The rockefeller university

Linear 2D- or 3D-structured illumination microscopy (SIM or 3D-SIM, respectively) enables multicolor volumetric imaging of fixed and live specimens with subdiffraction resolution in all spatial dimensions. However, the reliance of SIM on algorithmic post-processing renders it particularly sensitive to artifacts that may reduce resolution, compromise data and its interpretations, and drain resources in terms of money and time spent. Here we present a protocol that allows users to generate high-quality SIM data while accounting and correcting for common artifacts. The protocol details preparation of calibration bead slides designed for SIM-based experiments, the acquisition of calibration data, the documentation of typically encountered SIM artifacts and corrective measures that should be taken to reduce them. It also includes a conceptual overview and checklist for experimental design and calibration decisions, and is applicable to any commercially available or custom platform. This protocol, plus accompanying guidelines, allows researchers from students to imaging professionals to create an optimal SIM imaging environment regardless of specimen type or structure of interest. The calibration sample preparation and system calibration protocol can be executed within 1-2 d.

Recapitulation of lung development from human pluripotent stem cells (hPSCs) in three dimensions (3D) would allow deeper insight into human development, as well as the development of innovative strategies for disease modelling, drug discovery and regenerative medicine(1). We report here the generation from hPSCs of lung bud organoids (LBOs) that contain mesoderm and pulmonary endoderm and develop into branching airway and early alveolar structures after xenotransplantation and in Matrigel 3D culture. Expression analysis and structural features indicated that the branching structures reached the second trimester of human gestation. Infection in vitro with respiratory syncytial virus, which causes small airway obstruction and bronchiolitis in infants(2), led to swelling, detachment and shedding of infected cells into the organoid lumens, similar to what has been observed in human lungs(3). Introduction of mutation in HPS1, which causes an early-onset form of intractable pulmonary fibrosis(4,5), led to accumulation of extracellular matrix and mesenchymal cells, suggesting the potential use of this model to recapitulate fibrotic lung disease in vitro. LBOs therefore recapitulate lung development and may provide a useful tool to model lung disease.

Myosin Ic is thought to be the principal constituent of the motor that adjusts mechanical responsiveness during adaptation to prolonged stimuli by hair cells, the sensory receptors of the inner ear. In this context myosin molecules operate neither as filaments, as occurs in muscles, nor as single or few molecules, as characterizes intracellular transport. Instead, myosin Ic molecules occur in a complex cluster in which they may exhibit cooperative properties. To better understand the motor's remarkable function, we introduce a theoretical description of myosin Ic's chemomechanical cycle based on experimental data from recent single-molecule studies. The cycle consists of distinct chemical states that the myosin molecule stochastically occupies. We explicitly calculate the probabilities of the occupancy of these states and show their dependence on the external force, the availability of actin, and the nucleotide concentrations as required by thermodynamic constraints. This analysis highlights that the strong binding of myosin Ic to actin is dominated by the ADP state for small external forces and by the ATP state for large forces. Our approach shows how specific parameter values of the chemomechanical cycle for myosin Ic result in behaviors distinct from those of other members of the myosin family. Integrating this single-molecule cycle into a simplified ensemble description, we predict that the average number of bound myosin heads is regulated by the external force and nucleotide concentrations. The elastic properties of such an ensemble are determined by the average number of myosin cross-bridges. Changing the binding probabilities and myosin's stiffness under a constant force results in a mechanical relaxation which is large enough to account for fast adaptation in hair cells.

A CD1d-binding, invariant (i) natural killer T (NICE)-cell stimulatory glycolipid, alpha-Galactosylceramide (alpha GalCer), has been shown to act as an adjuvant. We previously identified a fluorinated phenyl ring modified aGalCer analog, 7DW8-5, displaying a higher binding affinity for CD1d molecule and more potent adjuvant activity than aGalCer. In the present study, 7DW8-5 co-administered intramuscularly (i.m.) with a recombinant adenovirus expressing a Plasmodium yoelii circumsporozoite protein (PyCSP), AdPyCS, has led to a co-localization of 7DW8-5 and a PyCSP in draining lymph nodes (dLNs), particularly in dendritic cells (DCs). This occurrence initiates a cascade of events, such as the recruitment of DCs to dLNs and their activation and maturation, and the enhancement of the ability of DCs to prime CD8+ T cells induced by AdPyCS and ultimately leading to a potent adjuvant effect and protection against malaria. (C) 2017 Elsevier Ltd. All rights reserved.

The liver plays a pivotal role in metabolism and xenobiotic detoxification, processes that must be particularly efficient when animals are active and feed. A major question is how the liver adapts to these diurnal changes in physiology. Here, we show that, in mice, liver mass, hepatocyte size, and protein levels follow a daily rhythm, whose amplitude depends on both feeding-fasting and light-dark cycles. Correlative evidence suggests that the daily oscillation in global protein accumulation depends on a similar fluctuation in ribosome number. Whereas rRNA genes are transcribed at similar rates throughout the day, some newly synthesized rRNAs are polyadenylated and degraded in the nucleus in a robustly diurnal fashion with a phase opposite to that of ribosomal protein synthesis. Based on studies with cultured fibroblasts, we propose that rRNAs not packaged into complete ribosomal subunits are polyadenylated by the poly(A) polymerase PAPD5 and degraded by the nuclear exosome.

High levels of immune activation are reported for people who inject drugs. Studies of the relationship between injection behaviors and immune activation have yielded mixed results, in part due to lack of control for hepatitis C virus in analyses. This study, of 48 HIV-seronegative people who inject drugs, examines this relationship controlling for hepatitis C virus viremia. Frequency of injection was positively related to markers of immune activation (soluble CD14, %CD8(+)CD38(+)HLADR(+) T cells), as was duration of injection (high-specificity C-reactive protein and D-dimer). Sharing injection equipment was not related to markers studied. Findings suggest that efforts to encourage injection cessation or reduction in frequency can have positive health benefits through reducing immune activation.

Organizing data about patterning and morphogenesis into a coherent framework remains a challenge in developmental biology. Reporting in Science, Corson et al. (2017) apply innovative analysis to an old problem of bristle patterns in Drosophila, reducing the nonlinear interactions among tens of cells to a succinct model with quantitative predictions.