ilastik: interactive machine learning for (bio)image analysis.

We present ilastik, an easy-to-use interactive tool that brings machine-learning-based (bio)image analysis to end users without substantial computational expertise. It contains pre-defined workflows for image segmentation, object classification, counting and tracking. Users adapt the workflows to the problem at hand by interactively providing sparse training annotations for a nonlinear classifier. ilastik can process data in up to five dimensions (3D, time and number of channels). Its computational back end runs operations on-demand wherever possible, allowing for interactive prediction on data larger than RAM. Once the classifiers are trained, ilastik workflows can be applied to new data from the command line without further user interaction. We describe all ilastik workflows in detail, including three case studies and a discussion on the expected performance.

Impaired jump landing after exercise in recreational and in high-performance athletes.

The risk of sustaining injuries increases with fatigue. The aim of this study was to analyze the influence of fatigue on dynamic postural control in jump landing and stabilization (ST) in athletes of different levels. In all, 18 high-performance ball sports athletes and 24 recreationally active subjects performed a jump test (JT) before and at 1, 5, 10, 15, and 20 minutes after a 30-minute treadmill run at the individual anaerobic threshold. An overhead ball switch hit during a forward jump triggered indicator lamps on either side of a force plate. After landing on the plate, ST on 1 leg (no light cue) or a second jump sideways (toward a light cue) was required. The ST force integral index was calculated for the ST trials. Dynamic postural control was significantly impaired in jump landing and ST in the first minute after the run: mean difference ± SD: 0.25 ± 0.48 m·s-1 (95% confidence interval: 0.10-0.40 m·s-1, p = 0.043; analysis of variance). No significant group differences were found. Under fatigued conditions, dynamic postural control in jump landing was impaired in an unexpected ST task. Not only recreational but also high-performance athletes were affected. Ball sports athletes could add a training exercise to their workout, which alternates between periods of high effort and neuromuscular training. Resistance to fatigue effects should be checked on a regular basis using JTs.

Temporal control of the integrated stress response by a stochastic molecular switch.

Stress granules (SGs) are formed in the cytosol as an acute response to environmental cues and activation of the integrated stress response (ISR), a central signaling pathway controlling protein synthesis. Using chronic virus infection as stress model, we previously uncovered a unique temporal control of the ISR resulting in recurrent phases of SG assembly and disassembly. Here, we elucidate the molecular network generating this fluctuating stress response by integrating quantitative experiments with mathematical modeling and find that the ISR operates as a stochastic switch. Key elements controlling this switch are the cooperative activation of the stress-sensing kinase PKR, the ultrasensitive response of SG formation to the phosphorylation of the translation initiation factor eIF2α, and negative feedback via GADD34, a stress-induced subunit of protein phosphatase 1. We identify GADD34 messenger RNA levels as the molecular memory of the ISR that plays a central role in cell adaptation to acute and chronic stress.

Experimental and computational analyses reveal that environmental restrictions shape HIV-1 spread in 3D cultures.

Pathogens face varying microenvironments in vivo, but suitable experimental systems and analysis tools to dissect how three-dimensional (3D) tissue environments impact pathogen spread are lacking. Here we develop an Integrative method to Study Pathogen spread by Experiment and Computation within Tissue-like 3D cultures (INSPECT-3D), combining quantification of pathogen replication with imaging to study single-cell and cell population dynamics. We apply INSPECT-3D to analyze HIV-1 spread between primary human CD4 T-lymphocytes using collagen as tissue-like 3D-scaffold. Measurements of virus replication, infectivity, diffusion, cellular motility and interactions are combined by mathematical analyses into an integrated spatial infection model to estimate parameters governing HIV-1 spread. This reveals that environmental restrictions limit infection by cell-free virions but promote cell-associated HIV-1 transmission. Experimental validation identifies cell motility and density as essential determinants of efficacy and mode of HIV-1 spread in 3D. INSPECT-3D represents an adaptable method for quantitative time-resolved analyses of 3D pathogen spread.