Impaired arginine/ornithine metabolism drives severe HFMD by promoting cytokine storm.

Introduction: The Hand, Foot and Mouth Disease (HFMD), caused by enterovirus 71 infection, is a global public health emergency. Severe HFMD poses a significant threat to the life and well-being of children. Numerous studies have indicated that the occurrence of severe HFMD is associated with cytokine storm. However, the precise molecular mechanism underlying cytokine storm development remains elusive, and there are currently no safe and effective treatments available for severe HFMD in children. Methods: In this study, we established a mouse model of severe HFMD to investigate the molecular mechanisms driving cytokine storm. We specifically analyzed metabolic disturbances, focusing on arginine/ornithine metabolism, and assessed the potential therapeutic effects of spermine, an ornithine metabolite. Results: Our results identified disturbances in arginine/ornithine metabolism as a pivotal factor driving cytokine storm onset in severe HFMD cases. Additionally, we discovered that spermine effectively mitigated the inflammatory injury phenotype observed in mice with severe HFMD. Discussion: In conclusion, our findings provide novel insights into the molecular mechanisms underlying severe HFMD from a metabolic perspective while offering a promising new strategy for its safe and effective treatment.

Microbiota alert: Proteobacteria consume arginine to dampen omental antitumor immunity.

The microbiota can impact antitumor immunity, but whether the microbiota regulates omental antitumor immunity remains elusive. In this issue of Cell Host & Microbe, Meza-Perez et al. demonstrated that Proteobacteria consume arginine to increase Treg cell suppressive capacity and inhibit antitumor immune responses, promoting tumor growth in the omentum.

PRMT6-mediated ADMA promotes p62 phase separation to form a negative feedback loop in ferroptosis.

Purpose: Due to intrinsic defensive response, ferroptosis-activating targeted therapy fails to achieve satisfactory clinical benefits. Though p62-Keap1-Nrf2 axis is activated to form a negative feedback loop during ferroptosis induction, how p62 is activated remains largely unknown. Methods: MTS assay was applied to measure cell growth. Lipid ROS was detected with C11-BODIPY reagent by flow cytometer. Quantitative real-time PCR (qPCR) and western blotting were performed to determine mRNA and protein level. Immunofluorescence (IF) was performed to examine the distribution of proteins. Fluorescence recovery after photobleaching (FRAP) was adopted to evaluate p62 phase separation. Immunoprecipitation (IP), co-IP and Proximal ligation assay (PLA) were performed to detected protein posttranslational modifications and protein-protein interactions. Tumor xenograft model was employed to inspect in vivo growth of pancreatic cancer cells. Results: Upon ferroptosis induction, Nuclear Factor E2 Related Factor 2 (Nrf2) protein and its downstream genes such as HMOX1 and NQO1 were upregulated. Knockdown of p62 significantly reversed Nrf2 upregulation and Keap1 decrease after ferroptosis induction. Knockdown of either p62 or Nrf2 remarkably sensitized ferroptosis induction. Due to augmented p62 phase separation, formation of p62 bodies were increased to recruit Keap1 after ferroptosis induction. Protein arginine methyltransferase 6 (PRMT6) mediated asymmetric dimethylarginine (ADMA) of p62 to increase its oligomerization, promoting p62 phase separation and p62 body formation. Knockdown of p62 or PRMT6 notably sensitized pancreatic cancer cells to ferroptosis both in vitro and in vivo through suppressing Nrf2 signaling. Conclusion: During ferroptosis induction, PRMT6 mediated p62 ADMA to promote its phase separation, sequestering Keap1 to activate Nrf2 signaling and inhibit ferroptosis. Therefore, targeting PRMT6-mediated p62 ADMA could be a new option to sensitize ferroptosis for cancer treatment.

Nitric oxide brings innate immune resistance to M. tuberculosis infection among high-risk household contacts of pulmonary tuberculosis patients.

Nitric oxide (NO) and iNOS are crucial host factors in innate immunity against intracellular pathogens. However, the role of NO in Mycobacterium tuberculosis (M. tb) infection in humans remains controversial, unlike in the murine model of TB. To investigate this, levels of NO, iNOS, and L-arginine, as well as the NOS2A gene polymorphism rs57234985 at the promoter region of NOS2A, were evaluated in pulmonary TB (PTB) patients and their household contacts (HHCs). Increased levels of NO and iNOS expression in HHCs indicated exposure to M. tb infection which was confirmed by higher levels of iNOS and NO in Mantouxpositive individuals. Furthermore, higher levels of arginine were detected in HHCs, suggesting its potential role in promoting optimal NO synthesis. PTB patients had higher levels of these analytes due to ongoing active infection. Interestingly, iNOS and NO levels were inversely related to bacterial burden, suggesting their antimicrobial role. NOS2A gene polymorphism was found to be associated with disease susceptibility, with the TT genotype linked to increased iNOS expression. To conclude, iNOS plays a crucial role in controlling early M. tb infection in HHCs by inducing optimal NO production with help of L-arginine. Further longitudinal studies are needed to better understand the role of these host factors upon disease activation.

Proton Transfer via Arginine with Suppressed pKa Mediates Catalysis by Gentisate and Salicylate Dioxygenase.

Gentisate and salicylate 1,2-dioxygenases (GDO and SDO) facilitate aerobic degradation of aromatic rings by inserting both atoms of dioxygen into their substrates, thereby participating in global carbon cycling. The role of acid-base catalysts in the reaction cycles of these enzymes is debatable. We present evidence of the participation of a proton shuffler during catalysis by GDO and SDO. The pH dependence of Michaelis-Menten parameters demonstrates that a single proton transfer is mandatory for the catalysis. Measurements at variable temperatures and pHs were used to determine the standard enthalpy of ionization (ΔHion°) of 51 kJ/mol for the proton transfer event. Although the observed apparent pKa in the range of 6.0-7.0 for substrates of both enzymes is highly suggestive of a histidine residue, ΔHion° establishes an arginine residue as the likely proton source, providing phylogenetic relevance for this strictly conserved residue in the GDO family. We propose that the atypical 3-histidine ferrous binding scaffold of GDOs contributes to the suppression of arginine pKa and provides support for this argument by employing a 2-histidine-1-carboxylate variant of the enzyme that exhibits elevated pKa. A reaction mechanism considering the role of the proton source in stabilizing key reaction intermediates is proposed.

Metabolomic changes in Cryptocaryon irritans from Larimichthys crocea after exposure to copper plate.

Cryptocaryon irritans is a highly detrimental parasite in mariculture, causing significant economic losses to the aquaculture industry of Larimichthys crocea. In recent years, copper and copper alloy materials have been used to kill parasites. In this study, the effect of copper plates on the tomont period of C. irritans was explored. The findings indicated that copper plates effectively eradicated tomonts, resulting in a hatching rate of 0. The metabolomic analysis revealed that a total of 2,663 differentially expressed metabolites (1,032 up-regulated and 1,631 down-regulated) were screened in the positive ion mode, and 2,199 differentially expressed metabolites (840 up-regulated and 1,359 down-regulated) were screened in the negative ion mode. L-arginine and L-aspartic acid could be used as potential biomarkers. Copper plate treatment affected 25 metabolic pathways in the tomont, most notably influencing histidine metabolism, retinol metabolism, the biosynthesis of phenylalanine, tyrosine, and tryptophan, as well as arginine and proline metabolism. It was shown that high concentrations of copper ions caused a certain degree of disruption to the metabolome of tomonts in C. irritans, thereby impacting their metabolic processes. Consequently, this disturbance ultimately leads to the rapid demise of tomonts upon exposure to copper plates. The metabolomic changes observed in this study elucidate the lethal impact of copper on C. irritans tomonts, providing valuable reference data for the prevention and control of C. irritans in aquaculture.

How arginine inhibits substrate-binding domain 2 elucidated using molecular dynamics simulations.

The substrate-binding domain 2 (SBD2) is an important part of the bacterial glutamine (GLN) transporter and mediates binding and delivery of GLN to the transporter translocation subunit. The SBD2 consists of two domains, D1 and D2, that bind GLN in the space between domains in a closed structure. In the absence of ligand, the SBD2 adopts an open conformation with larger space between domains. The GLN binding and closing are essential for the subsequent transport into the cell. Arginine (ARG) can also bind to SBD2 but does not induce closing and inhibits GLN transport. We use atomistic molecular dynamics (MD) simulations in explicit solvent to study ARG binding in the presence of the open SBD2 structure and observed reversible binding to the native GLN binding site with similar contacts but no transition to a closed SBD2 state. Absolute binding free energy simulations predict a considerable binding affinity of ARG and GLN to the binding site on the D1 domain. Free energy simulations to induce subsequent closing revealed a strong free energy penalty in case of ARG binding in contrast to GLN binding that favors the closed SBD2 state but still retains a free energy barrier for closing. The simulations allowed the identification of the molecular origin of the closing penalty in case of bound ARG and suggested a mutation of lysine at position 373 to alanine that strongly reduced the penalty and allowed closing even in the presence of bound ARG. The study offers an explanation of the molecular mechanism of how ARG competitively inhibits GLN from binding to SBD2 and from triggering the transition to a closed conformation. The proposed Lys373Ala mutation shows promise as a potential tool to validate whether a conformational mismatch between open SBD2 and the translocator is responsible for preventing ARG uptake to the cell.

The ArgR-Regulated ADI Pathway Facilitates the Survival of Vibrio fluvialis under Acidic Conditions.

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.

Myricetin ameliorates the severity of pancreatitis in mice by regulating cathepsin B activity and inflammatory cytokine production.

Cathepsin B (CTSB) and inflammatory cytokines are critical in initiating and developing pancreatitis. Calcineurin, a central calcium (Ca2+)-responsive signaling molecule, mediates acinar cell death and inflammatory responses leading to pancreatitis. However, the detailed mechanisms for regulating CTSB activity and inflammatory cytokine production are unknown. Myricetin (MC) exhibits various biological activities, including anti-inflammatory effects. Here, we aimed to investigate MC effects on pancreatitis and the underlying mechanisms. Prophylactic and therapeutic MC treatment ameliorated the severity of cerulein-, L-arginine-, and PDL-induced acute pancreatitis (AP). The inhibition of CTSB activity by MC was mediated via decreased calcineurin activity and macrophage infiltration, not neutrophils infiltration, into the pancreas. Additionally, calcineurin activity inhibition by MC prevented the phosphorylation of Ca2+/CaM-dependent protein kinase kinase 2 (CaMKK2) during AP, resulting in the inhibition of CaMKIV phosphorylation and adenosine monophosphate-activated protein kinase (AMPK) dephosphorylation. Furthermore, MC reduced nuclear factor-κB activation by modulating the calcineurin-CaMKIV-IKKα/ß-Iκ-Bα and calcineurin-AMPK-sirtuin1 axes, resulting in reduced production of tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6. Our results showed that MC alleviated AP severity by inhibiting acinar cell death and inflammatory responses, suggesting that MC as a calcineurin and CaMKK2 signaling modulator may be a potential treatment for AP.

A PRMT5-ZNF326 axis mediates innate immune activation upon replication stress.

DNA replication stress (RS) is a widespread phenomenon in carcinogenesis, causing genomic instability and extensive chromatin alterations. DNA damage leads to activation of innate immune signaling, but little is known about transcriptional regulators mediating such signaling upon RS. Using a chemical screen, we identified protein arginine methyltransferase 5 (PRMT5) as a key mediator of RS-dependent induction of interferon-stimulated genes (ISGs). This response is also associated with reactivation of endogenous retroviruses (ERVs). Using quantitative mass spectrometry, we identify proteins with PRMT5-dependent symmetric dimethylarginine (SDMA) modification induced upon RS. Among these, we show that PRMT5 targets and modulates the activity of ZNF326, a zinc finger protein essential for ISG response. Our data demonstrate a role for PRMT5-mediated SDMA in the context of RS-induced transcriptional induction, affecting physiological homeostasis and cancer therapy.

Proteobacteria impair anti-tumor immunity in the omentum by consuming arginine.

Gut microbiota influence anti-tumor immunity, often by producing immune-modulating metabolites. However, microbes consume a variety of metabolites that may also impact host immune responses. We show that tumors grow unchecked in the omenta of microbe-replete mice due to immunosuppressive Tregs. By contrast, omental tumors in germ-free, neomycin-treated mice or mice colonized with altered Schaedler's flora (ASF) are spontaneously eliminated by CD8+ T cells. These mice lack Proteobacteria capable of arginine catabolism, causing increases in serum arginine that activate the mammalian target of the rapamycin (mTOR) pathway in Tregs to reduce their suppressive capacity. Transfer of the Proteobacteria, Escherichia coli (E. coli), but not a mutant unable to catabolize arginine, to ASF mice reduces arginine levels, restores Treg suppression, and prevents tumor clearance. Supplementary arginine similarly decreases Treg suppressive capacity, increases CD8+ T cell effectiveness, and reduces tumor burden. Thus, microbial consumption of arginine alters anti-tumor immunity, offering potential therapeutic strategies for tumors in visceral adipose tissue.

Positive charges promote the recognition of proteins by the chaperone SlyD from Escherichia coli.

SlyD is a widely-occurring prokaryotic FKBP-family prolyl isomerase with an additional chaperone domain. Often, such as in Escherichia coli, a third domain is found at its C-terminus that binds nickel and provides it for nickel-enzyme biogenesis. SlyD has been found to bind signal peptides of proteins that are translocated by the Tat pathway, a system for the transport of folded proteins across membranes. Using peptide arrays to analyze these signal peptide interactions, we found that SlyD interacted only with positively charged peptides, with a preference for arginines over lysines, and large hydrophobic residues enhanced binding. Especially a twin-arginine motif was recognized, a pair of highly conserved arginines adjacent to a stretch of hydrophobic residues. Using isothermal titration calorimetry (ITC) with purified SlyD and a signal peptide-containing model Tat substrate, we could show that the wild type twin-arginine signal peptide was bound with higher affinity than an RR>KK mutated variant, confirming that positive charges are recognized by SlyD, with a preference of arginines over lysines. The specific role of negative charges of the chaperone domain surface and of hydrophobic residues in the chaperone active site was further analyzed by ITC of mutated SlyD variants. Our data show that the supposed key hydrophobic residues of the active site are indeed crucial for binding, and that binding is influenced by negative charges on the chaperone domain. Recognition of positive charges is likely achieved by a large negatively charged surface region of the chaperone domain, which is highly conserved although individual positions are variable.

Arginine Regulates Skeletal Muscle Fiber Type Formation via mTOR Signaling Pathway.

The composition of skeletal muscle fiber types affects the quality of livestock meat and human athletic performance and health. L-arginine (Arg), a semi-essential amino acid, has been observed to promote the formation of slow-twitch muscle fibers in animal models. However, the precise molecular mechanisms are still unclear. This study investigates the role of Arg in skeletal muscle fiber composition and mitochondrial function through the mTOR signaling pathway. In vivo, 4-week C56BL/6J male mice were divided into three treatment groups and fed a basal diet supplemented with different concentrations of Arg in their drinking water. The trial lasted 7 weeks. The results show that Arg supplementation significantly improved endurance exercise performance, along with increased SDH enzyme activity and upregulated expression of the MyHC I, MyHC IIA, PGC-1α, and NRF1 genes in the gastrocnemius (GAS) and quadriceps (QUA) muscles compared to the control group. In addition, Arg activated the mTOR signaling pathway in the skeletal muscle of mice. In vitro experiments using cultured C2C12 myotubes demonstrated that Arg elevated the expression of slow-fiber genes (MyHC I and Tnnt1) as well as mitochondrial genes (PGC-1α, TFAM, MEF2C, and NRF1), whereas the effects of Arg were inhibited by the mTOR inhibitor rapamycin. In conclusion, these findings suggest that Arg modulates skeletal muscle fiber type towards slow-twitch fibers and enhances mitochondrial functions by upregulating gene expression through the mTOR signaling pathway.

Dual topology of equine arteritis virus GP3 protein and the role of arginine motif RXR in GP3 ER retention.

Glycoprotein 3 (GP3) serves as a structural protein in equine arteritis virus (EAV), forming a heterotrimeric complex that plays a pivotal role in virus tropism. In this study, we tested the membrane topology of GP3, both when expressed separately and during infection with recombinant tagged EAV GP3-HA. In our antibody accessibility experiment, we made a noteworthy discovery: GP3, when expressed separately, exhibits a dual topology. We introduced an additional N-glycosylation site, which was only partially used, providing further evidence for the dual topology of GP3. Intriguingly, this mutated GP3 was secreted into the medium, a result of the disruption of the ER retention motif RXR. The additional glycosylation site was not used when we examined the recombinant EAV virus with the same mutation. Despite the fact of higher expression levels of mutant GP3-HA, the protein was not secreted, and the recombinant mutant virus did not have growth delay compared to the EAV wild-type virus. This finding suggests that GP3 has a single type one membrane topology in virus infected cells, whereas the expression of GP3 in trans results in the dual topology of this protein. The RXR motif in the C-terminus is a co-factor of ER retention of the protein, but the main retention signal remains elusive.

Levels of asymmetric dimethylarginine in plasma and aqueous humor: a key risk factor for the severity of fibrovascular proliferation in proliferative diabetic retinopathy.

Introduction: Proliferative diabetic retinopathy (PDR) is a common diabetes complication, significantly impacting vision and quality of life. Previous studies have suggested a potential link between arginine pathway metabolites and diabetic retinopathy (DR). Connective tissue growth factor (CTGF) plays a role in the occurrence and development of fibrovascular proliferation (FVP) in PDR patients. However, the relationship between arginine pathway metabolites and FVP in PDR remains undefined. This study aimed to explore the correlation between four arginine pathway metabolites (arginine, asymmetric dimethylarginine[ADMA], ornithine, and citrulline) and the severity of FVP in PDR patients. Methods: In this study, plasma and aqueous humor samples were respectively collected from 30 patients with age-related cataracts without diabetes mellitus (DM) and from 85 PDR patients. The PDR patients were categorized as mild-to-moderate or severe based on the severity of fundal FVP. The study used Kruskal-Wallis test to compare arginine, ADMA, ornithine, and citrulline levels across three groups. Binary logistic regression identified risk factors for severe PDR. Spearman correlation analysis assessed associations between plasma and aqueous humor metabolite levels, and between ADMA and CTGF levels in aqueous humor among PDR patients. Results: ADMA levels in the aqueous humor were significantly greater in patients with severe PDR than in those with mild-to-moderate PDR(P=0.0004). However, the plasma and aqueous humor levels of arginine, ornithine, and citrulline did not significantly differ between mild-to-moderate PDR patients and severe PDR patients (P>0.05). Binary logistic regression analysis indicated that the plasma (P=0.01) and aqueous humor (P=0.006) ADMA levels in PDR patients were risk factors for severe PDR. Furthermore, significant correlations were found between plasma and aqueous humor ADMA levels (r=0.263, P=0.015) and between aqueous humor ADMA and CTGF levels (r=0.837, P<0.001). Conclusion: Elevated ADMA levels in plasma and aqueous humor positively correlate with the severity of FVP in PDR, indicating ADMA as a risk factor for severe PDR.

Arginine, glycine, and creatine supplementation improves symptoms in a female with creatine transporter deficiency.

X-linked creatine transporter deficiency is caused by hemizygous or heterozygous pathogenic variants in SLC6A8 that cause neuropsychiatric symptoms because of impaired uptake of creatine into tissues throughout the body. Small cohorts have suggested that supplementation of creatine, arginine, and glycine can stop disease progression in males, but only six cases of supplementation in females have been published. Here, we present a female with a de-novo pathogenic SLC6A8 variant who had ongoing weight loss, mild intellectual disability, and neuropsychiatric symptoms. Magnetic resonance spectroscopy of the brain showed reduced creatine on all acquired spectra. The patient was started on creatine-monohydrate, l -arginine, and l -glycine supplementation, and she had significant symptomatic improvement within the following 3 weeks. After 8 months of supplementation, magnetic resonance spectroscopy showed improved creatine concentrations with normalizing semiquantitative ratios with other brain metabolites. Current data supports clinicians trialing creatine, arginine, and glycine supplements for female patients with creatine transporter deficiency.

DDAH-1 maintains endoplasmic reticulum-mitochondria contacts and protects dopaminergic neurons in Parkinson's disease.

The loss of dopaminergic neurons in the substantia nigra is a hallmark of pathology in Parkinson's disease (PD). Dimethylarginine dimethylaminohydrolase-1 (DDAH-1) is the critical enzyme responsible for the degradation of asymmetric dimethylarginine (ADMA) which inhibits nitric oxide (NO) synthase and has been implicated in neurodegeneration. Mitochondrial dysfunction, particularly in the mitochondria-associated endoplasmic reticulum membrane (MAM), plays a critical role in this process, although the specific molecular target has not yet been determined. This study aims to examine the involvement of DDAH-1 in the nigrostriatal dopaminergic pathway and PD pathogenesis. The distribution of DDAH-1 in the brain and its colocalization with dopaminergic neurons were observed. The loss of dopaminergic neurons and aggravated locomotor disability after rotenone (ROT) injection were showed in the DDAH-1 knockout rat. L-arginine (ARG) and NO donors were employed to elucidate the role of NO respectively. In vitro, we investigated the effects of DDAH-1 knockdown or overexpression on cell viability and mitochondrial functions, as well as modulation of ADMA/NO levels using ADMA or ARG. MAM formation was assessed by the Mitofusin2 oligomerization and the mitochondrial ubiquitin ligase (MITOL) phosphorylation. We found that DDAH-1 downregulation resulted in enhanced cell death and mitochondrial dysfunctions, accompanied by elevated ADMA and reduced NO levels. However, the recovered NO level after the ARG supplement failed to exhibit a protective effect on mitochondrial functions and partially restored cell viability. DDAH-1 overexpression prevented ROT toxicity, while ADMA treatment attenuated these protective effects. The declines of MAM formation in ROT-treated cells were exacerbated by DDAH-1 downregulation via reduced MITOL phosphorylation, which was reversed by DDAH-1 overexpression. Together, the abundant expression of DDAH-1 in nigral dopaminergic neurons may exert neuroprotective effects by maintaining MAM formation and mitochondrial function probably via ADMA, indicating the therapeutic potential of targeting DDAH-1 for PD.

Adaptive Effects of Intermittent Hypoxia Training on Oxygen-Dependent Processes as a Potential Therapeutic Strategy Tool.

BACKGROUND/AIMS: Important benefits of intermittent hypoxic training (IHT) have emerged as an effective tool for enhancing adaptive potential in different pathological states, among which acute hypoxia dominates. Therefore, the aim of our study was to evaluate the mechanisms related to the effects of the nitric oxide system (nitrites, nitrates, carbamide, and total polyamine content) on ADP-stimulated oxygen consumption and oxidative phosphorylation in heart and liver mitochondria and biomarkers of oxidative stress in the blood, heart, and liver of rats exposed to the IHT method and acute hypoxia and treated with the amino acid L-arginine (600 mg/kg, 30 min) or the NO synthase inhibitor L-NNA (35 mg/kg, 30 min) prior to each IHT session. METHODS: We analysed the modulation of the system of oxygen-dependent processes (mitochondrial respiration with the oxygraphic method, microsomal oxidation, and lipoperoxidation processes using biochemical methods) in tissues during IHT in the formation of short-term and long-term effects (30, 60, and 180 days after the last IHT session) with simultaneous administration of L-arginine. In particular, we investigated how mitochondrial functions are modulated during intermittent hypoxia with the use of oxidation substrates (succinate or α-ketoglutarate) in bioenergetic mechanisms of cellular stability and adaptation. RESULTS: The IHT method is associated with a significant increase in the production of endogenous nitric oxide measured by the levels of its stable metabolite, nitrite anion, in both plasma (almost 7-fold) and erythrocytes (more than 7-fold) of rats. The intensification of nitric oxide-dependent pathways of metabolic transformations in the energy supply processes in the heart and liver, accompanied by oscillatory mechanisms of adaptation in the interval mode, causes a probable decrease in the production of urea and polyamines in plasma and liver, but not in erythrocytes. The administration of L-arginine prior to the IHT sessions increased the level of the nitrite-reducing component of the nitric oxide cycle, which persisted for up to 180 days of the experiment. CONCLUSION: Thus, the efficacy of IHT and its nitrite-dependent component shown in this study is associated with the formation of long-term adaptive responses by preventing the intensification of lipoperoxidation processes in tissues due to pronounced changes in the main enzymes of antioxidant defence and stabilisation of erythrocyte membranes, which has a pronounced protective effect on the system of regulation of oxygen-dependent processes as a whole.

Evaluation of endothelial glycocalyx injury biomarkers in feline hemotropic mycoplasmosis.

The present study aimed to investigate endothelial glycocalyx (eGCx) damage in cats with feline hemotropic mycoplasmosis caused by Mycoplasma haemofelis using selected biomarkers and to determine the diagnostic and prognostic significance of these biomarkers. The study included 25 cats with feline hemotropic mycoplasmosis and 10 healthy cats. Clinical examination, blood gas analysis, complete blood count, and biochemical analysis were performed. Hemotropic mycoplasmosis diagnosed by microscopic examination and molecularly confirmed by PCR targeting the Mycoplasma haemofelis 16s rRNA gene. To evaluate endothelial glycocalyx damage, syndecan-1, endothelin-1 (ET-1), asymmetric dimethylarginine (ADMA), and vascular endothelial growth factor-A (VEGF-A) concentrations were measured using cat-specific commercial ELISA kits. Of the cats with feline hemotropic mycoplasmosis, 14 (56%) survived and 11 (44%) died. While syndecan-1 and ET-1 concentrations were significantly higher in cats with hemotropic mycoplasmosis compared to the control group (p < 0.001), no statistically significant difference was found for ADMA and VEGF-A concentrations (p > 0.05). Endothelial glycocalyx biomarkers showed significant correlations with each other and with hematological parameters (p < 0.01). The results of the ROC analysis showed that ET-1 with area under the curve (AUC) of 0.821 (p < 0.01) and VEGF-A with AUC of 0.805 (p < 0.010) were found to be significant prognostic indicators. In conclusion, this study demonstrated that serum syndecan-1 and ET-1 can be used as diagnostic and serum ET-1 and VEGF-A as prognostic biomarkers in cats with hemotropic mycoplasmosis. Our results indicate the development of eGCx damage in feline hemotropic mycoplasmosis and suggest that glycocalyx disruption may contribute to the pathogenesis of the disease.

Targeted Quantification of Glutathione/Arginine Redox Metabolism Based on a Novel Paired Mass Spectrometry Probe Approach for the Functional Assessment of Redox Status.

Glutathione (GSH) redox control and arginine metabolism are critical in regulating the physiological response to injury and oxidative stress. Quantification assessment of the GSH/arginine redox metabolism supports monitoring metabolic pathway shifts during pathological processes and their linkages to redox regulation. However, assessing the redox status of organisms with complex matrices is challenging, and single redox molecule analysis may not be accurate for interrogating the redox status in cells and in vivo. Herein, guided by a paired derivatization strategy, we present a new ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS)-based approach for the functional assessment of biological redox status. Two structurally analogous probes, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and newly synthesized 2-methyl-6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (MeAQC), were set for paired derivatization. The developed approach was successfully applied to LPS-stimulated RAW 264.7 cells and HDM-induced asthma mice to obtain quantitative information on GSH/arginine redox metabolism. The results suggest that the redox status was remarkably altered upon LPS and HDM stimulation. We expect that this approach will be of good use in a clinical biomarker assay and potential drug screening associated with redox metabolism, oxidative damage, and redox signaling.