A lava-inspired proteolytic enzyme therapy on cancer with a PEG-based hydrogel enhances tumor distribution and penetration of liposomes.

The enhanced permeability and retention (EPR) effect has become the guiding principle for nanomedicine against cancer for a long time. However, several biological barriers severely resist therapeutic agents' penetration and retention into the deep tumor tissues, resulting in poor EPR effect and high tumor mortality. Inspired by lava, we proposed a proteolytic enzyme therapy to improve the tumor distribution and penetration of nanomedicine. A trypsin-crosslinked hydrogel (Trypsin@PSA Gel) was developed to maintain trypsin's activity. The hydrogel postponed trypsin's self-degradation and sustained the release. Trypsin promoted the cellular uptake of nanoformulations in breast cancer cells, enhanced the penetration through endothelial cells, and degraded total and membrane proteins. Proteomic analysis reveals that trypsin affected ECM components and down-regulated multiple pathways associated with cancer progression. Intratumoral injection of Trypsin@PSA Gel significantly increased the distribution of liposomes in tumors and reduced tumor vasculature. Combination treatment with intravenous injection of gambogic acid-loaded liposomes and intratumoral injection of Trypsin@PSA Gel inhibited tumor growth. The current study provides one of the first investigations into the enhanced tumor distribution of liposomes induced by a novel proteolytic enzyme therapy.

Antioxidant and Antimicrobial Potential of Peptide from Rabbit Meat Hydrolysate Prepared by Trypsin and Zingibain.

<b>Background and Objective:</b> Rabbit meat is a livestock product potentially viable as a protein source to obtain peptides. Antioxidant and antimicrobial peptides are ingredients extracted from various foods through enzymatic hydrolysis, chemical hydrolysis and fermentation to produce health-promoting foods. This research aims to investigate the potential of rabbit meat as a source of antioxidant and antimicrobial peptides through hydrolysis using trypsin and zingibain enzymes. <b>Materials and Methods:</b> This research conducted an explorative-descriptive approach, focusing on antioxidant and antimicrobial activity. Rabbit meat was extracted using trypsin, zingibain and a combination of trypsin and crude extract zingibain. The hydrolyzed rabbit meat extract was tested at intervals of 0, 2, 6, 16, 24, 40 and 48 hrs to determine the degree of hydrolysis and the profile of hydrolyzed proteins with electrophoresis SDS PAGE. The antioxidant activity was tested using the DPPH method and the antimicrobial activity using agar well diffusion method. <b>Results:</b> The degree of hydrolysis increased with the hydrolysis time. The highest protein content of rabbit meat extract hydrolyzed with trypsin was 287.65 mg/mL, observed during 12 hrs hydrolysis. The optimum conditions for the hydrolysis of rabbit meat protein were obtained at 24 hrs, with an IC₅₀ value of 52.45% hydrolyzed by trypsin. As per antimicrobial activities, <i>Escherichia coli</i> and <i>Salmonella</i> sp. were more effective in inhibiting rabbit meat hydrolysates compared to <i>Pseudomonas aeruginosa</i> and <i>Staphylococcus aureus</i>. The inhibition of all pathogen increased until 12 hrs hydrolysis but decreased in 24 hrs hydrolysis. <b>Conclusion:</b> The combination zingibain enzyme and trypsin is feasible for hydrolyzing rabbit meat and the optimum hydrolysis time was 24 hrs with IC₅₀ 52.45 ppm, although accompanied by reduction in antibacterial activities.

A photoelectrochemical biosensor based on self-calibration platform of carbon-rich plasmonic probe with near-infrared driving signal amplification.

Exploring the photochemical (PEC) method induced by low-energy light source makes great significance to achieve high stability and accurate analysis. A sensing platform driven by near-infrared (NIR) light was designed by making the biochemically encoded carbon rich plasmonic hybrid (CPH) probe, the peptide@C-Mo2C. The inherent plasmonic effect of C-Mo2C CPH can directly absorb NIR light, thus starting effective electronic-hole pairs separation. Moreover, the photothermal effect of C-Mo2C CPH also promoted the reaction yield of photothermal catalyst reaction on sensing interface to assist the PEC signal amplification. In the presence of target trypsin, it cleaves the peptides, resulting in the release of peptide@C-Mo2C probe from interface, which leads to a relative decrease in PEC signal. More importantly, a self-calibration system consisting of two independent PEC test channels attempted to eliminate the influence of background signal and baseline drift. The test channel was used to specify the recognition target, while the blank channel was used as a reference. Therefore, the signal difference between two channels was recorded, so as to obtain results with less error and higher stability. In this NIR driven PEC sensor, the carbon rich probe with direct and efficient NIR light conversion promoted the sensitivity and a self-calibration system guaranteed the stability which provided innovative thoughts for developing ingenious PEC sensor.

Integration of caveolin-mediated cytosolic delivery and enzyme-responsive releasing of squalenoyl nanoparticles enhance the anti-cancer efficacy of chidamide in pancreatic cancer.

We explored the potential of overcoming the dense interstitial barrier in pancreatic cancer treatment by enhancing the uptake of hydrophilic chemotherapeutic drugs. In this study, we synthesized the squalenoyl-chidamide prodrug (SQ-CHI), linking lipophilic squalene (SQ) with the hydrophilic antitumor drug chidamide (CHI) through a trypsin-responsive bond. Self-assembled nanoparticles with sigma receptor-bound aminoethyl anisamide (AEAA) modification, forming AEAA-PEG-SQ-CHI NPs (A-C NPs, size 116.6 ± 0.4 nm), and reference nanoparticles without AEAA modification, forming mPEG-SQ-CHI NPs (M-C NPs, size 88.3 ± 0.3 nm), were prepared. A-C NPs exhibited significantly higher in vitro CHI release (74.7 %) in 0.5 % trypsin medium compared to release (20.2 %) in medium without trypsin. In vitro cell uptake assays revealed 3.6 and 2.3times higher permeation of A-C NPs into tumorspheres of PSN-1/HPSC or CFPAC-1/HPSC, respectively, compared to M-C NPs. Following intraperitoneal administration to subcutaneous tumor-bearing nude mice, the A-C NPs group demonstrated significant anti-pancreatic cancer efficacy, inducing cancer cell apoptosis and inhibiting proliferation in vivo. Mechanistic studies revealed that AEAA surface modification on nanoparticles promoted intracellular uptake through caveolin-mediated endocytosis. This nanoparticle system presents a novel therapeutic approach for pancreatic cancer treatment, offering a delivery strategy to enhance efficacy through improved tumor permeation, trypsin-responsive drug release, and specific cell surface receptor-mediated intracellular uptake.

Improved Detection of Tryptic Peptides from Tissue Sections Using Desorption Electrospray Ionization Mass Spectrometry Imaging.

DESI-MSI is an ambient ionization technique used frequently for the detection of lipids, small molecules, and drug targets. Until recently, DESI had only limited use for the detection of proteins and peptides due to the setup and needs around deconvolution of data resulting in a small number of species being detected at lower spatial resolution. There are known differences in the ion species detected using DESI and MALDI for nonpeptide molecules, and here, we identify that this extends to proteomic species. DESI MS images were obtained for tissue sections of mouse and rat brain using a precommercial heated inlet (approximately 450 °C) to the mass spectrometer. Ion mobility separation resolved spectral overlap of peptide ions and significantly improved the detection of multiply charged species. The images acquired were of pixel size 100 µm (rat brain) and 50 µm (mouse brain), respectively. Observed tryptic peptides were filtered against proteomic target lists, generated by LC-MS, enabling tentative protein assignment for each peptide ion image. Precise localizations of peptide ions identified by DESI and MALDI were found to be comparable. Some spatially localized peptides ions were observed in DESI that were not found in the MALDI replicates, typically, multiply charged species with a low mass to charge ratio. This method demonstrates the potential of DESI-MSI to detect large numbers of tryptic peptides from tissue sections with enhanced spatial resolution when compared to previous DESI-MSI studies.

Advances in Understanding the Antioxidant and Antigenic Properties of Egg-Derived Peptides.

Pepsin, trypsin and proteinase K were used in the present study to hydrolyse the proteins from whole eggs, yolks or whites, and the resulting hydrolysates were characterised in terms of antioxidant and IgE-binding properties, using a combination of in vitro and in silico methods. Based on the degree of hydrolysis (DH) results, the egg yolk proteins are better substrates for all the tested enzymes (DH of 6.2-20.1%) compared to those from egg whites (DH of 2.0-4.4%). The SDS-PAGE analysis indicated that pepsin and proteinase K were more efficient compared to trypsin in breaking the intramolecular peptide bonds of the high molecular weight egg proteins. For all the tested substrates, enzyme-assisted hydrolysis resulted in a significant increase in antioxidant activity, suggesting that many bioactive peptides are encrypted in inactive forms in the parent proteins. The hydrolysates obtained with proteinase K exhibited the highest DPPH radical scavenging activity (124-311 µM Trolox/g protein) and the lowest residual IgE-binding capacity. The bioinformatics tools revealed that proteinase K is able to break the integrity of the main linear IgE-binding epitopes from ovalbumin and ovomucoid. It can be concluded that proteinase K is a promising tool for modulating the intrinsic properties of egg proteins.

Expanding the scope of self-assembled supramolecular biosensors: a highly selective and sensitive enzyme-responsive AIE-based fluorescent biosensor for trypsin detection and inhibitor screening.

Trypsin, a pancreatic enzyme associated with diseases like pancreatic cancer and cystic fibrosis, requires effective diagnostic tools. Current detection systems seldom utilize macrocyclic molecules and tetraphenyl ethylene (TPE) derivative-based supramolecular assemblies, known for their biocompatibility and aggregation-induced emission (AIE) properties, for trypsin detection. This study presents an enzyme-responsive, AIE-based fluorescence 'Turn-On' sensing platform for trypsin detection, employing sulfated-ß-cyclodextrin (S-ßCD), an imidazolium derivative of TPE (TPE-IM), and protamine sulfate (PrS). The anionic S-ßCD and cationic TPE-IM formed a strongly fluorescent supramolecular aggregation complex in an aqueous buffer. However, PrS suppresses fluorescence because of its strong binding affinity with S-ßCD. The non-fluorescent TPE-IM/S-ßCD/PrS supramolecular assembly system exhibits trypsin-responsive properties, as PrS is a known trypsin substrate. Trypsin restores fluorescence in the TPE-IM/S-ßCD system through the enzymatic cleavage of PrS, correlating linearly with trypsin catalytic activity in the 0-10 nM concentration range. The limit of detection is 10 pM. This work contributes to the development of self-assembled supramolecular biosensors using charged TPE derivatives and ß-cyclodextrin-based host-guest chemistry, offering an innovative fluorescence 'Turn-On' trypsin sensing platform. The sensing system is highly stable under various conditions, selective for trypsin, and demonstrates potential for biological analysis and disease diagnosis in human serum. Additionally, it shows promise for the screening of trypsin inhibitors.

Role of leucine-rich repeat kinase 2 in severe acute pancreatitis.

Introduction: Intrapancreatic activation of trypsinogen caused by alcohol or high-fat intake and the subsequent autodigestion of the pancreas tissues by trypsin are indispensable events in the development of acute pancreatitis. In addition to this trypsin-centered paradigm, recent studies provide evidence that innate immune responses triggered by translocation of intestinal bacteria to the pancreas due to intestinal barrier dysfunction underlie the immunopathogenesis of acute pancreatitis. Although severe acute pancreatitis is often associated with pancreatic colonization by fungi, the molecular mechanisms linking fungus-induced immune responses to the development of severe acute pancreatitis are poorly understood. Leucine-rich repeat kinase 2 (LRRK2) is a multifunctional protein that mediates innate immune responses to fungi and bacteria. Mutations in Lrrk2 is a risk factor for Parkinson's disease and Crohn's disease, both of which are driven by innate immune responses to gut organisms. Discussion: In this Minireview article, we discuss how activation of LRRK2 by the recognition of fungi induces severe acute pancreatitis.

Effect of moxibustion on colonic low-grade inflammatory response in rats with diarrhea-predominant irritable bowel syndrome based on mast cell degranulation. / åŸºäºŽè‚¥å¤§ç»†èƒžè„±é¢—ç²’æŽ¢è®¨è‰¾ç¸å¯¹è ¹æ³»åž‹è‚ æ˜“æ¿€ç»¼åˆå¾å¤§é¼ ç»“è‚ ä½Žåº¦ç‚Žæ€§ååº”çš„å½±å“.

OBJECTIVES: To observe the effects of moxibustion on colonic mast cell degranulation and inflammatory factor expression in rats with diarrhea-predominant irritable bowel syndrome (IBS-D), and explore the potential mechanism of moxibustion in treating IBS-D. METHODS: Forty-five rat pups born from 5 healthy SPF-grade pregnant SD rats, with 8 rats were randomly selected as the normal group. The remaining 37 rats were intervened with maternal separation, acetic acid enema, and chronic restraint stress to establish the IBS-D model. The successfully modeled 32 rats were then randomly assigned to a model group, a ketotifen group, a moxibustion group, and a moxibustion-medication group, with 8 rats in each group. The rats in the ketotifen group were intervened with intragastric administration of ketotifen solution (10 mL/kg); the rats in the moxibustion group were intervened with suspended moxibustion on bilateral "Tianshu" (ST 25) and "Shangjuxu" (ST 37); the rats in the moxibustion-medication group were intervened with suspended moxibustion combined with intragastric administration of ketotifen solution. All interventions were administered once daily for 7 consecutive days. The diarrhea rate and minimum volume threshold of abdominal withdrawal reflex (AWR) were calculated before and after modeling, as well as after intervention. After intervention, colonic tissue morphology was observed using HE staining; colonic mucosal ultrastructure was examined by scanning electron microscopy; colonic mast cell ultrastructure was observed using transmission electron microscopy; mast cell degranulation was assessed by toluidine blue staining; serum and colonic levels of histamine, interleukin (IL)-1ß, IL-6, IL-1α, trypsin-like enzyme, and protease-activated receptor 2 (PAR-2) were measured by ELISA; the Western blot and real-time quantitative PCR were employed to evaluate the protein and mRNA expression of colonic IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2; the immunofluorescence was used to detect the positive expression of histamine, IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 in the colonic tissue. RESULTS: Compared to the normal group, the rats in the model group exhibited extensive infiltration of inflammatory cells in colonic tissue, severe damage to the colonic mucosa, disordered arrangement of villi, reduced electron density, and a significant decrease in granule quantity within mast cells. The diarrhea rate and mast cell degranulation rate were increased (P<0.01), AWR minimum volume threshold was decreased (P<0.01); the serum and colonic levels of histamine, IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 were elevated (P<0.01); the positive expression of histamine, as well as protein, mRNA and positive expression of IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 in the colon were all elevated (P<0.01). Compared to the model group, the rats in the ketotifen group, the moxibustion group, and the moxibustion-medication group exhibited significantly reduced infiltration of inflammatory cells in colonic tissue, relatively intact colonic mucosa, orderly arranged villi, increased electron density, and an augmented number of mast cell granules; the diarrhea rate and mast cell degranulation rate were decreased (P<0.01), and AWR minimum volume threshold was increased (P<0.01); the serum and colonic levels of histamine, IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 were reduced (P<0.01); the positive expression of histamine, as well as protein, mRNA and positive expression of IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 in the colon were all decreased (P<0.01). Compared to the ketotifen group, the moxibustion group showed decreased serum levels of histamine, IL-6, and trypsin-like enzyme (P<0.01, P<0.05), as well as reduced colonic levels of IL-1ß and IL-6 (P<0.01, P<0.05); the protein expression of colonic IL-1ß, IL-1α, and PAR-2 was reduced (P<0.05), and the positive expression of colonic IL-1ß and trypsin-like enzyme was reduced (P<0.01, P<0.05). Compared to both the ketotifen group and the moxibustion group, the moxibustion-medication group exhibited decreased diarrhea rate and mast cell degranulation rate (P<0.01), an increased AWR minimum volume threshold (P<0.01), reduced serum and colonic levels of histamine, IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 (P<0.01), decreased protein expression of colonic IL-1ß, trypsin-like enzyme, and PAR-2 (P<0.01, P<0.05), reduced mRNA and positive expression of colonic IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 (P<0.01, P<0.05), and decreased positive expression of colonic histamine (P<0.01). CONCLUSIONS: Moxibustion on "Tianshu" (ST 25) and "Shangjuxu" (ST 37) might inhibit low-grade inflammatory reactions in the colon of IBS-D model rats. The mechanism may be related to the inhibition of histamine and trypsin-like enzyme secreted by mast cell, thereby reducing the expression of related inflammatory factors.

An in silico scheme for optimizing the enzymatic acquisition of natural biologically active peptides based on machine learning and virtual digestion.

BACKGROUND: As a potential natural active substance, natural biologically active peptides (NBAPs) are recently attracting increasing attention. The traditional proteolysis methods of obtaining effective NBAPs are considerably vexing, especially since multiple proteases can be used, which blocks the exploration of available NBAPs. Although the development of virtual digesting brings some degree of convenience, the activity of the obtained peptides remains unclear, which would still not allow efficient access to the NBAPs. It is necessary to develop an efficient and accurate strategy for acquiring NBAPs. RESULTS: A new in silico scheme named SSA-LSTM-VD, which combines a sparrow search algorithm-long short-term memory (SSA-LSTM) deep learning and virtually digested, was presented to optimize the proteolysis acquisition of NBAPs. Therein, SSA-LSTM reached the highest Efficiency value reached 98.00 % compared to traditional machine learning algorithms, and basic LSTM algorithm. SSA-LSTM was trained to predict the activity of peptides in the proteins virtually digested results, obtain the percentage of target active peptide, and select the appropriate protease for the actual experiment. As an application, SSA-LSTM was employed to predict the percentage of neuroprotective peptides in the virtual digested result of walnut protein, and trypsin was ultimately found to possess the highest value (85.29 %). The walnut protein was digested by trypsin (WPTrH) and the peptide sequence obtained was analyzed closely matches the theoretical neuroprotective peptide. More importantly, the neuroprotective effects of WPTrH had been demonstrated in nerve damage mouse models. SIGNIFICANCE: The proposed SSA-LSTM-VD in this paper makes the acquisition of NBAPs efficient and accurate. The approach combines deep learning and virtually digested skillfully. Utilizing the SSA-LSTM-VD based strategy holds promise for discovering and developing peptides with neuroprotective properties or other desired biological activities.

Addressing common challenges of biotherapeutic protein peptide mapping using recombinant trypsin.

Peptide mapping is the key method for characterization of primary structure of biotherapeutic proteins. This method relies on digestion of proteins into peptides that are then analyzed for amino acid sequence and post-translational modifications. Owing to its high activity and cleavage specificity, trypsin is the protease of choice for peptide mapping. In this study, we investigated critical requirements of peptide mapping and how trypsin affects these requirements. We found that the commonly used MS-grade trypsins contained non-specific, chymotryptic-like cleavage activity causing generation of semi-tryptic peptides and degradation of tryptic-specific peptides. Furthermore, MS-grade trypsins contained pre-existing autoproteolytic peptides and, moreover, additional autoproteolytic peptides were resulting from prominent autoproteolysis during digestion. In our long-standing quest to improve trypsin performance, we developed novel recombinant trypsin and evaluated whether it could address major trypsin drawbacks in peptide mapping. The study showed that the novel trypsin was free of detectable non-specific cleavage activity, had negligible level of autoproteolysis and maintained high activity over the course of digestion reaction. Taking advantage of the novel trypsin advanced properties, especially high cleavage specificity, we established the application for use of large trypsin quantities to digest proteolytically resistant protein sites without negative side effects. We also tested trypsin/Lys-C mix comprising the novel trypsin and showed elimination of non-specific cleavages observed in the digests with the commonly used trypsins. In addition, the improved features of the novel trypsin allowed us to establish the method for accurate and efficient non-enzymatic PTM analysis in biotherapeutic proteins.

A validated LC-MS/MS method for the quantitation of daratumumab in rat serum using rapid tryptic digestion without IgG purification and reduction.

Daratumumab, a humanized monoclonal antibody utilized in treating immunoglobulin light-chain amyloidosis and relapsed/refractory multiple myeloma, was quantified in rat serum through a simple, economical and effective liquid chromatography tandem-mass spectrometry (LC-MS/MS) method. A surrogate peptide, LLIYDASNR, derived from trypsin hydrolysis, was quantitatively analyzed with LLIYDASN [13C6, 15N4] RAT as an internal standard. This corrected variations from sample pretreatment and mass spectrometry response, involving denaturation and trypsin hydrolysis in a two-step process lasting approximately 1 hour. Methodological validation demonstrated a linear range of 1 µg/mL to 1000 µg/mL in rat serum. Precision, accuracy, matrix effect, sensitivity, stability, selectivity, carryover, and interference met acceptance criteria. The validated LC-MS/MS approach was successfully applied to a pharmacokinetic study of daratumumab in rats at an intravenous dose of 15 mg/kg.

High-throughput method for Peptide mapping and Amino acid sequencing for Calcitonin Salmon in Calcitonin Salmon injection using Ultra High Performance Liquid Chromatography - High Resolution Mass Spectrometry (UHPLC-HRMS) with the application of Bioinformatic tools.

BACKGROUND: Tandem mass spectrometry (MS/MS) can provide direct and accurate sequence characterization of synthetic peptide drugs, and peptide drug products including side chain modifications in the Peptide drugs. This article explains a step-by-step guide to developing a high-throughput method using high resolution mass spectrometry for characterization of Calcitonin Salmon injection containing high proportion of UV-active excipients. METHODS: The major challenge in the method development of Amino acid sequencing and Peptide mapping was presence of phenol in drug product. Phenol is a UV-active excipient and reacts with both Dithiothreitol (DTT) and Trypsin. Hence Calcitonin Salmon was extracted from the Calcitonin Salmon injection using solid phase extraction after the extraction, Amino acid sequencing and peptide mapping study was performed. Upon incubation of Calcitonin Salmon with Trypsin and DTT, digested fragments were generated which were separated by mass compatible reverse phase chromatography and the molecular mass of each fragment was determined using HRMS. RESULTS: A reverse phase chromatographic method was developed using UHPLC-HRMS for the determination of direct mass, peptide mapping and to determine the amino acid sequencing in the Calcitonin Salmon injection. The method was found Specific and fragments after trypsin digest are well resolved from each other and the molecular mass of each fragment was determined using HRMS. Sequencing was performed using automated identification of b and y ions annotation and identifications based on MS/MS spectra using Biopharma finder and Proteome discoverer software. CONCLUSION: Using this approach 100% protein coverage was obtained and protein was identified as Calcitonin Salmon and the observed masses of tryptic digest of peptide was found similar with theoretical masses. The method can be used for both UV and MS based Peptide mapping and whereas the UV based peptide mapping method can be used as identification test for Calcitonin Salmon drug substance and drug product in quality control.

Ligand-Based Design of Selective Peptidomimetic uPA and TMPRSS2 Inhibitors with Arg Bioisosteres.

Trypsin-like serine proteases are involved in many important physiological processes like blood coagulation and remodeling of the extracellular matrix. On the other hand, they are also associated with pathological conditions. The urokinase-pwlasminogen activator (uPA), which is involved in tissue remodeling, can increase the metastatic behavior of various cancer types when overexpressed and dysregulated. Another member of this protease class that received attention during the SARS-CoV 2 pandemic is TMPRSS2. It is a transmembrane serine protease, which enables cell entry of the coronavirus by processing its spike protein. A variety of different inhibitors have been published against both proteases. However, the selectivity over other trypsin-like serine proteases remains a major challenge. In the current study, we replaced the arginine moiety at the P1 site of peptidomimetic inhibitors with different bioisosteres. Enzyme inhibition studies revealed that the phenylguanidine moiety in the P1 site led to strong affinity for TMPRSS2, whereas the cyclohexylguanidine derivate potently inhibited uPA. Both inhibitors exhibited high selectivity over other structurally similar and physiologically important proteases.

House dust mite allergens induce Ca2+ signalling and alarmin responses in asthma airway epithelial cells.

Type 2 inflammation in asthma develops with exposure to stimuli to include inhaled allergens from house dust mites (HDM). Features include mucus hypersecretion and the formation of pro-secretory ion transport characterised by elevated basal Cl- current. Studies using human sinonasal epithelial cells treated with HDM extract report a higher protease activated receptor-2 (PAR-2) agonist-induced calcium mobilisation that may be related to airway sensitisation by allergen-associated proteases. Herein, this study aimed to investigate the effect of HDM on Ca2+ signalling and inflammatory responses in asthmatic airway epithelial cells. Primary bronchial epithelial cells (hPBECs) from asthma donors cultured at air-liquid interface were used to assess electrophysiological, Ca2+ signalling and inflammatory responses. Differences were observed regarding Ca2+ signalling in response to PAR-2 agonist 2-Furoyl-LIGRLO-amide (2-FLI), and equivalent short-circuit current (Ieq) in response to trypsin and 2-FLI, in ALI-asthma and healthy hPBECs. HDM treatment led to increased levels of intracellular cations (Ca2+, Na+) and significantly reduced the 2-FLI-induced change of Ieq in asthma cells. Apical HDM-induced Ca2+ mobilisation was found to mainly involve the activation of PAR-2 and PAR-4-associated store-operated Ca2+ influx and TRPV1. In contrast, PAR-2, PAR-4 antagonists and TRPV1 antagonist only showed slight impact on basolateral HDM-induced Ca2+ mobilisation. HDM trypsin-like serine proteases were the main components leading to non-amiloride sensitive Ieq and also increased interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP) from asthma hPBECs. These studies add further insight into the complex mechanisms associated with HDM-induced alterations in cell signalling and their relevance to pathological changes within asthma.

Optimal conditions for carrying out trypsin digestions on complex proteomes: From bulk samples to single cells.

To identify proteins by the bottom-up mass spectrometry workflow, enzymatic digestion is essential to break down proteins into smaller peptides amenable to both chromatographic separation and mass spectrometric analysis. Trypsin is the most extensively used protease due to its high cleavage specificity and generation of peptides with desirable positively charged N- and C-terminal amino acid residues that are amenable to reverse phase HPLC separation and MS/MS analyses. However, trypsin can yield variable digestion profiles and its protein cleavage activity is interdependent on trypsin source and quality, digestion time and temperature, pH, denaturant, trypsin and substrate concentrations, composition/complexity of the sample matrix, and other factors. There is therefore a need for a more standardized, general-purpose trypsin digestion protocol. Based on a review of the literature we delineate optimal conditions for carrying out trypsin digestions of complex proteomes from bulk samples to limiting amounts of protein extracts. Furthermore, we highlight recent developments and technological advances used in digestion protocols to quantify complex proteomes from single cells. SIGNIFICANCE: Currently, bottom-up MS-based proteomics is the method of choice for global proteome analysis. Since trypsin is the most utilized protease in bottom-up MS proteomics, delineating optimal conditions for carrying out trypsin digestions of complex proteomes in samples ranging from tissues to single cells should positively impact a broad range of biomedical research.

Correlation of Experimental and Calculated Inhibition Constants of Protease Inhibitor Complexes.

Predicting the potency of inhibitors is key to in silico screening of promising synthetic or natural compounds. Here we describe a predictive workflow that provides calculated inhibitory values, which concord well with empirical data. Calculations of the free interaction energy ΔG with the YASARA plugin FoldX were used to derive inhibition constants Ki from PDB coordinates of protease-inhibitor complexes. At the same time, corresponding KD values were obtained from the PRODIGY server. These results correlated well with the experimental values, particularly for serine proteases. In addition, analyses were performed for inhibitory complexes of cysteine and aspartic proteases, as well as of metalloproteases, whereby the PRODIGY data appeared to be more consistent. Based on our analyses, we calculated theoretical Ki values for trypsin with sunflower trypsin inhibitor (SFTI-1) variants, which yielded the more rigid Pro14 variant, with probably higher potency than the wild-type inhibitor. Moreover, a hirudin variant with an Arg1 and Trp3 is a promising basis for novel thrombin inhibitors with high potency. Further examples from antibody interaction and a cancer-related effector-receptor system demonstrate that our approach is applicable to protein interaction studies beyond the protease field.

Genetic Factors Associated With Adverse Pregnancy Outcomes in Chronic Pancreatitis.

INTRODUCTION: The effects of genetic factors on pregnancy outcomes in chronic pancreatitis (CP) patients remain unclear. We evaluated the impacts of clinical features and mutations in main CP-susceptibility genes ( SPINK1 , PRSS1 , CTRC , and CFTR ) on pregnancy outcomes in Chinese CP patients. METHODS: This was a prospective cohort study with 14-year follow-up. The sample comprised female CP patients with documented pregnancy and known genetic backgrounds. Adverse pregnancy outcomes were compared between patients with and without gene mutations. Univariate and multivariate analyses were performed to determine the impact factors for adverse pregnancy outcomes. RESULTS: Totally, 160 female CP patients with a pregnancy history were enrolled; 59.4% of patients carried pathogenic mutations in CP-susceptibility genes. Adverse pregnancy outcomes occurred in 38 patients (23.8%); the prevalence of adverse outcomes was significantly higher in those harboring gene mutations than those without (30.5% vs 13.8%, P = 0.015). Notably, the rates of preterm delivery (12.6% vs 3.1%, P = 0.036) and abortion (17.9% vs 4.6%, P = 0.013) were remarkably higher in patients with gene mutations (especially SPINK1 mutations) than those without. In multivariate analyses, both CP-susceptibility gene mutations (odds ratio, 2.52; P = 0.033) and SPINK1 mutations (odds ratio, 2.60; P = 0.037) significantly increased the risk of adverse pregnancy outcomes. Acute pain attack during pregnancy was another risk factor for adverse pregnancy outcomes. DISCUSSION: Pathogenic mutations in CP-susceptibility genes, especially SPINK1 , were independently related to adverse pregnancy outcomes in CP patients. Significant attention should be paid to pregnant females harboring CP-susceptibility gene mutations (ClinicalTrials.gov: NCT06055595).

The antibacterial activity and mechanism of a novel peptide MR-22 against multidrug-resistant Escherichia coli.

Introduction: Bacterial infections have become serious threats to human health, and the excessive use of antibiotics has led to the emergence of multidrug-resistant (MDR) bacteria. E. coli is a human bacterial pathogen, which can cause severe infectious. Antimicrobial peptides are considered the most promising alternative to traditional antibiotics. Materials and methods: The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and hemolytic activity were determined by the microdilution method. The antimicrobial kinetics of MR-22 against E. coli were studied by growth curves and time-killing curves. The cytotoxicity of MR-22 was detected by the CCK-8 assay. The antimicrobial activity of MR-22 in salt, serum, heat and trypsin was determined by the microdilution method. The antimicrobial mechanism of MR-22 against drug-resistant E. coli was studied by Scanning Electron Microscope, laser confocal microscopy, and Flow Cytometry. The in vivo antibacterial activity of MR-22 was evaluated by the mice model of peritonitis. Results and discussion: In this study, MR-22 is a new antimicrobial peptide with good activity that has demonstrated against MDR E. coli. The antimicrobial activity of MR-22 exhibited stability under conditions of high temperature, 10% FBS, and Ca2+. However, a decline of the activity was observed in the presence of Na+, serum, and trypsin. MR-22 had no significant cytotoxicity or hemolysis in vitro. SEM and fluorescent images revealed that MR-22 could disrupt the integrity of cell membrane. DCFH-DA indicated that MR-22 increased the content of reactive oxygen species, while it decreased the content of intracellular ATP. In mice model of peritonitis, MR-22 exhibited potent antibacterial activity in vivo. These results indicated that MR-22 is a potential drug candidate against drug-resistant E. coli.

Trypsin-induced aggregation of transthyretin Valine 30 variants associated with hereditary amyloidosis.

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