Otoliths developed in microgravity

Little is known about mechanisms that regulate the development of the otoliths in the gravity-sensing organs. Several reported experiments suggest that the growth of the otoliths is adjusted to produce a test mass of the appropriate weight. If this is the case, larger than normal otoliths would be expected in animals reared in reduced gravity and a reduced mass, relative to 1-g controls, would be expected in animals reared at elevated g. In gastropod mollusks, the gravity-sensing organ is the statocyst, a spherical organ whose wall is made largely of sensory receptor cells with motile cilia facing the lumen. Dense statoconia in the cyst lumen interact with cilia of receptor cells at the bottom of the cyst and action potentials in their axons carry information on direction and magnitude of gravity and linear acceleration. In the marine mollusk, Aplysia californica, larvae reared at 2 to 5-g, the volume of statoconia was reduced in a graded manner, compared to 1-g control animals. In the statocyst of the fresh-water pond snail, Biomphalaria glabrata, reared in space in the Closed Equilibrated Biological Aquatic System (CEBAS), the number and total volume of statoconia was increased approximately 50%, relative to ground-reared controls. Lychakov found the utricular otolith to be 30% larger in space-reared Xenopus, whereas we found the saccular otolith to be significantly larger in newt larvae reared in space. In cichlid fish reared on a centrifuge, the saccular otolith was smaller than in 1-g controls. Here, we demonstrate that the otoliths of late-stage embryos of the swordtail fish, Xiphophorus helleri, reared in space on STS-89 and STS-90 (Neurolab) were significantly larger than those of ground-controls reared in functionally identical hardware.     

Physiology of a microgravity environment invited review: microgravity and skeletal muscle.

Spaceflight (SF) has been shown to cause skeletal muscle atrophy; a loss in force and power; and, in the first few weeks, a preferential atrophy of extensors over flexors. The atrophy primarily results from a reduced protein synthesis that is likely triggered by the removal of the antigravity load. Contractile proteins are lost out of proportion to other cellular proteins, and the actin thin filament is lost disproportionately to the myosin thick filament. The decline in contractile protein explains the decrease in force per cross-sectional area, whereas the thin-filament loss may explain the observed postflight increase in the maximal velocity of shortening in the type I and IIa fiber types. Importantly, the microgravity-induced decline in peak power is partially offset by the increased fiber velocity. Muscle velocity is further increased by the microgravity-induced expression of fast-type myosin isozymes in slow fibers (hybrid I/II fibers) and by the increased expression of fast type II fiber types. SF increases the susceptibility of skeletal muscle to damage, with the actual damage elicited during postflight reloading. Evidence in rats indicates that SF increases fatigability and reduces the capacity for fat oxidation in skeletal muscles. Future studies will be required to establish the cellular and molecular mechanisms of the SF-induced muscle atrophy and functional loss and to develop effective exercise countermeasures.     

Cell-wall structural changes in wheat straw pretreated for bioethanol production

Background  

Pretreatment is an essential step in the enzymatic hydrolysis of biomass and subsequent production of bioethanol. Recent results indicate that only a mild pretreatment is necessary in an industrial, economically feasible system. The Integrated Biomass Utilisation System hydrothermal pretreatment process has previously been shown to be effective in preparing wheat straw for these processes without the application of additional chemicals. In the current work, the effect of the pretreatment on the straw cell-wall matrix and its components are characterised microscopically (atomic force microscopy and scanning electron microscopy) and spectroscopically (attenuated total reflectance Fourier transform infrared spectroscopy) in order to understand this increase in digestibility.     

Cell-wall composition and taxonomy of Cephaloascus fragrans and some Ophiostomataceae

Carbohydrates of intact cells and cell walls were studied by gas-liquid chromatographical analysis after acid hydrolysis. Isolated cellulose was determined by infrared spectrophotometry, pyrolysis mass spectrometry and histochemistry. Biochemical characters do not support an assumed relationship between Ophiostoma (including Europhium) and Cephaloascus fragrans. Cephaloascus fragrans differs from Ophiostoma by a high mannose content and by the absence of cellulose and rhamnose. A relationship between Cephaloascus fragrans and Ceratocystis cannot be excluded on the basis of the biochemical characters, although there is a marked difference in conidiogenesis. Saprolegnia ferax (Oomycetes) was included as a cellulose-containing fungus for comparison.     

The mechanisms underlying neuromuscular changes in microgravity environment.

We reported that the levels of electromyogram in soleus muscle and the afferent neurogram recorded at L5 segmental level of the spinal cord were instantly decreased in response to exposure to microgravity (micro-G) environment created during a parabolic flight, although these activities were constantly presented at 1-G. It was also observed that the soleus muscle length was passively shortened in micro-G due to the plantarflexion of ankle joint. Similar phenomena were also induced by acute hindlimb suspension at 1-G. Further, the soleus muscle atrophied, if the dorsal root at L5 was transected. These results suggested that the unloading-related effects on muscle are closely associated with the inhibition of the afferent input. However, effects of gravity on most of the cells in the whole body can not be removed, although hindlimb suspension can inhibit the antigravity activity of rat hindlimb muscles. And parabolic flight can create micro-G only for a short period of time. Further, effects of hypergravity before and after micro-G are unavoidable. Therefore, further experiments utilizing space environment are essential.     

Cell-wall structure and composition of Penicillium janczewskii as affected by inulin

Penicillium janczewskii, a filamentous fungus isolated from the rhizosphere of Vernonia herbacea (Asteraceae), grows rapidly on media containing either sucrose or inulin as carbon sources. Maintenance of P. janczewskii on inulin medium induces secretion of proteins with high inulinase activity but results in a mycelium that easily collapses and breaks. We evaluated the influence of inulin on fungal growth and colony morphology and on cell-wall structure and composition in comparison with growth and wall characteristics on sucrose-containing medium. P. janczewskii grown on Czapek medium with agar containing 1% (w/v) sucrose or inulin showed differences in the color and morphology of the colonies, although growth rates were similar on both carbon sources. Scanning-electron microscopy revealed that the hyphae from fungus grown on inulin-containing medium are much thinner than those from fungus cultivated on sucrose. Ultrastructural analysis of 5 d old cultures using transmission-electron microscopy indicated significant differences in the cell-wall thickness between hyphae grown on inulin or sucrose media. No differences were detected in the overall carbohydrate and protein contents of cell walls isolated from cultures grown on the two carbon sources. Glycosyl composition analyses showed glucose and galactose as the predominant neutral monosaccharides in the walls but showed no differences attributable to the carbon source. Glycosyl linkage composition analyses indicated a predominance of 3-linked glucopyranosyl in the hyphal walls when P. janczewskii was grown on inulin-containing medium. Our results suggest that growth on inulin as the sole carbon source results in structural changes in the mycelia of P. janczewskii that lead to mycelial walls with altered physical and biological properties.     

Effects of hypergravity environment on lignin formation in Arabidopsis.

Lignin and the secondary wall formation are essential for evolution of land plants. In this study, effects of hypergravity environment on the morphology of the secondary wall and the lignin content were examined in Arabidopsis thaliana. Xylem vessels showed intense staining with phloroglucinol-HCl and autofluorescence under UV light at the basal region of the flower stalk when seedlings grown for 3 days after hypergravity treatment for 24 hours. And, the flower stalk exposed to hypergravity showed slight increase in the lignin content. These results suggest that the lignin formation is positively regulated under hypergravity.     

Cortical microtubules in sweet clover columella cells developed in microgravity

Electron micrographs of columella cells from sweet clover seedlings grown and fixed in microgravity revealed longitudinal and cross sectioned cortical microtubules. This is the first report demonstrating the presence and stability of this network in plants in microgravity.     

Responses of neuromuscular systems under gravity or microgravity environment.

Hindlimb suspension of rats induces induces fiber atrophy and type shift of muscle fibers. In contrast, there is no change in the cell size or oxidative enzyme activity of spinal motoneurons innervating muscle fibers. Growth-related increases in the cell size of muscle fibers and their spinal motoneurons are inhibited by hindlimb suspension. Exposure to microgravity induces atrophy of fibers (especially slow-twitch fibers) and shift of fibers from slow- to fast-twitch type in skeletal muscles (especially slow, anti-gravity muscles). In addition, a decrease in the oxidative enzyme activity of spinal motoneurons innervating slow-twitch fibers and of sensory neurons in the dorsal root ganglion is observed following exposure to microgravity. It is concluded that neuromuscular activities are important for maintaining metabolism and function of neuromuscular systems at an early postnatal development and that gravity effects both efferent and afferent neural pathways.     

Enzymic chemical reaction under microgravity environment in space

In recent years, some papers have reported synergism in the biological effects of space radiation and microgravity. However, there is no direct evidence for these phenomena. As one possible mechanism, we investigated whether DNA ligation in the final step of DSBs repair of DNA molecules induced by radiation is depressed by microgravity. Therefore, we have scheduled the space experiments of the effects of microgravity on repair activity of T4 DNA ligase for DSBs prepared with digestion of a restriction enzyme (Sma I) to plasmid DNA. As another possible mechanism, the high mutation frequency may be induced from abnormal base-incorporation during DNA replication under microgravity. Using the Taq polymerase and polymerase III, we have also scheduled whether mutation frequency is affected by microgravity during DNA replication for a damaged DNA base induced by an alkylating agent (N-methyl-N-nitrosourea, MNU).     

Analysis of peg formation in cucumber seedlings grown on clinostats and in a microgravity (space) environment.

In young cucumber seedlings, the peg is a polar out-growth of tissue that functions by snagging the seed coat, thereby freeing the cotyledons. Previous studies have indicated that peg formation is gravity dependent. In this study we analyzed peg formation in cucumber seedlings (Cucumis sativus L. cv Burpee Hybrid II) grown under conditions of normal gravity, microgravity, and simulated microgravity (clinostat rotation). Seeds were germinated on the ground, in clinostats and on board the space shuttle (STS 95) for 1-2 days, frozen and subsequently examined for their stage of development, degree of hook formation, number of pegs formed, and peg morphology. The frequency of peg formation in space grown seedlings was found to be nearly identical to that of clinostat grown seedlings and to differ from that of seedlings germinated under normal gravity only in a minority of cases; approximately 6% of the seedlings formed two pegs and nearly 2% of the seedlings lacked pegs, whereas such abnormalities did not occur in ground controls. The degree of hook formation was found to be less pronounced for space grown seedlings, compared to clinostat grown seedlings, indicating a greater degree of decoupling between peg formation and hook formation in space. Nonetheless, in all seedlings having single pegs and a hook, the peg was found to be positioned correctly on the inside of the hook, showing that there is coordinate development even in microgravity environments. Peg morphologies were altered in space grown samples, with the pegs having a blunt appearance and many pegs showing alterations in expansion, with the peg extending out over the edges of the seed coat and downwards. These phenotypes were not observed in clinostat or ground grown seedlings.     

Proteomic analysis of mice hippocampus in simulated microgravity environment

Space travel induces many deleterious effects on the flight crew due to the '0' g environment. The brain experiences a tremendous fluid shift, which is responsible for many of the detrimental changes in physical behavior seen in astronauts. It therefore indicates that the brain may undergo major changes in its protein levels in a '0' g environment to counteract the stress. Analysis of these global changes in proteins may explain to better understand the functioning of brain in a '0' g condition. Toward such an effort, we have screened proteins in the hippocampus of mice kept in simulated microgravity environment for 7 days and have observed a few changes in major proteins as compared to control mice. Essentially, the results show a major loss of proteins in the hippocampus of mice subjected to simulated microgravity. These changes occur in structural proteins such as tubulin, coupled with the loss of proteins involved in metabolism. This preliminary investigation leads to an understanding of the alteration of proteins in the hippocampus in response to the microgravity environment.     

Nastic curvatures of wheat coleoptiles that develop in true microgravity

Dark-grown wheat coleoptiles developed strong curvatures within 5 h of being transferred in orbit from a 1 g centrifuge to microgravity during an experiment flown on the IML-1 shuttle mission. The curving tendency was strongest in seedlings that were immature, with coleoptiles shorter than 10 mm at the time of transfer. The curvature direction was non-random, and directed away from the caryopsis (the coleptile face adjacent to the caryopsis becoming convex). The curvatures were most marked in the basal third of the coleoptiles, contrasting with phototropic responses, which occur in the apical third. We interpret these curvatures as being nastic, and related to the curvatures commonly reported to occur during clinostat rotation treatments.     

Change in composition of hydrolyzed lignin during composting

Microbial assemblages were composed for composting hydrolysed lignin. Data on bioconversion of aromatic compounds with various types of substitution in the ring were used for this purpose. Composting of hydrolysed lignin reduced the contents of lignin, low-hydrolyzable polysaccharides, resins, and low-molecular-weight phenols and resulted in accumulation of humic acids. The resulting compost showed no phytotoxicity.     

Influence of simulated microgravity on human skeletal muscle architecture and function

Ten male volunteers underwent a period of prolonged bed rest. Four subjects performed exercise countermeasures 2-3 times per week, while 6 subjects received no countermeasures. After bed rest plantarflexor force declined significantly (P < 0.001) in both exercise (-42%) and control (-55%) groups. The internal architecture of the gastrocnemius medialis (GM) muscle was significantly altered. This was associated with a reduction in fascicle shortening during isometric contraction. Exercise countermeasures partially mitigated the loss of muscle force and function following 90 days of bed rest.     

Artificial humification of lignin architecture: Top-down and bottom-up approaches

Humic substances readily identifiable in the environment are involved in several biotic and abiotic reactions affecting carbon turnover, soil fertility, plant nutrition and stimulation, xenobiotic transformation and microbial respiration. Inspired by natural roles of humic substances, several applications of these substances, including crop stimulants, redox mediators, anti-oxidants, human medicines, environmental remediation and fish feeding, have been developed. The annual market for humic substances has grown rapidly for these reasons and due to eco-conscious features, but there is a limited supply of natural coal-related resources such as lignite and leonardite from which humic substances are extracted in bulk. The structural similarity between humic substances and lignin suggests that lignocellulosic refinery resulting in lignin residues as a by-product could be a potential candidate for a bulk source of humic-like substances, but structural differences between the two polymeric materials indicate that additional transformation procedures allowing lignin architecture to fully mimic commercial humic substances are required. In this review, we introduce the emerging concept of artificial humification of lignin-related materials as a promising strategy for lignin valorization. First, the core structural features of humic substances and the relationship between these features and the physicochemical properties, natural functions and versatile applications of the substances are described. In particular, the mechanism by which humic substances stimulate the growth of plants and hence can improve crop productivity is highlighted. Second, top-down and bottom-up transformation pathways for scalable humification of small lignin-derived phenols, technical lignins and lignin-containing plant residues are described in detail. Finally, future directions are suggested for research and development of artificial lignin humification to achieve alternative ways of producing customized analogues of humic substances.     

Generation of a tumor spheroid in a microgravity environment as a 3D model of melanoma

An in vitro 3D model was developed utilizing a synthetic microgravity environment to facilitate studying the cell interactions. 2D monolayer cell culture models have been successfully used to understand various cellular reactions that occur in vivo. There are some limitations to the 2D model that are apparent when compared to cells grown in a 3D matrix. For example, some proteins that are not expressed in a 2D model are found up-regulated in the 3D matrix. In this paper, we discuss techniques used to develop the first known large, free-floating 3D tissue model used to establish tumor spheroids. The bioreactor system known as the High Aspect Ratio Vessel (HARVs) was used to provide a microgravity environment. The HARVs promoted aggregation of keratinocytes (HaCaT) that formed a construct that served as scaffolding for the growth of mouse melanoma. Although there is an emphasis on building a 3D model with the proper extracellular matrix and stroma, we were able to develop a model that excluded the use of matrigel. Immunohistochemistry and apoptosis assays provided evidence that this 3D model supports B16.F10 cell growth, proliferation, and synthesis of extracellular matrix. Immunofluorescence showed that melanoma cells interact with one another displaying observable cellular morphological changes. The goal of engineering a 3D tissue model is to collect new information about cancer development and develop new potential treatment regimens that can be translated to in vivo models while reducing the use of laboratory animals.     

Sleep restriction does not affect orthostatic tolerance in the simulated microgravity environment.

Orthostatic intolerance (OI) is a major problem following spaceflight, and, during flight, astronauts also experience sleep restriction. We hypothesized that sleep restriction will compound the risk and severity of OI following simulated microgravity and exaggerate the renal, cardioendocrine, and cardiovascular adaptive responses to it. Nineteen healthy men were equilibrated on a constant diet, after which they underwent a tilt-stand test. They then completed 14-16 days of simulated microgravity [head-down tilt bed rest (HDTB)], followed by repeat tilt-stand test. During HDTB, 11 subjects were assigned to an 8-h sleep protocol (non-sleep restricted), and 8 were assigned to a sleep-restricted protocol with 6 h of sleep per night. During various phases, the following were performed: 24-h urine collections, hormonal measurements, and cardiovascular system identification. Development of presyncope or syncope defined OI. There was a significant decrease in time free of OI (P = 0.02) and an increase in OI occurrence (P = 0.06) after HDTB among all subjects. However, the increase in OI occurrence did not differ significantly between the two groups (P = 0.60). The two groups also experienced similar physiological changes with HDTB (initial increase in sodium excretion; increased excretion of potassium at the end of HDTB; increase in plasma renin activity secretion without a change in serum or urine aldosterone). No significant change in autonomic function or catecholamines was noted. Simulated microgravity leads to increased OI, and sleep restriction does not additively worsen OI in simulated microgravity. Furthermore, conditions of sleep restriction and nonsleep restriction are similar with respect to renal, cardioendocrine, and cardiovascular responses to simulated microgravity.