Biochemical and biophysical research communications最新文献

Resistance to lenvatinib has become a major obstacle in the clinical treatment of liver cancer, highlighting the significant research value and translational potential of developing synergistic drug combinations. In this study, deep learning models (MARSY and MatchMaker) were employed to predict potential synergistic partners for lenvatinib, with vincristine identified as a promising candidate. In vitro experiments confirmed that the combination synergistically inhibited the proliferation, migration, and clonogenic formation of liver cancer cells: CCK-8 and colony formation assays demonstrated a significant reduction in cell viability and clonogenic ability, while wound healing and Transwell assays indicated effective suppression of cell migration. The synergistic effect was quantitatively validated using the ZIP model. Furthermore, flow cytometry and Western blot analyses confirmed that the combination effectively induced apoptosis. Mechanistic studies revealed that the co-treatment led to excessive accumulation of intracellular reactive oxygen species (ROS), which activated the TNF-α/Caspase-8 signaling pathway, thereby inducing apoptosis in liver cancer cells. The cytotoxicity and pro-apoptotic effects were significantly attenuated by the ROS scavenger NAC. These findings provide a solid preclinical foundation for the further development of this combination therapy and underscore the importance of the "computational prediction-mechanistic validation" strategy in advancing cancer drug discovery.

We previously reported that murine fecal microRNAs (miRNAs) alter the composition of cultured gut microbiota and selectively increase Enterococcus. Since host miRNA expression is influenced by microbial colonization, this study investigated whether fecal miRNAs derived from germ-free (GF) and specific pathogen-free (SPF) mice differentially affect the composition and metabolic activity of cultured gut microbiota. Fecal miRNAs isolated from GF and SPF mice were added to cultures of murine gut microbiota. 16S rRNA gene sequencing showed that GF mice- and SPF mice-derived miRNAs altered microbial community structures in distinct ways. ANCOM identified Enterococcus as the only taxon significantly increased by miRNA treatment. GF mice-derived miRNAs significantly enhanced fermentation, as evidenced by a lower culture pH and higher concentrations of acetate, propionate, and lactate compared with SPF mice-derived miRNAs or vehicle controls. Correlation analysis demonstrated positive associations between organic acid levels and secondary fermenters, including Alloprevotella, Muribaculum, Lachnoclostridium, and Peptococcaceae. In contrast, typical saccharolytic butyrate producers such as Blautia, Lachnospiraceae, and Oscillospiraceae showed negative correlations. Microarray analysis revealed differences in the fecal miRNA profiles of GF and SPF mice, supporting the hypothesis that microbial exposure modulates fecal miRNA composition. Fecal miRNAs derived from antibiotic-treated mice did not reproduce the broad fermentation-promoting effects observed with GF mice-derived miRNAs, although lactate concentration increased. These observations suggest that fecal miRNAs promote fermentation by stimulating Enterococcus and downstream cross-feeding networks, and that prior microbial exposure attenuates this effect. Consequently, fecal miRNAs appear to represent a coevolved host mechanism that modulates microbial fermentation to maintain intestinal homeostasis.

Cardiac fibroblasts (CFs) are the predominant non-myocyte cell type in the heart and play central roles in extracellular matrix remodeling and intercellular signaling during cardiac physiology and pathology. However, the bioenergetic basis underlying CF functions remains poorly understood, mainly due to the lack of tools for visualizing intracellular adenosine triphosphate (ATP) dynamics with high spatiotemporal resolution. Here, we established immortalized murine cardiac fibroblasts stably expressing the genetically encoded ATP indicator GO-ATeam2 based on Förster Resonance Energy Transfer (FRET). The resulting CF7/GO-ATeam2 cell line allows real-time and quantitative monitoring of cytosolic ATP levels in living cells. CF7/GO-ATeam2 cells exhibited robust proliferation and quick responses to change of cytosolic ATP level. We demonstrated dynamic cytosolic ATP imaging upon pharmacological perturbations of oxidative phosphorylation and glycolysis, as well as under growth factor stimulation. Our work provides the CF7/GO-ATeam2 platform, a versatile cellular resource for dissecting the metabolic regulation of cardiac fibroblasts, offering new opportunities to explore energy dynamics in cardiac physiology and disease.

This study evaluates the radiobiological effectiveness comparing superficial X-rays to Cs-137 in V79 and U2OS cells cultured in T25 flasks using colony formation assays. Additional dosimetric care was given to maintaining equal absolute dose for all beam qualities, correcting for absorption and scattering in the flask, and quantifiable interpretation due to dose of the spatial differences in cell survival with significantly higher survival near the flask's edges. Ion chamber measurements quantified flask lid attenuation (6.9 %, 2.4 %, and 1.9 % for 50, 70, and 100 kVp), enabling dose correction for accurate survival analysis. CT-based Monte Carlo simulations revealed thicker flask walls and the meniscus effect created a 35 % dose reduction from flask center to edges, directly explaining the observed survival patterns. Monte Carlo simulation also predicted an increased secondary electron production at higher energies. These results emphasize the necessity of precise dosimetry in low-energy X-ray studies. Combined experimental and computational approaches enhanced reliability of radiobiological assessments. Survival curves yielded Relative Biological Effectiveness (RBE) values for 50 kVp X-ray of 1.45 (V79) and 1.64 (U2OS); 1.41 (V79) and 1.55 (U2OS) at 70 kVp; and 1.23 (V79) and 1.36 (U2OS) at 100 kVp, relative to Cs-137.

Chemotherapy is a significant treatment for cancer; however, it is frequently associated with deleterious side effects. Thymoquinone (TQ), a naturally occurring compound, has been reported to exhibit notable anti-cancer potential. The present study focuses on the in vitro effects of TQ-loaded nanoemulsion (TQ-NE) as an independent treatment and alongside the prevalent chemotherapeutic agents, 5-fluorouracil (5-FU) and capecitabine (CAP), on a colorectal cancer (HT-29) cell line. A TQ-NE formulation was synthesized and characterized, revealing an average particle size of 129.5 nm. The IC₅₀ values for CAP, 5-FU, TQ, NE (carrier), and TQ-NE were 39.57, 61.01, 21.81, 218.4, and 15.12 μM, respectively. Cytotoxic effects were examined in both HT-29 cells and fibroblasts, revealing that TQ-NE enhanced the sensitivity of cancer cells to chemotherapeutic agents. When combined with 5-FU or CAP, TQ-NE produced a greater reduction in cell viability than TQ alone, indicating synergistic interaction. Flow cytometry analysis further revealed that TQ-NE induced distinct apoptotic responses compared to the control, whereas its combination with chemotherapy drugs showed a different pattern. These findings suggest that TQ-NE can enhance the cytotoxic activities of 5-FU and CAP in vitro. Furthermore, using lower concentrations of TQ-NE could potentiate the inhibitory effects of 5-FU and CAP, presenting a promising strategy for improving colorectal cancer (CRC) treatment outcomes while potentially reducing the adverse effects related to higher doses of conventional chemotherapy.

Extracellular vesicles (EVs) are lipid bilayer-encased nano-size carriers that orchestrate molecular exchanges in the tumor microenvironment (TME) and carry significant information about the tumor development, progression and aggressiveness. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) mediates Helicobacter pylori adhesion to gastric epithelial cells and is markedly upregulated in gastric cancer (GC). This study identified that EVs secreted by H. pylori-infected gastric cancer cells (GCCs) were loaded with CEACAM6. In order to examine the tumorigenic potential of EVs released by H. pylori-infected cells with and without CEACAM6 overexpression, EVs were thoroughly characterised and several functional assays were conducted. CEACAM6 overexpressed cell-derived EVs mimicked the elevated status of CEACAM6 as in their source cells which were found to be further enhanced in EVs collected from infected cells. As revealed by the population-doubling, clonogenicity, wound-healing and matrigel invasion assays, CEACAM6-enriched EVs promoted oncogenic properties of recipient cells while EVs from H. pylori-infected CEACAM6-expressing cells further amplified these tumorigenic abilities. A novel approach of EV-sonication and fractionation identified that CEACAM6 were mainly located in the EV membrane. Interestingly, aligned with the finding of elevated CEACAM6 protein in the H. pylori infection-led metastatic GC tissue samples, sera from those GC patients exhibited significantly high CEACAM6 compared to those from the healthy volunteers. Collectively, these findings highlight CEACAM6-containing EVs as mediators of tumorigenesis. This study also showcases the technical, translational and clinical advantages of considering CEACAM6 as a diagnostic biomarker for the detection of GC in a minimally-invasive manner.

Background: Intrauterine adhesions (IUAs) are defined by excessive fibrotic remodeling, failure of endometrial regeneration, and impaired fertility, and that have not yet had any effective treatment options. Human amniotic fluid-derived extracellular vesicles (hAF-EVs) have inherent regenerative potential and represent a promising platform for targeted delivery of antifibrotic microRNAs such as miR-29.

Methods: A murine IUA model was established after mechanical damage of the endometrium. Unmodified hAF-derived EVs or miR-29-enriched hAF-EVs were administered to mice. Histological and immunohistochemical methods, quantitative PCR, and fertility tests were used to determine endometrial regeneration, fibrosis, angiogenesis, and molecular fibrogenic marker expression.

Results: Both hAF-EVs and miR-29-enriched hAF-EVs significantly improved endometrial architecture, reduced collagen deposition, and enhanced fertility outcomes compared with untreated IUA controls. While unmodified EVs exerted robust regenerative and antifibrotic effects, miR-29 enrichment resulted in greater suppression of TGF-β/SMAD3-associated fibrogenic signaling, enhanced angiogenesis, and selective improvement in implantation-related parameters. Multiple histological results revealed similar recovery in the two EV-treated groups.

Conclusion: hAF-derived EVs constitute an effective cell-free therapeutic strategy for attenuating fibrosis and promoting endometrial repair following intrauterine adhesions. Enrichment with miR-29 provides selective enhancement of molecular and functional outcomes, supporting its role as a targeted modulatory component within an EV-based regenerative platform.

Functional monoclonal antibodies (mAbs) are crucial for the development of effective control and therapeutic strategies against lumpy skin disease (LSD), a cattle disease caused by Lumpy skin disease virus (LSDV) that results in significant economic losses. In this study, the soluble recombinant ORF117 protein of LSDV was expressed in 293F cells. It was then employed as an immunogen to generate two mAbs, designated 11C4 and 28B5. Bio-layer interferometry measurements indicated high binding affinities, with equilibrium dissociation constants (Kd) values of 0.05 nM for 11C4 and 0.8 nM for 28B5. Indirect ELISA revealed half-maximal effective concentration (EC₅₀) value of 6.69 × 10^-3μg/mL for 11C4 and 11.22 × 10^-3μg/mL for 28B5. Immunofluorescence assays confirmed that both mAbs recognized the native viral ORF117. Competition ELISA demonstrated that the mAbs preferentially binded to viral particles rather than to recombinant ORF117 in a co-incubation system. Neutralization assays showed that 11C4 achieved 45.1 % neutralization of LSDV infection, while 28B5 exhibited comparable activity. Epitope mapping indicated both mAbs recognized novel distinct epitopes. 11C4 targeted the epitope (IFPGDDDETNERNINHREKT) with loop structure, whereas 28B5 bound to a novel distinct region (KKIINERYSNYISIDDDEISDILKDSFISNEEMQI) with helix structure. Structural docking simulations revealed that 11C4 recognized a conserved segment IFPGDDDET among Chordopoxvirinae subfamily, with Asp12 and Glu14 identified as critical residues forantibody binding. This study provided valuable tools to advance the development of diagnostic and therapeutic measures against LSDV and related poxviruses.

Metabolic enzymes are increasingly recognized to perform functions beyond their canonical roles. Here, we report that Escherichia coli argininosuccinate lyase (ArgH), a central enzyme in arginine biosynthesis, directly binds duplex DNA in vitro. This study was prompted by reproducible observations of a nucleic acid-like component while isolating ArgH by anion-exchange chromatography. To characterize ArgH-DNA interactions, six duplex DNAs were selected based on structure-guided docking simulations (PADA1 and HDOCK), and their dissociation constants (K_D) were determined by fluorescence spectroscopy. The K_D values ranged from 60 to 200 nM, suggesting that the duplex DNAs interacted with ArgH strongly. These observations suggested ArgH as a previously unrecognized DNA-binding protein and provided a quantitative basis for exploring additional roles of this metabolic enzyme in nucleic acid-associated cellular processes.