Combined ROS Sensitive Folate Receptor Targeted Micellar Formulations of Curcumin Effective Against Rheumatoid Arthritis in Rat Model.

Introduction: Rheumatoid arthritis (RA) is an inflammatory immune-mediated disease that involves synovitis, cartilage destruction, and even joint damage. Traditional agents used for RA therapy remain unsatisfactory because of their low efficiency and obvious adverse effects. Therefore, we here established RA microenvironment-responsive targeted micelles that can respond to the increase in reactive oxygen species (ROS) levels in the joint and improve macrophage-specific targeting of loaded drugs. Methods: We here prepared ROS-responsive folate-modified curcumin micelles (TK-FA-Cur-Ms) in which thioketal (TK) was used as a ROS-responsive linker for modifying polyethylene glycol 5000 (PEG5000) on the micellar surface. When micelles were in the ROS-overexpressing inflammatory microenvironment, the PEG5000 hydration layer was shed, and the targeting ligand FA was exposed, thereby enhancing cellular uptake by macrophages through active targeting. The targeting, ROS sensitivity and anti-inflammatory properties of the micelles were assessed in vitro. Collagen-induced arthritis (CIA) rats model was utilized to investigate the targeting, expression of serum inflammatory factors and histology change of the articular cartilage by micelles in vivo. Results: TK-FA-Cur-Ms had a particle size of 90.07 ± 3.44 nm, which decreased to 78.87 ± 2.41 nm after incubation with H2O2. The micelles exhibited in vitro targeting of RAW264.7 cells and significantly inhibited inflammatory cytokine levels. Pharmacodynamic studies have revealed that TK-FA-Cur-Ms prolonged the drug circulation and exhibited augmented cartilage-protective and anti-inflammatory effects in vivo. Conclusion: The unique ROS-responsive targeted micelles with targeting, ROS sensitivity and anti-inflammatory properties were successfully prepared and may offer an effective therapeutic strategy against RA.

Investigation of folate-modified EGCG-loaded thermosensitive nanospheres inducing immunogenic cell death and damage-associated molecular patterns in hepatocellular carcinoma.

BACKGROUND: The systemic treatment of advanced hepatocellular carcinoma is currently facing a bottleneck. EGCG, the primary active compound in green tea, exhibits anti-tumor effects through various pathways. However, there is a lack of study on EGCG-induced immunogenic cell death (ICD) in hepatocellular carcinoma. METHODS: In a previous study, we successfully synthesized folate-modified thermosensitive nano-materials, encapsulated EGCG within nanoparticles using a hydration method, and established the EGCG nano-drug delivery system. The viability of HepG2 cells post-EGCG treatment was assessed via the MTT and EdU assays. Cell migration and invasion were evaluated through wound healing experiments, Transwell assays, and Annexin V-FITC/PI assay for apoptosis detection. Additionally, the expression levels of damage-associated molecular patterns (DAMPs) were determined using immunofluorescence, ATP measurement, RT-qPCR, and Western Blot. RESULTS: The drug sensitivity test revealed an IC50 value of 96.94 µg/mL for EGCG in HepG2 cells after 48 h. EGCG at a low concentration (50 µg/mL) significantly impeded the migration and invasion of HepG2 cells, showing a clear dose-dependent response. Moreover, medium to high EGCG concentrations induced cell apoptosis in a dose-dependent manner and upregulated DAMPs expression. Immunofluorescence analysis demonstrated a notable increase in CRT expression following low-concentration EGCG treatment. As EGCG concentration increased, cell viability decreased, leading to CRT exposure on the cell membrane. EGCG also notably elevated ATP levels. RT-qPCR and Western Blot analyses indicated elevated expression levels of HGMB1, HSP70, and HSP90 following EGCG intervention. CONCLUSION: EGCG not only hinders the proliferation, migration, and invasion of hepatocellular carcinoma cells and induces apoptosis, but also holds significant clinical promise in the treatment of malignant tumors by promoting ICD and DAMPs secretion.

Developmental Windows for Effects of Choline and Folate on Excitatory and Inhibitory Neurotransmission During Human Gestation.

Choline and folate are critical nutrients for fetal brain development, but the timing of their influence during gestation has not been previously characterized. At different periods during gestation, choline stimulation of α7-nicotinic receptors facilitates conversion of γ-aminobutyric acid (GABA) receptors from excitatory to inhibitory and recruitment of GluR1-R2 receptors for faster excitatory responses to glutamate. The outcome of the fetal development of inhibition and excitation was assessed in 159 newborns by P50 cerebral auditory-evoked responses. Paired stimuli, S1, S2, were presented 500 msec apart. Higher P50 amplitude in response to S1 (P50S1microV) assesses excitation, and lower P50S2microV assesses inhibition in this paired-stimulus paradigm. Development of inhibition was related solely to maternal choline plasma concentration and folate supplementation at 16 weeks' gestation. Development of excitation was related only to maternal choline at 28 weeks. Higher maternal choline concentrations later in gestation did not compensate for earlier lower concentrations. At 4 years of age, increased behavior problems on the Child Behavior Checklist 1½-5yrs were related to both newborn inhibition and excitation. Incomplete development of inhibition and excitation associated with lower choline and folate during relatively brief periods of gestation thus has enduring effects on child development.

Assessment of the in Vitro Effects of Folate Core-Shell Conjugated Iron Oxide Nanoparticles as a Potential Agent for Acute Leukemia Treatment.

BACKGROUND AND OBJECTIVE: There is a growing need to comprehend the potential outcomes of nanoparticles (NPs) on human well-being, including their potential for detecting and treating leukemia. This study examined the role of iron folate core-shell and iron oxide nanoparticles in inducing apoptosis and altering the expression of the B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X-protein (Bax), and Caspase-3 genes in leukemia cells. METHODS: The obtained iron oxide and iron folate core-shell nanoparticles were analyzed using a variety of analytical techniques, including ultraviolet-visible (UV-Vis) absorption spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), dynamic light scattering (DLS), zeta potential, and transmission electron microscopy (TEM). Additionally, FTIR and UV-Vis were used to characterize doxorubicin. The MTT test was utilized to investigate the cytotoxicity of iron oxide and iron folate core-shell nanoparticles. The expression of the apoptotic signaling proteins Bcl-2, Bax, and Caspase-3 was evaluated using the real-time reverse transcription polymerase chain reaction (RT-qPCR) method. Additionally, flow cytometry was performed to gauge the degrees of necrosis and apoptosis. RESULTS: UV-Visible spectroscopy analysis showed that the generated iron oxide and iron folate core-shell NPs had a distinctive absorption curve in the 250-300 nm wavelength range. The XRD peaks were also discovered to index the spherical form with a size of less than 50 nm, which validated the crystal structure. The FTIR analysis determined the bonds and functional groups at wavenumbers between 400 and 4000 cm-1. A viable leukemia treatment approach is a nanocomposite consisting of iron and an iron folate core-shell necessary for inhibiting and activating cancer cell death. The nearly resistant apoptosis in the CCRF-CEM cells may have resulted from upregulating Bax and Casepase-3 while downregulating Bcl-2 expression. CONCLUSIONS: Our study documents the successful synthetization and characterization of iron oxide, which has excellent anticancer activities. A metal oxide conjugation with the nanoparticles' core-shell enhanced the effect against acute leukemia.

A multi-mode Rhein-based nano-platform synergizing ferrotherapy/chemotherapy-induced immunotherapy for enhanced tumor therapy.

Ferroptosis has emerged as a promising strategy for treating triple-negative breast cancer (TNBC) due to bypassing apoptosis and triggering immunogenic cell death (ICD) of tumor cells. However, the antitumor efficacy has been limited by the insufficient intracellular ferrous iron concentration required for ferroptosis and inadequate antitumor immune response. To address these limitations, we designed a multi-mode nano-platform (MP-FA@R-F NPs), which exhibited a synergistic effect of ferroptosis, apoptosis and induced immune response for enhanced antitumor therapy. MP-FA@R-F NPs target folate receptors, which are over-expressed on the tumor cell's surface to promote intracellular uptake. The cargoes, including Rhein and Fe3O4, would be released in intracellular acid, accelerating by NIR laser irradiation. The released Rhein induced apoptosis of tumor cells mediated by the caspase 3 signal pathway, while the released Fe3O4 triggered ferroptosis through the Fenton reaction and endowed the nanoplatform with magnetic resonance imaging (MRI) capabilities. In addition, ferroptosis-dying tumor cells could release damage-associated molecular patterns (DAMPs) to promote T cell activation and infiltration for immune response and induce immunogenic cell death (ICD) for tumor immunotherapy. Together, MP-FA@R-F NPs represent a potential synergistic ferro-/chemo-/immuno-therapy strategy with MRI guidance for enhanced antitumor therapy. STATEMENT OF SIGNIFICANCE: The massive strategies of cancer therapy based on ferroptosis have been emerging in recent years, which provided new insights into designing materials for cancer therapy. However, the antitumor efficacy of ferroptosis is still unsatisfactory, mainly due to insufficient intracellular pro-ferroptotic stimuli. In the current study, we designed a multi-mode nano-platform (MP-FA@R-F NPs), which represented a potential synergistic ferro-/chemo-/immuno-therapy strategy with MRI guidance for enhanced antitumor therapy.

Immunoantitumor Activity and Oxygenation Effect Based on Iron-Copper-Doped Folic Acid Carbon Dots.

Cancer metastasis and recurrence are closely associated with immunosuppression and a hypoxic tumor microenvironment. Chemodynamic therapy (CDT) and photothermodynamic therapy (PTT) have been shown to induce immunogenic cell death (ICD), effectively inhibiting cancer metastasis and recurrence when combined with immune adjuvants. However, the limited efficacy of Fenton's reaction and suboptimal photothermal effect present significant challenges for successfully inducing ICD through CDT and PTT. This paper described the synthesis and immunoantitumor activity of the novel iron-copper-doped folic acid carbon dots (CFCFB). Copper-doped folic acid carbon dots (Cu-FACDs) were initially synthesized via a hydrothermal method, using folic acid and copper gluconate as precursors. Subsequently, the nanoparticles CFCFB were obtained through cross-linking and self-assembly of Cu-FACDs with ferrocene dicarboxylic acid (FeDA) and 3-bromopyruvic acid (3BP). The catalytic effect of carbon dots in CFCFB enhanced the activity of the Fenton reaction, thereby promoting CDT-induced ICD and increasing the intracellular oxygen concentration. Additionally, 3BP inhibited cellular respiration, further amplifying the oxygen concentration. The photothermal conversion efficiency of CFCFB reached 55.8%, which significantly enhanced its antitumor efficacy through photothermal therapy. Immunofluorescence assay revealed that treatment with CFCFB led to an increased expression of ICD markers, including calreticulin (CRT) and ATP, as well as extracellular release of HMGB-1, indicating the induction of ICD by CFCFB. Moreover, the observed downregulation of ARG1 expression indicates a transition in the tumor microenvironment from an immunosuppressive state to an antitumor state following treatment with CFCFB. The upregulation of IL-2 and CD8 expression facilitated the differentiation of effector T cells, resulting in an augmented population of CD8+ T cells, thereby indicating the activation of systemic immune response.

Folic acid supplementation prevents high body fat-induced bone loss through TGR5 signaling pathways.

Osteoporosis caused by bone loss is one of the serious global public health problems. Folic acid is a B vitamin with multiple physiological functions such as lipid regulation and antioxidant capacity, and its potential to improve bone loss has attracted our attention. Through NHANES database analysis, we found that folic acid intake was significantly correlated with whole-body bone mineral density (BMD) in people aged 20-60 years, and the association may be mediated by the body fat rate. Male C57Bl/6 mice were fed either a normal diet or a high-fat diet, and folic acid was added to drinking water for supplementation. Our results indicated that mice with high body fat showed bone microstructure damage and bone loss, while folic acid supplementation improved bone quality. At the same time, we found that mice with high body fat exhibited abnormal blood lipids, dysregulation of intestinal flora, and metabolic disorders. Folic acid supplementation improved these phenomena. Through the network analysis of intestinal flora and metabolites, we found that LCA and TGR5 may play important roles. The results showed that folic acid promoted the expression of LCA and TGR5 in mice, increased the phosphorylation of AMPK, and decreased the phosphorylation of NF-κB and ERK, thereby reducing bone loss. In summary, folic acid intake is closely related to BMD, and folic acid supplementation can prevent high body fat-induced bone loss. Our study provides new ideas and an experimental basis for preventing bone loss and osteoporosis.

Effects of folate-chicory acid liposome on macrophage polarization and TLR4/NF-κB signaling pathway in ulcerative colitis mouse.

BACKGROUND: Chichoric acid (CA) is a major active ingredient found in chicory and Echinacea. As a derivative of caffeic acid, it has various pharmacological effects. PURPOSE: Due to the unclear etiology and disease mechanisms, effective treatment methods for ulcerative colitis (UC) are currently lacking. The study investigated the therapeutic effects of the folate-chicory acid liposome on both LPS-induced macrophage inflammation models and dextran sulfate sodium (DSS)-induced mouse UC models. METHODS: Folate-chicory acid liposome was prepared using the double emulsion ultrasonic method with the aim of targeting folate receptors specifically expressed on macrophages. The study investigated the therapeutic effects of the folate-chicory acid liposome on both LPS-induced macrophage inflammation models and DSS -induced mouse UC models. Furthermore, the effects of the liposomes on macrophage polarization and their underlying mechanisms in UC were explored. RESULTS: The average particle size of folate-chicory acid liposome was 120.4 ± 0.46 nm, with an encapsulation efficiency of 77.32 ± 3.19 %. The folate-chicory acid liposome could alleviate macrophage apoptosis induced by LPS, decrease the expression of inflammatory factors in macrophages, enhance the expression of anti-inflammatory factors, inhibit macrophage polarization towards the M1 phenotype, and mitigate cellular inflammation in vetro. In vivo test, folate-chicory acid liposome could attenuate clinical symptoms, increased colon length, reduced DAI scores, CMDI scores, and alleviated the severity of colonic histopathological damage in UC mice. Furthermore, it inhibited the polarization of macrophages towards the M1 phenotype in the colon and downregulated the TLR4/NF-κB signaling pathway, thereby ameliorating UC in mice. CONCLUSION: Folate-chicory acid liposome exhibited a uniform particle size distribution and high encapsulation efficiency. It effectively treated UC mice by inhibiting the polarization of macrophages towards the M1 phenotype in the colon and downregulating the TLR4/NF-κB signaling pathway.

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.

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.

One-carbon metabolite supplementation to nutrient-restricted beef heifers affects placental vascularity during early pregnancy.

We hypothesized that restricted maternal nutrition and supplementation of one-carbon metabolites (OCM; methionine, folate, choline, and vitamin B12) would affect placental vascular development during early pregnancy. A total of 43 cows were bred, and 32 heifers successfully became pregnant with female calves, leading to the formation of four treatment groups: CON - OCM (n = 8), CON + OCM (n = 7), RES - OCM (n = 9), and RES + OCM (n = 8). The experimental design was a 2 × 2 factorial, with main factors of dietary intake affecting average daily gain: control (CON; 0.6 kg/d ADG) and restricted (RES; -0.23 kg/d ADG); and OCM supplementation (+OCM) in which the heifers were supplemented with rumen-protected methionine (7.4 g/d) and choline (44.4 g/d) and received weekly injections of 320 mg of folate and 20 mg of vitamin B12, or received no supplementation (-OCM; corn carrier and saline injections). Heifers were individually fed and randomly assigned to treatment at breeding (day 0). Placentomes were collected on day 63 of gestation (0.225 of gestation). Fluorescent staining with CD31 and CD34 combined with image analysis was used to determine the vascularity of the placenta. Images were analyzed for capillary area density (CAD) and capillary number density (CND). Areas evaluated included fetal placental cotyledon (COT), maternal placental caruncle (CAR), whole placentome (CAR + COT), intercotyledonary fetal membranes (ICOT, or chorioallantois), intercaruncular endometrium (ICAR), and endometrial glands (EG). Data were analyzed with the GLM procedure of SAS, with heifer as the experimental unit and significance at P ≤ 0.05 and a tendency at P > 0.05 and P < 0.10. Though no gain × OCM interactions existed (P ≥ 0.10), OCM supplementation increased (P = 0.01) CAD of EG, whereas nutrient restriction tended (P < 0.10) to increase CAD of ICOT and CND of COT. Additionally, there was a gain × OCM interaction (P < 0.05) for CAD within the placentome and ICAR, such that RES reduced and supplementation of RES with OCM restored CAD. These results indicate that maternal rate of gain and OCM supplementation affected placental vascularization (capillary area and number density), which could affect placental function and thus the efficiency of nutrient transfer to the fetus during early gestation.

In cow­calf production, periods of poor forage availability or quality can result in nutrient restriction during pregnancy. Previous studies have shown that even moderate maternal feed restriction during pregnancy, including very early in pregnancy, has profound effects on fetal and placental development, potentially having lasting impacts on calf growth and body composition later in life. One-carbon metabolites (OCM) in the diet are biomolecules required for methylation reactions and participate in the regulation of gene expression. Our objective was to evaluate the effects of nutrient restriction and OCM supplementation (specifically methionine, choline, folate, and vitamin B12) on placental vascular development during early pregnancy. Proper placental vascular development is necessary for healthy pregnancy outcomes, reflected by normal birth weight and healthy offspring. Our results indicated that maternal rate of gain and OCM supplementation affect placental vascularization, which could affect placental function and thereby fetal development throughout gestation. In the context of beef cattle production, our study sheds light on strategies that could enhance placental vascular development during early pregnancy. However, it is essential to recognize the nuances in our data, highlighting the need for further research to fully comprehend these intricate processes.

Impact of folic acid supplementation on ischemia‒reperfusion-induced kidney injury in rats: folic acid prophylactic role revisited.

Folic acid (FA), with its anti-inflammatory and antioxidant properties, may offer protection against ischemia-reperfusion (IR) injury. This study investigated whether FA safeguards rat kidneys from IR by targeting high mobility group box-1 (HMGB1), a key inflammatory mediator. Fifty adult male Wistar rats were randomly allocated into four groups: control, IR, IR + FA pretreatment, and FA alone. Compared to controls, IR significantly impaired renal function and elevated levels of malondialdehyde, HMGB1, NF-κB, and caspase 3. FA pretreatment effectively reversed these detrimental changes, protecting renal function and minimizing tissue damage. The FA-alone group showed no significant differences compared to the control group, indicating no adverse effects of FA treatment. Mechanistically, FA inhibited HMGB1 expression and its downstream activation of NF-κB and caspase 3, thereby quelling inflammation and cell death. FA shields rat kidneys from IR-induced injury by suppressing HMGB1-mediated inflammation and apoptosis, suggesting a potential therapeutic avenue for IR-associated kidney damage.

Sustained effects of small-quantity lipid-based nutrient supplements provided during the first 1000 days on child growth at 9-11 y in a randomized controlled trial in Ghana.

BACKGROUND: There is limited research on whether nutritional supplementation in the first 1000 d affects long-term child outcomes. We previously demonstrated that pre- and postnatal small-quantity lipid-based nutrient supplements (SQ-LNS) increased birth weight and child length at 18 mo of age in Ghana. OBJECTIVES: We aimed to investigate the effect of pre- and postnatal SQ-LNS on child growth and blood pressure at 9-11 y. METHODS: In the International Lipid-Based Nutrient Supplements (iLiNS)-DYAD-Ghana trial, 1320 females ≤20 weeks of gestation were randomly assigned to receive daily: iron and folic acid (IFA) during pregnancy and placebo during 6 mo postpartum or multiple micronutrients (MMNs) during pregnancy and 6 mo postpartum, or SQ-LNS during pregnancy and 6 mo postpartum and for their children aged from 6 to 18 mo. We re-enrolled 966 children aged 9-11 y and assessed child blood pressure, height-for-age z-score (HAZ), body mass index (BMI)-for-age z-score, waist-to-height ratio, triceps skinfold, and midupper arm circumference. We compared SQ-LNS with control (IFA + MMN) groups adjusting for child's age. RESULTS: Mean (standard deviation [SD]) HAZ in SQ-LNS and control group was -0.04 (0.96) and -0.16 (0.99); P = 0.060. There were no indications of group differences in the other outcomes (P > 0.10). Effects on HAZ varied by child sex (P-interaction = 0.075) and maternal prepregnancy BMI (kg/m2; P-interaction = 0.007). Among females, HAZ was higher in the SQ-LNS [0.08 (1.04)] than in the control group [-0.16 (1.01)] (P = 0.010); among males, SQ-LNS [-0.16 (0.85)] and control groups [-0.16 (0.96)] did not differ (P = 0.974). Among children of females with BMI of <25, HAZ was higher in the SQ-LNS [-0.04 (1.00)] than in the control group [-0.29 (0.94)] (P = 0.004); among females with BMI of ≥25, SQ-LNS [-0.04 (0.91)] and control groups [0.07 (1.00)] did not differ (P = 0.281). CONCLUSIONS: There is a sustained impact of prenatal and postnatal SQ-LNS on linear growth among female children and children whose mothers were not overweight. This trial was registered at clinicaltrials.gov as NCT00970866 (https://clinicaltrials.gov/ct2/show/record/NCT00970866).

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.

Efficient Sequential Co-Delivery Nanosystem for Inhibition of Tumor and Tumor-Associated Fibroblast-Induced Resistance and Metastasis.

Purpose: Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer. However, the effect of current treatment strategies by inducing tumor cell apoptosis alone is not satisfactory. The growth, metastasis and treatment sensitivity of tumors can be strongly influenced by cancer-associated fibroblasts (CAFs) in the microenvironment. Effective cancer therapies may need to target not only the tumor cells directly but also the CAFs that protect them. Methods: Celastrol and small-sized micelles containing betulinic acid were co-encapsulated into liposomes using the thin-film hydration method (CL@BM). Folic acid was further introduced to modify liposomes as the targeting moiety (F/CL@BM). We established a novel NIH3T3+4T1 co-culture model to mimic the tumor microenvironment and assessed the nanocarrier's inhibitory effects on CAFs-induced drug resistance and migration in the co-culture model. The in vivo biological distribution, fluorescence imaging, biological safety evaluation, and combined therapeutic effect evaluation of the nanocarrier were carried out based on a triple-negative breast cancer model. Results: In the present study, a novel multifunctional nano-formulation was designed by combining the advantages of sequential release, co-loading of tretinoin and betulinic acid, and folic acid-mediated active targeting. As expected, the nano-formulation exhibited enhanced cytotoxicity in different cellular models and effectively increased drug accumulation at the tumor site by disrupting the cellular barrier composed of CAFs by tretinoin. Notably, the co-loaded nano-formulations proved to be more potent in inhibiting tumor growth in mice and also showed better anti-metastatic effects in lung metastasis models compared to the formulations with either drug alone. This novel drug delivery system has the potential to be used to develop more effective cancer therapies. Conclusion: Targeting CAFs with celastrol sensitizes tumor cells to chemotherapy, increasing the efficacy of betulinic acid. The combination of drugs targeting tumor cells and CAFs may lead to more effective therapies against various cancers.

Sperm DNA methylation defects in a new mouse model of the 5,10-methylenetetrahydrofolate reductase 677C>T variant and correction with moderate dose folic acid supplementation.

5,10-Methylenetetrahydrofolate reductase (MTHFR) is an enzyme that plays a key role in providing methyl groups for DNA methylation, including during spermatogenesis. A common genetic variant in humans (MTHFR 677C>T) results in reduced enzyme activity and has been linked to various disorders, including male infertility. A new animal model has been created by reproducing the human equivalent of the polymorphism in mice using CRISPR/Cas9. Biochemical parameters in the Mthfr 677TT mice recapitulate alterations found in MTHFR 677TT men. Our aims were to characterize the sperm DNA methylome of the Mthfr 677CC and TT mice on a control diet (2 mg folic acid/kg diet) and assess the effects of folic acid supplementation (10 mg/kg diet) on the sperm DNA methylome. Body and reproductive organ weights, testicular sperm counts, and histology were examined. DNA methylation in sperm was assessed using bisulfite pyrosequencing and whole-genome bisulfite sequencing (WGBS). Reproductive parameters and locus-specific imprinted gene methylation were unaffected by genotype or diet. Using WGBS, sperm from 677TT mice had 360 differentially methylated tiles as compared to 677CC mice, predominantly hypomethylation (60% of tiles). Folic acid supplementation mostly caused hypermethylation in sperm of males of both genotypes and was found to partially correct the DNA methylation alterations in sperm associated with the TT genotype. The new mouse model will be useful in understanding the role of MTHFR deficiency in male fertility and in designing folate supplementation regimens for the clinic.

Developing Folate-Conjugated miR-34a Therapeutic for Prostate Cancer: Challenges and Promises.

Prostate cancer (PCa) remains a common cancer with high mortality in men due to its heterogeneity and the emergence of drug resistance. A critical factor contributing to its lethality is the presence of prostate cancer stem cells (PCSCs), which can self-renew, long-term propagate tumors, and mediate treatment resistance. MicroRNA-34a (miR-34a) has shown promise as an anti-PCSC therapeutic by targeting critical molecules involved in cancer stem cell (CSC) survival and functions. Despite extensive efforts, the development of miR-34a therapeutics still faces challenges, including non-specific delivery and delivery-associated toxicity. One emerging delivery approach is ligand-mediated conjugation, aiming to achieve specific delivery of miR-34a to cancer cells, thereby enhancing efficacy while minimizing toxicity. Folate-conjugated miR-34a (folate-miR-34a) has demonstrated promising anti-tumor efficacy in breast and lung cancers by targeting folate receptor α (FOLR1). Here, we first show that miR-34a, a TP53 transcriptional target, is reduced in PCa that harbors TP53 loss or mutations and that miR-34a mimic, when transfected into PCa cells, downregulated multiple miR-34a targets and inhibited cell growth. When exploring the therapeutic potential of folate-miR-34a, we found that folate-miR-34a exhibited impressive inhibitory effects on breast, ovarian, and cervical cancer cells but showed minimal effects on and targeted delivery to PCa cells due to a lack of appreciable expression of FOLR1 in PCa cells. Folate-miR-34a also did not display any apparent effect on PCa cells expressing prostate-specific membrane antigen (PMSA) despite the reported folate's binding capability to PSMA. These results highlight challenges in the specific delivery of folate-miR-34a to PCa due to a lack of target (receptor) expression. Our study offers novel insights into the challenges and promises within the field and casts light on the development of ligand-conjugated miR-34a therapeutics for PCa.

Effect of lipid-based nutrient supplements on micronutrient status and hemoglobin among children with stunting: secondary analysis of a randomized controlled trial in Uganda.

BACKGROUND: Micronutrient deficiencies and anemia are widespread among children with stunting. OBJECTIVES: We assessed the effects of lipid-based nutrient supplements (LNS) containing milk protein (MP) and/or whey permeate (WP) on micronutrient status and hemoglobin (Hb) among children with stunting. METHODS: This was a secondary analysis of a randomized controlled trial. Children aged 12-59 mo with stunting were randomly assigned to LNS (100 g/d) with milk or soy protein and WP or maltodextrin for 12 wk, or no supplement. Hb, serum ferritin (S-FE), serum soluble transferrin receptor (S-TfR), plasma cobalamin (P-Cob), plasma methylmalonic acid (P-MMA), plasma folate (P-Fol), and serum retinol-binding protein (S-RBP) were measured at inclusion and at 12 wk. Data were analyzed using linear and logistic mixed-effects models. RESULTS: Among 750 children, with mean age ± SD of 32 ± 11.7 mo, 45% (n = 338) were female and 98% (n = 736) completed follow-up. LNS, compared with no supplementation, resulted in 43% [95% confidence interval (CI): 28, 60] greater increase in S-FE corrected for inflammation (S-FEci), 2.4 (95% CI: 1.2, 3.5) mg/L greater decline in S-TfR, 138 (95% CI: 111, 164) pmol/L greater increase in P-Cob, 33% (95% CI: 27, 39) reduction in P-MMA, and 8.5 (95% CI: 6.6, 10.3) nmol/L greater increase in P-Fol. There was no effect of LNS on S-RBP. Lactation modified the effect of LNS on markers of cobalamin status, reflecting improved status among nonbreastfed and no effects among breastfed children. LNS increased Hb by 3.8 (95% CI: 1.7, 6.0) g/L and reduced the odds of anemia by 55% (odds ratio: 0.45, 95% CI: 0.29, 0.70). MP compared with soy protein increased S-FEci by 14% (95% CI: 3, 26). CONCLUSIONS: LNS supplementation increases Hb and improves iron, cobalamin, and folate status, but not vitamin A status among children with stunting. LNS should be considered for children with stunting. This trial was registered at ISRCTN as 13093195.

Fecal microbiota transplantation revealed the function of folic acid on reducing abdominal fat deposition in broiler chickens mediated by gut microbiota.

Excess abdominal fat reduces carcass yield and feed conversion ratio, thereby resulting in significant economic losses in the poultry industry. Our previous study demonstrated that dietary addition of folic acid reduced fat deposition and changed gut microbiota and short-chain fatty acid. However, whether folic acid regulating abdominal fat deposition was mediated by gut microbiota was unclear. A total of 210 one-day-old broiler chickens were divided into 3 groups including the control (CON), folic acid (FA), and fecal microbiota transplantation (FMT) groups. From 14th day, broiler chickens in CON and FA groups were given perfusion administration with 1 mL diluent daily, while 1 mL fecal microbiota transplantation suspension from FA group prepared before was perfusion in FMT group receiving control diets. The result showed that abdominal fat percentage was significantly lower in FA and FMT groups when compared with CON group (P < 0.05). Morphology analysis revealed that the villus height of jejunum and ileum were significantly higher in FMT group (P < 0.05), and the villus height of jejunum was also significantly higher in FA group (P < 0.05), while the diameter and cross-sectional area (CSA) of adipocytes were significantly decreased in FA and FMT groups when compared with CON group (P < 0.05). Western blot results indicated that the expression levels of FOXO1 and PLIN1 in FMT group were significantly increased (P < 0.05), whereas the expression levels of PPARγ, C/EBPα, and FABP4 were significantly decreased (P < 0.05). Additionally, the Chao1, Observed-species, Shannon and Simpson indexes in FA and FMT groups were significantly higher (P < 0.05), but the microbiota were similar between FMT and FA groups (P < 0.05). LEfSe analysis determined that Lactobacillus, Clostridium and Dehalobacterium were found to be predominant in FA group, while Oscillospira, Shigella, and Streptococcus were the dominant microflora in FMT group. Furthermore, these cecal microbiota were mostly involved in infectious disease, cellular community prokaryotes, cell motility and signal transduction in FA group (P < 0.05), whereas functional capacities involved in signal transduction, cell motility, infectious disease and environment adaptation were enriched significantly of cecal microbiota in FMT group (P < 0.05). In summary, both fecal microbiota transplantation from the broiler chickens of dietary added folic acid and dietary folic acid addition effectively reduced abdominal fat deposition, indicating that the regulatory effect of folic acid on abdominal fat deposition was mediated partly by gut microbiota in broiler chickens.