Eigenvalues of Dirichlet Laplacian within the class of open sets with constant diameter

This paper is about a shape optimization problem related to the Dirichlet Laplacian eingevalues in the Euclidean plane. More precisely we study the shape of the minimizer in the class of open sets of constant width. We prove that the disk is not a local minimizer except for a limited number of eigenvalues

Canonical TGFβ signaling in SSc skin.

<p>(A) Representative images of double immunofluorescent staining performed to detect phospho-Smad2/3 (red) and K14 (green) in the epidermis of SSc patients and controls forearm skin sections (both n = 6, means of 5 high power views for each individual patient or control). DAPI (blue) was used to stain nuclei. Nuclear translocation of phospho-Smad2/3 was seen in SSc epidermal cells extending to suprabasal and granular layers. K14 expression was found to extend into suprabasal layers abnormally in SSc consistent with altered differentiation. (B) Also phospho-Smad2/3 nuclear translocation was seen in cells within the papillary dermis, increased in SSc sections (blue DAPI, red pSMAD2/3, double positive cells indicated with arrows). (C) When quantified the mean number of phospho-Smad2/3 positive cells was increased in SSc both in the epidermis (p<0.001) and in the adjacent papillary dermis (p<0.05) consistent with active TGFβ signaling. (D) qPCR of whole epidermal sheets obtained during suction blister formation revealed increased expression of SNAI1 transcription factor downstream of TGFβ. However SNAI2 was not increased. (ED = epidermis, PD = papillary dermis, RD = reticular dermis, ** = P<0.001, * = P<0.05).</p

TGFβ stimulated EMT in HaCat cells.

<p>(A) HaCat cells cultured for 72hrs with TGFβ1 4ng/ml became elongated, lost cytokeratin and induced FSP-1 expression consistent with transition to a mesenchymal phenotype. (B) Culture with TGFβ1 also led to induction of both <i>SNAI1</i> and <i>SNAI2</i> mRNA maximal with TGFβ1 2 ng/ml and consistent with fully evoked EMT.</p

Epithelial and mesenchymal markers in SSc skin.

<p>(A) Representative images of double immunofluorescent staining of forearm skin sections performed to detect basement membrane protein collagen IV (red) and mesenchymal cell marker FSP-1 (green). The basement membrane collagen IV was not seen to be compromised in SSc, whereas some abnormal expression of FSP-1 in keratinocytes was seen in the SSc sections. (B) Additional stains for E-cadherin (green) and vimentin (red) were performed showing no loss of E-cadherin in SSc epidermal cells. Some expression of vimentin was however observed in the SSc epidermis. (C)Further analysis of FSP-1 positive cells was confirmed, since Langerhans cells within the epidermis are known to stain positive for mesenchymal markers. Immunostaining for FSP-1 (green) and Langerin (red) revealed that at least some of the FSP-1 positive cells were indeed from the Langerhans cell population (double positive cells shown with arrow). (BM = basement membrane, BV = blood vessels).</p

Identification of autoantigens and their potential post-translational modification in EGPA and severe eosinophilic asthma

BACKGROUND: The chronic airway inflammation in severe eosinophilic asthma (SEA) suggests potential autoimmune aetiology with unidentified autoantibodies analogous to myeloperoxidase (MPO) in ANCA-positive EGPA (eosinophilic granulomatosis with polyangiitis). Previous research has shown that oxidative post-translational modification (oxPTM) of proteins is an important mechanism by which autoantibody responses may escape immune tolerance. Autoantibodies to oxPTM autoantigens in SEA have not previously been studied. METHODS: Patients with EGPA and SEA were recruited as well as healthy control participants. Autoantigen agnostic approach: Participant serum was incubated with slides of unstimulated and PMA-stimulated neutrophils and eosinophils, and autoantibodies to granulocytes were identified by immunofluorescence with anti-human IgG FITC antibody. Target autoantigen approach: Candidate proteins were identified from previous literature and FANTOM5 gene set analysis for eosinophil expressed proteins. Serum IgG autoantibodies to these proteins, in native and oxPTM form, were detected by indirect ELISA. RESULTS: Immunofluorescence studies showed that serum from patients with known ANCA stained for IgG against neutrophils as expected. In addition, serum from 9 of 17 tested SEA patients stained for IgG to PMA-stimulated neutrophils undergoing NETosis. Immunofluorescent staining of eosinophil slides was evident with serum from all participants (healthy and with eosinophilic disease) with diffuse cytoplasmic staining except for one SEA individual in whom subtle nuclear staining was evident. FANTOM5 gene set analysis identified TREM1 (triggering receptor expressed on myeloid cells 1) and IL-1 receptor 2 (IL1R2) as eosinophil-specific targets to test for autoantibody responses in addition to MPO, eosinophil peroxidase (EPX), and Collagen-V identified from previous literature. Indirect ELISAs found high concentrations of serum autoantibodies to Collagen-V, MPO, and TREM1 in a higher proportion of SEA patients than healthy controls. High concentrations of serum autoantibodies to EPX were evident in serum from both healthy and SEA participants. The proportion of patients with positive autoantibody ELISAs was not increased when examining oxPTM compared to native proteins. DISCUSSION: Although none of the target proteins studied showed high sensitivity for SEA, the high proportion of patients positive for at least one serum autoantibody shows the potential of more research on autoantibody serology to improve diagnostic testing for severe asthma. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, identifier, NCT04671446.</p

Fibril diameters in the KO reticular dermis are smaller and more uniform than WT fibrils.

<p><b>A.</b> Representative micrographs of skin in deep dermal areas near subcutaneous fat. (Scale bar = 0.5 μm) <b>B.</b> Histogram of the frequency of collagen fibrils with a given diameter range from WT (white bars) and KO (black bars). At least 200 fibrils from 3 WT and 3 KO animals at 5 months of age were used for analysis. P<0.05.</p

Stiff surfaces and TGFβ induce MRTF-A nuclear accumulation.

<p>SSc and control dermal fibroblast lines (both N = 3) were cultured on 6 well plates with collagen type I coated soft substrates (5 kPa Softwell), or hard substrate (50 kPa). <b>B. Induction of collagen transcription on hard surfaces requires MRTF-A.</b> Mouse fibroblasts from wild type (WT) and loss-of-function (KO) mice with collagen promoter driving GFP were cultured on fibronectin coated soft and hard surfaces. Pictures taken 24 hours after plating. Fluorescence was quantified using ImageJ. The numbers of cells in each image was counted (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126015#pone.0126015.s004" target="_blank">S4 Fig</a>) to determine the luminance per cell. <b>C.&D.</b> Normal control fibroblasts and SSc fibroblasts were cultured without serum for 16 hours then treated with TGFβ (4 ng/ml) for 0, 8 and 24 hours or treated with either saline or TGFβ (4 ng/ml) with or without CCG1423 (10μM) or NSC2376 (50 μM). Proteins (20 μg) from cytoplasm and nuclei were extracted using NE-PER Nuclear Protein Extraction Kit and separated on 4–12% gradient gel and visualized using MRTF-A antibody.</p

Mouse KO skin and lung are less stiff than WT skin and lung.

<p>A 3x1x1 mm strip of dorsal skin or lung tissue was placed longitudinally into a computer-controlled dual-mode lever arm force transducer system and stretched intermittently. <b>A.</b> Young’s module plotted at each strain. The Young’s module is the slope of the stress-strain curve. <b>B.</b> Dynamic stretch storage modulus describes the ability of the material to store elastic energy during the loading phase of a cyclic stretch <b>C</b>. Dynamic stretch loss modulus—The amount of energy lost (usually as heat) during a cycle. Vertical brackets denote the overall group differences using 2-way repeated measure ANOVA (*: <i>p</i><0.05, **: <i>p</i><0.01 and ***: <i>p</i><0.001) whereas horizontal brackets show Tukey’s post hoc differences between WT and KO at the same strain (Panel A) or frequency (Panels B and C). For the Young’s and loss moduli of lung tissue, there are also significant interactions between strain and group (panel A) as well as frequency and group (panel C, p<0.05). For the skin, there is a significant interaction between frequency and group only for the loss modulus (p<0.05).</p

Nuclear MRTF-A expression is more prominent in SSc skin then healthy control.

<p><b>A.</b> Representative pictures of healthy control and SSc sections of biopsy. Histological samples of human skin were stained with MRTF-A antibody (1:2000). Higher magnification of epithelium and small vessels (20X) in papillary dermis. Brown arrows = MRTF-A nuclei, Blue arrow = hematoxylin stained nuclei without MRTF-A. Graphical representations of % nuclei in epidermis, vasculature, and interstitial cells in papillary dermis. Histological samples of 5 healthy control and 9 scleroderma human skin were evaluated for nuclear staining. B. Total cells with MRTF-A nuclear localization in the epidermis, vasculature, and interstitial papillary dermal layers were counted and compared with the total amount of nuclei in the epidermal/papillary dermal layer. White bars = healthy controls, Black bars = SSc (* = p<0.01 using nonparametric Mann-Whitney U, two-tailed).</p

Increased expression of MRTF-A in the SSc epidermis and at the epidermal dermal junction in established SSc correlates with increased intracellular procollagen, and SMA.

<p>The expression of MRTF-A, procollagen type I, and SMA was detected by immunohistochemical staining in the epidermis and papillary dermis of early and established SSc patients and controls healthy control patients. Arrows point to staining in vascular cells (V), epidermis (e), and fibroblast (F).</p