Boise State University NASA Microgravity University Updated: Gravitational Modulation of
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Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 1 of 41 NASA Reduced Gravity Education Flight Program Microgravity University 2011 Final Report Gravitational Modulation of Calcium Signaling in Bone Boise State University Department of Biological Sciences College of Engineering 1910 University Dr. Boise, ID 83725-2100 Student Lead Investigator: Jake Forsberg iorbit.earth@gmail.com (208) 571-2011 sondramiller1@boisestate.edu (208) 426-2894 Dr. Brian Crucian Kami Faust Mayra Nelman Heather Quiriarte Dr. Clarence Sams JSC Immunology Laboratory Benjamin Davis Stephanie Frahs Ron Pierce Dawn Mikelonis David Connolly BSU Principle Investigator: Dr. Sondra Miller NASA Associate Investigators: Undergraduate Investigators: Professional Advisors: Dr. Robert Hay Dr. Julia Oxford Dr. Barbara Morgan Alex Miller Dr. Tom Glass Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 2 of 41 Table of Contents REVISION HISTORY................................................................................................................................ 4 LIST OF ACRONYMS AND ABBREVIATIONS .................................................................................. 5 Acronyms .............................................................................................................................................. 5 Abbreviations........................................................................................................................................ 5 INTRODUCTION ....................................................................................................................................... 6 ABSTRACT ................................................................................................................................................. 6 STATEMENT OF RESEARCH PROBLEM........................................................................................... 7 BACKGROUND INFORMATION........................................................................................................... 7 METHOD..................................................................................................................................................... 8 EXPERIMENTAL OBJECTIVES .................................................................................................................... 8 HYPOTHESIS .............................................................................................................................................. 8 EXPERIMENTAL APPROACH............................................................................................................... 9 PREPARATIVE RESEARCH........................................................................................................................ 10 Conceptual Development and Literature Review............................................................................... 10 Product Review and Determination of Need for Customized Solution .............................................. 10 Instrument Research and Development.............................................................................................. 10 Synchronous Dual-Detection Fluorometer Prototype........................................................................ 11 EXPERIMENTAL SYSTEMS ....................................................................................................................... 13 Cell Samples ....................................................................................................................................... 13 Ratiometric Fluorometry .................................................................................................................... 14 Environmental Monitoring ................................................................................................................. 15 Optics.................................................................................................................................................. 15 Electronics .......................................................................................................................................... 17 Software .............................................................................................................................................. 18 Flow Cytometry .................................................................................................................................. 21 Cell Fixation ....................................................................................................................................... 21 RESULTS................................................................................................................................................... 22 EXPERIMENTAL SAMPLES ....................................................................................................................... 23 CONTROLS ............................................................................................................................................... 24 PORTABLE FLOW CYTOMETRY ............................................................................................................... 31 FIXATION ................................................................................................................................................. 31 DISCUSSION ............................................................................................................................................ 32 TIME ........................................................................................................................................................ 32 EXPERTISE ............................................................................................................................................... 32 FAILURES ................................................................................................................................................ 33 Non-Ideal Temperature Measurement ............................................................................................... 33 Inflexible Fluorometer Gain............................................................................................................... 33 Cell Culture Optimization .................................................................................................................. 33 Electronics Design.............................................................................................................................. 33 SUCCESSES .............................................................................................................................................. 34 Professional Development and Interaction ........................................................................................ 34 Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 3 of 41 Encouraging Results and Follow-On Promise................................................................................... 35 CONCLUSION.......................................................................................................................................... 35 LEARNING AND RECOMMENDATIONS ..................................................................................................... 35 Science Results ................................................................................................................................... 35 Time Management .............................................................................................................................. 35 Software .............................................................................................................................................. 35 Temperature Sensing .......................................................................................................................... 35 Variable Fluorometer Gain ................................................................................................................ 35 Increasing Sample Number Capability and Reducing Variability ..................................................... 36 RESEARCH CONTRIBUTIONS TO NASA .................................................................................................. 36 OUTREACH.............................................................................................................................................. 36 IDAHO SCIENCE AND AEROSPACE SCHOLARS EVENT ............................................................................. 36 SCIENCE CAFÉ ......................................................................................................................................... 37 BOISE STATE UNIVERSITY 8TH ANNUAL UNDERGRADUATE RESEARCH AND SCHOLARSHIP CONFERENCE........................................................................................................................................... 37 IDAHO ACADEMY OF SCIENCE 2011 SYMPOSIUM .................................................................................. 37 SUMMER RESEARCH COMMUNITY EVENING SEMINAR .......................................................................... 37 IGNITE BOISE ........................................................................................................................................... 37 DR. NANDIKOLLA ................................................................................................................................... 38 SPACE DAY’S EVENT ............................................................................................................................... 38 TEAM BLOG............................................................................................................................................. 38 “D4K” ..................................................................................................................................................... 38 “NASA DIGITAL LEARNING NETWORK”................................................................................................ 38 NASA FIT EXPLORER CHALLENGE “LIVING BONES, STRONG BONES” ................................................. 39 PRESS RELEASES................................................................................................................................... 39 REFERENCES .......................................................................................................................................... 40 ACKNOWLEDGEMENTS...................................................................................................................... 41 Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Revision History 1.0 Initial Submission to NASA RGO (8-2-2011) Updated: 2 August 2011 Page: 4 of 41 Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 List of Acronyms and Abbreviations Acronyms AC ASCR BSU D4K DC DCI DLN EGTA HEPES Alternating Current Astronaut Strength Conditioning and Rehabilitation Boise State University Dialogue For Kids Direct Current Discovery Center of Idaho Digital Learning Network Ethylene Glycol Tetraacetic Acid IAS 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Idaho Academy of Science JSC LED NASA NCBI RGO PID STEM USB WBSD Johnson Space Center Light Emitting Diode National Aeronautics and Space Administration National Center for Biotechnology Information Reduced Gravity Office Proportional Integral Derivative (control) Science Technology Engineering and Math Universal Serial Bus Whole Blood Staining Device Abbreviations DAQ Data Acquisition g Standard Earth gravitational acceleration in. OR “ Inch ft. OR ‘ Foot Hz Hertz mg milligram mL milliliter mM milliMolar nm Nanometer ug microgram uM microMolar UV Ultraviolet Updated: 2 August 2011 Page: 5 of 41 Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 6 of 41 Introduction The following is a report on a project prepared by the Boise State University Microgravity Team as part of the NASA Reduced Gravity Office (RGO) Student Education Flight Program’s 2011 Microgravity University campaign. The students and advisors on the project represent diverse backgrounds ranging across Biological Sciences, Chemistry, Computer Science, and Engineering. The interdisciplinary nature of this investigation provided all participants with valuable experience working with Biomedical Research and Molecular and Cell Biology, as well as Electrical, Mechanical, Optical, and Software Engineering. The team began with a basic scientific question relevant to astronaut safety and space biology. In the process of evaluating experimental approaches, a need was identified to develop custom instrumentation for the investigation. The June 2011 flights and resulting data presented herein are projected to serve as a basis to guide further development of the experimental system and protocols by future members of the Boise State Microgravity Team. Abstract In vitro investigations of the mechanobiology of bone are often limited in their ability to provide real-time data on immediate biochemical responses to changes in the gravitational environment. Further, current methods for bench-top investigations of cellular responses to changes in the gravitational environment cannot produce opposing departures from homeostatic gravity. That is, one can transition cells conditioned to a 1G environment to model microgravity or hyper gravity using bioreactors, but there is no available technology that can explore both extremes in a single experiment. The result of these limitations is a dearth of studies that evaluate how bone integrates information on mechanical environments that are highly complex and likely provide contrasting inputs over short periods of time. An example of such an environment would be the body in motion. And while over time, mechanical stimulus has been shown to elicit committed responses in the form of changes in gene expression, it remains unclear how such responses are regulated under dynamic conditions. Parabolic flight is a testing environment that oscillates between periods of hyper and microgravity, and is thus ideal for identification of early points of regulation. Calcium signaling is known to act up stream within pathways that affect committed responses to mechanical stimulation. Further, ion flow does not require polymerization and therefore is positioned as an energetically conservative mode of rapidly integrating contrasting stimuli. To evaluate this possibility, a custom fluorometer system capable of synchronously monitoring the ratiometric calcium indicator Indo-1 in real-time was flown on the Zero-G Corporation’s G-Force One aircraft, under contract by the RGO. Calcium flux as it related to changes in the gravitational environment was monitored in MLO-Y4 osteocytes, a line representative of the putative primary mechanosensory cells in bone. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 7 of 41 Statement of Research Problem The primary purpose of this study is to understand how calcium ion flux signaling in bone cells relates to the mechanical stimulation of cyclical application of hyper- and microgravity environments. The understanding of calcium as a signaling factor may help develop therapeutic solutions to undesirable bone remodeling trends, which itself may result from a variety of environmental or pathological conditions. Background Information Bone remodeling refers to the continuous turnover of mineralized matrix that maintains tissue integrity. It is accomplished by sensor and effector cell populations that use mechanical information to direct deposition and resorption of matrix material.1,2 This process can generally be described as the translation of mechanical information acquired from the local environment (mechanosensation) into biochemical information that defines cellular behavior (mechanotransduction).3,4 Under most circumstances, the informed nature of these activities leads to mineral distributions optimized to the biomechanical requirements placed on the bone.3,4 However, some pathological conditions have been associated with deregulated remodeling activities; most notably, significant decreases in mineral density are observed in patients suffering from osteoporosis and in astronauts returning from chronic exposure to microgravity.5 In order to design effective therapies for diseases of pathological remodeling, it is important to develop a clear biomolecular understanding of how cells integrate dynamic and complex sets of mechanical stimuli. A simplified paradigm of mechanically-regulated remodeling refers to a ‘mechanostat’ model wherein loading and unloading work in simple opposition to promote deposition and resorption, respectively.3 However, data exists that supports a more complex scenario in which the application of identical stimuli can produce varied responses over time, suggesting at least some capacity for attenuation, sensitization, or saturation of mechanotransducing pathways. In reality, the local mechanical environment experienced by bone cells changes rapidly even during simple actions such as walking.6 One might imagine that corresponding mechanostat-like adjustments made to each specific stimulus could be energetically very costly, particularly in light of studies demonstrating that sustained mechanical perturbations stimulate protein and nucleic acid synthesis.2,7 In the nervous system, stimuli are integrated through the summation of inhibitory and excitatory ion flow that ultimately determines whether or not a signal is passed on. Similarly, ion flow, in particular calcium flux, may provide a level of integration for contrasting stimuli that is rapid and relatively energetically inexpensive. Changes in calcium concentration can be enacted on the order of microseconds, yet due to calcium’s pleiotropic role in a wide array of signaling pathways, calcium flux can direct long term responses such as cellular proliferation and differentiation.2,7 To investigate this possibility, calcium flux must be monitored under the influence of changing mechanical environments. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 8 of 41 While conventional laboratory based techniques can model either hyper- or microgravity, no such technique can seamlessly expose cells to transitions between opposing departures from the homeostatic gravitational environment. Parabolic flight is such an environment, thereby providing ideal conditions for direct evaluation of how closely the behavior of known mediators of mechonsensitive remodeling, such as calcium ions, reflect the mechanostat model. Method This experiment aimed to acquire baseline data on the effects of the oscillating gravitational accelerations produced by parabolic flight on intracellular free calcium concentrations in bone cell cultures. These assays where designed to indicate the extent to which calcium flux events correlate with cyclical exposure to alternating gravitational conditions (i.e., hyper- and microgravity) and the extent to which these events remain consistent over time. Several specific objectives were defined for this experiment in order to fulfill this purpose. Experimental Objectives • • • • • • • Expose osteocyte cultures to gravitational oscillations produced in parabolic flight. Monitor and record changes in cytosolic calcium concentrations in real-time as indicated by calcium responsive fluorescent dyes via portable ratiometric fluorometry and flow cytometry systems. Collect environmental data at regular high-resolution time intervals to associate with fluorescence data via 3-axis accelerometer. Establish quantitative correlations between fluorescence data and in-flight environmental conditions. Fixate cellular samples at regular time intervals for post-flight analysis of calciumdependent activation of cell signaling intermediates via immunofluorescent flow cytometry and microscopy assays. Perform in-fight tests in steady 1G environment both before take off and immediately after the flight to obtain experimental control data. Construct potential qualitative relationships between gravity-induced calcium mobilization and mechanotransduction pathways to direct future testing. Hypothesis Since calcium is an established mediator of the coupling of cellular sensation of mechanical stimuli to cellular biochemical responses, it was anticipated that calcium mobilization would be observed in MLO-Y4 osteocyte cultures exposed to environmental conditions oscillating between hyper- and microgravity. If changes in calcium flux that correspond consistently with changes in environmental conditions over the duration of the experiment, it would support the notion that, at the level of calcium flux, mechanosensitive responses are directed in a straightforward manner by opposing stimuli. In contrast, if calcium responses showed variance over the course of cycles through gravitational oscillation, it would suggest a more complex role for calcium-meditated mechanotransduction. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 9 of 41 Experimental Approach The experimental approach consists of three primary actions: environmental data collection, realtime calcium monitoring, and cellular fixation. Real-time data was collected in-flight for gravitational acceleration via a 3-axis accelerometer, and for calcium flux through ratiometric fluorometry and flow cytometry. Simultaneously, a chronological set of fixated cellular samples was generated for post-flight assessment of calcium sensitive signaling activation by flow cytometry and microscopy. All these data sets were correlated temporally. Figure 1 illustrates the high-level experiment structure. Figure 1: Experiment breakdown structure diagram. The comparison of primary interest is between the real-time fluorescence and gravitational data, as this provides the greatest temporal resolution with respect to the central scientific question of how parabolic flight affects calcium flux over repeated gravitational oscillations. The team constructed an application-specific instrument to acquire this data set due to limitations identified in commercially-available fluorescence detection systems. The novelty of the instrument and the testing environment increased the chance that results from the first set of flights was not ideal. With this in mind, the cytometry and fixation experiments were performed as parallel modes to assess whether or not gravitationally-sensitive calcium flux occurred, and if the fluorometry system successfully identified any changes. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 10 of 41 Preparative Research A large amount of time was spent conducting preparative research preceding the selection of a final experimental system design and fabrication. The research focus and concurrent designs changed as efforts progressed throughout this time, and a brief overview of activities leading up to the concluded design is presented in the following sections. Conceptual Development and Literature Review Over the course of the project development, extensive literature review was performed. Identification of a relevant experimental question was accomplished through study of peerreviewed mechanobiology manuscripts, curated by organizations such as the National Center for Biotechnology Information (NCBI) in public databases such as PubMed. Review of biomedical literature also served to provide considerable information on methodological approaches to investigating the research question. Methods for monitoring calcium flux and cell culture were identified in this fashion. The MLO-Y4 cell line was also identified through literature review, as was a source to obtain the cells (Bonewald Lab). Product Review and Determination of Need for Customized Solution In addition to peer-reviewed publications, significant time was spent researching product information on supplies that could be integrated into the experimental system. The original intent of the team was to use off-the-shelf instrumentation, in particular, a microplate reader to obtain fluorescent data on calcium flux in cell cultures. The team decided that the capability to simultaneously monitor both emission wavelengths of the Indo-1 dye, for multiple samples at the same time, was necessary to adequately address the scientific goals of the investigation. Examination of product manuals and conversations with sales representatives regarding the functionality of such systems made it clear that a commercial instrument could not provide this level of resolution. Instrument Research and Development The need to design, construct and implement a custom fluorometry system required extensive commitment to research and development. Review of literature pertaining to ‘lab on a chip’ portable fluorometric technology for analysis of biological samples was performed. From this research it was learned that sensitive, portable, and relatively in expensive fluorometric instruments could be constructed with Light Emitting Diode (LED) excitation and photo diode based detection systems. The team then identified some suppliers of relevant products, and began building a prototype system to investigate the feasibility of this kind of system for the experimental apparatus. Figure 2 shows an image of the first proof of concept LED-excitation, photodiode detection system developed as part of design validation. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 11 of 41 Figure 2: Op-Amp and photodiode detector design validation prototype. Synchronous Dual-Detection Fluorometer Prototype A prototype dual emission, synchronous detection fluorometer module was quickly developed using a drilled-out wooden block to arrange excitation and emission filters around mock samples of free calcium in solution with Indo-1 dye. A high-gain photodiode detector setup and an ultraviolet transilluminator table were also borrowed for the construction of this prototype. Evaluations of different filter elements, calcium/dye concentration standard solutions, and photodiode detectors were conducted. An image of the dual emission, synchronous detector prototype module is shown in Figure 3, along with the second phase prototype constructed for PCB and structural design validation. Figure 3: Dual emission, synchronous detection fluorometer module prototype phase 1 and 2. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 12 of 41 Cell-free samples of were prepared in a dilution series of dye and calcium concentration and instrument responsiveness was evaluated using a voltmeter. These tested showed that the basic design did allow for ratio shifts to be detected across physiologically relevant calcium concentration ranges as well as literature-recommended dye working concentration ranges. Figure 4 shows data from one such evaluation test. Multiple platforms for sample containment were also explored in the context of culturing cells, including microplate insert membranes and various microcarrier systems. Figure 4: Prototype validation results demonstrating shift in ratio from low to high calcium concentration. Conceptualization of the optical layout of the fluorometer system involved several methods for imaging a large number of samples which shared excitation sources and detection setups, and using a dedicated excitation source and detector system for each sample. Eventually, the choice was made to use self-contained excitation and detection systems for each sample, based on simplifying the complexity of each fluorometer setup, while trading off the number of samples that could be economically monitored. Having dedicated excitation LEDs, photodiodes, and filters for each sample also simplified the control system and data logging requirements, but the number of individual filters required became the dominating factor, financially limiting the number of samples that could be monitored at a given time. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 13 of 41 Given the preferred optical design of the fluorometer apparatus decided, the physical layout of the cell samples was relaxed from a 96-well microplate in favor of optical-glass cuvettes which allowed for further simplification of the optical layout. Cells cultured on Cytopore-2 microcarriers were selected as the sample format, and various methods were explored for seeding of cells on to the carriers in the absence of the manufacturer-recommended stir flasks. The relative success of seeding procedures was evaluated through microscopy; sample image provided in Figure 5. Figure 5: Fluorescent microscopy of seeded Cytopre-2 microcarrier. Green represents phalloiden cytoskeletal staining of actin filaments, blue represents Hoecke's nuclear staining. Carriers are shown to be wellpopulated by cells. Experimental Systems The final experimental design comprises the same elements of the overall experimental approach in the way of onboard flow cytometry and cell fixation, but containes the ultimately-refined design of the ratiometric fluorometer system. An overview of the experimental systems which were flown on the reduced gravity flight is provided in this section. Cell Samples The fluorometer samples are composed of MLO-Y4 osteocyte-like cells loaded with fluorescent calcium detecting dyes Indo-1 (flight day 1) or Mag-Indo-1 (flight day 2) at 10 uM concentration. The cells were adhered to Cytopore-2 microcarrier beads (200,000 cells per milligram of microcarrier), which provided the three-dimensional matrix necessary to maintain proper cell phenotype. Prior to flight, the MLO-Y4 osteocyte-like cell lines were maintained by the staff of the JSC Immunology Lab using standard cell-culture protocols. The cells were seeded onto the Cytopore-2 microcarrier beads 24 hours prior to flight and then infused with fluorescent dye one hour prior to flight. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 14 of 41 The microcarriers are suspended at 10 mg/mL in Minimal Essential Media-alpha growth medium with 5% fetal bovine serum, 5% calf serum, the antibiotics penicillin and streptomycin, 25mM HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), and 0.1% low melting point agarose to create the viscosity necessary to prevent sample shifting during flight. Three experimental samples per flight have the previously-described basic composition, while two other samples contain additional control reagents. The sample herein referred to as the negative control contains 8 mM EGTA (ethylene glycol tetraacetic acid), a calcium chealtor that out-competes fluorescent dye for calcium binding thus buffering against ratio shifts, and ideally representing a maximum unbound fluorescent dye signal. The sample herein referred to as the positive control contains 1 ug/mL ionomycin, an ionophore that non-specifically permeabilizes cellular membranes to calcium ion flow with the anticipated effect of releasing of all intracellular calcium stores for a maximum bound fluorescent dye signal. The control samples are otherwise prepared identically to the experimental samples, and all samples are contained within 6 mL optical glass cuvettes. Ratiometric Fluorometry The team developed a synchronous, dual emission fluorometry system to monitor ratiometric calcium indicators with high temporal resolution. This required several sub-systems including the samples themselves, sample containment, temperature control (requisite to maintain a physiological environment for sample viability), excitation and detection optics, electronics, and software to facilitate data acquisition and analysis. The finalized fluorometer with integrated subsystems is shown in Figure 6. Figure 6: Synchronous, dual emission fluorometer assembled for microgravity flight testing. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 15 of 41 Environmental Monitoring In order to generate a precise map of the gravitational conditions which the cell samples are subjected to, gravitational acceleration data is collected with corresponding time stamps during the entire flight at approximately 10 Hz. Ratiometric and flow cytometric fluorescence data, as well as fixation time points are correlated to this acceleration data set via timestamps recorded from the same clock. The cell culture sample wells are maintained at a physiological temperature of 37ºC during flight by the temperature control sub-system of the ratiometric fluorometer. A commercial USB-toserial temperature probe from Quality Thermistor, Inc. interfaces with the flight software and monitors the underside of the aluminum warming plate to allow real-time temperature measurements in the LabVIEW software. A pulse-width modulated PID control loop triggers the active duty cycle of four 3-Ohm resistor modules mounted to the bottom of the warming plate surface to maintain the desired temperature setpoint. Temperature measurements made for control loop feedback are stored as a timestamped data set to correlate temperature variations to any observed changes in calcium concentration. Optics The optical design of the ratiometric fluorometer revolves around the requirement of exciting the Indo-1 dye at its responsive peak in the ultraviolet range, 355 nm, and simultaneously detecting the two emission peaks of the dye at 405 nm and 485 nm. A diagram of the Indo-1 excitation and emission spectral response curves is provided in Figure 7. The most direct method of separating out these desired wavelengths from a broad-spectrum source is using optical filters. A large amount of design time was spent in selecting the optimized filter set for these purposes, and the optical filters also comprised a large fraction of the cost of the total fluorometer system. Figure 7: Excitation and fluorescent emission spectra of Indo-1 dye. A represents measurements in high calcium concentrations and B in calcium-free solutions. (http://www.invitrogen.com/site/us/en/home/support/Product-Technical-Resources/Product-Spectra.1202ca.html) Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 16 of 41 Three different narrow-band-pass optical filters are used for each of the five constructed fluorometer modules. Each filter is tuned to pass a particular wavelength at peak transmittance, and the full-width at half-maximum transmission window for most of the filters used is 15 nm. The narrow pass-band of the excitation filter is primarily required to keep the very bright excitation LEDs from coupling into the photodiode detectors and saturating out the comparatively dimmer Indo-1 dye fluorescence signal. The remaining two filters were each placed ahead of the photodiodes to detect the 405 nm and 485 nm emission spectral peaks, respectively. The narrow pass-band of the two detection filters removed any remaining transmitted light in the 355 nm range, and also blocked out any signal from the other dye emission mode, so that each detector should only be observing the light generated by the particular emission mode of the dye. The rest of the sample holder well in each fluorometer module was made to minimize stray light admittance into the interior where it might affect measurements, although an electronic noisecancelling solution was also used to mitigate any effects from that potential situation. Figure 8 provides a schematic of the primary optical components in each fluorometer module. Figure 8: Synchronous, dual emission module schematic. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 17 of 41 Electronics The fluorometer is powered by two identical 12-volt power supplies, separated in order to limit noise between the emission and detection circuits. The electronics in this system cycles ultraviolet (UV) LEDs on and off at an approximately 1 kHz rate with a 50% duty cycle. This allows for running the LEDs at a higher current than they can continuously endure, keeping them from overheating while exposing the cell samples to as much UV radiation as possible in the 355 nm range. Photodiodes are placed on either side of the cell sample, with optical filters blocking out all but the 405nM spectrum on one side and the 485nM spectrum on the other. The photodiodes absorb the light emitted by the dye as it fluoresces and convert the light to a very small electric current. This current is then amplified by a three stage amplifier in order to get a strong enough signal to be read by the Data Acquisition (DAQ) system. The DAQ then converts the voltages obtained to digital values that are passed to the LabVIEW software. The system is powered by the 115V AC electrical service of the aircraft. All power from the supply cord passes through an emergency kill switch that will instantly de-energize the experiment apparatus in the event of an emergency. From the kill switch power is routed to a power strip where the laptop and DC power supplies for the fluorometer are connected. An overview diagram of the electrical system is shown in Figure 9. Figure 9: High-level schematic of experiment electrical system. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 18 of 41 Software The experiment software is developed as a LabVIEW Virtual Instrument (VI) and performs synchronous analog and digital IO no the fluorometer device through a National Instruments DAQ module. The architecture of the experiment software for in-flight fluorometry is implemented as five main components that execute in parallel for hardware control and data storage. The overarching software system design is illustrated in Figure 10. Figure 10: High-level diagram of the flight system software design and data paths. The application provides a graphical user interface (GUI) for simplified user control and visualization of experiment data in real-time during ground and flight operation. An image of the in-flight experiment VI is shown in Figure 11. The GUI is the primary device through which acceleration, temperature, and fluorescence data is synchronized and displayed. The fluorometer is controlled through the National Instruments DAQmx driver, which provides synchronization of analog and digital devices through high-level API calls. Dual emission fluorescence measurements are made on the five analog output pins at approximately 50Hz through a software initialized clock. The bound and unbound channel outputs are multiplexed by a digital DAQ output line. Each dual fluorescence measurement requires an analog read of the first channel, a digital MUX select, an adequate settling time delay, and an analog read of the second channel. Digital logic in the control loop alternates the first channel read on each iteration. The dual fluorescence measurement control sequence is provided in Figure 12. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 19 of 41 Figure 11: In-flight fluorometry graphical user interface VI. The UV excitation source can be pulsed or made inactive by a digital software switch. Every 60 seconds the excitation source is turned off and a dual emission measurement recorded. This “dark data” is stored as a separate data set for post-flight analysis of dark current drift. Acceleration data was collected during flight by a serial-to-USB 3-axis accelerometer. This device runs continuously in parallel with the fluorometer controller as a separate execution thread and stores acceleration changes in a time stamped file at approximately 60Hz. Figure 12: Software control sequence diagram for synchronous dual emission measurements. Boise State University NASA Microgravity University 2011 Final Report Rev. 1.0 Gravitational Modulation of Calcium Signaling in Bone Updated: 2 August 2011 Page: 20 of 41 A pulse-width modulated proportional integral derivative (PID) controller was implemented and tuned for precise temperature control of the device. The control loop triggers the active duty cycle of four 3-Ohm resistor modules mounted to the bottom of the warming plate surface to maintain the desired temperature setpoint. The control logic for combining calculated PID terms is shown in the block diagram of Figure 13. The duty cycle is updated on each control loop iteration by calculating the P, I, and D terms as Pterm = (S - T) x Pgain Iterm = [(S - T) + Istate] x Igain Dterm = (T - Dstate) x Dgain Where: S = desired temperature setpoint T = actual temperature measured Figure 13: Update PID controller block diagram. To correlate fixation samples with experiment duration, a time stamped data set was collected during microgravity flight. The experiment operator pushes the ‘Save Time Point’ graphical button on the user interface simultaneously with manual sample collection, which flags the fixation time stamp controller to retrieve the current time from the system clock and append it to the fixation time stamp file. Fixative samples are labeled and collected in numerical order to correspond with saved time points. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 21 of 41 Flow Cytometry In order to evaluate the performance of the custom fluorometer system, a portable flow cytometry system developed by the JSC Immunology Laboratory was used to collect fluorescent data on calcium flux. In contrast to the fluorometry system, data was collected for limited time periods (~20 seconds) during microgravity portions of parabolic flight and compared to samples processed in the 1-g laboratory environment. The calcium sensitive dye Rhod-2 was used as its optical properties are compatible with the instrument. In contrast to the ratiometric Indo dyes, Rhod-2 does not fluoresce until it binds calcium allowing calcium flux to be evaluated by corresponding increases and decreases in fluorescent intensity. In flow cytometry, cellular samples are passed through a microfluidic system that produces laminar flow, presenting microcarriers to a laser inspection system in single-file. Optical detection of forward and side light scatter provides information on sample size and shape. This information is combined with fluorescent intensity measurements to determine what is fluorescing and to what extent. Cell Fixation As a third data set, cellular samples were fixed using Whole Blood Staining Devices (WBSDs), developed by the JSC Immunology laboratory for collection of biological samples aboard the International Space Station, which were generously donated for this project’s use. WBSDs provide sterile containment of ~5 mL of fluid. Using a plastic clip, half the volume can be loaded with doubly-concentrated fixative solutions and sequestered from the sample until the clip is removed, at which point mixing leads to sample fixation. Three fixative conditions were employed for subsequent analysis by flow cytometry (2% paraformaldehyde), fluorescent microscopy (4 mM EGTA, 6% formaldehyde), and electron microscopy (4% gluteraldehyde, 6% formaldehyde). Seven sets of the three conditions were collected at different times during each flight for a total of 21 samples per flight. The fixated sample set spanned the entire test period so as to be correlated to the gravitational data, albeit with decreased temporal resolution. This provided a limited set of ‘snapshots’ of the biochemical responses of the cells to gravitational stimuli and will be used to assess whether calcium signaling events were induced by the flight environment and if the fluorometer successfully identified them. The schematic in Figure 14 represents the reduced gravity aircraft flight path. Three cellular samples were fixated at each time point indicated (numbers 1-7). Sample 1 was taken immediately prior to flight take-off; sample 2 was fixed at altitude before beginning parabolic maneuvers. Sample 3 was fixed during the first set of 16 parabolas, sample 4 during the turnaround period, sample 4 during the second set of 16 parabolas and sample 6 at the conclusion of parabolic maneuvers. Finally, sample 7 was taken following landing and completion of aircraft taxi. An image describing the relevant components of the WBSDs as well as the in-flight procedure for their use is also shown in Figure 15. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 22 of 41 Figure 14: Flight path with planned time points for cellular fixation Figure 15: Whole Blood Staining Device (WBSD) used for fixating cellular samples Results The prototype instrument was not capable of collecting data over time and thus was not suitable for testing of the hypothesis in 1G prior to departure from Boise State. Further, the experimental instrument was not ready for testing prior to flight in Houston. Therefore, all ground testing was scheduled to be performed upon return to Idaho. At the time of this report such testing has not taken place due to obstacles encountered in getting the fluorescence system software running in the lab. It is clear that any definitive interpretation of observed fluctuations in signal ratios during flight must be interpreted using results from ground based runs as this controls for all variables except for parabolic flight. In the absence of such data, ratio changes in parabolic flight have been compared to signal activity prior to take-off and after landing. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 23 of 41 Results for the two flights varied considerably and this is interpreted as a consequence of the different dyes used for the different flights. For the first flight, the standard Indo-1 was used as this was identified as the most common ratiometric calcium indicator in the literature. For the second flight, Mag-Indo-1 was chosen to explore the consequences of altered affinity on calcium flux observation in parabolic flight. Mag-Indo-1 has considerably lower binding affinity for calcium ion than standard Indo-1 (Kd ~35nM vs. 230nM), that is ions bind and disassociate at much higher rates. Ultimately this decreases the likelihood of calcium flux buffering in cells overloaded with dye. Further it increases the chance of observing rapidly changing events as the dye signal is more likely to ‘re-set’ along with any active transport of calcium associated with oscillating gravity. Experimental Samples Results for the first flight were generally inconclusive with little change in ratio observed in association with parabolic flight; the exception was experimental sample 3. All samples showed a steady increase in ratio over the course of the experiment in flight 1. This was consistent in flight 2 with the exception of sample 3, which showed a steady decrease in ratio over time; interestingly this was recorded in the same module that appeared to record gravity sensitive calcium flux in flight 1 and showed overall increase in ratio. The ratio represents the signal, in volts, from the 485nm channel divided by that from the 405nm channel; therefore it can be thought of as unbound over bound. In this way a positive slope indicates a relative increase in unbound dye and by extension a decrease in free calcium. An early biological interpretation then could be that, in general, it appeared that free calcium decreased over time. However, if non biological factors (electronic performance for example)led to either an increase in signal from 485nm channels, decrease in signal from the 405nm channels, or some combination of the two, the ratio would increase. Ground testing for comparable periods to flight testing (~2 hours) should help shed light on this question. Examination of ratio response during parabolic maneuvering as compared to measurements obtained on the ground and during non-parabolic periods of the flight suggest that gravitationally sensitive calcium flux was observed on the second flight using Mag-Indo-1. As is evident in the following figures, ratios consistently increased immediately following the initiation of microgravity periods, while ratios consistently decreased immediately following initiation of hypergravity periods. This would suggest that free calcium decreases in response to microgravity and increases in response to hypergravity. Additionally, ignoring overall changes in slope, this behavior remained relatively consistent throughout the entire 32 parabolas. Taking the results of flight 2 into account, calcium flux does appear to occur in response to parabolic flight, but did not appear to show regulative behavior over the time course of this experiment. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 24 of 41 Controls In the first flight the control sample results did not behave significantly different than experimental samples 1 and 2. It was anticipated that the ratios would remain relatively constant, with the negative control maintaining a higher ratio value due to its anticipated increase in the proportion of unbound dye. In the second flight the positive control still behaved like the experimental samples (with the exception of an opposite overall slope to sample 3). In contrast the negative control sample did seem to maintain a relatively stable, although low, ratio even in the presence of the lower affinity dye. The results of the negative control in flight 2 are encouraging that the experiment functioned as anticipated while the results from the positive control are less supportive of this interpretation. The positive condition did require use of a regent with which the team did not have prior experience and thus the possibility that it was not properly prepared or delivered is an area of consideration in future sample preparation optimization exercises. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Flight 1 Fluorometry Results A B Updated: 2 August 2011 Page: 25 of 41 Boise State University Gravitational Modulation of Calcium Signaling in Bone C D NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 26 of 41 Boise State University Gravitational Modulation of Calcium Signaling in Bone E F NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 27 of 41 Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Flight 2 Fluorometry Results Updated: 2 August 2011 Page: 28 of 41 Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 29 of 41 Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 30 of 41 Figure 16: Flight 2 Figures A=(-),B=(+), C-E= experimental replicates 1-3. Axes: x= time (s), Y left= ratio signal 485nm/signal 405nm (unbound/bound), Y right= acceleration (G’s). Lines: Blue = ratio, green = acceleration. arrows: green= begin hyper, red= begin micro. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 31 of 41 Portable Flow Cytometry Flow cytometry demonstrated a decrease in microgravity relative to 1G with Mean Fluorescence Units (MFI) going from 1321.6 in 1G to 1165.2 in Rhod-2 stained cells in microgravity. These measurements were taken during the first flight in which the fluorometry results were inconclusive, but these results (displayed in Figure 17) are in accordance with flight 2 fluorometry results and support the notion that the calcium flux suggested by ratio shifts in MagIndo-1 signals were not artifactual. Figure 17: Scatter plot analysis of portable flow cytometry in microgravity. Fixation Samples fixed for analysis by microscopy and cytometry have not yet been processed. This work will be ongoing in the late summer and fall of 2011. Results will be included in any potential publications as well as in proposals for follow-up flights. In addition to lengthy protocols for processing of samples, the novelty of the work for team members requires time for protocol optimization on representative samples before valuable experimental material is processed. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 32 of 41 Samples from the second flight are expected to yield more insightful results for two reasons. The first is that the fixation time point plan was excecuted correctly on the second flight, but not on the first. On the first flight samples for time points 1 and 2 were not fixed until parabolic flight began due to a lack of familiarity with the flight experience and the fact that samples were stowed by the ground team and were not immediately accessible. Drawing on this experience, flight team 1 instructed flight team 2 in how to make sure samples were available and fixated samples for time point 1 of the second flight prior to the flight team boarding the aircraft and taking their seats. The second reason is that the flight 2 samples correlate to the fluorometry data in which the dye choice allowed for observation of calcium flux. Discussion Time The greatest challenge was the amount of time available for instrument research and development. Despite careful planning, due to the complexity and novelty of our instrument, the experimental system was not functional until the days just prior to flight, making quality testing and improvement impossible. In effect, this made the flight instrument to be somewhat of a prototype, and the flight served as a system test. Various elements had to come together in the final phases of development, including software and electronics. Since these subsystems were integrated at JSC where sample preparation materials were in limited supply (cellular stocks, dye, etc.) as well as adequate time for sample preparation, a process that in total took at least 48 hours, it was not possible to optimize dye concentrations, incubation times etc. to the performance of the flight-ready instrument. These parameters can seriously affect experimental results. Due to its interactions with calcium ions, the dye itself can buffer calcium flux and its downstream biochemical consequences; this affect is increased by higher dye affinity, higher loading concentrations, and longer incubation periods. Therefore, minimal dye concentration and incubation time are ideal. However, doubts pertaining to the sensitivity of the instrument and strength of signal from samples led to the decision to lean on the side of maximizing signal through relatively high loading concentration (10uM) and long incubation periods (30min). Expertise Time would not have been as much of an issue had it been clear sooner during the process that it would be necessary to fabricate an instrument. Since no team member had expertise in design of optical systems, the design phase took longer than ideally planned. Further, inexperience limited the sense of design options and led to considerable need for compromises to be made without adequate time to consider consequences and alternatives. For example, deciding against purchasing a commercial fluorometer was accompanied by deviating from the use of standardized cell culture vessels for sample preparation and containment. Final choice of cuvettes as a vessel left little time for consideration of how to best prepare adherence dependent cells in an environment that presented no suitable surfaces for adherence. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 33 of 41 Failures Several aspects of the experimental system could be considered inadequate. Non-Ideal Temperature Measurement The temperature control system needs improvement as the current placement of the sensor loop on the exterior of the aluminum block produces readings that are generally lower than actual sample port temperatures due to exposure to the ambient environment. Inflexible Fluorometer Gain Another area for improvement of the current system design would be integration of variable gain control. The current system requires gain to be adjusted in solid state through changing resistors etc. Variable gain would aid particularly in sample preparation optimization as it would allow one to evaluate minimal dye concentration and incubation periods without having to re-solder PCBs every time the signals are decreased. In the absence of such functionality, attempts where made to create representative samples to adjust gain to. This required costly materials, such as dye, to be consumed to obtain approximate ranges on performance requirements. A more flexible system would allow adjustment of the instrument to samples, rather than having to adjust samples to the instrument. Iteration toward the ideal testing conditions would be considerably aided with this setup. Cell Culture Optimization Some obvious failures of sample preparation include the choice of standard Indo-1 for the first flight. It was also desired to explore more complicated sample compositions, such as those involving more cell types in an effort to create a more biologically relevant system. Due to time constraints and the inability to evaluate samples prior to departure to Houston it was deemed prudent to simply test mono-cultures of osteocytes this year. Electronics Design The key requirement of the electronics system was to detect a very small amount of light, and to convert that light into a useful signal that accurately and constantly reflects how much the sample fluoresces at a specific wavelength. There are many difficulties in the design and construction of such a circuit. Dr. Hay proved to be an incredible resource to our team in developing and simulating a feasible circuit design. Once the design and simulation was complete, members of the team had to lay out the design for the printed circuit boards. Because of the need to have separate optical filters on the emission and detection elements, and to prevent any source light from reaching the detectors directly, it was necessary to make 3 separate circuit boards for each of the 5 modules. Two prototype versions of these boards were manufactured and tested for a single module. From these we gathered as much information as possible about needed changes and improvements. There was not enough time for another prototype revision, so all of the final boards, 16 in all, were manufactured despite the circuit performance not being as completely optimized and proven as we would have liked. The boards were manufactured in a lab at Boise State. All of the surface-mount components were then hand Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 34 of 41 soldered to the boards by members of our team. This required many hours of soldering on each board, followed by hours of testing and troubleshooting. In order to amplify the fluorescent light from the dye into a large enough signal for the DAQ to process, the signal had to be amplified more than a billion times. This is very problematic since any noise introduced prior to the early amplification stages will result in a useless signal. It was also critical to have just the right amount of gain so that we would obtain a good signal without saturating the output. If we had the time, we would have implemented an easy way to adjust the circuit gains, such as a trim pot so that variations in cell samples could be adjusted for in a short time. As it was, adjusting the gain required removing and adding surface mount resistors to the boards by soldering. Each channel was adjusted so that a cell sample deemed to be ideal caused a signal around 5 volts, midway in the output range. None of the channels were exactly the same, but by testing a calibration sample in each module prior to flight it was hoped that any variations could be accounted for and adjusted in the output data. We experienced problems with humidity and with static damage. We found that when dealing with amplification levels of this magnitude, and input signals so small, even the smallest leakage path for current would render the circuit inoperable. This became especially apparent when we brought our experiment into the humid environment of the outdoor hangar in Houston and several of the channels stopped functioning the day before our flight. We found that solder flux paste used during the board soldering process, became slightly conductive with moisture and created undesirable current flow in the sensor circuit. We cleaned off as much flux as we could with rubbing alcohol and swabs, getting some of the boards to function this way. On a few of the boards, we had to actually cut and remove conductive traces from the circuit board and replace them with insulated wire. We also had several of the operational amplifiers on our sensor boards become non-functional after handling, specifically when the modules were being assembled together. We had to replace several of the amplifier chips and implement new handling procedures to prevent this damage which was likely due to static discharge Successes Due to the complexity of the experiment, the limited background of undergraduate researchers, and the aforementioned time constraints, simply delivering a functional system and producing data that addressed the scientific question was a momentous success. Professional Development and Interaction As a professional development exercise, the project was highly beneficial. Even the challenges encountered in the form of managing time, budgets, delegation of duties, etc. were highly instructive. Students experienced first hand how challenging and how rewarding it can be to work on a project where the scope is beyond any individuals’ skill set. Appropriately, working in the JSC Immunology laboratory while in Houston provided wonderful opportunities to reflect on such a process with researchers in the life sciences who constantly work with engineers, administrators, and collaborators from other scientific fields to complete projects. It was clear that this project was quite representative of the balance of compromise and patient resolve required to work in such a context. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 35 of 41 Encouraging Results and Follow-On Promise The encouraging results from the flight along with the significant increase in familiarity with concepts and skills required to complete this investigation set the stage for increased chance of success in the event of follow up flights. Future teams will benefit greatly from our successes and failures. At the very least they will be able to begin testing on the current instrument that can serve as a basis for improvement or a prototype. Conclusion Lessons Learned and Recommendations Science Results We learned a great deal through this experiment. Foremost, we learned scientific concepts. Each team member, including the biology students, learned more about cell signaling. Likewise, we learned a great deal about engineering practices. Beyond the knowledge gained through practice, we gained information about how osteocytes react to fluctuating gravity. Namely, we observed that parabolic flight elicits calcium flux. Time Management We experienced the consequences of a condensed timeline schedule with little room for sliding deadlines. Multiple unforeseen challenges arose with design and component integration, which could have been prevented or mitigated with adequate time for design and ground tests. Software We would like to refine data acquisition software for easy transfer to different machines to avoid problems encountered with running the instrument in the lab using desktop machines. Furthermore, we would like to develop a comprehensive data analysis scripts before a follow-up flight so that results can be immediately evaluated post flight and any necessary adjustments can be made between flights. These scripts would have certain functionalities not currently available, such as the ability to plot multiple fluormetric data sets on the same axes for comparison of, for example, experimental results to control results. Temperature Sensing As previously mentioned, temperature sensing design must be re-evaluated to obtain accurate readings of sample port temperatures. This will likely require repositioning of the temperature sensing loop to the interior of the warming block. Variable Fluorometer Gain Given the chance to improve the circuit in the future, we would definitely like to make two changes. One is to implement an easy and quick gain adjustment for each channel. The other would be to obtain industry quality circuit boards which have a protective solder-mask layer to eliminate problems due to moisture and metallic changes such as oxidation over time that will certainly occur with our bare copper circuit boards. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 36 of 41 Increasing Sample Number Capability and Reducing Variability We would like to explore the possibility of transitioning from photodiode based, modular detection to a lens based system in which a single CCD could monitor a larger area covering multiple samples. Such a system might increase sample capacity and reduce variability observed across replicates that is likely due to a lack of uniformity across modules. We would modify sample preparation, perhaps in ways dependent on a new design, such as employing standard microplates with well inserts as sample containment to increase uniformity, increase sample number, and decrease the amount of material per sample, which currently is very large by biomedical research standards (~6 mL vs. ~200 uL). Higher sample number, reduced sample size, and greater uniformity in analysis would make it possible to perform more complex experiments with an increased number of variables to introduce to identify underlying molecular mechanisms of gravitationally induced calcium flux. Examples include the introduction of samples in calcium free media to evaluate the contribution of influx of extracellular calcium as well as the inclusion of inhibitors and stimulators of specific mediators of calcium flux to determine which pathway constituents are involved. We would continue to use lower affinity dye for all flights, perhaps exploring other reduced affinity Indo derivatives such as Indo-5F. Further we explore reducing dye loading concentrations as suggested concentrations range from 1-10 uM and we loaded at the high end. We would also evaluate shortening incubation times which can range from 15minutes to one hour; in this experiment we used 30 minute incubations. We would like to explore co-culturing osteocytes with osteoblasts as in tissue these cell populations represent sensors and effectors of mechanical stimulation and mechanically directed remodeling, respectively. Calcium flux might behave differently in this more complex, and representative, system. Research Contributions to NASA Astronauts can lose bone mass at rates that make long term mission to destinations such as the moon, mars, and beyond unfeasible. Additionally, astronauts experience damage to their immune systems, musculature, vasculature, and even nervous tissues. Much work remains to be done to identify effective strategies for mitigation of the negative health consequences of exposure to non-homeostatic gravitational environments. This project and the technologies it produces can be of use in evaluating any number of tissue types and biomolecular activities. Outreach Idaho Science and Aerospace Scholars event The Idaho Science and Aerospace Scholars (ISAS) is a program in which 144 of Idaho’s top high school Juniors were accepted to take a 16 week course from Idaho Digital Learning Academy in preparation to design a mission to Mars. At the end of the 16 week course the top 88 students were selected to continue the program and design the actual mission. During this portion of the program students participated in events such as touring NASA’s Ames research Center and Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 37 of 41 talking with engineers. On July 18 2011 BSU’s Microgravity team presented to the first group of Scholars about designing, building, and finally testing a project on NASA’s zero-g flight. Another presentation will be made to the second group of Scholars on August 1st 2011. Science Café The Science Café in Boise, sponsored by the Discovery Center of Idaho (DCI), is a monthly meeting place for informal science discussion. A guest scientist begins each meeting by presenting a scientific topic for conversation and debate. Although local researchers and scientists often attend the Science Café, these meetings are intended for people with any level of science background. On May 17 2011 Boise State research students presented a synopsis of the bone remodeling process with particular emphasis on how it is affected by changes in the mechanical environment, such as gravity. Discussion points emphasized gaps in the current model of how diverse and potentially conflicting mechanical inputs are integrated by bone cells, and the team's approach to investigating a potential regulative role for calcium signaling during a reduced gravity research flight in June at Johnson Space Center. Boise State University 8th Annual Undergraduate Research and Scholarship Conference Boise State University’s annual Undergraduate Research and Scholarship Conference exhibits the research and artwork of select undergraduate students from each College in the form of posters, lectures, or visual presentations. The Boise State Microgravity University Team presented a poster at this conference on April 11, 2011. Idaho Academy of Science 2011 Symposium The Idaho Academy of Science (IAS) advocates for further scientific education across the State. The IAS Symposium was hosted by the College of Idaho, and featured poster and lecture presentations from collegiate researchers in science or engineering fields. The event was open to the public. The Boise State Microgravity University Team presented a poster at this conference on April 1, 2011. Summer Research Community Evening Seminar The Summer Research Community coordinates social events and educational activities for students involved in STEM field summer research fellowships at Boise State. Team member Benjamin Davis was the invited speaker for the July 14th Evening Seminar; his talk “Teeter Boards, Mass Spectrometers, and the Vomit Comet” included discussion of his experience as a student participant in the Microgravity University program. Ignite Boise Ignite Boise is a local community function recognized as part of the global network of Ignite presentation events. A member of BSU’s microgravity team presented 20 slides in 5 minutes on April 21 2011 before a community audience of 700 discussing the team’s success up to that point of designing a biological experiment which would measure calcium flux within bone cells while receiving successively different gravitational inputs. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 38 of 41 Dr. Nandikolla Dr. Nandikolla was an outreach item that was taken down to Houston and flew on the zero-g flight along with the team members and experiment. This item was a model skeleton which visually demonstrated the effects of the gravitational changes during the flight. Dr. Nandikolla will be presented to the Discovery Center of Idaho along with a video showing its reactions to the continually changing gravitational environment. He will be used as a display in DCI to further the knowledge of its visitors. Space Day’s event The Space Day’s event, at Discovery Center of Idaho, will be open to the general public and will be held August 5th-10th 2011. During this event the BSU Microgravity team will present their experience of flying a biological study on a zero-g flight through NASA’s Microgravity program. Team Blog The team has created an outreach website which is used for promoting the experiment to the public and marketing outreach activities. The website has a blog, showcasing the day-to-day activities of the team leading up to flight week. It documents the process of building and testing the apparatus as well as outreach activities. This student-built and maintained site is hosted and linked from the Boise State University College of Engineering website at: http://coen.boisestate.edu/MicrogravityU2010/Bioengineering/index.html. “D4K” Dialogue for Kids (“D4K”) is a live television show that is broadcast on Idaho Public Television which features guest scientists. The scientists introduce a topic and children watching from their Elementary School classroom may call in with questions. The Boise State Microgravity Team will be featured in “D4K” during the upcoming 2011-2012 school year and will outline bone loss in microgravity. “NASA Digital Learning Network” The Boise State Microgravity Team is requesting to utilize NASA Digital Learning Network (DLN) to host an interactive videoconference with the students at Garfield Elementary. The team would like to feature a former astronaut and co-faculty advisor, Barbara Morgan, who has experienced bone loss as a result of spaceflight. As an equally exciting alternative, the team would like to request an Astronaut Strength Conditioning and Rehabilitation (ASCR) specialist to partake in the event as the NASA educator. This event would take place in the upcoming 2011-2012 school year. Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 39 of 41 NASA Fit Explorer Challenge “Living Bones, Strong Bones” The Boise State Microgravity Team will introduce Garfield Elementary teachers to NASApromoted educational hands-on classroom activities. The NASA Fit Explorer Challenge assists teachers in student engagement through physical activities related to health and physical fitness while incorporating specific science topics; in this case, astronaut bone loss (cause, prevention, and treatment). This outreach event will take place in the upcoming 2011-2012 school year, and may serve as an extension on NASA DNL. Press Releases The Boise State Microgravity University Team was featured in several local articles, which are outlined below. “Microgravity University Team awarded NASA Special Project Grant” by Boise State University undergraduate student, Katie Ammar, was published on the Boise State Biomolecular Research Center website (http://brc.boisestate.edu/brc/news/) in May, 2011. “Boise State Research Team Among 14 Nationwide Selected for NASA’s Microgravity University 2011” by Erin Ryan was featured in Boise State University’s UPDATE online newsletter on December 23, 2010 (http://news.boisestate.edu/update/2010/12/23/boise-stateresearch-team-among-14-nationwide-selected-for-nasas-microgravity-university-2011/). Additionally, the Boise State Microgravity University Team was mentioned in the Discovery Center of Idaho’s July/August/September newsletter in an article titled “Idaho Space Days 2011-August 5th through 10th” (http://scidaho.org/Library/Newsletters/Newsletter_Q3_2011.pdf). Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 40 of 41 References 1. Ingber D. 1999. How cells (might) sense microgravity. FASEB Journal 13 (Suppl.): 3-15. 2. Liedert A, Kaspar D, Blakytny R, Claes L, Ignatius A. 2006. Signal transduction pathways involved in mechanotransduction in bone cells. Biochem Biophys Res Comm 349:1–5. 3. Zhang P, Hamamura K, Yokota H. 2008. A Brief Review of Bone Adaptation to Unloading. Geno Prot Bioinfo 6(1): 4-7. 4. Turner CH. 2006. Bone Strength: Current Concepts. Ann N.Y. Acad Sci 1068: 429–446. 5. LeBlanc AD, Spector ER, Evans HJ, Sibonga JD. 2007. Skeletal responses to space flight and the bed rest analog: A review. J Musculoskelet Neuronal Interact 7(1):33-47. 6. Kufhal RH, Saha S. 1990. A theoretical model for stress-generated fluid flow in canaliculilacunae network in bone tissue. J Biomech 23(2):171-180. 7. Kurpinski K, Park J, Thakar RG, Li S. 2006. Regulation of Vascular Smooth Muscle Cells and Mesenchymal Stem Cells by Mechanical Strain. MCB 3(1): 21-34 Boise State University Gravitational Modulation of Calcium Signaling in Bone NASA Microgravity University 2011 Final Report Rev. 1.0 Updated: 2 August 2011 Page: 41 of 41 Acknowledgements This project was funded by the National Institute of Health/National Center for Research Resources (NIH/NCRR; P20RR16454), Idaho State Board of Education for the Musculoskeletal Research Institute, NASA/EPSCoR (NNX10AM75H), Boise State University Office of Research, and the Idaho Space Grant Consortium (ISGC; 128G106040). The osteocytes used in this study were generously donated by Dr. Linda Bonewald and her laboratory at the University of Kansas City, Missouri. We would like to thank Heather Quiriarte, Dr. Brian Crucian, Mayra Nelman, Kami Faust, Dr. Clarance Sams and the entire Johnson Space Center Immunology Laboratory team for use of their facilities, and their invaluable technical assistance. We would also like to thank Dr. Julia Oxford, Barbara Jibben, Raquel Brown and Diane Smith at the Boise State University Biomolecular Research Center, as well as Kim Long and Linda Georgiev at the Boise State University College of Engineering for their support. We graciously acknowledge Dr. Thomas Glass and Sapidyne Instruments for their professional guidance, and the Discovery Center of Idaho and Ignite Boise 6 for the outreach opportunities. Special thanks to Dr. Vidya Nandikolla, Kameryn Williams, Jason Griswold, Chris Barbie, Dr. Ken Cornell, Dr. Roy Langton, and Kellen Moore for their collaboration.
