Biotechnology and applied biochemistry最新文献

Cyclooxygenase-2 (COX-2) plays a crucial role in inflammatory responses and has been implicated in neuroinflammatory processes associated with neurological disorders such as Alzheimer's disease. While selective COX-2 inhibitors (coxib class of drugs) have been developed, their use is limited by adverse effects, necessitating the exploration of alternative therapeutic approaches. This study investigates the potential of curcumin derivatives as COX-2 inhibitors and their possible therapeutic applications in neurological disorders. Previous in silico studies show various amino acids interacting with naturally occurring curcumin derivatives. We explored the potential of 2561 curcumin derivatives as COX-2 enzyme inhibitors by examining their binding affinity to the protein. Using molecular docking, we assessed their interactions with two regions of COX-2, identifying five standout compounds with powerful binding affinities. The binding energies of these compounds lie around -10.7 and -10.6 kcal/mol. To better understand how these top candidates behave in a dynamic biological environment, we ran molecular dynamics simulations focusing on their interactions with one part of the enzyme. These simulations revealed that the compounds formed stable complexes with COX-2, maintaining consistent hydrogen bonds and hydrophobic contacts throughout. RMSD and RMSF graphs exhibit greater stability of these compounds as compared to the control molecule. Finally, energy calculations confirmed that these interactions were not only stable but also energetically favorable, suggesting that several curcumin derivatives could be promising COX-2 inhibitors. This study provides valuable insights into the potential of curcumin derivatives as COX-2 inhibitors and their possible therapeutic applications in neurological disorders. The identified compounds warrant further investigation through in vitro and in vivo studies to validate their efficacy and safety as potential alternatives to current selective COX-2 inhibitors in the treatment of neuroinflammatory conditions.

Oxidation of biologically active 3-mercapto-L-alanine for the formation of disulphide bond (cystine) catalyzed by Copper(II) in alkaline borate buffer and oxygenated condition has been carried out. The progress of the reaction was monitored using an optical spectrophotometer at 355 nm. Kinetic observations and initial rate data reveal a second-order dependence of rate of the oxidation process on 3-mercapto-L-alanine concentrations with k₂ value of 7.52×10^-4 mol^-1 dm^-1 s^-1. In the range of concentration of the metal ion, Copper(II), the finding is unit order dependence of rate on copper catalyst. Over the range of acidity conditions maintained using borate buffer in the present work, the reaction order exhibits a complex dependence on acid concentration. The influence of ionic strength on the initial rate of reaction is conclusive of the fact that there was formation of intermediate complex possibly involving ions of opposite charge. Based on all the kinetic observations and the derived data, the reactive species of 3-mercapto-L-alanine under our reaction conditions are predicted and a suitable mechanism for the occurrence of electron transfer process is suggested. The good agreement of the computed dissociation constant (pK₂ = 8.23) of the substrate leading to its formation of different forms when compared with the literature value validates our proposal of the mechanism of the 3-mercapto-L-alanine to cystine conversion.

Rolling-circle amplification (RCA) is a completely synthetic and rapid mechanism for scaling-up bulk DNA that eliminates requirements for large-scale bacterial fermentation. Several reports have described the surprising ability of large unprocessed (hyperbranched) rolling circle-amplified DNA (RCA DNA) to effectively transfect cultured cells; however, to our knowledge a comprehensive analysis of transfection conditions has not been conducted for this unique type of large synthetic DNA. Herein, we present comparative transfection data for two of the most common mammalian cell types used in biopharmaceutical manufacturing (suspension Chinese hamster ovary CHO cells and adherent HEK293 cells) and a panel of commercial transfection reagents that represent three general compositions (i.e., cationic lipid, cationic polymer, and complex blends). We show the efficiency of RCA DNA delivery is highly influenced by the transfection reagent and cell line, but expression patterns generally track with plasmid controls. Reagents comprising complex blends provide some of the best transfection results for RCA DNA, although additional reagent types work well. We also show it is possible to transfect two different RCA DNA products into CHO cells to produce monoclonal antibodies at high titer. We conclude that large and unique synthetic DNA produced by RCA is capable of being used in place of supercoiled plasmid for downstream transfection workflows.

Prostatic obstruction is a blockage of the urethra caused by benign prostatic hyperplasia, a common condition in aging men that often results in significant urinary complications. Polyurethane prostatic stents are widely used to alleviate this obstruction. However, their susceptibility to biofilm formation and subsequent bacterial infections by pathogens such as Escherichia coli and Proteus mirabilis remains a major clinical challenge. This study investigates the polyurethane prostatic stents (PS) that are surface-immobilized with gentamicin-loaded chitosan nanoparticles (GMCSNPs) to combat these issues. GMCSNPs were synthesized via the ionic gelation method, achieving high drug encapsulation efficiency (92.32%) and exhibiting a spherical morphology, as confirmed by field emission scanning electron microscopy (FESEM); particle size was determined using a Zetasizer. The nanoparticles had a size range of 200-350 nm. The surface of polyurethane prostatic stents was activated and covalently immobilized with GMCSNPs, as confirmed through attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and FESEM analyses. The antimicrobial efficacy of GMCSNPs and GMCSNPs-immobilized polyurethane prostatic stents (GMCSNPs-PS) was evaluated against E. coli and P. mirabilis. Bacterial viability assays, flow cytometry, and biofilm inhibition studies revealed significant antibacterial activity and a marked reduction in biofilm formation. The sustained release of gentamicin, combined with the intrinsic antimicrobial properties of chitosan, demonstrated a synergistic effect, successfully inhibiting bacterial growth and biofilm development.

Background: Ran GTPase plays a pivotal role in cell fate determination and is frequently overexpressed in various cancers due to dysregulated signaling pathways such as PI3K/Akt and ERK/MEK.

Problem statement: There are very limited drugs that specifically target Ran GTPase. Thus, in order to minimize the metastatic potential of cancer cells, it is important to identify molecules that inhibit the activity of Ran GTPase.

Methods: To explore novel therapeutic options, we performed a virtual screening of the SuperNatural database targeting Ran GTPase using the Schrödinger platform. Molecular docking was performed using Schrödinger Maestro Academic, and molecular dynamics simulations (100 ns) confirmed the stability of these complexes through sustained hydrogen bonding and other key interactions. Additionally, density functional theory (DFT)-based electronic structure analysis, including frontier molecular orbital (FMO) and molecular electrostatic potential (MEP) evaluations, supported their reactivity and binding potential. ADME&T analysis plays a pivotal role in evaluating pharmacokinetic properties.

Results: Ten top-ranking natural compounds were identified with docking scores ranging from -13.85 to -10.48 kcal/mol, indicating strong binding affinity at the active site. Virtual screening was further evaluated via molecular docking and intermolecular interaction analysis. Furthermore, molecular dynamics simulations (100 ns) confirmed the stability of these complexes through sustained hydrogen bonding and other key interactions, while three compounds (CID_11194, CID_16220, and CID_6220) displayed high stability and were further analyzed by using DFT and ADME/toxicity profiling, among them, CID_11194 and CID_16220 exhibited favorable pharmacokinetic properties, being non-toxic and non-carcinogenic. These findings suggest that the compound CID_16220 may serve as a promising candidate for breast cancer therapy.

Endometrial carcinoma (EC) is a common gynecological malignancy whose incidence has been increasing globally. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) has been implicated in several cancers, but its clinicopathological and prognostic significance in EC remains unclear. A total of 66 EC patients who underwent surgical treatment between September 2017 and September 2018 were included. Paired tumor and adjacent normal tissues (> 3 cm from the tumor margin) were analyzed by immunohistochemistry using a validated anti-ROR1 antibody. ROR1 expression was semi-quantitatively scored based on staining intensity and the percentage of positive cells, with scores ≥ 6 indicating high expression. Associations between ROR1 expression and clinicopathological features were evaluated using χ² tests and logistic regression. Univariate and multivariate logistic regression models identified independent predictors of high ROR1 expression, and model fit was assessed using the Hosmer-Lemeshow test and ROC analysis. Kaplan-Meier and Cox proportional hazards models were used to assess the prognostic impact of ROR1 expression on overall survival (OS), with subgroup analyses stratified by clinical stage and histological type. ROR1 was highly expressed in 65.15% (43/66) of EC tissues compared with 22.73% (15/66) of adjacent normal tissues (p < 0.05). The mean number of ROR1-positive cells was significantly greater in high-expression tumors (86.15 ± 9.79) than in normal endometrial tissue (3.13 ± 1.25) or low-expression tumors (6.08 ± 3.37) (p < 0.05). High ROR1 expression was significantly associated with advanced clinical stage (III-IV), Type II histology, lymph node metastasis, and poor prognosis (all p < 0.05). Multivariate logistic regression confirmed that ROR1 overexpression was independently associated with advanced stage (OR = 11.59, p = 0.004), Type II histology (OR = 4.68, p = 0.031), and poor prognosis (OR = 5.68, p = 0.036). Kaplan-Meier analysis demonstrated that patients with high ROR1 expression had a shorter median OS (46 vs. 59 months, p < 0.001). Subgroup analyses revealed that the prognostic value of ROR1 was most evident in Stage I-II and Type I EC, where high expression remained an independent predictor of poor survival after multivariate adjustment (HR = 4.07, 95% CI: 1.71-9.66, p = 0.0015). ROR1 is markedly overexpressed in endometrial carcinoma and independently associated with advanced stage, Type II histology, and adverse prognosis. High ROR1 expression predicts shorter overall survival, particularly in early-stage and Type I EC, supporting its potential as a prognostic biomarker. Given the retrospective single-center design, limited sample size, and absence of molecular validation, further multicenter and mechanistic studies are warranted to confirm these findings.

Phospholipase A₂ (PLA₂) is a multifunctional enzyme involved in diverse physiological processes and industrial applications, including phospholipid modification, oil refining, and feed additive production. Consequently, the exogenous expression of PLA₂ from various species holds significant biotechnological potential. In this study, we aimed to heterologous express the PLA₂ gene from Apostichopus japonicus (AjPLA₂) in prokaryotic systems. Four plasmid vectors (pET-28a, pCold II, pMALc2x, and pET32a) were selected for constructing recombinant Aj-PLA₂-expressing strains. Among these constructs, only Rosetta gami (DE3)/pET32a-Aj-PLA₂ produced soluble and catalytically active recombinant TrxA-Aj-PLA₂ fusion protein upon induction. Optimal expression conditions were determined as follows: 0.1 mM IPTG induction for 2.5 h at 25°C, followed by 16 h of post-induction incubation. The active TrxA-Aj-PLA₂ and pure Aj-PLA₂ enzymes were subsequently purified via Ni²⁺-NTA affinity chromatography using a 300 mM imidazole elution buffer. Enzymatic characterization revealed that the TrxA-Aj-PLA₂ exhibited maximal activity at approximately 40°C and pH 9.0 in the presence of 5 mM Ca²⁺ ions. Gas chromatography-mass spectrometry (GC-MS) analysis confirmed that TrxA-Aj-PLA₂ specifically hydrolyzed the sn-2 ester bond of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), releasing oleic acid (C18:0). Furthermore, turbidimetric assays demonstrated the antimicrobial potential of TrxA-Aj-PLA₂ against marine bacteria. Collectively, this study achieved the soluble expression of the TrxA-Aj-PLA₂ enzyme possessing dual functionalities: phospholipid hydrolysis and marine bacterial growth inhibition in vitro. These findings establish a critical foundation for future mechanistic investigations and biotechnological applications of Aj-PLA₂.

Age-related mitochondrial dysfunction is a primary cause of muscle degeneration. We aimed to identify mitochondria-related differentially expressed genes (MR-DEGs) and construct a diagnostic model to improve the diagnosis of sarcopenia. Transcriptomic datasets GSE226151 (training) and GSE1428 (validation) were downloaded from GEO. Mitochondria-related genes (MRGs) were sourced from human MitoCarta 3.0, and MR-DEGs were screened as intersections of sarcopenia-related genes, DEGs, and MRGs. Optimal biomarkers were selected using univariate logistic regression and LASSO regression. A diagnostic model was further estimated, and the diagnostic value was determined using receiver operating characteristic (ROC) curves. Finally, interaction networks and immune correlation analyses of the optimal MR-DEGs were assessed. Six optimal MR-DEGs (ACOT11, BCO2, MRPL4, NDUFB9, UQCR10, and CASP8) were identified. The model achieved area under the curve (AUC) values of 0.935 and 0.899, respectively, in the GSE226151 and GSE1428. Significant immune correlations emerged; CD56^bright natural killer cells and neutrophils were correlated with MR-DEGs expression (all p < 0.05). Network analysis revealed 20 co-regulated genes that were significantly enriched in functions such as ATP synthesis-coupled electron transport, mitochondrial respiration, and oxidative phosphorylation. Our six-MR-DEG diagnostic model demonstrated a robust clinical potential for sarcopenia diagnosis, with validation across platforms and significant pathophysiological relevance to mitochondrial immune dysregulation.

Bacterial leaf spot caused by Xanthomonas citri pv. viticola (Xcv) in India is of economic importance. The objective of the investigation was to isolate and characterize bacterial antagonists from different tissues of the grapevine. Fifty endophytes were isolated from the leaves, stems, and roots of 15 different varieties of grapevine. The primary identification of the isolates was done by microscopic and biochemical characterizations. These endophytes were promoting the growth of the plants through both direct and indirect mechanisms. They were capable of producing various plant growth-promoting traits, such as indole acetic acid and hydrolytic enzymes like cellulase, amylase, protease, and lipase. They were assessed for additional traits like phosphate solubilization, hydrogen cyanide production, and ammonia production. Further, these endophytes were screened for antagonistic activity against six X. citri pv. viticola isolated from different locations. Eleven out of 50 endophytic bacteria significantly inhibited Xcv, as evident from well-diffusion methods. Three isolates, that is, EB7, EB26, and EB45, manifested the highest inhibition and were further screened for antibacterial activity using the detached leaf assay. The results showed that all three isolates have the highest potential to inhibit the Xcv, causing bacterial leaf spot. The molecular identification on the basis of 16S rRNA sequencing of the isolates EB7, EB26, and EB45 revealed that they were Providencia vermicola, Bacillus australimaris, and Bacillus zhangzhouensis, respectively. B. australimaris and B. zhangzhouensis could be effectively used for the biological control of Xcv as they exhibited effective control in field trials.

Murraya koenigii (MK) is a medicinal plant known for its diverse phytochemical constituents and therapeutic properties. This study presents the first comprehensive analysis of MK leaf and stem whole extracts obtained using toluene, a non-polar solvent that enabled selective extraction of lipophilic phytochemicals. LC-MS profiling revealed 832 unique compounds including lipids, triterpenoids, flavonoids, alkaloids, and benzenoids. Despite lower yields of phenolics and antioxidants compared to methanolic extracts, qualitative and quantitative assays demonstrated distinct antioxidant, antimicrobial, and cytotoxic properties. The stem extract showed greater antioxidant activity in the DPPH assay, while only the leaf extract inhibited S. aureus and E. coli growth. Both extracts exhibited cytotoxic effects against PA1 ovarian cancer cells, with the leaf extract demonstrating higher potency (IC₅₀ = 52±1.25 µg/mL) and apoptosis induction. In silico ADMET screening of 832 compounds predicted 41 drug-like compounds with high GI absorption (≥95%), good bioavailability (≥0.55), moderate solubility, and no violations of Lipinski's Rule. Thirteen were predicted to be P-glycoprotein substrates, and a few showed weak inhibition of CYP2C9 and CYP3A4, indicating acceptable metabolic stability. Among these, five compounds, PPB-FMID (PubChem ID:24204170), ABM-PPM (PubChem ID:267684), Estrone (PubChem ID:5870), MFCD00019066 (PubChem ID:12553), and MPA-PPM (PubChem ID:319793), were predicted to exhibit strong binding affinities (-10.3 to -9.1 kcal/mol) towards TNFα, CDK4, and PRAP1 from a panel of 12 key targets involved in apoptosis and cell signalling. The study underscores the utility of toluene-based extraction in expanding MK's phytochemical landscape and supports its therapeutic promise in oncology and infectious disease contexts. As this study represents a preliminary evaluation, further mechanistic validation, compound isolation, and broad-spectrum screening are essential to confirm the bioactivity and pharmacological relevance of these leads.