The favorable IFNL3 genotype escapes mRNA decay mediated by AU-rich elements and hepatitis C virus-induced microRNAs.

IFNL3, which encodes interferon-λ3 (IFN-λ3), has received considerable attention in the hepatitis C virus (HCV) field, as many independent genome-wide association studies have identified a strong association between polymorphisms near IFNL3 and clearance of HCV. However, the mechanism underlying this association has remained elusive. In this study, we report the identification of a functional polymorphism (rs4803217) in the 3' untranslated region (UTR) of IFNL3 mRNA that dictated transcript stability. We found that this polymorphism influenced AU-rich element (ARE)-mediated decay (AMD) of IFNL3 mRNA, as well as the binding of HCV-induced microRNAs during infection. Together these pathways mediated robust repression of the unfavorable IFNL3 polymorphism. Our data reveal a previously unknown mechanism by which HCV attenuates the antiviral response and indicate new potential therapeutic targets for HCV treatment.

Personalized Inflammatory Bowel Disease Care Reduced Hospitalizations.

BACKGROUND AND AIMS: IBD patients with inadequately treated disease often relapse and require hospitalizations for further management. The purpose of this practice review was to determine whether personalized IBD care improved patient outcomes as measured by IBD-related hospitalizations. METHODS: A dedicated IBD clinic was created for personalized patient care in a tertiary veterans health care center in 2014. In the first year, the care program consisted of patient-centered medical home (PCMH). In the second year, personalized biologic therapy was incorporated into the program, based on the severity of mucosal barrier dysfunction measured by probe-based confocal laser endomicroscopy (pCLE) analysis of the terminal ileum during colonoscopy. IBD-related hospitalizations during these 2 years were compared to the year before the care program. RESULTS: The IBD-related admissions at baseline, year 1 and 2 of the program were: total number of admissions of 25, 24, 8 (P = 0.03) per year, total number of hospital days of 177, 144, 31 days per year (P < 0.01), median length of stay 7, 4, and 2 days per visit (P = 0.013), respectively. Patients had significant increases in serum hemoglobin (11.5 ± 2.7, 11.9 ± 2.6, 14.0 ± 1.4 g/dl; P = 0.035), albumin (2.7 ± 0.7, 3.0 ± 0.6 g/dl 3.7 ± 0.8 g/dl; P = 0.031) and body mass index (26.6 ± 2.9, 28.1 ± 5.9; 34.0 ± 10.8; P = 0.047). CONCLUSIONS: Personalized IBD care incorporating a PCMH model and tailored biologic therapy based on pCLE findings of mucosal barrier dysfunction significantly reduced IBD-related hospitalizations.

IL-1ß production through the NLRP3 inflammasome by hepatic macrophages links hepatitis C virus infection with liver inflammation and disease.

Chronic hepatitis C virus (HCV) infection is a leading cause of liver disease. Liver inflammation underlies infection-induced fibrosis, cirrhosis and liver cancer but the processes that promote hepatic inflammation by HCV are not defined. We provide a systems biology analysis with multiple lines of evidence to indicate that interleukin-1ß (IL-1ß) production by intrahepatic macrophages confers liver inflammation through HCV-induced inflammasome signaling. Chronic hepatitis C patients exhibited elevated levels of serum IL-1ß compared to healthy controls. Immunohistochemical analysis of healthy control and chronic hepatitis C liver sections revealed that Kupffer cells, resident hepatic macrophages, are the primary cellular source of hepatic IL-1ß during HCV infection. Accordingly, we found that both blood monocyte-derived primary human macrophages, and Kupffer cells recovered from normal donor liver, produce IL-1ß after HCV exposure. Using the THP-1 macrophage cell-culture model, we found that HCV drives a rapid but transient caspase-1 activation to stimulate IL-1ß secretion. HCV can enter macrophages through non-CD81 mediated phagocytic uptake that is independent of productive infection. Viral RNA triggers MyD88-mediated TLR7 signaling to induce IL-1ß mRNA expression. HCV uptake concomitantly induces a potassium efflux that activates the NLRP3 inflammasome for IL-1ß processing and secretion. RNA sequencing analysis comparing THP1 cells and chronic hepatitis C patient liver demonstrates that viral engagement of the NLRP3 inflammasome stimulates IL-1ß production to drive proinflammatory cytokine, chemokine, and immune-regulatory gene expression networks linked with HCV disease severity. These studies identify intrahepatic IL-1ß production as a central feature of liver inflammation during HCV infection. Thus, strategies to suppress NLRP3 or IL-1ß activity could offer therapeutic actions to reduce hepatic inflammation and mitigate disease.

HCV infection selectively impairs type I but not type III IFN signaling.

A stable and persistent Hepatitis C virus (HCV) replication cell culture model was developed to examine clearance of viral replication during long-term treatment using interferon-α (IFN-α), IFN-λ, and ribavirin (RBV). Persistently HCV-infected cell culture exhibited an impaired antiviral response to IFN-α+RBV combination treatment, whereas IFN-λ treatment produced a strong and sustained antiviral response that cleared HCV replication. HCV replication in persistently infected cells induced chronic endoplasmic reticulum (ER) stress and an autophagy response that selectively down-regulated the functional IFN-α receptor-1 chain of type I, but not type II (IFN-γ) or type III (IFN-λ) IFN receptors. Down-regulation of IFN-α receptor-1 resulted in defective JAK-STAT signaling, impaired STAT phosphorylation, and impaired nuclear translocation of STAT. Furthermore, HCV replication impaired RBV uptake, because of reduced expression of the nucleoside transporters ENT1 and CNT1. Silencing ER stress and the autophagy response using chemical inhibitors or siRNA additively inhibited HCV replication and induced viral clearance by the IFN-α+RBV combination treatment. These results indicate that HCV induces ER stress and that the autophagy response selectively impairs type I (but not type III) IFN signaling, which explains why IFN-λ (but not IFN-α) produced a sustained antiviral response against HCV. The results also indicate that inhibition of ER stress and of the autophagy response overcomes IFN-α+RBV resistance mechanisms associated with HCV infection.

Direct visualization of hepatitis C virus-infected Huh7.5 cells with a high titre of infectious chimeric JFH1-EGFP reporter virus in three-dimensional Matrigel cell cultures.

Identification of the hepatitis C virus (HCV) JFH1 isolate enabled the development of infectious HCV cell culture systems. However, the relatively low virus titres and instability of some chimeric JFH1 reporter viruses restricts some uses of this system. We describe a higher-titre JFH1-EGFP reporter virus where the NS5A V3 region was replaced with the EGFP gene and adapted by serial passage in Huh7.5 cells. Six adaptive mutants were identified: one each in E2, P7 and NS4B, plus three in the NS5A region. These adaptive mutants increased the reporter virus titres to 1×10(6) immunofluorescent focus-forming units ml(-1), which is the highest titre of JFH1-EGFP reporter virus reported to our knowledge. This chimeric virus did not lose EGFP expression following 40 days of passage and it can be used to test the activity of HCV antivirals by measuring EGFP fluorescence in 96-well plates. Moreover, this reporter virus allows living infected Huh7.5 cells in Matrigel three-dimensional (3D) cultures to be visualized and produces infectious viral particles in these 3D cultures. The chimeric NS5A-EGFP infectious JFH1 reporter virus described should enable new studies of the HCV life cycle in 3D cell cultures and will be useful in identifying antivirals that interfere with HCV release or entry.

Genome-wide transcriptome analysis identifies novel gene signatures implicated in human chronic liver disease.

The molecular mechanisms behind human liver disease progression to cirrhosis remain elusive. Nuclear receptor small heterodimer partner (SHP/Nr0b2) is a hepatic tumor suppressor and a critical regulator of liver function. SHP expression is diminished in human cirrhotic livers, suggesting a regulatory role in human liver diseases. The goal of this study was to identify novel SHP-regulated genes that are involved in the development and progression of chronic liver disease. To achieve this, we conducted the first comprehensive RNA sequencing (RNA-seq) analysis of Shp(-/-) mice, compared the results with human hepatitis C cirrhosis RNA-seq and nonalcoholic steatohepatitis (NASH) microarray datasets, and verified novel results in human liver biospecimens. This approach revealed new gene signatures associated with chronic liver disease and regulated by SHP. Several genes were selected for validation of physiological relevance based on their marked upregulation, novelty with regard to liver function, and involvement in gene pathways related to liver disease. These genes include peptidoglycan recognition protein 2, dual specific phosphatase-4, tetraspanin 4, thrombospondin 1, and SPARC-related modular calcium binding protein-2, which were validated by qPCR analysis of 126 human liver specimens, including steatosis, fibrosis, and NASH, alcohol and hepatitis C cirrhosis, and in mouse models of liver inflammation and injury. This RNA-seq analysis identifies new genes that are regulated by the nuclear receptor SHP and implicated in the molecular pathogenesis of human chronic liver diseases. The results provide valuable transcriptome information for characterizing mechanisms of these diseases.

Baseline Features and Reasons for Nonparticipation in the Colonoscopy Versus Fecal Immunochemical Test in Reducing Mortality From Colorectal Cancer (CONFIRM) Study, a Colorectal Cancer Screening Trial.

Importance: The Colonoscopy Versus Fecal Immunochemical Test in Reducing Mortality From Colorectal Cancer (CONFIRM) randomized clinical trial sought to recruit 50 000 adults into a study comparing colorectal cancer (CRC) mortality outcomes after randomization to either an annual fecal immunochemical test (FIT) or colonoscopy. Objective: To (1) describe study participant characteristics and (2) examine who declined participation because of a preference for colonoscopy or stool testing (ie, fecal occult blood test [FOBT]/FIT) and assess that preference's association with geographic and temporal factors. Design, Setting, and Participants: This cross-sectional study within CONFIRM, which completed enrollment through 46 Department of Veterans Affairs medical centers between May 22, 2012, and December 1, 2017, with follow-up planned through 2028, comprised veterans aged 50 to 75 years with an average CRC risk and due for screening. Data were analyzed between March 7 and December 5, 2022. Exposure: Case report forms were used to capture enrolled participant data and reasons for declining participation among otherwise eligible individuals. Main Outcomes and Measures: Descriptive statistics were used to characterize the cohort overall and by intervention. Among individuals declining participation, logistic regression was used to compare preference for FOBT/FIT or colonoscopy by recruitment region and year. Results: A total of 50 126 participants were recruited (mean [SD] age, 59.1 [6.9] years; 46 618 [93.0%] male and 3508 [7.0%] female). The cohort was racially and ethnically diverse, with 748 (1.5%) identifying as Asian, 12 021 (24.0%) as Black, 415 (0.8%) as Native American or Alaska Native, 34 629 (69.1%) as White, and 1877 (3.7%) as other race, including multiracial; and 5734 (11.4%) as having Hispanic ethnicity. Of the 11 109 eligible individuals who declined participation (18.0%), 4824 (43.4%) declined due to a stated preference for a specific screening test, with FOBT/FIT being the most preferred method (2820 [58.5%]) vs colonoscopy (1958 [40.6%]; P < .001) or other screening tests (46 [1.0%] P < .001). Preference for FOBT/FIT was strongest in the West (963 of 1472 [65.4%]) and modest elsewhere, ranging from 199 of 371 (53.6%) in the Northeast to 884 of 1543 (57.3%) in the Midwest (P = .001). Adjusting for region, the preference for FOBT/FIT increased by 19% per recruitment year (odds ratio, 1.19; 95% CI, 1.14-1.25). Conclusions and Relevance: In this cross-sectional analysis of veterans choosing nonenrollment in the CONFIRM study, those who declined participation more often preferred FOBT or FIT over colonoscopy. This preference increased over time and was strongest in the western US and may provide insight into trends in CRC screening preferences.

Role of nuclear receptor SHP in metabolism and cancer.

Small heterodimer partner (SHP, NR0B2) is a unique member of the nuclear receptor (NR) superfamily that contains the dimerization and ligand-binding domain found in other family members, but lacks the conserved DNA-binding domain. The ability of SHP to bind directly to multiple NRs is crucial for its physiological function as a transcriptional inhibitor of gene expression. A wide variety of interacting partners for SHP have been identified, indicating the potential for SHP to regulate an array of genes in different biological pathways. In this review, we summarize studies concerning the structure and target genes of SHP and discuss recent progress in understanding the function of SHP in bile acid, cholesterol, triglyceride, glucose, and drug metabolism. In addition, we review the regulatory role of SHP in microRNA (miRNA) regulation, liver fibrosis and cancer progression. The fact that SHP controls a complex set of genes in multiple metabolic pathways suggests the intriguing possibility of developing new therapeutics for metabolic diseases, including fatty liver, dyslipidemia and obesity, by regulating SHP with small molecules. To achieve this goal, more progress regarding SHP ligands and protein structure will be required. Besides its metabolic regulatory function, studies by us and other groups provide strong evidence that SHP plays a critical role in the development of cancer, particularly liver and breast cancer. An increased understanding of the fundamental mechanisms by which SHP regulates the development of cancers will be critical in applying knowledge of SHP in diagnostic, therapeutic or preventive strategies for specific cancers. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.

Genome-wide characterization of methylguanosine-capped and polyadenylated small RNAs in the rice blast fungus Magnaporthe oryzae.

Small RNAs are well described in higher eukaryotes such as mammals and plants; however, knowledge in simple eukaryotes such as filamentous fungi is limited. In this study, we discovered and characterized methylguanosine-capped and polyadenylated small RNAs (CPA-sRNAs) by using differential RNA selection, full-length cDNA cloning and 454 transcriptome sequencing of the rice blast fungus Magnaporthe oryzae. This fungus causes blast, a devastating disease on rice, the principle food staple for over half the world's population. CPA-sRNAs mapped primarily to the transcription initiation and termination sites of protein-coding genes and were positively correlated with gene expression, particularly for highly expressed genes including those encoding ribosomal proteins. Numerous CPA-sRNAs also mapped to rRNAs, tRNAs, snRNAs, transposable elements and intergenic regions. Many other 454 sequence reads could not be mapped to the genome; however, inspection revealed evidence for non-template additions and chimeric sequences. CPA-sRNAs were independently confirmed using a high affinity variant of eIF-4E to capture 5'-methylguanosine-capped RNA followed by 3'-RACE sequencing. These results expand the repertoire of small RNAs in filamentous fungi.

Effect of vaccination on the case fatality rate for COVID-19 infections 2020-2021: multivariate modelling of data from the US Department of Veterans Affairs.

OBJECTIVES: To evaluate the benefits of vaccination on the case fatality rate (CFR) for COVID-19 infections. DESIGN, SETTING AND PARTICIPANTS: The US Department of Veterans Affairs has 130 medical centres. We created multivariate models from these data-339 772 patients with COVID-19-as of 30 September 2021. OUTCOME MEASURES: The primary outcome for all models was death within 60 days of the diagnosis. Logistic regression was used to derive adjusted ORs for vaccination and infection with Delta versus earlier variants. Models were adjusted for confounding factors, including demographics, comorbidity indices and novel parameters representing prior diagnoses, vital signs/baseline laboratory tests and outpatient treatments. Patients with a Delta infection were divided into eight cohorts based on the time from vaccination to diagnosis. A common model was used to estimate the odds of death associated with vaccination for each cohort relative to that of unvaccinated patients. RESULTS: 9.1% of subjects were vaccinated. 21.5% had the Delta variant. 18 120 patients (5.33%) died within 60 days of their diagnoses. The adjusted OR for a Delta infection was 1.87±0.05, which corresponds to a relative risk (RR) of 1.78. The overall adjusted OR for prior vaccination was 0.280±0.011 corresponding to an RR of 0.291. Raw CFR rose steadily after 10-14 weeks. The OR for vaccination remained stable for 10-34 weeks. CONCLUSIONS: Our CFR model controls for the severity of confounding factors and priority of vaccination, rather than solely using the presence of comorbidities. Our results confirm that Delta was more lethal than earlier variants and that vaccination is an effective means of preventing death. After adjusting for major selection biases, we found no evidence that the benefits of vaccination on CFR declined over 34 weeks. We suggest that this model can be used to evaluate vaccines designed for emerging variants.

Further insights into the roles of GTP and the C terminus of the hepatitis C virus polymerase in the initiation of RNA synthesis.

The hepatitis C virus (HCV) NS5b protein is an RNA-dependent RNA polymerase essential for replication of the viral RNA genome. In vitro and presumably in vivo, NS5b initiates RNA synthesis by a de novo mechanism. Different structural elements of NS5b have been reported to participate in RNA synthesis, especially a so-called "ß-flap" and a C-terminal segment (designated "linker") that connects the catalytic core of NS5b to a transmembrane anchor. High concentrations of GTP have also been shown to stimulate de novo RNA synthesis by HCV NS5b. Here we describe a combined structural and functional analysis of genotype 1 HCV-NS5b of strains H77 (subtype 1a), for which no structure has been previously reported, and J4 (subtype 1b). Our results highlight the linker as directly involved in lifting the first boundary to processive RNA synthesis, the formation of the first dinucleotide primer. The transition from this first dinucleotide primer state to processive RNA synthesis requires removal of the linker and of the ß-flap with which it is shown to strongly interact in crystal structures of HCV NS5b. We find that GTP specifically stimulates this transition irrespective of its incorporation in neosynthesized RNA.

Efficient 5' cap-dependent RNA purification : use in identifying and studying subsets of RNA.

Microarray-based screening technologies have revealed a larger than expected diversity of gene expression profiles for many cells, tissues, and organisms. The complexity of RNA species, defined by their molecular structure, represents a major new development in biology. RNA not only carries genetic information in the form of templates and components of the translational machinery for protein synthesis but also directly regulates gene expression as exemplified by micro-RNAs (miRNAs). Recent evidence has demonstrated that 5' capped and 3' polyadenylated ends are not restricted to mRNAs, but that they are also present in precursors of both miRNAs and some antisense RNA transcripts. In addition, as many as 40% of transcribed RNAs may lack 3' poly(A) ends. In concert with the presence of a 5' cap (m7 GpppN), the length of the 3' poly(A) end plays a critical role in determining the translational efficiency, stability, and the cellular distribution of a specific mRNA. RNAs with short or lacking 3' poly(A) ends, that escape isolation and amplification with oligo(dT)-based methods, provide a challenge in RNA biology and gene expression studies. To circumvent the limitations of 3' poly(A)-dependent RNA isolation methods, we developed an efficient RNA purification system that binds the 5' cap of RNA with a high-affinity variant of the cap-binding protein eIF4E. This system can be used in differential selection approaches to isolate subsets of RNAs, including those with short 3' poly(A) ends that are likely targets of post-transcriptional regulation of gene expression. The length of the 3' poly(A) ends can be defined using a rapid polymerase chain reaction (PCR)- based approach.

Adapted HCV JFH1 variant is capable of accommodating a large foreign gene insert and allows lower level HCV replication and viral production.

Infectious HCV carrying reporter genes have further applications in understanding the HCV life cycle including replication, viral assembly and release. In this study, a full-length 3039bp LacZ gene was inserted into the derivative of JFH1-AM120 to develop an additional reporter virus. The results showed that the recombinant reporter virus JFH1-AM120-LacZ can replicate and produce lower titers of infectious virus. However, insertion of the LacZ gene in the C-terminal region of the NS5A in HCV JFH1-AM120-LacZ decreased viral replication and dramatically impaired the production of infectious viral particles. Moreover, the JFH1-AM120-LacZ reporter virus lost the LacZ gene after serial passage. Nevertheless, the JFH1-AM120-LacZ reporter virus displayed the entire life cycle of HCV, from replication to production of infectious virus, in Huh7.5 cells. This study demonstrates that the NS5A region of HCV JFH1-AM120 has the capacity to accommodate large foreign genes up to 3,039 bp and suggests that other relatively large gene inserts can be accommodated at this site.

NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis.

Angiotensin II (Ang II) is a pro-oxidant and fibrogenic cytokine. We investigated the role of NADPH oxidase in Ang II-induced effects in hepatic stellate cells (HSCs), a fibrogenic cell type. Human HSCs express mRNAs of key components of nonphagocytic NADPH oxidase. Ang II phosphorylated p47phox, a regulatory subunit of NADPH oxidase, and induced reactive oxygen species formation via NADPH oxidase activity. Ang II phosphorylated AKT and MAPKs and increased AP-1 DNA binding in a redox-sensitive manner. Ang II stimulated DNA synthesis, cell migration, procollagen alpha1(I) mRNA expression, and secretion of TGF-beta1 and inflammatory cytokines. These effects were attenuated by N-acetylcysteine and diphenylene iodonium, an NADPH oxidase inhibitor. Moreover, Ang II induced upregulation of genes potentially involved in hepatic wound-healing response in a redox-sensitive manner, as assessed by microarray analysis. HSCs isolated from p47phox-/- mice displayed a blunted response to Ang II compared with WT cells. We also assessed the role of NADPH oxidase in experimental liver fibrosis. After bile duct ligation, p47phox-/- mice showed attenuated liver injury and fibrosis compared with WT counterparts. Moreover, expression of smooth muscle alpha-actin and expression of TGF-beta1 were reduced in p47phox-/- mice. Thus, NADPH oxidase mediates the actions of Ang II on HSCs and plays a critical role in liver fibrogenesis.

Laminar shear stress and 3' polyadenylation of eNOS mRNA.

The 3' poly(A) tail is important in messenger RNA stability and translational efficiency. In somatic tissues, 3' polyadenylation of mRNAs has been thought to largely be a constitutively active process. We have reported that laminar shear stress causes a brief increase in endothelial nitric oxide synthase (eNOS) transcription, followed by a prolonged increase in eNOS mRNA stability. We sought to determine whether shear stress and other stimuli affected eNOS 3' polyadenylation in endothelial cells. Under basal (static) conditions, eNOS mRNA possessed short 3' poly(A) tails of <25 nt. In contrast, laminar shear stress increased expression of eNOS transcripts with long poly(A) tails. ENOS transcripts with longer poly(A) tails had prolonged half-lives (6 hours in static cells versus 18 hours in sheared cells). Polysome analysis revealed that eNOS mRNA from sheared cells was shifted into more translationally active polysome fractions compared with eNOS mRNA from static cells. Shear-induced lengthening of the eNOS 3' poly(A) tail was the result of increased nuclear polyadenylation. Furthermore, hydrogen peroxide and HMG Co-A reductase inhibitors, other stimuli known to modulate eNOS expression posttranscriptionally, also induced eNOS 3' poly(A) tail lengthening. These results support the concept that shear stress modulates eNOS mRNA stability and translation via increased 3' polyadenylation. We suggest that mRNA 3' polyadenylation is a posttranscriptional mechanism used by endothelial cells to regulate gene expression.

Platform-independent gene expression signature differentiates sessile serrated adenomas/polyps and hyperplastic polyps of the colon.

BACKGROUND: Sessile serrated adenomas/polyps are distinguished from hyperplastic colonic polyps subjectively by their endoscopic appearance and histological morphology. However, hyperplastic and sessile serrated polyps can have overlapping morphological features resulting in sessile serrated polyps diagnosed as hyperplastic. While sessile serrated polyps can progress into colon cancer, hyperplastic polyps have virtually no risk for colon cancer. Objective measures, differentiating these types of polyps would improve cancer prevention and treatment outcome. METHODS: RNA-seq training data set and Affimetrix, Illumina testing data sets were obtained from Gene Expression Omnibus (GEO). RNA-seq single-end reads were filtered with FastX toolkit. Read mapping to the human genome, gene abundance estimation, and differential expression analysis were performed with Tophat-Cufflinks pipeline. Background correction, normalization, and probe summarization steps for Affimetrix arrays were performed using the robust multi-array method (RMA). For Illumina arrays, log2-scale expression data was obtained from GEO. Pathway analysis was implemented using Bioconductor package GSAR. To build a platform-independent molecular classifier that accurately differentiates sessile serrated and hyperplastic polyps we developed a new feature selection step. We also developed a simple procedure to classify new samples as either sessile serrated or hyperplastic with a class probability assigned to the decision, estimated using Cantelli's inequality. RESULTS: The classifier trained on RNA-seq data and tested on two independent microarray data sets resulted in zero and three errors. The classifier was further tested using quantitative real-time PCR expression levels of 45 blinded independent formalin-fixed paraffin-embedded specimens and was highly accurate. Pathway analyses have shown that sessile serrated polyps are distinguished from hyperplastic polyps and normal controls by: up-regulation of pathways implicated in proliferation, inflammation, cell-cell adhesion and down-regulation of serine threonine kinase signaling pathway; differential co-expression of pathways regulating cell division, protein trafficking and kinase activities. CONCLUSIONS: Most of the differentially expressed pathways are known as hallmarks of cancer and likely to explain why sessile serrated polyps are more prone to neoplastic transformation than hyperplastic. The new molecular classifier includes 13 genes and may facilitate objective differentiation between two polyps.

Activation of PERK-Nrf2 oncogenic signaling promotes Mdm2-mediated Rb degradation in persistently infected HCV culture.

The mechanism of how chronic hepatitis C virus (HCV) infection leads to such a high rate of hepatocellular carcinoma (HCC) is unknown. We found that the PERK axis of endoplasmic reticulum (ER) stress elicited prominent nuclear translocation of Nrf2 in 100% of HCV infected hepatocytes. The sustained nuclear translocation of Nrf2 in chronically infected culture induces Mdm2-mediated retinoblastoma protein (Rb) degradation. Silencing PERK and Nrf2 restored Mdm2-mediated Rb degradation, suggesting that sustained activation of PERK/Nrf2 axis creates oncogenic stress in chronically infected HCV culture model. The activation of Nrf2 and its nuclear translocation were prevented by ER-stress and PERK inhibitors, suggesting that PERK axis is involved in the sustained activation of Nrf2 signaling during chronic HCV infection. Furthermore, we show that HCV clearance induced by interferon-α based antiviral normalized the ER-stress response and prevented nuclear translocation of Nrf2, whereas HCV clearance by DAAs combination does neither. In conclusion, we report here a novel mechanism for how sustained activation of PERK axis of ER-stress during chronic HCV infection activates oncogenic Nrf2 signaling that promotes hepatocyte survival and oncogenesis by inducing Mdm2-mediated Rb degradation.

Bcl2 is a critical regulator of bile acid homeostasis by dictating Shp and lncRNA H19 function.

Bile acid (BA) metabolism is tightly controlled by nuclear receptor signaling to coordinate regulation of BA synthetic enzymes and transporters. Here we reveal a molecular cascade consisting of the antiapoptotic protein BCL2, nuclear receptor Shp, and long non-coding RNA (lncRNA) H19 to maintain BA homeostasis. Bcl2 was overexpressed in liver of C57BL/6J mice using adenovirus mediated gene delivery for two weeks. Hepatic overexpression of Bcl2 caused drastic accumulation of serum BA and bilirubin levels and dysregulated BA synthetic enzymes and transporters. Bcl2 reactivation triggered severe liver injury, fibrosis and inflammation, which were accompanied by a significant induction of H19. Bcl2 induced rapid SHP protein degradation via the activation of caspase-8 pathway. The induction of H19 in Bcl2 overexpressed mice was contributed by a direct loss of Shp transcriptional repression. H19 knockdown or Shp re-expression largely rescued Bcl2-induced liver injury. Strikingly different than Shp, the expression of Bcl2 and H19 was hardly detectable in adult liver but was markedly increased in fibrotic/cirrhotic human and mouse liver. We demonstrated for the first time a detrimental effect of Bcl2 and H19 associated with cholestatic liver fibrosis and an indispensable role of Shp to maintain normal liver function.

Interferon lambda 4 expression is suppressed by the host during viral infection.

Interferon (IFN) lambdas are critical antiviral effectors in hepatic and mucosal infections. Although IFNλ1, IFNλ2, and IFNλ3 act antiviral, genetic association studies have shown that expression of the recently discovered IFNL4 is detrimental to hepatitis C virus (HCV) infection through a yet unknown mechanism. Intriguingly, human IFNL4 harbors a genetic variant that introduces a premature stop codon. We performed a molecular and biochemical characterization of IFNλ4 to determine its role and regulation of expression. We found that IFNλ4 exhibits similar antiviral activity to IFNλ3 without negatively affecting antiviral IFN activity or cell survival. We show that humans deploy several mechanisms to limit expression of functional IFNλ4 through noncoding splice variants and nonfunctional protein isoforms. Furthermore, protein-coding IFNL4 mRNA are not loaded onto polyribosomes and lack a strong polyadenylation signal, resulting in poor translation efficiency. This study provides mechanistic evidence that humans suppress IFNλ4 expression, suggesting that immune function is dependent on other IFNL family members.