Adult Human Keratinocytes Research Articles

Silver and Carbon Nanomaterials/Nanocomplexes as Safe and Effective ACE2-S Binding Blockers on Human Skin Cell Lines.

(1) Background: Angiotensin-converting enzyme 2 (ACE2) is a crucial functional receptor of the SARS-CoV-2 virus. Although the scale of infections is no longer at pandemic levels, there are still fatal cases. The potential of the virus to infect the skin raises questions about new preventive measures. In the context of anti-SARS-CoV-2 applications, the interactions of antimicrobial nanomaterials (silver, Ag; diamond, D; graphene oxide, GO and their complexes) were examined to assess their ability to affect whether ACE2 binds with the virus. (2) Methods: ACE2 inhibition competitive tests and in vitro treatments of primary human adult epidermal keratinocytes (HEKa) and primary human adult dermal fibroblasts (HDFa) were performed to assess the blocking capacity of nanomaterials/nanocomplexes and their toxicity to cells. (3) Results: The nanocomplexes exerted a synergistic effect compared to individual nanomaterials. HEKa cells were more sensitive than HDFa cells to Ag treatments and high concentrations of GO. Cytotoxic effects were not observed with D. In the complexes, both carbonic nanomaterials had a soothing effect against Ag. (4) Conclusions: The Ag5D10 and Ag5GO10 nanocomplexes seem to be most effective and safe for skin applications to combat SARS-CoV-2 infection by blocking ACE2-S binding. These nanocomplexes should be evaluated through prolonged in vivo exposure. The expected low specificity enables wider applications.

Short cell cycle duration is a phenotype of human epidermal stem cells

BackgroundA traditional view is that stem cells (SCs) divide slowly. Meanwhile, both embryonic and pluripotent SCs display a shorter cell cycle duration (CCD) in comparison to more committed progenitors (CPs).MethodsWe examined the in vitro proliferation and cycling behavior of somatic adult human cells using live cell imaging of passage zero keratinocytes and single-cell RNA sequencing.ResultsWe found two populations of keratinocytes: those with short CCD and protracted near exponential growth, and those with long CCD and terminal differentiation. Applying the ergodic principle, the comparative numbers of cycling cells in S phase in an enriched population of SCs confirmed a shorter CCD than CPs. Further, analysis of single-cell RNA sequencing of cycling adult human keratinocyte SCs and CPs indicated a shortening of both G1 and G2M phases in the SC.ConclusionsContrary to the pervasive paradigm, SCs progress through cell cycle more quickly than more differentiated dividing CPs. Thus, somatic human adult keratinocyte SCs may divide infrequently, but divide rapidly when they divide. Additionally, it was found that SC-like proliferation persisted in vitro.

Human keratin matrices promote wound healing by modulating skin cell expression of cytokines and growth factors.

A wide variety of biomaterials has been developed to assist in wound healing, including acellular animal and human-derived protein matrices. However, millions of patients worldwide still suffer from non-healing chronic wounds, demonstrating a need for further innovation in wound care. To address this need, a novel biomaterial, the human keratin matrix (HKM), was developed, characterised, and tested in vitro and in vivo. HKM was found to be degradation-resistant, and a proteomics analysis showed it to be greater than 99% human keratin proteins. PCR revealed adult human epidermal keratinocytes (HEKa) grown in contact with HKM showed increased gene expression of keratinocyte activations markers such as Epidermal Growth Factor (EGF). Additionally, a cytokine microarray demonstrated culture on HKM increased the release of cytokines involved in wound inflammatory modulation by both HEKa cells and adult human dermal fibroblasts (HDFa). Finally, in a murine chronic wound model, full-thickness wounds treated weekly with HKM were smaller through the healing process than those treated with human amniotic membrane (AM), bovine dermis (BD), or porcine decellularized small intestinal submucosa (SIS). HKM-treated wounds also closed significantly faster than AM- and SIS-treated wounds. These data suggest that HKM is an effective novel treatment for chronic wounds.

Oxidative Stress Induces Skin Pigmentation in Melasma by Inhibiting Hedgehog Signaling.

There is growing evidence that oxidative stress plays a role in melasma and disrupts primary cilia formation. Additionally, primary cilia have been suggested to have an inhibitory role in melanogenesis. This study examined the potential link between oxidative stress, skin hyperpigmentation, and primary cilia. We compared the expression levels of the nuclear factor E2-related factor 2 (NRF2), intraflagellar transport 88 (IFT88), and glioma-associated oncogene homologs (GLIs) in skin samples from patients with melasma, both in affected and unaffected areas. We also explored the roles of NRF2, IFT88, and GLIs in ciliogenesis and pigmentation using cultured adult human keratinocytes, with or without melanocytes. Our findings revealed decreased levels of NRF2, heme oxygenase-1, IFT88, and GLIs in lesional skin from melasma patients. The knockdown of NRF2 resulted in reduced expressions of IFT88 and GLI1, along with fewer ciliated cells. Furthermore, NRF2, IFT88, or GLI1 knockdown led to increased expressions in protease-activated receptor-2 (PAR2), K10, involucrin, tyrosinase, and/or melanin. These effects were reversed by the smoothened agonist 1.1. Calcium also upregulated these proteins, but not NRF2. The upregulation of involucrin and PAR2 after NRF2 knockdown was mitigated with a calcium chelator. In summary, our study suggests that oxidative stress in NRF2-downregulated melasma keratinocytes impedes ciliogenesis and related molecular processes. This inhibition stimulates keratinocyte differentiation, resulting in melanin synthesis and melanosome transfer, ultimately leading to skin hyperpigmentation.

Aquaporin-3 Downregulation in Vitiligo Keratinocytes Increases Oxidative Stress of Melanocytes.

Oxidative stress-induced melanocyte apoptosis is linked to the immune system and plays a critical role in the pathogenesis of vitiligo. Aquaporin-3 (AQP3), which is downregulated in vitiligo keratinocytes, regulates intracellular H2O2 accumulation. However, the role of AQP3 in oxidative stress is uncertain in vitiligo. This study investigated the effect of downregulated AQP3 on oxidative stress in vitiligo using lesional and non-lesional skin specimen sets from vitiligo patients and primary cultured adult normal human epidermal keratinocytes, with or without downregulation and overexpression of AQP3 in the presence or absence of H2O2 treatment. The levels of nuclear factor E2-related factor 2 (NRF2) and/or its main target, NAD(P)H quinone dehydrogenase 1 (NQO-1), were lower in the lesional keratinocytes and cultured keratinocytes with AQP3 knockdown, but were increased in keratinocytes upon AQP3 overexpression. Ratios of NRF2 nuclear translocation and NQO-1 expression levels were further reduced in AQP3-knockdown keratinocytes following H2O2 treatment. The conditioned media from AQP3-knockdown keratinocytes treated with H2O2 contained higher concentrations of reactive oxygen species (ROS). Moreover, the number of viable melanocytes was reduced when the conditioned media were added to the culture media. Overall, AQP3 downregulation in the keratinocytes of patients with vitiligo can induce oxidative stress in neighboring melanocytes, leading to melanocyte death.

Dual targeting of mTOR/IL-17A and autophagy by fisetin alleviates psoriasis-like skin inflammation.

Psoriasis is a chronic autoimmune inflammatory skin disorder characterized by epidermal hyperplasia and aberrant immune response. In addition to aberrant cytokine production, psoriasis is associated with activation of the Akt/mTOR pathway. mTOR/S6K1 regulates T-lymphocyte activation and migration, keratinocytes proliferation and is upregulated in psoriatic lesions. Several drugs that target Th1/Th17 cytokines or their receptors have been approved for treating psoriasis in humans with variable results necessitating improved therapies. Fisetin, a natural dietary polyphenol with anti-oxidant and anti-proliferative properties, covalently binds mTOR/S6K1. The effects of fisetin on psoriasis and its underlying mechanisms have not been clearly defined. Here, we evaluated the immunomodulatory effects of fisetin on Th1/Th17-cytokine-activated adult human epidermal keratinocytes (HEKa) and anti-CD3/CD28-stimulated inflammatory CD4+ T cells and compared these activities with those of rapamycin (an mTOR inhibitor). Transcriptomic analysis of HEKa revealed 12,713 differentially expressed genes (DEGs) in the fisetin-treated group compared to 7,374 DEGs in the rapamycin-treated group, both individually compared to a cytokine treated group. Gene ontology analysis revealed enriched functional groups related to PI3K/Akt/mTOR signaling pathways, psoriasis, and epidermal development. Using in silico molecular modeling, we observed a high binding affinity of fisetin to IL-17A. In vitro, fisetin significantly inhibited mTOR activity, increased the expression of autophagy markers LC3A/B and Atg5 in HEKa cells and suppressed the secretion of IL-17A by activated CD4+ T lymphocytes or T lymphocytes co-cultured with HEKa. Topical administration of fisetin in an imiquimod (IMQ)-induced mouse psoriasis model exhibited a better effect than rapamycin in reducing psoriasis-like inflammation and Akt/mTOR phosphorylation and promoting keratinocyte differentiation and autophagy in mice skin lesions. Fisetin also significantly inhibited T-lymphocytes and F4/80+ macrophage infiltration into skin. We conclude that fisetin potently inhibits IL-17A and the Akt/mTOR pathway and promotes keratinocyte differentiation and autophagy to alleviate IMQ-induced psoriasis-like disease in mice. Altogether, our findings suggest fisetin as a potential treatment for psoriasis and possibly other inflammatory skin diseases.

Evaluation of photochemically cross-linked collagen/gold nanoparticle composites as potential skin tissue scaffolds.

Collagen type I is the main structural unit in skin tissue and is therefore used preferentially in skin tissue scaffolds. However, collagen-based 3D scaffolds have weak aqueous stability and degradation profiles in their uncross-linked states and chemical cross-linking reagents arise toxicity concerns, which generally restrict the spectrum of their biomedical applicability. Here, the research goal is to photochemically cross-link collagen type I with rose bengal (RB) when subjected to green laser light and to investigate the effect of silk sericin-capped gold nanoparticles (S-AuNP) when incorporated into scaffolds on the cross-linking process and thus on the scaffold properties. All the collagen scaffolds, that is plain collagen (C), collagen/S-AuNP (C-Au), cross-linked collagen (C-RBL), and cross-linked collagen/S-AuNP (C-AuRBL) were characterized for their potential as skin tissue scaffolds. C-AuRBL group had the best thermal stability, resistance to enzymatic degradation, and more uniform pore size distribution. None of the groups had cytotoxicity (cell viability > 70%) regarding the microscopic observations and MTT cell viability assays for L929 fibroblasts. L929 fibroblasts and primary adult human epidermal keratinocytes (HEKa) were also separately seeded on C-AuRBL scaffolds and according to microscopy results, they could support the stimulation of adhesion, morphological changes, and spreading of both cells, thereby encouraging the usage of this fabrication strategy for prospective skin tissue scaffolds.

Application of an instructive hydrogel accelerates re-epithelialization of xenografted human skin wounds

Poor quality (eg. excessive scarring) or delayed closure of skin wounds can have profound physical and pyschosocial effects on patients as well as pose an enormous economic burden on the healthcare system. An effective means of improving both the rate and quality of wound healing is needed for all patients suffering from skin injury. Despite wound care being a multi-billion-dollar industry, effective treatments aimed at rapidly restoring the skin barrier function or mitigating the severity of fibrotic scar remain elusive. Previously, a hydrogel conjugated angiopoietin-1 derived peptide (QHREDGS; Q-peptide) was shown to increase keratinocyte migration and improve wound healing in diabetic mice. Here, we evaluated the effect of this Q-Peptide Hydrogel on human skin wound healing using a mouse xenograft model. First, we confirmed that the Q-Peptide Hydrogel promoted the migration of adult human keratinocytes and modulated their cytokine profile in vitro. Next, utilizing our human to mouse split-thickness skin xenograft model, we found improved healing of wounded human epidermis following Q-Peptide Hydrogel treatment. Importantly, Q-Peptide Hydrogel treatment enhanced this wound re-epithelialization via increased keratinocyte migration and survival, rather than a sustained increase in proliferation. Overall, these data provide strong evidence that topical application of QHREDGS peptide-modified hydrogels results in accelerated wound closure that may lead to improved outcomes for patients.

Effects of mucopolysaccharide polysulphate on tight junction barrier in human epidermal keratinocytes.

Tight junctions (TJs) play important roles in epidermal barrier function and their dysfunction is involved in the pathogenesis of various skin diseases, including atopic dermatitis (AD). Mucopolysaccharide polysulphate (MPS) is the active ingredient of a moisturizing agent used to treat xerosis in patients with AD; however, its mechanism of action on TJ barrier function remains unclear. To elucidate the effects of MPS on TJs, adult human epidermal keratinocyte (HEKa) cells were exposed to MPS, subjected to Western blotting and quantitative PCR analyses for the investigation of TJ-related factors. MPS treatment significantly increased the mRNA and protein expression of claudin-1 (CLDN1) and zonula occludens-1, and significantly increased transepithelial electrical resistance (TEER), which indicates TJ integrity. Conversely, the sulphated and non-sulphated glycosaminoglycans, chondroitin sulphate and hyaluronic acid, respectively, had little effect on TEER or the expression of mRNAs or TJ-related proteins. Interestingly, MPS treatment also inactivated the extracellular signal-regulated kinase signalling pathway, which is known to negatively regulate CLDN1 expression. Furthermore, MPS notably improved the reduction in CLDN1 expression and TEER caused by histamine, which is upregulated in the skin of patients with AD and is known to disrupt the TJ barrier function. Taken together, these findings demonstrate that treatment with the moisturizing agent, MPS, can repair TJ dysfunction and could therefore represent a new therapeutic option for treating patients with AD.

Recovery of DNA from fired and unfired cartridge casings: comparison of two DNA collection methods

For over 10 years, various studies have attempted to increase the recovery of DNA from ammunition by modifying the DNA collection, extraction, purification, and amplification procedures, with varying levels of success. This study focused on the “soaking” method of Montpetit & O’Donnell [1] and the “rinse-and-swab" method of Bille et al. [2]. First, testing for the presence of exogenous DNA, 210 boxed cartridges (brass, steel, and nickel-plated) from nine manufacturers were swabbed and DNA was extracted, concentrated, and quantified. Extracts that quantified > 0 ng/µL (44 of 210) were amplified and genotyped with GlobalFiler™. Of those, only one extract yielded two alleles indicating that the manufacturing and packaging of ammunition was virtually DNA free. Next, to obtain a baseline comparison of two DNA collection methods on a non-metallic substrate and identify a suitable number of cells to spot on cartridges, different DNA input amounts of primary human adult epidermal keratinocytes (HEKa) were tested. Thereafter, 300 brass and 300 nickel-plated, cartridges were spotted with HEKa cells containing ~5 ng of DNA, fired or unfired, and processed with either method. Finally, five methods representing hybrids of the soaking and rinse-and-swab methods were tested to determine if variations of those methods could be used to increase DNA yield and recovery. The results show that the soaking method consistently yielded more DNA than the rinse-and-swab method from a non-metallic substrate. However, the comparison study demonstrated that both methods performed comparably for cartridges. On average, the soaking method recovered 0.25 ng of DNA (5.1% recovery) and the rinse-and-swab method recovered 0.28 ng (5.8% recovery). However, average recoveries were significantly different among three analysts and considerable variation in yields were observed, possibly due to storage time. Furthermore, consistent with prior reports, the DNA recovered from brass casings was only 16% of that recovered from nickel-plated casings and the average yield of DNA from fired casings was reduced to 67% of unfired casings. Moreover, DNA extracts from brass or nickel-plated casings did not appear to contain amplification inhibitors and only 30/596 appeared severely degraded. Finally, both the published rinse-and-swab and soaking methods yielded more DNA than all modifications of the two methods. Overall, both methods yielded equivalent DNA quantities. Additionally, recovery of DNA from any given cartridge casing may be dependent on storage time as well as the skill, proficiency, and experience of the analyst and may reflect stochastic effects, particularly for casings containing low copy and/or degraded DNA.

24 The CD34+ Adipose-derived Stem Cells from Fat Grafts Accelerates Keratinocyte Wound Closure

IntroductionAdipose derived stem cells (ADSCs) possess high regenerative potential and therefore are postulated to enhance re-epithelialization via recruitment of keratinocytes into the wound bed, and thusly improve healing of severe burns. Although ADSC’s are enriched in the stromal vascular fraction (SVF) of fat grafts, the ADSC frequency in these samples is very low (1%) which has hindered their further characterization. So far, the CD34+ subset of SVF cells has shown to contain up to 75% of all the ADSCs present in SVF samples but their ADSC frequency remains low (2.5%). Unfortunately, while the ADSC frequency increases in passaged SVF cells, the expression of CD34 is lost in these cells. Therefore, there is a pressing need to identify new ADSC biomarkers to study ADSC mechanisms that accelerate wound closure. In this study, we aim to identify new ADSC biomarkers and to examine the impact of ADSC-enriched cells on skin wound closure.MethodsSingle cells from SVFs were obtained from disassociated fat tissue, collected from excess lipoaspirates used in patients undergoing mastectomies. Native cells (P0), and passaged cells (P3) were placed in cell cultures and the ADSC numbers were obtained using the colony forming unit-fibroblast assays. The expression of 244 cell surface receptors was examined in P0 (low ADSCs) and P3 (high ADSCs) SVF cells to obtain new ADSC biomarkers. In another experiment, the impact of ADSC-enriched CD34+ SVF cells on wound closure was assessed. Using transwell inserts containing adult human epidermal keratinocyte (HEKa) cells in the bottom and the bulk SVF or CD34+ SVF cells in the top chamber, a scratch was induced in the HEKa cell layer and the scratch area was examined at 0h and 12h.ResultsOur cell surface antibody array experiments revealed 10 potentially new ADSC biomarkers that will be used to examine their ability to enrich for ADSCs. Both the bulk SVF and the CD34+ SVF cells showed a 2-fold increase in would closure. It is noteworthy that ADSCs frequency in the CD34+ SVF cells is still very low, and these experiments need to be conducted using the new SVF subsets with potentially higher ADSC frequencies based on our cell surface array.ConclusionsThe ADSC-enriched CD34+ SVF enhance keratinocyte wound closure, a function that is important to healing of severe burns. Further studies are needed to validate our findings using more purified SVF subsets to identify ADSC-regulated keratinocyte wound closure mechanisms.

Overexpression of Flii during Murine Embryonic Development Increases Symmetrical Division of Epidermal Progenitor Cells.

Epidermal progenitor cells divide symmetrically and asymmetrically to form stratified epidermis and hair follicles during late embryonic development. Flightless I (Flii), an actin remodelling protein, is implicated in Wnt/β-cat and integrin signalling pathways that govern cell division. This study investigated the effect of altering Flii on the divisional orientation of epidermal progenitor cells (EpSCs) in the basal layer during late murine embryonic development and early adolescence. The effect of altering Flii expression on asymmetric vs. symmetric division was assessed in vitro in adult human primary keratinocytes and in vivo at late embryonic development stages (E16, E17 and E19) as well as adolescence (P21 day-old) in mice with altered Flii expression (Flii knockdown: Flii+/−, wild type: WT, transgenic Flii overexpressing: FliiTg/Tg) using Western blot and immunohistochemistry. Flii+/− embryonic skin showed increased asymmetrical cell division of EpSCs with an increase in epidermal stratification and elevated talin, activated-Itgb1 and Par3 expression. FliiTg/Tg led to increased symmetrical cell division of EpSCs with increased cell proliferation rate, an elevated epidermal SOX9, Flap1 and β-cat expression, a thinner epidermis, but increased hair follicle number and depth. Flii promotes symmetric division of epidermal progenitor cells during murine embryonic development.

Medicated multi-targeted alginate-based dressings for potential treatment of mixed bacterial-fungal infections in diabetic foot ulcers

Recently developed medicated dressings target either bacterial or fungal infection only, which is not effective for the treatment of mixed infections common in diabetic foot ulcers (DFUs). This study aimed to develop advanced bioactive alginate-based dressings (films and wafers) to deliver therapeutically relevant doses of ciprofloxacin (CIP) and fluconazole (FLU) to target mixed bacterial and fungal infections in DFUs. The alginate compatibility with the drugs was confirmed by SEM, XRD, FTIR and texture analysis, while the medicated wafers showed better fluid handling properties than the films in the presence of simulated wound fluid. The dressings showed initial fast release of FLU followed by sustained release of CIP which completely eradicated E. coli, S. aureus, P. aeruginosa and reduced fungal load (C. albicans) by 10-fold within 24 h. Moreover, the medicated dressings were biocompatible (>70% cell viability over 72 h) with human primary adult keratinocytes and in-vitro scratch assay showed 65–68% wound closure within 7 days.

Inhibition of dipeptidyl-peptidase 4 induces upregulation of the late cornified envelope cluster in keratinocytes.

Dipeptidyl-peptidase 4 (DPP4) is a multifunctional type II transmembrane glycoprotein that is expressed on various cell surfaces. While DPP4 inhibitors have a therapeutic role in the treatment of diabetes mellitus, they are an independent risk factor in the development of bullous pemphigoid. Contrarily, there are reports of improvement in psoriasis with DPP4 inhibition. We investigated the effect of DPP4 inhibition on primary human keratinocytes to determine whether DPP4 modulates keratinocyte inflammatory signaling and keratinocyte homeostasis. We performed RNA sequencing of primary adult human keratinocytes treated with DPP4 inhibitor, identifying 424 differentially expressed genes. Gene ontology analysis revealed significant enrichment of epidermal differentiation and cornified envelope genes. Using three-dimensional organotypic cultures and a pan-late cornified envelope 2 (LCE2) antibody, we demonstrate a dose dependent relationship between DPP4 inhibition and increased expression of LCE2 during epidermal development. The late cornified envelope gene clusters are expressed at the late stages of epithelial development, responding tostimuli such as calcium and ultraviolet light. While its biologic function is not fully understood, mutations in LCE3B/LCE3C confer a 40% increased risk in the development of plaque psoriasis. While we did not identify significant modulation of keratinocyte inflammatory markers, DPP4 inhibition increased expression of the late cornified envelope may offer a potential alternative therapeutic mechanism in psoriasis.

Three-Dimensionally Printed Skin Substitute Using Human Dermal Fibroblasts and Human Epidermal Keratinocytes.

Wound healing affects millions of people annually. After injury, keratinocytes from the wound edge proliferate, migrate, and differentiate to recapitulate the 3-dimensional (3D) structure needed to provide a barrier function. If the wound is too large, skin grafting may be required. We are interested in discovering novel strategies to enhance the wound healing process. It may be possible to recreate a viable and histologically accurate skin tissue using 3D printing. We hypothesize that keratinocytes and dermal fibroblasts can be bioprinted into a viable skin substitute. Adult human dermal fibroblasts (HDFa) and adult human epidermal keratinocytes (HEKa) were cultured and subsequently printed with a 3D bioprinter within a hydrogel scaffold. After printing the HDFa and HEKa separately, cell viability and histological appearance were determined by sectioning the printed tissue and performing hematoxylin and eosin staining. The stained histological sections were analyzed for tissue morphology. The HEKa and HDFa cells suspended in the hydrogel were successfully printed into 3D scaffolds that resembled skin with hematoxylin and eosin staining. The HEKa and HDFa cells can be grown on 3D-printed hydrogels successfully. In addition, HEKa and HDFa cells can survive and grow when suspended in a hydrogel and 3D printed. Future potential applications of these results could lead to the creation of viable skin tissue for wound healing and surgical repair.

IL-36 and IL-17A Cooperatively Induce a Psoriasis-Like Gene Expression Response in Human Keratinocytes

IL-36 and IL-17A Cooperatively Induce a Psoriasis-Like Gene Expression Response in Human Keratinocytes

Superenhanced Removal of Fungal Biofilms by Protease‐Functionalized Amphotericin B Nanocarriers

A strong enhancement in the antifungal activity of amphotericin B (AmpB) encapsulated into shellac nanoparticles (NPs) surface functionalized with protease is reported. These AmpB‐loaded shellac NPs are fabricated by pH‐induced nucleation of aqueous solutions of shellac and AmpB in the presence of Poloxamer 407 (P407) as a steric stabilizer. The AmpB‐loaded shellac NPs are surface coated with the cationic protease Alcalase 2.4L FG. The performance of the AmpB‐encapsulated NPs against Candida albicans is evaluated. The AmpB‐loaded shellac NPs show a remarkable boost of their antifungal action compared to free AmpB when applied to C. albicans in both planktonic and biofilm forms. The surface functionalization with a cationic protease allows the NPs to adhere to the fungal cell walls, delivering AmpB directly to their membranes. Additionally, the hydrolyzing activity of the protease coating degrades the biofilm matrix, thus increasing the effectiveness of the encapsulated AmpB compared to free AmpB at the same concentration. The protease‐coated AmpB‐loaded shellac NPs show no greater toxicity to human adult keratinocyte cells (HaCaT) compared to the free AmpB. These AmpB nanocarriers demonstrat increased efficacy against C. albicans and can be potentially used to treat fungal biofilm infection in the clinic, for example, in recalcitrant chronic wounds.

Unexplored regulatory sequences of divergently paired GLA and HNRNPH2 loci pertinent to Fabry disease in human kidney and skin cells: Presence of an active bidirectional promoter.

Fabry disease (FD) is a rare hereditary disorder characterized by a wide range of symptoms caused by a variety of mutations in the galactosidase α (GLA) gene. The heterogeneous nuclear ribonucleoprotein (HNRNPH2) gene is divergently paired with GLA on chromosome X and is thought to be implicated in FD. However, insufficient information is available on the regulatory mechanisms associated with the expression of HNRNPH2 and the GLA loci. Therefore, the current study performed bioinformatics analyses to assess the GLA and HNRNPH2 loci and investigate the regulatory mechanisms involved in the expression of each gene. The regulatory mechanisms underlying GLA and HNRNPH2 were revealed. The expression of each gene was associated with a bidirectional promoter (BDP) characterized by the absence of TATA box motifs and the presence of specific transcription factor binding sites (TFBSs) and a CpG Island (CGI). The nuclear run-on transcription assay confirmed the activity of BDP GLA and HNRNPH2 transcription in 293T. Methylation-specific PCR analysis demonstrated a statistically significant variation in the DNA methylation pattern of BDP in several cell lines, including human adult epidermal keratinocytes (AEKs), human renal glomerular endothelial cells, human renal epithelial cells and 293T cells. The highest observed significance was demonstrated in AEKs (P<0.05). The results of the chromatin-immunoprecipitation assay using 293T cells identified specific TFBS motifs for Yin Yang 1 and nuclear respiratory factor 1 transcription factors in BDPs. The National Center for Biotechnology Information-single nucleotide polymorphism database revealed pathogenic variants in the BDP sequence. Additionally, a previously reported variant associated with a severe heterozygous female case of GLA FD was mapped in BDP. The results of the present study suggested that the expression of the divergent paired loci, GLA and HNRNPH2, were controlled by BDP. Mutations in BDP may also serve a role in FD and may explain clinical disease diversity.

Laminarin Effects, a β-(1,3)-Glucan, on Skin Cell Inflammation and Oxidation

Laminarin, a β-(1,3)-glucan from the seaweed Laminaria digitata, is a polysaccharide which provides anti-inflammatory and anti-oxidative properties. Its influence on both human dermal fibroblasts adult (HDFa) and normal human epidermal keratinocytes (NHEK) has not been established yet. Herein, laminarin effects were examined on skin cells’ mitochondrial and antioxidant activities. Cytokines, hyaluronic acid, and procollagen type I secretions and interaction mechanisms were explored after a maximum of 72 h treatment with laminarin. Our results demonstrated a decrease in mitochondrial activities with 72 h treatment with laminarin from 500 µg.mL−1 for NHEK cells and from 100 µg.mL−1 for HDFa cells without cytotoxicity. No variation of hyaluronic acid or type I procollagen was observed for all laminarin concentrations, while an antioxidant effect was found against reactive oxygen species (ROS) from 1 µg.mL−1 for HDFa cells in both H2O2 and UVA radiation conditions, and from 10 µg.mL−1 and 1 µg.mL−1 for NHEK cells in both H2O2 and UVA radiation conditions, respectively. Laminarin treatment modulated both cells surface glycosylation and cytokine secretions of skin cells. Overall, our data suggest a positive effect of β-(1,3)-glucan on skin cells on oxidative stress and inflammation induced by environmental factors. Of note, these effects are through the modulation of glycan and receptors interactions at the skin cells surface.

Terminal keratinocyte differentiation in vitro is associated with a stable DNA methylome.

The epidermal compartment of the skin is regenerated constantly by proliferation of epidermal keratinocytes. Differentiation of a subset of these keratinocytes allows the epidermis to retain its barrier properties. Regulation of keratinocyte fate—whether to remain proliferative or terminally differentiate—is complex and not fully understood. The objective of our study was to assess if DNA methylation changes contribute to the regulation of keratinocyte fate. We employed genome‐wide MethylationEPIC beadchip array measuring approximately 850 000 probes combined with RNA sequencing of in vitro cultured non‐differentiated and terminally differentiated adult human primary keratinocytes. We did not observe a correlation between methylation status and transcriptome changes. Moreover, only two differentially methylated probes were detected, of which one was located in the TRIM29 gene. Although TRIM29 knock‐down resulted in lower expression levels of terminal differentiation genes, these changes were minor. From these results, we conclude that—in our in vitro experimental setup—it is unlikely that changes in DNA methylation have an important regulatory role in terminal keratinocyte differentiation.