Site‐specific and Spatially Controlled Addressability of a New Viral Nanobuilding Block: Sulfolobus islandicus Rod‐shaped Virus 2

Nanotechnology seeks to mimic what nature has achieved: self‐assembly at the nanometer scale. Viral nanoparticles (VNPs) provide natural examples of self‐assembled architectures with unique structural and chemical properties. Here, the utilization of an archaeal virus, Sulfolobus islandicus rod‐shaped virus 2 (SIRV2), as a template for site‐selective and spatially controlled bioconjugation is described. SIRV2 is a virus of a hyperthermophilic and acidophilic host, the archaeon S. islandicus growing optimally at 80 °C and pH 3, and is thus, by its nature, an extremely stable VNP. The stability of SIRV2 in different solvent/water mixtures is monitored, and it is found that in other, non‐natural harsh conditions the VNPs also remained intact. Further, the question of whether the particles offer attachment sites allowing for selective chemical modification and decoration with functional ligands using biotin as a probe is addressed. It is found that carboxylate‐, carbohydrate‐ and amine‐selective chemistries are applicable and various biotinylated SIRV2 formulations can be fabricated. Depending on the chemistry and hence attachment site used, the display of the biotin labels can be spatially controlled at the virus body and at the ends. Labeling studies also provide novel insights into the structural properties of SIRV2, indicating that the major coat protein (CP) forms the virus body while the minor CP is located in the tail fibers at the end of the particles. Overall, SIRV2 represents an extremely stable and structurally interesting VNP with the potential for novel nanobiotechnological applications.     

Local pH variation as an initial step in bacterial surface-sensing and biofilm formation

The development of surface-associated layers of matrix-embedded microbial populations, called biofilms, is a serious problem in many medical and industrial settings. These contaminations are difficult to eradicate because of the high resistance level acquired by the cells in their particular environment. From the very beginning of the colonization process, modifications of gene expression are observed and could, at least partially, explain biofilm resistance. In order to develop anti-biofilm molecules and surface treatments, it is of pivotal importance to identify the physico-chemical parameters which activate the sensor systems of pioneering microbes when they come into contact with a surface. The aim of our study was to examine the pH variations in the local micro-environment created between the cell layer and the surface after bacteria adhesion. Using an ion-sensitive field effect transistor (ISFET), as a substratum, colonized by a curli hyperproducing strain known to form biofilms, we observed that the evolution of the pH change was significantly different in the micro-compartment in contact with the electrochemical sensor compared to that within the liquid phase.     

Preparation and Reinforcement of Dual‐Porous Biocompatible Cellulose Scaffolds for Tissue Engineering

Biocompatible cellulose‐based aerogels composed of nanoporous struts, which embed interconnected voids of controlled micron‐size, have been prepared employing temporary templates of fused porogens, reinforcement by interpenetrating PMMA networks and supercritical carbon dioxide drying. Different combinations of cellulose solvent (Ca(SCN)₂/H₂O/LiCl or [EMIm][OAc]/DMSO) and anti‐solvent (EtOH), porogen type (paraffin wax or PMMA spheres) and porogen size (various fractions in the range of 100–500 μm) as well as intensity of PMMA reinforcement have been investigated to tailor the materials for cell scaffolding applications. All aerogels exhibited an open and dual porosity (micronporosity >100 μm and nanoporosity extending to the low micrometer range). Mechanical properties of the dual‐porous aerogels under compressive stress were considerably improved by introduction of interpenetrating PMMA networks. The effect of the reinforcing polymer on attachment, spreading, and proliferation of NIH 3T3 fibroblast cells, cultivated on selected dual‐porous aerogels to pre‐evaluate their biocompatibility was similarly positive.     

Comparing in situ particle monitoring to microscopic counts of plankton in a drinking water reservoir

In a one-year study, the multispecies assemblages of phytoplankton (picoplankton to microplankton) within a drinking water reservoir were counted, determined and evaluated in their size fractions using microscope enumeration (MC). The manual counts were compared with the size evaluation obtained by a light obscuring particle counter (PC) in order to evaluate its use for the monitoring practice of a drinking water reservoir. With this multispecies comparison we present a novel approach for the evaluation of automated counting systems.The picoplankton clearly remained uncounted by the PC even though its lower size limits imply an adequate match. The highest and most consistent count numbers of plankton (nano- and microplankton) and particles were obtained during the spring mass development. However, from the middle of the year onwards, the measured particle concentration surpassed the counted plankton abundances by two- to threefold indicating the rise of seston within the water column. This fraction would be missed if counted solely by MC. Further, the PC consistently undersized the biological counts, but not the minerogenic fraction represented by the manganese oxidising bacteria. Consequently, the rise and decline of Metallogenium bacteria was reliably monitored with the PC.The PC provides additional size information compared to other bulk optical sensors (turbidity, chlorophyll-a). The correlation of particles with probe measurement always exceeded the plankton coefficient, but all combinations of plankton, particle and probe measurement revealed significant linear regressions. However, the redundancy of the chlorophyll-a probes was also shown in order to explain plankton abundances.Our results indicate that background knowledge of the monitored system and cautious interpretation of data is required to allocate and understand automated particle counts. Therefore, only in combination with MC, the PC enables phytoplankton or minerogenic particle counts under frequent real-time monitoring conditions. As such it may serve as a helpful tool for example in critical situations in the management of drinking water reservoirs.     

Brookite Formation in TiO2?Ag Nanocomposites and Visible‐Light‐Induced Templated Growth of Ag Nanostructures in TiO2

TiO₂? Ag‐nanocomposites exhibit various desirable properties that make them suitable for a variety of applications, for example in photocatalysis and as bactericidal coatings. In this work, a new method for processing TiO₂? Ag nanocomposites is presented. The nanocomposite films are fabricated from one precursor solution with high silver loading of up to 50%. The resulting films exhibit a microstructure consisting of TiO₂? Ag_xO nanocomposites with a largely XRD‐amorphous TiO₂ matrix containing brookite nanocrystals. This specific microstructure absorbs in the visible range so that photoreduction of Ag ions can be accomplished by using visible light. The thin films can be patterned using simple shadow masks. The illuminated areas show a high density of self‐organized nanoparticles (SNPs) and nanorods (SNRs), which are templated by the TiO₂ porous network. The particle size can be tuned by varying the irradiation time. Most of the SNPs and SNRs form faceted crystals, which are mostly a combination of {111} and {110}. The application of these films as substrates for surface‐enhanced Raman scattering is shown. Enhancement factors as high as 4.6 × 10⁶ could be obtained using rhodamine 6G dye molecules. More applications should involve photocatalytic water purification using visible light.     

Quantification of clay dispersion in nanocomposites of styrenic polymers

Polymer‐clay nanocomposites are materials with many interesting structures, properties, and potential applications. Microstructural evaluation of a nanocomposite is not an easy task, as clay may form hierarchical structures which may look different when observed at various magnifications under a microscope, and also as the concepts of “intercalation” and “exfoliation” are not self‐sufficient to describe its morphology. In this work polymer‐clay nanocomposites of polystyrene and two styrene‐containing block copolymers (styrene‐butadiene‐styrene and styrene‐ethylene/butylene‐styrene) were prepared using three different techniques. Clay dispersion was evaluated by a recently developed microscopy image analysis procedure, combining the analysis of optical and transmission electron micrographs, and the characterization was complemented by X‐ray diffraction and rheological measurements. The results showed better clay dispersion for both block copolymers nanocomposites, mainly due to their molecular architectures. Moreover, the techniques which showed the best results involved mixing the materials in a solvent medium. POLYM. ENG. SCI., 2009. © 2009 Society of Plastics Engineers     

GC–MS analysis of oxysterols and their formation in cultivated liver cells (HepG2)

Oxysterols play a key role in many (patho)physiological processes and they are potential biomarkers for oxidative stress in several diseases. Here we developed a rapid gas chromatographic-mass spectrometry-based method for the separation and quantification of 11 biologically relevant oxysterols bearing hydroxy, epoxy, and dihydroxy groups. Efficient chromatographic separation (resolution ≥ 1.9) was achieved using a medium polarity 35%-diphenyl/65%-dimethyl polysiloxane stationary phase material (30 m × 0.25 mm inner diameter and 0.25 μm film thickness). Based on thorough analysis of the fragmentation during electron ionization we developed a strategy to deduce structural information of the oxysterols. Optimized sample preparation includes (i) extraction with a mixture of n-hexane/iso-propanol, (ii) removal of cholesterol by solid phase extraction with unmodified silica, and (iii) trimethylsilylation. The method was successfully applied on the analysis of brain samples, showing consistent results with previous studies and a good intra- and interday precision of ≤20%. Finally, we used the method for the investigation of oxysterol formation during oxidative stress in HepG2 cells. Incubation with tert-butyl hydroperoxide led to a massive increase in free radical formed oxysterols (7-keto-chol > 7β-OH-chol >> 7α-OH-chol), while 24 h incubation with the glutathione peroxidase 4 inhibitor RSL3 showed no increase in oxidative stress based on the oxysterol pattern. Overall, the new method described here enables the robust analysis of a biologically meaningful pattern of oxysterols with high sensitivity and precision allowing us to gain new insights in the biological formation and role of oxysterols.