Optogenetic Methods to Control Tissue Mechanics in Drosophila.

Optogenetics is a powerful technique that allows the control of protein function with high spatiotemporal precision using light. Here, we describe the application of this method to control tissue mechanics during Drosophila embryonic development. We detail optogenetic protocols to either increase or decrease cell contractility and analyze the interplay between cell-cell interaction, tissue geometry, and force transmission during gastrulation.

Cell division in tissues enables macrophage infiltration.

Cells migrate through crowded microenvironments within tissues during normal development, immune response, and cancer metastasis. Although migration through pores and tracks in the extracellular matrix (ECM) has been well studied, little is known about cellular traversal into confining cell-dense tissues. We find that embryonic tissue invasion by Drosophila macrophages requires division of an epithelial ectodermal cell at the site of entry. Dividing ectodermal cells disassemble ECM attachment formed by integrin-mediated focal adhesions next to mesodermal cells, allowing macrophages to move their nuclei ahead and invade between two immediately adjacent tissues. Invasion efficiency depends on division frequency, but reduction of adhesion strength allows macrophage entry independently of division. This work demonstrates that tissue dynamics can regulate cellular infiltration.

Visible-Light Induced Sustainable Water Treatment Using Plasmo-Semiconductor Nanogap Bridge Array, PNA.

The development of sustainable methods for energy-intensive water treatment processes continues to be a challenging issue. Plasmonic-semiconductor nanoparticles, which absorb large amounts of sunlight in the visible range for conversion into chemical energy efficiently, can form the basis of a sustainable water treatment method. However, the potential uses of plasmonic semiconductor particles for water treatment have not been fully explored yet because of the limitations associated with the imbalance between light capture, charge transfer, and the required recycling steps for the particles themselves. Herein, a significantly improved visible-light-induced water treatment method that uses a plasmo-semiconductor nanogap bridge array (PNA) is reported. As an arrangement of antenna-reactors, the PNA enables the balancing of the largely enhanced electromagnetic field in the plasmonic nanogap coupling region and optimal separation of charge carriers in the semiconductor. The simultaneous effects of visible-light absorption and charge transfer lead to the generation of a highly enhanced visible-light-induced OH radical (•OH). Consequently, visible-light-induced 5-log N/N0 water disinfection and 100% chemical decomposition for sustainable water treatment were demonstrated. Owing to the large light absorption, charge carrier utilization, and array-oriented scalability, the PNA will be valuable in various sustainable energy and environmental applications.

Clinical classification of scoliosis patients using machine learning and markerless 3D surface trunk data.

Markerless 3D surface topography for scoliosis diagnosis and brace treatment can avoid repeated radiation known from standard X-ray analysis and possible side effects. Combined with the method of torso asymmetry analysis, curve severity and progression can be evaluated with high reliability. In the current study, a machine learning approach was utilised to classify scoliosis patients based on their trunk surface asymmetry pattern. Frontal X-ray and 3D scanning analysis with a clinical classification based on Cobb angle and spinal curve pattern were performed with 50 patients. Similar as in a previous study, each patient's trunk 3D reconstruction was used for an elastic registration of a reference surface mesh with fixed number of vertices. Subsequently, an asymmetry distance map between original and reflected torso was calculated. A fully connected neural network was then utilised to classify patients regarding their Cobb angle (mild, moderate, severe) and an Augmented Lehnert-Schroth (ALS) classification based on their full torso asymmetry distance map. The results reveal a classification success rate of 90% (SE: 80%, SP: 100%) regarding the curve severity (mild vs moderate-severe) and 50-72% regarding the ALS group. Identifying patient curve severity and treatment group was reasonably possible allowing for a decision support during diagnosis and treatment planning. Graphical abstract.

ßH-spectrin is required for ratcheting apical pulsatile constrictions during tissue invagination.

Actomyosin-mediated apical constriction drives a wide range of morphogenetic processes. Activation of myosin-II initiates pulsatile cycles of apical constrictions followed by either relaxation or stabilization (ratcheting) of the apical surface. While relaxation leads to dissipation of contractile forces, ratcheting is critical for the generation of tissue-level tension and changes in tissue shape. How ratcheting is controlled at the molecular level is unknown. Here, we show that the actin crosslinker ßH-spectrin is upregulated at the apical surface of invaginating mesodermal cells during Drosophila gastrulation. ßH-spectrin forms a network of filaments which co-localize with medio-apical actomyosin fibers, in a process that depends on the mesoderm-transcription factor Twist and activation of Rho signaling. ßH-spectrin knockdown results in non-ratcheted apical constrictions and inhibition of mesoderm invagination, recapitulating twist mutant embryos. ßH-spectrin is thus a key regulator of apical ratcheting during tissue invagination, suggesting that actin cross-linking plays a critical role in this process.

Innovative decision support for scoliosis brace therapy based on statistical modelling of markerless 3D trunk surface data.

Recently markerless 3D scanning methods receive an increased interest for therapy planning and brace treatment of patients with scoliosis. This avoids repeated radiation known from standard X-Ray analysis. Several authors introduced the method of asymmetry distance maps in order to classify curve severity and progression. The current work extends this approach by statistical mean shape 3D models of the human trunk in order to classify patients. 50 patients were included in this study performing frontal X-ray and 3D scanning analysis. All patients were classified by a clinician according to their Cobb angle and spinal curve pattern (Augmented-Lehnert-Schroth ALS). 3D reconstructions of each patient trunk were processed in a way to elastically register a reference surface mesh with fixed number of data points. Mean 3D shape models were generated for each curve pattern. An asymmetry distance map was then calculated for each patient and mean shape model. Single patient 3D reconstructions were classified according to severity and ALS treatment group. Optimal sensitivity and specificity was 97%/39% thoracic and 87%/42% lumbar respectively for detecting mild and moderate-severe patients. Identifying a treatment group was possible for three combined groups allowing to support decisions during diagnosis and therapy planning.

Using optogenetics to tackle systems-level questions of multicellular morphogenesis.

Morphogenesis of multicellular systems is governed by precise spatiotemporal regulation of biochemical reactions and mechanical forces which together with environmental conditions determine the development of complex organisms. Current efforts in the field aim at decoding the system-level principles underlying the regulation of developmental processes. Toward this goal, optogenetics, the science of regulation of protein function with light, is emerging as a powerful new tool to quantitatively perturb protein function in vivo with unprecedented precision in space and time. In this review, we provide an overview of how optogenetics is helping to address system-level questions of multicellular morphogenesis and discuss future directions.

Optogenetic inhibition of Delta reveals digital Notch signalling output during tissue differentiation.

Spatio-temporal regulation of signalling pathways plays a key role in generating diverse responses during the development of multicellular organisms. The role of signal dynamics in transferring signalling information in vivo is incompletely understood. Here, we employ genome engineering in Drosophila melanogaster to generate a functional optogenetic allele of the Notch ligand Delta (opto-Delta), which replaces both copies of the endogenous wild-type locus. Using clonal analysis, we show that optogenetic activation blocks Notch activation through cis-inhibition in signal-receiving cells. Signal perturbation in combination with quantitative analysis of a live transcriptional reporter of Notch pathway activity reveals differential tissue- and cell-scale regulatory modes. While at the tissue-level the duration of Notch signalling determines the probability with which a cellular response will occur, in individual cells Notch activation acts through a switch-like mechanism. Thus, time confers regulatory properties to Notch signalling that exhibit integrative digital behaviours during tissue differentiation.

Principles and applications of optogenetics in developmental biology.

The development of multicellular organisms is controlled by highly dynamic molecular and cellular processes organized in spatially restricted patterns. Recent advances in optogenetics are allowing protein function to be controlled with the precision of a pulse of laser light in vivo, providing a powerful new tool to perturb developmental processes at a wide range of spatiotemporal scales. In this Primer, we describe the most commonly used optogenetic tools, their application in developmental biology and in the nascent field of synthetic morphogenesis.

Cross-linker-mediated regulation of actin network organization controls tissue morphogenesis.

Contraction of cortical actomyosin networks driven by myosin activation controls cell shape changes and tissue morphogenesis during animal development. In vitro studies suggest that contractility also depends on the geometrical organization of actin filaments. Here we analyze the function of actomyosin network topology in vivo using optogenetic stimulation of myosin-II in Drosophila embryos. We show that early during cellularization, hexagonally arrayed actomyosin fibers are resilient to myosin-II activation. Actomyosin fibers then acquire a ring-like conformation and become contractile and sensitive to myosin-II. This transition is controlled by Bottleneck, a Drosophila unique protein expressed for only a short time during early cellularization, which we show regulates actin bundling. In addition, it requires two opposing actin cross-linkers, Filamin and Fimbrin. Filamin acts synergistically with Bottleneck to facilitate hexagonal patterning, while Fimbrin controls remodeling of the hexagonal network into contractile rings. Thus, actin cross-linking regulates the spatio-temporal organization of actomyosin contraction in vivo, which is critical for tissue morphogenesis.

Self-Organized Nuclear Positioning Synchronizes the Cell Cycle in Drosophila Embryos.

The synchronous cleavage divisions of early embryogenesis require coordination of the cell-cycle oscillator, the dynamics of the cytoskeleton, and the cytoplasm. Yet, it remains unclear how spatially restricted biochemical signals are integrated with physical properties of the embryo to generate collective dynamics. Here, we show that synchronization of the cell cycle in Drosophila embryos requires accurate nuclear positioning, which is regulated by the cell-cycle oscillator through cortical contractility and cytoplasmic flows. We demonstrate that biochemical oscillations are initiated by local Cdk1 inactivation and spread through the activity of phosphatase PP1 to generate cortical myosin II gradients. These gradients cause cortical and cytoplasmic flows that control proper nuclear positioning. Perturbations of PP1 activity and optogenetic manipulations of cortical actomyosin disrupt nuclear spreading, resulting in loss of cell-cycle synchrony. We conclude that mitotic synchrony is established by a self-organized mechanism that integrates the cell-cycle oscillator and embryo mechanics.

Downregulation of basal myosin-II is required for cell shape changes and tissue invagination.

Tissue invagination drives embryo remodeling and assembly of internal organs during animal development. While the role of actomyosin-mediated apical constriction in initiating inward folding is well established, computational models suggest relaxation of the basal surface as an additional requirement. However, the lack of genetic mutations interfering specifically with basal relaxation has made it difficult to test its requirement during invagination so far. Here we use optogenetics to quantitatively control myosin-II levels at the basal surface of invaginating cells during Drosophila gastrulation. We show that while basal myosin-II is lost progressively during ventral furrow formation, optogenetics allows the maintenance of pre-invagination levels over time. Quantitative imaging demonstrates that optogenetic activation prior to tissue bending slows down cell elongation and blocks invagination. Activation after cell elongation and tissue bending has initiated inhibits cell shortening and folding of the furrow into a tube-like structure. Collectively, these data demonstrate the requirement of myosin-II polarization and basal relaxation throughout the entire invagination process.

Ultrafast tissue staining with chemical tags.

Genetically encoded fluorescent proteins and immunostaining are widely used to detect cellular and subcellular structures in fixed biological samples. However, for thick or whole-mount tissue, each approach suffers from limitations, including limited spectral flexibility and lower signal or slow speed, poor penetration, and high background labeling, respectively. We have overcome these limitations by using transgenically expressed chemical tags for rapid, even, high-signal and low-background labeling of thick biological tissues. We first construct a platform of widely applicable transgenic Drosophila reporter lines, demonstrating that chemical labeling can accelerate staining of whole-mount fly brains by a factor of 100. Using viral vectors to deliver chemical tags into the mouse brain, we then demonstrate that this labeling strategy works well in mice. Thus this tag-based approach drastically improves the speed and specificity of labeling genetically marked cells in intact and/or thick biological samples.

Development of a primary care physician task list to evaluate clinic visit workflow.

BACKGROUND: Interventions designed to improve the delivery of primary care, including Patient-Centered Medical Homes and electronic health records, require an understanding of clinical workflow to be successfully implemented. However, there is a lack of tools to describe and study primary care physician workflow. We developed a comprehensive list of primary care physician tasks that occur during a face-to-face patient visit. METHODS: A validated list of tasks performed by primary care physicians during patient clinic visits was developed from a secondary data analysis of observation data from two studies evaluating primary care workflow. Thirty primary care physicians participated from a convenience sample of 17 internal medicine and family medicine clinics in Wisconsin and Iowa across rural and urban settings and community and academic settings. RESULTS: The final task list has 12 major tasks, 189 subtasks, and 191 total tasks. The major tasks are: Enter Room, Gather Information from Patient, Review Patient Information, Document Patient Information, Perform, Recommend / Discuss Treatment Options, Look Up, Order, Communicate, Print / Give Patient (advice, instructions), Appointment Wrap-up, and Leave Room. Additional subcodes note use of paper or EHR and the presence of a caregiver or medical student. CONCLUSIONS: The task list presented here is a tool that will help clinics study their workflows so they can plan for changes that will take place because of EHR implementation and/or transformation to a patient centered medical home.

Identifying and responding to technical assistance and training needs in tobacco prevention and control.

The Master Settlement Agreement generated expectations that significant, long-term funding would be available to the 46 participating states in reparation for the health costs incurred by tobacco use. Facing intense pressure to use anticipated funds for effective programming, states and national organizations considered how to supply the technical assistance and training at the state and local levels. This article reviews assessments by the American Legacy Foundation, the Tobacco Technical Assistance Consortium, and selected states of the current needs for support, technical assistance, and training in tobacco prevention and control. Key findings indicated the need for information, information exchange, mentoring, and training targeted to new staff, with advanced skill sets for experienced staff. As future funding is uncertain, all these organizations are exploring innovative ways to maintain infrastructure and programming at the state and local levels. Training and technical assistance can be the solution to sustaining impetus in the movement.