Molecular Biotechnology最新文献

Helicobacter pylori (H. pylori) is an infectious agent colonized in gastric epithelium and leads to serious diseases such as ulcers and gastric carcinoma. H. pylori infection requires rapid and effective treatment options however existing therapies gradually diminish in efficacy due to the development of resistance. Type II fatty acid synthesis (FAS-II) pathway is a potent target for drug discovery studies because of its absence in humans and vital necessity for bacteria. In the last step of the synthesis, trans-2-enoyl-ACP is reduced to acyl-ACP with cofactor of NADH by enoyl-ACP reductase, FabI. In this study, recombinant HpFabI was successfully produced using an aLICator ligation-independent cloning and expression vector system for the first time. HpFabI gene was cloned, and then expressed, and the protein was purified in high yield. Recombinant HpFabI with a molecular mass of ~ 30 kDa was confirmed with Western Blot analysis and its concentration was determined in the range of 1.406-3.9495 mg/ml by Bradford Assay. The enzyme-specific activity of HpFabI was determined as 1.5871 nmol min^-1 μg^-1 by using NADH and crotonoyl-CoA as cofactor and substrate, respectively. HpFabI was produced in high yield to facilitate future inhibition studies including high throughput screening studies for FabI inhibition to contribute novel drug development studies fighting against H. pylori infection.

Viruses, microscopic menace that transcends time leaving its mark on every era have been silent predators since the dawn of civilization, evolving with us and shaping our history. Chandipura virus (CHPV), a potent member of the Rhabdoviridae family poses a significant threat in India with rapid neuroinvasive potential leading to fatal encephalitis, particularly in children. Given the scarcity of research, our study consolidates critical information regarding its lifecycle, fusion process, and reviewed the LRP1 and GRP78 as CHPV target receptors. With no FDA-approved drugs currently available for CHPV prevention, our research focuses on identifying potential molecules that can disrupt the virus at its most critical juncture, the fusion stage. The results derived from compounds screening indicated Silibinin, 3-(2,3-Dihydroxy-3-Methylbutyl)-6-Hydroxy-2-[(1E,5E)-3,4,10-Trihydroxyundeca-1,5-Dienyl] Benzaldehyde, Budmunchiamine L5, and L4 as a leading molecule may efficaciously inhibit G ectodomain fusion. By analyzing pharmacokinetic properties through radar graph, outcomes support the nomination of four compounds as potential inhibitory molecules and ensure they possess the optimal balance of drug-like characteristics. Working with the CHPV presents significant challenges, making the in silico parameters crucial in guiding future research. Our study sought to pioneer the discovery of therapeutic molecules against the CHPV, providing a foundational framework for developing effective antiviral strategies.

Hepatocellular carcinoma (HCC) is one of the five most common cancers and the second leading cause of cancer-related death worldwide. In this study, three monoclonal antibodies were developed for the early detection of HCC. The enzyme-linked immunosorbent assay (ELISA) method is used to detect antigens causing HCC. The final working dilutions of the coated antigen, monoclonal antibody, and enzyme-labeled secondary antibody were determined to be 1:5, 1:100, and 1:15,000, respectively. The optimal dilution of blocking buffer was 1.5% BSA phosphate buffer. The cutoff values were determined to be 0.1989, 0.2539, and 0.3059 for the Des-gamma carboxyprothrombin (DCP), Alpha-fetoprotein (AFP) and Thioredoxin (TXN) antigens, respectively. There is no cross-reaction between antigens and antibodies of different types. The coincidence rates between the indirect ELISA and commercial kits for detecting DCP, AFP, and TXN antigens were 95.24%, 95.24%, and 96.83%, respectively. In addition, a procedure to detect genes encoding TXN, DCP, and AFP via PCR has been developed. The results of the indirect ELISA and PCR methods are similar. In summary, we successfully constructed an indirect ELISA method to detect HCC-causing antigens via three monoclonal antibodies and designed primers to amplify HCC-causing gene fragments, which can be used for diagnosis and screening in clinical medicine.

Biotransformation, a dynamic process conducted by microorganisms, holds promise in modifying natural compounds for enhanced therapeutic potential. In this study, we leverage bacterial systems to catalyze the transformation of hesperidin, obtained from Citrus sinensis, aiming for structural modifications that could optimize its bioactivity and computational targeting against Helicobacter pylori. Multiple bacterial species were employed to perform biotransformation reactions. HPLC and FTIR analyses were used to determine structural modifications and bio-transformed products. The reaction in which hesperidin metabolite was formed was catalyzed by Bacillus spp. The transformed products, along with the original compound, underwent rigorous bioactivity testing to evaluate their potential in combating oxidative stress, inflammation, and diabetes. Employing well-established in vitro methods, we assessed the bio-transformed derivatives for antioxidant efficacy, revealing an impressive 94% inhibition of free radicals compared to hesperidin. In terms of anti-inflammatory activity, the results showcased a substantial 92% inhibition, while the assessment of antidiabetic activity demonstrated a notable 85% inhibition. The hesperidin metabolites were more active than hesperidin in biological activity evaluations. So, the bio-transformed compound derived from hesperidin, along with pure compound, was used to design a computational drug targeting the bacterium H. pylori. Among these two compounds, the bio-transformed product of hesperidin with an alkyl amine exhibited the highest docking energy of - 180.26 kJ/mol, as compared to pure compound. SwissADME provided valuable insights into the compound's drug-likeness like 0.55 bioavailability and 8.41 synthetic accessibility. ProTox-II evaluated different toxicity endpoints with a 0.96 probability of being inactive in cytotoxicity. These findings support the potential of the bio-transformed compound as a promising candidate for further investigation and development as a drug against H. pylori.

As a component of the alternative replication factor C (RFC) complex, DSCC1 plays a significant role in cancer progression due to its aberrant expression. However, the potential function of DSCC1 in a pan-cancer context remains unclear. In this study, we conducted a comprehensive analysis of DSCC1's role in tumors by integrating multi-omics bioinformatics tools. First, we utilized various databases to compare the expression of DSCC1 between tumor and normal tissues, revealing a strong association between its dysregulated expression and clinical diagnosis, prognosis, and staging. Additionally, we investigated different mutation types of DSCC1 and their contributions to cancer progression, finding that DSCC1 expression is regulated by epigenetics and RNA modifications. Furthermore, we explored the correlation between DSCC1 and immune-infiltrating cells, as well as immunotherapeutic biomarkers, suggesting that its expression influences the tumor immune microenvironment. By employing single-cell and spatial transcriptome data through platforms such as SingleCellBase, CancerSEA, and CROST, we further uncovered the heterogeneity of DSCC1 across various cancer types. Finally, we validated the significant upregulation of DSCC1 mRNA in multiple tumor cell lines using q-RTPCR, and demonstrated through CCK8 assays that silencing DSCC1 expression effectively suppressed cell proliferation. Our findings establish a foundational understanding of DSCC1's potential as a biomarker for cancer diagnosis, prognosis, and immunotherapy.

The major facilitator superfamily domain containing 8 (MFSD8) belongs to an orphan transporter protein expressed in a wide range of tissues. Nevertheless, the specific role of MFSD8 in human health and disease remains unknown. This study aimed to evaluate the role of MFSD8 protein on metabolic function using untargeted metabolomics analysis in human umbilical vein endothelial cells (HUVECs). HUVECs overexpressing MFSD8 were subjected to metabolomics analysis to evaluate changes in endogenous small molecules using LC-MS/MS analysis. In the positive scan mode, 634 metabolites from 1583 compounds were identified. In the negative scan mode, 169 metabolites from 405 compounds were identified. According to the established criteria for identifying differential metabolites, 96 metabolites exhibited significant differences between the MFSD8 and Vector groups. Among them, 62 metabolites were found to be up-regulated, whereas 34 metabolites were classified as down-regulated. Bioinformatics pipeline analysis revealed three common metabolic pathways, including arginine biosynthesis, beta-alanine metabolism, and pyrimidine metabolism, were found under the positive and negative scan modes. The semi-quantitative analysis was conducted on the differential metabolites, revealing that overexpression of MFSD8 resulted in increased levels of L-citrulline, L-aspartic acid, ornithine, N-acetyl-l-aspartic acid, L-histidine, beta-alanine metabolites and exhibited decreased levels of cytidine. The findings of our study indicated that MFSD8 had the most significant role in arginine biosynthesis, beta-alanine metabolism, and pyrimidine metabolism pathways within endothelial cells. The metabolomics data provide new insights into studying potential features of MFSD8 protein in the future.

Hepatitis C virus (HCV) infects approximately 58 million individuals worldwide, often progressing to chronic liver disease, cirrhosis, and hepatocellular carcinoma. The viral envelope glycoproteins E1 and E2 are critical for HCV entry and serve as primary targets for neutralizing antibodies. Recent advancements in cryo-electron tomography have provided high-resolution structures (3.5 Å) of the E1E2 heterodimer, revealing interactions between the E1 and E2 ectodomains, as well as neutralizing antibody complexes (e.g., AR4A, AT1209, IGH505). This structural information facilitates the design of a synthetic peptide vaccine targeting conserved E1 and E2 regions. We suggest developing a vaccine tailored to the HLA-A*24:02 allele, the most prevalent in the Indian population. Epitope candidates will be screened using immunoinformatics tools, incorporating epitopes derived from epitope mapping with 7t6x protein structure modeling. Molecular docking studies will identify high-affinity interactions with human MHC-Class I alleles, using tools such as AutoDock and HADDOCK. GROMACS molecular dynamics simulations will assess peptide-HLA binding stability and dynamics. Among ten screened epitopes, KWEYVVLLF and QWQVLPCSF emerged as the most promising based on their toxicity profiles, conservation, and docking scores with HLA-A*24:02 (- 9.3 kcal/mol for KWEYVVLLF and - 225.34 kcal/mol for QWQVLPCSF). Molecular dynamics simulations indicated that the KWEY segment of KWEYVVLLF underwent structural changes, while the VVLLF region maintained stable binding to Chain A, suggesting immunogenic potential. These epitopes represent strong candidates for T-cell-based vaccines, and the reverse vaccinology approach, supported by computational tools, offers a population-specific strategy for HCV vaccine development, advancing precision immunotherapy.

Kānuka (Kunzea ericoides) has been traditionally recognised for its health-promoting properties. However, limited comprehensive data on its bioactive profile hinders its potential industrial applications. Therefore, this study investigated the bioactive constituents of the kānuka subcritical water extract, exhibiting maximum antioxidant potential, using LC-MS/MS QTOF. Experiments were performed at different extraction temperatures and times to determine antioxidant content and capacity. The data were statistically analysed to select extracts with the maximum antioxidant response for bioactive screening. The highest recovery of polyphenolics and flavonoids occurred at 170 °C, 5 min (319.96 ± 22.67 mg GAE/g dw and 163.57 ± 9.7 mg QE/g dw), correlating with increased antioxidant activity (DPPH: 87.77 ± 1.74%; ABTS: 97.62 ± 0.12; FRAP: 166.43 ± 25.54 mg TE/g dw). Extracts obtained at 170 °C, 180 °C, 200 °C, and 220 °C with shorter extraction times (5, 10, and 15 min) were selected for bioactive screening using multivariate principal component analysis. Consequently, a rich composition of phenylpropanoids, including flavonoids, phenolic acids, stilbenes, and monolignols, was identified in the kānuka extracts using LC-MS/MS QTOF. Additionally, bioactives previously not found in kānuka extracts were identified, including 2',6'-Dihydroxy-4'-methoxydihydrochalcone, resveratrol, scopoletin, and naringin, underscoring its potential as a source of valuable metabolites to the pharmaceutical and food industries.

Our previous study uncovered that long-term abuse of caffeine sodium benzoate (CSB) could lead to dysfunction in human umbilical vein endothelial cells (HUVECs). However, the mechanism by which CSB induced endothelial dysfunction remains largely unstudied. CSB containing serum (CSB-CS) was collected from patients under long-term CSB inhalation. RAW264.7 cells were treated with different concentrations of CSB-CS, after which the conditioned medium (CM) was collected and cultured with HUVECs. The migration, tube formation, and senescence of HUVECs were evaluated. CSB-CS could induce polarization of RAW264.7 cells toward the M1 phenotype, as evidenced by the elevated CD86 and iNOS levels. Additionally, the CM from CSB-treated RAW264.7 cells notably suppressed the migration, tube formation, and induced cell senescence and endothelial dysfunction in HUVECs. Moreover, the CM from CSB-treated RAW264.7 cells greatly reduced mitochondrial membrane potential level, increased the ROS production, reduced OPA1 levels, but elevated DRP1 levels in HUVECs, leading to mitochondrial fission and dysfunction. Meanwhile, the CM from CSB-treated RAW264.7 cells remarkably reduced p-AKT and p-GSK3β levels in HUVECs. Notably, promotion of mitochondrial fusion by MASM7 could mitigate mitochondrial dysfunction and endothelial dysfunction in HUVECs induced by the CM from CSB-treated RAW264.7 cells. Collectively, we found that CSB could induce mitochondrial dysfunction in HUVECs by the polarization of pro-inflammatory M1 macrophages, resulting in endothelial dysfunction. These findings may provide a foundational basis for developing treatments for diseases associated with CSB.

Dengue fever, dengue hemorrhagic dengue fever and dengue shock syndrome were caused by Dengue mosquito bites. Common signs such as fever and headache, are connected to distinctive medical disorders. From the previous and ongoing studies, it is far unknown what genes or protein signaling pathway mechanism underlies the association between DF, DHF and DSS in Indian context. In our study, the gene expression dataset was retrieved from the GEO database with accession number GSE94892. Here, mRNA sequence analysis done of each DF, DHF and DSS patients from peripheral blood mononuclear cells sample. GEO2R became used to carry out differential gene expression analysis using a dengue data set. Protein-protein interaction networks have been built, gene set GO enrichment and KEGG Pathway enrichment done in SR plot, and cluster analyses have been performed in STRING and MCODE. During this study, we diagnosed 10 hub genes in all 3 condition. The gene set of showed that the ten hub genes diagnosed in each condition constituted the best range of common hub genes discovered beneath all 3 conditions in India. The conclusion of this study can be beneficial for treating DF, DHF and DSS conditions within the context of handling DEV in India.