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Mutations in the BRCA1 or BRCA2 tumor suppressor genes predispose individuals to breast and ovarian cancer. In the clinic, these cancers are treated with inhibitors that target poly(ADP-ribose) polymerase (PARP). We show that inhibition of DNPH1, a protein that eliminates cytotoxic nucleotide 5-hydroxymethyl-deoxyuridine (hmdU) monophosphate, potentiates the sensitivity of BRCA-deficient cells to PARP inhibitors (PARPi). Synthetic lethality was mediated by the action of SMUG1 glycosylase on genomic hmdU, leading to PARP trapping, replication fork collapse, DNA break formation, and apoptosis. BRCA1-deficient cells that acquired resistance to PARPi were resensitized by treatment with hmdU and DNPH1 inhibition. Because genomic hmdU is a key determinant of PARPi sensitivity, targeting DNPH1 provides a promising strategy for the hypersensitization of BRCA-deficient cancers to PARPi therapy. [ABSTRACT FROM AUTHOR]

Sepsis is the leading cause of acute kidney injury (AKI) and lung injury worldwide. Despite therapeutic advances, sepsis continues to be associated with high mortality. Because Brazilian green propolis (GP) has promising anti-inflammatory, antioxidant, and immunomodulatory properties, we hypothesized that it would protect kidneys and lungs in rats induced to sepsis by cecal ligation and puncture (CLP). Male Wistar rats were divided into groups—control (sham-operated); CLP (CLP only); and CLP + GP (CLP and treatment with GP at 6 h thereafter)—all receiving volume expansion and antibiotic therapy at 6 h after the procedures. By 24 h after the procedures, treatment with GP improved survival, attenuated sepsis-induced AKI, and restored renal tubular function. Whole-blood levels of reduced glutathione were higher in the CLP + GP group. Sepsis upregulated the Toll-like receptor 4/nuclear factor-kappa B axis in lung and renal tissues, as well as increasing inflammatory cytokine levels and macrophage infiltration; all of those effects were attenuated by GP. Treatment with GP decreased the numbers of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling-positive cells in renal and lung tissue, as well as protecting the morphology of the renal mitochondria. Our data open the prospect for clinical trials of the use of GP in sepsis. [ABSTRACT FROM AUTHOR]

The effects of modified deoxyuridine triphosphates (mod-dUTPs) with different substituents at the C5 position of the pyrimidine cycle on the kinetics of PCR with Taq and Vent (exo-) DNA polymerases are studied. Substituents in mod-dUTP include carboxamide group and groups that are part of the side chains of alanine, valine, leucine, phenylalanine, tryptophan, or tyrosine. For each mod-dUTP, the yields of the target product are measured with the full substitution of dTTP. A fragment of bacterial DNA with a certain nucleotide sequence and a synthetic combinatorial DNA library of random nucleotide sequences are used as templates for amplification. For each mod-dUTP–template–polymerase combination, the correlation between the amplification efficiencies and yields of the target product are investigated. PCR product accumulation curves are influenced by both the template used and the presence of a modified substrate. The catalytic activity of Taq polymerase is higher when mod-dUTPs with short aliphatic substituents are used and decreases when the derivatives with long aliphatic, phenyl, and indole substituents are utilized. Vent (exo-) polymerase is less sensitive to the chemical structure of mod-dUTP. The dynamic measuring of DNA accumulation may be useful for optimizing the temperature–time PCR profiles individually for each of the mod-dUTP. The derivatives may be used in combination with Vent (exo-) polymerase to obtain modified DNA sequences for the method of selection of modified aptamers (mod-SELEX). [ABSTRACT FROM AUTHOR]

Summary: To reveal the mode of morphogenesis of flattened unifacial leaves, we analysed the cell division direction and distribution on the leaf blade of Juncus prismatocarpus.Using the pulse–chase 5‐ethynyl‐2′‐deoxyuridine method, we quantified and mapped the cell division direction on the leaf blade of J. prismatocarpus and compared the distribution of thickening cell divisions with the expression pattern of DROOPING LEAF (DL), a key gene involved in leaf blade thickening.Thickening cell divisions were the most abundant (> 45%) among all cell division directions on the leaf blade of J. prismatocarpus from the early plastochron 2 stage through the plastochron 3 stage. Mapping of cell divisions indicated that cell divisions in a particular direction were not restricted to a particular domain but were distributed diffusely throughout the entire cross‐sectional area of the leaf blade. Gradient analysis indicated that the distribution of thickening cell divisions of the adaxial domain was denser than that of the abaxial domain. Contrary to the prolonged and diffuse distribution of thickening cell divisions, DL expression was transient and restricted in a narrow band.Our results suggest that a diffuse 'thickening meristem' plays the key role in the development of flattened unifacial leaves. [ABSTRACT FROM AUTHOR]

This study was aimed at exploring the underlying mechanisms of ketamine in the SV‐40 immortalized human ureteral epithelial (SV‐HUC‐1) cells. The viability and apoptosis of SV‐HUC‐1 cells treated with 0.01, 0.1, and 1 mM ketamine were respectively detected via cell counting kit‐8 (CCK‐8) assay and terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining. Reactive oxygen species (ROS) level was measured through ROS probe staining. Apoptosis‐related proteins (B‐cell lymphoma 2 [Bcl‐2] and Bax) and autophagy‐associated proteins (light chain 3‐I [LC3‐I] and LC3‐II) were determined by western blot or immunofluorescent assay. Additionally, transmission electron microscopy (TEM) was used to evaluate the formation of autophagosomes. After cotreatment of 3‐methyladenine (3‐MA) or N‐acetyl‐l‐cysteine (NAC), the biological functions of SV‐HUC‐1 cells were analyzed to determine the association of ROS with cell viability and autophagy. CCK‐8 assay and TUNEL staining indicated that ketamine effectively decreased the viability of SV‐HUC‐1 cells and accelerated apoptosis of SV‐HUC‐1 cells through regulating the expression level of IKBα (phospho), nuclear factor кB (P65), Bcl‐2, and Bax proteins. Enhanced ROS production was also confirmed in ketamine‐treated SV‐HUC‐1 cells treated with ketamine. Ketamine‐induced autophagosomes in SV‐HUC‐1 cells were observed by means of TEM, and increased levels of LC3 II/I ratio and Beclin 1 were examined through western blot and immunofluorescent assay. Furthermore, ketamine exerted effects on SV‐HUC‐1 cells in a dose‐dependent and time‐dependent manner. Additionally, cotreatment of NAC with 3‐MA significantly attenuated the ROS level and suppressed the cell autophagy. Ketamine promoted SV‐HUC‐1 cell autophagy and impaired the cell viability of SV‐HUC‐1 cells by inducing ROS. Ketamine increased ROS production and induced autophagy in SV‐HUC‐1 cells. ROS inhibitor NAC and autophagy inhibitor 3‐MA could resist effects of ketamine on SV‐HUC‐1 cells. Overproduction of ROS under various cellular stresses can lead to cell death. [ABSTRACT FROM AUTHOR]

Background: MNGIE is a rare and fatal disease in which absence of the enzyme thymidine phosphorylase induces systemic accumulation of thymidine and deoxyuridine and secondary mitochondrial DNA alterations. Gastrointestinal (GI) symptoms are frequently reported in MNGIE patients, however, they are not resolved with the current treatment interventions. Recently, our understanding of the GI pathology has increased, which rationalizes the pursuit of more targeted therapeutic strategies. In particular, interstitial cells of Cajal (ICC) play key roles in GI physiology and are involved in the pathogenesis of the GI dysmotility. However, understanding of the triggers of ICC deficits in MNGIE is lacking. Herein, we review the current knowledge about the pathology of GI dysmotility in MNGIE, discuss potential mechanisms in relation to ICC loss/dysfunction, remark on the limited contribution of the current treatments, and propose intervention strategies to overcome ICC deficits. Finally, we address the advances and new research avenues offered by organoids and tissue engineering technologies, and propose schemes to implement to further our understanding of the GI pathology and utility in regenerative and personalized medicine in MNGIE.Conclusion: Interstitial cells of Cajal play key roles in the physiology of the gastrointestinal motility. Evaluation of their status in the GI dysmotility related to MNGIE would be valuable for diagnosis of MNGIE. Understanding the underlying pathological and molecular mechanisms affecting ICC is an asset for the development of targeted prevention and treatment strategies for the GI dysmotility related to MNGIE. [ABSTRACT FROM AUTHOR]

The influence of Bacillus subtilis Ch9 on intestinal apoptosis of grass carp Ctenopharyngodon idella challenged with Aeromonas hydrophila was studied. Groups of grass carp were orally intubated with B. subtilis or phosphate buffered saline for 3 days and subjected to oral A. hydrophila challenge. Intestinal tissues were collected and prepared for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay, real-time quantitative polymerase chain reaction analysis of caspase-3 and -8 and b-cell lymphoma-2 protein (bcl-2), histopathological analyses and measurement of caspase-3 and -8 activities at 0, 12, 24, 48, 72 and 96 h post-challenge. TUNEL analyses demonstrated that B. subtilis induced intestinal cell apoptosis, and caspase-3 and -8 activities increased significantly compared to in the control and A. hydrophila groups. The intestinal damage due to A. hydrophila was also reduced. However, messenger RNA transcript levels of caspase-3 and -8 and bcl-2 were not markedly elevated. These results suggested that B. subtilis Ch9 increased intestinal apoptosis in fish challenged with A. hydrophila, and that B. subtilis Ch9-induced caspase-dependent apoptosis may play an important role in reducing intestinal damage following A. hydrophila challenge. [ABSTRACT FROM AUTHOR]

Abstract—: A modification of the enzymatic method for the preparation of combinatorial random DNA libraries, which combines amplification in isolated microvolumes with the simultaneous incorporation of modified nucleotides and subsequent separation of DNA strands, was developed. Deoxyuridine triphosphate with hydrophobic substituents such as structural analogues of amino acid side chains in the C5 position of the pyrimidine ring was used to introduce modifications into DNA. To prevent competitive amplification, which reduces the representativeness of combinatorial libraries, PCR in inverse emulsion was used. The separation of the strands of PCR products was carried out. There were six single-stranded DNA libraries with complete substitution of deoxythymidine via modified analogues with various functional groups. These DNA libraries are suitable for generating aptamers to protein targets through additional hydrophobic interactions from the introductions of appropriate modifications, and are completely compatible with the SELEX aptamer selection methodology. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide, and microRNAs (miRs) are considered to serve important functions in the pathogenesis of HCC by regulating the expression of specific target genes. The present study was conducted to investigate the role of miR-199 and its putative target X-box binding protein 1 (XBP1) in HCC, as well as of the downstream gene cyclin D. The expression levels of miR-199, XBP1 and cyclin D were detected in clinical HCC specimens. The effect of miR-199 on the regulation of HCC cell proliferation and its underlying mechanism were examined in Hep3B2.1–7 cells, through expression assays and measurement of cell proliferation (via Cell Counting Kit-8, and 5-ethynyl-2′-deoxyuridine and DAPI double-staining assays) coupled with gain- and lose- of function experiments. The expression of XBP1 and cyclin D was significantly increased in HCC tissues when compared with adjacent non-HCC tissues, while the expression of miR-199 was decreased. Exogenous miR-199 significantly suppressed the expression of XBP1 and cyclin D in Hep3B2.1–7 cells. However, the expression of XBP1 and cyclin D significantly increased on treatment with miR-199 inhibitor. Consistently, Hep3B2.1–7 cells co-transfected with a wild type reporter plasmid [XBP1-3′untranslated region (UTR)-WT] and exogenous miR-199 exhibited lower relative luciferase enzyme activity than cells co-transfected with negative control miRNA and XBP1-3′UTR-WT, while cells co-transfected with mutated plasmid (XBP1-3′UTR-MU) and miR-199 exhibited no change. It was further observed that knockdown of XBP1 by small interfering RNA significantly decreased the expression of cyclin D in Hep3B2.1–7 cells. Additionally, exogenous miR-199 decreased the proliferation of Hep3B2.1–7 cells, which was contrary to the effect of miR-199 inhibitor. In conclusion, it was demonstrated that miR-199 negatively regulated the expression of XBP1 by directly binding to its 3′UTR and that XBP1 impacted cyclin D expression, which was associated with the cell cycle regulation in Hep3B2.1–7 cells. These findings suggested that a miR-199/XBP1/cyclin D axis may serve an important role in the pathogenesis of HCC. [ABSTRACT FROM AUTHOR]

Hepatocyte growth factor (HGF) signals mediate mouse skeletal muscle stem cell, or satellite cell (SC), reentry into the cell cycle and myoblast proliferation. Because the athletic horse experiences exercise-induced muscle damage, the objective of the experiment was to determine the effect of HGF on equine SC (eqSC) bioactivity. Fresh isolates of adult eqSC were incubated with increasing concentrations of HGF and the initial time to DNA synthesis was measured. Media supplementation with HGF did not shorten (P > 0.05) the duration of G0/G1 transition suggesting the growth factor does not affect activation. Treatment with 25 ng/mL HGF increased (P < 0.05) eqSC proliferation that was coincident with phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and AKT serine/threonine kinase 1 (AKT1). Chemical inhibition of the upstream effectors of ERK1/2 or AKT1 elicited no effect (P > 0.05) on HGF-mediated 5-ethynyl- 2′-deoxyuridine (EdU) incorporation. By contrast, treatment of eqSC with 2 μm Gö6983, a pan-protein kinase C (PKC) inhibitor, blocked (P < 0.05) HGF-initiated mitotic activity. Geneexpression analysis revealed that eqSC express PKCα, PKCδ, and PKCε isoforms. Knockdown of PKCδ with a small interfering RNA (siRNA) prevented (P > 0.05) HGF-mediated EdU incorporation. The siPKCδ was specific to the kinase and did not affect (P > 0.05) expression of either PKCα or PKCε. Treatment of confluent eqSC with 25 ng/mL HGF suppressed (P < 0.05) nuclear myogenin expression during the early stages of differentiation. These results demonstrate that HGF may not affect activation but can act as a mitogen and modest suppressor of differentiation. [ABSTRACT FROM AUTHOR]

5-Hydroxymethylcytosine and 5-formylcytosine are stable DNA base modifications generated from 5-methylcytosine by the ten-eleven translocation protein family that function as epigenetic markers. 5-Hydroxymethyluracil may also be generated from thymine by ten-eleven translocation enzymes. Here, we asked if these epigenetic changes accumulate in senescent cells, since they are thought to be inversely correlated with proliferation. Testing this in ERCC1-XPF-deficient cells and mice also enabled discovery if these DNA base changes are repaired by nucleotide excision repair. Epigenetic marks were measured in proliferating, quiescent and senescent wild-type (WT) and Ercc1-/- primary mouse embryonic fibroblasts. The pattern of epigenetic marks depended more on the proliferation status of the cells than their DNA repair capacity. The cytosine modifications were all decreased in senescent cells compared to quiescent or proliferating cells, whereas 5-(hydroxymethyl)-2'-deoxyuridine was increased. In vivo, both 5-(hydroxymethyl)-2'-deoxyuridine and 5-(hydroxymethyl)-2'-deoxycytidine were significantly increased in liver tissues of aged WT mice compared to young adult WT mice. Livers of Ercc1-deficient mice with premature senescence and aging had reduced level of 5-(hydroxymethyl)-2'-deoxycytidine and 5-formyl-2'-deoxycytidine compared to aged-matched WT controls. Taken together, we demonstrate for the first time, that 5-(hydroxymethyl)-2'-deoxycytidine is significantly reduced in senescent cells and tissue, potentially yielding a novel marker of senescence. [ABSTRACT FROM AUTHOR]

A series of analogues of potent antiviral perylene nucleoside dUY11 with methylthiomethyl (MTM), azidomethyl (AZM) and HO-C 1–4 -alkyl-1,2,3-triazol-1,4-diyl groups at 3′- O -position as well as the two products of copper-free alkyne-azide cycloaddition of the AZM derivative were prepared and evaluated against tick-borne encephalitis virus (TBEV). Four compounds ( 4 , 6 , 8a , 8b ) showed EC 50  ≤ 10 nM, thus appearing the most potent TBEV inhibitors to date. Moreover, these nucleosides have higher lipophilicity (clogP) and increased solubility in aq. DMSO vs. parent compound dUY11 . [ABSTRACT FROM AUTHOR]

As a primary active ingredient of safflor yellow, hydroxysafflor yellow A (HSYA) exhibits notable antioxidative and neuroprotective effects. The aim of the present study was to investigate the protective effects of HSYA in mesenchymal stem cells (MSCs) exposed to hypoxia (5% O2) and serum deprivation (H/SD), and to explore the mechanisms underlying HSYA-mediated protection. Under H/SD conditions, HSYA was applied to protect MSCs against injury. Cell viability, proliferation, apoptosis and reactive oxygen species (ROS) levels were determined using an 5-ethynyl-2'-deoxyuridine assay, MTT assay, Hoechst 33342/propidium iodide and 2',7'-dichlorodihydrofluorescein diacetate staining, respectively. The results revealed that 160 mg/l HSYA significantly reduced apoptosis and ROS levels compared with the H/SD group; however, HSYA demonstrated minimal effects on cell proliferation. A western blot assay demonstrated that HSYA reduced cleaved caspase-3 expression and cytC release from the mitochondria to the cytoplasm when compared with the H/SD group. In addition, western blotting and RT-qPCR analyses revealed that HSYA treatment significantly increased the expression of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). In conclusion, the results of the current study demonstrated that HSYA exerts protective effects against H/SD-induced apoptosis in MSCs potentially via activation of the HIF-1α/VEGF signaling pathway and stabilization of the mitochondrial membrane. [ABSTRACT FROM AUTHOR]

The main goal was to evaluate the correlation between sperm parameters and chromatin quality with embryo kinetics via time-lapse monitoring system (TLM). A total of 40 couples involved in the ICSI program as a result of male infertility. For assessment of sperm chromatin and DNA quality, we used aniline blue, toluidine blue, chromomycin A3, acridine orange and terminal transferase-mediated deoxyuridine triphosphate biotin end labelling assays. All mature oocytes were injected, and the generated zygotes (2PNs) were cultured in TLM. In day 3 after injection, single embryo transfer (SET) was carried out according to the morphology and morphokinetics. The patients were followed up until delivery. There were positive significant correlations between sperm count with CC2 (r = .330, p = .049), T4 (r = .329, p = .038), T6 (r = .342, p = .035) and T7 (r = .374, p = .025). Also, there were positive significant correlations between nonprogressive motility and T2 (r = 0.323, p = .042), T3 (r = .411, p = .013) and T4 (r = .418, p = .007). Regarding the sperm chromatin quality assays, there were negative significant correlations between CMA3 and CC2 (r = -.272, p = .049) and between acridine orange and T5 (r = -.221, p = .040). It seems that the abnormal sperm parameters and chromatin alteration affect the normal embryo kinetics in ICSI program. [ABSTRACT FROM AUTHOR]

A conjugate of triphosphorylated 2′,3′-dideoxyuridine (ddU) with SiO 2 nanoparticles was obtained via the CuAAC click chemistry between a γ-alkynyl ddU triphosphate and azido-modified SiO 2 nanoparticles. Assessment of cytotoxicity in human breast adenocarcinoma MCF7 cells demonstrated that ddU triphosphate conjugated to SiO 2 nanoparticles exhibited a 50% decrease in cancer cell growth at a concentration of 183 ± 57 µg/mL, which corresponds to 22 ± 7 µM of the parent nucleotide, whereas the parent nucleoside, nucleotide and alkynyl triphosphate precursor do not show any cytotoxicity. The data provide an example of remarkable potential of novel conjugates of SiO 2 nanoparticles with phosphorylated nucleoside analogues, even those, which have not been used previously as therapeutics, for application as new anticancer agents. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa (P. aeruginosa) is an important opportunistic pathogen that causes serious infections in humans, including keratitis in contact lens wearers. Therefore, establishing a rapid, specific and sensitive method for the identification of P. aeruginosa is imperative. In the present study, the uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification combined with nanogold labeled hybridization probe (UDG-LAMP-AuNP) was developed for the detection of P. aeruginosa. UDG-LAMP was performed to prevent carry over contamination and the LAMP reactions can be readily observed using the nanogold probe. A set of 4 primers and a hybridization probe were designed based on the ecfX gene. The UDG-LAMP reactions were performed at 65˚C for 60 min using the ratio of 40% deoxyuridine triphosphate to 60% deoxythymidine triphosphate. The detection of UDG-LAMP products using the nanogold labeled hybridization probe, which appeared as a red-purple color, was examined at 65˚C for 5 min with 40 mM MgSO4. The UDG-LAMP-AuNP demonstrated specificity to all tested isolates of P. aeruginosa without cross reaction to other bacteria. The sensitivity for the detection of pure culture was 1.6x103 colony-forming units (CFU) ml-1 or equivalent to 3 CFU per reaction while that of polymerase chain reaction was 30 CFU per reaction. The detection limit of spiked contact lenses was 1.1x103 CFU ml-1 or equivalent to 2 CFU per reaction. In conclusion, the UDG-LAMP-AuNP assay was rapid, simple, specific and was effective for the identification of P. aeruginosa in contaminated samples. [ABSTRACT FROM AUTHOR]

Apoptosis of excessive or defective germ cells is a natural process occurring in mammalian testes. Tumour suppressor protein p53 is involved in this process both in developing and adult male gonads. Its contribution to testicular physiology is known to be modified by genetic background. The aim of this study was to evaluate the combined influence of the p53 and Y chromosome long arm genes on male germ cell apoptosis. Knockout of the transformation related protein 53 (Trp53) gene was introduced into congenic strains: B10.BR (intact Y chromosome) and B10.BR-Ydel (Y chromosome with a deletion in the long arm). The level of apoptosis in the testes of 19-day-old and 3-month-old male mice was determined using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick-end labelling (TUNEL) method. The study revealed that although p53 is involved in germ cell apoptosis in peripubertal testes, this process can also be mediated by p53-independent mechanisms. However, activation of p53-independent apoptotic pathways in the absence of the p53 protein requires engagement of the multicopy Yq genes and was not observed in gonads of B10.BR-Ydel-p53 -/- males. The role of Yq genes in the regulation of testicular apoptosis seems to be restricted to the initial wave of spermatogenesis and is not evident in adult gonads. The study confirmed, instead, that p53 does participate in spontaneous apoptosis in mature testes. [ABSTRACT FROM AUTHOR]

Barley is one of the cereals that are most sensitive to aluminum (Al). Al in acid soils limits barley growth and development and, as a result, its productivity. The inhibition of root growth is a widely accepted indicator of Al stress. Al toxicity is affected by many factors including the culture medium, pH, Al concentration and the duration of the treatment. However, Al can act differently in different species and still Al toxicity in barley deserves study. Since the mechanism of Al toxicity is discussed we cytogenetically describe the effects of different doses of bioavailable Al on the barley nuclear genome—mitotic activity, cell cycle profile and DNA integrity. At the same time, we tested an established deep-water culture (DWC) hydroponics system and analyzed the effects of Al on the root system parameters using WinRHIZO software. We demonstrated the cytotoxic and genotoxic effect of Al in barley root cells. We showed that Al treatment significantly reduced the mitotic activity of the root tip cells and it also induced micronuclei and damaged nuclei. The DNA-damaging effect of Al was observed using the TUNEL test. We define the inhibitory influence of Al on DNA replication in barley. Analysis with the labelling and detection of 5-ethynyl-2‘-deoxyuridin (EdU) showed that the treatment with Al significantly decreased the frequency of S phase cells. We also demonstrated that Al exposure led to changes in the cell cycle profile of barley root tips. The delay of cell divisions observed as increased frequency of cells in G2/M phase after Al treatment was reported using flow cytometry. [ABSTRACT FROM AUTHOR]

We have designed and synthesized new 5-fluoro-2′-deoxyuridine 5′-phosphate pronucleotides which can function as potential agents against the glioblastoma multiforme tumor. Their anti-malignant potency has been tested against T98G, U-118 MG, U-87 MG gliomas, HeLa, and Caco-2 cancer cell lines, using MRC-5 healthy cells as a reference. Five of the sixteen compounds ( 4c , 4f-i ) exhibited significant anticancer potency and high selectivity indices (SI 12–66). It is likely that these zwitterionic pronucleotides may function in a similar manner to zwitterionic phospholipids, by inducing cell membrane charge disorder, making the cell permeable to bioactive agents. The most promising therapeutic pronucleotides 4c , 4f-h , have high intestinal-blood uptake potency (Caco-2 cell line), and may be considered as potential, orally administrated, anticancer drugs. [ABSTRACT FROM AUTHOR]

A surface-enhanced Raman scattering (SERS) method for in situ detection and analysis of the intranuclear biomolecular information of a cell has been developed based on a small, biocompatible, nuclear-targeting alkyne-tagged deoxyribonucleic acid (DNA) probe (5-ethynyl-2′-deoxyuridine, EDU) that can specially accumulate in the cell nucleus during DNA replications to precisely locate the nuclear region without disturbance in cell biological activities and functions. Since the specific alkyne group shows a Raman peak in the Raman-silent region of cells, it is an interior label to visualize the nuclear location synchronously in real time when measuring the SERS spectra of a cell. Because no fluorescent-labeled dyes were used for locating cell nuclei, this method is simple, nondestructive, non- photobleaching, and valuable for the in situ exploration of vital physiological processes with DNA participation in cell organelles. [ABSTRACT FROM AUTHOR]

Mammalian nucleotide excision repair (NER) eliminates the broadest diversity of bulky lesions from DNA with wide specificity. However, the double incision efficiency for structurally different adducts can vary over several orders of magnitude. Therefore, great attention is drawn to the question of the relationship among structural properties of bulky DNA lesions and the rate of damage elimination. This paper studies the properties of several structurally diverse synthetic (model) DNAs containing bulky modifications. Model DNAs have been designed using modified nucleosides (exo- N -{2- N -[ N -(4-azido-2,5-difluoro-3-chloropyridin-6-yl)-3-aminopropionyl]aminoethyl}-2′-deoxycytidine (Fap-dC) and 5-{1-[6-(5[6]-fluoresceinylcarbomoyl)hexanoyl]-3-aminoallyl}-2′-deoxyuridine (Flu-dU)) and the nonnucleosidic reagent N -[6-(9-antracenylcarbomoyl)hexanoyl]-3-amino-1,2-propandiol (nAnt). The impact of these lesions on spatial organization and stability of the model DNA was evaluated. Their affinity for the damage sensor XPC was also studied. It was expected, that the values of melting temperature decrease, bending angles and K D values clearly define the row of model DNA substrate properties such as Flu-dU-DNA >> nAnt ≈ Fap-dC-DNA. Unexpectedly the experimentally estimated levels of the substrate properties were actually in the row: nAnt-DNA >> Flu-dU-DNA >> Fap-dC-DNA. Molecular dynamics simulations have revealed structural and energetic bases for the discrepancies observed. DNA destabilization patterns plotted explain these results on a structural basis in terms of differences in dynamic perturbations of stacking interactions. [ABSTRACT FROM AUTHOR]

In this study, we prepared 2‐fluorenyl‐1,2,3‐triazol‐4‐yl‐2′‐deoxyuridine (UFT) and inserted it at the central position of an oligodeoxynucleotide (ODN) to investigate whether the triazole group is an efficient linker between fluorene and uracil. UFT displays different fluorescence intensities and emission maxima in different solvents, indicating its potential for application as an environmentally sensitive probe. Moreover, excimer emission from UFT was observed in water. However, UFT containing ODN did not exhibit selective thermal stability and fluorescence changes upon duplex formation with matched and single‐base mismatched targets. This highlighted the importance of selecting the appropriate linker to connect the fluorophore to the nucleobase when developing such DNA probes. [ABSTRACT FROM AUTHOR]

Background: Several lines of evidence have indicated that growth arrest-specific transcript 5 (GAS5) functions as a tumor suppressor and is aberrantly expressed in multiple cancers. GAS5 was found to be downregulated in gastric cancer (GC) tissues, and ectopic expression of GAS5 inhibited GC cell proliferation.Aims: The present study aimed to explore the underlying mechanisms of GAS5 involved in GC cell proliferation.Methods: GAS5 and miR-222 expressions in GC cell lines were estimated by quantitative real-time polymerase chain reaction. The effects of GAS5 and miR-222 on GC cell proliferation were assessed by MTT assay and 5-bromo-2-deoxyuridine (BrdU) incorporation assays. The interaction between GAS5 and miR-222 was confirmed by luciferase reporter assay and RNA immunoprecipitation assay. The protein levels of the phosphatase and tensin homolog (PTEN), phosphorylated protein kinase B (Akt) (p-Akt), Akt, phosphorylated mammalian target of rapamycin (mTOR) (p-mTOR), and mTOR were determined by western blot.Results: GAS5 was downregulated and miR-222 was upregulated in GC cells. GAS5 directly targeted and suppressed miR-222 expression. GAS5 overexpression and miR-222 inhibition suppressed cell proliferation, increased PTEN protein level and decreased p-Akt and p-mTOR protein levels in GC cells while GAS5 knockdown and miR-222 overexpression exhibited the opposite effects. Moreover, mechanistic analyses revealed that GAS5 regulated GC cell proliferation through the PTEN/Akt/mTOR pathway by negatively regulating miR-222.Conclusions: GAS5/miR-222 axis regulated proliferation of GC cells through the PTEN/Akt/mTOR pathway, which facilitated the development of lncRNA-directed therapy against this deadly disease. [ABSTRACT FROM AUTHOR]

Background: Immune dysregulation has been linked to occlusive vascular remodeling in pulmonary arterial hypertension (PAH) that is hereditary, idiopathic, or associated with other conditions. Circulating autoantibodies, lung perivascular lymphoid tissue, and elevated cytokines have been related to PAH pathogenesis but without a clear understanding of how these abnormalities are initiated, perpetuated, and connected in the progression of disease. We therefore set out to identify specific target antigens in PAH lung immune complexes as a starting point toward resolving these issues to better inform future application of immunomodulatory therapies.Methods: Lung immune complexes were isolated and PAH target antigens were identified by liquid chromatography tandem mass spectrometry, confirmed by enzyme-linked immunosorbent assay, and localized by confocal microscopy. One PAH antigen linked to immunity and inflammation was pursued and a link to PAH pathophysiology was investigated by next-generation sequencing, functional studies in cultured monocytes and endothelial cells, and hemodynamic and lung studies in a rat.Results: SAM domain and HD domain-containing protein 1 (SAMHD1), an innate immune factor that suppresses HIV replication, was identified and confirmed as highly expressed in immune complexes from 16 hereditary and idiopathic PAH versus 12 control lungs. Elevated SAMHD1 was localized to endothelial cells, perivascular dendritic cells, and macrophages, and SAMHD1 antibodies were prevalent in tertiary lymphoid tissue. An unbiased screen using metagenomic sequencing related SAMHD1 to increased expression of human endogenous retrovirus K (HERV-K) in PAH versus control lungs (n=4). HERV-K envelope and deoxyuridine triphosphate nucleotidohydrolase mRNAs were elevated in PAH versus control lungs (n=10), and proteins were localized to macrophages. HERV-K deoxyuridine triphosphate nucleotidohydrolase induced SAMHD1 and proinflammatory cytokines (eg, interleukin 6, interleukin 1β, and tumor necrosis factor α) in circulating monocytes, pulmonary arterial endothelial cells, and also activated B cells. Vulnerability of pulmonary arterial endothelial cells (PAEC) to apoptosis was increased by HERV-K deoxyuridine triphosphate nucleotidohydrolase in an interleukin 6-independent manner. Furthermore, 3 weekly injections of HERV-K deoxyuridine triphosphate nucleotidohydrolase induced hemodynamic and vascular changes of pulmonary hypertension in rats (n=8) and elevated interleukin 6.Conclusions: Our study reveals that upregulation of the endogenous retrovirus HERV-K could both initiate and sustain activation of the immune system and cause vascular changes associated with PAH. [ABSTRACT FROM AUTHOR]

5-Bromo-2′-deoxyuridine (BrdU) photosensitizes DNA to strand break formation. However, this type of photodamage is completely quenched by the presence of triethylamine (TEA) which originates from RP-HPLC purification commonly employed by oligonucleotide providers. While the presence of TEA in oligonucleotide samples does not interfere with PCR or other molecular biology applications, the mechanism of photochemical reaction proceeding in the labeled DNA is dramatically changed due to the photoinduced electron transfer (PET) between the photoexcited BrdU and the ground state TEA. For the first time, we demonstrated that the latter process produces 2′-deoxyuridne2′-deoxyuridine (debromination) in the labeled DNA instead of the expected strand break. PET between TEA and BrdU was additionally confirmed by the UV irradiations of aqueous solutions containing both species. Indeed, the efficient formation of 2′-deoxyuridine was observed in the studied photolytes. Moreover, we showed the formation of an additional product in these binary mixtures, i.e. imidazole derivative, that is not formed in DNA and was reported in the literature in the context of dark rather than photochemical processes. Using mass spectrometry we demonstrated that the amount of TEA impurity in the commercial samples of oligos exceeds up to 3 orders of magnitude that of the purchased DNA. [ABSTRACT FROM AUTHOR]

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare, autosomal-recessive mitochondrial disorder caused by TYMP mutations presenting with a multisystemic, often lethal syndrome of progressive leukoencephalopathy, ophthalmoparesis, demyelinating neuropathy, cachexia and gastrointestinal dysmotility. Hemodialysis (HMD) has been suggested as a treatment to reduce accumulation of thymidine and deoxyuridine. However, all studies so far have failed to measure the toxic metabolites in cerebrospinal fluid (CSF), which is the crucial compartment for CNS damage.Our study is the first prospective, longitudinal investigation, exploiting detailed serial testing of predefined clinical and molecular outcome parameters (including serial CSF assessments) in a 29-year-old MNGIE patient undergoing 1 year of extensive HMD. We demonstrate that HMD only transiently restores increased serum and urine levels of thymidine and deoxyuridine, but fails to reduce CSF levels of the toxic metabolites and is ineffective to influence neurological function. These findings have direct important implications for clinical practice: They prevent a burdensome, long-term invasive, but ultimately probably ineffective procedure in future MNGIE patients. [ABSTRACT FROM AUTHOR]

Background: RNA-Sequencing (RNA-seq) is now commonly used to reveal quantitative spatiotemporal snapshots of the transcriptome, the structures of transcripts (splice variants and fusions) and landscapes of expressed mutations. However, standard approaches for library construction typically require relatively high amounts of input RNA, are labor intensive, and are time consuming. Methods: Here, we report the outcome of a systematic effort to optimize and streamline steps in strand-specific RNA-seq library construction. Results: This work has resulted in the identification of an optimized messenger RNA isolation protocol, a potent reverse transcriptase for cDNA synthesis, and an efficient chemistry and a simplified formulation of library construction reagents. We also present an optimization of bead-based purification and size selection designed to maximize the recovery of cDNA fragments. Conclusions: These developments have allowed us to assemble a rapid high throughput pipeline that produces high quality data from amounts of total RNA as low as 25 ng. While the focus of this study is on RNA-seq sample preparation, some of these developments are also relevant to other next-generation sequencing library types. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) can infect extrahepatic tissues, including lymphocytes, creating reservoir of the virus. Moreover, HCV proteins can interact with DNA damage response proteins of infected cells. In this article we investigated the influence of the virus infection and a new ombitasvir/paritaprevir/ritonavir ± dasabuvir ± ribavirin (OBV/PTV/r ± DSV ± RBV) anti-HCV therapy on the PBMCs (peripheral blood mononuclear cells, mainly lymphocytes) DNA base excision repair (BER) system. BER protein activity was analyzed in the nuclear and mitochondrial extracts (NE and ME) of PBMC isolated from patients before and after therapy, and from subjects without HCV, using modeled double-strand DNA, with 2′-deoxyuridine substitution as the DNA damage. The NE and ME obtained from patients before therapy demonstrated lower efficacy of 2′-deoxyuridine removal and DNA repair polymerization than those of the control group or patients after therapy. Moreover, the extracts from the patients after therapy had similar activity to those from the control group. However, the efficacy of apurinic/apyrimidinic site excision in NE did not differ between the studied groups. We postulate that infection of lymphocytes by the HCV can lead to a decrease in the activity of BER enzymes. However, the use of novel therapy results in the improvement of glycosylase activity as well as the regeneration of endonuclease and other crucial repair enzymes. [ABSTRACT FROM AUTHOR]

Here we report one new case each of an X-autosome translocation (maternally derived), and an X-Y-chromosome translocation. Besides characterizing the involved breakpoints and/or imbalances in detail by molecular cyto-genetics, also skewed X-chromosome inactivation was determined on single cell level using 5-ethynyl-2-deoxyuridine (EdU). Thus, we confirmed that the recently suggested EdU approach can be simply adapted for routine diagnostic use. The latter is important, as only by knowing the real pattern of the skewed X-chromosome inactivation, correct interpretation of obtained results and subsequent reliable genetic counseling, can be done. [ABSTRACT FROM AUTHOR]

Here, we describe the synthesis and purification of six deoxyuridine triphosphate derivatives that contain protein-like functional groups and alkene linkers of various lengths. Using KOD XL and Deep Vent polymerases, these derivatives have been incorporated into single-stranded DNA, achieving a high degree of DNA modification. These polymerases are able to utilize highly modified DNA strands as templates for synthesizing unmodified DNA. The synthesized deoxyuridine triphosphate derivatives are promising as substrates for producing modified aptamers to various target proteins using, e.g., the systematic evolution of ligands by exponential enrichment (SELEX) methodology. [ABSTRACT FROM AUTHOR]

5-Bromo-2′-deoxyuridine (BrdU) labelling and immunostaining is commonly used for the detection of DNA replication using specific antibodies. Previously, we found that these antibodies significantly differ in their affinity to BrdU. Our present data showed that one of the reasons for the differences in the replication signal is the speed of antibody dissociation. Whereas highly efficient antibodies created stable complexes with BrdU, the low efficiency antibodies were unstable. A substantial loss of the signal occurred within several minutes. The increase of the complex stability can be achieved by i) formaldehyde fixation or ii) a quick reaction with a secondary antibody. These steps allowed the same or even higher signal/background ratio to be reached as in the highly efficient antibodies. Based on our findings, we optimised an approach for the fully enzymatic detection of BrdU enabling the fast detection of replicational activity without a significant effect on the tested proteins or the fluorescence of the fluorescent proteins. The method was successfully applied for image and flow cytometry. The speed of the method is comparable to the approach based on 5-ethynyl-2′-deoxyuridine. Moreover, in the case of short labelling pulses, the optimised method is even more sensitive. The approach is also applicable for the detection of 5-trifluoromethyl-2'-deoxyuridine. [ABSTRACT FROM AUTHOR]

The study of cell proliferation is a useful tool in the fi elds of toxicology, pathophysiology and pharmacology. Cell proliferation and its degree can be evaluated using 5-bromo-2'- deoxyuridine which is incorporated into the newly synthesized DNA. The aim of this study was the optimization of subcutaneous application of 5-bromo-2'-deoxyuridine implantation for continuous and persistent marking of proliferating cells in the rat forestomach. 3-tert-Butyl-4-hydroxyanisole was used as the agent that ensures cell proliferation. In order to determine the optimal dose for proliferating cells labeling, 5-bromo-2'-deoxyuridine doses of 50 mg, 100 mg, 200 mg or 350 mg were implemented 2 days prior to sacrifi ce by fl at-faced cylindrical matrices. Immunohistochemical analysis using 5-bromo-2'-deoxyuridine in situ detection kit was performed for the detection of 5-bromo-2'-deoxyuridine labeled cells. The results showed that for adult rats, the optimum 5-bromo-2'-deoxyuridine dose is 200 mg per animal for subcutaneous application. The here described manner of 5-bromo-2'-deoxyuridine in vivo labeling provides a simple, effi cient, and reliable method for cell labeling, and at the same minimizes stress to animals. [ABSTRACT FROM AUTHOR]

Controlling extracellular glutamate level in a physiological range is important to maintain normal sensory transmission. Here, we investigated the paradoxical action of glutamate transporters in the rat formalin test to elucidate a possible role of inversely operating transporters in its analgesic mechanism. The effects of glutamate transporter inhibitor on formalin-induced pain behavior were examined. Then we performed a microdialysis study to clarify the differential change in extracellular glutamate concentration by intrathecal administration of transportable and non-transportable blockers. And we further investigated the mechanism pharmacologically via pretreatment with antagonists of various receptors and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining. Intrathecally-injected glutamate transporter inhibitors, non-transportable DL-threo-β-benzyloxyaspartat (TBOA) and transportable trans-pyrrolidine-2,4-dicarboxylic acid ( t -PDC), produced paradoxical antinociception in the formalin test. In normal rats, inhibition of the glutamate transporter increased extracellular glutamate. In the formalin model rats, TBOA suppressed while t-PDC enhanced glutamate release. When tPDC was pretreated 30 min prior to formalin injection, glutamate release was blocked. Blocking α-2 adrenergic receptors reversed the tPDC analgesia. Increased apoptosis was not apparent in the spinal dorsal horn of tPDC-treated rats compared to the control group. These data suggest that glutamate transporters in a formalin-induced pain state work in a reverse mode and can be blocked from releasing glutamate by TBOA and preloaded tPDC. The analgesic mechanism of TBOA may be related to the blockade of inversely operating transporter, and that of tPDC may be associated with the activation of noradrenergic neurotransmission but not with dorsal horn neurotoxicity. [ABSTRACT FROM AUTHOR]

Fine-tuned regulation of the cellular nucleotide pools is indispensable for faithful replication of Deoxyribonucleic Acid (DNA). The genetic information is also safeguarded by DNA damage recognition and repair processes. Uracil is one of the most frequently occurring erroneous bases in DNA; it can arise from cytosine deamination or thyminereplacing incorporation. Two enzyme activities are primarily involved in keeping DNA uracil-free: dUTPase (dUTP pyrophosphatase) activity that prevent thymine-replacing incorporation and uracil-DNA glycosylase activity that excise uracil from DNA and initiate uracil-excision repair. Both dUTPase and the most efficient uracil-DNA glycosylase (UNG) is thought to be ubiquitous in free-living organisms. In the present work, we have systematically investigated the genotype of deposited fully sequenced bacterial and Archaeal genomes. We have performed bioinformatic searches in these genomes using the already well described dUTPase and UNG gene sequences. For dUTPases, we have included the trimeric all-beta and the dimeric all-alpha families and also, the bifunctional dCTP (deoxycytidine triphosphate) deaminase-dUTPase sequences. Surprisingly, we have found that in contrast to the generally held opinion, a wide number of bacterial and Archaeal species lack all of the previously described dUTPase gene(s). The dut-genotype is present in diverse bacterial phyla indicating that loss of this (or these) gene(s) has occurred multiple times during evolution. We discuss potential survival strategies in lack of dUTPases, such as simultaneous lack or inhibition of UNG and possession of exogenous or alternate metabolic enzymes involved in uracil-DNA metabolism. The potential that genes previously not associated with dUTPase activity may still encode enzymes capable of hydrolyzing dUTP is also discussed. Our data indicate that several unicellular microorganisms may efficiently cope with a dut-genotype lacking all of the previously described dUTPase genes, and potentially leading to an unusual uracil-enrichment in their genomic DNA. [ABSTRACT FROM AUTHOR]

A series of 5-(4-alkyl-1,2,3-triazol-1-yl)methyl derivatives of 2′-deoxyuridine have been synthesized by the interaction of 3′,5′-diacetyl-5-azidomethyl-2′-deoxyuridine with the corresponding 1-alkynes in a biphasic methylene chloride-water system catalyzed by Cu(I) followed by the deblocking with a water-alcohol ammonia solution. A low cytotoxicity of the compounds in Vero, Jurkat, and A549 cell cultures has been shown. The 2′-deoxyuridine derivatives exhibited an antiherpetic activity in vitro toward two laboratory strains of human herpes simplex virus type 1 (HSV-1): acyclovir-sensitive (HSV-1/L) and acyclovir-resistant (HSV-1/LR). They also inhibited the growth of some bacteria ( Mycobacterium smegmatis, Staphylococcus aureus, Micrococcus luteus, and Leuconostoc mesenteroides) and yeasts Saccharomyces cerevisiae in vitro. [ABSTRACT FROM AUTHOR]

Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) is essential for genome integrity. Interestingly, this enzyme from Drosophila virilis has an unusual form, as three monomer repeats are merged with short linker sequences, yielding a fused trimer-like dUTPase fold. Unlike homotrimeric dUTPases that are encoded by a single repeat dut gene copy, the three repeats of the D. virilis dut gene are not identical due to several point mutations. We investigated the potential evolutionary pathway that led to the emergence of this extant fused trimeric dUTPase in D. virilis. The herein proposed scenario involves two sequential gene duplications followed by sequence divergence amongst the dut repeats. This pathway thus requires the existence of a transient two-repeat-containing fused dimeric dUTPase intermediate. We identified the corresponding ancestral dUTPase single repeat enzyme together with its tandem repeat evolutionary intermediate and characterized their enzymatic function and structural stability. We additionally engineered and characterized artificial single or tandem repeat constructs from the extant enzyme form to investigate the influence of the emergent residue alterations on the formation of a functional assembly. The observed severely impaired stability and catalytic activity of these latter constructs provide a plausible explanation for evolutionary persistence of the extant fused trimeric D. virilis dUTPase form. For the ancestral homotrimeric and the fused dimeric intermediate forms, we observed strong catalytic and structural competence, verifying viability of the proposed evolutionary pathway. We conclude that the progression along the herein described evolutionary trajectory is determined by the retained potential of the enzyme for its conserved three-fold structural symmetry. [ABSTRACT FROM AUTHOR]

Measurement of the direction of cell division is an important, yet difficult, task to analyse how a plant organ acquires its final shape from an initially small group of cells. We introduce a method that rapidly and easily quantifies cell division direction and is applicable to all plant species., A pulse-chase strategy for 5-ethynyl-2′-deoxyuridine (EdU) labelling assay was established and was shown to be successful for leaves of Arabidopsis thaliana (Arabidopsis) and Juncus prismatocarpus., By optimization of the pulse and chase periods, most of the signals obtained were sets of daughter nuclei. For Arabidopsis, the optimal time was a 45-min pulse and a 7-h chase. For J. prismatocarpus, the optimal time was a 2-h pulse and a 13.5-h chase. The positions of the daughter nuclei were used to quantify cell division direction in the Arabidopsis leaf primordia. Overall, cell division along the proximal-distal axis was more frequent than along the medial-lateral axis. In petiole, major vein, minor vein and margin areas, the major cell division direction seemed to be coincident with the direction of auxin flow., The advantages of our method over the few methods used previously are discussed. We anticipate that it will provide opportunities to study plant development in the near future. [ABSTRACT FROM AUTHOR]

The characterization of DNA synthesis and cell proliferation in vitro by 5-ethynyl-20-deoxyuridine (EdU) incorporation is widely used, but this has not been optimized for in vivo use. In this study, the administration route, dose (0-50 mg kg-1), and incorporation duration (0-72 h) of EdU and the procedure of cell preparation for flow cytometry were optimized for in vivo proliferation assay in bone marrow cells. The percentage of EdU-positive bone marrow cells was higher following intravenous than intraperitoneal injection (p <0.05); the percentage of positive cells increased in a dose-dependent manner from 0 to 50 mg kg-1 (p <0.05); and the optimal dose was 25 mg kg-1. However, the percentage of positive cells was not affected by the duration of incubation in vivo from 12 to 72 h (p >0.05). False-positive signals observed during flow cytometry were markedly reduced by subtracting the signals generated by nonviable cells stained with 7-amino-actinomycin. In addition, the percentage and mean fluorescence intensity of EdU-positive cells were increased by adding a signal enhancer to block background staining. In conclusion, the optimized procedures for in vivo incorporation of EdU and cell preparation for flow cytometry markedly improved the specificity and sensitivity of the proliferation assay of bone marrow cells. [ABSTRACT FROM AUTHOR]

Artificially modified nucleotides, in the form of nucleoside analogues, are widely used in the treatment of cancers and various other diseases, and have become important tools in the laboratory to characterise DNA repair pathways. In contrast, the role of endogenously occurring nucleotide modifications in genome stability is little understood. This is despite the demonstration over three decades ago that the cellular DNA precursor pool is orders of magnitude more susceptible to modification than the DNA molecule itself. More recently, underscoring the importance of this topic, oxidation of the cellular nucleotide pool achieved through targeting the sanitation enzyme MTH1, appears to be a promising anti-cancer strategy. This article reviews our current understanding of modified DNA precursors in genome stability, with a particular focus upon oxidised nucleotides, and outlines some important outstanding questions. [ABSTRACT FROM AUTHOR]

Lung exposure to sulfur mustard (SM) results in pulmonary complications, which is the main cause of long-term disability and morbidity. Up to now, the precise mechanisms of SM-induced lung complications has not been identified. The aim of this study was to evaluate apoptosis in the lung tissue of SM-exposed individuals. The study was performed on archived lung paraffin-embedded tissue specimens of21 patients suffering from pulmonary complications due to previous SM exposure and 9 unexposed patients who had undergone lung resections for another lung disease. Evaluation of apoptosis in paraffin-embedded lung tissue sections was performed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay and the cleaved caspase-3 immunohistochemistry assays. TUNEL-positive apoptotic features and caspase-3 expression of specimens were significantly higher in the SM-exposed group compared with the control group. This result demonstrates higher apoptosis rate in the SM-exposed group. Furthermore, the majority of positive cells consisted of alveolar epithelial cells in both methods. In conclusion,it seems that exposure to SM may result in increased apoptosis in respiratory epithelium. More studies are needed to evaluate the role of apoptosis in SM-induced lung complications in order to design new and effective therapeutic protocols. [ABSTRACT FROM AUTHOR]

We previously demonstrated that NG2-positive oligodendrocyte precursor cells (OPCs) do not express DM- 20 mRNA and identified a distinct DM- 20 mRNA-positive cell population expressing glutathione-S-transferase pi isoform (GST-pi) in the nucleus (GST-pi(Nuc)) of the adult rat spinal cord. As GST-pi intranuclear localization correlates with progenitor cell properties, we examined the differentiation status of this cell population under the intensive 5-bromo-2′-deoxyuridine (BrdU) administration method, consisting of intraperitoneal BrdU injections every 2 h for 48 h. We observed that a certain population of proliferating/proliferated cells expressed DM- 20 mRNA, and sometimes two proliferating/proliferated cells were observed still attached to each other. We performed triple staining for BrdU, DM- 20 mRNA, and NG2 and found pairs of neighboring BrdU-positive cells, which were considered to originate from the same progenitor cells and where both cells expressed DM- 20 mRNA. Triple staining for BrdU, DM- 20 mRNA, and GST-pi detected proliferating/proliferated cells exhibiting the GST-pi(Nuc)/ DM- 20 mRNA-positive expression pattern. These findings suggested the presence of a GST-pi(Nuc)/ DM- 20 mRNA-positive oligodendrocyte-lineage progenitor cell population in the adult rat spinal cord. However, we did not find any pair of neighboring BrdU-positive cells with this expression pattern. These observations collectively support the idea that GST-pi(Nuc)/ DM- 20 mRNA-expressing cells are the progeny of NG2-positive OPCs rather than a novel type of oligodendrocyte-lineage progenitor cells and that DM- 20 mRNA expression is dynamically regulated during differentiation of OPCs into oligodendrocytes. [ABSTRACT FROM AUTHOR]

A novel deoxyuridine (dU) benzothiazolium (BZ) derivative, referred to as dU-BZ, is reported that was synthesized via Sonogashira coupling reaction methodology. The deoxyuridine building block was introduced to enhance hydrophilicity, while an alkynylated benzothiazolium dye was incorporated for long wavelength absorption to reduce potential phototoxicity that is characteristic of using UV light to excite common fluorphores, better discriminate from native autofluorescence, and potentially facilitate deep tissue imaging. An impressive 30-fold enhancement of fluorescence intensity of dU-BZ was achieved upon increasing viscosity. Fluorescence quantum yields in 99% glycerol/1% methanol (v/v) solution as a function of temperature (293-343 K), together with viscosity-dependent fluorescence lifetimes and radiative and non-radiative rate constants in glycerol/methanol solutions (ranging from 4.8 to 950 cP) were determined. Both fluorescence quantum yields and lifetimes increased with increased viscosity, consistent with results predicted by theory. This suggests that the newly-designed compound, dU-BZ, is capable of functioning as a probe of local microviscosity, an aspect examined by in vitro bioimaging experiments. [ABSTRACT FROM AUTHOR]

Background: Label-retaining cells (LRCs) have been recognized as rare stem and progenitor-like cells, but their complex biological features in renal repair at the cellular level have never been reported. This study was conducted to evaluate whether LRCs in kidney are indeed renal stem/progenitor cells and to delineate their potential role in kidney regeneration. Methods: We utilized a long-term pulse chase of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells in C57BL/6J mice to identify renal LRCs. We tracked the precise morphological characteristics and locations of BrdU+LRCs by both immunohistochemistry and immunofluorescence. To examine whether these BrdU+LRCs contribute to the repair of acute kidney injury, we analyzed biological characteristics of BrdU+LRCs in mice after ischemia/reperfusion (I/R) injury. Results: The findings revealed that the nuclei of BrdU+ LRCs exhibited different morphological characteristics in normal adult kidneys, including nuclei in pairs or scattered, fragmented or intact, strongly or weakly positive. Only 24.3 ± 1.5 % of BrdU+ LRCs co-expressed with Ki67 and 9.1 ± 1.4 % of BrdU+ LRCs were positive for TUNEL following renal I/R injury. Interestingly, we found that newly regenerated cells formed a niche-like structure and LRCs in pairs tended to locate in this structure, but the number of those LRCs was very low. We found a few scattered LRCs co-expressed Lotus tetragonolobus agglutinin (LTA) in the early phase of injury, suggesting differentiation of those LRCs in mouse kidney. Conclusions: Our findings suggest that LRCs are not a simple type of slow-cycling cells in adult kidneys, indicating a limited role of these cells in the regeneration of I/R injured kidney. Thus, LRCs cannot reliably be considered stem/ progenitor cells in the regeneration of adult mouse kidney. When researchers use this technique to study the cellular basis of renal repair, these complex features of renal LRCs and the purity of real stem cells among renal LRCs should be considered. [ABSTRACT FROM AUTHOR]

We report a series of novel O-(substituted benzyl) phosphoramidate prodrugs of 5-fluoro-2′-deoxyuridine for the treatment of hepatocellular carcinoma. Through structure optimization, the o-methylbenzyl analog (1t) was identified as an orally bioavailable and liver-targeted lead compound. This lead prodrug is well-tolerated at a dose up to 3 g/kg in Kuming mice via oral administration. An efficacy study demonstrated that it possesses good inhibitory effect (61.67% and 72.50%, respectively) on tumor growth in a mouse xenograft model. A metabolism study in Sprague-Dawley rats suggested that 1t can release the desired 5′-monophosphate in the liver with high liver-targeting index. [ABSTRACT FROM AUTHOR]