Probabilistic Tracking and Behavior Identification of Fluorescent Particles

Explicit and tractable characterizations of the dynamical behavior of virus particles are pivotal for a thorough understanding of the infection mechanisms of viruses. This thesis deals with the problem of extracting symbolic representations of the dynamical behavior of fluorescent particles from fluorescence microscopy image sequences. The focus is on the behavior of virus particles such as fusion with the cell membrane. A numerical representation is obtained by tracking the particles in the image sequences. We have investigated probabilistic tracking approaches, including approaches based on the Kalman filter as well as based on particle filters. For reasons of efficiency and robustness, we developed a tracking approach based on the probabilistic data association (PDA) algorithm in combination with an ellipsoidal sampling scheme that exploits effectively the image data via parametric appearance models. To track objects in close proximity, we compute the support that each image position provides to each tracked object relative to the support provided to the object's neighbors. After tracking, the problem of mapping the trajectory information computed by the tracking approaches to symbolic representations of the behavior arises. To compute symbolic representations of behaviors related to the fusion of single virus particles with the cell membrane based on their intensity over time, we developed a layered probabilistic approach based on stochastic hybrid systems as well as hidden Markov models (HMMs). We use a maxbelief strategy to efficiently combine both representations. The layered approach describes the intensity, intensity models, and behaviors of single virus particles. We introduce models for the evolution of the intensity and the behavior. To compute estimates for the intensity, intensity models, and behaviors we use a hybrid particle filter and the Viterbi algorithm. The developed approaches have been applied to synthetic images as well as to real microscopy image sequences displaying human immunodeficiency virus (HIV-1) particles. We have performed an extensive quantitative evaluation of the performance and a comparison with several existing approaches. It turned out that our approaches outperform previous ones, thus yielding more accurate and more reliable information about the behavior of virus particles. Moreover, we have successfully applied our tracking approaches to 3D image sequences displaying herpes simplex virus (HSV) replication compartments. We also applied the tracking approaches to image data displaying microtubule tips and analyzed their motion. In addition, our tracking approaches were successfully applied to the 2D and 3D image data of a Particle Tracking Challenge

Dynamics of HIV-1 Assembly and Release

Assembly and release of human immunodeficiency virus (HIV) occur at the plasma membrane of infected cells and are driven by the Gag polyprotein. Previous studies analyzed viral morphogenesis using biochemical methods and static images, while dynamic and kinetic information has been lacking until very recently. Using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated the assembly and release of fluorescently labeled HIV-1 at the plasma membrane of living cells with high time resolution. Gag assembled into discrete clusters corresponding to single virions. Formation of multiple particles from the same site was rarely observed. Using a photoconvertible fluorescent protein fused to Gag, we determined that assembly was nucleated preferentially by Gag molecules that had recently attached to the plasma membrane or arrived directly from the cytosol. Both membrane-bound and cytosol derived Gag polyproteins contributed to the growing bud. After their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over 1–2 hours. Assembly kinetics were rapid: the number of Gag molecules at a budding site increased, following a saturating exponential with a rate constant of ∼5×10−3 s−1, corresponding to 8–9 min for 90% completion of assembly for a single virion. Release of extracellular particles was observed at ∼1,500±700 s after the onset of assembly. The ability of the virus to recruit components of the cellular ESCRT machinery or to undergo proteolytic maturation, or the absence of Vpu did not significantly alter the assembly kinetics

Objective comparison of particle tracking methods

Particle tracking is of key importance for quantitative analysis of intracellular dynamic processes from time-lapse microscopy image data. Because manually detecting and following large numbers of individual particles is not feasible, automated computational methods have been developed for these tasks by many groups. Aiming to perform an objective comparison of methods, we gathered the community and organized an open competition in which participating teams applied their own methods independently to a commonly defined data set including diverse scenarios. Performance was assessed using commonly defined measures. Although no single method performed best across all scenarios, the results revealed clear differences between the various approaches, leading to notable practical conclusions for users and developers

Chromosome segregation by the Escherichia coli

The mechanisms underlying chromosome segregation in prokaryotes remain a subject of debate and no unifying view has yet emerged. Given that the initial disentanglement of duplicated chromosomes could be achieved by purely entropic forces, even the requirement of an active prokaryotic segregation machinery has been questioned. Using computer simulations, we show that entropic forces alone are not sufficient to achieve and maintain full separation of chromosomes. This is, however, possible by assuming repeated binding of chromosomes along a gradient of membrane-associated tethering sites toward the poles. We propose that, in Escherichia coli, such a gradient of membrane tethering sites may be provided by the oscillatory Min system, otherwise known for its role in selecting the cell division site. Consistent with this hypothesis, we demonstrate that MinD binds to DNA and tethers it to the membrane in an ATP-dependent manner. Taken together, our combined theoretical and experimental results suggest the existence of a novel mechanism of chromosome segregation based on the Min system, further highlighting the importance of active segregation of chromosomes in prokaryotic cell biology

Herpesviral Replication Compartments Move and Coalesce at Nuclear Speckles to Enhance Export of Viral Late mRNA

The role of the intranuclear movement of chromatin in gene expression is not well understood. Herpes simplex virus forms replication compartments (RCs) in infected cell nuclei as sites of viral DNA replication and late gene transcription. These structures develop from small compartments that grow in size, move and coalesce. Quantitative analysis of RC trajectories, derived from 4D images, show that most RCs move by directed motion. Directed movement is impaired in the presence of actin and myosin inhibitors as well as a transcription inhibitor. In addition, RCs coalesce at and reorganize nuclear speckles. Lastly, distinct effects of actin and myosin inhibitors on viral gene expression suggest that RC movement is not required for transcription but rather, movement results in the bridging of transcriptionally active RCs with nuclear speckles to form structures that enhance export of viral late mRNAs

Improving Prevention, Early Recognition and Management of Acute Kidney Injury after Major Surgery: Results of a Planning Meeting with Multidisciplinary Stakeholders

Purpose of review: Acute kidney injury (AKI) is common after major surgery, and is associated with morbidity, mortality, increased length of hospital stay, and high health care costs. Although recent guidelines for AKI provide recommendations for identification of patients at risk, monitoring, diagnosis, and management of AKI, there is lack of understanding to guide successful implementation of these recommendations into clinical practice. Sources of information: We held a planning meeting with multidisciplinary stakeholders to identify barriers, facilitators, and strategies to implement recommendations for prevention, early identification, and management of AKI after major surgery. Barriers and facilitators to knowledge use for peri-operative AKI prevention and care were discussed. Findings: Stakeholders identified barriers in knowledge (how to identify high-risk patients, what criteria to use for diagnosis of AKI), attitudes (self-efficacy in preventive care and management of AKI), and behaviors (common use of diuretics, non-steroidal anti-inflammatory drugs, withholding of intravenous fluids, and competing time demands in peri-operative care). Educational, informatics, and organizational interventions were identified by stakeholders as potentially useful elements for future interventions for peri-operative AKI. Limitation: Meeting participants were from a single centre. Implications: The information and recommendations obtained from this stakeholder's meeting will be useful to design interventions to improve prevention and early care for AKI after major surgery