Advancements in non-invasive skin sampling: Clinical conditions characterization via the assessment of skin surface cytokine biomarkers.

The skin is increasingly recognized as a biological active organ interacting with the immune system. Given that the epidermal skin layer actively releases various cytokines, non-invasive skin sampling methods could detect these cytokines, offering insights into clinical conditions. This study aims non-invasively measuring cytokine levels directly from the skin surface to characterize different inflammatory chronic disorders in the adult and elderly population: psoriasis, diabetes type 2, rosacea, chronic kidney disease (CKD) and aging. Cytokines IL-1ß, IL-8 and IL-10 were sampled from healthy subjects and patients aged 18-80 using skin surface wash technique. A well with sterile phosphate-buffered saline solution was placed on the skin for 30 min, and the extracted solution was collected from the well for further cytokine levels analysis using ELISA assay. Results show distinct cytokine profiles in different pathological processes, healthy controls, affected and unaffected areas. Aging was associated with increased IL-1ß, IL-8, and IL-10 levels in skin. In diabetes, IL-1ß and IL-8 levels were elevated in lesional areas, while IL-10 levels were decreased in non-lesional skin. Psoriatic lesions showed elevated levels of IL-1ß and IL-8. Rosacea patients had lower IL-10 levels in both lesional and non-lesional areas. CKD patients exhibited significantly lower IL-10 levels compared to healthy individuals. In conclusion, skin surface wash-derived cytokine profiles could serve as "alert biomarkers" for disease prediction, enabling early detection. Additionally, this method's cost-effectiveness allows pre-screening of molecules in clinical studies and holds potential as a tool for biomarkers and omics analysis, enhancing disorder characterization and disease management.

Development of Powdery Mildew Resistance in Cucumber Using CRISPR/Cas9-Mediated Mutagenesis of CsaMLO8.

Powdery mildew (PM) diseases may severely limit the production of various crops, including members of the family Cucurbitaceae. Successful PM infection relies on the Mildew Resistance Locus O (MLO) plant gene family, which encodes susceptibility factors essential for fungus penetration into the host cell. In cucumber (Cucumis sativus), natural mutations in CsaMLO8 confer resistance to the PM pathogen Podosphaera xanthii. Here, we used CRISPR/Cas9-mediated mutagenesis to generate PM resistance in the susceptible cucumber cultivar Ilan. Two transgene-free Csamlo8 CRISPR mutant lines (Csamlo-cr-1 and Csamlo-cr-2) were isolated, the first with a 5-bp deletion in exon 1, and the second harboring a 1,280-bp deletion and 10-bp insertion between exons 1 and 5. Both lines showed high resistance to PM under semicommercial growth conditions in the summer growing seasons of 2019 and 2021. These results provide the basis for generating transgene-free powdery mildew resistance in cucumber in any genetic background. This method can directly be employed on commercial cultivars and hybrid parental lines, and thereby substantially shorten and simplify the breeding process for PM resistance in cucumber.

Molecular epidemiology of non-syndromic autosomal recessive congenital ichthyosis in a Middle-Eastern population.

Autosomal recessive congenital ichthyosis (ARCI) is a rare and heterogeneous skin cornification disorder presenting with generalized scaling and varying degrees of erythema. Clinical manifestations range from lamellar ichthyosis (LI), congenital ichthyosiform erythroderma (CIE) through the most severe form of ARCI, Harlequin ichthyosis (HI). We used homozygosity mapping, whole-exome and direct sequencing to delineate the relative distribution of pathogenic variants as well as identify genotype-phenotype correlations in a cohort of 62 Middle Eastern families with ARCI of various ethnic backgrounds. Pathogenic variants were identified in most ARCI-associated genes including TGM1 (21%), CYP4F22 (18%), ALOX12B (14%), ABCA12 (10%), ALOXE3 (6%), NIPAL4 (5%), PNPLA1 (3%), LIPN (2%) and SDR9C7 (2%). In 19% of cases, no mutation was identified. Our cohort revealed a higher prevalence of CYP4F22 and ABCA12 pathogenic variants and a lower prevalence of TGM1 and NIPAL4 variants, as compared to data obtained in other regions of the world. Most variants (89%) in ALOX12B were associated with CIE and were the most common cause of ARCI among patients of Muslim origin (26%). Palmoplantar keratoderma associated with fissures was exclusively a result of pathogenic variants in TGM1. To our knowledge, this is the largest cohort study of ARCI in the Middle-Eastern population reported to date. Our data demonstrate the importance of population-tailored mutation screening strategies and shed light upon specific genotype-phenotype correlations.

Dead Sea minerals: New findings on skin and the biology beyond.

BACKGROUND: Therapeutic effects of Dead Sea (DS) minerals are well established, and their unique combination is analysed and reported. DS water (DSW) is a key source for DS minerals, and various studies report the capability of DSW to alleviate symptoms of different skin disorders and to contribute to skin maintenance. However, the biological mechanisms beyond reported effects are not fully understood yet. OBJECTIVE: To elucidate the effect of topically applied DSW via the expression of different skin biomarkers related to barrier function, homeostasis, inflammation and irritation. METHODS: In vitro skin equivalents and ex vivo human skin organ culture were used to assess the biological effects of DSW. Epidermal barrier protein expression and DSW ions transdermal penetration were analysed on skin equivalents. ß-endorphin secretion was tested on human skin organ culture. The capability of DSW to protect against skin inflammation and irritation was tested on ex vivo human skin organ culture by lipopolysaccharides and sodium dodecyl sulphate addition, respectively. RESULTS: Topical application of DSW encouraged the expression of the barrier-related proteins: filaggrin, involucrin and transglutaminase, while transdermal penetration of calcium ions was not detected. Additionally, DSW application had increased skin secretion of ß-endorphin and attenuated the expression of inflammatory and irritation-related cytokines. CONCLUSIONS: This study reports new findings of DSW effects on skin. Signalling pathway activation is proposed as a key step that may result in a vast range of proven biological activities following skin exposure to DS minerals, and specifically DSW.

Integration of Inverse Supercritical Fluid Extraction and Miniaturized Asymmetrical Flow Field-Flow Fractionation for the Rapid Analysis of Nanoparticles in Sunscreens.

We report the use of inverse supercritical fluid extraction (SFE) and miniaturized asymmetrical flow field-flow fractionation (mAF4) for the preparation and subsequent analysis of titanium dioxide nanoparticles in model and commercial sunscreens. The approach allows for the fast and reliable fractionation and sizing of TiO2 nanoparticles and their quantitation in commercial products. This new method represents a powerful and efficient tool for the verification of nanoparticle content in a wide range of matrixes, as demanded by recently introduced regulatory requirements. Furthermore, the use of carbon dioxide as an environmentally friendly solvent is in line with the increasing need for ecologically compatible analytical techniques.

Pancreatic Beta-Cell Proliferation Induced by Estradiol-17ß is Foxo1 Dependent.

Estradiol-17ß (E2) and the Foxo1 transcription factor have each been implicated in the regulation of ß-cell proliferation. Interaction between Foxo1and estrogen receptor alpha (ERα), effecting cell cycle, has been demonstrated in breast cancer cells, but has not been studied thus far in ß-cells. Using human islets and the INS1-E ß-cell line, this study investigated the contribution of Foxo1 to E2-mediated ß-cell replication. Foxo1 expression was knocked down in INS1-E cells using siRNA and Foxo1 activity was inhibited in human islets with a specific Foxo1 inhibitor (AS1842856). Cells were treated with E2 and the ERα agonist PPT and evaluated for proliferation by 3[H]-thymidine incorporation and for transcriptional activity through the estrogen response element by the luciferase assay. As Foxo1 activity is regulated by post-translational modifications, the effect of E2 on phosphorylation was also assessed. In INS1-E cells, knock down of Foxo1 abrogated the proliferative response to E2 and PPT. In human islets, inhibition of Foxo1 abrogated E2-mediated proliferation and attenuated the response to PPT. Foxo1 knock down and inhibition reduced activity through the estrogen response element by 25% (p<0.05) and 50% (p<0.01) respectively, in INS1-E cells. E2 increased Foxo1 phosphorylation in a time dependent manner in INS1-E and human islets (p<0.01, p<0.05, respectively). These findings suggest that Foxo1 is involved in E2-mediated proliferation in INS1-E cells and human islets. This may have implications vis-à-vis variations in circulating endogenous E2 concentrations in diabetes.

SVEP1 plays a crucial role in epidermal differentiation.

SVEP1 is a recently identified multidomain cell adhesion protein, homologous to the mouse polydom protein, which has been shown to mediate cell-cell adhesion in an integrin-dependent manner in osteogenic cells. In this study, we characterized SVEP1 function in the epidermis. SVEP1 was found by qRT-PCR to be ubiquitously expressed in human tissues, including the skin. Confocal microscopy revealed that SVEP1 is normally mostly expressed in the cytoplasm of basal and suprabasal epidermal cells. Downregulation of SVEP1 expression in primary keratinocytes resulted in decreased expression of major epidermal differentiation markers. Similarly, SVEP1 downregulation was associated with disturbed differentiation and marked epidermal acanthosis in three-dimensional skin equivalents. In contrast, the dispase assay failed to demonstrate significant differences in adhesion between keratinocytes expressing normal vs low levels of SVEP1. Homozygous Svep1 knockout mice were embryonic lethal. Thus, to assess the importance of SVEP1 for normal skin homoeostasis in vivo, we downregulated SVEP1 in zebrafish embryos with a Svep1-specific splice morpholino. Scanning electron microscopy revealed a rugged epidermis with perturbed microridge formation in the centre of the keratinocytes of morphant larvae. Transmission electron microscopy analysis demonstrated abnormal epidermal cell-cell adhesion with disadhesion between cells in Svep1-deficient morphant larvae compared to controls. In summary, our results indicate that SVEP1 plays a critical role during epidermal differentiation.

Safety evaluation of traces of nickel and chrome in cosmetics: The case of Dead Sea mud.

BACKGROUND: Metal impurities such as nickel and chrome are present in natural ingredients-containing cosmetic products. These traces are unavoidable due to the ubiquitous nature of these elements. Dead Sea mud is a popular natural ingredient of cosmetic products in which nickel and chrome residues are likely to occur. OBJECTIVE: To analyze the potential systemic and local toxicity of Dead Sea mud taking into consideration Dead Sea muds' natural content of nickel and chrome. METHODS: The following endpoints were evaluated: (Regulation No. 1223/20, 21/12/2009) systemic and (SCCS's Notes of Guidance) local toxicity of topical application of Dead Sea mud; health reports during the last five years of commercial marketing of Dead Sea mud. RESULTS AND CONCLUSIONS: Following exposure to Dead Sea mud, MoS (margin of safety) calculations for nickel and chrome indicate no toxicological concern for systemic toxicity. Skin sensitization is also not to be expected by exposure of normal healthy skin to Dead Sea mud. Topical application, however, is not recommended for already nickel-or chrome-sensitized persons. As risk assessment of impurities present in cosmetics may be a difficult exercise, the case of Dead Sea mud is taken here as an example of a natural material that may contain traces of unavoidable metals.

Non-invasive evaluation of skin cytokines secretion: an innovative complementary method for monitoring skin disorders.

In this review, a novel non-invasive approach based on skin surface wash sampling is described. Since the epidermis possesses a high metabolic activity, the secretion of various biomarkers can be exploited to develop non-invasive procedures for skin measurement to monitor disorders and to define a therapeutic strategy. Thus, we developed a method for the quantification of skin surface compounds. In this procedure, a well is placed on skin surface and is attached using an adhesive pad. Extraction buffer is introduced into the well for 30 min incubation period and the secretion of different biomarkers on skin surface can be measured: cytokines, antioxidants, peptides, RNA, DNA volatile organic compounds etc. Here, the focus is on cytokine measurement. After collecting skin samples cytokines can be quantified using ELISA assay. Since so far cytokine levels in skin have been evaluated mostly by invasive and prolonged procedures (punch biopsy, blister fluid and scrapping), employing this method has important implications, because it allows assessing cytokine amount with minimal invasion and high accuracy. We have already applied skin surface wash sampling for cytokine quantification in different clinical conditions: psoriasis, atopic dermatitis and chronic renal failure. A distinct pattern of cytokine secretion has been demonstrated for each disorder. Differences were also observed between lesional and non-lesional areas. The obtained results shed a new light on cutaneous cytokine expression in different clinical conditions. Moreover, the interplay between cytokines and other soluble compounds can give an added value in understanding the mechanism of skin pathologies.

Cutaneous Nrf2-Keap1 pathway modulation by environmental factors: The Dead Sea area as a test case.

The skin is constantly exposed to exogenous environmental stressors and has to cope with excessive oxidative stress and tissue damage. However, exposure to moderate environmental stressors may be beneficial for the cutaneous tissue and assist in protecting against oxidative damage via the enhanced activation of the nuclear factor erythroid 2-related factor 2-Kelch-like ECH-associated protein 1 (Nrf2-Keap1) pathway. Such moderate stressors can be found in various locations around the globe. In this manuscript, we chose to focus on the Dead Sea (DS) area as a test case to study the effect of moderate stressors on the cutaneous tissue because of the unique combinations of moderate stressors in this area. The exceptional location of the DS at an altitude of -438 meters below sea level (the lowest place on earth) is responsible for its rare accumulation of moderate stressors such as high-water salinity, high atmospheric pressure, and unique solar radiation. In this manuscript, we hypothesized that the unique solar radiation in the DS area generates moderate oxidative stress in the skin leading to the induction of intracellular electrophiles, which in turn can activate the protecting Nrf2-Keap1 pathway. We showed that exposure of human skin organ culture from the same donor to solar radiation at the DS resulted in significant activation of the Nrf2-Keap1 pathway, induction of phase II enzymes, and lower apoptotic activity compared to a nearby location at a higher altitude (Jerusalem +700 m). This remarkable effect of activating the Nrf2 protecting pathway and the importance and characteristics of the solar irradiation at the DS is discussed.

A novel "reactomics" approach for cancer diagnostics.

Non-invasive detection and monitoring of lethal diseases, such as cancer, are considered as effective factors in treatment and survival. We describe a new disease diagnostic approach, denoted "reactomics", based upon reactions between blood sera and an array of vesicles comprising different lipids and polydiacetylene (PDA), a chromatic polymer. We show that reactions between sera and such a lipid/PDA vesicle array produce chromatic patterns which depend both upon the sera composition as well as the specific lipid constituents within the vesicles. The chromatic patterns were processed through machine-learning algorithms, and the bioinformatics analysis could distinguish both between cancer-bearing and healthy patients, respectively, as well between two types of cancers. Size-separation and enzymatic digestion experiments indicate that lipoproteins are the primary components in sera which react with the chromatic biomimetic vesicles. This colorimetric reactomics concept is highly generic, robust, and does not require a priori knowledge upon specific disease markers in sera. Therefore, it could be employed as complementary or alternative approach for disease diagnostics.

Skin organ culture as a model to study oxidative stress, inflammation and structural alterations associated with UVB-induced photodamage.

BACKGROUND: Ultraviolet (UV) irradiation is a major cause of skin damage, of long-term alteration of skin metabolism, homoeostasis and physical structure. The analysis of UV-induced pathogenic processes requires in vitro models allowing biochemical studies, and appropriate for the development of novel, accurate diagnosis methods based on non-invasive procedures. OBJECTIVES: This work was aimed to reproduce the effects of UVB on whole-skin explants ex vivo and to study underlying biochemical mechanisms, especially in correlation with skin autofluorescence. METHODS: Human skin organ cultures were irradiated with UVB and subjected to enzyme assays, Western blots, solid-phase ELISA, HPLC and fluorescence measurements. RESULTS: UVB irradiation was found to enhance ROS production, to deplete the pool of low-molecular-weight antioxidants and to decrease the overall antioxidant capacity in the epidermis, in a manner dependent on xanthine-oxidase activity. Epidermal cell proliferation and mitochondrial activity were transiently stimulated. IκB-α was degraded, and the secretion of inflammatory cytokines was drastically increased. Inducible nitric oxide synthase activity was increased in non-irradiated controls, probably due to the mechanical stress of skin excision, and this phenomenon was suppressed by UVB. Autofluorescence measurements revealed alterations of dermal protein crosslinks following UVB irradiation. CONCLUSIONS: Skin organ culture proved to be an integrated model appropriate for in vitro analysis of UVB biologic effects and their correlations, and for the study of non-invasive diagnostic methods in cellular and molecular terms.

Cell cycle oscillators underlying orderly proteolysis of E2F8.

E2F8 is a transcriptional repressor that antagonizes E2F1 at the crossroads of the cell cycle, apoptosis, and cancer. Previously, we discovered that E2F8 is a direct target of the APC/C ubiquitin ligase. Nevertheless, it remains unknown how E2F8 is dynamically controlled throughout the entirety of the cell cycle. Here, using newly developed human cell-free systems that recapitulate distinct inter-mitotic and G1 phases and a continuous transition from prometaphase to G1, we reveal an interlocking dephosphorylation switch coordinating E2F8 degradation with mitotic exit and the activation of APC/CCdh1. Further, we uncover differential proteolysis rates for E2F8 at different points within G1 phase, accounting for its accumulation in late G1 while APC/CCdh1 is still active. Finally, we demonstrate that the F-box protein Cyclin F regulates E2F8 in G2-phase. Altogether, our data define E2F8 regulation throughout the cell cycle, illuminating an extensive coordination between phosphorylation, ubiquitination and transcription in mammalian cell cycle.

Protective effects of a cream containing Dead Sea minerals against UVB-induced stress in human skin.

BACKGROUND: Dead Sea (DS) mud and water are known for their unique composition of minerals, and for their therapeutic properties on psoriasis and other inflammatory skin diseases. Their mode of action, however, remains poorly known. OBJECTIVES: To analyse the ability of Dermud, a leave-on skin preparation containing DS mud and other ingredients like DS water, zinc oxide, aloe-vera extract, pro-vitamin B5 and vitamin E, to antagonize biological effects induced by UVB irradiation in skin when topically applied in organ cultures. METHODS: We have used human skin organ cultures as a model to assess the biological effects of UVB irradiation and of Dermud cream topical application. Skin pieces were analysed for mitochondrial activity by MTT assay, for apoptosis by caspase 3 assay, for cytokine secretion by solid phase ELISA, for overall antioxidant capacity by ferric reducing antioxidant power and Oxygen radical absorbance capacity assays (epidermis) or by cyclic voltammetry (external medium), and for uric acid (UA) content by HPLC. RESULTS: We report that UVB irradiation decreases cell viability, total antioxidant capacity and UA contents in the epidermis of skin organ cultures, while increasing the levels of apoptosis in cells and their cytokine secretion. Topical application of Dermud decreased all these effects significantly. CONCLUSIONS: Our results clearly show that Dermud has protective, anti-oxidant and anti-inflammatory properties that can antagonize biological effects of UVB irradiation in skin. It may therefore be able to reduce skin photodamage and photoaging, and more generally to reduce oxidative stress and inflammation in skin pathologies.

A randomized controlled clinical trial comparing the efficacy of dead sea mineral-enriched body lotion versus two types of placebo in the treatment of cutaneous dryness, itching, peeling and tightness in hemodialysis patients (EDIT).

BACKGROUND/AIMS: The present study was designed to investigate the short-term safety and efficacy of topical application with body lotion enriched with minerals from the Dead Sea versus 2 different placebo treatments in reducing symptoms of uremic pruritus. METHODS: In this single-center, randomized, double placebo-controlled clinical trial, 78 hemodialysis patients with self-reported uremic pruritus were randomized to twice-daily topical treatment with body lotion enriched with minerals from the Dead Sea (DS) or to each of 2 types of placebo: (1) lotion with no Dead Sea minerals but otherwise identical to DS (P1) or (2) lotion with no active ingredients (P2). Symptoms of uremic pruritus (itching, dryness, peeling, tightness) were evaluated at baseline and 2 weeks (14 days) after treatment intervention using a 5-point Likert scale. RESULTS: Following treatment, significant differences in symptom severity scores between DS and P1 and, separately, between group DS and P2, were not detected. Additionally, when DS was compared to the combined placebo groups (P1 and P2 together), significant post-treatment differences in symptom severity scores were not observed. Symptoms were less severe post-treatment regardless of treatment assignment. CONCLUSIONS: DS was not superior to either of the placebo treatments in the symptomatic relief of uremic pruritus.

Antipollution skin protection - a new paradigm and its demonstration on two active compounds.

BACKGROUND: Urban pollution is a major source of concern for human health and is a complex of many environmental factors. The topical exposure to pollution activates cutaneous stress. OBJECTIVE: In this study, we tested the antipollution protection of two active components: Dead Sea minerals (Dead Sea mineral-rich water [DSW]) and anionic polysaccharide (PolluStop® [PS]). MATERIALS AND METHODS: Two representative pollution models were studied using reconstructed epidermis: 1) mixture of pollutants (MOP) containing heavy metals and atmospheric particulate matter and 2) ozone exposure. DSW and PS were topically applied alone or in combination, and their protection against pollution was assessed by testing the levels of the inflammation markers interleukin 1α (IL-1α) and prostaglandin E2 (PGE2). RESULTS: MOP exposure induced IL-1α release, which was attenuated following pre-application with DSW and PS alone or in combination. Ozone exposure induced IL-1α and PGE2 release. Pre-application with DSW or PS alone did not inhibit IL-1α and PGE2 overproduction. Only when DSW and PS were mixed together, inhibition of these inflammatory markers was observed. CONCLUSION: The observations reveal the potential use of active agents in combination for a selective mode of protection from urban pollution. This is because many active materials cannot solely provide a broad protection against different types of pollutants. This strategy might be beneficial for future antipollution regimen formulated in both pharmaceutical and cosmetic products.

Inverse supercritical fluid extraction as a sample preparation method for the analysis of the nanoparticle content in sunscreen agents.

We demonstrate the use of inverse supercritical carbon dioxide (scCO2) extraction as a novel method of sample preparation for the analysis of complex nanoparticle-containing samples, in our case a model sunscreen agent with titanium dioxide nanoparticles. The sample was prepared for analysis in a simplified process using a lab scale supercritical fluid extraction system. The residual material was easily dispersed in an aqueous solution and analyzed by Asymmetrical Flow Field-Flow Fractionation (AF4) hyphenated with UV- and Multi-Angle Light Scattering detection. The obtained results allowed an unambiguous determination of the presence of nanoparticles within the sample, with almost no background from the matrix itself, and showed that the size distribution of the nanoparticles is essentially maintained. These results are especially relevant in view of recently introduced regulatory requirements concerning the labeling of nanoparticle-containing products. The novel sample preparation method is potentially applicable to commercial sunscreens or other emulsion-based cosmetic products and has important ecological advantages over currently used sample preparation techniques involving organic solvents.

Peptidyl cyclopropenones: reversible inhibitors, irreversible inhibitors, or substrates of cysteine proteases?

Peptidyl cyclopropenones were previously introduced as selective cysteine protease reversible inhibitors. In the present study we synthesized one such peptidyl cyclopropenone and investigated its interaction with papain, a prototype cysteine protease. A set of kinetics, biochemical, HPLC, MS, and (13)C-NMR experiments revealed that the peptidyl cyclopropenone was an irreversible inhibitor of the enzyme, alkylating the catalytic cysteine. In parallel, this cyclopropenone also behaved as an alternative substrate of the enzyme, providing a product that was tentatively suggested to be either a spiroepoxy cyclopropanone or a gamma-lactone. Thus, a single family of compounds exhibits an unusual variety of activities, being reversible inhibitors, irreversible inhibitors and alternative substrates towards enzymes of the same family.

Unbiased transcriptome signature of in vivo cell proliferation reveals pro- and antiproliferative gene networks.

Different types of mature B-cell lymphocytes are overall highly similar. Nevertheless, some B cells proliferate intensively, while others rarely do. Here, we demonstrate that a simple binary classification of gene expression in proliferating vs. resting B cells can identify, with remarkable selectivity, global in vivo regulators of the mammalian cell cycle, many of which are also post-translationally regulated by the APC/C E3 ligase. Consequently, we discover a novel regulatory network between the APC/C and the E2F transcription factors and discuss its potential impact on the G1-S transition of the cell cycle. In addition, by focusing on genes whose expression inversely correlates with proliferation, we demonstrate the inherent ability of our approach to also identify in vivo regulators of cell differentiation, cell survival, and other antiproliferative processes. Relying on data sets of wt, non-transgenic animals, our approach can be applied to other cell lineages and human data sets.