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After heart disease, cancer is the second-leading cause of death worldwide. The most effective method of cancer treatment is target therapy. One of the potential goals of therapy could be DT-diaphorase, which reduces quinone moiety to hydroquinone, and reactive oxygen species are create as a byproduct. The obtaining of hybrid compounds containing the quinone moiety and other bioactive compounds leads to new derivatives which can activate DT-diaphorase. The aim of this research was the synthesis and characterization of new hybrids of 5,8-quinolinedione with thymidine derivatives. The analysis of the physicochemical properties shows a strong relationship between the structure and properties of the tested compounds. The enzymatic assay shows that hybrids are good substrates of NQO1 protein. The analysis of the structure-activity relationship shows that the localization of nitrogen atoms influences the enzymatic conversion rate. The analysis was supplemented by a molecular docking study. Comparing the results of the enzymatic assay and the molecular docking presents a strong correlation between the enzymatic conversion rate and the scoring value.

Thymidine, as a crucial precursor of anti-AIDS drugs (e.g., zidovudine and stavudine), has wide application potential in the pharmaceutical industry. In this study, we introduced the thymidine biosynthesis pathway into the wild-type Escherichia coli MG1655 by systems metabolic engineering to improve the thymidine production in E . coli . Firstly, deoA , tdk , udp , rihA , rihB , and rihC were successively deleted to block the thymidine degradation pathway and salvage pathway in the wild-type E . coli MG1655. Then, the pyrimidine nucleoside operons from Bacillus subtilis F126 were introduced to enlarge the metabolic flux of the uridylic acid synthesis pathway. Finally, the expression of uridylate kinase, ribonucleoside diphosphate reductase, thymidine synthase, and 5'-nucleotidase in the thymidine biosynthesis pathway was optimized to enhance the metabolic flux from uridylic acid to thymidine. The engineered THY6-2 strain produced 11.10 g/L thymidine in a 5 L bioreactor with a yield of 0.04 g/g glucose and productivity of 0.23 g/(L·h). In this study, we constructed a strain that used glucose as the only carbon source for efficient production of thymidine and did not harbor plasmids, which provided a reference for the research on other pyrimidine nucleosides.

Background: DNA polymerase gamma (POLG)-related disorders are a group of rare neurodegenerative mitochondrial diseases caused by pathogenic variants in POLG , the gene encoding POLG. Patients may experience a range of signs and symptoms, including seizures, vision loss, myopathy, neuropathy, developmental impairment or regression, and liver failure. The diseases follow a progressive, degenerative course, with most affected individuals dying within 3 months-12 years of diagnosis. At present, there are no effective treatments for POLG-related disorders., Methods: In this study we report the interim 6-month data from a long term open-label, single arm phase 2 trial, in which we assessed the safety and efficacy of combination therapy with deoxycytidine and deoxythymidine (dC/dT) in children with POLG-related disorders. dC/dT was given enterally in powder form, dissolved in water. The primary outcome measures included Newcastle Mitochondrial Disease Scale (NMDS) score, serum growth differentiation factor 15 (GDF-15; a biomarker of mitochondrial dysfunction), electroencephalography (EEG), seizure diary, and blood and urine tests to assess end organ and mitochondrial function. Secondary outcome measures included recording of all adverse events to evaluate the safety of the intervention. The trial is registered with ClinicalTrials.gov, NCT04802707 (https://clinicaltrials.gov/ct2/show/NCT04802707). Data were collected from 14 October, 2021 to 13 December, 2023., Findings: We present 6-month interim data from the first ten people with POLG-related disorders enrolled in the trial, six with Alpers-Huttenlocher syndrome, two with ataxia-neuropathy spectrum, and two who do not fit into a classical POLG-related phenotype. During the 6 months of treatment, NMDS score improved from a mean of 27.3 at baseline to 20.7 at 6 months (estimated difference 6.0; 95% CI 2.5-∞). GDF-15 values remained stable or decreased in all patients; the mean decreased from 1031 pg/ml to 729 pg/ml (estimated difference 200; 95% CI 12-∞). 8/10 patients had abnormal baseline EEG; improvement in EEG was seen in 5 of these 8. There were no significant changes in other blood and urine testing. Regarding adverse events, two patients experienced diarrhea that spontaneously resolved., Interpretation: dC/dT is a promising treatment option for people with POLG-related disorders. Further research is needed to assess the long-term safety and efficacy in POLG-related disorders, as well as safety and efficacy in other mitochondrial DNA depletion disorders., Funding: This study was primarily funded by the Liam Foundation, with additional funding from the Savoy Foundation, Grand Défi Pierre Lavoie Foundation, and Fonds de Recherche du Québec - Santé., Competing Interests: KAM reports a consulting or advisory role with Jazz Pharmaceuticals and research grants paid to institution from Liam Foundation, Savoy Foundation, Fonds de Recherche du Québec—Santé, Grand Défi Pierre Lavoie Foundation, Pediatric Research Foundation, Epilepsy Canada, Ultragenyx and LivaNova. PJW reports a leadership role with the Garrod Association. HP, SB, CD, ACTC, TG, RE, DB report no competing interests., (© 2024 The Author(s).)

Three compounds with fluorescence quantum yields in the range of 10 - 5 to 10 - 4 and emission spectra covering the UV/Vis spectral range are suggested as new references for the determination of small fluorescence quantum yields. The compounds are thymidine (dT) in water, dibenzoylmethane (DBM) in ethanol, and malachite green chloride (MG) in water, representing the blue, green, and red regions of the spectrum, respectively. All compounds are easily handled, photostable, and commercially available. Furthermore, these compounds exhibit a mirror-image symmetry between their absorption and fluorescence spectra. This symmetry, along with closely aligned fluorescence excitation and absorption spectra, confirms that the observed emissions originate from the compounds themselves. The fluorescence quantum yields were determined via a relative approach as well as Strickler-Berg analysis in conjunction with time resolved fluorescence spectroscopy. Within the respective error margins, the two approaches yielded identical results., (© 2024. The Author(s).)

In this study, we designed the 4'- C -acetamidomethyl-2'- O -methoxyethyl (4'- C -ACM-2'- O -MOE) uridine and thymidine modifications, aiming to test them into small interfering RNAs. Thermal melting studies revealed that incorporating a single 4'- C -ACM-2'- O -MOE modification in the DNA duplex reduced thermal stability. In contrast, an increase in thermal stability was observed when the modification was introduced in DNA:RNA hybrid and in siRNAs. Thermal destabilization in DNA duplex was attributed to unfavorable entropy, which was mainly compensated by the enthalpy factor to some extent. A single 4'- C -ACM-2'- O -MOE thymidine modification at the penultimate position of the 3'-end of dT 20 oligonucleotides in the presence of 3'-specific exonucleases, snake venom phosphodiesterase (SVPD), demonstrated significant stability as compared to monomer modifications including 2'- O -Me, 2'- O -MOE, and 2'-F. In gene silencing studies, we found that the 4'- C -ACM-2'- O -MOE uridine or thymidine modifications at the 3'-overhang in the passenger strand in combination with two 2'-F modifications exhibited superior RNAi activity. The results suggest that the dual modification is well tolerated at the 3'-end of the passenger strand, which reflects better siRNA stability and silencing activity. Interestingly, 4'- C -ACM-2'- O -MOE-modified siRNAs showed considerable gene silencing even after 96 h posttransfection; it showed that our modification could induce prolonged gene silencing due to improved metabolic stability. Molecular modeling studies revealed that the introduction of the 4'- C -ACM-2'- O -MOE modification at the 3'-end of the siRNA guide strand helps to anchor the strand within the PAZ domain of the hAgo2 protein. The overall results indicate that the 4'- C -ACM-2'- O -MOE uridine and thymidine modifications are promising modifications to improve the stability, potency, and hAgo2 binding of siRNAs.

5-Fluorouracil is now routinely used in chemo- and radiotherapy. Incorporated within DNA, the molecule is bound to the sugar backbone, forming the 5-fluorouridine sub-unit investigated in the present work. For the clinical usage of the latter, no information exists on the mechanisms that control the radiosensitizing effect at the molecular level. As low energy (< 12 eV) electrons are abundantly produced along the radiation tracks during cancer treatment using beams of high energy particles, we study how these ballistic secondary electrons damage the sensitizing molecule. The salient result from our study shows that the N -glycosidic bonds are principally affected with a cross-section of approximately two orders of magnitude higher than the canonical thymidine, reflecting to some degree the surviving factor of radiation-treated carcinoma cells with and without 5-fluorouracil incorporation. This result may help in the comprehension of the radiosensitizing effect of the fluoro-substituted thymidine in DNA.

Sophisticated gene circuits built by synthetic biology can enable bacteria to sense their environment and respond predictably. Engineered biosensing bacteria outfitted with such circuits can potentially probe the human gut microbiome to prevent, diagnose, or treat disease. To provide robust biocontainment for engineered bacteria, we devised a Cas9-assisted auxotrophic biocontainment system combining thymidine auxotrophy, an Engineered Riboregulator (ER) for controlled gene expression, and a CRISPR Device (CD). The CD prevents the engineered bacteria from acquiring thyA via horizontal gene transfer, which would disrupt the biocontainment system, and inhibits the spread of genetic elements by killing bacteria harboring the gene cassette. This system tunably controlled gene expression in the human gut commensal bacterium Bacteroides thetaiotaomicron, prevented escape from thymidine auxotrophy, and blocked transgene dissemination. These capabilities were validated in vitro and in vivo. This biocontainment system exemplifies a powerful strategy for bringing genetically engineered microorganisms safely into biomedicine., (© 2024. The Author(s).)

To understand the regioselectivity observed in the allylation of pyrimidine nucleosides and to identify the factors directing the reaction, a theoretical study of the regioselective allylation was carried out. Several key points were considered such as: the structure of the deprotonated nucleobase in the presence of Na + ; the effect of the solvent on the dissociation and aggregation reactions of thymidine/Na + ion pair; and the likely allylation reaction mechanisms involved. The results showed that the regioselectivity observed experimentally can be attributed to a greater stability of a dimeric form coupled to an increase of the reaction barrier in THF due to larger Na + binding to the nucleobase., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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.

Importance: New antibacterial agents are urgently needed to counter increasingly resistant bacteria. One approach to this problem is library screening for new antibacterial agents. Library screening efforts can be improved by increasing the information content of the screening effort. In this study, we screened the National Cancer Institute diversity set V against methicillin-resistant Staphylococcus aureus (MRSA) with several enhancements. One of these is to screen the library before and after microsomal metabolism as means to identify potential active metabolites. A second enhancement is to screen the library in the absence and presence of sub-minimum inhibitory concentration levels of another antibiotic, such as cefoxitin in this study. This identified four agents with synergistic activity with cefoxitin out of 16 agents with good MRSA activity alone. Finally, active agents from this effort were counter-screened in the presence of thymidine, which quickly identified three folate/thymidine biosynthesis inhibitors, and also screened for bactericidal vs bacteriostatic activity., Competing Interests: The authors declare no conflict of interest.

DNA alkylation is caused by long-term exposure of cells to the environmental and endogenous alkylating agents, which can also lead to DNA mutations and therefore trigger some cancers. Since O4-methylthymidine (O4-meT), mismatched with guanine (G), is the most common but not easily repaired alkylated nucleoside, monitoring O4-meT can help to effectively reduce the occurrence of carcinogenesis. In this work, the modified G-analogues are selected as the fluorescence probe to monitor the existence of O4-meT according to its pairing characteristics. The photo-physical properties of considered G-analogues formed by ring expansion or addition of fluorophores were studied in detail. It is found that, compared with natural G, the absorption peaks of these fluorescence analogues are red-shifted (>55 nm) and the luminescence is enhanced by π-conjugation. Especially, the xG has a large Stokes shift (65 nm) with fluorescence insensitive to natural cytosine (C) and retains efficient emission after pairing, while it is sensitive to O4-meT and the quenching phenomenon occurs due to the excited state intermolecular charge transfer. Accordingly, the xG can be used as a fluorescent probe to identify the O4-meT in solution. In addition, the direct use of deoxyguanine fluorescent analogue for monitoring O4-meT was evaluated by the effects of ligating deoxyribose on absorption and fluorescence emission., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

UV-damaged DNA-binding protein (UV-DDB) is a heterodimeric protein, consisting of DDB1 and DDB2 subunits, that works to recognize DNA lesions induced by UV damage during global genome nucleotide excision repair (GG-NER). Our laboratory previously discovered a non-canonical role for UV-DDB in the processing of 8-oxoG, by stimulating 8-oxoG glycosylase, OGG1, activity 3-fold, MUTYH activity 4-5-fold, and APE1 (apurinic/apyrimidinic endonuclease 1) activity 8-fold. 5-hydroxymethyl-deoxyuridine (5-hmdU) is an important oxidation product of thymidine which is removed by single-strand selective monofunctional DNA glycosylase (SMUG1). Biochemical experiments with purified proteins indicated that UV-DDB stimulates the excision activity of SMUG1 on several substrates by 4-5-fold. Electrophoretic mobility shift assays indicated that UV-DDB displaced SMUG1 from abasic site products. Single-molecule analysis revealed that UV-DDB decreases the half-life of SMUG1 on DNA by ∼8-fold. Immunofluorescence experiments demonstrated that cellular treatment with 5-hmdU (5 μM for 15 min), which is incorporated into DNA during replication, produces discrete foci of DDB2-mCherry, which co-localize with SMUG1-GFP. Proximity ligation assays supported a transient interaction between SMUG1 and DDB2 in cells. Poly(ADP)-ribose accumulated after 5-hmdU treatment, which was abrogated with SMUG1 and DDB2 knockdown. These data support a novel role for UV-DDB in the processing of the oxidized base, 5-hmdU., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

With the intent to discover new antituberculosis (TB) compounds, coumarin-thymidine analogs were synthesized using second-order nucleophilic substitution reactions of bromomethyl coumarin with thymidine. The newly synthesized coumarin-thymidine conjugates (1a-l) were characterized using IR, NMR, GC-MS, and CHN elemental analysis. The novel conjugates were found to exhibit potent anti-TB activity against the Mycobacterium tuberculosis H 37 Rv strain, with minimum inhibitory concentrations (MIC) of the active compounds ranging between 0.012 and 0.482 µM. Compound 1k was established as the most active candidate with a MIC of 0.012 µM. The toxicity study on HEK cells confirmed the nontoxic nature of compounds 1e, 1h, 1i, 1j, and 1k. Also, the most active compounds (1k, 1j, and 1e) were stable in the pH range from 2.5 to 10, indicating compatibility with the biophysical environment. Based on the pK a studies, compounds 1k, 1j, and 1e are capable of crossing lipid-membrane barriers and acting on target cells. Molecular docking studies on the M. tuberculosis β-oxidation trifunctional enzyme (PDB ID: 7O4V) were conducted to investigate the mechanisms of anti-TB activity. All compounds showed excellent hydrogen binding interactions and exceptional docking scores against M. tuberculosis, which was in accordance with the results. Compounds 1a-l possessed excellent affinity to proteins, with binding energies ranging from -7.4 to -8.7 kcal/mol., (© 2023 Deutsche Pharmazeutische Gesellschaft.)

The primary method for probing DNA replication dynamics is DNA fiber analysis, which utilizes thymidine analog incorporation into nascent DNA, followed by immunofluorescent microscopy of DNA fibers. Besides being time-consuming and prone to experimenter bias, it is not suitable for studying DNA replication dynamics in mitochondria or bacteria, nor is it adaptable for higher-throughput analysis. Here, we present mass spectrometry-based analysis of nascent DNA (MS-BAND) as a rapid, unbiased, quantitative alternative to DNA fiber analysis. In this method, incorporation of thymidine analogs is quantified from DNA using triple quadrupole tandem mass spectrometry. MS-BAND accurately detects DNA replication alterations in both the nucleus and mitochondria of human cells, as well as bacteria. The high-throughput capability of MS-BAND captured replication alterations in an E. coli DNA damage-inducing gene library. Therefore, MS-BAND may serve as an alternative to the DNA fiber technique, with potential for high-throughput analysis of replication dynamics in diverse model systems., (© 2023 Ashour et al.)

Telomere length in humans is associated with lifespan and severe diseases, yet the genetic determinants of telomere length remain incompletely defined. Here we performed genome-wide CRISPR-Cas9 functional telomere length screening and identified thymidine (dT) nucleotide metabolism as a limiting factor in human telomere maintenance. Targeted genetic disruption using CRISPR-Cas9 revealed multiple telomere length control points across the thymidine nucleotide metabolism pathway: decreasing dT nucleotide salvage via deletion of the gene encoding nuclear thymidine kinase (TK1) or de novo production by knockout of the thymidylate synthase gene (TYMS) decreased telomere length, whereas inactivation of the deoxynucleoside triphosphohydrolase-encoding gene SAMHD1 lengthened telomeres. Remarkably, supplementation with dT alone drove robust telomere elongation by telomerase in cells, and thymidine triphosphate stimulated telomerase activity in a substrate-independent manner in vitro. In induced pluripotent stem cells derived from patients with genetic telomere biology disorders, dT supplementation or inhibition of SAMHD1 promoted telomere restoration. Our results demonstrate a critical role of thymidine metabolism in controlling human telomerase and telomere length, which may be therapeutically actionable in patients with fatal degenerative diseases., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Plasma thymidine levels in rodents are higher than in other mammals including humans, possibly due to a different pattern and lower level of thymidine phosphorylase expression. Here, we generated a novel knock-in (KI) mouse line with high systemic expression of human thymidine phosphorylase to investigate this difference in nucleotide metabolism in rodents. The KI mice showed growth retardation around weaning and died by 4 weeks of age with a decrease in plasma thymidine level compared with the litter-control WT mice. These phenotypes were completely or partially rescued by administration of the thymidine phosphorylase inhibitor 5-chloro-6-(2-iminopyrrolidin-1-yl) methyl-2,4(1H,3H)-pyrimidinedione hydrochloride or thymidine, respectively. Interestingly, when thymidine phosphorylase inhibitor administration was discontinued in adult animals, KI mice showed deteriorated grip strength and locomotor activity, decreased bodyweight, and subsequent hind-limb paralysis. Upon histological analyses, we observed axonal degeneration in the spinal cord, muscular atrophy with morphologically abnormal mitochondria in quadriceps, retinal degeneration, and abnormality in the exocrine pancreas. Moreover, we detected mitochondrial DNA depletion in multiple tissues of KI mice. These results indicate that the KI mouse represents a new animal model for mitochondrial diseases and should be applicable for the study of differences in nucleotide metabolism between humans and mice., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Heavy metal pollution has become a major problem in environmental pollution. Ion imprinted polymers with specific identification and wide practicality have gradually become an important tool for wastewater treatment. In this work, ion-imprinted polymer-grafted modified nanocellulose was designed as an adsorbent for the serious hazard of Pb(II) and Hg(II) in wastewater. This work used medical cotton wool as raw material to prepare a nanocellulose suspension by acid-catalyzed hydrolysis. The high reactivity of carbonyl diimidazole (CDI) was utilized to react with acrylic acid (AA) to generate reactive intermediates, which then reacted with nanocellulose to form activated nanocellulose (AA-CDI-NC). Crown ether was used as functional monomers to synthesize Pb(II) ion-imprinted polymers and grafted onto the AA-CDI-NC surface (Pb(II)-MIP-NC). Meanwhile, Hg(II) ion-imprinted polymer was synthesized and grafted onto the AA-CDI-NC surface (Hg(II)-MIP-NC) using thymine as a functional monomer. The experimental results showed that Pb(II)-MIP-NC and Hg(II)-MIP-NC could effectively adsorb Pb(II) and Hg(II), respectively. Their adsorption behaviors for Pb(II) and Hg(II) were consistent with the secondary kinetic model and Langmuir adsorption isotherm model. The adsorption capacities of Pb (II)-MIP-NC and Hg (II)-MIP-NC for Pb (II) and Hg (II) were 27.55 mg/g and 161.31, respectively., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Tropolone is a non-benzenoid aromatic scaffold with unique photophysical and metal-chelating properties. Recently, it has been conjugated with DNA, and the photophysical properties of this conjugate have been explored. Tropolonyl-deoxyuridine (tr-dU) is a synthetic fluorescent DNA nucleoside analogue that exhibits pH-dependent emissions. However, its solvent-dependent fluorescence properties are unexplored owing to its poor solubility in most organic solvents. It would be interesting to incorporate it into DNA primer enzymatically. This report describes the solvent-dependent fluorescence properties of the silyl-derivative, and enzymatic incorporation of its triphosphate analogue. For practical use, its cell-internalization and cytotoxicity are also explored. tr-dU nucleoside was found to be a potential analogue to design DNA probes and can be explored for various therapeutic applications in the future., (© 2022 Wiley-VCH GmbH.)

Due to the rapid spread of the monkeypox virus and rise in the number of cases, there is an urgent need for the development of effective drugs against the infection. Serine/threonine protein kinase (Ser/Thr kinase) and Thymidine Kinase (TK) plays an imperative role in the replication and virulence of monkeypox virus and thus is deliberated as an attractive target in anti-viral drug development. In the present study, the 3D structure of monkeypox virus Ser/Thr kinase and TK was generated via molecular modeling techniques and performed their thorough structural analysis. We have screened potent anti-viral phytochemicals from the literature to inhibit Ser/Thr kinase and TK. As part of the initial screening, the physicochemical properties of the compounds were examined. Following this, a structure-based molecular docking technique was used to select compounds based on their binding affinity towards Ser/Thr kinase and TK. In order to find more potent hits against Ser/Thr kinase and TK, further examinations of ADMET properties, PAINS patterns and blood-brain barrier permeability were conducted. As a result, thalimonine and galanthamine were identified from the screening process bearing appreciable binding affinity towards Ser/Thr kinase and TK respectively, which showed a worthy set of drug-like properties. In the end, molecular dynamics simulations were performed for 100 ns, which showed decent stability of both protein-ligand complex throughout the trajectory. Due to the possibility that both monkeypox virus target proteins may be inhibited by thalimonine and galanthamine, our study highlights the need to investigate in vivo effects of thalimonine and galanthamine.Communicated by Ramaswamy H. Sarma.

Clinical variability and substantial overlap between mitochondrial disorders and other genetic disorders and inborn errors make the clinical and metabolic diagnosis of mitochondrial disorders quite challenging. Evaluating specific laboratory markers is essential in the diagnostic process, but mitochondrial disease can be present in the absence of any abnormal metabolic markers. In this chapter, we share the current consensus guidelines for metabolic investigations, including investigations in blood, urine, and the cerebral spinal fluid and discuss different diagnostic approaches. As personal experience might significantly vary and there are different recommendations published as diagnostic guidelines, the Mitochondrial Medicine Society developed a consensus approach based on literature review for metabolic diagnostics in a suspected mitochondrial disease. According to the guidelines, the work-up should include the assessment of complete blood count, creatine phosphokinase, transaminases, albumin, postprandial lactate and pyruvate (lactate/pyruvate ratio when the lactate level is elevated), uric acid, thymidine, amino acids, acylcarnitines in blood, and urinary organic acids (especially screening for 3-methylglutaconic acid). Urine amino acid analysis is recommended in mitochondrial tubulopathies. CSF metabolite analysis (lactate, pyruvate, amino acids, and 5-methyltetrahydrofolate) should be included in the presence of central nervous system disease. We also suggest a diagnostic strategy based on the mitochondrial disease criteria (MDC) scoring system in mitochondrial disease diagnostics; evaluating muscle-, neurologic-, and multisystem involvement, and the presence of metabolic markers and abnormal imaging. The consensus guideline encourages a primary genetic approach in diagnostics and only suggests a more invasive diagnostic approach with tissue biopsies (histology, OXPHOS measurements, etc.) after nonconclusive genetic testing., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Nucleoside analogs are an important, well-established class of clinically useful medicinal agents that exhibit potent antimicrobial activity. Thus, we designed to explore the synthesis and spectral characterization of 5'- O- (myristoyl)thymidine esters (2-6) for in vitro antimicrobial, molecular docking, molecular dynamics, SAR, and POM analyses. An unimolar myristoylation of thymidine under controlled conditions furnished the 5'- O- (myristoyl)thymidine and it was further converted into four 3'- O -(acyl)-5'- O -(myristoyl)thymidine analogs. The chemical structures of the synthesized analogs were ascertained by analyzing their physicochemical, elemental, and spectroscopic data. In vitro antimicrobial tests along with PASS, prediction indicated expectant antibacterial functionality of these thymidine esters compared to the antifungal activities. In support of this observation, their molecular docking studies have been performed against lanosterol 14α-demethylase (CYP51A1) and Aspergillus flavus (1R51) and significant binding affinities and non-bonding interactions were observed. The stability of the protein-ligand complexes was monitored by a 100 ns MD simulation and found the stable conformation and binding mode in a stimulating environment of thymidine esters. Pharmacokinetic predictions were studied to assess their ADMET properties and showed promising results in silico . SAR investigation indicated that acyl chains, lauroyl (C-12) and myristoyl (C-14), combined with deoxyribose, were most effective against the tested bacterial and fungal pathogens. The POM analyses provide the structural features responsible for their combined antibacterial/antifungal activity and provide guidelines for further modifications, with the aim of improving each activity and selectivity of designed drugs targeting potentially drug-resistant microorganisms. It also opens avenues for the development of newer antimicrobial agents targeting bacterial and fungal pathogens.

Galli Gigerii Endothelium Corneum(GGEC), the dried gizzard membrane of Gallus gallus domesticus is a Chinese medicinal material commonly used for digestion. However, due to the particularity of texture and composition, its active ingre-dients have not been clarified so far, and there is also a lack of quality evaluation indicators. In this study, UPLC-Q-TOF-MS was used to analyze the chemical components from the water extract of GGEC, and ten nucleosides were identified for the first time. HPLC fingerprints of the water extracts of GGEC were established and the content of seven nucleosides was determined. The fingerprint similarities of 40 batches of GGEC samples ranged from 0.765 to 0.959, indicating that there were great differences among the GGEC products processed with different methods. In addition, SPSS 22.0 and SIMCA 14.1 were used for hierarchical cluster analysis(HCA) and principal component analysis(PCA) on the 19 common peaks of the HPLC fingerprints of GGEC, and the 40 batches of samples were divided into three categories: raw GGEC, fried GGEC and vinegar-processed GGEC. Eight differential components in GGEC were marked by orthogonal partial least squares discrimination analysis(OPLS-DA), two of which were adenine and thymine. The results of content determination showed that the total content of the seven nucleosides in raw GGEC, fried GGEC and vinegar-processed GGEC were 182.5-416.8, 205.3-368.7, and 194.2-283.0 μg·g~(-1), respectively. There were significant differences in the content of hypoxanthine, thymine and thymidine among the GGEC products processed with different methods(P&lt;0.05), which were graded in the order of fried GGEC&gt;vinegar-processed GGEC&gt;raw GGEC. This suggested that the content of hypoxanthine, thymine and thymidine tended to increase during the frying process, and the variation range might be related to the degree of heat exposure. The established methods in this study were simple and reproducible, and could be used for qualitative and quantitative analysis of GGEC and its processed pro-ducts. This study also provided reference for the establishment of quality standards of GGEC with chemical components as control index.

N4-acetylcytidine (ac4C) is an mRNA modification catalyzed by the enzyme N-acetyltransferase 10 (NAT10), with position-dependent effects on mRNA translation. This protocol details a procedure to map ac4C at base resolution using NaBH 4 -induced reduction of ac4C and conversion to thymidine followed by sequencing (RedaC:T-seq). Total RNA is ribodepleted and then treated with NaBH 4 to reduce ac4C to tetrahydro-ac4C, which specifically alters base pairing during cDNA synthesis, allowing the detection of ac4C at positions called as thymidine following Illumina sequencing. For complete details on the use and execution of this protocol, please refer to Arango et al. (2022). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Molecular docking of N-1,4-disubstituted-1,2,3-triazole tethered carboxymethylated thymidine and uridine with ribonuclease A, indicated their possible binding with the P 1 , B 1 and P 2 subsites with varied efficiencies. This theoretical study in combination of our earlier experimental observations was used as the guiding principles for designing a range of 1,4-disubstituted 1, 2, 3- triazole tethered carboxymethylated pyrimidine nucleosides. Triazoles are biologically important molecules and at the same time easily accessible through less complicated synthetic routes as reported about two decades back in the context of "click" reactions. Regioselective propargylation of the nucleosides under controlled conditions followed by the use of CuAAC strategy afforded mono-, bis-, tris- and tetratriazolyl pyrimidine nucleosides. Although the characteristics of nucleosides were lost in these densely functionalized polyheterocycles, the catalytic efficiency of ribonuclease A was significantly reduced by these molecules which were investigated experimentally and by docking studies. Triazoles as linkers helped one or more acidic groups to reach the P 1 subsite of ribonuclease A. Enzyme kinetics showed that the efficiency of inhibition reached the highest point with an optimum number of functional groups and were not linearly dependent on the number of triazole tethered carboxymethyl groups. The location of the triazole ring in the molecule affected the efficiency and nature of inhibition which were the result of the overall structure of the modified nucleosides. Thus, the tris-triazolylated thymidine derivative (T-3', 5', N-tris-CH 2 TzCH 2 COOH) as opposed to tetra-triazolylated uridine (U-2', 3', 5', N-tetrakis-CH 2 TzCH 2 COOH) emerged as the best inhibitor with an inhibition constant value of 2.3 ± 0.05 µM., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Due to the dynamic conformations of G-quadruplex structures (G4), determining the guanines that form G4 in a guanine-rich sequence is elusive. Here, we report a method for identifying deoxyguanines (dGs) forming antiparallel G4 by optical spectroscopy. The method, referred to as dG-to-deoxythymidine (dT) scanning, compares the spectra between a wild type and a single nucleobase dG-to-dT mutant at all dG positions. The most strongly involved dGs to form antiparallel G4 in the two model sequences were estimated using dG-to-dT scanning by circular dichroism (CD) and UV-Vis melting curve. This simple and robust method will facilitate understanding de novo antiparallel G4., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Among the scarce validated drug targets against Chagas disease (CD), caused by Trypanosoma cruzi , the parasite's nucleoside salvage system has recently attracted considerable attention. Although the trypanocidal activity of tubercidin (7-deazapurine) has long been known, the identification of a class of 7-substituted tubercidin analogs with potent in vitro and in vivo activity and much-enhanced selectivity has made nucleoside analogs among the most promising lead compounds against CD. Here, we investigate the recently identified TcrNT2 nucleoside transporter and its potential role in antimetabolite chemotherapy. TcrNT2, expressed in a Leishmania mexicana cell line lacking the NT1 nucleoside transporter locus, displayed very high selectivity and affinity for thymidine with a K m of 0.26 ± 0.05 µM. The selectivity was explained by interactions of 2-oxo, 4-oxo, 5-Me, 3'-hydroxy and 5'-hydroxy with the transporter binding pocket, whereas a hydroxy group at the 2' position was deleterious to binding. This made 5-halogenated 2'-deoxyuridine analogues good substrates but 5-F-2'-deoxyuridine displayed disappointing activity against T. cruzi trypomastigotes. By comparing the EC 50 values of tubercidin and its 7-substituted analogues against L. mexicana Cas9, Cas9 ΔNT1 and Cas9 ΔNT1+TcrNT2 it was shown that TcrNT2 can take up tubercidin and, at a minimum, a subset of the analogs.

Trace analysis of mercury ions (Hg 2+ ) is of great significance to human health and environmental protection. In this work, a novel photoelectrochemical (PEC) biosensor was developed for the ultrasensitive detection of Hg 2+ based on the MOFs-like composite/CdS quantum dots (QDs) as photoactive substrate materials. The MOFs-like composite in situ formed by hydrophobically modified alginate (HMA) with europium ion (Eu 3+ ) not only offered a friendly platform for bioconjugation but also resulted in enhancing sensor photocurrent response. Furthermore, the immobilized thymidine-rich probe DNA on the MOFs-like composite surface was bent to produce a T-Hg 2+ -T structure in the presence of Hg 2+ , resulting in enlarged steric hindrance on the electrode surface and decreased the proposed biosensor PEC response. This proposed photoelectrochemical sensing system displayed selective detection of Hg 2+ with a linear range from 0.1 pM to 1.0 μM with a detection limit of 0.067 pM. The utilization of semiconductor quantum dots as light-harvesting components and the MOFs-like composite as sensitizers broadens the possible design ideas for photoelectrochemical sensing systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The design of reversible-covalent molecules to selectively target the ε-amino functionality of lysine residues in enzymes or proteins is a highly desirable goal. Herein, we describe synthetic methodology used to prepare a series of 5'-thymidine-linked formylphenylboronic acids as probes to interrogate sugar nucleotide processing enzymes that recognize thymidine. The first synthetic strategy mitigated the need for protecting group manipulations of thymidine by capitalizing upon the straightforward preparation, isolation, and reactivity of 5'-azidothymidine. An alkyne cycloaddition partner was installed through either a propargyl or ethynyl phenyl ketone derived boronic acid. The second strategy directly linked formylphenylboronic acids to 5-thymidine through an ether linkage installed using Mitsunobu conditions with 3'- O ,3-dibenzoylthymidine. Iminoboronate formation was observed with a selected probe.

Objective: Endometrial carcinoma (EC) is a common malignant tumour in women. Berberin (BBR) is an alkaloid with anti-tumour activity, and circular RNA (circRNAs) has been extensively studied in cancers. However, whether BBR regulates the development of EC by regulating circular RNA zinc finger protein 608 (ZNF608) is unknown., Methods: Different concentrations of BBR were used to treat endometrial cancer cells. A quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to assess the expression of circ&#95;ZNF608, microRNA-377-3p (miR-377-3p) and cyclooxygenase 2 (COX2). The expression of COX2 protein was detected by western blot. The effect of circ&#95;ZNF608 in BBR-treated EC cells was verified by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) assay, colony formation assay, transwell, and flow cytometry. The effect of BBR and circ&#95;ZNF608 on tumour growth was evaluated by xenograft tumour model in vivo ., Results: Berberine can inhibit the proliferation and metastasis of EC cells and promote apoptosis, which is related to the concentration. Circ&#95;ZNF608 and COX2 were abnormally increased, while the levels of miR-377-3p were reversed in EC tissues and cells. Overexpression of circ&#95;ZNF608 can restore the inhibitory effect of BBR on EC cells. In addition, circ&#95;ZNF608 restored the inhibitory effect of BBR on EC cells by inhibiting the expression of miR-377-3p. Similarly, MiR-377-3p/COX2 can regulate the tumour progression of EC under BBR. Finally, BBR can inhibit the growth of endometrial carcinoma in vivo ., Conclusion: BBR was found to inhibit EC via the circ&#95;ZNF608/miR-377-3p/COX2 axis, which is helpful in endometrial carcinoma.

Background: Circular RNAs (circRNAs) have been reported to function as vital regulators in cancers, including triple-negative breast cancer (TNBC). This study aimed to explore the role of circ&#95;0062558 in TNBC., Methods: The real-time quantitative polymerase chain reaction (RT-qPCR) was conducted to quantify the expressions of circ&#95;0062558, microRNA-876-3p (miR-876-3p), and solute carrier family 1 (neutral amino acid transporter), member 5 (SLC1A5) in TNBC tissues and cells. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide (MTT), thymidine analog 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, wound healing, and Transwell assays were employed for cell phenotype analyses. Protein expression was tested by western blot analysis. Dual-luciferase reporter was used to confirm the association among circ&#95;0062558, miR-876-3p, and SLC1A5 in TNBC. Xenograft experiments were performed to elucidate the function of circ&#95;0062558 in vivo., Results: TNBC tissues and cells showed the higher level of circ&#95;0062558 when compared with control samples. Downregulation of circ&#95;0062558 inhibited proliferation, migration, invasion, and glutamine metabolism, while enhanced apoptosis of TNBC cells, and silencing of circ&#95;0062558 also inhibited the growth of tumor in vivo. MiR-876-3p was confirmed as a target of circ&#95;0062558, and circ&#95;0062558 knockdown repressed TNBC cell malignant behaviors by increasing miR-876-3p. Furthermore, miR-876-3p inhibited malignant behaviors of TNBC cells by down-regulating SLC1A5, a newly identified target of miR-876-3p., Conclusion: Circ&#95;0062558 promoted TNBC progression by enhancing proliferation, survival, migration, invasion, and glutamine metabolism via miR-876-3p/SLC1A5 axis, which was helpful for understanding the carcinogenic roles of circ&#95;0062558., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objective: We previously reported that alterations of the tumor microenvironment (TME) by programmed death receptor-1 (PD1) blockade affected tumor glucose metabolism and tumor 2-deoxy-2-[ 18 F]fluoro-D-glucose ([ 18 F]FDG) uptake. In cancer cells, high glycolysis allows cells to sustain rapid proliferation since glycolysis is closely related to the proliferation of cancer cells. Therefore, imaging of cellular proliferation may provide more detail of TME alterations. In this study, we investigated how TME alterations by PD1 blockade affects the uptake of 3'-deoxy-3'-[ 18 F]fluorothymidine ([ 18 F]FLT), which is a 18 F-radiolabeled thymidine derivative and is taken up by proliferating cells., Methods: Mice inoculated with murine colon carcinoma CT26 cells were intraperitoneally administered an anti-PD1 antibody on Day 0, when the tumor volume exceeded 50 mm 3 , and Day 5. [ 18 F]FLT-PET imaging was performed pre-treatment (Day 0) and post treatment (Day 7). Tumor infiltrating lymphocytes (TILs) were identified by flow cytometry. [ 18 F]FLT accumulation and localization in tumor tissue was evaluated by autoradiography and immunohistochemistry. The cell-cycle distribution of tumors and CT26 cells exposed to cytokines (interleukin-2, interferon [INF]-γ, and tumor necrosis factor [TNF]-α) was analyzed by flow cytometry., Results: PD1 blockade increased CD8 + and CD4 + T cells in tumor tissue and significantly suppressed tumor proliferation; however, tumor [ 18 F]FLT uptake remained unchanged. Autoradiography and immunohistochemistry showed that [ 18 F]FLT was mainly taken up by cancer cells, but not TILs. Flow cytometric analysis demonstrated that the population of cells in G2/M phase increased after PD1 blockade. Moreover, INF-γ and TNF-α significantly increased cells in G2/M phase in vitro., Conclusion: PD1 blockade-induced alteration of the TME increased CT26 tumor cells in the G2/M phase, which have high thymidine kinase 1 activity. Therefore, [ 18 F]FLT is taken up by tumor cells even if tumor proliferation is suppressed. This observation may be useful for evaluating the response to immunotherapy., (© 2022. The Author(s) under exclusive licence to The Japanese Society of Nuclear Medicine.)

CircRNAs have been found to be participated in the development of numerous cancers. Nevertheless, the role of circRNAs in the progression of nonsmall cell lung cancer (NSCLC) has not been fully made clear. The purpose of our study was to study and understand the mechanism of circ&#95;0007841 regulating the progression of NSCLC. NSCLC tissue samples and adjacent normal tissue samples used were obtained from 53 NSCLC patients. The expressions of circ&#95;0007841, miR-199a-5p and SphK2 in all samples were detected by the real-time quantitative PCR. Then luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay were used to analyze the relevance between circ&#95;0007841, miR-199a-5p and SphK2. Cell Counting Kit-8, colony-forming, thymidine analog 5-ethynyl-2'-deoxyuridine assays, and transwell assay detect the effects of these three biomolecules on NSCLC carcinogenesis by western blot. We evaluate the effect of circ&#95;0007841 on the growth of NSCLC by establishing the xenograft mice model. Experimental studies have shown that the higher expression of circ&#95;0007841 in NSCLC tissues, and circ&#95;0007841 strengthen cell viability, cell proliferation and cell adhesion. In addition, miR-199a-5p exerts an inhibitory effect in NSCLC cells by inhibiting SphK2. And Sphk2 regulates cell proliferation and adhesion. In addition, in-vivo silencing of circ&#95;0007841 was found to inhibit the growth of NSCLC tumors. This research demonstrated that circ&#95;0007841 had a positive influence in improving NSCLC development by targeting miR-199a-5p and upregulating oncogene SphK2., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Alzheimer's disease (AD) is a progressive disease with a long duration and complicated pathogenesis. Thymidine (Thy) and 2'-deoxyuridine (2'-De) are pyrimidines nucleotides that are associated with nervous system diseases. However, it remains unclear whether Thy and 2'-De exert neuroprotective effects in AD. Therefore, this study was conducted to explore the interventional effects and mechanisms of Thy and 2'-De on the Aβ 25-35 -induced brain injury. Donepezil (Do, 10 mg/kg/d), Thy (20 mg/kg/d), and 2'-De (20 mg/kg/d) were administered for 4 weeks after the injection of Aβ 25-35 peptides (200 μM, i.c.v.) to mice. UPLC-MS/MS method was performed to quantify Thy and 2'-De in the hippocampus of mice brain. The cognition ability, neuronal and mitochondria damage, and levels of Aβ 1-42 /Aβ 1-40 , p-Tau, Na + K + -ATPase, apoptosis, oxidative stress, immune cells, and Iba 1 + were measured in Aβ 25-35 -induced mice. The oxygen consumption (OCR) and extracellular acidification rate (ECAR) were measured using a seahorse analyzer in Aβ 25-35 -induced N9 cells. Moreover, 2-Deoxy-D-glucose (2-DG), a glycolysis inhibitor, was added to explore the mechanisms underlying the effects of Thy and 2'-De on Aβ 25-35 -induced N9 cells. The expression of Iba 1 + and levels of CD11b + and reactive oxygen species (ROS) were measured after treatment with Thy (5 μM) and 2'-De (10 μM) against 2-DG (5 mM) in Aβ 25-35 -induced N9 cells. The results suggested that Do, Thy, and 2'-De improved the cognition ability, attenuated the damage to hippocampus and mitochondria, downregulated the levels of Aβ 1-42 /Aβ 1-40 , p-Tau, Na + K + -ATPase, apoptosis, oxidative stress, and Iba 1 + , and regulated the immune response induced by Aβ 25-35 against the brain injury. Furthermore, Do, Thy, and 2'-De increased ATP production and inhibited glycolysis in Aβ 25-35 -induced N9 cells. Moreover, 2-DG enhanced the effects of drugs, reduced microglial activation, and attenuated oxidative stress to interfere with Aβ 25-35 -induced N9 cells. In conclusion, Thy and 2'-De reduced microglial activation and improved oxidative stress damage by modulating glycolytic metabolism on the Aβ 25-35 -induced brain injury., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

A chemical study of the CH 2 Cl 2 -MeOH (1:1) extract from the sponge Ernsta naturalis collected in Rodrigues (Mauritius) based on a molecular networking dereplication strategy highlighted one novel aminopyrimidone alkaloid compound, ernstine A ( 1 ), seven new aminoimidazole alkaloid compounds, phorbatopsins D-E ( 2 , 3 ), calcaridine C ( 4 ), naamines H-I ( 5 , 7 ), naamidines J-K ( 6 , 8 ), along with the known thymidine ( 9 ). Their structures were established by spectroscopic analysis (1D and 2D NMR spectra and HRESIMS data). To improve the investigation of this unstudied calcareous marine sponge, a metabolomic study by molecular networking was conducted. The isolated molecules are distributed in two clusters of interest. Naamine and naamidine derivatives are grouped together with ernstine in the first cluster of twenty-three molecules. Phorbatopsin derivatives and calcaridine C are grouped together in a cluster of twenty-one molecules. Interpretation of the MS/MS spectra of other compounds of these clusters with structural features close to the isolated ones allowed us to propose a structural hypothesis for 16 compounds, 5 known and 11 potentially new.

Thymidine Kinase 1 (TK1) plays an important role in DNA precursor synthesis and serum TK1 activity has been used as a biomarker for prognosis and therapy monitoring of different malignancies. AroCell has developed a dual monoclonal antibody ELISA for determination of TK1 protein in clinical samples. The purpose of the study is to validate the ELISA analytically in relation to the gold standard, [3H]-deoxythymidine (dThd) phosphorylation assay for TK1 activity using sera from patients with different malignancies. The colorimetric TK 210 ELISA was validated analytically by assessment of precision, linearity, interfering substances, and stability. For the clinical validation, serum samples from patients with hematological malignancies (n = 100), breast cancer (n = 56), prostate cancer (n = 70) and blood donors (n = 159) were analyzed using TK 210 ELISA and TK1 activity by [3H]-deoxythymidine (dThd) phosphorylation assay. The sandwich TK 210 ELISA was highly specific for TK1 protein having a detection limit of 0.12 ng/mL, with a functional sensitivity of 0.25 ng/mL. Within-run CVs ranged from 5.5% to 10% and between-run CVs ranged from 5% to 15%. The ratio of observed to expected dilutional parallelism of 5 serum samples was in the range of 80-120%. Samples exhibited stability through four freeze/thaw cycles and 5 days at 4°C. Further, the ROC curve analysis showed that TK 210 ELISA and [3H]-dThd phosphorylation assay had similar sensitivity (62% vs 59%) in hematological malignancies. However, in the case of breast and prostate cancer sera, TK 210 ELISA had higher sensitivity (59% and 44%) compared to [3H]-dThd phosphorylation assay (47% and 25%) at a specificity of 98%. These data demonstrate that the dual monoclonal antibody based AroCell TK 210 ELISA is a robust, accurate and precise tool for measuring TK1 protein in different malignancies that can improve the clinical applications of TK1 as a biomarker in cancer management., Competing Interests: Staffan Eriksson is an inventor of a TK1 patent licensed to DiaSorin Inc and is a shareholder in AroCell AB. The other authors have declared that no competing interest and this does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background/aim: Solar ultraviolet radiation (UVR) is a carcinogen and irradiation of the skin results in DNA damage. Cyclobutane pyrimidine dimers (CPDs), including thymidine dimers, are among the most frequent forms of DNA damage. When CPDs are formed, the nucleotide excision repair system is activated and CPDs are excreted in the urine. Here, we developed a mass spectrometry-based method to quantify thymidine dimers in the urine and tested the method on a small group of volunteers after whole-body UVR exposure., Patients and Methods: Years of research resulted in a method based on the "dilute-and-shoot" principle and ultra-performance liquid chromatography (UPLC) coupled to mass spectrometry. The whole body of each of eight healthy volunteers was exposed to 1.5-2.0 standard erythema doses (SEDs) of UVR for 3 consecutive days. Morning urine was collected on Day 1 (before irradiation) and on the following 7-9 days. Prior to analysis, sample preparation consisted of a simple dilution. A tandem quadrupole mass spectrometer coupled to UPLC was used for quantitative analysis in the multiple reaction monitoring mode., Results: After 3 consecutive days of 1.5-2 SEDs, the highest level of thymidine dimer excretion occurred on Day 6 (0.7 ng/ml urine). Compared with baseline, significantly more thymidine dimers were excreted every day until Day 8 (p<0.016). Our method quantifies thymidine dimers that are excreted as dimers (i.e., not degraded further) after nucleotide excision repair., Conclusion: This is the first published mass spectrometry-based method for quantifying thymidine dimers in the urine after whole-body UVR exposure., (Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

This current review is focused on the generation and settled patterns of mouse Purkinje cells (PCs) and deep cerebellar nuclei (DCN) neurons. By mean of progressively delayed comprehensive labeling procedure, I will show, with the technique of [ 3 H] thymidine autoradiography, the quantitative determination of PCs and DCN neurons production along the mediolateral and rostrocaudal axes of the cerebellum. The procedure consists of injecting groups of pregnant mice, on specific embryonic (E) days, with two doses of [ 3 H] thymidine in an overlapping series with 24 h delays between groups (E11-12, E12-13, E13-14, E14-15). The analysis of the autoradiograms revealed that PCs and DCN neurons are sequentially generated following precise neurogenetic timetables. PCs are born somewhat later than the DCN neurons. Both macroneurons are produced following two gradients. The first of these is mediolateral and the second is rostrocaudal. On the other hand, it will be also shown that PCs and DCN neurons were settled in the cerebellum following accurate neurogenetic gradients. These data have suggested that the chronological sequence of neuron production is a key factor in facilitating, in the adulthood, the cytoarchitecture of the cerebellum, and the establishment of patterns of orderly connections between PCs and DCN neurons., Competing Interests: Declaration of competing interest The author reports no declarations of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

In this study, a novel, rapid, and sensitive fluorescence sensing platform was developed for the detection of coralyne (COR) by the conjugation of coumarin 6H (C6H) fluorescent dye with oligonucleotide-modified silver nanoparticles [(dT) 32 -AgNPs]. In the presence of COR, a remarkable and rapid decrease in the fluorescence signal of the probe with a quenching efficiency of around 62% was observed. The quenching response of the system towards COR was possibly due to the displacement of thymidine-rich deoxyoligonucleotides by COR on the surface of AgNPs. The complementary experiments with an adenine-rich single strand as well as with two different secondary structures (i.e., duplex and triplex) revealed a favorable sequence specificity of the sensing platform. The influence of key parameters including the incubation time and temperature was evaluated and optimized to achieve the highest performance. The linear range of 10-183 nM with a correlation coefficient of R = 0.9982 and a limit of detection of 5.24 nM were obtained under the optimized conditions. The selectivity of the proposed probe towards COR was revealed by the evaluation of its response to other small molecules that have molecular structures similar to COR. Finally, the successful applicability of the system was shown with the obtained average recoveries in the range of 87.28-104.52% in human urine samples., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

Previous studies have suggested that circular RNAs (circRNAs) are engaged in the progression of papillary thyroid carcinoma (PTC). However, the mechanism of circ&#95;0002111 in PTC is still unclear. In this study, quantitative real-time PCR was carried out to measure the expressions of circ&#95;0002111, microRNAs (miRNAs) and high-mobility group box 1 (HMGB1). Immunohistochemistry assay and western blot were applied for the determination of protein levels. The assays of 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide and thymidine analog 5-ethynyl-2'-deoxyuridine were deployed to assess PTC cell viability and proliferation, respectively. Besides, the capacities of cell apoptosis, invasion and angiogenesis were determined by flow cytometry, transwell and tube formation assays, respectively. Moreover, the interaction between miR-363-3p and circ&#95;0002111 or HMGB1 was confirmed using a dual-luciferase reporter assay. Lastly, we established a xenograft model for the examination of the function of circ&#95;0002111 in vivo. It was found that the expression of circ&#95;0002111 was enhanced in PTC tissues and cells. Silencing circ&#95;0002111 apparently retarded the viability, proliferation, invasion and tube formation, as well as expedited the apoptosis of PTC cells. Besides, circ&#95;0002111 knockdown impeded the growth of the tumor in vivo. For mechanism analysis, circ&#95;0002111 adjusted the expression of HMGB1 by sponge adsorption of miR-363-3p. Moreover, miR-363-3p inhibitor regained the influence of cellular malignant phenotype caused by circ&#95;0002111 knockdown. Additionally, miR-363-3p overexpression impacted the cell functions by targeting HMGB1 in PTC. Thus, silencing circ&#95;0002111 constrained the progression of PTC by the miR-363-3p/HMGB1 axis, which perhaps provided a novel idea of the therapeutic in PTC., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Efficient protocols were developed for the synthesis of a new compounds - nucleoside 5'-α-iminophosphates using the Staudinger reaction. These substances are alpha-phosphate mimetic nucleotide in which an oxygen atom is replaced by a corresponding imino (=N-R)-group. Various 5'-iminomonophosphates of nucleosides were obtained. A chemical method for the synthesis of triphosphate derivatives based on the iminomonophosphates has been designed. Thymidine 5'-(1,3-dimethylimidazolidin-2-ylidene)-triphosphate (ppp(DMI)T) was synthesized, its hydrolytic stability and substrate properties in relation to some DNA polymerases was firstly studied. It was shown that ppp(DMI)T can serve as substrate for enzyme catalyzed template-independent DNA synthesis by human terminal deoxynucleotidyltransferase TdT., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In this study, the efficiency of RNA interference of small interfering RNAs (siRNAs) bearing 5'- O -methyl-2'-deoxythymidine (X) and 5'-amino-2', 5'-dideoxythymidine (Z) at the 5'-end of the sense strand and the antisense strand of siRNA was investigated in HeLa cells stably expressing enhanced green fluorescent protein. The results indicated that when one strand of siRNA was modified with X or Z and the other was unmodified, the X or Z modification was predominant in the process of strand selection and the unmodified strand was selected as a guide strand. When both strands are modified with X or Z, the modified antisense strand with X or Z will be selected as a guide strand with a certain probability. The resulting mature RNA-induced silencing complex exerted reduced, but still moderate silencing activity remained. These results suggest that the modification of the sense strand with X or Z eliminates the off-target effects caused by the sense strand without affecting the silencing efficiency of the siRNA.

Elaiophylin (Ela), a unique 16-membered symmetric macrodiolide antibiotic, displays broad biological activity. Two rare 2-deoxy-L-fucose moieties at the ends of Ela are critical for its activity. Previously, elaiophylin glycosyltransferase (ElaGT) was identified as the enzyme that is responsible for the symmetric glycosylation of Ela, acting as a potential enzymatic tool for enhancing the diversity and activity of Ela. However, a symmetric catalytic mechanism has never been reported for a glycosyltransferase (GT). To explore the catalytic mechanism, the structure of ElaGT was determined in four forms: the apo form and Ela-bound, thymidine diphosphate-bound and uridine diphosphate-bound forms. In the Ela-bound structure, two ElaGTs form a `face-to-face' C2-symmetric homodimer with a continuous acceptor-binding pocket, allowing a molecule of Ela to shuffle through. Interestingly, this dimer interface resembles that of the activator-dependent GT EryCIII with its activator EryCII. Sequence analysis also indicates that ElaGT belongs to the activator-dependent GT family, but no putative activator has been identified in the Ela gene cluster. It was then found that the ElaGT homodimer may utilize this `face-to-face' arrangement to stabilize the Ela-binding loops on the interface and to simultaneously allosterically regulate the catalytic center. Therefore, these structures present a novel self-activating model for symmetric sugar transfer in the GT family and a new potential regulation site for substrate specificity.

Hydrogen bonding between the DNA nucleobases and small organic molecules, such as melamine, is a new strategy for the design of novel DNA materials. Poly(thymidine) DNA and melamine self-assemble into a duplex structure containing two antiparallel DNA strands hydrogen bonded to central melamine units. In this Article, molecular dynamics simulations rationalize the observed antiparallel duplex structure. Alternative duplex and triplex structures with parallel and antiparallel strand orientations are shown to be unstable because of the increase in unfavorable interactions between the DNA backbones.

Antisense oligonucleotide (ASO) therapeutics target the pathogenic mRNA directly and modulate protein expression. Novel chemical modifications help to improve the action of ASOs with better thermal stability and resistance against nucleases. Oligodeoxynucleotides (ODNs) containing 4'-C-(aminoethyl)thymidine modifications exhibit efficient and stable hybridization with complementary DNA as well as RNA strands showing remarkably improved resistance against nucleolytic hydrolysis, which makes them promising candidates for antisense therapeutics. This article describes the synthesis of a novel nucleoside analog, 4'-C-[(N-methyl)aminoethyl]-thymidine (4'-MAE-T), 3, and previously reported 4'-C-aminoethyl-thymidine (4'-AE-T), 2, through a newly designed synthetic route to obtain a high overall yield. This has been established by changing the starting material from thymidine to diacetone-D-glucofuranose and synthesizing the known 4-C-hydroxyethyl pentofuranose. Conversion of the hydroxy group to an azide functional group through Mitsunobu azidation and performing acetolysis, provide the common intermediate 4-C-(2-azidoethyl)-ribofuranose. Subsequent coupling of the thymine nucleobase with the common intermediate under Vorbrüggen glycosylation conditions provides the corresponding modified nucleoside in high yield. It was subjected for conversion of the azide to an amine by Staudinger reaction and 2'-deoxygenation using Barton-McCombie conditions. Debenzylation with Lewis acid and mono-dimethoxytritylation of the 5'-OH afforded a fully protected 3'-OH intermediate for phosphitylation to give the corresponding phosphoramidites. In the case of 4'-MAE-T, benzyloxymethyl protection of the N 3 -position and methylation were carried out prior to debenzylation. These phosphoramidite monomers were suitable with conventional oligonucleotide synthesis, and imparted ameliorated nuclease resistance, and competent RNase H activity, suggesting its potential utilization in ASO drugs. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of 4-C-(2-azidoethyl)-ribofuranose (6) Basic Protocol 2: Synthesis of 4'-C-aminoethyl thymidine phosphoramidite (15) Basic Protocol 3: Synthesis of 4'-C-(N-methyl)aminoethyl thymidine phosphoramidite (20)., (© 2022 Wiley Periodicals LLC.)

Background: Several new generation CDK4/6 inhibitors have been developed and approved for breast cancer therapy in combination with endocrine therapeutics. Application of these inhibitors either alone or in combination in other solid tumors has been proposed, but no imaging biomarkers of response have been reported in non-breast cancer animal models. The purpose of this study was to evaluate 3'-[ 18 F]fluoro-3'-deoxythymidine ([ 18 F]FLT) Positron Emission Tomography (PET) as in vivo biomarker of response to palbociclib in a non-breast cancer model., Methods: Twenty-four NSG mice bearing patient derived xenografts (PDX) of a well-characterized bladder tumor were randomized into 4 treatment groups: vehicle (n = 6); palbociclib (n = 6); temozolomide (n = 6); and palbociclib plus temozolomide (n = 6) and treated with two cycles of therapy or vehicle. Tumor uptake of [ 18 F]FLT was determined by micro-PET/CT at baseline, 3 days, and 9 days post initiation of therapy. Following the second cycle of therapy, the mice were maintained until their tumors reached a size requiring humane termination., Results: [ 18 F]FLT uptake decreased significantly in the palbociclib and combination arms (p = 0.0423 and 0.0106 respectively at day 3 and 0.0012 and 0.0031 at day 9) with stable tumor volume. In the temozolomide arm [ 18 F]FLT uptake increased with day 9 uptake significantly different than baseline (p = 0.0418) and progressive tumor growth was observed during the treatment phase. All groups exhibited progressive disease after day 22, 10 days following cessation of therapy., Conclusion: Significant decreases in [ 18 F]FLT uptake as early as three days post initiation of therapy with palbociclib, alone or in combination with temozolomide, in this bladder cancer model correlates with an absence of tumor growth during therapy that persists until day 18 for the palbociclib group and day 22 for the combination group (6 days and 10 days) following cessation of therapy. These results support early modulation of [ 18 F]FLT as an in vivo biomarker predictive of palbociclib therapy response in a non-breast cancer model., (© 2022. The Author(s).)

The linear synthesis of 4'-C-aminoethoxy thymidine (AEoT) nucleoside phosphoramidite was accomplished using deoxythymidine as the starting material. This analog was incorporated into several oligonucleotides, the applicability of which as antisense oligonucleotides (ASOs) was then evaluated. The AEoT-modified DNA/RNA duplex exhibited improved thermal stability compared to unmodified and 4'-C-aminoethyl thymidine (4'-AET) modified heteroduplexes. The serum stability of AEoT-modified DNA was notably increased by several-folds compared to that of unmodified DNA. Furthermore, RNase H-dependent cleavage of the modified-DNA/RNA hybrids was found to be sustained. In addition, the modified antisense and unmodified oligonucleotides also displayed relatively comparable inhibition of the KRAS gene in human lung cancer cells. This study strengthens our understanding of the potential application of 4'-C-aminoethoxy-modified nucleotides as ASO therapeutics., (© 2022 Wiley-VHCA AG, Zurich, Switzerland.)

Antimicrobials can impact bacterial physiology and host immunity with negative treatment outcomes. Extensive exposure to antifolate antibiotics promotes thymidine-dependent Staphylococcus aureus small colony variants (TD-SCVs), commonly associated with worse clinical outcomes. We show that antibiotic-mediated disruption of thymidine synthesis promotes elevated levels of the bacterial second messenger cyclic di-AMP (c-di-AMP), consequently inducing host STING activation and inflammation. An initial antibiotic screen in Firmicutes revealed that c-di-AMP production was largely driven by antifolate antibiotics targeting dihydrofolate reductase (DHFR), which promotes folate regeneration required for thymidine biosynthesis. Additionally, TD-SCVs exhibited excessive c-di-AMP production and STING activation in a thymidine-dependent manner. Murine lung infection with TD-SCVs revealed STING-dependent elevation of proinflammatory cytokines, causing higher airway neutrophil infiltration and activation compared with normal-colony S. aureus and hemin-dependent SCVs. Collectively, our results suggest that thymidine metabolism disruption in Firmicutes leads to elevated c-di-AMP-mediated STING-dependent inflammation, with potential impacts on antibiotic usage and infection outcomes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Mycoplasma hominis is an opportunistic urogenital pathogen in vertebrates. It is a non-glycolytic species that produces energy via arginine degradation. Among genital mycoplasmas, M. hominis is the most commonly reported to play a role in systemic infections and can persist in the host for a long time. However, it is unclear how M. hominis proceeds under arginine limitation. The recent metabolic reconstruction of M. hominis has demonstrated its ability to catabolize deoxyribose phosphate to produce ATP. In this study, we cultivated M. hominis on two different energy sources (arginine and thymidine) and demonstrated the differences in growth rate, antibiotic sensitivity, and biofilm formation. Using label-free quantitative proteomics, we compared the proteome of M. hominis under these conditions. A total of 466 proteins were identified from M. hominis , representing approximately 85% of the predicted proteome, while the levels of 94 proteins changed significantly. As expected, we observed changes in the levels of metabolic enzymes. The energy source strongly affects the synthesis of enzymes related to RNA modifications and ribosome assembly. The translocation of lipoproteins and other membrane-associated proteins was also impaired. Our study, the first global characterization of the proteomic switching of M. hominis in arginine-deficiency media, illustrates energy source-dependent control of pathogenicity factors and can help to determine the mechanisms underlying the interaction between the growth rate and fitness of genome-reduced bacteria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Evsyutina, Semashko, Galyamina, Kovalchuk, Ziganshin, Ladygina, Fisunov and Pobeguts.)

Pharmaceutical grade 3'-deoxy-3'-[ 18 F]fluorothymidine [ 18 F]FLT was synthesized using 3-N-Boc-5'-O-dimethoxytrityl-3'-O-nosyl-thymidine (BOC-Nosyl) precursor, in the general purpose TRACERlab FX modules. Purification of [ 18 F]FLT, via solid phase extraction (SPE) after radiosynthesis, using a combination of different SPE cartridges, yielded satisfactory results, with radiochemical and chemical purity >99%. While the non-decay corrected radiochemical yield (RCY) with 20 mg (24 μmole) of BOC-Nosyl precursor was found to be 6.80 ± 0.16%, the decay corrected radiochemical yield (RCY) was 9.95 ± 0.24%. Residual acetone, acetonitrile, and ethanol levels were found to be 22.97 ± 0.76, 109.08 ± 0.93, and 7,666.45 ± 3.7 ppm, respectively. A simplified method for solid-phase purification of [ 18 F]FLT was developed, circumventing the need for HPLC purification. Biodistribution in C57BL/6 mice with B16F10 cell line-induced melanoma showed tumor to blood ratio of ~3.8 at 90 min. PET/CT imaging of normal rabbit injected with [ 18 F]FLT shows selective uptake in the bone marrow and small intestine. [ 18 F]FLT was found to be excreted through the kidneys and get collected in the urinary bladder, 120 min post injection. PET/CT imaging performed in rabbit model at 30, 60, 90, and 120 min post [ 18 F]FLT injections showed concordance with tissue distribution kinetics of mice tumor model., (© 2022 John Wiley & Sons, Ltd.)