Direct massively parallel sequencing of SARS-CoV-2 genome was undertaken from nasopharyngeal and oropharyngeal swab samples of infected individuals in Eastern India. Seven of the isolates belonged to ...the A2a clade, while one belonged to the B4 clade. Specific mutations, characteristic of the A2a clade, were also detected, which included the P323L in RNA-dependent RNA polymerase and D614G in the Spike glycoprotein. Further, our data revealed emergence of novel subclones harbouring nonsynonymous mutations, viz. G1124V in Spike (S) protein, R203K, and G204R in the nucleocapsid (N) protein. The N protein mutations reside in the SR-rich region involved in viral capsid formation and the S protein mutation is in the S
2
domain, which is involved in triggering viral fusion with the host cell membrane. Interesting correlation was observed between these mutations and travel or contact history of COVID-19 positive cases. Consequent alterations of miRNA binding and structure were also predicted for these mutations. More importantly, the possible implications of mutation D614G (in S
D
domain) and G1124V (in S
2
subunit) on the structural stability of S protein have also been discussed. Results report for the first time a bird’s eye view on the accumulation of mutations in SARS-CoV-2 genome in Eastern India.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Utrophin, the autosomal homologue of dystrophin can functionally compensate for dystrophin deficiency. Utrophin upregulation could therefore be a therapeutic strategy in Duchenne Muscular Dystrophy ...(DMD) that arises from mutation in dystrophin gene. In contrast to its transcriptional regulation, mechanisms operating at post-transcriptional level of utrophin expression have not been well documented. Although utrophin-A 5'-UTR has been reported with internal ribosome entry site (IRES), its inhibitory effect on translation is also evident. In the present study we therefore aimed to compare relative contribution of cap-independent and cap-dependent translation with mouse utrophin-A 5'-UTR through m7G-capped and A-capped mRNA transfection based reporter assay. Our results demonstrate that cap-independent translation with utrophin-A 5'-UTR is not as strong as viral IRES. However, cap-independent mode has significant contribution as cap-dependent translation is severely repressed with utrophin-A 5'-UTR. We further identified two sequence elements and one upstream open reading frame in utrophin-A 5'-UTR responsible for repression. The repressor elements in utrophin-A 5'-UTR may be targeted for utrophin upregulation.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The evolution of viral variants and their impact on viral transmission have been an area of considerable importance in this pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ...We analyzed the viral variants in different phases of the pandemic in West Bengal, a state in India that is important geographically, and compared the variants with other states like Delhi, Maharashtra, and Karnataka, located in other regions of the country. We have identified 57 pango-lineages in 3,198 SARS-CoV-2 genomes, alteration in their distribution, as well as contrasting profiles of amino acid mutational dynamics across different waves in different states. The evolving characteristics of Delta (B.1.617.2) sublineages and alterations in hydrophobicity profiles of the viral proteins caused by these mutations were also studied. Additionally, implications of predictive host miRNA binding/unbinding to emerging spike or nucleocapsid mutations were highlighted. Our results throw considerable light on interesting aspects of the viral genomic variation and provide valuable information for improved understanding of wave-defining mutations in unfolding the pandemic. IMPORTANCE Multiple waves of infection were observed in many states in India during the coronavirus disease 2019 (COVID19) pandemic. Fine-scale evolution of major SARS-CoV-2 lineages and sublineages during four wave-window categories: Pre-Wave 1, Wave 1, Pre-Wave 2, and Wave 2 in four major states of India: Delhi (North), Maharashtra (West), Karnataka (South), and West Bengal (East) was studied using large-scale virus genome sequencing data. Our comprehensive analysis reveals contrasting molecular profiles of the wave-defining mutations and their implications in host miRNA binding/unbinding of the lineages in the major states of India.
Commercially available reagents and published protocols are widely used for RNA isolation. However, genomic DNA contamination in isolated RNA is a potential problem. Here we describe a simple, ...inexpensive method for eliminating genomic DNA contamination beyond the level of PCR-based detection through reduction of the guanidine thiocyanate concentration (1.5M) in a single monophasic solution based on Chomczynski–Sacchi reagents. The new method can be used to isolate small and large RNA species of high quality and can be completed within an hour.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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•Molecular size and surface charge specific sericin nanoparticles were prepared.•Sericin nanoparticles (SNPs) were highly stable and non-cytotoxic to human cells.•Positively charged ...SNPs from 30−50 kDa sericin showed highest antibacterial activity.•ROS generation and membrane damage are responsible for antibacterial activity.
Investigating the role of molecular size and interfacial potential dependent antimicrobial propensity of nanoparticles (NPs) against bacteria is the important goal for secure usage of NPs to any living systems. In this study, crude silk sericin protein of Antheraea mylitta cocoon was fractionated into three different molecular size-ranges fractions such as fraction-1 (50−300 kDa), fraction-2 (30−50 kDa) and fraction-3 (10−30 kDa), and used to prepare crude sericin nanoparticles (CRSNPs), as well as fraction specific negative surface potential nanoparticles : n-SNP1, n-SNP2 and n-SNP3, respectively. SNPs were coated with poly-l-lysine to make the surface potential positive (p-SNPs) and confirmed through UV–vis spectroscopy, FTIR, zeta sizer and zeta potential measurement. The shape and sizes of all SNPs were determined by electron microscopy and found spherical in shape having diameter ranging from 110−165 nm (CRSNPs), 66−85 nm (SNP1), 33−49 nm (SNP2) and 14−24 nm (SNP3) for n-SNPs and p-SNPs, respectively. Evaluation of antibacterial activity using different concentrations (50, 100, 200 μg/mL) of all these SNPs showed significantly more activity of p-SNPs than n-SNPs against Staphylococcus aureus and Escherichia coli. Among these, SNP2 showed the strongest antibacterial activity followed by SNP3, SNP1 and CRSNPs. Relatively higher amounts of reactive oxygen species (ROS) generation were observed after treatment of bacteria with p-SNP2 (50 μg/mL) which is non-toxic to human cells. FE-SEM analysis showed more disruption of bacterial cell membrane after treatment with p-SNPs than n-SNPs. All these data suggested that molecular size and interfacial potential of SNPs enhance ROS generation to exert their antibacterial activity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Antheraea mylitta, a tropical non-mulberry silkworm, is cultivated for tasar silk production in India. Several defense molecules including few antimicrobial peptides (AMPs) and proteins have been ...identified from this insect. Here, we have isolated and purified an antimicrobial tri-peptide by sequential chromatographic separation procedures. The amino acid sequence of the peptide was determined as NH2-Gln-Ala-Lys-COOH (QAK) using MALDI MS/MS fragmentation analysis. Further, the peptide was synthesized in vitro following solid phase chemistry of peptide synthesis and acetylated by acetic anhydride reaction. Antimicrobial activities of non-acetylated and acetylated QAK were tested against both Escherichia coli and Staphylococcus aureus bacteria. Acetylated peptide inhibited bacterial growth more effectively and its minimum inhibitory concentration (MICs) was found lower than non-acetylated peptide. SEM studies revealed more membrane damage and release of intracellular materials like β-galactosidase enzyme from acetylated peptide treated bacteria in comparison to non-acetylated QAK. At MIC, acetylated peptide did not show any significant hemolytic activity against rabbit erythrocytes. The results suggest that acetylated-QAK is a promising new antimicrobial peptide and can be used for therapeutic purpose.
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•Purification of an antimicrobial peptide from hemolymph of A. mylitta.•Sequence determination as Q-A-K and chemical synthesis of this peptide.•QAK was acetylated and acetylated form showed higher antibacterial activity.•No significant hemolytic activity was observed for the peptide.•Peptide disrupts bacterial membrane and releases intracellular materials.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Antimicrobial proteins (AMPs) are small, cationic proteins that exhibit activity against bacteria, viruses, parasites, fungi as well as boost host‐specific innate immune responses. Insects produce ...these AMPs in the fat body and hemocytes, and release them into the hemolymph upon microbial infection. Hemolymph was collected from the bacterially immunized fifth instar larvae of tasar silkworm, Antheraea mylitta, and an AMP was purified by organic solvent extraction followed by size exclusion and reverse‐phase high‐pressure liquid chromatography. The purity of AMP was confirmed by thin‐layer chromatography and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis. The molecular mass was determined by matrix‐assisted laser desorption ionization–time of flight mass spectrometry as 14 kDa, and hence designated as AmAMP14. Peptide mass fingerprinting of trypsin‐digested AmAMP14 followed by de novo sequencing of one peptide fragment by tandem mass spectrometry analysis revealed the amino acid sequences as CTSPKQCLPPCK. No homology was found in the database search and indicates it as a novel AMP. The minimum inhibitory concentration of the purified AmAMP14 was determined against Escherichia coli, Staphylococcus aureus, and Candida albicans as 30, 60, and 30 µg/ml, respectively. Electron microscopic examination of the AmAMP14‐treated cells revealed membrane damage and release of cytoplasmic contents. All these results suggest the production of a novel 14 kDa AMP in the hemolymph of A. mylitta to provide defense against microbial infection.
Purification of one antimicrobial protein from the bacterially immunized Antheraea mylitta larva and analysis of its antimicrobial activity. (a) Escherichia coli, (b) Staphylococcus aureus, and (c) Candida albicans.
Highlights
Purification of a proline‐rich 14‐kDa antimicrobial protein from the hemolymph of Antheraea mylitta.
It inhibits growth of Escherichia coli, Staphylococcus aureus, and Candida albicans by disrupting membrane and releasing intracellular material.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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