An energy-conserving reaction in amino acid metabolism catalyzed by arginine synthetase.

All forms of life are presumed to synthesize arginine from citrulline via a two-step pathway consisting of argininosuccinate synthetase and argininosuccinate lyase using citrulline, adenosine 5'-triphosphate (ATP), and aspartate as substrates. Conversion of arginine to citrulline predominantly proceeds via hydrolysis. Here, from the hyperthermophilic archaeon Thermococcus kodakarensis, we identified an enzyme which we designate "arginine synthetase". In arginine synthesis, the enzyme converts citrulline, ATP, and free ammonia to arginine, adenosine 5'-diphosphate (ADP), and phosphate. In the reverse direction, arginine synthetase conserves the energy of arginine deimination and generates ATP from ADP and phosphate while releasing ammonia. The equilibrium constant of this reaction at pH 7.0 is [Cit][ATP][NH3]/[Arg][ADP][Pi] = 10.1 ± 0.7 at 80 °C, corresponding to a ΔG°' of -6.8 ± 0.2 kJ mol-1. Growth of the gene disruption strain was compared to the host strain in medium composed of amino acids. The results suggested that arginine synthetase is necessary in providing ornithine, the precursor for proline biosynthesis, as well as in generating ATP. Growth in medium supplemented with citrulline indicated that arginine synthetase can function in the direction of arginine synthesis. The enzyme is widespread in nature, including bacteria and eukaryotes, and catalyzes a long-overlooked energy-conserving reaction in microbial amino acid metabolism. Along with ornithine transcarbamoylase and carbamate kinase, the pathway identified here is designated the arginine synthetase pathway.

The Role of Citrullination Modification in CD4+ T Cells in the Pathogenesis of Immune-Related Diseases.

The post-translational modifications (PTMs) of proteins play a crucial role in increasing the functional diversity of proteins and are associated with the pathogenesis of various diseases. This review focuses on a less explored PTM called citrullination, which involves the conversion of arginine to citrulline. This process is catalyzed by peptidyl arginine deiminases (PADs). Different members of the PAD family have distinct tissue distribution patterns and functions. Citrullination is a post-translational modification of native proteins that can alter their structure and convert them into autoantigens; thus, it mediates the occurrence of autoimmune diseases. CD4+ T cells, including Th1, Th2, and Th17 cells, are important immune cells involved in mediating autoimmune diseases, allergic reactions, and tumor immunity. PADs can induce citrullination in CD4+ T cells, suggesting a role for citrullination in CD4+ T cell subset differentiation and function. Understanding the role of citrullination in CD4+ T cells may provide insights into immune-related diseases and inflammatory processes.

Effect of Natural Osmolytes on Recombinant Tau Monomer: Propensity of Oligomerization and Aggregation.

The pathological misfolding and aggregation of the microtubule associated protein tau (MAPT), a full length Tau2N4R with 441aa, is considered the principal disease relevant constituent in tauopathies including Alzheimer's disease (AD) with an imbalanced ratio in 3R/4R isoforms. The exact cellular fluid composition, properties, and changes that coincide with tau misfolding, seed formation, and propagation events remain obscure. The proteostasis network, along with the associated osmolytes, is responsible for maintaining the presence of tau in its native structure or dealing with misfolding. In this study, for the first time, the roles of natural brain osmolytes are being investigated for their potential effects on regulating the conformational stability of the tau monomer (tauM) and its propensity to aggregate or disaggregate. Herein, the effects of physiological osmolytes myo-inositol, taurine, trimethyl amine oxide (TMAO), betaine, sorbitol, glycerophosphocholine (GPC), and citrulline on tau's aggregation state were investigated. The overall results indicate the ability of sorbitol and GPC to maintain the monomeric form and prevent aggregation of tau, whereas myo-inositol, taurine, TMAO, betaine, and citrulline promote tau aggregation to different degrees, as revealed by protein morphology in atomic force microscopy images. Biochemical and biophysical methods also revealed that tau proteins adopt different conformations under the influence of these osmolytes. TauM in the presence of all osmolytes expressed no toxicity when tested by a lactate dehydrogenase assay. Investigating the conformational stability of tau in the presence of osmolytes may provide a better understanding of the complex nature of tau aggregation in AD and the protective and/or chaotropic nature of osmolytes.

L-Citrulline Ameliorates Iron Metabolism and Mitochondrial Quality Control via Activating AMPK Pathway in Intestine and Improves Microbiota in Mice with Iron Overload.

SCOPE: Oxidative stress caused by iron overload tends to result in intestinal mucosal barrier dysfunction and intestinal microbiota imbalance. As a neutral and nonprotein amino acid, L-Citrulline (L-cit) has been implicated in antioxidant and mitochondrial amelioration properties. This study investigates whether L-cit can alleviate iron overload-induced intestinal injury and explores the underlying mechanisms. METHODS AND RESULTS: C57BL/6J mice are intraperitoneally injected with iron dextran, then gavaged with different dose of L-cit for 2 weeks. L-cit treatment significantly alleviates intestine pathological injury, oxidative stress, ATP level, and mitochondrial respiratory chain complex activities, accompanied by ameliorating mitochondrial quality control. L-cit-mediated protection is associated with the upregulation of Glutathione Peroxidase 4 (GPX4) expression, inhibition Nuclear Receptor Coactivator 4 (NCOA4)-mediated ferritinophagy and ferroptosis, and improvement of gut microbiota. To investigate the underlying molecular mechanisms, Intestinal Porcine Epithelial Cell line-J2 (IPEC-J2) cells are treated with L-cit or AMP-activated Protein Kinase (AMPK) inhibitor. AMPK signaling has been activated by L-cit. Notably, Compound C abolishes L-cit's protection on intestinal barrier, mitochondrial function, and antioxidative capacity in IPEC-J2 cells. CONCLUSION: L-cit may restrain ferritinophagy and ferroptosis to regulate iron metabolism, and induce AMPK pathway activation, which contributes to exert antioxidation, ameliorate iron metabolism and mitochondrial quality control, and improve intestinal microbiota. L-cit is a promising therapeutic strategy for iron overload-induced intestinal injury.

GnRH Induces Citrullination of the Cytoskeleton in Murine Gonadotrope Cells.

Peptidylarginine deiminases (PADs or PADIs) catalyze the conversion of positively charged arginine to neutral citrulline, which alters target protein structure and function. Our previous work established that gonadotropin-releasing hormone agonist (GnRHa) stimulates PAD2-catalyzed histone citrullination to epigenetically regulate gonadotropin gene expression in the gonadotrope-derived LßT2 cell line. However, PADs are also found in the cytoplasm. Given this, we used mass spectrometry (MS) to identify additional non-histone proteins that are citrullinated following GnRHa stimulation and characterized the temporal dynamics of this modification. Our results show that actin and tubulin are citrullinated, which led us to hypothesize that GnRHa might induce their citrullination to modulate cytoskeletal dynamics and architecture. The data show that 10 nM GnRHa induces the citrullination of ß-actin, with elevated levels occurring at 10 min. The level of ß-actin citrullination is reduced in the presence of the pan-PAD inhibitor biphenyl-benzimidazole-Cl-amidine (BB-ClA), which also prevents GnRHa-induced actin reorganization in dispersed murine gonadotrope cells. GnRHa induces the citrullination of ß-tubulin, with elevated levels occurring at 30 min, and this response is attenuated in the presence of PAD inhibition. To examine the functional consequence of ß-tubulin citrullination, we utilized fluorescently tagged end binding protein 1 (EB1-GFP) to track the growing plus end of microtubules (MT) in real time in transfected LßT2 cells. Time-lapse confocal microscopy of EB1-GFP reveals that the MT average lifetime increases following 30 min of GnRHa treatment, but this increase is attenuated by PAD inhibition. Taken together, our data suggest that GnRHa-induced citrullination alters actin reorganization and MT lifetime in gonadotrope cells.

Oral post-treatment supplementation with a combination of glutamine, citrulline, and antioxidant vitamins additively mitigates jejunal damage, oxidative stress, and inflammation in rats with intestinal ischemia and reperfusion.

INTRODUCTION: Intestinal ischemia and reperfusion (IIR) injury is closely associated with oxidative stress. Evidence shows that oral supplementation with glutamine and citrulline alleviates IIR-induced jejunal damage. We investigated the effects of a combination of glutamine, citrulline, and antioxidant vitamins on IIR-induced jejunal damage, oxidative stress, and inflammation. METHOD: Male Wistar rats that underwent 60 min of superior mesenteric artery occlusion were orally administered glutamine plus citrulline (GC), vitamin C plus E (CE), or a combination of GC and CE 15 min before and 3, 9, and 21 h after reperfusion. Healthy rats without IIR were used as controls. RESULTS: After reperfusion for 24 h, rats with IIR showed lower levels of red blood cells, hemoglobin, serum glucose, and jejunal DNA and increased white blood cell counts compared to controls (1-way ANOVA with the least significant difference, P < 0.05). The IIR-induced decrease in serum albumin and increase in plasma interleukin-6 and jejunal thiobarbituric acid-reactive substances (TBARS) were significantly reversed by GC and/or CE. The results of the 2-way ANOVA indicated that GC was the main factor that increased jejunal villus height and muscularis DNA, and CE was the main factor that increased jejunal muscularis protein and decreased jejunal proinflammatory cytokine levels and myeloperoxidase activity. In addition, GC and CE are the main factors that decrease plasma proinflammatory cytokine levels and the jejunal apoptotic index. CONCLUSION: Oral post-treatment supplementation with glutamine and citrulline, combined with vitamins C and E, may alleviate IIR-induced oxidative stress, inflammation, and jejunal damage.

The roles of gut microbiome and metabolites associated with skin photoaging in mice by intestinal flora sequencing and metabolomics.

Photoaging of skin, a chronic disease, can produce the appearance changes and cancer lesions of skin. Therefore, it is of great significance to investigate the mechanisms and explore effective methods to treat the disorder. Gut microbiota and intestinal metabolisms have critical roles in a variety of diseases. However, their roles on photoaging of skin were not well tested. In the present work, the results showed that compared with control group, the levels of MDA, SOD and CAT associated with oxidative stress, the levels of COL I, CER, and HA associated with skin function, and the mRNA levels of IL-1ß, IL-6, TNF-α associated with inflammation after long-term exposure to ultraviolet radiation in mice were significantly changed. Skin pathological tissue was also seriously damaged. The protein levels of AQP3 and FLG were significantly decreased. Ultraviolet exposure also promoted skin photoaging by activating TNFR1/TRAF2-mediated MAPK pathway, in which the protein levels of P38/P-P38, c-FOS/P-c-FOS, MMP1, TNFR1 and TRAF2 were significantly increased in model mice compared with control group. In fecal microbiota transplantation (FMT) experiment, we found that the intestinal microbiome of control mice alleviated skin photoaging via adjusting the protein levels of P38/P-P38, c-FOS/P-c-FOS, MMP1, TNFR1 and TRAF2. 16S rRNA sequencing found that 1639 intestinal bacteria were found, in which 15 bacteria including norank_f_Ruminococcaceae, Lachnospirac -eae_NK4A136_group, Lachnoclostridium, etc., were significantly different at the genus level. Untargeted GC-TOF/MS and UHPLC-MS/MS metabolomics showed 72 and 188 metabolites including taurine, ornithine, L-arginine, L-histidine, sucrose with significant differences compared with control group. Then, amino acid targeting assay showed 10 amino acids including L-ornithine, L-arginine and L-citrulline with higher levels in control group compared with model group. In addition, we also found that the variation of Lachnoclostridium abundance may regulate L-arginine metabolism to affect skin photoaging. Some intestinal bacteria and metabolites including amino acids may be closely related to skin photoaging, which should provide new methods to treat skin photoaging in the future.

Spontaneous Liquid Droplet-to-Gel Transition of Citrulline Polypeptide Complexed with Nucleic Acids.

Inside cells, proteins complex with nucleic acids to form liquid droplets resulting from liquid-liquid phase separation. The presence of mutated proteins can change the state of these liquid droplets to solids or gels, triggering neurodegenerative diseases. The mechanism of the liquid to solid or gel transition is still unclear. Solutions of poly(l-ornithine-co-l-citrulline) (PLOC) copolymers, which exhibit upper critical solution temperature-type behavior, change state upon cooling. In this study, we evaluated the effect of nucleic acids complexed with PLOC on phase changes. In the presence of nucleic acids, such as polyC and polyU, PLOC formed liquid droplets at low temperatures. The droplets dissolved at temperatures above the phase separation temperature. The phase separation temperature depended on the chemical structure of the nucleobase, implying that electrostatic and hydrogen bonding interactions between the nucleic acid and PLOC influenced phase separation. Furthermore, the liquid droplets spontaneously changed to gel-like precipitates due to spontaneous release of nucleic acids from the complex. The rate of the liquid droplet-to-gel transition depended on the magnitude of electrostatic and hydrogen bonding interactions between PLOC and nucleic acid. PLOC complexed with mRNA also underwent a liquid droplet-to-gel transition upon the release of mRNA. This work provides insights into the mechanism of pathogenic transitions of the cellular droplets.

Development of a Two-Dimensional Liquid Chromatographic Method for Analysis of Urea Cycle Amino Acids.

The urea cycle has been found to be closely associated with certain types of cancers and other diseases such as cardiovascular disease and chronic kidney disease. An analytical method for the precise quantification of urea cycle amino acids (arginine, ornithine, citrulline, and argininosuccinate) by off-line two-dimensional liquid chromatography (2D-LC) combined with fluorescence-based detection was developed. Before analysis, the amino acids were derivatised with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to obtain NBD-amino acids. The first dimension involved the reversed-phase separation, in which NBD derivatives of urea cycle amino acids were completely separated from each other and mostly separated from the 18 NBD-proteinogenic amino acids. The samples were eluted with stepwise gradient using 0.02% trifluoroacetic acid in water-acetonitrile as the mobile phase. In the second dimension, an amino column was used for the separation of NBD-ornithine, -citrulline, and -argininosuccinate, while a sulfonic acid column was used to separate NBD-arginine. The developed 2D-LC system was used to analyse human plasma samples. The fractions of NBD-urea cycle amino acids obtained in the first dimension were collected manually and introduced into the second dimension. By choosing appropriate mobile phases for the second dimension, each NBD-urea cycle amino acid eluted in the first dimension was well separated from the other proteinogenic amino acids and interference from endogenous substance. This could not be achieved in the first dimension. The urea cycle amino acids in human plasma sample were quantified, and the method was well validated. The calibration curves for each NBD-urea cycle amino acid showed good linearity from 3 (ASA) or 15 (Orn, Cit, and Arg) to 600 nM, with correlation coefficients higher than 0.9969. The intraday and interday precisions were less than 7.9% and 15%, respectively. The 2D-LC system is expected to be useful for understanding the involvement of the urea cycle in disease progression.

Effect of supplementing a Bacillus subtilis-based probiotic on performance, intestinal integrity, and serum antioxidant capacity and metabolites concentrations of heat-stressed growing pigs.

Exposing pigs to heat stress (HS) seems to modify the intestinal microbiota which may compromise the integrity of the small intestine epithelia. Probiotics, live microorganisms, can help pigs to maintain a healthy intestinal environment. Eighty pigs (21.6 ±â€…3.4 kg body weight) exposed to HS or thermal neutral (TN) conditions were used to evaluate the effect of a Bacillus subtilis-based probiotic on performance, body temperature, and intestinal integrity. Treatments were: TN pigs fed a control diet without (TN-C) or with 1 × 106 CFU probiotic/g of feed (TN-P), and HS pigs fed a control without (HS-C) or with probiotic (HS-P). The control diet was formulated with wheat, soybean meal, and free amino acids (AA). Feed and water were freely available during the 21-d study. At completion, samples from duodenum, jejunum, and ileum were collected to analyze epithelial histology and tight junction protein expression; antioxidant activity, and free AA and metabolites in serum. Relative abundance of Lactobacillus, Bifidobacterium, Escherichia coli, and Bacillus in ileal content was analyzed. Ambient temperature in the TN room ranged from 19 to 25 °C, and in HS room from 30 to 38.5 °C. Intestinal temperature in HS-P pigs was lower than in HS-C pigs. Weight gain and feed intake reduced, but feed:gain and respiration rate increased in HS compared to TN pigs, regardless of diet (P < 0.01). Probiotic increased weight gain and improved feed:gain (P < 0.05) in both TN and HS pigs, but feed intake did not differ. Heat stress decreased villi height in jejunum and villi height:crypt depth in duodenum and jejunum (P < 0.05). Probiotic increased villi height in duodenum and ileum, and villi height:crypt depth in all small intestine segments (P < 0.05). Relative abundance of Lactobacillus and Bifidobacterium tended to reduce, and E. coli tended to increase (P < 0.10) in ileal content of HS-C pigs. Ileal relative abundance of Bacillus was higher (P < 0.01) in HS-P pigs than in HS-C and TN-C pigs. Cystathionine, homocysteine, hydroxylysine, α-amino-adipic acid, citrulline, α-amino-n-butyric acid, P-Ser, and taurine were higher in HS than in TN pigs (P < 0.05). These data confirm the negative effect of HS on performance, body temperature, and intestinal integrity of pigs. These data suggest that supplementing 1 × 106 CFU probiotic/g of feed based on Bacillus subtilis DSM 32540 may help to counteract the negative effects of HS on the performance and intestinal integrity of pigs.

High ambient temperature provokes heat stress in animals, and pigs are highly affected showing low performance, and intestinal integrity and microbiota compromised. Probiotics could help pigs to maintain a healthy intestinal environment. We analyzed the effect of a probiotic based on Bacillus subtilis on growth performance, intestinal integrity of small intestine, intestinal microbiota, antioxidant activity, and serum concentration of amino acids in pigs under heat stress (HS) and thermal neutral (TN) conditions. Ambient temperature under TN and HS conditions was 19­25 and 30­38.5 °C, respectively. Weight gain and feed intake reduced in HS pigs compared to TN pigs, but probiotic increased weight gain in both TN and HS pigs. Heat stress decreased villi size in duodenum and jejunum, and probiotic restored them. Relative abundance of Lactobacillus and Bifidobacterium reduced and Escherichia coli increased in the small intestine content of HS pigs; probiotic increased the abundance of Bacillus. The activity of antioxidant enzymes increased in HS pigs fed the probiotic diet. Serum concentration of amino acid metabolites was affected by HS. These data suggest that supplementing a Bacillus subtilis-based probiotic may help to counteract the negative effects of HS on the performance and intestinal integrity of pigs.

Heteroplasmic pathogenic m.12315G>A variant in MT-TL2 presenting with MELAS syndrome and depletion of nitric oxide donors.

The MT-TL2 m.12315G>A pathogenic variant has previously been reported in five individuals with mild clinical phenotypes. Herein we report the case of a 5-year-old child with heteroplasmy for this variant who developed neurological regression and stroke-like episodes similar to those observed in mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Biochemical evaluation revealed depletion of arginine on plasma amino acid analysis and low z-scores for citrulline on untargeted plasma metabolomics analysis. These findings suggested that decreased availability of nitric oxide may have contributed to the stroke-like episodes. The use of intravenous arginine during stroke-like episodes and daily enteral L-citrulline supplementation normalized her biochemical values of arginine and citrulline. Untargeted plasma metabolomics showed the absence of nicotinamide and 1-methylnicotinamide, and plasma total glutathione levels were low; thus, nicotinamide riboside and N-acetylcysteine therapies were initiated. This report expands the phenotype associated with the rare mitochondrial variant MT-TL2 m.12315G>A to include neurological regression and a MELAS-like phenotype. Individuals with this variant should undergo in-depth biochemical analysis to include untargeted plasma metabolomics, plasma amino acids, and glutathione levels to help guide a targeted approach to treatment.

Biological importance of arginine: A comprehensive review of the roles in structure, disorder, and functionality of peptides and proteins.

Arginine shows Jekyll and Hyde behavior in several respects. It participates in protein folding via ionic and H-bonds and cation-pi interactions; the charge and hydrophobicity of its side chain make it a disorder-promoting amino acid. Its methylation in histones; RNA binding proteins; chaperones regulates several cellular processes. The arginine-centric modifications are important in oncogenesis and as biomarkers in several cardiovascular diseases. The cross-links involving arginine in collagen and cornea are involved in pathogenesis of tissues but have also been useful in tissue engineering and wound-dressing materials. Arginine is a part of active site of several enzymes such as GTPases, peroxidases, and sulfotransferases. Its metabolic importance is obvious as it is involved in production of urea, NO, ornithine and citrulline. It can form unusual functional structures such as molecular tweezers in vitro and sprockets which engage DNA chains as part of histones in vivo. It has been used in design of cell-penetrating peptides as drugs. Arginine has been used as an excipient in both solid and injectable drug formulations; its role in suppressing opalescence due to liquid-liquid phase separation is particularly very promising. It has been known as a suppressor of protein aggregation during protein refolding. It has proved its usefulness in protein bioseparation processes like ion-exchange, hydrophobic and affinity chromatographies. Arginine is an amino acid, whose importance in biological sciences and biotechnology continues to grow in diverse ways.

Multi-dose enteral L-citrulline administration in premature infants at risk of developing pulmonary hypertension associated with bronchopulmonary dysplasia.

OBJECTIVE: Information is needed to guide the design of randomized controlled trials (RCTs) evaluating L-citrulline therapy for premature infants with pulmonary hypertension associated with bronchopulmonary dysplasia (BPD-PH). Based on our single-dose pharmacokinetic study, we evaluated the ability of a multi-dose enteral L-citrulline strategy to achieve a target trough steady-state L-citrulline plasma concentration and its tolerability in premature infants. STUDY DESIGN: Plasma L-citrulline concentrations were measured in six premature infants receiving 60 mg/kg L-citrulline every 6 h for 72 h before the first and last L-citrulline doses. L-citrulline concentrations were compared to concentration-time profiles from our previous study. RESULTS: Target trough plasma L-citrulline concentrations were achieved in 2/6 subjects. No serious adverse events occurred. CONCLUSIONS: Multi-dose L-citrulline was well tolerated. These results will assist in the design of phase II RCTs evaluating L-citrulline dosage strategies to achieve target plasma L-citrulline concentrations in infants at risk for BPD-PH. CLINICAL TRIALS: gov ID: NCT03542812.

PSMA-targeted small-molecule drug-conjugates with valine-citrulline and phosphoramidate cleavable linkers.

In this study, we present a modular synthesis and evaluation of two prostate-specific membrane antigen (PSMA) targeted small molecule drug conjugates (SMDCs) incorporating the potent chemotherapeutic agent monomethyl auristatin E (MMAE). These SMDCs are distinguished by their cleavable linker modules: one utilizing the widely known valine-citrulline linker, susceptible to cleavage by cathepsin B, and the other featuring a novel acid-labile phosphoramidate-based (PhosAm) linker. Both SMDCs maintained nanomolar affinity to PSMA. Furthermore, we confirmed the selective release of the payload and observed chemotherapeutic efficacy specifically within PSMA-positive prostate cancer cells, while maintaining cell viability in PSMA-negative cells. These findings not only validate the efficacy of our approach but also highlight the potential of the innovative pH-responsive PhosAm linker. This study contributes significantly to the field and also paves the way for future advancements in targeted cancer therapy.

Development of a Multi-Organ Radiation Injury Model with Precise Dosimetry with Focus on GI-ARS.

The goal of this study was to establish a model of partial-body irradiation (PBI) sparing 2.5% of the bone marrow (BM2.5-PBI) that accurately recapitulates radiological/nuclear exposure scenarios. Here we have reported a model which produces gastrointestinal (GI) damage utilizing a clinical linear accelerator (LINAC) with precise dosimetry, which can be used to develop medical countermeasures (MCM) for GI acute radiation syndrome (ARS) under the FDA animal rule. The PBI model (1 hind leg spared) was developed in male and female C57BL/6 mice that received radiation doses ranging from 12-17 Gy with no supportive care. GI pathophysiology was assessed by crypt cell loss and correlated with peak lethality between days 4 and 10 after PBI. The radiation dose resulting in 50% mortality in 30 days (LD50/30) was determined by probit analysis. Differential blood cell counts in peripheral blood, colony forming units (CFU) in bone marrow, and sternal megakaryocytes were analyzed between days 1-30, to assess the extent of hematopoietic ARS (H-ARS) injury. Radiation-induced GI damage was also assessed by measuring: 1. bacterial load (16S rRNA) by RT-PCR on days 4 and 7 after PBI in liver, spleen and jejunum, 2. liposaccharide binding protein (LBP) levels in liver, and 3. fluorescein isothiocyanate (FITC)-dextran, E-selectin, sP-selectin, VEGF, FGF-2, MMP-9, citrulline, and serum amyloid A (SAA) levels in serum. The LD50/30 of male mice was 14.3 Gy (95% confidence interval 14.1-14.7 Gy) and of female mice was 14.5 Gy (95% confidence interval 14.3-14.7 Gy). Secondary endpoints included loss of viable crypts, higher bacterial loads in spleen and liver, higher LBP in liver, increased FITC-dextran and SAA levels, and decreased levels of citrulline and endothelial biomarkers in serum. The BM2.5-PBI model, developed for the first time with precise dosimetry, showed acute radiation-induced GI damage that is correlated with lethality, as well as a response to various markers of inflammation and vascular damage. Sex-specific differences were observed with respect to radiation dose response. Currently, no MCM is available as a mitigator for GI-ARS. This BM2.5-PBI mouse model can be regarded as the first high-throughput PBI model with precise dosimetry for developing MCMs for GI-ARS under the FDA animal rule.

Markers of intestinal mucositis to predict blood stream infections at the onset of fever during treatment for childhood acute leukemia.

Despite chemotherapy-induced intestinal mucositis being a main risk factor for blood stream infections (BSIs), no studies have investigated mucositis severity to predict BSI at fever onset during acute leukemia treatment. This study prospectively evaluated intestinal mucositis severity in 85 children with acute leukemia, representing 242 febrile episodes (122 with concurrent neutropenia) by measuring plasma levels of citrulline (reflecting enterocyte loss), regenerating islet-derived-protein 3α (REG3α, an intestinal antimicrobial peptide) and CCL20 (a mucosal immune regulatory chemokine) along with the general neutrophil chemo-attractants CXCL1 and CXCL8 at fever onset. BSI was documented in 14% of all febrile episodes and in 20% of the neutropenic febrile episodes. In age-, sex-, diagnosis- and neutrophil count-adjusted analyses, decreasing citrulline levels and increasing REG3α and CCL20 levels were independently associated with increased odds of BSI (OR = 1.6, 1.5 and 1.7 per halving/doubling, all p < 0.05). Additionally, higher CXCL1 and CXCL8 levels increased the odds of BSI (OR = 1.8 and 1.7 per doubling, all p < 0.0001). All three chemokines showed improved diagnostic accuracy compared to C-reactive protein and procalcitonin. These findings underline the importance of disrupted intestinal integrity as a main risk factor for BSI and suggest that objective markers for monitoring mucositis severity may help predicting BSI at fever onset.

Dose-response effect of L-citrulline on skeletal muscle damage after acute eccentric exercise: an in vivo study in mice.

Background: Eccentric exercise may trigger mechanical stress, resulting in muscle damage that may decrease athletic performance. L-citrulline potentially prevents skeletal muscle damage after acute eccentric exercise. This study aimed to assess the dose-response effect of L-citrulline as a preventive therapy for skeletal muscle damage in mice after acute eccentric exercise. Methods: This is a controlled laboratory in vivo study with a post-test-only design. Male mice (BALB/c, n = 25) were randomized into the following groups: a normal control (C1) (n = 5); a negative control (C2) with downhill running and placebo intervention (n = 5); treatment groups: T1 (n = 5), T2 (n = 5), and T3 (n = 5), were subjected to downhill running and 250, 500, and 1,000 mg/kg of L-citrulline, respectively, for seven days. Blood plasma was used to determine the levels of TNNI2 and gastrocnemius muscle tissue NOX2, IL-6, and caspase 3 using ELISA. NF-κB and HSP-70 expressions were determined by immunohistochemistry. Results: Skeletal muscle damage (plasma TNNI2 levels) in mice after eccentric exercise was lower after 250 and 500 mg/kg of L-citrulline. Further, changes in oxidative stress markers, NOX2, were reduced after a 1,000 mg/kg dose. However, a lower level of change has been observed in levels of cellular response markers (NF-κB, HSP-70, IL-6, and caspase 3) after administration of L-citrulline doses of 250, 500, and 1,000 mg/kg. Conclusion: L-citrulline may prevent skeletal muscle damage in mice after acute eccentric exercise through antioxidant effects as well as inflammatory and apoptotic pathways. In relation to dose-related effects, it was found that L-citrulline doses of 250, 500, and 1,000 mg/kg significantly influenced the expression of NF-κB and HSP-70, as well as the levels of IL-6 and caspase 3. Meanwhile, only doses of 250 and 500 mg/kg had an impact on TNNI2 levels, and the 1,000 mg/kg dose affected NOX2 levels.

From Fruit Waste to Medical Insight: The Comprehensive Role of Watermelon Rind Extract on Renal Adenocarcinoma Cellular and Transcriptomic Dynamics.

Cancer researchers are fascinated by the chemistry of diverse natural products that show exciting potential as anticancer agents. In this study, we aimed to investigate the anticancer properties of watermelon rind extract (WRE) by examining its effects on cell proliferation, apoptosis, senescence, and global gene expression in human renal cell adenocarcinoma cells (HRAC-769-P) in vitro. Our metabolome data analysis of WRE exhibited untargeted phyto-constituents and targeted citrulline (22.29 µg/mg). HRAC-769-P cells were cultured in RPMI-1640 media and treated with 22.4, 44.8, 67.2, 88.6, 112, 134.4, and 156.8 mg·mL-1 for 24, 48, and 72 h. At 24 h after treatment, (88.6 mg·mL-1 of WRE) cell proliferation significantly reduced, more than 34% compared with the control. Cell viability decreased 48 and 72 h after treatment to 45% and 37%, respectively. We also examined poly caspase, SA-beta-galactosidase (SA-beta-gal), and wound healing activities using WRE. All treatments induced an early poly caspase response and a significant reduction in cell migration. Further, we analyzed the transcript profile of the cells grown at 44.8 mg·mL-1 of WRE after 6 h using RNA sequencing (RNAseq) analysis. We identified 186 differentially expressed genes (DEGs), including 149 upregulated genes and 37 downregulated genes, in cells treated with WRE compared with the control. The differentially expressed genes were associated with NF-Kappa B signaling and TNF pathways. Crucial apoptosis-related genes such as BMF, NPTX1, NFKBIA, NFKBIE, and NFKBID might induce intrinsic and extrinsic apoptosis. Another possible mechanism is a high quantity of citrulline may lead to induction of apoptosis by the production of increased nitric oxide. Hence, our study suggests the potential anticancer properties of WRE and provides insights into its effects on cellular processes and gene expression in HRAC-769-P cells.

Amino acid profile in overweight and obese prepubertal children - can simple biochemical tests help in the early prevention of associated comorbidities?

Background: It is accepted that plasma branched-chain amino acids (BCAAs) and aromatic amino acids (AAAs) are closely related to metabolic risk. Arterial hypertension, metabolic syndrome, endothelial dysfunction, inflammation, and metabolic dysfunction-associated fatty liver disease (MAFLD) are frequently seen in obese patients. Many attempts have been made to find biochemical indicators for the early detection of metabolic complications in children. It is not known if different amino acid profiles and BCAA and AA concentrations in overweight and obese children correlate with chemerin, proinflammatory, and simple biochemical markers. Thus, the study aimed to find out the early markers of cardiovascular disease and MAFLD in overweight and obese children. Materials and methods: The study included 20 overweight and obese children (M/F 12/8; mean age 7.7 ± 2.3 years; BMI 26.8 ± 5.0 kg/m2) and 12 non-obese children (control group) (M/F 4/8; mean age 6.5 ± 2.2 years; BMI 14.8 ± 1.5 kg/m2). The following plasma amino acids were measured: aspartic acid, glutamic acid, serine, asparagine, glycine, glutamine, taurine, histidine, citrulline, threonine, alanine, arginine, proline, tyrosine, methionine, valine, isoleucine, leucine, phenylalanine, tryptophan, ornithine, and lysine. Chemerin, high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), and basic biochemistry parameters were measured. Results: The mean plasma levels of leucine, isoleucine, valine, phenylalanine, tyrosine, glutamic acid, and alanine were significantly higher in overweight and obese children than in the control group (p<0.03-p<0.0004). Conversely, the mean values of serine, asparagine, glutamine, and citrulline were significantly lower in overweight and obese children than in the control group (p<0.03-p<0.0007). Isoleucine, leucine, valine (BCAAs) tyrosine, and phenylalanine (AAAs) levels showed a positive correlation with uric acid, ALT, hs-CRP, and chemerin (r=0.80-0.36; p<0.05-p<0.00001), but not with IL-6. The mean values of glucose, IL-6, hs-CRP, chemerin, uric acid, and ALT were significantly higher in overweight and obese children than in the control group (p<0.03-p<0.00002). In contrast, the lipid profile did not differ between groups. Conclusion: An abnormal amino acid profile in overweight and obese pre-pubertal children, accompanied by elevated ALT and UA observed in the studied cohort, may suggest early metabolic disturbances that can potentially lead to metabolic syndrome, or MAFLD, and increased cardiovascular risk.