Swagata Majumdar

Abstract Diagnosis of hepatocellular carcinoma (HCC) remains challenging to clinicians, particularly in a patient with low alpha‐fetoprotein. Here, in silico, ex vivo and in vitro data were combined to identify liver‐specific exosomal miRNAs as an early diagnostic marker for HCC. Transcriptome profiling for mRNA and small RNA in same HCV‐HCC and normal liver tissues followed by cross‐validation of 41 deregulated miRNAs (log 2 FoldChange > 1.5, P adj < .1) with GEO/TCGA datasets of HCV/HBV related HCC vs normal/adjacent tissue revealed three miRNAs were commonly deregulated (miR‐10b/miR‐21/miR‐182) among all HCC irrespective of viral etiology. Targets of top deregulated miRNAs were identified by TargetScan/miRwalk and validated in mRNA transcriptome data followed by Panther/Gene Ontology enrichment/Cytoscape analysis suggested that targets were mostly from carcinogenesis pathways. Hence, those miRNAs were validated in normal and HCV‐HCC tissues by qRT‐PCR and subsequently in plasma‐derived‐exosomes of both HBV/HCV infected non‐HCC (chronic hepatitis [CH]/liver cirrhosis [LC]) and HCC samples, and in liver‐specific Anti‐Asgr2 immuno‐enriched exosomes. Exosomes were verified using Nanosight/TEM/immune‐blotting with anti‐Alix/anti‐GRP78/anti‐Asgr2. Along with miR‐21‐5p, miR‐10b‐5p/miR‐221‐3p/miR‐223‐3p was found significantly upregulated in the exosome of HCC patients than CH/non‐HCC. The comparable expression pattern was seen in anti‐Asgr2 immuno‐precipitated exosomes. Interestingly, the AFP level was found below 250 ng/mL in about 94% of HCV‐HCC and 62% of HBV‐HCC patients. ROC analysis showed that miR‐10b‐5p + miR‐221‐3p + miR‐223‐3p + miR‐21‐5p could differentiate CH/non‐HCC(CH + LC) from HCC with AUROC: 0.86 (97.5% CI: 0.77‐0.94)/0.80 (97.5% CI: 0.70‐0.89), sensitivity: 74%/58% and specificity: 86%/95% while miR‐10b‐5p + miR‐221‐3p + miR‐223‐3p showed AUROC: 0.84 (97.5% CI: 0.74‐0.94)/0.74 (97.5% CI: 0.63‐0.84), sensitivity: 86%/86% and specificity:66%/53% for low AFP‐HCC vs CH/non‐HCC, respectively, having better sensitivity than the combination of four miRNAs. Multivariate analysis further revealed low Albumin and high miR‐21‐5p as probable independent risk factor for HCC.

Based on the recommendation of the WHO's Technical Advisory Group on Virus Evolution, WHO designated the variant B.1.1.529 as a variant of concern (VOC) and named Omicron on 26 November, 2021. Omicron was first observed in Africa in mid-November 2021.1 The infection by this variant has been rapidly spreading and 85 countries have already reported the cases of human infection with this variant as of 15 December 2021.2 The rapid spread of Omicron has again fueled the fears of COVID-19 all around the world like other four VOCs (Alpha, Beta, Gamma, and Delta). Recently, the Omicron variant has classified into two different lineages BA.1 and BA.2 based on the mutations, some of which are common and some are unique to both lineages.3, 4 Still, there is no clear evidence or published article on the mutational diversity and phylogenetic analysis of these two lineages. Therefore, in the present study, we have performed the whole-genome mutational mapping and phylogenetic analysis of BA.1 and BA.2 lineages. We have downloaded 6 genome sequences each of BA.1 and BA.2, and also the genome sequence of the prototype strain (hCoV-19/Wuhan/WIV04/2019) from the Global Initiative on Sharing All Influenza Data (GISAID) and performed whole-genome mutational analysis according to the protocol described in Sarkar et al.5, 6 Each of the six genomes of both BA.1 and BA.2 lineages was found to have 51 mutations dispersed throughout the genome, 32 of which are common to both lineages, whereas each lineage has 19 signature mutations. Among 32 common mutations, 21 are present in the S glycoprotein and the rest 11 are present in the other four coding regions (ORF1ab, E, M, and N). Nineteen unique mutations of BA.1 include 13 in the S glycoprotein and that of BA.2 includes 7 in the S glycoprotein (Table 1). Phylogenetic analysis of 12 genome sequences of Omicron variant, encompassing 6 genomes each of BA.1 and BA.2, along with the 2000 genomes of 25 different clades by Ultrafast Sample placement of Existing tRees,6, 7 revealed that genomes of Omicron variant formed a new cluster that emerged from the 20B clade (also known as GR) and also subdivided into two different subclusters (BA.1 and BA.2) based on the unique mutations (Figure 1). The S glycoprotein mediates virus attachment to ACE2 receptor, membrane fusion, and entry into the host cell, and also acts as a primary target for neutralizing antibodies elicited by the host immune response.8 Presence of 34 and 28 mutations in the S glycoprotein of BA.1 and BA.2, respectively, raising concern whether these lineages have increased transmissibility, immune escape potential, and virulence compared to other circulating SARS-CoV-2 strains especially Delta which is currently dominating worldwide. Seven mutations of both BA.1 and BA.2 (G142D, K417N, T478K, N501Y, D614G, H655Y, and P681H) and three mutations of BA.1 (∆HV69del, T95I, and ∆YY144del) overlap four other VOCs (Alpha, Beta, Gamma, and Delta) and have previously been linked with high transmissibility, increased viral binding affinity, and immune evasion.9-12 Functional implication of the remaining mutations of the S glycoprotein and other coding regions of BA.1 and BA.2 still unknown, leaving a question of how the whole set of mutations of the two lineages will affect viral fitness. Preliminary evidence indicated that Omicron has increased infectivity and a high transmission rate compared to Delta.13-15 However, whether the rapid spread of Omicron in countries with increased population immunity is due to increased transmissibility and/or immune evasion remains unclear. Though, some recent studies have claimed the immune evasion properties of the Omicron.14-17 Based on this existing evidence, Omicron is anticipated to overtake Delta in areas where community transmission occurs. The severity of Omicron infection still remains elusive. Preliminary studies from South Africa suggested that Omicron may be less severe than Delta,18 and all COVID-19 patients, infected with Omicron, from countries of EU and EEA either showed mild symptoms or were asymptomatic.19 Detection accuracy of routinely used PCR and antigen-based rapid diagnostic test (Ag-RDT) assays was not found to be influenced by most of the Omicron strains. However, the BA.1 lineage showed S gene target failure (SGTF) in RT-PCR assay due to multiple deletions in the NTD of S glycoprotein, whereas BA.2 lineage may skip SGTF due to lack of deletions in the NTD. Overall, the global threat related to Omicron remains very high for its potential to escape humoral immune response and high transmissibility, which may lead to another wave of COVID-19 with severe consequences.19 The authors acknowledge the University Grants Commission (UGC) of India for providing fellowship to Swagata Majumdar. The authors would also like to acknowledge GISAID (https://www.gisaid.org/) for facilitating open data sharing. The authors declare that there are no conflict of interests. Swagata Majumdar and Rakesh Sarkar conceived the study. Swagata Majumdar performed sequence retrieval of SARS-CoV-2 and whole-genome mutational analysis. Rakesh Sarkar performed the phylogenetic analysis and drafted the manuscript. All authors revised the manuscript and approved the final manuscript for submission. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Background and aims: Alcoholic liver disease (ALD) is the leading cause of the liver cirrhosis related death worldwide. Excessive alcohol consumption resulting enhanced gut permeability which trigger sensitization of inflammatory cells to bacterial endotoxins and induces secretion of cytokines, chemokines leading to activation of stellate cells, neutrophil infiltration and hepatocyte injury followed by steatohepatitis, fibrosis and cirrhosis. But all chronic alcoholics are not susceptible to ALD. This study investigated the causes of differential immune responses among ALD patients and alcoholic controls (ALC) to identify genetic risk factors and assessed the therapeutic potential of a microRNA, miR-124-3p. Materials and methods: Bio-Plex Pro™ Human Chemokine analysis/qRT-PCR array was used for identification of deregulated immune genes. Sequencing/luciferase assay/ELISA detected and confirmed the polymorphisms. THP1 co-cultured with HepG2/LX2/HUVEC and apoptosis assay/qRT-PCR/neutrophil migration assay were employed as required. Results: The combined data analysis of the GSE143318/Bio-Plex Pro™ Human Chemokine array and qRT-PCR array revealed that six genes (TNFα/IL1β/IL8/MCP1/IL6/TGFβ) were commonly overexpressed in both serum/liver tissue of ALD-patients compared to ALC. The promoter sequence analysis of these 6 genes among ALD (n=322)/ALC (n=168) samples revealed that only two SNPs, rs361525(G/A) at -238 in TNF-α/rs1143627(C/T) at -31 in IL1β were independently associated with ALD respectively. To evaluate the functional implication of these SNPs on ALD development, the serum level of TNF-α/IL1β was verified and observed significantly higher in ALD patients with risk genotypes TNF-α-238GA/IL1β-31CT+TT than TNF-α-238GG/IL1β-31CC. The TNF-α/IL1β promoter Luciferase-reporter assays showed significantly elevated level of luciferase activities with risk genotypes -238AA/-31TT than -238GG/-31CC respectively. Furthermore, treatment of conditioned medium of TNF-α/IL1β over-expressed THP1 cells to HepG2/LX2/HUVEC cells independently showed enhanced level of ER stress and apoptosis in HepG2/increased TGFβ and collagen-I production by LX2/huge neutrophil infiltration through endothelial layer. However, restoration of miR-124-3p in THP1 attenuated such inter-cellular communications and hepatocyte damage/collagen production/neutrophil infiltration were prohibited. Target analysis/luciferase-reporter assays revealed that both TNF-α/IL1β were inhibited by miR-124-3p along with multiple genes from TLR4 signaling/apoptosis/fibrogenesis pathways including MYD88, TRAF3/TRADD, Caspase8/PDGFRA, TGFβR2/MCP1, and ICAM1 respectively. Conclusion: Thus, rs361525(G/A) in TNF-α and rs1143627(C/T) in IL1β gene may be used as early predictors of ALD susceptibility among East Indian population. Impeding overexpressed TNF-α/IL1β and various genes from associated immune response pathways, miR-124-3p exhibits robust therapeutic potential for ALD patients.

Abstract Diverse mechanisms have been established to understand the chemoresistance of hepatocellular carcinoma (HCC), but the contribution of non-coding RNAs is not surveyed well. Here, we aimed to explore the lncRNA-miRNA axis in Hepatitis C and B virus (HCV and HBV) infected HCC to investigate the molecular mechanism of chemoresistance and to identify a potential therapeutic target for HCC. The small RNA transcriptome analysis followed by qRT-PCR validation with the liver tissues of both HCV and HBV infected HCC patients revealed that miR-424-5p, miR-136-3p, miR-139-5p, miR-223-3p, and miR-375-3p were the most downregulated miRNAs in HCC compared to normal (log 2 fold change ≤−1.5, P adj ≤ 0.05). In silico pathway analysis with the validated targets of each miRNA revealed that the signalling pathway regulating pluripotency of stem cells is commonly targeted by these five miRNAs. Subsequent validation by 3′UTR-luciferase assay and western blot analysis unveiled that these five miRNAs impeded either same or diverse genes, but all linked to BMP signalling pathway such as BMPR1A/BMPR1B by miR-139-5p, miR-136-3p, and miR-375-3p, and ACVR2A/ACVR2B by miR-424-5p and miR-223-3p. Furthermore, restoration of each miRNA in Huh7/SNU449 cells inhibited phosphorylation of downstream SMAD1/5 and ERK1/2, and attenuated Epithelial-mesenchymal transition, stemness, spheroid formation, chemoresistance, invasion and migration of cells. To investigate the mechanism of suppression of these miRNAs, “DIANA” tool was employed and lncRNA-KCNQ1OT1 was retrieved as interacting partner of all the five miRNAs. In vitro RNA pull-down assay revealed that lncRNA-KCNQ1OT1 physically interacted and sequestered these five miRNAs in the cytoplasm. Hence, KCNQ1OT1 was suppressed in Huh7/SNU449 cells using CRISPR technology and observed regression of oncogenic properties with enhanced chemosensitivity and reduced metastasis in cancer cells. Shrinkage of tumour size and volume in NOD-SCID mice injected with KCNQ1OT1-sgRNA cells further strengthened our observations. Thus, lncRNA-KCNQ1OT1 is the main regulator, which reduces the level of beneficiary miRNAs in the tumour milieu and modulates BMP signalling pathway to promote chemoresistance in HCC, suggesting lncRNA-KCNQ1OT1 might have robust potential to be a therapeutic target in HCC.

Abstract Uncontrolled high transmission is driving the continuous evolution of SARS-CoV-2, leading to the nonstop emergence of the new variants with varying sensitivity to the neutralizing antibodies and vaccines.Wehave analysed of 8,82,740 SARS-CoV-2 genome sequences, collected and sequenced during late December 2019 to 25 March 2021 from all across the world. The findings revealed differences in temporal and spatial distribution,and predominance of various clades/variants among six different continents.We found no clear association between the pathogenic potential of the various clades by comparing the case fatality rate (CFR) of 170 countries with the predominant SARS-CoV-2 clades in those countries, demonstrating the insignificance of the clade specific mutations on case fatality. Overall, relying on a large-scale dataset,this study illustratedthe time-basedevolution andprevalence of various clades/variantsamong different geographic regions.The study may help in designing continent specific vaccines in the future.