New approach for hair keratin characterization: Use of the confocal Raman spectroscopy to assess the effect of thermal stress on human hair fibre.

OBJECTIVE: The objective of our research was to investigate the heat-protecting effect of a product ex vivo and in vivo on human hair fibres. METHODS: A preparatory study was carried out in order to determine an optimal threshold of thermal stress. For this, the structure of cross-sections of the hair fibre was observed by optical microscopy. Then, Scanning Electron Microscopy (SEM) and Confocal Raman Spectroscopy (CRS) were applied to analyse ex vivo and in vivo morphological and molecular damage in hair structure after heat stress. Finally, in vivo tests were used to collect consumer perception. RESULTS: The preparatory study enabled us to determine an optimal stress threshold of 10 heating cycle for SEM and 5 heating cycle for CRS. Based on spectral hierarchical classification using Ward's clustering algorithm, the ex vivo Raman results show that the spectral signature of the hair treated and heated is very close to the negative control. This shows that the product preserves the keratin structure after thermal stress. These results were also confirmed by an in vivo Raman analysis performed on hair samples from 5 donors. In concordance with Raman results, SEM shows that treated hair presents lesser "bubbles" and "crackling" on the hair surface. Finally, the in vivo studies proved that hair was more protected from heat. CONCLUSION: The authors concluded that the product shows protective properties with respect to morphological and molecular heat damage. We also demonstrate that the product promotes the α-helix keratin conformation and preserves the S-S disulfide bands.

OBJECTIF: L'objectif de notre étude est d'évaluer ex vivo et in vivo l'effet thermoprotecteur d'un produit sur les fibres capillaires humaines. MÉTHODES: Une étude préparatoire a été réalisée afin de déterminer un seuil optimal du stress thermique. Pour cela, la structure des coupes transversales des cheveux a été observée par microscopie optique. Ensuite, la microscopie électronique à balayage (MEB) et la spectroscopie confocale Raman (SCR) ont été appliquées pour analyser les dommages morphologiques et moléculaires (ex vivo et in vivo) de la structure du cheveu après un stress thermique. Enfin, des tests in vivo ont été réalisés pour recueillir la perception des consommateurs. RÉSULTATS: L'étude préparatoire nous a permis de déterminer un seuil de stress thermique optimal correspondant à 10 cycles de chauffage pour la MEB et 5 cycles de chauffage pour la SCR. Basés sur une classification hiérarchique utilisant l'algorithme de Ward, les résultats Raman « ex vivo ¼ montrent que la signature spectrale des cheveux traités et chauffés est très proche du témoin négatif. Cela montre que le produit préserve la structure de la kératine après un stress thermique. Ces résultats ont également été confirmés par une analyse Raman « in vivo ¼ réalisée sur des échantillons de cheveux de 5 donneurs. En concordance avec les résultats Raman, la MEB montre que les cheveux traités présentent moins de « bulles ¼ et de « craquelures ¼ à la surface des cheveux. Enfin, l'étude in vivo a prouvé que les cheveux sont mieux protégés de la chaleur. CONCLUSION: Les auteurs ont conclu que le produit présente des propriétés protectrices vis-à-vis des dommages thermiques morphologiques et moléculaires. Nous avons démontré également que le produit favorise la conformation de la kératine en hélice-α et préserve les bandes disulfures S-S.

Combining Raman imaging and MCR-ALS analysis for monitoring retinol permeation in human skin.

BACKGROUND: Monitoring the transcutaneous permeation of exogenous molecules using conventional techniques generally requires long pre-analytical preparation or labelling of samples. However, Raman spectroscopy is a label-free and non-destructive method which provides spatial distribution of tracked actives in skin. The aim of our study was to prove the interest of Raman imaging coupled with multivariate curve resolution alternating least square (MCR-ALS) analysis in monitoring retinol penetration into frozen and living human skin. MATERIALS AND METHODS: After topical treatment of skin samples by free or encapsulated retinol, thin cross sections were analysed by Raman imaging (up to 100 µm depth). Mann-Whitney test was used to identify retinol spectroscopic markers in skin. MCR-ALS was used to estimate retinol contribution in Raman spectral images. Heat maps were constructed to compare the distribution of free and encapsulated retinol in skin models. RESULTS: We identified the bands at 1158, 1196 and 1591 cm-1 as specific features for monitoring retinol in skin. Moreover, our MCR-ALS results showed an improvement of retinol penetration (up to 30 µm depth) with the encapsulated form as well as storage reservoir formation in stratum corneum, for each skin model. Finally, greater retinol penetration into living skin was observed. CONCLUSION: This study shows a proof of concept for the evaluation of retinol penetration in skin using Raman imaging coupled with MCR-ALS. This concept needs to be validated on more subjects to include inter-individual variability but also other factors affecting skin permeation (age, sex, pH, etc). Our study can be extended to other actives.

Atopic skin: In vivo Raman identification of global molecular signature, a comparative study with healthy skin.

Atopic dermatitis (AD) is the most common skin inflammatory disease, affecting up to 3% of adults and 20% of children. Skin barrier impairment is thought to be the primary factor in this disease. Currently, there is no method proposed to monitor non-invasively the different molecular disorders involved in the upper layer of AD skin. Raman microspectroscopy has proved to be a powerful tool to characterize some AD molecular descriptors such as lipid content, global hydration level, filaggrin and its derivatives. Our investigations aimed to extend the use of in vivo Raman microspectroscopy as a rapid and non-invasive diagnostic technique for lipid conformation and organization, protein secondary structure and bound water content analysis in atopic skin. Our approach was based on the analysis of Raman data collected on the stratum corneum (SC) of 11 healthy and 10 mild-to-moderate atopic patients. Atopic skin revealed a modification of lipid organization and conformation in addition to the decrease of the lipid-to-protein ratio. This study also highlighted a reduction of the bound water and an increase in protein organized secondary structure in atopic skin. All these descriptors worsen the barrier function, state and appearance of the skin in AD. This precise and relevant information will allow an in vivo follow-up of the pathology and a better evaluation of the pharmacological activity of therapeutic molecules for the treatment of AD.

Epidemiological investigation and typing of Candida glabrata clinical isolates by FTIR spectroscopy.

Candida glabrata has emerged as one of the leading agents of fungal infections and strain typing is essential for epidemiological investigation that is generally achieved by molecular techniques. In this work, we studied twenty-nine C. glabrata strains isolated from different patients, using a phenotypic approach based on Fourier Transform Infrared (FTIR) spectroscopy, which has been in a previous study successfully applied as a rapid typing method for Candida albicans. A two-step procedure was used for the analysis. The first step included sixteen strains for the internal validation phase, which aimed at finding the spectral windows that would provide the best differentiation between strains. In this phase, hierarchical cluster analysis (HCA) carried out using three spectral windows (900-1200, 1540-1800, 2800-3000 cm(-1)) allowed to obtain the best classification, where each patient strains could be clustered together. A genotypic technique based on randomly amplified polymorphic DNA-analysis (RAPD) confirmed these results. In a second step, the external validation phase, thirteen other clinical strains of C. glabrata isolated from multiple sites in four ICU patients, were tested by FTIR spectroscopy. The analysis was based on the spectral regions previously found in the first step. HCA classification of the strains gave four groups, one group per patient. These results suggest that no inter-human transmission took place. This study shows the potential of FTIR approach for typing of C. glabrata with several advantages compared to other techniques. FTIR typing is fast, effective, and reagent free. Moreover, it is applicable to all micro-organisms and requires a small quantity of biomass.

Rapid identification of Candida species by FT-IR microspectroscopy.

Due to the continuous increase of human candidiasis and the great diversity of yeasts of the Candida genera, it is indispensable to identify this yeast as early as possible. Early identification enables an early diagnostic and patient-adapted anti-fungal therapy, thus reducing morbidity and mortality related to these infections. In view of this, we have in this study investigated microcolonies using a method based on Fourier transform-infrared microspectroscopy (FTIRM) for a rapid and early identification of the most frequent Candida species encountered in human pathology. FTIR spectroscopy is a whole-cell "fingerprinting" method by which microorganisms can be identified. By exploiting the huge discriminating capacity of this technique, we identified 6 species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, and Candida kefyr) from a collection of 57 clinical strains of Candida, isolated from hospitalised patients. Data obtained on 10- to 18-h-old microcolonies were compared to cultures of 24 h. Our results clearly show the efficiency and the robustness of FTIR (micro)spectroscopy in identifying species with a classification rate of 100% for both microcolonies and 24-h cultures. FTIR microspectroscopy is thus a promising clinical approach, because compared to conventional and molecular techniques, it is time and money saving, has great identification and discriminating potentials, and is amenable to an automated high-throughput routine system.

FTIR spectroscopy in medical mycology: applications to the differentiation and typing of Candida.

The incidence of fungal infections, in particular candidiasis and aspergillosis, has considerably increased during the last three decades. This is mainly due to advances in medical treatments and technologies. In high risk patients (e.g. in haematology or intensive care), the prognosis of invasive candidiasis is relatively poor. Therefore, a rapid and correct identification of the infectious agent is important for an efficient and prompt therapy. Most clinical laboratories rely on conventional identification methods that are based on morphological, physiological and nutritional characteristics. However, these have their limitations because they are time-consuming and not always very accurate. Moreover, molecular methods may be required to determine the genetic relationship between the infectious strains, for instance in Candida outbreaks. In addition, the latter methods require time, expensive consumables and highly trained staff to be performed adequately. In this study, we have applied the FTIR spectroscopic approach to different situations encountered in routine mycological diagnosis. We show the potentials of this phenotypic approach, used in parallel with routine identification methods, for the differentiation of 3 frequently encountered Candida species (C. albicans, C. glabrata and C. krusei) by using both suspensions and microcolonies. This approach, developed for an early discrimination, may help in the initial choice of antifungal treatment. Furthermore, we demonstrate the feasibility of the method for intraspecies comparison (typing) of 3 Candida species (C. albicans, C. glabrata and C. parapsilosis), particularly when an outbreak is suspected.

Osmoadaptative responses in the rhizobia nodulating Acacia isolated from south-eastern Moroccan Sahara.

Four strains of rhizobia nodulating Acacia were isolated from the Moroccan desert soil by trapping with seedlings of Acacia gummifera and Acacia raddiana, and were studied for their ability to tolerate high salinity and dryness conditions. The strains MDSMC 2, MDSMC 18 and MDSMC 50 were halotolerant (they tolerated up to 1 M NaCl) and they accumulated glutamate and mannosucrose. The synthesis of the latter solute, which is the major endogenous osmolyte, is partially repressed in the presence of glycine betaine. The strain MDSMC 34 was less halotolerant (growth inhibited by a concentration greater than 0.5 M NaCl), and accumulated trehalose (as the main endogenous osmolyte) and glutamate. Rhizobia from the Moroccan desert soil were highly resistant to desiccation and their tolerance to dryness was stimulated by osmotic pretreatment. Thus, the accumulation of mannosucrose or trehalose by desert rhizobia represents both an osmoadaptative response and a part of a desiccation tolerance mechanism.