Pyrimidine Ribonucleotides Research Articles

Baseline Gut Microbiota Was Associated with Long-Term Immune Response at One Year Following Three Doses of BNT162b2.

This study explored neutralizing IgG antibody levels against COVID-19 decline over time post-vaccination. We conducted this prospective cohort study to investigate the function of gut microbiota in the host immune response following three doses of BNT162b2. Subjects who received three doses of BNT162b2 were recruited from three centers in Hong Kong. Blood samples were obtained before the first dose and at the one-year timepoint for IgG ELISA to determine the level of neutralizing antibody (NAb). The primary outcome was a high immune response (NAb > 600 AU/mL). We performed shotgun DNA metagenomic sequencing on baseline fecal samples to identify bacterial species and metabolic pathways associated with high immune response using linear discriminant analysis effect size analysis. A total of 125 subjects were recruited (median age: 52 years [IQR: 46.2-59.0]; male: 43 [34.4%]), and 20 were regarded as low responders at the one-year timepoint. Streptococcus parasanguinis (log10LDA score = 2.38, p = 0.003; relative abundance of 2.97 × 10-5 vs. 0.03%, p = 0.001), Bacteroides stercoris (log10LDA score = 4.29, p = 0.024; relative abundance of 0.14% vs. 2.40%, p = 0.014) and Haemophilus parainfluenzae (log10LDA score = 2.15, p = 0.022; relative abundance of 0.01% vs. 0, p = 0.010) were enriched in low responders. Bifidobacterium pseudocatenulatum (log10LDA score = 2.99, p = 0.048; relative abundance of 0.09% vs. 0.36%, p = 0.049) and Clostridium leptum (log10LDA score = 2.38, p = 0.014; relative abundance of 1.2 × 10-5% vs. 0, p = 0.044) were enriched in high responders. S. parasanguinis was negatively correlated with the superpathway of pyrimidine ribonucleotides de novo biosynthesis (log10LDA score = 2.63), which contributes to inflammation and antibody production. H. parainfluenzae was positively correlated with pathways related to anti-inflammatory processes, including the superpathway of histidine, purine, and pyrimidine biosynthesis (log10LDA score = 2.14). Among three-dose BNT162b2 recipients, S. parasanguinis, B. stercoris and H. parainfluenzae were associated with poorer immunogenicity at one year, while B. pseudocatenulatum and C. leptum was associated with a better response.

Ornithine aspartate effects on bacterial composition and metabolic pathways in a rat model of steatotic liver disease.

Metabolic-dysfunction associated steatotic liver disease (MASLD) is a hepatic manifestation of metabolic syndrome. Studies suggest ornithine aspartate (LOLA) as drug therapy. To analyze the influence of LOLA intake on gut microbiota using a nutritional model of MASLD. Adult male Sprague Dawley rats were randomized into three groups: Control (10 rats fed with a standard diet), MASLD (10 rats fed with a high-fat and choline-deficient diet), and LOLA (10 rats receiving 200 mg/kg/d LOLA, after the 16th week receiving high-fat and choline-deficient diet). After 28 wk of the experiment, animals were euthanized, and feces present in the intestine were collected. Following fecal DNA extraction, the V4 region of the 16S rRNA gene was amplified followed by sequencing in an Ion S5™ system. Alpha and beta diversity metrics were comparable between MASLD and LOLA. 3 OTUs were differentially abundant between MASLD and LOLA, which belong to the species Helicobacter rodentium, Parabacteroides goldsteinii, and Parabacteroides distasonis. The functional prediction provided two different metabolic profiles between MASLD and LOLA. The 9 pathways differentially abundant in MASLD are related to a change in energy source, adenosine/purine nucleotides degradation as well as guanosine and adenosine deoxyribonucleotides biosynthesis. The 14 pathways differentially abundant in LOLA are associated with four major metabolic functions primarily influenced by L-aspartate, including tricarboxylic acid cycle pathways, purine/guanosine nucleotides biosynthesis, pyrimidine ribonucleotides biosynthesis and salvage as well as lipid IVA biosynthesis. Although LOLA had no influence on alpha and beta diversity in this nutritional model of MASLD, it was associated with changes in specific gut microbes and their related metabolic pathways.

Selection and Characterization of DNA Aptamers for Cytidine and Uridine.

Cytidine and uridine are two essential pyrimidine ribonucleotides, and accurate detection of these nucleosides holds significant biological importance. While many aptamers were reported to bind purines, little success was achieved for pyrimidine binding. This study employs the library-immobilization capture-SELEX technique to isolate aptamers capable of selectively binding to cytidine and uridine. First, a selection was performed using a mixture of cytidine and uridine as the target. This selection led to the isolation of a highly selective aptamer for cytidine with a dissociation constant (Kd ) of 0.9 μM as determined by isothermal titration calorimetry (ITC). In addition, a dual-recognition aptamer was also discovered, which exhibited selective binding to both cytidine and uridine. Subsequently, a separate selection was carried out using uridine as the sole target, and the resulting uridine aptamer displayed a Kd of 4 μM based on a thioflavin T fluorescence assay and a Kd of 102 μM based on ITC. These aptamers do not have a strict requirement of metal ions for binding, and they showed excellent selectivity since no binding was observed with their nucleobases or nucleotides. This study has resulted three aptamers for pyrimidines, which can be employed in biosensors and DNA switches.

Ribonucleotides differentially modulate oral glutamate detection thresholds.

The savory or umami taste of the amino acid glutamate is synergistically enhanced by the addition of the purines inosine 5'-monophosphate (IMP) and guanosine 5'-monophosphate (GMP) disodium salt. We hypothesized that the addition of purinergic ribonucleotides, along with the pyrimidine ribonucleotides, would decrease the absolute detection threshold of (increase sensitivity to) l-glutamic acid potassium salt (MPG). To test this, we measured both the absolute detection threshold of MPG alone and with a background level (3mM) of 5 different 5'-ribonucleotides. The addition of the 3 purines IMP, GMP, and adenosine 5'-monophosphate (AMP) lowered the MPG threshold in all participants (P < 0.001), indicating they are positive modulators or enhancers of glutamate taste. The average detection threshold of MPG was 2.08mM, and with the addition of IMP, the threshold was decreased by approximately 1.5 orders of magnitude to 0.046mM. In contrast to the purines, the pyrimidines uridine 5'-monophosphate (UMP) and cytidine 5'-monophosphate (CMP) yielded different results. CMP reliably raised glutamate thresholds in 10 of 17 subjects, suggesting it is a negative modulator or diminisher of glutamate taste for them. The rank order of effects on increasing sensitivity to glutamate was IMP > GMP> AMP >> UMP// CMP. These data confirm that ribonucleotides are modulators of glutamate taste, with purines enhancing sensitivity and pyrimidines displaying variable and even negative modulatory effects. Our ability to detect the co-occurrence of glutamate and purines is meaningful as both are relatively high in evolutionarily important sources of nutrition, such as insects and fermented foods.

Retinoblastoma vulnerability to combined de novo and salvage pyrimidine ribonucleotide synthesis pharmacologic blockage

Retinoblastoma is an eye cancer that commonly affects young children. Despite significant advances, current treatments cause side effects even when administered locally, and patients may still have to undergo enucleation. This is particularly disheartening in cases of bilateral retinoblastoma. Hence, there is an urgent need for novel therapeutic strategies. Inhibitors of the enzyme dihydroorotate dehydrogenase (DHODH), which is involved in the de novo pyrimidine ribonucleotide synthesis pathway, have proven to be effective in preclinical trials against several cancers including pediatric cancers. Here we tested whether blocking pyrimidine ribonucleotide synthesis promotes retinoblastoma cell death. Cultured retinoblastoma cell lines were treated with small molecule inhibitors of DHODH alone or in combination with inhibitors of nucleoside uptake to also block the salvage pathway for pyrimidine ribonucleotide formation. On their own, DHODH inhibitors had a moderate killing effect. However, the combination with nucleoside uptake inhibitors greatly enhanced the effect of DHODH inhibition. In addition, we observed that pyrimidine ribonucleotide synthesis blockage can cause cell death in a p53 mutant retinoblastoma cell line derived from a patient with metastasis. Explaining these results, the analysis of a published patient cohort revealed that loss of chr16q22.2 (containing the DHODH gene) is amongst the most frequent alterations in retinoblastoma and that these tumors often show gains in chromosome regions expressing pyrimidine ribonucleotide salvage factors. Furthermore, these genome alterations associate with malignancy. These results indicate that targeting pyrimidine ribonucleotide synthesis may be an effective therapeutic strategy to consider as a treatment for retinoblastoma.

MRNA incorporation of C(5)-halogenated pyrimidine ribonucleotides and induced high expression of corresponding protein for the development of mRNA vaccine

In this report, we present our studies on mRNA, which was modified by introducing various halogen substituents at the C(5) position of the pyrimidine base. Specifically, we synthesized C(5)-halogenated (F, Cl, Br, I) pyrimidine ribonucleoside triphosphates and incorporated them into mRNA during in-vitro transcription. The efficiency of the in-vitro transcription reaction of halogenated pyrimidine was observed to decrease as the size of the halogen substituent increased and the electronegativity thereof decreased (F > Cl > Br) except for iodine. Interestingly, we found that, among the C(5)-halogenated pyrimidine ribonucleotides, mRNA incorporating C(5)-halogenated cytidine (5-F rCTP and 5-Cl rCTP) exhibited more prominent protein expression than mRNA modified with C(5)-halogenated uridine and unmodified mRNA. In particular, in the case of mRNA to which fluorine (5-F rCTP) and chlorine (5-Cl rCTP) were introduced, the protein was dramatically expressed about 4 to 5 times more efficiently than the unmodified mRNA, which was similar to pseudouridine (ψ). More interestingly, when pseudouridine(ψ) and fluorocytidine nucleotides (5-F rCTP), were simultaneously introduced into mRNA for dual incorporation, the protein expression efficiency dramatically increased as much as tenfold. The efficiency of cap-dependent protein expression is much higher than the IRES-dependent (internal ribosome entry site) expression with mRNA incorporating C(5)-halogenated pyrimidine ribonucleotide. We expect these results to contribute meaningfully to the development of therapeutics based on modified mRNA.

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.

Long non-coding RNA AC099850.4 correlates with advanced disease state and predicts worse prognosis in triple-negative breast cancer.

Our understanding of the function of long non-coding RNAs (lncRNAs) in health and disease states has evolved over the past decades due to the many advances in genome research. In the current study, we characterized the lncRNA transcriptome enriched in triple-negative breast cancer (TNBC, n = 42) and estrogen receptor (ER+, n = 42) breast cancer compared to normal breast tissue (n = 56). Given the aggressive nature of TNBC, our data revealed selective enrichment of 57 lncRNAs in TNBC. Among those, AC099850.4 lncRNA was chosen for further investigation where it exhibited elevated expression, which was further confirmed in a second TNBC cohort (n = 360) where its expression correlated with a worse prognosis. Network analysis of AC099850.4high TNBC highlighted enrichment in functional categories indicative of cell cycle activation and mitosis. Ingenuity pathway analysis on the differentially expressed genes in AC099850.4high TNBC revealed the activation of the canonical kinetochore metaphase signaling pathway, pyridoxal 5'-phosphate salvage pathway, and salvage pathways of pyrimidine ribonucleotides. Additionally, upstream regulator analysis predicted the activation of several upstream regulator networks including CKAP2L, FOXM1, RABL6, PCLAF, and MITF, while upstream regulator networks of TP53, NUPR1, TRPS1, and CDKN1A were suppressed. Interestingly, elevated expression of AC099850.4 correlated with worse short-term relapse-free survival (log-rank p = 0.01). Taken together, our data are the first to reveal AC099850.4 as an unfavorable prognostic marker in TNBC, associated with more aggressive clinicopathological features, and suggest its potential utilization as a prognostic biomarker and therapeutic target in TNBC.

Ribonucleotide synthesis by NME6 fuels mitochondrial gene expression

Replication of the mitochondrial genome and expression of the genes it encodes both depend on a sufficient supply of nucleotides to mitochondria. Accordingly, dysregulated nucleotide metabolism not only destabilises the mitochondrial genome, but also affects its transcription. Here, we report that a mitochondrial nucleoside diphosphate kinase, NME6, supplies mitochondria with pyrimidine ribonucleotides that are necessary for the transcription of mitochondrial genes. Loss of NME6 function leads to the depletion of mitochondrial transcripts, as well as destabilisation of the electron transport chain and impaired oxidative phosphorylation. These deficiencies are rescued by an exogenous supply of pyrimidine ribonucleosides. Moreover, NME6 is required for the maintenance of mitochondrial DNA when the access to cytosolic pyrimidine deoxyribonucleotides is limited. Our results therefore reveal an important role for ribonucleotide salvage in mitochondrial gene expression.

Computer-Based Design of a Cell Factory for High-Yield Cytidine Production.

Pyrimidine ribonucleotide de novo biosynthesis pathway (PRdnBP) is an important pathway to produce pyrimidine nucleosides. We attempted to systematically investigate PRdnBP in Escherichia coli with genome-scale metabolic models and utilized the models to guide strain design. The balance of central carbon metabolism and PRdnBP affected the production of cytidine from glucose. Using Bayesian metabolic flux analysis, the effect of modified PRdnBP on the metabolic network was analyzed. The acetate overflow became coupled with PRdnBP flux, while they were originally independent under oxygen-sufficient conditions. The coupling between cytidine production and acetate secretion in the modified strain was weakened by arcA deletion, which resulted in further improving the efficient accumulation of cytidine. In total, 1.28 g/L of cytidine with a yield of 0.26 g/g glucose was produced. The yield of cytidine produced by E. coli is higher than previous reports. Our strategy provides an effective attempt to find metabolic bottlenecks in genetically engineered bacteria by using flux coupling analysis.

Proof-of-principle studies on a strategy to enhance nucleotide imbalance specifically in cancer cells

Highly specific and potent inhibitors of dihydroorotate dehydrogenase (DHODH), an essential enzyme of the de novo pyrimidine ribonucleotide synthesis pathway, are in clinical trials for autoimmune diseases, viral infections and cancer. However, because DHODH inhibitors (DHODHi) are immunosuppressants they may reduce the anticancer activity of the immune system. Therefore, there may be a need to improve the therapeutic index of DHODHi in cancer patients. The aim of this study was to find strategies to protect activated T cells from DHODHi and to identify cancer types hypersensitive to these inhibitors. First, we observed that like uridine supplementation, adding cytidine to the culture medium protects T cells from DHODH blockage. Next, we identified tumor types with altered expression of pyrimidine ribonucleotide synthesis enzymes. In this regard, we detected that the expression of cytidine deaminase (CDA), which converts cytidine into uridine, is low in an important proportion of cancer cell lines and consistently low in neuroblastoma samples and in cell lines from neuroblastoma and small cell lung carcinoma. This suggested that in the presence of a DHODHi, an excess of cytidine would be deleterious for low CDA expressing cancer cell lines. We show that this was the case (as could be seen almost immediately after treatment) when cells were cultured with fetal bovine serum but, was significantly less evident when cultures contained human serum. One interesting feature of CDA is that aside from acting intracellularly, it is also present in human plasma/serum. Altogether, experiments using recombinant CDA, human serum, pharmacologic inhibition of CDA and T cell/cancer cell co-cultures suggest that the therapeutic index of DHODHi could be improved by selecting patients with low-CDA expressing cancers in combination with strategies to increase cytidine or the cytidine/uridine ratio in the extracellular environment. Collectively, this proof-of-principle study warrants the discovery of agents to deplete extracellular CDA.

Early changes in gene expression profiles in AML patients during induction chemotherapy

BackgroundElucidation of the genetic mechanisms underlying treatment response to standard induction chemotherapy in AML patients is warranted, in order to aid in risk-adapted treatment decisions as novel treatments are emerging. In this pilot study, we explored the treatment-induced expression patterns in a small cohort of AML patients by analyzing differential gene expression (DGE) over the first 2 days of induction chemotherapy.MethodsBlood samples were collected from ten AML patients at baseline (before treatment initiation) and during the first 2 days of treatment (Day 1; approximately 24 h, and Day 2; approximately 48 h after treatment initiation, respectively) and RNA was extracted for subsequent RNA sequencing. DGE between time points were assessed by pairwise analysis using the R package edgeR version 3.18.1 in all patients as well as in relation to treatment response (complete remission, CR, vs non-complete remission, nCR). Ingenuity Pathway Analysis (Qiagen) software was used for pathway analysis and visualization.ResultsAfter initial data quality control, two patients were excluded from further analysis, resulting in a final cohort of eight patients with data from all three timepoints. DGE analysis demonstrated activation of pathways with genes directly or indirectly associated with NF-κB signaling. Significant activation of the NF-κB pathway was seen in 50% of the patients 2 days after treatment start, while iNOS pathway effects could be identified already after 1 day. nCR patients displayed activation of pathways associated with cell cycle progression, oncogenesis and anti-apoptotic behavior, including the STAT3 pathway and Salvage pathways of pyrimidine ribonucleotides. Notably, a significant induction of cytidine deaminase, an enzyme responsible for the deamination of Ara-C, could be observed between baseline and Day 2 in the nCR patients but not in patients achieving CR.ConclusionsIn conclusion, we show that time-course analysis of gene expression represents a feasible approach to identify relevant pathways affected by standard induction chemotherapy in AML patients. This poses as a potential method for elucidating new drug targets and biomarkers for categorizing disease aggressiveness and evaluating treatment response. However, more studies on larger cohorts are warranted to elucidate the transcriptional basis for drug response.

RNA Sequencing of Intestinal Enterocytes Pre- and Post-Roux-en-Y Gastric Bypass Reveals Alteration in Gene Expression Related to Enterocyte Differentiation, Restitution, and Obesity with Regulation by Schlafen 12.

Background: The intestinal lining renews itself in a programmed fashion that can be affected by adaptation to surgical procedures such as gastric bypass. Methods: To assess adaptive mechanisms in the human intestine after Roux-en-Y gastric bypass (RYGB), we biopsied proximal jejunum at the anastomotic site during surgery to establish a baseline and endoscopically re-biopsied the same area 6–9 months after bypass for comparison. Laser microdissection was performed on pre- and post-RYGB biopsies to isolate enterocytes for RNA sequencing. Results: RNA sequencing suggested significant decreases in gene expression associated with G2/M DNA damage checkpoint regulation of the cell cycle pathway, and significant increases in gene expression associated with the CDP-diacylglycerol biosynthesis pathway TCA cycle II pathway, and pyrimidine ribonucleotide salvage pathway after RYGB. Since Schlafen 12 (SLFN12) is reported to influence enterocytic differentiation, we stained mucosa for SLFN12 and observed increased SLFN12 immunoreactivity. We investigated SLFN12 overexpression in HIEC-6 and FHs 74 Int intestinal epithelial cells and observed similar increased expression of the following genes that were also increased after RYGB: HES2, CARD9, SLC19A2, FBXW7, STXBP4, SPARCL1, and UTS. Conclusions: Our data suggest that RYGB promotes SLFN12 protein expression, cellular mechanism and replication pathways, and genes associated with differentiation and restitution (HES2, CARD9, SLC19A2), as well as obesity-related genes (FBXW7, STXBP4, SPARCL1, UTS).

Structure-Based Prototyping of Allosteric Inhibitors of Human Uridine/Cytidine Kinase 2 (UCK2).

Pyrimidine nucleotide biosynthesis in humans is a promising chemotherapeutic target for infectious diseases caused by RNA viruses. Because mammalian cells derive pyrimidine ribonucleotides through a combination of de novo biosynthesis and salvage, combined inhibition of dihydroorotate dehydrogenase (DHODH; the first committed step in de novo pyrimidine nucleotide biosynthesis) and uridine/cytidine kinase 2 (UCK2; the first step in salvage of exogenous nucleosides) strongly attenuates viral replication in infected cells. However, while several pharmacologically promising inhibitors of human DHODH are known, to date there are no reports of medicinally viable leads against UCK2. Here, we use structure-based drug prototyping to identify two classes of promising leads that noncompetitively inhibit UCK2 activity. In the process, we have identified a hitherto unknown allosteric site at the intersubunit interface of this homotetrameric enzyme. By reducing the kcat of human UCK2 without altering its KM, these new inhibitors have the potential to enable systematic dialing of the fractional inhibition of pyrimidine salvage to achieve the desired antiviral effect with minimal host toxicity.

Control of a pyrimidine ribonucleotide salvage pathway in Pseudomonas oleovorans.

The control of a pyrimidine ribonucleotide salvage pathway in the bacterium Pseudomonas oleovorans ATCC 8062 was studied. This bacterium is important for its ability to synthesize polyesters as well as for its increasing clinical significance in humans. The pyrimidine salvage pathway enzymes pyrimidine nucleotide N-ribosidase and cytosine deaminase were investigated in P. oleovorans ATCC 8062 under selected culture conditions. Initially, the effect of carbon source on the two pyrimidine salvage enzymes in ATCC 8062 cells was examined and it was observed that cell growth on the carbon source succinate generally produced higher enzyme activities than did glucose or glycerol as a carbon source when ammonium sulfate served as the nitrogen source. Using succinate as a carbon source, growth on dihydrouracil as nitrogen source caused a 1.9-fold increase in the pyrimidine nucleotide N-ribosidase activity and a 4.8-fold increase in cytosine deaminase activity compared to the ammonium sulfate-grown cells. Growth of ATCC 8062 cells on cytosine or dihydrothymine as a nitrogen source elevated deaminase activity by more than double that observed for ammonium sulfate-grown cells. The findings indicated a relationship between this pyrimidine salvage pathway and the pyrimidine reductive catabolic pathway since growth on dihydrouracil appeared to increase the degradation of the pyrimidine ribonucleotide monophosphates to uracil. The uracil produced could be degraded by the pyrimidine base reductive catabolic pathway to β-alanine as a source of nitrogen. This investigation could prove helpful to future work examining the metabolic relationship between pyrimidine salvage pathways and pyrimidine reductive catabolism in pseudomonads.

Profiling Ribonucleotide and Deoxyribonucleotide Pools Perturbed by Remdesivir in Human Bronchial Epithelial Cells.

Remdesivir (RDV) has generated much anticipation for its moderate effect in treating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, the unsatisfactory survival rates of hospitalized patients limit its application to the treatment of coronavirus disease 2019 (COVID-19). Therefore, improvement of antiviral efficacy of RDV is urgently needed. As a typical nucleotide analog, the activation of RDV to bioactive triphosphate will affect the biosynthesis of endogenous ribonucleotides (RNs) and deoxyribonucleotides (dRNs), which are essential to RNA and DNA replication in host cells. The imbalance of RN pools will inhibit virus replication as well. In order to investigate the effects of RDV on cellular nucleotide pools and on RNA transcription and DNA replication, cellular RNs and dRNs concentrations were measured by the liquid chromatography-mass spectrometry method, and the synthesis of RNA and DNA was monitored using click chemistry. The results showed that the IC50 values for BEAS-2B cells at exposure durations of 48 and 72 h were 25.3 ± 2.6 and 9.6 ± 0.7 μM, respectively. Ten (10) μM RDV caused BEAS-2B arrest at S-phase and significant suppression of RNA and DNA synthesis after treatment for 24 h. In addition, a general increase in the abundance of nucleotides and an increase of specific nucleotides more than 2 folds were observed. However, the variation of pyrimidine ribonucleotides was relatively slight or even absent, resulting in an obvious imbalance between purine and pyrimidine ribonucleotides. Interestingly, the very marked disequilibrium between cytidine triphosphate (CTP) and cytidine monophosphate might result from the inhibition of CTP synthase. Due to nucleotides which are also precursors for the synthesis of viral nucleic acids, the perturbation of nucleotide pools would block viral RNA replication. Considering the metabolic vulnerability of endogenous nucleotides, exacerbating the imbalance of nucleotide pools imparts great promise to enhance the efficacy of RDV, which possibly has special implications for treatment of COVID-19.

DHODH inhibition modulates glucose metabolism and circulating GDF15, and improves metabolic balance

SummaryDihydroorotate dehydrogenase (DHODH) is essential for the de novo synthesis of pyrimidine ribonucleotides, and as such, its inhibitors have been long used to treat autoimmune diseases and are in clinical trials for cancer and viral infections. Interestingly, DHODH is located in the inner mitochondrial membrane and contributes to provide ubiquinol to the respiratory chain. Thus, DHODH provides the link between nucleotide metabolism and mitochondrial function. Here we show that pharmacological inhibition of DHODH reduces mitochondrial respiration, promotes glycolysis, and enhances GLUT4 translocation to the cytoplasmic membrane and that by activating tumor suppressor p53, increases the expression of GDF15, a cytokine that reduces appetite and prolongs lifespan. In addition, similar to the antidiabetic drug metformin, we observed that in db/db mice, DHODH inhibitors elevate levels of circulating GDF15 and reduce food intake. Further analysis using this model for obesity-induced diabetes revealed that DHODH inhibitors delay pancreatic β cell death and improve metabolic balance.

NMR analysis of nucleotide \u03c0-stacking in prebiotically relevant crowded environment

The prebiotic soup of a putative ‘RNA World’ would have been replete with a plethora of molecules resulting from complex chemical syntheses and exogeneous delivery. The presence of background molecules could lead to molecular crowding, potentially affecting the course of the reactions facilitated therein. Using NMR spectroscopy, we have analyzed the effect of crowding on the stacking ability of RNA monomers. Our findings corroborate that the purines stack more efficiently than the pyrimidine ribonucleotides. This competence is further enhanced in the presence of a crowding agent. This enhanced stacking could result in greater sequestration of the purine monomers, putting their ready availability for relevant nonenzymatic reactions into question. Thus, this study demonstrates the need for systematic characterization of molecular crowding in the context of prebiotically pertinent processes. Unraveling such phenomena is essential for our understanding of the transition from abiotic to biotic, during the origin of life.

Metabolomic Description of Ivacaftor Elevating Polymyxin B Mediated Antibacterial Activity in Cystic Fibrosis Pseudomonas aeruginosa.

We have demonstrated that ivacaftor displays synergistic antibacterial activity in combination with polymyxin B against polymyxin-resistant Pseudomonas aeruginosa that commonly colonizes the lungs of people with cystic fibrosis (CF). However, the underlying mechanism(s) remain unclear. In the present study, we employed untargeted metabolomics to investigate the synergistic killing mechanism of polymyxin B in combination with ivacaftor against a polymyxin-susceptible P. aeruginosa FADDI-PA111 (polymyxin B MIC = 2 mg/L) and a polymyxin-resistant CF P. aeruginosa FADDI-PA006 (polymyxin B MIC = 8 mg/L). Metabolites were extracted at 3 h after treatments with polymyxin B alone (2 μg/mL for FADDI-PA111 and 4 μg/mL FADDI-PA006 P. aeruginosa), ivacaftor alone (8 μg/mL), and in combination. Polymyxin B monotherapy induced significant perturbations in the glycerophospholipid and fatty acid metabolism pathways against FADDI-PA111 and to a lesser extent in FADDI-PA006. In both strains, treatment with ivacaftor alone induced more pronounced perturbations in glycerophospholipid and fatty acid metabolism pathways than that with polymyxin B alone. This highlights the unique antimicrobial mode of action of ivacaftor. Pathway analysis revealed that in combination treatment, polymyxin B mediated killing is elevated by ivacaftor, largely due to the inhibition of cell envelope biogenesis via suppression of key membrane lipid metabolites (e.g., sn-glycerol 3-phosphate and sn-glycero-3-phosphoethanolamine) as well as perturbations in peptidoglycan and lipopolysaccharide biosynthesis. Furthermore, significant perturbations in the levels of amino sugars and nucleotide sugars, glycolysis, the tricarboxylic acid cycle, and pyrimidine ribonucleotide biogenesis were observed with the combination treatment. These findings provide novel mechanistic information on the synergistic antibacterial activity of polymyxin-ivacaftor combination.

Ultraviolet-Driven Deamination of Cytidine Ribonucleotides Under Planetary Conditions.

A previously proposed synthesis of pyrimidine ribonucleotides makes use of ultraviolet (UV) light to convert β-d-ribocytidine-2',3'-cyclic phosphate to β-d-ribouridine-2',3'-cyclic phosphate, while simultaneously selectively degrading synthetic byproducts. Past studies of the photochemical reactions of pyrimidines have employed mercury arc lamps, characterized by narrowband emission centered at 254 nm, which is not representative of the UV environment of the early Earth. To further assess this process under more realistic circumstances, we investigated the wavelength dependence of the UV-driven conversion of β-d-ribocytidine-2',3'-cyclic phosphate to β-d-ribouridine-2',3'-cyclic phosphate. We used constraints provided by planetary environments to assess the implications for pyrimidine nucleotides on the early Earth. We found that the wavelengths of light (255-285 nm) that most efficiently drive the deamination of β-d-ribocytidine-2',3'-cyclic phosphate to β-d-ribouridine-2',3'-cyclic phosphate are accessible on planetary surfaces such as those of the Hadean-Archaean Earth for CO2-N2-dominated atmospheres. However, continued irradiation could eventually lead to low levels of ribocytidine in a low-temperature, highly irradiated environment, if production rates are slow.