Misinterpretation of ISO 4049 standard recommendations: Impact on Young's modulus and conversion degree of dental composites.

OBJECTIVES: The purpose of this study was to study the effects on Young's modulus and conversion degree of variations in polymerization conditions during the 3-point bending test of composite samples in accordance with the ISO 4049 standard. METHODS: Three nanocomposites were used in the 3-point bending test based on the conditions described in the ISO 4049 standard. Samples of 2 mm × 2 mm x 25 mm were fabricated and tested with a different number of irradiation points and irradiation time. Conversion degree of the samples were also measured by micro-Raman spectroscopy and correlated with the Young's modulus values obtained for each one. RESULTS: The variations in curing protocol during specimen's realization influenced the Young's modulus and degree of conversion of all composites. These two properties correlated well. The ISO 4049 standard defines the conditions for performing the properties tests of composites to allow reproducibility and comparison of different studies. Concerning the 3-point bending test, even a minimal change in the state causes differences in the results obtained. The standard should thus clarify the tools that can be used when producing samples in order to minimize discrepancies. SIGNIFICANCE: The influence of the parameters surrounding the design of the samples should be controlled and defined so as not to include bias in the studies carried out. This will allow literature studies to be compared with more accuracy.

Human skin penetration of hyaluronic acid of different molecular weights as probed by Raman spectroscopy.

BACKGROUND: Topical delivery of molecules into the human skin is one of the main issues in dermatology and cosmetology. Several techniques were developed to study molecules penetration into the human skin. Although widely accepted, the conventional methods such as Franz diffusion cells are unable to provide the accurate localization of actives in the skin layers. A different approach based on Raman spectroscopy has been proposed to follow-up the permeation of actives. It presents a high molecular specificity to distinguish exogenous molecules from skin constituents. METHODS: Raman micro-imaging was applied to monitor the skin penetration of hyaluronic acids (HA) of different molecular weights. The first step, was the spectral characterization of these HA. After, we have determined spectral features of HA by which they can be detected in the skin. In the second part, transverse skin sections were realized and spectral images were recorded. RESULTS: Our results show a difference of skin permeation of the three HA. Indeed, HA with low molecular weight (20-300 kDa) passes through the stratum corneum in contrast of the impermeability of high molecular weight HA (1000-1400 kDa). CONCLUSION: Raman spectroscopy represents an analytical, non-destructive, and dynamic method to evaluate the permeation of actives in the skin layers.

Development of a shear force scanning near-field fluorescence microscope for biological applications.

In this paper, a shear force scanning near-field fluorescence microscope combined with a confocal laser microspectrofluorometer is described. The shear force detection is realized based on a bimorph cantilever, which provides a very sensitive, reliable, and easy to use method to control the probe-sample distance during scanning. With the system, high-quality shear force imaging of various samples has been carried out. Furthermore, simultaneous shear force and near-field fluorescence imaging of biological cells has also been realized. As an example, we especially present the result on the distribution of P-glycoprotein in the plasma membrane of human small cell lung cancer cells, suggesting that the system would be a promising tool for biological applications.

Changes in adsorption and permeability of mitoxantrone on plasma membrane of BCRP/MXR resistant cells.

A selective analysis of adsorbed mitoxantrone (MTX) was performed by surface-enhanced Raman scattering (SERS) at the range of cellular membrane. Disruption of the membrane fluidity was carried out to appraise changes in membrane adsorption of MTX and drug uptake in sensitive (HCT-116 S) and resistant BCRP/MXR (HCT-116 R) cells. Based on spectral MTX modifications, micro-SERS spectroscopy discriminated clearly drug adsorption phenomena on plasma membrane from drug in solution. A 3-fold higher SERS intensity of MTX for HCT-116 R was observed concluding to a higher drug adsorption on resistant membrane. The increase of membrane fluidity with benzyl alcohol (BA) or chloroform (CF) resulted in a 3-fold decrease of MTX adsorption on HCT-116 R, exclusively. BA and CF improved intracellular accumulation of MTX (e.g., 823 and 191 pmol MTX/10(6) HCT-116 R incubated with or without BA). At 4 degrees C, drug accumulation measurements showed a decrease of MTX permeability in resistant membrane (42 pmol MTX/10(6) cells), restored with fluidizers (e.g., 342 pmol MTX/10(6) cells with BA). Fluorescence confocal microscopy involved an exclusive MTX emission around the plasma membrane of resistant cells whereas fluidizers increased the intracellular uptake of MTX in both cell lines at the same time with less drug emission around the plasma membrane. Changes of the membrane structure of resistant cells should modify both drug adsorption and membrane permeation.

Shear force near-field optical microscope based on Q-controlled bimorph sensor for biological imaging in liquid.

Shear force near-field microscopy on biological samples in their physiological environment loses considerable sensitivity and resolution as a result of liquid viscous damping. Using a bimorph-based cantilever sensor incorporating force feedback, as recently developed by us, gives an alternative force detection scheme for biological imaging in liquid. The dynamics and sensitivity of this sensor were theoretically and experimentally discussed. Driving the bimorph cantilever close to its resonance frequency with appropriate force feedback allows us to obtain a quality factor (Q-factor) of up to 10(3) in water, without changing its intrinsic resonance frequency and spring constant. Thus, the force detection sensitivity is improved. Shear force imaging on mouse brain sections and human skin tissues in liquid with an enhanced Q-factor of 410 have shown a high sensitivity and stability. A resolution of about 50 nm has been obtained. The experimental results suggest that the system is reliable and particularly suitable for biological cell imaging in a liquid environment.

Surface-enhanced Raman scattering reveals adsorption of mitoxantrone on plasma membrane of living cells.

Surface-enhanced Raman scattering (SERS) spectroscopy was applied to analyze mitoxantrone (MTX) adsorption on the plasma membrane microenvironment of sensitive (HCT-116 S) or BCRP/MXR-type resistant (HCT-116 R) cells. The addition of silver colloid to MTX-treated cells revealed an enhanced Raman scattering of MTX. Addition of extracellular DNA induced a total extinction of MTX Raman intensity for both cell lines, which revealed an adsorption of MTX on plasma membrane. A threefold higher MTX Raman intensity was observed for HCT-116 R, suggesting a tight MTX adsorption in the plasma membrane microenvironment. Fluorescence confocal microscopy confirmed a relative MTX emission around plasma membrane for HCT-116 R. After 30 min at 4 degrees C, a threefold decrease of the MTX Raman scattering was observed for HCT-116 R, contrary to HCT-116 S. Permeation with benzyl alcohol revealed a threefold decrease of membrane MTX adsorption on HCT-116 R, exclusively. This additional MTX adsorption should correspond to the drug bound to an unstable site on the HCT-116 R membrane. This study showed that SERS spectroscopy could be a direct method to reveal drug adsorption to the membrane environment of living cells.

Characterization of island films as surface-enhanced Raman spectroscopy substrates for detecting low antitumor drug concentrations at single cell level.

Gold and silver vacuum-deposited island films were characterized by studying deposition variables such as film thickness, evaporation rate, and substrate temperature. For both metals, these parameters were correlated with the surface-enhanced Raman spectroscopy (SERS) effect and an increase in film thickness and low evaporation rates were shown to upshift the wavelength at maximum optical density (lambda max) and increase the optical density of the substrates. In contrast, pre- and postdeposition annealing of gold films led to the formation of substrates that exhibited a downshift of lambda max. Our spectral data also indicated that silver films are substrates that are more suited for SERS applications where high frequency visible excitations are used. Measurements on gold films classified them into two groups: thin Au films (10-50 A) well adapted for red excitations and thicker ones that are operative in the near infrared. SERS results, which were obtained from a single HL60 cell treated with micromolar drug quantities, placed on thin gold island films indicated that these island films could be future promising substrates for SERS imaging at the cellular level.

Quantitative determination of free calcium in subcellular compartments, as probed by Indo-1 and confocal microspectrofluorometry.

Confocal UV-microspectrofluorometry has been applied to measure fluorescence emission spectra of Indo-1 for the intracellular determination of free calcium ([Ca2+]i). To perform in situ calibrations of [Ca2+]i in the nucleus and the cytoplasm, Indo-1 has been loaded into living cells under its esterified form Indo-1/AM. For each controlled [Ca2+]i, intranuclear and intracytoplasmic spectra show a systematic blue shift, as compared with spectra in solution at the same [Ca2+]. In the Ca2+ saturated condition, the intranuclear spectra are more blue-shifted than in the cytoplasm. Thus, distinct in situ calibration curves have been distinguished for the nucleus and the cytoplasm. To calculate [Ca2+]i, intracellular spectra of Indo-1 have been characterized by two distinct methods. First, the ratio of emission intensities at 410 and 500 nm has been determined. Secondly, the analyzed spectrum has been decomposed into a linear combination of in situ free and Ca-bound Indo-1 reference spectra from the considered cellular compartment. Satisfactory spectral decompositions have been observed for each [Ca2+]i. The subcellular calibration curves allow one to determine the product of both intracellular constants, i.e. the ratio of quantum yields between free and Ca-bound Indo-1 and the apparent dissociation constant for Ca2+. The product of these constants has been shown to be similar in both subcellular compartments and in a buffered solution. The two methods used for the [Ca2+]i determination lead to equivalent results on unpermeabilized KB cells. They show a 1.3 times higher [Ca2+]i in the cytoplasm than in the nucleus (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Monitoring of bacterial growth and structural analysis as probed by FT-IR spectroscopy.

Fourier-transform infrared spectroscopy was used to explore structural changes in bacteria under different incubation conditions. In particular, differences between Bradyrhizobium japonicum (BRJ) grown in liquid and on solid media were investigated, as well as the rearrangement of BRJ after transfer from one medium to the other. The FT-IR absorption bands located between 1200 and 900 cm-1 region, vary in spectral shape and intensity when BRJ were suspended in solution medium or plated on solid medium. In agreement with the electronic micrograph data, these spectroscopic changes are due to the changes involving the bacterial wall (peptidoglycan) when BRJ are plated in agar medium. By means of this FT-IR ultrastructural study of Bradyrhizobium japonicum bacteria, it has been possible to follow and to evaluate the rate of the molecular change in bacteria without any destructive interference. This indicates that FT-IR spectroscopy can prove to be a valuable technique in the monitoring of metabolic events in bacterial cells relevant to agriculture as well as environmental and health sciences.

Determination of vinorelbine (Navelbine) in tumour cells by high-performance liquid chromatography.

A high-performance liquid chromatographic method has been developed for the determination within tumour cells of a new vinca alkaloid, vinorelbine. Extractions of vinorelbine from cells were carried out using absolute ethanol. The extracts were injected into a reversed-phase system consisting of two Novapak C18 columns connected in series. The mobile phase was acetonitrile-phosphate buffer, pH 2.7 (60:40, v/v). Using a fluorescence detection, the limit of determination was 8 pmol injected. This method would be suitable for studying the cellular pharmacokinetics of vinorelbine in patients.

Possible role of water structure in biological magnesium systems.

Magnesium chloride-water solutions have been studied by Fourier Transform Infrared Spectroscopy (FT-IR) in the near infrared region, 5000-10,000 cm-1. The effect of the concentration of magnesium chloride and temperature on the solutions has been studied from the spectra and it is concluded that magnesium chloride modifies the structure of the bulk water. The important absorption bands of water at 5200 and 7020 cm-1 may be assigned to combination vibrations and overtones. They are shifted either by increasing the magnesium chloride concentration or the temperature. The hydrated magnesium ions, [Mg(H2O)6]2+, will most probably break important hydrogen bonds in the clusters of water (H2O)n, where n = 2, 3, 4, 5, 6.../forming new hydrogen bonds in the presence of hexa-aquated magnesium cations. FAB mass spectra also suggest the formation of hydrated magnesium cations, Mg (H2O)6(2+).

Candida albicans--adriamycin interactions: ultrastructural and spectrofluorometric study of whole yeasts and spheroplasts.

The occurrence of candidiasis in cancer patients who undergo chemotherapy requires the interrelation of Candida albicans and the antimitotic drug Adriamycin (ADM) which is well known as an intercalating agent. The whole yeasts were not affected by 2 h of contact with the drug at 10(-4) M neither for their growth curve nor for their ultrastructure, despite the presence of free ADM on their surface. Spheroplasts displayed a delay in their growth and exhibited altered nucleoli with segregation of their granular and fibrillar components. The modified emission spectrum of ADM, determined by spectrofluorometry, corresponded neither to the free ADM nor to the DNA-bound drug, but it could be related to a metabolite of the drug. The cell wall appeared to be one of the main sites for ADM resistance of Candida albicans in vitro.