Interplay between Artemis and TDP1 in sensitivity to radiomimetic agents

DNA double-strand breaks containing unligatable termini are potent cytotoxic lesions leading to cell death or growth arrest. Artemis, which is associated with the Non-Homologous End Joining (NHEJ) pathway, is the major end processing nuclease that resolves unligatable termini, especially the 3′ blocks, by nucleolytic trimming. Tyrosyl-DNA Phosphodiesterase 1 (TDP1) is an enzyme which is biochemically competent in 3′-phosphoglycolate processing. The purpose of this study is to investigate if TDP1 is an end-processing enzyme involved in the NHEJ pathway. Clonogenic Survival assays using shRNA-mediated TDP1 knockdown and Artemis knockout (Artemis-/-) in HCT116 cells showed increased sensitivity to Neocarzinostatin (NCS) and Calicheamicin, radiomimetic drugs that produce 3′-phosphoglycolate-terminated double-strand breaks. Thus, a cell line with combined deficiency in Artemis and TDP1 was generated by infecting Artemis-/- single mutants with a lentivirus expressing a TDP1 shRNA. Positive clones were screened for maximum TDP1 knockdown which was found to be 10X. Clonogenic survival assays carried out on shTDP1 & Artemis-/- single mutants and the Artemis-/-.shTDP1 double mutants showed similar sensitivity to Calicheamicin and NCS. Immunofluorescence studies on Art-/- and Art-/-.shTDP1 mutants also showed a similar increase in persistent 53BP1 foci, a measure of DNA damage, after treatment with NCS. Cell cycle analysis studies showed all these mutants arrest in G1 phase of the cell cycle after treatment with NCS. Thus, taken all together, surprisingly, these experiments suggest that TDP1 functions are epistatic with Artemis in the NHEJ pathway for repair of Calicheamicin- and NCS-mediated DNA damage

PROCESSING OF 3′-BLOCKED DNA DOUBLE-STRAND BREAKS BY TYROSYL-DNA PHOSPHODIESTERASE 1, ARTEMIS AND POLYNUCLEOTIDE KINASE/ PHOSPHATASE

DNA double-strand breaks (DSBs) containing unligatable termini are potent cytotoxic lesions leading to growth arrest or cell death. The Artemis nuclease and tyrosyl-DNA phosphodiesterase (TDP1) are each capable of resolving protruding 3′-phosphoglycolate (PG) termini of DNA double-strand breaks (DSBs). Consequently, a knockout of Artemis and a knockout/knockdown of TDP1 rendered cells sensitive to the radiomimetic agent neocarzinostatin (NCS), which induces 3′-PG-terminated DSBs. Unexpectedly, however, a knockdown or knockout of TDP1 in Artemis-null cells did not confer any greater sensitivity than either deficiency alone, indicating a strict epistasis between TDP1 and Artemis. Moreover, a deficiency in Artemis, but not TDP1, resulted in a fraction of unrepaired DSBs, which were assessed as 53BP1 foci. Conversely, a deficiency in TDP1, but not Artemis, resulted in a dramatic increase in dicentric chromosomes following NCS treatment. An inhibitor of DNA-dependent protein kinase, a key regulator of the classical nonhomologous end joining (C-NHEJ) pathway sensitized cells to NCS but eliminated the sensitizing effects of both TDP1 and Artemis deficiencies. Moreover, Polynucleotide Kinase/ Phosphatase (PNKP) is known to process 3′-phosphates and 5′-hydroxyls during DSB repair. PNKP-deficiency sensitized both HCT116 and HeLa cells to 3′-phosphate ended DSBs formed upon radiation and radiomimetic drug treatment. The increased cytotoxicity in the absence of PNKP was synonymous with persistent, un-rejoined 3′-phosphate-ended DSBs. However, DNA-PK deficiency sensitized PNKP-/- cells to low doses of NCS suggesting that, in the absence of PNKP, alternative enzyme(s) can remove 3′-phosphates in a DNA-PK-dependent manner. These results suggest that TDP1 and Artemis perform different functions in the repair of terminally blocked DSBs by the C-NHEJ pathway, and that whereas an Artemis deficiency prevents end joining of some DSBs, a TDP1 deficiency tends to promote DSB mis-joining. In addition, loss of PNKP significantly sensitizes cells to 3′-phosphate-ended DSBs due to a defect in 3′-dephosphorylation

Factors associated with retention in Option B+ in Malawi: a case control study.

IntroductionThere are limited data on factors associated with retention in Option B+. We sought to explore the characteristics of women retained in Option B+ in Malawi, with a focus on the role of HIV disclosure, awareness of partner HIV status, and knowledge around the importance of Option B+ for maternal-child health. Methods We performed a case-control study of HIV-infected women in Malawi initiated on antiretroviral therapy (ART) under Option B+. Cases were enrolled if they met criteria for default from Option B+ (out of ART for >60 days), and controls were enrolled in approximately 3:1 ratio if they were retained in care for at least 12 months. We surveyed socio-demographic characteristics, HIV disclosure and awareness of partner HIV status, self-report about receiving pre-ART education, and knowledge of Option B+. Univariate logistic regression was performed to determine factors associated with retention. Multivariate logistic regression model was used to evaluate the relationship between HIV disclosure, Option B+ knowledge, and retention after adjusting for age, schooling, and travel time to clinic.ResultsWe enrolled 50 cases and 153 controls. Median age was 30 years (interquartile range (IQR) 25-34), and the majority (82%) initiated ART during pregnancy at a median gestational age of 24 weeks (IQR 16-28). Ninety-one per cent of the cases (39/43) who started ART during pregnancy defaulted by three months postpartum. HIV disclosure to the primary sex partner was more common among women retained in care (100% versus 78%, p < 0.001). Odds of retention were significantly higher among women with: age >25 years (odds ratio (OR) 2.44), completion of primary school (OR 3.06), awareness of partner HIV status (OR 5.20), pre-ART education (OR 6.17), higher number of correct answers to Option B+ knowledge questions (OR 1.82), and support while taking ART (OR 3.65). Pre-ART education and knowledge were significantly correlated (r = 0.43, p < 0.001). In multivariate analysis, awareness of partner HIV status (OR 4.07, 95% confidence interval (CI) 1.51-10.94, p = 0.02) and Option B+ knowledge (OR 1.60, 95% CI 1.15-2.23, p = 0.004) remained associated with retention.ConclusionsInterventions that address partner disclosure and strengthen pre-ART education around the benefits of ART for maternal and child health should be evaluated to improve retention in Malawi's Option B+ programme

Carbon nanotube: An indirect ~ 0 eV band gap material

Thin film of carbon was synthesized from camphor (C10H16O) by CVD technique in hydrogen atmosphere. For the first time it is confirmed the presence of almost zero indirect band gap in addition to its direct band gap.. Carrier concentration with intrinsic carbon is found to be around 1021 n/cm3. It is suggested that unless the zero indirect band gap is increased carbon thin film cannot be used for making a p:n junction. XRD, Raman and SEM analysis are performed

Highly effective and isotropic pinning in epitaxial Fe(Se,Te) thin films grown on CaF2 substrates

We report on the isotropic pinning obtained in epitaxial Fe(Se,Te) thin films grown on CaF2 (001) substrate. High critical current density values larger than 1 MA/cm2 in self field in liquid helium are reached together with a very weak dependence on the magnetic field and a complete isotropy. Analysis through Transmission Electron Microscopy evidences the presence of defects looking like lattice disorder at a very small scale, between 5 and 20 nm, which are thought to be responsible for such isotropic behavior in contrast to what observed on SrTiO3, where defects parallel to the c-axis enhance pinning in that directio

INVESTIGATION OF THE NEUROPROTECTIVE EFFECT OF LINAGLIPTIN AND CELIPROLOL IN RESERPINE-INDUCED OROFACIAL DYSKINESIA AND ROTENONE-INDUCED NEURODEGENERATION IN RATS

Objective: Linagliptin, an anti-diabetic agent, proven to play an important role in regulating neuronal plasticity and reduce apoptosis and neuroinflammation by activating downstream AMPK/Sirt 1 pathway, which protects mitochondrial function and suppresses intracellular ROS accumulation and shows antioxidant action. Celiprolol, a β-1selective adrenoceptor blocker used as an anti-hypertensive agent, possesses a direct scavenging activity on oxygen radicals with antioxidant properties. The current study was designed to investigate the combined neuroprotective effect of linagliptin and celiprolol. Methods: Wistar rats of either sex were divided into different groups (n = 6). Eight groups each for Reserpine induced orofacial dyskinesia model and Rotenone induced neurodegeneration model to mimic Parkinson’s like conditions and treated or not with different doses of linagliptin and celiprolol. 24 h after the last dose, animals were subjected to behavioral, biochemical and histopathological evaluations. The data were analyzed by ANOVA and Bonferroni multiple comparison test. Results: Reserpine treatment increased VCMs, tongue protrusion and decreased locomotor activity. Rotenone treatment decreases the motor activity and exploratory ability of the animals. Reserpine as well as rotenone treatments decrease catalase, GSH, SOD and increase the LPO levels as compared to sham group animals. Reserpine and rotenone also showed the presence of ghost cells and vacuolated cytoplasm. Linagliptin and celiprolol alone as well as in combination normalized the behavioral, biochemical and histopathological complications. Conclusion: Linagliptin and Celiprolol showed neuroprotection by antioxidant activity as well as improved reserpine and rotenone-induced behavioral deficits. Both drugs have tenacious potential and can be used clinically with some further investigations

PROTECTIVE EFFECT OF NEBIVOLOL ON ALUMINIUM-INDUCED NEUROBEHAVIORAL AND BIOCHEMICAL ALTERATIONS IN RATS

Objective: The present study was designed to investigate the neuroprotective potential of nebivolol, a β1 adrenergic blocker on aluminium-induced neurobehavioral and biochemical alterations in rats. Methods: The neurotoxicity was induced by administration of aluminium (50 mg/kg/day, p.o.) for 5 weeks. Nebivolol was administered at a dose of 10 mg/kg, p.o. for 5 weeks. Behavioral assessments were done by using open field test and modified elevated plus maze (mEPM) test. At the end of the study, oxidative stress parameters were determined and histopathological studies of cerebral cortex of rat brains were performed. Results: Aluminium chloride treated rats showed significant reduction in motor activity in open field test and memory impairment in mEPM test as compared to control group. Nebivolol significantly reversed these parameters and restored brain antioxidant defensive enzymes with reduction in lipid peroxidation. The neurotoxicity was confirmed by the histopathological analysis of cerebral cortex of rat brains. Aluminium treated animals showed presence of ghost cells, vacuolated cytoplasm and haemorrhage in rat cerebral cortex, indicating neurotoxicity. Nebivolol attenuated all these changes. Thus, the potential of nebivolol to prevent aluminium-induced neurotoxicity was also reflected at microscopic level, indicative of its neuroprotective effects. Conclusion: Nebivolol showed significant antioxidant and neuroprotective activities against aluminium-induced neuronal degeneration. The results of the present study strengthen oxidative stress hypothesis of aluminium-induced neurotoxicity and suggest beneficial role of nebivolol in the treatment of neurodegenerative disorders

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National Research Foundation (NRF) Singapore under its International Research Centres in Singapore Funding Initiativ