Effect of microgravity on mammalian embryo development evaluated at the International Space Station.

Mammalian embryos differentiate into the inner cell mass (ICM) and trophectoderm at the 8-16 cell stage. The ICM forms a single cluster that develops into a single fetus. However, the factors that determine differentiation and single cluster formation are unknown. Here we investigated whether embryos could develop normally without gravity. As the embryos cannot be handled by an untrained astronaut, a new device was developed for this purpose. Using this device, two-cell frozen mouse embryos launched to the International Space Station were thawed and cultured by the astronauts under microgravity for 4 days. The embryos cultured under microgravity conditions developed into blastocysts with normal cell numbers, ICM, trophectoderm, and gene expression profiles similar to those cultured under artificial-1 g control on the International Space Station and ground-1 g control, which clearly demonstrated that gravity had no significant effect on the blastocyst formation and initial differentiation of mammalian embryos.

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.

Comprehensive analyses of plant hormones in etiolated pea and maize seedlings grown under microgravity conditions in space: Relevance to the International Space Station experiment "Auxin Transport".

Functional relationships between endogenous levels of plant hormones in the growth and development of shoots in etiolated Alaska pea and etiolated Golden Cross Bantam maize seedlings under different gravities were investigated in the "Auxin Transport" experiment aboard the International Space Station (ISS). Comprehensive analyses of 31 species of plant hormones of pea and maize seedlings grown under microgravity (µg) in space and 1 g conditions were conducted. Principal component analysis (PCA) and a multiple regression analysis with the dataset from the plant hormone analysis of the etiolated pea seedlings grown under µg and 1 g conditions in the presence and absence of 2,3,5-triiodobenzoic acid (TIBA) revealed endogenous levels of auxin correlated positively with bending and length of epicotyls. Endogenous cytokinins correlated negatively with them. These results suggest an interaction of auxin and cytokinins in automorphogenesis and growth inhibition of etiolated Alaska pea epicotyls grown under µg conditions in space. Less polar auxin transport with reduced endogenous levels of auxin increased endogenous levels of cytokinins, resulting in changing the growth direction of epicotyls and inhibiting growth. On the other hand, almost no close relationship between endogenous plant hormone levels and growth and development in etiolated maize seedlings grown was observed under µg conditions in space, as per Schulze et al. (1992). However, endogenous levels of IAA in the seedlings grown under µg conditions in space were significantly higher than those grown on Earth, similar to the cases of polar auxin transport already reported.

Muscle synergies of multidirectional postural control in astronauts on Earth after a long-term stay in space.

Movements of the human biological system have adapted to the physical environment under the 1-g gravitational force on Earth. However, the effects of microgravity in space on the underlying functional neuromuscular control behaviors remain poorly understood. Here, we aimed to elucidate the effects of prolonged exposure to a microgravity environment on the functional coordination of multiple muscle activities. The activities of 16 lower limb muscles of 5 astronauts who stayed in space for at least 3 mo were recorded while they maintained multidirectional postural control during bipedal standing. The coordinated activation patterns of groups of muscles, i.e., muscle synergies, were estimated from the muscle activation datasets using a factorization algorithm. The experiments were repeated a total of five times for each astronaut, once before and four times after spaceflight. The compositions of muscle synergies were altered, with a constant number of synergies, after long-term exposure to microgravity, and the extent of the changes was correlated with the increased velocity of postural sway. Furthermore, the muscle synergies extracted 3 mo after the return were similar in their activation profile but not in their muscle composition compared with those extracted in the preflight condition. These results suggest that the modularity in the neuromuscular system became reorganized to adapt to the microgravity environment and then possibly reoptimized to the new sensorimotor environment after the astronauts were reexposed to a gravitational force. It is expected that muscle synergies can be used as physiological markers of the status of astronauts with gravity-dependent change.NEW & NOTEWORTHY The human neuromuscular system has adapted to the gravitational environment on Earth. Here, we demonstrated that prolonged exposure to a microgravity environment in space changes the functional coordination of multiple muscle activities regarding multidirectional standing postural control. Furthermore, the amount of change led to a greater regulatory balancing activity needed for postural control immediately after returning to Earth and differences in muscular coordination before space flight and 3 mo after the return to Earth.

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.

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.

Evaluating the long-term effect of space radiation on the reproductive normality of mammalian sperm preserved on the International Space Station.

Space radiation may cause DNA damage to cells and concern for the inheritance of mutations in offspring after deep space exploration. However, there is no way to study the long-term effects of space radiation using biological materials. Here, we developed a method to evaluate the biological effect of space radiation and examined the reproductive potential of mouse freeze-dried spermatozoa stored on the International Space Station (ISS) for the longest period in biological research. The space radiation did not affect sperm DNA or fertility after preservation on ISS, and many genetically normal offspring were obtained without reducing the success rate compared to the ground-preserved control. The results of ground x-ray experiments showed that sperm can be stored for more than 200 years in space. These results suggest that the effect of deep space radiation on mammalian reproduction can be evaluated using spermatozoa, even without being monitored by astronauts in Gateway.

Findings from recent studies by the Japan Aerospace Exploration Agency examining musculoskeletal atrophy in space and on Earth.

The musculoskeletal system provides the body with correct posture, support, stability, and mobility. It is composed of the bones, muscles, cartilage, tendons, ligaments, joints, and other connective tissues. Without effective countermeasures, prolonged spaceflight under microgravity results in marked muscle and bone atrophy. The molecular and physiological mechanisms of this atrophy under unloaded conditions are gradually being revealed through spaceflight experiments conducted by the Japan Aerospace Exploration Agency using a variety of model organisms, including both aquatic and terrestrial animals, and terrestrial experiments conducted under the Living in Space project of the Japan Ministry of Education, Culture, Sports, Science, and Technology. Increasing our knowledge in this field will lead not only to an understanding of how to prevent muscle and bone atrophy in humans undergoing long-term space voyages but also to an understanding of countermeasures against age-related locomotive syndrome in the elderly.

Altered localisation of ZmPIN1a proteins in plasma membranes responsible for enhanced-polar auxin transport in etiolated maize seedlings under microgravity conditions in space.

In the International Space Station experiment 'Auxin Transport', polar auxin transport (PAT) in shoots of etiolated maize (Zea mays L. cv. Golden Cross Bantam) grown under microgravity in space was substantially enhanced compared with those grown on Earth. To clarify the mechanism, the effects of microgravity on expression of ZmPIN1a encoding essential auxin efflux carrier and cellular localisation of its products were investigated. The amounts of ZmPIN1a mRNA in the coleoptiles and the mesocotyls in space-grown seedlings were almost the same as those in 1 g-grown seedlings, but its products were not. Immunohistochemical analysis with anti-ZmPIN1a antibody revealed a majority of ZmPIN1a localised in the basal side of plasma membranes of endodermal cells in the coleoptiles and the mesocotyls, and in the basal and lateral sides of plasma membranes in coleoptile parenchymatous cells, in which it directed towards the radial direction, but not towards the vascular bundle direction. Microgravity dramatically altered ZmPIN1a localisation in plasma membranes in coleoptile parenchymatous cells, shifting mainly towards the vascular bundle direction. These results suggest that mechanism of microgravity-enhanced PAT in maize shoots is more likely to be due to the enhanced ZmPIN1a accumulation and the altered ZmPIN1a localisation in parenchymatous cells of the coleoptiles.

Microarray profile of gene expression in etiolated Pisum sativum seedlings grown under microgravity conditions in space: Relevance to the International Space Station experiment "Auxin Transport".

This paper introduces the use of microarray data technology with Medicago (Medicago truncatula) microarrays to characterize global changes in the transcript abundance of etiolated Alaska pea (Pisum sativum L.) seedlings grown under microgravity (µg) conditions in comparison with those under artificial 1 g conditions on the International Space Station. Of the 44,000 genes of the Medicago microarray platform, more than 25,000 transcripts of pea seedlings were hybridized, suggesting that the microarray platform for Medicago could be useful in the study of gene expression of etiolated pea seedlings grown under µg conditions in space. Gene array data were analyzed according to stringent criteria that restricted the scored genes for specific hybridization values at least twofold. Expression of 1362 and 1558 genes in proximal side (the proximal side) and distal side of the epicotyl to the cotyledons (the distal side), respectively, were highly affected by µg conditions in space. Of the genes analyzed, 407 of 1362 transcripts in the proximal side and 740 of 1558 transcripts in the distal side were expressed at ratios at least twofold. However, in the presence of the auxin transport inhibitor TIBA, 212 of 399 transcripts and 255 of 477 transcripts were expressed at ratios at least twofold as high in the proximal and the distal sides of epicotyls in the seedlings grown under µg conditions, respectively. Based on Venn diagram analysis, 31 transcripts and 24 transcripts were found to commonly increase and decrease, respectively, under µg conditions in space. Venn analysis revealed six auxin-related genes and three water channel AQUAPORIN genes that were responsive to gravity. Among 6 auxin-related genes, the accumulation of transcripts of Auxin-induced protein 5NG4 and Indole-3-acetic acid-amido synthetase GH3.3 tended to increase, and that of Auxin-induced protein, Auxin response factor, SAUR-like auxin-responsive family protein and Auxin response factor tended to decrease under µg conditions, whereas there were no statistic differences between under µg and artificial 1 g conditions. Similarly there were no statistic differences between under µg conditions and artificial 1 g, but the accumulation of NIP3-1 and Plasma membrane intrinsic protein11, and AQUAPORIN1/Tonoplast intrinsic protein tended to increase and decrease, respectively. A possible role of auxin-related genes and AQUAPORIN genes in regulating growth of etiolated pea seedlings grown under µg conditions in space is discussed.

Author Correction: Changes in mitochondrial homeostasis and redox status in astronauts following long stays in space.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Gravity-regulated localization of PsPIN1 is important for polar auxin transport in etiolated pea seedlings: Relevance to the International Space Station experiment.

To clarify the mechanism of gravity-controlled polar auxin transport, we conducted the International Space Station (ISS) experiment "Auxin Transport" (identified by NASA's operation nomenclature) in 2016 and 2017, focusing on the expression of genes related to auxin efflux carrier protein PsPIN1 and its localization in the hook and epicotyl cells of etiolated Alaska pea seedlings grown for three days in the dark under microgravity (µg) and artificial 1 g conditions on a centrifuge in the Cell Biology Experiment Facility (CBEF) in the ISS, and under 1 g conditions on Earth. Regardless of gravity conditions, the accumulation of PsPIN1 mRNA in the proximal side of epicotyls of the seedlings was not different, but tended to be slightly higher as compared with that in the distal side. 2,3,5-Triiodobenzoic acid (TIBA) also did not affect the accumulation of PsPIN1 mRNA in the proximal and distal sides of epicotyls. However, in the apical hook region, TIBA increased the accumulation of PsPIN1 mRNA under µg conditions as compared with that under artificial 1 g conditions in the ISS. The accumulation of PsPIN1 proteins in epicotyls determined by western blotting was almost parallel to that of mRNA of PsPIN1. Immunohistochemical analysis with a specific polyclonal antibody of PsPIN1 revealed that a majority of PsPIN1 in the apical hook and subapical regions of the seedlings grown under artificial 1 g conditions in the ISS localized in the basal side (rootward) of the plasma membrane of the endodermal tissues. Conversely, in the seedlings grown under µg conditions, localization of PsPIN1 was greatly disarrayed. TIBA substantially altered the cellular localization pattern of PsPIN1, especially under µg conditions. These results strongly suggest that the mechanisms by which gravity controls polar auxin transport are more likely to be due to the membrane localization of PsPIN1. This physiologically valuable report describes a close relationship between gravity-controlled polar auxin transport and the localization of auxin efflux carrier PsPIN1 in etiolated pea seedlings based on the µg experiment conducted in space.

Polar auxin transport is essential to maintain growth and development of etiolated pea and maize seedlings grown under 1 g conditions: Relevance to the international space station experiment.

We conducted "Auxin Transport" space experiments in 2016 and 2017 in the Japanese Experiment Module (JEM) on the International Space Station (ISS), with the principal objective being integrated analyses of the growth and development of etiolated pea (Pisum sativum L. cv Alaska) and maize (Zea mays L. cv Golden Cross Bantam) seedlings under true microgravity conditions in space relative to auxin dynamics. Etiolated pea seedlings grown under microgravity conditions in space for 3 days showed automorphogenesis. Epicotyls and roots bent ca. 45° and 20° toward the direction away from the cotyledons, respectively, whereas those grown under artificial 1 g conditions produced by a centrifuge in the Cell Biology Experimental Facility (CBEF) in space showed negative and positive gravitropic response in epicotyls and in roots, respectively. On the other hand, the coleoptiles of 4-day-old etiolated maize seedlings grew almost straight, but the mesocotyls curved and grew toward a random direction under microgravity conditions in space. In contrast, the coleoptiles and mesocotyls of etiolated maize seedlings grown under 1 g conditions on Earth were almost straight and grew upward or toward the direction against the gravity vector. The polar auxin transport activity in etiolated pea epicotyls and in maize shoots was significantly inhibited and enhanced, respectively, under microgravity conditions in space as compared with artificial 1 g conditions in space or 1 g conditions on Earth. An inhibitor of polar auxin transport, 2,3,5-triiodobenzoic acid (TIBA) substantially affected the growth direction and polar auxin transport activity in etiolated pea seedlings grown under both artificial 1 g and microgravity conditions in space. These results strongly suggest that adequate polar auxin transport is essential for gravitropic response in plants. Possible mechanisms enhancing polar auxin transport in etiolated maize seedlings grown under microgravity conditions in space are also proposed.

Effectiveness of endothelial progenitor cell culture under microgravity for improved angiogenic potential.

Endothelial progenitor cell (EPC) transplantation is beneficial for ischemic diseases such as critical limb ischemia and ischemic heart disease. The scarcity of functional EPCs in adults is a limiting factor for EPC transplantation therapy. The quality and quantity culture (QQc) system is an effective ex vivo method for enhancing the number and angiogenic potential of EPCs. Further, microgravity environments have been shown to enhance the functional potential of stem cells. We therefore hypothesized that cells cultured with QQc under microgravity may have enhanced functionality. We cultured human peripheral blood mononuclear cells using QQc under normal (E), microgravity (MG), or microgravity followed by normal (ME) conditions and found that ME resulted in the most significant increase in CD34+ and double positive Dil-Ac-LDL-FITC-Ulex-Lectin cells, both EPC markers. Furthermore, angiogenic potential was determined by an EPC-colony forming assay. While numbers of primitive EPC-colony forming units (pEPC-CFU) did not change, numbers of definitive EPC-CFU colonies increased most under ME conditions. Gene-expression profiling also identified increases in angiogenic factors, including vascular endothelial growth factor, under MG and ME conditions. Thus, QQc along with ME conditions could be an efficient system for significantly enhancing the number and angiogenic potential of EPCs.

Procedures for chemical fixation in immunohistochemical analyses of PIN proteins regulating polar auxin transport: Relevance to spaceflight experiments.

The mechanism by which gravity controls the polar transport of auxin, a plant hormone regulating multiple physiological processes in higher plants, remains unclear, although an important role of PIN proteins as efflux carriers/facilitators in polar auxin transport is suggested. We are going to study the effect of microgravity on the polar transport of auxin, focusing on the cellular localization of its efflux carrier, PsPIN1 in etiolated pea seedlings and ZmPIN1a in etiolated maize seedlings grown under microgravity conditions on the International Space Station (ISS) using immunohistochemical analyses according to space experimental plans (Ueda, 2016). To obtain adequate results regarding the cellular localization of functional proteins, prolonged chemical fixation processes as well as chemical fixatives should be well-matched to the properties of functional proteins as antigens since experimental analyses will be performed on the ground after keeping samples for a long duration on the ISS. As a result of ground verification, clear detection of the cellular localization of PsPIN1 and ZmPIN1a immunohistochemically was successful based on the results of several kinds of chemical fixation tested, even when etiolated pea and maize seedlings were fixed by immersion in chemical fixative for a long duration. The addition of 0.1% (w/v) Nonidet P-40 to chemical fixative composed of 50% (v/v) ethanol and 5% (v/v) acetic acid or that of 50% (v/v) methanol and 5% (v/v) acetic acid has led to a significant improvement in the immunohistochemical detection of PsPIN1 or ZmPIN1a. These chemical fixatives were also shown to be storage-stable for a long time before use. In this study, adequate chemical fixatives and fixation protocols were developed, which can be used to detect localization of PsPIN1 and ZmPIN1a proteins in young etiolated pea and maize seedlings, respectively, using anti PsPIN1 and ZmPIN1a antibodies. These protocols can be used in spaceflight experiments to investigate the effects of the microgravity environment on the ISS on PIN protein localization in pea and maize seedlings.

Regulation of asymmetric polar auxin transport by PsPIN1 in endodermal tissues of etiolated Pisum sativum epicotyls: focus on immunohistochemical analyses.

This manuscript reports the production of specific polyclonal antibodies for PsPIN1, a putative auxin efflux carrier in Alaska pea (Pisum sativum L.) plants, and the cellular immunolocalization of PsPIN1. When pea seeds were set with the seed axis horizontal to the upper surface of a rockwool block, and allowed to germinate and grow for 3 days in the dark, the epicotyl grew upward. On the other hand, the application of 2,3,5-triiodobenzoic acid (TIBA) inhibited graviresponse. In the subapical epicotyl regions, PsPIN1 has been found to localize in the basal side of the plasma membrane of cells in endodermal tissues. Asymmetric PsPIN1 localization between the proximal and distal sides of the epicotyl was observed, the total amounts of PsPIN1 being more abundant in the proximal side. The asymmetric PsPIN1 distribution between the proximal and distal sides of the epicotyl was well correlated with unequal polar auxin transport as well as asymmetric accumulation of mRNA of PsPIN1 (Ueda et al. in Biol Sci Space 26:32-41, 2012; Ueda et al. in Plant Biol 16(suppl 1):43-49, 2014). In the proximal side of an apical hook, PsPIN1 localized in the basal side of the plasma membrane of cells in endodermal tissues, whereas in the distal side, the abundant distribution of PsPIN1 localized in the basal-lower (endodermal) side of the basal plasma membrane, suggesting possible lateral auxin movement from the distal side to the proximal side in this region. The application of TIBA significantly reduced the amount of PsPIN1 in the proximal side of epicotyls, but little in the distal side. These results suggest that unequal auxin transport in epicotyls during the early growth stage of etiolated pea seedlings is derived from asymmetric PsPIN1 localization in the apical hook and subapical region of epicotyls, and that asymmetric transport between the proximal and distal sides of epicotyls is required for the graviresponse of epicotyls.

Reactive oxygen species upregulate expression of muscle atrophy-associated ubiquitin ligase Cbl-b in rat L6 skeletal muscle cells.

Unloading-mediated muscle atrophy is associated with increased reactive oxygen species (ROS) production. We previously demonstrated that elevated ubiquitin ligase casitas B-lineage lymphoma-b (Cbl-b) resulted in the loss of muscle volume (Nakao R, Hirasaka K, Goto J, Ishidoh K, Yamada C, Ohno A, Okumura Y, Nonaka I, Yasutomo K, Baldwin KM, Kominami E, Higashibata A, Nagano K, Tanaka K, Yasui N, Mills EM, Takeda S, Nikawa T. Mol Cell Biol 29: 4798-4811, 2009). However, the pathological role of ROS production associated with unloading-mediated muscle atrophy still remains unknown. Here, we showed that the ROS-mediated signal transduction caused by microgravity or its simulation contributes to Cbl-b expression. In L6 myotubes, the assessment of redox status revealed that oxidized glutathione was increased under microgravity conditions, and simulated microgravity caused a burst of ROS, implicating ROS as a critical upstream mediator linking to downstream atrophic signaling. ROS generation activated the ERK1/2 early-growth response protein (Egr)1/2-Cbl-b signaling pathway, an established contributing pathway to muscle volume loss. Interestingly, antioxidant treatments such as N-acetylcysteine and TEMPOL, but not catalase, blocked the clinorotation-mediated activation of ERK1/2. The increased ROS induced transcriptional activity of Egr1 and/or Egr2 to stimulate Cbl-b expression through the ERK1/2 pathway in L6 myoblasts, since treatment with Egr1/2 siRNA and an ERK1/2 inhibitor significantly suppressed clinorotation-induced Cbl-b and Egr expression, respectively. Promoter and gel mobility shift assays revealed that Cbl-b was upregulated via an Egr consensus oxidative responsive element at -110 to -60 bp of the Cbl-b promoter. Together, this indicates that under microgravity conditions, elevated ROS may be a crucial mechanotransducer in skeletal muscle cells, regulating muscle mass through Cbl-b expression activated by the ERK-Egr signaling pathway.

Root-tip-mediated inhibition of hydrotropism is accompanied with the suppression of asymmetric expression of auxin-inducible genes in response to moisture gradients in cucumber roots.

In cucumber seedlings, gravitropism interferes with hydrotropism, which results in the nearly complete inhibition of hydrotropism under stationary conditions. However, hydrotropic responses are induced when the gravitropic response in the root is nullified by clinorotation. Columella cells in the root cap sense gravity, which induces the gravitropic response. In this study, we found that removing the root tip induced hydrotropism in cucumber roots under stationary conditions. The application of auxin transport inhibitors to cucumber seedlings under stationary conditions suppressed the hydrotropic response induced by the removal of the root tip. To investigate the expression of genes related to hydrotropism in de-tipped cucumber roots, we conducted transcriptome analysis of gene expression by RNA-Seq using seedlings exhibiting hydrotropic and gravitropic responses. Of the 21 and 45 genes asymmetrically expressed during hydrotropic and gravitropic responses, respectively, five genes were identical. Gene ontology (GO) analysis indicated that the category auxin-inducible genes was significantly enriched among genes that were more highly expressed in the concave side of the root than the convex side during hydrotropic or gravitropic responses. Reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) analysis revealed that root hydrotropism induced under stationary conditions (by removing the root tip) was accompanied by the asymmetric expression of several auxin-inducible genes. However, intact roots did not exhibit the asymmetric expression patterns of auxin-inducible genes under stationary conditions, even in the presence of a moisture gradient. These results suggest that the root tip inhibits hydrotropism by suppressing the induction of asymmetric auxin distribution. Auxin transport and distribution not mediated by the root tip might play a role in hydrotropism in cucumber roots.

The effects of heat stress on morphological properties and intracellular signaling of denervated and intact soleus muscles in rats.

The effects of heat stress on the morphological properties and intracellular signaling of innervated and denervated soleus muscles were investigated. Heat stress was applied to rats by immersing their hindlimbs in a warm water bath (42°C, 30 min/day, every other day following unilateral denervation) under anesthesia. During 14 days of experimental period, heat stress for a total of seven times promoted growth-related hypertrophy in sham-operated muscles and attenuated atrophy in denervated muscles. In denervated muscles, the transcription of ubiquitin ligase, atrogin-1/muscle atrophy F-box (Atrogin-1), and muscle RING-finger protein-1 (MuRF-1), genes was upregulated and ubiquitination of proteins was also increased. Intermittent heat stress inhibited the upregulation of Atrogin-1, but not MuRF-1 transcription. And the denervation-caused reduction in phosphorylated protein kinase B (Akt), 70-kDa heat-shock protein (HSP70), and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which are negative regulators of Atrogin-1 and MuRF-1 transcription, was mitigated. In sham-operated muscles, repeated application of heat stress did not affect Atrogin-1 and MuRF-1 transcription, but increased the level of phosphorylated Akt and HSP70, but not PGC-1α Furthermore, the phosphorylation of Akt and ribosomal protein S6, which is known to stimulate protein synthesis, was increased immediately after a single heat stress particularly in the sham-operated muscles. The effect of a heat stress was suppressed in denervated muscles. These results indicated that the beneficial effects of heat stress on the morphological properties of muscles were brought regardless of innervation. However, the responses of intracellular signaling to heat stress were distinct between the innervated and denervated muscles.

Gravitropism interferes with hydrotropism via counteracting auxin dynamics in cucumber roots: clinorotation and spaceflight experiments.

Roots of land plants show gravitropism and hydrotropism in response to gravity and moisture gradients, respectively, for controlling their growth orientation. Gravitropism interferes with hydrotropism, although the mechanistic aspects are poorly understood. Here, we differentiated hydrotropism from gravitropism in cucumber roots by conducting clinorotation and spaceflight experiments. We also compared mechanisms regulating hydrotropism and auxin-regulated gravitropism. Clinorotated or microgravity (µG)-grown cucumber seedling roots hydrotropically bent toward wet substrate in the presence of moisture gradients, but they grew straight in the direction of normal gravitational force at the Earth's surface (1G) on the ground or centrifuge-generated 1G in space. The roots appeared to become hydrotropically more sensitive to moisture gradients under µG conditions in space. Auxin transport inhibitors significantly reduced the hydrotropic response of clinorotated seedling roots. The auxin efflux protein CsPIN5 was differentially expressed in roots of both clinorotated and µG-grown seedlings; with higher expression in the high-humidity (concave) side than the low-humidity (convex) side of hydrotropically responding roots. Our results suggest that roots become hydrotropically sensitive in µG, and CsPIN5-mediated auxin transport has an important role in inducing root hydrotropism. Thus, hydrotropic and gravitropic responses in cucumber roots may compete via differential auxin dynamics established in response to moisture gradients and gravity.