Collin E. LeFrois, Mingqi Zhou, D. Amador, Natasha J. L. Sng, A. Paul, R. Ferl
Abstract Spaceflight has a unique set of abiotic conditions to which plants respond by orchestrating genome-wide alterations to their transcriptome. The methods for preserving plants for RNA analysis are well-established and proven over multiple missions, but, methods for investigating the possible epigenetic mechanisms that may contribute to the transcriptome alteration are not well-developed for the confining limitations of the International Space Station (ISS). Currently, the methods used to isolate genomic DNA and to perform epigenetic analyses are ideal for frozen plants, as opposed to plants stored in RNAlater®—a high salt solution that chemically suspends all cellular activity and is typically used on the ISS. Therefore, we developed a method for extracting high-quality genomic DNA suitable for epigenetic analysis from Arabidopsis thaliana (Arabidopsis) plants that were preserved with the current preservation system aboard the ISS—fixation in RNAlater® using Kennedy Space Center Fixation Tubes (KFTs).
{"title":"Enabling the Spaceflight Methylome: DNA Isolated from Plant Tissues Preserved in RNAlater® Is Suitable for Bisulfite PCR Assay of Genome Methylation","authors":"Collin E. LeFrois, Mingqi Zhou, D. Amador, Natasha J. L. Sng, A. Paul, R. Ferl","doi":"10.2478/GSR-2016-0010","DOIUrl":"https://doi.org/10.2478/GSR-2016-0010","url":null,"abstract":"Abstract Spaceflight has a unique set of abiotic conditions to which plants respond by orchestrating genome-wide alterations to their transcriptome. The methods for preserving plants for RNA analysis are well-established and proven over multiple missions, but, methods for investigating the possible epigenetic mechanisms that may contribute to the transcriptome alteration are not well-developed for the confining limitations of the International Space Station (ISS). Currently, the methods used to isolate genomic DNA and to perform epigenetic analyses are ideal for frozen plants, as opposed to plants stored in RNAlater®—a high salt solution that chemically suspends all cellular activity and is typically used on the ISS. Therefore, we developed a method for extracting high-quality genomic DNA suitable for epigenetic analysis from Arabidopsis thaliana (Arabidopsis) plants that were preserved with the current preservation system aboard the ISS—fixation in RNAlater® using Kennedy Space Center Fixation Tubes (KFTs).","PeriodicalId":90510,"journal":{"name":"Gravitational and space research : publication of the American Society for Gravitational and Space Research","volume":"28 1","pages":"28 - 37"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83480603","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Abstract Spaceflight experiments offer a unique environment for fundamental research in biology. Utilization of microgravity environments has provided insights into how plants and animals perceive and respond to gravity, or the lack thereof. However, performing spaceflight experiments on the International Space Station (ISS) requires years of planning and testing before execution. A few of the complex steps preceding the experiment include: development of the experimental timeline and programming of experimental equipment, testing hardware for biocompatibility, planning the logistics of sending samples to NASA or ESA centers for testing, and launching samples to the ISS. In this paper, using the Seedling Growth-2 spaceflight experiment as an example, we cover the entire timeline leading up to a flight experiment. These events include the Schedule Test, the Operations and Validations Test (OVT), and the Flight Build and Experiment, as well as the post-flight sample processing.
{"title":"Spaceflight Procedures and Operations Utilized During the Seedling Growth Experiments","authors":"Joshua Vandenbrink, J. Kiss","doi":"10.2478/GSR-2016-0011","DOIUrl":"https://doi.org/10.2478/GSR-2016-0011","url":null,"abstract":"Abstract Spaceflight experiments offer a unique environment for fundamental research in biology. Utilization of microgravity environments has provided insights into how plants and animals perceive and respond to gravity, or the lack thereof. However, performing spaceflight experiments on the International Space Station (ISS) requires years of planning and testing before execution. A few of the complex steps preceding the experiment include: development of the experimental timeline and programming of experimental equipment, testing hardware for biocompatibility, planning the logistics of sending samples to NASA or ESA centers for testing, and launching samples to the ISS. In this paper, using the Seedling Growth-2 spaceflight experiment as an example, we cover the entire timeline leading up to a flight experiment. These events include the Schedule Test, the Operations and Validations Test (OVT), and the Flight Build and Experiment, as well as the post-flight sample processing.","PeriodicalId":90510,"journal":{"name":"Gravitational and space research : publication of the American Society for Gravitational and Space Research","volume":"39 1","pages":"38 - 46"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73369771","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sarahann Hutchinson, Proma Basu, S. Wyatt, D. R. Luesse
Abstract Large-scale omics approaches make excellent choices for research aboard the International Space Station (ISS) because they provide large amounts of data that can be continually mined even after the original research has been completed. A proteomic approach can provide information about which proteins are produced, degraded, or post-translationally modified, potentially shedding light on cellular strategies that cannot be discerned from transcriptomic data. To collect sufficient tissue from a Biological Research In Canisters (BRIC)-grown experiment on the ISS for proteomic analysis, several modifications were made to existing protocols. Approximately 800–1000 seeds were housed in each Petri Dish Fixation Units (PDFU). These were germinated up to 120 h after planting by transferring the BRIC from cold stasis to room temperature. Growth continued for only 72 h after germination to allow sufficient tissue for extraction, and to minimize the impact of ethylene and crowding stress. Seedlings were then exposed to RNAlater®. Results indicate that RNAlater® - treated Arabidopsis seedlings yield an equal amount of protein to those flash-frozen in liquid nitrogen.
{"title":"Methods for On-Orbit Germination of Arabidopsis thaliana for Proteomic Analysis","authors":"Sarahann Hutchinson, Proma Basu, S. Wyatt, D. R. Luesse","doi":"10.2478/GSR-2016-0009","DOIUrl":"https://doi.org/10.2478/GSR-2016-0009","url":null,"abstract":"Abstract Large-scale omics approaches make excellent choices for research aboard the International Space Station (ISS) because they provide large amounts of data that can be continually mined even after the original research has been completed. A proteomic approach can provide information about which proteins are produced, degraded, or post-translationally modified, potentially shedding light on cellular strategies that cannot be discerned from transcriptomic data. To collect sufficient tissue from a Biological Research In Canisters (BRIC)-grown experiment on the ISS for proteomic analysis, several modifications were made to existing protocols. Approximately 800–1000 seeds were housed in each Petri Dish Fixation Units (PDFU). These were germinated up to 120 h after planting by transferring the BRIC from cold stasis to room temperature. Growth continued for only 72 h after germination to allow sufficient tissue for extraction, and to minimize the impact of ethylene and crowding stress. Seedlings were then exposed to RNAlater®. Results indicate that RNAlater® - treated Arabidopsis seedlings yield an equal amount of protein to those flash-frozen in liquid nitrogen.","PeriodicalId":90510,"journal":{"name":"Gravitational and space research : publication of the American Society for Gravitational and Space Research","volume":"174 1","pages":"20 - 27"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86459172","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Abstract The NASA GeneLab Data System (GLDS) was recently developed to facilitate cross-experiment comparisons in order to understand the response of microorganisms to the human spaceflight environment. However, prior spaceflight experiments have been conducted using a wide variety of different hardware, media, culture conditions, and procedures. Such confounding factors could potentially mask true differences in gene expression between spaceflight and ground control samples. In an attempt to mitigate such confounding factors, we describe here the development of a standardized set of hardware, media, and protocols for liquid cultivation of microbes in Biological Research in Canisters (BRIC) spaceflight hardware, using the model bacteria Bacillus subtilis strain 168 and Staphylococcus aureus strain UAMS-1 as examples.
{"title":"Establishing Standard Protocols for Bacterial Culture in Biological Research in Canisters (BRIC) Hardware","authors":"P. Fajardo-Cavazos, W. Nicholson","doi":"10.2478/gsr-2016-0013","DOIUrl":"https://doi.org/10.2478/gsr-2016-0013","url":null,"abstract":"Abstract The NASA GeneLab Data System (GLDS) was recently developed to facilitate cross-experiment comparisons in order to understand the response of microorganisms to the human spaceflight environment. However, prior spaceflight experiments have been conducted using a wide variety of different hardware, media, culture conditions, and procedures. Such confounding factors could potentially mask true differences in gene expression between spaceflight and ground control samples. In an attempt to mitigate such confounding factors, we describe here the development of a standardized set of hardware, media, and protocols for liquid cultivation of microbes in Biological Research in Canisters (BRIC) spaceflight hardware, using the model bacteria Bacillus subtilis strain 168 and Staphylococcus aureus strain UAMS-1 as examples.","PeriodicalId":90510,"journal":{"name":"Gravitational and space research : publication of the American Society for Gravitational and Space Research","volume":"2015 1","pages":"58 - 69"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87087776","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
C. Fitzgerald, Richard Barker, Won-Gyu Choi, S. Swanson, S. D. Stephens, Colleen Huber, A. Nimunkar, S. Gilroy
Abstract In order to use plants as part of a bioregenerative life support system capable of sustaining long-term human habitation in space, it is critical to understand how plants adapt to the stresses associated with extended growth in spaceflight. Optimally, dormant seeds would be germinated on orbit to divorce the effects of spaceflight from the one-time stresses of launch. At an operational level, it is also important to develop experiment protocols that are flexible in timing so they can adapt to crew schedules and unexpected flight-related delays. Arabidopsis thaliana is widely used for investigating the molecular responses of plants to spaceflight. Here we describe the development of a far-red light seed treatment device that suppresses germination of Arabidopsis seeds for periods of ≥12 weeks. Germination can then be induced when the seeds encounter red light, such as transfer to the illumination from on orbit plant growth hardware. This device allows for up to twelve 10×10 cm square Petri dishes containing seeds on nutrient gel to be irradiated simultaneously. The far-red device is contained within a light-proof fabric tent allowing the user to wrap the Petri dishes in aluminum foil in the dark, preventing room lights from reversing the far-red treatment. Long-term storage of the wrapped plates is accomplished using foil storage bags. The throughput of this device facilitates robust, high-replicate biological experiment design, while providing the long-term pre-experiment storage required for maximum mission flexibility.
{"title":"Development of Equipment that Uses Far-Red Light to Impose Seed Dormancy in Arabidopsis for Spaceflight","authors":"C. Fitzgerald, Richard Barker, Won-Gyu Choi, S. Swanson, S. D. Stephens, Colleen Huber, A. Nimunkar, S. Gilroy","doi":"10.2478/GSR-2016-0008","DOIUrl":"https://doi.org/10.2478/GSR-2016-0008","url":null,"abstract":"Abstract In order to use plants as part of a bioregenerative life support system capable of sustaining long-term human habitation in space, it is critical to understand how plants adapt to the stresses associated with extended growth in spaceflight. Optimally, dormant seeds would be germinated on orbit to divorce the effects of spaceflight from the one-time stresses of launch. At an operational level, it is also important to develop experiment protocols that are flexible in timing so they can adapt to crew schedules and unexpected flight-related delays. Arabidopsis thaliana is widely used for investigating the molecular responses of plants to spaceflight. Here we describe the development of a far-red light seed treatment device that suppresses germination of Arabidopsis seeds for periods of ≥12 weeks. Germination can then be induced when the seeds encounter red light, such as transfer to the illumination from on orbit plant growth hardware. This device allows for up to twelve 10×10 cm square Petri dishes containing seeds on nutrient gel to be irradiated simultaneously. The far-red device is contained within a light-proof fabric tent allowing the user to wrap the Petri dishes in aluminum foil in the dark, preventing room lights from reversing the far-red treatment. Long-term storage of the wrapped plates is accomplished using foil storage bags. The throughput of this device facilitates robust, high-replicate biological experiment design, while providing the long-term pre-experiment storage required for maximum mission flexibility.","PeriodicalId":90510,"journal":{"name":"Gravitational and space research : publication of the American Society for Gravitational and Space Research","volume":"16 1","pages":"19 - 8"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85036133","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Prashant J. Parmar, R. Perry, Greta M. Cesarz, Alex Roberts, Houston Hardman, J. Caruso
Abstract The deleterious effects of spaceflight encompass numerous physiological effects that undermine long-term goals of manned round-trip missions to Mars. Among the greater losses are to the human musculoskeletal system due to limited mechanical/load-bearing activity. In-flight exercise and nutritional countermeasures seek to reduce physiological losses. Restoration of mechanical/load-bearing activity in microgravity is achieved with flywheel-based exercise hardware. Research with spaceflight analogs showed exercise done with flywheel-based devices abated muscle mass and strength losses with modest increases in net energy costs. This led to the installment of flywheel-based hardware on The International Space Station (ISS). To date, exercise with flywheel-based hardware has reduced musculoskeletal losses, with more success achieved for muscle-, versus bone-based, outcomes. In-flight exercise may better address bone losses with hardware that imparts high rates of impulse loading to the engaged musculoskeleton.
{"title":"Physiological Effects of Spaceflight/Unloading and the Mitigating Effects of Flywheel-Based Resistive Exercise","authors":"Prashant J. Parmar, R. Perry, Greta M. Cesarz, Alex Roberts, Houston Hardman, J. Caruso","doi":"10.2478/gsr-2016-0006","DOIUrl":"https://doi.org/10.2478/gsr-2016-0006","url":null,"abstract":"Abstract The deleterious effects of spaceflight encompass numerous physiological effects that undermine long-term goals of manned round-trip missions to Mars. Among the greater losses are to the human musculoskeletal system due to limited mechanical/load-bearing activity. In-flight exercise and nutritional countermeasures seek to reduce physiological losses. Restoration of mechanical/load-bearing activity in microgravity is achieved with flywheel-based exercise hardware. Research with spaceflight analogs showed exercise done with flywheel-based devices abated muscle mass and strength losses with modest increases in net energy costs. This led to the installment of flywheel-based hardware on The International Space Station (ISS). To date, exercise with flywheel-based hardware has reduced musculoskeletal losses, with more success achieved for muscle-, versus bone-based, outcomes. In-flight exercise may better address bone losses with hardware that imparts high rates of impulse loading to the engaged musculoskeleton.","PeriodicalId":90510,"journal":{"name":"Gravitational and space research : publication of the American Society for Gravitational and Space Research","volume":"34 1","pages":"64 - 77"},"PeriodicalIF":0.0,"publicationDate":"2016-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72964816","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Abstract Recently, the increase in incidence of cardiovascular degeneration associated with weightlessness has drawn much attention to the detrimental effects of space travel on cardiovascular health. Particularly, the regulatory role of the endothelium in cardiovascular degeneration has been studied extensively. The goal of this study was to understand the effects of simulated microgravity on the proliferative, secretory, and anti-thrombogenic functions of endothelial cells differentiated from human blood-derived progenitor cells. Exposure to simulated microgravity enhanced proliferation, as well as the release of soluble nitric oxide while downregulating the release of pro-inflammatory cytokines, such as interleukin-6 (IL-6). Interestingly, the cells also upregulated gene expression of heat shock protein 70 (hsp70), which may be a potential adaptation mechanism of the cells to altered gravity conditions. However, the secretory and proliferative functions had no effect on the anti-thrombogenic functions of these cells. Their anti-coagulative and anti-thrombogenic abilities, as assessed by both upregulation of tissue plasminogen activator (tPA) and their ability to delay plasma clotting, were impaired on exposure to simulated microgravity. These results collectively provide a useful insight into various mechanisms involved in regulating anti-thrombogenic ability of the endothelium, as well as cardiovascular health in altered gravity conditions.
{"title":"Altered Functions of Human Blood-Derived Vascular Endothelial Cells by Simulated Microgravity","authors":"Vidhya Ramaswamy, A. Goins, J. Allen","doi":"10.2478/GSR-2016-0001","DOIUrl":"https://doi.org/10.2478/GSR-2016-0001","url":null,"abstract":"Abstract Recently, the increase in incidence of cardiovascular degeneration associated with weightlessness has drawn much attention to the detrimental effects of space travel on cardiovascular health. Particularly, the regulatory role of the endothelium in cardiovascular degeneration has been studied extensively. The goal of this study was to understand the effects of simulated microgravity on the proliferative, secretory, and anti-thrombogenic functions of endothelial cells differentiated from human blood-derived progenitor cells. Exposure to simulated microgravity enhanced proliferation, as well as the release of soluble nitric oxide while downregulating the release of pro-inflammatory cytokines, such as interleukin-6 (IL-6). Interestingly, the cells also upregulated gene expression of heat shock protein 70 (hsp70), which may be a potential adaptation mechanism of the cells to altered gravity conditions. However, the secretory and proliferative functions had no effect on the anti-thrombogenic functions of these cells. Their anti-coagulative and anti-thrombogenic abilities, as assessed by both upregulation of tissue plasminogen activator (tPA) and their ability to delay plasma clotting, were impaired on exposure to simulated microgravity. These results collectively provide a useful insight into various mechanisms involved in regulating anti-thrombogenic ability of the endothelium, as well as cardiovascular health in altered gravity conditions.","PeriodicalId":90510,"journal":{"name":"Gravitational and space research : publication of the American Society for Gravitational and Space Research","volume":"11 1","pages":"16 - 2"},"PeriodicalIF":0.0,"publicationDate":"2016-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90787235","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
P. Neuhaus, Chris Jumonville, R. Perry, R. Edwards, Jake L. Martin, Ahlam M. Alarbi, W. Potter, J. Caruso
Abstract To assess the comparative similarity of squat data collected as they wore a robotic exoskeleton, female athletes (n=14) did two exercise bouts spaced 14 days apart. Data from their exoskeleton workout was compared to a session they did with free weights. Each squat workout entailed a four-set, four-repetition paradigm with 60-second rest periods. Sets for each workout involved progressively heavier (22.5, 34, 45.5, 57 kg) loads. The same physiological, perceptual, and exercise performance dependent variables were measured and collected from both workouts. Per dependent variable, Pearson correlation coefficients, t-tests, and Cohen's d effect size compared the degree of similarity between values obtained from the exoskeleton and free weight workouts. Results show peak O2, heart rate, and peak force data produced the least variability. In contrast, far more inter-workout variability was noted for peak velocity, peak power, and electromyography (EMG) values. Overall, an insufficient amount of comparative similarity exists for data collected from both workouts. Due to the limited data similarity, the exoskeleton does not exhibit an acceptable degree of validity. Likely the cause for the limited similarity was due to the brief amount of familiarization subjects had to the exoskeleton prior to actual data collection. A familiarization session that accustomed subjects to squats done with the exoskeleton prior to actual data collection may have considerably improved the validity of data obtained from that device.
{"title":"Comparative Responses to Squats Completed with Free Weights and an Exoskeleton","authors":"P. Neuhaus, Chris Jumonville, R. Perry, R. Edwards, Jake L. Martin, Ahlam M. Alarbi, W. Potter, J. Caruso","doi":"10.2478/gsr-2016-0005","DOIUrl":"https://doi.org/10.2478/gsr-2016-0005","url":null,"abstract":"Abstract To assess the comparative similarity of squat data collected as they wore a robotic exoskeleton, female athletes (n=14) did two exercise bouts spaced 14 days apart. Data from their exoskeleton workout was compared to a session they did with free weights. Each squat workout entailed a four-set, four-repetition paradigm with 60-second rest periods. Sets for each workout involved progressively heavier (22.5, 34, 45.5, 57 kg) loads. The same physiological, perceptual, and exercise performance dependent variables were measured and collected from both workouts. Per dependent variable, Pearson correlation coefficients, t-tests, and Cohen's d effect size compared the degree of similarity between values obtained from the exoskeleton and free weight workouts. Results show peak O2, heart rate, and peak force data produced the least variability. In contrast, far more inter-workout variability was noted for peak velocity, peak power, and electromyography (EMG) values. Overall, an insufficient amount of comparative similarity exists for data collected from both workouts. Due to the limited data similarity, the exoskeleton does not exhibit an acceptable degree of validity. Likely the cause for the limited similarity was due to the brief amount of familiarization subjects had to the exoskeleton prior to actual data collection. A familiarization session that accustomed subjects to squats done with the exoskeleton prior to actual data collection may have considerably improved the validity of data obtained from that device.","PeriodicalId":90510,"journal":{"name":"Gravitational and space research : publication of the American Society for Gravitational and Space Research","volume":"86 1","pages":"51 - 63"},"PeriodicalIF":0.0,"publicationDate":"2016-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79928609","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
T. Hammond, L. Stodieck, P. Koenig, J. Hammond, Margaret A. Gunter, P. Allen, H. Birdsall
Abstract To evaluate effects of microgravity on virulence, we studied the ability of four common clinical pathogens—Klebsiella, Streptococcus, Proteus, and Pseudomonas—to kill wild type Caenorhabditis elegans (C. elegans) nematodes at the larval and adult stages. Simultaneous studies were performed utilizing spaceflight, rotation in a 2D clinorotation device, and static ground controls. Nematodes, microbes, and growth media were separated until exposed to true or modeled microgravity, then mixed and grown for 48 hours. Experiments were terminated by paraformaldehyde fixation, and optical density measurements were used to assay residual microorganisms. Spaceflight was associated with reduced virulence for Klebsiella and Streptococcus, but had negligible effect on Enterococcus and Pseudomonas. Clinorotation generated very different results with all four organisms showing significantly reduced virulence. We conclude that clinorotation is not a consistent model of the changes that actually occur under microgravity conditions. Further, bacteria virulence is unchanged or reduced, not increased during spaceflight.
{"title":"Effects of Microgravity and Clinorotation on the Virulence of Klebsiella, Streptococcus, Proteus, and Pseudomonas","authors":"T. Hammond, L. Stodieck, P. Koenig, J. Hammond, Margaret A. Gunter, P. Allen, H. Birdsall","doi":"10.2478/GSR-2016-0004","DOIUrl":"https://doi.org/10.2478/GSR-2016-0004","url":null,"abstract":"Abstract To evaluate effects of microgravity on virulence, we studied the ability of four common clinical pathogens—Klebsiella, Streptococcus, Proteus, and Pseudomonas—to kill wild type Caenorhabditis elegans (C. elegans) nematodes at the larval and adult stages. Simultaneous studies were performed utilizing spaceflight, rotation in a 2D clinorotation device, and static ground controls. Nematodes, microbes, and growth media were separated until exposed to true or modeled microgravity, then mixed and grown for 48 hours. Experiments were terminated by paraformaldehyde fixation, and optical density measurements were used to assay residual microorganisms. Spaceflight was associated with reduced virulence for Klebsiella and Streptococcus, but had negligible effect on Enterococcus and Pseudomonas. Clinorotation generated very different results with all four organisms showing significantly reduced virulence. We conclude that clinorotation is not a consistent model of the changes that actually occur under microgravity conditions. Further, bacteria virulence is unchanged or reduced, not increased during spaceflight.","PeriodicalId":90510,"journal":{"name":"Gravitational and space research : publication of the American Society for Gravitational and Space Research","volume":"81 1","pages":"39 - 50"},"PeriodicalIF":0.0,"publicationDate":"2016-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90387852","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
H. Birdsall, P. Allen, J. Hammond, Margaret A. Gunter, T. Hammond
Abstract Giant yeast colonies develop a low redox potential, which mimics the electrophilic milieu of both the mitochondrial drug metabolizing compartment and the hypoxic core of many tumors. The major metabolic mediators of low redox potential include: ATP, glutathione, NAD+/NADH, and NADP+/NADPH. Ammonia signaling is the critical mechanism that induces stratification of the giant yeast colonies to allow a low redox potential. A comparison of two powerful investigative models for drug pathways using Saccharomyces cerevisiae have been compounded by the use of different growth media and stimuli to the system. Chemogenetic profiling, which uses a pool of yeast deletion mutants to determine survival changes, is heavily slanted to the use of rich media. Giant yeast colonies studies are heavily slanted to the use of poor media. The current study answers the question “what is the difference over time in redox potential, and its major metabolic mediators, between giant yeast colonies grown on rich and poor media?” Using gene deletion tools, we show that cell death in giant yeast colonies is ammonia-dependent. In poor nutrient, ammonia-depleted (Sok2 deletion mutants) giant yeast cultures, rotation can allow manipulation of reactive oxygen species, providing a model to compare high and low redox states without chemical administration. Mechanistically, these changes are not due to detectable NAD/NAPH or NADP/NADPH changes, but are related in changes in glutathione and ATP concentration.
{"title":"Establishing a Low Redox Potential in Giant Yeast Colonies: Effects of Media and Rotation","authors":"H. Birdsall, P. Allen, J. Hammond, Margaret A. Gunter, T. Hammond","doi":"10.2478/GSR-2016-0003","DOIUrl":"https://doi.org/10.2478/GSR-2016-0003","url":null,"abstract":"Abstract Giant yeast colonies develop a low redox potential, which mimics the electrophilic milieu of both the mitochondrial drug metabolizing compartment and the hypoxic core of many tumors. The major metabolic mediators of low redox potential include: ATP, glutathione, NAD+/NADH, and NADP+/NADPH. Ammonia signaling is the critical mechanism that induces stratification of the giant yeast colonies to allow a low redox potential. A comparison of two powerful investigative models for drug pathways using Saccharomyces cerevisiae have been compounded by the use of different growth media and stimuli to the system. Chemogenetic profiling, which uses a pool of yeast deletion mutants to determine survival changes, is heavily slanted to the use of rich media. Giant yeast colonies studies are heavily slanted to the use of poor media. The current study answers the question “what is the difference over time in redox potential, and its major metabolic mediators, between giant yeast colonies grown on rich and poor media?” Using gene deletion tools, we show that cell death in giant yeast colonies is ammonia-dependent. In poor nutrient, ammonia-depleted (Sok2 deletion mutants) giant yeast cultures, rotation can allow manipulation of reactive oxygen species, providing a model to compare high and low redox states without chemical administration. Mechanistically, these changes are not due to detectable NAD/NAPH or NADP/NADPH changes, but are related in changes in glutathione and ATP concentration.","PeriodicalId":90510,"journal":{"name":"Gravitational and space research : publication of the American Society for Gravitational and Space Research","volume":"1 1","pages":"27 - 38"},"PeriodicalIF":0.0,"publicationDate":"2016-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84936943","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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