Simultaneous surface acoustic wave and surface plasmon resonance measurements: electrodeposition and biological interactions monitoring

We present results from an instrument combining surface acoustic wave (SAW) propagation and surface plasmon resonance (SPR) measurements. The objective is to use two independent methods, the former based on adsorbed mass change measurements and the latter on surface dielectric properties variations, to identify physical properties of protein layers, and more specifically their water content. We display mass sensitivity calibration curves using electrodeposition of copper leading to a sensitivity in liquid of 150$\pm15$ $cm^2/g$ for the Love mode device used here, and the application to monitoring biological processes. The extraction of protein layer thickness and protein to water content ratio is also presented for S-layer proteins under investigation. We obtain respectively 4.7$\pm$0.7 nm and 75$\pm$15%.Comment: 13 pages, 4 figure

Design rules for the self-assembly of a protein crystal

Theories of protein crystallization based on spheres that form close-packed crystals predict optimal assembly within a `slot' of second virial coefficients and enhanced assembly near the metastable liquid-vapor critical point. However, most protein crystals are open structures stabilized by anisotropic interactions. Here, we use theory and simulation to show that assembly of one such structure is not predicted by the second virial coefficient or enhanced by the critical point. Instead, good assembly requires that the thermodynamic driving force be on the order of the thermal energy and that interactions be made as nonspecific as possible without promoting liquid-vapor phase separation.Comment: 5 pages, 4 figure

Journal of Nanobiotechnology / Characterization of CurcuEmulsomes: nanoformulation for enhanced solubility and delivery of curcumin

Background: Curcumin is a polyphenolic compound isolated from the rhizomes of the plant Curcuma longa and shows intrinsic anti-cancer properties. Its medical use remains limited due to its extremely low water solubility and bioavailability. Addressing this problem, drug delivery systems accompanied by nanoparticle technology have emerged. The present study introduces a novel nanocarrier system, so-called CurcuEmulsomes, where curcumin is encapsulated inside the solid core of emulsomes. Results: CurcuEmulsomes are spherical solid nanoparticles with an average size of 286 nm and a zeta potential of 37 mV. Encapsulation increases the bioavailability of curcumin by up to 10,000 fold corresponding to a concentration of 0.11 mg/mL. Uptaken by HepG2 human liver carcinoma cell line, CurcuEmulsomes show a significantly prolonged biological activity and demonstrated therapeutic efficacy comparable to free curcumin against HepG2 in vitro - with a delay in response, as assessed by cell viability, apoptosis and cell cycle studies. The delay is attributed to the solid character of the nanocarrier prolonging the release of curcumin inside the HepG2 cells. Conclusions: Incorporation of curcumin into emulsomes results in water-soluble and stable CurcuEmulsome nanoformulations. CurcuEmulsomes do not only successfully facilitate the delivery of curcumin into the cell in vitro, but also enable curcumin to reach its effective concentrations inside the cell. The enhanced solubility of curcumin and the promising in vitro efficacy of CurcuEmulsomes highlight the potential of the system for the delivery of lipophilic drugs. Moreover, high degree of compatibility, prolonged release profile and tailoring properties feature CurcuEmulsomes for further therapeutic applications in vivo

Expression of an endotoxin-free S-layer/allergen fusion protein in gram-positive Bacillus subtilis 1012 for the potential application as vaccines for immunotherapy of atopic allergy

<p>Abstract</p> <p>Background</p> <p>Genetic fusion of the major birch pollen allergen (Bet v1) to bacterial surface-(S)-layer proteins resulted in recombinant proteins exhibiting reduced allergenicity as well as immunomodulatory capacity. Thus, S-layer/allergen fusion proteins were considered as suitable carriers for new immunotherapeutical vaccines for treatment of Type I hypersensitivity. Up to now, endotoxin contamination of the fusion protein which occurred after isolation from the gram-negative expression host <it>E. coli </it>had to be removed by an expensive and time consuming procedure. In the present study, in order to achieve expression of pyrogen-free, recombinant S-layer/allergen fusion protein and to study the secretion of a protein capable to self-assemble, the S-layer/allergen fusion protein rSbpA/Bet v1 was produced in the gram-positive organism <it>Bacillus subtilis </it>1012.</p> <p>Results</p> <p>The chimaeric gene encoding the S-layer protein SbpA of <it>Lysinibacillus sphaericus </it>CCM 2177 as well as Bet v1 was cloned and expressed in <it>B. subtilis </it>1012. For that purpose, the <it>E. coli-B. subtilis </it>shuttle vectors pHT01 for expression in the <it>B. subtilis </it>cytoplasm and pHT43 for secretion of the recombinant fusion protein into the culture medium were used. As shown by western blot analysis, immediately after induction of expression, <it>B. subtilis </it>1012 was able to secret rSbpA/Bet v1 mediated by the signal peptide amyQ of <it>Bacillus amyloliquefaciens</it>. Electron microscopical investigation of the culture medium revealed that the secreted fusion protein was able to form self-assembly products in suspension but did not recrystallize on the surface of the <it>B. subtilis </it>cells. The specific binding mechanism between the N-terminus of the S-layer protein and a secondary cell wall polymer (SCWP), located in the peptidoglycan-containing sacculi of <it>Ly. sphaericus </it>CCM 2177, could be used for isolation and purification of the secreted fusion protein from the culture medium. Immune reactivity of rSbpA/Bet v1 could be demonstrated in immunoblotting experiments with Bet v1 specific IgE containing serum samples from patients suffering birch pollen allergy.</p> <p>Conclusions</p> <p>The impact of this study can be seen in the usage of a gram-positive organism for the production of pyrogen-free self-assembling recombinant S-layer/allergen fusion protein with great relevance for the development of vaccines for immunotherapy of atopic allergy.</p

Identification and characterization of domains responsible for self-assembly and cell wall binding of the surface layer protein of Lactobacillus brevis ATCC 8287

Background: Lactobacillus brevis ATCC 8287 is covered by a regular surface (S-) layer consisting of a 435 amino acid protein SlpA. This protein is completely unrelated in sequence to the previously characterized S-layer proteins of Lactobacillus acidophilus group. Results: In this work, the self-assembly and cell wall binding domains of SlpA were characterized. The C-terminal self-assembly domain encompassed residues 179435 of mature SlpA, as demonstrated by the ability of N-terminally truncated recombinant SlpA to form a periodic structure indistinguishable from that formed by full length SlpA. Furthermore, a trypsin degradation analysis indicated the existence of a protease resistant C-terminal domain of 214 amino acids. By producing a set of C-terminally truncated recombinant SlpA (rSlpA) proteins the cell wall binding region was mapped to the N-terminal part of SlpA, where the first 145 amino acids of mature SlpA alone were sufficient for binding to isolated cell wall fragments of L. brevis ATCC 8287. The binding of full length rSlpA to the cell walls was not affected by the treatment of the walls with 5% trichloroacetic acid (TCA), indicating that cell wall structures other than teichoic acids are involved, a feature not shared by the Lactobacillus acidophilus group S-layer proteins characterized so far. Conserved carbohydrate binding motifs were identified in the positively charged N-terminal regions of six Lactobacillus brevis S-layer proteins. Conclusion: This study identifies SlpA as a two-domain protein in which the order of the functional domains is reversed compared to other characterized Lactobacillus S-layer proteins, and emphasizes the diversity of potential cell wall receptors despite similar carbohydrate binding sequence motifs in Lactobacillus S-layer proteins.(VLID)90437

Characterization of greywater from urban and peri-urban areas of Nakuru Municipality, Kenya

Kenya faces serious challenges regarding water and sanitation services. Despite many years of government investment, existing facilities continue to deteriorate and have also failed to meet the demand of increasing population. These challenges are particularly severe in rapidly growing settlements of urban poor. One such settlement is Nakuru municipality which has an average annual population growth of about 8%. The municipality’s sewerage connection is inadequate (11% coverage) and only serves middle and high income areas. This study used a semi-structured questionnaire aiming at characterizing and determining the composition of greywater, besides identifying existing water supply and lifestyle characteristics

Nanorheology of viscoelastic shells: Applications to viral capsids

We study the microrheology of nanoparticle shells [Dinsmore et al. Science 298, 1006 (2002)] and viral capsids [Ivanovska et al. PNAS 101, 7600 (2004)] by computing the mechanical response function and thermal fluctuation spectrum of a viscoelastic spherical shell that is permeable to the surrounding solvent. We determine analytically the damped dynamics of the shear, bend, and compression modes of the shell coupled to the solvent both inside and outside the sphere in the zero Reynolds number limit. We identify fundamental length and time scales in the system, and compute the thermal correlation function of displacements of antipodal points on the sphere and the mechanical response to pinching forces applied at these points. We describe how such a frequency-dependent antipodal correlation and/or response function, which should be measurable in new AFM-based microrheology experiments, can probe the viscoelasticity of these synthetic and biological shells constructed of nanoparticles.Comment: 17 page