Genomic epidemiology of SARS-CoV-2 from Uttar Pradesh, India.

The various strains and mutations of SARS-CoV-2 have been tracked using several forms of genomic classification systems. The present study reports high-throughput sequencing and analysis of 99 SARS-CoV-2 specimens from Western Uttar Pradesh using sequences obtained from the GISAID database, followed by phylogeny and clade classification. Phylogenetic analysis revealed that Omicron lineages BA-2-like (55.55%) followed by Delta lineage-B.1.617.2 (45.5%) were predominantly circulating in this area Signature substitution at positions S: N501Y, S: D614G, S: T478K, S: K417N, S: E484A, S: P681H, and S: S477N were commonly detected in the Omicron variant-BA-2-like, however S: D614G, S: L452R, S: P681R and S: D950N were confined to Delta variant-B.1.617.2. We have also identified three escape variants in the S gene at codon position 19 (T19I/R), 484 (E484A/Q), and 681 (P681R/H) during the fourth and fifth waves in India. Based on the phylogenetic diversification studies and similar changes in other lineages, our analysis revealed indications of convergent evolution as the virus adjusts to the shifting immunological profile of its human host. To the best of our knowledge, this study is an approach to comprehensively map the circulating SARS-CoV-2 strains from Western Uttar Pradesh using an integrated approach of whole genome sequencing and phylogenetic analysis. These findings will be extremely valuable in developing a structured approach toward pandemic preparedness and evidence-based intervention plans in the future.

Prioritization and Meta-analysis of regulatory SNPs identified IL6, TGFB1, TLR9 and MMP7 as significantly associated with cervical cancer.

Cervical cancer is a leading women cancer globally with respect to both incidence and mortality. Its increased risk has been linked with HPV infection and genetic variations like single nucleotide polymorphisms (SNPs). Although, studies have been published which evaluates the effect of SNPs in a few candidate genes, however the role of number of regulatory SNPs (rSNPs) in cervical cancer is not available. As literature evidence has shown that non-coding rSNPs are related with increasing cervical cancer risk, we undertook this study to prioritize the important rSNPs and elucidate their role. A search was conducted in PubMed up to December 2020, which led to the identification of 263 articles and 969 SNPs in the non-coding region. These 969 SNPs were analysed through rSNPBase and RegulomeDB, leading to identification of 105 rSNPs. Afterwards, a regulatory module was constructed using protein-protein interaction data and a hub of highly interacting 23 target genes (corresponding to 34 rSNPs) was identified using MCODE. To further understand the mechanism of action of the 34 rSNPs, their transcription factor information with respect to cervical cancer was retrieved. To evaluate the pooled effect of these prioritized polymorphisms in cervical cancer patients, a meta-analysis was performed on 10,537 cases and 11,252 controls from 30 studies corresponding to 8 rSNPs. It led to identification of polymorphisms in IL6 (rs2069837), TGFB1 (rs1800469), TLR9 (rs187084) and MMP7 (rs11568818) which are significantly (p < 0.05) associated with increased cervical cancer risk at the population level. Overall, the study demonstrates that rSNPs targeting immune and inflammatory genes (IL1B, IL6, IL10, IL18, TGFB1, CCR5, CD40, TLR9, and MMP7) are associated with cervical cancer.

Enhanced Detection of Desmoplasia by Targeted Delivery of Iron Oxide Nanoparticles to the Tumour-Specific Extracellular Matrix.

Diagnostic imaging of aggressive cancer with a high stroma content may benefit from the use of imaging contrast agents targeted with peptides that have high binding affinity to the extracellular matrix (ECM). In this study, we report the use of superparamagnetic iron-oxide nanoparticles (IO-NP) conjugated to a nonapeptide, CSGRRSSKC (CSG), which specifically binds to the laminin-nidogen-1 complex in tumours. We show that CSG-IO-NP accumulate in tumours, predominantly in the tumour ECM, following intravenous injection into a murine model of pancreatic neuroendocrine tumour (PNET). In contrast, a control untargeted IO-NP consistently show poor tumour uptake, and IO-NP conjugated to a pentapeptide. CREKA that bind fibrin clots in blood vessels show restricted uptake in the angiogenic vessels of the tumours. CSG-IO-NP show three-fold higher intratumoral accumulation compared to CREKA-IO-NP. Magnetic resonance imaging (MRI) T2-weighted scans and T2 relaxation times indicate significant uptake of CSG-IO-NP irrespective of tumour size, whereas the uptake of CREKA-IO-NP is only consistent in small tumours of less than 3 mm in diameter. Larger tumours with significantly reduced tumour blood vessels show a lack of CREKA-IO-NP uptake. Our data suggest CSG-IO-NP are particularly useful for detecting stroma in early and advanced solid tumours.

In Silico Analysis of HSP70 Gene Family in Bovine Genome.

Heat shock proteins (HSPs), members of molecular chaperones families fulfill essential roles under normal conditions and provide protection and adaptation during and after stress. Among different HSPs, HSP70 kDa family of proteins is most abundant and well-studied in human and mouse but has not yet been characterized in bovines. In silico analysis was performed to characterize members of HSP70 gene family in bovine genome and a total of 17 genes of bovine HSP70 gene family were identified. The members of HSP70 family were distributed over 12 chromosomes with gene size ranging from 1911 (HSPA2) to 54,017 bp (HSPA4). Five genes were intronless, while rest of 12 genes were multiexonic. Phylogenetic analysis of HSP70 gene family distinguished them into eight major evolutionary groups wherein members of group 1 were most divergent and quite dissimilar than from rest of the HSP70 sequences. Domain structure of all bovine HSP70 genes was conserved and three signature patterns HSP70_1, HSP70_2, and HSP70_3 were identified. HSPA8, HSP9, and HSPA1A showed comparatively higher expression in majority of tissues. Like humans, bovine HSP70 family was characterized by remarkable evolutionary diversity. The analysis also suggested resemblance of bovine HSP70 family to that of human compared to mouse. Overall, the study indicates the presence of diversity for structure, function, localization, and expression in the bovine HSP70 family chaperons which could form the basis to understand thermotolerance/adaptive changes in the bovines.

Immune-mediated ECM depletion improves tumour perfusion and payload delivery.

High extracellular matrix (ECM) content in solid cancers impairs tumour perfusion and thus access of imaging and therapeutic agents. We have devised a new approach to degrade tumour ECM, which improves uptake of circulating compounds. We target the immune-modulating cytokine, tumour necrosis factor alpha (TNFα), to tumours using a newly discovered peptide ligand referred to as CSG. This peptide binds to laminin-nidogen complexes in the ECM of mouse and human carcinomas with little or no peptide detected in normal tissues, and it selectively delivers a recombinant TNFα-CSG fusion protein to tumour ECM in tumour-bearing mice. Intravenously injected TNFα-CSG triggered robust immune cell infiltration in mouse tumours, particularly in the ECM-rich zones. The immune cell influx was accompanied by extensive ECM degradation, reduction in tumour stiffness, dilation of tumour blood vessels, improved perfusion and greater intratumoral uptake of the contrast agents gadoteridol and iron oxide nanoparticles. Suppressed tumour growth and prolonged survival of tumour-bearing mice were observed. These effects were attainable without the usually severe toxic side effects of TNFα.

Biocompatibility and Targeting Efficiency of Encapsulated Quinapyramine Sulfate-Loaded Chitosan-Mannitol Nanoparticles in a Rabbit Model of Surra.

Quinapyramine sulfate (QS) produces trypanocidal effects against the parasite Trypanosoma evansi but is often poorly tolerated and causes serious reactions in animals. The encapsulation of QS in chitosan-mannitol to provide sustained release would improve both the therapeutic effect of QS and the quality of life of animals treated with this formulation. QS was encapsulated into a nanoformulation prepared from chitosan, tripolyphosphate, and mannitol nanomatrix (ChQS-NPs). ChQS-NPs were well ordered in shape, with nanoparticle size, as determined by transmission electron microscopy and atomic force microscopy. Our research revealed dose-dependent effects on biosafety and DNA damage in mammalian cells treated with ChQS-NPs. ChQS-NPs were absolutely risk-free at effective as well as many times higher doses against T. evansi ChQS-NPs were effective in rabbits, as they killed the parasites, relieving the animals from the clinical symptoms of the disease. The extent of this protection was similar to that observed with the conventional drug at higher dosages (5 mg QS/kg of body weight). ChQS-NPs are safe, nontoxic, and more effective than QS and offer a promising alternative to drug delivery against surra in animal models. ChQS-NPs may be useful for the treatment of surra due to reduced dosages and frequency of administration.

Author Correction: Transcriptomic signature of drought response in pearl millet (Pennisetum glaucum (L.) and development of web-genomic resources.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Transcriptomic signature of drought response in pearl millet (Pennisetum glaucum (L.) and development of web-genomic resources.

Pearl millet, (Pennisetum glaucum L.), an efficient (C4) crop of arid/semi-arid regions is known for hardiness. Crop is valuable for bio-fortification combating malnutrition and diabetes, higher caloric value and wider climatic resilience. Limited studies are done in pot-based experiments for drought response at gene-expression level, but field-based experiment mimicking drought by withdrawal of irrigation is still warranted. We report de novo assembly-based transcriptomic signature of drought response induced by irrigation withdrawal in pearl millet. We found 19983 differentially expressed genes, 7595 transcription factors, gene regulatory network having 45 hub genes controlling drought response. We report 34652 putative markers (4192 simple sequence repeats, 12111 SNPs and 6249 InDels). Study reveals role of purine and tryptophan metabolism in ABA accumulation mediating abiotic response in which MAPK acts as major intracellular signal sensing drought. Results were validated by qPCR of 13 randomly selected genes. We report the first web-based genomic resource ( http://webtom.cabgrid.res.in/pmdtdb/ ) which can be used for candidate genes-based SNP discovery programs and trait-based association studies. Looking at climatic change, nutritional and pharmaceutical importance of this crop, present investigation has immense value in understanding drought response in field condition. This is important in germplasm management and improvement in endeavour of pearl millet productivity.

Sodium alginate and gum acacia hydrogels of ZnO nanoparticles show wound healing effect on fibroblast cells.

An ideal biomaterial for wound dressing applications should possess antibacterial and anti-inflammatory properties without any toxicity to the host cells while providing the maximum healing activity. Zinc oxide nanoparticles (ZnONPs) possess antimicrobial activity and enhance wound healing, but the questions regarding their safety arise before application to the biological systems. We synthesized ZnONPs-loaded-sodium alginate-gum acacia hydrogels (SAGA-ZnONPs) by cross linking hydroxyl groups of the polymers sodium alginate and gum acacia with the aldehyde group of gluteradehyde. Here, we report the wound healing properties of sodium alginate/gum acacia/ZnONPs, circumventing the toxicity of ZnONPs simultaneously. We demonstrated the concentration-dependent zones of inhibition in treated cultures of Pseudomonas aerigunosa and Bacillus cereus and biocompatability on peripheral blood mononuclear/fibroblast cells. SAGA-ZnONPs hydrogels showed a healing effect at a low concentration of ZnONPs using sheep fibroblast cells. Our findings suggest that high concentrations of ZnONPs were toxic to cells but SAGA-ZnONPs hydrogels significantly reduced the toxicity and preserved the beneficial antibacterial and healing effect.

Synthesis and evaluation of isometamidium-alginate nanoparticles on equine mononuclear and red blood cells.

Isometamidium hydrochloride (ISMM) is an effective drug for the treatment of trypanosomosis, but it causes local and systemic toxicity. Isometamidium hydrochloride has limited therapeutic index and exhibit considerable variation in their prophylactic activities. We developed a trypanocidal nanoformulation using ISMM and polymers sodium alginate/gum acacia to enhance the efficacy of the drug at lower doses, while minimizing undesirable side effects. It was characterized by transmission electron microscopy and infrared spectroscopy for evaluation of size, morphology, functional groups, etc. In vitro cytotoxicity studies were performed by metabolic resazurin assay at different concentrations of isometamidium-loaded alginate/gum acacia nanoparticles using equine peripheral blood mononuclear cells. Hemolytic assay revealed significantly less toxicity compared to the conventional drug. The results demonstrate that the developed drug delivery module can be evaluated in suitable animal models to evaluate its potency.

Cytotoxicity and genotoxicity of a trypanocidal drug quinapyramine sulfate loaded-sodium alginate nanoparticles in mammalian cells.

We synthesized quinapyramine sulfate loaded-sodium alginate nanoparticles (QS-NPs) to reduce undesirable toxic effects of QS against the parasite Trypanosoma evansi, a causative agent of trypanosomosis. To determine the safety of the formulated nanoparticles, biocompatibility of QS-NPs was determined using Vero, Hela cell lines and horse erythrocytes in a dose-dependent manner. Our experiments unveiled a concentration-dependent safety/cytotoxicity (metabolic activity), genotoxicity (DNA damage, chromosomal aberrations), production of reactive oxygen species and hemolysis in QS-NPs treated cells. Annexin-V propidium iodide (PI) staining showed no massive apoptosis or necrosis. However, at very high doses (more than 300 times than the effective doses), we observed more toxicity in QS-NPs treated cells as compared to QS treated cells. QS-NPs were safe at effective trypanocidal doses and even at doses several times higher than the effective dose.

Diversity, Antimicrobial Action and Structure-Activity Relationship of Buffalo Cathelicidins.

Cathelicidins are an ancient class of antimicrobial peptides (AMPs) with broad spectrum bactericidal activities. In this study, we investigated the diversity and biological activity of cathelicidins of buffalo, a species known for its disease resistance. A series of new homologs of cathelicidin4 (CATHL4), which were structurally diverse in their antimicrobial domain, was identified in buffalo. AMPs of newly identified buffalo CATHL4s (buCATHL4s) displayed potent antimicrobial activity against selected Gram positive (G+) and Gram negative (G-) bacteria. These peptides were prompt to disrupt the membrane integrity of bacteria and induced specific changes such as blebing, budding, and pore like structure formation on bacterial membrane. The peptides assumed different secondary structure conformations in aqueous and membrane-mimicking environments. Simulation studies suggested that the amphipathic design of buCATHL4 was crucial for water permeation following membrane disruption. A great diversity, broad-spectrum antimicrobial action, and ability to induce an inflammatory response indicated the pleiotropic role of cathelicidins in innate immunity of buffalo. This study suggests short buffalo cathelicidin peptides with potent bactericidal properties and low cytotoxicity have potential translational applications for the development of novel antibiotics and antimicrobial peptidomimetics.

Comparative genomic analysis of buffalo (Bubalus bubalis) NOD1 and NOD2 receptors and their functional role in in-vitro cellular immune response.

Nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs) are innate immune receptors that recognize bacterial cell wall components and initiate host immune response. Structure and function of NLRs have been well studied in human and mice, but little information exists on genetic composition and role of these receptors in innate immune system of water buffalo--a species known for its exceptional disease resistance. Here, a comparative study on the functional domains of NOD1 and NOD2 was performed across different species. The NOD mediated in-vitro cellular responses were studied in buffalo peripheral blood mononuclear cells, resident macrophages, mammary epithelial, and fibroblast cells. Buffalo NOD1 (buNOD1) and buNOD2 showed conserved domain architectures as found in other mammals. The domains of buNOD1 and buNOD2 showed analogy in secondary and tertiary conformations. Constitutive expressions of NODs were ubiquitous in different tissues. Following treatment with NOD agonists, peripheral lymphocytes showed an IFN-γ response along-with production of pro-inflammatory cytokines. Alveolar macrophages and mammary epithelial cells showed NOD mediated in-vitro immune response through NF-κB dependent pathway. Fibroblasts showed pro-inflammatory cytokine response following agonist treatment. Our study demonstrates that both immune and non-immune cells could generate NOD-mediated responses to pathogens though the type and magnitude of response depend on the cell types. The structural basis of ligand recognition by buffalo NODs and knowledge of immune response by different cell types could be useful for development of non-infective innate immune modulators and next generation anti-inflammatory compounds.

Splice variants and seasonal expression of buffalo HSF genes.

In eukaryotes, the heat shock factors (HSFs) are recognized as the master regulator of the heat shock response. In this respect, the genes encoding the heat shock factors seem to be important for adaptation to thermal stress in organisms. Despite this, only few mammalian HSFs has been characterized. In this study, four major heat shock factor genes viz. HSF-1, 2, 4, and 5 were studied. The main objective of the present study was to characterize the cDNA encoding using conserved gene specific primers and to investigate the expression status of these buffalo HSF genes. Our RT-PCR analysis uncovered two distinct variants of buffalo HSF-1 and HSF-2 gene transcripts. In addition, we identified a variant of the HSF5 transcript in buffalo lacking a DNA-binding domain. In silico analysis of deduced amino acid sequences for buffalo HSF genes showed domain architecture similar to other mammalian species. Changes in the gene expression profile were noted by quantitative real-time PCR (qRT-PCR) analysis. We detected the transcript of buffalo HSF genes in different tissues. We also evaluated the seasonal changes in the expression of HSF genes. Interestingly, the transcript level of HSF-1 gene was found upregulated in months of high and low ambient temperatures. In contrast, the expression of the HSF-4 and 5 genes was found to be downregulated in months of high ambient temperature. This suggests that the intricate balance of different HSFs is adjusted to minimize the effect of seasonal changes in environmental conditions. These findings advance our understanding of the complex, context-dependent regulation of HSF gene expression under normal and stressful conditions.

Alginate/gum acacia bipolymeric nanohydrogels--promising carrier for zinc oxide nanoparticles.

Zinc oxide nanoparticles (ZnO nps) are known to be effective against a wide array of microorganisms. At nanoscale, they have higher toxicity and they need to be rendered less toxic and more biocompatible. To achieve this, ZnO nps were incorporated in nanohydrogel particles made out of sodium alginate/gum acacia and cross-linker glutaraldehyde in order to ensure their gradual and sustained release instead of burst release, and hence lowering their toxicity. The particles synthesized were in the nano-range, i.e., 70-100 nm size and their in vitro release studies indicated that release of upto 68% of ZnO nps was prolonged to over 2 weeks following the Higuchi model. Cytotoxicity studies on vero cell line (African green monkey kidney cell line) revealed that toxicity of ZnO nps-loaded nanohydrogels was substantially lower as compared to ZnO nps. At the same time, it demonstrated desired level of antibiotic activity against Pseudomonas aeruginosa, an antibiotic resistant microbial model. In conclusion, this work led to successful preparation of novel formulation of ZnO incorporated in nanohydrogels that are not only safer but also retain adequate antibacterial activity due to their ability for gradual and sustained release of the active constituent.

Quinapyramine sulfate-loaded sodium alginate nanoparticles show enhanced trypanocidal activity.

AIM: To reduce the dose, toxic effects and to ensure sustained release of quinapyramine sulfate (QS), a highly effective drug against Trypanosoma evansi. MATERIALS & METHODS: QS-loaded sodium alginate nanoparticles (QS-NPs) were formed by emulsion-crosslinking technology using dioctyl-sodium-sulfosuccinate and sodium alginate. The formulation was characterized for size, stability, morphology and functional groups by a zetasizer, scanning electron microscopy, atomic force microscopy, transmission electron microscopy and Fourier transform infrared spectroscopy. In vitro safety and toxicity studies were performed by metabolic assay in Vero cell lines, and in vivo efficacy was evaluated in mice. RESULTS: QS-NPs were <60 nm with 96.48% entrapment efficiency and 3.70% drug loading. The formulation showed an initial burst effect followed by slow drug release in accordance with quasi-Fickian Higuchi diffusion mechanism. QS-NPs were much less toxic and able to clear the parasite at a much lower concentration than QS. CONCLUSION: The QS-NPs synthesized are safe, less toxic and highly effective compared with QS.