Design and probing of efflux functions of EGFP fused ABC membrane transporters in live cells using fluorescence spectroscopy

We have designed and constructed fusion genes of C-terminal (Ct) or N-terminal (Nt) bmrA with EGFP vectors and successfully expressed them in ΔBmrA (BmrA deletion strain of Bacillus subtilis), generating two new strains of B. subtilis (Ct-BmrA-EGFP and Nt-BmrA-EGFP). The fusion genes were characterized using gel electrophoresis and DNA sequencing. Their expression in live cells was determined by measuring the fluorescence of EGFP in single live cells using fluorescence microscopy and spectroscopy. The efflux function of the new strains was studied by measuring their accumulation kinetics of intracellular Hoechst dye molecules (a pump substrate) using fluorescence spectroscopy, which were compared with wild-type (WT-BmrA) and ΔBmrA strains. Both new strains show lower accumulation rates than ΔBmrA, and their efflux kinetics are inhibited by a pump inhibitor (orthovanadate). The results suggest that both strains extrude the dye molecules and the fusion proteins retain the efflux function of BmrA (ATP-binding cassette, ABC, transporter). Notably, Nt-BmrA-EGFP strain shows lower accumulation rates (higher efflux rates) than Ct-BmrA-EGFP. Modeled structures of the fusion proteins illustrate a highly flexible linker region connecting EGFP with BmrA, suggesting a minimal obstruction of EGFP to the BmrA. A closer distance of two C termini (∼14 ?) than two N termini (47.9 ?) of the “closed” BmrA dimer depicts the larger steric effect of C-terminal fusion. This study also shows that glucose affects the fluorescence study of efflux function of BmrA, suggesting that efflux kinetics of ABC membrane transporters in live cells must be characterized in the absence of glucose.     

Stability of DNA containing a structural water mimic in an A-T rich sequence

We describe here the synthesis and properties of A-T rich DNA containing covalently bound water mimics located in the DNA minor groove.     

Dissecting alkynes: full cleavage of polarized C≡C moiety via sequential bis-Michael addition/retro-Mannich cascade

The reaction of diaryl ketoalkynes with 1,2-diamino ethane leads to the full scission of the triple bond with the formation of acetophenone and imidazoline fragments. In this transformation, one of the alkyne carbons undergoes formal reduction with the formation of three C-H bonds, whereas the other carbon undergoes formal oxidation via the formation of three C-N bonds (one π and two σ). Computational analysis confirmed that the key fragmentation step proceeds via a six-membered TS in a concerted manner. Both amines are involved in the fragmentation: the N-H moiety of one amine transfers a proton to the developing negative charge at the enolate oxygen, while the other amine provides direct stereoelectronic assistance to the C-C bond cleavage via a hyperconjugative n(N) → σ*(C-C) interaction.     

Increasing risk aversion and life-cycle investing

Mathematics and Financial Economics - We derive the optimal portfolio for an investor with increasing relative risk aversion in a complete continuous-time securities market. The IRRA assumption...     

Hydrodynamics of a particle impact on a wall

The problem of a particle impacting on a wall, a common phenomenon in particle-laden flows in the minerals and process industries, is investigated computationally using a spectral-element method with the grid adjusting to the movement of the particle towards the wall. Remeshing is required at regular intervals to avoid problems associated with mesh distortion and the constantly reducing maximum time-step associated with integration of the non-linear convective terms of the Navier–Stokes equations. Accurate interpolation between meshes is achieved using the same high-order interpolation employed by the spectral-element flow solver. This approach allows the full flow evolution to be followed from the initial approach, through impact and afterwards as the flow relaxes. The method is applied to the generic two-dimensional and three-dimensional bluff body geometries, the circular cylinder and the sphere. The principal case reported here is that of a particle colliding normally with a wall and sticking. For the circular cylinder, non-normal collisions are also considered. The impacts are studied for moderate Reynolds numbers up to approximately 1200. A cylindrical body impacting on a wall produces two vortices from its wake that convect away from the cylinder along the wall before stalling while lifting induced wall vorticity into the main flow. The situation for a sphere impact is similar, except in this case a vortex ring is formed from the wake vorticity. Again, secondary vorticity from the wall and particle plays a role. At higher Reynolds number, the secondary vorticity tends to form a semi-annular structure encircling the primary vortex core. At even higher Reynolds numbers, the secondary annular structure fragments into semi-discrete structures, which again encircle and orbit the primary core. Vorticity fields and passive tracer particles are used to characterize the interaction of the vortical structures. The evolution of the pressure and viscous drag coefficients during a collision are provided for a typical sphere impact. For a Reynolds number greater than approximately 1000 for a sphere and 400 for a cylinder, the primary vortex core produced by the impacting body undergoes a short-wavelength instability in the azimuthal/spanwise direction. Experimental visualisation using dye carried out in water is presented to validate the predictions.     

Helium cosmic ray flux measurements at Mars

The helium energy spectrum in Martian orbit has been observed by the MARIE charged particle spectrometer aboard the 2001 Mars Odyssey spacecraft. The orbital data were taken from March 13, 2002 to October 28, 2003, at which time a very intense Solar Particle Event caused a loss of communication between the instrument and the spacecraft. The silicon detector stack in MARIE is optimized for the detection of protons and helium in the energy range below , which typically includes almost all of the flux during SPEs. This also makes MARIE an efficient detector for GCR helium in the energy range of 50–. We will present the first fully normalized flux results from MARIE, using helium ions in this energy range.     

Detection of mycotic pseudoaneurysm of the ascending aorta using MRI

Mycotic pseudoaneurysms of the ascending aorta are rare cardiovascular lesions that carry the risk of potentially disastrous complications such as aortic rupture or widespread sepsis. Regardless of its location in the arterial system, this lesion carries a high mortality rate. Early diagnosis of mycotic pseudoaneurysm is paramount for optimizing the chances of surgical therapy. While imaging strategies of this lesion have included aortography, echocardiography, and computed tomography, the information gained from each of these individual studies may be acquired in a single MR imaging session. MRI offers the fundamental advantages of noninvasiveness, nonionization, and multiplanar imaging capabilities. Additionally, MRI provides excellent soft tissue contrast, a wide field of view, qualitative and quantitative flow analysis, and an independence of operator expertise or patient body habitus.     

Immobilization of a sulfide-oxidizing bacterium in a novel adsorbent biocatalyst support

Applied Biochemistry and Biotechnology - Preliminary experiments have shown thatT. denitrificans strain F can be immobilized in DuPont BIO-SEP beads and used to treat refinery spent sulfidic...     

Session 5 Environmental biotechnology

Applied Biochemistry and Biotechnology -