Agrobacterium-mediated transformation of the temperate grass Brachypodium distachyon (genotype Bd21) for T-DNA insertional mutagenesis

Brachypodium distachyon is a promising model system for the structural and functional genomics of temperate grasses because of its physical, genetic and genome attributes. The sequencing of the inbred line Bd21 ( http://www.brachypodium.org ) started in 2007. However, a transformation method remains to be developed for the community standard line Bd21. In this article, a facile, efficient and rapid transformation system for Bd21 is described using Agrobacterium -mediated transformation of compact embryogenic calli (CEC) derived from immature embryos. Key features of this system include: (i) the use of the green fluorescent protein (GFP) associated with hygromycin selection for rapid identification of transgenic calli and plants; (ii) the desiccation of CEC after inoculation with Agrobacterium ; (iii) the utilization of Bd21 plants regenerated from tissue culture as a source of immature embryos; (iv) the control of the duration of the selection process; and (v) the supplementation of culture media with CuSO₄ prior to and during the regeneration of transgenic plants. Approximately 17% of CEC produced transgenic plants, enabling the generation of hundreds of T-DNA insertion lines per experiment. GFP expression was observed in primary transformed Bd21 plants (T₀) and their progeny (T₁). The Mendelian inheritance of the transgenes was confirmed. An adaptor-anchor strategy was developed for efficient retrieval of flanking sequence tags (FSTs) of T-DNA inserts, and the resulting sequences are available in public databases. The production of T-DNA insertion lines and the retrieval of associated FSTs reported here for the reference inbred line Bd21 will facilitate large-scale functional genomics research in this model system.     

Intravital microscopy of the murine pulmonary microcirculation.

Intravital microscopy (IVM) is considered as the gold standard for in vivo investigations of dynamic microvascular regulation. The availability of transgenic and knockout animals has propelled the development of murine IVM models for various organs, but technical approaches to the pulmonary microcirculation are still scarce. In anesthetized and ventilated BALB/c mice, we established a microscopic access to the surface of the right upper lung lobe by surgical excision of a window of 7- to 10-mm diameter from the right thoracic wall. The window was covered by a transparent polyvinylidene membrane and sealed with alpha-cyanoacrylate. Removal of intrathoracic air via a trans-diaphragmal intrapleural catheter coupled the lung surface to the window membrane. IVM preparations were hemodynamically stable for at least 120 min, with mean arterial blood pressure above 70 mmHg, and mean arterial Po(2) and arterial Pco(2) in the range of 90-100 Torr and 30-40 Torr, respectively. Imaged lungs did not show any signs of acute lung injury or edema. Following infusion of FITC dextran, subpleural pulmonary arterioles and venules of up to 50-microm diameter and alveolar capillary networks could be visualized during successive expiratory plateau phases over a period of at least 2 h. Vasoconstrictive responses to hypoxia (11% O(2)) or infusion of the thromboxane analog U-46619 were prominent in medium-sized arterioles (30- to 50-microm diameter), minor in small arterioles <30 microm, and absent in venules. The presented IVM model may constitute a powerful new tool for investigations of pulmonary microvascular responses in mice.     

The effects of feeding by the rice water weevil, Lissorhoptrus oryzophilus Kuschel, on the growth and yield components of rice, Oryza sativa

Abstract 1 The effects of feeding by larvae of the rice water weevil, Lissorhoptrus oryzophilus Kuschel, on the growth and yield components of rice, Oryza sativa, were evaluated using four varieties of rice, ‘Cocodrie’, ‘Cypress’, ‘Bengal’ and ‘XP1003’, over 2 years. 2 Both adults and larvae of L. oryzophilus feed on rice plants, but it is feeding by the larval stage that causes economic losses. Feeding by L. oryzophilus larvae resulted in extensive damage to root systems. Pruning of root systems resulted in a decrease in tiller number and shoot biomass of rice plants in the vegetative stage of growth. 3 Yield losses were due to a combination of decreases in panicle densities, numbers of grains per panicle, and grain weights. Decreases in panicle densities were a direct result of the reductions in tiller numbers. Reductions in numbers of grains per panicle and grain weights probably resulted from decreases in shoot biomass. 4 Injury by rice water weevil larvae is chronic. The tillering stage of rice suffered the majority of weevil damage, but the growth effects were not manifested until later.     

Involvement of SSAO-mediated deamination in adipose glucose transport and weight gain in obese diabetic KKAy mice

Semicarbazide-sensitive amine oxidase (SSAO) is located on outer surfaces of adipocytes and endothelial and vascular smooth muscle cells. This enzyme catalyzes deamination of methylamine and aminoacetone, leading to production of toxic formaldehyde and methylglyoxal, respectively, as well as hydrogen peroxide and ammonium. Several lines of evidence suggest that increased SSAO activity is related to chronic inflammation and vascular disorders related to diabetic complications. We found that a highly potent and selective SSAO inhibitor, (E)-2-(4-fluorophenethyl)-3-fluoroallylamine (FPFA), was capable of reducing numbers of atherosclerotic lesions as well as weight gain in obese KKAy mice fed an atherogenic diet. SSAO inhibitors cause a moderate and long-lasting hyperglycemia. Such an increase in serum glucose is a result of reduction of glucose uptake by adipocytes. SSAO-mediated deamination of endogenous methylamine substrates induces adipocyte glucose uptake and lipogenesis. Highly selective SSAO inhibitors can effectively block induced glucose uptake. The results suggest that increased SSAO-mediated deamination may be concomitantly related to obesity and vascular disorders associated with type 2 diabetes.     

Load-shift--numerical evaluation of a new design philosophy for uncemented hip prostheses

All hip replacement prostheses alter the load transfer from the hip joint into the femur by changing the mechanical loading of the proximal femur from an externally to an internally loaded system. This alteration of the load transfer causes stress shielding and might lead to severe bone loss, especially with uncemented prostheses. To minimize these effects, load transfer to the femur should occur as proximal as possible. In order to support sufficient primary stability, however, directly post operative (PO) distal stabilization is reasonable. Consequently, the prostheses have to alter its mechanical characteristics after implantation. This concept is referred to as load-shift concept. Primary stability during the early PO state is provided by a prosthesis shaft, which is widened at the tip by a biodegradable pin. After resorption of the pin load transfer occurs no longer distally. The objective of this study was the numerical evaluation of the load-shift concept. The analysis was performed with a finite element model. Three-dimensional non-linear dynamic gait analyses data were used to evaluate whether the load transfer during walking can be altered effectively by insertion and resorption of a distal pin. Directly PO 38% of the transverse load is transferred through the prosthesis shaft and micromotion of the proximal prostheses tip is below 55 microm. After resorption of the pin, no transverse loads are transferred through the prosthesis shaft. Therefore, the loading of the proximal bone tissue is far more pronounced than in the case of a standard prosthesis, demonstrating the feasibility of the load-shift concept. A balanced degradation of the pin simultaneously with the ingrowth of the prosthesis is expected to reduce hip replacement complications.     

Observations on interactions between naturally-collected bacteria and several species of algae

Interactions between a naturally-collected algal species and strains of bacteria with which it was closely associated were examined under controlled conditions. Three strains of bacteria, Pseudomonas, Xanthomonas and Flavobacterium, were isolated from Oscillatoria. These bacteria were grown in combination with axenic cultures of the Oscillatoria culture as well as with several additional algal species. Oscillatoria growth was stimulated by all of the bacteria, but other algal species varied in their response. Some were stimulated, but others were inhibited or unaffected by exposure to the bacterial strains. There were also observations indicating that some algae may be able to develop resistance to antagonistic bacteria. These data suggest that succession and dominance of individual algal species may be influenced by interactions with bacteria.     

Production and evaluation of dry alginate-chitosan microcapsules as an enteric delivery vehicle for probiotic bacteria

This study investigates the production of alginate microcapsules, which have been coated with the polysaccharide chitosan, and evaluates some of their properties with the intention of improving the gastrointestinal viability of a probiotic ( Bifidobacterium breve ) by encapsulation in this system. The microcapsules were dried by a variety of methods, and the most suitable was chosen. The work described in this Article is the first report detailing the effects of drying on the properties of these microcapsules and the viability of the bacteria within relative to wet microcapsules. The pH range over which chitosan and alginate form polyelectrolyte complexes was explored by spectrophotometry, and this extended into swelling studies on the microcapsules over a range of pHs associated with the gastrointestinal tract. It was shown that chitosan stabilizes the alginate microcapsules at pHs above 3, extending the stability of the capsules under these conditions. The effect of chitosan exposure time on the coating thickness was investigated for the first time by confocal laser scanning microscopy, and its penetration into the alginate matrix was shown to be particularly slow. Coating with chitosan was found to increase the survival of B. breve in simulated gastric fluid as well as prolong its release upon exposure to intestinal pH.     

Recent amplification of the kangaroo endogenous retrovirus, KERV, limited to the centromere

Mammalian retrotransposons, transposable elements that are processed through an RNA intermediate, are categorized as short interspersed elements (SINEs), long interspersed elements (LINEs), and long terminal repeat (LTR) retroelements, which include endogenous retroviruses. The ability of transposable elements to autonomously amplify led to their initial characterization as selfish or junk DNA; however, it is now known that they may acquire specific cellular functions in a genome and are implicated in host defense mechanisms as well as in genome evolution. Interactions between classes of transposable elements may exert a markedly different and potentially more significant effect on a genome than interactions between members of a single class of transposable elements. We examined the genomic structure and evolution of the kangaroo endogenous retrovirus (KERV) in the marsupial genus Macropus. The complete proviral structure of the kangaroo endogenous retrovirus, phylogenetic relationship among relative retroviruses, and expression of this virus in both Macropus rufogriseus and M. eugenii are presented for the first time. In addition, we show the relative copy number and distribution of the kangaroo endogenous retrovirus in the Macropus genus. Our data indicate that amplification of the kangaroo endogenous retrovirus occurred in a lineage-specific fashion, is restricted to the centromeres, and is not correlated with LINE depletion. Finally, analysis of KERV long terminal repeat sequences using massively parallel sequencing indicates that the recent amplification in M. rufogriseus is likely due to duplications and concerted evolution rather than a high number of independent insertion events.