The stretch-activated channel blocker Gd(3+) reduces palytoxin toxicity in primary cultures of skeletal muscle cells.

Palytoxin (PLTX) is one of the most toxic seafood contaminants ever isolated. Reports of human food-borne poisoning ascribed to PLTX suggest skeletal muscle as a primary target site. Primary cultures of mouse skeletal muscle cells were used to study the relationship between Ca(2+) response triggered by PLTX and the development of myotoxic insult. Ca(2+) imaging experiments revealed that PLTX causes a transitory intracellular Ca(2+) response (transient phase) followed by a slower and more sustained Ca(2+) increase (long-lasting phase). The transient phase is due to Ca(2+) release from intracellular stores and entry through voltage-dependent channels and the Na(+)/Ca(2+) exchanger (reverse mode). The long-lasting phase is due to a massive and prolonged Ca(2+) influx from the extracellular compartment. Sulforhodamine B assay revealed that the long-lasting phase is the one responsible for the toxicity in skeletal muscle cells. Our data analyzed, for the first time, pathways of PLTX-induced Ca(2+) entry and their correlation with PLTX-induced toxicity in skeletal muscle cells. The cellular morphology changes induced by PLTX and the sensitivity to gadolinium suggest a role for stretch-activated channels.

Contraction stress, elastic modulus, and degree of conversion of three flowable composites.

The aim of this study was to measure the contraction stress of three flowable resin composites and to correlate the stress with the elastic modulus and the degree of conversion. One low-shrinkage (Venus Diamond Flow) and two conventional (Tetric EvoFlow and X-Flow) flowable composites were polymerized for 40s with a light-emitting diode (LED) curing unit. Contraction force was continuously recorded for 300s using a stress-analyser, and stress values were calculated at 40s and at 300s. The maximum stress rate was also calculated for each specimen. The elastic modulus of each composite was assayed using a biaxial flexural test, and degree of conversion was analysed with Raman spectroscopy. X-Flow exhibited higher stress values than the other tested materials. Venus Diamond Flow showed the lowest stress values at 40s and at 300s, and the lowest maximum stress rate. Stress values were correlated with elastic modulus but not with degree of conversion, which was comparable among all tested materials.

The effect of ageing on the elastic modulus and degree of conversion of two multistep adhesive systems.

During the curing reaction, the monomers of dentine bonding systems should cross-link sufficiently to strengthen an adhesive so that it is clinically reliable. This study evaluated how different storage conditions (air vs. water storage) affect the elastic modulus (E-modulus) and degree of conversion (DC) of a three-step etch-and-rinse adhesive and a two-step self-etch adhesive. The biaxial flexural test and Raman microscopy were performed on resin disks made from the bonding agents Adper Scotchbond Multi-Purpose (SBMP; 3M ESPE) and Clearfil Protect Bond (CPB; Kuraray). The measurements were repeated after storage in either air or water for 15 and 30 min and for 1, 24, and 72 h. At time 0, the E-modulus was not affected by the adhesive system, whilst the degree of cure of CPB was higher than that of SBMP. Air storage increased the E-modulus at each ageing interval. Storage in water increased the E-modulus until it reached a maximum at 24 h, after which it decreased significantly at 72 h. No linear correlation between the percentage DC and E-modulus of the two adhesives was found when stored in water. The results of this study indicate that the mechanical properties and polymerization kinetics of SBMP and CPB are affected by storage time and medium.

Degree of conversion and interfacial nanoleakage expression of three one-step self-etch adhesives.

Suboptimally polymerized monomers may be responsible for the reduced material properties of simplified adhesives and their inherent bonded interface instability. This study was performed to determine the degree of cure within the hybrid layers produced by three one-step self-etch adhesives in situ using Raman microspectroscopy and to investigate nanoleakage expression. Dentin disks were bonded with AdheSE One, Adper Prompt L-Pop, or iBond. Composite layers of 2-mm thickness were built up in bulk on the polymerized adhesive surfaces and then the adhesive-dentin interfaces were exposed to a micro-Raman beam. Adhesive penetration was calculated using the relative intensities of bands associated with mineral and adhesive, and the degree of conversion (DC) was evaluated. Interfacial nanoleakage expression was evaluated on the same specimens. The DC values for the tested adhesives were found to increase in the following order: AdheSE One (48 +/- 16%) < Adper Prompt L-Pop (83 +/- 2%) = iBond (90 +/- 6%; P < 0.05). AdheSE One showed greater nanoleakage expression than iBond or Adper Prompt L-Pop. Increased nanoleakage expression was associated with AdheSE One that showed the lowest DC. This suggests that a low DC may affect the quality and the long-term stability of the adhesive interface owing to the elution of unreacted monomers forming a porous and highly permeable hybrid layer.

Inhibition of adhesion, migration and of α5ß1 integrin in the HCT-116 colorectal cancer cells treated with the ruthenium drug NAMI-A.

NAMI-A, imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate, is a ruthenium-based drug characterised by the selective activity against tumour metastases. Previously we have shown the influence of the hepatic microenvironment to direct the arrest of the metastatic cells of colorectal cancer. Here we used the experimental model of HCT-116 colorectal cancer cells in vitro to explore whether the interference with α5ß1 integrin may mechanistically explain the anti-metastatic effect of NAMI-A. NAMI-A inhibits two important steps of the tumour metastatic progression of colorectal cancer, i.e. the adhesion and migration of the tumour cells on the extracellular matrix proteins. The fibronectin receptor α5ß1 integrin is likely involved in the anti-adhesive effects of NAMI-A on the HCT-116 colorectal cancer cells during their interaction with the extracellular matrix. Mechanistically, NAMI-A decreases the α5ß1 integrin expression, and reduces FAK (Focal Adhesion Kinase) auto-phosphorylation on Tyr397, an important signalling event, involved in α5ß1 integrin activation. These effects were validated by siRNA-induced knock down of the α5 integrin subunit and/or by the use of specific blocking mAbs against the active site of the integrin. Our results demonstrate the relevance of α5ß1 integrin for colorectal cancer. We also show that the anti-metastatic effect of NAMI-A depends on the modulation of this integrin. Thus, our data on NAMI-A support the new concept that metal-based drugs can inhibit tumour metastases through targeting of integrins and of other proteins which mediate tumour progression-related cell functions such as adhesion and migration.

The Cardiomyopathy Lamin A/C D192G Mutation Disrupts Whole-Cell Biomechanics in Cardiomyocytes as Measured by Atomic Force Microscopy Loading-Unloading Curve Analysis.

Atomic force microscopy (AFM) cell loading/unloading curves were used to provide comprehensive insights into biomechanical behavior of cardiomyocytes carrying the lamin A/C (LMNA) D192G mutation known to cause defective nuclear wall, myopathy and severe cardiomyopathy. Our results suggested that the LMNA D192G mutation increased maximum nuclear deformation load, nuclear stiffness and fragility as compared to controls. Furthermore, there seems to be a connection between this lamin nuclear mutation and cell adhesion behavior since LMNA D192G cardiomyocytes displayed loss of AFM probe-to-cell membrane adhesion. We believe that this loss of adhesion involves the cytoskeletal architecture since our microscopic analyses highlighted that mutant LMNA may also lead to a morphological alteration in the cytoskeleton. Furthermore, chemical disruption of the actin cytoskeleton by cytochalasin D in control cardiomyocytes mirrored the alterations in the mechanical properties seen in mutant cells, suggesting a defect in the connection between the nucleoskeleton, cytoskeleton and cell adhesion molecules in cells expressing the mutant protein. These data add to our understanding of potential mechanisms responsible for this fatal cardiomyopathy, and show that the biomechanical effects of mutant lamin extend beyond nuclear mechanics to include interference of whole-cell biomechanical properties.

A study on the cellular structure during stress solicitation induced by BioMEMS.

The investigation of single cells is a topic in continuous evolution. The complexity of the cellular matrix, the huge variety of cells, the interaction of one cell with the other are all factors that must be taken into consideration in the study of the cellular structure and mechanics. In this project, we developed different types of bioMEMS for cell's stretching, both transparent devices based on silicon nitride and non-transparent silicon based. While the use of silicon devices is limited to reflection microscopes, transparent bioMEMS can be used with transmission and reflection microscopes but can also be easily coupled with other tools such as patch clamp analyzers or atomic force microscope. This improvement will open brand new possibilities in the biological investigation field. We used these two BioMEMS to stretch a single cell in a controlled way and, as a first investigation, we focused on its morphology. We noticed that during a controlled stretch, cells react to the applied deformation. A hysteretic behavior on the ratio between area and perimeter has been highlighted.

Influence of different initiators on the degree of conversion of experimental adhesive blends in relation to their hydrophilicity and solvent content.

OBJECTIVES: The aim of this study was to compare the curing reaction of five experimental adhesive blends containing different photo-initiating systems. The hypothesis tested was that degree of conversion (DC) of resin blends is affected by resin type, solvent content and photo-initiating system. METHODS: The experimental methacrylate resin blends were ranked from hydrophobic (R2) to hydrophilic (R3 and R4) and tested as neat, or solvated with 10% or 20% ethanol, or 10% ethanol and 10% water. Three different photo-initiators were used: IS-1=0.25% CQ (camphorquinone)+1% EDMAB (ethyl 4-dimethylaminobenzoate); IS-2=1.25% TPO (diphenyl(2,4,6-trimethylbenzoyl)-phosphine oxide); IS-3=0.25% CQ+0.50% EDMAB+0.50% TPO. DC of resin blends was measured with a differential scanning calorimeter. Data were analyzed with a three-way ANOVA. RESULTS: Neat resin type influenced DC, as R4 showed the highest values compared to R2 and R3 (p<0.05). Solvent had a significant effect on DC (p<0.05): dilution of resin blends with 10% or 20% ethanol or 10% ethanol+10% water increased the DC of all resins, except for R4. Initiators influenced the polymerization since neat resins and mixtures solvated with 10% or 20% ethanol showed their highest DC values when polymerized with IS-1 or IS-3 (p>0.05), while IS-2 or IS-3 increased the DC values of resins diluted with 10% ethanol and 10% water (p<0.05). CONCLUSIONS: Water-compatible photo-initiators such as TPO should be included in the hydrophilic solvated adhesive formulation to ensure an appropriate DC of the adhesive layer.

Implementation of maskless laser lithography using a Raman spectroscopy microprobe.

A new use of a Raman spectroscopy microprobe is described. This kind of instrument uses an intense light source, usually a laser, to obtain chemical information from the scattered light sent to the sample through a microscope objective lens. A Raman microprobe is most often equipped with a motorized stage. The coupling of a focused laser light with the possibility of moving the stage under the very same light can be used to perform maskless photolithography as a laser writer. The versatility and resolution of this application are tested and described, producing different arrays of holes on photoresist. Holes as small as 1 mum are produced, thus demonstrating a lateral resolution sufficient for several applications. As an example of application, a pattern of a human protein, fibronectin, has been produced on a gold surface, which can then be used for the study of cell adhesion.