCR4 2.63E-05 4.1577 TSC22D3 6.39E-09 4.16131 ARG2 7.58E-06 4.17119 TMPRSS2 1.53E-05 4.17652 IER2 1.32E-05 4.18241 KDM6A 1.94E-07 4.18349 CDH17 1.02E-06 4.18484 STX3 6.34E-08 4.19155 RSRC2 1.72E-05 4.19805 RIT1 1.20E-08 4.20747 CCL4 1.04E-05 4.21764 FOXD4L2 8.23E-09 4.21842 WASF2 0.00028954 4.22815 LOC392288 1.22E-08 4.22929 RIOK3 1.07E-05 4.22959 DUSP5 0.00032123 4.23174 PPP1R1C 0.00064279 4.23842 BRD2 9.15E-08 4.24092 EP300 0.00010317 4.25038 NUFIP2 2.06E-07 4.25862 WDR47 1.91E-07 4.26888 STYK1 3.24E-05 4.27926 SPEN 0.00050537 4.28409 C3orf58 1.31E-07 4.28493 MEX3B 7.22E-06 4.28567 ZNF425 2.21E-05 4.28927 PELI1 5.24E-05 4.29432 SERTAD1 0.00068638 4.3041 SPDYA 6.20E-10 4.3067 PPP1R10 4.50E-05 4.3159 ANKRD37 1.91E-09 4.32629 CLK4 5.64E-05 4.32856 RPL13P5 8.37E-05 4.33166 NDRG1 2.32E-05 4.33428 LNP1 1.05E-07 4.33429 NSUN7 5.57E-07 4.34484 PTPLB 0.00048005 4.36222 FOXD4L1 2.28E-08 4.36373 FILIP1L 5.53E-06 4.36515 C1orf114 2.80E-06 4.36681 ATF3 1.92E-07 4.37612 INTS2 2.29E-10 4.3766 HES1 1.44E-08 4.38009 RICTOR 1.88E-07 4.38715 GKAP1 2.83E-05 4.3882 LINC00312 1.46E-07 4.40234 MLL5 4.32E-06 4.40278 YPEL2 7.37E-09 4.40807 RIOK3 2.90E-07 4.40843 C16orf72 2.58E-08 4.40898 SLC2A10 2.53E-06 4.41801 KLF2 7.12E-05 4.41809 PIP5K1B 1.21E-06 4.41853 NFKBIA 1.69E-05 4.44081 CDK19 1.01E-05 4.44384 ACVR1C 2.52E-06 4.45121 PCDHA2 6.92E-07 4.45402 MXI1 1.58E-05 4.4762 EIF4A2 4.79E-08 4.47866 ASXL1 1.11E-06 4.48783 WAC 4.81E-05 4.49227 RNASE4 4.05E-09 4.50121 ZFP36 1.65E-07 4.5024 LGALSL 7.37E-10 4.51215 DNAJB1 4.39E-06 4.52189 KRT4 5.54E-08 4.52559 EPHA2 3.03E-05 4.52589 BCAS1 5.79E-05 4.52617 MAP1LC3B 4.82E-10 4.52652 DNAJB4 5.21E-08 4.54149 IDI2 1.04E-08 4.55904 FAM175A 4.15E-05 4.56156 ZNF304 9.61E-05 4.56965 ABHD5 2.10E-06 4.57152 RDH10 1.01E-06 4.62095 ZNF503 7.79E-06 4.63796 DEFB135 5.39E-05 4.64573 CDKN2AIP 2.88E-07 4.65861 NDRG1 0.00013098 4.66441 DIRAS3 4.53E-06 4.66536 PNRC1 4.94E-11 4.66617 CLK1 5.54E-06 4.67533 VEGFA 1.22E-07 4.67854 SNORA14B 9.61E-06 4.67967 FAM214A 1.80E-08 4.68632 PTRF 6.09E-05 4.69322 GAB1 5.66E-07 4.70366 RBM47 5.24E-08 4.7198 ID3 2.99E-07 4.72777 SPEN 0.00089761 4.73531 MMP28 1.29E-05 4.73571 TOB1 7.25E-06 4.73713 LEP 1.97E-06 4.75505 CCNL1 5.35E-05 4.75823 CDH17 9.45E-08 4.7645 AVIL 4.57E-05 4.77581 SNX31 5.31E-10 4.78985 DDX26B 3.21E-06 4.8007 EML6 1.11E-05 4.83783 SEPP1 4.08E-08 4.83931 RYBP 1.31E-05 4.84151 CD14 1.00E-05 4.84214 ARG2 9.76E-06 4.84985 RDH10 2.97E-07 4.86769 OVOL1 9.06E-05 4.87451 BTG1 4.79E-09 4.87782 ING1 6.91E-10 4.88184 GTF2IRD2 3.53E-06 4.89709 PPP1R10 8.65E-07 4.89933 PTPRN2 1.88E-06 4.90343 POU2F2 2.40E-08 4.90407 HSPH1 2.98E-06 4.90936 PBX1 6.65E-05 4.91386 TDP2 2.60E-07 4.91534 BCL2L11 1.04E-09 4.91598 TRIB1 1.73E-06 4.92548 NUFIP2 1.59E-08 4.9342 PCDHA1 3.20E-07 4.94477 HEG1 2.38E-07 4.94539 CYP2U1 4.11E-06 4.94686 NFAT5 3.98E-08 4.94902 IDI1 5.83E-07 4.95145 FRY 0.00013107 4.95643 ARL5B 0.00010006 4.96531 C16orf72 2.02E-06 4.96628 DDX3X 6.42E-05 4.97612 PTGER4 0.0001235 5.00728 NR4A1 0.0045056 5.01208 SPDYE6 5.16E-07 5.0167 ELL2 5.24E-07 5.02976 MED26 3.61E-07 5.05223 CDKN1C 3.49E-05 5.05482 TUBB8 8.90E-06 5.05936 PMAIP1 3.58E-07 5.06392 NUFIP2 0.00058049 5.07325 CLC 7.03E-07 5.07583 GIMAP8 2.66E-05 5.08783 LINC00341 4.90E-08 5.10624 INTS6 9.25E-08 5.11893 SLC3A1 2.06E-09 5.1241 SLC6A13 6.26E-06 5.13146 DEPTOR 5.30E-05 5.14613 ZFAND5 2.07E-09 5.17766 EPHA2 6.07E-05 5.18646 BAMBI 6.41E-08 5.19047 C1orf63 1.72E-09 5.19435 HSPA1L 3.22E-08 5.20236 KDM6A 1.14E-09 5.20983 EREG 0.00017579 5.21254 TSC22D2 4.45E-08 5.21447 BTG1 7.43E-10 5.21742 GADD45B 4.95E-07 5.21843 C1orf63 5.23E-10 5.22219 SLC30A1 1.05E-10 5.22228 FOXA3 5.60E-05 5.24276 ABHD5 1.89E-05 5.24428 RND1 7.37E-06 5.28386 ELOVL3 5.32E-09 5.2851 USP12 1.33E-06 5.29303 DIP2C 4.31E-06 5.29586 IQCJ-SCHIP1 3.52E-06 5.31533 INTS6 6.16E-08 5.32474 ZNF295 1.40E-06 5.3396 HAVCR1 7.34E-05 5.35415 VEGFA 7.76E-07 5.35538 CCNYL1 4.53E-06 5.38598 EIF4A2 2.20E-09 5.40277 DUSP10 2.36E-06 5.41332 BCL2L11 0.00014494 5.44923 USP12 6.98E-08 5.45115 PDE6C 8.11E-05 5.46893 PTP4A1 8.26E-07 5.4752 RBBP6 1.22E-08 5.48013 ARL5B 4.78E-05 5.48905 SLC19A3 0.00018659 5.49905 NR4A1 0.00585083 5.5023 CATSPERG 9.02E-09 5.5357 ABHD5 5.84E-07 5.56055 RTN4 0.00011162 5.57753 CDKN1C 4.99E-06 5.59122 FLNC 8.86E-07 5.59233 SLA 2.95E-11 5.59491 C1orf114 6.64E-06 5.59563 CDKN2B 1.78E-06 5.61301 TRNAU1AP 6.20E-08 5.61902 GPR83 1.90E-07 5.62173 ZBTB1 4.24E-06 5.62799 GAB1 4.89E-05 5.63173 ZNF34 3.18E-06 5.63312 INTS6 3.17E-08 5.64325 IRS2 1.59E-09 5.6437 TSC22D3 5.65E-06 5.64569 BCL6 7.98E-07 5.64873 PNRC1 1.29E-07 5.65356 DDR2 6.03E-08 5.68926 LARP6 1.44E-06 5.71097 BAG3 2.72E-08 5.72669 RGS16 2.02E-06 5.72949 FLJ20518 5.61E-10 5.7361 AKAP7 3.01E-05 5.74206 KCNQ1OT1 2.33E-06 5.76314 NXF1 7.94E-12 5.76567 GPR64 2.84E-05 5.83899 MSMO1 1.31E-06 5.84247 MXI1 1.74E-05 5.85607 KRT4 5.38E-10 5.87873 RASL11A 0.00028442 5.886 GPX3 8.93E-07 5.89415 FRMD4B 5.78E-06 5.89501 NDRG1 5.00E-05 5.90877 DUSP1 5.06E-09 5.93444 LARP6 7.23E-08 5.94832 SLC7A9 2.26E-08 5.96841 NDRG1 3.71E-05 5.98572 FBXL17 4.02E-05 5.98899 AHR 6.69E-09 6.00747 ZSWIM6 2.03E-09 6.04473 LINC00281 1.45E-08 6.08391 ARL5B 5.97E-06 6.11407 SEPP1 1.57E-09 6.12256 LETMD1 4.65E-06 6.13785 FKBP14 6.63E-07 6.15465 FAM134B 0.00030111 6.16205 NDRG1 1.63E-05 6.16421 CXCL13 1.87E-08 6.17364 PELI1 5.29E-05 6.22638 JUN 1.47E-06 6.23384 RDH10 9.30E-09 6.24271 CXCL3 5.16E-07 6.26349 GDF15 1.26E-06 6.27649 TIPARP 0.00033865 6.30392 SMAD7 4.75E-07 6.32547 FXYD3 1.68E-05 6.33679 SEMA6A 4.96E-06 6.35036 EIF5 3.32E-07 6.37693 KCNH2 8.19E-06 6.38604 FAM108B1 9.60E-06 6.41358 ELL2 6.13E-08 6.4266 RASD1 8.82E-08 6.42713 TNFSF11 4.45E-06 6.43573 EFNB2 5.57E-06 6.45131 DUSP10 8.64E-08 6.46688 FAM22D 6.17E-06 6.4739 RBBP6 1.08E-06 6.48622 SLC3A1 1.66E-07 6.49717 TSPAN12 3.41E-08 6.50083 EIF4E 3.79E-06 6.54363 DNAJB1 1.25E-07 6.54596 MYLIP 1.52E-09 6.55031 ZNF439 2.41E-05 6.57876 RND3 7.79E-11 6.63115 FKBP14 2.97E-08 6.6655 FOXD4 2.45E-08 6.73723 CPEB4 3.31E-05 6.79429 ITGAM 8.45E-07 6.86874 RBBP6 2.58E-07 6.87928 HIST1H2BK 1.06E-10 6.93756 LDHAL6B 5.20E-09 6.97849 MYLIP 1.95E-10 6.99735 SLC38A2 1.52E-07 7.02279 ADM 8.62E-09 7.03094 ELL2 1.64E-06 7.04167 DDIT3 4.73E-10 7.0557 PLSCR4 8.04E-08 7.06772 NXF1 2.79E-06 7.07105 ATF3 2.79E-05 7.07277 ING1 1.24E-10 7.0775 DDR2 4.33E-08 7.09397 AADAC 3.15E-06 7.13475 C4orf47 2.56E-08 7.14427 ASXL1 7.95E-08 7.14457 SPDYE1 4.13E-05 7.1706 BRD2 1.05E-05 7.18802 HSPH1 2.69E-06 7.21138 HIST1H2AG 2.49E-05 7.21768 FILIP1L 2.12E-06 7.3244 TNFSF15 5.34E-08 7.34917 CXCL3 2.76E-07 7.38732 SLC38A2 7.68E-07 7.39534 CPEB2 5.86E-08 7.42192 NDRG1 0.00019185 7.44941 GATA2 2.45E-07 7.46095 TAF7L 4.39E-08 7.56659 JUN 4.98E-09 7.6043 CDK5R1 2.08E-06 7.70502 GDF15 2.64E-06 7.7787 CCNYL1 5.15E-10 7.78566 S100A7 6.55E-10 7.78639 DDIT3 5.04E-10 7.82871 RAB30 8.16E-07 7.91706 SLC38A2 2.66E-06 7.93323 TSC22D2 5.71E-09 7.97459 GADD45A 2.96E-07 8.0303 IL11 1.12E-11 8.06652 USP38 1.98E-08 8.11592 KLHL15 2.71E-08 8.13594 PPM1D 7.28E-07 8.17047 DNAJB9 5.24E-05 8.21276 TIGD3 3.72E-08 8.2231 DDR2 8.69E-09 8.2657 CCNG2 1.55E-05 8.28025 BCL6 1.09E-07 8.2945 ITPR1 1.50E-07 8.52954 SNX31 5.42E-08 8.61761 CPEB2 9.58E-09 8.65764 NUFIP2 5.03E-07 8.80873 JUN 1.94E-07 8.81318 BCL6 8.56E-11 8.84516 MYLIP 1.70E-06 8.87696 HSPA2 2.28E-08 8.92543 LOC729974 2.30E-08 8.95921 FOS 2.31E-08 9.12045 SIX4 8.21E-07 9.16375 MGARP 4.55E-09 9.17524 TSC22D2 2.08E-10 9.30512 ITGAM 2.06E-06 9.3096 IRS2 1.44E-08 9.33457 GATA2 4.43E-10 9.35042 NUFIP2 2.92E-10 9.53514 CBX4 7.97E-09 9.55449 CITED2 1.45E-08 9.57062 OVOL1 7.44E-05 9.611 RBM47 1.24E-07 9.66099 MYBL1 4.77E-07 9.68806 NR4A3 5.27E-07 9.69223 HIST1H2AG 2.91E-06 9.81615 ARL4D 3.50E-06 9.85032 EID3 3.95E-07 10.0055 MCL1 2.14E-06 10.0344 DDX3X 0.00012398 10.2062 ID3 1.27E-10 10.2668 CTGF 4.89E-08 10.3196 BTG1 2.43E-07 10.4197 DUSP1 6.63E-08 10.4742 FGF18 3.72E-10 10.5133 RND3 1.26E-09 10.5359 GADD45G 4.08E-09 10.5372 MIR22 1.86E-09 10.5374 EGR1 8.80E-06 10.549 TSC22D2 5.01E-09 10.5593 JUN 3.45E-08 10.6039 HEY1 6.82E-11 10.7461 ZSWIM6 1.09E-06 10.7743 HEXIM1 1.46E-08 10.8649 KLHL24 1.37E-07 10.9238 FOS 1.84E-08 11.0673 CDKN1C 3.94E-08 11.1222 SKIL 2.25E-07 11.1825 SLC16A10 4.85E-08 11.2013 TSC22D2 4.49E-11 11.2699 SLA 1.27E-07 11.3869 TUBB7P 3.63E-09 11.3946 NFAT5 7.98E-06 11.4059 KRT12 6.73E-09 11.4824 WSB1 7.25E-08 11.572 FAM117A 6.65E-10 11.6477 GPR64 1.93E-05 11.7205 CRTAM 8.45E-08 11.7335 GDF15 1.53E-06 11.7675 GDF9 1.06E-07 11.803 TRIM69 1.48E-09 11.8276 CYP2U1 1.93E-06 12.0693 PPM1D 2.48E-11 12.2122 ARL14 1.26E-08 12.3434 KCNE4 2.96E-08 12.7518 RND3 4.59E-08 12.7594 C3orf58 3.61E-07 12.8049 CBX4 1.40E-11 12.9514 PPM1D 5.19E-10 12.9614 NR4A2 3.19E-08 13.0252 BCL6 6.99E-09 13.1002 ADM 3.85E-12 13.5239 CITED2 1.42E-05 13.5315 SKIL 5.18E-08 13.6462 PCDHA1 9.22E-07 13.8481 PPP1R15A 2.17E-11 13.8565 C1orf63 1.73E-08 14.1159 THBS1 1.57E-06 14.5019 ARL4D 3.79E-10 14.6443 RAB30 5.16E-08 14.6971 FOS 1.18E-08 14.8274 S100A7 4.70E-10 14.8652 PPM1D 1.99E-09 14.8855 NR4A1 0.00128418 15.2494 IL20 1.11E-08 15.5155 SKIL 7.01E-08 15.7276 MAFB 6.39E-09 16.3042 THBS1 9.10E-09 16.3444 C7orf53 1.83E-10 16.3862 PCK1 0.00072946 16.678 MEX3B 9.92E-10 16.6927 HEXIM1 3.13E-08 16.7646 DDR2 1.27E-09 17.014 NR4A2 4.47E-09 17.5743 FOS 8.14E-10 17.7463 MCL1 3.87E-07 17.8411 SNAI1 1.45E-11 18.0173 ADM 7.25E-13 18.2656 NR4A2 1.23E-09 18.3035 FPR2 5.23E-08 18.5427 CYR61 1.24E-10 18.6906 CTGF 2.83E-10 18.8584 HEXIM1 1.19E-11 19.6499 EGR3 5.64E-05 19.6948 PDK4 4.12E-08 19.7129 KLF4 3.51E-12 19.7363 HBEGF 4.84E-09 20.4206 CSRNP1 0.00022353 20.6759 C1S 7.90E-10 21.9537 CD55 1.47E-08 21.9871 KLF4 1.46E-11 22.0876 HEXIM1 2.09E-11 23.1251 THBS1 3.16E-08 23.4806 DDR2 1.55E-10 23.5079 HEXIM1 9.21E-09 23.5434 EGR1 9.24E-08 26.0004 HBEGF 1.89E-10 27.7547 FOSB 1.13E-06 27.998 CTGF 5.52E-09 28.0556 MIR22 7.85E-10 28.0988 PCK1 0.00044064 28.9244 EGR1 1.90E-07 28.9641 MAFB 6.49E-09 29.7213 PCK1 0.00081135 31.485 MIR22 3.60E-10 31.6836 MIR22 4.97E-10 32.3044 MAFB 1.11E-09 33.4953 GADD45B 9.38E-11 36.0615 ATF3 2.12E-05 39.5912 DLX2 9.37E-11 40.526 FAM171B 2.59E-11 45.0896 MIR22 5.41E-09 46.0157 EGR1 3.20E-08 46.1002 CYR61 9.18E-12 50.9559 EGR1 1.07E-08 59.3485 GADD45B 3.83E-13 62.5888 ERVK3-2 3.46E-11 66.6743 EGR2 5.47E-07 74.2688 FAM46C 4.97E-12 91.0784 GEM 1.82E-12 129.518 FAM46C 1.32E-10 142.744