Syk and Src family kinases regulate C-type lectin receptor 2 (CLEC-2)-mediated clustering of podoplanin and platelet adhesion to lymphatic endothelial cells

The interaction of C-type lectin receptor 2 (CLEC-2) on platelets with Podoplanin on lymphatic endothelial cells initiates platelet signaling events that are necessary for prevention of blood-lymph mixing during development. In the present study, we show that CLEC-2 signaling via Src family and Syk tyrosine kinases promotes platelet adhesion to primary mouse lymphatic endothelial cells at low shear. Using supported lipid bilayers containing mobile Podoplanin, we further show that activation of Src and Syk in platelets promotes clustering of CLEC-2 and Podoplanin. Clusters of CLEC-2-bound Podoplanin migrate rapidly to the center of the platelet to form a single structure. Fluorescence lifetime imaging demonstrates that molecules within these clusters are within 10 nm of one another and that the clusters are disrupted by inhibition of Src and Syk family kinases. CLEC-2 clusters are also seen in platelets adhered to immobilized Podoplanin using direct stochastic optical reconstruction microscopy. These findings provide mechanistic insight by which CLEC-2 signaling promotes adhesion to Podoplanin and regulation of Podoplanin signaling, thereby contributing to lymphatic vasculature development.

Surface-enhanced resonance Raman scattering: Single-molecule detection in a Langmuir-Blodgett monolayer

Surface-enhanced resonance Raman scattering (SERRS) is used for single-molecule detection from spatially resolved 1-mum(2) sections of a Langmuir-Blodgett (LB) monolayer deposited onto a Ag film. The target molecule, his (benzimidazo) thioperylene (BZP), is dispersed in an arachidic acid monomolecular layer containing one BZP molecule per mum(2) which is also the probing area of the Raman microscope. For concentrated samples (attomole quantities in the field of view), average SERRS, surface-enhanced fluorescence (SEF), and Raman imaging, including line mapping and global images at different temperatures, were recorded. Single-molecule SERRS spectra, obtained using an LB monolayer, present changes in bandwidth and relative intensities, highlighting the properties of single-molecule SERRS that are lost in average SERRS measurements of mixed LB monolayers obtained at the same temperatures. Also, the dilute system phenomenon of blinking is discussed with regard to results obtained from LB monolayers. The dilution process used in the single-molecule LB SERRS work is independently supported by fluorescence results obtained from very dilute solutions with monomer concentrations down to 10(-12) M.

Fibroblast functionality on novel Ti-30Ta nanotube array

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Molecular adaptability of nucleoside diphosphate kinase b from trypanosomatid parasites: stability, oligomerization and structural determinants of nucleotide binding

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Application of dissociation curve analysis to radiation hybrid panel marker scoring: generation of a map of river buffalo (B-bubalis) chromosome 20

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Traditional and novel methods for occlusal caries detection: Performance on primary teeth

This study aimed to assess the performance of International Caries Detection and Assessment System (ICDAS), radiographic examination, and fluorescence-based methods for detecting occlusal caries in primary teeth. One occlusal site on each of 79 primary molars was assessed twice by two examiners using ICDAS, bitewing radiography (BW), DIAGNOdent 2095 (LF), DIAGNOdent 2190 (LFpen), and VistaProof fluorescence camera (FC). The teeth were histologically prepared and assessed for caries extent. Optimal cutoff limits were calculated for LF, LFpen, and FC. At the D 1 threshold (enamel and dentin lesions), ICDAS and FC presented higher sensitivity values (0.75 and 0.73, respectively), while BW showed higher specificity (1.00). At the D 2 threshold (inner enamel and dentin lesions), ICDAS presented higher sensitivity (0.83) and statistically significantly lower specificity (0.70). At the D 3 threshold (dentin lesions), LFpen and FC showed higher sensitivity (1.00 and 0.91, respectively), while higher specificity was presented by FC (0.95), ICDAS (0.94), BW (0.94), and LF (0.92). The area under the receiver operating characteristic (ROC) curve (Az) varied from 0.780 (BW) to 0.941 (LF). Spearman correlation coefficients with histology were 0.72 (ICDAS), 0.64 (BW), 0.71 (LF), 0.65 (LFpen), and 0.74 (FC). Inter- and intraexaminer intraclass correlation values varied from 0.772 to 0.963 and unweighted kappa values ranged from 0.462 to 0.750. In conclusion, ICDAS and FC exhibited better accuracy in detecting enamel and dentin caries lesions, whereas ICDAS, LF, LFpen, and FC were more appropriate for detecting dentin lesions on occlusal surfaces in primary teeth, with no statistically significant difference among them. All methods presented good to excellent reproducibility. © 2012 Springer-Verlag London Ltd.

Study on the Curcumin dynamics and distribution through living biofilms

Human oral cavity is colonized by a wide range of microorganisms, often organized in biofilms. These biofilms are responsible for the pathogenesis of caries and most periodontal diseases. A possible alternative to reduce biofilms is the photodynamic inactivation (PDI). The success of the PDI depends on different factors. The time required by the PS to remain in contact with the target cells prior to illumination is determinant for the technique's efficacy. This study aimed to assess the interaction between the PS and the biofilm prior to the PDI. We used confocal microscopy and FLIM to evaluate the interaction between the PS and the biofilm's microorganism during the pre-irradiation time (PIT). The study of this dynamics can lead to the understanding of why only some PSs are effective and why is necessary a long PIT for some microorganisms. Our results showed that are differences for each PIT. These differences can be the determinate for the efficacy of the PDI. We observed that the microorganism needs time to concentrate and/or transport the PS within the biofilm. We presented preliminary results for biofilms of Candida albicans and Streptococcus mutans in the presence of Curcumin and compared it with the literature. We observed that the effectiveness of the PDI might be directly correlated to the position of the PS with the biofilm. Further analyses will be conducted in order to confirm the potential of FLIM to assess the PS dynamics within the biofilms. © 2013 SPIE.

Interaction between mesenchymal stem cells and Ti-30Ta alloy after surface treatment

In this study, in vitro cytocompatibility was investigated in the Ti-30Ta alloy after two kinds of surfaces treatments: alkaline and biomimetic treatment. Each condition was evaluated by scanning electron microscopy/energy-dispersive X-ray spectroscopy. Cellular adhesion, viability, protein expression, morphology, and differentiation were evaluated with Bone marrow stromal cells (MSCs) to investigate the short and long-term cellular response by fluorescence microscope imaging and colorimetric assays techniques. Two treatments exhibited similar results with respect to total protein content and enzyme activity as compared with alloy without treatment. However, it was observed improved of the biomineralization, bone matrix formation, enzyme activity, and MSCs functionality after biomimetic treatment. These results indicate that the biomimetic surface treatment has a high potential for enhanced osseointegration. © 2013 Wiley Periodicals, Inc.

Caracterização da superfície da liga Ti-30Ta após crescimento de nanotubos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Detecção e avaliação da atividade de lesões de cárie primária e secundária em superfícies oclusais: Estudos in vitro e in vivo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Detecção de lesões de cárie oclusal por meio de métodos baseados na medição da fluorescência induzida pela luz

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Green tea extract and its major constituent epigallocatechin-3-gallate inhibit growth and halitosis-related properties of Solobacterium moorei

Background: Solobacterium moorei is a volatile sulfide compound (VSC)-producing Gram-positive anaerobic bacterium that has been associated with halitosis. The aim of this study was to investigate the effects of green tea extract and its major constituent epigallocatechin-3-gallate (EGCG) on growth and severalhalitosis-related properties of S. moorei.Methods: A microplate dilution assay was used to determine the antibacterial activity of green tea extract and EGCG against S. moorei. Their effects on bacterial cell membrane integrity were investigated by transmission electron microscopy and a fluorescence-based permeability assay. Biofilm formation was quantified by crystal violet staining. Adhesion of FITC-labeled S. moorei to oral epithelial cells was monitored by fluorometry. The modulation of beta-galactosidase gene expression in S. moorei was evaluated by quantitative RT-PCR.Results: The green tea extract as well as EGCG inhibited the growth of S. moorei, with MIC values of 500 and 250 mu g/ml, respectively. Transmission electron microscopy analysis and a permeabilization assay brought evidence that the bacterial cell membrane was the target of green tea polyphenols. Regarding the effects of green tea polyphenols on the S. moorei colonization properties, it was found that biofilm formation on EGCG-treated surfaces was significantly affected, and that green tea extract and EGCG can cause the eradication of pre-formed S. moorei biofilms. Moreover, both the green tea extract and EGCG were found to reduce the adherence of S. moorei to oral epithelial cells. The beta-galactosidase activity of S. moorei, which plays a key role in VSC production, was dose-dependently inhibited by green tea polyphenols. In addition, EGCG at 1/2 MIC significantly decreased the beta-galactosidase gene expression.Conclusion: Our study brought evidence to support that green tea polyphenols possess a number of properties that may contribute to reduce S. moorei-related halitosis. Therefore, these natural compounds may be of interest to be used to supplement oral healthcare products.

Expression of NADPH Oxidase by Trophoblast Cells: Potential Implications for the Postimplanting Mouse Embryo

Cytochemical localization of hydrogen peroxide-generating sites suggests NADPH (nicotinamide adenine dinucleotide 3-phosphate [ reduced form]) oxidase expression at the maternal-fetal interface. To explore this possibility, we have characterized the expression and activity of the NADPH oxidase complex in trophoblast cells during the postimplantation period. Implantation sites and ectoplacental cones (EPCs) from 7.5-gestational day embryos from CD1 mice were used as a source for expression analyses of NADPH oxidase catalytic and regulatory subunits. EPCs grown in primary culture were used to investigate the production of superoxide anion through dihydroxyethidium oxidation in confocal microscopy and immunohistochemical assays. NADPH subunits Cybb (gp91phox), Cyba (p22phox), Ncf4 (p40phox), Ncf1 (p47phox), Ncf2 (p67phox), and Rac1 were expressed by trophoblast cells. The fundamental subunits of membrane CYBB and cytosolic NCF2 were markedly upregulated after phorbol-12-myristate-13-acetate (PMA) treatment, as detected by quantitative real-time PCR, Western blotting, and immunohistochemistry. Fluorescence microscopy imaging showed colocalization of cytosolic and plasma membrane NADPH oxidase subunits mainly after PMA treatment, suggesting assembly of the complex after enzyme activation. Cultured EPCs produced superoxide in a NADPH-dependent manner, associating the NADPH oxidase-mediated superoxide production with postimplantation trophoblast physiology. NADPH-oxidase cDNA subunit sequencing showed a high degree of homology between the trophoblast and neutrophil isoforms of the oxidase, emphasizing a putative role for reactive oxygen species production in phagocytic activity and innate immune responses.

Anwendungen von Rezeptoren auf Triphenylenketal-Basis

Im Rahmen dieser Arbeit wurde die Bindung von Koffein und verwandten Oxopurinen in C¬3-symmetrischen Rezeptoren auf der Basis von Triphenylenketalen untersucht. Dabei stand vor allem die Evaluierung für eine spätere Anwendung im Vordergrund. Für die Anwendung als Chemosensor wurden mehrere optische Verfahren getestet. Die Verwendung von UV/Vis-Spektroskopie gelingt nur unter Einsatz eines elektronenarmen Konkurrenzgastes, welcher durch das stärker bindende Koffein unter Entfärbung verdrängt wird. Obwohl dieser Effekt sogar mit bloßem Auge zu erkennen ist und somit eine einfache Untersuchung ermöglichen würde, machen die besondere Reaktivität des Konkurrenzgastes und dessen geringe Affinität zum Rezeptor eine weitere Anwendung als Chemosensor für Koffein unwahrscheinlich. Den entscheidenden Durchbruch lieferte der Wechsel auf Fluoreszenzspektroskopie. Die Bindung von Gästen lässt sich mit dieser Methode direkt beobachten und für quantitative Studien nutzen. Die Signalzunahme bei Zugabe von Koffein liegt bei maximal 30%. Durch Verwendung eines vom Koffein abgeleiteten Konkurrenzgastes können weitere Verbesserungen erzielt werden. So konnte eine maximale Signaldynamik von fast 400% erzielt werden. Durch die Entwicklung eines geeigneten Probenvorbereitungsprotokolls war es möglich, mit dem fluoreszenzbasierten System einen Nachweis von Koffein an kommerziell verfügbaren Getränkeproben durchzuführen. Die Ergebnisse waren in guter Übereinstimmung mit HPLC-Kontrollexperimenten. Die Eignung von Rezeptoren auf Triphenylenketalbasis für die enantiofaciale Differenzierung an Heteroaromaten wurde durch Untersuchung verschiedener Wirt-Gast-Komplexe mittels CD-Spektroskopie und Tieftemperatur-NMR systematisch demonstriert. Rezeptoren mit Menthyl-Substituenten liefern laut NMR die stärkste Seitendifferenzierung. Anhand des CD wird ein vollständiges und schlüssiges Bild über den Zusammenhang zwischen dem Raumbedarf am Gast, der Ausrichtung der chiralen Gruppen am Wirt und dem erhaltenen CD hergestellt. Durch umfangreiche molekulardynamische Simulationen und nachfolgende semiempirische Berechnungen wurden Referenzspektren berechnet, welche die Zuordnung der Stereochemie anhand des CD eindeutig belegen. Die Ergebnisse sind zudem in guter Übereinstimmung mit den Ergebnissen aus röntgenkristallographischen Untersuchungen. (Diese Methode ließ sich erfolgreich auf die helicale Faltung von Alkanen in Kapseln von Rebek, jr. umsetzen.) Obwohl die Energieunterschiede zwischen den diastereomeren Komplexen klein sind, konnte anhand der CD-Spektroskopie somit erstmalig die enantiofaciale Differenzierung an einem heterocyclischen System bei Raumtemperatur beobachtet werden. Die beste enantiofaciale Differenzierung erzielen die Menthyl-abgeleiteten Rezeptoren. Diese sind hinsichtlich einer möglichen Anwendung als chirales „Auxiliar“ ungeeignet, da sie mit den sperrigen Cyclohexylgruppen auch den Raum oberhalb des gebundenen Gastes blockieren. Daher wird für die weitere Entwicklung auf die praktische Einführung chiraler Information in Form des Isocyanats verzichten werden müssen. Stattdessen zielen aktuelle Bemühungen auf den Aufbau chiraler Rückgrate, welche den Raum in der unteren Peripherie des Gastes beeinflussen.

Functional dna nanostructures for in vitro biosensing in live cells

DNA is a fascinating biomolecule that is well known for its genetic role in living systems. The emerging area of DNA nanotechnology provides an alternative view that exploits unparallel self-assembly ability of DNA molecules for material use of DNA. Although many reports exist on the results of DNA self-assembling systems, still few of them focus on the in vitro study about the function of such DNA nanostructures in live cells. Due to this, there are still a limited research about the in vitro functionality of such designs. To address an aspect of this issue, we have designed, synthesized and characterized two multifunctional fluorescencent nanobiosensors by DNA self-assembling. Each structure was designed and implemented to be introduced in live cells in order to give information on their functioning in real-time. Computational tools were used in order to design a graphic model of two new DNA motifs and also to obtain the specific sequences to all the ssDNA molecules. By thermal self-assembly techniques we have successfully synthesized the structure and corroborate their formation by the PAGE technique. In addition, we have established the conditions to characterize their structural conformation change when they perform their sensor response. The sensing behavior was also accomplished by fluorescence spectroscopy techniques; FRET evaluation and fluorescence microscopy imaging. Providing the evidence about their adequate sensing performance outside and inside the cells detected in real-time. In a preliminary evaluation we have tried to show the in vitro functionality of our structures in different cancer cell lines with the ability to perform local sensing responses. Our findings suggest that DNA sensor nanostructures could serve as a platform to exploit further therapeutic achievements in live cells.