New cytochalasans from an endophytic Xylaria species associated with Costa Rican Palicourea elata (Rubiaceae).

Four new leucine-derived cytochalasans, possessing a 5,6,5,8-ring (1) and a 5,6,11-ring core (2-4), were isolated from a cultivated endophytic fungus Xylaria sp. strain WH2D4 (Xylariaceae). This fungus was isolated from leaves of the neotropical tree species Palicourea elata (Sw.) Borhidi (Rubiaceae) collected in Costa Rica. The chemical structures were determined by employing IR, MS as well as 1D- and 2D-NMR experiments. The stereochemistry at C-15 of compound 4 was determined by quantum calculations. The isolated compounds did not affect germination and growth of Trichoderma reesei and the opportunistic human fungal pathogen T. longibrachiatum.

Raman and scanning probe microscopy for differentiating surface imprints of E. coli and B. cereus.

Molecularly imprinted polymers (MIPs) are artificial recognition materials mimicking biological recognition entities such as antibodies. The general model of imprinting assumes that functional monomers interact with functional groups present on the target species which leads to cavities complementing the template in surface chemistry and shape thus ensuring recognition. However, to date there is little independent experimental evidence supporting that the surface chemistry in the imprints is tailored to analyte recognition and thus differs from the surface chemistry of the surrounding polymer. Herein, we investigate such chemical differences between imprints of Escherichia coli and Bacillus cereus in poly(styrene-co-DVB) and a commercial acrylate-based polymer by the means of confocal Raman microscopy and PLS-DA. The MIPs were generated using a stamping approach. Peak-force QNM measurements were conducted to rule out residues of bacterial cells in the imprints. While imprints of E. coli and B. cereus could be distinguished based on their Raman spectra in the acrylate-based polymer, differentiation in poly(styrene-co-DVB) was not significant. This could be a result of a higher potential of acrylate functional groups for interacting with lipopolysaccharides and peptidoglycans on bacteria surfaces compared to the phenyl groups of poly(styrene-co-DVB) and emphasizes the importance of the right choice of functional monomers for a specific target analyte.

Raman Studies on Surface-Imprinted Polymers to Distinguish the Polymer Surface, Imprints, and Different Bacteria.

Molecularly imprinted polymers (MIPs) are widely used as robust biomimetic recognition layers in sensing devices targeting a wide variety of analytes including microorganisms such as bacteria. Assessment of imprinting success and selectivity toward the target is of great importance in MIP quality control. We generated Escherichia coli-imprinted poly(styrene-co-DVB) as a model system for bacteria-imprinted polymers via surface imprinting using a glass stamp with covalently immobilized E. coli. Confocal Raman Microscopy was successfully employed to visualize bacteria, imprints, and polymer and to distinguish them from each other. The method has proven highly feasible for assessing if imprinting had been successful. In addition, we developed a method for selectivity investigation of bacteria MIPs based on combining Confocal Raman Microscopy and Partial Least Squares Discriminant Analysis (PLS-DA). The Raman spectra of E. coli and Bacillus cereus were acquired on E. coli-imprinted poly(styrene-co-DVB) and used to establish a PLS-DA model for differentiating between the bacteria species. Model validation demonstrated a correct classification of 95% of Raman spectra, indicating sufficient accuracy of the model for future use in MIP selectivity studies. Simultaneous differentiation of 3 bacteria species (E. coli, B. cereus, and Lactococcus lactis) on E. coli-imprinted poly(styrene-co-DVB) proved more difficult, which might be due to the limited depth resolution of the confocal Raman microscope resulting in the presence of interfering signals from the polymer substrate. It might be possible to overcome this obstacle by selective enhancement of the Raman signals originating from bacteria surfaces, such as tip enhanced Raman spectroscopy.

Modified amino-dextrans as carriers of Gd-chelates for retrograde transport and visualization of peripheral nerves by magnetic resonance imaging (MRI).

Amino-dextrans (AD) conjugated with gadolinium (Gd3+) were developed as neuro-specific contrast agents (CA) for the visualization of the sciatic nerve in rats by magnetic resonance imaging (MRI). AD with 3, 10, and 70 kDa molecular weights were assessed as carrier molecules known to be transported with various speed by axonal microtubules. Detailed spectroscopic characterizations, analyses by Fast Protein Liquid Chromatography (FPLC), Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), and inductively coupled plasma-mass spectrometry (ICP-MS), were carried out. For MRI, the paramagnetic Gd3+ ion was coupled as a T1 signal enhancer. The well-established linear chelator, diethylenetriaminepentaacetic acid (DTPA), was used and subsequently replaced by the more stable cyclic chelator 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In addition, a fluorescently labeled AD-DTPA-Gd was prepared to demonstrate an active transport to the spinal cord by histochemistry. After successful synthesis and characterization, molecular migration of the AD-DTPA-Gd in the sciatic nerve of healthy Sprague Dawley rats was monitored by MRI for up to seven days. Enhancement of nerve structures was evaluated by MRI and correlated with ICP-MS analyses. To investigate the distribution of CA along the neuraxis, all animals were sacrificed after the final MRI monitoring. Nerves, spinal ganglions, and corresponding spinal cord sections were harvested, to determine the localization and concentration of the paramagnetic element. This is the first report that demonstrates the active uptake and transport of AD-Gd conjugates within the sciatic nerve. This new concept may serve as a potential diagnostic tool for the direct visualization and monitoring of the continuity of injured nerves.

Penetration monitoring of drugs and additives by ATR-FTIR spectroscopy/tape stripping and confocal Raman spectroscopy - A comparative study.

Vibrational spectroscopy is a useful tool for analysis of skin properties and to confirm the penetration of drugs and other formulation compounds into the skin. In particular, attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and confocal Raman spectroscopy (CRS) have been optimised for skin analysis. Despite an impressive amount of data on these techniques, a comparative methodological assessment for skin penetration monitoring of model substances is still amiss. Thus, in vitro skin penetration studies were conducted in parallel using the same porcine material and four model substances, namely sodium laureth sulfate (SLES), sodium dodecyl sulfate (SDS), sulfathiazole sodium (STZ) and dimethyl sulfoxide (DMSO). ATR-FTIR spectroscopy in combination with tape stripping and CRS were employed to evaluate the skin penetration of the applied substances. In addition, the skin hydration status or change in skin hydration after application was investigated. The results show that both methods provide valuable information on the skin penetration potential of applied substances. The penetration profiles determined by CRS or ATR-FTIR/tape stripping were comparable for all substances; a slow decrease in relative substance concentration was visible from the skin surface inwards within the stratum corneum (SC). In general, deeper penetration into the SC was observed with CRS, which may be related to the depth resolution of the employed device. However, when related to the respective total SC thickness of each experiment, the penetration depths determined by parallel CRS and ATR-FTIR analysis were in good agreement for all model substances. The observed order of the penetration depth was DMSO > SDS > SLES > STZ with both techniques. A decrease of the relative concentration to 10% of the maximum value was found approximately between 34 and 89% of total SC thickness. Summarising these findings, advantages and drawbacks of the two techniques for in vitro skin penetration studies are discussed.

Novel concentrated water-in-oil emulsions based on a non-ionic silicone surfactant: Appealing application properties and tuneable viscoelasticity.

Silicone excipients are non-irritating ingredients that are extensively used in topical formulations. In the present study, innovative water-in-oil emulsions with a high water content stabilised by a non-ionic silicone surfactant were developed. Effects of formulation composition on its properties and stability were investigated. It was possible to prepare highly stable emulsions with a water volume fraction of up to 80%. The emulsions exhibited desirable application properties such as non-sticky and cooling qualities. A dependency of the viscosity on the water fraction was found; this offers the opportunity to create emulsions with fine-tuned rheological properties. Furthermore, it could be shown in skin studies that the in vitro release of a hydrophilic model drug is influenced by the configuration of the oil phase. The penetration of the silicone surfactant and the other deployed additives was monitored using combined tape stripping and ATR-FTIR experiments, revealing that the compounds remain in the superficial layers of the stratum corneum, thus minimising the risk for skin irritation.

Distribution of phospholipid based formulations in the skin investigated by combined ATR-FTIR and tape stripping experiments.

The spatial distribution of exogenous substances in the stratum corneum (SC) could have an influence on their skin irritation potential. In this study it was possible to monitor the distribution of phospholipids with their phosphatidylcholine scaffold on porcine ear skin by combining tape stripping and in vitro ATR-FTIR spectroscopy. Significant vibrational modes in the spectra could be successfully assigned to the functional groups of the molecules. Thus it was possible to track the phospholipids without the need of their deuterated form by calculating difference spectra from the treated - untreated skin samples. The correlation between four characteristic bands (R2≥0.9909) revealed the excellent suitability of this semi-quantitative method for deep profiling analysis. The penetration capabilities of aqueous suspensions of the different phospholipid compositions as well as two monoacyl-phosphatidylcholine based liposome formulations were investigated using this method. Nevertheless, differences in the distribution of the investigated phospholipid species, having different amounts of monoacyl-phosphatidylcholine, could not be found. It could be clearly shown that the deepest skin penetration was seen in the irritating anionic SDS (sodium dodecyl sulfate) out of the aqueous solution. The aqueous suspensions based on different phospholipid surfactants showed the same range of penetration depth (10-15% of SC), whereas the smallest skin penetration depth was observed after the application of liposomal formulations.

pH and Potential Transients of the bc1 Complex Co-Reconstituted in Proteo-Lipobeads with the Reaction Center from Rb. sphaeroides.

His-tag technology is employed to bind membrane proteins, such as the bc1 complex and the reaction center (RC) from Rhodobacter sphaeroides, to spherical as well as planar surfaces in a strict orientation. Subsequently, the spherical and planar surfaces are subjected to in situ dialysis to form proteo-lipobeads (PLBs) and protein-tethered bilayer membranes, respectively. PLBs based on Ni-nitrileotriacetic acid-functionalized agarose beads that have diameters ranging from 50 to 150 µm are used to assess proton release and membrane potential parameters by confocal laser-scanning microscopy. The pH and potential transients are thus obtained from bc1 activated by the RC. To assess the turnover of bc1 excited by the RC in a similar setting, we used the planar surface of an attenuated total reflection crystal modified with a thin gold layer to carry out time-resolved surface-enhanced IR absorption spectroscopy triggered by flash lamp excitation. The experiments suggest that both proteins interact in a cyclic manner in both environments. The activity of the proteins seems to be preserved in the same manner as that in chromatophores or reconstituted in liposomes.

Complete Exchange of the Hydrophobic Dispersant Shell on Monodisperse Superparamagnetic Iron Oxide Nanoparticles.

High-temperature synthesized monodisperse superparamagnetic iron oxide nanoparticles are obtained with a strongly bound ligand shell of oleic acid and its decomposition products. Most applications require a stable presentation of a defined surface chemistry; therefore, the native shell has to be completely exchanged for dispersants with irreversible affinity to the nanoparticle surface. We evaluate by attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) and thermogravimetric analysis/differential scanning calorimetry (TGA/DSC) the limitations of commonly used approaches. A mechanism and multiple exchange scheme that attains the goal of complete and irreversible ligand replacement on monodisperse nanoparticles of various sizes is presented. The obtained hydrophobic nanoparticles are ideally suited for magnetically controlled drug delivery and membrane applications and for the investigation of fundamental interfacial properties of ultrasmall core-shell architectures.

Monitoring the distribution of surfactants in the stratum corneum by combined ATR-FTIR and tape-stripping experiments.

Combined ATR-FTIR (attenuated total reflection-Fourier transform infrared) spectroscopy and tape-stripping experiments in vitro on porcine ear skin were used to investigate the spatial distribution of different surfactants in the stratum corneum (SC). To reveal a possible connection between the size of the formed micelles and skin penetration, dynamic light-scattering measurements of the aqueous surfactant solutions were also taken. Compared to an alkyl polyglycoside and sucrose laurate, a deeper skin penetration of the anionic surfactants sodium dodecyl sulfate (SDS) und sodium lauryl ether sulfate (SLES) could be related to a smaller size of the formed micelles. Beside the differences in spatial distribution, a link between the physical presence of anionic surfactants in the SC and a decrease of skin hydration was found. Furthermore, the incorporation of SDS and SLES into the SC, even after a brief, consumer-orientated washing procedure with commercially available hair shampoos, was confirmed.

Validation of the combined ATR-FTIR/tape stripping technique for monitoring the distribution of surfactants in the stratum corneum.

The physical presence of surfactants in the skin is linked to their skin irritation potential. Combined ATR-FTIR spectroscopy and tape stripping experiments in vitro on porcine ear skin were used to investigate the spatial distribution of sodium lauryl ether sulfate (SLES) in the stratum corneum and to assess its effects on conformational order of stratum corneum intercellular lipids, secondary structure of keratin and skin hydration. It was possible to monitor the spatial distribution of SLES in the stratum corneum for the first time by subtracting spectra of untreated from treated skin samples and without the need of a perdeuterated form. This method of analysis was evaluated by addressing potential error sources such as differences in removed amounts of corneocytes and intra-individual changes in stratum corneum composition as a function of depth. The obtained results indicate a penetration of SLES into deep layers of the stratum corneum. Furthermore, SLES treatment led to significantly decreased skin hydration levels, whereas the secondary structure of keratin remained nearly unaffected. The reliability of this semi-quantitative method of analysis was confirmed by receiving a coefficient of determination of 0.9963 after making a correlation of deep depended absorbances of two different characteristic bands with different absorption coefficients.

Determination of surface concentrations of individual molecule-layers used in nanoscale biosensors by in situ ATR-FTIR spectroscopy.

For the development of nanowire sensors for chemical and medical detection purposes, the optimal functionalization of the surface is a mandatory component. Quantitative ATR-FTIR spectroscopy was used in situ to investigate the step-by-step layer formation of typical functionalization protocols and to determine the respective molecule surface concentrations. BSA, anti-TNF-α and anti-PSA antibodies were bound via 3-(trimethoxy)butylsilyl aldehyde linkers to silicon-oxide surfaces in order to investigate surface functionalization of nanowires. Maximum determined surface concentrations were 7.17 × 10(-13) mol cm(-2) for BSA, 1.7 × 10(-13) mol cm(-2) for anti-TNF-α antibody, 6.1 × 10(-13) mol cm(-2) for anti-PSA antibody, 3.88 × 10(-13) mol cm(-2) for TNF-α and 7.0 × 10(-13) mol cm(-2) for PSA. Furthermore we performed antibody-antigen binding experiments and determined the specific binding ratios. The maximum possible ratio of 2 was obtained at bulk concentrations of the antigen in the µg ml(-1) range for TNF-α and PSA.

S-layer templated bioinspired synthesis of silica.

The current understanding of the molecular mechanisms involved in the bioinspired formation of silica structures laid foundation for investigating the potential of the S-layer protein SbpA from Lysinibacillus sphaericus CCM 2177 as catalyst, template and scaffold for the generation of novel silica architectures. SbpA reassembles into monomolecular lattices with square (p4) lattice symmetry and a lattice constant of 13.1 nm. Silica layers on the S-layer lattice were formed using tetramethoxysilane (TMOS) and visualized by transmission electron microscopy. In situ quartz crystal microbalance with dissipation monitoring (QCM-D) measurements showed the adsorption of silica in dependence on the presence of phosphate in the silicate solution and on the preceding chemical modification of the S-layer. An increased amount of precipitated silica could be observed when K2HPO4/KH2PO4 was present in the solution (pH 7.2). Further on, independent of the presence of phosphate the silica deposition was higher on S-layer lattices upon activation of their carboxyl groups with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) compared to native S-layers or EDC treated S-layers when the activated carboxyl groups were blocked with ethylene diamine (EDA). Fourier transform infrared attenuated total reflectance (FTIR-ATR) spectroscopy revealed the formation of an amorphous silica gel (SiO2)x.yH2O on the S-layer. The silica surface concentrations on the S-layer was 4 x 10(-9) to 2 x 10(-8) mol cm(-2) depending on the modification of the protein layer and corresponded to 4-21 monolayers of SiO2.

Evidence for heterodimers of 2,4,5-trichlorophenol on planar lipid layers. A FTIR-ATR investigation.

Trichlorophenols are weak acids of high hydrophobicity and are able to transport protons across the mitochondrial membrane. Thus the proton motive force is dissipated and the ATP production decreased. In situ Fourier Transform Infrared-Attenuated Total Reflection (FTIR-ATR) experiments with 2,4,5-trichlorophenol (TCP) adsorbed to model membranes resulted in good evidence for the formation of the TCP-heterodimer. Two surfaces were examined: a dipalmitoyl phosphatidic acid (DPPA) monolayer and a planar DPPA/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. TCP was adsorbed from 1 to 3 mM solutions at pH 6.0 to the lipid layers leading to surface layers at the water/lipid interface. Difference spectra showed an effect on DPPA acyl chains even when it was covered with POPC. Time-resolved measurements revealed two distinct adsorption processes, which were assigned to TCP and its deprotonated anion (phenoxide), respectively. For DPPA/POPC bilayers, the adsorption of TCP was faster than that of its phenoxide, whereas adsorption of both species to DPPA monolayers proceeded with similar velocity. In both cases, phenoxide formation at the membrane was found to be delayed with respect to phenol adsorption. Phenoxide and phenol were retained after replacing the TCP solution with buffer. For the retained species, we estimated a phenol/phenoxide molar ratio of 1 at pH 6.0 (pKa=6.94 for TCP), demonstrating strong evidence for heterodimer formation.

Electric field-induced changes in lipids investigated by modulated excitation FTIR spectroscopy.

The effect of electric fields on dry oriented multibilayers of dimyristoylphosphatidylcholine (DMPC) was investigated by transmission Fourier transform infrared electric field modulated excitation (E-ME) spectroscopy. A periodic rectangular electric potential (0-150 V, 1.25 Hz, 28.4 degrees C +/- 0.2 degrees C) was applied across the sample. To discriminate electric field-induced effects from possible temperature-induced effects resulting from a current flow (<1 pA) across the sample, corresponding temperature-modulated excitation (T-ME) measurements within the temperature uncertainty limits of +/-0.2 degrees C at 28.4 degrees C were performed. T-ME induced reversible gauche defects in the hydrocarbon chains, whereas E-ME resulted in reversible compression of dry DMPC bilayers. Periodic variation of the tilt angle of the hydrocarbon chains is suggested. The degree of absorbance modulation in the CH-stretching region was found to be in the order of 1:700, corresponding to a variation of the bilayer thickness of Deltaz = 0.0054 nm. Using a series connection of capacitors as equivalent circuit of the cell resulted in E = (1.2 +/- 0.7) x 10(7) V/m for the electric field in DMPC. Young's elasticity modulus of DMPC could be calculated to be E( perpendicular ) = 2.2 x 10(6) Pa +/- 1.8 x 10(6) Pa, which is in good agreement with published data obtained by electric field-dependent capacitance measurements.