Reactive Oxygen Species and Neutrophil Function.

Neutrophils are essential for killing bacteria and other microorganisms, and they also have a significant role in regulating the inflammatory response. Stimulated neutrophils activate their NADPH oxidase (NOX2) to generate large amounts of superoxide, which acts as a precursor of hydrogen peroxide and other reactive oxygen species that are generated by their heme enzyme myeloperoxidase. When neutrophils engulf bacteria they enclose them in small vesicles (phagosomes) into which superoxide is released by activated NOX2 on the internalized neutrophil membrane. The superoxide dismutates to hydrogen peroxide, which is used by myeloperoxidase to generate other oxidants, including the highly microbicidal species hypochlorous acid. NOX activation occurs at other sites in the cell, where it is considered to have a regulatory function. Neutrophils also release oxidants, which can modify extracellular targets and affect the function of neighboring cells. We discuss the identity and chemical properties of the specific oxidants produced by neutrophils in different situations, and what is known about oxidative mechanisms of microbial killing, inflammatory tissue damage, and signaling.

Proteomic Analysis of Unbounded Cellular Compartments: Synaptic Clefts.

Cellular compartments that cannot be biochemically isolated are challenging to characterize. Here we demonstrate the proteomic characterization of the synaptic clefts that exist at both excitatory and inhibitory synapses. Normal brain function relies on the careful balance of these opposing neural connections, and understanding how this balance is achieved relies on knowledge of their protein compositions. Using a spatially restricted enzymatic tagging strategy, we mapped the proteomes of two of the most common excitatory and inhibitory synaptic clefts in living neurons. These proteomes reveal dozens of synaptic candidates and assign numerous known synaptic proteins to a specific cleft type. The molecular differentiation of each cleft allowed us to identify Mdga2 as a potential specificity factor influencing Neuroligin-2's recruitment of presynaptic neurotransmitters at inhibitory synapses.

Bromine is an essential trace element for assembly of collagen IV scaffolds in tissue development and architecture.

Bromine is ubiquitously present in animals as ionic bromide (Br(-)) yet has no known essential function. Herein, we demonstrate that Br(-) is a required cofactor for peroxidasin-catalyzed formation of sulfilimine crosslinks, a posttranslational modification essential for tissue development and architecture found within the collagen IV scaffold of basement membranes (BMs). Bromide, converted to hypobromous acid, forms a bromosulfonium-ion intermediate that energetically selects for sulfilimine formation. Dietary Br deficiency is lethal in Drosophila, whereas Br replenishment restores viability, demonstrating its physiologic requirement. Importantly, Br-deficient flies phenocopy the developmental and BM defects observed in peroxidasin mutants and indicate a functional connection between Br(-), collagen IV, and peroxidasin. We establish that Br(-) is required for sulfilimine formation within collagen IV, an event critical for BM assembly and tissue development. Thus, bromine is an essential trace element for all animals, and its deficiency may be relevant to BM alterations observed in nutritional and smoking-related disease. PAPERFLICK:

Transcription factor GmAlfin09 regulates endoplasmic reticulum stress in soybean via peroxidase GmPRDX6.

Soybean (Glycine max [L.] Merr.) is a valuable oil crop but is also highly susceptible to environmental stress. Thus, developing approaches to enhance soybean stress resistance is vital to soybean yield improvement. In previous studies, transcription factor Alfin has been shown to serve as an epigenetic regulator of plant growth and development. However, no studies on Alfin have yet been reported in soybean. In this study, the endoplasmic reticulum (ER) stress- and reactive oxygen species (ROS)-related GmAlfin09 was identified. Screening of genes co-expressed with GmAlfin09 unexpectedly led to the identification of soybean peroxidase 6 (GmPRDX6). Further analyses revealed that both GmAlfin09 and GmPRDX6 were responsive to ER stress, with GmPRDX6 localizing to the ER under stress. Promoter binding experiments confirmed the ability of GmAlfin09 to bind to the GmPRDX6 promoter directly. When GmAlfin09 and GmPRDX6 were overexpressed in soybean, enhanced ER stress resistance and decreased ROS levels were observed. Together, these findings suggest that GmAlfin09 promotes the upregulation of GmPRDX6, and GmPRDX6 subsequently localizes to the ER, reduces ROS levels, promotes ER homeostasis, and ensures the normal growth of soybean even under ER stress. This study highlights a vital target gene for future molecular breeding of stress-resistant soybean lines.

Causal relationship between immune cells and the risk of myeloperoxidase antineutrophil cytoplasmic antibody-associated vasculitis: A Mendelian randomization study.

Eosinophilic granulomatosis with polyangiitis (EGPA) is a rare inflammatory disease categorized as antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. The majority of patients are ANCA-positive, predominantly against myeloperoxidase (MPO). Previous studies have predominantly concentrated on the association between EGPA and neutrophils, but recent research has emphasized the role of lymphocytes in the development of EGPA. The objective of our research was to examine the causal association between immune cells and MPO + ANCA EGPA. A two-sample bidirectional Mendelian randomization (MR) analysis was performed, which included 159 MPO + ANCA EGPA cases and 6688 controls and utilized Genome-Wind Associaton Studies (GWAS) summary statistics of immune traits from approximately 3757 individuals, encompassing around 22 million single nucleotide polymorphisms (SNPs). Our findings revealed that 23 immunophenotypes were associated with MPO + ANCA EGPA. Furthermore, the reverse MR analysis showed that MPO + ANCA EGPA had significant causal effects on three immunophenotypes within the Treg panel. By integrating existing research, our study unveiled the contributions of Tregs, B cells, and monocytes to the development of EGPA. Subgroup analysis specifically examined the roles of lymphocyte subtypes, cytokines, and their surface molecules in the pathogenic mechanisms of the disease. This comprehensive approach provides a novel perspective on the biological mechanisms and early intervention strategies for MPO + ANCA EGPA by focusing on immune cells.

Identification of biomarkers in multiple myeloma: A comprehensive study combining microarray analysis and Mendelian randomization.

Despite remarkable advancements in the treatment of multiple myeloma (MM), relapse remains a challenge. However, the mechanisms underlying this disease remain unclear. This study aimed to identify potential biomarkers that could open new avenues for MM treatment. Microarray data and clinical characteristics of patients with MM were obtained from the Gene Expression Omnibus database. Differential expression analysis and protein-protein interaction (PPI) network construction were used to identify hub genes associated with MM. Predictive performance was further assessed using receiver operating characteristic curves and nomogram construction. Functional enrichment analysis was conducted to investigate possible mechanisms. Mendelian randomization (MR) was used to evaluate the causal relationship between the crucial gene and MM risk. Topological analysis of the PPI network revealed five hub genes associated with MM, with myeloperoxidase (MPO) being the key gene owing to its highest degree and area under the curve values. MPO showed significant differences between patients with MM and controls across all datasets. Functional enrichment analysis revealed a strong association between MPO and immune-related pathways in MM. MR analysis confirmed a causal relationship between MPO and the risk of MM. By integrating microarray analysis and MR, we successfully identified and validated MPO as a promising biomarker for MM that is potentially implicated in MM pathogenesis and progression through immune-related pathways.

Transcription factor Nrf2 activation regulates NETosis, endothelial injury, and kidney disease in myeloperoxidase-positive antineutrophil cytoplasmic antibody-associated vasculitis.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic autoimmune disease pathologically characterized by vascular necrosis with inflammation. During AAV development, activated neutrophils produce reactive oxygen species (ROS), leading to the aberrant formation of neutrophil extracellular traps (NETs) via NETosis and subsequent fibrinoid vascular necrosis. Nuclear factor-erythroid 2-related factor 2 (Nrf2) functions as an intracellular defense system to counteract oxidative stress by providing antioxidant properties. Herein, we explored the role of Nrf2 in the pathogenesis of AAV. The role and mechanism of Nrf2 in ANCA-stimulated neutrophils and subsequent endothelial injury were evaluated in vitro using Nrf2 genetic deletion and Nrf2 activator treatment. In corresponding in vivo studies, the role of Nrf2 in ANCA-transfer AAV and spontaneous AAV murine models was examined. Pharmacological activation of Nrf2 in vitro suppressed ANCA-induced NET formation via the inhibition of ROS. In contrast, NET formation was enhanced in Nrf2-deficient neutrophils. Furthermore, Nrf2 activation protected endothelial cells from ANC-induced NETs-mediated injury. In vivo, Nrf2 activation ameliorated glomerulonephritis in two AAV models by upregulating antioxidants and inhibiting ROS-mediated NETs. Furthermore, Nrf2 activation restrained the expansion of splenic immune cells, including T lymphocytes and limited the infiltration of Th17 cells into the kidney. In contrast, Nrf2 genetic deficiency exacerbated vasculitis in a spontaneous AAV model. Thus, the pathophysiological process in AAV may be downregulated by Nrf2 activation, potentially leading to a new therapeutic strategy by regulating NETosis.

Role of neutrophil myeloperoxidase in the development and progression of high-altitude pulmonary edema.

BACKGROUND: Neutrophil infiltration and hypoxic pulmonary vasoconstriction induced by hypobaric hypoxic stress are vital in high-altitude pulmonary edema (HAPE). Myeloperoxidase (MPO), an important enzyme in neutrophils, is associated with inflammation and oxidative stress and is also involved in the regulation of nitric oxide synthase (NOS), an enzyme that catalyzes the production of the vasodilatory factor nitric oxide (NO). However, the role of neutrophil MPO in HAPE's progression is still uncertain. Therefore, we hypothesize that MPO is involved in the development of HAPE via NOS. METHODS: In Xining, China (altitude: 2260 m), C57BL/6 N wild-type and mpo-/- mice served as normoxic controls, while a hypobaric chamber simulated 7000 m altitude for hypoxia. L-NAME, a nitric oxide synthase (NOS) inhibitor to inhibit NO production, was the experimental drug, and D-NAME, without NOS inhibitory effects, was the control. After measuring pulmonary artery pressure (PAP), samples were collected and analyzed for blood neutrophils, oxidative stress, inflammation, vasoactive substances, pulmonary alveolar-capillary barrier permeability, and lung tissue morphology. RESULTS: Wild-type mice's lung injury scores, permeability, and neutrophil counts rose at 24 and 48 h of hypoxia exposure. Under hypoxia, PAP increased from 12.89 ± 1.51 mmHg under normoxia to 20.62 ± 3.33 mmHg significantly in wild-type mice and from 13.24 ± 0.79 mmHg to 16.50 ± 2.07 mmHg in mpo-/- mice. Consistent with PAP, inducible NOS activity, lung permeability, lung injury scores, oxidative stress response, and inflammation showed more significant increases in wild-type mice than in mpo-/- mice. Additionally, endothelial NOS activity and NO levels decreased more pronouncedly in wild-type mice than in mpo-/- mice. NOS inhibition during hypoxia led to more significant increases in PAP, permeability, and lung injury scores compared to the drug control group, especially in wild-type mice. CONCLUSION: MPO knockout reduces oxidative stress and inflammation to preserve alveolar-capillary barrier permeability and limits the decline in endothelial NOS activity to reduce PAP elevation during hypoxia. MPO inhibition emerges as a prospective therapeutic strategy for HAPE, offering avenues for precise interventions.

Characterization of Amycolatopsis 75iv2 dye-decolorizing peroxidase on O-glycosides.

Dye-decolorizing peroxidases are heme peroxidases with a broad range of substrate specificity. Their physiological function is still largely unknown, but a role in the depolymerization of plant cell wall polymers has been widely proposed. Here, a new expression system for bacterial dye-decolorizing peroxidases as well as the activity with previously unexplored plant molecules are reported. The dye-decolorizing peroxidase from Amycolatopsis 75iv2 (DyP2) was heterologously produced in the Gram-positive bacterium Streptomyces lividans TK24 in both intracellular and extracellular forms without external heme supplementation. The enzyme was tested on a series of O-glycosides, which are plant secondary metabolites with a phenyl glycosidic linkage. O-glycosides are of great interest, both for studying the compounds themselves and as potential models for studying specific lignin-carbohydrate complexes. The primary DyP reaction products of salicin, arbutin, fraxin, naringin, rutin, and gossypin were oxidatively coupled oligomers. A cleavage of the glycone moiety upon radical polymerization was observed when using arbutin, fraxin, rutin, and gossypin as substrates. The amount of released glucose from arbutin and fraxin reached 23% and 3% of the total substrate, respectively. The proposed mechanism suggests a destabilization of the ether linkage due to the localization of the radical in the para position. In addition, DyP2 was tested on complex lignocellulosic materials such as wheat straw, spruce, willow, and purified water-soluble lignin fractions, but no remarkable changes in the carbohydrate profile were observed, despite obvious oxidative activity. The exact action of DyP2 on such lignin-carbohydrate complexes therefore remains elusive. IMPORTANCE: Peroxidases require correct incorporation of the heme cofactor for activity. Heterologous overproduction of peroxidases often results in an inactive enzyme due to insufficient heme synthesis by the host organism. Therefore, peroxidases are incubated with excess heme during or after purification to reconstitute activity. S. lividans as a production host can produce fully active peroxidases both intracellularly and extracellularly without the need for heme supplementation. This reduces the number of downstream processing steps and is beneficial for more sustainable production of industrially relevant enzymes. Moreover, this research has extended the scope of dye-decolorizing peroxidase applications by studying naturally relevant plant secondary metabolites and analyzing the formed products. A previously overlooked artifact of radical polymerization leading to the release of the glycosyl moiety was revealed, shedding light on the mechanism of DyP peroxidases. The key aspect is the continuous addition, rather than the more common approach of a single addition, of the cosubstrate, hydrogen peroxide. This continuous addition allows the peroxidase to complete a high number of turnovers without self-oxidation.

Peroxidase gene TaPrx109-B1 enhances wheat tolerance to water deficit via modulating stomatal density.

Increasing the tolerance of crops to water deficit is crucial for the improvement of crop production in water-restricted regions. Here, a wheat peroxidase gene (TaPrx109-B1) belonging to the class III peroxidase gene family was identified and its function in water deficit tolerance was revealed. We demonstrated that overexpression of TaPrx109-B1 reduced leaf H2O2 level and stomatal density, increased leaf relative water content, water use efficiency, and tolerance to water deficit. The expression of TaEPF1 and TaEPF2, two key negative regulators of stomatal development, were significantly upregulated in TaPrx109-B1 overexpression lines. Furthermore, exogenous H2O2 downregulated the expression of TaEPF1 and TaEPF2 and increased stomatal density, while exogenous application of diphenyleneiodonium chloride, a potent NADPH oxidase inhibitor that repressed the synthesis of H2O2, upregulated the expression of TaEPF1 and TaEPF2, decreased stomatal density, and enhanced wheat tolerance to water deficit. These findings suggest that TaPrx109-B1 influences leaf stomatal density by modulation of H2O2 level and the expression of TaEPF1 and TaEPF2. The results of the field trial showed that overexpressing TaPrx109-B1 increased grain number per spike, which reduced the yield loss caused by water deficiency. Therefore, TaPrx109-B1 has great potential in breeding wheat varieties with improved water deficit tolerance.

Tuning the peroxidase activity of artificial P450 peroxygenase by engineering redox-sensitive residues.

Cytochrome P450 monooxygenases (P450s) are well recognized as versatile bio-oxidation catalysts. However, the catalytic functions of P450s are highly dependent on NAD(P)H and redox partner proteins. Our group has recently reported the use of a dual-functional small molecule (DFSM) for generating peroxygenase activity of P450BM3, a long-chain fatty acid hydroxylase from Bacillus megaterium. The DFSM-facilitated P450BM3 peroxygenase system exhibited excellent peroxygenation activity and regio-/enantioselectivity for various organic substrates, such as styrenes, thioanisole, small alkanes, and alkylbenzenes. Very recently, we demonstrated that the DFSM-facilitated P450BM3 peroxygenase could be switched to a peroxidase by engineering the redox-sensitive tyrosine residues in P450BM3. Given the great potential of P450 peroxidase for C-H oxyfunctionalization, we herein report scrutiny of the effect of mutating redox-sensitive residues on peroxidase activity by deeply screening all redox-sensitive residues of P450BM3, namely methionines, tryptophans, cysteines, and phenylalanines. As a result, six beneficial mutations at positions M212, F81, M112, F173, M177, and F77 were screened out from 78 constructed mutants, and significantly enhanced the peroxidase activity of P450BM3 in the presence of Im-C6-Phe, a typical DFSM molecule. Further combination of the beneficial mutations resulted in a more than 100-fold improvement in peroxidase activity compared with that of the combined parent enzyme and DFSM, comparable to or better than most natural peroxidases. In addition, mutations of redox-sensitive residues even dramatically increased, by more than 300-fold, the peroxidase activity of the starting F87A enzyme in the absence of the DFSM, despite the far lower apparent catalytic turnover number compared with the DFSM-P450 system. This study provides new insights and a potential strategy for regulating the catalytic promiscuity of P450 enzymes for multiple functional oxidations.

Transcriptomics Combined with Physiology and Metabolomics Reveals the Mechanism of Tolerance to Lead Toxicity in Maize Seedling.

Lead (Pb) exposure can induce molecular changes in plants, disrupt metabolites, and impact plant growth. Therefore, it is essential to comprehend the molecular mechanisms involved in Pb tolerance in plants to evaluate the long-term environmental consequences of Pb exposure. This research focused on maize as the test subject to study variations in biomass, root traits, genes, and metabolites under hydroponic conditions under Pb conditions. The findings indicate that high Pb stress significantly disrupts plant growth and development, leading to a reduction in catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) activities by 17.12, 5.78, and 19.38%, respectively. Conversely, Pb stress led to increase malondialdehyde (MDA) contents, ultimately impacting the growth of maize. The non-targeted metabolomics analysis identified 393 metabolites categorized into 12 groups, primarily consisting of organic acids and derivatives, organ heterocyclic compounds, lipids and lipid-like molecules and benzenoids. Further analysis indicated that Pb stress induced an accumulation of 174 metabolites mainly enriched in seven metabolic pathways, for example phenylpropanoid biosynthesis and flavonoid biosynthesis. Transcriptome analysis revealed 1933 shared differentially expressed genes (DEGs), with 1356 upregulated and 577 downregulated genes across all Pb treatments. Additionally, an integrated analysis identified several DEGs and differentially accumulated metabolites (DAMs), including peroxidase, alpha-trehalose, and D-glucose 6-phosphate, which were linked to cell wall biosynthesis. These findings imply the significance of this pathway in Pb detoxification. This comprehensive investigation, employing multiple methodologies, provides a detailed molecular-level insight into maize's response to Pb stress.

Genetic analysis of different subtypes of aseptic pustulosis in the Chinese population.

Aseptic pustulosis involves inflammatory skin conditions with nonbacterial pustules on erythema, accompanied by neutrophil and eosinophil infiltration in the epidermis. Dysregulation of the interleukin (IL)-36 pathway leads to neutrophil aggregation and pustule formation. Variants in IL36RN, CARD14, AP1S3, MPO, SERPINA3 and BTN3A3 have been identified in generalized pustular psoriasis (GPP) in the past. Some patients with acrodermatitis continua of Hallopeau (ACH), palmoplantar pustulosis and acute generalized exanthematous pustulosis (AGEP) also exhibit mutations in IL36RN, CARD14 and AP1S3, albeit with regional and population-specific variations. This study aims to explore a shared genetic foundation among those with aseptic pustulosis. We performed Sanger sequencing on six genes in 126 patients with aseptic pustulosis. Genetic analysis identified IL36RN variants strongly associated with ACH, AGEP and subcorneal pustular dermatosis (SPD). Immunohistochemistry revealed elevated inflammatory cytokines in all subtypes. This study establishes a significant association between IL36RN variants and ACH, AGEP and SPD, emphasizing the IL-1/IL-36-chemokine-neutrophil axis as a common pathogenic mechanism. Targeting this axis holds promise for therapeutic interventions for aseptic pustulosis.

Myeloperoxidase Gene Deletion Causes Drastic Microbiome Shifts in Mice and Does Not Mitigate Dextran Sodium Sulphate-Induced Colitis.

Neutrophil-myeloperoxidase (MPO) is a heme-containing peroxidase which produces excess amounts of hypochlorous acid during inflammation. While pharmacological MPO inhibition mitigates all indices of experimental colitis, no studies have corroborated the role of MPO using knockout (KO) models. Therefore, we investigated MPO deficient mice in a murine model of colitis. Wild type (Wt) and MPO-deficient mice were treated with dextran sodium sulphate (DSS) in a chronic model of experimental colitis with three acute cycles of DSS-induced colitis over 63 days, emulating IBD relapse and remission cycles. Mice were immunologically profiled at the gut muscoa and the faecal microbiome was assessed via 16S rRNA amplicon sequencing. Contrary to previous pharmacological antagonist studies targeting MPO, MPO-deficient mice showed no protection from experimental colitis during cyclical DSS-challenge. We are the first to report drastic faecal microbiota shifts in MPO-deficient mice, showing a significantly different microbiome profile on Day 1 of treatment, with a similar shift and distinction on Day 29 (half-way point), via qualitative and quantitative descriptions of phylogenetic distances. Herein, we provide the first evidence of substantial microbiome shifts in MPO-deficiency, which may influence disease progression. Our findings have significant implications for the utility of MPO-KO mice in investigating disease models.

The Increase in the Peroxidase Activity of the Cytochrome C with Substitutions in the Universal Binding Site Is Associated with Changes in the Ability to Interact with External Ligands.

Cytochrome c (CytC), a one-electron carrier, transfers electrons from complex bc1 to cytochrome c oxidase (CcO) in the electron-transport chain. Electrostatic interaction with the partners, complex bc1 and CcO, is ensured by a lysine cluster near the heme forming the Universal Binding Site (UBS). We constructed three mutant variants of mitochondrial CytC with one (2Mut), four (5Mut), and five (8Mut) Lys->Glu substitutions in the UBS and some compensating Glu->Lys substitutions at the periphery of the UBS for charge compensation. All mutants showed a 4-6 times increased peroxidase activity and accelerated binding of cyanide to the ferric heme of CytC. In contrast, decomposition of the cyanide complex with ferrous CytC, as monitored by magnetic circular dichroism spectroscopy, was slower in mutants compared to WT. Molecular dynamic simulations revealed the increase in the fluctuations of Cα atoms of individual residues of mutant CytC compared to WT, especially in the Ω-loop (70-85), which can cause destabilization of the Fe…S(Met80) coordination link, facilitation of the binding of exogenous ligands cyanide and peroxide, and an increase in peroxidase activity. It was found that only one substitution K72E is enough to induce all these changes, indicating the significance of K72 and the Ω-loop (70-85) for the structure and physiology of mitochondrial CytC. In this work, we also propose using a ferro-ferricyanide buffer as a substrate to monitor the peroxidase activity of CytC. This new approach allows us to determine the rate of peroxidase activity at moderate (200 µM) concentrations of H2O2 and avoid complications of radical formation during the reaction.

Heterologous expression and characterization of a dye-decolorizing peroxidase from Ganoderma lucidum, and its application in decolorization and detoxifization of different types of dyes.

Dye-decolorizing peroxidases (DyPs) belong to a novel superfamily of heme peroxidases that can oxidize recalcitrant compounds. In the current study, the GlDyP2 gene from Ganoderma lucidum was heterologously expressed in Escherichia coli, and the enzymatic properties of the recombinant GlDyP2 protein were investigated. The GlDyP2 protein could oxidize not only the typical peroxidase substrate ABTS but also two lignin substrates, namely guaiacol and 2,6-dimethoxy phenol (DMP). For the ABTS substrate, the optimum pH and temperature of GlDyP2 were 4.0 and 35 °C, respectively. The pH stability and thermal stability of GlDyP2 were also measured; the results showed that GlDyP2 could function normally in the acidic environment, with a T50 value of 51 °C. Moreover, compared to untreated controls, the activity of GlDyP2 was inhibited by 1.60 mM of Mg2+, Ni2+, Mn2+, and ethanol; 0.16 mM of Cu2+, Zn2+, methanol, isopropyl alcohol, and Na2EDTA·2H2O; and 0.016 mM of Fe2+ and SDS. The kinetic constants of recombinant GlDyP2 for oxidizing ABTS, Reactive Blue 19, guaiacol, and DMP were determined; the results showed that the recombination GlDyP2 exhibited the strongest affinity and the most remarkable catalytic efficiency towards guaiacol in the selected substrates. GlDyP2 also exhibited decolorization and detoxification capabilities towards several dyes, including Reactive Blue 19, Reactive Brilliant Blue X-BR, Reactive Black 5, Methyl Orange, Trypan Blue, and Malachite Green. In conclusion, GlDyP2 has good application potential for treating dye wastewater.

[Optimization and verification of conditions for induction of neutrophil extracellular traps in vitro].

This study aims to optimize the conditions for the formation of neutrophil extracellular traps(NETs) in vitro, so as to establish a relatively stable experimental research platform. Different conditions were compared, including commonly used laboratory animals(rats and mice) and a variety of cell sources(bone marrow neutrophils and peripheral blood neutrophils separated by percoll density gradient centrifugation). Different inducers like lipopolysaccharide(LPS) and phorbol 12-myristate 13-acetate(PMA) were used for induction in vitro. Myeloperoxidase(MPO)/citrullinated histone H3(CitH3)/DAPI immunofluorescence and cell free DNA(cf-DNA) content determination were used for comprehensive evaluation to screen the optimal conditions for the formation of NETs induced in vitro. Furthermore, the stability of the selected conditions for inducing the formation of NETs in vitro was evaluated by tetramethylpyrazine(TMP), an active component in Chinese herbal medicines. The results showed that coated poly-D-lysine(PDL) induced the formation of NETs in bone marrow neutrophils of mice to a certain extent. Both LPS and PMA significantly up-regulated the protein levels of MPO and CitH3 in mouse bone marrow neutrophils and elevated the cfDNA level in the supernatant of rat peripheral blood neutrophils. The cfDNA level in the PMA-induced group increased more significantly than that in the LPS-induced group(P<0.05). The results of immunofluorescence staining showed that the expression of MPO and CitH3 in mouse bone marrow neutrophils, rat bone marrow neutrophils, and rat peripheral blood neutrophils were significantly increased after PMA induction, especially in rat peripheral blood neutrophils. TMP significantly down-regulated the protein levels of MPO, CitH3, and neutrophil elastase(NE) in rat peripheral blood neutrophils induced by PMA. In conclusion, treating the peripheral blood neutrophils of rats with PMA is the optimal condition for inducing the formation of NETs in vitro. This study provides an optimal platform for in vitro studies based on NETs and a basis for studying the effects of traditional Chinese medicines targeting NETs.

Putative NAD(P)-Binding Rossmann Fold Protein Is Involved in Chitosan-Induced Peroxidase Activity and Lipoxygenase Expression in Physcomitrium patens.

Oxidative burst, the rapid production of high levels of reactive oxygen species in response to external stimuli, is an early defense reaction against pathogens. The fungal elicitor chitosan causes an oxidative burst in the moss Physcomitrium patens (formerly Physcomitrella patens), mainly due to the peroxidase enzyme Prx34. To better understand the chitosan responses in P. patens, we conducted a screen of part of a P. patens mutant collection to isolate plants with less peroxidase activity than wild-type (WT) plants after chitosan treatment. We isolated a P. patens mutant that affected the gene encoding NAD(P)-binding Rossmann fold protein (hereafter, Rossmann fold protein). Three Rossmann fold protein-knockout (KO) plants (named Rossmann fold KO lines) were generated and used to assess extracellular peroxidase activity and expression of defense-responsive genes, including alternative oxidase, lipoxygenase (LOX), NADPH oxidase, and peroxidase (Prx34) in response to chitosan treatment. Extracellular (apoplastic) peroxidase activity was significantly lower in Rossmann fold KO lines than in WT plants after chitosan treatments. Expression of the LOX gene in Rossmann fold KO plants was significantly lower before and after chitosan treatment when compared with WT. Peroxidase activity assays together with gene expression analyses suggest that the Rossmann fold protein might be an important component of the signaling pathway leading to oxidative burst and basal expression of the LOX gene in P. patens. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A Peroxide-Responding sRNA Evolved from a Peroxidase mRNA.

Small RNAs (sRNAs) are important gene regulators in bacteria, but it is unclear how new sRNAs originate and become part of regulatory networks that coordinate bacterial response to environmental stimuli. Using a covariance modeling-based approach, we analyzed the presence of hundreds of sRNAs in more than a thousand genomes across Enterobacterales, a bacterial order with a confluence of factors that allows robust genome-scale sRNA analyses: several well-studied organisms with fairly conserved genome structures, an established phylogeny, and substantial nucleotide diversity within a narrow evolutionary space. We discovered that a majority of sRNAs arose recently, and uncovered protein-coding genes as a potential source from which new sRNAs arise. A detailed investigation of the emergence of OxyS, a peroxide-responding sRNA, revealed that it evolved from a fragment of a peroxidase messenger RNA. Importantly, although it replaced the ancestral peroxidase, OxyS continues to be part of the ancestral peroxide-response regulon, indicating that an sRNA that arises from a protein-coding gene would inherently be part of the parental protein's regulatory network. This new insight provides a fresh framework for understanding sRNA origin and regulatory integration in bacteria.

Regulation of neutrophil myeloperoxidase inhibitor SPIN by the small RNA Teg49 in Staphylococcus aureus.

Teg49 is a Staphylococcus aureus trans-acting regulatory sRNA derived from cleavage of the sarA P3 transcript. We showed by RNA-Seq here that the 5' trident-like structure in Teg49 regulates transcriptionally (direct and indirect) 22 genes distinct from sarA. Among these, Teg49 was noted to repress spn, encoding a 102 residue preprotein which yields the mature 73 residue peptide which inhibits the catalytic activity of myeloperoxidase in human neutrophils. Teg49 was found to regulate spn mRNA post-transcriptionally in strain SH1000 through 9-nt base-pairing between hairpin loop 2 of Teg49 and an exposed bulge of the spn mRNA. Mutations of the Teg49 binding site disrupted the repression of spn, leading to reduced degradation, and increased half-life of spn mRNA in the Teg49 mutant. The spn-Teg49 interaction was also confirmed with a synonymous spn mutation to yield enhanced spn expression in the mutant vs. the parent. The Teg49 mutant with increased spn expression exhibited enhanced resistance to MPO activity in vitro. Killing assays with human neutrophils showed that the Teg49 mutant was more resistant to killing after phagocytosis. Altogether, this study shows that Teg49 in S. aureus has a distinct and important regulatory profile whereby this sRNA modulates resistance to myeloperoxidase-mediated killing by human neutrophils.