Radiation Biophysics Laboratory

UNLABELLED: Bone loss is a current limitation for long-term space exploration. Bone markers, calcitropic hormones, and calcium kinetics of crew members on space missions of 4-6 months were evaluated. Spaceflight-induced bone loss was associated with increased bone resorption and decreased calcium absorption. INTRODUCTION: Bone loss is a significant concern for the health of astronauts on long-duration missions. Defining the time course and mechanism of these changes will aid in developing means to counteract these losses during space flight and will have relevance for other clinical situations that impair weight-bearing activity. MATERIALS AND METHODS: We report here results from two studies conducted during the Shuttle-Mir Science Program. Study 1 was an evaluation of bone and calcium biochemical markers of 13 subjects before and after long-duration (4-6 months) space missions. In study 2, stable calcium isotopes were used to evaluate calcium metabolism in six subjects before, during, and after flight. Relationships between measures of bone turnover, biochemical markers, and calcium kinetics were examined. RESULTS: Pre- and postflight study results confirmed that, after landing, bone resorption was increased, as indicated by increases in urinary calcium (p < 0.05) and collagen cross-links (N-telopeptide, pyridinoline, and deoxypyridinoline were all increased >55% above preflight levels, p < 0.001). Parathyroid hormone and vitamin D metabolites were unchanged at landing. Biochemical markers of bone formation were unchanged at landing, but 2-3 weeks later, both bone-specific alkaline phosphatase and osteocalcin were significantly (p < 0.01) increased above preflight levels. In studies conducted during flight, bone resorption markers were also significantly higher than before flight. The calcium kinetic data also validated that bone resorption was increased during flight compared with preflight values (668 +/- 130 versus 427 +/- 153 mg/day; p < 0.001) and clearly documented that true intestinal calcium absorption was significantly lower during flight compared with preflight values (233 +/- 87 versus 460 +/- 47 mg/day; p < 0.01). Weightlessness had a detrimental effect on the balance in bone turnover such that the daily difference in calcium retention during flight compared with preflight values approached 300 mg/day (-234 +/- 102 versus 63 +/- 75 mg/day; p < 0.01). CONCLUSIONS: These bone marker and calcium kinetic studies indicated that the bone loss that occurs during space flight is a consequence of increased bone resorption and decreased intestinal calcium absorption.

In their normal in vivo matrix milieu, tissues assume complex well-organized three-dimensional architectures. Therefore, the primary aim in the tissue engineering design process is to fabricate an optimal analog of the in vivo scenario. This challenge can be addressed by applying emerging layered biofabrication approaches in which the precise configuration and composition of cells and bioactive matrix components can recapitulate the well-defined three-dimensional biomimetic microenvironments that promote cell-cell and cell-matrix interactions. Furthermore, the advent of and refinements in microfabricated systems can present physical and chemical cues to cells in a controllable and reproducible fashion unmatched with conventional cultures, resulting in the precise construction of engineered biomimetic microenvironments on the cellular length scale in geometries that are readily parallelized for high throughput in vitro models. As such, the convergence of layered solid freeform fabrication (SFF) technologies along with microfabrication techniques enables the creation of a three-dimensional micro-organ device to serve as an in vitro platform for cell culture, drug screening or to elicit further biological insights, particularly for NASA's interest in a flight-suitable high-fidelity microscale platform to study drug metabolism in space and planetary environments. The proposed model in this paper involves the combinatorial setup of an automated syringe-based, layered direct cell writing bioprinting process with micro-patterning techniques to fabricate a microscale in vitro device housing a chamber of bioprinted three-dimensional liver cell-encapsulated hydrogel-based tissue constructs in defined design patterns that biomimic the cell's natural microenvironment for enhanced biological functionality. In order to assess the structural formability and biological feasibility of such a micro-organ, reproducibly fabricated tissue constructs were biologically characterized for liver cell-specific function. Another key facet of the in vivo microenvironment that was recapitulated with the in vitro system included the necessary dynamic perfusion of the three-dimensional microscale liver analog with cells probed for their collective drug metabolic function and suitability as a drug metabolism model. This paper details the principles and methods that undergird the direct cell writing biofabrication process development and adaptation of microfluidic devices for the creation of a drug screening model, thereby establishing a novel drug metabolism study platform for NASA's interest to adopt a microfluidic microanalytical device with an embedded three-dimensional microscale liver tissue analog to assess drug pharmacokinetic profiles in planetary environments.

We compare scaling properties of the cardiac dynamics during sleep and wake periods for healthy individuals, cosmonauts during orbital flight, and subjects with severe heart disease. For all three groups, we find a greater degree of anticorrelation in the heartbeat fluctuations during sleep compared to wake periods. The sleep-wake difference in the scaling exponents for the three groups is comparable to the difference between healthy and diseased individuals. The observed scaling differences are not accounted for simply by different levels of activity, but appear related to intrinsic changes in the neuroautonomic control of the heartbeat.

RNA interference holds tremendous potential as a therapeutic approach, especially in the treatment of malignant tumors. However, efficient and biocompatible delivery methods are needed for systemic delivery of small interfering RNA (siRNA). To maintain a high level of growth, tumor cells scavenge high-density lipoprotein (HDL) particles by overexpressing its receptor: scavenger receptor type B1 (SR-B1). In this study, we exploited this cellular characteristic to achieve efficient siRNA delivery and established a novel formulation of siRNA by incorporating it into reconstituted HDL (rHDL) nanoparticles. Here, we demonstrate that rHDL nanoparticles facilitate highly efficient systemic delivery of siRNA in vivo, mediated by the SR-B1. Moreover, in therapeutic proof-of-concept studies, these nanoparticles were effective in silencing the expression of two proteins that are key to cancer growth and metastasis (signal transducer and activator of transcription 3 and focal adhesion kinase) in orthotopic mouse models of ovarian and colorectal cancer. These data indicate that an rHDL nanoparticle is a novel and highly efficient siRNA carrier, and therefore, this novel technology could serve as the foundation for new cancer therapeutic approaches.

The objective of this paper is to introduce a novel cell printing and microfluidic system to serve as a portable ground model for the study of drug conversion and radiation protection of living liver tissue analogs. The system is applied to study behavior in ground models of space stress, particularly radiation. A microfluidic environment is engineered by two cell types to prepare an improved higher fidelity in vitro micro-liver tissue analog. Cell-laden Matrigel printing and microfluidic chips were used to test radiation shielding to liver cells by the pro-drug amifostine. In this work, the sealed microfluidic chip regulates three variables of interest: radiation exposure, anti-radiation drug treatment and single- or dual-tissue culture environments. This application is intended to obtain a scientific understanding of the response of the multi-cellular biological system for long-term manned space exploration, disease models and biosensors.

PURPOSE: Increased adrenergic activity in response to chronic stress is known to promote tumor growth by stimulating the tumor microenvironment. The focus of the current study was to determine whether dopamine, an inhibitory catecholamine, could block the effects of chronic stress on tumor growth. EXPERIMENTAL DESIGN: Expression of dopamine receptors (DR1-DR5) was analyzed by reverse transcriptase-PCR and by Western blotting. In vitro effects of dopamine on cell viability, apoptosis, and migration were examined. For in vivo therapy, murine and human DR2-siRNAs were incorporated into chitosan nanoparticles (CH-NP). RESULTS: In this model of chronic stress, tumoral norepinephrine levels remained elevated whereas dopamine levels were significantly decreased compared with nonstressed animals. Daily restraint stress resulted in significantly increased tumor growth in both immunodeficient (SKOV3ip1 and HeyA8) and immunocompetent (ID8) ovarian cancer models. This increase was completely blocked with daily dopamine treatment. Dopamine treatment also blocked the stress-induced increase in angiogenesis. Endothelial and ovarian cancer cells expressed all dopamine receptors except for the lack of DR3 expression in ovarian cancer cells. DR2 was responsible for the inhibitory effects of dopamine on tumor growth and microvessel density as well as the stimulatory effect on apoptosis, as the DR2 antagonist eticlopride reversed these effects. Dopamine significantly inhibited cell viability and stimulated apoptosis in vitro. Moreover, dopamine reduced cyclic AMP levels and inhibited norepinephrine and vascular permeability factor/VEGF-induced Src kinase activation. CONCLUSIONS: Dopamine depletion under chronic stress conditions creates a permissive microenvironment for tumor growth that can be reversed by dopamine replacement.

Medications have been taken since the first Mercury flight in 1967 and, since then, have been used for several indications such as space motion sickness, sleeplessness, headache, nausea, vomiting, back pain, and congestion. As the duration of space missions get longer, it is even more likely that astronauts will encounter some of the acute illnesses that are frequently seen on Earth. Microgravity environment induces several physiological changes in the human body. These changes include cardiovascular degeneration, bone decalcification, decreased plasma volume, blood flow, lymphocyte and eosinophil levels, altered hormonal and electrolyte levels, muscle atrophy, decreased blood cell mass, increased immunoglobulin A and M levels, and a decrease in the amount of microsomal P-450 and the activity of some of its dependent enzymes. These changes may be expected to have severe implications on the pharmacokinetic and pharmacodynamic properties of drug substances.

Stress granules are macromolecular aggregates of mRNA and proteins assembling in response to stresses that promote the repression of protein synthesis. Most of the work characterizing stress granules has been done under acute stress conditions or during viral infection. Comparatively less work has been done to understand stress granule assembly during chronic stress, specifically regarding the composition and function of stress granules in this alternative context. Here, we describe key aspects of stress granule biology under acute stress, and how these stress granule hallmarks differ in the context of chronic stress conditions. We will provide perspective for future work aimed at further uncovering the form and function of both acute and chronic stress granules and discuss aspects of stress granule biology that have the potential to be exploited in human disease.

UNLABELLED: Counteracting bone loss is required for future space exploration. We evaluated the ability of treadmill exercise in a LBNP chamber to counteract bone loss in a 30-day bed rest study. Eight pairs of identical twins were randomly assigned to sedentary control or exercise groups. Exercise within LBNP decreased the bone resorption caused by bed rest and may provide a countermeasure for spaceflight. INTRODUCTION: Bone loss is one of the greatest physiological challenges for extended-duration space missions. The ability of exercise to counteract weightlessness-induced bone loss has been studied extensively, but to date, it has proven ineffective. We evaluated the effectiveness of a combination of two countermeasures-treadmill exercise while inside a lower body negative pressure (LBNP) chamber-on bone loss during a 30-day bed rest study. MATERIALS AND METHODS: Eight pairs of identical twins were randomized into sedentary (SED) or exercise/LBNP (EX/LBNP) groups. Blood and urine samples were collected before, several times during, and after the 30-day bed rest period. These samples were analyzed for markers of bone and calcium metabolism. Repeated measures ANOVA was used to determine statistical significance. Because identical twins were used, both time and group were treated as repeated variables. RESULTS: Markers of bone resorption were increased during bed rest in samples from sedentary subjects, including the collagen cross-links and serum and urinary calcium concentrations. For N-telopeptide and deoxypyridinoline, there were significant (p < 0.05) interactions between group (SED versus EX/LBNP) and phase of the study (sample collection point). Pyridinium cross-links were increased above pre-bed rest levels in both groups, but the EX/LBNP group had a smaller increase than the SED group. Markers of bone formation were unchanged by bed rest in both groups. CONCLUSIONS: These data show that this weight-bearing exercise combined with LBNP ameliorates some of the negative effects of simulated weightlessness on bone metabolism. This protocol may pave the way to counteracting bone loss during spaceflight and may provide valuable information about normal and abnormal bone physiology here on Earth.

Half of the nucleotide substitutions during the evolutionary divergence of genes in animals, bacteria, and viruses are silent changes. These result from an inherent biochemical property of DNA and are fixed by genetic drift. Evolution may be viewed as a device for protecting DNA molecules from extinction.

Exposure to radiation provokes cellular responses controlled in part by gene expression networks. MicroRNAs (miRNAs) are small non-coding RNAs which mostly regulate gene expression by degrading the messages or inhibiting translation. Here, we investigated changes in miRNA expression patterns after low (0.1 Gy) and high (2.0 Gy) doses of X-ray in human fibroblasts. At early (0.5 h) and late (6 and 24 h) time points, irradiation caused qualitative and quantitative differences in the down-regulation of miRNA levels, including miR-92b, 137, 660, and 656. A transient up-regulation of miRNAs was observed after 2 h post-irradiation following high doses of radiation, including miR-558 and 662. MicroRNA levels were inversely correlated with targets from mRNA and proteomic profiling after 2.0 Gy of radiation. MicroRNAs miR-579, 608, 548-3p, and 585 are noted for targeting genes involved in radioresponsive mechanisms, such as cell cycle checkpoint and apoptosis. We suggest here a model in which miRNAs may act as "hub" regulators of specific cellular responses, immediately down-regulated so as to stimulate DNA repair mechanisms, followed by up-regulation involved in suppressing apoptosis for cell survival. Taken together, miRNAs may mediate signaling pathways in sequential fashion in response to radiation, and may serve as biodosimetric markers of radiation exposure.

Multi-satellite sensor and ground observation data were analysed soon after the Wenchuan Earthquake of magnitude 7.9 (according to the USGS) or 8.0 (according to Xinhua, the Chinese News Agency) that occurred on 12 May 2008. This earthquake was felt by millions of people living in a number of countries; it took 65 000 lives and made millions of people homeless, damaged small and large infrastructure, and large surface manifestations were observed on the surface. Soon after the earthquake multi-satellite sensor and ground observatory data were analysed to study changes in ground, meteorological and atmospheric parameters. A detailed analysis of these data shows pronounced anomalous changes prior to the Wenchuan Earthquake event. Analysis of Atmospheric InfraRed Sounder (AIRS) data at different pressure levels clearly shows characteristic behaviour of the air temperature and relative humidity compared to other days. Such changes are observed up to 500 hPa, with maximal change at the lower levels. Changes in the surface, atmosphere, meteorological and ionospheric parameters prior to the Wenchuan Earthquake are complementary to one another and show the existence of a strong coupling between land, atmosphere and ionosphere, associated with the Wenchuan Earthquake.

Abstract Previous models which describe the water‐content potential relationship for plant tissue do not account for die presence of apoplastic water in the tissue and its variation with water potential. This could cause errors in determination of tissue water parameters which are sensitive to plant water deficits. The theory was modified to account for apoplastic water fraction and used to determine the osmotic potential at full and zero turgor and tissue elastic modulus from moisture release curves for leaves of three winter wheat ( Triticum aestivum L.) cultivars grown under moderately high and low stress environments in eastern Washington. Moisture release curves for individual leaves were obtained using a pressure chamber. Apoplastic water content was obtained independently from moisture release curves of frozen leaves using a hydraulic press. Osmotic potential at full and zero turgor showed significant responses to environment with leaves from the drier location having osmotic potentials 5 bars or more lower than leaves from the more moist site. Shifts in osmotic potential from wet to dry portions of the season were also about 5 bars. Apoplastic water fraction and elastic modulus were relatively insensitive to environment, averaging around 0.3 and 200 bars, respectively. Significant cultivar differences were found in tissue osmotic potential, but these did not correlate with performance of the cultivars in droughty environments. The study showed that apoplastic water in tissue, if not corrected for, markedly influences osmotic potential measurements using expressed cell sap since this water, which is essentially solute‐free, dilutes the cell sap when the membranes are destroyed. The tissue water parameters studied provide further insight on physiological factors important in plant resistance to moisture stress.

PURPOSE: To investigate how cell-cycle delays in human peripheral lymphocytes affect the expression of complex chromosome damage in metaphase following high- and low-LET radiation exposure. MATERIALS AND METHODS: Whole blood was irradiated in vitro with a low and a high dose of 1 GeV u(-1) iron particles, 400MeV u(-1) neon particles or y-rays. Lymphocytes were cultured and metaphase cells were collected at different time points after 48-84h in culture. Interphase chromosomes were prematurely condensed using calyculin-A, either 48 or 72 h after exposure to iron particles or gamma-rays. Cells in first division were analysed using a combination of FISH whole-chromosome painting and DAPI/ Hoechst 33258 harlequin staining. RESULTS: There was a delay in expression of chromosome damage in metaphase that was LET- and dose-dependant. This delay was mostly related to the late emergence of complex-type damage into metaphase. Yields of damage in PCC collected 48 h after irradiation with iron particles were similar to values obtained from cells undergoing mitosis after prolonged incubation. CONCLUSION: The yield of high-LET radiation-induced complex chromosome damage could be underestimated when analysing metaphase cells collected at one time point after irradiation. Chemically induced PCC is a more accurate technique since problems with complicated cell-cycle delays are avoided.

2D-NOE and 1H NMR chemical shift data obtained for the title oligonucleotides were compared with similar data previously reported [Broido et al. (1985) Eur. J. Biochem. 150, 117-128] for the unmodified "parent" structure, [d(GGAATTCC)]2. The spectroscopically detectable structural perturbations caused by replacement of phosphate oxygen with sulfur were mostly localized within the GsA moiety, and were greater for the Rp configuration wherein sulfur is oriented into the major groove of the B-helix. UV-derived Tm measurements gave the following order of stability for the duplexes in 0.4 M NaCl: unmodified (33.9 +/- 0.1 degrees C) approximately Sp-Sp (34.1 degrees C) greater than Rp-Rp (31.7 degrees C). The title compounds were prepared by a new and convenient synthetic route which utilized HPLC to separate the diastereomeric O-ethyl phosphorothioate precursors, (Rp)- and (Sp)-d[GG(S,Et)AATTCC], for subsequent de-ethylation by ammonia in water.

High-protein and acidogenic diets induce hypercalciuria. Foods or supplements with excess sulfur-containing amino acids increase endogenous sulfuric acid production and therefore have the potential to increase calcium excretion and alter bone metabolism. In this study, effects of an amino acid/carbohydrate supplement on bone resorption were examined during bed rest. Thirteen subjects were divided at random into two groups: a control group (Con, n = 6) and an amino acid-supplemented group (AA, n = 7) who consumed an extra 49.5 g essential amino acids and 90 g carbohydrate per day for 28 days. Urine was collected for n-telopeptide (NTX), deoxypyridinoline (DPD), calcium, and pH determinations. Bone mineral content was determined and potential renal acid load was calculated. Bone-specific alkaline phosphatase was measured in serum samples collected on day 1 (immediately before bed rest) and on day 28. Potential renal acid load was higher in the AA group than in the Con group during bed rest (P < 0.05). For all subjects, during bed rest urinary NTX and DPD concentrations were greater than pre-bed rest levels (P < 0.05). Urinary NTX and DPD tended to be higher in the AA group (P = 0.073 and P = 0.056, respectively). During bed rest, urinary calcium was greater than baseline levels (P < 0.05) in the AA group but not the Con group. Total bone mineral content was lower after bed rest than before bed rest in the AA group but not the Con group (P < 0.05). During bed rest, urinary pH decreased (P < 0.05), and it was lower in the AA group than the Con group. These data suggest that bone resorption increased, without changes in bone formation, in the AA group.

PURPOSE: Src is an attractive target because it is overexpressed in a number of malignancies, including ovarian cancer. However, the effect of Src silencing on other Src family kinases (SFKs) is not known. We hypothesized that other SFK members could compensate for the lack of Src activity. EXPERIMENTAL DESIGN: Cell viability after either Src or Fgr silencing was examined in ovarian cancer cell lines by MTT assay. Expression of SFKs after Src silencing in ovarian cancer cells was examined by real-time reverse transcriptase (RT)-PCR. Therapeutic effect of in vivo Src and/or Fgr silencing was examined using siRNA incorporated into chitosan nanoparticles (siRNA/CH-NP). Microvessel density, cell proliferation, and apoptosis markers were determined by immunohistochemical staining in ovarian tumor tissues. RESULTS: Src silencing enhanced cytotoxicity of docetaxel in both SKOV3ip1 and HeyA8 cells. In addition, Src silencing using siRNA/CH-NP in combination with docetaxel resulted in significant inhibition of tumor growth compared with control siRNA/CH-NP (81.8% reduction in SKOV3ip1, P = 0.017; 84.3% reduction in HeyA8, P < 0.005). These effects were mediated by decreased tumor cell proliferation and angiogenesis, and increased tumor cell apoptosis. Next, we assessed the effects of Src silencing on other SFK members in ovarian cancer cell lines. Src silencing resulted in significantly increased Fgr levels. Dual Src and Fgr silencing in vitro resulted in increased apoptosis that was mediated by increased caspase and AKT activity. In addition, dual silencing of Src and Fgr in vivo using siRNA/CH-NP resulted in the greatest reduction in tumor growth compared with silencing of either Src or Fgr alone in the HeyA8 model (68.8%, P < 0.05). CONCLUSIONS: This study demonstrates that, in addition to Src, Fgr plays a biologically significant role in ovarian cancer growth and might represent an important target.

Sulfonylurea receptors SUR1 and SUR2 are the regulatory subunits of K(ATP) channels. Their differential affinity for hypoglycemic sulfonylureas provides a basis for the selectivity of these compounds for different K(ATP) channel isoforms. Sulfonylureas have a 100- to 1000-fold greater affinity for SUR1 vs. SUR2. Structure-activity studies suggested a bipartite binding pocket. Chimeric SUR1 approximately SUR2 receptors have shown TMD2, the third bundle of transmembrane helices, to be part of an "A" site that confers SUR1 selectivity for sulfonylureas. The purpose of this study is to determine the position of the "B" site. Previous photoaffinity labeling studies have placed the B site on the amino-terminal third of SUR and colabeled the associated K(IR). In our study, deletion of TMD0, the first bundle of transmembrane helices, did not compromise labeling. Further deletions into the cytoplasmic linker, L0, eliminated binding and labeling. Alanine substitutions in L0 identified a limited number of conserved residues, Y230 and W232, important for affinity labeling. A fragment of K(IR)6.2, missing M2 and the entire carboxyl terminal, assembles with SUR1 and is affinity labeled, while deletion of 10 or more amino-terminal residues compromises labeling. These studies indicate that the B site involves L0 and the K(IR) amino terminus, elements that are critical for control of channel gating.

PURPOSE: To study the frequency of true incomplete exchanges in radiation-induced chromosome aberrations. MATERIALS AND METHODS: Human lymphocytes were exposed to 2 Gy and 5 Gy of gamma-rays. Chromosome aberrations were studied using the fluorescence in situ hybridization (FISH) technique with whole chromosome-specific probes, together with human telomere probes. Chromosomes 2 and 4 were chosen in the present study. RESULTS: The percentage of incomplete exchanges was 27% when telomere signals were not considered. After excluding false incomplete exchanges identified by the telomere signals, the percentage of incomplete exchanges decreased to 11%. Since telomere signals appear on about 82% of the telomeres, the percentage of true incomplete exchanges should be even lower and was estimated to be 3%. This percentage was similar for chromosomes 2 and 4 and for doses of both 2 Gy and 5 Gy. CONCLUSIONS: The percentage of true incomplete exchanges is significantly lower in gamma-irradiated human lymphocytes than the frequencies reported in the literature.

Transposable elements (TEs) comprise a large proportion of long non-coding RNAs (lncRNAs). Here, we employed CRISPR to delete a short interspersed nuclear element (SINE) in Malat1, a cancer-associated lncRNA, to investigate its significance in cellular physiology. We show that Malat1 with a SINE deletion forms diffuse nuclear speckles and is frequently translocated to the cytoplasm. SINE-deleted cells exhibit an activated unfolded protein response and PKR and markedly increased DNA damage and apoptosis caused by dysregulation of TDP-43 localization and formation of cytotoxic inclusions. TDP-43 binds stronger to Malat1 without the SINE and is likely 'hijacked' by cytoplasmic Malat1 to the cytoplasm, resulting in the depletion of nuclear TDP-43 and redistribution of TDP-43 binding to repetitive element transcripts and mRNAs encoding mitotic and nuclear-cytoplasmic regulators. The SINE promotes Malat1 nuclear retention by facilitating Malat1 binding to HNRNPK, a protein that drives RNA nuclear retention, potentially through direct interactions of the SINE with KHDRBS1 and TRA2A, which bind to HNRNPK. Losing these RNA-protein interactions due to the SINE deletion likely creates more available TDP-43 binding sites on Malat1 and subsequent TDP-43 aggregation. These results highlight the significance of lncRNA TEs in TDP-43 proteostasis with potential implications in both cancer and neurodegenerative diseases.