In silico analysis of genomic landscape of SARS-CoV-2 and its variant of concerns (Delta and Omicron) reveals changes in the coding potential of miRNAs and their target genes.

COVID-19 related morbidities and mortalities are still continued due to the emergence of new variants of SARS-CoV-2. In the last few years, viral miRNAs have been the centre of study to understand the disease pathophysiology. In this work, we aimed to predict the change in coding potential of the viral miRNAs in SARS-CoV-2's VOCs, Delta and Omicron compared to the Reference (Wuhan origin) strain using bioinformatics tools. After ab-intio based screening by the Vmir tool and validation, we retrieved 22, 6, and 6 pre-miRNAs for Reference, Delta, and Omicron. Most of the predicted unique pre-miRNAs of Delta and Omicron were found to be encoded from the terminal and origin of the genomic sequence, respectively. Mature miRNAs identified by MatureBayes from the unique pre-miRNAs were used for target identification using miRDB. A total of 1786, 216, and 143 high-confidence target genes were captured for GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. The GO and KEGG pathways terms analysis revealed the involvement of Delta miRNAs targeted genes in the pathways such as Human cytomegalovirus infection, Breast cancer, Apoptosis, Neurotrophin signaling, and Axon guidance whereas the Sphingolipid signaling pathway was found for the Omicron. Furthermore, we focussed our analysis on target genes that were validated through GEO's (Gene Expression Omnibus) DEGs (Differentially Expressed Genes) dataset, in which FGL2, TNSF12, OGN, GDF11, and BMP11 target genes were found to be down-regulated by Reference miRNAs and YAE1 and RSU1 by Delta. Few genes were also observed to be validated among in up-regulated gene set of the GEO dataset, in which MMP14, TNFRSF21, SGMS1, and TMEM192 were related to Reference whereas ZEB2 was detected in all three strains. This study thus provides an in-silico based analysis that deciphered the unique pre-miRNAs in Delta and Omicron compared to Reference. However, the findings need future wet lab studies for validation.

Chlorhexidine and SARS-CoV-2 main protease: Molecular docking study.

As the beginning of the COVID-19 pandemic, numerous attempts have been made to identify specific antiviral mouth rinses which may help reduce the salivary viral load of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). Although the results from in vivo well-controlled clinical studies are yet pending, many contemporary antimicrobial mouth rinses have been explored for potential antiviral properties with respect to SARS-CoV-2. The most widely used compounds such as povidone-iodine, chlorhexidine, hydrogen peroxide, and essential oils have been known to have antiviral activity by targeting the outer lipid membrane or by denaturing the capsid proteins of enveloped virus SARS-CoV. Until now, there has been scanty scientific evidence on the molecular basis of interaction of the gold standard antimicrobial mouth rinse as an underlying mechanism of its anti-SARS-CoV-2 effect. The current communication reports the findings of our in silico docking study pertaining to understand the interactions of chlorhexidine with the most well-studied target of the SARS-CoV main protease.

Multiple-reaction monitoring (MRM) LC-MS/MS quantitation of venlafaxine and its O-desmethyl metabolite for a preclinical pharmacokinetic study in rabbits.

Preclinical pharmacokinetic (PK) studies in animal models during the formulation development phase give preliminary evidence and near clear picture of the PK behavior of drug and/or its dosage forms before clinical studies on humans and help in the tailoring of the dosage form according to the expected and requisite clinical behavior. The present work reports a first of its kind preclinical PK study on extended-release (ER) solid oral dosage forms of venlafaxine (VEN) in New Zealand White rabbits. The VEN is a highly prescribed and one of the safest and most effective therapeutic agents used in the treatment of different types of depression disorders worldwide. The multiple-reaction monitoring (MRM) LC-MS/MS method developed for this purpose demonstrated enough reliability in simultaneously quantitating VEN and its equipotent metabolite O-desmethylvenlafaxine (ODV) in rabbit plasma. The method described uses solid-phase extraction for sample preparation followed by an ultrafast LC-MS/MS analysis. The chromatographic separation was achieved isocratically with a predominantly polar mobile phase by employing RPLC. The triple quadrupole LC/MS/MS system operated in MRM mode used an ESI probe as an ion source in positive polarity. The validation results are within the permissible limits of US FDA recommendations and acceptance criteria for bioanalytical method validation.

Deciphering the functional role of hypothetical proteins from Chloroflexus aurantiacs J-10-f1 using bioinformatics approach.

Chloroflexus aurantiacus J-10-f1 is an anoxygenic, photosynthetic, facultative autotrophic gram negative bacterium found from hot spring at a temperature range of 50-60°C. It can sustain itself in dark only if oxygen is available thereby exhibiting a dark orange color, however display a dark green color when grown in sunlight. Genome of the organism contains total of 3853 proteins out of which 785 (~20%) proteins are uncharacterised or hypothetical proteins (HPs). Therefore in this work we have characterized the 785 hypothetical proteins of Chloroflexus aurantiacus J-10-f1 using bioinformatics tools and databases. HPs annotated by more than five domain prediction tools were filtered and named high confidence-hypothetical proteins (HC-HPs). These HC-HPs were further annotated by calculating their physiochemical properties, homologous, subcellular locations, signal peptides and transmembrane regions. We found most of the HC-HPs were involved in photosynthesis, carbohydrate metabolism, biofuel production and cellulose synthesis processes. Furthermore, few of these HC-HPs could provide resistance to bacteria at high temperature due to their thermophilic nature. Hence these HC-HPs have the potential to be used in industrial as well as in biomedical needs. To conclude, the bioinformatics approach used in this study provides an insight to better understand the nature and role of Chloroflexus aurantiacus J-10-f1 hypothetical proteins.

Genome-wide computational prediction of miRNAs in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) revealed target genes involved in pulmonary vasculature and antiviral innate immunity.

The current outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China threatened humankind worldwide. The coronaviruses contains the largest RNA genome among all other known RNA viruses, therefore the disease etiology can be understood by analyzing the genome sequence of SARS-CoV-2. In this study, we used an ab-intio based computational tool VMir to scan the complete genome of SARS-CoV-2 to predict pre-miRNAs. The potential pre-miRNAs were identified by ViralMir and mature miRNAs were recognized by Mature Bayes. Additionally, predicted mature miRNAs were analysed against human genome by miRDB server to retrieve target genes. Besides that we also retrieved GO (Gene Ontology) terms for pathways, functions and cellular components. We predicted 26 mature miRNAs from genome of SARS-CoV-2 that targets human genes involved in pathways like EGF receptor signaling, apoptosis signaling, VEGF signaling, FGF receptor signaling. Gene enrichment tool analysis and substantial literature evidences suggests role of genes like BMPR2 and p53 in pulmonary vasculature and antiviral innate immunity respectively. Our findings may help research community to understand virus pathogenesis.

Computational prediction of miRNAs in Nipah virus genome reveals possible interaction with human genes involved in encephalitis.

Current re-emergence of Nipah virus (NiV) in India caused 11 deaths so far and many patients were kept in quarantine. A thorough study of previous outbreaks occurred in Malaysia, Bangladesh and India represents cases with high rate of fatality due to acute encephalitis. Our work involves genome analysis of NiV for prediction of miRNAs and their targeted genes in human in order to understand encephalitis origin. Ab-intio program-VMir was used for initial screening of genome, obtained nine pre-miRNAs was analyzed by ViralMir to check for any pseudo pre-miRNAs. Eighteen functional mature miRNAs were extracted from pre-miRNAs by using Mature-Bayes tool, which targets 669 genes in human genome as retrieved by miRDB. Gene ontology terms by PANTHER provide important pathways in which target genes were involved like Axon guidance, T cell activation, and nicotinic acetylcholine receptor signaling. Significant outcome was obtained after NCBI Gene and OMIM database mining and literature search for predicted target genes. TLR3, TJP1, NOTCH2, FHL1, and GRIA3 target genes obtained showed their involvement in host defense, blood brain barrier, neurogenesis, mental retardation and encephalitis. To conclude, we predicted significant genes in human that can be inhibited by miRNAs of NiV and results in etiology of encephalitis.

In silico mutational analysis and identification of stability centers in human interleukin-4.

Interleukin-4 (IL-4) is a multifunctional cytokine that plays a critical role in apoptosis, differentiation and proliferation. The intensity of IL4 response depends upon binding to its receptor, IL-4R. The therapeutic efficiency of interleukins can be increased by generating structural mutants having greater stability. In the present work, attempts were made to increase the stability of human IL-4 using in-silico site directed mutagenesis. Different orthologous sequences of IL4 from Pan troglodytes, Aotusnigriceps, Macacamulatta, Papiohamadryas, Chlorocebusaethiops, Vicugnapacos, Susscrofa and Homo sapiens were aligned using Clustal Omega that revealed the conserved and non-conserved positions. For each non-conserved position, possible favorable and stabilizing mutations were found using CUPSAT with predicted ΔΔG (kcal/mol). The one with highest ΔΔG (kcal/mol) among all possible mutations, for each non-conserved position was selected and introduced manually in human IL-4 sequence resulting in multiple mutants of IL-4. Mutant proteins were modeled using structure of IL4 (PDB ID: 2B8U) as a template by SWISS MODEL. The mutants A49L and Q106T were identified to have stability centre using SCide. Molecular dynamics and docking analysis also confirmed the mutants stability and binding respectively. Mutants A49L and Q106T had -7.580079 kcal/mol and -39.418124 kcal/mol respectively lesser energy value than the wild type IL4. The result suggested that, the stability of human IL-4 has been increased by mutation.

Role of P-Glycoprotein Inhibitors in the Bioavailability Enhancement of Solid Dispersion of Darunavir.

OBJECTIVE: The aim of the present study was to improve bioavailability of an important antiretroviral drug, Darunavir (DRV), which has low water solubility and poor intestinal absorption through solid dispersion (SD) approach incorporating polymer with P-glycoprotein inhibitory potential. METHODS: A statistical approach where design of experiment (DoE) was used to prepare SD of DRV with incorporation of P-glycoprotein inhibitors. Using DoE, different methods of preparation, like melt, solvent evaporation, and spray drying method, utilizing carriers like Kolliphor TPGS and Soluplus were evaluated. The optimized SD was characterized by DSC, FTIR, XRD, and SEM and further evaluated for enhancement in absorption using everted gut sac model, effect of food on absorption of DRV, and in vivo prospect. RESULTS AND DISCUSSION: DSC, FTIR, XRD, and SEM confirmed the amorphicity of drug in SD. Oral bioavailability studies revealed better absorption of DRV when given with food. Absorption studies and in vivo study findings demonstrated great potential of Kolliphor TPGS as P-glycoprotein inhibitor for increasing intestinal absorption and thus bioavailability of DRV. CONCLUSION: It is concluded that SD of DRV with the incorporation of Kolliphor TPGS was potential and promising approach in increasing bioavailability of DRV as well as minimizing its extrusion via P-glycoprotein efflux transporters.

Child who presented with hematohidrosis (sweating blood) with oppositional defiant disorder.

Hematohidrosis is a very rare condition of sweating blood. A child's case who presented to us with hematohidrosis is reported. There are only few reports in the literature. A 10-year-old boy presented to our hospital with a history of repeated episodes of oozing of blood from navel, eyes, ear lobules, and nose. During the examination, it disappeared as soon as it was mopped leaving behind no sign of trauma only to reappear within a few seconds. Bleeding time, clotting time, and prothrombin time were normal. Patient was diagnosed with hematohidrosis and oppositional defiant disorder clinically. Management of this condition at our center is discussed below.

Non-high-density lipoprotein cholesterol calculation and goal awareness among physicians-in-training.

BACKGROUND: Non-high density lipoprotein cholesterol (non-HDL-C) goal attainment per Adult Treatment Panel III (ATP III) guidelines remains low. OBJECTIVE: To understand gaps in knowledge and practices of physicians-in-training (internal medicine, family medicine, cardiology, endocrinology) towards non-HDL-C. METHODS: A survey based on a conceptual model to assess the trainee's knowledge, attitudes, and practice regarding non-HDL-C was developed and administered to physicians-in-training (n = 655) at 26 training programs in the United States. Responses of those in internal medicine and family medicine (residents-in-training; n = 418) were compared with those in cardiology and endocrinology (fellows-in-training; n = 124). RESULTS: Response rate was 83.7%. Fifty-three percent of residents and 31% of fellows-in-training had not read the ATP III guidelines (P < .001). Thirty-three percent of the residents and 35% fellows-in-training could not calculate non-HDL-C from a standard lipid panel (P = .7). Sixty-seven percent of the residents and 52% of fellows were not aware of treatment goals for non-HDL-C (P = .004 for comparison between residents and fellows). Both residents and fellows reported infrequent calculation of non-HDL-C levels in patients with elevated triglycerides (≥200 mg/dL; 32.5% vs 35.4%, respectively, P = .6). Lack of familiarity with ATP III guidelines, lack of knowledge regarding importance of non-HDL-C, lack of institutional mandate to calculate non-HDL-C, and lack of emphasis on non-HDL-C by teaching staff were reported as barriers to non-HDL-C use in routine clinical practice. CONCLUSIONS: At least one-third of physicians-in-training could not calculate non-HDL-C from a standard lipid panel, and a large number were not aware of ATP III treatment goals pertaining to non-HDL-C. This area represents one for improvement if non-HDL-C is to be retained as a treatment target in the forthcoming ATP-IV guidelines.

Protective efficacy and immunogenicity of Escherichia coli K13 diphtheria toxoid conjugate against experimental ascending pyelonephritis.

In the present study, protective efficacy of Escherichia coli capsular antigen, K13, was evaluated in a mouse model of pyelonephritis. Unconjugated capsular polysaccharide failed to provide any protection. However, coupling of K13 to diphtheria toxoid (DT) enhanced its immunogenicity and led to significant production of anticapsular antibodies in mice. Immunization of animals with K13-DT conjugate also caused significant improvement in cell-mediated immune response as indicated by an increase in lymphoblastogenic response and in the CD4+/CD8+ cell ratio of splenic lymphocytes. Significant decrease in bacterial load and renal severity scores were observed in K13-DT immunized animals. Suitability of K13-DT conjugate as an effective vaccine candidate against urinary tract infections caused by E. coli has been discussed.