Efficient directional cloning of recombinant adenovirus vectors using DNA-protein complex

We describe an efficient cloning system utilizing adenoviral DNA-protein complexes which allows the directional cloning of genes into adenoviral expression vectors in a single step. DNA-protein complexes derived from a recombinant adenovirus (AVC2.null) were isolated by sequential use of CsCl step gradients followed by isopycnic centrifugation in a mixture of CsCl and guanidine HCl. AVC2.null is an adenoviral expression vector containing unique restriction sites between the human CMV-IE promoter and the SV40 intron/polyadenylation site. Transgenes were prepared for cloning into this vector by introduction of compatible restriction sites by PCR. A vector expressing rat granulocyte-macrophage colony-stimulating factor (GM-CSF) was constructed using DNA-protein complex as well as by traditional recombination techniques. The efficacy of our adenoviral cloning system utilizing DNA-protein complex was two logs higher than that seen using homologous recombination. All viruses generated by directional ligation of the insert into the vector DNA-protein complexes contained the desired transgene in the correct orientation. This technique greatly simplifies and accelerates the generation of recombinant adenoviral vectors.     

Effect of Product Conductivity on Field-Activated Combustion Synthesis

Experimental and modeling results on the field-activated combustion synthesis (FACS) of Nb₅Si₃ are reported. In the absence of an electric field and without reactant preheating, Nb₅Si₃ cannot be prepared by self-propagating high-temperature synthesis (SHS). Under the influence of a field a self-sustaining combustion wave is established whose rate of propagation decreases with traveled distance. For relatively low field values, the wave propagation mode changes from a continuous (smooth) to a spin mode. The product of synthesis depends on the mode of propagation. Synthesis during continuous wave propagation results in the formation of Nb₅Si₃, primarily in the α-modification. In contrast, when the wave propagates in a spin mode, the product is NbSi₂ with unreacted niobium. The present observations demonstrate a case where the field effect is not localized, as was the case in previous studies. The difference in behavior is attributed to differences in the electrical conductivities of the product phases.     

Video background modeling: recent approaches,issues and our proposed techniques

Effective and efficient background subtraction is important to a number of computer vision tasks. We introduce several new techniques to address key challenges for background modeling using a Gaussian mixture model (GMM) for moving objects detection in a video acquired by a static camera. The novel features of our proposed model are that it automatically learns dynamics of a scene and adapts its parameters accordingly, suppresses ghosts in the foreground mask using a SURF features matching algorithm, and introduces a new spatio-temporal filter to further refine the foreground detection results. Detection of abrupt illumination changes in the scene is dealt with by a model shifting-based scheme to reuse already learned models and spatio-temporal history of foreground blobs is used to detect and handle paused objects. The proposed model is rigorously tested and compared with several previous models and has shown significant performance improvements.     

Consolidation and properties of binderless sub-micron tungsten carbide by field-activated sintering

The rapid sintering of nano-structured WC hard materials in a short time is introduced with a focus on the manufacturing potential of this spark plasma sintering process. The advantage of this process allows very quick densification to near theoretical density and prohibition of grain growth in nano-structured materials. A dense pure WC hard material with a relative density of up to 97.6% was produced with simultaneous application of 60 MPa pressure and electric current of 2800 A within 2 min. A larger current caused a higher rate of temperature increase and therefore a higher densification rate of the WC powder. The finer the initial WC powder size the higher is the density and the better are the mechanical properties. The fracture toughness and hardness values obtained were 6.6 MPa m^1/2 and 2480 kg/mm², respectively under 60 MPa pressure and 2800 A using 0.4 μm WC powder.     

Characterisation of flucloxacillin and 5-hydroxymethyl flucloxacillin haptenated HSA in vitro and in vivo

Flucloxacillin is a synthetic penicillin used in the treatment of Staphylococcal infections. Adverse reactions to the drug are believed to arise through covalent modification of proteins, with tissue damage occurring secondary to an immune reaction. Serum proteins have been shown by adduct-specific antibodies to be modified by flucloxacillin, but the nature and sites of modification have not been characterised. Here, in vitro studies on HSA have shown by MS that the modification of protein lysine residues occurs in a dose-, time- and site-dependent manner. Affinity, cation exchange and reversed phase chromatography prior to MS revealed in vivo modification of HSA with flucloxacillin in tolerant patients, with up to nine modified lysine residues being detected in each patient, and with modification of Lys190 and Lys212 being detected in 8/8 patients. It was also revealed for the first time that plasma proteins could be modified with the 5-hydroxymethyl metabolite of flucloxacillin, and that essentially the same Lys residues were targeted by both the parent drug and its metabolite. This study provides a detailed characterisation of sites of chemical modification of an endogenous target and reveals candidate peptides for T-cell and antibody assays of flucloxacillin hypersensitivity.     

Role of disorder-order transformation in consolidation of ceramics

The ordering of stacking-disordered silicon carbide prepared from the elements by high energy ball milling was investigated during sintering. A sharp increase in density in the temperature region 1700–1800°C was associated with a decrease in the disorder. Samples which had low disorder density showed a more continuous sintering behavior with temperature. Highly dense (up to 99% relative density) SiC can be obtained at 1900°C under a pressure of 70 MPa with no hold time. Similar results were observed for structurally disordered carbon with 10 at% of boron. The sintering behavior exhibited an abrupt density increase in the narrow temperature region of 1450–1600°C and was associated with disorder-order transformation.     

Effect of fortification on physico-chemical and microbiological stability of whole wheat flour

Stability of fortified whole wheat flour (WWF) was evaluated using NaFeEDTA, elemental iron, ZnSO₄ and ZnO as fortificants. Fortified WWF was stored in tin boxes and polypropylene bags for 60 days under ambient storage condition (ASC) and controlled storage condition (CSC). Fortification significantly (p ? 0.05) decreased moisture and protein content and increased ash content to 5.44%, 6% and 23%, as compared to control. Fortified WWF, assayed periodically for mould contamination manifested a significant inhibition (∼1 log reduction) in flours containing elemental iron. Low storage temperature and relative humidity (RH) indicated lower level of mould count during extended storage time. Tin boxes, as storage material, exhibited a better protection against mould attack, acting as an effective barrier for moisture. Fortificants exerted a slight deteriorative effect on texture characteristics of the chapattis made of these flours but chapattis were still accepted by the judges. Zinc fortificants seemed like having little or no effect on the quality of the flours and chapattis, made of such flours. Shelf life of fortified flour may be extended by using elemental iron as fortificant and storing the product in tin boxes under relatively low temperature and RH.