Search Results (229)

X-ray crystallography is still the most widely used and versatile method for structural studies of biological macromolecules. This study concerns the application of nanogels to facilitate protein crystallization, a prerequisite for X-ray crystallography. Nanogels (NGs) are nano-sized, highly crosslinked polymeric particles that have been extensively studied for chemical catalysis and drug delivery but not for protein crystal nucleation. The efficacy of six types of nanogels (three N-isopropylacrylamide-based and three acrylamide-based) was tested, with promising results. They were subsequently functionalised with active hydroxyl groups for further testing. Both functionalised and non-functionalised nanogels were tested on model (trypsin, thaumatin, proteinase K, ferritin and catalase) and target proteins (glulisine, α-crustacyanin and acriflavine resistance protein subunit AcrB) using both manual and automated techniques. All nanogels were found to be effective in promoting protein crystallization in both screening and optimization trials, giving crystal ‘hits’ that would have otherwise been missed. Overall, the functionalised nanogels were more effective. Nanogel effects are proposed to be due to a combination of surface porosity and surface chemistry. Full article

This study investigates the degradation characteristics, pathways, and mechanisms of ochratoxin A (OTA) by Weizmannia coagulans CGMCC 9951 (W. coagulans CGMCC 9951), as well as its detoxification effect on Cornus officinalis pulp through fermentation. The strain efficiently degraded 300 ng/mL of OTA within 72 h (98% degradation) under optimal conditions of 37 °C, pH 5.0, and 180 rpm. Active degradation substances were primarily localized in the cell-free supernatant (CF). The degradation activity was significantly inhibited by heat treatment, proteinase K, EDTA, Cu²⁺, and organic reagents, suggesting an enzymatic mechanism. UHPLC-MS and MS/MS analysis indicated that OTA appears to be degraded to a product consistent with ochratoxin α (OTα). Based on homology to known OTA-degrading carboxypeptidases, the gene encoding WGU28473.1 was selected, expressed in E. coli, and confirmed to possess OTA-degrading activity. Molecular docking suggested potential interactions between the enzyme and OTA. Under optimal conditions, co-fermentation with Cornus officinalis pulp contaminated with 300 ng/mL OTA for 96 h resulted in a 74% degradation of OTA. The fermentation process increased the pulp’s sugar content and ABTS⁺ free radical scavenging capacity, reduced acidity, and improved the safety of the pulp. These findings demonstrate that W. coagulans CGMCC 9951 efficiently degrades OTA and improves pulp quality, highlighting its potential as a starter culture for detoxifying OTA-contaminated food. Full article

A simple, fast, and cost-effective organic solvent-based protocol was developed for DNA extraction from deer antlers and prepared trophy skulls, eliminating the need for commercial kits or cryogenic grinding. The method combines bead-based mechanical homogenization with a 4 h enzymatic digestion in EDTA buffer containing N-lauryl sarcosine and Proteinase K, followed by phenol–chloroform–isoamyl alcohol purification and centrifugal filtration. DNA quality and quantity were evaluated using agarose gel electrophoresis, Qubit fluorometry, and Nanodrop spectrophotometry. The protocol was tested on 60 samples, comprising 30 antlers and 30 pedicle parts from prepared trophy skulls of roe deer (Capreolus capreolus), fallow deer (Dama dama), and red deer (Cervus elaphus). To assess suitability for downstream applications, species-specific microsatellite markers were amplified using multiplex PCR, successfully generating complete genotypes from all 60 samples. These results, along with a demonstrated case study, confirm that the developed protocol provides high-quality DNA suitable for molecular genetic investigations, enabling reliable genotyping from small amounts of both antler and processed trophy materials in forensic and conservation contexts. Full article

Salmonella remains a leading cause of foodborne illness worldwide, with poultry products representing a major source of human exposure, underscoring the need for rapid and reliable detection methods. Although qPCR offers sensitive and timely pathogen detection, assay performance is highly dependent on sample preparation efficiency and nucleic acid purity, particularly in complex food matrices. In this study, we systematically optimized the sample preparation workflow of a SYBR Green based qPCR assay for enrichment-free detection of Salmonella enterica in poultry. Multiple lysis chemistries, incubation times, DNA extraction methods, centrifugation strategies, inoculum sources, and magnetic nanoparticle (MNP) assisted workflows were evaluated using phosphate-buffered saline and chicken rinsate matrices. Among the conditions tested, lysis with 20 µL Proteinase K and 400 µL PrepMan™ for 20 min produced the lowest and most consistent Cq values. Although Promega Wizard^® produced slightly lower mean Cq values than PrepMan™, statistical analysis showed no significant differences between extraction methods or centrifugation protocols, indicating comparable overall performance. Broth-derived inocula yielded earlier and more reproducible Cq values than colony-derived preparations. In contrast, inclusion of MNP processing resulted in higher Cq values in both matrices compared to the non-MNP workflow. Overall, these findings demonstrate that optimized lysis, extraction, and centrifugation workflows enhances the consistency and analytical reliability of direct qPCR detection of Salmonella in poultry matrices, supporting laboratory-based rapid detection applications. Full article

Grain is highly vulnerable to contamination by fungi during storage, leading to reduced product quality and substantial economic losses. Lactic acid bacteria (LAB) have the potential to be used as antifungal agents; however, strains aimed at inhibiting stored grain molds remain limited, and the inhibitory mechanisms require further investigation. To solve this problem, 71 LAB strains were isolated from various samples. Among these, 17 strains exhibiting inhibitory activity against A. flavus, A. niger and P. citrinum were selected using a dual-layer plate assay. Based on morphological characterization and 16S rRNA gene sequence analysis, these strains were classified as Weissella cibaria, Pediococcus pentosaceus, and Lactiplantibacillus paraplantarum. Further investigations involving pH adjustment, catalase, and proteinase K treatments confirmed that organic acids were the primary antifungal substances in LAB cell-free supernatant (CFS). HPLC quantification identified acetic acid, malic acid, lactic acid, phenyllactic acid and citric acid contained in the CFS. Antifungal assays verified that acetic acid and lactic acid exhibited the strongest inhibitory effects against P. citrinum and A. flavus, whereas phenyllactic acid and acetic acid demonstrated the most potent suppression against A. niger. These findings established a theoretical basis for the application of LAB CFS in grain storage. Full article

Space conditions offer new insights into fundamental biological and molecular mechanisms. The study aimed to evaluate the enzymatic activity of proteinase K (PK) under extreme conditions relevant to space environments: simulated microgravity, hypergravity, and gamma radiation. PK activity was tested using azocasein (AZO) as a chromogenic substrate, with enzymatic reactions monitored spectrophotometrically at 450 nm. A rotating wall vessel (RWV) simulated microgravity, centrifugation at 1000× g (3303 rpm) generated hypergravity, and gamma radiation exposure used cesium-137 as the ionizing source. PK activity showed no remarkable changes under microgravity after 16 or 48 h; however, higher absorbance values after 96 h indicated enhanced AZO proteolysis compared to 1 g (Earth gravity) controls. In hypergravity, low PK concentrations exhibited slightly increased activity, while higher concentrations led to reduced activity. Meanwhile, gamma radiation caused a dose-dependent decline in PK activity; samples exposed to deep-space equivalent doses showed reduced substrate degradation. PK retained enzymatic activity under all tested conditions, though the type and duration of stress modulated its efficiency. The results suggest that enzyme-based systems may remain functional during space missions and, in some cases, exhibit enhanced activity. Nevertheless, their behavior must be evaluated in a context-dependent manner. These findings may be significant to advance biotechnology, diagnostics, and the development of enzyme systems for space applications. Full article

Acinetobacter baumannii relies on biofilms for antibiotic resistance, but the role of planktonic aggregates in drug tolerance is uncharacterized. We studied 103 clinical isolates to explore how the RND efflux pump gene adeG regulates aggregation. Non-MDR strains (with RND deletions) formed aggregates more frequently (13.79%, 4/29) than MDR strains (1.35%, 1/74), driven by residual RND efflux activity (not just deletions). adeG deletion induced 1–2 mm aggregates in a strain with combined adeR/ΔadeABC defects (via upregulated adhesion genes/hydrophobicity) but not in one with only ΔadeC. Aggregates boosted antibiotic tolerance (2–4-fold higher survival vs. disaggregated/parental strains) via metabolic dormancy (5-fold lower ATP), maintained growth in human serum, and promoted persistent bacteremia in immunosuppressed mice. Proteinase K disrupted aggregates, confirming protein matrices’ role. These findings identify planktonic aggregates as pivotal adaptive and virulence-related targets for combating refractory non-MDR A. baumannii infections while also revealing an association between adeG-related genetic contexts and aggregate formation in the bacterium. Full article

Polylactic acid (PLA), a widely used biobased biopolymer, is highly resistant to biodegradation under ambient conditions, contributing to persistent plastic pollution and posing potential environmental and health risks. This study investigates the enzymatic degradation of PLA by Proteinase K, a proteolytic hydrolase enzyme with the ability to degrade PLA, and explores the underlying mechanisms for degradation. Both amorphous and semi-crystalline PLA were treated with Proteinase K (2 mg/mL) at 37 °C over varying time periods. PLA degradation was evaluated using multiple techniques, including weight loss measurement, pH reduction, quantification of lactic acid monomer release by High-Performance Liquid Chromatography (HPLC), surface morphology analysis through Scanning Electron Microscopy (SEM), changes in thermal properties by Differential Scanning Calorimetry (DSC), and structural changes by X-Ray Diffraction (XRD). The data revealed that the degradation of amorphous regions of the PLA polymer was faster and more extensive than the crystalline regions of the polymer. Repeated enzymatic treatments significantly enhanced the degradation rate. Furthermore, Proteinase K showed a clear preference for degrading amorphous regions of the PLA, as evidenced by higher weight loss, sharper pH decline, higher lactic acid production, and more pronounced surface disruptions, such as visible gaps between degraded oligomer structures. Full article

Due to the high toxicity and widespread distribution of aflatoxin B1 (AFB1), there is significant interest in efficient, safe, and environmentally friendly microbial degradation methods. Rhodococcus opacus PD630 cell-free supernatant (RCFS) shows excellent activity in degrading AFB1, but its active components and mechanisms remain unclear. We assessed the feasibility of ethanol precipitation to enrich active components in RCFS and characterized the ethanol precipitate (RCFSC-EP). Metabolomics and proteomics were used to elucidate the active components, mechanisms, and products of AFB1 degradation by RCFS. The results indicate that ethanol precipitation enriches over 80% of the active components for AFB1 degradation in RCFS. RCFSC-EP exhibits excellent heat resistance, and inhibitors like EDTA-2Na and proteinase K significantly inhibit its activity. Multi-omics analysis suggests that active components in RCFS metabolize AFB1 into six products through four potential pathways, three of which withstand 135 °C for 20 min. The AFB1-degrading activity of RCFS is an intrinsic, constitutive trait of R. opacus PD630 during normal growth. The active components are diverse proteins or enzymes, including glutathione S-transferases, aldo/keto reductase, peroxidases, and carbonyl reductases. This study enriches and reveals the active components, pathways, and products of AFB1 degradation by RCFS, providing a basis for developing RCFS as a biological agent for AFB1 degradation. Full article

The global rise of antimicrobial resistance represents a critical challenge to public health, with Escherichia coli emerging as one of the most significant contributors due to its high adaptability and prevalence of extended-spectrum β-lactamase (ESBL) production. Outer membrane vesicles (OMVs), nanoscale structures released by Gram-negative bacteria, have recently been implicated in the dissemination of resistance determinants and direct antibiotic inactivation. This study investigated the role of OMVs derived from ESBL-producing E. coli in mediating resistance to ampicillin. Clinical strains harboring CTX-M-15 resistance genes were cultured under selective pressure, and OMVs were purified via size-exclusion chromatography. Characterization using tunable resistive pulse sensing (TRPS) and cryo-transmission electron microscopy confirmed vesicle integrity, with sizes ranging from 80 to 150 nm. DNA quantification and PCR analysis revealed the presence of CTX-M-15 genes within vesicles, which remained protected from DNase digestion, confirming encapsulation. Functional assays demonstrated β-lactamase activity within OMVs, with proteinase K treatment indicating localization primarily within vesicles rather than on their surface. Importantly, OMVs inactivated ampicillin in a dose-dependent manner, significantly reducing its efficacy against susceptible E. coli. Disc diffusion and microtiter plate assays confirmed that β-lactamase-positive OMVs protected susceptible strains from antibiotic killing, promoting bacterial survival and growth. This study uniquely demonstrates that OMVs from CTX-M-15–producing Escherichia coli carry both resistance genes and active β-lactamase enzymes, thereby facilitating both genetic dissemination and direct antibiotic inactivation. Targeting OMV biogenesis may represent a novel strategy to combat antimicrobial resistance. Full article

The understanding and control of protein crystallization are crucial in structural biology, drug development, and biomaterial design. This study introduces a unified framework for modeling and comparing crystallization kinetics using selected growth functions. Experimental datasets from the literature for four proteins, Lysozyme, Thaumatin, Ribonuclease A, and Proteinase K, under Hanging Drop and Langmuir–Blodgett conditions were analyzed. Five kinetic models, Avrami, Kashchiev, Hill, Logistic, and Generalized Sigmoid (GSM), were fitted to size–time data of the four benchmark proteins. From each fit, four descriptors were extracted: crystallization half-time, time of maximum growth, width at half-maximum, and peak growth rate. These metrics summarize crystallization dynamics and enable cross-comparison of proteins and methods. Langmuir–Blodgett templating accelerated onset and improved synchrony, though the effect varied by protein and model. Logistic, Hill, and GSM models provided consistent fits across most conditions, while Avrami and Kashchiev were more sensitive to early or late deviations. Notably, descriptor extraction remained reliable even with limited or uneven sampling, revealing kinetic regimes such as synchrony, asymmetry, or prolonged nucleation, not evident in raw data. This transferable analytical framework supports quantitative evaluation of crystallization behavior, aiding screening, process optimization, and time-resolved structural studies. Full article

Expansion microscopy (ExM) enables conventional light microscopes to achieve nanoscale resolution by physically enlarging biological specimens. While ExM has been widely applied in neurobiology, it has not been adapted for the enteric nervous system (ENS). Here, we provide a detailed and reproducible protocol for applying ExM to mouse colonic ENS tissue. The procedure includes preparation of the external muscle layers with the myenteric plexus, histochemical staining for NADPH-diaphorase, immunostaining for glial fibrillary acidic protein (GFAP), anchoring of biomolecules, gelation, proteinase K digestion, and isotropic expansion in a swellable polymer matrix. Step-by-step instructions, required reagents, and critical parameters are described to ensure robustness and reproducibility. Using this protocol, tissues expand 3–5-fold, allowing neuronal somata, fibers, and glial cell processes to be clearly visualized by standard brightfield or fluorescence microscopy. The tissue architecture is preserved, with distortion in the X–Y plane of about 7%. This protocol provides a reliable framework for high-resolution structural analysis of the ENS and can be readily adapted to other peripheral tissues. Full article

Caenorhabditis elegans, a free-living nematode model, secretes neuropeptides, but the ecological roles of its peptide exudates in regulating rhizosphere microbial activity remain largely unexplored. We identified six short peptides (P1, P9, P19, P20, P25, and P26) from C. elegans exudates that significantly enhanced indole-3-acetic acid (IAA) production by the plant growth-promoting bacterium Arthrobacter pascens ZZ21. These peptides were heat-labile and proteinase K-sensitive but unaffected by DNase I or RNase A, confirming their proteinaceous (peptide) nature rather than nucleic acid origin. The retention of bioactivity in n-butanol extracts further supported their hydrophilic, peptide-like properties. LC-MS/MS identified 30 linear peptides, including the six bioactive ones, which exhibited distinct dose-dependent effects, suggesting diverse regulatory mechanisms. Despite their relatively low abundance, these peptides strongly promoted IAA production in the bacterial culture system across multiple concentrations. These findings reveal an unrecognized mechanism whereby free-living nematodes regulate rhizobacterial metabolism via secreted peptides, offering new insights into nematode-mediated chemical signaling. Therefore, this study advances understanding of plant–microbe–nematode interactions and highlights strategies for manipulating rhizosphere microbiota in sustainable agriculture. Full article

This study aimed to identify a probiotic bacterium with antagonistic activity against the foodborne pathogen Staphylococcus aureus (S. aureus) and investigate the mechanism of its antibacterial components. Growth kinetics were analyzed to assess bacterial proliferation. Acid and bile salt tolerance are vital indicators for evaluating probiotic survival in the gastrointestinal tract. The results indicated that Companilactobacillus farciminis (C. farciminis) YLR-1 not only had high tolerance to salt conditions (0.03%, 0.3%, and 0.5%) but also has a high survival rate at pH 3–4. The bacteriocin-like inhibitory substance (BLIS) isolated from C. farciminis YLR-1 was dialyzed using a membrane with a molecular weight cut-off (MWCO) of 500 Da, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The results indicate that the BLIS produced by C. farciminis YLR-1 is a small-molecule peptide. BLIS displayed pH tolerance within acidic and neutral environments (4–8) and exhibited thermostability. When treated with proteinase K, the antibacterial action of BLIS was found to be inactivated. Membrane disruption mechanisms were examined using fluorescence imaging and scanning electron microscopy (SEM). SEM and fluorescence imaging revealed that BLIS-induced membrane damage in S. aureus ATCC 25923 causes cytoplasmic leakage and cell death. Full article

Icariin (ICA) is widely recognized for its health benefits. In this work, we examined the intermolecular interactions between ICA and proteinase K (PK) via multi-spectroscopic techniques and molecular simulations. The experimental findings revealed that ICA quenched the fluorescence emission of PK by forming a noncovalent complex. Both hydrogen bonding and van der Waals interactions are essential for the complex’s formation. Then Förster resonance energy transfer (FRET), competitive experiments, and synchronous fluorescence spectroscopy were adopted to verify the formation of the complex. Molecular docking studies demonstrated that ICA could spontaneously bind to PK by hydrogen bonding and hydrophobic interactions, which is consistent with the spectroscopic results. The PK-ICA complex’s dynamic stability was evaluated using a 50 ns molecular dynamics (MD) simulation. The simulation results revealed no significant structural deformation or positional changes throughout the entire simulation period. The complex appears to be rather stable, as seen by the average root-mean-square deviation (RMSD) fluctuations for the host protein in the PK-ICA complex of 1.08 Å and 3.09 Å. These outcomes of molecular simulations suggest that ICA interacts spontaneously and tightly with PK, consistent with the spectroscopic findings. The approach employed in this research presents a pragmatic and advantageous method for examining protein–ligand interactions, as evidenced by the concordance between empirical and theoretical findings. Full article