Journal: JACC: Basic to Translational Science

Article Title: Bioprosthetic Valve Deterioration

doi: 10.1016/j.jacbts.2023.01.003

Figure Lengend Snippet: Sparse Collagen Fibers and Fewer Mesenchymal Cells in Bioprosthetic Valves (A) Masson’s trichrome staining and immunohistochemical staining for vimentin and anti-α-smooth muscle actin (SMA) of explanted bioprosthetic valves (eBVs) and normal native and aortic valve stenosis (AS) valves. The left panels are whole views of the valve leaflets. Scale bars, 100 μm. The right panels show colocalizations of vimentin and αSMA in the valves. (B) Quantitative data of the ratios (%) of red/blue areas of Masson’s trichrome staining, vimentin-positive cells, and α-SMA-positive cells in the valves. Statistical analyses were performed using the Kruskal-Wallis test with Dunn’s multiple-comparisons test (all comparisons). ∗∗ P < 0.01 and ∗∗∗ P < 0.001.

Article Snippet: Anti–α-smooth muscle actin (SMA) antibody was obtained from Novus Biologicals.

Techniques: Staining, Immunohistochemical staining

Journal: JACC: Basic to Translational Science

Article Title: Bioprosthetic Valve Deterioration

doi: 10.1016/j.jacbts.2023.01.003

Figure Lengend Snippet: Protein Profiling in Tissue Lysates From the Explanted Native Valves (AS and Normal) and eBVs (A) Two-dimensional gel electrophoresis of tissue lysates from native and bioprosthetic valves. The left panels are the enlarged figures of the areas surrounded by black broken lines in the right panels . (B) Western blot analyses for fibrinogen, plasminogen (PLG), apolipoprotein E (APOE), and anti–serum amyloid P component (APCS) in tissue lysates from the explanted native and bioprosthetic valves. (C) Statistical quantifications of the western blot results are presented. Analysis of variance, followed by Tukey’s multiple-comparisons test (all comparisons), was used for statistical analyses of the fibrinogen, APCS, and APOE levels. The Kruskal-Wallis test (all comparisons) was used for statistical analyses of PLG levels. ∗ P < 0.05 and ∗∗∗ P < 0.001. FGB = fibrinogen β-chain; other abbreviations as in Figure 1 .

Article Snippet: Anti–α-smooth muscle actin (SMA) antibody was obtained from Novus Biologicals.

Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Western Blot

Journal: JACC: Basic to Translational Science

Article Title: Bioprosthetic Valve Deterioration

doi: 10.1016/j.jacbts.2023.01.003

Figure Lengend Snippet: Tissue Localization of Fibrinogen, PLG, Anti-APOE, and Anti-APCS (A) Whole–valve leaflet views of the immunohistochemical images of the preimplanted and explanted bioprosthetic valves (BVs) and normal valve (left panels) . Porcine-derived eBV6, which strongly detected fibrinogen and PLG by western blotting, was stained using antifibrinogen or PLG antibodies. Bovine-derived eBV5, which was strongly detected by APOE and APCS using western blotting, was stained using anti-APOE and APCS antibodies. Preimplanted porcine- and bovine-derived BVs were used according to immunohistochemical staining against each species. The right panels show high magnification of the areas surrounded by lines in the whole view of the left panels . Scale bars, 200 μm. Arrowheads indicate strongly stained areas. (B) Immunofluorescence costaining of fibrinogen/CD68 or PLG/CD68 in the BV. Scale bars, 20 μm. Abbreviations as in Figures 1 , , and .

Article Snippet: Anti–α-smooth muscle actin (SMA) antibody was obtained from Novus Biologicals.

Techniques: Immunohistochemical staining, Derivative Assay, Western Blot, Staining, Immunofluorescence

Journal: JACC: Basic to Translational Science

Article Title: Bioprosthetic Valve Deterioration

doi: 10.1016/j.jacbts.2023.01.003

Figure Lengend Snippet: Histologic Evaluations of Explanted IBTA Valves (A) HE- and Masson’s trichrome–stained whole-leaflet views of the preimplanted Biosheet and explanted in-body tissue architecture (IBTA) valves (implantation term 3 and 12 months) (top panel) . Scale bars, 500 μm. Macroscopic views of the explanted valves (bottom panel) . (B) Masson’s trichrome, HE, and IHC for vimentin and FGB of the areas surrounded by lines in the whole view of right panels in A . Scale bars, 200 μm. (C) Surface structures of Masson’s trichrome–stained IBTA and pericardium-derived bioprosthetic valves for 12 months of implantation. Arrowheads indicate digested collagen fibers. (D) Quantifications of the infiltration foci per leaflet (top panel) and stained areas of immunohistochemistry against FGB (bottom panel) . Ratios were calculated by dividing brown-stained areas by the entire area of the leaflets. Statistical analysis was conducted using analysis of variance, followed by Sidak’s multiple-comparisons test. (E) Whole-leaflet images of the explanted normal, IBTA (3 and 12 months), and pericardium-derived valves (3 and 12 months). All leaflets were immune stained with anti-FGB antibodies. Scale bar, 1 mm. (F) Schematic diagram of molecular mechanism regarding circulating proteins’ and cells’ infiltration. ∗ P < 0.05 and ∗∗∗ P < 0.001. PGB = plasminogen; other abbreviations as in Figures 3 and .

Article Snippet: Anti–α-smooth muscle actin (SMA) antibody was obtained from Novus Biologicals.

Techniques: Staining, Derivative Assay, Immunohistochemistry

Journal: Virology

Article Title: Proteomic analysis reveals the actin cytoskeleton as cellular target for the human papillomavirus type 8.

doi: 10.1016/j.virol.2009.01.036

Figure Lengend Snippet: Fig. 1. (A) 2D gel electrophoresis pattern of proteins from adult keratinocytes retrovirally transduced for HPV8-E7, HPV8-CER or empty vector. For the first dimension IEF, 24 cm pH 3–10 (linear) Immobiline DryStrip (IPG) strips (GE Healthcare) were cuploaded with 150 μg of CyDye labeled proteins (50 μg of each fluor Cy2, Cy3 and Cy5 — for sample combinations see Table 1). Isoelectric focusing was performed on an Ettan IPGphor IEF System using the Ettan IPGphor Cup Loading Manifold (both GE Healthcare). Equilibrated IPG strips were transferred onto 1 mm thick 25×20 cm 12% polyacrylamide gels without a stacking gel, cast between low-fluorescence glass plates. Gels were electrophoresed in an Ettan Dalt six gel tank (GE Healthcare) at 20 °C, at 1.5 W/gel constant power overnight. Following SDS-PAGE, fluorescently labeled proteins were visualized while still between the low-fluorescence glass plates using a Typhoon 9400 Variable Mode Imager and Typhoon Scanner Control Software, Version 3.0 (GE Healthcare). For the Cy2 images a 488 nm laser and an emission filter of 520 nm with a band pass (BP) of 40 nm was used. Cy3 images were obtained using a 532 nm laser and an emission filter of 580 nm/BP30. Cy5 images were obtained using a 633 nm laser and an emission filter of 670 nm/BP30. (B) Protein association network of the proteins differentially expressed due to HPV8 expression based on STRING Network Display. Protein–protein interaction prediction from the identified proteins. Stronger associations are represented by thicker lines. ACTA1: Actin, alpha skeletal muscle; KRT14: Keratin-14; VCL: Vinculin; KRT5: Keratin-5; IMMT: Mitochondrial inner membrane protein (Mitofilin); CNN2: Calponin-2; ENSP00000264785: WD repeat domain 1 (WDR1); MSN: Moesin; GSN: Gelsolin; FLNB: Filamin B; HSPA4: Heat shock 70; TBCA: Tubulin-specific chaperone A TCP1; PDCD6IP: Programmed cell death 6-interacting protein; LMNA: Lamin A/C; RDX: Radixin; TPM1: Tropomyosin-1; TPM3: Tropomyosin-3; TPM4: Tropomyosin-4.

Article Snippet: The fact, that less alpha-actinin is detected in extracts of E7 cells by the anti alpha-actinin antibody (clone H2, Santa Cruz, Heidelberg, Germany) (Fig. 3) further supports the observation that actin fibers are diffusely organized in HPV8 positive cells.

Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Plasmid Preparation, Labeling, SDS Page, Control, Software, Expressing, Membrane

Journal: Virology

Article Title: Proteomic analysis reveals the actin cytoskeleton as cellular target for the human papillomavirus type 8.

doi: 10.1016/j.virol.2009.01.036

Figure Lengend Snippet: Fig. 3. Western blots showing the protein expressions of alpha-actinin, alpha-actin, Tropomyosin-1, Keratin-5 and GAPDH in total cell extracts of adult keratinocytes retrovirally transduced for HPV8-E7 or empty vector pLXSN.

Article Snippet: The fact, that less alpha-actinin is detected in extracts of E7 cells by the anti alpha-actinin antibody (clone H2, Santa Cruz, Heidelberg, Germany) (Fig. 3) further supports the observation that actin fibers are diffusely organized in HPV8 positive cells.

Techniques: Western Blot, Plasmid Preparation

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Modeling human endometrial decidualization from the interaction between proteome and secretome.

doi: 10.1210/jc.2010-1825

Figure Lengend Snippet: FIG. 2. 2D-DIGE of decidualized (D) vs. control (C) ESCs and validation. Individual proteins of control cells, decidual cells, and a pooled internal standard were labeled with CyDyes, Cy3, Cy5, and Cy2, respectively, and were mixed and separated on a 2D gel using 24 cm pH 3–11 NL (left to right) strips in the first dimension and 10% PAGE-SDS gels in the second dimension. Gels were scanned to obtain images of the overlay of the two dyes (Cy3, Cy5) (A.1, A.2). The same gels, after fluorescence imaging, were silver-stained and scanned (A.3, A.4). The 53 protein spots found to be significantly deregulated in the decidualizated vs. the control samples (P 0.05) are encircled. The proteins identified by in-gel trypsin digestion and MALDI-TOF/TOF are summarized in Table 1. Validation of the DIGE experiments of -actinin, transgelin, cathepsinB, and transglutaminase by Western blot. B, Protein extracts from endometrial samples used in the DIGE experiments; bands correspond to 107-kDa actinin and 23-kDa transgelin. Both significantly reduced in the decidualized vs. the control cells (densitometry values of 4.5 and 6.1, respectively); 38-kDa cathepsin B and 90-kDa transglutaminase proteins were found up-regulated (densitometry values of 1.4 and 1.7, respectively). C, Validation experiments were also performed in a new set of samples which had not been previously analyzed in the DIGE cohort. The results matched the previous ones. GAPDH was used as a control to normalize protein abundance in all the experiments performed. The densitometric results are provided as the normalized values between the indicated markers and GAPDH.

Article Snippet: PVDF membranes were blocked in PBS and 5% dry nonfat milk at room temperature for 1 h. Immunoblotting was performed with primary antibodies; 2.5 g/ml mouse monoclonal transgelin, 2 g/ml mouse monoclonal actinin, 2 g /ml mouse monoclonal transglutaminase2, and0.25 g/mlmousemonoclonal cathepsinB (all of Abcam, Cambridge, UK) at 4 C. Then membranes were incubatedwith the1:2000dilutionof thepolyclonalgoatantirabbit IgG-HRP (DakoCytomation, CA) and the polyclonal rabbit antigoat IgG-HRP (DakoCytomation) at room temperature for 1 h. PVDF membranes were analyzed with the ECL Western Blotting Analysis System (GE Healthcare, Freiburg, Germany) and were photographed using Fujifilm LAS-300.

Techniques: Control, Biomarker Discovery, Labeling, Two-Dimensional Gel Electrophoresis, Fluorescence, Imaging, Staining, Western Blot, Quantitative Proteomics