Expression patterns of folate metabolism-related enzymes in the bovine oviduct: estrous cycle-dependent modulation and responsiveness to folic acid.

Folate metabolism is required for important biochemical processes that regulate cell functioning, but its role in female reproductive physiology in cattle during peri- and post-conceptional periods has not been thoroughly explored. Previous studies have shown the presence of folate in bovine oviductal fluid, as well as finely regulated gene expression of folate receptors and transporters in bovine oviduct epithelial cells (BOECs). Additionally, extracellular folic acid (FA) affects the transcriptional levels of genes important for the functioning of BOECs. However, it remains unknown whether the anatomical and cyclic features inherent to the oviduct affect regulation of folate metabolism. The present study aimed to characterize the gene expression pattern of folate cycle enzymes in BOECs from different anatomical regions during the estrous cycle and to determine the transcriptional response of these genes to increasing concentrations of exogenous FA. A first PCR screening showed the presence of transcripts encoding dihydrofolate reductase (DHFR), methylenetetrahydrofolate reductase (MTHFR), and methionine synthase (MTR) in bovine reproductive tissues (ovary, oviduct and uterus), with expression levels in oviductal tissues comparable to, or even higher than, those detected in ovarian and uterine tissues. Moreover, expression analysis through RT-qPCR in BOECs from the ampulla and isthmus during different stages of the estrous cycle demonstrated that folate metabolism-related enzymes exhibited cycle-dependent variations. In both anatomical regions, DHFR was upregulated during the preovulatory stage, while MTHFR and MTR exhibited increased expression levels during the postovulatory stage. Under in vitro culture conditions, ampullary and isthmic cells were cultured in the presence of 10, 50, and 100 µM FA for 24 h. Under these conditions, isthmus epithelial cells exhibited a unique transcriptional response to exogenous FA, showing a pronounced increase in MTR expression levels. Our results suggest that the expression of folate metabolism-related genes in BOECs is differentially regulated during the estrous cycle and may respond to exogenous levels of folate. This offers a new perspective on the transcriptional regulation of genes associated with the folate cycle in oviductal cells and provides groundwork for future studies on their functional and epigenetic implications within the oviductal microenvironment.

Raman spectroscopy study of 7,8-dihydrofolate inhibition on the Wuhan strain SARS-CoV-2 binding to human ACE2 receptor.

Emerging evidence suggests that elevated levels of folic acid in the bloodstream may confer protection against Wuhan-SARS-CoV-2 infection and mitigate its associated symptoms. Notably, two comprehensive studies of COVID-19 patients in Israel and UK uncovered a remarkable trend, wherein individuals with heightened folic acid levels exhibited only mild symptoms and necessitated no ventilatory support. In parallel, research has underscored the potential connection between decreased folic acid levels and the severity of Covid-19 among hospitalized patients. Yet, the underlying mechanisms governing this intriguing inhibition remain elusive. In a quest to elucidate these mechanisms, we conducted a molecular dynamics simulation approach followed by a Raman spectroscopy study to delve into the intricate interplay between the folic acid metabolite, 7,8-dihydrofolate (DHF), and the angiotensin-converting enzyme ACE2 receptor, coupled with its interaction with the receptor-binding domain (RBD) of the Wuhan strain of SARS-CoV-2. Through a meticulous exploration, we scrutinized the transformation of the ACE2 + RBD complex, allowing these reactants to form bonds. This was juxtaposed with a similar investigation where ACE2 was initially permitted to react with DHF, followed by the exposure of the ACE2 + DHF complex to RBD. We find that DHF, when bonded to ACE2, functions as a physical barrier, effectively inhibiting the binding of the Wuhan strain RBD. This physicochemical process offers a cogent explanation for the observed inhibition of host cell infection in subjects receiving supplementary folic acid doses, as epidemiologically substantiated in multiple studies. This study not only sheds light on a potential avenue for mitigating SARS-CoV-2 infection but also underscores the crucial role of folic acid metabolites in host-virus interactions. This research paves the way for novel therapeutic strategies in the battle against COVID-19 and reinforces the significance of investigating the molecular mechanisms underlying the protective effects of folic acid in the context of viral infections.

Paternal methotrexate exposure affects sperm small RNA content and causes craniofacial defects in the offspring.

Folate is an essential vitamin for vertebrate embryo development. Methotrexate (MTX) is a folate antagonist that is widely prescribed for autoimmune diseases, blood and solid organ malignancies, and dermatologic diseases. Although it is highly contraindicated for pregnant women, because it is associated with an increased risk of multiple birth defects, the effect of paternal MTX exposure on their offspring has been largely unexplored. Here, we found MTX treatment of adult medaka male fish (Oryzias latipes) causes cranial cartilage defects in their offspring. Small non-coding RNA (sncRNAs) sequencing in the sperm of MTX treated males identify differential expression of a subset of tRNAs, with higher abundance for specific 5' tRNA halves. Sperm RNA methylation analysis on MTX treated males shows that m5C is the most abundant and differential modification found in RNAs ranging in size from 50 to 90 nucleotides, predominantly tRNAs, and that it correlates with greater testicular Dnmt2 methyltransferase expression. Injection of sperm small RNA fractions from MTX-treated males into normal fertilized eggs generated cranial cartilage defects in the offspring. Overall, our data suggest that paternal MTX exposure alters sperm sncRNAs expression and modifications that may contribute to developmental defects in their offspring.

Folate Pathway Inhibitors, An Underestimated and Underexplored Molecular Target for New Anti-tuberculosis Agents.

The folate metabolic cycle is an important biochemical process for the maintenance of cellular homeostasis, and is a widely studied pathway of cellular replication control in all organisms. In microorganisms such as M. tuberculosis (Mtb), for instance, dihydrofolate reductase (MtDHFR) is the enzyme commonly explored as a molecular target for the development of new antibiotics. In the same way, dihydropteroate synthase (MtDHPS) was studied extensively until the first multidrug-resistant strains of mycobacteria that could not be killed by sulfonamides were found. However, the other enzymes belonging to the metabolic cycle, until recently less explored, have drawn attention as potential molecular targets for obtaining new antituberculosis agents. Recent structural determinations and mechanism of action studies of Mtb flavin-dependent thymidylate synthase (MtFDTS) and MtRv2671, enzymes that acts on alternative metabolic pathways within the folate cycle, have greatly expanded the scope of potential targets that can be screened in drug design process. Despite the crystallographic elucidation of most cycle proteins, some enzymes, such as dihydrofolate synthase (MtDHFS) and serine hydroxylmethyltransferase (MtSHMT), remain underexplored. In this review, we highlight recent efforts towards the inhibitor design to achieve innovative antituberculosis agents and a brief history of all enzymes present in the folate metabolic cycle. In the final section of this work, we have presented the main synthetic strategies used to obtain the most promising inhibitors.

Folate Metabolism in Hepatocellular Carcinoma. What Do We Know So Far?

Cancer cells are characterized by accelerated proliferation and an outstanding adaptation of their metabolic pathways to meet energy demands. The folate cycle, also known as folate metabolism or one-carbon metabolism, through enzymatic interconversions, provides metabolites necessary for nucleotide synthesis, methylation, and reduction power, helping to maintain the high rate of proliferation; therefore, the study of this metabolic pathway is of great importance in the study of cancer. Moreover, multiple enzymes involved in this cycle have been implicated in different types of cancer, corroborating the cell's adaptations under this pathology. During the last decade, nonalcoholic fatty liver disease has emerged as the leading etiology related to the rise in the incidence and deaths of hepatocellular carcinoma. Specifically, cholesterol accumulation has been a determinant promoter of tumor formation, with solid evidence that an enriched-cholesterol diet plays a crucial role in accelerating the development of an aggressive subtype of hepatocellular carcinoma compared to other models. In this review, we will discuss the most recent findings to understand the contribution of folate metabolism to cancer cells and tumor microenvironment while creating a link between the dynamics given by cholesterol and methylenetetrahydrofolate dehydrogenase 1-like, a key enzyme of the cycle located in the mitochondrial compartment.

Folate metabolism abnormalities in infertile patients with endometriosis.

Background: Homocysteine levels can be impacted by enzymes variations. Aim: To correlate MTHFR, MTR and MTRR variants with homocysteine levels in the blood and follicular fluid and assisted reproduction results. Material & methods:MTHFR (rs2274976, rs1801131, rs1801133), MTR (rs1805087) and MTRR (rs1801394) genotyping was performed by TaqMan assays and compared with homocysteine levels, measured by ELISA, to oocytes retrieved and to the pregnancy status of women with endometriosis and controls. Results: The MTR G allele and GG genotype were more common in patients with endometriosis. They also showed lower levels of homocysteine and more clinical gestations. Epistasis analysis showed a model associated with gestational results, composed of MTHFR+MTR variants (CC+AG). Conclusion: The summation effect of variants in genes participating in folate metabolism was associated with pregnancy status in Brazilian women. MTR variants were more observed in endometriosis patients, as well as lower follicular Hcy levels and increased clinical pregnancy results.

What was the aim of the study? To correlate genetic variants to homocysteine levels in the blood and oocyte surrounding fluid, and the results of assisted reproduction techniques. How was the study done? A total of 152 women with endometriosis and controls with male infertility were evaluated. DNA was extracted from blood for genetic analysis, and homocysteine levels were measured from the blood and oocyte surrounding fluid. Genetic results were correlated to homocysteine levels, oocyte quality and pregnancy status. What were the results? A specific genetic marker occurred more in endometriosis patients. They also showed lower levels of homocysteine and a tendency to more clinical gestations than controls. What do the results of the study mean? Endometriosis patients showed specific genetic markers and different levels of homocysteine compared with controls. These results can be helpful to predict gestational results.

One-carbon metabolism pathway genes and their non-association with the development of amyotrophic lateral sclerosis.

Although of unknown etiology, some mechanisms associated with the metabolic cycle of folate are speculated to be related to the genesis of amyotrophic lateral sclerosis (ALS). Thus, the aim of the study was to analyze the role of genetic polymorphisms rs1051266 in SLC19A1 gene and rs1805087 in MTR gene and their associations with ALS development. A case-control study was conducted with 101 individuals with ALS and 119 individuals without diagnosis of neurodegenerative diseases, from the Brazilian central population. The polymorphisms were determined using the polymerase chain reaction-restriction fragment length polymorphism technique. The results showed no statistically significant differences, even when genotypes were analyzed by the dominant, recessive, codominant, and overdominant inheritance models. It was observed a statistical significance relating alcohol consumption with individuals in the case group (p = 0.01). Therefore, the need for more studies to evaluate the influence of genetic variants is highlighted, seeking to provide information on the etiopathogenesis of ALS.

Screening of six polymorphisms related with folate metabolism in parents of individuals with Down syndrome.

OBJECTIVE: The study aim to investigate MTHFR C677T, MTHFR A1298C, RFC1 A80G, MTR A2756G, CBS 844ins68, MTRR A66G polymorphisms in Down syndrome (DS) parents. METHODS: Polymorphisms were evaluated in 35 mothers and 24 fathers of individuals with free trisomy of chromosome 21 confirmed by karyotype. The control group included 26 mothers and 26 fathers who had no children with DS. The molecular analysis was performed by polymerase chain reaction and restriction fragment length polymorphism (reaction chain polymerase restriction fragment length polymorphism) or polymerase chain reaction. The χ2 test (chi-square) was used to compare allele's differences among the study and the control group. Hardy-Weinberg equilibrium model was performed by χ2 testing. Multiple logistic regression models and binary logistic regression used to determine the association between polymorphisms and parental DS risk. RESULTS: This study did not reveal any significant difference in frequencies of polymorphisms. The haplotype analysis did not reveal linkage disequilibrium. The logistic regression analysis did not demonstrate differences between the groups. However, the binary logistic regression showed a higher frequency of the polymorphic homozygote genotype in DS parent group to codominant and dominant model in the RFC1 A80G. CONCLUSION: In conclusion, although the screening results were significant only to the RFC1 A80G polymorphism, the other determinations of the genetic factors associated with abnormal chromosome segregation could be helpful in future studies, including other polymorphisms involved in folate metabolism.

pH-responsive and folate-coated liposomes encapsulating irinotecan as an alternative to improve efficacy of colorectal cancer treatment.

Irinotecan (IRN) is a semisynthetic derivative of camptothecin that acts as a topoisomerase I inhibitor. IRN is used worldwide for the treatment of several types of cancer, including colorectal cancer, however its use can lead to serious adverse effects, as diarrhea and myelosuppression. Liposomes are widely used as drug delivery systems that can improve chemotherapeutic activity and decrease side effects. Liposomes can also be pH-sensitive to release its content preferentially in acidic environments, like tumors, and be surface-functionalized for targeting purposes. Herein, we developed a folate-coated pH-sensitive liposome as a drug delivery system for IRN to reach improved tumor therapy without potential adverse events. Liposomes were prepared containing IRN and characterized for particle size, polydispersity index, zeta potential, concentration, encapsulation, cellular uptake, and release profile. Antitumor activity was investigated in a murine model of colorectal cancer, and its toxicity was evaluated by hematological/biochemical tests and histological analysis of main organs. The results showed vesicles smaller than 200 nm with little dispersion, a surface charge close to neutral, and high encapsulation rate of over 90%. The system demonstrated prolonged and sustained release in pH-dependent manner with high intracellular drug delivery capacity. Importantly, the folate-coated pH-sensitive formulation had significantly better antitumor activity than the pH-dependent system only or the free drug. Tumor tissue of IRN-containing groups presented large areas of necrosis. Furthermore, no evidence of systemic toxicity was found for the groups investigated. Thus, our developed nanodrug IRN delivery system can potentially be an alternative to conventional colorectal cancer treatment.

One-carbon metabolism and global DNA methylation in mothers of individuals with Down syndrome.

Down syndrome (DS) is the most common chromosomal disorder, resulting from the failure of normal chromosome 21 segregation. Studies have suggested that impairments within the one-carbon metabolic pathway can be of relevance for the global genome instability observed in mothers of individuals with DS. Based on the association between global DNA hypomethylation, genome instability, and impairments within the one-carbon metabolic pathway, the present study aimed to identify possible predictors, within the one-carbon metabolism, of global DNA methylation, measured by methylation patterns of LINE-1 and Alu repetitive sequences, in mothers of individuals with DS and mothers of individuals without the syndrome. In addition, we investigated one-carbon genetic polymorphisms and metabolites as maternal predisposing factors for the occurrence of trisomy 21 in children. Eighty-three samples of mothers of children with DS with karyotypically confirmed free trisomy 21 (case group) and 84 of mothers who had at least one child without DS or any other aneuploidy were included in the study. Pyrosequencing assays were performed to access global methylation. The results showed that group affiliation (case or control), betaine-homocysteine methyltransferase (BHMT) G742A and transcobalamin 2 (TCN2) C776G polymorphisms, and folate concentration were identified as predictors of global Alu DNA methylation values. In addition, thymidylate synthase (TYMS) 28-bp repeats 2R/3R or 3R/3R genotypes are independent maternal predisposing factors for having a child with DS. This study adds evidence that supports the association of impairments in the one-carbon metabolism, global DNA methylation, and the possibility of having a child with DS.

Maternal Folic Acid Intake and Methylation Status of Genes Associated with Ventricular Septal Defects in Children: Case-Control Study.

BACKGROUND: DNA methylation is the best epigenetic mechanism for explaining the interactions between nutrients and genes involved in intrauterine growth and development programming. A possible contributor of methylation abnormalities to congenital heart disease is the folate methylation regulatory pathway; however, the mechanisms and methylation patterns of VSD-associated genes are not fully understood. OBJECTIVE: To determine if maternal dietary intake of folic acid (FA) is related to the methylation status (MS) of VSD-associated genes (AXIN1, MTHFR, TBX1, and TBX20). METHODS: Prospective case-control study; 48 mothers and their children were evaluated. The mothers' dietary variables were collected through a food frequency questionnaire focusing on FA and the consumption of supplements with FA. The MS of promoters of genes was determined in the children. RESULTS: The intake of FA supplements was significantly higher in the control mothers. In terms of maternal folic acid consumption, significant differences were found in the first trimester of pregnancy. Significant differences were observed in the MS of MTHFR and AXIN1 genes in VSD and control children. A correlation between maternal FA supplementation and MS of AXIN1 and TBX20 genes was found in control and VSD children, respectively. CONCLUSIONS: A lower MS of AXIN1 genes and a higher MS of TBX20 genes is associated with FA maternal supplementation.

Relevancia del metabolismo del folato en el contexto de enfermedades neurodegenerativas / Relevance of folate metabolism in neurodegenerative disorders

Introducción: Varias enfermedades neurodegenerativas están asociadas a alteraciones en el metabolismo del folato, lo que tiene sustanciales implicaciones fisiopatológicas, clínicas y terapéuticas potenciales. Objetivo: Reflejar la relevancia del metabolismo del folato para enfermedades neurodegenerativas, destacando su significación fisiopatológica y clínica, y sus implicaciones terapéuticas. Material y métodos: Se consultaron las bases de datos especializadas en busca de artículos publicados hasta marzo de 2020. Se emplearon descriptores específicos y operadores booleanos. Se empleó la estrategia de búsqueda avanzada para la selección de los artículos, teniendo en cuenta la calidad metodológica o validez de los estudios. Desarrollo: Fueron identificadas evidencias de asociación entre alteraciones del metabolismo del folato y enfermedades neurodegenerativas. Se han identificado variantes en genes que codifican enzimas involucradas en el metabolismo del folato, y modificaciones en patrones de metilación de ADN, asociadas al riesgo o a la gravedad clínica de las enfermedades de Alzheimer, Parkinson, Huntington, Temblor Esencial y Ataxia Espinocerebelosa tipo 2. Fueron encontradas asociaciones entre enfermedades neurodegenerativas y alteraciones en los niveles de metabolitos del folato, y la frecuencia de micronúcleos. Se han realizado varios estudios observacionales o experimentales que indican que la suplementación con ácido fólico y vitaminas B6 y B12, tiene utilidad terapéutica potencial en el contexto de enfermedades neurodegenerativas. Conclusiones: El metabolismo del folato es de relevancia fisiopatológica, clínica y terapéutica para enfermedades neurodegenerativas. El uso de estrategias dirigidas a restaurar los niveles normales de folatos o de co-factores enzimáticos involucrados en el metabolismo del folato, o a reducir la acumulación de homocisteína, tiene potenciales aplicaciones terapéuticas en el contexto de estas enfermedades(AU)

Introduction: Several neurodegenerative disorders are associated with alterations in folate metabolism, having essential physiopathological, clinical and therapeutic implications. Objective: To assess the relevance of folate metabolism in neurodegenerative disorders, highlighting its physiopathological, clinical and therapeutic significance. Material and Methods: Specialized biomedical databases were searched for studies published up to March 2020. Descriptors and Boolean operators were used. Advanced search strategy was used for the selection of articles, taking into account the methodological quality and validity of the studies. Results: Strong evidence of the association between folate metabolism and neurodegenerative disorders were identified. Enzyme-coding genes involved in folate metabolism and epigenetic DNA modifications associated with increased risk or disease severity in Alzheimer´s, Parkinson´s, and Huntington´s diseases, Essential Tremor, and Spinocerebellar ataxia type 2 were also identified. Associations between neurodegenerative disorders and altered levels of folate metabolites and the frequency of micronuclei were found. A number of observational and experimental studies have demonstrated that the supplementation with folic acid and vitamin B6 and B12 has therapeutic potential in the context of neurodegenerative disorders. Conclusions: Folate metabolism is of physiopathological, clinical and therapeutic relevance for neurodegenerative disorders. The use of strategies to normalize folate levels or enzyme cofactors involved in folate metabolism or to reduce homocysteine levels has potential therapeutic applications for these disorders(AU)

MTHFR C677T and A1298C Polymorphisms in Breast Cancer, Gliomas and Gastric Cancer: A Review.

Folate (vitamin B9) is found in some water-soluble foods or as a synthetic form of folic acid and is involved in many essential biochemical processes. Dietary folate is converted into tetrahydrofolate, a vital methyl donor for most methylation reactions, including DNA methylation. 5,10-methylene tetrahydrofolate reductase (MTHFR) is a critical enzyme in the folate metabolism pathway that converts 5,10-methylenetetrahydrofolate into 5-methyltetrahydrofolate, which produces a methyl donor for the remethylation of homocysteine to methionine. MTHFR polymorphisms result in reduced enzyme activity and altered levels of DNA methylation and synthesis. MTHFR polymorphisms have been linked to increased risks of several pathologies, including cancer. Breast cancer, gliomas and gastric cancer are highly heterogeneous and aggressive diseases associated with high mortality rates. The impact of MTHFR polymorphisms on these tumors remains controversial in the literature. This review discusses the relationship between the MTHFR C677T and A1298C polymorphisms and the increased risk of breast cancer, gliomas, and gastric cancer. Additionally, we highlight the relevance of ethnic and dietary aspects of population-based studies and histological stratification of highly heterogeneous tumors. Finally, this review discusses these aspects as potential factors responsible for the controversial literature concerning MTHFR polymorphisms.

Study of Endogen Substrates, Drug Substrates and Inhibitors Binding Conformations on MRP4 and Its Variants by Molecular Docking and Molecular Dynamics.

Multidrug resistance protein-4 (MRP4) belongs to the ABC transporter superfamily and promotes the transport of xenobiotics including drugs. A non-synonymous single nucleotide polymorphisms (nsSNPs) in the ABCC4 gene can promote changes in the structure and function of MRP4. In this work, the interaction of certain endogen substrates, drug substrates, and inhibitors with wild type-MRP4 (WT-MRP4) and its variants G187W and Y556C were studied to determine differences in the intermolecular interactions and affinity related to SNPs using protein threading modeling, molecular docking, all-atom, coarse grained, and umbrella sampling molecular dynamics simulations (AA-MDS and CG-MDS, respectively). The results showed that the three MRP4 structures had significantly different conformations at given sites, leading to differences in the docking scores (DS) and binding sites of three different groups of molecules. Folic acid (FA) had the highest variation in DS on G187W concerning WT-MRP4. WT-MRP4, G187W, Y556C, and FA had different conformations through 25 ns AA-MD. Umbrella sampling simulations indicated that the Y556C-FA complex was the most stable one with or without ATP. In Y556C, the cyclic adenosine monophosphate (cAMP) and ceefourin-1 binding sites are located out of the entrance of the inner cavity, which suggests that both cAMP and ceefourin-1 may not be transported. The binding site for cAMP and ceefourin-1 is quite similar and the affinity (binding energy) of ceefourin-1 to WT-MRP4, G187W, and Y556C is greater than the affinity of cAMP, which may suggest that ceefourin-1 works as a competitive inhibitor. In conclusion, the nsSNPs G187W and Y556C lead to changes in protein conformation, which modifies the ligand binding site, DS, and binding energy.

Inhibition of MTOR signaling impairs rat embryo organogenesis by affecting folate availability.

Mechanistic target of rapamycin (MTOR) is essential for embryo development by acting as a nutrient sensor to regulate cell growth, proliferation and metabolism. Folate is required for normal embryonic development and it was recently reported that MTOR functions as a folate sensor. In this work, we tested the hypothesis that MTOR functions as a folate sensor in the embryo and its inhibition result in embryonic developmental delay affecting neural tube closure and that these effects can be rescued by folate supplementation. Administration of rapamycin (0.5 mg/kg) to rats during early organogenesis inhibited embryonic ribosomal protein S6, a downstream target of MTOR Complex1, markedly reduced embryonic folate incorporation (-84%, P < 0.01) and induced embryo developmental impairments, as shown by an increased resorption rate, reduced embryo somite number and delayed neural tube closure. These alterations were prevented by folic acid administered to the dams. Differently, although an increased rate of embryonic rotation defects was observed in the rapamycin-treated dams, this alteration was not prevented by maternal folic acid supplementation. In conclusion, MTOR inhibition during organogenesis in the rat resulted in decreased folate levels in the embryo, increased embryo resorption rate and impaired embryo development. These data suggest that MTOR signaling influences embryo folate availability, possibly by regulating the transfer of folate across the maternal-embryonic interface.

Starch nanofibers as vehicles for folic acid supplementation: thermal treatment, UVA irradiation and in vitro simulation of digestion.

BACKGROUND: The supplementation of folic acid in food is essential in the human diet. The present study aimed to encapsulate folic acid at different concentrations (5, 10 and 15% (w/w) on a dry basis) in potato starch nanofibers produced through electrospinning. The starch/folic acid nanofibers were evaluated through morphology, Fourier transform infrared (FTIR) spectra, thermal properties, encapsulation efficiency (EE) and in vitro simulation of the human digestion. The nanofibers were also evaluated based on the folic acid content after thermal treatment (100 and 180 °C) and UVA irradiation (1 and 24 h). RESULTS: Folic acid incorporation influenced the morphology of the nanofibers to display a homogeneous and beadless morphology for nanofibers containing 15% folic acid compared with the other nanofibers (0, 5 and 10% folic acid). The mean diameter varied from 75 to 81 nm. Folic acid characteristic bands and peaks were not found in the nanofiber FTIR spectra and thermograms, respectively. The EE was 73, 87 and 95% for nanofibers with 5, 10 and 15% folic acid, respectively. CONCLUSIONS: The starch nanofibers protected folic acid from high temperature and UVA irradiation and during in vitro digestion, showing a release of the vitamin at the end of the simulation (intestinal conditions). The supplementation of folic acid in foods can be effectively achieved by its encapsulation into starch nanofibers, to ensure its protection and controlled release. © 2020 Society of Chemical Industry.

Evaluation of vitamin-producing and immunomodulatory lactic acid bacteria as a potential co-adjuvant for cancer therapy in a mouse model.

AIMS: To evaluate a mixture of selected lactic acid bacteria (LAB) (a riboflavin-producer, a folate-producer and an immunomodulatory strain) as co-adjuvant for 5-fluorouracil (5-FU) chemotherapy in cell culture and using a 4T1 cell animal model of breast cancer. METHODS AND RESULTS: The viability of Caco-2 cells exposed to 5-FU and/or LAB was analysed. Mice bearing breast tumour were treated with 5-FU and/or LAB. Tumour growth was measured. Intestinal mucositis (IM) was evaluated in small intestine; haematological parameters and plasma cytokines were determined. The bacterial mixture did not negatively affect the cytotoxic activity of 5-FU on Caco-2 cells. The LAB mixture attenuated the IM and prevented blood cell decreases associated with 5-FU treatment. Mice that received 5-FU and LAB mixture decreased tumour growth and showed modulation of systemic cytokines modified by both tumour growth and 5-FU treatment. The LAB mixture by itself delayed tumour growth. CONCLUSIONS: The mixture of selected LAB was able to reduce the side-effects associated with chemotherapy without affecting its primary anti-tumour activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This bacterial mixture could prevent the interruption of conventional oncologic therapies by reducing undesirable side-effects. In addition, this blend would provide essential nutrients (vitamins) to oncology patients.

Optimized PAH/Folic acid layer-by-layer films as an electrochemical biosensor for the detection of folate receptors.

Folate receptor alpha (FR-α) is a glycoprotein overexpressed in tumor cell surfaces, especially in gynecologic cancers, and can be used as a biomarker for diagnostics. Currently, FRα is quantified by positron emission tomography (PET) or fluorescence imaging techniques. However, these methods are costly and time-consuming. We report on the development of an electrochemical biosensor for FRα detection based on the use of nanostructured layer-by-layer (LbL) films as modified electrodes. Multilayer films were deposited on indium tin oxide (ITO) electrodes by the alternately assembling of positively charged polyallylamine hydrochloride (PAH) and negatively charged folic acid (FA), used as the biorecognition element. UV-vis and FTIR spectroscopies revealed the successful PAH and FA adsorption on ITO. Devices performance was evaluated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The [PAH/FA] films presented a good reproducibility (RSD of 1.12%) and stability when stored in the Tris-HCl solution (RSD 6.7%). The biosensor electrochemical response exhibited a linear relationship with FRα concentration in the range from 10 to 40 nM. The limit of detection reached for CV and EIS measurements were 0.7 and 1.5 nM, respectively. As a proof-of-concept, we show that the devices can differenciate tumor cells from healthy cell, showing an excellent selectivity. The biosensor device based on [PAH/FA] films represents a promising strategy for a simple, rapid, and low-cost cancer diagnosis through FRα quantification on the surface of cancer cells.

Gene Polymorphisms Involved in Folate Metabolism and DNA Methylation with the Risk of Head and Neck Cancer.

BACKGROUND: Folate is essential for DNA synthesis, repair, and methylation. Polymorphisms in genes associated with folate metabolism may alter these processes and, consequently, modulate cancer development. AIM: We aimed to assess DNMT3B -149C/T (rs2424913), DNMT3B -283T/C (rs6087990), DNMT3B -579G/T (rs2424909), DHFR 19-pb ins/del (rs70991108), SHMT1 1420C/T (rs1979277), and TYMS 28-bp tandem repeat (rs34743033) polymorphisms with risk of head and neck cancer. METHODS: A case-control study was conducted in 1,086 Brazilian individuals. Real-time and conventional polymerase chain reactions-PCR were performed for genotyping the polymorphisms. RESULTS: The single nucleotide polymorphism (SNP), DNMT3B -283T/C, revealed a higher risk of head and neck squamous cell carcinoma (HNSCC) when compared with the C group in the codominant (p < 0.001), dominant (p <0.001), and overdominant (p= 0.001) models for T/C and C/C genotypes. DNMT3B -149C/T and DNMT3B -579G/T revealed no association between groups in any model. The DHFR 19-pb ins/del polymorphism protected against HNSCC development compared to the C group by the codominant (p < 0.001), dominant (p < 0.001), and overdominant (p < 0.001) models. In the TYMS, the 3R/3R genotype had a protective effect against HNSCC development compared with the C group by the recessive models (p= 0.009). In contrast, SHMT1 1420 C/T presented no association between the HNSCC and C groups. DHFR 19-pb ins/del polymorphisms protected against oral cavity cancer (p= 0.003), and only TYMS-28 3R/3R decreased the risk of tumor progression (p= 0.023). In the Kaplan-Meier curve, an association was found between DHFR ins/ins and TYMS -28 3R carriers with respect to relapse-free time; further, DNMT3B -579 T and TYMS-28 2R/2R carriers had longer survival times. CONCLUSION: DNMT3B -283T/C is associated with higher risk, whereas DHFR 19-pb ins/del and TYMS 28 3R/3R protect against head and neck cancer. We also highlighted the association of TYMS 3R/3R genotype carriers with relapse-free cancer protection and survival time.

A comprehensive analysis of AHRR gene as a candidate for cleft lip with or without cleft palate.

Cleft lip and palate (CL/P) is among the most common congenital malformations and affects 1 in 700 newborns. CL/P is caused by genetic and environmental factors (maternal smoking, alcohol or drug use and others). Many genes and loci were associated with cleft lip/palate but the amount of heterogeneity justifies identifying new causal genes and variants. AHRR (Aryl-Hydrocarbon Receptor Repressor) gene has recently been related to CL/P however, few functional studies analyze the genotypephenotype interaction of AHRR with CL/P. Several studies associate the molecular pathway of AHRR to CL/P which indicates this gene as a functional candidate in CL/P etiology. METHODS: Systematic Literature Review was performed using PUBMED database with the keywords cleft lip, cleft palate, orofacial cleft, AHRR and synonyms. SLR resulted in 37 included articles. RESULTS: AHRR is a positional and functional candidate gene for CL/P. In silico analysis detected interactions with other genes previously associated to CL/P like ARNT and CYP1A1. AHRR protein regulates cellular toxicity through TCDD mediated AHR pathway. Exposure to TCDD in animal embryos is AHR mediated and lead to cleft palate due to palate fusion failure and post fusion rupture. AHRR regulates cellular growth and differentiation, fundamental to lip and palatogenesis. AHRR decreases carcinogenesis and recently a higher tumor risk has been described in CL/P patients and families. AHRR is also a smoking biomarker due to changed methylation sites found in smokers DNA although folate intake may partially revert these methylation alterations. This corroborates the role of maternal smoking and lack of folate supplementation as risk factors for CL/P. CONCLUSION: This research identified the importance of AHRR in dioxin response and demonstrated an example of genetic and environmental interaction, indispensable in the development of many complex diseases.