Comprehensive bioinformatics-based annotation and functional characterization of bovine chymosin protein revealed novel biological insights.

Chymosin, an aspartic protease present in the stomachs of young ruminants like cows (bovine), causes milk coagulation and cheese production through the breakdown of κ-casein peptide bonds at the Met105-Phe106 site. Bovine chymosin is first synthesized as a pre-prochymosin that is cleaved to produce the mature chymosin protein. Despite significant strides in research, our understanding of this crucial enzyme remains incomplete. The purpose of this work was to perform in silico evolutionary and functional analysis and to gain unique insights into the structure of this protein. For this, the sequence of Bos taurus chymosin from UniProt database was subjected to various bioinformatics analyses. We found that bovine chymosin is a low molecular weight and hydrophilic protein that has homologs in other Bovidae species. Two active sites of aspartic peptidases, along with a functional domain, were identified. Gene Ontology analysis further confirmed chymosin's involvement in proteolysis and aspartic endopeptidase activity. Potential disordered residues and post-translational modification sites were also uncovered. It was revealed that the secondary structure of bovine chymosin is comprised of beta strands (44.27%), coils (43.65%), and alpha helices (12.07%). A highly optimized 3D structure was also obtained. Moreover, crucial protein-protein interactions were unveiled. Altogether, these findings provide valuable insights that could guide future research on bovine chymosin and its biological roles.

Immunoinformatics-Aided Analysis of RSV Fusion and Attachment Glycoproteins to Design a Potent Multi-Epitope Vaccine.

Respiratory syncytial virus (RSV) usually causes respiratory tract infections of upper airways in infants and young children. Despite recent medical advances, no approved vaccine is available to control RSV infections. Therefore, we conducted an immunoinformatics study to design and evaluate a potential multi-epitope vaccine against RSV. Sequence-based analyses of the glycoproteins F and G revealed a total of eight CD8 T-cell and three CD4 T-cell epitopes after considering antigenicity, binding affinity and other parameters. Molecular docking analysis confirmed that these T-cell epitopes developed strong structural associations with HLA allele(s). By integrating these prioritized epitopes with linkers and a cholera toxin-derived adjuvant, a multi-epitope vaccine was designed. The developed vaccine was found to be stable, non-allergenic, flexible and antigenic. Molecular docking analysis revealed a striking mean HADDOCK score (-143.3) of top-ranked vaccine-TLR cluster and a Gibbs free energy change (ΔG) value of -11.3 kcal mol-1. As per computational immune simulation results, the vaccine generated a high titer of antibodies (especially IgM) and effector T-cells. Also, codon optimization and in silico cloning ensured the increased expression of vaccine in Escherichia coli. Altogether, we anticipate that the multi-epitope vaccine reported in this study will stimulate humoral and cellular responses against RSV infection, subject to follow-up experimental validation.

Genomic Investigation of Methicillin-Resistant Staphylococcus aureus ST113 Strains Isolated from Tertiary Care Hospitals in Pakistan.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multi-drug resistant and opportunistic pathogen. The emergence of new clones of MRSA in both healthcare settings and the community warrants serious attention and epidemiological surveillance. However, epidemiological data of MRSA isolates from Pakistan are limited. We performed a whole-genome-based comparative analysis of two (P10 and R46) MRSA strains isolated from two provinces of Pakistan to understand the genetic diversity, sequence type (ST), and distribution of virulence and antibiotic-resistance genes. The strains belong to ST113 and harbor the SCCmec type IV encoding mecA gene. Both the strains contain two plasmids, and three and two complete prophage sequences are present in P10 and R46, respectively. The specific antibiotic resistance determinants in P10 include two aminoglycoside-resistance genes, aph(3')-IIIa and aad(6), a streptothrin-resistance gene sat-4, a tetracycline-resistance gene tet(K), a mupirocin-resistance gene mupA, a point mutation in fusA conferring resistance to fusidic acid, and in strain R46 a specific plasmid associated gene ant(4')-Ib. The strains harbor many virulence factors common to MRSA. However, no Panton-Valentine leucocidin (lukF-PV/lukS-PV) or toxic shock syndrome toxin (tsst) genes were detected in any of the genomes. The phylogenetic relationship of P10 and R46 with other prevailing MRSA strains suggests that ST113 strains are closely related to ST8 strains and ST113 strains are a single-locus variant of ST8. These findings provide important information concerning the emerging MRSA clone ST113 in Pakistan and the sequenced strains can be used as reference strains for the comparative genomic analysis of other MRSA strains in Pakistan and ST113 strains globally.

Designing a multi-epitope vaccine against Mycobacteroides abscessus by pangenome-reverse vaccinology.

Mycobacteroides abscessus (Previously Mycobacterium abscessus) is an emerging microorganism of the newly defined genera Mycobacteroides that causes mainly skin and tissue diseases in humans. The recent availability of total 34 fully sequenced genomes of different strains belonging to this species has provided an opportunity to utilize this genomics data to gain novel insights and guide the development of specific antimicrobial therapies. In the present study, we collected collectively 34 complete genome sequences of M. abscessus from the NCBI GenBank database. Pangenome analysis was conducted on these genomes to understand the genetic diversity and to obtain proteins associated with its core genome. These core proteins were then subjected to various subtractive filters to identify potential antigenic targets that were subjected to multi-epitope vaccine design. Our analysis projected the open pangenome of M. abscessus containing 3443 core genes. After applying various stepwise filtration steps on the core proteins, a total of four potential antigenic targets were identified. Utilizing their constituent CD4 and CD8 T-cell epitopes, a multi-epitope based subunit vaccine was computationally designed. Sequence-based analysis as well as structural characterization revealed the immunological effectiveness of this designed vaccine. Further molecular docking, molecular dynamics simulation and binding free energy estimation with Toll-like receptor 2 indicated strong structural associations of the vaccine with the immune receptor. The promising results are encouraging and need to be validated by additional wet laboratory studies for confirmation.

Multiepitope Subunit Vaccine Design against COVID-19 Based on the Spike Protein of SARS-CoV-2: An In Silico Analysis.

The global health crisis caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, has resulted in a negative impact on human health and on social and economic activities worldwide. Researchers around the globe need to design and develop successful therapeutics as well as vaccines against the novel COVID-19 disease. In the present study, we conducted comprehensive computer-assisted analysis on the spike glycoprotein of SARS-CoV-2 in order to design a safe and potent multiepitope vaccine. In silico epitope prioritization shortlisted six HLA I epitopes and six B-cell-derived HLA II epitopes. These high-ranked epitopes were all connected to each other via flexible GPGPG linkers, and at the N-terminus side, the sequence of Cholera Toxin ß subunit was attached via an EAAAK linker. Structural modeling of the vaccine was performed, and molecular docking analysis strongly suggested a positive association of a multiepitope vaccine with Toll-like Receptor 3. The structural investigations of the vaccine-TLR3 complex revealed the formation of fifteen interchain hydrogen bonds, thus validating its integrity and stability. Moreover, it was found that this interaction was thermodynamically feasible. In conclusion, our data supports the proposition that a multiepitope vaccine will provide protective immunity against COVID-19. However, further in vivo and in vitro experiments are needed to validate the immunogenicity and safety of the candidate vaccine.

In Vitro Antioxidant, Anti-inflammatory, and In Vivo Anticolitis Effects of Combretin A and Combretin B on Dextran Sodium Sulfate-Induced Ulcerative Colitis in Mice.

Combretum fragrans (Combretaceae) is a Cameroonian medicinal plant containing various secondary metabolites and traditionally used for the treatment of several pathologies. Two cycloartane-type triterpenes, Combretin A and Combretin B, were isolated from this plant. This study was aimed at evaluating the anti-inflammatory, antioxidant, and anticolitis effects of these compounds. In vitro anti-inflammatory properties were evaluated by inhibition of cyclooxygenase, 5-lipoxygenase, and denaturation of the protein; antioxidant properties were assessed by using 1,1-diphenyl-2-picrylhydrazyl (DPPH), (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) ABTS•+, capacity tests ferric reducing antioxidant (FRAP), and trapping nitric oxide. For in vivo analysis, we used the model of ulcerative colitis induced by Dextran Sulfate Sodium (DSS). Studies of the anti-inflammatory activity showed that Combretin A and Combretin B had maximal inhibitory activity on cyclooxygenase (71.92% and 89.59%), 5-lipoxygenase (76.68% and 91.21%), and protein denaturation (63.93% and 87.78%). Antioxidant activity on DPPH, ABTS•+, ferric reducing antioxidant capacity (FRAP), and nitric oxide scavenging showed that Combretin A and Combretin B showed good antioxidant activities. These compounds significantly reduced the signs of DSS-induced colitis in the treated animals by preventing the weight loss of the animals, by significantly reducing the disease activity index, improving the condition of the stool, preventing the reduction of the length of the colon, and preventing the degradation of the colon. This study revealed that Combretin A and Combretin B have anti-inflammatory, antioxidant, and curative properties against colitis experimentally induced by DSS in rats.

Pangenome Analysis of Mycobacterium tuberculosis Reveals Core-Drug Targets and Screening of Promising Lead Compounds for Drug Discovery.

Tuberculosis, caused by Mycobacterium tuberculosis (M. tuberculosis), is one of the leading causes of human deaths globally according to the WHO TB 2019 report. The continuous rise in multi- and extensive-drug resistance in M. tuberculosis broadens the challenges to control tuberculosis. The availability of a large number of completely sequenced genomes of M. tuberculosis has provided an opportunity to explore the pangenome of the species along with the pan-phylogeny and to identify potential novel drug targets leading to drug discovery. We attempt to calculate the pangenome of M. tuberculosis that comprises a total of 150 complete genomes and performed the phylo-genomic classification and analysis. Further, the conserved core genome (1251 proteins) is subjected to various sequential filters (non-human homology, essentiality, virulence, physicochemical parameters, and pathway analysis) resulted in identification of eight putative broad-spectrum drug targets. Upon molecular docking analyses of these targets with ligands available at the DrugBank database shortlisted a total of five promising ligands with projected inhibitory potential; namely, 2'deoxy-thymidine-5'-diphospho-alpha-d-glucose, uridine diphosphate glucose, 2'-deoxy-thymidine-beta-l-rhamnose, thymidine-5'-triphosphate, and citicoline. We are confident that with further lead optimization and experimental validation, these lead compounds may provide a sound basis to develop safe and effective drugs against tuberculosis disease in humans.

Anti-COVID-19 multi-epitope vaccine designs employing global viral genome sequences.

BACKGROUND: The coronavirus SARS-CoV-2 is a member of the Coronaviridae family that has caused a global public health emergency. Currently, there is no approved treatment or vaccine available against it. The current study aimed to cover the diversity of SARS-CoV-2 strains reported from all over the world and to design a broad-spectrum multi-epitope vaccine using an immunoinformatics approach. METHODS: For this purpose, all available complete genomes were retrieved from GISAID and NGDC followed by genome multiple alignments to develop a global consensus sequence to compare with the reference genome. Fortunately, comparative genomics and phylogeny revealed a significantly high level of conservation between the viral strains. All the Open Reading Frames (ORFs) of the reference sequence NC_045512.2 were subjected to epitope mapping using CTLpred and HLApred, respectively. The predicted CTL epitopes were then screened for antigenicity, immunogenicity and strong binding affinity with HLA superfamily alleles. HTL predicted epitopes were screened for antigenicity, interferon induction potential, overlapping B cell epitopes and strong HLA DR binding potential. The shortlisted epitopes were arranged into two multi-epitope sequences, Cov-I-Vac and Cov-II-Vac, and molecular docking was performed with Toll-Like Receptor 8 (TLR8). RESULTS: The designed multi-epitopes were found to be antigenic and non-allergenic. Both multi-epitopes were stable and predicted to be soluble in an Escherichia coli expression system. The molecular docking with TLR8 also demonstrated that they have a strong binding affinity and immunogenic potential. These in silico analyses suggest that the proposed multi-epitope vaccine can effectively evoke an immune response.

Whole-genome sequencing of a new sequence type (ST5352) strain of community-acquired methicillin-resistant Staphylococcus aureus from a hospital in Pakistan.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) is an important drug-resistant pathogen causing a number of diseases, resulting in increased mortality. Therefore, whole-genome sequencing of an MRSA strain isolated from a patient admitted to a hospital in Lahore, Pakistan, was performed to better characterise the strain and to understand the genetic components of antimicrobial resistance and virulence. METHODS: MRSA isolate Lr12 was sequenced on an Illumina HiSeq 2500 platform. The genome was assembled with SPAdes and was annotated using PGAP v.4.3. The strain was characterised using spaTyper 1.0, SCCmecFinder v.1.2 and MLST 2.0 server. Plasmids, antimicrobial resistance determinants and virulence factors were identified using PlasmidFinder v.2.0, CARD and VFDB, respectively. RESULTS: MRSA strain Lr12 has an estimated genome size of 2 769 144bp with a GC content of 32.7% and harbours 1 plasmid, 2 prophages, 11 antimicrobial resistance determinants and several virulence factors. The allelic profile of seven housekeeping genes was unique and the sequence type (ST) was classified as unknown, hence a novel sequence type (ST5352) was assigned. CONCLUSION: MRSA strain Lr12 has a novel sequence type (ST5352) and could be used as a reference strain for comparative genomic analysis of other MRSA strains belong to ST5352.

Immunoinformatics-Aided Design and Evaluation of a Potential Multi-Epitope Vaccine against Klebsiella Pneumoniae.

Klebsiella pneumoniae is an opportunistic gram-negative bacterium that causes nosocomial infection in healthcare settings. Despite the high morbidity and mortality rate associated with these bacterial infections, no effective vaccine is available to counter the pathogen. In this study, the pangenome of a total of 222 available complete genomes of K. pneumoniae was explored to obtain the core proteome. A reverse vaccinology strategy was applied to the core proteins to identify four antigenic proteins. These proteins were then subjected to epitope mapping and prioritization steps to shortlist nine B-cell derived T-cell epitopes which were linked together using GPGPG linkers. An adjuvant (Cholera Toxin B) was also added at the N-terminal of the vaccine construct to improve its immunogenicity and a stabilized multi-epitope protein structure was obtained using molecular dynamics simulation. The designed vaccine exhibited sustainable and strong bonding interactions with Toll-like receptor 2 and Toll-like receptor 4. In silico reverse translation and codon optimization also confirmed its high expression in E. coli K12 strain. The computer-aided analyses performed in this study imply that the designed multi-epitope vaccine can elicit specific immune responses against K. pneumoniae. However, wet lab validation is necessary to further verify the effectiveness of this proposed vaccine candidate.

Revisiting the AIDS Epidemic in Pakistan: Where We Stand and What We Must Aim For.

Human immunodeficiency virus type 1 (HIV-1) is the major cause of acquired immunodeficiency syndrome (AIDS) in humans, where the immune system totally succumbs to the virus. A large proportion of the AIDS infected belong to developing countries and AIDS prevalence is intensified by severe poverty, malnutrition, and famine; fatal illnesses with a scorn shortage of medical amenities complemented with the lack of education and development. Current Pakistani health system setting is in a dire need of improvement. Low literacy rates, high birth rates, and associated maternal mortality plus a lack of clean drinking water and appropriate sanitation system have a serious impact on general living conditions contributing to a relatively short lifespan. HIV is, therefore, becoming a growing health concern in Pakistan with a rapid rise in the reported cases. AIDS is most prevalent among injection drug users (IDUs), male/female sex workers, and unchecked deported migrant workforce. To combat this virus, the Pakistan Government has been working hard over the past few years with local bodies and international organizations in an effort to combat this menace. This review aims to discuss the risk factors for the rise of this epidemic in the country and the recommendations, efforts to be done to address this alarming issue.

Structural analysis and insight into Zika virus NS5 mediated interferon inhibition.

The Zika virus outbreak in 2015-2016 is the largest of its kind for which WHO declared a Public Health Emergency of International Concerns. No FDA approved drug is available for the treatment of the viral infection. The interaction of flavivirus NS5 protein with SIAH2 ubiquitin ligase has been previously known. NS5 of Zika virus has been implicated in the degradation of STAT2 protein, which activates interferon-stimulated antiviral activity. Based on our proposition that NS5 utilizes SIAH2-mediated proteasomal degradation of STAT2, an in-silico study was carried out to characterize the protein-protein interactions between NS5, SIAH2 and STAT2 proteins. The aim of our study was to identify the amino acid residues of NS5 involved in IFN antagonism as well as to find the association between NS5, SIAH2 and STAT2 to predict the interaction pattern of these proteins. Analysis proposed that NS5 recruits SIAH2 for the ubiquitination-dependent degradation of STAT2. NS5 residues involved in interaction with SIAH2 and/or STAT2 were found to be mostly conserved across related flaviviruses. These are novel findings regarding the Zika virus and require confirmation through experimental approaches.

Potential link between Complement 5a Receptor and mood disorders in mouse exposed to experimental malaria in utero.

In Africa, a large number of pregnancies are exposed to Plasmodium falciparum infection. The in-utero environment extremely influences childhood neurodevelopment and behaviour. The complement 5a receptor (C5aR) is linked to several disease conditions. However, the influence of Plasmodium berghei during pregnancy on maternal complement 5a receptor and subsequently on fetal behaviour is unknown. Pregnant mice were intra-peritoneally inoculated on gestational day 13 with 1.02x105 infected red blood cells (iRBCs). iRBCs used in this experiment were gotten by in vivo passage of P. berghei in mice when the level of iRBCs have gotten to about 10-20%. A section of pregnant mice (both test and control groups) were earmarked to give birth and their offspring monitored up to postnatal day 42 when depression-like behaviour was evaluated using tail suspension test model. The other pregnant mice were subjected to cardiac puncture on gestational day 19 for C5a receptor estimation using Elisa assay. Results showed that pregnant mice infected with P. berghei had elevated C5a receptor compared with uninfected pregnant females. It was also shown that P. berghei-exposed offspring presented a depressive-like behaviour compared to unexposed controls. It may be concluded from this study, that complement 5a receptor demonstrates a pathogenic role in signaling and its possible role in mediating depression linked to Plasmodium berghei exposure in utero.

Prediction of promiscuous T-cell epitopes in the Zika virus polyprotein: An in silico approach.

OBJECTIVE: To predict immunogenic promiscuous T cell epitopes from the polyprotein of the Zika virus using a range of bioinformatics tools. To date, no epitope data are available for the Zika virus in the IEDB database. METHODS: We retrieved nearly 54 full length polyprotein sequences of the Zika virus from the NCBI database belonging to different outbreaks. A consensus sequence was then used to predict the promiscuous T cell epitopes that bind MHC 1 and MHC II alleles using PorPred1 and ProPred immunoinformatic algorithms respectively. The antigenicity predicted score was also calculated for each predicted epitope using the VaxiJen 2.0 tool. RESULTS: By using ProPred1, 23 antigenic epitopes for HLA class I and 48 antigenic epitopes for HLA class II were predicted from the consensus polyprotein sequence of Zika virus. The greatest number of MHC class I binding epitopes were projected within the NS5 (21%), followed by Envelope (17%). For MHC class II, greatest number of predicted epitopes were in NS5 (19%) followed by the Envelope, NS1 and NS2 (17% each). A variety of epitopes with good binding affinity, promiscuity and antigenicity were predicted for both the HLA classes. CONCLUSION: The predicted conserved promiscuous T-cell epitopes examined in this study were reported for the first time and will contribute to the imminent design of Zika virus vaccine candidates, which will be able to induce a broad range of immune responses in a heterogeneous HLA population. However, our results can be verified and employed in future efficacious vaccine formulations only after successful experimental studies.