The design and development of a release mechanism for space shuttle life-science experiments

The design, development, and testing of a release mechanism for use in two life science experiments on the Spacelab 1, 4, and D1 missions is described. The mechanism is a self latching ball lock device actuated by a linear solenoid. An unusual feature is the tapering of the ball lock plunger to give it a near constant breakout force for release under a wide range of loads. The selection of the design, based on the design requirements, is discussed. A number of problems occurred during development and test, including problems caused by human factors that became apparent after initial delivery for crewtraining sessions. These problems and their solutions are described to assist in the design and testing of similar mechanisms

Stable Expression of Gal/GalNAc Lectin of Entamoeba Histolytica in Transgenic Chloroplasts and Immunogenicity in Mice Towards Vaccine Development for Amoebiasis

Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance and multigene engineering in a single transformation event. Entamoeba histolytica infects 50 million people, causing about 100 000 deaths annually, but there is no approved vaccine against this pathogen. LecA, a potential target for blocking amoebiasis, was expressed for the first time in transgenic plants. Stable transgene integration into chloroplast genomes and homoplasmy were confirmed by polymerase chain reaction and Southern blot analyses. LecA expression was evaluated by Western blots and quantified by enzyme-linked immunosorbent assay (up to 6.3% of total soluble protein or 2.3 mg LecA/g leaf tissue). Subcutaneous immunization of mice with crude extract of transgenic leaves resulted in higher immunoglobulin G titres (up to 1 : 10 000) than in previous reports. An average yield of 24 mg of LecA per plant should produce 29 million doses of vaccine antigen per acre of transgenic plants. Such high levels of expression and immunogenicity should facilitate the development of a less expensive amoebiasis vaccine

Novel pathways for glycoprotein import into chloroplasts

Although the chloroplast contains its own genome, majority of its protein components are encoded by nuclear genes and must be imported post-translationally. In general, proteins synthesized by cytosolic ribosomes are post-translationally targeted to the chloroplast through interactions between their N-terminal transit sequence and protein translocon Toc/Tic complexes in the chloroplast membranes. An alternative pathway that mediates post-translational delivery of proteins to the chloroplast via the secretory pathway was recently described. This pathway provides new opportunities for complementation of the chloroplast protein maturation machinery with chaperones needing endoplasmic reticulum and/or Golgi typical maturations such as N-glycosylation for their biological activity or using chloroplasts as a storage compartment for glycoproteins

A posteriori teleportation

The article by Bouwmeester et al. on experimental quantum teleportation constitutes an important advance in the burgeoning field of quantum information. The experiment was motivated by the proposal of Bennett et al. in which an unknown quantum state is `teleported' by Alice to Bob. As illustrated in Fig. 1, in the implementation of this procedure, by Bouwmeester et al., an input quantum state is `disembodied' into quantum and classical components, as in the original protocol. However, in contrast to the original scheme, Bouwmeester et al.'s procedure necessarily destroys the state at Bob's receiving terminal, so a `teleported' state can never emerge as a freely propagating state for subsequent examination or exploitation. In fact, teleportation is achieved only as a postdiction.Comment: 1 page LaTeX including 1 figure. Scientific Correspondence about: "Experimental quantum teleportation" Nature 390, 575 (1997

High fidelity imaging and high performance computing in nonlinear EIT

We show that nonlinear EIT provides images with well defined characteristics when smoothness of the image is used as a constraint in the reconstruction process. We use the gradient of the logarithm of resistivity as an effective measure of image smoothness, which has the advantage that resistivity and conductivity are treated with equal weight. We suggest that a measure of the fidelity of the image to the object requires the explicit definition and application of such a constraint. The algorithm is applied to the simulation of intra-ventricular haemorrhaging (IVH) in a simple head model. The results indicate that a 5% increase in the blood content of the ventricles would be easily detectable with the noise performance of contemporary instrumentation. The possible implementation of the algorithm in real time via high performance computing is discussed

Molecular Strategies for Gene Containment in Transgenic Crops

The potential of genetically modified (GM) crops to transfer foreign genes through pollen to related plant species has been cited as an environmental concern. Until more is known concerning the environmental impact of novel genes on indigenous crops and weeds, practical and regulatory considerations will likely require the adoption of gene-containment approaches for future generations of GM crops. Most molecular approaches with potential for controlling gene flow among crops and weeds have thus far focused on maternal inheritance, male sterility, and seed sterility. Several other containment strategies may also prove useful in restricting gene flow, including apomixis (vegetative propagation and asexual seed formation), cleistogamy (self-fertilization without opening of the flower), genome incompatibility, chemical induction/deletion of transgenes, fruit-specific excision of transgenes, and transgenic mitigation (transgenes that compromise fitness in the hybrid). As yet, however, no strategy has proved broadly applicable to all crop species, and a combination of approaches may prove most effective for engineering the next generation of GM crops

Expression of Bacillus Anthracis Protective Antigen in Transgenic Chloroplasts of Tobacco, a Non-Food/Feed Crop

The Centers for Disease Control (CDC) lists Bacillus anthracis as a category A agent and estimates the cost of an anthrax attack to exceed US$ 26 billion per 100,000 exposed individuals. Concerns regarding anthrax vaccine purity, a requirement for multiple injections, and a limited supply of the protective antigen (PA), underscore the urgent need for an improved vaccine. Therefore, the 83 kDa immunogenic Bacillus anthracis protective antigen was expressed in transgenic tobacco chloroplasts. The PA gene (pag) was cloned into a chloroplast vector along with the psbA regulatory signals to enhance translation. Chloroplast integration of the transgenes was confirmed by PCR and Southern blot analyses. Crude plant extracts contained up to 2.5 mg full length PA/g of fresh leaf tissue and this showed exceptional stability for several months in stored leaves or crude extracts. Maximum levels of expression were observed in mature leaves under continuous illumination. Co-expression of the ORF2 chaperonin from Bacillus thuringiensis did not increase PA accumulation or induce folding into cuboidal crystals in transgenic chloroplasts. Trypsin, chymotrypsin and furin proteolytic cleavage sites present in PA were protected in transgenic chloroplasts because only full length PA 83 was observed without any degradation products. Both CHAPS and SDS detergents extracted PA with equal efficiency and PA was observed in the soluble fraction. Chloroplast-derived PA was functionally active in lysing mouse macrophages when combined with lethal factor (LF). Crude leaf extracts contained up to 25 μg functional PA/ml. With an average yield of 172 mg of PA per plant using an experimental transgenic cultivar grown in a greenhouse, 400 million doses of vaccine (free of contaminants) could be produced per acre, a yield that could be further enhanced 18-fold using a commercial cultivar in the field

Expression of Cholera Toxin B–Proinsulin Fusion Protein in Lettuce and Tobacco Chloroplasts – Oral Administration Protects Against Development of Insulitis in Non-Obese Diabetic Mice

Lettuce and tobacco chloroplast transgenic lines expressing the cholera toxin B subunit–human proinsulin (CTB-Pins) fusion protein were generated. CTB-Pins accumulated up to ~16% of total soluble protein (TSP) in tobacco and up to ~2.5% of TSP in lettuce. Eight milligrams of powdered tobacco leaf material expressing CTB-Pins or, as negative controls, CTB–green fluorescent protein (CTB-GFP) or interferon–GFP (IFN-GFP), or untransformed leaf, were administered orally, each week for 7 weeks, to 5-week-old female non-obese diabetic (NOD) mice. The pancreas of CTB-Pins-treated mice showed decreased infiltration of cells characteristic of lymphocytes (insulitis); insulin-producing β-cells in the pancreatic islets of CTB-Pins-treated mice were significantly preserved, with lower blood or urine glucose levels, by contrast with the few β-cells remaining in the pancreatic islets of the negative controls. Increased expression of immunosuppressive cytokines, such as interleukin-4 and interleukin-10 (IL-4 and IL-10), was observed in the pancreas of CTB-Pins-treated NOD mice. Serum levels of immunoglobulin G1 (IgG1), but not IgG2a, were elevated in CTB-Pins-treated mice. Taken together, T-helper 2 (Th2) lymphocyte-mediated oral tolerance is a likely mechanism for the prevention of pancreatic insulitis and the preservation of insulin-producing β-cells. This is the first report of expression of a therapeutic protein in transgenic chloroplasts of an edible crop. Transplastomic lettuce plants expressing CTB-Pins grew normally and transgenes were maternally inherited in T1 progeny. This opens up the possibility for the low-cost production and delivery of human therapeutic proteins, and a strategy for the treatment of various other autoimmune diseases

DNA Barcoding and Morphological Identification of Benthic Nematodes Assemblages of Estuarine Intertidal Sediments: Advances in Molecular Tools for Biodiversity Assessment

Concerns regarding the status of marine ecosystems have increased in part due to traditional and emerging human activities in marine waters, driving a demand for approaches with high sample throughput capability to improve ecosystem monitoring. Nematodes are already used as indicator species in biodiversity assessments and biomonitoring of terrestrial and marine systems, with molecular approaches offering the opportunity to utilize these organisms further in large scale ecological surveys and environmental assessments. Based on an available nematode dataset for estuarine sediments of the Mira estuary (SW coast, Portugal), we evaluated the diversity of the nematode community of this system, using the molecular markers 18S rRNA and COI genes. These approaches were compared to voucher specimens from a morphological characterization of the same samples allowing validation and comparison between nematode communities. The spatial and temporal variability of the density and diversity of the nematode assemblages was analyzed based on morphological characterization to allow the validation and efficiency of the genetic characterization. A PCO ordination plot showed a distinct separation of the assemblages between sampling occasions confirmed by PERMANOVA analysis, which showed significant differences, although no significant differences were detected between sampling sites. The morphological characterization identified 50 genera of which only 26 and 25 distinct 18S rRNA and COI DNA barcodes, respectively, were obtained. 90.2% of the morphologically identified specimens representing eleven different genera, successfully generated DNA barcodes for both 18S rRNA and COI genes. This study confirmed that the success of the 18S rRNA gene PCR amplification is higher than of COI gene with 43 species amplified against 34. The study highlights a limitation of available sequences for both targets in databases when compared to the known diversity of marine nematodes. The gene sequences of this study enriched the databases, contributing gene sequences from 7 to 16 new genera for the 18S rRNA and COI genes, respectively. A robust database of gene sequences is a prerequisite for the development of robust high sample throughput techniques to be applied in marine assessing and monitoring programs

Catalytic combustion of methane on Co/MgO: characterisation of active cobalt sites

A series of Co/MgO catalysts with 3–12 wt.% Co were prepared by impregnation and calcined at 1073 K for 10 h. The catalytic behaviour of these samples toward CH4 combustion was found to increase with cobalt loading, though a plateau was reached at ca. 9 wt.% Co content. Bulk characterisation was carried out using XRD, TPR and Raman spectroscopy, and showed that the solids were made up of a CoO–MgO solid solution and a MgO phase. A detailed examination of their surfaces was achieved through FTIR spectroscopy of adsorbed CO probe molecules, which indicated that at low cobalt loadings only a small proportion of the Co going into the solid solution was present on exposed faces as either Co2+ oxo-species or pentacoordinated Co2+. However, as the cobalt content of the samples increased, a larger amount was exposed on the surface. This effect levelled off at 9 wt.% Co, after which the increase in exposed Co2+ sites was countered by the masking effect of islands of MgO. In addition, at high cobalt loadings (9 and 12 wt.%) Co formed small clusters which showed bulk CoO-like behaviour. Consequently, the benefit of having surface Co2+ species was balanced by the clustering effect of these species and the presence of MgO islands, negating their contribution to the overall catalytic activity of the samples.Fil: Ulla, Maria Alicia del H.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Investigaciones en Catálisis y Petroquímica ; ArgentinaFil: Spretz, R.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Investigaciones en Catálisis y Petroquímica ; ArgentinaFil: Lombardo, Eduardo Agustin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Investigaciones en Catálisis y Petroquímica ; ArgentinaFil: Daniell, W.. Ludwig Maximilians Universitat; AlemaniaFil: Knözinger, H.. Ludwig Maximilians Universitat; Alemani