![]() Unusual Rifle Steyr AUG leather weapons for the game cs 1.6.The authors of this model of weapons did a good job. Appearance, texture, animation and sound - everything is done with dignity. I advise you to download and evaluate it. Abstract The main structural component of connective tissues is fibrillar, cross-linked collagen whose fibrillogenesis can be modulated by Small Leucine-Rich Proteins/Proteoglycans (SLRPs). Not all SLRPs’ effects on collagen and extracellular matrix in vivo have been elucidated; one of the less investigated SLRPs is asporin. Here we describe the successful generation of an Aspn -/- mouse model and the investigation of the Aspn -/- skin phenotype. Functionally, Aspn -/- mice had an increased skin mechanical toughness, although there were no structural changes present on histology or immunohistochemistry. Electron microscopy analyses showed 7% thinner collagen fibrils in Aspn -/- mice (not statistically significant). Several matrix genes were upregulated, including collagens ( Col1a1, Col1a2, Col3a1), matrix metalloproteinases ( Mmp2, Mmp3) and lysyl oxidases ( Lox, Loxl2), while lysyl hydroxylase ( Plod2) was downregulated. Intriguingly no differences were observed in collagen protein content or in collagen cross-linking-related lysine oxidation or hydroxylation. The glycosaminoglycan content and structure in Aspn -/- skin was profoundly altered: chondroitin/dermatan sulfate was more than doubled and had an altered composition, while heparan sulfate was halved and had a decreased sulfation. Also, decorin and biglycan were doubled in Aspn -/- skin. Overall, asporin deficiency changes skin glycosaminoglycan composition, and decorin and biglycan content, which may explain the changes in skin mechanical properties. Introduction The bulk of the extracellular matrix (ECM) is composed of collagens woven into a fibrillar network that accommodates water-retaining proteoglycans and provides a cellular niche. Collagen fibrillogenesis and cross-linking can be modulated by Small Leucine-Rich Proteins/Proteoglycans (SLRPs); this constitutes a mechanism that contributes to the mechanical and physiological tissue properties [–]. Cs Skins Download![]() The effects of SLRPs on collagen fibrillogenesis are evident in the organ-specific phenotypes of the various SLRP knockout mice: lumican-deficient mice have fragile skin and opaque cornea [–], fibromodulin-deficient mice have abnormally cross-linked and ill-fused collagen fibrils in tendons [, ], decorin-deficient mice have fragile skin, weak tendons and lower lung airway resistance [–], keratocan-deficient mice have flattened cornea (mimicking human cornea plana disorder caused by KERA mutations) [, ], and biglycan-deficient mice have osteoporotic bones []. Compound SLRP knockout mice have even graver multi-organ abnormities with more pronounced collagen fibril phenotypes [, ]. Not all SLRP knockout mouse phenotypes have been reported, but the collective knowledge of SLRPs’ tissue-specific effects and collagen interaction redundancy [–] will aid in understanding the complex mechanisms underlying the shaping of extracellular matrices. In this paper, we present an initial report on the phenotype of asporin-deficient mice. ![]() Compared with other SLRPs, asporin (ASPN or PLAP1) lacks glycosaminoglycan (GAG) chains [] and carries a polymorphic calcium-binding polyaspartate sequence [, ]. Asporin expression has been detected in dermis, perichondrium and periosteum, tendon, and eye sclera [, ]. Concluded from several in vitro studies, asporin function has been related to collagen fibrillogenesis and collagen mineralization [, –]. Asporin has also been suggested to modulate cellular response to FGF-2 [], BMP-2 [], and TGF-β []. Asporin expression is induced by TGF-β [, ] and suppressed by IL-1β and TNF-α []. It can interact and inhibit TGF-β [] and consequently act as a tumor suppressor [], but other studies propose asporin to be an invasion-promoting protein [–]. In this paper we hypothesized that asporin functions in formation of a structurally coherent extracellular matrix in vivo. To test this hypothesis we generated an asporin knockout mouse model ( Aspn -/-). We focused on analyzing the effect of asporin deficiency on skin extracellular matrix, where asporin has been detected [, ] and where we, in the current study, observed a biomechanical phenotype. To elucidate the underlying cause of this phenotype we also analyzed skin collagen, proteoglycans and GAGs. This report contributes to the overall understanding of the unique functions of SLRPs in connective tissues. Aspn -/- mouse generation. (A) Strategy for the creation of Aspn-null allele. The diagram shows wild-type, targeted, floxed, and excised alleles. Exons 1–4 (out of 8 present in the gene) are shown as rectangles. Primers used for genotyping are targeted to P1, P2, P3 sites and the expected PCR products’ sizes are denoted. (B) Genotyping PCR of Aspn -/- mice using primers targeted to sites P1, P2, P3. (C) Confirmation of Aspn protein expression loss in Aspn -/- mice by immunobloting tail SDS extracts for asporin. Skin histology and immunohistochemistry Mouse skins were fixed in HistoChoice (Sigma) for 24 h, dehydrated, embedded in paraffin and sectioned. Sections were stained with Masson Trichrome (Sigma) or processed for immunohistochemistry: rehydrated sections were washed 2 x 5 min in Tris-buffered saline (TBS) plus 0. Trapcode particular plugin after effects. 025% Triton X-100 and blocked with 10% goat serum and 1% bovine serum albumin (BSA) for 2 h. Sections were then incubated for 16 h at 4°C with rat anti-mouse CD31 (Optistain, clone SZ31) or rat anti-mouse F4/80 (BioRad, clone CI:A3-1) diluted to 10 μg/mL in 1% BSA in TBS. Sections were rinsed 2 x 5 min with TBS plus 0.025% Triton X-100 and quenched with 0.3% hydrogen peroxide in TBS for 15 min. After rinsing with TBS, sections were stained with ultrasensitive ABC staining kit (ThermoFisher). Sections were washed 3 x 5 min with TBS and developed using diaminobenzidine peroxidase substrate (Vector Labs). Skin tensile strength determination Mice were euthanized and kept on ice before shaving and gently removing the dorsal skin samples. Two parallel strips of skin (3 mm x 30 mm) were punched out in the craniocaudal direction, symmetrically on the left and right side of the spine. The samples were washed briefly in PBS, the central 5 mm were wrapped in PBS soaked gauze and the ends were allowed to dry for 90 min while keeping the central part moist with PBS. The dry ends were glued onto clamps using cyanoacrylate glue and after curing for 40 min the sample was placed in a PBS bath and mechanically tested (200-N tensile stage, petri dish version, Deben, Suffolk, UK). The test consisted of 4 preconditioning cycles to 15% strain, followed finally by a stretch to failure. All tests were at a rate of 6 mm/min with data recorded at 10 Hz. Sample width and length were measured by microscopy images. To avoid mechanically damaging the strips, thickness was measured with a constant-pressure caliper (model 293-334-30, Mitutoyo, Kawasaki, Japan) on a piece of skin adjacent to the tested strip.
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