Search Results (889)

Despite the widespread use of cryopreserved dog semen, reliable predictors of individual semen freezability are still not available. This study investigated the relationship between post-ejaculatory blood plasma concentrations of canine prostate-specific esterase (CPSE) and semen quality following cryopreservation. The CPSE levels were quantified using a canine-specific sandwich-type immunoassay, and sperm quality was evaluated in fresh and frozen–thawed samples using motility, kinematic, and morphological sperm parameters. Cryopreservation resulted in a significant decline in sperm motility and morphology, with marked variability in the magnitude of post-thaw changes among individuals. CPSE showed significant correlations with several morpho-functional parameters in both fresh and frozen–thawed sperm (p ≤ 0.05). However, its strongest predictive value for freezability was observed for total motility (p = 0.004), with a minimum threshold of 53 ng/mL identifying dogs showing a ≥20% post-thaw motility decline (AUC = 0.785, p = 0.010). These findings suggest that post-ejaculatory CPSE levels in blood plasma may reflect sperm cryoresilience and serve as a potential biomarker of canine sperm freezability. Full article

PET biodegradation remains limited due to its intrinsic properties—high crystallinity, hydrophobicity, and strong chemical stability. These characteristics lead to extremely slow degradation rates and contribute to PET’s persistence in the environment. Understanding how microorganisms respond at the molecular level when exposed to such a recalcitrant polymer is therefore essential. Living organisms express genes in response to their needs during development. When microbes are under critical conditions, such as when contaminants are present, they express genes encoding specific enzymes that attack the pollutant. In this study, a fungus isolated from the infected fruit of the plant Randia monantha was identified as Aspergillus terreus. It was tested for polyethylene terephthalate (PET) degradation, and the fungus Aspergillus nidulans was evaluated due to its previously reported recombinant cutinases for PET degradation. A microplastic polyethylene terephthalate (PET-MP) particle size of <355 μm for degradation was established, and a PET weight loss of 1.62% for A. nidulans and 1.01% for A. terreus was found. Additionally, the degradation of PET was confirmed by FTIR and SEM. This study also compares the transcriptomic profiles of Aspergillus nidulans and Aspergillus terreus during cultivation with PET-MP residues, which serve as a replacement for the carbon source. We present the first evidence of chitinase overexpression during direct exposure of PET to Aspergillus fungi. Interestingly, chitinase expression was detected in the crude extracts of A. nidulans and A. terreus during culture in the presence of PET residues, which replaced the carbon source. The chitinase produced by each fungus has a similar molecular weight of approximately 44 kDa. Chitinase activity was monitored over a 14-day cultivation period; from day 2, chitinase activity was detected in both cultures and continued to increase until day 14, when the highest values reported in this work were 24.88 ± 4.17 U mg⁻¹ and 10.41 ± 0.47 U mg⁻¹ for A. nidulans and A. terreus, respectively. Finally, we proposed a pathway for PET degradation by Aspergillus fungi that involves mycelial adherence and the secretion of hydrophobins, followed by the production of intermediates and monomers via esterase hydrolysis, and ultimately, the entry of monomers to the ethylene glycol (EG) and terephthalic acid (TPA) pathways, further suggesting these Aspergillus as candidates to produce valuable compounds under these conditions, such as muconic acid, gallic acid, and vanillic acid. Full article

To address the limited functional diversity of traditional kombucha, this study utilized red yeast rice (RYR) as an alternative substrate and prepared three samples: black tea kombucha (KBT), black tea-red yeast rice mixed kombucha (KBL, at a 1:1 ratio), and red yeast rice kombucha (KRY). After 9 days of fermentation, KRY exhibited the lowest pH, the highest total acidity, and notable sugar metabolic activity. It exhibited in vitro inhibition rates of 82.8%, 78.2%, 70.3%, and 76.9% against cholesterol esterase, pancreatic lipase, α-glucosidase, and α-amylase, respectively, indicating potential hypoglycemic and hypolipidemic activities. In contrast, KBT maintained the strongest antioxidant capacity, with scavenging rates exceeding 90% against both 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS). A total of 72 volatile flavor compounds (VFCs) were identified, with 7 key compounds enriched in KRY, which enhanced its sensory acceptance and received the highest scores in color, clarity, and aroma. Microbial community analysis revealed the post-fermentation dominance of Komagataeibacter, Acetobacter, and Saccharomyces, which correlated positively with key VFCs. These findings indicate that RYR as a substrate enhances functional microbial growth, sugar metabolism, organic acid production, flavor enrichment, and in vitro inhibitory activity of enzymes associated with hypoglycemic and hypolipidemic effects. Full article

Heroin is a highly addictive drug that exerts its primary effects through activation of μ-opioid receptors. Its principal active metabolite, 6-monoacetylmorphine (6-MAM), significantly contributes to heroin’s neurological effects and acute toxicity. Current pharmacotherapies for heroin use disorder, employing opioid receptor agonist or antagonist, are often limited by risks of dependence, tolerance, and/or adverse side effects. In this context, enzyme-based therapy emerges as a promising alternative by rapidly converting drugs into inactive or less harmful metabolites in the blood. As a macromolecule, the enzyme does not cross the blood–brain barrier, thereby avoiding side effects in CNS. Through structure-based computational screening, Xoo-PepA (PDB ID: 3JRU), a leucine aminopeptidase from Xanthomonas oryzae pv. oryzae, was identified as a potential enzyme capable of hydrolyzing heroin and 6-MAM. Computational and experimental analyses confirm that Xoo-PepA hydrolyzes heroin sequentially to 6-MAM and subsequently to morphine. Enzymatic properties including dependence on metal ions, optimal pH, thermal stability, and substrate specificity were characterized accordingly. Notably, supplementation with Ni²⁺ or Zn²⁺ and TCEP extended Xoo-PepA’s half-life at 37 °C from 1 h to over 24 h, highlighting the essential role of metal ions in maintaining structural stability. Moreover, Ni²⁺ enhanced Xoo-PepA’s hydrolysis toward peptidase substrate L-leucine-p-nitroaniline by 770-fold, yet conferred no significant activation toward heroin. Mutations in metal ion-coordination residues (e.g., K262A, D267A/E346L) exhibited different activity profiles toward these two types of substrates, suggesting a distinct regulatory mechanism of metal ions may be involved in these activities. This study provides the first demonstration that Xoo-PepA, a non-mammalian, metal-dependent aminopeptidase, can hydrolyze heroin and 6-MAM, shedding light on its functional versatility and biochemical characteristics. Full article

Insect hemocytic cell lines offer substantial advantages over primary, in vivo hemocyte cultures, fundamentally transforming experimental approaches in cellular immunology and related fields. Selected Malacosoma disstria cell lines were characterized for optimal growth temperatures, morphogenesis, blebbing, extracellular enzyme profiles, and their interactions with material (polystyrene) and microbial (Bacillus subtilis) surfaces. The adhesive hemocyte lines UA-Md221 and Md108 showed optimal growth at 28 °C, whereas UA-Md203 and Md66 grew best at 21 °C, with Md66 tolerating 21–28 °C. Md108 demonstrated a broader temperature tolerance than other adherent cultures. Both Md108 and UA-Md221 adhered to polystyrene within 24 h post-subculturing, although protease-induced morphological changes in modified Grace’s medium continued through 48 h and 72 h, respectively. Culture quality was monitored by assessing the release of multiple enzymes, including alkaline and acid phosphatases, esterases and lipases, aminopeptidases, proteases, glycosidases, and hydrolases from the cell lines at 50% confluency in modified Grace’s medium. Fetal bovine serum showed elevated esterase lipase (C8) and phosphoamidase activities when diluted in Grace’s medium and phosphate buffered saline (PBS). Exposure to dead B. subtilis suspended in PBS induced quantitative and qualitative alterations in the enzyme secretion profiles of Md66 and Md108 cultures. We conclude that semi-quantitative assessments of hemocytic cell lines can provide valuable insights for the time window of each enzyme release, revealing immune and metabolic signaling patterns. Full article

Sorghum (Sorghum bicolor L. Moench) is a climate-resilient cereal with significant potential as a functional food due to its distinctive polyphenolic profile, including rare 3-deoxyanthocyanidins (3-DXAs). Broader utilisation of sorghum is limited by low protein digestibility and the presence of anti-nutritional factors, such as condensed tannins and phytates. This review consolidates current evidence on germination as a bioprocessing strategy to address these limitations and enhance the bioactivity of sorghum polyphenols. Germination activates endogenous hydrolytic enzymes, such as phytases and esterases, and upregulates the phenylpropanoid pathway through phenylalanine ammonia-lyase, which promotes the release of cell wall-bound phenolic acids and the de novo synthesis of flavonoids. A “germination paradox” is identified, in which qualitative shifts toward lower-molecular-weight, more bioaccessible aglycones enhance antioxidant and anti-inflammatory efficacy, even when total phenolic content fluctuates. The review also examines the effects of germination on digestive release, transepithelial transport, and colonic microbial transformation of phenolics. Finally, genotype- and process-dependent optimisation windows, typically 48–72 h, are delineated to balance anti-nutrient reduction with phytochemical retention, providing a basis for the development of germinated sorghum-based functional foods and nutraceuticals. Full article

Monitoring the soil–plant system in forest ecosystems is crucial for preserving their ecological functions and services. This study assessed carbon and nitrogen stable isotopes and ecoenzymatic stoichiometry as suitable indicators for characterizing the soil–plant system as a functional unit of ecological processes. To this end, in June 2021 six plots (1 m² each) were selected in two typical Mediterranean forest ecotypes: a coastal stone pine forest (Pinus pinea L., PF) and a meso-hygrophilous broadleaf forest (RV). Soil samples (0–15 and 15–30 cm depth) and litter samples (40 × 40 cm) were collected and characterized in terms of physical, chemical and biochemical properties. t-tests revealed significant differences between RV and PF, indicating distinct microbial nutrient acquisition strategies. The higher C:N ratio in PF suggested lower litter quality and greater recalcitrance to microbial decomposition. Consistently, RV showed a more pronounced ¹³C and ¹⁵N enrichment from litter to SOM down to a 30 cm depth, confirming faster organic matter decomposition and mineralization. Enzyme activity patterns supported these findings. The higher β-glucosidase and butyrate esterase activities in RV reflected its greater microbial potential to activate biogeochemical cycles. Both forests exhibited a higher microbial demand for C and P than for N to maintain ecological stoichiometric balance, with stronger C limitation at the surface and P limitation in the subsoil, particularly in RV soil. This integrated monitoring approach provides insights into nutrient cycling and ecosystem resilience and offers tools to evaluate ecosystem functionality under changing environmental conditions, supporting sustainable forest management. Full article

Esterases catalyze the hydrolysis and transesterification of short-chain fatty acid esters, and microbial esterases are used in the production of biofuels, cosmetics, food, and pharmaceuticals. The soil strain Bacillus paralicheniformis T7 secretes enzymes with esterase activity; however, many bacterial enzymes remain insufficiently studied. Therefore, this study aimed to identify and characterize novel GDSL esterases produced by B. paralicheniformis. Protein mass spectrometry, combined with proteomics and genomics, identified genes encoding two GDSL esterases, which were cloned into the pET-28c(+) vector. The resulting proteins were obtained in Escherichia coli BL21(DE3) as the recombinant esterases rEST-24 and rEST-28. These recombinant GDSL esterases showed maximum activity at 40 °C and pH 7.0. Moreover, Ca²⁺, Zn²⁺, Cu²⁺, and Fe²⁺ ions inhibited their activity, and rEST-28 was resistant to the detergents Tween-20, Tween-80, and Triton X-100. High-yield esterase activity was detected in bacteria cultured on feather medium and nutrient broth, and submerged fermentation of the B. paralicheniformis T7 strain on feather medium enabled the production of an esterase extract exhibiting activity of 17,618 ± 610 U/g. These results suggest that the B. paralicheniformis T7 strain can produce esterases and shows promising potential for application in technologies that degrade fatty acid esters using hydrolytic enzymes. Full article

Efficient inorganic carbon supply is a common limitation in microalgal cultivation, particularly in waste-derived media such as anaerobic digestate. Carbonic anhydrase (CA) accelerates the interconversion of CO₂ and bicarbonate and may therefore enhance carbon utilisation under conditions where inorganic carbon is abundant but not readily available. In this study, crude CA-containing extracts (aCA) were prepared from Scenedesmus-dominated algal biomass, and CA activity was quantified using an esterase assay (EAA). Although EAA activities varied depending on biomass pretreatment (0.15–0.47 U g⁻¹ DW), the physiological response to extract addition was consistent. In batch cultures of Chlorella sorokiniana grown in diluted digestate, aCA supplementation increased the specific growth rate (SGR) by 21–82%. In contrast, stimulation in a mineral medium was minimal, indicating that the benefit of aCA addition is most apparent under reduced inorganic carbon availability. In semi-continuous cultivation, repeated extract addition sustained a higher biomass productivity over time (rather than a specific growth rate). These results demonstrate that crude microalgal extracts containing CA can improve growth performance in digestate-based cultures and may offer a simple, low-cost approach to enhancing inorganic carbon utilisation in waste-integrated algal production systems. Full article

This study examined the role of vaginolysin (VLY), a virulence factor of the bacterium Gardnerella vaginalis (GV), in bacterial vaginosis (BV). In a group of 112 women with BV (diagnosis on the Nugent scale ≥7 points) and 122 control cases with normal microbiota, VLY levels, the state of the vaginal microecology (colposcopy, laboratory markers, pH), GV genotypes (clades 1–4), and clinical symptoms were assessed. It was found that GV also occurs in healthy women, but VLY levels are significantly higher in BV and correlate with inflammatory markers (e.g., leukocyte esterase) and symptom severity. However, the relationship is nonlinear: low and moderate VLY levels have little effect on symptoms, while high levels cause a sharp increase in symptoms. Thus, VLY is potentially important for the pathophysiology and clinical assessment of BV. Full article

The thermophilic bacterium Thermus thermophilus represents a crucial genetic reservoir for exploring thermostable enzymes as valuable biocatalysts for industrial and biotechnology applications. Here, we identify, clone, and characterize Ces1-ET, Est1-ET, and Plp1-ET, three lipolytic enzymes obtained from T. thermophilus strain ET-1 isolated from El Tatio Geothermal Field in Northern Chile. To enable recombinant expression, we constructed the pTGT-1 expression system, a versatile bifunctional shuttle vector compatible with both Escherichia coli and T. thermophilus. The three thermoenzymes Ces1-ET, Est1-ET, and Plp1-ET, were successfully cloned, expressed, and purified using the pTGT-1 system, with a molecular mass of 25 kDa, 36 kDa, and 28 kDa, respectively. The recombinant purified enzymes displayed optimal temperatures at 60 °C, 80 °C, and 70 °C and optimal pH of 7.5, 9.0, and 8.0 for Ces1-ET, Est1-ET, and Plp1-ET, respectively. Functional biochemical assays revealed a broad tolerance to surfactants, detergents, divalent cations, and high salinity, relevant properties for their application in an industrial setting. These thermostable esterases expand the repertoire of thermozymes from Thermus spp., introducing pTGT-1 as an innovative tool for thermophilic protein expression and highlighting T. thermophilus strain ET-1 from El Tatio Geothermal Field as a valuable source of thermostable enzymes for industrial and biotechnology applications. Full article

Influenza D virus (IDV) and co-circulating respiratory pathogens were investigated using RT-qPCR in 316 samples collected from 210 Spanish cattle farms with bovine respiratory disease (BRD) outbreaks between July 2023 and September 2024. Thirty-eight IDV-positive samples, from 30 farms across 15 provinces throughout Spain, were identified. IDV was significantly more frequent in bronchoalveolar lavage samples (22.1%) and nasal swabs (13.5%) compared to lung tissues (5%) and other sample mixtures (5%). All IDV-positive specimens exhibited co-infections, with most (94.7%) harboring three to seven viral and/or bacterial pathogens, highlighting the complexity of BRD outbreaks. Cramer’s V analysis revealed moderate IDV association with Mycoplasma bovis (V = 0.255) and Pasteurella multocida (V = 0.223), and a weaker IDV-bovine coronavirus association (V = 0.202), while IDV association with Histophilus somni (V = 0.025) and bovine herpesvirus 1 (V = 0.000) was negligible. Partial sequences of the hemagglutinin-esterase (HEF) gene from a subset of 13 IDV-positive samples (Ct < 32) were obtained. This confirmed the presence of the two major genetic lineages detected among cattle in Europe, D/OK and D/660, with D/660 exhibiting higher genetic diversity, as determined by DNAsp 6.12 software. This is the first report of IDV infection in Spanish cattle, confirming the circulation of the D/OK and D/660 lineages within the cattle population. Full article

In this study, a Nocardia niigatensis strain was isolated from boron-rich mining soils in the Bigadiç region of Türkiye and comprehensively characterized. The primary aim of this study was to isolate boron-tolerant Nocardia species and evaluate their physiological, enzymatic, and biochemical profiles. Selective isolation techniques were employed to obtain Nocardia isolates, and species-level identification was achieved using both 16S rRNA gene sequencing and MALDI-TOF MS analysis, which consistently confirmed the isolate as N. niigatensis. In addition to molecular identification, the morphological, physiological, and biochemical characteristics of the strain were extensively investigated. The strain demonstrated notable boron tolerance, exhibiting robust growth at concentrations up to 50 mM, highlighting its potential applicability in the bioremediation of boron-contaminated environments. Physiological assays further revealed moderate halotolerance and a mesophilic growth profile, with optimal growth observed at 27–37 °C. Enzymatic screening indicated positive L-glutaminase activity, an enzyme of considerable industrial relevance. Moreover, API ZYM profiling revealed a broad enzymatic spectrum, including esterases, arylamidases, phosphatases, and glucosidases, suggesting substantial metabolic versatility. Antibiotic susceptibility testing showed sensitivity to doxycycline, tobramycin, and erythromycin, whereas resistance was observed against imipenem and several β-lactam antibiotics. Metagenomic analysis of boron-rich soils from two distinct mining sites revealed marked differences in microbial community composition, with variations in Actinobacteria abundance associated with mineral type. Overall, these findings emphasize the adaptive capacity and biotechnological potential of environmental Nocardia strains inhabiting chemically stressful ecosystems, warranting further genomic and metabolomic investigations. Full article

Candida spp. are important opportunistic human fungal pathogens. This study aimed to identify and characterize Candida spp. obtained from patients admitted to an Intensive Care Unit (ICU), focusing on virulence attributes and susceptibility to antifungal agents. A total of 131 isolates from oral and tracheobronchial secretions of adult ICU patients were evaluated. Phenotypic identification was performed using chromogenic culture media for Candida, followed by MALDI-TOF mass spectrometry, with representative isolates confirmed by ITS sequencing. Antifungal susceptibility to fluconazole, ketoconazole, and amphotericin B was determined only by the agar disk diffusion method, and virulence was assessed through esterase, DNase, protease, and hemolytic activity assays. C. albicans was the prevalent species, followed by C. tropicalis, C. krusei, C. glabrata, C. parapsilosis, C. dubliniensis, C. lusitaniae, and C. guilliermondii. Antifungal resistance rates reached 51.1% for fluconazole, 42.7% for ketoconazole, and 19.1% for amphotericin B, as determined by disk diffusion method. Overall, 64.9% of the isolates exhibited esterase activity, 18.3% DNase, 45.8% protease, and 67.2% exhibited hemolytic activity. Oral isolates were more frequent than tracheal isolates and demonstrated a higher prevalence of antifungal resistance and virulence traits. These findings underscore the epidemiological importance of characterizing Candida species in hospitals to better understand the yeast profile and to support adequate clinical management. Full article

Background/Objectives: The global prevalence and incidence of inflammatory bowel diseases have risen in the past two decades. Among them, Crohn’s disease and ulcerative colitis are still challenging to treat due to vascular and proliferative alterations. Studies in rats suggest that blocking histamine receptors (H1–H4) can improve colitis progression. However, the specific histamine receptor responsible for this effect remains debated. The experiment aimed to assess the role of specific histamine receptor subtypes in colitis development, focusing on oxidative stress markers in the liver and skeletal muscle. Methods: The study involved 60 adult male Wistar rats, divided into control and colitis experimental groups. Colitis was induced through intracolonic administration of 2,4,6-trinitrobenzenesulfonic acid. Animals in both experimental groups received intramuscular injections of NaCl (non-treated, NT) or H1, H2, H3, and H4 receptor antagonists (10 study subgroups in total). On day eight, the animals were re-anesthetized and euthanized via exsanguination. Then, liver and skeletal muscle (m. soleus) samples were collected for analysis of oxidative stress markers. Results: The analyses of skeletal muscle samples showed that using the H1 and H2 receptor antagonists increased superoxide dismutase (SOD) and catalase (CAT) activities, as well as parameters related to glutathione metabolism (reduced glutathione (GSH), glutathione S-transferase (GST)) in rats from the control groups, indicating enhanced antioxidant defense. In rats with chemically induced colitis, we observed that H1 receptor antagonists elevated CAT activity, whereas β-esterase (β-EST) activity remained elevated across all colitis subgroups. In the liver, histamine receptor antagonists produced receptor-specific redox effects: the H2 receptor antagonist reduced oxidative damage (malondialdehyde (MDA)); the H1 receptor antagonist attenuated SOD hyperactivity, but depleted GSH; and the H4 receptor antagonist increased GSH while elevating MDA. Chemically induced colitis increased α- and β-EST activities, whereas administration of the H1 or H3 antagonist reduced β-EST levels. Conclusions: Histamine receptor antagonists modulated oxidative stress responses in both liver and skeletal muscle tissues in a receptor-dependent manner. Among them, the H2 receptor antagonist most effectively mitigated hepatic oxidative injury, highlighting its potential as a therapeutic target in colitis-associated systemic oxidative stress. Full article