Structural and functional insights into a novel homozygous missense pathogenic variant in CUL7 identified in consanguineous Pakistani family.

3M syndrome is a rare genetic familial disorder characterized by short stature, growth retardation, facial dysmorphism, skeletal abnormalities, fleshy protruding heels, and normal intelligence, caused by mutations in the CUL7, OBSL1 and CCDC8 genes. In the present study, a novel homozygous missense variant of CUL7 (NP_001161842.1, c.4493T > C, p.L1498P) has been identified in a consanguineous Pakistani family by whole exome sequencing. In silico structural evaluation, molecular docking and simulation studies of mutant CUL7 provides substantial evidence about its crucial role in the progression of discussed ailment. The newly discovered variant significantly altered the protein's three dimensional structure, leading to abnormal interaction with binding proteins. This computational and experimental investigation provides useful information to drug developers for the synthesis of novel therapeutics against the discussed ailment.Communicated by Ramaswamy H. Sarma.

Discovering Potential Bacteriocins Against Pseudomonas fragi: a Subtractive Proteomics and Molecular Dynamic Simulation Study for Food Preservation.

Food preservation is a schematic and scientific procedure employed for the maintenance and improvement of food's quality, shelf life, and nutritional value. Although, on one hand, ancient conventional methods such as freezing, pasteurization, canning, and chemical methods have the potential to lengthen the shelf life of edible substances, but on the other hand, they can deteriorate its nutritional value as well. Present research focuses on the identification of promising bacteriocins against Pseudomonas fragi via subtractive proteomics pipeline as an alternative approach for food preservation. Bacteriocins are small peptides produced by certain microbes to naturally defend themselves by destroying other closely related bacteria residing in their neighborhood. P. fragi lies among the most notable microbes responsible for the elicitation of food spoilage. Due to increasing emergence and prevalence of multidrug resistance bacteria, there is a need to unravel novel drug targets, crucially involved in food decay process. Based on subtractive scrutinization, UDP-N-acetylglucosamine O-acyltransferase (LpxA) was chosen as promising therapeutic protein target that could play a significant role in progression of food spoilage. Subtilosin A, thuricin-CD, and mutacin B-NY266 were found as the most robust inhibitors of LpxA according to the molecular docking assay results. Molecular dynamic simulations and binding energy calculations via MM/PBSA method of LpxA and three top hit docked complexes, i.e., LpxA-subtilosin A, LpxA-thuricin-CD, and LpxA-mutacin B-NY266, revealed stability throughout simulations and ensured that shortlisted bacteriocins had strong affinity for LpxA.

Discovering potential stabilizers for KRAS22RT G-quadruplex DNA: an alternative next generation approach to treat pancreatic cancer.

KRAS is the signature gene responsible for the occurrence of pancreatic cancer, which is a complex, multifactorial and intractable lethal malignancy. Prevention and treatment of the ailment have always been a key motivation behind the search for new therapeutic drug molecules. G-quadruplexes are non-canonical guanine-rich secondary structures, commonly formed at eukaryotic telomeric ends, oncogenic promotors and G-rich regions of the DNA. These G-quadruplexes play a crucial role in the regulation of gene expression and maintenance of genome integrity, therefore, they are considered as emerging potential therapeutic drug targets. The present study is concerned with the discovery of a potential stabilizer for KRAS22RT G-quadruplex DNA, located in the NHE region of the promotor, while inhibiting the upregulation of KRAS proto-oncogene, as an alternative approach for the treatment of pancreatic cancer. Various chemical libraries have been virtually screened against the targeted G4 structure and 143 compounds showed promising results. However, molecular dynamic studies, ADME and toxicity analyses predicted that three compounds belonging to the class of tetra-substituted phenanthrolines (i.e., 7i, 7j and 7k) can not only effectively stabilize KRAS22RT G4 structure but also have least toxic effects in the in vivo system. Therefore, it is highly recommended to further investigate their effectiveness and efficacy through experimental analysis in laboratory.Communicated by Ramaswamy H. Sarma.

Immuno-informatics profiling of monkeypox virus cell surface binding protein for designing a next generation multi-valent peptide-based vaccine.

Monkeypox is a viral etiological agent with hallmarks analogous to those observed in smallpox cases in the past. The ongoing outbreak of Monkeypox viral infection is becoming a global health problem. Multi-valent peptide based next generation vaccines provides us a promising solution to combat these emerging infectious diseases by eliciting cell-mediated and humoral immune response. Considering the success rate of subtractive proteomics pipeline and reverse vaccinology approach, in this study, we have developed a novel, next-generation, multi-valent, in silico peptide based vaccine construct by employing cell surface binding protein. After analyzing physiochemical and biological properties of the selected target, the protein was subjected to B cell derived T cell epitope mapping. Iterative scrutinization lead to the identification of two highly antigenic, virulent, non-allergic, non-toxic, water soluble, and Interferon-gamma inducer epitopes i.e. HYITENYRN and TTSPVRENY. We estimated that the shortlisted epitopes for vaccine construction, roughly correspond to 99.74% of the world's population. UK, Finland and Sweden had the highest overall population coverage at 100% which is followed by Austria (99.99%), Germany (99.99%), France (99.98%), Poland (99.96), Croatia (99.93), Czech Republic (99.87%), Belgium (99.87), Italy (99.86%), China (97.83%), India (97.35%) and Pakistan (97.13%). The designed vaccine construct comprises of 150 amino acids with a molecular weight of 16.97242 kDa. Molecular docking studies of the modelled MEMPV (Multi-epitope Monkeypox Vaccine) with MHC I (PDB ID: 1I1Y), MHC II (PDB ID: 1KG0), and other immune mediators i.e. toll like receptors TLR3 (PDB ID: 2A0Z), and TLR4 (PDB ID: 4G8A) revealed strong binding affinity with immune receptors. Host immune simulation results predicted that the designed vaccine has strong potency to induce immune responses against target pathogen in the form of cellular and antibody-dependent immunity. Our findings suggest that the hypothesized vaccine candidate can be utilized as a potential therapeutic against Monkeypox however experimental study is required to validate the results and safe immunogenicity.

Design of Multi-Epitope Vaccine for Staphylococcus saprophyticus: Pan-Genome and Reverse Vaccinology Approach.

Staphylococcus saprophyticus is a Gram-positive coccus responsible for the occurrence of cystitis in sexually active, young females. While effective antibiotics against this organism exist, resistant strains are on the rise. Therefore, prevention via vaccines appears to be a viable solution to address this problem. In comparison to traditional techniques of vaccine design, computationally aided vaccine development demonstrates marked specificity, efficiency, stability, and safety. In the present study, a novel, multi-epitope vaccine construct was developed against S. saprophyticus by targeting fully sequenced proteomes of its five different strains, which were examined using a pangenome and subtractive proteomic strategy to characterize prospective vaccination targets. The three immunogenic vaccine targets which were utilized to map the probable immune epitopes were verified by annotating the entire proteome. The predicted epitopes were further screened on the basis of antigenicity, allergenicity, water solubility, toxicity, virulence, and binding affinity towards the DRB*0101 allele, resulting in 11 potential epitopes, i.e., DLKKQKEKL, NKDLKKQKE, QDKLKDKSD, NVMDNKDLE, TSGTPDSQA, NANSDGSSS, GSDSSSSNN, DSSSSNNDS, DSSSSDRNN, SSSDRNNGD, and SSDDKSKDS. All these epitopes have the efficacy to cover 99.74% of populations globally. Finally, shortlisted epitopes were joined together with linkers and three different adjuvants to find the most stable and immunogenic vaccine construct. The top-ranked vaccine construct was further scrutinized on the basis of its physicochemical characterization and immunological profile. The non-allergenic and antigenic features of modeled vaccine constructs were initially validated and then subjected to docking with immune receptor major histocompatibility complex I and II (MHC-I and II), resulting in strong contact. In silico cloning validations yielded a codon adaptation index (CAI) value of 1 and an ideal percentage of GC contents (46.717%), indicating a putative expression of the vaccine in E. coli. Furthermore, immune simulation demonstrated that, after injecting the proposed MEVC, powerful antibodies were produced, resulting in the sharpest peaks of IgM + IgG formation (>11,500) within 5 to 15 days. Experimental testing against S. saprophyticus can evaluate the safety and efficacy of these prophylactic vaccination designs.

Identification of Novel and Safe Fungicidal Molecules against Fusarium oxysporum from Plant Essential Oils: In Vitro and Computational Approaches.

Phytopathogenic fungi are serious threats in the agriculture sector especially in fruit and vegetable production. The use of plant essential oil as antifungal agents has been in practice from many years. Plant essential oils (PEOs) of Cuminum cyminum, Trachyspermum ammi, Azadirachta indica, Syzygium aromaticum, Moringa oleifera, Mentha spicata, Eucalyptus grandis, Allium sativum, and Citrus sinensis were tested against Fusarium oxysporum. Three phase trials consist of lab testing (MIC and MFC), field testing (seed treatment and foliar spray), and computer-aided fungicide design (CAFD). Two concentrations (25 and 50 µl/ml) have been used to asses MIC while MFC was assessed at four concentrations (25, 50, 75, and 100 µl/ml). C. sinensis showed the largest inhibition zone (47.5 and 46.3 m2) for both concentrations. The lowest disease incidence and disease severity were recorded in treatments with C. sinensis PEO. Citrus sinensis that qualified in laboratory and field trials was selected for CAFD. The chemical compounds of C. sinensis PEO were docked with polyketide synthase beta-ketoacyl synthase domain of F. oxysporum by AutoDock Vina. The best docked complex was formed by nootkatone with -6.0 kcal/mol binding affinity. Pharmacophore of the top seven C. sinensis PEO compounds was used for merged pharmacophore generation. The best pharmacophore model with 0.8492 score was screened against the CMNP database. Top hit compounds from screening were selected and docked with polyketide synthase beta-ketoacyl synthase domain. Four compounds with the highest binding affinity and hydrogen bonding were selected for confirmation of lead molecule by doing MD simulation. The polyketide synthase-CMNPD24498 showed the highest stability throughout 80 ns run of MD simulation. CMNPD24498 (FW054-1) from Verrucosispora was selected as the lead compound against F. oxysporum.

Design of a Multi-Epitopes Vaccine against Hantaviruses: An Immunoinformatics and Molecular Modelling Approach.

Hantaviruses are negative-sense, enveloped, single-stranded RNA viruses of the family Hantaviridae. In recent years, rodent-borne hantaviruses have emerged as novel zoonotic viruses posing a substantial health issue and socioeconomic burden. In the current research, a reverse vaccinology approach was applied to design a multi-epitope-based vaccine against hantavirus. A set of 340 experimentally reported epitopes were retrieved from Virus Pathogen Database and Analysis Resource (ViPR) and subjected to different analyses such as antigenicity, allergenicity, solubility, IFN gamma, toxicity, and virulent checks. Finally, 10 epitopes which cleared all the filters used were linked with each other through specific GPGPG linkers to construct a multi-antigenic epitope vaccine. The designed vaccine was then joined to three different adjuvants-TLR4-agonist adjuvant, ß-defensin, and 50S ribosomal protein L7/L12-using an EAAAK linker to boost up immune-stimulating responses and check the potency of vaccine with each adjuvant. The designed vaccine structures were modelled and subjected to error refinement and disulphide engineering to enhance their stability. To understand the vaccine binding affinity with immune cell receptors, molecular docking was performed between the designed vaccines and TLR4; the docked complex with a low level of global energy was then subjected to molecular dynamics simulations to validate the docking results and dynamic behaviour. The docking binding energy of vaccines with TLR4 is -29.63 kcal/mol (TLR4-agonist), -3.41 kcal/mol (ß-defensin), and -11.03 kcal/mol (50S ribosomal protein L7/L12). The systems dynamics revealed all three systems to be highly stable with a root-mean-square deviation (RMSD) value within 3 Å. To test docking predictions and determine dominant interaction energies, binding free energies of vaccine(s)-TLR4 complexes were calculated. The net binding energy of the systems was as follows: TLR4-agonist vaccine with TLR4 (MM-GBSA, -1628.47 kcal/mol and MM-PBSA, -37.75 kcal/mol); 50S ribosomal protein L7/L12 vaccine with TLR4 complex (MM-GBSA, -194.62 kcal/mol and MM-PBSA, -150.67 kcal/mol); ß-defensin vaccine with TLR4 complex (MM-GBSA, -9.80 kcal/mol and MM-PBSA, -42.34 kcal/mol). Finally, these findings may aid experimental vaccinologists in developing a very potent hantavirus vaccine.