Folate Biosynthesis Pathway Research Articles

Bisubstrate inhibitors of 6-hydroxymethyl-7,8-dihydroptein pyrophosphokinase: Toward cell permeability

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a key enzyme in the folate biosynthesis pathway. It catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). HPPK is essential for microorganisms but is absent in mammals. Yet, it is not the target of any existing antibiotics. Hence, this enzyme is an attractive target for developing novel antimicrobial agents. A wealth of structural and mechanistic information has provided solid basis for structure-based design of HPPK inhibitors. Our bisubstrate inhibitors were initially created by linking 6-hydroxymethylpterin to adenosine through 2, 3, or 4 phosphate groups (HPnA, n = 2, 3, or 4), among which HP4A exhibited the highest binding affinity (Kd = 0.47 ± 0.04 μM). Further development was carried out based on high-resolution structures of HPPK in complex with HP4A. Replacing the phosphate bridge with a piperidine linked thioether eliminated multiple negative charges of the bridge. Substituting the pterin moiety with 7,7-dimethyl-7,8-dihydropterin improved the binding affinity. Arming the piperidine ring with a carboxyl group and oxidizing the thioether further enhanced the potency, resulting in a druglike inhibitor of HPPK (Kd = 0.047 ± 0.007 μM). None of these inhibitors, however, exhibits bacterial cell permeability. It is most likely due to the lack of active folate transporters in bacteria. Replacing the pterin moiety with a 7-deazagaunine moiety, we have obtained a novel bisubstrate inhibitor (HP-101) showing observable cell permeability toward a Gram-positive bacterium. Here, we report the in vitro activity of HP-101 and its structure in complex with HPPK, providing a framework for structure-based further development.

Control of biofilm formation by an Agrobacterium tumefaciens pterin-binding periplasmic protein conserved among diverse Proteobacteria

Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen Agrobacterium tumefaciens produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase, is crucial in control of UPP production and surface attachment. DcpA is regulated by PruR, a protein with distant similarity to enzymatic domains known to coordinate the molybdopterin cofactor (MoCo). Pterins are bicyclic nitrogen-rich compounds, several of which are produced via a nonessential branch of the folate biosynthesis pathway, distinct from MoCo. The pterin-binding protein PruR controls DcpA activity, fostering c-di-GMP breakdown and dampening its synthesis. Pterins are excreted, and we report here that PruR associates with these metabolites in the periplasm, promoting interaction with the DcpA periplasmic domain. The pteridine reductase PruA, which reduces specific dihydro-pterin molecules to their tetrahydro forms, imparts control over DcpA activity through PruR. Tetrahydromonapterin preferentially associates with PruR relative to other related pterins, and the PruR-DcpA interaction is decreased in a pruA mutant. PruR and DcpA are encoded in an operon with wide conservation among diverse Proteobacteria including mammalian pathogens. Crystal structures reveal that PruR and several orthologs adopt a conserved fold, with a pterin-specific binding cleft that coordinates the bicyclic pterin ring. These findings define a pterin-responsive regulatory mechanism that controls biofilm formation and related c-di-GMP-dependent phenotypes in A. tumefaciens and potentially acts more widely in multiple proteobacterial lineages.

Effect of folA gene in human breast milk-derived Limosilactobacillus reuteri on its folate biosynthesis.

Folate supplementation is crucial for the human body, and the chemically synthesized folic acid might have undesirable side effects. The use of molecular breeding methods to modify the genes related to the biosynthesis of folate by probiotics to increase folate production is currently a focus of research. In this study, the folate-producing strain of Limosilactobacillus reuteri B1-28 was isolated from human breast milk, and the difference between B1-28 and folA gene deletion strain ΔFolA was investigated by phenotyping, in vitro probiotic evaluation, metabolism and transcriptome analysis. The results showed that the folate producted by the ΔFolA was 2-3 folds that of the B1-28. Scanning electron microscope showed that ΔFolA had rougher surface, and the acid-producing capacity (p = 0.0008) and adhesion properties (p = 0.0096) were significantly enhanced than B1-28. Transcriptomic analysis revealed that differentially expressed genes were mainly involved in three pathways, among which the biosynthesis of ribosome and aminoacyl-tRNA occurred in the key metabolic pathways. Metabolomics analysis showed that folA affected 5 metabolic pathways, involving 89 different metabolites. In conclusion, the editing of a key gene of folA in folate biosynthesis pathway provides a feasible pathway to improve folate biosynthesis in breast milk-derived probiotics.

Transcriptome profiles of Trypanosoma brucei rhodesiense in Malawi reveal focus specific gene expression profiles associated with pathology.

Sleeping sickness caused by Trypanosoma brucei rhodesiense is a fatal disease and endemic in Southern and Eastern Africa. There is an urgent need to develop novel diagnostic and control tools to achieve elimination of rhodesiense sleeping sickness which might be achieved through a better understanding of trypanosome gene expression and genetics using endemic isolates. Here, we describe transcriptome profiles and population structure of endemic T. b. rhodesiense isolates in human blood in Malawi. Blood samples of r-HAT cases from Nkhotakota and Rumphi foci were collected in PaxGene tubes for RNA extraction before initiation of r-HAT treatment. 100 million reads were obtained per sample, reads were initially mapped to the human genome reference GRCh38 using HiSat2 and then the unmapped reads were mapped against Trypanosoma brucei reference transcriptome (TriTrypDB54_TbruceiTREU927) using HiSat2. Differential gene expression analysis was done using the DeSeq2 package in R. SNP calling from reads that were mapped to the T. brucei genome was done using GATK in order to identify T.b. rhodesiense population structure. 24 samples were collected from r-HAT cases of which 8 were from Rumphi and 16 from Nkhotakota foci. The isolates from Nkhotakota were enriched with transcripts for cell cycle arrest and stumpy form markers, whereas isolates in Rumphi focus were enriched with transcripts for folate biosynthesis and antigenic variation pathways. These parasite focus-specific transcriptome profiles are consistent with the more virulent disease observed in Rumphi and a less symptomatic disease in Nkhotakota associated with the non-dividing stumpy form. Interestingly, the Malawi T.b. rhodesiense isolates expressed genes enriched for reduced cell proliferation compared to the Uganda T.b. rhodesiense isolates. PCA analysis using SNPs called from the RNAseq data showed that T. b. rhodesiense parasites from Nkhotakota are genetically distinct from those collected in Rumphi. Our results suggest that the differences in disease presentation in the two foci is mainly driven by genetic differences in the parasites in the two major endemic foci of Rumphi and Nkhotakota rather than differences in the environment or host response.

Repurposing FDA-approved drugs to target malaria through inhibition of dihydrofolate reductase in the folate biosynthesis pathway: A prospective approach.

Dihydrofolate reductase (DHFR) is a ubiquitous enzyme that regulates the biosynthesis of tetrahydrofolate among various species of Plasmodium parasite. It is a validated target of the antifolate drug pyrimethamine (Pyr) in Plasmodium falciparum (Pf), but its clinical efficacy has been hampered due to the emergence of drug resistance. This has made the attempt to screen Food & Drug Administration-approved drugs against wild- and mutantPfDHFR by employing an in-silico pipeline to identify potent candidates. The current study has followed a virtual screening approach for identifying potential DHFR inhibitors from DrugBank database, based on a structure similarity search of candidates, followed by absorption, distribution, metabolism, and excretion estimation. The screened drugs were subjected to various parameters like docking, molecular mechanics with generalized born and surface area solvation calculations, and molecular simulations. We have thus identified twopotential drug candidates, duloxetine and guanethidine, which can be repurposed to be tested for their efficacy against wild type and drug resistant falciparum malaria.

Transcriptome Analysis Reveals the Mechanisms of Accumulation and Conversion of Folate Derivatives during Germination of Quinoa (Chenopodium quinoa Willd.) Seeds.

Folate was enriched during quinoa germination, while molecular mechanisms were not well understood. In this study, three quinoa varieties were selected for germination, and changes in substrate content and enzyme activity of the folate biosynthesis pathway were monitored. 5-Methyltetrahydrofolate (5-CH3-THF) and 5-formyltetrahydrofolate (5-CHO-THF) were significantly enriched in quinoa sprouts. Among the selected varieties, QL-2 exhibited the lowest content of the oxidation product MeFox and the highest total folate content. Based on transcriptome analysis, the p-ABA branch was found to be crucial for folate accumulation, while the pterin branch served as a key control point for the one carbon pool by folate pathway, which limited further folate biosynthesis. In the one carbon pool by folate pathway, genes CqMTHFR and CqAMT significantly contributed to the enrichment of 5-CH3-THF and 5-CHO-THF. Findings gained here would facilitate the potential application of quinoa sprouts as an alternative strategy for folate supplementation.

Urine proteomic analysis of the rat e-cigarette model.

We were curious if the urinary proteome could reflect the effects of e-cigarettes on the organism. Urine samples were collected from a rat e-cigarette model before, during, and after two weeks of e-cigarette smoking. Urine proteomes before and after smoking of each rat were compared individually, while the control group was set up to rule out differences caused by rat growth and development. Fetuin-B, a biomarker of chronic obstructive pulmonary disease (COPD), and annexin A2, which is recognized as a multiple tumour marker, were identified as differential proteins in five out of six smoking rats on day 3. To our surprise, odourant-binding proteins expressed in the olfactory epithelium were also found and were significantly upregulated. Pathways enriched by the differential proteins include the apelin signalling pathway, folate biosynthesis pathway, arachidonic acid metabolism, chemical carcinogenesis-DNA adducts and chemical carcinogenesis-reactive oxygen species. They have been reported to be associated with immune system, cardiovascular system, respiratory system, etc. Urinary proteome could reflect the effects of e-cigarettes in rats.

Identification of novel targets and mechanisms of wogonin on lung cancer, bladder cancer, and colon cancer

Wogonin (WOG) has been demonstrated to have anti-cancer activity, but the mechanisms remain unclear. In this study, new targets of WOG were predicted for lung cancer, bladder cancer, and colon cancer by using bioinformatics methods. WOG might primarily suppress cancers via regulating arachidonic acid, Ras, MAPK, linoleic acid, PI3K-Akt, and folate biosynthesis pathways. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay showed that WOG inhibited the proliferation of A549 cells. Real-time quantitative reverse transcription PCR (RT-qPCR) results indicated that anti-lung cancer effect of WOG was achieved by regulating the expression of 18 target genes, including AKR1B10, AKR1C3, BDNF, CAV1, CXCL2, CYP2B6, CYP4F3, DAO, EGF, ENO3, IL6, PLA2G1B, PLA2G2F, PLA2G4A, PTGES, SLCO1B1, SLCO1B3, and TFAP2A. The Kaplan-Meier survival curves further confirmed that DAO, PLA2G1B, SLCO1B3 and TFAP2A were essential targets via which WOG affected lung cancer survival. Moreover, BDNF, FGF2, and PTGS1 were predicted to be the targets via which WOG alleviated cancer proliferation and invasion in bladder cancer. As for colon cancer, WOG might induce autophagy and inhibit proliferation by down-regulating NTF4 and TH. The study will provide clue for using WOG as a promising antineoplastic agent in basic and translational research, and bring light to the application of herbs containing WOG as food supplements.

Silibinin reduces in vitro methane production by regulating the rumen microbiome and metabolites.

This study used Silibinin as an additive to conduct fermentation experiments, wherein its effects on rumen gas production, fermentation, metabolites, and microbiome were analyzed in vitro. The silibinin inclusion level were 0 g/L (control group), 0.075 g/L, 0.15 g/L, 0.30 g/L, and 0.60 g/L (experimental group). Fermentation parameters, total gas production, carbon dioxide (CO2), methane (CH4), hydrogen (H2), and their percentages were determined. Further analysis of the rumen microbiome's relative abundance and α/β diversity was performed on the Illumina NovaSeq sequencing platform. Qualitative and quantitative metabolomics analyses were performed to analyze the differential metabolites and metabolic pathways based on non-targeted metabolomics. The result indicated that with an increasing dose of silibinin, there was a linear reduction in total gas production, CO2, CH4, H2 and their respective percentages, and the acetic acid to propionic acid ratio. Concurrent with a linear increase in pH, when silibinin was added at 0.15 g/L and above, the total volatile fatty acid concentration decreased, the acetic acid molar ratio decreased, the propionic acid molar ratio increased, and dry matter digestibility decreased. At the same time, the relative abundance of Prevotella, Isotricha, Ophryoscolex, unclassified_Rotifera, Methanosphaera, Orpinomyces, and Neocallimastix in the rumen decreased after adding 0.60 g/L of silibinin. Simultaneously, the relative abundance of Succiniclasticum, NK4A214_group, Candidatus_Saccharimonas, and unclassified_Lachnospiraceae increased, altering the rumen species composition, community, and structure. Furthermore, it upregulated the ruminal metabolites, such as 2-Phenylacetamide, Phlorizin, Dalspinin, N6-(1,2-Dicarboxyethyl)-AMP, 5,6,7,8-Tetrahydromethanopterin, Flavin mononucleotide adenine dinucleotide reduced form (FMNH), Pyridoxine 5'-phosphate, Silibinin, and Beta-D-Fructose 6-phosphate, affecting phenylalanine metabolism, flavonoid biosynthesis, and folate biosynthesis pathways. In summary, adding silibinin can alter the rumen fermentation parameters and mitigate enteric methane production by regulating rumen microbiota and metabolites, which is important for developing novel rumen methane inhibitors.

Effects of Chromium Exposure on the Gene Expression of the Midgut in Silkworms, Bombyx mori.

Chromium is a severe heavy metal pollutant with significant environmental risks. The effects of Chromium on the digestion of Bombyx mori (silkworms) are of particular importance due to their ecological and economic significance. Herein, RNA sequencing was conducted on nine midgut samples from silkworms exposed to control, 12 g/kg and 24 g/kg Chromium chemical diets. Comparative transcriptomics revealed that under moderate Chromium exposure, there was a significant increase in up-regulated genes (1268 up-regulated to 857 down-regulated), indicating a stimulation response. At higher stress levels, a weakened survival response was observed, with a decrease in up-regulated genes and an increase in down-regulated genes (374 up-regulated to 399 down-regulated). A notable shift in cellular responses under medium chromium exposure was exposed, signifying the activation of crucial metabolic and transport systems and an elevation in cellular stress and toxicity mechanisms. The observation of up-regulated gene expression within xenobiotic metabolism pathways suggests a heightened defense against Chromium-induced oxidative stress, which was primarily through the involvement of antioxidant enzymes. Conversely, high-dose Chromium exposure down-regulates the folate biosynthesis pathway, indicating biological toxicity. Two novel genes responsive to pressure were identified, which could facilitate future stress adaptation understanding. The findings provide insights into the molecular mechanisms underlying silkworms' digestion response to Chromium exposure and could inform its biological toxicity.

Functional analysis of Rickettsia monacensis strain humboldt folA dihydrofolate reductase gene via complementation assay

Nutritive symbiosis between bacteria and ticks is observed across a range of ecological contexts; however, little characterization on the molecular components responsible for this symbiosis has been done. Previous studies in our lab demonstrated that Rickettsia monacensis str. Humboldt (strain Humboldt) can synthesize folate de novo via the folate biosynthesis pathway involving folA, folC, folE, folKP, and ptpS genes. In this study, expression of the strain Humboldt folA gene within a folA mutant Escherichia coli construct was used to functionally characterize the strain Humboldt folA folate gene in vivo. The strain Humboldt folA folate gene was subcloned into a TransBac vector and transformed into a folA mutant E. coli construct. The mutant containing strain Humboldt folA subclone and a pFE604 clone of the knocked-out folA gene was cured of pFE604. Curing of the folA mutant E. coli construct was successful using acridine orange and 43.5 °C incubation temperature. The plasmid curing assay showed curing efficiency of the folA mutant at 100%. Functional complementation was assessed by growth phenotype on minimal media with and without IPTG between strain Humboldt folA and E. coli folA. Large and homogenous wild-type colony growth was observed for both strain Humboldt and E. coli folA on minimal media with 0.1 mM IPTG, wild-type growth for strain Humboldt folA and pin-point growth for E. coli folA on 0.01 mM IPTG, and pin-point growth without IPTG for both strain Humboldt and E. coli folA. This study provides evidence substantiating the in vivo functionality of strain Humboldt folA in producing functional gene products for folate biosynthesis.

Systems-wide analysis of A. fumigatus using kinetic modeling of metabolic pathways to identify putative drug targets

Aspergillosis is a major causative factor for morbidity in those with impaired immune systems, often caused by Aspergillus fumigatus. The diagnosis and treatment are difficult due to the diversity of individuals and risk factors and still pose a challenge for medical professionals. To understand the pathogenicity of any organism, it is critical to identify the significant metabolic pathways that are involved. Our work focused on developing kinetic models of critical pathways crucial for the survival of A. fumigatus using COPASI. While focusing on the folate biosynthesis, ergosterol biosynthesis and glycolytic pathway; sensitivity, time-course and steady-state analysis were performed to find the proteins/enzymes that are essential in the pathway and can be considered as potential drug targets. For further analysis of the interaction of drug targets identified, a protein–protein interaction (PPI) network was built, and hub nodes were identified using the Cytohubba package from Cytoscape. Based on the findings, dihydropteroate-synthase, dihydrofolate-reductase, 4-amino-4-deoxychorismate synthase, HMG-CoA-reductase, PG-isomerase and hexokinase could act as potential drug targets. Further, molecular docking and MM-GBSA analysis were performed with ligands chosen from DrugBank, and PubChem, and validated by experimental evidence and existing literature based on results from kinetic modeling and PPI network analysis. Based on docking scores and MM-GBSA results, molecular simulations were carried out for 1AJ2-dapsone, 1DIS-sulfamethazine, 1T02-lovastatin and 70YL-3-bromopyruvic acid complexes, which validated our findings. Our study provides a deeper insight into the mechanisms of A. fumigatus’s metabolism to reveal dapsone, sulfamethazine, lovastatin and 3-bromopyruvic acid as potential drugs for the treatment of Aspergillosis.

SGLT2 Inhibition Alters the Hepatic and Circulating Metabolome of Mice Under a High-Milk-Fat Diet

Recent studies have shown that the inhibition of Sodium-glucose cotransporter 2 (SGLT2), which reabsorbs glucose in the renal proximal tubule, can aid in glycemic control under conditions of type 2 diabetes (T2D). Though it acts directly on the kidney, these inhibitors also lead to great improvement in liver function. Previously, we challenged TallyHo mice, an inbred polygenetic mice model that recapitulates T2D, with a high milk fat diet (HMFD) for 12 weeks in the continued absence or presence of Empagliflozin (EMPA), an FDA-approved SGLT2 inhibitor. Male mice showed a decrease in circulating triglycerides, hyperglycemia, and hepatic lipid and cholesterol esters (Kurtz et al 2022). However, the mechanisms that underlie this improved liver phenotype is unknown. Thus, we sought to characterize the hepatic and circulating metabolic and lipidomic profiles that are altered and/or restored in the presence of EMPA using targeted and untargeted LC-MS. Control, HMFD, and HMFD + EMPA liver tissues and plasma samples were dissolved in a methanol/water 50/50 extraction buffer that contained debrisoquine (DBQ) for the positive mode standard and 4-nitrobenzoic acid for the negative mode standard. For the untargeted liver and plasma samples, a UPLC-QTOF analysis was performed. MetaboAnalyst was used to quantify metabolite changes with inclusion criteria set to a fold change of 2 and a p-value <0.05. Of note, previous studies have shown that EMPA promotes the upregulation of ketone bodies including β-Hydroxybutyrate (BHB). We noted a similar upregulation of BHB in our metabolomic analysis and confirmed that EMPA led to a ~1.5-fold increase (p < 0.022) in this circulating ketone body. In addition, of those metabolites that met the inclusion criteria, we identified 12 that were downregulated and 9 that were upregulated +EMPA in the male samples. Out of these, orotate, sarcosine, tartrate, and dihydrofolate levels were restored to those of control levels. In the female samples, 6 were downregulated and 3 were upregulated +EMPA. Indoxyl sulfate, butylcarnitine, and valerylcarnetine were all restored to control levels. Of particular interest is the finding that EMPA treatment restores levels of key metabolites that are involved in the folate and pyrimidine biosynthesis pathways. Previous studies have determined that folate deficiency can promote insulin resistance and the urea cycle, a pathway that is integrated with pyrimidine biosynthesis, is upregulated under conditions of T2D and non-alcoholic fatty liver disease. Future studies are aimed at identifying the role of EMPA's hepatic protection in these metabolic pathways. NIDDK This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

RTP4, a Biomarker Associated with Diagnosing Pulmonary Tuberculosis and Pan-Cancer Analysis.

Pulmonary tuberculosis (PTB) is a global epidemic of infectious disease; the purpose of our study was to explore new potential biomarkers for the diagnosis of pulmonary tuberculosis and to use the biomarkers for further pan-cancer analysis. Four microarray gene expression sets were downloaded from the GEO public databases and conducted for further analysis. Healthy control (HC) samples and samples of pulmonary tuberculosis (PTB) were calculated with enrichment scores in folate biosynthesis pathways. The scores acted as a new phenotype combined with clinical information (control or PTB) for subsequent analysis. Weight gene coexpression network analysis (WGCNA) was used to seek the modules mostly related to PTB and folate biosynthesis in training sets. Twenty-nine coexistence genes were screened by intersecting the genes in the green-yellow module of GSE28623 and the brown module of GSE83456. We used the protein-protein interaction network analysis to narrow the gene range to search for hub genes. Then, we downloaded the unified and standardized pan-cancer data set from the UCSC database for correlations between biomarkers and prognosis and tumor stage differences. Eventually, RTP4 was selected as a biomarker. To verify the reliability of this biomarker, an area under the ROC (AUC) was calculated in gene sets (GSE28623, GSE83456, and GSE34608). Lastly, to explore the difference in RTP4 expression before and after antituberculosis treatment, the GSE31348 gene set was enrolled to compare the expressions in weeks 0 and 26. The results showed significant differences between these two time points (p < 0.001). RTP4 was significantly upregulated in the pulmonary tuberculosis group compared to the healthy control group in three gene sets and downregulated after antituberculosis therapy in one gene set. These results suggest that RTP4 can be used as a potential biomarker in diagnosing tuberculosis. The results of pan-cancer analysis showed that high expression of RTP4 in 4 tumor types was positively correlated with poor prognosis and high expression of RTP4 in 6 tumor types was negatively correlated with poor prognosis. We found significant differences in the expression of the RTP4 gene at different stages in 5 types of tumors. RTP4 might be a new potential biomarker for diagnosing pulmonary tuberculosis.

Inhibitory effect of natural compounds on Dihydropteroate synthase of Mycobacterium leprae: Molecular dynamic study

Leprosy is a chronic infectious disease caused by a bacillus, Mycobacterium leprae. According to official data from 139 countries in the 6 WHO Regions, there were 127558 new leprosy cases worldwide in 2020. Leprosy mainly affects the skin, the peripheral nerves, mucosa of the upper respiratory tract, and the eyes. If this disease is left untreated, can harm the skin, nerves, limbs, eyes, and skin permanently. The disease is curable with multidrug therapy. Over a period of time Mycobacterium leprae has become resistant to these drugs. Therefore, new therapeutic molecules are warranted. This study was aimed to carry out the in-silico analysis to determine the inhibitory effect of natural compounds on Dihydropteroate synthase (DHPS) of Mycobacterium leprae. The DHPS is a key enzyme in the folate biosynthesis pathway in M. leprae and acts as a competitive inhibitor of PABA. The 3D structure of DHPS protein was modeled using homology modeling and was validated. Molecular docking and simulation along with other in-silico methods were employed to determine the inhibitory effect of ligand molecules towards DHPS target protein. Results revealed ZINC03830554 molecule as a potential inhibitor of DHPS. Binding experiments and bioassays utilizing this strong inhibitor molecule against purified DHPS protein are necessary to validate these early findings.Communicated by Ramaswamy H. Sarma

Role of miRNAs in regulation of SA-mediated upregulation of genes involved in folate and methionine metabolism in foxtail millet.

The effect of exogenous salicylic acid (SA) on folate metabolism and the related gene regulatory mechanisms is still unclear. In this study, the panicle of foxtail millet treated with different SA concentrations showed that 6 mM SA doubled the 5-methyltetrahydrofolate content compared to that of the control. An untargeted metabolomic analysis revealed that 275 metabolites were enriched in amino acid metabolic pathways. Significantly, the relative content of methionine (Met) after 6 mM SA treatment was 3.14 times higher than the control. Transcriptome analysis revealed that differentially expressed genes were mainly enriched in the folate and amino acid biosynthesis pathways (including Met, Cys, Pro, Ser etal.). The miRNA-mRNA interactions related to the folate and Met metabolic pathways were analyzed and several likely structural gene targets for miRNAs were identified, miRNA-seq analysis revealed that 33 and 51 miRNAs targeted 11 and 15 genes related to the folate and Met pathways, respectively. Eight key genes in the folate metabolism pathway were likely to be up-regulated by 14 new miRNAs and 20 new miRNAs up-regulated the 9 key genes in the Met metabolism pathway. The 6 miRNA-mRNA interactions related to the folate and Met metabolism pathways were verified by qRT-PCR, and consistent with the prediction. The results showed that DHFR1 gene expression level related to folate synthesis was directly up-regulated by Nov-m0139-3p with 3.8 times, but DHFR2 was down-regulated by Nov-m0731-5p with 0.62 times. The expression level of CYSC1 and APIP related to Met synthesis were up-regulated by Nov-m0461-5p and Nov-m0664-3p with 4.27 and 1.32 times, respectively. Our results suggested that exogenous SA could induce the folate and Met accumulated in the panicle of foxtail millet. The higher expression level of DHFR1, FTHFD, CYSC1 and APIP in the folate and Met metabolism pathway and their regulators, including Nov-m0139-3p, Nov-m0717-5p, Nov-m0461-5p and Nov-m0664-3p, could be responsible for these metabolites accumulation. This study lays the theoretical foundation for elucidating the post-transcription regulatory mechanisms of folate and Met metabolism.

A genome-wide association study of folates in sweet corn kernels.

Folate is commonly synthesized in natural plants and is an essential water-soluble vitamin of great importance inhuman health. Although the key genes involved in folate biosynthesis and transformation pathways have been identified in plants, the genetic architecture of folate in sweet corn kernels remain largely unclear. In this study, an association panel of 295 inbred lines of sweet corn was constructed. Six folate derivatives were quantified in sweet corn kernels at 20 days after pollination and a total of 95 loci were identified for eight folate traits using a genome-wide association study. A peak GWAS signal revealed that natural variation in ZmFCL, encoding a 5-formyltetrahydrofolate cyclo-ligase, accounted for 30.12% of phenotypic variation in 5-FTHF content. Further analysis revealed that two adjacent SNPs on the second exon resulting in an AA-to-GG in the gene and an Asn-to-Gly change in the protein could be the causative variant influencing 5-FTHF content. Meanwhile, 5-FTHF content was negatively correlated with ZmFCL expression levels in the population. These results extend our knowledge regarding the genetic basis of folate and provide molecular markers for the optimization of folate levels in sweet corn kernels.

Deep metagenomic characterization of gut microbial community and function in preeclampsia.

Preeclampsia (PE) is a pregnancy complication characterized by severe hypertension and multiple organ damage. Gut microbiota has been linked to PE by previous amplicon sequencing studies. To resolve the PE gut microbiota in a higher taxonomy resolution, we performed shotgun metagenomic sequencing on the fecal samples from 40 early-onset PE and 37 healthy pregnant women. We recovered 1,750 metagenome-assembled genomes (representing 406 species) from the metagenomic dataset and profiled their abundances. We found that PE gut microbiota had enriched in some species belonging to Blautia, Pauljensenia, Ruminococcus, and Collinsella and microbial functions such as the bacitracin/lantibiotics transport system, maltooligosaccharide transport system, multidrug efflux pump, and rhamnose transport system. Conversely, the gut microbiome of healthy pregnant women was enriched in species of Bacteroides and Phocaeicola and microbial functions including the porphyrin and chlorophyll metabolism, pyridoxal-P biosynthesis, riboflavin metabolism, and folate biosynthesis pathway. PE diagnostic potential of gut microbial biomarkers was developed using both species and function profile data. These results will help to explore the relationships between gut bacteria and PE and provide new insights into PE early warning.

Development of Novel Anti-Leishmanials: The Case for Structure-Based Approaches.

The neglected tropical disease (NTD) leishmaniasis is the collective name given to a diverse group of illnesses caused by ~20 species belonging to the genus Leishmania, a majority of which are vector borne and associated with complex life cycles that cause immense health, social, and economic burdens locally, but individually are not a major global health priority. Therapeutic approaches against leishmaniasis have various inadequacies including drug resistance and a lack of effective control and eradication of the disease spread. Therefore, the development of a rationale-driven, target based approaches towards novel therapeutics against leishmaniasis is an emergent need. The utilization of Artificial Intelligence/Machine Learning methods, which have made significant advances in drug discovery applications, would benefit the discovery process. In this review, following a summary of the disease epidemiology and available therapies, we consider three important leishmanial metabolic pathways that can be attractive targets for a structure-based drug discovery approach towards the development of novel anti-leishmanials. The folate biosynthesis pathway is critical, as Leishmania is auxotrophic for folates that are essential in many metabolic pathways. Leishmania can not synthesize purines de novo, and salvage them from the host, making the purine salvage pathway an attractive target for novel therapeutics. Leishmania also possesses an organelle glycosome, evolutionarily related to peroxisomes of higher eukaryotes, which is essential for the survival of the parasite. Research towards therapeutics is underway against enzymes from the first two pathways, while the third is as yet unexplored.

Assay Development and Identification of the First Plasmodium falciparum 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase Inhibitors.

In the fight towards eradication of malaria, identifying compounds active against new drug targets constitutes a key approach. Plasmodium falciparum 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (PfHPPK) has been advanced as a promising target, as being part of the parasite essential folate biosynthesis pathway while having no orthologue in the human genome. However, no drug discovery efforts have been reported on this enzyme. In this study, we conducted a three-step screening of our in-house antifolate library against PfHPPK using a newly designed PfHPPK-GFP protein construct. Combining virtual screening, differential scanning fluorimetry and enzymatic assay, we identified 14 compounds active against PfHPPK. Compounds’ binding modes were investigated by molecular docking, suggesting competitive binding with the HMDP substrate. Cytotoxicity and in vitro ADME properties of hit compounds were also assessed, showing good metabolic stability and low toxicity. The most active compounds displayed low micromolar IC50 against drug-resistant parasites. The reported hit compounds constitute a good starting point for inhibitor development against PfHPPK, as an alternative approach to tackle the malaria parasite.