Project description:Metabolic changes in immune cells contribute to both physiological and pathophysiological outcomes of immune reactions. Here, by comparing protein expression, transcriptome, and salivary metabolome profiles of uninfected and HIV+ individuals, we found perturbations of polyamine metabolism in the oral mucosa of HIV+ patients. Mechanistic studies using an in vitro human tonsil organoid infection model revealed that HIV infection of T cells also resulted in increased polyamine synthesis, which was dependent on the activities of caspase-1, IL-1β, and ornithine decarboxylase-1. HIV-1 also led to a heightened expression of polyamine synthesis intermediates including ornithine decarboxylase-1 as well as an elevated dysfunctional regulatory T cell (TregDys)/T helper 17 (Th17) cell ratios. Blockade of caspase-1 and polyamine synthesis intermediates reversed the TregDys phenotype showing the direct role of polyamine pathway in altering T cell functions during HIV-1 infection. Lastly, oral mucosal TregDys/Th17 ratios and CD4 hyperactivation positively correlated with salivary putrescine levels, which were found to be elevated in the saliva of HIV+ patients. Thus, by revealing the role of aberrantly increased polyamine synthesis during HIV infection, our study unveils a mechanism by which chronic viral infections could drive distinct T cell effector programs and Treg dysfunction.
Project description:Acrolein, a respiratory irritant, induces systemic neuroendocrine stress. However, peripheral metabolic effects have not been examined. Male and female WKY rats were exposed to air (0 ppm) or acrolein (3.16 ppm) for 4 h, followed by immediate serum and liver tissue collection. Serum metabolomics in both sexes and liver transcriptomics in males were evaluated to characterize the systemic metabolic response. Of 887 identified metabolites, > 400 differed between sexes at baseline. An acrolein biomarker, 3-hydroxypropyl mercapturic acid, increased 18-fold in males and 33-fold in females, indicating greater metabolic detoxification in females than males. Acrolein exposure changed 174 metabolites in males but only 50 in females. Metabolic process assessment identified higher circulating free-fatty acids, glycerols, and other lipids in male but not female rats exposed to acrolein. In males, acrolein also increased branched-chain amino acids, which was linked with metabolites of nitrogen imbalance within the gut microbiome. The contribution of neuroendocrine stress was evident by increased corticosterone in males but not females. Male liver transcriptomics revealed acrolein-induced over-representation of lipid and protein metabolic processes, and pathway alterations including Sirtuin, insulin-receptor, acute-phase, and glucocorticoid signaling. In sum, acute acrolein inhalation resulted in sex-specific serum metabolomic and liver transcriptomic derangement, which may have connections to chronic metabolic-related diseases.
Project description:Background Cardiac arrest (CA) has been a leading cause of death for many decades. Despite years of research, we still do not understand how each organ responds to the reintroduction of blood flow after prolonged CA. Following changes in metabolites of individual organs after CA and resuscitation gives context to the efficiency and limitations of current resuscitation protocols. Methods and Results Adult male Sprague-Dawley rats were arbitrarily assigned into 3 groups: control, 20 minutes of CA, or 20 minutes of CA followed by 30 minutes of cardiopulmonary bypass resuscitation. The rats were euthanized by decapitation to harvest brain, heart, kidney, and liver tissues. The obtained tissue samples were analyzed by ultra-high-performance liquid chromatography-high-accuracy mass spectrometry for comprehensive metabolomics evaluation. After resuscitation, the brain showed decreased glycolysis metabolites and fatty acids and increased amino acids compared with control. Similarly, the heart displayed alterations mostly in amino acids. The kidney showed decreased amino acid and fatty acid pools with severely increased tricarboxylic acid cycle metabolites following resuscitation, while the liver showed minimal alterations with slight changes in the lipid pool. Each tissue has a distinct pattern of metabolite changes after ischemia/reperfusion. Furthermore, resuscitation worsens the metabolic dysregulation in the brain and kidney, while it normalizes metabolism in the heart. Conclusions Developing metabolic profiles using a global metabolome analysis identifies the variable nature of metabolites in individual organs after CA and reperfusion, establishing a stark contrast between the normalized heart and liver and the exacerbated brain and kidney, only after the reestablishment of blood circulation.
Project description:BackgroundPichia pastoris (Komagataella phaffii) is a model organism widely used for the recombinant expression of eukaryotic proteins, and it can metabolize methanol as its sole carbon and energy source. Methanol is oxidized to formaldehyde by alcohol oxidase (AOX). In the dissimilation pathway, formaldehyde is oxidized to CO2 by formaldehyde dehydrogenase (FLD), S-hydroxymethyl glutathione hydrolase (FGH) and formate dehydrogenase (FDH).ResultsThe transcriptome and metabolome of P. pastoris were determined under methanol cultivation when its dissimilation pathway cut off. Firstly, Δfld and Δfgh were significantly different compared to the wild type (GS115), with a 60.98% and 23.66% reduction in biomass, respectively. The differential metabolites between GS115 and Δfld were mainly enriched in ABC transporters, amino acid biosynthesis, and protein digestion and absorption. Secondly, comparative transcriptome between knockout and wild type strains showed that oxidative phosphorylation, glycolysis and the TCA cycle were downregulated, while alcohol metabolism, proteasomes, autophagy and peroxisomes were upregulated. Interestingly, the down-regulation of the oxidative phosphorylation pathway was positively correlated with the gene order of dissimilation pathway knockdown. In addition, there were significant differences in amino acid metabolism and glutathione redox cycling that raised our concerns about formaldehyde sorption in cells.ConclusionsThis is the first time that integrity of dissimilation pathway analysis based on transcriptomics and metabolomics was carried out in Pichia pastoris. The blockage of dissimilation pathway significantly down-regulates the level of oxidative phosphorylation and weakens the methanol assimilation pathway to the point where deficiencies in energy supply and carbon fixation result in inefficient biomass accumulation and genetic replication. In addition, transcriptional upregulation of the proteasome and autophagy may be a stress response to resolve formaldehyde-induced DNA-protein crosslinking.
Project description:Pheochromocytomas (PCCs) are tumors arising from chromaffin cells in the adrenal medulla, and paragangliomas (PGLs) are tumors derived from extra-adrenal sympathetic or parasympathetic paraganglia; these tumors are collectively referred to as PPGL cancer. Treatment for PPGL primarily involves surgical removal of the tumor, and only limited options are available for treatment of the disease once it becomes metastatic. Human carriers of the heterozygous mutations in the succinate dehydrogenase subunit B (SDHB) gene are susceptible to the development of PPGL. A physiologically relevant PCC patient-derived cell line hPheo1 was developed, and SDHB_KD cells carrying a stable short hairpin knockdown of SDHB were derived from it. An untargeted metabolomic approach uncovered an overactive polyamine pathway in the SDHB_KD cells that was subsequently fully validated in a large set of human SDHB-mutant PPGL tumor samples. We previously reported that treatment with the polyamine metabolism inhibitor N1,N11-diethylnorspermine (DENSPM) drastically inhibited growth of these PCC-derived cells in culture as well as in xenograft mouse models. Here we explored the mechanisms underlying DENSPM action in hPheo1 and SDHB_KD cells. Specifically, by performing an RNAseq analysis, we have identified gene expression changes associated with DENSPM treatment that broadly interfere with all aspects of lipid metabolism, including fatty acid (FA) synthesis, desaturation, and import/uptake. Furthermore, by performing an untargeted lipidomic liquid chromatography-mass spectrometry (LC/MS)-based analysis we uncovered specific groups of lipids that are dramatically reduced as a result of DENSPM treatment. Specifically, the bulk of plasmanyl ether lipid species that have been recently reported as the major determinants of cancer cell fate are notably decreased. In summary, this work suggests an intersection between active polyamine and lipid pathways in PCC cells.
Project description:New therapeutic targets for oral squamous cell carcinoma (OSCC) are urgently needed. We conducted genome-wide CRISPR-Cas9 screens in 21 OSCC cell lines, primarily derived from Asians, to identify genetic vulnerabilities that can be explored as therapeutic targets. We identify known and novel fitness genes and demonstrate that many previously identified OSCC-related cancer genes are non-essential and could have limited therapeutic value, while other fitness genes warrant further investigation for their potential as therapeutic targets. We validate a distinctive dependency on YAP1 and WWTR1 of the Hippo pathway, where the lost-of-fitness effect of one paralog can be compensated only in a subset of lines. We also discover that OSCCs with WWTR1 dependency signature are significantly associated with biomarkers of favorable response toward immunotherapy. In summary, we have delineated the genetic vulnerabilities of OSCC, enabling the prioritization of therapeutic targets for further exploration, including the targeting of YAP1 and WWTR1.
Project description:BackgroundMale fertility impaired by exogenous toxins is a serious worldwide issue threatening the health of the new-born and causing infertility. However, the metabolic connection between toxic exposures and testicular dysfunction remains unclear.ResultsIn the present study, the metabolic disorder of testicular dysfunction was investigated using triptolide-induced testicular injury in mice. We found that triptolide induced spermine deficiency resulting from disruption of polyamine biosynthesis and uptake in testis, and perturbation of the gut microbiota. Supplementation with exogenous spermine reversed triptolide-induced testicular dysfunction through increasing the expression of genes related to early and late spermatogenic events, as well as increasing the reduced number of offspring. Loss of gut microbiota by antibiotic treatment resulted in depletion of spermine levels in the intestine and potentiation of testicular injury. Testicular dysfunction in triptolide-treated mice was reversed by gut microbial transplantation from untreated mice and supplementation with polyamine-producing Parabacteroides distasonis. The protective effect of spermine during testicular injury was largely dependent on upregulation of heat shock protein 70s (HSP70s) both in vivo and in vitro.ConclusionsThe present study linked alterations in the gut microbiota to testicular dysfunction through disruption of polyamine metabolism. The diversity and dynamics of the gut microbiota may be considered as a therapeutic option to prevent male infertility. Video Abstract.
Project description:The polyamines putrescine, spermidine and spermine are nutrient-like polycationic molecules involved in metabolic processes and signaling pathways linked to cell growth and cancer. One important pathway is the PI3K/Akt pathway where studies have shown that polyamines mediate downstream growth effects. Downstream of PI3K/Akt is the mTOR signaling pathway, a nutrient-sensing pathway that regulate translation initiation through 4EBP1 and p70S6K phosphorylation and, along with the PI3K/Akt, is frequently dysregulated in breast cancer. In this study, we investigated the effect of intracellular polyamine modulation on mTORC1 downstream protein and general translation state in two breast cancer cell lines, MCF-7 and MDA-MB-231. The effect of mTORC1 pathway inhibition on the growth and intracellular polyamines was also measured. Results showed that polyamine modulation alters 4EBP1 and p70S6K phosphorylation and translation initiation in the breast cancer cells. mTOR siRNA gene knockdown also inhibited cell growth and decreased putrescine and spermidine content. Co-treatment of inhibitors of polyamine biosynthesis and mTORC1 pathway induced greater cytotoxicity and translation inhibition in the breast cancer cells. Taken together, these data suggest that polyamines promote cell growth in part through interaction with mTOR pathway. Similarly intracellular polyamine content appears to be linked to mTOR pathway regulation. Finally, dual inhibition of polyamine and mTOR pathways may provide therapeutic benefits in some breast cancers.
Project description:BackgroundFruit skin color play important role in commercial value of cucumber, which is mainly determined by the content and composition of chlorophyll and anthocyanins. Therefore, understanding the related genes and metabolomics involved in composition of fruit skin color is essential for cucumber quality and commodity value.ResultsThe results showed that chlorophyll a, chlorophyll b and carotenoid content in fruit skin were higher in Lv (dark green skin) than Bai (light green skin) on fruit skin. Cytological observation showed more chloroplast existed in fruit skin cells of Lv. A total of 162 significantly different metabolites were found between the fruit skin of the two genotypes by metabolome analysis, including 40 flavones, 9 flavanones, 8 flavonols, 6 anthocyanins, and other compounds. Crucial anthocyanins and flavonols for fruit skin color, were detected significantly decreased in fruit skin of Bai compared with Lv. By RNA-seq assay, 4516 differentially expressed genes (DEGs) were identified between two cultivars. Further analyses suggested that low expression level of chlorophyll biosynthetic genes, such as chlM, por and NOL caused less chlorophylls or chloroplast in fruit skin of Bai. Meanwhile, a predicted regulatory network of anthocyanin biosynthesis was established to illustrate involving many DEGs, especially 4CL, CHS and UFGT.ConclusionsThis study uncovered significant differences between two cucumber genotypes with different fruit color using metabolome and RNA-seq analysis. We lay a foundation to understand molecular regulation mechanism on formation of cucumber skin color, by exploring valuable genes, which is helpful for cucumber breeding and improvement on fruit skin color.
Project description:Nucleotide sugar-dependent glycosyltransferases (UGTs) are critical to the homeostasis of endogenous metabolites and the detoxification of xenobiotics. Their impact on the cell metabolome remains unknown. Cellular metabolic changes resulting from human UGT expression were profiled by untargeted metabolomics. The abundant UGT1A1 and UGT2B7 were studied as UGT prototypes along with their alternative (alt.) splicing-derived isoforms displaying structural differences. Nineteen biochemical routes were modified, beyond known UGT substrates. Significant variations in glycolysis and pyrimidine pathways, and precursors of the co-substrate UDP-glucuronic acid were observed. Bioactive lipids such as arachidonic acid and endocannabinoids were highly enriched by up to 13.3-fold (p < 0.01) in cells expressing the canonical enzymes. Alt. UGT2B7 induced drastic and unique metabolic perturbations, including higher glucose (18-fold) levels and tricarboxylic acid cycle (TCA) cycle metabolites and abrogated the effects of the UGT2B7 canonical enzyme when co-expressed. UGT1A1 proteins promoted the accumulation of branched-chain amino acids (BCAA) and TCA metabolites upstream of the mitochondrial oxoglutarate dehydrogenase complex (OGDC). Alt. UGT1A1 exacerbated these changes, likely through its interaction with the OGDC component oxoglutarate dehydrogenase-like (OGDHL). This study expands the breadth of biochemical pathways associated with UGT expression and establishes extensive connectivity between UGT enzymes, alt. proteins and other metabolic processes.