Investigating the role of Tyrosyl-DNA Phosphodiesterase 1 in nuclear and mitochondrial DNA repair

Damages to the genetic materials arise throughout the lifespan of a cell, and elicit upregulation of DNA repair factors. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is part of a DNA repair protein complex that specialises in the repair of DNA base modifications and single-strand breaks (SSBs). TDP1 removes a broad spectrum of chemical adducts from the 3’ end of a DNA strand break, including topoisomerase 1 (TOP1) peptide, during DNA transcription and replication. Inactivation or deletion of TDP1 is associated with cerebellar dysfunction and degeneration, with remarkably little extraneurological manifestation. The reason for the selective dependence of the cerebellar neurons on TDP1 activity is not clear. It was hypothesised that the TDP1 activity is upregulated in tissues with high levels of SSBs, either from DNA transcriptional activity, or reactive oxygen species (ROS)-induced damage. The aim of this doctoral project was therefore to identify and characterise the cellular mechanisms that regulate TDP1 activity. Our lab has previously shown that the Nterminus domain (NTD) of TDP1 covalently interacts with DNA ligase 3α. In this thesis, evidence has been presented to show that this interaction is regulated by the putative ATM/ATR/DNA-PK phosphorylation site, serine 81, to prolong TDP1 half-life, and enhance cellular survival after genotoxic stress. A second post-translational modification in the NTD by SUMOylation of the K111 residue was identified, enlightening a mechanism by which TDP1 is recruited to sites of transcription-mediated SSBs. To investigate the requirement for TDP1 in cells under high levels of oxidative stress, I have developed a mouse cellular model whereby the levels of endogenous ROS can be modulated by overexpression of the human anti-oxidant enzyme superoxide dismutase 1 (SOD1) or its toxic mutant SOD1G93A. Overexpression of SOD1G93A in Tdp1-/- MEFs induces accumulation of chromosomal SSBs and decreases survival after H2O2 challenge, while overexpression of SOD1 has a protective effect. Besides repair of ROS-induced TOP1-cc in the nucleus, TDP1 also repairs mitochondrial topoisomerase 1-mediated DNA breaks. This role is required during transcription and assembly of mitochondrial subunits of the electron transfer chain complexes, and has direct impact on mitochondrial respiration and ROS production. Collectively, these data provide mechanistic insights into regulation of TDP1-mediated chromosomal and mitochondrial DNA repair

ATM deficiency results in accumulation of DNA-Topoisomerase I covalent intermediates in neural cells

Accumulation of peptide-linked DNA breaks contributes to neurodegeration in humans. This is typified by defects in tyrosyl DNA phosphodiesterase 1 (TDP1) and human hereditary ataxia. TDP1 primarily operates at single-strand breaks (SSBs) created by oxidative stress or by collision of transcription machinery with topoisomerase I intermediates (Top1-CCs). Cellular and cell-free studies have shown that Top1 at stalled Top1-CCs is first degraded to a small peptide resulting in Top1-SSBs, which are the primary substrates for TDP1. Here we established an assay to directly compare Top1-SSBs and Top1-CCs. We subsequently employed this assay to reveal an increased steady state level of Top1-CCs in neural cells lacking Atm; the protein mutated in ataxia telangiectasia. Our data suggest that the accumulation of endogenous Top1-CCs in Atm-/- neural cells is primarily due to elevated levels of reactive oxygen species. Biochemical purification of Top1-CCs from neural cell extract and the use of Top1 poisons further confirmed a role for Atm during the formation/resolution of Top1-CCs. Finally, we report that global transcription is reduced in Atm-/- neural cells and fails to recover to normal levels following Top1-mediated DNA damage. Together, these data identify a distinct role for ATM during the formation/resolution of neural Top1-CCs and suggest that their accumulation contributes to the neuropathology of ataxia telangiectasia

Spermatic Cord Metastasis of Primary Hepatocellular Carcinoma Presenting as an Inguinal Mass: A Case Report

Most spermatic cord masses are benign, and malignant spermatic cord tumors are uncommon. Spermatic cord metastases originating from hepatocellular carcinoma (HCC) have not been previously reported in the English language literature as determined by a PubMed search. We report a male patient who presented with a painful palpable mass in the right inguinal area. The patient was diagnosed with HCC in 2004 and undertook a nonsurgical approach to control the cancer. A radical orchiectomy was performed, and the pathological report showed metastatic HCC in the spermatic cord. The patient received palliative radiation therapy because of a positive surgical margin. No recurrence was noted after 6 months of followup

UNDERSTANDING COMPETITIVE PERFORMANCE OF SOFTWARE-AS-A-SERVICE (SAAS)—THE COMPETITIVE DYNAMICS PERSPECTIVE

Understanding the antecedents and consequences of a firm’s agility in cloud software applications is important. This papers draws on the competitive dynamics perspective to develop a model that explains the relationships between collaboration with vendors, agility, and competitive performance in software-as-a-service (SaaS) context. Collaboration reflects a firm’s ability to leverage interfirm resources, characterized as knowledge sharing and process alignment. Agility is measured by a firm’s strategy-oriented agility and service-oriented agility. This study also investigates the moderating effect of environmental turbulence. The proposed hypotheses are supported by the empirical data. The results show that competitive performance is affected by ability, which, in turn, is impacted by collaboration. Environmental turbulence positively moderates the relationship between agility and performance. Finally, we discuss the implications of our results

Expression of a pathogenic mutation of SOD1 sensitizes aprataxin-deficient cells and mice to oxidative stress and triggers hallmarks of premature ageing

Aprataxin (APTX) deficiency causes progressive cerebellar degeneration, ataxia and oculomotor apraxia in man. Cell free assays and crystal structure studies demonstrate a role for APTX in resolving 5'-adenylated nucleic acid breaks, however, APTX function in vertebrates remains unclear due to the lack of an appropriate model system. Here, we generated a murine model in which a pathogenic mutant of superoxide dismutase 1 (SOD1(G93A)) is expressed in an Aptx-/- mouse strain. We report a delayed population doubling and accelerated senescence in Aptx-/- primary mouse fibroblasts, which is not due to detectable telomere instability or cell cycle deregulation but is associated with a reduction in transcription recovery following oxidative stress. Expression of SOD1(G93A) uncovers a survival defect ex vivo in cultured cells and in vivo in tissues lacking Aptx. The surviving neurons feature numerous and deep nuclear envelope invaginations, a hallmark of cellular stress. Furthermore, they possess an elevated number of high-density nuclear regions and a concomitant increase in histone H3 K9 trimethylation, hallmarks of silenced chromatin. Finally, the accelerated cellular senescence was also observed at the organismal level as shown by down-regulation of insulin-like growth factor 1 (IGF-1), a hallmark of premature ageing. Together, this study demonstrates a protective role of Aptx in vivo and suggests that its loss results in progressive accumulation of DNA breaks in the nervous system, triggering hallmarks of premature ageing, systemically

UVA-induced carbon-centered radicals in lightly pigmented cells detected using ESR spectroscopy

Ultraviolet-A and melanin are implicated in melanoma, but whether melanin in vivo screens or acts as a UVA photosensitiser is debated. Here, we investigate the effect of UVA-irradiation on non-pigmented, lightly and darkly pigmented melanocytes and melanoma cells using electron spin resonance (ESR) spectroscopy. Using the spin trap 5,5 Dimethyl-1-pyrroline N-oxide (DMPO), carbon adducts were detected in all cells. However, higher levels of carbon adducts were detected in lightly pigmented cells than in non-pigmented or darkly pigmented cells. Nevertheless, when melanin levels were artificially increased in lightly pigmented cells by incubation with L-Tyrosine, the levels of carbon adducts decreased significantly. Carbon adducts were also detected in UVA-irradiated melanin-free cell nuclei, DNA-melanin systems, and the nucleoside 2’-deoxyguanosine combined with melanin, whereas they were only weakly detected in irradiated synthetic melanin and not at all in irradiated 2’-deoxyguanosine. The similarity of these carbon adducts suggests they may be derived from nucleic acid– guanine – radicals. These observations suggest that melanin is not consistently a UVA screen against free-radical formation in pigmented cells, but may also act as a photosensitizer for the formation of nucleic acid radicals in addition to superoxide. The findings are important for our understanding of the mechanism of damage caused by the UVA component of sunlight in non-melanoma and melanoma cells, and hence the causes of skin cancer

The dimer interface of the SARS coronavirus nucleocapsid protein adapts a porcine respiratory and reproductive syndrome virus-like structure

AbstractWe have employed NMR to investigate the structure of SARS coronavirus nucleocapsid protein dimer. We found that the secondary structure of the dimerization domain consists of five α helices and a β-hairpin. The dimer interface consists of a continuous four-stranded β-sheet superposed by two long α helices, reminiscent of that found in the nucleocapsid protein of porcine respiratory and reproductive syndrome virus. Extensive hydrogen bond formation between the two hairpins and hydrophobic interactions between the β-sheet and the α helices render the interface highly stable. Sequence alignment suggests that other coronavirus may share the same structural topology

Protective effects of Scoparia dulcis L. extract on high glucose-induced injury in human retinal pigment epithelial cells

Diabetic retinopathy (DR) is a major cause of vision loss in diabetic patients. Hyperglycemia-induced oxidative stress and the accumulation of inflammatory factors result in blood-retinal barrier dysfunction and the pathogenesis of DR. Scoparia dulcis L. extract (SDE), a traditional Chinese medicine, has been recently recognized for its various pharmacological effects, including anti-diabetic, anti-hyperlipidemia, anti-inflammatory, and anti-oxidative activities. However, there is no relevant research on the protective effect of SDE in DR. In this study, we treated high glucose (50 mM) in human retinal epithelial cells (ARPE-19) with different concentrations of SDE and analyzed cell viability, apoptosis, and ROS production. Moreover, we analyzed the expression of Akt, Nrf2, catalase, and HO-1, which showed that SDE dose-dependently reduced ROS production and attenuated ARPE-19 cell apoptosis in a high-glucose environment. Briefly, we demonstrated that SDE exhibited an anti-oxidative and anti-inflammatory ability in protecting retinal cells from high-glucose (HG) treatment. Moreover, we also investigated the involvement of the Akt/Nrf2/HO-1 pathway in SDE-mediated protective effects. The results suggest SDE as a nutritional supplement that could benefit patients with DR