Publications Resulting from ISS National Lab Sponsored Projects

Below, explore peer-reviewed journal articles related to ISS National Lab investigations. For a more extensive list of spaceflight-related publications (not limited to ISS National Lab sponsorship), see the International Space Station Research Results Citations on the NASA website.

Planarian Regeneration in Space: Persistent Anatomical, Behavioral, and Bacteriological Changes Induced by Space Travel

Morokuma J, Durant FR, Williams KB, Finkelstein JM, Blackiston DJ, Clements T, Reed DW, Roberts M, Jain M, Kimel K, Trauger SA, Wolfe BE, Levin M. Planarian regeneration in space: Persistent anatomical, behavioral, and bacteriological changes induced by space travel. Regeneration. 2017;4(2):85-102.

Regeneration is regulated not only by chemical signals but also by physical processes, such as bioelectric gradients. How these may change in the absence of the normal gravitational and geomagnetic fields is largely unknown. Planarian flatworms were moved to the International Space Station for 5 weeks, immediately after removing their heads and tails. A control group in spring water remained on Earth. No manipulation of the planaria occurred while they were in orbit, and space-exposed worms were returned to our laboratory for analysis. One animal out of 15 regenerated into a double-headed phenotype?normally an extremely rare event. Remarkably, ampu- tating this double-headed worm again, in plain water, resulted again in the double-headed phenotype. Moreover, even when tested 20 months after return to Earth, the space-exposed worms displayed significant quantitative differences in behavior and microbiome composition. These obser- vations may have implications for human and animal space travelers, but could also elucidate how microgravity and hypomagnetic environments could be used to trigger desired morphological, neurological, physiological, and bacteriomic changes for various regenerative and bioengineering applications.

Assembly of Hepatocyte Spheroids Using Magnetic 3D Cell Culture for CYP450 Inhibition/Induction

Desai PK, Tseng H, Souza GR. Assembly of hepatocyte spheroids using magnetic 3D cell culture for CYP450 inhibition/induction. J Mol Sci. 2017;18(5):1085.

There is a significant need for in vitro methods to study drug-induced liver injury that are rapid, reproducible, and scalable for existing high-throughput systems. However, traditional monolayer and suspension cultures of hepatocytes are difficult to handle and risk the loss of phenotype. Generally, three-dimensional (3D) cell culture platforms help recapitulate native liver tissue phenotype, but suffer from technical limitations for high-throughput screening, including scalability, speed, and handling. Here, we developed a novel assay for cytochrome P450 (CYP450) induction/inhibition using magnetic 3D cell culture that overcomes the limitations of other platforms by aggregating magnetized cells with magnetic forces. With this platform, spheroids can be rapidly assembled and easily handled, while replicating native liver function. We assembled spheroids of primary human hepatocytes in a 384-well format and maintained this culture over five days, including a 72 h induction period with known CYP450 inducers/inhibitors. CYP450 activity and viability in the spheroids were assessed and compared in parallel with monolayers. CYP450 activity was induced/inhibited in spheroids as expected, separate from any toxic response. Spheroids showed a significantly higher baseline level of CYP450 activity and induction over monolayers. Positive staining in spheroids for albumin and multidrug resistance-associated protein (MRP2) indicates the preservation of hepatocyte function within spheroids. The study presents a proof-of-concept for the use of magnetic 3D cell culture for the assembly and handling of novel hepatic tissue models.

The Active Modulation of Drug Release by an Ionic Field Effect Transistor for an Ultra-low Power Implantable Nanofluidic System

Bruno G, Canavese G, Liu X, Filguera CS, Sacco A, Demarchi A, Ferrari M, Grattoni A. The active modulation of drug release by an ionic field effect transitor for an ultra-low power implantable nanofluidic system. Nanoscale. 2016;8(44):18718-18725.

We report an electro-nanofluidic membrane for tunable, ultra-low power drug delivery employing an ionic field effect transistor. Therapeutic release from a drug reservoir was successfully modulated, with high energy efficiency, by actively adjusting the surface charge of slit-nanochannels 50, 110, and 160 nm in size, by the polarization of a buried gate electrode and the consequent variation of the electrical double layer in the nanochannel. We demonstrated control over the transport of ionic species, including two relevant hypertension drugs, atenolol and perindopril, that could benefit from such modulation. By leveraging concentration-driven diffusion, we achieve a 2 to 3 order of magnitude reduction in power consumption as compared to other electrokinetic phenomena. The application of a small gate potential (±5 V) in close proximity (150 nm) of 50 nm nanochannels generated a sufficiently strong electric field, which doubled or blocked the ionic flux depending on the polarity of the voltage applied. These compel- ling findings can lead to next generation, more reliable, smaller, and longer lasting drug delivery implants with ultra-low power consumption.

Magnetically Bioprinted Human Myometrial 3D Cell Rings as a Model for Uterine Contractility

Souza GR, Tseng H, Gage JA, Mani A, Desai P, Leonard F, Liao A, Longo M, Refuerzo JS, Godin B. Magnetically bioprinted human myometrial 3D cell rings as a model for uterine contractility. Int J Mol Sci. 2017;18(4):683.

Deregulation in uterine contractility can cause common pathological disorders of the female reproductive system, including preterm labor, infertility, inappropriate implantation, and irregular menstrual cycle. A better understanding of human myometrium contractility is essential to designing and testing interventions for these important clinical problems. Robust studies on the physiology of human uterine contractions require in vitro models, utilizing a human source. Importantly, uterine contractility is a three-dimensionally (3D)-coordinated phenomenon and should be studied in a 3D environment. Here, we propose and assess for the first time a 3D in vitro model for the evaluation of human uterine contractility. Magnetic 3D bioprinting is applied to pattern human myometrium cells into rings, which are then monitored for contractility over time and as a function of various clinically relevant agents. Commercially available and patient-derived myometrium cells were magnetically bioprinted into rings in 384-well formats for throughput uterine contractility analysis. The bioprinted uterine rings from various cell origins and patients show different patterns of contractility and respond differently to clinically relevant uterine contractility inhibitors, indomethacin and nifedipine. We believe that the novel system will serve as a useful tool to evaluate the physiology of human parturition while enabling high-throughput testing of multiple agents and conditions.

Direct Evidence That an Extended Hydrogen-bonding Network Influences Activation of Pyridoxal 5′-phosphate in Aspartate Aminotransferase

Dajnowicz S, Parks JM, Hu X, Gesler K, Kovalevsky AY, Mueser TC. Direct evidence that an extended hydrogen-bonding network influences activation of pyridoxal 5'-phosphate in aspartate aminotransferase. J Biol Chem. 2017;292(14):5970-5980.

Pyridoxal 5 -phosphate (PLP) is a fundamental, multifunc- tional enzyme cofactor used to catalyze a wide variety of chemical reactions involved in amino acid metabolism. PLP-de- pendent enzymes optimize specific chemical reactions by mod- ulating the electronic states of PLP through distinct active site environments. In aspartate aminotransferase (AAT), an extended hydrogen bond network is coupled to the pyridinyl nitrogen of the PLP, influencing the electrophilicity of the cofactor. This network, which involves residues Asp-222, His- 143, Thr-139, His-189, and structural waters, is located at the edge of PLP opposite the reactive Schiff base. We demonstrate that this hydrogen bond network directly influences the proto- nation state of the pyridine nitrogen of PLP, which affects the rates of catalysis. We analyzed perturbations caused by sin- gle- and double-mutant variants using steady-state kinetics, high resolution X-ray crystallography, and quantum chemi- cal calculations. Protonation of the pyridinyl nitrogen to form a pyridinium cation induces electronic delocalization in the PLP, which correlates with the enhancement in cata- lytic rate in AAT. Thus, PLP activation is controlled by the proximity of the pyridinyl nitrogen to the hydrogen bond microenvironment. Quantum chemical calculations indicate that Asp-222, which is directly coupled to the pyridinyl nitro- gen, increases the pKa of the pyridine nitrogen and stabilizes the pyridinium cation. His-143 and His-189 also increase the pKa of the pyridine nitrogen but, more significantly, influ- ence the position of the proton that resides between Asp-222 and the pyridinyl nitrogen. These findings indicate that the second shell residues directly enhance the rate of catalysis in AAT.

Neutron Structures of the Helicobacter pylori 5′-methylthioadenosine Nucleosidase Highlight Proton Sharing and Protonation States

Banco MT, Mishra V, Ostermann A, Schrader TE, Evans GB, Kovalevsky A, Ronning DR. Neutron structures of the Helicobacter pylori 5'-methylthioadenosine nucleosidase highlight proton sharing and protonation states. PNAS. 2016;113(48):13756-13761.

MTAN (5?-methylthioadenosine nucleosidase) catalyzes the hydrolysis of the N-ribosidic bond of a variety of adenosine-containing metabolites. The Helicobacter pylori MTAN (HpMTAN) hydrolyzes 6-amino-6-deoxyfutalosine in the second step of the alternative menaquinone biosynthetic pathway. Substrate binding of the adenine moiety is mediated almost exclusively by hydrogen bonds, and the proposed catalytic mechanism requires multiple proton-transfer events. Of particular interest is the protonation state of residue D198, which possesses a pKa above 8 and functions as a general acid to initiate the enzymatic reaction. In this study we present three corefined neutron/X-ray crystal structures of wild-type HpMTAN cocrystallized with S-adenosylhomocysteine (SAH), Formycin A (FMA), and (3R,4S)-4-(4-Chlorophenylthiomethyl)-1-[(9-deaza-adenin-9-yl)methyl]-3-hydroxypyrrolidine (p-ClPh-Thio-DADMe-ImmA) as well as one neutron/X-ray crystal structure of an inactive variant (HpMTAN-D198N) cocrystallized with SAH. These results support a mechanism of D198 pKa elevation through the unexpected sharing of a proton with atom N7 of the adenine moiety possessing unconventional hydrogen-bond geometry. Additionally, the neutron structures also highlight active site features that promote the stabilization of the transition state and slight variations in these interactions that result in 100-fold difference in binding affinities between the DADMe-ImmA and ImmA analogs.

Spaceflight and Simulated Microgravity Cause a Significant Reduction of Key Gene Expression in Early T-cell Activation

Martinez EM, Yoshida MC, Candelario TLT, Hughes-Fulford M. Spaceflight and simulated microgravity cause a significant reduction of key gene expression in early T-cell activation. Am J Physiol Regul Integr Comp Physiol. 2015;308(6):R480-R488.

Healthy immune function depends on precise regulation of lymphocyte activation. During the National Aeronautics and Space Administration (NASA) Apollo and Shuttle eras, multiple spaceflight studies showed depressed lymphocyte activity under microgravity (μg) conditions. Scientists on the ground use two models of simulated μg (sμg): 1) the rotating wall vessel (RWV) and 2) the random positioning machine (RPM), to study the effects of altered gravity on cell function before advancing research to the true μg when spaceflight opportunities become available on the International Space Station (ISS). The objective of this study is to compare the effects of true μg and sμg on the expression of key early T-cell activation genes in mouse splenocytes from spaceflight and ground animals. For the first time, we compared all three conditions of microgravity spaceflight, RPM, and RWV during immune gene activation of Il2, Il2rα, Ifnγ, and Tagap; moreover, we confirm two new early T-cell activation genes, Iigp1 and Slamf1. Gene expression for all samples was analyzed using quantitative real-time PCR (qRT-PCR). Our results demonstrate significantly increased gene expression in activated ground samples with suppression of mouse immune function in spaceflight, RPM, and RWV samples. These findings indicate that sμg models provide an excellent test bed for scientists to develop baseline studies and augment true μg in spaceflight experiments. Ultimately, sμg and spaceflight studies in lymphocytes may provide insight into novel regulatory pathways, benefiting both future astronauts and those here on earth suffering from immune disorders.

The Effects of Thermal Precondition on Oncogenic and Intraspinal Cord Growth Features of Human Glioma Cells

Zeng X, Han I, Abd-El-Barr M, Anderson JE, Chi JH, Zafonte RD, Teng YD. The effects of thermal precondition on oncogenic and intraspinal cord growth features of human glioma cells. Cell Transplant. 2016;25(12):2099-2109.

The adult rodent spinal cord presents an inhibitory environment for donor cell survival, impeding efficiency for xenograft-based modeling of gliomas. We postulated that mild thermal precondition may influence the fate of the implanted tumor cells. To test this hypothesis, high grade human astrocytoma G55 and U87 cells were cultured under 37°C and 38.5°C, to mimic regular experimental or core body temperature of rodents, respectively. In vitro, 38.5°C-conditioned cells, relative to 37°C, grew slightly faster. Comparing to U87, G55 demonstrated greater response to the temperature difference. Hyperthermal culture markedly increased production of HSP27 in most G55 but only promoted transient expression of cancer stem cell marker CD133 in a small cell subpopulation. We subsequently transplanted G55 cells following 37°C or 38.5°C culture into the C2 or T10 spinal cord of adult female immunodeficient rats (3 rats/each locus/per temperature; total: 12 rats). Systematical analyses revealed that 38.5°C-preconditioned G55 grew more malignantly at either C2 or T10 as determined by tumor size, outgrowth profile, resistance to bolus intratumor administration of 5-fluorouracil (0.1 micromole), and post-tumor survival (P < 0.05; n = 6/group). Therefore, thermal precondition of glioma cells may be an effective way to influence the in vitro and in vivo oncological contour of glioma cells. Future studies are needed for assessing potential oncogenic modifying effect of hyperthermia regimens on glioma cells.

Genetic and Environmental Models of Circadian Disruption Link SRC-2 Function to Hepatic Pathology

Fleet T, Stashi E, Zhu B, Rajapakshe K, Marcelo KL, Kettner NM, Gorman BK, Coarfa C, Fu L, O'Malley BW, York B. Genetic and environmental models of circadian disruption link SRC-2 function to hepatic pathology. Journal of Biological Rythms. 2016;31(5):443-460.

Circadian rhythmicity is a fundamental process that synchronizes behavioral cues with metabolic homeostasis. Disruption of daily cycles due to jet lag or shift work results in severe physiological consequences including advanced aging, metabolic syndrome, and even cancer. Our understanding of the molecular clock, which is regulated by intricate positive feedforward and negative feedback loops, has expanded to include an important metabolic transcriptional coregulator, Steroid Receptor Coactivator-2 (SRC-2), that regulates both the central clock of the suprachiasmatic nucleus (SCN) and peripheral clocks including the liver. We hypothesized that an environmental uncoupling of the light-dark phases, termed chronic circadian disruption (CCD), would lead to pathology similar to the genetic circadian disruption observed with loss of SRC-2 We found that CCD and ablation of SRC-2 in mice led to a common comorbidity of metabolic syndrome also found in humans with circadian disruption, non-alcoholic fatty liver disease (NAFLD). The combination of SRC-2(-/-) and CCD results in a more robust phenotype that correlates with human non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) gene signatures. Either CCD or SRC-2 ablation produces an advanced aging phenotype leading to increased mortality consistent with other circadian mutant mouse models. Collectively, our studies demonstrate that SRC-2 provides an essential link between the behavioral activities influenced by light cues and the metabolic homeostasis maintained by the liver.

Simulated Microgravity and 3D Culture Enhance Induction, Viability, Proliferation and Differentiation of Cardiac Progenitors from Human Pluripotent Stem Cells

Jha R, Wu Q, Singh M, Preininger MK, Han P, Ding G, Cho HC, Jo H, Maher KO, Wagner MB, Xu C. Simulated microgravity and 3D culture enhance induction, viability, proliferation and differentiation of cardiac progenitors from human pluripotent stem cells. Sci Rep. 2016;6(30956).

Efficient generation of cardiomyocytes from human pluripotent stem cells is critical for their regenerative applications. Microgravity and 3D culture can profoundly modulate cell proliferation and survival. Here, we engineered microscale progenitor cardiac spheres from human pluripotent stem cells and exposed the spheres to simulated microgravity using a random positioning machine for 3 days during their differentiation to cardiomyocytes. This process resulted in the production of highly enriched cardiomyocytes (99% purity) with high viability (90%) and expected functional properties, with a 1.5 to 4-fold higher yield of cardiomyocytes from each undifferentiated stem cell as compared with 3D-standard gravity culture. Increased induction, proliferation and viability of cardiac progenitors as well as up-regulation of genes associated with proliferation and survival at the early stage of differentiation were observed in the 3D culture under simulated microgravity. Therefore, a combination of 3D culture and simulated microgravity can be used to efficiently generate highly enriched cardiomyocytes.

A Human Pluripotent Stem Cell Model of Catecholaminergic Polymoprhic Ventricular Tachyardia Recapitulates Patient-Specific Drug Responses

Preininger MK, Jha R, Maxwell JT, Wu Q, Singh M, Wang B, Dalal A, Mceachin Z, Rossoll W, Hales CM, Fischbach PS, Wagner MB, Xu C. A human pluripotent stem cell model of catecholaminergic polymorphic ventricular tachyardia recapitulates patient-specific drug responses. Dis Model Mech. 2016;9(9):927-939.

β-blockers are unsuccessful in eliminating stress-induced ventricular arrhythmias in approximately 25% of patients with catecholaminergic polymorphic ventricular tachycardia (CPVT). Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from these patients have potential for investigating the phenomenon, but it remains unknown whether they can recapitulate patient-specific drug responses to β-blockers. This study assessed whether the inadequacy of β-blocker therapy in an individual can be observed in vitro using patient-derived CPVT iPSC-CMs. A CPVT patient harboring a novel mutation in the type 2 cardiac ryanodine receptor (RyR2) was identified whose persistent ventricular arrhythmias during β-blockade with nadolol were abolished during flecainide treatment. iPSC-CMs generated from this patient and two control individuals expressed comparable levels of excitation-contraction genes, but assessment of the sarcoplasmic reticulum Ca2+ leak and load relationship revealed intracellular Ca2+ homeostasis was altered in CPVT iPSC-CMs. β-adrenergic stimulation potentiated spontaneous Ca2+ waves and unduly frequent, large, and prolonged Ca2+ sparks in CPVT compared to control iPSC-CMs, validating the disease phenotype. Pursuant to the patient's in vivo responses, nadolol treatment during β-adrenergic stimulation achieved negligible reduction of Ca2+ wave frequency and failed to rescue Ca2+ spark defects in CPVT iPSC-CMs. In contrast, flecainide reduced both frequency and amplitude of Ca2+ waves and restored the frequency, width, and duration of Ca2+ sparks to baseline levels. By recapitulating a CPVT patient's improved response to flecainide compared to β-blocker therapy in vitro, these data provide new evidence that iPSC-CMs can capture basic components of patient-specific drug responses.

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

Shi J, Lee S, Uyeda M, Tanjaya J, Kim JK, Pan HC, Reese P, Stodieck L, Lin A, TingK, Kwak JH, Soo C. 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. Tissue Eng Part C Methods. 2016;22(5):451-463.

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.

SRC-2 Is an Essential Coactivator for Orchestrating Metabolism and Circadian Rhythm

Stashi E, Lanz R, Mao J, Michailidis G, Zhu B, Kettner N, Putluri N, Reineke E, Reineke L, Dasgupta S, Dean A, Stevenson C, Sivasubramanian N, Sreekumar A, DeMayo F, York B, Fu L, O'Malley B. SRC-2 is an essential coactivator for orchestrating metabolism and circadian rhythm. Cell Rep. 2014;6(4):633-645.

Synchrony of the mammalian circadian clock is achieved by complex transcriptional and translational feedback loops centered on the BMAL1: CLOCK heterodimer. Modulation of circadian feedback loops is essential for maintaining rhythmicity, yet the role of transcriptional coactivators in driving BMAL1:CLOCK transcriptional networks is largely unexplored. Here, we show diurnal hepatic steroid receptor coactivator 2 (SRC-2) recruitment to the genome that extensively overlaps with the BMAL1 cistrome during the light phase, targeting genes that enrich for circadian and metabolic processes. Notably, SRC-2 ablation impairs wheel-running behavior, alters circadian gene expression in several peripheral tissues, alters the rhythmicity of the hepatic metabolome, and deregulates the synchronization of cell-autonomous metabolites. We identify SRC-2 as a potent coregulator of BMAL1:CLOCK and find that SRC-2 targets itself with BMAL1:CLOCK in a feedforward loop. Collectively, our data suggest that SRC-2 is a transcriptional coactivator of the BMAL1:CLOCK oscillators and establish SRC-2 as a critical positive regulator of the mammalian circa-dian clock.

SRC-2 Orchestrates Polygenic Inputs for Fine-tuning Glucose Homeostasis

Fleet T, Bin Z, Lin F, Zhu B, Dasgupta S, Stashi E, Tackett B, Thevananther S, Rajapakshe K, Gonzales N, Dean A, Mao J, Timchenko N, Malovannaya A, Qin J, Coarfa C, DeMayo F, Dacso C, Foulds C, O'Malley B and York B. SRC-2 Orchestrates polygenic inputs for fine-tuning glucose homeostasis. PNAS. 2015;112(44):E6068-E6077.

Despite extensive efforts to understand the monogenic contributions to perturbed glucose homeostasis, the complexity of genetic events that fractionally contribute to the spectrum of this pathology remain poorly understood. Proper maintenance of glucose homeostasis is the central feature of a constellation of comorbidities that define the metabolic syndrome. The ability of the liver to balance carbohydrate uptake and release during the feeding-to-fasting transition is essential to the regulation of peripheral glucose availability. The liver coordinates the expression of gene programs that control glucose absorption, storage, and secretion. Herein, we demonstrate that Steroid Receptor Coactivator 2 (SRC-2) orchestrates a hierarchy of nutritionally responsive transcriptional complexes to precisely modulate plasma glucose availability. Using DNA pull-down technology coupled with mass spectrometry, we have identified SRC-2 as an indispensable integrator of transcriptional complexes that control the rate-limiting steps of hepatic glucose release and accretion. Collectively, these findings position SRC-2 as a major regulator of polygenic inputs to metabolic gene regulation and perhaps identify a previously unappreciated model that helps to explain the clinical spectrum of glucose dysregulation.

Pulse Transit Time Measured by Photoplethysmography Improves the Accuracy of Heart Rate as a Surrogate Measure of Cardiac Output, Stroke Volume and Oxygen Uptake in Response to Graded Exercise

Pollonini L, Padhye NS, Re R, Torricelli A, Simpson RJ, Dacso, CC. Pulse transit time measured by photoplethysmography improves the accuracy of heart rate as a surrogate measure of cardiac output, stroke volume and oxygen uptake in response to graded exercise. Physiol Meas. 2015;2015;36(5):911-924.

Heart rate (HR) is a valuable and widespread measure for physical training programs, although its description of conditioning is limited to the cardiac response to exercise. More comprehensive measures of exercise adaptation include cardiac output (dot Q), stroke volume (SV) and oxygen uptake (dot VO2), but these physiological parameters can be measured only with cumbersome equipment installed in clinical settings. In this work, we explore the ability of pulse transit time (PTT) to represent a valuable pairing with HR for indirectly estimating dot Q, SV and dot VO2 non-invasively. PTT was measured as the time interval between the peak of the electrocardiographic (ECG) R-wave and the onset of the photoplethysmography (PPG) waveform at the periphery (i.e. fingertip) with a portable sensor. Fifteen healthy young subjects underwent a graded incremental cycling protocol after which HR and PTT were correlated with dot Q, SV and dot VO2 using linear mixed models. The addition of PTT significantly improved the modeling of dot Q, SV and dot VO2 at the individual level ($R_1^2 = 0.419$ for SV, 0.548 for dot Q, and 0.771 for dot VO2) compared to predictive models based solely on HR ($R_1^2 = 0.379$ for SV, 0.503 for dot Q, and 0.745 for dot VO2). While challenges in sensitivity and artifact rejection exist, combining PTT with HR holds potential for development of novel wearable sensors that provide exercise assessment largely superior to HR monitors.

Leveraging Electrokinetics for the Active Control of Dendritic Fullerene-1 Release Across a Nanochannel Membrane

Bruno G, Geninatti T, Hood RL, Fine D, Scorrano G, Schmulen J, Hosali S, Ferrari M, Grattoni A. Leveraging electrokinetics for the active control of dendritic fullerene-1 release across a nanochannel membrane. Nanoscale. 2015;7(12):5240-5248.

General adoption of advanced treatment protocols such as chronotherapy will hinge on progress in drug delivery technologies that provide precise temporal control of therapeutic release. Such innovation is also crucial to future medicine approaches such as telemedicine. Here we present a nanofluidic membrane technology capable of achieving active and tunable control of molecular transport through nanofluidic channels. Control was achieved through application of an electric field between two platinum electrodes positioned on either surface of a 5.7 nm nanochannel membrane designed for zero-order drug delivery. Two electrode configurations were tested: laser-cut foils and electron beam deposited thin-films, configurations capable of operating at low voltage (≤1.5 V), and power (100 nW). Temporal, reproducible tuning and interruption of dendritic fullerene 1 (DF-1) transport was demonstrated over multi-day release experiments. Conductance tests showed limiting currents in the low applied potential range, implying ionic concentration polarization (ICP) at the interface between the membrane's micro- and nanochannels, even in concentrated solutions (≤1 M NaCl). The ability of this nanotechnology platform to facilitate controlled delivery of molecules and particles has broad applicability to next-generation therapeutics for numerous pathologies, including autoimmune diseases, circadian dysfunction, pain, and stress, among others.

Spaceflight Alters Expression of microRNA during T-cell Activation

Hughes-Fulford M, Chang T, Martinez E, Li C. Spaceflight alters expression of microRNA during t-cell activation. FASEB J. 2015;29(12):4893-4900.

Altered immune function has been demonstrated in astronauts during spaceflights dating back to Apollo and Skylab; this could be a major barrier to long-term space exploration. We tested the hypothesis that spaceflight causes changes in microRNA (miRNA) expression. Human leukocytes were stimulated with mitogens on board the International Space Station using an onboard normal gravity control. Bioinformatics showed that miR-21 was significantly up-regulated 2-fold during early T-cell activation in normal gravity, and gene expression was suppressed under microgravity. This was confirmed using quantitative real-time PCR (n = 4). This is the first report that spaceflight regulates miRNA expression. Global microarray analysis showed significant (P < 0.05) suppression of 85 genes under microgravity conditions compared to normal gravity samples. EGR3, FASLG, BTG2, SPRY2, and TAGAP are biologically confirmed targets and are co-up-regulated with miR-21. These genes share common promoter regions with pre-mir-21; as the miR-21 matures and accumulates, it most likely will inhibit translation of its target genes and limit the immune response. These data suggest that gravity regulates T-cell activation not only by transcription promotion but also by blocking translation via noncoding RNA mechanisms. Moreover, this study suggests that T-cell activation itself may induce a sequence of gene expressions that is self-limited by miR-21.—Hughes-Fulford, M., Chang, T. T., Martinez, E. M., Li, C.-F. Spaceflight alters expression of microRNA during T-cell activation.

Anti-PolyQ Antibodies Recognize a Short PolyQ Stretch in Both Normal and Mutant Huntingtin Exon 1

Owens G, New D, West A, Bjorkman P. Anti-polyQ antibodies recognize a short polyQ stretch in both normal and mutant Huntingtin exon 1. J Mol Biol. 2015;427(15):2507-2519.

Huntington's disease is caused by expansion of a polyglutamine (polyQ) repeat in the huntingtin protein. A structural basis for the apparent transition between normal and disease-causing expanded polyQ repeats of huntingtin is unknown. The "linear lattice" model proposed random-coil structures for both normal and expanded polyQ in the preaggregation state. Consistent with this model, the affinity and stoichiometry of the anti-polyQ antibody MW1 increased with the number of glutamines. An opposing "structural toxic threshold" model proposed a conformational change above the pathogenic polyQ threshold resulting in a specific toxic conformation for expanded polyQ. Support for this model was provided by the anti-polyQ antibody 3B5H10, which was reported to specifically recognize a distinct pathologic conformation of soluble expanded polyQ. To distinguish between these models, we directly compared binding of MW1 and 3B5H10 to normal and expanded polyQ repeats within huntingtin exon 1 fusion proteins. We found similar binding characteristics for both antibodies. First, both antibodies bound to normal, as well as expanded, polyQ in huntingtin exon 1 fusion proteins. Second, an expanded polyQ tract contained multiple epitopes for fragments antigen-binding (Fabs) of both antibodies, demonstrating that 3B5H10 does not recognize a single epitope specific to expanded polyQ. Finally, small-angle X-ray scattering and dynamic light scattering revealed similar binding modes for MW1 and 3B5H10 Fab-huntingtin exon 1 complexes. Together, these results support the linear lattice model for polyQ binding proteins, suggesting that the hypothesized pathologic conformation of soluble expanded polyQ is not a valid target for drug design.

The Effect of Spaceflight on the Gravity-sensing Auxin Gradient of Roots: GFP Reporter Gene Microscopy on Orbit

Ferl RJ, Paul A-L. The effect of spaceflight on the gravity-sensing auxin gradient of roots: GFP reporter gene microscopy on orbit. npj Microgravity. 2016;2(15023).

Our primary aim was to determine whether gravity has a direct role in establishing the auxin-mediated gravity-sensing system in primary roots. Major plant architectures have long been thought to be guided by gravity, including the directional growth of the primary root via auxin gradients that are then disturbed when roots deviate from the vertical as a gravity sensor. However, experiments on the International Space Station (ISS) now allow physical clarity with regard to any assumptions regarding the role of gravity in establishing fundamental root auxin distributions. We examined the spaceflight green fluorescent protein (GFP)-reporter gene expression in roots of transgenic lines of Arabidopsis thaliana: pDR5r::GFP, pTAA1::TAA1–GFP, pSCR::SCR–GFP to monitor auxin and pARR5::GFP to monitor cytokinin. Plants on the ISS were imaged live with the Light Microscopy Module (LMM), and compared with control plants imaged on the ground. Preserved spaceflight and ground control plants were examined post flight with confocal microscopy. Plants on orbit, growing in the absence of any physical reference to the terrestrial gravity vector, displayed typically “vertical” distribution of auxin in the primary root. This confirms that the establishment of the auxin-gradient system, the primary guide for gravity signaling in the root, is gravity independent. The cytokinin distribution in the root tip differs between spaceflight and the ground controls, suggesting spaceflight-induced features of root growth may be cytokinin related. The distribution of auxin in the gravity-sensing portion of the root is not dependent on gravity. Spaceflight appears benign to auxin and its role in the development of the primary root tip, whereas spaceflight may influence cytokinin-associated processes.