Molecular Biotechnology最新文献

Renal ischemia-reperfusion injury (RIRI) is a primary cause of acute kidney injury (AKI), frequently resulting in high mortality rates and progression to chronic kidney disease (CKD). This study aimed to investigate the therapeutic potential of total saponins from Panax notoginseng (PNS) in the context of RIRI. Utilizing a murine RIRI model, the efficacy of PNS was evaluated, demonstrating a significant reduction in renal inflammation and cellular pyroptosis. Furthermore, PNS was found to modulate the ROCK2/NF-κB signaling pathway, thereby attenuating the inflammatory response. Importantly, in vitro experiments with hypoxia/reoxygenation cell models corroborated these findings, showing that PNS inhibited pyroptosis and regulated the ROCK2/NF-κB pathway. This research underscores the therapeutic potential of PNS in the treatment of RIRI, providing a robust scientific basis for its consideration as a prospective clinical therapy.

Lasiodiplodia theobromae is an emerging threat and the main pathogenic fungi associated with basal stem rot of passion fruit in Guangxi Zhuang Autonomous Region, China. Current pathogen identification protocols are labor-intensive and time-consuming, emphasizing the need for more efficient methods to enable precise surveillance of L. theobromae for early detection and warning. The present study sought to develop a rapid colorimetric LAMP assay for early detection and surveillance of L. theobromae in passion fruit plants. For that, amplifications of ITS locus were performed on fungal genomic DNA using conventional PCR, with the specific primer pair ITS1 and ITS4. The hydroxy naphthol blue (HNB)-dependent colorimetric LAMP assay was then optimized by varying primer sets, inner primers concentration, reaction temperatures and incubation time. A microbial lysis buffer was employed to extract genomic DNA from stems infected with L. theobromae. The prime LAMP primer set targeting the ITS region of L. theobromae was designed and an HNB based colorimetric LAMP assay was optimized. Various optimization parameters were evaluated, with the optimal conditions determined as 1.6 μM of each FIB and BIP, 0.2 μM of each F3 and B3, and incubation at 65 °C for 40 min. This ITS-based LAMP assay could effectively distinguish L. theobromae from less dominant pathogens in passion fruits with a detection limit of 3 pg for ITS locus amplicons. Our proposed method utilizing a microbial lysis buffer enables rapid and cost-effective detection of L. theobromae DNA in early-infected passion fruit plants, eliminating the need for microbial cultivation and DNA purification.

Oncolytic viral-based therapy and specific gene expression by promoters are modern targeted oncotherapy approaches that have gained significant attention in recent years. In this study, both strategies were combined by designing cancer-specific activation of vesicular stomatitis virus matrix expression under the survivin promoter. The matrix sequence was cloned downstream of the survivin promoter (pM). After transfecting MCF-7 cells with pM, cell proliferation and apoptosis induction were assessed. Additionally, the transcript levels of matrix and apoptosis-related genes in response to pM was assessed. The proliferation of MCF-7 cells was significantly reduced by the constructed matrix-expressing plasmid at 48 and 72 h post-transfection (p < 0.05). Enhanced matrix expression resulted in the down-regulation of MMP-9, TP53, and NF-kB, while simultaneously up-regulating Bax transcripts. Evaluating the effect of pM vector on apoptosis induction revealed a significant increase in the MCF-7 cells compared to untreated cells (p < 0.05). The absence of significant matrix gene expression in HDF cells, relative to MCF-7 cells, further underscores the specific function of the Survivin promoter in cancer cells. These findings suggest that the matrix may have various biological functions in a diverse set of non-apoptotic pathways. Further research on the association of the matrix with other genes could provide insights into the biomedical significance and future perspectives of the matrix in cancer gene therapy.

Glucanases are widely applied in industrial applications such as brewing, biomass conversion, food, and animal feed. Glucanases catalyze the hydrolysis of glucan to produce the sugar hemiacetal through hydrolytic cleavage of glycosidic bonds. Current study aimed to investigate structural insights of a glucanase from Clostridium perfringens through blind molecular docking, site-specific molecular docking, molecular dynamics (MD) simulation, and binding energy calculation. Furthermore, we aimed to enhance structural stabilization through formation of hydrophobic interaction network. The molecular docking results illustrated that residues Glu222 and Asp187 may act as nucleophile acid/base catalyst. Moreover, the MM/PBSA results illustrated a high binding affinity of 108.71 ± 8.5 kJ/mol between glucanase and barely glucan during 100 ns simulation. The RMSF analysis illustrated a high flexible surface loop with the highest mobility at position D130. Therefore, the structural engineering was carried out through introducing a double-mutant S125Y/D130P, and the structural stability was improved by forming the hydrophobic interaction network and one π-π aromatic interaction. The spatial distance between the mutation sites and the catalytic pocket attenuates their direct impact on binding interactions within the catalytic pocket.

Despite significant advancements in gene delivery and CRISPR technology, several challenges remain. Chief among these are overcoming serum inhibition and achieving high transfection efficiency with minimal cytotoxicity. To address these issues, there is a need for novel vectors that exhibit lower toxicity, maintain stability in serum-rich environments, and effectively deliver plasmids of various sizes across diverse cell types. In this study, to convert common polyethylenimine (PEI_1.8k) into high-performance DNA delivery vectors, an innovative multifunctional vector was constructed based on histidine linked to PEI_1.8k by redox-responsive disulfide bonds. Apart from highly efficient transfection of both small and large plasmids into HEK 293T (Human Embryonic Kidney 293T cells) with negligible cytotoxicity, PEI_1.8k-S-S-His showed great transfection potential even at low plasmid doses (0.5 µg), as well as at serum concentrations ranging from 5 to 30% into HEK 293T cells, and achieved excellent plasmid transfection into NIH/3T3 (Mouse Embryonic Fibroblast cells), and MCF7 (Human Breast Cancer cells). Additionally, several metals were tested (Co, Cu, Cd, Ni, Zn, and Mn) to promote the plasmid packaging functionality and improve transfection efficiency. We observed that, in comparison to PEI_1.8k-S-S-His, the manganese-functionalized nanocarrier (PEI_1.8k-S-S-His-Mn) could transfect a large plasmid with equal efficiency (~ 30%) into MSCs (Mesenchymal Stem Cells). Interestingly, PEI_1.8k-S-S-His-Mn showed higher transfection efficiency with the small plasmid (~ 90%) and the large one (~ 80%) into HEK 293T cells, even better than its backbone. We propose that the presence of metal-coordinated His ligand, redox-responsive S-S bonds, and the cationic polymer can synergistically provide robust DNA binding, efficient endosomal disruption, tolerance of serum protein adsorption, and low cytotoxicity. These new vectors could be promising for gene delivery and may be therapeutically relevant.

Clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system is a new gene editing tool that represents a revolution in gene therapy. This study aimed to review the clinical trials conducted to evaluate the efficacy and safety of the CRISPR/Cas9 system in treating thalassemia and sickle cell disease (SCD). We searched relevant literature using "CRISPR Cas", "thalassemia", "sickle cell" and "clinical trial" as subject terms in PubMed, Cochrane, Web of Science, and Google Scholar up to December 3rd, 2023. Following the PIO format (Patients, Intervention, Outcome), PRISMA guidelines were followed in the study selection, data extraction, and quality assessment processes. Out of 110 publications, 6 studies met our eligibility criteria with a total of 115 patients involved. CRISPR/Cas9 system was used to disrupt BCL11A gene enhancer in 4 studies and to disrupt γ-globin gene promoters in 2 studies. Patients demonstrated significant activation of fetal hemoglobin, elevated total hemoglobin, transfusion independence in thalassemia, and repression of vaso-occlusive episodes in SCD. Using CRISPR/Cas9 system to directly disrupt genes provides a safe and potential one-time functional cure for thalassemia and SCD, suggesting CRISPR/Cas9 as a potential therapeutic tool for the treatment of inherited hematological disorders.

The etiological agent for the coronavirus disease 2019 (COVID-19), the SARS-CoV-2, caused a global pandemic. Although mRNA, viral-vectored, DNA, and recombinant protein vaccine candidates were effective against the SARS-CoV-2 Wuhan strain, the emergence of SARS-CoV-2 variants of concern (VOCs) reduced the protective efficacies of these vaccines. This necessitates the need for effective and accelerated vaccine development against mutated VOCs. The development of multi-epitope vaccines against SARS-CoV-2 based on in silico identification of highly conserved and immunogenic epitopes is a promising strategy for future SARS-CoV-2 vaccine development. Considering the evolving landscape of the COVID-19 pandemic, we have conducted a bibliometric analysis to consolidate current findings and research trends in multi-epitope vaccine development to provide insights for future vaccine development strategies. Analysis of 102 publications on multi-epitope vaccine development against SARS-CoV-2 revealed significant growth and global collaboration, with India leading in the number of publications, along with an identification of the most prolific authors. Key journals included the Journal of Biomolecular Structure and Dynamics, while top collaborations involved Pakistan-China and India-USA. Keyword analysis showed a prominent focus on immunoinformatics, epitope prediction, and spike glycoprotein. Advances in immunoinformatics, including AI-driven epitope prediction, offer promising avenues for the development of safe and effective multi-epitope vaccines. Immunogenicity may be further improved through nanoparticle-based systems or the use of adjuvants along with real-time genomic surveillance to tailor vaccines against emerging variants.

Antibodies have specific binding capabilities and therapeutic potential for treating various diseases, including viral infections. The amino acid composition of the hypervariable complementarity determining regions (CDR) loops and the framework regions (FR) are the determining factors for the affinity and therapeutic efficacy of the antibodies. In this study selected and curated, 77 viral antigen-human antibody complexes from Protein data bank from the Thera-SAbdab database were analyzed. The results revealed diversity indices within specific CDR regions, amino acid frequencies, paratope-epitope interactions, bond formations, and bond types among the analyzed viral Ag-Ab complexes. The finding revealed that Ser, Gly, Tyr, Thr, and Phe are prominently present in the antibody CDRs. Analysis of CDR profiles indicated an average amino acid diversity of 60-80% in heavy chain CDRs and 45-60% in light chain CDRs. Aromatic residues, particularly Tyr, Phe, and Trp showed significant involvement in the paratope-epitope interactions in the heavy chain, while Tyr, Ser, and Thr were key contributors in the light chain. Furthermore, the study examined the occurrence of amino acids in both light and heavy chains of viral Ag- human Ab complexes, analyzing the presence of amino acids as single residues, dipeptides and tripeptides. The analysis is crucial for enhancing the antibody engineering processes including, design, optimization, affinity enhancement, and overall antibody development.

Nine homologous Cold Shock Proteins (Csps) have been recognized in the E.coli Cold Shock Domain gene family. These Csps function as RNA chaperones. This study aims to establish the evolutionary relationships among these genes by identifying and classifying their paralogous counterparts. It focuses on the physicochemical, structural, and functional analysis of the genes to explore the phylogeny of the Csp gene family. Computational tools were employed for protein molecular modeling, conformational analysis, functional studies, and duplication-divergence assessments. The research also examined amino acid conservation, protein mutations, domain-motif patterns, and evolutionary residue communities to better understand residual interactions, evolutionary coupling, and co-evolution. H33, M5, W11 and F53 residues were highly conserved within the protein family. It was further seen that residues M5, G17, G58, G61, P62, A64, V67 were intolerant to any kind of mutation whereas G3, D40, G41, Y42, S44, T54, T68, S69 were most tolerable towards substitutions. The study of residue communities displayed that the strongest residue coupling was observed in N13, F18, S27, F31, and W11. It was observed that all the gene pairs except CspF/CspH had new motifs generated over time. It was ascertained that all the gene pairs underwent asymmetric expression divergence after duplication. The K_a/ K_s ratio also revealed that all residues undertook neutral and purifying selection pressure. New functions were seen to develop in gene pairs evident from generation of new motifs. The discovery of new motifs and functions in Csps highlights their adaptive versatility, crucial for E. coli's resilience to environmental stressors and valuable for understanding bacterial stress response mechanisms. These findings will pave the way for future investigations into Csp evolution, with potential applications in microbial ecology and antimicrobial strategy development.

Mitochondrial ribosomal protein L21 (MRPL21) is essential for normal cell function and may play a significant role in cancer development. In this study, we performed a comprehensive pan-cancer analysis to explore MRPL21's function across different cancers, utilizing multiple online data platforms such as TCGA. Our analysis covered its clinical significance and biological functions, including expression levels, survival and diagnostic analysis, gene mutations, multidimensional immune-correlation analysis, tumor heterogeneity, and cancer-associated signaling pathways. Additionally, we constructed a prognostic nomogram for lung adenocarcinoma (LUAD) patients based on MRPL21 and validated its biological function through in vitro experiments. Our findings revealed that MRPL21 is commonly overexpressed in various cancers and is associated with poor prognosis. It significantly impacts cancer-related pathways, particularly those related to cell cycle activation. Moreover, MRPL21 is critical in the tumor microenvironment and is closely linked to immune infiltration across several cancer types. Its expression correlates with essential factors such as tumor mutational burden, microsatellite instability, immune checkpoint, and methylation patterns. In LUAD, MRPL21 was identified as an independent risk factor and demonstrated that MRPL21 promotes LUAD progression. Overall, MRPL21 holds potential as both a diagnostic and prognostic marker in cancer and could serve as a promising therapeutic target, particularly for LUAD.