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Supplementary Tables 1-3 Supplementary Table 1: Characteristics of P/LP BRCA1, BRCA2, PALB2, and ATM carriers who underwent EUS. Supplementary Table 2: Non-EUS imaging in P/LP BRCA1, BRCA2, PALB2, and ATM carriers without a family history of PDAC. Supplementary Table 3: Pancreatic findings from MRI/CT in P/LP BRCA1, BRCA2, PALB2, and ATM carriers.

Carriers of a pathogenic/likely pathogenic (P/LP) BRCA1/BRCA2/ATM/PALB2 variant are at increased risk of pancreatic ductal adenocarcinoma (PDAC), yet current guidelines recommend surveillance only for those with a family history of PDAC. We aimed to investigate outcomes of endoscopic ultrasound (EUS)–based PDAC surveillance in BRCA1/BRCA2/ATM/PALB2 carriers without a family history of PDAC. We performed a retrospective analysis of all P/LP BRCA1/BRCA2/ATM/PALB2 carriers who underwent EUS at a tertiary care center. Of 194 P/LP BRCA1/BRCA2/ATM/PALB2 carriers who underwent EUS, 64 (33%) had no family history of PDAC and had at least 1 EUS for PDAC surveillance. These individuals underwent 143 total EUSs, were predominantly female (72%), and BRCA2 carriers (73%), with the majority having a personal history of cancer other than PDAC (67%). The median age at time of first EUS was 62 years [interquartile range (IQR), 53–67 years] and a median of 2 EUSs (IQR 1–3) were performed per patient, with a median of 3 years (IQR 2–4.5 years) between the first and last EUS for those with more than 1 EUS. Pancreatic abnormalities were detected in 44%, including cysts in 27%, and incidental luminal abnormalities in 41%. Eight percent developed a new pancreatic mass or cyst during surveillance, 2 individuals developed PDAC, and no serious complications resulted from surveillance. After discussion of the risks, limitations, and potential benefits, PDAC surveillance can be considered in BRCA1/BRCA2/ATM/PALB2 carriers without a family history of PDAC; however, the effectiveness of PDAC surveillance in this population requires further study.Prevention Relevance:BRCA1/BRCA2/ATM/PALB2 carriers have increased pancreatic ductal adenocarcinoma (PDAC) risk, yet are typically not eligible for PDAC surveillance in the absence of PDAC family history. Herein we describe outcomes of PDAC surveillance in BRCA1/BRCA2/ATM/PALB2 carriers without a family history of PDAC, showing that PDAC surveillance can be considered in this high-risk group.

BACKGROUND Lynch syndrome (LS) is a hereditary cancer predisposition syndrome associated with increased risk of multiple cancers. While colorectal cancer surveillance decreases mortality in LS and is recommended by guidelines, there is lack of evidence for the efficacy of surveillance for extra-colonic cancers associated with LS, including small intestinal cancer (SIC) and urinary tract cancer (UTC). Given the limited evidence, guidelines do not consistently recommend surveillance for SIC and UTC, and it remains unclear how often individuals will choose to undergo and follow through with extra-colonic surveillance recommendations. AIM To study factors associated with SIC and UTC surveillance uptake and outcomes in LS. METHODS This is an IRB-approved retrospective analysis of individuals with LS seen at a tertiary care referral center. Included individuals had a pathogenic or likely pathogenic variant in MLH1, MSH2, MSH6, PMS2, or EPCAM, or were a confirmed obligate carrier, and had at least one documented visit to our center. Information regarding SIC and UTC surveillance was captured for each individual, and detailed personal and family history was obtained for individuals who had an initial LS management visit in our center’s dedicated high-risk LS clinic between January 1, 2017 and October 29, 2020. During these initial management visits, all patients had in-depth discussions of SIC and UTC surveillance with 1 of 3 providers experienced in LS management to promote informed decision-making about whether to pursue SIC and/or UTC surveillance. Statistical analysis using Pearson’s chi-squared test and Wilcoxon rank-sum test was completed to understand the factors associated with pursuit and completion of SIC and UTC surveillance, and a P value below 0.05 was deemed statistically significant. RESULTS Of 317 individuals with LS, 86 (27%) underwent a total of 105 SIC surveillance examinations, with 5 leading to additional work-up and no SICs diagnosed. Additionally, 99 (31%) patients underwent a total of 303 UTC surveillance examinations, with 19 requiring further evaluation and 1 UTC identified. Of 155 individuals who had an initial LS management visit between January 1, 2017 and October 29, 2020, 63 (41%) chose to undergo SIC surveillance and 58 (37%) chose to undergo UTC surveillance. However, only 26 (41%) and 32 (55%) of those who initially chose to undergo SIC or UTC surveillance, respectively, successfully completed their surveillance examinations. Individuals with a pathogenic variant in MSH2 or EPCAM were more likely to initially choose to undergo SIC surveillance (P = 0.034), and older individuals were more likely to complete SIC surveillance (P = 0.007). Choosing to pursue UTC surveillance was more frequent among older individuals (P = 0.018), and females more frequently completed UTC surveillance (P = 0.002). Personal history of cancer and family history of SIC or UTC were not significantly associated with electing nor completing surveillance. Lastly, the provider discussing SIC/UTC surveillance was significantly associated with subsequent surveillance choices. CONCLUSION Pursuing and completing SIC/UTC surveillance in LS is influenced by several factors, however broad incorporation in LS management is likely unhelpful due to low yield and frequent false positive results.

Carriers of a pathogenic/likely pathogenic (P/LP) BRCA1/BRCA2/ATM/PALB2 variant are at increased risk of pancreatic ductal adenocarcinoma (PDAC), yet current guidelines recommend surveillance only for those with a family history of PDAC. We aimed to investigate outcomes of endoscopic ultrasound (EUS)–based PDAC surveillance in BRCA1/BRCA2/ATM/PALB2 carriers without a family history of PDAC. We performed a retrospective analysis of all P/LP BRCA1/BRCA2/ATM/PALB2 carriers who underwent EUS at a tertiary care center. Of 194 P/LP BRCA1/BRCA2/ATM/PALB2 carriers who underwent EUS, 64 (33%) had no family history of PDAC and had at least 1 EUS for PDAC surveillance. These individuals underwent 143 total EUSs, were predominantly female (72%), and BRCA2 carriers (73%), with the majority having a personal history of cancer other than PDAC (67%). The median age at time of first EUS was 62 years [interquartile range (IQR), 53–67 years] and a median of 2 EUSs (IQR 1–3) were performed per patient, with a median of 3 years (IQR 2–4.5 years) between the first and last EUS for those with more than 1 EUS. Pancreatic abnormalities were detected in 44%, including cysts in 27%, and incidental luminal abnormalities in 41%. Eight percent developed a new pancreatic mass or cyst during surveillance, 2 individuals developed PDAC, and no serious complications resulted from surveillance. After discussion of the risks, limitations, and potential benefits, PDAC surveillance can be considered in BRCA1/BRCA2/ATM/PALB2 carriers without a family history of PDAC; however, the effectiveness of PDAC surveillance in this population requires further study. Prevention Relevance: BRCA1/BRCA2/ATM/PALB2 carriers have increased pancreatic ductal adenocarcinoma (PDAC) risk, yet are typically not eligible for PDAC surveillance in the absence of PDAC family history. Herein we describe outcomes of PDAC surveillance in BRCA1/BRCA2/ATM/PALB2 carriers without a family history of PDAC, showing that PDAC surveillance can be considered in this high-risk group.

Pruritus (itch) is a debilitating symptom in liver diseases with cholestasis, which severely affects patients' quality of life. Limited treatment options are available for cholestatic itch, largely due to the incomplete understanding of the underlying molecular mechanisms. Several factors have been proposed as pruritogens for cholestatic itch, such as bile acids, bilirubin, lysophosphatidic acid, and endogenous opioids. Recently, two research groups independently identified Mas-related G protein-coupled receptor X4 (MRGPRX4) as a receptor for bile acids and bilirubin and demonstrated its likely role in cholestatic itch. This discovery not only opens new avenues for understanding the molecular mechanisms in cholestatic itch but provides a promising target for developing novel anti-itch treatments. In this review, we summarize the current theories and knowledge of cholestatic itch, emphasizing MRGPRX4 as a bile acid and bilirubin receptor mediating cholestatic itch in humans. We also discuss some future perspectives in cholestatic itch research.

Preclinical and clinical studies provide evidence for aspirin as a preventative agent for cancer. Compelling direct evidence supports a chemopreventive effect of aspirin in individuals at high risk of developing colorectal cancer due to Lynch syndrome, while indirect evidence indicates that aspirin may reduce the risk of and mortality from sporadic colorectal cancer. There is weaker evidence for a protective effect of aspirin against all cancers taken as a group. Nevertheless, the results of recent retrospective cohort studies consistently indicate a beneficial effect of aspirin as a chemopreventive or adjuvant chemotherapeutic agent in hepatocellular carcinoma (HCC). Epidemiologic studies conducted in the general population or in selected populations at higher risk for HCC reveal that regular aspirin use is associated with reduced HCC incidence. In addition, aspirin may act as an adjuvant to other therapies in reducing HCC recurrence. According to studies in animal models, the cancer-preventative effect of aspirin may be related to its antiplatelet and anti-inflammatory activities. Prospective studies are warranted to determine whether aspirin should be recommended to diverse populations of patients at risk for HCC.

Lynch syndrome is a prevalent hereditary cancer predisposition syndrome. While colorectal cancer is the most common gastrointestinal (GI) cancer in Lynch syndrome, there is also increased risk of gastric and small intestinal cancers. Recommendations for upper GI cancer surveillance in Lynch syndrome vary widely with limited data supporting effectiveness. Herein, we collected data on individuals with a diagnosis of Lynch syndrome seen at our tertiary care referral center. We identified individuals who underwent upper endoscopy and those with upper GI cancers, and associated demographics, genetic testing results, and endoscopic information. Standard statistical analyses were performed. Among 295 individuals with Lynch syndrome seen at our center, 217 (73.6%) underwent 660 total upper endoscopies. Of these 217, precancerous upper endoscopy findings included Barrett's esophagus (7, 3.2%), gastric intestinal metaplasia (18, 8.3%), and duodenal adenomas (4, 1.8%), and Helicobacter pylori was identified in 6 (2.8%). Upper GI cancers were diagnosed in 11 individuals (3.7%), including esophageal in 1, gastric in 6, and duodenal in 4. Five (1.7%) of these upper GI cancers were identified on surveillance. Individuals with upper GI cancers identified on surveillance were older at first surveillance endoscopy, with median age 63.3 versus 44.9 years (P < 0.001). Of the upper GI cancers detected on surveillance, 80% (4/5) occurred within 2 years of last upper endoscopy and 80% were stage I. In conclusion, upper endoscopy surveillance in Lynch syndrome identifies upper GI cancers. For individuals with Lynch syndrome who undergo upper GI surveillance, a short surveillance interval may be warranted.

Chronic hepatitis B and C viral infections are major risk factors for hepatocellular carcinoma (HCC) in the United States and worldwide. Direct and indirect mechanisms of viral infection lead to the development of HCC. Chronic viral infection leads to inflammation and liver damage, culminating in cirrhosis, the penultimate step in the progression toward HCC. Host, viral, and environmental factors likely interact to promote oncogenesis. Clinical considerations include recommendations for screening for HCC in persons at risk, treatment with antivirals, and an emerging role for immunotherapy in HCC. We pose unanswered questions regarding HCC susceptibility and pathogenesis in the setting of chronic hepatitis B and C.

PURPOSE Hepatocellular carcinoma (HCC) has well-defined environmental risk factors. In addition, epidemiologic studies have suggested hereditary risk factors. The goals of this study were to determine the rate of pathogenic and likely pathogenic (P/LP) germline variants in cancer predisposition genes in patients with HCC, possible enrichment of P/LP variants in particular genes, and potential impact on clinical management. MATERIALS AND METHODS A prospective study at a tertiary medical center enrolled 217 patients with a personal history of HCC. Multigene panel testing was performed for 134 cancer predisposition genes in all patients. The rate of P/LP variants was compared with population rates. A separate retrospective cohort included 219 patients with HCC who underwent testing at a commercial laboratory. RESULTS In the prospective cohort, P/LP germline variants were identified in 25 of 217 patients with HCC (11.5%). Four patients (1.8%) had P/LP variants in the highly penetrant cancer genes BRCA2 (n = 2), MSH6 (n = 1), and PMS2 (n = 1). In addition, multiple patients had P/LP variants in FANCA (n = 5) and BRIP1 (n = 4), which were significantly enriched in HCC compared with the general population. Detection of P/LP variants led to changes in clinical management in regard to therapy selection, screening recommendations, and cascade testing of relatives. In a separate retrospective analysis of 219 patients with HCC, 30 (13.7%) were positive for P/LP variants including 13 (5.9%) with highly penetrant genes APC (n = 2), BRCA1 (n = 1), BRCA2 (n = 6), MSH2 (n = 2), or TP53 (n = 2). CONCLUSION P/LP germline variants in cancer predisposition genes were detected in 11%-14% of patients with HCC. Inherited genetics should not be overlooked in HCC as there are important implications for precision treatment, future risk of cancers, and familial cancer risk.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 activation (CRISPRa) systems have enabled genetic screens in cultured cell lines to discover and characterize drivers and inhibitors of cancer cell growth. We adapted this system for use in vivo to assess whether modulating endogenous gene expression levels can result in functional outcomes in the native environment of the liver. We engineered the catalytically dead CRISPR-associated 9 (dCas9)-positive mouse, cyclization recombination-inducible (Cre) CRISPRa system for cell type-specific gene activation in vivo. We tested the capacity for genetic screening in live animals by applying CRISPRa in a clinically relevant model of liver injury and repopulation. We targeted promoters of interest in regenerating hepatocytes using multiple single guide RNAs (gRNAs), and employed high-throughput sequencing to assess enrichment of gRNA sequences during liver repopulation and to link specific gRNAs to the initiation of carcinogenesis. All components of the CRISPRa system were expressed in a cell type-specific manner and activated endogenous gene expression in vivo. Multiple gRNA cassettes targeting a proto-oncogene were significantly enriched following liver repopulation, indicative of enhanced division of cells expressing the proto-oncogene. Furthermore, hepatocellular carcinomas developed containing gRNAs that activated this oncogene, indicative of cancer initiation events. Also, we employed our system for combinatorial cancer genetics in vivo as we found that while clonal hepatocellular carcinomas were dependent on the presence of the oncogene-inducing gRNAs, they were depleted for multiple gRNAs activating tumor suppressors.ConclusionThe in vivo CRISPRa platform developed here allows for parallel and combinatorial genetic screens in live animals; this approach enables screening for drivers and suppressors of cell replication and tumor initiation. (Hepatology 2017).

Tissues that undergo rapid cellular turnover, such as the mammalian haematopoietic system or the intestinal epithelium, are dependent on stem and progenitor cells that proliferate to provide differentiated cells to maintain organismal health. Stem and progenitor cells, in turn, are thought to rely on signals and growth factors provided by local niche cells to support their function and self-renewal. Several cell types have been hypothesized to provide the signals required for the proliferation and differentiation of the intestinal stem cells in intestinal crypts1-6. Here we identify subepithelial telocytes as an important source of Wnt proteins, without which intestinal stem cells cannot proliferate and support epithelial renewal. Telocytes are large but rare mesenchymal cells that are marked by expression of FOXL1 and form a subepithelial plexus that extends from the stomach to the colon. While supporting the entire epithelium, FOXL1+ telocytes compartmentalize the production of Wnt ligands and inhibitors to enable localized pathway activation. Conditional genetic ablation of porcupine (Porcn), which is required for functional maturation of all Wnt proteins, in mouse FOXL1+ telocytes causes rapid cessation of Wnt signalling to intestinal crypts, followed by loss of proliferation of stem and transit amplifying cells and impaired epithelial renewal. Thus, FOXL1+ telocytes are an important source of niche signals to intestinal stem cells.

The liver’s extraordinary ability to regenerate has been known since the myth of Prometheus, but the mechanisms involved are still being discovered. Various small animal models have been used in this quest. Two of the most popular include partial hepatectomy (PHx), in which two-thirds of the liver mass is surgically removed to evoke a massive, immediate stimulus for regeneration, and prolonged exposure to toxins that kill liver cells more gradually, provoking chronic regenerative activity. In either case, multiple types of cells must interact effectively to repopulate the organ with functional mature hepatocytes and thus assure ultimate restoration of healthy liver structure and function. This complexity has confounded efforts to distinguish specific changes that occur in cells that repopulate the hepatocyte compartment from changes in other cell populations, including subpopulations of hepatocytes or hepatocyte precursors that do not become regenerative. In the current issue of the JCI, Wang et al. used translating ribosome affinity purification followed by high-throughput RNA sequencing (TRAP-seq) to isolate mRNAs from repopulating hepatocytes in order to profile gene expression specifically in the hepatocytes that regenerate the liver following toxic injury imposed by inherent byproducts of tyrosine metabolism. This innovative methodology can potentially be used to design therapeutic strategies for liver regeneration.

Prostate cancer is commonly diagnosed by using a transrectal ultrasound (TRUS)-guided biopsy. Although this procedure is usually well tolerated, rarely it may be complicated by massive rectal bleeding. We report a case of a 77-year-old male who underwent a TRUS biopsy and subsequently developed recurrent episodes of rectal bleeding with syncope and anemia requiring the transfusion of multiple units of blood. A sigmoidoscopy revealed the source of the bleeding: a large hemorrhoid on the anterior wall of the rectum with an overlying ulceration. We successfully applied a band to ligate the hemorrhoid, and the patient's condition improved. To our knowledge, this case represents the first report of a successful band ligation to treat massive bleeding from a hemorrhoid that had been punctured in the course of the TRUS biopsy procedure.

Chromatin is traditionally viewed as a nuclear entity that regulates gene expression and silencing. However, we recently discovered the presence of cytoplasmic chromatin fragments that pinch off from intact nuclei of primary cells during senescence, a form of terminal cell-cycle arrest associated with pro-inflammatory responses. The functional significance of chromatin in the cytoplasm is unclear. Here we show that cytoplasmic chromatin activates the innate immunity cytosolic DNA-sensing cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway, leading both to short-term inflammation to restrain activated oncogenes and to chronic inflammation that associates with tissue destruction and cancer. The cytoplasmic chromatin-cGAS-STING pathway promotes the senescence-associated secretory phenotype in primary human cells and in mice. Mice deficient in STING show impaired immuno-surveillance of oncogenic RAS and reduced tissue inflammation upon ionizing radiation. Furthermore, this pathway is activated in cancer cells, and correlates with pro-inflammatory gene expression in human cancers. Overall, our findings indicate that genomic DNA serves as a reservoir to initiate a pro-inflammatory pathway in the cytoplasm in senescence and cancer. Targeting the cytoplasmic chromatin-mediated pathway may hold promise in treating inflammation-related disorders.

Existing drug therapies for hepatocellular carcinoma (HCC), including sorafenib, extend patient survival by only three months. We sought to identify novel druggable targets for use in combination with sorafenib to increase its efficacy. We implemented an in vivo genetic screening paradigm utilizing a library of 43 genes-of-interest expressed in the context of repopulation of the injured livers of Fumarylacetoacetate Hydrolase-deficient (Fah−/−) mice, which led to highly penetrant HCC. We then treated mice with vehicle or sorafenib to discover genetic determinants of sensitivity and resistance. Liver X Receptor alpha (LXRα) emerged as a potential target. To examine LXRα agonism in combination with sorafenib treatment, we added varying concentrations of sorafenib and LXRα agonist drugs to HCC cell lines. We performed transcriptomic analysis to elucidate the mechanisms of HCC death. Fah−/− mice injected with the screening library developed HCC tumor clones containing Myc cDNA plus various other cDNAs. Treatment with sorafenib resulted in sorafenib-resistant HCCs that were significantly depleted in Nr1h3 cDNA, encoding LXRα, suggesting that LXRα activation is incompatible with tumor growth in the presence of sorafenib treatment in vivo. The combination of sorafenib and LXR agonism led to enhanced cell death as compared to monotherapy in multiple HCC cell lines, due to reduced expression of cell cycle regulators and increased expression of genes associated with apoptosis. Combination therapy also enhanced cell death in a sorafenib-resistant primary human HCC cell line. Our novel in vivo screen led to the discovery that LXR agonist drugs potentiate the efficacy of sorafenib in treating HCC.

Liver regeneration is impaired in mice with hepatocyte-specific deficiencies in microRNA (miRNA) processing; yet the roles of individual miRNAs or their combinatorial effects in this process are largely unknown. In this study, we sought to identify miRNAs that regulate hepatocyte repopulation following toxic liver injury in a high-throughput manner using the Fah−/− mouse. We constructed plasmid pools encoding over 30,000 tough decoy (TuD) miRNA inhibitors designed to target hepatocyte miRNAs in a pairwise manner. Plasmid libraries were delivered to hepatocytes of Fah−/− mice at the time of liver injury via hydrodynamic tail vein injection and integrated transgene-containing transposons were quantified following repopulation via high-throughput sequencing. Changes in polysome-bound transcripts following miRNA inhibition were determined using translating ribosome affinity purification followed by high-throughput sequencing. Analysis of TuD abundance in hepatocyte genomic DNA and input plasmid pools identified several thousand miRNA inhibitors that were significantly altered following repopulation. We classified a subset of miRNA-binding sites (MBSs) as having strong effect on liver repopulation, thus implicating the targeted hepatocyte miRNAs as regulators of this process. Furthermore, we generated a high-content map of pairwise interactions between 171 MBSs and identified both synergistic and redundant effects. Our study highlights the power of higher-order screens to uncover miRNA functions that would go undetected by individual miRNA perturbations, and provides a new paradigm for the study of epistasis of miRNA activities.

Background & Aims The adult liver is the main detoxification organ and routinely is exposed to environmental insults but retains the ability to restore its mass and function upon tissue damage. However, extensive injury can lead to liver failure, and chronic injury causes fibrosis, cirrhosis, and hepatocellular carcinoma. Currently, the transcriptional regulation of organ repair in the adult liver is incompletely understood. Methods We isolated nuclei from quiescent as well as repopulating hepatocytes in a mouse model of hereditary tyrosinemia, which recapitulates the injury and repopulation seen in toxic liver injury in human beings. We then performed the assay for transposase accessible chromatin with high-throughput sequencing specifically in repopulating hepatocytes to identify differentially accessible chromatin regions and nucleosome positioning. In addition, we used motif analysis to predict differential transcription factor occupancy and validated the in silico results with chromatin immunoprecipitation followed by sequencing for hepatocyte nuclear factor 4α (HNF4α) and CCCTC-binding factor (CTCF). Results Chromatin accessibility in repopulating hepatocytes was increased in the regulatory regions of genes promoting proliferation and decreased in the regulatory regions of genes involved in metabolism. The epigenetic changes at promoters and liver enhancers correspond with the regulation of gene expression, with enhancers of many liver function genes showing a less accessible state during the regenerative process. Moreover, increased CTCF occupancy at promoters and decreased HNF4α binding at enhancers implicate these factors as key drivers of the transcriptomic changes in replicating hepatocytes that enable liver repopulation. Conclusions Our analysis of hepatocyte-specific epigenomic changes during liver repopulation identified CTCF and HNF4α as key regulators of hepatocyte proliferation and regulation of metabolic programs. Thus, liver repopulation in the setting of toxic injury makes use of both general transcription factors (CTCF) for promoter activation, and reduced binding by a hepatocyte-enriched factor (HNF4α) to temporarily limit enhancer activity. All sequencing data in this study were deposited to the Gene Expression Omnibus database and can be downloaded with accession number GSE109466., Graphical abstract

Background & Aims Liver regeneration is impaired in mice with hepatocyte-specific deficiencies in microRNA (miRNA) processing, but it is not clear which miRNAs regulate this process. We developed a high-throughput screen to identify miRNAs that regulate hepatocyte repopulation after toxic liver injury using fumarylacetoacetate hydrolase–deficient mice. Methods We constructed plasmid pools encoding more than 30,000 tough decoy miRNA inhibitors (hairpin nucleic acids designed to specifically inhibit interactions between miRNAs and their targets) to target hepatocyte miRNAs in a pairwise manner. The plasmid libraries were delivered to hepatocytes in fumarylacetoacetate hydrolase–deficient mice at the time of liver injury via hydrodynamic tail-vein injection. Integrated transgene-containing transposons were quantified after liver repopulation via high-throughput sequencing. Changes in polysome-bound transcripts after miRNA inhibition were determined using translating ribosome affinity purification followed by high-throughput sequencing. Results Analyses of tough decoy abundance in hepatocyte genomic DNA and input plasmid pools identified several thousand miRNA inhibitors that were significantly depleted or increased after repopulation. We classified a subset of miRNA binding sites as those that have strong effects on liver repopulation, implicating the targeted hepatocyte miRNAs as regulators of this process. We then generated a high-content map of pairwise interactions between 171 miRNA-binding sites and identified synergistic and redundant effects. Conclusions We developed a screen to identify miRNAs that regulate liver repopulation after injury in live mice.

Hepatocellular carcinoma (HCC) is a devastating and prevalent cancer with limited treatment options. Technological advances have enabled genetic screens to be employed in HCC model systems to characterize genes regulating tumor initiation and growth. Relative to traditional methods for studying cancer biology, such as candidate gene approaches or expression analysis, genetic screens have several advantages: they are unbiased, with no a priori selection; can directly annotate gene function; and can uncover gene–gene interactions. In HCC, three main types of screens have been conducted and are reviewed here: (1) transposon-based mutagenesis screens, (2) knockdown screens using RNA interference (RNAi) or the CRISPR/Cas9 system, and (3) overexpression screens using CRISPR activation (CRISPRa) or cDNAs. These methods will be valuable in future genetic screens to delineate the mechanisms underlying drug resistance and to identify new treatments for HCC.

Given the increase of nonalcoholic fatty liver disease (NAFLD) in the general population, a similar rise might be expected in autoimmune hepatitis (AIH) patients.We sought to determine the clinical outcome of patients with coincident AIH and NAFLD.We identified all intradepartmental AIH cases, and those meeting study criteria were placed into one of three cohorts: AIH only, AIH and simple steatosis (SS), and AIH and nonalcoholic steatohepatitis (NASH). The following outcome and clinical data were analyzed: incidence of all-cause mortality, incidence of liver-related mortality, incidence of liver-related adverse outcomes, and prevalence of cirrhosis at index biopsy.Out of a total 73 study patients, 14 % classified as AIH with SS and 16 % as AIH and NASH. Fifty percent of AIH and NASH patients had cirrhosis at index biopsy as compared to 18 % of AIH-only patients (p = 0.032). Patients with AIH and NASH had a relative risk of 7.65 (95 % CI 1.43-40.8) for liver-related mortality and 2.55 (95 % CI 0.92-7.09) for liver-related adverse outcomes, as compared to the AIH-only cohort. No significant difference in outcome measures existed in comparing (AIH only) with (AIH and SS) cohorts.Patients with coincident AIH and NASH were more likely to present with cirrhosis and more likely to develop adverse clinical outcome with decreased survival as compared to AIH-only patients. These findings suggest that simultaneous exposure confers a clinically significant increased risk, which may warrant closer follow-up and surveillance.

The expression of biliary/progenitor markers by hepatocellular carcinoma (HCC) is often associated with poor prognosis and stem cell-like behaviors of tumor cells. Hepatocellular adenomas (HCAs) also often express biliary/progenitor markers and frequently act as precursor lesions for HCC. However, the cell of origin of HCA and HCC that expresses these markers remains unclear. Therefore, to evaluate if mature hepatocytes give rise to HCA and HCC tumors and to understand the molecular pathways involved in tumorigenesis, we lineage-labeled hepatocytes by injecting adeno-associated virus containing thyroxine-binding globulin promoter-driven causes recombination (AAV-TBG-Cre) into RosaYFP mice. Yellow fluorescent protein (YFP) was present in >96% of hepatocytes before exposure to carcinogens. We treated AAV-TBG-Cre; RosaYFP mice with diethylnitrosamine (DEN), followed by multiple injections of carbon tetrachloride to induce carcinogenesis and fibrosis and found that HCA and HCC nodules were YFP+ lineage-labeled; positive for osteopontin, SRY (sex determining region Y)-box 9, and epithelial cell adhesion molecule; and enriched for transcripts of biliary/progenitor markers such as prominin 1, Cd44, and delta-like 1 homolog. Next, we performed the converse experiment and lineage-labeled forkhead box protein L1(Foxl1)-positive hepatic progenitor cells simultaneously with exposure to carcinogens. None of the tumor nodules expressed YFP, indicating that Foxl1-expressing cells are not the origin for hepatotoxin-induced liver tumors. We confirmed that HCA and HCC cells are derived from mature hepatocytes and not from Foxl1-Cre-marked cells in a second model of toxin-induced hepatic neoplasia, using DEN and 3,3′,5,5′-tetrachloro-1,4-bis(pyridyloxy)benzene (TCPOBOP). Conclusion: Hepatocytes are the cell of origin of HCA and HCC in DEN/carbon tetrachloride and DEN/TCPOBOP induced liver tumors. (Hepatology 2016;64:1163-1177)

It is well known that induction of hepatocyte senescence could inhibit the development of hepatocellular carcinoma (HCC). Until now, it is still unclear how the degree of liver injury dictates hepatocyte senescence and carcinogenesis. In this study, we investigated whether the severity of injury determines cell fate decisions between hepatocyte senescence and carcinogenesis. After testing of different degrees of liver injury, we found that hepatocyte senescence is strongly induced in the setting of severe acute liver injury. Longer-term, moderate liver injury, on the contrary did not result into hepatocyte senescence, but led to a significant incidence of HCC instead. In addition, carcinogenesis was significantly reduced by the induction of severe acute injury after chronic moderate liver injury. Meanwhile, immune surveillance, especially the activations of macrophages, was activated after re-induction of senescence by severe acute liver injury. We conclude that severe acute liver injury leads to hepatocyte senescence along with activating immune surveillance and a low incidence of HCC, whereas chronic moderate injury allows hepatocytes to proliferate rather than to enter into senescence, and correlates with a high incidence of HCC. This study improves our understanding in hepatocyte cell fate decisions and suggests a potential clinical strategy to induce senescence to treat HCC.

Hepatocyte transplantation holds great promise as an alternative to orthotopic organ transplantation in the treatment of liver diseases. However, obtaining clinically meaningful levels of liver repopulation has not been achieved because the mechanisms regulating hepatocyte proliferation in recipient livers have not yet been well characterized. In the mouse model of Hereditary Tyrosinemia Type I, the fumarylacetoacetate hydrolase-deficient (Fah−/−) mouse, we found gradually increasing expression level of insulin-like growth factor 2 (IGF2) in the hepatocytes of host livers. Similarly, high levels of IGF2 were found in the livers of patients with deficient FAH activity. Recombinant IGF2 directly promotes proliferation of primary hepatocytes in vitro. Inhibition on IGF2 expression through the interruption of PI3K/Akt and MAPK pathways significantly reduced the level of liver repopulation in Fah−/− mice. Interestingly, treatment with IGF2 before hepatocyte transplantation generally improved the amount of liver repopulation seen in various mice models of liver injury. Altogether, these findings underscore the underlying mechanisms of therapeutic liver repopulation in Fah−/− mice, and indicate that IGF2 is a potential hepatocyte mitogen for liver cell transplantation therapies.

Interferons are a set of cytokines that activate antiviral responses by the body's immune cells and have been a mainstay of treatment of hepatitis C. Well-known neuropsychiatric effects of interferons include depression, irritability, and impaired concentration. A condition reported rarely in association with this treatment is parkinsonism. We report 2 patients who developed parkinsonism in conjunction with treatment of hepatitis C with alpha interferons. The first is a 51-year-old man who developed intermittent rest and postural tremor during treatment with pegylated interferon alpha ribavirin, and amantadine, with resolution of the symptoms after completing a 36-week course. Similar tremor recurred 3 years later with progressive parkinsonism, compatible with Parkinson disease (PD). The second patient is a 71-year-old man who developed postural tremor 8 weeks into a regimen of consensus interferon. Tremor resolved at completion of 48 weeks of interferon. Pegylated interferon alpha and ribavirin were started 2 years later because of lack of sustained virologic response. At 24 weeks of treatment, postural tremor returned along with features and a progressive course compatible with PD. Thus, both patients presented here developed (rest and/or postural) tremor during interferon therapy followed by delayed onset of parkinsonism. We identified 10 other cases in the literature of parkinsonism/PD associated with interferon administration. This report reviews the clinical presentation and potential pathophysiological mechanisms and recommends that physicians who prescribe interferon be vigilant for symptoms of PD in their patients.

Background The revolutionary concept of “jumping genes” was conceived by McClintock in the late 1940s while studying the Activator/Dissociation (Ac/Ds) system in maize. Transposable elements (TEs) represent the most abundant component of many eukaryotic genomes. Mobile elements are a driving force of eukaryotic genome evolution. McClintock’s Ac, the autonomous element of the Ac/Ds system, together with hobo from Drosophila and Tam3 from snapdragon define an ancient and diverse DNA transposon superfamily named hAT. Other members of the hAT superfamily include the insect element Hermes and Tol2 from medaka. In recent years, genetic tools derived from the ‘cut’ and ‘paste’ Tol2 DNA transposon have been widely used for genomic manipulation in zebrafish, mammals and in cells in vitro. Results We report the purification of a functional recombinant Tol2 protein from E.coli. We demonstrate here that following microinjection using a zebrafish embryo test system, purified Tol2 transposase protein readily catalyzes gene transfer in both somatic and germline tissues in vivo. We show that purified Tol2 transposase can promote both in vitro cutting and pasting in a defined system lacking other cellular factors. Notably, our analysis of Tol2 transposition in vitro reveals that the target site preference observed for Tol2 in complex host genomes is maintained using a simpler target plasmid test system, indicating that the primary sequence might encode intrinsic cues for transposon integration. Conclusions This active Tol2 protein is an important new tool for diverse applications including gene discovery and molecular medicine, as well as for the biochemical analysis of transposition and regulation of hAT transposon/genome interactions. The measurable but comparatively modest insertion site selection bias noted for Tol2 is largely determined by the primary sequence encoded in the target sequence as assessed through studying Tol2 protein-mediated transposition in a cell-free system. Electronic supplementary material The online version of this article (doi:10.1186/s13100-016-0062-z) contains supplementary material, which is available to authorized users.

Functional human hepatocytes xeno-engrafted in mouse liver can be used as a model system to study hepatitis virus infection and vaccine efficacy. Significant liver xeno-repopulation has been reported in two kinds of genetically modified mice that have both immune deficiency and liver injury-induced donor hepatocyte selection: the uPA/SCID mice and Fah(-/-) Rag2(-/-)Il2rg(-/-) mice. The lack of hardy breeding and the overly elaborated technique in these two models may hinder the potential future application of these models to hepatitis virus infection and vaccination studies. Improving the transplantation protocol for liver xeno-repopulation from human hepatocytes will increase the model efficiency and application. In this study, we successfully apply immunosuppressive drug treatments of anti-asialo GM1 and FK506 in Fah(-/-)Rag2(-/-) mice, resulting in significant liver xeno-repopulation from human hepatocytes and human fetal liver cells. This methodology decreases the risk of animal mortality during breeding and surgery. When infected with hepatitis B virus (HBV) sera, Fah(-/-)Rag2(-/-) mice with liver xeno-repopulation from human hepatocytes accumulate significant levels of HBV DNA and HBV proteins. Our new protocol for humanized liver could be applied in the study of human hepatitis virus infection in vivo, as well as the pharmacokinetics and efficacy of potential vaccines.

Change history: In this Letter, the surname of author Efi E. Massasa was misspelled ‘Massassa’. This error has been corrected online.

Insertional mutagenesis screens play an integral part in the annotating of functional data for all sequenced genes in the postgenomic era. Chemical mutagenesis screens are highly efficient but identifying the causative gene can be a laborious task. Other mutagenesis platforms, such as transposable elements, have been successfully applied for insertional mutagenesis screens in both the mouse and rat. However, relatively low transposition efficiency has hampered their use as a high-throughput forward genetic mutagenesis screen. Here we report the first evidence of germline activity in the mouse using a naturally active DNA transposon derived from the medaka fish called Tol2, as an alternative system for high-throughput forward genetic mutagenesis screening tool.

Advances in drug therapy have made it possible to cure HCV infection. This article describes how best to screen, diagnose, and counsel these patients.

The fundamental question of which genes are most important in controlling liver regeneration remains unanswered. We employed a parallel screen to test the impact of 43 selected genes on liver repopulation in the Fah−/− mouse model of hereditary tyrosinemia. We discovered that the transcription factor Foxa3 was a strong promoter of liver regeneration, while tumor necrosis factor receptor 1 (TNFR1) was the most significant suppressor of repopulation among all of the genes tested. Our approach enabled the identification of these factors as important regulators of liver repopulation and potential drug targets for the promotion of liver repopulation.

Idiopathic myointimal hyperplasia of the mesenteric veins (IMHMV) of the colon is a very rare disease that in previously reported cases was often mistaken for inflammatory bowel disease because of similar clinical characteristics. In our review of the literature, we found a total of 15 cases described, generally featuring sigmoid and rectal colitis and symptoms of abdominal pain, diarrhea, and hematochezia refractory to treatment with immunosuppressants. In all previously reported cases, the diagnosis was achieved only after surgical resection of the affected area. Herein, we report a case of IMHMV that was diagnosed preoperatively based on clinical information and endoscopy with biopsies. This led to the withdrawal of immunosuppression before a carefully planned surgical resection, with confirmation of the diagnosis in the resected tissue. To our knowledge, our case of IMHMV is the first to be diagnosed preoperatively.

A better understanding of hepatocyte senescence could be used to treat age-dependent disease processes of the liver. Whether continuously proliferating hepatocytes could avoid or reverse senescence has not yet been fully elucidated. We confirmed that the livers of aged mice accumulated senescent and polyploid hepatocytes, which is associated with accumulation of DNA damage and activation of p53-p21 and p16ink4a-pRB pathways. Induction of multiple rounds continuous cell division is hard to apply in any animal model. Taking advantage of serial hepatocyte transplantation assays in the fumarylacetoacetate hydrolase-deficient (Fah−/−) mouse, we studied the senescence of hepatocytes that had undergone continuous cell proliferation over a long time period, up to 12 rounds of serial transplantations. We demonstrated that the continuously proliferating hepatocytes avoided senescence and always maintained a youthful state. The reactivation of telomerase in hepatocytes after serial transplantation correlated with reversal of senescence. Moreover, senescent hepatocytes harvested from aged mice became rejuvenated upon serial transplantation, with full restoration of proliferative capacity. The same findings were also true for human hepatocytes. After serial transplantation, the high initial proportion of octoploid hepatocytes decreased to match the low level of youthful liver. Conclusion: These findings suggest that the hepatocyte “ploidy conveyer” is regulated differently during aging and regeneration. The findings of reversal of hepatocyte senescence could enable future studies on liver aging and cell therapy. (Hepatology 2014;60:349–361)

Tsai et al.1 reported an association between hepatitis C virus (HCV) infection and Parkinson disease (PD) in their study of nearly 50,000 patients with HCV, hepatitis B virus (HBV), or HCV/HBV coinfection from the Taiwan National Health Insurance Research Database (2000–2010). Another recent and comparably large study found a similar association between PD and HCV infection.2 Both studies lacked an analysis of the association between treatment of …