The role of extracellular matrix components in pin bone attachments during storage—a comparison between farmed Atlantic salmon (Salmo salar) and cod (Gadus morhua L.)

Pin bones represent a major problem for processing and quality of fish products. Development of methods of removal requires better knowledge of the pin bones’ attachment to the muscle and structures involved in the breakdown during loosening. In this study, pin bones from cod and salmon were dissected from fish fillets after slaughter or storage on ice for 5 days, and thereafter analysed with molecular methods, which revealed major differences between these species before and after storage. The connective tissue (CT) attaches the pin bone to the muscle in cod, while the pin bones in salmon are embedded in adipose tissue. Collagens, elastin, lectin-binding proteins and glycosaminoglycans (GAGs) are all components of the attachment site, and this differ between salmon and cod, resulting in a CT in cod that is more resistant to enzymatic degradation compared to the CT in salmon. Structural differences are reflected in the composition of transcriptome. Microarray analysis comparing the attachment sites of the pin bones with a reference muscle sample showed limited differences in salmon. In cod, on the other hand, the variances were substantial, and the gene expression profiles suggested difference in myofibre structure, metabolism and cell processes between the pin bone attachment site and the reference muscle. Degradation of the connective tissue occurs closest to the pin bones and not in the neighbouring tissue, which was shown using light microscopy. This study shows that the attachment of the pin bones in cod and salmon is different; therefore, the development of methods for removal should be tailored to each individual species. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10695-016-0309-0) contains supplementary material, which is available to authorized users

The role of extracellular matrix components in pin bone attachments during storage-a comparison between farmed Atlantic salmon (Salmo salar) and cod (Gadus morhua L.).

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Processed eggshell membrane powder regulates cellular functions and increase MMP-activity important in early wound healing processes.

Avian eggshell membrane (ESM) is a natural biomaterial that has been used as an alternative natural bandage to cure wounds, and is available in large quantities from egg industries. We have previously demonstrated that processed eggshell membrane powder (PEP), aiming to be used in a low cost wound healing product, possesses anti-inflammatory properties. In this study, we further investigated effects of PEP on MMP activities in vitro (a dermal fibroblast cell culture system) and in vivo (a mouse skin wound healing model). Three days incubation with PEP in cell culture led to rearrangement of the actin-cytoskeleton and vinculin in focal adhesions and increased syndecan-4 shedding. In addition, we observed increased matrix metalloproteinase type 2 (MMP-2) enzyme activation, without effects on protein levels of MMP-2 or its regulators (membrane type 1 (MT1)-MMP and tissue inhibitor of matrix metalloproteinase type 2 (TIMP-2). Longer incubation (10 days) led to increased protein levels of MMP-2 and its regulators. We also observed an increased alpha-smooth muscle actin (α-SMA) production, suggesting an effect of PEP on myofibroblast differentiation. In vivo, using the mouse skin wound healing model, PEP treatment (3 days) increased MMP activity at the wound edges, along with increased MMP-2 and MMP-9 protein levels, and increased keratinocyte cell proliferation. Altogether, our data suggest PEP stimulates MMP activity, and with a positive effect on early cellular events during wound healing

Epitope mapping of syndecan-4 antibody (sc-12766) and design of a specific blocking peptide.

<p>A) The antibody epitope was identified by overlaying an array of immobilized overlapping 20-mer human syndecan-4 peptides with anti-syndecan-4 (upper panel). Amino acids relevant for anti-syndecan-4 recognition are given. Underlined amino acids indicate the core epitope. Immunoblotting without anti-syndecan-4 was used as control (lower panel). B) Alignment of human vs. bovine and mouse syndecan-4 protein sequences. Black boxes indicate identical amino acids (DNA Star, Madison, Wisconsin). C) Amino acid sequences relevant for anti-syndecan-4 binding were synthesized on a membrane and overlaid with anti-syndecan-4 pre-incubated without (left panel) or with the blocking peptide (middle panel). Anti-syndecan-4 was omitted in the negative control (right panel). D) Staining of myoblasts and myotubes after three days in differentiating medium. Cells were fixed with ice-cold ethanol and immunostained with anti-syndecan 4 (sc-12766) alone (diluted in Pierce Immunostain Enhancer for increased antibody binding), or in combination with various concentrations of blocking peptide (2-330-660 μM), followed by Alexa 546-conjugated goat anti-mouse (red) before fluorescence microscopy analysis (ZEISS Axio Observer Z1 microscope). Scale bar: 50 μm. E) Control experiment with secondary antibody (SA) alone show little unspecific binding. Cells stained with anti-syndecan-4 followed by Alexa 546-conjugated goat anti-mouse (upper panel), or with secondary antibody (Alexa 546-conjugated goat anti-mouse) alone (lower panel) before fluorescence microscopy analysis (ZEISS Axio Observer Z1 microscope). Nuclei were stained with DAPI (blue). All images were captured using the same settings.</p

Tentative model illustrating how syndecan-4 may regulate muscle differentiation.

<p>During muscle cell proliferation cell surface syndecan-4 function as a co-receptor for FGF2 and its receptor (1), thus increasing their (FGF2 and FGFR) local concentration and leading to enhanced cell growth. At the same time the intracellular domain of syndecan-4 exhibit a negative control of myoblast fusion. Upon differentiation (2) syndecan-4 is internalized from the plasma membrane (3) into endocytic compartments. The recruitment of specific (unknown) adaptor proteins to syndecan-4 enables transport of syndecan-4 from endosomal compartments to the nuclear membrane (4) thus escaping recycling of syndecan-4 back to plasma membrane (5), exosome formation (6) or degradation of syndecan-4 (7). This re-localization may then affect gene transcription and muscle differentiation (8).</p

Syndecan-4 co-localizes with the nuclear protein Lamin A/C.

<p>A-B) Myoblasts and myotubes induced to differentiate for three days were fixed with 4% PFA and immunostained with mouse anti-syndecan-4 and goat-anti Lamin A/C, followed by Alexa 546-conjugated goat anti-mouse (green) and Alexa 647-conjugated donkey anti goat (red) before confocal fluorescence microscopy analysis (ZEISS Axio Observer Z1 microscope). The micrographs show the middle section (A) and the top section (B) of the nuclei. The inserts in the right panel show higher magnification of the framed areas. Arrows denote areas within the nuclear membrane which appear positive for both syndecan-4 and Lamin A/C. C) Muscle cells transiently transfected with SDC4-HA were immunostained with mouse anti-HA and goat Lamin A/C, followed by Alexa 647-conjugated donkey-anti goat (green) and Alexa 546-conjugated goat-anti mouse (red) before fluorescence microscopy analysis. Arrow indicates co-localization of Lamin A/C and anti-HA in the nuclear membrane. The inserts in the right panel shows higher magnification of the framed area. Scale bars: 20 μm.</p

The cytoplasmic domain of syndecan-4 is involved in myoblast fusion.

<p>A-B) Confluent muscle cells incubated without (left panel), or with either the cell-penetrating peptide Arg₉-Syn-4 cyt (middle panel) or the cell-penetrating Arg₉ control peptide (right panel) for 24 h in differentiation media. A) Fluorescence microscopy analysis of cells stained with anti-desmin (green) and DAPI (nuclei, blue) after fixation with 4% PFA. Scale bars: 50 μm. Dashed area show myotubes in untreated cells (left) and in cells treated with control peptide (right panel), but no myotubes are observed in cells treated with Arg₉-Syn-4 cyt (middle panel). B) Light microscopy analysis of muscle cells treated as in A. Arrows indicate myotubes in left and right panels (untreated cells and cell treated with the cell-penetrating Arg₉ control peptide). Note that myotubes cultured in dishes will have a mixture of morphological characteristics, both branched and unbranched. It should be emphasized that the important observation in this experiment is the complete absence of myotubes in Arg₉-Syn-4 cyt treated cells (Fig 8A and 8B, middle panels). Scale bar: 5 μm. C) The fusion index (FI) (the number of cells with more than 2 nuclei) was calculated based on scoring at least four randomly chosen regions with nuclei and myotubes stained as in A in three independent experiments. The FI was calculated as the percentage of total nuclei incorporated into myotubes. Asterisk denote significant differences between untreated and Arg₉-Syn-4 cyt treated cells (**p<0.01, n>50 cells). Arg₉ was used as a control peptide. D) Media from untreated muscle cells, muscle cells treated with Arg₉-Syn-4 cyt and with Arg₉ control peptide for 24 hours were subjected to LDH release analysis. Incubation with 2% Triton X-100 for 2 h was used as a positive control for the assay. Differences in release was tested by Mann Whitney U test (*p<0.05, n = 3–6). Error bars indicate SEM.</p

Syndecan-4 is internalized from the plasma membrane to the nuclear membrane.

<p>Muscle cells transiently transfected with SDC4-HA were either left untreated (A) or incubated with mouse anti-HA antibody for 30 min on ice to allow binding of antibody to SDC4-HA at the plasma membrane. The cells were then washed with ice-cold PBS and either fixed immediately (B), or chased for 30 min (C), 3 h (D), 24 h (E) and 48 h (F) at 37°C in differentiation media before fixation. After permeabilization, localization of anti-HA was detected using an Alexa 488-conjugated anti-mouse antibody (green). Nuclei were stained with DAPI (blue) (A-F). The right panels show only the anti-HA staining. This allows a better visualization of internalized anti-HA localized to intracellular compartments and the nuclear membrane. The inserts (framed areas at high magnification) show staining for HA in the nuclear membrane (denoted by arrows). Scale bars as indicated.</p

List of primers and probes used for quantitative real-time PCR.

<p>List of primers and probes used for quantitative real-time PCR.</p

Localization of syndecan-4 to the perinuclear area during muscle cell differentiation.

<p>Myoblasts and myotubes induced to differentiate for three days were fixed with 4% PFA and immunostained with mouse anti-syndecan-4, followed by Alexa 488-conjugated goat anti-mouse (green) before confocal microscopy analysis. Nuclei were stained with DAPI (blue). The framed areas (A-C) are presented at high magnification in the right panels. Arrows denote nuclei with syndecan-4 localized to the nuclear membrane in early differentiated muscle cells (B) and in complex myotubes (C). Scale bar 20 μm.</p