Differential One-Carbon Metabolites among Children with Autism Spectrum Disorder: A Case-Control Study.

BACKGROUND: Driven by the complex multifactorial etiopathogenesis of autism spectrum disorder (ASD), a growing interest surrounds the disturbance in folate-dependent one-carbon metabolism (OCM) in the pathology of ASD, whereas the evidence remained inconclusive. OBJECTIVES: The study aims to investigate the association of OCM metabolism and ASD and characterize differential OCM metabolites among children with ASD. METHODS: Plasma OCM metabolites were investigated in 59 children with ASD and 40 neurotypical children using ultra-performance liquid chromatography tandem mass spectrometry technology. Differences (significance level < 0.001) were tested in each OCM metabolite between cases and controls. Multivariable models were also performed after adjusting for covariates. RESULTS: Ten out of 22 examined OCM metabolites were significantly different in children with ASD, compared with neurotypical controls. Specifically, S-adenosylmethionine (SAM), oxidized glutathione (GSSG), and glutathione (GSH) levels were increased, whereas S-adenosylhomocysteine (SAH), choline, glycine, L-serine, cystathionine, L-cysteine, and taurine levels were significantly decreased. Children with ASD showed significantly higher SAM/SAH ratio (3.87 ± 0.93 compared with 2.00 ± 0.76, P = 0.0001) and lower GSH/GSSG ratio [0.58 (0.46, 0.81) compared with 1.71 (0.93, 2.99)] compared with the neurotypical controls. Potential interactive effects between SAM/SAH ratio, taurine, L-serine, and gastrointestinal syndromes were further observed. CONCLUSIONS: OCM disturbance was observed among children with ASD, particularly in methionine methylation and trans-sulfuration pathways. The findings add valuable insights into the mechanisms underlying ASD and the potential of ameliorating OCM as a promising therapeutic of ASD, which warrant further validation.

Hydrogen sulfide-mediated persulfidation regulates homocysteine metabolism and enhances ferroptosis in non-small cell lung cancer.

Hydrogen sulfide (H2S), a metabolite of the transsulfuration pathway, has been implicated in ferroptosis, a unique form of cell death caused by lipid peroxidation. While the exact mechanisms controlling ferroptosis remain unclear, our study reveals that H2S sensitizes human non-small cell lung cancer (NSCLC) cells to this process, particularly when cysteine levels are low. Combining H2S with cystine depletion significantly enhances the effectiveness of ferroptosis-based cancer therapy. Mechanistically, H2S persulfidates the 195th cysteine on S-adenosyl homocysteine hydrolase (SAHH), reducing its enzymatic activity. This leads to decreased homocysteine levels, subsequently lowering cysteine and glutathione concentrations under cystine depletion conditions. These changes ultimately increase the vulnerability of NSCLC cells to ferroptosis. Our findings establish H2S as a key regulator of homocysteine metabolism and a critical factor in determining NSCLC cell susceptibility to ferroptosis. These results highlight the potential of H2S-based therapies to improve the efficacy of ferroptosis-targeted cancer treatments for NSCLC.

Targeting the sulfur-containing amino acid pathway in leukemia.

sulfur-containing amino acids have been reported to patriciate in gene regulation, DNA methylation, protein synthesis and other physiological or pathological processes. In recent years, metabolism-related molecules of sulfur-containing amino acids affecting the occurrence, development and treatment of tumors have been implicated in various disorders, especially in leukemia. Here, we summarize current knowledge on the sulfur-containing amino acid metabolism pathway in leukemia and examine ongoing efforts to target this pathway, including treatment strategies targeting (a) sulfur-containing amino acids, (b) metabolites of sulfur-containing amino acids, and (c) enzymes and cofactors related to sulfur-containing amino acid metabolism in leukemia. Future leukemia therapy will likely involve innovative strategies targeting the sulfur-containing amino acid metabolism pathway.

Longitudinal Analysis of One-Carbon Metabolism-Related Metabolites in Maternal and Cord Blood of Japanese Pregnant Women.

One-carbon metabolism (OCM) is a complex and interconnected network that undergoes drastic changes during pregnancy. In this study, we investigated the longitudinal distribution of OCM-related metabolites in maternal and cord blood and explored their relationships. Additionally, we conducted cross-sectional analyses to examine the interrelationships among these metabolites. This study included 146 healthy pregnant women who participated in the Chiba Study of Mother and Child Health. Maternal blood samples were collected during early pregnancy, late pregnancy, and delivery, along with cord blood samples. We analyzed 18 OCM-related metabolites in serum using stable isotope dilution liquid chromatography/tandem mass spectrometry. We found that serum S-adenosylmethionine (SAM) concentrations in maternal blood remained stable throughout pregnancy. Conversely, S-adenosylhomocysteine (SAH) concentrations increased, and the total homocysteine/total cysteine ratio significantly increased with advancing gestational age. The betaine/dimethylglycine ratio was negatively correlated with total homocysteine in maternal blood for all sampling periods, and this correlation strengthened with advances in gestational age. Most OCM-related metabolites measured in this study showed significant positive correlations between maternal blood at delivery and cord blood. These findings suggest that maternal OCM status may impact fetal development and indicate the need for comprehensive and longitudinal evaluations of OCM during pregnancy.

Metabolic engineering of CHO cells towards cysteine prototrophy and systems analysis of the ensuing phenotype.

Chinese hamster ovary (CHO) cells require cysteine for growth and productivity in fed-batch cultures. In intensified processes, supplementation of cysteine at high concentrations is a challenge due to its limited solubility and instability in solution. Methionine can be converted to cysteine (CYS) but key enzymes, cystathionine beta-synthase (Cbs) and cystathionine gamma-lyase (Cth), are not active in CHO cells resulting in accumulation of an intermediate, homocysteine (HCY), in cell culture milieu. In this study, Cbs and Cth were overexpressed in CHO cells to confer cysteine prototrophy, i.e., the ability to grow in a cysteine free environment. These pools (CbCt) needed homocysteine and beta-mercaptoethanol (ßME) to grow in CYS-free medium. To increase intracellular homocysteine levels, Gnmt was overexpressed in CbCt pools. The resultant cell pools (GnCbCt), post adaptation in CYS-free medium with decreasing residual HCY and ßME levels, were able to proliferate in the HCY-free, ßME-free and CYS-free environment. Interestingly, CbCt pools were also able to be adapted to grow in HCY-free and CYS-free conditions, albeit at significantly higher doubling times than GnCbCt cells, but couldn't completely adapt to ßME-free conditions. Further, single cell clones derived from the GnCbCt cell pool had a wide range in expression levels of Cbs, Cth and Gnmt and, when cultivated in CYS-free fed-batch conditions, performed similarly to the wild type (WT) cell line cultivated in CYS supplemented fed-batch culture. Intracellular metabolomic analysis showed that HCY and glutathione (GSH) levels were lower in the CbCt pool in CYS-free conditions but were restored closer to WT levels in the GnCbCt cells cultivated in CYS-free conditions. Transcriptomic analysis showed that GnCbCt cells upregulated several genes encoding transporters as well as methionine catabolism and transsulfuration pathway enzymes that support these cells to biosynthesize cysteine effectively. Further, 'omics analysis suggested CbCt pool was under ferroptotic stress in CYS-free conditions, which, when inhibited, enhanced the growth and viability of these cells in CYS-free conditions.

Metformin-induced oxidative stress inhibits LNCaP prostate cancer cell survival.

BACKGROUND: Preclinical and clinical studies over the past several decades have indicated the potential value of metformin, a widely utilized treatment for Type 2 diabetes, in prostate cancer therapy. Notably, these studies demonstrated metformin's pleiotropic effects on several molecular and metabolic pathways, such as androgen signaling, cell cycle, and cellular bioenergetics. In this study we investigated the role of metformin in regulating intracellular redox status and cell survival in LNCaP prostate cancer cells. METHODS AND RESULTS: The cytotoxic effects of metformin with or without the presence of SBI0206965 (AMPK inhibitor) on LNCaP cells were determined using MTT and trypan blue exclusion assays. Seahorse XP extracellular analysis, Liquid Chromatography/ Mass Spectrophotometry (LC/MS), and 2,7- and Dichlorofluoresin diacetate (DCFDA) assay were used to assess the effects of metformin on cellular bioenergetics, redox status, and redox-related metabolites. mRNA expression and protein concentration of redox-related enzymes were measured using Real Time-qPCR and ELISA assay, respectively. Independently of AMP-activated protein kinase, metformin exhibited a dose- and time-dependent inhibition of LNCaP cell survival, a response mitigated by glutathione or N-acetylcysteine (ROS scavengers) treatment. Notably, these findings were concomitant with a decline in ATP levels and the inhibition of oxidative phosphorylation. The results further indicated metformin's induction of reactive oxygen species, which significantly decreased glutathione levels and the ratio of reduced to oxidized glutathione, as well as the transsulfuration metabolite, cystathionine. Consistent with an induction of oxidative stress condition, metformin increased mRNA levels of the master redox transcription factor Nrf-2 (nuclear factor erythroid-derived 2-like), as well as transsulfuration enzymes cystathionine beta-synthase and cystathionase and GSH synthesis enzymes γ-glutamylcysteine synthetase and glutathione synthetase. CONCLUSION: Our findings highlight multiple mechanisms by which metformin-induced formation of reactive oxygen species may contribute to its efficacy in prostate cancer treatment, including promotion of oxidative stress, Nrf2 activation, and modulation of redox-related pathways, leading to its anti-survival action.

Dietary sulfur amino acid restriction in humans with overweight and obesity: Evidence of an altered plasma and urine sulfurome, and a novel metabolic signature that correlates with loss of fat mass and adipose tissue gene expression.

BACKGROUND: In animals, dietary sulfur amino acid restriction (SAAR) improves metabolic health, possibly mediated by altering sulfur amino acid metabolism and enhanced anti-obesogenic processes in adipose tissue. AIM: To assess the effects of SAAR over time on the plasma and urine SAA-related metabolites (sulfurome) in humans with overweight and obesity, and explore whether such changes were associated with body weight, body fat and adipose tissue gene expression. METHODS: Fifty-nine subjects were randomly allocated to SAAR (∼2 g SAA, n = 31) or a control diet (∼5.6 g SAA, n = 28) consisting of plant-based whole-foods and supplemented with capsules to titrate contents of SAA. Sulfurome metabolites in plasma and urine at baseline, 4 and 8 weeks were measured using HPLC and LC-MS/MS. mRNA-sequencing of subcutaneous white adipose tissue (scWAT) was performed to assess changes in gene expression. Data were analyzed with mixed model regression. Principal component analyses (PCA) were performed on the sulfurome data to identify potential signatures characterizing the response to SAAR. RESULTS: SAAR led to marked decrease of the main urinary excretion product sulfate (p < 0.001) and plasma and/or 24-h urine concentrations of cystathionine, sulfite, thiosulfate, H2S, hypotaurine and taurine. PCA revealed a distinct metabolic signature related to decreased transsulfuration and H2S catabolism that predicted greater weight loss and android fat mass loss in SAAR vs. controls (all pinteraction < 0.05). This signature correlated positively with scWAT expression of genes in the tricarboxylic acid cycle, electron transport and ß-oxidation (FDR = 0.02). CONCLUSION: SAAR leads to distinct alterations of the plasma and urine sulfurome in humans, and predicted increased loss of weight and android fat mass, and adipose tissue lipolytic gene expression in scWAT. Our data suggest that SAA are linked to obesogenic processes and that SAAR may be useful for obesity and related disorders. TRIAL IDENTIFIER: https://clinicaltrials.gov/study/NCT04701346.

TRP14 is the rate-limiting enzyme for intracellular cystine reduction and regulates proteome cysteinylation.

It has remained unknown how cells reduce cystine taken up from the extracellular space, which is a required step for further utilization of cysteine in key processes such as protein or glutathione synthesis. Here, we show that the thioredoxin-related protein of 14 kDa (TRP14, encoded by TXNDC17) is the rate-limiting enzyme for intracellular cystine reduction. When TRP14 is genetically knocked out, cysteine synthesis through the transsulfuration pathway becomes the major source of cysteine in human cells, and knockout of both pathways becomes lethal in C. elegans subjected to proteotoxic stress. TRP14 can also reduce cysteinyl moieties on proteins, rescuing their activities as here shown with cysteinylated peroxiredoxin 2. Txndc17 knockout mice were, surprisingly, protected in an acute pancreatitis model, concomitant with activation of Nrf2-driven antioxidant pathways and upregulation of transsulfuration. We conclude that TRP14 is the evolutionarily conserved enzyme principally responsible for intracellular cystine reduction in C. elegans, mice, and humans.

Chronic Exposure to Arsenic and Fluoride Starting at Gestation Alters Liver Mitochondrial Protein Expression and Induces Early Onset of Liver Fibrosis in Male Mouse Offspring.

The presence of arsenic (As) and fluoride (F-) in drinking water is of concern due to the enormous number of individuals exposed to this condition worldwide. Studies in cultured cells and animal models have shown that As- or F-induced hepatotoxicity is primarily associated with redox disturbance and altered mitochondrial homeostasis. To explore the hepatotoxic effects of chronic combined exposure to As and F- in drinking water, pregnant CD-1 mice were exposed to 2 mg/L As (sodium arsenite) and/or 25 mg/L F- (sodium fluoride). The male offspring continued the exposure treatment up to 30 (P30) or 90 (P90) postnatal days. GSH levels, cysteine synthesis enzyme activities, and cysteine transporter levels were investigated in liver homogenates, as well as the expression of biomarkers of ferroptosis and mitochondrial biogenesis-related proteins. Serum transaminase levels and Hematoxylin-Eosin and Masson trichrome-stained liver tissue slices were examined. Combined exposure at P30 significantly reduced GSH levels and the mitochondrial transcription factor A (TFAM) expression while increasing lipid peroxidation, free Fe 2+, p53 expression, and serum ALT activity. At P90, the upregulation of cysteine uptake and synthesis was associated with a recovery of GSH levels. Nevertheless, the downregulation of TFAM continued and was now associated with a downstream inhibition of the expression of MT-CO2 and reduced levels of mtDNA and fibrotic liver damage. Our experimental approach using human-relevant doses gives evidence of the increased risk for early liver damage associated with elevated levels of As and F- in the diet during intrauterine and postnatal period.

Vitamin B6 alleviates chronic sleep deprivation-induced hippocampal ferroptosis through CBS/GSH/GPX4 pathway.

Several studies have found that sleep deprivation (SD) can lead to neuronal ferroptosis and affect hippocampal function. However, there are currently no effective interventions. Vitamin B6 is a co-factor for key enzymes in the transsulfuration pathway which is critical for maintaining cell growth in the presence of cysteine deprivation. The results showed that SD inhibited cystine-glutamate antiporter light chain subunit xCT protein expression and caused cysteine deficiency, which reduced the synthesis of the glutathione (GSH) to trigger neuronal ferroptosis. Nissl staining further revealed significant neuronal loss and shrinkage in the CA1 and CA3 regions of the hippocampus in SD mice. Typical ferroptotic indicators characterized by lipid peroxidation and iron accumulation were showed in the hippocampus after sleep deprivation. As expected, vitamin B6 could alleviate hippocampal ferroptosis by upregulating the expression of cystathionine beta-synthase (CBS) in the transsulfuration pathway, thereby replenishing the intracellular deficient GSH and restoring the expression of GPX4. Similar anti-ferroptotic effects of vitamin B6 were demonstrated in HT-22 cells treated with ferroptosis activator erastin. Furthermore, vitamin B6 had no inhibitory effect on erastin-induced ferroptosis in CBS-knockout HT22 cells. Our findings suggested chronic sleep deprivation caused hippocampal ferroptosis by disrupting the cyst(e)ine/GSH/GPX4 axis. Vitamin B6 alleviated sleep deprivation-induced ferroptosis by enhancing CBS expression in the transsulfuration pathway.

Versatile roles of cysteine persulfides in tumor biology.

Rewiring the transsulfuration pathway is recognized as a rapid adaptive metabolic response to environmental conditions in cancer cells to support their increased cysteine demand and to produce Reactive Sulfur Species (RSS) including hydrogen sulfide (H2S) and cysteine persulfide. This can directly (via RSS) or indirectly (by supplying Cys) trigger chemical or enzyme catalyzed persulfidation on critical protein cysteine residues to protect them from oxidative damage and to orchestrate protein functions, and thereby contribute to cancer cell plasticity. In this review key aspects of persulfide-mediated biological processes are highlighted and critically discussed in relation to cancer cell survival, bioenergetics, proliferation as well as in tumor angiogenesis, adaptation to hypoxia and oxidative stress, and regulation of epithelial to mesenchymal transition.

Breast cancer cells have an increased ferroptosis risk induced by system xc- blockade after deliberately downregulating CYTL1 to mediate malignancy.

Cytokine-like protein 1 (CYTL1) expression is deliberately downregulated during the progression of multiple types of cancers, especially breast cancer. However, the metabolic characteristics of cancer progression remain unclear. Here, we uncovered a risk of breast cancer cells harboring low CYTL1 expression, which is metabolically controlled during malignant progression. We performed metabolism comparison and revealed that breast cancer cells with low CYTL1 expression have highly suppressed transsulfuration activity that is driven by cystathionine ß-synthase (CBS) and contributes to de novo cysteine synthesis. Mechanistically, CYTL1 activated Nrf2 by promoting autophagic Keap1 degradation, and Nrf2 subsequently transactivated CBS expression. Due to the lack of cellular cysteine synthesis, breast cancer cells with low CYTL1 expression showed hypersensitivity to system xc- blockade-induced ferroptosis in vitro and in vivo. Silencing CBS counteracted CYTL1-mediated ferroptosis resistance. Our results show the importance of exogeneous cysteine in breast cancer cells with low CYTL1 expression and highlight a potential metabolic vulnerability to target.

The unfolded protein response-glutathione metabolism axis: A novel target of a cycloruthenated complexes bypassing tumor resistance mechanisms.

Platinum-based drugs remain the reference treatment for gastric cancer (GC). However, the frequency of resistance, due to mutations in TP53 or alterations in the energy and redox metabolisms, impairs the efficacy of current treatments, highlighting the need for alternative therapeutic options. Here, we show that a cycloruthenated compound targeting the redox metabolism, RDC11, induces higher cytotoxicity than oxaliplatin in GC cells and is more potent in reducing tumor growth in vivo. Detailed investigations into the mode of action of RDC11 indicated that it targets the glutathione (GSH) metabolism, which is an important drug resistance mechanism. We demonstrate that cycloruthenated complexes regulate the expression of enzymes of the transsulfuration pathway via the Unfolded Protein Response (UPR) and its effector ATF4. Furthermore, RDC11 induces the expression of SLC7A11 encoding for the cystine/glutamate antiporter xCT. These effects lead to a lower cellular GSH content and elevated oxygen reactive species production, causing the activation of a caspase-independent apoptosis. Altogether, this study provides the first evidence that cycloruthenated complexes target the GSH metabolism, neutralizing thereby a major resistance mechanism towards platinum-based chemotherapies and anticancer immune response.

Role of circadian clock system in the mitochondrial trans-sulfuration pathway and tissue remodeling.

Previous studies from our laboratory revealed that the gaseous molecule hydrogen sulfide (H2S), a metabolic product of epigenetics, involves trans-sulfuration pathway for ensuring metabolism and clearance of homocysteine (Hcy) from body, thereby mitigating the skeletal muscle's pathological remodeling. Although the master circadian clock regulator that is known as brain and muscle aryl hydrocarbon receptor nuclear translocator like protein 1 (i.e., BMAL 1) is associated with S-adenosylhomocysteine hydrolase (SAHH) and Hcy metabolism but how trans-sulfuration pathway is influenced by the circadian clock remains unexplored. We hypothesize that alterations in the functioning of circadian clock during sleep and wake cycle affect skeletal muscle's biology. To test this hypothesis, we measured serum matrix metalloproteinase (MMP) activities using gelatin gels for analyzing the MMP-2 and MMP-9. Further, employing casein gels, we also studied MMP-13 that is known to be influenced by the growth arrest and DNA damage-45 (GADD45) protein during sleep and wake cycle. The wild type and cystathionine ß synthase-deficient (CBS-/+) mice strains were treated with H2S and subjected to measurement of trans-sulfuration factors from skeletal muscle tissues. The results suggested highly robust activation of MMPs in the wake mice versus sleep mice, which appears somewhat akin to the "1-carbon metabolic dysregulation", which takes place during remodeling of extracellular matrix during muscular dystrophy. Interestingly, the levels of trans-sulfuration factors such as CBS, cystathionine γ lyase (CSE), methyl tetrahydrofolate reductase (MTHFR), phosphatidylethanolamine N-methyltransferase (PEMT), and Hcy-protein bound paraoxonase 1 (PON1) were attenuated in CBS-/+ mice. However, treatment with H2S mitigated the attenuation of the trans-sulfuration pathway. In addition, levels of mitochondrial peroxisome proliferator-activated receptor-gamma coactivator 1-α (PGC 1-α) and mitofusin-2 (MFN-2) were significantly improved by H2S intervention. Our findings suggest participation of the circadian clock in trans-sulfuration pathway that affects skeletal muscle remodeling and mitochondrial regeneration.

Disease-causing cystathionine ß-synthase linker mutations impair allosteric regulation.

Cystathionine ß-synthase (CBS) catalyzes the committing step in the transsulfuration pathway, which is important for clearing homocysteine and furnishing cysteine. The transsulfuration pathway also generates H2S, a signaling molecule. CBS is a modular protein with a heme and pyridoxal phosphate-binding catalytic core, which is separated by a linker region from the C-terminal regulatory domain that binds S-adenosylmethionine (AdoMet), an allosteric activator. Recent cryo-EM structures reveal that CBS exists in a fibrillar form and undergoes a dramatic architectural rearrangement between the basal and AdoMet-bound states. CBS is the single most common locus of mutations associated with homocystinuria, and, in this study, we have characterized three clinical variants (K384E/N and M391I), which reside in the linker region. The native fibrillar form is destabilized in the variants, and differences in their limited proteolytic fingerprints also reveal conformational alterations. The crystal structure of the truncated K384N variant, lacking the regulatory domain, reveals that the overall fold of the catalytic core is unperturbed. M391I CBS exhibits a modest (1.4-fold) decrease while the K384E/N variants exhibit a significant (∼8-fold) decrease in basal activity, which is either unresponsive to or inhibited by AdoMet. Pre-steady state kinetic analyses reveal that the K384E/N substitutions exhibit pleiotropic effects and that the differences between them are expressed in the second half reaction, that is, homocysteine binding and reaction with the aminoacrylate intermediate. Together, these studies point to an important role for the linker in stabilizing the higher-order oligomeric structure of CBS and enabling AdoMet-dependent regulation.

An Open-Label Case Series of Glutathione Use for Symptomatic Management in Children with Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a type of neurodevelopmental disorder that has been diagnosed in an increasing number of children around the world. The existing data suggest that early diagnosis and intervention can improve ASD outcomes. The causes of ASD remain complex and unclear, and there are currently no clinical biomarkers for autism spectrum disorder. There is an increasing recognition that ASD might be associated with oxidative stress through several mechanisms including abnormal metabolism (lipid peroxidation) and the toxic buildup of reactive oxygen species (ROS). Glutathione acts as an antioxidant, a free radical scavenger and a detoxifying agent. This open-label pilot study investigates the tolerability and effectiveness of oral supplementation with OpitacTM gluthathione as a treatment for patients with ASD. The various aspects of glutathione OpitacTM glutathione bioavailability were examined when administered by oral routes. The absorption of glutathione from the gastrointestinal tract has been recently investigated. The results of this case series suggest that oral glutathione supplementation may improve oxidative markers, but this does not necessarily translate to the observed clinical improvement of subjects with ASD. The study reports a good safety profile of glutathione use, with stomach upset reported in four out of six subjects. This article discusses the role of the gut microbiome and redox balance in ASD and notes that a high baseline oxidative burden may make some patients poor responders to glutathione supplementation. In conclusion, an imbalance in redox reactions is only one of the many factors contributing to ASD, and further studies are necessary to investigate other factors, such as impaired neurotransmission, immune dysregulation in the brain, and mitochondrial dysfunction.

Trypanosoma brucei proliferates normally even after losing all S-adenosylhomocysteine hydrolase genes.

S-adenosylhomocysteine (SAH) hydrolase is the enzyme responsible for breaking down SAH into adenosine and homocysteine. It has long been believed that a deficiency of this enzyme leads to SAH accumulation, subsequently inhibiting methyltransferases responsible for nucleic acids and proteins, which severely affects cell proliferation. To investigate whether targeting this enzyme could be a viable strategy to combat Trypanosoma brucei, the causative agent of human African trypanosomiasis, we created a null mutant of the SAH hydrolase gene in T. brucei using the Cre/loxP system and conducted a phenotype analysis. Surprisingly, the null mutant, where all five SAH hydrolase gene loci were deleted, exhibited normal proliferation despite the observed SAH accumulation. These findings suggest that inhibiting SAH hydrolase may not be an effective approach to suppressing T. brucei proliferation, making the enzyme a less promising target for antitrypanosome drug development.

Metabolic control analysis of the transsulfuration pathway and the compensatory role of the cysteine transport in Trypanosoma cruzi.

Trypanosoma cruzi is the causal agent of American Trypanosomiasis or Chagas Disease in humans. The current drugs for its treatment benznidazole and nifurtimox have inconveniences of toxicity and efficacy; therefore, the search for new therapies continues. Validation through genetic strategies of new drug targets against the parasite metabolism have identified numerous essential genes. Target validation can be further narrowed by applying Metabolic Control Analysis (MCA) to determine the flux control coefficients of the pathway enzymes. That coefficient is a quantitative value that represents the degree in which an enzyme/transporter determines the flux of a metabolic pathway; those with the highest coefficients can be promising drug targets. Previous studies have demonstrated that cysteine (Cys) is a key precursor for the synthesis of trypanothione, the main antioxidant metabolite in the parasite. In this research, MCA was applied in an ex vivo system to the enzymes of the reverse transsulfuration pathway (RTP) for Cys synthesis composed by cystathionine beta synthase (CBS) and cystathionine gamma lyase (CGL). The results indicated that CGL has 90% of the control of the pathway flux. Inhibition of CGL with propargylglycine (PAG) decreased the levels of Cys and trypanothione and depleted those of glutathione in epimastigotes (proliferative stage in the insect vector); these metabolite changes were prevented by supplementing with Cys, suggesting a compensatory role of the Cys transport (CysT). Indeed, Cys supplementation (but not PAG treatment) increased the activity of the CysT in epimastigotes whereas in trypomastigotes (infective stage in mammals) CysT was increased when they were incubated with PAG. Our results suggested that CGL could be a potential drug target given its high control on the RTP flux and its effects on the parasite antioxidant defense. However, the redundant Cys supply pathways in the parasite may require inhibition of the CysT as well. Our findings also suggest differential responses of the Cys supply pathways in different parasite stages.

Arginine, Transsulfuration, and Folic Acid Pathway Metabolomics in Chronic Obstructive Pulmonary Disease: A Systematic Review and Meta-Analysis.

There is an increasing interest in biomarkers of nitric oxide dysregulation and oxidative stress to guide management and identify new therapeutic targets in patients with chronic obstructive pulmonary disease (COPD). We conducted a systematic review and meta-analysis of the association between circulating metabolites within the arginine (arginine, citrulline, ornithine, asymmetric, ADMA, and symmetric, SDMA dimethylarginine), transsulfuration (methionine, homocysteine, and cysteine) and folic acid (folic acid, vitamin B6, and vitamin B12) metabolic pathways and COPD. We searched electronic databases from inception to 30 June 2023 and assessed the risk of bias and the certainty of evidence. In 21 eligible studies, compared to healthy controls, patients with stable COPD had significantly lower methionine (standardized mean difference, SMD = -0.50, 95% CI -0.95 to -0.05, p = 0.029) and folic acid (SMD = -0.37, 95% CI -0.65 to -0.09, p = 0.009), and higher homocysteine (SMD = 0.78, 95% CI 0.48 to 1.07, p < 0.001) and cysteine concentrations (SMD = 0.34, 95% CI 0.02 to 0.66, p = 0.038). Additionally, COPD was associated with significantly higher ADMA (SMD = 1.27, 95% CI 0.08 to 2.46, p = 0.037), SDMA (SMD = 3.94, 95% CI 0.79 to 7.08, p = 0.014), and ornithine concentrations (SMD = 0.67, 95% CI 0.13 to 1.22, p = 0.015). In subgroup analysis, the SMD of homocysteine was significantly associated with the biological matrix assessed and the forced expiratory volume in the first second to forced vital capacity ratio, but not with age, study location, or analytical method used. Our study suggests that the presence of significant alterations in metabolites within the arginine, transsulfuration, and folic acid pathways can be useful for assessing nitric oxide dysregulation and oxidative stress and identifying novel treatment targets in COPD. (PROSPERO registration number: CRD42023448036.).

A Study on the Amelioration of Circadian Rhythm Disorders in Fat Mice Using High-Protein Diets.

This innovative study investigates the effects of high-protein diets (milk protein) on the circadian rhythm of hepatic lipid metabolism. We aimed to understand how high-protein interventions regulate biological clock genes, maintain lipid metabolism balance, and affect the circadian rhythm of antioxidant levels in vivo. We divided 120 SPF-class C57BL/6J mice into the control, high-fat/low-protein (HF-LP), and high-fat/high-protein (HF-HP) groups. Mice were sacrificed during active (2 a.m. and 8 a.m.) and rest periods (2 p.m. and 8 p.m.). In the HF-LP group, hepatic lipid anabolic enzymes were consistently expressed at high levels, while key lipolytic enzymes slowly increased after feeding with no significant diurnal differences. This led to an abnormal elevation in blood lipid levels, a slow increase in and low levels of superoxide dismutase, and a rapid increase in malondialdehyde levels, deviating from the diurnal trend observed in the control group. However, high-protein interventions in the HF-HP group restored lipid synthase activity and the expression of key catabolic enzymes, exhibiting a precise circadian rhythm. It also improved the lipid-metabolism rhythm, which was disrupted by the high-fat diet. Overall, high-protein interventions restored the expression of key enzymes involved in lipid metabolism, improving the lipid-metabolism rhythm, which was disrupted by the high-fat diet.