Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage

<p>Abstract</p> <p>Background</p> <p>The DNA-dependent protein kinase (DNA-PK) is a nuclear complex composed of a large catalytic subunit (DNA-PKcs) and a heterodimeric DNA-targeting subunit Ku. DNA-PK is a major component of the non-homologous end-joining (NHEJ) repair mechanism, which is activated in the presence of DNA double-strand breaks induced by ionizing radiation, reactive oxygen species and radiomimetic drugs. We have recently reported that down-regulation of protein kinase CK2 by siRNA interference results in enhanced cell death specifically in DNA-PKcs-proficient human glioblastoma cells, and this event is accompanied by decreased autophosphorylation of DNA-PKcs at S2056 and delayed repair of DNA double-strand breaks.</p> <p>Results</p> <p>In the present study, we show that CK2 co-localizes with phosphorylated histone H2AX to sites of DNA damage and while CK2 gene knockdown is associated with delayed DNA damage repair, its overexpression accelerates this process. We report for the first time evidence that lack of CK2 destabilizes the interaction of DNA-PKcs with DNA and with Ku80 at sites of genetic lesions. Furthermore, we show that CK2 regulates the phosphorylation levels of DNA-PKcs only in response to direct induction of DNA double-strand breaks.</p> <p>Conclusions</p> <p>Taken together, these results strongly indicate that CK2 plays a prominent role in NHEJ by facilitating and/or stabilizing the binding of DNA-PKcs and, possibly other repair proteins, to the DNA ends contributing to efficient DNA damage repair in mammalian cells.</p

Co-expression of adjacent genes in yeast cannot be simply attributed to shared regulatory system

<p>Abstract</p> <p>Background</p> <p>Adjacent gene pairs in the yeast genome have a tendency to express concurrently. Sharing of regulatory elements within the intergenic region of those adjacent gene pairs was often considered the major mechanism responsible for such co-expression. However, it is still in debate to what extent that common transcription factors (TFs) contribute to the co-expression of adjacent genes. In order to resolve the evolutionary aspect of this issue, we investigated the conservation of adjacent pairs in five yeast species. By using the information for TF binding sites in promoter regions available from the MYBS database <url>http://cg1.iis.sinica.edu.tw/~mybs/</url>, the ratios of TF-sharing pairs among all the adjacent pairs in yeast genomes were analyzed. The levels of co-expression in different adjacent patterns were also compared.</p> <p>Results</p> <p>Our analyses showed that the proportion of adjacent pairs conserved in five yeast species is relatively low compared to that in the mammalian lineage. The proportion was also low for adjacent gene pairs with shared TFs. Particularly, the statistical analysis suggested that co-expression of adjacent gene pairs was not noticeably associated with the sharing of TFs in these pairs. We further proposed a case of the PAC (polymerase A and C) and RRPE (rRNA processing element) motifs which co-regulate divergent/bidirectional pairs, and found that the shared TFs were not significantly relevant to co-expression of divergent promoters among adjacent genes.</p> <p>Conclusion</p> <p>Our findings suggested that the commonly shared <it>cis</it>-regulatory system does not solely contribute to the co-expression of adjacent gene pairs in yeast genome. Therefore we believe that during evolution yeasts have developed a sophisticated regulatory system that integrates both TF-based and non-TF based mechanisms(s) for concurrent regulation of neighboring genes in response to various environmental changes.</p

A high-throughput fluorescence polarization assay for discovering inhibitors targeting the DNA-binding domain of signal transducer and activator of transcription 3 (STAT3)

Anti-cancer drug discovery efforts to directly inhibit the signal transducer and activator of transcription 3 (STAT3) have been active for over a decade following the discovery that 70% of cancers exhibit elevated STAT3 activity. The majority of research has focused on attenuating STAT3 activity through preventing homo-dimerization by targeting the SH2 or transcriptional activation domains. Such dimerization inhibitors have not yet reached the market. However, an alternative strategy focussed on preventing STAT3 DNA-binding through targeting the DNA-binding domain (DBD) offers new drug design opportunities. Currently, only EMSA and ELISA-based methods have been implemented with suitable reliability to characterize STAT3 DBD inhibitors. Herein, we present a new orthogonal, fluorescence polarization (FP) assay suitable for high-throughput screening of molecules. This assay, using a STAT3127-688construct, was developed and optimized to screen molecules that attenuate the STAT3:DNA association with good reliability (Z' value > 0.6) and a significant contrast (signal-tonoise ratio > 15.0) at equilibrium. The assay system was stable over a 48 hour period. Significantly, the assay is homogeneous and simple to implement for high-throughput screening compared to EMSA and ELISA. Overall, this FP assay offers a new way to identify and characterize novel molecules that inhibit STAT3:DNA association

Combination of cyclic nucleotide modulators with P2Y12 receptor antagonists as anti-platelet therapy

"This is the peer reviewed version of the following article: Armstrong, PC, Ferreira, PM, Chan, MV, et al. Combination of cyclic nucleotide modulators with P2Y12 receptor antagonists as anti‐platelet therapy. J Thromb Haemost. 2020 https://doi.org/10.1111/jth.14826 which has been published in final form at   https://doi.org/10.1111/jth.14826BACKGROUND: Endothelium-derived prostacyclin and nitric oxide elevate platelet cyclic nucleotide levels and maintain quiescence. We previously demonstrated that a synergistic relationship exists between cyclic nucleotides and P2Y12 receptor inhibition. A number of clinically approved drug classes can modulate cyclic nucleotide tone in platelets including activators of NO-sensitive guanylyl cyclase (GC) and phosphodiesterase (PDE) inhibitors. However, the doses required to inhibit platelets produce numerous side effects including headache. OBJECTIVE: We investigated using GC-activators in combination with P2Y12 receptor antagonists as a way to selectively amplify the anti-thrombotic effect of both drugs. METHODS: In vitro light transmission aggregation and platelet adhesion under flow were performed on washed platelets and platelet rich plasma. Aggregation in whole blood and a ferric chloride-induced arterial thrombosis model were also performed. RESULTS: The GC-activator BAY-70 potentiated the action of the P2Y12 receptor inhibitor prasugrel active metabolite in aggregation and adhesion studies and was associated with raised intra-platelet cyclic nucleotide levels. Furthermore, mice administered sub-maximal doses of the GC activator cinaciguat together with the PDE inhibitor dipyridamole and prasugrel, showed significant inhibition of ex vivo platelet aggregation and significantly reduced in vivo arterial thrombosis in response to injury without alteration in basal carotid artery blood flow. CONCLUSIONS: Using in vitro, ex vivo, and in vivo functional studies, we show that low dose GC activators synergize with P2Y12 inhibition to produce powerful anti-platelet effects without altering blood flow. Therefore, modulation of intra-platelet cyclic nucleotide levels alongside P2Y12 inhibition can provide a strong, focused anti-thrombotic regimen while minimizing vasodilator side effects

Relationship between maximal incremental and high-intensity interval exercise performance in elite athletes

This descriptive study aimed to explore the physiological factors that determine tolerance to exertion during high-intensity interval effort. Forty-seven young women (15–28 years old) were enrolled: 23 athletes from Taiwan national or national reserve teams and 24 moderately active females. Each participant underwent a maximal incremental INC (modified Bruce protocol) cardiopulmonary exercise test on the first day and high-intensity interval testing (HIIT) on the second day, both performed on a treadmill. The HIIT protocol involved alternation between 1-min effort at 120% of the maximal speed, at the same slope reached at the end of the INC, and 1-min rest until volitional exhaustion. Gas exchange, heart rate (HR), and muscle oxygenation at the right vastus lateralis, measured by near-infrared spectroscopy, were continuously recorded. The number of repetitions completed (Rlim) by each participant was considered the HIIT tolerance index. The results showed a large difference in the Rlim (range, 2.6–12.0 repetitions) among the participants. Stepwise linear regression revealed that the variance in the Rlim within the cohort was related to the recovery rates of oxygen consumption (), HR at the second minute after INC, and muscle tissue saturation index at exhaustion (R = 0.644). In addition, age was linearly correlated with Rlim (adjusted R = −0.518, p \u3c 0.0001). In conclusion, the recovery rates for and HR after the incremental test, and muscle saturation index at exhaustion, were the major physiological factors related to HIIT performance. These findings provide insights into the role of the recovery phase after maximal INC exercise testing. Future research investigating a combination of INC and HIIT testing to determine training-induced performance improvement is warranted

No entropy enigmas for N=4 dyons

We explain why multi-centered black hole configurations where at least one of the centers is a large black hole do not contribute to the indexed degeneracies in theories with N=4 supersymmetry. This is a consequence of the fact that such configurations, although supersymmetric, belong to long supermultiplets. As a result, there is no entropy enigma in N=4 theories, unlike in N=2 theories.Comment: 14 page

Optimizing Human Synovial Fluid Preparation for Two-Dimensional Gel Electrophoresis

<p>Abstract</p> <p>Background</p> <p>Proteome analysis is frequently applied in identifying the proteins or biomarkers in knee synovial fluids (SF) that are associated with osteoarthritis and other arthritic disorders. The 2-dimensional gel electrophoresis (2-DE) is the technique of choice in these studies. Disease biomarkers usually appear in low concentrations and may be masked by high abundant proteins. Therefore, the main aim of this study was to find the most suitable sample preparation method that can optimize the expression of proteins on 2-DE gels that can be used to develop a reference proteome picture for non-osteoarthritic knee synovial fluid samples. Proteome pictures obtained from osteoarthritic knee synovial fluids can then be compared with the reference proteome pictures obtained in this study to assist us in identifying the disease biomarkers more correctly.</p> <p>Results</p> <p>The proteomic tool of 2-DE with immobilized pH gradients was applied in this study. A total of 12 2-DE gel images were constructed from SF samples that were free of osteoarthritis. In these samples, 3 were not treated with any sample preparation methods, 3 were treated with acetone, 3 were treated with 2-DE Clean-Up Kit, and 3 were treated with the combination of acetone and 2-D Clean-Up Kit prior to 2-DE analysis. Gel images were analyzed using the PDQuest Basic 8.0.1 Analytical software. Protein spots that were of interest were excised from the gels and sent for identification by mass spectrometry. Total SF total protein concentration was calculated to be 21.98 ± 0.86 mg/mL. The untreated SF samples were detected to have 456 ± 33 protein spots on 2-DE gel images. Acetone treated SF samples were detected to have 320 ± 28 protein spots, 2-D Clean-Up Kit treated SF samples were detected to have 413 ± 31 protein spots, and the combined treatment method of acetone and 2-D Clean-Up Kit was detected to have 278 ± 26 protein spots 2-DE gel images. SF samples treated with 2-D Clean-Up Kit revealed clearer presentation of the isoforms and increased intensities of the less abundant proteins of haptoglobin, apolipoprotein A-IV, prostaglandin-D synthase, alpha-1B-glycoprotein, and alpha-2-HS-glycoprotein on 2-DE gel images as compared with untreated SF samples and SF samples treated with acetone.</p> <p>Conclusions</p> <p>The acetone precipitation method and the combined treatment effect of acetone and 2-DE Clean-Up Kit are not preferred in preparing SF samples for 2-DE analysis as both protein intensities and numbers decrease significantly. On the other hand, 2-D Clean-Up Kit treated SF samples revealed clearer isoforms and higher intensities for the less abundant proteins of haptoglobin, apolipoprotein A-IV, prostaglandin-D synthase, alpha-1B-glycoprotein, and alpha-2-HS-glycoprotein on 2-DE gels. As a result, it is recommended that SF samples should be treated with protein clean up products such as 2-D Clean-Up Kit first before conducting proteomic research in searching for the relevant biomarkers associated with knee osteoarthritis.</p

Conducting Research with Stigmatized Populations: Practices, Challenges, and Lessons Learned

Conducting research with communities who are at risk of being stigmatized can be a challenging endeavor. It is often difficult to reach and recruit individuals for research purposes regarding a stigmatized condition or situation. Yet, researchers in our field have recognized the importance of work in this area and have individually developed a range of strategies to reach, recruit, and work with these populations. This workshop will invite researchers and practitioners to present, discuss, and compare strategies and experiences when working with stigmatized communities in the context of the ever-evolving nature of technology. The outcomes of the workshop will include an outline for an article that will summarize the strategies and practices discussed as well as identify the approaches that have led to the best outcomes across different populations

The regional differences in prevalence, medical expenditures and risk factors for injury in Taiwanese teenagers

BACKGROUND: Injury is the leading cause of death in teenagers worldwide. In Taiwan, people in mountainous areas have a 4 to 8 years shorter life span than the general population. Injury among teenagers is likely a major cause. The objective of this study was to investigate the regional differences in the prevalence, the risk factors, and the medical expenditures for injury among Taiwanese teenagers. METHOD: An equal probability national sample was used. In addition, representative samples from mountainous areas and offshore islands were used. Only those who aged between 12 and 21 years, and signed the consent form permitting us to link their National Health Insurance (NHI) claim data were included in the analysis. Injury-related visits and expenditures in outpatient services were extracted from the NHI data. Logistic regression was used to examine the factors associated with injury. For those who had injury related outpatient visits, mixed model was used to examine the factors associated with medical expenditures accounting for multiple visits by the same individual. RESULTS: The prevalence of nonfatal injury was around 30% of teenagers in Taiwan. It was 10% higher in mountainous areas. Factors associated with injury were those who lived in mountainous areas (adjusted odds ratio [OR]: 1.7; 95%; confidence interval [CI]: 1.3–2.3), males (OR: 1.3; 95%; CI: 1.1–1.6), older teens (18–21 years old), and those with risk behavior were positively associated with injury. These factors were also associated with the number of injury-related outpatient visits. However, teenagers in mountainous areas did not spend more on medical care than those who lived in metropolitan Taiwan. CONCLUSION: Around 30% of the teenagers were injured in a year, not including the dead. Each of the injured spent at least 851.4NTD (~27USD) for outpatient visits. The scope of the problem was not trivial. Hazardous environments and high-risk behaviors were the universal causes. In remote areas, lack of medical resources was another possibility. Empowering local people to design prevention programs according to their needs is recommended