Photonic crystal nanostructures for optical biosensing applications

We present the design, fabrication and optical investigation of photonic crystal (PhC) nanocavity drop filters for use as optical biosensors. The resonant cavity mode wavelength and Q-factor are studied as a function of the ambient refractive index and as a function of adsorbed proteins (bovine serum albumin) on the sensor surface. Experiments were performed by evanescent excitation of the cavity mode via a PhC waveguide. This in turn is coupled to a ridge waveguide that allows the introduction of a fluid flow cell on a chip. A response of ∂λ/∂c=(4.54±0.66)×10⁵ nm/M is measured leading to a measured detection limit as good as  fg or  pg/mm²in the sensitive area.     

Host-specific symbiotic requirement of BdeAB, a RegR-controlled RND-type efflux system in Bradyrhizobium japonicum

Multidrug efflux systems not only cause resistance against antibiotics and toxic compounds but also mediate successful host colonization by certain plant-associated bacteria. The genome of the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum encodes 24 members of the family of resistance/nodulation/cell division (RND) multidrug efflux systems, of which BdeAB is genetically controlled by the RegSR two-component regulatory system. Phylogenetic analysis of the membrane components of these 24 RND-type transporters revealed that BdeB is more closely related to functionally characterized orthologs in other bacteria, including those associated with plants, than to any of the other 23 paralogs in B. japonicum. A mutant with a deletion of the bdeAB genes was more susceptible to inhibition by the aminoglycosides kanamycin and gentamicin than the wild type, and had a strongly decreased symbiotic nitrogen-fixation activity on soybean, but not on the alternative host plants mungbean and cowpea, and only very marginally on siratro. The host-specific role of a multidrug efflux pump is a novel feature in the rhizobia-legume symbioses. Consistent with the RegSR dependency of bdeAB, a B. japonicum regR mutant was found to have a greater sensitivity against the two tested antibiotics and a symbiotic defect that is most pronounced for soybean.     

Effect of collagen I and fibronectin on the adhesion, elasticity and cytoskeletal organization of prostate cancer cells

The adipokine adiponectin circulates in high concentration, and activates the classical pathway of complement by binding C1q, leading to the activation of C3 and formation of the membrane attack complex. Such behaviour is potentially pathophysiological. However, we showed adiponectin captured the complement inhibitor Factor H both as a pure protein and from human serum. Both heparin and a homologue of C3b, substrates binding to the C-terminus of Factor H, were inhibitory of the interaction, as was EDTA. Factor H bound equivalently to high and low molecular weight serum adiponectin, and to an N-terminal 16 kDa cyanogen bromide cleavage product of adiponectin. The binding of Factor H inhibited both the C3 and C5 convertases generated from complement activation by adiponectin, so reducing potentially pathophysiological consequences such as the deposition of C5b-9, while allowing opsonisation of target molecules with C3b.     

Isolation of RNA from bacterial samples of the human gastrointestinal tract

The human gastrointestinal (GI) tract contains a complex microbial community that consists of numerous uncultured microbes. Therefore, nucleic-acid-based approaches have been introduced to study microbial diversity and activity, and these depend on the proper isolation of DNA, rRNA and mRNA. Here, we present an RNA isolation protocol that is suitable for a wide variety of GI tract samples. The procedure for isolating DNA from GI tract samples is described in another Nature Protocols article. One of the benefits of our RNA isolation protocol is that sampling can be performed outside the laboratory, which offers possibilities for implementation in large intervention studies. The RNA isolation is based on mechanical disruption, followed by isolation of nucleic acids using phenol:chloroform:isoamylalcohol extraction and removal of DNA. In our laboratory, this protocol has resulted in the isolation of rRNA and mRNA of sufficient quality and quantity for microbial diversity and activity studies. Depending on the number of samples, the sample type and the quenching procedure chosen, the whole procedure can be performed within 2.5-4 h.     

Structural studies on a twin-arginine signal sequence

Translocation of folded proteins across biological membranes can be mediated by the so-called ‘twin-arginine translocation’ (Tat) system. To be translocated, Tat substrates require N-terminal signal sequences which usually contain the eponymous twin-arginine motif. Here we report the first structural analysis of a twin-arginine signal sequence, the signal sequence of the high potential iron-sulfur protein from Allochromatium vinosum. Nuclear magnetic resonance (NMR) analyses of amide proton resonances did not indicate a signal sequence structure. Accordingly, data from H/D exchange matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry showed that the amide protons of the signal sequence exchange rapidly, indicating the absence of secondary structure in the signal sequence up to L29. We conclude that the conserved twin-arginine motif does not form a structure by itself or as a result of intramolecular interactions.     

Optimization of single-base-pair mismatch discrimination in oligonucleotide microarrays

The discrimination between perfect-match and single-base-pair-mismatched nucleic acid duplexes was investigated by using oligonucleotide DNA microarrays and nonequilibrium dissociation rates (melting profiles). DNA and RNA versions of two synthetic targets corresponding to the 16S rRNA sequences of Staphylococcus epidermidis (38 nucleotides) and Nitrosomonas eutropha (39 nucleotides) were hybridized to perfect-match probes (18-mer and 19-mer) and to a set of probes having all possible single-base-pair mismatches. The melting profiles of all probe-target duplexes were determined in parallel by using an imposed temperature step gradient. We derived an optimum wash temperature for each probe and target by using a simple formula to calculate a discrimination index for each temperature of the step gradient. This optimum corresponded to the output of an independent analysis using a customized neural network program. These results together provide an experimental and analytical framework for optimizing mismatch discrimination among all probes on a DNA microarray.     

Cell-type specific targeting and gene expression using a variant of polyoma VP1 virus-like particles

The variant VP1-Z of the polyomavirus coat protein VP1 has been recently described as an engineered fusion protein of VP1 and the antibody binding domain protein Z. This construct is able to specifically bind and functionally present antibodies on the surface of virus-like particles of VP1-Z. Here we demonstrate that with the binding of Herceptin, an antibody directed against the receptor tyrosine kinase ErbB2, a cell type-specific targeting was established. ErbB2-positive cell lines were transduced with different plasmids encoding eGFP or beta-galactosidase. With both reporter systems functional gene expression in transduced cells could be observed. The transduction was strictly dependent on the use of a ternary complex formed of VLPs of VP1-Z, Herceptin, and the reporter plasmid DNA. The use of single components or ErbB2-negative cell lines did not result in functional gene transfer. The transduction was also completely dependent on the use of chloroquine, a lysosomotropic reagent. This indicates that the complex is internalized by ErbB2-mediated endocytosis.