Mechanics of a mosquito bite with applications to microneedle design

The mechanics of a fascicle insertion into the skin by a mosquito of the type aedes aegypti has been studied experimentally using high-speed video (HSV) imaging, and analytically using a mathematical model. The fascicle is a polymeric microneedle composed of a ductile material, chitin. It has been proposed that the mosquito applies a non-conservative follower force component in addition to the Euler compressive load in order to prevent buckling and penetrate the skin. In addition, the protective sheath surrounding the fascicle (labium) provides lateral support during insertion. The mechanics model presented approximates the fascicle as a slender column supported on an elastic foundation (labium) subjected to non-conservative (Beck) and conservative Euler loads simultaneously at the end. Results show that the lateral support of the fascicle provided by the labium is essential for successful penetration by increasing the critical buckling load by a factor of 5. The non-conservative follower force application increases the buckling load by an additional 20% and may or may not be necessary for successful penetration. Experimental results showing the importance of the labium have been cited to validate the model predictions, in addition to the video observations presented in this work. This understanding may be useful in designing painless needle insertion systems as opposed to miniaturized hypodermic needles.     

Determination of kinetic constants of hybrid textile wastewater treatment system

The present study is related to treatment of textile wastewater in microaerophilic–aerobic hybrid reactor. The study showed the effectiveness of biological treatment of wastewater involving appropriate microorganism and suitable reactors. COD and color were reduced to 82–94%, and 99% respectively for textile wastewater. The reactor was operated at highest loading of 16.4 g COD g l⁻¹ d⁻¹ and obtained 80% COD and 72% color removal. Biokinetic models were applied to data obtained from experimental studies in continuously operated hybrid reactor. Treatment efficiencies of the reactor were investigated at different hydraulic retention times (2.3–9.1 d) and organic loading rates (2.6–16.4 g COD l⁻¹ d⁻¹). Second-order and a Stover–Kincannon models were best fitted to the hybrid column reactor. The second-order substrate removal rate constant (k_2(S)) was found as 41.44 d⁻¹ for hybrid reactor. Applying the modified Stover–Kincannon model to the hybrid reactor, the maximum removal rate constant (U_max) and saturation value constant (K_B) were found to be 212 g l⁻¹ d⁻¹ and 22.89 g l⁻¹ d⁻¹, respectively.     

Comparative studies on removal of Congo red by native and modified mycelial pellets of Trametes versicolor in various reactor modes

Aerated and rotated mode adsorption experiments were carried out for the removal of Congo red from aqueous solution using native and pre-treated mycelial pellets/biomass of Trametes versicolor. The effect of process parameters like contact time, dosage of adsorbent, adsorbate concentration and pH on adsorption was investigated. Higher the dye concentration lower was the adsorption. Equilibrium time was attained at 90 min. Increase in biomass dosage increased the adsorption. Experimental data were analyzed by the Langmuir and Temkin isotherms. Adsorption capacity (Q(0)) of autoclaved biomass was 51.81 mg/g, which was higher than other biomass studied. The second order kinetic model by Ho and Mckay described well the experimental data. Acidic pH was favorable for the adsorption of Congo red. Studies on pH effect and desorption show that chemisorption seems to play a major role in the adsorption process. Among the native and pre-treated biomass studied, autoclaved biomass showed a better adsorption capacity. Utilization of autoclaved biomass is much safer as it does not pose any threat to environment. Aerated mode showed a better adsorption capacity when compared to rotated mode.     

A custom-designed recombinant multiepitope protein as a dengue diagnostic reagent

Currently, dengue fever is the most important re-emerging mosquito-borne viral disease, with the major proportion of the target population residing in the developing countries of the world. In endemic areas, potentially fatal secondary dengue infections, characterized by high anti-dengue IgG antibody titers, are most common. Most currently available commercial dengue diagnostic kits rely on the use of whole virus antigens and are consequently associated with false positives due to serologic cross-reactivity, high cost of antigen production, and biohazard risk. This has prompted the need to develop an alternate antigen to replace the whole virus antigen in diagnostic tests. We have designed and expressed a novel recombinant protein antigen by assembling key immunodominant linear IgG-specific dengue virus epitopes, chosen on the basis of pepscan analysis, phage display, and computer predictions. The recombinant dengue multiepitope protein was expressed to high levels in Escherichia coli, purified in a single step, yielding >25 mg pure protein per liter culture. We developed an in-house enzyme-linked immunosorbent assay (ELISA) to detect anti-dengue antibodies in a panel of 20 patient sera using the purified recombinant dengue multiepitope protein as the capture antigen. The ELISA results were in excellent agreement with those obtained using a commercially available diagnostic test, Dengue Duo rapid strip test from PanBio, Australia. The high epitope density, careful choice of epitopes, and the use of E. coli system for expression, coupled to simple purification, jointly have the potential to lead to the development of an inexpensive diagnostic test with a high degree of sensitivity and specificity.     

Magnitude of the hydrophobic effect at central versus peripheral sites in protein-protein interfaces

Hydrophobic interactions are essential for stabilizing protein-protein complexes, whose interfaces generally consist of a central cluster of hot spot residues surrounded by less important peripheral residues. According to the O-ring hypothesis, a condition for high affinity binding is solvent exclusion from interacting residues. This hypothesis predicts that the hydrophobicity at the center is significantly greater than at the periphery, which we estimated at 21 cal mol(-1) A(-2). To measure the hydrophobicity at the center, structures of an antigen-antibody complex where a buried phenylalanine was replaced by smaller hydrophobic residues were determined. By correlating structural changes with binding free energies, we estimate the hydrophobicity at this central site to be 46 cal mol(-1) A(-2), twice that at the periphery. This context dependence of the hydrophobic effect explains the clustering of hot spots at interface centers and has implications for hot spot prediction and the design of small molecule inhibitors.     

Structural basis of ordered binding of donor and acceptor substrates to the retaining glycosyltransferase,alpha-1,3-galactosyltransferase

Bovine alpha-1,3-galactosyltransferase (alpha3GT) catalyzes the synthesis of the alpha-galactose (alpha-Gal) epitope, the target of natural human antibodies. It represents a family of enzymes, including the histo blood group A and B transferases, that catalyze retaining glycosyltransfer reactions of unknown mechanism. An initial study of alpha3GT in a crystal form with limited resolution and considerable disorder suggested the possible formation of a beta-galactosyl-enzyme covalent intermediate (Gastinel, L. N., Bignon, C., Misra, A. K., Hindsgaul, O., Shaper, J. H., and Joziasse, D. H. (2001) EMBO J. 20, 638-649). Highly ordered structures are described for complexes of alpha3GT with donor substrate, UDP-galactose, UDP- glucose, and two acceptor substrates, lactose and N-acetyllactosamine, at resolutions up to 1.46 A. Structural and calorimetric binding studies suggest an obligatory ordered binding of donor and acceptor substrates, linked to a donor substrate-induced conformational change, and the direct participation of UDP in acceptor binding. The monosaccharide-UDP bond is cleaved in the structures containing UDP-galactose and UDP-glucose, producing non-covalent complexes containing buried beta-galactose and alpha-glucose. The location of these monosaccharides and molecular modeling suggest that binding of a distorted conformation of UDP-galactose may be important in the catalytic mechanism of alpha3GT.     

Recombinant dengue virus type 2 envelope/hepatitis B surface antigen hybrid protein expressed in Pichia pastoris can function as a bivalent immunogen

A truncated version of the dengue virus type 2 envelope protein (Den2E) encoding the first 395 amino acid (aa) residues, and Den2E fused in-frame with the full-length 226-aa hepatitis B surface antigen (Den2E-HBsAg) protein were expressed in the methylotrophic yeast, Pichia pastoris. Both the recombinant proteins showed evidence of the capacity to form high molecular weight aggregates. Electron microscopic analysis of the purified proteins showed that while Den2E displayed an amorphous morphology, Den2E-HBsAg existed as well-structured virus-like particles (VLPs). Using immuno-gold electron microscopy, these VLPs were demonstrated to contain both components of the Den2E-HBsAg hybrid protein. Seroanalysis showed that the hybrid VLPs could function in vivo as bivalent immunogens, which could elicit immune responses directed against both components of the hybrid protein, as evidenced by ELISA, immunoprecipitation and immunofluorescence data.