Dolutegravir and Folic Acid Interaction during Neural System Development in Zebrafish Embryos.

Dolutegravir (DTG) is one of the most prescribed antiretroviral drugs for treating people with HIV infection, including women of child-bearing potential or pregnant. Nonetheless, neuropsychiatric symptoms are frequently reported. Early reports suggested that, probably in relation to folic acid (FA) shortage, DTG may induce neural tube defects in infants born to women taking the drug during pregnancy. Subsequent reports did not definitively confirm these findings. Recent studies in animal models have highlighted the association between DTG exposure in utero and congenital anomalies, and an increased risk of neurologic abnormalities in children exposed during in utero life has been reported. Underlying mechanisms for DTG-related neurologic symptoms and congenital anomalies are not fully understood. We aimed to deepen our knowledge on the neurodevelopmental effects of DTG exposure and further explore the protective role of FA by the use of zebrafish embryos. We treated embryos at 4 and up to 144 h post fertilization (hpf) with a subtherapeutic DTG concentration (1 µM) and observed the disruption of the anterior-posterior axis and several morphological malformations in the developing brain that were both prevented by pre-exposure (2 hpf) and rescued by post-exposure (10 hpf) with FA. By whole-mount in situ hybridization with riboprobes for genes that are crucial during the early phases of neurodevelopment (ntl, pax2a, ngn1, neurod1) and by in vivo visualization of the transgenic Tg(ngn1:EGFP) zebrafish line, we found that DTG induced severe neurodevelopmental defects over time in most regions of the nervous system (notochord, midbrain-hindbrain boundary, eye, forebrain, midbrain, hindbrain, spinal cord) that were mostly but not completely rescued by FA supplementation. Of note, we observed the disruption of ngn1 expression in the dopaminergic regions of the developing forebrain, spinal cord neurons and spinal motor neuron projections, with the depletion of the tyrosine hydroxylase (TH)+ dopaminergic neurons of the dorsal diencephalon and the strong reduction in larvae locomotion. Our study further supports previous evidence that DTG can interfere with FA pathways in the developing brain but also provides new insights regarding the mechanisms involved in the increased risk of DTG-associated fetal neurodevelopmental defects and adverse neurologic outcomes in in utero exposed children, suggesting the impairment of dopaminergic pathways.

The Role of One-Carbon Metabolism and Methyl Donors in Medically Assisted Reproduction: A Narrative Review of the Literature.

One-carbon (1-C) metabolic deficiency impairs homeostasis, driving disease development, including infertility. It is of importance to summarize the current evidence regarding the clinical utility of 1-C metabolism-related biomolecules and methyl donors, namely, folate, betaine, choline, vitamin B12, homocysteine (Hcy), and zinc, as potential biomarkers, dietary supplements, and culture media supplements in the context of medically assisted reproduction (MAR). A narrative review of the literature was conducted in the PubMed/Medline database. Diet, ageing, and the endocrine milieu of individuals affect both 1-C metabolism and fertility status. In vitro fertilization (IVF) techniques, and culture conditions in particular, have a direct impact on 1-C metabolic activity in gametes and embryos. Critical analysis indicated that zinc supplementation in cryopreservation media may be a promising approach to reducing oxidative damage, while female serum homocysteine levels may be employed as a possible biomarker for predicting IVF outcomes. Nonetheless, the level of evidence is low, and future studies are needed to verify these data. One-carbon metabolism-related processes, including redox defense and epigenetic regulation, may be compromised in IVF-derived embryos. The study of 1-C metabolism may lead the way towards improving MAR efficiency and safety and ensuring the lifelong health of MAR infants.

A folate inhibitor exploits metabolic differences in Pseudomonas aeruginosa for narrow-spectrum targeting.

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections for which the development of antibiotics is urgently needed. Unlike most enteric bacteria, P. aeruginosa lacks enzymes required to scavenge exogenous thymine. An appealing strategy to selectively target P. aeruginosa is to disrupt thymidine synthesis while providing exogenous thymine. However, known antibiotics that perturb thymidine synthesis are largely inactive against P. aeruginosa.Here we characterize fluorofolin, a dihydrofolate reductase (DHFR) inhibitor derived from Irresistin-16, that exhibits significant activity against P. aeruginosa in culture and in a mouse thigh infection model. Fluorofolin is active against a wide range of clinical P. aeruginosa isolates resistant to known antibiotics. Metabolomics and in vitro assays using purified folA confirm that fluorofolin inhibits P. aeruginosa DHFR. Importantly, in the presence of thymine supplementation, fluorofolin activity is selective for P. aeruginosa. Resistance to fluorofolin can emerge through overexpression of the efflux pumps MexCD-OprJ and MexEF-OprN, but these mutants also decrease pathogenesis. Our findings demonstrate how understanding species-specific genetic differences can enable selective targeting of important pathogens while revealing trade-offs between resistance and pathogenesis.

Two maize homologs of mammalian proton-coupled folate transporter, ZmMFS_1-62 and ZmMFS_1-73, are essential to salt and drought tolerance.

Folates are essential to the maintenance of normal life activities in almost all organisms. Proton-coupled folate transporter (PCFT), belonging to the major facilitator superfamily, is one of the three major folate transporter types widely studied in mammals. However, information about plant PCFTs is limited. Here, a genome-wide identification of maize PCFTs was performed, and two PCFTs, ZmMFS_1-62 and ZmMFS_1-73, were functionally investigated. Both proteins contained the typical 12 transmembrane helixes with N- and C-termini located in the cytoplasm, and were localized in the plasma membrane. Molecular docking analysis indicated their binding activity with folates via hydrogen bonding. Interference with ZmMFS_1-62 and ZmMFS_1-73 in maize seedlings through virus-induced gene silencing disrupted folate homeostasis, mainly in the roots, and reduced tolerance to drought and salt stresses. Moreover, a molecular chaperone protein, ZmHSP20, was found to interact with ZmMFS_1-62 and ZmMFS_1-73, and interference with ZmHSP20 in maize seedlings also led to folate disruption and increased sensitivity to drought and salt stresses. Overall, this is the first report of functional identification of maize PCFTs, which play essential roles in salt and drought stress tolerance, thereby linking folate metabolism with abiotic stress responses in maize.

Association of Increased Homocysteine Levels with Impaired Folate Metabolism and Vitamin B Deficiency in Early-Onset Multiple Sclerosis.

The contents of homocysteine (HCy), cyanocobalamin (vitamin B12), folic acid (vitamin B9), and pyridoxine (vitamin B6) were analyzed and the genotypes of the main gene polymorphisms associated with folate metabolism (C677T and A1298C of the MTHFR gene, A2756G of the MTR gene and A66G of the MTRR gene) were determined in children at the onset of multiple sclerosis (MS) (with disease duration of no more than six months), healthy children under 18 years (control group), healthy adults without neurological pathology, adult patients with MS at the onset of disease, and adult patients with long-term MS. A significant increase in the HCy levels was found in children at the MS onset compared to healthy children of the corresponding age. It was established that the content of HCy in children has a high predictive value. At the same time, an increase in the HCy levels was not accompanied by the deficiency of vitamins B6, B9, and B12 in the blood. The lack of correlation between the laboratory signs of vitamin deficiency and HCy levels may be due to the polymorphic variants of folate cycle genes. An increased HCy level should be considered as a marker of functional disorders of folate metabolism accompanying the development of pathological process in pediatric MS. Our finding can be used to develop new approaches to the prevention of demyelination in children and treatment of pediatric MS.

Folic Acid-Conjugated Brush Polymers Show Enhanced Blood-Brain Barrier Crossing in Static and Dynamic In Vitro Models Toward Brain Cancer Targeting Therapy.

Over the past decades, evidence has consistently shown that treatment of central nervous system (CNS)-related disorders, including Alzheimer's disease, Parkinson's disease, stroke, multiple sclerosis, and brain cancer, is limited due to the presence of the blood-brain barrier (BBB). To assist with the development of new therapeutics, it is crucial to engineer a drug delivery system that can cross the BBB efficiently and reach target cells within the brain. In this study, we present a potentially efficient strategy for targeted brain delivery through utilization of folic acid (FA)-conjugated brush polymers, that specifically target the reduced folate carrier (RFC, SLC19A1) expressed on brain endothelial cells. Here, azide (N3)-decorated brush polymers were prepared in a straightforward manner coupling a heterotelechelic α-NH2, ω-N3-poly(2-ethyl-2-oxazoline) (NH2-PEtOx-N3) to N-acylated poly(amino ester) (NPAE)-based brushes. Strain-promoted azide-alkyne cycloaddition (SPAAC) 'click chemistry' with DBCO-folic acid (FA) yielded FA-brush polymers. Interestingly, while azide functionalization of the brush polymers dramatically reduced their association to brain microvascular endothelial cells (hCMEC/D3), the introduction of FA to azide led to a substantial accumulation of the brush polymers in hCMEC/D3 cells. The ability of the polymeric brush polymers to traverse the BBB was quantitatively assessed using different in vitro BBB models including static Transwell and microfluidic platforms. FA-brush polymers showed efficient transport across hCMEC/D3 cells in a manner dependent on FA composition, whereas nonfunctionalized brush polymers exhibited limited trafficking under the same conditions. Further, cellular uptake inhibition studies suggested that the interaction and transport pathway of FA-brush polymers across BBB relies on the RFC-mediated pathways. The potential application of the developed FA-brush polymers in brain cancer delivery was also investigated in a microfluidic model of BBB-glioblastoma. Brush polymers with more FA units successfully presented an enhanced accumulation into U-87 MG glioma cells following its BBB crossing, compared to controls. These results demonstrate that FA-modified brush polymers hold a great potential for more efficient delivery of future brain therapeutics.

Altered placental immune cell composition and gene expression with isolated fetal spina bifida.

PROBLEM: Fetal spina bifida (SB) is more common in pregnant people with folate deficiency or anomalies of folate metabolism. It is also known that fetuses with SB have a higher risk of low birthweight, a condition that is typically placental-mediated. We therefore hypothesized that fetal SB would associate with altered expression of key placental folate transporters and an increase in Hofbauer cells (HBCs), which are folate-dependent placental macrophages. METHOD OF STUDY: Folate receptor-α (FRα), proton coupled folate receptor (PCFT), and reduced folate carrier (RFC) protein localization and expression (immunohistochemistry) and HBC phenotypes (HBC abundance and folate receptor-ß [FRß] expression; RNA in situ hybridization) were assessed in placentae from fetuses with SB (cases; n = 12) and in term (n = 10) and gestational age (GA) - and maternal body mass index - matched (n = 12) controls without congenital anomalies. RESULTS: Cases had a higher proportion of placental villous cells that were HBCs (6.9% vs. 2.4%, p = .0001) and higher average HBC FRß expression (3.2 mRNA molecules per HBC vs. 2.3, p = .03) than GA-matched controls. HBCs in cases were largely polarized to a regulatory phenotype (median 92.1% of HBCs). In sex-stratified analyses, only male cases had higher HBC levels and HBC FRß expression than GA-matched controls. There were no differences between groups in the total percent of syncytium and stromal cells that were positive for FRα, PCFT, or RFC protein immunolabeling. CONCLUSIONS: HBC abundance and FRß expression by HBCs are increased in placentae of fetuses with SB, suggesting immune-mediated dysregulation in placental phenotype, and could contribute to SB-associated comorbidities.

Role of folate receptor α in the partial rejuvenation of dentate gyrus cells: Improvement of cognitive function in 21-month-old aged mice.

Neuronal aging may be, in part, related to a change in DNA methylation. Thus, methyl donors, like folate and methionine, may play a role in cognitive changes associated to neuronal aging. To test the role of these metabolites, we performed stereotaxic microinjection of these molecules into the dentate gyrus (DG) of aged mice (an average age of 21 month). Folate, but not S-Adenosyl-Methionine (SAM), enhances cognition in aged mice. In the presence of folate, we observed partial rejuvenation of DG cells, characterized by the expression of juvenile genes or reorganization of extracellular matrix. Here, we have also tried to identify the mechanism independent of DNA methylation, that involve folate effects on cognition. Our analyses indicated that folate binds to folate receptor α (FRα) and, upon folate binding, FRα is transported to cell nucleus, where it is acting as transcription factor for expressing genes like SOX2 or GluN2B. In this work, we report that a FRα binding peptide also replicates the folate effect on cognition, in aged mice. Our data suggest that such effect is not sex-dependent. Thus, we propose the use of this peptide to improve cognition since it lacks of folate-mediated side effects. The use of synthetic FRα binding peptides emerge as a future strategy for the study of brain rejuvenation.

Simultaneous Determination of One-Carbon Folate Metabolites and One-Carbon-Related Amino Acids in Biological Samples Using a UHPLC-MS/MS Method.

One-carbon folate metabolites and one-carbon-related amino acids play an important role in human physiology, and their detection in biological samples is essential. However, poor stability as well as low concentrations and occurrence in different species in various biological samples make their quantification very challenging. The aim of this study was to develop a simple, fast, and sensitive ultra-high-performance liquid chromatography MS/MS (UHPLC-MS/MS) method for the simultaneous quantification of various one-carbon folate metabolites (folic acid (FA), tetrahydrofolic acid (THF), p-aminobenzoyl-L-glutamic acid (pABG), 5-formyltetrahydrofolic acid (5-CHOTHF), 5-methyltetrahydrofolic acid (5-CH3THF), 10-formylfolic acid (10-CHOFA), 5,10-methenyl-5,6,7,8-tetrahydrofolic acid (5,10-CH+-THF), and 4-α-hydroxy-5-methyltetrahydrofolate (hmTHF)) and one-carbon-related amino acids (homocysteine (Hcy), methionine (Met), S-ade-L-homocysteine (SAH), and S-ade-L-methionine (SAM)). The method was standardized and validated by determining the selectivity, carryover, limits of detection, limits of quantitation, linearity, precision, accuracy, recovery, and matrix effects. The extraction methods were optimized with respect to several factors: protease-amylase treatment on embryos, deconjugation time, methanol precipitation, and proteins' isoelectric point precipitation on the folate recovery. Ten one-carbon folate metabolites and four one-carbon-related amino acids were detected using the UHPLC-MS/MS technique in various biological samples. The measured values of folate in human plasma, serum, and whole blood (WB) lay within the concentration range for normal donors. The contents of each analyte in mouse plasma were as follows: pABG (864.0 nmol/L), 5-CH3THF (202.2 nmol/L), hmTHF (122.2 nmol/L), Met (8.63 µmol/L), and SAH (0.06 µmol/L). The concentration of each analyte in mouse embryos were as follows: SAM (1.09 µg/g), SAH (0.13 µg/g), Met (16.5 µg/g), 5,10-CH+THF (74.3 ng/g), pABG (20.6 ng/g), and 5-CH3THF (185.4 ng/g). A simple and rapid sample preparation and UHPLC-MS/MS method was developed and validated for the simultaneous determination of the one-carbon-related folate metabolites and one-carbon-related amino acids in different biological samples.

How food matrices modulate folate bioaccessibility: A comprehensive overview of recent advances and challenges.

The incomplete absorption of dietary folate makes it crucial to understand how food matrices affect folate bioaccessibility. Bioavailability encompasses bioaccessibility, which depicts the proportion that is liberated from the food matrix during digestion and becomes available for absorption. Bioavailability studies are expensive and difficult to control, whereas bioaccessibility studies utilize in vitro digestion models to parameterize the complex digestion, allowing the evaluation of the effect of food matrices on bioaccessibility. This review covers the folate contents in various food matrices, the methods used to determine and the factors affecting folate bioaccessibility, and the advances and challenges in understanding how food matrices affect folate bioaccessibility. The methods for determining bioaccessibility have been improved in the last decade. Current research shows that food matrices modulate folate bioaccessibility by affecting the liberation and stability of folate during digestion but do not provide enough information about folate and food component interactions at the molecular level. In addition, information on folate interconversion and degradation during digestion is scant, hindering our understanding of the impact of food matrices on folate stability. Moreover, the role of conjugase inhibitors should not be neglected when evaluating the nutritional value of food folates. Due to the complexity of food digestion, holistic methods should be applied to investigate bioaccessibility. By synthesizing the current state of knowledge on this topic, this review highlights the lack of in-depth understanding of the mechanisms of how food matrices modulate folate bioaccessibility and provides insights into potential strategies for accurate evaluation of the nutritional value of dietary folate.

Toxic effects of triclosan on hepatic and intestinal lipid accumulation in zebrafish via regulation of m6A-RNA methylation.

Triclosan (TCS), recognized as an endocrine disruptor, has raised significant concerns due to its widespread use and potential health risks. To explore the impact of TCS on lipid metabolism, both larval and adult zebrafish were subjected to acute and chronic exposure to TCS. Through analyzes of biochemical and physiological markers, as well as Oil Red O (ORO) and hematoxylin and eosin (H&E) staining, our investigation revealed that TCS exposure induced hepatic and intestinal lipid accumulation in larval and adult zebrafish, leading to structural damage and inflammatory responses in these tissues. The strong affinity of TCS with PPARγ and subsequent pathway activation indicate that PPARγ pathway plays a crucial role in TCS-induced lipid buildup. Furthermore, we observed a decrease in m6A-RNA methylation levels in the TCS-treated group, which attributed to the increased activity of the demethylase FTO and concurrent suppression of the methyltransferase METTL3 gene expression by TCS. The alteration in methylation dynamics is identified as a potential underlying mechanism behind TCS-induced lipid accumulation. To address this concern, we explored the impact of folic acid-a methyl donor for m6A-RNA methylation-on lipid accumulation in zebrafish. Remarkably, folic acid administration partially alleviated lipid accumulation by restoring m6A-RNA methylation. This restoration, in turn, contributed to a reduction in inflammatory damage observed in both the liver and intestines. Additionally, folic acid partially mitigates the up-regulation of PPARγ and related genes induced by TCS. These findings carry substantial implications for understanding the adverse effects of environmental pollutants such as TCS. They also emphasize the promising potential of folic acid as a therapeutic intervention to alleviate disturbances in lipid metabolism induced by environmental pollutants.

Synthesis and biological evaluation of folic acid-rotenol conjugate as a potent targeted anticancer prodrug.

Rotenone, a plant-based agricultural insecticide, has been shown to have anti-tumor activity through targeting mitochondrial complex I in cancer cells. However, off-target toxic side effect on nervous systems have greatly restricted the application of rotenone as anticancer drugs. Here, a folic acid-rotenol (FA-rotenol) conjugate was prepared by covalent coupling of the tumor-targeting ligand folic acid with rotenone derivative-rotenol to enhance its accumulation at tumor site. FA-rotenol conjugates present high in vitro cytotoxicties against several cell lines by inducing mitochondrial membrane potential depolarization and increasing the level of intracellular reactive oxygen species (ROS) to activate the mitochondrial pathway of apoptosis and enhance the G2/M cell cycle arrest. Because of the high affinity with over-expressed folate receptors, FA-rotenol conjugate demonstrated more effective in vivo therapeutic outcomes in 4T1 tumor-bearing mice than rotenone and rotenol. In addition, FA-rotenol conjugate can markedly inhibit the cell migration and invasion of HepG-2 cells. These studies confirm the feasibility of tumor-targeted ligand conjugated rotenone derivatives for targeted antitumor therapy; likewise, they lay the foundations for the development of other rotenol-conjugates with antitumor potential.

Inhibition of mitochondrial folate metabolism drives differentiation through mTORC1 mediated purine sensing.

Supporting cell proliferation through nucleotide biosynthesis is an essential requirement for cancer cells. Hence, inhibition of folate-mediated one carbon (1C) metabolism, which is required for nucleotide synthesis, has been successfully exploited in anti-cancer therapy. Here, we reveal that mitochondrial folate metabolism is upregulated in patient-derived leukaemic stem cells (LSCs). We demonstrate that inhibition of mitochondrial 1C metabolism through impairment of de novo purine synthesis has a cytostatic effect on chronic myeloid leukaemia (CML) cells. Consequently, changes in purine nucleotide levels lead to activation of AMPK signalling and suppression of mTORC1 activity. Notably, suppression of mitochondrial 1C metabolism increases expression of erythroid differentiation markers. Moreover, we find that increased differentiation occurs independently of AMPK signalling and can be reversed through reconstitution of purine levels and reactivation of mTORC1. Of clinical relevance, we identify that combination of 1C metabolism inhibition with imatinib, a frontline treatment for CML patients, decreases the number of therapy-resistant CML LSCs in a patient-derived xenograft model. Our results highlight a role for folate metabolism and purine sensing in stem cell fate decisions and leukaemogenesis.

Epigenetic Genome Modifications during Pregnancy: The Impact of Essential Nutritional Supplements on DNA Methylation.

Pregnancy is an extremely stressful period in a pregnant woman's life. Currently, women's awareness of the proper course of pregnancy and its possible complications is constantly growing. Therefore, a significant percentage of women increasingly reach for various dietary supplements during gestation. Some of the most popular substances included in multi-ingredient supplements are folic acid and choline. Those substances are associated with positive effects on fetal intrauterine development and fewer possible pregnancy-associated complications. Recently, more and more attention has been paid to the impacts of specific environmental factors, such as diet, stress, physical activity, etc., on epigenetic modifications, understood as changes occurring in gene expression without the direct alteration of DNA sequences. Substances such as folic acid and choline may participate in epigenetic modifications by acting via a one-carbon cycle, leading to the methyl-group donor formation. Those nutrients may indirectly impact genome phenotype by influencing the process of DNA methylation. This review article presents the current state of knowledge on the use of folic acid and choline supplementation during pregnancy, taking into account their impacts on the maternal-fetal unit and possible pregnancy outcomes, and determining possible mechanisms of action, with particular emphasis on their possible impacts on epigenetic modifications.

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.

Noncanonical function of folate through folate receptor 1 during neural tube formation.

Folate supplementation reduces the occurrence of neural tube defects (NTDs), birth defects consisting in the failure of the neural tube to form and close. The mechanisms underlying NTDs and their prevention by folate remain unclear. Here we show that folate receptor 1 (FOLR1) is necessary for the formation of neural tube-like structures in human-cell derived neural organoids. FOLR1 knockdown in neural organoids and in Xenopus laevis embryos leads to NTDs that are rescued by pteroate, a folate precursor that is unable to participate in metabolism. We demonstrate that FOLR1 interacts with and opposes the function of CD2-associated protein, molecule essential for apical endocytosis and turnover of C-cadherin in neural plate cells. In addition, folates increase Ca2+ transient frequency, suggesting that folate and FOLR1 signal intracellularly to regulate neural plate folding. This study identifies a mechanism of action of folate distinct from its vitamin function during neural tube formation.

Vitamin D promotes the folate transport and metabolism in zebrafish (Danio rerio).

Vitamin D (VD) is a fat-soluble sterol that possesses a wide range of physiological functions. The present study aimed to evaluate the effects of VD on folate metabolism in zebrafish and further investigated the underlying mechanism. Wild-type (WT) zebrafish were fed with a diet containing 0 IU/kg VD3 or 800 IU/kg VD3 for 3 wk. Meanwhile, cyp2r1 mutant zebrafish with impaired VD metabolism was used as another model of VD deficiency. Our results showed that VD deficiency in zebrafish suppressed the gene expression of folate transporters, including reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) in the intestine. Moreover, VD influenced the gene expression of several enzymes related to cellular folate metabolism in the intestine and liver of zebrafish. Importantly, VD-deficient zebrafish contained a remarkably lower level of folate content in the liver. Notably, VD was incapable of altering folate metabolism in zebrafish when gut microbiota was depleted by antibiotic treatment. Further studies proved that gut commensals from VD-deficient fish displayed a lower capacity to produce folate than those from WT fish. Our study revealed the potential correlation between VD and folate metabolism in zebrafish, and gut microbiota played a key role in VD-regulated folate metabolism in zebrafish.NEW & NOTEWORTHY Our study has identified that VD influences intestinal uptake and transport of folate in zebrafish while also altering hepatic folate metabolism and storage. Interestingly, the regulatory effects of VD on folate transport and metabolism diminished after the gut flora was interrupted by antibiotic treatment, suggesting that the regulatory effects of VD on folate metabolism in zebrafish are most likely dependent on the intestinal flora.

Tumor-specific activation of folate receptor beta enables reprogramming of immune cells in the tumor microenvironment.

Folate receptors can perform folate transport, cell adhesion, and/or transcription factor functions. The beta isoform of the folate receptor (FRß) has attracted considerable attention as a biomarker for immunosuppressive macrophages and myeloid-derived suppressor cells, however, its role in immunosuppression remains uncharacterized. We demonstrate here that FRß cannot bind folate on healthy tissue macrophages, but does bind folate after macrophage incubation in anti-inflammatory cytokines or cancer cell-conditioned media. We further show that FRß becomes functionally active following macrophage infiltration into solid tumors, and we exploit this tumor-induced activation to target a toll-like receptor 7 agonist specifically to immunosuppressive myeloid cells in solid tumors without altering myeloid cells in healthy tissues. We then use single-cell RNA-seq to characterize the changes in gene expression induced by the targeted repolarization of tumor-associated macrophages and finally show that their repolarization not only changes their own phenotype, but also induces a proinflammatory shift in all other immune cells of the same tumor mass, leading to potent suppression of tumor growth. Because this selective reprogramming of tumor myeloid cells is accompanied by no systemic toxicity, we propose that it should constitute a safe method to reprogram the tumor microenvironment.

Synthesis and Characterization of Graphene Oxide/Polyethylene Glycol/Folic Acid/Brucine Nanocomposites and Their Anticancer Activity on HepG2 Cells.

Background: Liver cancer is the sixth most prevalent form of cancer and the second major cause of cancer-associated mortalities worldwide. Cancer nanotechnology has the ability to fundamentally alter cancer treatment, diagnosis, and detection. Objective: In this study, we explained the development of graphene oxide/polyethylene glycol/folic acid/brucine nanocomposites (GO/PEG/Bru-FA NCs) and evaluated their antimicrobial and anticancer effect on the liver cancer HepG2 cells. Methodology: The GO/PEG/Bru-FA NCs were prepared using the co-precipitation technique and characterized using various techniques. The cytotoxicity of the GO/PEG/Bru-FA NCs was tested against both liver cancer HepG2 and non-malignant Vero cells using an MTT assay. The antimicrobial activity of the GO/PEG/Bru-FA NCs was tested against several pathogens using the well diffusion technique. The effects of GO/PEG/Bru-FA NCs on endogenous ROS accumulation, apoptosis, and MMP levels were examined using corresponding fluorescent staining assays, respectively. The apoptotic protein expressions, such as Bax, Bcl-2, and caspases, were studied using the corresponding kits. Results: The findings of various characterization assays revealed the development of GO/PEG/Bru-FA NCs with face-centered spherical morphology and an agglomerated appearance with an average size of 197.40 nm. The GO/PEG/Bru-FA NCs treatment remarkably inhibited the growth of the tested pathogens. The findings of the MTT assay evidenced that the GO/PEG/Bru-FA NCs effectively reduced the HepG2 cell growth while not showing toxicity to the Vero cells. The findings of the fluorescent assay proved that the GO/PEG/Bru-FA NCs increased ROS generation, reduced MMP levels, and promoted apoptosis in the HepG2 cells. The levels of Bax, caspase-9, and -3 were increased, and Bcl-2 was reduced in the GO/PEG/Bru-FA NCs-treated HepG2 cells. Conclusion: The results of this work demonstrate that GO/PEG/Bru-FA NCs suppress viability and induce apoptosis in HepG2 cells, indicating their potential as an anticancer candidate.

Effect of folic acid on isoprenaline-induced myocardial injury in rats.

Background: Isoprenaline (ISO), a synthetic catecholamine and a ß-adrenoceptor agonist, is widely used to develop an experimental model of myocardial injury (MI) in rats. The leading hypothesis for ISO-induced MI in rats is that it results from catecholamine overstimulation, oxidative stress, inflammatory responses, and development of cardiomyopathy during ISO administration. Folic acid (FA) reduces oxidative stress, improves endothelial function and prevents apoptosis, thereby contributing to cardiovascular protection. This study aimed to investigate the potentially protective effect of FA pretreatment on ISO-induced MI in rats. Methods: For 7 days, adult male Wistar albino rats were pretreated with 5 mg/kg/day of FA. On the sixth and seventh days, MI in rats was induced by administering 85 mg/kg/day of ISO. Prooxidant markers in plasma samples, antioxidant capacity in erythrocyte lysates, cardiac damage markers, lipid profile, electrocardiography (ECG) and histopathological analysis were evaluated. Results: FA pretreatment significantly alleviated changes induced by ISO; it decreased the homocysteine and high-sensitivity troponin I level. FA moderately decreased the reactive oxygen species (ROS) levels (superoxide anion radical, hydrogen peroxide and thiobarbituric acid reactive substances) and improved the antioxidant activities of catalase, superoxide dismutase and reduced glutathione. ISO reduced the nitrite level and FA significantly alleviated this change. Conclusion: It can be concluded that FA, as a mild antioxidant, could be an appropriate cardioprotective substance in the rat model of ISO-induced MI.