The rockefeller university

Background: Vitamin D deficiency, defined as a serum 25-hydroxy-vitamin D [25(OH) D] concentration,20 ng/mL, is correlated with a more atherogenic lipid profile. However, oral vitamin D supplementation does not lower LDL-cholesterol concentrations or raise HDL-cholesterol concentrations. This uncoupling between association and causation may result from a failure of oral vitamin D to mimic the effect of dermally synthesized vitamin D in response to ultraviolet type B (UVB) light. Objective: We tested the hypothesis that, in vitamin D-deficient adults, the replenishment of vitamin D with UVB exposure would lower LDL-cholesterol concentrations compared with the effect of oral vitamin D-3 supplementation. Design: We performed a randomized clinical trial in vitamin D-deficient adults and compared vitamin D replenishment between subjects who received oral vitamin D-3 (n = 60) and those who received narrow-band UVB exposure (n = 58) <= 6 mo. Results: There was no difference in the change from baseline LDL-cholesterol concentrations between oral vitamin D-3 and UVB groups (difference in median of oral vitamin D-3 minus that of UVB: 1.5 mg/dL; 95% CI: -5.0, 7.0 mg/dL). There were also no differences within groups or between groups for changes in total or HDL cholesterol or triglycerides. Transcriptional profiling of skin and blood, however, revealed significant upregulation of immune pathway signaling with oral vitamin D-3 but significant downregulation with UVB. Conclusions: Correcting vitamin D deficiency with either oral vitamin D-3 or UVB does not improve the lipid profile. Beyond cholesterol, these 2 modalities of raising 25(OH) D have disparate effects on gene transcription.

Cytoplasmic dyneins are motor proteins in the AAA+ superfamily that transport cellular cargos toward microtubule minus-ends. Recently, ciliobrevins were reported as selective cell-permeable inhibitors of cytoplasmic dyneins. As is often true for first-in-class inhibitors, the use of ciliobrevins has in part been limited by low potency. Moreover, suboptimal chemical properties, such as the potential to isomerize, have hindered efforts to improve ciliobrevins. Here, we characterized the structure of ciliobrevins and designed conformationally constrained isosteres. These studies identified dynapyrazoles, inhibitors more potent than ciliobrevins. At single-digit micromolar concentrations dynapyrazoles block intraflagellar transport in the cilium and lysosome motility in the cytoplasm, processes that depend on cytoplasmic dyneins. Further, we find that while ciliobrevins inhibit both dynein's microtubule-stimulated and basal ATPase activity, dynapyrazoles strongly block only microtubule-stimulated activity. Together, our studies suggest that chemical-structure-based analyses can lead to inhibitors with improved properties and distinct modes of inhibition.

Although several progestins have been tested for hormonal male contraception, the effects of dosage and nature of various progestins on gonadotropin suppression combined with and without additional testosterone has not been performed in a comparative trial. The aim of this study was to evaluate the differential impact of four oral or transdermal progestins on the suppression of gonadotropins in healthy men: oral: cyproterone acetate (CPA), levonorgestrel (LNG), norethisterone acetate (NETA), and transdermal: Nestorone((R)) (NES), all in combination with transdermal testosterone (T). Randomized clinical trial testing was performed with four progestins at two doses each. After a 2-week progestin-only treatment, transdermal T was added for further 4weeks and was followed by a 3-week recovery period. Progestin-dose per day: CPA 10mg/20mg, NES 2mg/3mg/dose e.g. 200/300g/day absorbed, NETA 5mg/10mg, LNG 120g/240g. From an andrology outpatient clinic, 56 healthy men aged 18-50years, with body mass index 33kg x m(-2) were included in the study. Serum concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were studied. Secondary outcome measure included were serum testosterone concentrations, sperm concentrations, and safety parameters. Intergroup comparisons demonstrated that CPA and LNG had the strongest effect on LH/FSH suppression. Nevertheless, every substance showed significant inhibitory effects on gonadotropin secretion, especially in combination with transdermal T. A decrease in hematocrit and insulin sensitivity as well as cholesterol subfractions and triglycerides was uniformly seen for every group. The combination of oral or transdermal progestins with a transdermal testosterone preparation is able to suppress gonadotropins. Further dose titration studies with sperm suppression as an end-point should be conducted to determine the lowest effective dose for hormonal male contraception.

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.