Molecular Biotechnology最新文献

Recently biocementation has got attention of many researchers worldwide as one of the most potent techniques for sustainable construction. Several studies have been carried out worldwide on biocementation by urea hydrolysis. Biocementation by bacterially induced calcium carbonate precipitation by different bacterial species has been among the most widely researched areas in this field. Biocementation has proved efficient in enhancing the strength and durability of cement-based materials. However, no significant work has been carried out to determine the performance of biocemented specimens at elevated temperatures. This study primarily focuses on the effects of high temperatures (300, 450, and 600 °C) on the compressive strength of two types of biocemented specimens prepared by using ureolytic bacteria and rich in urease watermelon seeds. The motive behind testing these two types is to know how the enzyme induced or microbially induced react to temperature elevation. Also, the effect of different cooling techniques (viz., natural cooling, water spray cooling and fire extinguishing foam spray cooling) were studied. These cooling techniques were selected so as to check which cooling technique should be preferred in case of fire situation in a cement-based structure. Results show that biocemented specimens can perform very good up to the temperature 300 °C as compared to control specimens in terms of compressive strength. At 450 °C temperature, there is no significant difference in compressive strengths of control and biocemented specimens. When the specimens were subjected to 600 °C, biocemented specimens showed lower strength than control specimens at the same temperature due to denser microstructures. Thus, biocemented cement mortar should not be used in reactors, muffles and ovens where temperature would go above 450 °C.

The field of gene therapy has witnessed significant advancements in the utilization of Adeno-associated virus (AAV) owing to its inherent biological advantages. Targeted AAV vectors are generated through genetic or chemical modification of the capsid for user-directed purposes. However, this process can result in imbalances in viral protein sequence homogeneity, stoichiometry, and functional transduction vector units, thereby introducing new challenges. This mini review focuses on the ongoing efforts to develop targeted vectors, which inadvertently present unsolicited obstacles for clinical application and provided perspectives on future directions.

Azaleas (Rhododendron simsii) are popular ornamental woody plants known for their bright colors; however, very limited studies have been reported on the process of flower petal pigmentation. In this study, we found significant differences in the anthocyanin contents of petals from different colored azaleas, and the results of quantitative real-time PCR indicated that the R2R3 MYB genes, RsMYB12, RsMYB90, and RsMYB123, showed significant expression changes during the petal coloration in azalea petals; therefore, we hypothesized that RsMYB12, RsMYB90, and RsMYB123 might involve in the coloring process of azalea petals by regulating anthocyanin synthesis. This work provides insights into the underlying mechanisms of petal pigmentation in R. simsii and provides candidate genes for flower color breeding of azaleas and other ornamental flowers.

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.

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.

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.

ADP-glucose pyrophosphorylase (AGPase; E.C. 2.7.7.27) is the rate-limiting enzyme catalyzing the first committed step of starch biosynthesis in higher plants. The enzyme functions as a heterotetramer comprising two large (LS) and two small (SS) subunits that share 47.02% sequence identity in wheat. To elucidate the structural mechanism underlying heterotetramer assembly, we generated six possible dimeric conformations based on two-fold symmetry, three side-by-side (D1, D2, D3), and three upside-down (D4, D5, D6) orientations and evaluated their stability through all-atom molecular dynamics (MD) simulations combined with MM-GBSA and MM-PBSA free energy analyses. Among all configurations, the D2 heterodimer emerged as the most stable, exhibiting the lowest binding free energy (-15.2 kcal·mol⁻¹), largest interface area (1757.2 Ų), and strongest predicted affinity (Kd = 2.1 × 10⁻¹¹ M). Interaction energy analysis revealed that D2 stability is primarily governed by an extensive network of 25 hydrogen bonds and seven salt bridges at the LS-SS interface. Together, these results provide the first comprehensive molecular insight into the assembly and stabilization of wheat AGPase, a central determinant of starch biosynthetic efficiency. These results provide the first deep-learning-based molecular model of wheat AGPase, offering detailed structural insight into its subunit assembly and stability mechanisms. By identifying key interfacial residues that govern complex formation, this study establishes a foundation for rational protein engineering aimed at enhancing AGPase thermostability and catalytic efficiency, traits directly linked to improved starch accumulation and grain yield in cereal crops.

Lipase/phospholipase (Lip/Plip) from thraustochytrids is a bifunctional enzyme capable of hydrolyzing both triglycerides and phospholipids and is characterized by an unusual 150-residue N-terminal domain whose function remains unclear. To elucidate the role of this region, we engineered an N-terminally truncated enzyme (S-270) and compared it with the full-length enzyme (L-420) from Aurantiochytrium sp. MG91 heterologously expressed in Escherichia coli. The present research is limited and directed toward comparing enzyme activities (specific activity, substrate selectivity, and growth-related evaluation) and bioinformatics evidence to offer an extensive overview of the biological effects that occur. The 18S rRNA sequence analysis revealed that MG91 isolate is closely related to Aurantiochytrium limacinum and A. mangrovei. Both constructs were successfully expressed in E. coli BL21 (DE3), yielding proteins of ~ 45 kDa (L-420) and ~ 30 kDa (S-270). Although the substrate specificity remained constant favoring pNP-C12 and sunflower-derived phosphatidylcholine, the S-270 demonstrated a marked enhancement in specific activity, showing a 4-6-fold rise in lipase and a 1.4-fold increase in phospholipase. Consistent with the growth assessments, the S-270 appeared to alleviate the harmful effects Lip/Plip observed in L-420, as evidenced by its normal colony morphology and higher growth relative to the L-420 under IPTG-induced overexpression in E. coli. Nevertheless, the 3D Structural modeling revealed no major conformational differences between L-420 and S-270 apart from the N-terminal membrane-associated region present only in the full-length enzyme. The G-Y-S-R-G lipase motif, containing the catalytic residues Ser311, Asp368, and His382, was annotated and phylogenetic analysis positioned Lip/Plip MG91 within a miscellaneous cluster that encompasses both Y-type and GX-type lipases. Our findings demonstrate that the L-420 exhibits notably higher toxicity in E. coli expression system, as indicated by its diminished expression, decreased specific activity, suppressed cellular growth, and the appearance of smaller colonies compared with S-270. This is the first report showing that removing the N-terminal domain of thraustochytrid Lip/Plip increases its specific activity, highlighting its biotechnological potential.

The study aimed to generate a simple and valid scheme for biosynthesis of Hypecoum pendulum L. (HP) extract-based silver nanoparticles (HP-AgNPs) and to assess their in vivo anti-hyperglycemic and in vitro antimicrobial potency. Characterization by UV spectroscopy verified the existence of HP-AgNPs at 417 nm. The functional moieties that help in the HP-AgNPs stabilization and reduction were analyzed by the FTIR technique. The face-centered cubic crystal nature of HP-AgNPs (size: 36.3 nm) was assessed using the XRD technique. Surface morphologies with predicted nanoscale size (80 nm) were confirmed by SEM examination. EDX revealed a sharp peak (3.2 keV) that confirmed Ag as a leading element (49%). Alloxan was applied to induce diabetes in female Sprague-Dawely rats (age: 1.5-2 months, body weight: 120-150 g). The 21-day treatment of diabetic rats with HP extract and HP-AgNPs resulted in a significant gain in average body weight, average organ weight, and hemoglobin level, as well as a decline in HbA1c, blood sugar, lipid, and liver profile levels in comparison to the diabetic-untreated group. HP-AgNPs showed promising antibacterial efficacy against all tested strains (Gram-positive and negative) in a positive trend with concentration. Green-synthesized HP-AgNPs also showed considerable inhibition of all tested fungal strains as compared to HP extract. The phytochemical analysis of the HP plant confirmed the phytochemicals attributed to the antimicrobial and anti-diabetic power of HP extract and HP-AgNPs. These outcomes showed that HP-AgNPs have demonstrated promising anti-diabetic and antimicrobial action than HP-extract.

The genus Equus, encompassing horses, donkeys, and extinct relatives, has evolved over approximately 55 million years from small, multi-toed ancestors to the modern horse. Selective breeding has produced over 600 distinct horse breeds optimized for diverse traits such as size, conformation, performance, and adaptability. In the past two decades, rapid advances in equine genomics have significantly deepened our understanding of the molecular basis of these traits. The integration of high-throughput sequencing, genome-wide association studies (GWAS), and single-nucleotide polymorphism (SNP) genotyping has revealed key genes and genomic regions associated with body size, coat color and texture, performance, behavior, and environmental adaptation. Variants in genes such as MC1R, ASIP, KIT, PAX3, and KRT25 govern pigmentation and coat characteristics, while DRD4, COMT, and SLC6A4 are associated with behavioral attributes like trainability, fear response, and sociability. Athletic traits arise from complex genetic interactions affecting muscle composition, gait, speed, and stamina. Furthermore, genomic studies highlight adaptations to diverse environments, including hypoxia tolerance, heat resistance, and endurance in harsh terrains, demonstrating the species' remarkable plasticity. Collectively, these findings emphasize how evolutionary processes and human-driven selection have shaped the genetic diversity, adaptability, and enduring success of equines across ecological and functional landscapes.