THE PML PROTEIN IS NOT ESSENTIAL FOR TELOMERE MAINTENANCE BY THE ALTERNATIVE LENGTHENING OF TELOMERES PATHWAY (ALT) AND ITS DELETION MODIFIES ALT BIOMARKERS

All cancers must maintain their chromosomal ends (telomeres) in order to continue to proliferate indefinitely and to prevent intolerable levels of chromosomal instability. In ~95% of cancers, telomeres are maintained, or extended, through the reactivation of the telomerase enzyme. For the remaining 5% of cancers, a homologous recombination-dependent mechanism called alternative lengthening of telomeres (ALT) is utilized. ALT has several associated characteristics, but no one biomarker is defining. It becomes necessary then to examine a body of data on the presence of these characteristics to confirm a cancer as having ALT as a telomere maintenance mechanism (TMM). One such characteristic is the presence of large ultra-bright telomere DNA foci visible by fluorescence in situ hybridization (FISH), and the co-localization of these foci with immunoaffinity signals for the promyelocytic leukemia protein (PML) which together are referred to as ALT associated PML bodies (APBs). Repeated reports of this phenomenon in the literature, coupled with experiments demonstrating that large ultra-bright telomere FISH foci are a likely site of significant ALT-associated activity have elevated the level of interest in APBs. The body of work described in this thesis addresses the question of whether PML is required for the ALT mechanism and the related question if the presence of APBs is a defining ALT characteristic. CRISPR/Cas9 was used to generate PML knockouts in the ALT-positive human cancer-derived cell lines U2OS and SAOS2. Cell viability and maintenance of telomeres over long term culture, the presence of telomere DNA c-circles, homologous recombination at telomeres, and the absence of telomerase activity were all tested. The results were consistent with ALT representing a TMM in PML-knockout U2OS and SAOS2 cells. Large telomere ultra-bright foci still formed in cells lacking PML, and were found to co-localize with the APB components SP100 and DAXX. However, the extent to which SP100, DAXX and large telomere foci co-localized relative to the total number nuclear bodies formed by each of these proteins was found to be significantly reduced. Therefore, while it is not required for ALT, PML does significantly impact the frequency of co-localization between SP100, DAXX and large telomere foci, which suggests that the function of PML in ALT is as a mediator of nuclear trafficking at APBs

ImageJ2: ImageJ for the next generation of scientific image data

ImageJ is an image analysis program extensively used in the biological sciences and beyond. Due to its ease of use, recordable macro language, and extensible plug-in architecture, ImageJ enjoys contributions from non-programmers, amateur programmers, and professional developers alike. Enabling such a diversity of contributors has resulted in a large community that spans the biological and physical sciences. However, a rapidly growing user base, diverging plugin suites, and technical limitations have revealed a clear need for a concerted software engineering effort to support emerging imaging paradigms, to ensure the software's ability to handle the requirements of modern science. Due to these new and emerging challenges in scientific imaging, ImageJ is at a critical development crossroads. We present ImageJ2, a total redesign of ImageJ offering a host of new functionality. It separates concerns, fully decoupling the data model from the user interface. It emphasizes integration with external applications to maximize interoperability. Its robust new plugin framework allows everything from image formats, to scripting languages, to visualization to be extended by the community. The redesigned data model supports arbitrarily large, N-dimensional datasets, which are increasingly common in modern image acquisition. Despite the scope of these changes, backwards compatibility is maintained such that this new functionality can be seamlessly integrated with the classic ImageJ interface, allowing users and developers to migrate to these new methods at their own pace. ImageJ2 provides a framework engineered for flexibility, intended to support these requirements as well as accommodate future needs

Initial United States experience with the Paracor HeartNet⁎⁎Paracor Medical, Inc, Sunnyvale, Calif. myocardial constraint device for heart failure

ObjectiveThis study was undertaken to review the initial results and surgical safety data for the US Food and Drug Administration safety and feasibility trial of the Paracor HeartNet (Paracor Medical, Inc, Sunnyvale, Calif.) myocardial constraint device.MethodsPatients with New York Heart Association functional class II or III heart failure underwent device implantation (n = 21) through a left minithoracotomy.ResultsThe average age was 53 years (31–72 years). There were 18 men and 3 women, and 17 patients had nonischemic etiology of heart failure. Mean heart failure duration was 8.3 years (1.4-18.8 years). Average ejection fraction was 22% (11%-33%), with an average left ventricular end-diastolic dimension of 74 mm (55-94 mm). Previous medical therapy included angiotensin-converting enzyme inhibitors, β-blockers, diuretics, digoxin, and aldosterone receptor blockers. At implantation, 17 patients had implantable electronic devices: 1 biventricular pacemaker, 11 biventricular pacemakers with cardioverter-defibrillators, and 5 implantable cardioverter-defibrillators. Patient comorbidities included hypertension in 10 cases, diabetes mellitus in 8, myocardial infarction in 1, and ventricular tachycardia in 8. Mean operative time was 68 minutes (42–102 minutes), and implantation time averaged 15 minutes (5–51 minutes). The average time to ambulation was 1.6 days (1–4 days). The intensive care unit stay averaged 3.3 days (1–16 days), and hospital stay averaged 6.3 days (4–16 days). Atrial fibrillation occurred in 2 patients, and there were 2 in-hospital deaths.ConclusionsThe Paracor device can be implanted in patients with heart failure and reduced left ventricular function with a high degree of success. Significant surgical complications were infrequent. The initial US experience supports the conduct of a randomized, controlled, pivotal trial

Expression of paramyxovirus V proteins promotes replication and spread of hepatitis C virus in cultures of primary human fetal liver cells

Here we demonstrate that primary cultures of human fetal liver cells (HFLC) reliably support infection with laboratory strains of hepatitis C virus (HCV), although levels of virus replication vary significantly between different donor cell preparations and frequently decline in a manner suggestive of active viral clearance. To investigate possible contributions of the interferon (IFN) system to control HCV infection in HFLC, we exploited the well-characterized ability of paramyxovirus (PMV) V proteins to counteract both IFN induction and antiviral signaling. The V proteins of measles virus (MV) and parainfluenza virus 5 (PIV5) were introduced into HFLC using lentiviral vectors encoding a fluorescent reporter for visualization of HCV-infected cells. V protein-transduced HFLC supported enhanced (10 to 100-fold) levels of HCV infection relative to untransduced or control vector-transduced HFLC. Infection was assessed by measurement of virus-driven luciferase, by assays for infectious HCV and viral RNA, and by direct visualization of HCV-infected hepatocytes. Live cell imaging between 48 and 119 hours postinfection demonstrated little or no spread of infection in the absence of PMV V protein expression. In contrast, V protein-transduced HFLC showed numerous HCV infection events. V protein expression efficiently antagonized the HCV-inhibitory effects of added IFNs in HFLC. In addition, induction of the type III IFN, IL29, following acute HCV infection was inhibited in V protein-transduced cultures. Conclusion: These studies suggest that the cellular IFN response plays a significant role in limiting the spread of HCV infection in primary hepatocyte cultures. Strategies aimed at dampening this response may be key to further development of robust HCV culture systems, enabling studies of virus pathogenicity and the mechanisms by which HCV spreads in its natural host cell population.National Institutes of Health (U.S.) (NIH Roadmap for Medical Research Grant 1 R01 DK085713-01)Greenberg Institute for Medical ResearchStarr Foundatio

Schmallenberg virus pathogenesis, tropism and interaction with the innate immune system of the host

Schmallenberg virus (SBV) is an emerging orthobunyavirus of ruminants associated with outbreaks of congenital malformations in aborted and stillborn animals. Since its discovery in November 2011, SBV has spread very rapidly to many European countries. Here, we developed molecular and serological tools, and an experimental in vivo model as a platform to study SBV pathogenesis, tropism and virus-host cell interactions. Using a synthetic biology approach, we developed a reverse genetics system for the rapid rescue and genetic manipulation of SBV. We showed that SBV has a wide tropism in cell culture and “synthetic” SBV replicates in vitro as efficiently as wild type virus. We developed an experimental mouse model to study SBV infection and showed that this virus replicates abundantly in neurons where it causes cerebral malacia and vacuolation of the cerebral cortex. These virus-induced acute lesions are useful in understanding the progression from vacuolation to porencephaly and extensive tissue destruction, often observed in aborted lambs and calves in naturally occurring Schmallenberg cases. Indeed, we detected high levels of SBV antigens in the neurons of the gray matter of brain and spinal cord of naturally affected lambs and calves, suggesting that muscular hypoplasia observed in SBV-infected lambs is mostly secondary to central nervous system damage. Finally, we investigated the molecular determinants of SBV virulence. Interestingly, we found a biological SBV clone that after passage in cell culture displays increased virulence in mice. We also found that a SBV deletion mutant of the non-structural NSs protein (SBVΔNSs) is less virulent in mice than wild type SBV. Attenuation of SBV virulence depends on the inability of SBVΔNSs to block IFN synthesis in virus infected cells. In conclusion, this work provides a useful experimental framework to study the biology and pathogenesis of SBV

Measurement of νˉμ\bar{\nu}_{\mu} and νμ\nu_{\mu} charged current inclusive cross sections and their ratio with the T2K off-axis near detector

We report a measurement of cross section $\sigma(\nu_{\mu}+{\rm nucleus}\rightarrow\mu^{-}+X)$ and the first measurements of the cross section $\sigma(\bar{\nu}_{\mu}+{\rm nucleus}\rightarrow\mu^{+}+X)$ and their ratio $R(\frac{\sigma(\bar \nu)}{\sigma(\nu)})$ at (anti-)neutrino energies below 1.5 GeV. We determine the single momentum bin cross section measurements, averaged over the T2K $\bar{\nu}/\nu$-flux, for the detector target material (mainly Carbon, Oxygen, Hydrogen and Copper) with phase space restricted laboratory frame kinematics of $\theta_{\mu}$500 MeV/c. The results are $\sigma(\bar{\nu})=\left( 0.900\pm0.029{\rm (stat.)}\pm0.088{\rm (syst.)}\right)\times10^{-39}$ and $\sigma(\nu)=\left( 2.41\ \pm0.022{\rm{(stat.)}}\pm0.231{\rm (syst.)}\ \right)\times10^{-39}$in units of cm$^{2}$/nucleon and$R\left(\frac{\sigma(\bar{\nu})}{\sigma(\nu)}\right)= 0.373\pm0.012{\rm (stat.)}\pm0.015{\rm (syst.)}$.Comment: 18 pages, 8 figure

Outcome Prediction of Postanoxic Coma:A Comparison of Automated Electroencephalography Analysis Methods

BACKGROUND: To compare three computer-assisted quantitative electroencephalography (EEG) prediction models for the outcome prediction of comatose patients after cardiac arrest regarding predictive performance and robustness to artifacts. METHODS: A total of 871 continuous EEGs recorded up to 3 days after cardiac arrest in intensive care units of five teaching hospitals in the Netherlands were retrospectively analyzed. Outcome at 6 months was dichotomized as "good" (Cerebral Performance Category 1-2) or "poor" (Cerebral Performance Category 3-5). Three prediction models were implemented: a logistic regression model using two quantitative features, a random forest model with nine features, and a deep learning model based on a convolutional neural network. Data from two centers were used for training and fivefold cross-validation (n = 663), and data from three other centers were used for external validation (n = 208). Model output was the probability of good outcome. Predictive performances were evaluated by using receiver operating characteristic analysis and the calculation of predictive values. Robustness to artifacts was evaluated by using an artifact rejection algorithm, manually added noise, and randomly flattened channels in the EEG. RESULTS: The deep learning network showed the best overall predictive performance. On the external test set, poor outcome could be predicted by the deep learning network at 24 h with a sensitivity of 54% (95% confidence interval [CI] 44-64%) at a false positive rate (FPR) of 0% (95% CI 0-2%), significantly higher than the logistic regression (sensitivity 33%, FPR 0%) and random forest models (sensitivity 13%, FPR, 0%) (p < 0.05). Good outcome at 12 h could be predicted by the deep learning network with a sensitivity of 78% (95% CI 52-100%) at a FPR of 12% (95% CI 0-24%) and by the logistic regression model with a sensitivity of 83% (95% CI 83-83%) at a FPR of 3% (95% CI 3-3%), both significantly higher than the random forest model (sensitivity 1%, FPR 0%) (p < 0.05). The results of the deep learning network were the least affected by the presence of artifacts, added white noise, and flat EEG channels. CONCLUSIONS: A deep learning model outperformed logistic regression and random forest models for reliable, robust, EEG-based outcome prediction of comatose patients after cardiac arrest

A Humanized Mouse Model to Study Hepatitis C Virus Infection, Immune Response, and Liver Disease

Studies of hepatitis C virus (HCV) infection, immunopathogenesis, and resulting liver diseases have been hampered by the lack of a small animal model. We developed humanized mice with human immune system and liver tissues to improve the studies of hepatitis C pathogenesis and treatment