Characterization of AHL-mediated quorum sensing in Pseudomonas gessardii from raw milk and insights into control of proteolytic activity.

Dairy environment and seasons affect the microbiome of a traditional artisanal cheese.

Plant-derived proteolytic enzymes are widely used in biochemistry and food processing. For example, bromelain, ficin, and papain serve as meat tenderizers, while cardosin A is used as a plant-based rennet in cheese production. A cysteine protease has been identified in grapevine products such as fresh juice, wine, and wine vinegar. The enzyme (CYSP) shares sequence similarity with RD21A from Arabidopsis and other plant cysteine endopeptidases. This mass spectrometry-based study investigated the proteolytic activity of wine and wine vinegars, including both commercial and laboratory-prepared samples. Vinegar types examined included white wine vinegar, balsamic vinegar, and red wine vinegar produced through grape juice fermentation and spontaneous acetification. Protease activity and specificity were assessed using substrates such as pure protein standards, casein, and minced beef proteins. The activity assay also included spectrophotometry with azocasein and electrophoresis followed by gelatin zymography. Results confirmed the presence of CYSP and indicated aspartic protease involvement. Digestion experiments coupled with mass spectrometry identified peptide cleavage sites, with C-terminal residues frequently being L, F, R, Y, K, and D/E. This pattern reflects the combined specificity of CYSP and pepsin-like proteases. Notably, CYSP activity was higher in wine, whereas aspartic protease activity predominated in vinegar.

CRISPR/Cas9-mediated development of Penicillium roqueforti strains deficient in roquefortine C and mycophenolic acid enables toxin-free blue cheese production.

Effect of phenolic compounds on proteolytic activity and spoilage potential of Pseudomonas aeruginosa in milk

From aging to space: A comparative biology of skeletal muscle degeneration.

Transcriptome analysis of Crandell Rees Feline Kidney (CRFK) cells infected with Feline calicivirus strain 023 (FCV 023).

Review: Comprehending the influence of enzyme sources, environmental parameters, and storage conditions on the proteolytic activity of cow milk

Enhancing the sensory and nutritional properties of faba bean protein through fermentation with lactic acid bacteria and Bacillus spp.

Mechanistic insights into the structure-toxicity relationship of α-aminononanophenone derivatives: Role of CYP3A4 and oxidative stress.

Cholesterol homeostasis in mammalian cells relies on the interaction between two endoplasmic reticulum (ER) sterol-sensing membrane proteins, Scap and Insig. Their interaction regulates activation of transcription factors called sterol regulatory element-binding proteins (SREBPs) that control genes for cholesterol biosynthesis and uptake. Previous studies suggested a model where cholesterol sensing by Scap involves communication across the ER membrane between two functional domains, a cholesterol-binding domain on the luminal side and a COPII-binding domain on the cytosolic side. When ER cholesterol is low, Scap binds COPII adapter proteins to facilitate ER-to-Golgi transport and proteolytic activation of SREBPs. When ER cholesterol is above a threshold concentration, this transport is blocked, a process that requires Insigs. However, the precise molecular mechanisms by which cholesterol and Insigs control the conformations of Scap remain unknown. Here, we elucidate the 3.2 Å cryo-EM structure of a Scap/Insig complex in the presence of saturating amounts of cholesterol. Structure-guided mutagenesis of the Scap/Insig interface indicates that Scap's transmembrane helix 7 (TM7) plays a critical role in transducing conformational changes between the luminal and cytosolic sides of the ER membrane to control Scap/SREBP transport from ER to Golgi. An intramembrane cholesterol bound at the Scap/Insig interface competes with the intramolecular interaction of Scap's TM7 at this interface to modulate cholesterol sensing. These results further advance our understanding of how Scap senses cholesterol and provides additional targets for controlling cholesterol and lipid synthesis in the context of metabolic diseases and even some cancers.

With the rise in antimicrobial resistance, understanding the virulence factors utilized by pathogenic E. coli is essential for the development of alternative therapeutics. While previous work has shown that disruption of the E. coli rhomboid protease gene glpG leads to defects in bacterial colonization, here we provide mechanistic insight into the loss of fitness. We show GlpG is essential for the assembly of type 1 pili, a virulence factor required for the colonization of eukaryotic cells. Since pili are critical for biofilm formation and bacterial persistence, the absence of GlpG proteolytic activity reduces the production of biofilm. Working towards new potential antimicrobial targets for treating infections, we show that biofilm formation is hampered by GlpG inhibition. Our data demonstrates that GlpG plays a key role in protein quality control of type 1 pili and alters the paradigm for GlpG proteolysis, previously implicated in the cleavage of only membrane embedded substrates.

Currently, earthworm extracts and products derived from earthworms are aimed at improving ecosystem stability as well as discovering new therapeutic applications. This study aimed to isolate, characterise and assess the antimicrobial and antitumor properties of the earthworm (Lampito mauritii) extract against pathogenic microbes and cancer cells. Characterisation of the earthworm extracts was executed using Ultraviolet (UV)-Vis spectroscopy, Fourier Transform Infrared (FT-IR) and ionexchange chromatography, accompanied by the evaluation of cytotoxicity, initially using MTT assay. The earthworm extracts were then evaluated for wound healing potential. Additionally, Acridine Orange (AO)/Ethidium Bromide (EB) and nuclear staining techniques were performed, followed by flow cytometry and gene expression analysis with MDA-MB231 cell lines. The antimicrobial assays of L. mauritii extract exerted a substantial increase in the zone of inhibition as perceived in terms of bactericidal and fungicidal activity. Earthworm extract’s diverse activities are attributed to bioactive signatures in the extract. It was found that earthworm extracts at a concentration of 10 μg/mL inhibited 50% of tumour cell growth (IC50). When exposed to IC50 concentrations of earthworm extract, excellent wound healing and cell migration were observed. These findings displayed promising antiproliferative, antimigratory and apoptotic effects on MDA-MB231 cells compared to untreated control, against a pan of malignant cells, which correlates with gene expression levels of key molecules like AKT involved in growth and proliferation. It is possible to develop an effective therapeutic molecule from L. mauritii extract for the treatment of breast cancer, pathogenic bacterial and fungal infections with further research. Major/Key findings: 1. Isolation and characterisation of earthworm extract; 2. Cytotoxicity of extract against MDA-MB231 cancer cell line; 3. Antimicrobial, proteolytic and wound healing activities of earthworm extract.

The object of research is the process of complex formation of elastic leather using enzymes of proteolytic and hydrolytic action. One of the most problematic areas is the decrease in the elasticity of the leather semi-finished product at the stage of its dehydration. Enzyme treatment increases the mobility of the microfibrillar structure of the semi-finished product due to the destruction of physical intermolecular bonds. During the study, a proteolytic enzyme was used at the stage of bating the pelt and enzymes of hydrolytic action were used to treat the tanned chrome semi-finished product. A semi-finished product was obtained, which is characterized by an increase in porosity compared to the original semi-finished product. The porosity of the semi-finished product increases by 22% in the case of using enzyme treatment at the bating stage and by 67% with repeated treatment of the tanned semi-finished product with enzymes. This is due to the fact that the proposed enzyme treatment promotes the removal of glycosaminoglycans from the dermis at the bating stage. Further use of enzymes after tanning of the semi-finished product contributes to the destruction of carbohydrate bonds with collagen macromolecules, which ensures an increase in its physicochemical properties. The peculiarity of this effect can be explained by the presence of an active center in enzymes, which forms enzyme-hydrocarbon-collagen complexes with carbohydrates and collagen macromolecules. Inside the formed complexes, the destruction of existing bonds occurs and the separation of carbohydrates from the collagen of the dermis. This provides the possibility of obtaining a leather semi-finished product, which is characterized by an increase in the tensile strength and elongation at 9.8 MPa by 8.4 and 23.0%, respectively, and these indicators reach 20.7 MPa and 48.0% compared to the indicators of the tanned semi-finished product.

Traditional dairy products (TDP) are a valuable source of lactic acid bacteria (LAB) for use in sustainable and natural food systems. This study examined the technology and biopreservative properties of LAB isolated from 167 dairy samples across 11 regions of Morocco. Among the 2,672 pure isolates, 531 presumptive LAB were selected for laboratory phenotypic evaluation, focusing on milk acidity, enzyme activities, stress resistance, and antibacterial efficacy against foodborne microorganisms. The acidity test revealed considerable intraspecies variability; notably, the top-performing outliers were isolates of Enterococcus faecalis and Lactococcus lactis, predominantly derived from Lben and the Tetouan region. Significant heterogeneity was also observed in enzymatic activity, with approximately 70% exhibiting proteolytic activity, 22% lipolytic activity, 43% esterolytic activity, and 42% amylolytic activity. In terms of inhibitory activity, 32% of the LAB isolates showed inhibition against the tested pathogens, with noteworthy suppression observed against Staphylococcus aureus (13%) and Listeria monocytogenes (15%). The most potent antibacterial activity was identified in isolates of Enterococcus faecium, Lactiplantibacillus plantarum, and L. lactis, which were isolated from Lben (Agadir), Milk (Fes), and Raib (Fes), respectively. Principal component analysis (PCA) and clustering identified three distinct functional clusters of isolates, shaped by geographical origin and dairy products type, with specialized traits in acidification, stress resilience, or enzymatic activity. Representative isolates (n = 84) from key clusters were identified by 16S rRNA sequencing, encompassing 15 LAB species, dominated by L. lactis, Leuconostoc mesenteroides, Enterococcus spp. and Lpb. plantarum. This work represents the first extensive screening of LAB from Morocco, providing a valuable collection of strains and a foundational framework for developing tailored autochthonous starter or adjunct cultures for improved dairy fermentation and biopreservation.

The study aimed to evaluate bottom sediments as a promising source of probiotic bacteria for aquaculture applications. Bacillus strains were selected as the most suitable bacterial species for application in the food industry. Initially, seven Bacillus spp. strains were isolated from the intestinal contents of healthy sterlet specimens; however, none of them demonstrated high potential probiotic properties. Subsequently, bottom sediments were considered as a source of probiotic strains. In the bottom sediments, bacilli exist in a vegetative form and constitute an integral part of the microbial community. A total of 120 Bacillus spp. strains were isolated and comprehensively analyzed. Proteolytic and amylolytic activities were detected at moderate levels in almost all isolated strains. Most isolates exhibited low or negligible antioxidant, DNA-protective, and antimicrobial activities; however, a small group of strains showed high values of these properties. Principal component and cluster analyses indicated the co-existence of three life strategies of bacilli in bottom sediments. These findings highlight the high probiotic potential of bacilli from bottom sediments and support their suitability as novel probiotics for enhancing the health and productivity of aquatic organisms in aquaculture.

Background. Chronic venous disease (CVD) with chronic venous insufficiency (CVI) involves disruption of the proteolytic balance in the venous wall driven by an imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors (TIMPs), particularly MMP-2 and TIMP-4. Aim. To assess changes in MMP-2, TIMP-4 and their ratio before and after surgical treatment of CVD and to compare the effectiveness of different surgical techniques. Materials and Methods. A total of 139 patients with chronic venous disease (CEAP C3–C6), aged 18–75 years, were enrolled and allocated into three groups based on the extent of surgical intervention performed. All participants underwent standard clinical assessment and duplex ultrasound. Serum MMP-2 and TIMP-4 concentrations were measured before and after treatment using validated ELISA assays under identical laboratory conditions. Thirty age-matched healthy volunteers served as the control group. Results. Before treatment, patients showed elevated MMP-2 and reduced TIMP-4 compared with controls. Combined intervention (RFA + miniphlebectomy + perforator ligation) resulted in the most significant decrease in MMP-2, increase in TIMP-4 and near-normalisation of their ratio. Less pronounced but significant changes occurred in subgroup 2A, whereas subgroup 2B and the RFA-only group retained an abnormal MMP-2/TIMP-4 ratio. Conclusions. Combined surgical treatment most effectively restores proteolytic balance in CVD. Dynamic assessment of MMP-2, TIMP-4 and their ratio may serve as a laboratory indicator of treatment efficiency.

Ribosomally synthesized and post translationally modified peptides (RiPPs) rely on a diverse array of enzymes to tailor peptide backbones and side chains. In this study, we characterized enzymes from two different biosynthetic gene clusters (BGCs) from Pseudomonas strains (pfl and pos) that catalyze new transformations in RiPP biosynthesis. Two α-ketoglutarate-dependent HEXXH enzymes, PflC and PosC, perform hydroxylation of multiple consecutive glutamine residues and selectively recognize a C-terminal ARMD tetrapeptide to trigger oxidative backbone cleavage that generates an amide terminus. Mutational analysis pinpoints the first position of this motif as a critical determinant. Notably, PflC displays proteolytic activity in the absence of the leader peptide, indicating that leader peptide-enzyme interactions modulate the observed reaction selectivity. The biosynthetic gene clusters also encode a unique MNIO-nitroreductase fusion enzyme that installs a rare Z-dehydrophenylalanine and hydroxylates an Asp residue. Collectively, this work expands both the catalytic repertoire and structural diversity accessible through bacterial RiPP biosynthesis.

In our previous work, we reported for the first time the presence of enterocin-encoding genes in novel Lacticaseibacillus paracasei (L. paracasei) and Levilactobacillus brevis (Lev. brevis) strains isolated from artisanal dairy products made from raw cow milk. The aim of this study was to isolate enterocin-positive lactic acid bacteria (LAB) from artisanal dairy products and assess their technological characteristics and safety for potential application in food systems. LAB isolates were characterized using phenotypic tests, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) identification, and PCR detection of enterocin genes, followed by evaluation of their physiological and technological properties and a comprehensive safety assessment, including antimicrobial resistance, virulence, biogenic amine, and integron genes. Two strains, L. paracasei S2 and Lev. brevis S62, carried enterocin genes (entAS-48 and entQ) and exhibited strong acidifying and proteolytic activities, along with antibacterial effects against foodborne pathogens and reference strains. Both isolates tolerated environmental stresses, including low pH, and lacked virulence factors, clinically relevant antibiotic resistance genes, biogenic amine production, and integron elements. These results indicate that the strains are safe, multifunctional, and suitable for developing regionally adapted dairy products, highlighting artisanal dairy products as a valuable source of novel LAB with promising biotechnological applications.

The symbiotic association of legumes with rhizobia results in the formation of new root organs called nodules. However, the lifespan of nodules is limited by the senescence process. Increased proteolytic activity is one of the hallmarks of nodule senescence. In Medicago truncatula, a papain cysteine protease encoding gene, MtCP6, is a marker for the onset of nodule senescence under both developmental and stress-induced pathways. To identify the promoter regions responsible for the senescence-related expression of MtCP6, progressive MtCP6 promoter deletions were generated and fused with the GUS reporter for promoter::GUS activity analysis in transgenic M. truncatula roots. In planta, a minimal promoter sequence of 67bp was identified as sufficient for specific spatiotemporal transcriptional activation of MtCP6 in nodules. The functionality of this promoter regulatory module, thereafter named 'nodule senescence (NS) promoter regulatory module', was validated by both gain- and loss-of-function approaches in M. truncatula. A yeast-one-hybrid (Y1H) screen identified the AP2/ERF transcription factor ERF091, shown to positively regulate nodulation in Lotus japonicus, as an NS- interacting factor. Further Y1H and Nicotiana transactivation assays demonstrated the specificity of ERF91 to interact with and mediate transcription activation of the NS promoter regulatory motif. This work has uncovered a new senescence-related nodule-specific regulatory region and provides evidence for the likely involvement of a stress-related ERF family member in the regulation of MtCP6, at the onset of nodule senescence.