NOTE: End date changed to 12/31/2025 per JSC Grants Office (Ed., 8/5/25).

NOTE: End date changed to 12/31/2024 per NSSC information (Ed., 9/2/24).

NOTE: End date changed to 05/31/2024 per T. Sirmons/JSC (Ed., 5/16/23).

NOTE: End date changed to 12/31/2023 per PI and NSSC information (Ed., 3/21/23).

NOTE: End date changed to 04/26/2023 per T. Sirmons/JSC (Ed., 3/3/23).

NOTE: End date changed to 12/31/2022 per NSSC information (Ed., 1/4/22)

NOTE: End date changed to 12/31/2021 per NSSC information (Ed., 12/31/20)

NOTE: End date changed to 12/31/2020 per NSSC information (Ed., 6/12/20)

NOTE: End date changed to 3/31/2020 per NSSC information (Ed., 3/25/19)

Space flight results in exposure of astronauts to several stressors, such as space radiation, microgravity, and physiological stress, that could exacerbate the risks of adverse health effects. To protect astronauts, we must improve our understanding of molecular changes that influence immunological conditions associated with increased astronaut health risks. The in vivo response to the space environment is complex, involving multiple proteins associated with various signal transduction cascades, resulting in different outcomes. Molecular mechanisms responsible for such diverse consequences are poorly understood. It is, therefore, essential to characterize the protein signatures of responses to the space environment in blood plasma samples from astronauts, collected at pre-, in-, and post-flights. Such analyses should help to reveal a particular set of proteins causing adverse immunological changes and to develop methods that help to prevent, or at least to counteract, these effects.

In this flight definition project, we will use cutting age proteomic technology to determine protein alterations, qualitatively and quantitatively, in plasma samples collected from astronauts before, during, and after space flights. Our findings will help to provide an understanding of the time course and etiology of immune changes induced by the space environment. Furthermore, since pre- and post-flight samples, in addition to the in-flight samples, will be evaluated in the same astronaut, the direct effects of the space environment can be determined. Hence, our findings will provide high-priority and highly relevant information to NASA. We will further correlate protein expression profiles to the available data on immune dysfunction detected in each astronaut. This approach makes it possible to determine potential predictive biomarkers for space-flight-induced immune dysregulation. Consequently, effective countermeasures against such harmful effects of the space environment can be identified.

The biological functions of the proteins that were significantly altered in expression were classified into 10 categories: acute inflammatory response, actin skeleton organization, blood coagulation, defense response to other organisms, extracellular matrix organization, negative regulation of endopeptidase activity (metabolic process), phagocytosis, platelet degranulation, positive regulation of cytokine secretion, and tissue homeostasis. Some proteins have multiple biological functions, e.g., APOL1, SAA1, and TE.

Following the pre-flight, the levels of certain proteins, such as Apolipoprotein L1 (APOL1) and inter-alpha-trypsin inhibitor heavy chain H2 (ITIH2), returned to or close to their pre-flight levels. Meanwhile, the levels of some proteins involved in the actin cytoskeleton (e.g., pleckstrin or PLEK) and coagulation (e.g., platelet glycoprotein 1b alpha chain or GP1BA) decreased postflight. Only two proteins increased postflight as compared to the preflight level, i.e., Brain acid soluble protein 1 (BASP1) and Insulin-like growth factor-binding protein 4 (IGFBP4).

Although the mechanisms behind the downregulation and upregulation of these proteins remain unclear, they may have important roles in response to spaceflight or during the process of readjustment to Earth. This could potentially impact cellular and tissue integrity, as well as homeostasis, leading to long-term health risks.

We created a heatmap and the dendrogram of these 16 proteins, comparing their expressions and relative changes (not absolute values) of expression of eight astronauts collected preflight, in-flight, and post-flight. Circle size and color represent the Z-score. Each row represents a temporal expression of a protein. Each column represents a time point of sample collection. In the heatmap, red color represents high expression levels of proteins, and green represents very low expression levels. Changes in the expression level (either increased or decreased) of each protein were first detected in the in-flight samples (early responses).

We identified 693 non-redundant proteins and observed significant expression changes in 16 of them, first noted in in-flight samples. This indicates that the space environment affects physiological systems. The expression levels of these proteins at day 90 post-flight (R+90) can be categorized into three groups: Group A: Seven proteins exhibited similar or minimal changes (or slight increases or decreases) in expression levels between pre-flight and post-flight. This group includes APOL1, PEPD, THBS4, ITIH2, SERPIND1, SAA1, TNXB, and TF; Group B: Seven proteins displayed lower expression levels in postflight samples compared to preflight samples. This group includes GP1BA, Q9UL85, PGD, CPB2, CRP, PLEK, and TF; Group C: Two proteins showed higher expression levels in postflight samples compared to preflight samples, which are BASP1 and IGFBP.

Each group consists of proteins with diverse functions, as reflected in the sub-cluster classifications. Our data suggests that proteins in Group A were able to readapt to Earth's gravity 90 days after returning, although it is still unclear how this return to near preflight levels occurred. It is possible that the repair systems played a role. It remains unknown whether the levels of these proteins will remain consistent over time post-flight. Several proteins with altered expression were previously reported in the NASA Twin Study or in the Space Omics and Medical Atlas (SOMA) and the international astronaut biobank. However, some proteins, such as BASP1 and Q9UL85, were not reported in either study, while others, like PEPD and THBS4, were detected only in the Twin Study and not in the SOMA databank. It is important to note that in-flight samples were not included in the proteomic analyses in the SOMA databank; however, our study and the Twin Study did include such samples for proteomic analyses.

Our results show that several immune-related proteins (e.g., CPB2, CRP, TF, and Q9UL85) had significantly reduced expression levels at R+90, indicating that the space environment profoundly affects the immune system. This reduction, first noted during the flight, persisted in post-flight samples and suggests an imbalance in astronauts' immune systems, which has been linked to immune dysfunctions like altered cytokine production, phagocytosis, and T cell signaling. These issues may increase chronic inflammation and health risks for crewmembers.

The reduced expression level of the PLEK protein in the R+90 post-flight samples, compared to pre-flight levels, indicates that the space environment may downregulate actin-binding proteins. Microgravity and space radiation might lead to DNA methylation of the genes encoding these proteins, resulting in decreased expression. It remains unclear if these low protein levels will persist or return to pre-flight levels. Notably, we observed changes in SERPIN family proteins, particularly SERPIND1 (Heparin cofactor II), which is important for coagulation. Disruption of vascular homeostasis poses a significant health risk for astronauts. Our previous studies also noted a decrease in SERPINC1, another SERPIN family member related to coagulation. Thus, the suppression of specific SERPIN proteins may indicate spaceflight-induced coagulation dysfunction and could inform therapeutic strategies.

A significant finding is the increased levels of BASP1 (brain-abundant, membrane-attached signal protein 1) and IGFBP4 (insulin-like growth factor binding protein 4) in the R+90 samples. BASP1 is crucial for signal processing, synaptic plasticity, and neurite outgrowth, being associated with the cell membrane and cytoskeleton. BASP1 is found in the brain as well as in other tissues, including the kidney, lymphoid tissues, neck, prostate, and epididymis. The observed increase in plasma BASP1 levels after space missions suggests a potential link to tissue injuries from spaceflight, warranting further investigation. Elevated BASP1 levels have been noted in various solid tumors. Such findings indicate its potential significance in carcinogenesis. While its exact role in this context remains unclear, BASP1 may have a prognostic value in several types of cancer. However, a tumor-suppressor role of BASP1 was recently reported in MYC-related breast cancer. The finding of increased IGFBP4 levels is important because this protein is associated with senescence. Exposure to toxic agents such as radiation (X-rays) has been found to enhance the level of IGFBP4 and trigger its release into the bloodstream. Senescence is associated with impairments in the immune, cardiovascular, and nervous systems, as well as increased cancer risk in astronauts. While the mechanisms of IGFBP4 upregulation are unclear, the spaceflight environment may contribute to its overexpression. The increased IGFBP4 levels found in astronauts' plasma could serve as a biomarker for space stressors and aid in developing biological countermeasures.

NOTE: End date changed to 12/31/2024 per NSSC information (Ed., 9/2/24).

NOTE: End date changed to 05/31/2024 per T. Sirmons/JSC (Ed., 5/16/23).

NOTE: End date changed to 12/31/2023 per PI and NSSC information (Ed., 3/21/23).

NOTE: End date changed to 04/26/2023 per T. Sirmons/JSC (Ed., 3/3/23).

NOTE: End date changed to 12/31/2022 per NSSC information (Ed., 1/4/22)

NOTE: End date changed to 12/31/2021 per NSSC information (Ed., 12/31/20)

NOTE: End date changed to 12/31/2020 per NSSC information (Ed., 6/12/20)

NOTE: End date changed to 3/31/2020 per NSSC information (Ed., 3/25/19)

Space flight results in exposure of astronauts to several stressors, such as space radiation, microgravity, and physiological stress, that could exacerbate the risks of adverse health effects. To protect astronauts, we must improve our understanding of molecular changes that influence immunological conditions associated with increased astronaut health risks. The in vivo response to the space environment is complex, involving multiple proteins associated with various signal transduction cascades, resulting in different outcomes. Molecular mechanisms responsible for such diverse consequences are poorly understood. It is, therefore, essential to characterize the protein signatures of responses to the space environment in blood plasma samples from astronauts, collected at pre-, in-, and post-flights. Such analyses should help to reveal a particular set of proteins causing adverse immunological changes and to develop methods that help to prevent, or at least to counteract, these effects.

In this flight definition project, we will use cutting age proteomic technology to determine protein alterations, qualitatively and quantitatively, in plasma samples collected from astronauts before, during, and after space flights. Our findings will help to provide an understanding of the time course and etiology of immune changes induced by the space environment. Furthermore, since pre- and post-flight samples, in addition to the in-flight samples, will be evaluated in the same astronaut, the direct effects of the space environment can be determined. Hence, our findings will provide high-priority and highly relevant information to NASA. We will further correlate protein expression profiles to the available data on immune dysfunction detected in each astronaut. This approach makes it possible to determine potential predictive biomarkers for space-flight-induced immune dysregulation. Consequently, effective countermeasures against such harmful effects of the space environment can be identified.

NOTE: End date changed to 05/31/2024 per T. Sirmons/JSC (Ed., 5/16/23).

NOTE: End date changed to 12/31/2023 per PI and NSSC information (Ed., 3/21/23).

NOTE: End date changed to 04/26/2023 per T. Sirmons/JSC (Ed., 3/3/23).

NOTE: End date changed to 12/31/2022 per NSSC information (Ed., 1/4/22)

NOTE: End date changed to 12/31/2021 per NSSC information (Ed., 12/31/20)

NOTE: End date changed to 12/31/2020 per NSSC information (Ed., 6/12/20)

NOTE: End date changed to 3/31/2020 per NSSC information (Ed., 3/25/19)

Space flight results in exposure of astronauts to several stressors, such as space radiation, microgravity, and physiological stress, that could exacerbate the risks of adverse health effects. To protect astronauts, we must improve our understanding of molecular changes that influence immunological conditions associated with increased astronaut health risks. The in vivo response to the space environment is complex, involving multiple proteins associated with various signal transduction cascades, resulting in different outcomes. Molecular mechanisms responsible for such diverse consequences are poorly understood. It is, therefore, essential to characterize the protein signatures of responses to the space environment in blood plasma samples from astronauts, collected at pre-, in-, and post-flights. Such analyses should help to reveal a particular set of proteins causing adverse immunological changes and to develop methods that help to prevent, or at least to counteract, these effects.

In this flight definition project, we will use cutting age proteomic technology to determine protein alterations, qualitatively and quantitatively, in plasma samples collected from astronauts before, during, and after space flights. Our findings will help to provide an understanding of the time course and etiology of immune changes induced by the space environment. Furthermore, since pre- and post-flight samples, in addition to the in-flight samples, will be evaluated in the same astronaut, the direct effects of the space environment can be determined. Hence, our findings will provide high-priority and highly relevant information to NASA. We will further correlate protein expression profiles to the available data on immune dysfunction detected in each astronaut. This approach makes it possible to determine potential predictive biomarkers for space-flight-induced immune dysregulation. Consequently, effective countermeasures against such harmful effects of the space environment can be identified.

We grouped the samples based on the sampling times. There was one preflight sampling time (L45, i.e., 45 days before flight), one in-flight sampling time (In-flight, ~2-4 months in the International Space Station/ISS), and three post-flight sampling times, i.e., within 24 hrs of returning to Earth (R+0), 30 days after returning to Earth (R+30), and 90 days after returning to Earth. We identified 694 unique and non-redundant proteins (at > 99% confidence). Numbers of unique and non-redundant proteins in each group are:

315 proteins from the L45 sample group 315 proteins from the In-flt sample group 320 proteins from the R+0 sample group 249 proteins from the R +30 sample group 287 proteins from the R+90 sample group

These proteins are involved in inflammatory response, actin cytoskeleton organization, defense responses, phagocytosis, extracellular matrix organization, platelet degradation, and tissue homeostasis. Such changes are pronounced in the in-flight samples. Subsequently, the levels of few proteins return to the preflight levels, e.g., cluster of differentiation 14 (CD14). However, we found that the expression levels of proteins involved in actin cytoskeleton and platelet degradation (e.g., filamin A or FLNA), and platelet releasate cytosolic proteins (PLEK) organization were decreased in samples collected at post-flight, while the expression levels of some proteins are increased in samples collected post-flight, e.g., insulin-like growth factor-binding protein 4 (IGFBP4) and tenascin XB (TNXB). Thus, changes in these proteins (i.e., increased or decreased) may potentially have functional roles in response to space flight or re-adaptation to returning to Earth and may affect cell/tissue integrity and homeostasis, leading to late-occurring health risks.

In summary, the highlights of our findings are:

• Proteins with positive regulation of cytokine secretion are highly expressed in samples collected in-flight. These include: CD14 (Cluster of differentiation 14), COTL1 (Coactosin-like protein-1, an actin binding protein), ORM1 (Orosomucoid or Alpha-1-acid glycoprotein 1, acute phase protein) SERPINA4 (Serine protease inhibitor A4, one of proteins in the Serpin family), SAA1 (Serum amyloid A1 or SAA1, acute phase protein), and MASP1 (Mannan-binding lectin serine protease 1, innate immunity).

However, the levels of these proteins return to or near the preflight level at d-90 post-flight.

• The expression levels of some proteins involved in anti-inflammation, controlling the homeostasis of cytoskeleton, and normal blood coagulation are reduced at d 90 post-flight. Examples of such proteins are: FLNA (Filamin A), PLEK (Platelet releasate cytosolic proteins), and TLN1 (TLN1).

• At 90 days post-flight, expression levels of some proteins are higher than those of the preflight levels: IGFBP4 (Insulin-like growth factor-binding protein4 or IGFBP4), TNXB (Tenascin or TNXB, involved in cytoskeleton), and BASP1 (Brain acid soluble protein 1 or BASP1).

NOTE: End date changed to 12/31/2021 per NSSC information (Ed., 12/31/20)

NOTE: End date changed to 12/31/2020 per NSSC information (Ed., 6/12/20)

NOTE: End date changed to 3/31/2020 per NSSC information (Ed., 3/25/19)

Space flight results in exposure of astronauts to several stressors, such as space radiation, microgravity, and physiological stress, that could exacerbate the risks of adverse health effects. To protect astronauts, we must improve our understanding of molecular changes that influence immunological conditions associated with increased astronaut health risks. The in vivo response to the space environment is complex, involving multiple proteins associated with various signal transduction cascades, resulting in different outcomes. Molecular mechanisms responsible for such diverse consequences are poorly understood. It is, therefore, essential to characterize the protein signatures of responses to the space environment in blood plasma samples from astronauts, collected at pre-, in-, and post-flights. Such analyses should help to reveal a particular set of proteins causing adverse immunological changes and to develop methods that help to prevent, or at least to counteract, these effects.

In this flight definition project, we will use cutting age proteomic technology to determine protein alterations, qualitatively and quantitatively, in plasma samples collected from astronauts before, during, and after space flights. Our findings will help to provide an understanding of the time course and etiology of immune changes induced by the space environment. Furthermore, since pre- and post-flight samples, in addition to the in-flight samples, will be evaluated in the same astronaut, the direct effects of the space environment can be determined. Hence, our findings will provide high-priority and highly relevant information to NASA. We will further correlate protein expression profiles to the available data on immune dysfunction detected in each astronaut. This approach makes it possible to determine potential predictive biomarkers for space-flight-induced immune dysregulation. Consequently, effective countermeasures against such harmful effects of the space environment can be identified.

Our contribution to this Flight Definition is to identify and characterize plasma proteins in the blood plasma of astronauts that can be used as predictive biomarkers of immunological dysfunction due to space flight. Specifically, we characterize the proteome of blood plasma collected from the same astronauts at different times, i.e. pre-, in-, and post-flight.

Previously, we analyzed the data by comparing pre-vs-in-flight and in-vs-post-flight separately. During the past few months, we analyzed the data by studying the temporal changes of each protein in samples collected at pre, in-, and post-flight from the same astronauts. The results from this approach not only help us to understand the temporal changes of proteins during space flights but also are highly relevant to the effects of space flight on protein expression. Using this approach, we detected new low abundance proteins with altered expression levels that have never been reported.

Our data demonstrate that there are 19 proteins with significant changes, i.e., increased or decreased, in expression levels due to space flight. Among the 19 proteins with significant changes in expression levels, there were seven proteins with significantly decreased levels. These include lumican (LUM), extracellular matrix protein 1 (ECM1), vitronectin (VTN), filaggrin-2 (FLG2), ceruloplasmin (CP), Desmoplakin (DSP), CD5 antigen-like (CD5L). These proteins are involved in cell defense responses, inflammatory responses, and cell/tissue repair. Hence, a reduction in the expression levels of these proteins may be associated with the impairment of the immune system and the repair of damaged tissues. The deficiency of filaggrin-2 (FLG2) is involved in skin inflammation. Although further studies are needed, a reduction in the expression level of FLG2 may play a role in the occurrence of skin rashes in some astronauts. It is known that lumican (LUM) is a major keratan sulfate proteoglycan of the cornea responsible for circumferential growth, corneal transparency, epithelial cell migration, and tissue repair. Hence, LUM is critical in maintaining corneal clarity. Further, a loss of LUM expression is associated with corneal inflammation. Hence, persistent decreases in the level of LUM after space flight would impair the homeostasis of the eyes. Taken together, it is plausible to hypothesize that a reduction in LUM level may be associated with vision impairment that has been observed in many astronauts after space flight. Our findings warrant further investigation of the potential role of LUM in vision impairment detected in astronauts.

The levels of the remaining 12 proteins were significantly increased. These are antithrombin-II (SERPINC1), fibronectin (FN1), protein disulfide-isomerase A (PDIA 3), titin (TTN), tropomodulin (TMOD3), zyxin (ZYN), bridging integrator 2 (BIN2), apolipoprotein A-II (APOA2), platelet factor 4 variant (PF4V1), beta-Ala-His dipeptidase (CNDP1), alpha-2-HS-glycoprotein (AHSG), pigment epithelium-derived factor (SERPINF1). The majority of these proteins are involved in inflammatory responses, cytoskeleton organization, and aging. Increased expression levels of CNDP1 may be indicative of increased oxidative stress in the brain and the muscle since CNDP1 degrades carnosine and homocarnosine (proteins with anti-oxidative activity mostly concentrated in the brain and the muscle). The protein in the SERPIN (serine protease inhibitor) family, i.e., SERPINC1, and SERPINF1, is the majority of those with increased expression levels. The SERPIN protease inhibitors comprise a large family of molecules involved in inflammation, immune response, blood clotting, hormone transport, and complement activation, dementia, and tumorigenesis. Hence, our findings suggest that dysregulation of these proteins may affect cell/tissue integrity and homeostasis, leading to late occurring health risks.

Based on these findings, we are preparing a manuscript to be submitted for possible publication in Acta Astronautica. In addition to the preparation of a manuscript, we are currently using Orbitrap Fusion Lumos Tribrid MS coupled with Thermo Ultimate 3000 HPLC system to investigate the pattern of protein expression profiles of astronauts’ plasma. This strategy helps us to achieve more in-depth coverage of the proteome than the MudPIT. However, it was not available in our laboratory in the past. We are currently analyzing the data generated by this highly sensitive and accurate mass spectrometer system. We anticipate another publication from this analysis.

NOTE: End date changed to 12/31/2021 per NSSC information (Ed., 12/31/20)

NOTE: End date changed to 12/31/2020 per NSSC information (Ed., 6/12/20)

NOTE: End date changed to 3/31/2020 per NSSC information (Ed., 3/25/19)

Space flight results in exposure of astronauts to several stressors, such as space radiation, microgravity, and physiological stress, that could exacerbate the risks of adverse health effects. To protect astronauts, we must improve our understanding of molecular changes that influence immunological conditions associated with increased astronaut health risks. The in vivo response to the space environment is complex, involving multiple proteins associated with various signal transduction cascades, resulting in different outcomes. Molecular mechanisms responsible for such diverse consequences are poorly understood. It is, therefore, essential to characterize the protein signatures of responses to the space environment in blood plasma samples from astronauts, collected at pre-, in-, and post-flights. Such analyses should help to reveal a particular set of proteins causing adverse immunological changes and to develop methods that help to prevent, or at least to counteract, these effects.

In this flight definition project, we will use cutting age proteomic technology to determine protein alterations, qualitatively and quantitatively, in plasma samples collected from astronauts before, during, and after space flights. Our findings will help to provide an understanding of the time course and etiology of immune changes induced by the space environment. Furthermore, since pre- and post-flight samples, in addition to the in-flight samples, will be evaluated in the same astronaut, the direct effects of the space environment can be determined. Hence, our findings will provide high-priority and highly relevant information to NASA. We will further correlate protein expression profiles to the available data on immune dysfunction detected in each astronaut. This approach makes it possible to determine potential predictive biomarkers for space-flight-induced immune dysregulation. Consequently, effective countermeasures against such harmful effects of the space environment can be identified.

In the last annual report, we reported that a total of 453 unique and non-redundant proteins were identified at >99% confidence. Changes in protein concentrations during the pre-, in-, and post-flight timeline were determined by Students’ t-test analysis of comparisons between groups (p<0.1 is considered significant). Our data demonstrate that there are 14 proteins with significant changes, i.e., increased or decreased, in expression levels in plasma samples collected in-flight as compared to those collected pre-flight. We also reported that there were 16 proteins identified with significant changes, i.e., increased in green or decreased in red, in expression levels found astronauts’ plasma collected post-flight, in relation to those collected in-flight. Last January, we presented our results at the Annual NASA Human Research Program (HRP) workshop in Galveston.

During the past year, we use Principal Component Analysis (PCA) to analyze these 453 unique and non-redundant proteins identified by LTQ Orbitrap XL Ion trap mass spectrometer. The PCA is frequently used in the global analysis of the “omic” datasets. It provides fully unsupervised information on the dominant directions of highest variability in the data and can, therefore, be used to investigate similarities between individual samples, or the formation of clusters. The PCA is a dimensionality reduction method used to reduce the dimensionality of large data sets by transforming a large set of variables into a smaller one while preserving as much information as possible. The new variables are called the principal components and they are linear combinations of the actual variables. The first principal component is a linear combination of all the actual variables that have maintained the greatest amount of variation. The second principal component is a linear combination of the remaining variables to give the second greatest amount of variation and this can continue for third, fourth, and so on components but usually the first and second components carry the most important information. A scatter plot of the first and second components (a score plot) will often display samples sharing similar characteristics being grouped together apart from samples with different characteristics (i.e., it shows clustering based on similarity). Another plot is also generated in this analysis called a loading plot. The loading plot shows how strongly each original variable influences the principal component. Often, most points in a loading plot will be clustered around the center and the outlier points may be associated with the variables making the largest contribution to the data variation. In summary, the PCA indicates the differences in the pattern of protein expression profiles between samples collected pre- and in-flight.

Moreover, we constructed a heatmap to visualize the result of a hierarchical clustering calculation of the 14 proteins differentially expressed in samples collected at pre- or in-flight from each astronaut. The results from the heatmap show that the intensities (level of expression) of tropomodulin-3 (TMOD3), SERPINA7, SERPING1, and SERPRINC1 were higher in samples collected in-flight, in relation to those collected pre-flight. Notably, the protein in the SERPIN (serine protease inhibitor) family, i.e., SERPINA7, SERPINC1, and ISERPING1, is the majority of those with increased intensities (expression levels). However, the data also demonstrated individual variability in the intensities of these proteins. It should be noted that SERPIN protease inhibitors comprise a large family of molecules involved in inflammation, immune response, blood clotting, hormone transport, and complement activation, dementia, and tumorigenesis. Our heatmap data also showed high intensities of CP, FLG2, KRT2, F13A1, DMKN, LUM in samples collected pre-flight. Subsequently, the intensities of expression of these proteins were declined in samples collected in-flights. Likewise, the intensities of another set of proteins (i.e., CFB, FBLN1, AHSG, and ECM1) were high in samples collected pre-flight; this followed by a reduction in intensities in samples collected in-flight, with one exception of increased intensity of FBLN1 protein in sample4s collected mid-flight from one astronaut.

Overall, the heatmap of our dataset demonstrated that the intensities of not only proteins in the SERPIN family but also TMOD3 (an actin filament pointed-end capping protein with multifunctional roles, including cell proliferation, cell migration, inflammation, and carcinogenesis) are consistently high in the in-flight samples. In contrast, low intensities of lumican (LUM) have been repeatedly detected in the in-flight samples. Of note, LUM is a major keratan sulfate proteoglycan of the cornea responsible for the circumferential growth, corneal transparency, epithelial cell migration, and tissue repair. Hence, it is possible to speculate that a reduction in LUM level may be associated with vision impairment that has been observed in many astronauts after spaceflight. In summary, our findings suggest that dysregulation of the SERPIN, TMOD3, and LUM may affect cell/tissue integrity and homeostasis, leading to late occurring health risks. Thus, our results may represent a foundation for the identification of countermeasures against the harmful effects of spaceflights.

NOTE: End date changed to 3/31/2020 per NSSC information (Ed., 3/25/19)

Space flight results in exposure of astronauts to several stressors, such as space radiation, microgravity, and physiological stress, that could exacerbate the risks of adverse health effects. To protect astronauts, we must improve our understanding of molecular changes that influence immunological conditions associated with increased astronaut health risks. The in vivo response to the space environment is complex, involving multiple proteins associated with various signal transduction cascades, resulting in different outcomes. Molecular mechanisms responsible for such diverse consequences are poorly understood. It is, therefore, essential to characterize the protein signatures of responses to the space environment in blood plasma samples from astronauts, collected at pre-, in-, and post-flights. Such analyses should help to reveal a particular set of proteins causing adverse immunological changes and to develop methods that help to prevent, or at least to counteract, these effects.

In this flight definition project, we will use cutting age proteomic technology to determine protein alterations, qualitatively and quantitatively, in plasma samples collected from astronauts before, during, and after space flights. Our findings will help to provide an understanding of the time course and etiology of immune changes induced by the space environment. Furthermore, since pre- and post-flight samples, in addition to the in-flight samples, will be evaluated in the same astronaut, the direct effects of the space environment can be determined. Hence, our findings will provide high-priority and highly relevant information to NASA. We will further correlate protein expression profiles to the available data on immune dysfunction detected in each astronaut. This approach makes it possible to determine potential predictive biomarkers for space-flight-induced immune dysregulation. Consequently, effective countermeasures against such harmful effects of the space environment can be identified.

Subsequently, the raw data files were interrogated and searched against a current UniProt human proteome database using the Andromeda search engine within MaxQuant. This produced a list of proteins for each sample which was quantitated by extracted ion chromatogram intensity by the MaxQuant program. The raw data from all the samples were searched simultaneously to reduce the occurrence of homology redundancy. The data from all the samples were compiled into a spreadsheet and the protein quantity was normalized based on the fractional signal strength within each sample. The samples were grouped according to the timeline stages of collection (i.e., pre-, in-, and post-flight). A total of 453 unique and non-redundant proteins were identified at >= 99% confidence. Changes in protein concentrations during the timeline were determined by Student’s t-test analysis of comparisons between groups (p<0.1 is considered significant). Those proteins displaying statistically significant changes were subjected to interacting network analysis using the Genemania app within the Cytoscape software package.

Our data demonstrate that there are 14 proteins with significant changes, i.e., increased or decreased, in expression levels in plasma samples collected in-flight as compared to those collected pre-flight. Four proteins with significant increases were detected in plasma collected in-flight, as compared to the plasma collected pre-flight. These are antithrombin-II (SERPINC1), plasma protease C1 inhibitor (SERPING1), Tropomodulin-3 (TMOD3), thyroxine-binding globulin (SERPINA7). The levels of the remaining 10 proteins were decreased in plasma collected in-flight as compared to those collected pre-flight. Notably, the protein in the SERPIN (serine protease inhibitor) family, i.e., SERPINA7, SERPINC1, and ISERPING1, is the majority of those with increased expression levels. The SERPIN protease inhibitors comprise a large family of molecules involved in inflammation, immune response, blood clotting, hormone transport, and complement activation, dementia, and tumorigenesis. Hence, our findings suggest that dysregulation of these proteins may affect cell/tissue integrity and homeostasis, leading to late occurring health risks. It has been suggested that overexpression of tropomodulin- 3 (TMOD3, actin pointed end-capping protein) leads to decreased endothelial motility. The majority of proteins with decreased levels are those involved in immune response and cytoskeleton systems.

Further, there were 16 proteins with significant changes in expression levels in astronauts’ plasma collected post-flight, in relation to those collected in-flight. There are eight proteins with significantly increased expression levels, while another set of eight proteins showed significantly decreased levels. The majority of proteins with changes in expression are those involved in immune and cytoskeleton systems. A new set of proteins with significant changes in expression levels was found in astronauts’ plasma collected post-flight, e.g., vitronectin, ceruloplasmin, Zyxin, and tubulin beta chain. This set of proteins may be involved in the re-adaptation to the Earth environment. Importantly, our results show that a decreased level of lumican (LUM) and an increased level of antithrombin-III (SERPINC1) persisted in astronauts’ plasma collected in- and post-flight. Such a prolonged increase in the level of SERPINC1 may contribute to coagulopathy, the phenomenon observed in individuals exposed to radiation. It is known that LUM is a major keratan sulfate proteoglycan of the cornea responsible for the circumferential growth, corneal transparency, epithelial cell migration, and tissue repair. Hence, LUM has been found to be critical in maintaining corneal clarity. Further, a loss of LUM expression has been found to be associated with corneal inflammation. Hence, persistent decreases in the level of LUM after spaceflight would impair the homeostasis of the eyes. Taken together, it is plausible to hypothesize that a reduction in LUM level may be associated with vision impairment that has been observed in many astronauts after spaceflight. Our findings warrant further investigation of the potential role of LUM in vision impairment detected in astronauts.

Highlights: The highlights of the findings from our study on the proteome of astronauts’ plasma collected at pre-, in-, and post-flights are:

• The majority of proteins with altered expression levels detected in plasma samples collected in- or post-flight are those involved in immune and cytoskeleton systems.

• A new set of proteins with altered expression levels was detected in plasma collected post-flight as compared to the samples collected in-flight.

• An increased level of antithrombin-III (SERPINC1) and a decreased level of Lumican (LUM, an important protein involved in maintaining corneal clarity protein) persisted for a long time post-flight, suggesting the potential role in late-occurring health risks.

Space flight results in exposure of astronauts to several stressors, such as space radiation, microgravity, and physiological stress, that could exacerbate the risks of adverse health effects. To protect astronauts, we must improve our understanding of molecular changes that influence immunological conditions associated with increased astronaut health risks. The in vivo response to the space environment is complex, involving multiple proteins associated with various signal transduction cascades, resulting in different outcomes. Molecular mechanisms responsible for such diverse consequences are poorly understood. It is, therefore, essential to characterize the protein signatures of responses to the space environment in blood plasma samples from astronauts, collected at pre-, in-, and post-flights. Such analyses should help to reveal a particular set of proteins causing adverse immunological changes and to develop methods that help to prevent, or at least to counteract, these effects.

In this flight definition project, we will use cutting age proteomic technology to determine protein alterations, qualitatively and quantitatively, in plasma samples collected from astronauts before, during, and after space flights. Our findings will help to provide an understanding of the time course and etiology of immune changes induced by the space environment. Furthermore, since pre- and post-flight samples, in addition to the in-flight samples, will be evaluated in the same astronaut, the direct effects of the space environment can be determined. Hence, our findings will provide high-priority and highly relevant information to NASA. We will further correlate protein expression profiles to the available data on immune dysfunction detected in each astronaut. This approach makes it possible to determine potential predictive biomarkers for space-flight-induced immune dysregulation. Consequently, effective countermeasures against such harmful effects of the space environment can be identified.

Our data on protein concentrations show no effects of time in keeping samples at room temperature prior to storage at -80oC. Additionally, there was no clear difference between the ACD and EDTA samples. We also optimized the procedures for sample preparation prior to mass spectrometry analyses. Such optimal conditions will be used to study the proteome of astronauts’ plasma samples which will be the focus of our project in the next fiscal year.

Space flight results in exposure of astronauts to several stressors, such as space radiation, microgravity, and physiological stress, that could exacerbate the risks of adverse health effects. To protect astronauts, we must improve our understanding of molecular changes that influence immunological conditions associated with increased astronaut health risks. The in vivo response to the space environment is complex, involving multiple proteins associated with various signal transduction cascades, resulting in different outcomes. Molecular mechanisms responsible for such diverse consequences are poorly understood. It is, therefore, essential to characterize the protein signatures of responses to the space environment in blood plasma samples from astronauts, collected at pre-, in-, and post-flights. Such analyses should help to reveal a particular set of proteins causing adverse immunological changes and to develop methods that help to prevent, or at least to counteract, these effects.

In this flight definition project, we will use cutting age proteomic technology to determine protein alterations, qualitatively and quantitatively, in plasma samples collected from astronauts before, during, and after space flights. Our findings will help to provide an understanding of the time course and etiology of immune changes induced by the space environment. Furthermore, since pre- and post-flight samples, in addition to the in-flight samples, will be evaluated in the same astronaut, the direct effects of the space environment can be determined. Hence, our findings will provide high-priority and highly relevant information to NASA. We will further correlate protein expression profiles to the available data on immune dysfunction detected in each astronaut. This approach makes it possible to determine potential predictive biomarkers for space-flight-induced immune dysregulation. Consequently, effective countermeasures against such harmful effects of the space environment can be identified.

Space flight results in exposure of astronauts to several stressors, such as space radiation, microgravity, and physiological stress, that could exacerbate the risks of adverse health effects. To protect astronauts, we must improve our understanding of molecular changes that influence immunological conditions associated with increased astronaut health risks. The in vivo response to the space environment is complex, involving multiple proteins associated with various signal transduction cascades, resulting in different outcomes. Molecular mechanisms responsible for such diverse consequences are poorly understood. It is, therefore, essential to characterize the protein signatures of responses to the space environment in blood plasma samples from astronauts, collected at pre-, in-, and post-flights. Such analyses should help to reveal a particular set of proteins causing adverse immunological changes and to develop methods that help to prevent, or at least to counteract, these effects.

In this flight definition project, we will use cutting age proteomic technology to determine protein alterations, qualitatively and quantitatively, in plasma samples collected from astronauts before, during, and after space flights. Our findings will help to provide an understanding of the time course and etiology of immune changes induced by the space environment. Furthermore, since pre- and post-flight samples, in addition to the in-flight samples, will be evaluated in the same astronaut, the direct effects of the space environment can be determined. Hence, our findings will provide high-priority and highly relevant information to NASA. We will further correlate protein expression profiles to the available data on immune dysfunction detected in each astronaut. This approach makes it possible to determine potential predictive biomarkers for space-flight-induced immune dysregulation. Consequently, effective countermeasures against such harmful effects of the space environment can be identified.