Loss of ATRX Confers DNA Repair Defects and PARP Inhibitor Sensitivity in Glioma

Alpha Thalassemia/Mental Retardation Syndrome X-Linked (ATRX) is mutated frequently in gliomas and represents a potential target for cancer therapeutics development. ATRX is known to function as a histone chaperone that incorporates the histone variant, H3.3, into the genome. Studies have implicated ATRX in key DNA damage response (DDR) pathways, including non-homologous end joining (NHEJ), homologous recombination (HR), and replication stress response, but a distinct role for this protein in DNA repair has yet to be fully elucidated. To further investigate ATRX function in glioma, I created CRISPR knockout clones in multiple glioma cell lines. These cell lines were found to have DNA repair defects that indicated increased replication stress. I then determined that the immortalized astrocyte ATRX KO cell line is sensitive to PARP inhibitors. This sensitivity is due to an increase in replication stress identified through increased ATR activation. The DNA repair defect in these cells overlaps with a frequently co-occurring mutation in gliomas, IDH1 R132H, and so leads to equal sensitivity compared to either mutation alone. I also performed a CRISPR screen which highlighted the importance of the chromatin modifications made by ATRX and how knockout cells are more susceptible to chromatin instability. Taken together, these data reveal that ATRX may be used as a molecular marker for DDR defects and PARP inhibitor sensitivity, which is independent of IDH1/2 mutations. These data highlight the important role of common glioma-associated mutations in the regulation of DDR, and they highlight novel avenues for molecularly guided therapeutic intervention

Willingness to work in rural areas and the role of intrinsic versus extrinsic professional motivations - a survey of medical students in Ghana

<p>Abstract</p> <p>Background</p> <p>Retaining health workers in rural areas is challenging for a number of reasons, ranging from personal preferences to difficult work conditions and low remuneration. This paper assesses the influence of intrinsic and extrinsic motivation on willingness to accept postings to deprived areas among medical students in Ghana.</p> <p>Methods</p> <p>A computer-based survey involving 302 fourth year medical students was conducted from May-August 2009. Logistic regression was used to assess the association between students' willingness to accept rural postings and their professional motivations, rural exposure and family parental professional and educational status (PPES).</p> <p>Results</p> <p>Over 85% of students were born in urban areas and 57% came from affluent backgrounds. Nearly two-thirds of students reported strong intrinsic motivation to study medicine. After controlling for demographic characteristics and rural exposure, motivational factors did not influence willingness to practice in rural areas. High family PPES was consistently associated with lower willingness to work in rural areas.</p> <p>Conclusions</p> <p>Although most Ghanaian medical students are motivated to study medicine by the desire to help others, this does not translate into willingness to work in rural areas. Efforts should be made to build on intrinsic motivation during medical training and in designing rural postings, as well as favour lower PPES students for admission.</p

Switchgrass (Panicum virgatum L.) polyubiquitin gene (PvUbi1 and PvUbi2) promoters for use in plant transformation

<p>Abstract</p> <p>Background</p> <p>The ubiquitin protein is present in all eukaryotic cells and promoters from ubiquitin genes are good candidates to regulate the constitutive expression of transgenes in plants. Therefore, two switchgrass (<it>Panicum virgatum </it>L.) ubiquitin genes (<it>PvUbi1 </it>and <it>PvUbi2</it>) were cloned and characterized. Reporter constructs were produced containing the isolated 5' upstream regulatory regions of the coding sequences (i.e. <it>PvUbi1 </it>and <it>PvUbi2 </it>promoters) fused to the <it>uidA </it>coding region (<it>GUS</it>) and tested for transient and stable expression in a variety of plant species and tissues.</p> <p>Results</p> <p><it>PvUbi1 </it>consists of 607 bp containing <it>cis</it>-acting regulatory elements, a 5' untranslated region (UTR) containing a 93 bp non-coding exon and a 1291 bp intron, and a 918 bp open reading frame (ORF) that encodes four tandem, head -to-tail ubiquitin monomer repeats followed by a 191 bp 3' UTR. <it>PvUbi2 </it>consists of 692 bp containing <it>cis</it>-acting regulatory elements, a 5' UTR containing a 97 bp non-coding exon and a 1072 bp intron, a 1146 bp ORF that encodes five tandem ubiquitin monomer repeats and a 183 bp 3' UTR. <it>PvUbi1 </it>and <it>PvUbi2 </it>were expressed in all examined switchgrass tissues as measured by qRT-PCR. Using biolistic bombardment, <it>PvUbi1 </it>and <it>PvUbi2 </it>promoters showed strong expression in switchgrass and rice callus, equaling or surpassing the expression levels of the CaMV <it>35S, 2x35S, ZmUbi1</it>, and <it>OsAct1 </it>promoters. GUS staining following stable transformation in rice demonstrated that the <it>PvUbi1 </it>and <it>PvUbi2 </it>promoters drove expression in all examined tissues. When stably transformed into tobacco (<it>Nicotiana tabacum</it>), the <it>PvUbi2+3 </it>and <it>PvUbi2+9 </it>promoter fusion variants showed expression in vascular and reproductive tissues.</p> <p>Conclusions</p> <p>The <it>PvUbi1 </it>and <it>PvUbi2 </it>promoters drive expression in switchgrass, rice and tobacco and are strong constitutive promoter candidates that will be useful in genetic transformation of monocots and dicots.</p

Biochemical Characterization of the Interaction between Rab Proteins and Myosin XIa in the moss Physcomitrella patens

In Physcomitrella patens, actin-mediated trafficking and myosin XIa are essential for polarized growth. In other model organisms, the association between myosin and its cargo is mediated by Rab-GTPases. We cloned and purified wild type and mutants of moss myosin XIa that fail to complement an RNAi myosin XIa loss-of-polarity phenotype. Through binding assays, we identified RabA21 as having potential myosin XIa binding activity. The disruption of this interaction may be responsible for the lack of rescue of the phenotype

Repair with Hair

Oil spills have been greatly impacting the ecosystem and drinking water in the Niger Delta, causing adverse health effects on the Nigerian people. We propose the use of hair mats to remove oil from contaminated water. Two filter systems were made to attempt to remove oil from the water. Though precise data could not be collected, we were able to visually show the effectiveness of hair mats

The Future of Financial Messaging

Financial telecommunication governs the exchange of information between the computer systems of banks, their clients and other financial institutions. The purpose of this project was to inform a major Swiss bank, Credit Suisse, of market trends and future potential in the sector of financial messaging systems. By interviewing internal and external clients of Credit Suisse, financial software vendors, and competing banks, we identified potential future improvements to Credit Suisse’s current financial messaging system. After analyzing our interview and survey results we developed a list of major findings and a set of recommendations regarding ‘Big Data,’ infrastructure development, the adoption of the ISO 20022 international standards, and the process and impacts of organizational change

Loss of ATRX confers DNA repair defects and PARP inhibitor sensitivity

Alpha Thalassemia/Mental Retardation Syndrome X-Linked (ATRX) is mutated frequently in gliomas and represents a potential target for cancer therapies. ATRX is known to function as a histone chaperone that helps incorporate histone variant, H3.3, into the genome. Studies have implicated ATRX in key DNA damage response (DDR) pathways but a distinct role in DNA repair has yet to be fully elucidated. To further investigate the function of ATRX in the DDR, we created isogenic wild-type (WT) and ATRX knockout (KO) model cell lines using CRISPR-based gene targeting. These studies revealed that loss of ATRX confers sensitivity to poly(ADP)-ribose polymerase (PARP) inhibitors, which was linked to an increase in replication stress, as detected by increased activation of the ataxia telangiectasia and Rad3-related (ATR) signaling axis. ATRX mutations frequently co-occur with mutations in isocitrate dehydrogenase-1 and -2 (IDH1/2), and the latter mutations also induce HR defects and PARP inhibitor sensitivity. We found that the magnitude of PARP inhibitor sensitivity was equal in the context of each mutation alone, although no further sensitization was observed in combination, suggesting an epistatic interaction. Finally, we observed enhanced synergistic tumor cell killing in ATRX KO cells with ATR and PARP inhibition, which is commonly seen in HR-defective cells. Taken together, these data reveal that ATRX may be used as a molecular marker for DDR defects and PARP inhibitor sensitivity, independent of IDH1/2 mutations. These data highlight the important role of common glioma-associated mutations in the regulation of DDR, and novel avenues for molecularly guided therapeutic intervention

Insights into the inhibition of platelet activation by omega-3 polyunsaturated fatty acids: Beyond aspirin and clopidogrel

We sought to examine the effects of escalating doses of omega-3 polyunsaturated fatty acid (PUFA) supplements on platelet function using light transmission aggregometry (LTA) and electrophoretic quasi-elastic light scattering technology (EQELS). PUFA may inhibit platelet function through fatty acid substitution in the platelet membrane by changing the surface charge density and causing decreased production of thromboxane A2. EQELS can measure platelet surface charge density and determine whether the platelet is in resting or activated state. A total of 30volunteers were divided in 3 groups of 10 as follows: Group A, no antiplatelet agent; Group B, daily aspirin only, and Group C, daily aspirin and clopidogrel. All patients received escalating doses of omega-3PUFA from 1 to 8g daily over 24weeks. Platelet function was measured by template bleeding time, LTA, and EQELS at baseline and at 6, 12, 18 and 24weeks. Mean bleeding time increased in a dose-dependent manner with escalating omega-3 PUFA doses. LTA confirmed expected antiplatelet effects of aspirin and clopidogrel, but did not detect any additional antiplatelet effects of omega-3 PUFA. EQELS showed a significant increase in the negative resting platelet charge compared to baseline and an attenuated response to arachidonic acid mediated platelet activation. No bleeding events were observed. In this pilot study we were able to successfully measure platelet surface charge variation as a measure of omega-3 PUFA effect on platelets. Our results suggest that omega-3 PUFA increase the total platelet surface charge and, therefore, attenuate platelet activation, even among patients taking aspirin or aspirin plus clopidogrel. Further studies are needed to determine the clinical significance of these measured effects and EQELS results