The zebrafish (Danio rerio) is one of the most widely used research model organisms funded by the United States' National Institutes of Health, second only to the mouse. Here, we discuss the advantages and unique qualities of this model organism. Additionally, we discuss key aspects of experimental design and statistical approaches that apply to studies using the zebrafish model organism. Finally, we list critical details that should be considered in the design of zebrafish experiments to enhance rigor and data reproducibility. These guidelines are designed to aid new researchers, journal editors, and manuscript reviewers in supporting the publication of the highest-quality zebrafish research.

Abstract Intratumoral hypoxia correlates with metastasis and poor survival in patients with sarcoma. Using an impedance sensing assay and a zebrafish intravital microinjection model, we demonstrated here that the hypoxia-inducible collagen-modifying enzyme lysyl hydroxylase PLOD2 and its substrate collagen type VI (COLVI) weaken the lung endothelial barrier and promote transendothelial migration. Mechanistically, hypoxia-induced PLOD2 in sarcoma cells modified COLVI, which was then secreted into the vasculature. Upon reaching the apical surface of lung endothelial cells, modified COLVI from tumor cells activated integrin β1 (ITGβ1). Furthermore, activated ITGβ1 colocalized with Kindlin2, initiating their interaction with F-actin and prompting its polymerization. Polymerized F-actin disrupted endothelial adherens junctions and induced barrier dysfunction. Consistently, modified and secreted COLVI was required for the late stages of lung metastasis in vivo. Analysis of patient gene expression and survival data from The Cancer Genome Atlas (TCGA) revealed an association between the expression of both PLOD2 and COLVI and patient survival. Furthermore, high levels of COLVI were detected in surgically resected sarcoma metastases from patient lungs and in the blood of tumor-bearing mice. Together, these data identify a mechanism of sarcoma lung metastasis, revealing opportunities for therapeutic intervention. Significance: Collagen type VI modified by hypoxia-induced PLOD2 is secreted by sarcoma cells and binds to integrin β1 on endothelial cells to induce barrier dysfunction, which promotes sarcoma vascular dissemination and metastasis.

Phospho Antibody Array Data

Supplementary figures and legends

The past 5 years have seen tremendous growth at Cellular and Molecular Gastroenterology and Hepatology (CMGH), the official basic science research journal of the American Gastroenterological Association (AGA) Institute. When we took over from the founding editorial team in July of 2019, it was our goal to increase the number of yearly submissions to 300—a goal that we exceeded by more than 3-fold, with nearly 1000 submissions in the past year. We thank the authors and readers of CMGH for accepting CMGH as their go-to journal to make this success possible.

Neural crest (NC) is a unique vertebrate cell type arising from the border of the neural plate and epidermis that gives rise to diverse tissues along the entire body axis. Roberto Mayor and colleagues have made major contributions to our understanding of NC induction, delamination, and migration. We report that a truncating mutation of the classical tumor suppressor Adenomatous Polyposis Coli (apc) disrupts craniofacial development in zebrafish larvae, with a marked reduction in the cranial neural crest (CNC) cells that contribute to mandibular and hyoid pharyngeal arches. While the mechanism is not yet clear, the altered expression of signaling molecules that guide CNC migration could underlie this phenotype. For example, apcmcr/mcr larvae express substantially higher levels of complement c3, which Mayor and colleagues showed impairs CNC cell migration when overexpressed. However, we also observe reduction in stroma-derived factor 1 (sdf1/cxcl12), which is required for CNC migration into the head. Consistent with our previous work showing that APC directly enhances the activity of glycogen synthase kinase 3 (GSK-3) and, independently, that GSK-3 phosphorylates multiple core mRNA splicing factors, we identify 340 mRNA splicing variations in apc mutant zebrafish, including a splice variant that deletes a conserved domain in semaphorin 3f (sema3f), an axonal guidance molecule and a known regulator of CNC migration. Here, we discuss potential roles for apc in CNC development in the context of some of the seminal findings of Mayor and colleagues.

See “Deleterious variants in ABCC12 are detected in idiopathic chronic cholestasis and cause of intrahepatic bile duct loss in model organisms,” by Pham D-H, Kudira R, Xu L, et al, on page 287. See “Deleterious variants in ABCC12 are detected in idiopathic chronic cholestasis and cause of intrahepatic bile duct loss in model organisms,” by Pham D-H, Kudira R, Xu L, et al, on page 287. Neonatal cholestasis is an important cause of pediatric liver disease.1Fawaz R. Baumann U. Ekong U. et al.Guideline for the evaluation of cholestatic jaundice in infants: joint recommendations of the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition and the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition.J Pediatr Gastroenterol Nutr. 2017; 64: 154-168Crossref PubMed Scopus (170) Google Scholar,2Feldman A.G. Sokol R.J. Neonatal cholestasis: emerging molecular diagnostics and potential novel therapeutics.Nat Rev Gastroenterol Hepatol. 2019; 16: 346-360Crossref PubMed Scopus (37) Google Scholar Clinical evaluation at presentation is focused on distinguishing extrahepatic biliary system disorders from intrahepatic causes. A significant percentage of the latter group have a genetic etiology affecting either bile acid metabolism or intrahepatic biliary development, both of which lead to retention of toxic bile acids within hepatocytes.1Fawaz R. Baumann U. Ekong U. et al.Guideline for the evaluation of cholestatic jaundice in infants: joint recommendations of the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition and the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition.J Pediatr Gastroenterol Nutr. 2017; 64: 154-168Crossref PubMed Scopus (170) Google Scholar, 2Feldman A.G. Sokol R.J. Neonatal cholestasis: emerging molecular diagnostics and potential novel therapeutics.Nat Rev Gastroenterol Hepatol. 2019; 16: 346-360Crossref PubMed Scopus (37) Google Scholar, 3Goldberg A. Mack C.L. Inherited Cholestatic Diseases in the Era of Personalized Medicine.Clin Liver Dis (Hoboken). 2020; 15: 105-109Crossref PubMed Scopus (3) Google Scholar Over time, chronic hepatocyte injury and inflammation triggered by bile acid retention leads to hepatic fibrosis and, in some instances, cirrhosis and liver failure.4Li M. Cai S.Y. Boyer J.L. Mechanisms of bile acid mediated inflammation in the liver.Mol Aspects Med. 2017; 56: 45-53Crossref PubMed Scopus (85) Google Scholar Monogenic disorders that cause infantile cholestasis account for a significant percentage of pediatric liver transplants3Goldberg A. Mack C.L. Inherited Cholestatic Diseases in the Era of Personalized Medicine.Clin Liver Dis (Hoboken). 2020; 15: 105-109Crossref PubMed Scopus (3) Google Scholar,5Nicastro E. Di Giorgio A. Marchetti D. et al.Diagnostic yield of an algorithm for neonatal and infantile cholestasis integrating next-generation sequencing.J Pediatr. 2019; 211: 54-62Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar; thus, a timely diagnosis is important. Advances in molecular analyses have streamlined the diagnostic approach to infants with cholestasis considered to have a genetic origin. Currently, this involves the combined use of DNA sequencing panels (to interrogate established candidate genes) and whole-genome exome sequencing for individuals with nondiagnostic panel findings.5Nicastro E. Di Giorgio A. Marchetti D. et al.Diagnostic yield of an algorithm for neonatal and infantile cholestasis integrating next-generation sequencing.J Pediatr. 2019; 211: 54-62Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 6Lipiński P. Ciara E. Jurkiewicz D. et al.Targeted next-generation sequencing in diagnostic approach to monogenic cholestatic liver disorders-single-center experience.Front Pediatr. 2020; 8: 414Crossref PubMed Scopus (5) Google Scholar, 7Maddirevula S. Alhebbi H. Alqahtani A. et al.Identification of novel loci for pediatric cholestatic liver disease defined by KIF12, PPM1F, USP53, LSR, and WDR83OS pathogenic variants.Genet Med. 2019; 21: 1164-1172Crossref PubMed Scopus (30) Google Scholar In addition to identifying pathogenic variants in recognized cholestasis genes, most commonly those responsible for Alpha-1 antitrypsin deficiency, Alagille Syndrome, and progressive familial intrahepatic cholestasis (PFIC) types 1–6, this approach can identify novel gene variants, often of unknown clinical significance, thus presenting a diagnostic dilemma for physicians. Predictive algorithms can often determine which variants will have functional consequences and, thus, are likely to be causative; however, experimental analysis in model systems can play an important confirmatory role. The article published by Pham et al8Pham D.-H. Kudira R. Xu L. et al.Deleterious variants in ABCC12 are detected in idiopathic chronic cholestasis and cause of intrahepatic bile duct loss in model organisms.Gastroenterology. 2021; 161: 287-300Abstract Full Text Full Text PDF Scopus (2) Google Scholar in this issue of Gastroenterology reports the use of exome sequencing in 4 patients with unexplained neonatal cholestasis and is a significant contribution to our understanding of this disorder. Three of the 4 patients analyzed were found to be homozygous for recessive pathogenic variants in 2 established PFIC genes, TJP2 and ABCB11. The fourth was homozygous for a novel single nucleotide deletion that led to a frame shift and premature termination codon in the ABCC12 gene, which encodes MRP9, an ABC transporter of unknown function. Targeted sequencing subsequently identified 5 additional rare heterozygous ABCC12 coding variants in 89 subjects with unexplained cholestasis. One of the ABCC12 variants was predicted to truncate MRP9, like the index patient’s variant, and was therefore deemed pathogenic. The remaining 4 ABCC12 variants were considered to be of unknown significance. The presence of 2 pathogenic mutations in this patient cohort, one of which was biallelic (homozygous), argued in favor of causality despite little understanding of the ABCC12/MRP9 function. To gain additional insight into causality, Pham et al8Pham D.-H. Kudira R. Xu L. et al.Deleterious variants in ABCC12 are detected in idiopathic chronic cholestasis and cause of intrahepatic bile duct loss in model organisms.Gastroenterology. 2021; 161: 287-300Abstract Full Text Full Text PDF Scopus (2) Google Scholar studied the hepatic expression of MRP9 via immunohistochemistry, documenting its presence in large and small intrahepatic bile ducts and zone 1 hepatocytes. Interestingly, a liver biopsy specimen from the index patient showed bile duct paucity, thus suggesting a role for MRP9 in cholangiocytes. Supporting this hypothesis, male zebrafish engineered to carry a homozygous Mrp9 protein truncation mutation developed pronounced bile duct paucity accompanied by growth delay and splenomegaly. Subsequent analyses determined that bile duct paucity arose from cholangiocyte cell death (via apoptosis) rather than other plausible explanations, such as defects in cholangiocyte proliferation or maintenance of cell fate. Female mutants developed normally without bile duct paucity; the basis of the sex predilection was not determined. Having generated elegant data in the zebrafish model supporting a role for ABCC12 in the index patient’s cholestasis syndrome, Pham et al8Pham D.-H. Kudira R. Xu L. et al.Deleterious variants in ABCC12 are detected in idiopathic chronic cholestasis and cause of intrahepatic bile duct loss in model organisms.Gastroenterology. 2021; 161: 287-300Abstract Full Text Full Text PDF Scopus (2) Google Scholar established a mouse model that enabled them to study MRP9 function in mammalian cholangiocytes. Mice homozygous for an Mrp9 truncating mutation had biochemical evidence of liver injury and cholestasis along with histologic evidence of intrahepatic bile duct paucity. Interestingly, bile duct injury was increased, and cholangiocyte survival was reduced (ex vivo) as a result of apoptotic cell death in homozygous and heterozygous ABCC12 mutants treated with cholic acid, a toxic human bile acid present at only low levels in mice, which normally have a more polar and, hence, comparatively nontoxic bile acid pool.9de Boer J.F. Verkade E. Mulder N.L. et al.A human-like bile acid pool induced by deletion of hepatic Cyp2c70 modulates effects of FXR activation in mice.J Lipid Res. 2020; 61: 291-305Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar,10de Boer J.F. de Vries H.D. Palmiotti A. et al.Cholangiopathy and biliary fibrosis in Cyp2c70-deficient mice are fully reversed by ursodeoxycholic acid.Cell Mol Gastroenterol Hepatol. 2020; 11: 1045-1069Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar These data argue that ABCC12/MRP9 plays a constitutive role in the cholangiocyte injury response. In summary, the data generated by Pham et al8Pham D.-H. Kudira R. Xu L. et al.Deleterious variants in ABCC12 are detected in idiopathic chronic cholestasis and cause of intrahepatic bile duct loss in model organisms.Gastroenterology. 2021; 161: 287-300Abstract Full Text Full Text PDF Scopus (2) Google Scholar are significant for 2 reasons, First, they argue that ABCC12/MRP9 is a bona fide cholestasis susceptibility gene that should be included in diagnostic DNA sequencing panels. Second, the data offer novel insights into the function of MRP9, which appears to prevent cholangiocyte injury caused, at least in part, by their absorption of free, nonmicellar bile acids. Given continuous bile exposure, cholangiocytes have developed adaptive mechanisms to limit bile acid toxicity, such as alkalization of bile, which deprotonates free bile acids (preventing uptake via passive diffusion) and the expression of transporters that excrete bile acids for transport into the vasculature.11Banales J.M. Huebert R.C. Karlsen T. Strazzabosco M. LaRusso N.F. Gores G.J. Cholangiocyte pathobiology.Nat Rev Gastroenterol Hepatol. 2019; 16: 269-281Crossref PubMed Scopus (117) Google Scholar,12Trampert D.C. van de Graaf S.F.J. Jongejan A. Beuers U. Oude Elferink RPJHepatobiliary acid-base homeostasis: insights from analogous secretory epithelia.J Hepatol. 2021; 74: 428-441Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar Further experimental studies will be required to determine whether MRP9 normally contributes to these or novel adaptive mechanisms and whether this knowledge can be exploited for therapeutic purposes. An example of the latter is the recently reported protective role of redox and proteomic stress response in extrahepatic biliary atresia, the most common cause of neonatal cholestasis.13Zhao X. Lorent K. Escobar-Zarate D. et al.Impaired redox and protein homeostasis as risk factors and therapeutic targets in toxin-induced biliary atresia.Gastroenterology. 2020; 159: 1068-1084Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar,14Luo Z. Shivakumar P. Mourya R. Gutta S. Bezerra J.A. Gene expression signatures associated with survival times of pediatric patients with biliary atresia identify potential therapeutic agents.Gastroenterology. 2019; 157: 1138-1152Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar Like the current study, the combined used of zebrafish and cell culture models played a central role in these discoveries, additionally identifying modulators of cGMP signaling as a potential way to augment cholangiocyte survival in response to exogenous stressors.13Zhao X. Lorent K. Escobar-Zarate D. et al.Impaired redox and protein homeostasis as risk factors and therapeutic targets in toxin-induced biliary atresia.Gastroenterology. 2020; 159: 1068-1084Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar Also to be considered in future studies is whether cholestasis induced by ABCC12/MRP9 mutation arises from a nonhepatic function of MRP9, as was recently proposed for ABCC7/CFTR in cystic fibrosis cholangiopathy,15Fiorotto R. Strazzabosco M. Pathophysiology of cystic fibrosis liver disease: a channelopathy leading to alterations in innate immunity and in microbiota.Cell Mol Gastroenterol Hepatol. 2019; 8: 197-207Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar and whether loss of MRP9 affects cholangiocyte heterogeneity.16Maroni L. Haibo B. Ray D. et al.Functional and structural features of cholangiocytes in health and disease.Cell Mol Gastroenterol Hepatol. 2015; 1: 368-380Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar,17Tulasi D.Y. Castaneda D.M. Wager K. et al.Sox9EGFP defines biliary epithelial heterogeneity downstream of Yap activity.Cell Mol Gastroenterol Hepatol. 2021; 11: 1437-1462Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar From a clinical perspective, the most important unanswered question raised by the work of Pham et al8Pham D.-H. Kudira R. Xu L. et al.Deleterious variants in ABCC12 are detected in idiopathic chronic cholestasis and cause of intrahepatic bile duct loss in model organisms.Gastroenterology. 2021; 161: 287-300Abstract Full Text Full Text PDF Scopus (2) Google Scholar is whether the heterozygous ABCC12 missense variants they report are pathogenic. These genotype-phenotype correlations will be clarified over time as more variants are identified. Indeed, it is conceivable that ABCC12 variants cause a spectrum of cholestatic disorders, similar to ABCB4, which has been linked to PFIC type 3 and other conditions.18Stättermayer A.F. Halilbasic E. Wrba F. Ferenci P. Trauner M. Variants in ABCB4 (MDR3) across the spectrum of cholestatic liver diseases in adults.J Hepatol. 2020; 73: 651-663Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar Until then, and in the absence of in vitro assays of MRP9 function, the most direct approach may be to analyze patient variants using in vivo (zebrafish, mouse) and/or cell culture models, a strategy that is greatly facilitated by the ever-improving arsenal of CRISPR/Cas9 genome-editing methods.19Anzalone A.V. Koblan L.W. Liu D.R. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors.Nat Biotechnol. 2020; 38: 824-844Crossref PubMed Scopus (278) Google Scholar Given the efficiency of these methods, it is conceivable that this approach may even become an integral part of the neonatal cholestasis diagnostic workup.

Dear Contributors, Reviewers and Readers of CMGH, As we approach the half-way point in our tenure as co-Editors-in-Chief of CMGH, we are happy to share new information demonstrating the significant impact CMGH continues to have within the digestive disease research community, and CMGH’s ever improving journal metrics, which highlight its continued growth and overall success. Perhaps most important to our readership is our second Clarivate Impact Factor, which was released in June 2021. As you may recall, our inaugural impact factor was 7.076, which is outstanding for a new journal. We are delighted to tell you now that our 2020 Impact Factor climbed to 9.225, which again is a remarkable achievement for a new journal. Many factors have contributed to this rapid acceptance of the journal to our research community. The first is the outstanding work of our former Board of Editors and reviewers that oversaw articles published in 2018 and 2019. Secondly, the efforts of the journal’s current Associate Editors, editorial board and of course the talented authors allowed us to maintain this high level of performance in 2020. Thirdly, the continued superb administrative support from the AGA and our publisher Elsevier is an important contributor to the success of the journal. Thank you to all of our former and current colleagues for enabling CMGH to thrive in the highly competitive world of scientific publishing. While citation data that underly the Impact Factor are an important indicator of a journal’s success, other metrics are equally useful measures by which to gauge impact. Another metric of interest is CiteScore. The 2020 Cite Score considers the citations received in 2017-2020 to articles and reviews published in 2017-2020. Remarkably, CMGH already ranks 11th among 271 gastroenterology and hepatology journals, with a 2020 CiteScore of 11.6. Additionally, CMGH has seen a progressive increase in manuscript submissions of the last three years. For 2021 we are on track to receive over 1,000 manuscripts, compared to 600 in 2020 and only 250 in 2019. The large number of high-quality submissions we are now receiving is an important indicator of the journal’s standing within our field. Furthermore, we have maintained an impressive turnaround time for new article submissions, which now averages 13 days for a first decision, and 28 days to a first decision for those manuscripts undergoing external review. As co-Editors-in-Chief, we pledge to do our best to shorten these decision times even further while maintaining an open dialog with our authors when conflicts arise. Overall, we consider it our responsibility to help our readership gain access to high quality, high impact original research as quickly as possible. We are indebted to our Associate Editors for helping us make the difficult decisions needed to select manuscripts for publication. Sadly, earlier this year we had to say “Auf Wiedersehen” to Dr. Tom Lüdde, whose new responsibilities as Director of Gastroenterology, Hepatology and Infectious Diseases at the University Hospital in Düsseldorf, Germany precluded his continued work for the journal. Tom was an outstanding Associate Editor, and we are grateful that he has agreed to be a member of our editorial board and continue to review manuscripts related to hepatology. We wish Tom the best of luck in his new position! While we will certainly miss Dr. Lüdde’s insights and expertise, we are lucky to have recruited an equally talented and highly regarded hepatology researcher as a new Associate Editor. Dr. Irene Ng, Chair of Pathology and Director of the State Key Laboratory for Liver Research at the University of Hong Kong, has joined our Board of Editors as of July 1. Dr. Ng has extensive experience in many aspects of liver research, particularly hepatocellular carcinoma, for which she is recognized internationally as a thought leader. We are looking forward to receiving Dr. Ng’s guidance on the many liver-related manuscripts we consider for publication. As the number of manuscripts submitted to CMGH has grown, it became necessary to recruit a fourth Associate Editor to help the Board of Editors evaluate the ever-increasing submissions related to gut immunology. Dr. Alison Simmons, Professor of Gastroenterology at Oxford University, has done a remarkable job handling these submissions for the past two years. As of July 1, Dr. Simmons was joined by Dr. Nadine Cerf-Bensussan, Research Director at the French National Institute of Health and Medical Research (INSERM), and head of the Laboratory of Intestinal Immunity at Imagine Institute and Paris University Paris. Dr. Cerf-Bensussan is one of the world’s leading authorities on celiac disease and mucosal immunology and we are excited to have her as a colleague on the Board of Editors. Several years ago, AGA began an editorial fellowship program for its journals, Gastroenterology, CGH,CMGH and TIGE. The goal of this program is to provide talented junior researchers the opportunity to gain editorial experience at an early stage of their career through participation in Board of Editors meetings and providing assistance to Associate Editors evaluating manuscripts within the fellow’s area of scientific expertise. Our first two years in the program brought to us three outstanding fellows who provided valuable contributions to the journal: Dr. Cambrian Liu from Children’s Hospital Los Angeles, Dr. Tirthadipa Pradhan from the University of Pittsburgh, and Dr. Sam Hinman from the University of Washington. This year, we welcome two new fellows, Dr. Vivian Ortiz, who is currently completing basic science training in hepatology as part of her GI fellowship at the University of Pennsylvania, and Dr. Lindsey Kennedy, who is an Assistant Research Professor at the Indiana University School of Medicine. Drs. Ortiz and Kennedy have expertise in many aspects of liver biology, and we look forward to having the opportunity to interact with and mentor them this year. In closing, we thank our authors and reviewers, and you, the CMGH readers, for allowing us to work on your behalf. We also thank our outstanding colleagues with whom we share a vision for the future of CMGH. Looking forward, we will do our best to ensure that CMGH meets your expectations in the upcoming year. Sincerely, Klaus and Michael

Biliary atresia (BA) is a rapidly progressive cholangiopathy of neonates notable for pronounced fibro-obliteration of the bileducts, particularly extra hepatic bile ducts, which in the majority of patients results in end-stage liver disease. This chapter reviews the data suggesting that BA has an environmental cause with a particular emphasis on the evidence that toxins contribute to other human biliary diseases and to other forms of human organ fibrosis. Cholangiocytes are metabolically active cells that have important roles in normal liver physiology including modification of bile, antigen presentation, and immune signaling. The most extensively characterized toxicant associated with biliary injury in humans is 4,4'-methylenedianiline. Alpha-naphthylisothiocyanate is well-characterized toxicant that induces biliary cell injury. 3,5-Diethoxycarbonyl-1,4-dihydro-collidine is a third xenobiotic with well-characterized biliary toxicity. Environmental toxins are well-established causes of organ fibrosis.

BACKGROUND AND AIMS Extra-hepatic biliary atresia (BA) is a pediatric liver disease with no approved medical therapy. Recent studies using human samples and experimental modeling suggest that glutathione redox metabolism and heterogeneity play a role in disease pathogenesis. We sought to dissect the mechanistic basis of liver redox variation and explore how other stress responses affect cholangiocyte injury in BA. METHODS We performed quantitative in situ hepatic glutathione redox mapping in zebrafish larvae carrying targeted mutations in glutathione metabolism genes and correlated these findings with sensitivity to the plant-derived BA-linked toxin biliatresone. We also determined whether genetic disruption of HSP90 protein quality control pathway genes implicated in human BA altered biliatresone toxicity in zebrafish and human cholangiocytes. An in vivo screen of a known drug library was performed to identify novel modifiers of cholangiocyte injury in the zebrafish experimental BA model with subsequent validation. RESULTS Glutathione metabolism gene mutations caused regionally distinct changes in the redox potential of cholangiocytes that differentially sensitized them to biliatresone. Disruption of human BA-implicated HSP90 pathway genes sensitized zebrafish and human cholangiocytes to biliatresone-induced injury independent of glutathione. Phosphodiesterase-5 inhibitors (PDE5i) and other cGMP signaling activators worked synergistically with the glutathione precursor N-acetylcysteine (NAC) in preventing biliatresone-induced injury in zebrafish and human cholangiocytes. PDE5i enhanced proteasomal degradation and required intact HSP90 chaperone. CONCLUSION Regional variation in glutathione metabolism underlies sensitivity to the biliary toxin biliatresone, and may account for the reported association between BA transplant-free survival and glutathione metabolism gene expression. Human BA can be causatively linked to genetic modulation of protein quality control. Combined treatment with NAC and cGMP signaling enhancers warrants further investigation as therapy for BA.

The field of neuropharmacology has not yet achieved a full understanding of how the brain transitions between states of consciousness and drug-induced unconsciousness, or anesthesia. Many small molecules are used to alter human consciousness, but the repertoire of underlying molecular targets, and thereby the genes, are incompletely understood. Here we describe a robust larval zebrafish model of anesthetic action, from sedation to general anesthesia. We use loss of movement under three different conditions, spontaneous movement, electrical stimulation or a tap, as a surrogate for sedation and general anesthesia, respectively. Using these behavioral patterns, we find that larval zebrafish respond to inhalational and IV anesthetics at concentrations similar to mammals. Additionally, known sedative drugs cause loss of spontaneous larval movement but not to the tap response. This robust, highly tractable vertebrate model can be used in the detection of genes and neural substrates involved in the transition from consciousness to unconsciousness.

Gastrointestinal motility disorders include a spectrum of mild to severe clinical phenotypes that are caused by smooth muscle dysfunction. We investigated the genetic etiology of severe esophageal, gastric, and colonic dysmotility in two unrelated families with autosomal dominant disease presentation. Using exome sequencing, we identified a 2 base pair insertion at the end of the myosin heavy chain 11 (MYH11) gene in all affected members of Family 1 [NM_001040113:c.5819_5820insCA(p.Gln1941Asnfs*91)] and a 1 base pair deletion at the same genetic locus in Proband 2 [NM_001040113:c.5819del(p.Pro1940Hisfs*91)]. Both variants are predicted to result in a similarly elongated protein product. Heterozygous dominant negative MYH11 pathogenic variants have been associated with thoracic aortic aneurysm and dissection while biallelic null alleles have been associated with megacystis microcolon intestinal hypoperistalsis syndrome. This report highlights heterozygous protein-elongating MYH11 variants affecting the SM2 isoforms of MYH11 as a cause for severe gastrointestinal dysmotility, and we hypothesize that the mechanistic pathogenesis of this disease, dominant hypercontractile loss-of-function, is distinct from those implicated in other diseases involving MYH11 dysfunction.

It is with great pride that we announce the first Impact Factor of Cellular and Molecular Gastroenterology and Hepatology (CMGH), which is an astonishing 7.076, thus placing the journal 15th among 88 journals in the field. The Impact Factor is a measure of the frequency with which the average article in a journal has been cited in the 2 years after its publication. Although not the only way to assess the success of a scientific journal, the Impact Factor has become a standard metric frequently used to measure the rank of a journal in its field. For certain, a high Impact Factor such as 7.076 is a testament to the quality of the work being published in CMGH, the high visibility of the articles and the website on which they are published, and the relevance of the content the journal provides. CMGH’s impressive inaugural Impact Factor further attests to the journal’s success and standing within the gastroenterology research community as we began our tenure as new Editors-in-Chief in July 2019. CMGH was founded in 2015 by Jerrold Turner, Editor-in-Chief, with Associate Editors Rebecca Wells and James Goldenring, and later joined by Maria Rescigno, as a forum for novel, cutting-edge science, thought-provoking editorials, and insightful focused reviews of diverse topics in gastrointestinal, hepatobiliary, and pancreatology research. At its outset, CMGH was established as a fully digital publication, with all articles freely available to researchers around the world. Under Jerry Turner’s leadership, the journal also has been successful in providing authors a very short turnaround time from submission to online publication while maintaining rigorous and fair peer-review. We and our Associate Editors, Alison Simmons, Thomas Luedde, and Jonathan Katz, are extremely grateful to the prior CMGH Board of Editors for establishing such an impactful platform for the rapid dissemination of high-quality, peer-reviewed research in our field. We are working hard to maintain and possibly even improve the reach and impact of the journal. As we celebrate the remarkable success and achievement of CMGH, we remind our readers, contributors, reviewers, and friends that all credit goes to the founding Board of Editors: Jerry, Rebecca, and James, and Maria. Thank you!

Abstract High-grade sarcomas are metastatic and pose a serious threat to patient survival. Undifferentiated pleomorphic sarcoma (UPS) is a particularly dangerous and relatively common sarcoma subtype diagnosed in adults. UPS contains large quantities of extracellular matrix (ECM) including hyaluronic acid (HA), which is linked to metastatic potential. Consistent with these observations, expression of the HA receptor, hyaluronan-mediated motility receptor (HMMR/RHAMM), is tightly controlled in normal tissues and upregulated in UPS. Moreover, HMMR expression correlates with poor clinical outcome in these patients. Deregulation of the tumor-suppressive Hippo pathway is also linked to poor outcome in these patients. YAP1, the transcriptional regulator and central effector of Hippo pathway, is aberrantly stabilized in UPS and was recently shown to control RHAMM expression in breast cancer cells. Interestingly, both YAP1 and RHAMM are linked to TGFβ signaling. Therefore, we investigated crosstalk between YAP1 and TGFβ resulting in enhanced RHAMM-mediated cell migration and invasion. We observed that HMMR expression is under the control of both YAP1 and TGFβ and can be effectively targeted with small-molecule approaches that inhibit these pathways. Furthermore, we found that RHAMM expression promotes tumor cell proliferation and migration/invasion. To test these observations in a robust and quantifiable in vivo system, we developed a zebrafish xenograft assay of metastasis, which is complimentary to our murine studies. Importantly, pharmacologic inhibition of the TGFβ–YAP1–RHAMM axis prevents vascular migration of tumor cells to distant sites. Implications: These studies reveal key metastatic signaling mechanisms and highlight potential approaches to prevent metastatic dissemination in UPS.YAP1 and TGFβ cooperatively enhance proliferation and migration/invasion of UPS and fibrosarcomas.

Traditionally, drug dosing is based on a concentration-response relationship estimated in a population. Yet, in specific individuals, decisions based on the population-level effects frequently result in over or under-dosing. Here, we interrogate the relationship between population-based and individual-based responses to anesthetics in mice and zebrafish. The anesthetic state was assessed by quantifying responses to simple stimuli. Individual responses dynamically fluctuated at a fixed drug concentration. These fluctuations exhibited resistance to state transitions. Drug sensitivity varied dramatically across individuals in both species. The amount of noise driving transitions between states, in contrast, was highly conserved in vertebrates separated by 400 million years of evolution. Individual differences in anesthetic sensitivity and stochastic fluctuations in responsiveness complicate the ability to appropriately dose anesthetics to each individual. Identifying the biological substrate of noise, however, may spur novel therapies, assure consistent drug responses, and encourage the shift from population-based to personalized medicine.

To identify mutations that promote the early stages of UV-induced carcinogenesis, we performed whole exome sequencing on 10 paired libraries derived from laser-captured micro-dissected squamous cell carcinoma in situ (SCCIS) and adjacent epidermis. Data analysis demonstrated a high frequency of UV-signature single/double nucleotide variations and deletions in nucleoporins (Nups) which would likely alter the structure and function of the nuclear pore complex (NPC). In total, thirteen of twenty-eight NUPs were mutated in either the epidermis or SCCIS; 25 independent mutations detected in the epidermal libraries and 14 mutations were found in the SCCIS libraries. No analogous mutations were found in 4000 genes which included “housekeeping’ genes such as GAPDH, B2M, PGK1 and PP1A. These data raise the hypothesis that UV-signature mutations in Nups could promote skin cancer. To address this hypothesis, we developed a murine model with heterozygous deletion of Elys in the epidermis. Elys is a nuclear-basket Nup and the only Nup with a DNA-binding domain. Elys heterozygous mice were subjected to three doses of 200mJ/cm2 UVB/A and demonstrated prominent epidermal necrosis followed by prominent hyperplasia; minimal hyperplasia was seen in control mice. The epidermal hyperplasia in Elys heterozygous mice demonstrated prominent dysplasia resembling actinic keratoses. Notch and its target genes, including HES1 were downregulated in Elys heterozygous mice. siRNA mediated knockdown of Nup 153, a frequently mutated Nup, demonstrated increased production of UVB-induced CPD in HaCaT cells. Together, these data implicate loss of Nup function in the early stages UV-induced carcinogenesis.

Chronic kidney disease (CKD) is a complex gene-environmental disease affecting close to 10% of the US population. Genome-wide association studies (GWASs) have identified sequence variants, localized to non-coding genomic regions, associated with kidney function. Despite these robust observations, the mechanism by which variants lead to CKD remains a critical unanswered question. Expression quantitative trait loci (eQTL) analysis is a method to identify genetic variation associated with gene expression changes in specific tissue types. We hypothesized that an integrative analysis combining CKD GWAS and kidney eQTL results can identify candidate genes for CKD. We performed eQTL analysis by correlating genotype with RNA-seq-based gene expression levels in 96 human kidney samples. Applying stringent statistical criteria, we detected 1,886 genes whose expression differs with the sequence variants. Using direct overlap and Bayesian methods, we identified new potential target genes for CKD. With respect to one of the target genes, lysosomal beta A mannosidase (MANBA), we observed that genetic variants associated with MANBA expression in the kidney showed statistically significant colocalization with variants identified in CKD GWASs, indicating that MANBA is a potential target gene for CKD. The expression of MANBA was significantly lower in kidneys of subjects with risk alleles. Suppressing manba expression in zebrafish resulted in renal tubule defects and pericardial edema, phenotypes typically induced by kidney dysfunction. Our analysis shows that gene-expression changes driven by genetic variation in the kidney can highlight potential new target genes for CKD development.

To identify genomic mutations that promote the early stages of UV-induced carcinogenesis, we performed whole exome sequencing on 10 paired libraries derived from laser-captured micro-dissected squamous cell carcinoma in situ (SCCIS) and adjacent epidermis. Prior work has shown that the nuclear pore complex (NPC) plays a key role in maintaining genomic stability in yeast and xenopus. However, the role of the NPC in maintaining genomic stability in mammalian cells, especially during carcinogenesis, remains unclear. Sequence data from the 10 paired libraries were analyzed using a customized software package to identify UV-signature single/double nucleotide variations, deletions, and splice variants. High quality sequence data with a Phred score of >30 was obtained for all libraries. The NUP genes, 28 genes that form the NPC, demonstrated a high frequency of disruptive mutations which could impact protein structure and stability. No analogous mutations were found in a 4505 genes which included many "housekeeping" genes such as GAPDH, B2M, PGK1 and PP1A. Analysis of the SCCIS libraries demonstrated 81 mutations in NUP proteins with 10 mutations in the core NUP 107-160 sub-complex. In SCCIS, the mean allele frequency (MAP) for the NUP 107-160 subcomplex mutations was 49.5%. Epidermal libraries demonstrated 6 mutations in this same gene set with a MAF of 40%. A Chi-square analysis comparing the incidence of NUP mutations in SCCIS or epidermis vs non-mutated genes yields a p < 0.0001. These data indicate that NUP mutations may play a role in promoting the early stages UV-induced carcinogenesis.