Search Results (6,677)

Listeria innocua is a bacterium frequently detected in food and food production plants (FPPs). Understanding the heterogeneity of L. innocua food isolates is essential for predicting potential food safety threats and developing preventive and control measures. This study aimed to characterize L. innocua isolated from raw drinking milk by investigating the genomic features related to virulence, antimicrobial resistance, and persistence using whole-genome sequencing (WGS), along with phenotypic antimicrobial susceptibility testing using the disk diffusion method. All ten isolates analyzed in this study belonged to sequence type (ST) 492 and were distantly related to the reference strain. A total of 80 virulence-associated genes were identified, including the complete Listeria Pathogenicity Islands-3 (LIPI-3) and LIPI-4 clusters typically found in virulent L. monocytogenes clones, as well as 66 additional genes involved in adhesion, invasion, motility, post-translational modification, regulation, immune modulation, and stress survival. Stress survival islet 2 (SSI-2) and genes encoding the Clp protease complex (clpC, clpE, clpP), which support both persistence and virulence, were also detected, whereas LIPI-1 and internalin genes were not detected. The antimicrobial resistance determinants included fosX, lin, norB, sul, and three multidrug efflux pumps (lde, mdrL and mdrM). Mobile genetic elements (plasmids, prophages, or transposons) were not detected. All isolates were phenotypically susceptible to benzylpenicillin, ampicillin, meropenem, erythromycin, and trimethoprim–sulfamethoxazole. These findings underscore the importance of ongoing genomic surveillance of L. innocua in food environments and highlight the need to assess the potential risk posed by specific lineages, such as ST492, to food safety. Full article

Sterol regulatory element-binding proteins (SREBPs) regulate lipid homeostasis and coordinate sterol metabolism and carotenogenesis in the astaxanthin-producing yeast Phaffia rhodozyma. While Sre1, the SREBP ortholog, and the site-2 protease Stp1 have been identified as essential components of this pathway in P. rhodozyma, additional factors involved in Sre1 processing or regulation remain unknown. In Aspergillus species, a signal peptide peptidase contributes to the activation of the SREBP ortholog, raising the possibility of a similar role in this yeast. In this work, we identified and characterized the P. rhodozyma signal peptide peptidase (SppA) homolog. Sequence analysis, domain prediction, and phylogenetic analyses supported its classification within the SPP family of intramembrane aspartyl proteases. To evaluate its functional role, ΔsppA mutants were constructed in genetic backgrounds with constitutive Sre1 activity, including the cyp61⁻ mutant and a strain expressing the active form of Sre1 (Sre1N). Deletion of SPPA did not alter sensitivity to clotrimazole or cobalt chloride, nor affect pigmentation, indicating that SppA is not required for Sre1 activation in P. rhodozyma. Transcriptomic analyses further showed that expression of SRE1 and of its known target genes remained unchanged upon SPPA deletion. Interestingly, the loss of SppA in the Sre1N background caused marked downregulation of genes associated with protein refolding and unfolded protein binding. In agreement with these transcriptional changes, the Sre1NΔsppA strain displayed increased sensitivity to dithiothreitol. These findings suggest that, although SppA is not involved in Sre1 activation in P. rhodozyma, it may play a role in protein stress-related processes. Future studies will be required to define the molecular mechanisms underlying this role and its integration with protein homeostasis networks. Full article

Halophilic proteases are valuable in industrial applications due to their resistance to harsh conditions. HtrA2 serine protease is widely distributed and conserved among eukaryotes and prokaryotes. However, HtrA2 proteases from archaea have been poorly characterized. In this study, htrA2 from haloarcheon Haloarcula sp. TG1 was cloned and corresponding nucleotide and amino acid sequences were analyzed. Recombinant HtrA2 was produced in Escherichia coli, and biochemical properties of purified HtrA2 were characterized. HtrA2 was immobilized for the first time using polyhydroxybutyrate (PHB) nanoparticles. Additionally, potential of HtrA2 as a detergent additive was evaluated by its bloodstain removal activity. Recombinant HtrA2 showed its optimum activity at 50 °C, pH 7.0, and 3.0 M NaCl. HtrA2 activity was highly retained over wide temperature (40 to 60 °C) and pH ranges (pH 5.0 to 11.0). Moreover, various organic solvents, inhibitors and metal ions were well tolerated by the enzyme. Acetone and Fe²⁺ significantly increased HtrA2 activity, while it was not inhibited by phenylmethylsulfonyl fluoride and sodium dodecyl sulfate. Also, immobilization of HtrA2 onto PHB nanoparticles improved its reusability. Furthermore, HtrA2 successfully removed the bloodstain from cotton fabric. This comprehensive characterization of HtrA2 demonstrates that recombinant HtrA2 obtained from Haloarcula sp. TG1 is promising for industrial applications. Full article

The SARS-CoV-2 public health challenges have highlighted the urgent need for coronavirus-targeting life-saving therapeutics. Given the emergence of drug-resistant strains, the development of antivirals against viral proteins beyond the commonly targeted main protease or RNA-dependent RNA polymerase is critical. The SARS-CoV-2 nonstructural protein 13 (nsp13) is a highly conserved RNA helicase and an essential component of the viral replication–transcription complex (RTC). It unwinds double-stranded RNA to facilitate viral transcription and replication, making it a strong target for drug development. To identify nsp13 inhibitors, we used an ultra-high-throughput nucleic acid unwinding assay to screen a library of FDA-approved drugs and bioactive compounds. We identified forty inhibitors with IC₅₀ values ranging from 1.4 to 10 μM. Ten were further selected for biochemical and biophysical characterization. Four of these are bound to nsp13 without interacting with the nucleic acid substrate and without inhibiting the ATPase activity of nsp13. Hydrogen–deuterium exchange coupled with Mass Spectrometry (HDX-MS) studies show compound binding causes differential exchange in two regions of nsp13. Furthermore, these compounds have antiviral activity against infectious SARS-CoV-2 in multiple cell lines, with cytotoxicity affecting, in some cases, the apparent antiviral effect. Future optimization efforts could help develop therapeutics against SARS-CoV-2 and other potential coronavirus threats. Full article

Objectives: This experiment was conducted to investigate the effects of adding walnut (Juglans regia L.) green husk (WGH) on the quality of alfalfa mixed silage, protein degradation, microbial community, and their interrelationships. Methods: Alfalfa (Medicago sativa L.) fresh grass and WGH dried powder were used as raw materials to prepare three mixed silages of alfalfa fresh grass with 80 g/kg (A1), 120 g/kg (A2), and 160 g/kg (A3) of WGH dried powder, respectively, with alfalfa fresh grass silage as the control group (CK). After 60 days of ensilage, samples were taken and analyzed, with three replicates per treatment. Results: WGH treatment significantly improved alfalfa silage fermentation and nutritional quality. It reduced undesirable fermentation products while promoting beneficial lactic acid bacteria and preventing mold growth. Increasing the WGH ratio enhanced dry matter content and digestibility, with only a minor effect on crude protein. These results suggest that WGH is an effective silage additive for improving both fermentation characteristics and feed value. With the increase in the proportion of WGH, the proportions of rapidly degradable protein (PB1) and medium rate degradable protein (PB2) increased linearly, while the proportions of free amino acid nitrogen (FAA-N), peptide nitrogen (Peptide-N), slow degradable protein (PB3) and binding protein (PC) decreased linearly and the protease activity decreased significantly (p < 0.05). Bacterial community analysis showed that the relative abundance of Lactiplantibacillus and Levilactobacillus in the silage increased after WGH was added, while the relative abundance of Acetobacter, Pantoea, Weissella and Serratia decreased. Conclusions: Compared with pure alfalfa silage, the addition of WGH has a positive effect on silage quality, protein degradation and bacterial community structure, and the addition of WGH with 120 g/kg is more suitable. Full article

Cheese ripening is slow and costly, driving interest in accelerating maturation. This study aimed to isolate a safe, efficient adjunct starter from traditional Sichuan yak yoghurt, a niche rich in stress-adapted lactic acid bacteria. From 295 isolates, 15 strains tolerant to high salt, low pH, and low temperature were selected. Using acidification, autolysis, proteolysis, and peptidase activity as indices, principal component analysis identified Limosilactobacillus fermentum 270 as the best candidate. Phenotypic assays showed no haemolysis, gelatin liquefaction, indole production, or amino acid decarboxylase activity. Whole-genome sequencing confirmed species identity and revealed 52 protease/peptidase genes, complete pathways for diacetyl/acetoin biosynthesis and branched-chain amino acid conversion, and no functional biogenic amine synthesis genes. Stress-related genes (F-ATPase, glycine-betaine transport, cold-shock proteins) support cheese adaptability. Antibiotic resistance gene homologs were mainly chromosomal and unlinked to mobile genetic elements; a functional CRISPR-Cas system lowers horizontal transfer risk. The strain was developed as a freeze-dried direct-vat starter (97.3% viability). Orthogonal optimisation of yak Gouda cheese-making defined best conditions: 0.018% adjunct, 45 min acidification, pH 5.8, and 30% curd washing. L. fermentum 270 thus combines proteolytic, flavour-enhancing, genetic safety, and processing traits, offering a promising adjunct for accelerated cheese ripening. Full article

Kupffer cells (KCs) make up the predominant population of resident innate immune cells in the liver, serving as key immune sentinels that maintain local immune surveillance and immunoregulatory homeostasis. However, their functional involvement and phenotypic dynamics during radiation-induced liver damage (RILD) remain insufficiently explored. Therefore, we established a mouse model of RILD and, through systematic single-cell-level profiling of hepatic immune cell populations, found that KCs play a critical role in hepatic immune responses and undergo a pronounced radiation-induced shift toward a pro-inflammatory M1 phenotype. Further KC depletion/reconstitution, molecular assays, and coculture experiments consistently demonstrated that M1-polarized KCs exacerbate liver damage, with secretory leukocyte protease inhibitor (SLPI) being identified as a key molecular mediator driving this polarization and its pathogenic effects. To further substantiate these findings, we designed a liposome-based delivery strategy to selectively inhibit SLPI in KCs, which effectively suppressed M1 polarization and alleviated radiation-induced liver damage, underscoring the therapeutic relevance and translational potential of this approach in RILD. Overall, these findings demonstrate that radiation drives KCs toward an SLPI-dependent pro-inflammatory M1 state, thereby exacerbating liver injury. Moreover, targeted liposomal suppression of SLPI effectively reverses this polarization and protects against RILD, highlighting SLPI-modulated KC reprogramming as a promising therapeutic approach. Full article

The choroid plexus (CP) has traditionally been regarded as a cerebrospinal fluid-producing structure; however, increasing evidence indicates that it functions as a dynamic regulatory interface involved in immune surveillance, metabolic homeostasis, and brain clearance. Neuroimaging studies consistently report CP enlargement across aging and diverse neurological and neuropsychiatric disorders, yet the underlying cellular mechanisms remain poorly integrated. In this review, we synthesize morphological, molecular, and imaging evidence to propose a sequential degenerative model of the CP epithelium. This model comprises: (1) regulated epithelial cell loss via apical extrusion, (2) compensatory hypertrophy of residual cells, (3) mitochondrial remodeling with oncocytic-like change, and (4) progressive blood–cerebrospinal fluid barrier dysfunction. At the molecular level, alterations in epithelial adhesion systems—particularly SPINT1-mediated protease regulation and E-cadherin–based adherens junction stability—may initiate epithelial instability. Hypertrophic epithelial cells exhibit increased mitochondrial burden, reflected by Tom20 expression, which may initially support metabolic adaptation but ultimately contribute to oxidative stress and functional decline. At the macroscopic level, the cumulative effects of cell loss, hypertrophy, and mitochondrial remodeling likely underlie CP enlargement detectable by magnetic resonance imaging. This framework positions CP enlargement as an imaging-visible manifestation of epithelial stress and provides a structural–molecular basis for interpreting CP alterations in brain aging and neurodegenerative disorders. Full article

Comparative studies report inconsistent peptide yields, bioactivities, and sensory outcomes for Alcalase across substrates, creating uncertainty about when it should be favored over other proteases. This study mapped research on hydrolysis of food proteins with Alcalase to quantify scientific output, organize thematic trends, and identify gaps relevant to peptide-based functional foods. A bibliometric analysis of Web of Science records (2004–2024) was performed in R (bibliometrix), using co-occurrence networks, temporal overlays, and conceptual mapping. The dataset comprised 203 documents from 78 sources, exhibiting a 10.3% annual growth rate and a 36.9% international co-authorship rate. Themes clustered around antioxidant and angiotensin-converting enzyme (ACE) inhibitory peptides, particularly in dairy and marine matrices, are supported by workflows combining Alcalase hydrolysis with size-guided ultrafiltration, RP-HPLC (Reverse Phase High-Performance Liquid Chromatography), and, more recently, in silico analyses and encapsulation studies. Recurrent limitations were identified: heterogeneous hydrolysates and uneven reporting that hinder sequence–activity correlations, gastrointestinal degradation and bitterness affecting applicability, and scale-up and purification choices influencing feasibility. The mapping clarified where Alcalase enables bioactive peptide generation and highlighted practical priorities, including protocol standardization and enzyme benchmarking, the integration of peptidomics and machine learning with targeted assays, and formulation-focused validation (encapsulation, stability, and delivery) to bridge in vitro activity to real-world use. These directions support the production of reproducible, application-ready peptide ingredients. Full article

Alpha-2-macroglobulin (α₂M) is a conserved plasma glycoprotein traditionally known for its broad-spectrum protease inhibitory activity. However, emerging evidence indicates that its activated form, α₂M*, generated via proteolytic cleavage or nucleophilic attack, functions as a versatile signaling ligand. By engaging specific cell-surface receptors, most notably low-density lipoprotein receptor-related protein 1 (LRP1) and glucose-regulated protein 78 (GRP78), α₂M* orchestrates a diverse array of intracellular programs, including the PI3K/Akt/mTOR, MAPK/ERK, and JAK/STAT cascades, as well as mechanosensitive YAP/TAZ signaling. These pathways collectively govern fundamental cellular processes such as proliferation, metabolic reprogramming, cytoskeletal remodeling, and inflammatory adaptation across various cell types, including macrophages, cardiomyocytes, and malignant cells. Altogether, this review synthesizes current knowledge on α₂M activation, structural transitions, receptor interactions, and downstream signaling, highlighting the expanding functional landscape of α₂M* as a potent regulator of intracellular communication with implications for physiology and disease. Full article

The valorization of poultry bone by-products into high-value bioactive ingredients aligns with the principles of a sustainable circular bioeconomy. This study established an integrated process for the production, identification, and validation of bioactive antioxidant peptides from Xuefeng black-bone chicken bones (BCB). Alcalase was selected as the optimal protease due to its superior performance in both the degree of hydrolysis and antioxidant activity under the optimized conditions. Using response surface methodology (RSM), the optimal hydrolysis conditions were determined as 50 °C, pH 10.18, and 4.2 h, resulting in a hydrolysate with a hydrolysis degree of 25.10% and ABTS radical scavenging activity of 84.36%. Upon ultrafiltration, the <3 kDa fraction demonstrated a significantly higher antioxidant capacity than the crude hydrolysate. Further purification through gel filtration chromatography yielded the F3 sub-fraction (predominantly <1 kDa peptides), which exhibited the most potent activity across all four antioxidant assays conducted (ABTS, DPPH, hydroxyl radical scavenging, and reducing power). A liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis of F3 led to the identification of 21 peptide sequences. An in silico screening based on bioactivity and toxicity predictions pinpointed three promising candidates: DYPF, WDY, and FGYK. These peptides were chemically synthesized and validated to possess significant in vitro radical scavenging activities against both DPPH and hydroxyl radicals. Molecular docking simulations revealed that all three peptides could spontaneously bind to the Keap1 protein with a high affinity (binding energy < −7.0 kcal/mol), primarily through hydrogen bonds and hydrophobic interactions, suggesting a possible molecular mechanism that may involve the Keap1-Nrf2-ARE antioxidant pathway. This computational insight provides a testable hypothesis for their bioactivity, the verification of which is contingent upon future studies demonstrating their cellular delivery and intracellular action. This work not only provides a sustainable strategy for BCB utilization but also identifies potent antioxidant peptides with potential applications in functional foods and nutraceuticals. Full article

Endocytic trafficking in Candida albicans is a fundamental cellular process that is crucial for its secretion, filamentation, and virulence-related processes. We have previously demonstrated that loss of the key endocytosis-related C. albicans gene END3 disrupts clathrin-mediated endocytosis, leading to impairments in actin patch formation, filamentation, biofilm formation, cell wall integrity, and extracellular protease secretion. The end3 null mutant also exhibits altered antifungal susceptibility and reduced host-cell damage in an in vitro keratinocyte infection model. To ascertain whether endocytosis is required for virulence in vivo, we assessed virulence of the C. albicans end3 null mutant in a murine model of disseminated candidiasis. After infection via the tail vein, and analysis of host survival over 28 days, the end3 null mutant was markedly hypovirulent compared to corresponding control strains. These results indicate that endocytosis mediated by END3 in C. albicans contributes to pathogenesis in vivo. Full article

Background/Objectives: Selection of polymer carriers for targeted drug delivery is typically guided by material availability or trigger responsiveness rather than disease-specific evidence. However, successful preclinical formulations may already encode implicit design rules linking polymer composition to particular pathological environments. This study aimed to identify reproducible material-disease associations across biodegradable polymer systems and to derive formulation-oriented guidance for disease-calibrated carrier selection. Methods: A structured synthesis of 65 preclinical in vivo studies (2020–2025) covering inflammatory bowel disease, arthritis, cardiovascular inflammation, and solid tumors was performed. Extracted variables included polymer family, backbone chemistry, stimulus responsiveness, disease model, and reported therapeutic benefit relative to controls. Associations between polymer composition, trigger mechanisms, and disease categories were analyzed using cross-tabulation, chi-square statistics, Cramér’s V, and direction-of-effect synthesis. Results: Distinct material-disease clustering patterns emerged. Ionizable polysaccharide and methacrylate systems (e.g., alginate, chitosan, Eudragit) were strongly associated with intestinal inflammatory models, reflecting reliance on pH- and ion-mediated mechanisms. Enzyme-degradable hyaluronic acid matrices were concentrated in joint and cartilage disorders characterized by protease overexpression. Oxidation-sensitive polyether systems (e.g., PEG-PPS) and redox-active hybrid platforms predominated in atherosclerosis and tumor models, where oxidative stress is a defining pathological feature. Composite and multi-responsive systems were disproportionately represented in tumors, consistent with microenvironmental heterogeneity. Across studies, therapeutic improvement was consistently reported when polymer functional motifs aligned with dominant biochemical drivers of the disease. Conclusions: Successful biodegradable polymer carriers exhibit disease-specific compatibility patterns rather than universal applicability. These recurring associations suggest that polymer selection can be guided by pathological context even in the absence of direct outcome comparisons. The resulting formulation-oriented framework supports rational carrier choice for personalized drug delivery based on disease-specific microenvironment signatures. Full article

Enhancing plant protein structure, functionality, and digestion-associated bioactivity is pivotal to advancing sustainable food applications. In this study, a controlled thermal treatment was applied to Xanthoceras sorbifolium seed meal protein (XSMP) to characterize alterations in structural features, functional performance, and digestion-related bioactivity. Structural analyses showed that moderate heating induced partial unfolding and disaggregation, leading to reduced particle size and improved colloidal stability, with optimal performance observed at 65 °C. Accordingly, foaming capacity and emulsifying activity index reached their highest values under moderate heat pretreatment (71.43% and 27.21 m²/g, respectively). Simulated in vitro gastrointestinal digestion revealed that moderate heat pretreatment enhanced protease accessibility and was associated with increased formation of low-molecular-weight fragments. As a result, digestion products from optimally treated XSMP exhibited significantly enhanced antioxidant activities during the intestinal phase, including higher reducing power, Fe²⁺-chelating capacity (up to 51.21%), and lipid peroxidation inhibition (82.83%). In contrast, insufficient unfolding at lower temperatures or excessive aggregation at higher temperatures reduced the susceptibility to digestive proteases and the associated functional performance. These findings demonstrate that controlled heat treatment provides a simple and eco-friendly strategy to enhance the functional potential of XSMP, supporting its application as a functional protein ingredient. Full article

Antimicrobial resistance (AMR) poses an escalating global health crisis driven by multidrug-resistant ESKAPE pathogens and emerging fungal threats such as Candida auris (C. auris). In response to this urgent need for new therapeutic strategies, antimicrobial peptides (AMPs) represent a mechanistically distinct alternative to conventional antibiotics due to their membrane-targeting mechanisms and a reduced propensity for resistance development; however, clinical translation has been hindered by toxicity, instability and manufacturing constraints. Recent advances in artificial intelligence (AI) are reshaping AMP discovery and optimization. Machine learning (ML), deep learning (DL) and transformer-based protein language models now enable improved prediction of antimicrobial activity, selectivity, protease stability and host toxicity. Generative approaches, including variational autoencoders, diffusion models and reinforcement learning, facilitate de novo multi-objective peptide design and pathogen-directed optimization against resistant bacteria and multidrug-resistant fungal pathogens. Integrated design–test–learn pipelines are accelerating iterative peptide engineering by tightly coupling computational prediction with experimental validation. Clinically used peptide-derived antibiotics such as polymyxins and daptomycin demonstrate the therapeutic feasibility of peptide-based antimicrobials, while investigational peptides, including pexiganan, illustrate ongoing translational progress. Although no fully AI-designed AMP has yet achieved regulatory approval, the accelerating convergence of computational modeling and experimental validation suggests a rapidly evolving translational landscape. Advancing scalable, surveillance-informed AI frameworks that integrate resistance data, predictive safety modeling and delivery optimization will be essential to accelerate the clinical translation of next-generation, multi-objective AMPs against high-risk resistant pathogens. Full article