At P17, a single dose of uridine 5′ monophosphate (Sigma cat. U1752, St. Louis, MO, USA) was administered subcutaneously (s.c.) at a concentration of either 330 mg/kg (n = 5, low-dose Ur group) or 1000 mg/kg (n = 5, high-dose Ur group). Blood samples (150–200 μL; anticoagulated with EDTA) were obtained from the saphenous vein at 0 time point (baseline, n = 8) prior to the administration of Ur or saline vehicle. Subsequent blood samples were collected at 30 min, 1 h, 2 h, and 4 h. Circulating levels of uridine and uracil were measured using high pressure liquid chromatography-mass spectrometry (Supplementary Methods ). Our goal was to determine a Ur dose that resulted in circulating concentrations similar to those conferring neuroprotection in a rodent model of neonatal brain injury: 10,000 ng/μL [14 (link)].
Uridine 5 monophosphate
Manufactured by Merck Group
Sourced in Poland, India, United States
Uridine 5'-monophosphate is a nucleotide that consists of the nucleoside uridine and a phosphate group. It is a key intermediate in the de novo biosynthesis of pyrimidine nucleotides and plays a role in various cellular processes.
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Lab products found in correlation
Cytidine 5 monophosphate, by Merck Group (6 mentions)
Adenosine 5 monophosphate, by Merck Group (3 mentions)
Guanosine 5 monophosphate, by Merck Group (3 mentions)
Leflunomide, by Merck Group (2 mentions)
Progesterone, by Merck Group (2 mentions)
A77 1726, by Enzo Life Sciences (2 mentions)
Esomeprazole, by Enzo Life Sciences (2 mentions)
Aphidicolin, by Enzo Life Sciences (2 mentions)
A77 1726, by Merck Group (2 mentions)
Inosine 5 monophosphate, by Merck Group (1 mentions)
Masslynx software, by Waters Corporation (1 mentions)
Masslynx, by Waters Corporation (1 mentions)
Acquity uplc hss t3 column, by Waters Corporation (1 mentions)
1 palmitoyl 2 oleoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions)
2 deoxycytidine 5 monophosphate, by Merck Group (1 mentions)
Adenosine, by Merck Group (1 mentions)
2 deoxyadenosine, by Merck Group (1 mentions)
2 deoxycytidine, by Merck Group (1 mentions)
Milli q water purification system, by Merck Group (1 mentions)
Ammonium acetate, by Merck Group (1 mentions)
8 protocols using uridine 5 monophosphate
1
Uridine Dose-response Evaluation
2
Quantitative Analysis of Monophosphate Nucleotides
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The qualitative analysis of monophosphate nucleotides was performed as reported by Yamaoka et al. [19 (link)]. LC–MS was performed under the following conditions: instrument, Waters UPLC™ and system with an electrospray ionization (ESI) interface and a quadrupole mass detection system (Waters, Milford, MA, USA); software, MassLynxTM; column, ACQUITY UPLC™ Column HSS T3 (2.1 mm ID and 100 mm length, 1.8-μm particle size) (Waters, Milford, MA, USA). The ESI source was operated at 120 °C with a desolvation temperature of 450 °C, 800 L/h desolvation gas flow rate, and a capillary voltage set at 3.5 kV. The cone voltage was 30 V and collision energies 30 eV. Integration and quantitation were performed using the Waters MassLynxTM software. Detection of these compounds was performed in the negative ionization. Monophosphates nucleotides (xanthosine 5’-monophosphate (XMP), cytidine 5’-monophosphate (CMP), uridine-5’-monophosphate (UMP), thymidin-5’-monophosphate (TMP), adenosine-5’-monophosphate (AMP), inosine 5’-monophosphate (IMP), guanosine-5’-monophosphate (GMP)), were purchased from Sigma–Aldrich (Wrocław, Poland). All data were obtained in triplicate. The results were expressed as mg/100 g of dry weight (d.w.).
3
Nucleotide Synthesis and Lipid Purification
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Adenosine 5′-monophosphate (AMP), uridine 5′-monophosphate (UMP), guanosine 5′-monophosphate (GMP), cytidine 5′-monophosphate (CMP), and ribose 5′-monophosphate (rMP) were purchased as disodium salts from Sigma-Aldrich (Bangalore, India) and used without further purification. The phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), was purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). All other reagents used were of analytical grade and purchased from Sigma-Aldrich (Bangalore, India).
4
Bacillus subtilis Growth Conditions
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B. subtilis 168 trpC2 strains (Table 1 ) were grown on Lysogeny Broth (LB). E. coli MC1061 was used as a cloning host. All plasmids are listed in Table 2 and the oligonucleotides in Table 3 . The C-medium contains 70 mM K2HPO4, 30 mM KH2PO4, 25 mM (NH4)2SO4, 0.5 mM Mg2SO4, 10 μM MnSO4, 22 mg/l Ferric Ammonium Citrate, 250 μM L-Tryptophan, and 0.4% (w/v) glucose. C-medium supplemented with glutamate contains 0.03% (w/v) L-Glutamate and when it was also supplemented with branched chain amino acids it contained 0.25% (w/v) L-Isoleucine, 0.25% (w/v) L-Leucine, 2.5% (w/v) L-Valine, and 2.5% (w/v) L-Methionine. Uridine 5′-monophosphate (Sigma) was added to C-medium in a final concentration of 20 mg/l.
5
Nucleotide Metabolism Assay Protocol
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The list of the ANPs and chemotherapeutics used in this study is provided in Tables S1 and Figure S9 . The source of other chemical reagents was as follows: Uridine (Urd) and adenosine-5′-triphosphate (ATP) Calbiochem; cytidine (Cyd), Valeant Pharmaceuticals; cytidine-5′-monophosphate (CMP), uridine-5′-monophosphate (UMP), 2′-deoxycytidine-5′-monophosphate (dCMP) and 2′-deoxyuridine-5′-monophosphate (dUMP), Sigma-Aldrich; arabinocytidine-5′-monophosphate (araCMP), [5-3H]-radiolabeled CDV and [5-3H]-uridine, Moravek Biochemicals; and [5-3H]-radiolabeled cytidine, MP Biochemicals.
6
Quantitative Analysis of Matsutake Metabolites
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Eighty dried samples of T. matsutake were collected in accordance with official sampling requirements [19 ] from the mountain areas of Xiaojin County, Jiulong County, Yajiang County, Kangding County, Muli County, and Lixian County in Sichuan Province, China. Fifteen authentic standards of adenosine (A), cytidine (C), guanosine (G), inosine (I), thymidine (T), uridine (U), xanthosine dehydrate (X), 2′-deoxyadenosine (dA), 2′-deoxycytidine (dC), 2′-deoxyguanosine (dG), 2′-deoxyuridine (dU), adenosine 5′-monophosphate (AMP), cytidine 5′-monophosphate (CMP), guanosine 5′-monophosphate (GMP), and uridine 5′-monophosphate (UMP) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The Milli-Q water purification system was used to prepare ultra-pure water for the UPLC analysis (Millipore, Bedford, MA, USA). The solvent ammonium acetate, acetonitrile, and diethylamine with LC-MS grade for UPLC-MS analysis were also purchased from Sigma-Aldrich (Sigma-Aldrich, St.Louis, MO, USA). Other chemicals and solvents of analytical grade were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
7
Zebrafish Embryo Drug Screening
8
Zebrafish Embryo Drug Screening
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Wild-type zebrafish embryos were treated with leflunomide from 50% epiboly until 24 hours post-fertilization in E3 embryo media and fixed with 4% Formaldehyde. Chemical libraries screened: LOPAC (n= 1280), ICCBL (n= 480) and NIH clinical collection (n=450). Other chemical used include leflunomide (Sigma L5025), Progesterone (Sigma, P0130), Esomeprazole (Enzo life sciences), Aphidicolin (Enzo life sciences, BML-CC101–0001) and A77 1726 (Enzo Life Sciences, ALX-430–096-M025). For nucleotide rescue experiments, a cocktail of 10 μg/ml uridine 5′-monophosphate (Sigma, UMP) and 10 μg/ml cytidine 5′-monophosphate (Sigma, CMP) was added to A77 1726. For in situ hybridizations, we followed the methods as described by Thisse C, and Thisse, B28 ,29 .
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