Scalp-Ear-Nipple syndrome: first report of a Potassium channel tetramerization domain-containing 1 in-frame insertion and review of the literature.

OBJECTIVES: Pathogenic missense variants in the potassium channel tetramerization domain-containing 1 (KCTD1) gene are associated with autosomal dominant Scalp-Ear-Nipple syndrome (SENS), a type of ectodermal dysplasia characterized by aplasia cutis congenita of the scalp, hairless posterior scalp nodules, absent or rudimentary nipples, breast aplasia and external ear anomalies. We report a child with clinical features of an ectodermal dysplasia, including sparse hair, dysmorphic facial features, absent nipples, 2-3 toe syndactyly, mild atopic dermatitis and small cupped ears with overfolded helices. We also review the published cases of SENS with molecularly confirmed KCTD1 variants. METHODS AND RESULTS: Using whole-exome sequencing, we identified a novel, de novo in-frame insertion in the broad-complex, tramtrack and bric-a-brac (BTB) domain of the KCTD1 gene. By comparing to the previously reported patients, we found that our patient's clinical features and molecular variant are consistent with a diagnosis of SENS. CONCLUSIONS: This is only the 13th KCTD1 variant described and the first report of an in-frame insertion causing clinical features, expanding the mutational spectrum of KCTD1 and SENS.

KCTD1 mutants in scalp­ear­nipple syndrome and AP­2α P59A in Char syndrome reciprocally abrogate their interactions, but can regulate Wnt/ß­catenin signaling.

Potassium­channel tetramerization-domain-containing 1 (KCTD1) mutations are reported to result in scalp­ear­nipple syndrome. These mutations occur in the conserved broad­complex, tramtrack and bric a brac domain, which is associated with inhibited transcriptional activity. However, the mechanisms of KCTD1 mutants have not previously been elucidated; thus, the present study aimed to investigate whether KCTD1 mutants affect their interaction with transcription factor AP­2α and their regulation of the Wnt pathway. Results from the present study demonstrated that none of the ten KCTD1 mutants had an inhibitory effect on the transcriptional activity of AP­2α. Co­immunoprecipitation assays demonstrated that certain mutants exhibited changeable localization compared with the nuclear localization of wild­type KCTD1, but no KCTD1 mutant interacted with AP­2α. Almost all KCTD1 mutants, except KCTD1 A30E and H33Q, exhibited differential inhibitory effects on regulating TOPFLASH luciferase reporter activity. In addition, the interaction region of KCTD1 to the PY motif (amino acids 59­62) in AP­2α was identified. KCTD1 exhibited no suppressive effects on the transcriptional activity of the AP­2α P59A mutant, resulting in Char syndrome, a genetic disorder characterized by a distinctive facial appearance, heart defect and hand abnormalities, by altered protein cellular localization that abolished protein interactions. However, the P59A, P60A, P61R and 4A AP­2α mutants inhibited TOPFLASH reporter activity. Moreover, AP­2α and KCTD1 inhibited ß­catenin expression levels and SW480 cell viability. The present study thus identified a putative mechanism of disease­related KCTD1 mutants and AP­2α mutants by disrupting their interaction with the wildtype proteins AP­2α and KCTD1 and influencing the regulation of the Wnt/ß­catenin pathway.

Pigment epithelium-derived factor attenuates angiogenesis and collagen deposition in hypertrophic scars.

Scar forming wounds are often characterized by higher levels of vascularity than non-scarring wounds and normal skin, and inhibition of angiogenesis has been shown to inhibit scar formation in some model systems. The rabbit ear hypertrophic scar (HS) model has been widely used to study the mechanisms that underlie the development of HS as well as the effectiveness of various treatments. Although the rabbit ear HS model is well characterized in terms of scar formation, the rate and level of angiogenesis has not been investigated in this model, and the cause-effect relationship between angiogenesis and rabbit HSs has not been examined. In the current study, full-thickness excisional wounds were created on the ventral side of New Zealand White rabbit ears to induce HS formation, and the dynamic pattern of angiogenesis and the expression of angiogenic regulatory factors were examined over time. Blood vessel density was found to peak at 2.7% on day 14 post-wounding, decreasing to 1.7% by day 28. mRNA levels of the proangiogenic factor VEGF-A peaked at day 14, while the expression of the antiangiogenic factors pigment epithelium-derived factor (PEDF) and thrombospondin 1 (TSP1) peaked at day 28 post-wounding. To examine whether inhibition of angiogenesis influences HS formation in this model, wounds were treated with exogenous soluble antiangiogenic agents including recombinant PEDF (rPEDF) and a functional PEDF peptide (PEDF-335). rPEDF and PEDF-335 were administered intradermally from day 4 post-wounding every 3 days until day 19. Intradermal injection of rPEDF or PEDF-335 both led to decreased angiogenesis and decreased collagen deposition at the wound site. The results support the utility of antiangiogenic therapies, including rPEDF/PEDF-335, as a potential new strategy for the prevention or treatment of HSs.

Fluorescence Probes Exhibit Photoinduced Structural Planarization: Sensing In Vitro and In Vivo Microscopic Dynamics of Viscosity Free from Polarity Interference.

We demonstrate the construction of wavelength λ-ratiometric images that allow visualizing the distribution of microscopic dynamics within living cells and tissues by using the newly developed principle of fluorescence response. The bent-to-planar motion in the excited state of incorporated fluorescence probes leads to elongation of the π-delocalization, resulting in microviscosity-dependent but polarity-insensitive interplay between well-separated blue and red bands in emission spectra. This allows constructing the exceptionally contrasted images of cellular dynamics. Moreover, the application of probes with increased affinity toward biological membranes allowed detecting the differences in dynamics between the plasma membrane and intracellular membrane structures. Such λ-ratiometric microviscosity imaging was extended for mapping the living tissues and observing their inflammation-dependent changes.

Liposome-encapsulated statins reduce hypertrophic scarring through topical application.

Hypertrophic scar is an important clinical problem with limited therapeutic options. Aside from their roles as 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, statins have also been demonstrated to decrease scarring by reducing connective tissue growth factor (CTGF) expression. However, poor penetrative ability limits their utility as topical treatments for hypertrophic scar. Here, we aim to develop novel statin formulations using liposomes to enhance dermal penetrative ability and to evaluate their efficacy against formation of hypertrophic scar utilizing our validated rabbit ear hypertrophic scar model. Liposomal simvastatin or pravastatin were compounded using a novel, flexible liposomal formulation and applied topically to rabbit ear hypertrophic scars daily from postoperation day (POD) 14 until POD 25. Scar color, including erythema and melanin, was measured using reflectance spectrophotometry on POD 28, and scar tissue was harvested for evaluation of scar elevation index as well as gene and protein expression. Human foreskin fibroblasts were also treated with statin formulations and CCN2 expression was determined by quantitative PCR. Both simvastatin and pravastatin were efficiently encapsulated in liposomes, forming nanometer-scale particles possessing highly negative charges. Topical treatment with liposomal simvastatin and pravastatin at 6.5% concentration significantly reduced scar elevation index and decreased type I/III collagen content and myofibroblast persistence in the wound. The erythema/vascularity of scars was reduced by liposomal statin treatment, with concomitant decrease of CD31 expression as measured histologically. Expression levels of transcripts encoding CTGF, collagen I, and collagen III collagen in scar tissue were also decreased by liposomal pravastatin treatment, as were myofibroblast persistence and the type I/III collagen ratio as assessed by immunofluorescence and picrosirus red staining, respectively. Treatment of human foreskin fibroblasts with simvastatin or with liposome-encapsulated pravastatin resulted in decreased expression of transcript encoding CTGF. Overall, our novel statin formulations encapsulated in liposomes were successfully delivered through topical application, significantly reducing hypertrophic scarring in a rabbit ear model.

The role of viscosity on skin penetration from cellulose ether-based hydrogels.

BACKGROUND: The rheological properties of dermal drug delivery systems are of importance when designing new formulations. Viscosity not only affects features such as spreadability and skin feel, but may also affect the skin penetration of incorporated actives. Data on the latter aspect are controversial. Our objective was to elucidate the relation between viscosity and drug delivery performance of different model hydrogels assuming that enhanced microviscosity might delay drug release and penetration. MATERIALS AND METHODS: Hydrogels covering a broad viscosity range were prepared by adding either HPMC or HEC as gelling agents in different concentrations. To investigate the ability of the gels to deliver a model drug into the skin, sulphadiazine sodium was incorporated and its in vitro skin penetration was monitored using tape stripping/HPLC analysis and non-invasive confocal Raman spectroscopy. RESULTS: The trends observed with the two different experimental setups were comparable. Drug penetration depths decreased slightly with increasing viscosity, suggesting slower drug release due to the increasingly dense gel networks. However, the total penetrated drug amounts were independent of the exact formulation viscosity. CONCLUSION: Drug penetration was largely unaffected by hydrogel viscosity. Moderately enhanced viscosity is advisable when designing cellulose ether hydrogels to allow for convenient application.

Quercetin and 3-O-methylquercetin in vitro skin layers permeation/retention from hydrogels: why only a methoxy group difference determines different behaviors?

OBJECTIVES: The present study was designed to verify if quercetin (QCT), a flavonoid with antioxidant and antiviral activity, and 3-O-methylquercetin (3OMQ), a quercetin C3-methoxylated derivative, present differences in their behavior against complexation with ß-cyclodextrin (ß-CD) and the corresponding permeation/retention trhough porcine ear skin, when incorporated into hydroxypropyl methylcellulose (HPMC) or chitosan (CS) hydrogels. METHODS: The influence of ß-CD on the skin permeation/retention of QCT and 3OMQ from hydrogels is comparatively evaluated for both flavonoids using porcine ear skin in Franz cells model. The properties of the two flavonoids using the semi-empirical method Recife Model was studied. KEY FINDINGS: Quercetin presented higher skin retention compared with its C3-methoxy derivative 3OMQ. The best permeation/retention of QCT was observed when it was incorporated into CS hydrogel containing 5% ß-CD, whereas, for 3OMQ, the HPMC hydrogel containing 5% ß-CD was the best formulation. The flavonoids complexation with ß-CD in water occurred preferentially with the insertion of the B ring through the secondary OH rim. CONCLUSIONS: The dynamic molecular modeling revealed that the methyl group at C3 in 3OMQ molecule determined significant difference in its complexation with ß-CD, in comparison to its analogous QCT and that difference is coincident with the permeation behavior of these flavonoids, denoting a possible relationship with their molecular dynamics.

Expression of cytokines and chemokines in mouse skin treated with sulfur mustard.

Sulfur mustard (2,2'-dichlorodiethyl sulfide, SM) is a chemical warfare agent that generates an inflammatory response in the skin and causes severe tissue damage and blistering. In earlier studies, we identified cutaneous damage induced by SM in mouse ear skin including edema, erythema, epidermal hyperplasia and microblistering. The present work was focused on determining if SM-induced injury was associated with alterations in mRNA and protein expression of specific cytokines and chemokines in the ear skin. We found that SM caused an accumulation of macrophages and neutrophils in the tissue within one day which persisted for at least 7 days. This was associated with a 2-15 fold increase in expression of the proinflammatory cytokines interleukin-1ß, interleukin-6, and tumor necrosis factor α at time points up to 7 days post-SM exposure. Marked increases (20-1000 fold) in expression of chemokines associated with recruitment and activation of macrophages were also noted in the tissue including growth-regulated oncogene α (GROα/CXCL1), monocyte chemoattractant protein 1 (MCP-1/CCL2), granulocyte-colony stimulating factor (GCSF/CSF3), macrophage inflammatory protein 1α (MIP1α/CCL3), and IFN-γ-inducible protein 10 (IP10/CXCL10). The pattern of cytokines/chemokine expression was coordinate with expression of macrophage elastase/MMP12 and neutrophil collagenase/MMP8 suggesting that macrophages and neutrophils were, at least in part, a source of cytokines and chemokines. These data support the idea that inflammatory cell-derived mediators contribute to the pathogenesis of SM induced skin damage. Modulating the infiltration of inflammatory cells and reducing the expression of inflammatory mediators in the skin may be an important strategy for mitigating SM-induced cutaneous injury.

Silkworm dropping extract ameliorate trimellitic anhydride-induced allergic contact dermatitis by regulating Th1/Th2 immune response.

Allergic contact dermatitis (ACD) is an inflammatory skin disease caused by hapten-specific immune response. Silkworm droppings are known to exert beneficial effects during the treatment of inflammatory diseases. Here, we studied whether topical treatment and oral administration of silkworm dropping extract (SDE) ameliorate trimellitic anhydride (TMA)-induced ACD. In ACD mice model, SDE treatment significantly suppressed the increase in both ear thickness and serum IgE levels. Furthermore, IL-1ß and TNF-α levels were reduced by SDE. In allergic responses, SDE treatment significantly attenuated the production of the Th2-associated cytokine IL-4 in both ear tissue and draining lymph nodes. However, it increased the production of the Th1-mediated cytokine IL-12. Thus, these results showed that SDE attenuated TMA-induced ACD symptoms through regulation of Th1/Th2 immune response. Taken together, we suggest that SDE treatment might be a potential agent in the prevention or therapy of Th2-mediated inflammatory skin diseases such as ACD and atopic dermatitis. ABBREVIATIONS: ACD: allergic contact dermatitis; AD: atopic dermatitis; APC: antigen presenting cells; CCL: chemokine (C-C motif) ligand; CCR: C-C chemokine receptor; Dex: dexamethasone; ELISA: enzyme-linked immunosorbent assay; IFN: interferon; Ig: immunoglobulin; IL: interleukin; OVA: ovalbumin; PS: prednisolone; SDE: silkworm dropping extract; Th: T helper; TMA: trimellitic anhydride; TNF: tumor necrosis factor.

Development and characterization of clay facial mask containing turmeric extract solid dispersion.

OBJECTIVE: To develop clay facial mask containing turmeric extract solid dispersion (TESD) for enhancing curcumin water solubility and permeability and to determine suitable clay based facial mask. METHODS: The TESD were prepared by solvent and melting solvent method with various TE to polyvinylpyrrolidone (PVP) K30 mass ratios. The physicochemical properties, water solubility, and permeability were examined. The effects of clay types on physical stability of TESD, water adsorption, and curcumin adsorption capacity were evaluated. RESULTS: The TESD prepared by solvent method with a TE to PVP K30 mass ratio of 1:2 showed physically stable, dry powders, when mixed with clay. When TESD was dissolved in water, the obtained TESD micelles showed spherical shape with mean size of ∼100 nm resulting in a substantial enhancement of curcumin water solubility, ∼5 mg/ml. Bentonite (Bent) and mica (M) showed the highest water adsorption capacity. The TESD's color was altered when mixed with Bent, titanium dioxide (TiO2) and zinc oxide (ZnO) indicating curcumin instability. Talcum (Talc) showed the greatest curcumin adsorption followed by M and kaolin (K), respectively. Consequently, in vitro permeation studies of the TESD mixed with Talc showed lowest curcumin permeation, while TESD mixed with M or K showed similar permeation profile as free TESD solutions. The developed TESD-based clay facial mask showed lower curcumin permeation as compared to those formulations with Tween 80. CONCLUSION: The water solubility and permeability of curcumin in clay based facial mask could be improved using solid dispersion technique and suitable clay base composed of K, M, and Talc.

Protective effects of ginsenoside F2 on 12-O-tetradecanoylphorbol-13-acetate-induced skin inflammation in mice.

Topical use of ginsenosides, the major bioactive substances in Panax ginseng, has been used for the treatment of irritated skin complaints. However, the protective mechanisms of ginsenosides remain unclear. In the present study, we investigated the anti-inflammatory role of ginsenoside F2 (GF2) on the skin inflammation. To induce irritant dermatitis, 12-O-tetradecanoylphorbol-13-acetate (TPA) was applied on the surface of the mouse ears with or without treatments of GF2 and dexamethasone for 24 h. Protective effects of GF2 on edema and inflammation were assessed by measuring ear thickness, weights of skin punch, and inflammatory responses. In gross findings, treatments with GF2 significantly decreased skin thickness and weight compared to those of TPA-treated groups, which was comparable with the protective effects of dexamethasone. In addition, expression of inflammatory mediators was remarkably reduced in GF2-treated ears compared to that of vehicle-treated ears of mice. Interestingly, immunohistochemistry and flow cytometry analyses revealed that TPA treatment significantly increased infiltration of interleukin-17 (IL-17) producing dermal γδ T cells, while frequencies of γδ T cells was decreased by GF2 treatment, subsequently ameliorating inflammation in skin. Concomitantly, TPA-mediated skin inflammation was significantly ameliorated in IL-17A knock out mice. Furthermore, GF2 treatment inhibited infiltration and generation of reactive oxygen species (ROS) of neutrophils in damaged ears compared with vehicle-treated mice. These results clearly suggest that GF2 treatment ameliorates TPA-induced dermal inflammation by inhibiting production of IL-17 and ROS in γδ T cells and neutrophils, respectively. Therefore, as a natural compound, application of GF2 may be a novel therapeutic approach for treating skin inflammation.

Comparative evaluation of rivastigmine permeation from a transdermal system in the Franz cell using synthetic membranes and pig ear skin with in vivo-in vitro correlation.

In the present study, in vitro permeation experiments in a Franz diffusion cell were performed using different synthetic polymeric membranes and pig ear skin to evaluate a rivastigmine (RV) transdermal drug delivery system. In vitro-in vivo correlations (IVIVC) were examined to determine the best model membrane. In vitro permeation studies across different synthetic membranes and skin were performed for the Exelon(®) Patch (which contains RV), and the results were compared. Deconvolution of bioavailability data using the Wagner-Nelson method enabled the fraction of RV absorbed to be determined and a point-to-point IVIVC to be established. The synthetic membrane, Strat-M™, showed a RV permeation profile similar to that obtained with pig ear skin (R(2)=0.920). Studies with Strat-M™ resulted in a good and linear IVIVC (R(2)=0.991) when compared with other synthetic membranes that showed R(2) values less than 0.90. The R(2) for pig ear skin was 0.982. Strat-M™ membrane was the only synthetic membrane that adequately simulated skin barrier performance and therefore it can be considered to be a suitable alternative to human or animal skin in evaluating transdermal drug transport, potentially reducing the number of studies requiring human or animal samples.

Comparative analysis of ear-hole closure identifies epimorphic regeneration as a discrete trait in mammals.

Why mammals have poor regenerative ability has remained a long-standing question in biology. In regenerating vertebrates, injury can induce a process known as epimorphic regeneration to replace damaged structures. Using a 4-mm ear punch assay across multiple mammalian species, here we show that several Acomys spp. (spiny mice) and Oryctolagus cuniculus completely regenerate tissue, whereas other rodents including MRL/MpJ 'healer' mice heal similar injuries by scarring. We demonstrate ear-hole closure is independent of ear size, and closure rate can be modelled with a cubic function. Cellular and genetic analyses reveal that injury induces blastema formation in Acomys cahirinus. Despite cell cycle re-entry in Mus musculus and A. cahirinus, efficient cell cycle progression and proliferation only occurs in spiny mice. Together, our data unite blastema-mediated regeneration in spiny mice with regeneration in other vertebrates such as salamanders, newts and zebrafish, where all healthy adults regenerate in response to injury.

Expression Profile of Long Noncoding RNAs in Human Earlobe Keloids: A Microarray Analysis.

Background. Long noncoding RNAs (lncRNAs) play key roles in a wide range of biological processes and their deregulation results in human disease, including keloids. Earlobe keloid is a type of pathological skin scar, and the molecular pathogenesis of this disease remains largely unknown. Methods. In this study, microarray analysis was used to determine the expression profiles of lncRNAs and mRNAs between 3 pairs of earlobe keloid and normal specimens. Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to identify the main functions of the differentially expressed genes and earlobe keloid-related pathways. Results. A total of 2068 lncRNAs and 1511 mRNAs were differentially expressed between earlobe keloid and normal tissues. Among them, 1290 lncRNAs and 1092 mRNAs were upregulated, and 778 lncRNAs and 419 mRNAs were downregulated. Pathway analysis revealed that 24 pathways were correlated to the upregulated transcripts, while 11 pathways were associated with the downregulated transcripts. Conclusion. We characterized the expression profiles of lncRNA and mRNA in earlobe keloids and suggest that lncRNAs may serve as diagnostic biomarkers for the therapy of earlobe keloid.

Exacerbated and prolonged inflammation impairs wound healing and increases scarring.

Altered inflammation in the early stage has long been assumed to affect subsequent steps of the repair process that could influence proper wound healing and remodeling. However, the lack of explicit experimental data makes the connection between dysregulated wound inflammation and poor wound healing elusive. To bridge this gap, we used the established rabbit ear hypertrophic scar model for studying the causal effect of dysregulated inflammation. We induced an exacerbated and prolonged inflammatory state in these wounds with the combination of trauma-related stimulators of pathogen-associated molecular patterns from heat-killed Pseudomonas aeruginosa and damage-associated molecular patterns from a dermal homogenate. In stimulated wounds, a heightened and lengthened inflammation was observed based on quantitative measurements of IL-6 expression, tissue polymorphonuclear leukocytes infiltration, and tissue myeloperoxidase activity. Along with the high level of inflammation, wound healing parameters (epithelial gap and others) at postoperative day 7 and 16 were significantly altered in stimulated wounds compared to unstimulated controls. By postoperative day 35, scar elevation of stimulated wounds was higher than that of control wounds (scar elevation index: 1.90 vs. 1.39, p < 0.01). Moreover, treatment of these inflamed wounds with Indomethacin (at concentrations of 0.01, 0.1, and 0.4%) reduced scar elevation but with adverse effects of delayed wound closure and increased cartilage hypertrophy. In summary, successful establishment of this inflamed wound model provides a platform to understand these detrimental aspects of unchecked inflammation and to further test agents that can modulate local inflammation to improve wound outcomes.

Importance of a suitable working protocol for tape stripping experiments on porcine ear skin: Influence of lipophilic formulations and strip adhesion impairment.

The tape stripping method is a very important tool for dermopharmacokinetic experiments in vitro and the accurate measurement of the removed corneocytes is key for a reliable calculation of a drug's skin penetration behavior. Therefore, various methods to quantify the amount of corneocytes removed with each tape strip have been employed, ranging from gravimetric approaches to protein assays and recently near infrared densitometry (NIR) has become very widely used. As this method is based on a reduction of light intensity, interference of formulation components seems conceivable, as they could scatter light and change the results. In this study, NIR measurements were compared to a protein assay and in addition, the influence of highly lipophilic formulations on the results of tape stripping experiments was investigated as impairment of the adherence of strips has been reported. To this end, different tape stripping protocols were employed. The obtained results ensure suitability of the NIR method and moreover suggest a more pronounced influence on adherence with increasing lipophilicity in applied formulations. The results show that adaptation of the tape stripping protocol to the specifications of envisioned experiments is important for reliable results. Two protocols were found favorable and are presented in this work.

A role for microRNA-155 expression in microenvironment associated to HPV-induced carcinogenesis in K14-HPV16 transgenic mice.

Human Papillomavirus cause a number of diseases most notably cervical cancer. K14-HPV16 transgenic mice expressing the HPV16 early genes in squamous epithelial cells provide a suitable experimental model for studying these diseases. MicroRNAs are small non-coding RNAs that play an important role in regulating gene expression and have been suggested to play an important role in cancer development. The role of miR-155 in cancer remains controversial and there is limited evidence linking this miRNA to HPV- associated diseases. We hypothesized that miR-155 expression modulates each tissue's susceptibility to develop HPV-associated carcinogenesis. In this study, we analyzed miR-155 expression in ear and chest skin samples from 22-26 weeks old, female K14-HPV16 transgenic (HPV16+/-) and wild-type (HPV-/-) mice. Among wild-type mice the expression of miR-155 was lower in ear skin compared with chest skin (p = 0.028). In transgenic animals, in situ carcinoma was present in all ear samples whereas chest tissues only showed epidermal hyperplasia. Furthermore, in hyperplastic chest skin samples, miR-155 expression was lower than in normal chest skin (p = 0,026). These results suggest that miR-155 expression may modulate the microenvironmental susceptibility to cancer development and that high miR155 levels may be protective against the carcinogenesis induced by HPV16.

External ear microRNA expression profiles during mouse development.

MicroRNAs (miRNAs) comprise a class of approximately 22 nucleotide regulatory non-coding RNAs that play several roles in diverse biological processes. Recent reports suggest that embryonic development in mammals is accompanied by dynamic changes in miRNA expression; however, there is no information regarding the role of miRNAs in the development of the external ear. The aim of this study was to determine the stage-specific expression of miRNAs during mouse external ear development in order to identify potentially implicated miRNAs along with their possible targets. miRNA expression profiles from fetal mice pinnae and back skin tissues at 13.5 dpc and 14.5 dpc were obtained using an Affymetrix GeneChip miRNA 3.0 array. Biological triplicates for both tissues, each collected from a litter averaging 16 fetuses, were analyzed. The results were analyzed with Affymetrix's Transcriptome Analysis Console software to identify differentially expressed miRNAs. We observed differential expression of 40 miRNAs including some predicted to target genes implicated in external ear development, such as mmu-miR-10a, an miRNA known to modulate Hoxa1 mRNA levels, and mmu-miR-200c and mmu-miR-205. To our knowledge, this is the first miRNA expression profiling study of external ear development in mammals. These data could set the basis to understand the implications of miRNAs in normal external ear development.

CXCL1 gene silencing in skin using liposome-encapsulated siRNA delivered by microprojection array.

The barrier morphology of skin provides major obstacles for the application of siRNA for gene silencing, which current delivery technologies do not effectively overcome. Emerging technologies utilise microprojection array devices to penetrate into the skin epidermis and dermis for delivery of drug payloads. Delivery of siRNA by such devices has been proven in principle, yet requires optimisation for clinical applications. Herein, we demonstrate the use of Nanopatch™ microprojection arrays to deliver liposome-encapsulated siRNA to overcome skin barrier, and in vivo siRNA delivery hurdles. This application provided effective silencing of CXCL1 expression induced by the co-delivery of Fluvax 2012® by microprojection array. Liposomes encapsulating siRNA were dry-coated onto microprojection arrays, and remained intact after elution from arrays in vitro. Microprojection arrays facilitated the delivery of fluorescently-labelled nucleic acids through murine ear stratum corneum to the epidermis and dermis, with diffusion from microprojections into adjacent skin evident within 30s. CXCL1 mRNA, induced by delivery of Fluvax by microprojection array, was reduced by 75% up to 20 h post-treatment by co-delivery of liposome-encapsulated CXCL1-specific siRNA, but not by arrays co-delivering liposome-encapsulated control siRNA. CXCL1 protein expression in explant cultures from skin treated with arrays bearing CXCL1 specific or control siRNA was similarly reduced. These results as a test case have many implications for gene silencing in skin and inflammation, with the benefit of targeted delivery using microprojection arrays to deliver liposome-encapsulated siRNA.