Protease Activity Research Articles

Complete genome sequence provides information on quorum sensing related spoilage and virulence of Aeromonas salmonicida GMT3 isolated from spoiled sturgeon

Foodborne bacteria can pose a threat to the public health due to their spoilage and virulence potential, which can be regulated by quorum sensing (QS) system. In the study, we isolated a spoilage bacteria strain Aeromonas salmonicida GMT3 from refrigerated sturgeon. The complete genome of A. salmonicida GMT3 was sequenced, and the QS related genes were assigned. QS signal molecules N-acyl-homoserine lactones (AHLs) and AI-2 were detected. Genes regulating the spoilage-related metabolic pathways, including protease and lipase secretion, amines metabolism, sulfur metabolism, motility and biofilm formation were analyzed. Furthermore, genes encoding for several virulence factors, e.g. hemolysin, aerolysin, type II secretion system (T2SS), type VI secretion system (T6SS), antibiotic and multidrug resistance were also identified. In addition, the spoilage and virulence phenotypes associated with QS including protease, swimming and swarming activity, biofilm and hemolytic activity were detected. This study provided new insights into spoilage and virulence mechanisms correlated with QS of A. salmonicida GMT3, which might promote development of new approaches for spoilage and virulence control based on QS target.

The use of Moringa leaves extract in rabbit diets: its effect on performance, lipid profile, kidney and liver function, immunity, antioxidant, digestive enzymes, and cecal microbiota

Abstract This study evaluated the role of Moringa oleifera leaves extract (MOLEx) in improving the performance and health of rabbits during the fattening period. The growth, carcass measurements, serum immunity, lipid profile, liver and kidney functions, digestive enzymes, antioxidant condition, cecal microbiota of rabbits have been examined. A total of 100 New Zealand White male rabbits (5-week-old) were randomly distributed into five groups and were fed on the basal diet only or the basal diet supplemented with graded levels of MOLEx (1, 2, 3 or 4 g/kg diet) for 8 weeks. Animals in each group were divided into 10 replicates, with two animals each. Results showed that dietary supplementation of MOLEx at levels of 1, 3 and 4 g/kg feed improved growth performance (p < 0.05) including LBW (7, 9, 11, 13 weeks), BWG (5-13 weeks) and FCR (11-13 and 5-13 weeks). The inclusion of MOLEx in rabbits feed (3 and 4 g/kg) increased carcass%, dressing% and spleen% and decreased duodenum pH and cecal pH. Abdominal fat% was significantly lowered after the treatment with MOLEx. Lipid profile, liver and kidney functions, and cecal microbiota (increasing beneficial bacteria and reducing harmful bacteria) of growing rabbits were positively affected by MOLEx levels. The dietary supplementation of MOLEx improved most of antioxidant biomarkers of growing rabbits. The dietary supplementation of MOLEx (2, 3 and 4 g/kg) increased immunological indicators (IgM, IgG, IgA, lysozyme and complement 3), and lowered cortisol level compared with the control group. Digestive enzymes (protease, lipase and amylase) activities of rabbits positively affected by MOLEx levels (3 and 4 g/kg feed). In conclusion, the inclusion of MOLEx in rabbits feed can be effective in improving productive performance, kidney and liver functions, digestive enzymes, antioxidant biomarkers, immunological indicators, cecal microbiota and carcass traits.

Spirulina sebagai Pengganti Artemia untuk Meningkatkan Produksi Larva Ikan Patin Pangasius hypophthalmus

The provision of quality and sufficient shrimp fry for production is largely determined by the management of maintenance during larval stadia. This study aims to obtain the best treatment of natural feed in the form of Spirulina sp. and Artemia sp. as feed supplements to improve the growth performance and survival rate of patin fish larvae. The experiment used a Complete Randomized Design consisting of 4 feed treatments and 3 replicates: A (Artemia sp. 100%), B (Artemia sp. 100% and Spirulina sp. 100%), C (Artemia sp. 50% and Spirulina sp. 50%), and D (Spirulina sp. 100%). The larvae with a density of 50 heads/L were kept in the aquarium and fed according to the treatment. The density of Spirulina sp. administered was 104–105 cells/mL maintained until day 7 according to the treatment. The parameters measured included growth performance, which included specific growth rate (SGR), survival (SR), absolute length growth (PPM), and analysis of larval digestive enzymes (amylase, lipase, and protease). The data were analyzed using one way-analysis of variance and continued with Duncan's Multiple Range Test with a 95% confidence interval using SPSS version 22.0. The results showed that the administration of Spirulina sp. produced better SGR, SR, PPM, amylase enzyme, lipase, and protease activities compared to the control (100% administration of Artemia sp.). The best treatment was given by 100% Spirulina sp. treatment. Keywords: Artemia sp., digestive enzymes, patin fish larvae, Spirulina sp.

Non-lethal biomarkers as promising tools for fish health assessment: In situ exposure to bifenthrin as a case study

Non-lethal biomonitoring should provide an innovative approach to establish bioethical protocols for the management of both aquaculture and wild fisheries resources. We aimed to assess non-lethal biomarkers in Piaractus mesopotamicus caged in a rice field during a bifenthrin (BF) application. We analyzed parameters related to the immune system, energy metabolism and oxidative stress in fish skin mucus and blood plasma. Fish exposed to BF showed a significant increase in skin mucus glucose levels and the enzymatic activities of protease, alkaline phosphatase and superoxide dismutase. Regarding plasmatic parameters, BF increased the levels of glucose, total protein and albumin, but decreased triglycerides. In addition, increased activities of lysozyme and alkaline phosphatase were found in the blood plasma of exposed fish. Our results indicated an increased energy demand, altered immune function and a mild oxidative stress response in fish exposed in situ to BF. We have shown that skin mucus and blood plasma are very promising matrices for the development of non-lethal biomarkers to assess fish health in a stressed environment.

Intrinsic Proteolytic Activities from Cancer Cells Are Sufficient to Activate Alkoxyamine Prodrugs and Induce Cell Death.

In search of better specificity and lower chances of resistance, protease-activatable alkoxyamine prodrugs to fight cancer have been proposed. These molecules are made of a peptide linked to an alkoxyamine. Proteolysis of the peptide converts the stable prodrug at 37 °C to a metastable alkoxyamine that spontaneously homolyzes into two free radicals: a stable nitroxide and a very reactive alkyl radical. The alkyl radical induces apoptosis in the surrounding cells by inducing random chemical alterations. Here, we show that varying the peptide moiety from succinyl-Ala-Ala-Pro-Val- to PyroGlu-Gly-Arg- or PyroGlu-Gly-Lys- is effective in switching the activating enzyme from elastase to urokinase. Furthermore, these prodrugs induce the death of HT-1080 cells, a cell line that secretes several active proteases in culture. This cytotoxic activity can be suppressed by protease inhibitors and does not affect cell lines devoid of active urokinase. We thus provide examples of alkoxyamine prodrugs that are efficiently activated by the limited intrinsic protease activity and that succeed in the destruction of cancer cell lines and cancer cells from tumor explants.

A Gaussia luciferase reporter assay for the evaluation of coronavirus Nsp5/3CLpro activity

Human coronaviruses (hCoVs) infect millions of people every year. Among these, MERS, SARS-CoV-1, and SARS-CoV-2 caused significant morbidity and mortality and their emergence highlights the risk of possible future coronavirus outbreaks. Therefore, broadly-active anti-coronavirus drugs are needed. Pharmacological inhibition of the hCoV protease Nsp5 (3CLpro) is clinically beneficial as shown by the wide and effective use of Paxlovid (nirmatrelvir, ritonavir). However, further treatment options are required due to the risk of drug resistance. To facilitate the assessment of coronavirus protease function and its pharmacological inhibition, we developed an assay allowing rapid and reliable quantification of Nsp5 activity under biosafety level 1 conditions. It is based on an ACE2-Gal4 transcription factor fusion protein separated by a Nsp5 recognition site. Cleavage by Nsp5 releases the Gal4 transcription factor, which then induces the expression of Gaussia luciferase. Our assay is compatible with Nsp5 proteases from all hCoVs and allows simultaneous measurement of inhibitory and cytotoxic effects of the tested compounds. Proof-of-concept measurements confirmed that nirmatrelvir, GC376 and lopinavir inhibit SARS-CoV-2 Nsp5 function. Furthermore, the assay accurately predicted the impact of Nsp5 mutations on catalytic activity and inhibitor sensitivity. Overall, the reporter assay is suitable for evaluating viral protease activity.

Immunogenicity and Neutralization Potential of Recombinant Chimeric Protein Comprising the Catalytic Region of Gp63 of Leishmania and LTB against Leishmania donovani

Aim: To study the inhibition potential of antibody against a recombinant chimera comprising of the catalytic epitope of gp63 of Leishmania donovani and B subunit of heat-labile enterotoxin (LTB) in the functional activity of L. donovani. Background: Visceral leishmaniasis, caused by the protozoan parasite Leishmania donavani, is a major health problem and causes mortality in tropical regions. Protozoan proteases play a crucial role in the pathogenesis of the disease and in establishing infection by countering the host's innate immune responses, namely complement-mediated lysis and phagocytosis. A surface-bound metalloprotease (gp63) has been reported to be a major virulence factor resulting in the evasion of complement- mediated lysis, cleaving host extracellular and intracellular substrates, resulting in intra- phagolysosomal survival. Method: The epitope corresponding to the catalytic motif of gp63 of Leishmania donovani was fused with the B subunit of heat-labile enterotoxin, which is known to be immunogenic. The chimera was cloned to a prokaryotic expression vector and purified using Ni NTA affinity chromatography. Antibodies were generated against the purified fusion protein and analyzed for its ability to bind to the gp63 catalytic motif peptide by ELISA. The effect of fusion protein antibody on the functional activity of gp63 was evaluated by assessing the effect of purified IgGs on the protease activity and complement-mediated lysis of L. donovani promastigotes in vitro. Results: The present study reports that a recombinant chimera of the catalytic epitope of gp63 and B subunit of heat-labile enterotoxin (LTB) of E. coli, a potent adjuvant of humoral response can mount significant immune response towards the catalytic epitope. ELISA and Western blot analysis showed that the anti-fusion protein antiserum could recognize the native gp63. Also, it significantly inhibited the protease activity of promastigotes and subsequently increased complement-mediated lysis of the promastigotes in vitro. Conclusion: It could be concluded that the hybrid protein containing catalytic motif L. donovani gp63 protein and carrier protein (LTB) could elicit antibodies that could neutralise the functional activity of gp63 and thus could be a potential candidate for subunit leishmaniasis vaccine.

Comparative venom analysis between melanistic and normally coloured phenotypes of the common adder (Vipera berus).

Snake venom is an ecologically relevant functional trait directly linked with a snake's fitness and survival, facilitating predation and defence. Snake venom variation occurs at all taxonomic levels, but the study at the intraspecific level is still in its early stages. The common adder (Vipera berus) exhibits considerable variation in colour phenotypes across its distribution range. Melanistic (fully black) individuals are the subject of myths and fairytales, and in German folklore such 'hell adders' are considered more toxic than their normally coloured conspecifics despite any formal investigation. Here, we provide the first comparative analysis of venoms from melanistic and normally coloured common adders. Specifically, we compared the venom profiles by sodium dodecylsulfate polyacrylamide gel electrophoresis and reversed-phase high-performance liquid chromatography and tested the venoms' protease, phospholipase A2 and cytotoxic activities. Phospholipase A2 activity was similar in both phenotypes, whereas general protease activity was higher in the melanistic venom, which was also more cytotoxic at two concentrations (6.25 and 12.5 µg ml-1). These minor differences between the venoms of melanistic and normally coloured adders are unlikely to be of clinical relevance in the context of human envenomation. In light of our results, the claim that melanistic adders produce more toxic venom than their normally coloured conspecifics appears rooted entirely in folklore.

Discovery of β-nitrostyrene derivatives as potential quorum sensing inhibitors for biofilm inhibition and antivirulence factor therapeutics against Serratia marcescens.

Quorum sensing (QS) inhibition has emerged as a promising target for directed drug design, providing an appealing strategy for developing antimicrobials, particularly against infections caused by drug-resistant pathogens. In this study, we designed and synthesized a total of 33 β-nitrostyrene derivatives using 1-nitro-2-phenylethane (NPe) as the lead compound, to target the facultative anaerobic bacterial pathogen Serratia marcescens. The QS-inhibitory effects of these compounds were evaluated using S. marcescens NJ01 and the reporter strain Chromobacterium violaceum CV026. Among the 33 new β-nitrostyrene derivatives, (E)-1-methyl-4-(2-nitrovinyl)benzene (m-NPe, compound 28) was proven to be a potent inhibitor that reduced biofilm formation of S. marcescens NJ01 by 79%. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) results revealed that treatment with m-NPe (50 μg/ml) not only enhanced the susceptibility of the formed biofilms but also disrupted the architecture of biofilms by 84%. m-NPe (50 μg/ml) decreased virulence factors in S. marcescens NJ01, reducing the activity of protease, prodigiosin, and extracellular polysaccharide (EPS) by 36%, 72%, and 52%, respectively. In S. marcescens 4547, the activities of hemolysin and EPS were reduced by 28% and 40%, respectively, outperforming the positive control, vanillic acid (VAN). The study also found that the expression levels of QS- and biofilm-related genes (flhD, fimA, fimC, sodB, bsmB, pigA, pigC, and shlA) were downregulated by 1.21- to 2.32-fold. Molecular dynamics analysis showed that m-NPe could bind stably to SmaR, RhlI, RhlR, LasR, and CviR proteins in a 0.1 M sodium chloride solution. Importantly, a microscale thermophoresis (MST) test revealed that SmaR could be a target protein for the screening of a quorum sensing inhibitor (QSI) against S. marcescens. Overall, this study highlights the efficacy of m-NPe in suppressing the virulence factors of S. marcescens, identifying it as a new potential QSI and antibiofilm agent capable of restoring or improving antimicrobial drug sensitivity.

A homo-FRET assay for patatin-specific proteolytic activity

The potato protein patatin embeds bioactive peptides that require targeted hydrolysis to be released as promising food additives. This study presents a patatin-specific protease assay for assessing a wide range of protease activities in high-throughput format. Conjugating patatin to the amine reactive fluorogenic BODIPY FL dye provided a stable protease substrate with efficient homo-FRET quenching at a low degree (7–8) of labeling. Compared to commercial BODIPY-casein, BODIPY-patatin provided higher fluorescence enhancement (by de-quenching) at high protease concentrations, while the sensitivity was generally comparable for both highly specific (e.g. Trypsin) and industrial relevant proteases (e.g. Alcalase and Neutrase) at low doses. For Chymotrypsin, BODIPY-patatin provided a 39 % response improvement at 5 ng dose. A peptide-centric analysis of mass spectrometry-based bottom-up proteomics data identified several BODIPY-labeling sites with varying occupancies in patatin, indicating heterogenous labeling under the applied conjugation conditions. BODIPY-labeled patatin complements commercial BODIPY-labeled casein as a globular, plant-based alternative for screening of proteolytic activity.

Chronic intermittent hypoxia facilitates the development of angiotensin II-induced abdominal aortic aneurysm in male mice.

Obstructive sleep apnea (OSA), characterized by episodes of intermittent hypoxia (IH), is highly prevalent in patients with abdominal aortic aneurysm (AAA). However, whether IH serves as an independent risk factor for AAA development remains to be investigated. Here, we determined the effects of chronic (6 mo) IH on angiotensin (Ang II)-induced AAA development in C57BL/6J male mice and investigated the underlying mechanisms of IH in cultured vascular smooth muscle cells (SMCs). IH increased the susceptibility of mice to develop AAA in response to Ang II infusion by facilitating the augmentation of the abdominal aorta's diameter as assessed by transabdominal ultrasound imaging. Importantly, IH with Ang II augmented aortic elastin degradation and the expression of matrix metalloproteinases (MMPs), mainly MMP8, MMP12, and a disintegrin and metalloproteinase-17 (ADAM17) as measured by histology and immunohistochemistry. Mechanistically, IH increased the activities of MMP2, MMP8, MMP9, MMP12, and ADAM17, while reducing the expression of the MMP regulator reversion-inducing cysteine-rich protein with Kazal motifs (RECK) in cultured SMCs. Aortic samples from human AAA were associated with decreased RECK and increased expression of ADAM17 and MMPs. These data suggest that IH facilitates AAA development when additional stressors are superimposed and that this occurs in association with an increased presence of aortic MMPs and ADAM17, potentially due to IH-induced modulation of RECK expression. These findings support a plausible synergistic link between OSA and AAA and provide a better understanding of the molecular mechanisms underlying the pathogenesis of AAA.NEW & NOTEWORTHY IH facilitates Ang II-induced abdominal aortic diameter expansion and AAA development in C57BL/6J male mice. IH upregulates the expression of specific MMPs such as MMP8, MMP12, and ADAM17. IH directly suppresses RECK expression and increases MMPs activity in SMCs. Human AAA tissues exhibit a downregulation of RECK and an upregulation of ADAM17 and MMPs.

Characterization of a Halostable Metalloprotease from the Halophilic Bacterium Bacillus clausii J1G-0%B

Protein plays a crucial role as a biocatalyst in various industries, particularly in breaking down proteins into amino acids. The demand for proteases capable of functioning under extreme conditions, such as high salinity, temperature, and pH, is increasing. To address this, the exploration of bacteria that produce stable proteases in such environments is essential. Bacillus clausii J1G-0%B, a halophilic bacterium isolated from Madura salt ponds, thrives in salinity levels of 0-20% NaCl. This study aims to obtain and characterize the protease produced by Bacillus clausii J1G-0%B, focusing on its activity and stability under extreme conditions. The research involved screening, production, and purification of the protease using ammonium sulfate fractionation and dialysis. Protease activity was measured using the Kunitz method, and protein content was determined using the Lowry method. Characterization included optimizing enzymatic conditions (pH, temperature, NaCl concentration), identifying metalloprotease types, and analyzing enzyme kinetics and thermodynamics. The study successfully produced protease using a halophilic medium with casein and 5% NaCl. After 96 hours of incubation, the protease exhibited a specific activity of 654.737 U/mg. Optimal activity was observed at pH 7, 50°C, and 10% NaCl, with stability between 2.5% and 15% NaCl concentration. Enzyme kinetics revealed a high affinity for casein, with a KM value of 0.164 mg/mL and Vmax of 13.182 µmol/mL·min. Thermodynamic analysis indicated high stability, as shown by a positive ΔGi value (+105.84 kJ/mol), a low inactivation constant (ki = 0.0031 min-1), and a long half-life (t½ = 223.548 minutes). EDTA chelation tests confirmed that the protease is a metalloprotease. The halostable protease from Bacillus clausii J1G-0%B shows significant potential for industrial applications and bioremediation in high-salinity environments.

Effects of Foliar Application of Plant Growth Regulators on germination Enzyme Activities in the Wild Barely (<i>Hordeum spontanium</i>)

Wild barley, from the Gramineae family, due to primary dormancy, is one of the most important weed in wheat. We hypothesized that foliar application of plant growth regulators (PGRs) affects breaking dormancy in wild barley via germination enzyme activities and seed germination immediately on the mother plant to reduce soil seed bank. To measure the activity of enzymes alpha-amylase, protease, and invertase, two experiments were conducted based on a completely randomized design with 3 replications at the University of Kurdistan farm and Laboratory in 2016 and 2017. In the first experiment the highest and lowest alpha-amylase activity was obtained in gibberellin treatment at a concentration of 100 mgL<sup>-1</sup> and control treatment on hull seed, respectively. The activity of protease enzyme in applied treatments had a similar trend as 100 mgL<sup>-1</sup> of gibberellin treatment. The highest and the lowest protease activities were 12.62 and 3.82 Ug<sup>-1</sup> related to gibberellin treatment at a concentration of 100 mgL<sup>-1</sup> and control treatment, respectively. The second experiment was conducted to investigate the effect of time of PGRs foliar application on the parent plant on the activity of enzymes. Gibberellin 100 mg<sup>-1</sup>, salicylic acid 0.5, and 1 mM treatment on the mother plant produced the highest alpha-amylase, invertase, and protease activities when used after 50 days after pollination, there was no uniform trend in enzymes activity. In general, gibberellin treatment at a concentration of 100 mgL<sup>-1</sup> 50 days after pollination produced the highest activities of germination enzyme activities.

Effects of water temperature on growth, health and digestive processes of the thicklip grey mullet Chelon labrosus

This study aimed to identify the effects of water temperature on the rearing of the omnivorous teleost Chelon labrosus and to determine the optimal temperature for the culture of this interesting candidate for the diversification of European aquaculture. The fish (initial weight 26.81 ± 6.2 g) were held at four water temperatures (18, 22, 26 and 30 °C) in triplicate for 90 days in 100 L recirculating tanks (n = 14 / tank). The effects of said temperatures were assessed on growth, intestinal health and the digestive processes of the fish. At 18 °C, energy consumption was low, leading to a high accumulation of reserves and slow growth, a typical overwintering strategy. The best growth results were obtained at 22 °C, followed closely by 26 °C, which resulted in low energy reserves. The fact that the fish at 26 °C grew less than the ones at 22 °C while having similar energy consumption, suggested that this temperature was close to the end of the optimal range, but no signs of stress were detected. However, at 30 °C the fish had lower energy reserves but grew significantly less, which can be considered indicative of stress. Health impairment at this temperature was confirmed by the epithelial lesions found in this group. The growth and energy availability of the different groups is discussed in the light of the activities of α-amylase, pepsin and alkaline proteases. As an integration of all these results, a quadratic regression model resulting from growth values (SGR) in relation to water temperature was performed, and it allowed predicting an actual optimal water temperature of 22.8 °C for growth of C. labrosus. This is the first assessment of the optimal water temperature for the culture of C. labrosus, and it will be highly valuable for the development of its intensive culture.

Efficacy and safety of serrapeptase on ankle sprain cases: A single center prospective comparative study

BackgroundNon-steroid anti-inflammatory drugs (NSAIDs) rank among the frequently prescribed medications for addressing pain and inflammation. Although they are powerful pain relievers, their side effects are indisputable. Serrapeptase, a serine protease, exhibits anti-inflammatory and anti-oedemic activity without the side effects of NSAIDs. We aim to assess the efficacy and safety of serrapeptase in the treatment of pain and edema in ankle sprains. MethodsIn a single-centre prospective comparative study, 76 patients aged 18–53 with Grade II ankle sprains were assigned to either a serrapeptase intervention group (n = 38) receiving 5 mg serrapeptase (two tablets, three times per day) for ten days, or a control group (n = 38) receiving 500 mg paracetamol (three times per day) for the same duration. Ankle joint edema was assessed using both Figure-of-Eight and water-displacement methods. Pain was assessed with Visual Analogue Scale (V.A.S.). Within-groups and between-groups analyses were performed in the 3rd and 10th day. ResultsBoth groups exhibited reduced edema and pain over time. Serrapeptase demonstrated a superior reduction in ankle joint edema on the third and tenth day compared to paracetamol, while pain management did not differ significantly. ConclusionSerrapeptase exhibited better efficacy than paracetamol in reducing ankle joint edema, highlighting its potential as an alternative treatment. Level of evidenceLevel II, prospective comparative trial without randomization.

Potential of Coffee Cherry Pulp Extract against Polycyclic Aromatic Hydrocarbons in Air Pollution Induced Inflammation and Oxidative Stress for Topical Applications.

Airborne particulate matter (PM) contains polycyclic aromatic hydrocarbons (PAHs) as primary toxic components, causing oxidative damage and being associated with various inflammatory skin pathologies such as premature aging, atopic dermatitis, and psoriasis. Coffee cherry pulp (CCS) extract, rich in chlorogenic acid, caffeine, and theophylline, has demonstrated strong antioxidant properties. However, its specific anti-inflammatory effects and ability to protect macrophages against PAH-induced inflammation remain unexplored. Thus, this study aimed to evaluate the anti-inflammatory properties of CCS extract on RAW 264.7 macrophage cells exposed to atmospheric PAHs, compared to chlorogenic acid (CGA), caffeine (CAF), and theophylline (THP) standards. The CCS extract was assessed for its impact on the production of nitric oxide (NO) and expression of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Results showed that CCS extract exhibited significant antioxidant activities and effectively inhibited protease and lipoxygenase (LOX) activities. The PAH induced the increase in intracellular reactive oxygen species, NO, TNF-α, IL-6, iNOS, and COX-2, which were markedly suppressed by CCS extract in a dose-dependent manner, comparable to the effects of chlorogenic acid, caffeine, and theophylline. In conclusion, CCS extract inhibits PAH-induced inflammation by reducing pro-inflammatory cytokines and reactive oxygen species (ROS) production in RAW 264.7 cells. This effect is likely due to the synergistic effects of its bioactive compounds. Chlorogenic acid showed strong antioxidant and anti-inflammatory activities, while caffeine and theophylline enhanced anti-inflammatory activity. CCS extract did not irritate the hen's egg chorioallantoic membrane. Therefore, CCS extract shows its potential as a promising cosmeceutical ingredient for safely alleviating inflammatory skin diseases caused by air pollution.

The effect of peanut meal on the physicochemical properties, antioxidant activities, and flavour of soy sauce

SummaryTo solve the serious problem of homogenising commercial soy sauce, peanut meal, a high‐quality plant protein resource, was used to replace soybean meal to ferment soy sauce. In this study, we investigated the effects of different raw material ratios (w/w) and starters on the physicochemical and antioxidant activities, sensory characteristics and flavour of soy sauce. The results showed that peanut meal helped to increase the protease activity of koji, deepen the colour of soy sauce, improve the antioxidant activity of soy sauce, and enrich the aroma of soy sauce. The total phenol and total flavonoid content of PMSS (2:2) with starters were increased by 20% and 36%, respectively, compared to soybean meal soy sauce. The results of SPME‐GC–MS and recombination and omission experiments showed that the aroma of peanut meal soy sauce was more complex and more abundant in esters, pyrazines, and phenols. Ethyl 2‐methylbutanoate, Apricolin, 2‐Methyl‐1‐butanol, and Isovaleraldehyde in peanut meal had high OAV values. In addition, PMSS (2:2) with starters had the highest sensory score, with intense fruity, caramel, malty, and smoky aromas. These results show that peanut meal can improve the quality and flavour of soy sauce and provide a theoretical basis for the development of peanut soy sauce.

Clinical, Laboratory, and Molecular Aspects of Factor VII Deficiency.

Congenital factor VII (FVII) deficiency, the most frequent among the recessively inherited disorders of blood coagulation, is characterized by a wide range of symptoms, from mild mucosal bleeds to life-threatening intracranial hemorrhage. Complete FVII deficiency may cause perinatal lethality. Clinically relevant thresholds of plasma levels are still uncertain, and modest differences in low FVII levels are associated with large differences in clinical phenotypes. Activated FVII (FVIIa) expresses its physiological protease activity only in a complex with tissue factor (TF), which triggers clotting at a very low concentration. Knowledge of the FVIIa-TF complex helps to interpret the clinical findings associated with low FVII activity as compared with other rare bleeding disorders and permits effective management, including prophylaxis, with recombinant FVIIa, which, however, displays a short half-life. Newly devised substitutive and nonsubstitutive treatments, characterized by extended half-life properties, may further improve the quality of life of patients. Genetic diagnosis has been performed in thousands of patients with FVII deficiency, and among the heterogeneous F7 mutations, mostly missense changes, several recurrent variants show geographical distribution and identity by descent. In the general population, common F7 polymorphisms explain a large proportion of FVII level variance in plasma through FVII-lowering effects. Their combination with pathogenic variants may impact on the frequent detection of FVII coagulant levels lower than normal, as well as on mild bleeding conditions. In the twenties of this century, 70 years after the first report of FVII deficiency, more than 200 studies/reports about FVII/FVII deficiency have been published, with thousands of FVII-deficient patients characterized all over the world.

Agro-industrial Waste Utilization, Medium Optimization, and Immobilization of Economically Feasible Halo-Alkaline Protease Produced by Nocardiopsis dassonvillei Strain VCS-4.

The oceanic actinobacteria have strong potential to secrete novel enzymes with unique properties useful for biotechnological applications. The Nocardiopsis dassonvillei strain VCS-4, associated with seaweed Caulerpa scalpeliformis, was a halo-alkaline protease producer. Further investigation focuses on medium optimization and the use of agro-industrial waste for economically feasible, high-yield protease production. A total of 12 experimental runs were designed using Minitab-20 software and Placket-Burman design. Among the 7 physicochemical parameters analyzed, incubation time and gelatin were detected as significant factors responsible for higher protease production. Incubation time and gelatin were further analyzed using OVATs. Optimal protease production was achieved with 2% gelatin, 0.1% yeast extract, 0.1% bacteriological peptone, 7% NaCl, pH 8, 5% inoculum, and a 7-day incubation period, resulting in a maximum protease activity (Pmax) of 363.97 U/mL, generation time of 11.9h, specific growth rate of 0.161g/mL/h, and protease productivity (Qp) of 61.65 U/mL/h. Moreover, utilizing groundnut cake as an agro-industrial waste led to enhanced production parameters: Pmax of 408.42 U/mL, generation time of 9.74h, specific growth rate of 0.361g/mL/h, and Qp of 68.07 U/mL/h. The immobilization of crude protease was achieved using Seralite SRC 120 as a support matrix resulting in 470.38 U/g immobilization, 88.20% immobilization yield, and 28.90% recovery activity. Characterization of both crude and immobilized proteases revealed optimal activity at pH 10 and 70°C. Immobilization enhanced the shelf-life, reusability, and stability of VCS-4 protease under extreme conditions.

Biofilms from the Kapova Cave Walls as a Source of Hydrolase Producers

The studies of bacterial communities from extreme econiches are presently aimed mainly at analyzing the biodiversity of microorganisms using molecular biology methods. Cultivated bacteria from karst caves represent a unique group of microorganisms, the biochemical potential of which has been poorly studied. In the present work, bacteria from biofilms on the walls of the Kapova Cave (Shulgan-Tash Nature Reserve, Bashkortostan) were isolated and characterized in order to assess the ability of identified isolates to produce extracellular hydrolytic enzymes. Most of the isolates (89%) were members of the phylum Proteobacteria, with the remaining ones belonging to the phyla Actinobacteria, Firmicutes, and Bacteroidetes, which accounted for 5, 4, and 2% of the isolates, respectively. Strains with high levels of secreted protease, RNase, and amylase activity were identified as Stenotrophomonas rhizophila, Lysinibacillus fusiformis, and Pseudomonas stutzeri, respectively.