One-carbon Metabolism Research Articles

Metabolic Programming of Hematopoietic Stem Cell Function By Prenatal Folate

Hematopoietic stem cells (HSCs) are programmed by metabolic changes resulting in alterations to HSC function. Here, we discovered that varying prenatal folate status metabolically programs HSCs during development and affects adult HSC self-renewal and engraftment potential into adulthood. Folate status varies greatly in the global population based on nutritional intake, common genetic polymorphisms, and widespread supplementation. Folate-derived one-carbon metabolism (OCM) regulates cellular methylation, de novo nucleotide biosynthesis and mitochondrial metabolism, processes critical to HSC function and establishment. Despite immense therapeutic potential, there is limited understanding of mechanisms driving HSC developmental programming. We hypothesize that varying prenatal folate status affects risk for hematologic dysfunction by developmentally programming HSCs. To test how prenatal folate modulated HSC function, female mice were assigned to one of three experimental diets to model population-wide folate consumption: 0mg/kg (deficient, FD), 2mg/kg (control, FC) and 8mg/kg (supplemented, FS). Profiling of fetal liver (FL) hematopoietic cells revealed that FD diet caused expansion of long term (LT)-HSCs that was propagated across the hematopoietic hierarchy, including myeloid and lymphoid progenitors and mature myeloid cells. In contrast, FS decreased FL cell output. We next determined if these changes were driven by metabolic activity. FD fetal HSPCs exhibited higher mitochondrial membrane potential and lower glycolytic activity, whereas FS fetal HSPCs displayed both higher glycolytic activity and oxygen consumption despite impaired production of downstream hematopoietic cells. Together, these data suggest that HSPC metabolic programming initiates during fetal development and is folate-dose dependent. To determine if prenatal folate programs hematopoiesis into adulthood, we quantified bone marrow (BM) hematopoietic cells in adult offspring after weaning onto standard chow. Surprisingly, FD expanded lymphoid progenitors in adult offspring with no impact on mature lymphoid cell output. In contrast, FS diet expanded myeloid and lymphoid progenitors in the BM as well as downstream mature cells, including monocytes and B-cells. To determine if these effects were HSC cell-intrinsic, we performed competitive transplantation assays of LT-HSCs isolated from adult offspring. FD offspring HSCs showed worse engraftment by chimerism across all peripheral blood (PB) populations compared to FC. In comparison, FS offspring HSCs showed enhanced total cellular output by donor cells/uL across all PB mature lineages except platelets. Secondary whole BM transplantation revealed that FS HSCs retained enhanced total cellular output and superior engraftment compared to FC, whereas FD HSCs demonstrated complete exhaustion. These data indicate that adult HSC function is programmed by prenatal folate status. To investigate molecular alterations driving metabolic programming of adult hematopoiesis by prenatal folate, we performed single-cell RNA sequencing (Scseq) and metabolomics on adult offspring HSPCs. Scseq analysis revealed most differential gene expression (DE) in HSC clusters, with less DE in progenitors. Re-clustering of all HSCs revealed distinct heterogeneity within this population, where both FD and FS HSCs were identified as distinct clusters compared to FC. Metabolomic profiling further suggested that FD HSCs exhibited signs of mitochondrial dysfunction through, 1) accumulating glucose and lactate suggesting reliance on glycolysis and, 2) accumulating succinate and glutamine suggesting complex II impairment and inadequate utilization of mitochondrial metabolism. Surprisingly, the same changes associated with mitochondrial dysfunction in FD HSCs persisted downstream into lymphoid-biased multipotent progenitors. In contrast, limited metabolic changes were observed in FS HSCs, suggesting alternative pathways for sustained programming. These changes indicate that FD and FS offspring have distinct metabolic programming. Together, our data suggest that prenatal folate status programs hematopoietic stem cell function into adulthood and is driven by alterations to metabolic activity. Ongoing studies examine how prenatal folate status affects the adult hematological and immune response to challenge.

One-Carbon Metabolites Promote Systemic Inflammation, Gut Dysbiosis, and a Myeloid Lineage Differentiation Bias

TET methylcytosine dioxygenase 2 ( TET2) loss-of-function mutations induce a pre-malignant state known as clonal hematopoiesis of indeterminate potential (CHIP). CHIP occurs in approximately 10% of people over 65 years of age and confers a 10-fold greater risk of developing hematological malignancy. Several environmental factors, including radiation, sleep deprivation, atherosclerosis, and diet, have been associated with the expansion of pre-malignant clones in CHIP patients. It has previously been shown that hematopoietic Tet2 deficiency in mice triggers a pro-inflammatory state with increased intestinal permeability, gut bacterial translocation, and accelerated clonal expansion. Gut microbes themselves can exert an influence on myeloid leukemia progression through synthesis of compounds including short-chain fatty acids (SCFAs), which promote intestinal barrier integrity. Dietary levels of one-carbon metabolites and cofactors have been found to alter gut microbial composition, affecting SCFA production and intestinal permeability, in disease-free adults. Given the connection between diet, SCFAs and gut permeability, we sought to determine the impact of dietary one-carbon metabolites on gut microbial composition and function in CHIP progression. We performed competitive bone marrow transplantation assays with Tet2+/- cells in mice supplemented with altered one-carbon metabolites: a control diet, high or low methionine diets, high or low folate diets, and high or low vitamin B12 supplementation. Mice were treated for a total of 7 months before timed sacrifice. Altered supplementation of the one carbon metabolites tested did not influence the competitiveness of Tet2-deficient cells in peripheral blood, spleen or bone marrow, however, high vitamin B12 levels promoted a myeloid differentiation bias in the spleen and bone marrow. Plasma cytokine analysis also showed that high vitamin B12 supplementation caused increased circulating inflammatory cytokines (IL-1b, IL-23, IL-27, CCL3/4, CXCL2). Based on this strong inflammatory phenotype, we performed scRNA-sequencing of splenic Tet2+/- CD11b+ cells. These data confirmed a neutrophilic cell expansion in the spleens of vitamin B12-treated mice along with increased expression of innate inflammatory genes S100a8/9 and Lyz2 as well as IL-1 b and Cxcl2 in the neutrophil clusters. Gut microbial-dependent inflammation is known to drive TET2-dependent myeloproliferation, thus we collected fecal samples before timed sacrifice and performed shotgun sequencing. Vitamin B12 supplementation decreased alpha diversity and the composition of genera that produce butyrate, an SCFA known to promote intestinal barrier integrity. Metagenomic analysis revealed increased amino acid metabolism and fatty acid biosynthesis in the gut microbiome of vitamin B12-supplemented mice, along with decreased butanoate metabolism, suggesting an alteration in microbial function that could influence SCFA content. To test whether these alterations in SCFA-producers led to increased gut permeability, we performed fluorescence in situ hybridization (FISH) against bacterial ribosomal 16S in livers from our mouse cohorts and observed increased levels of bacterial dissemination in mice with vitamin B12 supplementation. Our findings suggest that vitamin B12 supplementation in mice exacerbates the myeloid lineage differentiation bias of Tet2+/- hematopoiesis, possibly due to decreased gut barrier integrity which contributes to a heightened innate inflammatory response. This study highlights the potential for micronutrients of one-carbon metabolism to influence CHIP progression through maintenance of proper gut microbial homeostasis.

Cerebral Spinal Fluid Attenuates the Efficacy of Methotrexate Against Acute Lymphoblastic Leukemia Cells

The anti-folate methotrexate is a critical component of acute lymphoblastic leukemia (ALL) therapy. In addition to systemic administration, methotrexate is given intrathecally, or directly into the cerebral spinal fluid (CSF) via lumbar puncture, for central nervous system (CNS) leukemia treatment and prophylaxis. While IT methotrexate played a critical role in improving outcomes for ALL, CNS ALL relapse remains a major cause of treatment failure and therapy related morbidity. However, attempts to develop more efficacious and less toxic CNS ALL therapies have been largely unsuccessful and IT methotrexate has remained the standard of care for 60+ years. Based on our prior work showing that ALL cells are more poised to undergo apoptosis in nutrient poor CSF, we initially hypothesized that ALL chemosensitivity will be increased in CSF (Basile et al., 2020). While this was confirmed for several ALL drugs including cytarabine and anthracyclines, we strikingly found that methotrexate was significantly less efficacious and potent against ALL cells in CSF relative to standard tissue culture media or the more physiologically relevant human plasma-like media (HPLM). CSF also attenuated the sensitivity of ALL cells to other anti-folates including raltitrexed, trimetrexate, and pralatrexate. To define the mechanism(s) underlying this methotrexate resistance induced by CSF, we explored previously well-described mechanisms of methotrexate resistance in leukemia and other cancers. However, to date this work suggests that the molecular etiology of this CSF-induced relative methotrexate resistance is not entirely explained by diminished leukemia proliferation in CSF, altered methotrexate cellular influx/efflux, methotrexate metabolism, folate analog rescue, dysregulated expression of methotrexate target proteins, or compensation by nucleotide salvage pathways. To complement our work examining known mechanisms of methotrexate resistance, we also used a more discovery-based approach and compared the gene expression patterns of leukemia cells in CSF versus tissue culture media. Preliminary bioinformatic analyses identified dysregulation of multiple genes involved in the integrated stress response (ISR) and unfolded protein response (UPR) pathways in leukemia cells in CSF. The ISR/UPR are mechanisms by which cells adapt to diverse stress stimuli in the microenvironment, including potentially nutrient poor CSF which is low in glucose, proteins, and lipids relative to plasma. The hallmark event in this pathway is the phosphorylation of the translation initiation factor eIF2α, which inhibits global protein synthesis and enables the translation of selected genes that together enable cell survival. Supporting our gene expression data, ALL cells in CSF showed increased phosphorylation of eIF2α and a decrease in global protein synthesis. Intriguingly, it has been shown that activation of the UPR can divert metabolites from glycolysis to mitochondrial one-carbon metabolism with resulting resistance to anti-folates such as methotrexate (Reich et al., 2020). Accordingly, we next tested the effect of UPR/ISR activation on leukemia cell sensitivity to methotrexate in tissue culture media. Activation of the UPR with thapsigargin, a sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) inhibitor that triggers ER stress, increased methotrexate resistance in leukemia cells. Similarly, BtdCPU a small-molecule activator of heme regulated inhibitor (HRI) kinase that phosphorylates eIF2α and activates the ISR, also enhanced leukemia resistance to methotrexate. Building upon these results, current investigations are focused on testing the role of UPR/IST activation in methotrexate resistance in leukemia cells in CSF and modulating this pathway to overcome resistance. While methotrexate is clearly an efficacious and critical component of CNS leukemia treatment and prophylaxis, we have shown that CSF attenuates the overall efficacy of methotrexate in targeting leukemia cells. Moreover, we anticipate that our current work defining the mechanisms driving this resistance may identify novel approaches for maximizing methotrexate efficacy and more completely eradicating leukemia cells in CSF and the CNS. Finally, this work highlights the importance of critically evaluating even long-established standards of care.

Vitamin B12 is a limiting factor for induced cellular plasticity and tissue repair

Transient reprogramming by the expression of OCT4, SOX2, KLF4 and MYC (OSKM) is a therapeutic strategy for tissue regeneration and rejuvenation, but little is known about its metabolic requirements. Here we show that OSKM reprogramming in mice causes a global depletion of vitamin B12 and molecular hallmarks of methionine starvation. Supplementation with vitamin B12 increases the efficiency of reprogramming both in mice and in cultured cells, the latter indicating a cell-intrinsic effect. We show that the epigenetic mark H3K36me3, which prevents illegitimate initiation of transcription outside promoters (cryptic transcription), is sensitive to vitamin B12 levels, providing evidence for a link between B12 levels, H3K36 methylation, transcriptional fidelity and efficient reprogramming. Vitamin B12 supplementation also accelerates tissue repair in a model of ulcerative colitis. We conclude that vitamin B12, through its key role in one-carbon metabolism and epigenetic dynamics, improves the efficiency of in vivo reprogramming and tissue repair.

Nobiletin inhibits hypoxia-induced placental redox imbalance and subsequent offspring brain damage by regulating the one-carbon metabolism

Nobiletin (NOB) is one of the major polymethoxyflavone in citrus peels, which has antioxidant activities. The purpose of this study was to investigate the effects of NOB on hypoxia-induced placental oxidative stress and its mechanism. In vitro experiments, JEG-3 hypoxic model was constructed, and treated with 100 μM NOB. In vivo experiment, pregnant rats were injected intraperitoneally with 50 mg/kg body weight NOB daily, and the placental hypoxia model was established by RUPP. NOB hampered the reducing of reducing power in hypoxic cells, promoted NADPH to provide hydrogen donors to regenerate antioxidants GPX and GSH, thereby promoting the decomposition of ROS. NOB alleviated the adverse reaction of hypoxic placental one-carbon metabolism disorder by regulating the levels of homocysteine, DNMTs and TETs. Moreover, NOB suppressed offspring brain damage induced by RUPP. Taken together, NOB attenuated hypoxia-induced placental redox perturbation and subsequent offspring brain injury by modulating one-carbon metabolism.

72 Effects of Differing Planes of Nutrition and one-Carbon Metabolite Supplementation of Beef Heifers on B12, Folate, and Folate Intermediate Concentrations in the dam and Fetus

Abstract Vitamin B12 and folate are involved in the metabolic process known as one-carbon metabolism, which among other things, has a role in DNA, RNA, and protein methylation. This methylation connects one-carbon metabolism to epigenetics, which influences fetal development. Thus, an adequate supply of one-carbon metabolites (OCM) is important during fetal development. We hypothesized that differing planes of nutrition, along with supplementation of OCM, would impact concentrations of B12, folate, and folate intermediates in maternal liver, maternal serum, fetal liver, allantoic fluid (ALF), and amniotic fluid (AMF). Specifically, OCM supplementation would increase circulating and hepatic B12 and folate concentrations compared with non-supplemented heifers. Heifers (n = 72) were individually fed on one of two planes of nutrition: control (CON; 0.45 kg/d gain) or restricted (RES; -0.23 kg/d gain). To complete the 2 × 2 factorial arrangement, heifers were also given either OCM supplementation (10 g/d rumen-protected methionine + 60 g/d rumen-protected choline in the diet plus 20 mg B12 + 320 mg folic acid; weekly intramuscular injections) or no supplementation (grain carrier + saline injection). Thus, the final treatments were CON-OCM, CON+OCM, RES-OCM, and RES+OCM. Heifers were bred to a single sire via artificial insemination with sexed semen. Heifers pregnant with female calves (n = 31) were selected for slaughter on d 63. Samples of maternal liver, ALF, AMF, and fetal liver were collected at slaughter. Maternal serum was collected at breeding (d 0) and at slaughter. For B12 and folate concentration analysis, maternal serum was sent to IDEXX BioAnalytics (N. Grafton, MA). For analysis of folate intermediate concentrations in maternal and fetal liver, ALF, and AMF, samples were analyzed using LC-MS/MS. There were treatment × day interactions (P ≤ 0.05) for B12 and folate in maternal serum; on d 63, R+OCM was greater compared with C-OCM and R-OCM and all treatments at d 0 (Table 1). For folate intermediate concentrations, no plane of nutrition × supplement interactions (P ≥ 0.06) were found in fetal liver, ALF, or AMF; however, 5-methyl THF in ALF tended (P = 0.06) to be greater in R+OCM compared with all other treatments (Table 2). No interactions were found for concentrations in maternal liver when corrected for total liver weight; however, there was a supplement effect (P ≤ 0.05) where the concentrations of folic acid, dihydrofolate, tetrahydrofolate, 5-methyltetrahydrofolate, 5,10-methylenetetrahydrofolate and 5,10-methenyltetrahydrofolate were increased in +OCM compared with -OCM, and a plane of nutrition effect (P ≤ 0.05) with increased 5,10-methenyltetrahydrofolate and 5,10-methylenetetrahydrofolate concentrations in RES. We conclude that supplementing OCM increases concentrations of B12, folate and its intermediates in maternal liver and serum. USDA is an equal opportunity provider and employer.

Time-resolved concentrations of serum amino acids, one-carbon metabolites and B-vitamin biomarkers during the postprandial and fasting state: the Postprandial Metabolism in Healthy Young Adults (PoMet) Study.

Metabolomics has been utilised in epidemiological studies to investigate biomarkers of nutritional status and metabolism in relation to non-communicable diseases. However, little is known about the effect of prandial status on several biomarker concentrations. Therefore, the aim of this intervention study was to investigate the effect of a standardised breakfast meal followed by food abstinence for 24 h on serum concentrations of amino acids, one-carbon metabolites and B-vitamin biomarkers. Thirty-four healthy subjects (eighteen males and sixteen females) aged 20-30 years were served a breakfast meal (∼500 kcal) after which they consumed only water for 24 h. Blood samples were drawn before and at thirteen standardised timepoints after the meal. Circulating concentrations of most amino acids and metabolites linked to one-carbon metabolism peaked within the first 3 h after the meal. The branched-chain amino acids steadily increased from 6 or 8 hours after the meal, while proline decreased in the same period. Homocysteine and cysteine concentrations immediately decreased after the meal but steadily increased from 3 and 4 hours until 24 h. FMN and riboflavin fluctuated immediately after the meal but increased from 6 h, while folate increased immediately after the meal and remained elevated during the 24 h. Our findings indicate that accurate reporting of time since last meal is crucial when investigating concentrations of certain amino acids and one-carbon metabolites. Our results suggest a need for caution when interpretating studies, which utilise such biomarkers, but do not strictly control for time since the last meal.

Optic disc edema during strict 6° head-down tilt bed rest is related to one-carbon metabolism pathway genetics and optic cup volume.

Some astronauts on International Space Station missions experience neuroophthalmological pathologies as part of spaceflight associated neuro-ocular syndrome (SANS). Strict head-down tilt bed rest (HDTBR) is a spaceflight analog that replicates SANS findings and those who had 3-4 risk alleles (G and C alleles from the methionine synthase reductase [MTRR] A66G and serine hydroxymethyltransferase [SHMT1] C1420T, respectively) as compared to 1-2 risk alleles, had a greater increase in total retinal thickness (TRT). The objective of this study was to identify factors that contribute to the individual variability of the development of SANS in a 60d HDTBR at the German Aerospace Center's:envihab facility, Cologne Germany. 22 of 24 subjects who participated in the HDTBR study provided blood samples for genetic analysis. Total retinal thickness and optic cup volume were measured before and after bed rest. Subjects with 3-4 versus 0-2 risk alleles had greater ΔTRT during and after bed rest, and the model improved with the addition of baseline optic cup volume. This bed rest study confirms that variants of MTRR and SHMT1 are associated with ocular pathologies. Subjects with more risk alleles had the greatest HDTBR-induced ΔTRT, reaffirming that genetics predispose some individuals to developing SANS. Preflight optic cup volume and genetics better predict ΔTRT than either one alone. Whether nutritional supplements can override the genetic influences on biochemistry, physiology, and pathophysiology remains to be tested. These findings have significant implications for both aerospace and terrestrial medicine.

The Heterogeneous Impact of Prediagnostic Folate Intake for Fluorouracil-Containing Induction Chemotherapy for Head and Neck Cancer.

Fluorouracil (FU) exerts its antitumor activity by inhibiting folate-mediated one-carbon metabolism. Evidence that folate may play a role in the carcinogenic process via folate-mediated one-carbon metabolism has given rise to the hypothesis that pre-diagnostic folate intake may induce heterogeneous chemosensitivity to FU-containing induction chemotherapy (IC) in head and neck cancer. To assess this hypothesis, we conducted a cohort study to investigate whether the association between prediagnostic dietary folate intake and cancer survival differed between treatment regimens with and without FU-containing IC in 504 cases of locally advanced (stage III/IV) HNSCC, using an epidemiologic database combined with clinical data. In total, 240 patients were treated with FU-containing IC followed by definitive treatment, and 264 patients were treated with definitive treatment alone. Definitive treatment is defined as (1) the surgical excision of a tumor with clear margins, with or without neck lymph node dissection; or (2) radiotherapy with or without chemotherapy. In the overall cohort of the FU-containing IC group, a higher folate intake was significantly associated with better overall survival (adjusted hazard ratios (HRs) for the highest compared to the lowest folate tertiles (HRT3-T1) = 0.42, 95%CI, 0.25-0.76, Ptrend = 0.003). Conversely, no apparent association between prediagnostic folate intake and survival was observed with definitive treatment alone (HRT3-T1: 0.83, 95%CI, 0.49-1.42, Ptrend = 0.491)). A consideration of the cumulative dose of FU-containing IC showed that the survival impact of prediagnostic folate intake differed statistically significantly by treatment regimen (Pinteraction = 0.012). In conclusion, an association between prediagnostic folate intake and HNSCC survival significantly differed by FU-containing IC. This finding indicates that in the carcinogenic process, folate status causes HNSCC to be heterogenous in terms of one-carbon metabolism.

Quantitative profiling and diagnostic potential of one-carbon and central metabolism pools in MODY2 and T1DM

BackgroundMaturity-onset diabetes of the young type 2 (MODY2) is a rare genetic disorder characterized as mild fasting hyperglycemia with low risk of vascular complications caused by glucokinase gene mutation. This study aims to investigate metabolites alteration associated with MODY2, exploring possible mechanism underlying characteristic clinical manifestations and low cardiovascular risks of MODY2 and providing serum metabolite biomarkers to facilitating MODY2 diagnosis.MethodsFasting serum samples from MODY2, type 1 diabetes (T1DM) and healthy individuals were collected. By using targeted metabolomics via liquid chromatography–tandem mass spectrometry platform, we quantified the metabolites involved in tricarboxylic acid (TCA) cycle and one-carbon metabolism.ResultsMetabolomic profiling revealed significant difference of intermediates from central metabolism cycle, methionine cycle and several amino acids between MODY2 and T1DM groups. Among these, serum citrate, α-ketoglutaric acid, serine, glycine, glutamine and homocysteine were significantly elevated in MODY2 patients compared with T1DM patients; and compared with healthy subjects, malate and methionine levels were significantly increased in the two groups of diabetic patients. The correlation analysis with clinical indexes showed that α- ketoglutarate, serine, glycine, and glutamine were negatively correlated with blood glucose indicators including fasting blood glucose, HbA1c, and GA, while citrate was positively correlated with C-peptide. And homocysteine displayed positive correlation with HDL and negative with C-reactive protein, which shed light on the mechanism of mild symptoms and low risk of cardiovascular complications in MODY2 patients. A panel of 4 metabolites differentiated MODY2 from T1DM with AUC of 0.924, and a combination of clinical indices and metabolite also gained good diagnostic value with AUC 0.948.ConclusionIn this research, we characterized the metabolite profiles of TCA cycle and one-carbon metabolism in MODY2 and T1DM and identified promising diagnostic biomarkers for MODY2. This study may provide novel insights into the pathogenesis and clinical manifestations of MODY2.

Biological Pathways Associated with Vitamins in Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is characterized by early-appearing social communication deficits, with genetic and environmental factors potentially playing a role in its etiology, which remains largely unknown. During pregnancy, certain deficiencies in critical nutrients are mainly associated with central nervous system impairment. The vitamin B9 (folate) is primarily related to one-carbon and methionine metabolism, participating in methyl donor generation. In addition, supplementation with folic acid (FA) is recommended by the World Health Organization (WHO) in the first three gestational months to prevent neural tube defects. Vitamin B12 is related to folate regeneration, converting it into an active form. Deficiencies in this vitamin have a negative impact on cognitive function and brain development since it is involved in myelin synthesis. Vitamin D is intimately associated with Ca2+ levels, acting in bone development and calcium-dependent signaling. This vitamin is associated with ASD at several levels since it has a relation with ASD genes and oxidative stress environment. This review carries the recent literature about the role of folate, vitamin B12, and vitamin D in ASD. In addition, we discuss the possible impact of nutrient deficiency or hypersupplementation during fetal development. On the other hand, we explore the biases of vitamin supplementation studies such as the loss of participants in retrospective studies, as well as multiple variants that are not considered in the conclusion, like dietary intake or auto-medication during pregnancy. In this regard, we aim to contribute to the discussion about the role of vitamins in ASD currency, but also in pregnancy and fetal development as well. Furthermore, stress during pregnancy can be an ASD predisposition, with cortisol as a regulator. In this view, we propose that cortisol is the bridge of susceptibility between vitamin disorders and ASD prevalence.

Anticancer Activity of Alangium Salvifolium on Human Breast Cancer Cell Lines MCF-7, MDA-MB-468

Alangium salvifolium is a plant that grows in the alangium family. The study's plant species were chosen.The present study was implemented for revealing the anticancer effect of the leaves of methanolic extract of Alangium salvifolium. Phytochemical Screening of A.Salvifolium leaf extract was done which showed the presence of Alkaloids, Flavonoids,Terpenoids. Human Breast Cancer cell lines MCF-7, MDA-MB-468 cells were grown in RPMI media and the 5-10- methylenetetrahydrofolate reductase (MTHFR) Enzyme inhibitory activity was investigated. MTHFR is a one-carbon metabolism enzyme that redirects the pool of folate from DNA synthesis and repair to methylation. The enzyme is vital for cellular homeostasis due to its key functions in the one carbon cycle,which include methionine and protein,DNA and RNA synthesis. Cancer cell lines MCF-7 and MDA-MB-468 treated with Alangium Salvifolium compounds AS1- Deoxytubulosine and AS2 - carboline Hermaline.

Structure of full-length cobalamin-dependent methionine synthase and cofactor loading captured in crystallo

Cobalamin-dependent methionine synthase (MS) is a key enzyme in methionine and folate one-carbon metabolism. MS is a large multi-domain protein capable of binding and activating three substrates: homocysteine, folate, and S-adenosylmethionine for methylation. Achieving three chemically distinct methylations necessitates significant domain rearrangements to facilitate substrate access to the cobalamin cofactor at the right time. The distinct conformations required for each reaction have eluded structural characterization as its inherently dynamic nature renders structural studies difficult. Here, we use a thermophilic MS homolog (tMS) as a functional MS model. Its exceptional stability enabled characterization of MS in the absence of cobalamin, marking the only studies of a cobalamin-binding protein in its apoenzyme state. More importantly, we report the high-resolution full-length MS structure, ending a multi-decade quest. We also capture cobalamin loading in crystallo, providing structural insights into holoenzyme formation. Our work paves the way for unraveling how MS orchestrates large-scale domain rearrangements crucial for achieving challenging chemistries.

A transgenic mice model of retinopathy of cblG-type inherited disorder of one-carbon metabolism highlights epigenome-wide alterations related to cone photoreceptor cells development and retinal metabolism

BackgroundMTR gene encodes the cytoplasmic enzyme methionine synthase, which plays a pivotal role in the methionine cycle of one-carbon metabolism. This cycle holds a significant importance in generating S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), the respective universal methyl donor and end-product of epigenetic transmethylation reactions. cblG type of inherited disorders of vitamin B12 metabolism due to mutations in MTR gene exhibits a wide spectrum of symptoms, including a retinopathy unresponsive to conventional therapies.MethodsTo unveil the underlying epigenetic pathological mechanisms, we conducted a comprehensive study of epigenomic-wide alterations of DNA methylation by NGS of bisulfited retinal DNA in an original murine model with conditional Mtr deletion in retinal tissue. Our focus was on postnatal day 21, a critical developmental juncture for ocular structure refinement and functional maturation.ResultsWe observed delayed eye opening and impaired visual acuity and alterations in the one-carbon metabolomic profile, with a notable dramatic decline in SAM/SAH ratio predicted to impair DNA methylation. This metabolic disruption led to epigenome-wide changes in genes involved in eye development, synaptic plasticity, and retinoid metabolism, including promoter hypermethylation of Rarα, a regulator of Lrat expression. Consistently, we observed a decline in cone photoreceptor cells and reduced expression of Lrat, Rpe65, and Rdh5, three pivotal genes of eye retinoid metabolism.ConclusionWe introduced an original in vivo model for studying cblG retinopathy, which highlighted the pivotal role of altered DNA methylation in eye development, cone differentiation, and retinoid metabolism. This model can be used for preclinical studies of novel therapeutic targets.

SFXN3 is Associated with Poor Clinical Outcomes and Sensitivity to the Hypomethylating Therapy in Non-M3 Acute Myeloid Leukemia Patients.

DNA hypermethylation plays a critical role in the occurrence and progression of acute myeloid leukemia (AML). The mitochondrial serine transporter, SFXN3, is vital for onecarbon metabolism and DNA methylation. However, the impact of SFXN3 on the occurrence and progression of AML has not been reported yet. In this study, we hypothesized that SFXN3 indicates a poor prognosis and suggested tailored treatment for AML patients. We used GEPIA and TCGA repository data to analyze the expression of SFXN3 and its correlation with survival in AML patients. RT-qPCR was used to detect the SFXN3 level in our enrolled AML patients and volunteers. Additionally, Whole Genome Bisulfite Sequencing (WGBS) was used to detect the genomic methylation level in individuals. Through the TCGA and GEPIA databases, we found that SFXN3 was enriched in AML patients, predicting shorter survival. Furthermore, we confirmed that SFXN3 was primarily overexpressed in AML patients, especially non-M3 patients, and that high SFXN3 in non-M3 AML patients was found to be associated with poor outcomes and frequent blast cells. Interestingly, non-M3 AML patients with high SFXN3 levels who received hypomethylating therapy showed a higher CR ratio. Finally, we found that SFXN3 could promote DNA methylation at transcription start sites (TSS) in non-M3 AML patients. These sites were found to be clustered in multiple vital cell functions and frequently accompanied by mutations in DNMT3A and NPM1. In conclusion, SXFN3 plays an important role in the progression and hypermethylation in non-M3 AML patients and could be a potential biomarker for indicating a high CR rate for hypomethylating therapy.

Longitudinal characterization of the metabolome of dairy cows transitioning from one lactation to the next: Investigations in blood serum

The objective of this study was to characterize changes in the serum metabolome and various indicators of oxidative balance in dairy cows starting 2 wk before dry-off and continuing until wk 16 of lactation. Twelve Holstein dairy cows (body weight 745 ± 71 kg, body condition score 3.43 ± 0.66; mean ± SD) were housed in a tiestall barn from 10 wk before to 16 wk after parturition. Cows were dried off 6 wk before the expected calving date (mean dry period length = 42 d). From 8 wk before calving to 16 wk after calving, blood samples were taken weekly to study redox metabolism by determining antioxidant capacity, measured as the ferric-reducing ability of plasma, reactive oxidative metabolites, oxidative stress index, oxidative damage of lipids, measured as thiobarbituric acid reactive substances, and glutathione peroxidase activity. According to these results, dairy cows had the lowest serum antioxidant capacity and greater levels of oxidative stress during the dry-off period and the early postpartum period. For metabolomics, a subset of serum samples including wk -7 (before dry-off), -5 (after dry-off), -1, 1, 5, 10, and 15 relative to calving were used. A targeted metabolomics approach was performed using liquid chromatography and flow injection with electrospray ionization triple quadrupole mass spectrometry using the MxP Quant 500 kit (Biocrates Life Sciences AG). A total of 240 metabolites in serum were used in the final data analysis. Principal component analysis revealed a clear separation by days of sampling, indicating a remarkable shift in metabolic phenotype between the dry period and late and early lactation. Changes in many non-lipid metabolites associated with one-carbon metabolism, the tricarboxylic acid cycle, the urea cycle, and AA catabolism were observed in the study, with changes in AA serum concentrations likely related to factors such as energy and nitrogen balance, digestive efficiency, and changing diets. The study confirmed an extensive remodeling of the serum lipidome in peripartum dairy cows, highlighting the importance of changes in acylcarnitine (acylCN), phosphatidylcholines (PC), and triacylglycerols (TG), as they play a crucial role in lipid metabolism. Results showed that short-chain acylCN increased after dry-off and decreased thereafter, whereas lipid-derived acylCN increased around parturition, suggesting that more fatty acids could enter mitochondria. Phospholipids and sphingolipids in serum showed changes during lactation. In particular, concentrations of sphingomyelins, PC, and lysoPC decreased around calving but increased in mid- and late lactation. In contrast, concentrations of TG remained consistently low after parturition. The serum concentrations of bile acids fluctuated during the dry period and lactation, with glycocholic acid, cholic acid, glycodeoxycholic acid, and taurocholic acid showing the greatest concentrations. These changes are likely due to the interplay of diet, liver function, and the ability of the gut microbiota to convert primary to secondary bile acids. Overall, these descriptive results may aid in hypothesis generation and in the design and interpretation of future metabolite-based studies in dairy cows. Furthermore, they contribute to our understanding of the physiological ranges in serum metabolites relative to the lactation cycle of the dairy cow.

Optimal energy and redox metabolism in the cyanobacterium Synechocystis sp. PCC 6803

Understanding energy and redox homeostasis and carbon partitioning is crucial for systems metabolic engineering of cell factories. Carbon metabolism alone cannot achieve maximal accumulation of metabolites in production hosts, since an efficient production of target molecules requires energy and redox balance, in addition to carbon flow. The interplay between cofactor regeneration and heterologous production in photosynthetic microorganisms is not fully explored. To investigate the optimality of energy and redox metabolism, while overproducing alkenes—isobutene, isoprene, ethylene and 1-undecene, in the cyanobacterium Synechocystis sp. PCC 6803, we applied stoichiometric metabolic modelling. Our network-wide analysis indicates that the rate of NAD(P)H regeneration, rather than of ATP, controls ATP/NADPH ratio, and thereby bioproduction. The simulation also implies that energy and redox balance is interconnected with carbon and nitrogen metabolism. Furthermore, we show that an auxiliary pathway, composed of serine, one-carbon and glycine metabolism, supports cellular redox homeostasis and ATP cycling. The study revealed non-intuitive metabolic pathways required to enhance alkene production, which are mainly driven by a few key reactions carrying a high flux. We envision that the presented comparative in-silico metabolic analysis will guide the rational design of Synechocystis as a photobiological production platform of target chemicals.

Exploring the Interplay Between Vitamin B12-related Biomarkers, DNA Methylation, and Gene-Nutrition Interaction in Esophageal Precancerous Lesions

Vitamin B12 depletion has been suggested to be associated with esophageal precancerous lesions (EPL). However, the potential mechanisms remain unclear. This study aims to evaluate the role of vitamin B12 and its regulated epigenetic modification in EPL and provide preliminary information on the identification of potential molecular biomarkers for the early prediction of EPL. We collected information and samples from the Early Diagnosis and Early Treatment Project of Esophageal Cancer database from 200 EPL cases and 200 matched controls. Vitamin B12, one-carbon metabolism biomarkers, genetic polymorphism of TCN2 C776G, and DNA methylation were compared. Preliminarily identified candidate promoters of differentially methylated CpG positions were further verified by targeted bisulfite sequencing. EPL cases had significantly lower serum levels of vitamin B12 and transcobalamin II, and higher serum levels of homocysteine and 5-methyltetrahydrofolate than controls. The TCN2 C776G polymorphism was found to be associated with susceptibility to EPL and may interact with vitamin B12 nutritional status to influence the risk of EPL in male subjects. In addition, global hypomethylation related to vitamin B12 depletion was observed in EPL cases, along with region-specific hypermethylation of UGT2B15 and FGFR2 promoters. This study suggests that vitamin B12 depletion may be associated with aberrant DNA methylation and increased risk of EPL through the one-carbon metabolism pathway, presents that the TCN2 C776G polymorphism may interact with vitamin B12 nutritional status to affect EPL risk in males, and also identifies specific locations in the UGT2B15 and FGFR2 promoters with potential as promising molecular biomarkers.

Association between polymorphisms of one-carbon metabolism genes and oral cancer risk

Association between polymorphisms of one-carbon metabolism genes and oral cancer risk

Genome-wide promoter responses to CRISPR perturbations of regulators reveal regulatory networks in Escherichia coli

Elucidating genome-scale regulatory networks requires a comprehensive collection of gene expression profiles, yet measuring gene expression responses for every transcription factor (TF)-gene pair in living prokaryotic cells remains challenging. Here, we develop pooled promoter responses to TF perturbation sequencing (PPTP-seq) via CRISPR interference to address this challenge. Using PPTP-seq, we systematically measure the activity of 1372 Escherichia coli promoters under single knockdown of 183 TF genes, illustrating more than 200,000 possible TF-gene responses in one experiment. We perform PPTP-seq for E. coli growing in three different media. The PPTP-seq data reveal robust steady-state promoter activities under most single TF knockdown conditions. PPTP-seq also enables identifications of, to the best of our knowledge, previously unknown TF autoregulatory responses and complex transcriptional control on one-carbon metabolism. We further find context-dependent promoter regulation by multiple TFs whose relative binding strengths determined promoter activities. Additionally, PPTP-seq reveals different promoter responses in different growth media, suggesting condition-specific gene regulation. Overall, PPTP-seq provides a powerful method to examine genome-wide transcriptional regulatory networks and can be potentially expanded to reveal gene expression responses to other genetic elements.