Copurification of the Lac repressor with polyhistidine-tagged proteins in immobilized metal affinity chromatography.

One of the commonly used resins for immobilized metal affinity purification of polyhistidine-tagged recombinant proteins is TALON resin, a cobalt (II)--carboxymethylaspartate-based matrix linked to Sepharose CL-6B. Here, we show that TALON resin efficiently purifies the native form of Lac repressor, which represents the major contaminant when (His)(6)-tagged proteins are isolated from Escherichia coli host cells carrying the lacI(q) gene. Inspection of the crystal structure of the repressor suggests that three His residues (residues 163, 173, and 202) in each subunit of the tetramer are optimally spaced on an exposed face of the protein to allow interaction with Co(II). In addition to establishing a more efficient procedure for purification of the Lac repressor, these studies indicate that non-lacI(q)-based expression systems yield significantly purer preparations of recombinant polyhistidine-tagged proteins.

Tunable hydrodynamic chromatography of microparticles localized in short microchannels.

This paper describes a new way to perform hydrodynamic chromatography (HDC) for the size separation of particles based on a unique recirculating flow pattern. Pressure-driven (PF) and electro-osmotic flows (EOF) are opposed in narrow glass microchannels that expand at both ends. The resulting bidirectional flow turns into recirculating flow because of nonuniform microchannel dimensions. This hydrodynamic effect, combined with the electrokinetic migration of the particles themselves, results in a trapping phenomenon, which we have termed flow-induced electrokinetic trapping (FIET). In this paper, we exploit recirculating flow and FIET to perform a size-based separation of samples of microparticles trapped in a short separation channel using a HDC approach. Because these particles have the same charge (same zeta potential), they exhibit the same electrophoretic mobility, but they can be separated according to size in the recirculating flow. While trapped, particles have a net drift velocity toward the low-pressure end of the channel. When, because of a change in the externally applied PF or electric field, the sign of the net drift velocity reverses, particles can escape the separation channel in the direction of EOF. Larger particles exhibit a larger net drift velocity opposing EOF, so that the smaller particles escape the separation channel first. In the example presented here, a sample plug containing 2.33 and 2.82 microm polymer particles was introduced from the inlet into a 3-mm-long separation channel and trapped. Through tuning of the electric field with respect to the applied PF, the particles could be separated, with the advantage that larger particles remained trapped. The separation of particles with less than 500 nm differences in diameter was performed with an analytical resolution comparable to that of baseline separation in chromatography. When the sample was not trapped in the separation channel but located further downstream, separations could be carried out continuously rather than in batch. Smaller particles could successfully pass through the separation channel, and particles were separated by size. One of the main advantages of exploiting FIET for HDC is that this method can be applied in quite short (a few millimeters) channel geometries. This is in great contrast to examples published to date for the separation of nanoparticles in much longer micro- and nanochannels.

Analysis of in-cylinder hydrocarbons in a multi-cylinder gasoline HCCI engine using gas chromatography

Gasoline Homogeneous Charge Compression Ignition (HCCI) combustion has been studied widely in the past decade. However, in HCCI engines using negative valve overlap (NVO), there is still uncertainty as to whether the effect of pilot injection during NVO on the start of combustion is primarily due to heat release of the pilot fuel during NVO or whether it is due to pilot fuel reformation. This paper presents data taken on a 4-cylinder gasoline direct injection, spark ignition/HCCI engine with a dual cam system, capable of recompressing residual gas. Engine in-cylinder samples are extracted at various points during the engine cycle through a high-speed sampling system and directly analysed with a gas chromatograph and flame ionisation detector. Engine parameter sweeps are performed for different pilot injection timings and quantities at a medium load point. Results show that for lean engine running conditions, earlier pilot injection timing leads to partial oxidation of the injected pilot fuel during NVO, while the fraction of light hydrocarbons remains constant for all parameter variations investigated. The same applies for a variation in pilot fuel amount. Thus there is evidence that in lean conditions, pilot injection-related NVO effects are dominated by heat release rather than fuel reformation. © 2009 SAE International.

Discrete element calculations of the impact of a sand column against rigid structures

Discrete particle simulations of column of an aggregate of identical particles impacting a rigid, fixed target and a rigid, movable target are presented with the aim to understand the interaction of an aggregate of particles upon a structure. In most cases the column of particles is constrained against lateral expansion. The pressure exerted by the particles upon the fixed target (and the momentum transferred) is independent of the co-efficient of restitution and friction co-efficient between the particles but are strongly dependent upon the relative density of the particles in the column. There is a mild dependence on the contact stiffness between the particles which controls the elastic deformation of the densified aggregate of particles. In contrast, the momentum transfer to a movable target is strongly sensitive to the mass ratio of column to target. The impact event can be viewed as an inelastic collision between the sand column and the target with an effective co-efficient of restitution between 0 and 0.35 depending upon the relative density of the column. We present a foam analogy where impact of the aggregate of particles can be modelled by the impact of an equivalent foam projectile. The calculations on the equivalent projectile are significantly less intensive computationally and yet give predictions to within 5% of the full discrete particle calculations. They also suggest that "model" materials can be used to simulate the loading by an aggregate of particles within a laboratory setting. © 2012 Elsevier Ltd. All rights reserved.

Concrete column recognition in images and videos

The automated detection of structural elements (e.g., columns and beams) from visual data can be used to facilitate many construction and maintenance applications. The research in this area is under initial investigation. The existing methods solely rely on color and texture information, which makes them unable to identify each structural element if these elements connect each other and are made of the same material. The paper presents a novel method of automated concrete column detection from visual data. The method overcomes the limitation by combining columns’ boundary information with their color and texture cues. It starts from recognizing long vertical lines in an image/video frame through edge detection and Hough transform. The bounding rectangle for each pair of lines is then constructed. When the rectangle resembles the shape of a column and the color and texture contained in the pair of lines are matched with one of the concrete samples in knowledge base, a concrete column surface is assumed to be located. This way, one concrete column in images/videos is detected. The method was tested using real images/videos. The results are compared with the manual detection ones to indicate the method’s validity.