Evolutionary and structural analysis of SARS-CoV-2 specific evasion of host immunity.

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading fast worldwide. There is a pressing need to understand how the virus counteracts host innate immune responses. Deleterious clinical manifestations of coronaviruses have been associated with virus-induced direct dysregulation of innate immune responses occurring via viral macrodomains located within nonstructural protein-3 (Nsp3). However, no substantial information is available concerning the relationship of macrodomains to the unusually high pathogenicity of SARS-CoV-2. Here, we show that structural evolution of macrodomains may impart a critical role to the unique pathogenicity of SARS-CoV-2. Using sequence, structural, and phylogenetic analysis, we identify a specific set of historical substitutions that recapitulate the evolution of the macrodomains that counteract host immune response. These evolutionary substitutions may alter and reposition the secondary structural elements to create new intra-protein contacts and, thereby, may enhance the ability of SARS-CoV-2 to inhibit host immunity. Further, we find that the unusual virulence of this virus is potentially the consequence of Darwinian selection-driven epistasis in protein evolution. Our findings warrant further characterization of macrodomain-specific evolutionary substitutions in in vitro and in vivo models to determine their inhibitory effects on the host immune system.

Evolutionary relevance of single nucleotide variants within the forebrain exclusive human accelerated enhancer regions.

BACKGROUND: Human accelerated regions (HARs) are short conserved genomic sequences that have acquired significantly more nucleotide substitutions than expected in the human lineage after divergence from chimpanzees. The fast evolution of HARs may reflect their roles in the origin of human-specific traits. A recent study has reported positively-selected single nucleotide variants (SNVs) within brain-exclusive human accelerated enhancers (BE-HAEs) hs1210 (forebrain), hs563 (hindbrain) and hs304 (midbrain/forebrain). By including data from archaic hominins, these SNVs were shown to be Homo sapiens-specific, residing within transcriptional factors binding sites (TFBSs) for SOX2 (hs1210), RUNX1/3 (hs563), and FOS/JUND (hs304). Although these findings suggest that the predicted modifications in TFBSs may have some role in present-day brain structure, work is required to verify the extent to which these changes translate into functional variation. RESULTS: To start to fill this gap, we investigate the SOX2 SNV, with both forebrain expression and strong signal of positive selection in humans. We demonstrate that the HMG box of SOX2 binds in vitro with Homo sapiens-specific derived A-allele and ancestral T-allele carrying DNA sites in BE-HAE hs1210. Molecular docking and simulation analysis indicated highly favourable binding of HMG box with derived A-allele containing DNA site when compared to site carrying ancestral T-allele. CONCLUSION: These results suggest that adoptive changes in TF affinity within BE-HAE hs1210 and other HAR enhancers in the evolutionary history of Homo sapiens might. have brought about changes in gene expression patterns and have functional consequences on forebrain formation and evolution. METHODS: The present study employ electrophoretic mobility shift assays (EMSA) and molecular docking and molecular dynamics simulations approaches.

Visceral Leishmaniasis (Kala-Azar): A Triumph Against a Trickster Disease.

Leishmaniasis, a protozoan parasitic pathology, is caused by infection with a parasite known as Leishmania donovani, which is transmitted to humans through the bite of a sand fly. The disease has various manifestations, including cutaneous leishmaniasis, visceral leishmaniasis (VL), and disseminated cutaneous leishmaniasis. Visceral leishmaniasis (VL), also known as kala-azar, is mostly seen in the Asian and African regions and can be deadly if left untreated. We present the case of a 10-month-old male infant who was brought to the office with the chief complaints of recurrent fever for the past one month, along with generalized fatigue and poor feeding. A comprehensive history, detailed physical examination, and laboratory testing, including bone marrow aspiration, were performed, and visceral leishmaniasis was diagnosed.

Niemann-Pick Disease Type A: A Rare Disease With a Fatal Outcome.

Niemann-Pick disease (NPD) type A is a fatal autosomal recessive lysosomal storage disorder. This rare condition impairs the metabolization of lipids, leading to their accumulation within the cells. Consequently, it causes growth retardation, pancytopenia, and cellular malfunctioning in various organ systems, including ocular, hepatic, pulmonary, brain, and neuronal tissues. Although rare, these patients present in both emergency and outpatient settings. Here, we report the case of a seven-month-old male infant who presented to the emergency department with continuous fever for one week, poor feeding, and failure to thrive. After a thorough history, examination, and laboratory workup, NPD type A was diagnosed. The patient received symptomatic therapy with the continuation of conservative management. In addition, the parents received detailed counseling regarding the genetics, progressive disease course, and prognosis of this condition.

Core amino acid substitutions in HCV-3a isolates from Pakistan and opportunities for multi-epitopic vaccines.

Hepatitis C virus (HCV), which infected 71 million worldwide and about 5%-6% are from Pakistan, is an ssRNA virus, responsible for end-stage liver disease. To date, no effective therapy is available to cure this disease. Hence, it is important to study the most prevalent genotypes infecting human population and design novel vaccine or small molecule inhibitors to control the infections associated with HCV. Therefore, in this study clinical samples (n = 35; HCV-3a) from HCV patients were subjected to Sanger sequencing method. The sequencing of the core gene, which is generally considered as conserved, involved in the detection, quantitation and genotyping of HCV was performed. Multiple mutations, that is, R46C, R70Q, L91C, G60E, N/S105A, P108A, N110I, S116V, G90S, A77G and G145R that could be linked with response to antiviral therapies were detected. Phylogenetic analysis suggests emerging viral isolates are circulating in Pakistan. Using ab initio modelling technique, we predicted the 3D structure of core protein and subjected to molecular dynamics simulation to extract the most stable conformation of the structure for further analysis. Immunoinformatic approaches were used to propose a multi-epitopes vaccine against HCV by using core protein. The vaccine constructs consist of nine CTL and three HTL epitopes joined by different linkers were docked against the two reported Toll-like receptors (TLR-3 and TLR-8). Docking of vaccine construct with TLR-3 and TLR-8 shows proper binding and in silico expression of the vaccine resulted in a CAI value of 0.93. These analyses suggest that specific immune responses may be produced by the proposed vaccine.Communicated by Ramaswamy H. Sarma.

Overlap of diabetic ketoacidosis and hyperosmolar hyperglycemic state.

Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemia state (HHS) are two life-threatening metabolic complications of diabetes that significantly increase mortality and morbidity. Despite major advances, reaching a uniform consensus regarding the diagnostic criteria and treatment of both conditions has been challenging. A significant overlap between these two extremes of the hyperglycemic crisis spectrum poses an additional hurdle. It has well been noted that a complete biochemical and clinical patient evaluation with timely diagnosis and treatment is vital for symptom resolution. Worldwide, there is a lack of large-scale studies that help define how hyperglycemic crises should be managed. This article will provide a comprehensive review of the pathophysiology, diagnosis, and management of DKA-HHS overlap.

Proteome-wide mapping and reverse vaccinology-based B and T cell multi-epitope subunit vaccine designing for immune response reinforcement against Porphyromonas gingivalis.

Porphyromonas gingivalis, a prominent pathogen responsible for acute periodontal diseases, is widely studied by the scientific community for its successful evasion of the host immune system. P. gingivalis is associated with rheumatoid arthritis, dementia, and Alzheimer's. The pathogen successfully survives itself against the heavy load of conventional antibiotics because of its ability to evade the host immune system. Subtractive proteomics and reverse vaccinology approaches were employed in order to prioritize the best proteins for vaccine designing. Three vaccine candidates with Uniprot ID: Q7MWZ2 (histidine Kinase), Q7MVL1 (Fe (2+) transporter), and Q7MWZ2 (Capsular polysaccharide transport protein) were identified for vaccine designing. These proteins are antigenic and essential for pathogen survival. A wide range of immunoinformatics tools was applied for the prediction of epitopes, B, and T cells, for the vaccine candidate proteins. Molecular docking of the predicted epitopes against the MHC molecules were carried out. In-silico vaccine was constructed using carefully evaluated epitopes and consequently modeled for docking with human Toll-like receptor 2. Chain C of Pam3CSK4 (PDB ID; 2Z7X) was linked to the vaccine as an adjuvant to boost immune response towards the vaccine. For stability evaluation of the vaccine-TLR-2 docked complex, Molecular Dynamics simulations were performed. The reverse-translated nucleotide sequence cloned in Eschericia coli to attain the maximal expression of the vaccine protein. The maximal expression was ensured by CAI score of 0.96. The current vaccine requires future experimental validation to confirm its effectiveness. The vaccine developed will be helpful to protect against P. gingivalis associated infections.Communicated by Ramaswamy H. Sarma.

Recurrent Episodes of Paroxysmal Supraventricular Tachycardia Triggered by Dyspepsia: A Rare Case of Gastrocardiac Syndrome.

Supraventricular tachycardia (SVT) refers to the narrow complex tachycardia originating at or above the bundle of His. Several risk factors are associated with the development and recurrence of SVT, but its association with gastric problems, especially dyspepsia, is relatively rare. We report the case of a 54-year-old female who presented to the emergency room (ER) with palpitations, which were diagnosed as an episode of paroxysmal supraventricular tachycardia (PSVT). She had a history of PSVT in the past, along with hypertension and dyspepsia. After thorough history and examination, dyspepsia was identified as the common trigger of her PSVT episodes, pointing towards the likelihood of gastrocardiac symptoms. Therefore, an appropriate regimen of beta-blockers, proton pump inhibitors (PPIs), and anti-foaming agents (simethicone) was prescribed to manage her symptoms with the plan to perform a catheter ablation later.

Mutational Landscape of Pirin and Elucidation of the Impact of Most Detrimental Missense Variants That Accelerate the Breast Cancer Pathways: A Computational Modelling Study.

Pirin (PIR) protein is highly conserved in both prokaryotic and eukaryotic organisms. Recently, it has been identified that PIR positively regulates breast cancer cell proliferation, xenograft tumor formation, and metastasis, through an enforced transition of G1/S phase of the cell cycle by upregulation of E2F1 expression at the transcriptional level. Keeping in view the importance of PIR in many crucial cellular processes in humans, we used a variety of computational tools to identify non-synonymous single-nucleotide polymorphisms (SNPs) in the PIR gene that are highly deleterious for the structure and function of PIR protein. Out of 173 SNPs identified in the protein, 119 are non-synonymous, and by consensus, 24 mutations were confirmed to be deleterious in nature. Mutations such as V257A, I28T, and I264S were unveiled as highly destabilizing due to a significant stability fold change on the protein structure. This observation was further established through molecular dynamics (MD) simulation that demonstrated the role of the mutation in protein structure destability and affecting its internal dynamics. The findings of this study are believed to open doors to investigate the biological relevance of the mutations and drugability potential of the protein.

Immunogenomics guided design of immunomodulatory multi-epitope subunit vaccine against the SARS-CoV-2 new variants, and its validation through in silico cloning and immune simulation.

Reports of the novel and more contagious strains of SARS-CoV-2 originating in different countries have further aggravated the pandemic situation. The recent substitutions in spike protein may be critical for the virus to evade the host's immune system and therapeutics that have already been developed. Thus, this study has employed an immunoinformatics pipeline to target the spike protein of this novel strain to construct an immunogenic epitope (CTL, HTL, and B cell) vaccine against the new variant. Our investigation revealed that 12 different epitopes imparted a critical role in immune response induction. This was validated by an exploration of physiochemical properties and experimental feasibility. In silico and host immune simulation confirmed the expression and induction of both primary and secondary immune factors such as IL, cytokines, and antibodies. The current study warrants further lab experiments to demonstrate its efficacy and safety.

Combined drug repurposing and virtual screening strategies with molecular dynamics simulation identified potent inhibitors for SARS-CoV-2 main protease (3CLpro).

The current coronavirus (SARS-COV-2) pandemic and phenomenal spread to every nook and cranny of the world has raised major apprehensions about the modern public health care system. So far as a result of this epidemic, 4,434,653 confirmed cases and 302,169 deaths are reported. The growing infection rate and death toll demand the use of all possible approaches to design novel drugs and vaccines to curb this disease. In this study, we combined drugs repurposing and virtual drug screening strategies to target 3CLpro, which has an essential role in viral maturation and replication. A total of 31 FDA approved anti-HIV drugs, and Traditional Chinese medicines (TCM) database were screened to find potential inhibitors. As a result, Saquinavir, and five drugs (TCM5280805, TCM5280445, TCM5280343, TCM5280863, and TCM5458190) from the TCM database were found as promising hits. Furthermore, results from molecular dynamics simulation and total binding free energy revealed that Saquinavir and TCM5280805 target the catalytic dyad (His41 and Cys145) and possess stable dynamics behavior. Thus, we suggest that these compounds should be tested experimentally against the SARS-COV-2 as Saquinavir has been reported to inhibit HIV protease experimentally. Considering the intensity of coronavirus dissemination, the present research is in line with the idea of discovering the latest inhibitors against the coronavirus essential pathways to accelerate the drug development cycle.Communicated by Ramaswamy H. Sarma.

Structural insights into the mechanism of RNA recognition by the N-terminal RNA-binding domain of the SARS-CoV-2 nucleocapsid phosphoprotein.

The emergence of recent SARS-CoV-2 has become a global health issue. This single-stranded positive-sense RNA virus is continuously spreading with increasing morbidities and mortalities. The proteome of this virus contains four structural and sixteen nonstructural proteins that ensure the replication of the virus in the host cell. However, the role of phosphoprotein (N) in RNA recognition, replicating, transcribing the viral genome, and modulating the host immune response is indispensable. Recently, the NMR structure of the N-terminal domain of the Nucleocapsid Phosphoprotein has been reported, but its precise structural mechanism of how the ssRNA interacts with it is not reported yet. Therefore, here, we have used an integrated computational pipeline to identify the key residues, which play an essential role in RNA recognition. We generated multiple variants by using an alanine scanning strategy and performed an extensive simulation for each system to signify the role of each interfacial residue. Our analyses suggest that residues T57A, H59A, S105A, R107A, F171A, and Y172A significantly affected the dynamics and binding of RNA. Furthermore, per-residue energy decomposition analysis suggests that residues T57, H59, S105 and R107 are the key hotspots for drug discovery. Thus, these residues may be useful as potential pharmacophores in drug designing.

Phylogenetic Analysis and Structural Perspectives of RNA-Dependent RNA-Polymerase Inhibition from SARs-CoV-2 with Natural Products.

Most recently, an outbreak of severe pneumonia caused by the infection of SARS-CoV-2, a novel coronavirus first identified in Wuhan, China, imposes serious threats to public health. Upon infecting host cells, coronaviruses assemble a multi-subunit RNA-synthesis complex of viral non-structural proteins (nsp) responsible for the replication and transcription of the viral genome. Therefore, the role and inhibition of nsp12 are indispensable. A cryo-EM structure of RdRp from SARs-CoV-2 was used to identify novel drugs from Northern South African medicinal compounds database (NANPDB) by using computational virtual screening and molecular docking approaches. Considering Remdesivir as the control, 42 compounds were shortlisted to have docking score better than Remdesivir. The top 5 hits were validated by using molecular dynamics simulation approach and free energy calculations possess strong inhibitory properties than the Remdesivir. Thus, this study paved a way for designing novel drugs by decoding the architecture of an important enzyme and its inhibition with compounds from natural resources. This disclosing of necessary knowledge regarding the screening and the identification of top hits could help to design effective therapeutic candidates against the coronaviruses and design robust preventive measurements.

Subtractive proteomics and immunoinformatics approaches to explore Bartonella bacilliformis proteome (virulence factors) to design B and T cell multi-epitope subunit vaccine.

Bartonella bacilliformis a gram-negative facultative aerobe responsible for the Carrion's disease widely distributed in Ecuador, Peru, and Colombia with a high mortality rate when no specific treatment is received. B bacilliformis is transmitted by Sand fly (Lutzomyia verrucarum) to healthy individuals. Immunoinformatic and subtractive proteomics approaches were employed in this study to prioritize the best candidates for vaccine designing. These approaches resulted in five vaccine candidates, flagellar biosynthetic protein (Uniprot ID: A1UTU1), heme exporter protein C (UniProt ID: A1UU82), Cytochrome c-type biogenesis protein (Uniprot ID: A1URZ7), Hemin ABC transporter (Uniprot ID: A1US20) and Phosphatidate cytidylyltransferase (Uniprot ID: A1USE3). The mentioned proteins are antigenic and essential for pathogen survival. A range of immune-informatics tools was applied for the prediction of B and T cell epitopes for the vaccine candidate proteins. In-silico vaccine was constructed using carefully evaluated epitopes and consequently modeled for docking with human Toll-like receptor 4. TLR-4 agonist 50S ribosomal protein L7/L12 (UniproKB ID; P9WHE3) was linked to the vaccine as an adjuvant to boost immune response towards the vaccine. For stability evaluation of the vaccine-TLR-4 docked complex, MD simulations were performed. The final vaccine was back-translated and cloned in Eschericia coli to attain the maximal expression of the vaccine protein. The maximal expression was ensured, and the CAI score of 0.96 was reported. The current vaccine requires future experimental validation to confirm its effectiveness. The vaccine developed will be helpful to protect against B bacilliformis associated infections.

Immunoinformatic and systems biology approaches to predict and validate peptide vaccines against Epstein-Barr virus (EBV).

Epstein-Barr virus (EBV), also known as human herpesvirus 4 (HHV-4), is a member of the Herpesviridae family and causes infectious mononucleosis, Burkitt's lymphoma, and nasopharyngeal carcinoma. Even in the United States of America, the situation is alarming, as EBV affects 95% of the young population between 35 and 40 years of age. In this study, both linear and conformational B-cell epitopes as well as cytotoxic T-lymphocyte (CTL) epitopes were predicted by using the ElliPro and NetCTL.1.2 webservers for EBV proteins (GH, GL, GB, GN, GM, GP42 and GP350). Molecular modelling tools were used to predict the 3D coordinates of peptides, and these peptides were then docked against the MHC molecules to obtain peptide-MHC complexes. Studies of their post-docking interactions helped to select potential candidates for the development of peptide vaccines. Our results predicted a total of 58 T-cell epitopes of EBV;  where the most potential were selected based on their TAP, MHC binding and C-terminal Cleavage score. The top most peptides were subjected to MD simulation and stability analysis. Validation of our predicted epitopes using a 0.45 µM concentration was carried out by using a systems biology approach. Our results suggest a panel of epitopes that could be used to immunize populations to protect against multiple diseases caused by EBV.

Dynamics Insights Into the Gain of Flexibility by Helix-12 in ESR1 as a Mechanism of Resistance to Drugs in Breast Cancer Cell Lines.

Incidents of breast cancer (BC) are on the rise on a daily basis and have proven to be the most prevelant cause of death for women in both developed and developing countries. Among total BC cases diagnosed after menopause, 70% of cases are Estrogen Receptor (ER) positive (ER-positive or ER+). Mutations in the LBD (ligand-binding domain) of the ER have recently been reported to be the major cause of resistance to potent antagonists. In this study, the experimentally reported mutations K303R, E380Q, V392I, S463P, V524E, P535H, P536H, Y537C, Y537N, Y537S, and D538G were analyzed, and the most significant mutations were shortlisted based on multiple analyses. Initial analyses, such as mCSM stability, occluded depth analysis, mCSM-binding affinity, and FoldX energy changes shortlisted only six mutations as being highly resistant. Finally, simulations of force field-based molecular dynamics (MD on wild type (WT) ERα) on six mERα variants (E380Q, S463P, Y537S, Y537C, Y537N, and D538G) were carried out to justify mechanism of the resistance. It was observed that these mutations increased the flexibility of the H12. A bonding analysis suggested that previously reported important residue His524 lost bonding upon mutation. Other parameters, such as PCA (principal component analysis), DCCM (dynamics cross-correlation), and FEL (free energy landscape), verified that the shortlisted mutations affect the H12 helix, which opens up the co-activator binding conformation. These results provide deep insight into the mechanism of relative resistance posed to fulvestrant due to mutations in breast cancer. This study will facilitate further understanding of the important aspects of designing specific and more effective drugs.

Allosteric ligands for the pharmacologically important Flavivirus target (NS5) from ZINC database based on pharmacophoric points, free energy calculations and dynamics correlation.

Dengue virus belongs to a group of human pathogens, which causes different diseases, dengue hemorrhagic fever and dengue shock syndrome in humans. It possesses RNA as a genetic material and is replicated with the aid of NS5 protein. RNA-dependent RNA polymerase (RdRp) is an important domain of NS5 in the replication of that virus. The catalytic process activity of RdRp is making it an important target for antiviral chemical therapy. To date, No FDA drug has been approved and marketed for the treatment of diseases caused by Dengue virus. So, there is a dire need to advance an area of active antiviral inhibitors that is safe, less expensive and widely available. An experimentally validated complex of Dengue NS5 and compound 27 (6LS) were used as pharmacophoric input and hits were identified. We also used Molecular dynamics (MD) simulations alongside free energy and dynamics of the internal residues of the apo and holo systems to understand the binding mechanism. Our analysis resulted that the three inhibitors (ZINC72070002, ZINC6551486, and ZINC39588257) greatly affected the interior dynamics and residual signaling to dysfunction the replicative role of NS5. The interaction of these inhibitors caused the loss of the correlated motion of NS5 near to the N terminus and helped the stability of the binding complex. This investigation provided a methodological route to discover allosteric inhibitors against the epidemics of this Flaviviruses. Allosteric inhibitors are important and major assets in considering the development of the competitive and robust antiviral agents such as against Dengue viral infection.

In-vitro analysis of selective nutraceuticals binding to human transcription factors through computer aided molecular docking predictions.

The contest of cancer couldn't be completed without novel drug with novel modes of action, improved efficacy and acceptable pharmacokinetic properties. Transcription factors are attractive targets to develop anti-cancerous drugs. 6-Gingerol, Anethol analogues, Capsaicinoids, Curcumin, Dibenzoylmethane, Diosgenin, Eugenol, Gambogic acid, Thymoquinone, Ursolic acid, Xanthohumol, Zerumbone are the promising nutraceuticals that help in the prevention of cancer. These nutraceuticals showed promising activity in invitro tests. In this study In-silico tools were applied to confirm the activity of these nutraceuticals against the transcription factors including Nuclear Factor-Kappa B (NF-κB), AP-1, NRF2, PPAR-γ, ß-catenin/Wnt and Sonic Hedgehog. This studied followed molecular docking based approach to verify the in-vitro activities of the said nutraceuticals against the cancer. Molecular Docking based approached provide a path towards the identification of novel ligands against these transcription factors. Based on the interaction of Cardamoninand capsaicin it was found to have an influencing role against the transcription factor like NF-κB andPPAR-γ. The interaction of Cardamoninwith NF- κBand capsaicinwith PPAR-γ provide a way toward structure-based virtual screening to identify novel ligands against the targets which could be very help full in successful chemotherapy of cancer. This study delivers structural features of nutraceuticals and its interactions against different transcription factors and gives a theoretical entry to use these compounds as a potential inhibitor against the transcription factors involved in cancer.