Characterizing arginine, ornithine, and putrescine pathways in enteric pathobionts.

Arginine-ornithine metabolism plays a crucial role in bacterial homeostasis, as evidenced by numerous studies. However, the utilization of arginine and the downstream products of its metabolism remain undefined in various gut bacteria. To bridge this knowledge gap, we employed genomic screening to pinpoint relevant metabolic targets. We also devised a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics method to measure the levels of arginine, its upstream precursors, and downstream products in cell-free conditioned media from enteric pathobionts, including Escherichia coli, Klebsiella aerogenes, K. pneumoniae, Pseudomonas fluorescens, Acinetobacter baumannii, Streptococcus agalactiae, Staphylococcus epidermidis, S. aureus, and Enterococcus faecalis. Our findings revealed that all selected bacterial strains consumed glutamine, glutamate, and arginine, and produced citrulline, ornithine, and GABA in our chemically defined medium. Additionally, E. coli, K. pneumoniae, K. aerogenes, and P. fluorescens were found to convert arginine to agmatine and produce putrescine. Interestingly, arginine supplementation promoted biofilm formation in K. pneumoniae, while ornithine supplementation enhanced biofilm formation in S. epidermidis. These findings offer a comprehensive insight into arginine-ornithine metabolism in enteric pathobionts.

Amino Sugar-Enriched Fraction of Korean Red Ginseng Extract Induces the Priming Step of NLRP3 Inflammasome.

Intracellular protein complexes, known as inflammasomes, activate caspase-1 and induce the secretion of pro-inflammatory cytokines, namely interleukin (IL)-1ß and -18. Korean Red Ginseng extract (RGE) is a known immunomodulator and a potential candidate for the regulation of inflammasomes. The saponins, such as ginsenosides, of RGE inhibit inflammasome signaling, while non-saponin substances containing amino sugars promote the priming step, up-regulating inflammasome components (pro-IL-1ß, NLRP3, caspase-1, and Asc). In this study, the amino sugar-enriched fraction (ASEF), which increases only non-saponin components, including amino sugars, without changing the concentration of saponin substances, was used to investigate whether saponin or non-saponin components of RGE would have a greater impact on the priming step. When murine macrophages were treated with ASEF, the gene expression of inflammatory cytokines (IL-1α, TNFα, IL-6, and IL-10) increased. Additionally, ASEF induced the priming step but did not affect the inflammasome activation step, such as the secretion of IL-1ß, cleavage of caspase-1, and formation of Asc pyroptosome. Furthermore, the upregulation of gene expression of inflammasome components by ASEF was blocked by inhibitors of Toll-like receptor 4 signaling. Maltol, the main constituent of ASEF, promoted the priming step but inhibited the activation step of the inflammasome, while arginine, sugars, arginine-fructose-glucose, and fructose-arginine, the other main constituents of ASEF, had no effect on either step. Thus, certain amino sugars in RGE, excluding maltol, are believed to be the components that induce the priming step. The priming step that prepares the NLRP3 inflammasome for activation appears to be induced by amino sugars in RGE, thereby contributing to the immune-boosting effects of RGE.

Arginine Supplementation in MELAS Syndrome: What Do We Know about the Mechanisms?

MELAS syndrome, characterized by mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes, represents a devastating mitochondrial disease, with the stroke-like episodes being its primary manifestation. Arginine supplementation has been used and recommended as a treatment for these acute attacks; however, insufficient evidence exists to support this treatment for MELAS. The mechanisms underlying the effect of arginine on MELAS pathophysiology remain unclear, although it is hypothesized that arginine could increase nitric oxide availability and, consequently, enhance blood supply to the brain. A more comprehensive understanding of these mechanisms is necessary to improve treatment strategies, such as dose and regimen adjustments; identify which patients could benefit the most; and establish potential markers for follow-up. This review aims to analyze the existing evidence concerning the mechanisms through which arginine supplementation impacts MELAS pathophysiology and provide the current scenario and perspectives for future investigations.

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.

Arginine alleviates Clostridium perfringens α toxin-induced intestinal injury in vivo and in vitro via the SLC38A9/mTORC1 pathway.

Introduction: Clostridium perfringens α toxin is a main virulence factor responsible for gut damage in animals. Arginine is a functional amino acid exhibiting significant immunoregulatory activities. However, the effects and immunoregulatory mechanisms of arginine supplementation on α toxin-induced intestinal injury remain unclear. Methods: In vivo, 256 male Arbor Acres chickens were randomly assigned to a 2×2 factorial arrangement, involving diet treatments (with or without 0.3% arginine supplementation) and immunological stress (with or without α toxin challenge). In vitro, IEC-6 cells were treated with or without arginine in the presence or absence of α toxin. Moreover, IEC-6 cells were transfected with siRNA targeting mTOR and SLC38A9 to explore the underlying mechanisms. Results and discussion: The results showed that in vivo, arginine supplementation significantly alleviated the α toxin-induced growth performance impairment, decreases in serum immunoglobulin (Ig)A and IgG levels, and intestinal morphology damage. Arginine supplementation also significantly reduced the α toxin-induced increase in jejunal proinflammatory cytokines interleukin (IL)-1ß, IL-6 and IL-17 mRNA expression. Clostridium perfringens α toxin significantly decreased jejunal mechanistic target of rapamycin (mTOR) and solute carrier family 38 member 9 (SLC38A9) mRNA expression, while arginine supplementation significantly increased mTOR and SLC38A9 mRNA expression. In vitro, arginine pretreatment mitigated the α toxin-induced decrease in cell viability and the increase in cytotoxicity and apoptosis. Arginine pretreatment also alleviated the α toxin-induced upregulation of mRNA expression of inflammation-related cytokines IL-6, C-X-C motif chemokine ligand (CXCL)10, CXCL11 and transforming growth factor-ß (TGF-ß), as well as apoptosis-related genes B-cell lymphoma-2 associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-extra large (Bcl-XL) and cysteinyl aspartate specific proteinase 3 (Caspase-3) and the ratio of Bax to Bcl-2. Arginine pretreatment significantly increased the α toxin-induced decrease in mTOR, SLC38A9, eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1) and ribosomal protein S6 kinase (S6K) mRNA expression. Knockdown SLC38A9 and mTOR largely abrogated the positive effects of arginine pretreatment on α toxin-induced intracellular changes. Furthermore, SLC38A9 silencing abolished the increased mTOR mRNA expression caused by arginine pretreatment. In conclusion, arginine administration attenuated α toxin-induced intestinal injury in vivo and in vitro, which could be associated with the downregulation of inflammation via regulating SLC38A9/mTORC1 pathway.

Impact of L-Arginine on diabetes-induced neuropathy and myopathy: Roles of PAI-1, Irisin, oxidative stress, NF-κß, autophagy and microRNA-29a.

BACKGROUND: T2DM is a chronic disorder with progressive neuromuscular alterations. L-arginine (ARG) is the most common semi-essential amino acid having several metabolic functions. AIM: to investigate the impact of L-arginine in combating diabetic-induced neuromyopathy and its possible mechanisms. MATERIALS & METHODS: 24 rats were divided into CON, CON+ARG, DC, DC+ARG. Behavioral tests, Body weight (BW), fasting blood glucose (FBG), insulin, total antioxidant capacity (TAC), malondialdehyde (MDA), plasminogen activator inhibitor-1 (PAI-1), and irisin were done. Creatine kinase-MM (CK-MM), interleukin 4 (IL-4), interleukin 6 (IL-6), TAC, MDA, expression of microRNA-29a mRNA & light chain 3 protein were determined in muscle. Histological and NF-κß immunohistochemical expression in muscle and nerve were assessed. RESULTS: ARG supplementation to diabetic rats improved altered behavior, significantly increased BW, insulin, TAC, irisin and Il-4, decreased levels of glucose, microRNA-29a, NF-κß and LC3 expression, PAI-1, CK-MM and restored the normal histological appearance. CONCLUSIONS: ARG supplementation potently alleviated diabetic-induced neuromuscular alterations.

Anti-inflammatory mechanism of the non-volatile ingredients originated from Guanghuoxiang () based on high performance liquid chromatography-heated electron spray ionization-high resolution mass spectroscope and cell metabolomics.

OBJECTIVE: To explore the anti-inflammatory components and mechanism of the non-volatile ingredients of patchouli. METHODS: High performance liquid chromatography-heated electron spray ionization-high resolution mass spectroscope (HPLC-HESI-HRMS) was used to analyze the chemical constituents of the non-volatile ingredients of patchouli. The anti-inflammatory activity of ingredients was evaluated using lipopolysaccharide (LPS) induced RAW264.7 cell inflammation model, and the anti-inflammatory mechanism was investigated using multivariate statistical analysis of cell metabolomics. RESULTS: The non-volatile ingredients of patchouli were characterized by HPLC-HESI-HRMS, and 36 flavonoids and 18 other components were identified. These ingredients of patchouli not only had a good protective effect on the LPS-induced inflammation model of RAW264.7 cells, but also regulated the expression levels of arginine, L-leucine, cholesterol, fructose and sorbitol by down-regulating arginine metabolism, aminoacyl-tRNA biosynthesis, polyol/sorbitol pathway, so as to reduce inflammation and reduce cell damage. CONCLUSION: The non-volatile ingredients of patchouli had good anti-inflammatory effect and exerted its curative effect by regulating endogenous metabolic pathway to reduce inflammatory response.

Effects of Exercise Training and L-Arginine Loaded Chitosan Nanoparticles on Hippocampus Histopathology, ß-Secretase Enzyme Function, APP, Tau, Iba1and APOE-4 mRNA in Aging Rats.

The objective of this study was to evaluate the combined and independent effects of exercise training and L-Arginine loaded chitosan nanoparticles (LA CNPs) supplementation on hippocampal Tau, App, Iba1, and ApoE gene expression, oxidative stress, ß-secretase enzyme activity, and hippocampus histopathology in aging rats. Thirty-five male Wistar rats were randomly assigned to five groups (n = 7 in each): Young (8 weeks old), Old (20 months old), old + L-arginine supplementation (Old Sup), old + exercise (Old Exe) and old + L-arginine supplementation + exercise (Old Sup + Exe). LA CNPs were administered to the supplement groups through gavage at a dosage of 500 mg/kg/day for 6-weeks. Exercise groups were subjected to a swimming exercise program five days/week for the same duration. Upon the completion of their interventions, the animals underwent behavioral and open-field task tests and were subsequently sacrificed for hippocampus genetic and histopathological evaluation. For histopathological analysis of brain, Cresyl violet staining was used. Congo Red staining was employed to confirm amyloid plaques in the hippocampus. Expressions of Tau, App, Iba1, and ApoE genes were determined by real-time PCR. In contrast to the Old group, Old Exe and Old Sup + Exe groups spent more time in the central space in the open field task (p < 0.05) and have more live cells in the hippocampus. Old rats (Old, Old Sup and Old Exe groups) exhibited a significant Aß peptide accumulation and increases in APP, Tau, Iba1, APOE-4 mRNA and MDA, along with decreases in SOD compared to the young group (p < 0.05). However, LA CNPs supplementation, exercise, and their combination (Old Sup, Old Exe and Old Sup + Exe) significantly reduced MDA, Aß plaque as well as APP, Tau, Iba1, and APOE-4 mRNA compared to the Old group (p < 0.05). Consequently, the administration of LA CNPs supplements and exercise might regulate the risk factors of hippocampus cell and tissue.

A metabolomics approach reveals metabolic disturbance of human cholangiocarcinoma cells after parthenolide treatment.

ETHNOPHARMACOLOGICAL RELEVANCE: Tanacetum parthenium (L.) Schultz-Bip, commonly known as feverfew, has been traditionally used to treat fever, migraines, rheumatoid arthritis, and cancer. Parthenolide (PTL), the main bioactive ingredient isolated from the shoots of feverfew, is a sesquiterpene lactone with anti-inflammatory and antitumor properties. Previous studies showed that PTL exerts anticancer activity in various cancers, including hepatoma, cholangiocarcinoma, acute myeloid leukemia, breast, prostate, and colorectal cancer. However, the metabolic mechanism underlying the anticancer effect of PTL remains poorly understood. AIM OF THE STUDY: To explore the anticancer activity and underlying mechanism of PTL in human cholangiocarcinoma cells. MATERIAL AND METHODS: In this investigation, the effects and mechanisms of PTL on human cholangiocarcinoma cells were investigated via a liquid chromatography/mass spectrometry (LC/MS)-based metabolomics approach. First, cell proliferation and apoptosis were evaluated using cell counting kit-8 (CCK-8), flow cytometry analysis, and western blotting. Then, LC/MS-based metabolic profiling along with orthogonal partial least-squares discriminant analysis (OPLS-DA) has been constructed to distinguish the metabolic changes between the negative control group and the PTL-treated group in TFK1 cells. Next, enzyme-linked immunosorbent assay (ELISA) was applied to investigate the changes of metabolic enzymes associated with significantly alerted metabolites. Finally, the metabolic network related to key metabolic enzymes, metabolites, and metabolic pathways was established using MetaboAnalyst 5.0 and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Database. RESULTS: PTL treatment could induce the proliferation inhibition and apoptosis of TFK1 in a concentration-dependent manner. Forty-three potential biomarkers associated with the antitumor effect of PTL were identified, which primarily related to glutamine and glutamate metabolism, alanine, aspartate and glutamate metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, arginine biosynthesis, arginine and proline metabolism, glutathione metabolism, nicotinate and nicotinamide metabolism, pyrimidine metabolism, fatty acid metabolism, phospholipid catabolism, and sphingolipid metabolism. Pathway analysis of upstream and downstream metabolites, we found three key metabolic enzymes, including glutaminase (GLS), γ-glutamyl transpeptidase (GGT), and carnitine palmitoyltransferase 1 (CPT1), which mainly involved in glutamine and glutamate metabolism, glutathione metabolism, and fatty acid metabolism. The changes of metabolic enzymes associated with significantly alerted metabolites were consistent with the levels of metabolites, and the metabolic network related to key metabolic enzymes, metabolites, and metabolic pathways was established. PTL may exert its antitumor effect against cholangiocarcinoma by disturbing metabolic pathways. Furthermore, we selected two positive control agents that are considered as first-line chemotherapy standards in cholangiocarcinoma therapy to verify the reliability and accuracy of our metabolomic study on PTL. CONCLUSION: This research enhanced our comprehension of the metabolic profiling and mechanism of PTL treatment on cholangiocarcinoma cells, which provided some references for further research into the anti-cancer mechanisms of other drugs.

Deciphering alternative splicing events and their therapeutic implications in colorectal Cancer.

Colorectal cancer (CRC) is one of the most common malignant tumors with complex molecular regulatory mechanisms. Alternative splicing (AS), a fundamental regulatory process of gene expression, plays an important role in the occurrence and development of CRC. This study analyzed AS Percent Spliced In (PSI) values from 49 pairs of CRC and normal samples in the TCGA SpliceSeq database. Using Lasso and SVM, AS features that can differentiate colorectal cancer from normal were screened. Univariate COX regression analysis identified prognosis-related AS events. A risk model was constructed and validated using machine learning, Kaplan-Meier analysis, and Decision Curve Analysis. The regulatory effect of protein arginine methyltransferase 5 (PRMT5) on poly(RC) binding protein 1 (PCBP1) was verified by immunoprecipitation experiments, and the effect of PCBP1 on the AS of Obscurin (OBSCN) was verified by PCR. Five AS events, including HNF4A.59461.AP and HNF4A.59462.AP, were identified, which can distinguish CRC from normal tissue. A machine learning model using 21 key AS events accurately predicted CRC prognosis. High-risk patients had significantly shorter survival times. PRMT5 was found to regulate PCBP1 function and then influence OBSCN AS, which may drive CRC progression. The study concluded that some AS events is significantly different in CRC and normal tissues, and some of these AS events are related to the prognosis of CRC. In addition, PRMT family-driven arginine modifications play an important role in CRC-specific AS events.

Polyamine-mediated ferroptosis amplification acts as a targetable vulnerability in cancer.

Targeting ferroptosis, an iron-dependent form of regulated cell death triggered by the lethal overload of lipid peroxides, in cancer therapy is impeded by our limited understanding of the intersection of tumour's metabolic feature and ferroptosis vulnerability. In the present study, arginine is identified as a ferroptotic promoter using a metabolites library. This effect is mainly achieved through arginine's conversion to polyamines, which exerts their potent ferroptosis-promoting property in an H2O2-dependent manner. Notably, the expression of ornithine decarboxylase 1 (ODC1), the critical enzyme catalysing polyamine synthesis, is significantly activated by the ferroptosis signal--iron overload--through WNT/MYC signalling, as well as the subsequent elevated polyamine synthesis, thus forming a ferroptosis-iron overload-WNT/MYC-ODC1-polyamine-H2O2 positive feedback loop that amplifies ferroptosis. Meanwhile, we notice that ferroptotic cells release enhanced polyamine-containing extracellular vesicles into the microenvironment, thereby further sensitizing neighbouring cells to ferroptosis and accelerating the "spread" of ferroptosis in the tumour region. Besides, polyamine supplementation also sensitizes cancer cells or xenograft tumours to radiotherapy or chemotherapy through inducing ferroptosis. Considering that cancer cells are often characterized by elevated intracellular polyamine pools, our results indicate that polyamine metabolism exposes a targetable vulnerability to ferroptosis and represents an exciting opportunity for therapeutic strategies for cancer.

Effectiveness of glutamine and arginine in wound healing of pressure ulcers: A systematic review protocol.

INTRODUCTION: Various nutrients play a physiological role in the healing process of pressure ulcers (PUs). Nutritional interventions include the administration of enteral nutritional supplements and formulas containing arginine, glutamine, and micronutrients. The aim of this systematic review is to evaluate the effectiveness of enteral nutritional supplements and formulas containing arginine and glutamine on wound-related outcomes. These include (1) time to healing, (2) changes in wound size, (3) local wound infection, (4) PU recurrence, and (5) PU-related pain. MATERIALS AND METHODS: This protocol was developed according to the guidelines of the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P). A search will be conducted in the Cochrane Library, EMBASE, PubMed (MEDLINE), CINAHL (EBSCOhost interface) and Web of Science. In addition, a manual search will be conducted to identify relevant records. Except for systematic reviews, no restrictions will be placed on the study design, the population studied or the setting. Studies that do not address PUs, in vitro studies and studies that do not report wound-related outcomes will be excluded. Study selection, risk of bias assessment and data extraction will be performed independently by three researchers. Depending on the extent of heterogeneity of interventions, follow-up time and populations, results will be summarised either by meta-analysis or narrative synthesis. CONCLUSIONS: This is the first systematic review to identify, evaluate and summarise the current evidence for enteral arginine and glutamine supplementation on wound-related outcomes in PUs. The review will provide a solid basis for deriving valid and clinically relevant conclusions in this area.

Tangshen Formula alleviates inflammatory injury against aged diabetic kidney disease through modulating gut microbiota composition and related amino acid metabolism.

Diabetic kidney disease (DKD) is leading causes and one of the fastest growing causes of chronic kidney disease worldwide, and leads to high morbidity and mortality. Emerging evidences have revealed gut microbiota dysbiosis and related metabolism dysfunction play a dominant role in DKD progression and treatment through modulating inflammation. Our previous studies showed that Tangshen Formula (TSF), a Chinese herbal prescription, exhibited anti-inflammatory effect on DKD, but underlying mechanism that involved gut microbiota and related metabolism in aged model remained obscure. Here, BTBR ob/ob mice were used to establish aged DKD model, and 16S rRNA sequence and untargeted metabolomic analyses were employed to investigate the correlation between colonic microbiota and serum metabolism. The aged ob/ob mice exhibited obvious glomerular and renal tubule injury and kidney function decline in kidney, while TSF treatment significantly attenuated these abnormalities. TSF also exhibited potent anti-inflammatory effect in aged ob/ob mice indicating by reduced proinflammatory factor IL-6 and TNF-α, MCP-1 and COX-2 in serum, kidney and intestine, which suggested the involvement of gut microbiota with TSF effect. The 16S rDNA sequencing of the colonic microbiome and untargeted serum metabolomics analysis revealed significant differences in gut microbiota structure and serum metabolomic profiles between WT and ob/ob mice. Notably, TSF treatment reshaped the structure of gut microbiota and corrected the disorder of metabolism especially tryptophan metabolism and arginine biosynthesis. TSF increased Anaeroplasma and Barnesiella genera and decreased Romboutsia, Akkermansia, and Collinsella genera, and further elevated tryptophan, 5-hydroxyindoleacetate, glutamic acid, aspartate and reduced 4-hydroxy-2-quinolinecarboxylic acid, indole-3-acetic acid, xanthurenic acid, glutamine. Further correlation analysis indicated that disturbed gut microbiota was linked to tryptophan metabolism and arginine biosynthesis to regulate inflammation in aged DKD. Our data revealed that TSF attenuated renal inflammation by modulating gut microbiota and related amino acid metabolism in aged DKD model, highlighting gut microbiota and related metabolism functioned as potential therapeutic target for DKD in elderly patients.

Analysis of heterologous expression of phaCBA promotes the acetoin stress response mechanism in Bacillus subtilis using transcriptomics and metabolomics approaches.

Acetoin, a versatile platform chemical and popular food additive, poses a challenge to the biosafety strain Bacillus subtilis when produced in high concentrations due to its intrinsic toxicity. Incorporating the PHB synthesis pathway into Bacillus subtilis 168 has been shown to significantly enhance the strain's acetoin tolerance. This study aims to elucidate the molecular mechanisms underlying the response of B. subtilis 168-phaCBA to acetoin stress, employing transcriptomic and metabolomic analyses. Acetoin stress induces fatty acid degradation and disrupts amino acid synthesis. In response, B. subtilis 168-phaCBA down-regulates genes associated with flagellum assembly and bacterial chemotaxis, while up-regulating genes related to the ABC transport system encoding amino acid transport proteins. Notably, genes coding for cysteine and D-methionine transport proteins (tcyB, tcyC and metQ) and the biotin transporter protein bioY, are up-regulated, enhancing cellular tolerance. Our findings highlight that the expression of phaCBA significantly increases the ratio of long-chain unsaturated fatty acids and modulates intracellular concentrations of amino acids, including L-tryptophan, L-tyrosine, L-leucine, L-threonine, L-methionine, L-glutamic acid, L-proline, D-phenylalanine, L-arginine, and membrane fatty acids, thereby imparting acetoin tolerance. Furthermore, the supplementation with specific exogenous amino acids (L-alanine, L-proline, L-cysteine, L-arginine, L-glutamic acid, and L-isoleucine) alleviates acetoin's detrimental effects on the bacterium. Simultaneously, the introduction of phaCBA into the acetoin-producing strain BS03 addressed the issue of insufficient intracellular cofactors in the fermentation strain, resulting in the successful production of 70.14 g/L of acetoin through fed-batch fermentation. This study enhances our understanding of Bacillus's cellular response to acetoin-induced stress and provides valuable insights for the development of acetoin-resistant Bacillus strains.

The potential of Rhodobacter sphaeroides extract as an alternative supplement for cell culture systems.

It is essential to identify suitable supplements that enhance cell growth, viability, and functional development in cell culture systems. The use of fetal bovine serum (FBS) has been common, but it has limitations, such as batch-to-batch variability, ethical concerns, and risks of environmental contamination. In this study, we explore the potential of Rhodobacter sphaeroides extract, derived from a probiotic photosynthetic bacterium, as an alternative supplement. Our results demonstrate that the extract from R. sphaeroides significantly improves various aspects of cell behavior compared to serum-free conditions. It enhances cell growth and viability to a greater extent than FBS supplementation. Additionally, the extract alleviates oxidative stress by reducing intracellular levels of reactive oxygen species and stimulates lysosomal activity, contributing to cellular processes. The presence of abundant amino acids, glycine and arginine, in the extract may play a role in promoting cell growth. These findings emphasize the potential of R. sphaeroides extract as a valuable supplement for cell culture, offering advantages over the use of FBS.IMPORTANCEThe choice of supplements for cell culture is crucial in biomedical research, but the widely used fetal bovine serum (FBS) has limitations in terms of variability, ethics, and environmental risks. This study explores the potential of an extract from Rhodobacter sphaeroides, a probiotic bacterium, as an alternative supplement. The findings reveal that the R. sphaeroides extract surpasses FBS in enhancing cell growth, viability, and functionality. It also mitigates oxidative stress and stimulates lysosomal activity, critical for cellular health. The extract's abundance of glycine and arginine, amino acids with known growth-promoting effects, further highlights its potential. By providing a viable substitute for FBS, the R. sphaeroides extract addresses the need for consistent, ethical, and environmentally friendly cell culture supplements. This research paves the way for sustainable and reliable cell culture systems, revolutionizing biomedical research and applications in drug development and regenerative medicine.

CLPP-Null Eukaryotes with Excess Heme Biosynthesis Show Reduced L-arginine Levels, Probably via CLPX-Mediated OAT Activation.

The serine peptidase CLPP is conserved among bacteria, chloroplasts, and mitochondria. In humans and mice, its loss causes Perrault syndrome, which presents with growth deficits, infertility, deafness, and ataxia. In the filamentous fungus Podospora anserina, CLPP loss leads to longevity. CLPP substrates are selected by CLPX, an AAA+ unfoldase. CLPX is known to target delta-aminolevulinic acid synthase (ALAS) to promote pyridoxal phosphate (PLP) binding. CLPX may also influence cofactor association with other enzymes. Here, the evaluation of P. anserina metabolomics highlighted a reduction in arginine/histidine levels. In Mus musculus cerebellum, reductions in arginine/histidine and citrulline occurred with a concomitant accumulation of the heme precursor protoporphyrin IX. This suggests that the increased biosynthesis of 5-carbon (C5) chain deltaALA consumes not only C4 succinyl-CoA and C1 glycine but also specific C5 delta amino acids. As enzymes responsible for these effects, the elevated abundance of CLPX and ALAS is paralleled by increased OAT (PLP-dependent, ornithine delta-aminotransferase) levels. Possibly as a consequence of altered C1 metabolism, the proteome profiles of P. anserina CLPP-null cells showed strong accumulation of a methyltransferase and two mitoribosomal large subunit factors. The reduced histidine levels may explain the previously observed metal interaction problems. As the main nitrogen-storing metabolite, a deficiency in arginine would affect the urea cycle and polyamine synthesis. Supplementation of arginine and histidine might rescue the growth deficits of CLPP-mutant patients.

Elevated SLC7A2 expression is associated with an abnormal neuroinflammatory response and nitrosative stress in Huntington's disease.

We previously identified solute carrier family 7 member 2 (SLC7A2) as one of the top upregulated genes when normal Huntingtin was deleted. SLC7A2 has a high affinity for L-arginine. Arginine is implicated in inflammatory responses, and SLC7A2 is an important regulator of innate and adaptive immunity in macrophages. Although neuroinflammation is clearly demonstrated in animal models and patients with Huntington's disease (HD), the question of whether neuroinflammation actively participates in HD pathogenesis is a topic of ongoing research and debate. Here, we studied the role of SLC7A2 in mediating the neuroinflammatory stress response in HD cells. RNA sequencing (RNA-seq), quantitative RT-PCR and data mining of publicly available RNA-seq datasets of human patients were performed to assess the levels of SLC7A2 mRNA in different HD cellular models and patients. Biochemical studies were then conducted on cell lines and primary mouse astrocytes to investigate arginine metabolism and nitrosative stress in response to neuroinflammation. The CRISPR-Cas9 system was used to knock out SLC7A2 in STHdhQ7 and Q111 cells to investigate its role in mediating the neuroinflammatory response. Live-cell imaging was used to measure mitochondrial dynamics. Finally, exploratory studies were performed using the Enroll-HD periodic human patient dataset to analyze the effect of arginine supplements on HD progression. We found that SLC7A2 is selectively upregulated in HD cellular models and patients. HD cells exhibit an overactive response to neuroinflammatory challenges, as demonstrated by abnormally high iNOS induction and NO production, leading to increased protein nitrosylation. Depleting extracellular Arg or knocking out SLC7A2 blocked iNOS induction and NO production in STHdhQ111 cells. We further examined the functional impact of protein nitrosylation on a well-documented protein target, DRP-1, and found that more mitochondria were fragmented in challenged STHdhQ111 cells. Last, analysis of Enroll-HD datasets suggested that HD patients taking arginine supplements progressed more rapidly than others. Our data suggest a novel pathway that links arginine uptake to nitrosative stress via upregulation of SLC7A2 in the pathogenesis and progression of HD. This further implies that arginine supplements may potentially pose a greater risk to HD patients.

Regulation of Gut Microbiota and Microbial Metabolome of Kefir Supernatant against Fusobacterium nucleatum and DSS-Coinduced Colitis.

The aim of this study was to investigate the intervention effect of kefir supernatant (KS) on the initiation and progression of an ulcerative colitis (UC) murine model. We established an UC murine model by orally administrating with 109 CFUs of Fusobacterium nucleatum for 3 weeks and 3% dextran sulfate sodium (DSS) treatment in the third week. KS was used to intervene in this colitis model. Our results showed that KS supplementation ameliorated the symptoms, restrained the secretion of pro-inflammatory cytokines (TNF-α, IL-6, and IL-17F), promoted the release of anti-inflammatory cytokines (IL-4 and IL-10), and ameliorated oxidative stress. Furthermore, the increased number of goblet cells and upregulated expression of MUC2, occludin and claudin-1 indicated that the colon barrier was protected by KS. Additionally, KS supplementation mitigated gut microbiota dysbiosis in the UC murine model, leading to an increase in the abundance of Blautia and Akkermansia and a decrease in the level of Bacteroides. The altered gut microbiota also affected colon metabolism, with differential metabolites mainly associated with the biosynthesis of the l-arginine pathway. This study revealed that KS supplementation restored the community structure of gut microbiota, altered the biosynthesis of l-arginine, and thereby modulated the process of colonic inflammation.

Targeted arginine metabolomics combined with metagenomics revealed the potential mechanism of Pueraria lobata extract in treating myocardial infarction.

The extraction methods for traditional Chinese medicine (TCM) may have varying therapeutic effects on diseases. Currently, Pueraria lobata (PL) is mostly extracted with ethanol, but decoction, as a TCM extraction method, is not widely adopted. In this study, we present a strategy that integrates targeted metabolomics, 16 s rDNA sequencing technology and metagenomics for exploring the potential mechanism of the water extract of PL (PLE) in treating myocardial infarction (MI). Using advanced analytical techniques like ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), we comprehensively characterized PLE's chemical composition. Further, we tested its efficacy in a rat model of MI induced by ligation of the left anterior descending branch of the coronary artery (LAD). We assessed cardiac enzyme levels and conducted echocardiograms. UPLC-MS/MS was used to compare amino acid differences in serum. Furthermore, we investigated fecal samples using 16S rDNA sequencing and metagenomic sequencing to study intestinal flora diversity and function. This study demonstrated PLE's effectiveness in reducing cardiac injury in LAD-ligated rats. Amino acid metabolomics revealed significant improvements in serum levels of arginine, citrulline, proline, ornithine, creatine, creatinine, and sarcosine in MI rats, which are key compounds in the arginine metabolism pathway. Enzyme-linked immunosorbent assay (ELISA) results showed that PLE significantly improved arginase (Arg), nitric oxide synthase (NOS), and creatine kinase (CK) contents in the liver tissue of MI rats. 16 s rDNA and metagenome sequencing revealed that PLE significantly improved intestinal flora imbalance in MI rats, particularly in taxa such as Tuzzerella, Desulfovibrio, Fournierella, Oscillibater, Harryflintia, and Holdemania. PLE also improved the arginine metabolic pathway in the intestinal microorganisms of MI rats. The findings indicate that PLE effectively modulates MI-induced arginine levels and restores intestinal flora balance. This study, the first to explore the mechanism of action of PLE in MI treatment considering amino acid metabolism and intestinal flora, expands our understanding of the potential of PL in MI treatment. It offers fresh insights into the mechanisms of PL, guiding further research and development of PL-based medicines.

Arginase-induced cell death pathways and metabolic changes in cancer cells are not altered by insulin.

Arginine, a semi-essential amino acid, is critical for cell growth. Typically, de novo synthesis of arginine is sufficient to support cellular processes, however, it becomes vital for cancer cells that are unable to synthesise arginine due to enzyme deficiencies. Targeting this need, arginine depletion with enzymes such as arginase (ARG) has emerged as a potential cancer therapeutic strategy. Studies have proposed using high dose insulin to induce a state of hypoaminoacidaemia in the body, thereby further reducing circulating arginine levels. However, the mitogenic and metabolic properties of insulin could potentially counteract the therapeutic effects of ARG. Our study examined the combined impact of insulin and ARG on breast, lung, and ovarian cell lines, focusing on cell proliferation, metabolism, apoptosis, and autophagy. Our results showed that the influence of insulin on ARG uptake varied between cell lines but failed to promote the proliferation of ARG-treated cells or aid recovery post-ARG treatment. Moreover, insulin was largely ineffective in altering ARG-induced metabolic changes and did not prevent apoptosis. In vitro, at least, these findings imply that insulin does not offer a growth or survival benefit to cancer cells being treated with ARG.