Dr Camelia Dijkstra

Spaceflight experiments have suggested a possible effect of microgravity on the plasmid transfer among strains of the Gram-positive Bacillus thuringiensis, as opposed to no effect recorded for Gram-negative conjugation. To investigate these potential effects in a more affordable experimental setup, three ground-based microgravity simulators were tested: the Rotating Wall Vessel (RWV), the Random Positioning Machine (RPM), and a superconducting magnet. The bacterial conjugative system consisted in biparental matings between two B. thuringiensis strains, where the transfer frequencies of the conjugative plasmid pAW63 and its ability to mobilize the nonconjugative plasmid pUB110 were assessed. Specifically, potential plasmid transfers in a 0 g position (simulated microgravity) were compared to those obtained under 1 g (normal gravity) condition in each device. Statistical analyses revealed no significant difference in the conjugative and mobilizable transfer frequencies between the three different simulated microgravitational conditions and our standard laboratory condition. These important ground-based observations emphasize the fact that, though no stimulation of plasmid transfer was observed, no inhibition was observed either. In the case of Gram-positive bacteria, this ability to exchange plasmids in weightlessness, as occurs under Earth's conditions, should be seen as particularly relevant in the scope of spread of antibiotic resistances and bacterial virulence. © Copyright 2009 Mary Ann Liebert, Inc.

Background: Many biological systems respond to the presence or absence of gravity. Since experiments performed in space are expensive and can only be undertaken infrequently, Earth-based simulation techniques are used to investigate the biological response to weightlessness. A high gradient magnetic field can be used to levitate a biological organism so that its net weight is zero.Results: We have used a superconducting magnet to assess the effect of diamagnetic levitation on the fruit fly D. melanogaster in levitation experiments that proceeded for up to 22 consecutive days. We have compared the results with those of similar experiments performed in another paradigm for microgravity simulation, the Random Positioning Machine (RPM). We observed a delay in the development of the fruit flies from embryo to adult. Microarray analysis indicated changes in overall gene expression of imagoes that developed from larvae under diamagnetic levitation, and also under simulated hypergravity conditions. Significant changes were observed in the expression of immune-, stress-, and temperature-response genes. For example, several heat shock proteins were affected. We also found that a strong magnetic field, of 16.5 Tesla, had a significant effect on the expression of these genes, independent of the effects associated with magnetically-induced levitation and hypergravity.Conclusions: Diamagnetic levitation can be used to simulate an altered effective gravity environment in which gene expression is tuned differentially in diverse Drosophila melanogaster populations including those of different age and gender. Exposure to the magnetic field per se induced similar, but weaker, changes in gene expression. © 2012 Herranz et al; licensee BioMed Central Ltd.

Gibberellins (GAs) are endogenous hormones that play a predominant role in regulating plant stature by increasing cell division and elongation in stem internodes. The product of the GA 2-oxidase gene from Phaseolus coccineus (PcGA2ox1) inactivates C19-GAs, including the bioactive GAs GA 1 and GA4, by 2β-hydroxylation, reducing the availability of these GAs in plants. The PcGA2ox1 gene was introduced into Solanum melanocerasum and S. nigrum (Solanaceae) by Agrobacterium-mediated transformation with the aim of decreasing the amounts of bioactive GA in these plants and thereby reducing their stature. The transgenic plants exhibited a range of dwarf phenotypes associated with a severe reduction in the concentrations of the biologically active GA1 and GA4. Flowering and fruit development were unaffected. The transgenic plants contained greater concentrations of chlorophyll b (by 88%) and total chlorophyll (11%), although chlorophyll a and carotenoid contents were reduced by 8 and 50%, respectively. This approach may provide an alternative to the application of chemical growth retardants for reducing the stature of plants, particularly ornamentals, in view of concerns over the potential environmental and health hazards of such compounds. © 2007 Springer-Verlag.

Abstract

Potato is one of the main targets for genetic improvement by gene transfer. The aim of the present study was to establish a robust protocol for the genetic transformation of three dihaploid and four economically important cultivars of potato using Agrobacterium tumefaciens carrying the in vivo screenable reporter gene for green fluorescent protein (gfp) and the marker gene for neomycin phosphotransferase (nptII). Stem and leaf explants were used for transformation by Agrobacterium tumefaciens strain LBA4404 carrying the binary vector pHB2892. Kanamycin selection, visual screening of GFP by epifluorescent microscopy, PCR amplification of nptII and gfp genes, as well as RT-PCR and Southern blotting of gfp and Northern blotting of nptII, were used for transgenic plant selection, identification and analysis. Genetic transformation was optimized for the best performing genotypes with a mean number of shoots expressing gfp per explant of 13 and 2 (dihaploid line 178/10 and cv. 'Baltica', respectively). The nptII marker and gfp reporter genes permitted selection and excellent visual screening of transgenic tissues and plants. They also revealed the effects of antibiotic selection on organogenesis and transformation frequency, and the identification of escapes and chimeras in all potato genotypes. Silencing of the gfp transgene that may represent site-specific inactivation during cell differentiation, occurred in some transgenic shoots of tetraploid cultivars and in specific chimeric clones of the dihaploid line 178/10. The regeneration of escapes could be attributed to either the protection of non-transformed cells by neighbouring transgenic cells, or the persistence of Agrobacterium cells in plant tissues after co-cultivation. © 2006 Springer-Verlag.

Diamagnetic levitation is a technique that uses a strong, spatially varying magnetic field to reproduce aspects of weightlessness, on the Earth. We used a superconducting magnet to levitate growing bacterial cultures for up to 18 h, to determine the effect of diamagnetic levitation on all phases of the bacterial growth cycle. We find that diamagnetic levitation increases the rate of population growth in a liquid culture and reduces the sedimentation rate of the cells. Further experiments and microarray gene analysis show that the increase in growth rate is owing to enhanced oxygen availability. We also demonstrate that the magnetic field that levitates the cells also induces convective stirring in the liquid. We present a simple theoretical model, showing how the paramagnetic force on dissolved oxygen can cause convection during the aerobic phases of bacterial growth. We propose that this convection enhances oxygen availability by transporting oxygen around the liquid culture. Since this process results from the strong magnetic field, it is not present in other weightless environments, e.g. in Earth orbit. Hence, these results are of significance and timely to researchers considering the use of diamagnetic levitation to explore effects of weightlessness on living organisms and on physical phenomena.

Abstract

Understanding the effects of gravity on biological organisms is vital to the success of future space missions. Previous studies in Earth orbit have shown that the common fruitfly (Drosophila melanogaster) walks more quickly and more frequently in microgravity, compared with its motion on Earth. However, flight preparation procedures and forces endured on launch made it difficult to implement on the Earth's surface a control that exposed flies to the same sequence of major physical and environmental changes. To address the uncertainties concerning these behavioural anomalies, we have studied the walking paths of D. melanogaster in a pseudo-weightless environment (0g*) in our Earth-based laboratory. We used a strong magnetic field, produced by a superconducting solenoid, to induce a diamagnetic force on the flies that balanced the force of gravity. Simultaneously, two other groups of flies were exposed to a pseudo-hypergravity environment (2g*) and a normal gravity environment (1g*) within the spatially varying field. The flies had a larger mean speed in 0g* than in 1g*, and smaller in 2g*. The mean square distance travelled by the flies grew more rapidly with time in 0g* than in 1g*, and slower in 2g*. We observed no other clear effects of the magnetic field, up to 16.5 T, on the walks of the flies. We compare the effect of diamagnetically simulated weightlessness with that of weightlessness in an orbiting spacecraft, and identify the cause of the anomalous behaviour as the altered effective gravity.

Gravity is an important environmental factor that controls plant growth and development. Studies have shown that the perception of gravity is not only a property of specialized cells, but can also be performed by undifferentiated cultured cells. In this investigation, callus of Arabidopsis thaliana cv. Columbia was used to investigate the initial steps of gravity-related signalling cascades, through altered expression of transcription factors (TFs). TFs are families of small proteins that regulate gene expression by binding to specific promoter sequences. Based on microarray studies, members of the gene families WRKY, MADS-box, MYB, and AP2/EREBP were selected for investigation, as well as members of signalling chains, namely IAA 19 and phosphoinositol-4-kinase. Using qRT-PCR, transcripts were quantified within a period of 30 min in response to hypergravity (8g), clinorotation [2-D clinostat and 3-D random positioning machine (RPM)] and magnetic levitation (ML). The data indicated that (1) changes in gravity induced stress-related signalling, and (2) exposure in the RPM induced changes in gene expression which resemble those of magnetic levitation. Two dimensional clinorotation resulted in responses similar to those caused by hypergravity. It is suggested that RPM and ML are preferable to simulate microgravity than clinorotation. © 2007.

Changes have been reported in the pattern of gene expression in Arabidopsis on exposure to microgravity. Plant cell growth and proliferation are functions that are potentially affected by such changes in gene expression. In the present investigation, the cell proliferation rate, the regulation of cell cycle progression and the rate of ribosome biogenesis (this latter taken to estimate cell growth) have been studied using morphometric markers or parameters evaluated by light and electron microscopy in real microgravity on the International Space Station (ISS) and in ground-based simulated microgravity, using the Random Positioning Machine and the Magnetic Levitation Instrument. Results showed enhanced cell proliferation but depleted cell growth in both real and simulated microgravity, indicating that the two processes are uncoupled, unlike the situation under normal gravity on Earth in which they are strictly co-ordinated events. It is concluded that microgravity is an important stress condition for plant cells compared to normal ground gravity conditions. © 2008 Springer Science+Business Media B.V.

The aim of the present study was to produce marker-free transgenic potato resistant to PVY by using a construct generating self-complementary hairpin RNAs. The method has two steps: 1) establishing a robust protocol for the genetic transformation of economically important cultivars of potato by using gfp and nptII genes; 2) applying the protocol for the transfer of marker-free hairpin construct and analysis of transgenic plants by PCR. In the first step internodes were transformed by Agrobacterium tumefaciens LBA4404 with pHB2892 construct. Kanamycin, visual screening of GFP, PCR of nptII and gfp as well as RT-PCR and Southern blotting of gfp and Northern blotting of nptII were used for transgenic plant selection. Good regeneration and transformation efficiency were obtained for the cvs. Baltica and Désirée while the cvs. Agave and Delikat showed lower figures. In the second step A. tumefaciens with construct: 35SCaMV enhancer and promoter, two repeated inverted PVY-CP sequences separated by an intron and pAnos terminator was used. Regenerated plants without selection, were analysed by PCR using primers in the: 35S promoter, promoter and hairpin region, or in both repeated PVY-CP sequences. A large number of transgenic plants were identified for the most responsive cv. Baltica (17 out of 33) and Désirée (23 out of 66), but only 1 for the cv. Agave (out of 3) and none for the cv. Delikat. This simple two-step strategy might be applied to generate marker-free transgenic plants in other cvs. or plant species. © 2010 University of Bucharest.

Abstract

Withania sominifera (Indian ginseng) was transformed by Agrobacterium rhizogenes. Explants from seedling roots, stems, hypocotyls, cotyledonary nodal segments, cotyledons and young leaves were inoculated with A. rhizogenes strain R1601. Hairy (transformed) roots were induced from cotyledons and leaf explants. The transgenic status of hairy roots was confirmed by polymerase chain reaction using nptII and rolB specific primers and, subsequently, by Southern analysis for the presence of nptII and rolB genes in the genomes of transformed roots. Four clones of hairy roots were established; these differed in their morphology. The doubling time of faster growing cultures was 8–14 d with a fivefold increase in biomass after 28 d compared with cultured, non‐transformed seedling roots. MS‐based liquid medium was superior for the growth of transformed roots compared with other culture media evaluated (SH, LS and N6), with MS‐based medium supplemented with 40 g/L sucrose being optimal for biomass production. Cultured hairy roots synthesized withanolide A, a steroidal lactone of medicinal and therapeutic value. The concentration of withanolide A in transformed roots (157.4 μg/g dry weight) was 2.7‐fold more than in non‐transformed cultured roots (57.9 μg/g dry weight).

Previous experiments in space (unmanned satellites, space shuttle and the International Space Station, ISS),have shown that adult Drosophila flies change their motile behaviour in microgravity. A consistent increase in motility in space was found in these experiments, but mature flies (two weeks old) showed less increase than recently hatched flies. In the case of relatively long exposure to microgravity, the aging of male flies measured upon return to Earth was increased, with flies dying earlier than the corresponding in-flight 1g centrifuge or ground controls. The older flies, which experienced a smaller increase in motility, did not show this acceleration in the aging process. More recently we have performed comparative experiments using ground simulation facilities. Preliminary experiments using a random positioning machine (RPM) indicate that the effects of this simulation approach on the behavior of Drosophila are of smaller magnitude than the corresponding exposure to real microgravity. Further experiments are in progress to confirm this effect. However, when exposed to magnetic levitation, flies exposed to simulated weightlessness increased markedly their motile behavior compared with 1g controls both inside and outside the magnet. This altered gravity-related increase in motility was also less pronounced in more mature flies. This motility effect at the levitation position reproduces the results in real microgravity indicating the interest for space science of this simulation approach. Similar experiments are being performed in the Larger Diameter Centrifuge (LDC) located in ESTEC (the Netherlands) and indicate that 6g, 12g and 20g are key points in the hypergravity response in flies. Our experiments have shown that developmental processes from embryo to adult proceeded normally in the magnet, the RPM and the LDC. In terms of gene expression, preliminary results indicate that the affected set of genes under hypergravity responds in general in an opposite direction than that induced by the real or simulated microgravity exposure. The interest in conducting comparative parallel experiments in the complete spectrum of ground simulation methods is shown in the above studies and will be achieved in the near future.

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.

Cell cultures of Arabidopsis thaliana (cv. Columbia) were used to screen for early changes in gene expression in response to altered gravitational fields. Genes of interest (mainly components of signalling chains) were selected from a larger group, the expression of which was affected under hypergravity [Martzivanou M. and Hampp R., Physiol. Plant., 118, 221-231,2003]. Transcriptional changes of these genes were studied within a period of up to 10 min of exposure to clinorotation (random positioning machine), magnetophoresis, and hypergravity (8 g). Microarrays identified a set of transcription factor genes which responded in a treatment-specific way. The respective transcripts were quantified by real time RT PCR. As most responses occurred within 10 min of treatment, such genes can be used for the investigation of microgravity-related alterations in gene expression under sounding rocket conditions (TEXUS, MAXUS).

This policy review summarises the existing policy discussion with regard to Place. There are several themes, such as the salience of the Local, the importance of economic factors, and the role of Levelling Up, that emerge throughout and they will be discussed in the course of the review. This Introduction will also discuss definitions and the concept of Place. What is Place? As a concept, and in the sense we are using it here, Place can be defined as a specific position, location, or area in space. As an area, it is by implication bounded, with a border and boundary delineating its extent; the former with regard to what is outside and the latter to what is within, denoting a territory or bounded space of almost any size. These are social constructions evolving through complex socio-political processes. It is those dimensions of being a territorially-bound location that contextualises definitions of what is ‘devolved’ and ‘regional’. The first is a location where a governing entity has greater powers devolved to it from the centre. In the second instance, regions exist in a space between the local and the national, devolved or otherwise, and often have areas within them that are, themselves, subject to urban mayoral and/or other arrangements. Other aspects inform ideas about Place: language, identity, nation, and the ideal of a homeland all graft comparatively neatly onto it as a territorially-bound location and aspects of all of these apply as readily in parts of England as they do in the devolved nations. These are worth considering carefully because they underpin arrangements locally that are likely to be specific. Good examples are the role and importance of the Welsh language in Wales, or pride in a national identity in Wales, Scotland, and in Northern Ireland. Language and identity are also factors in Cornwall and a consideration for policy there. Other local identities lead to pride of place in locales and regions across England. The following sections of this review report will consider themes arising from the academic literature, such as space and identity, before going on to the discussion of the Policy environment. That discussion will cover the following themes: Place in Central Government: the development of Place Policy and the Levelling Up Agenda; Place in Devolved Administrations outside England; Place in Regional Government, Pan-Regional Entities, and Freeports; Place in Combined Authorities; Place in Local Government; Thinking about Place beyond the State: The Private and Third Sectors; and Place in Policy as a problematising factor. The Conclusion will discuss the changing character of the Place.