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BACKGROUND & AIMS: Heterozygous SPINK1 mutations are strong risk factors for chronic pancreatitis in humans, yet heterozygous disruption of mouse Spink1 yielded no pancreatic phenotype. To resolve this contradiction, we used CRISPR/Cas9-mediated genome editing to generate heterozygous Spink1-deleted mice (Spink1-KOhet) in the C57BL/6N strain and studied the effect of this allele in trypsin-independent and trypsin-dependent pancreatitis models. METHODS: We investigated severity of acute pancreatitis and progression to chronic pancreatitis in Spink1-KOhet mice after transient (10 injections) and prolonged (2 × 8 injections) cerulein hyperstimulation. We crossed Spink1-KOhet mice with T7D23A and T7D22N,K24R mice that carry strongly autoactivating trypsinogen mutants and exhibit spontaneous chronic pancreatitis. RESULTS: Prolonged but not transient cerulein stimulation resulted in increased intrapancreatic trypsin activity and more severe acute pancreatitis in Spink1-KOhet mice relative to the C57BL/6N control strain. After the acute episode, Spink1-KOhet mice developed progressive disease with chronic pancreatitis-like features, whereas C57BL/6N mice recovered rapidly. Trypsinogen mutant mice carrying the Spink1-KOhet allele exhibited strikingly more severe chronic pancreatitis than the respective parent strains. CONCLUSIONS: Heterozygous Spink1 deficiency caused more severe acute pancreatitis after prolonged cerulein stimulation and promoted chronic pancreatitis after the cerulein-induced acute episode, and in two strains of trypsinogen mutant mice with spontaneous disease. In contrast, acute pancreatitis induced with limited cerulein hyperstimulation was unaffected by heterozygous Spink1 deletion, in agreement with recent observations that trypsin activity does not mediate pathologic responses in this model. Taken together, the findings strongly support the notion that loss-of-function SPINK1 mutations in humans increase chronic pancreatitis risk in a trypsin-dependent manner.

Acute pancreatitis (AP) is a multifaceted inflammatory disorder stemming from the aberrant activation of trypsin within the pancreas. Despite the contribution of various factors to the pathogenesis of AP, such as trypsin activation, dysregulated increases in cytosolic Ca2+ levels, inflammatory cascade activation, and mitochondrial dysfunction, the precise molecular mechanisms underlying the disease are still not fully understood. Mitophagy, a cellular process that preserves mitochondrial homeostasis under stress, has emerged as a pivotal player in the context of AP. Research suggests that augmenting mitophagy can mitigate pancreatic injury by clearing away malfunctioning mitochondria. Elucidating the role of mitophagy in AP may pave the way for novel therapeutic strategies. This review article aims to synthesize the current research findings on mitophagy in AP and underscore its significance in the clinical management of the disorder. [ABSTRACT FROM AUTHOR]

The crystal structure of Michaelis complex of the bovine trypsin with N‐α‐benzoyl‐l‐arginine ethyl ester (BAEE) was determined at 2.30 Å resolution. The structure of enzyme‐substrate complex was solved in space group H32. The active site of trypsin was found to be occupied with the N‐α‐benzoyl‐l‐arginine ethyl ester. The hydrolyzed product of substrate molecules was also crystallized with trypsin. The substrate was embedded within the S1 binding site of the enzyme, and showed contacts with Gly‐195, Ser‐216, and Ser‐192. Some water molecules were also found within the vicinity of catalytic residues of enzyme. Interestingly, the hydrolyzed product present within the crystal lattice was found to be with inverted configuration. The crystal structure of trypsin in‐complex with N‐α‐benzoyl‐l‐arginine ethyl ester has never been reported previously. [ABSTRACT FROM AUTHOR]

Renal cell carcinoma (RCC) is a common malig- nancy of the urinary system. Although traditional therapies, such as surgery assisted with chemotherapy have improved the quality of life and survival time of patients with RCC, patients with metastasis or recurrence benefit little from such therapies. At present, little is known about the underlying mechanisms of RCC, rendering treatment selection and implementation challenging. Therefore, investigating the cause and underlying mechanisms of RCC remain of importance to explore poten- tial new avenues for its treatment. Inter-α-trypsin inhibitor heavy chain 1 (ITIH1) is an inflammation-associated gene reported to suppress the progression of liver cancer. However, its role in RCC remains poorly understood. Therefore, the present study aimed to investigate the role and mechanism of ITIH1 in RCC. Based on data obtained from The Cancer Genome Atlas database, ITIH1 expression was demonstrated to be significantly higher in tumor tissues compared with normal tissues, which was in turn negatively associated with the survival of patients with RCC. However, in RCC cells, ITIH1 was shown to be expressed at significantly lower levels compared with those in HK-2 cells. The discrepancy between tissues and cell lines might be due to the different environment of cell growth. ITIH1 knockdown in RCC cells significantly increased cell proliferation and invasion whilst significantly decreasing the apoptosis rate, compared with those in control cells (without ITIH1 knockdown). By contrast, overexpression of ITIH1 significantly inhibited cell proliferation and inva- sion in RCC cells. In terms of western blotting results, the phosphorylation levels of NF-κB were significantly increased following ITIH1 knockdown. The protein expression level of IκB significantly decreased whereas that of IKK, Cyclin D1, proliferating cell nuclear antigen and α-smooth muscle actin were significantly increased in ITIH1-knockdown cells, compared with those in the control cells (without ITIH1 knockdown). This suggests that the NF-κB pathway may be activated after ITIH1 knockdown. Following treatment with the NF-κB pathway inhibitor JSH-23 in combination with ITIH1 knockdown, RCC cell proliferation and invasion were significantly reduced compared with those after ITIH1 knockdown alone. In summary, results from the present study suggest that ITIH1 can serve an inhibitory role in the progres- sion of RCC, which could potentially be inhibited through the NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

Plant peptidase inhibitors play crucial roles in plant defence mechanisms and physiological processes. In this study, we isolated and characterised a Kunitz trypsin inhibitor from Enterolobium gummiferum seeds named EgPI (E. gummiferum peptidase inhibitor). The purification process involved two chromatography steps using size exclusion and hydrophobic resins, resulting in high purity and yield. EgPI appeared as a single band of ~20 kDa in SDS-PAGE. Under reducing conditions, the inhibitor exhibited two polypeptide chains, with 15 and 5 kDa. Functional characterisation revealed that EgPI displayed an inhibition stoichiometry of 1:1 against trypsin, with a dissociation constant of 8.4 × 10−9 mol·L−1. The amino-terminal sequencing of EgPI revealed the homology with Kunitz inhibitors. Circular dichroism analysis provided insights into the secondary structure of EgPI, which displayed the signature typical of Kunitz inhibitors. Stability studies demonstrated that EgPI maintained the secondary structure necessary to exhibit its inhibitory activity up to 70 °C and over a pH range from 2 to 8. Microbiological screening revealed that EgPI has antibiofilm properties against pathogenic yeasts at 1.125 μmol·L−1, and EgPI reduced C. albicans biofilm formation by 82.7%. The high affinity of EgPI for trypsin suggests potential applications in various fields. Furthermore, its antibiofilm properties recommended its usefulness in agriculture and antimicrobial therapy research, highlighting the practical implications of our research. [ABSTRACT FROM AUTHOR]

Serine proteases are important environmental contributors of enterovirus biocontrol. However, the structural features of molecular interaction accounting for the susceptibility of enteroviruses to proteases remains unexplained. Here, we describe the molecular mechanisms involved in the recruitment of serine proteases to viral capsids. Among the virus types used, coxsackievirus A9 (CVA9), but not CVB5 and echovirus 11 (E11), was inactivated by Subtilisin A in a host-independent manner, while Bovine Pancreatic Trypsin (BPT) only reduced CVA9 infectivity in a host-dependent manner. Predictive interaction models of each protease with capsid protomers indicate the main targets as internal disordered protein (IDP) segments exposed either on the 5-fold vertex (DE loop VP1) or at the 5/2-fold intersection (C-terminal end VP1) of viral capsids. We further show that a functional binding protease/capsid depends on both the strength and the evolution over time of protease-VP1 complexes, and lastly on the local adaptation of proteases on surrounding viral regions. Finally, we predicted three residues on CVA9 capsid that trigger cleavage by Subtilisin A, one of which may act as a sensor residue contributing to enzyme recognition on the DE loop. Overall, this study describes an important biological mechanism involved in enteroviruses biocontrol. A predictive molecular model sheds light on serine proteases and Enterovirus capsids interaction leading to viral inactivation. [ABSTRACT FROM AUTHOR]

The current investigation focused on separating Cerastes cerastes venom to produce the first Kunitz-type peptide. Based on its anti-trypsin effect, Cerastokunin, a 7.75 kDa peptide, was purified until homogenity by three steps of chromatography. Cerastokunin was found to include 67 amino acid residues that were obtained by de novo sequencing using LC-MALDI-MSMS. Upon alignment with Kunitz-type peptides, there was a high degree of similarity. Cerastokunin's 3D structure had 12% α-helices and 21% β-strands with pI 8.48. Cerastokunin showed a potent anticoagulant effect by inhibiting the protease activity of thrombin and trypsin as well as blocking the intrinsic and extrinsic coagulation pathways. In both PT and aPPT, Cerastokunin increased the blood clotting time in a dose-dependent way. Using Lys48 and Gln192 for direct binding, Cerastokunin inhibited thrombin, Factor Xa and trypsin as shown by molecular docking. Cerastokunin exhibited a dose–response blockade of PARs-dependent pathway platelet once stimulated by thrombin. An increased concentration of Cerastokunin resulted in a larger decrease of tail thrombus in the mice-carrageenan model in an in vivo investigation when compared to the effects of antithrombotic medications. At all Cerastokunin doses up to 6 mg/kg, no in vivo toxicity was seen in challenged mice over the trial's duration. [ABSTRACT FROM AUTHOR]

Enzymes are challenging to formulate due to their inherent instability, particularly in solution. This study aims to use the design of experiment (DOE) approach to develop spray-dried encapsulated trypsin nano-powder (TrySP) with maximum activity and stability using a Büchi B-90 nano spray dryer. A 53 full factorial design with 26 randomly ordered experiments was created to study the impact of process variables on the quality attributes of TrySP. The additive concentration (%), mesh cap size (μm), inlet temperature (Tinlet) (°C), trypsin concentration (%), and additive type (Mannitol or trehalose) were selected as the independent variables. The dependent parameters were yield value, particle size, residual enzyme activity, and moisture content. Further, comparative physicochemical and kinetic characterization of TrySP was performed. TrySP had diverse qualities (yield value 60.2–96.8%, residual activity >90–50%, particle size 314–1030 nm, and moisture content 0.7–2.2%). Optimized TrySP exhibited 89% yield value and 95% residual activity in the presence of 8% (w/w) mannitol. Kcat of TypSP increased from 22.5/s to 25.6/s with improved operational and storage stabilities. The half-life (t1/2) of TrySP showed 6 folds increase. Furthermore, TrySP activation energy increased from 45,918 J/mol to 94,491 J/mol. The sustainable model presented in this study enables the development of a thermostable, encapsulated trypsin nano-powder with physical and chemical properties that are optimized for various biotechnological industrial applications. [ABSTRACT FROM AUTHOR]

Microglial cells, or brain immune cells, are highly dynamic and continuously migrate in pathophysiological conditions. Their adhesion, as a physical characteristic, plays a key role in migration. In this study, we presented a microfluidic chip combination of two assays: a microglial BV2 adhesion assay and a wound-healing migration assay. The chip could create the cell-free area (wound) under chemical stimuli with trypsin (chemical assay) and also mechanical stimuli with the PBS flow (mechanical assay). The microfluidic chip functioned as the cell adhesion assay during wounding, when the cell adhesion of microglia BV2 cells was characterized by the cell removal time under various shear stress ranges. The cell detachment pattern on the glass substrate was found under physiological conditions. After wounding, the chip operated as a migration assay; it was shown that cell migration in the cell-free area generated chemically with trypsin was highly improved compared to mechanical cell-free area creations with PBS flow and the scratch assay. Our findings indicated that the increase in inlet flow rate in the mechanical assay led to a reduced experiment time and mechanical force on the cells, which could improve cell migration. Furthermore, the study on the effect of the device geometry showed that the increased channel width had an inhibitory effect on cell migration. The bi-functional chip offers an opportunity for the development of new models for a better understanding of cellular adhesion and migration in in vitro microenvironments. [ABSTRACT FROM AUTHOR]

Copyright of China Condiment is the property of China Condiment and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Quantifying the formation and decomposition of amyloid is a crucial issue in the development of new drugs and therapies for treating amyloidosis. The current technologies for grasping amyloid formation and decomposition include fluorescence analysis using thioflavin-T, secondary structure analysis using circular dichroism, and image analysis using atomic force microscopy or transmission electron microscopy. These technologies typically require spectroscopic devices or expensive nanoscale imaging equipment and involve lengthy analysis, which limits the rapid screening of amyloid-degrading drugs. In this study, we introduce a technology for rapidly assessing amyloid decomposition using capillary flow-based paper (CFP). Amyloid solutions exhibit gel-like physical properties due to insoluble denatured polymers, resulting in a shorter flow distance on CFP compared to pure water. Experimental conditions were established to consistently control the flow distance based on a hen-egg-white lysozyme amyloid solution. It was confirmed that as amyloid is decomposed by trypsin, the flow distance increases on the CFP. Our method is highly useful for detecting changes in the gel properties of amyloid solutions within a minute, and we anticipate its use in the rapid, large-scale screening of anti-amyloid agents in the future. [ABSTRACT FROM AUTHOR]

This study investigated the effect of natural compounds from Nile tilapia (Oreochromis niloticus) skin on wound healing in IL-10 knockout mice. The healing fraction, Fraction T19, was obtained through hydrolysis with trypsin. In vitro, T19 was not cytotoxic to RAW 264.7 macrophage cells, promoting increased cell proliferation and migration. In vivo, mice (n = 30) were divided into three groups with 12 mm wounds in the dorsal region: control (distilled water), T1 (T19 at 125 μg/mL), and T2 (T19 at 250 μg/mL). Daily applications were performed, with tissue removal after nine days. The results showed that T19 increased the production of nitric oxide (NO) and hydrogen peroxide (H2O2), preventing wound contamination. There was an increase in pro-inflammatory (IL-2 and IFNγ) and anti-inflammatory (IL-4) cytokines, as well as cell proliferation markers (PCNA and KI67). Antibodies CD31, CD163, and COX-2 indicated an increase in the formation of new vessels and a reduction in inflammation. Both groups treated with T19 showed better healing results, with better effects observed at higher doses. It was concluded that T19 can effectively modulate the skin repair process and represent an alternative therapeutic for improving the quality of wound skin, especially in the clinical context. Formulations using tilapia skin are safe and effective for accelerating wound healing. [ABSTRACT FROM AUTHOR]

Serine proteases (SPs) are distributed among all living cells accounting for almost one-third of all proteases. Dysregulation of SPs during inflammation and/or infection can result in devastating consequences, such as skin and lung inflammation, neuroinflammation, arthritis, as well as metastasis of cancerous cells. Such activities are tightly regulated by various inhibitors known as serine protease inhibitors (SERPIN). The thermodynamic investigations previously revealed that L-ascorbic acid binds to trypsin more firmly than pepsin and the binding force of L-ascorbic acid is driven by hydrogen bonds and van der Waals forces. However, the physiochemical effects of such interaction on trypsin and/or pepsin have not yet been reported. Ascorbic acid, also known as vitamin C, is one of the essential nutrients and most common food supplements, fortificants, and preservatives. The aim of this study was to explore the inhibitory effects of ascorbic acid on serine proteases at various concentrations on the in-vitro digestion and/or hydrolysis of intercellular matrix of cell monolayer and human serum albumin (HSA). The inhibitory effects of ascorbic on trypsin are investigated by qualitative and quantitative analysis using SDS-PAGE imaging and NIH densitometric software. Upon the addition of ascorbic acid in both indicator systems, the detachment and/or dissociation of cell monolayer and the digestion of HSA were inhibited in the presence of EDTA-Trypsin. The inhibitory effect of ascorbic acid on the digestion of intercellular matrix and/or hydrolysis of HSA showed a dose-dependent trend until it reached the maximum extent of inhibition. At an equal concentration (2.5mg/mL) ascorbic acid and EDTA-Trypsin exhibited the most potent inhibitory effect on the in vitro digestion of protein either in the form of intercellular matrix in cell monolayer and/or HSA respectively. Overall, our results based on two indicator systems strongly indicate that ascorbic acid may function as a serine protease inhibitor (SERPIN) beyond other important functions. [ABSTRACT FROM AUTHOR]

Abrin toxin, highly dangerous with an estimated human lethal dose of 0.1–1 μg per kg body weight, has attracted much attention regarding criminal and terroristic misuse over the past decade. Therefore, developing a rapid detection method for abrin toxin is of great significance in the field of biosecurity. In this study, based on the specific dissociation method of an immobilized enzyme reactor, the trypsin immobilized reactor Fe3O4@CTS–GA–Try was prepared to replace free trypsin, and the immobilized enzyme digestion process was systematically investigated and optimized by using bovine serum albumin as the simulant of abrin. After 5 min one-step denaturation and reduction, a satisfactory peptide number and coverage were yielded with only 15 s assisted by an ultrasound probe to identify model proteins. Subsequently, abrin was rapidly digested using the established method, resulting in a stable and highly reproducible characteristic peptide number of 39, which can be analyzed by nanoelectrospray ionization coupled with high-resolution mass spectrometry. With the acquisition mode of full MS scan coupled with PRM, not only MS spectroscopy of total abrin peptides but also the corresponding MS/MS spectroscopy of specific abrin peptides can achieve the characteristic detection of abrin toxin and its different isoforms in less than 10 minutes, with high repeatability. This assay provides a universal platform and has great potential for the development of on-site detection and rapid mass spectrometric analysis techniques for macromolecular protein toxins and can further be applied to the integrated detection of chemical and biological agents. [ABSTRACT FROM AUTHOR]

Background: Alpha-1 antitrypsin deficiency (AATD) is a genetic condition resulting from mutations in the alpha-1 antitrypsin (AAT) protein, a major systemic antiproteinase, resulting in reduced/no release of AAT, disrupting the proteinase/antiproteinase balance. A sustained imbalance can cause structural changes to the lung parenchyma, leading to emphysema. Predicting and assessing human responses to potential therapeutic candidates from preclinical animal studies have been challenging. Our aims were to develop a more physiologically relevant in vitro model of the proteinase/antiproteinase balance and assess whether the data generated could better predict the efficacy of pharmacological candidates to inform decisions on clinical trials, together with expected biomarker responses. Methods: We developed an in vitro model assessing the proteinase/antiproteinase balance by the changes in the fibrinogen cleavage products of neutrophil elastase (NE) and proteinase 3 (PR3). This allowed the assessment of physiological and pharmaceutical neutrophil serine proteinase (NSP) inhibitors to determine the putative threshold at which the maximal effect is achieved. Results: AAT significantly reduced NE and PR3 activity footprints, with themaximal reduction achieved at concentrations above 10 µM. The inhibitor MPH966 alone also significantly reduced NE footprint generation in a concentration-dependent manner, leveling out above 100 nM but had no effect on the PR3 footprint. At levels of AAT consistent with AATD, MPH966 had an additive effect, reducing the NE activity footprint more than either inhibitor alone. Conclusion: Our results support an inhibitor threshold above which the activity footprint generation appears resistant to increasing dosage. Our model can support the testing of inhibitors, confirming activity biomarkers as indicators of likely pharmaceutical efficacy, the assessment of NSP activity in the pathophysiology of emphysema, and the likely function of biological or pharmacological inhibitors in disease management. [ABSTRACT FROM AUTHOR]

Objective: Retinal vascular endothelial cell (RVECs) injury is a major cause of morbidity and mortality among the patients with diabetes. RVECs dysfunction is the predominant pathological manifestation of vascular complication in diabetic retinopathy. N6-methyladenosine (m6A) serves as the most prevalent modification in eukaryotic mRNAs. However, the role of m6A RNA modification in RVECs dysfunction is still unclear. Methods: RT-qPCR analysis and western blot were conducted to detect the change of m6A RNA modification in diabetic retinopathy. CCK-8 assay, transwell experiment, wound healing assay, tube formation experiment, m6A-IP-qPCR were performed to determine the role of YTHDC1 in RVECs. Retinal trypsin digestion test and H&E staining were used to evaluate histopathological changes. Results: The levels of m6A RNA methylation were significantly up-regulated in HG-induced RVECs, which were caused by increased expression of YTHDC1. YTHDC1 regulated the viability, proliferation, migration and tube formation ability in vitro. YTHDC1 overexpression impaired RVECs function by repressing CDK6 expression, which was mediated by YTHDC1-dependent mRNA decay. Moreover, it showed sh-YTHDC1 inhibited CDK6 nuclear export. Sh-YTHDC1 promotes the mRNA degradation of CDK6 in the nucleus but does not affect the cytoplasmic CDK6 mRNA. In vivo experiments showed that overexpression of CDK6 reversed the protective effect of sh-YTHDC1 on STZ-induced retinal tissue damage. Conclusion: YTHDC1-mediated m6A methylation regulates diabetes-induced RVECs dysfunction. YTHDC1-CDK6 signaling axis could be therapeutically targeted for treating DR. [ABSTRACT FROM AUTHOR]

In recent years, a few asparaginyl endopeptidases (AEPs) from certain higher plants have been identified as efficient peptide ligases with wide applications in protein labeling and cyclic peptide synthesis. Recently, we developed a NanoLuc Binary Technology (NanoBiT)‐based peptide ligase activity assay to identify more AEP‐type peptide ligases. Herein, we screened 61 bamboo species from 16 genera using this assay and detected AEP‐type peptide ligase activity in the crude extract of all tested bamboo leaves. From a popular bamboo species, Bambusa multiplex, we identified a full‐length AEP‐type peptide ligase candidate (BmAEP1) via transcriptomic sequencing. After its zymogen was overexpressed in Escherichia coli and self‐activated in vitro, BmAEP1 displayed high peptide ligase activity, but with considerable hydrolytic activity. After site‐directed mutagenesis of its ligase activity determinants, the mutant zymogen of [G238V]BmAEP1 was normally overexpressed in E. coli, but failed to activate itself. To resolve this problem, we developed a novel protease‐assisted activation approach in which trypsin was used to cleave the mutant zymogen and was then conveniently removed via ion‐exchange chromatography. After the noncovalently bound cap domain was dissociated from the catalytic core domain under acidic conditions, the recombinant [G238V]BmAEP1 displayed high peptide ligase activity with much lower hydrolytic activity and could efficiently catalyze inter‐molecular protein ligation and intramolecular peptide cyclization. Thus, the engineered bamboo‐derived peptide ligase represents a novel tool for protein labeling and cyclic peptide synthesis. [ABSTRACT FROM AUTHOR]

BACKGROUND: Olive and sunflower seeds are by‐products generated in large amounts by the plant oil industry. The technological and biological properties of plant‐based substrates, especially protein hydrolysates, have increased their use as functional ingredients for food matrices. The present study evaluates the physical and oxidative stabilities of 50 g kg−1 fish oil‐in‐water emulsions where protein hydrolysates from olive and sunflower seeds were incorporated at 20 g kg−1 protein as natural emulsifiers. The goal was to investigate the effect of protein source (i.e. olive and sunflower seeds), enzyme (i.e. subtilisin and trypsin) and degree of hydrolysis (5%, 8% and 11%) on the ability of the hydrolysate to stabilize the emulsion and retard lipid oxidation over a 7‐day storage period. RESULTS: The plant protein hydrolysates displayed different emulsifying and antioxidant capacities when incorporated into the fish oil‐in‐water emulsions. The hydrolysates with degrees of hydrolysis (DH) of 5%, especially those from sunflower seed meal, provided higher physical stability, regardless of the enzymatic treatment. For example, the average D [2, 3] values for the emulsions containing sunflower subtilisin hydrolysates at DH 5% only slightly increased from 1.21 ± 0.02 μm (day 0) to 2.01 ± 0.04 μm (day 7). Moreover, the emulsions stabilized with sunflower or olive seed hydrolysates at DH 5% were stable against lipid oxidation throughout the storage experiment, with no significant variation in the oxidation indices between days 0 and 4. CONCLUSION: The results of the present study support the use of sunflower seed hydrolysates at DH 5% as natural emulsifiers for fish oil‐in‐water emulsions, providing both physical and chemical stability against lipid oxidation. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: The present study was conducted to investigate the effects of dietary novel alkaline protease from Bacillus licheniformis on the growth performance, meat quality, antioxidant status and intestinal morphology of broilers. In total, 4000 broilers were randomly assigned into five groups and treated with normal control, normal control + 100 mg kg−1 protease, normal control + 200 mg kg−1 protease, normal control + 300 mg kg−1 protease and normal control + 400 mg kg−1 protease. RESULTS: Supplementing protease impacted final body weight (linear, P = 0.003; quadratic, P = 0.006) and decreased feed conversion rate (linear, P = 0.036) in broilers. Moreover, dietary protease significantly increased breast muscle rate (linear, P = 0.005; quadratic, P = 0.021) and decreased drip loss (linear, P < 0.001; quadratic, P < 0.001). In addition, dietary protease notably increased protein digestibility (linear, P = 0.001; quadratic, P = 0.006) and trypsin activity (linear, P = 0.002; quadratic, P = 0.009) in jejunum. Light microscopy revealed that the jejunum villi in the 300 mg kg−1 and 400 mg kg−1 groups exhibited greater height and a denser arrangement compared to those in the control group. The addition of protease decreased malondialdehyde content (linear, P < 0.001; quadratic, P < 0.001) and increased total antioxidant capacity (linear, P = 0.001; quadratic, P < 0.001) in pectoral muscles. CONCLUSION: The results of the present study suggest that dietary novel alkaline protease from B. licheniformis improved growth performance by affecting trypsin activity, protein digestibility, antioxidant capacity and intestinal health. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The purpose of this study was to evaluate the antibacterial efficacy of using 2.5% NaOCl, 2% chlorhexidine (CHX), Irritrol, and chitosan-coated silver nanoparticles (AgCNPs) alone or in combination with deoxyribonuclease I (DNase I) and trypsin pre-enzyme applications in dentin samples contaminated with Enterococcus faecalis (E. faecalis) by CLSM. 144 dentin blocks with confirmed E. faecalis biofilm formation were divided randomly according to the irrigation protocol (n = 12): NaOCl, CHX, Irritrol, AgCNPs, trypsin before NaOCl, CHX, Irritrol, AgCNPs, and DNase I before NaOCl, CHX, Irritrol, AgCNPs. Dentin blocks were stained with the Live/Dead BacLight Bacterial Viability Kit and viewed with CLSM after irrigation applications. The percentage of dead and viable bacteria was calculated using ImageJ software on CLSM images. At a significance level of p < 0.05, the obtained data were analyzed using one-way Anova and post-hoc Tukey tests. In comparison with NaOCl, CHX had a higher percentage of dead bacteria, both when no pre-enzyme was applied and when DNase I was applied as a pre-enzyme (p < 0.05). There was no difference in the percentage of dead bacteria between the irrigation solutions when trypsin was applied as a pre-enzyme (p > 0.05). AgCNPs showed a higher percentage of dead bacteria when trypsin was applied as a pre-enzyme compared to other irrigation solutions (p < 0.05), while the pre-enzyme application did not affect the percentage of dead bacteria in NaOCl, CHX, and Irritrol (p > 0.05). No irrigation protocol tested was able to eliminate the E. faecalis biofilm. While the application of trypsin as a pre-enzyme improved the antimicrobial effect of AgCNPs, it did not make any difference over other irrigation solutions. [ABSTRACT FROM AUTHOR]

Optical coherence elastography (OCE) demonstrated impressive abilities for diagnosing tissue types/states using differences in their biomechanics. Usually, OCE visualizes tissue deformation induced by some additional stimulus (e.g., contact compression or auxiliary elastic‐wave excitation). We propose a new variant of OCE with osmotically induced straining (OIS‐OCE) and demonstrate its application to assess various stages of proteoglycan content degradation in cartilage. The information‐bearing signatures in OIS‐OCE are the magnitude and rate of strains caused by the application of osmotically active solutions onto the sample surface. OCE examination of the induced strains does not require special tissue preparation, the osmotic stimulation is highly reproducible, and strains are observed in noncontact mode. Several minutes suffice to obtain a conclusion. These features are promising for intraoperative method usage when express assessment of tissue state is required during surgical operations. The "waterfall" images demonstrate the development of cumulative osmotic strains in control and degraded cartilage samples. [ABSTRACT FROM AUTHOR]

Copyright of Food Research & Development is the property of Food Research & Development Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Digestion is driven by digestive enzymes and digestive enzyme gene copy number can provide insights on the genomic underpinnings of dietary specialization. The Adaptive Modulation Hypothesis (AMH) proposes that digestive enzyme activity, which increases with increased gene copy number, should correlate with substrate quantity in the diet. To test the AMH and reveal some of the genetics of herbivory vs carnivory, we sequenced, assembled, and annotated the genome of Anoplarchus purpurescens, a carnivorous prickleback fish in the family Stichaeidae, and compared the gene copy number for key digestive enzymes to that of Cebidichthys violaceus, a herbivorous fish from the same family. A highly contiguous genome assembly of high quality (N50 = 10.6 Mb) was produced for A. purpurescens, using combined long-read and short-read technology, with an estimated 33,842 protein-coding genes. The digestive enzymes that we examined include pancreatic α-amylase, carboxyl ester lipase, alanyl aminopeptidase, trypsin, and chymotrypsin. Anoplarchus purpurescens had fewer copies of pancreatic α-amylase (carbohydrate digestion) than C. violaceus (1 vs. 3 copies). Moreover, A. purpurescens had one fewer copy of carboxyl ester lipase (plant lipid digestion) than C. violaceus (4 vs. 5). We observed an expansion in copy number for several protein digestion genes in A. purpurescens compared to C. violaceus, including trypsin (5 vs. 3) and total aminopeptidases (6 vs. 5). Collectively, these genomic differences coincide with measured digestive enzyme activities (phenotypes) in the two species and they support the AMH. Moreover, this genomic resource is now available to better understand fish biology and dietary specialization.

Enterotoxemia caused by Clostridium perfringens type D usually affects sheep and goats ≥ 2-wk-old. The main clinical signs and lesions of the disease are produced by the epsilon toxin (ETX) elaborated by this microorganism. However, ETX is produced in the form of a mostly inactive prototoxin that requires protease cleavage for activation. It has traditionally been believed that younger animals are not affected by type D enterotoxemia given the low trypsin activity in the intestinal content associated with the trypsin-inhibitory action of colostrum. Two Nigerian dwarf goat kids, 2- and 3-d-old, with a history of acute diarrhea followed by death, were submitted for postmortem examination and diagnostic workup. Autopsy and histopathology revealed mesocolonic edema, necrosuppurative colitis, and protein-rich pulmonary edema. Alpha toxin and ETX were detected in intestinal content, and C. perfringens type D was isolated from the colon of both animals. The isolates encoded the gene for lambda toxin, a protease that has been shown previously to activate ETX in vitro. Type D enterotoxemia has not been reported previously in neonatal kids, to our knowledge, and we suggest that lambda toxin activated the ETX.

Trypsin production from skipjack tuna (Katsuwonus pelamis) viscera is one significant way to increase the value of fish’s industrial waste. The present work reports the biochemical properties of trypsin from skipjack tuna viscera. The trypsin was fractionated using 0–60% ammonium sulfate and dialyzed. The enzyme was characterized to find the optimum temperature and pH for the substrate N-α-benzoyl-dl-arginine-p-nitroanilide. The 40–50% ammonium sulfate fractionation showed the highest activity at a specific activity of 1.66 U/mg and yield of 69.91%. Specific activity increased after dialysis to 2.17 U/mg with 4.49 times purity and yield of 39.20%. The molecular weights of the enzymes were estimated as 25, 29, and 35 kDa based on the enzyme activity separated by electrophoresis. The enzyme worked optimally at a temperature and pH of 50–60°C and 8.0, respectively. Metal ions (Ca2+, K+, Na+, Mg2+) at a concentration of 20 mM showed no influence on the activity. Enzyme activity was inhibited by Zn2+ at 20 mM, phenyl methyl sulfonyl fluoride (PMSF), benzamidine, and soybean trypsin inhibitor (SBTI), which confirmed the characteristics of a serine protease.

Simple Summary: This study focused on finding effective anticancer peptides (ACPs) from induced tumor-suppressing cells (iTSCs) to combat breast cancer metastasis to the skeletal system. This study identified P18 as the most potent ACP from a pool of candidates, derived from a protein called Arhgdia. P18 showed significant inhibitory effects on breast cancer cell viability, migration, and invasion, as well as interfering with certain cancer-promoting proteins and GTPase signaling. Importantly, P18 demonstrated enhanced effectiveness when combined with traditional chemotherapy drugs, without significantly affecting healthy cells. Moreover, it mitigated the bone loss associated with breast cancer spread to bones. This study suggests that P18, especially in its modified form (Ac-P18-NH2), could be a promising candidate for breast cancer treatment and preventing bone destruction by regulating specific cellular signaling pathways. Background: The skeletal system is a common site for metastasis from breast cancer. In our prior work, we developed induced tumor-suppressing cells (iTSCs) capable of secreting a set of tumor-suppressing proteins. In this study, we examined the possibility of identifying anticancer peptides (ACPs) from trypsin-digested protein fragments derived from iTSC proteomes. Methods: The efficacy of ACPs was examined using an MTT-based cell viability assay, a Scratch-based motility assay, an EdU-based proliferation assay, and a transwell invasion assay. To evaluate the mechanism of inhibitory action, a fluorescence resonance energy transfer (FRET)-based GTPase activity assay and a molecular docking analysis were conducted. The efficacy of ACPs was also tested using an ex vivo cancer tissue assay and a bone microenvironment assay. Results: Among the 12 ACP candidates, P18 (TDYMVGSYGPR) demonstrated the most effective anticancer activity. P18 was derived from Arhgdia, a Rho GDP dissociation inhibitor alpha, and exhibited inhibitory effects on the viability, migration, and invasion of breast cancer cells. It also hindered the GTPase activity of RhoA and Cdc42 and downregulated the expression of oncoproteins such as Snail and Src. The inhibitory impact of P18 was additive when it was combined with chemotherapeutic drugs such as Cisplatin and Taxol in both breast cancer cells and patient-derived tissues. P18 had no inhibitory effect on mesenchymal stem cells but suppressed the maturation of RANKL-stimulated osteoclasts and mitigated the bone loss associated with breast cancer. Furthermore, the P18 analog modified by N-terminal acetylation and C-terminal amidation (Ac-P18-NH2) exhibited stronger tumor-suppressor effects. Conclusions: This study introduced a unique methodology for selecting an effective ACP from the iTSC secretome. P18 holds promise for the treatment of breast cancer and the prevention of bone destruction by regulating GTPase signaling. [ABSTRACT FROM AUTHOR]

Porcine epidemic diarrhea virus (PEDV) is a single-stranded RNA virus with a capsid membrane that causes acute infectious gastrointestinal disease characterized by vomiting, diarrhea, and dehydration in swine. Piglets are more susceptible to PEDV than adults, with an infection rate reaching 90% and a fatality rate as high as 100%. Moreover, PEDV has a rapid transmission rate and broad transmission range. Consequently, PEDV has caused considerable economic losses and negatively impacted the sustainability of the pig industry. The surface spike (S) glycoprotein is the largest structural protein in PEDV virions and is closely associated with host cell fusion and virus invasion. As such, the S protein is an important target for vaccine development. In this article, we review the genetic variation, immunity, apoptosis-induction function, virulence, vaccine potential, and other aspects of the PEDV S protein. This review provides a theoretical foundation for preventing and controlling PEDV infection and serves as a valuable resource for further research and development of PEDV vaccines. [ABSTRACT FROM AUTHOR]

Background: Recombinant peptide production in Escherichia coli provides a sustainable alternative to environmentally harmful and size-limited chemical synthesis. However, in-vivo production of disulfide-bonded peptides at high yields remains challenging, due to degradation by host proteases/peptidases and the necessity of translocation into the periplasmic space for disulfide bond formation. Results: In this study, we established an expression system for efficient and soluble production of disulfide-bonded peptides in the periplasm of E. coli. We chose model peptides with varying complexity (size, structure, number of disulfide bonds), namely parathyroid hormone 1–84, somatostatin 1–28, plectasin, and bovine pancreatic trypsin inhibitor (aprotinin). All peptides were expressed without and with the N-terminal, low molecular weight CASPON™ tag (4.1 kDa), with the expression cassette being integrated into the host genome. During BioLector™ cultivations at microliter scale, we found that most of our model peptides can only be sufficiently expressed in combination with the CASPON™ tag, otherwise expression was only weak or undetectable on SDS-PAGE. Undesired degradation by host proteases/peptidases was evident even with the CASPON™ tag. Therefore, we investigated whether degradation happened before or after translocation by expressing the peptides in combination with either a co- or post-translational signal sequence. Our results suggest that degradation predominantly happened after the translocation, as degradation fragments appeared to be identical independent of the signal sequence, and expression was not enhanced with the co-translational signal sequence. Lastly, we expressed all CASPON™-tagged peptides in two industry-relevant host strains during C-limited fed-batch cultivations in bioreactors. We found that the process performance was highly dependent on the peptide-host-combination. The titers that were reached varied between 0.6–2.6 g L−1, and exceeded previously published data in E. coli. Moreover, all peptides were shown by mass spectrometry to be expressed to completion, including full formation of disulfide bonds. Conclusion: In this work, we demonstrated the potential of the CASPON™ technology as a highly efficient platform for the production of soluble peptides in the periplasm of E. coli. The titers we show here are unprecedented whenever parathyroid hormone, somatostatin, plectasin or bovine pancreatic trypsin inhibitor were produced in E. coli, thus making our proposed upstream platform favorable over previously published approaches and chemical synthesis. [ABSTRACT FROM AUTHOR]

Natural compounds are known as a resource of anti-cancer agents. Anti-cancer capacity toward human epithelial lung cancer cell lines (A549, H460) was examined with and without treatment of trypsin on the extract of Anoplophora glabripennis. IC50 values without trypsin treatment were about 21.3 and 25.0 μg/mL for H460 and A549, respectively. When the extract was treated with trypsin, the IC50 values were 16.0 and 15.6 μg/mL for H460 and A549, respectively, indicating that the trypsin treatment increased the anti-cancer capacity. Because trypsin treatment increased the capacity, the extract was treated with trypsin to isolate the peptide W-0803 which has lysin (K) in C-terminal and α-helix structures. With treatment of W-0803, the cell viability decreased dose-dependently for H460 and A549 cells. Apoptosis analysis showed that the cell death with the treatment of peptide W-0803 was mainly by apoptosis. The wound-healing assay also showed that the peptide W-0803 has an inhibitory capability on cell migration of H460 and A549 cells. All these results suggest that the peptide W-0803 is an anti-cancer agent for lung cancer cell treatment. [ABSTRACT FROM AUTHOR]

Although trypsin is a protease naturally applicable in many processes, it is still possible to increase its efficiency and forms of use. Processes that increase their resilience to different conditions in the reaction medium can expand and/or refine their range of applications. Its performance in the presence of organic solvents, such as acetonitrile (ACN), has been indicated as a promising way to increase the efficiency of digestion processes, such as in sample treatment for MALDI-MS peptide mapping. The maintenance of the activity of trypsin immobilized on glyoxyl-agarose was herein demonstrated in different temperature and ACN ranges. Compared to the soluble, the immobilized enzyme was able to remain active above 50% ACN, where the soluble trypsin showed no activity. Although low, it was still possible to detect about 7% activity at 70% ACN when immobilized form was used. With 7.5% of ACN, the soluble enzyme has already shown loss of activity. The same only occurred with immobilized trypsin at 20% or more. Although the immobilized enzyme may have lower specific activity compared to the soluble one, as confirmed by the determined kinetic parameters, its possibility of reuse was confirmed, with no loss of activity for 5 cycles at 0% and 5% ACN. Added to this is the simplicity that the reaction can be interrupted, through the removal of the enzyme from the medium. What could prevent its own protein chain from contaminating the final product. With these characteristics added, trypsin in its immobilized form presents itself as a promising biocatalyst. [ABSTRACT FROM AUTHOR]

Summary: In the present study, rice bran protein (RBP) from the PB1121 variety was hydrolysed using trypsin and subjected to non‐targeted IMS profiling to identify antioxidant and antihypertensive amino acids in the resulting RBP hydrolysates. The hydrolysate was then separated into three fractions using ultrafiltration, and each fraction was tested for antioxidant and ACE inhibitory activities. The ACE activity was assessed using the tripeptide hippuryl‐histidyl‐leucine (HHL) as a model peptide via HPLC‐DAD. The fraction with the molecular weight (>3000 Da) displayed the strongest antioxidant and ACE inhibitory activity. This fraction was further divided into five fractions using gel Sephadex G‐25, and the greatest levels of antioxidant and ACE inhibitory activity were found in fraction F3B. Fraction F3B was then fractionated using HPLC, and the fraction with ACE inhibitory activity (IC50 of 43 mg mL−1) was analysed using MALDI‐TOF‐TOF mass spectrometry to determine its exact molecular mass and amino acid sequence. The amino acid sequence FMKSK (phe‐met‐lys‐ser‐lys) with a Mw of 655.376 Da was detected, and the molecular docking investigation revealed that FMKSK suppresses ACE by forming strong hydrogen bonds with the active pockets of human ACE. These findings suggest that rice bran contains bioactive peptides with antioxidant and ACE inhibitory effects, making it a promising raw material for the production of beneficial products. [ABSTRACT FROM AUTHOR]

Objectives: Dysregulation of proteolysis underlies diseases like cancer. Protease inhibitors (PIs) regulate many biological functions and hence have potential anticancer properties. With this background, the current study aimed to identify the PI from natural sources such as plants and microbes against trypsin (a protease), which was assayed against casein, using an ultraviolet spectrophotometer-based methodology. Materials and Methods: PI extracted from a few plants and microbial samples were screened for their PI activity against trypsin. The PI from the most promising source in our study, Tinospora cordifolia (Willd.) Hook. f. and Thoms. stem, was partially purified using ammonium sulfate precipitation followed by dialysis. The PI activity of the partially purified inhibitor was analyzed against chymotrypsin and collagenase enzymes, and the cytotoxic effect of the PI was checked on HepG2 (liver carcinoma) cells by MTT-[3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide]-assay. Liquid Chromatograography Mass Spectrometry-based proteomic studies were performed on HepG2 cells to understand the signaling pathways affected by the PIs in the liver cancer cell line. Results: Among the samples tested the PIs from T. cordifolia stem extract had the highest inhibitory activity (72.0%) against trypsin along with cytotoxicity to HepG2 cells. After partial purification by 80.0% ammonium sulfate precipitation, PI had increased inhibitory activity (83.0%) against trypsin and enhanced cytotoxicity (47.0%) to HepG2 cells. Proteomic analysis of the PI-treated HepG2 cells revealed that BAG2 and FAT10 signaling pathways were affected, which may have caused the inhibition of cancer cell proliferation. Conclusion: PI from T. cordifolia stem has promising anticancer potential and hence can be used for further purification and characterization studies toward cancer drug development. [ABSTRACT FROM AUTHOR]

The emergence of antibiotic-resistant bacteria is a serious threat to public health. Antimicrobial peptides (AMP) are a powerful alternative to antibiotics due to their low propensity to induce bacterial resistance. However, cytotoxicity and short half-lives have limited their clinical translation. To overcome these problems, AMP conjugation has gained relevance in the biomaterials field. Nevertheless, few studies describe the influence of conjugation on enzymatic protection, mechanism of action and antimicrobial efficacy. This review addresses this gap by providing a detailed comparison between conjugated and soluble AMP. Additionally, commonly employed chemical reactions and factors to consider when promoting AMP conjugation are reviewed. The overall results suggested that AMP conjugated onto biomaterials are specifically protected from degradation by trypsin and/or pepsin. However, sometimes, their antimicrobial efficacy was reduced. Due to limited conformational freedom in conjugated AMP, compared to their soluble forms, they appear to act initially by creating small protuberances on bacterial membranes that may lead to the alteration of membrane potential and/or formation of holes, triggering cell death. Overall, AMP conjugation onto biomaterials is a promising strategy to fight infection, particularly associated to the use of medical devices. Nonetheless, some details need to be addressed before conjugated AMP reach clinical practice. Covalent conjugation of antimicrobial peptides (AMP) has been one of the most widely used strategies by bioengineers, in an attempt to not only protect AMP from proteolytic degradation, but also to prolong their residence time at the target tissue. However, an explanation for the mode of action of conjugated AMP is still lacking. This review extensively gathers works on AMP conjugation and puts forward a mechanism of action for AMP when conjugated onto biomaterials. The implications of AMP conjugation on antimicrobial activity, cytotoxicity and resistance to proteases are all discussed. A thorough review of commonly employed chemical reactions for this conjugation is also provided. Finally, details that need to be addressed for conjugated AMP to reach clinical practice are discussed. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: Hawm Gra Dang Ngah 59 rice (HGDN 59) is a pure line grown in the Takbai district, Narathiwat, located in the southern border province of Thailand. The previously reported HGDN 59 rice bran's potential as a component rich in nutrients-especially high-quality protein, has not been exploited. There is also insufficient research data on biological activity and tested methods of exposing and releasing bioactive hydrolysates from HGDN 59 rice bran hydrolysates (HGDN 59-RBH). Objectives: This study aimed to determine the antioxidative activity of three different pretreatments following ultrafiltration fractionation of HGDN 59-RBH. Methods: HGDN 59 rice bran was defatted and pretreated with acid, alpha amylase, and water prior to digestion with consecutive pepsin and trypsin. HGDN 59-RBH was further fractionated by ultrafiltration into three molecular weight (MW) fractions: < 3 kDa, 3-5 kDa, and > 5 kDa. The whole RBH and three MW fractions were assessed to determine protein yield, protein recovery, total phenolic content, and the following antioxidative assays: 2,2'-azino-bis (3- ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferrous ion chelating activity, and ferric ion reducing antioxidation power (FRAP). Results: The highest protein content was obtained using pretreatment with acid, yielding 17.93± 0.08 g protein/100 g hydrolysates with 23.21 g/100 g protein recovery in defatted rice bran (DRB). The > 5 kDa fraction from the acid pretreatment-assisted enzymatic extraction of HGDN 59-RBH showed the highest phenolic content and scavenging activity against ABTS radical (2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid)) (EC50 0.669 mg/mL) and DPPH radical (2,2-diphenyl-1-picrylhydrazyl) (EC50 0.065 mg/mL) (P<0.05). Conclusions: The findings reveal that subjecting HGDN 59-RBH to acid treatment before enzymatic breakdown is a successful approach to extracting peptides with potent antioxidant properties. Consequently, the resulting hydrolysates, rich in antioxidants, hold promise as valuable additions to food and nutraceutical formulations, enhancing their functional attributes. [ABSTRACT FROM AUTHOR]

The objective: To delay the digestion of carbohydrates by inhibiting the activity of carbohydrate digestive enzymes, thereby preventing and controlling the effects of diabetes has become a research hotspot across multiple fields. j8 Conglycinin is the most abundant functional protein in soy protein, but it is unknown whether the peptides produced after gastrointestinal digestion possess the activity to inhibit carbohydrate digestive enzymes. Method: Conglycinin was subjected to simulated gastrointestinal digestion, and glucosidase inhibitory peptides and amylase inhibitory peptides were selected through virtual screening and ADMET prediction, and the inhibitory effects of the peptides on " --glucosi-dase and " --amylase were tested. Results: ! --Conglycinin was virtually digested into 95 small molecular peptides by pepsin, trypsin, and chymotrypsin; eight "--glucosidase inhibitory peptides and "--amylase inhibitory peptides were selected; it was found that hydrogen bonds and electrostatic interactions play an important role in the binding of peptides with glucosidase and amylase; in vitro verification found that the tetrapeptide EASY has a strong inhibitory effect on glucosidase, with an IC0 value of 208.6 g/mL, and no obvious inhibitory effect on amylase was observed. Conclusion: The glucosidase inhibitory peptide EASY possesses the activity to inhibit carbohydrate digestive enzymes and may become a potential inhibitor for controlling diabetes. [ABSTRACT FROM AUTHOR]

During postmortem storage, fluoride in Antarctic krill can be enriched in the muscle. Trypsin, as the most important digestive enzyme in Antarctic krill with a high activity in low temperature, plays a potential role in this process. In this study, endogenous trypsin was purified and its properties were investigated. The involvement of trypsin in the generation of free fluoride from Antarctic krill cuticle was explored. Cuticle microstructure before and after hydrolysis was compared with scanning electron microscopy, and the ash samples of the hydrolyzed Antarctic krill cuticle were analyzed with X-ray diffraction, Fourier transform infrared spectroscopy, and electron dispersive spectroscopy, respectively. Mass spectrometry analysis and inhibition tests confirmed that the purified enzyme was endogenous trypsin. Results of the present study indicated that trypsin digestion caused the increases of the concentrations of both fluoride ions and free amino N simultaneously, while the protein coated on the cuticle surface was dissolved too. However, no compositional change was detected in the cuticle inorganic salts. These findings suggest that trypsin triggered free fluoride release from Antarctic krill cuticle. In addition, the kinetics of free fluoride release could be described by the equation CW = (1−0.97−0006t −0.03e0.0558t)×337.53+10.50. The present study improved the understanding of the role of trypsin in free fluoride release from Antarctic krill cuticle, facilitating future studies aimed at reducing the fluoride content in krill protein during Antarctic krill processing. [ABSTRACT FROM AUTHOR]

Cystine-knot peptides (CKPs) are naturally occurring peptides that exhibit exceptional chemical and proteolytic stability. We leveraged the CKP carboxypeptidase A1 inhibitor as a scaffold to construct phage-displayed CKP libraries and subsequently screened these collections against HTRA1, a trimeric serine protease implicated in age-related macular degeneration and osteoarthritis. The initial hits were optimized by using affinity maturation strategies to yield highly selective and potent picomolar inhibitors of HTRA1. Crystal structures, coupled with biochemical studies, reveal that the CKPs do not interact in a substrate-like manner but bind to a cryptic pocket at the S1' site region of HTRA1 and abolish catalysis by stabilizing a non-competent active site conformation. The opening and closing of this cryptic pocket is controlled by the gatekeeper residue V221, and its movement is facilitated by the absence of a constraining disulfide bond that is typically present in trypsin fold serine proteases, thereby explaining the remarkable selectivity of the CKPs. Our findings reveal an intriguing mechanism for modulating the activity of HTRA1, and highlight the utility of CKP-based phage display platforms in uncovering potent and selective inhibitors against challenging therapeutic targets. Here the authors use phage display to develop cystine-knot peptides that inhibit the trimeric serine protease HTRA1. Structural and biochemical characterisation uncovered binding of the peptides to a cryptic pocket that locked the active site in a noncompetent state. [ABSTRACT FROM AUTHOR]

Introduction: Hyperglycaemia can occur because of decreased insulin secretion due to pancreatic dysfunction or insulin resistance. The different amino acid content of black soldier fly (Hermetia illucens) larvae can protect pancreatic beta cells from oxidative stress in patients with diabetes. Protein hydrolysate is a product of protein breakdown catalysed by enzymes into smaller peptides. Insulinotropic amino acids have several pathways that induce insulin synthesis. Methods: Sample preparation of larval protein hydrolysate through dissolution, homogenisation, sonication, enzyme digestion, centrifugation, and lyophilisation. Identify amino acids using LC-MS/MS by dissolving the hydrolysate and ionising the molecules to be separated by the mass analyser. The results were interpreted using the MassLynx software. The α-glucosidase inhibitory assay was conducted for hydrolysate with trypsin incubated for 7 hours. An oral glucose tolerance test was conducted to evaluate hypoglycaemic ability. The selected protein hydrolysate and sham controls were fed orally to male Sprague–Dawley rats using oral gavage. Results: The selected larval protein hydrolysate was a trypsin catalyst incubated for 7 hours. The LC-MS/MS results showed that the chosen protein hydrolysate was based on the types of insulinotropic amino acids, including lysine, methionine, histidine, proline, and ornithine. Hydrolysate insignificantly inhibits glucose absorption in the intestine through the α-glucosidase enzyme. The selected dose was 300 mg/kg BW. Conclusion: The selected hydrolysate was hydrolysed by trypsin enzyme for 7 hours, with an optimum dose of 300 mg/kg BW, to produce the desired hypoglycaemic effect. [ABSTRACT FROM AUTHOR]

Background: White Velvet bean (Mucuna pruriens) is a very valuable plant due to its medical potential. Different parts of the plant have been studied by various researchers. However, the seeds, which contain a lot of bioactive compounds, have been considered of substantial medicinal importance. Methods: The total phenolic content, total flavonoid content, antioxidant and trypsin inhibition properties in the methanol extract of white Velvet bean (WVB) seeds and its fractions (methanol, n-hexane, ethyl acetate, n-butanol and aqueous) were studied. Result: The highest total phenolic content was recorded in the methanol extract (206.3 mg GAE g-1). The highest total flavonoid content was in the n-hexane fraction (7.75 mg rutin g-1). The methanol extract showed the highest total antioxidant potential (261.5 mg AAE g-1). The methanol extract also had the strongest DPPH radical scavenging activity and the highest level of trypsin inhibitory activity compared to the other fractions with an IC50 value (35.3 µg mL-1). [ABSTRACT FROM AUTHOR]

Peptides with antimicrobial activity or protease inhibitory activity are potential candidates to supplement traditional antibiotics or cancer chemotherapies. However, the potential of many peptides are limited by drawbacks such as cytotoxicity or susceptibility to hydrolysis. Therefore, strategies to modify the structure of promising peptides may represent an effective approach for developing more promising clinical candidates. In this study, the mature peptide OSTI−1949, a Kunitz-type inhibitor from Odorrana schmackeri, and four designed analogues were successfully synthesised. In contrast to the parent peptide, the analogues showed impressive multi-functionality including antimicrobial, anticancer, and trypsin inhibitory activities. In terms of safety, there were no obvious changes observed in the haemolytic activity at the highest tested concentration, and the analogue OSTI−2461 showed an increase in activity against cancer cell lines without cytotoxicity to normal cells (HaCaT). In summary, through structural modification of a natural Kunitz-type peptide, the biological activity of analogues was improved whilst retaining low cytotoxicity. The strategy of helicity enhancement by forming an artificial α-helix and ß-sheet structure provides a promising way to develop original bioactive peptides for clinical therapeutics. [ABSTRACT FROM AUTHOR]

Trypsin, a novel superoxide scavenger, significantly enhances the storage quality of Hylocereus undatus (H. undatus). To elucidate the preservation mechanism of trypsin on H. undatus, a widely targeted metabolomic analysis, and transcriptomics analysis were conducted. Firstly, a total of 453 metabolites were identified, with organic acids and their derivatives constituting the largest proportion (25%). Amino acids and their metabolites, prominent among organic acids, were further analyzed. Among them, 73 metabolites were associated with amino acids, and 37 exhibited significant differences. The most enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was arginine biosynthesis (map00220), with polyamine metabolites showing the most pronounced differences, particularly spermine (FC = 1.7594). Compared with the control group, 4-hydroxy-2-oxoglutaric acid was significantly upregulated (FC = 2.117) in the process of spermine biosynthesis. Furthermore, the results of Gene Ontology (GO) and KEGG enrichment analysis of the H. undatus transcriptome profile revealed that trypsin treatment led to 187 differentially expressed genes associated with arginine. Both GO and KEGG analyses exhibited significant enrichment in the spermine biosynthetic process (GO:0006597) (map:00220) within the arginine biosynthesis pathway. Moreover, most enzymes and metabolites within the spermine biosynthesis pathway in H. undatus were upregulated. The results of the PPI network highlighted that ADC, SPDS, and SAMDC, among others, were pivotal proteins involved in trypsin-regulated arginine metabolism and spermine synthesis. This study revealed that trypsin could significantly delay postharvest senescence of H. undatus at room temperature. This effect might be attributed to trypsin triggering the synthesis of 4-hydroxy-2-oxoglutaric acid in the fruit peel, thereby promoting the biosynthesis of spermine and other polyamines. [ABSTRACT FROM AUTHOR]

A novel humic acid-based biomass-derived magnetic nanocomposite (Fe3O4-HA@[TEAC][G]), which was modified by a tetraethylammonium chloride/glycerol ([TEAC][G]) deep eutectic solvent, was synthesized. Combined with magnetic solid phase extraction technology, Fe3O4-HA@[TEAC][G] was applied to separate trypsin (Tryp) after being systematically characterized. Under optimal adsorption conditions, the adsorption capacity of Fe3O4-HA@[TEAC][G] for Tryp was higher than 1300 mg g−1. Simultaneously, the adsorption isotherm fitting test showed that the relationship between Tryp and Fe3O4-HA@[TEAC][G] matched well with the Langmuir adsorption model, which belonged to monolayer adsorption. Methodological research verified that the assay work for Tryp separation had good limits of detection (LOD) and quantification (LOQ), which were 0.033 mg mL−1 and 0.11 mg mL−1, respectively. In addition, methodological results showed that the extraction method had excellent stability, instrumental precision and repeatability. SDS-PAGE electrophoresis results proved that the Fe3O4-HA@[TEAC][G] extractant could extract Tryp from the actual crude extract of bovine pancreas. In conclusion, the humic acid-based biomass-derived magnetic nanocomposite prepared in this study has an exciting potential application value in the pretreatment of Tryp samples. [ABSTRACT FROM AUTHOR]

Amyloid fibrils of transthyretin (TTR) consist of full‐length TTR and C‐terminal fragments starting near residue 50. However, the molecular mechanism underlying the production of the C‐terminal fragment remains unclear. Here, we investigated trypsin‐induced aggregation and urea‐induced unfolding of TTR variants associated with hereditary amyloidosis. Trypsin strongly induced aggregation of variants V30G and V30A, in each of which Val30 in the hydrophobic core of the monomer was mutated to less‐bulky amino acids. Variants V30L and V30M, in each of which Val30 was mutated to bulky amino acids, also exhibited trypsin‐induced aggregation. On the other hand, pathogenic variant I68L as well as the nonpathogenic V30I did not exhibit trypsin‐induced aggregation. The V30G variant was extremely unstable compared with the other variants. The V30G mutation caused the formation of a cavity and the rearrangement of Leu55 in the hydrophobic core of the monomer. These results suggest that highly destabilized transthyretin variants are more susceptible to trypsin digestion. [ABSTRACT FROM AUTHOR]

A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15 kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617 μM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24 h of treatment at 70 °C, after 1 h treatments with different pH values, and β-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma. [ABSTRACT FROM AUTHOR]

This study sought to purify and identify antioxidant peptides from sheep (Ovis aries) plasma protein hydrolysates and assess their protective impacts on H2O2‐induced Caco‐2 cells. The purification process involved reversed high‐performance liquid chromatography, anion‐exchange chromatography, and Sephadex G‐25. Three peptides, namely Trp‐Glu‐Glu‐Pro‐Ala‐Met (WEEPAM), Ser‐Leu‐His‐Phe‐Met‐Glu (SLHFME), and His‐Cys‐Thr‐Thr‐Phe‐Met‐Ile, with molecular weights of 761.84, 762.87, and 852.03 Da, respectively, were identified by liquid chromatography with tandem mass spectrometry. Among the three antioxidant peptides, superoxide radical (O2−) radical scavenging capacity of WEEPAM and SLHFME was not significantly different from glutathione (GSH) (p > 0.05), while their 1,1‐diphenyl‐2‐picrylhydrazyl radical scavenging capacity was greater than GSH (p < 0.05). WEEPAM revealed increased antioxidant activity after pepsin and trypsin hydrolysis under an in vitro digestion model. In addition, WEEPAM inhibited oxidative damage in Caco‐2 cells by significantly reducing reactive oxygen species accumulation, early apoptosis, malondialdehyde formation, and increasing intracellular superoxide dismutase, glutathione peroxidase, and catalase activities. [ABSTRACT FROM AUTHOR]

Bating animal hides with trypsin is an essential step for making leathers. But the mechanism why the calcium in the hide influences the bating performance remains unclear. Here, the enzyme activity of trypsin was determined using N-alpha-benzoyl-L-arginine ethyl ester, bovine albumin (BSA) and collagen fibres as substrates in the presence and absence of calcium salts. The interactions of calcium with trypsin, BSA and collagen were analysed by fluorescence spectroscopy and molecular docking. Data reveal that calcium which binds to BSA and collagen improves the structural stability and enzyme resistance of hide proteins. As a result, a high calcium content of delimed hide is useful to obtain an intact surface of bated hide. However, a low calcium content of hide for pickling is necessary for producing leathers with high physical properties and uniform colour. According to these results, a novel bating system involving deliming, bating and redeliming operations was developed. This system provides a high calcium content of hide for bating and a low calcium content of hide for subsequent pickling and tanning, which effectively reduces the risk of hide damage and improves the physical properties and colour uniformity of leather. [Display omitted] • How calcium salts affect hydrolysis of hide proteins with trypsin in bating was revealed. • A high calcium content of delimed hide protects the grain surface of hide. • A low calcium content of hide is necessary for high physical properties of leather. • A novel system involving deliming, bating, and redeliming operations was designed. • This system effectively reduces the risk of hide damage and can ensure leather quality. [ABSTRACT FROM AUTHOR]

The current work aimed to study the impacts of supplementation of Bacillus subtilis (BS) and Saccharomyces cerevisiae (SC) on egg production of Japanese quail, blood biochemical parameters, and the specific activity of some digestive enzymes (trypsin, chymotrypsin, and amylase). A total of 150 female Japanese quail were randomly divided into three groups. First group (control group) had the basal diet contained the recommended requirements; where the 2nd group fed on the basal diet enriched with BS (1×108 CFU/kg feed); and the third group, the basal diet enriched with SC (3×108 CFU/kg feed). The group supplemented with BS showed a significant improvement in egg production compared to the other groups (P ≤0.05). There was a significant decreased in serum cholesterol, LDL, and increased in HDL in the BS and SC supplemented groups. In the BS and SC-enriched groups, the serum levels of urea and creatinine were significantly decreased in supplemented groups (P ≤0.05) than the control group. Serum total protein, albumin, and globulin levels were not affected. Levels of liver function markers (AST and ALT) were decreased (P ≤ 0.05) in the second group. BS improved the activity of digestive enzymes, particularly α-amylase. In conclusion, adding BS and SC to the layer quail diets has a potential effect on egg production, digestion, and general health. [ABSTRACT FROM AUTHOR]