Characterization of rhinovirus subviral A particles via capillary electrophoresis, electron microscopy and gas-phase electrophoretic mobility molecular analysis: Part I

During infection, enteroviruses, such as human rhinoviruses (HRVs), convert from the native, infective form with a sedimentation coefficient of 150S to empty subviral particles sedimenting at 80S (B particles). B particles lack viral capsid protein 4 (VP4) and the single-stranded RNA genome. On the way to this end stage, a metastable intermediate particle is observed in the cell early after infection. This subviral A particle still contains the RNA but lacks VP4 and sediments at 135S. Native (150S) HRV serotype 2 (HRV2) as well as its empty (80S) capsid have been well characterized by capillary electrophoresis. In the present paper, we demonstrate separation of at least two forms of subviral A particles on the midway between native virions and empty 80S capsids by CE. For one of these intermediates, we established a reproducible way for its preparation and characterized this particle in terms of its electrophoretic mobility and its appearance in transmission electron microscopy (TEM). Furthermore, the conversion of this intermediate to 80S particles was investigated. Gas-phase electrophoretic mobility molecular analysis (GEMMA) yielded additional insights into sample composition. More data on particle characterization including its protein composition and RNA content (for unambiguous identification of the detected intermediate as subviral A particle) will be presented in the second part of the publication.     

Dynamic assembly of porphyrin wires trapped on a highly oriented pyrolitic graphite surface

Carefully designed porphyrin building blocks assemble through selective imidazole binding in various solvents to form linear multiporphyrin objects. From a dynamic mixture of monomers, dimers, and oligomers, linear objects were observed on a highly oriented pyrolitic graphite (HOPG) surface. On the surface, the objects' morphology clearly depended on the solvent used for deposition and was modified upon heating.     

Phototoxic effect of visible light on Porphyromonas gingivalis and Fusobacterium nucleatum: an in vitro study

The antibacterial effect of visible light irradiation combined with photosensitizers has been reported. The objective of this was to test the effect of visible light irradiation without photosensitizers on the viability of oral microorganisms. Strains of Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mutans and Streptococcus faecalis in suspension or grown on agar were exposed to visible light at wavelengths of 400-500 nm. These wavelengths are used to photopolymerize composite resins widely used for dental restoration. Three photocuring light sources, quartz-tungsten-halogen lamp, light-emitting diode and plasma-arc, at power densities between 260 and 1300 mW/cm2 were used for up to 3 min. Bacterial samples were also exposed to a near-infrared diode laser (wavelength, 830 nm), using identical irradiation parameters for comparison. The results show that blue light sources exert a phototoxic effect on P. gingivalis and F. nucleatum. The minimal inhibitory dose for P. gingivalis and F. nucleatum was 16-62 J/cm2, a value significantly lower than that for S. mutans and S. faecalis (159-212 J/cm2). Near-infrared diode laser irradiation did not affect any of the bacteria tested. Our results suggest that visible light sources without exogenous photosensitizers have a phototoxic effect mainly on Gram-negative periodontal pathogens.     

Analysis of natural flavonoids by microchip-micellar electrokinetic chromatography with pulsed amperometric detection

Catechins (catechin and other derivatives) are naturally occurring flavonoids present in a number of plants and foods. They are also part of numerous nutraceutical formulations because they are believed to have antioxidant, cancer chemo-preventative, anti-inflammatory and antimicrobial properties. The determination of catechins has traditionally been performed by HPLC. However, this methodology is both time and sample intensive and generates large amounts of organic solvent waste. In the current report, an application of MEKC using a PDMS microchip is presented for the analysis of catechins. The system uses pulsed amperometric detection for direct analysis of important naturally occurring catechins. The effect of pH, surfactant concentration, detection potential and signal stability were analyzed. Linear relationships were found between the concentration and peak current, with good stability and limits of detection of 8 [micro sign]M for catechin, epigallocatechin gallate and epicatechin, and 14 [micro sign]M for epicatechin gallate. Optimum conditions were applied to the detection of selected catechins in a commercially available green tea extract nutraceutical and the results were compared to HPLC analysis. The analysis using microchip micellar electrokinetic chromatography and pulsed amperometric detection was completed in 4.5 min, 10 times faster than the HPLC analysis.     

Anticalins versus antibodies: made-to-order binding proteins for small molecules

Engineering proteins to bind small molecules presents a challenge as daunting as drug discovery, for both hinge upon our understanding of receptor-ligand molecular recognition. However, powerful techniques from combinatorial molecular biology can be used to rapidly select artificial receptors. While traditionally researchers have relied upon antibody technologies as a source of new binding proteins, the lipocalin scaffold has recently emerged as an adaptable receptor for small molecule binding. 'Anticalins', engineered lipocalin variants, offer some advantages over traditional antibody technology and illuminate features of molecular recognition between receptors and small molecule ligands.     

Synthesis of New Regioselectively Sulfated Hyaluronans for Biomedical Application

Sulfated glycosaminoglycans (GAGs) display various biological effects which are strongly influenced by the degree of sulfation and the position of sulfate groups within the polymer. Hyaluronan, a non-sulfated GAG, represents a readily accessible educt to synthesize structural analogues of sulfated GAGs mimicking their biological activity. Different strategies were developed and evaluated to synthesize hyaluronan sulfates with a free primary hydroxyl group at C-6' and sulfated secondary hydroxyl groups. Applying selective desulfation methods of high-sulfated hyaluronan by means of silylating agents, products regioselectively desulfated at the primary C-6' but also partly the C-4' position were obtained. A pathway using benzoyl ester protecting groups to block the primary hydroxyl function of Hya during the sulfation resulted in a high-sulfated product, functionalized only at the secondary hydroxyl groups.     

Electrophoresis on a microfluidic chip for analysis of fluorescence-labeled human rhinovirus

We report the analysis of human rhinovirus serotype 2 (HRV2) on a commercially available lab-on-a-chip instrument. Due to lack of sufficient native fluorescence, the proteinaceous capsid of HRV2 was labeled with Cy5 for detection by the red laser (lambda ex 630 nm) implemented in the instrument. On the microdevice, electrophoresis of the labeled virus was possible in a BGE without stabilizing detergents, which is in contrast to conventional CE; moreover, analysis times were drastically shortened to the few 10 s range. Resolution of the sample constituents (virions, a contaminant present in all virus preparations, and excess dye) was improved upon adaptation of the separation conditions, mainly by adjusting the SDS concentration of the BGE. Purity of fractions from size-exclusion chromatography after labeling of virus was assessed, and affinity complex formation of the labeled virus with various recombinant very-low-density lipoprotein receptor derivatives differing in the number of concatenated V3 ligand binding repeats was monitored. Virus analysis on microchip devices is of particular interest for experiments with infectious material because of easy containment and disposal of samples. Thus, the employment of microchip devices in routine analysis of viruses appears to be exceptionally attractive.     

Exploring Coordination Modes: Late Transition Metal Complexes with a Methylene‐bridged Macrocyclic Tetra‐NHC Ligand

A tetranuclear silver(I) N‐heterocyclic carbene (NHC) complex bearing a macrocyclic, exclusively methylene‐bridged, tetracarbene ligand was synthesized and employed as transmetalation agent for the synthesis of nickel(II), palladium(II), platinum(II), and gold(I) derivatives. The transition metal complexes exhibit different coordination geometries, the coinage metals being bound in a linear fashion forming molecular box‐type complexes, whereas the group 10 metals adapt an almost ideal square planar coordination geometry within the ligand's cavity, resulting in saddle‐shaped complexes. Both the Ag^I and the Au^I complexes show ligand‐induced metal–metal contacts, causing photoluminescence in the blue region for the gold complex. Distinct metal‐dependent differences of the coordination behavior between the group 10 transition metals were elucidated by low‐temperature NMR spectroscopy and DFT calculations.     

(R)‐12‐Hydroxystearic Acid Hydrazides as Very Efficient Gelators: Diffusion,Partial Thixotropy,and Self‐Healing in Self‐Standing Gels

The gelation properties of derivatives of N‐alkylated (R)‐12‐hydroxystearic acid hydrazide (n‐HSAH, n=0, 2, 6, 10; n is the length of an n‐alkyl chain on the terminal nitrogen atom) in a wide variety of liquids is reported. The n‐HSAH compounds were derived from a naturally occurring alkanoic acid, (R)‐12‐hydroxystearic acid (R‐12HSA), and although they differ from the analogous N‐alkyl (R)‐12‐hydroxystearamides (n‐HSAA) only by the presence of one N?H group, their behavior as gelators is very different. For example, the parent molecule (0‐HSAH) is a supergelator in ethylene glycol, in which it forms self‐standing gels that are self‐healing, partially thixotropic, moldable, and load‐bearing; gels of 0‐HSAA are not self‐standing. 0‐HSAH is structurally the simplest molecular gelator of which we are aware that is capable of forming both self‐standing and partially thixotropic gels. Also, diffusion of the cationic dye erythrosine B and the anionic dye methylene blue in 0‐HSAH/ethylene glycol gel blocks is much slower than the self‐diffusion of ethylene glycol. Polarizing optical microscopy, X‐ray diffraction, and FTIR studies revealed that the self‐assembled fibrillar networks (SAFINs) of the gels are crystalline, and that 0‐HSAH molecules may be arranged in a triclinic subcell with bilayer stacking. The SAFINs are stabilized by strong hydrogen‐bonding interactions between the hydrazide groups of adjacent molecules and a perpendicular hydrogen‐bonding network between the pendent hydroxyl groups of 0‐HSAH. The other n‐HSAH (n=2, 6, 10) molecules appear to be arranged in orthorhombic subcells with monolayers and strong hydrogen‐bonding interactions between the hydrazide group of one gelator molecule and the hydroxyl group of a neighboring one. These results show how small structural modifications of structurally simple gelator molecules can be exploited to form gels with novel properties that can lead potentially to valuable applications, such as in drug delivery.     

Radon as a tracer to determine the mean residence times of groundwater in decontamination reactors

The groundwater at a former gasoline production site in Germany is heavily contaminated with aromatic hydrocarbons (mostly benzene) and is currently being treated in bioreactors under anaerobic conditions. To determine the reaction kinetics it is essential to know the mean residence time of the groundwater in these reactors. Most of the commonly used tracers (dyes and salts) did not give reliable results because of their interaction with the mineral matrix in the reactors. In this study radon (²²²Rn) dissolved in the groundwater is used as the tracer. The flow rate of groundwater through the reactors is 1 l/h. Over a period of 8 hours the radon-spiked groundwater was injected into the natural groundwater which has a very low radon concentration. The radon concentration of the discharged water is measured online at the reactor outlet. An increasing radon concentration at the reactor exit indicates the shortest residence time of the water. The time-dependent progress of the radon concentration provides detailed information about the flow behavior and residence times of water in the reactor.