BIOLAB, EPU and EMCS for cell culture experiments on the ISS

Two ESA facilities are under development for biological research on the International Space Station: BIOLAB as part of the European "Columbus" Laboratory and the European Modular Cultivation System (EMCS), foreseen for accommodation in the US Lab "Destiny". Both facilities have an incubator (18-40 degrees C) and use standard Experiment Containers, mounted on two centrifuge rotors providing either microgravity or variable g-levels from 0.001 x g to 2.0 x g. Standard interface plates supply each container with power and data lines, with gas (controlled CO2, O2 and water vapour concentration; trace gas removal), and--for EMCS only--with water. The degree of automation is higher in BIOLAB: it contains a robotic Handling Mechanism for automatic sampling and handling of liquids, which can be stored at cool or cold temperatures or injected for automatic on-board analysis into a microscope or a spectrophotometer. For analyses on the running centrifuge, small automatic microscopes can be installed in the Experiment Containers. Several designs for supporting cell culture experiments have been studied for BIOLAB and EMCS. BIOLAB has in addition a Bio-Glovebox, which can be sterilised and where new cell cultures may be prepared under 1 x g conditions from deep-frozen samples in the Experiment Preparation Unit (EPU): the cryo-protectant will be removed by automatic washing cycles. Both facilities, EMCS and BIOLAB (with EPU), have also provisions for telescience operations through video, data and command lines, either operated by the crew or by the experimenter on ground.     

Plant growth using EMCS hardware on the ISS

Under separate contracts with ESA (FUMO and ERM Study) and as a link in the development of the European Modular Cultivation System's (EMCS) functionality and biocompatibility, plant studies have been performed at The Plant Biocentre in Trondheim, Norway. The main goal was to test whether the breadboards containing the major components planned for use in the EMCS would be optimal for space experiments with plant material. The test plans and the experimental set-up for the verification of biocompatibility and biological functionality included the use of a few model plant species including cress (Lepidium sativum L.) and Arabidopsis thaliana. The plants were tested at different developmental levels of morphological and physiological complexity (illumination, life support, humidity control, water supply, observation, short- and long-term plant growth experiments and contamination prevention). Results from the tests show that the EMCS concept is useful for long duration plant growth on the ISS.     

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.     

Automated culture system experiments hardware: developing test results and design solutions

The experiment proposed by Prof. Ricci University of Milan is funded by ASI with Laben as industrial Prime Contractor. ACS-EH (Automated Culture System-Experiment Hardware) will support the multigenerational experiment on weightlessness with rotifers and nematodes within four Experiment Containers (ECs) located inside the European Modular Cultivation System (EMCS) facility..Actually the Phase B is in progress and a concept design solution has been defined. The most challenging aspects for the design of such hardware are, from biological point of view the provision of an environment which permits animal's survival and to maintain desiccated generations separated and from the technical point of view, the miniaturisation of the hardware itself due to the reduce EC provided volume (160mmx60mmx60mm). The miniaturisation will allow a better use of the available EMCS Facility resources (e.g. volume. power etc.) and to fulfil the experiment requirements. ACS-EH, will be ready to fly in the year 2005 on boar the ISS.     

Toxicity and extrachromosomal DNA in strains of the cyanobacterium Microcystis aeruginosa

Abstract Correlations were sought between toxicity and the presence of plasmids in toxic and non-toxic strains of Microcystis aeruginosa . Plasmids were present in toxic and non-toxic cultures. Cultivation of the toxic Microcystis PCC7820 in the presence of novobiocin did not influence toxicity, although extrachromosomal DNA was no longer detected after novobiocin treatment. The data indicate that it is unlikely that plasmids are involved in the toxicity of Microcystis PCC7820.     

Proton Irradiation Facility and space radiation monitoring at the Paul Scherrer Institute

The Proton Irradiation Facility (PIF) has been designed and constructed, in cooperation between Paul Scherrer Institute (PSI) and European Space Agency (ESA), for terrestrial proton testing of components and materials for spacecraft. Emphasis has been given to generating realistic proton spectra encountered by space-flights at any potential orbit. The facility, designed in a user-friendly manner, can be readily adapted to the individual requirements of experimenters. It is available for general use serving also in testing of radiation monitors and for proton experiments in different scientific disciplines. The Radiation Environment Monitor REM has been developed for measurements of the spacecraft radiation conditions. Two instruments were launched into space, one into a Geo-stationary Transfer Orbit on board of the STRV-1b satellite and one into a Low Earth Orbit on the Russian MIR station. The next generation of monitors (SREMs--Standard REMs) is currently under development in partnership of ESA, PSI and Contraves-Space. They will operate both as minimum intrusive monitors, which provide radiation housekeeping data and alert the spacecraft when the radiation level crosses allowed limits and as small scientific devices measuring particle spectra and fluxes. Future missions as e.g. INTEGRAL, STRV-1c and PROBA will be equipped with new SREMs.     

A rapid controller of temperature for use in determining Arrhenius profiles in biomembrane systems

To minimize artifacts in temperature-velocity (Arrhenius) profiles due to aging of preparations of biological membranes, a rapid controller of temperature was developed for spectrophotometric or polarographic (O₂ electrode) measurements. The reaction mixture is cooled or heated through contact with Peltier elements. One Pt temperature sensor in the cuvette or electrode holder controls current flow into the Peltier units, and another Pt temperature sensor in the reaction mixture is used to read out the sample temperature on a meter or recorder, and to provide feedback control. The sample temperature can be reproducibly set to within 0.1°C, with a noise level of 0.04°C or less; a change of 4°C takes 1 min.On leave at the Arrhenius Laboratory, University of Stockholm.     

Detection of Staphylococcus aureus enterotoxin B at femtomolar levels with a miniature integrated two-channel surface plasmon resonance (SPR) sensor

Surface plasmon resonance (SPR) biosensors offer the capability for continuous real-time monitoring. The commercial instruments available have been large in size, expensive, and not amenable to field applications. We report here an SPR sensor system based on a prototype two-channel system similar to the single channel Spreeta devices. This system is an ideal candidate for field use. The two-channel design provides a reference channel to compensate for bulk refractive index (RI), non-specific binding and temperature variations. The SPR software includes a calibration function that normalizes the response from both channels, thus enabling accurate referencing. In addition, a temperature-controlled enclosure utilizing a thermo-electric module based on the Peltier effect provides the temperature stability necessary for accurate measurements of RI. The complete SPR sensor system can be powered by a 12V battery. Pre-functionalized, disposable, gold-coated thin glass slides provide easily renewable sensor elements for the system. Staphylococcus aureus enterotoxin B (SEB), a small protein toxin was directly detectable at sub-nanomolar levels and with amplification at femtomolar levels. A regeneration procedure for the sensor surface allowed for over 60 direct detection cycles in a 1-month period.     

The human serum paraoxonase/arylesterase polymorphism.

The heterozygous human serum paraoxonase phenotype can be clearly distinguished from both homozygous phenotypes on the basis of its distinctive ratio of paraoxonase to arylesterase activities. A trimodal distribution of the ratio values was found with 348 individual serum samples, measuring the ratio of paraoxonase activity (with 1 M NaCl in the assay) to arylesterase activity, using phenylacetate. The three modes corresponded to the three paraoxonase phenotypes, A, AB, and B (individual genotypes), and the expected Mendelian segregation of the trait was observed within families. The paraoxonase/arylesterase activity ratio showed codominant inheritance. We have defined the genetic locus determining the aromatic esterase (arylesterase) responsible for the polymorphic paraoxonase activity as esterase-A (ESA) and have designated the two common alleles at this locus by the symbols ESA*A and ESA*B. The frequency of the ESA*A allele was estimated to be .685, and that of the ESA*B allele, 0.315, in a sample population of unrelated Caucasians from the United States. We postulate that a single serum enzyme, with both paraoxonase and arylesterase activity, exists in two different isozymic forms with qualitatively different properties, and that paraoxon is a "discriminating" substrate (having a polymorphic distribution of activity) and phenylacetate is a "nondiscriminating" substrate for the two isozymes. Biochemical evidence for this interpretation includes the cosegregation of the degree of stimulation of paraoxonase activity by salt and paraoxonase/arylesterase activity ratio characteristics; the very high correlation between both the basal (non-salt stimulated) and salt-stimulated paraoxonase activities with arylesterase activity; and the finding that phenylacetate is an inhibitor for paraoxonase activities in both A and B types of enzyme.     

Can <Emphasis Type="Italic">Bradyrhizobium</Emphasis> strains inoculation reduce water deficit effects on peanuts?

Drought is one of the environmental factors that most affects peanut cultivation in semi-arid regions, resulting in economic losses to growers. However, growth promoting bacteria are able to reduce water deficit damage in some plant species. In this context, this study aimed to evaluate the interaction of Bradyrhizobium strains reducing water stress effects on peanut genotypes by antioxidant enzymes activities, leaf gas exchanges and vegetative growth, as well as to determine the taxonomic positioning of strain ESA 123. The 16S rRNA gene of ESA 123 was amplified by PCR and sequenced by dideoxy Sanger sequencing method. An experiment was performed in greenhouse with three peanut genotypes (BRS Havana, CNPA 76 AM and 2012-4), two Bradyrhizobium strains (SEMIA 6144 and ESA 123), a mineral source of N and an absolute control (without N) under two water regimes (with and without irrigation). Seeds of peanut were sown and the plants were grown until 30 days after emergence. On the 20th day, the water deficit plants group had their irrigation suspended for 10 days. At in silico analyzes, ESA 123 presented 98.97% similarity with the type strain of B. kavangense. Leaf gas exchange was affected by water deficit; as well as alteration of antioxidant activities and reduction of vegetative growth variables. However, some plants inoculated with SEMIA 6144 and ESA 123 strains presented lower reductions and increment of some evaluated variables, mainly the ones inoculated with the ESA 123 strain, Bradyrhizobium sp. from the semi-arid region of Northeast Brazil. This data suggests beneficial effects of the peanut-Bradyrhizobium interaction in a water stress condition, specially with the ESA 123 strain.     

The study of the genetic effects in generation of pea plants cultivated during the whole cycle of ontogenesis on the board of RS ISS

Results of studies on growth and development of offspring of two genetically marked dwarf pea lines planted during the whole ontogenesis cycle in the Lada space greenhouse on board of Russian Segment of International Space Station (RS ISS) are presented. The offspring of M1 and M2 plants grown from seeds formed during space flight was examined under conditions of Earth-based. Cultivation. It had been shown that growth and developmental characteristics, frequency of chromosome aberrations in primary root meristem and level of molecular polymorphism revealed in individual plants via RAPD method show no significant differences between offspring of "space-grown" and control seeds.     

Finite Element Study of the Mechanical Response in Spinal Cord during the Thoracolumbar Burst Fracture

Background

The mechanical response of the spinal cord during burst fracture was seldom quantitatively addressed and only few studies look into the internal strain of the white and grey matters within the spinal cord during thoracolumbar burst fracture (TLBF). The aim of the study is to investigate the mechanical response of the spinal cord during TLBF and correlate the percent canal compromise (PCC) with the strain in the spinal cord.

Methodology/Principal Findings

A three-dimensional (3D) finite element (FE) model of human T12-L1 spinal cord with visco-elastic property was generated based on the transverse sections images of spinal cord, and the model was validated against published literatures under static uniaxial tension and compression. With the validated model, a TLBF simulation was performed to compute the mechanical strain in the spinal cord with the PCC. Linear regressions between PCC and strain in the spinal cord show that at the initial stage, with the PCC at 20%, and 45%, the corresponding mechanical strains in ventral grey, dorsal grey, ventral white, dorsal white matters were 0.06, 0.04, 0.12, 0.06, and increased to 0.14, 0.12, 0.23, and 0.13, respectively. At the recoiled stage, when the PCC was decreased from 45% to 20%, the corresponding strains were reduced to 0.03, 0.02, 0.04 and 0.03. The strain was correlated well with PCC.

Conclusions/Significance

The simulation shows that the strain in the spinal cord correlated well with the PCC, and the mechanical strains in the ventral regions are higher than those in the dorsal regions of spinal cord tissue during burst fracture, suggesting that the ventral regions of the spinal cord may susceptible to injury than the dorsal regions.     

Peptide switch is essential for Sirt1 deacetylase activity

In mammals, the Sirtuins are composed of seven Sir2 orthologs (Sirt1-7) with a conserved deacetylase core that utilizes NAD(+) as a cofactor. Interestingly, the deacetylase core of Sirt1 by itself has no catalytic activity. We found within the C-terminal domain a 25 aa sequence that is essential for Sirt1 activity (ESA). Our results indicate that the ESA region interacts with and functions as an "on switch" for the deacetylase core. The endogenous Sirt1 inhibitor DBC1, which also binds to the deacetylase core, competes with and inhibits the ESA region from interacting with the deacetylase core. We discovered an ESA mutant peptide that can bind to the deacetylase core and inhibit Sirt1 in trans. By using this mutant peptide, we were able to inhibit Sirt1 activity and to increase the chemosensitivity of androgen-refractory prostate cancer cells. Therefore, the ESA region is a potential target for development of therapies to regulate Sirt1.     

A novel plasmid recombination mechanism of the marine cyanobacterium Synechococcus sp. PCC7002.

We describe a novel mechanism of site-specific recombination in the unicellular marine cyanobacterium Synechococcus sp. PCC7002. The specific recombination sites on the smallest plasmid pAQ1 were localized by studying the properties of pAQ1-derived shuttle-vectors. We found that a palindromic element, the core sequence of which is G(G/A)CGATCGCC, functions as a resolution site for site-specific plasmid recombination. Furthermore, site-directed mutagenesis analysis of the element show that the site-specific recombination in the cyanobacterium requires sequence specificity, symmetry in the core sequence and, in part, the spacing between the elements. Interestingly, this element is over-represented not only in pAQ1 and in the genome of the cyanobacterium, but also in the accumulated cyanobacterial sequences from Synechococcus sp. PCC6301, PCC7942, vulcanus and Synechocystis sp. PCC6803 within GenBank and EMBL databases. Thus, these findings strongly suggest that the site-specific recombination mechanism based on the palindromic element should be common in these cyanobacteria.     

Evaluation of immunization with tachyzoite excreted-secreted proteins in a novel susceptible mouse model (A/Sn) for Toxoplasma gondii

Toxoplasma gondii is an important food-borne parasite transmitted primarily from animals to humans through meat consumption, mainly pork and lamb, as well as through oocysts shed by cats. Infection in humans can cause severe neonatal malformations, ocular complications or encephalitis. Toxoplasmosis infection during pregnancy, especially in sheep, often results in abortion, representing considerable economic loss. The aim of this study was to investigate whether Toxoplasma gondii pooled excreted-secreted antigens (ESA), recovered from infected culture supernatants with tachyzoites used as immunogen, can protect experimental mice against T. gondii infection. For immunization experiments, we evaluated A/Sn inbred mice, a novel susceptible mouse model for T. gondii and a virulent strain (RH) for challenge experiments. The antigen selection was based on those produced by tachyzoites since they are responsible for disseminating the infection as well as stimulating the humoral and cellular immune responses. ESA were recovered from VERO cell-culture supernatants infected with virulent RH strain tachyzoites harvested after 48 h. Groups of 5 female mice were intraperitoneally (i.p.) immunized with 4 doses at 2 week intervals with 20 μg of ESA adsorbed to 0.5 mg of alum. The control group received only the adjuvant in PBS on the same dates. Pooled serum collected from chronically infected mice was used as positive control. Blood samples were collected from tail veins 14 days after each immunization. Antibody was detected using ELISA, indirect immunofluorescence and immunoblotting. Anti-ESA antibodies were also evaluated by agglutination, complement-mediated lysis and antibody-mediated cellular toxicity. Fifteen days after the last immunization, both groups were challenged (i.p.) with 1 × 10³ RH strain tachyzoites. The parasitemia was evaluated by PCR, and survival was followed daily. The results showed an increase of antibody levels after each immunization. Anti-ESA antibodies also reacted with a crude tachyzoite antigen and bonded on the parasite surface, with particularly high intensity at the apical region. Anti-ESA antibodies were also able to agglutinate and kill tachyzoites in vitro through interactions with complement and cellular pathways. Even though the tachyzoite challenge was lethal to the mice, PCR results suggested that immunized mice had lower parasitemia as well as longer survival (72 h) than mice from the control group.     

Non-consensus heptamer sequences destabilize the RAG post-cleavage complex,making ends available to alternative DNA repair pathways

V(D)J recombination entails double-stranded DNA cleavage at the antigen receptor loci by the RAG1/2 proteins, which recognize conserved recombination signal sequences (RSSs) adjoining variable (V), diversity (D) and joining (J) gene segments. After cleavage, RAG1/2 remain associated with the coding and signal ends (SE) in a post-cleavage complex (PCC), which is critical for their proper joining by classical non-homologous end joining (NHEJ). Certain mutations in RAG1/2 destabilize the PCC, allowing DNA ends to access inappropriate repair pathways such as alternative NHEJ, an error-prone pathway implicated in chromosomal translocations. The PCC is thus thought to discourage aberrant rearrangements by controlling repair pathway choice. Since interactions between RAG1/2 and the RSS heptamer element are especially important in forming the RAG-SE complex, we hypothesized that non-consensus heptamer sequences might affect PCC stability. We find that certain non-consensus heptamers, including a cryptic heptamer implicated in oncogenic chromosomal rearrangements, destabilize the PCC, allowing coding and SEs to be repaired by non-standard pathways, including alternative NHEJ. These data suggest that some non-consensus RSS, frequently present at chromosomal translocations in lymphoid neoplasms, may promote genomic instability by a novel mechanism, disabling the PCC’s ability to restrict repair pathway choice.