Effects of flowable liners on the shrinkage vectors of bulk-fill composites.

OBJECTIVES: This investigation evaluated the effect of flowable liners beneath a composite restoration applied via different methods on the pattern of shrinkage vectors. METHODS: Forty molars were divided into five groups (n = 8), and cylindrical cavities were prepared and bonded with a self-etch adhesive (AdheSe). Tetric EvoCeram Bulk Fill (TBF) was used as the filling material in all cavities. The flowable liners Tetric EvoFlow Bulk Fill (TEF) and SDR were used to line the cavity floor. In gp1-TBF, the flowable composite was not used. TEF was applied in a thin layer in gp2-fl/TEF + TBF and gp3-fl/TEF + TBFincremental. Two flowable composites with a layer thickness of 2 mm were compared in gp4-fl/TEF + TBF and gp5-fl/SDR + TBF. TEF and SDR were mixed with radiolucent glass beads, while air bubbles inherently present in TBF served as markers. Each material application was scanned twice by micro-computed tomography before and after light curing. Scans were subjected to image segmentation for calculation of the shrinkage vectors. RESULTS: The absence of a flowable liner resulted in the greatest shrinkage vectors. A thin flowable liner (gp2-fl/TEF + TBFbulk) resulted in larger overall shrinkage vectors for the whole restoration than a thick flowable liner (gp4-fl/TEF + TBF). A thin flowable liner and incremental application (gp3-fl/TEF + TBFincremental) yielded the smallest shrinkage vectors. SDR yielded slightly smaller shrinkage vectors for the whole restoration than that observed in gp4-fl/TEF + TBF. CONCLUSIONS: Thick flowable liner layers had a more pronounced stress-relieving effect than thin layers regardless of the flowable liner type. CLINICAL RELEVANCE: It is recommended to apply a flowable liner (thin or thick) beneath bulk-fill composites, preferably incrementally.

IgE and allergen-specific immunotherapy-induced IgG4 recognize similar epitopes of Bet v 1, the major allergen of birch pollen.

BACKGROUND: Allergen-specific immunotherapy (AIT) with birch pollen generates Bet v 1-specific immunoglobulin (Ig)G4 which blocks IgE-mediated hypersensitivity mechanisms. Whether IgG4 specific for Bet v 1a competes with IgE for identical epitopes or whether novel epitope specificities of IgG4 antibodies are developed is under debate. OBJECTIVE: We sought to analyze the epitope specificities of IgE and IgG4 antibodies from sera of patients who received AIT. METHODS: 15 sera of patients (13/15 received AIT) with Bet v 1a-specific IgE and IgG4 were analyzed. The structural arrangements of recombinant (r)Bet v 1a and rBet v 1a_11x , modified in five potential epitopes, were analyzed by circular dichroism and nuclear magnetic resonance spectroscopy. IgE binding to Bet v 1 was assessed by ELISA and mediator release assays. Competitive binding of monoclonal antibodies specific for Bet v 1a and serum IgE/IgG4 to rBet v 1a and serum antibody binding to a non-allergenic Bet v 1-type model protein presenting an individual epitope for IgE was analyzed in ELISA and western blot. RESULTS: rBet v 1a_11x had a Bet v 1a - similar secondary and tertiary structure. Monomeric dispersion of rBet v 1a_11x was concentration and buffer-dependent. Up to 1500-fold increase in the EC50 for IgE-mediated mediator release induced by rBet v 1a_11x was determined. The reduction of IgE and IgG4 binding to rBet v 1a_11x was comparable in 67% (10/15) of sera. Bet v 1a-specific monoclonal antibodies inhibited binding of serum IgE and IgG4 to 66.1% and 64.9%, respectively. Serum IgE and IgG4 bound specifically to an individual epitope presented by our model protein in 33% (5/15) of sera. CONCLUSION AND CLINICAL RELEVANCE: Patients receiving AIT develop Bet v 1a-specific IgG4 which competes with IgE for partly identical or largely overlapping epitopes. The similarities of epitopes for IgE and IgG4 might stimulate the development of epitope-specific diagnostics and therapeutics.

The conformational IgE epitope profile of soya bean allergen Gly m 4.

BACKGROUND: Birch pollen-related soya allergy is mediated by Gly m 4. Conformational IgE epitopes of Gly m 4 are unknown. OBJECTIVE: To identify the IgE epitope profile of Gly m 4 in subjects with birch pollen-related soya allergy utilizing an epitope library presented by Gly m 4-type model proteins. METHODS: Sera from patients with (n = 26) and without (n = 19) allergy to soya as determined by oral provocation tests were studied. Specific IgE (Bet v 1/Gly m 4) was determined by ImmunoCAP. A library of 59 non-allergenic Gly m 4-type model proteins harbouring individual and multiple putative epitopes for IgE was tested in IgE binding assays. Primary, secondary and tertiary protein structures were assessed by mass spectrometry, circular dichroism and nuclear magnetic resonance spectroscopy. RESULTS: All subjects were sensitized to Gly m 4 and Bet v 1. Allergen-specific serum IgE levels ranged from 0.94 to > 100 kUA /L. The avidities of serum IgE were 5.06 ng (allergic) and 1.8 ng (tolerant) as determined by EC50 for IgE binding to Gly m 4. 96% (46/48) of the protein variants bound IgE. Model proteins had Gly m 4-type conformation and individual IgE binding clustered in six major surface areas. Gly m 4-specific IgE binding could be inhibited to up to 80% by model proteins harbouring individual IgE binding sites in an epitope-wise equimolar fashion. Receiver operating curve analysis revealed an area under fitted curve of up to 0.88 for model proteins and 0.66 for Gly m 4. CONCLUSION AND CLINICAL RELEVANCE: Serum levels and avidity of Gly m 4-specific IgE do not correlate with clinical reactivity to soya. Six IgE-binding areas, represented by 23 amino acids, account for more than 80% of total IgE binding capacity of Gly m 4. Model proteins may be used for epitope-resolved diagnosis to differentiate birch-soya allergy from clinical tolerance.

Identification of Bacillus anthracis via Raman spectroscopy and chemometric approaches.

Raman micro-spectroscopy was applied to compile a large-scale database of Raman spectra of single Bacillus endospores and to calculate classification functions, which were trained to discriminate between endospores of 66 strains from 13 Bacillus and Bacillus-related species including B. anthracis. The developed two-stage classification system comprising two support vector machines and one linear discriminant analysis classifier was then challenged by a test set of 27 samples to simulate the case of a real-world-scenario, when "unknown samples" are to be identified. In the end, all 27 test set samples including six B. anthracis strains were identified correctly. The samples thereby covered a diverse selection of species within the phylogenetically broad Bacillus genus and also included strains, which were not incorporated in the database before. All of them were correctly identified on the species level with accuracies between 88 and 100%. The sample analysis itself requires no biomass enrichment step prior to the analysis and qualifies the presented Raman spectroscopic approach to be a rapid analysis system in term of Bacillus endospore typing.

Raman spectroscopy-compatible inactivation method for pathogenic endospores.

Micro-Raman spectroscopy is a fast and sensitive tool for the detection, classification, and identification of biological organisms. The vibrational spectrum inherently serves as a fingerprint of the biochemical composition of each bacterium and thus makes identification at the species level, or even the subspecies level, possible. Therefore, microorganisms in areas susceptible to bacterial contamination, e.g., clinical environments or food-processing technology, can be sensed. Within the scope of point-of-care-testing also, detection of intentionally released biosafety level 3 (BSL-3) agents, such as Bacillus anthracis endospores, or their products is attainable. However, no Raman spectroscopy-compatible inactivation method for the notoriously resistant Bacillus endospores has been elaborated so far. In this work we present an inactivation protocol for endospores that permits, on the one hand, sufficient microbial inactivation and, on the other hand, the recording of Raman spectroscopic signatures of single endospores, making species-specific identification by means of highly sophisticated chemometrical methods possible. Several physical and chemical inactivation methods were assessed, and eventually treatment with 20% formaldehyde proved to be superior to the other methods in terms of sporicidal capacity and information conservation in the Raman spectra. The latter fact has been verified by successfully using self-learning machines (such as support vector machines or artificial neural networks) to identify inactivated B. anthracis-related endospores with adequate accuracies within the range of the limited model database employed.

Vibrational spectroscopy--a powerful tool for the rapid identification of microbial cells at the single-cell level.

Rapid microbial detection and identification with a high grade of sensitivity and selectivity is a great and challenging issue in many fields, primarily in clinical diagnosis, pharmaceutical, or food processing technology. The tedious and time-consuming processes of current microbiological approaches call for faster ideally on-line identification techniques. The vibrational spectroscopic techniques IR absorption and Raman spectroscopy are noninvasive methods yielding molecular fingerprint information; thus, allowing for a fast and reliable analysis of complex biological systems such as bacterial or yeast cells. In this short review, we discuss recent vibrational spectroscopic advances in microbial identification of yeast and bacterial cells for bulk environment and single-cell analysis. IR absorption spectroscopy enables a bulk analysis whereas micro-Raman-spectroscopy with excitation in the near infrared or visible range has the potential for the analysis of single bacterial and yeast cells. The inherently weak Raman signal can be increased up to several orders of magnitude by applying Raman signal enhancement methods such as UV-resonance Raman spectroscopy with excitation in the deep UV region, surface enhanced Raman scattering, or tip-enhanced Raman scattering.

Design and characterization of a soluble fragment of the extracellular ligand-binding domain of the peptide hormone receptor guanylyl cyclase-C.

The intestinal guanylyl cyclase-C (GC-C) was originally identified as an Escherichia coli heat-stable enterotoxin (STa) receptor. STa stimulates GC-C to much higher activity than the endogenous ligands guanylin and uroguanylin, causing severe diarrhea. To investigate the interactions of the endogenous and bacterial ligands with GC-C, we designed and characterized a soluble and properly folded fragment of the extracellular ligand-binding domain of GC-C. The membrane-bound guanylyl cyclases exhibit a single transmembrane spanning helix and a globularly folded extracellular ligand-binding domain that comprises about 410 of 1050 residues. Based on the crystal structure of the dimerized-binding domain of the guanylyl cyclase-coupled atrial natriuretic peptide receptor and a secondary structure-guided sequence alignment, we generated a model of the extracellular domain of GC-C comprised of two subdomains. Mapping of mutational and cross-link data onto this structural model restricts the ligand-binding region to the membrane proximal subdomain. We thus designed miniGC-C, a 197 amino acid fragment that mimics the ligand-binding membrane proximal subdomain. Cloning, expression and spectroscopic studies reveal miniGC-C to be a soluble and properly folded protein with a distinct secondary and tertiary structure. MiniGC-C binds STa with nanomolar affinity.

Minimal invasive gender determination of birds by means of UV-resonance Raman spectroscopy.

The identification of avian gender is important for prosperous breeding of birds. Since birds do not possess external genital organs, endoscopic investigations, blood analysis, and molecular biological methods are applied to determine the gender in monomorphic species. However, anesthesia and blood sampling impose stress on the examined bird and should be avoided in terms of animal protection. Here we report on the application of UV-resonance Raman spectroscopy as a minimal invasive method for gender determination of birds via an evaluation of feather pulp samples. Sample preparation for this investigation method is simple and facilitates a quick and easy analysis. The UV-resonance Raman spectra of the feather pulp sample extracts are dominated by DNA and protein signals. The different DNA content in male and female chicken allows for gender differentiation via its characteristic Raman fingerprint. The classification either to male or female chicken is ideally accomplished by support vector machines due to the fact that no unknown classes are involved. Recognition rates of about 95% were compared to less effective results of the unsupervised hierarchical cluster analysis. Within the scope of our investigations, principal component analysis was also applied to determine the important spectral regions for the classification of chicken's feather pulp samples.

UV-resonance Raman spectroscopic study of human plasma of healthy donors and patients with thrombotic microangiopathy.

Various diseases shift the composition of human plasma; hence, the relative quantification of plasma constituents offers the opportunity to use the dynamic and complex composition of plasma to gain information on novel diagnostic and prognostic factors. Since plasma contains, besides water, mostly proteins, UV-resonance Raman spectroscopy (UVRR) seems to be a suitable method for investigating plasma. With this method the signals of aromatic amino acids and proteins are selectively enhanced. In this study an UV-resonance Raman approach was used for the investigation of human plasma of healthy volunteers and patients with thrombotic microangiopathy. For comparison, selected plasma components were analyzed for a more detailed characterization of cryoprecipitates from human plasma.

Classification of lactic acid bacteria with UV-resonance Raman spectroscopy.

UV-resonance Raman spectroscopy is applied as a method for the identification of lactic acid bacteria from yogurt. Eight different strains of bacteria from Lactobacillus acidophilus, L. delbrueckii ssp. bulgaricus, and Streptococcus thermophilus were investigated. At an excitation wavelength of 244 nm signals from nucleic acids and proteins are selectively enhanced. Classification was accomplished using different chemometric methods. In a first attempt, the unsupervised methods hierarchical cluster analysis and principal component analysis were applied to investigate natural grouping in the data. In a second step the spectra were analyzed using several supervised methods: K-nearest neighbor classifier, nearest mean classifier, linear discriminant analysis, and support vector machines.

Identification of single eukaryotic cells with micro-Raman spectroscopy.

For a fast identification of eukaryotic cells such as yeast species without a cultivation step it should be possible to perform the investigation on only one single cell. Since yeasts as eukaryotes are heterogeneous and their Raman spectra are therefore dependent on the measuring position, one Raman spectra is not representative of the whole cell. In this contribution we demonstrate the application of average Raman spectra of a line scan over single yeast cells. These average spectra are used for classification with the help of a support vector machine.

Micro-Raman spectroscopic identification of bacterial cells of the genus Staphylococcus and dependence on their cultivation conditions.

Microbial contamination is not only a medical problem, but also plays a large role in pharmaceutical clean room production and food processing technology. Therefore many techniques were developed to achieve differentiation and identification of microorganisms. Among these methods vibrational spectroscopic techniques (IR, Raman and SERS) are useful tools because of their rapidity and sensitivity. Recently we have shown that micro-Raman spectroscopy in combination with a support vector machine is an extremely capable approach for a fast and reliable, non-destructive online identification of single bacteria belonging to different genera. In order to simulate different environmental conditions we analyzed in this contribution different Staphylococcus strains with varying cultivation conditions in order to evaluate our method with a reliable dataset. First, micro-Raman spectra of the bulk material and single bacterial cells that were grown under the same conditions were recorded and used separately for a distinct chemotaxonomic classification of the strains. Furthermore Raman spectra were recorded from single bacterial cells that were cultured under various conditions to study the influence of cultivation on the discrimination ability. This dataset was analyzed both with a hierarchical cluster analysis (HCA) and a support vector machine (SVM).

Quality control of Harpagophytum procumbens and its related phytopharmaceutical products by means of NIR-FT-Raman spectroscopy.

NIR-FT-Raman spectroscopy was used for identification and quantification of harpagoside in secondary roots of Harpagophytum procumbens as well as in related phytopharmaceutical products, e.g., ethanolic extracts and tablets. Applied Raman mappings reveal the spatial distribution of this valuable iridoid glycoside within the different samples. The same technique can be used for quality control purposes beginning from the plant to its final products. Based on the obtained spectral data and reference HPLC values of harpagoside, a reliable multivariate calibration model was developed.

On the way to a quality control of the essential oil of fennel by means of Raman spectroscopy.

Essential oils are one of the most valuable natural products. The price of special essential oils that can be purchased on the market strongly depends on the quality of the product. The quality, which depends on the quantitative and qualitative variation of different monoterpenes, varies with respect of the origin and the harvesting period. This contribution reports on a Raman spectroscopic study on the essential oil occurring in fennel. Cross-sections of fennel seed were investigated by use of Raman spectroscopy and Raman mapping to localize the essential oil and to analyze its chemical composition directly in the plant. Furthermore the practicability of a home-built mobile transportable Raman spectrometer to perform on-site measurements was successfully tested.

The application of a SERS fiber probe for the investigation of sensitive biological samples.

The applicability of an etched and silver or gold coated SERS fiber probe in combination with a commercially available laboratory micro-Raman setup or a home built mobile micro-Raman setup to perform on-site field measurements was evaluated and successfully tested on different biological samples. The SERS fiber probe allows one to perform measurements with high spatial resolution. Simultaneously, the laser power used for Raman spectroscopy on biological samples as compared with conventional Raman experiments can be reduced by more than two orders of magnitude. This experimental arrangement was tested to investigate sensitive biological samples like mint plants (Bergamot mint, spear mint) and citrus fruits (kumquat). Furthermore, traces of fungicides on wine leaves were detected by means of such a SERS fiber probe setup.

Identification of biotic and abiotic particles by using a combination of optical tweezers and in situ Raman spectroscopy.

A highly versatile setup, which introduces an optical gradient trap into a Raman spectrometer, is presented. The particular configuration, which consists of two lasers, makes trapping independent from the Raman excitation laser and allows a separate adjustment of the trapping and excitation wavelengths. Thus, the excitation wavelength can be chosen according to the needs of the application. We describe the successful application of an optical gradient trap on transparent as well as on reflective, metal-coated microparticles. Raman spectra were recorded from optically trapped polystyrene beads and from single biological cells (e.g., erythrocytes, yeast cells). Also, metal-coated microparticles were trapped and used as surface enhanced Raman spectroscopy (SERS) substrates for tests on yeast cells. Furthermore, the optical gradient trap was combined with a SERS fiber probe. Raman spectra were recorded from trapped red blood cells using the SERS fiber probe for excitation.

In situ Raman investigation of single lipid droplets in the water-conducting xylem of four woody plant species.

A micro-Raman spectroscopy approach was used for the direct in situ characterization of lipid bodies in the water-conducting branch xylem of an African resurrection plant and three deciduous European tree species. Because of average diameters of at least 1 microm, the lipid bodies of all investigated species proved to be easily accessible by this technique. All vesicle-forming xylem lipids were identified as fatty acid esters, which may correspond to phospholipids. Whereas in the resurrection plant saturated lipids were dominant, the lipid bodies of the European trees consisted of highly unsaturated fatty acids. A comparison of the spectra of lipid droplets of lime obtained in situ and from isolated xylem sap revealed slightly different signatures. This finding suggests that micro-Raman spectroscopy may be used to detect modifications of the chemical composition of biological substances as a result of the extraction mode.