Molecular Biotechnology最新文献

Bacterial cellulose (BC) is one of the biodegradable materials that is produced by BC-producing bacteria and is widely used in various industries. In previous studies, we isolated high-yield BC-producing Komagataeibacter diospyri MI 2 and analyzed its genomes. In this study, we aimed to improve the BC production ability of K. diospyri MI 2 by UV light and to investigate BC-regulating genes by comparative genomics. Of the 17 surviving colonies after UV irradiation, most produced significantly more BC higher than the wild type, while K. diospyri mutants B, G, and O had the highest BC yield. The properties of BC were analyzed by SEM and X-ray diffraction. The results showed that the BC produced by the K. diospyri mutants was denser than that of the wild type but did not significantly change crystallinity. The whole-genome sequencing and comparative genomics were performed to explore the genes involved in the improvement of BC in the K. diospyri mutants. The result showed that the descriptive statistics of the assembled genomes of the K. diospyri mutants were similar to that of the wild type. When comparing their genomes, we found that genes, including galE, aes, and bfr, which were likely involved in BC biosynthesis, disappeared in the K. diospyri mutants. In addition, variant analysis was performed, and SNPs and InDels located on the CDS of genes, including the response regulator, efflux transporter outer membrane subunit, chloride channel protein, and ribonuclease E, could be potential biomarkers for higher BC production. Our study provided the K. diospyri mutants by UV mutagenesis and explored the set of genes possibly involved in BC production.

Herein, nine coumarins were isolated and characterized from Ammi visnaga, prompting a comprehensive evaluation of their pharmacological potential. An in silico target selection approach was initially employed to identify plausible protein targets, which, combined with a repurposing rationale, prioritized squalene epoxidase (SQLE) for detailed investigation. The in vitro SQLE inhibition assay demonstrated that khellin, khellol, and visnagin exhibited low-micromolar IC₅₀ values (3.48 ± 0.23, 2.83 ± 0.18, and 2.74 ± 0.15µM, respectively), rivaling the reference inhibitor (2.75 ± 0.20µM). Enzyme- kinetic studies confirmed a competitive inhibition mechanism, with K_i values in the low micromolar range. Subsequent docking and molecular dynamics simulations corroborated these findings, revealing that each active coumarin remains stably engaged within the SQLE active site. Free-energy analyses (MM/PBSA) further underscored their favorable binding energetics, while free-energy landscape (FEL) calculations indicated well-defined and energetically accessible conformations for the inhibitor-enzyme complexes. Moreover, key structural and energetic MD parameters collectively demonstrated stable interactions and minimal perturbations to the enzyme's global fold. An ADMET assessment revealed high oral absorption potential, no immediate concerns regarding P-glycoprotein efflux, and generally favorable physicochemical characteristics, despite some predicted inhibitory activity against specific cytochromeP450 isoforms. Overall, these data suggest that naturally derived coumarins from Ammi visnaga-especially khellol, khellin, and visnagin-can effectively target SQLE, highlighting their potential for antifungal and possibly anticancer applications. This study illustrates the value of integrating phytochemical isolation, in silico repurposing, enzyme-based screening, and advanced MD simulation workflows to accelerate the discovery of promising new inhibitors from medicinal plants.

Evidence for the microbiome's role in human health and disease has been piling up ever since the human microbiome project. The composition of one's microbiome can have a major effect on one's risk of developing cancer and the nature of how cancer develops. Several estimates suggest the percentage of cancer cases that can be attributed to microorganisms at around 15%. In addition, researchers are still trying to figure out how the microbiota, and the gut microbiota in particular, affects how a patient responds to chemotherapy, immunotherapy, and radiotherapy. In this light, we conducted an in-depth bioinformatics analysis of the gut microbiota- RCCstem cells axis, utilizing python-based programme and enrichment databases to analyses data from many sources, including clinical data, transcription factors, kinases and gene expression profile of RCCstem cells. Five genes, including SLC16A6, CPNE5, AFAP1L1, SCARF1, and NOTCH4, were shown to be shared by the hub gut microbiota and extracellular proteins. Patients with RCCstem cells had a disproportionately high number of certain types of bacteria. In patients expression profile have high CPNE5, AFAP1L1, SCARF1, and NOTCH4 expression. RCCsurvival rates are reduced by roughly 50% due to all of the genes involved. Also, the Actinobacteria and Gammaproteobacteria possible role in renal cancer development via relation to cancer stem cells. The gut microbiota and its components were considered for their possible relevance in the development of RCC.

Sepsis-associated acute kidney injury (SA-AKI) is a severe complication of sepsis, primarily driven by immune-inflammatory dysregulation and oxidative stress, and 1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) shows renoprotective potential, yet its molecular targets and mechanisms remain unclear. Transcriptomic data of SA-AKI from GEO were analyzed by ssGSEA for immune infiltration, differential expression analysis, WGCNA, and machine learning (LASSO, SVM-RFE, Random Forest) to identify the biomarkers. In vivo, SA-AKI was induced by cecal ligation and puncture (CLP) in mice treated with 1,25(OH)₂D_3, and renal function, histology, inflammatory cytokines, oxidative stress markers, and CMKLR1 expression were evaluated. In vitro, LPS-stimulated HK-2 cells were treated with 1,25(OH)₂D₃ with or without CMKLR1 overexpression to assess cytokines, cell viability, apoptosis, and oxidative stress. Four key biomarkers-CMKLR1, COL1A1, RPS19, and THBS1-were identified by machine learning, with CMKLR1 as the key target. In SA-AKI mice, 1,25(OH)₂D₃ downregulated CMKLR1, improved renal function, and reduced inflammation and oxidative stress. In LPS-stimulated HK-2 cells, it dose-dependently suppressed cytokine release, restored cell viability, and alleviated oxidative stress, while CMKLR1 overexpression partially reversed these effects. CMKLR1 was identified as a key immune inflammation target in SA-AKI. 1,25(OH)₂D₃ was protected against renal injury by suppressing CMKLR1, and mitigating inflammation, oxidative stress, and apoptosis, while CMKLR1 overexpression partially reversed these effects. These findings highlight CMKLR1 as a potential therapeutic target for SA-AKI.

Ferroptosis, a novel form of programmed cell death, is closely linked to the imbalance between cellular oxidative stress and antioxidant defenses. Studies have shown that the occurrence and progression of ferroptosis are associated with Nrf2 and ROS levels, providing important guidance for ferroptosis research and the development of novel therapeutic strategies targeting Nrf2/ROS-related mechanisms. Despite its potential in treating various diseases, the clinical application of ferroptosis modulation is limited due to an insufficient interventional strategy and mechanism. Sestrin proteins are highly conserved family proteins induced by stress and injury. They function in mTORC regulation, ROS inhibition, and leucine binding. Emerging direct evidences suggest sestrin proteins inhibit ferroptosis in various pathological contexts, and numerous studies of sestrin proteins and Nrf2/ROS (the core to ferroptosis) have provided a series of indirect evidence that sestrin proteins and ferroptosis are strongly correlated. In the present study, the regulatory effects of sestrin proteins on ferroptosis/Nrf2/ROS were reviewed to describe the interplay between sestrin proteins and ferroptosis, alongside exploring their potential mechanisms. The present study represents the first comprehensive review dedicated to examining the relationship between ferroptosis and sestrin proteins. This work offers clinicians and researchers a thorough framework for assessing the dynamics of ferroptosis and sestrin proteins.

Histidine phosphotransfer proteins (HP/Hpts) are key components in the two-component system (TCS) involved in cytokinin signalling. In rice, five Hpt genes have been identified, including two authentic and three pseudo-Hpts. This study focused on isolating and characterizing the OsHpt4/PHP2-like gene from three O. indica rice varieties, and on exploring the evolutionary and regulatory features of the Hpt gene family across Oryza species. Hence, we isolated the OsHpt4/PHP2-like gene (456 bp; 151 amino acids) from three O. indica rice varieties- ASD-16 (Parentage- ADT 31/Co39), ADT-54 (Parentage-I.W.Ponni/ Banskathi) and CO-52 (Parentage- BPT 5204/ Co (R) 50) and compared their physicochemical properties. To gain broader insights, a genome-wide analysis was conducted across six Oryza species (Oryza sativa ssp. japonica, Oryza sativa ssp. indica, Oryza rufipogon, Oryza nivara, Oryza glumaepatula, and Oryza barthii), identifying 35 Hpt isoforms. Studies on gene structure and conserved motifs showed that intron and exon organization, as well as motif composition, are well preserved. Phylogenetic analysis grouped Hpts into four main clusters with other monocots. Both segmental and tandem gene duplications contributed to gene family expansion. Promoter analysis revealed cis-elements responsive to phytohormones, abiotic stresses, light, and development. These results offer new insights into the evolution and regulation of Hpt genes in rice, and it paves a strong avenue for future research on stress tolerance and crop improvement.

Bone remodeling relies on balanced osteoclast and osteoblast activity, with dysregulated osteoclast differentiation contributing to pathologies like osteoporosis. While RANKL/M-CSF signaling and master regulators (NFATc1, c-Fos) are established, comprehensive temporal dynamics of gene networks governing progressive osteoclastogenesis stages remain poorly characterized. We employed an integrated approach combining RANKL-induced osteoclast differentiation from murine bone marrow-derived macrophages (BMMs) with rigorous functional validation (TRAP staining, podosome visualization via rhodamine-phalloidin/DAPI, Western blotting for NFATc1/c-Fos/CTSK) and high-throughput RNA sequencing across critical time points (day 0, 1, 3, and 5). Subsequent bioinformatic analyses included differential expression profiling (DESeq2/edgeR), Gene Ontology (GO) and KEGG pathway enrichment, Gene Set Enrichment Analysis (GSEA), and temporal clustering using Mfuzz. Key transcriptional findings were confirmed by RT-qPCR. GO/KEGG/GSEA analyses revealed significant enrichment of DEGs in FA signaling, PI3K-Akt pathway, and cytoskeletal organization. Mfuzz clustering delineated distinct gene expression trajectories. We documented stage-specific expression kinetics: 23 FA-related genes showed dynamic shifts (e.g., upregulated Itgb3, Src, Pik3r3, Fn1; downregulated Itga6, Parvg); multiple MMPs (Mmp2, Mmp9, Mmp13-17) and TIMPs (Timp1-3) were progressively induced, while Mmp8, Mmp12, Mmp27 were suppressed. Cell fusion involved upregulated Tnfrsf11a, Nfatc1, Plcg1, Xkr8 alongside downregulated Trem2, Tyrobp, Ccr2, Rhoa, Casp3. This study provides the first comprehensive temporal transcriptome atlas of RANKL-induced osteoclastogenesis. It delineates stage-specific molecular reprogramming, revealing dynamic regulation of FA signaling components promoting adhesion/migration, complex MMP/TIMP induction balancing ECM degradation, and coordinated transcriptional networks enabling fusion. These findings significantly expand understanding beyond the core RANKL-NFATc1 axis, identifying novel stage-specific regulatory hubs and potential therapeutic targets within FA, PI3K-Akt, MMP/TIMP, and fusion pathways for osteoclast-driven bone diseases.

The CRISPR/Cas9 genome editing technology has had a significant impact on cancer research and therapeutic development, providing unprecedented precision in manipulating cancer-associated genes. Although this review focuses on Cas9, we situate it within the broader CRISPR landscape that includes DNA-targeting effectors (Cas9/Cas12), RNA-targeting systems such as Cas13, and type III systems with dual DNA and RNA activity, modalities that expand both experimental and therapeutic possibilities. This comprehensive review examines the current applications of CRISPR/Cas9 in oncology, including its mechanisms and the challenges associated with its clinical translation. Knockout, interference, and activation CRISPR screening platforms have transformed functional genomics by systematically interrogating gene function, identifying therapeutic vulnerabilities, and clarifying resistance mechanisms across diverse cancer phenotypes. This technology has also reshaped cancer modeling, enabling precise recapitulation of disease-relevant mutations from engineered cell lines to patient-derived xenografts that capture tumor heterogeneity and microenvironmental interactions. Notably, the integration of CRISPR/Cas9 with CAR-T therapy has enabled multiplex editing to eliminate alloreactivity, overcome checkpoint-mediated exhaustion, and engineer universal CAR-T cells. Emerging in vivo strategies that directly generate or reprogram CAR-T cells in patients via targeted viral and nonviral delivery underscore accelerating translational momentum. However, significant challenges, including off-target mutagenesis, delivery barriers, p53-mediated selective pressure favoring potentially oncogenic populations, and Cas9 immunogenicity, continue to hinder clinical translation. These limitations necessitate high-fidelity nucleases, optimized guide designs, and improved delivery systems. The future of CRISPR/Cas9 in cancer therapy will depend on technological innovation, comprehensive safety frameworks, and rigorous clinical evaluation as next-generation editing modalities advance toward transformative precision oncology.

Oxidative stress (OS) is thought to mediate the processes of glycolipid disorders of a number of metabolic diseases and recent data suggest that OS may be involved in the pathophysiology of hyperlipidemia. The gene expression profiles of hyperlipidemia samples were downloaded from the Gene Expression Omnibus (GEO) database. Oxidative stress-related genes (ORGs) was the intersection of all valid data of discovery dataset and the ORGs in Genecards. The Differentially expressed genes (DEGs) between hyperlipidemia and control samples were obtained via "limma" R package, and differentially expressed oxidative stress-related genes (DEORGs) associated with hyperlipidemia were screened via OS gene sets. Gene Ontology (GO) and Kyoto encyclopaedia of Genes and Genomes (KEGG) enrichment analyses were performed to study the biological function of DEORGs, and protein-protein interaction (PPI) network analysis was conducted to screen hub genes. Then we constructed microRNA (miRNA), transcription factor (TF) and drug component targets network to explain the regulatory mechanism of ORGs in hyperlipidemia. After screening and evaluating we took GSE1010 as the discovery dataset and the GSE13985 as the validation set. There were 395 ORGs and 14 DEORGs retained from the hyperlipidemia. GO and KEGG results showed that DEORGs were mostly related to OS and lipid metabolism. Then, we used miRNA, TF, and drug component targets network to reveal the regulatory mechanism of hub genes. Finally, we verified expression of DEGs and hub gene in validation set. Our study has further confirmed the relationships between OS and hyperlipidemia, providing oxidative stress-related hub genes with possible function analysis and pathways summarized. These molecules might play a crucial role in the progression of hyperlipidemia and serve as potential biomarkers and therapeutic targets, giving us additional insight into the genes and the mechanism linking the OS system and metabolic disorders. We have not only proved hyperlipidemia is associated with OS but also gave foundation and reference for future researches.

The highly conserved alpha crystallin domain of the small heat shock proteins is essential for dimerization and also implicated in substrate interaction. In this study, we designed four novel mini-peptides from alpha crystallin domain of archaeal Small Heat Shock Protein Tpv HSP 14.3. Among the peptide designs, the mini-peptides ₃₈SDLVLEAEMAGFDKKNIKVS₅₇ and ₄₀LVLEAEMAGFD₅₀ overlapped to the sequences of β3-β4 region. The other two peptides ₇₇YIDQRVDKVYKVVKLPVE₉₄ and ₁₀₇GILTVRMK₁₁₄ correspond to β6-β7 region and β9, respectively. Functional activity of the peptides was evaluated by monitoring heat-induced aggregation of the model substrates alcohol dehydrogenase at 43 °C and citrate synthase at 45 °C. Our results showed that the (38-57) and the (77-94) fragments exhibited chaperone activity with both of the substrate proteins. The (40-50) fragment while exhibiting a noticeable protective effect (> 90%) when tested with citrate synthase showed an anti-chaperone property toward alcohol dehydrogenase. Unlike the (40-50) fragment, the (107-114) fragment did not show any chaperone activity with citrate synthase but exhibited the highest chaperone efficiency among four mini-peptides with alcohol dehydrogenase. The selectivity of the (40-50) and the (107-114) fragments in targeting the client proteins is most likely dependent on their surface hydrophobicity and/or charge as revealed by the sequence and exposed surface analyses.