Effects of acute low-moderate dose ionizing radiation to human brain organoids.

Human exposure to low-to-moderate dose ionizing radiation (LMD-IR) is increasing via environmental, medical, occupational sources. Acute exposure to LMD-IR can cause subclinical damage to cells, resulting in altered gene expression and cellular function within the human brain. It has been difficult to identify diagnostic and predictive biomarkers of exposure using traditional research models due to factors including lack of 3D structure in monolayer cell cultures, limited ability of animal models to accurately predict human responses, and technical limitations of studying functional human brain tissue. To address this gap, we generated brain/cerebral organoids from human induced pluripotent stem cells to study the radiosensitivity of human brain cells, including neurons, astrocytes, and oligodendrocytes. While organoids have become popular models for studying brain physiology and pathology, there is little evidence to confirm that exposing brain organoids to LMD-IR will recapitulate previous in vitro and in vivo observations. We hypothesized that exposing brain organoids to proton radiation would (1) cause a time- and dose-dependent increase in DNA damage, (2) induce cell type-specific differences in radiosensitivity, and (3) increase expression of oxidative stress and DNA damage response genes. Organoids were exposed to 0.5 or 2 Gy of 250 MeV protons and samples were collected at 30 minute, 24 hour, and 48 hour timepoints. Using immunofluorescence and RNA sequencing, we found time- and dose-dependent increases in DNA damage in irradiated organoids; no changes in cell populations for neurons, oligodendrocytes, and astrocytes by 24 hours; decreased expression of genes related to oligodendrocyte lineage, astrocyte lineage, mitochondrial function, and cell cycle progression by 48 hours; increased expression of genes related to neuron lineage, oxidative stress, and DNA damage checkpoint regulation by 48 hours. Our findings demonstrate the possibility of using organoids to characterize cell-specific radiosensitivity and early radiation-induced gene expression changes within the human brain, providing new avenues for further study of the mechanisms underlying acute neural cell responses to IR exposure at low-to-moderate doses.

Transcriptomic Effects on the Mouse Heart Following 30 Days on the International Space Station.

Efforts to understand the impact of spaceflight on the human body stem from growing interest in long-term space travel. Multiple organ systems are affected by microgravity and radiation, including the cardiovascular system. Previous transcriptomic studies have sought to reveal the changes in gene expression after spaceflight. However, little is known about the impact of long-term spaceflight on the mouse heart in vivo. This study focuses on the transcriptomic changes in the hearts of female C57BL/6J mice flown on the International Space Station (ISS) for 30 days. RNA was isolated from the hearts of three flight and three comparable ground control mice and RNA sequencing was performed. Our analyses showed that 1147 transcripts were significantly regulated after spaceflight. The MAPK, PI3K-Akt, and GPCR signaling pathways were predicted to be activated. Transcripts related to cytoskeleton breakdown and organization were upregulated, but no significant change in the expression of extracellular matrix (ECM) components or oxidative stress pathway-associated transcripts occurred. Our results indicate an absence of cellular senescence, and a significant upregulation of transcripts associated with the cell cycle. Transcripts related to cellular maintenance and survival were most affected by spaceflight, suggesting that cardiovascular transcriptome initiates an adaptive response to long-term spaceflight.

Characterization of gene expression profiles in the mouse brain after 35 days of spaceflight mission.

It has been proposed that neuroinflammatory response plays an important role in the neurovascular remodeling in the brain after stress. The goal of the present study was to characterize changes in the gene expression profiles associated with neuroinflammation, neuronal function, metabolism and stress in mouse brain tissue. Ten-week old male C57BL/6 mice were launched to the International Space Station (ISS) on SpaceX-12 for a 35-day mission. Within 38 ± 4 h of splashdown, mice were returned to Earth alive. Brain tissues were collected for analysis. A novel digital color-coded barcode counting technology (NanoStringTM) was used to evaluate gene expression profiles in the spaceflight mouse brain. A set of 54 differently expressed genes (p < 0.05) significantly segregates the habitat ground control (GC) group from flight (FLT) group. Many pathways associated with cellular stress, inflammation, apoptosis, and metabolism were significantly altered by flight conditions. A decrease in the expression of genes important for oligodendrocyte differentiation and myelin sheath maintenance was observed. Moreover, mRNA expression of many genes related to anti-viral signaling, reactive oxygen species (ROS) generation, and bacterial immune response were significantly downregulated. Here we report that significantly altered immune reactions may be closely associated with spaceflight-induced stress responses and have an impact on the neuronal function.

An Analysis of the Effects of Spaceflight and Vaccination on Antibody Repertoire Diversity.

Ab repertoire diversity plays a critical role in the host's ability to fight pathogens. CDR3 is partially responsible for Ab-Ag binding and is a significant source of diversity in the repertoire. CDR3 diversity is generated during VDJ rearrangement because of gene segment selection, gene segment trimming and splicing, and the addition of nucleotides. We analyzed the Ab repertoire diversity across multiple experiments examining the effects of spaceflight on the Ab repertoire after vaccination. Five datasets from four experiments were analyzed using rank-abundance curves and Shannon indices as measures of diversity. We discovered a trend toward lower diversity as a result of spaceflight but did not find the same decrease in our physiological model of microgravity in either the spleen or bone marrow. However, the bone marrow repertoire showed a reduction in diversity after vaccination. We also detected differences in Shannon indices between experiments and tissues. We did not detect a pattern of CDR3 usage across the experiments. Overall, we were able to find differences in the Ab repertoire diversity across experimental groups and tissues.

Immunological and hematological outcomes following protracted low dose/low dose rate ionizing radiation and simulated microgravity.

Using a ground-based model to simulate spaceflight [21-days of single-housed, hindlimb unloading (HLU) combined with continuous low-dose gamma irradiation (LDR, total dose of 0.04 Gy)], an in-depth survey of the immune and hematological systems of mice at 7-days post-exposure was performed. Collected blood was profiled with a hematology analyzer and spleens were analyzed by whole transcriptome shotgun sequencing (RNA-sequencing). The results revealed negligible differences in immune differentials. However, hematological system analyses of whole blood indicated large disparities in red blood cell differentials and morphology, suggestive of anemia. Murine Reactome networks indicated majority of spleen cells displayed differentially expressed genes (DEG) involved in signal transduction, metabolism, cell cycle, chromatin organization, and DNA repair. Although immune differentials were not changed, DEG analysis of the spleen revealed expression profiles associated with inflammation and dysregulated immune function persist to 1-week post-simulated spaceflight. Additionally, specific regulation pathways associated with human blood disease gene orthologs, such as blood pressure regulation, transforming growth factor-ß receptor signaling, and B cell differentiation were noted. Collectively, this study revealed differential immune and hematological outcomes 1-week post-simulated spaceflight conditions, suggesting recovery from spaceflight is an unremitting process.

Brain organoids: A promising model to assess oxidative stress-induced central nervous system damage.

Oxidative stress (OS) is one of the most significant propagators of systemic damage with implications for widespread pathologies such as vascular disease, accelerated aging, degenerative disease, inflammation, and traumatic injury. OS can be induced by numerous factors such as environmental conditions, lifestyle choices, disease states, and genetic susceptibility. It is tied to the accumulation of free radicals, mitochondrial dysfunction, and insufficient antioxidant protection, which leads to cell aging and tissue degeneration over time. Unregulated systemic increase in reactive species, which contain harmful free radicals, can lead to diverse tissue-specific OS responses and disease. Studies of OS in the brain, for example, have demonstrated how this state contributes to neurodegeneration and altered neural plasticity. As the worldwide life expectancy has increased over the last few decades, the prevalence of OS-related diseases resulting from age-associated progressive tissue degeneration. Unfortunately, vital translational research studies designed to identify and target disease biomarkers in human patients have been impeded by many factors (e.g., limited access to human brain tissue for research purposes and poor translation of experimental models). In recent years, stem cell-derived three-dimensional tissue cultures known as "brain organoids" have taken the spotlight as a novel model for studying central nervous system (CNS) diseases. In this review, we discuss the potential of brain organoids to model the responses of human neural cells to OS, noting current and prospective limitations. Overall, brain organoids show promise as an innovative translational model to study CNS susceptibility to OS and elucidate the pathophysiology of the aging brain.

Spaceflight induces oxidative damage to blood-brain barrier integrity in a mouse model.

Many factors contribute to the health risks encountered by astronauts on missions outside Earth's atmosphere. Spaceflight-induced potential adverse neurovascular damage and late neurodegeneration are a chief concern. The goal of the present study was to characterize the effects of spaceflight on oxidative damage in the mouse brain and its impact on blood-brain barrier (BBB) integrity. Ten-week-old male C57BL/6 mice were launched to the International Space Station (ISS) for 35 days as part of Space-X 12 mission. Ground control (GC) mice were maintained on Earth in flight hardware cages. Within 38 ± 4 hours after returning from the ISS, mice were euthanized and brain tissues were collected for analysis. Quantitative assessment of brain tissue demonstrated that spaceflight caused an up to 2.2-fold increase in apoptosis in the hippocampus compared to the control group. Immunohistochemical analysis of the mouse brain revealed an increased expression of aquaporin4 (AQP4) in the flight hippocampus compared to the controls. There was also a significant increase in the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) and a decrease in the expression of the BBB-related tight junction protein, Zonula occludens-1 (ZO-1). These results indicate a disturbance of BBB integrity. Quantitative proteomic analysis showed significant alterations in pathways responsible for neurovascular integrity, mitochondrial function, neuronal structure, protein/organelle transport, and metabolism in the brain after spaceflight. Changes in pathways associated with adhesion and molecular remodeling were also documented. These data indicate that long-term spaceflight may have pathological and functional consequences associated with neurovascular damage and late neurodegeneration.

Spaceflight influences gene expression, photoreceptor integrity, and oxidative stress-related damage in the murine retina.

Extended spaceflight has been shown to adversely affect astronaut visual acuity. The purpose of this study was to determine whether spaceflight alters gene expression profiles and induces oxidative damage in the retina. Ten week old adult C57BL/6 male mice were flown aboard the ISS for 35 days and returned to Earth alive. Ground control mice were maintained on Earth under identical environmental conditions. Within 38 (+/-4) hours after splashdown, mice ocular tissues were collected for analysis. RNA sequencing detected 600 differentially expressed genes (DEGs) in murine spaceflight retinas, which were enriched for genes related to visual perception, the phototransduction pathway, and numerous retina and photoreceptor phenotype categories. Twelve DEGs were associated with retinitis pigmentosa, characterized by dystrophy of the photoreceptor layer rods and cones. Differentially expressed transcription factors indicated changes in chromatin structure, offering clues to the observed phenotypic changes. Immunofluorescence assays showed degradation of cone photoreceptors and increased retinal oxidative stress. Total retinal, retinal pigment epithelium, and choroid layer thickness were significantly lower after spaceflight. These results indicate that retinal performance may decrease over extended periods of spaceflight and cause visual impairment.

Effects of skeletal unloading on the bone marrow antibody repertoire of tetanus toxoid and/or CpG treated C57BL/6J mice.

Spaceflight is known to impact the immune system in multiple ways. However, its effect on the antibody repertoire, especially in response to challenge, has not been well characterized. The development of the repertoire has multiple steps that could be affected by spaceflight, including V-(D-)J-gene segment rearrangement and the selection of complementarity determining regions (CDRs); specifically, CDR3, responsible for much of the diversity in the repertoire. We used skeletal unloading with the antiorthostatic suspension (AOS) model to simulate some of the physiological effects associated with spaceflight. Animals ± AOS were challenged with tetanus toxoid (TT) and/or CpG, an adjuvant. Two weeks after challenge, bone marrow was collected and sequenced using the Illumina MiSeq 2 × 300 platform. The resulting antibody repertoire was characterized, including V-, D- (heavy only), and J-gene segment usage, constant region usage, CDR3 length, and V(D)J combinations. We detected changes in gene-segment usage in response to AOS, TT, and CpG treatment in both the heavy and light chains. Additionally, changes were seen in the class-switched VH-gene repertoire. Alterations were also detected in V/J pairing for both the heavy and light chains, and changes in CDR3 length. We also detected lower levels of CDR3 AA overlap than detected in the splenic repertoire. These results demonstrate that AOS, TT, and CpG alter the bone marrow antibody repertoire however, it is still unclear from the data whether there is a loss of host antigen-specific responsiveness because of the change in gene use.

Characterization of mouse ocular response to a 35-day spaceflight mission: Evidence of blood-retinal barrier disruption and ocular adaptations.

The health risks associated with spaceflight-induced ocular structural and functional damage has become a recent concern for NASA. The goal of the present study was to characterize the effects of spaceflight and reentry to 1 g on the structure and integrity of the retina and blood-retinal barrier (BRB) in the eye. To investigate possible mechanisms, changes in protein expression profiles were examined in mouse ocular tissue after spaceflight. Ten week old male C57BL/6 mice were launched to the International Space Station (ISS) on Space-X 12 at the Kennedy Space Center (KSC) on August, 2017. After a 35-day mission, mice were returned to Earth alive. Within 38 +/- 4 hours of splashdown, mice were euthanized and ocular tissues were collected for analysis. Ground control (GC) and vivarium control mice were maintained on Earth in flight hardware or normal vivarium cages respectively. Repeated intraocular pressure (IOP) measurements were performed before the flight launch and re-measured before the mice were euthanized after splashdown. IOP was significantly lower in post-flight measurements compared to that of pre-flight (14.4-19.3 mmHg vs 16.3-20.3 mmHg) (p < 0.05) for the left eye. Flight group had significant apoptosis in the retina and retinal vascular endothelial cells compared to control groups (p < 0.05). Immunohistochemical analysis of the retina revealed that an increased expression of aquaporin-4 (AQP-4) in the flight mice compared to controls gave strong indication of disturbance of BRB integrity. There were also a significant increase in the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) and a decrease in the expression of the BRB-related tight junction protein, Zonula occludens-1 (ZO-1). Proteomic analysis showed that many key proteins and pathways responsible for cell death, cell cycle, immune response, mitochondrial function and metabolic stress were significantly altered in the flight mice compared to ground control animals. These data indicate a complex cellular response that may alter retina structure and BRB integrity following long-term spaceflight.

A comparison of unamplified and massively multiplexed PCR amplification for murine antibody repertoire sequencing.

Sequencing antibody repertoires has steadily become cheaper and easier. Sequencing methods usually rely on some form of amplification, often a massively multiplexed PCR prior to sequencing. To eliminate potential biases and create a data set that could be used for other studies, our lab compared unamplified sequencing results from the splenic heavy-chain repertoire in the mouse to those processed through two commercial applications. We also compared the use of mRNA vs total RNA, reverse transcriptase, and primer usage for cDNA synthesis and submission. The use of mRNA for cDNA synthesis resulted in higher read counts but reverse transcriptase and primer usage had no statistical effects on read count. Although most of the amplified data sets contained more antibody reads than the unamplified data set, we detected more unique V-gene segments in the unamplified data set. Although unique CDR3 detection was much lower in the unamplified data set, RNASeq detected 98% of the high frequency CDR3s. We have shown that unamplified profiling of the antibody repertoire is possible, detects more V-gene segments, and detects high frequency clones in the repertoire.

Effects of skeletal unloading on the antibody repertoire of tetanus toxoid and/or CpG treated C57BL/6J mice.

Spaceflight affects the immune system, but the effects on the antibody repertoire, responsible for humoral immunity, has not been well explored. In particular, the complex gene assembly and expression process; including mutations, might make this process vulnerable. Complementarity determining region 3 (CDR3), composed of parts of the V-(D-)J-gene segments, is very important for antigen binding and can be used as an important measure of variability. Skeletal unloading, and the physiological effects of it, parallel many impacts of space flight. Therefore, we explored the impact of skeletal unloading using the antiorthostatic suspension (AOS) model. Animals were experimentally challenged with tetanus toxoid (TT) and/or the adjuvant CpG. Blood was analyzed for anti-TT antibody and corticosterone concentrations. Whole spleen tissue was prepared for repertoire characterization. AOS animals showed higher levels of corticosterone levels, but AOS alone did not affect anti-TT serum antibody levels. Administration of CpG significantly increased the circulating anti-TT antibody concentrations. AOS did alter constant gene usage resulting in higher levels of IgM and lower levels of IgG. CpG also altered constant gene region usage increasing usage of IgA. Significant changes could be detected in multiple V-, D-, and J-gene segments in both the heavy and light chains in response to AOS, TT, and CpG treatments. Analysis of class-switched only transcripts revealed a different pattern of V-gene segment usage than detected in the whole repertoire and also showed significant alterations in gene segment usage after challenge. Alterations in V/J pairing were also detected in response to challenge. CDR3 amino acid sequence overlaps were similar among treatment groups, though the addition of CpG lowered overlap in the heavy chain. We isolated 3,045 whole repertoire and 98 potentially TT-specific CDR3 sequences for the heavy chain and 569 for the light chain. Our results demonstrate that AOS alters the repertoire response to challenge with TT and/or CpG.

A comparison of unamplified and massively multiplexed PCR amplification for murine antibody repertoire sequencing.

Sequencing antibody repertoires has steadily become cheaper and easier. Sequencing methods usually rely on some form of amplification, often a massively multiplexed PCR prior to sequencing. To eliminate potential biases and create a data set that could be used for other studies, our laboratory compared unamplified sequencing results from the splenic heavy-chain repertoire in the mouse to those processed through two commercial applications. We also compared the use of mRNA vs total RNA, reverse transcriptase, and primer usage for cDNA synthesis and submission. The use of mRNA for cDNA synthesis resulted in higher read counts but reverse transcriptase and primer usage had no statistical effects on read count. Although most of the amplified data sets contained more antibody reads than the unamplified data set, we detected more unique variable (V)-gene segments in the unamplified data set. Although unique CDR3 detection was much lower in the unamplified data set, RNASeq detected 98% of the high-frequency CDR3s. We have shown that unamplified profiling of the antibody repertoire is possible, detects more V-gene segments, and detects high-frequency clones in the repertoire.

Proteomic Analysis of Mouse Brain Subjected to Spaceflight.

There is evidence that spaceflight poses acute and late risks to the central nervous system. To explore possible mechanisms, the proteomic changes following spaceflight in mouse brain were characterized. Space Shuttle Atlantis (STS-135) was launched from the Kennedy Space Center (KSC) on a 13-day mission. Within 3⁻5 h after landing, brain tissue was collected to evaluate protein expression profiles using quantitative proteomic analysis. Our results showed that there were 26 proteins that were significantly altered after spaceflight in the gray and/or white matter. While there was no overlap between the white and gray matter in terms of individual proteins, there was overlap in terms of function, synaptic plasticity, vesical activity, protein/organelle transport, and metabolism. Our data demonstrate that exposure to the spaceflight environment induces significant changes in protein expression related to neuronal structure and metabolic function. This might lead to a significant impact on brain structural and functional integrity that could affect the outcome of space missions.

Cardiovascular progenitor cells cultured aboard the International Space Station exhibit altered developmental and functional properties.

The heart and its cellular components are profoundly altered by missions to space and injury on Earth. Further research, however, is needed to characterize and address the molecular substrates of such changes. For this reason, neonatal and adult human cardiovascular progenitor cells (CPCs) were cultured aboard the International Space Station. Upon return to Earth, we measured changes in the expression of microRNAs and of genes related to mechanotransduction, cardiogenesis, cell cycling, DNA repair, and paracrine signaling. We additionally assessed endothelial-like tube formation, cell cycling, and migratory capacity of CPCs. Changes in microRNA expression were predicted to target extracellular matrix interactions and Hippo signaling in both neonatal and adult CPCs. Genes related to mechanotransduction (YAP1, RHOA) were downregulated, while the expression of cytoskeletal genes (VIM, NES, DES, LMNB2, LMNA), non-canonical Wnt ligands (WNT5A, WNT9A), and Wnt/calcium signaling molecules (PLCG1, PRKCA) was significantly elevated in neonatal CPCs. Increased mesendodermal gene expression along with decreased expression of mesodermal derivative markers (TNNT2, VWF, and RUNX2), reduced readiness to form endothelial-like tubes, and elevated expression of Bmp and Tbx genes, were observed in neonatal CPCs. Both neonatal and adult CPCs exhibited increased expression of DNA repair genes and paracrine factors, which was supported by enhanced migration. While spaceflight affects cytoskeletal organization and migration in neonatal and adult CPCs, only neonatal CPCs experienced increased expression of early developmental markers and an enhanced proliferative potential. Efforts to recapitulate the effects of spaceflight on Earth by regulating processes described herein may be a promising avenue for cardiac repair.

Impact of Spaceflight and Artificial Gravity on the Mouse Retina: Biochemical and Proteomic Analysis.

Astronauts are reported to have experienced some impairment in visual acuity during their mission on the International Space Station (ISS) and after they returned to Earth. There is emerging evidence that changes in vision may involve alterations in ocular structure and function. To investigate possible mechanisms, changes in protein expression profiles and oxidative stress-associated apoptosis were examined in mouse ocular tissue after spaceflight. Nine-week-old male C57BL/6 mice (n = 12) were launched from the Kennedy Space Center on a SpaceX rocket to the ISS for a 35-day mission. The animals were housed in the mouse Habitat Cage Unit (HCU) in the Japan Aerospace Exploration Agency (JAXA) "Kibo" facility on the ISS. The flight mice lived either under an ambient microgravity condition (µg) or in a centrifugal habitat unit that produced 1 g artificial gravity (µg + 1 g). Habitat control (HC) and vivarium control mice lived on Earth in HCUs or normal vivarium cages, respectively. Quantitative assessment of ocular tissue demonstrated that the µg group induced significant apoptosis in the retina vascular endothelial cells compared to all other groups (p < 0.05) that was 64% greater than that in the HC group. Proteomic analysis showed that many key pathways responsible for cell death, cell repair, inflammation, and metabolic stress were significantly altered in µg mice compared to HC animals. Additionally, there were more significant changes in regulated protein expression in the µg group relative to that in the µg + 1 g group. These data provide evidence that spaceflight induces retinal apoptosis of vascular endothelial cells and changes in retinal protein expression related to cellular structure, immune response and metabolic function, and that artificial gravity (AG) provides some protection against these changes. These retinal cellular responses may affect blood⁻retinal barrier (BRB) integrity, visual acuity, and impact the potential risk of developing late retinal degeneration.

Vive la radiorésistance!: converging research in radiobiology and biogerontology to enhance human radioresistance for deep space exploration and colonization.

While many efforts have been made to pave the way toward human space colonization, little consideration has been given to the methods of protecting spacefarers against harsh cosmic and local radioactive environments and the high costs associated with protection from the deleterious physiological effects of exposure to high-Linear energy transfer (high-LET) radiation. Herein, we lay the foundations of a roadmap toward enhancing human radioresistance for the purposes of deep space colonization and exploration. We outline future research directions toward the goal of enhancing human radioresistance, including upregulation of endogenous repair and radioprotective mechanisms, possible leeways into gene therapy in order to enhance radioresistance via the translation of exogenous and engineered DNA repair and radioprotective mechanisms, the substitution of organic molecules with fortified isoforms, and methods of slowing metabolic activity while preserving cognitive function. We conclude by presenting the known associations between radioresistance and longevity, and articulating the position that enhancing human radioresistance is likely to extend the healthspan of human spacefarers as well.

Effects of spaceflight on the immunoglobulin repertoire of unimmunized C57BL/6 mice.

Spaceflight has been shown to suppress the adaptive immune response, altering the distribution and function of lymphocyte populations. B lymphocytes express highly specific and highly diversified receptors, known as immunoglobulins (Ig), that directly bind and neutralize pathogens. Ig diversity is achieved through the enzymatic splicing of gene segments within the genomic DNA of each B cell in a host. The collection of Ig specificities within a host, or Ig repertoire, has been increasingly characterized in both basic research and clinical settings using high-throughput sequencing technology (HTS). We utilized HTS to test the hypothesis that spaceflight affects the B-cell repertoire. To test this hypothesis, we characterized the impact of spaceflight on the unimmunized Ig repertoire of C57BL/6 mice that were flown aboard the International Space Station (ISS) during the Rodent Research One validation flight in comparison to ground controls. Individual gene segment usage was similar between ground control and flight animals, however, gene segment combinations and the junctions in which gene segments combine was varied among animals within and between treatment groups. We also found that spontaneous somatic mutations in the IgH and Igκ gene loci were not increased. These data suggest that space flight did not affect the B cell repertoire of mice flown and housed on the ISS over a short period of time.

Characterization of the naive murine antibody repertoire using unamplified high-throughput sequencing.

Antibody specificity and diversity are generated through the enzymatic splicing of genomic gene segments within each B cell. Antibodies are heterodimers of heavy- and light-chains encoded on separate loci. We studied the antibody repertoire from pooled, splenic tissue of unimmunized, adult female C57BL/6J mice, using high-throughput sequencing (HTS) without amplification of antibody transcripts. We recovered over 90,000 heavy-chain and over 135,000 light-chain immunoglobulin sequences. Individual V-, D-, and J-gene segment usage was uniform among the three mouse pools, particularly in highly abundant gene segments, with low frequency V-gene segments not being detected in all pools. Despite the similar usage of individual gene segments, the repertoire of individual B-cell CDR3 amino acid sequences in each mouse pool was highly varied, affirming the combinatorial diversity in the B-cell pool that has been previously demonstrated. There also was some skewing in the V-gene segments that were detected depending on chromosomal location. This study presents a unique, non-primer biased glimpse of the conventionally housed, unimmunized antibody repertoire of the C57BL6/J mouse.

Spaceflight Activates Protein Kinase C Alpha Signaling and Modifies the Developmental Stage of Human Neonatal Cardiovascular Progenitor Cells.

Spaceflight impacts cardiovascular function in astronauts; however, its impact on cardiac development and the stem cells that form the basis for cardiac repair is unknown. Accordingly, further research is needed to uncover the potential relevance of such changes to human health. Using simulated microgravity (SMG) generated by two-dimensional clinorotation and culture aboard the International Space Station (ISS), we assessed the effects of mechanical unloading on human neonatal cardiovascular progenitor cell (CPC) developmental properties and signaling. Following 6-7 days of SMG and 12 days of ISS culture, we analyzed changes in gene expression. Both environments induced the expression of genes that are typically associated with an earlier state of cardiovascular development. To understand the mechanism by which such changes occurred, we assessed the expression of mechanosensitive small RhoGTPases in SMG-cultured CPCs and observed decreased levels of RHOA and CDC42. Given the effect of these molecules on intracellular calcium levels, we evaluated changes in noncanonical Wnt/calcium signaling. After 6-7 days under SMG, CPCs exhibited elevated levels of WNT5A and PRKCA. Similarly, ISS-cultured CPCs exhibited elevated levels of calcium handling and signaling genes, which corresponded to protein kinase C alpha (PKCα), a calcium-dependent protein kinase, activation after 30 days. Akt was activated, whereas phosphorylated extracellular signal-regulated kinase levels were unchanged. To explore the effect of calcium induction in neonatal CPCs, we activated PKCα using hWnt5a treatment on Earth. Subsequently, early cardiovascular developmental marker levels were elevated. Transcripts induced by SMG and hWnt5a-treatment are expressed within the sinoatrial node, which may represent embryonic myocardium maintained in its primitive state. Calcium signaling is sensitive to mechanical unloading and directs CPC developmental properties. Further research both in space and on Earth may help refine the use of CPCs in stem cell-based therapies and highlight the molecular events of development.