During dysregulated inflammation and sepsis, there is a sudden surge of cytokines, such as TNF alpha, IL6, and IL10, which disrupts the body homeostasis. This sudden and rapid increase in cytokine gene expression cannot be explained by the conventional central dogma mechanism. DNA methylation is one of the most significant epigenetic modifications. DNA methylation is mostly associated with the suppression of gene activity, and DNA demethylation is associated with the activation of gene activity. There are numerous transcription factors, such as CREB1, c-FOS, AP-1, IRF1, and EGR1, that play pivotal roles in regulating inflammation. Thus, it was hypothesized that changes in the DNA methylation patterns of cytokine promoter regions or in the promoter regions of these specific transcription factors might be the potential reasons for the increase in cytokine levels during sepsis. During endotoxin stimulation, DNA methyltransferases were suppressed, and DNA methylation levels were altered at the global level. Bisulfite sequencing revealed no change in the DNA methylation patterns of the TNF-alpha, IL6 and IL10 cytokine promoters. The CREB1 and c-FOS transcription factor promoters were demethylated after LPS stimulation and in clinical sepsis samples. Overall, this study highlights the importance of the role of DNA methylation in sepsis. ### Competing Interest Statement The authors have declared no competing interest.

Antivirals such as nucleotide analogs (NAs) are potent inhibitors of hepatitis B virus (HBV) replication. However, NAs fail to diminish the signaling and mitogenic activities of the transactivator HBx protein. Earlier we have shown that thiourea derivative IR-415 (DSA-00) targeted HBx to down-regulate its target viral and host genes. However, the molecular mechanism of its antiviral action is poorly understood. Here we investigated the anti-HBV properties of DSA-00 and its new derivatives in cell culture models. DSA-00 and its derivatives DSA-02 and DSA-09 not only suppressed HBV DNA levels similar to well-known antiviral Entecavir but also diminished the expression of pgRNA and secretion of HBsAg and HBeAg. Apparently, the three DSA derivatives inhibited the viral pregenomic RNA expression by stabilizing the episomal DNA silencing protein SMC5, suppressed transcription from viral and host gene promoters, and normalized intracellular CDK2 activity. As none the compounds are reportedly cytotoxic, thiourea derivatives could be good candidates for developing future antivirals for a functional cure of hepatitis B infection.

Nucleot(s)ide analogues, the current antiviral treatments against chronic hepatitis B (CHB) infection, are non-curative due to their inability to eliminate covalently closed circular DNA (cccDNA) from the infected hepatocytes. Preclinical studies have shown that coumarin derivatives can effectively reduce the HBV DNA replication. We evaluated the antiviral efficacy of thirty new coumarin derivatives in cell culture models for studying HBV. Furanocoumarins Fc-20 and Fc-31 suppressed the levels of pre-genomic RNA as well as cccDNA, and reduced the secretion of virions, HBsAg and HBeAg. The antiviral efficacies of Fc-20 and Fc31 improved further when used in combination with the hepatitis B antiviral drug Entecavir. There was a marked reduction in the intracellular HBx level in the presence of these furanocoumarins due to proteasomal degradation resulting in the down-regulation of HBx-dependent viral genes. Importantly, both Fc-20 and Fc-31 were non-cytotoxic to cells even at high concentrations. Further, our molecular docking studies confirmed a moderate to high affinity interaction between furanocoumarins and viral HBx via residues Ala3, Arg26 and Lys140. These data suggest that furanocoumarins could be developed as a new therapeutic for CHB infection.

BACKGROUND AND AIMS:Hepatitis B virus X protein (HBx) play a key role in pathogenesis of HBV-induced hepatocellular carcinoma (HCC) by promoting epithelial to mesenchymal transition (EMT). In this study, we hypothesized that inhibition of HBx is an effective strategy to combat HCC. METHODOLOGY AND RESULTS:We designed and synthesized novel HBx gene specific single guide RNA (sgRNA) with CRISPR/Cas9 system and studied its in vitro effects on tumour properties of HepG2-2.15. Full length HBx gene was excised using HBx-CRISPR that resulted in significant knockdown of HBx expression in hepatoma cells. HBx-CRISPR also decreased levels of HBsAg and HBV cccDNA expression. A decreased expression of mesenchymal markers, proliferation and tumorigenic properties was observed in HBx-CRISPR treated cells as compared to controls in both two- and three- dimensional (2D and 3D) tumour models. Transcriptomics data showed that out of 1159 differentially expressed genes in HBx-CRISPR transfected cells as compared to controls, 70 genes were upregulated while 1089 genes associated with cell proliferation and EMT pathways were downregulated. CONCLUSION:Thus, targeting of HBx by CRISPR/Cas9 gene editing system reduces covalently closed circular DNA (cccDNA) levels, HBsAg production and mesenchymal characteristics of HBV-HCC cells. We envision inhibition of HBx by CRISPR as a novel therapeutic approach for HBV-induced HCC.

The primary mechanism in cancer metastasis, collective invasion is becoming more widely recognized. Leader cells are specially trained cancer cells that invading the tumors. They are crucial in establishing invasion pathways, organizing follower cells, and facilitating the survival of cancer cells throughout the metastatic cascade. To aid group invasion, these leader cells activate a variety of mechanical, genomic, and metabolomic pathways. Leader cell development and function are influenced by stromal cells, matrix properties, genetic and epigenetic factors, and more.

DSA-00 and its two derivatives (DSA-02 & DSA-09) are new thiourea derivatives that exhibit strong antiviral activity against hepatitis B virus comparable to Entecavir. Here these compounds were evaluated for in-vitro cytotoxicity and genotoxicity, and in vivo acute toxicity for their potential therapeutic use. The cytotoxicity of thiourea derivatives was assessed in HepG2 and HepG2.2.15 cells by MTT assay whereas their genotoxicity was measured by Ames II test. The acute toxicity study was carried out in the Sprague-Dawley rats by observing the following parameters: mortality, clinical symptoms, hematological parameters, urine and changes in animal body & organs weight, gross necropsy and histopathology. DSA-00, DSA-02, and DSA-09 were non-cytotoxic even at the 320uM concentration with respective CC50 values of 329.6, 323.5, and 349.7 uM. The Ames II test revealed that these molecules were non-mutagenic at a ~1M concentration. The acute toxicity studies revealed LD50 values belong from the moderate range of toxicity of DSA-00, DSA-02 and DSA-09. Importantly, there were no abnormal change in body weight and organs weight. Moreover, no abnormal clinical signs such as hematological parameter, urinalysis, gross necropsy and or histopathological in any of the animals after receiving oral doses of thiourea derivatives. Thiourea derivatives DSA-00, DSA-02, and DSA-09 appear to have a moderate range of acute toxicity at dosages used and thus, appear to be generally safe.

Chronic hepatitis B (CHB) infection and the Hepatitis B virus protein X (HBx) are a major risk factor for the development of hepatocellular carcinoma (HCC). In CHB, HBx induces mitochondrial dysfunction, exhaustion and impaired function in hepatocytes. Restoring hepatocyte health along with reduction in virus replication could be an ideal treatment of CHB. Thiourea derivatives are well known for their antiviral property though their effect on mitochondrial and/ or hepatocyte health is obscure. The current pilot study focus on the repurposing of DSA-00, DSA-02, and DSA-09 on mitochondrial health and hepatocyte replenishment. HepG2.2.15 cells were treated with these, alongside Entecavir (ETV). The proteomics analysis showed DSA-00 and ETV were enriched with proteins associated with antiviral responses. DSA-00 additionally showed increase in proteins linked to mitochondrial response. DSA-02 showed association with innate immune system and citric acid cycle. DSA-09 displayed pathways similar to DSA-00 and ETV. The treated groups exhibited enhanced bio-energetic and antiviral response compared to the untreated group. FACS analysis, validated thiourea derivatives restored exhausted hepatocytes by targeting mitochondrial potential and function. Our findings suggest that (DSA-00, DSA-02, and DSA-09) may hold potential as a novel treatment strategy restoring mitochondrial healthy along with anti-viral response in CHB.

The chronic infection of the hepatitis B virus (CHB) represents a major public health problem worldwide. Despite the availability of an effective prophylactic vaccine, millions of hepatitis B patients are at increased risk of developing chronic liver disease. The currently available treatments for HBV infection include interferon and nucleos(t)ide analogues that are effective at suppressing viral load and preventing or delaying the progression of liver disease. However, these treatments offer somewhat unsatisfactory clinical cures due to the persistence of the intrahepatic pool of covalently closed circular DNA (cccDNA) that serves as a reservoir for viral progenies and a potential source of recurring infections. Elimination of viral cccDNA remains a challenge for scientists and pharmaceutical industries in order to achieve the eradication and control of HBV infection. This would involve a detailed understanding of the molecular mechanisms of cccDNA formation, its intracellular stability, and regulation during replication and transcription. Recent advances in drug therapy have heralded a new horizon of novel therapeutic approaches for CHB infection, with several promising antiviral and immunomodulatory agents currently in preclinical or clinical testing. However, approval of any new curative therapy would involve rigorous evaluation of the efficacy and safety of each treatment and defining correct endpoints associated with improved clinical outcomes. This article summarizes the current landscape of HBV treatments, and drugs in clinical trials and highlights the most recent anti-HBV small molecules designed to directly target HBV or to improve immune response during chronic infection.

A candidate molecule 1-(3-(1H-imidazol-1-yl) propyl)-3-(2,4-difluorophenyl) thiourea (coded as 'IR-415') exhibiting excellent antiviral efficacy in cell culture model was identified after a high-throughput screening of the Maybridge library.([7]) Twenty derivatives of IR-415 hereafter referred to as DSA-00 were synthesized and evaluated for their antiviral activity against hepatitis B virus (HBV) in cell culture. The HBV-expressing HepG2.2.15 cells or HBV-permissive HepG2-hNTCP-C4 cells were treated with DSA-00 or its new derivatives. A significant and improved inhibition in viral DNA replication and secretion of hepatitis B surface antigen were observed in the presence two derivatives, viz., 1-(2,4-Difluoro-phenyl)-3-(4-imidazol-1-yl-butyl)-thiourea and 1-(3,5-Difluoro-phenyl)-3-(4-imidazol-1-yl-butyl)-thiourea. Consistent with these antiviral properties, our molecular docking studies predicted a high affinity interaction of these derivatives with HBx protein. Importantly, DSA-00 and its derivatives exhibited minimal toxicity at higher concentrations. Thus, these derivatives have the potential to be developed as new therapeutics for mono or combination therapy for the management of HBV infection.

Chronic hepatitis B (CHB) infection and the Hepatitis B virus X protein (HBx) are major risk factors associated with hepatocellular carcinoma (HCC). In CHB infection, HBx induces mitochondrial dysfunction, exhaustion and impaired function in hepatocytes. Restoring hepatocyte health along with reduction in virus replication could be an ideal treatment for CHB. Thiourea derivatives are well known for their antiviral property though their effect on mitochondrial and/ or hepatocyte health remains obscure. This study focus on the repurposing of thiourea derivatives (DSA-00, DSA-02, and DSA-09) on hepatocyte replenishment. HepG2.2.15 cells were treated with thiourea derivatives, alongside Entecavir (ETV). The proteomics analysis showed both DSA-00 and ETV were enriched with proteins associated with antiviral responses. In addition, DSA-00 additionally showed increase in proteins linked to mitochondrial response. Whereas DSA-02 exhibited association with innate immune system and citric acid cycle and DSA-09 displayed pathways similar to DSA-00 and ETV. Treated groups exhibited enhanced bio-energetic and antiviral response as compared to the untreated group. FACS analysis revealed the restoration of exhausted hepatocytes by thiourea derivatives through targeting mitochondria. Our findings suggest that thiourea derivatives hold potential as a novel therapeutic agent that seems to restore mitochondrial health along with anti-viral response in CHB.

Severe alcoholic hepatitis (SAH) has a high mortality and corticosteroid therapy is effective only in 60% patients. A reliable indicator of therapy response, and early mortality in needed. A total of 223 SAH patients, 70 in derivative [50 responders (R) and 10 non-responders (NR)] and 153 in validation cohort [136 R, 17 NR] were studied. Baseline plasma metabolome/meta-proteome can stratify pre-therapy steroid response. NR showed significant increase in alpha, beta diversity and COG functions; biosynthesis of secondary metabolites, energy metabolism (p<0.05). Temporal metabolite expression post corticosteroid therapy showed metabolic inactivity in NR. Plasma urobilinogen [AUC=0.94] predicted NR with HR 1.5(1.1-1.6) and cut-off >0.07mg/ml segregated non-survivors which on Machine learning validation showed >98% accuracy/sensitivity/specificity. Plasma urobilinogen correlated with bacterial peptide known for converting bilirubin to urobilinogen (r2>0.7; p<0.05). Additionally, urobilinogen activated neutrophils, promoted inflammation, increased GRb expression mediated resistance, modulates intestinal permeability and predicted outcomes (log-rank<0.05) in Severe Alcoholic Hepatitis.

One complete unit of an 80S eukaryotic ribosome contains nearly 88 ribosomal proteins (RPs). These proteins have a high ratio of arginine/lysine and specific extension tails to facilitate protein-RNA and protein-protein interactions, respectively. The RPs are encoded by ribosomal protein genes (RPGs) that are evenly distributed in the genome and have common features with diverse promoters range. The RPs can be divided into two groups—those present in the small ribosomal subunit and those present in the large subunit. The small ribosomal subunit has around 35 proteins whereas the large subunit has 53 RPs. Ribosome biogenesis is an energy-consuming process required for the translation of mRNA into proteins. It is crucial for cell survival. The expression of RPs is well synchronized at the levels of transcription and posttranscriptional modification, translation, and posttranslational modification to produce an accurate stoichiometric ratio during ribosome biogenesis.