Chronic hepatitis C is characterized by a highly variable clinical course. Approximately 20% of all patients who either do not receive antiviral therapy or fail to respond develop liver cirrhosis within 20 years. Currently, serum markers for hepatic fibrosis diagnosis show a limited sensitivity and specificity so that liver biopsy with high sampling error rate is still used (1). Clinical application of current biomarkers is therefore not feasible. There is an absolute demand on high-throughput methods allowing the reliable assessment of the severity and/or progression of hepatic fibrogenesis.

Aims: TGF-β1-mediated transdifferentiation of hepatic stellate cells (HSC) to myofibroblast-like cells (MFB) is the key event to initiate the fibrogenic program in the liver. This comprises upregulation of contractile elements, e.g. α-SMA, increased expression of collagen type I and connective tissue growth factor (CTGF) as well as a higher proliferation and migration rate [1]. To determine the factors that govern myoblast conversion upon TGF-β1-treatment in more detail, we analyzed a model system for TGF-β1-signaling, i.e. myoblasts, which display a myofibroblast-like phenotype when cultured in the presence of TGF-β1 [2].

BACKGROUND: A functional population of adipocyte precursors, termed adipose-derived stromal/stem cells (ASCs), is crucial for proper adipose tissue (AT) expansion, lipid handling, and prevention of lipotoxicity in response to chronic positive energy balance. We previously showed that obese human subjects contain a dysfunctional pool of ASCs. Elucidation of the mechanisms underlying abnormal ASC function might lead to therapeutic interventions for prevention of lipotoxicity by improving the adipogenic capacity of ASCs. METHODS: Using epigenome-wide association studies, we explored the impact of obesity on the methylation signature of human ASCs and their differentiated counterparts. Mitochondrial phenotyping of lean and obese ASCs was performed. TBX15 loss- and gain-of-function experiments were carried out and western blotting and electron microscopy studies of mitochondria were performed in white AT biopsies from lean and obese individuals. RESULTS: We found that DNA methylation in adipocyte precursors is significantly modified by the obese environment, and adipogenesis, inflammation, and immunosuppression were the most affected pathways. Also, we identified TBX15 as one of the most differentially hypomethylated genes in obese ASCs, and genetic experiments revealed that TBX15 is a regulator of mitochondrial mass in obese adipocytes. Accordingly, morphological analysis of AT from obese subjects showed an alteration of the mitochondrial network, with changes in mitochondrial shape and number. CONCLUSIONS: We identified a DNA methylation signature in adipocyte precursors associated with obesity, which has a significant impact on the metabolic phenotype of mature adipocytes.

So far, CRC cell lines have contributed to descriptions of 2 patterns of genetic instability, affecting either microsatellite sequences or chromosome number and structure. Often, these patterns are mutually exclusive; while near-diploid karyotypes usually appear with MSI and chromosomal stability, near-triploid or tetraploid cells display a high degree of CIN and are stable at the microsatellite level. In the present study, we describe the genomic instability pattern of KM12 CRC cells. KM12C and derived cell lines with different metastatic properties were analyzed by conventional cytogenetics, CGH and M-FISH. Results were compared to 5 cell lines usually used as model of MSI and CIN. Concordance between our results and previously published SKY data are also reviewed. Interestingly, the poorly metastatic KM12C cell line displayed a near-diploid karyotype with high levels of structural chromosome instability and microsatellite instability. The highly metastatic KM12SM and KM12L4A cell lines showed polyploid karyotypes and maintained CIN and MSI. A comparison between karyotypes of poorly and highly metastatic KM12 cell lines allowed us to delineate a cytogenetic evolution pathway. Our results clearly demonstrated that endoreduplication was the origin of the polyploid dosages in the highly metastatic forms following the monosomic model postulated for CRC. Therefore, we demonstrate that KM12C cells and their metastatic derivates, KM12SM and KM12L4A, are a useful model of chromosomal evolution where MSI may coexist with CIN.

Widespread genetic alterations are a common feature of most colorectal cancers. While specific recurrent alterations may reveal the involvement of a gene or set of genes in the biology of the disease, the cumulated genomic damage is likely to reflect the biological history of the neoplastic cells. Furthermore, the functional implications behind many of these genetic changes may show the evolutionary potential of the neoplastic cells. Different approaches, ranging from the gross determination of total nuclear DNA content to cytogenetic and molecular approaches, reveal different types of chromosomal and subchromosomal alterations and have been applied to measure generalized genomic damage in colorectal carcinomas. High levels of genomic damage usually appear associated with increased aggressiveness in colorectal cancer, and the use of different assessments of genomic damage as independent prognostic factors has been proposed. Therefore, appropriate definition of the extent of cumulated alterations and their functional consequences may be of interest in the understanding and management of cancer. The different methodologies and clues to the interpretation and integration of the results obtained with each technique are discussed in this review.

ABSTRACT While somatic mutations in colorectal cancer (CRC) are well characterized, little is known about the accumulation of cancer mutations in the normal colon prior to cancer. Here we have developed and applied an ultra-sensitive, single-molecule mutational test based on CRISPR-DS technology, which enables mutation detection at extremely low frequency (<0.001) in normal colon from patients with and without CRC. We found oncogenic KRAS mutations in the normal colon of about one third of patients with CRC but in none of the patients without CRC. Patients with CRC also carried more TP53 mutations than patients without cancer, and these mutations were more pathogenic and formed larger clones, especially in patients with early onset CRC. Most mutations in normal colon were different from the driver mutations in tumors suggesting that the occurrence of independent clones with pathogenic KRAS and TP53 mutations is a common event in the colon of individuals that develop CRC. SIGNIFICANCE Our results suggest a prevalent process of somatic mutation and evolution in the normal colon of patients with CRC, which can be detected by ultra-sensitive sequencing of driver genes and potentially employed clinically for CRC risk prediction.

The NLRP3 inflammasome is a multi-protein complex that, upon activation by a large range of stimuli, activates Caspase-1 that in turn mediates the maturation of the pro-inflammatory cytokines IL-1β and IL-18. In a previous study we have shown that LPS stimulation induces a clear time- and concentration-dependent induction of inflammasome-associated genes in the immortalized hepatic stellate cell line CFSC-2G [1]. Although there are actually many unclear and inconsistent data for TGF-β1, it was suggested that TGF-β expression is stimulated by the activity of the different inflammasome branches in hepatic stellate cells [2] and renal tubular epithelium [3].

With the increasing incidence of obesity, diabetes and hypertriglyceridemia in Western countries, metabolic liver disease, in particular nonalcoholic fatty liver disease (NAFLD) has become a growing problem (Angulo and Lindor, 2002). Hepatocellular lipid accumulation which is mainly caused by excess dietary intake, increased rates of de novo lipogenesis and/ or decreased lipid export from hepatocytes is known to be the initial stage in the pathogenesis of NAFLD (Farrell and Larter, 2006). Steatosis hepatis in itself is a reversible condition but can progress to severe acute and chronic liver disease, including nonalcoholic steatohepatitis, fibrosis and cirrhosis (Friedman, 2008). The aim of the present study was to elucidate the role of acyl CoA synthetase 5 (ACSL5), a key enzyme in lipid metabolism, in the association of steatosis hepatis and hepatocellular apoptosis. Our current data reveal that ACSL5, a member of the ACSL gene family that is localized in the outer mitochondrial membrane, showed a significantly elevated expression level in fatty liver and concurrently induced an increased sensitization to hepatocellular apoptosis. By examining HepG2 cells as well as human liver tissue, we could show that the ACSL5 expression is significantly increased in both in vitro induced fatty degeneration of HepG2 and in vivo adipose liver tissue. Since ACSL5 is shown to be a sensitizing signal for apoptosis in enterocytes along the crypt villus axis (Gassler et al, 2008), ACSL5-overexpressing hepatocytes as well as control-transfected hepatocytes were treated with a panel of proapoptotic agents representing diverse cell death stimuli. The analysis revealed that ACSL5-overexpressing cells were substantially more sensitive to the apoptosis ligands TNFα, TRAIL and FAS than control-transfected cells. In conclusion, our findings give possible hints that ACSL5 could be a sensitizing signal for the induction of hepatocellular apoptosis in the pathogenesis of NAFLD.

PURPOSE: To study bone marrow micrometastases from colorectal cancer patients for the presence of K-ras mutations and to compare their genotype with that of the corresponding primary tumor. PATIENTS AND METHODS: Bilateral iliac crest aspiration was performed in 51 patients undergoing surgery for colorectal cancer, and bone marrow micrometastases were detected by immunohistochemistry. The presence of K-ras mutations was determined by single-strand conformation polymorphism analysis on both primary tumors and paired bone marrow samples and was confirmed by sequencing. RESULTS: In six patients with primary tumor mutations, it was possible to amplify a mutated K-ras gene also from the bone marrow sample. In three of those patients the pattern of K-ras mutations differed between both samples, in two patients the mutation was identical between the bone marrow and its primary tumor, and in one patient the same mutation plus a different one were found. Fifteen of 17 K-ras mutations found in primary tumors were located in codon 12, whereas in bone marrow, five of seven mutations were found in codon 13 (P =.003). CONCLUSION: Our results demonstrate that, at least for K-ras mutations, disseminated epithelial cells are not always clonal with the primary tumor and they question the malignant genotype of bone marrow micrometastases. They also indicate that different tumoral clones may be circulating simultaneously or sequentially in the same patient. Analysis of the type of mutations suggests that cell dissemination might be an early event in colorectal carcinogenesis.

The advent of various 'omic' technologies has increased expectations in the field of biomarkers. In an attempt to clarify how different strategies may contribute to improving prognostic classification and to identify new predictors of patient outcome we analyzed genomic and transcriptomic profiles in a series of R0 Dukes B and C colorectal carcinomas. We have compared the predictive capability of each approach against conventional clinicopathological and molecular parameters. At a genomic level, gains at 11q including amplification at 11q13 were an indicator of poorer outcome. In transcriptomic analyses we identified 68 genes whose expression levels correlated with survival (p<0.01) and included overexpression of WASF1, NFE2L2, and MMP9, and underexpression of ITGAL, TSC2, and SDF2. Gene expression levels paralleled chromosomal changes only in 56% of the genes, suggesting that, as a general trend, the direct effect of chromosomal copy number changes on gene expression levels is minimal. Classification of tumors by genomic and transcriptomic signatures resulted in non-overlapping subgroups and was not of prognostic value. We conclude that genomic and transcriptomic profiling of colorectal carcinomas may contribute as novel prognostic markers, but it does not improve outcome prediction when global profiles or signatures are considered.

We compared patterns of mitochondrial DNA (mtDNA) differentiation in three host-specific lice (Halipeurus abnormis, Austromenopon echinatum and Saemundssonia peusi) and one generalist flea (Xenopsylla gratiosa), parasitizing 22 colonies of Cory's and Cape Verde shearwater (Calonectris). The shearwater hosts show distinct phylogeographic structure corresponding to the three taxa Calonectris d. diomedea, C. d. borealis, and C. edwardsii. The host-specific lice appeared undifferentiated among the three Calonectris taxa, whereas the more generalist flea displayed significant levels of population differentiation. Neither genetic distances among host populations, nor their spatial distribution explained the patterns of genetic variability observed in the ectoparasites. The lack of differentiation among lice is unexpected, given that previous work has found evidence of cospeciation between procellariiform seabirds and their lice, and lice typically have an elevated rate of mtDNA evolution with respect to their hosts. Our results suggest that either rates of evolution in seabird lice are not always as high as previously thought, or that the magnitude of movement of lice between seabird hosts has been substantially underestimated.

Histone deacetylase 11 (HDAC11) is the latest identified member of the histone deacetylase family of enzymes. It is highly expressed in brain, heart, testis, kidney, and skeletal muscle, although its role in these tissues is poorly understood. Here, we investigate for the first time the consequences of HDAC11 genetic impairment on skeletal muscle regeneration, a process principally dependent on its resident stem cells (satellite cells) in coordination with infiltrating immune cells and stromal cells. Our results show that HDAC11 is dispensable for adult muscle growth and establishment of the satellite cell population, while HDAC11 deficiency advances the regeneration process in response to muscle injury. This effect is not caused by differences in satellite cell activation or proliferation upon injury, but rather by an enhanced capacity of satellite cells to differentiate at early regeneration stages in the absence of HDAC11. Infiltrating HDAC11-deficient macrophages could also contribute to this accelerated muscle regenerative process by prematurely producing high levels of IL-10, a cytokine known to promote myoblast differentiation. Altogether, our results show that HDAC11 depletion advances skeletal muscle regeneration and this finding may have potential implications for designing new strategies for muscle pathologies coursing with chronic damage. DATABASE: Data were deposited in NCBI's Gene Expression Omnibus accessible through GEO Series accession number GSE147423.

The COVID-19 pandemic posed a global health crisis, with new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants weakening vaccine-driven protection. Trained immunity could help tackle COVID-19 disease. Our objective was to analyze whether heat-killed Mycobacterium manresensis (hkMm), an environmental mycobacterium, induces trained immunity and confers protection against SARS-CoV-2 infection. To this end, THP-1 cells and primary monocytes were trained with hkMm. The increased secretion of tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, IL-1β, and IL-10, metabolic activity, and changes in epigenetic marks suggested hkMm-induced trained immunity in vitro. Healthcare workers at risk of SARS-CoV-2 infection were enrolled into the MANRECOVID19 clinical trial (NCT04452773) and were administered Nyaditum resae (NR, containing hkMm) or placebo. No significant differences in monocyte inflammatory responses or the incidence of SARS-CoV-2 infection were found between the groups, although NR modified the profile of circulating immune cell populations. Our results show that M. manresensis induces trained immunity in vitro but not in vivo when orally administered as NR daily for 14 days.