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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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At least 235 records · Page 13
All sourcesNASA NTRSDOE OSTI
NASA NTRS2003

The Zebrafish G12 Gene is required for Nuclear Positioning and Cell Migrations during Early Development

After fertilization Zebrafish embryos undergo synchronous cleavage to form a blastula of cells sitting upon a single multinucleate yolk cell. At the beginning of gastrulation these cells undergo extensive cell migrations to form the major body axes. We have discovered a gene, G12, which is required for cell migrations and positioning of nuclei in the large syncytial yolk cell. Overexpression of a G12-GFP fusion protein is not toxic and shows that the protein localizes inside the yolk cell to the yolk nuclei, microtubules, and to the margin between the blastomeres and the large yolk cell. Morpholino (MO) injection into the 1-cell embryo or into just the yolk syncytium conipletely inhibits cell migrations, doming of the yolk cell, and positioning of nuclei around the margin. This effect can be partially rescued by injection of G12-GFP encoding RNA. Given the known role of microtubules in nuclear positioning of yolk nuclei in Zebrafish, we investigated the microtubules in morpholiiio injected and rescued embryos. We find that microtubules are sparse and disorganized in MO-injected embryos and are restored to normal organization upon G12-GFP rescue. G12 plays a pivotal role in organization of inicrotubules during early development. G12 is highly conserved in vertebrates and two homologues exist in the human genome. One of the human hoinologues is amplified in aggressive breast tumors.

Reinsch, S. S.↗
NASA NTRS2004

Modeled microgravity-induced protein kinase C isoform expression in human lymphocytes

In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited in both microgravity and modeled microgravity (MMG) as reflected by diminished DNA synthesis in peripheral blood lymphocytes and their locomotion through gelled type I collagen. Direct activation of protein kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas the calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 1 g and MMG culture. Human lymphocytes were cultured and harvested at 24, 48, 72, and 96 h, and serial samples were assessed for locomotion by using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta, and -epsilon was assessed by RT-PCR, flow cytometry, and immunoblotting. Results indicated that PKC isoforms delta and epsilon were downregulated by >50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 1-g controls. Events upstream of PKC, such as phosphorylation of phospholipase Cgamma in MMG, revealed accumulation of inactive enzyme. Depressed calcium-independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than PKC, but after ligand-receptor interaction.

Lymphocytes/enzymology↗
NASA NTRS2005

SAFER Inspection of Space Shuttle Thermal Protection System

In the aftermath of the space shuttle Columbia accident, it quickly became clear that new methods would need to be developed that would provide the capability to inspect and repair the shuttle's thermal protection system (TPS). A boom extension to the Remote Manipulator System (RMS) with a laser topography sensor package was identified as the primary means for measuring the damage depth in acreage tile as well as scanning Reinforced Carbon- Carbon (RCC) surfaces. However, concern over the system's fault tolerance made it prudent to investigate alternate means of acquiring close range photographs and contour depth measurements in the event of a failure. One method that was identified early was to use the Simplified Aid For EVA Rescue (SAFER) propulsion system to allow EVA access to damaged areas of concern. Several issues were identified as potential hazards to SAFER use for this operation. First, the ability of an astronaut to maintain controlled flight depends upon efficient technique and hardware reliability. If either of these is insufficient during flight operations, a safety tether must be used to rescue the crewmember. This operation can jeopardize the integrity of the Extra-vehicular Mobility Unit (EMU) or delicate TPS materials. Controls were developed to prevent the likelihood of requiring a tether rescue, and procedures were written to maximize the chances for success if it cannot be avoided. Crewmember ability to manage tether cable tension during nominal flight also had to be evaluated to ensure it would not negatively affect propellant consumption. Second, although propellant consumption, flight control, orbital dynamics, and flight complexity can all be accurately evaluated in Virtual Reality (VR) Laboratory at Johnson Space Center, there are some shortcomings. As a crewmember's hand is extended to simulate measurement of tile damage, it will pass through the vehicle without resistance. In reality, this force will push the crewmember away from the vehicle, and could induce a moment which, if strong enough, could saturate the attitude control system in SAFER. This raises the concern that additional propellant will be consumed to maintain controlled flight. To account for this, the fidelity of the Virtual Reality simulation was improved to include the effect of crewmember contact with the vehicle during SAFER flight. In addition, while participating in VR simulations, the subject is in shirt sleeves and sits in a chair. This does not provide a flight-like representation of body position awareness. To prevent inadvertent contact with tile or RCC, other facilities were utilized to establish crew preferences for body attitude and tool configuration. Finally, a study was performed to determine if attitude constraints are needed for the Space shuttle and International Space Station to reduce SAFER flight difficulty.

Scoville, Zebulon C.↗
NASA NTRS2008

Sea State and Weather Capability for NASA's Constellation Program

Marine weather and related parameters such as wind, ocean wave height and period, air temperature, sea surface temperature, visibility, and potential for icing are critical to the design, operation, and safety of crewed space vehicles. The National Aeronautics and Space Administration's (NASA's) Constellation Program requires detailed assessment of marine weather related parameters that may be encountered during launch, abort, landing, and crew rescue operations for the crewed Axes/Orion space vehicles. This information is required for both space vehicle design and operational purposes. The space vehicles must be designed such that they cam withstand the environment they are likely to encounter. The crewed Axes/Orion space vehicles will launch from NASA's Kennedy Space Center (KSC), Florida for both International Space Station (ISS) missions with 51.6deg inclination orbits and lunar missions with approximately 280 inclination orbits. Since both missions will fly ever the Atlantic Ocean on ascent to orbit and will fly over the Pacific Ocean on descent from orbit, an unlikely but possible emergency abort could require parachuting the Orion capsule and crew into the ocean. This situation could potentially put the crew in an isolated and hazardous environment for severn hours while they await rescue. Therefore, abort, landing, and crew rescue elements of the Constellation Program must address weather related parameters on a global scale. This paper describes buoy measurement data, sea surface temperature satellite data, and sea state computer model data that are being utilized by the Constellation Program to address these design and operational issues.

Barbre, R. E.↗
NASA NTRS2008

Sea State and Weather Assessment Capability for NASA's Constellation Program

Marine weather and related parameters such as wind, ocean wave height and period, air temperature, sea surface temperature, visibility, and potential for icing are critical to the design, operation, and safety of crewed space vehicles. The National Aeronautics and Space Administration's (NASA's) Constellation Program requires detailed assessment of marine weather related parameters that may be encountered during launch, abort, landing, and crew rescue operations for the crewed Ares/Orion space vehicles. This information is required for both space vehicle design and operational purposes. The space vehicles must be designed such that they can withstand the environment they are likely to encounter. The crewed Ares/Orion space vehicles will launch from NASA's Kennedy Space Center (KSC), Florida for both International Space Station (ISS) missions with 51.6 o inclination orbits and lunar missions with approximately 28 inclination orbits. Since both missions will fly over the Atlantic Ocean on ascent to orbit and will fly over the Pacific Ocean on descent from orbit, an unlikely but possible emergency abort could require parachuting the Orion capsule and crew into the ocean. This situation could potentially put the crew in an isolated and hazardous environment for several hours while they await rescue. Therefore, abort, landing, and crew rescue elements of the Constellation Program must address weather related parameters on a global scale. This paper describes buoy measurement data, sea surface temperature satellite data, and sea state computer model data that are being utilized by the Constellation Program to address these design and operational issues.

Barbre, B. J.↗
NASA NTRS2003

Modeled Microgravity-Induced Protein Kinase C Isoform Expression in Human Lymphocytes

In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited both in microgravity and modeled microgravity (MMG) as reflected in diminished DNA synthess in peripheral blood lymphocytes and their locomotion through gelled type 1 collagen. Direct activation of Protein Kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 19 and MMG-culture. Human lymphocytes were cultured and harvested at 24, 48, 72 and 96 hours and serial samples assessed for locomotion using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta and -epsilon was assessed by RT-PCR, flow cytometry and immunoblotting. Results indicated that PKC isoforms delta and epsilon were down-regulated by more than 50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 19 controls. Events upstream of PKC such as phosphorylation of Phospholipase C(gamma) (PLC-gamma) in MMG, revealed accumulation of inactive enzyme. Depressed Ca++ -independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than, but after ligand-receptor interaction. Keywords: Signal transduction, locomotion, immunity

Sundaresan, A.↗
NASA NTRS2012

13 Things That Saved Apollo 13

Perhaps, the most exciting rescue, terrestrial or extra-terrestrial, is the successful return of the Apollo 13 crew to Earth in April of 1970. The mission s warning system engineer, Jerry Woodfill, who remains a NASA employee after 47 years of government service has examined facets of the rescue for the past 42 years. He will present "13 Things That Saved Apollo 13" from the perspective of his real time experience as well as two score years of study. Many are recent discoveries never before published in mission reports, popular books or documentary and Hollywood movies depicting the rescue.

Woodfill, Jared↗
NASA NTRS2012

Hands-Free Control Interfaces for an Extra Vehicular Jetpack

The National Aeronautics and Space Administration (NASA) strategic vision includes, as part of its long-term goals, the exploration of deep space and Near Earth Asteroids (NEA). To support these endeavors, funds have been invested in research to develop advanced exploration capabilities. To enable the human mobility necessary to effectively explore NEA and deep space, a new extravehicular activity (EVA) Jetpack is under development at the Johnson Space Center. The new design leverages knowledge and experience gained from the current astronaut rescue device, the Simplified Aid for EVA Rescue (SAFER). Whereas the primary goal for a rescue device is to return the crew to a safe haven, in-space exploration and navigation requires an expanded set of capabilities. To accommodate the range of tasks astronauts may be expected to perform while utilizing the Jetpack, it was desired to offer a hands-free method of control. This paper describes the development and innovations involved in creating two hands-free control interfaces and an experimental test platform for a suited astronaut flying the Jetpack during an EVA.

Zumbado, Jennifer Rochlis↗
NASA NTRS2016

Preserving, Enhancing, and Continuing the Scientific Legacy of the Apollo Sample Suite

From 1969 to 1972, Apollo astronauts collected 382 kg of rocks, soils, and core samples from six geologically diverse locations on the Moon. In the nearly 50 years since the samples were collected, over 3000 different studies have been conducted using the nearly 2200 different Apollo samples. Despite the maturity of the sample collection, many new studies of lunar samples are undertaken each year, with an average of more than 55 requests and more than 600 distinct subsamples allocated annually over the past five years. The Apollo samples are a finite resource, however. Although new studies are encouraged, it is important that new studies do not duplicate previous studies, and where possible, leverage previous results to inform and enhance the current studies. This helps to preserve the samples and scientific funding, both of which are precious resources. We have initiated several new efforts to rescue some of the early analyses from these samples, including unpublished analytical data. We are actively scanning NASA documentation in paper form that is related to the Apollo missions and sample processing, and we are collaborating with IEDA to establish a geochemical data base called MoonDB. To populate this database, we are actively working with about a dozen prominent lunar PIs to organize and transcribe years of both published and unpublished data, making it available to all researchers. This effort will also take advantage of new online analytical tools like PetDB. There have already been tangible results from the MoonDB data rescue effort. A pilot project involving the rescue of geochemical data of John Delano on Apollo pyroclastic glasses has already been referenced in multiple Apollo sample requests, and in fact, the compiled data was used as part of one of the new studies. Similarly, scanned sample handling reports have been utilized to find previously analyzed samples that were appropriate to fulfill new sample requests. We have also begun to image the Apollo samples using (1) micro‐CT scanning to document the interior structure of samples, and (2) comprehensive high resolution photography of samples, enabling high resolution 3D reconstructions of the samples. Both efforts will provide comprehensive access to these samples and allow for more targeted requests, and thus better curation of the samples for decades to come.

Zeigler, Ryan↗
NASA NTRSDate not supplied

The Wizard Staff: A Mobile Tool Supporting Human Exploration on The Lunar Surface

The Wizard Staff is a rechargeable, battery powered mobile tool with multiple uses for lunar surface exploration. It supports human activity as a surface survey tool, provides external illumination to enhance safety, delivers networking, communications, and navigation features to remote sites, and assists crew rescue with an emergency beacon and rigid splint structure. As a survey tool, it assists crewed geologic field site exploration with external lights, cameras, and LiDAR to illuminate, map, and record the sample collection process. As a relay station, it provides a surface intranet communications and navigation node for extended exploration far from the lander or habitation module. A separately developed Search-and-Rescue (SAR) handheld device can broadcast a signal to the Gandalf Staff containing crew position and biometric data. The staff then formats the message and sends it orbiting satellite(s) for emergency response via the LunaNET on a dedicated frequency similar to terrestrial applications using an Emergency Position-Indicating Radiobeacon (EPIRB). The staff is designed with an upper and lower segment. Components in the upper segment are the LiDAR, external lighting arrays, cameras, communications equipment, intelligence devices, and user interface for crew operations. The lower segment is a set of FeLiPO4 battery cells packed into the 2” diameter carbon fiber tube. When the battery pack is discharged, a spring connector (designed for the dusty lunar environment) easily disconnects the two segments for replacement with another fully charged battery pack. The lower segment can be used for Incapacitated Crew Rescue (ICR) as a rigid splint. As a stand-alone science platform, the Wizard Staff provides infrastructure for instruments and sensors to record the lunar environment over long periods of time. After a crew installs the components on the lunar surface, the platform delivers data to the lunar lander or base station for analysis. The stand-alone mode requires an auxiliary power source (e.g. solar array) and energy storage system (e.g. battery), so it could also be used as an electrical recharging station. A working prototype of Wizard Staff has been developed with collaboration between NASA (JSC, MSFC, and GSFC), Texas Space Technology Applications and Research (T STAR), and Texas A&M University (TAMU) using $300k of innovation funding in FY’21 and FY’22. The functional staff weights 36# on Earth (6# on the Moon), stands 2.1m tall and delivers external lighting with an array of 40 LED bulbs generating 1460 Lux at 2m range (near field), or 2 Lux at 50m (far field).

Gandalf Staff↗
NASA NTRSDate not supplied

EVA Planning: Using Neutral Buoyancy Laboratory (NBL) Training to Predict in-Flight Energy Expenditure

Metabolic rate (“met rate”) is the amount of energy expended over a period of time and is influenced by many factors including body composition, level of physical activity, resting metabolic rate, sex, age, and food intake. Met rate is measured during Extravehicular Activity (EVA) training at the Neutral Buoyancy Laboratory (NBL) and during in-flight EVAs through indirect calorimetry, calculating energy expenditure from respiratory measurements of O 2 consumption and/or CO 2 production. During Extravehicular Activity (EVA) planning, metabolic cost is important to consider and is used to inform EVA duration based on spacesuit consumables associated with life support systems. Currently, NBL and previous ISS EVA met rate data for specified crewmembers are utilized to predict in-flight EVA metabolic costs based on a proposed EVA timeline. Timeline data collected during training is used to relate met rates to specific EVA activities, which are in turn assigned to more generalized EVA task categories, categorizing by both task type and restraint type. EVA task categories include EVA Setup/Cleanup, Worksite Setup/Cleanup, Cable Routing, Bolts, Fluid Connectors, Electrical Connectors, R&R Work, Miscellaneous Work, Incapacitated Crew Rescue (being rescued or performing), Assisted Crew Rescue (being assisted or performing), and Translation. Restraint types consist of Free-Float, Body Restraint Tether (BRT), Articulating Portable Foot Restraint (APFR), and Space Station Remote Manipulator System (SSRMS). From a crewmember’s historical data, individualized 10th, 50th and 90th percentile met rate estimates are generated for each task category and used to estimate the proposed EVA timeline metabolic cost. In-flight metabolic data (“As-Executed”) from recent ISS US EVAs 85-88 (totaling eight EVA crewmember met rates) was compared with their predicted metabolic cost (“As-Planned”) to evaluate the accuracy of the current met rate estimation method. Across the four EVAs, As-Executed Cumulative EVA Total Metabolic Cost (M = 5893.78 BTU, SD = 816.60) was not significantly different compared to As-Planned Cumulative EVA Total Metabolic Cost (M = 6106.60 BTU, SD = 826.62; t(7) = 0.751 , p = .477). Though not a significant difference, generally, As-Planned total estimates were slightly higher than As-Executed total metabolic cost. Relative Error for Cumulative EVA Total Metabolic Cost ranged from -33% to 14.7%, depending on the crewmember and EVA. When comparing As-Planned to A-Executed EVA task categories for Bolts, Electrical Connectors, EVA Cleanup, EVA Setup, Miscellaneous Work, Translation, Worksite Cleanup, and Worksite Setup during these EVAs, no significant differences were observed, however, there was a significant difference in As-Planned (M = 783.71 BTU, SD = 408.88) compared to As-Executed (M = 608.87 BTU, SD = 425.29) metabolic cost for the task category of Repair-and-Replace (R&R) Work (t(17) = 3.21 , p = .005). Looking closer within the R&R Work task category, As-Executed R&R Work with Free-Float restraint type (M = 706.17 BTU, SD = 352.18) was significantly less than As-Planned R&R Work with Free-Float restraint type (M= 887.96 BTU, SD = 381.42; t(12) = 2.59, p < .024). As-Executed R&R Work with SSRMS Restraint type (M = 355.89 BTU, SD = 534.65) was not significantly different from As-Planned values (M = 512.69 BTU, SD = 383.34; t(4) = 1.88, p = 0.132). These findings suggest that the energy expended performing R&R Work (Free-Float) is lower in flight than predicted. Accurate predictions of the metabolic cost of EVA are essential for planning and executing successful ISS EVAs. Overall, the current met rate prediction method is similar to actual in-flight values, slightly erring on the side of overestimation. Future work includes analysis of more historical in-flight EVA data to increase the power of the analysis, evaluating the NBL-ISS met rate conversion factor between NBL and ISS tasks, as well as exploring methods of substitution when crewmembers are missing prior task category data.

Lauren Cox↗
DOE OSTI2022

DNA Sequence Analysis of an Inversion Hot Spot in Lobeliaceae Plastomes

The evolution of plastid genomes (plastomes) in land plants is typically conservative, with extensive structural rearrangements present in only a few groups. Early Southern blot analysis identified two Lobelia species that minimally required deletion of the plastid gene accD and five inversions to account for their plastome arrangement relative to the ancestral organization. Sixty alternative 5-step inversion scenarios could account for the observed arrangement, but only one scenario was consistent with the criterion of ‘common cause’ attributable to a putative rearrangement hot spot at the accD deletion-site. Plastome sequencing demonstrated that this previously hypothesized inversion order is historically accurate. Detailed reconstructions of the ancestral plastome organization before and after each inversion are presented herein. Stem-loop and disruption-rescue models were evaluated for each inversion. One inversion has an obvious stem-loop basis, but the other four inversions were primarily caused by serial insertion of foreign (extra-plastid) DNA bearing large open-reading frames that disrupted plastome organization at the accD deletion-site, and complete plastomes were rescued by seemingly arbitrary ligation or fortuitous recombination at the other inversion endpoint. Transposed copies of DNA segments from elsewhere in the plastome are frequently inserted at inversion junctions, and four junctions are consistent with the stem-loop ligation model.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The mitochondrially targeted peptide elamipretide (SS-31) improves ADP sensitivity in aged mitochondria by increasing uptake through the adenine nucleotide translocator (ANT)

Aging muscle experiences functional decline in part mediated by impaired mitochondrial ADP sensitivity. Elamipretide (ELAM) rapidly improves physiological and mitochondrial function in aging and binds directly to the mitochondrial ADP transporter ANT. We hypothesized that ELAM improves ADP sensitivity in aging leading to rescued physiological function. We measured the response to ADP stimulation in young and old muscle mitochondria with ELAM treatment, in vivo heart and muscle function, and compared protein abundance, phosphorylation, and S-glutathionylation of ADP/ATP pathway proteins. ELAM treatment increased ADP sensitivity in old muscle mitochondria by increasing uptake of ADP through the ANT and rescued muscle force and heart systolic function. Protein abundance in the ADP/ATP transport and synthesis pathway was unchanged, but ELAM treatment decreased protein s-glutathionylation incuding of ANT. Mitochondrial ADP sensitivity is rapidly modifiable. This research supports the hypothesis that ELAM improves ANT function in aging and links mitochondrial ADP sensitivity to physiological function.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

RAN and YBX1 are required for cell proliferation and IL-4 expression and linked to poor prognosis in oral squamous cell carcinoma

Highlights: • RAN and YBX1 are co-expressed and required for cell proliferation in OSCC cells. • RAN and YBX1 are positively associated with the immune infiltrates of CD4{sup +} Th2 cells. • RAN and YBX1 can promote IL-4 expression. • RAN is a direct target of YBX1. • RAN and YBX1 can serve as potential co-biomarkers for poor prognosis in OSCC. Oral squamous cell carcinoma (OSCC) is one of the most common malignancies in the world, with a high mortality rate. RAN is a member of the Ras GTPase family and is overexpressed in a range of cancers, however, the relationship between RAN and OSCC is rarely reported. In this study, we found that RAN is overexpressed in OSCC tissues. RAN inhibition retarded OSCC cell proliferation and led to apoptosis and cell cycle arrest. Knockdown of RAN inhibited tumor growth in vivo. Strikingly, we found that RAN and oncogene Y-box binding protein-1 (YBX1) are positively associated with the immune infiltrates of CD4{sup +} Th2 cells in multiple types of cancer, and can promote IL-4 expression. IL-4 treatment can partially rescue RAN knockdown-induced cell apoptosis in OSCC cells. Moreover, overexpression of RAN could rescue cell growth inhibition caused by knockdown of YBX1. Furthermore, patients with low expression of both RAN and YBX1 had better overall survival than others. Collectively, these findings indicate that RAN is a target of YBX1. RAN and YBX1 are required for cell proliferation and IL-4 expression. RAN and YBX1 are co-expressed and can serve as potential co-biomarkers for poor prognosis in OSCC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Selenomethionine protects oxidative-stress-damaged bone-marrow-derived mesenchymal stem cells via an antioxidant effect and the PTEN/PI3K/AKT pathway

Highlights: • Oxidative stress stimuli by H{sub 2}O{sub 2} suppressed osteogenic differentiation of BMSCs. • Selenomethionine could alleviate osteogenic dysfunction of oxidative-stress-damaged BMSCs. • Selenomethionine rescued osteogenic ability partly through the modulation of the PTEN/PI3K/AKT pathway. Dental implant surgery is currently a routine therapy for the repair of missing dentition or dentition defects. Both clinical and basic research have elucidated that oxidative stress caused by the accumulation of reactive oxygen species (ROS) for various reasons impairs the process of osteointegration after dental implantation. Therefore, the osteogenic micro-environment must be ameliorated to decrease the damage caused by oxidative stress. Selenomethionine (SEMET) has been reported to play an important role in alleviating oxidative stress and accelerating cell viability and growth. However, it remains unclear whether it exerts protective effects on bone-marrow-derived mesenchymal stem cells (BMSCs) under oxidative stress. In this study, we explored the influence of selenomethionine on the viability and osteogenic differentiation of BMSCs under oxidative stress and the underlying mechanisms. Results showed that 1 μM selenomethionine was the optimum concentration for BMSCs under H{sub 2}O{sub 2} stimulation. H{sub 2}O{sub 2}-induced oxidative stress suppressed the viability and osteogenic differentiation of BMSCs, manifested by the increases in ROS production and cell apoptosis rates, and by the decrease of osteogenic differentiation-related markers. Notably, the aforementioned oxidative damage and osteogenic dysfunction induced by H{sub 2}O{sub 2} were rescued by selenomethionine. Furthermore, we found that the PTEN expression level was suppressed and its downstream PI3K/AKT pathway was activated by selenomethionine. However, when PTEN was stimulated, the PI3K/AKT pathway was down-regulated, and the protective effects of selenomethionine on BMSC osteogenic differentiation diminished, while the inhibition of PTEN up-regulated the protective effects of selenomethionine. Together, these results revealed that selenomethionine could attenuate H{sub 2}O{sub 2}-induced BMSC dysfunction through an antioxidant effect, modulated via the PTEN/PI3K/AKT pathway, suggesting that selenomethionine is a promising antioxidant candidate for reducing oxidative stress during the process of dental implant osteointegration.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Assessment of TMT Labeling Efficiency in Large-Scale Quantitative Proteomics: The Critical Effect of Sample pH

Isobaric labeling via tandem mass tag (TMT) reagents enables sample multiplexing prior to LC–MS/MS, facilitating high-throughput large-scale quantitative proteomics. Consistent and efficient labeling reactions are essential to achieve robust quantification; therefore, embedded in our clinical proteomic protocol is a quality control (QC) sample that contains a small aliquot from each sample within a TMT set, referred to as “Mixing QC.” This Mixing QC enables the detection of TMT labeling issues by LC–MS/MS before combining the full samples to allow for salvaging of poor TMT labeling reactions. While TMT labeling is a valuable tool, factors leading to poor reactions are not fully studied. We observed that relabeling does not necessarily rescue TMT reactions and that peptide samples sometimes remained acidic after resuspending in 50 mM HEPES buffer (pH 8.5), which coincided with low labeling efficiency (LE) and relatively low median reporter ion intensities (MRIIs). To obtain a more resilient TMT labeling procedure, we investigated LE, reporter ion missingness, the ratio of mean TMT set MRII to individual channel MRII, and the distribution of log 2 reporter ion ratios of Mixing QC samples. We discovered that sample pH is a critical factor in LE, and increasing the buffer concentration in poorly labeled samples before relabeling resulted in the successful rescue of TMT labeling reactions. Moreover, resuspending peptides in 500 mM HEPES buffer for TMT labeling resulted in consistently higher LE and lower missing data. By better controlling the sample pH for labeling and implementing multiple methods for assessing labeling quality before combining samples, we demonstrate that robust TMT labeling for large-scale quantitative studies is achievable.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

The cholesterol transporter NPC1 is essential for epigenetic regulation and maturation of oligodendrocyte lineage cells

The intracellular cholesterol transporter NPC1 functions in late endosomes and lysosomes to efflux unesterified cholesterol, and its deficiency causes Niemann–Pick disease Type C, an autosomal recessive lysosomal disorder characterized by progressive neurodegeneration and early death. Here, we use single-nucleus RNA-seq on the forebrain of Npc1 –/– mice at P16 to identify cell types and pathways affected early in pathogenesis. Our analysis uncovers significant transcriptional changes in the oligodendrocyte lineage during developmental myelination, accompanied by diminished maturation of myelinating oligodendrocytes. We identify upregulation of genes associated with neurogenesis and synapse formation in Npc1 –/– oligodendrocyte lineage cells, reflecting diminished gene silencing by H3K27me3. Npc1 –/ – oligodendrocyte progenitor cells reproduce impaired maturation in vitro, and this phenotype is rescued by treatment with GSK-J4, a small molecule inhibitor of H3K27 demethylases. Moreover, mobilizing stored cholesterol in Npc1 –/– mice by a single administration of 2-hydroxypropyl-β-cyclodextrin at P7 rescues myelination, epigenetic marks, and oligodendrocyte gene expression. Our findings highlight an important role for NPC1 in oligodendrocyte lineage maturation and epigenetic regulation, and identify potential targets for therapeutic intervention.

59 BASIC BIOLOGICAL SCIENCES↗