Genome-wide RNA-seq, DNA methylation and small RNA-seq analysis unraveled complex gene regulatory networks in psoriasis pathogenesis.

Psoriasis is a complex inflammatory skin disease characterized by reversible albeit relapsing red scaly plaques in the skin of a patient. In addition to the genetic predisposition, involvement of epigenetic and non-coding RNAs have also been liked with the disease. Nevertheless, any comprehensive study involving transcriptomic, small-RNA and DNA methylation at the genomic level from same patients is lacking. To investigate the complex regulation of molecular pathways in psoriasis, we carried out multi-omics integrative analysis of RNA-sequencing, small RNA-sequencing and DNA methylation profiling from the psoriatic and adjacent normal skin tissues. Our multi-omics analysis identified the genes and biological processes regulated either independently or in combination by DNA methylation and microRNAs. We identified miRNAs that specifically regulated keratinocyte hyper-proliferation, and cell cycle progression and checkpoint signaling in psoriasis. On contrary, DNA methylation was found to be more predominant in regulating immune and inflammatory responses, another causative factor in psoriasis pathogenesis. Many characteristic pathways in psoriasis e.g., Th17 cell differentiation and JAK-STAT signaling, were found to be regulated by both miRNAs and DNA methylation. We carried out functional characterization of a downregulated miRNA hsa-let-7c-5p, predicted to target upregulated genes in psoriasis involved in cell cycle processes, Th17 cell differentiation and JAK-STAT signaling pathways. Overexpression of hsa-let-7c-5p in keratinocytes caused the downregulation of its target genes, resulting in reduced cell proliferation and migration rates, demonstrating potential of miRNAs in regulating psoriasis pathogenesis. In conclusion, our findings identified distinct and shared gene-networks regulated by DNA methylation and miRNAs of a complex disease with reversible phenotype.

An Observational Study to Find the Association of Viral Load, NS1 Antigen, IgG Antibody, and Other Laboratory Parameters With the Outcome of Dengue Patients in Eastern India.

Background Dengue, the mosquito-borne febrile disease caused by the dengue virus, has become one of the major concerns of public health. It may present with only fever, or there may be a hemorrhagic manifestation or septic shock. As there is no specific treatment for dengue, early detection of the disease, assessment of progression, and prediction of outcome by studying the laboratory markers will help guide the management of cases and lower morbidity and mortality. Methodology This clinico-observational study was conducted at the Department of Microbiology in a tertiary care hospital in Kolkata, India, from February 2020 to August 2022 to determine the outcome of dengue patients in correlation with viral load, NS1 antigen, IgM and IgG antibodies, ferritin level, platelet count, and other laboratory parameters. Results Out of 316 samples from fever patients, 103 (32.5%) were NS1 antigen reactive. We followed up the dengue patients (n = 103) for 15 days and divided them into three groups according to their duration of symptoms (group A suffered for ≤5 days, group B for 5 to 10 days, and group C for >10 days) and per the WHO classification of disease severity, namely dengue without warning signs (DOS), dengue with warning signs (DWS), and severe dengue (SD). Based on severity, 65 (63.1%) patients had DOS, whereas 31 (30.09%) patients had DWS, and seven (6.79%) patients had SD. Secondary infection was present in 83.33% of patients in group C, 71% of DWS cases, and 57% of SD cases, which positively correlates with liver enzymes, viral load (mean value 102195 in secondary infection vs. 1195 copies/10 µl in primary infection), and negatively correlates with platelet counts (mean value 60,213 in secondary infection vs. 1,25,516 in primary infection). Patients in group C had higher liver enzymes, a lower platelet count, and a higher initial viral load than groups A and B. Similarly, SD cases had a higher ferritin level (9215 ug/l), a lower platelet count (mean value 23,250), and a higher initial viral load (mean value 2,74,257 copies/10 µl). An increase in hematocrit value considering the peak value and its baseline value is an important marker for disease severity rather than its absolute value. Conclusion Poor outcome of dengue infection, i.e., an increase in the duration of symptoms and disease severity depends on concurrent associations between high serum ferritin, increased hematocrit level, thrombocytopenia, secondary infection, increasing liver enzymes, and increased initial viral load.

Identifying the genetic associations among the psoriasis patients in eastern India.

Psoriasis is a multifactorial genetic disorder manifested by hyperproliferation and abnormal differentiation of epidermal keratinocytes, along with the infiltration of inflammatory cells into the skin. Although ~80 genetic susceptibility variants were reported in psoriasis, many loci showed population-specific associations, warranting the need for more population-specific association studies in psoriasis. We determined the association of forty single nucleotide polymorphisms (SNPs) among 2136 psoriasis patients and normal individuals from eastern India. We investigated the expression of corresponding genes and evaluated the protein structure stability for the genes with susceptible coding variants. We found fifteen SNPs significantly associated with psoriasis, while additional three SNPs showed significant association when we classified the patients based on the presence of HLA-Cw6 allele. Epistatic interaction between HLA-Cw6 and other associated loci showed significant association with the SNPs at PSORS1 region, along with other five SNPs outside PSORS1. Three genes showed significant differential expression in psoriatic tissues compared to the adjacent normal skin tissues but were not differential when classified the patients based on their genotypes. SNP rs495337 at SPATA2 (Spermatogenesis Associated 2) showed a 1.2-fold increased risk among the HLA-Cw6 patients compared to combined samples. We found significant downregulation of SPATA2 among the patients with risk genotypes and HLA-Cw6 allele compared to the non-risk genotypes. Protein structure stability analysis showed reduced structural stability for all the mutant residues caused by the associated coding variants. Our study evaluated the genetic associations of psoriasis-susceptible variants in India and evaluated the possible functional significance of these associated variants in psoriasis.

Functional Mapping of Genetic Interactions between HLA-Cw6 and LCE3A in Psoriasis.

Functional studies to delineate the molecular mechanisms of causal genetic variants are the main focus in the post-GWAS era. Previous GWASs have identified >50 susceptibility loci associated with psoriasis. Functional understanding of the biology underlying the disease risk of most of these associated loci is unclear. In this study, we identified a regulatory SNP at the putative enhancer of the LCE3A gene within the epidermal differentiation complex that showed epistatic interaction with HLA-Cw6. The variant allele disrupted signal transducer and activator of transcription 3 binding to the region, thereby regulating the expression of the downstream LCE3A gene. Electrophoretic mobility shift and pulldown assay confirmed the preferential binding of signal transducer and activator of transcription 3 to the DNA with a wild-type allele compared with the DNA with a variant allele. The reporter assay further validated the IL-6‒stimulated phosphorylated signal transducer and activator of transcription 3‒mediated LCE3A activation in the presence of the wild-type allele. Interestingly, the presence of the HLA-Cw6 allele leads to IL-6‒mediated phosphorylation of signal transducer and activator of transcription 3, followed by its nuclear localization in the epidermal keratinocytes of psoriatic skin, suggesting indirect interaction of the HLA-Cw6 allele and a regulatory SNP upstream of the LCE3A gene. This study reflects an interesting approach to dissecting the molecular mechanism underlying the genetic interaction observed between HLA-Cw6 and LCE3A in psoriasis pathogenesis.

Dysbiosis of Oral Microbiota During Oral Squamous Cell Carcinoma Development.

Infection with specific pathogens and alterations in tissue commensal microbial composition are intricately associated with the development of many human cancers. Likewise, dysbiosis of oral microbiome was also shown to play critical role in the initiation as well as progression of oral cancer. However, there are no reports portraying changes in oral microbial community in the patients of Indian subcontinent, which has the highest incidence of oral cancer per year, globally. To establish the association of bacterial dysbiosis and oral squamous cell carcinoma (OSCC) among the Indian population, malignant lesions and anatomically matched adjacent normal tissues were obtained from fifty well-differentiated OSCC patients and analyzed using 16S rRNA V3-V4 amplicon based sequencing on the MiSeq platform. Interestingly, in contrast to the previous studies, a significantly lower bacterial diversity was observed in the malignant samples as compared to the normal counterpart. Overall our study identified Prevotella, Corynebacterium, Pseudomonas, Deinococcus and Noviherbaspirillum as significantly enriched genera, whereas genera including Actinomyces, Sutterella, Stenotrophomonas, Anoxybacillus, and Serratia were notably decreased in the OSCC lesions. Moreover, we demonstrated HPV-16 but not HPV-18 was significantly associated with the OSCC development. In future, with additional validation, this panel could directly be applied into clinical diagnostic and prognostic workflows for OSCC in Indian scenario.

Characterizations of SARS-CoV-2 mutational profile, spike protein stability and viral transmission.

The recent pandemic of SARS-CoV-2 infection has affected more than 3.0 million people worldwide with more than 200 thousand reported deaths. The SARS-CoV-2 genome has the capability of gaining rapid mutations as the virus spreads. Whole-genome sequencing data offers a wide range of opportunities to study mutation dynamics. The advantage of an increasing amount of whole-genome sequence data of SARS-CoV-2 intrigued us to explore the mutation profile across the genome, to check the genome diversity, and to investigate the implications of those mutations in protein stability and viral transmission. We have identified frequently mutated residues by aligning ~660 SARS-CoV-2 genomes and validated in 10,000 datasets available in GISAID Nextstrain. We further evaluated the potential of these frequently mutated residues in protein structure stability of spike glycoprotein and their possible functional consequences in other proteins. Among the 11 genes, surface glycoprotein, nucleocapsid, ORF1ab, and ORF8 showed frequent mutations, while envelop, membrane, ORF6, ORF7a and ORF7b showed conservation in terms of amino acid substitutions. Combined analysis with the frequently mutated residues identified 20 viral variants, among which 12 specific combinations comprised more than 97% of the isolates considered for the analysis. Some of the mutations across different proteins showed co-occurrences, suggesting their structural and/or functional interaction among different SARS-COV-2 proteins, and their involvement in adaptability and viral transmission. Analysis of protein structure stability of surface glycoprotein mutants indicated the viability of specific variants and are more prone to be temporally and spatially distributed across the globe. A similar empirical analysis of other proteins indicated the existence of important functional implications of several variants. Identification of frequently mutated variants among COVID-19 patients might be useful for better clinical management, contact tracing, and containment of the disease.

Genome-wide regulatory gene-derived SSRs reveal genetic differentiation and population structure in fiber flax genotypes.

We designed a set of 580 simple sequence repeat markers; 506 from transcription factor-coding genes, and 74 from long non-coding RNAs and designated them as regulatory gene-derived simple sequence repeat (ReG-SSR) markers. From this set, we could anchor 559 ReG-SSR markers on 15 flax chromosomes with an average marker distance of 0.56 Mb. Thirty-one polymorphic ReG-SSR primers, amplifying SSR loci length of at least 20 bp were chosen from 134 screened primers. This primer set was used to characterize a diversity panel of 93 flax accessions. The panel included 33 accessions from India, including released varieties, dual-purpose lines and landraces, and 60 fiber flax accessions from the global core collection. Thirty-one ReG-SSR markers generated 76 alleles, with an average of 2.5 alleles per primer and a mean allele frequency of 0.77. These markers recorded 0.32 average gene diversity, 0.26 polymorphism information content and 1.35% null alleles. All the 31 ReG-SSR loci were found selectively neutral and showed no evidence of population reduction. A model-based clustering analysis separated the flax accessions into two sub-populations-Indian and global, with some accessions showing admixtures. The distinct clustering pattern of the Indian accessions compared to the global accessions, conforms to the principal coordinate analysis, genetic dissimilarity-based unweighted neighbor-joining tree and analysis of molecular variance. Fourteen flax accessions with 99.3% allelic richness were found optimum to adopt in breeding programs. In summary, the genome-wide ReG-SSR markers will serve as a functional marker resource for genetic and phenotypic relationship studies, marker-assisted selections, and provide a basis for selection of accessions from the Indian and global gene pool in fiber flax breeding programs.