Noninvasive evaluation of the skin barrier in reconstructed human epidermis using speckle analysis: Correlation with Raman microspectroscopy.

BACKGROUND: Reconstructed epidermis models, obtained from 3D keratinocytes culture, have gained significant prominence as prototypes for safety and efficacy testing in skin research. To effectively evaluate these models, it is essential to perform molecular and functional characterization. The skin's barrier function is one of the essential aspects of the epidermis that needs to be assessed. A noninvasive method is thus required for the evaluation of the skin barrier in these models. With this perspective, the aim of this feasibility study is to apply the speckle technique for the assessment of the skin barrier in the Reconstructed Human Epidermis (RHE). MATERIALS AND METHODS: Speckle analysis as well as Raman microspectroscopy were performed on RHE samples at two maturation days, D17 and D20. RESULTS: Between D17 and D20, our study showed an increase in various Raman parameters, including stratum corneum percentage, lateral lipid packing, lipid-to-protein ratio, and protein secondary structure. Furthermore, the degree of light polarization and the speckle grain size also increased over this period. CONCLUSION: The speckle technique proved to be effective for evaluating the skin barrier in Reconstructed Human Epidermis (RHE) models. Comparison with Raman validates this approach and provides comprehensive molecular and functional characterization of reconstructive skin models.

DNA adduct formation in Saccharomyces cerevisiae following exposure to environmental pollutants, as in vivo model for molecular toxicity studies.

DNA adduction in the model yeast Saccharomyces cerevisiae was investigated after exposure to the fungicide penconazole and the reference genotoxic compound benzo(a)pyrene, for validating yeasts as a tool for molecular toxicity studies, particularly of environmental pollution. The effect of the toxicants on the yeast's growth kinetics was determined as an indicator of cytotoxicity. Fermentative cultures of S. cerevisiae were exposed to 2 ppm of Penconazole during different phases of growth; while 0.2 and 2 ppm of benzo(a)pyrene were applied to the culture medium before inoculation and on exponential cultures. Exponential respiratory cultures were also exposed to 0.2 ppm of B(a)P for comparison of both metabolisms. Penconazole induced DNA adducts formation in the exponential phase test; DNA adducts showed a peak of 54.93 adducts/109 nucleotides. Benzo(a)pyrene induced the formation of DNA adducts in all the tests carried out; the highest amount of 46.7 adducts/109 nucleotides was obtained in the fermentative cultures after the exponential phase exposure to 0.2 ppm; whereas in the respiratory cultures, 14.6 adducts/109 nucleotides were detected. No cytotoxicity was obtained in any experiment. Our study showed that yeast could be used to analyse DNA adducts as biomarkers of exposure to environmental toxicants.

A simple and fast experimental protocol for the extraction of xanthophylls from microalga Chlorella luteoviridis.

This study aimed to optimize the key parameters of extraction methods and to increase the recovery yields of intact xanthophylls (violaxanthin, zeaxanthin, astaxanthin) from microalgae (Chlorella luteoviridis). An effective, simple, and fast extraction protocol is described. It consists of a grinding pretreatment followed by a microwave-assisted extraction, using ethanol 90% as an environmentally preferable extraction solvent. Xanthopylls were quantified using high performance liquid chromatography. Irradiation time of 6 s only resulted in the extraction of violaxanthin (4.479 ± 0.009 mg/g), astaxanthin (4.154 ± 0.013 mg/g), and zeaxanthin (4.776 ± 0.120 mg/g). The described protocol seems to be the fastest extraction method of xantophylls compared to the literature and could be an advantage for industrial scale, while saving time and energy.

Interpretation of the bacterial growth process based on the analysis of the speckle field generated by calibrated scattering media.

The speckle imaging technique has been proven to be a reliable and effective method for real-time monitoring of the growth kinetics of any bacterium in suspension. To understand the interaction between the light and the bacterial density, a simulation of the bacterial growth of Bacillus thuringiensis was performed using calibrated microspheres of different concentrations and sizes. Results show that the decrease of speckle grain size with the increase of the medium scattering coefficient reveals the two essential phases of the bacterial growth: the exponential phase where the number of the bacteria increases and the stationary phase where sporulation and cell lysis occur.

Antityrosinase Activity of Combretum micranthum, Euphorbia hirta and Anacardium occidentale Plants: Ultrasound Assisted Extraction Optimization and Profiling of Associated Predominant Metabolites.

Tyrosinase is an important component of the enzyme polyphenol oxidase, which upon contact with the phenolic substrates forms the pigment melanin and induces undesirable food browning. The phenolic and triterpenoid compounds that naturally occur in plants are well known as tyrosinase inhibitors. Combretum micranthum (CM) leaves, Euphorbia hirta (EH) plant, and Anacardium occidentale (AO) fruits are traditionally known to have potential anti-tyrosinase activities. The aim of this study was to optimize the ultrasound-assisted extraction of secondary metabolites from these matrices, and to evaluate in tubo the antityrosinase activity of these extracts. Efforts were also taken to profile the secondary metabolites, mainly the phenolic and triterpenoid compounds, in order to understand their probable association with tyrosinase inhibition. The optimal ultrasound-assisted extraction conditions for simultaneous extraction of phenolic, and triterpenoid compounds were determined. The aqueous fraction of these extracts showed significant antityrosinase activity, with the CM leaves exhibiting the strongest inhibitory effect (IC50 of 0.58 g·L-1). The predominant metabolic compounds from these natural extracts were putatively identified by using a high-resolution quadrupole-time of flight (QToF) LC-MS instrument. The high-resolution accurate mass-based screening resulted in identification of 88 predominant metabolites, which included dihydrodaidzein-7-O-glucuronide, micromeric acid, syringic acid, morin, quercetin-3-O-(6″-malonyl-glucoside), 4-hydroxycoumarin, dihydrocaffeic acid-3-O-glucuronide, to name some, with less than 5 ppm of mass error.

Bioremediation of Ni, Al and Pb by the living cells of a resistant strain of microalga.

The microalgae treatment system is an economically and environmentally friendly option for wastewater treatment. However, the effects of heavy metal toxicity on microalgae cells can limit the use of microalgae in the treatment of industrial effluents rich in heavy metals. In this work, we studied the effect of Ni, Cu, Al, Hg and Pb, added as single-metal solutions to the microalgae culture medium, on the growth of 20 indigenous strains belonging to a wide variety of microalgae genera. Ni and Cu were the most toxic to the strains tested. A highly tolerant strain of the Phacus genera was selected. We determined the effect of multiple combinations of Ni, Al and Pb on the cell growth of the selected strain and on the removal capacity of each metal from the microalgae culture medium. Phacus was able to grow in the multi-metal solution (Ni, 5.00 mg/L; Al, 9.94 mg/L and Pb 1.00 mg/L) and to efficiently remove the metals, with removal capacities of 8.82 ±0.16 mg/g for Ni, 2.09 ± 0.05 mg/g for Pb and 16.90 ± 0.53 mg/g for Al. The reductions of Ni, Al and Pb concentrations were 66.67, 64.28 and 79.17% respectively.

Secondary metabolism in Penicillium expansum: Emphasis on recent advances in patulin research.

The plant pathogenic fungus Penicillium expansum is a major concern of the global food industry due to its wide occurrence and ability to produce various mycotoxins, of which the most significant is patulin. Relatively less highlighted in the literature, in comparison with the other food-borne mycotoxins, patulin is one of the main factors in economic losses of vegetables and fruits. Otherwise, patulin is a health hazard which results in both short-term and long-term risks. This review includes knowledge on the biosynthetic mechanisms used for secondary metabolite production in P. expansum, with special emphasis on patulin biosynthesis. The abiotic factors triggering the production of patulin and the strategies developed to reduce or prevent the contamination by this mycotoxin are comprehensively discussed. The database presented in this review would be useful for the prioritization and development of future research.

Patulin transformation products and last intermediates in its biosynthetic pathway, E- and Z-ascladiol, are not toxic to human cells.

Patulin is the main mycotoxin contaminating apples. During the brewing of alcoholic beverages, this mycotoxin is degraded to ascladiol, which is also the last precursor of patulin. The present study aims (1) to characterize the last step of the patulin biosynthetic pathway and (2) to describe the toxicity of ascladiol. A patE deletion mutant was generated in Penicillium expansum. In contrast to the wild strain, this mutant does not produce patulin but accumulates high levels of E-ascladiol with few traces of Z-ascladiol. This confirms that patE encodes the patulin synthase involved in the conversion of E-ascladiol to patulin. After purification, cytotoxicities of patulin and E- and Z-ascladiol were investigated on human cell lines from liver, kidney, intestine, and immune system. Patulin was cytotoxic for these four cell lines in a dose-dependent manner. By contrast, both E- and Z-ascladiol were devoid of cytotoxicity. Microarray analyses on human intestinal cells treated with patulin and E-ascladiol showed that the latter, unlike patulin, did not alter the whole human transcription. These results demonstrate that E- and Z-ascladiol are not toxic and therefore patulin detoxification strategies leading to the accumulation of ascladiol are good approaches to limit the patulin risk.

Assessing White Wine Viscosity Variation Using Polarized Laser Speckle: A Promising Alternative to Wine Sensory Analysis.

In this paper, we report measurements of wine viscosity, correlated to polarized laser speckle results. Experiments were performed on white wine samples produced with a single grape variety. Effects of the wine making cellar, the grape variety, and the vintage on wine Brix degree, alcohol content, viscosity, and speckle parameters are considered. We show that speckle parameters, namely, spatial contrast and speckle decorrelation time, as well as the inertia moment extracted from the temporal history speckle pattern, are mainly affected by the alcohol and sugar content and hence the wine viscosity. Principal component analysis revealed a high correlation between laser speckle results on the one hand and viscosity and Brix degree values on the other. As speckle analysis proved to be an efficient method of measuring the variation of the viscosity of white mono-variety wine, one can therefore consider it as an alternative method to wine sensory analysis.

Development of a real-time PCR assay for Penicillium expansum quantification and patulin estimation in apples.

Due to the occurrence and spread of the fungal contaminants in food and the difficulties to remove their resulting mycotoxins, rapid and accurate methods are needed for early detection of these mycotoxigenic fungi. The polymerase chain reaction and the real time PCR have been widely used for this purpose. Apples are suitable substrates for fungal colonization mostly caused by Penicillium expansum, which produces the mycotoxin patulin during fruit infection. This study describes the development of a real-time PCR assay incorporating an internal amplification control (IAC) to specifically detect and quantify P. expansum. A specific primer pair was designed from the patF gene, involved in patulin biosynthesis. The selected primer set showed a high specificity for P. expansum and was successfully employed in a standardized real-time PCR for the direct quantification of this fungus in apples. Using the developed system, twenty eight apples were analyzed for their DNA content. Apples were also analyzed for patulin content by HPLC. Interestingly, a positive correlation (R(2) = 0.701) was found between P. expansum DNA content and patulin concentration. This work offers an alternative to conventional methods of patulin quantification and mycological detection of P. expansum and could be very useful for the screening of patulin in fruits through the application of industrial quality control.

EBT3 Gafchromic® film as a new substrate for in vitro evaluation of sun protection factor.

There exist different methods for the determination of sun protection factor (SPF) values for sunscreens. We aimed to develop a new in vitro method using EBT3 Gafchromic® film as a substrate. The colour of EBT3 Gafchromic® film changes when exposed to UV light. Films were covered by sunscreen preparations of different SPF values ranging from 0 to 50. Uncovered and covered films were exposed to different solar light energies and their colour change was compared. Absorbance spectra of films was measured at 633 nm using a UV-VIS spectrophotometer apparatus. The colour of the film darkens when ultraviolet energy increases, which means that absorbance increases with exposure time. However, when films are covered by sunscreens, the colour change is less visible and the absorbance significantly decreases with increasing SPF value. There is a linear correlation between the absorbance of EBT3 Gafchromic® film and SPF value of sunscreens covering the film. Statistical analysis demonstrated that the SPF value of a sunscreen can be predicted using EBT3 Gafchromic® film as a substrate. This is the first report of an in vitro method based on colour change of a substrate which takes into consideration exposure time, and relates more closely to conditions of real-life. Based on these parameters, this is a reliable in vitro method for SPF testing.

Real-time monitoring of bacterial growth kinetics in suspensions using laser speckle imaging.

In microbiology, monitoring the growth of any microorganism in culture is important for studying and optimizing the growth kinetics, the biomass and the metabolite production. In this work, we show that laser speckle imaging is a reliable technique that can be used to perform real-time monitoring of bacteria growth kinetic in liquid culture media. Speckle parameters, specifically speckle grain size and the spatial contrast of the speckle images, and standard analytical parameters (optical density, pH and colony forming units) were measured during the culture of different strains of Bacillus thuringiensis. Our results show that both speckle grain size and spatial contrast decrease with bacterial growth. Furthermore, speckle parameters are sensitive to the fermentation conditions. Statistical analysis revealed a relatively high correlation between speckle and analytical parameters.

Skin lightening effect of natural extracts coming from Senegal botanical biodiversity.

BACKGROUND: Skin depigmentation is increasingly oriented toward plant extracts because of harmfulness of depigmenting active ingredients used in cosmetics and dermatology. Reconstructed human pigmented epidermis (RHPE) is the closest in vitro model to human skin and offers the possibility to test the global depigmenting effect of a plant extract. These co-cultures of keratinocytes and melanocytes are the most advanced and newest models for testing depigmentation, and until now very few studies have been done with these cultures. We investigated the cytotoxicity and the inhibitory effect on tyrosinase and melanogenesis of four extracts from Combretum micranthum (G. Don) leaves, Anacardium occidentale (L.) fruits, Moringa oleifera (Lam.) seeds, and Adansonia digitata (L.) seeds. METHODS: The vegetal extracts were obtained by ultrasound-assisted extraction and the vegetal oils by maceration. Anti-tyrosinase properties of two aqueous extracts were evaluated. Then, the cytotoxicity and depigmenting effects of these plant extracts were tested in vitro with RHPE model delivered by SkinEthic® . RESULTS: Antityrosinase activities were found to be 84.58% and 31.02% for C. micranthum and A. occidentale, respectively. All extracts, except A. occidentale, showed to be nontoxic. C. micranthum, M. oleifera, A. digitata, and mixture of M. oleifera and A. digitata extracts have shown, for the first time, an in vitro depigmenting activity equivalent or even more important than kojic acid. CONCLUSIONS: These natural extracts coming from Senegal botanical biodiversity could be used in cosmetic and dermatology as alternative agents to achieve skin depigmentation. Further study should be focused on the mechanism of action of these plant extracts.

Origanum essential oils reduce the level of melanin in B16-F1 melanocytes.

BACKGROUND: Hyperpigmentation disorders are considered signs of skin aging and are aesthetically unpleasant. Most active ingredients used against hyperpigmentation disorders predominantly target tyrosinase activity. OBJECTIVES: To study the effect of two Origanum essential oils on the melanogenic activity of B16-F1 murine melanocytes. The main component of these oils, carvacrol, was also investigated and a model for anti-melanogenic activity is proposed. MATERIALS AND METHODS: B16-F1 melanocytes were exposed to different concentrations of essential oils and carvacrol. The level of tyrosinase and melanin was determined using spectrophotometric measurements. RESULTS: Essential oils of Origanum syriacum and Origanum ehrenbergii led to a significant 14% and 17% reduction in melanin level at 40 µg mL-1, respectively. However, neither demonstrated a significant effect on the level of intracellular tyrosinase. The same effects were found for carvacrol which led to a 30% reduction in melanin at 45 µg mL-1. CONCLUSION: Our results indicate that the oils studied are anti-melanogenic. We propose a mechanism, similar to that for hydroquinone, whereby carvacrol functions as a competitive inhibitor of tyrosinase, thus inhibiting oxidation of tyrosine and causing a deregulation of melanogenesis.

Phytochemical screening and antityrosinase activity of carvacrol, thymoquinone, and four essential oils of Lebanese plants.

OBJECTIVE: In our study, we aim to explore the ability of four essential oils (EO) of Lebanese plants to inhibit the tyrosinase activity and to correlate their efficiency level to their phytochemical compositions. METHODS: The EO have been extracted by hydrodistillation using a Clevenger apparatus and have been studied by GC-MS analysis. Active compounds of Origanum species were identified and antityrosinase activities of EO and active molecules (carvacrol and thymoquinone) have been tested in tubo. RESULTS: Antityrosinase activities were obtained as follows: EO of Origanum syriacum (80.41% ± 2.00%), EO of Origanum ehrenbergii (45.33% ± 2.20%), EO of Salvia fruticosa (14.62% ± 2.30%), EO of Calamintha origanifolia (16.51% ± 5.80%), Carvacrol (56.55% ± 3.10%), and Thymoquinone (19.49% ± 1.50%). CONCLUSION: Origanum essential oils resulted in the highest antityrosinase activity due to their high content in carvacrol. However, when present together with carvacrol, thymoquinone decreases the efficiency of carvacrol, which is the case of O. ehrenbergii essential oil. Thus, for improved antityrosinase activity, O. syriacum and O. ehrenbergii should be harvested during flowering stage where carvacrol is present at its highest dosage and thymoquinone at its lowest.

Fungal contamination and Aflatoxin B1 and Ochratoxin A in Lebanese wine-grapes and musts.

Five hundred and ten strains of filamentous fungi were isolated from Lebanese grapes during 2005 at veraison and harvesting periods. Four hundred eighty-seven isolates belonged to the Aspergillus spp. (95.5%) and 23 belonged to the Penicillium spp. (4.5%). Black aspergilli constituted 56.9% (52.2% Aspergillus niger aggregates, 2.9% Aspergillus japonicus and 1.8% Aspergillus carbonarius) while the isolation rate of Aspergillus flavus the none habitual member of grape mycobiota was 43.1% of the total Aspergillus spp. isolated. All isolates were tested for the ability to produce the Ochratoxin A (OTA) and the Aflatoxin B1 (AFB1). A. carbonarius showed that it is the only species able to produce the OTA with a production ability of 100% and a maximum concentration reaching 8.38microg/g CYA. As for the aflatoxigenic ability, 43.4% of A. flavus isolates produced this mycotoxin with a maximum production reaching 22.6microg/g CYA while none of the other isolates showed a production capacity of this mycotoxin. Forty-seven samples of must produced from the collected grapes were also analyzed. None of these samples was contaminated by OTA at a detectable limit while 40% of these same samples were found to contain AFB1 with concentrations ranging from 0.01 to 0.46microgl(-1).

Effects of penconazole on two yeast strains: growth kinetics and molecular studies.

The aim of this study consisted to evaluate the impact of a pesticide (penconazole) on the growth kinetics and genotoxicity on two yeast strains (Saccharomyces cerevisiae and Metschnikowia pulcherrima). When the penconazole was added at different phases of the growth of M. pulcherrima, no effect was noticed on the kinetics of yeast growth but DNA adducts were observed when penconazole was added in the exponential phase. Increasing doses (1-15 maximum residue limit) of the pesticide added at the beginning of the fermentation did not induce DNA adducts while kinetics were affected.

Occurrence of ochratoxin A- and aflatoxin B1-producing fungi in Lebanese grapes and ochratoxin a content in musts and finished wines during 2004.

This paper reports the results of an extensive survey on the occurrence of filamentous fungi isolated from wine-grapes in Lebanon and to test their ability to produce ochratoxin A (OTA) and aflatoxin B1 (AFB1) on CYA culture medium, in order to assess their potential for producing these mycotoxins on grapes. From the 470 grapes samples taken during season 2004, 550 fungi strains were isolated with 490 belonging to Aspergillus spp. and 60 belonging to Penicillium spp. All these isolated fungi starins were tested for their ability to produce OTA and AFB1. Aspergillus carbonarius shows that it is the only species able to produce OTA with a production percentage reaching 100% and a maximum concentration of 52.8 microg/g of Czapek yeast extract agar (CYA). In its turn, Aspergillus flavus was considered as the only AFB1-producing species with production percentage of 45.3% and a maximum concentration reaching 40 microg/g CYA. A total of 47 handmade musts produced from the collected grapes were also analyzed in order to correlate the presence of OTA in must and the occurrence of filamentous fungi on grapes; 57.4% were contaminated with OTA at low level with concentrations ranging between 0.011 and 0.221 microg OTA L(-1). The analysis of these must samples was not performed with regard to AFB1. Seventy samples of finish red wine were also assayed for OTA content. The results showed that 42 of the tested samples (60%) were found to be positive for OTA with low levels (0.012-0.126 microg OTA L(-1)).

Comparison of steam sterilization conditions efficiency in the treatment of Infectious Health Care Waste.

Many studies show that the treatment of Infectious Health Care Waste (IHCW) in steam sterilization devices at usual operating standards does not allow for proper treatment of Infectious Health Care Waste (IHCW). Including a grinding component before sterilization allows better waste sterilization, but any hard metal object in the waste can damage the shredder. The first objective of the study is to verify that efficient IHCW treatment can occur at standard operating parameters defined by the contact time-temperature couple in steam treatment systems without a pre-mixing/fragmenting or pre-shredding step. The second objective is to establish scientifically whether the standard operation conditions for a steam treatment system including a step of pre-mixing/fragmenting were sufficient to destroy the bacterial spores in IHCW known to be the most difficult to treat. Results show that for efficient sterilization of dialysis cartridges in a pilot 60L steam treatment system, the process would require more than 20 min at 144°C without a pre-mixing/fragmenting step. In a 720L steam treatment system including pre-mixing/fragmenting paddles, only 10 min at 144°C are required to sterilize IHCW proved to be sterilization challenges such as dialysis cartridges and diapers in normal conditions of rolling.

A study on the physicochemical parameters for Penicillium expansum growth and patulin production: effect of temperature, pH, and water activity.

Penicillium expansum is among the most ubiquitous fungi disseminated worldwide, that could threaten the fruit sector by secreting patulin, a toxic secondary metabolite. Nevertheless, we lack sufficient data regarding the growth and the toxigenesis conditions of this species. This work enables a clear differentiation between the favorable conditions to the P. expansum growth and those promising for patulin production. A mathematical model allowing the estimation of the P. expansum growth rate according to temperature, a W, and pH, was also developed. An optimal growth rate of 0.92 cm/day was predicted at 24°C with pH level of 5.1 and high a W level of 0.99. The model's predictive capability was tested successfully on artificial contaminated apples. This model could be exploited by apple growers and the industrialists of fruit juices in order to predict the development of P. expansum during storage and apple processing.