Search

Histone modifications are catalyzed and recognized by specific proteins to regulate dynamic DNA metabolism processes. NSD2 is a histone H3 lysine 36 (H3K36)‐specific methyltransferase that is associated with both various transcription regulators and DNA repair factors. Specifically, it has been implicated in the repair of DNA double‐strand breaks (DSBs); however, the role of NSD2 during DSB repair remains enigmatic. Here, we show that NSD2 does not accumulate at DSB sites and that it is not further mobilized by DSB formation. Using three different DSB repair reporter systems, which contained the endonuclease site in the active thymidine kinase gene (TK) locus, we demonstrated separate dose‐dependent effects of NSD2 on homologous recombination (HR), canonical‐non‐homologous end joining (c‐NHEJ), and non‐canonical‐NHEJ (non‐c‐NHEJ). Endogenous NSD2 has a role in repressing non‐c‐NHEJ, without affecting DSB repair efficiency by HR or total NHEJ. Furthermore, overexpression of NSD2 promotes c‐NHEJ repair and suppresses HR repair. Therefore, we propose that NSD2 has functions in chromatin integrity at the active regions during DSB repair. [ABSTRACT FROM AUTHOR]

Dual bioorthogonal labeling enables the investigation and understanding of interactions in the biological environment that are not accessible by a single label. However, applying two bioorthogonal reactions in the same environment remains challenging due to cross‐reactivity. We developed a pair of differently modified 2′‐deoxynucleosides that solved this issue for dual and orthogonal labeling of DNA. Inverse‐electron demand Diels–Alder and photoclick reactions were combined to attach two different fluorogenic labels to genomic DNA in cells. Using a small synthetic library of 1‐ and 3‐methylcyclopropenyl‐modified 2′‐deoxynucleosides, two 2′‐deoxyuridines were identified to be the fastest‐reacting ones for each of the two bioorthogonal reactions. Their orthogonal reactivity could be evidenced in vitro. Primer extension experiments were performed with both 2′‐deoxyuridines investigating their replication properties as substitutes for thymidine and evaluating subsequent labeling reactions on the DNA level. Finally, dual, orthogonal and metabolic fluorescent labeling of genomic DNA was demonstrated in HeLa cells. An experimental procedure was developed combining intracellular transport and metabolic DNA incorporation of the two 2′‐deoxyuridines with the subsequent dual bioorthogonal labeling using a fluorogenic cyanine‐styryl tetrazine and a fluorogenic pyrene‐tetrazole. These results are fundamental for advanced metabolic labeling strategies for nucleic acids in the future, especially for live cell experiments. [ABSTRACT FROM AUTHOR]

This article explores the use of dual bioorthogonal and orthogonal metabolic labeling techniques to study interactions in biological environments. The researchers developed modified 2'-desoxynucleosides to overcome the challenge of cross-reactivity and successfully linked genomic DNA in cells with two different fluorogenic markers. The study demonstrates the potential for advanced metabolic labeling strategies for nucleic acids, particularly in living cells. The authors conducted experiments to investigate the reactivity and selectivity of certain nucleosides in two different reactions, IEDDA and photoclick, for potential use in DNA labeling. While successful DNA marking was achieved using the IEDDA reaction, attempts to mark DNA using the photoclick reaction were unsuccessful. The authors also tested the nucleosides for metabolic labeling of cellular DNA, but no successful labeling was observed. Overall, this study provides a foundation for future research in metabolic labeling strategies for nucleic acids, including experiments in living cells. [Extracted from the article]

Background: An accurate and easily accessible method for diagnosing malignancies in local veterinary clinics has not yet been established. Objectives: To investigate the usefulness of serum thymidine kinase 1 (TK1) protein and its autoantibody as tumor biomarkers in dogs. Animals: Serum samples from 1702 dogs were collected from local animal hospitals and referral animal medical centers in South Korea. Methods: TK1 protein OD value and TK1 autoantibody ratio (TK1 autoantibody OD/total IgG OD) in serum samples of dogs classified into healthy controls, group with nontumor disease, group with benign and group with malignant tumors were measured using lateral flow immunochromatographic assay methods. Results: TK1 autoantibody levels were significantly higher in malignant tumor group (median 0.71) than in healthy controls (median 0.34), group with nontumor disease (median 0.34), and group with benign tumor (median 0.32, Welch t test, P <.0001). They were also significantly different among dogs with carcinomas (median 0.77), hematopoietic tumors (median 0.71), and sarcomas (median 0.56) than in healthy controls (median 0.34, post hoc Games‐Howell test, P <.0001). In the receiver operating characteristic curve of TK1 protein, AUC was 0.633 (95% CI: 0.592‐0.675, P <.0001). The AUC of TK1 autoantibody ratio was 0.758 (95% CI: 0.723‐0.793, P <.0001). Conclusions and Clinical Importance: TK1 autoantibody is a potentially useful biomarker for differentiating between healthy and tumor‐bearing dogs, better than TK1 protein measurement. However, both were inadequate when used as single biomarkers for screening dogs to discover occult malignant tumors. [ABSTRACT FROM AUTHOR]

5‐Hydroxymethyl‐pyrimidine‐based nucleobases have attracted attention in the last years due to their important role in gene regulation. It prompted the development of efficient routes to the corresponding nucleoside building blocks for further incorporation into oligonucleotides. Several pathways have been employed in recent years to access properly protected 5‐hydroxymethyl‐pyrimidine based nucleosides, including hydroxymethylation of 2'‐deoxyuridine, radical bromination of thymidine followed by bromine substitution, or carbonylative coupling through Stille conditions starting from 5‐iodo‐2'‐deoxyuridine. In this study, we review the different approaches currently used to introduce the 5‐hydroxymethyl moiety, we reinvestigate some of them, and finally, we propose an alternative route based on the oxidation of the methyl of thymidine followed by its reduction that allows access to protected 5‐hydroxymethyl‐2'‐deoxyuridine in a simple way with good yields. Finally, we present some examples of application including the synthesis of base J and 5‐azidomethyl‐2'‐deoxyuridine. [ABSTRACT FROM AUTHOR]

Introduction: Hepatocellular carcinoma (HCC) is one of the most serious cancers. Circular RNA (circRNA) has been reported to regulate the progression of HCC. Herein, the role of circ_0102543 in HCC tumorigenesis was investigated. Materials: The expression levels of circ_0102543, microRNA‐942‐5p (miR‐942‐5p), and small glutamine rich tetratricopeptide repeat co‐chaperone beta (SGTB) were detected by quantitative real‐time PCR (qRT‐PCR). 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium Bromide (MTT) assay, thymidine analog 5‐ethynyl‐2'‐deoxyuridine (EDU) assay, transwell assay, and flow cytometry were conducted to explore the function of circ_0102543 in HCC cells and the regulatory mechanism among circ_0102543, miR‐942‐5p and SGTB in HCC cells. Western blot examined the related protein levels. Results: The expression of circ_0102543 and SGTB was decreased in HCC tissues, while the expression of miR‐942‐5p was increased. Circ_0102543 acted as a sponge for miR‐942‐5p, and SGTB was the target of miR‐942‐5p. Circ_0102543 up‐regulation hindered tumor growth in vivo. In vitro experiments showed that overexpression of circ_0102543 significantly repressed the malignant behaviors of HCC cells, while co‐transfection of miR‐942‐5p partially attenuated these effects mediated by circ_0102543. In addition, SGTB knockdown increased the proliferation, migration, and invasion of HCC cells inhibited by miR‐942‐5p inhibitor. Mechanically, circ_0102543 regulated SGTB expression in HCC cells by sponging miR‐942‐5p. Conclusion: Overexpression of circ_0102543 suppressed proliferation, migration, and invasion of HCC cells by regulating the miR‐942‐5p/SGTB axis, suggesting that circ_0102543/miR‐942‐5p/SGTB axis may be a potential therapeutic target for HCC. [ABSTRACT FROM AUTHOR]

Diffuse large B‐cell lymphoma (DLBCL) is frequently treated with chemotherapy incorporating cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP), which induces remission in 80% to 95% of cases. However, not all dogs derive meaningful benefit from CHOP, and prognostic factors for dogs with DLBCL are poorly defined. Serum thymidine kinase 1 (TK1) activity, a marker of tumour cell proliferation, has shown promising initial results as a prognostic biomarker in dogs with multicentric lymphomas. The purpose of this study was to determine if baseline serum TK1 activity is associated with clinical outcome in dogs with CHOP‐treated DLBCL. Baseline serum TK1 activity was measured in banked sera from 98 dogs with CHOP‐treated DLBCL using a commercially available ELISA kit. Data on other potential prognostic factors were abstracted retrospectively from electronic medical records. Multivariable statistical methods were used to identify associations between TK1 and other potential prognostic factors with progression‐free survival (PFS) and attainment of complete remission. TK1 activity at baseline was not associated with PFS (p =.299) or attainment of complete remission (p =.910) following CHOP chemotherapy. Of the other prognostic factors analysed, only purebred (vs. mixed breed) status (HR 8.81, 95% CI 1.68–46.30, p =.010), attainment of complete (vs. partial) remission (HR 0.09, 95% CI 0.02–0.49, p =.006), and baseline serum C‐reactive protein concentration (HR 1.19, 95% CI 1.07–1.32, p =.001) were independently associated with PFS. Based on these findings, baseline serum TK1 activity does not appear to be a useful prognostic biomarker in dogs with CHOP‐treated DLBCL. [ABSTRACT FROM AUTHOR]

Controlling the pairing strength of nucleobases in DNA through reactions with compounds found inside the cell is a formidable challenge. Here we report how a thiazolyl substituent turns a strongly pairing ethynylpyridone C‐nucleoside into a reactive residue in oligonucleotides. The thiazolyl‐bearing pyridone reacts with soft nucleophiles, such as glutathione, but not with hard nucleophiles like hydroxide or carbonate. The addition products pair much more weakly with adenine in a complementary strand than the starting material, and also change their fluorescence. This makes oligonucleotides containing the new deoxynucleoside interesting for controlled release. Due to its reactivity toward N, P, S, and Se‐nucleophiles, and the visual signal accompanying chemical conversion, the fluorescent nucleotide reported here may also have applications in chemical biology, sensing and diagnostics. [ABSTRACT FROM AUTHOR]

5‐Bromodeoxyuridine (BrdU), a thymidine analogue, is an interesting reagent that modulates various biological phenomena. BrdU, upon incorporation into DNA, causes destabilized nucleosome positioning which leads to changes in heterochromatin organization and gene expression in cells. We have previously shown that BrdU effectively induces cellular senescence, a phenomenon of irreversible growth arrest in mammalian cells. Identification of the mechanism of action of BrdU would provide a novel insight into the molecular mechanisms of cellular senescence. Here, we showed that a basic domain in the histone H2B N‐terminal tail, termed the HBR (histone H2B repression) domain, is involved in the action of BrdU. Notably, deletion of the HBR domain causes destabilized nucleosome positioning and derepression of gene expression, as does BrdU. We also showed that the genes up‐regulated by BrdU significantly overlapped with those by deletion of the HBR domain, the result of which suggested that BrdU and deletion of the HBR domain act in a similar way. Furthermore, we showed that decreased HBR domain function induced cellular senescence or facilitated the induction of cellular senescence. These findings indicated that the HBR domain is crucially involved in the action of BrdU, and also suggested that disordered nucleosome organization may be involved in the induction of cellular senescence. [ABSTRACT FROM AUTHOR]

A click chemistry‐based protocol has been utilized for the synthesis of a series of twelve novel tert‐butyldiphenylsilylated N1‐(coumarin‐4′′′‐yl)‐C4‐(2′,3′‐di‐deoxyuridin‐3′‐yl)‐oxymethyl‐1,2,3‐triazoles and N1‐(coumarin‐4′′′‐yl)‐C4‐(3′‐deoxythymin‐3′‐yl)‐oxymethyl‐1,2,3‐triazoles by utilising Cu(I)‐catalyzed azide‐alkyne cycloaddition reaction of 4‐azidocoumarins and 3′‐O‐(prop‐1‐yn‐3‐yl)‐5′‐O‐(tert‐butyldiphenylsilyl)‐2′,3′‐dideoxyuridin or 3′‐O‐(prop‐1‐yn‐3‐yl)‐5′‐O‐(tert‐butyldiphenylsilyl)‐3′‐deoxythymidin in 70–82 % and 68–82 % yields, respectively. The deprotection of resulted silylated uridine and thymidine conjugate with TBAF afforded title compounds in 81–91 % yield. The photophysical studies have been carried out on the synthesized coumarin‐triazolylmethyl nucleoside conjugates which revealed a very high Stokes shift value ranging from 82–215 nm. [ABSTRACT FROM AUTHOR]

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE; OMIM 603041) is a rare inherited metabolic disorder mostly caused by mutations in TYMP gene encoding thymidine phosphorylase (TP) protein that affects the mitochondrial nucleotide metabolism. TP, functionally active as a homodimer, is involved in the salvage pathway of pyrimidine nucleosides. MNGIE-like syndrome having an overlapping phenotype of MNGIE was also described and has been associated with mutations in POLG and RRM2B genes. In the present study, we report the molecular investigation of a consanguineous family including two patients with clinical features suggestive of MNGIE syndrome. Bioinformatics analyses were carried out in addition to mtDNA deletion screening and copy number quantification in the blood of the two patients. Whole exome sequencing and Sanger sequencing analyses revealed the segregation in the affected family a novel mutation c.1205T>A (p.L402Q) within the exon 9 of the TYMP gene. In addition, mtDNA analysis revealed the absence of mtDNA deletions and a decrease of the copy number in the blood of the two patients of the studied family. The p.Leu402Gln mutation was located in a conserved amino acid within the α/β domain of the TP protein and several software supported its pathogenicity. In addition, and based on docking and molecular dynamic simulation analyses, results revealed that L402Q caused a conformational change in TP mutated structure and could therefore alter its flexibility and stability. These changes prevent also the formation of stable homodimer leading to non-functional protein with partial or complete loss of its catalytic activity. [ABSTRACT FROM AUTHOR]

Thymine or thymidine starvation induces robust chromosomal fragmentation in Escherichia coli thyA deoCABD mutants and is proposed to be the cause of thymineless death (TLD). However, fragmentation kinetics challenges the idea that fragmentation causes TLD, by peaking before the onset of TLD and disappearing by the time TLD accelerates. Quantity and kinetics of fragmentation also stay unchanged in hyper‐TLD‐exhibiting recBCD mutant, making its faster and deeper TLD independent of fragmentation as well. Elimination of fragmentation without affecting cellular metabolism did not abolish TLD in the thyA mutant, but reduced early TLD in the thyA recBCD mutant, suggesting replication‐dependent, but undetectable by pulsed‐field gel, double‐strand breaks contributed to TLD. Chromosomal fragmentation, but not TLD, was eliminated in both the thyA and thyA recBCD mutants harboring deoCABD operon. The expression of a single gene, deoA, encoding thymidine phosphorylase, was sufficient to abolish fragmentation, suggesting thymidine‐to‐thymine interconversion during T‐starvation being a key factor. Overall, this study reveals that chromosomal fragmentation, a direct consequence of T‐starvation, is either dispensable or redundant for the overall TLD pathology, including hyper‐TLD in the recBCD mutant. Replication forks, unlike chromosomal fragmentation, may provide a minor contribution to TLD, but only in the repair‐deficient thyA deoCABD recBCD mutant. [ABSTRACT FROM AUTHOR]

High fidelity base pairing is important for the transmission of genetic information. Weak base pairs can lower fidelity, complicating sequencing, amplification and replication of DNA. Thymidine 5′‐monophosphate (TMP) is the most weakly pairing nucleotide among the canonical deoxynucleotides, causing high errors rates in enzyme‐free primer extension. Here we report the synthesis of an ethynylpyridone C‐nucleoside analog of 3′‐amino‐2′,3′‐dideoxythymidine monophosphate and its incorporation in a growing strand by enzyme‐free primer extension. The ethynylpyridone C‐nucleotide accelerates extension more than five‐fold, reduces misincorporation and readily displaces TMP in competition experiments. The results bode well for the use of the C‐nucleoside as replacements for thymidine in practical applications. [ABSTRACT FROM AUTHOR]

Objective: Autosomal recessive human thymidine kinase 2 (TK2) mutations cause TK2 deficiency, which typically manifests as a progressive and fatal mitochondrial myopathy in infants and children. Treatment with pyrimidine deoxynucleosides deoxycytidine and thymidine ameliorates mitochondrial defects and extends the lifespan of Tk2 knock‐in mouse (Tk2KI) and compassionate use deoxynucleoside therapy in TK2 deficient patients have shown promising indications of efficacy. To augment therapy for Tk2 deficiency, we assessed gene therapy alone and in combination with deoxynucleoside therapy in Tk2KI mice. Methods: We generated pAAVsc CB6 PI vectors containing human TK2 cDNA (TK2). Adeno‐associated virus (AAV)‐TK2 was administered to Tk2KI, which were serially assessed for weight, motor functions, and survival as well as biochemical functions in tissues. AAV‐TK2 treated mice were further treated with deoxynucleosides. Results: AAV9 delivery of human TK2 cDNA to Tk2KI mice efficiently rescued Tk2 activity in all the tissues tested except the kidneys, delayed disease onset, and increased lifespan. Sequential treatment of Tk2KI mice with AAV9 first followed by AAV2 at different ages allowed us to reduce the viral dose while further prolonging the lifespan. Furthermore, addition of deoxycytidine and deoxythymidine supplementation to AAV9 + AAV2 treated Tk2KI mice dramatically improved mtDNA copy numbers in the liver and kidneys, animal growth, and lifespan. Interpretation: Our data indicate that AAV‐TK2 gene therapy as well as combination deoxynucleoside and gene therapies is more effective in Tk2KI mice than pharmacological alone. Thus, combination of gene therapy with substrate enhancement is a promising therapeutic approach for TK2 deficiency and potentially other metabolic disorders. ANN NEUROL 2021;90:640–652 [ABSTRACT FROM AUTHOR]

Background: Antemortem definitive diagnosis of lymphoma in horses is often difficult. Thymidine kinase 1 (TK1) assay is a potentially useful biomarker for lymphoma in horses. Hypothesis/objectives: To report the clinical performance of a commercially available TK1 assay for diagnosis of lymphoma in horses. We hypothesized that there would be no association between serum TK1 activity and a diagnosis of lymphoma in horses. Animals: Forty‐two hospitalized horses, 14 with a definitive diagnosis of lymphoma, 4 with other neoplasia, and 24 with inflammatory disease. Methods: Retrospective medical record review, groups were compared via Kruskal‐Wallis and Mann‐Whitney tests, and logistic regression was performed. Results: Median (range) TK1 was 3 U/L (0.4‐17.7 U/L) in horses with lymphoma and 3.9 U/L (0.8‐94 U/L) in horses without lymphoma (P =.59). There was no significant difference in total protein between horses with and without lymphoma (6.6 g/dL [5.5‐8.3 g/dL] vs 6.6 g/dL [4.7‐10.4 g/dL]; P =.83). There was no significant difference in fibrinogen between horses with and without lymphoma (447 [100‐1364] mg/dL vs 433 [291‐2004] mg/dL; P =.47). On logistic regression, serum TK1 activity was not associated with a diagnosis of lymphoma (odds ratio, 0.97; 95% confidence interval, 0.9‐1.05, P =.48). Conclusion and Clinical Importance: Serum TK1 values were not predictive of lymphoma diagnosis in this cohort of horses. [ABSTRACT FROM AUTHOR]

Macrophages are generally thought to play a key role in the pathogenesis of aseptic loosening through initiating periprosthetic inflammation and pathological bone resorption. The aim of this study was to identify macrophage‐derived factors that promote osteoclast differentiation and periprosthetic bone destruction. To achieve this, we examined the effects of 12 macrophage‐derived factors that were identified by RNA‐seq analysis of stimulated macrophages on osteoclast differentiation. Surprisingly, thymidine phosphorylase (TYMP) was found to trigger significant number of osteoclasts that exhibited resorbing activities on dentine slices. Functionally, TYMP knockdown reduced the number of osteoclasts in macrophages that had been stimulated with polyethylene debris. TYMP were detected in serum and synovial tissues of patients that had been diagnosed with aseptic loosening. Moreover, the administration of TYMP onto calvariae of mice induced pathological bone resorption that was accompanied by an excessive infiltration of inflammatory cells and osteoclasts. The RNA‐seq for TYMP‐induced‐osteoclasts was then performed in an effort to understand action mode of TYMP. TYMP stimulation appeared to activate the tyrosine kinase FYN signaling associated with osteoclast formation. Oral administration of saracatinib, a FYN kinase inhibitor, significantly suppressed formation of bone osteolytic lesions in a polyethylene debris‐induced osteolysis model. Our findings highlight a novel molecular target for therapeutic intervention in periprosthetic osteolysis. [ABSTRACT FROM AUTHOR]

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an inherited disease caused by a deficiency in thymidine phosphorylase and characterized by elevated systemic deoxyribonucleotides and gastrointestinal (GI) and neurological manifestations. We report the clinical and biochemical manifestations that were evaluated in a single patient before, during, and after pregnancy, over a period of 7 years. GI symptoms significantly improved, and plasma deoxyribonucleotide concentrations decreased during pregnancy. Within days after delivery, the patient's digestive symptoms recurred, coinciding with a rapid increase in plasma deoxyribonucleotide concentrations. We hypothesize that the clinico‐metabolic improvements could be attributed to the enzyme replacement action of the placental thymidine phosphorylase. [ABSTRACT FROM AUTHOR]

This study shows that double thymidine block treatment efficiently arrests the EO771 cells in the S‐phase without altering cell growth or survival. A long‐term analysis of cell behavior, using 5(6)‐carboxyfluorescein diacetate N‐succinimidyl ester (CFSE) staining, show synchronization to be stable and consistent over time. The EO771 cell line is a medullary breast‐adenocarcinoma cell line isolated from a spontaneous murine mammary tumor, and can be used to generate murine tumor implantation models. Different biological (serum or amino acid deprivation), physical (elutriation, mitotic shake‐off), or chemical (colchicine, nocodazole, thymidine) treatments are widely used for cell synchronization. Of the different methods tested, the double thymidine block is the most efficient for synchronization of murine EO771 cells if a large quantity of highly synchronized cells is recommended to study functional and biochemical events occurring in specific points of cell cycle progression. [ABSTRACT FROM AUTHOR]

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer, and thymidine phosphorylase (TP) is a regulator of angiogenesis. To investigate the biological activities of TP in ICC, we established human cholangiocarcinoma RBE cell lines overexpressing TP or silencing TP. Overexpression of TP enhanced viability, suppressed apoptosis and increased tube formation in human umbilical vein endothelial cells, while downregulation of TP reversed these effects. Moreover, an orthotopic xenograft mouse model of ICC was built to further explore TP's function in ICC in vivo. Histological analysis using H&E, TUNEL and Ki67 staining showed that TP promoted tumour growth and inhibited cell apoptosis. Immunostaining for CD31 revealed an elevation in microvessel density in the presence of TP. Besides, upregulation of TP increased the expression of vascular endothelial growth factor, basic fibroblast growth factor, interleukin‐8 and tumour necrosis factor alpha. In contrast, TP knockdown inhibited tumour growth, suppressed microvessel formation and decreased the expression of angiogenesis‐related proteins. Therefore, we suggest that TP promotes angiogenesis and tumour growth in ICC, which can be a potent therapeutic target for ICC treatment. [ABSTRACT FROM AUTHOR]

Here we report the synthesis of two‐an acyclic and a macrocyclic‐analogues of thymidine linked to a triazole moiety. The new molecules were synthesized through an intramolecular 1,3‐dipolar cycloaddition reaction of the diazido derivative of thymidine with a suitable alkyne. The acyclic nucleoside showed dose dependent cytotoxicity against glioblastoma and breast cancer cells by causing significant cellular damage as studied with confocal laser scanning microscopy. In silico induced fit molecular docking studies showed poly(ADP‐ribose) polymerase 1 (PARP1) as a potential target of the acyclic nucleoside. On the other hand, both the analogues showed no toxicity against normal fibroblast cells. [ABSTRACT FROM AUTHOR]

The gas‐phase hydration of Mg2+ complexes with deprotonated uracil (U), thymine (T), uridine (Ur, uracil riboside), and thymidine (Tdr, thymine deoxyriboside) was studied. The aim of the work was to analyze the hydration of product ions (eg, [2U‐H+Mg]+) formed as a result of the collision induced dissociation of the respective parent ion (eg, [3Ur‐H+Mg]+). The efficiency of gas‐phase hydration of the ions [2U‐H+Mg]+ and [2T‐H+Mg]+ was similar. However, the efficiency of gas‐phase hydration of the ion [U+Ur‐H+Mg]+ was much higher than that of gas‐phase hydration of the ion [T+Tdr‐H+Mg]+. On the basis of the mass spectra obtained and the performed molecular modelling, it was concluded that in the ion [T+Tdr‐H+Mg]+, we deal with a steric hindrance due to the presence of a sugar moiety, which affects water attachment. In the ion [U+Ur‐H+Mg]+, the position of the sugar moiety does not affect water attachment. [ABSTRACT FROM AUTHOR]

By applying caged thymidine residues, DNA duplexes were created in which HgII‐mediated base pair formation can be triggered by irradiation with light. When a bidentate ligand was used as the complementary nucleobase, an unprecedented stepwise formation of different metal‐mediated base pairs was achieved. [ABSTRACT FROM AUTHOR]

Thymidine phosphorylase (TPase), a dimeric proteinaceous enzyme is responsible for elevation of drug resistance and cell apoptosis. Increased TPase levels lead to angiogenesis which is a major culprit for the metastasis and cancer in the body. Hence, an efficient approach for anticancer drug discovery is based up on inhibition of thymidine phosphorylase enzyme. Solving of the crystal structure of TPase made the drug discovery easier. Legion of drugs was designed and synthesized having a wide variety of structural moieties and different functionalities such as phosphonic acid, amines, arylamines, acetamides, benzoyl, esters, amides, and so forth. Among the structural designs and models engineered, uracil is one of the structural motifs utilized for drug design. In this review, uracil is chosen where in structural unit with its structural modifications were summarized. Uracil unit could be structurally modified at its N1, N3, C5, and C6 positions to afford innumerable uracil derivatives. In general consensus, N1‐ and N3‐substituted uracil derivatives resulted in diminished/lower inhibitory activities which might be attributed to reduced acidic character. Whereas, substitution at 5‐ and 6‐positions have yielded fruitful results and excellent thymidine phosphorylase inhibitors prevailed. Furthermore, uracil possessing nucleoside uridine/deoxyuridine was also derivatized to afford potential TPase inhibitors. 3′‐ & 5′‐Hydroxyl groups of uridine/deoxyuridine were also exploited for derivative preparation and some of them were found to be potent TPase inhibitors. [ABSTRACT FROM AUTHOR]

Objective: Thymidine kinase 2, encoded by the nuclear gene TK2, is required for mitochondrial DNA maintenance. Autosomal recessive TK2 mutations cause depletion and multiple deletions of mtDNA that manifest predominantly as a myopathy usually beginning in childhood and progressing relentlessly. We investigated the safety and efficacy of deoxynucleoside monophosphate and deoxynucleoside therapies.Methods: We administered deoxynucleoside monophosphates and deoxynucleoside to 16 TK2-deficient patients under a compassionate use program.Results: In 5 patients with early onset and severe disease, survival and motor functions were better than historically untreated patients. In 11 childhood and adult onset patients, clinical measures stabilized or improved. Three of 8 patients who were nonambulatory at baseline gained the ability to walk on therapy; 4 of 5 patients who required enteric nutrition were able to discontinue feeding tube use; and 1 of 9 patients who required mechanical ventilation became able to breathe independently. In motor functional scales, improvements were observed in the 6-minute walk test performance in 7 of 8 subjects, Egen Klassifikation in 2 of 3, and North Star Ambulatory Assessment in all 5 tested. Baseline elevated serum growth differentiation factor 15 levels decreased with treatment in all 7 patients tested. A side effect observed in 8 of the 16 patients was dose-dependent diarrhea, which did not require withdrawal of treatment. Among 12 other TK2 patients treated with deoxynucleoside, 2 adults developed elevated liver enzymes that normalized following discontinuation of therapy.Interpretation: This open-label study indicates favorable side effect profiles and clinical efficacy of deoxynucleoside monophosphate and deoxynucleoside therapies for TK2 deficiency. ANN NEUROL 2019;86:293-303. [ABSTRACT FROM AUTHOR]

Objective: Osteogenesis is a multiple‐step process through which osteoblasts are derived from bone marrow mesenchymal stem cells (MSCs) with multilineage differentiation potential. This study aimed to analyze gene expression profiling during osteogenic differentiation of MSCs. Materials and Methods: Human MSCs were isolated and induced for differentiation in osteogenic medium. Full‐genome gene expression microarrays and gene ontology analysis were performed. Results: A total of 1,680 differentially expressed genes in differentiated MSCs were identified including 430 upregulated and 1,250 downregulated. Moreover, pathway‐act‐network analysis showed that cell cycle, p53 signaling pathway and focal adhesion, had high degree (>5). The ribonucleotide reductase M1, thymidine kinase 1 and histone cluster 1 H3e also showed high degree (>10). Polymerase chain reaction analysis confirmed the differential expression of insulin‐like growth factor binding protein 3, SMAD family member 3, transforming growth factor beta 2, and fibroblast growth factor 14 in differentiated MSCs. Conclusions: Gene expression profiling provides a foundation to reveal the mechanisms that regulate osteogenic differentiation of MSCs. We applied microarray and bioinformatics technology to perform gene expression profiling of human bone marrow mesenchymal stem cells during osteogenic differentiation. Our findings of the differential genes and significant pathways provide new insight into osteogenic differentiation of hMSC. [ABSTRACT FROM AUTHOR]

Genetic defects in the pyrimidine nucleoside transporters of the CNT transporter family have not yet been reported. Metabolic investigations in a patient with infantile afebrile tonic‐clonic seizures revealed increased urinary uridine and cytidine excretion. Segregation of this metabolic trait in the family showed the same biochemical phenotype in a healthy older brother of the index. Whole exome sequencing revealed biallelic mutations in SLC28A1 encoding the pyrimidine nucleoside transporter CNT1 in the index and his brother. Both parents and unaffected sibs showed the variant in heterozygous state. The transporter is expressed in the kidneys. Compelling evidence is available for the disrupting effect of the mutation on the transport function thus explaining the increased excretion of the pyrimidine nucleosides. The exome analysis also revealed a pathogenic mutation in PRRT2 in the index, explaining the epilepsy phenotype in infancy. At present, both the index (10 years) and his older brother are asymptomatic. Mutations in SLC28A1 cause a novel inborn error of metabolism that can be explained by the disrupted activity of the pyrimidine nucleoside transporter CNT1. This is the first report describing a defect in the family of CNT concentrative pyrimidine nucleoside transporter proteins encoded by the SLC28 gene family. In all likelihood, the epilepsy phenotype in the index is unrelated to the SLC28A1 defect, as this can be fully explained by the pathogenic PRRT2 variant. Clinical data on more patients are required to prove whether pathogenic mutations in SLC28A1 have any clinical consequences or are to be considered a benign metabolic phenotype. [ABSTRACT FROM AUTHOR]

Summary: Nucleotide biosynthesis proceeds through a de novo pathway and a salvage route. In the salvage route, free bases and/or nucleosides are recycled to generate the corresponding nucleotides. Thymidine kinase (TK) is the first enzyme in the salvage pathway to recycle thymidine nucleosides as it phosphorylates thymidine to yield thymidine monophosphate. The Arabidopsis genome contains two TK genes −TK1a and TK1b− that show similar expression patterns during development. In this work, we studied the respective roles of the two genes during early development and in response to genotoxic agents targeting the organellar or the nuclear genome. We found that the pyrimidine salvage pathway is crucial for chloroplast development and genome replication, as well as for the maintenance of its integrity, and is thus likely to play a crucial role during the transition from heterotrophy to autotrophy after germination. Interestingly, defects in TK activity could be partially compensated by supplementation of the medium with sugar, and this effect resulted from both the availability of a carbon source and the activation of the nucleotide de novo synthesis pathway, providing evidence for a compensation mechanism between two routes of nucleotide biosynthesis that depend on nutrient availability. Finally, we found differential roles of the TK1a and TK1b genes during the plant response to genotoxic stress, suggesting that different pools of nucleotides exist within the cells and are required to respond to different types of DNA damage. Altogether, our results highlight the importance of the pyrimidine salvage pathway, both during plant development and in response to genotoxic stress. Significance Statement: The Arabidopsis genome contains two genes coding for thymidine kinase (TK1a and TK1b) that share 58% amino acid identity. This study presents evidence on the distinct roles of TK1a and TK1b genes during the DNA damage response to genotoxic stress, and finds that the thymidine salvage pathway is critical for chloroplast development, genome replication and integrity. Sugars compensate for the lack of TK activity by inducing the nucleotide de novo synthesis pathway. [ABSTRACT FROM AUTHOR]

Luminescent materials are of great interest in many fields, such as fluorescent sensing and medical imaging. Here, the construction of lanthanide‐based luminescent ultralong microfibers through supramolecular self‐assembly (SSA) is reported. Nucleosides (thymidine in particular), the building blocks of nucleic acids, were used as new ligands to mediate the formation of luminescent microfibers in water. The length of microfibers from thymidine–lanthanide ion (Eu and Tb) SSA was on the centimeter scale. Notably, the microfibers exhibited strong luminescence because the lanthanide ions had been chelated, sensitized and effectively protected by thymidine molecules in water. Only when the stoichiometric ratio of lanthanide ion to thymidine was 1:3 and the pH of the solution was 7, are luminescent microfibers formed. Other nucleosides, such as adenosine, cytidine, and guanosine, could not form microfibers with the lanthanide ions. This work opens a new avenue for constructing nucleoside–lanthanide SSA architectures, which hold great potential in biological and optical related applications. It was that long!! Luminescent ultralong microfibers were prepared with lanthanide and thymidine by supramolecular self‐assembly (SSA). The microfibers exhibited strong luminescence because the lanthanide ions had been chelated, sensitized and effectively protected by thymidine molecules in water. This work opens a new avenue for constructing nucleoside/lanthanide SSA architectures, which hold great potential for biological and optical‐related applications. [ABSTRACT FROM AUTHOR]

A novel C3′‐functionalized thymidine dithiocarbamate derivative (3’DTC‐TdR) was successfully synthesized and labelled using [99mTcO]3+ core and [99mTc(CO)3(H2O)3]+ core with high yields. The structures of the 99mTc complexes were verified by preparation and characterization of the corresponding stable rhenium complexes. Both of the complexes were lipophilic and stable in vitro. Cell internalization experiments indicated that the uptakes of 99mTcO‐3’DTC‐TdR were related to nucleoside transporters. Biodistribution of these complexes in mice bearing tumor showed that they had high tumor uptakes, good tumor/muscle ratios and tumor/blood ratios. Especially for 99mTcO‐3’DTC‐TdR, it exhibited the highest tumor/muscle ratio and tumor/blood ratio at 4 h post‐injection. SPECT/CT imaging studies indicated clear accumulation in tumor, suggesting 99mTcO‐3’DTC‐TdR would be a promising candidate for tumor imaging. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: Thymidine kinase 1 (TK1) is one of the salvage enzymes engaged in the synthesis of DNA. Although a pro‐carcinogenetic role of TK1 has been reported in various types of cancers, its role in pancreatic ductal adenocarcinoma (PDAC) is still unknown. The study is aimed to elaborate the function of TK1 in PDAC and the potential mechanisms in the following study. Materials and methods: TK1 expression was analysed by immunohistochemistry, real‐time PCR and Western blot, and its relationship with clinicopathological characteristics of PDAC patients was further investigated. To verify the function of TK1 and potential mechanism, TK1 siRNA was used to transfect PDAC cells and performed a series of assays in cell and animal models. Results: The level of TK1 expression was higher in cancerous tissues compared with matched adjacent tissues. TK1 overexpression was associated with progression of PDAC and poor prognosis. Knockdown of TK1 could suppress cell proliferation via inducing S phase arrest mediated by upregulation of P21. Further mechanism investigation suggested that transcription factor E2F‐1 could directly regulate the TK1 and promote tumour proliferation. Conclusions: The results suggested that TK1 might be involved in the development and progression of PDAC by regulating cell proliferation and show that TK1 may work as a promising therapeutic target in patients with PDAC. [ABSTRACT FROM AUTHOR]

The modulation of expression levels of fluorescent fusion proteins (FFPs) is central for recombinant DNA technologies in modern biology as overexpression of proteins contributes to artifacts in biological experiments. In addition, some microscopy techniques such as fluorescence correlation spectroscopy (FCS) and single‐molecule‐based techniques are very sensitive to high expression levels of FFPs. To reduce the levels of recombinant protein expression in comparison with the commonly used, very strong CMV promoter, the herpes simplex virus thymidine kinase (TK) gene promoter, and mutants thereof were analyzed. Deletion mutants of the TK promoter were constructed and introduced into the Gateway® system for ectopic expression of enhanced green fluorescent protein (eGFP), monomeric cherry (mCherry), and FFPs containing these FPs. Two promoter constructs, TK2ST and TKTSC, were established, which have optimal low expression levels suitable for FCS studies in U2OS, HeLa CCL2, NIH 3T3, and BALB/c cells. Interestingly, when tested in these four cell lines, promoter constructs having a deletion within TK gene 5′‐UTR showed significantly higher protein expression levels than the equivalent constructs lacking this deletion. This suggests that a negative regulatory element is localized within the TK gene 5′‐UTR. [ABSTRACT FROM AUTHOR]

Abstract: Bioorthogonal metabolic DNA labeling with fluorochromes is a powerful strategy to visualize DNA molecules and their functions. Here, we report the development of a new DNA metabolic labeling strategy enabled by the catalyst‐free bioorthogonal ligation using vinyl thioether modified thymidine and o‐quinolinone quinone methide. With the newly designed vinyl thioether‐modified thymidine (VTdT), we added labeling tags on cellular DNA, which could further be linked to fluorochromes in cells. Therefore, we successfully visualized the DNA localization within cells as well as single DNA molecules without other staining reagents. In addition, we further characterized this bioorthogonal DNA metabolic labeling using DNase I digestion, MS characterization of VTdT as well as VTdT‐oQQF conjugate in cell nuclei or mitochondria. This technique provides a powerful strategy to study DNA in cells, which paves the way to achieve future spatiotemporal deciphering of DNA synthesis and functions. [ABSTRACT FROM AUTHOR]

Thymidylate kinase is an important enzyme in DNA synthesis. It catalyzes the conversion of thymidine monophosphate to thymidine diphosphate, with ATP as the preferred phosphoryl donor, in the presence of Mg2+. In this study, the dynamics of the active site and the communication paths between the substrates, ATP and TMP, are reported for thymidylate kinase from Thermus thermophilus. Conformational changes upon ligand binding and the path for communication between the substrates and the protein are important in understanding the catalytic mechanism of the enzyme. High‐resolution X‐ray crystal structures of thymidylate kinase in apo and ligand‐bound states were solved. This is the first report of structures of binary and ternary complexes of thymidylate kinase with its natural substrates ATP and ATP–TMP, respectively. Distinct conformations of the active‐site residues, the P‐loop and the LID region observed in the apo and ligand‐bound structures revealed that their concerted motion is required for the binding and proper positioning of the substrate TMP. Structural analyses provide an insight into the mode of substrate binding at the active site. The residues involved in communication between the substrates were identified through network analysis using molecular‐dynamics simulations. The residues identified showed high sequence conservation across species. Biochemical analyses show that mutations of these residues either resulted in a loss of activity or affected the thermal stability of the protein. Further, molecular‐dynamics analyses of mutants suggest that the proper positioning of TMP is important for catalysis. These data also provide an insight into the phosphoryl‐transfer mechanism. [ABSTRACT FROM AUTHOR]

Abstract: Nasopharyngeal carcinoma (NPC), which is closely associated with Epstein–Barr virus (EBV), is one of the most prevalent cancers in southeast China. Most NPC patients are diagnosed at late stage due to inconspicuous symptoms at the early stage, and the prognosis of these patients is poor. The early diagnosis rate of NPC could be significantly increased by serological screening, but the positive predictive value (PPV) is relatively low. A simple two‐step serological screening scheme was established to improve the PPV of the screening strategy and was validated by a prospective cohort. Serum antibodies specific for EBNA1, Zta, Thymidine Kinase (TK), EAD, EAR, and VCA were detected by enzyme‐linked immunosorbent assay. The combination of EBNA1/IgA and VCA/IgA was used in the first step of screening, and anti‐early antigens (EAs) were used in the second step of screening. EAD/IgA was the most prominent marker in the second step of screening, and other anti‐EAs were complementary to EAD/IgA. As validated by a prospective cohort including 4200 participants, using the combination of EAD/IgA and TK/IgA in the second step decreased the number of high‐risk participants from 128 to 27, and increased the PPV from 4.69% to 18.52%, with only one very early‐stage case missed. The two‐step screening scheme provides a standardized approach for NPC screening with an improved PPV and may be used in future field studies. With this two‐step serological screening method, more people benefit from the screening program without increasing the need for fiberoptic endoscopy. [ABSTRACT FROM AUTHOR]

Background Relapses of immune-mediated hemolytic anemia ( IMHA), thrombocytopenia ( ITP), or polyarthropathy ( IMPA) occur despite normal hematologic and cytologic parameters. Thymidine kinase 1 ( TK1), canine C-reactive protein (c- CRP), haptoglobin ( HPT), and 25-Hydroxyvitamin- D (25( OH)D) might be adjunct to current monitoring strategies. Hypothesis/Objectives Compare serum concentrations of TK1, c- CRP, HPT, and 25( OH)D in dogs with well- and poorly controlled primary IMHA, ITP, or IMPA. Animals Thirty-eight client-owned dogs. Methods Prospective descriptive study. Dogs diagnosed with IMHA, ITP, or IMPA had serum biomarker concentrations measured commercially. Disease control was assessed by hematocrit/ PCV and reticulocyte count, platelet count, and synovial fluid cytology for IMHA, ITP, and IMPA, respectively. Statistical analysis performed by Mann-Whitney rank-sum tests and receiver operating characteristic curves. Results TK1 and c- CRP, but not HPT significantly decreased with well- versus poorly controlled IMHA ( P = 0.047, P = 0.028, P = 0.37). C- CRP, but not TK or HPT was significantly lower with well- versus poorly controlled IMPA ( P = 0.05, P = 0.28, P = 0.84). Sensitivity and specificity of TK and c- CRP (simultaneously) for detecting dogs with poorly controlled IMHA were 88 and 100%, respectively. Sensitivity and specificity of c- CRP for detecting poorly controlled dogs with IMPA were 13 and 100%, respectively. 92% of dogs were vitamin D insufficient (<100 ng/mL) regardless of disease control. Conclusions and Clinical Importance Combining TK1 and c- CRP might act markers of disease control in dogs with IMHA. Canine- CRP cannot be recommended as an independent marker of disease control in IMPA. 25( OH)D insufficiency in immune-mediated disorders might benefit from further study to determine if supplementation could improve therapeutic response or reduce disease risk. [ABSTRACT FROM AUTHOR]

Here, we examined the potential of blocking the thymidine de novo synthesis pathways for sensitizing melanoma cells to the nucleoside salvage pathway targeting endogenous DNA irradiation. Expression of key nucleotide synthesis and proliferation enzymes thymidylate synthase ( TS) and thymidine kinase 1 ( TK1) was evaluated in differentiated ( MITFhigh [microphthalmia-associated transcription factor] IGR1) and invasive ( MITFmedium IGR37) melanoma cells. For inhibition of de novo pathways cells were incubated either with an irreversible TS inhibitor 5-fluoro-2′-deoxyuridine (FdUrd) or with a competitive dihydrofolate-reductase ( DHFR) inhibitor methotrexate ( MTX). Salvage pathway was addressed by irradiation-emitting thymidine analog [123/125I]-5-iodo-4′-thio-2′-deoxyuridine (123/125I- ITdU). The in vivo targeting efficiency was visualized by single-photon emission computed tomography. Pretreatment with FdUrd strongly increased the cellular uptake and the DNA incorporation of 125I- ITdU into the mitotically active IGR37 cells. This effect was less pronounced in the differentiated IGR1 cells. In vivo, inhibition of TS led to a high and preferential accumulation of 123I- ITdU in tumor tissue. This preclinical study presents profound rationale for development of therapeutic approach by highly efficient and selective radioactive targeting one of the crucial salvage pathways in melanomas. [ABSTRACT FROM AUTHOR]

Telbivudine, an FDA pregnancy category B drug, has been found to reduce hepatitis B virus (HBV) perinatal transmission with no safety concerns in infants aged up to 1 year. This study evaluated the long-term efficacy and safety of telbivudine in 214 infants born to 210 pregnant women with chronic hepatitis B infection who were treated with telbivudine during pregnancy (weeks 20-32 of gestation). The infants were followed for up to 5 years after birth. The efficacy endpoint was the rate of perinatal transmission, which was established by HBsAg and HBV DNA levels at 7 and 12 months. Safety endpoints included head circumference, weight, height, congenital abnormality and hospitalization rates. In addition, the Denver Developmental Screening Test was performed in 92 randomly selected infants. None of the 214 infants born to these women were infected with HBV, and all had effective serum hepatitis B surface antibody (HBsAb) levels. Compared with Chinese standard values, there were few differences in the infants' mean head circumference, weight, and height values. No birth defects were diagnosed, and the congenital abnormality rate was 0.934%. Serious adverse events requiring hospitalization occurred in 20 infants (9.35%). The qualified Denver Developmental Screening Test rate in 92 infants was 97.82%, which was comparable to a rate of 92% in normal Chinese children. Thus, treatment with telbivudine during the second or third trimesters of pregnancy safely blocked perinatal transmission of HBV. Infants born to telbivudine-treated mothers showed normal growth and development during long-term follow-up of up to 5 years. [ABSTRACT FROM AUTHOR]

Objective: Thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial pyrimidine salvage pathway, is essential for mitochondrial DNA (mtDNA) maintenance. Mutations in the nuclear gene, TK2, cause TK2 deficiency, which manifests predominantly in children as myopathy with mtDNA depletion. Molecular bypass therapy with the TK2 products, deoxycytidine monophosphate (dCMP) and deoxythymidine monophosphate (dTMP), prolongs the life span of Tk2-deficient (Tk2-/- ) mice by 2- to 3-fold. Because we observed rapid catabolism of the deoxynucleoside monophosphates to deoxythymidine (dT) and deoxycytidine (dC), we hypothesized that: (1) deoxynucleosides might be the major active agents and (2) inhibition of deoxycytidine deamination might enhance dTMP+dCMP therapy.Methods: To test these hypotheses, we assessed two therapies in Tk2-/- mice: (1) dT+dC and (2) coadministration of the deaminase inhibitor, tetrahydrouridine (THU), with dTMP+dCMP.Results: We observed that dC+dT delayed disease onset, prolonged life span of Tk2-deficient mice and restored mtDNA copy number as well as respiratory chain enzyme activities and levels. In contrast, dCMP+dTMP+THU therapy decreased life span of Tk2-/- animals compared to dCMP+dTMP.Interpretation: Our studies demonstrate that deoxynucleoside substrate enhancement is a novel therapy, which may ameliorate TK2 deficiency in patients. Ann Neurol 2017;81:641-652. [ABSTRACT FROM AUTHOR]

Background/Purpose Facial lipoatrophy in HIV patients, secondary to antiretroviral therapy ( ART) with thymidine analogs, has been related to important psychosocial alterations and poor adherence to treatment. Polyacrylamide gel ( PAAG) is a filler that has been used for treating facial lipoatrophy in HIV patients. The aim was to assess the clinical and sonographic anatomical changes after injection of PAAG in HIV patients with facial lipoatrophy secondary to ART. Methods HIV patients receiving ART and suffering from severe facial lipoatrophy were recruited and underwent clinical and color Doppler ultrasound evaluation prior to PAAG application ( AQUAMID®) and sonographically monitored at 18 months and clinically followed up for 36 months after the procedure. Adverse effects were recorded based on occurrence and complexity. Results A total of 33 patients were evaluated, 30 men (91%) and 3 women (9%) with an average age of 49.6 years (±8.4). Clinical improvement assessed by a dermatologist had an average score of 5.9 (±0.7) on a scale of 1-7. On color Doppler ultrasound there was a significant increase of the thickness of the subcutaneous tissue (SCT) in both nasofold lines when comparing before and after PAAG injection ( P < 0.01) and no signs of inflammation (hypervascularity). User satisfaction was qualified as excellent or good in all cases. Only two patients experienced adverse effects (hematoma and puncture site infection), which was successfully managed without consequences. Conclusion Treatment of facial lipoatrophy with PAAG seems to be effective in HIV patients and no signs of complications were observed in the monitoring at 36 months after injection. Color Doppler ultrasound can identify the filler deposits and the anatomical changes of the SCT non-invasively. [ABSTRACT FROM AUTHOR]

Radical S-adenosyl-L-methionine ( SAM) enzymes have emerged as one of the last superfamilies of enzymes discovered to date. Arguably, it is the most versatile group of enzymes involved in at least 85 biochemical transformations. One of the founding members of this enzyme superfamily is the spore photoproduct ( SP) lyase, a DNA repair enzyme catalyzing the direct reversal repair of a unique DNA lesion, the so-called spore photoproduct, back into two thymidine residues. Discovered more than 20 years ago in the bacterium Bacillus subtilis, SP lyase has been shown to be widespread in the endospore-forming Firmicutes from the Bacilli and Clostridia classes and to use radical-based chemistry to perform C-C bond breakage, a chemically challenging reaction. This review describes how the work on SP lyase has illuminated a unique strategy for DNA repair and provided major advances in our understanding of the emerging radical SAM superfamily of enzymes, from a biochemical and structural perspective. [ABSTRACT FROM AUTHOR]

Topoisomerases catalyze changes in DNA topology by directing the movement of DNA strands through consecutive cleavage-rejoining reactions of the DNA backbone. We describe the use of a phenylselenyl-modified thymidine incorporated into a specific position of a partially unwound DNA substrate in crosslinking studies of Escherichia coli topoisomerase I to gain new insights into its catalytic mechanism. Crosslinking of the phenylselenyl-modified thymidine to the topoisomerase protein was achieved by the addition of a mild oxidant. Following nuclease and trypsin digestion, lysine residues on topoisomerase I crosslinked to the modified thymidine were identified by mass spectrometry. The crosslinked sites may correspond to proximal sites for the unwound DNA strand as it interacts with enzyme in the different stages of the catalytic cycle. [ABSTRACT FROM AUTHOR]

Systematic NMR characterization of 4-thio-5-furan-pyrimidine nucleosides or 4-thio-5-thiophene-pyrimidine nucleosides (ribonucleosides and 2′-deoxynucleosides) was performed. All proton and carbon signals of 4-thio-5-thiophene-ribouridine and related analogues were unambiguously assigned. The orientations of the base (4-thiouridine or its deoxy analogue) relative to the ring (furan or thiophene) are explored by a NMR approach and further supported by X-ray crystallographic studies. The procedures presented here would be applicable to other modified nucleosides and nucleotides. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Thymidine kinase (TK) is a key enzyme in the pyrimidine salvage pathway which catalyzes the transfer of the γ-phosphate of ATP to 2′-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). Unlike other type II TKs, the Trypanosoma brucei enzyme ( TbTK) is a tandem protein with two TK homolog domains of which only the C-terminal one is active. In this study, we establish that TbTK is essential for parasite viability and cell cycle progression, independently of extracellular pyrimidine concentrations. We show that expression of TbTK is cell cycle regulated and that depletion of TbTK leads to strongly diminished dTTP pools and DNA damage indicating intracellular dThd to be an essential intermediate metabolite for the synthesis of thymine-derived nucleotides. In addition, we report the X-ray structure of the catalytically active domain of TbTK in complex with dThd and dTMP at resolutions up to 2.2 Å. In spite of the high conservation of the active site residues, the structures reveal a widened active site cavity near the nucleobase moiety compared to the human enzyme. Our findings strongly support TbTK as a crucial enzyme in dTTP homeostasis and identify structural differences within the active site that could be exploited in the process of rational drug design. [ABSTRACT FROM AUTHOR]

Koi herpesvirus ( KHV) is the aetiological agent of an emerging disease ( KHVD) associated with mass mortalities in koi and common carp and reported from at least 30 countries. We report the first detection of KHV from koi in Iran using clinical, histopathological and molecular studies. KHV-infected fish showed reduced swimming activity, sunken eyes and increased mucus production on skin and fins. On post-mortem examination, gill necrosis was observed in the majority of fish. Histopathologically, the gill showed diffuse necrosis of the branchial epithelial cells. Margination of chromatin was detected in gills, kidney, heart, spleen, intestine and brain. In addition, sequence analyses of the TK gene, ORF 136 and marker I and II, demonstrates that Iranian KHV isolates were identical and classified as variant A1 of TUSMT1 (J strain) and displayed the I++ II+ allele of this Asian genotype. [ABSTRACT FROM AUTHOR]

The usage of recombinant lactic acid bacteria for delivery of therapeutic proteins to the mucosa has been emerging. In the present study, an attempt was made to engineer a thyA mutant of Lactobacillus plantarum ( L. plantarum) using lactococcal group II intron Ll.LtrB for the development of biologically contained recombinant L. plantarum for prevention of calcium oxalate stone disease. The 3 kb Ll.LtrB intron donor cassettes from the source vector pACD4C was PCR amplified, ligated into pSIP series of lactobacillus vector pLp_3050sAmyA, yielding a novel vector pLpACD4C (8.6 kb). The quantitative real-time PCR experiment shows 94-fold increased expression of Ll.LtrB intron and 14-fold increased expression of ltrA gene in recombinant L. plantarum containing pLpACD4C. In order to target the thyA gene, the potential intron RNA binding sites in the thyA gene of L. plantarum was predicted with help of computer algorithm. The insertion location 188|189s of thyA gene (lowest E-0.134) was chosen and the wild type intron Ll.LtrB was PCR modified, yielding a retargeted intron of pLpACDthyA. The retargeted intron was expressed by using induction peptide (sppIP), subsequently the integration of intron in thyA gene was identified by PCR screening and finally ThyA− mutant of L. plantarum (ThyA18) was detected. In vitro growth curve result showed that in the absence of thymidine, colony forming units of mutant ThyA18 was decreased, whereas high thymidine concentration (10 μM) supported the growth of the culture until saturation. In conclusion, ThyA− mutant of L. plantarum (ThyA18) constructed in this study will be used as a biologically contained recombinant probiotic to deliver oxalate decarboxylase into the lumen for treatment of hyperoxaluria and calcium oxalate stone deposition. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the in vitro steroid sensitivity as a predictor of clinical response to glucocorticoids in childhood idiopathic nephrotic syndrome (INS). Seventy-four patients (median age 4.33, interquartile range [IQR] 2.82-7.23; 63.5% male) were enrolled in a prospective multicenter study: in vitro steroid inhibition of patients' peripheral blood mononuclear cell proliferation was evaluated by [methyl-3H] thymidine incorporation assay at disease onset (T0) and after 4 weeks (T4) of treatment. Steroid dependence was associated with increased in vitro sensitivity at T4 assessed both as drug concentration inducing 50% of inhibition (IC50; odds ratio [OR] = 0.48, 95% confidence interval [CI] = 0.24-0.85; P = 0.0094) and maximum inhibition at the highest drug concentration (Imax; OR = 1.13, 95% CI = 1.02-1.31; P = 0.017). IC50 > 4.4 nM and Imax < 92% at T4 were good predictors for optimal clinical response. These results suggest that this test may be useful for predicting the response to glucocorticoid therapy in pediatric INS. [ABSTRACT FROM AUTHOR]

Background Serum thymidine kinase type 1 ( TK1) and canine C-Reactive Protein ( cCRP) might be useful in detecting dogs with cancer. Algorithms combining biomarkers are sometimes more accurate than results of individual tests. Objectives The aim of this study was to compare serum TK1 and cCRP and Neoplasia Index ( NI) in healthy and tumor-bearing dogs. Animals Client-owned dogs with (n = 253) and without (n = 156) cancer. Methods Retrospective case-control study. Dogs with cancer were identified after submission of samples for commercial assay and case details were retrospectively collected. Healthy dogs (control) were identified through breed groups and health status was confirmed by health questionnaire for a minimum of 6 months. Serum TK1 activity was measured using a quantitative chemiluminescent assay and serum cCRP was measured using a quantitative ELISA assay. Results TK1 activity in the cancer (n = 253) and control group (n = 156) were 7.0 μ/L (median, range <0.5 to >100) and 1.8 μ/L (median, range 0.4 to 55.3), respectively ( P < .001). cCRP concentrations in the cancer and control group were 6.0 mg/L (median, range <0.5 to >50) and 1.6 mg/L (median, range 0.09 to >50), respectively ( P < .001). The NI in the cancer and control group were 6.4 (median, range 0-9.9) and 0.9 (median, range 0-7.6), respectively ( P < .001). ROC AUCs of the NI and TK1 for all cancers were greater than 0.8, highest for lymphoma and histiocytic sarcoma. Conclusions and Clinical Importance Increased concentrations of TK1 and cCRP, when present in dogs with cancer, might be useful in confirming a diagnosis and monitoring response to treatment. [ABSTRACT FROM AUTHOR]

Chemical modifications of the nucleotides can improve the stability of aptamers against enzyme degradation in serum, but it is not clear whether these methods are effective in snake venom. In this study, a DNA aptamer, βB-1, which specifically recognize β-bungarotoxin and Bungarus multicinctus venom was chosen, and the key binding sequence of the aptamer was determined. Based on the secondary structure of the truncated aptamer, locked nucleic acids and 2′-O-methyl nucleotides were applied to modify the stem and loop sequences, respectively. In addition, a 3′-3′-thymidine cap was also adopted to block the 3′ end. It was shown that these chemical modifications can all enhance the stability of the aptamer in snake venom. Simultaneously, modified aptamer with the above modifications in one sequence exhibited a significantly elevated biostability, with the half-life improved from several minutes to 210 min while maintaining its binding affinity to the target. [ABSTRACT FROM AUTHOR]

In vitro transcriptional signatures that predict toxicities can facilitate chemical screening. We previously developed a transcriptomic biomarker (known as TGx-28.65) for classifying agents as genotoxic (DNA damaging) and non-genotoxic in human lymphoblastoid TK6 cells. Because TK6 cells do not express cytochrome P450s, we confirmed accurate classification by the biomarker in cells co-exposed to 1% 5,6 benzoflavone/phenobarbital-induced rat liver S9 for metabolic activation. However, chemicals may require different types of S9 for activation. Here we investigated the response of TK6 cells to higher percentages of Aroclor-, benzoflavone/phenobarbital-, or ethanol-induced rat liver S9 to expand TGx-28.65 biomarker applicability. Transcriptional profiles were derived 3 to 4 hr following a 4 hr co-exposure of TK6 cells to test chemicals and S9. Preliminary studies established that 10% Aroclor- and 5% ethanol-induced S9 alone did not induce the TGx-28.65 biomarker genes. Seven genotoxic and two non-genotoxic chemicals (and concurrent solvent and positive controls) were then tested with one of the S9s (selected based on cell survival and micronucleus induction). Relative survival and micronucleus frequency was assessed by flow cytometry in cells 20 hr post-exposure. Genotoxic/non-genotoxic chemicals were accurately classified using the different S9s. One technical replicate of cells co-treated with dexamethasone and 10% Aroclor-induced S9 was falsely classified as genotoxic, suggesting caution in using high S9 concentrations. Even low concentrations of genotoxic chemicals (those not causing cytotoxicity) were correctly classified, demonstrating that TGx-28.65 is a sensitive biomarker of genotoxicity. A meta-analysis of datasets from 13 chemicals supports that different S9s can be used in TK6 cells, without impairing classification using the TGx-28.65 biomarker. Environ. Mol. Mutagen. 57:243-260, 2016. © 2016 Her Majesty the Queen in Right of Canada. Environmental and Molecular Mutagenesis © 2016 Environmental Mutagen Society [ABSTRACT FROM AUTHOR]

The metabolic conversion of nucleoside analogues into their triphosphates often proceeds insufficiently. Rate-limitations can be at the mono-, but also at the di- and triphosphorylation steps. We developed a nucleoside triphosphate (NTP) delivery system (Tri PPPro-approach). In this approach, NTPs are masked by two bioreversible units at the γ-phosphate. Using a procedure involving H-phosphonate chemistry, a series of derivatives bearing approved, as well as potentially antivirally active, nucleoside analogues was synthesized. The enzyme-triggered delivery of NTPs was demonstrated by pig liver esterase, in human T-lymphocyte cell extracts and by a polymerase chain reaction using a prodrug of thymidine triphosphate. The Tri PPPro-compounds of some HIV-inactive nucleoside analogues showed marked anti-HIV activity. For cellular uptake studies, a fluorescent Tri PPPro-compound was prepared that delivered the triphosphorylated metabolite to intact CEM cells. [ABSTRACT FROM AUTHOR]