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We explore how a protein's native structure determines its unfolding process. We examine how the local structural features, like shear, and the global structural properties, like the number of soft modes, change during unfolding. Simulations are performed using a Gaussian Network Model (GNM) with bond breaking for both thermal and force-induced unfolding scenarios. We find that unfolding starts in areas of high shear in the native structure and progressively spreads to the low shear regions. Interestingly, analysis of single domain protein families (Chymotrypsin inhibitor and Barnase) reveal that proteins with distinct unfolding pathways exhibit divergent behavior of the number of soft modes during unfolding. This suggests that the number of soft modes might be a valuable tool for understanding thermal unfolding pathways. Additionally, we found a strong link between a protein's overall structural similarity (TM-score) and its unfolding pathways, highlighting the importance of the native structure in determining how a protein unfolds. [ABSTRACT FROM AUTHOR]

Background: Serine proteases represent over 1% of all proteins in humans. This family of proteins is found on cell surfaces, subcellular organelles like lysosomes or mitochondria, within the nucleus and the protoplasm. Among them, trypsin, chymotrypsin and elastase have aroused great interest because of their numerous functions in pathophysiological processes. Altered expression of these enzymes in experimental animal models and humans has been related to various pathologies, like developmental defects, metabolic dysfunctions, cancer, peripheral vascular diseases and infectious diseases. Trypsin and chymotrypsin-like proteases activate, or less oftentimes inactivate, numerous substrates, together with growth factors, receptors, adhesion molecules, angiogenic factors and metalloproteases. Among these substrates, a number of them are key factors in cancer progression, metastasis and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease. Elastin-degrading enzyme- elastase, slowly damages elastin over the lifetime of an organism. The physiological processes triggered by elastase leads to the progression of different conditions such as cancer, metabolic syndrome, pulmonary emphysema, atherosclerosis, and chronic obstructive pulmonary disease. These serine proteases are currently considered to be targets for the development of new potent therapeutics. Short conclusion: The cumulative knowledge that outlined the physiological functions and pathological implications of these proteases and the proposed strategies to regulate a number of their activities and their targeting for therapeutic application and validation in selected disease states are highlighted. These should enhance our appreciation of their roles in aetiology of some diseases as well as the chemotherapeutic benefits of their inhibition or modulation. [ABSTRACT FROM AUTHOR]

The serine protease chymotrypsin protects the pancreas against pancreatitis by degrading trypsinogen, the precursor to the digestive protease trypsin. Taking advantage of previously generated mouse models with either the Ctrb1 gene (encoding chymotrypsin B1) or the Ctrl gene (encoding chymotrypsin-like protease) disrupted, here we generated the novel Ctrb1-del × Ctrl-KO strain in the C57BL/6N genetic background, which harbors a naturally inactivated Ctrc gene (encoding chymotrypsin C). The newly created mice are devoid of chymotrypsin, yet the animals develop normally, breed well, and show no spontaneous phenotype, indicating that chymotrypsin is dispensable under laboratory conditions. When given cerulein, the Ctrb1-del × Ctrl-KO strain exhibited markedly increased intrapancreatic trypsin activation and more severe acute pancreatitis, relative to wild-type C57BL/6N mice. After the acute episode, Ctrb1-del × Ctrl-KO mice spontaneously progressed to chronic pancreatitis, whereas C57BL/6N mice recovered rapidly. The cerulein-induced pancreas pathology in Ctrb1-del × Ctrl-KO mice was highly similar to that previously observed in Ctrb1-del mice; however, trypsin activation was more robust and pancreatitis severity was increased. Taken together, the results confirm and extend prior observations demonstrating that chymotrypsin safeguards the pancreas against pancreatitis by limiting pathologic trypsin activity. In mice, the CTRB1 isoform, which constitutes about 90% of the total chymotrypsin content, is responsible primarily for the anti-trypsin defenses and protection against pancreatitis; however, the minor isoform CTRL also contributes to an appreciable extent. NEW & NOTEWORTHY: Chymotrypsins defend the pancreas against the inflammatory disorder pancreatitis by degrading harmful trypsinogen. This study demonstrates that mice devoid of pancreatic chymotrypsins are phenotypically normal but become sensitized to secretagogue hyperstimulation and exhibit increased intrapancreatic trypsin activation, more severe acute pancreatitis, and rapid progression to chronic pancreatitis. The observations confirm and extend the essential role of chymotrypsins in pancreas health. [ABSTRACT FROM AUTHOR]

Background and Purpose: Chymotrypsin is a pancreatic protease secreted into the lumen of the small intestine to digest food proteins. We hypothesized that chymotrypsin activity may be found close to epithelial cells and that chymotrypsin signals to them via protease‐activated receptors (PARs). We deciphered molecular pharmacological mechanisms and gene expression regulation for chymotrypsin signalling in intestinal epithelial cells. Experimental Approach: The presence and activity of chymotrypsin were evaluated by Western blot and enzymatic activity tests in the luminal and mucosal compartments of murine and human gut samples. The ability of chymotrypsin to cleave the extracellular domain of PAR1 or PAR2 was assessed using cell lines expressing N‐terminally tagged receptors. The cleavage site of chymotrypsin on PAR1 and PAR2 was determined by HPLC–MS analysis. The chymotrypsin signalling mechanism was investigated in CMT93 intestinal epithelial cells by calcium mobilization assays and Western blot analyses of (ERK1/2) phosphorylation. The transcriptional consequences of chymotrypsin signalling were analysed on colonic organoids. Key Results: We found that chymotrypsin was present and active in the vicinity of the colonic epithelium. Molecular pharmacological studies have shown that chymotrypsin cleaves both PAR1 and PAR2 receptors. Chymotrypsin activated calcium and ERK1/2 signalling pathways through PAR2, and this pathway promoted interleukin‐10 (IL‐10) up‐regulation in colonic organoids. In contrast, chymotrypsin disarmed PAR1, preventing further activation by its canonical agonist, thrombin. Conclusion and Implications: Our results highlight the ability of chymotrypsin to signal to intestinal epithelial cells via PARs, which may have important physiological consequences in gut homeostasis. [ABSTRACT FROM AUTHOR]

Characteristics of the digestive enzymes of the fifth instar larvae of the beet moth were determined to achieve a superior understanding of the digestive physiology of the pest. After dissecting the larval digestive tube, optimum acidity for the activity of α-amylase and α-glucosidase was obtained 8–9, and 8 for β-glucosidase, respectively. The optimum temperatures were 35 °C and 35–40 °C for α-amylase activity and for α- and β-glucosidase, respectively. The zymogram displayed the existence of three α-amylase isoforms in the digestive tract of this insect. The maximum activity of general protease was determined in the digestive tract of this pest using azocasein substrate in the pH range of 10–11. Furthermore, the optimum temperature was 35 °C for general protease. The metal ions including iron, magnesium, manganese, zinc, and copper at 5 and 10mM concentrations had a significant influence on the activity of α-amylase, α- and β-glucosidase, trypsin, chymotrypsin, and elastase enzymes. Zymogram confirmed the significant inhibitory effect of PMSF and TLCK on the activity of serine proteases, especially trypsin. The results of this research have determined to some extent the digestive system physiology and the digestive enzymes activity range in S. ocellatella. Certainly, Study on the characteristics of digestive enzymes of beet moth can be the starting point for further studies on the use of enzyme inhibitors and the possibility of preparing new methods to control this key sugar beet pest. [ABSTRACT FROM AUTHOR]

Chymotrypsin C (CTRC) protects the pancreas against unwanted intrapancreatic trypsin activity through degradation of trypsinogen. Loss-of-function CTRC variants increase the risk for chronic pancreatitis (CP). The aim of the present study was to characterize novel CTRC variants found during genetic testing of CP cases at a pediatric pancreatitis center. We used next-generation sequencing to screen patients. We analyzed the functional effects of CTRC variants in HEK 293T cells and using purified enzymes. In 5 separate cases, we detected 5 novel heterozygous CTRC variants: c.407C>T (p.Thr136Ile), c.550G>A (p.Ala184Thr), c.627Cdup (p.Ser210Leufs∗?, where the naming indicates a frame shift with no stop codon), c.628T>C (p.Ser210Pro), and c.779A>G (p.Asp260Gly). Functional studies revealed that with the exception of p.Ser210Leufs∗?, the CTRC variants were secreted normally from transfected cells. Enzyme activity of purified variants p.Thr136Ile, p.Ala184Thr, and p.Asp260Gly was similar to that of wild-type CTRC, whereas variant p.Ser210Pro was inactive. The frame-shift variant p.Ser210Leufs∗? was not secreted but accumulated intracellularly, and induced endoplasmic reticulum stress, as judged by elevated mRNA levels of HSPA5 and DDIT3 , and increased mRNA splicing of XBP1. CTRC variants p.Ser210Pro and p.Ser210Leufs∗? abolish CTRC function and should be classified as pathogenic. Mechanistically, variant p.Ser210Pro directly affects the amino acid at the bottom of the substrate-binding pocket while the frame-shift variant promotes misfolding and thereby blocks enzyme secretion. Importantly, 3 of the 5 novel CTRC variants proved to be benign, indicating that functional analysis is indispensable for reliable determination of pathogenicity and the correct interpretation of genetic test results. [ABSTRACT FROM AUTHOR]

The main cost in aquaculture production is feed, so management practices and feeding strategies aim to optimize both feed intake and efficiency. This study aimed to evaluate the growth performance of beluga, Huso huso, affected by periodic changes in dietary protein sources and hence identify possible innovative feeding strategies toward more efficient use of dietary protein. This study was designed with two experimental diets, namely fish meal (diet A) and plant protein blend (diet B) (protein 40%, fat 16%), which were used in different food schedules for feeding beluga (initial weight 82.08 ± 4.1 g) during 8 weeks. The experimental treatments included: continuously feeding a fish meal diet (A); continuously feeding a plant protein diet (B); alternating between diet A in the morning and diet B in the afternoon (A am/B pm); alternating between diet B in the morning and diet A in the afternoon (B am/A pm); and alternating between diet A for one day and diet B for one day (1A/1B). At the end of the eight‐week trial, superior growth performance and feed utilization were respectively observed in fish receiving A, Bam/Apm, and Aam/Bpm diets compared with the remaining treatments. The specific activity of trypsin and chymotrypsin was significantly high in fish fed Aam/Bpm and Bam/Apm. Feeding the fish‐meal‐based diet (A) continuously, led to decreased protein and increased fat deposition in the carcass. Among the mixed feeding schedules, the highest cost‐saving was recorded with the Aam/Bpm and Bam/Apm schedules. Based on economic analysis, applying Aam/Bpm and Bam/Apm schedules led to the reduced cost of beluga production compared with the other treatments. Therefore, the optimal feeding schedules for beluga are Aam/Bpm and Bam/Apm, which should be used as a practical feeding guideline for H. huso production. [ABSTRACT FROM AUTHOR]

Serine protease inhibitors (serpins) participate in the regulation of inflammation, blood coagulation, and complement activation in humans. This research aimed to identify and characterize such inhibitors of the human liver fluke Opisthorchis viverrini. Parasite proteins that might contribute to the modulation of host physiology are of particular interest, especially as chronic opisthorchiasis increases the risk of developing biliary cancer. BLAST was used to find hypothetical serpins predicted from the parasite genome data. RNA extraction and reverse transcriptase PCR were used to isolate a serpin cDNA and to determine developmental transcript abundance. The evolutionary relation to other trematode serpins was revealed by phylogenetic analysis. Recombinant serpin was expressed in Escherichia coli and used to test the immunoreactivity of human opisthorchiasis sera and the inhibition of human serine proteases. A substantial serpin family with high sequence divergence among the members was found in the genus Opisthorchis. A serpin, different from previously analyzed trematode serpins, was cloned. The transcript was only detected in metacercariae and newly excysted juveniles. Human opisthorchiasis sera showed statistically significant reactivity to recombinant serpin. The serpin caused moderate inhibition of thrombin and low inhibition of kallikrein and chymotrypsin. This parasite serpin could be further evaluated as a diagnostic tool for early infection. Kallikrein and thrombin are involved in fibrinolysis; therefore, further research should explore the effects of the parasite serpin on this process. [ABSTRACT FROM AUTHOR]

The use of pork obtained from intensively growing hybrid animals as the main raw material in meat processing, in particular sausage production, is accompanied by undesirable variability of consumer characteristics of sausage products and a decrease in the economic indicators of enterprise performance. The main technological disadvantages of processed pork are reduced water-binding capacity and significant loss of meat fluid, which in practice is usually compensated by the increased use of food additives and non-meat ingredients. The aim of the study was to assess the possibility of targeted improvement of the technological properties of minced pork using a proteolytic enzyme of animal origin. Model samples of minced meat were developed and cured for 24 hours at a temperature of 4 °C. The following control samples were used: minced meat with the addition of 2% edible salt and minced meat with the addition of 2% edible salt and 0.2% sodium bicarbonate. Moreover, 0.0001% chymotrypsin was added to the test samples containing similar curing ingredients. During electrophoretic study, in samples with the enzyme, an increase in low-molecular fractions (20 kDa, 15 kDa and lower) was observed, which indicated the manifestation of proteolytic activity of chymotrypsin in model systems. Enzymatic treatment led to an increase in pH and water-binding capacity. Cooking loss was reduced by 3 to 6 times, compared to Control 1. After cooking, histological studies of model systems showed that the test samples subjected to enzymatic treatment were characterized by a denser arrangement of structural elements, less pronounced cellular components of muscle tissue and the presence of glutin formed as a result of protein breakdown, filling the microcapillaries. Thus, the use of an enzyme preparation provides an opportunity for targeted improvement of the technological properties of pork obtained from intensively growing hybrid animals.

Abstract Background Serine proteases represent over 1% of all proteins in humans. This family of proteins is found on cell surfaces, subcellular organelles like lysosomes or mitochondria, within the nucleus and the protoplasm. Main body of the abstract Among them, trypsin, chymotrypsin and elastase have aroused great interest because of their numerous functions in pathophysiological processes. Altered expression of these enzymes in experimental animal models and humans has been related to various pathologies, like developmental defects, metabolic dysfunctions, cancer, peripheral vascular diseases and infectious diseases. Trypsin and chymotrypsin-like proteases activate, or less oftentimes inactivate, numerous substrates, together with growth factors, receptors, adhesion molecules, angiogenic factors and metalloproteases. Among these substrates, a number of them are key factors in cancer progression, metastasis and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease. Elastin-degrading enzyme- elastase, slowly damages elastin over the lifetime of an organism. The physiological processes triggered by elastase leads to the progression of different conditions such as cancer, metabolic syndrome, pulmonary emphysema, atherosclerosis, and chronic obstructive pulmonary disease. These serine proteases are currently considered to be targets for the development of new potent therapeutics. Short conclusion The cumulative knowledge that outlined the physiological functions and pathological implications of these proteases and the proposed strategies to regulate a number of their activities and their targeting for therapeutic application and validation in selected disease states are highlighted. These should enhance our appreciation of their roles in aetiology of some diseases as well as the chemotherapeutic benefits of their inhibition or modulation.

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]

Copyright of Ege Journal of Medicine is the property of Ege University, Faculty of Medicine 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.)

The spotted seatrout (Cynoscion nebulosus)—an important commercial species—has a high potential for aquaculture in the Gulf of Mexico. To optimize its feeding during larval rearing, this study aims to evaluate the primary gastric (pepsin), intestinal (leucine aminopeptidase and alkaline phosphatase), and pancreatic (alkaline protease, trypsin, chymotrypsin, amylase, and lipase) enzyme activities from hatching to day 30. A multivariate analysis identified three digestive enzyme development stages during the spotted seatrout larval transformation. The first stage occurred between 1 (mean ± standard error (SE) = 1.73 ± 0.14 millimeter (mm) standard length (SL)) and 3 (2.14 ± 0.07 mm SL) days after hatching (DAH); a period of digestive stability showed the highest activity in amylase and bile salt-dependent lipase. The second stage (from 4 (2.53 ± 0.09 mm SL) to 20 (10.92 ± 0.51 mm SL) DAH) was a period of digestive transition, during which leucine aminopeptidase, chymotrypsin, and alkaline proteases were identified as the predominant enzymes from 4 to 5 DAH. In the third stage—a period of digestive stability—pepsin was the major enzyme that occurred between 25 (16.51 ± 0.81 mm SL) and 30 (25.91 ± 0.82 mm SL) DAH. These results indicate that the spotted seatrout larvae have a digestive system adapted to lipids and carbohydrates at the onset of feeding, with an immediate transition to protein digestion when exogenous feeding begins. Additionally, the digestive system of the spotted seatrout may be considered mature at 25 DAH. Further research is needed to elucidate the mechanisms of digestive tract development in the spotted seatrout larvae. [ABSTRACT FROM AUTHOR]

Chymotrypsin from the digestive organs of bigfin reef squid, a major commercial squid species in Thailand, was purified to 41-fold with 5.7% yield by a sequential purification process including ammonium sulfate precipitation, size exclusion, and ion exchange chromatography. According to SDS-PAGE, the molecular weight of purified chymotrypsin was 43 kDa. Native-PAGE analysis revealed a single band for this purified enzyme. The optimum pH and temperature for chymotrypsin activity of the purified enzyme were a pH of 7.0 with a temperature of 55 °C. The purified chymotrypsin remained stable throughout a wide range of pH levels (6–11) and at relatively high temperature (55 °C). It was significantly inhibited by PMSF and TPCK. The values of the kinetic constants Km and Kcat were found to be 1.33 mM and 31.46 s−1, respectively. The purified chymotrypsin has the N-terminal amino acid IVGGQEATPGEWPWQAALQV. This study provided new information about the biochemical properties of pure chymotrypsin from bigfin reef squid, which will be useful in the future investigation, aquaculture, and application of bigfin reef squid. [ABSTRACT FROM AUTHOR]

Long-lived proteins (LLPs), although less common than their short-lived counterparts, are increasingly recognized to play important roles in age-related diseases such as Alzheimers. In particular, spontaneous chemical modifications can accrue over time that serve as both indicators of and contributors to disrupted autophagy. For example, isomerization in LLPs is common and occurs in the absence of protein turnover while simultaneously interfering with the protein turnover by impeding proteolysis. In addition to the biological implications this creates, isomerization may also interfere with its own analysis. To clarify, bottom-up proteomics experiments rely on protein digestion by proteases, most commonly trypsin, but the extent to which isomerization might interfere with trypsin digestion is unknown. Here, we use a combination of liquid chromatography and mass spectrometry to examine the effect of isomerization on proteolysis by trypsin and chymotrypsin. Isomerized aspartic acid and serine residues (which represent the most common sites of isomerization in LLPs) were placed at various locations relative to the preferred protease cleavage point to evaluate the influence on digestion efficiency. Trypsin was found to be relatively tolerant of isomerization, except when present at the residue immediately C-terminal to Arg/Lys. For chymotrypsin, the influence of isomerization on digestion was less predictable, resulting in long-range interference for some isomer/peptide combinations. Given the trypsin- and chymotrypsin-like behaviors of the 20S proteasome, and to further establish the biological relevance of isomerization in LLPs, substrates with isomerized sites were also tested against proteasomal degradation. Significant disruption of 20S proteolysis was observed, suggesting that if LLPs persist long enough to isomerize, it will be difficult for the cells to digest them.

Secondary metabolites produced by primary producers have a wide range of functions as well as indirect effects outside the scope of their direct target. Research suggests that protease inhibitors produced by cyanobacteria influence grazing by herbivores and may also protect against parasites of cyanobacteria. In this study, we asked whether those same protease inhibitors produced by cyanobacteria could also influence the interactions of herbivores with their parasites. We used the Daphnia‐Metschnikowia zooplankton host‐fungal parasite system to address this question because it is well documented that cyanobacteria protease inhibitors suppress trypsin and chymotrypsin in the gut of Daphnia, and because it is known that Metschnikowia infects via the gut. We tested the hypothesis that Daphnia gut proteases are necessary for Metschnikowia spores to be released from their asci. We then also tested whether diets that decrease trypsin and chymotrypsin activity in the guts of Daphnia lead to lower levels of infection. Our results show that chymotrypsin promotes the release of the fungal spores from their asci. Moreover, a diet that strongly inhibited chymotrypsin activity in Daphnia decreased infection levels, particularly in the most susceptible Daphnia clones. Our results support the growing literature that cyanobacterial diets can be beneficial to zooplankton hosts when challenged by parasites and uncover a mechanism that contributes to the protective effect of cyanobacterial diets. Specifically, we demonstrate that host chymotrypsin enzymes promote the dehiscence of Metschnikowia spores; when cyanobacteria inhibit the activity of chymotrypsin in hosts, this most likely traps the spore inside the ascus, preventing the parasite from puncturing the gut and beginning the infection process. This study illustrates how secondary metabolites of phytoplankton can protect herbivores against their own enemies. [ABSTRACT FROM AUTHOR]

Background: Lung cancer diagnosis, staging and treatment may be enhanced by multidisciplinary participation and presentation in multidisciplinary meetings (MDM). We performed a systematic review and meta-analysis to explore literature evidence of clinical impacts of MDM exposure. Methods: A study protocol was registered (PROSPERO identifier CRD42021258069). Randomised controlled trials and observational cohort studies including adults with nonsmall cell lung cancer and who underwent MDM review, compared to no MDM, were included. MEDLINE, CENTRAL, Embase and ClinicalTrials.gov were searched on 31 May 2021. Studies were screened and extracted by two reviewers. Outcomes included time to diagnosis and treatment, histological confirmation, receipt of treatments, clinical trial participation, survival and quality of life. Risk of bias was assessed using the ROBINS-I (Risk of Bias in Non-randomised Studies – of Interventions) tool. Results: 2947 citations were identified, and 20 studies were included. MDM presentation significantly increased histological confirmation of diagnosis (OR 3.01, 95% CI 2.30–3.95; p<0.00001) and availability of clinical staging (OR 2.55, 95% CI 1.43–4.56; p=0.002). MDM presentation significantly increased likelihood of receipt of surgery (OR 2.01, 95% CI 1.29–3.12; p=0.002) and reduced the likelihood of receiving no active treatment (OR 0.32, 95% CI 0.21–0.50; p=0.01). MDM presentation was protective of both 1-year survival (OR 3.23, 95% CI 2.85–3.68; p<0.00001) and overall survival (hazard ratio 0.63, 95% CI 0.55–0.72; p<0.00001). Discussion: MDM presentation was associated with increased likelihood of histological confirmation of diagnosis, documentation of clinical staging and receipt of surgery. Overall and 1-year survival was better in those presented to an MDM, although there was some clinical heterogeneity in participants and interventions delivered. Further research is required to determine the optimal method of MDM presentation, and address barriers to presentation. [ABSTRACT FROM AUTHOR]

Chymotrypsin, a crucial enzyme in human digestion, catalyzes the breakdown of milk proteins, underscoring its significance in both health diagnostics and dairy quality assurance. Addressing the critical need for rapid, cost-effective detection methods, we introduce a groundbreaking approach utilizing far-red technology and HOMO-Förster resonance energy transfer (FRET). Our novel probe, SQ-122 PC, features a unique molecular design that includes a squaraine dye (SQ), a peptide linker, and SQ moieties synthesized through solid-phase peptide synthesis. Demonstrating a remarkable quenching efficiency of 93.75% in a tailored H2O:DMSO (7:3) solvent system, our probe exhibits absorption and emission properties within the far-red spectrum, with an unprecedented detection limit of 0.130 nM. Importantly, our method offers unparalleled selectivity towards chymotrypsin, ensuring robust and accurate enzyme detection. This pioneering work underscores the immense potential of far-red-based homo-FRET systems in enabling the sensitive and specific detection of chymotrypsin enzyme activity. By bridging the gap between cutting-edge technology and biomedical diagnostics, our findings herald a new era of enzyme sensing, promising transformative advancements in disease diagnosis and dairy quality control. [ABSTRACT FROM AUTHOR]

Antibiotic resistance (AR) is one of the most serious global health issues today. Antibiotic abuse in the medical, veterinary, and agricultural industries, including incorrect antibiotic prescribing, overuse in the livestock industry, and poor cleanliness practices in hospitals, all contribute to the emergence of AR. Antibacterial drugs are derived from bacteria or molds or are synthesized de novo. In the present study, S‐substituted derivatives of 5‐(4‐methoxyphenyl)‐1,3,4‐oxadiazole‐2‐thiol (6 a‐p) were synthesized, fully characterized, and assessed for their inhibitory potential against various Gram‐negative and positive bacterial strains. The compounds demonstrated potent inhibition activity against all selected bacterial strains P. aeroginosa (−), E. coli (−), K. pneumonae (−), S. typhi (−), S. aureus (+), and B. subtilis (+) in comparison to standard ciprofloxacin drug. Among the synthesized derivatives, compounds 6 e and 6 o exhibited high selectivity and potency against the S. typhi (−) strain. The library 6 a‐o was also evaluated for in vitro chymotrypsin enzyme inhibitory activity, and all derivatives displayed weak inhibition potential in contrast to standard chymostatin (IC50 = 8.24±0.11 μM). Studies such as this one may aid in designing and discovering potential antibacterial agents for treating various bacterial infections. [ABSTRACT FROM AUTHOR]

Simnotrelvir is a selective 3-chymotrypsin-like oral protease inhibitor with activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). On 18 January 2024, results of a double-blind, randomized, placebo-controlled trial of simnotrelvir as a treatment for mild-to moderate COVID-19-were published, indicating the drug, when given in combination with ritonavir, shortened the time to resolution of symptoms. Treatment options for most outpatients with mild-to-moderate COVID-19 are limited. The protease inhibitor nirmatrelvir in combination with ritonavir has proven effective in patients who are high risk for progression to severe COVID-19, but there are no approved therapies for standard-risk patients, who now comprise the majority of the population. Simnotrelvir appears to be effective in standard-risk patients, including those who have completed primary vaccination against COVID-19 and have received a booster dose. This manuscript examines the rationale for the development of simnotrelvir and explores how this drug may be used in the future to treat COVID-19. [ABSTRACT FROM AUTHOR]

Although the risk of complications due to postoperative pancreatic fistula (POPF) have been evaluated based on the amylase level in drained ascitic fluid, this method has much room for improvement regarding diagnostic accuracy and facility of the measurement. This study aimed to investigate the clinical value of measuring pancreatic chymotrypsin activity for rapid and accurate prediction of POPF after pancreaticoduodenectomy. In 52 consecutive patients undergoing pancreaticoduodenectomy, the chymotrypsin activity in pancreatic juice was measured by calculating the increase in fluorescence intensity during the first 5 min after activation with an enzyme-activatable fluorophore. The predictive value for clinically relevant POPF (CR-POPF) was compared between this technique and the conventional method based on the amylase level. According to receiver operating characteristic analyses, pancreatic chymotrypsin activity on postoperative day (POD) 3 measured with a multiplate reader had the highest predictive value for CR-POPF (area under the curve [AUC], 0.752; P < 0.001), yielding 77.8 % sensitivity and 68.8 % specificity. The AUC and sensitivity/specificity of the amylase level in ascitic fluid on POD 3 were 0.695 (P = 0.053) and 77.8 %/41.2 %, respectively. Multivariable analysis identified high pancreatic chymotrypsin activity on POD 3 as an independent risk factor for CR-POPF. Measurement of pancreatic chymotrypsin activity with a prototype portable fluorescence photometer could significantly predict CR-POPF (AUC, 0.731; P = 0.010). Measurement of pancreatic chymotrypsin activity enabled accurate and rapid prediction of CR-POPF after pancreaticoduodenectomy. This can help surgeons to implement appropriate drain management at the patient's bedside without delay. [ABSTRACT FROM AUTHOR]

It has been reported that patients with egg allergy can ingest baked eggs without the induction of allergy symptoms. Ovomucoid (OM) is a soluble protein that is resistant to heat; however, OM in baked eggs becomes insoluble. The aim of this study was to analyze the digestibility of OM and its properties (including solubility) to clarify the mechanism by which changes in its properties remove its capacity to induce allergic symptoms. We prepared purified OM, heated OM, non-baked OM, and baked OM and digested them using pepsin, trypsin, and chymotrypsin. We analyzed the digested samples using the Lowry method, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting. The soluble protein concentrations of purified, heated, and non-baked OM remained unchanged following digestion. The soluble protein concentration in the supernatant of the digested, baked OM was low; however, in pepsin and chymotrypsin digestion, the soluble protein concentration increased during digestion. The immunoblotting results showed that the protein bands detected in the digested supernatant of baked OM were thinner than those of purified, heated, and non-baked OM, even though the protein concentrations were the same. Furthermore, intact OM was detected in the precipitate of baked OM. In trypsin digestion, little change was observed after digestion in either sample. This study suggests that baked OM may have lower antibody binding capacity than other samples, and intact OM remains in the precipitate, which may affect allergy symptom induction. [ABSTRACT FROM AUTHOR]

Serine protease inhibitors (serpins) participate in the regulation of inflammation, blood coagulation, and complement activation in humans. This research aimed to identify and characterize such inhibitors of the human liver fluke Opisthorchis viverrini. Parasite proteins that might contribute to the modulation of host physiology are of particular interest, especially as chronic opisthorchiasis increases the risk of developing biliary cancer. BLAST was used to find hypothetical serpins predicted from the parasite genome data. RNA extraction and reverse transcriptase PCR were used to isolate a serpin cDNA and to determine developmental transcript abundance. The evolutionary relation to other trematode serpins was revealed by phylogenetic analysis. Recombinant serpin was expressed in Escherichia coli and used to test the immunoreactivity of human opisthorchiasis sera and the inhibition of human serine proteases. A substantial serpin family with high sequence divergence among the members was found in the genus Opisthorchis. A serpin, different from previously analyzed trematode serpins, was cloned. The transcript was only detected in metacercariae and newly excysted juveniles. Human opisthorchiasis sera showed statistically significant reactivity to recombinant serpin. The serpin caused moderate inhibition of thrombin and low inhibition of kallikrein and chymotrypsin. This parasite serpin could be further evaluated as a diagnostic tool for early infection. Kallikrein and thrombin are involved in fibrinolysis; therefore, further research should explore the effects of the parasite serpin on this process.

An amperometric bienzymatic biosensor was developed for the determination of aspartame in a flow injection analysis (FIA) system, consisting of two enzyme reactor columns packed with immobilized α-chymotrypsin (CHY) and alcohol oxidase (AOX) beads and a hydrogen peroxide electrode, connected in series. The CHY and AOX were separately immobilized on glutaraldehyde (GA)-activated beads through covalent bonding. The biosensor fabrication and operational conditions were optimized. The optimal fabrication conditions were: 2% GA with 120 min activation time; and 250 U/mL CHY and 100 U/mL AOX, with 180 min enzyme immobilization time. The optimal operational conditions were a flow rate of 0.5 mL/min and pH 8.0 at room temperature. The developed biosensor showed linearity over the aspartame concentration range 0.01–1.2 mM, with a detection limit of 0.005 mM. The developed biosensor was satisfactorily applied for detecting aspartame in beverage samples without any excessive pretreatments. [ABSTRACT FROM AUTHOR]

Brycon moorei is an opportunistic omnivorous species. It is not known what its nutritional requirements are at any stage of its development; this study determined the influence of diet on the zootechnical parameters and enzyme activity of these fish. In a completely randomized quintuplicate design, an ad libitum dietary protocol was applied, which included Prochilodus magdalenae larvae, Artemia salina and balanced feed with increasing inclusion levels (35 to 55% crude protein (CP)). Larvae 22 h post-hatching (HPH), with an average initial weight and length of 3.07 ± 0.69 mg and 6.069 ± 0.22 mm, respectively, were seeded at a density of 17 larvae L−1 in tanks with an effective volume of 60 L. The main zootechnical parameters, water quality and enzyme activity were analyzed with respect to the experimental diets. Significantly, the best diet was that of 45% CP when compared with the other CP levels used. Specific serum enzyme activity was found from hatching, with fluctuating values, the specific pepsin-like activity started at 111 HPH. B. moorei larvae can receive a dry diet from 124 HPH and their requirement corresponds to 48.5% CP. [ABSTRACT FROM AUTHOR]

We determined the effects of sodium propionate (SP) added to the diets of Atractosteus tropicus juveniles with respect to the growth, survival, digestive enzyme activity, and expression of genes that are associated with the immune system. Five treatments (0, 0.5, 1.0, 1.5, and 2.0%) were evaluated in triplicate on 180 fish (3.65 ± 0.12 g) distributed among 15 (70 L) tanks. The juveniles were fed five times a day with 5% feed in relation to the biomass of the organism. The treatment with 0.5% SP showed a final weight value of 25.7 ± 4.5 g, absolute weight of gain (AWG) of 21.93 ± 4.39 g, and specific growth rate (SGR) of 3.1 ± 0.26. Treatments with 1.5 and 2.0% SP showed the highest survival (91.6%). The control group (0%) showed a greater activity of lipases. There was a tendency that the highest activity of alkaline proteases and chymotrypsin occurred in the 0 and 0.5% treatments. The maximum relative expression of the genes ocln, muc2, and nod2 occurred in the 1.5% treatment. The inclusion of SP in the diet of A. tropicus juveniles could benefit the activity of some digestive enzymes as well as the expression of genes related to the function of the intestinal barrier, therefore benefitting the survival of the organisms. [ABSTRACT FROM AUTHOR]

Simple Summary: During the pupal stage, the midgut of Bombyx mori undergoes remarkable changes, condensing into the midgut lumen and, subsequently, undergoing decomposition. To comprehensively analyze this process of change, we utilized mass spectrometry to examine the midgut contents in the early and later stages of pupation. We detected a large number of enzymes involved in metabolism, proteases, inhibitors, chitin-binding proteins, chitinases, and storage proteins in the midgut contents during the pupal stage, indicating significant alterations within the pupal midgut lumen. Additionally, we have identified six proteases with potential gelatinolytic activity, which may be involved in the degradation of the midgut contents during the late stages of pupation. This research provides valuable insights into the mechanisms of tissue digestion and midgut remodeling in Bombyx mori pupae. The results serve as an important reference for comprehending the intricate processes taking place during this developmental stage in lepidopteran insects. The silkworm Bombyx mori, a lepidopteran insect, possesses an 8–10-day pupal stage, during which significant changes occur in the midgut, where it first condenses into the yellow body, and then undergoes decomposition. To gain insights into this transformation process, proteomics was performed on Bombyx mori midgut contents on day 2 and day 7 after pupation. The results revealed the identification of 771 proteins with more than one unique peptide. An analysis using AgriGO demonstrated that these proteins were predominantly associated with catalytic activity. Among the identified proteins, a considerable number were found to be involved in carbohydrate metabolism, amino acid metabolism, lipid metabolism, nucleic acid degradation, and energy support. Additionally, variations in the levels of certain proteases were observed between the midgut contents on day 2 and day 7 after pupation. An in-depth analysis of the two-dimensional electrophoresis of the midgut contents on day 7 after pupation led to the identification of twelve protein spots with potential gelatinolytic activity. Among these, six proteases were identified through mass spectrometry, including the p37k protease, vitellin-degrading protease, chymotrypsin-2, etc. These proteases may be responsible for the digestion of the yellow body during the later stages of pupal development. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The European catfish Silurus glanis is attracting growing interest as an object of fisheries and aquaculture, which is reinforced by the expansion of its natural range under climate change. Shaping the effective exploitation strategy for this valuable species requires detailed knowledge of its biology, including feeding and digestion processes, especially near the natural limits of the species range. Meanwhile, the digestion physiology of the European catfish remains poorly explored, including the activity of major digestive enzymes and the possible effect of intestinal parasites on this activity. In this regard, the activity of proteinases and α‐amylase in the intestinal mucosa of the catfish was studied. Adult catfish were collected in the Rybinsk reservoir (Upper Volga) located close to the northern limit of the species range. It was shown that all subclasses of intestinal digestive proteinases, including serine proteinases, metalloproteases and cysteine (thiol) proteinases, function in the gut mucosa of the catfish. The mucosal levels of total proteolytic activity depended on fish size, in contrast to those of trypsin, chymotrypsin and α‐amylase. The level of chymotrypsin activity was significantly higher than that of trypsin activity. It was also found that the incubation medium and extract of the cestodes Silurotaenia siluri parasitizing the catfish gut had a significant inhibitory effect on the activity of serine proteases (trypsin and chymotrypsin) operating in the intestines of the host fish. [ABSTRACT FROM AUTHOR]

Chymotrypsin from the digestive organs of bigfin reef squid, a major commercial squid species in Thailand, was purified to 41-fold with 5.7% yield by a sequential purification process including ammonium sulfate precipitation, size exclusion, and ion exchange chromatography. According to SDS-PAGE, the molecular weight of purified chymotrypsin was 43 kDa. Native-PAGE analysis revealed a single band for this purified enzyme. The optimum pH and temperature for chymotrypsin activity of the purified enzyme were a pH of 7.0 with a temperature of 55 °C. The purified chymotrypsin remained stable throughout a wide range of pH levels (6–11) and at relatively high temperature (55 °C). It was significantly inhibited by PMSF and TPCK. The values of the kinetic constants Km and Kcat were found to be 1.33 mM and 31.46 s−1, respectively. The purified chymotrypsin has the N-terminal amino acid IVGGQEATPGEWPWQAALQV. This study provided new information about the biochemical properties of pure chymotrypsin from bigfin reef squid, which will be useful in the future investigation, aquaculture, and application of bigfin reef squid.

Spodoptera exigua (Hübner), a highly destructive pest of agricultural crops, is often managed through the use of chemical pesticides. However, the use of chemical pesticides can have negative impacts on human health and the environment, making it important to find sustainable and alternative approaches to effectively manage this pest. One promising solution is the use of inhibitors that hinder the activity of proteases in the insect's digestive tract. Many digestive proteases, such as chymotrypsin, are produced as inactive zymogens with N-terminal pro-regions, which act as intramolecular chaperones and inhibitors of cognate catalytic regions. The present study aimed to utilize various bioinformatics tools to identify the physicochemical properties, secondary structures, and topology of chymotrypsin from S. exigua. Furthermore, we evaluated the potential of peptide inhibitors derived from the pro-region of zymogens that could be utilized as mimetic inhibitors for chymotrypsin in S. exigua. Homology molecular modeling was performed using SWISS-MODEL, with the predicted model validated by various programs. Molecular docking studies between five homologous species-derived protease inhibitors and the predicted model following by molecular dynamics (MD) simulation coupled with Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) calculations were also applied. The analysis of peptides/enzyme interactions revealed the antagonistic capacity of two screened inhibitors against S. exigua chymotrypsin. Protein-protein interaction networks demonstrated that chymotrypsin from S. exigua interacted with eleven other proteins in a high confidence score. Active site analysis revealed that S219, D230, and H232 serve as catalytic residues. These findings demonstrate a central role for computational methods in designing a potent inhibitor peptide that is highly specific and selective to its mature enzyme. Our study could be promising for future insecticide designs used in S. exigua control. [ABSTRACT FROM AUTHOR]

The effects of heating duration on digestibility of the muscle powder of three fish (pollock [WP], yellowtail [YT], mackerel [CM]) and the whole-body powder from mackerel (WM) were assessed in vitro using a digestive enzyme complex (EC) extracted from yellowtail pyloric caeca. This research will contribute to establishing a theoretical basis for feed processing conditions for yellowtail. Transcriptomic analysis showed that the most dominantly expressed genes in the pyloric caeca examined were those for trypsin and that their activity was well maintained in the EC. To assess heating effects, the powders were heated at 99 °C for 0, 3, 20, 72 and 240 min, followed by hydrolysis with the EC. After 3 min of heating the WP, YT and CM, the average production of essential amino acids (EAAs) decreased to 81.3%, 72.0% and 66.9%, respectively, compared to that detected in controls without heating. The production further decreased with increased heating duration from 3 to 240 min, but much of the decrease was within the first 3 min in WP, YT and CM. The decreasing rate of EAA production was higher in WM than CM, and increased with heating time. These results suggest that WP is the most suitable feed material for yellowtail of the four samples tested and that the shortening of heating time and removal of viscera are also important factors. [ABSTRACT FROM AUTHOR]

This paper is a comprehensive review of the techniques for the detection of pancreatic enzymes, which are common biochemical indicators of pancreatitis, including amylase, trypsin, chymotrypsin, elastase, and lipase. Pancreatitis is a disease with self-digestion due to the abnormal activation of digestive enzymes in the pancreas. Hospitalization is often required due to the lack of convenient therapeutic agents. The main recent results are reported in this review, especially the techniques that enable portability and Point-of-Care testing (POCT). This is because timely diagnosis at the early stage and avoiding recurrence after recovery are the keys to treatment. It is also important to reduce the rate of misdiagnosis and to avoid overtreatment. Various detection methods are discussed, with particular attention given to the implementation of chemical sensing and probe design. The new sensing technology for digestive enzymes makes it possible to perform early screening for pancreatitis in remote areas or in one's own home. [ABSTRACT FROM AUTHOR]

Background: The malaria parasite Plasmodium falciparum utilizes multiple alternative receptor-ligand interactions for the invasion of human erythrocytes. While some P. falciparum clones make use of sialic acid (SA) residues on the surface of the human glycophorin receptors to invade the erythrocyte, others use alternative receptors independent of sialic acid residues. We hypothesized that over the years, intensified malaria control interventions and declining prevalence in The Gambia have resulted in a selection of parasites with a dominant invasion pathways and ligand expression profiles. Methods: Blood samples were collected from 65 malaria-infected participants with uncomplicated malaria across 3 years (2015, 2016, and 2021). Genetic diversity was determined by genotyping the merozoite surface protein 2 (msp2) polymorphic gene of P. falciparum. Erythrocyte invasion phenotypes were determined using neuraminidase, trypsin, and chymotrypsin enzymes, known to cleave different receptors from the surface of the erythrocyte. Schizont-stage transcript levels were obtained for a panel of 6 P. falciparum invasion ligand genes (eba175, eba181, Rh2b, Rh4, Rh5, and clag2) using 48 successfully cultured isolates. Results: Though the allelic heterozygosity of msp2 repeat region decreased as expected with reduced transmission, there was an increase in infections with more than a single msp2 allelotype from 2015 to 2021. The invasion phenotypes of these isolates were mostly SA independent with a continuous increase from 2015 to 2021. Isolates from 2021 were highly inhibited by chymotrypsin treatment compared to isolates from 2015 and 2016. Higher invasion inhibition for 2021 isolates was further obtained following erythrocyte treatment with a combination of chymotrypsin and trypsin. The transcript levels of invasion ligand genes varied across years. However, levels of clag2, a rhoptry-associated protein, were higher in 2015 and 2016 isolates than in 2021 isolates, while Rh5 levels were higher in 2021 compared to other years. Conclusions: Overall, these findings suggest increasing mixed infections with an increase in the use of sialic-acid independent invasion pathways by P. falciparum clinical isolates in the Western part of Gambia. [ABSTRACT FROM AUTHOR]

We report the histopathological, immunohistochemical (IHC), and molecular findings in 3 patients with adult pancreatoblastoma, including 2 with autopsy features. The tumors were located in the tail and body of the pancreas, and the 2 autopsy examinations revealed liver and lung metastases. Histopathologically the neoplasms were composed of solid epithelial elements with nested or trabecular growth patterns, fibrous stroma, and squamoid clusters. Keratin 19 was positive mainly in squamoid corpuscles, and trypsin or chymotrypsin was positive in the acinar component. Neuroendocrine differentiation was observed in all tumors, and nuclear β-catenin expression in 2 tumors. Despite nuclear β-catenin expression, CTNNB1 mutation was found only in tumor 2. APC mutation was detected in tumor 1, and SMAD4 as well as MEN1 mutations in tumor 3. This last tumor also revealed chromosomal instability with many chromosomal losses and gains. The follow-up showed regional or distant metastases in all patients. Two patients died of disease after 3 and 26 months of follow-up and 1 patient is alive with no evidence of disease 6 years and 2 months after surgery. Adult pancreatoblastoma can display genetic heterogeneity, diverse histological appearance, and overlapping IHC findings. As a result, the differential diagnosis with other adult pancreatic tumors, such as acinar cell carcinoma, neuroendocrine neoplasm, solid pseudopapillary neoplasm, and mixed tumors may be challenging, especially when dealing with limited tumor tissue. The identification of squamoid corpuscles is essential for diagnosis. Although molecular findings might provide useful information, the integration of clinical, radiological, and histopathological findings is essential in pancreatoblastoma diagnosis. [ABSTRACT FROM AUTHOR]

The 3-Chymotrypsine-Like Protease (3CLpro) is an enzyme that has a major function in the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) life cycle. It can be utilized as a protein target in search for new medications. Herbal plants are expected to have major contributions to the prevention and treatment of the 2019 Corona Virus Disease (COVID-19), as many herbal plants might have a strong affinity for 3CLpro in the treatment of COVID-19. The purpose of this study is to examine the inhibitory activity, pytochemical components, and antioxidant of extracts from nine herbal plants extracted by the Ultrasound Assisted Extraction (UAE) method that have the potential to inhibit recombinant SARS-CoV-2 3CLpro using an in vitro method as a result, it can be used to find novel medication candidates for COVID-19 therapy. Determining antioxidant activity using DPPH and ABTS method, total polyphenol and total flavonoid content using Folin Ciocalteu and quercetin method. From nine herbal plants tested, Java Plum leaves (Syzygium cumini) had the most active inhibitory activity with an inhibitory value of 3CLpro (IC50 = 226 μg/ml) with a total polyphenol 413±1.83 mg GAE/g extract and total flavonoid as 12.091±0.037 mg QE/g extract. Measurement of antioxidant activity with the DPPH obtained an IC50 value of 3.751 ± 0.0149 μg/ml, and with an ABTS gives IC50 value 4.43±0.06 μg/ml. This suggests that Java Plum leaves could be a potential source of anti-COVID. [ABSTRACT FROM AUTHOR]

The antioxidative proteolytic fraction, MA-1, was partially purified from Mycoleptodonoides aitchisonii. MA-1 was purified to homogeneity using a two-step procedure, which resulted in an 89-fold increase in specific activity and 42.5% recovery. SDS-PAGE revealed two proteins with a molecular weight of 48 kDa. The zymography results revealed proteolytic activity based on the MA-1 band. MA-1 was found to be stable in the presence of Na+, Ca2+, Fe3+, K+, and Mg2+. MA-1 was also stable in methanol, ethanol, and acetone, and its enzyme activity increased by 15% in SDS. MA-1 was inhibited by ethylenediaminetetra-acetic acid or ethylene glycol tetraacetic acid and exerted the highest specificity for the substrate, MeO-Suc-Arg-Pro-Tyr-pNA, for chymotrypsin. Accordingly, MA-1 belongs to the family of chymotrypsin-like metalloproteins. The optimum temperature was 40 °C and stability was stable in the range of 20 to 35 °C. The optimum pH and stability were pH 5.5 and pH 4–11. MA-1 exhibited stronger fibrinolytic activity than plasmin. MA-1 hydrolyzed the Aα, Bβ, and γ chains of fibrinogen within 2 h. MA-1 exhibited an antithrombotic effect in animal models. MA-1 was devoid of hemorrhagic activity at a dose of 80,000 U/kg. Overall, our results show that M. aitchisonii produces an acid-tolerant and antioxidative chymotrypsin-like fibrinolytic enzyme, and M. aitchisonii containing MA-1 could be a beneficial functional material for the prevention of cardiovascular diseases and possible complications. [ABSTRACT FROM AUTHOR]

Chymotrypsin C (CTRC) is a digestive serine protease produced by the pancreas that regulates intrapancreatic trypsin activity and provides a defensive mechanism against chronic pancreatitis (CP). CTRC exerts its protective effect by promoting degradation of trypsinogen, the precursor to trypsin. Loss-of-function missense and microdeletion variants of CTRC are found in around 4% of CP cases and increase disease risk by approximately 3-7-fold. In addition, a commonly occurring synonymous CTRC variant c.180C>T (p.Gly60=) was reported to increase CP risk in various cohorts but a global analysis of its impact has been lacking. Here, we analyzed the frequency and effect size of variant c.180C>T in Hungarian and pan-European cohorts, and performed meta-analysis of the new and published genetic association data. When allele frequency was considered, meta-analysis revealed an overall frequency of 14.2% in patients and 8.7% in controls (allelic odds ratio (OR) 2.18, 95% confidence interval (CI) 1.72–2.75). When genotypes were examined, c.180TT homozygosity was observed in 3.9% of CP patients and in 1.2% of controls, and c.180CT heterozygosity was present in 22.9% of CP patients and in 15.5% of controls. Relative to the c.180CC genotype, the genotypic OR values were 5.29 (95% CI 2.63–10.64), and 1.94 (95% CI 1.57–2.38), respectively, indicating stronger CP risk in homozygous carriers. Finally, we obtained preliminary evidence that the variant is associated with reduced CTRC mRNA levels in the pancreas. Taken together, the results indicate that CTRC variant c.180C>T is a clinically relevant risk factor, and should be considered when genetic etiology of CP is investigated. [ABSTRACT FROM AUTHOR]

Emulsions offer the means to miniaturize and parallelize high-throughput screening but require a robust method to localize activity-based fluorescent probes in each droplet. Multiplexing probes in droplets is impractical, though highly desirable for identifying library members that possess very specific activity. Here, we present multiplexed probe immobilization on library beads for emulsion screening. During library bead preparation, we quantitated ∼106 primers per bead by fluorescence in situ hybridization, however emulsion PCR yielded only ∼103 gene copies per bead. We leveraged the unextended bead-bound primers to hybridize complementary probe-oligonucleotide heteroconjugates to the library beads. The probe-hybridized bead libraries were then used to program emulsion in vitro transcription/translation reactions and analyzed by FACS to perform multiplexed activity-based screening of trypsin and chymotrypsin mutant libraries for novel proteolytic specificity. The approach's modularity should permit a high degree of probe multiplexing and appears extensible to other enzyme classes and library types.

Digestive enzyme activities of Yellow Rasbora (Rasbora lateristriata) had been reported, but only focused on a daily feeding strategy. The effect of starvation and refeeding on the growth, body composition, and digestive enzyme activities in Yellow Rasbora has never been reported. This study aimed to know the most efficient feeding strategy for growth, body composition, and digestive enzyme activities of Yellow Rasbora. The experiment was conducted in 20 aquariums using two months old fingerling. Four different feeding strategies were tested; twice feeding a day, one-day starvation and six days refeeding, two days starvation and five days refeeding, and four days starvation and ten days refeeding. The result showed that different feeding strategies had no significant effect on all growth parameters. The result proved that Yellow Rasbora could compensate for starvation through absolute growth, with the highest value achieved in two days of starvation and five refeeding. No significant value was also observed on crude protein, and lipid contents indicate that starvation does not affect the utilization of protein and lipid reserves. This result was consistent with fish condition factors which did not differ among treatments. The feeding strategy did not significantly affect total protease, trypsin, and amylase activities, but significantly affected chymotrypsin, lipase, and alkaline phosphatase activities. All parameter indicated that two days starvation and five days refed duration showed similar values to daily feeding. Therefore, it is concluded that two days starvation and five days refeeding method is the most efficient feeding strategy for Yellow Rasbora cultivation. [ABSTRACT FROM AUTHOR]

This study evaluated the effect of replacing Glycine max with processed Lemna paucicostata on haematological parameters and enzyme activity of Oreochromis niloticus. The processing methods employed were blanching and sun-drying. Nine diets (D1-D9) were formulated using the least cost feed formulation software. Soybean meal was replaced by blanched and sun-dried L. paucicostata at 25%, 50%, 75% and 100% each. 27 nets measuring 1m² each were used in outdoor concrete ponds of 5m x 3.5m (l × b) and depth of 1.5m each. Ten fingerlings of O. niloticus were stocked per Hapa and fed three times daily at 5% body weight. The fish fed 75% blanched L. paucicostata obtained the best concentration of the packed cell volume (32.01%) in the blood while a poor concentration of 27.01% was observed in the fish fed 100% blanched L. paucicostata. The fish fed 75% blanched L. paucicostata and 50% blanched L. paucicostata gave higher total enzyme activities of 4,675.13(TU) and 4,648.06(TU), respectively. While the fish fed 100% blanched L. paucicostata gave the lowest value of 3,558.33(TU). The digestive enzyme activities were observed to be the main factors that helped the experimental fish to digest the blanched and sun-dried Lemna paucicostata efficiently. [ABSTRACT FROM AUTHOR]

Antimicrobial peptide (AMP), as a new antibiotic agent, has promising prospects in overcoming the problem of resistance. AMP production can be carried out by proteolytic enzymes. Protein from castor bean (R. communis) is toxic, so it can potentially be a source of AMP. This study aims to obtain protein hydrolysate from the castor bean (R. communis) using several extraction methods, including SDS dialysis, SDS-gel filtration, trifluoroacetic acid (TFA), and acetone precipitation. Chymotrypsin enzyme was used to hydrolyze the protein, and the peptide hydrolysate was fractionated using RP-HPLC. The peptide fraction was tested for its antibacterial activity by agar diffusion and microdilution methods, and the most active fraction was identified for its amino acid sequence by LC-HRMS. The results showed that the acetone precipitation extraction method was the best method, with a degree of hydrolysis of 83.9%. The active fractions 6 and 10 of RP-HPLC had IC50 values of 14.1 and 14.5 µg/mL for E. coli and 13.3 and 14.4 µg/mL for S. aureus, respectively. NVLRGKGMASL peptides were found in fraction 10, and GIILLSSK, NMIAKR, and LLDILTKK peptides were found in fraction 6 with an alpha helix secondary structure that can cause membrane damage. The peptides NVLRGKGMASL, GIILLSSK, NMIAKR, and LLDILTKK are thought to have potential as antibacterial compounds. [ABSTRACT FROM AUTHOR]

The experiment was conducted to study the effect of Medicago sativa on growth performance, digestive enzyme activity and gene expression of metabolic related factors of Eriocheir sinensis. The experiment was randomly divided into four groups with three replicates per group and 10 crabs per replicate. Control group was the bait, 5% group, 10% group and 15% group supplemented with 5%, 10% and 15% Medicago sativa in basic bait, respectively. The results showed that with the increase of Medicago sativa content in bait, a tendency to increase and then decrease of the survival rate, weight gain rate and specific growth rate of Eriocheir sinensis. The weight gain rate and specific growth rate of Eriocheir sinensis in 10% Medicago sativa group were significantly higher than those in control group and 5% alfalfa group (P<0.05), and the feed coefficient was significantly lower than those in other groups (P<0.05). Dietary 10% Medicago sativa significantly increased the activity of protease, amylase and lipase in hepatopancreas (P<0.05), and significantly increased the activity of protease, lipase and cellulase in stomach (P<0.05) and lipase and cellulase in intestinal tract (P<0.05). The EcR, mTOR, Chymotrypsin, Es-AMY, Es-ATGL, Es-COX, Es-ATP, Es-GPx and Es-CAT of 10% Medicago sativa group were significantly up-regulated (P<0.05). The resuls indicates that the optimum content of Medicago sativa is 10% in the bait of Eriocheir sinensis. [ABSTRACT FROM AUTHOR]

In order to improve the rearing of Coccinella septempunctata L. (Coleoptera: Coccinellidae), nutrients such as shrimp, pollen, honey, and lard were added to the basic artificial diet, and the effects of the artificial diet on biological parameters and digestive enzymes were evaluated. The results show that beetles feeding on the supplemented diet exhibited pupation, emergence, fecundity, and hatching rates that were 102.69%, 125.02%, 162.33%, and 119.90% of those supplied with the basic diet, respectively. The addition of shrimp and pollen to the basal diet improved protease, trypsin, chymotrypsin, and aminopeptidase activity in larvae and female adults. The addition of lard improved lipase activity in female adults, and the addition of honey improved invertase activity in adults of both sexes. This study provides guidance for improving the nutritional benefits of ladybug artificial diets. [ABSTRACT FROM AUTHOR]

Copyright of Mersin University School of Medicine Lokman Hekim Journal of History of Medicine & Folk Medicine is the property of Mersin University School of Medicine 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.)

Copyright of Journal of Faculty of Pharmacy of Ankara University / Ankara Üniversitesi Eczacilik Fakültesi Dergisi is the property of Ankara University 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.)

We previously reported that Lys175 in the region of the active site of chymotrypsin (Csin) could be site-selectively modified by using an N-hydroxy succinimide (NHS) ester of the peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester [NHS-Suc-Ala-Ala-PheP(OPh)2]. In this study, the Lys175-selective modification method was expanded to incorporate functional groups into Lys 175 in Csin. Two types of peptidyl phosphonate derivatives with the dansyl group (Dan) as a functional molecule, Dan-β-Ala-[Asp(NHS) or Glu(NHS)]-Ala-Ala-(R)-PheP(OPh)2 (DanD and DanE, respectively), were synthesized, and their action was evaluated when modifying Lys175 in Csin. Ion-exchange chromatography (IEC), fluorescence spectroscopy, and LC-MS/MS were used to analyze the products from the reaction of Csin with DanD or DanE. By IEC and LC-MS/MS, the results showed that DanE reacted with Csin more effectively than DanD to produce the modified Csin (DanMCsin) bearing Dan at Lys175. DanMCsin exhibited an enzymatic activity corresponding to 1/120 of Csin against Suc-Ala-Ala-Phe-pNA. In addition, an effect of Lys175 modification on the access of the proteinaceous Bowman–Birk inhibitor to the active site of DanMCsin was investigated. In conclusion, by using a peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester, we demonstrated that a functional group could be incorporated into Lys175 in Csin. [ABSTRACT FROM AUTHOR]

Simple Summary: Panax notoginseng (Burk) F. H. Chen is a traditional Chinese herbal medicine commonly used in clinical applications. This study examined the effects of the Panax notoginseng water extract (PNWE) on the immune responses and digestive enzyme activity of Litopenaeus vannamei (L. vannamei). The results showed that different concentrations of PNWE significantly increased the total haemocyte count, granular haemocytes, emi-granular haemocytes, phenoloxidase and respiratory burst activity, phagocytic ratio, and the phagocytic index of L. vannamei. In addition, PNWE also significantly increased the chymotrypsin, trypsin, and amylase activity of L. vannamei. Additionally, different concentrations of PNWE significantly reduced the Vibrio numbers in the intestine without damaging the hepatopancreas and intestine tissues. These results indicated that PNWE improves the immune responses of L. vannamei by increasing the haemocyte count and regulating intestinal digestive enzymes. Panax notoginseng (Burk) F. H. Chen is a traditional Chinese herbal medicine commonly used in clinical applications. This study examined the effects of the Panax notoginseng water extract (PNWE) on the immune responses and digestive enzyme activity of Litopenaeus vannamei (L. vannamei). The PNWE (50, 100, and 200 μg (g shrimp)−1) was injected into L. vannamei to analyze the immune response parameters, including the total haemocyte count (THC), granular haemocytes (GC), semi-granular haemocytes (SGC), hialin haemocyte (HC), the respiratory burst (RB), the phagocytic ratio (PR), the phagocytic index (PI), and phenoloxidase (PO). We evaluated the activity of the intestinal digestive enzymes (trypsin, chymotrypsin, amylase, and lipase), the histopathology, and the intestine Vibrio numbers. The results showed that different concentrations of the PNWE significantly increased THC, GC, SGC, PO and RB activity, the PR, and the PI of L. vannamei while reducing the HC. In addition, the PNWE also significantly increased the chymotrypsin, trypsin, and amylase activity of L. vannamei. Furthermore, 50 µg (g shrimp)−1 of PNWE regulated the lipase activity. Additionally, different concentrations of the PNWE significantly reduced the Vibrio numbers in the intestine without damaging the hepatopancreas and intestine tissues. These results indicate that the PNWE improves the immune responses of L. vannamei by increasing the haemocyte count and regulating intestinal digestive enzymes. [ABSTRACT FROM AUTHOR]