Publications Resulting from ISS National Lab Sponsored Projects

The attenuation of sedimentation and convection in microgravity can sometimes decrease irregularities formed during macromolecular crystal growth. Current terrestrial protein crystal growth (PCG) capabilities are very different than those used during the Shuttle era and that are currently on the International Space Station (ISS). The focus of this experiment was to demonstrate the use of a commercial off-the-shelf, high throughput, PCG method in microgravity. Using Protein BioSolutions’ microfluidic Plug Maker™/CrystalCard™ system, we tested the ability to grow crystals of the regulator of glucose metabolism and adipogenesis: peroxisome proliferator-activated receptor gamma (apo-hPPAR-γ LBD), as well as several PCG standards. Overall, we sent 25 CrystalCards™ to the ISS, containing ~10,000 individual microgravity PCG experiments in a 3U NanoRacks NanoLab (1U = 103 cm.). After 70 days on the ISS, our samples were returned with 16 of 25 (64%) microgravity cards having crystals, compared to 12 of 25 (48%) of the ground controls. Encouragingly, there were more apo-hPPAR-γ LBD crystals in the microgravity PCG cards than the 1g controls. These positive results hope to introduce the use of the PCG standard of low sample volume and large experimental density to the microgravity environment and provide new opportunities for macromolecular samples that may crystallize poorly in standard laboratories.

?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.

Heart diseases cause over 17.9 million total deaths globally, making them the leading source of mortality. The aim of this review is to describe the characteristic mechanical, chemical and cellular properties of human cardiac tissue and how these properties can be mimicked in 3D bioprinted tissues. Furthermore, the authors review how current healthy cardiac models are being 3D bioprinted using extrusion-, laser- and inkjet-based printers. The review then discusses the pathologies of cardiac diseases and how bioprinting could be used to fabricate models to study these diseases and potentially find new drug targets for such diseases. Finally, the challenges and future directions of cardiac disease modeling using 3D bioprinting techniques are explored.

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.

Long noncoding RNAs (lncRNAs) are crucial in many cellular processes, yet relatively few have been shown to regulate human cardiomyocyte differentiation. Here, we demonstrate an essential role of GATA6 antisense RNA 1 (GATA6‐AS1) in cardiomyocyte differentiation from human pluripotent stem cells (hPSCs). GATA6‐AS1 is adjacent to cardiac transcription factor GATA6. We found that GATA6‐AS1 was nuclear‐localized and transiently upregulated along with GATA6 during the early stage of cardiomyocyte differentiation. The knockdown of GATA6‐AS1 did not affect undifferentiated cell pluripotency but inhibited cardiomyocyte differentiation, as indicated by no or few beating cardiomyocytes and reduced expression of cardiomyocyte‐specific proteins. Upon cardiac induction, the knockdown of GATA6‐AS1 decreased GATA6 expression, altered Wnt‐signaling gene expression, and reduced mesoderm development. Further characterization of the intergenic region between genomic regions of GATA6‐AS1 and GATA6 indicated that the expression of GATA6‐AS1 and GATA6 were regulated by a bidirectional promoter within the intergenic region. Consistently, GATA6‐AS1 and GATA6 were co‐expressed in several human tissues including the heart, similar to the mirror expression pattern of GATA6‐AS1 and GATA6 during cardiomyocyte differentiation. Overall, these findings reveal a previously unrecognized and functional role of lncRNA GATA6‐AS1 in controlling human cardiomyocyte differentiation.

Polymer-derived silicon oxycarbide (SiOC) materials enable the formation of homogeneous microstructures and high temperature stable properties. However, the relationships between the processing parameters and microstructures/properties have not been clearly understood. In this study, a materials informatics approach was employed to the SiOC materials to analyze and estimate the relationships. Datasets were constructed from results of previously reported literature about SiOC. The correlation analysis provided processing parameter ranking regarding the corresponding influences on the properties and microstructures. Such an understanding can be utilized for desired material fabrication. Machine learning models with high accuracy were proposed using the ranked features obtained from the correlation analysis. In addition, important points on the data collection, correlation analysis, and machine learning as well as limitations of the current dataset were discussed. The proposed workflow for the SiOC materials can be extended to different types of polymer-derived ceramics by incorporating various features and targets involved in the processing variables, microstructures, and properties.

This paper describes a novel miniature microcontroller based curve tracing circuit, which was designed to monitor the environmental effects on Silicon Carbide Junction Field Effect Transistor (SiC JFET) device performance, while exposed to the low earth orbit environment onboard the International Space Station (ISS) as a resident experiment on the 7th Materials on the International Space Station Experiment (MISSE7). Specifically, the microcontroller circuit was designed to operate autonomously and was flown on the external structure of the ISS for over a year. This curve tracing circuit is capable of measuring current vs. voltage (I-V) characteristics of transistors and diodes. The circuit is current limited for low current devices and is specifically designed to test high temperature, high drain-to-source resistance SiC JFETs. The results of each I-V data set are transmitted serially to an external telemetered communication interface. This paper discusses the circuit architecture, its design, and presents example results.

Bacteria behave differently in space, as indicated by reports of reduced lag phase, higher final cell counts, enhanced biofilm formation, increased virulence, and reduced susceptibility to antibiotics. These phenomena are theorized, at least in part, to result from reduced mass transport in the local extracellular environment, where movement of molecules consumed and excreted by the cell is limited to diffusion in the absence of gravity-dependent convection. However, to date neither empirical nor computational approaches have been able to provide sufficient evidence to confirm this explanation. Molecular genetic analysis findings, conducted as part of a recent spaceflight investigation, support the proposed model. This investigation indicated an overexpression of genes associated with starvation, the search for alternative energy sources, increased metabolism, enhanced acetate production, and other systematic responses to acidity all of which can be associated with reduced extracellular mass transport.

Understanding and quantifying the natural processes that occur along coasts are critical components of managing environmental resources and planning and executing coastal operations, from humanitarian relief to military actions. However, the coastal ocean is complicated, with dissolved and suspended matter that hinders water transparency, phytoplankton blooms that can be toxic, and bathymetry and bottom types that vary over spatial scales of tens of meters, all of which affect processes in an area that spans millions of square kilometers. A hyperspectral imager collects the spectrum of the light received from each pixel in an image. For environmental characterization the wavelength range typically spans the visible and shortwave infrared wavelengths, and the spectrum is collected in contiguous spectral intervals 1–10 nanometers wide. This spectral information is exploited to provide significantly more information about vegetation, minerals, and other components in the scene than can be retrieved from panchromatic or even multispectral imagery, which rely primarily on the shape of the object for detection [Goetz et al., 1985]. Such technology can also work over shallow seas. Over the past 2 decades, experiments with hyperspectral imagers on airborne platforms have demonstrated the ability to characterize the coastal environment [Davis et al., 2002, Davis et al. 2006] and produce maps of coastal bathymetry, in‐water constituents, and bottom type.

An ongoing challenge in biomedical research is the search for simple, yet robust assays using 3D cell cultures for toxicity screening. This study addresses that challenge with a novel spheroid assay, wherein spheroids, formed by magnetic 3D bioprinting, contract immediately as cells rearrange and compact the spheroid in relation to viability and cytoskeletal organization. Thus, spheroid size can be used as a simple metric for toxicity. The goal of this study was to validate spheroid contraction as a cytotoxic endpoint using 3T3 fibroblasts in response to 5 toxic compounds (all-trans retinoic acid, dexamethasone, doxorubicin, 5′-fluorouracil, forskolin), sodium dodecyl sulfate (+control), and penicillin-G (−control). Real-time imaging was performed with a mobile device to increase throughput and efficiency. All compounds but penicillin-G significantly slowed contraction in a dose-dependent manner (Z’ = 0.88). Cells in 3D were more resistant to toxicity than cells in 2D, whose toxicity was measured by the MTT assay. Fluorescent staining and gene expression profiling of spheroids confirmed these findings. The results of this study validate spheroid contraction within this assay as an easy, biologically relevant endpoint for high-throughput compound screening in representative 3D environments.

The Hyperspectral Imager for the Coastal Ocean (HICO) instrument currently on board the International Space Station is a new sensor designed specifically for the studies of turbid coastal waters and large inland lakes and rivers. It covers the wavelength range between 0.4 and 0.9 μm with a spectral resolution of 5.7 nm and a spatial resolution of approximately 90 m. The HICO sensor is not equipped with a second-order blocking filter in front of the focal plane array. As a result, the second-order light from the shorter visible spectral region falls onto the detectors covering the near-IR spectral region above 0.8 μm. In order to have accurate radiometric calibration of the near-IR channels, the second-order light contribution needs to be removed. The water-leaving radiances of these near-IR channels over clear ocean waters are close to zero because of strong liquid water absorption above 0.8 μm. Through analysis of HICO imaging data containing features of shallow underwater objects, such as coral reefs, we have developed an empirical technique to correct for the second-order light effects in near-IR channels. HICO data acquired over Midway Island in the Pacific Ocean and the Bahamas Banks in the Atlantic Ocean are used to demonstrate the effectiveness of the new technique.

A vascularized human proximal tubule model in a dual-channel microphysiological system (VPT-MPS) was developed, representing an advance over previous, single-cell-type kidney microphysiological systems. Human proximal tubule epithelial cells (PTECs) and human umbilical vein endothelial cells (HUVECs) were cocultured in side-by-side channels. Over 24 h of coculturing, PTECs maintained polarized expression of Na+/K+ ATPase, tight junctions (ZO-1), and OAT1. HUVECs showed the absence of ZO-1 but expressed endothelial cell marker (CD-31). In time-lapse imaging studies, fluorescein isothiocyanate (FITC)-dextran passed freely from the HUVEC vessel into the supporting extracellular matrix, confirming the leakiness of the endothelium (at 80 min, matrix/intravessel fluorescence ratio = 0.2). Dextran-associated fluorescence accumulated in the matrix adjacent to the basolateral aspect of the PTEC tubule with minimal passage of the compound into the tubule lumen observed (at 80 min, tubule lumen/matrix fluorescence ratio = 0.01). This demonstrates that the proximal tubule compartment is the rate-limiting step in the secretion of compounds in VPT-MPS. In kinetic studies with radiolabeled markers, p-aminohippuric acid (PAH) exhibited greater output into the tubule lumen than did paracellular markers mannitol and FITC-dextran (tubule outflow/vessel outflow concentration ratio of 7.7% vs 0.5 and 0.4%, respectively). A trend toward reduced PAH secretion by 45% was observed upon coadministration of probenecid. This signifies functional expression of renal transporters in PTECs that normally mediate the renal secretion of PAH. The VPT-MPS holds the promise of providing an in vitro platform for evaluating the renal secretion of new drug candidates and investigating the dysregulation of tubular drug secretion in chronic kidney disease.

Manatakis DV, VanDevender A, Manolakos ES. An information-theoretic approach for measuring the distance of organ tissue samples using their transcriptomic signatures [published online ahead of print July 19, 2020]. Bioinformatics. doi: 10.1093/bioinformatics/btaa654

Microgravity facilitates the opportunistic infections by augmenting the pathogenic virulence and suppressing the host resistance. Hence the extraterrestrial infections may activate potentially novel bionetworks different from the terrestrial equivalent, which could only be probed by investigating the host-pathogen relationship with a minimum of terrestrial bias.

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.

A precision measurement by AMS of the antiproton flux and the antiproton-to-proton flux ratio in primary cosmic rays in the absolute rigidity range from 1 to 450 GV is presented based on 3.49×105 antiproton events and 2.42×109 proton events. The fluxes and flux ratios of charged elementary particles in cosmic rays are also presented. In the absolute rigidity range ∼60 to ∼500  GV, the antiproton ¯p, proton p, and positron e+ fluxes are found to have nearly identical rigidity dependence and the electron e− flux exhibits a different rigidity dependence. Below 60 GV, the (¯p/p), (¯p/e+), and (p/e+) flux ratios each reaches a maximum. From ∼60 to ∼500  GV, the (¯p/p), (¯p/e+), and (p/e+) flux ratios show no rigidity dependence. These are new observations of the properties of elementary particles in the cosmos.

As a demonstrator for technologies for the next generation of ocean color sensors, the Hyperspectral Imager for the Coastal Ocean (HICO) provides enhanced spatial and spectral resolution that is required to understand optically complex aquatic environments. In this study we apply HICO, along with satellite remote sensing and in situ observations, to studies of phytoplankton ecology in a dynamic coastal upwelling environment—Monterey Bay, CA, USA. From a spring 2011 study, we examine HICO-detected spatial patterns in phytoplankton optical properties along an environmental gradient defined by upwelling flow patterns and along a temporal gradient of upwelling intensification. From a fall 2011 study, we use HICO’s enhanced spatial and spectral resolution to distinguish a small-scale “red tide” bloom, and we examine bloom expansion and its supporting processes using other remote sensing and in situ data. From a spectacular HICO image of the Monterey Bay region acquired during fall of 2012, we present a suite of algorithm results for characterization of phytoplankton, and we examine the strengths, limitations, and distinctions of each algorithm in the context of the enhanced spatial and spectral resolution.

There is growing appreciation that architectural components of the nucleus regulate gene accessibility by altering chromatin organization. While nuclear membrane connector proteins link the mechanosensitive actin cytoskeleton to the nucleoskeleton, actin’s contribution to the inner architecture of the nucleus remains enigmatic. Control of actin transport into the nucleus, plus the presence of proteins that control actin structure (the actin tool-box) within the nucleus, suggests that nuclear actin may support biomechanical regulation of gene expression. Cellular actin structure is mechanoresponsive: actin cables generated through forces experienced at the plasma membrane transmit force into the nucleus. We posit that dynamic actin remodeling in response to such biomechanical cues provides a novel level of structural control over the epigenetic landscape. We here propose to bring awareness to the fact that mechanical forces can promote actin transfer into the nucleus and control structural arrangements as illustrated in mesenchymal stem cells, thereby modulating lineage commitment.

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.

Several ocean color earth observation satellite sensors are presently collecting daily imagery, including the Hyperspectral Imager for the Coastal Ocean (HICO). HICO has been operating aboard the International Space Station since its installation on September 24, 2009. It provides high spatial resolution hyperspectral imagery optimized for the coastal ocean. Atmospheric correction, however, still remains a challenge for this sensor, particularly in optically complex coastal waters. In this paper, we assess the application of the cloud-shadow atmospheric correction approach on HICO data and validate the results with the in situ data. We also use multiple sets of cloud, shadow, and sunlit pixels to correct a single image multiple times and intercompare the results to assess variability in the retrieved reflectance spectra. Retrieved chlorophyll values from this intercomparison are similar and also agree well with the in situ chlorophyll measurements.