Techniques to Quantify cGMP Dysregulation as a Common Pathway Associated with Photoreceptor Cell Death in Retinitis Pigmentosa.

The targeted development of neuroprotective therapies for retinitis pigmentosa (RP) depends upon a better understanding of the mechanisms of photoreceptor cell death. Nucleotide metabolite-associated photoreceptor cell death is an emerging area of research that is important in multiple models of RP, yet the exact pathophysiology remains to be elucidated. One common pathway of photoreceptor cell death in RP is cGMP dysregulation, which is underscored by its potential to be relevant in up to 30% of patients with RP. Optimizing tools for detecting and quantifying nucleotide metabolites in the retina is vital to expanding this area of research. Immunohistochemistry is useful for localizing abnormally high levels of cGMP in a cell-specific manner, while enzyme-linked immunosorbent assay and liquid chromatography-mass spectrometry are quantitative and more sensitive. These techniques can form the basis for more sophisticated experiments to elucidate upstream events in photoreceptor cell death, which will hopefully lead to the development of novel therapies for patients with RP.

Metformin promotes anti-tumor immunity in STK11 mutant NSCLC through AXIN1-dependent upregulation of multiple nucleotide metabolites.

Background: Metformin has pleiotropic effects beyond glucose reduction, including tumor inhibition and immune regulation. It enhanced the anti-tumor effects of programmed cell death protein 1 (PD-1) inhibitors in serine/threonine kinase 11 (STK11) mutant non-small cell lung cancer (NSCLC) through an axis inhibition protein 1 (AXIN1)-dependent manner. However, the alterations of tumor metabolism and metabolites upon metformin administration remain unclear. Methods: We performed untargeted metabolomics using liquid chromatography (LC)-mass spectrometry (MS)/MS system and conducted cell experiments to verify the results of bioinformatics analysis. Results: According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, most metabolites were annotated into metabolism, including nucleotide metabolism. Next, the differentially expressed metabolites in H460 (refers to H460 cells), H460_met (refers to metformin-treated H460 cells), and H460_KO_met (refers to metformin-treated Axin1 -/- H460 cells) were distributed into six clusters based on expression patterns. The clusters with a reversed expression pattern upon metformin treatment were selected for further analysis. We screened out metabolic pathways through KEGG pathway enrichment analysis and found that multiple nucleotide metabolites enriched in this pathway were upregulated. Furthermore, these metabolites enhanced the cytotoxicity of activated T cells on H460 cells in vitro and can activate the stimulator of the interferon genes (STING) pathway independently of AXIN1. Conclusion: Relying on AXIN1, metformin upregulated multiple nucleotide metabolites which promoted STING signaling and the killing of activated T cells in STK11 mutant NSCLC, indicating a potential immunotherapeutic strategy for STK11 mutant NSCLC.

TIR-catalyzed nucleotide signaling molecules in plant defense.

Toll and interleukin-1 receptor (TIR) domain is a conserved immune module in prokaryotes and eukaryotes. Signaling regulated by TIR-only proteins or TIR domain-containing intracellular immune receptors is critical for plant immunity. Recent studies demonstrated that TIR domains function as enzymes encoding a variety of activities, which manifest different mechanisms for regulation of plant immunity. These enzymatic activities catalyze metabolism of NAD+, ATP and other nucleic acids, generating structurally diversified nucleotide metabolites. Signaling roles have been revealed for some TIR enzymatic products that can act as second messengers to induce plant immunity. Herein, we summarize our current knowledge about catalytic production of these nucleotide metabolites and their roles in plant immune signaling. We also highlight outstanding questions that are likely to be the focus of future investigations about TIR-produced signaling molecules.

Adenine Nucleotide Metabolites in Uremic Erythrocytes as Metabolic Markers of Chronic Kidney Disease in Children.

Chronic kidney disease (CKD) is associated with multifaceted pathophysiological lesions including metabolic pathways in red blood cells (RBC). The aim of the study was to determine the concentration of adenine nucleotide metabolites, i.e., nicotinamide adenine dinucleotide (NAD)-oxidized form, nicotinamide adenine dinucleotide hydrate (NADH)-reduced form, nicotinic acid mononucleotide (NAMN), ß-nicotinamide mononucleotide (NMN), nicotinic acid adenine dinucleotide (NAAD), nicotinic acid (NA) and nicotinamide (NAM) in RBC and to determine a relationship between NAD metabolites and CKD progression. Forty-eight CKD children and 33 age-matched controls were examined. Patients were divided into groups depending on the CKD stages (Group II-stage II, Group III- stage III, Group IV- stage IV and Group RRT children on dialysis). To determine the above-mentioned metabolites concentrations in RBC liquid chromatography-mass spectrometry was used. Results: the only difference between the groups was shown concerning NAD in RBC, although the values did not differ significantly from controls. The lowest NAD values were found in Group II (188.6 ± 124.49 nmol/mL, the highest in group IV (324.94 ± 63.06 nmol/mL. Between Groups II and IV, as well as III and IV, the differences were statistically significant (p < 0.032, p < 0.046 respectively). Conclusions. CKD children do not have evident abnormalities of RBC metabolism with respect to adenine nucleotide metabolites. The significant differences in erythrocyte NAD concentrations between CKD stages may suggest the activation of adaptive defense mechanisms aimed at erythrocyte metabolic stabilization. It seems that the implementation of RRT has a positive impact on RBC NAD metabolism, but further research performed on a larger population is needed to confirm it.

Direct and indirect quantification of phosphate metabolites of nucleoside analogs in biological samples.

Nucleoside reverse transcriptase inhibitors (NRTIs) are prodrugs that require intracellular phosphorylation to active triphosphate nucleotide metabolites (NMs) for their pharmacological activity. However, monitoring these pharmacologically active NMs is challenging due to their instability, high hydrophilicity, and their low concentrations in blood and tissues. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the gold standard technique for the quantification of NRTIs and their phosphorylated NMs. In this review, an overview of the publications describing the quantitative analysis of intracellular and total tissue concentration of NMs is presented. The focus of this review is the comparison of the different approaches and challenges associated with sample collection, tissue homogenization, cell lysis, cell counting, analyte extraction, sample storage conditions, and LC-MS analysis. Quantification methods of NMs via LC-MS can be categorized into direct and indirect methods. In the direct LC-MS methods, chromatographic retention of the NMs is accomplished by ion-exchange (IEX), ion-pairing (IP), hydrophilic interaction (HILIC), porous graphitic carbon (PGC) chromatography, or capillary electrophoresis (CE). In indirect methods, parent nucleosides are 1st generated from the dephosphorylation of NMs during sample preparation and are then quantified by reverse phase LC-MS as surrogates for their corresponding NMs. Both approaches have advantages and disadvantages associated with them, which are discussed in this review.

Postmortem nucleotide degradation in turbot mince during chill and partial freezing storage.

Nucleotide degradation in fish is an important biochemical change after death, which is closely related to freshness and sensory quality. However, except ATP-relative nucleotides, it remains unclear about changes in other nucleotide metabolites during postmortem stage. In this study, a strategy for the simultaneous quantification of 28 nucleobases, nucleosides, and nucleotides using hydrophilic interaction chromatography coupled with tandem mass spectrometry (HILIC-MS/MS) with positive/negative ion switching was developed. This method showed good linearity, precision, repeatability, and recovery. Furthermore, it was successfully applied to monitor the postmortem nucleotide degradation of turbot mince during chill (4 °C) and partial freezing (-3 °C) storage for 168 h. It was noted that the patterns of the changes in nucleotide metabolites differed considerably depending on the storage temperature. Meanwhile, the different pathway and speed of nucleotide catabolism in turbot mince was summarized based on the quantification data.