Phosphoribosyl pyrophosphate synthetase from Escherichia coli was cloned, purified, and crystal- lized. Single crystals of the enzyme were grown under microgravity. The X-ray diffraction data set was col- lected at the Spring-8 synchrotron facility and used to determine the three-dimensional structure of the enzyme by the molecular-replacement method at 2.71 Å resolution. The active and regulatory sites in the molecule of E. coli phosphoribosyl pyrophosphate synthetase were revealed by comparison with the homol- ogous protein from Bacillus subtilis, the structure of which was determined in a complex with functional ligands. The conformations of polypeptide-chain fragments surrounding and composing the active and reg- ulatory sites were shown to be identical in both proteins.
Research Containing: Microgravity
Three-dimensional structure of carboxypeptidase T from Thermoactinomyces vulgaris in complex with N-BOC-L-leucine
The 3D structure of recombinant bacterial carboxypeptidase T (CPT) in complex with N-BOC-L-leucine was determined at 1.38 A resolution. Crystals for the X-ray study were grown in microgravity using the counter-diffusion technique. N-BOC-L-leucine and SO4(2-) ion bound in the enzyme active site were localized in the electron density map. Location of the leucine side chain in CPT-N-BOC-L-leucine complex allowed identification of the S1 subsite of the enzyme, and its structure was determined. Superposition of the structures of CPT-N-BOC-L-leucine complex and complexes of pancreatic carboxypeptidases A and B with substrate and inhibitors was carried out, and similarity of the S1 subsites in these three carboxypeptidases was revealed. It was found that SO4(2-) ion occupies the same position in the S1′ subsite as the C-terminal carboxy group of the substrate.
We present a phase-field study of oscillatory breathing modes observed during the solidification of three-dimensional cellular arrays in microgravity. Directional solidification experiments conducted onboard the International Space Station have allowed us to observe spatially extended homogeneous arrays of cells and dendrites while minimizing the amount of gravity-induced convection in the liquid. In situ observations of transparent alloys have revealed the existence, over a narrow range of control parameters, of oscillations in cellular arrays with a period ranging from about 25 to 125 min. Cellular patterns are spatially disordered, and the oscillations of individual cells are spatiotemporally uncorrelated at long distance. However, in regions displaying short-range spatial ordering, groups of cells can synchronize into oscillatory breathing modes. Quantitative phase-field simulations show that the oscillatory behavior of cells in this regime is linked to a stability limit of the spacing in hexagonal cellular array structures. For relatively high cellular front undercooling (i.e., low growth velocity or high thermal gradient), a gap appears in the otherwise continuous range of stable array spacings. Close to this gap, a sustained oscillatory regime appears with a period that compares quantitatively well with experiment. For control parameters where this gap exists, oscillations typically occur for spacings at the edge of the gap. However, after a change of growth conditions, oscillations can also occur for nearby values of control parameters where this gap just closes and a continuous range of spacings exists. In addition, sustained oscillations at to the opening of this stable gap exhibit a slow periodic modulation of the phase-shift among cells with a slower period of several hours. While long-range coherence of breathing modes can be achieved in simulations for a perfect spatial arrangement of cells as initial condition, global disorder is observed in both three-dimensional experiments and simulations from realistic noisy initial conditions. In the latter case, erratic tip-splitting events promoted by large-amplitude oscillations contribute to maintaining the long-range array disorder, unlike in thin-sample experiments where long-range coherence of oscillations is experimentally observable.
We examine the dynamics of a binary mixture in a cubic cell subjected to a temperature differential and oscillatory forcing. The Soret effect, which is negative in the present study, provides a coupling mechanism by which a temperature gradient establishes a concentration gradient in a mixture. We present the results of experiments that were performed on the International Space Station (ISS) and compare the observations with the results of direct numerical simulations. The evolution of temperature and concentration fields is investigated by optical digital interferometry. One advantage of the experimental technique is the observation of the fields along two perpendicular directions of the cell, allowing us to restore the three-dimensional field. Experimental evidence disproves speculations that the ISS microgravity environment always affects diffusion-controlled processes. Furthermore, we demonstrate that imposed vibrations with constant frequency and amplitude create slow mean flows and that they do influence the diffusion kinetics. The perturbation of the diffusive fields scales as the square of the vibrational velocity. In addition to calculations of the full three-dimensional Navier–Stokes equations, a two-time-scale computational methodology is used for situations in which the forcing period is very small compared to the natural time scales of the problem. The simulations show excellent agreement with experimental observations.
Guidelines for Dual Energy X-Ray Absorptiometry Analysis of Trabecular Bone-Rich Regions in Mice: Improved Precision, Accuracy, and Sensitivity for Assessing Longitudinal Bone Changes
Trabecular bone is frequently studied in osteoporosis research because changes in trabecular bone are the most common cause of osteoporotic fractures. Dual energy X-ray absorptiometry (DXA) analysis specific to trabecular bone-rich regions is crucial to longitudinal osteoporosis research. The purpose of this study is to define a novel method for accurately analyzing trabecular bone-rich regions in mice via DXA. This method will be utilized to analyze scans obtained from the International Space Station in an upcoming study of microgravity-induced bone loss. Thirty 12-week-old BALB/c mice were studied. The novel method was developed by preanalyzing trabecular bone-rich sites in the distal femur, proximal tibia, and lumbar vertebrae via high-resolution X-ray imaging followed by DXA and micro-computed tomography (micro-CT) analyses. The key DXA steps described by the novel method were (1) proper mouse positioning, (2) region of interest (ROI) sizing, and (3) ROI positioning. The precision of the new method was assessed by reliability tests and a 14-week longitudinal study. The bone mineral content (BMC) data from DXA was then compared to the BMC data from micro-CT to assess accuracy. Bone mineral density (BMD) intra-class correlation coefficients of the new method ranging from 0.743 to 0.945 and Levene’s test showing that there was significantly lower variances of data generated by new method both verified its consistency. By new method, a Bland-Altman plot displayed good agreement between DXA BMC and micro-CT BMC for all sites and they were strongly correlated at the distal femur and proximal tibia (r=0.846, p<0.01; r=0.879, p<0.01, respectively). The results suggest that the novel method for site-specific analysis of trabecular bone-rich regions in mice via DXA yields more precise, accurate, and repeatable BMD measurements than the conventional method.
The effect of weightlessness on the human skeletal system is one of the greatest concerns in safely extending space missions [1–11]. The ability to understand and counteract weightlessness-induced bone mineral loss will be vital to crew health and safety during and after extended-duration space sta- tion and exploration missions [1–7]. Research on bone mineral loss during space flight has gone on for more than half a century, and recent studies have shown significant progress in developing coun- termeasures that have proved to be effective, including good nutrition and exercise. We review the history of this research here and provide a summary of recent and ongoing studies, including efforts to counteract bone and calcium loss resulting from weightlessness. Unfortunately, the most obvious nutritional countermeasure—providing excess calcium—does not protect against bone loss . This result is likely related to the decreased calcium absorption observed in space flight and in ground-based models [13–16]. Phosphate supplementation was also ineffective at reducing calcium excretion . Combination therapy with calcium and phosphorus was also unsuccessful at mitigating bone loss and hypercalciuria . Other nutrients, specifically sodium, protein, potassium, vitamin K, and omega-3 fatty acids, have also been proposed and/or tested as bone loss countermeasures , and are discussed in more detail below.
Bone loss and renal stone risk are longstanding concerns for astronauts. Bone resorption brought on by spaceflight elevates urinary calcium and the risk of renal stone formation. Loss of bone calcium leads to concerns about fracture risk and increased long-term risk of osteoporosis. Bone metabolism involves many factors and is interconnected with muscle metabolism and diet. We report here bone biochemistry and renal stone risk data from astronauts on 4- to 6-month International Space Station missions. All had access to a type of resistive exercise countermeasure hardware, either the Advanced Resistance Exercise Device (ARED) or the Interim Resistance Exercise Device (iRED). A subset of the ARED group also tested the bisphosphonate alendronate as a potential anti-resorptive countermeasure (Bis+ARED). While some of the basic bone marker data have been published, we provide here a more comprehensive evaluation of bone biochemistry with a larger group of astronauts. Regardless of exercise, the risk of renal stone formation increased during spaceflight. A key factor in this increase was urine volume, which was lower during flight in all groups at all time points. Thus, the easiest way to mitigate renal stone risk is to increase fluid consumption. ARED use increased bone formation without changing bone resorption, and mitigated a drop in parathyroid hormone in iRED astronauts. Sclerostin, an osteocyte-derived negative regulator of bone formation, increased 10-15% in both groups of astronauts who used the ARED (p<0.06). IGF-1, which regulates bone growth and formation, increased during flight in all 3 groups (p<0.001). Our results are consistent with the growing body of literature showing that the hyper-resorptive state of bone that is brought on by spaceflight can be countered pharmacologically or mitigated through an exercise-induced increase in bone formation, with nutritional support. Key questions remain about the effect of exercise-induced alterations in bone metabolism on bone strength and fracture risk.
Magnesium is an essential nutrient for muscle, cardiovascular, and bone health on Earth, and during space flight. We sought to evaluate magnesium status in 43 astronauts (34 male, 9 female; 47 +/- 5 years old, mean +/- SD) before, during, and after 4-6-month space missions. We also studied individuals participating in a ground analog of space flight (head-down-tilt bed rest; n = 27 (17 male, 10 female), 35 +/- 7 years old). We evaluated serum concentration and 24-h urinary excretion of magnesium, along with estimates of tissue magnesium status from sublingual cells. Serum magnesium increased late in flight, while urinary magnesium excretion was higher over the course of 180-day space missions. Urinary magnesium increased during flight but decreased significantly at landing. Neither serum nor urinary magnesium changed during bed rest. For flight and bed rest, significant correlations existed between the area under the curve of serum and urinary magnesium and the change in total body bone mineral content. Tissue magnesium concentration was unchanged after flight and bed rest. Increased excretion of magnesium is likely partially from bone and partially from diet, but importantly, it does not come at the expense of muscle tissue stores. While further study is needed to better understand the implications of these findings for longer space exploration missions, magnesium homeostasis and tissue status seem well maintained during 4-6-month space missions.
Cerium oxide (CeO2) was prepared using a controlled-precipitation method under microgravity at the International Space Station (ISS). For comparison, ceria was also synthesized under normal-gravity conditions (referred as control). The Brunauer-Emmett-Teller (BET) surface area, pore volume and pore size analysis results indicated that the ceria particles grown in space had lower surface area and pore volume compared to the control samples. Furthermore, the space samples had a broader pore size distribution ranging from 30–600 Å, whereas the control samples consisted of pore sizes from 30–50 Å range. Structural information of the ceria particles were obtained using TEM and XRD. Based on the TEM images, it was confirmed that the space samples were predominantly nano-rods, on the other hand, only nano-polyhedra particles were seen in the control ceria samples. The average particle size was larger for ceria samples synthesized in space. XRD results showed higher crystallinity as well as larger mean crystal size for the space samples. The effect of sodium hydroxide concentration on synthesis of ceria was also examined using 1 M and 3 M solutions. It was found that the control samples, prepared in 1 M and 3 M sodium hydroxide solutions, did not show a significant difference between the two. However, when the ceria samples were prepared in a more basic medium (3 M) under microgravity, a decrease in the particle size of the nano-rods and appearances of nano-polyhedra and spheres were observed.
Abstract: Hackney, KJ, Scott, JM, Hanson, AM, English, KL, Downs, ME, and Ploutz-Snyder, LL. The astronaut-athlete: optimizing human performance in space. J Strength Cond Res 29(12): 3531–3545, 2015—It is well known that long-duration spaceflight results in deconditioning of neuromuscular and cardiovascular systems, leading to a decline in physical fitness. On reloading in gravitational environments, reduced fitness (e.g., aerobic capacity, muscular strength, and endurance) could impair human performance, mission success, and crew safety. The level of fitness necessary for the performance of routine and off-nominal terrestrial mission tasks remains an unanswered and pressing question for scientists and flight physicians. To mitigate fitness loss during spaceflight, resistance and aerobic exercise are the most effective countermeasure available to astronauts. Currently, 2.5 h·d−1, 6–7 d·wk−1 is allotted in crew schedules for exercise to be performed on highly specialized hardware on the International Space Station (ISS). Exercise hardware provides up to 273 kg of loading capability for resistance exercise, treadmill speeds between 0.44 and 5.5 m·s−1, and cycle workloads from 0 and 350 W. Compared to ISS missions, future missions beyond low earth orbit will likely be accomplished with less vehicle volume and power allocated for exercise hardware. Concomitant factors, such as diet and age, will also affect the physiologic responses to exercise training (e.g., anabolic resistance) in the space environment. Research into the potential optimization of exercise countermeasures through use of dietary supplementation, and pharmaceuticals may assist in reducing physiological deconditioning during long-duration spaceflight and have the potential to enhance performance of occupationally related astronaut tasks (e.g., extravehicular activity, habitat construction, equipment repairs, planetary exploration, and emergency response).