Reduced Folate Carrier Research Articles

Investigating the diagnostic and prognostic significance of genes related to fatty acid metabolism in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancers worldwide, with death rates increasing by approximately 2–3% per year. The high mortality and poor prognosis of HCC are primarily due to inaccurate early diagnosis and lack of monitoring when liver transplantation is not feasible. Fatty acid (FA) metabolism is a critical metabolic pathway that provides energy and signaling factors in cancer, particularly in HCC, and promotes malignancy. Therefore, it is essential to explore specific FA metabolism-related diagnostic and prognostic signatures that can enable the effective early diagnosis and monitoring of HCC.MethodsIn this study, we used genes associated with FA metabolism pathway and weighted gene co-expression network analysis (WGCNA) to establish a gene co-expression network and identify hub genes related to HCC (disease WGCNA) and FA clusters (cluster WGCNA). A diagnostic model was constructed using data downloaded from the Gene Expression Omnibus database (GSE25097), and a prognostic model was established using The Cancer Genome Atlas cohort, in which Univariate Cox regression analysis, multivariate Cox risk model, and LASSO Cox regression analysis were applied. The immune infiltration of HCC cells was evaluated using CIBERSORT. The function of the key SLC22A1 gene was experimentally verified in vitro and in vivo.ResultsTwelve overlapping genes (CPEB3, ASPDH, DEPDC7, ETFDH, UGT2B7, GYS2, F11, ANXA10, CYP2C8, GLYATL1, C6, and SLC22A1) from disease and cluster WGCNA were identified as key genes and used in the construction of the diagnostic and prognostic models. The RF model had the highest area under the ROC curve (AUC) of 0.994 was identified as the most effective for distinguishing patients with HCC with different features. The top five important genes (C6, UGT2B7, SLC22A1, F11, and CYP2C8) from the RF model were selected as diagnostic genes for further analysis (ROC curves: AUC value = 0.986, 95% confidence interval [95% CI] = 0.967–0.999). Moreover, a risk score formula consisting of four genes (GYS2, F11, ANXA10 and SLC22A1) was established and its independent prognostic ability was further demonstrated (univariate Cox regression analysis: hazard ratio [HR] = 3.664%, 95% CI = 2.033–6.605, P < 0.001; multivariate Cox regression analysis: HR = 2.801%, 95% CI = 1.553–5.049, P < 0.001). Additionally, in vitro and in vivo experiments demonstrated that SLC22A1 inhibits HCC tumor development, suggesting it may be a potential therapeutic target for HCC.ConclusionsThese findings indicate a considerable value of specific FA metabolism-related genes in the diagnostic and prognostic evaluation of HCC, which provide novel insights into the disease’s management, as well as has potential implications for personalized treatment strategies. However, further investigation of the effects of these model genes on HCC is required.

Abstract A018: Alternative Polyadenylation Regulates Pancreatic Cancer Fibroblast Metabolism

Abstract Dense, fibrotic stroma is a defining feature of pancreatic ductal adenocarcinoma (PDAC), comprising up to 90% of tumor volume. Multiple studies have shown that this stroma strongly influences cancer progression and response to treatment. Cancer Associated Fibroblasts (CAFs) make up the majority of the Tumor Microenvironment (TME) and directly alter the tumor landscape and response to therapeutics. CAFs are a heterogenous population comprising of a mixture of inflammatory, myofibroblastic, and antigen-presenting CAFs, with both pro- and anti- tumorigenic functions. While we have been able to define CAFs phenotypically, we lack a deeper understanding of the mechanisms underlying their transcriptional heterogeneity and ultimately their contribution to driving PDAC progression. Our lab has previously shown 3’UTR-Alternative Polyadenylation (APA) to be an important driver of genetic dysregulation in PDAC. APA is an important process regulating mRNA stability, protein localization, and expression, ultimately determining cellular processes such as proliferation, metastasis, and migration. We have also shown that increased 3’ UTR shortening is associated with the pro- tumorigenic inflammatory CAF subtype. However, how APA regulates CAF function is unknown. We are now trying to understand the possible mechanisms of these changes in vitro using human-derived CAF cell lines and treating them with a known pharmacological modulator of APA. We performed PAC-Seq analyses on these samples and show that important drivers of CAF metabolism are hindered by APA, ultimately affecting the ability of CAFs to support cancer cell growth. We find that inhibition of APA drives CAF migration and senescence phenotype and alters gene expression of critical genes required for CAF function, and metabolism. We identified a novel mechanism by which a key metabolic gene SLC7A11 is regulated by APA. To our knowledge, our work is one of the first studies to study how APA is an important regulator of CAF function. Citation Format: Aditi H Chaubey, Michael E Feigin, Eric J Wagner. Alternative Polyadenylation Regulates Pancreatic Cancer Fibroblast Metabolism [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A018.

Combined Analysis of Metabolomics and Transcriptome Revealed the Effect of Bacillus thuringiensis on the 5th Instar Larvae of Dendrolimus kikuchii Matsumura.

Dendrolimus kikuchii Matsumura (D. kikuchii) is a serious pest of coniferous trees. Bacillus thuringiensis (Bt) has been widely studied and applied as a biological control agent for a variety of pests. Here, we found that the mortality rate of D. kikuchii larvae after being fed Bt reached 95.33% at 24 h; the midgut membrane tissue was ulcerated and liquefied, the MDA content in the midgut tissue decreased and the SOD, CAT and GPx enzyme activities increased, indicating that Bt has toxic effects on D. kikuchii larvae. In addition, transmission electron microscopy showed that Bt infection caused severe deformation of the nucleus of the midgut tissue of D. kikuchii larvae, vacuoles in the nucleolus, swelling and shedding of microvilli, severe degradation of mitochondria and endoplasmic reticulum and decreased number. Surprisingly, metabolomics and transcriptome association analysis revealed that four metabolic-related signaling pathways, Nicotinate and nicotinamide metabolism, Longevity regulating pathway-worm, Vitamin digestion and absorption and Lysine degradation, were co-annotated in larvae. More surprisingly, Niacinamide was a common differential metabolite in the first three signaling pathways, and both Niacinamide and L-2-Aminoadipic acid were reduced. The differentially expressed genes involved in the four signaling pathways, including NNT, ALDH, PNLIP, SETMAR, GST and RNASEK, were significantly down-regulated, but only SLC23A1 gene expression was up-regulated. Our results illustrate the effects of Bt on the 5th instar larvae of D. kikuchii at the tissue, cell and molecular levels, and provide theoretical support for the study of Bt as a new biological control agent for D. kikuchii.

The reverse transsulfuration pathway affects the colonic microbiota and contributes to colitis in mice

Cystathionine γ-lyase (CTH) is a critical enzyme in the reverse transsulfuration pathway, the major route for the metabolism of sulfur-containing amino acids, notably converting cystathionine to cysteine. We reported that CTH supports gastritis induced by the pathogen Helicobacter pylori. Herein our aim was to investigate the role of CTH in colonic inflammation. First, we found that CTH is induced in the colon mucosa in mice with dextran sulfate sodium-induced colitis. Expression of CTH was completely absent in the colon of Cth–/– mice. We observed that clinical and histological parameters are ameliorated in Cth-deficient mice compared to wild-type animals. However, Cth deletion had no effect on tumorigenesis and the level of dysplasia in mice treated with azoxymethane-DSS, as a reliable model of colitis-associated carcinogenesis. Mechanistically, we determined that the deletion of the gene Slc7a11 encoding for solute carrier family 7 member 11, the transporter of the anionic form of cysteine, does not affect DSS colitis. Lastly, we found that the richness and diversity of the fecal microbiota were significantly increased in Cth–/– mice compared to both WT and Slc7a11–/– mice. In conclusion, our data suggest that the enzyme CTH represents a target for clinical intervention in patients with inflammatory bowel disease, potentially by beneficially reshaping the composition of the gut microbiota.

The population-specific Thr44Met OCT3 coding variant affects metformin pharmacokinetics with subsequent effects on insulin sensitivity in C57Bl/6J mice.

Metformin is an important first-line treatment for type 2 diabetes and acts by increasing the body's ability to dispose of glucose. Metformin's efficacy can be affected by genetic variants in the transporters that regulate its uptake into cells. The SLC22A3 gene (also known as EMT; EMTH; OCT3) codes for organic cation transporter 3 (OCT3), which is a broad-specificity cation transporter that also transports metformin. Most SLC22A3 variants reduce the rate of metformin transport but the rs8187715 variant (p.Thr44Met) is reported to increase uptake of metformin in vitro. However, the impact of this on in vivo metformin transport and efficacy is unknown. Very few carriers of this variant have been reported globally, but, notably, all were of Pacific Island descent. Therefore, this study aims to understand the prevalence of this variant in Polynesian peoples (Māori and Pacific peoples) and to understand its impact on metformin transport and efficacy in vivo. rs8187715 was genotyped in 310 individuals with Māori and Pacific ancestry recruited in Aotearoa New Zealand. To study this variant in a physiological context, an orthologous knockin mouse model with C57BL/6J background was used. Pharmacokinetic analysis compared uptake rate of metformin into tissues. Plasma growth/differentiation factor 15 (GDF-15) was also measured as a marker of metformin efficacy. Glucose and insulin tolerance was assessed after acute or sustained metformin treatment in knockin and wild-type control mice to examine the impact of the variant on metformin's glycaemic control. The minor allele frequency of this variant in the Māori and Pacific participants was 15.4%. There was no association of the variant with common metabolic parameters including diabetes status, BMI, blood pressure, lipids, or blood glucose and HbA1c. However, in the orthologous knockin mouse model, the rate of metformin uptake into the blood and tissues was increased. Acute metformin dosing increased insulin sensitivity in variant knockin mice but this effect was lost after longer-term metformin treatment. Metformin's effects on GDF-15 levels were also lost in variant knockin mice with longer-term metformin treatment. These data provide evidence that the SLC22A3 rs8187715 variant accelerates metformin uptake rate in vivo. While this acutely improves insulin sensitivity, there was no increased effect of metformin with longer-term dosing. Thus, our finding of a high prevalence of this variant specifically in Māori and Pacific peoples identifies it as a potential population-specific pharmacogenetic marker with potential to guide metformin therapy in these peoples.

Association of SLC19A1 Gene Polymorphisms and Its Regulatory miRNAs with Methotrexate Toxicity in Children with Acute Lymphoblastic Leukemia.

Methotrexate (MTX) is an anti-folate chemotherapeutic agent that is considered to be a gold standard in Acute Lymphoblastic Leukemia (ALL) therapy. Nevertheless, toxicities induced mainly due to high doses of MTX are still a challenge for clinical practice. MTX pharmacogenetics implicate various genes as predictors of MTX toxicity, especially those that participate in MTX intake like solute carrier family 19 member 1 (SLC19A1). The aim of the present study was to evaluate the association between SLC19A1 polymorphisms and its regulatory miRNAs with MTX toxicity in children with ALL. A total of 86 children with ALL were included in this study and were all genotyped for rs2838958, rs1051266 and rs1131596 SLC19A1 polymorphisms as well as the rs56292801 polymorphism of miR-5189. Patients were followed up (48, 72 and 96 h) after treatment with MTX in order to evaluate the presence of MTX-associated adverse events. Our results indicate that there is a statistically significant correlation between the rs1131596 SLC19A1 polymorphism and the development of MTX-induced hepatotoxicity (p = 0.03), but there is no significant association between any of the studied polymorphisms and mucositis or other side effects, such as nausea, emesis, diarrhea, neutropenia, skin rash and infections. In addition, when genotype TT of rs1131596 and genotype AA of rs56292801 are both present in a patient then there is a higher risk of developing severe hepatotoxicity (p = 0.0104).

FMRP protects breast cancer cells from ferroptosis by promoting SLC7A11 alternative splicing through interacting with hnRNPM

Ferroptosis is a unique modality of regulated cell death that is driven by iron-dependent phospholipid peroxidation. N6-methyladenosine (m6A) RNA modification participates in varieties of cellular processes. However, it remains elusive whether m6A reader Fragile X Mental Retardation Protein (FMRP) are involved in the modulation of ferroptosis in breast cancer (BC). In this study, we found that FMRP expression was elevated and associated with poor prognosis and pathological stage in BC patients. Overexpression of FMRP induced ferroptosis resistance and exerted oncogenic roles by positively regulating a critical ferroptosis defense gene SLC7A11. Mechanistically, upregulated FMRP catalyzes m6A modification of SLC7A11 mRNA and further influences the SLC7A11 translation through METTL3-dependent manner. Further studies revealed that FMRP interacts with splicing factor hnRNPM to recognize the splice site and then modulated the exon skip splicing event of SLC7A11 transcript. Interestingly, SLC7A11-S splicing variant can effectively promote FMRP overexpression-induced ferroptosis resistance in BC cells. Moreover, our clinical data suggested that FMRP/hnRNPM/SLC7A11 expression were significantly increased in the tumor tissues, and this signal axis was important evaluation factors closely related to the worse survival and prognosis of BC patients. Overall, our results uncovered a novel regulatory mechanism by which high FMRP expression protects BC cells from undergoing ferroptosis. Targeting the FMRP–SLC7A11 axis has a dual effect of inhibiting ferroptosis resistance and tumor growth, which could be a promising therapeutic target for treating BC.

Functional Characterization of Reduced Folate Carrier and Protein-Coupled Folate Transporter for Antifolates Accumulation in Non-Small Cell Lung Cancer Cells.

Antifolates are important for chemotherapy in non-small cell lung cancer (NSCLC). They mainly rely on reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) to enter cells. PCFT is supposed to be the dominant transporter of the two in tumors, as it operates optimally at acidic pH and has limited transport activity at physiological pH, whereas RFC operates optimally at neutral pH. In this study, we found RFC showed a slightly pH-dependent uptake of antifolates, with similar affinity values at pH 7.4 and 6.5. PCFT showed a highly pH-dependent uptake of antifolates, with an optimum pH of 6.0 for pemetrexed and 5.5 for methotrexate. The Michaelis-Menten constant (Km ) value of PCFT for pemetrexed at pH 7.4 was more than 10 times higher than that at pH 6.5. Interestingly, we found that antifolate accumulations mediated by PCFT at acidic pH were significantly affected by the efflux transporter, breast cancer resistance protein (BCRP). The highest pemetrexed concentration was observed at pH 7.0-7.4 after a 60-minute accumulation in PCFT-expressing cells, which was further evidenced by the cytotoxicity of pemetrexed, with the IC50 value of pemetrexed at pH 7.4 being one-third of that at pH 6.5. In addition, the in vivo study indicated that increasing PCFT and RFC expression significantly enhanced the antitumor efficacy of pemetrexed despite the high expression of BCRP. These results suggest that both RFC and PCFT are important for antifolates accumulation in NSCLC, although there is an acidic microenvironment and high BCRP expression in tumors. SIGNIFICANCE STATEMENT: Evaluating the role of reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) on antifolates accumulation in non-small cell lung cancer (NSCLC) is necessary for new drug designs. By using cell models, we found both RFC and PCFT were important for antifolates accumulation in NSCLC. Breast cancer resistance protein (BCRP) significantly affected PCFT-mediated antifolates accumulation at acidic pH but not RFC-mediated pemetrexed accumulation at physiological pH. High expression of PCFT or RFC enhanced the cytotoxicity and antitumor effect of pemetrexed.

A Homozygous Variant in NAA60 Is Associated with Primary Familial Brain Calcification.

Primary familial brain calcification (PFBC) is a monogenic disorder characterized by bilateral calcifications in the brain. The genetic basis remains unknown in over half of the PFBC patients, indicating the existence of additional novel causative genes. NAA60 was a recently reported novel causative gene for PFBC. The aim was to identify the probable novel causative gene in an autosomal recessive inherited PFBC family. We performed a comprehensive genetic study on a consanguineous Chinese family with 3 siblings diagnosed with PFBC. We evaluated the effect of the variant in a probable novel causative gene on the protein level using Western blot, immunofluorescence, and coimmunoprecipitation. Possible downstream pathogenic mechanisms were further explored in gene knockout (KO) cell lines and animal models. We identified a PFBC co-segregated homozygous variant of c.460_461del (p.D154Lfs*113) in NAA60. Functional assays showed that this variant disrupts NAA60 protein localization to Golgi and accelerated protein degradation. The mutant NAA60 protein alters its interaction with the PFBC-related proteins PiT2 and XPR1, affecting intracellular phosphate homeostasis. Further mass spectrometry analysis in NAA60 KO cell lines revealed decreased expression of multiple brain calcification-associated proteins, including reduced folate carrier (RFC), a folate metabolism-related protein. Our study replicated the identification of NAA60 as a novel causative gene for autosomal recessive PFBC, demonstrating our causative variant leads to NAA60 loss of function. The NAA60 loss of function disrupts not only PFBC-related proteins (eg, PiT2 and XPR1) but also a wide range of other brain calcification-associated membrane protein substrates (eg, RFC), and provided a novel probable pathogenic mechanism for PFBC. © 2024 International Parkinson and Movement Disorder Society.

Abstract P136: Phosphorylation and regulation of URAT1 by insulin and NaCl: a possible mechanism linking insulin resistance and excessive salt intake to hyperuricemia

Objective: Insulin resistance (IR) is the key environmental factor associated with hyperuricemia; however, the underlying mechanisms are not fully understood. Although several studies suggested that excessive salt intake results in hyperuricemia, the association still remains controversial. Focusing on urate transporter 1 (URAT1), we here investigated the mechanisms by which insulin and salt intake contribute to hyperuricemia. Methods: In a large UK Biobank (UKB) dataset involving 377,358 participants, we evaluated the association of TyG index (a surrogate of IR) and habitual salt intake with serum uric acid levels using multivariable regression models. We addressed whether single nucleotide polymorphisms (SNPs) that influence URAT1 expression (eQTL) modify the association of TyG index with serum uric acid levels. We also analyzed underlying mechanisms by using HEK cells expressing hURAT1. Results: In UKB dataset, TyG index and habitual salt intake were significantly and independently associated with elevated serum uric acid levels (P &lt; 0.05). From previous studies and imputation accuracy, we extracted an eQTL SNP for SLC22A12 gene and found that there is a positive interaction between TyG Index and the eQTL SNP. As post-translational modification is the key mechanism that enables adaptation to environmental changes, we evaluated the contribution of phosphorylation by AGC kinases, such as Akt, as possible downstream effectors of insulin signaling and salt-sensing pathway. Western blotting with phospho-Akt substrate motif antibody confirmed URAT1 phosphorylation and identified T350 and T408 as candidate sites. Among them, URAT1 carrying non-phosphorylatable A408 substitution (T408A-URAT1) suppressed glycosylation and plasma membrane translocation, while T350A had no effect. By using kinase screen assay involving 53 AGC and related Ser/Thr kinases, we found that SGK1, PKA, and PKG also phosphorylated URAT1-T408. Insulin and NaCl loading induced Akt and SGK1 expression respectively, which resulted in URAT1 phosphorylation and increased cell-surface membrane expression; this effect was abolished in T408A-URAT1. Finally, we found that URAT1-T408 phosphorylation was also detected in human kidney sections. Conclusion: IR and excessive salt intake contribute to hyperuricemia, and the effect is in part mediated by hURAT1. Mechanistically, insulin and NaCl promote membrane trafficking of URAT1 by inducing T408 phosphorylation through Akt and SGK1, respectively.

Disulfidptosis-related gene SLC7A11 predicts prognosis and indicates tumor immune infiltration in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is closely associated with factors such as smoking and metabolic disorders. A unique form of cell death known as disulfidptosis, which is regulated by genes like SLC7A11, has emerged as an area of interest; however, its effect on the immune microenvironment in the context of cancer remains largely unexplored. The aim of this study was to analyze the immunoregulatory role of disulfidptosis-related genes in LUAD to unveil and underscore their significance in the process of immune regulation. This study examined the role of disulfidptosis-related genes in LUAD using data from The Cancer Genome Atlas (TCGA) with a particular focus on immune infiltration and the function of SLC7A11. The research employed a clustering analysis, survival analysis, and immune function assessment, integrating both bulk and single-cell RNA sequencing data, to gain a comprehensive understanding of disulfidptosis in LUAD. The analysis revealed three distinct LUAD clusters, each characterized by different survival rates and patterns of immune cell infiltration. Notably, high expression levels of SLC7A11 were associated with a poor prognosis and mechanisms of immune evasion. High SLC7A11 expression is correlated with a poor prognosis and immune evasion in LUAD. These results underscore the significant role of SLC7A11 in the progression of disulfidptosis and LUAD. This study sheds new light on the role of disulfidptosis in LUAD, particularly highlighting the immunoregulatory effects of SLC7A11. The findings suggest that targeting SLC7A11 could lead to the development of novel therapeutic strategies aimed at enhancing the response to immunotherapy in LUAD patients. To substantiate these results, further experimental validation is needed.

Unraveling the potential effects of non-synonymous single nucleotide polymorphisms (nsSNPs) on the Protein structure and function of the human SLC30A8 gene on type 2 diabetes and colorectal cancer: An In silico approach

Background and aimsThe single nucleotide polymorphisms (SNPs) in SLC30A8 gene have been recognized as contributing to type 2 diabetes (T2D) susceptibility and colorectal cancer. This study aims to predict the structural stability, and functional impacts on variations in non-synonymous SNPs (nsSNPs) in the human SLC30A8 gene using various computational techniques. Materials and methodsSeveral in silico tools, including SIFT, Predict-SNP, SNPs&GO, MAPP, SNAP2, PhD-SNP, PANTHER, PolyPhen-1,PolyPhen-2, I-Mutant 2.0, and MUpro, have been used in our study. ResultsAfter data analysis, out of 336 missenses, the eight nsSNPs, namely R138Q, I141N, W136G, I349N, L303R, E140A, W306C, and L308Q, were discovered by ConSurf to be in highly conserved regions, which could affect the stability of their proteins. Project HOPE determines any significant molecular effects on the structure and function of eight mutated proteins and the three-dimensional (3D) structures of these proteins. The two pharmacologically significant compounds, Luzonoid B and Roseoside demonstrate strong binding affinity to the mutant proteins, and they are more efficient in inhibiting them than the typical SLC30A8 protein using Autodock Vina and Chimera. Increased binding affinity to mutant SLC30A8 proteins has been determined not to influence drug resistance. Ultimately, the Kaplan-Meier plotter study revealed that alterations in SLC30A8 gene expression notably affect the survival rates of patients with various cancer types. ConclusionFinally, the study found eight highly deleterious missense nsSNPs in the SLC30A8 gene that can be helpful for further proteomic and genomic studies for T2D and colorectal cancer diagnosis. These findings also pave the way for personalized treatments using biomarkers and more effective healthcare strategies.

Single nucleotide polymorphisms (SNPs) that are associated with obesity and type 2 diabetes among Asians: a systematic review and meta-analysis

Single nucleotide polymorphisms (SNPs) could increase the susceptibility of individuals to develop obesity and type 2 diabetes (T2DM). Obesity and T2DM are closely related pathophysiologically, thus similar SNPs could mediate both these diseases, but this is rarely reported. Furthermore, limited studies have been performed to summarize SNP data in the Asian population compared to the Western population. In this study, we aimed to summarize SNPs that are associated with the development of obesity and T2DM among Asian populations. We searched six literature databases and Review Manager (RevMan) was used for meta-analysis. The pooled odds ratios (ORs) and 95% CIs were calculated with a random effects model for the heterogeneity among studies. The pooled analysis showed that rs9939609 (FTO gene) and rs17782313 and rs571312 (MC4R gene) are associated with obesity with an odd ratio (OR) of 1.37, 1.36 and 1.29 respectively. For T2DM, five SNPs, rs7903146 and rs12255372 (TCF7L2 gene), rs13266634 and rs11558471 (SLC30A8 gene) and rs2283228 (KCNQ1 gene) have also shown strong associations with T2DM at OR of 1.64, 1.61, 1.22, 1.29 and 1.60 respectively. This data could be used to develop a gene screening panel for assessing obesity and T2DM susceptibility.

Regulation of folate transport at the mouse arachnoid barrier

BackgroundFolates are a family of B9 vitamins essential for normal growth and development in the central nervous system (CNS). Transport of folates is mediated by three major transport proteins: folate receptor alpha (FRα), proton-coupled folate transporter (PCFT), and reduced folate carrier (RFC). Brain folate uptake occurs at the choroid plexus (CP) epithelium through coordinated actions of FRα and PCFT, or directly into brain parenchyma at the vascular blood–brain barrier (BBB), mediated by RFC. Impaired folate transport can occur due to loss of function mutations in FRα or PCFT, resulting in suboptimal CSF folate levels. Our previous reports have demonstrated RFC upregulation by nuclear respiratory factor-1 (NRF-1) once activated by the natural compound pyrroloquinoline quinone (PQQ). More recently, we have identified folate transporter localization at the arachnoid barrier (AB). The purpose of the present study was to further characterize folate transporters localization and function in AB cells, as well as their regulation by NRF-1/PGC-1α signaling and folate deficiency.MethodsIn immortalized mouse AB cells, polarized localization of RFC and PCFT was assessed by immunocytochemical analysis, with RFC and PCFT functionality examined with transport assays. The effects of PQQ treatment on changes in RFC functional expression were also investigated. Mouse AB cells grown in folate-deficient conditions were assessed for changes in gene expression of the folate transporters, and other key transporters and tight junction proteins.ResultsImmunocytochemical analysis revealed apical localization of RFC at the mouse AB epithelium, with PCFT localized on the basolateral side and within intracellular compartments. PQQ led to significant increases in RFC functional expression, mediated by activation of the NRF-1/PGC-1α signalling cascade. Folate deficiency led to significant increases in expression of RFC, MRP3, P-gp, GLUT1 and the tight junction protein claudin-5.ConclusionThese results uncover the polarized expression of RFC and PCFT at the AB, with induction of RFC functional expression by activation of the NRF-1/PGC-1α signalling pathway and folate deficiency. These results suggest that the AB may contribute to the flow of folates into the CSF, representing an additional pathway when folate transport at the CP is impaired.

Mitochondrial related variants associated with cardiovascular traits.

Cardiovascular disease (CVD) is responsible for over 30% of mortality worldwide. CVD arises from the complex influence of molecular, clinical, social, and environmental factors. Despite the growing number of autosomal genetic variants contributing to CVD, the cause of most CVDs is still unclear. Mitochondria are crucial in the pathophysiology, development and progression of CVDs; the impact of mitochondrial DNA (mtDNA) variants and mitochondrial haplogroups in the context of CVD has recently been highlighted. We investigated the role of genetic variants in both mtDNA and nuclear-encoded mitochondrial genes (NEMG) in CVD, including coronary artery disease (CAD), hypertension, and serum lipids in the UK Biobank, with sub-group analysis for diabetes. We investigated 371,542 variants in 2,527 NEMG, along with 192 variants in 32 mitochondrial genes in 381,994 participants of the UK Biobank, stratifying by presence of diabetes. Mitochondrial variants showed associations with CVD, hypertension, and serum lipids. Mitochondrial haplogroup J was associated with CAD and serum lipids, whereas mitochondrial haplogroups T and U were associated with CVD. Among NEMG, variants within Nitric Oxide Synthase 3 (NOS3) showed associations with CVD, CAD, hypertension, as well as diastolic and systolic blood pressure. We also identified Translocase Of Outer Mitochondrial Membrane 40 (TOMM40) variants associated with CAD; Solute carrier family 22 member 2 (SLC22A2) variants associated with CAD and CVD; and HLA-DQA1 variants associated with hypertension. Variants within these three genes were also associated with serum lipids. Our study demonstrates the relevance of mitochondrial related variants in the context of CVD. We have linked mitochondrial haplogroup U to CVD, confirmed association of mitochondrial haplogroups J and T with CVD and proposed new markers of hypertension and serum lipids in the context of diabetes. We have also evidenced connections between the etiological pathways underlying CVDs, blood pressure and serum lipids, placing NOS3, SLC22A2, TOMM40 and HLA-DQA1 genes as common nexuses.

Association of Functional Genetic Variations in Uric Acid Transporters with the Risk of Idiopathic Male Infertility: A Genetic Association Study and Bioinformatic Analysis.

Uric acid plays an important role in sustaining and improving sperm morphology, viability, and motility. It is known that SLC2A9 and ABCG2 protein are the main urate transporter and genetic variations in these genes could be associated with the levels of serum uric acid. This study aimed to investigate the association between single-nucleotide polymorphisms (SNPs) SLC2A9-rs16890979, SLC2A9-rs3733591, ABCG2-rs2231142, and ABCG2-rs2231137 with male infertility. Additionally, the correlation of these SNPs with the uric acid level in seminal plasma of infertile men was examined. Subsequently, an in silico analysis was performed. In a case-control study, 193 infertile and 154 healthy controls were recruited. After semen sample collection, the uric acid level of seminal plasma was measured by a commercial kit. After genomic DNA extraction from sperm samples, SNPs genotyping was performed by PCR-RFLP method. Lastly, the effects of SNPs on the SLC2A9 and ABCG2 gene function were evaluated by bioinformatics tools. The genetic association study revealed that there are significant associations between rs16890979, rs3733591, rs2231142, and rs2231137 genetic variations and increased risk of male infertility. Also, these variations were associated with oligozoospermia and teratozoospermia, and sometimes with asthenozoospermia. Also, we found that four studied SNPs could be associated with a decreased level of uric acid of seminal plasma in teratozoospermia and asthenozoospermia. Bioinformatic analysis revealed that the mentioned polymorphisms could affect molecular aspects of SLC2A9 and ABCG2 genes. In this preliminary study, the rs16890979, rs3733591, rs2231142, and rs2231137 genetic variations could be considered as genetic risk factors for male infertility by interfering with the uric acid level of seminal plasma.

Microsporidian EnP1 alters host cell H2B monoubiquitination and prevents ferroptosis facilitating microsporidia survival

Microsporidia are intracellular eukaryotic pathogens that pose a substantial threat to immunocompromised hosts. The way these pathogens manipulate host cells during infection remains poorly understood. Using a proximity biotinylation strategy we established that microsporidian EnP1 is a nucleus-targeted effector that modifies the host cell environment. EnP1's translocation to the host nucleus is meditated by nuclear localization signals (NLSs). In the nucleus, EnP1 interacts with host histone H2B. This interaction disrupts H2B monoubiquitination (H2Bub), subsequently impacting p53 expression. Crucially, this inhibition of p53 weakens its control over the downstream target gene SLC7A11, enhancing the host cell's resilience against ferroptosis during microsporidian infection. This favorable condition promotes the proliferation of microsporidia within the host cell. These findings shed light on the molecular mechanisms by which microsporidia modify their host cells to facilitate their survival.

The Human OCTN Sub-Family: Gene and Protein Structure, Expression, and Regulation.

OCTN1 and OCTN2 are membrane transport proteins encoded by the SLC22A4 and SLC22A5 genes, respectively. Even though several transcripts have been predicted by bioinformatics for both genes, only one functional protein isoform has been described for each of them. Both proteins are ubiquitous, and depending on the physiopathological state of the cell, their expression is regulated by well-known transcription factors, although some aspects have been neglected. A plethora of missense variants with uncertain clinical significance are reported both in the dbSNP and the Catalogue of Somatic Mutations in Cancer (COSMIC) databases for both genes. Due to their involvement in human pathologies, such as inflammatory-based diseases (OCTN1/2), systemic primary carnitine deficiency (OCTN2), and drug disposition, it would be interesting to predict the impact of variants on human health from the perspective of precision medicine. Although the lack of a 3D structure for these two transport proteins hampers any speculation on the consequences of the polymorphisms, the already available 3D structures for other members of the SLC22 family may provide powerful tools to perform structure/function studies on WT and mutant proteins.

Clinical profile and thiamine transporter gene (SLC19A2 and SLC19A3) variations in infants with thiamine-responsive pulmonary hypertension and acute respiratory infection

Abstract Maternal thiamine deficiency is prevalent in low- and middle-income countries. Thiamine-responsive pulmonary hypertension (TRPHTN) in exclusively breastfed infants is reported in India. Thiamine transporter gene (ThTR) variations have not been studied. This study compared the presentation of exclusively breastfed infants with respiratory distress diagnosed as TRPHTN or acute respiratory infection (ARI). We investigated pathogenic variations in the SLC19A2 and SLC19A3 ThTr genes in a representative sample. Observational study. Tertiary care pediatric unit of a teaching hospital in southern India. Data collection was prospective. We included exclusively breastfed infants between 1 and 6 months of age with respiratory distress. Infants with PHTN in echocardiography and lactic acidosis (LA) received thiamine. TRPHTN was diagnosed based on response within 72 h. Infants with fever, chest findings, and positive microbiology were managed as ARI. The ThTr genes were sequenced and analyzed. Chi-square and stratified analysis were done to determine TRPHTN risk. Forty infants with TRPHTN and 42 with ARI were included. The median pulmonary arterial pressure in the TRPHTN group was 51.5 mmHg. Mild PHTN was seen in 65%, moderate in 22.5%, and severe in 12.5%. Cardiac failure (P &amp;lt; .001), stridor and aphonia (P &amp;lt; .001), encephalopathy (P = .024), LA (P &amp;lt; .001), and PHTN (P &amp;lt;.001) facilitated the diagnosis. The adjusted risk was 17.3 (95% confidence interval 7.8–38.3; P &amp;lt;.001). The ThTR sequencing showed wild-type genotypes. TRPHTN has a distinct, identifiable presentation. Lactate and pulmonary pressure estimations are useful investigations in thiamine deficiency endemic areas. We could not demonstrate a genetic variation that determines susceptibility.

The role of single nucleotide variants of folate cycle genes of a mother with epilepsy in the occurrence of congenital malformations of the fetus

Aim - To study the frequency of single-nucleotide variants (ONV) rs1801133 and rs1801131 of the MTHFR gene; rs1801394 of the MTRR gene, rs1805087 of the MTR gene and rs1051266 of the SLC19A1 gene in women with epilepsy and to evaluate their associations with major congenital malformations (MCM) of the fetus. Material and methods. The study included 61 women with epilepsy who have children: 20 had different fetal MCM (main group), 41 patients had children born without MCM (comparison group). DNA was extracted from blood, and the genotyping of five ONV into four genes was analyzed by polymerase chain reaction. The frequencies of genotypes and alleles in the mothers of the main and the comparison group were determined, the differences were assessed using Pearson's chi-squared criterion (χ2) and Fisher's exact criterion. Results. There were no statistically significant differences in the frequencies of genotypes and alleles for all analyzed ONV between the main group and the comparison group. There were no statistically significant differences in the frequencies of genotypes and alleles of ONV genes of the folate cycle in mothers of children with malformations (n = 14) and without malformations (n = 22), taking valproic acid. A statistically significant relationship was revealed between the carrier of a certain haplogroup of the mother and the formation of fetal MCM. Conclusion. The MCM in a child is a multifactorial phenomenon in which genetic factors with a small effect size can play a significant role only in the case of certain unfavorable combinations.