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Publications and source records attributed to Sharma, M. K..
Withdrawal statementThe authors have withdrawn this manuscript because the conclusions have been updated with the addition of new data for further publication. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.
BackgroundHepatitis B virus (HBV) modulates epigenetic landscape by epigenetic regulators. HBsAg seroconversion is possible with immune activation, therefore we aimed to investigate epigenetic modulation in HBV reactivation (rHBV) for viral clearance and seroconversion. MethodsSixteen retrospectively collected rHBV patients [Seroconverters (SC, n=7, HBsAg loss and anti-HBs>10 IU/ml), non- seroconverters (NSC, n=9)], chronic hepatitis B treatment naive (nCHBV, n=7) patients and healthy controls (HC, n=7) were included in this study. Genome methylation, gene expression, plasma-cytokines, and immune cell profiling was analysed by Reduced Representation Bisulfite Sequencing (RRBS), QRT-PCR, multiplex-cytokine-bead array and flow-cytometry. ResultsrHBV patients having high HBV DNA and ALT showed epigenetic remodellers; KDM2B, NCOR2 and GATA6, immune and metabolic genes; TGF-{beta}, IL-6, IRF8, RPTOR, HK3 significantly (p<0.05) hypomethylated at specific CpG islands compared to nCHBV. TOX was hypomethylated in nCHBV suggesting immune-exhaustion. At-baseline, seroconverters showed hypomethylation of KDM2B, COX19, IRF8, TLR5 and hypermethylation of LAG3 compared to non-seroconverters. Further, in seroconverters at week-24, IL17RA, IFN-{gamma}, TGF-{beta}, and STAT5B (p<0.05) were additionally hypomethylated at specific CpG islands suggesting immune activation. Cytokine-bead analysis revealed increased IL-6 (p=0.009) and decreased LAG3 plasma levels (p=0.01) also imply on significantly differentiated HBV specific CD8, Tfh and Th1/17 cells in seroconverters at baseline and week-24. However, both nCHBV and non-seroconverters had consistent hypomethylation of LAG3 and TOX, which leads to immune exhaustion. ConclusionIn rHBV, seroconversion is driven by position specific CpG islands methylation in epigenetic remodellers, immune and metabolic genes. Immune metabolic reprograming is reflected by Th1/17 differentiation, extensive interleukin production for HBsAg seroconversion. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/554133v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@48e206org.highwire.dtl.DTLVardef@ef0fa7org.highwire.dtl.DTLVardef@ecb710org.highwire.dtl.DTLVardef@e6b34e_HPS_FORMAT_FIGEXP M_FIG C_FIG Lay summaryEpigenetic landscape in nCHBV depicts exhaustion and immune dysfunction. Out of many hypermethylated CpG islands of nCHBV, few become hypomethylated in rHBV and drives immune and metabolic reprogramming. This study provides insights into the cellular and molecular basis of epigenomic programs that regulate the differentiation and activation of immune cells leading to viral clearance and seroconversion. Targeting epigenetic mechanism could be promising strategy for the treatment of nCHBV and non-seroconverters.
BackgroundIn Mycobacterium tuberculosis, molecular predictions of ethambutol resistance rely primarily on the detection of mutations within embB. However, discordance between embB406 mutations and phenotypic drug sensitivity questions its clinical significance. This study aims to decipher the association of embB406 mutations with ethambutol resistance in M. tuberculosis. MethodsAll M. tuberculosis isolates from our culture collection containing embB406 mutations (n=16) and pan-sensitive control isolates (n=10) were selected for this study. Phenotypic drug susceptibility testing for ethambutol was performed in duplicate on the BACTEC MGIT 960 at concentrations of 2, 3, 4, and 5 g/mL with strain H37Rv as assay control. Whole genome sequencing was performed on Illumina Miseq for drug resistance predictions (MyKrobe Predictor v.0.7.0), phylogenomics (SNVPhyl v.1.2.3) and single nucleotide polymorphism analysis (Snippy). ResultsTwo embB406 mutation subtypes were found among 16 strains: Gly406Asp and Gly406Ala. MyKrobe predicted all strains of either subtype to be ethambutol resistant. However, 12 of 16 strains appear phenotypically sensitive at 5 g/mL but exhibit variable resistance between 2-4 g/mL. Of these 12 strains, a newly described frameshift mutation in regulator embR (Gln258fs) was found in 9 strains. ConclusionsMutations in embB406 are associated with low-level ethambutol resistance currently undetectable by the critical concentration of 5 g/mL for ethambutol. Novel mutations are predicted to exacerbate variability in ethambutol resistance. We suggest amendment to molecular and phenotypic drug susceptibility testing to improve ethambutol DST sensitivity and specificity as well as concordance between rapid and gold standard methods.
A recently described member of the Mycobacterium tuberculosis complex (MTBC) is Mycobacterium orygis, which can cause disease primarily in animals but also in humans. Although M. orygis has been reported from different geographic regions around the world, due to a lack of proper identification techniques, the contribution of this emerging pathogen to the global burden of zoonotic tuberculosis is not fully understood. In the present work, we report single nucleotide polymorphisms (SNPs) analysis using whole genome sequencing (WGS) that can accurately identify M. orygis and differentiate it from other members of MTBC species. WGS-based SNPs analysis was performed for 61 isolates from different Provinces in Canada that were identified as M. orygis. A total of 56 M. orygis sequences from the public databases were also included in the analysis. Several unique SNPs in gyrB, PPE55, Rv2042c, leuS, mmpL6, and mmpS6 genes were used to determine their effectiveness as genetic markers for the identification of M. orygis. To the best of our knowledge, five of these SNPs, viz., gyrB277 (A[->]G), gyrB1478 (T[->]C), leuS1064 (A[->]T), mmpL6486 (T[->]C), and mmpS6334 (C[->]G) are reported for the first time in this study. Our results also revealed several SNPs specific to other species within MTBC. The phylogenetic analysis shows that studied genomes were genetically diverse and clustered with M. orygis sequences of human and animal origin reported from different geographic locations. Therefore, the present study provides a new insight into the high confidence identification of M. orygis from MTBC species based on WGS data, which can be useful for reference and diagnostic laboratories.
The role of foot-and-mouth disease virus (FMDV) persistently infected ruminants in initiating new outbreaks remains controversial, and the perceived threat posed by such animals hinders international trade in FMD-endemic countries. In this study we report longitudinal analyses of genetic and antigenic variations of FMDV serotype O/ME-SA/Ind2001d sublineage during naturally occurring, persistent infection in cattle and buffalo at an organised dairy farm in India. The proportion of animals from which FMDV RNA was recovered was not significantly different between convalescent (post-clinical) and sub-clinically infected animals or between cattle and buffalo across the sampling period. However, infectious virus was isolated from a higher proportion of buffalo samples and for a longer duration compared to cattle. Analysis of the P1 sequences from recovered viruses indicated fixation of mutations at the rate of 1.816 x 10-2 substitution/site/year (s/s/y) (95% CI 1.362-2.31 x 10-2 s/s/y). However, the majority of point mutations were transitional substitutions. Within individual animals, the mean dN/dS ({omega}) value for the P1 region varied from 0.076 to 0.357, suggesting the selection pressure acting on viral genomes differed substantially across individual animals. Statistical parsimony analysis indicated that all of the virus isolates from carrier animals originated from the outbreak virus. The antigenic relationship value as determined by 2D-VNT assay revealed fluctuation of antigenic variants within and between carrier animals during the carrier state which suggested that some carrier viruses had diverged substantially from the protection provided by the vaccine strain. This study contributes to understanding the extent of within-host and within-herd evolution that occurs during the carrier state of FMDV.
Accurate breast region segmentation is an important step in various automated algorithms involving detection of lesions like masses and microcalcifications, and efficient telemammography. Existing segmentation algorithms underperform due to variations in image quality and shape of the breast region. In this paper, we propose to segment breast region by combining data-driven clustering with deformable image registration. In the first phase of the approach, we identify atlas images from a dataset of mammograms using data-driven clustering. Then, we segment these atlas images and use in the next phase of the algorithm. The second phase is atlas-based registration. For a candidate image, we find the most similar atlas image from the set of atlases identified in phase one. We deform the selected atlas image to match the given test image using the Demon's registration algorithm. Then, the segmentation mask of the deformed atlas is transferred to the mammogram in consideration. Finally, we refine the segmentation mask with some morphological operations in order to obtain accurate breast region boundary. We evaluated the performance of our method using ground-truth segmentation masks verified by an expert radiologist. We compared the proposed method with three existing state-of-the-art algorithms for breast region segmentation and the proposed approach outperformed all three in most of the cases.