Evaluation of therapeutic potential of recombinant buffalo lactoferrin N-lobe expressed in E. coli.

Lactoferrin (Lf) is a multifunctional bi-lobate iron-binding glycoprotein belonging to transferrin family with a mass of approximately 80 kD. Being ubiquitously present in almost all biological secretions, it performs important biological functions. One of the earliest and very well-documented functions of Lf is the antibacterial effect against broad spectrum Gram-negative and Gram-positive bacteria. In this study, buffalo Lf N-lobe cDNA was amplified, cloned and expressed as a fusion protein in Escherichia coli cells using pQE30 expression vector. After post-induction confirmation of expressed protein by SDS-PAGE, purification of recombinant protein using Ni-NTA was attempted and the yield of recombinant buffalo N-lobe Lf was estimated to be 1 mg/ml. Antibacterial activity of recombinant buffalo Lf N-lobe was assessed on pathogenic E. coli and Staphylococcus aureus strains. Peptic digest of recombinant N-lobe buffalo Lf showed antibacterial activity comparable to commercially available bovine Lf. The successful expression and characterization of functional recombinant N-lobe of buffalo Lf expressed in E. coli opens new vistas for developing alternate therapeutics, particularly against the diseases caused by Gram-negative microbes such as septicemia and diarrhea in newborn calves and mastitis in dairy animals.

RUNX1 induces DNA replication independent active DNA demethylation at SPI1 regulatory regions.

BACKGROUND: SPI1 is an essential transcription factor (TF) for the hematopoietic lineage, in which its expression is tightly controlled through a -17-kb upstream regulatory region and a promoter region. Both regulatory regions are demethylated during hematopoietic development, although how the change of DNA methylation status is performed is still unknown. RESULTS: We found that the ectopic overexpression of RUNX1 (another key TF in hematopoiesis) in HEK-293T cells induces almost complete DNA demethylation at the -17-kb upstream regulatory region and partial but significant DNA demethylation at the proximal promoter region. This DNA demethylation occurred in mitomycin-C-treated nonproliferating cells at both regulatory regions, suggesting active DNA demethylation. Furthermore, ectopic RUNX1 expression induced significant endogenous SPI1 expression, although its expression level was much lower than that of natively SPI1-expressing monocyte cells. CONCLUSIONS: These results suggest the novel role of RUNX1 as an inducer of DNA demethylation at the SPI1 regulatory regions, although the mechanism of RUNX1-induced DNA demethylation remains to be explored.

Identification of polymorphism in fatty acid binding protein 3 (FABP3) gene and its association with milk fat traits in riverine buffalo (Bubalus bubalis).

The fatty acid binding protein 3 (FABP3) gene, known to be associated with fat percentage of milk and meat in bovines, was screened among swamp and riverine buffaloes for polymorphism detection and further association with milk fat contents. An SNP g.307C > T was identified in the intron 2 (+53 exon 2) region of FABP3 gene of Indian buffaloes. The SNP identified was genotyped in 692 animals belonging to 15 riverine, swamp and hybrid (riverine × swamp) buffalo populations of diverse phenotypes and utilities, by PCR-RFLP. A marked contrast was observed between the C and T allele frequencies in three types of buffaloes. The frequency of C allele ranged from 0.67 to 0.96 in pure swamp buffalo populations, with the highest in Mizoram (0.96). Whereas the frequency of T allele was high across all the Indian riverine buffalo breeds, ranging from 0.57 to 0.96. None of the genotypes at FABP3 g.307C > T locus was found to have significant association with milk fat and other production traits in Mehsana dairy buffalo breed. Our study revealed marked differences in the allele frequencies between riverine and swamp buffaloes at FABP3 g.307C > T locus, without any significant association with different milk traits in riverine buffaloes.

Ascertaining variations in the activity of larval midgut enzymes of Helicoverpa armigera by dietary emamectin benzoate through biochemical and in silico docking study.

Helicoverpa armigera, a ubiquitous polyphagous pest, poses a significant threat to global agriculture, causing substantial economic losses and demonstrating resistance to synthetic pesticides. This study investigates the potential of emamectin benzoate (EMB), an avermectin derivative, as an effective control agent against H. armigera. The larvae of the NBII-MP-NOC-01 strain of H. armigera were reared on an artificial diet. The impact of dietary EMB was examined on four midgut enzymes; alanine aminotransferase (ALT), aspartate aminotransferase (AST), acid phosphatase (ACP), and alkaline phosphatase (ALP). Results showed a dose-dependent and time-dependent reduction in ALT and AST activity, while an initial increase and subsequent decline in ACP and ALP activity at higher EMB concentrations. Computational modelling of enzyme structures and molecular docking studies revealed differential binding of EMB with the midgut enzymes. The strongest interaction was observed between EMB and ALT residues, contrasting with weakest interactions observed with AST. The study also showed that decreased activity of transaminases in H. armigera caused by EMB may be because of stability-activity trade-off, while in phosphatases reverse may be the case. This research provides crucial insights into the biochemical responses and the intricate insecticide-enzyme interactions in H. armigera caused by EMB exposure. This study lays the foundation for further research aimed at developing environmentally friendly approaches for managing H. armigera, addressing the challenges associated with conventional pesticides.

Sequence and topological characterization of Toll-like receptor 8 gene of Indian riverine buffalo (Bubalus bubalis).

In this study, buffalo (Bubalus bubalis) Toll-like receptor 8 (TLR8) gene has been characterized by sequence analysis and detecting polymorphism. Complete ORF of buffalo TLR8 gene was amplified using the RNA isolated from spleen tissue, which was found to be 3,102 nucleotides long encoding a 1,033 amino acid protein. Buffalo TLR8 had 10 nucleotide changes as compared to other livestock species resulting in six unique amino acid changes, four of them lying within leucine-rich repeat (LRR) domains. As compared to cattle (Bos indicus and Bos taurus), out of fifteen cysteine residues, fourteen were conserved and Cys at position 521 was replaced by Arg. Nine of the LRR domains had no amino acid change as compared to cattle, whereas LRR-C-terminus had maximum, five amino acid changes. Sequence characterization of 12 riverine and swamp buffaloes revealed presence of four polymorphic nucleotides, two of them were non-synonymous, one synonymous and one site in 3'UTR. PCR-RFLP genotyping of non-synonymous SNP 2758A>G (ILeu920Val) in Toll-interleukin-1 receptor domain of 463 swamp and riverine buffaloes showed a higher frequency of allele A in swamp (95 %) as compared to riverine (9.84 %) buffaloes.

Detection of polymorphism and sequence characterization of Toll-like receptor 7 gene of Indian goat revealing close relationship between ruminant species.

In this study, approximately 3.4 kb nucleotide sequence of caprine TLR7 (Toll-like receptor 7) gene was generated from twelve different Indian goat breeds belonging to different geographical regions. Goat TLR7 gene ORF (Open Reading Frame) was found to be 3141 nucleotides long coding for 1046 amino acids similar to sheep. The sequence analysis at nucleotide level revealed goat TLR7 having 99.5% homology with sheep, followed by other livestock species. Simple Modular Architecture Research Tool (SMART) was used for the structural analysis of goat TLR7 that showed the presence of 22 leucine rich repeats (LRRs) along with single Toll/interleukin-1 receptor (TIR) domains. TIR domain, when compared, was found to be similar in ruminant species, goat, sheep, cattle, and buffalo. The phylogenetic analysis also revealed grouping of all ruminant species together, goat being closer to sheep followed by cattle and buffalo. A total of 22 polymorphic sites were observed in TLR7 gene of 24 goats representing 12 different breeds, out of which 19 were present within the coding region and three in 3'UTR. Out of the seven nonsynonymous SNPs, two were in ectodomains and one in TIR domain. Overall our results indicate substantial variation within goat TLR7 gene, which could be exploited for association with disease susceptibility.

In-silico screening of plant-derived antivirals against main protease, 3CLpro and endoribonuclease, NSP15 proteins of SARS-CoV-2.

Novel Coronavirus or SARS-CoV-2 outbreak has developed a pandemic condition all over the world. The virus is highly infectious and spreads by human to human local transmission mode. Till date, there is no vaccination or drugs been approved for the treatment by the World Health Organisation. Henceforth, the discovery of the potential drugs is an urgent and utmost requirement for the medical fraternity. Since, the side effects of plant-derived compounds will be lower compared to synthetic/chemical drugs. The Main protease (3CLpro or NSP5) and endoribonuclease (NSP15) proteins are necessity for viral replication and its survival in the host cell. In the present study, in-silico approach of drug development was used to search for potential antiviral plant-derived compounds as inhibitors against SARS-CoV-2 replication proteins. Eight plant-derived compounds of which the antiviral activity was known and available, and two reported drugs against SARS-CoV-2 selected for the molecular docking analysis. The docking results suggested that bisdemethoxycurcumin, demethoxycurcumin, scutellarin, quercetin and myricetin showed least binding energy, i.e., greater than -6.5 Kcal/mol against 3CLpro and endoribonuclease of SARS-CoV-2. Further studies of ADME-Tox and bioavailability of drugs were also performed that exhibited efficient parameters of drug likeness. Molecular dynamics simulation calculations were performed for the most negative binding affinity of the compound to evaluate the dynamic behavior,and stability of protein-ligand complex. Our findings suggest that these compounds could be potential inhibitors of SARS-CoV-2 main protease and endoribonuclease. However, further in-vitro and pre-clinical experiments would validate the potential inhibitors of SARS-CoV-2 proteins.

Efficacy and Safety of COVID-19 Vaccines-An Update.

A few centuries ago, the first vaccine vial was formulated, and since then, they have resulted in an eminent reduction in infectious diseases associated morbidity and mortality. The discovery of the novel SARS-CoV-2 virus and the COVID-19 disease and its steady progression to a global pandemic with 603,711,760 confirmed cases and 6,484,136 reported deaths according to the World Health Organization (WHO) on 7 September 2022 was exceedingly catastrophic. This brought about an unexpected need for preventative and cost-effective measures to curb the devastating impact of the virus, followed by accelerated competition within the pharma giants to manufacture and dispense vaccines at an exponential rate. Non-pharmaceutical medications such as mandated face mask policies, the imposition of travel limitations and generalized disinfectant use were somewhat successful in mitigating the catastrophic effect, but the onus fell upon vaccination strategies and other medical interventions to counteract and subdue this international health threat. The need to ensure current and future pandemic preparedness, however, presents multiple hurdles, among which are equitable vaccine access and the rising trend of vaccine hesitancy at an individual and international level, which are beyond the scope of this discussion. With this review article, we seek to draw perspective on current COVID-19 virus variants, in-hand vaccine types with their mechanism of action along with their effectiveness and safety profile. We also aim to discuss substantial side effects while adding a segment on the booster dose controversy.

Psychological impact of mass quarantine on population during pandemics-The COVID-19 Lock-Down (COLD) study.

BACKGROUND: Quarantine often is an unpleasant experience. The aim of this study is to explore the degree of psychological distress in terms of-Depression, Anxiety and Stress among the adult population in India during the strict 21 days mandatory lockdown. We hypothesize that quantification of psychological impact of current situation will help us to modify the policies and implementation strategies. This assessment might also help in future to keep targeted services in place, to cope up with the psychological distress of the quarantined population. METHOD: A cross sectional survey design was adopted to assess the psychological state of general population in India, during the COVID-19 mandatory lockdown period, with the help of a validated questionnaire. FINDINGS: The reported prevalence of depression was around 30.5%, which was the highest among the variables of psychological health. Anxiety was reported by 22.4%, followed by stress which was seen in 10.8% of respondents. In the third week the incidence of depression (37.8% versus 23.4%; p<0.001), anxiety (26.6% versus 18.2%; p<0.001) and stress (12.2% versus 9.3%; p<0.045) was reported to be significantly higher as compared to second week. INTERPRETATION: Our results suggest a progressively detrimental impact of lockdown on various aspects of psychological health. We noticed around eight to ten fold increase in the prevalence of depression (30.5%) and anxiety (22.4%) during lockdown, as compared to baseline statistics in Indian population (3·1-3·6% for depressive disorders and 3·0-3·5% for anxiety disorders).

Sequence based structural characterization and genetic diversity analysis across coding and promoter regions of goat Toll-like receptor 5 gene.

The exploration of candidate immune response genes in goat may be vital in improving further our understanding about the species specific response to pathogens specifically among the ruminants. In this study, approximately 3.7 kb long genomic sequence of Toll-like receptor 5 (TLR5) covering the entire coding and 5'upstream regions of the gene, was characterized in the Indian goat breeds. Sequence analysis revealed a 2577-nucleotide long open reading frame (ORF) of goat TLR5, encoding 858 amino acids from single exon, similar to other ruminants. The domain structure analysis of goat TLR5 showed the presence of 13 leucine rich repeats (LRRs) in extracellular domain (amino acid position 1-634), single transmembrane domain (position 644-666), and a Toll/interleukin-1 receptor (position 692-837) in cytoplasmic domain, similar to other species. A total of 87 putative transcription factor binding sites were observed within the 5' upstream region of TLR5 gene in goat, 106 in cattle, and 103 in buffalo. Sixteen polymorphic sites were observed in goat TLR5 gene, out of which 10 non-synonymous SNPs were in the functionally important regions. However, none of the amino acid substitutions was found to be potentially damaging to the structure and function of the receptor protein. Further, one of the SNPs in the transmembrane region was genotyped by a TETRA-ARMS PCR in 444 goats of nine breeds from different geographical regions and having different utilities. A significant variation in allelic frequencies was observed across the milch and other types of goat breeds. The comparative modeling of goat TLR5 followed by molecular dynamics simulation gave an insight into its 3D structural arrangements. The molecular docking of Salmonella flagellin and TLR5 dimer elucidated LRRNT (N-terminal) to LRR4 as the key flagellin binding domains region in goat TLR5. The study shows that, although being highly conserved among the ruminants, comparatively high variations in goat TLR5 might give an opportunity to host for recognizing the wider spectrum of pathogens.

Exploration of the binding modes of buffalo PGRP1 receptor complexed with meso-diaminopimelic acid and lysine-type peptidoglycans by molecular dynamics simulation and free energy calculation.

The peptidoglycan recognition proteins (PGRPs) are the key components of innate-immunity, and are highly specific for the recognition of bacterial peptidoglycans (PGN). Among different mammalian PGRPs, the PGRP1 binds to murein PGN of Gram-positive bacteria (lysine-type) and also have bactericidal activity towards Gram-negative bacteria (diaminopimelic acid or Dap-type). Buffaloes are the major sources of milk and meat in Asian sub-continents and are highly exposed to bacterial infections. The PGRP activates the innate-immune signaling, but their studies has been confined to limited species due to lack of structural and functional information. So, to understand the structural constituents, 3D model of buffalo PGRP1 (bfPGRP1) was constructed and conformational and dynamics properties of bfPGRP1 was studied. The bfPGRP1 model highly resembled human and camel PGRP structure, and shared a highly flexible N-terminus and centrally placed L-shaped cleft. Docking simulation of muramyl-tripeptide, tetrapeptide, pentapeptide-Dap-(MTP-Dap, MTrP-Dap and MPP-Dap) and lysine-type (MTP-Lys, MTrP-Lys and MPP-Lys) in AutoDock 4.2 and ArgusLab 4.0.1 anticipated ß1, α2, α4, ß4, and loops connecting ß1-α2, α2-ß2, ß3-ß4 and α4-α5 as the key interacting domains. The bfPGRP1-ligand complex molecular dynamics simulation followed by free binding energy (BE) computation conceded BE values of -18.30, -35.53, -41.80, -25.03, -24.62 and -22.30 kJ mol(-1) for MTP-Dap, MTrP-Dap, MPP-Dap, MTP-Lys, MTrP-Lys and MPP-Lys, respectively. The groove-surface and key binding residues involved in PGN-Dap and Lys-type interaction intended by the molecular docking, and were also accompanied by significant BE values directed their importance in pharmacogenomics, and warrants further in vivo studies for drug targeting and immune signaling pathways exploration.

Structural and dynamic investigation of bovine folate receptor alpha (FOLR1), and role of ultra-high temperature processing on conformational and thermodynamic characteristics of FOLR1-folate complex.

The folate receptor alpha (FOLR1) present in milk has widely been studied to investigate the effects of pasteurization, ultra-high temperature (UHT) processing and fermentation on net folate concentration. However, the folate binding mechanism with FOLR1, and effect of temperature on FOLR1-folate complex is poorly explored till now in bovine milk which is a chief resource of folate. Despite of enormous importance of folic acid and the routine intake of bovine milk, folic acid deficiency diseases are common in human race. To understand the folate deficiency in milk after processing, in absence of experimental structure, 3D model of bovine FOLR1 (bvFOLR1) was built followed by 40ns molecular dynamics (MD) simulation. The folate and its derivatives binding sites in bvFOLR1 were anticipated by molecular docking using AutoDock 4.2. Essential MD studies suggested the presence of a longer signal peptide (22 residues) and a short propeptide (7 residues) at the C-terminus that may cleaved during post-translational modification. MD analysis of bvFOLR1-folate complex at 298, 323, 353, 373 and 408K followed by binding energy (BE) calculation showed maximum binding affinity at ∼353K. However, at 373K and UHT (408K), the folate BE is significantly decreased with substantial conformational alteration. Heating at UHT followed by cooling within 298-408K range demoed no structural reformation with temperature reduction, and the folate was displaced from the active site. This study presented the disintegration of folate from bvFOLR1 during high temperature processing and revealed a lower folate concentration in UHT milk and dairy products.

Clinical aspects of acute post-operative pain management & its assessment.

Management of postoperative pain relieve suffering and leads to earlier mobilization, shortened hospital stay, reduced hospital costs, and increased patient satisfaction. An effective postoperative management is not a standardized regime rather is tailored to the needs of the individual patient, taking into account medical, psychological, and physical condition; age; level of fear or anxiety; surgical procedure; personal preference; and response to therapeutic agents given. The major goal in the management of postoperative pain is to minimize the dose of medications to lessen side effects & provide adequate analgesia. Postoperative pain is still under managed due to obstacles in implementation of Acute Pain Services due to insufficient education, fear of complications associated with available analgesic drugs, poor pain assessment and inadequate staff. This review reflects the clinical aspects of postoperative pain & its assessment & management with an emphasis on research for new analgesic molecules & delivery system.