Phytochemical and biological screening of Berberis aristata

Background: Berberis aristata occupies significant position as a medicinal plant. Given its clinical applications and the grave concern of weed based crop damage in Pakistan, the plant was investigated for its antimicrobial and allelopathic activities.Methods: Fresh Berberis aristata plant was obtained from Rawalakot and Hajeera (District Poonch) Azad Kashmir. Methanolic extract preparation and phytochemical analysis was done using standard procedures. Antibacterial and antifungal activities of the root, stem and leaf extracts of the plant were assayed against the bacterial strains E. coli, S. typhi, S. aureus, Shigella, Citrobacter, P. vulgaris, Enterobacter, S. pyrogenes, V. cholera and Klebsiella spp. and fungal strains A. niger,Cladosporium, Rhizoctonia, Alternaria, Trichoderma, Penicillium, Curvularia, Paecilomyces andRhizopus using disc diffusion method. Also, the phytoxicity of the extracts was evaluated againstLemna minor and the data was recorded after seven days.Results: Phytochemical screening of the three extracts identified the presence of alkaloids, reducing sugars, steroids, flavonoids, terpenoids, glycosides and saponins while tannins were found to be absent. The leaf extract also showed negative tests for alkaloids and steroids. The extracts significantly inhibited the growth of the employed microbial isolates. The leaf extract, however, was not active against A. niger, Curvularia, Paecilomyces and Rhizopus. For most of the tested strains, the effectiveness of the extracts was much higher than that of Amoxicillin and Fluconazole; the positive controls used for bacterial and fungal cultures, respectively. All the extracts demonstrated 100% phytotoxicity against Lemna minor at 1000 μg/mL while low activity (10-20%) was observed at 10 μg/mL and 100 μg/mL, respectively.Conclusion: The results strongly support the profound ethnobotanical applications of this plant and also demonstrate its potential for use in weed control strategies

Protein Engineering of Endoglucanase CelR of Clostridium thermocellum for Enhanced Expression

Background: Enhanced production and improved properties of cellulases for a greater activity on plant biomass would rank amongst the top priorities for second-generation ethanol production. Based on the emergence of protein engineering as a cutting-edge technology for enhancing enzyme activity and expression level, the present study is aimed at the application of this technique to the major cellulosomal processing endoglucanase of C. thermocellum, CelR for refining enzyme characteristics. Methods: The full-length native enzyme gene (CelR) and a truncated version without the docking domains at C-terminus (CelR-CB) were PCR amplified using gene specific primers. The amplified PCR products were T/A cloned in the vector pTZ57 R/T and transformed in E. coli DH5α. The cellulase genes from the confirmed transformed plasmids were sub-cloned in T7 promoter-based expression vector pET-28a and expression analysis was done in E. coli (DE3) BL21 codon Plus. Results: An SDS PAGE analysis of both the CelR derivatives revealed that the truncated version i.e. CelR-CB showed a two-fold increase in expression level as compared to the full-length enzyme. Conclusion: The increased expression level of CelR in E. coli coupled with its increased production therefore makes it a promising method for augmenting the recombinant enzyme production for potential applications.

Antigenic Peptide Prediction From E6 and E7 Oncoproteins of HPV Types 16 and 18 for Therapeutic Vaccine Design Using Immunoinformatics and MD Simulation Analysis

Human papillomavirus (HPV) induced cervical cancer is the second most common cause of death, after breast cancer, in females. Three prophylactic vaccines by Merck Sharp & Dohme (MSD) and GlaxoSmithKline (GSK) have been confirmed to prevent high-risk HPV strains but these vaccines have been shown to be effective only in girls who have not been exposed to HPV previously. The constitutively expressed HPV oncoproteins E6 and E7 are usually used as target antigens for HPV therapeutic vaccines. These early (E) proteins are involved, for example, in maintaining the malignant phenotype of the cells. In this study, we predicted antigenic peptides of HPV types 16 and 18, encoded by E6 and E7 genes, using an immunoinformatics approach. To further evaluate the immunogenic potential of the predicted peptides, we studied their ability to bind to class I major histocompatibility complex (MHC-I) molecules in a computational docking study that was supported by molecular dynamics (MD) simulations and estimation of the free energies of binding of the peptides at the MHC-I binding cleft. Some of the predicted peptides exhibited comparable binding free energies and/or pattern of binding to experimentally verified MHC-I-binding epitopes that we used as references in MD simulations. Such peptides with good predicted affinity may serve as candidate epitopes for the development of therapeutic HPV peptide vaccines

An Immunoinformatics Approach to Design a Potent Multi-Epitope Vaccine against Asia-1 Genotype of Crimean–Congo Haemorrhagic Fever Virus Using the Structural Glycoproteins as a Target

Crimean–Congo haemorrhagic fever (CCHF), caused by Crimean–Congo haemorrhagic fever virus (CCHFV), is a disease of worldwide importance (endemic yet not limited to Asia, Middle East, and Africa) and has triggered several outbreaks amounting to a case fatality rate of 10–40% as per the World Health Organization. Genetic diversity and phylogenetic data revealed that the Asia-1 genotype of CCHFV remained dominant in Pakistan, where 688 confirmed cases were reported between the 2012–2022 period. Currently, no approved vaccine is available to tackle the viral infection. Epitope-based vaccine design has gained significant attention in recent years due to its safety, timeliness, and cost efficiency compared to conventional vaccines. In the present study, we employed a robust immunoinformatics-based approach targeting the structural glycoproteins G1 and G2 of CCHFV (Asia-1 genotype) to design a multi-epitope vaccine construct. Five B-cells and six cytotoxic T-lymphocytes (CTL) epitopes were mapped and finalized from G1 and G2 and were fused with suitable linkers (EAAAK, GGGS, AAY, and GPGPG), a PADRE sequence (13 aa), and an adjuvant (50S ribosomal protein L7/L12) to formulate a chimeric vaccine construct. The selected CTL epitopes showed high affinity and stable binding with the binding groove of common human HLA class I molecules (HLA-A*02:01 and HLA-B*44:02) and mouse major histocompatibility complex class I molecules. The chimeric vaccine was predicted to be an antigenic, non-allergenic, and soluble molecule with a suitable physicochemical profile. Molecular docking and molecular dynamics simulation indicated a stable and energetically favourable interaction between the constructed antigen and Toll-like receptors (TLR2, TLR3, and TLR4). Our results demonstrated that innate, adaptive, and humoral immune responses could be elicited upon administration of such a potent muti-epitope vaccine construct. These results could be helpful for an experimental vaccinologist to develop an effective vaccine against the Asia-1 genotype of CCHFV