Mouse Skin Cells Research Articles

Estimating and correcting index hopping misassignments in single-cell RNA-seq data.

Index hopping causes errors in multiplexed high-throughput sequencing data whereby inaccurate barcoding leads to the misassignment of sequence reads to an incorrect source. This can result in gene expression assignments to the wrong cell in single-cell RNA sequencing (scRNA-seq). We performed scRNA-seq on sorted mouse skin cells and identified four distinct cell types based on gene expression profiles. Using genes unique to each cell type, we discovered that approximately 0.65% of total reads per gene were incorrectly assigned to another cell due to index hopping, regardless of gene expression levels. To correct for the misassigned reads, we proportionally adjusted each cell's gene expression data. Applying this correction improved analyses allowing for enhanced cell clustering accuracy and refining the identification of intermediate cell states during cell differentiation. Our study emphasizes the significance of index hopping in scRNA-seq experiments and presents a practical correction approach to remove misassigned reads that can be applied to any scRNA-seq study involving cells with distinct gene expression profiles or where non-expressed genes are introduced as a quality control measure during library preparation.

P2RY6 deletion promotes UVB-induced skin carcinogenesis by activating the PI3K/AKT signal pathway.

Our previous research has demonstrated that P2RY6 functions as an oncogene in DMBA/TPA-induced two-stage chemical skin carcinogenesis in mice. However, considering that human skin cancer is predominantly attributed to UV radiation from sunlight, additional investigations are needed to elucidate the role of P2RY6 in UVB-induced skin carcinogenesis. Surprisingly, we found that P2ry6-deficient mice exhibited marked promotion to UVB-induced skin papilloma formation compared with wild-type mice, suggesting its tumor-suppressive role in UVB-induced skin cancer. Additionally, a P2ry6 gene knockout promoted skin hyperplasia induced by short-term UVB irradiation, while UDP, the ligand of P2RY6, could inhibit the short-term UVB-induced increase of epiderma thickness in mouse skin. Furthermore, UVB irradiation could significantly upregulate P2RY6 expression in human and mouse skin cells. These results indicated that P2RY6 may play a crucial protective role in resisting the UVB-induced formation of skin tumors. At the molecular level, the loss of the P2RY6 gene inhibits the ubiquitination modification and expression of XPC after UVB irradiation in skin keratinocytes, resulting in the accumulation of CPDs (cyclobutane pyrimidine dimers). We have also demonstrated that P2RY6 deletion activates the PI3K/AKT signaling pathway both invitro and invivo. The CPD accumulation and acute inflammatory response enhanced by the loss of the P2RY6 gene can be reversed by an AKT inhibitor. These findings suggest that P2RY6 may act as a tumor suppressor in UVB-induced skin cancer by regulating the PI3K/AKT signaling pathway.

Malassezia globosa Induces Differentiation of Pathogenic Th17 Cells by Inducing IL-23 Secretion by Keratinocytes.

Malassezia, the most abundant fungal commensal on the mammalian skin, has been linked to several inflammatory skin diseases such as atopic dermatitis, seborrheic dermatitis and psoriasis. This study reveals that epicutaneous application with Malassezia globosa (M. globosa) triggers skin inflammation in mice. RNA-sequencing of the resulting mouse lesions indicates activation of Interleukin-17 (IL-17) signaling and T helper 17 (Th17) cells differentiation pathways by M. globosa. Furthermore, our findings demonstrate a significant upregulation of IL-23, IL-23R, IL-17A, and IL-22 expressions, along with an increase in the proportion of Th17 and pathogenic Th17 cells in mouse skin exposed to M. globosa. In vitro experiments illustrate that M. globosa prompts human primary keratinocytes to secrete IL-23 via TLR2/MyD88/NF-κB signaling. This IL-23 secretion by keratinocytes is shown to be adequate for inducing the differentiation of pathogenic Th17 cells in the skin. Overall, these results underscore the significant role of Malassezia in exacerbating skin inflammation by stimulating IL-23 secretion by keratinocytes and promoting the differentiation of pathogenic Th17 cells.

Innate lymphoid cell-based immunomodulatory hydrogel microspheres containing Cutibacterium acnes extracellular vesicles for the treatment of psoriasis

Psoriasis is a chronic skin inflammation influenced by dysregulated skin microbiota, with the role of microbiota in psoriasis gaining increasing prominence. Bacterial extracellular vesicles (bEVs) serve as crucial regulators in the interaction between hosts and microbiota. However, the mechanism underlying the therapeutic potential of bEVs from commensal bacteria in psoriasis remains unclear. Here, we investigated the therapeutic role of Cutibacterium acnes (C. acnes)-derived extracellular vesicles (CA-EVs) in psoriasis treatment. To prolong the active duration of CA-EVs, we encapsulated them in gelatin methacrylate (GelMA) to fabricate hydrogel microspheres (CA-EVs@GHM) with sustained release properties. As GelMA degraded, CA-EVs were gradually released, maintaining a high concentration in mouse skin even 96 h post-treatment. In human keratinocyte cells (HaCaT), CA-EVs@GHM enhanced resistance to Staphylococcus aureus (S. aureus), promoted proliferation and migration of HaCaT cells exposed to S. aureus, and significantly reduced the expression of inflammatory genes such as interleukin (IL)-6 and C-X-C motif chemokine ligand 8 (CXCL8). In vivo, CA-EVs@GHM, more potent than CA-EVs alone, markedly attenuated proinflammatory gene expression, including tumor necrosis factor (TNF), Il6, Il17a, Il22 and Il23a in imiquimod (IMQ)-induced psoriasis-like mice, and restored skin barrier function. 16S rRNA sequencing revealed that CA-EVs@GHM might provide therapeutic effects against psoriasis by restoring microbiota diversity on the back skin of mice, reducing Staphylococcus colonization, and augmenting lipid metabolism. Furthermore, flow cytometry analysis showed that CA-EVs@GHM prevented the conversion of type 2 innate lymphoid cells (ILC2) to type 3 innate lymphoid cells (ILC3) in psoriasis-like mouse skin, reducing the pathogenic ILC3 population and suppressing the secretion of IL-17 and IL-22. In summary, our findings demonstrate that the long-term sustained release of CA-EVs alleviated psoriasis symptoms by controlling the transformation of innate lymphoid cells (ILCs) subgroups and restoring skin microbiota homeostasis, thus offering a promising therapy for psoriasis treatment. Statement of significanceCutibacterium acnes, which is reduced in psoriasis skin, has been reported to promote skin homeostasis by regulating immune balance. Compared to live bacteria, bacterial extracellular vesicles (bEVs) are less prone to toxicity and safety concerns. bEVs play a pivotal role in maintaining bacterial homeostasis and modulating the immune system.However,bEVs without sustained release materials are unable to function continuously in chronic diseases. Therefore, we utilized hydrogel microspheres to encapsulate Cutibacterium acnes (C. acnes)-derived extracellular vesicles (CA-EVs), enabling long term sustained release. Our findings indicate that, CA-EVs loaded gelatin methacrylate hydrogel microspheres (CA-EVs@GHM) showed superior therapeutic effects in treating psoriasis compared to CA-EVs. CA-EVs@GHM exhibited a more significant regulation of pathological type 3 innate lymphoid cells (ILC3) and skin microbiota, providing a promising approach for microbiota-derived extracellular vesicle therapy in the treatment of skin inflammation.

Imidazole propionate ameliorates atopic dermatitis-like skin lesions by inhibiting mitochondrial ROS and mTORC2.

Imidazole propionate (IMP) is a histidine metabolite produced by some gut microorganisms in the human colon. Increased levels of IMP are associated with intestinal inflammation and the development and progression of cardiovascular disease and diabetes. However, the anti-inflammatory activity of IMP has not been investigated. This study aimed to elucidate the role of IMP in treating atopic dermatitis (AD). To understand how IMP mediates immunosuppression in AD, IMP was intraperitoneally injected into a Dermatophagoides farinae extract (DFE)/1-chloro-2,4 dinitrochlorobenzene (DNCB)-induced AD-like skin lesions mouse model. We also characterized the anti-inflammatory mechanism of IMP by inducing an AD response in keratinocytes through TNF-α/IFN-γ or IL-4 stimulation. Contrary to the prevailing view that IMP is an unhealthy microbial metabolite, we found that IMP-treated AD-like skin lesions mice showed significant improvement in their clinical symptoms, including ear thickness, epidermal and dermal thickness, and IgE levels. Furthermore, IMP antagonized the expansion of myeloid (neutrophils, macrophages, eosinophils, and mast cells) and Th cells (Th1, Th2, and Th17) in mouse skin and prevented mitochondrial reactive oxygen species production by inhibiting mitochondrial energy production. Interestingly, we found that IMP inhibited AD by reducing glucose uptake in cells to suppress proinflammatory cytokines and chemokines in an AD-like in vitro model, sequentially downregulating the PI3K and mTORC2 signaling pathways centered on Akt, and upregulating DDIT4 and AMPK. Our results suggest that IMP exerts anti-inflammatory effects through the metabolic reprogramming of skin inflammation, making it a promising therapeutic candidate for AD and related skin diseases.

Targeting phosphorylation circuits on CREB and CRTCs as the strategy to prevent acquired skin hyperpigmentation.

Background: The cAMP response element-binding protein (CREB) and CREB-regulated transcription coactivators (CRTCs) cooperate in the transcriptional activation of microphthalmia-associated transcription factor subtype M (MITF-M) that is a master regulator in the biogenesis, pigmentation and transfer of melanosomes at epidermal melanocytes. Here, we propose the targeting of phosphorylation circuits on CREB and CRTCs in the expression of MITF-M as the rationale to prevent skin hyperpigmentation by elucidating the inhibitory activity and mechanism of yakuchinone A (Yaku A) on facultative melanogenesis. Methods: We employed human epidermal melanocyte cell, mouse skin, and mouse melanoma cell, and applied Western blotting, reverse transcription-polymerase chain reaction, immunoprecipitation and confocal microscopy to conduct this study. Results: This study suggested that α-melanocyte stimulating hormone (α-MSH)-induced melanogenic programs could switch on the axis of protein kinase A-salt inducible kinases (PKA-SIKs) rather than that of PKA-AMP activated protein kinase (PKA-AMPK) during the dephosphorylation of CRTCs in the expression of MITF-M. SIK inhibitors rather than AMPK inhibitors stimulated melanin production in melanocyte cultures in the absence of extracellular melanogenic stimuli, wherein SIK inhibitors increased the dephosphorylation of CRTCs but bypassed the phosphorylation of CREB for the expression of MITF-M. Treatment with Yaku A prevented ultraviolet B (UV-B)-irradiated skin hyperpigmentation in mice and inhibited melanin production in α-MSH- or SIK inhibitor-activated melanocyte cultures. Mechanistically, Yaku A suppressed the expression of MITF-M via dually targeting the i) cAMP-dependent dissociation of PKA holoenzyme at the upstream from PKA-catalyzed phosphorylation of CREB coupled with PKA-SIKs axis-mediated dephosphorylation of CRTCs in α-MSH-induced melanogenic programs, and ii) nuclear import of CRTCs after SIK inhibitor-induced dephosphorylation of CRTCs. Conclusions: Taken together, the targeting phosphorylation circuits on CREB and CRTCs in the expression of MITF-M could be a suitable strategy to prevent pigmentary disorders in the skin.

Arsenic is a potent co-mutagen of ultraviolet light

Arsenic enhances the carcinogenicity of ultraviolet radiation (UVR). However, the mechanisms of arsenic-driven oncogenesis are not well understood. Here, we utilize experimental systems to investigate the carcinogenic and mutagenic properties of co-exposure to arsenic and UVR. In vitro and in vivo exposures indicate that, by itself, arsenic is not mutagenic. However, in combination with UVR, arsenic exposure has a synergistic effect leading to an accelerated mouse skin carcinogenesis and to more than 2-fold enrichment of UVR mutational burden. Notably, mutational signature ID13, previously found only in UVR-associated human skin cancers, is observed exclusively in mouse skin tumors and cell lines jointly exposed to arsenic and UVR. This signature was not observed in any model system exposed purely to arsenic or purely to UVR, making ID13, to the best of our knowledge, the first co-exposure signature to be reported using controlled experimental conditions. Analysis of existing skin cancer genomics data reveals that only a subset of cancers harbor ID13 and these exhibit an elevated UVR mutagenesis. Our results report a unique mutational signature caused by a co-exposure to two environmental carcinogens and provide comprehensive evidence that arsenic is a potent co-mutagen and co-carcinogen of UVR.

MiR-21 Expressed by Dermal Fibroblasts Enhances Skin Wound Healing Through the Regulation of Inflammatory Cytokine Expression.

The management of skin wound healing is still a challenge. MicroRNA-21 (miR-21) has been reported to play important roles in wound repair; however, the underlying mechanism needs to be further clarified. The present study aimed to study the direct role of miR-21 in skin wound healing in miR-21 KO mice and to investigate the role of miR-21 in controlling the migration and proliferation of primary human skin cells and its underlying mechanism(s). miR-21 KO and wild-type (WT) mice were used for in vivo wound healing assays, while mouse and human primary skin cells were used for in vitro assays. miR-21 inhibitors or mimics or negative control small RNAs were transfected to either inhibit or enhance miR-21 expression in the human primary dermal fibroblasts or epidermal cells. RNA sequencing analysis was performed to identify the potential molecular pathways involved. We found that the loss of miR-21 resulted in slower wound healing in miR-21 KO mouse skin and especially delayed the healing of dermal tissue. In vitro assays demonstrated that the reduced expression of miR-21 caused by its inhibitor inhibited the migration of human primary dermal fibroblasts, which could be enhanced by increased miR-21 expression caused by miR-21 mimics. RNA-sequence analysis revealed that the inhibition of miR-21 expression downregulated the inflammatory response pathways associated with the decreased expression of inflammatory cytokines, and the addition of IL-1β into the culture medium enhanced the migration and proliferation of dermal fibroblasts in vitro. In conclusion, miR-21 in dermal fibroblasts can promote the migration and growth of epidermal and dermal cells to enhance skin wound healing through controlling the expression of inflammatory cytokines.

Antibacterial and biofilm-inhibiting cotton fabrics decorated with copper nanoparticles grown on graphene nanosheets.

Infectious pathogens can be transmitted through textiles. Therefore, additional efforts are needed to develop functional fabrics containing antimicrobial substances to prevent the growth of antibiotic-resistant bacteria and their biofilms. Here, we developed a cotton fabric coated with reduced graphene oxide (rGO) and copper nanoparticles (Cu NPs), which possessed hydrophobic, antimicrobial, and anti-biofilm properties. Once the graphene oxide was dip-coated on a cellulose cotton fabric, Cu NPs were synthesized using a chemical reduction method to fabricate an rGO/Cu fabric, which was analyzed through FE-SEM, EDS, and ICP-MS. The results of our colony-forming unit assays indicated that the rGO/Cu fabric possessed high antibacterial and anti-biofilm properties against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Corynebacterium xerosis, and Micrococcus luteus. Particularly, the fabric could inhibit the growth of E. coli, C. xerosis, and M. luteus with a 99% efficiency. Furthermore, our findings confirmed that the same concentrations of rGO/Cu had no cytotoxic effects against CCD-986Sk and Human Dermal Fibroblast (HDF), human skin cells, and NIH/3T3, a mouse skin cell. The developed rGO/Cu fabric thus exhibited promising applicability as a cotton material that can maintain hygienic conditions by preventing the propagation of various bacteria and sufficiently suppressing biofilm formation while also being harmless to the human body.

GRB2 serves as a viable target against skin fibrosis in systemic sclerosis by regulating endothelial cell apoptosis

BackgroundSystemic Sclerosis (SSc) is an autoimmune disease characterized by vascular and immune system dysfunction, along with tissue fibrosis. Our previous study found GRB2 was downregulated by salvianolic acid B, a small molecule drug that attenuated skin fibrosis of SSc. ObjectivesHere we aim to investigate the role of GRB2 in SSc. MethodsThe microarray data of SSc skin biopsies in Caucasians were obtained from the Gene Expression Omnibus (GEO) database. The expression of GRB2 was further detected in Chinese SSc and healthy controls. Bleomycin (BLM)-induced skin fibrosis mice were used to explore how GRB2 downregulation affected fibrosis. The apoptosis of EA.hy926 endothelial cells was induced by H2O2 and apoptosis ratio was measured by flow cytometric. Transcriptome and phosphoproteomic analyses were performed to explore the regulated pathway. ResultsThe expression of GRB2 was significantly enhanced in SSc patient skin, 1.51-fold in Caucasians and 1.40-fold in Chinese. Double immunofluorescence staining showed the endothelial cells of SSc patient’s skin highly expressed GRB2. The in vivo study revealed that GRB2 knockdown alleviated skin fibrosis and apoptosis of endothelial cells in BLM mouse skin. The in vitro study showed that GRB2 downregulation inhibited the apoptosis of EA.hy926 and protected them from H2O2-induced hyperpermeability. Moreover, transcriptome and phosphoproteomic analysis suggested the focal adhesion pathway was enriched in GRB2 siRNA transfected endothelial cells. ConclusionsOur results demonstrated GRB2 highly expressed in endothelial cells of SSc skin, and inhibiting GRB2 could effectively attenuate BLM-induced skin fibrosis and endothelial cell apoptosis. GRB2 is expected to be a new therapeutic target for SSc.

Peptide Supramolecular Hydrogels with Sustained Release Ability for Combating Multidrug-Resistant Bacteria.

Chronic wound infection caused by multidrug-resistant bacteria is a major threat globally, leading to high mortality rates and a considerable economic burden. To address it, an innovative supramolecular nanofiber hydrogel (Hydrogel-RL) harboring antimicrobial peptides was developed based on the novel arginine end-tagging peptide (Pep 6) from our recent study, triggering cross-linking. In vitro results demonstrated that Hydrogel-RL can sustain the release of Pep 6 up to 120 h profiles, which is biocompatible and exhibits superior activity for methicillin-resistant Staphylococcus aureus (MRSA) biofilm inhibition and elimination. A single treatment of supramolecular Hydrogel-RL on an MRSA skin infection model revealed formidable antimicrobial activity and therapeutic effects in vivo. In the chronic wound infection model, Hydrogel-RL promoted mouse skin cell proliferation, reduced inflammation, accelerated re-epithelialization, and regulated muscle and collagen fiber formation, rapidly healing full-thickness skin wounds. To show its vehicle property for wound infection combined therapy, etamsylate, an antihemorrhagic drug, was loaded into the porous network of Hydrogel-RL, which demonstrated improved hemostatic activity. Collectively, Hydrogel-RL is a promising clinical candidate agent for functional supramolecular biomaterials designed for combating multidrug-resistant bacteria and rescuing stalled healing in chronic wound infections.

Matrix prior for data transfer between single cell data types in latent Dirichlet allocation.

Single cell ATAC-seq (scATAC-seq) enables the mapping of regulatory elements in fine-grained cell types. Despite this advance, analysis of the resulting data is challenging, and large scale scATAC-seq data are difficult to obtain and expensive to generate. This motivates a method to leverage information from previously generated large scale scATAC-seq or scRNA-seq data to guide our analysis of new scATAC-seq datasets. We analyze scATAC-seq data using latent Dirichlet allocation (LDA), a Bayesian algorithm that was developed to model text corpora, summarizing documents as mixtures of topics defined based on the words that distinguish the documents. When applied to scATAC-seq, LDA treats cells as documents and their accessible sites as words, identifying "topics" based on the cell type-specific accessible sites in those cells. Previous work used uniform symmetric priors in LDA, but we hypothesized that nonuniform matrix priors generated from LDA models trained on existing data sets may enable improved detection of cell types in new data sets, especially if they have relatively few cells. In this work, we test this hypothesis in scATAC-seq data from whole C. elegans nematodes and SHARE-seq data from mouse skin cells. We show that nonsymmetric matrix priors for LDA improve our ability to capture cell type information from small scATAC-seq datasets.

Extracellular vesicles from IFN-γ-primed mesenchymal stem cells repress atopic dermatitis in mice

Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by immune dysregulation, pruritus, and abnormal epidermal barrier function. Compared with conventional mesenchymal stem cell (MSC), induced pluripotent stem cell (iPSC)-derived mesenchymal stem cell (iMSC) is recognized as a unique source for producing extracellular vesicles (EVs) because it can be obtained in a scalable manner with an enhanced homogeneity. Stimulation of iMSCs with inflammatory cytokines can improve the immune-regulatory, anti-inflammatory, and tissue-repairing potential of iMSC-derived EVs. Proteome analysis showed that IFN-γ-iMSC-EVs are enriched with protein sets that are involved in regulating interferon responses and inflammatory pathways. In AD mice, expression of interleukin receptors for Th2 cytokines (IL-4Rα/13Rα1/31Rα) and activation of their corresponding intracellular signaling molecules was reduced. IFN-γ-iMSC-EVs decreased itching, which was supported by reduced inflammatory cell infiltration and mast cells in AD mouse skin; reduced IgE receptor expression and thymic stromal lymphopoietin and NF-kB activation; and recovered impaired skin barrier, as evidenced by upregulation of key genes of epidermal differentiation and lipid synthesis. IFN-γ-iMSC-EVs inhibit Th2-induced immune responses, suppress inflammation, and facilitate skin barrier restoration, contributing to AD improvement.

Autofluorescence identifies highly phagocytic tissue-resident macrophages in mouse and human skin and cutaneous squamous cell carcinoma.

Macrophages from human and mouse skin share phenotypic and functional features, but remain to be characterized in pathological skin conditions. Skin-resident macrophages are known to derive from embryonic precursors or from adult hematopoiesis. In this report, we investigated the origins, phenotypes and functions of macrophage subsets in mouse and human skin and in cutaneous squamous cell carcinoma (cSCC) using the spectral flow cytometry technology that enables cell autofluorescence to be considered as a full-fledged parameter. Autofluorescence identifies macrophage subsets expressing the CD206 mannose receptor in human peri-tumoral skin and cSCC. In mouse, all AF+ macrophages express the CD206 marker, a subset of which also displaying the TIM-4 marker. While TIM-4-CD206+ AF+ macrophages can differentiate from bone-marrow monocytes and infiltrate skin and tumor, TIM-4 identifies exclusively a skin-resident AF+ macrophage subset that can derive from prenatal hematopoiesis which is absent in tumor core. In mouse and human, AF+ macrophages from perilesional skin and cSCC are highly phagocytic cells contrary to their AF- counterpart, thus identifying autofluorescence as a bona fide marker for phagocytosis. Our data bring to light autofluorescence as a functional marker characterizing subsets of phagocytic macrophages in skin and cSCC. Autofluorescence can thus be considered as an attractive marker of function of macrophage subsets in pathological context.

Photodissociation of nitric oxide from designed ruthenium nitrosyl complex: Studies on wound healing and antibacterial activity

A photoactivable NO releasing complex [Ru(L1-2)(PPh3)(NO)Cl2](PF6)(1a) have been synthesized by complex [RuL1-2(PPh3)2Cl2](1). Newly designed bidentate ligands, i.e., 4-methoxy-N′-phenyl-N'-(pyridin-2-ylmethyl)benzohydrazide(L1) and 4-nitro-N′-phenyl-N'-(pyridin-2-ylmethyl)benzohydrazide (L2) were utilized to synthesize complex (1). Complex (1) was characterized by ESI-MS, and the solid structure of the complex [1a](PF6) was acquired by X-ray crystallography. Different spectroscopic techniques were employed for the identification of ligands (L1 and L2) and complexes (1 and [1a](PF6)). Calculations employing DFT and TD-DFT were made better to understand the electronic properties of the complex [1a](PF6). The photo liberation experiments were screened in the presence of visible light lamp. Griess assay experiment was used to quantify the photo released amount to NO. The photo liberated NO was successfully transferred to reduced myoglobin (Mb). The complex [1a](PF6) at 50 μg/mL concentration was used for wound healing and antimicrobial activity on B16F1 mouse skin cells and Escherichia coli bacteria, respectively. In results, we observed a considerable wound healing activity of [1a](PF6) complex after 36 h of incubation in the light-treated cells compared to the control medium, and also it shows more than 99% inhibition of bacterial cells after 1.5 h of treatment in the presence of light. These study suggested that this complex 1a](PF6) could be utilized for topical delivery of NO for combating several dermatological infections.

Design, Development, and Testing of a Device for Gene Electrotransfer to Skin Cells In Vivo.

Gene electrotransfer (GET) is considered one of the most efficient, safe, reproducible, and cost-effective methods of gene therapy, in which a gene is delivered to the cells in the form of a plasmid DNA vector by a method known as electroporation. To achieve successful electroporation, cells must be exposed to sufficiently high electric fields generated by short-duration, high-voltage electrical pulses that result in a temporary increase in plasma membrane permeability. The electrical pulses are generated by pulse generators (electroporators) and delivered to the cells via electrodes (applicators). However, there is a lack of standardized pulse delivery protocols as well as certified clinical pulse generators and applicators for gene delivery. In this paper, the development of a new pulse generator, applicator, and pulse delivery protocol for GET to skin cells is presented. A numerical model of electroporated skin developed and tested for two electrode configurations and two different pulse delivery protocols is also presented. An alternative pulse delivery protocol was proposed. The developed pulse generator, applicator, and the proposed pulse delivery protocol were then used in vivo for GET to skin cells in mice. The results showed high efficiency of the proposed pulse delivery protocol for the purpose of GET in mouse skin cells. Specifically, electroporation with the developed pulse generator, applicator, and proposed pulse delivery protocol resulted in higher gene expression in skin cells compared to the currently used pulse generator, applicator, and pulse delivery protocol.

Vasculature atrophy causes a stiffened microenvironment that augments epidermal stem cell differentiation in aged skin.

Stem cell loss causes tissue deterioration associated with aging. The accumulation of genomic and oxidative stress-induced DNA damage is an intrinsic cue for stem cell loss1,2; however, whether there is an external microenvironmental cue that triggers stem cell loss remains unclear. Here we report that the involution of skin vasculature causes dermal stiffening that augments the differentiation and hemidesmosome fragility of interfollicular epidermal stem cells (IFESCs) in aged mouse skin. Aging-related IFESC dysregulation occurs in plantar and tail skin, and is correlated with prolonged calcium influx, which is contributed by the mechanoresponsive ion channel Piezo1 (ref. 3). Epidermal deletion of Piezo1 ameliorated IFESC dysregulation in aged skin, whereas Piezo1 activation augmented IFESC differentiation and hemidesmosome fragility in young mice. The dermis stiffened with age, which was accompanied by dermal vasculature atrophy. Conversely, induction of the dermal vasculature softened the dermis and ameliorated IFESC dysregulation in aged skin. Single-cell RNA sequencing of dermal fibroblasts identified an aging-associated anti-angiogenetic secretory molecule, pentraxin 3 (ref. 4), which caused dermal sclerotization and IFESC dysregulation in aged skin. Our findings show that the vasculature softens the microenvironment for stem cell maintenance and provide a potential mechanobiology-based therapeutic strategy against skin disorders in aging.

The Effectiveness of Topical 5-fluorouracil Treatment on Mouse Skin Squamous Cell Precancerous Lesions through Caspase-3 Expression

Skin cancer is a disease that develops in the epidermis of the skin and can be invasive, such as squamous cell carcinoma (SCC). Early detection of squamous cell precancerous can prevent these lesions from progressing to invasive SCC and increase the effectiveness of therapy. 5-fluorouracil (5-FU) is an antimetabolite compound as a pyrimidine DNA/RNA antagonist molecule that induces cell apoptosis. The main objective of this study was to evaluate the effectiveness of the topical 5-FU cream (Dharmais NCH) compared to imiquimod 5% on apoptosis through the expression of caspase-3 in precancerous squamous cells of mouse skin induced by 7,12-dimethylbenzen[a]-anthracene (DMBA)/croton oil treatment. This research assess three differences concentration of 5-FU include 1%, 2%, and 5% on 24 wild type mouse divided into 6 groups including positive control (with carcinogenesis but without treatment), negative control (without treatment; normal), carcinogenesis with treatment 5-FU cream (1%, 2%, and 5%) or 5% imiquimod cream. Two-stages carcinogenesis induced by DMBA and followed by croton oil. The expression of caspase-3 was analyzed using immunohistochemistry. Statistical analysis was performed by one-way ANOVA using SPSS version 23. The induction of two-stages of carcinogenesis (weeks 1 to 10) caused papilloma lesions on the skin of mouse. Furthermore, 5-FU treatment for 4 weeks (weeks 11 to 14) showed a decrease in the cumulative number of papillomas (p<0.05) and immunohistochemical analysis showed caspase-3 expression on 5-FU treatments (1%, 2%, and 5%) was not significantly different from the imiquimod treatment (p>0.05). The apoptotic effect of 5-FU treatment on precancerous skin squamous cell lesions in mouse was not significantly different from the standard treatment using imiquimod. This suggests that 5-FU treatment has potential as a future therapy in squamous cell precancerous skin lesions.Keywords: 5-fluorouracil, caspase-3, squamous cell precancerous, skin, topical treatment.

Resistance to chemical carcinogenesis induction via a dampened inflammatory response in naked mole-rats

Naked mole-rats (NMRs) have a very low spontaneous carcinogenesis rate, which has prompted studies on the responsible mechanisms to provide clues for human cancer prevention. However, it remains unknown whether and how NMR tissues respond to experimental carcinogenesis induction. Here, we show that NMRs exhibit extraordinary resistance against potent chemical carcinogenesis induction through a dampened inflammatory response. Although carcinogenic insults damaged skin cells of both NMRs and mice, NMR skin showed markedly lower immune cell infiltration. NMRs harbour loss-of-function mutations in RIPK3 and MLKL genes, which are essential for necroptosis, a type of necrotic cell death that activates strong inflammation. In mice, disruption of Ripk3 reduced immune cell infiltration and delayed carcinogenesis. Therefore, necroptosis deficiency may serve as a cancer resistance mechanism via attenuating the inflammatory response in NMRs. Our study sheds light on the importance of a dampened inflammatory response as a non-cell-autonomous cancer resistance mechanism in NMRs.

Single-cell transcriptome profile of mouse skin undergoing antigen-driven allergic inflammation recapitulates findings in atopic dermatitis skin lesions

Allergic skin inflammation elicited in mice by epicutaneous (EC) sensitization with antigen shares characteristics with human atopic dermatitis (AD). We characterized gene expression by single cells in mouse skin undergoing antigen-driven allergic inflammation and compared the results with findings in AD skin lesions. Mice were EC sensitized by application of ovalbumin (OVA) or saline to tape-stripped skin. Single-cell RNA sequencing was performed on skin cells 12 days later. Flow cytometry analysis was performed to validate results. Sequencing identified 7 nonhematopoietic and 6 hematopoietic cell subsets in EC-sensitized mouse skin. OVA sensitization resulted in the expansion in the skin of T cells, dendritic cells, macrophages, mast cells/basophils, fibroblasts, and myocytes cell clusters, and in upregulation of TH2 cytokine gene expression in CD4+ T cells and mast cells/basophils. Genes differentially expressed in OVA-sensitized skin included genes important for inflammation in dendritic cells and macrophages, collagen deposition, and leukocyte migration in fibroblasts, chemotaxis in endothelial cells and skin barrier integrity, and differentiation in KCs-findings that recapitulate those in AD skin lesions. Unexpectedly, mast cells/basophils, rather than T cells, were the major source of Il4 and ll13 in OVA-sensitized mouse skin. In addition, our results suggest novel pathways in fibroblast and endothelial cells that may contribute to allergic skin inflammation. The gene expression profile of single cells in mouse skin undergoing antigen-driven shares many features with that in AD skin lesions and unveils novel pathways that may be involved in allergic skin inflammation.