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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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All sourcesNASA NTRSDOE OSTI
NASA NTRS2021

Optimizing GRACE/GRACE-FO Data and A priori Hydrological Knowledge for Improved Global Terrestial Water Storage Component Estimates

The comprehensive information of global terrestrial water storage (TWS) components (soil moisture, groundwater, snow, surface water) is essential for effective assessment of water resource availability, climate variation, and disaster mitigation measures. Observational data provided by the Gravity Recovery And Climate Experiment (GRACE) and GRACE Follow-On satellite missions offer global TWS variation (ΔTWS) in terms of an integrated water column. However, GRACE spatial resolution is relatively coarse (i.e., 3°), and the vertically integrated value cannot be separated into ΔTWS components directly. This study demonstrates the feasibility to estimate ΔTWS components at any desired spatial-vertical resolution by effectively maintaining the native resolution of the employed hydrological knowledge. It utilizes a least-squares with constraints (LSC) approach to rigorously incorporate GRACE and GRACE-FO data and a priori hydrological knowledge, with the aim to improve global ΔTWS components’ accuracy and spatial resolution. The 3°×3° GRACE mascon derived ΔTWS data is disaggregated into the 0.5°×0.5° anomalous soil moisture storage (ΔSMS), groundwater storage (ΔGWS), snow water equivalent (ΔSWE), and surface water storage (ΔSWS) based on the covariance information obtained from the Community Atmosphere Biosphere Land Exchange (CABLE) and the PCRaster Global Water Balance (PCR-GLOBWB) models. Evaluation with different ground measurements and satellite products between 2002 and 2019 exhibits significantly improved accuracy in all individual ΔTWS components. This improvement is of particular note in ΔGWS and ΔSWS, where the LSC approach increases the globally averaged correlation values by approximately 0.13 and 0.05, respectively. Reliable prior knowledge leads to a more accurate ΔTWS component estimate, and the use of ensemble-mean knowledge yields the best result.

GRACE↗
NASA NTRSDate not supplied

The maize rough endosperm6 (rgh6) mutant encodes a predicted DEAD-box RNA helicase

Maize rough endosperm (rgh) mutants have defective seeds with a rough, etched, or pitted endosperm surface. Molecular genetic analysis of this mutant class has identified multiple RNA processing proteins critical to endosperm development. We isolated the rgh6 locus from the UniformMu transposon tagging population. Mutant kernels have reduced grain-fill with defective embryos that fail to germinate. Self-pollination of rgh6 heterozygotes produces mutant seeds at frequency consistent with a single recessive mutation. Molecular mapping localized rgh6 to a 60 kbp interval on chromosome 5. PCR analysis of gene models within the fine-map interval identified a Mutator (Mu) transposon insertion within a predicted DEAD-box RNA helicase gene. A second allele of rgh6 was identified from UniformMu population. Reciprocal crosses between rgh6-umu1 and rgh6-umu2 heterozygotes produces mutant seeds at a frequency consistent with a 3:1 ratio indicating these mutants are allelic. Both rgh6-umu1 and rgh6-umu2 have Mu element insertions at the same insertion site within the DEAD-box RNA helicase gene. However, the terminal inverted repeat sequences are polymorphic between the alleles showing the alleles are independent mutations. We conclude that mutation of the DEAD-box RNA helicase causes the rgh6 phenotype. Further characterization of the rgh6 mutant phenotype and biochemical investigation of the RGH6 protein is expected to give further insights on the roles of RNA processing in endosperm development.

maize↗
NASA NTRSDate not supplied

A physician’s working group guides COVID-19 prevention in a mission critical environment

Background A team was assembled at the National Aeronautics and Space Administration’s (NASA) Johnson Space Center (JSC) to help navigate the shifting COVID-19 pandemic. Their goal was to mitigate the impact of COVID on astronauts, other essential personnel, and the human spaceflight program. This team supplemented the JSC Pandemic Incident Response Group, whose function is to set policy for the overall management of JSC’s COVID-19 mitigations for all onsite mission-essential and mission-critical activities, including health policy. Context The first mission to return to Earth from the International Space Station at the start of the pandemic returned when there was limited information about the transmission and prevention of COVID-19. The medical leadership at JSC determined that a dedicated team was required to navigate the uncharted waters of the pandemic. Description of Activities Issues tackled by this panel included: prevention of COVID-19 transmission during altitude, dive, and spacecraft training – activities that require prolonged close contact between multiple individuals who often cannot safely wear face masks; mitigating the risks of domestic and international travel for essential personnel; securing COVID-19 testing from local and foreign hospital systems; acquiring antigen and PCR analyzers for in-house testing; and the development of quarantine policies for crewed spacecraft launches and landings in Kazakhstan and the United States. The panel also provides clinical return-to-work guidance for cases of COVID-19 in essential personnel.

covid↗
NASA NTRS2021

Impact of Recent Climate Extremes on Mosquito-borne Disease Transmission in Kenya

Climate change and variability influence temperature and rainfall, which impact vector abundance and the dynamics of vector-borne disease transmission. Climate change is projected to increase the frequency and intensity of extreme climate events. Mosquito-borne diseases, such as dengue fever, are primarily transmitted by Aedes aegypti mosquitoes. Freshwater availability and temperature affect dengue vector populations via a variety of biological processes and thus influence the ability of mosquitoes to effectively transmit disease. However, the effect of droughts, floods, heat waves, and cold waves is not well understood. Using vector, climate, and dengue disease data collected between 2013 and 2019 in Kenya, this retrospective cohort study aims to elucidate the impact of extreme rainfall and temperature on mosquito abundance and the risk of arboviral infections. To define extreme periods of rainfall and land surface temperature (LST), we calculated monthly anomalies as deviations from long-term means (1983–2019 for rainfall, 2000–2019 for LST) across four study locations in Kenya. We classified extreme climate events as the upper and lower 10% of these calculated LST or rainfall deviations. Monthly Ae. aegypti abundance was recorded in Kenya using four trapping methods. Blood samples were also collected from children with febrile illness presenting to four field sites and tested for dengue virus using an IgG enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). We found that mosquito eggs and adults were significantly more abundant one month following an abnormally wet month. The relationship between mosquito abundance and dengue risk follows a non-linear association. Our findings suggest that early warnings and targeted interventions during periods of abnormal rainfall and temperature, especially flooding, can potentially contribute to reductions in risk of viral transmission

Aedes aegypti mosquitoes↗
NASA NTRSDate not supplied

Method Development for Multiplex, In-Situ, and Real-Time Detection of Herpesvirus Reactivation in Spaceflight Crews using Nanopore Sequencing

Reactivation of latent herpesviruses in crews onboard the International Space Station (ISS) is a well-established spaceflight-associated phenomenon and has been linked to overall immune stress. Beyond an indicator of an altered immune state, this stress-induced reactivation of viruses such as herpesvirus simplex virus 1 (HSV-1), Epstein-Barr virus (EBV), and Varicella-Zoster virus (VZV) may cause clinical symptoms in crew. There is currently no established protocol for in-flight monitoring, and samples are analyzed post-flight using ground-based assays. A real-time, in-flight method for herpesvirus detection followed by stress-mitigation strategies would be a significant advance. In this study, we developed a real-time assay for the multiplex detection of HSV-1, EBV, and VZV from saliva that could be implemented for in-situ monitoring of ISS crew. This method builds on previously validated spaceflight-compatible portable molecular protocols and platforms, such as the miniPCR thermal cycler and the MinION sequencer. Since a metagenomic approach is not currently permitted for crew samples (NASA policy), we employed multiplexing-ready primers directed toward targeted regions within the HSV-1, EBV, and VZV genomes. We also used primers for the human gene, Statherin (STATH), as an internal control. These primers were validated using spiked-in, positive control HSV-1, EBV, and VZV from virus-negative saliva extracted using the Zymo-Research Quick-DNA/RNA Viral MagBead Kit. The PCR Barcoding Kit was used to prepare DNA libraries that were then sequenced on the MK1C and analyzed against known reference genomes using minimap2. Following validation of this method with spiked saliva samples, suspected herpesvirus-positive clinical saliva samples were successfully tested. Prior to use onboard the ISS, this method will be deployed to an analog environment during overwintering at Palmer Station, Antarctica in 2023. This work represents the successful application of molecular technologies currently onboard the ISS for the real-time monitoring of latent herpesvirus reactivation from saliva samples. This assay, in combination with stress-reduction strategies, holds the potential to manage herpesvirus reactivation in ISS crew, thereby improving health outcomes and overall immunity.

Patrick M. Rydzak↗
NASA NTRS2023

Electronic Nose Development and Preliminary Human Breath Testing for Rapid, Non-Invasive COVID-19 Detection

We adapted an existing, spaceflight-proven, robust “electronic nose” (E-Nose) that uses an array of electrical resistivity-based nanosensors mimicking aspects of mammalian olfaction to conduct on-site, rapid screening for COVID-19 infection by measuring the pattern of sensor responses to volatile organic compounds (VOCs) in exhaled human breath. We built and tested multiple copies of a hand-held prototype E-Nose sensor system, composed of 64 chemically sensitive nanomaterial sensing elements tailored to COVID-19 VOC detection; data acquisition electronics; a smart tablet with software (App) for sensor control, data acquisition and display; and a sampling fixture to capture exhaled breath samples and deliver them to the sensor array inside the E-Nose. The sensing elements detect the combination of VOCs typical in breath at parts-per-billion (ppb) levels, with repeatability of 0.02% and reproducibility of 1.2%; the measurement electronics in the E-Nose provide measurement accuracy and signal-to-noise ratios comparable to benchtop instrumentation. Preliminary clinical testing at Stanford Medicine with 63 participants, their COVID-19-positive or COVID-19-negative status determined by concomitant RT-PCR, discriminated between these two categories of human breath with a 79% correct identification rate using “leave-one-out” training-and-analysis methods. Analyzing the E-Nose response in conjunction with body temperature and other non-invasive symptom screening using advanced machine learning methods, with a much larger database of responses from a wider swath of the population, is expected to provide more accurate on-the-spot answers. Additional clinical testing, design refinement, and a mass manufacturing approach are the main steps toward deploying this technology to rapidly screen for active infection in clinics and hospitals, public and commercial venues, or at home.

COVID-19↗
NASA NTRS2022

Biological Oxidation of Fe(II)-Bearing Smectite by Microaerophilic Iron Oxidizer Sideroxydans lithotrophicus Using Dual Mto and Cyc2 Iron Oxidation Pathways

Fe(II) clays are common across many environments, making them a potentially significant microbial substrate, yet clays are not well established as an electron donor. Therefore, we explored whether Fe(II)-smectite supports the growth of Sideroxydans lithotrophicus ES-1, a microaerophilic Fe(II)-oxidizing bacterium (FeOB), using synthesized trioctahedral Fe(II)-smectite and 2% oxygen. S. lithotrophicus grew substantially and can oxidize Fe(II)-smectite to a higher extent than abiotic oxidation, based on X-ray near-edge spectroscopy (XANES). Sequential extraction showed that edge-Fe(II) is oxidized before interior-Fe(II) in both biotic and abiotic experiments. The resulting Fe(III) remains in smectite, as secondary minerals were not detected in biotic and abiotic oxidation products by XANES and Mössbauer spectroscopy. To determine the genes involved, we compared S. lithotrophicus grown on smectite versus Fe(II)-citrate using reverse-transcription quantitative PCR and found that cyc2 genes were highly expressed on both substrates, while mtoA was upregulated on smectite. Proteomics confirmed that Mto proteins were only expressed on smectite, indicating that ES-1 uses the Mto pathway to access solid Fe(II). We integrate our results into a biochemical and mineralogical model of microbial smectite oxidation. This work increases the known substrates for FeOB growth and expands the mechanisms of Fe(II)-smectite alteration in the environment.

smectite↗
NASA NTRS2022

Examining the Effects of 4He Exposure on the Gut-Brain Axis

Beyond low earth orbit, space radiation poses significant risks to astronaut health. Previous studies have shown that the microbial composition of the gastrointestinal (GI) microbiome changes upon exposure to high linear energy transfer radiation. Interestingly, radiation-induced shifts in GI microbiota composition are linked to various neuropsychological disorders. Herein, we aimed to study changes in GI microbiota and behaviors of rats exposed to whole-body radiation (0, 5, or 25 cGy 4 He, 250 MeV/n) at approximately 6 months of age. Fecal samples were collected 24 hours prior to 4 He radiation and 24 hours and 7 days post-exposure for quantitative PCR analyses to assess fecal levels of spore-forming bacteria (SFB), Bifidobacterium, Lactobacillus, and Akkermansia. Rats were also tested in the social odor recognition memory (SORM) test at 7 days following 4 He exposure. A subset of rats was euthanized 90 minutes following completion of the SORM test, and GI tissue from small intestine to colon were prepared for examining overall histological changes and immunohistochemical staining for serotonin (5-HT). No notable pathological changes were observed in GI tissues. Akkermansia spp. and SFB were significantly decreased the 25 cGy group at 24 hours and 7 days post-exposure compared to pre-exposure, respectively. Bifidobacterium and Lactobacillus spp. showed no significant changes. 5-HT production was significantly higher in the proximal small intestine and the cecum in the 25 cGy group compared to the sham group. The 25 cGy group exhibited deficits in recognition in SORM testing at 7 days post-exposure. Taken together, these results suggest a connection between GI microbiome 3 composition, serotonin production, and neurobehavioral performance, and that this connection may be disrupted upon exposure to 25 cGy of 4 He ions

Carli B Jones↗
NASA NTRSDate not supplied

Efficacy of Dried Plum to Countermeasure Simulated Spaceflight Exposure

Human exploration of the space environment has been a topic of interest for decades; however, the threat to physiological health posed by ionizing radiation and microgravity effects remains a concern. Previous research utilizing combined hindlimb unloading and irradiation to simulate spaceflight effects found that a dried plum diet can mitigate adverse effects of simulated spaceflight on skeletal health. Though the mechanism is not fully understood we believe this protective effect is due to its high antioxidant capacity and polyphenolic content. This study seeks to extend these findings to investigate if dried plum is an effective countermeasure in other tissues relating to aging and neurotoxicity. Male C57BL/6 J mice were assigned to one of eight groups (n = 10/group) and were fed either a controlled diet or dried plum diet, underwent normal loading or hindlimb unloading as well as exposure 2 Gy gamma radiation or sham exposure. At the end of the experiment, mice were euthanized, and RNA was extracted from brain and bone marrow tissue and PCR was employed to analyze significant changes in gene expression profiles. Analysis of five house-keeping genes revealed genes B2M (neurotoxicity) and GUSB (aging) to be the most stable genes across all treatments and were used to find the fold change within our genes of interest. Through this, and analysis via ANOVA, we were able to identify genes with significant altered expression levels within brain and bone marrow tissues related to neurotoxicity and aging. Considering previous research, we hypothesize that the dried plum diet’s antioxidative properties ameliorated the oxidative stress from irradiation and microgravity via altered levels of gene expression. Identified expression changes will provide insights into molecular mechanisms behind the observed protective effects. These findings present potential avenues for future interventions to alleviate negative impacts of spaceflight on astronaut health.

Faisal Ahmad↗
NASA NTRSDate not supplied

Gene Expression of Peripheral Blood Mononuclear Cells of Crew Members During Long-Duration Space Missions Indicate Dysregulation of Immunological and Cell Survival Mechanisms

Lymphocytes are naturally exposed to genotoxic stresses. DNA damage occurs during the entire lymphocyte’s life span and is induced mainly by reactive oxygen species (ROS), replication fork collapse, or telomere shortening during the immune response or intense cell proliferation phases. Strong evidence for the influence of immune function on DNA repair comes from studies of SCID disease. SCID mice not only have a deficient V(D)J recombination but are also unable to repair double strand breaks, leading to increased radiation sensitivity. The leukocytes’ transcriptome of 8 ISS crew members revels a dysregulated immune function and activation of cellular survival pathways in response to space environment. We have performed PCR analysis in peripheral mononuclear cells from the same crew members. A list of 62 genes were carefully selected addressing immunological and cell survival pathways. Differentially expressed genes indicated changes in chemokine receptor activity, chemokine binding, toll-like receptors, adhesion molecules and cellular response to DNA damage.

Maria Moreno-Villanueva↗
NASA NTRSDate not supplied

Strategies for Determining Safety of Fermented Foods Produced in Space

The BioNutrients experiments examine the feasibility of using microbially produced foods – yogurt, kefir, fermented food products– to supplement the current prepackaged food system to provide freshly synthesized nutrients and genetically engineered protein therapeutics to support crew health for sustainable and long duration crewed exploration. Safety of the products must be established as direct consumption of such products is a risk due to potential contamination with pathogenic organisms. Identifying pathogen contamination is particularly challenging as the fermenting cultures can mask the presence of contaminating organisms. Here we consider current methods of detecting coliforms, aerobic colonies, non-lactic acid bacteria, molds (yeasts), and specific pathogens including Staphylococcus aureus and salmonella. We also consider alternate and additional testing including sequencing and quantitative PCR. We investigate the use of the E-Nose developed by NASA Ames Research Center. This portable device senses volatile organic compounds in real time and can be trained to recognize “good” uncontaminated products from contaminated ones as a means of predicting food safety. Finally, we consider methods of in-flight pasteurization to increase safety and decrease overall microbial load of these products. This presentation will summarize testing status and outline the strategy that will be employed in the BioNutrients-3 ISS fermented foods experiment.

Natalie Ball↗
NASA NTRSDate not supplied

The Efficacy of Dried Plum to Counter Spaceflight Microgravity and Radiation

Human exploration of the space environment has been a topic of interest for decades; however, the threat to physiological health posed by ionizing radiation and microgravity effects remains a concern. Previous research utilizing combined hindlimb unloading and irradiation to simulate spaceflight effects found that a dried plum diet can mitigate adverse effects of simulated spaceflight on skeletal health. Though the mechanism is not fully understood we believe this protective effect is due to its high antioxidant capacity and polyphenolic content. This study seeks to extend these findings to investigate if dried plum is an effective countermeasure in other tissues relating to aging and neurotoxicity. Male C57BL/6 J mice were assigned to one of eight groups (n = 10/group) and were fed either a controlled diet or dried plum diet, underwent normal loading or hindlimb unloading as well as exposure 2 Gy gamma radiation or sham exposure. At the end of the experiment, mice were euthanized, and RNA was extracted from brain and bone marrow tissue and PCR was employed to analyze significant changes in gene expression profiles. Analysis of five house-keeping genes revealed genes B2M (neurotoxicity) and GUSB (aging) to be the most stable genes across all treatments and were used to find the fold change within our genes of interest. Through this, and analysis via ANOVA, we were able to identify genes with significant altered expression levels within brain and bone marrow tissues related to neurotoxicity and aging. Considering previous research, we hypothesize that the dried plum diet’s antioxidative properties ameliorated the oxidative stress from irradiation and microgravity via altered levels of gene expression. Identified expression changes will provide insights into molecular mechanisms behind the observed protective effects. These findings present potential avenues for future interventions to alleviate negative impacts of spaceflight on astronaut health.

Faisal Ahmad↗
NASA NTRSDate not supplied

Strategies for Determining Safety of Fermented Foods Produced in Space

The BioNutrients experiments examine the feasibility of using microbially produced foods – yogurt, kefir, fermented food products– to supplement the current prepackaged food system to provide freshly synthesized nutrients and genetically engineered protein therapeutics to support crew health for sustainable and long duration crewed exploration. Safety of the products must be established as direct consumption of such products is a risk due to potential contamination with pathogenic organisms. Identifying pathogen contamination is particularly challenging as the fermenting cultures can mask the presence of contaminating organisms. Here we consider current methods of detecting coliforms, aerobic colonies, non-lactic acid bacteria, molds (yeasts), and specific pathogens including Staphylococcus aureus and salmonella. We also consider alternate and additional testing including sequencing and quantitative PCR. We investigate the use of the E-Nose developed by NASA Ames Research Center. This portable device senses volatile organic compounds in real time and can be trained to recognize “good” uncontaminated products from contaminated ones as a means of predicting food safety. Finally, we consider methods of in-flight pasteurization to increase safety and decrease overall microbial load of these products. This presentation will summarize testing status and outline the strategy that will be employed in the BioNutrients-3 ISS fermented foods experiment.

Food↗
NASA NTRS2024

Gene Expression of Peripheral Blood Mononuclear Cells of Crew Members During Long-Duration Space Missions Indicate Dysregulation of Immunological and Cell Survival Mechanisms

Lymphocytes are naturally exposed to genotoxic stresses. DNA damage occurs during the entire lymphocyte’s life span and is induced mainly by reactive oxygen species (ROS), replication fork collapse, or telomere shortening during the immune response or intense cell proliferation phases. Strong evidence for the influence of immune function on DNA repair comes from studies of SCID disease. SCID mice not only have a deficient V(D)J recombination but are also unable to repair double strand breaks, leading to increased radiation sensitivity. The leukocytes’ transcriptome of 8 ISS crew members revels a dysregulated immune function and activation of cellular survival pathways in response to space environment. We have performed PCR analysis in peripheral mononuclear cells from the same crew members. A list of 62 genes were carefully selected addressing immunological and cell survival pathways. Differentially expressed genes indicated changes in chemokine receptor activity, chemokine binding, toll-like receptors, adhesion molecules and cellular response to DNA damage.

María Moreno-Villanueva↗
NASA NTRSDate not supplied

Validation of Multisystem Countermeasures Protocol for Spaceflight during Antarctica Winter-over at Palmer Station (Palmer Countermeasures)

Stressors associated with spaceflight induce persistent immune compromise in astronauts which increase subclinical latent virus reactivation. In select crews, adverse clinical events have been documented. Antarctica winter-over (AWO) mission most closely reproduces these mission stressors: prolonged deployment, extreme environment, circadian misalignment, isolation, station lifestyle, and personal risk. The US maintains three primary stations in Antarctica: South Pole Station, McMurdo, and Palmer. Previous studies suggest that stations located near the interior of Antarctica (South Pole, McMurdo) have confounding effects on the immune system due to persistent hypobaric hypoxia. We hypothesized that winter-over at a coastal station (Palmer) would be more akin to spaceflight due to its normoxic but still extreme environment. Therefore, AWO at Palmer Station was selected, and validated in a pilot study [2], as the platform for testing and validating the effectiveness of an immune-restorative countermeasure protocol designed for deep space missions. Specifics include diet modifications, nutritional supplementation (vitamin D, probiotic, etc.), prescribed aerobic and resistive exercise, and a protocol of stress relieving virtual reality exercises. A multitude of biological sample types, including blood, saliva, and hair will be collected in tandem with the countermeasures in order to examine the combined effectiveness of the countermeasures. Samples and logs from subjects will be transported from Palmer Station to Johnson Space Center for further processing and distribution to co-investigators at the end of each winter-over. Extracted samples will be analyzed by appropriate testing platforms (Multiplex, qPCR, ELISA, etc.) to monitor alterations in leukocyte distribution, T cell and NK function, cytokine profiles, reactivation of latent herpesviruses, and nutritional factors. The data collected will be compared to a control year in which no countermeasures were deployed to evaluate the overall effectiveness of the analog and to validate the candidate immune countermeasure strategy. AWO 2023 concluded with the 4th in-mission timepoint conducted in September 2023. Samples for 16 subjects, including blood, saliva, hair, surveys, and PCR data, were all successfully returned from Antarctica to NASA/JSC mid-November 2023. Samples have since been distributed to co-investigators for further processing and analysis. With the completion of the first countermeasure year, preliminary data on the effectiveness of the deep-space protocol is being evaluated, however, no conclusions can be drawn yet until the completion of the second AWO countermeasure year, AWO 2024. AWO 2024 commenced in late-March 2024, with 13 subjects consenting and performing their baseline data collections (BDCs). Unique to the 2024 deployment, NSF lifted certain COVID restrictions and rallied the crewmembers in Punta Arenas, Chile. All NSF activities were transferred to this location and NASA was allowed, for the first time, to perform consent briefings, baseline samplings and training in person. This augment greatly increased the likelihood of success for the overwinter activities.

Cody L Gutierrez↗
NASA NTRSDate not supplied

Validation of Multisystem Countermeasures Protocol for Spaceflight during Antarctica Winter-over at Palmer Station (Palmer Countermeasures)

Stressors associated with spaceflight induce persistent immune compromise in astronauts which increase subclinical latent virus reactivation. In select crews, adverse clinical events have been documented. Antarctica winter-over (AWO) mission most closely reproduces these mission stressors: prolonged deployment, extreme environment, circadian misalignment, isolation, station lifestyle, and personal risk. The US maintains three primary stations in Antarctica: South Pole Station, McMurdo, and Palmer. Previous studies suggest that stations located near the interior of Antarctica (South Pole, McMurdo) have confounding effects on the immune system due to persistent hypobaric hypoxia. We hypothesized that winter-over at a coastal station (Palmer) would be more akin to spaceflight due to its normoxic but still extreme environment. Therefore, AWO at Palmer Station was selected, and validated in a pilot study, as the platform for testing and validating the effectiveness of an immune-restorative countermeasure protocol designed for deep space missions. Specifics include diet modifications, nutritional supplementation (vitamin D, probiotic, etc.), prescribed aerobic and resistive exercise, and a protocol of stress relieving virtual reality exercises. A multitude of biological sample types, including blood, saliva, and hair will be collected in tandem with the countermeasures in order to examine the combined effectiveness of the countermeasures. Samples and logs from subjects will be transported from Palmer Station to Johnson Space Center for further processing and distribution to co-investigators at the end of each winter-over. Extracted samples will be analyzed by appropriate testing platforms (Multiplex, qPCR, ELISA, etc.) to monitor alterations in leukocyte distribution, T cell and NK function, cytokine profiles, reactivation of latent herpesviruses, and nutritional factors. The data collected will be compared to a control year in which no countermeasures were deployed to evaluate the overall effectiveness of the analog and to validate the candidate immune countermeasure strategy. AWO 2023 concluded with the 4th in-mission timepoint conducted in September 2023. Samples for 16 subjects, including blood, saliva, hair, surveys, and PCR data, were all successfully returned from Antarctica to NASA/JSC mid-November 2023. Samples have since been distributed to co-investigators for further processing and analysis. With the completion of the first countermeasure year, preliminary data on the effectiveness of the deep-space protocol is being evaluated, however, no conclusions can be drawn yet until the completion of the second AWO countermeasure year, AWO 2024. AWO 2024 commenced in late-March 2024, with 13 subjects consenting and performing their baseline data collections (BDCs). Unique to the 2024 deployment, NSF lifted certain COVID restrictions and rallied the crewmembers in Punta Arenas, Chile. All NSF activities were transferred to this location and NASA was allowed, for the first time, to perform consent briefings, baseline samplings and training in person. This augment greatly increased the likelihood of success for the overwinter activities.

Cody L Gutierrez↗
DOE OSTI2024

CRISPR-Cas9/Cas12a systems for efficient genome editing and large genomic fragment deletions in Aspergillus niger

CRISPR technology has revolutionized fungal genetic engineering by accelerating the pace and expanding the feasible scope of experiments in this field. Among various CRISPR-Cas systems, Cas9 and Cas12a are widely used in genetic and metabolic engineering. In filamentous fungi, both Cas9 and Cas12a have been utilized as CRISPR nucleases. In this work we first compared efficacies and types of genetic edits for CRISPR-Cas9 and -Cas12a systems at the polyketide synthase (albA) gene locus in Aspergillus niger. By employing a tRNA-based gRNA polycistronic cassette, both Cas9 and Cas12a have demonstrated equally remarkable editing efficacy. Cas12a showed potential superiority over Cas9 protein when one gRNA was used for targeting, achieving an editing efficiency of 86.5% compared to 31.7% for Cas9. Moreover, when employing two gRNAs for targeting, both systems achieved up to 100% editing efficiency for single gene editing. In addition, the CRISPR-Cas9 system has been reported to induce large genomic deletions in various species. However, its use for engineering large chromosomal segments deletions in filamentous fungi still requires optimization. Here, we engineered Cas9 and -Cas12a-induced large genomic fragment deletions by targeting various genomic regions of A. niger ranging from 3.5 kb to 40 kb. Our findings demonstrate that targeted engineering of large chromosomal segments can be achieved, with deletions of up to 69.1% efficiency. Furthermore, by targeting a secondary metabolite gene cluster, we show that fragments over 100 kb can be efficiently and specifically deleted using the CRISPR-Cas9 or -Cas12a system. Overall, in this paper, we present an efficient multi-gRNA genome editing system utilizing Cas9 or Cas12a that enables highly efficient targeted editing of genes and large chromosomal regions in A. niger.

59 BASIC BIOLOGICAL SCIENCES↗