Production of recombinant bleaching enzymes from thermophilic microorganisms in fungal hosts

Cost-effective production of enzymes for industrial processes makes the appropriate selection of the host-vector expression system critical. We have developed two systems for the bulk production of bleaching enzymes from thermophiles. Kluyveromyces lactis has been developed as a secretion host employing expression vectors based on the 2μ-like plasmid pKD1 of Kluyveromyces drosophilarium. Our second system involves the filamentous fungus Trichoderma reesei. Fusion and nonfusion vectors have been constructed using the strong cellobiohydrolase 1 (cbh1) promoter. The KEX2 protease cleavage site and a 6 × HIS-tag have been incorporated to facilitate both cleavage and purification of the mature foreign proteins.     

Amide Moieties Modulate the Antimicrobial Activities of Conjugated Oligoelectrolytes against Gram-negative Bacteria

Cationic conjugated oligoelectrolytes (COEs) are a class of compounds that can be tailored to achieve relevant in vitro antimicrobial properties with relatively low cytotoxicity against mammalian cells. Three distyrylbenzene-based COEs were designed containing amide functional groups on the side chains. Their properties were compared to two representative COEs with only quaternary ammonium groups. The optimal compound, COE2−3C−C3-Apropyl , has an antimicrobial efficacy against Escherichia coli with an MIC=2 μg mL⁻¹, even in the presence of human serum albumin low cytotoxicity (IC₅₀=740 μg mL⁻¹) and minimal hemolytic activity. Moreover, we find that amide groups increase interactions between COEs and a bacterial lipid mimic based on calcein leakage assay and allow COEs to readily permeabilize the cytoplasmic membrane of E. coli. These findings suggest that hydrogen bond forming moieties can be further applied in the molecular design of antimicrobial COEs to further improve their selectivity towards bacteria.     

Longitudinal-transverse separations of deep-inelastic structure functions at low Q2 for hydrogen and deuterium

We report on a study of the longitudinal to transverse cross section ratio, R=sigmaL/sigmaT, at low values of x and Q2, as determined from inclusive inelastic electron-hydrogen and electron-deuterium scattering data from Jefferson Laboratory Hall C spanning the four-momentum transfer range 0.06相似文献   

Efficiency enhancement of high power vacuum BWO's using nonuniformslow wave structures

The Sinus-6, a high-power relativistic repetitively-pulsed electron beam accelerator, is used to drive various slow wave structures in a BWO configuration in vacuum. Peak output power of about 550 MW at 9.45 GHz was radiated in an 8-ns pulse. We describe experiments which study the relative efficiencies of microwave generation from a two-stage nonuniform amplitude slow wave structure and its variations without an initial stage. Experimental results are compared with 2.5 D particle-in-cell computer simulations. Our results suggest that prebunching the electron beam in the initial section of the nonuniform BWO results in increased microwave generation efficiency, Furthermore, simulations reveal that, in addition to the backward propagating surface harmonic of the TM₀₁ mode, backward and forward propagating volume harmonics with phase velocity twice that of the surface harmonic play an important role in high-power microwave generation and radiation     

Optimization of spin-valve parameters for magnetic bead trapping and manipulation

Magneto-optic Kerr effect (MOKE) and magnetoresistance (MR) measurements were used to measure the switching characteristics of spin-valve (SV) arrays currently being developed to trap and release superparamagnetic beads within a fluid medium. The effect of SV size on switching observed by MOKE showed that a 1 μm×8 μm SV element was found to have optimal switching characteristics. MR measurements on a single 1 μm×8 μm SV switched with either an external applied magnetic field or a local magnetic field generated by an integrated write wire (current density ranging from 10⁶ to 10⁷ A/cm²) confirmed the MOKE findings. The 1 μm×8 μm SV low field switching was observed to be +8 and −2 mT with two stable states at zero field; the high field switching was observed to be −18 mT. The low switching fields and the large magnetic moment of the SV trap along with our observation of minimal magnetostatic effects for dense arrays are necessary design characteristics for high-force, “switchable-magnet,” microfluidic bead trap applications.     

RNA unrestrained molecular dynamics ensemble improves agreement with experimental NMR data compared to single static structure: a test case

Nuclear magnetic resonance (NMR) provides structural and dynamic information reflecting an average, often non-linear, of multiple solution-state conformations. Therefore, a single optimized structure derived from NMR refinement may be misleading if the NMR data actually result from averaging of distinct conformers. It is hypothesized that a conformational ensemble generated by a valid molecular dynamics (MD) simulation should be able to improve agreement with the NMR data set compared with the single optimized starting structure. Using a model system consisting of two sequence-related self-complementary ribonucleotide octamers for which NMR data was available, 0.3 ns particle mesh Ewald MD simulations were performed in the AMBER force field in the presence of explicit water and counterions. Agreement of the averaged properties of the molecular dynamics ensembles with NMR data such as homonuclear proton nuclear Overhauser effect (NOE)-based distance constraints, homonuclear proton and heteronuclear ¹H–³¹P coupling constant (J) data, and qualitative NMR information on hydrogen bond occupancy, was systematically assessed. Despite the short length of the simulation, the ensemble generated from it agreed with the NMR experimental constraints more completely than the single optimized NMR structure. This suggests that short unrestrained MD simulations may be of utility in interpreting NMR results. As expected, a 0.5 ns simulation utilizing a distance dependent dielectric did not improve agreement with the NMR data, consistent with its inferior exploration of conformational space as assessed by 2-D RMSD plots. Thus, ability to rapidly improve agreement with NMR constraints may be a sensitive diagnostic of the MD methods themselves.     

Molecular design of antimicrobial conjugated oligoelectrolytes with enhanced selectivity toward bacterial cells

A series of cationic conjugated oligoelectrolytes (COEs) was designed to understand how variations in molecular dimensions impact the relative activity against bacteria and mammalian cells. These COEs kept a consistent distyrylbenzene framework but differed in the length of linker between the core and the cationic site and the length of substitute on the quaternary ammonium functioned group. Their antimicrobial efficacy, mammalian cell cytotoxicity, hemolytic activity, and cell association were determined. We find that hydrophobicity is a factor that controls the degree of COE association to cells, but in vitro efficacy and cytotoxicity depend on more subtle structural features. COE2-3C-C4butyl was found to be the optimal structure with a minimum inhibitory concentration (MIC) of 4 μg mL⁻¹ against E. coli K12, low cytotoxicity against HepG2 cells and negligible hemolysis of red blood cells, even at 1024 μg mL⁻¹. A time-kill kinetics study of COE2-3C-C4butyl against E. coli K12 demonstrates bactericidal activity. These findings provide the first systematic investigation of how COEs may be modulated to achieve low mammalian cell cytotoxicity with the long-range perspective of finding candidates suitable for developing a broad-spectrum antimicrobial agent.

A series of cationic conjugated oligoelectrolytes (COEs) was designed to understand how variations in molecular dimensions impact the relative activity against bacteria and mammalian cells.