Serine Protease Research Articles

Granzyme K provides protection for Nile tilapia (Oreochromis niloticus) from acute bacterial infection.

Granzyme K (GzmK) is a trypsin-like serine protease that functions as a pro-apoptotic protease, has non-cytotoxic functions, and is involved in various physiopathological processes. However, the impact of GzmK on the immunological response of Nile tilapia against bacterial challenge is still poorly explored. In this study, we identified the GzmK gene from Nile tilapia (On-GzmK) and investigated its immunologic roles through in vivo experiments. On-GzmK has an open reading frame of 786 bp and encodes 255 amino acids. On-GzmK has over 60 % genetic similarity with other fish and over 30 % similarity with mammals. Its transcript levels were highest in the liver and vastly increased after being challenged with Streptococcus agalactiae and Aeromonas hydrophila. In vivo experiments implied that On-GzmK might promote inflammatory responses and regulate apoptosis and pyroptosis by participating in the activation of multiple signaling pathways, such as JAK-STAT, MAPK, and NF-κB. The present observations offer additional theoretical support for investigating the processes by which GzmK shields fish against bacterial infections.

Supplementation of polyclonal antibodies, developed against epitope-string toxin-specific peptide immunogens, to commercial polyvalent antivenom, shows improved neutralization of Indian Big Four and Naja kaouthia snake venoms

Snakebites profoundly impact the rural population of tropical nations, leading to significant socio-economic repercussions. Polyvalent antivenom (PAV) therapy faces several limitations, including intra-specific variations and poor efficacy against some major toxins and low molecular mass, poorly immunogenic toxins, which contribute to increased mortality and morbidity rates. Innovative strategies for developing novel antivenoms are continuously explored to address these challenges. The present study focuses on designing of 17 epitope-string toxin-specific peptide immunogens from pharmacologically active major and/or poorly immunogenic toxins (snake venom metalloprotease, Kunitz-type serine protease inhibitor, phospholipase A2, three-finger toxin) from the venom of the ‘Big Four’ venomous snakes and Naja kaouthia (NK) in India. These custom peptide antibodies demonstrated robust immuno-reactivity against the venoms ‘Big Four’ and NK. When these antibodies were supplemented with commercial PAV at a defined ratio (formulated polyvalent antivenom or FPAV), it significantly enhanced the neutralization of snake venom enzymes and in vivo neutralization of lethality and pharmacological activities such as haemorrhage, necrosis, pro-coagulant, defibrinogenation, and myotoxicity of ‘Big Four’ and NK venoms compared to PAV in mice. The present study highlights a promising strategy for developing next-generation antivenoms using synthetic peptide-based immunogens, offering a targeted approach to address the limitations of current antivenom therapy.

Bioinformatic study of keratinases from the S01 family from Streptomyces microflavus strain Dsm 40593

Aim. Determination and study of the primary structures and domain organization of keratinase precursors (serine proteases from the S01 family) of S. microflavus DSM 40593 using bioinformatic methods. Methods. In the research information presented in databases on the NCBI server was analyzed. Computerized sequence analysis was performed using the BLAST program on the NCBI server. Results. BLAST analysis of the genome sequence of the strain S. microflavus DSM 40593 was carried out using the sequence of the precursor AGK81825 as a query. Thanks to bioinformative analysis, seven genes encoding precursors of serine proteases from the S01 family were found in the genome of the strain S. microflavus DSM 40593. Conclusions. In the genomes of streptomycetes, there may be more than one gene encoding precursors of keratinases from the S01 family. It was established that the amino acid sequences of the enzymatic domains of keratinase precursors from the S01 family are characterized by higher similarity indices than the sequences of foldase domains.

Formulation and Evaluation of Haemocoagulase Gel for Dental Pulp- an In-vitro study.

This study aims to develop a novel hemostatic gel of hemocoagulase, a snake venom-derived serine protease enzyme, by incorporating it into the matrix of bioadhesive polymers to prevent damage due to hemorrhage. Haemocoagulase was combined with polymers Carbopol 940 and HPMC K 100, and these formulations were evaluated for physical properties, efficacy, safety and stability. Batches F3 and F6 were selected for further studies on animal models, which were found to be safe without any skin irritation effect and effective as hemostats with blood clotting times of 1.24 ± 0.007 min and 1.36 ± 0.014 min, respectively, in a rat liver incision model. From this study, it can be suggested that the combination of hemocoagulase with bioadhesive polymer can provide a novel hemostatic tool for controlling bleeding.

Salivary proteins modulate Candida albicans virulence and may prevent oropharingeal candidiasis.

Oral candidiasis can be presented in different ways due to the virulence factors of its etiology such as Candida albicans that have developed an effective set of these factors that are able to improve its pathogenesis. The role of salivary immunological components in the development of candidiasis can provide insights for the development of new methodologies aiming to control this disease. The aim of this study was to evaluate the antifungal activity of two salivary components, histatin 5 and lactoferrin on C. albicans viability and virulence using a fluconazole resistant C. albicans clinical strain. Results showed that histatin 5 and lactoferrin decreased cell viability, and the cell surface hydrophobicity was increased by 18% in presence of 151µg/mL of histatin 5 but was not altered by lactoferrin. It was observed the reduction of 69.3% in the expression of mannoproteins on C. albicans surface in the presence of 151µg/mL of histatin, but proteolytic activity of serine proteinases was not inhibited by any of the proteins. Histatin 5 altered cell ultrastructure predominantly in the cytoplasmic compartment. However, this peptide does not interfere with mitochondrial function neither in membrane permeability of the yeasts. The association index between C. albicans and epithelial cells was increased by 51% in presence of 151µg/mL of histatin. Results suggest that histatin 5 and lactoferrin affects viability and virulence of C. albicans at physiological levels, and the maintenance of these levels may be essential in the prevention of oropharyngeal candidiasis. Exogenous administration of these proteins may become a therapeutic alternative for resistant strains of C. albicans, circumventing toxicity issues, considering their constitutive features.

The influence of сestodes parasitizing in the small intestine of herring gull chicks on the activity of the host proteases

The activity of proteases and subclasses of proteases in small intestines of the herring gull (Larus argentatus (Pontoppidan, 1763)) chicks were determined. Changes of the protease activity in chicks infested by cestodes Tetrabothrius erostris (Loennberg, 1889) (Cestoda: Tetrabothriidae), Microsomacanthus ductilus (Linton, 1927) (Cestoda: Hymenolepididae), Wardium cirrosa (Krabbe, 1869) (Cestoda: Aploparaksidae), Arctotaenia tetrabothrioides (Loennberg, 1890) and Alcataenia larina (Krabbe, 1869) (Cestoda: Dilepididae) were studied. The activity of serine proteases decreased in the small intestine of gulls at the sites of cestode parasitism with high infestation values (M. ductilus in the proximal segment, W. cirrosa in the distal segment, A. tetrabothrioides, T. erostris and A. larina in the medial segment). The activity of cysteine proteases increased in the sites of localization of cestodes M. ductilus, W. cirrosa and A. larina. All the studied cestodes adsorbed proteases on the surface of the tegument, most of those were represented by serine proteases. Cestode homogenates of W. cirrosa, M. ductilus, A. tetrabothrioides, T. erostris and A. larina inhibited the activity of host proteases and the activity of commercial trypsin.

Glial-Cell-Line-Derived Neurotrophic Factor Promotes Glioblastoma Cell Migration and Invasion via the SMAD2/3-SERPINE1-Signaling Axis.

Glial-cell-line-derived neurotrophic factor (GDNF) is highly expressed and is involved in the malignant phenotype in glioblastomas (GBMs). However, uncovering its underlying mechanism for promoting GBM progression is still a challenging work. In this study, we found that serine protease inhibitor family E member 1 (SERPINE1) was a potential downstream gene of GDNF. Further experiments confirmed that SERPINE1 was highly expressed in GBM tissues and cells, and its levels of expression and secretion were enhanced by exogenous GDNF. SERPINE1 knockdown inhibited the migration and invasion of GBM cells promoted by GDNF. Mechanistically, GDNF increased SERPINE1 by promoting the phosphorylation of SMAD2/3. In vivo experiments demonstrated that GDNF facilitated GBM growth and the expressions of proteins related to migration and invasion via SERPINE1. Collectively, our findings revealed that GDNF upregulated SERPINE1 via the SMAD2/3-signaling pathway, thereby accelerating GBM cell migration and invasion. The present work presents a new mechanism of GDNF, supporting GBM development.

High resolution analysis of proteolytic substrate processing

Members of the widely conserved high temperature requirement A (HtrA) family of serine proteases are involved in multiple aspects of protein quality control. In this context, they have been shown to efficiently degrade misfolded proteins or protein fragments. However, recent reports suggest that folded proteins can also be native substrates. To gain a deeper understanding of how folded proteins are initially processed and subsequently degraded into short peptides by human HTRA1, we established an integrated and quantitative approach using time-resolved mass spectrometry, CD spectroscopy, and bioinformatics. The resulting data provide high-resolution information on up to 178 individual proteolytic sites within folded ANXA1 (consisting of 346 amino acids), the relative frequency of cuts at each proteolytic site, the preferences of the protease for the amino acid sequence surrounding the scissile bond, as well as the degrees of sequential structural relaxation and unfolding of the substrate that occur during progressive degradation. Our workflow provides precise molecular insights into protease-substrate interactions, which could be readily adapted to address other posttranslational modifications such as phosphorylation in dynamic protein complexes.

An integrative pan-cancer analysis of MASP1 and the potential clinical implications for the tumor immune microenvironment

Mannose-binding lectin-associated serine protease 1 (MASP1) plays a crucial role in the complement lectin pathway and the mediation of immune responses. However, comprehensive research on MASP1 across various cancer types has not been performed to date. This study aimed to evaluate the significance of MASP1 in pan-cancer.The Cancer Genome Atlas (TCGA), UCSC Xena and Genotype Tissue Expression (GTEx) databases were used to evaluate the expression profiles, genomic features, prognostic relevance, and immune microenvironment associations of MASP1 across 33 cancer types. We observed significant dysregulation of MASP1 expression in multiple cancers, with strong associations between MASP1 expression levels and diagnostic value as well as patient prognosis. Mechanistic insights revealed significant correlations between MASP1 levels and various immunological and genomic factors, including tumor-infiltrating immune cells (TIICs), immune-related genes, mismatch repair (MMR), tumor mutation burden (TMB), and microsatellite instability (MSI), highlighting a critical regulatory function of MASP1 within the tumor immune microenvironment (TIME). In vitro and in vivo experiments demonstrated that MASP1 expression was markedly decreased in liver hepatocellular carcinoma (LIHC). Moreover, the overexpression of MASP1 in hepatocellular carcinoma (HCC) cell lines significantly inhibited their proliferation, invasion and migration. In conclusion, MASP1 exhibits differential expression in the pan-cancer analyses and might play an important role in TIME. MASP1 is a promising prognostic biomarker and a potential target for immunological research, particularly in LIHC.

A randomized phase I study of the safety and pharmacokinetics of BI 1291583 in healthy Japanese male subjects

AimsBronchiectasis patients face an unmet need for treatment options that reduce inflammation. Cathepsin C inhibition is expected to achieve this by reducing the activation of neutrophil‐derived serine proteases. Here, we present safety and pharmacokinetic (PK) data from a Phase I trial evaluating the novel cathepsin C inhibitor BI 1291583 in healthy Japanese male subjects.MethodsThis randomized, double‐blind, placebo‐controlled, parallel‐group study investigated BI 1291583 in healthy Japanese male subjects (jRCT2071210111) and consisted of a single‐rising‐dose (SRD) part and a multiple‐dose (MD) part. The primary endpoint was the percentage of subjects with drug‐related treatment‐emergent adverse events (AEs). Secondary PK endpoints (SRD: AUC0‐∞ and Cmax; MD: AUCτ,1 and Cmax,1 after first dose and AUCτ,ss and Cmax,ss after last dose), as well as further safety and PK endpoints, were also assessed.ResultsOverall, 36 subjects (n = 24 for SRD part; n = 12 for MD part) entered this Phase I trial. BI 1291583 was safe and well tolerated across the doses tested. All AEs were of mild intensity, with no drug‐related treatment‐emergent AEs, deaths, serious AEs or AEs of special interest reported in either part of the trial. Following both SRD and MD administration, BI 1291583 was readily absorbed, and PK was supraproportional over the doses assessed.ConclusionThe results show that BI 1291583 has an appropriate benefit–risk ratio for Japanese patients, with no safety or exposure concerns at the doses studied. Japanese patients with bronchiectasis can be safely integrated into future global clinical trials of BI 1291583, with no dose adjustment required.

The fibroblast activation protein alpha as a biomarker of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a rare, chronic, and progressive interstitial lung disease with an average survival of approximately 3 years. The evolution of IPF is unpredictable, with some patients presenting a relatively stable condition with limited progression over time, whereas others deteriorate rapidly. In addition to IPF, other interstitial lung diseases can lead to pulmonary fibrosis, and up to a third have a progressive phenotype with the same prognosis as IPF. Clinical, biological, and radiological risk factors of progression were identified, but no specific biomarkers of fibrogenesis are currently available. A recent interest in the fibroblast activation protein alpha (FAPα) has emerged. FAPα is a transmembrane serine protease with extracellular activity. It can also be found in a soluble form, also named anti-plasmin cleaving enzyme (APCE). FAPα is specifically expressed by activated fibroblasts, and quinoline-based specific inhibitors (FAPI) were developed, allowing us to visualize its distribution in vivo by imaging techniques. In this review, we discuss the use of FAPα as a useful biomarker for the progression of lung fibrosis, by both its assessment in human fluids and/or its detection by imaging techniques and immunohistochemistry.

The Proteolytic Activity of Neutrophil-Derived Serine Proteases Bound to the Cell Surface Arming Lung Epithelial Cells for Viral Defense.

The collaboration between cellular proteases and host cells is pivotal in mounting an effective innate immune defense. Of particular interest is the synergistic interaction between cathepsin G (CatG) and neutrophil elastase (NE), which are proteases secreted by activated neutrophils, and the human alveolar basal epithelial cell line (A549) and the human lung epithelial-like cell line (H1299), because of the potential implications for viral infection. Our study aimed to investigate the binding capacity of CatG and NE on the surface of A549 and H1299 cells through preincubation with purified CatG and NE; thereby, the proteolytic activity could be detected using activity-based probes. Both CatG and NE were capable of binding to the cell surface and exhibited proteolytic activity, leading to increased cell surface levels of MHC I molecules, which is crucial for displaying the endogenous antigenic repertoire. In addition, CatG cleaved the S2' site of the SARS-CoV-2 spike protein at two specific sites (815RS816 and 817FI818) as well as NE (813SK814 and 818IE819), which potentially leads to the destruction of the fusion peptide. Additionally, furin required the presence of Ca2+ ions for the distinct cleavage site necessary to generate the fusion peptide. Overall, the findings suggest that CatG and NE can fortify target cells against viral entry, underscoring the potential significance of cell surface proteases in protecting against viral invasion.

Missense variant rs75603675 within TMPRSS2 gene is associated with the increased risk of severe form of COVID-19

Host genetics among other factors play an important role in COVID-19 disease severity. TMPRSS2, a serine protease, facilitates the priming of the SARS-CoV-2 spike protein, which is essential for the virus to enter the host cell. Several studies had targeted the TMPRSS2 polymorphisms with respect to SARS-CoV-2 infection and COVID-19 disease severity. Initially, the Whole Exome Sequencing (WES) of 7 healthy individuals and 22 COVID-19 patients with different degrees of disease severity was conducted to find the mutational landscape in different genes. A total of 26 single nucleotide polymorphisms (SNPs) were identified of which six were within the exons of TMPRSS2 (four synonymous and two nonsynonymous variants) while the rest of the 20 SNPs were recognized within the flanking intronic regions of TMPRSS2 gene. The nonsynonymous SNPs, rs75603675 and rs12329760, were further evaluated for association with disease severity in a larger sample size of 120 individuals by PCR followed by Sanger sequencing. Neither allelic nor genotypic distributions of rs12329760 were significantly associated with COVID-19 disease severity. However, individuals harboring the A allele of rs75603675 was found to have higher risk of severe COVID-19 compared to the C allele [OR (95%CI): 1.95 (1.11–3.39), p = 0.019]. Also, the genotype A/A [OR (95%CI): 5.13 (1.00–26.38), p = 0.033] of rs75603675 was associated with increased risk of severe COVID-19 under the recessive genetic inheritance model. Although the impact of COVID-19 pandemic has waned due to vaccination and public health measures, continued research on the association of COVID-19 disease severity and infection susceptibility with host genetics is required to shed valuable insights on the future long-term health effects of COVID-19 and impact of new variants on different populations and enable implication of proper informed healthcare strategies during future public health crises.

Hepsin as a potential therapeutic target for alleviating acetaminophen-induced hepatotoxicity via gap-junction regulation and oxidative stress modulation

Acetaminophen (APAP) overdose is a leading cause of drug-induced liver damage, highlighting the limitations of current emergency treatments that primarily involve administering the glutathione precursor N-acetylcysteine and supportive therapy. This study highlights the essential protective role of the type II transmembrane serine protease (TTSP), hepsin, in mitigating acetaminophen-induced liver injury, particularly through its regulation of gap junction (GJ) abundance in response to reactive oxygen stress in the liver. We previously reported that reduced levels of activated hepatocyte growth factor and the c-Met receptor tyrosine kinase—both of which are vital for maintaining cellular redox balance—combined with increased expression of GJ proteins in hepsin-deficient mice. Here, we show that hepsin deficiency in mice exacerbates acetaminophen toxicity compared to wild-type mice, leading to more severe liver pathology, elevated oxidative stress, and greater mortality within 6 h after exposure. Administering hepsin had a protective effect in both mouse models, reducing hepatotoxicity by modulating GJ abundance. Additionally, transcriptome analysis and a functional GJ inhibitor have highlighted hepsin's mechanism for managing oxidative stress. Combining hepsin with relatively low doses of N-acetylcysteine had a synergistic effect that was more efficacious than high-dose N-acetylcysteine alone. Our results illustrate the crucial role of hepsin in modulating the abundance of hepatic GJs and reducing oxidative stress, thereby offering early protection against acetaminophen-induced hepatotoxicity and a new, combination approach. Emerging as a promising therapeutic target, hepsin holds potential for combination therapy with N-acetylcysteine, paving the way for novel approaches in managing drug-induced liver injury.Graphical 1. Hepsin−/− mice exhibit exacerbated APAP toxicity, resulting in more severe liver damage, elevated oxidative stress, and higher mortality.2. Hepsin is crucial in protecting against APAP-induced liver injury by regulating gap junctions and reducing oxidative stress.3. Combining hepsin with low doses of N-acetylcysteine provides greater protection against APAP-induced hepatotoxicity than high-dose NAC alone.

Harringtonine metabolites: 5'-de-O-methylharringtonine and cephalotaxine, targeting spike protein and TMPRSS2 to double block membrane fusion of SARS-CoV-2 and its variants

Membrane fusion is the main pathway for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to invade host cells. Harringtonine (HT), derived from cephalotaxus fortunei Hook. f., has been recognized as an effective antagonist of SARS-CoV-2. It can directly block the active binding of spike (S) protein to host angiotensin converting enzyme 2 (ACE2), as well as hinder the enzymolysis of transmembrane serine proteases 2 (TMPRSS2). This study examined the potential of HT metabolites, 5'-de-O-methylharringtonine and cephalotaxine, as the membrane fusion inhibitors for SARS-CoV-2. 5'-De-O-methylharringtonine was synthesized and subsequently characterized by high resolution mass spectrometry and nuclear magnetic resonance to be structurally consistent, with a purity of 92.677% determined by reverse phase high performance liquid chromatography. Both 5'-de-O-methylharringtonine and cephalotaxine can specifically bind to SARS-CoV-2 S protein and TMPRSS2 using cell membrane chromatography. They can form hydrogen bonds with key sites that correlated highly with the enhanced binding affinity of SARS-CoV-2 and its variants to ACE2 or nafamostat to TMPRSS2. Moreover, 5'-de-O-methylharringtonine and cephalotaxine can inhibit pseudotyped virus entry and membrane fusion in a dose-dependent manner, with enhanced effectiveness upon elevated expression of TMPRSS2. Importantly, they displayed low cytotoxic effects on human normal cell lines. Our study suggested that 5'-de-O-methylharringtonine and cephalotaxine were of low toxicity and safety for humans as potential antagonists of SARS-CoV-2 and its variants, which deserve further validation in a biosafety level 3 facility.

Small leucine zipper protein negatively regulates liver fibrosis by suppressing the expression of plasminogen activator inhibitor-1

Liver fibrosis, which is caused by viral infection, toxic exposure, and autoimmune diseases, is a chronic liver disease. Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor of tissue-type plasminogen activator (tPA) and urokinase plasminogen activator, which convert plasminogen into plasmin. Therefore, PAI-1 suppresses fibrinolysis by blocking plasmin synthesis and is involved in liver fibrosis via extracellular matrix deposition. Small leucine zipper protein (sLZIP) acts as a transcription factor and plays critical roles in many cellular processes. However, the role of sLZIP in liver fibrosis remains unclear. In this study, we investigated the role of sLZIP in regulating PAI-1 transcription and liver fibrosis. sLZIP knockdown enhanced the expression of PAI-1 at the mRNA and protein levels. sLZIP knockdown also increased PAI-1 secretion and suppressed blood clot lysis by blocking tPA activity. Moreover, conditioned medium derived from sLZIP knockdown cells downregulated the expression of matrix metalloprotease (MMP)-2 and MMP-9 in the presence of tPA in hepatic stellate cells (HSCs). Liver-specific sLZIP knockout mice showed deteriorated liver fibrosis compared to control mice in a bile duct ligation-induced fibrosis model. These findings demonstrate that sLZIP functions as a negative regulator of liver fibrosis by suppressing PAI-1 transcription and HSC activation.

Pharmacological inhibition of cathepsin S and of NSPs-AAP-1 (a novel, alternative protease driving the activation of neutrophil serine proteases)

An uncontrolled activity of neutrophil serine proteases (NSPs) contributes to inflammatory diseases. Cathepsin C (CatC) is known to activate NSPs during neutrophilic differentiation and represents a promising pharmacological target in NSP-mediated diseases. In humans, Papillon-Lefèvre syndrome (PLS) patients have mutations in theirCTSC gene, resulting in the complete absence of CatC activity. Despite this, low residual NSP activities are detected in PLS neutrophils (<10% vs healthy individuals), suggesting the involvement of CatC-independent proteolytic pathway(s) in the activation of proNSPs. This prompted us to characterize CatC-independent NSP activation pathways by blocking proCatC maturation. In this study, we show that inhibition of intracellular CatS almost completely blocked CatC maturation in human promyeloid HL-60 cells. Despite this, NSP activation was not significantly reduced, confirming the presence of a CatC-independent activation pathway involving a CatC-like protease that we termed NSPs-AAP-1. Similarly, when human CD34+ progenitor cells were treated with CatS inhibitors during neutrophilic differentiation in vitro, CatC activity was nearly abrogated but ∼30% NSP activities remained, further supporting the existence of NSPs-AAP-1. Our data indicate that NSPs-AAP-1 is a cysteine protease that is inhibited by reversible nitrile compounds designed for CatC inhibition. We further established a proof of concept for the indirect, although incomplete, inhibition of NSPs by pharmacological targeting of CatC maturation using CatS inhibitors. This emphasizes the potential of CatS as a therapeutic target for inflammatory diseases. Thus, preventing proNSP maturation using a CatS inhibitor, alone or in combination with a CatC/NSPs-AAP-1 inhibitor, represents a promising approach to efficiently control the extent of tissue injury in neutrophil-mediated inflammatory diseases.

Comparative Genomic Analyses of Colletotrichum lindemuthianum Pathotypes with Different Virulence Levels and Lifestyles.

Colletotrichum lindemuthianum is the most frequent pathogenic fungus of the common bean Phaseolus vulgaris. This filamentous fungus employs a hemibiotrophic nutrition/infection strategy, which is characteristic of many Colletotrichum species. Due to host-pathogen coevolution, C. lindemuthianum includes pathotypes with a diversity of virulence against differential common bean varieties. In this study, we performed comparative genomic analyses on three pathotypes with different virulence levels and a non-pathogenic pathotype, isolated from different geographical areas in Mexico. Our results revealed large genomes with high transposable element contents that have undergone expansions, generating intraspecific diversity. All the pathotypes exhibited a similar number of clusters of orthologous genes (COGs) and Gene Ontology (GO) terms. TFomes contain families that are typical in fungal genomes; however, they show different contents between pathotypes, mainly in transcription factors with the fungal-specific TF and Zn2Cys6 domains. Peptidase families mainly contain abundant serine peptidases, metallopeptidases, and cysteine peptidases. In the secretomes, the number of genes differed between the pathotypes, with a high percentage of candidate effectors. Both the virulence gene and CAZyme gene content for each pathotype was abundant and diverse, and the latter was enriched in hemicellulolytic enzymes. We provide new insights into the nature of intraspecific diversity among C. lindemuthianum pathotypes and the origin of their ability to rapidly adapt to genetic changes in its host and environmental conditions.

Nutrient starvation activates ECM remodeling gene enhancers associated with inflammatory bowel disease risk in fibroblasts.

Nutrient deprivation induces a reversible cell cycle arrest state termed quiescence, which often accompanies transcriptional silencing and chromatin compaction. Paradoxically, nutrient deprivation is associated with activated fibroblast states in pathological microenvironments in which fibroblasts drive extracellular matrix (ECM) remodeling to alter tissue environments. The relationship between nutrient deprivation and fibroblast activation remains unclear. Here, we report that serum deprivation extensively activates transcription of ECM remodeling genes in cultured fibroblasts, despite the induction of quiescence. Starvation-induced transcriptional activation accompanied large-scale histone acetylation of putative distal enhancers, but not promoters. The starvation-activated putative enhancers were enriched for non-coding genetic risk variants associated with inflammatory bowel disease (IBD), suggesting that the starvation-activated gene regulatory network may contribute to fibroblast activation in IBD. Indeed, the starvation-activated gene PLAU, encoding uPA serine protease for plasminogen and ECM, was upregulated in inflammatory fibroblasts in the intestines of IBD patients. Furthermore, the starvation-activated putative enhancer at PLAU, which harbors an IBD risk variant, gained chromatin accessibility in IBD patient fibroblasts. This study implicates nutrient deprivation in transcriptional activation of ECM remodeling genes in fibroblasts and suggests nutrient deprivation as a potential mechanism for pathological fibroblast activation in IBD.

Retesting Intervals for Tryptase

What Is the Issue? Tryptase is a serine protease primarily produced and released by mast cells. Tryptase measures are most used to diagnose and manage anaphylaxis and mastocytosis, and the timing of tryptase measurement is crucial for its utility. The clinical utility of tryptase remains unclear, and available evidence showed that more than 30% of patients with acute anaphylaxis did not exhibit an elevated tryptase level, which suggests that repeated tryptase testing may be overused in this population. Appropriate retesting intervals or frequency for tryptase retesting in patients with anaphylaxis or mastocytosis remain unclear. What Did We Do? To inform decisions about the use and timing of repeat tryptase testing for patients with confirmed or suspected anaphylaxis and patients with cutaneous or systemic mastocytosis, we identified and summarized related recommendations and publications on the potential biological or physiological factors that may impact retesting intervals in these populations. We searched key resources, including journal citation databases, and conducted a focused internet search for relevant evidence published since 2014. One reviewer screened articles for inclusion based on predefined criteria, critically appraised the included evidence-based guidelines, and narratively summarized the findings. What Did We Find? For patients with suspected anaphylaxis, identified guidelines suggest measuring tryptase levels 1 to 2 times during the event onset (e.g., immediately or within 4 hours) and 1 to 2 times after resolution of symptoms (e.g., after 24 hours) to establish baseline tryptase levels. These recommendations were based on very low certainty of evidence. For patients with systemic mastocytosis, 1 evidence-based guideline in Brazil recommends testing tryptase once a year, which is based on very low certainty of evidence. Baseline tryptase level varies very little over time within the same individual and is determined by their genetic background. For patients with anaphylaxis, the timing of the peak tryptase level is from 1 minute to 6 hours (median 30 minutes), and the half-life of serum tryptase is about 1.5 hours to 2.5 hours and needs up to 24 hours to return to baseline level. We did not find any recommendations that met our inclusion criteria regarding the minimum retesting interval for tryptase testing or the use of repeat or serial tryptase testing in patients with confirmed or suspected anaphylaxis or with cutaneous or systemic mastocytosis. What Does It Mean? The available recommendations suggest testing blood tryptase at the acute (1 or 2 times) and baseline phases (1 or 2 times) for patients with suspected anaphylaxis. There is little rationale for the ongoing monitoring of tryptase levels beyond the recommended 4 time points for patients with anaphylaxis. For patients with mastocytosis, the guidelines recommend retesting tryptase only once a year and do not support frequent repeat tryptase testing within a year. Decision-makers and clinicians should consider the cost of tryptase testing, potential burden on health care resources, local lab capacity, or accessibility of testing facilities when making decisions regarding tryptase retesting. Given the very low-quality evidence regarding measuring tryptase and the unclear utility of tryptase testing for patients with anaphylaxis, future research that focuses on the clinical utility of tryptase relative to clinical assessment for anaphylaxis is needed.