Locus specific epigenetic modalities of random allelic expression imbalance

Most autosomal genes are thought to be expressed from both alleles, with some notable exceptions, including imprinted genes and genes showing random monoallelic expression (RME). The extent and nature of RME has been the subject of debate. Here we investigate the expression of several candidate RME genes in F1 hybrid mouse cells before and after differentiation, to define how they become persistently, monoallelically expressed. Clonal monoallelic expression is not present in embryonic stem cells, but we observe high frequencies of monoallelism in neuronal progenitor cells by assessing expression status in more than 200 clones. We uncover unforeseen modes of allelic expression that appear to be gene-specific and epigenetically regulated. This non-canonical allelic regulation has important implications for development and disease, including autosomal dominant disorders and opens up therapeutic perspectives

New Lidocaine-Based Pharmaceutical Cocrystals: Preparation, Characterization, and Influence of the Racemic vs. Enantiopure Coformer on the Physico-Chemical Properties

This study describes the preparation, characterization, and influence of the enantiopure vs. racemic coformer on the physico-chemical properties of a pharmaceutical cocrystal. For that purpose, two new 1:1 cocrystals, namely lidocaine:dl-menthol and lidocaine:d-menthol, were prepared. The menthol racemate-based cocrystal was evaluated by means of X-ray diffraction, infrared spectroscopy, Raman, thermal analysis, and solubility experiments. The results were exhaustively compared with the first menthol-based pharmaceutical cocrystal, i.e., lidocaine:l-menthol, discovered in our group 12 years ago. Furthermore, the stable lidocaine/dl-menthol phase diagram has been screened, thoroughly evaluated, and compared to the enantiopure phase diagram. Thus, it has been proven that the racemic vs. enantiopure coformer leads to increased solubility and improved dissolution of lidocaine due to the low stable form induced by menthol molecular disorder in the lidocaine:dl-menthol cocrystal. To date, the 1:1 lidocaine:dl-menthol cocrystal is the third menthol-based pharmaceutical cocrystal, after the 1:1 lidocaine:l-menthol and the 1:2 lopinavir:l-menthol cocrystals reported in 2010 and 2022, respectively. Overall, this study shows promising potential for designing new materials with both improved characteristics and functional properties in the fields of pharmaceutical sciences and crystal engineering

Cohesin Protects Genes against γH2AX Induced by DNA Double-Strand Breaks

Chromatin undergoes major remodeling around DNA double-strand breaks (DSB) to promote repair and DNA damage response (DDR) activation. We recently reported a high-resolution map of γH2AX around multiple breaks on the human genome, using a new cell-based DSB inducible system. In an attempt to further characterize the chromatin landscape induced around DSBs, we now report the profile of SMC3, a subunit of the cohesin complex, previously characterized as required for repair by homologous recombination. We found that recruitment of cohesin is moderate and restricted to the immediate vicinity of DSBs in human cells. In addition, we show that cohesin controls γH2AX distribution within domains. Indeed, as we reported previously for transcription, cohesin binding antagonizes γH2AX spreading. Remarkably, depletion of cohesin leads to an increase of γH2AX at cohesin-bound genes, associated with a decrease in their expression level after DSB induction. We propose that, in agreement with their function in chromosome architecture, cohesin could also help to isolate active genes from some chromatin remodelling and modifications such as the ones that occur when a DSB is detected on the genome

Endoplasmic reticulum stress inhibition protects steatotic and non-steatotic livers in partial hepatectomy under ischemia–reperfusion

During partial hepatectomy, ischemia–reperfusion (I/R) is commonly applied in clinical practice to reduce blood flow. Steatotic livers show impaired regenerative response and reduced tolerance to hepatic injury. We examined the effects of tauroursodeoxycholic acid (TUDCA) and 4-phenyl butyric acid (PBA) in steatotic and non-steatotic livers during partial hepatectomy under I/R (PH+I/R). Their effects on the induction of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress were also evaluated. We report that PBA, and especially TUDCA, reduced inflammation, apoptosis and necrosis, and improved liver regeneration in both liver types. Both compounds, especially TUDCA, protected both liver types against ER damage, as they reduced the activation of two of the three pathways of UPR (namely inositol-requiring enzyme and PKR-like ER kinase) and their target molecules caspase 12, c-Jun N-terminal kinase and C/EBP homologous protein-10. Only TUDCA, possibly mediated by extracellular signal-regulated kinase upregulation, inactivated glycogen synthase kinase-3β. This is turn, inactivated mitochondrial voltage-dependent anion channel, reduced cytochrome c release from the mitochondria and caspase 9 activation and protected both liver types against mitochondrial damage. These findings indicate that chemical chaperones, especially TUDCA, could protect steatotic and non-steatotic livers against injury and regeneration failure after PH+I/R

Levosimendan: a cardiovascular drug to prevent liver ischemia-reperfusion injury?

INTRODUCTION: Temporary occlusion of the hepatoduodenal ligament leads to an ischemic-reperfusion (IR) injury in the liver. Levosimendan is a new positive inotropic drug, which induces preconditioning-like adaptive mechanisms due to opening of mitochondrial KATP channels. The aim of this study was to examine possible protective effects of levosimendan in a rat model of hepatic IR injury. MATERIAL AND METHODS: Levosimendan was administered to male Wistar rats 1 hour (early pretreatment) or 24 hours (late pretreatment) before induction of 60-minute segmental liver ischemia. Microcirculation of the liver was monitored by laser Doppler flowmeter. After 24 hours of reperfusion, liver and blood samples were taken for histology, immuno- and enzyme-histochemistry (TUNEL; PARP; NADH-TR) as well as for laboratory tests. Furthermore, liver antioxidant status was assessed and HSP72 expression was measured. RESULTS: In both groups pretreated with levosimendan, significantly better hepatic microcirculation was observed compared to respective IR control groups. Similarly, histological damage was also reduced after levosimendan administration. This observation was supported by significantly lower activities of serum ALT (pearly = 0.02; plate = 0.005), AST (pearly = 0.02; plate = 0.004) and less DNA damage by TUNEL test (pearly = 0.05; plate = 0.034) and PAR positivity (pearly = 0.02; plate = 0.04). Levosimendan pretreatment resulted in significant improvement of liver redox homeostasis. Further, significantly better mitochondrial function was detected in animals receiving late pretreatment. Finally, HSP72 expression was increased by IR injury, but it was not affected by levosimendan pretreatment. CONCLUSION: Levosimendan pretreatment can be hepatoprotective and it could be useful before extensive liver resection

Determinants of SARS-CoV-2 receptor gene expression in upper and lower airways

The recent outbreak of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has led to a worldwide pandemic. One week after initial symptoms develop, a subset of patients progresses to severe disease, with high mortality and limited treatment options. To design novel interventions aimed at preventing spread of the virus and reducing progression to severe disease, detailed knowledge of the cell types and regulating factors driving cellular entry is urgently needed. Here we assess the expression patterns in genes required for COVID-19 entry into cells and replication, and their regulation by genetic, epigenetic and environmental factors, throughout the respiratory tract using samples collected from the upper (nasal) and lower airways (bronchi). Matched samples from the upper and lower airways show a clear increased expression of these genes in the nose compared to the bronchi and parenchyma. Cellular deconvolution indicates a clear association of these genes with the proportion of secretory epithelial cells. Smoking status was found to increase the majority of COVID-19 related genes including ACE2 and TMPRSS2 but only in the lower airways, which was associated with a significant increase in the predicted proportion of goblet cells in bronchial samples of current smokers. Both acute and second hand smoke were found to increase ACE2 expression in the bronchus. Inhaled corticosteroids decrease ACE2 expression in the lower airways. No significant effect of genetics on ACE2 expression was observed, but a strong association of DNA- methylation with ACE2 and TMPRSS2- mRNA expression was identified in the bronchus.</p

Small-molecule-induced DNA damage identifies alternative DNA structures in human genes.

Guanine-rich DNA sequences that can adopt non-Watson-Crick structures in vitro are prevalent in the human genome. Whether such structures normally exist in mammalian cells has, however, been the subject of active research for decades. Here we show that the G-quadruplex-interacting drug pyridostatin promotes growth arrest in human cancer cells by inducing replication- and transcription-dependent DNA damage. A chromatin immunoprecipitation sequencing analysis of the DNA damage marker γH2AX provided the genome-wide distribution of pyridostatin-induced sites of damage and revealed that pyridostatin targets gene bodies containing clusters of sequences with a propensity for G-quadruplex formation. As a result, pyridostatin modulated the expression of these genes, including the proto-oncogene SRC. We observed that pyridostatin reduced SRC protein abundance and SRC-dependent cellular motility in human breast cancer cells, validating SRC as a target of this drug. Our unbiased approach to define genomic sites of action for a drug establishes a framework for discovering functional DNA-drug interactions

Cellular Phenotype-Dependent and -Independent Effects of Vitamin C on the Renewal and Gene Expression of Mouse Embryonic Fibroblasts

Vitamin C has been shown to delay the cellular senescence and was considered a candidate for chemoprevention and cancer therapy. To understand the reported contrasting roles of vitamin C: growth-promoting in the primary cells and growth-inhibiting in cancer cells, primary mouse embryonic fibroblasts (MEF) and their isogenic spontaneously immortalized fibroblasts with unlimited cell division potential were used as the model pair. We used microarray gene expression profiling to show that the immortalized MEF possess human cancer gene expression fingerprints including a pattern of up-regulation of inflammatory response-related genes. Using the MEF model, we found that a physiological treatment level of vitamin C (10−5 M), but not other unrelated antioxidants, enhanced cell growth. The growth-promoting effect was associated with a pattern of enhanced expression of cell cycle- and cell division-related genes in both primary and immortalized cells. In the immortalized MEF, physiological treatment levels of vitamin C also enhanced the expression of immortalization-associated genes including a down-regulation of genes in the extracellular matrix functional category. In contrast, confocal immunofluorescence imaging of the primary MEF suggested an increase in collagen IV protein upon vitamin C treatment. Similar to the cancer cells, the growth-inhibitory effect of the redox-active form of vitamin C was preferentially observed in immortalized MEF. All effects of vitamin C required its intracellular presence since the transporter-deficient SVCT2−/− MEF did not respond to vitamin C. SVCT2−/− MEF divided and became immortalized readily indicating little dependence on vitamin C for the cell division. Immortalized SVCT2−/− MEF required higher concentration of vitamin C for the growth inhibition compared to the immortalized wildtype MEF suggesting an intracellular vitamin C toxicity. The relevance of our observation in aging and human cancer prevention was discussed

HIV-1 Residual Viremia Correlates with Persistent T-Cell Activation in Poor Immunological Responders to Combination Antiretroviral Therapy

BACKGROUND:The clinical significance and cellular sources of residual human immunodeficiency virus type 1 (HIV-1) production despite suppressive combination antiretroviral therapy (cART) remain unclear and the effect of low-level viremia on T-cell homeostasis is still debated. METHODOLOGY/PRINCIPAL FINDINGS:We characterized the recently produced residual viruses in the plasma and short-lived blood monocytes of 23 patients with various immunological responses to sustained suppressive cART. We quantified the residual HIV-1 in the plasma below 50 copies/ml, and in the CD14(high) CD16(-) and CD16+ monocyte subsets sorted by flow cytometry, and predicted coreceptor usage by genotyping V3 env sequences. We detected residual viremia in the plasma of 8 of 10 patients with poor CD4+ T-cell reconstitution in response to cART and in only 5 of 13 patients with good CD4+ T-cell reconstitution. CXCR4-using viruses were frequent among the recently produced viruses in the plasma and in the main CD14(high) CD16(-) monocyte subset. Finally, the residual viremia was correlated with persistent CD4+ and CD8+ T-cell activation in patients with poor immune reconstitution. CONCLUSIONS:Low-level viremia could result from the release of archived viruses from cellular reservoirs and/or from ongoing virus replication in some patients. The compartmentalization of the viruses between the plasma and the blood monocytes suggests at least two origins of residual virus production during effective cART. CXCR4-using viruses might be produced preferentially in patients on cART. Our results also suggest that low-level HIV-1 production in some patients may contribute to persistent immune dysfunction despite cART