Assessing the Impact of Polyethylene Nano/Microplastic Exposure on Human Vaginal Keratinocytes.

The global rise of single-use throw-away plastic products has elicited a massive increase in the nano/microplastics (N/MPLs) exposure burden in humans. Recently, it has been demonstrated that disposable period products may release N/MPLs with usage, which represents a potential threat to women's health which has not been scientifically addressed yet. By using polyethyl ene (PE) particles (200 nm to 9 µm), we showed that acute exposure to a high concentration of N/MPLs induced cell toxicity in vaginal keratinocytes after effective cellular uptake, as viability and apoptosis data suggest, along with transmission electron microscopy (TEM) observations. The internalised N/MPLs altered the expression of junctional and adherence proteins and the organisation of the actin cortex, influencing the level of genes involved in oxidative stress signalling pathways and that of miRNAs related to epithelial barrier function. When the exposure to PE N/MPLs was discontinued or became chronic, cells were able to recover from the negative effects on viability and differentiation/proliferation gene expression in a few days. However, in all cases, PE N/MPL exposure prompted a sustained alteration of DNA methyltransferase and DNA demethylase expression, which might impact epigenetic regulation processes, leading to accelerated cell ageing and inflammation, or the occurrence of malignant transformation.

Non-invasive scoring of cellular atypia in keratinocyte cancers in 3D LC-OCT images using Deep Learning.

Diagnosis based on histopathology for skin cancer detection is today's gold standard and relies on the presence or absence of biomarkers and cellular atypia. However it suffers drawbacks: it requires a strong expertise and is time-consuming. Moreover the notion of atypia or dysplasia of the visible cells used for diagnosis is very subjective, with poor inter-rater agreement reported in the literature. Lastly, histology requires a biopsy which is an invasive procedure and only captures a small sample of the lesion, which is insufficient in the context of large fields of cancerization. Here we demonstrate that the notion of cellular atypia can be objectively defined and quantified with a non-invasive in-vivo approach in three dimensions (3D). A Deep Learning (DL) algorithm is trained to segment keratinocyte (KC) nuclei from Line-field Confocal Optical Coherence Tomography (LC-OCT) 3D images. Based on these segmentations, a series of quantitative, reproducible and biologically relevant metrics is derived to describe KC nuclei individually. We show that, using those metrics, simple and more complex definitions of atypia can be derived to discriminate between healthy and pathological skins, achieving Area Under the ROC Curve (AUC) scores superior than 0.965, largely outperforming medical experts on the same task with an AUC of 0.766. All together, our approach and findings open the door to a precise quantitative monitoring of skin lesions and treatments, offering a promising non-invasive tool for clinical studies to demonstrate the effects of a treatment and for clinicians to assess the severity of a lesion and follow the evolution of pre-cancerous lesions over time.

Proteomic analysis of human keratinocytes under UVA-induced stress / Análise proteômica de queratinócitos humanos sob estresse induzido por luz UVA

The solar ultraviolet (UV) radiation that reaches the Earth is composed of 95% of UVA (320 to 400 nm) and 5% of UVB (280 to 320 nm) radiation. UVB is carcinogenic, generating potentially mutagenic DNA lesions. The solar UVA radiation also causes DNA damage, but this fact does not fully account for its biological impact. UVA is absorbed by non-DNA cellular chromophores, generating reactive oxygen species such as singlet oxygen. Knowing the proteome mediates stress responses in cells, here we investigated the cellular effects of a non-cytotoxic dose of UVA radiation, equivalent to about 20 minutes of midday sun exposure, on the proteome of human keratinocytes. Using a combination of mass spectrometry-based proteomics, bioinformatics, and conventional biochemical assays, we analyzed two aspects of UVA-induced stress: spatial remodeling of the proteome in subcellular compartments 30 minutes after stress and long-term changes in protein levels and secretion (24 hours and 7 days postirradiation). In the first part of this thesis, we quantified and assigned subcellular localization for over 3000 proteins, of which about 600 potentially redistribute upon UVA exposure. Protein redistributions were accompanied by redox modulations, mitochondrial fragmentation and DNA damage. In the second part of the work, our results showed that primary human keratinocytes enter senescence upon exposure to a single dose of UVA, mounting antioxidant and inflammatory responses. Cells under UVA-induced senescence further elicit paracrine responses in neighboring premalignant HaCaT epithelial cells via inflammatory mediators. Altogether, these results reiterate the role of UVA radiation as a potent metabolic stressor in the skin

A radiação ultravioleta (UV) solar que atinge a superfície terrestre é composta por 95% de radiação UVA (320 a 400 nm) e 5% de radiação UVB (280 a 320 nm). A radiação UVB é carcinogênica e gera lesões potencialmente mutagênicas no DNA. A radiação UVA solar também gera danos no DNA, mas a genotoxicidade dessa radiação não explica inteiramente o seu impacto biológico. Atualmente, sabe-se que a radiação UVA é absorvida por cromóforos celulares, gerando espécies reativas de oxigênio, como o oxigênio singlete. Sabendo que o proteoma é um mediador de respostas ao estresse celular, nós investigamos os efeitos celulares de uma dose não-citotóxica de radiação UVA, equivalente a cerca de 20 minutos de exposição ao sol, no proteoma de queratinócitos humanos. Utilizando espectrometria de massas, bioinformática e ensaios bioquímicos convencionais, nós analisamos dois aspectos do estresse induzido por radiação UVA: o remodelamento espacial do proteoma 30 minutos depois do estresse e alterações nos níveis e na secreção de proteínas no longo prazo (24 horas e 7 dias depois da irradiação). Na primeira parte desta tese, nós quantificamos e atribuímos classificações de localização subcelular a mais de 3000 proteínas. Dentre essas proteínas, 600 tem potencialmente a sua distribuição subcelular alterada em resposta à radiação. As redistribuições subcelulares são acompanhadas de modulações redox, fragmentação mitocondrial e danos no DNA. Na segunda parte da tese, os nossos resultados mostraram que queratinócitos humanos primários entram em senescência sob exposição a uma única dose de radiação UVA, montando respostas antioxidantes e pró-inflamatórias. Células sob senescência induzida por UVA, por sua vez, desencadeiam respostas parácrinas em queratinócitos pré-tumorais (células HaCaT) por meio de mediadores inflamatórios. Em conjunto, esses resultados reiteram o papel da radiação UVA como um potente estressor metabólico em células da pele

Overriding native cell coordination enhances external programming of collective cell migration.

As collective cell migration is essential in biological processes spanning development, healing, and cancer progression, methods to externally program cell migration are of great value. However, problems can arise if the external commands compete with strong, preexisting collective behaviors in the tissue or system. We investigate this problem by applying a potent external migratory cue-electrical stimulation and electrotaxis-to primary mouse skin monolayers where we can tune cell-cell adhesion strength to modulate endogenous collectivity. Monolayers with high cell-cell adhesion showed strong natural coordination and resisted electrotactic control, with this conflict actively damaging the leading edge of the tissue. However, reducing preexisting coordination in the tissue by specifically inhibiting E-cadherin-dependent cell-cell adhesion, either by disrupting the formation of cell-cell junctions with E-cadherin-specific antibodies or rapidly dismantling E-cadherin junctions with calcium chelators, significantly improved controllability. Finally, we applied this paradigm of weakening existing coordination to improve control and demonstrate accelerated wound closure in vitro. These results are in keeping with those from diverse, noncellular systems and confirm that endogenous collectivity should be considered as a key quantitative design variable when optimizing external control of collective migration.

Nuclear IL-33 Plays an Important Role in the Suppression of FLG, LOR, Keratin 1, and Keratin 10 by IL-4 and IL-13 in Human Keratinocytes.

IL-33 is a chromatin-associated multifunctional cytokine implicated in the pathogenesis of atopic dermatitis (AD), an inflammatory skin disorder characterized by skin barrier dysfunction. The previous reports show that IL-33 is highly detected in the nucleus of epidermal keratinocytes in AD lesions compared with that in unaffected or normal skin. However, it is unclear whether intracellular IL-33 directly contributes to the pathogenesis of AD. T helper type 2 cytokines IL-4 and IL-13 that are elevated in AD lesions suppress keratinocyte differentiation to impair skin barrier function. We investigated whether intracellular IL-33 is involved in IL-4 and IL-13 function. In monolayer culture and living skin equivalent analyses, IL-4 and IL-13 increased the expression of full-length IL-33 in the nucleus of keratinocytes by activating the MAPK/extracellular signal‒regulated kinase kinase/extracellular signal‒regulated kinase signaling pathway, which is necessary for the inhibition of differentiation markers FLG, LOR, keratin 1, and keratin 10. The nuclear IL-33 functions as a transcription cofactor of signal transducer and activator of transcription 3, increasing the binding of phosphorylated signal transducer and activator of transcription 3 to FLG promoter, thereby inhibiting its transcription, and it inhibits the expression of transcription factor RUNX1 by signal transducer and activator of transcription 3 and signal transducer and activator of transcription 6, thereby downregulating LOR, keratin 1, and keratin 10. Thus, the elevated nuclear IL-33 in the epidermis of AD lesions may be involved in the pathogenesis of AD by inhibiting keratinocyte differentiation and skin barrier function.

Anti-Inflammatory and Proliferative Properties of Luteolin-7-O-Glucoside.

Flavonoids display a broad range of structures and are responsible for the major organoleptic characteristics of plant-derived foods and beverages. Recent data showed their activity, and in particular of luteolin-7-O-glucoside (LUT-7G), in reduction of oxidative stress and inflammatory mechanisms in different physiological systems. In this paper, we tried to elucidate how LUT-7G could exert both antioxidant and anti-inflammatory effects in endothelial cells cultured in vitro. Here, we showed that LUT-7G is able to inhibit the STAT3 pathway, to have an antiproliferative action, and an important antioxidant property in HUVEC cells. These properties are exerted by the flavone in endothelial through the transcriptional repression of a number of inflammatory cytokines and their receptors, and by the inhibition of ROS generation. ROS and STAT3 activation has been correlated with the production of oxysterols and other hydroxylated fatty acids, and they have been recognized important as players of atherogenesis and cardiocirculatory system diseases. The analysis of the general production pathway of these hydroxylated species, showed a strong decrease of cholesterol hydroxylated species such as 7-alpha-hydroxicholesterol, 7-beta-hydroxicholesterol by the treatment with LUT-7G. This confirms the anti-inflammatory properties of LUT-7G also in the endothelial district, showing for the first time the molecular pathway that verify previous postulated cardiovascular benefits of this flavone.

Hyaline Inclusion Acanthoma.

ABSTRACT: Eosinophilic hyaline inclusions (EHIs) or globules have been reported in various cutaneous tumors including vascular lesions, myoepithelial neoplasms, and basal cell carcinoma. In basal cell carcinoma, the presence of intracytoplasmic inclusions is reportedly associated with myoepithelial differentiation. In this regard, EHI has not been conclusively documented in a cutaneous lesion of genuine squamous cell lineage without aberrant differentiation. In the current case, a biopsy from the right thigh of a 71-year-old male patient demonstrated a relatively well-demarcated intraepidermal squamous lesion featured an admixture of predominantly enlarged keratinocytes harboring distinct eccentric intracytoplasmic EHI and a smaller population of keratinocytes displaying pale cytoplasm. Cytologic atypia, mitotic activity, and inflammatory cells were not identified. The intracytoplasmic EHI stained red with Masson's trichrome and were negative with periodic-acid Schiff with and without diastase. Immunologically, the lesion was strongly and diffusely positive for various cytokeratins but negative for ubiquitin and myoepithelial markers. Only cytokeratin AE1 revealed a differential staining pattern as the suprabasal lesional cells displayed significantly stronger immunoreactivity in comparison with the adjacent normal keratinocytes. Polymerase chain reaction for low-risk and high-risk human papillomavirus was negative. Molecular studies did not reveal any mutations commonly encountered in seborrheic or lichenoid keratoses. As an analogous lesion has not previously reported in the literature, the term hyaline inclusion acanthoma is proposed for this peculiar lesion.

How many cells are required for successful DNA profiling?

Through advances in fluorescent nucleic acid dye staining and visualisation, targeted collection of cellular material deposited, for example by touch or within a saliva deposit, is possible. In regard to the potential evidentiary value of the deposit the questions remain: 'How many cells are required to generate an informative DNA profile?'; 'How many visualised corneocytes within a touch deposit compared to typical nucleated cells are required in order to achieve successful DNA profiling?'. Diamond TM Nucleic Acid Dye (DD) staining of cellular material, and subsequent visualisation utilising portable fluorescence microscopy, was performed for touch and saliva samples to target defined numbers of cells for collection, by swab and tapelift, and subsequent processing via direct PCR and PCR post-extraction. The resulting DNA quantification data and alleles generated within subsequent DNA profiles could be correlated to the number of cells initially collected to determine cellular threshold requirements for DNA profile generation for each workflow. Full profiles were consistently generated using direct PCR when the template was ≥40 buccal cells collected by either a swab or tapelift. By contrast ≥800 corneocytes collected by swabbing or ≥4,000 corneocytes collected by a tapelift were required to generate same number of STR alleles from touch samples. When samples were processed through a DNA extraction workflow, ≥80 buccal cells were required to generate full profiles from both swab and tapelift, while touch samples required ≥4,000 corneocytes collected by a swab and >8,000 corneocytes collected by a tapelift. The data presented within this study allow for informative sample triage and workflow decisions to be made to optimise STR amplification based on the presence and visual quantification of stained cellular material.

Gene expression profile of human follicle dermal papilla cells in response to Camellia japonica phytoplacenta extract.

Camellia japonica L. is a flowering tree with several medicinal and cosmetic applications. Here, we investigated the efficacy of C. japonica placenta extract (CJPE) as a potential therapeutic agent for promotion of hair growth and scalp health by using various in vitro and in vivo assays. Moreover, we performed transcriptome analysis to examine the relative expression of human follicle dermal papilla cells (HFDPC) in response to CJPE by RNA-sequencing (RNA-seq). In vitro assays revealed upregulation of the expression of hair growth marker genes in HFDPC after CJPE treatment. Moreover, in vivo clinical tests with 42 adult female participants showed that a solution containing 0.5% CJPE increased the moisture content of the scalp and decreased the scalp's sebum content, dead scalp keratin, and erythema. Furthermore, RNA-seq analysis revealed key genes in HFDPC which are associated with CJPE. Interestingly, genes associated with lipid metabolism and cholesterol efflux were upregulated. Genes upregulated by CJPE are associated with several hormones, including parathyroid, adrenocorticotropic hormone, α-melanocyte-stimulating hormone (alpha-MSH), and norepinephrine, which are involved in hair follicle biology. Furthermore, some upregulated genes are associated with the regulation of axon guidance. In contrast, many genes downregulated by CJPE are associated with structural components of the cytoskeleton. In addition, CJPE suppressed genes associated with muscle structure and development. Taken together, this study provides extensive evidence that CJPE may have potential as a therapeutic agent for scalp treatment and hair growth promotion.

Cell Membrane-Coated Electrospun Fibers Enhance Keratinocyte Growth through Cell-Type Specific Interactions.

Although cell membrane-coated fiber scaffolds can be useful for regenerative medicine by presenting both cell surface antigens and topographical cues, it remains unknown whether changes in cellular behavior on cell membrane-coated scaffolds are due to specific cell-cell interactions. In this work, the effects of scaffold fiber diameters and surface charges on the cell membrane coating efficiency were explored. Furthermore, fibroblast membrane-coated scaffolds improved the growth of human keratinocytes as compared to red blood cell membrane-coated and plain scaffolds. These results suggest the biofunctionality of cell membrane-coated scaffolds and the specific cell-cell interactions that are preserved to modulate cellular response.

Effect of Collagen Nanofibers and Silanization on the Interaction of HaCaT Keratinocytes and 3T3 Fibroblasts with Alumina Nanopores.

During wound healing, a complex cascade of cellular and molecular events occurs, which is governed by topographical and biochemical cues. Therefore, optimal tissue repair requires scaffold materials with versatile structural and biochemical features. Nanoporous anodic aluminum oxide (AAO) membranes exhibit good biocompatibility along with customizable nanotopography and antimicrobial properties, which has brought them into the focus of wound treatment. However, despite their good permeability, such bioinert ceramic nanopores cannot actively promote cell growth as they lack biochemical cues to support specific ligand-receptor interactions. Therefore, we modified AAO nanopores with the biochemical features of collagen nanofibers or amino groups provided by silanization with (3-aminopropyl)triethoxysilane (APTES) to design a permeable scaffold material that can additionally promote cell adhesion. Viability assays revealed that the metabolic activity of both 3T3 fibroblasts and HaCaT keratinocytes on bare and silanized AAO pores was comparable to glass controls until 72 h. Interestingly, both cell types showed a reduced proliferation on AAO with collagen nanofibers. Nevertheless, scanning electron and fluorescence microscopy revealed that 3T3 fibroblasts exhibited a well-spread morphology with filopodia attached to the nanoporous surface of the underlying AAO membranes or nanofibrous collagen networks, thus indicating a close interaction with the composites. Keratinocytes, although growing in clusters on bare and APTES-modified AAO, also adhered well on collagen-modified AAO membranes. When in contact with Escherichia coli suspensions for 20 h, the AAO membranes successfully prevented bacteria penetration irrespective of the biochemical functionalization. In summary, both functionalization strategies have high potential to specifically control molecular signaling and cell migration to further develop alumina nanopores for wound healing.

Diffusion through skin in the light of a fractional derivative approach: progress and challenges.

This review is focussed on modelling the transport processes of different drugs across the intact human skin by introducing a memory formalism based on the fractional derivative approach. The fundamental assumption of the classic transport equation in the light of the Fick's law is that the skin barrier behaves as a pseudo-homogeneous membrane and that its properties, summarized by the diffusion coefficient D,  do not vary with time and position. This assumption does not hold in the case of a highly heterogeneous system as the skin is, whose outermost layer (the stratum corneum) is comprised of a multi-layered structure of keratinocytes embedded in a lamellar matrix of hydrophobic lipids, followed by the dermis that contains a network of capillaries that connect to the systemic circulation. A possible way to overcome these difficulties resides in the introduction of mathematical models which involve fractional derivatives to describe complex systems with interactions in space and time, following the model originally developed by Caputo in order to consider the memory effects in materials. Although the introduction of fractional derivatives to model memory effects is completely phenomenological, i.e., characterized by a single parameter, i.e., the fractional derivative order [Formula: see text] a number of authors have found that this approach can provide a better comparison to experimental data and that this technique may be alternative to integer-order derivative models. In this review, we aim to summarize some our recent results, concerning the transport of different diffusing compounds of different structural complexity across the intact skin.

Expression of invadopodia markers can identify oral lesions with a high risk of malignant transformation.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the oral cavity and is usually preceded by a range of premalignant tissue abnormalities termed oral potentially malignant disorders. Identifying malignant transformation is critical for early treatment and consequently improved survival and decreased morbidity. Invadopodia (INV) are specialized subcellular structures required for cancer cell invasion. We developed a new method to visualize INV in keratinocytes using fluorescent immunohistochemistry (FIHC) and semi-automated images analysis. The presence of INV was used to determine the risk of malignant transformation. We analyzed 145 formalin-fixed, paraffin-embedded (FFPE) oral biopsy samples from 95 patients diagnosed as nondysplastic, dysplastic, and OSCC including 49 patients whose lesions transformed to OSCC (progressing) and 46 cases that did not transform to OSCC (control). All samples were stained for Cortactin, tyrosine kinase substrate with five SH3 domains (Tks5) and matrix metallopeptidase 14 (MMP14) using FIHC, imaged using confocal microscopy and analyzed using a multichannel colocalization analysis. The areas of colocalization were used to generate an INV score. Using the INV score, we were able to identify progressing lesions with a sensitivity of 75-100% and specificity of 72-76%. A positive INV score was associated with increased risk of progression to OSCC. Our results suggest that INV markers can be used in conjunction with the current diagnostic standard for early detection of OSCC.

Corneocyte lysis and fragmented DNA considerations for the cellular component of forensic touch DNA.

DNA deposited by individuals' hands is a routine part of forensic analysis, yet little is understood about the precise cellular contents left by handling. "Dead" skin cells known as corneocytes make up the majority of the cellular material left in touch deposits by people's hands but are known to lack nuclei, making their DNA content ambiguous. Here we measure DNA released from anucleate corneocytes following various lysis methods to determine how much DNA may be present in these cells and how best to recover it from inside the cornified envelope. We demonstrate that enhanced lysis methods using a reducing agent and longer incubation may be valuable for hand deposit samples. Corneocyte DNA can be characterized as highly degraded based on the quantification, STR profiling and fluorescence microscopy of the cells from freshly washed hands. Purification to target shorter DNA fragments is demonstrated. DNA from the washed corneocyte cells is shown to constitute the majority of recoverable DNA with these methods. We consider the use of new methods adapted to cornified cells and fragmented DNA for future research into this sample type.

The Whey Acidic Protein WFDC12 Is Specifically Expressed in Terminally Differentiated Keratinocytes and Regulates Epidermal Serine Protease Activity.

WFDC proteins such as peptidase inhibitor 3 and SLPI inhibit proteases in the epidermis and other tissues. In this study, we tested the hypothesis that further WFDC protein family members might contribute to epidermal homeostasis. We found that in addition to peptidase inhibitor 3 and SLPI, WFDC5 and WFDC12 were expressed in human epidermis. In contrast to WFDC5, the expression of WFDC12 was induced during the late differentiation of keratinocytes and was restricted to the outermost layer of live cells. Single-cell RNA sequencing demonstrated that WFDC12-positive keratinocytes were characterized by the upregulation of LCE mRNA expression and downregulated the expression of keratins and claudins. Immunogold-electron microscopy revealed the colocalization of WFDC12 with corneodesmosomes in the lower stratum corneum. WFDC12 was elevated in the affected skin of patients with psoriasis, atopic dermatitis, and Darier disease. By contrast, WFDC12 expression was strongly upregulated not only in the affected but even more so in clinically normal-appearing skin of patients with Netherton syndrome. Finally, functional analysis showed distinct inhibitory activity of WFDC12 on neutrophil elastase and epidermal kallikrein‒related peptidase. Altogether, our study identified WFDC12 as a marker of the last stage of epidermal keratinocyte differentiation and suggests that WFDC12 contributes to the control of protease activity in the stratum corneum.

The ceramide [NP]/[NS] ratio in the stratum corneum is a potential marker for skin properties and epidermal differentiation.

BACKGROUND: Specific species of ceramides (Cer), major constituents of lipids in the stratum corneum (SC), are decreased and are correlated with SC barrier and water-holding functions in the skin of patients with atopic dermatitis (AD) or psoriasis (Pso). However, possible correlations between Cer subclass ratios and skin properties in barrier-disrupted skin and in healthy skin remain unclear. The objective of this study was to identify a new marker to evaluate skin properties and epidermal differentiation in SC not only in barrier-disrupted skin but also in healthy skin. METHODS: The Cer subclass ratios in the SC of healthy control subjects and in patients with AD or Pso were evaluated. Correlations with candidate markers and facial skin features of healthy Japanese females (20-74 years old, n = 210) were investigated. Variations of markers during epidermal differentiation were studied in human epidermis and in cultured keratinocytes. RESULTS: The ratios of Cer [NP]/[NS], Cer [NH]/[NS], Cer [NP]/[AS], Cer [NH]/[NS], Cer [NDS]/[AS], Cer [AH]/[AS] and Cer [EOP]/[AS] showed significant differences between non-lesional skin of AD patients and normal skin of healthy control subjects, as well as Pso patients and their healthy control subjects. The Cer [NP]/[NS] ratio was correlated with SC functional parameters (transepidermal water loss and capacitance) and with skin appearance (texture, scaling and color) even in the cheek skin of healthy female subjects. The Cer [NP]/[NS] ratio in the SC was approximately 18-times higher than in living keratinocytes, and it increased as they differentiated. CONCLUSIONS: The Cer [NP]/[NS] ratio in the SC is a potential marker for skin properties and epidermal differentiation in barrier-disrupted skin as well as in healthy skin.

Discovery of 4-Anilinoquinolinylchalcone Derivatives as Potential NRF2 Activators.

Activation of nuclear factor erythroid-2-related factor 2 (NRF2) has been proven to be an effective means to prevent the development of cancer, and natural curcumin stands out as a potent NRF2 activator and cancer chemopreventive agent. In this study, we have synthesized a series of 4-anilinoquinolinylchalcone derivatives, and used a NRF2 promoter-driven firefly luciferase reporter stable cell line, the HaCaT/ARE cells, to screen a panel of these compounds. Among them, (E)-3-{4-[(4-acetylphenyl)amino]quinolin-2-yl}-1-(4-fluorophenyl)prop-2-en-1-one (13b) significantly increased NRF2 activity in the HaCaT cell with a half maximal effective concentration (EC50) value of 1.95 µM. Treatment of compound 13b upregulated HaCaT cell NRF2 expression at the protein level. Moreover, the mRNA level of NRF2 target genes, heme oxygenase-1 (HO-1), glutamate-cysteine ligase catalytic subunit (GCLC), and glucose-6-phosphate dehydrogenase (G6PD) were significantly increased in HaCaT cells upon the compound 13b treatment. The molecular docking results exhibited that the small molecule 13b is well accommodated by the bound region of Kelch-like ECH-associated protein 1 (Keap1)-Kelch and NRF2 through stable hydrogen bonds and hydrophobic interaction, which contributed to the enhancement of affinity and stability between the ligand and receptor. Compound 13b has been identified as the lead compound for further structural optimization.

MiR-23b Promotes the Migration of Keratinocytes Through Downregulating TIMP3.

BACKGROUND: Wound healing is a complex process aiming at repairing the damaged skin. MiR-23b has been reported to be upregulated during wound healing. In this study, we intended to explore the working mechanism of miR-23b during wound healing. METHODS: Quantitative real-time polymerase chain reaction was performed to detect the enrichment of miR-23b and tissue inhibitor of metalloproteinase-3 (TIMP3) in HaCaT cells. Scratch wound assay was carried out to measure the migration of HaCaT cells. The target of miR-23b was predicted by microT-CDS software, and the combination was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay. The abundance of TIMP3 protein was detected by Western blot assay. RESULTS: The abundance of miR-23b was positively related to the concentration and time of transforming growth factor ß1 treatment in HaCaT cells. MiR-23b promoted the migration of keratinocytes. TIMP3 was a direct target of miR-23b and was negatively regulated by miR-23b. TIMP3 inhibited the migration of keratinocytes. MiR-23b accelerated the migration of keratinocytes by downregulating the abundance of TIMP3. CONCLUSIONS: MiR-23b promoted the migration of keratinocytes partly through reducing the enrichment of TIMP3. MiR-23b might be a promising target for the treatment of wound healing-associated diseases.

LOC285194 inhibits proliferation of human keratinocytes through regulating miR-616/GATA3 pathway.

LncRNA LOC285194 has been associated with the occurrence of psoriasis. However, the underlying mechanisms that lead to psoriasis remain unclear. In this study, the expression of LOC285194, miR-616, and GATA3 was determined by western blotting and quantitative real-time PCR, and their relationships were assessed using dual-luciferase reporter assays. The effects of LOC285194 on the proliferation and apoptosis of keratinocytes were investigated using cell counting kit-8 assays and flow cytometry, respectively. Reduced expression of LOC285194 was detected in the skin lesion samples from patients with psoriasis. Overexpression of LOC285194 led to reduced cell viability, cell cycle arrest, and increased cell apoptosis in keratinocytes, whereas LOC285194 silencing resulted in opposite effects. In addition, LOC285194 was found to negatively regulate miR-616, which modulated GATA3 expression through its direct binding to the 3'-untranslated region of GATA3. Knockdown of GATA3 rescued LOC285194 overexpression-mediated cell viability reduction, cell cycle arrest and apoptosis induction in keratinocytes. Taken together, LOC285194 was found to inhibit keratinocyte growth by sponging miR-616 that regulates GATA3.

Electric field down-regulates CD9 to promote keratinocytes migration through AMPK pathway.

Endogenous electric field (EF)-directed keratinocytes migration is known to play a key role in the wound re-epithelialization process. Although many molecules and signaling pathways are reported important for directional keratinocytes migration under EF, the underlying mechanism remains unclear. Our previous research found that CD9, a trans-membrane protein, is involved in wound re-epithelialization and CD9 downregulation contributes to keratinocytes migration. In this study, we observed the effect of EF on CD9 expression and keratinocytes migration. The keratinocytes migrated directionally toward the cathode and CD9 expression was down-regulated under EF (200mV/mm). In addition, CD9 overexpression reversed EF-induced migratory speed and the electrotactic response of keratinocytes. Also, we found that EF reduced AMP-activated protein kinase (AMPK) activity. Furthermore, AICAR, an AMPK activator, increased CD9 expression under EF, while compound C, an AMPK inhibitor, decreased CD9 expression in keratinocytes. Our results demonstrate that EF regulates CD9 expression and keratinocytes directional migration, in which AMPK pathway plays an important role.