Non-alcoholic steatohepatitis (NASH, also known as MASH) is a severe form of non-alcoholic fatty liver disease (NAFLD, also known as MASLD). Emerging data indicate that the progression of the disease to MASH is higher in postmenopausal women and that genetic susceptibility increases the risk of MASH-related cirrhosis. This study aimed to investigate the association between genetic polymorphisms in MASH and sexual dimorphism. We applied whole-exome sequencing (WES) to identify gene variants in 8 age-adjusted matched pairs of livers from both male and female patients. Sequencing alignment, variant calling, and annotation were performed using standard methods. Polymerase chain reaction (PCR) coupled with Sanger sequencing and immunoblot analysis were used to validate specific gene variants. cBioPortal and Gene Set Enrichment Analysis (GSEA) were used for actionable target analysis. We identified 148,881 gene variants, representing 57,121 and 50,150 variants in the female and male cohorts, respectively, of which 251 were highly significant and MASH sex-specific (p < 0.0286). Polymorphisms in CAPN14, SLC37A3, BAZ1A, SRP54, MYH11, ABCC1, and RNFT1 were highly expressed in male liver samples. In female samples, Polymorphisms in RGSL1, SLC17A2, HFE, NLRC5, ACTN4, SBF1, and ALPK2 were identified. A heterozygous variant 1151G>T located on 18q21.32 for ALPK2 (rs3809983) was validated by Sanger sequencing and expressed only in female samples. Immunoblot analysis confirmed that the protein level of β-catenin in female samples was 2-fold higher than normal, whereas ALPK2 expression was 0.5-fold lower than normal. No changes in the protein levels of either ALPK2 or β-catenin were observed in male samples. Our study suggests that the perturbation of canonical Wnt/β-catenin signaling observed in postmenopausal women with MASH could be the result of polymorphisms in ALPK2.

The prevalence of non‐alcoholic fatty liver disease (NAFLD) and its progression to non‐alcoholic steatohepatitis (NASH) is higher in postmenopausal women than men. The aim of this study was to determine the molecular mechanisms underlying this sexual dimorphism in NAFLD.

Introduction: To better elucidate the impact of cooperative learning outside the classroom, a student-initiated research project was conducted to explore the effects of participating in peer-led study groups (PLSGs) on student examination scores and perceptions. Methods: First-year pharmacy students were given the opportunity to participate in weekly PLSGs for a pharmacogenomics course during spring 2016 and spring 2017. Student exam performance was stratified by those who attended vs. those who did not. Optional pre- and post-course surveys examined student perceptions of PLSGs. Results: No significant differences were seen between the attendance groups in spring 2016. In spring 2017, student attendees were significantly more likely to pass two of their six exams (p = .04, p = .0029) and to have higher exam scores on one exam (p = .02) in comparison to non-attendees. Overall exam score averages were significantly different between attendees and non-attendees during spring 2017 (p = .03) but not during spring 2016 (p = .38). Perception surveys indicated students believed participation helped them to demonstrate competency and build confidence. Additionally, students reported they felt more comfortable clarifying questions during the study groups vs. during class time. Conclusions: The impact of study group participation on student exam performance was minimal over the two years of data collection, but there were instances where exam scores were positively impacted. Students perceived value in study group participation even if it did not translate directly to improved exam performance on all exams.

Hepatocyte nuclear factor 4-alpha (HNF4α) is a highly conserved member of nuclear receptor superfamily of ligand-dependent transcription factors that is expressed in liver and gastrointestinal organs (pancreas, stomach, and intestine). In liver, HNF4α is best known for its role as a master regulator of liver-specific gene expression and essential for adult and fetal liver function. Dysregulation of HNF4α expression has been associated with many human diseases such as ulcerative colitis, colon cancer, maturity-onset diabetes of the young, liver cirrhosis, and hepatocellular carcinoma. However, the precise role of HNF4α in the etiology of these human pathogenesis is not well understood. Limited information is known about the role of HNF4α isoforms in liver and gastrointestinal disease progression. There is, therefore, a critical need to know how disruption of the expression of these isoforms may impact on disease progression and phenotypes. In this review, we will update our current understanding on the role of HNF4α in human liver and gastrointestinal diseases. We further provide additional information on possible use of HNF4α as a target for potential therapeutic approaches.

Hepatocyte nuclear factor 4-alpha (HNF4α) is a highly conserved member of nuclear receptor superfamily of ligand-dependent transcription factors that is expressed in liver and gastrointestinal organs (pancreas, stomach, and intestine). In liver, HNF4α is best known for its role as a master regulator of liver-specific gene expression and essential for adult and fetal liver function. Dysregulation of HNF4α expression has been associated with many human diseases such as ulcerative colitis, colon cancer, maturity-onset diabetes of the young, liver cirrhosis, and hepatocellular carcinoma. However, the precise role of HNF4α in the etiology of these human pathogenesis is not well understood. Limited information is known about the role of HNF4α isoforms in liver and gastrointestinal disease progression. There is, therefore, a critical need to know how disruption of the expression of these isoforms may impact on disease progression and phenotypes. In this review, we will update our current understanding on the role of HNF4α in human liver and gastrointestinal diseases. We further provide additional information on possible use of HNF4α as a target for potential therapeutic approaches.

Chronic liver diseases are one of the major public health issues in United States, and there are substantial racial disparities in liver cancer-related mortality. We previously identified racially distinct alterations in the expression of transcripts and proteins of hepatitis C (HCV)-induced hepatocellular carcinoma (HCC) between Caucasian (CA) and African American (AA) subgroups. Here, we performed a comparative genome-wide analysis of normal vs. HCV+ (cirrhotic state), and normal adjacent tissues (HCCN) vs. HCV+HCC (tumor state) of CA at the gene and alternative splicing levels using Affymetrix Human Transcriptome Array (HTA2.0). Many genes and splice variants were abnormally expressed in HCV+ more than in HCV+HCC state compared with normal tissues. Known biological pathways related to cell cycle regulations were altered in HCV+HCC, whereas acute phase reactants were deregulated in HCV+ state. We confirmed by quantitative RT-PCR that SAA1, PCNA-AS1, DAB2, and IFI30 are differentially deregulated, especially in AA compared with CA samples. Likewise, IHC staining analysis revealed altered expression patterns of SAA1 and HNF4α isoforms in HCV+ liver samples of AA compared with CA. These results demonstrate that several splice variants are primarily deregulated in normal vs. HCV+ stage, which is certainly in line with the recent observations showing that the pre-mRNA splicing machinery may be profoundly remodeled during disease progression, and may, therefore, play a major role in HCV racial disparity. The confirmation that certain genes are deregulated in AA compared to CA tissues also suggests that there is a biological basis for the observed racial disparities.

Objective. To design and implement a pharmacogenomics course that focuses on analysis and integration of pharmacogenomic data into clinical practice and to explore how participation in the course influences student self-confidence. Design. The Basic and Clinical Pharmacogenomics course content was divided into three modules: genetic-based didactic sessions, genomic techniques and self-genotype/phenotype laboratory exercise, and clinical-based case studies. Student learning assessment included knowledge- and application-based tests and performance on a group project. Assessment. Effectiveness of the course was evaluated using results of student performance on coded test questions, student perceptions on pre- and post-course self-assessments, performance on a group project, and course evaluation results. Student pharmacists successfully demonstrated competency in pharmacogenomics knowledge-based learning, demonstrated their abilities to apply learned skills in clinical-based scenarios, and reported improved confidence in analyzing patient-based genomic testing results. Conclusions. This course appears to have contributed to student learning and positively influenced student self-confidence in pharmacogenomics.

Hepatocellular carcinoma (HCC) is the most rapidly increasing type of cancer in the United States. HCC is a highly malignant cancer, accounting for at least 14000 deaths in the United States annually, and it ranks third as a cause of cancer mortality in men. One major difficulty is that most patients with HCC are diagnosed when the disease is already at an advanced stage, and the cancer cannot be surgically removed. Furthermore, because almost all patients have cirrhosis, neither chemotherapy nor major resections are well tolerated. Clearly there is need of a multidisciplinary approach for the management of HCC. For example, there is a need for better understanding of the fundamental etiologic mechanisms that are involved in hepatocarcinogenesis, which could lead to the development of successful preventive and therapeutic modalities. It is also essential to define the cellular and molecular bases for malignant transformation of hepatocytes. Such knowledge would: (1) greatly facilitate the identification of patients at risk; (2) prompt efforts to decrease risk factors; and (3) improve surveillance and early diagnosis through diagnostic imaging modalities. Possible benefits extend also to the clinical management of this disease. Because there are many factors involved in pathogenesis of HCC, this paper reviews a multidisciplinary perspective of recent advances in basic and clinical understanding of HCC that include: molecular hepatocarcinogenesis, non-invasive diagnostics modalities, diagnostic pathology, surgical modality, transplantation, local therapy and oncological/target therapeutics.

Hepatocellular carcinoma (HCC) is one of the few tumors in which the incidence is on the rise worldwide, especially in the US [1]. The increasing incidence of this disease is associated with the rise in hepatitis C virus (HCV) infection [2]. It is estimated that 3.2 million people in the US are infected with HCV; a blood-borne disease linked to 12,000 US deaths a year [3]. More concerning are projections that rate will quadruple in the next 10 years, to over 40,000 cases per year. Therefore, there is a great need to understand the molecular mechanisms for HCV-induced malignant transformation. Many studies have shown that malignant transformation of HCV infected hepatocytes occurs through a pathway of increased liver cell turnover induced by chronic liver injury and regeneration in a context of inflammation and oxidative DNA damage. In this context, the host immune response against the various HCV-encoded proteins (HCV core, E2, NS3, and NS5A) becomes relevant for HCC development and tumor progression. HCV proteins interact with many host cells factors and affect a wide range of cellular and humoral activities, including cell signaling, transcriptional modulation, transformation, apoptosis, membrane rearrangements, vesicular trafficking and cytokine & chemokine production. Inflammatory cells and immunomodulatory mediators present in the microenvironment polarize host immune response toward specific phenotypes impacting initiation and progression of the disease [4].

Clustering has become the norm in analyzing genomic and proteomic data. There are various algorithms for which graphs and software are easily available. However, often there is little agreement among these methods; prompting the question of which algorithm should one choose? In this manuscript, we consider eleven different methods of clustering, including partitioning, hierarchical and model based methods, to look at proteomic data from a colon cancer study. We use a pairwise index to evaluate these eleven clustering methods by comparing the clustering results. In an effort to comprehensively understand and determine the best method under a given structure, a simulation study is performed to compare the eleven methods under different distributional structures. The data analysis and the simulation study results agree that clustering is dependent on the method chosen. However, our study suggests that k-means and model based methods tend to perform reasonably well under all data structures used.

We studied promoter methylation (PM) of 11 genes in Peripheral Blood Lymphocytes (PBLs) and tissues of hepatitis C virus (HCV) associated hepatocellular carcinoma (HCC) and chronic hepatitis (CH) Egyptian patients. The present study included 31 HCC with their ANT, 38 CH and 13 normal hepatic tissue (NHT) samples. In all groups, PM of APC, FHIT, p15, p73, p14, p16, DAPK1, CDH1, RARβ, RASSF1A, O6MGMT was assessed by methylation-specific PCR (MSP). APC and O6-MGMT protein expression was assessed by immunohistochemistry (IHC) in the studied HCC and CH (20 samples each) as well as in a different HCC and CH set for confirmation of MSP results. PM was associated with progression from CH to HCC. Most genes showed high methylation frequency (MF) and the methylation index (MI) increased with disease progression. MF of p14, p73, RASSF1A, CDH1 and O6MGMT was significantly higher in HCC and their ANT. MF of APC was higher in CH. We reported high concordance between MF in HCC and their ANT, MF in PBL and CH tissues as well as between PM and protein expression of APC and O6MGMT. A panel of 4 genes (APC, p73, p14, O6MGMT) classifies the cases independently into HCC and CH with high accuracy (89.9%), sensitivity (83.9%) and specificity (94.7%). HCV infection may contribute to hepatocarcinogenesis through enhancing PM of multiple genes. PM of APC occurs early in the cascade while PM of p14, p73, RASSF1A, RARB, CDH1 and O6MGMT are late changes. A panel of APC, p73, p14, O6-MGMT could be used in monitoring CH patients for early detection of HCC. Also, we found that, the methylation status is not significantly affected by whether the tissue was from the liver or PBL, indicating the possibility of use PBL as indicator to genetic profile instead of liver tissue regardless the stage of disease.

Abstract Cancer health disparities represent a major public health concern in the US. Even when factors such as socioeconomic status, carcinogen exposure, and access to care are accounted for, disparities persist in the form of higher overall incidence rates and worse clinical outcomes for minority populations than for overall population. A case of point is the racial disparities in liver cancer-related mortality. Hepatitis C virus (HCV) is the most significant contributing factor in the development of hepatocellular carcinoma (HCC). In the US, African Americans (AAs) have twice the prevalence of HCV/genotype 1 infection, and develop HCC at more than twice the rate as Caucasian (CA) counterparts. African Americans are, however, less likely to respond to interferon-based therapy than CAs, and have considerably lower likelihood of receiving liver transplantation. While it is evident that viral infection with HCV is associated with the development of HCC, there are critical gaps in our understanding of the biological basis for this racial disparity. The aim of the current study was to define the molecular signatures of HCV disease progression in liver & tumor tissue samples obtained from AA & CA patients using 8-plex iTRAQ-based proteomics coupled with bioinformatics data analyses. The raw data were analyzed by the ProteinPilot v3.0 using the paragon algorithm. Searches were performed against a comprehensive database generated from SwissPort, Refseq, and Tremble protein sequences. The data were normalized for loading error & background correction. The proteins with confidence score > 90% and with at least 1 peptide of 95% identification confidence were used for further quality control & differential expression analysis. The quality control analysis was performed using pairwise correlation plots, boxplots, principal component analysis (PCA), and unsupervised hierarchical clustering. Supervised analysis was performed to identify differentially expressed proteins (DEP), where the relative protein expression values were compared between groups (Normal vs. Cirrhosis (CIR), Normal vs. HCC, CIR vs. HCC). Based on our experimental design, 787 unique proteins were identified. Of those, 32 were differentially expressed between normal, cirrhosis & HCC groups. Targets validation using real-time PCR (RT-PCR) or western blotting (WB) shows racially distinct alteration in the expression of certain targets. For example, the mRNA expression levels of TF were 2 and18-fold higher in CIR & HCC, respectively in AAs compared to CAs. Similarly, the expression of APOA1 mRNA levels was 7-fold higher in HCC of AAs compared to CAs. This trend was similar to their protein expression levels using WB. However, the level of HNF4α protein was down regulated in AAs compared to CAs. This indicates that HNF4α does not regulate TF & APOA1 expression in HCC of AA samples. Citation Format: Simon T. Dillon, Manoj K. Bhasin, Xiaoxing (Stella) Feng, David Koh, Sayed Salih Daoud. Biomarkers discovery and racial disparity in hepatitis C-associated hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1150. doi:10.1158/1538-7445.AM2013-1150

The incidence and mortality of hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) is higher in African Americans (AA) than other racial/ethnic groups in the U.S., but the reasons for this disparity are unknown. There is an urgent need for the discovery of novel molecular signatures for HCV disease progression to understand the underlying biological basis for this cancer rate disparity to improve the clinical outcome.

Copyright: © 2013 Daoud SS. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Over the past 20 years or so, there have been tremendous advances in our understanding of how normal cells transform to diseased/cancer cells and the importance of signaling pathways in cancer initiation and progression. This progress in scientific knowledge has resulted in the development of “personalized medicine concept” and as a proof-ofconcept, the approval of Gleevec and Herceptin. In addition, technologic advances and the use of high-throughput approaches, particularly in the last 10 years, are beginning to impact our knowledge in genome science and medical technologies. For example, advances in genotyping technologies have made it possible for the field of pharmacogenetics (the study of genetic variability in individual responses to drugs) to assume its place of importance in the medical field. Then pharmacogenetics has evolved into pharmacogenomics (the study of the roles on inherited and acquired genetic variations in drug responses) with a shift from a focus on individual candidate genes to a more comprehensive approach using Genome-Wide Association Studies (GWAS). This shift has led to the identification of genetic variations of Single Nucleotide Polymorphisms (SNPs) in the region of many genes that may play a critical role in predicting treatment response in patients at various disease states, among them patients infected with Hepatitis C Virus (HCV) [1].

The global rising incidence of hepatocellular carcinoma (HCC), which parallels the increase of hepatitis C virus (HCV) prevalence, has sparked a renewed interest in discovering additional HCC serum markers. In this study, we investigated the clinical use of serum E-cadherin, ICAM, MMP-2, VEGF, OPN and β-catenin as potential diagnostic makers for HCV/genotype 4-associated HCC. Twenty cases of healthy subjects, 11 cases with asymptomatic HCV/genotype 4 carriers (ASC), 28 chronic hepatitis (CH) cases and 32 patients with HCC were enrolled in this study. Serum levels of proteins were measured by a sandwich-enzyme-linked (ELISA) assay. The diagnostic accuracy of each candidate marker was evaluated using receiver-operating characteristic (ROC) curve analysis, reporting the area under the curve (AUC) and its 95% confidence interval (CI). We demonstrated that serum β-catenin levels were significantly elevated in patients with HCC compared to those with CH, ASC and healthy controls. Among the six studied markers, β-catenin was also found to be the only marker that can significantly discriminate between patients with HCC and those with CH; therefore, β-catenin could be considered as a potential marker for early diagnosis of HCV-associated HCC in patients infected with HCV genotype 4.

Abstract Hepatocellular carcinoma (HCC) is one of the few tumors in which the incidence is on the rise worldwide. The increasing incidence of HCC is associated with the rise in hepatitis C virus (HCV) infection. It is estimated that 2.0% of the US general population are infected with HCV. Egypt has the highest prevalence of HCV/genotype 4 infection in the world, with 14% of the population infected. Little is known about the role(s) of epigenetic changes that may participate in HCV-induced HCC? Epigenetic studies directed at mapping the etiology of HCV disease progression to HCC and how they correlate with patients clinicopathological parameters are expected to provide new insights that will aid in the design of effective strategies for earlier detection and management of the disease, and therefore of great public/global health interest. The aim of the current study was to investigate the aberrant methylation of 11 genes in liver tissue samples obtained from Egyptian patients infected with HCV/genotype 4, and to explore possible relationships between aberrant methylation and clinicopathological features in HCC. Thirty-eight HCC and matching non-tumor tissues, thirty-one chronic hepatitis liver tissues, and thirteen normal liver tissues were analyzed for the methylation status of APC, FHIT, p15, p73, p14, p16, DAPK1, CDH1, RARβ, RASSF1A and O6MGMT genes by quantitative methylation-specific PCR. Based on methylation profiles using McNemar's test for paired binary data, a methylation of only 7 genes was found to be highly significant, which is dependent on disease state. For chronic hepatitis tissues, promoter methylation of p14 was highly significant in both liver tissues and peripheral blood leukocytes (PBL) (P < 0.0047 and <0.003, respectively), whereas methylation of RASSF1A was highly significant in normal liver tissues (P < 0.0005). For HCC, promoter methylation of p15, p14 and APC was highly significant in tumor tissues, non-tumor tissues, and PBL, respectively (P < 0.0001, < 0.0076 and < 0.0001, respectively). To account for the clinical variables of patients in the methylation status of a gene and disease state, a logistic regression analysis was performed. For HCC, promoter methylation of APC, DAPK1, and p14 was highly significant in addition to the blood profiles of patients (e.g., platelets, hemoglobin, WBCs levels). For patients with chronic hepatitis, methylation of CDH1 (E-Cadherin) was highly significant in addition to the patient's liver enzymes level (e.g., AST, ALT). Our data indicate that DNA methylation may serve as a marker for HCV-associated HCC, and demonstrate the importance of considering the clinicopathological variables as predicators of disease state. Quantitative analysis of methylated p14 may serve as a powerful molecular marker in detecting HCV disease progression to HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 321. doi:10.1158/1538-7445.AM2011-321

BACKGROUND:To understand the complex and largely not well-understood apoptotic pathway and immune system evasion mechanisms in hepatitis C virus (HCV)-associated hepatocellular carcinoma (HCC) and HCV associated chronic hepatitis (CH), we studied the expression patterns of a number of pro-apoptotic and anti-apoptotic genes (Fas, FasL, Bcl-2, Bcl-xL and Bak) in HepG2 cell line harboring HCV- genotype-4 replication. For confirmation, we also assessed the expression levels of the same group of genes in clinical samples obtained from 35 HCC and 34 CH patients.METHODS:Viral replication was assessed in the tissue culture medium by RT-PCR, quantitative Real-Time PCR (qRT-PCR); detection of HCV core protein by western blot and inhibition of HCV replication with siRNA. The expression level of Fas, FasL, Bcl-2, Bcl-xL and Bak was assessed by immunohistochemistry and RT-PCR whereas caspases 3, 8 and 9 were assessed by colorimetric assay kits up to 135 days post infection.RESULTS:There was a consistent increase in apoptotic activity for the first 4 weeks post-CV infection followed by a consistent decrease up to the end of the experiment. The concordance between the changes in the expression levels of Fas, FasL, Bcl-2, Bcl-xL and Bak in vitro and in situ was statistically significant (p < 0.05). Fas was highly expressed at early stages of infection in cell lines and in normal control liver tissues followed by a dramatic reduction post-HCV infection and an increase in the expression level of FasL post HCV infection. The effect of HCV infection on other apoptotic proteins started very early post-infection, suggesting that hepatitis C modulating apoptosis by modulating intracellular pro-apoptotic signals.CONCLUSIONS:Chronic HCV infection differently modulates the apoptotic machinery during the course of infection, where the virus induces apoptosis early in the course of infection, and as the disease progresses apoptosis is modulated. This study could open a new opportunity for understanding the various signaling of apoptosis and in the developing a targeted therapy to inhibit viral persistence and HCC development.

There are few reports describing the role of p21-dependent protein repression in cell death.To identify such cell death-associated proteins and to shed the light into the molecular mechanisms by which p21 is responding to pharmacological stress, we used a subcellular proteomic approach for the analysis of protein expression profiles of fractionated nuclei, mitochondria, and cytosols of isogenic p21 null (p21-/-) and wild-type human HCT-116 cells following treatment with sublethal doses (1μM) of the topoisomerase I inhibitor, topotecan (TPT).In total, 174 unique deregulated proteins were identified in HCT-116 cells following treatment with TPT, whereas only 146 proteins were identified in p21-/-cells.They contributed to multiple functional activities of stress signaling pathways, and that p21-/-cells are accelerated to be more responsive to topotecan-induced cell death due to the following: 1) down regulation of proteins involved in the transcriptional and replication machinery of cells like DNA (cytosine-5)-methyltransferase 1, Matrin 3, DNA replication licensing factor MCM4, heterogeneous nuclear ribonucleoprotein Q, poly(Rc)-binding protein 1 and splicing factor arginine serine rich 7; 2) the activation of a caspase-independent apoptosis by the upregulation of the Bcl2 inhibitor of transcription (Bit1) protein; and 3) the activation of TNF signaling by the upregulation of macrophage immigration inhibitory factor (MIF), and 26S proteosome non-ATPase regulatory subunit 2 (TRAP2) proteins.We suggest that the upregulation of these proteins are contributing factors to the molecular mechanisms of topotecan-induced cell death in p21-/-cells; and that the data present an opportunity for developing new therapeutic approaches for selective targeting of p21signaling pathways.