S. aureus drives itch and scratch-induced skin damage through a V8 protease-PAR1 axis.

Itch is an unpleasant sensation that evokes a desire to scratch. The skin barrier is constantly exposed to microbes and their products. However, the role of microbes in itch generation is unknown. Here, we show that Staphylococcus aureus, a bacterial pathogen associated with itchy skin diseases, directly activates pruriceptor sensory neurons to drive itch. Epicutaneous S. aureus exposure causes robust itch and scratch-induced damage. By testing multiple isogenic bacterial mutants for virulence factors, we identify the S. aureus serine protease V8 as a critical mediator in evoking spontaneous itch and alloknesis. V8 cleaves proteinase-activated receptor 1 (PAR1) on mouse and human sensory neurons. Targeting PAR1 through genetic deficiency, small interfering RNA (siRNA) knockdown, or pharmacological blockade decreases itch and skin damage caused by V8 and S. aureus exposure. Thus, we identify a mechanism of action for a pruritogenic bacterial factor and demonstrate the potential of inhibiting V8-PAR1 signaling to treat itch.

Cryo-EM structures show the mechanistic basis of pan-peptidase inhibition by human α2-macroglobulin.

Human α2-macroglobulin (hα2M) is a multidomain protein with a plethora of essential functions, including transport of signaling molecules and endopeptidase inhibition in innate immunity. Here, we dissected the molecular mechanism of the inhibitory function of the ∼720-kDa hα2M tetramer through eight cryo­electron microscopy (cryo-EM) structures of complexes from human plasma. In the native complex, the hα2M subunits are organized in two flexible modules in expanded conformation, which enclose a highly porous cavity in which the proteolytic activity of circulating plasma proteins is tested. Cleavage of bait regions exposed inside the cavity triggers rearrangement to a compact conformation, which closes openings and entraps the prey proteinase. After the expanded-to-compact transition, which occurs independently in the four subunits, the reactive thioester bond triggers covalent linking of the proteinase, and the receptor-binding domain is exposed on the tetramer surface for receptor-mediated clearance from circulation. These results depict the molecular mechanism of a unique suicidal inhibitory trap.

IdeS, a secreted proteinase of Streptococcus pyogenes, is bound to a nuclease at the bacterial surface where it inactivates opsonizing IgG antibodies.

The important bacterial pathogen Streptococcus pyogenes secretes IdeS (immunoglobulin G-degrading enzyme of S. pyogenes), a proteinase that cleaves human immunoglobulin G (IgG) antibodies in the hinge region resulting in Fc (fragment crystallizable) and F(ab')2 (fragment antigen-binding) fragments and protects the bacteria against phagocytic killing. Experiments with radiolabeled IdeS and flow cytometry demonstrated that IdeS binds to the surface of S. pyogenes, and the interaction was most prominent in conditions resembling those in the pharynx (acidic pH and low salt), the habitat for S. pyogenes. SpnA (S. pyogenes nuclease A) is a cell wall-anchored DNase. A dose-dependent interaction between purified SpnA and IdeS was demonstrated in slot binding and surface plasmon resonance spectroscopy experiments. Gel filtration showed that IdeS forms proteolytically active complexes with SpnA in solution, and super-resolution fluorescence microscopy revealed the presence of SpnA-IdeS complexes at the surface of S. pyogenes. Finally, specific IgG antibodies binding to S. pyogenes surface antigens were efficiently cleaved by surface-associated IdeS. IdeS is secreted by all S. pyogenes isolates and cleaves IgG antibodies with a unique degree of specificity and efficiency. These properties and the finding here that the proteinase is present and fully active at the bacterial surface in complex with SpnA implicate an important role for IdeS in S. pyogenes biology and pathogenesis.

Neutrophil elastase decreases SARS-CoV-2 spike protein binding to human bronchial epithelia by clipping ACE-2 ectodomain from the epithelial surface.

Patients with cystic fibrosis (CF) have decreased severity of severe acute respiratory syndrome-like coronavirus-2 (SARS-CoV-2) infections, but the underlying cause is unknown. Patients with CF have high levels of neutrophil elastase (NE) in the airway. We examined whether respiratory epithelial angiotensin-converting enzyme 2 (ACE-2), the receptor for the SARS-CoV-2 spike protein, is a proteolytic target of NE. Soluble ACE-2 levels were quantified by ELISA in airway secretions and serum from patients with and without CF, the association between soluble ACE-2 and NE activity levels was evaluated in CF sputum. We determined that NE activity was directly correlated with increased ACE-2 in CF sputum. Additionally, primary human bronchial epithelial (HBE) cells, exposed to NE or control vehicle, were evaluated by Western analysis for the release of cleaved ACE-2 ectodomain fragment into conditioned media, flow cytometry for the loss of cell surface ACE-2, its impact on SARS-CoV-2 spike protein binding. We found that NE treatment released ACE-2 ectodomain fragment from HBE and decreased spike protein binding to HBE. Furthermore, we performed NE treatment of recombinant ACE-2-Fc-tagged protein in vitro to assess whether NE was sufficient to cleave recombinant ACE-2-Fc protein. Proteomic analysis identified specific NE cleavage sites in the ACE-2 ectodomain that would result in loss of the putative N-terminal spike-binding domain. Collectively, data support that NE plays a disruptive role in SARS-CoV-2 infection by catalyzing ACE-2 ectodomain shedding from the airway epithelia. This mechanism may reduce SARS-CoV-2 virus binding to respiratory epithelial cells and decrease the severity of COVID19 infection.

Constitutive and induced forms of membrane-bound proteinase 3 interact with antineutrophil cytoplasmic antibodies and promote immune activation of neutrophils.

Proteinase 3 (PR3) is the main target antigen of antineutrophil cytoplasmic antibodies (ANCAs) in PR3-ANCA-associated vasculitis. A small fraction of PR3 is constitutively exposed on the surface of quiescent blood neutrophils in a proteolytically inactive form. When activated, neutrophils expose an induced form of membrane-bound PR3 (PR3mb) on their surface as well, which is enzymatically less active than unbound PR3 in solution due to its altered conformation. In this work, our objective was to understand the respective role of constitutive and induced PR3mb in the immune activation of neutrophils triggered by murine anti-PR3 mAbs and human PR3-ANCA. We quantified immune activation of neutrophils by the measurement of the production of superoxide anions and secreted protease activity in the cell supernatant before and after treatment of the cells by alpha-1 protease inhibitor that clears induced PR3mb from the cell surface. Incubation of TNFα-primed neutrophils with anti-PR3 antibodies resulted in a significant increase in superoxide anion production, membrane activation marker exposition, and secreted protease activity. When primed neutrophils were first treated with alpha-1 protease inhibitor, we observed a partial reduction in antibody-induced neutrophil activation, suggesting that constitutive PR3mb is sufficient to activate neutrophils. The pretreatment of primed neutrophils with purified antigen-binding fragments used as competitor significantly reduced cell activation by whole antibodies. This led us to the conclusion that PR3mb promoted immune activation of neutrophils. We propose that blocking and/or elimination of PR3mb offers a new therapeutic strategy to attenuate neutrophil activation in patients with PR3-ANCA-associated vasculitis.

In vitro functional analysis and in silico structural modelling of pathogen-secreted polyglycine hydrolases.

Polyglycine hydrolases are fungal effectors composed of an N-domain with unique sequence and structure and a C-domain that resembles ß-lactamases, with serine protease activity. These secreted fungal proteins cleave Gly-Gly bonds within a polyglycine sequence in corn ChitA chitinase. The polyglycine hydrolase N-domain (PND) function is unknown. In this manuscript we provide evidence that the PND does not directly participate in ChitA cleavage. In vitro analysis of site-directed mutants in conserved residues of the PND of polyglycine hydrolase Es-cmp did not specifically impair protease activity. Furthermore, in silico structural models of three ChitA-bound polyglycine hydrolases created by High Ambiguity Driven protein-protein DOCKing (HADDOCK) did not predict significant interactions between the PND and ChitA. Together these results suggest that the PND has another function. To determine what types of PND-containing proteins exist in nature we performed a computational analysis of Foldseek-identified PND-containing proteins. The analysis showed that proteins with PNDs are present throughout biology as either single domain proteins or fused to accessory domains that are diverse but are usually proteases or kinases.

Computer-assisted semi-rational design enhanced the enzymatic activity and protein stability of Proteinase K in calcium-free conditions.

Wild-type Proteinase K binds to two Ca2+ ions, which play an important role in regulating enzymaticactivity and maintaining protein stability. Therefore, a predetermined concentration of Ca2+ must be added during the use of Proteinase K, which increases its commercial cost. Herein, we addressed this challenge using a computational strategy to engineer a Proteinase K mutant that does not require Ca2+ and exhibits high enzymatic activity and protein stability. In the absence of Ca2+, the best mutant, MT24 (S17W-S176N-D260F), displayed an activity approximately 9.2-fold higher than that of wild-type Proteinase K. It also exhibited excellent protein stability, retaining 56.2 % of its enzymatic activity after storage at 4 °C for 5 days. The residual enzymatic activity was 65-fold higher than that of the wild-type Proteinase K under the same storage conditions. Structural analysis and molecular dynamics simulations suggest that the introduction of new hydrogen bond and π-π stacking at the Ca2+ binding sites due to the mutation may be the reasons for the increased enzymatic activity and stability of MT24.

Enterovirus-Cardiomyocyte Interactions: Impact of Terminally Deleted Genomic RNAs on Viral and Host Functions.

Group B enteroviruses, including coxsackievirus B3 (CVB3), can persistently infect cardiac tissue and cause dilated cardiomyopathy. Persistence is linked to 5' terminal deletions of viral genomic RNAs that have been detected together with minor populations of full-length genomes in human infections. In this study, we explored the functions and interactions of the different viral RNA forms found in persistently infected patients and their putative role(s) in pathogenesis. Since enterovirus cardiac pathogenesis is linked to the viral proteinase 2A, we investigated the effect of different terminal genomic RNA deletions on 2A activity. We discovered that 5' terminal deletions in CVB3 genomic RNAs decreased the levels of 2A proteinase activity but could not abrogate it. Using newly generated viral reporters encoding nano-luciferase, we found that 5' terminal deletions resulted in decreased levels of viral protein and RNA synthesis in singly transfected cardiomyocyte cultures. Unexpectedly, when full-length and terminally deleted forms were cotransfected into cardiomyocytes, a cooperative interaction was observed, leading to increased viral RNA and protein production. However, when viral infections were carried out in cells harboring 5' terminally deleted CVB3 RNAs, a decrease in infectious particle production was observed. Our results provide a possible explanation for the necessity of full-length viral genomes during persistent infection, as they would stimulate efficient viral replication compared to that of the deleted genomes alone. To avoid high levels of viral particle production that would trigger cellular immune activation and host cell death, the terminally deleted RNA forms act to limit the production of viral particles, possibly as trans-dominant inhibitors. IMPORTANCE Enteroviruses like coxsackievirus B3 are able to initiate acute infections of cardiac tissue and, in some cases, to establish a long-term persistent infection that can lead to serious disease sequelae, including dilated cardiomyopathy. Previous studies have demonstrated the presence of 5' terminally deleted forms of enterovirus RNAs in heart tissues derived from patients with dilated cardiomyopathy. These deleted RNAs are found in association with very low levels of full-length enterovirus genomic RNAs, an interaction that may facilitate continued persistence while limiting virus particle production. Even in the absence of detectable infectious virus particle production, these deleted viral RNA forms express viral proteinases at levels capable of causing viral pathology. Our studies provide mechanistic insights into how full-length and deleted forms of enterovirus RNA cooperate to stimulate viral protein and RNA synthesis without stimulating infectious viral particle production. They also highlight the importance of targeting enteroviral proteinases to inhibit viral replication while at the same time limiting the long-term pathologies they trigger.

Role of Yme1 in mitochondrial protein homeostasis: from regulation of protein import, OXPHOS function to lipid synthesis and mitochondrial dynamics.

Mitochondria are essential organelles of eukaryotic cells and thus mitochondrial proteome is under constant quality control and remodelling. Yme1 is a multi-functional protein and subunit of the homo-hexametric complex i-AAA proteinase. Yme1 plays vital roles in the regulation of mitochondrial protein homeostasis and mitochondrial plasticity, ranging from substrate degradation to the regulation of protein functions involved in mitochondrial protein biosynthesis, energy production, mitochondrial dynamics, and lipid biosynthesis and signalling. In this mini review, we focus on discussing the current understanding of the roles of Yme1 in mitochondrial protein import via TIM22 and TIM23 pathways, oxidative phosphorylation complex function, as well as mitochondrial lipid biosynthesis and signalling, as well as a brief discussion of the role of Yme1 in modulating mitochondrial dynamics.

Isolation and characterisation of PR3-specific B cells and their immunoglobulin sequences.

BACKGROUND: PR3 autoantibodies are essential to the diagnosis and monitoring of granulomatosus with polyangiitis, but to date no PR3 autoantibody sequences have been published. OBJECTIVES: To identify and characterise PR3-specific B cells from the peripheral blood of patients with PR3 autoantibodies. METHODS: Peripheral blood mononuclear cells from seven patients with PR3 autoantibodies were stained with PR3. B cells that bound PR3 underwent single cell sorting, transcriptome sequencing, and their immunoglobulin sequences expressed as antibodies and tested for PR3-specificity by ELISA. RESULTS: We identified 19 PR3-specific B cells from only one PR3-seropositive patient at a low frequency (0.0075 % of B cells) in the peripheral blood. These were polyclonal, IgG+ and enriched for IgG4, lambda pairing, IGHJ6 gene usage, CDRH3 length, IGHE and CD71 expression. They demonstrated relatively low levels of somatic hypermutation and variably reduced PR3 binding when reverted to germline. CONCLUSIONS: Identifying PR3-specific B cells in the peripheral blood is possible but challenging and those we did identify exhibited features suggesting that PR3-self reactivity may occur early in B-cell development.

Saliva-based Proteinase K method: A rapid and reliable diagnostic tool for the detection of SARS-COV-2 in children.

Early and accurate detection of viruses in children might help prevent transmission and severe diseases. In this study, the severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) detection in children was evaluated using saliva specimens with a Proteinase K (PTK)-based RNA preparation, as saliva collection is a simple and noninvasive procedure, even in young children, with fewer concerns about sample contamination. The saliva-based PTK and the conventional paired nasopharyngeal aspiration (NPA)-based detection methods were compared between COVID-19-positive and -negative children. In addition, the detection rate for SARS-COV-2 and the difference between admission and discharge by the saliva-based PTK method was tested in COVID-19 patients. The diagnostic accuracy of the saliva-based PTK method was 98.8% compared to NP swab-based reverse transcriptase polymerase chain reaction. Saliva samples showed high sensitivity (94.1%) and specificity (100%) when using the PTK method. Furthermore, the saliva-based PTK method significantly reduced the test processing time by 2 h. Notably, Ct values at discharge increased in saliva samples compared with those at admission, which might indicate patients' clinical conditions or virus activity. In conclusion, the saliva-based PTK implemented in this study streamlines RNA extraction, making the process faster, safer, and more cost-effective, demonstrating that this method is a rapid and reliable diagnostic tool for SARS-CoV-2 detection in children.

Extracellular BSA-degrading SAPs in the rare pathogen Meyerozyma guilliermondii strain SO as potential virulence factors in candidiasis.

Meyerozyma guilliermondii (Candida guilliermondii) is one of the Candida species associated with invasive candidiasis. With the potential for expressing industrially important enzymes, M. guilliermondii strain SO possessed 99 % proteome similarity with the clinical ATCC 6260 isolate and showed pathogenicity towards zebrafish embryos. Recently, three secreted aspartyl proteinases (SAPs) were computationally identified as potential virulence factors in this strain without in vitro verification of SAP activity. The quantification of Candida SAPs activity in liquid broth were also scarcely reported. Thus, this study aimed to characterize M. guilliermondii strain SO's ability to produce SAPs (MgSAPs) in different conditions (morphology and medium) besides analyzing its growth profile. MgSAPs' capability to cleave bovine serum albumin (BSA) was also determined to propose that MgSAPs as the potential virulence factors compared to the avirulent Saccharomyces cerevisiae. M. guilliermondii strain SO produced more SAPs (higher activity) in yeast nitrogen base-BSA-dextrose broth compared to yeast extract-BSA-dextrose broth despite insignificantly different SAP activity in both planktonic and biofilm cells. FeCl3 supplementation significantly increased the specific protein activity (∼40 %). The BSA cleavage by MgSAPs at an acidic pH was proven through semi-quantitative SDS-PAGE, sharing similar profile with HIV-1 retropepsin. The presented work highlighted the MgSAPs on fungal cell wall and extracellular milieu during host infection could be corroborated to the quantitative production in different growth modes presented herein besides shedding lights on the potential usage of retropepsin's inhibitors in treating candidiasis. Molecular and expression analyses of MgSAPs and their deletion should be further explored to attribute their respective virulence effects.

Elevated first-trimester neutrophil elastase and proteinase 3 increase the risk of gestational diabetes mellitus and adverse fetal outcomes.

BACKGROUND: Chronic inflammation plays a vital role in the development of gestational diabetes mellitus (GDM). Studies in mouse models show that neutrophil serine proteases (NSPs), neutrophil elastase (NE) and proteinase-3 (PR3) are important drivers of chronic inflammation with consequent metabolic disturbances. This study evaluated the association of NE and PR3 with GDM development and adverse fetal outcomes. METHOD(S): This was a prospective cohort study. Serum PR3 and NE concentration was measured in all enrolled pregnant women in the first and the second trimester to determine the connection between NSPs and GDM and adverse fetal outcomes. Logistic regression, spline regression and linear regression analyses were applied to investigate the association of NE or PR3 with GDM development and adverse fetal outcomes. The concentration of NE and PR3 in placental biopsies was evaluated by semi-quantitative analysis of immunohistochemistry staining. RESULT(S): NE or PR3 concentration in the first trimester, rather than the second, increased more significantly in women with GDM than in those without, regardless of pre-pregnancy body mass index and age. There was a stepwise increase in GDM occurrence as well as comprehensive adverse fetal outcomes across tertiles of NE and PR3. NE and PR3 were positively associated with neutrophil count, pre-pregnancy BMI, plasma glucose level and newborn weight. Logistic regression revealed NE or PR3 to be independent risk factors for the development of GDM and comprehensive adverse fetal outcomes. Spline regression showed a significant increased risk of GDM occurrence and comprehensive adverse fetal outcomes when serum NE concentration exceeded 417.60 ng/mL and a similar result for PR3 and GDM occurrence when the latter exceeded 88.52 ng/mL. Immunohistochemistry data confirmed that enriched NE and PR3 content in placental tissue may have contributed to the development of GDM. CONCLUSION(S): This work demonstrates that excessive first-trimester NE and PR3 increase the risk of GDM development and comprehensive adverse fetal outcomes.

High serum proteinase-3 levels predict poor progression-free survival and lower efficacy of bevacizumab in metastatic colorectal cancer.

BACKGROUND: To improve the prognosis of patients with metastatic colorectal cancer (mCRC), investigating predictive biomarkers of their prognosis and chemotherapeutic responsiveness is necessary. This study aimed to analyze the clinical significance of serum proteinase-3 (PRTN3) as a predictor for prognosis and chemosensitivity, especially to bevacizumab therapy, in mCRC. METHODS: This single-center retrospective observational study enrolled 79 patients with mCRC in our hospital and 353 patients with colorectal cancer in the TCGA database. Preoperative serum PRTN3 levels were measured using an enzyme-linked immunosorbent assay. The clinicopathological characteristics and prognosis according to serum PRTN3 levels were then evaluated. PRTN3 expression in tumor and stromal cells was evaluated immunohistochemically. The impact of PRTN3 levels on angiogenesis and bevacizumab sensitivity was evaluated using the tube formation assay. RESULTS: Serum PRTN3 levels were an independent poor prognostic factor for progression-free survival (PFS) (hazard ratio, 2.082; 95% confidence interval, 1.118-3.647; P=0.010) in patients with mCRC. Similarly, prognostic analysis with TCGA data sets showed poorer overall survival in patients with PRTN3 expression than that in patients without PRTN3 expression, especially in patients with stage IV. Immunohistochemical analysis of resected specimens revealed that stromal neutrophils expressed PRTN3, and their expression level was significantly correlated with serum PRTN3 levels. Interestingly, the effectiveness of first-line chemotherapy was significantly poorer in the high serum PRTN3 level group. High serum PRTN3 was significantly associated with poor PFS (hazard ratio, 3.027; 95% confidence interval, 1.175-7.793; P=0.0161) in patients treated with bevacizumab, an anti-angiogenic inhibitor. The tube formation assay revealed that PRTN3 administration notably augmented angiogenesis while simultaneously attenuating the anti-angiogenic influence exerted by bevacizumab therapy. CONCLUSIONS: Serum PRTN3 levels could be a novel predictive biomarker of PFS of first-line chemotherapy, especially for bevacizumab therapy, in patients with mCRC.

Suitability of reduced dose glucocorticoids therapy regimen for antibody-associated vasculitis patients with TB: a retrospective study.

INTENTION: Immunosuppressive therapy is the major treatment approach for patients with anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV). Due to impaired cellular immunological function and the use of glucocorticoids and immunosuppressants, AAV patients are predisposed to opportunistic infections, including tuberculosis (TB). This retrospective study aims to analyze the clinical characteristics of patients with AAV and TB and explore suitable glucocorticoid regimens for them. So as to provide a basis for future clinical guidelines and have important value for guiding clinical treatment. METHODS: This study retrospectively reviewed 58 AAV patients (18-80 years old) with TB admitted to Changsha Central Hospital Affiliated with the University of South China from 2016.1 to 2023.4 Patients were divided into standard-dose and reduced-dose glucocorticoid groups before retrospectively analyzing their medical records. RESULTS: A total of 58 AAV patients with TB were enrolled, with 15 dying throughout the monitoring period. Through analysis data, compared with the standard-dose group, the reduced group had less proteinuria and hematuria. In survival analysis, the reduced-dose glucocorticoid group had lower mortality than the standard-dose group (P = 0.03); however, no significant difference was noted in the use of immunoglobulin (P = 0.39), tuberculosis activity (P = 0.64), and age stratification (P = 0.40). The BVAS score before treatment and 6 months post-treatment suggest that the two regimens cause the same risk of ESKD (P > 0.05). CONCLUSION: In conclusion, the reduced glucocorticoid dose group can achieve the same curative effect as the standard dose group and has less damage to the kidney in hematuria and proteinuria. Therefore, the reduced glucocorticoid dose treatment regimen may be more suitable for AAV patients with TB.

PAR4 Inhibition Reduces Coronary Artery Atherosclerosis and Myocardial Fibrosis in SR-B1/LDLR Double Knockout Mice.

BACKGROUND: SR-B1 (scavenger receptor class B type 1)/LDLR (low-density lipoprotein receptor) double knockout mice fed a high-fat, high-cholesterol diet containing cholate exhibit coronary artery disease characterized by occlusive coronary artery atherosclerosis, platelet accumulation in coronary arteries, and myocardial fibrosis. Platelets are involved in atherosclerosis development, and PAR (protease-activated receptor) 4 has a prominent role in platelet function in mice. However, the role of PAR4 on coronary artery disease in mice has not been tested. METHODS: We tested the effects of a PAR4 inhibitory pepducin (RAG8) on diet-induced aortic sinus and coronary artery atherosclerosis, platelet accumulation in atherosclerotic coronary arteries, and myocardial fibrosis in SR-B1/LDLR double knockout mice. SR-B1/LDLR double knockout mice were fed a high-fat, high-cholesterol diet containing cholate and injected daily with 20 mg/kg of either the RAG8 pepducin or a control reverse-sequence pepducin (SRQ8) for 20 days. RESULTS: Platelets from the RAG8-treated mice exhibited reduced thrombin and PAR4 agonist peptide-mediated activation compared with those from control SRQ8-treated mice when tested ex vivo. Although aortic sinus atherosclerosis levels did not differ, RAG8-treated mice exhibited reduced coronary artery atherosclerosis, reduced platelet accumulation in atherosclerotic coronary arteries, and reduced myocardial fibrosis. These protective effects were not accompanied by changes in circulating lipids, inflammatory cytokines, or immune cells. However, RAG8-treated mice exhibited reduced VCAM-1 (vascular cell adhesion molecule 1) protein levels in nonatherosclerotic coronary artery cross sections and reduced leukocyte accumulation in atherosclerotic coronary artery cross sections compared with those from SRQ8-treated mice. CONCLUSIONS: The PAR4 inhibitory RAG8 pepducin reduced coronary artery atherosclerosis and myocardial fibrosis in SR-B1/LDLR double knockout mice fed a high-fat, high-cholesterol diet containing cholate. Furthermore, RAG8 reduced VCAM-1 in nonatherosclerotic coronary arteries and reduced leukocyte and platelet accumulation in atherosclerotic coronary arteries. These findings identify PAR4 as an attractive target in reducing coronary artery disease development, and the use of RAG8 may potentially be beneficial in cardiovascular disease.

Recent advances in the knowledge of the mechanism of reflux hypersensitivity.

Reflux hypersensitivity (RH) is a subtype of gastroesophageal reflux disease. The Rome IV criteria separated RH from the original nonerosive reflux disease subgroup and classified it as a new functional oesophageal disease. Recently, the pathogenesis of RH has become the focus of research. According to the latest research reports, upregulation of acid-sensitive receptors, distribution of calcitonin gene-related peptide-positive nerve fibres, and psychiatric comorbidity have key roles in the pathogenesis of RH. This work reviews the latest findings regarding RH mechanisms.

Functional characterization of serine proteinase inhibitor Kazal-Type in the red claw crayfish Cherax quadricarinatus.

Serine protease inhibitors Kazal type (SPINKs) function in physiological and immunological processes across multicellular organisms. In the present study, we identified a SPINK gene, designated as CqSPINK, in the red claw crayfish Cherax quadricarinatus, which is the ortholog of human SPINK5. The deduced CqSPINK contains two Kazal domains consisting of 45 amino acid residues with a typical signature motif C-X3-C-X5-PVCG-X5-Y-X3-C-X6-C-X12-14-C. Each Kazal domain contains six conserved cysteine residues forming three pairs of disulfide bonds, segmenting the structure into three rings. Phylogenetic analysis revealed CqSPINK as a homolog of human SPINK5. CqSPINK expression was detected exclusively in hepatopancreas and epithelium, with rapid up-regulation in hepatopancreas upon Vibrio parahaemolyticus E1 challenge. Recombinant CqSPINK protein (rCqSPINK) was heterologously expressed in Escherichia coli and purified for further study. Proteinase inhibition assays demonstrated that rCqSPINK could potently inhibit proteinase K and subtilisin A, weakly inhibit α-chymotrypsin and elastase, but extremely weak inhibit trypsin. Furthermore, CqSPINK inhibited bacterial secretory proteinase activity from Bacillus subtilis, E. coli, and Staphylococcus aureus, and inhibited B. subtilis growth. These findings suggest CqSPINK's involvement in antibacterial immunity through direct inhibition of bacterial proteases, contributing to resistance against pathogen invasion.

Verification, implementation and harmonization of automated chemiluminescent immunoassays for MPO- and PR3-ANCA detection.

OBJECTIVES: Antineutrophil cytoplasmic antibody (ANCA) testing assists clinicians diagnose ANCA-associated vasculitis (AAV). We aimed to verify and harmonize chemiluminescent immunoassays for the detection of myeloperoxidase (MPO)- and proteinase 3 (PR3)-ANCA. METHODS: An in-house ELISA, a capture ELISA, and a chemiluminescent assay QUANTA Flash on a BIO-FLASH analyzer were used to detect MPO- and PR3-ANCA in sera from 39 patients with AAV, 55 patients with various non-AAV, and 66 patients with connective tissue diseases. The results of the assays were evaluated, and their clinical performance was assessed. The precision and linearity of the QUANTA Flash assays were determined, and likelihood ratios (LRs) for AAV at diagnosis were calculated. RESULTS: The precision and linearity of the QUANTA Flash assays were confirmed. Overall agreement between 97.5 and 98.8 % and Cohen's kappa coefficients between 0.861 and 0.947 were observed for the results of the QUANTA Flash assays and ELISAs. The diagnostic sensitivity, specificity, and ROC analysis of the assays for AAV were statistically similar (in-house ELISA 89.7 %, 95.0 %, and 0.937; capture ELISA 92.3 %, 98.3 %, and 0.939; and QUANTA Flash 89.7 %, 95.9 %, and 0.972). For the QUANTA Flash assay results, the interval-specific LRs for AAV at diagnosis were: 0-8 CU had LR 0.08, 8-29 CU had LR 1.03, 29-121 CU had LR 7.76, 121-191 CU had LR 12.4, and >191 CU had LR ∞. CONCLUSIONS: The QUANTA Flash MPO and PR3 assays provide precise and consistent results and have comparable clinical utility for AAV. The calculated LRs were consistent with published LRs, confirming the utility of LRs for harmonization of ANCA results.

Silkworm pupae protein co-degradation by magnetic nanoparticles immobilized proteinase K and Mucor circinelloides aspartic protease for further utilization of sericulture by-products.

Silkworm pupae, by-product of sericulture industry, is massively discarded. The degradation rate of silkworm pupae protein is critical to further employment, which reduces the impact of waste on the environment. Herein, magnetic Janus mesoporous silica nanoparticles immobilized proteinase K mutant T206M and Mucor circinelloides aspartic protease were employed in the co-degradation. The thermostability of T206M improved by enhancing structural rigidity (t1/2 by 30 min and T50 by 5 °C), prompting the degradation efficiency. At 65 °C and pH 7, degradation rate reached the highest of 61.7%, which improved by 26% compared with single free protease degradation. Besides, the immobilized protease is easy to separate and reuse, which maintains 50% activity after 10 recycles. Therefore, immobilized protease co-degradation was first applied to the development and utilization of silkworm pupae resulting in the release of promising antioxidant properties and reduces the environmental impact by utilizing a natural and renewable resource.