Treatment of Autosomal Dominant Hypocalcemia Type 1 With the Calcilytic NPSP795 (SHP635).

Autosomal dominant hypocalcemia type 1 (ADH1) is a rare form of hypoparathyroidism caused by heterozygous, gain-of-function mutations of the calcium-sensing receptor gene (CAR). Individuals are hypocalcemic with inappropriately low parathyroid hormone (PTH) secretion and relative hypercalciuria. Calcilytics are negative allosteric modulators of the extracellular calcium receptor (CaR) and therefore may have therapeutic benefits in ADH1. Five adults with ADH1 due to four distinct CAR mutations received escalating doses of the calcilytic compound NPSP795 (SHP635) on 3 consecutive days. Pharmacokinetics, pharmacodynamics, efficacy, and safety were assessed. Parallel in vitro testing with subject CaR mutations assessed the effects of NPSP795 on cytoplasmic calcium concentrations (Ca2+i ), and ERK and p38MAPK phosphorylation. These effects were correlated with clinical responses to administration of NPSP795. NPSP795 increased plasma PTH levels in a concentration-dependent manner up to 129% above baseline (p = 0.013) at the highest exposure levels. Fractional excretion of calcium (FECa) trended down but not significantly so. Blood ionized calcium levels remained stable during NPSP795 infusion despite fasting, no calcitriol supplementation, and little calcium supplementation. NPSP795 was generally safe and well-tolerated. There was significant variability in response clinically across genotypes. In vitro, all mutant CaRs were half-maximally activated (EC50 ) at lower concentrations of extracellular calcium (Ca2+o ) compared to wild-type (WT) CaR; NPSP795 exposure increased the EC50 for all CaR activity readouts. However, the in vitro responses to NPSP795 did not correlate with any clinical parameters. NPSP795 increased plasma PTH levels in subjects with ADH1 in a dose-dependent manner, and thus, serves as proof-of-concept that calcilytics could be an effective treatment for ADH1. Albeit all mutations appear to be activating at the CaR, in vitro observations were not predictive of the in vivo phenotype or the response to calcilytics, suggesting that other parameters impact the response to the drug. © 2019 American Society for Bone and Mineral Research.

Stochastic fracture of additively manufactured porous composites.

Extrusion-based fused deposition modeling (FDM) introduces inter-bead pores into dense materials, which results in part-to-part mechanical property variations, i.e., low mechanical reliability. In addition, the internal structure of FDMed materials can be made porous intentionally to tailor mechanical properties, introduce functionality, reduce material consumption, or decrease production time. Despite these potential benefits, the effects of porosity on the mechanical reliability of FDMed composites are still unclear. Accordingly, we investigated the stochastic fracture of 241 FDMed short-carbon-fiber-reinforced-ABS with porosity ranging from 13 to 53 vol.% under tensile load. Weibull analysis was performed to quantify the variations in mechanical properties. We observed an increase in Weibull modulus of fracture/tensile strength for porosity higher than ~40 vol.% and a decrease in Weibull modulus of fracture strain for an increase in porosity from 25 to 53 vol.%. Micromechanics-based 2D simulations indicated that the mechanical reliability of FDMed composites depends on variations in bead strength and elastic modulus of beads. The change in raster orientation from 45°/-45° to 0° more than doubled the Weibull modulus. We identified five different types of pores via high-resolution X-ray computed tomography. A 22% and 48% decrease in carbon fiber length due to extrusion was revealed for two different regions of the filament.

Three-Dimensional Morphological and Chemical Evolution of Nanoporous Stainless Steel by Liquid Metal Dealloying.

Nanoporous materials, especially those fabricated by liquid metal dealloying processes, possess great potential in a wide range of applications due to their high surface area, bicontinuous structure with both open pores for transport and solid phase for conductivity or support, and low material cost. Here, we used X-ray nanotomography and X-ray fluorescence microscopy to reveal the three-dimensional (3D) morphology and elemental distribution within materials. Focusing on nanoporous stainless steel, we evaluated the 3D morphology of the dealloying front and established a quantitative processing-structure-property relationship at a later stage of dealloying. The morphological differences of samples created by liquid metal dealloying and aqueous dealloying methods were also discussed. We concluded that it is particularly important to consider the dealloying, coarsening, and densification mechanisms in influencing the performance-determining, critical 3D parameters, such as tortuosity, pore size, porosity, curvature, and interfacial shape.

Multifunctional Hydrogels with Reversible 3D Ordered Macroporous Structures.

Three-dimensionally ordered macroporous (3DOM) hydrogels prepared by colloidal crystals templating display highly reversible shape memory properties, as confirmed by indirect electron microscopy imaging of their inverse replicas and direct nanoscale resolution X-ray microscopy imaging of the hydrated hydrogels. Modifications of functional groups in the 3DOM hydrogels result in various materials with programmed properties for a wide range of applications.

Differential growth of and nanoscale TiO2 accumulation in Tetrahymena thermophila by direct feeding versus trophic transfer from Pseudomonas aeruginosa.

Nanoscale titanium dioxide (TiO2) is increasingly used in consumer goods and is entering waste streams, thereby exposing and potentially affecting environmental microbes. Protozoans could either take up TiO2 directly from water and sediments or acquire TiO2 during bactivory (ingestion of bacteria) of TiO2-encrusted bacteria. Here, the route of exposure of the ciliated protozoan Tetrahymena thermophila to TiO2 was varied and the growth of, and uptake and accumulation of TiO2 by, T. thermophila were measured. While TiO2 did not affect T. thermophila swimming or cellular morphology, direct TiO2 exposure in rich growth medium resulted in a lower population yield. When TiO2 exposure was by bactivory of Pseudomonas aeruginosa, the T. thermophila population yield and growth rate were lower than those that occurred during the bactivory of non-TiO2-encrusted bacteria. Regardless of the feeding mode, T. thermophila cells internalized TiO2 into their food vacuoles. Biomagnification of TiO2 was not observed; this was attributed to the observation that TiO2 appeared to be unable to cross the food vacuole membrane and enter the cytoplasm. Nevertheless, our findings imply that TiO2 could be transferred into higher trophic levels within food webs and that the food web could be affected by the decreased growth rate and yield of organisms near the base of the web.

Laboratory-based cryogenic soft x-ray tomography with correlative cryo-light and electron microscopy.

Here we present a novel laboratory-based cryogenic soft X-ray microscope for whole cell tomography of frozen hydrated samples. We demonstrate the capabilities of this compact cryogenic microscope by visualizing internal subcellular structures of Saccharomyces cerevisiae cells. The microscope is shown to achieve better than 50 nm half-pitch spatial resolution with a Siemens star test sample. For whole biological cells, the microscope can image specimens up to 5 µm thick. Structures as small as 90 nm can be detected in tomographic reconstructions following a low cumulative radiation dose of only 7.2 MGy. Furthermore, the design of the specimen chamber utilizes a standard sample support that permits multimodal correlative imaging of the exact same unstained yeast cell via cryo-fluorescence light microscopy, cryo-soft X-ray microscopy, and cryo-transmission electron microscopy. This completely laboratory-based cryogenic soft X-ray microscope will enable greater access to three-dimensional ultrastructure determination of biological whole cells without chemical fixation or physical sectioning.

A 30 nm-resolution hard X-ray microscope with X-ray fluorescence mapping capability at BSRF.

A full-field transmission X-ray microscope (TXM) operating continuously from 5 keV to 12 keV with fluorescence mapping capability has been designed and constructed at the Beijing Synchrotron Radiation Facility, a first-generation synchrotron radiation facility operating at 2.5 GeV. Spatial resolution better than 30 nm has been demonstrated using a Siemens star pattern in both absorption mode and Zernike phase-contrast mode. A scanning-probe mode fluorescence mapping capability integrated with the TXM has been shown to provide 50 p.p.m. sensitivity for trace elements with a spatial resolution (limited by probing beam spot size) of 20 µm. The optics design, testing of spatial resolution and fluorescence sensitivity are presented here, including performance measurement results.

Soybean susceptibility to manufactured nanomaterials with evidence for food quality and soil fertility interruption.

Based on previously published hydroponic plant, planktonic bacterial, and soil microbial community research, manufactured nanomaterial (MNM) environmental buildup could profoundly alter soil-based food crop quality and yield. However, thus far, no single study has at once examined the full implications, as no studies have involved growing plants to full maturity in MNM-contaminated field soil. We have done so for soybean, a major global commodity crop, using farm soil amended with two high-production metal oxide MNMs (nano-CeO(2) and -ZnO). The results provide a clear, but unfortunate, view of what could arise over the long term: (i) for nano-ZnO, component metal was taken up and distributed throughout edible plant tissues; (ii) for nano-CeO(2), plant growth and yield diminished, but also (iii) nitrogen fixation--a major ecosystem service of leguminous crops--was shut down at high nano-CeO(2) concentration. Juxtaposed against widespread land application of wastewater treatment biosolids to food crops, these findings forewarn of agriculturally associated human and environmental risks from the accelerating use of MNMs.

The study of the reconstructed three-dimensional structure of a solid-oxide fuel-cell cathode by X-ray nanotomography.

The microstructure and morphology of solid-oxide fuel-cell electrodes are very complex but important because they strongly affect the electrical performance of the cell. In this work the high-resolution X-ray nanotomography technique is applied to reconstruct the three-dimensional microstructure of a (La(0.8)Sr(0.2))(0.95)MnO(3) yttria-stabilized zirconia composite cathode. Some key microstructural parameters, such as the porosity, representative elementary volume, connected pore volume and pore phase tortuosity, were obtained based on the three-dimensional reconstruction volume data with a spatial resolution of sub-60 nm. These parameters bear intimate correlation with the efficiency of the electrochemical conversion process, and provide valuable information for optimizing the manufacturing processes and improving the device's reliability.

Nanoscale X-ray microscopic imaging of mammalian mineralized tissue.

A novel hard transmission X-ray microscope (TXM) at the Stanford Synchrotron Radiation Lightsource operating from 5 to 15 keV X-ray energy with 14 to 30 microm2 field of view has been used for high-resolution (30-40 nm) imaging and density quantification of mineralized tissue. TXM is uniquely suited for imaging of internal cellular structures and networks in mammalian mineralized tissues using relatively thick (50 microm), untreated samples that preserve tissue micro- and nanostructure. To test this method we performed Zernike phase contrast and absorption contrast imaging of mouse cancellous bone prepared under different conditions of in vivo loading, fixation, and contrast agents. In addition, the three-dimensional structure was examined using tomography. Individual osteocytic lacunae were observed embedded within trabeculae in cancellous bone. Extensive canalicular networks were evident and included processes with diameters near the 30-40 nm instrument resolution that have not been reported previously. Trabecular density was quantified relative to rod-like crystalline apatite, and rod-like trabecular struts were found to have 51-54% of pure crystal density and plate-like areas had 44-53% of crystal density. The nanometer resolution of TXM enables future studies for visualization and quantification of ultrastructural changes in bone tissue resulting from osteoporosis, dental disease, and other pathologies.

High resolution hard x-ray microscope on a second generation synchrotron source.

A full-field, transmission x-ray microscope (TXM) operating in the energy range of 7-11 keV has been installed at the U7A beamline at the National Synchrotron Radiation Laboratory, a second generation synchrotron source operating at 0.8 GeV. Although the photon flux at sample position in the operating energy range is significantly low due to its relatively large emittance, the TXM can get high quality x-ray images with a spatial resolution down to 50 nm with acceptable exposure time. This TXM operates in either absorption or Zernike phase contrast mode with similar resolution. This TXM is a powerful analytical tool for a wide range of scientific areas, especially studies on nanoscale phenomena and structural imaging in biology, materials science, and environmental science. We present here the property of the x-ray source, beamline design, and the operation and key optical components of the x-ray TXM. Plans to improve the throughput of the TXM will be discussed.