Exploration of plant growth and development using the European Modular Cultivation System facility on the International Space Station.

Space experiments provide a unique opportunity to advance our knowledge of how plants respond to the space environment, and specifically to the absence of gravity. The European Modular Cultivation System (EMCS) has been designed as a dedicated facility to improve and standardise plant growth in the International Space Station (ISS). The EMCS is equipped with two centrifuges to perform experiments in microgravity and with variable gravity levels up to 2.0 g. Seven experiments have been performed since the EMCS was operational on the ISS. The objectives of these experiments aimed to elucidate phototropic responses (experiments TROPI-1 and -2), root gravitropic sensing (GRAVI-1), circumnutation (MULTIGEN-1), cell wall dynamics and gravity resistance (Cell wall/Resist wall), proteomic identification of signalling players (GENARA-A) and mechanism of InsP3 signalling (Plant signalling). The role of light in cell proliferation and plant development in the absence of gravity is being analysed in an on-going experiment (Seedling growth). Based on the lessons learned from the acquired experience, three preselected ISS experiments have been merged and implemented as a single project (Plant development) to study early phases of seedling development. A Topical Team initiated by European Space Agency (ESA), involving experienced scientists on Arabidopsis space research experiments, aims at establishing a coordinated, long-term scientific strategy to understand the role of gravity in Arabidopsis growth and development using already existing or planned new hardware.

Angiolymphoid hyperplasia with eosinophilia of the eyelid and orbit: the Western cousin of Kimura's disease?

Angiolymphoid hyperplasia with eosinophilia (ALHE) is an uncommon disease most frequently occurring in the head and neck region. It occurs in all races. In spite of a clear distinction between ALHE and Kimura's disease on histological grounds for almost 20 years now, confusion and misdiagnosis still exist to date. Three Spanish patients presented to our institution over a 6 year period with mass lesions in the superior orbit, medial orbito-nasal wall and eyelid, respectively. All patients were of Caucasian extraction and were diagnosed with epithelioid haemangioma on histological tissue analysis. We report on the history and management of these patients which resulted in good functional and cosmetic outcome with no subsequent evidence of disease recurrence.

Cell proliferation and plant development under novel altered gravity environments.

Gravity is a key factor for life on Earth. It is the only environmental factor that has remained constant throughout evolution, and plants use it to modulate important physiological activities; gravity removal or alteration produces substantial changes in essential functions. For root gravitropism, gravity is sensed in specialised cells, which are capable of detecting magnitudes of the g vector lower than 10(-3) . Then, the mechanosignal is transduced to upper zones of the root, resulting in changes in the lateral distribution of auxin and in the rate of auxin polar transport. Gravity alteration has consequences for cell growth and proliferation rates in root meristems, which are the basis of the developmental programme of a plant, in which regulation via auxin is involved. The effect is disruption of meristematic competence, i.e. the strict coordination between cell proliferation and growth, which characterises meristematic cells. This effect can be related to changes in the transport and distribution of auxin throughout the root. However, similar effects of gravity alteration have been found in plant cell cultures in vitro, in which neither specialised structures for gravity sensing and signal transduction, nor apparent gravitropism have been described. We postulate that gravity resistance, a general mechanism of cellular origin for developing rigid structures in plants capable of resisting the gravity force, could also be responsible for the changes in cell growth and proliferation parameters detected in non-specialised cells. The mechanisms of gravitropism and graviresistance are complementary, the first being mostly sensitive to the direction of the gravity vector, and the second to its magnitude. At a global molecular level, the consequence of gravity alteration is that the genome should be finely tuned to counteract a type of stress that plants have never encountered before throughout evolution. Multigene families and redundant genes present an advantage in that they can experience changes without the risk of being deleterious and, for this reason, they should play a key role in the response to gravitational stress.

Neuroprotective effects of extremely low-frequency electromagnetic fields on a Huntington's disease rat model: effects on neurotrophic factors and neuronal density.

There is evidence to suggest that the neuroprotective effect of exposure of extremely low-frequency electromagnetic fields (ELF-EMF) may be due, at least in part, to the effect of these fields on neurotrophic factors levels and cell survival, leading to an improvement in behavior. This study was undertaken to investigate the neuroprotective effects of ELFEF in a rat model of 3-nitropropionic acid (3NP)-induced Huntington's disease. Behavior patterns were evaluated, and changes in neurotrophic factor, cell damage, and oxidative stress biomarker levels were monitored in Wistar rats. Rats were given 3NP over four consecutive days (20 mg/kg body weight), whereas ELFEF (60 Hz and 0.7 mT) was applied over 21 days, starting after the last injection of 3NP. Rats treated with 3NP exhibited significantly different behavior in the open field test (OFT) and the forced swim test (FST), and displayed significant differences in neurotrophic factor levels and oxidative stress biomarkers levels, together with a neuronal damage and diminished neuronal density, with respect neuronal controls. ELFEF improved neurological scores, enhanced neurotrophic factor levels, and reduced both oxidative damage and neuronal loss in 3NP-treated rats. ELFEF alleviates 3NP-induced brain injury and prevents loss of neurons in rat striatum, thus showing considerable potential as a therapeutic tool.

Cerebral fat embolism detection with susceptibility-weighted images in sickle cell disease.

Cerebral fat embolism is a known but feared complication of sickle cell disease, which leads to significant morbidity. Although trauma-related fat embolism is more frequent and its diagnosis is straightforward, fat embolism secondary to bone infarcts after vaso-occlusive events can be unrecognized. Lack of criteria make the clinical diagnosis of sickle cell disease related fat embolism challenging. The neuroimaging findings are helpful to support the clinical diagnosis. Magnetic resonance imaging findings of fat embolism have been described including those seen on susceptibility-weighted imaging. Even though susceptibility-weighted imaging has been used for the diagnosis of cerebral fat embolism due to trauma, to the best of our knowledge its use has not been described in patients with sickle cell disease. We report an uncommon case of a 60-year-old woman with sickle cell disease who developed cerebral fat embolism and in which the findings on susceptibility-weighted imaging were characteristic and established the diagnosis.

Huntingtons Disease: The Value of Transcranial Meganetic Stimulation

Huntington's disease (HD) is a genetic neurodegenerative process whose etiology is based on a localized disturbance in the short arm of chromosome 4 that encodes the huntingtin protein (Htt). The elongation of triple CAG for glutamine characterizes this change. Mutated Htt (mHtt) causes the appearance of intracellular aggregates inducing alterations in mitochondrial metabolism in the form of reactive oxygen species (ROS) and ATP depletion. The oxidative imbalance caused by mHtt leads the neurons to a state of oxidative stress resulting in damage to macromolecules and cellular death. Since the discovery of certain mechanisms underlying the pathogenesis of HD, several therapeutic procedures have been shown to delay or slow the evolution of the condition and have demonstrated the biochemical and molecular mechanism involved. The studies have reported that transcranial magnetic stimulation (TMS) may improve motor and other symptoms associated with neurodegenerative and neuropsychiatric processes such as major depression, schizophrenia, epilepsy, neuropathic pain, amyotrophic lateral sclerosis, progressive muscle atrophy, multiple sclerosis, stroke, Alzheimer's disease, Parkinson's disease or HD. This study focuses on the effect of TMS on oxidative stress and neurogenesis in studies and its possible usefulness in HD.

Olfactory bulbectomy induced oxidative and cell damage in rat: protective effect of melatonin.

In this study we analyzed the effects of melatonin (Mel, 1 mg/kg ip) on behavioral changes as well as cell and oxidative damage prompted by bilaterally olfactory bulbectomy. Olfactory bulbectomy caused an increase in lipid peroxidation products and caspase-3, whereas it prompted a decrease of reduced glutathione (GSH) content and antioxidative enzymes activities. Additionally, olfactory bulbectomy induced behavioral changes characterized by the enhancement of immobility time in the forced swim test and hyperactivity in the open field test. All these changes were normalized by treatment of Mel (14 days). Our data show that Mel has a beneficial neuropsychiatric action against oxidative stress, cell damage and behavior alterations.

Neuroprotective effect of carvedilol and melatonin on 3-nitropropionic acid-induced neurotoxicity in neuroblastoma.

In neurodegenerative diseases, progressive oxidative stress is a major event that precedes neuronal death. Oxidative stress is characterized by an imbalance between oxidants and antioxidants. This imbalance induced oxidative molecular and cell damage, reducing cellular viability. 3-Nitropropionic acid (3NP) causes oxidative stress and other molecular and cellular changes similar to those observed in neurons of patients with Huntington's disease. Since carvedilol and melatonin act as free-radical scavengers, this study examined the effect of carvedilol (10(-5) M) and melatonin (10(-5) M) on oxidative and cell damage induced by 3NP in N1E-115 neuroblastoma cells. Carvedilol and melatonin prevented the increases in lipid peroxidation and total LDH activity, as well as the depletion of reduced glutathione (GSH) and the reduction of antioxidative enzymes activities in N1E-115 cells incubated with 100 mM 3NP. All these carvedilol and melatonin effects were more intense when the drugs were added before rather than after inducing the damage by 3NP. These results also provided evidence supporting the hypothesis that carvedilol and melatonin can be useful for treating neurodegenerative diseases, such as Huntington's disease.

Direct association between C-reactive protein serum levels and endothelial dysfunction in patients with claudication.

OBJECTIVES: To evaluate the relationship between C-Reactive Protein (hsCRP), a serum marker of inflammation, and endothelial dysfunction in patients with intermittent claudication. DESIGN, PATIENTS AND METHODS: Cross-sectional study with stratified sampling on dependent variables of age, genre, hypertension, hyperlipidemia, diabetes, smoking status and ankle-brachial index (ABI) to select 156 patients from a target population of 4,100 patients with claudication. We assessed the flow-mediated arterial dilation (FMAD) as a reporter of endothelial function and plasma levels of hsCRP and fibrinogen. RESULTS: Patients with a FMAD<3% (range for the lowest 5% of healthy subjects) had increased levels of plasma hsCRP (6.3 vs 2.3mg/L; p<0.05) and fibrinogen (351vs 302mg/L; p<0.05) in comparison to those with FMAD>3%. There was a negative correlation between hsCRP and FMAD(r=-0.465; p<0.05). CONCLUSION: Impaired endothelial dysfunction is association with increased plasma concentrations of inflammatory markers, and both may have a role in the aetiopathogenesis of peripheral arterial disease.

Comparative analysis of Drosophila melanogaster and Caenorhabditis elegans gene expression experiments in the European Soyuz flights to the International Space Station.

The European Soyuz missions have been one of the main routes for conducting scientific experiments onboard the International Space Station, which is currently in the construction phase. A relatively large number of life and physical sciences experiments as well as technology demonstrations have been carried out during these missions. Included among these experiments are the Gene experiment during the Spanish "Cervantes" Soyuz mission and the ICE-1st experiment during the Dutch "Delta" mission. In both experiments, full genome microarray analyses were carried out on RNA extracted from whole animals recovered from the flight. These experiments indicated relatively large scale changes in gene expression levels in response to spaceflight for two popular model systems, Drosophila melanogaster (Gene) and Caenorabditis elegans (ICE-1st). Here we report a comparative analysis of results from these two experiments. Finding orthologous genes between the fruit fly and the nematode was far from straightforward, reducing the number of genes that we could compare to roughly 20% of the full comparative genome. Within this sub-set of the data (2286 genes), only six genes were found to display identical changes between species (decreased) while 1809 genes displayed no change in either species. Future experiments using ground simulation techniques will allow producing a better, more comprehensive picture of the putative set of genes affected in multicellular organisms by changes in gravity and getting a deeper understanding of how animals respond and adapt to spaceflight.

Changes in the two-dimensional proteome of the soluble fraction of nuclear proteins from Lepidium sativum root meristematic cells grown under clinorotation.

The changes in the fundamental biological processes of nuclear RNA transcription and splicing under altered gravity conditions are still unclear. The quantitative and qualitative characteristics of the proteins involved in nuclear RNA metabolism in control and under clinorotation were investigated by two-dimensional gel electrophoresis. We revealed firstly a decrease in the isoelectric point range of nuclear soluble proteins, which are known to be actively engaged in nuclear RNA metabolism, and a shortening in the molecular weight range of them under clinorotation. Moreover, minor and major proteins in clinorotated samples had decreased optical densities in comparison with control ones. Our results are in agreement with the hypothesis that a rearrangement of the pattern of nuclear proteins involved in gene expression processes occurs in seedlings grown and developed in altered gravity.

Drosophila melanogaster, a model system for comparative studies on the responses to real and simulated microgravity.

A key requirement to enhance our understanding of the response of biological organisms to different levels of gravity is the availability of experimental systems that can simulate microgravity and hypergravity in ground-based laboratories. This paper compares the results obtained from analysing gene expression profiles of Drosophila in space versus those obtained in a random position machine (RPM) and by centrifugation. The correlation found validates the use of the RPM simulation technique to establish the effects of real microgravity on biological systems. This work is being extended to investigate Drosophila development in another gravity modifying instrument, the levitation magnet.

[Exploration of the anterior segment by optical coherence tomography-3]. / Exploración del segmento anterior mediante tomografía óptica de coherencia 3.

PURPOSE: To show the utility of optical coherence tomography (OCT) for studying the anterior segment and to explain its potential advantages as compared to ultrasonic biomicroscopy (UBM). MATERIALS AND METHODS: We have described the findings in 5 patients with different pathologies of the anterior segment, all of whom were studied with UBM and OCT-3 adjusted for the anterior segment. There were 2 cases of an acute attack of closed angle glaucoma, 1 case of rubeosis iridis, 1 case of penetrating ocular trauma and 1 case of a primary stromal iris cyst. RESULTS: OCT was found to be as useful as UBM in detecting angle closure in patients affected by an acute attack of glaucoma. In addition, OCT was more comfortable for the patient and faster than UBM in obtaining images, with the exploration mean time using OCT being less than five minutes while that with UBM was over ten minutes. OCT has also been demonstrated to be a safe and valuable non-contact examination in other iris pathologies such as rubeosis iridis, for checking the permeability of iridotomies and even for studying iris masses such as primary stromal iris cysts. In these entities OCT may reach a higher resolution than ultrasonic biomicroscopy. CONCLUSION: OCT-3, with an adjustable focus, can obtain images from the scleral angle, as well as from other ocular structures like the iris, thus assisting in the diagnosis of numerous pathologies.

Subnucleolar location of fibrillarin and NopA64 in Lepidium sativum root meristematic cells is changed in altered gravity.

Fibrillarin and the plant nucleolin homolog NopA64 are two important nucleolar proteins involved in pre-rRNA processing. In order to determine the effects of the altered gravity environment on the nucleolus, we have investigated the location of fibrillarin and NopA64 in nucleolar subcomponents of cress (Lepidium sativum L.) root meristematic cells grown under clinorotation, which reproduces an important feature of microgravity, namely, the absence of the orienting action of a gravity vector, and compared it to the location in control cells grown in normal 1 g conditions. Prior to these experiments, we report here the characterization of cress fibrillarin as a 41 kDa protein which can be isolated from meristematic cells in three nuclear fractions, namely, the soluble ribonucleoprotein fraction, the chromatin fraction, and the nuclear-matrix fraction. Furthermore, as reported for other species, the location of both fibrillarin and NopA64 in the cress cell nucleolus was in zones known to contain complex ribonucleoprotein particles involved in early pre-rRNA processing, i.e., processomes. Under altered gravity, a decrease in the quantity of both fibrillarin and NopA64 compared to controls was observed in the transition zone between fibrillar centers and the dense fibrillar component, as well as in the bulk of the dense fibrillar component. These data suggest that altered (reduced) gravity results in a lowered level of functional activity in the nucleolus.

[Altered gravity affects subnucleolus localization of fibrillarin and NopA64, the most important proteins of rRNA processing].

Fibrillarin and plant nucleolin homologue NopA64 are two important nucleolar proteins involved in pre-rRNA processing. To understand better the effects of the altered gravity environment on the nucleolus functioning we have investigated the location of fibrillarin and NopA64 in nucleolar subcomponents of cress (Lepidium sativum L.) root meristematic cells grown under simulated microgravity that was compared to the control cells grown in normal conditions at I g. Cress fibrillarin was first shown to have the molecular weight 41 kDa. Both fibrillarin and NopA64 in the cress cell nucleolus are located in the zones known to contain processing pre-rRNA molecules as it has been previously reported in other species. The data confirm participation of these proteins in processomes--RNP complex particles involved in pre-rRNA processing. Under altered gravity a decrease in the quantity of both fibrillarin and NopA64 in the transition zone between fibrillar centres and the dense fibrillar component was observed, compared to control, which could point out to a lowering of the level of early pre-rRNA processing in these experimental conditions. This decrease was also detected in the bulk of the dense fibrillar component. These data support the idea that altered (reduced) gravity results in lowering the level of functional activity of the nucleolus.

Effect of fluorogestone acetate on embryo recovery and quality in eCG-superovulated goats with premature luteal regression.

The objective of this study was to evaluate if treatment of eCG-superovulated goats with fluorogestone acetate (FGA) would increase the number and quality of embryos recovered. Goats (n = 25) were given an intravaginal sponge containing 45 mg FGA for 12 days, with 1000 IU eCG and 7.5mg of Luprostiol (a PGF(2 alpha) analog) given -48 and 0 h relative to sponge removal. Goats were mated by natural service every 12h during estrus and surgical embryo collection was done 6 days after the last mating. There were two treatment groups; those in the FGA group (n = 13) had a FGA sponge from 8h after mating to embryo collection, whereas goats in the control group (n = 12) did not receive any post-mating treatment. Premature luteal regression occurred in 61.5% (8/13) and 83.3% (10/12) of the goats in the FGA and the control groups, respectively (P > 0.05). Corpus luteum life span averaged 4 days in goats with premature luteolysis. The mean (+/- S.E.) number of transferable embryos was 5.7 +/- 1.6 in the FGA group and 0.1 +/- 0.1 in the control group (P < 0.05). Within the FGA group, the embryo recovery rate was similar in goats with premature luteal regression compared to those with normal luteal function, although non-transferable embryos were only found in goats with premature luteal regression. In conclusion, post-breeding treatment with FGA increased embryonic survival in eCG-superovulated goats, even though it did not prevent premature luteal regression.

Alterations of the intranucleolar DNA localization caused by simulated microgravity.

The functions of the nucleolus responsible for the biosynthesis of ribosomes in altered gravity are still unclear. The location of nucleolar DNA both in control and simulated microgravity with fluorescent and immunogold cytochemistry was investigated. We determined firstly the redistribution of rDNA in the nucleolar components accompanied with the changes in their ultrastructure. According to it, lowering the level of rDNA transcription in simulated microgravity was firstly proposed.

Towards the establishment of a permanent colony of Drosophila in the International Space Station: hardware tests and adaptation of techniques.

Our group has the long-term goal of establishing a permanent colony of Drosophila melanogaster in the International Space Station (ISS) to study its long-term adaptation to this unusual environment. We are currently building an enhanced version of an automatic cultivation unit (ESE1) in which we can grow flies with minimal crew involvement. We are working on a fixation unit as well. We are also adapting current protocols to maintain fixed animals during at least three months, the minimal refurbishment shuttle flight time, with minimal handling and maximal automatization, that can be used for inmuno-cytochemistry, ultrastructure or molecular experiments.

The functional organization of the nucleolus in proliferating plant cells.

The nucleolus is a prominent nuclear organelle which morphologically expresses all functional steps necessary for the synthesis of ribosomes, from transcription of rRNA genes to the assembly and maturation of preribosomal particles and their transport to the cytoplasm. Structurally, the nucleolus contains some basic components common to practically all cell types, namely fibrillar centers (FCs), the dense fibrillar component (DFC), and the granular component (GC); however, the organization and distribution of these components is highly variable, depending on cell identity and functional status. The different steps of ribosome biogenesis are not strictly correlated with the structural components of the nucleolus. Thus, FCs are most likely the anchoring sites for the accumulation of rDNA, and the sites where the assembly of transcription complexes takes place, but transcription of rRNA genes actually occurs at discrete points in the transition zone between FCs and the DFC. The DFC is a structurally homogeneous, but functionally heterogeneous component in which transcription and some early and advanced steps of pre-rRNA processing develop successively in a gradual fashion, from transition with FCs to transition with the GC. Finally, the GC is the site of the later steps of preribosomal processing, including the final assembly of ribosomal proteins for the export of mature particles to the cytoplasm. The rate of ribosome biogenesis, as well as the structure of the nucleolus, are highly influenced by the proliferation status of the cell, and by factors regulating cell cycle progression. These factors are nucleolar proteins, such as nucleolin, which are targets of signal transduction mechanisms, being at the same time regulators of key steps in preribosome synthesis and processing. Thus, many features of the nucleolus, such as the structural organization of its components, the level and distribution of certain nucleolar proteins and, in general, the rate of ribosome biogenesis, show profound variations throughout cell cycle periods. Particularly interesting is the behavior of the nucleolus during mitosis, in which its structure is disorganized and its activity is stopped, even though the individual transcription and processing complexes are not disassembled, but carried from one cell generation to the next one in such a way that the daughter-cell nucleoli are built with materials coming from the parent-cell nucleolus. Transcription complexes remain assembled at the chromosomal nucleolar organizer in which the rRNA genes are clustered, and processing complexes are carried at the chromosome periphery, and then they are organized into discrete entities called prenucleolar bodies, whose fusion, together with the resumption of transcription and processing, originates the new nucleolus.

Simultaneous localization of transcription and early processing markers allows dissection of functional domains in the plant cell nucleolus.

Nucleolar transcription in isolated onion cell nuclei was visualized, after Br-UTP incorporation, under the conventional fluorescence microscope, the confocal microscope, and the transmission electron microscope. The confocal microscopy study of transcription was combined with immunodetection of fibrillarin, a component of the RNP complex involved in the early processing of pre-rRNA. Superposition of transcription and fibrillarin images from the same optical section showed some small "black holes" in the nucleolus, around which a lateral and radial differentiation of labeling was observed: laterally, zones corresponding to transcription labeling alternated with zones of fibrillarin labeling; radially, areas of transcription gradually became areas of colocalization of transcription and fibrillarin, and, further outward, of fibrillarin alone, which occupied the major part of the labeled nucleolar area. Three-dimensional reconstruction of the nucleolar transcription labeling, from confocal optical sections, showed clusters of foci arranged around an area of low or no labeling. Thin labeled extensions, connecting single foci, were observed. Visualization of transcription at the ultrastructural level identified the black holes as fibrillar centers, in view of their size and the absence of labeling in them. In fact, most of the labeling was observed in discrete areas of the dense fibrillar component, near fibrillar centers, including the transition area between these two components. This observation was supported by a quantitative study. Otherwise, the outline of fibrillar centers did not appear entirely surrounded by particles, and a minor proportion of particles was detected dispersed throughout the dense fibrillar component. As a complementary study, the transcription factor upstream binding factor (UBF) and the protein NopA64, a plant nucleolin homologue, were immunolocalized. Small foci of UBF localization alone and other foci in which the two protein markers overlapped were observed. The outer areas of the nucleolus showed the exclusive presence of NopA64. Under the electron microscope, UBF labeling, quantitatively assessed, appeared as clusters of particles, most of them surrounding fibrillar centers. A graphic model is presented to give a molecular interpretation of these data.