Combining recombinase polymerase amplification with tyrosine modified 2'-deoxyuridine-5'-triphosphate for direct voltammetric detection of double-stranded DNA: Application to potato pathogen Dickeya solani.

The approach based on a combination of isothermal recombinase polymerase amplification (RPA), 2'-deoxyuridine-5'-triphosphate modified with tyrosine aromatic group (dUTP-Y1), and direct voltammetric detection of RPA product carrying electroactive labels was successfully applied to the potato pathogen Dickeya solani. The artificial nucleotide dUTP-Y1 demonstrated a good compatibility with RPA, enabling by targeting a section of D. solani genome with a unique sequence to produce the full-size modified products at high levels of substitution of dTTP by dUTP-Y1 (up to 80-90 %) in the reaction mixture. The optimized procedure of square wave voltammetry allowed to reliably detect the product generated by RPA at 80 % substitution of dTTP by dUTP-Y1 (dsDNA-Y1) in microliter sample volumes on the surface of disposable carbon screen printed electrodes at the potential of about 0.6 V. The calibration curve for the amplicon detection was linear in coordinates 'Ip, A vs. Log (c, M)' within the 0.05-1 µM concentration range. The limit of detection for dsDNA-Y1 was estimated as 8 nM. The sensitivity of the established electrochemical approach allowed to detect amplicons generated in a single standard 50 µL RPA reaction after their purification with silica-coated magnetic beads. The overall detectability of D. solani with the suggested combination of RPA and voltammetric registration of dsDNA-Y1 can be as low as a few copies of bacterial genome per standard reaction. In total, amplification, purification, and electrochemical detection take about 120-150 min. Considering the potential of direct electrochemical analysis for miniaturization, as well as compliance with low-cost and low-power requirements, the findings provide grounds for future development of microfluidic devices integrating isothermal amplification, amplicon purification and detection based on the tyrosine modified nucleotide for the purpose of 'on-site' detection of various pathogens.

Polymerase incorporation of 4-nitrophenyl modified 2'-deoxyuridine-5'-triphosphates into double-stranded DNA for direct electrochemical detection.

Three novel 2'-deoxyuridine-5'-triphosphates modified with 4-nitrophenyl groups via various linkers (dUTP-N1, dUTP-N2, and dUTP-N3) were tested as bearers of reducible electroactive labels as well as substrates suitable for enzymes used in polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA) with a potential application to direct electrochemical detection of double-stranded deoxyribonucleic acid (dsDNA). In cyclic and square wave voltammograms on carbon screen printed electrodes, the labeled dUTP have demonstrated distinct reduction peaks at potentials of -0.7 V to -0.9 V (phosphate buffer, pH 7.4). The reduction peak currents of dUTP-N derivatives were found to increase with their molar concentrations. The dUTP-N3 with a double bond in the linker had the lowest reduction potential (about 100 mV less negative) among the derivatives studied. Further, dUTP-N nucleotides were tested as substrates in PCR and RPA to incorporate the electroactive labels into 90, 210, or 206 base pair long dsDNA amplicons. However, only a dUTP-N1 derivative with a shorter linker without the double bond demonstrated satisfactory compatibility with both PCR and RPA, though with a low reaction output of modified dsDNA amplicons (at 100% substitution of dTTP). The dsDNA amplicons produced by PCR with 85% substitution of dTTP by the dUTP-N1 in the reaction mixture were successfully detected by square wave voltammetry at micromolar concentrations at high square wave frequency.

Thymidine Kinase-Independent Click Chemistry DNADetect Probes for DNA Proliferation Assessment in Malaria Parasites.

Metabolic chemical probes are small-molecule reagents that utilize naturally occurring biosynthetic enzymes for in situ incorporation into biomolecules of interest. These reagents can be used to label, detect, and track important biological processes within living cells including protein synthesis, protein glycosylation, and nucleic acid proliferation. A limitation of current chemical probes, which have largely focused on mammalian cells, is that they often cannot be applied to other organisms due to metabolic differences. For example, the thymidine derivative 5-ethynyl-2'-deoxyuridine (EdU) is a gold standard metabolic chemical probe for assessing DNA proliferation in mammalian cells; however, it is unsuitable for the study of malaria parasites due to Plasmodium species lacking the thymidine kinase enzyme that is essential for metabolism of EdU. Herein, we report the design and synthesis of new thymidine-based probes that sidestep the requirement for a thymidine kinase enzyme in Plasmodium. Two of these DNADetect probes exhibit robust labeling of replicating asexual intraerythrocytic Plasmodium falciparum parasites, as determined by flow cytometry and fluorescence microscopy using copper-catalyzed azide-alkyne cycloaddition to a fluorescent azide. The DNADetect chemical probes are synthetically accessible and thus can be made widely available to researchers as tools to further understand the biology of different Plasmodium species, including laboratory lines and clinical isolates.

Enzyme kinetics of deoxyuridine triphosphatase from Western corn rootworm.

OBJECTIVE: The Western corn rootworm (WCR), Diabrotica virgifera virgifera, is a highly adaptable insect pest that has evolved resistance to a variety of control strategies, including insecticides. Therefore, it is interesting to examine how housekeeping proteins in WCR have been changed under WCR-controlling strategies. In this study, we focused on one of such proteins in WCR, a ubiquitous enzyme 5'-triphosphate nucleotidohydrolase (dUTPase). In the thymidine synthetic pathway, dUTPase hydrolyzes deoxyuridine triphosphate (dUTP) and supplies the substrate, deoxyuridine monophosphate, for the thymidylate synthase (TS). It decreases the cellular content of uracil, reducing uracil misincorporation into DNA. Suppressing the dUTPase activity, therefore, contributes to thymineless death. In this study, we investigated the enzymatic properties of dUTPase. RESULTS: The WCR dUTPase gene (DUT) was synthesized with the addition of His-tag corresponding DNA sequence and then cloned and expressed in Escherichia coli, and the protein product was purified. The product of WCR DUT hydrolyzed dUTP and was designated as dUTPase. WCR dUTPase did not hydrolyze dATP, dTTP, dCTP, or dGTP. WCR dUTPase was analyzed via size-exclusion chromatography and exhibited a molecular weight corresponding to that of trimer. The present format can be interpreted as nuclear trimer type. Possible isomers will be examined once transcriptome analyses are conducted.

Comparative Transcriptomic and Metabolomic Analyses of Differences in Trunk Spiral Grain in Pinus yunnanensis.

Having a spiral grain is considered to be one of the most important wood properties influencing wood quality. Here, transcriptome profiles and metabolome data were analyzed in the straight grain and twist grain of Pinus yunnanensis. A total of 6644 differential expression genes were found between the straight type and the twist type. A total of 126 differentially accumulated metabolites were detected. There were 24 common differential pathways identified from the transcriptome and metabolome, and these pathways were mainly annotated in ABC transporters, arginine and proline metabolism, flavonoid biosynthesis, isoquinoline alkaloid biosynthesis, linoleic acid metabolism, phenylpropanoid, tryptophan metabolism, etc. A weighted gene coexpression network analysis showed that the lightblue4 module was significantly correlated with 2'-deoxyuridine and that transcription factors (basic leucine zipper (bZIP), homeodomain leucine zipper (HD-ZIP), basic helix-loop-helix (bHLH), p-coumarate 3-hydroxylase (C3H), and N-acetylcysteine (NAC)) play important roles in regulating 2'-deoxyuridine, which may be involved in the formation of spiral grains. Meanwhile, the signal transduction of hormones may be related to spiral grain, as previously reported. ARF7 and MKK4_5, as indoleacetic acid (IAA)- and ethylene (ET)-related receptors, may explain the contribution of plant hormones in spiral grain. This study provided useful information on spiral grain in P. yunnanensis by transcriptome and metabolome analyses and could lay the foundation for future molecular breeding.

Novel ProTide prodrugs of 5-fluoro-2'-deoxyuridine for the treatment of liver cancer.

ProTide prodrug technology has emerged as a promising way for the development of anti-viral and anti-tumor drugs, whereas, there are fewer applications for the treatment of liver cancer. Herein, a series of distinct 3'-ester ProTide prodrugs of 5-fluoro-2'-deoxyuridine (FdUR) were synthesized and evaluated for their anti-liver cancer activity. The most efficient prodrug 11b reached a sub-micromolar activity (IC50 = 0.42 ± 0.13 µM) against HepG2 and over 100-fold and 200-fold improvements compared to 5-FU, respectively. 11b also demonstrated favorable selectivity towards normal liver cells L-02 (IC50 > 100 µM). In vitro metabolic stability studies revealed that 11b is stable in the plasma and could be activated rapidly in the liver, which supported that 11b is liver-targeted. Importantly, to more accurately evaluate the anti-HCC activity of 11b, the liver orthotopic model was built and 11b significantly suppressed tumor growth (TGI = 75.5%) at a dose of 60 mg/kg/2d in vivo without obvious toxicity. Overall, these promising results indicated that 11b could serve as a safe and effective prodrug of 5-FU nucleoside for liver cancer therapy.

Synthesis, Base Pairing Properties, and Biological Activity Studies of Platinum(II) Complexes Based on Uracil Nucleosides.

The synthesis and base pairing properties of platinum complexes based on uridine and deoxyuridine nucleosides and preliminary studies of their antiproliferative activity are described. Platinum(II) uridine and deoxyuridine complexes were synthesized by C-I oxidative addition to Pt(0)(PPh3)4. First, the synthesis was performed with protected nucleosides to generate complexes 1 and 2, which were deprotected under basic conditions, affording complexes 3 and 4 in good yields. The synthesis with the unprotected nucleosides was also performed and provided complexes 3 and 4 effectively. Base pairing interactions were measured for complex 1, either for self-base pairing or for the Watson-Crick base pair. Complex 1 undergoes self-base pairing in CDCl3, and this aggregation was found not to be dependent on metalation. Contrastingly, for the Watson-Crick base pair with adenine, base pairing was also observed, but metalation was found to affect hydrogen bonding considerably. Complexes 3 and 4 and the corresponding ligand precursors were evaluated for their antiproliferative activity against human glioblastoma cell line U-251. The compounds showed IC50 values of 3.30 (3) and 1.84 (4) µM but are also toxic for nontumorous cell lines.

Polymerase incorporation of fluorescein or rhodamine modified 2'-deoxyuridine-5'-triphosphates into double-stranded DNA for direct electrochemical detection.

The 2'-deoxyuridine-5'-triphosphates modified with fluorescein (dUTP-Fl) or rhodamine (dUTP-Rh) were tested as bearers of electroactive labels and as proper substrates for polymerases used in polymerase chain reaction (PCR) and isothermal recombinase polymerase amplification (RPA) with the aim of electrochemical detection of double-stranded DNA (dsDNA) amplification products. For this purpose, electrochemical behavior of free fluorescein and rhodamine as well as the modified nucleotides, dUTP-Fl and dUTP-Rh, was studied by cyclic (CV) and square wave (SWV) voltammetry on carbon screen printed electrodes. Both free fluorescein and dUTP-Fl underwent a two-step oxidation at the peak potentials (Ep) of 0.6-0.7 V and 0.8-0.9 V (phosphate buffer, pH 7.4). The reduction peaks of fluorescein and dUTP-Fl were registered between -0.9 V and -1 V, but they did not depend on concentration. The free rhodamine and dUTP-Rh have demonstrated the well-defined oxidation peaks at 0.8-0.9 V. In addition, the distinct reduction peaks at Ep between -0.8 V and -0.9 V were registered for both rhodamine and dUTP-Rh. The dUTP-Fl and dUTP-Rh were further tested as substrates to incorporate an electroactive label into 210 or 206 base pair long dsDNA amplicons generated either by PCR or RPA. Among two dUTP derivatives tested, dUTP-Fl revealed significantly better compatibility with PCR and RPA, producing the full-size amplicons at 50-90% substitution of dTTP in the reaction mixture. In the PCR, the best compromise between amplicon output and labeling was achieved at the dUTP-Fl : dTTP and dUTP-Rh : dTTP molar ratios of 70% : 30% and 20% : 80% in the PCR mixture, respectively, allowing the direct electrochemical detection of amplicons at micromolar concentrations. Alongside with fluorescence DNA assays, the fluorescein and rhodamine modified dUTP appear as promising electroactive labels to develop direct electrochemical DNA assays for detecting PCR and RPA products.

Human 2'-Deoxynucleoside 5'-Phosphate N-Hydrolase 1: Mechanism of 2'-Deoxyuridine 5'-Monophosphate Hydrolysis.

The enzyme 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 (DNPH1) catalyzes the N-ribosidic bond cleavage of 5-hydroxymethyl-2'-deoxyuridine 5'-monophosphate to generate 2-deoxyribose 5-phosphate and 5-hydroxymethyluracil. DNPH1 accepts other 2'-deoxynucleoside 5'-monophosphates as slow-reacting substrates. DNPH1 inhibition is a promising strategy to overcome resistance to and potentiate anticancer poly(ADP-ribose) polymerase inhibitors. We solved the crystal structure of unliganded human DNPH1 and took advantage of the slow reactivity of 2'-deoxyuridine 5'-monophosphate (dUMP) as a substrate to obtain a crystal structure of the DNPH1:dUMP Michaelis complex. In both structures, the carboxylate group of the catalytic Glu residue, proposed to act as a nucleophile in covalent catalysis, forms an apparent low-barrier hydrogen bond with the hydroxyl group of a conserved Tyr residue. The crystal structures are supported by functional data, with liquid chromatography-mass spectrometry analysis showing that DNPH1 incubation with dUMP leads to slow yet complete hydrolysis of the substrate. A direct UV-vis absorbance-based assay allowed characterization of DNPH1 kinetics at low dUMP concentrations. A bell-shaped pH-rate profile indicated that acid-base catalysis is operational and that for maximum kcat/KM, two groups with an average pKa of 6.4 must be deprotonated, while two groups with an average pKa of 8.2 must be protonated. A modestly inverse solvent viscosity effect rules out diffusional processes involved in dUMP binding to and possibly uracil release from the enzyme as rate limiting to kcat/KM. Solvent deuterium isotope effects on kcat/KM and kcat were inverse and unity, respectively. A reaction mechanism for dUMP hydrolysis is proposed.

Modification of N-hydroxycytidine yields a novel lead compound exhibiting activity against the Venezuelan equine encephalitis virus.

Nucleoside and nucleobase analogs capable of interfering with nucleic acid synthesis have played essential roles in fighting infectious diseases. However, many of these agents are associated with important and potentially lethal off-target intracellular effects that limit their use. Based on the previous discovery of base-modified 2'-deoxyuridines, which showed high anticancer activity while exhibiting lower toxicity toward rapidly dividing normal human cells compared to antimetabolite chemotherapeutics, we hypothesized that a similar modification of the N4-hydroxycytidine (NHC) molecule would provide novel antiviral compounds with diminished side effects. This presumption is due to the substantial structural difference with natural cytidine leading to less recognizability by host cell enzymes. Among the 42 antimetabolite species that have been synthesized and screened against VEEV, one hit compound was identified. The structural features of the modifying moiety were similar to those of the anticancer lead 2'-deoxyuridine derivative reported previously, providing an opportunity to pursue further structure-activity relationship (SAR) studies directed to lead improvement, and obtain insight into the mechanism of action, which can lead to identifying drug candidates against a broad spectrum of RNA viral infections.

Inner Amino Acid Contacts Are Key Factors of Multistage Structural Rearrangements of DNA and Affect Substrate Specificity of Apurinic/Apyrimidinic Endonuclease APE1.

Apurinic/apyrimidinic endonuclease 1 (APE1) is one of the most important enzymes in base excision repair. Studies on this enzyme have been conducted for a long time, but some aspects of its activity remain poorly understood. One such question concerns the mechanism of damaged-nucleotide recognition by the enzyme, and the answer could shed light on substrate specificity control in all enzymes of this class. In the present study, by pulsed electron-electron double resonance (DEER, also known as PELDOR) spectroscopy and pre-steady-state kinetic analysis along with wild-type (WT) APE1 from Danio rerio (zAPE1) or three mutants (carrying substitution N253G, A254G, or E260A), we aimed to elucidate the molecular events in the process of damage recognition. The data revealed that the zAPE1 mutant E260A has much higher activity toward DNA substrates containing 5,6-dihydro-2'-deoxyuridine (DHU), 2'-deoxyuridine (dU), alpha-2'-deoxyadenosine (αA), or 1,N6-ethenoadenosine (εA). Examination of conformational changes in DNA clearly revealed multistep DNA rearrangements during the formation of the catalytic complex. These structural rearrangements of DNA are directly associated with the capacity of damaged DNA for enzyme-induced bending and unwinding, which are required for eversion of the damaged nucleotide from the DNA duplex and for its placement into the active site of the enzyme. Taken together, the results experimentally prove the factors that control substrate specificity of the AP endonuclease zAPE1.

Palladium-Catalyzed C-H Olefination for Nucleic Acid Production.

The palladium-catalyzed direct C-H olefination of unprotected uridine, 2'-deoxyuridine, uridine monophosphate, and uridine analogues are described here. This protocol provides an efficient, atom-economical, and environmentally friendly method for the introduction of an alkenyl group at the C5 position of the uracil without pre-functionalization. A series of C5-alkenylated uridine analogues, including some biologically significant compounds and potential pharmaceutical candidates, were synthesized with exposed hydroxyl groups on the ribose. © 2023 Wiley Periodicals LLC. Basic Protocol 1: The reaction of uridine, 2'-deoxyuridine, and sofosbuvir for the C-H olefination with methyl acrylate Basic Protocol 2: The reaction of uridine and 2'-deoxyuridine for the C-H olefination with styrene.

Formal [4 + 2] Cycloadditions of Maleimides on Duplex DNA.

Near-quantitative DNA bioconjugation and detailed mechanistic investigations of reactions involving 5-(vinyl)-2'-deoxyuridine (VdU) and maleimides are reported. According to accelerated reaction rates in solvents with increasing polarity and trends in product stereochemistry, VdU-maleimide reactions proceed via a formal [4 + 2] stepwise cycloaddition. In contrast, 5-(1,3-butadienyl)-2'-deoxyuridine (BDdU) reacts with maleimides in a concerted [4 + 2] Diels-Alder cycloaddition. VdU-maleimide reactions enable high-yielding bioconjugation of duplex DNA in vitro (>90%) as well as metabolic labeling experiments in cells.

Evaluation of Neurotoxicity With Human Pluripotent Stem Cell-Derived Cerebral Organoids.

The recent development of human cerebral organoids provides an invaluable in vitro model of human brain development to assess the toxicity of natural or man-made toxic substances. By recapitulating key aspects of early human neurodevelopment, investigators can evaluate with this three-dimensional (3D) model the effect of certain compounds on the formation of neuronal networks and their electrophysiological properties with more physiological relevance than neurons grown in monolayers and in cultures composed of a unique cell type. This promising potential has contributed to the development of a large number of diverse protocols to generate human cerebral organoids, making interlaboratory comparisons of results difficult. Based on a previously published protocol to generate human cortical organoids (herein called cerebral organoids), we detail several approaches to evaluate the effect of chemicals on neurogenesis, apoptosis, and neuronal function when exogenously applied to cultured specimens. Here, we take as an example 4-aminopyridine, a potassium channel blocker that modulates the activity of neurons and neurogenesis, and describe a simple and cost-effective way to test the impact of this agent on cerebral organoids derived from human induced pluripotent stem cells. We also provide tested protocols to evaluate neurogenesis in cerebral organoids with ethynyl deoxyuridine labeling and neuronal activity with live calcium imaging and microelectrode arrays. Together, these protocols should facilitate the implementation of cerebral organoid technologies in laboratories wishing to evaluate the effects of specific compounds or conditions on the development and function of human neurons with only basic cell culture equipment. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of human cerebral organoids from pluripotent stem cells Support Protocol 1: Human pluripotent stem cell culture Basic Protocol 2: Evaluation of neurogenesis in cerebral organoids with ethynyl deoxyuridine labeling Basic Protocol 3: Calcium imaging in cerebral organoids Basic Protocol 4: Electrophysiological evaluation of cerebral organoids with microelectrode arrays Support Protocol 2: Immunostaining of cerebral organoids.

Why 6-Iodouridine Cannot Be Used as a Radiosensitizer of DNA Damage? Computational and Experimental Studies.

Previous density functional theory (DFT) studies on 6-brominated pyrimidine nucleosides suggest that 6-iodo-2'-deoxyuridine (6IdU) should act as a better radiosensitizer than its 5-iodosubstituted 2'-deoxyuridine analogue. In this work, we show that 6IdU is unstable in an aqueous solution. Indeed, a complete disappearance of the 6IdU signal was observed during its isolation by reversed-phase high-performance liquid chromatography (RP-HPLC). As indicated by the thermodynamic characteristics for the SN1-type hydrolysis of 6IdU obtained at the CAM-B3LYP/DGDZVP++ level and the polarizable continuum model (PCM) of water, 6-iodouracil (6IU) was already released quantitatively at ambient temperatures. The simulation of the hydrolysis kinetics demonstrated that a thermodynamic equilibrium was reached within seconds for the title compound. To assess the reliability of the calculations carried out, we synthesized 6-iodouridine (6IUrd), which was, unlike 6IdU, sufficiently stable in an aqueous solution at room temperature. The activation barrier for the N-glycosidic bond dissociation in 6IUrd was estimated experimentally using an Arrhenius plot. The stabilities in water calculated for 6IdU, 6IUrd, and 5-iodo-2'-deoxyuridine (5IdU) could be explained by the electronic and steric effects of the 2'-hydroxy group present in the ribose moiety. Our studies highlight the issue of the hydrolytic stability of potentially radiosensitizing nucleotides which, besides having favorable dissociative electron attachment (DEA) characteristics, must be stable in water to have any practical application.

In Situ Analysis of Mitochondrial DNA Synthesis Using Metabolic Labeling Coupled to Fluorescence Microscopy.

Metabolic labeling with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU) enables the selective labeling of DNA synthesis in live cells. Newly synthesized EdU-containing DNA can be covalently modified after extraction or in fixed cells using copper-catalyzed azide-alkyne cycloaddition "click chemistry" reactions, enabling bioconjugation to various substrates including fluorophores for imaging studies. While often used to study nuclear DNA replication, EdU labeling can also be leveraged to detect the synthesis of organellar DNA in the cytoplasm of Eukaryotic cells. In this chapter, we outline methods for the application of EdU labeling to the study of mitochondrial genome synthesis in fixed cultured human cells, using fluorescent labeling and superresolution light microscopy.

Actively replicating gut bacteria identified by 5-ethynyl-2'-deoxyuridine (EdU) click chemistry and cell sorting.

The composition of the intestinal bacterial community is well described, but recent research suggests that the metabolism of these bacteria plays a larger role in health than which species are present. One fundamental aspect of gut bacterial metabolism that remains understudied is bacterial replication. Indeed, there exist few techniques which can identify actively replicating gut bacteria. In this study, we aimed to address this gap by adapting 5-ethynyl-2'-deoxyuridine (EdU) click chemistry (EdU-click), a metabolic labeling method, coupled with fluorescence-activated cell sorting and sequencing (FACS-Seq) to characterize replicating gut bacteria. We first used EdU-click with human gut bacterial isolates and show that many of them are amenable to this technique. We then optimized EdU-click and FACS-Seq for murine fecal bacteria and reveal that Prevotella UCG-001 and Ileibacterium are enriched in the replicating fraction. Finally, we labeled the actively replicating murine gut bacteria during exposure to cell wall-specific antibiotics in vitro. We show that regardless of the antibiotic used, the actively replicating bacteria largely consist of Ileibacterium, suggesting the resistance of this taxon to perturbations. Overall, we demonstrate how combining EdU-click and FACSeq can identify the actively replicating gut bacteria and their link with the composition of the whole community in both homeostatic and perturbed conditions. This technique will be instrumental in elucidating in situ bacterial replication dynamics in a variety of other ecological states, including colonization and species invasion, as well as for investigating the relationship between the replication and abundance of bacteria in complex communities.

The bacteria that live in our guts are known to influence our intestinal and overall health. Though we know a lot about which kinds of bacteria are in our guts, we still don't know much about which bacteria are actually alive and growing. This is important to know, because bacteria that are growing, or replicating, are more likely to impact our health than bacteria which are not replicating. Our research group aimed to address this issue by developing a new technique that can identify which gut bacteria are actively replicating. We first tested this technique on specific gut bacteria, and then we made sure the technique worked when it was used on the gut bacteria of mice. By using this technique, we identified several types of mouse gut bacteria that were actively replicating. We also demonstrated one possible application of this technique by using it to identify mouse gut bacteria that were able to replicate after they were grown with antibiotics. Overall, we have introduced a new technique to identify replicating gut bacteria and show how it can be used to increase our knowledge on which bacteria are growing in the gut. This technique will help us identify which bacteria may be more important to our health due to their active growth.

An in silico comparative study of curcumin and 2-deoxyuridine nucleoside derivatives: Reveals the role of angiogenin in ER stress-induced apoptosis signaling.

Angiogenin (ANG) protein plays a crucial role in angiogenesis, neovascularization, and cancer metastasis in NSCLC (non-small cell lung cancer) via non-coding tiRNA. It protects the cell under ER (endoplasmic reticulum) stress-induced apoptosis through the translational reprogramming process. Although B82 (Curcumin derivatives) induces ER stress-induced apoptosis, its mechanism of action was not studied. Therefore, it was hypothesized that the ribonucleolytic activity of ANG may be regulated by B82, resulting in modulated ER stress signaling for apoptosis. Hence, we designed and proposed a synthesis scheme for RNA-based anti-angiogenic derivatives of 2-deoxyuridine nucleoside forming peptide bond with amino acids like serine (Ser-3) and para-hydroxy-phenyl glycine (Normtyr-1) and compared B82 with them to know the binding affinity with ANG, anti-angiogenic potential, and its probable mechanism of anti-RNase activity through MD simulation study. Therefore, using Gromos96 43a1 and 43a2 force fields, MD simulation was performed to investigate binding affinity, ligand-induced molecular surface area change, conformational change, and dynamics of catalytic site residues to predict ligand binding to ANG in this study. The obtained binding free energy (∆Gbind ) result showed the total average ∆Gbind as -113.480 ± 1.682 (Normtyr-1) > -53.038 ± 33.069 (B82) > -27.909 ± 16.438 (Ser-3) kJ/mole specify role of B82 in regulating ER stress signaling induced apoptosis through ANG ribonucleolytic activity inhibition, suitability of 43a2 force fields and methodology in ligand screening. It shows the crucial role of Leu115 and His13 residue involvement in total ∆Gbind contribution. Hence, based on the MD result, novel conformation of catalytic residues, and ∆Gbind , a promising combination candidate could be proposed for metastatic NSCLC therapy.

Fluorescent DNA analog: 2-aminotroponyl-pyrrolyl-2'-deoxyuridinyl DNA oligo enhance fluorescence in DNA-duplex as compared to 2-aminotroponyl-ethynyl-2'-deoxyuridinyl DNA oligo.

The nucleobase modified fluorescent DNA and RNA analogs are synthesized by the conjugation of aromatic scaffolds through linkers, comprising mostly ethyne/ethene or fused ring residues at the pyrimidine/purine ring. These scaffolds are mainly derived from the benzenoid aromatic molecules comprising electron withdrawing/donating characters. However, non-benzenoid aromatic scaffolds such as tropolone and related derivatives are constituents of various troponoid natural products. The conjugation of nucleobases with a troponyl moiety is underutilized for the synthesis of fluorescent DNA analogs. This report describes the synthesis and photophysical studies of 2-aminotroponyl conjugated deoxyuridine nucleosides and their DNA analogs. 2-Aminotropone derivatives are conjugated at the C-5 position of uridine through an ethynyl linker/pyrrolyl ring fusion and their DNA analogs. Their photophysical studies reveal that aminotroponyl deoxyuridine analogs exhibit solvent-dependent fluorescence properties. Moreover, pyrrolyl ring-fused aminotroponyl DNA oligonucleotides enhance the fluorescence after formation of duplexation with complementary sequences of native DNA oligonucleotides. Hence, these modified nucleosides and DNA are promising fluorescent analogs which could be useful to design the sequence-specific DNA probes.

Pyrimidine salvage in Toxoplasma gondii as a target for new treatment.

Toxoplasmosis is a common protozoan infection that can have severe outcomes in the immunocompromised and during pregnancy, but treatment options are limited. Recently, nucleotide metabolism has received much attention as a target for new antiprotozoal agents and here we focus on pyrimidine salvage by Toxoplasma gondii as a drug target. Whereas uptake of [3H]-cytidine and particularly [3H]-thymidine was at most marginal, [3H]-uracil and [3H]-uridine were readily taken up. Kinetic analysis of uridine uptake was consistent with a single transporter with a Km of 3.3 ± 0.8 µM, which was inhibited by uracil with high affinity (Ki = 1.15 ± 0.07 µM) but not by thymidine or 5-methyluridine, showing that the 5-Me group is incompatible with uptake by T. gondii. Conversely, [3H]-uracil transport displayed a Km of 2.05 ± 0.40 µM, not significantly different from the uracil Ki on uridine transport, and was inhibited by uridine with a Ki of 2.44 ± 0.59 µM, also not significantly different from the experimental uridine Km. The reciprocal, complete inhibition, displaying Hill slopes of approximately -1, strongly suggest that uridine and uracil share a single transporter with similarly high affinity for both, and we designate it uridine/uracil transporter 1 (TgUUT1). While TgUUT1 excludes 5-methyl substitutions, the smaller 5F substitution was tolerated, as 5F-uracil inhibited uptake of [3H]-uracil with a Ki of 6.80 ± 2.12 µM (P > 0.05 compared to uracil Km). Indeed, we found that 5F-Uridine, 5F-uracil and 5F,2'-deoxyuridine were all potent antimetabolites against T. gondii with EC50 values well below that of the current first line treatment, sulfadiazine. In vivo evaluation also showed that 5F-uracil and 5F,2'-deoxyuridine were similarly effective as sulfadiazine against acute toxoplasmosis. Our preliminary conclusion is that TgUUT1 mediates potential new anti-toxoplasmosis drugs with activity superior to the current treatment.