Pyrimidine Pathway Research Articles

Integrate Analysis of Eyestalk Proteome and Metabolome in Precocious and Formal Juvenile Female Eriocheir sinensis

The Chinese mitten crab (Eriocheir sinensis) is an economically important crustacean. With the development of the E. sisnensis industry, precocity has become a significant challenge in juvenile crab culturing. In this study, the eyestalks of female E. sinensis from precocious (PE) and normal juvenile (NE) groups were used for proteome and metabolome analyses. In total, 731 up-regulated and 657 down-regulated differentially expressed proteins (DEPs) were identified in the PE and NE groups. In addition, 110 differentially expressed metabolites (DMs) were up-regulated and 256 were down-regulated in the PE group. An integrated analysis showed 5667 significant correlations between the metabolites and proteins and 109 common pathways in the proteome and metabolome. The proteins were mostly associated with the mechanistic target of rapamycin (mTOR) pathway, longevity regulation, autophagy, and the pyrimidine and purine metabolism pathways. The metabolites were primarily enriched in amino acid and lipid metabolisms. These results demonstrated the differences in the PE and NE groups at two omics levels and will be useful for the E. sinensis industry.

Metabolomic profiles differentiate between porto-sinusoidal vascular disorder, cirrhosis, and healthy individuals

Background & AimsPorto-sinusoidal vascular disorder (PSVD) is a rare and diagnostically challenging vascular liver disease. This study aimed to identify distinct metabolomic signatures in patients with PSVD, cirrhosis, and healthy volunteers (HV) to facilitate non-invasive diagnosis and elucidate perturbed metabolic pathways. MethodsSerum samples from 20 HV and patients with histologically confirmed PSVD or cirrhosis were analyzed. Metabolites were measured using liquid chromatography-mass spectrometry (LC-MS). Differential abundance was evaluated with Limma’s moderated t-statistics. Artificial neural network (ANN) and support vector machine (SVM) models were developed to classify PSVD against cirrhosis or HV metabolomic profiles. An independent cohort was used for validation. ResultsA total of 283 metabolites were included for downstream analysis. Clustering effectively separated PSVD metabolomes from HV, although a subset of PSVD patients (n=5, 25%) overlapped with cirrhosis. Differential testing revealed significant PSVD-linked metabolic perturbations, including taurocholic and adipic acids, distinguishing PSVD from both HV and cirrhosis. Alterations in pyrimidine, glycine, serine, and threonine pathways were exclusively associated with PSVD. Machine learning models utilizing selected metabolic signatures reliably differentiated PSVD from HV or cirrhosis using only 4 to 6 metabolites. Validation in an independent cohort demonstrated the high discriminative ability of taurocholic acid (AUROC 0.899) for PSVD vs HV and the taurocholic acid/aspartic acid ratio (AUROC 0.720) for PSVD vs cirrhosis. ConclusionsHigh-throughput metabolomics enabled the identification of distinct metabolic profiles for differentiating PSVD, cirrhosis, and healthy individuals. Unique alterations in the glycine, serine, and threonine pathways suggest their potential involvement in PSVD pathogenesis. Impact And ImplicationsPorto-sinusoidal vascular disorder (PSVD) is a vascular liver disease that can lead to pre-sinusoidal portal hypertension in the absence of cirrhosis, with poorly understood pathophysiology and no established treatment. Our study demonstrates that analyzing the serum metabolome could reveal distinct metabolic signatures in PSVD patients, including alterations in the pyrimidine, glycine, serine and threonine pathways and potentially shedding light on the disease's underlying pathways. These findings hold potential for earlier and non-invasive diagnosis of PSVD, potentially reducing reliance on invasive procedures like liver biopsy and guiding diagnostic pathways.

Effects of Lactocaseibacillus paracasei subsp. paracasei NTU 101 on gut microbiota: a randomized, double-blind, placebo-controlled clinical study.

Lactocaseibacillus paracasei subsp. paracasei NTU 101 (NTU101) is a well-known commercial probiotic with multiple health beneficial effects. In this study, the gut microbiota modulation effect of an NTU 101 product, Vigiis 101-LAB, on healthy human was investigated in a randomized, double-blind, placebo-controlled human trial. Vigiis 101-LAB significantly modulated human gut microbiota at fourth and sixth weeks of trial (anosim analysis, P = 0.001). It also significantly improved peristalsis (P = 0.003) and shortened defecation interval of subjects. The shift of gut microbiota is significantly fit with defecation interval (P = 0.009) and stool shape (P = 0.001) of subjects. Our results suggest that Vigiis 101-LAB promotes human intestinal health with improvement of peristalsis and fecal quality. The gut modulation effects of Vigiis 101-LAB subsequently raised the abundance of vitamin B7, vitamin K, pyrimidine and purine biosynthesis pathways. Vigiis 101-LAB may promote peristalsis via purinergic pathway and possibly conferring prophylactic benefits against irritable bowel syndrome with constipation. © 2024 Society of Chemical Industry.

Role of vaccinia virus growth factor in stimulating the mTORC1-CAD axis of the de novo pyrimidine pathway under different nutritional cues.

Vaccinia virus (VACV), the prototype poxvirus, actively reprograms host cell metabolism upon infection. However, the nature and molecular mechanisms remain largely elusive. Given the diverse nutritional exposures of cells in different physiological contexts, it is essential to understand how VACV may alter various metabolic pathways in different nutritional conditions. In this study, we established the importance of de novo pyrimidine biosynthesis in VACV infection. We elucidated the significance of vaccinia growth factor (VGF), a viral early protein and a homolog of cellular epidermal growth factor, in enabling VACV to phosphorylate the key enzyme CAD of the de novo pyrimidine pathway at serine 1859, a site known to positively regulate CAD activity. While nutrient-poor conditions typically inhibit mTORC1 activation, VACV activates CAD via mTORC1-S6K1 signaling axis, in conditions where glutamine and asparagine are absent. However, unlike its cellular homolog, epidermal growth factor (EGF), VGF peptide alone in the absence of VACV infection has minimal ability to activate CAD, suggestive of the involvement of other viral factor(s) and differential functions to EGF acquired during poxvirus evolution. Our research provides a foundation for understanding the regulation of a significant metabolic pathway, namely, de novo pyrimidine synthesis during VACV infection, shedding new light on viral regulation under distinct nutritional environments. This study not only has the potential to contribute to the advancement of antiviral treatments but also improve the development of VACV as an oncolytic agent and vaccine vector.

The DNA damage response of Escherichia coli, revisited: Differential gene expression after replication inhibition

In 1967, in this journal, Evelyn Witkin proposed the existence of a coordinated DNA damage response in Escherichia coli, which later came to be called the "SOS response." We revisited this response using the replication inhibitor azidothymidine (AZT) and RNA-Seq analysis and identified several features. We confirm the induction of classic Save our ship (SOS) loci and identify several genes, including many of the pyrimidine pathway, that have not been previously demonstrated to be DNA damage-inducible. Despite a strong dependence on LexA, these genes lack LexA boxes and their regulation by LexA is likely to be indirect via unknown factors. We show that the transcription factor "stringent starvation protein" SspA is as important as LexA in the regulation of AZT-induced genes and that the genes activated by SspA change dramatically after AZT exposure. Our experiments identify additional LexA-independent DNA damage inducible genes, including 22 small RNA genes, some of which appear to activated by SspA. Motility and chemotaxis genes are strongly down-regulated by AZT, possibly as a result of one of more of the small RNAs or other transcription factors such as AppY and GadE, whose expression is elevated by AZT. Genes controlling the iron siderophore, enterobactin, and iron homeostasis are also strongly induced, independent of LexA. We confirm that IraD antiadaptor protein is induced independent of LexA and that a second antiadaptor, IraM is likewise strongly AZT-inducible, independent of LexA, suggesting that RpoS stabilization via these antiadaptor proteins is an integral part of replication stress tolerance.

The energy metabolism and transcriptomic responses of the Manila clam (Ruditapes philippinarum) under the low-temperature stress

Low temperature in winter poses a threat to the Manila clam Ruditapes philippinarum in North China. However, a number of low-temperature-tolerant clams could survive such condition. It is therefore of interest to explore the survival mechanisms underlying the cold tolerance of R. philippinarum. The Zebra II population of R. philippinarum (Zebra II) from North China and the native Putian population from South China were used as experimental materials. Both populations were stressed with low-temperature and the differences in their survival rates, energy metabolism and transcriptional responses were compared. The results shown that after cold treatment at −1.9 °C, survival rate of Zebra II was higher than that of the Putian group. For both groups, the respiration, ammonia excretion, and ingestion rates continuously decreased till 0 with reductions temperature. In addition, RNA-seq revealed that as compared with the Putian group, there were 3682 up-regulated differentially expressed genes (DEGs) and 3361 down-regulated DEGs in Zebra II group. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that these DEGs were mostly enriched in the purine, pyrimidine, and pyruvate metabolism pathways in Zebra II under low-temperature stress. Furthermore, qRT–PCR analysis further confirmed that Zebra II responded to low-temperature stress through upregulating genes involved in purine, pyrimidine, and pyruvate metabolism pathways. Taken together, all these results indicated that Zebra II has higher cold tolerance than the Putian group. Therefore, Zebra II is capable for overwintering in the intertidal zone of North China.

Uracil as a biomarker for spatial pyrimidine metabolism in the development of gingivobuccal oral squamous cell carcinoma

No biomarker has yet been identified that allows accurate diagnosis and prognosis of oral cancers. In this study, we investigated the presence of key metabolites in oral cancer using proton nuclear magnetic resonance (NMR) spectroscopy to identify metabolic biomarkers of gingivobuccal oral squamous cell carcinoma (GB-OSCC). NMR spectroscopy revealed that uracil was expressed in 83.09% of tumor tissues and pyrimidine metabolism was active in GB-OSCC; these results correlated well with immunohistochemistry (IHC) and RNA sequencing data. Based on further gene and protein analyses, we proposed a pathway for the production of uracil in GB-OSCC tissues. Uridinetriphosphate (UTP) is hydrolyzed to uridine diphosphate (UDP) by CD39 in the tumor microenvironment (TME). We hypothesized that UDP enters the cell with the help of the UDP-specific P2Y6 receptor for further processing by ENTPD4/5 to produce uracil. As the ATP reserves diminish, the weakened immune cells in the TME utilize pyrimidine metabolism as fuel for antitumor activity, and the same mechanism is hijacked by the tumor cells to promote their survival. Correspondingly, the differential expression of ENTPD4 and ENTPD5 in immune and tumor cells, respectively, indicatedtheir involvement in disease progression. Furthermore, higher uracil levels were detected in patients with lymph node metastasis, indicating that metastatic potential is increased in the presence of uracil. The presence of uracil and/or expression patterns of intermediate molecules in purine and pyrimidine pathways, such asCD39, CD73, and P2Y6 receptors together with ENTPD4 and ENTPD5, hold promise as biomarker(s) for oral cancer diagnosis and prognosis.

Prognostic and Immunotherapeutic Predictive Value of CAD Gene in Hepatocellular Carcinoma: Integrated Bioinformatics and Experimental Analysis.

Hepatocellular carcinoma (HCC) is a common type of cancer that ranks first in cancer-associated death worldwide. Carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) are the key components of the pyrimidine pathway, which promotes cancer development. However, the function of CAD in HCC needs to be clarified. In this study, the clinical and transcriptome data of 424 TCGA-derived HCC cases were analyzed. The results demonstrated that high CAD expression was associated with poor prognosis in HCC patients. The effect of CAD on HCC was then investigated comprehensively using GO annotation analysis, KEGG enrichment analysis, Gene Set Enrichment Analysis (GSEA), and CIBERSORT algorithm. The results showed that CAD expression was correlated with immune checkpoint inhibitors and immune cell infiltration. In addition, low CAD levels in HCC patients predicted increased sensitivity to anti-CTLA4 and PD1, while HCC patients with high CAD expression exhibited high sensitivity to chemotherapeutic and molecular-targeted agents, including gemcitabine, paclitaxel, and sorafenib. Finally, the results from clinical sample suggested that CAD expression increased remarkably in HCC compared with non-cancerous tissues. Loss of function experiments demonstrated that CAD knockdown could significantly inhibit HCC cell growth and migration both in vitro and in vivo. Collectively, the results indicated that CAD is a potential oncogene during HCC metastasis and progression. Therefore, CAD is recommended as a candidate marker and target for HCC prediction and treatment.

Microbiome profiling and Co-metabolism pathway analysis in cervical cancer patients with acute radiation enteritis

BackgroundIntestinal bacteria significantly contribute to the metabolism of intestinal epithelial tissues. As the occurrence and development of radiation enteritis (RE) depend on the “co-metabolism” microenvironment formed by the host and intestinal microbiota, which involves complex influencing factors and strong correlations, ordinary techniques struggle to fully explain the underlying mechanisms. However, given that it is based on systems biology, metabolomics analysis is well-suited to address these issues. This study aimed to analyze the metabolomic changes in urine, serum, and fecal samples during volumetric modulated arc therapy (VMAT) for cervical cancer and screen for characteristic metabolites of severe acute radiation enteritis (SARE) and RE. MethodsWe enrolled 50 patients who received radiotherapy for cervical cancer. Urine, serum, and fecal samples of patients were collected at one day before radiotherapy and the second week, fourth week, and sixth week after the start of radiotherapy. Control group samples were collected during the baseline period. Differential metabolites were identified by metabolomics analysis; co-metabolic pathways were clarified. We used the mini-SOM library for incorporating characteristic metabolites, and established metabolite classification models for predicting SARE and RE. ResultsUrine and serum sample data showed remarkable clustering effect; metabolomics data of the fecal supernatant were evidently disturbed. Patient sample analyses during VMAT revealed the following. Urine samples: Downregulation of the pyrimidine and riboflavin metabolism pathways as well as initial upregulation followed by downregulation of arginine and proline metabolism pathways and the arginine biosynthesis pathway. Fecal samples: Upregulation of linoleic acid and phenylalanine metabolic pathways and initial downregulation followed by upregulation of arachidonic acid (AA) metabolic pathways. Serum samples: Initial upregulation followed by downregulation of the arginine biosynthesis pathway and downregulation of glutathione, AA, and arginine and proline metabolic pathways. ConclusionPatients with cervical cancer exhibited characteristic metabolic pathways and characteristic metabolites predicting RE and SARE were screened out. An effective RE mini-SOM classification model was successfully established.

A novel large intragenic DPYD deletion causing dihydropyrimidine dehydrogenase deficiency: a case report

BackgroundDihydropyrimidine dehydrogenase (DPD), is the initial and rate-limiting enzyme in the catabolic pathway of pyrimidines. Deleterious variants in the DPYD gene cause DPD deficiency, a rare autosomal recessive disorder. The clinical spectrum of affected individuals is wide ranging from asymptomatic to severely affected patients presenting with intellectual disability, motor retardation, developmental delay and seizures. DPD is also important as the main enzyme in the catabolism of 5-fluorouracil (5-FU) which is extensively used as a chemotherapeutic agent. Even in the absence of clinical symptoms, individuals with either complete or partial DPD deficiency face a high risk of severe and even fatal fluoropyrimidine-associated toxicity. The identification of causative genetic variants in DPYD is therefore gaining increasing attention due to their potential use as predictive markers of fluoropyrimidine toxicity.MethodsA male infant patient displaying biochemical features of DPD deficiency was investigated by clinical exome sequencing. Bioinformatics tools were used for data analysis and results were confirmed by MLPA and Sanger sequencing.ResultsA novel intragenic deletion of 71.2 kb in the DPYD gene was identified in homozygosity. The deletion, DPYD(NM_000110.4):c.850 + 23455_1128 + 8811del, eliminates exons 9 and 10 and may have resulted from a non-homologous end-joining event, as suggested by in silico analysis.ConclusionsThe study expands the spectrum of DPYD variants associated with DPD deficiency. Furthermore, it raises the concern that patients at risk for fluoropyrimidine toxicity due to DPYD deletions could be missed during pre-treatment genetic testing for the currently recommended single nucleotide polymorphisms.

Adrenal pheochromocytoma impacts three main pathways: cysteine-methionine, pyrimidine, and tyrosine metabolism.

Pheochromocytomas and paragangliomas (PPGLs) cause symptoms by altering the circulation levels of catecholamines and peptide hormones. Currently, the diagnosis of PPGLs relies on diagnostic imaging and the detection of catecholamines. In this study, we used ultra-performance liquid chromatography (UPLC)/quadrupole time-of-flight mass spectrometry (Q-TOF MS) analysis to identify and measure the perioperative differential metabolites in the plasma of adrenal pheochromocytoma patients. We identified differentially expressed genes by comparing the transcriptomic data of pheochromocytoma with the normal adrenal medulla. Through conducting two steps of metabolomics analysis, we identified 111 differential metabolites between the healthy group and the patient group, among which 53 metabolites were validated. By integrating the information of differential metabolites and differentially expressed genes, we inferred that the cysteine-methionine, pyrimidine, and tyrosine metabolism pathways were the three main metabolic pathways altered by the neoplasm. The analysis of transcription levels revealed that the tyrosine and cysteine-methionine metabolism pathways were downregulated in pheochromocytoma, whereas the pyrimidine pathway showed no significant difference. Finally, we developed an optimized diagnostic model of two metabolites, L-dihydroorotic acid and vanylglycol. Our results for these metabolites suggest that they may serve as potential clinical biomarkers and can be used to supplement and improve the diagnosis of pheochromocytoma.

Machine learning identifies fatigue as a key symptom of fibromyalgia reflected in tyrosine, purine, pyrimidine, and glutaminergic metabolism.

Fibromyalgia patients vary in clinical phenotype and treatment can be challenging. The pathophysiology of fibromyalgia is incompletely understood but appears to involve metabolic changes at rest or in response to stress. We enrolled 54 fibromyalgia patients and 31 healthy controls to this prospective study. Symptoms were assessed using the Fibromyalgia Impact Questionnaire (FIQ) and blood samples were collected for metabolomics analysis at baseline and after an oral glucose tolerance test and a cardiopulmonary exercise test. We identified key symptoms of fibromyalgia and related them to changes in metabolic pathways with supervised and unsupervised machine learning methods. Algorithms trained with the FIQ information assigned the fibromyalgia diagnosis in new data with balanced accuracy of 88% while fatigue alone already provided the diagnosis with 86% accuracy. Supervised analyses reduced the metabolomic information from 77 to 13 key markers. With these metabolites, fibromyalgia could be identified in new cases with 79% accuracy. In addition, 5-hydroxyindole-3-acetic acid and glutamine levels correlated with the severity of fatigue. Patients differed from controls at baseline in tyrosine and purine pathways, and in the pyrimidine pathway after the stress challenges. Several key markers are involved in glutaminergic neurotransmission. This data-driven analysis highlights fatigue as a key symptom of fibromyalgia. Fibromyalgia is associated with metabolic changes which also reflect the degree of fatigue. Responses to metabolic and physical stresses result in a metabolic pattern that allows discrimination of fibromyalgia patients from controls and narrows the focus on key pathophysiological processes in fibromyalgia as treatment targets.

Metabolomics and gut microbiota analysis reveal the differential efficacy of areca nut and charred areca nut in treating constipation.

Areca nut (AN) is a traditional Chinese herbal medicine used for centuries to treat gastrointestinal (GI) disorders. Charred AN (CAN) is a processed product of AN with similar therapeutic effects. This study aimed to investigate the therapeutic mechanisms of AN and CAN for constipation via metabolomics and gut microbiota analysis. In this study, the rats were randomly divided into 5 groups (n = 6): control, constipation model, positive drug, AN treatment, and CAN treatment groups. Constipation was induced by intragastric administration of loperamide hydrochloride, followed by 14-day treatment with mosapride, AN, or CAN. The efficacy difference between AN and CAN was assessed by evaluating the weight gain, fecal water content, GI transit rate, colonic histopathology, serum levels of GI hormones, gut microbiota, and fecal metabolites. The results demonstrated that both AN and CAN could alleviate loperamide-induced constipation. Furthermore, they significantly elevated the serum levels of motilin, vasoactive intestinal peptide, substance P, and acetylcholine. 16S rRNA analysis revealed that AN regulated the relative abundance of Bacillus, UCG-005, norank_f_Muribaculaceae, Candidatus_Saccharimonas, and Ruminococcus, whereas CAN modulate the relative abundance of Lactobacillus, Bacillus, norank_f_Muribaculaceae, Ruminococcus, unclassified_f_Oscillospiraceae, and unclassified_f_Prevotellaceae. Moreover, the metabolic profile of AN- and CAN-treated rats was also different, where AN treatment involved pathways of citrate cycle (TCA) and tyrosine, alanine, aspartate, and glutamate metabolisms. Whereas CAN treatment involved pathways of steroid and primary bile acid biosynthesis, as well as pyrimidine and purine metabolisms. Spearman correlation analysis indicated a close relationship between gut microbiota and fecal metabolites. In summary, this study revealed that AN may protect GI mucosa, enhance GI motility, and alleviate constipation symptoms by regulating the relative abundance of specific gut microbiota (Bacillus, UCG-005, norank_f_Muribaculaceae, Candidatus_Saccharimonas, Ruminococcus) as well as citrate cycle or tyrosine, alanine, aspartate, and glutamate metabolic pathways. Furthermore, CAN was observed to promote gastric emptying and intestinal propulsion, thereby alleviating constipation, by modulating the relative abundance of specific gut microbiota (Lactobacillus, Bacillus, norank_f_Muribaculaceae, Ruminococcus, unclassified_f_Oscillospiraceae, unclassified_f_Prevotellaceae) as well as steroid and primary bile acid biosynthesis, as well as pyrimidine and purine metabolic pathways.

Plasma metabolomics and quantitative interstitial abnormalities in ever-smokers

BackgroundQuantitative interstitial abnormalities (QIA) are an automated computed tomography (CT) finding of early parenchymal lung disease, associated with worse lung function, reduced exercise capacity, increased respiratory symptoms, and death. The metabolomic perturbations associated with QIA are not well known. We sought to identify plasma metabolites associated with QIA in smokers. We also sought to identify shared and differentiating metabolomics features between QIA and emphysema, another smoking-related advanced radiographic abnormality.MethodsIn 928 former and current smokers in the Genetic Epidemiology of COPD cohort, we measured QIA and emphysema using an automated local density histogram method and generated metabolite profiles from plasma samples using liquid chromatography–mass spectrometry (Metabolon). We assessed the associations between metabolite levels and QIA using multivariable linear regression models adjusted for age, sex, body mass index, smoking status, pack-years, and inhaled corticosteroid use, at a Benjamini–Hochberg False Discovery Rate p-value of ≤ 0.05. Using multinomial regression models adjusted for these covariates, we assessed the associations between metabolite levels and the following CT phenotypes: QIA-predominant, emphysema-predominant, combined-predominant, and neither- predominant. Pathway enrichment analyses were performed using MetaboAnalyst.ResultsWe found 85 metabolites significantly associated with QIA, with overrepresentation of the nicotinate and nicotinamide, histidine, starch and sucrose, pyrimidine, phosphatidylcholine, lysophospholipid, and sphingomyelin pathways. These included metabolites involved in inflammation and immune response, extracellular matrix remodeling, surfactant, and muscle cachexia. There were 75 metabolites significantly different between QIA-predominant and emphysema-predominant phenotypes, with overrepresentation of the phosphatidylethanolamine, nicotinate and nicotinamide, aminoacyl-tRNA, arginine, proline, alanine, aspartate, and glutamate pathways.ConclusionsMetabolomic correlates may lend insight to the biologic perturbations and pathways that underlie clinically meaningful quantitative CT measurements like QIA in smokers.

Integrative metabolomic and network pharmacological analysis reveals potential mechanisms of Cardamine circaeoides Hook.f. & Thomson in alleviating potassium oxonate-induced asymptomatic hyperuricemia in rats.

Cardamine circaeoides Hook.f. & Thomson (CC), a herb of the genus Cardamine (family Brassicaceae), has a rich historical usage in China for both culinary and medicinal purposes. It is distinguished by its remarkable ability to hyperaccumulate selenium (Se). CC has demonstrated efficacy in the prevention of metabolic disorders. However, investigations into the effects of CC on asymptomatic hyperuricemia remain scarce. The objective of this study is to elucidate the mechanism by which CC aqueous extract (CCE) exerts its anti-hyperuricemic effects on asymptomatic hyperuricemic rats induced by potassium oxonate (PO) by integrating metabolomics and network pharmacological analysis. Asymptomatic hyperuricemia was induced by feeding rats with PO (1000mg/kg) and CCE (0.75, 1.5, or 3g/kg) once daily for 30days. Various parameters, including body weight, uric acid (UA) levels, histopathology of renal tissue, and inflammatory factors (IL-1β, IL-6, IL-8, and TNF-α) were assessed. Subsequently, metabolomic analysis of kidney tissues was conducted to explore the effects of CCE on renal metabolites and the related pathways. Furthermore, network pharmacology was employed to explicate the mechanism of action of CCE components identified through UPLC-Q-TOF-MS analysis. Finally, metabolomic and network-pharmacology analyses were performed to predict crucial genes dysregulated in the disease model and rescued by CCE, which were then subjected to verification by RT-qPCR. The findings revealed that CCE significantly inhibited the UA levels from the 21stday to the 30thday. Moreover, CCE exhibited significant inhibition of IL-1β, IL-6, IL-8, and TNF-α levels in renal tissues. The dysregulation of 18 metabolites and the tyrosine, pyrimidine, cysteine, methionine, sphingolipid, and histidine metabolism pathways was prevented by CCE treatment. A joint analysis of targets predicted using the network pharmacology approach and the differential metabolites found in metabolics predicted 8 genes as potential targets of CCE, and 3 of them (PNP gene, JUN gene, and ADA gene) were verified at the mRNA level by RT-qPCR. We conclude that CCE has anti-hyperuricemia effects and alleviates renal inflammation in a rat model of hyperuricemia, and these efficacies are associated with the reversal of increased ADA, PNP, and JUN mRNA expression in renal tissues.

A conserved metabolic signature associated with response to fast-acting anti-malarial agents.

In malaria drug discovery, understanding the mode of action of lead compounds is important as it helps in predicting the potential emergence of drug resistance in the field when these drugs are eventually deployed. In this study, we have employed metabolomics technologies to characterize the potential targets of anti-malarial drug candidates in the developmental pipeline at NITD. We show that NITD fast-acting leads belonging to spiroindolone and imidazothiadiazole class induce a common biochemical theme in drug-exposed malaria parasites which is similar to another fast-acting, clinically available drug, DHA. These biochemical features which are absent in a slower acting NITD lead (GNF17) point to hemoglobin digestion and inhibition of the pyrimidine pathway as potential action points for these drugs. These biochemical themes can be used to identify and inform on the mode of action of fast drug candidates of similar profiles in future drug discovery programs.

Perturbation and mechanism of non-antibiotic drug in regulating resistome and metabolome of anammox consortia: An overlooked and underrated cause of multiresistance

Given that antibiotic resistance genes (ARGs) become emerging pollutants, their accumulation and transfer caused by extensive use of antibiotic and non-antibiotic drugs raise concerns. In contrast, the impacts of non-antibiotic drugs have been largely overlooked, and their regulation mechanism in anammox system remains unclear. In this study, the single and combined effects of typical antibiotic (doxycycline) and non-antibiotic drug (propranolol) on anammox consortia were evaluated and compared from process performance, community succession, metabolic and gene regulation perspectives. Significant reduction in the nitrogen removal efficiency (49.0 ± 0.5%) was only observed in the anammox process stressed by propranolol and doxycycline, but it was reversible. Metabolome results showed that anammox consortia upregulated amino acid metabolism under propranolol stress, while the abundance of pyrimidine and purine metabolic pathways significantly increased under doxycycline and multidrug stress. Microorganisms adapted to these stresses by changing the utilization preference for metabolites. Metagenomic analyses indicated that doxycycline and propranolol induced the high frequency of gene transfer through increasing oxidative stress level, membrane permeability, pili and efflux pumps. Significant correlations between ARGs and mobile genetic elements (MGEs), especially intI1, demonstrated the conjugative transfer of ARGs. Furthermore, structural equation models showed that microbial community, bacterial metabolism, MGEs, reactive oxygen species (ROS) and corresponding SOS response were key driving factors for ARGs transfer in the anammox systems. These findings fill the gap in the effect of non-antibiotic drugs on anammox system and their regulation mechanism, which further provides a theoretical guidance for anammox-based process to treat multidrug-containing wastewater.

Modularized reconstruction of pyrimidine pathway in Escherichia coli for enhanced pyrimidine nucleosides biosynthesis

Pyrimidine is a fundamental component of genetic material and a precursor for synthesis of various antiviral and antitumor drugs. In this study, we constructed a plasmid-free Escherichia coli strain HD11 as a platform for cytidine production. Compared to strain CR23-p21-p31, a higher titer of cytidine was achieved by overexpressing uridylate kinase from Sulfurovum lithotrophicum and the newly discovered cytidine triphosphate synthetase of Aquifex aeolicus in HD11. To enable efficient cytosine biosynthesis, a modularized metabolic pathway was integrated into HD11. Two different pathways based on the type of precursor were compared, and ribosome binding sites were optimized to enhance cytosine production. For pyrimidine ribonucleotide de novo biosynthesis pathway, an enzyme constrained metabolic model was utilized to guide media optimization. Finally, HD11-pDA20 produced 3.6 g/L cytosine and HD11 produced 8.1 g/L cytidine in fed-batch fermentation. The application of an enzyme-constrained metabolic model in cytidine/cytosine producing strain represents a novel approach to modular engineering.

Water extract of Pingchuan formula ameliorated murine asthma through modulating metabolites and gut microbiota

BackgroundPingchuan formula is a traditional Chinese herbal prescription for asthma, but its components and underlying mechanisms remain unclear. Here, we evaluated its anti-asthmatic actvity and regulatory effects on the gut microbiota in mice based on the traditional Chinese medicine Zang-Fu theory, which proposed the exterior–interior relationship between the lung and the large intestine. MethodsMouse model withovalbumin (OVA)-induced asthma was used to assess the protective effect of the water extract of Pingchuan formula (PC). The chemical compounds of PC and mouse serum metabolites were identified by Ultraperformance liquid chromatography-Q Exactive HF-X spectrometry. Gut microbiota was evaluated by 16 S rRNA gene sequencing. The gut microbiota was depleted with a broad-spectrum antibiotic mixture (Abx) to explore whether it plays a role in the protective effects of PC. ResultsPC mainly contains phenols, flavonoids, alkaloids, carboxylic acids, and their derivatives. PC attenuated OVA-induced asthma in mice by alleviating inflammatory infiltration, indicated by decreased levels of IL-18, IL-6, IL-4, and Eotaxin in lung tissues. PC treatment altered the serum metabolites and affected the pyrimidine pathway. In addition, our results showed that acacetin and abscisic acid were the key serum metabolites PC treatment changed the composition of gut microbiota by increasing the relative abundance of Clostridia_UCG_014 and Akkermansia while decreasing Blautia, Barnesiella, and Clostridium_Ⅲ at the genus level. Importantly, the Abx treatment partly abolished the anti-asthmatic effect of PC. ConclusionWe demonstrated that PC could alleviate OVA-induced asthma in mice and protect against inflammatory infiltration in lungs via modulating the serum metabolites and gut microbiota, thereby providing a new reference for the therapeutic effect of PC.

Combined analysis of the proteome and metabolome provides insight into microRNA-1174 function in Aedes aegypti mosquitoes

BackgroundPathogenic viruses can be transmitted by female Aedes aegypti (Ae. aegypti) mosquitoes during blood-meal acquisition from vertebrates. Silencing of mosquito- and midgut-specific microRNA (miRNA) 1174 (miR-1174) impairs blood intake and increases mortality. Determining the identity of the proteins and metabolites that respond to miR-1174 depletion will increase our understanding of the molecular mechanisms of this miRNA in controlling blood-feeding and nutrient metabolism of mosquitoes.MethodsAntisense oligonucleotides (antagomirs [Ant]) Ant-1174 and Ant-Ct were injected into female Ae. aegypti mosquitoes at 12–20 h posteclosion, and depletion of miR-1174 was confirmed by reverse transcription quantitative real-time PCR (RT-qPCR). Ant-1174-injected and control mosquitoes were collected before the blood meal at 72 h post-injection for tandem mass tag-based proteomic analysis and liquid chromatography-tandom mass spectrometry non-target metabolomic analysis to identify differentially expressed proteins and metabolites, respectively. RNA interference (RNAi) using double-stranded RNA (dsRNA) injection was applied to investigate the biological roles of these differentially expressed genes. The RNAi effect was verified by RT-qPCR and western blotting assays. Triglyceride content and ATP levels were measured using the appropriate assay kits, following the manufacturers’ instructions. Statistical analyses were conducted with GraphPad7 software using the Student’s t-test.ResultsUpon depletion of mosquito- and midgut-specific miR-1174, a total of 383 differentially expressed proteins (DEPs) were identified, among which 258 were upregulated and 125 were downregulated. Functional analysis of these DEPs using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment suggested that miR-1174 plays important regulatory roles in amino acid metabolism, nucleotide metabolism, fatty acid metabolism and sugar metabolism pathways. A total of 292 differential metabolites were identified, of which 141 were upregulated and 151 were downregulated. Integrative analysis showed that the associated differential proteins and metabolites were mainly enriched in a variety of metabolic pathways, including glycolysis, citrate cycle, oxidative phosphorylation and amino acid metabolism. Specifically, the gene of one upregulated protein in miR-1174-depleted mosquitoes, purine nucleoside phosphorylase (PNP; AAEL002269), was associated with the purine, pyrimidine and niacin-nicotinamide metabolism pathways. PNP knockdown seriously inhibited blood digestion and ovary development and increased adult mortality. Mechanically, PNP depletion led to a significant downregulation of the vitellogenin gene (Vg); in addition, some important genes in the ecdysone signaling and insulin-like peptide signaling pathways related to ovary development were affected.ConclusionsThis study demonstrates differential accumulation of proteins and metabolites in miR-1174-depleted Ae. aegypti mosquitoes using proteomic and metabolomic techniques. The results provide functional evidence for the role of the upregulated gene PNP in gut physiological activities. Our findings highlight key molecular changes in miR-1174-depleted Ae. aegypti mosquitoes and thus provide a basis and novel insights for increased understanding of the molecular mechanism involved in a lineage-specific miRNA in mosquito vectors.Graphical