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All sourcesNASA NTRSDOE OSTI
NASA NTRS2013

Development Status of the WetLab-2 Project: New Tools for On-orbit Real-time Quantitative Gene Expression.

The primary objective of NASA Ames Research Centers WetLab-2 Project is to place on the ISS a research platform to facilitate gene expression analysis via quantitative real-time PCR (qRT-PCR) of biological specimens grown or cultured on orbit. The WetLab-2 equipment will be capable of processing multiple sample types ranging from microbial cultures to animal tissues dissected on-orbit. In addition to the logistical benefits of in-situ sample processing and analysis, conducting qRT-PCR on-orbit eliminates the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms. The system can also validate terrestrial analyses of samples returned from ISS by providing quantitative on-orbit gene expression benchmarking prior to sample return. The ability to get on orbit data will provide investigators with the opportunity to adjust experimental parameters for subsequent trials based on the real-time data analysis without need for sample return and re-flight. Finally, WetLab-2 can be used for analysis of air, surface, water, and clinical samples to monitor environmental contaminants and crew health. The verification flight of the instrument is scheduled to launch on SpaceX-5 in Aug. 2014.Progress to date: The WetLab-2 project completed a thorough study of commercially available qRT-PCR systems and performed a downselect based on both scientific and engineering requirements. The selected instrument, the Cepheid SmartCycler, has advantages including modular design (16 independent PCR modules), low power consumption, and rapid ramp times. The SmartCycler has multiplex capabilities, assaying up to four genes of interest in each of the 16 modules. The WetLab-2 team is currently working with Cepheid to modify the unit for housing within an EXPRESS rack locker on the ISS. This will enable the downlink of data to the ground and provide uplink capabilities for programming, commanding, monitoring, and instrument maintenance. The project is currently designing a module that will lyse the cells and extract RNA of sufficient quality for use in qRT-PCR reactions while using a housekeeping gene to normalize RNA concentration and integrity. Current testing focuses on two promising commercial products and chemistries that allow for RNA extraction with minimal complexity and crew time.

quantitative PCR↗
NASA NTRS2014

WetLab-2: Tools for Conducting On-Orbit Quantitative Real-Time Gene Expression Analysis on ISS

The objective of NASA Ames Research Centers WetLab-2 Project is to place on the ISS a research platform capable of conducting gene expression analysis via quantitative real-time PCR (qRT-PCR) of biological specimens sampled or cultured on orbit. The project has selected a Commercial-Off-The-Shelf (COTS) qRT-PCR system, the Cepheid SmartCycler and will fly it in its COTS configuration. The SmartCycler has a number of advantages including modular design (16 independent PCR modules), low power consumption, rapid ramp times and the ability to detect up to four separate fluorescent channels at one time enabling multiplex assays that can be used for normalization and to study multiple genes of interest in each module. The team is currently working with Cepheid to enable the downlink of data from the ISS to the ground and provide uplink capabilities for programming, commanding, monitoring, and instrument maintenance. The project has adapted commercial technology to design a module that can lyse cells and extract RNA of sufficient quality and quantity for use in qRT-PCR reactions while using a housekeeping gene to normalize RNA concentration and integrity. The WetLab-2 system is capable of processing multiple sample types ranging from microbial cultures to animal tissues dissected on-orbit. The ability to conduct qRT-PCR on-orbit eliminates the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples. The system can be used to validate terrestrial analyses of samples returned from ISS by providing on-orbit gene expression benchmarking prior to sample return. The ability to get on orbit data will provide investigators with the opportunity to adjust experiment parameters for subsequent trials based on the real-time data analysis without need for sample return and re-flight. Researchers will also be able to sample multigenerational changes in organisms. Finally, the system can be used for analysis of air, surface, water, and clinical samples to monitor environmental contaminants and crew health. The verification flight of the instrument is scheduled to launch on SpaceX-7 in June 2015.

quantitative PCR↗
NASA NTRS2015

On-Orbit Quantitative Real-Time Gene Expression Analysis Using the Wetlab-2 System

NASA Ames Research Center's WetLab-2 Project enables on-orbit quantitative Reverse Transcriptase PCR (qRT-PCR) analysis without the need for sample return. The WetLab-2 system is capable of processing sample types ranging from microbial cultures to animal tissues dissected on-orbit. The project developed a RNA preparation module that can lyse cells and extract RNA of sufficient quality and quantity for use as templates in qRT-PCR reactions. Our protocol has the advantage of using non-toxic chemicals and does not require alcohols or other organics. The resulting RNA is dispensed into reaction tubes that contain all lyophilized reagents needed to perform qRT-PCR reactions. System operations require simple and limited crew actions including syringe pushes, valve turns and pipette dispenses. The project selected the Cepheid SmartCycler (TradeMark), a Commercial-Off-The-Shelf (COTS) qRT-PCR unit, because of its advantages including rugged modular design, low power consumption, rapid thermal ramp times and four-color multiplex detection. Single tube multiplex assays can be used to normalize for RNA concentration and integrity, and to study multiple genes of interest in each module. The WetLab-2 system can downlink data from the ISS to the ground after a completed run and uplink new thermal cycling programs. The ability to conduct qRT-PCR and generate results on-orbit is an important step towards utilizing the ISS as a National Laboratory facility. Specifically, the ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. On orbit gene expression analysis can also eliminate the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples and provide on-orbit gene expression benchmarking prior to sample return. Finally, the system can also be used for analysis of air, surface, water, and clinical samples to monitor environmental pathogens and crew health. The validation flight of the WetLab-2 system using E. coli bacteria and mouse liver launched on SpaceX-7 in June 2015 and will remain on the ISS National Laboratory.

ISS tools↗
NASA NTRS2011

PMA-PhyloChip DNA Microarray to Elucidate Viable Microbial Community Structure

Since the Viking missions in the mid-1970s, traditional culture-based methods have been used for microbial enumeration by various NASA programs. Viable microbes are of particular concern for spacecraft cleanliness, for forward contamination of extraterrestrial bodies (proliferation of microbes), and for crew health/safety (viable pathogenic microbes). However, a "true" estimation of viable microbial population and differentiation from their dead cells using the most sensitive molecular methods is a challenge, because of the stability of DNA from dead cells. The goal of this research is to evaluate a rapid and sensitive microbial detection concept that will selectively estimate viable microbes. Nucleic acid amplification approaches such as the polymerase chain reaction (PCR) have shown promise for reducing time to detection for a wide range of applications. The proposed method is based on the use of a fluorescent DNA intercalating agent, propidium monoazide (PMA), which can only penetrate the membrane of dead cells. The PMA-quenched reaction mixtures can be screened, where only the DNA from live cells will be available for subsequent PCR reaction and microarray detection, and be identified as part of the viable microbial community. An additional advantage of the proposed rapid method is that it will detect viable microbes and differentiate from dead cells in only a few hours, as opposed to less comprehensive culture-based assays, which take days to complete. This novel combination approach is called the PMA-Microarray method. DNA intercalating agents such as PMA have previously been used to selectively distinguish between viable and dead bacterial cells. Once in the cell, the dye intercalates with the DNA and, upon photolysis under visible light, produces stable DNA adducts. DNA cross-linked in this way is unavailable for PCR. Environmental samples suspected of containing a mixture of live and dead microbial cells/spores will be treated with PMA, and then incubated in the dark. Thereafter, the sample is exposed to visible light for five minutes, so that the DNA from dead cells will be cross-linked. Following this PMA treatment step, the sample is concentrated by centrifugation and washed (to remove excessive PMA) before DNA is extracted. The 16S rRNA gene fragments will be amplified by PCR to screen the total microbial community using PhyloChip DNA microarray analysis. This approach will detect only the viable microbial community since the PMA intercalated DNA from dead cells would be unavailable for PCR amplification. The total detection time including PCR reaction for low biomass samples will be a few hours. Numerous markets may use this technology. The food industry uses spore detection to validate new alternative food processing technologies, sterility, and quality. Pharmaceutical and medical equipment companies also detect spores as a marker for sterility. This system can be used for validating sterilization processes, water treatment systems, and in various public health and homeland security applications.

Venkateswaran, Kasthuri J.↗
NASA NTRS1993

Spatiotemporal behavior and nonlinear dynamics in a phase conjugate resonator

The work described can be divided into two parts. The first part is an investigation of the transient behavior and stability property of a phase conjugate resonator (PCR) below threshold. The second part is an experimental and theoretical study of the PCR's spatiotemporal dynamics above threshold. The time-dependent coupled wave equations for four-wave mixing (FWM) in a photorefractive crystal, with two distinct interaction regions caused by feedback from an ordinary mirror, was used to model the transient dynamics of a PCR below threshold. The conditions for self-oscillation were determined and the solutions were used to define the PCR's transfer function and analyze its stability. Experimental results for the buildup and decay times confirmed qualitatively the predicted behavior. Experiments were carried out above threshold to study the spatiotemporal dynamics of the PCR as a function of Pragg detuning and the resonator's Fresnel number. The existence of optical vortices in the wavefront were identified by optical interferometry. It was possible to describe the transverse dynamics and the spatiotemporal instabilities by modeling the three-dimensional-coupled wave equations in photorefractive FWM using a truncated modal expansion approach.

Liu, Siuying Raymond↗
NASA NTRS2009

Discrimination of Spore-Forming Bacilli Using spoIVA

A method of discriminating between spore-forming and non-spore-forming bacteria is based on a combination of simultaneous sporulation-specific and non-sporulation-specific quantitative polymerase chain reactions (Q-PCRs). The method was invented partly in response to the observation that for the purposes of preventing or reducing biological contamination affecting many human endeavors, ultimately, only the spore-forming portions of bacterial populations are the ones that are problematic (or, at least, more problematic than are the non-spore-forming portions). In some environments, spore-forming bacteria constitute small fractions of the total bacterial populations. The use of sporulation-specific primers in Q-PCR affords the ability to assess the spore-forming fraction of a bacterial population present in an environment of interest. This assessment can provide a more thorough and accurate understanding of the bacterial contamination in the environment, thereby making it possible to focus contamination- testing, contamination-prevention, sterilization, and decontamination resources more economically and efficiently. The method includes the use of sporulation-specific primers in the form of designed, optimized deoxyribonucleic acid (DNA) oligonucleotides specific for the bacterial spoIVA gene (see table). [In "spoIVA," "IV" signifies Roman numeral four and the entire quoted name refers to gene A for the fourth stage of sporulation.] These primers are mixed into a PCR cocktail with a given sample of bacterial cells. A control PCR cocktail into which are mixed universal 16S rRNA primers is also prepared. ["16S rRNA" denotes a ribosomal ribonucleic acid (rRNA) sequence that is common to all organisms.] Following several cycles of heating and cooling according to the PCR protocol to amplify amounts of DNA molecules, the amplification products can be analyzed to determine the types of bacterial cells present within the samples. If the amplification product is strong, relative to the product of a control PCR sequence, then it is concluded that the bacterial population in the sample consists predominantly of spore-forming cells. If the amplification product is weak or nonexistent, then it is concluded that the bacterial population in the sample consists predominantly or entirely of non-spore-forming cells.

Venkateswaran, Kasthuri↗
NASA NTRS2015

Deriving Scaling Factors Using a Global Hydrological Model to Restore GRACE Total Water Storage Changes for China's Yangtze River Basin

This study used a global hydrological model (GHM), PCR-GLOBWB, which simulates surface water storage changes, natural and human induced groundwater storage changes, and the interactions between surface water and subsurface water, to generate scaling factors by mimicking low-pass filtering of GRACE signals. Signal losses in GRACE data were subsequently restored by the scaling factors from PCR-GLOBWB. Results indicate greater spatial heterogeneity in scaling factor from PCR-GLOBWB and CLM4.0 than that from GLDAS-1 Noah due to comprehensive simulation of surface and subsurface water storage changes for PCR-GLOBWB and CLM4.0. Filtered GRACE total water storage (TWS) changes applied with PCR-GLOBWB scaling factors show closer agreement with water budget estimates of TWS changes than those with scaling factors from other land surface models (LSMs) in China's Yangtze River basin. Results of this study develop a further understanding of the behavior of scaling factors from different LSMs or GHMs over hydrologically complex basins, and could be valuable in providing more accurate TWS changes for hydrological applications (e.g., monitoring drought and groundwater storage depletion) over regions where human-induced interactions between surface water and subsurface water are intensive.

water↗
NASA NTRS2013

Detection of Only Viable Bacterial Spores Using a Live/Dead Indicator in Mixed Populations

This method uses a photoaffinity label that recognizes DNA and can be used to distinguish populations of bacterial cells from bacterial spores without the use of heat shocking during conventional culture, and live from dead bacterial spores using molecular-based methods. Biological validation of commercial sterility using traditional and alternative technologies remains challenging. Recovery of viable spores is cumbersome, as the process requires substantial incubation time, and the extended time to results limits the ability to quickly evaluate the efficacy of existing technologies. Nucleic acid amplification approaches such as PCR (polymerase chain reaction) have shown promise for improving time to detection for a wide range of applications. Recent real-time PCR methods are particularly promising, as these methods can be made at least semi-quantitative by correspondence to a standard curve. Nonetheless, PCR-based methods are rarely used for process validation, largely because the DNA from dead bacterial cells is highly stable and hence, DNA-based amplification methods fail to discriminate between live and inactivated microorganisms. Currently, no published method has been shown to effectively distinguish between live and dead bacterial spores. This technology uses a DNA binding photoaffinity label that can be used to distinguish between live and dead bacterial spores with detection limits ranging from 109 to 102 spores/mL. An environmental sample suspected of containing a mixture of live and dead vegetative cells and bacterial endospores is treated with a photoaffinity label. This step will eliminate any vegetative cells (live or dead) and dead endospores present in the sample. To further determine the bacterial spore viability, DNA is extracted from the spores and total population is quantified by real-time PCR. The current NASA standard assay takes 72 hours for results. Part of this procedure requires a heat shock step at 80 degC for 15 minutes before the sample can be plated. Using a photoaffinity label would remove this step from the current assay as the label readily penetrates both live and dead bacterial cells. Secondly, the photoaffinity label can only penetrate dead bacterial spores, leaving behind the viable spore population. This would allow for rapid bacterial spore detection in a matter of hours compared to the several days that it takes for the NASA standard assay.

Behar, Alberto E.↗
NASA NTRS2017

Microbial Monitoring from the Frontlines to Space: Department of Defense Small Business Innovation Research Technology Aboard the International Space Station

The RAZOR (trademark) EX, a quantitative Polymerase Chain Reaction (qPCR) instrument, is a portable, ruggedized unit that was designed for the Department of Defense (DoD) with its reagent chemistries traceable to a Small Business Innovation Research (SBIR) contract beginning in 2002. The PCR instrument's primary function post 9/11 was to enable frontline soldiers and first responders to detect biological threat agents and bioterrorism activities in remote locations to include field environments. With its success for DoD, the instrument has also been employed by other governmental agencies including Department of Homeland Security (DHS). The RAZOR (Trademark) EX underwent stringent testing by the vendor, as well as through the DoD, and was certified in 2005. In addition, the RAZOR (trademark) EX passed DHS security sponsored Stakeholder Panel on Agent Detection Assays (SPADA) rigorous evaluation in 2011. The identification and quantitation of microbial pathogens is necessary both on the ground as well as during spaceflight to maintain the health of astronauts and to prevent biofouling of equipment. Currently, culture-based monitoring technology has been adequate for short-term spaceflight missions but may not be robust enough to meet the requirements for long-duration missions. During a NASA-sponsored workshop in 2011, it was determined that the more traditional culture-based method should be replaced or supplemented with more robust technologies. NASA scientists began investigating innovative molecular technologies for future space exploration and as a result, PCR was recommended. Shortly after, NASA sponsored market research in 2012 to identify and review current, commercial, cutting edge PCR technologies for potential applicability to spaceflight operations. Scientists identified and extensively evaluated three candidate technologies with the potential to function in microgravity. After a thorough voice-of-the-customer trade study and extensive functional and safety evaluations, the RAZOR (trademark) EX PCR instrument(Bio-Fire Defense, Salt Lake City, UT) was selected as the most promising current technology for spaceflight monitoring applications.

Cherie M Oubre↗
NASA NTRS1994

A Custom Robotic System for Inspecting HEPA Filters in the Payload Changeout Room at the NASA Kennedy Space Center

In this paper, the prime objective is to describe a custom 4-dof (degree-of-freedom) robotic arm capable of autonomously or telerobotically performing systematic HEPA filter inspection and certification in the Shuttle Launch Pad Payload Changeout Rooms (PCR's) on pads A and B at the Kennedy Space Center, Florida. This HEPA filter inspection robot (HFIR) has been designed to be easily deployable and is equipped with the necessary sensory devices, control hardware, software and man-machine interfaces needed to implement HEPA filter inspection reliably and efficiently without damaging the filters or colliding with existing PCR structures or filters. The main purpose of the HFIR is to implement an automated positioning system to move special inspection sensors in pre-defined or manual patterns for the purpose of verifying filter integrity and efficiency. This will ultimately relieve NASA Payload Operations from significant problems associated with time, cost and personnel safety, impacts realized during non-automated PCR HFIR filter certification.

Spencer, James E., Jr.↗
NASA NTRS1999

Latent Viruses: A Space Travel Hazard??

A major issue associated with long-duration space flight is the possibility of infectious disease causing an unacceptable medical risk to crew members. Our proposal is designed to gain information that addresses several issues outlined in the Immunology/Infectious disease critical path. The major hypothesis addressed is that space flight causes alterations in the immune system that may allow latent viruses which are endogenous in the human population to reactivate and shed to higher levels than normal which can affect the health of crew members during a long term space-flight mission. We will initially focus our studies on the human herpesviruses and human polyomaviruses which are important pathogens known to establish latent infections in the human population. Both primary infection and reactivation from latent infection with this group of viruses can cause a variety of illnesses that result in morbidity and occasionally mortality of infected individuals. Effective vaccines exist for only one of the eight known human herpesviruses and the vaccine itself can still reactivate from latent infection. Available antivirals are of limited use and are effective against only a few of the human herpesviruses. Although most individuals display little if any clinical consequences from latent infection, events which alter immune function such as immunosuppressive therapy following solid organ transplantation are known to increase the risk of developing complications as a result of latent virus reactivation. This proposal will measure both the frequency and magnitude of viral shedding and genome loads in the blood from humans participating in activities that serve as ground based models of space flight conditions. Our initial goal is to develop sensitive quantitative competitive PCR- based assays (QC-PCR) to detect the herpesvirus Epstein-Barr virus (EBV), and the polyomaviruses SV40, BKV, and JCV. Using these assays we will establish baseline patterns of viral genome load in the blood and viral shedding from normal volunteers in a longitudinal study over I year in length. As a comparison, we will measure patterns of viral genome loads and shedding from individuals who are severely immunosuppressed, in whom herpesvirus reactivation or primary infection with a herpesvirus is known to cause complications. In addition, we will proceed to testing ground based analogs in collaboration with Dr. Duane Pierson (Lyndon B. Johnson Space Center). This will include measuring samples obtained from individuals living and working in the extreme environment of Antarctica. We expect to detect viral shedding or reactivation from most of the test groups, although the magnitude of shedding or reactivation cannot be predicted. The data accumulated from studies in this proposal should allow us to evaluate whether events that simulate certain aspects of space flight reactivate viral infections severe enough in nature that they may compromise the success of long-term space flight missions. These studies will also provide a foundation to monitor viral reactivation and shedding from crew members participating in actual space flight missions. We will present data showing the establishment of our QC-PCR assay for detection of EBV.

Ling, P. D.↗
NASA NTRS2002

Energetics of muscle contraction: the whole is less than the sum of its parts

Understanding muscle energetics is a problem in optimizing supply of ATP to the demands of ATPases. The complexity of reactions and their fluxes to achieve this balance is greatly reduced by recognizing constraints imposed by the integration of common metabolites at fixed stoichiometry among modular units. ATPase is driven externally. Oxidative phosphorylation and glycogenolysis are the suppliers. We focus on their regulation which involves different controls, but reduces to two principles that enable facile experimental analysis of the supply and demand fluxes. The ratio of concentration of phosphocreatine (PCr) to ATP, not their individual values, sets the range of achievable concentrations of ADP in resting and active muscle (at fixed pH) in different cell types. This principle defines the fraction of available flux of oxidative phosphorylation utilized (at fixed enzyme activities). Then the kinetics of PCr recovery defines the kinetics of oxygen supply and substrate utilization. The second principle is the constancy of PCr and H(+) (lactate) production by glycogenolysis due to the coupling of ATPase and glycolysis. This principle enables glycogenolytic flux to be measured from intracellular proton loads. Further simplification occurs because the magnitude of the interacting fluxes and metabolite concentrations are specified within narrow limits when both the resting and active fluxes are quantified. Thus there is a small set of rules for assessing and understanding the thermodynamics and kinetics of muscle energetics.

NASA Program Biomedical Research and Countermeasur↗
NASA NTRS2007

Subclinical Reactivation and Shed of Infectious Varicella Zoster Virus in Saliva of Astronauts

We have previously detected VZV in healthy astronauts both during spaceflight and shortly after landing. Herein, we show that VZV shed in seropositive astronauts is infectious. A total of 40 saliva samples were obtained from each of the 3 astronauts. From each astronaut, 14 samples were taken 109 to 133 days before liftoff, 1 sample was taken every day during 12 days in space, and one sample was taken for 14 consecutive days beginning the second day after landing. Quantitative PCR was used to detect VZV DNA in saliva. None of 42 preflight saliva samples contained VZV DNA. VZV DNA was detected in saliva from 2 of 3 astronauts. In 1 astronaut, 6 of 12 samples obtained during space flight contained 120 to 2,500 copies of VZV DNA per ml; after landing, 1250 copies of VZV DNA were present on day 2, 45 copies on day 3, and 110 copies on day 5. All samples taken 6 to 15 days after touchdown were negative for VZV DNA. In the second astronaut, 5 of 12 samples obtained during space flight contained 18 to 650 copies of VZV DNA per ml; after landing, 560 copies of VZV DNA were present in saliva on day 2, 340 copies on day 4, 45 copies on day 5, and 23 copes on day 6. All samples taken 7 to 15 days after touchdown were negative for VZV DNA. Saliva taken 2 to 6 days after landing from all 3 astronauts was cultured on human fetal lung cells. After one subcultivation, a cytopathic effect developed in cultures inoculated with saliva from the two astronauts whose saliva contained VZV DNA. Both PCR and immunostaining identified the isolates to be VZV and not HSV-1. Importantly, the astronaut in whom no VZV was detected had a history of zoster 9 years earlier. It is possible that a boost in cell-mediated immunity to VZV which is known to develop after zoster protected him from subclinical reactivation. The genotype of the two VZV isolates was determined by VZV ORF22-based PCR/sequencing along with FRET-based PCR assays that target specific nucleotide polymorphisms. Both VZV isolates were found to be the European genotype which also contained a rare MspI restriction enodnuclease site in VZV ORF62 at position 107,252. These findings extend our previous demonstration of VZV DNA in saliva of astronauts by showing that infectious VZV is also present. Thus, like HSV-1 and HSV-2, VZV can reactivate and shed infectious virus in the absence of clinical disease.

Cohrs, Randall J.↗
NASA NTRS2008

Incidence of Latent Virus Shedding during Space Flight

Measurements of immune parameters of both cellular and innate immunity indicate alterations in immune function in astronauts. Immune changes are due to stress and perhaps other factors associated with launch, flight, and landing phases. Medical relevance of observed changes is not known. The reactivation of latent viruses has been identified as an important in vivo indicator of clinically relevant immune changes. The polymerase chain reaction (PCR) was used to detect the presence of specific viral DNA in body fluids. Initial studies demonstrated Epstein-Barr virus (EBV) reactivation during all 3 mission phases. EBV is shed in saliva following reactivation from B-cells. Incidence of EBV in saliva was higher than control subjects during all 3 mission phases. However, quantitative PCR revealed 10-fold higher levels of EBV DNA present in saliva collected during flight than found in pre- and post flight specimens. To determine if other latent viruses showed similar effects, cytomegalovirus (CMV), another herpes virus, shed in urine following reactivation was studied. A very low incidence (less than 2%) of CMV in urine is found in healthy, lowstressed individuals. However, 25-50% of astronauts shed CMV in their urine before, during, or after flight. Our studies are now focused on varicella-zoster virus (VZV), the etiological agent of chicken-pox during childhood and shingles later in life. We demonstrated reactivation of VZV and shedding of the virus during and after spaceflight in saliva of astronauts with no sign of active infection or symptoms. The maximum shedding of VZV occurred during the flight phase and diminishes rapidly during the first five days after landing. We have utilized the same PCR assay for VZV in a clinical study of shingles patients. Generally, shingles patients shed much more VZV in saliva than astronauts. However, the VZV levels in astronauts overlap with the lower range of VZV numbers in shingles patients. Saliva from shingles patients and astronauts were cultured and infectious VZV was recovered from both groups. We have concluded that multiple latent viruses do reactivate before, during, and after spaceflight and serve as very sensitive indicators for diminished cellular immunity. Future plans will be focused on the clinical risks posed by the reactivation of these viruses. Initial efforts will determine the effect of longer missions on the International Space Station on the reactivation patterns of these viruses.

Mehta, Satish K.↗
NASA NTRS2011

Standardization of Spore Inactivation Method for PMA-PhyloChip Analysis

In compliance with the Committee on Space Research (COSPAR) planetary protection policy, National Aeronautics and Space Administration (NASA) monitors the total microbial burden of spacecraft as a means for minimizing the inadvertent transfer of viable contaminant microorganisms to extraterrestrial environments (forward contamination). NASA standard assay-based counts are used both as a proxy for relative surface cleanliness and to estimate overall microbial burden as well as to assess whether forward planetary protection risk criteria are met for a given mission, which vary by the planetary body to be explored and whether or not life detection missions are present. Despite efforts to reduce presence of microorganisms from spacecraft prior to launch, microbes have been isolated from spacecraft and associated surfaces within the extreme conditions of clean room facilities using state of the art molecular technologies. Development of a more sensitive method that will better enumerate all viable microorganisms from spacecraft and associated surfaces could support future life detection missions. Current culture-based (NASA standard spore assay) and nucleic-acid-based polymerase chain reaction (PCR) methods have significant shortcomings in this type of analysis. The overall goal of this project is to evaluate and validate a new molecular method based on the use of a deoxyribonucleic acid (DNA) intercalating agent propidium monoazide (PMA). This is used in combination with DNA microarray (PhyloChip) which has been shown to identify very low levels of organisms on spacecraft associated surfaces. PMA can only penetrate the membrane of dead cells. Once penetrated, it intercalates the DNA and, upon photolysis using visible light it produces stable DNA monoadducts. This allows DNA to be unavailable for further PCR analysis. The specific aim of this study is to standardize the spore inactivation method for PMA-PhyloChip analysis. We have used the bacterial spores Bacillus subtilis 168 (standard laboratory isolate) as a test organism.

spore inactivation↗
NASA NTRS1989

An advanced telerobotic system for shuttle payload changeout room processing applications

To potentially alleviate the inherent difficulties in the ground processing of the Space Shuttle and its associated payloads, a teleoperated, semi-autonomous robotic processing system for the Payload Changeout Room (PCR) is now in the conceptual stages. The complete PCR robotic system as currently conceived is described and critical design issues and the required technologies are discussed.

Sklar, M.↗
NASA NTRS2002

Insertional Mutagenesis for Genes involved in Otic/Vestibular Development and Function in Xenopus Tropicalis

Sensitivity to gravity is essential for spatial orientation. Consequently, the gravity receptor system is one of the phylogenetically oldest sensory systems, and the special adaptations that enhance sensitivity to gravity are highly conserved. The main goal of this project is to use Xenopus (frog) to identify genes expressed during vestibular and auditory development. These studies will lead a better understanding of the molecular mechanisms involved in vestibular and auditory development and function. We are using a gene-trap approach in Xenopus tropicalis with the green fluorescent protein (GFP) gene as the transgene reporter. GFP expression occurs only when the GFP gene is correctly integrated in actively transcribed genes. Using the GFP as a tag we can easily identify and clone the mutated gene. In addition, we can study the function of the mutated gene by analyzing the defects generated by insertion of the GFP transgene. To date we have tissue specific GFP expression in X. tropicalis including expression in ear, neural tube, kidney, muscle, eyes and nose. Our transgenic animals will soon reach maturity so that we can outcross them and analyze their progeny. Our next goal is to isolate RNA from our transgenics and clone the tagged genes using RACE-PCR. Currently we are optimizing the RACE-PCR method using transgenics with crystallin GFP expression.

Torrejon, Marcela↗
NASA NTRS2003

Carbon Nanotube Nanoelectrode Array as an Electronic Chip for Ultrasensitive Label-free DNA Detection

A reliable nanoelectrode array based on vertically aligned multi-walled carbon nanotubes (MWNTs) embedded in SiO2 is used for ultrasensitive DNA detection. Characteristic nanoelectrode behavior is observed using low-density MWNT arrays for measuring both bulk and surface immobilized redox species such as K4Fe(CN)6 and ferrocene derivatives. The open-end of MWNTs are found to present similar properties as graphite edge-plane electrodes with wide potential window, flexible chemical functionalities, and good biocompatibility. BRCA1 related oligonucleotide probes with 18 bp are selectively functionalized at the open ends of the nanotube array and specifically hybridized with oligonucleotide targets incorporated with a polyG tag. The guanine groups are employed as the signal moieties in the electrochemical measurements. R(bpy)(sup 2+, sub 3) mediator is used to further amplify the guanine oxidation signal. The hybridization of sub-attomoles of DNA targets is detected electrochemically by combining the MWNT nanoelectrode array with the R(bpy)(sup 2+, sub 3) amplification mechanism. This technique was employed for direct electrochemical detection of label-free PCR amplicon from a healthy donor through specific hybridization with the BRCA1 probe. The detection limit is estimated to be less than 1000 DNA molecules since abundant guanine bases in the PCR amplicon provides a large signal. This system provides a general platform for rapid molecular diagnostics in applications requiring ultrahigh sensitivity, high-degree of miniaturization, and simple sample preparation, and low-cost operation.

Li, Jun↗