Dietary Folates Research Articles

Breast Milk Microbiota: A Systematic Review on the Influence of Maternal Characteristics on Its Composition

Objective:The objective of this study was to identify current evidence on the impact of maternal characteristics (nutritional status, diet, geographic location/cultural influences, and stress) on breast milk microbiota (BMM) composition and diversity.Methods:A systematic review was conducted by searching articles in five different databases. Quality and risk of bias were assessed for each article using RevMan software.Results:Fifteen articles were selected. Maternal obesity had only a transient effect on microbiota diversity. Dietary fatty acids and folates were the main dietary factors influencing BMM. Maternal geographic location strongly influences BMM composition and ultimately its diversity. Available data on maternal stress were insufficient to draw conclusions regarding the impact of this factor.Conclusions:Geographic location of the mother and dietary factors are the main determinants of BMM composition. Further studies are needed to assess the impact of BMM on infant health.

Abstract 1792: Synthetic lethal vulnerabilities stemming from inhibition of one carbon metabolism in anaplastic thyroid cancer

Abstract Reprogramming of metabolic processes by tumor cells is essential to cope with their increased proliferative activity, and with the challenges of the unique microenvironment they inhabit. These metabolic alterations are attractive targets to design novel therapeutic approaches.The most aggressive subtype of thyroid cancer, Anaplastic Thyroid Cancer (ATC), is often unresectable at presentation, highly resistant to therapy and usually lethal. We have recently discovered that ATCs overexpress several genes encoding enzymes involved in one-carbon metabolism (1C-Met), which utilizes serine and dietary folates to produce glycine and tetrahydrofolate-bound one-carbon units, required for nucleotide biosynthesis and for NADPH and glutathione (GSH) production. We have shown that i) the increased activity of the 1C-Met pathway supports the high purine demand of ATC; ii) inhibition of 1C-Met impairs tumor growth in vitro and in vivo, leading to growth arrest; iii) inhibition of 1C-Met renders thyroid cancer cells glycine-auxotroph.We now demonstrate that the profound addiction of ATC cells to 1C-Met creates a series of targetable vulnerabilities that can be harnessed to induce massive ATC cell death.Our new data demonstrate that:1- The purine depletion consequent impaired 1C-Met induces replicative stress and triggers the DNA Damage Response (DDR). Activation of DDR, coupled with the absence of functional TP53 in the vast majority of ATCs, leads to synthetic lethality between inhibition of 1C-Met and G2/M checkpoint kinases.2- Inhibition of 1C-Met in ATC cells generates an absolute dependence on extracellular glycine uptake. We have identified and validated the main glycine transporter(s) in ATC cells and show that inhibition of 1C-Met is synthetic lethal with inhibition of glycine uptake.3- Impaired 1C-Met leads to increased oxidative stress and decrease of NADPH and glutathione levels. These alterations create a synthetic lethality relationship with the glutathione-depleting activity of PRIMA-1 (APR246), a small molecule originally identified as a compound restoring mutant p53 conformation and function, but later shown to be converted within cells into the reactive electrophile methylene quinuclidinone, which acts in a p53-independent manner through a variety of mechanisms, including reduction of cellular GSH levels.Our data shed light on the molecular consequences of 1C-Met upregulation in ATC and support the efficacy of novel rationally designed therapeutic approaches with curative intent not only for ATC, but also for other aggressive, dedifferentiated solid tumors. Citation Format: Alexander Forrest, Anil Ahsan, Antonio Di Cristofano. Synthetic lethal vulnerabilities stemming from inhibition of one carbon metabolism in anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1792.

Unmetabolized folic acid and organ fibrosis: Are they nefarious partners?

Recent publications raise the possibility that folic acid (FA), specifically unmetabolized folic acid (UMFA), might promote the progression of organ fibrosis via signaling through folate receptors, leading to excessive extracellular matrix production by myofibroblasts and other cell types. Clinical research over the past two decades adds credence to this largely unexplored contention. FA is a synthetic form of folate, first made in 1945, that has been added to the food supply in the US since 1998 to decrease neural tube defects; subsequently, other countries have followed suit. Notably, synthetic FA, compared to natural dietary folates, is metabolized differently, and potentially activates cellular signaling pathways, leading to fibrosis progression. Although FA food supplementation programs have resulted in a roughly 2-fold increase in serum folate concentrations and a decrease in the prevalence of neural tube defects, other concerns have surfaced. As such, due to the incomplete metabolism of ingested FA, before it can enter cellular processes, most individuals exhibit concentrations of unmetabolized folic acid (UMFA) in the circulation. We hypothesize that it is UMFA, but not natural occurring food folates, which in part promotes the progression of organ fibrosis in many systems. We feel the issue is relevant since organ fibrosis is involved in nearly half of all mortality in industrialized nations. Finally, this proposed nexus of UMFA and organ fibrosis might have preventive and therapeutic implications, and more research is needed on the topic.

The evolving biology of the proton-coupled folate transporter: New insights into regulation, structure, and mechanism.

The human proton‐coupled folate transporter (PCFT; SLC46A1) or hPCFT was identified in 2006 as the principal folate transporter involved in the intestinal absorption of dietary folates. A rare autosomal recessive hereditary folate malabsorption syndrome is attributable to human SLC46A1 variants. The recognition that hPCFT was highly expressed in many tumors stimulated substantial interest in its potential for cytotoxic drug targeting, taking advantage of its high‐level transport activity under acidic pH conditions that characterize many tumors and its modest expression in most normal tissues. To better understand the basis for variations in hPCFT levels between tissues including human tumors, studies have examined the transcriptional regulation of hPCFT including the roles of CpG hypermethylation and critical transcription factors and cis elements. Additional focus involved identifying key structural and functional determinants of hPCFT transport that, combined with homology models based on structural homologies to the bacterial transporters GlpT and LacY, have enabled new structural and mechanistic insights. Recently, cryo‐electron microscopy structures of chicken PCFT in a substrate‐free state and in complex with the antifolate pemetrexed were reported, providing further structural insights into determinants of (anti)folate recognition and the mechanism of pH‐regulated (anti)folate transport by PCFT. Like many major facilitator proteins, hPCFT exists as a homo‐oligomer, and evidence suggests that homo‐oligomerization of hPCFT monomeric proteins may be important for its intracellular trafficking and/or transport function. Better understanding of the structure, function and regulation of hPCFT should facilitate the rational development of new therapeutic strategies for conditions associated with folate deficiency, as well as cancer.

Targeting the epigenome to treat neurodegenerative diseases or delay their onset: a perspective

Targeting the epigenome to treat neurodegenerative diseases or delay their onset: a perspective

Impact of nanodisc lipid composition on cell-free expression of proton-coupled folate transporter.

The Proton-Coupled Folate Transporter (PCFT) is a transmembrane transport protein that controls the absorption of dietary folates in the small intestine. PCFT also mediates uptake of chemotherapeutically used antifolates into tumor cells. PCFT has been identified within lipid rafts observed in phospholipid bilayers of plasma membranes, a micro environment that is altered in tumor cells. The present study aimed at investigating the impact of different lipids within Lipid-protein nanodiscs (LPNs), discoidal lipid structures stabilized by membrane scaffold proteins, to yield soluble PCFT expression in an E. coli lysate-based cell-free transcription/translation system. In the absence of detergents or lipids, we observed PCFT quantitatively as precipitate in this system. We then explored the ability of LPNs to support solubilized PCFT expression when present during in-vitro translation. LPNs consisted of either dimyristoyl phosphatidylcholine (DMPC), palmitoyl-oleoyl phosphatidylcholine (POPC), or dimyristoyl phosphatidylglycerol (DMPG). While POPC did not lead to soluble PCFT expression, both DMPG and DMPC supported PCFT translation directly into LPNs, the latter in a concentration dependent manner. The results obtained through this study provide insights into the lipid preferences of PCFT. Membrane-embedded or solubilized PCFT will enable further studies with diverse biophysical approaches to enhance the understanding of the structure and molecular mechanism of folate transport through PCFT.

Development of a Zebrafish Model for Studies of the Interaction of Methylenetetrahydrofolate Reductase Deficiency and Dietary Folates on Metabolic Regulation

ObjectivesMethylenetetrahydrofolate reductase (MTHFR) is required for 5-methyltetrahydrofolate (5MTHF) synthesis, and common variants reduces its efficiency and associate with metabolic disorders. High folic acid (FA) intakes, commonly consumed by pregnant women in North America, may further inhibit MTHFR enzyme; programming long-term metabolic dysregulation in offspring. The zebrafish (Danio rerio) is a valuable model for study of embryonic development and high-throughput nutrient × gene interactions. The objective of this study was to characterize a zebrafish model of mthfr deficiency and assess the interaction between mthfr and FA intakes on early-life metabolic dysregulation. MethodsZebrafish were co-injected with a set of 4 guide RNAs (gRNAs) or cas9 protein alone and F0 embryos were assayed for a high-throughput phenotypic screen. Germline F1 knock-out homozygous mutants (mthfr –/–) were made by co-injecting cas9 mRNA with 2 gRNAs targeting the transcriptional start site of the mthfr gene. Embryos were raised up to 5 days post-fertilization (dpf) and folate and 1-carbon metabolites measured by LC-MS/MS. Lipid accumulation was assessed at 5dpf and after feeding a high cholesterol diet (HDC) with cholesteryl-ester (CE)-BoDipy-C12® from 5–15dpf. A subset of embryos were exposed to no (0μM) or high (100μM) FA from 0–5dpf and whole-body lipids measured. Resultsmthfr disruption in zebrafish reduced (80%) mthfr mRNA and 5MTHF levels (90%) compared to controls (P < 0.0001). They had lower 1-carbon metabolites including betaine, methionine, s-adenosylmethionine, and higher choline, s-adenosylhomocysteine, cystathionine and homocysteine (P < 0.01). As well, neutral lipid accumulation was higher in liver, heart and vasculature at 5 and 15 dpf along with higher CE altered cholesterol transport/metabolism. High FA exposure ameliorated lipid accumulation in mthfr mutants at 5 dpf (P = 0.06), but increased lipids accumulation in controls compared to no exposure (P = 0.03). ConclusionsThe zebrafish mthfr deficient model exhibits a similar alteration to 1-carbon metabolites as in humans with severe MTHFR deficiency. This zebrafish model has potential for understanding the interaction of mthfr deficiency and dietary folates on metabolism. Funding SourcesCIHR-INMD, EP by NSERC-CGS

Abstract PR14: Dietary folate and prostate cancer tumor aggressiveness differences between African Americans and European Americans

Abstract Introduction: Folate is a water-soluble B vitamin, which is involved in DNA synthesis and repair and in regulation of gene expression through DNA methylation as a methyl donor. Despite the confirmed beneficial effect on the prevention of neural tube defect, concerns have been raised on high intakes of folate and its synthetic form, folic acid, may promote carcinogenesis or cancer progression. African American (AA) males tend to have a more aggressive prostate cancer tumor diagnosis compared to European American (EA) males. Objective: This aim of this study is to examine the association between folic acid intake in the year prior to PCa diagnosis among AAs and EAs. Using a population-based case-only study, an examination of folic acid was conducted to evaluate the effects of dietary folate on prostate cancer aggressiveness. Methods: Data from the North Carolina-Louisiana Prostate Cancer Project (PCaP) questionnaire were used to evaluate 1,497 participants (AA n = 722 and EA n = 775) with a low or high aggressiveness prostate cancer to assess dietary folate intake one year prior to PCa diagnosis using the National Cancer Institute Dietary History Questionnaire. High-aggressive disease was defined as Gleason sum ≥8, or prostate-specific antigen (PSA) &amp;gt;20 ng/mL, or Gleason score ≥7 and clinical stage T3-T4, while low-aggressive disease was defined as Gleason sum &amp;lt;7 and stage T1—T2 and PSA &amp;lt;10 ng/mL. All four variables for dietary folate (natural folate, synthetic folate, folate, and dietary folates) were examined. Multivariate logistic regression was used to assess dietary folate and prostate cancer aggressiveness. Confounding variables considered for this analysis include age, BMI, total energy (kcal), education level, and first-degree family history of PCa. Additionally, dietary folate was categorized into tertiles. Models were stratified by race and the dose-response relationship was evaluated. Results: Folate intakes, regardless natural folate (mean = 354.5 vs 304.1), synthetic folate (176.3 vs 157.5), or total dietary folate equivalent (654.0 vs 571.5), were higher among AA than EA, respectively. Based on the tertile categorization, the highest dietary folate was significantly associated with high-aggressive prostate cancer when compared to the lowest intake group among AA and EA combined (odds ratio (OR) = 1.41, 95% confidence interval (CI) = 1.04 – 1.90) after adjusted for confounders. Stratified model by race showed that there is an increased trend in PCa aggressiveness and increased folate intake (2nd tertile OR = 1.03, 95%CI = 0.67 – 1.56; 3rd tertile OR = 1.41, CI = 1.11 – 2.48), p-value for trend = 0.01. The association was not observed among EA. The trend is very similar regardless of natural or synthetic folate. Conclusion: It appeared that AA with high folic acid intake had a greater chance of being diagnosed with high-aggressiveness PCa, while the association was not observed among EA. The finding suggests that the metabolism of folate may be different between AA and EA, possibly due to genetic polymorphisms. This abstract is also being presented as Poster C034. Citation Format: Daniela Ramirez Aguilar, Susan E Steck, Hui-Yi Lin, LJ Su. Dietary folate and prostate cancer tumor aggressiveness differences between African Americans and European Americans [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr PR14.

Plasma Homocysteine and Polymorphisms of Genes Involved in Folate Metabolism Correlate with DNMT1 Gene Methylation Levels.

DNA methyltransferase 1 (DNMT1) is responsible for the maintenance of DNA methylation patterns during cell division. Several human diseases are characterized by impaired DNMT1 gene methylation, but less is known about the factors that regulate DNMT1 promoter methylation levels. Dietary folates and related B-vitamins are essential micronutrients for DNA methylation processes, and we performed the present study to investigate the contribution of circulating folate, vitamin B12, homocysteine, and common polymorphisms in folate pathway genes to the DNMT1 gene methylation levels. We investigated DNMT1 gene methylation levels in peripheral blood DNA samples from 215 healthy individuals. All the DNA samples were genotyped for MTHFR 677C > T (rs1801133) and 1298A > C (rs1801131), MTRR 66A > G (rs1801394), MTR 2756A > G (rs1805087), SLC19A1 (RFC1) 80G > A (rs1051266), TYMS 28-bp tandem repeats (rs34743033) and 1494 6-bp insertion/deletion (indel) (rs34489327), DNMT3A -448A > G (rs1550117), and DNMT3B -149C > T (rs2424913) polymorphisms. Circulating homocysteine, folate, and vitamin B12 levels were available from 158 of the recruited individuals. We observed an inverse correlation between plasma homocysteine and DNMT1 methylation levels. Furthermore, both MTR rs1805087 and TYMS rs34743033 polymorphisms showed a statistically significant effect on DNMT1 methylation levels. The present study revealed several correlations between the folate metabolic pathway and DNMT1 promoter methylation that could be of relevance for those disorders characterized by altered DNA methylation.

Buriti of the cerrado of Minas Gerais, Brazil: physical and chemical characterization and content of carotenoids and vitamins

Abstract The fruit species of the Brazilian Cerrado, such as buriti (Mauritia vinifera ), occupy a prominent position in the ecosystem since they contribute to the economic potential of the region and have high nutritional value. We aimed to evaluate the physical characteristics, chemical composition and content of vitamin C, carotenoids, vitamin E and folates in pulp of buriti from the Brazilian Cerrado of Minas Gerais, Brazil. The length, diameter, mass and fruit yield were evaluated. The titratable acidity was determined by volumetric neutralization; pH by potentiometry; soluble solids by refractometry; moisture using an oven; ash using a muffle furnace; proteins by the micro-Kjeldhal method; total dietary fiber by the gravimetric non-enzymatic method and lipids with a Soxhlet extractor. Vitamin C and carotenoids were analyzed by high performance liquid chromatography with detection by diode array (HPLC-DAD) and vitamin E and folates by HPLC with fluorescence detection. Buriti pulp showed 12.13 g.100g -1 of lipids and 74.86 g.100g-1 of moisture. Also, the fruit showed high content of dietary fiber, carotenoids, vitamin C, vitamin E and folates. Buriti can contribute to the adequate intake of fiber and vitamins and to preventing the incidence of disorders, especially those more frequent in native populations of the Cerrado.

Dissecting the role of Folr1 and Folh1 genes in the pathogenesis of metabolic syndrome in spontaneously hypertensive rats.

Increased levels of plasma cysteine predispose to obesity and metabolic disturbances. Our recent genetic analyses in spontaneously hypertensive rats (SHR) revealed mutated Folr1 (folate receptor 1) on chromosome 1 as a quantitative trait gene associated with reduced folate levels, hypercysteinemia and metabolic disturbances. The Folr1 gene is closely linked to the Folh1 (folate hydrolase 1) gene which codes for an enzyme involved in the hydrolysis of dietary polyglutamyl folates in the intestine. In the current study, we obtained evidence that Folh1 mRNA of the BN (Brown Norway) origin is weakly but significantly expressed in the small intestine. Next we analyzed the effects of the Folh1 alleles on folate and sulfur amino acid levels and consecutively on glucose and lipid metabolism using SHR-1 congenic sublines harboring either Folr1 BN and Folh1 SHR alleles or Folr1 SHR and Folh1 BN alleles. Both congenic sublines when compared to SHR controls, exhibited significantly reduced folate clearance and lower plasma cysteine and homocysteine levels which was associated with significantly decreased serum glucose and insulin concentrations and reduced adiposity. These results strongly suggest that, in addition to Folr1, the Folh1 gene also plays an important role in folate and sulfur amino acid levels and affects glucose and lipid metabolism in the rat.

Improved Stable Isotope Dilution Assay for Dietary Folates Using LC-MS/MS and Its Application to Strawberries.

Folates play an important role in the human body and a deficiency of this vitamin can cause several diseases. Therefore, a reliable analytical method is crucial for the determination of folate vitamers in strawberries and other dietary folate sources. A stable isotope dilution LC-MS/MS method for analyzing folates in food was developed and validated. The folate vitamers Pteroylmonoglutamic acid, tetrahydrofolate, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate were quantified using 13C-labeled internal standards. Validation of the assay was accomplished by determining linearity, precision, recovery, limit of detection, and limit of quantification and revealed to be a precise, sensitive, and accurate method to determine folate vitamers. Strawberries are worldwide consumed and known to be a good dietary source of nutritive compounds. Using this method, folate concentrations in selected commercial strawberry cultivars and experimental breeding lines grown in Germany and Australia were investigated. Total folates varied from 59 to 153 μg/100 g on fresh weight basis. Furthermore, folate content after lyophilizing or freezing did not show any significant differences compared to fresh strawberries. However, significant losses of total folates in pureed strawberries could be observed after 5 days of storage with only 16% of the original concentration retained. In summary, some of the investigated strawberry cultivars/breeding lines can be considered as rich dietary sources of natural folates.

Human proton coupled folic acid transporter is a monodisperse oligomer in the lauryl maltose neopentyl glycol solubilized state

The human proton coupled folic acid transporter PCFT is the major import route for dietary folates. Mutations in the gene encoding PCFT cause hereditary folic acid malabsorption, which manifests itself by compromised folate absorption from the intestine and also in impaired folate transport into the central nervous system. Since its recent discovery, PCFT has been the subject of numerous biochemical studies aiming at understanding its structure and mechanism. One major focus has been its oligomeric state, with some reports supporting oligomers and others a monomer. Here, we report the overexpression and purification of recombinant PCFT. Following detergent screening, n-Dodecyl β-D-maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG) were chosen for further work as they exhibited the most optimal solubilization. We found that purified detergent solubilized PCFT was able to bind folic acid, thus indicating a functionally active protein. Size exclusion chromatography showed that PCFT in DDM was polydisperse; the LMNG preparation was clearly monodisperse but with shorter retention time than the major DDM peak. To assess the oligomeric state negative stain electron microscopy was performed which showed a particle with the size of a PCFT dimer.

Is High Folic Acid Intake a Risk Factor for Autism?—A Review

Folate is required for metabolic processes and neural development. Insuring its adequate levels for pregnant women through supplementation of grain-based foods with synthetic folic acid (FA) in order to prevent neural tube defects has been an ongoing public health initiative. However, because women are advised to take multivitamins containing FA before and throughout pregnancy, the supplementation together with natural dietary folates has led to a demographic with high and rising serum levels of unmetabolized FA. This raises concerns about the detrimental effects of high serum synthetic FA, including a rise in risk for autism spectrum disorder (ASD). Some recent studies have reported a protective effect of FA fortification against ASD, but others have concluded there is an increased risk for ASD and other negative neurocognitive development outcomes. These issues are accompanied by further health questions concerning high, unmetabolized FA levels in serum. In this review, we outline the reasons excess FA supplementation is a concern and review the history and effects of supplementation. We then examine the effects of FA on neuronal development from tissue culture experiments, review recent advances in understanding of metabolic functional blocks in causing ASD and treatment for these with alternative forms such as folinic acid, and finally summarize the conflicting epidemiological findings regarding ASD. Based on the evidence evaluated, we conclude that caution regarding over supplementing is warranted.

The promise and challenges of exploiting the proton-coupled folate transporter for selective therapeutic targeting of cancer.

This review considers the "promise" of exploiting the proton-coupled folate transporter (PCFT) for selective therapeutic targeting of cancer. PCFT was discovered in 2006 and was identified as the principal folate transporter involved in the intestinal absorption of dietary folates. The recognition that PCFT was highly expressed in many tumors stimulated substantial interest in using PCFT for cytotoxic drug targeting, taking advantage of its high level transport activity under the acidic pH conditions that characterize many tumors. For pemetrexed, among the best PCFT substrates, transport by PCFT establishes its importance as a clinically important transporter in malignant pleural mesothelioma and non-small cell lung cancer. In recent years, the notion of PCFT-targeting has been extended to a new generation of tumor-targeted 6-substituted pyrrolo[2,3-d]pyrimidine compounds that are structurally and functionally distinct from pemetrexed, and that exhibit near exclusive transport by PCFT and potent inhibition of de novo purine nucleotide biosynthesis. Based on compelling preclinical evidence in a wide range of human tumor models, it is now time to advance the most optimized PCFT-targeted agents with the best balance of PCFT transport specificity and potent antitumor efficacy to the clinic to validate this novel paradigm of highly selective tumor targeting.

Identification and determination of naturally occurring folates in grains of rice (Oryza sativa L.) by UPLC-MS/MS analysis

The genetic potential and biofortification of India-grown rice with bioavailable folate has not been studied yet. The objectives of this study were to determine the folates concentration in four cultivars of rice through UPLC–MS/MS. Total folate concentration in rice cultivars ranged from 11.0 to 51 μg/100 g with a mean of 26.0 μg/100 g. Among the four rice cultivars, the pigmented grain cultivar Nootripathu possesses two-fold rich sources of total folates than the other three non-pigmented grain cultivars. The average value of 100 g serving of rice grains could provide the amount of recommended daily allowance (% RDA) of dietary folates (6.5%) for adults, which ranged from 2.7–12.7%. Among the 5 individual forms of folates, 5-methyltetrahydrofolate was most abundant in rice cultivars followed by 10-Formylfolic acid and folic acid. The result of this study has been useful for biofortification of folates in rice.

Safety and benefits of interventions to increase folate status in malaria-endemic areas.

SummaryFor decades, folic acid has routinely been given to prevent or treat anaemia in children, pregnant women and people with sickle cell disease. However, there is no conclusive evidence that folate deficiency anaemia constitutes a public health problem in any of these groups. Industrial flour fortification is recommended and implemented in many countries to combat neural tube defects. Dietary folates or folic acid can antagonise the action of antifolate drugs that play a critical role in the prevention and treatment of malaria. Randomised trials have shown that folic acid supplementation increases the rate of treatment failures with sulfadoxine‐pyrimethamine. The efficacy of antifolate drugs against Plasmodium is maximized in the absence of exogenous folic acid, suggesting that there is no safe minimum dose of ingested folic acid. We here review the safety and benefits of interventions to increase folate status in malaria‐endemic countries. We conclude that formal cost‐benefit analyses are required.

Molecular characterization of dihydroneopterin aldolase and aminodeoxychorismate synthase in common bean-genes coding for enzymes in the folate synthesis pathway.

Common beans (Phaseolus vulgaris) are excellent sources of dietary folates, but different varieties contain different amounts of these compounds. Genes coding for dihydroneopterin aldolase (DHNA) and aminodeoxychorismate synthase (ADCS) of the folate synthesis pathway were characterized by PCR amplification, BAC clone sequencing, and whole genome sequencing. All DHNA and ADCS genes in the Mesoamerican cultivar OAC Rex were isolated and compared with those genes in the genome of Andean genotype G19833. Both genotypes have two functional DHNA genes and one pseudo gene. PvDHNA1 and PvDHNA2 proteins have similar secondary structures and conserved residues as DHNA homologs in Staphylococcus aureus and Arabidopsis. Sequence analysis and synteny mapping indicated that PvDHNA1 might be a duplicated and transposed copy of PvDHNA2. There is only one ADCS gene (PvADCS) identified in the bean genome and it is identical in OAC Rex and G19833. PvADCS has the conserved motifs required for catalytic activity similar to other plant ADCS homologs. DHNA and ADCS gene-specific markers were developed, mapped, and compared to their physical locations on chromosomes 1 and 7, respectively. The gene-specific markers developed in this study should be useful for detection and selection of varieties with enhanced folate contents in bean breeding programs.

Computational modelling folate metabolism and DNA methylation: implications for understanding health and ageing.

Dietary folates have a key role to play in health, as deficiencies in the intake of these B vitamins have been implicated in a wide variety of clinical conditions. The reason for this is folates function as single carbon donors in the synthesis of methionine and nucleotides. Moreover, folates have a vital role to play in the epigenetics of mammalian cells by supplying methyl groups for DNA methylation reactions. Intriguingly, a growing body of experimental evidence suggests that DNA methylation status could be a central modulator of the ageing process. This has important health implications because the methylation status of the human genome could be used to infer age-related disease risk. Thus, it is imperative we further our understanding of the processes which underpin DNA methylation and how these intersect with folate metabolism and ageing. The biochemical and molecular mechanisms, which underpin these processes, are complex. However, computational modelling offers an ideal framework for handling this complexity. A number of computational models have been assembled over the years, but to date, no model has represented the full scope of the interaction between the folate cycle and the reactions, which governs the DNA methylation cycle. In this review, we will discuss several of the models, which have been developed to represent these systems. In addition, we will present a rationale for developing a combined model of folate metabolism and the DNA methylation cycle.

Expression, Purification and Functional Characterization of Human Proton-Coupled Folate Transporter (SLC46A1)

Folate cofactors play important roles in hundreds of metabolic reactions in a cell such as synthesis of protein and DNA precursors. The human proton-coupled folate transporter (PCFT) is the only means for absorption of dietary folates. PCFT also transports anti-cancer agents such as Pemetrexed and Methotrexate. Inadequate supply of folates and impaired PCFT function is associated with many disorders such as cancer, hereditary folate malabsorption, hypercysteinemia, heart diseases, obesity, anemia, neural tube defects and Alzheimer's disease. It is therefore important to study structural and functional characteristics of PCFT, to understand folate-homeostasis mechanisms and to develop better folate-based therapies. The primary requirement in structural and functional characterization of mammalian membrane proteins such as PCFT is the availability of good expression systems and methods to obtain large quantities of purified protein. To address this primary concern, we are establishing an efficient system to over-express PCFT and developing methods to obtain purified PCFT in good yields. We have expressed human PCFT in insect, yeast, and bacterial cells. We have successfully purified PCFT to homogeneity. Importantly, we also demonstrate that the heterologously-expressed PCFT is functional. Our findings are significant because 12-transmembrane helical, mammalian membrane proteins such as PCFT are notoriously difficult to express in versatile prokaryotic and single-celled eukaryotic systems. Furthermore, due to their predominant hydrophobic character and requirement of detergent and/or lipid environment for their stability, such membrane proteins are difficult to purify. Each of the three PCFT expression systems that we have developed offers unique advantages over another depending on the intended use of PCFT. Our findings will be significant for the advancement of knowledge concerning both the structure and function of PCFT through basic science research as well as for industrial applications including design and screening of PCFT-targeted agents.