Levels Of Guanosine Research Articles

Mechanism of ginsenoside Rb3 against OGD/R damage based on metabonomic and PCR array analyses.

In order to study the mechanisms of ginsenoside Rb3 (G-Rb3) against oxygen-glucose deprivation/reoxygenation (OGD/R) injury in HT22 cells based on metabolomics and PCR array, HT22 cells were randomly divided into control group, model group, G-Rb3 high-dose group (10 µmol/l) and G-Rb3 low-dose group (5 µmol/l). Except for the control group, which was left untreated, the remaining groups were incubated with 10 mmol/l Na2S2O4 in sugar-free DMEM medium for 2 h and then replaced with serum-free high-sugar DMEM medium for 2 h in order to replicate in vitro OGD/R model. Trypan blue staining was used to detect the cell viability; flow cytometry was used to detect apoptosis; western blotting was used to detect the protein expression levels of Bax, Bcl-2 and caspase-3. The metabolomics were used to analyze the differential metabolites of G-Rb3 affecting OGD/R in order to find the relevant metabolic pathways. PCR array assay was performed to identify the expression of the differential genes. G-Rb3 could inhibit HT22 apoptosis according to the result of cell morphology, trypan blue staining and flow cytometry. The levels of Bax and caspase-3 protein expression were decreased, whereas the level of Bcl-2 protein expression was increased after the treatment of G-Rb3. Metabolomics results showed that a total of 31 differential metabolites between OGD/R group and G-Rb3 group, such as guanosine level, was downregulated, the levels of enalaprilat and sorbitol were upregulated, affecting ABC transporters, galactose metabolism, citrate cycle and other related metabolic pathways; according to the result of PCR array, it was observed that G-Rb3 significantly downregulated Trp63, Trp73, Dapk1, Casp14 and Cd70 pro-apoptotic genes. In conclusion, G-Rb3 has a significant protective effect on the OGD/R model simulated in vitro, and the mechanism may be related to the inhibition of apoptosis by affecting metabolites.

Prevotella copri transplantation promotes neurorehabilitation in a mouse model of traumatic brain injury

BackgroundThe gut microbiota plays a critical role in regulating brain function through the microbiome-gut-brain axis (MGBA). Dysbiosis of the gut microbiota is associated with neurological impairment in Traumatic brain injury (TBI) patients. Our previous study found that TBI results in a decrease in the abundance of Prevotella copri (P. copri). P. copri has been shown to have antioxidant effects in various diseases. Meanwhile, guanosine (GUO) is a metabolite of intestinal microbiota that can alleviate oxidative stress after TBI by activating the PI3K/Akt pathway. In this study, we investigated the effect of P. copri transplantation on TBI and its relationship with GUO-PI3K/Akt pathway.MethodsIn this study, a controlled cortical impact (CCI) model was used to induce TBI in adult male C57BL/6J mice. Subsequently, P. copri was transplanted by intragastric gavage for 7 consecutive days. To investigate the effect of the GUO-PI3K/Akt pathway in P. copri transplantation therapy, guanosine (GUO) was administered 2 h after TBI for 7 consecutive days, and PI3K inhibitor (LY294002) was administered 30 min before TBI. Various techniques were used to assess the effects of these interventions, including quantitative PCR, neurological behavior tests, metabolite analysis, ELISA, Western blot analysis, immunofluorescence, Evans blue assays, transmission electron microscopy, FITC-dextran permeability assay, gastrointestinal transit assessment, and 16 S rDNA sequencing.ResultsP. copri abundance was significantly reduced after TBI. P. copri transplantation alleviated motor and cognitive deficits tested by the NSS, Morris’s water maze and open field test. P. copri transplantation attenuated oxidative stress and blood-brain barrier damage and reduced neuronal apoptosis after TBI. In addition, P. copri transplantation resulted in the reshaping of the intestinal flora, improved gastrointestinal motility and intestinal permeability. Metabolomics and ELISA analysis revealed a significant increase in GUO levels in feces, serum and injured brain after P. copri transplantation. Furthermore, the expression of p-PI3K and p-Akt was found to be increased after P. copri transplantation and GUO treatment. Notably, PI3K inhibitor LY294002 treatment attenuated the observed improvements.ConclusionsWe demonstrate for the first time that P. copri transplantation can improve GI functions and alter gut microbiota dysbiosis after TBI. Additionally, P. copri transplantation can ameliorate neurological deficits, possibly via the GUO-PI3K/Akt signaling pathway after TBI.

The causal relationship between genetically predicted blood metabolites and idiopathic pulmonary fibrosis: A bidirectional two-sample Mendelian randomization study.

Numerous metabolomic studies have confirmed the pivotal role of metabolic abnormalities in the development of idiopathic pulmonary fibrosis (IPF). Nevertheless, there is a lack of evidence on the causal relationship between circulating metabolites and the risk of IPF. The potential causality between 486 blood metabolites and IPF was determined through a bidirectional two-sample Mendelian randomization (TSMR) analysis. A genome-wide association study (GWAS) involving 7,824 participants was performed to analyze metabolite data, and a GWAS meta-analysis involving 6,257 IPF cases and 947,616 control European subjects was conducted to analyze IPF data. The TSMR analysis was performed primarily with the inverse variance weighted model, supplemented by weighted mode, MR-Egger regression, and weighted median estimators. A battery of sensitivity analyses was performed, including horizontal pleiotropy assessment, heterogeneity test, Steiger test, and leave-one-out analysis. Furthermore, replication analysis and meta-analysis were conducted with another GWAS dataset of IPF containing 4,125 IPF cases and 20,464 control subjects. Mediation analyses were used to identify the mediating role of confounders in the effect of metabolites on IPF. There were four metabolites associated with the elevated risk of IPF, namely glucose (odds ratio [OR] = 2.49, 95% confidence interval [95%CI] = 1.13-5.49, P = 0.024), urea (OR = 6.24, 95% CI = 1.77-22.02, P = 0.004), guanosine (OR = 1.57, 95%CI = 1.07-2.30, P = 0.021), and ADpSGEGDFXAEGGGVR (OR = 1.70, 95%CI = 1.00-2.88, P = 0.0496). Of note, the effect of guanosine on IPF was found to be mediated by gastroesophageal reflux disease. Reverse Mendelian randomization analysis displayed that IPF might slightly elevate guanosine levels in the blood. Conclusively, hyperglycemia may confer a promoting effect on IPF, highlighting that attention should be paid to the relationship between diabetes and IPF, not solely to the diagnosis of diabetes. Additionally, urea, guanosine, and ADpSGEGDFXAEGGGVR also facilitate the development of IPF. This study may provide a reference for analyzing the potential mechanism of IPF and carry implications for the prevention and treatment of IPF.

A multi-omics investigation of the lung injury induced by PM2.5 at environmental levels via the lung-gut axis

Long-term exposure to fine particulate matter (PM2.5) posed injury for gastrointestinal and respiratory systems, ascribing with the lung-gut axis. However, the cross-talk mechanisms remain unclear. Here, we attempted to establish the response networks of lung-gut axis in mice exposed to PM2.5 at environmental levels. Male Balb/c mice were exposed to PM2.5 (dose of 0.1, 0.5, and 1.0 mg/kg) collected from Chengdu, China for 10 weeks, through intratracheally instillation, and examined the effect of PM2.5 on lung functions of mice. The changes of lung and gut microbiota and metabolic profiles of mice in different groups were determined. Furthermore, the results of multi-omics were conjointly analyzed to elucidate the primary microbes and the associated metabolites in lung and gut responsible for PM2.5 exposure. Accordingly, the cross-talk network and key pathways between lung-gut axis were established. The results indicated that exposed to PM2.5 0.1 mg/kg induced obvious inflammations in mice lung, while emphysema was observed at 1.0 mg/kg. The levels of metabolites guanosine, hypoxanthine, and hepoxilin B3 increased in the lung might contribute to lung inflammations in exposure groups. For microbiotas in lung, PM2.5 exposure significantly declined the proportions of Halomonas and Lactobacillus. Meanwhile, the metabolites in gut including L-tryptophan, serotonin, and spermidine were up-regulated in exposure groups, which were linked to the decreasing of Oscillospira and Helicobacter in gut. Via lung-gut axis, the activations of pathways including Tryptophan metabolism, ABC transporters, Serotonergic synapse, and Linoleic acid metabolism contributed to the cross-talk between lung and gut tissues of mice mediated by PM2.5. In summary, the microbes including Lactobacillus, Oscillospira, and Parabacteroides, and metabolites including hepoxilin B3, guanosine, hypoxanthine, L-tryptophan, and spermidine were the main drivers. In this lung-gut axis study, we elucidated some pro- and pre-biotics in lung and gut microenvironments contributed to the adverse effects on lung functions induced by PM2.5 exposure.

Hypothermia increases adenosine monophosphate and xanthosine monophosphate levels in the mouse hippocampus, preventing their reduction by global cerebral ischemia

Global cerebral ischemia (GCI) caused by clinical conditions such as cardiac arrest leads to delayed neuronal death in the hippocampus, resulting in physical and mental disability. However, the mechanism of delayed neuronal death following GCI remains unclear. To elucidate the mechanism, we performed a metabolome analysis using a mouse model in which hypothermia (HT) during GCI, which was induced by the transient occlusion of the bilateral common carotid arteries, markedly suppressed the development of delayed neuronal death in the hippocampus after reperfusion. Fifteen metabolites whose levels were significantly changed by GCI and 12 metabolites whose levels were significantly changed by HT were identified. Furthermore, the metabolites common for both changes were narrowed down to two, adenosine monophosphate (AMP) and xanthosine monophosphate (XMP). The levels of both AMP and XMP were found to be decreased by GCI, but increased by HT, thereby preventing their decrease. In contrast, the levels of adenosine, inosine, hypoxanthine, xanthine, and guanosine, the downstream metabolites of AMP and XMP, were increased by GCI, but were not affected by HT. Our results may provide a clue to understanding the mechanism by which HT during GCI suppresses the development of delayed neuronal death in the hippocampus.

Purine Metabolism and Hexosamine Biosynthetic Pathway Abnormalities in Diarrheal Weaned Piglets Identified Using Metabolomics.

Post-weaning diarrhea significantly contributes to the high mortality in pig production, but the metabolic changes in weaned piglets with diarrhea remain unclear. This study aimed to identify the differential metabolites in the urine of diarrheal weaned piglets and those of healthy weaned piglets to reveal the metabolic changes associated with diarrhea in weaned piglets. Nine 25-day-old piglets with diarrhea scores above 16 and an average body weight of 5.41 ± 0.18 kg were selected for the diarrhea group. Corresponding to the body weight and sex of the diarrhea group, nine 25-month-old healthy piglets with similar sex and body weights of 5.49 ± 0.21 kg were selected as the control group. Results showed that the serum C-reactive protein and cortisol of piglets in the diarrhea group were higher than those in the control group (p < 0.05). The mRNA expression of TNF-α, IFN-γ in the jejunum and colon, and IL-1β in the jejunum were increased in diarrhea piglets (p < 0.05), accompanied by a reduction in the mRNA expression of ZO-1, ZO-2, and CLDN1 in the jejunum and colon (p < 0.05); mRNA expression of OCLN in the colon also occurred (p < 0.05). Metabolomic analysis of urine revealed increased levels of inosine, hypoxanthine, guanosine, deoxyinosin, glucosamine, glucosamine-1-p, N-Acetylmannosamine, chitobiose, and uric acid, identified as differential metabolites in diarrhea piglets compared to the controls. In summary, elevated weaning stress and inflammatory disease were associated with the abnormalities of purine metabolism and the hexosamine biosynthetic pathway of weaned piglets. This study additionally indicated the presence of energy metabolism-related diseases in diarrheal weaned piglets.

A novel strain of Levilactobacillus brevis PDD-5 isolated from salty vegetables has beneficial effects on hyperuricemia through anti-inflammation and improvement of kidney damage

Hyperuricemia is a metabolic disorder caused by abnormal purine metabolism, resulting in abnormally high serum uric acid. In this study, a novel Levilactobacillus brevis PDD-5 isolated from salty vegetables was verified with the function of alleviating hyperuricemia. The relevant effects of L. brevis PDD-5 in lowering uric acid were analyzed by in vitro and in vivo experiments. The results showed that the L. brevis PDD-5 has (68.86 ± 15.46)% of inosine uptake capacity and (95.75 ± 3.30)% of guanosine uptake capacity in vitro. Oral administration of L. brevis PDD-5 to hyperuricemia rats reduced uric acid, creatinine, and urea nitrogen in serum, as well as decreased inosine and guanosine levels in the intestinal contents of rats. Analysis of relevant markers in the kidney by ELISA kits revealed that L. brevis PDD-5 alleviated oxidative stress and inflammation. Moreover, the gene expression of uric acid transporter 1 (URAT1) and glucose transporter 9 (GLUT9) was down-regulated, and the gene expression of organic anion transporter 1 (OAT1) was up-regulated after treatment with L. brevis PDD-5. Western blot analysis showed that L. brevis PDD-5 alleviated hyperuricemia-induced kidney injury through the NLRP3 pathway. These findings suggest that L. brevis PDD-5 can lower uric acid, repair kidney damage, and also has the potential to prevent uric acid nephropathy.

High temperature stress induced oxidative stress, gut inflammation and disordered metabolome and microbiome in tsinling lenok trout

Tsinling lenok trout (Brachymystax lenok tsinlingensis Li) is a species of cold-water salmon that faces serious challenges due to global warming. High temperature stress has been found to damage the gut integrity of cold-water fish, impacting their growth and immunity. However, limited research exists on the causal relationship between gut microbial disturbance and metabolic dysfunction in cold-water fish induced by high temperature stress. To address this gap, we conducted a study to investigate the effects of high temperature stress (24 °C) on the gut tissue structure, antioxidant capacity, gut microorganisms, and metabolome reactions of tsinling lenok trout. Our analysis using 16 S rDNA gene sequencing revealed significant changes in the gut microbial composition and metabolic profile. Specifically, the abundance of Firmicutes and Gemmatimonadetes decreased significantly with increasing temperature, while the abundance of Bacteroidetes increased significantly. Metabolic analysis revealed a significant decrease in the abundance of glutathione, which is synthesized from glutamate and glycine, under high temperature stress. Additionally, there was a notable reduction in the levels of adenosine, inosine, xanthine, guanosine, and deoxyguanosine, which are essential for DNA/RNA synthesis. Conversely, there was a significant increase in the abundance of D-glucose 6 P. Furthermore, high temperature stress adversely affects intestinal structure and barrier function. Our findings provide valuable insights into the mechanism of high temperature stress in cold-water fish and serve as a foundation for future research aimed at mitigating the decline in production performance caused by such stress.

Dysregulated metabolites and lipids in serum of patients with acute hepatitis E: A longitudinal study.

Hepatitis E is a disease associated with acute inflammation of the liver. It is related to several dysregulated metabolic pathways and alterations in the concentration of several metabolites. However, longitudinal analysis of the alterations in metabolites and lipids is generally lacking. This study investigated the changes in levels of metabolites and lipids over time in sera from men with acute hepatitis E compared to healthy controls similar in age and gender. Untargeted measurement of levels of various metabolites and lipids was done using mass spectrometry on 65 sera sequentially sampled from 14 patients with acute hepatitis E and 25 serum samples from five controls. Temporal changes in intensities of metabolites and lipids were determined over different times at 3-day periods for the hepatitis E virus (HEV) group. In carbohydrate metabolism, glucose levels, fructose 1-6-bisphosphate and ribulose-5-phosphate were increased in the HEV-infected persons compared to the healthy controls. HEV infection is significantly associated with decreased levels of inosine, guanosine, adenosine and urate in purine metabolism and thymine, uracil and β-aminoisobutyrate in pyrimidine metabolism. Glutamate, alanine and valine levels were significantly lower in the HEV group than in healthy individuals. Homogentisate of tyrosine metabolism and cystathionine of serine metabolism were increased, whereas kynurenate of tryptophan metabolism decreased in the HEV group. Metabolites of the bile acid biosynthesis, urea cycle (arginine and citrulline) and ammonia recycling (urocanate) were significantly altered. Co-enzymes, pantothenate and pyridoxal, and co-factors, lipoamide and FAD, were elevated in the HEV group. The acylcarnitines, sphingomyelins, phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysoPC and lysoPE tended to be lower in the HEV group. In conclusion, acute hepatitis E is associated with altered metabolite and lipid profiles, significantly increased catabolism of carbohydrates, purines/pyrimidines and amino acids, and decreased levels of several glycerophospholipids.

Effects of different temperatures and diets on nutritional and bio‐functional components in the juvenile lined seahorse (Hippocampus erectus)

AbstractA two‐factorial experiment (temperature with three levels: 23°C, 25.5°C and 28°C; diet with four kinds: live Artemia sp., copepods, live Artemia sp. + copepods and frozen Neomysis awatschensis [FNA]) was carried out to evaluate the effects of different temperatures and diets on biochemistry in the juvenile lined seahorse (Hippocampus erectus) for thirty days. The juveniles (n = 1260, body height: 5.1 ± 0.48 cm, initial wet weight: 0.42 ± 0.14 g) were distributed in 36 experimental aquaria (35 individuals each). Each treatment had 3 replicates. On day 30, the seahorses were vacuum freeze‐dried at −56°C before chemical analysis. Amino acids, fatty acids, Se, Zn, nucleosides, and nucleotides were measured in the present study. The results show that temperature, diet, and their interaction significantly affected all components. The content of amino acids and fatty acids in the juveniles fed FNA at 23°C was the highest. The content of Zn in the juveniles fed FNA at 23°C, and the content of Se fed Artemia sp. + copepod at 25.5°C were the highest, respectively. The content of uracil, adenine, and thymidine in the juveniles at 23°C was significantly lower than those treatments at 25.5°C and 28°C. The levels of uracil, guanine, cytosine, uridine, and guanosine at 28°C were significantly higher than those at 23°C or 25.5°C when fed Artemia sp. + copepod. The juveniles fed Artemia sp. + copepod and Artemia sp. at 28°C has the highest comprehensive scores of nucleoside and nucleotide. In conclusion, FNA is the best feed to improve the content of amino acids, fatty acids, and Zn at 23°C.

Identification of potential serum biomarkers for congenital heart disease children with pulmonary arterial hypertension by metabonomics

BackgroundPulmonary arterial hypertension is a common complication in patients with congenital heart disease. In the absence of early diagnosis and treatment, pediatric patients with PAH has a poor survival rate. Here, we explore serum biomarkers for distinguishing children with pulmonary arterial hypertension associated with congenital heart disease (PAH-CHD) from CHD.MethodsSamples were analyzed by nuclear magnetic resonance spectroscopy-based metabolomics and 22 metabolites were further quantified by ultra-high-performance liquid chromatography–tandem mass spectroscopy.ResultsSerum levels of betaine, choline, S-Adenosyl methionine (SAM), acetylcholine, xanthosine, guanosine, inosine and guanine were significantly altered between CHD and PAH-CHD. Logistic regression analysis showed that combination of serum SAM, guanine and N-terminal pro-brain natriuretic peptide (NT-proBNP), yielded the predictive accuracy of 157 cases was 92.70% with area under the curve of the receiver operating characteristic curve value of 0.9455.ConclusionWe demonstrated that a panel of serum SAM, guanine and NT-proBNP is potential serum biomarkers for screening PAH-CHD from CHD.

8-Aminoguanine and Its Actions on Renal Excretory Function.

The endogenous purine 8-aminoguanine induces diuresis/natriuresis/glucosuria by inhibiting PNPase (purine nucleoside phosphorylase); however, mechanistic details are unknown. Here, we further explored in rats 8-aminoguanine's effects on renal excretory function by combining studies using intravenous 8-aminoguanine, intrarenal artery infusions of PNPase substrates (inosine and guanosine), renal microdialysis, mass spectrometry, selective adenosine receptor ligands, adenosine receptor knockout rats, laser doppler blood flow analysis, cultured renal microvascular smooth muscle cells, HEK293 cells expressing A2B receptors and homogeneous time resolved fluorescence assay for adenylyl cyclase activity. Intravenous 8-aminoguanine caused diuresis/natriuresis/glucosuria and increased renal microdialysate levels of inosine and guanosine. Intrarenal inosine, but not guanosine, exerted diuretic/natriuretic/glucosuric effects. In 8-aminoguanine-pretreated rats, intrarenal inosine did not induce additional diuresis/natriuresis/glucosuria. 8-Aminoguanine did not induce diuresis/natriuresis/glucosuria in A2B-receptor knockout rats, yet did so in A1- and A2A-receptor knockout rats. Inosine's effects on renal excretory function were abolished in A2B knockout rats. Intrarenal BAY 60-6583 (A2B agonist) induced diuresis/natriuresis/glucosuria and increased medullary blood flow. 8-Aminoguanine increased medullary blood flow, a response blocked by pharmacological inhibition of A2B, but not A2A, receptors. In HEK293 cells expressing A2B receptors, inosine activated adenylyl cyclase, and this was abolished by MRS 1754 (A2B antagonist). In renal microvascular smooth muscle cells, 8-aminoguanine and forodesine (PNPase inhibitor) increased inosine and 3',5'-cAMP; however, in cells from A2B knockout rats, 8-aminoguanine and forodesine did not augment 3',5'-cAMP yet increased inosine. 8-Aminoguanine induces diuresis/natriuresis/glucosuria by increasing renal interstitial levels of inosine which, via A2B receptor activation, increases renal excretory function, perhaps in part by increasing medullary blood flow.

Antifungal activity and metabolomics analysis of Piper sarmentosum extracts against Fusarium graminearum.

Fusarium graminearum is a toxic fungus that affects food and feed crops. Piper sarmentosum extract (PSE) is a potential source of anti-mildew natural products for the food and feed industry due to its various pharmacological properties. In this study, we evaluated the antifungal activity and untargeted metabolomics analysis of PSE against F. graminearum. Antifungal activity was evaluated using the mycelium growth rate method. Untargeted metabolomics analysis of PSE was performed using ultra high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The results showed that PSE (1 and 2 mg ml-1) possesses inhibitory activity against F. graminearum, and a total of 17 compounds that including 8 alkaloids, 3 phenols, 3 lipids, and 3 organic acids might be the antifungal markers in PSE. Metabolomics analysis further revealed that PSE could significantly increase the levels of guanosine, guanine, adenosine, and L-isoleucine in fungi, which are related to purine and L-isoleucine metabolic pathways. PSE is a promising anti-mildew agent that inhibits the growth of F. graminearum in food and feed. PSE (1 and 2 mg ml-1) may exert antifungal properties by inhibiting fungal purine nucleotide synthesis and enhancing the level of L-isoleucine compared with the control groups.

Hepatoprotective effects of parsley (Petroselinum Crispum) extract in rats with bile duct ligation

Background and study aimsThis study aimed to investigate the possible protective effects of parsley extract (Petroselinum Crispum; PC) against oxidative liver damage caused by bile obstruction in rats. Material and methodsBile duct ligation (BDL) method was used to induce liver injury in rats. The rats were divided into the three groups each consisting of 8 rats; Sham-operated control (C), bile duct ligated + saline treated (BDL), and BDL + PC treated groups. PC extract was given at a dose of 2 g/kg orally for 28 days. Aspartate amino transferase (AST), alanin amino transferase (ALT), and bilirubin levels were analyzed in sera. In order to determine free radicals in liver injury, luminol and lucigenin chemiluminescence tests used. Oxidative stress was evaluated through superoxide dismutase, glutathione, malondialdehyde, Na+/K+-ATPase and 8-hydroxy guanosine levels. Furthermore, inflammation marker myeloperoxidase, apoptosis marker caspase-3, and fibrosis markers TGF- β and hydoxyproline were investigated. The liver tissues were also examined for histological evaluations. ResultsWhile PC treatment decreased AST and ALT levels which increased with BDL, oxidant damage parameters also decreased with this treatment. ConclusionThe present study, which is the first research for PC extract on cholestasis induced liver damage, demonstrated that PC extract could be a potential therapeutic agent against liver fibrosis and need further studies.

Suppressed renoprotective purines in COVID-19 patients with acute kidney injury

Acute kidney injury (AKI) is common in patients hospitalized for COVID-19, complicating their clinical course and contributing to worse outcomes. Animal studies show that adenosine, inosine and guanosine protect the kidney against some types of AKI. However, until now there was no evidence in patients supporting the possibility that abnormally low kidney levels of adenosine, inosine and guanosine contribute to AKI. Here, we addressed the question as to whether these renoprotective purines are altered in the urine of COVID-19 patients with AKI. Purines were measured by employing ultra-high-performance liquid chromatography-tandem mass spectrometry with stable-isotope-labeled internal standards for each purine of interest. Compared with COVID-19 patients without AKI (n = 23), COVID-19 patients with AKI (n = 20) had significantly lower urine levels of adenosine (P < 0.0001), inosine (P = 0.0008), and guanosine (P = 0.0008) (medians reduced by 85%, 48% and 61%, respectively) and lower levels (P = 0.0003; median reduced by 67%) of the 2nd messenger for A2A and A2B adenosine receptors, i.e., 3’,5’-cAMP. Moreover, in COVID-19 patients with AKI, urine levels of 8-aminoguanine (endogenous inhibitor of inosine and guanosine metabolism) were nearly abolished (P < 0.0001). In contrast, the “upstream” precursors of renoprotective purines, namely 5’-AMP and 5’-GMP, were not significantly altered in COVID-19 patients with AKI, suggesting defective conversion of these precursors by CD73 (converts 5’-AMP to adenosine and 5’-GMP to guanosine). These findings imply that an imbalance in renoprotective purines may contribute to AKI in COVID-19 patients and that pharmacotherapy targeted to restore levels of renoprotective purines may attenuate the risk of AKI in susceptible patients with COVID-19.

Regulator Of G Protein Signaling 14 Disruption Affects The Gut Microbiota And Metabolome In Mice

The gut microbiota is linked to brown adipose tissue (BAT), but the mechanisms and microbes facilitating BAT production or function are unknown. A novel mouse model containing a gene knock-out of regulator of G protein signaling 14 (RGS14 KO) has increased BAT. Our early studies found distinct microbes in RGS14 KO mice and BAT-specific metabolites. PURPOSE: To identify key gut microbial species and uncover BAT-specific metabolites in RGS14 KO mice. Further, we aim to identify and associate metabolites produced in specific tissue samples with these key gut microbes. METHODS: Twenty-two mice (N = 13 RGS14 KO, N = 9 Wild type (WT)) were used to identify predominant microbes and metabolites. Gut microbiota profiles were obtained by sequencing bacterial ribosomal operons. Metabolomics used ultra-high performance liquid chromatography to evaluate polar and nonpolar untargeted metabolites in fecal, cecal, brain, and BAT samples. Microbiome analysis used Kulczynski distance to compare WT to RGS14 KO reads in two-dimensional non-metric multidimensional scaling (NMDS) plots. Two-tailed t-tests were used to compare WT and RGS14 KO metabolite means (p < 0.05). MetaboAnalyst 5.0 was used to identify significant metabolite pathways in tissue samples and generate pathway plots. RESULTS: Approximately 500 k rRNA reads post QA/QC ((84% identify; >1000 bp alignment) were obtained from all samples by MegaBlast. NMDS plots showed significant bacterial community differences (Genus: p = 0.035; species: p = 0.028; strain: p = 0.037) between WT and RGS14 KO mice. Specifically, RGS14 KO mice housed two unique strains of Akkermansia muciniphilia (A. muciniphilia BIOML-A22 and A. muciniphilia AN78) while WT animals contained A. muciniphilia EB-AMDK-1. Untargeted metabolomics identified 82 significantly different (p < 0.05) unique metabolites were between RGS14 KO and WT mice in one of the four samples. Specifically, RGS14 KO animals had significantly higher levels of G6P, glycyl-l-proline, glyercophosphocholine, isoleucine, pipecolic acid, thymidine, UDP-D-glucose, malate, leucic acid, NADP+, and guanosine in BAT compared to WT animals. CONCLUSION: The gut microbiome differs between RGS14 KO and WT mice from genus to strain level. Unique metabolites found in RGS14 KO BAT may be linked to BAT function. Supported by ONR Grant 826640

P1312: FORODESINE AMPLIFIES HOST INNATE IMMUNE RESPONSE THROUGH TOLL-LIKE RECEPTOR 7 ACTIVATION WHILE PREVENTING EXPERIMENTAL GRAFT-VERSUS-HOST DISEASE

Background: Forodesine, a novel inhibitor of purine nucleoside phosphorylase (PNP), causes nucleotide pool imbalances by accumulating intracellular deoxyguanosine, thereby resulting in selective apoptosis of T-cells. Recent evidence indicates that specific types of nuclear acids including guanosine and its derivatives act as natural ligands for toll-like receptor 7 (TLR7). So far, it is unclear whether and how forodesine can modulate host innate immunity, including antigen-presenting cells (APCs) such as macrophages and dendritic cells (DCs), which express TLR7 abundantly. Aims: The aim of this study is to investigate the role of forodesine in host APC function as well as donor T-cell activation both in vivo graft-versus-host disease (GVHD) models and in vitro cell assays. Methods: NOG mice were subjected to 250 cGy total body irradiation followed by intravenous injection of human isolated T-cells. Mice were treated with forodesine (20 mg/kg) or vehicle daily after transplantation. Results: Despite the mice without treatment developing severe GVHD after transplantation, guanosine and deoxyguanosine were hardly detected in the plasma, suggesting that purine nucleosides released from damaged tissues were rapidly degraded by PNP. In fact, inhibition of PNP activity by forodesine markedly increased plasma guanosine levels, whereas deoxyguanosine was still undetectable. This was also the case in GVHD target organs, including liver and lungs. At the same level of guanosine in the liver, forodesine promoted TNF-a production from in vitro alveolar macrophage cultures. Preincubation with a TLR7 antagonist, ODN20958, attenuated the TNF-a production from macrophages stimulated by guanosine and forodesine. Nuclease treatment did not reduce macrophage TNF-a production, suggesting that single-strand RNA, a well-known TLR7 ligand, is not involved in this response. Similar results were observed in Flt3-ligand-induced plasmacytoid DCs. Not only TNF-a but also IL-12 production was induced by the combination of guanosine and forodesine, and was comparable to that in pDCs pulsed with R848, a potent agonist of TLR7/8. These observations suggest that a sustained high level of guanosine caused by forodesine treatment lead to continuous activation of TLR7 signaling, resulting in cytokine production from APCs. Meanwhile, as reported previously, forodesine inhibited the proliferation of human T-cells activated with CD3 and CD28 antibodies in the presence of deoxyguanosine. Of note, however, this combination did not impair but rather stimulated IFN-g production from activated T-cells cocultured with pDCs. This suggests that T-cell priming capacity mediated by the interaction with pDCs is conserved through TLR7 activation. Finally, we investigated the in vivo effects of forodesine on survival, body weight change, and disease severity of GVHD mice. Forodesine treatment significantly prolonged survival of the mice compared with control mice, with a median survival of 17.5 days vs 14 days, respectively (p = 0.029). The mean body weight loss percentage at day 9 was 11.5% vs 19.0% in the forodesine group and control, respectively (p = 0.003). Furthermore, the clinical GVHD scores were also lower in the forodesine group vs control (4.9 vs 0.6 points, p = 0.001). Image:Summary/Conclusion: This study demonstrates that forodesine amplifies host innate immune response through TLR7 signaling, while ameliorating T-cell GVH reaction. Further studies are needed to investigate whether forodesine can provide host protection against infections in a transplant setting.

A fly GWAS for purine metabolites identifies human FAM214 homolog medusa, which acts in a conserved manner to enhance hyperuricemia-driven pathologies by modulating purine metabolism and the inflammatory response.

Elevated serum urate (hyperuricemia) promotes crystalline monosodium urate tissue deposits and gout, with associated inflammation and increased mortality. To identify modifiers of uric acid pathologies, we performed a fly Genome-Wide Association Study (GWAS) on purine metabolites using the Drosophila Genetic Reference Panel strains. We tested the candidate genes using the Drosophila melanogaster model of hyperuricemia and uric acid crystallization ("concretion formation") in the kidney-like Malpighian tubule. Medusa (mda) activity increased urate levels and inflammatory response programming. Conversely, whole-body mda knockdown decreased purine synthesis precursor phosphoribosyl pyrophosphate, uric acid, and guanosine levels; limited formation of aggregated uric acid concretions; and was sufficient to rescue lifespan reduction in the fly hyperuricemia and gout model. Levels of mda homolog FAM214A were elevated in inflammatory M1- and reduced in anti-inflammatory M2-differentiated mouse bone marrow macrophages, and influenced intracellular uric acid levels in human HepG2 transformed hepatocytes. In conclusion, mda/FAM214A acts in a conserved manner to regulate purine metabolism, promotes disease driven by hyperuricemia and associated tissue inflammation, and provides a potential novel target for uric acid-driven pathologies.

Metabolomic Profiling of Cardiac Allografts After Controlled Circulatory Death

<h3>Purpose</h3> Assessment of myocardial viability during ex situ heart perfusion (ESHP) is only based on lactate trends. Metabolomic profiling of hearts donated after circulatory death (DCD) was performed to investigate new biomarkers of myocardial viability during ESHP. <h3>Methods</h3> Eighteen piglets were randomly allocated in three groups (N=6/per group): donor hearts procured either after 15 minutes of warm ischemia (DCD group); after brain-stem death (BSD group); or without DCD nor BSD (Control group). Blood normothermic ESHP was then initiated for 240 minutes. Plasma and myocardial samples were procured respectively every 30 and 60 minutes, and analyzed by liquid chromatography coupled to high-resolution mass spectrometry. <h3>Results</h3> Median duration of agonal phase was 24 minutes [22-29] in DCD animals, including 15 minutes of functional warm ischemia. Mean coronary flow and mean aortic pressure during ESHP ranged from 720 to 740 mL/min, and from 68 to 73 mmHg respectively, without differences between groups (P=0.544 and P=0.203). At T0, arterial lactate was higher in DCD (7.0±0.9) than in BSD (1.1±0.2) and Control groups (2.0±0.8 mmol/L, P<0.01), and progressively decreased until T240. Lactate level within myocardial biopsies was not significantly different between groups at T0 (P=0.281), and remained stable over the 4-hour period of ESHP. Three different metabolomic profiles corresponding to DCD, BSD and Control groups were remarkable from T30 to T240, including 300 different metabolites in plasma and tissue samples. Marked nucleotide degradation was observed in DCD group compared to BSD and Control groups (P<0.01). Plasma levels of inosine and guanosine were significantly higher in DCD group and progressively decreased over the time to reach control level after 210 minutes of ESHP (Figure 1). <h3>Conclusion</h3> A specific metabolomic profile was observed in donor hearts exposed to warm ischemia. Considering plasma levels of inosine and guanosine, nucleotides could be relevant biomarkers for viability assessment of DCD hearts.

Cerebrospinal fluid purinomics as a biomarker approach to predict outcome after severe traumatic brain injury.

Severe traumatic brain injury (TBI) is associated with high rates of mortality and long-term disability linked to neurochemical abnormalities. Although purine derivatives play important roles in TBI pathogenesis in preclinical models, little is known about potential changes in purine levels and their implications in human TBI. We assessed cerebrospinal fluid (CSF) levels of purines in severe TBI patients as potential biomarkers that predict mortality and long-term dysfunction. This was a cross-sectional study performed in 17 severe TBI patients (Glasgow Coma Scale <8) and 51 controls. Two to 4h after admission to ICU, patients were submitted to ventricular drainage and CSF collection for quantification of adenine and guanine purine derivatives by HPLC. TBI patients' survival was followed up to 3days from admission. A neurofunctional assessment was performed through the modified Rankin Scale (mRS) 2 years after ICU admission. Purine levels were compared between control and TBI patients, and between surviving and non-surviving patients. Relative to controls, TBI patients presented increased CSF levels of GDP, guanosine, adenosine, inosine, hypoxanthine, and xanthine. Further, GTP, GDP, IMP, and xanthine levels were different between surviving and non-surviving patients. Among the purines, guanosine was associated with improved mRS (p=0.042; r=-0.506). Remarkably, GTP displayed predictive value (AUC=0.841, p=0.024) for discriminating survival versus non-survival patients up to 3days from admission. These results support TBI-specific purine signatures, suggesting GTP as a promising biomarker of mortality and guanosine as an indicator of long-term functional disability.