Improved performance of the SH test as an in vitro skin sensitization test with a new predictive model and decision tree.

Demands for the elimination and replacement of animal experiments for cosmetic safety assessment have increased in recent years. Evaluation of skin sensitization, however, is a critical issue in cosmetic safety assessment. The SH test is an in vitro skin sensitization test method that evaluates protein binding of chemical substances, which is an important event in skin sensitization. We previously verified the technical transferability and between-laboratory reproducibility of the SH test, a domestic test method for which no scientific research has been conducted, and improved the protocol, but also noted some unresolved issues. Therefore, in the present study, we successfully improved the operational efficiency and clarity of the final judgment of the SH test by (i) developing a new decision-making system that can make a final judgment without statistical processing, (ii) changing the statistical method, and (iii) evaluating and determining the maximum number of repetitions necessary for optimal efficiency. The improved SH test was verified by comparing it with existing test methods already adopted by the Organization for Economic Cooperation and Development. The results of this study demonstrated excellent performance of the improved SH test, with high reproducibility, reliable predictability, and good operational efficiency. The predictive performance of the improved method does not differ significantly from that of the conventional method, although it is clearer and more efficient. Therefore, the results of the present improved method are consistent with those obtained using the conventional method, with higher efficiency.

Development of a skin microbiome diagnostic method to assess skin condition in healthy individuals: Application of research on skin microbiomes and skin condition.

OBJECTIVE: Skin microbiomes vary across individuals. They are known to play essential roles in maintaining homeostasis and preventing infectious pathogens. In recent years, cosmetic product development has begun to focus on the relationship between skin microbiomes and skin conditions. However, the statistical methods used in many studies include the standard t-test and small-scale correlation analysis, which do not take into account the internal correlation structure in data on skin microbiomes and skin features. In this study, we aimed to understand the relationship between skin microbiomes and skin features by analysing complex microbiomes and skin data. METHODS: We obtained data on 19 skin characteristics and skin microbiomes based on 16s ribosomal RNA (16S rRNA) gene analysis of 276 healthy Japanese women. We then performed the principal component analysis (PCA), a method that takes into account the internal correlation structure, on 234 panels of them that did not contain outliers or missing values. We confirmed the relationship between skin microbiomes and skin features with principal component regression analysis and hierarchical clustering analysis (HCA). RESULTS: The principal component regression analysis showed strong relationships between skin microbiomes and sebum-related skin characteristics and skin pH. In the HCA, the female panel was classified into two major groups based on the skin microbiome. Furthermore, there were significant differences in sebum-related skin characteristics and the way skin condition changes with ageing between those groups, suggesting the possibility of measuring skin condition and age-related skin risk based on microbiome data. In addition, sebum-related characteristics differed significantly among middle-aged participants, suggesting a strong relationship between skin microbiomes and sebum-related characteristics. CONCLUSION: Analysis of skin condition and skin microbiome in Japanese women, taking into account the correlation between variables, showed that skin microbiome was significantly related to the number of pores and the amount of sebum. Furthermore, it was suggested that the skin condition and the way the skin condition changes with ageing may differ depending on the type of skin microbiome. The finding of a relationship between skin condition and skin microbiome suggests the possibility of proposing a new beauty method focusing on the skin microbiome in the future.

OBJECTIF: Les microbiomes de la peau varient selon les individus. Ils sont connus pour jouer des rôles essentiels dans le maintien de l'homéostasie et la prévention des agents pathogènes infectieux. Ces dernières années, le développement de produits cosmétiques a commencé à se concentrer sur la relation entre les microbiomes cutanés et les conditions de la peau. Cependant, les méthodes statistiques utilisées dans de nombreuses études comprennent le t-test standard et l'analyse de corrélation à petite échelle, qui ne tiennent pas compte de la structure de corrélation interne dans les données sur les microbiomes cutanés et les caractéristiques de la peau. Dans cette étude, nous avons cherché à comprendre la relation entre les microbiomes cutanés et les caractéristiques de la peau en analysant des données complexes sur les microbiomes et la peau. MÉTHODES: Nous avons obtenu des données sur 19 caractéristiques de la peau et sur les microbiomes cutanés à partir de l'analyse du gène de l'ARNr 16S (16S rRNA) de 276 femmes japonaises en bonne santé. Nous avons ensuite effectué l'analyse en composantes principales (PCA: principal component analysis), une méthode qui prend en compte la structure de corrélation interne, sur 234 d'entre elles qui ne contenaient pas de valeurs aberrantes ou manquantes. Nous avons confirmé la relation entre les microbiomes cutanés et les caractéristiques de la peau à l'aide d'une analyse de régression en composantes principales et d'une analyse de regroupement hiérarchique (HCA: hierarchical clustering analysis). RÉSULTATS: L'analyse de régression en composantes principales a montré des relations fortes entre les microbiomes cutanés et les caractéristiques de la peau liées au sébum et au pH de la peau. Dans l'étude HCA, le panel de femmes a été classé en deux grands groupes sur la base du microbiome cutané. En outre, il y avait des différences significatives dans les caractéristiques de la peau liées au sébum et dans la façon dont l'état de la peau change avec l'âge entre ces groupes, ce qui suggère la possibilité de mesurer l'état de la peau et le risque cutané lié à l'âge à partir des données du microbiome. En outre, les caractéristiques liées au sébum différaient de manière significative chez les participants d'âge moyen, ce qui suggère une forte relation entre les microbiomes cutanés et les caractéristiques liées au sébum. CONCLUSION: L'analyse de l'état de la peau et du microbiome cutané chez les femmes japonaises, en tenant compte de la corrélation entre les variables, a montré que le microbiome cutané était significativement lié au nombre de pores et à la quantité de sébum. En outre, il a été suggéré que l'état de la peau et la façon dont l'état de la peau évolue avec le vieillissement peuvent différer en fonction du type de microbiome cutané. La découverte d'une relation entre l'état de la peau et le microbiome cutané suggère la possibilité de proposer à l'avenir une nouvelle méthode de beauté axée sur le microbiome cutané.

Enhancing between-facility reproducibility of the SH test as an in vitro skin sensitization test by the improved test method.

There has been an increased demand to eliminate animal experiments and to replace the experiments with alternative tests for assessing the safety of cosmetics. The SH test is an in vitro skin sensitization test that evaluates the protein binding abilities of a test substance. Skin sensitization must be evaluated by multiple test methods. The SH test uses the same cell line and measuring instruments as the human Cell-Line Activation Test (h-CLAT), which is one of the test methods used to evaluate different key events and is listed in the OECD test guidelines. There are cost advantages to usher the SH test into facilities that are already running the h-CLAT. The SH test is conducted only at a facility that has developed the SH test because studies on the between-facility reproducibility and validity have not been performed. Therefore, to verify the transferability of the SH test and the between-facilities reproducibility, we evaluated the reproducibility of the SH test results at three facilities, including the development facility. After an initial round of testing, the protocol was refined as follows to improve reproducibility among the three facilities: i) determine the optimum pH range, ii) change the maximum applicable concentration of water-soluble substances, and iii) define the appropriate dispersion conditions for evaluating hydrophobic substances. These refinements markedly enhanced the between-facility reproducibility (from 76.0% to 96.0%) for the 25 substances evaluated in this study. This study confirmed that the SH test is an effective skin sensitization test method with high technical transferability and between-facility reproducibility.

Prophage-triggered membrane vesicle formation through peptidoglycan damage in Bacillus subtilis.

Bacteria release membrane vesicles (MVs) that play important roles in various biological processes. However, the mechanisms of MV formation in Gram-positive bacteria are unclear, as these cells possess a single cytoplasmic membrane that is surrounded by a thick cell wall. Here we use live cell imaging and electron cryo-tomography to describe a mechanism for MV formation in Bacillus subtilis. We show that the expression of a prophage-encoded endolysin in a sub-population of cells generates holes in the peptidoglycan cell wall. Through these openings, cytoplasmic membrane material protrudes into the extracellular space and is released as MVs. Due to the loss of membrane integrity, the induced cells eventually die. The vesicle-producing cells induce MV formation in neighboring cells by the enzymatic action of the released endolysin. Our results support the idea that endolysins may be important for MV formation in bacteria, and this mechanism may potentially be useful for the production of MVs for applications in biomedicine and nanotechnology.It is unclear how Gram-positive bacteria, with a thick cell wall, can release membrane vesicles. Here, Toyofuku et al. show that a prophage-encoded endolysin can generate holes in the cell wall through which cytoplasmic membrane material protrudes and is released as vesicles.

Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms.

Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs.

Bacterial membrane vesicles, an overlooked environmental colloid: Biology, environmental perspectives and applications.

Phospholipid vesicles play important roles in biological systems. Bacteria are one of the most abundant organisms on Earth, and bacterial membrane vesicles (MVs) were first observed 50 years ago. Many bacteria release MVs to the environment that mainly consist of the cell membrane and typically range from 20 to 400 nm in size. Bacterial MVs are involved in several biological functions, such as delivery of cargo, virulence and gene transfer. MVs can be isolated from laboratory culture and directly from the environment, indicating their high abundance in and impact on ecosystems. Many colloidal particles in the environment ranging in size from 1 nm to 1 µm have been reported but not characterized at the molecular level, and MVs remain to be explored. Hence, MVs can be considered terra incognita in environmental colloid research. Although MV biogenesis and biological roles are yet to be fully understood, the accumulation of knowledge has opened new avenues for their applications. Via genetic engineering, the MV yield can be greatly increased, and the components of MVs can be tailored. Recent studies have demonstrated that MVs have promising potential for applications such as drug delivery systems and nanobiocatalysts. For instance, MV vaccines have been extensively studied and have already been approved in Europe. Recent MV studies have evoked great interest in the fields of biology and biotechnology, but fundamental questions, such as their transport in the environment or physicochemical features of MVs, remain to be addressed. In this review, we present the current understanding of bacterial MVs and environmental perspectives and further introduce their applications.