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Peptidylarginine deiminase 4 (PAD4) is an enzyme that modifies proteins by converting positively charged arginine residues to neutral citrulline residues. This process, termed citrullination, has been known to trigger NETosis, a neutrophil cell death pathway involving the release of neutrophil extracellular traps (NETs). Abnormal PAD4 activity and protein citrullination have been linked to various diseases, including those affecting the central nervous system. Herein we investigated the profile of PAD4 expression in an animal model of stroke induced by middle cerebral artery occlusion (MCAO). PAD4 levels were significantly elevated in the ischemic core and penumbra of the affected hemisphere at 3–6 and 6–48 h post-MCAO, respectively. Notably, NETosis induction, indicated by the upregulation of CitH3 (citrullinated histone H3, a NETosis marker), was observed between 48 and 96 h post-MCAO, peaking at 96 h. While PAD4 was present in most brain cell types of sham controls, strong PAD4 induction was primarily observed in neurons during the peak PAD4 induction period (12–24 h post-MCAO). Importantly, intranasal administration of the PAD4 inhibitor BB-Cl-amidine (BBCA) significantly reduced infarct volume and improved neurological and functional outcomes at 24 h post-MCAO, demonstrating a strong protective effect of PAD4 inhibition in ischemic stroke. Staining with an antibody that recognizing citrullinated proteins (F95) revealed an accumulation of these proteins, especially degenerating neurons, however, BBCA treatment significantly suppressed this accumulation in dying neurons. These findings indicate that PAD4-mediated protein citrullination in neurons plays a critical role in promoting ischemic brain damage. Furthermore, delayed administration of BBCA (at 48/72 h post-MCAO) suppresses the NETosis induction observed at 96 h post-MCAO, potentially ameliorating repair processes such as blood vessel regeneration. Collectively, these findings suggest a complex role of PAD4 in cerebral ischemia, with neuroprotective effects (NETosis-independent function) during the acute to subacute period and NETosis-suppressive effects at later time points. [ABSTRACT FROM AUTHOR]

The main purpose of this study was to optimize the L-citrulline production process using Plackett-Burman and Box-Behnken designs. L-citrulline-producing bacterium BH-01 was isolated from raw buffalo milk. The isolate was tested for probiotic activities such as tolerance to simulated gastric and intestinal juices, antagonistic activity against six antibiotic-resistant bacteria, and temperature tolerance. L-citrulline production and arginine deiminase (ADI) activity were optimized using statistical designs. The bacterial isolate was molecularly identified as Bacillus subtilis strain AUMC B-498 (accession number PP574248.1). The strain exhibited resistance at pH 2.0 and bile salt 0.5% for a two-hour exposure period. It could inhibit the growth of Escherichia coli, Klebsiella pneumonia, Serratia sp., Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and Streptococcus pneumoniae. From the results of statistical optimization, the Plackett-Burman design identified temperature, L-arginine, incubation period, and peptone as the most effective factors among the eight selected variables. Based on these, the Box-Behnken design was used to optimize the factors required to maximize citrulline production. The maximum L-citrulline was 632.5 µg/L, and ADI activity was 1.42 U/mL. Therefore, BH-01 isolated from Buffalo milk might be a promising candidate in food, biotechnological, and pharmaceutical applications due to its dual functionality for citrulline production and probiotic characteristics. [ABSTRACT FROM AUTHOR]

Previous work reported that deletion of the Enzyme IIAB subunits (EIIABMan and manL) of the glucose phosphotransferase system (PTS) (glucose-PTS, manLMNO) in Streptococcus sanguinis impacted carbon catabolite repression and bacterial fitness. Here, a single-nucleotide polymorphism in ManN, ManNA91E, produced the unusual phenotype of increased excretion of organic acids and H2O2 yet elevated PTS activities. To characterize the contributions of each component of the glucose-PTS to bacterial fitness, we performed genetic analyses by deleting from S. sanguinis SK36 the entire operon and each EIIMan subunit individually; and genes encoding the catabolite control protein A (ΔccpA) and the redox regulator Rex (Δrex) for comparison. Deletion of each subunit incurred a growth defect on glucose partly due to elevated excretion of H2O2; when supplemented with catalase, this defect was rescued, instead resulting in a significantly higher yield than the parent. All glucose-PTS deletion mutants presented an increased antagonism against the oral pathobiont Streptococcus mutans, a phenotype absent in ΔccpA despite increased H2O2 output. A shift in the pyruvate node toward mixed acid fermentation and increased arginine deiminase activity enhanced pH homeostasis in glucose-PTS mutants but not ΔccpA. Despite the purported ability of Rex to regulate central carbon metabolism, deletion of rex had no significant impact on most of the phenotypes discussed here. These findings place glucose-PTS in the pivotal position of controlling central carbon flux in streptococci, with critical outcomes impacting acidogenicity, aciduricity, pH homeostasis, and antagonism, highlighting its potential as a therapeutic target for treating diseases with a dysbiotic microbiome. [ABSTRACT FROM AUTHOR]

【Objective】The objective of this study is to explore the influencing elements of hyaluronic acid (HA) molecular weight, and to construct Streptococcus zooepidemicus mutants that synthesize HA with different molecular weights.【Method】When Streptococcus zooepidemicus S12 was cultured using different carbon sources to produce HA, it was found that the molecular weight of HA fermented by 10 g/Lfructose as the carbon source was 1.48×106 Da, which was 29.52% lower than that of the original fermentation medium (50 g/L sucrose as the carbon source, 2.10×106 Da) . Subsequently, transcriptomic sequencing of S12 fermentation broth with 50 g/L sucrose and 10 g/L fructose as carbon sources showed that the transcription levels of the two key genes of arginine deiminase pathway, arcA (encoding arginine deiminase) and argF (encoding ornithine carbamyltransferase), increased by 16.29 and 11.27 times, respectively, in addition to the fructose metabolism-related genes. In order to explore the effects of these two genes on HA synthesis, the genes arcA and argF were knocked out or overexpressed in S12, respectively.【Result】The HA molecular weight of arcA overexpressing strains was 2.96×106 Da in CDM medium, which was 50.25% higher than that of S12 (1.97×106 Da), and the molecular weight of HA in the other three mutant strains did not show conspicuous changes. Further RT-qPCR showed that the transcription level of glnA, the synthase-encoding gene of glutamine (amino donor in HA synthesis), was up-regulated by 2.0 times in arcA overexpressing strain, which may be one of the reasons for the increase of the HA molecular weight.【Conclusion】Two genes related to arginine metabolism were found to regulate the molecular weight of HA, which provides new targets for other strains to control the molecular weight of HA. [ABSTRACT FROM AUTHOR]

Neutrophils interact with Leishmania when the sandfly vector inoculates these parasites in the host with saliva and promastigotes-derived extracellular vesicles (EVs). It has been shown that this co-injection induces inflammation and exacerbates leishmaniasis lesions. EVs are a heterogeneous group of vesicles released by cells that play a crucial role in intercellular communication. Neutrophils are among the first cells to interact with the parasites and release neutrophil extracellular traps (NETs) that ensnare and kill the promastigotes. Here, we show that Leishmania amazonensis EVs induce NET formation and identify molecular mechanisms involved. We showed the requirement of neutrophils' toll-like receptors for EVs-induced NET. EVs carrying the virulence factors lipophosphoglycan and the zinc metalloproteases were endocytosed by some neutrophils and snared by NETs. EVs-induced NET formation required reactive oxygen species, myeloperoxidase, elastase, peptidyl arginine deiminase, and Ca++. The proteomic analysis of the EVs cargo revealed 1,189 proteins; the 100 most abundant identified comprised some known Leishmania virulent factors. Importantly, L. amazonensis EVs-induced NETs lead to the killing of promastigotes and could participate in the exacerbated inflammatory response induced by the EVs, which may play a role in the pathogenesis process. [ABSTRACT FROM AUTHOR]

Covalent drugs can achieve high potency with long dosing intervals. However, concerns remain about side-effects associated with off-target reactivity. Combining macrocyclic peptides with covalent warheads provides a solution to minimise off-target reactivity: the peptide enables highly specific target binding, positioning a weakly reactive warhead proximal to a suitable residue in the target. Here we demonstrate the direct discovery of covalent cyclic peptides using encoded libraries containing a weakly electrophilic cysteine-reactive fluoroamidine warhead. We combine direct incorporation of the warhead into peptide libraries using the flexible in vitro translation system with a peptide selection approach that identifies only covalent target binders. Using this approach, we identify potent and selective covalent inhibitors of the peptidyl arginine deiminase, PADI4 or PAD4, that react exclusively at the active site cysteine. We envisage this approach will enable covalent peptide inhibitor discovery for a range of related enzymes and expansion to alternative warheads in the future. Covalent peptide drugs can achieve high potency and selectivity with long dosing intervals, however, methods to discover them are limited. Here, the authors incorporate a genetically encoded, weakly electrophilic cysteine-reactive fluoroamidine warhead into mRNA display libraries and screen these to discover potent and selective covalent cyclic peptide inhibitors of peptidyl arginine deiminase 4. [ABSTRACT FROM AUTHOR]

Background: BG is a novel bioactive peptide derived from bitter gourd (Momordica charantia), known for its anti‐inflammatory and immunomodulatory properties. In the present study, our objective is to investigate the functional roles and mechanisms of BG in the context of rheumatoid arthritis (RA). Methods: A rat model of adjuvant‐induced arthritis (AIA) was established by administering complete Freund's adjuvant (CFA). The viability of BG‐mediated AIA was evaluated by assessing changes in rat body weight, joint swelling, ankle joint pathology, inflammation, necroptosis, the formation of neutrophil extracellular traps (NETs), and gut microbiota. Results: The results of the study showed that peptide BG was effective in improving weight loss, joint swelling, serum IgM‐rheumatoid factor (IgM‐RF) level, and pathological injury of ankle joint in rats with AIA. BG administration resulted in a decrease in erythrocyte sedimentation rate, serum C‐reactive protein (CRP), and inflammatory factor (interferon‐γ (IFN‐γ), interleukin‐1β (IL‐1β), and tumor necrosis factor‐α (TNF‐α)) in AIA rats. Additionally, the administration of CFA resulted in an increase in the protein levels of myeloperoxidase (MPO), neutrophil elastase (NE), citrullinated histone H3 (CitH3), peptidyl arginine deiminase 4 (PAD4), p‐mixed lineage kinase domain‐like (p‐MLKL), and cleaved caspase 8. However, this increase was found to be inhibited by BG treatment. Furthermore, it has been found that peptide BG possesses the capacity to regulate the species composition structure of the intestinal microbiota, thereby, facilitating the reestablishment of microbial diversity and equilibrium. Conclusion: Peptide BG has demonstrated efficacy in ameliorating AIA through its regulation of the necroptosis/NETs/inflammation axis and the gut microbiota. This finding underscores the potential of BG as a promising therapeutic intervention for RA. [ABSTRACT FROM AUTHOR]

Purpose: Early embryo arrest is characterized by premature termination of development in preimplantation embryos. Human subcortical maternal complex (SCMC) is a protein complex that is specifically expressed in mammalian oocytes and early embryos and is essential for embryonic cell division. Peptidyl arginine deiminase 6 (PADI6) is proven to be a member of SCMC. Variants in the PADI6 gene have been shown to induce early embryo arrest. In this study, we performed genetic analysis in patients with female infertility due to early embryo arrest to identify the disease-causing gene variants. Methods: Whole-exome sequencing and Sanger sequencing were used to identify the variants in the patients and their families. Western blotting and immunofluorescence staining were used to check the effects of the variants on expression and function of PADI6. Results: We identified a novel homozygous variant (c.358A > C [p.Thr120Pro]) and novel compound-heterozygous variants (c.2044C > T [p.Arg682Trp] and c.707dupT [p.Leu237Alafs*24]) in PADI6 in two infertile individuals with early embryo arrest. We found that these variants resulted in a decrease in the expression level of PADI6, which may lead to abnormal protein function. Immunofluorescence staining also suggested that these variants affected the expression of PADI6. Conclusion: Our study expands the spectrum of genetic defects in female early embryo arrest and further supports the causality between PADI6 variants and female infertility. [ABSTRACT FROM AUTHOR]

In the present study, nutraceutical citrulline was produced using immobilization of permeabilized whole cells of Pseudomonas furukawaii , an efficient producer of ADI. Since arginine deiminase (ADI) is intracellularly localized, various additives such as SDS (Sodium dodecyl sulfate), Triton X-100, and EDTA (Ethylenediaminetetraacetic Acid) were used to permeabilize the cell to improve substrate accessibility and ADI activity. The maximum ADI activity was observed with 0.25 mg ml−1 biomass concentration treated with 0.5 mmol l−1 EDTA for 15 min using OFAT (One factor at a time) approach. Optimization of permeabilization conditions of P. furukawaii cells using novel neural networks and particle swarm optimization led to maximum ADI activity with 0.10 mmol l−1 EDTA and 0.30 mg ml−1 biomass. Further, the morphological characterization of immobilized cells was assessed by field emission scanning electron microscopy and FTIR (Fourier transform infrared spectroscopy). An optimal citrulline production of 1.19 mmol l−1 was achieved at 2.5% sodium alginate with 20 mmol l−1 arginine at 38°C, and 180 min of incubation. The immobilized cells retained 90.3% productivity after seven reuse cycles. Thus, the formulated immobilized whole-cell biocatalyst, with higher stability offers cost-effective methods of citrulline production. [ABSTRACT FROM AUTHOR]

AbstractNanocarrier-based immobilization has created new avenues for enhancing the biophysical properties of enzymes. Nanomatrices such as magnetite nanoparticles (MNPs), chitin, and chitosan with large surface areas and tunable morphology have been developed to circumvent the bottlenecks of free enzymes. The present study used MNPs to immobilize the enzyme arginine deiminase (ADI) for improved morphological control, recovery, operational stability, and easy recyclability. Hybrid magnetic arginine deiminase cross-linked enzyme aggregate (mADI-CLEA) was developed for the first time by co-aggregating ADI with magnetite nanocomposites, followed by its cross-linkage with glutaraldehyde. Structural analysis by DLS/ZETA, SEM, and FT-IR revealed their highly stable and robust nature. The resulting mADI-CLEA exhibited higher pH resistivity and thermostability than ADI-CLEA. Reusability and storage stability assay indicated that mADI-CLEA maintained more than 60% residual activity even after seven batch cycles and was stable for more than 70 days. These hybrid magnetic aggregates of ADI offer an economical and stable alternative for biomedical applications of ADI. [ABSTRACT FROM AUTHOR]

Due to the possible coexistence of Klebsiella pneumoniae (KP) and Candida albicans (CA), strains of KP and CA with biofilm production properties clinically isolated from patients were tested. The production of biofilms from the combined organisms (KP+CA) was higher than the biofilms from each organism alone, as indicated by crystal violet and z-stack immunofluorescence. In parallel, the bacterial abundance in KP + CA was similar to KP, but the fungal abundance was higher than CA (culture method), implying that CA grows better in the presence of KP. Proteomic analysis was performed to compare KP + CA biofilm to KP biofilm alone. With isolated mouse neutrophils (thioglycolate induction), KP + CA biofilms induced less prominent responses than KP biofilms, as determined by (i) neutrophilic supernatant cytokines (ELISA) and (ii) neutrophil extracellular traps (NETs), using immunofluorescent images (neutrophil elastase, myeloperoxidase, and citrullinated histone 3), peptidyl arginine deiminase 4 (PAD4) expression, and cell-free DNA. Likewise, intratracheal KP + CA in C57BL/6 mice induces less severe pneumonia than KP alone, as indicated by organ injury (serum creatinine and alanine transaminase) (colorimetric assays), cytokines (ELISA), bronchoalveolar lavage fluid parameters (bacterial culture and neutrophil abundances using a hemocytometer), histology score (H&E stains), and NETs (immunofluorescence on the lung tissue). In conclusion, the biofilm biomass of KP + CA was mostly produced from CA with less potent neutrophil activation and less severe pneumonia than KP alone. Hence, fungi in the respiratory tract might benefit the host in some situations, despite the well-known adverse effects of fungi. [ABSTRACT FROM AUTHOR]

In the realm of epigenetic regulation, Protein arginine deiminase 2 (PAD2) stands out as a therapeutic target due to its significant role in neurological disorders, rheumatoid arthritis (RA), multiple sclerosis (MS), and various cancers. To date, no in silico studies have focused on PAD2 for lead compound identification. Therefore, we conducted structure-based pharmacophore modeling, virtual screening, molecular docking, molecular dynamics (MD) simulations, and essential dynamics studies using PCA and free energy landscape analyses to identify repurposed drugs and selective inhibitors against PAD2. The best pharmacophore model, 'Pharm_01,' had a selectivity score of 10.485 and an excellent ROC curve quality of 0.972. Pharm1 consisted of three hydrogen bond donors (HBD) and two hydrophobic (Hy) features (DDDHH). A virtual screening of about 9.2 million compounds yielded 2575 hits using a fit value threshold of 2.5 and drug-likeness criteria. Molecular docking identified the top ten molecules, which were verified using MD simulations. Stability was verified using MM-PBSA studies, whereas conformational differences were investigated using PCA and free energy landscape analyses. Two hits (Leads 1 and 2) from the DrugBank dataset showed promise for repurposing as PAD2 inhibitors, while one hit compound (Lead 8) from the ZINC database emerged as a novel PAD2 inhibitor. These findings indicate that the discovered compounds may be potent PAD2 inhibitors, necessitating additional preclinical and clinical research to produce viable treatments for cancer and neurological disorders. [ABSTRACT FROM AUTHOR]

Background: Hepatocellular carcinoma (HCC) is one of the most common as well as leading causes of mortality worldwide, and sorafenib is the first-line treatment in HCC patients. Unfortunately, drug resistance to sorafenib often develops. However, the underlying mechanism remains unclear. Here, we reveal the important role of macrophage extracellular traps (METs)-mediated crosstalk between macrophages and tumor cells in sorafenib resistance. Methods: METs in HCC tumor tissues were detected using immunofluorescence. The concentrations of MPO-DNA, elastase and cytokines were measured using ELISA. The mRNA expression levels of genes were confirmed by qRT-PCR. The siRNAs were conducted to knock ARHGDIG in Hepa1-6 and Hep3B cells. Western Blot assay was performed to determine protein expression of Rho GDP dissociation inhibitor gamma (ARHGDIG, or RHOGDI-3), PADI2, and PADI4. Cell viability and migration were evaluated by CCK-8 assay and transwell assay, respectively. Cell ferroptosis was assessed by measurement of Fe2+ concentration, flow cytometry assay of lipid ROS, and western blot assay of GPX4. The functions of sorafenib, DNase I, IL4 neutralization antibody and GPX4 in tumor growth were explored through in vivo experiments. Results: Sorafenib induced MET formation in M2 macrophages rather than M1 macrophages derived from both human and mice. In Hepa1-6 HCC mice, METs clearance by DNase I improved response to sorafenib therapy, detected by tumor weight, tumor growth curve, tumor volume, and survival. By screening candidate cytokines that affect macrophage function, we found that sorafenib-promoting IL4 secretion by HCC cells plays a crucial role in sorafenib-induced MET formation. Understanding the critical role of IL4 in sorafenib-induced MET formation led us to find that IL4 neutralization significantly improved the efficiency of sorafenib in HCC models. Mechanistically, we discovered that sorafenib increased the expression of ARHGDIG in HCC cells, which led to the release of IL4. In M2 macrophages, IL4 triggered MET formation by elevating the mRNA and protein expression of peptidyl arginine deiminase 4 (PADI4) rather than PADI2. In HCC models, GSK484 inhibition of PADI4 could consistently weaken sorafenib resistance and improve sorafenib efficiency. Importantly, we discovered that METs contribute to sorafenib resistance by inhibiting the ferroptosis of HCC cells. Meanwhile, PADI4 inhibition or DNase I could reverse the sorafenib resistance caused by METs-inhibiting ferroptosis of HCC cells. Conclusion: Our study concludes that sorafenib-induced METs inhibit the ferroptosis of tumor cells, suggesting that targeting the IL4/PADI4/METs axis in HCC could reduce or prevent sorafenib resistance. [ABSTRACT FROM AUTHOR]

Excessive formation of macrophage extracellular trap (MET) has been implicated in several autoimmune disease pathogeneses; however, its impact on type 1 diabetes (T1D) and related mechanisms remains enigmatic. We demonstrated the pivotal role of peptidyl arginine deiminase 4 (PAD4) in driving profuse MET formation and macrophage M1 polarization in intestinal inflammation in NOD mice. Genetic knockout of PAD4 or adoptive transfer of METs altered the proportion of proinflammatory T cells in the intestine, subsequently influencing their migration to the pancreas. Combining RNA sequencing and CUT&Tag analysis, we found activated PAD4 transcriptionally regulated CXCL10 expression. This study comprehensively investigated how excessive PAD4-mediated MET formation in the colon increases the aggravation of intestinal inflammation and proinflammatory T-cell migration and finally is involved in T1D progression, suggesting that inhibition of MET formation may be a potential therapeutic target in T1D. Article Highlights: The role of macrophage extracellular trap (MET) formation in the pathology of type 1 diabetes (T1D) remains unclear. We aimed to investigate the impact of MET formation on intestinal inflammation and its subsequent effect on the pathogenesis of T1D in NOD mice. Activated peptidyl arginine deiminase 4 (PAD4) facilitates MET formation and transcriptionally regulates CXCL10 expression, leading to accelerated T1D through the activation and migration of proinflammatory T cells via the gut/pancreas axis. Findings suggest that targeting PAD4-mediated MET formation could emerge as a potential therapeutic strategy. [ABSTRACT FROM AUTHOR]

Integrin α9β1 is known to stabilize leukocyte adhesion to the activated endothelium. We determined the role of myeloid cell α9β1 in early atherosclerosis in two models: α9Mye-KOApoe−/− or the Ldlr−/− mice transplanted with bone marrow (BM) from α9Mye-KO mice fed a high-fat "Western" diet for 4 wk. α9Mye-KOApoe−/− mice exhibited reduced early lesions in the aortae and aortic sinuses (P < 0.05 vs α9WT Apoe−/− mice). Similar results were obtained in α9Mye-KO BM→Ldlr−/− mice (P < 0.05 vs α9WT BM→Ldlr−/− mice). Reduced early atherosclerosis in α9Mye-KOApoe−/− mice was associated with decreased neutrophil and neutrophil extracellular traps (NETs) content in the aortic lesions (P < 0.05 vs α9WTApoe−/−). Vascular cell adhesion molecule-1-stimulated neutrophils from α9Mye-KO mice exhibited reduced adhesion, transmigration, and NETs formation (NETosis) (P < 0.05 vs α9WT neutrophils). Reduced NETosis was associated with decreased extracellular signal-regulated kinase phosphorylation, peptidyl arginine deiminase 4, and citrullinated histone H3 expression. In summary, genetic ablation of myeloid cell-specific α9 reduces early atherosclerosis, most likely by reducing neutrophil adhesion, transmigration, and NETosis. [ABSTRACT FROM AUTHOR]

The pursuit of novel therapeutics is a complex and resource-intensive endeavor marked by significant challenges, including high costs and low success rates. In response, drug repositioning strategies leverage existing FDA-approved compounds to predict their efficacy across diverse diseases. Peptidyl arginine deiminase 4 (PAD4) plays a pivotal role in protein citrullination, a process implicated in the autoimmune pathogenesis of rheumatoid arthritis (RA). Targeting PAD4 has thus emerged as a promising therapeutic approach. This study employs computational and enzyme inhibition strategies to identify potential PAD4-targeting compounds from a library of FDA-approved drugs. In silico docking analyses validated the binding interactions and orientations of screened compounds within PAD4's active site, with key residues such as ASP350, HIS471, ASP473, and CYS645 participating in crucial hydrogen bonding and van der Waals interactions. Molecular dynamics simulations further assessed the stability of top compounds exhibiting high binding affinities. Among these compounds, Saquinavir (SQV) emerged as a potent PAD4 inhibitor, demonstrating competitive inhibition with a low IC50 value of 1.21 ± 0.04 μM. In vitro assays, including enzyme kinetics and biophysical analyses, highlighted significant changes in PAD4 conformation upon SQV binding, as confirmed by circular dichroism spectroscopy. SQV induced localized alterations in PAD4 structure, effectively occupying the catalytic pocket and inhibiting enzymatic activity. These findings underscore SQV's potential as a therapeutic candidate for RA through PAD4 inhibition. Further validation through in vitro and in vivo studies is essential to confirm SQV's therapeutic benefits in autoimmune diseases associated with dysregulated citrullination. [ABSTRACT FROM AUTHOR]

Elastin is an extracellular matrix protein (ECM) that supports elasticity of the lung, and in patients with chronic obstructive pulmonary disease (COPD) and emphysema, the structural changes that reduce the amount of elastic recoil, lead to loss of pulmonary function. We recently demonstrated that elastin is a target of peptidyl arginine deiminase (PAD) enzyme-induced citrullination, thereby leading to enhanced susceptibility of this ECM protein to proteolysis. This study aimed to investigate the impact of PAD activity in vivo and furthermore assessed whether pharmacological inhibition of PAD activity protects against pulmonary emphysema. Using a Serpina1a-e knockout mouse model, previously shown to develop inflammation-mediated emphysema, we validated the involvement of PADs in airway disease. In line with emphysema development, intratracheal administration of lipopolysaccharide in combination with PADs provoked significant airspace enlargement (P < 0.001) and diminished lung function, including loss of lung tissue elastance (P = 0.0217) and increases in lung volumes (P = 0.0463). Intraperitoneal treatment of mice with the PAD inhibitor, BB-Cl-amidine, prevented PAD/LPS-mediated lung function decline and emphysema and reduced levels of citrullinated airway elastin (P = 0.0199). These results provide evidence for the impact of PADs on lung function decline, indicating promising potential for the future development of PAD-based therapeutics for preserving lung function in patients with COPD. NEW & NOTEWORTHY: This study provides evidence for the impact of peptidyl arginine deiminase (PAD) enzymes on lung function decline, indicating promising potential for the future development of PAD-based therapeutics for preserving lung function in patients with COPD. [ABSTRACT FROM AUTHOR]

Arginine deimination by Tetragenococcus halophilus , a halophilic lactic acid bacterium, is an undesirable reaction in soy sauce brewing because it is responsible for the production of ethyl carbamate, a potential carcinogen. Therefore, arginine deiminase system-deficient mutants have been generated and used as starter cultures. However, the pre-existing screening method for arginine deiminase system-deficient mutants was time consuming. To reduce the burden of this screening process, we established a method to isolate mutants incapable of arginine deimination using the arginine analog canavanine. Strains lacking arginine deiminase system were less sensitive to canavanine than wild type strain, which is likely because arginine deiminase consumes arginine in the cytoplasm and increases the relative concentration of canavanine in the cells and enhances its toxicity. This report provides an industrially useful method to efficiently obtain arginine deiminase system-deficient mutants. [ABSTRACT FROM AUTHOR]

Current clinical research suggests that inhibitors of protein arginine deiminase 4 (PAD4), major histocompatibility complex (MHC) class II HLA-DQ-ALPHA chain, and thyrotropin receptor (or TSH receptor) which are of biological and therapeutic interest, may show potential in treating rheumatoid arthritis, type 1 diabetes, Graves' disease and other autoimmune disorder. In the present study, a comprehensive analysis was conducted on a collection of 32 compounds concerning their anti-rheumatoid arthritis activity as inhibitors of PAD4. This analysis represents the first instance in which these compounds were computationally examined, employing an in-silico approach that considered 2D-3D QSAR modeling, and molecular docking and was further validated through molecular dynamics and ADMET properties assessment. A credible 2D QSAR (Q_LOO^2 = 0.6611 and R^2 = 0.7535) model was constructed and verified using an external validation test set, Y-randomization, variance inflation factor (VIF), mean effect (MF), and William's plot applicability domain (AD). Ligand-based alignment was implemented in the 3D-QSAR examination. The outcomes demonstrated that CoMFA (uvepls) (Q²LOO = 0.5877; R² = 0.9983) possess remarkable stability and foresight. The internal validation indicated that CoMFA (uvepls) MIFs display superior predictive capability compared to COMFA (ffdsel). Structural criteria determined by the contour maps of the model and molecular docking simulations were strategically employed to computationally develop 10 new, nontoxic autoimmune disease inhibitors with increased efficacy. Docking tests were done on the newly developed compounds to illustrate their binding mechanism and to identify critical interaction residues inside the active [ABSTRACT FROM AUTHOR]

Context: Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is the central system in epigenomic exploration. Chromatin immunoprecipitation coupled with sequencing (ChIP-seq) is an important technology to identify the genome-wide location of DNA-binding proteins such as histones proteins, transcription factors, RNA polymerase, or any protein of interest. ChIP-seq has been used to study the binding sites and efficacy of drugs in cancer cell lines etc. Aims: In current research, breast cancer cell line data have been used to study the effect PADI2 (peptidyl arginine deiminase) gene in the progression of breast cancer. Further, this ChIP-seq data have also been used to study the binding site of Amanitin drug in breast cancer. Settings and Design: Breast cancer ChIP-seq data have been retrieved from the European Nucleotide Archive database with project Id PRJNA415426 short read archive. Four samples of FASTQ files were used and analyzed for the genome-wide analysis. Materials and Methods: Galaxy server (https://usegalaxy.org/) was used for complete ChIP-seq data analysis; different tools such as fast-quality control (QC), multi-QC, Bowtie2, model-based analysis of ChIP-sequencing, and ChIPseeker tools were used for motif enrichment and functional analysis. Motif analysis was done through the Multiple Expectation maximizations for Motif Elicitation database (https://meme-suite.org/meme/db/motifs). Results: Computational investigation demonstrates the binding sequences of the T47-D breast cancer cell line as TTTTGTATTTTTAGT, and this motif occurs 2123 times in the Homo Sapiens reference genome that is hg19. Conclusions: This research classifies the binding site and affinity of the T47-D human breast cancer cell line. Further, wet laboratory studies are required to verify the function of the predicted motifs and their importance in drug development or research in breast cancer. [ABSTRACT FROM AUTHOR]

Lower urinary tract infection (UTI) is common but only rarely complicated by pyelonephritis. However, the mechanisms preventing extension to the kidney are unclear. Here, we identified neutrophil extracellular traps (NETs) in healthy human urine that provide an antibacterial defense strategy within the urinary tract. In both in vivo murine models of UTI where uropathogenic E. coli are inoculated into the bladder and ex vivo human urine models, NETs interacted with uromodulin to form large webs that entrapped the bacteria. Peptidyl arginine deiminase 4 (PADI4) inhibition in mice blocked NETosis and resulted in progression of cystitis into pyelonephritis, suggesting that NETosis of urinary neutrophils acts to prevent bacterial ascent into the kidney. Analysis of UK Biobank data revealed that genetic variants in PADI4 that associated with increased risk of rheumatoid arthritis in multiple genome-wide association studies were consistently associated with reduced susceptibility to UTI. Last, we showed that urine dipstick testing for leukocyte esterase was negative in the presence of intact blood neutrophils but became positive when neutrophils were stimulated to NET, and this could be prevented by selective PADI4 inhibition, demonstrating that this test does not detect absolute neutrophil count, as has long been assumed, but specifically detects neutrophils that have undergone NETosis. These findings highlight the role of NETosis in preventing ascending infections in the urinary tract and improve our understanding of one of the most common clinical tests in medicine. Editor's summary: Lower urinary tract infections (UTIs) rarely ascend to the kidney, but the mechanisms underlying this protection are not fully understood. Here, Stewart and colleagues identified neutrophil extracellular traps (NETs) in the urine of healthy individuals that interacted with uromodulin to form webs and entrap bacteria. Inhibition of peptidyl arginine deiminase type 4 (PADI4) reduced NETosis and increased pyelonephritis in mice challenged with uropathogenic E. coli, and an analysis of UK Biobank data revealed that variants in PADI4 were associated with reduced incidence of UTI. The authors also showed that the leukocyte esterase urine dipstick test specifically detects the presence of NETosing neutrophils, highlighting the importance of NETs in both the prevention and diagnosis of UTI. —Melissa L. Norton [ABSTRACT FROM AUTHOR]

Background: Arginine deprivation therapy (ADT) hinders glioma cells' access to nutrients by reducing peripheral blood arginine, showing great efficacy in various studies, which suggests it as a potentially promising treatment for glioma. The aim of this systematic review was to explore the mechanism of ADT for gliomas, the therapeutic effect based on existing research, and possible combination therapies. Methods: We performed a systematic literature review of PubMed, ScienceDirect and Web of Science databases according to PRISMA guidelines, searching for articles on the efficacy of ADT in glioma. Results: We identified 17 studies among 786 search results, among which ADT therapy mainly based on Arginine free condition, Arginine Deiminase and Arginase, including three completed clinical trials. ADT therapy has shown promising results in vivo and in vitro, with its safety confirmed in clinical trials. In the early phase of treatment, glioblastoma (GBM) cells develop protective mechanisms of stress and autophagy, which eventually evolve into caspase dependent apoptosis or senescence, respectively. The immunosuppressive microenvironment is also altered by arginine depletion, such as the transformation of microglia into a pro-inflammatory phenotype and the activation of T-cells. Thus, ADT therapy demonstrates glioma-killing effect in the presence of a combination of mechanisms. In combination with various conventional therapies and investigational drugs such as radiotherapy, temozolomide (TMZ), cyclin-dependent kinase inhibitors (CDK) inhibitors and autophagy inducers, ADT therapy has been shown to be more effective. However, the phenomenon of drug resistance due to re-expression of ASS1 rather than stem cell remains to be investigated. Conclusion: Despite the paucity of studies in the literature, the available data demonstrate the therapeutic potential of arginine deprivation therapy for glioma and encourage further research, especially the exploration of its combination therapies and the extrapolation of what we know about the effects and mechanisms of ADT from other tumors to glioma. [ABSTRACT FROM AUTHOR]

Citrullination plays an essential role in various physiological or pathological processes, however, whether citrullination is involved in regulating tumour progression and the potential therapeutic significance have not been well explored. Here, we find that peptidyl arginine deiminase 4 (PADI4) directly interacts with and citrullinates hypoxia-inducible factor 1α (HIF-1α) at R698, promoting HIF-1α stabilization. Mechanistically, PADI4-mediated HIF-1αR698 citrullination blocks von Hippel-Lindau (VHL) binding, thereby antagonizing HIF-1α ubiquitination and subsequent proteasome degradation. We also show that citrullinated HIF-1αR698, HIF-1α and PADI4 are highly expressed in hepatocellular carcinoma (HCC) tumour tissues, suggesting a potential correlation between PADI4-mediated HIF-1αR698 citrullination and cancer development. Furthermore, we identify that dihydroergotamine mesylate (DHE) acts as an antagonist of PADI4, which ultimately suppresses tumour progression. Collectively, our results reveal citrullination as a posttranslational modification related to HIF-1α stability, and suggest that targeting PADI4-mediated HIF-1α citrullination is a promising therapeutic strategy for cancers with aberrant HIF-1α expression. Post-translational modifications on HIF-1α can regulate its stability and activity in tumoral processes. Here, the authors show that PADI4-mediated citrullination avoids HIF-1α degradation to promote hepatocellular carcinoma progression and this can be prevented by a PADI4 antagonist [ABSTRACT FROM AUTHOR]

Background: Arginine is a conditionally essential amino acid that is depleted in critically ill or surgical patients. In pediatric and adult patients, sepsis results in an arginine-deficient state, and the depletion of plasma arginine is associated with greater mortality. However, direct supplementation of arginine can result in the excessive production of nitric oxide (NO), which can contribute to the hypotension and macrovascular hypo-reactivity observed in septic shock. Pegylated arginine deiminase (ADI-PEG20, pegargiminase) reduces plasma arginine and generates citrulline that can be transported intracellularly to generate local arginine and NO, without resulting in hypotension, while maintaining microvascular patency. The objective of this study was to assess the efficacy of ADI-PEG20 with and without supplemental intravenous citrulline in mitigating hypovolemic shock, maintaining tissue levels of arginine, and reducing systemic inflammation in an endotoxemic pediatric pig model. Methods: Twenty 3-week-old crossbred piglets were implanted with jugular and carotid catheters as well as telemetry devices in the femoral artery to measure blood pressure, body temperature, heart rate, and respiration rate. The piglets were assigned to one of three treatments before undergoing a 5 h lipopolysaccharide (LPS) infusion protocol. Twenty-four hours before LPS infusion, control pigs (LPS; n=6) received saline, ADI-PEG20 pigs (n=7) received an injection of ADI-PEG20, and seven pigs (ADI-PEG20 + CIT pigs [n=7]) received ADI-PEG20 and 250 mg/kg citrulline intravenously. Pigs were monitored throughout LPS infusion and tissue was harvested at the end of the protocol. Results: Plasma arginine levels decreased and remained low in ADI-PEG20 + CIT and ADI-PEG20 pigs compared with LPS pigs but tissue arginine levels in the liver and kidney were similar across all treatments. Mean arterial pressure in all groups decreased from 90 mmHg to 60 mmHg within 1 h of LPS infusion but there were no significant differences between treatment groups. ADI-PEG20 and ADIPEG20 + CIT pigs had less CD45+ infiltrate in the liver and lung and lower levels of pro-inflammatory cytokines in the plasma. Conclusion: ADI-PEG20 and citrulline supplementation failed to ameliorate the hypotension associated with acute endotoxic sepsis in pigs but reduced systemic and local inflammation in the lung and liver. [ABSTRACT FROM AUTHOR]

Neutrophil extracellular trap formation has been identified as a new cell death mediator, termed NETosis, which is distinct from apoptosis and necrosis. NETs capture foreign substances, such as bacteria, by releasing DNA into the extracellular environment, and have been associated with inflammatory diseases and altered immune responses. Short-chain fatty acids, such as acetate, are produced by the gut microbiota and reportedly enhance innate immune responses; however, the underlying molecular mechanisms remain unclear. Here, we investigated the effects of sodium acetate, which has the highest SCFA concentration in the blood and gastrointestinal tract, on NETosis by focusing on the mechanisms associated with histone acetylation in neutrophil-like HL-60 cells. Sodium acetate enhanced NETosis, as shown by fluorescence staining with SYTOX green, and the effect was directly proportional to the treatment duration (16–24 h). Moreover, the addition of sodium acetate significantly enhanced the acetylation of Ace-H3, H3K9ace, and H3K14ace. Sodium acetate-induced histone acetylation rapidly decreased upon stimulation with the calcium ionophore A23187, whereas histone citrullination markedly increased. These results demonstrate that sodium acetate induces NETosis via histone acetylation in neutrophil-like HL-60 cells, providing new insights into the therapeutic effects based on the innate immunity-enhancing effect of dietary fiber. [ABSTRACT FROM AUTHOR]

Protein expression is regulated through multiple mechanisms, including post-translational modifications (PTMs), which can alter protein structure, stability, localization, and function. Among these, citrullination stands out due to its ability to convert arginine residues into citrulline, altering protein charge and mass. This modification is catalyzed by calcium-dependent protein arginine deiminases (PADs), enzymes implicated in various inflammatory diseases. We have recently shown that human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) exploit these enzymes to enhance their replication capabilities. Although the role of PADs in HCMV and HSV-1 infections is well documented, their involvement in HSV-2 infection has not yet been thoroughly investigated. Here, we demonstrate that HSV-2 manipulates the overall protein citrullination profile by activating three PAD isoforms: PAD2, PAD3, and PAD4. However, as previously observed during HSV-1 infection, PAD3 is the most significantly upregulated isoform, both at the mRNA and protein levels. Consistently, we demonstrate that inhibiting PAD3, either through the specific inhibitor CAY10727 or via CRISPR/Cas9-mediated gene silencing, markedly reduces HSV-2 replication and viral protein expression. Lastly, we show that CAY10727 displays an IC50 value of 0.3 μM, which is extremely close to what was previously observed for HSV-1. Overall, our findings highlight the crucial role of PAD3 in the life cycle of HSV-2 and suggest that the targeted inhibition of PAD3 may represent a promising approach for treating HSV-2 infections, especially in cases resistant to existing antiviral therapies. [ABSTRACT FROM AUTHOR]

Background: Protein arginine deiminase 3 (PADI3) is involved in various biological processes of human disease. PADI3 has recently received increasing attention due to its role in tumorigenesis. In a previous study, we found that PADI3 plays a tumor suppressor role in colon cancer by inducing cell cycle arrest, but its critical role and mechanism in cancer metastasis remain obscure. In this study, we fully studied the role of PADI3 in colon cancer cell metastasis. Methods: The expression levels of related proteins were detected by Western blotting, and Transwell and wound healing assays were used to examine the cell migration ability. Flow cytometry was used to measure and exclude cell apoptosis-affected cell migration. Both overexpression and rescue experiments were employed to elucidate the molecular mechanism of CKS1 in colon cancer cells. Results: The expression levels of PADI3 and CKS1 are negatively related, and PADI3 can promote CKS1 degradation in a ubiquitin-dependent manner. PADI3 can suppress colon cancer cell migration and reduce the wound healing speed by inhibiting CKS1 expression. The molecular mechanism showed that CKS1 can promote EMT by increasing Snail and N-cadherin expression and suppressing E-cadherin expression. PADI3, as a suppressor of CKS1, can block the process of EMT by impairing CKS1-induced Snail upregulation and E-cadherin downregulation; however, the expression of N-cadherin cannot be rescued. Conclusions: CKS1 promotes EMT in colon cancer by regulating Snail/E-cadherin expression, and this effect can be reversed by PADI3 via the promotion of CKS1 degradation in a ubiquitylation-dependent manner. [ABSTRACT FROM AUTHOR]

The present study reports on the development, adaptation, and optimization of a novel monoenzyme conductometric biosensor based on a recombinant arginine deiminase (ADI) for the determination of arginine in dietary supplements with a high accuracy of results. Aiming for the highly sensitive determination of arginine in real samples, we studied the effect of parameters of the working buffer solution (its pH, buffer capacity, ionic strength, temperature, and protein concentration) on the sensitivity of the biosensor to arginine. Thus, it was determined that the optimal buffer is a 5 mM phosphate buffer solution with pH 6.2, and the optimal temperature is 39.5 °C. The linear functioning range is 2.5–750 µM of L-arginine with a minimal limit of detection of 2 µM. The concentration of arginine in food additive samples was determined using the developed ADI-based biosensor. Based on the obtained results, the most effective method of biosensor analysis using the method of standard additions was chosen. It was also checked how the reproducibility of the biosensor changes during the analysis of pharmaceutical samples. The results of the determination of arginine in real samples using a conductometric biosensor based on ADI clearly correlated with the data obtained using the method of ion-exchange chromatography and enzymatic spectrophotometric analysis. We concluded that the developed biosensor would be effective for the accurate and selective determination of arginine in dietary supplements intended for the prevention and/or elimination of arginine deficiency. [ABSTRACT FROM AUTHOR]

Neutrophils release neutrophil extracellular traps (NETs) as a defense strategy in response to broad-spectrum infections and sterile triggers. NETs consist of a DNA scaffold decorated with antimicrobial peptides (AMPs) and enzymatically active proteases, including peptidyl arginine deiminase type 4 (PAD4). Susceptibility to infections and inflammatory dysregulation are hallmarks of alcohol-related liver disease (ALD). Sixty-two patients with ALD were prospectively recruited, and they were followed for 90 days. Twenty-four healthy volunteers served as the control group. PAD4 concentrations were quantified using immunoenzymatic ELISAs. Correlation coefficients between PAD4 blood concentrations and markers of systemic inflammation; liver dysfunction severity scores; and ALD complications were calculated. The receiver operating curves (ROCs) and their areas under the curve (AUCs) were checked in order to assess the accuracy of PAD4 expression in predicting the degree of liver failure and the development of ALD complications. Systemic concentrations of PAD4 were significantly increased in the patients with ALD in comparison with controls. PAD4 levels correlated with the standard markers of inflammation and revealed a good predictive AUC (0.76) for survival in the whole ALD group. PAD4 seems to be an inflammatory mediator and may be potentially applied as a predictor of patient survival in ALD. [ABSTRACT FROM AUTHOR]

Sonodynamic therapy (SDT) is demonstrated to trigger the systemic immune response of the organism and facilitate the treatment of metastatic tumors. However, SDT‐mediated neutrophil extracellular traps (NETs) formation can promote tumor cell spread, thus weakening the therapeutic effectiveness of metastatic tumors. Herein, the amorphous CoW‐layered double hydroxide (a‐CoW‐LDH) nanosheets are functionalized with a peptidyl arginine deiminase 4 (PAD4) inhibitor, i.e., YW3‐56, to construct a multifunctional nanoagent (a‐LDH@356) for synergistic SDT/immunotherapy. Specifically, a‐CoW‐LDH nanosheets can act as a sonosensitizer to generate abundant reactive oxygen species (ROS) under US irradiation. After loading with YW3‐56, a‐LDH@356 plus US irradiation not only effectively induces ROS generation and immunogenic cell death, but also inhibits the elevation of citrullinated histone H3 (H3cit) and the release of NETs, enabling a synergistic enhancement of anti‐tumor metastasis effect. Using 4T1 tumor model, it is demonstrated that combining a‐CoW‐LDH with YW3‐56 stimulates an anti‐tumor response by upregulating the proportion of immune‐activated cells and inducing polarization of M1 macrophages, and inhibits immune escape by downregulating the expression of PD‐1 on immune cells under US irradiation, which not only arrests primary tumor progression with a tumor inhibition rate of 69.5% but also prevents tumor metastasis with the least number of lung metastatic nodules. [ABSTRACT FROM AUTHOR]

Objective: Chronic inflammation and oxidative stress mediate the pathological progression of diabetic complications, like diabetic retinopathy (DR), peripheral neuropathy (DPN) and impaired wound healing. Studies have shown that treatment with a stable form of arginase 1 that reduces l-arginine levels and increases ornithine and urea limits retinal injury and improves visual function in DR. We tested the therapeutic efficacy of PEGylated arginine deiminase (ADI-PEG20) that depletes l-arginine and elevates l-citrulline on diabetic complications in the db/db mouse model of type 2 diabetes (T2D). Methods: Mice received intraperitoneal (IP), intramuscular (IM), or intravitreal (IVT) injections of ADI-PEG20 or PEG20 as control. Effects on body weight, fasting blood glucose levels, blood-retinal-barrier (BRB) function, visual acuity, contrast sensitivity, thermal sensitivity, and wound healing were determined. Studies using bone marrow-derived macrophages (BMDM) examined the underlying signaling pathway. Results: Systemic injections of ADI-PEG20 reduced body weight and blood glucose and decreased oxidative stress and inflammation in db/db retinas. These changes were associated with improved BRB and visual function along with thermal sensitivity and wound healing. IVT injections of either ADI-PEG20, anti-VEGF antibody or their combination also improved BRB and visual function. ADI-PEG20 treatment also prevented LPS/IFNℽ-induced activation of BMDM in vitro as did depletion of l-arginine and elevation of l-citrulline. Conclusions/interpretation: ADI-PEG20 treatment limited signs of DR and DPN and enhanced wound healing in db/db mice. Studies using BMDM suggest that the anti-inflammatory effects of ADI-PEG20 involve blockade of the JAK2-STAT1 signaling pathway via l-arginine depletion and l-citrulline production.

Arginine metabolism plays an important role in regulating the functions of immune cells in mammals. Pathogenic microbes use the mechanism of arginine depletion to suppress the immune response during infection. Arginine deiminase is a microbial arginine-hydrolyzing enzyme important for survival at low pH in the focus of infection, or in phagolysosomes, as well as under low-glucose conditions. The effect of bacterial arginine deiminase on the functions of adaptive immune cells remains poorly understood. The aim of our study was to evaluate the effect of streptococcal arginine deiminase on the proliferation and autophagy of CD4+ and CD8+ human peripheral blood T lymphocytes.The enzyme effects were studied with supernates of ultrasonic lysates from parental Streptococcus pyogenes M49-16, and its isogenic mutant with inactivated arcA gene (Streptococcus pyogenes M49-16delarcA). The study was performed with blood samples of healthy donors. The fraction of mononuclear leukocytes was isolated by centrifugation in a Ficoll density gradient. To evaluate proliferation levels, a method based on the staining of intracellular proteins with vital fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) was used. The level of autophagy was studied using the fluorescent Lysotracker Green DND-26 dye. To analyze the proliferation and autophagy of T helper cells (CD3+CD4+) and cytotoxic T lymphocytes (CD3+CD4-), the obtained cell suspensions were stained with antibodies against CD4, CD45RA, and CD3. The proportion of necrotic cells was determined by staining with a fluorescent DNA-binding DAPI dye. The normality of the distribution was assessed by Shapiro–Wilk test. The data were analyzed using Kruskal–Wallis criterion, followed by Mann–Whitney criterion for pairwise comparisons and expressed as median and interquartile ranges (Q0.25-Q0.75).When comparing the effects of supernatants from the parental and mutant streptococcal strains, which differed in expression of arginine deiminase gene, we have shown that the bacterial enzyme had no effect on the functions of inactive lymphocytes. However, streptococcal arginine deiminase completely suppressed proliferation of CD4+ and CD8+T lymphocytes stimulated with anti-CD2/CD3/CD28 antibodies. These effects were accompanied by a decrease in the autophagy levels. At the same time, arginine deiminase did not exert cytotoxic effects upon lymphocytes. L-arginine applied at the doses exceeding physiological levels caused restoration of the cellular functions. There were no differences between the studied parameters of CD4+ and CD8+T lymphocyte subsets.The obtained data show that the antiproliferative effect of arginine demimnase may be associated with ability of the enzyme to inhibit autophagy and confirm an opportunity of the bacterial enzyme to suppress host adaptive immune responses.

Traumatic brain injuries (TBI) and stroke are major causes of morbidity and mortality in both developing and developed countries. The complex and heterogeneous pathophysiology of TBI and cerebral ischemia‐reperfusion injury (CIRI), in addition to the blood‐brain barrier (BBB) resistance, is a major barrier to the advancement of diagnostics and therapeutics. Clinical data showed that the severity of TBI and stroke is positively correlated with the number of neutrophils in peripheral blood and brain injury sites. Furthermore, neutrophil extracellular traps (NETs) released by neutrophils correlate with worse TBI and stroke outcomes by impairing revascularization and vascular remodeling. Therefore, targeting neutrophils to deliver NETs inhibitors to brain injury sites and reduce the formation of NETs can be an optimal strategy for TBI and stroke therapy. Herein, the study designs and synthesizes a reactive oxygen species (ROS)‐responsive neutrophil‐targeting delivery system loaded with peptidyl arginine deiminase 4 (PAD4) inhibitor, GSK484, to prevent the formation of NETs in brain injury sites, which significantly inhibited neuroinflammation and improved neurological deficits, and improved the survival rate of TBI and CIRI. This strategy may provide a groundwork for the development of targeted theranostics of TBI and stroke. [ABSTRACT FROM AUTHOR]

Background: Filaggrin (FLG) is an essential protein that plays a vital role in maintaining skin barrier function and moisture levels, allowing the skin to adapt to dry environments. However, the precise temporal dynamics of FLG metabolism in the human epidermis remain poorly understood, and suitable tools to study these time‐dependent effects are currently lacking. Objective: To investigate the molecular mechanisms and time course of FLG metabolism and skin barrier function under high‐ and low‐humidity conditions, utilizing a reconstructed epidermis model. Methods: EpiSkin specimens cultured under humid or dry conditions for varying durations (2–48 h) were compared by assessing FLG degradation and skin barrier formation using immunofluorescence staining and western blotting. Results: Under conditions of low humidity, the proteolysis of FLG in EpiSkin increased between 4 and 12 h and was accompanied by elevated levels of cysteine–aspartic protease (caspase)‐14. The expression of peptidyl arginine deiminase 1 and calpain 1 also increased at 4 h. However, after 24 h, the expression of these three FLG‐degrading proteins significantly decreased. Conversely, the levels of pyrrolidone‐5‐carboxylic acid and urocanic acid initially decreased at 2 h and then increased between 12 and 24 h. Additionally, the expression of skin barrier proteins, such as FLG, transglutaminase 5, loricrin and zonula occludens‐1, decreased starting from 12 h. Notably, epidermal cell viability and activity were also inhibited. Conclusion: We propose a reliable and ethical model to study the temporal dynamics of FLG metabolism and its role in skin barrier function. Using a commercially reconstructed epidermis to mimic dry skin formation obviates the need for animal and human testing. [ABSTRACT FROM AUTHOR]

Vibrio fluvialis is an emerging foodborne pathogenic bacterium that can cause severe cholera-like diarrhea and various extraintestinal infections, posing challenges to public health and food safety worldwide. The arginine deiminase (ADI) pathway plays an important role in bacterial environmental adaptation and pathogenicity. However, the biological functions and regulatory mechanisms of the pathway in V. fluvialis remain unclear. In this study, we demonstrate that L-arginine upregulates the expression of the ADI gene cluster and promotes the growth of V. fluvialis. The ADI gene cluster, which we proved to be comprised of two operons, arcD and arcACB, significantly enhances the survival of V. fluvialis in acidic environments both in vitro (in culture medium and in macrophage) and in vivo (in mice). The mRNA level and reporter gene fusion analyses revealed that ArgR, a transcriptional factor, is necessary for the activation of both arcD and arcACB transcriptions. Bioinformatic analysis predicted the existence of multiple potential ArgR binding sites at the arcD and arcACB promoter regions that were further confirmed by electrophoretic mobility shift assay, DNase I footprinting, or point mutation analyses. Together, our study provides insights into the important role of the ArgR-ADI pathway in the survival of V. fluvialis under acidic conditions and the detailed molecular mechanism. These findings will deepen our understanding of how environmental changes and gene expression interact to facilitate bacterial adaptations and virulence. [ABSTRACT FROM AUTHOR]

Background and Aim: Nonsteroidal anti‐inflammatory drugs (NSAIDs) damage the small intestine via neutrophil infiltration driven by the mucosal invasion of enterobacteria. The antimicrobial function of neutrophils is partially dependent on neutrophil extracellular traps (NETs). Excessive NET formation has been associated with several inflammatory diseases. Here, we aimed to investigate the role of NETs in NSAID‐induced small intestinal damage using human samples and an experimental mouse model. Methods: Human small intestine specimens were obtained from NSAID users during double‐balloon enteroscopy. Wild‐type, protein arginine deiminase 4 (PAD4) knockout, and antibiotic‐treated mice were administered indomethacin to induce small intestinal injury. The expression of NET‐associated proteins, including PAD4, citrullinated histone H3 (CitH3), cell‐free DNA, and myeloperoxidase (MPO), was evaluated. Results: The double‐positive stained area with CitH3 and MPO, which is specific for neutrophil‐derived extracellular traps, was significantly high in the injured small intestinal mucosa of NSAID users. In a mouse model, small intestinal damage developed at 6 h after indomethacin administration, accompanied by increased mRNA levels of interleukin‐1β and keratinocyte chemoattractant and elevated NET‐associated protein levels of PAD4, CitH3, and MPO in small intestine and serum levels of cell‐free DNA. Both genetic deletion and pharmacological inhibition of PAD4 attenuated this damage by reducing the mRNA expression of inflammatory cytokines and NET‐associated proteins. Furthermore, mice pretreated with antibiotics showed resistance to indomethacin‐induced small intestinal damage, with less NET formation. Conclusion: These results suggest that NETs aggravate NSAID‐induced small intestinal injury. Therefore, NET inhibition could be a potential treatment for NSAID‐induced small intestinal injury. [ABSTRACT FROM AUTHOR]

The human microbiome exists throughout the body, and it is essential for maintaining various physiological processes, including immunity, and dysbiotic events, which are associated with autoimmunity. Peptidylarginine deiminase (PAD) enzymes can citrullinate self-proteins related to rheumatoid arthritis (RA) that induce the production of anti-citrullinated protein antibodies (ACPAs) and lead to inflammation and joint damage. The present investigation was carried out to demonstrate the expression of homologs of PADs or arginine deiminases (ADs) and citrullinated proteins in members of the human microbiota. To achieve the objective, we used 17 microbial strains and specific polyclonal antibodies (pAbs) of the synthetic peptide derived from residues 100–200 of human PAD2 (anti-PAD2 pAb), and the recombinant fragment of amino acids 326 and 611 of human PAD4 (anti-PAD4 pAb), a human anti-citrulline pAb, and affinity ACPAs of an RA patient. Western blot (WB), enzyme-linked immunosorbent assay (ELISA), elution, and a test with Griess reagent were used. This is a cross-sectional case–control study on patients diagnosed with RA and control subjects. Inferential statistics were applied using the non-parametric Kruskal–Wallis test and Mann–Whitney U test generated in the SPSS program. Some members of phyla Firmicutes and Proteobacteria harbor homologs of PADs/ADs and citrullinated antigens that are reactive to the ACPAs of RA patients. Microbial citrullinome and homolog enzymes of PADs/ADs are extensive in the human microbiome and are involved in the production of ACPAs. Our findings suggest a molecular link between microorganisms of a dysbiotic microbiota and RA pathogenesis. [ABSTRACT FROM AUTHOR]

Nanomaterials, including ZIF‐8 nanoparticles (NPs), are shown to be effective antimicrobial agents against Methicillin‐resistant Staphylococcus aureus(MRSA). However, the antibiofilm properties and mechanisms of ZIF‐8 NPs remain uncertain. In this study, ZIF‐8 NPs are prepared using the room temperature solution reaction method and characterized. Biofilm formation inhibition test and biofilm eradication test are performed and the results show that ZIF‐8 NPs can inhibit the formation of MRSA biofilm as well as disperse established MRSA biofilm. Proteomics and real‐time fluorescence quantitative polymerase chain reaction (PCR) are conducted to prove that ZIF‐8 NPs reduce the expression of adhesion‐related proteins, namely the fibronectin‐binding proteins A and B (fnbA/fnbB), fibrinogen binding protein caking factors A and B (clfA/clfB), elastin binding protein (ebps), and fibrin binding protein (eno). ZIF‐8 NPs also inhibit the arginine biosynthesis pathway by affecting the activities of argininosuccinate lyase, ornithine carbamyl transferase, Glutamate dehydrogenase, carbamate kinase, and arginine deiminase. A conclusion can be drawn from the above results that ZIF‐8 NPs can inhibit bacterial adhesion and kill bacteria directly, ultimately destroying MRSA biofilm. This study provides a molecular basis for the treatment of MRSA biofilm with ZIF‐8 NPs. [ABSTRACT FROM AUTHOR]

Endophytic actinobacteria are a group of bacteria living inside plant tissue without harmful effects, and benefit the host plant. Many can inhibit plant pathogens and promote plant growth. This study aimed to identify a strain of Streptomyces as a novel species and study its antibiotics production. An endophytic actinobacterium, strain TML10T was isolated from a surface-sterilized leaf of a Thai medicinal plant (Terminalia mucronata Craib and Hutch). As a result of a polyphasic taxonomy study, strain TML10T was identified as a member of the genus Streptomyces. Strain TML10T was an aerobic actinobacterium with well-developed substrate mycelia with loop spore chains and spiny surface. Chemotaxonomic data, including cell wall components, major menaquinones, and major fatty acids, confirmed the affiliation of strain TML10T to the genus Streptomyces. The results of the phylogenetic analysis, including physiological and biochemical studies in combination with a genome comparison study, allowed the genotypic and phenotypic differentiation of strain TML10T and the closest related type strains. The digital DNA-DNA hybridization (dDDH), Average nucleotide identity Blast (ANIb), and ANIMummer (ANIm) values between strain TML10T and the closest type strain, Streptomyces musisoli CH5-8T were 38.8%, 88.5%, and 90.8%, respectively. The name proposed for the new species is Streptomyces naphthomycinicus sp. nov. (TML10T = TBRC 15050T = NRRL B-65638T). Strain TML10T was further studied for liquid and solid-state fermentation of antibiotic production. Solid-state fermentation with cooked rice provided the best conditions for antibiotic production against methicillin-resistant Staphylococcus aureus. The elucidation of the chemical structures from this strain revealed a known antimicrobial agent, naphthomycin A. Mining the genome data of strain TML10T suggested its potential as a producer of antbiotics and other valuable compounds such as ε-Poly-L-lysine (ε-PL) and arginine deiminase. Strain TML10T contains the arcA gene encoding arginine deiminase and could degrade arginine in vitro. [ABSTRACT FROM AUTHOR]

The anticancer potential of Levilactobacillus brevis KU15176 against the stomach cancer cell line AGS has been reported previously. In this study, we aimed to analyze the genome of L. brevis KU15176 and identify key genes that may have potential anticancer properties. Among potential anticancer molecules, the role of arginine deiminase (ADI) in conferring an antiproliferative functionality was confirmed. In vitro assay against AGS cell line confirmed that recombinant ADI from L. brevis KU15176 (ADI_br, 5 µg/mL), overexpressed in E. coli BL21 (DE3), exerted an inhibitory effect on AGS cell growth, resulting in a 65.32% reduction in cell viability. Moreover, the expression of apoptosis-related genes, such as bax, bad, caspase-7, and caspase-3, as well as the activity of caspase-9 in ADI_br-treated AGS cells, was higher than those in untreated (culture medium-only) cells. The cell-scattering behavior of ADI_br-treated cells showed characteristics of apoptosis. Flow cytometry analyses of AGS cells treated with ADI_br for 24 and 28 h revealed apoptotic rates of 11.87 and 24.09, respectively, indicating the progression of apoptosis in AGS cells after ADI_br treatment. This study highlights the potential of ADI_br as an effective enzyme for anticancer applications. [ABSTRACT FROM AUTHOR]

Chronic periodontitis (CP), an inflammatory disease of periodontal tissues driven by a dysbiotic subgingival bacterial biofilm, is also associated with several systemic diseases, including rheumatoid arthritis (RA). Porphyromonas gingivalis, one of the bacterial species implicated in CP as a keystone pathogen produces peptidyl arginine deiminase (PPAD) that citrullinates C-terminal arginine residues in proteins and peptides. Autoimmunity to citrullinated epitopes is crucial in RA, hence PPAD activity is considered a possible mechanistic link between CP and RA. Here we determined the PPAD enzymatic activity produced by clinical isolates of P. gingivalis, sequenced the ppad gene, and correlated the results with clinical determinants of CP in patients from whom the bacteria were isolated. The analysis revealed variations in PPAD activity and genetic diversity of the ppad gene in clinical P. gingivalis isolates. Interestingly, the severity of CP was correlated with a higher level of PPAD activity that was associated with the presence of a triple mutation (G231N, E232T, N235D) in PPAD in comparison to W83 and ATCC 33277 type strains. The relation between mutations and enhanced activity was verified by directed mutagenesis which showed that all three amino acid residue substitutions must be introduced into PPAD expressed by the type strains to obtain the super-active enzyme. Cumulatively, these results may lead to the development of novel prognostic tools to assess the progress of CP in the context of associated RA by analyzing the ppad genotype in CP patients infected with P. gingivalis. [ABSTRACT FROM AUTHOR]

Vaccine development against group A Streptococcus (GAS) has gained traction in the last decade, fuelled by recognition of the significant worldwide burden of the disease. Several vaccine candidates are currently being evaluated in preclinical and early clinical studies. Here, we investigate two conjugate vaccine candidates that have shown promise in mouse models of infection. Two antigens, the J8 peptide from the conserved C-terminal end of the M protein, and the group A carbohydrate lacking N-acetylglucosamine side chain (ΔGAC) were each conjugated to arginine deiminase (ADI), an anchorless surface protein from GAS. Both conjugate vaccine candidates combined with alum adjuvant were tested in a non-human primate (NHP) model of pharyngeal infection. High antibody titres were detected against J8 and ADI antigens, while high background antibody titres in NHP sera hindered accurate quantification of ΔGAC-specific antibodies. The severity of pharyngitis and tonsillitis signs, as well as the level of GAS colonisation, showed no significant differences in NHPs immunised with either conjugate vaccine candidate compared to NHPs in the negative control group. [ABSTRACT FROM AUTHOR]

Abstract Background Colorectal cancer (CRC) is one of the most commonly diagnosed cancers, posing a serious public health challenge that necessitates the development of new therapeutics, therapies, and prevention methods. Among the various therapeutic approaches, interventions involving lactic acid bacteria (LAB) as probiotics and postbiotics have emerged as promising candidates for treating and preventing CRC. While human-isolated LAB strains are considered highly favorable, those sourced from environmental reservoirs such as dairy and fermented foods are also being recognized as potential sources for future therapeutics. Results In this study, we present a novel and therapeutically promising strain, Lactococcus lactis ssp. lactis Lc4, isolated from dairy sources. Lc4 demonstrated the ability to release the cytostatic agent - arginine deiminase (ADI) - into the post-cultivation supernatant when cultured under conditions mimicking the human gut environment. Released arginine deiminase was able to significantly reduce the growth of HT-29 and HCT116 cells due to the depletion of arginine, which led to decreased levels of c-Myc, reduced phosphorylation of p70-S6 kinase, and cell cycle arrest. The ADI release and cytostatic properties were strain-dependent, as was evident from comparison to other L. lactis ssp. lactis strains. Conclusion For the first time, we unveil the anti-proliferative properties of the L. lactis cell-free supernatant (CFS), which are independent of bacteriocins or other small molecules. We demonstrate that ADI, derived from a dairy-Generally Recognized As Safe (GRAS) strain of L. lactis, exhibits anti-proliferative activity on cell lines with different levels of argininosuccinate synthetase 1 (ASS1) expression. A unique feature of the Lc4 strain is also its capability to release ADI into the extracellular space. Taken together, we showcase L. lactis ADI and the Lc4 strain as promising, potential therapeutic agents with broad applicability.

Abstract L-arginine deiminase (ADI, EC 3.5.3.6) hydrolyzes arginine to ammonia and citrulline which is a natural supplement in health care. ADI was purified from Penicillium chrysogenum using 85% ammonium sulfate, DEAE-cellulose and Sephadex G200. ADI was purified 17.2-fold and 4.6% yield with a specific activity of 50 Umg− 1 protein. The molecular weight was 49 kDa. ADI expressed maximum activity at 40oC and an optimum pH of 6.0. ADI thermostability was investigated and the values of both t0.5 and D were determined. Kd increased by temperature and the Z value was 38oC. ATP, ADP and AMP activated ADI up to 0.6 mM. Cysteine and dithiothreitol activated ADI up to 60 µmol whereas the activation by thioglycolate and reduced glutathione (GSH) prolonged to 80 µmol. EDTA, α,α-dipyridyl, and o-phenanthroline inactivated ADI indicating that ADI is a metalloenzyme. N-ethylmaleimide (NEM), N-bromosuccinimide (NBS), butanedione (BD), dansyl chloride (DC), diethylpyrocarbonate (DEPC) and N-acetyl-imidazole (NAI) inhibited ADI activity indicating the necessity of sulfhydryl, tryptophanyl, arginyl, lysyl, histidyl and tyrosyl groups, respectively for ADI catalysis. The obtained results show that ADI from P. chrysogenum could be a potential candidate for industrial and biotechnological applications.

Abstract Background and Objectives: This study was aimed to characterize arginine deiminase (ADI) purified from Klebsiella pneumoniae in vitro. Materials and Methods: Precipitation with 70% saturated ammonium sulphate, ion exchange chromatography with a DEAE-cellulose column, and gel filtration chromatography throughout sepharose-6B were the three steps taken to isolate the arginine-degrading enzyme from a K. pneumoniae clinical isolate, which is a potent anticancer source. Results: After 5.9 folds of purification and 38.7% enzyme recovery, the specific activity of the purified enzyme reached 164.2 U/mg. When biochemical characteristics of the purified enzyme were studied, results showed that the activity was maximum at pH 6 and is most stable in pH ranging from (5–9), the optimum temperature for enzyme activity was observed at 37ºC and reach 11.5 U/mL. In contrast, ethylenediaminetetraacetic acid (EDTA), NaNO3, and ZnSO4 slightly inhibited ADI activity, whereas MnCl2, increased the remaining activity of enzyme to 125%., as well as NaNO3, EDTA, ZnSO4, and FeCl3 were found that they inhibit enzyme activity by 90, 70, 88, and 110, respectively. Conclusion: A locally isolated strain of K. pneumoniae N1 is a useful and potent arginine deiminase producer.

Background: Colorectal cancer (CRC) is one of the most commonly diagnosed cancers, posing a serious public health challenge that necessitates the development of new therapeutics, therapies, and prevention methods. Among the various therapeutic approaches, interventions involving lactic acid bacteria (LAB) as probiotics and postbiotics have emerged as promising candidates for treating and preventing CRC. While human-isolated LAB strains are considered highly favorable, those sourced from environmental reservoirs such as dairy and fermented foods are also being recognized as potential sources for future therapeutics. Results: In this study, we present a novel and therapeutically promising strain, Lactococcus lactis ssp. lactis Lc4, isolated from dairy sources. Lc4 demonstrated the ability to release the cytostatic agent - arginine deiminase (ADI) - into the post-cultivation supernatant when cultured under conditions mimicking the human gut environment. Released arginine deiminase was able to significantly reduce the growth of HT-29 and HCT116 cells due to the depletion of arginine, which led to decreased levels of c-Myc, reduced phosphorylation of p70-S6 kinase, and cell cycle arrest. The ADI release and cytostatic properties were strain-dependent, as was evident from comparison to other L. lactis ssp. lactis strains. Conclusion: For the first time, we unveil the anti-proliferative properties of the L. lactis cell-free supernatant (CFS), which are independent of bacteriocins or other small molecules. We demonstrate that ADI, derived from a dairy-Generally Recognized As Safe (GRAS) strain of L. lactis, exhibits anti-proliferative activity on cell lines with different levels of argininosuccinate synthetase 1 (ASS1) expression. A unique feature of the Lc4 strain is also its capability to release ADI into the extracellular space. Taken together, we showcase L. lactis ADI and the Lc4 strain as promising, potential therapeutic agents with broad applicability. [ABSTRACT FROM AUTHOR]

Atractylenolide II (AT-II), the major bioactive compound of Atractylodes macrocephala, exhibits anti-cancer activity against many types of tumors, but the roles and the potential mechanisms in endometrial cancer remain unclear. In the present study, AT-II treatment was found to significantly suppress RL95-2 and AN3CA cell proliferation and glycolysis, and induced their apoptosis by inactivating the ERK signaling pathway, accompanied by the changing expression of the glycolytic key enzymes and apoptotic-related proteins. Peptidyl arginine deiminase 3 (PADI3), as the candidate target gene of AT-II, was highly expressed in the endometrial cancer tissues and associated with a poor prognosis according to bioinformatics analysis. PADI3 knockdown inhibited proliferation and glycolysis in endometrial cancer cells and induced cell apoptosis. Furthermore, AT-II negatively regulated the expression of PADI3, and PADI3 overexpression reversed the effects of AT-II on endometrial cancer cells. Our findings suggested that the anti-cancer function of AT-II is associated with the suppression of glycolysis and induction of apoptosis by blocking the PADI3-ERK signaling pathway. Thus, AT-II represents a novel therapeutic target for endometrial cancer and targeting AT-II may serve as a potential strategy for the clinical therapy of endometrial cancer. [ABSTRACT FROM AUTHOR]

Protein arginine deiminase 4 (PAD4) plays an important role in cancer progression by participating in gene regulation, protein modification, and neutrophil extracellular trap (NET) formation. Many reversible and irreversible PAD4 inhibitors have been reported recently. In this review, we summarize the structure–activity relationships of newly investigated PAD4 inhibitors to bring researchers up to speed by guiding and describing new scaffolds as optimization and development leads for new effective, safe, and selective cancer treatments. In addition, some recent reports have shown evidence that PAD4 inhibitors are expected to trigger antitumor immune responses, regulate immune cells and related immune factors, enhance the effects of immune checkpoint inhibitors, and enhance their antitumor efficacy. Therefore, PAD4 inhibitors may potentially change tumor immunotherapy and provide an excellent direction for the development and clinical application of immunotherapy strategies for related diseases. [ABSTRACT FROM AUTHOR]

Aristolochic acid nephropathy (AAN) is a progressive kidney disease caused by herbal medicines. Proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2) and neutrophil extracellular traps (NETs) play important roles in kidney injury and immune defense, respectively, but the mechanism underlying AAN regulation by PSTPIP2 and NETs remains unclear. We found that renal tubular epithelial cell (RTEC) apoptosis, neutrophil infiltration, inflammatory factor, and NET production were increased in a mouse model of AAN, while PSTPIP2 expression was low. Conditional knock-in of Pstpip2 in mouse kidneys inhibited cell apoptosis, reduced neutrophil infiltration, suppressed the production of inflammatory factors and NETs, and ameliorated renal dysfunction. Conversely, downregulation of Pstpip2 expression promoted kidney injury. In vivo, the use of Ly6G-neutralizing antibody to remove neutrophils and peptidyl arginine deiminase 4 (PAD4) inhibitors to prevent NET formation reduced apoptosis, alleviating kidney injury. In vitro, damaged RTECs released interleukin-19 (IL-19) via the PSTPIP2/nuclear factor (NF)-κB pathway and induced NET formation via the IL-20Rβ receptor. Concurrently, NETs promoted apoptosis of damaged RTECs. PSTPIP2 affected NET formation by regulating IL-19 expression via inhibition of NF-κB pathway activation in RTECs, inhibiting RTEC apoptosis, and reducing kidney damage. Our findings indicated that neutrophils and NETs play a key role in AAN and therapeutic targeting of PSTPIP2/NF-κB/IL-19/IL-20Rβ might extend novel strategies to minimize Aristolochic acid I-mediated acute kidney injury and apoptosis. [ABSTRACT FROM AUTHOR]