Advances in computational methods for ligand binding kinetics.

Binding kinetic parameters can be correlated with drug efficacy, which in recent years led to the development of various computational methods for predicting binding kinetic rates and gaining insight into protein-drug binding paths and mechanisms. In this review, we introduce and compare computational methods recently developed and applied to two systems, trypsin-benzamidine and kinase-inhibitor complexes. Methods involving enhanced sampling in molecular dynamics simulations or machine learning can be used not only to predict kinetic rates, but also to reveal factors modulating the duration of residence times, selectivity, and drug resistance to mutations. Methods which require less computational time to make predictions are highlighted, and suggestions to reduce the error of computed kinetic rates are presented.

Monitoring Both Extended and Tryptic Forms of Stable Isotope-Labeled Standard Peptides Provides an Internal Quality Control of Proteolytic Digestion in Targeted Mass Spectrometry-Based Assays.

Targeted mass spectrometry (MS)-based proteomic assays, such as multiplexed multiple reaction monitoring (MRM)-MS assays, enable sensitive and specific quantification of proteotypic peptides as stoichiometric surrogates for proteins. Efforts are underway to expand the use of MRM-MS assays in clinical environments, which requires a reliable strategy to monitor proteolytic digestion efficiency within individual samples. Towards this goal, extended stable isotope-labeled standard (SIS) peptides (hE), which incorporate native proteolytic cleavage sites, can be spiked into protein lysates prior to proteolytic (trypsin) digestion, and release of the tryptic SIS peptide (hT) can be monitored. However, hT measurements alone cannot monitor the extent of digestion and may be confounded by matrix effects specific to individual patient samples; therefore, they are not sufficient to monitor sample-to-sample digestion variability. We hypothesized that measuring undigested hE, along with its paired hT, would improve detection of digestion issues compared to only measuring hT. We tested the ratio of the SIS pair measurements, or hE/hT, as a quality control (QC) metric of trypsin digestion for two MRM assays: a direct-MRM (398 targets) and an immuno-MRM (126 targets requiring immunoaffinity peptide enrichment) assay, with extended SIS peptides observable for 54% (216) and 62% (78) of the targets, respectively. We evaluated the quantitative bias for each target in a series of experiments that adversely affected proteolytic digestion (e.g., variable digestion times, pH, and temperature). We identified a subset of SIS pairs (36 for the direct-MRM, 7 for the immuno-MRM assay) for which the hE/hT ratio reliably detected inefficient digestion that resulted in decreased assay sensitivity and unreliable endogenous quantification. The hE/hT ratio was more responsive to a decrease in digestion efficiency than a metric based on hT measurements alone. For clinical-grade MRM-MS assays, this study describes a ready-to-use QC panel and also provides a road map for designing custom QC panels.

The monodomain Kunitz protein EgKU-7 from the dog tapeworm Echinococcus granulosus is a high-affinity trypsin inhibitor with two interaction sites.

Typical Kunitz proteins (I2 family of the MEROPS database, Kunitz-A family) are metazoan competitive inhibitors of serine peptidases that form tight complexes of 1:1 stoichiometry, mimicking substrates. The cestode Echinococcus granulosus, the dog tapeworm causing cystic echinococcosis in humans and livestock, encodes an expanded family of monodomain Kunitz proteins, some of which are secreted to the dog host interface. The Kunitz protein EgKU-7 contains, in addition to the Kunitz domain with the anti-peptidase loop comprising a critical arginine, a C-terminal extension of ∼20 amino acids. Kinetic, electrophoretic, and mass spectrometry studies using EgKU-7, a C-terminally truncated variant, and a mutant in which the critical arginine was substituted by alanine, show that EgKU-7 is a tight inhibitor of bovine and canine trypsins with the unusual property of possessing two instead of one site of interaction with the peptidases. One site resides in the anti-peptidase loop and is partially hydrolyzed by bovine but not canine trypsins, suggesting specificity for the target enzymes. The other site is located in the C-terminal extension. This extension can be hydrolyzed in a particular arginine by cationic bovine and canine trypsins but not by anionic canine trypsin. This is the first time to our knowledge that a monodomain Kunitz-A protein is reported to have two interaction sites with its target. Considering that putative orthologs of EgKU-7 are present in other cestodes, our finding unveils a novel piece in the repertoire of peptidase-inhibitor interactions and adds new notes to the evolutionary host-parasite concerto.

Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) as a compensatory protease inhibitor in hereditary angioedema.

BACKGROUND: Hereditary angioedema (HAE) is a genetic disorder that manifests as recurrent angioedema attacks, most frequently due to absent or reduced C1 inhibitor (C1INH) activity. C1INH is a crucial regulator of enzymatic cascades in the complement, fibrinolytic, and contact systems. Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) is an abundant plasma protease inhibitor that can inhibit enzymes in the proteolytic pathways associated with HAE. Nothing is known about its role in HAE. OBJECTIVE: We investigated ITIH4 activation in HAE, establishing it as a potential biomarker, and explored its involvement in HAE-associated proteolytic pathways. METHODS: Specific immunoassays for noncleaved ITIH4 (intact ITIH4) and an assay detecting both intact and cleaved ITIH4 (total ITIH4) were developed. We initially tested serum samples from HAE patients (n = 20), angiotensin-converting enzyme inhibitor-induced edema patients (ACEI) (n = 20), and patients with HAE of unknown cause (HAE-UNK) (n = 20). Validation involved an extended cohort of 80 HAE patients (60 with HAE-C1INH type 1, 20 with HAE-C1INH type 2), including samples taken during attack and quiescent disease periods, as well as samples from 100 healthy controls. RESULTS: In 63% of HAE patients, intact ITIH4 assay showed lower signals than total ITIH4 assay. This difference was not observed in ACEI and HAE-UNK patients. Western blot analysis confirmed cleaved ITIH4 with low intact ITIH4 samples. In serum samples lacking intact endogenous ITIH4, we observed immediate cleavage of added recombinant ITIH4, suggesting continuous enzymatic activity in the serum. Confirmatory HAE cohort analysis revealed significantly lower intact ITIH4 levels in both type 1 and type 2 HAE patients compared to controls, with consistently low intact/total ITIH4 ratios during clinical HAE attacks. CONCLUSION: The disease-specific low intact ITIH4 levels highlight its unique nature in HAE. ITIH4 may exhibit compensatory mechanisms in HAE, suggesting its utility as a diagnostic and prognostic biomarker. The variations during quiescent and active disease periods raise intriguing questions about the dynamics of proteolytic pathways in HAE.

Impact of Surfactants on Cumulative Trypsin Activity in Bottom-Up Proteome Analysis.

Trypsin digestion plays a pivotal role in successful bottom-up peptide characterization and quantitation. While denaturants are often incorporated to enhance protein solubility, surfactants are recognized to inhibit enzyme activity. However, several reports have suggested that incorporating surfactants or other solvent additives may enhance digestion and MS detection. Here, we assess the impacts of ionic surfactants on cumulative trypsin activity and subsequently evaluate the total digestion efficiency of a proteome mixture by quantitative MS. Although low surfactant concentrations, such as 0.01% SDS or 0.2% SDC, significantly enhanced the initial trypsin activity (by 14 or 42%, respectively), time course assays revealed accelerated enzyme deactivation, evident by 10- or 40-fold reductions in trypsin activity half-life at these respective surfactant concentrations. Despite enhanced initial tryptic activity, quantitative MS analysis of a common liver proteome extract, digested with various surfactants (0.01 or 0.1% SDS, 0.5% SDC), consistently revealed decreased peptide counts and signal intensity, indicative of a lower digestion efficiency compared to a nonsurfactant control. Furthermore, including detergents for digestion did not improve the detection of membrane proteins, nor hydrophobic peptides. These results stress the importance of assessing cumulative enzyme activity when optimizing the digestion of a proteome mixture, particularly in the presence of denaturants.

Metformin inhibits digestive proteases and impairs protein digestion in mice.

Metformin is among the most prescribed medications worldwide and the first-line therapy for type 2 diabetes. However, gastrointestinal side effects are common and can be dose limiting. The total daily metformin dose frequently reaches several grams, and poor absorption results in high intestinal drug concentrations. Here, we report that metformin inhibits the activity of enteropeptidase and other digestive enzymes at drug concentrations predicted to occur in the human duodenum. Treatment of mouse gastrointestinal tissue with metformin reduces enteropeptidase activity; further, metformin-treated mice exhibit reduced enteropeptidase activity, reduced trypsin activity, and impaired protein digestion within the intestinal lumen. These results indicate that metformin-induced protein maldigestion could contribute to the gastrointestinal side effects and other impacts of this widely used drug.

Secretagogue-induced pancreatitis in mice devoid of chymotrypsin.

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.

Structural basis for the binding of famotidine, cimetidine, guanidine, and pimagedine with serine protease.

Serine proteases are among the important groups of enzymes having significant roles in cell biology. Trypsin is a representative member of the serine superfamily of enzymes, produced by acinar cells of pancreas. It is a validated drug target for various ailments including pancreatitis and colorectal cancer. Premature activation of trypsin is involved in the lysis of pancreatic tissues, which causes pancreatitis. It is also reported to be involved in colorectal carcinoma by activating other proteases, such as matrix metalloproteinase (MMPs). The development of novel trypsin inhibitors with good pharmacokinetic properties could play important roles in pharmaceutical sciences. This study reports the crystal structures of bovine pancreatic trypsin with four molecules; cimetidine, famotidine, pimagedine, and guanidine. These compounds possess binding affinity towards the active site (S1) of trypsin. The structures of all four complexes provided insight of the binding of four different ligands, as well as the dynamics of the active site towards the bind with different size ligands. This study might be helpful in designing of new potent inhibitors of trypsin and trypsin like serine proteases.

Identification of Embryonic Chicken Proteases Activating Newcastle Disease Virus and Their Roles in the Pathogenicity of Virus Used as In Ovo Vaccine.

In ovo vaccination is an attractive immunization approach for chickens. However, most live Newcastle disease virus (NDV) vaccine strains used safely after hatching are unsafe as in ovo vaccines due to their high pathogenicity for chicken embryos. The mechanism for viral pathogenicity in chicken embryos is poorly understood. Our previous studies reported that NDV strain TS09-C was a safe in ovo vaccine, and the F protein cleavage site (FCS) containing three basic amino acids (3B-FCS) was the crucial determinant of the attenuation of TS09-C in chicken embryos. Here, five trypsin-like proteases that activated NDV in chicken embryos were identified. The F protein with 3B-FCS was sensitive to the proteases Tmprss4, Tmprss9, and F7, was present in fewer tissue cells of chicken embryos, which limited the viral tropism, and was responsible for the attenuation of NDV with 3B-FCS, while the F protein with FCS containing two basic amino acids could be cleaved not only by Tmprss4, Tmprss9, and F7 but also by Prss23 and Cfd, was present in most tissue cells, and thereby was responsible for broad tissue tropism and high pathogenicity of virus in chicken embryos. Furthermore, when mixed with the protease inhibitors aprotinin and camostat, NDV with 2B-FCS exhibited greatly weakened pathogenicity in chicken embryos. Thus, our results extend the understanding of the molecular mechanism of NDV pathogenicity in chicken embryos and provide a novel molecular target for the rational design of in ovo vaccines, ensuring uniform and effective vaccine delivery and earlier induction of immune protection by the time of hatching. IMPORTANCE As an attractive immunization approach for chickens, in ovo vaccination can induce a considerable degree of protection by the time of hatching, provide support in closing the window in which birds are susceptible to infection, facilitate fast and uniform vaccine delivery, and reduce labor costs by the use of mechanized injectors. The commercial live Newcastle disease virus (NDV) vaccine strains are not safe for in ovo vaccination and cause the death of chicken embryos. The mechanism for viral pathogenicity in chicken embryos is poorly understood. In the present study, we identified five trypsin-like proteases that activate NDV in chicken embryos and elucidated their roles in the tissue tropism and pathogenicity of NDV used as in ovo vaccine. Finally, we revealed the molecular basis for the pathogenicity of NDV in chicken embryos and provided a novel strategy for the rational design of in ovo ND vaccines.

Inter-alpha-trypsin inhibitor heavy chain H3 is a potential biomarker for disease activity in myasthenia gravis.

Myasthenia gravis is a chronic antibody-mediated autoimmune disease disrupting neuromuscular synaptic transmission. Informative biomarkers remain an unmet need to stratify patients with active disease requiring intensified monitoring and therapy; their identification is the primary objective of this study. We applied mass spectrometry-based proteomic serum profiling for biomarker discovery. We studied an exploration and a prospective validation cohort consisting of 114 and 140 anti-acetylcholine receptor antibody (AChR-Ab)-positive myasthenia gravis patients, respectively. For downstream analysis, we applied a machine learning approach. Protein expression levels were confirmed by ELISA and compared to other myasthenic cohorts, in addition to myositis and neuropathy patients. Anti-AChR-Ab levels were determined by a radio receptor assay. Immunohistochemistry and immunofluorescence of intercostal muscle biopsies were employed for validation in addition to interactome studies of inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3). Machine learning identified ITIH3 as potential serum biomarker reflective of disease activity. Serum levels correlated with disease activity scores in the exploration and validation cohort and were confirmed by ELISA. Lack of correlation between anti-AChR-Ab levels and clinical scores underlined the need for biomarkers. In a subgroup analysis, ITIH3 was indicative of treatment responses. Immunostaining of muscle specimens from these patients demonstrated ITIH3 localization at the neuromuscular endplates in myasthenia gravis but not in controls, thus providing a structural equivalent for our serological findings. Immunoprecipitation of ITIH3 and subsequent proteomics lead to identification of its interaction partners playing crucial roles in neuromuscular transmission. This study provides data on ITIH3 as a potential pathophysiological-relevant biomarker of disease activity in myasthenia gravis. Future studies are required to facilitate translation into clinical practice.

Comparative evaluation of trypsin and elastase digestion techniques for isolation of murine retinal vasculature.

Dysfunctional pericytes and disruption of adherens or tight junctions are related to many microvascular diseases, including diabetic retinopathy. In this context, visualizing retinal vascular architecture becomes essential for understanding retinal vascular disease pathophysiology. Although flat mounts provide a demonstration of the retinal blood vasculature, they often lack a clear view of microaneurysms and capillary architecture. Trypsin and elastase digestion are the two techniques for isolating retinal vasculatures in rats, mice, and other animal models. Our observations in the present study reveal that trypsin digestion impacts the association between pericytes and endothelial cells. In contrast, elastase digestion effectively preserves these features in the blood vessels. Furthermore, trypsin digestion disrupts endothelial adherens and tight junctions that elastase digestion does not. Therefore, elastase digestion emerges as a superior technique for isolating retinal vessels, which can be utilized to collect reliable and consistent data to comprehend the pathophysiology of disorders involving microvascular structures.

Development of an effective method for purifying trypsin using a recombinant inhibitor.

A trypsin affinity material was prepared by covalently immobilizing buckwheat trypsin inhibitor (BTI) on epichlorohydrin-activated cross-linked agarose gel (Selfinose CL 6 B). The optimal conditions for activating Selfinose CL 6 B were 15 % epichlorohydrin and 0.8 M NaOH at 40 °C for 2 h. The optimal pH for immobilizing BTI was 9.5. BTI-Sefinose CL 6 B showed a maximum adsorption capacity of 2.25 mg trypsin/(g support). The material also displayed good reusability, retaining over 90 % of its initial adsorption capacity after 30 cycles. High-purity trypsin was obtained from locust homogenate using BTI-Selfinose CL 6 B through one-step affinity chromatography. The molecular mass and Km value of locust trypsin were determined as 27 kDa and 0.241 mM using N-benzoyl-DL-arginine-nitroanilide as substrate. The optimal temperature and pH of trypsin activity were 55 °C and 9.0, respectively. The enzyme exhibited good stability in the temperature range of 30-50 °C and pH range of 4.0-10.0. BTI-Selfinose CL 6 B demonstrates potential application in the preparation of high-purity trypsin and the discovery of more novel trypsin from various species.

A novel, robust peptidyl-lys metalloendopeptidase from Trametes coccinea recombinantly expressed in Komagataella phaffii.

A novel peptidyl-lys metalloendopeptidase (Tc-LysN) from Tramates coccinea was recombinantly expressed in Komagataella phaffii using the native pro-protein sequence. The peptidase was secreted into the culture broth as zymogen (~38 kDa) and mature enzyme (~19.8 kDa) simultaneously. The mature Tc-LysN was purified to homogeneity with a single step anion-exchange chromatography at pH 7.2. N-terminal sequencing using TMTpro Zero and mass spectrometry of the mature Tc-LysN indicated that the pro-peptide was cleaved between the amino acid positions 184 and 185 at the Kex2 cleavage site present in the native pro-protein sequence. The pH optimum of Tc-LysN was determined to be 5.0 while it maintained ≥60% activity between pH values 4.5-7.5 and ≥30% activity between pH values 8.5-10.0, indicating its broad applicability. The temperature maximum of Tc-LysN was determined to be 60 °C. After 18 h of incubation at 80 °C, Tc-LysN still retained ~20% activity. Organic solvents such as methanol and acetonitrile, at concentrations as high as 40% (v/v), were found to enhance Tc-LysN's activity up to ~100% and ~50%, respectively. Tc-LysN's thermostability, ability to withstand up to 8 M urea, tolerance to high concentrations of organic solvents, and an acidic pH optimum make it a viable candidate to be employed in proteomics workflows in which alkaline conditions might pose a challenge. The nano-LC-MS/MS analysis revealed bovine serum albumin (BSA)'s sequence coverage of 84% using Tc-LysN which was comparable to the sequence coverage of 90% by trypsin peptides. KEY POINTS: •A novel LysN from Trametes coccinea (Tc-LysN) was expressed in Komagataella phaffii and purified to homogeneity •Tc-LysN is thermostable, applicable over a broad pH range, and tolerates high concentrations of denaturants •Tc-LysN was successfully applied for protein digestion and mass spectrometry fingerprinting.

Unique hyaluronan structure of expanded oocyte-cumulus extracellular matrix in ovarian follicles.

In preovulatory follicles, after the endogenous gonadotropin surge, the oocyte-cumulus complexes (OCCs) produce hyaluronan (HA) in a process called "cumulus expansion". During this process, the heavy chains (HCs) of the serum-derived inter-alpha-trypsin inhibitor (IαI) family bind covalently to synthesized HA and form a unique structure of the expanded cumulus HA-rich extracellular matrix. Understanding the biochemical mechanism of the covalent linkage between HA and the HCs of the IαI family is one of the most significant discoveries in reproductive biology, since it explains basis of the cumulus expansion process running in parallel with the oocyte maturation, both essential for ovulation. Two recent studies have supported the above-mentioned findings: in the first, seven components of the extracellular matrix were detected by proteomic, evolutionary, and experimental analyses, and in the second, the essential role of serum in the process of cumulus expansion in vitro was confirmed. We have previously demonstrated the formation of unique structure of the covalent linkage of HA to HCs of IαI in the expanded gonadotropin-stimulated OCC, as well as interactions with several proteins produced by the cumulus cells: tumor necrosis factor-alpha-induced protein 6, pentraxin 3, and versican. Importantly, deletion of these genes in the mice produces female infertility due to defects in the oocyte-cumulus structure.

Peptide-Based Mass Spectrometry for the Investigation of Protein Complexes.

In the last two decades, biological mass spectrometry has become the gold standard for the identification of proteins in biological samples. The technological advancement of mass spectrometers and the development of methods for ionization, gas phase transfer, peptide fragmentation as well as for acquisition of high-resolution mass spectrometric data marked the success of the technique. This chapter introduces peptide-based mass spectrometry as a tool for the investigation of protein complexes. It provides an overview of the main steps for sample preparation starting from protein fractionation, reduction, alkylation and focus on the final step of protein digestion. The basic concepts of biological mass spectrometry as well as details about instrumental analysis and data acquisition are described. Finally, the most common methods for data analysis and sequence determination are summarized with an emphasis on its application to protein-protein complexes.

Synchronous reducing anti-nutritional factors and enhancing biological activity of soybean by the fermentation of edible fungus Auricularia auricula.

Auricularia auricula fermentation was performed to reduce anti-nutritional factors, improve nutritional components, and enhance biological activity of soybean. Results showed that the contents of raffinose, stachyose, and trypsin inhibitor were significantly decreased from initial 1.65 g L-1, 1.60 g L-1, and 284.67 µg g-1 to 0.14 g L-1, 0.35 g L-1, and 4.52 µg g-1 after 144 h of fermentation, respectively. Simultaneously, the contents of polysaccharide, total phenolics, and total flavonoids were increased, and melanin was secreted. The isoflavone glycosides were converted to their aglycones, and the contents of glyctin and genistin were decreased from initial 1107.99 µg g-1 and 2852.26 µg g-1 to non-detection after 72 h of fermentation, respectively. After 96 h of fermentation, the IC50 values of samples against DPPH and ABTS radicals scavenging were decreased from 17.61 mg mL-1 and 3.43 mg mL-1 to 4.63 mg mL-1 and 0.89 mg mL-1, and those of samples inhibiting α-glucosidase and angiotensin I-converting enzyme were decreased from 53.89 mg mL-1 and 11.27 mg mL-1 to 18.24 mg mL-1 and 6.78 mg mL-1, respectively, indicating the significant increase in these bioactivities. These results suggested A. auricula fermentation can enhance the nutritional quality and biological activity of soybean, and the fermented soybean products have the potential to be processed into health foods/food additives.

Observation of the Effects of Different Enzyme Digestion Time and Enzyme Digestion Methods on the Extraction of Primary Renal Tubular Epithelial Cells from Sprague-Dawley Suckling Mice.

Renal tubular epithelial cells are one of the essential functional cells in the kidney. Optimizing the isolation and culture method of primary renal tubular epithelial cells from SD mammary rats provides better experimental materials for renal tubule-related studies, which is essential for studying the pathogenesis of renal diseases, especially diabetic nephropathy and drug screening. SD rat renal tubular epithelial cells were isolated and purified by 2.5-mg/ml collagenase II or 2 mg/ml trypsin + 2.5 mg/ml collagenase II enzymatic digestion. The isolation and purification were observed at different time points (15 min, 30 min, 45 min, and 60 min) to determine the optimal extraction time for the enzymatic digestion method. After comparing the two enzymatic methods, it was determined that the trypsin + collagenase II enzymatic method was more effective. The primary renal tubular epithelial cells extracted by the trypsin + collagenase II digestion method were identified by the marker Cytokeratin 18 of renal tubular epithelial cells at 45 min of digestion with high purity. We established a simple, efficient, and reproducible method for isolation and culture of renal tubular epithelial cells in SD mammary gland rats.

Characteristics of Trypsin genes and their roles in insecticide resistance based on omics and functional analyses in the malaria vector Anopheles sinensis.

Trypsin is one of the most diverse and widely studied protease hydrolases. However, the diversity and characteristics of the Trypsin superfamily of genes have not been well understood, and their role in insecticide resistance is yet to be investigated. In this study, a total of 342 Trypsin genes were identified and classified into seven families based on homology, characteristic domains and phylogenetics in Anopheles sinensis, and the LY-Domain and CLECT-Domain families are specific to the species. Four Trypsin genes, (Astry2b, Astry43a, Astry90, Astry113c) were identified to be associated with pyrethroid resistance based on transcriptome analyses of three field resistant populations and qRT-PCR validation, and the knock-down of these genes significantly decrease the pyrethroid resistance of Anopheles sinensis based on RNAi. The activity of Astry43a can be reduced by five selected insecticides (indoxacarb, DDT, temephos, imidacloprid and deltamethrin); and however, the Astry43a could not directly metabolize these five insecticides, like the trypsin NYD-Tr did in earlier reports. This study provides the overall information frame of Trypsin genes, and proposes the role of Trypsin genes to insecticide resistance. Further researches are necessary to investigate the metabolism function of these trypsins to insecticides.

Characterization of the interactions between Fulvic acid and Trypsin with Spectroscopic and Molecular Docking technology.

The interaction mechanism between trypsin and fulvic acid was analyzed by multispectral method and molecular docking simulation. The fluorescence spectra showed that fulvic acid induced static quenching of trypsin. The validity of this conclusion was further substantiated through the computation of the binding constants. The thermodynamic parameters show that the reaction is mainly controlled by van der Waals force and hydrogen bond force, and the reaction is spontaneous. In addition, based on the obtained binding distance, there may be a non-radiative energy transfer between the two. The ultraviolet spectrum showed that fulvic acid could shift the absorption peak of trypsin, indicating that fulvic acid had an effect on the secondary structure of trypsin. According to the synchronous fluorescence spectrum results, fulvic acid primarily interacts with tryptophan residues in trypsin and induces alterations in their microenvironment. Three-dimensional fluorescence spectrum and circular dichroism further proves this conclusion. The molecular docking simulation reveals that the interaction between the two groups primarily arises from hydrogen bonding and van der Waals forces. The findings suggest that FA has the ability to induce conformational changes in trypsin's secondary structure.

The effect of the enzymes trypsin and DNase I on the antimicrobial efficiency of root canal irrigation solutions.

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.