Common Variants in One-Carbon Metabolism Genes (MTHFR, MTR, MTHFD1) and Depression in Gynecologic Cancers.

We investigated the association between methylenetetrahydrofolate reductase (gene MTHFR 677C>T, rs1801133), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR 2756A>G, rs1805087), and methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1 (gene MTHFD1 1958G>A, rs2236225)-well-studied functional variants involved in one-carbon metabolism-and gynecologic cancer risk, and the interaction between these polymorphisms and depression. A total of 200 gynecologic cancer cases and 240 healthy controls were recruited to participate in this study. Three single-nucleotide variants (SNVs) (rs1801133, rs1805087, rs2236225) were genotyped using the PCR-restriction fragment length polymorphism method. Depression was assessed in all patients using the Hamilton Depression Scale. Depression was statistically significantly more frequent in women with gynecologic cancers (69.5% vs. 34.2% in controls, p < 0.001). MTHFD1 rs2236225 was associated with an increased risk of gynecologic cancers (in dominant OR = 1.53, p = 0.033, and in log-additive models OR = 1.37, p = 0.024). Moreover, an association was found between depression risk and MTHFR rs1801133 genotypes in the controls but not in women with gynecologic cancers (in codominant model CC vs. TT: OR = 3.39, 95%: 1.49-7.74, p = 0.011). Cancers of the female reproductive system are associated with the occurrence of depression, and ovarian cancer may be associated with the rs2236225 variant of the MTHFD1 gene. In addition, in healthy aging women in the Polish population, the rs1801133 variant of the MTHFR gene is associated with depression.

Assaying plant formate-tetrahydrofolate ligase with monoglutamylated and polyglutamylated substrates using a fluorescence-HPLC based method.

Formate-tetrahydrofolate ligase catalyzes reversible, ATP-dependent conversion of tetrahydrofolate and formate to 10-formyltetrahydrofolate, simultaneously releasing ADP and inorganic phosphate. This enzyme has traditionally been assayed in the direction of 10-CHO-tetrahydrofolate formation by lowering pH of the reaction post-incubation, thus converting the product of the reaction to 5,10-methenyltetrahydrofolate, which is then quantified spectrophotometrically. To increase sensitivity of the product detection, which is particularly useful when determining the kinetic parameters of the enzyme with polyglutamylated substrates, we have replaced the spectrophotometric detection with HPLC separation and fluorescence detection. In addition to the modified enzyme assay protocol, we are also providing protocols for producing recombinant formate-tetrahydrofolate ligase from Arabidopsis in Escherichia coli cells, producing crude Arabidopsis leaf and root extracts suitable for assaying this enzyme, and for synthesis of polyglutamylated tetrahydrofolate substrates.

Antiviral strategies: What can we learn from natural reservoirs?

Viruses cause many severe diseases in both plants and animals, urging us to explore new antiviral strategies. In their natural reservoirs, viruses live and replicate while causing mild or no symptoms. Some animals, such as bats, are the predicted natural reservoir of multiple viruses, indicating that they possess broad-spectrum antiviral capabilities. Mechanisms of host defenses against viruses are generally studied independently in plants and animals. In this article, we speculate that some antiviral strategies of natural reservoirs are conserved between kingdoms. To verify this hypothesis, we created null mutants of 10-formyltetrahydrofolate synthetase (AtTHFS), an Arabidopsis thaliana homologue of methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1 (MTHFD1), which encodes a positive regulator of viral replication in bats. We found that disruption of AtTHFS enhanced plant resistance to three different types of plant viruses, including the tomato spotted wilt virus (TSWV), the cucumber mosaic virus (CMV) and the beet severe curly top virus (BSCTV). These results demonstrate a novel antiviral strategy for plant breeding. We further discuss the approaches used to identify and study natural reservoirs of plant viruses, especially those hosting many viruses, and highlight the possibility of discovering new antiviral strategies from them for plant molecular breeding and antiviral therapy.

Performance of Paracoccus pantotrophus MA3 in heterotrophic nitrification-anaerobic denitrification using formic acid as a carbon source.

Excess amount of nitrogen in wastewater has caused serious concerns, such as water eutrophication. Paracoccus pantotrophus MA3, a novel isolated strain of heterotrophic nitrification-anaerobic denitrification bacteria, was evaluated for nitrogen removal using formic acid as the sole carbon source. The results showed that the maximum ammonium removal efficiency was observed under the optimum conditions of 26.25 carbon to nitrogen ratio, 3.39% (v/v) inoculation amount, 34.64 °C temperature, and at 180 rpm shaking speed, respectively. In addition, quantitative real-time PCR technique analysis assured that the gene expression level of formate dehydrogenase, formate tetrahydrofolate ligase, 5,10-methylenetetrahydrofolate dehydrogenase, serine hydroxymethyltransferase, respiratory nitrate reductase beta subunit, L-glutamine synthetase, glutamate dehydrogenase, and glutamate synthase were up-regulated compared to the control group, and combined with nitrogen mass balance analysis to conclude that most of the ammonium was removed by assimilation. A small amount of nitrate and nearly no nitrite were accumulated during heterotrophic nitrification. MA3 exhibited significant denitrification potential under anaerobic conditions with a maximum nitrate removal rate of 4.39 mg/L/h, and the only gas produced was N2. Additionally, 11.50 ± 0.06 mg/L/h of NH4+-N removal rate from biogas slurry was achieved.

Arginine methylation of MTHFD1 by PRMT5 enhances anoikis resistance and cancer metastasis.

Metastasis accounts for the major cause of cancer-related mortality. How disseminated tumor cells survive under suspension conditions and avoid anoikis is largely unknown. Here, using a metabolic enzyme-centered CRISPR-Cas9 genetic screen, we identified methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1 (MTHFD1) as a novel suppressor of anoikis. MTHFD1 depletion obviously restrained the capacity of cellular antioxidant defense and inhibited tumor distant metastasis. Mechanistically, MTHFD1 was found to bind the protein arginine methyltransferase 5 (PRMT5) and then undergo symmetric dimethylation on R173 by PRMT5. Under suspension conditions, the interaction between MTHFD1 and PRMT5 was strengthened, which increased the symmetric dimethylation of MTHFD1. The elevated methylation of MTHFD1 largely augmented its metabolic activity to generate NADPH, therefore leading to anoikis resistance and distant organ metastasis. Therapeutically, genetic depletion or pharmacological inhibition of PRMT5 declined tumor distant metastasis. And R173 symmetric dimethylation status was associated with metastasis and prognosis of ESCC patients. In conclusion, our study uncovered a novel regulatory role and therapeutic implications of PRMT5/MTHFD1 axis in facilitating anoikis resistance and cancer metastasis.

Do methylenetetrahydrofolate dehydrogenase, cyclohydrolase, and formyltetrahydrofolate synthetase 1 polymorphisms modify changes in intelligence of school-age children in areas of endemic fluorosis?

BACKGROUND: Excessive exposure to fluoride can reduce intelligence. Methylenetetrahydrofolate dehydrogenase, cyclohydrolase, and formyltetrahydrofolate synthetase 1 ( MTHFD1 ) polymorphisms have important roles in neurodevelopment. However, the association of MTHFD1 polymorphisms with children's intelligence changes in endemic fluorosis areas has been rarely explored. METHODS: A cross-sectional study was conducted in four randomly selected primary schools in Tongxu County, Henan Province, from April to May in 2017. A total of 694 children aged 8 to 12 years were included in the study with the recruitment by the cluster sampling method. Urinary fluoride (UF) and urinary creatinine were separately determined using the fluoride ion-selective electrode and creatinine assay kit. Children were classified as the high fluoride group and control group according to the median of urinary creatinine-adjusted urinary fluoride (UF Cr ) level. Four loci of MTHFD1 were genotyped, and the Combined Raven's Test was used to evaluate children's intelligence quotient (IQ). Generalized linear model and multinomial logistic regression model were performed to analyze the associations between children's UF Cr level, MTHFD1 polymorphisms, and intelligence. The general linear model was used to explore the effects of gene-environment and gene-gene interaction on intelligence. RESULTS: In the high fluoride group, children's IQ scores decreased by 2.502 when the UF Cr level increased by 1.0 mg/L (ß = -2.502, 95% confidence interval [CI]:-4.411, -0.593), and the possibility for having "excellent" intelligence decreased by 46.3% (odds ratio = 0.537, 95% CI: 0.290, 0.994). Children with the GG genotype showed increased IQ scores than those with the AA genotype of rs11627387 locus in the high fluoride group ( P   <  0.05). Interactions between fluoride exposure and MTHFD1 polymorphisms on intelligence were observed (Pinteraction < 0.05). CONCLUSION: Our findings suggest that excessive fluoride exposure may have adverse effects on children's intelligence, and changes in children's intelligence may be associated with the interaction between fluoride and MTHFD1 polymorphisms.

AcetoBase Version 2: a database update and re-analysis of formyltetrahydrofolate synthetase amplicon sequencing data from anaerobic digesters.

AcetoBase is a public repository and database of formyltetrahydrofolate synthetase (FTHFS) sequences. It is the first systematic collection of bacterial FTHFS nucleotide and protein sequences from genomes and metagenome-assembled genomes and of sequences generated by clone library sequencing. At its publication in 2019, AcetoBase (Version 1) was also the first database to establish connections between the FTHFS gene, the Wood-Ljungdahl pathway and 16S ribosomal RNA genes. Since the publication of AcetoBase, there have been significant improvements in the taxonomy of many bacterial lineages and accessibility/availability of public genomics and metagenomics data. The update to the AcetoBase reference database described here (Version 2) provides new sequence data and taxonomy, along with improvements in web functionality and user interface. The evaluation of this latest update by re-analysis of publicly accessible FTHFS amplicon sequencing data previously analysed with AcetoBase Version 1 revealed significant improvements in the taxonomic assignment of FTHFS sequences. Database URL: https://acetobase.molbio.slu.se.

Vitamin B-related Gene Polymorphisms and Cardiovascular Disease.

Hyperhomocysteinemia is an independent risk factor for atherosclerosis, even in early childhood. A mutation in genes that code homocysteine metabolism enzymes or deficiency of specific vitamin cofactors may cause hyperhomocysteinemia. Vitamin B complex has been correlated with serum homocysteine levels. Any abnormality in its metabolism or nutritional deficiency may lead to hyperhomocysteinemia. Both vitamin B complex and homocysteine levels are partly genetically determined. Specifically, the most studied polymorphism is 677T-C in exon 5 of the 5,10- methylenetetrahydrofolate reductase (MTHFR) gene, which plays an important role in folate's metabolism. This polymorphism has been shown to be correlated with hypertension and cardiovascular disease. Polymorphisms in methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like (MTHFD1L) gene have also been correlated with increased risk for coronary artery disease. Other common serious polymorphisms regard the area with high linkage disequilibrium, including the neuroblastoma breakpoint family, NBPF3 gene, and ~ 12-50 kb upstream of the tissue nonspecific alkaline phosphatase gene. Finally, the polymorphisms which have been mostly associated with vitamin B12 concentration are the rs11254363 polymorphism at intron 52 of the intrinsic factor vitamin B12 receptor of the CUBN and the rs526934 polymorphism at intron 8 of transcobalamin I. To sum up, several polymorphisms have already been associated with vitamin B complexes and therefore homocysteine level, highlighting the complex nature of vitamin B genetics.

Integrative analysis reveals methylenetetrahydrofolate dehydrogenase 1-like as an independent shared diagnostic and prognostic biomarker in five different human cancers.

BACKGROUND: Defects in methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L) expression have earlier been examined in only a few human cancers. OBJECTIVES: Multi-omics profiling of MTHFD1L as a shared biomarker in distinct subtypes of human cancers. METHODS: In the current study, for the multi-omics analysis of MTHFD1L in 24 major subtypes of human cancers, a comprehensive in silico approach was adopted to mine different open access online databases including UALCAN, Kaplan-Meier (KM) plotter, LOGpc, GEPIA, Human Protein Atlas (HPA), Gene Expression across Normal and Tumor tissue (GENT2), MEXPRESS, cBioportal, STRING, DAVID, TIMER, and Comparative Toxicogenomics Database (CTD). RESULTS: We noticed that the expression of MTHFD1L was significantly higher in all the analyzed 24 subtypes of human cancers as compared with the normal controls. Moreover, MTHDF1L overexpression was also found to be significantly associated with the reduced overall survival (OS) duration of Bladder urothelial cancer (BLCA), Head and neck cancer (HNSC), Kidney renal papillary cell carcinoma (KIRP), Lung adenocarcinoma (LUAD), and Uterine corpus endometrial carcinoma (UCEC). This implies that MTHFD1L plays a significant role in the development and progression of these cancers. We further noticed that MTHFD1L was also overexpressed in BLCA, HNSC, KIRP, LUAD, and UCEC patients of different clinicopathological features. Pathways enrichment analysis revealed the involvement of MTHFD1L-associated genes in five diverse pathways. We also explored few interesting correlations between MTHFD1L expression and its promoter methylation, genetic alterations, CNVs, and between CD8+ T immune cells level. CONCLUSION: In conclusion, our results elucidated that MTHFD1L can serve as a shared diagnostic and prognostic biomarker in BLCA, HNSC, KIRP, LUAD, and UCEC patients of different clinicopathological features.

Serine catabolism generates liver NADPH and supports hepatic lipogenesis.

Carbohydrate can be converted into fat by de novo lipogenesis, a process upregulated in fatty liver disease. Chemically, de novo lipogenesis involves polymerization and reduction of acetyl-CoA, using NADPH as the electron donor. The feedstocks used to generate acetyl-CoA and NADPH in lipogenic tissues remain, however, unclear. Here we show using stable isotope tracing in mice that de novo lipogenesis in adipose is supported by glucose and its catabolism via the pentose phosphate pathway to make NADPH. The liver, in contrast, derives acetyl-CoA for lipogenesis from acetate and lactate, and NADPH from folate-mediated serine catabolism. Such NADPH generation involves the cytosolic serine pathway in liver running in the opposite direction to that observed in most tissues and tumours, with NADPH made by the SHMT1-MTHFD1-ALDH1L1 reaction sequence. SHMT inhibition decreases hepatic lipogenesis. Thus, liver folate metabolism is distinctively wired to support cytosolic NADPH production and lipogenesis. More generally, while the same enzymes are involved in fat synthesis in liver and adipose, different substrates are used, opening the door to tissue-specific pharmacological interventions.

Melatonin modulates metabolic remodeling in HNSCC by suppressing MTHFD1L-formate axis.

Metabolic remodeling is now widely recognized as a hallmark of cancer, yet its role in head and neck squamous cell carcinoma (HNSCC) remains largely unknown. In this study, metabolomic analysis of melatonin-treated HNSCC cell lines revealed that exogenous melatonin inhibited many important metabolic pathways including folate cycle in HNSCC cells. Methylenetetrahydrofolate dehydrogenase 1 like (MTHFD1L), a metabolic enzyme of the folate cycle regulating the production of formate, was identified as a downstream target of melatonin. MTHFD1L was found to be markedly upregulated in HNSCC, and MTHFD1L overexpression was significantly associated with unfavorable clinical outcome of HNSCC patients. In addition, MTHFD1L promoted HNSCC progression in vitro and in vivo and reversed the oncostatic effects of exogenous melatonin. More importantly, the malignant phenotypes suppressed by knockdown of MTHFD1L or exogenous melatonin could be partially rescued by formate. Furthermore, we found that melatonin inhibited the expression of MTHFD1L in HNSCC cells through the downregulation of cyclic AMP-responsive element-binding protein 1 (CREB1) phosphorylation. Lastly, this novel regulatory axis of melatonin-p-CREB1-MTHFD1L-formate was also verified in HNSCC tissues. Collectively, our findings have demonstrated that MTHFD1L-formate axis promotes HNSCC progression and melatonin inhibits HNSCC progression through CREB1-mediated downregulation of MTHFD1L and formate. These findings have revealed new metabolic mechanisms in HNSCC and may provide novel insights on the therapeutic intervention of HNSCC.

Oncogenic Ras expression increases cellular formate production.

One-carbon units, critical intermediates for cell growth, may be produced by a variety of means, one of which is via the production of formate. Excessive formate accumulation, known as formate overflow and a characteristic of oxidative cancer, has been observed in cancer cells. However, the basis for this high rate of formate production is unknown. We examined the effect of elevated expression of oncogenic Ras (RasV12), on formate production in NIH-3T3 cells (mouse fibroblasts) cultured with either labelled 13C-serine or 13C-glycine. Formate accumulation by the fibroblasts transformed by RasV12 was increased two-threefold over those by vector control (Babe) cells. The production of formate exceeded the rate of utilization in both cell types. 13C-formate was produced almost exclusively from the #3 carbon of 13C-serine. Virtually no labelled formate was produced from either the #2 carbon of serine or the #2 carbon of glycine. The increased formate production by RasV12 cells was associated with increased mRNA abundances for enzymes of formate production in both the mitochondria and the cytosol. Thus, we find the oncogenic RasV12 significantly increases formate overflow and may be one way for tumor cells to produce one-carbon units required for enhanced proliferation of these cells and/or for other processes which have not been identified.

Orthogonal genome-wide screens of bat cells identify MTHFD1 as a target of broad antiviral therapy.

Bats are responsible for the zoonotic transmission of several major viral diseases, including those leading to the 2003 SARS outbreak and likely the ongoing COVID-19 pandemic. While comparative genomics studies have revealed characteristic adaptations of the bat innate immune system, functional genomic studies are urgently needed to provide a foundation for the molecular dissection of the viral tolerance in bats. Here we report the establishment of genome-wide RNA interference (RNAi) and CRISPR libraries for the screening of the model megabat, Pteropus alecto. We used the complementary RNAi and CRISPR libraries to interrogate P. alecto cells for infection with two different viruses: mumps virus and influenza A virus, respectively. Independent screening results converged on the endocytosis pathway and the protein secretory pathway as required for both viral infections. Additionally, we revealed a general dependence of the C1-tetrahydrofolate synthase gene, MTHFD1, for viral replication in bat cells and human cells. The MTHFD1 inhibitor, carolacton, potently blocked replication of several RNA viruses, including SARS-CoV-2. We also discovered that bats have lower expression levels of MTHFD1 than humans. Our studies provide a resource for systematic inquiry into the genetic underpinnings of bat biology and a potential target for developing broad-spectrum antiviral therapy.

Association between SNPs and hepatotoxicity in patients with primary central nervous system lymphoma on high-dose methotrexate therapy.

OBJECTIVES: This study aims to evaluate the association between polymorphisms of methotrexate pathway genes and high-dose methotrexate-related hepatotoxicity in Chinese patients with primary central nervous system lymphoma. METHODS: Sixty-five patients in 411 treatment courses were enrolled and their toxicities were evaluated. The association between 30 candidate SNPs from 20 methotrexate pathway genes and high-dose methotrexate-related hepatotoxicity was analysed by PLINK and logistic regression. KEY FINDINGS: TYMS 6 bp DI + II (rs151264360; OR, 0.41; 95% CI, 0.25-0.66; P = 0.00029), MTHFD1 1958 GA + AA (rs2236225; OR, 0.55; 95% CI, 0.33-0.91; P = 0.020) and CCND1 870 GA + GG (rs9344; OR, 0.42; 95% CI, 0.24-0.73; P = 0.0024) had less risk of hepatotoxicity compared with their homozygotes (DD, GG and AA, respectively), while ABCC2 intron 29 GA + GG (rs3740065; OR, 3.14; 95% CI, 1.89-5.20; P = 0.00001) was more prevalent in patients with hepatotoxicity than TT. CONCLUSIONS: TYMS 6 bp DI + II, MTHFD1 1958 GA + AA, CCND1 870 GA + GG genotypes were associated with a lower probability of hepatotoxicity in patients with primary central nervous system lymphoma on high-dose methotrexate therapy, and ABCC2 intron 29 GA + GG was correlated with increased risk of hepatotoxicity.

Triglyceride regulate ACE2 level through MTHFD1.

Obesity has been followed with interest as a risk factor for COVID-19, with triglycerides as one of four common criteria used to define obesity, which have been used to study the mechanism of obesity. In this study, we showed that angiotensin-converting enzyme-2 (ACE2) is widely expressed in the mouse body, including the kidney, spleen, brain, heart, lung, liver, and testis, and that ACE2 levels increased after a high-fat diet. The ACE2 levels were recorded at 0 days, 3 days, 7 days, and 14 days after a high-fat diet, and they increased at 14 days after high-fat diet initiation. In addition, triglyceride levels were also significantly increased at 14 days after high-fat diet initiation, but body weight was not changed. Furthermore, we examined the ACE2 levels in Calu3 cells (a lung cancer cell line) after triglyceride treatment, and the results indicated that ACE2 levels were increased at 25 µM and reached their peak at 200 µM. Finally, we found that the mRNA level of mthfd1 was significantly increased in the high-fat diet group. Given these findings, we hypothesize that triglycerides can regulate the expression of ACE2 and Mthfd1.

Metabotype analysis of Mthfd1l-null mouse embryos using desorption electrospray ionization mass spectrometry imaging.

Mammalian folate-dependent one-carbon (1C) metabolism provides the building blocks essential during development via amino acid interconversion, methyl-donor production, regeneration of redox factors, and de novo purine and thymidylate synthesis. Folate supplementation prevents many neural tube defects (NTDs) that occur during the embryonic process of neurulation. The mechanism by which folate functions during neurulation is not well understood, and not all NTDs are preventable by folate supplementation. Mthfd1l is a mitochondrial 1C metabolism enzyme that produces formate, a 1C donor that fuels biosynthesis and the methyl cycle in the cytoplasm. Homozygous deletion of the Mthfd1l gene in mice (Mthfd1lz/z) causes embryonic lethality, developmental delay, and folate-resistant NTDs. These mice also have defects in cranial mesenchyme formation. In this work, mass spectrometry imaging was used to obtain ion maps of the cranial mesenchyme that identified the spatial distribution and relative abundance of metabolites in wild-type and Mthfd1lz/z embryos. The relative abundances of purine and thymidylate derivatives, as well as amino acids, were diminished in the cranial mesenchyme of Mthfd1lz/z embryos. Loss of Mthfd1l activity in this region also led to abnormal levels of methionine and dysregulated energy metabolism. These alterations in metabolism suggest possible approaches to preventing NTDs in humans.

Integrated bioinformatics analysis identified MTHFD1L as a potential biomarker and correlated with immune infiltrates in hepatocellular carcinoma.

Liver hepatocellular carcinoma (LIHC) is one of the most frequently occurring primary malignant liver tumors and seriously harms people's health in the world. Methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L) has been shown to be associated with colon cancer cell proliferation, colony formation and invasion. In the present study, a total of 370 LIHC and 51 normal samples data were downloaded from The Cancer Genome Atlas (TCGA) database. Bioinformatics and immunohistochemistry (IHC) analysis showed that MTHFD1L is highly expressed in liver tumors. Correlation analysis suggested the differences of vital status between high- and low-expression MTHFD1L groups of LIHC. Univariate and multivariate Cox proportional hazards regression were performed to identify the relationship between clinical characteristics and overall survival (OS). In addition, to explore whether MTHFD1L has an effect on the immune infiltration of LIHC. The correlation between MTHFD1L expression and 24 immune cells were analyzed by ImmuneCellAI database. Furthermore, we combined three databases CIBERSORT, TIMER and ImmuneCellAI to do a comprehensive validation and determined that dendritic cells (DCs) resting, macrophage M0 and macrophage M2 closely related to the expression of MTHFD1L. The results showed that MTHFD1L was a potential prognostic biomarker for LIHC, and could help to elucidate that how the immune microenvironment promotes liver cancer development.

Mild Choline Deficiency and MTHFD1 Synthetase Deficiency Interact to Increase Incidence of Developmental Delays and Defects in Mice.

Folate and choline are interconnected metabolically. The MTHFD1 R653Q SNP is a risk factor for birth defects and there are concerns that choline deficiency may interact with this SNP and exacerbate health risks. 80-90% of women do not meet the Adequate Intake (AI) for choline. The objective of this study was to assess the effects of choline deficiency on maternal one-carbon metabolism and reproductive outcomes in the MTHFD1-synthetase deficient mouse (Mthfd1S), a model for MTHFD1 R653Q. Mthfd1S+/+ and Mthfd1S+/- females were fed control (CD) or choline-deficient diets (ChDD; 1/3 the amount of choline) before mating and during pregnancy. Embryos were evaluated for delays and defects at 10.5 days gestation. Choline metabolites were measured in the maternal liver, and total folate measured in maternal plasma and liver. ChDD significantly decreased choline, betaine, phosphocholine, and dimethylglycine in maternal liver (p < 0.05, ANOVA), and altered phosphatidylcholine metabolism. Maternal and embryonic genotype, and diet-genotype interactions had significant effects on defect incidence. Mild choline deficiency and Mthfd1S+/- genotype alter maternal one-carbon metabolism and increase incidence of developmental defects. Further study is required to determine if low choline intakes contribute to developmental defects in humans, particularly in 653QQ women.

Formyl tetrahydrofolate deformylase affects hydrogen peroxide accumulation and leaf senescence by regulating the folate status and redox homeostasis in rice.

It is well established that an abnormal tetrahydrofolate (THF) cycle causes the accumulation of hydrogen peroxide (H2O2) and leaf senescence, however, the molecular mechanism underlying this relationship remains largely unknown. Here, we reported a novel rice tetrahydrofolate cycle mutant, which exhibited H2O2 accumulation and early leaf senescence phenotypes. Map-based cloning revealed that HPA1 encodes a tetrahydrofolate deformylase, and its deficiency led to the accumulation of tetrahydrofolate, 5-formyl tetrahydrofolate and 10-formyl tetrahydrofolate, in contrast, a decrease in 5,10-methenyl-tetrahydrofolate. The expression of tetrahydrofolate cycle-associated genes encoding serine hydroxymethyl transferase, glycine decarboxylase and 5-formyl tetrahydrofolate cycloligase was significantly down-regulated. In addition, the accumulation of H2O2 in hpa1 was not caused by elevated glycolate oxidation. Proteomics and enzyme activity analyses further revealed that mitochondria oxidative phosphorylation complex I and complex V were differentially expressed in hpa1, which was consistent with the H2O2 accumulation in hpa1. In a further feeding assay with exogenous glutathione (GSH), a non-enzymatic antioxidant that consumes H2O2, the H2O2 accumulation and leaf senescence phenotypes of hpa1 were obviously compensated. Taken together, our findings suggest that the accumulation of H2O2 in hpa1 may be mediated by an altered folate status and redox homeostasis, subsequently triggering leaf senescence.

Arbutin suppresses osteosarcoma progression via miR-338-3p/MTHFD1L and inactivation of the AKT/mTOR pathway.

Arbutin, a glycoside extracted from the plant Arctostaphylos uva-ursi, has been previously reported to possess antioxidant, anti-inflammatory and anticancer effects. Here, we investigated whether arbutin affects the proliferation of the cells of the osteosarcoma (OS) cell lines MG-63 and SW1353. Arbutin suppressed OS cell viability in a dose- and time-dependent manner, as shown by Cell Counting Kit-8 assay. Furthermore, arbutin exposure decreased the protein levels of MTHFD1L, CCND1 and phosphorylated-protein kinase B (AKT)/phosphorylated-mammalian target of rapamycin (mTOR). Potential upstream miRNAs of MTHFD1L were predicted using TargetScan, PICTAR5, miRanda and miRWalk. We performed luciferase activity assays to show that miR-338-3p directly targets and negatively regulates the expression of MTHFD1L. Knockdown of miR-338-3p promoted cell invasion, migration and proliferation in arbutin-treated OS cells via MTHFD1L. In summary, our data suggest that arbutin inhibits OS cell proliferation, migration and invasion via miR-338-3p/MTHFD1L and by inactivating the AKT/mTOR pathway.