The role of chronic hepatitis C virus (HCV) in the pathogenesis of HCV-associated hepatocellular carcinoma (HCC) remains controversial. To understand the transition from benign to malignant, we studied the gene expression patterns in liver tissues at different stages, including normal, cirrhosis, and different HCC stages. We studied 108 liver tissue samples obtained from 88 distinct patients (41 HCV-cirrhotic tissues, 17 HCV-cirrhotic tissues from patients with HCC, and 47 HCV-HCC tissues). Differentially expressed genes (DEG) were studied by use of high-density oligonucleotide arrays. Among probe sets identified as differentially expressed via the F test, all pairwise comparisons were performed. Cirrhotic tissues with and without concomitant HCC were further evaluated, and a classifier was used to predict whether the tissue type was associated with HCC. Differential expression profiles were analyzed using Interaction Networks and Functional Analysis. Characteristic gene signatures were identified when normal tissue was compared with cirrhosis, cirrhosis with early HCC, and normal with HCC. Pathway analysis classified the cellular and biological functions of the DEG as related to cellular growth and proliferation, cell death and inflammatory disease in cirrhosis; cell death, cell cycle, DNA replication, and immune response in early HCCs; and cell death, cell growth and proliferation, cell cycle, and DNA repair in advanced HCCs. Characteristic gene signatures were identified at different stages of HCV-HCC progression. A set of genes were identified to predict whether the cirrhotic tissue was associated with HCC.

Our aim was to evaluate the safety of transplanting kidneys from HCV-infected donors in HCV-uninfected recipients. Data collected from 53 recipients in a single center, observational study included donor and recipient characteristics, liver and kidney graft function, new infections and de novo donor-specific antibodies and renal histology. Treatment with a direct-acting antiviral regimen was initiated when HCV RNA was detected. The mean ± SD age of recipients was 53 ± 11 years, 34% were female, 19% and 79% of recipients were white and African American, respectively. The median and interquartile range (IQR) time between transplant and treatment initiation was 76 (IQR: 68-88) days. All 53 recipients became viremic (genotype: 1a [N = 34], 1b [N = 1], 2 [N = 3], and 3 [N = 15]). The majority (81%) of recipients did not experience clinically significant increases (>3 times higher than upper limit of the normal value) in aminotransferase levels and their HCV RNA levels were in | 3047 MOLNAR et AL.

Despite the advances in immunosuppression, renal allograft attrition over time remains unabated due to chronic allograft dysfunction (CAD) with interstitial fibrosis (IF) and tubular atrophy (TA). We aimed to evaluate microRNA (miRNA) signatures in CAD with IF/TA and appraise correlation with paired urine samples and potential utility in prospective evaluation of graft function. MicroRNA signatures were established between CAD with IF/TA vs. normal allografts by microarray. Validation of the microarray results and prospective evaluation of urine samples was performed using RT-qPCR. Fifty-six miRNAs were identified in samples with CAD-IF/TA. Five miRNAs were selected for further validation based on: array fold change, p-value and in silico predicted mRNA targets. We confirmed the differential expression of these 5 miRNAs by RT-qPCR using an independent set of samples. Differential expression was detected for miR-142-3p, miR-204, miR-107, and miR-211 (P<0.001) and miR-32 (p<0.05). Furthermore, differential expression of miR-142-3p (p<0.01), miR-204 (p<0.01) and miR-211 (p<0.05) was also observed between patient groups in urine samples. A characteristic miRNA signature for IF/TA that correlates with paired urine samples was identified. These results support the potential use of miRNAs as non-invasive markers of IF/TA and for monitoring graft function.

Background: Development of quality-control criteria to ensure reproducibility of microarray results for potential clinical application is still in its infancy. Methods:In the present studies we developed qualitycontrol criteria and evaluated their effect in microarray data analysis using total RNA from cell lines, frozen tumors, and a commercially available reference RNA. Quality-control criteria such as A 260 /A 280 ratios, percentage of rRNA, and median size of cDNA and cRNA synthesis products were evaluated for robustness in microarray analysis. Furthermore, precision studies using a reference material were performed on the Affymetrix ® HG-U133A high-density oligonucleotide microarrays. The same reference RNA sample was examined in 16 different chips run on 2 different days in the four different modules of the Affymetrix fluidics workstation. Fresh and frozen fragmented cRNAs were also compared. An ANOVA model was fit to identify the main sources of variation. Results: Good-quality samples showed >30% rRNA in the electropherograms and cDNA and cRNA synthesis products with median sizes of 2.0 and 3.0 kb, respectively. Precision studies showed that the main source of variation was the day-to-day variability, minimally affecting hybridization exogenous control genes. Altogether, the results showed that the Affymetrix Genechip ® system is highly reproducible when RNA that meet the quality-control criteria are used (overall P >0.01).

Lipid deposition in the liver is associated with metabolic disorders including fatty liver disease, type II diabetes, and hepatocellular cancer. The enzymes acetyl-CoA carboxylase 1 (ACC1) and ACC2 are powerful regulators of hepatic fat storage; therefore, their inhibition is expected to prevent the development of fatty liver. In this study we generated liver-specific ACC1 and ACC2 double knockout (LDKO) mice to determine how the loss of ACC activity affects liver fat metabolism and whole-body physiology. Characterization of LDKO mice revealed unexpected phenotypes of increased hepatic triglyceride and decreased fat oxidation. We also observed that chronic ACC inhibition led to hyper-acetylation of proteins in the extra-mitochondrial space. In sum, these data reveal the existence of a compensatory pathway that protects hepatic fat stores when ACC enzymes are inhibited. Furthermore, we identified an important role for ACC enzymes in the regulation of protein acetylation in the extra-mitochondrial space.

Urinary extracellular vesicles (ueVs) provide bio-markers for kidney and urogenital diseases.centrifugation is the most common method used to enrich ueVs. However, a majority of studies to date have focused on the ultracentrifugation pellet, potentially losing a novel source of important biomarkers that could be obtained at lower centrifugation. thus, the aim of this study is to rigorously characterize for the first time uEVs in the low speed pellet and determine the minimal volume of urine required for proteomic analysis (≥9.0 mL urine) and gene ontology classification identified 75% of the protein as extracellular exosomes. cryo-transmission electron Microscopy (≥3.0 mL urine) provided evidence of a heterogeneous population of eVs for size and morphology independent of uromodulin filaments. Western blot detected several specific uEV kidney and EV markers (≥4.5 mL urine per lane). microRNAs quantification by qPCR was possible with urine volume as low as 0.5 mL. Particle enumeration with tunable resistive pulse sensing, nano particles tracking analysis and single eV high throughput imaging flow cytometry are possible starting from 0.5 and 3.0 mL of urine respectively. this work characterizes a neglected source of ueVs and provides guidance with regard to volume of urine necessary to carry out multi-omic studies and reveals novel aspects of ueV analysis such as autofluorescence of podocyte origin.Urinary extracellular vesicles (uEVs) are a medley of exosomes, exosome-like vesicles and microparticles/ microvesicles 1-4 . Confusing nomenclature aside 5,6 , all uEVs secreted in urine transport proteins, nucleic acid and small metabolites from all epithelial cells forming the nephron and lower urinary tract 7,8 . Thus, uEVs have become a valuable source of biomarkers for identifying any changes in the physio-pathological state of their parental cell. Moreover, uEVs are also bio-activators in renal diseases 9,10 . The most common method in use to enrich uEVs is a 2 or 3 step centrifugation protocol [11][12][13] . While it has been commonly discarded, the pellet obtained at relative low centrifugation force has proved to be an additional source of uEVs 14,15 . However this pellet has not been thoroughly characterized.In addition, the concomitant presence of multiple biomarker in uEVs offers the possibility to integrate multi-omic data analysis to better understand mechanism and possibly identify key role molecules implicated in the onset and progression of the disease 16 . However, no study has reported the amount of volume of urine that is necessary to collect to support multiple analyses. Hence, this study aims to: (1) estimate the minimum volume of urine necessary to yield uEVs for characterization according to both minimal information for studies of extracellular vesicles (MISEV) 17 and downstream analysis applying a very rigorous approach using several control sets for each analysis; (2) test the limit of detection of the techniques employed for downstream analysis and EV characterization before and after eliminati...

Genes related to fibrosis, extracellular matrix deposition, and immune response were found up-regulated in CAN. Markers resulting from the microarray analysis were differentially expressed in Ur samples of the CAN patients and in concordance with the microarray profiles.

Non-invasive, cost-effective biomarkers that allow accurate monitoring of graft function are needed in kidney transplantation. Since microRNAs (miRNAs) have emerged as promising disease biomarkers we sought to establish an miRNA signature in urinary cell pellets comparing kidney transplant patients diagnosed with chronic allograft dysfunction (CAD) with interstitial fibrosis and tubular atrophy and those recipients with normal graft function. Overall, we evaluated 191 samples from 125 deceased donor primary kidney transplant recipients in the discovery, initial validation and the longitudinal validation studies for non-invasive monitoring of graft function. Of 1,733 mature miRNAs studied using microarrays, 22 were found to be differentially expressed between groups. Ontology and pathway analyses showed inflammation as the principal biological function associated with these miRNAs. Twelve selected miRNAs were longitudinally evaluated in urine samples of an independent set of 66 patients, at two time-points post-kidney transplant. A subset of these miRNAs was found to be differentially expressed between groups early post-kidney transplant before histological allograft injury was evident. Thus, a panel of urine miRNAs was identified as potential biomarkers for monitoring graft function and anticipating progression to CAD in kidney transplant patients.