Virulence and host specificity of staphylococci from Staphylococcus intermedius group of pigeon origin with an emphasis on Staphylococcus intermedius.

The Staphylococcus intermedius group (SIG) includes coagulase-positive staphylococci commonly found in animals. The taxonomic classification within the SIG has evolved with molecular techniques distinguishing five species. Despite their similarities, these species exhibit varied host affinities, with unclear implications for virulence and host interaction. This study aimed to investigate the presence of coagulase-positive staphylococci in pigeons and to detect genes encoding for selected virulence factors in isolated strains. Another goal was to determine the adhesion capabilities of randomly selected pigeon S. intermedius, S. delphini, and canine S. pseudintermedius strains to canine and pigeon corneocytes and their adhesion and invasion abilities to canine keratinocytes in vitro. In total, 121 coagulase-positive strains were isolated from domestic and feral pigeons. The most prevalent species were S. delphini B and S. intermedius in domestic and feral pigeons, respectively. We proved that pigeon strains carried genes encoding for exfoliative toxin SIET and leukotoxin Luk-I. Moreover, we found that S. intermedius showed higher adherence to pigeon than to canine corneocytes, aligning with its presumed natural host. No difference in adherence abilities of S. pseudintermedius to canine and pigeon corneocytes was observed. In this study, we also observed that S. pseudintermedius could successfully invade the canine keratinocytes, in contrary to S. delphini and S. intermedius. Moreover, only S. intermedius was not able to invade canine keratinocytes at all. These findings highlight the complex interplay between SIG bacteria, and their hosts, underscoring the need for further research to understand the mechanisms of host adaptation and pathogenicity within this group.

Staphylococcus aureus binds to the N-terminal region of corneodesmosin to adhere to the stratum corneum in atopic dermatitis.

Staphylococcus aureus colonizes the skin of the majority of patients with atopic dermatitis (AD), and its presence increases disease severity. Adhesion of S. aureus to corneocytes in the stratum corneum is a key initial event in colonization, but the bacterial and host factors contributing to this process have not been defined. Here, we show that S. aureus interacts with the host protein corneodesmosin. Corneodesmosin is aberrantly displayed on the tips of villus-like projections that occur on the surface of AD corneocytes as a result of low levels of skin humectants known as natural moisturizing factor (NMF). An S. aureus mutant deficient in fibronectin binding protein B (FnBPB) and clumping factor B (ClfB) did not bind to corneodesmosin in vitro. Using surface plasmon resonance, we found that FnBPB and ClfB proteins bound with similar affinities. The S. aureus binding site was localized to the N-terminal glycine-serine-rich region of corneodesmosin. Atomic force microscopy showed that the N-terminal region was present on corneocytes containing low levels of NMF and that blocking it with an antibody inhibited binding of individual S. aureus cells to corneocytes. Finally, we found that S. aureus mutants deficient in FnBPB or ClfB have a reduced ability to adhere to low-NMF corneocytes from patients. In summary, we show that FnBPB and ClfB interact with the accessible N-terminal region of corneodesmosin on AD corneocytes, allowing S. aureus to take advantage of the aberrant display of corneodesmosin that accompanies low NMF in AD. This interaction facilitates the characteristic strong binding of S. aureus to AD corneocytes.

Characterisation of topographical, biomechanical and maturation properties of corneocytes with respect to anatomical location.

BACKGROUND: The Stratum Corneum (SC) is the first barrier of the skin. The properties of individual cells are crucial in understanding how the SC at different anatomical regions maintains a healthy mechanical barrier. The aim of the current study is to present a comprehensive description of the maturation and mechanical properties of superficial corneocytes at different anatomical sites in the nominal dry state. MATERIALS AND METHODS: Corneocytes were collected from five anatomical sites: forearm, cheek, neck, sacrum and medial heel of 10 healthy young participants. The surface topography was analysed using Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM). The level of positive-involucrin cornified envelopes (CEs) and desmoglein-1 (Dsg1) were used as indirect measures of immature CEs and corneodesmosomes, respectively. In addition, AFM nanoindentation and stress-relaxation experiments were performed to characterise the mechanical properties. RESULTS: Volar forearm, neck and sacrum corneocytes presented similar topographies (ridges and valleys) and levels of Dsg1 (13-37%). In contrast, cheek cells exhibited circular nano-objects, while medial heel cells were characterized by villi-like structures. Additionally, medial heel samples also showed the greatest level of immature CEs (32-56%, p < 0.001) and Dsg1 (59-78%, p < 0.001). A large degree of inter-subject variability was found for the Young's moduli of the cells (0.19-2.03 GPa), which was correlated with the level of immature CEs at the cheek, neck and sacrum (p < 0.05). CONCLUSION: It is concluded that a comprehensive study of the mechanical and maturation properties of corneocytes may be used to understand the barrier functions of the SC at different anatomical sites.

Key Factors in the Complex and Coordinated Network of Skin Keratinization: Their Significance and Involvement in Common Skin Conditions.

The epidermis serves many vital roles, including protecting the body from external influences and healing eventual injuries. It is maintained by an incredibly complex and perfectly coordinated keratinization process. In this process, desquamation is essential for the differentiation of epidermal basal progenitor cells into enucleated corneocytes, which subsequently desquamate through programmed death. Numerous factors control keratinocyte differentiation: epidermal growth factor, transforming growth factor-α, keratinocyte growth factor, interleukins IL-1-ß and IL-6, elevated vitamin A levels, and changes in Ca2+ concentration. The backbone of the keratinocyte transformation process from mitotically active basal cells into fully differentiated, enucleated corneocytes is the expression of specific proteins and the creation of a Ca2+ and pH gradient at precise locations within the epidermis. Skin keratinization disorders (histologically characterized predominantly by dyskeratosis, parakeratosis, and hyperkeratosis) may be categorized into three groups: defects in the α-helical rod pattern, defects outside the α-helical rod domain, and disorders of keratin-associated proteins. Understanding the process of keratinization is essential for the pathogenesis of many dermatological diseases because improper desquamation and epidermopoiesis/keratinization (due to genetic mutations of factors or due to immune pathological processes) can lead to various conditions (ichthyoses, palmoplantar keratodermas, psoriasis, pityriasis rubra pilaris, epidermolytic hyperkeratosis, and others).

Characterisation of superficial corneocytes in skin areas of the face exposed to prolonged usage of respirators by healthcare professionals during COVID-19 pandemic.

INTRODUCTION: During the COVID-19 pandemic healthcare workers (HCWs) have used respiratory protective equipment for prolonged periods, which has been associated with detrimental effects on the underlying skin. The present study aims to evaluate changes in the main cells (corneocytes) of the stratum corneum (SC) following prolonged and consecutive use of respirators. METHODS: 17 HCWs who wore respirators daily during routine hospital practice were recruited to a longitudinal cohort study. Corneocytes were collected via tape stripping from a negative control site (area outside the respirator) and from the cheek which was in contact with the device. Corneocytes were sampled on three occasions and analysed for the level of positive-involucrin cornified envelopes (CEs) and the amount of desmoglein-1 (Dsg1), as indirect measurements of immature CEs and corneodesmosomes (CDs), respectively. These were compared to biophysical measurements (Transepidermal water loss, TEWL, and SC hydration) at the same investigation sites. RESULTS: A large degree of inter-subject variability was observed, with maximum coefficients of variation of 43% and 30% for the level of immature CEs and Dsg1, respectively. Although it was observed that there was not an effect of prolonged respirator usage on the properties of corneocytes, the level of CDs was greater at the cheek than the negative control site (p < 0.05). Furthermore, low levels of immature CEs correlated with greater TEWL values after prolonged respirator application (p < 0.01). It was also noted that a smaller proportion of immature CEs and CDs was associated with a reduced incidence of self-reported skin adverse reactions (p < 0.001). CONCLUSIONS: This is the first study that investigated changes in corneocyte properties in the context of prolonged mechanical loading following respirator application. Although differences were not recorded over time, the levels of CDs and immature CEs were consistently higher in the loaded cheek compared to the negative control site and were positively correlated with a greater number of self-reported skin adverse reactions. Further studies are required to evaluate the role of corneocyte characteristics in the evaluation of both healthy and damaged skin sites.

Accumulation of endogenous and exogenous nucleic acids in "Touch DNA" components on hands.

Successful forensic DNA profiling from handled items is increasingly routine in casework. This "touch DNA" is thought to contain both cellular and acellular nucleic acid sources. However, there is little clarity on the origins or characteristics of this material. The cellular component consists of anucleate, terminally differentiated corneocytes (assumed to lack DNA), and the occasional nucleated cell. The acellular DNA source is fragmentary, presumably cell breakdown products. This study examines the relative contributions each component makes to the hand-secretions (endogenous) and hand-accumulations (exogenous) by recovering rinses from the inside and outside of worn gloves. Additionally, cellular and acellular DNA was measured at timepoints up to 2 h after hand washing, both with and without interim contact. Microscopic examination confirmed cell morphology and presence of nucleic acids. Following the novel application of a hair keratinocyte lysis method and plasma-DNA fragment purification to hand rinse samples, DNA profiles were generated from both fractions. Exogenous cell-free DNA is shown to be a significant source of touch DNA, which reaccumulates quickly, although its amplifiable nuclear alleles are limited. Endogenous DNA is mostly cellular in origin and provides more allelic information consistently over time.

Tracking solvents in the skin through atomically resolved measurements of molecular mobility in intact stratum corneum.

Solvents are commonly used in pharmaceutical and cosmetic formulations and sanitary products and cleansers. The uptake of solvent into the skin may change the molecular organization of skin lipids and proteins, which may in turn alter the protective skin barrier function. We herein examine the molecular effects of 10 different solvents on the outermost layer of skin, the stratum corneum (SC), using polarization transfer solid-state NMR on natural abundance 13C in intact SC. With this approach it is possible to characterize the molecular dynamics of solvent molecules when present inside intact SC and to simultaneously monitor the effects caused by the added solvent on SC lipids and protein components. All solvents investigated cause an increased fluidity of SC lipids, with the most prominent effects shown for the apolar hydrocarbon solvents and 2-propanol. However, no solvent other than water shows the ability to fluidize amino acids in the keratin filaments. The solvent molecules themselves show reduced molecular mobility when incorporated in the SC matrix. Changes in the molecular properties of the SC, and in particular alternation in the balance between solid and fluid SC components, may have significant influences on the macroscopic SC barrier properties as well as mechanical properties of the skin. Deepened understanding of molecular effects of foreign compounds in SC fluidity can therefore have strong impact on the development of skin products in pharmaceutical, cosmetic, and sanitary applications.

In vivo Tracking of DNA for Precise Determination of the Stratum Corneum Thickness and Superficial Microbiome Using Confocal Raman Microscopy.

The skin barrier function is mostly provided by the stratum corneum (SC), the uppermost layer of the epidermis. To noninvasively analyze the physiological properties of the skin barrier functionin vivo, it is important to determine the SC thickness. Confocal Raman microscopy (CRM) is widely used for this task. In the present in vivo study, a new method based on the determination of the DNA concentration profile using CRM is introduced for determining the SC thickness. The obtained SC thickness values are compared with those obtained using other CRM-based methods determining the water and lipid depth profiles. The obtained results show almost no significant differences in SC thickness for the utilized methods. Therefore, the results indicate that it is possible to calculate the SC thickness by using the DNA profile in the fingerprint region, which is comparable with the SC thickness calculated by the water depth profiles (ANOVA test p = 0.77) and the lipid depth profile (ANOVA test p = 0.74). This provides the possibility to measure the SC thickness by using the DNA profile, in case the water or lipid profile analyses are influenced by a topically applied formulation. The increase in DNA concentration in the superficial SC (0-2 µm) is related to the DNA presence in the microbiome of the skin, which was not present in the SC depth below 4 µm.

Stratum corneum occlusion induces water transformation towards lower bonding state: a molecular level in vivo study by confocal Raman microspectroscopy.

OBJECTIVE: It is conventionally understood that occlusive effects are the retention of excessive water in the stratum corneum (SC), the increase of SC thickness (swelling) and a decrease of the transepidermal water loss. However, the influence of occlusion on water binding properties in the SC is unknown. METHODS: The action of plant-derived jojoba and almond oils, as well as mineral-derived paraffin oil and petrolatum topically applied on human skin, is investigated in vivo using confocal Raman microspectroscopy. To understand the oils' influence on the SC on the molecular level, the depth-dependent hydrogen bonding states of water in the SC and their relationship to the conformation of keratin, concentration of natural moisturizing factor (NMF) molecules and lipid organization were investigated. RESULTS: A significant SC swelling was observed only in petrolatum-treated skin. The water concentration was increased in oil-treated skin in the intermediate SC region (40-70% SC depth). Meanwhile, the amount of free, weakly and tightly bound water increased, and strongly bound water decreased in the uppermost SC region (0-30% SC depth). The NMF concentration of oil-treated skin was significantly lower at 50-70% SC depth. The lateral organization of lipids in oil-treated skin was lower at 0-30% SC depth. The secondary structure of keratin was changed towards an increase of ß-sheet content in mineral-derived oil-treated skin and changed towards an increase of α-helix content in plant-derived oil-treated skin. CONCLUSION: The occlusive properties can be summarized as the increase of free water and the transformation of water from a more strongly to a more weakly hydrogen bonding state in the uppermost SC, although some oils cause insignificant changes of the SC thickness. The accompanied changes in the keratin conformation at the intermediate swelling region of the SC also emphasize the role of keratin in the SC's water-transporting system, that is the water in the SC transports intercellularly and intracellularly in the intermediate swelling region and only intercellularly in the uppermost non-swelling region. Bearing this in mind, almond, jojoba and paraffin oils, which are not occlusive from the conventional viewpoint, have an occlusion effect similar to petrolatum on the SC.

OBJECTIF: Il est généralement entendu que les effets occlusifs consistent en la rétention d'un excès d'eau dans la couche cornée (stratum corneum, SC), l'augmentation d'épaisseur de la SC (gonflement) et une diminution de la perte d'eau trans-épidermique. Cependant, l'influence de l'occlusion sur les propriétés de fixation de l'eau dans le SC est inconnue. MÉTHODES: L'action des huiles de jojoba et d'amande d'origine végétale, ainsi que des huiles de paraffine et de pétrolatum d'origine minérale appliquées topiquement sur la peau humaine est étudiée in vivo à l'aide de la microspectroscopie Raman confocale. Pour comprendre l'influence des huiles sur le SC au niveau moléculaire, on a étudié les états de liaison hydrogène de l'eau dans le SC en fonction de la profondeur et leur relation avec la conformation de la kératine, la concentration des molécules du facteur naturel d'hydratation (NMF) et l'organisation des lipides. RÉSULTATS: Un gonflement significatif de le SC n'a été observé que dans la peau traitée au pétrolatum. La concentration en eau a été augmentée dans la peau traitée au pétrolatum dans la région SC intermédiaire (40-70% de profondeur du SC). En meme temps, la quantité d'eau libre, faiblement et fortement liée augmentait, tandis que l'eau fortement liée diminuait dans la région SC supérieure (0-30% de profondeur du SC). La concentration en NMF de la peau traitée à l'huile était plus basse d´une manière significative à 50-70% de profondeur du SC. L'organisation latérale des lipides dans la peau huilée était plus basse à une profondeur du SC de 0 à 30 %. La structure secondaire de la kératine a été modifiée pour augmenter la teneur en feuillet-ß dans les peaux huilées d'origine minérale et pour augmenter la teneur en hélice α dans les peaux huilées d'origine végétale. CONCLUSION: Les propriétés occlusives peuvent être résumées comme l'augmentation de l'eau libre et la transformation de l'eau d'un état de liaison hydrogène plus fort à un état de liaison hydrogène plus faible dans le SC supérieure, bien que certaines huiles provoquent des changements insignifiants de l'épaisseur de la SC. Les modifications de la conformation de la kératine dans la zone de gonflement intermédiaire du SC soulignent également le rôle de la kératine dans le système de transport de l'eau du SC, c'est-à-dire que l'eau est transportée du SC de manière intercellulaire et intracellulaire dans la zone de gonflement intermédiaire et seulement de manière intercellulaire dans la zone non gonflée la plus élevée. En considérant cela, les huiles d'amande, de jojoba et de paraffine, qui ne sont pas occlusives du point de vue conventionnel, ont un effet d'occlusion similaire à celui du pétrolatum sur le SC.

The impact of carbonylated proteins on the skin and potential agents to block their effects.

Carbonylated proteins (CPs) are synthesized by reactions between amino groups in proteins and reactive aldehyde compounds (RAC) yielded from lipid peroxidation initiated by reactive oxygen species (ROS). In the skin, CPs are detected in a higher frequency at sun-exposed sites of the skin in elderly subjects. Since CPs in the stratum corneum (SC) have been reported to correlate with skin water content and transepidermal water loss, it is considered that the accumulation of CPs in the SC involves the loss of skin moisture functions. However, the roles of CPs in the dermis on skin physiology are still unclear. The purpose of this study was to investigate the roles of CPs in the dermis during the progression of photoaged skin and to propose a method to prevent or reduce the synthesis of CPs. The exposure of human normal dermal fibroblasts to CPs increased intracellular ROS levels and the synthesis of intracellular CPs. In addition, CPs caused morphological changes of fibroblasts. Furthermore, CPs caused alterations of mRNA expression levels of dermal matrix-related proteins, such as upregulating MMP-1 and IL-8. These results indicated that CPs disrupt construction of the dermal matrix. On the other hand, α-tocopherol and ß-carotene suppressed the synthesis of RAC during lipid peroxidation which resulted in the reduction of UVA-induced CPs in the SC. From these results, we propose that extracellular CPs increase intracellular ROS levels and contribute to alterations of the dermal matrix. To prevent the synthesis of CPs, the application of α-tocopherol or ß-carotene could be effective.

Quantitative Analysis of Free Amino Acids and Urea Derived from Isolated Corneocytes of Healthy Young, Healthy Aged, and Diseased Skin.

BACKGROUND/AIMS: Free amino acids (FAAs) and urea, present inside the corneocytes, can be important indicators of skin condition. However, due to the lack of a standard extraction protocol for FAAs from corneocytes, conflicting research results have been reported. Therefore, the purpose of this study was (1) to standardize the extraction protocol and (2) to investigate FAA profiles in healthy young and healthy old volunteers, as well as in psoriasis and atopic dermatitis patients. METHODS: Skin samples were collected from four groups (healthy young, healthy old, and psoriasis and atopic dermatitis patients) with 5 volunteers per group. Corneocytes were isolated and examined microscopically. FAAs and urea were extracted from the isolated corneocytes, and their amounts were quantified using LC-ESI/MS/MS (after derivatization with Fmoc-Cl) and colorimetric methods, respectively. RESULTS: The micrographs of the corneocytes showed no morphological features attributable to age or disease conditions. The highest and lowest concentrations of total FAAs and urea were observed in the healthy old group and the healthy young group, respectively. Unlike the other FAAs and urea, citrulline was found at a higher level in the healthy young group than in the disease groups. CONCLUSION: This study suggests that the levels of FAAs and urea in the skin are affected by age and skin conditions (healthy/diseased). However, further studies are needed to show the effects of different skin conditions on the levels of FAAs and urea.

Clumping Factor B Promotes Adherence of Staphylococcus aureus to Corneocytes in Atopic Dermatitis.

Staphylococcus aureus skin infection is a frequent and recurrent problem in children with the common inflammatory skin disease atopic dermatitis (AD). S. aureus colonizes the skin of the majority of children with AD and exacerbates the disease. The first step during colonization and infection is bacterial adhesion to the cornified envelope of corneocytes in the outer layer, the stratum corneum. Corneocytes from AD skin are structurally different from corneocytes from normal healthy skin. The objective of this study was to identify bacterial proteins that promote the adherence of S. aureus to AD corneocytes. S. aureus strains from clonal complexes 1 and 8 were more frequently isolated from infected AD skin than from the nasal cavity of healthy children. AD strains had increased ClfB ligand binding activity compared to normal nasal carriage strains. Adherence of single S. aureus bacteria to corneocytes from AD patients ex vivo was studied using atomic force microscopy. Bacteria expressing ClfB recognized ligands distributed over the entire corneocyte surface. The ability of an isogenic ClfB-deficient mutant to adhere to AD corneocytes compared to that of its parent clonal complex 1 clinical strain was greatly reduced. ClfB from clonal complex 1 strains had a slightly higher binding affinity for its ligand than ClfB from strains from other clonal complexes. Our results provide new insights into the first step in the establishment of S. aureus colonization in AD patients. ClfB is a key adhesion molecule for the interaction of S. aureus with AD corneocytes and represents a target for intervention.

Multifactorial skin barrier deficiency and atopic dermatitis: Essential topics to prevent the atopic march.

Atopic dermatitis (AD) is the most common inflammatory skin disease in the industrialized world and has multiple causes. Over the past decade, data from both experimental models and patients have highlighted the primary pathogenic role of skin barrier deficiency in patients with AD. Increased access of environmental agents into the skin results in chronic inflammation and contributes to the systemic "atopic (allergic) march." In addition, persistent skin inflammation further attenuates skin barrier function, resulting in a positive feedback loop between the skin epithelium and the immune system that drives pathology. Understanding the mechanisms of skin barrier maintenance is essential for improving management of AD and limiting downstream atopic manifestations. In this article we review the latest developments in our understanding of the pathomechanisms of skin barrier deficiency, with a particular focus on the formation of the stratum corneum, the outermost layer of the skin, which contributes significantly to skin barrier function.

Lipidomic analysis of epidermal lipids: a tool to predict progression of inflammatory skin disease in humans.

INTRODUCTION: Lipidomics is the large-scale profiling and characterization of lipid species in a biological system using mass spectrometry. The skin barrier is mainly comprised of corneocytes and a lipid-enriched extracellular matrix. The major skin lipids are ceramides, cholesterol and free fatty acids (FFA). Lipid compositions are altered in inflammatory skin disorders with disrupted skin barrier such as atopic dermatitis (AD). AREAS COVERED: Here we discuss some of the recent applications of lipidomics in human skin biology and in inflammatory skin diseases such as AD, psoriasis and Netherton syndrome. We also review applications of lipidomics in human skin equivalent and in pre-clinical animal models of skin diseases to gain insight into the pathogenesis of the skin disease. Expert commentary: Skin lipidomics analysis could be a fast, reliable and noninvasive tool to characterize the skin lipid profile and to monitor the progression of inflammatory skin diseases such as AD.

Cell surface glycans in the human stratum corneum: distribution and depth-related changes.

During the formation of the stratum corneum (SC) barrier, the extracellular spaces of viable epidermis, rich in glycans, are filled with a highly organized lipid matrix and the plasma membranes of keratinocytes are replaced by cornified lipid envelopes. These structures comprise cross-linked proteins, including transmembrane glycoproteins and proteoglycans, covalently bound to a monolayer of cell surface ceramides. Little is known about the presence and distribution of glycans on the SC corneocytes despite their possible involvement in SC hydration, cohesion and desquamation. In this work, we visualized ultrastructurally and quantified the distribution of glycans on the surface of native and delipidated corneocytes. The cells were harvested at different depths of the SC, allowing us to define the relationship between the distribution of various glycans, proteoglycans and glycoproteins, and other changes occurring in SC. At the cell periphery, we found a correlation between the depth-related alterations of corneodesmosome glycoproteins and α-d-mannosyl and N-acetyl-d-glucosamine-labelling patterns. Elimination of the terminal sugars, α-linked fucose and α-(2,3) linked sialic acid, was less abrupt, but also the initial extent of their peripheral distribution was overall lower than that of concanavalin A and wheat germ agglutinin lectin-detected glycans. Diffuse labelling of heparan sulphate glycosaminoglycans disappeared completely from the outermost corneocytes, whereas that of several simple carbohydrates could be detected at all SC levels. Our results suggest that specific glycan distribution may participate in the progressive changes of SC, as it evolves from the SC compactum to the SC disjunctum, towards desquamation.

Staphylococcus aureus skin colonization is mediated by SasG lectin variation.

Staphylococcus aureus causes the majority of skin and soft tissue infections, but this pathogen only transiently colonizes healthy skin. However, this transient skin exposure enables S. aureus to transition to infection. The initial adhesion of S. aureus to skin corneocytes is mediated by surface protein G (SasG). Here, phylogenetic analyses reveal the presence of two major divergent SasG alleles in S. aureus: SasG-I and SasG-II. Structural analyses of SasG-II identify a nonaromatic arginine in the binding pocket of the lectin subdomain that mediates adhesion to corneocytes. Atomic force microscopy and corneocyte adhesion assays indicate that SasG-II can bind to a broader variety of ligands than SasG-I. Glycosidase treatment results in different binding profiles between SasG-I and SasG-II on skin cells. In addition, SasG-mediated adhesion is recapitulated using differentiated N/TERT keratinocytes. Our findings indicate that SasG-II has evolved to adhere to multiple ligands, conferring a distinct advantage to S. aureus during skin colonization.

Changing human hair fibre colour and shape from the follicle.

INTRODUCTION: Natural hair curvature and colour are genetically determined human traits, that we intentionally change by applying thermal and chemical treatments to the fibre. Presently, those cosmetic methodologies act externally and their recurrent use is quite detrimental to hair fibre quality and even to our health. OBJECTIVES: This work represents a disruptive concept to modify natural hair colour and curvature. We aim to model the fibre phenotype as it is actively produced in the follicle through the topical delivery of specific bioactive molecules to the scalp. METHODS: Transcriptome differences between curly and straight hairs were identified by microarray. In scalp samples, the most variable transcripts were mapped by in situ hybridization. Then, by using appropriate cellular models, we screened a chemical library of 1200 generic drugs, searching for molecules that could lead to changes in either fibre colour or curvature. A pilot-scale, single-centre, investigator-initiated, prospective, blind, bilateral (split-scalp) placebo-controlled clinical study with the intervention of cosmetics was conducted to obtain a proof of concept (RNEC n.92938). RESULTS: We found 85 genes transcribed significantly different between curly and straight hair, not previously associated with this human trait. Next, we mapped some of the most variable genes to the inner root sheath of follicles, reinforcing the role of this cell layer in fibre shape moulding. From the drug library screening, we selected 3 and 4 hits as modulators of melanin synthesis and gene transcription, respectively, to be further tested in 33 volunteers. The intentional specific hair change occurred: 8 of 14 volunteers exhibited colour changes, and 16 of 19 volunteers presented curvature modifications, by the end of the study. CONCLUSION: The promising results obtained are the first step towards future cosmetics, complementary or alternative to current methodologies, taking hair styling to a new level: changing hair from the inside out.

Detection of Latent DNA Using a DNA Binding Dye.

Latent DNA can be deposited every time a person holds or touches an item. This "touch DNA" can be crucial evidence if the item is of forensic significance. Until very recently, there were no means to visualize this DNA. The advent of using a dye that binds to DNA has opened up this possibility. The application of the dye is simple to perform, and a mobile microscope allows rapid visualization of the cellular material, even in ambient light. The dye can be applied in a solution of either 75% ethanol or water. As this is a solution-based dye, the application works best on non-absorbent surfaces.DNA within cellular material, such as dead skin cells, appears as green dots under 50X magnification; zooming to 220X magnification confirms that these are cells. The location and number of these cells can be photographed allowing a record of the presence of otherwise latent DNA.This chapter details the processes involved in the detection of latent DNA using Diamond™ Nucleic Acid Dye with both control samples (that act as very effective training samples) and the staining of evidential items. By developing skills in determining cell locations, a targeted approach to crime scene collection is now possible.

Characterisation of superficial corneocyte properties over category I pressure ulcers: Insights into topographical and maturation changes.

BACKGROUND: Pressure ulcers (PUs) are chronic wounds that are detrimental to the quality of life of patients. Despite advances in monitoring skin changes, the structure and function of skin cells over the site of pressure ulcers are not fully understood. OBJECTIVE: The present study aims to evaluate local changes in the properties of superficial corneocytes in category 1 PU sites sampled from a cohort of hospitalised patients. METHODS: Cells were collected from a PU-compromised site and an adjacent control area and their topographical, maturation and mechanical properties were analysed. RESULTS: Corneocytes at the PU-compromised site were characterised by higher levels of immature cornified envelopes (p < 0.001) and greater amounts of desmoglein-1 (corneodesmosomal protein) (p < 0.001) compared to the adjacent control area. The cells at the control site presented the typical ridges-and-valleys topographical features of sacrum corneocytes. By contrast, the PU cells presented circular nano-objects at the cell surface, and, for some patients, the cell topography was deformed. CEs at the PU site were also smaller than at the control site. Although differences were not observed in the mechanical properties of the cells, those of the elderly patients were much softer compared with young subjects. CONCLUSION: This is the first study investigating the changes in corneocyte properties in category I pressure ulcers. Superficial cells at the PU sites showed altered topographical and maturation characteristics. Further studies are required to elucidate if these changes are a consequence of early loss of skin integrity or a result of mechanical and microclimate insults to the skin surface.

Staphylococcal Corneocyte Adhesion: Assay Optimization and Roles of Aap and SasG Adhesins in the Establishment of Healthy Skin Colonization.

Staphylococcus aureus is an opportunistic pathogen that causes the majority of wound and soft tissue infections. The accumulation-associated protein (Aap) from S. epidermidis and surface protein G (SasG) from S. aureus are cell wall-anchored (CWA) proteins known to be important in adhesion to healthy corneocytes from human skin. We investigated the mechanisms by which S. aureus colonizes healthy human skin by developing an optimized corneocyte adhesion assay. Trypan blue was used for enhanced red autofluorescent visualization of corneocytes with an overlay of green-fluorescent bacteria. The percent area of bacterial adhesion for images acquired by a fluorescence microscope was quantified using Fiji ImageJ. Using this optimized imaging procedure, differences in adhesion between various species and strains of staphylococci were measured. The ability of purified SasG to reduce Staphylococcus epidermidis adhesion was investigated in order to determine if these CWA proteins can compete for binding sites. To further test CWA-mediated adhesion, we engineered a nonadhering S. carnosus strain to express full-length SasG from two methicillin-resistant S. aureus (MRSA) strains. Finally, we demonstrated that the SasG A domain was a critical region of this surface protein for adherence to healthy human corneocytes. The developed imaging and expression methods are useful for studying staphylococcal adhesion to healthy human skin and have the potential to be used with a wide variety of fluorescently labeled organisms on both healthy and disease-state (such as atopic dermatitis) corneocytes. IMPORTANCE The skin is the largest organ of the human body and acts as a shield against hazards such as harmful bacteria like Staphylococcus aureus. A diverse skin microbiota and immune cross talk control S. aureus numbers. S. aureus can bind to healthy skin and subsequently proliferate when the skin barrier is compromised, such as in a wound or in patients with atopic dermatitis (AD). It is important to understand these mechanisms in an effort to prevent pathogenic bacteria from causing infection. We describe an augmented corneocyte adhesion assay using fluorescence microscopy to study binding of various staphylococcal species to healthy human skin cells. In addition, we tested the ability of homologous proteins from different staphylococcal species to reduce binding, and developed a new S. carnosus expression system to test individual protein binding properties. Our newly developed methods and findings will enhance the understanding of how staphylococci bind to healthy human skin.