High-resolution melt curve analysis: An approach for variant detection in the TPO gene of congenital hypothyroid patients in Bangladesh.

TPO (Thyroid Peroxidase) is known to be one of the major genes involved in congenital hypothyroid patients with thyroid dyshormonogenesis. The present study aims to validate high-resolution melting (HRM) curve analysis as a substitute method for Sanger sequencing, focusing on the frequently observed non-synonymous mutations c.1117G>T, c.1193G>C, and c.2173A>C in the TPO gene in patients from Bangladesh. We enrolled 36 confirmed cases of congenital hypothyroid patients with dyshormonogenesis to establish the HRM method. Blood specimens were collected, and DNA was extracted followed by PCR and Sanger sequencing. Among the 36 specimens, 20 were pre-sequenced, and variants were characterized through Sanger sequencing. Following pre-sequencing, the 20 pre-sequenced specimens underwent real-time PCR-HRM curve analysis to determine the proper HRM condition for separating the three variations from the wild-type state into heterozygous and homozygous states. Furthermore, 16 unknown specimens were subjected to HRM analysis to validate the method. This method demonstrated a sensitivity and specificity of 100 percent in accurately discerning wild-type alleles from both homozygous and heterozygous states of c.1117G>T (23/36; 63.8%), c.1193G>C (30/36; 83.3%), and c.2173A>C (23/36; 63.8%) variants frequently encountered among 36 Bangladeshi patients. The HRM data was found to be similar to the sequencing result, thus confirming the validity of the HRM approach for TPO gene variant detection. In conclusion, HRM-based molecular technique targeting variants c.1117G>T, c.1193G>C, and c.2173A>C could be used as a high throughput, rapid, reliable, and cost-effective screening approach for the detection of all common mutations in TPO gene in Bangladeshi patients with dyshormonogenesis.

Association between DPP6 gene rs10260404 polymorphism and increased risk of sporadic amyotrophic lateral sclerosis (sALS): a meta-analysis.

BACKGROUND: Sporadic amyotrophic lateral sclerosis (sALS) is a severe neurodegenerative disease characterized by continuous diminution of motor neurons in the brain and spinal cord. Earlier studies indicated that the DPP6 gene variant has a role in the development of sALS. This meta-analysis was designed to uncover the role of rs10260404 polymorphism of the DPP6 gene and its association with sALS. METHODS: All case-control articles published prior to October 2022 on the association between DPP6 (rs10260404) polymorphism and sALS risk were systematically extracted from different databases which include PubMed, PubMed Central, and Google Scholar. Overall odds ratios (ORs) and "95% confidence intervals (CIs)" were summarized for various genetic models. Subgroup and heterogeneity assessments were performed. Egger's and "Begg's tests were applied to evaluate publication bias. Trial sequential analysis (TSA) and false-positive report probability (FPRP) were performed. RESULTS: Nine case-control studies containing 4202 sALS cases and 4444 healthy controls were included in the meta-analysis. A significant association of the DPP6 (rs10260404) variant with an increased sALS risk in overall pooled subjects under allelic model [C allele vs. T allele, OR = 1.149, 95% CI (1.010-1.307), p-value = 0.035], dominant model [CC + CT vs. TT, OR = 1.165, 95% CI (1.067-1.273), p-value = 0.001], and homozygote comparison [CC vs. TT, OR = 1.421, 95% CI (1.003-2.011), p-value = 0.048] were observed. Moreover, in subgroup analysis by nationality, remarkable associations were detected in Dutch, Irish, American, and Swedish under allelic, dominant, and homozygote models. Additionally, stratification analysis by ethnicity exhibited an association with sALS risk among Caucasians and Americans under different genetic models. Interestingly, none of the models found any significant association with Asians. CONCLUSION: The present meta-analysis indicates that DPP6 (rs10260404) polymorphism could be a candidate risk factor for sALS predisposition.

Decoding the Parkinson's Symphony: PARIS, Maestro of Transcriptional Regulation and Metal Coordination for Dopamine Release.

Parkin interacting substrate (PARIS) is a pivotal transcriptional regulator in the brain that orchestrates the activity of various enzymes through its intricate interactions with biomolecules, including nucleic acids. Notably, the binding of PARIS to insulin response sequences (IRSs) triggers a cascade of events that results in the functional loss in the substantia nigra, which impairs dopamine release and, subsequently, exacerbates the relentless neurodegeneration. Here, we report the details of the interactions of PARIS with IRSs via classical zinc finger (ZF) domains in PARIS, namely, PARIS(ZF2-4). Our biophysical studies with purified PARIS(ZF2-4) elucidated the binding partner of PARIS, which generates specific interactions with the IRS1 (5'-TATTTTT, Kd = 38.9 ± 2.4 nM) that is positioned in the promoter region of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α). Mutational and metal-substitution studies demonstrated that Zn(II)-PARIS(ZF2-4) could recognize its binding partner selectively. Overall, our work provides submolecular details regarding PARIS and shows that it is a transcriptional factor that regulates dopamine release. Thus, PARIS could be a crucial target for therapeutic applications.

Nitric oxide inhibits FTO demethylase activity to regulate N6-methyladenosine mRNA methylation.

N6-methyladenosine (m6A) is the most abundant internal modification on eukaryotic mRNAs. Demethylation of m6A on mRNA is catalyzed by the enzyme fat mass and obesity-associated protein (FTO), a member of the nonheme Fe(II) and 2-oxoglutarate (2-OG)-dependent family of dioxygenases. FTO activity and m6A-mRNA are dysregulated in multiple diseases including cancers, yet endogenous signaling molecules that modulate FTO activity have not been identified. Here we show that nitric oxide (NO) is a potent inhibitor of FTO demethylase activity by directly binding to the catalytic iron center, which causes global m6A hypermethylation of mRNA in cells and results in gene-specific enrichment of m6A on mRNA of NO-regulated transcripts. Both cell culture and tumor xenograft models demonstrated that endogenous NO synthesis can regulate m6A-mRNA levels and transcriptional changes of m6A-associated genes. These results build a direct link between NO and m6A-mRNA regulation and reveal a novel signaling mechanism of NO as an endogenous regulator of the epitranscriptome.

Unveiling clinically significant PPARγ mutations for thiazolidinedione treatment responsiveness through atomistic simulations.

In Type 2 diabetes, increased insulin sensitivity is induced by thiazolidinedione activation of the peroxisome proliferator-activated receptor gamma (PPARγ). Recent data indicate a relationship between SNPs in PPARγ and poor drug response. Therefore, understanding the pathogenic consequences of mutations in PPARγ-mediated protein-drug interactions will be prima-facie for establishing personalized medicine. The PPARG gene has 197 missense SNPs, 22 of which were determined to be both deleterious and destabilizing, employing in silico approaches. Molecular docking analysis suggested that the mutation influenced the binding energy of at least seven of the variants. The mutant R316H was identified as the most damaging and deleterious from the observed results. For a better understanding of the dynamic variation upon mutation at the atomic level, molecular dynamics simulations of the wild-type and R316H mutant PPARγ structure were performed. The analysis indicates that the mutation increased protein structural compactness while decreasing flexibility. The reduced dynamics in the mutant structure was further validated by principal component analysis. This mechanistic evaluation of the PPARγ protein variants provides insight into the relationship between genetic variation and interindividual variability of drug responsiveness and will facilitate the future studies for the development of tailored treatment regime for precision medicine.

Association of transcription factor 7-like 2 rs12255372 polymorphism with susceptibility of type 2 diabetes mellitus in Bangladeshi population.

Polymorphism of transcription factor 7-like 2 (TCF7L2) has a link with type 2 diabetes mellitus (T2DM) through ß cell dysfunction that causes defect in blood glucose homeostasis. This case-control study recruited 67 T2DM as cases and 65 age-matched healthy individuals as controls to determine whether the polymorphism rs12255372 (G > T) in the TCF7L2 gene have an association with T2DM in Bangladeshi population. Genomic DNA was purified from peripheral whole blood sample and direct Sanger sequencing was done for genotyping of SNP. Bivariate logistic regression was done to find out the association between genetic variant and T2DM. In our study, the minor T allele frequency was significantly more frequent in T2DM group than healthy controls (29.1% vs. 16.9%). After adjusting with confounding factors, heterozygous-genotype GT had higher odds of developing T2DM (OR 2.4; 95% CI: 1.0-5.5; p value = 0.04) and in dominant model, having SNP in TCF7L2 increased the risk of T2DM 2.3 times (95% CI: 1.0-5.2; p value = 0.04). In interaction model, genetic susceptible SNP cases interacted significantly with increasing age and BMI, female gender, and having family history of diabetes mellitus to develop T2DM (pinteraction < 0.001). Having minor T allele either in heterozygous or homozygous variant form of rs12255372 (G > T) TCF7L2 had significant association with T2DM. In conclusion, TCF7L2 gene variant increases risk of developing T2DM among the Bangladeshi population.

E2F regulation of the Phosphoglycerate kinase gene is functionally important in Drosophila development.

The canonical role of the transcription factor E2F is to control the expression of cell cycle genes by binding to the E2F sites in their promoters. However, the list of putative E2F target genes is extensive and includes many metabolic genes, yet the significance of E2F in controlling the expression of these genes remains largely unknown. Here, we used the CRISPR/Cas9 technology to introduce point mutations in the E2F sites upstream of five endogenous metabolic genes in Drosophila melanogaster. We found that the impact of these mutations on both the recruitment of E2F and the expression of the target genes varied, with the glycolytic gene, Phosphoglycerate kinase (Pgk), being mostly affected. The loss of E2F regulation on the Pgk gene led to a decrease in glycolytic flux, tricarboxylic acid cycle intermediates levels, adenosine triphosphate (ATP) content, and an abnormal mitochondrial morphology. Remarkably, chromatin accessibility was significantly reduced at multiple genomic regions in PgkΔE2F mutants. These regions contained hundreds of genes, including metabolic genes that were downregulated in PgkΔE2F mutants. Moreover, PgkΔE2F animals had shortened life span and exhibited defects in high-energy consuming organs, such as ovaries and muscles. Collectively, our results illustrate how the pleiotropic effects on metabolism, gene expression, and development in the PgkΔE2F animals underscore the importance of E2F regulation on a single E2F target, Pgk.

Isolation and characterization of blackish-brown BY2-melanin accumulated in cultured tobacco BY-2 cells.

The tobacco BY-2 cell line is one of the most utilized plant cell lines. After long-term culture, the cells turn brown to black, but the causal pigment is unknown. We successfully isolated a blackish-brown pigment from BY-2 cells cultured for 3 weeks. Morphological and spectroscopic analyses indicated that the pigment had similar features to a melanin-like substance reported previously. Furthermore, physicochemical analyses revealed that this pigment possessed most of the properties of melanin-like pigments. In addition, the high nitrogen content suggested that it differed from common plant melanins classified as allomelanins, suggesting a novel eumelanin-like pigment: "BY2-melanin". This is the first example showing that eumelanin-like pigments are produced in the cultures of plant cells for which the accumulation of melanin has not been reported. This tobacco BY-2 cell culture technique may represent a customizable and sustainable alternative to conventional melanin production platforms, with significant potential for industrial and pharmacological applications.

Targeting SARS-CoV-2 non-structural protein 13 via helicase-inhibitor-repurposing and non-structural protein 16 through pharmacophore-based screening.

Novel drug compound hunting was carried out for SARS-CoV-2 proteins with low mutation susceptibility. The probability of escape mutation and drug resistance is lower if conserved microbial proteins are targeted by therapeutic drugs. Mutation rate of all SARS-CoV-2 proteins were analyzed via multiple sequence alignment Non-Structural Protein 13 and Non-Structural Protein 16 were selected for the current study due to low mutation rate among viral strains and significant functionality. Cross-species mutation rate analysis for NSP13 and NSP16 showed these are well-conserved proteins among four coronaviral species. Viral helicase inhibitors, identified using literature-mining, were docked against NSP13. Pharmacophore-based screening of 11,375 natural compounds was conducted for NSP16. Stabilities of top compounds inside human body were confirmed via molecular dynamic simulation. ADME properties and LD50 values of the helicase inhibitors and Ambinter natural compounds were analyzed. Compounds against NSP13 showed binding affinities between -10 and -5.9 kcal/mol whereby ivermectin and scutellarein showed highest binding energies of -10 and -9.9 kcal/mol. Docking of 18 hit compounds against NSP16 yielded binding affinities between -8.9 and -4.1 kcal/mol. Hamamelitannin and deacyltunicamycin were the top compounds with binding affinities of -8.9 kcal/mol and -8.4 kcal/mol. The top compounds showed stable ligand-protein interactions in molecular dynamics simulation. The analyses revealed two hit compounds against each targeted protein displaying stable behavior, high binding affinity and molecular interactions. Conversion of these compounds into drugs after in vitro experimentation can become better treatment options to elevate COVID management.

Modeling of MT. P495, an mRNA-based vaccine against the phosphate-binding protein PstS1 of Mycobacterium tuberculosis.

Tuberculosis (TB) is a contagious disease that predominantly affects the lungs, but can also spread to other organs via the bloodstream. TB affects about one-fourth population of the world. With age, the effectiveness of Bacillus Calmette-Guérin (BCG), the only authorized TB vaccine, decreases. In the quest for a prophylactic and immunotherapeutic vaccine, in this study, a hypothetical mRNA vaccine is delineated, named MT. P495, implementing in silico and immunoinformatics approaches to evaluate key aspects and immunogenic epitopes across the PstS1, a highly conserved periplasmic protein of Mycobacterium tuberculosis (Mtb). PstS1 elicited the potential to generate 99.9% population coverage worldwide. The presence of T- and B-cell epitopes across the PstS1 protein were validated using several computational prediction tools. Molecular docking and dynamics simulation confirmed stable epitope-allele interaction. Immune cell response to the antigen clearance rate was verified by the in silico analysis of immune simulation. Codon optimization confirmed the efficient translation of the mRNA in the host cell. With Toll-like receptors, the vaccine exhibited stable and strong interactions. Findings suggest that the MT. P495 vaccine probably will elicit specific immune responses against Mtb. This mRNA vaccine model is a ready source for further wet-lab validation to confirm the efficacy of this proposed vaccine candidate.

Modeling mRNA-based vaccine YFV.E1988 against yellow fever virus E-protein using immuno-informatics and reverse vaccinology approach.

To surmount constraints of live-attenuated vaccines we have in silico designed mRNA vaccine using envelope protein as a target antigen. From the alignment of 216 envelope proteins, a consensus sequence was obtained which was used for codon optimization. The secondary structure was predicted using Mfold and RNAfold tool. IEDB server was used to predict T-cell and B-cell epitopes, epitope conservancy, immunogenicity, and population coverage. Antigenicity, allergenicity, and toxicity were predicted using Vaxijen, AllerTOP, and ToxinPred tools, respectively. Interactions between MHC and identified epitopes were confirmed by docking and molecular dynamics simulation. In silico immune simulation was done using the C-ImmSim server. Vaccine peptide 3D structure was predicted and validated based on the Ramachandran plot. Finally, we designed the vaccine construct for simulating restriction cloning using the SnapGene tool. Our optimization of consensus E protein is highly immunogenic, conserved, has immune-dominance characteristics, and suggests high translational efficiency in the host cell. We validated the presence of T and B cell epitopes and interestingly we found one CD4+ and four CD8+ T-cell epitopes that satisfied all the criteria of an effective vaccine candidate. We found high-affinity interactions between epitope and HLA alleles that can stimulate the T-cell response. The immune simulation verified the immune cell response to eliminate the antigen. To ensure effective expression of the vaccine, a circular plasmid has been designed using in silico cloning approach for the in vitro transcription process. Obtained results suggest that the vaccine YFV.E1988 will elicit specific immune responses against YFV and it is a potential model ready for laboratory testing. HighlightsThe envelope (E) protein was found to be highly conserved and it has the potential to protect individuals against YFV infection.YFV.E1988 vaccine has been capable to stimulate both the CD8+ and CD4+ T cell, solving the major limitations of the current live-attenuated vaccines against YFV.Presence of T- and B-cell epitopes across the antigen have been validated using several computational tools.Molecular docking ensured the epitope-allele binding and protein-TLR/MR interaction. The vaccine was found to be immune-stimulatory, safe, and stable.The codons were optimized for efficient translation and increased stability into the human host. The UTR regions and poly (A) tail used for the development of YFV.E1988 showed immune stimulatory potential in several experiments.Communicated by Ramaswamy H. Sarma.

Drug and Anti-Viral Peptide Design to Inhibit the Monkeypox Virus by Restricting A36R Protein.

Most recently, monkeypox virus (MPXV) has emanated as a global public health threat. Unavailability of effective medicament against MPXV escalates demand for new therapeutic agent. In this study, in silico strategies were conducted to identify novel drug against the A36R protein of MPXV. The A36R protein of MPXV is responsible for the viral migration, adhesion, and vesicle trafficking to the host cell. To block the A36R protein, 4893 potential antiviral peptides (AVPs) were retrieved from DRAMP and SATPdb databases. Finally, 57 sequences were screened based on peptide filtering criteria, which were then modeled. Likewise, 31 monkeypox virus A36R protein sequences were collected from NCBI protein database to find consensus sequence and to predict 3D protein model. The refined and validated models of the A36R protein and AVP peptides were used to predict receptor-ligand interactions using DINC 2 server. Three peptides that showed best interactions were SATPdb10193, SATPdb21850, and SATPdb26811 with binding energies -6.10, -6.10, and -6.30 kcal/mol, respectively. Small molecules from drug databases were also used to perform virtual screening against the A36R protein. Among databases, Enamine-HTSC showed strong affinity with docking scores ranging from -8.8 to 9.8 kcal/mol. Interaction of target protein A36R with the top 3 peptides and the most probable drug (Z55287118) examined by molecular dynamic (MD) simulation. Trajectory analyses (RMSD, RMSF, SASA, and Rg) confirmed the stable nature of protein-ligand and protein-peptide complexes. This work suggests that identified top AVPs and small molecules might interfere with the function of the A36R protein of MPXV.

Functional Analysis of Hypothetical Proteins of Vibrio parahaemolyticus Reveals the Presence of Virulence Factors and Growth-Related Enzymes With Therapeutic Potential.

Vibrio parahaemolyticus, an aquatic pathogen, is a major concern in the shrimp aquaculture industry. Several strains of this pathogen are responsible for causing acute hepatopancreatic necrosis disease as well as other serious illness, both of which result in severe economic losses. The genome sequence of two pathogenic strains of V. parahaemolyticus, MSR16 and MSR17, isolated from Bangladesh, have been reported to gain a better understanding of their diversity and virulence. However, the prevalence of hypothetical proteins (HPs) makes it challenging to obtain a comprehensive understanding of the pathogenesis of V. parahaemolyticus. The aim of the present study is to provide a functional annotation of the HPs to elucidate their role in pathogenesis employing several in silico tools. The exploration of protein domains and families, similarity searches against proteins with known function, gene ontology enrichment, along with protein-protein interaction analysis of the HPs led to the functional assignment with a high level of confidence for 656 proteins out of a pool of 2631 proteins. The in silico approach used in this study was important for accurately assigning function to HPs and inferring interactions with proteins with previously described functions. The HPs with function predicted were categorized into various groups such as enzymes involved in small-compound biosynthesis pathway, iron binding proteins, antibiotics resistance proteins, and other proteins. Several proteins with potential druggability were identified among them. In addition, the HPs were investigated in search of virulent factors, which led to the identification of proteins that have the potential to be exploited as vaccine candidate. The findings of the study will be effective in gaining a better understanding of the molecular mechanisms of bacterial pathogenesis. They may also provide an insight into the process of evaluating promising targets for the development of drugs and vaccines against V. parahaemolyticus.

Deciphering the role of predicted miRNAs of polyomaviruses in carcinogenesis.

Human polyomaviruses are relatively common in the general population. Polyomaviruses maintain a persistent infection after initial infection in childhood, acting as an opportunistic pathogen in immunocompromised populations and their association has been linked to carcinogenesis. A comprehensive understanding of the underlying molecular mechanisms of carcinogenesis in consequence of polyomavirus infection remains elusive. However, the critical role of viral miRNAs and their potential targets in modifying the transcriptome profile of the host remains largely unknown. Polyomavirus-derived miRNAs have the potential to play a substantial role in carcinogenesis. Employing computational approaches, putative viral miRNAs along with their target genes have been predicted and possible roles of the targeted genes in many significant biological processes have been obtained. Polyomaviruses have been observed to target intracellular signal transduction pathways through miRNA-mediated epigenetic regulation, which may contribute to cancer development. In addition, BKPyV-infected human renal cell microarray data was coupled with predicted target genes and analysis of the downregulated genes indicated that viruses target multiple signaling pathways (e.g. MAPK signaling pathway, PI3K-Akt signaling pathway, PPAR signaling pathway) in the host as well as turning off several tumor suppression genes (e.g. FGGY, EPHX2, CACNA2D3, CDH16) through miRNA-induced mechanisms, assuring cell transformation. This study provides a conceptual framework for the underlying molecular mechanisms involved in the course of carcinogenesis upon polyomavirus infection.

Functional Annotation of Hypothetical Proteins From the Enterobacter cloacae B13 Strain and Its Association With Pathogenicity.

Enterobacter cloacae B13 strain is a rod-shaped gram-negative bacterium that belongs to the Enterobacteriaceae family. It can cause respiratory and urinary tract infections, and is responsible for several outbreaks in hospitals. E. cloacae has become an important pathogen and an emerging global threat because of its opportunistic and multidrug resistant ability. However, little knowledge is present about a large portion of its proteins and functions. Therefore, functional annotation of the hypothetical proteins (HPs) can provide an improved understanding of this organism and its virulence activity. The workflow in the study included several bioinformatic tools which were utilized to characterize functions, family and domains, subcellular localization, physiochemical properties, and protein-protein interactions. The E. cloacae B13 strain has overall 604 HPs, among which 78 were functionally annotated with high confidence. Several proteins were identified as enzymes, regulatory, binding, and transmembrane proteins with essential functions. Furthermore, 23 HPs were predicted to be virulent factors. These virulent proteins are linked to pathogenesis with their contribution to biofilm formation, quorum sensing, 2-component signal transduction or secretion. Better knowledge about the HPs' characteristics and functions will provide a greater overview of the proteome. Moreover, it will help against E. cloacae in neonatal intensive care unit (NICU) outbreaks and nosocomial infections.

Transcriptional Repression by FoxM1 Suppresses Tumor Differentiation and Promotes Metastasis of Breast Cancer.

The transcription factor Forkhead box M1 (FoxM1) is overexpressed in breast cancers and correlates with poor prognosis. Mechanistically, FoxM1 associates with CBP to activate transcription and with Rb to repress transcription. Although the activating function of FoxM1 in breast cancer has been well documented, the significance of its repressive activity is poorly understood. Using CRISPR-Cas9 engineering, we generated a mouse model that expresses FoxM1-harboring point mutations that block binding to Rb while retaining its ability to bind CBP. Unlike FoxM1-null mice, mice harboring Rb-binding mutant FoxM1 did not exhibit significant developmental defects. The mutant mouse line developed PyMT-driven mammary tumors that were deficient in lung metastasis, which was tumor cell-intrinsic. Single-cell RNA-seq of the tumors revealed a deficiency in prometastatic tumor cells and an expansion of differentiated alveolar type tumor cells, and further investigation identified that loss of the FoxM1/Rb interaction caused enhancement of the mammary alveolar differentiation program. The FoxM1 mutant tumors also showed increased Pten expression, and FoxM1/Rb was found to activate Akt signaling by repressing Pten. In human breast cancers, expression of FoxM1 negatively correlated with Pten mRNA. Furthermore, the lack of tumor-infiltrating cells in FoxM1 mutant tumors appeared related to decreases in pro-metastatic tumor cells that express factors required for infiltration. These observations demonstrate that the FoxM1/Rb-regulated transcriptome is critical for the plasticity of breast cancer cells that drive metastasis, identifying a prometastatic role of Rb when bound to FoxM1. SIGNIFICANCE: This work provides new insights into how the interaction between FoxM1 and Rb facilitates the evolution of metastatic breast cancer cells by altering the transcriptome.

Immunoinformatics guided modeling of CCHF_GN728, an mRNA-based universal vaccine against Crimean-Congo hemorrhagic fever virus.

The Crimean-Congo hemorrhagic fever virus (CCHFV) is a lethal human pathogen belonging to the Nairoviridae family that causes Crimean-Congo hemorrhagic fever (CCHF), a tick-borne infection with an alarming mortality rate of up to 80%. CCHFV is the most widespread tick-borne virus with the potential to trigger a pandemic. To date, no vaccines or therapeutics for CCHF have been authorized. In this study, we implemented immunoinformatics approach for developing CCHF_GN728, a universal mRNA-based multi-epitope vaccine against CCHFV. Glycoprotein precursor (GPC) and nucleoprotein (NP) from the virus were selected and screened for potential immunogenic T- and B-cell epitopes. Our developed antigen exhibited the potential to generate 99.95% population coverage worldwide. Stable epitope-allele interaction was confirmed using molecular docking and dynamics simulation. In silico immune simulation corroborated immune cell response to antigen clearance rate. Optimized codons ensured efficient expression of the mRNA in the host cell. The vaccine exhibited stable and strong interactions with the Toll-like receptors. Our findings suggest that the CCHF_GN728 vaccine will trigger specific anti-CCHFV immune responses. Our model is ready for wet-lab experimentation to assess the efficacy of this putative vaccine candidate.

Fenugreek steroidal saponins hinder osteoclastogenic bone resorption by targeting CSF-1R which diminishes the RANKL/OPG ratio.

Osteoporosis is skeletal fragility caused by the excessive bone resorption due to osteoclastogenesis. But current drugs are less bioavailable and possess higher toxicity. Our study was conducted to identify safe oral bioavailable drugs from Fenugreek steroidal saponins and to delineate underlying mechanism of them to lower the osteoclastogenic bone resorption. We observed higher molecular docked binding affinities in finally selected eight hit compounds within the range of -11.0 to -10.1 kcal/mol which was greater than currently used drugs. Molecular Dynamics simulation with Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), Solvent Accessible Surface Area (SASA) and Gyration trajectory projection reinforced the stability of the protein-ligand complexes. Pharmacokinetics analysis confirmed bioavailability of seven compounds out of eight, and drug likeliness and bioavailability profile evaluation indicated that they all are eligible to be developed as a potent oral inhibitor of CSF-1R. By literature mining knowledge-driven analysis, RNAseq data and Molecular Dynamics Simulation, we proposed that, the hit derivatives block the CSF-1/CSF-1R induced phosphorylation signaling pathway in both osteoclast and osteoblast resulting in hindrance of RANK expression and formation of Reactive oxygen species (ROS) in osteoclast and osteoblast respectively, thus declines the RANKL/OPG ratio, lowering the osteoclast survival, proliferation and differentiation.

SARS-CoV-2 mutations altering regulatory properties: Deciphering host's and virus's perspectives.

Since the first recorded case of the SARS-CoV-2, it has acquired several mutations in its genome while spreading throughout the globe. In this study, we investigated the significance of these mutations by analyzing the host miRNA binding and virus's internal ribosome entry site (IRES). Strikingly, we observed that due to the acquired mutations, five host miRNAs lost their affinity for targeting the viral genome, and another five can target the mutated viral genome. Moreover, functional enrichment analysis suggests that targets of both of these miRNAs might be involved in various host immune signaling pathways. Remarkably, we detected that three particular mutations in the IRES can disrupt its secondary structure which can consequently make the virus less functional. These results could be valuable in exploring the functional importance of the mutations of SARS-CoV-2 and could provide novel insights into the differences observed different parts of the world.

Synthesis, in vitro bioassays, and computational study of heteroaryl nitazoxanide analogs.

Antiprotozoal drug nitazoxanide (NTZ) has shown diverse pharmacological properties and has appeared in several clinical trials. Herein we present the synthesis, characterization, in vitro biological investigation, and in silico study of four hetero aryl amide analogs of NTZ. Among the synthesized molecules, compound 2 and compound 4 exhibited promising antibacterial activity against Escherichia coli (E. coli), superior to that displayed by the parent drug nitazoxanide as revealed from the in vitro antibacterial assay. Compound 2 displayed zone of inhibition of 20 mm, twice as large as the parent drug NTZ (10 mm) in their least concentration (12.5 µg/ml). Compound 1 also showed antibacterial effect similar to that of nitazoxanide. The analogs were also tested for in vitro cytotoxic activity by employing cell counting kit-8 (CCK-8) assay technique in HeLa cell line, and compound 2 was identified as a potential anticancer agent having IC50 value of 172 µg which proves it to be more potent than nitazoxanide (IC50  = 428 µg). Furthermore, the compounds were subjected to molecular docking study against various bacterial and cancer signaling proteins. The in vitro test results corroborated with the in silico docking study as compound 2 and compound 4 had comparatively stronger binding affinity against the proteins and showed a higher docking score than nitazoxanide toward human mitogen-activated protein kinase (MAPK9) and fatty acid biosynthesis enzyme (FabH) of E. coli. Moreover, the docking study demonstrated dihydrofolate reductase (DHFR) and thymidylate synthase (TS) as probable new targets for nitazoxanide and its synthetic analogs. Overall, the study suggests that nitazoxanide and its analogs can be a potential lead compound in the drug development.