Publications

Steen PH, Chang C, Bostwick JB. Droplet motions fill a periodic table. PNAS. 2019;116(11):4849-4854.

Drawing parallels to the symmetry breaking of atomic orbitals used to explain the periodic table of chemical elements; here we introduce a periodic table of droplet motions, also based on symmetry breaking but guided by a recent droplet spectral theory. By this theory, higher droplet mode shapes are discovered and a wettability spectrometer is invented. Motions of a partially wetting liquid on a support have natural mode shapes, motions ordered by kinetic energy into the periodic table, each table characteristic of the spherical-cap drop volume and material parameters. For water on a support having a contact angle of about 60°, the first 35 predicted elements of the periodic table are discovered. Periodic tables are related one to another through symmetry breaking into a two-parameter family tree.

McNeill EP, Reese RW, Tondon A, Clough BH, Pan S, Froese J, Palmer D, Krause U, Loeb DM, Kaunas R, Gregory CA. Three-dimensional in vitro modeling of malignant bone disease recapitulates experimentally accessible mechanisms of osteoinhibition. Cell Death Dis. 2018;9(12):1161.

Malignant bone disease (MBD) occurs when tumors establish in bone, causing catastrophic tissue damage as a result of accelerated bone destruction and inhibition of repair. The resultant so-called osteolytic lesions (OL) take the form of tumor-filled cavities in bone that cause pain, fractures, and associated morbidity. Furthermore, the OL microenvironment can support survival of tumor cells and resistance to chemotherapy. Therefore, a deeper understanding of OL formation and MBD progression is imperative for the development of future therapeutic strategies. Herein, we describe a novel in vitro platform to study bone?tumor interactions based on three-dimensional co-culture of osteogenically enhanced human mesenchymal stem cells (OEhMSCs) in a rotating wall vessel bioreactor (RWV) while attached to micro-carrier beads coated with extracellular matrix (ECM) composed of factors found in anabolic bone tissue. Osteoinhibition was recapitulated in this model by co-culturing the OEhMSCs with a bone?tumor cell line (MOSJ-Dkk1) that secretes the canonical Wnt (cWnt) inhibitor Dkk-1, a tumor-borne osteoinhibitory factor widely associated with several forms of MBD, or intact tumor fragments from Dkk-1 positive patient-derived xenografts (PDX). Using the model, we observed that depending on the conditions of growth, tumor cells can biochemically inhibit osteogenesis by disrupting cWnt activity in OEhMSCs, while simultaneously co-engrafting with OEhMSCs, displacing them from the niche, perturbing their activity, and promoting cell death. In the absence of detectable co-engraftment with OEhMSCs, Dkk-1 positive PDX fragments had the capacity to enhance OEhMSC proliferation while inhibiting their osteogenic differentiation. The model described has the capacity to provide new and quantifiable insights into the multiple pathological mechanisms of MBD that are not readily measured using monolayer culture or animal models.

Krishnamurthy A, Ferl RJ, Paul AL. Comparing RNA-Seq and microarray gene expression data in two zones of the Arabidopsis root apex relevant to spaceflight. Appl Plant Sci. 2018;6(11):e01197.

The root apex is an important region involved in environmental sensing, but comprises a very small part of the root. Obtaining root apex transcriptomes is therefore challenging when the samples are limited. The feasibility of using tiny root sections for transcriptome analysis was examined, comparing RNA sequencing (RNA-Seq) to microarrays in characterizing genes that are relevant to spaceflight.

Montague TG, Almansoori A, Gleason EJ, Copeland DS, Foley K, Kraves S, Saavedra EA. Gene expression studies using a miniaturized thermal cycler system on board the International Space Station. PLoS ONE. 2018;13(10):e0205852.

The distance and duration of human spaceflight missions is set to markedly increase over the coming decade as we prepare to send astronauts to Mars. However, the health impact of long-term exposure to cosmic radiation and microgravity is not fully understood. In order to identify the molecular mechanisms underpinning the effects of space travel on human health, we must develop the capacity to monitor changes in gene expression and DNA integrity in space. Here, we report successful implementation of three molecular biology procedures on board the International Space Station (ISS) using a miniaturized thermal cycler system and C. elegans as a model organism: first, DNA extraction?the initial step for any type of DNA analysis; second, reverse transcription of RNA to generate complementary DNA (cDNA); and third, the subsequent semi-quantitative PCR amplification of cDNA to analyze gene expression changes in space. These molecular procedures represent a significant expansion of the budding molecular biology capabilities of the ISS and will permit more complex analyses of space-induced genetic changes during spaceflight missions aboard the ISS and beyond.

Aguilar M, Cavasonza LA, Ambrosi G, Arruda L, Attig N, Aupetit S, Azzarello P, Bachlechner A, Barao F, Barrau A, Barrin L. Observation of new properties of secondary cosmic rays lithium, beryllium, and boron by the Alpha Magnetic Spectrometer on the International Space Station. Phys Rev Lett. 2018;120(2):021101.

We report on the observation of new properties of secondary cosmic rays Li, Be, and B measured in the rigidity (momentum per unit charge) range 1.9 GV to 3.3 TV with a total of 5.4 × 10 6 nuclei collected by AMS during the first five years of operation aboard the International Space Station. The Li and B fluxes have an identical rigidity dependence above 7 GV and all three fluxes have an identical rigidity dependence above 30 GV with the Li / Be flux ratio of 2.0 ± 0.1 . The three fluxes deviate from a single power law above 200 GV in an identical way. This behavior of secondary cosmic rays has also been observed in the AMS measurement of primary cosmic rays He, C, and O but the rigidity dependences of primary cosmic rays and of secondary cosmic rays are distinctly different. In particular, above 200 GV, the secondary cosmic rays harden more than the primary cosmic rays.

Aguilar M, Cavasonza LA, Alpat B, Ambrosi G, Arruda L, Attig N, Aupetit S, Azzarello P, Bachlechner A, Barao F, Barrau A. Observation of fine time structures in the cosmic proton and helium fluxes with the Alpha Magnetic Spectrometer on the International Space Station. Physical Review Letters. 2018;121:051101.

We present the precision measurement from May 2011 to May 2017 (79 Bartels rotations) of the proton fluxes at rigidities from 1 to 60 GV and the helium fluxes from 1.9 to 60 GV based on a total of 1×109 events collected with the Alpha Magnetic Spectrometer aboard the International Space Station. This measurement is in solar cycle 24, which has the solar maximum in April 2014. We observed that, below 40 GV, the proton flux and the helium flux show nearly identical fine structures in both time and relative amplitude. The amplitudes of the flux structures decrease with increasing rigidity and vanish above 40 GV. The amplitudes of the structures are reduced during the time period, which started one year after solar maximum, when the proton and helium fluxes steadily increase. Above ∼3  GV the p/He flux ratio is time independent. We observed that below ∼3  GV the ratio has a long-term decrease coinciding with the period during which the fluxes start to rise.

Aguilar M, Cavasonza LA, Ambrosi G, Arruda L, Attig N, Aupetit S, Azzarello P, Bachlechner A, Barao F, Barrau A, Barrin L. Observation of complex time Structures in the cosmic-ray electron and positron fluxes with the Alpha Magnetic Spectrometer on the International Space Station. Physical Review Letters. 2018;121:051102.

We present high-statistics, precision measurements of the detailed time and energy dependence of the primary cosmic-ray electron flux and positron flux over 79 Bartels rotations from May 2011 to May 2017 in the energy range from 1 to 50 GeV. For the first time, the charge-sign dependent modulation during solar maximum has been investigated in detail by leptons alone. Based on 23.5×106 events, we report the observation of short-term structures on the timescale of months coincident in both the electron flux and the positron flux. These structures are not visible in the e+/e− flux ratio. The precision measurements across the solar polarity reversal show that the ratio exhibits a smooth transition over 830±30 days from one value to another. The midpoint of the transition shows an energy dependent delay relative to the reversal and changes by 260±30 days from 1 to 6 GeV.

Aguilar, M, Cavasonza LA, Alpat B, Ambrosi G, Arruda L, Attig N, Aupetit S, Azzarello P, Bachlechner A, Barao F, Barrau A. Precision Measurement of Cosmic-Ray Nitrogen and Its Primary and Secondary Components with the Alpha Magnetic Spectrometer on the International Space Station. Physical Review Letters. 121(5):051103.

A precision measurement of the nitrogen flux with rigidity (momentum per unit charge) from 2.2 GV to 3.3 TV based on 2.2×106 events is presented. The detailed rigidity dependence of the nitrogen flux spectral index is presented for the first time. The spectral index rapidly hardens at high rigidities and becomes identical to the spectral indices of primary He, C, and O cosmic rays above ∼700  GV. We observed that the nitrogen flux ΦN can be presented as the sum of its primary component ΦPN and secondary component ΦSN, ΦN=ΦPN+ΦSN, and we found ΦN is well described by the weighted sum of the oxygen flux ΦO (primary cosmic rays) and the boron flux ΦB (secondary cosmic rays), with ΦPN=(0.090±0.002)×ΦO and ΦSN=(0.62±0.02)×ΦB over the entire rigidity range. This corresponds to a change of the contribution of the secondary cosmic ray component in the nitrogen flux from 70% at a few GV to <30% above 1 TV.

Jha R, Xu R-H, Xu C. Efficient differentiation of cardiomyocytes from human pluripotent stem cells with growth factors. Methods in Molecular Biology. 2015;1299:115-131.

Human pluripotent stem cells have tremendous replicative capacity and demonstrated potential to generate functional cardiomyocytes. These cardiomyocytes represent a promising source for cell replacement therapy to treat heart disease and may serve as a useful tool for drug discovery and disease modeling. Efficient cardiomyocyte differentiation, a prerequisite for the application of stem cell-derived cardiomyocytes, can be achieved with a growth factor-guided method. Undifferentiated cells are sequentially treated with activin A and BMP4 in a serum-free and insulin-free medium and then maintained in a serum-free medium with insulin. This method yields as much as >75% cardiomyocytes in the differentiation culture within 2 weeks, and the beating cardiomyocytes have expected molecular, cellular and electrophysiological characteristics. In this chapter, we describe in detail the differentiation protocol and follow-up characterization focusing on immunocytochemistry, quantitative RT-PCR and flow cytometry analysis.

Li C, Zheng Z, Zhang X, Asatrian G, Chen E, Song R, Culiat C, Ting K, Soo C. Nfatc1 is a functional transcriptional factor mediating Nell-1-induced Runx3 upregulation in chondrocytes. Int J Mol Sci. 2018;19(1):168.

Neural EGFL like 1 (Nell-1) is essential for chondrogenic differentiation, maturation, and regeneration. Our previous studies have demonstrated that Nell-1’s pro-chondrogenic activities are predominantly reliant upon runt-related transcription factor 3 (Runx3)-mediated Indian hedgehog (Ihh) signaling. Here, we identify the nuclear factor of activated T-cells 1 (Nfatc1) as the key transcriptional factor mediating the Nell-1 → Runx3 signal transduction in chondrocytes. Using chromatin immunoprecipitation assay, we were able to determine that Nfatc1 binds to the −833–−810 region of the Runx3-promoter in response to Nell-1 treatment. By revealing the Nell-1 → Nfatc1 → Runx3 → Ihh cascade, we demonstrate the involvement of Nfatc1, a nuclear factor of activated T-cells, in chondrogenesis, while providing innovative insights into developing a novel therapeutic strategy for cartilage regeneration and other chondrogenesis-related conditions.

Baio J, Martinez AF, Silva I, Hoehn CV, Countryman S, Bailey L, Hasaniya N, Pecaut MJ, Kearns-Jonker M. Cardiovascular progenitor cells cultured aboard the International Space Station exhibit altered developmental and functional properties. NPJ Microgravity. 2018;4:13.

The heart and its cellular components are profoundly altered by missions to space and injury on Earth. Further research, however, is needed to characterize and address the molecular substrates of such changes. For this reason, neonatal and adult human cardiovascular progenitor cells (CPCs) were cultured aboard the International Space Station. Upon return to Earth, we measured changes in the expression of microRNAs and of genes related to mechanotransduction, cardiogenesis, cell cycling, DNA repair, and paracrine signaling. We additionally assessed endothelial-like tube formation, cell cycling, and migratory capacity of CPCs. Changes in microRNA expression were predicted to target extracellular matrix interactions and Hippo signaling in both neonatal and adult CPCs. Genes related to mechanotransduction (YAP1, RHOA) were downregulated, while the expression of cytoskeletal genes (VIM, NES, DES, LMNB2, LMNA), non-canonical Wnt ligands (WNT5A, WNT9A), and Wnt/calcium signaling molecules (PLCG1, PRKCA) was significantly elevated in neonatal CPCs. Increased mesendodermal gene expression along with decreased expression of mesodermal derivative markers (TNNT2, VWF, and RUNX2), reduced readiness to form endothelial-like tubes, and elevated expression of Bmp and Tbx genes, were observed in neonatal CPCs. Both neonatal and adult CPCs exhibited increased expression of DNA repair genes and paracrine factors, which was supported by enhanced migration. While spaceflight affects cytoskeletal organization and migration in neonatal and adult CPCs, only neonatal CPCs experienced increased expression of early developmental markers and an enhanced proliferative potential. Efforts to recapitulate the effects of spaceflight on Earth by regulating processes described herein may be a promising avenue for cardiac repair.

Zhu B, Dacso CC, O'Malley BW. Unveiling "Musica Universalis" of the cell: A brief history of biological 12-hour rhythms. J Endocr Soc. 2018;2(7):727-752.

?Musica universalis? is an ancient philosophical concept claiming the movements of celestial bodies follow mathematical equations and resonate to produce an inaudible harmony of music, and the harmonious sounds that humans make were an approximation of this larger harmony of the universe. Besides music, electromagnetic waves such as light and electric signals also are presented as harmonic resonances. Despite the seemingly universal theme of harmonic resonance in various disciplines, it was not until recently that the same harmonic resonance was discovered also to exist in biological systems. Contrary to traditional belief that a biological system is either at stead-state or cycles with a single frequency, it is now appreciated that most biological systems have no homeostatic ?set point,? but rather oscillate as composite rhythms consisting of superimposed oscillations. These oscillations often cycle at different harmonics of the circadian rhythm, and among these, the ~12-hour oscillation is most prevalent. In this review, we focus on these 12-hour oscillations, with special attention to their evolutionary origin, regulation, and functions in mammals, as well as their relationship to the circadian rhythm. We further discuss the potential roles of the 12-hour clock in regulating hepatic steatosis, aging, and the possibility of 12-hour clock?based chronotherapy. Finally, we posit that biological rhythms are also musica universalis: whereas the circadian rhythm is synchronized to the 24-hour light/dark cycle coinciding with the Earth?s rotation, the mammalian 12-hour clock may have evolved from the circatidal clock, which is entrained by the 12-hour tidal cues orchestrated by the moon.

Rytlewski JD, Childress PJ, Scofield DC, Khan F, Alvarez MB, Tucker AT, Harris JS, Peveler JL, Hickman DL, Chu TG, Kacena MA. Cohousing male mice with and without segmental bone defects. Comp Med. 2018;68(2):131-138.

Spaceflight results in bone loss like that associated with osteoporosis or decreased weight-bearing (for example, high-energy trauma such as explosive injuries and automobile accidents). Thus, the unique spaceflight laboratory on the International Space Station presents the opportunity to test bone healing agents during weightlessness. We are collaborating with NASA and the US Army to study bone healing in spaceflight. Given the unique constraints of spaceflight, study design optimization was required. Male mice were selected primarily because their femur is larger than females', allowing for more reproducible surgical outcomes. However, concern was raised regarding male mouse aggression. In addition, the original spaceflight study design included cohousing nonoperated control mice with mice that had undergone surgery to create a segmental bone defect. This strategy prompted the concern that nonoperated mice would exhibit aggressive behavior toward vulnerable operated mice. We hypothesized that operated and nonoperated male mice could be cohoused successfully when they were cagemates since birth and underwent identical anesthetic, analgesic, preoperative, and postoperative conditions. Using quantitative behavioral scoring, body weight, and organ weight analyses (Student t test and ANOVA), we found that nonoperated and operated C57BL/6 male mice could successfully be housed together. The male mice did not exhibit aggressive behavior toward cagemates, whether operated or nonoperated, and the mice did not show evidence of stress, as indicated by veterinary assessment, or change in body or proportional organ weights. These findings allowed our mission to proceed (launched February 2017) and may inform future surgical study designs, potentially increasing housing flexibility.

Scofield DC, Rytlewski JD, Childress P, Shah K, Tucker A, Khan F, Peveler J, Li D, McKinley TO, Chu TG, Hickman DL, Kacena MA. Development of a step-down method for altering male C57BL/6 mouse housing density and hierarchical structure: Preparations for spaceflight studies. Life Sci Space Res (Amst). 2018;17:44-50.

This study was initiated as a component of a larger undertaking designed to study bone healing in microgravity aboard the International Space Station (ISS). Spaceflight experimentation introduces multiple challenges not seen in ground studies, especially with regard to physical space, limited resources, and inability to easily reproduce results. Together, these can lead to diminished statistical power and increased risk of failure. It is because of the limited space, and need for improved statistical power by increasing sample size over historical numbers, NASA studies involving mice require housing mice at densities higher than recommended in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). All previous NASA missions in which mice were co-housed, involved female mice; however, in our spaceflight studies examining bone healing, male mice are required for optimal experimentation. Additionally, the logistics associated with spaceflight hardware and our study design necessitated variation of density and cohort make up during the experiment. This required the development of a new method to successfully co-house male mice while varying mouse density and hierarchical structure. For this experiment, male mice in an experimental housing schematic of variable density (Spaceflight Correlate) analogous to previously established NASA spaceflight studies was compared to a standard ground based housing schematic (Normal Density Controls) throughout the experimental timeline. We hypothesized that mice in the Spaceflight Correlate group would show no significant difference in activity, aggression, or stress when compared to Normal Density Controls. Activity and aggression were assessed using a novel activity scoring system (based on prior literature, validated in-house) and stress was assessed via body weights, organ weights, and veterinary assessment. No significant differences were detected between the Spaceflight Correlate group and the Normal Density Controls in activity, aggression, body weight, or organ weight, which was confirmed by veterinary assessments. Completion of this study allowed for clearance by NASA of our bone healing experiments aboard the ISS, and our experiment was successfully launched February 19, 2017 on SpaceX CRS-10.

Bruno G, Di Trani N, Hood RL, Zabre E, Filgueira CS, Canavese G, Jain P, Smith Z, Demarchi D, Hosali S, Pimpinelli A, Ferrari M, Grattoni A. Unexpected behaviors in molecular transport through size-controlled nanochannels down to the ultra-nanoscale. Nat Commun. 2018;9(1):1682.

Ionic transport through nanofluidic systems is a problem of fundamental interest in transport physics and has broad relevance in desalination, fuel cells, batteries, filtration, and drug delivery. When the dimension of the fluidic system approaches the size of molecules in solution, fluid properties are not homogeneous and a departure in behavior is observed with respect to continuum-based theories. Here we present a systematic study of the transport of charged and neutral small molecules in an ideal nanofluidic platform with precise channels from the sub-microscale to the ultra-nanoscale (<5?nm). Surprisingly, we find that diffusive transport of nano-confined neutral molecules matches that of charged molecules, as though the former carry an effective charge. Further, approaching the ultra-nanoscale molecular diffusivities suddenly drop by up to an order of magnitude for all molecules, irrespective of their electric charge. New theoretical investigations will be required to shed light onto these intriguing results.

Spees JL, Lee RH, Gregory CA. Mechanisms of mesenchymal stem/stromal cell function. Stem Cell Res Ther. 2016;7(1):125.

The past decade has seen an explosion of research directed toward better understanding of the mechanisms of mesenchymal stem/stromal cell (MSC) function during rescue and repair of injured organs and tissues. In addition to delineating cell?cell signaling and molecular controls for MSC differentiation, the field has made particular progress in defining several other mechanisms through which administered MSCs can promote tissue rescue/repair. These include: 1) paracrine activity that involves secretion of proteins/peptides and hormones; 2) transfer of mitochondria by way of tunneling nanotubes or microvesicles; and 3) transfer of exosomes or microvesicles containing RNA and other molecules. Improved understanding of MSC function holds great promise for the application of cell therapy and also for the development of powerful cell-derived therapeutics for regenerative medicine. Focusing on these three mechanisms, we discuss MSC-mediated effects on immune cell responses, cell survival, and fibrosis and review recent progress with MSC-based or MSC-derived therapeutics.

Clough BH, McCarley MR, Krause U, Zeitouni S, Froese JJ, McNeill EP, Chaput CD, Sampson HW, Gregory CA. Bone regeneration with osteogenically enhanced mesenchymal stem cells and their extracellular matrix proteins. J Bone Miner Res. 2015;30(1):83-94.

Although bone has remarkable regenerative capacity, about 10% of long bone fractures and 25% to 40% of vertebral fusion procedures fail to heal. In such instances, a scaffold is employed to bridge the lesion and accommodate osteoprogenitors. Although synthetic bone scaffolds mimic some of the characteristics of bone matrix, their effectiveness can vary because of biological incompatibility. Herein, we demonstrate that a composite prepared with osteogenically enhanced mesenchymal stem cells (OEhMSCs) and their extracellular matrix (ECM) has an unprecedented capacity for the repair of critical‐sized defects of murine femora.Furthermore, OEhMSCs do not cause lymphocyte activation, and ECM/OEhMSC composites retain their in vivo efficacy after cryopreservation. Finally, we show that attachment to the ECM by OEhMSCs stimulates the production of osteogenic and angiogenic factors. These data demonstrate that composites of OEhMSCs and their ECM could be utilized in the place of autologous bone graft for complex orthopedic reconstructions.

Aunins TR, Erickson KE, Prasad N, Levy SE, Jones A, Shrestha S, Mastracchio R, Stodieck L, Klaus D, Zea L, Chatterjee A. Spaceflight modifies Escherichia coli gene expression in response to antibiotic exposure and reveals role of oxidative stress response. Front. Microbiol. 2018;9:310.

Bacteria grown in space experiments under microgravity conditions have been found to undergo unique physiological responses, ranging from modified cell morphology and growth dynamics to a putative increased tolerance to antibiotics. A common theory for this behavior is the loss of gravity-driven convection processes in the orbital environment, resulting in both reduction of extracellular nutrient availability and the accumulation of bacterial byproducts near the cell. To further characterize the responses, this study investigated the transcriptomic response of Escherichia coli to both microgravity and antibiotic concentration. E. coli was grown aboard International Space Station in the presence of increasing concentrations of the antibiotic gentamicin with identical ground controls conducted on Earth. Here we show that within 49 h of being cultured, E. coli adapted to grow at higher antibiotic concentrations in space compared to Earth, and demonstrated consistent changes in expression of 63 genes in response to an increase in drug concentration in both environments, including specific responses related to oxidative stress and starvation response. Additionally, we find 50 stress-response genes upregulated in response to the microgravity when compared directly to the equivalent concentration in the ground control. We conclude that the increased antibiotic tolerance in microgravity may be attributed not only to diminished transport processes, but also to a resultant antibiotic cross-resistance response conferred by an overlapping effect of stress response genes. Our data suggest that direct stresses of nutrient starvation and acid-shock conveyed by the microgravity environment can incidentally upregulate stress response pathways related to antibiotic stress and in doing so contribute to the increased antibiotic stress tolerance observed for bacteria in space experiments. These results provide insights into the ability of bacteria to adapt under extreme stress conditions and potential strategies to prevent antimicrobial-resistance in space and on Earth.

Malley KR, Koroleva O, Miller I, Sanishvili R, Jenkins CM, Gross RW, Korolev S. The structure of iPLA 2 β reveals dimeric active sites and suggests mechanisms of regulation and localization. Nat Commun. 2018;9(1):765.

Calcium-independent phospholipase A 2 β (iPLA 2 β) regulates important physiological processes including inflammation, calcium homeostasis and apoptosis. It is genetically linked to neurodegenerative disorders including Parkinson?s disease. Despite its known enzymatic activity, the mechanisms underlying iPLA 2 β-induced pathologic phenotypes remain poorly understood. Here, we present a crystal structure of iPLA 2 βthat significantly revises existing mechanistic models. The catalytic domains form a tight dimer. They are surrounded by ankyrin repeat domains that adopt an outwardly flared orientation, poised to interact with membrane proteins. The closely integrated active sites are positioned for cooperative activation and internal transacylation. The structure and additional solution studies suggest that both catalytic domains can be bound and allosterically inhibited by a single calmodulin. These features suggest mechanisms of iPLA 2 β cellular localization and activity regulation, providing a basis for inhibitor development. Furthermore, the structure provides a framework to investigate the role of neurodegenerative mutations and the function of iPLA 2 β in the brain.

Childress P, Brinker A, Gong CS, Harris J, Olivos DJ 3rd, Rytlewski JD, Scofield DC, Choi SY, Shirazi-Fard Y, McKinley TO, Chu TG, Conley CL, Chakraborty N, Hammamieh R, Kacena MA. Forces associated with launch into space do not impact bone fracture healing. Life Sci Space Res (Amst). 2018;16:52-62.

Segmental bone defects (SBDs) secondary to trauma invariably result in a prolonged recovery with an extended period of limited weight bearing on the affected limb. Soldiers sustaining blast injuries and civilians sustaining high energy trauma typify such a clinical scenario. These patients frequently sustain composite injuries with SBDs in concert with extensive soft tissue damage. For soft tissue injury resolution and skeletal reconstruction a patient may experience limited weight bearing for upwards of 6 months. Many small animal investigations have evaluated interventions for SBDs. While providing foundational information regarding the treatment of bone defects, these models do not simulate limited weight bearing conditions after injury. For example, mice ambulate immediately following anesthetic recovery, and in most cases are normally ambulating within 1–3 days post-surgery. Thus, investigations that combine disuse with bone healing may better test novel bone healing strategies. To remove weight bearing, we have designed a SBD rodent healing study in microgravity (µG) on the International Space Station (ISS) for the Rodent Research-4 (RR-4) Mission, which launched February 19, 2017 on SpaceX CRS-10 (Commercial Resupply Services). In preparation for this mission, we conducted an end-to-end mission simulation consisting of surgical infliction of SBD followed by launch simulation and hindlimb unloading (HLU) studies. In brief, a 2 mm defect was created in the femur of 10 week-old C57BL6/J male mice (n = 9–10/group). Three days after surgery, 6 groups of mice were treated as follows: 1) Vivarium Control (maintained continuously in standard cages); 2) Launch Negative Control (placed in the same spaceflight-like hardware as the Launch Positive Control group but were not subjected to launch simulation conditions); 3) Launch Positive Control (placed in spaceflight-like hardware and also subjected to vibration followed by centrifugation); 4) Launch Positive Experimental (identical to Launch Positive Control group, but placed in qualified spaceflight hardware); 5) Hindlimb Unloaded (HLU, were subjected to HLU immediately after launch simulation tests to simulate unloading in spaceflight); and 6) HLU Control (single housed in identical HLU cages but not suspended). Mice were euthanized 28 days after launch simulation and bone healing was examined via micro-Computed Tomography (µCT). These studies demonstrated that the mice post-surgery can tolerate launch conditions. Additionally, forces and vibrations associated with launch did not impact bone healing (p = .3). However, HLU resulted in a 52.5% reduction in total callus volume compared to HLU Controls (p = .0003). Taken together, these findings suggest that mice having a femoral SBD surgery tolerated the vibration and hypergravity associated with launch, and that launch simulation itself did not impact bone healing, but that the prolonged lack of weight bearing associated with HLU did impair bone healing. Based on these findings, we proceeded with testing the efficacy of FDA approved and novel SBD therapies using the unique spaceflight environment as a novel unloading model on SpaceX CRS-10.

Baio J, Martinez AF, Bailey L, Hasaniya N, Pecaut MJ, Kearns-Jonker M. Spaceflight Activates Protein Kinase C Alpha Signaling and Modifies the Developmental Stage of Human Neonatal Cardiovascular Progenitor Cells. Stem Cells Dev. 2018;27(12):805-818.

Spaceflight impacts cardiovascular function in astronauts; however, its impact on cardiac development and the stem cells that form the basis for cardiac repair is unknown. Accordingly, further research is needed to uncover the potential relevance of such changes to human health. Using simulated microgravity (SMG) generated by two-dimensional clinorotation and culture aboard the International Space Station (ISS), we assessed the effects of mechanical unloading on human neonatal cardiovascular progenitor cell (CPC) developmental properties and signaling. Following 6-7 days of SMG and 12 days of ISS culture, we analyzed changes in gene expression. Both environments induced the expression of genes that are typically associated with an earlier state of cardiovascular development. To understand the mechanism by which such changes occurred, we assessed the expression of mechanosensitive small RhoGTPases in SMG-cultured CPCs and observed decreased levels of RHOA and CDC42. Given the effect of these molecules on intracellular calcium levels, we evaluated changes in noncanonical Wnt/calcium signaling. After 6-7 days under SMG, CPCs exhibited elevated levels of WNT5A and PRKCA. Similarly, ISS-cultured CPCs exhibited elevated levels of calcium handling and signaling genes, which corresponded to protein kinase C alpha (PKCα), a calcium-dependent protein kinase, activation after 30 days. Akt was activated, whereas phosphorylated extracellular signal-regulated kinase levels were unchanged. To explore the effect of calcium induction in neonatal CPCs, we activated PKCα using hWnt5a treatment on Earth. Subsequently, early cardiovascular developmental marker levels were elevated. Transcripts induced by SMG and hWnt5a-treatment are expressed within the sinoatrial node, which may represent embryonic myocardium maintained in its primitive state. Calcium signaling is sensitive to mechanical unloading and directs CPC developmental properties. Further research both in space and on Earth may help refine the use of CPCs in stem cell-based therapies and highlight the molecular events of development.

Hatzistergos KE, Jiang Z, Valasaki K, Takeuchi LM, Balkan W, Atluri P, Saur D, Seidler B, Tsinoremas N, DiFede D, Hare JM. Simulated microgravity impairs cardiac autonomic neurogenesis from neural crest cells. Stem Cells Dev. 2018;27(12),819-830.

Microgravity-induced alterations in the autonomic nervous system (ANS) contribute to derangements in both the mechanical and electrophysiologic function of the cardiovascular system, leading to severe symptoms in humans following space travel. Because the ANS forms embryonically from neural crest progenitors (NCs), we hypothesized that microgravity can impair NC derived cardiac structures. Accordingly, we conducted in vitro simulated microgravity experiments employing NC genetic lineage-tracing in mice with cKitCreERT2/+, Isl1nLacZ and Wnt1-Cre reporter alleles. Inducible fate-mapping in adult mouse hearts and pluripotent stem cells (iPSCs) demonstrated reduced cKitCreERT2/+-mediated labeling of both NC-derived cardiomyocytes and autonomic neurons (p<0.0005 vs. controls). Whole-transcriptome analysis, suggested that this effect was associated with repressed cardiac NC- and upregulated mesoderm-related gene-expression profiles, coupled with abnormal BMP/TGF-? and Wnt/?-catenin signaling. To separate the manifestations of simulated microgravity on NC- vs. mesodermal-cardiac derivatives, we conducted Isl1nLacZ lineage analyses which indicated a ~3-fold expansion (p<0.05) in mesoderm-derived Isl-1+ pacemaker sinoatrial nodal cells; and a ~3-fold reduction (p<0.05) in cardiac NC-derived ANS cells, including sympathetic nerves and Isl-1+ cardiac ganglia. Finally, NC-specific fate-mapping with a Wnt1-Cre reporter iPSC model of murine NC development confirmed that simulated microgravity directly impacted the in vitro development of cardiac NC progenitors and their contribution to the sympathetic and parasympathetic innervation of the iPSC-derived myocardium. Together these findings reveal an important role for gravity in the development of NCs and their postnatal derivatives; and have important therapeutic implications for human space exploration, providing insights into cellular and molecular mechanisms of microgravity-induced cardiomyopathies/ channelopathies.

Boguraev A-S, Christensen HC, Bonneau AR, Pezza JA, Nichols NM, Giraldez, A J, Alvarez Saavedra E. Successful amplification of DNA aboard the International Space Station. NPJ Microgravity. 2017;3:26.

As the range and duration of human ventures into space increase, it becomes imperative that we understand the effects of the cosmic environment on astronaut health. Molecular technologies now widely used in research and medicine will need to become available in space to ensure appropriate care of astronauts. The polymerase chain reaction (PCR) is the gold standard for DNA analysis, yet its potential for use on-orbit remains under-explored. We describe DNA amplification aboard the International Space Station (ISS) through the use of a miniaturized miniPCR system. Target sequences in plasmid, zebrafish genomic DNA, and bisulfite-treated DNA were successfully amplified under a variety of conditions. Methylation-specific primers differentially amplified bisulfite-treated samples as would be expected under standard laboratory conditions. Our findings establish proof of concept for targeted detection of DNA sequences during spaceflight and lay a foundation for future uses ranging from environmental monitoring to on-orbit diagnostics.

Brisset J, Colwell J, Dove A, Maukonen D. NanoRocks: Design and performance of an experiment studying planet formation on the International Space Station. arXiv. 2017;1706.08625:7. 

In an effort to better understand the early stages of planet formation, we have developed a 1.5U payload that flew on the International Space Station (ISS) in the NanoRacks NanoLab facility between September 2014 and March 2016. This payload, named NanoRocks, ran a particle collision experiment under long-term microgravity conditions. The objectives of the experiment were (a) to observe collisions between mm-sized particles at relative velocities of <1~cm/s, and (b) to study the formation and disruption of particle clusters for different particle types and collision velocities. Four types of particles were used: mm-sized acrylic, glass, and copper beads, and 0.75 mm-sized JSC-1 lunar regolith simulant grains. The particles were placed in sample cells carved out of an aluminum tray. This tray was attached to one side of the payload casing with three springs. Every 60~s, the tray was agitated and the resulting collisions between the particles in the sample cells were recorded by the experiment camera. During the 18 months the payload stayed on ISS, we obtained 158 videos, thus recording a great number of collisions. The average particle velocities in the sample cells after each shaking event were around 1 cm/s. After shaking stopped, the inter-particle collisions damped the particle kinetic energy in less than 20~s, reducing the average particle velocity to below 1 mm/s, and eventually slowing them to below our detection threshold. As the particle velocity decreased, we observed the transition from bouncing to sticking collisions. We recorded the formation of particle clusters at the end of each experiment run. This paper describes the design and performance of the NanoRocks ISS payload.

Tauber S, Lauber BA, Paulsen K, Layer LE, Lehmann M, Hauschild S, Shepherd NR, Polzer J, Segerer J, Thiel CS, Ullrich O. Cytoskeletal stability and metabolic alterations in primary human macrophages in long-term microgravity. PLOS ONE. 2017;12(4):e0175599.

The immune system is one of the most affected systems of the human body during space flight. The cells of the immune system are exceptionally sensitive to microgravity. Thus, serious concerns arise, whether space flight associated weakening of the immune system ultimately precludes the expansion of human presence beyond the Earth's orbit. For human space flight, it is an urgent need to understand the cellular and molecular mechanisms by which altered gravity influences and changes the functions of immune cells. The CELLBOX-PRIME (= CellBox-Primary Human Macrophages in Microgravity Environment) experiment investigated for the first time microgravity-associated long-term alterations in primary human macrophages, one of the most important effector cells of the immune system. The experiment was conducted in the U.S. National Laboratory on board of the International Space Station ISS using the NanoRacks laboratory and Biorack type I standard CELLBOX EUE type IV containers. Upload and download were performed with the SpaceX CRS-3 and the Dragon spaceship on April 18th, 2014 / May 18th, 2014. Surprisingly, primary human macrophages exhibited neither quantitative nor structural changes of the actin and vimentin cytoskeleton after 11 days in microgravity when compared to 1g controls. Neither CD18 or CD14 surface expression were altered in microgravity, however ICAM-1 expression was reduced. The analysis of 74 metabolites in the cell culture supernatant by GC–TOF–MS, revealed eight metabolites with significantly different quantities when compared to 1g controls. In particular, the significant increase of free fucose in the cell culture supernatant was associated with a significant decrease of cell surface–bound fucose. The reduced ICAM-1 expression and the loss of cell surface–bound fucose may contribute to functional impairments, e.g. the activation of T cells, migration and activation of the innate immune response. We assume that the surprisingly small and non-significant cytoskeletal alterations represent a stable “steady state” after adaptive processes are initiated in the new microgravity environment. Due to the utmost importance of the human macrophage system for the elimination of pathogens and the clearance of apoptotic cells, its apparent robustness to a low gravity environment is crucial for human health and performance during long-term space missions.