Comparative Analysis of Muscle Atrophy During Spaceflight, Nutritional Deficiency and Disuse in the Nematode Caenorhabditis elegans.

While spaceflight is becoming more common than before, the hazards spaceflight and space microgravity pose to the human body remain relatively unexplored. Astronauts experience muscle atrophy after spaceflight, but the exact reasons for this and solutions are unknown. Here, we take advantage of the nematode C. elegans to understand the effects of space microgravity on worm body wall muscle. We found that space microgravity induces muscle atrophy in C. elegans from two independent spaceflight missions. As a comparison to spaceflight-induced muscle atrophy, we assessed the effects of acute nutritional deprivation and muscle disuse on C. elegans muscle cells. We found that these two factors also induce muscle atrophy in the nematode. Finally, we identified clp-4, which encodes a calpain protease that promotes muscle atrophy. Mutants of clp-4 suppress starvation-induced muscle atrophy. Such comparative analyses of different factors causing muscle atrophy in C. elegans could provide a way to identify novel genetic factors regulating space microgravity-induced muscle atrophy.

Loss of physical contact in space alters the dopamine system in C. elegans.

Progressive neuromuscular decline in microgravity is a prominent health concern preventing interplanetary human habitation. We establish functional dopamine-mediated impairments as a consistent feature across multiple spaceflight exposures and during simulated microgravity in C. elegans. Animals grown continuously in these conditions display reduced movement and body length. Loss of mechanical contact stimuli in microgravity elicits decreased endogenous dopamine and comt-4 (catechol-O-methyl transferase) expression levels. The application of exogenous dopamine reverses the movement and body length defects caused by simulated microgravity. In addition, increased physical contact made comt-4 and dopamine levels rise. It also increased muscular cytoplasmic Ca2+ firing. In dop-3 (D2-like receptor) mutants, neither decrease in movement nor in body length were observed during simulated microgravity growth. These results strongly suggest that targeting the dopamine system through manipulation of the external environment (contact stimuli) prevents muscular changes and is a realistic and viable treatment strategy to promote safe human deep-space travel.

Simultaneous nodular lymphocyte-predominant Hodgkin lymphoma with papillary thyroid carcinoma: a case report.

We herein report the case of a 48-year-old man diagnosed with nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL, Stage IA) and papillary thyroid carcinoma (PTC, Stage I). Total thyroidectomy, left modified neck dissection and biopsy of the right cervical lymph node were performed. Postoperatively, NLPHL treatment was prioritized, and external radiation (30.6 Gy) was applied to the right neck. PTC was considered a high-risk category for recurrence due to extranodal invasion of lymph node metastasis, and radioactive iodine therapy (ablative dose, 1110 MBq) was administered. Both PTC and NLPHL showed no recurrence 18 months after surgery.

Histone deacetylase HDA-4-mediated epigenetic regulation in space-flown C. elegans.

Epigenetic changes during long-term spaceflight are beginning to be studied by NASA's twin astronauts and other model organisms. Here, we evaluate the epigenetic regulation of gene expression in space-flown C. elegans by comparing wild type and histone deacetylase (hda)-4 mutants. Expression levels of 39 genes were consistently upregulated in all four generations of adult hda-4 mutants grown under microgravity compared with artificial Earth-like gravity (1G). In contrast, in the wild type, microgravity-induced upregulation of these genes occurred a little. Among these genes, 11 contain the domain of unknown function 19 (DUF-19) and are located in a cluster on chromosome V. When compared with the 1G condition, histone H3 trimethylation at lysine 27 (H3K27me3) increased under microgravity in the DUF-19 containing genes T20D4.12 to 4.10 locus in wild-type adults. On the other hand, this increase was also observed in the hda-4 mutant, but the level was significantly reduced. The body length of wild-type adults decreased slightly but significantly when grown under microgravity. This decrease was even more pronounced with the hda-4 mutant. In ground-based experiments, one of the T20D4.11 overexpressing strains significantly reduced body length and also caused larval growth retardation and arrest. These results indicate that under microgravity, C. elegans activates histone deacetylase HDA-4 to suppress overregulation of several genes, including the DUF-19 family. In other words, the expression of certain genes, including negative regulators of growth and development, is epigenetically fine-tuned to adapt to the space microgravity.

Microgravity elicits reproducible alterations in cytoskeletal and metabolic gene and protein expression in space-flown Caenorhabditis elegans.

Although muscle atrophy is a serious problem during spaceflight, little is known about the sequence of molecular events leading to atrophy in response to microgravity. We carried out a spaceflight experiment using Caenorhabditis elegans onboard the Japanese Experiment Module of the International Space Station. Worms were synchronously cultured in liquid media with bacterial food for 4 days under microgravity or on a 1-G centrifuge. Worms were visually observed for health and movement and then frozen. Upon return, we analyzed global gene and protein expression using DNA microarrays and mass spectrometry. Body length and fat accumulation were also analyzed. We found that in worms grown from the L1 larval stage to adulthood under microgravity, both gene and protein expression levels for muscular thick filaments, cytoskeletal elements, and mitochondrial metabolic enzymes decreased relative to parallel cultures on the 1-G centrifuge (95% confidence interval (P⩽0.05)). In addition, altered movement and decreased body length and fat accumulation were observed in the microgravity-cultured worms relative to the 1-G cultured worms. These results suggest protein expression changes that may account for the progressive muscular atrophy observed in astronauts.

Fluid dynamics alter Caenorhabditis elegans body length via TGF-ß/DBL-1 neuromuscular signaling.

Skeletal muscle wasting is a major obstacle for long-term space exploration. Similar to astronauts, the nematode Caenorhabditis elegans displays negative muscular and physical effects when in microgravity in space. It remains unclear what signaling molecules and behavior(s) cause these negative alterations. Here we studied key signaling molecules involved in alterations of C. elegans physique in response to fluid dynamics in ground-based experiments. Placing worms in space on a 1G accelerator increased a myosin heavy chain, myo-3, and a transforming growth factor-ß (TGF-ß), dbl-1, gene expression. These changes also occurred when the fluid dynamic parameters viscosity/drag resistance or depth of liquid culture were increased on the ground. In addition, body length increased in wild type and body wall cuticle collagen mutants, rol-6 and dpy-5, grown in liquid culture. In contrast, body length did not increase in TGF-ß, dbl-1, or downstream signaling pathway, sma-4/Smad, mutants. Similarly, a D1-like dopamine receptor, DOP-4, and a mechanosensory channel, UNC-8, were required for increased dbl-1 expression and altered physique in liquid culture. As C. elegans contraction rates are much higher when swimming in liquid than when crawling on an agar surface, we also examined the relationship between body length enhancement and rate of contraction. Mutants with significantly reduced contraction rates were typically smaller. However, in dop-4, dbl-1, and sma-4 mutants, contraction rates still increased in liquid. These results suggest that neuromuscular signaling via TGF-ß/DBL-1 acts to alter body physique in response to environmental conditions including fluid dynamics.

Expression of p53-regulated proteins in human cultured lymphoblastoid TSCE5 and WTK1 cell lines during spaceflight.

The aim of this study was to determine the biological effects of space radiations, microgravity, and the interaction of them on the expression of p53-regulated proteins. Space experiments were performed with two human cultured lymphoblastoid cell lines: one line (TSCE5) bears a wild-type p53 gene status, and another line (WTK1) bears a mutated p53 gene status. Under 1 gravity or microgravity conditions, the cells were grown in the cell biology experimental facility (CBEF) of the International Space Station for 8 days without experiencing the stress during launching and landing because the cells were frozen during these periods. Ground control samples were simultaneously cultured for 8 days in the CBEF on the ground for 8 days. After spaceflight, protein expression was analyzed using a Panorama(TM) Ab MicroArray protein chips. It was found that p53-dependent up-regulated proteins in response to space radiations and space environment were MeCP2 (methyl CpG binding protein 2), and Notch1 (Notch homolog 1), respectively. On the other hand, p53-dependent down-regulated proteins were TGF-ß, TWEAKR (tumor necrosis factor-like weak inducer of apoptosis receptor), phosho-Pyk2 (Proline-rich tyrosine kinase 2), and 14-3-3θ/τ which were affected by microgravity, and DR4 (death receptor 4), PRMT1 (protein arginine methyltransferase 1) and ROCK-2 (Rho-associated, coiled-coil containing protein kinase 2) in response to space radiations. ROCK-2 was also suppressed in response to the space environment. The data provides the p53-dependent regulated proteins by exposure to space radiations and/or microgravity during spaceflight. Our expression data revealed proteins that might help to advance the basic space radiation biology.

The effectiveness of RNAi in Caenorhabditis elegans is maintained during spaceflight.

BACKGROUND: Overcoming spaceflight-induced (patho)physiologic adaptations is a major challenge preventing long-term deep space exploration. RNA interference (RNAi) has emerged as a promising therapeutic for combating diseases on Earth; however the efficacy of RNAi in space is currently unknown. METHODS: Caenorhabditis elegans were prepared in liquid media on Earth using standard techniques and treated acutely with RNAi or a vector control upon arrival in Low Earth Orbit. After culturing during 4 and 8 d spaceflight, experiments were stopped by freezing at -80°C until analysis by mRNA and microRNA array chips, microscopy and Western blot on return to Earth. Ground controls (GC) on Earth were simultaneously grown under identical conditions. RESULTS: After 8 d spaceflight, mRNA expression levels of components of the RNAi machinery were not different from that in GC (e.g., Dicer, Argonaute, Piwi; P>0.05). The expression of 228 microRNAs, of the 232 analysed, were also unaffected during 4 and 8 d spaceflight (P>0.05). In spaceflight, RNAi against green fluorescent protein (gfp) reduced chromosomal gfp expression in gonad tissue, which was not different from GC. RNAi against rbx-1 also induced abnormal chromosome segregation in the gonad during spaceflight as on Earth. Finally, culture in RNAi against lysosomal cathepsins prevented degradation of the muscle-specific α-actin protein in both spaceflight and GC conditions. CONCLUSIONS: Treatment with RNAi works as effectively in the space environment as on Earth within multiple tissues, suggesting RNAi may provide an effective tool for combating spaceflight-induced pathologies aboard future long-duration space missions. Furthermore, this is the first demonstration that RNAi can be utilised to block muscle protein degradation, both on Earth and in space.

Occurrence and characteristics of group 1 introns found at three different positions within the 28S ribosomal RNA gene of the dematiaceous Phialophora verrucosa: phylogenetic and secondary structural implications.

BACKGROUND: Group 1 introns (ribozymes) are among the most ancient and have the broadest phylogenetic distribution among the known self-splicing ribozymes. Fungi are known to be rich in rDNA group 1 introns. In the present study, five sequences of the 28S ribosomal RNA gene (rDNA) regions of pathogenic dematiaceous Phialophora verrucosa were analyzed using PCR by site-specific primers and were found to have three insertions, termed intron-F, G and H, at three positions of the gene. We investigated the distribution of group 1 introns in this fungus by surveying 34 strains of P. verrucosa and seven strains of Phialophora americana as the allied species. RESULTS: Intron-F's (inserted at L798 position) were found in 88% of P. verrucosa strains, while intron-G's (inserted at L1921) at 12% and intron-H's (inserted at L2563) at 18%. There was some correlation between intron distribution and geographic location. In addition, we confirmed that the three kinds of introns are group 1 introns from results of BLAST search, alignment analysis and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Prediction of secondary structures and phylogenetic analysis of intron sequences identified introns-F and G as belonging to subgroup IC1. In addition, intron-H was identified as IE. CONCLUSION: The three intron insertions and their insertion position in the 28S rDNA allowed the characterization of the clinical and environmental isolates of P. verrucosa and P. americana into five genotypes. All subgroups of introns-F and G and intron-H were characterized and observed for the first time in both species.

Frozen human cells can record radiation damage accumulated during space flight: mutation induction and radioadaptation.

To estimate the space-radiation effects separately from other space-environmental effects such as microgravity, frozen human lymphoblastoid TK6 cells were sent to the "Kibo" module of the International Space Station (ISS), preserved under frozen condition during the mission and finally recovered to Earth (after a total of 134 days flight, 72 mSv). Biological assays were performed on the cells recovered to Earth. We observed a tendency of increase (2.3-fold) in thymidine kinase deficient (TK(-)) mutations over the ground control. Loss of heterozygosity (LOH) analysis on the mutants also demonstrated a tendency of increase in proportion of the large deletion (beyond the TK locus) events, 6/41 in the in-flight samples and 1/17 in the ground control. Furthermore, in-flight samples exhibited 48% of the ground-control level in TK(-) mutation frequency upon exposure to a subsequent 2 Gy dose of X-rays, suggesting a tendency of radioadaptation when compared with the ground-control samples. The tendency of radioadaptation was also supported by the post-flight assays on DNA double-strand break repair: a 1.8- and 1.7-fold higher efficiency of in-flight samples compared to ground control via non-homologous end-joining and homologous recombination, respectively. These observations suggest that this system can be used as a biodosimeter, because DNA damage generated by space radiation is considered to be accumulated in the cells preserved frozen during the mission, Furthermore, this system is also suggested to be applicable for evaluating various cellular responses to low-dose space radiation, providing a better understanding of biological space-radiation effects as well as estimation of health influences of future space explores.

The next phase of life-sciences spaceflight research: Harnessing the power of functional genomics.

Recently we demonstrated that the effectiveness of RNAi interference (RNAi) for inhibiting gene expression is maintained during spaceflight in the worm Caenorhabditis elegans and argued for the biomedical importance of this finding. We also successfully utilized green fluorescent protein (GFP)-tagged proteins to monitor changes in GPF localization during flight. Here we discuss potential applications of RNAi and GFP in spaceflight studies and the ramifications of these experiments for the future of space life-sciences research.

The expression of p53-regulated genes in human cultured lymphoblastoid TSCE5 and WTK1 cell lines during spaceflight.

PURPOSE: The space environment contains two major biologically significant influences; space radiations and microgravity. The 53 kDa tumour suppressor protein (p53) plays a role as a guardian of the genome through the activity of p53-centered signal transduction pathways. The aim of this study was to clarify the biological effects of space radiations, microgravity, and the space environment on the gene expression of p53-regulated genes. MATERIALS AND METHODS: Space experiments were performed with two human cultured lymphoblastoid cell lines; one line (TSCE5) bears a wild-type p53 gene status, and another line (WTK1) bears a mutated p53 gene status. Under one gravity or microgravity conditions, the cells were grown in the cell biology experimental facility (CBEF) of the International Space Station for 8 days without experiencing stress during launching and landing because the cells were frozen during these periods. Ground control samples also were cultured for 8 days in the CBEF on the ground during the spaceflight. Gene expression was analysed using an Agilent Technologies 44 k whole human genome microarray DNA chip. RESULTS: p53-dependent up-regulated gene expression was observed for 111, 95, and 328 genes and p53-dependent down-regulated gene expression was found for 177, 16, and 282 genes after exposure to space radiations, to microgravity, and to both, respectively. CONCLUSIONS: The data provide the p53-dependent regulated genes by exposure to radiations and/or microgravity during spaceflight. Our expression data revealed genes that might help to advance the basic space radiation biology.

p53-dependent adaptive responses in human cells exposed to space radiations.

PURPOSE: It has been reported that priming irradiation or conditioning irradiation with a low dose of X-rays in the range of 0.02-0.1 Gy induces a p53-dependent adaptive response in mammalian cells. The aim of the present study was to clarify the effect of space radiations on the adaptive response. METHODS AND MATERIALS: Two human lymphoblastoid cell lines were used; one cell line bears a wild-type p53 (wtp53) gene, and another cell line bears a mutated p53 (mp53) gene. The cells were frozen during transportation on the space shuttle and while in orbit in the International Space Station freezer for 133 days between November 15, 2008 and March 29, 2009. After the frozen samples were returned to Earth, the cells were cultured for 6 h and then exposed to a challenging X-ray-irradiation (2 Gy). Cellular sensitivity, apoptosis, and chromosome aberrations were scored using dye-exclusion assays, Hoechst33342 staining assays, and chromosomal banding techniques, respectively. RESULTS: In cells exposed to space radiations, adaptive responses such as the induction of radioresistance and the depression of radiation-induced apoptosis and chromosome aberrations were observed in wtp53 cells but not in mp53 cells. CONCLUSION: These results have confirmed the hypothesis that p53-dependent adaptive responses are apparently induced by space radiations within a specific range of low doses. The cells exhibited this effect owing to space radiations exposure, even though the doses in space were very low.

C. elegans RNAi space experiment (CERISE) in Japanese Experiment Module KIBO.

We have started a space experiment using an experimental organism, the nematode Caenorhabditis elegans, in the Japanese Experiment Module, KIBO, of the International Space Station (ISS). The specimens were boarded by space shuttle Atlantis on mission STS-129 which launched from NASA Kennedy Space Center on November 16, 2009. The purpose of the experiment was several-fold: (i) to verify the efficacy of RNA interference (RNAi) in space, (ii) to monitor transcriptional and post-translational alterations in the entire genome in space, and (iii) to investigate mechanisms regulating and countermeasures for muscle alterations in response to the space environment. In particular, this will be the first study to utilize RNAi in space.