No abstract

Highly sensitive NATs require new confirmatory algorithms as presented optimally using different genomic regions or sequence generation. The introduction of immigration-related HBV genotypes may impact HBV epidemiology in the United States.

ABCA12 is known to be critical for skin barrier integrity. Mutations in this gene cause the most severe form of Autosomal Recessive Congenital Ichthyosis, Harlequin Ichthyosis (HI). HI patients have marked hyperkeratosis at birth with fissuring, leading to life-threatening complications due to increased risk of infection, trans-epidermal water and heat loss. The aim of this study was to identify essential pathways involved in the pathomechanisms of Harlequin Ichthyosis, responsible for aberrant epidermal differentiation. We performed RNA-seq on calcium induced primary keratinocytes with siRNA knockdown of ABCA12 and identified 118 genes significantly down-regulated and 36 genes significantly up-regulated (FDR < 0.05). Functional annotation clustering analysis showed changes in epidermal differentiation, fatty acid metabolism, cytokine and interferon signaling. The suppressor of cytokine signaling 3 (SOCS3), a negative feedback regulator of the JAK-STAT signaling pathway, was 2.5 fold downregulated whereas Interleukin-1 (IL1A and IL1B) were 2 fold increased. To investigate these findings further we engineered an ABCA12 CRISPR-Cas9 knockout keratinocyte cell line and compared this with a HI patient-derived cell line and wild type controls. Alterations in differentiation and lipid profile in the HI OT models were observed, recapitulating the HI epidermis phenotype. We found that phospho-STAT1 (Y701) was strongly upregulated in the HI model compared to control. In HI patient skin, the STAT1 expression pattern was altered compared to control skin. The secretion of IL-1a was increased in the HI model compared to control. Both STAT1 and IL-1 regulate the Nitric Oxide (NO) pathway upregulating transcription of inducible NO synthase (iNOS), which we found to be significantly upregulated in HI skin. These data provide insights into the pathogenesis of HI suggest that the NO signaling pathway may be a possible therapeutic target in this disorder.

The autosomal recessive congenital ichthyoses (ARCI) are a group of chronic conditions where there is unmet therapeutic need. Harlequin ichthyosis (HI) is the most severe form of ARCI, with aberrant differentiation of the epidermis leading to a severe barrier defect. It is caused by mutations in ABCA12, a lipid transporter involved in the transport of glucosylceramides from the lamellar body to the lipid lamellae. Lamellar ichthyosis is caused by mutations in TGM1. Several Janus kinase (JAK) inhibitors (JAKi) inhibiting the JAK–STAT pathway are now licensed for atopic dermatitis. Previous work from our group in an in vitro HI model showed that tofacitinib had a restorative effect on the lipid barrier. We hypothesized that JAKi A, B and C could restore the epidermal barrier in both HI three-dimensional (3D) organotypic models (OTs) made using ABCA12 CrispR-Cas9 knockout and wild-type keratinocytes and ARCI 3D OTs generated with short hairpin RNA knock down of TGM1. The JAKi were tested at three concentrations in the 3D models (20, 50 and 500 nmol L–1 for JAKiA; 10, 50 and 250 nmol L–1 for JAKiB; 2, 25 and 125 nmol L–1 for JAKiC) and vehicle only was used as the control. The OTs were stained with haematoxylin and eosin, Nile red and differentiation markers such as K10, K14, transglutaminase 1 (TGM1) and involucrin. All three JAKi had a positive effect on the epidermal barrier. The epidermal structure looked more organized and uniform, with an increase in neutral and polar lipids in the upper epidermis. The differentiation markers showed normalization of the expression of involucrin, K10 and TGM1 at the same drug concentrations. These results suggest that JAKi have a generalized positive effect on the skin barrier and will be further investigated in an in vivo mouse model of HI.

MicroRNAs are considered to play critical roles in the pathogenesis of psoriasis. The purpose of this study was to explore the molecular mechanism of miR-20a-3p in the pathogenesis of IL-22-induced psoriasis. We found that miR-20a-3p was down-regulated in HaCaT cells (human keratinocytes) treated by IL-22 stimulation and in psoriatic lesions. MiR-20a-3p mimics and inhibitors were transfected into HaCaT cells. CCK8 assay and 5-Ethynyl-2'deoxyuridine(EDU) incorporation assay were used for cell proliferation. Annexin fluorescein isothiocyanate/propidium iodide assay was performed to detect cell apoptosis. Results showed that overexpression of miR-20a-3p in HaCaT cells suppressed proliferation and induced apoptosis. Conversely, inhibition of endogenous miR-20a-3p promoted cell proliferation and reduces cell apoptosis. SFMBT1 was identified as one of the direct targets of miR-20a-3p by dual luciferase reporter assay. Knocked down of SFMBT1 by specific RNAi inhibited cell growth and induced apoptosis, which was consistent with the function of miR-20a-3p upregulation in HaCaT cells. In addition, results of western blot analysis showed that miR-20a-3p upregulation or SFMBT1 knockdown promoted the protein expression levels of TGF-b1 and P-Smad2/3. Our findings suggest that miR-20a-3p play important roles through targeting SFMBT1 and TGF-b/smad signalling pathway, and loss of miR-20a-3p in psoriasis may contribute to hyperproliferation and aberrant apoptosis of keratinocytes. Atopic Dermatitis (AD) is a complex disease involving causative effects from both intrinsic and extrinsic sources. Murine models of the disease often fall short in one of these components and as a result, do not fully encapsulate the disease mechanisms. We aimed to determine whether the protease-activated 2 receptor over-expressor mouse with topical house-dust mite application (PAR2 + HDM) is a comprehensive representation of clinical AD in a murine model. Comparison of control groups with the PAR2 + HDM mice was performed to assess AD clinical scoring, skin morphology, barrier function and inflammation. Skin morphology was visualised using H&E staining and barrier function was assessed by transepidermal water loss (TEWL) measurements. Inflammation was evaluated by histological staining for immune cells. Differentially expressed genes (DEGs) were compared in the PAR2 + HDM model with other murine AD models and human AD transcriptome. Our PAR2 + HDM model accurately displays the characteristic clinical symptoms, skin morphology and inflammation seen in AD patients. TEWL is significantly increased in the area of HDM application (p<0.01). There is a significant influx of mast cells (p<0.01) and eosinophils (p<0.0001) into the dermis. The PAR2 + HDM model also shows similar expression pattern of key DEGs (e.g. IL-4, FLG, IVL) as both human AD and other murine models. We found that the PAR2 + HDM model presents with a classic AD pathophysiology and is valuable in terms of reproducibility and overall disease representation. 658In vivo confocal Raman spectro...