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Bioculture System Validation

The Bioculture System first flight will be to validate the performance of the hardware and its automated and manual operational capabilities in the space flight environment of the International Space Station. Biology, Engineering, and Operations tests will be conducted in the Bioculture System fully characterize its automated and manual functions to support cell culturing for short and long durations. No hypothesis-driven research will be conducted with biological sample, and the science leads have all provided their concurrence that none of the data they collect will be considered as proprietary and can be free distributed to the science community. The outcome of the validation flight will be to commission the hardware for use by the science community. This presentation will provide non-proprietary details about the Bioculture System and information about the activities for the first flight

NASA Bioculture System: From Experiment Definition to Flight Payload

Starting in 2015, the NASA Bioculture System will be available to the science community to conduct cell biology and microbiology experiments on ISS. The Bioculture System carries ten environmentally independent Cassettes, which house the experiments. The closed loop fluids flow path subsystem in each Cassette provides a perfusion-based method for maintain specimen cultures in a shear-free environment by using a biochamber based on porous hollow fiber bioreactor technology. Each Cassette contains an incubator and separate insulated refrigerator compartment for storage of media, samples, nutrients and additives. The hardware is capable of fully automated or manual specimen culturing and processing, including in-flight experiment initiation, sampling and fixation, up to BSL-2 specimen culturing, and the ability to up to 10 independent cultures in parallel for statistical analysis. The incubation and culturing of specimens in the Bioculture System is a departure from standard laboratory culturing methods. Therefore, it is critical that the PI has an understanding the pre-flight test required for successfully using the Bioculture System to conduct an on-orbit experiment. Overall, the PI will conduct a series of ground tests to define flight experiment and on-orbit implementation requirements, verify biocompatibility, and determine base bioreactor conditions. The ground test processes for the utilization of the Bioculture System, from experiment selection to flight, will be reviewed. Also, pre-flight test schedules and use of COTS ground test equipment (CellMax and FiberCell systems) and the Bioculture System will be discussed

Prevention of glucocorticoid induced-apoptosis of osteoblasts and osteocytes by protecting against endoplasmic reticulum (ER) stress in vitro and in vivo in female mice

Endoplasmic reticulum (ER) stress is associated with increased reactive oxygen species (ROS), results from accumulation of misfolded/unfolded proteins, and can trigger apoptosis. ER stress is alleviated by phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), which inhibits protein translation allowing the ER to recover, thus promoting cell viability. We investigated whether osteoblastic cell apoptosis induced by glucocorticoids (GCs) is due to induction of ROS/ER stress and whether inhibition of eIF2α dephosphorylation promotes survival opposing the deleterious effects of GC in vitro and in vivo. Apoptosis of osteocytic MLO-Y4 and osteoblastic OB-6 cells induced by dexamethasone was abolished by ROS inhibitors. Like GC, the ER stress inducing agents brefeldin A and tunicamycin induced osteoblastic cell apoptosis. Salubrinal or guanabenz, specific inhibitors of eIF2α dephosphorylation, blocked apoptosis induced by either GC or ER stress inducers. Moreover, GC markedly decreased mineralization in OB-6 cells or primary osteoblasts; and salubrinal or guanabenz increased mineralization and prevented the inhibitory effect of GC. Furthermore, salubrinal (1 mg/kg/day) abolished osteoblast and osteocyte apoptosis in cancellous and cortical bone and partially prevented the loss of BMD at all sites and the decreased vertebral cancellous bone formation induced by treatment with prednisolone for 28 days (1.4 mg/kg/day). We conclude that part of the pro-apoptotic actions of GC on osteoblastic cells is mediated through ER stress, and that inhibition of eIF2α dephosphorylation protects from GC-induced apoptosis of osteoblasts and osteocytes in vitro and in vivo and from the deleterious effects of GC on the skeleton

A Review and Comparison of Mouse and Rat Responses to Micro Gravity, Hyper Gravity and Simulated Models of Partial Gravity; Species Differences, Gaps in the Available Data, and Consideration of the Advantages and Caveats of Each Model for Spaceflight

Laboratory strains of mice and rat are widely used to study mammalian responses to stimulus, and both have been studied under a variety of gravity conditions, including space flight. We compared results obtained from exposure to spaceflight and microgravity, hyper gravity via centrifugation, earth gravity, and models of simulated partial gravity (hind-limb unloading and partial weight bearing treatments). We examined the reported changes in survival, body mass, circadian rhythm (body temperature and activity levels), behavior, bone, muscle, immune, cardio-vasculature, vestibular, reproduction and neonate survival, microbiome, and the visual system. Not all categories have published data for both species, some have limited data, and there are variations in experiment design that allow for only relative comparisons to be considered. The data reveal species differences in both the level of gravity required to obtain a response, degree of response, and in temporal expression of responses. Examination of the data across the gravity levels allows consideration of the hypothesis that gravitational responses follow a continuum, and organ specific differences are noted. In summary, we present advantages and caveats of each model system as pertains to gravitational biology research and identify gaps in our knowledge of how these mammals respond to gravity

Glucocorticoid-Induced Bone Fragility Is Prevented in Female Mice by Blocking Pyk2/Anoikis Signaling

Excess of glucocorticoids (GCs) is a leading cause of bone fragility, and therapeutic targets are sorely needed. We report that genetic deletion or pharmacological inhibition of proline-rich tyrosine kinase 2 (Pyk2) prevents GC-induced bone loss by overriding GC effects of detachment-induced bone cell apoptosis (anoikis). In wild-type or vehicle-treated mice, GCs either prevented osteoclast apoptosis or promoted osteoblast/osteocyte apoptosis. In contrast, mice lacking Pyk2 [knockout (KO)] or treated with Pyk2 kinase inhibitor PF-431396 (PF) were protected. KO or PF-treated mice were also protected from GC-induced bone resorption, microarchitecture deterioration, and weakening of biomechanical properties. In KO and PF-treated mice, GC increased osteoclasts in bone and circulating tartrate-resistant acid phosphatase form 5b, an index of osteoclast number. However, bone surfaces covered by osteoclasts and circulating C-terminal telopeptides of type I collagen, an index of osteoclast function, were not increased. The mismatch between osteoclast number vs function induced by Pyk2 deficiency/inhibition was due to osteoclast detachment and anoikis. Further, GC prolongation of osteoclast lifespan was absent in KO and PF-treated osteoclasts, demonstrating Pyk2 as an intrinsic osteoclast-survival regulator. Circumventing Pyk2 activation preserves skeletal integrity by preventing GC effects on bone cell survival (proapoptotic for osteoblasts/osteocytes, antiapoptotic for osteoclasts) and GC-induced bone resorption. Thus, Pyk2/anoikis signaling as a therapeutic target for GC-induced osteoporosis

Breaking the icosahedra in boron carbide

Findings of laser-assisted atom probe tomography experiments on boron carbide elucidate an approach for characterizing the atomic structure and interatomic bonding of molecules associated with extraordinary structural stability. The discovery of crystallographic planes in these boron carbide datasets substantiates that crystallinity is maintained to the point of field evaporation, and characterization of individual ionization events gives unexpected evidence of the destruction of individual icosahedra. Statistical analyses of the ions created during the field evaporation process have been used to deduce relative atomic bond strengths and show that the icosahedra in boron carbide are not as stable as anticipated. Combined with quantum mechanics simulations, this result provides insight into the structural instability and amorphization of boron carbide. The temporal, spatial, and compositional information provided by atom probe tomography makes it a unique platform for elucidating the relative stability and interactions of primary building blocks in hierarchically crystalline materials

PTHrP-Derived Peptides Restore Bone Mass and Strength in Diabetic Mice: Additive Effect of Mechanical Loading

There is an unmet need to understand the mechanisms underlying skeletal deterioration in diabetes mellitus (DM) and to develop therapeutic approaches to treat bone fragility in diabetic patients. We demonstrate herein that mice with type 1 DM induced by streptozotocin exhibited low bone mass, inferior mechanical and material properties, increased bone resorption, decreased bone formation, increased apoptosis of osteocytes, and increased expression of the osteocyte-derived bone formation inhibitor Sost/sclerostin. Further, short treatment of diabetic mice with parathyroid hormone related protein (PTHrP)-derived peptides corrected these changes to levels undistinguishable from non-diabetic mice. In addition, diabetic mice exhibited reduced bone formation in response to mechanical stimulation, which was corrected by treatment with the PTHrP peptides, and higher prevalence of apoptotic osteocytes, which was reduced by loading or by the PTHrP peptides alone and reversed by a combination of loading and PTHrP peptide treatment. In vitro experiments demonstrated that the PTHrP peptides or mechanical stimulation by fluid flow activated the survival kinases ERKs and induced nuclear translocation of the canonical Wnt signaling mediator β-catenin, and prevented the increase in osteocytic cell apoptosis induced by high glucose. Thus, PTHrP-derived peptides cross-talk with mechanical signaling pathways to reverse skeletal deterioration induced by DM in mice. These findings suggest a crucial role of osteocytes in the harmful effects of diabetes on bone and raise the possibility of targeting these cells as a novel approach to treat skeletal deterioration in diabetes. Moreover, our study suggests the potential therapeutic efficacy of combined pharmacological and mechanical stimuli to promote bone accrual and maintenance in diabetic subjects

How metal films de-wet substrates - identifying the kinetic pathways and energetic driving forces

We study how single-crystal chromium films of uniform thickness on W(110) substrates are converted to arrays of three-dimensional (3D) Cr islands during annealing. We use low-energy electron microscopy (LEEM) to directly observe a kinetic pathway that produces trenches that expose the wetting layer. Adjacent film steps move simultaneously uphill and downhill relative to the staircase of atomic steps on the substrate. This step motion thickens the film regions where steps advance. Where film steps retract, the film thins, eventually exposing the stable wetting layer. Since our analysis shows that thick Cr films have a lattice constant close to bulk Cr, we propose that surface and interface stress provide a possible driving force for the observed morphological instability. Atomistic simulations and analytic elastic models show that surface and interface stress can cause a dependence of film energy on thickness that leads to an instability to simultaneous thinning and thickening. We observe that de-wetting is also initiated at bunches of substrate steps in two other systems, Ag/W(110) and Ag/Ru(0001). We additionally describe how Cr films are converted into patterns of unidirectional stripes as the trenches that expose the wetting layer lengthen along the W[001] direction. Finally, we observe how 3D Cr islands form directly during film growth at elevated temperature. The Cr mesas (wedges) form as Cr film steps advance down the staircase of substrate steps, another example of the critical role that substrate steps play in 3D island formation

Skeletal Protection and Promotion of Microbiome Diversity by Dietary Boosting of the Endogenous Antioxidant Response

There is an unmet need for interventions with better compliance that prevent the adverse effects of sex steroid deficiency on the musculoskeletal system. We identified a blueberry cultivar (Montgomerym [Mont]) that added to the diet protects female mice from musculoskeletal loss and body weight changes induced by ovariectomy. Mont, but not other blueberries, increased the endogenous antioxidant response by bypassing the traditional antioxidant transcription factor Nrf2 and without activating estrogen receptor canonical signaling. Remarkably, Mont did not protect the male skeleton from androgen-induced bone loss. Moreover, Mont increased the variety of bacterial communities in the gut microbiome (α-diversity) more in female than in male mice; shifted the phylogenetic relatedness of bacterial communities (β-diversity) further in females than males; and increased the prevalence of the taxon Ruminococcus1 in females but not males. Therefore, this nonpharmacologic intervention (i) protects from estrogen but not androgen deficiency; (ii) preserves bone, skeletal muscle, and body composition; (iii) elicits antioxidant defense responses independently of classical antioxidant/estrogenic signaling; and (iv) increases gut microbiome diversity toward a healthier signature. These findings highlight the impact of nutrition on musculoskeletal and gut microbiome homeostasis and support the precision medicine principle of tailoring dietary interventions to patient individualities, like sex.Fil: Sato, Amy Y.. University of Arkansas for Medical Sciences; Estados Unidos. Indiana University. School of Medicine; Estados UnidosFil: Pellegrini, Gretel Gisela. Indiana University. School of Medicine; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; ArgentinaFil: Cregor, Meloney. University of Arkansas for Medical Sciences; Estados Unidos. Indiana University. School of Medicine; Estados UnidosFil: McAndrews, Kevin. Indiana University. School of Medicine; Estados UnidosFil: Choi, Roy B. Indiana University. School of Medicine; Estados UnidosFil: Maiz, Maria. Purdue University; Estados UnidosFil: Johnson, Olivia. Indiana University. School of Medicine; Estados UnidosFil: McCabe, Linda D.. Purdue University; Estados UnidosFil: McCabe, George P.. Purdue University; Estados UnidosFil: Ferruzzi, Mario G.. North Carolina State University; Estados UnidosFil: Lila, Mary Ann. North Carolina State University; Estados UnidosFil: Peacock, Munro. Indiana University. School of Medicine; Estados UnidosFil: Burr, David B.. Indiana University. School of Medicine; Estados UnidosFil: Nakatsu, Cindy H.. Purdue University; Estados UnidosFil: Weaver, Connie M.. Purdue University; Estados UnidosFil: Bellido, Teresita. University of Arkansas for Medical Sciences; Estados Unidos. Indiana University. School of Medicine; Estados Unido