Automated Biochemical, Morphological, and Organizational Assessment of Precancerous Changes from Endogenous Two-Photon Fluorescence Images

Multi-photon fluorescence microscopy techniques allow for non-invasive interrogation of live samples in their native environment. These methods are particularly appealing for identifying pre-cancers because they are sensitive to the early changes that occur on the microscopic scale and can provide additional information not available using conventional screening techniques.In this study, we developed novel automated approaches, which can be employed for the real-time analysis of two-photon fluorescence images, to non-invasively discriminate between normal and pre-cancerous/HPV-immortalized engineered tissues by concurrently assessing metabolic activity, morphology, organization, and keratin localization. Specifically, we found that the metabolic activity was significantly enhanced and more uniform throughout the depths of the HPV-immortalized epithelia, based on our extraction of the NADH and FAD fluorescence contributions. Furthermore, we were able to separate the keratin contribution from metabolic enzymes to improve the redox estimates and to use the keratin localization as a means to discriminate between tissue types. To assess morphology and organization, Fourier-based, power spectral density (PSD) approaches were employed. The nuclear size distribution throughout the epithelial depths was quantified by evaluating the variance of the corresponding spatial frequencies, which was found to be greater in the normal tissue compared to the HPV-immortalized tissues. The PSD was also used to calculate the Hurst parameter to identify the level of organization in the tissues, assuming a fractal model for the fluorescence intensity fluctuations within a field. We found the range of organization was greater in the normal tissue and closely related to the level of differentiation.A wealth of complementary morphological, biochemical and organizational tissue parameters can be extracted from high resolution images that are acquired based entirely on endogenous sources of contrast. They are promising diagnostic parameters for the non-invasive identification of early cancerous changes and could improve significantly diagnosis and treatment for numerous patients

Mutations in HPV18 E1^E4 Impact Virus Capsid Assembly, Infectivity Competence, and Maturation.

The most highly expressed protein during the productive phase of the human papillomavirus (HPV) life cycle is E1^E4. Its full role during infection remains to be established. HPV E1^E4 is expressed during both the early and late stages of the virus life cycle and contributes to viral genome amplification. In an attempt to further outline the functions of E1^E4, and determine whether it plays a role in viral capsid assembly and viral infectivity, we examined wild-type E1^E4 as well as four E1^E4 truncation mutants. Our study revealed that HPV18 genomes containing the shortest truncated form of E1^E4, the 17/18 mutant, produced viral titers that were similar to wild-type virus and significantly higher compared to virions containing the three longer E1^E4 mutants. Additionally, the infectivity of virus containing the shortest E1^E4 mutation was equivalent to wild-type and significantly higher than the other three mutants. In contrast, infectivity was completely abrogated for virus containing the longer E1^E4 mutants, regardless of virion maturity. Taken together, our results indicate for the first time that HPV18 E1^E4 impacts capsid assembly and viral infectivity as well as virus maturation

GPRA annual performace report

Part I. CDC Context for Performance Measurement -- Part II. CDC Performance Report -- Appendix A. Approach to Performance Measurement -- Appendix B. Data Verification and Validation -- Appendix C. Key Improvements in the CDC FY 2003 Performance Plan -- Appendix D. Performance Measurement Linkages -- Appendix E. Change Chart for Goals and Measures.2002736

Human papillomavirus E7 oncoprotein induces KDM6A and KDM6B histone demethylase expression and causes epigenetic reprogramming

Despite the availability of vaccines, human papillomavirus (HPV) infections remain a cause of significant cancer morbidity and mortality. We have previously shown that HPV16 E7 associates with and diminishes E2F6-containing polycomb repressive complexes. Here, we show that repressive trimethyl marks on lysine 27 of histone 3, which are necessary for binding of polycomb repressive complexes, are decreased in HPV16 E7-expressing cells and HPV16-positive cervical lesions. This is caused by transcriptional induction of the KDM6A and KDM6B histone 3 lysine 27-specific demethylases. HPV16 E7-mediated KDM6B induction accounts for expression of the cervical cancer biomarker, p16INK4A. Moreover, KDM6A- and KDM6B-responsive Homeobox genes are expressed at significantly higher levels, suggesting that HPV16 E7 results in reprogramming of host epithelial cells. These effects are independent of the ability of E7 to inhibit the retinoblastoma tumor suppressor protein. Most importantly, these effects are reversed when E7 expression is silenced, indicating that this pathway may have prognostic and/or therapeutic significance

Human Papillomavirus Type 16 E7 Oncoprotein Associates with E2F6▿

The papillomavirus life cycle is intimately coupled to the differentiation state of the infected epithelium. Since papillomaviruses lack most of the rate-limiting enzymes required for genome synthesis, they need to uncouple keratinocyte differentiation from cell cycle arrest and maintain or reestablish a replication-competent state within terminally differentiated keratinocytes. The human papillomavirus (HPV) E7 protein appears to be a major determinant for this activity and induces aberrant S-phase entry through the inactivation of the retinoblastoma tumor suppressor and related pocket proteins. In addition, E7 can abrogate p21 and p27. Together, this leads to the activation of E2F1 to E2F5, enhanced expression of E2F-responsive genes, and increased cdk2 activity. E2F6 is a pRB-independent, noncanonical member of the E2F transcription factor family that acts as a transcriptional repressor. E2F6 expression is activated in S phase through an E2F-dependent mechanism and thus may provide a negative-feedback mechanism that slows down S-phase progression and/or exit in response to the activation of the other E2F transcription factors. Here, we show that low- and high-risk HPV E7 proteins, as well as simian virus 40 T antigen and adenovirus E1A, can associate with and inactivate the transcriptional repression activity of E2F6, thereby subverting a critical cellular defense mechanism. This may result in the extended S-phase competence of HPV-infected cells. E2F6 is a component of polycomb group complexes, which bind to silenced chromatin and are critical for the maintenance of cell fate. We show that E7-expressing cells show decreased staining for E2F6/polycomb complexes and that this is at least in part dependent on the association with E2F6

L’École nationale d’apprentissage par la marionnette : un théâtre vivant

Ce rapport propose les résultats d’une recherche évaluative participative qui s’est déroulée sur une période de trois ans (2011 à 2014) et qui visait à documenter la pratique de l’École nationale d’apprentissage par la marionnette (ÉNAM). La démarche qualitative empruntée pour dresser le portrait de ce modèle de création/intervention pour le moins innovateur et singulier, offre un panorama détaillé des différentes constituantes au cœur de ce projet qui s’active maintenant depuis 25 ans avec des personnes qui ont vécu ou vivent des problèmes de santé mentale. Au fil des sections, se retrouve l’ensemble des éléments qui montrent l’originalité, la pertinence et les effets qu’une telle initiative produit au quotidien. L’incursion des auteures au cœur de cette étonnante aventure permet de mieux saisir les logiques et la complexité que ce modèle donne à voir et le défi qu’il pose

KDM6A addiction is caused by the HPV E7 protein.

<p><i>(A</i>,<i>B)</i> KDM6A was depleted in the HPV16 positive SiHa and CaSki cervical carcinoma cell lines, the HPV39 positive cervical cancer cell line Me-180, and the HPV18 positive cervical cancer cell line HeLa; <i>(A)</i> Cell viability was measured by reduction of resazurin. Three independent KDM6A shRNA constructs (60, 61, and 62) were used in the initial experiments in SiHa cells; <i>(B)</i> KDM6A depletion was verified by quantitative real-time RT-PCR; <i>(C-F)</i>. KDM6A was depleted in HFKs expressing control vector, HPV16 E7, E6, or E6 and E7; <i>(C)</i> Cell viability was measured by reduction of resazurin; <i>(D)</i> HPV E7 expression was verified by quantitative real-time RT-PCR and compared to levels found in SiHa and CaSki cervical carcinoma cells; <i>(E)</i> Due to the absence of appropriate antibodies, HPV16 E6 expression was determined by assessing p53 levels, which are decreased in HPV16 E6-expressing cells because of E6-mediated proteasomal degradation [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006661#ppat.1006661.ref066" target="_blank">66</a>], by Western blot analysis of p53. Lysates were separated by SDS/PAGE, transferred, and probed for p53. An actin blot is included as a loading control; <i>(F)</i> KDM6A depletion was verified by quantitative real-time RT-PCR; <i>(G-I)</i> KDM6A was depleted in HFKs expressing HPV6, HPV11, or HPV18 E7; <i>(G)</i> Cell viability was measured by reduction of resazurin; <i>(H)</i> HPV6 E7, HPV11 E7, and HPV18 E7 mRNA levels were determined by qRT-PCR; <i>(I)</i> KDM6A depletion was verified by quantitative real-time RT-PCR; <i>(J</i>,<i>K)</i> KDM6A was depleted in U2OS-tet on cells with doxycycline-inducible expression of HPV16 E7 using a KDM6A shRNA construct or KDMA6A-specific siRNA duplexes. <i>(J)</i>. Cell viability was measured by reduction of resazurin; <i>(K)</i> Western blot analysis of HPV16 E7 and KDM6A. Lysates were separated by SDS/PAGE, transferred, and probed for HPV16 E7 and KDM6A. An actin blot is included as a loading control; <i>(L)</i> KDM6A was depleted in the HPV16 positive SiHa cervical carcinoma cell line. After 10 days of puromycin selection, live cells were stained with sulforhodamine B; and <i>(M)</i> Cell cycle profile determined by fluorescence-activated sorting from a representative experiment and percentage of sub-G1 phase from three independent experiments. Averages and SDs for three independent experiments are shown. Statistically significant changes are indicated, **<i>P</i> < 0.01.</p