RESUMO
TpXyl10B is a glycoside hydrolase family 10 xylanase of hyperthermophile Thermotoga petrophila RKU-1. This enzyme is of considerable importance due to its thermostability. However, in its native state, this enzyme does not possess any carbohydrate-binding module (CBM) for efficient binding to plant biomass. In this study CBM6 from Clostridium thermocellum was attached to the N- and C-termini of TpXyl10B, thereby producing the variants TpXyl10B-B6C and TpXyl10B-CB6, respectively. TpXyl10B-B6C showed 5-7 folds increased activity on Beechwood xylan and the different types of plant biomass as compared to that from the catalytic domain only. However, the activity of TpXyl10B-CB6 decreased 0.6-0.8 folds on Beechwood xylan and plant biomass compared to the catalytic domain. We explained these results through molecular modeling, which showed that binding residues of CBM6's cleft B, which were previously reported to show no contribution towards binding due to steric hindrance from a loop region, were exposed in a favorable position in TpXyl10B-B6C such that they efficiently bound the substrate. In contrast, these binding residues of CBM6 in TpXyl10B-CB6 were exposed opposite to the catalytic residues; thus, binding to the substrate resulted in decreased exposure of catalytic residues to the substrate. CD spectroscopy and thermostability assays showed that TpXyl10B-B6C was highly thermostable, having a melting point > 90 °C, which is relatively higher than that of the other variant, TpXyl10B-CB6. In addition, this xylanase variant showed synergism with cellulases for the hydrolysis of plant biomass. Therefore, TpXyl10B-B6C, an engineered xylanase in this study, can be a valuable candidate for industrial applications.
Assuntos
Celulases , Celulases/metabolismo , Xilanos/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Domínio Catalítico , Archaea , Especificidade por SubstratoRESUMO
LHPP gene encodes a phospholysine phosphohistidine inorganic pyrophosphate phosphatase, which functions as a tumor-suppressor protein. The tumor suppression by this protein has been confirmed in various cancers, including hepatocellular carcinoma (HCC). LHPP downregulation promotes cell growth and proliferation by modulating the PI3K/AKT signaling pathway. This study identifies potentially deleterious missense single nucleotide variants (SNVs) associated with the LHPP gene using multiple computational tools based on different algorithms. A total of 4 destabilizing mutants are identified as L22P, I212T, G227R, and G236R, from the conserved region of the phosphatase. The 3-dimensional (3D) modeling and structural comparison of variants with the native protein reveals significant structural and conformational variations after mutations, suggesting disruption in the function of phospholysine phosphohistidine inorganic pyrophosphate phosphatase. The identified mutations might, therefore, participate in the cause of HCC.
RESUMO
OBJECTIVES: The design of peptide-based vaccines for cancer is a promising immunotherapy that can induce a cancer-specific cytotoxic response in tumor cells. METHODS: Herein, we used the immunoinformatic approach in designing a multi-epitope vaccine targeting G-protein coupled receptor 87 (GPCR-87), cystine/glutamate transporter (SLC7A11), Immunoglobulin binding protein 1 (IGBP1), and thioredoxin domain-containing protein 5 (TXNDC5), which can potentially contribute to NSCLC. The MHC-I and MHC-II epitopes selected for the fusion construct were evaluated for their antigenic and non-allergenic natures via VaxiJen and AllerTop. RESULTS: A total of five epitopes, four class-I (FIFYLKNIV, CRYTSVLFY, RYLKVVKPF, and RQAKIQRYK), and one class-II (NQVRGYPTLLWFRDG), having combined USA population coverage of 100%, were used to make ten possible multi-epitope fusion constructs. In these constructs, PADRE, a universal T-helper epitope, and RSO9, a TLR4 agonist, were fused as adjuvants. The molecular docking analysis revealed that two constructs were showing significant binding affinities toward HLA-A*02:01, the most prevalent HLA allele in USA. Moreover, MD simulations marked one construct as a promising therapeutic candidate. CONCLUSION: The multi-epitope vaccine constructs designed using immunogenic, and non-allergenic peptides of NSCLS tumor-associated proteins are likely to pose significant therapeutic efficacies in cancer immunotherapy due to their high binding affinities toward HLA molecules.
Assuntos
Vacinas Anticâncer/imunologia , Carcinoma Pulmonar de Células não Pequenas , Antígenos HLA , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/terapia , Biologia Computacional , Epitopos de Linfócito B , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Simulação de Acoplamento Molecular , Estados Unidos , Vacinas de Subunidades AntigênicasRESUMO
Currently, the whole world is facing the coronavirus disease-19 pandemic. As of now, approximately 0.15 million people around the globe are infected with the novel coronavirus. In the last decade, two strains of the coronavirus family, severe acute respiratory syndrome-related coronavirus and Middle East respiratory syndrome coronavirus, also resulted in epidemics in south Asian and the Middle Eastern countries with high mortality rate. This scenario demands the development of a putative vaccine which may provide immunity against all current and new evolving coronavirus strains. In this study, we designed an epitope-based vaccine using an immunoinformatic approach. This vaccine may protect against all coronavirus strains. The vaccine is developed by considering the geographical distribution of coronavirus strains and host genetics (Chinese population). Nine experimentally validated epitopes sequences from coronavirus strains were used to derive the variants considering the conservancy in all strains. Further, the binding affinities of all derived variants were checked with most abundant human leukocyte antigen alleles in the Chinese population. Three major histocompatibility complex (MHC) Class I epitopes from spike glycoprotein and nucleoprotein showed sufficient binding while one MHC Class II epitope from spike glycoprotein was found to be an effective binder. A cocktail of these epitopes gave more than 95% population coverage in the Chinese population. Moreover, molecular dynamics simulation supported the aforementioned predictions. Further, in vivo studies are needed to confirm the immunogenic potential of these vaccines.
Assuntos
Infecções por Coronavirus/prevenção & controle , Coronavirus/imunologia , Vacinas Virais/imunologia , Alelos , Sequência de Aminoácidos , China , Coronavirus/química , Coronavirus/genética , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Antígenos HLA/genética , Antígenos HLA/imunologia , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/química , Vacinas Virais/genéticaRESUMO
Tuberculosis (TB), a chronic disease caused by Mycobacterium tuberculosis (Mtb), is a global health issue across the world. Pakistan ranks fifth among the countries, which are facing, a significantly great number of mortalities and morbidities due to TB. Unfortunately, all previously reported treatments are not successful for the eradication of TB. Here in this study, we report an emerging treatment option for this disease. We have applied immunoinformatics to predict highly conserved B and T-cell epitopes from Mtb, showing significant binding affinities to the frequent HLA alleles in the Pakistani population. A total of ten highly referenced and experimentally validated epitopes were selected from the Immune Epitope Database (IEDB), followed by their conservancy analysis using weblogos. The consensus sequences and variants derived from these sequences were examined, for their binding affinities, with prevalent HLA alleles of Pakistan. Moreover, the antigenic and allergenic natures of these peptides were also evaluated via Vaxijen and AllerTOP, respectively. Consequently, all potentially allergenic and non-antigenic, peptide fragments, were excluded from the analysis. Among all putative epitopes, three CD8 + T-cell epitopes were selected, as ideal vaccine candidates and, population coverage analysis revealed that the combination of these three peptides was covering, 67.28% Pakistani Asian and 57.15% mixed Pakistani populations. Likewise, eleven linear and six conformational or discontinuous B-cell epitopes were also marked as potential vaccine candidates based on their prediction score, non-allergenic nature, and antigenic properties. These epitopes, however, need the final validation via wet-lab studies. After their approval, these epitopes would be effective candidates for the future designing of epitope-based vaccines against Mtb infections in Pakistan.
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The concentrations of zinc, copper, lead and cadmium owing to vehicles were analyzed in roadside soils during all the four seasons from Faisalabad-Gojra road (M-4) and Gojra-Jhang road (GJR) in the Punjab, Pakistan. Results indicated that all the polluted sites along the roadside had comparatively higher metal concentrations as compared to control, nevertheless, spatio-temporal variations in metal concentrations were apparent in different soil samples. The spatial differences in metal concentrations were strongly associated with vehicle density at the Gojra Interchange site and Forest Park site along M-4 and GJR roads, respectively. Among seasons, metal ions exhibited the following order: summer > autumn > spring > winter, indicating that high temperature might have released more metal ions. The metals were recorded in contaminated soil in the order of Zn > Cu > Pb > Cd along both roads. This investigation emphasizes the need for proper environmental management particularly the improvement of roads and proper checking of vehicles running on these regards.
Assuntos
Monitoramento Ambiental , Metais Pesados/análise , Poluentes do Solo/análise , Cádmio/análise , Cobre/análise , Poluição Ambiental , Paquistão , Estações do Ano , Solo , Emissões de Veículos/análise , Zinco/análiseRESUMO
Gonorrheal infections are the second most prevalent sexually transmitted diseases STDs in the USA populations after Chlamydia. These infections have now become an urgent problem to address because Neisseria gonorrhoeae is capable of developing resistance to multiple antibiotic classes. Infection with these antibiotic-resistant strains has become the major public health concern. Although extensive researches are ongoing to control its transmission and to develop the productive treatments against this pathogen, no effective vaccine could develop till now. The present study will effectively contribute to the future designing of USA specific epitope-based vaccines. Through computational approaches, this study has highlighted putative epitopes from Neisseria gonorrhoeae which restrict maximally to the frequent HLA alleles in the USA populations. Antigenic and non-allergenic nature of predicted epitopes was verified using vexijen and AllerTOP tools respectively. Total seven epitopes, four from class-I and three from class-II were antigenic as well as non-allergenic. These epitopes showed USA population coverage of 99.3% with no allergenic response. Still, additional studies are needed to validate the immunogenic properties of the predicted epitopes which are likely vaccine target for gonorrhoea in the USA populations.