A human papillomavirus 16 E2-TopBP1 dependent SIRT1-p300 acetylation switch regulates mitotic viral and human protein levels and activates the DNA damage response.

An interaction between human papillomavirus 16 (HPV16) E2 and the cellular proteins TopBP1 and BRD4 is required for E2 plasmid segregation function. The E2-TopBP1 interaction promotes increased mitotic E2 protein levels in U2OS and N/Tert-1 cells, as well as in human foreskin keratinocytes immortalized by HPV16 (HFK + HPV16). SIRT1 deacetylation reduces E2 protein stability and here we demonstrate that increased E2 acetylation occurs during mitosis in a TopBP1 interacting-dependent manner, promoting E2 mitotic stabilization. p300 mediates E2 acetylation and acetylation is increased due to E2 switching off SIRT1 function during mitosis in a TopBP1 interacting-dependent manner, confirmed by increased p53 stability and acetylation on lysine 382, a known target for SIRT1 deacetylation. SIRT1 can complex with E2 in growing cells but is unable to do so during mitosis due to the E2-TopBP1 interaction; SIRT1 is also unable to complex with p53 in mitotic E2 wild-type cells but can complex with p53 outside of mitosis. E2 lysines 111 and 112 are highly conserved residues across all E2 proteins and we demonstrate that K111 hyper-acetylation occurs during mitosis, promoting E2 interaction with Topoisomerase 1 (Top1). We demonstrate that K112 ubiquitination promotes E2 proteasomal degradation during mitosis. E2-TopBP1 interaction promotes mitotic acetylation of CHK2, promoting phosphorylation and activation of the DNA damage response (DDR). The results present a new model in which the E2-TopBP1 complex inactivates SIRT1 during mitosis, and activates the DDR. This is a novel mechanism of HPV16 activation of the DDR, a requirement for the viral life cycle. IMPORTANCE: Human papillomaviruses (HPVs) are causative agents in around 5% of all human cancers. While there are prophylactic vaccines that will significantly alleviate HPV disease burden on future generations, there are currently no anti-viral strategies available for the treatment of HPV cancers. To generate such reagents, we must understand more about the HPV life cycle, and in particular about viral-host interactions. Here, we describe a novel mitotic complex generated by the HPV16 E2 protein interacting with the host protein TopBP1 that controls the function of the deacetylase SIRT1. The E2-TopBP1 interaction disrupts SIRT1 function during mitosis in order to enhance acetylation and stability of viral and host proteins. We also demonstrate that the E2-TopBP1 interaction activates the DDR. This novel complex is essential for the HPV16 life cycle and represents a novel anti-viral therapeutic target.

Fibroblast Stromal Support Model for Predicting Human Papillomavirus-Associated Cancer Drug Responses.

Currently, there are no specific antiviral therapeutic approaches targeting Human papillomaviruses (HPVs), which cause around 5% of all human cancers. Specific antiviral reagents are particularly needed for HPV-related oropharyngeal cancers (HPV+OPCs) whose incidence is increasing and for which there are no early diagnostic tools available. We and others have demonstrated that the estrogen receptor alpha (ERα) is overexpressed in HPV+OPCs, compared to HPV-negative cancers in this region, and that these elevated levels are associated with an improved disease outcome. Utilizing this HPV+ specific overexpression profile, we previously demonstrated that estrogen attenuates the growth and cell viability of HPV+ keratinocytes and HPV+ cancer cells in vitro. Expansion of this work in vivo failed to replicate this sensitization. The role of stromal support from the tumor microenvironment (TME) has previously been tied to both the HPV lifecycle and in vivo therapeutic responses. Our investigations revealed that in vitro co-culture with fibroblasts attenuated HPV+ specific estrogen growth responses. Continuing to monopolize on the HPV+ specific overexpression of ERα, our co-culture models then assessed the suitability of the selective estrogen receptor modulators (SERMs), raloxifene and tamoxifen, and showed growth attenuation in a variety of our models to one or both of these drugs in vitro. Utilization of these SERMs in vivo closely resembled the sensitization predicted by our co-culture models. Therefore, the in vitro fibroblast co-culture model better predicts in vivo responses. We propose that utilization of our co-culture in vitro model can accelerate cancer therapeutic drug discovery.

A human papillomavirus 16 E2-TopBP1 dependent SIRT1-p300 acetylation switch regulates mitotic viral and human protein levels.

An interaction between human papillomavirus 16 (HPV16) E2 and the cellular proteins TopBP1 and BRD4 is required for E2 plasmid segregation function. The E2-TopBP1 interaction promotes increased mitotic E2 protein levels in U2OS and N/Tert-1 cells, as well as in human foreskin keratinocytes immortalized by HPV16 (HFK+HPV16). SIRT1 deacetylation reduces E2 protein stability and here we demonstrate that increased E2 acetylation occurs during mitosis in a TopBP1 interacting dependent manner, promoting E2 mitotic stabilization. p300 mediates E2 acetylation and acetylation is increased due to E2 switching off SIRT1 function during mitosis in a TopBP1 interacting dependent manner, confirmed by increased p53 stability and acetylation on lysine 382, a known target for SIRT1 deacetylation. SIRT1 can complex with E2 in growing cells but is unable to do so during mitosis due to the E2-TopBP1 interaction; SIRT1 is also unable to complex with p53 in mitotic E2 wild type cells but can complex with p53 outside of mitosis. E2 lysines 111 and 112 are highly conserved residues across all E2 proteins and we demonstrate that K111 hyper-acetylation occurs during mitosis, promoting E2 interaction with Topoisomerase 1 (Top1). We also demonstrate that K112 ubiquitination promotes E2 proteasomal degradation during mitosis. The results present a model in which the E2-TopBP1 complex inactivates SIRT1 during mitosis and E2 acetylation on K111 by p300 increases, promoting interaction with Top1 that protects K112 from ubiquitination and therefore E2 proteasomal degradation. Importance: Human papillomaviruses are causative agents in around 5% of all human cancers. While there are prophylactic vaccines that will significantly alleviate HPV disease burden on future generations, there are currently no anti-viral strategies available for the treatment of HPV cancers. To generate such reagents, we must understand more about the HPV life cycle, and in particular about viral-host interactions. Here we describe a novel mitotic complex generated by the HPV16 E2 protein interacting with the host protein TopBP1 that controls the function of the deacetylase SIRT1. The E2-TopBP1 interaction disrupts SIRT1 function during mitosis in order to enhance acetylation and stability of viral and host proteins. This novel complex is essential for the HPV16 life cycle and represents a novel anti-viral therapeutic target.

Human Papillomavirus 16 replication converts SAMHD1 into a homologous recombination factor and promotes its recruitment to replicating viral DNA.

We have demonstrated that SAMHD1 (sterile alpha motif and histidine-aspartic domain HD-containing protein 1) is a restriction factor for the HPV16 life cycle. Here we demonstrate that in HPV negative cervical cancer C33a cells and human foreskin keratinocytes immortalized by HPV16 (HFK+HPV16), SAMHD1 is recruited to E1-E2 replicating DNA. Homologous recombination (HR) factors are required for HPV16 replication and viral replication promotes phosphorylation of SAMHD1, which converts it from a dNTPase to an HR factor independent from E6/E7 expression. A SAMHD1 phosphor-mimic (SAMHD1 T592D) reduces E1-E2 mediated DNA replication in C33a cells and has enhanced recruitment to the replicating DNA. In HFK+HPV16 cells SAMHD1 T592D is recruited to the viral DNA and attenuates cellular growth, but does not attenuate growth in isogenic HFK cells immortalized by E6/E7 alone. SAMHD1 T592D also attenuates the development of viral replication foci following keratinocyte differentiation. The results indicated that enhanced SAMHD1 phosphorylation could be therapeutically beneficial in cells with HPV16 replicating genomes. Protein phosphatase 2A (PP2A) can dephosphorylate SAMHD1 and PP2A function can be inhibited by endothall. We demonstrate that endothall reduces E1-E2 replication and promotes SAMHD1 recruitment to E1-E2 replicating DNA, mimicking the SAMHD1 T592D phenotypes. Finally, we demonstrate that in head and neck cancer cell lines with HPV16 episomal genomes endothall attenuates their growth and promotes recruitment of SAMHD1 to the viral genome. The results suggest that targeting cellular phosphatases has therapeutic potential for the treatment of HPV infections and cancers. Importance: Human papillomaviruses are causative agents in around 5% of all human cancers. The development of anti-viral therapeutics depends upon an increased understanding of the viral life cycle. Here we demonstrate that HPV16 replication converts SAMHD1 into an HR factor via phosphorylation. This phosphorylation promotes recruitment of SAMHD1 to viral DNA to assist with replication. A SAMHD1 mutant that mimics phosphorylation is hyper-recruited to viral DNA and attenuates viral replication. Expression of this mutant in HPV16 immortalized cells attenuates the growth of these cells, but not cells immortalized by the viral oncogenes E6/E7 alone. Finally, we demonstrate that the phosphatase inhibitor endothall promotes hyper-recruitment of endogenous SAMHD1 to HPV16 replicating DNA and can attenuate the growth of both HPV16 immortalized human foreskin keratinocytes and HPV16 positive head and neck cancer cell lines. We propose that phosphatase inhibitors represent a novel tool for combating HPV infections and disease.

A World Psychiatric Association Initiative to Increase Interest in Psychiatry as a Career in Qatar Medical Students: A Pilot Study.

INTRODUCTION: The World Health Organization identified a chronic shortage of psychiatrists in Low- Middle- and High-Income Countries. In Qatar, the situation is dire with reports there is one psychiatrist per 170,000 people in the population. A one-day, student-led mental-health conference was held in Doha, Qatar under the auspices of the World Psychiatric Association with the aim of increasing interest in psychiatry as a career. SUBJECTS AND METHODS: In this single-arm, pre-post comparison study, a questionnaire assessing interest in psychiatry as a career was administered on participants before and after attending the conference. Demographic information was obtained and response items were on a 5-point Likert scale. Statistical analysis was performed using a two-sample t-test with a significant p-value set at <0.05. RESULTS: The conference was attended by 102 individuals. Fifty-four attendees completed the pre-conference survey (53% response rate) and 36 participants completed the post-conference survey (35% response rate). Data for the 36 matched pre-post responses were included in our analyses. The average age of respondents was 22 years and 81% were female. There was improvement in post-conference results however these changes were not statistically significant. CONCLUSION: To the best of our knowledge this is the first intervention study on increasing interest in psychiatry as a career carried out in Qatar. It is likely that psychiatry enthusiasts attended the conference and therefore their interest in this medical specialty was already high as corroborated by the favorable pre-conference survey results. This might explain why there was a lack of statistical significance in comparison to post- intervention scores. We recommend that such an event be integrated into the medical school curricula throughout Qatar to include students with low baseline interest in psychiatry. Further research in this area with more robust methodology is urgently needed to help narrow the pervasive treatment gap.

Validated RP-HPLC Method for Simultaneous Determination of Ribavirin, Sofosbuvir and Daclatasvir in Human Plasma: A Treatment Protocol Administered to HCV Patients in Egypt.

Egypt has the highest prevalence of hepatitis C virus (HCV) in the world thus it launched a national program for eliminating HCV aiming to treat 300,000 HCV patients per year. Three anti-HCV co-administered drugs; ribavirin (RBV), sofosbuvir (SF) daclatasvir (DAC) were simultaneously determined in human plasma by a validated, simple and sensitive RP-HPLC method using propyl paraben as an internal standard. Liquid-liquid extraction using ethyl acetate was used for samples extraction. Chromatographic separation was achieved on Scharlau® C18 column (250 × 4.6 mm2, 5 µm). Gradient elution was employed with a mobile phase mixture of water and acetonitrile at a flow rate 1 mL/min. UV detection using photodiode array detector was carried out at 207, 260 and 312 nm for RBV, SF and DAC, respectively. Method validation was performed according to the FDA guidelines for bioanalytical method validation. The calibration curves were linear over the ranges (0.5-80, 0.1-40 and 0.5-80 µg/mL) with average recoveries (100.64-108.28%, 98.48-105.91% and 97.68-101.38%) for RBV, SF and DAC, respectively. The intra-day and inter-day precision and accuracy results were within the acceptable limits. Stability assays revealed that the three studied analytes were stable during sample storage, preparation and injection. The method can be successfully applied in routine analysis of plasma of HCV patients treated with this combination therapy which aids in therapeutic drug monitoring and patients' follow-up especially in Egypt and other developing countries fighting HCV.

A new technique to improve the mechanical and biological performance of ultra high molecular weight polyethylene using a nylon coating.

A new patent pending technique is proposed in this study to improve the mechanical and biological performance of ultra high molecular weight polyethylene (UHMWPE), i.e., to uniformly coat nylon onto the UHMWPE fiber (Firouzi et al., 2012). Mechanical tests were performed on neat and new nylon coated UHMWPE fibers to examine the tensile strength and creep resistance of the samples at different temperatures. Cytotoxicity and osteolysis induced by wear debris of the materials were investigated using (MTT) assay, and RT-PCR for tumor necrosis factor alpha (TNFα) and interleukin 6 (IL-6) osteolysis markers. Mechanical test results showed substantial improvement in maximum creep time, maximum breaking force, and toughness values of Nylon 6,6 and Nylon 6,12 coated UHMWPE fibers between average 15% and 60% at 25, 50, and 70°C. Furthermore, cytotoxicity studies have demonstrated significant improvement in cell viability using the nylon coated UHMWPE over the neat one (72.4% vs 54.8%) for 48h and (80.7 vs 5%) for 72h (P<0.01). Osteolysis test results have shown that the expression levels of TNFα and IL-6 markers induced by the neat UHMWPE fiber were significantly higher than those induced by the Nylon 6,6 coated UHMWPE (2.5 fold increase for TNFα at 48h, and three fold increase for IL-6 at 72h (P<0.01)). This study suggests that UHMWPE coated with nylon could be used as a novel material in clinical applications with lower cytotoxicity, less wear debris-induced osteolysis, and superior mechanical properties compared to neat UHMWPE.