Mechanically separated fish meat (MSFM) can be used for the manufacturing of ready-to-eat products, such as sausages; however, it is highly perishable. Several plant by-products, including onion peel, which is rich in polyphenol antioxidants, can be added to food to extend shelf life. This study investigated the effects of the addition of onion peel powder (OPP) to sausage made from MSFM. Sausages were divided into four groups with different amounts of added OPP: 0% (control), 1%, 2%, and 3%. Cooked sausages were stored for 28 days at 5 °C. Samples were analyzed for thiobarbituric acid reactive substances, antioxidant activity, total polyphenol content, pH, and organoleptic properties. The addition of OPP significantly increased antioxidant activity and total polyphenol content and decreased pH, indicating acidic nature of OPP. Polyphenols from OPP effectively suppressed lipid oxidation. A 1–2% addition of OPP enhanced sensory properties. After the 28-day storage, the control samples received the lowest sensory score, due to the presence of a strong fishy odor, which was not present in samples with OPP. HPLC–MS/MS analysis revealed that quercetin is the most dominant compound in OPP. Overall, the results indicate that the addition of OPP in amounts of 1–2% can extend shelf life, without the deterioration of sensory properties. Fish meat is a nutritionally valuable part of the human diet, and consuming it two times a week is recommended, mostly due to the content of long-chain polyunsaturated n-3 fatty acids with a positive impact on human health .In 2018, about 88%  of fish production was utilized for direct human consumption, primarily in live, fresh, or chilled form; however, the fish industry often extracts only fillets. The remaining 12% was used for non-food purposes, such as fish meal or fish oil .Fish processing is closely related to the production of a wide range of waste or by-products. The edible proportion of fish represents approximately 45% of total fish weight; therefore, 55% of the fish can be considered as a waste from processing, including heads, guts, bones, skin, fins, frame, and meat adhered to bones and skin [According to Vanhonacker et al. , some peUnfortunately, fish meat has very short shelf life. A far worse situation is in the case of the utilization of product specific waste. The reasons are high water content, suitable environment for microorganisms, high level of enzymatic activity, and fast oxidation of lipids. The changes start immediately after a fish’s death ,9.The incorporation of antioxidants is considered as an effective method to inhibit or delay the lipid oxidation that may result in negative sensory and nutritional changes of meat products, thereby extending the shelf life of products. Despite synthetic antioxidants having been used in recent years, the demand for natural antioxidants has been increasing mainly because of the potentially adverse effects of synthetic antioxidants on human health. Thus, it is becoming increasingly trendy to investigate the effects of natural antioxidants from plant sources  as food products additives ,11,12,13Allium cepa L.) is the second most cultivated vegetable in the world, and thousands of tons of waste, generated during its processing, are produced annually just in the European Union [These plant sources could be, for example, vegetable-processing by-products, such as onion peel. Onion  have increasingly attracted more attention during the last few years as a functional food ingredient which has been incorporated, for example, into wheat bread , gluten-Therefore, the aim of this study was to investigate the effects of the incorporation of onion peel powder (OPP) into a sausage prepared from mechanically separated fish meat on selected chemical, technological, and sensory properties.Dry and soil-free middle layers of onion peels of the yellow variety Hybelle were donated by a Czech grower of onions . Onion peels (water content approximately 12%) were ground into a fine onion peel powder (OPP) with particles equal to or smaller than 250 µm and stored at room temperature in the dark until further use. The OPP was then subjected to an analysis of its basic chemical composition, antioxidant activity, polyphenols, and water-holding capacity determination.Cyprinus carpio L.) was bought frozen, at −18 °C , from a local fish producer . The separated meat came from fish skeletons (including spine and ribs without head) with adhered meat that remained after filleting. The eggs and pork belly were bought fresh at local markets 1 day prior to the experiment and stored overnight at 5 °C. Several hours before sausage preparation, pork fatback was put into a freezer (−18 °C) to be frozen and easily chopped in a bowl cutter. The spices used for sausage production were purchased from GOLDEN WAY spol. s r. o., Plzeň, Czech Republic. Natural pork casing (diameter 34–36 mm) was purchased from HEROLD ŘEZNICKÉ POTŘEBY s.r.o., Rakovník, Czech Republic.Mechanically separated fish meat of the common carp (w/w): mechanically separated fish meat 49.2%, pork belly 32.8%, garlic 0.4%, salt 1.6%, black pepper 0.2%, caraway seeds 0.1%, marjoram 0.03%, chili pepper 0.2%, paprika 0.5%, egg 2.7%, and ice 12.3%. In the experimental groups, the control mixture was replaced by 1%, 2%, and 3% (w/w) of OPP  were selected to determine the effect of the addition of OPP on the physicochemical and sensory properties of fish–pork sausages. The control group of sausages was prepared according to this recipe  of OPP Table S1The fish and pork meat were chopped into small pieces and weighed separately, according to the recipe. The ingredients were then mixed in a bowl cutter  in the following order: Firstly, pork belly and mechanically separated fish meat were slowly minced with ice. Secondly, salt and spices were added, and, finally, OPP was added at the end of mixing. The total time of mixing of the ingredients was approximately five minutes, and the temperature during this process did not exceed 8 °C. The sausage batter was then stuffed into a natural pork casing. After this, the raw sausages were left for approximately for one hour in the room, to allow the proteins to dissolve. The raw sausages were then cooked in a water bath 1.5 h at 80 °C  and then smoked for one hour at 70 °C in a smoking chamber. All batches, after cooling down to 5 °C, were then vacuum-packed and stored at 5 °C for 28 days. Cooking loss was determined during processing. Antioxidant activity, total polyphenol content (TPC), and basic composition chemical composition were measured on the 1st day of storage. The pH, thiobarbituric acid reactive substances (TBARS), CIE L*a*b* color measurement, and microbiological and sensory analysis were assessed on the 1st, 7th, 14th, and 28th day of storage.The sausages were weighted before and after cooking, after cooling down. The cooking loss was calculated according to the following formula:Cooking loss was calculated for each treatment group in triplicate.g for 30 min). The supernatant was transferred to a graduated 10 mL cylinder, where the volume was measured. The result was expressed as mL of H2O held by 1 g of OPP. The analysis was conducted in triplicate.The water-holding capacity of OPP was determined according to the method described by Benítez et al.  with sli−1).The prepared fish sausages  were aseptically sampled (10 g/sample), mixed with 0.1% peptone (90 mL) in a sterile plastic bag, and homogenized for 1 min, using an electric stomacher . Serial 10-fold dilutions were prepared from each sample using 1 mL in 0.1% peptone (9 mL). The total viable counts (TVCs) were determined by using the pour plate method according to ISO 4833  and the O-diglucoside (purity ≥ 85%), quercetin-4′-O-glucoside (purity ≥ 95%), gallic acid (purity ≥ 99%), sodium acetate (purity ≥ 99%), acetic acid (purity ≥ 99%), sodium acetate (purity ≥ 99%), sodium carbonate, ferric chloride, hydrochloric acid , 2,4,4-tris(2-pyridyl)-1,3,5-triazine , 2,2-diphenyl-1-picrylhydrazyl (DPPH), Trolox (purity ≥ 97%), Trolox (purity ≥ 97%), Folin-Ciocalteau’s phenol reagent, thiobarbituric acid (purity ≥ 98%), butylated hydroxytoluene , 1,1,3,3-tetraethoxypropane (TEP), trichloroacetic acid , ortho-phosphoric acid , Formic acid (LC/MS grade purity 98–100%), acetonitrile and methanol LC/MS grade, and ethanol for spectroscopy were purchased from Sigma-Aldrich .All chemicals, namely quercetin dihydrate (purity ≥ 95%), quercetin-3,4′-Water content, ash, crude protein (CP), ether extract (EE), and non-soluble fiber fractions were analyzed in OPP. Moisture was determined by drying the sample, at 105 °C, in an oven, to a constant weight, ash content by combustion at 550 °C, for 16 h in a muffle furnace to obtain light gray ash . Crude pAll analyses of onion peels were assessed in triplicate.TM N500; BÜCHI, Flawil, Switzerland), according to the manufacturer’s instructions. Approximately 50 g of sample from each group was homogenized, put into a Petri dish, and analyzed by using the NIR MasterTM, which scanned samples over an NIR range of 4000–10,000 cm−1, with a resolution of 4 cm−1. Three independent samples from each group were analyzed.The basic chemical composition of sausages Table S1The pH of cooked sausages was measured by using a pH meter equipped with a spear-type electrode . Approximately 25 g of homogenized sample was used to directly measure pH. Three independent samples from each group were measured.Two spectrophotometric methods for measurement of antioxidant activity of samples were selected: DPPH assay, to assess free radical scavenging ability, and FRAP assay, to evaluate reducing ability.v/v), shaken in a laboratory shaker for 10 min, and then centrifuged at 7000 RPM, at 5 °C for 15 min. The collected supernatant was further used for DPPH and FRAP (Ferric Reducing Antioxidant Power) analyses. Two independent samples from each treatment groups were taken, and each sample was extracted twice and analyzed (for DPPH and FRAP) in duplicate (n = 8/group).Then, 0.2 g of homogenized sample (either OPP of fish sausages) was extracted in 9.8 mL of 90% methanol  under reflux (90 °C/120 min.). A modified method of Lachman et al. [n = 4). The results are expressed as gallic acid equivalents (GAE)/g FW.Firstly, 5 g of homogenized (either OPP of fish sausages) was extracted in 100 mL of 80% ethanol  presented in OPP were assessed according to the method described by Bedrníček et al. [Extraction and HPLC–MS/MS quantification of main flavonol compounds , nebulizer pressure of 35 psi, gas temperature 300 °C, capillary voltage −4 kV, and cell acceleration voltage of 7 V. Optimized Fragmentor voltages, collision energy voltages, and transitions were optimized for each compound (n = 4). The results are expressed as µg/g of FW.Then, 5 µL of the supernatant was injected into the HPLC–MS/MS system. The analysis was carried out on the HPLC Dionex UltiMate 3000 system coupled with an Agilent 6420 triple-quad mass spectrometer (MS) equipped with an electrospray ionization source. Phenomenex Kinetex C18 column with diameters of 150 × 2.1 mm, (particle size of 2.6 µm), maintained at 35 °C, was used for the separation. Mobile phases consisted of mobile phase A (5% acetonitrile and 0.5% formic acid) and B (100 acetonitrile); the flow rate of mobile phase was 0.2 mL/min. During the separation, mobile phase B linearly increased from 15% to 70% within 20 min, and then decreased back to 15%. The following step was column equilibration for 5 min prior to the next injection. Separated analytes were then introduced into the MS operating in negative mode with the following settings: 11 L/min of drying gas (Ncompound Table S2Products of secondary lipid peroxidation (TBARS) were measured according to the method of Miller . Then, 1CIE L*a*b* color measurement was measured, using a ColorEye XTH Spectrophotometer . Slices were chosen as a representative part of the sausages. Results are expressed in the L*a*b* scale. Results are presented as mean of three independent samples.The analysis was assessed by 9 trained panelists  from the Faculty of Agriculture, University of South Bohemia, in České Budějovice, who are familiar with the sensory evaluation of meat products. The sausages were heated in a water bath at 70 °C for 10 min prior to sensory analysis and were served warm. Each panelist received approximately 15 g of each sample (randomly marked with a three-digit number) on a plate, at the same time. Appearance, odor, taste, texture, and overall acceptability were evaluated on a 100 mm unstructured abscissa . The intensity of fishy odor was also assessed on the 28th day of storage . Sensory analysis was conducted in a room (temperature approximately 22 °C) equipped with a table for each panelist and daylight-type bulbs for balanced light. Water and bread were served as a taste neutralizer and were consumed between the judging of samples.ijk is the dependent variables, i.e., pH, TBARS, CIE L*a*b*, sensory characteristics, and cooking loss; μ is the mean; OPPi is the percentage of onion peel powder ; Sj = storage , εij = residual error. Fisher’s LSD test was used for group comparisons (post hoc test). Pearson’s correlation coefficient was used to estimate the association between percentage of OPP addition and antioxidant activity, and TPC, as well as between CIE L*a*b* color measurement and sensory analysis results.Data were analyzed with the program Statistica CZ 12 (Statsoft CR), using one-way analysis of variance (ANOVA) or two-way ANOVA, using the following model with a fixed effect of percentage of OPP, storage, and interaction OPP × storage:The basic chemical composition, including total polyphenol content, flavonols, antioxidant activity, and WHC, of OPP is summarized in Crude protein, ether extract and ash content in OPP is 2.41 ± 0.61%, 0.85 ± 0.03%, and 8.06 ± 0.01% of FW, respectively. Benítez et al.  reportedO-glucoside, and quercetin-3,4′-O-diglucoside is 4.11 ± 0.15, 3.40 ± 0.08, and 0.63 ± 0.03 mg/g, respectively. Quercetin is the most abundant flavonol in OPP which corresponds to the finding of Prokopov et al. [O-glucoside, we found a similar concentration as Suh et al. [O-diglucoside was found in OPP. It is a foreseeable result because, according to Takahama and Hirota [O-glucoside, in onion peel in the range between 2.6% and 6.5% by weight [Main flavonols in OPP were also determined by LC–MS/MS, together with a spectrophotometric determination of TPC. Content of quercetin, quercetin-4′-v et al. , who reph et al. , who alsd Hirota , quercety weight .The concentration of total polyphenols in OPP is 78.60 ± 1.46 mg GAE/g FW. This value is comparable, but slightly higher, than with Chung et al. , who rep2•− and HO• radicals. Onion peels are also capable to scavenge free radicals in ORAC  antioxidant activity measurement [The presence of flavonols in onion peel is tightly related to high antioxidant activity, using DPPH (84.97 ± 2.61 mg TE/g FW) and FRAP method (91.47 ± 2.81 mg TE/g FW). According to Ly et al. , quercetsurement .WHC of dietary fiber is an important technological feature that is important for the incorporation of fiber into a meat product recipe. WHC of OPP was found to be 4.20 ± 0.10 mL/g . This vaNevertheless, it is very hard to compare the content of various compounds in onion peel with other authors, since different authors use different layers of onion peel that are either closer to or further from the surface of the onion, and could differ, for example, in moisture content. According to Benítez et al.  and Chenp < 0.05). Control samples did not differ (p < 0.05) from samples with 1% and 2% of OPP.In general, weight loss of meat products during heat treatment could be lowered by the addition of plant fiber. According to Ktari et al. , cookingThis is very interesting, and the results indicate that a 2% addition of OPP seems to be a threshold in this particular fish sausage recipe and that, after further addition of OPP, cooking loss increases. There are several explanations that could clarify this phenomenon. It could be explained by the degradation of polysaccharides  in OPP during thermal treatment of fish sausages, which can lead to the lowered water-holding capacity of fiber. Another reason could be that the fiber presented in OPP can absorb water much faster than solubilized proteins of meat, but that is not stable at elevated temperatures, and it releases water during cooking and, hence, increases cooking loss. It indicates that the bond between water and fibre is not as strong as between meat protein matrix and water. According to Feiner , WHC is Unlike many experiments that resulted in enhanced WHC and/or cooking loss of meat products enriched with plant dietary fiber ,48, therVarious antioxidants from natural sources have been used to increase the antioxidant activity of meat or meat products, e.g., rosemary extract  or red gp < 0.05) on antioxidant activity (DPPH and FRAP) and TPC, compared to the control (p < 0.05) which was found between the percentage of OPP in fish sausages and DPPH (r = 0.993), FRAP (r = 0.991), and TPC (r = 0.993) values. It points out that OPP addition contributed significantly to the improvement of antioxidant activity and higher TPC. Despite the fact that no OPP was used in the control group, very low values of antioxidant activity and TPC were found. It could be caused by the presence of other antioxidants occurring in spices used in the recipe. Spices are also source of antioxidant, especially polyphenolic [The addition of OPP into fish sausages had a significantly positive effect , it can significantly increase the antioxidant activity of certain meat products, especially due to the presence of considerable amounts of quercetin and its derivatives .p < 0.05) lowered the pH of fortified fish sausages in a dose-dependent manner, compared to the control samples  affected pH of sausages. The pH of all the sausages continuously decreased during storage with the highest value for the control samples (5.91 ± 0.00) and the lowest for the samples with 3% of OPP (5.56 ± 0.03). This decrease of pH for all the samples might be due to the presence of spoilage lactic acid bacteria [p < 0.05) interaction between the percentage of OPP incorporation and storage time was found.Storage time also significantly (bacteria  that havp < 0.05) lowered the concentration of MDA. Even on the first day of storage (after cooking), control samples showed signs of oxidation (0.44 ± 0.06 µg MDA/g), compared to sausages with OPP, where the oxidation was suppressed (0.26 ± 0.10–0.32 ± 0.04 µg MDA/g). This oxidation is probably a result of the exposure of fats that are prone to oxidation at high temperatures during cooking. Similarly, this phenomenon was described by Kurt et al. [The concentration of secondary lipid oxidation products (TBARS) was also determined . The addt et al.  and by ot et al. .p < 0.05) affected TBARS values. During the 28-day storage period, the concentration of MDA in the control samples grew to values above 0.71 µg MDA/g, while the samples with OPP remained stably low and did not show any increasing trend. Our results are consistent with Kurt et al. [p < 0.05) negative correlation (r = −0.703) was found between % of OPP and MDA concentration in all days of storage. All of these facts indicate that OPP is a promising additive for meat products that is effective in the suppression of fat oxidation in meat products, owing its strong antioxidant properties to high amounts of quercetin.Storage time also significantly  on the lightness of fish sausages . The lightness of fish sausages proportionally decreased with an increasing percentage of OPP, except for the first day. The difference between samples with 1% and 2% of OPP was small; however, the sample with 3% of OPP had a very dark color. Lightness was affected also by storage time (p < 0.05). L* values of the control samples and samples with 1% and 2% of OPP changed with only small alterations, but the samples with 3% were increasingly lighter over time. Furthermore, a* value (redness) was affected by OPP (p < 0.05). Values on the first day slightly differed (between 15.72 ± 1.30 and 16.91 ± 0.47), but these differences among treatment groups were more obvious in the later days of storage, where these values were lower with the higher addition of OPP. Again, the samples with 1% and 2% of OPP were very similar. It indicates that OPP can cover the typical red color of a meat product. The same trend was also observed in the case of b* value (yellowness), which was also affected by the addition of OPP (p < 0.05). This value was also much lower in the samples with OPP. However, this is an unexpected result, to some extent, because our presumption was that the incorporation of OPP would increase the b* value because peels of yellow onion varieties contain quercetin (as mentioned in p > 0.05).The variation in color other than the expected norm may be due to the physical characteristics of meat, the concentration and chemical state of pigments therein, and the presence of non-meat ingredients . Color a−1 for TVCs and 5.52 ± 0.02 log CFU.g−1 for psychrothrophic bacteria. These relatively high amounts could probably influence the microbial levels in fish sausages before heat treatment, which ranged from 5.29 ± 0.04 log CFU.g−1 (3% OPP sample) to 6.03 ± 0.04 log CFU.g−1 (1% OPP sample) for TVCs, and from 5.66 ± 0.04 log CFU.g−1 (control sample) to 7.02 ± 0.03 log CFU.g−1 (3% OPP sample) for psychrothrophic bacteria (−1 (control sample) to an uncountable amount (3% OPP sample) for TVCs, and from 6.30 ± 0.10 log CFU.g−1 (control sample) to an uncountable amount (3% OPP sample) for psychrothrophic bacteria. These results suggest the development of spore-forming microorganisms. According to Raju et al. [−1) and psychrotrophic bacteria (5.71 ± 0.02 log CFU.g−1). On the 28th day of storage, all examined samples were found as uncountable.The initial microbial loads in separated fish meat were 5.76 ± 0.02 log CFU.gbacteria Table S3u et al. , fish sau et al. . On the p < 0.05) influenced all evaluated sensory parameters.Although a diet enhanced by antioxidants may have considerable health effects, sensory properties should not be neglected. Sensory characteristics, particularly taste and appearance, have a great impact on consumers’ preference. The results of the analysis of appearance, odor, taste, texture, and overall acceptability are presented in r = 0.762, p < 0.05), suggesting that the lighter the sausage is, the more pleasant appearance it has. Together with this, there was also found a significant correlation between appearance and redness . Even though there were slight changes in appearance over time, they were not statistically significant (p > 0.05).It is evident that the addition of more than 1% of OPP negatively influenced the appearance of fish sausage. The addition of OPP caused darker color, compared to the control group , which ip < 0.05) odor. The pleasantness of odor of control samples continuously decreased over time. This could be related to the presence of fishy odor. It is attributed to compounds including alcohols, aldehydes, ketones, pyrazine, furan and trimethylamine [r = 0.999; p < 0.001) with the concentration of MDA at 28th day of storage.The worst score in the case of odor evaluation received the control samples for all days. The addition of OPP positively affected odor pleasantness. Moreover, storage time significantly affected  differ from each other. The taste score of control samples decreased over time; however, these changes were not significant (p > 0.05).Taste is also a very important sensory parameter. The assessors regarded samples with 1% and 2% of OPP as the tastiest samples over the whole storage period. The control samples and samples with 3% of OPP received similar values and did not statistically (p > 0.05).Sensory evaluation showed that the most pleasant texture had the control samples and samples with 1% of OPP. The samples with 2% and 3% of OPP were evaluated very negatively. This study confirms with the previous findings of Kurt et al. . The texIn general, among all the samples, the sensory panel preferred those with 1% and 2% of OPP. These samples received the highest score of overall acceptability. This is a satisfactory result, since some consumers do not like the typical fish smell , which cBased on our results, it is evident that OPP is able to extend the shelf life of sausages prepared from separated fish meat. The formation of MDA was suppressed by the addition of OPP, and the overall acceptability was also prolonged during storage. Unfortunately, OPP with a particle size ≤250 µm is not an appropriate source of dietary fiber that could enhance the technological properties (such as WHC and cooking loss) of fish sausage, which is closely related to the economical aspect of production. However, this problem could be solved by, for example, the reformulation of the meat product recipe, such as the addition of polyphosphates or potato fiber. Overall, the results of this study can be a useful example of the valorization of fish processing by-product together with onion processing by-product that could show a possible way to prevent the loss of valuable nutrients and biologically active substances from the food chain.


Coxsackievirus B3 (CVB3) is the primary etiologic agent of viral myocarditis, a major heart disease that occurs predominantly in children and young adolescents. In the heart, intercalated disks (ICD) are important structural formations that connect adjacent cardiomyocytes to maintain cardiac architecture and mediate signal communication. Deficiency in ICD components, such as desmosome proteins, leads to heart dysfunction. γ-catenin, a component protein of desmosomes, normally binds directly to desmocollin-2 and desmoglein-2. In this study, we found that CVB3 infection downregulated γ-catenin at the protein level but not the mRNA level in mouse HL-1 cardiomyocytes. We further found that this reduction of γ-catenin protein is a result of ubiquitin proteasome-mediated degradation, since the addition of proteasome inhibitor MG132 inhibited γ-catenin downregulation. In addition, we found that desmocollin-2 and desmoglein-2 were cleaved by both viral protease 3C and virus-activated cellular caspase, respectively. These cleavages led to the release of bound γ-catenin from the desmosome into the cytosol, resulting in rapid degradation of γ-catenin. Since γ-catenin shares high sequence homology with β-catenin in binding the TCF/LEF transcription factor, we further studied the effect of γ-catenin degradation on Wnt/β-catenin signaling. Luciferase assay showed that γ-catenin expression inhibited Wnt/β-catenin signaling. This finding was substantiated by qPCR to show that overexpression of γ-catenin downregulated transcription of Wnt signal target genes, c-myc and MMP9, while silencing γ-catenin upregulated these target genes. Finally, we demonstrated that γ-catenin expression inhibited CVB3 replication. In search for the underlying mechanism, we found that silencing γ-catenin caused down-regulation of interferon-β and its stimulated antiviral genes MDA5, MAVS, and ISG15. Taken together, our results indicate, for the first time, that CVB3 infection causes cardiomyocyte death through, at least in part, direct damage to the desmosome structure and reduction of γ-catenin protein, which in return promotes Wnt/β-catenin signaling and downregulates interferon-β stimulated immune responses. Coxsackievirus B3 (CVB3) is a primary etiologic pathogen of viral myocarditis . Acute vPicornaviridae family. Its positive, single-stranded RNA genome can be directly translated into a polyprotein, which is cleaved into 11 mature viral proteins by viral proteases 2A and 3C. In addition to processing viral polyproteins to complete viral life cycle, proteases 2A and 3C also cleave a number of host proteins involved in host gene transcription and translation as well as signal transduction  are substantial structures that connect adjacent cardiomyocytes in the myocardium. The entire ICD structure is composed of three major complexes: desmosomes, gap junctions, and fascia adherens . DesmosoIn addition to localizing in the desmosome as a structural protein, γ-catenin also participates in cell signaling in the cytosol. This protein is critical for desmosome assembly, especially the vertical linkage of desmosomal cadherins (desmoglein and desmocollin) to desmoplakin . InteresIn this study, we first found that the reduction of γ-catenin was primarily due to the ubiquitin proteasome-mediated degradation caused by CVB3 infection. We further found that desmosome cadherins, desmocollin-2 (DSC-2) and desmoglein-2 (DSG-2), were cleaved by both CVB3 protease and virus-activated caspases. These cleavages promoted cellular relocalization and subsequent degradation of γ-catenin. To our knowledge, this is the first report on the cleavage of ICD proteins by cardiotropic viral proteases. In addition, we also found by luciferase assay that γ-catenin played an inhibitory role in Wnt/β-catenin signaling by competing with β-catenin to interact with the LEF/TCF transcription factor. As a result, overexpression of γ-catenin suppressed Wnt/β-catenin target gene transcription and also inhibited CVB3 replication. These data imply that CVB3-induced reduction of γ-catenin benefits CVB3 replication and generates a positive feedback effect on the destruction of ICD proteins and eventually cardiomyocyte death. Taken together, our studies revealed a previously unrecognized mechanism of CVB3-mediated cardiomyocyte destruction, based on reduction of cardiomyocyte structural integrity and inhibition of an integral signaling pathway.2. HL-1 cardiomyocytes, a mouse cardiac muscle cell line established from a cardiomyocyte tumor linage, were a gift from Dr. William C. Claycomb . These cells were cultured in Claycomb medium (Sigma) with 10% FBS, 100 μg/ml penicillin-streptomycin, 4 mM L-glutamine (Thermo Fisher) and 100 μM norepinephrine. For in vivo studies, 4-week-old male A/J mice were purchased from Jackson Laboratories (United States). Animal work was conducted by strictly following the Guide to the Care and Use of Experimental Animals – Canadian Council on Animal Care. All protocols were approved by the Animal Care Committee of Faculty of Medicine, University of British Columbia (protocol number A16-0093). The CVB3 (CG) strain was obtained from Dr. Charles Gauntt  and propagated in HeLa cells. Viral infection of cultured cells was conducted by incubation with CVB3 at a multiplicity of infection (MOI) of 10 for 1 h (for HeLa) or at a MOI of 50 for 2 h (for HL-1) in a serum free medium, washed with phosphate-buffered saline (PBS) (Thermo Fisher) twice, and then replenished with fresh medium containing 10% FBS. Mice were infected with CVB3 at a plaque forming unit (pfu) of 105 or sham-infected with PBS by intraperitoneal injection. Infected mice were then euthanized at day 6 post-infection and the ventricular walls of the hearts were collected for analysis of target protein expression. The indicated MOI or pfu amounts and infection procedures were used throughout the entire work, unless otherwise specified.HeLa cells (ATCC) were cultured in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Sigma), 100 μg/ml penicillin-streptomycin and 2 mM glutamine (Thermo Fisher) at 37°C in 5% COHeLa cells were pre-treated with proteasome inhibitor MG132 (Selleckchem) to block protein degradation at 5 μM or an equal volume of DMSO for 1 h and then infected with CVB3. Cellular proteins were then collected at 5 and 7 h post infection (pi) to detect target protein degradation by Western blot analysis. To inhibit caspases, HeLa cells were pre-treated with z-VAD-fmk (Selleckchem) at 50 μM or equal volume of DMSO for 1 h and then infected with CVB3. Cellular proteins were collected at 5 and 7 h pi to detect the blockage of caspase-3-mediated cleavage of target protein by Western blot analysis. To inhibit glucose synthase kinase-3 (GSK3), HL-1 cells were pre-treated with inhibitor SB216763 (Selleckchem) at 40 μM or equal volume of DMSO for 1 h and then infected with CVB3. Cellular proteins were then collected at 12 and 24 h pi to determine the effect of inhibition on γ-catenin level by Western blot analysis. All these inhibitors were used at a lower concentration that could not significantly affect CVB3 replication based on our previous experience .5) were seeded in 6-well plates and grown to 60∼80% confluence (for plasmid) or 30∼40% confluence (for siRNA) before transfection. Plasmid DNA was then transfected into the cells using Lipofectamine 2000 (Life Technologies) and Opti-MEMTM (Thermo Fisher) complex for 6 h, and then incubated for 48 h after replacing the medium with DMEM containing 10% FBS. The siRNA was transfected using the same procedures as those described for plasmid except that Oligofectamine was used as transfection reagent. Scrambled siRNA and empty vector were used as controls in these transfections, respectively. Further, CVB3 infection was conducted at 36 h (for plasmid) or 48 h (for siRNA) post transfection before final analysis.All the plasmids used in this study were purchased from Addgene (United States), including 1105-desmoglein2-FLAG-eGFP, 476-desmocollin2-myc and 330-plakoglobin-myc. The γ-catenin siRNA (human) and DSC-2 siRNA (human) were purchased from Santa Cruz (United States). To perform the transient transfection, HeLa cells (2 × 10TM RNA mini kit (Invitrogen) according to the manufacturer’s instructions. cDNAs were then synthesized by reverse transcription using 5 μg of RNA and the SuperScript IV First-Strand Synthesis System (Invitrogen). The target genes were detected by qPCR using 2 × SYBR Green qPCR Master Mix (Bimake), 100 ng of first-strand cDNA and 1 μM of each primer. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression level was used as the endogenous control to normalize the data. All PCR experiments were performed in triplicates and repeated twice. The PCR primer sequences are listed in Total cellular RNAs were isolated using the PureLinkg for 20 min at 4°C and the protein-containing supernatant was collected. The protein concentration was determined by BCA Protein Assay (Thermo Fisher). For preparing mouse heart tissue lysates, the heart tissue sections were first rinsed with PBS to remove the blood and then lysed in RIPA lysis buffer using the TissueLyser LT (Qiagen). Samples were briefly sonicated at 50 Hz for 5 min to release the proteins. The protein-containing supernatants were collected by centrifugation and protein concentrations were determined as described above. Western blot was conducted by following the standard method  for 1 h and then incubated overnight with one of the following primary antibodies: monoclonal rat anti-VP1 (Doka), polyclonal rabbit anti-γ-catenin , monoclonal mouse anti-desmocollin-2 and monoclonal rabbit anti-desmoglein-2 (Abcam), monoclonal mouse anti-cleaved-caspase-3 , monoclonal rabbit anti-c-myc (Abcam) and K48-linkage specific polyubiquitin rabbit antibody  and monoclonal mouse anti-β-actin-HRP (Santa Cruz). After three washes with TBST, each blot was further incubated with a corresponding secondary antibody (goat anti-mouse or donkey anti-rabbit IgG) conjugated to horseradish peroxidase (Amersham). Detection was carried out by enhanced chemiluminescence (Amersham) as per the manufacturer’s instructions. β-actin was used as the loading control. Signal intensities were quantified by densitometric analysis using the NIH ImageJ program and then normalized to the loading control.Cultured cells were washed with ice cold PBS twice and then lysed with an appropriate volume of RIPA Lysis and Extraction Buffer (Thermo Fisher) on ice for 20 min. Cell lysates were centrifuged at 13,000 × d method . Brieflyin vitro cleavage assay was conducted as described previously  and 10 μl of recombinant wild-type or mutant  CVB3 protease 2A or 3C  containing 10 μg of protease (The eviously . In brieprotease . This reg to collect the supernatant containing the viruses. HeLa cells were seeded onto 6-well plates (8 × 105 cells/well) and incubated at 37°C for 20 h to reach confluence of approximately 90% and then washed with PBS and overlaid with 800 μl of virus-containing samples serially diluted (10–1 to 10–7) in cell culture medium. After a viral adsorption period of 60 min at 37°C, the supernatant was removed and the cells were overlaid with 2 ml of sterilized soft (0.75%) Bacto-agar-minimal essential medium, cultured at 37°C for 72 h, fixed with Carnoy’s fixative for 30 min, stained with 1% crystal violet for 2 min and then washed with PBS twice. The number of plaques was counted, and the virus titer was calculated as the pfu/ml. All the assays were conducted three times.Viral titer was determined following the procedure published previously . BrieflyCells cultured in μ-Slide 8 Well ibiTreat:#1.5 polymer coverslip  were washed with PBS and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. After removing the fixative, cells were further washed with fresh 0.1 M glycine in PBS for three times (5 min each). Fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 2 min, washed with PBS three times and then blocked with 3% bovine serum albumin (BSA) in PBS for 1 h at room temperature, which was followed by removing the blocking buffer and incubating the cells with a corresponding primary antibody (same antibodies as for the Western blot) at 4°C overnight. The cells were then washed with PBS and incubated with corresponding secondary antibodies: goat anti-rabbit IgG labeled with Alexa Fluor 488 (green), goat anti-mouse IgG labeled with Alexa Flour 594 (red), or goat anti-rat IgG labeled with Alexa Flour 633 (purple) (Invitrogen). The nuclei were stained with 4′,6′-diamidine-2′-phenylindole dihydrochloride (DAPI) (Sigma). Images were captured using a Zeiss LSM 880 confocal microscope and merged using the Zen Black and Zen Blue programs. To quantify the intensity of fluorescence signals, the same imaging setting parameters were applied to all the samples. Relative protein levels were then quantified by converting the captured photos to 8 bit-pictures and measuring the mean gray value of the entire image using the NIH ImageJ program.g for 5 min at 4°C. After preclearing for 2 h, the supernatants were immunoprecipitated overnight using the anti-myc antibody and the Pierce Crosslink IP Kit (Thermo Fisher) according to the manufacturer’s instructions. The enriched proteins were separated by a 6% SDS-PAGE and then immunoblotted using the K48-linkage anti-polyubiquitin antibody .Cell lysates of CVB3-infected and sham-infected cells were prepared using lysis buffer containing 50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and protease inhibitor cocktail (Roche), and centrifuged at 14,000 × The β-catenin firefly reporter (M50 Super 8 × TOPFlash), the Renilla reporter (pcDNA-RLuc8) and 330-plakoglobin-myc plasmids were purchased from Addgene. The β-catenin firefly reporter plasmid contains a TCF/LEF binding motif upstream of a firefly luciferase reporter, while the Renilla reporter is a constitutively expressed reporter, serving as a control. Thus, the expression of firefly luciferase is regulated by the activity of TCF/LEF binding proteins (such as β-catenin). To determine the effect of γ-catenin on the TCF/LEF-regulated luciferase activity, HeLa cells were co-transfected with two luciferase reporters (M50 Super 8 × TOPFlash and pcDNA-RLuc8) and the 330-plakoglobin-myc plasmid. For the negative control group, an empty vector was used to replace the 330-plakoglobin-myc plasmid for co-transfection. At 36 h post transfection, cells were infected with CVB3 for 6 h and then the cell lysates were harvested for luciferase assay on a Tecan GENios luminescence reader to determine the relative luciferase activity (firefly/Renilla) using the Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s instructions. Each treatment was verified by three biological repeats.t-test was conducted to analyze the paired groups. Results are shown as means ± standard deviation (SD) of three independent experiments. A p-value less than 0.05 (indicated by ∗) was considered statistically significant. Additionally, ∗∗p < 0.01; ∗∗∗p < 0.001; ****p < 0.0001.Two-way ANOVA analysis of variances was performed for determining differences between groups on multiple variables, and Student’s Our previous studies demonstrated downregulation of γ-catenin during CVB3 infection . To furtSince the half-life of γ-catenin is known to be prolonged in the desmosome compared to that in the cytoplasm , we specThese findings were further verified by immunostaining using CVB3-infected HeLa cells . In addiin vitro cleavage assay using purified CVB3 wild-type and mutant (control) proteases 3C to treat the cell lysates containing DSC-2 protein , an important kinase in Wnt signaling regulation that works via phosphorylation and destabilization of β-catenin . We treaHaving identified the role of γ-catenin in inhibiting Wnt/β-catenin signaling using a luciferase assay, our next step was to further confirm this notion by detecting the expression of Wnt signaling target genes. To this end, qPCR was conducted to detect transcription of c-myc and MMP9 using total RNAs isolated from HeLa cells transfected with γ-catenin plasmid and infected with CVB3 for 6 h. Results demonstrated that CVB3 infection upregulated the transcription of these two genes, indicating that CVB3 infection induces activation of Wnt signaling. Overexpressing γ-catenin, however, decreased transcription of both c-myc and MMP9 compared to their respective vector-transfected cells . These rTo determine the effect of γ-catenin on viral replication, we transfected HeLa cells with a plasmid expressing myc-tagged γ-catenin for 36 h and then infected with CVB3. At 4 h pi, the cell culture was divided into two parts. One part was used to extract total RNAs for qPCR measurement of CVB3 RNA using primers targeting the CVB3 2A gene. The other part was used for (i) Western blotting analysis of CVB3 VP1 using total proteins, and (ii) plaque assay of viral particle formation using culture supernatants. Results showed that γ-catenin overexpression dramatically reduced the levels of 2A RNA , VP1 proTo further determine the mechanism by which γ-catenin inhibits CVB3 replication, we selected several mediators in type I interferon (IFN) immune responses for further investigation. We treated cells with γ-catenin siRNA or scrambled siRNA before CVB3 infection and then isolated the cellular RNAs to measure the transcription levels of IFNB1, MDA5 (melanoma differentiation-associated gene 5), MAVS  and ISG15 (interferon-stimulated gene of 15 kDa) using qPCR. Data demonstrated that silencing γ-catenin significantly decreased the CVB3-activated transcription of IFNB1  and its We found that both DSC-2 and DSG-2 were cleaved during CVB3 infection. These cleavages likely cause a domino effect, resulting in the destruction of other partner proteins, such as γ-catenin, in the desmosome complex. We speculated that cleavage of DSC-2 or DSG-2 led to the release of γ-catenin into the cytosol, where it is quickly degraded through the ubiquitin proteasome pathway. This speculation was verified by using proteasome inhibitor MG132. We further found that γ-catenin reduction benefited Wnt/β-catenin signaling and enhanced viral replication by suppressing the interferon-β antiviral immune response. These findings may provide a two-fold explanation of the pathogenesis of CVB3 infection : first, Previously, it was unknown which protease(s) is/are responsible for the cleavage of DSG-2. Several proteases, such as caspase-8  and ADAMIntercalated disk component proteins not only maintain the architecture of the myocardium, but also participate in signaling transduction pathways . Thus, oγ-Catenin and β-catenin share many common interacting protein partners, such as cadherins, LEF/TCF transcription factors, and the APC/Axin degradation machinery, and these proteins can fulfill certain common functions . FurtherIn searching for the central role of γ-catenin in viral pathogenesis, we found that γ-catenin overexpression reduced CVB3 particle formation, suggesting an antiviral effect for γ-catenin. To reveal the underlying mechanism by which γ-catenin activates an antiviral effect during CVB3 infection, we focused on the type I interferon response. This is because type I IFNs are produced early during viral infection; mice deficient in type I IFN receptor display delayed CVB3 clearance and accelerated myocardial disease, and die more rapidly than wild-type mice . More imWe have provided the first evidence on how CVB3 destroys the integrity of desmosome by cleaving DSC-2 and DSG-2. The destruction of desmosomal cadherins causes γ-catenin to be released into the cytosol for further degradation via the ubiquitin-proteasome pathway. Since γ-catenin executes its anti-CVB3 function through activation of the IFN-β immune response, virus-induced degradation of γ-catenin benefits CVB3 replication, enhancing viral pathogenicity. Based on this new understanding of the underlying mechanism by which CVB3 infection causes direct damage or death of cardiomyocytes, searching for novel inhibitors to block cleavage and prevent γ-catenin degradation could be an effective strategy for therapeutic intervention to protect the integrity of the myocardium and treat viral myocarditis.All datasets generated for this study are included in the article/The animal study was reviewed and approved by the Animal Care Committee, Faculty of Medicine, University of British Columbia. Animal work was conducted by strictly following the Guide to the Care and Use of Experimental Animals – Canadian Council on Animal Care.DY, XY, and GZ conceived the study. GZ, HZ, and YQ conducted the experiments and data analyses. GZ and DY wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


These effects were maintained for 10 weeks after the mice stopped exercising. Our study is the first report that exercise reduces the expression of senescence-related genes in the visceral adipose tissue of young mice, and that exercise causes the legacy effect.Obesity has become a global medical problem. The upregulation of senescence-related markers in adipose tissue may cause impairment of adipose tissue and disorders of systemic metabolism. Weight control through diet has been found to ameliorate senescence in the adipose tissue. Exercise is also important in maintaining a healthy lifestyle, however, very few researchers have examined the relationship between senescence-related markers in adipose tissue. Dietary restriction is also reported to have a legacy effect, wherein the effects are maintained for some periods after the termination of the intervention. However, very few researchers have examined the relationship between exercise and senescence-related markers in adipose tissue. Besides, there is no study on the long-term effects of exercise. Hence, we investigated whether the exercise could change the expression of senescence-related genes in the visceral adipose tissue of young mice and whether there was a legacy effect of exercise for 10 weeks after the termination of exercise. Four-week-old male ICR mice were assigned to one of the three groups: 20 weeks of sedentary condition, 20 weeks of voluntary wheel running exercise, or 10 weeks of exercise followed by 10 weeks of sedentary condition. The mice showed decreased expression in genes related to senescence and senescence-associated secretory phenotype, such as  Imbalanced energy intake and eating disorders, induced by genetic and environmental factors, can cause overweight and even obesity . ObesityRecently, Schosserer et al.  reviewedTrp53 in mice), p21 , and p16  [ob/ob mice. Later, several groups reported that senescence-related markers were increased in the white adipose tissue of genetically obese Ay mice, high-fat diet-induced obese mice and rats from young, 8 weeks old, to middle age, 10 months old [Cellular senescence is the irreversible arrest of cell growth, characterized by the senescence-associated secretory phenotype (SASP) and the staining of senescence-associated β-galactosidase  in cells ,5. Senesnths old ,10,11.MDM2, the upstream inhibitor of p53, in white adipose tissue led to metabolic disorders, including type 2 diabetes. On the other hand, the metabolic disorders were alleviated in the white adipose tissue of young, MDM2 and p53 double knockout mice aged 3–12 weeks. Further, Minamino et al. [p53 knockout Ay mice. Moreover, Homayounfar et al. [The upregulation of senescence-related markers in adipose tissue also causes the impairment of adipose tissues and disorder of systemic metabolism even in young animals; disrupting the senescence-related markers could reduce the disorder’s severity. For example, Liu et al.  showed to et al.  reportedr et al.  reportedp53, p21, IL-6, and MCP-1 genes in the visceral adipose tissue in the mice on a high-fat, fast-food diet. Meanwhile, exercise had no effect on the normal diet groups.On the other hand, weight control appears to ameliorate senescence in adipose tissue. Ishaq et al.  reportedTherefore, the effects of exercise on the expression of senescence-related genes in the visceral adipose tissue of normal young mice need to be elucidated further. Here, we used microarray analysis to compare the gene expression in normal young mice under sedentary control to those under a voluntary wheel running exercise regimen. Using the gene ontology (GO) analysis and the gene set enrichment analysis (GSEA), we found significant differences in a senescence-related gene set in the visceral adipose tissue between these two conditions . On the other hand, some dietary interventions reported metabolic memory or legacy effect, wherein the effects were maintained for some periods after the intervention’s termination ,22,23,24However, there is no study on the long-term effects, or the legacy effects, of exercise on the expression of senescence-related genes in adipose tissue. Therefore, we investigated whether the exercise could change the expression of senescence-related genes in the visceral adipose tissue of normal young mice and whether the effects of exercise could be maintained for 10 weeks after the exercise’s termination.Male ICR mice were purchased from Japan SLC Inc. . At 4 weeks of age, the mice were divided into three groups according to each group’s comparable mean averaged body weight. The groups were randomly assigned to one of the following 3 groups, 20-week normal control sedentary condition (Co-Co) group (n = 8), 20-week voluntary wheel running exercise (Ex-Ex) group (n = 8), or 10-week exercise followed by 10-week sedentary condition (Ex-Co) group (n = 8). The Ex-Ex and Ex-Co cages included freely accessible running wheels  with digital revolution counters. All mice groups were fed a regular control diet of 11.5% fat, 60.9% carbohydrate, 27.6% protein per kcal , and water ad libitum. All animals were housed individually at 24.5 °C ± 0.1 °C, with 50.3% ± 0.3% humidity and exposed to a 12:12-h light-dark cycle with lights from 8:00 to 20:00. Animal care and experimental procedures were conducted in accordance with the guidelines of Keio University and were approved by the laboratory animal committee .The mice’s body weights were measured once a week during the 20-week experimental period. At the end of the experimental period, the 24-week-old mice were euthanized by inhaling 1–5% isoflurane. Blood was collected immediately from the abdominal vena cava inferior. Abdominal fat tissue samples, epididymal and retroperitoneal fats, were collected and weighed immediately after euthanasia. We also calculated the sum of these fat-pad weights  × 2 + retroperitoneal fat-pad weight (g) × 2). For mRNA measurement, small pieces of epididymal fat tissue were soaked overnight with the RNA stabilization Solution  at 4 °C. After removing the solution, the specimens were stored at –80 °C before analysis.Total RNA was isolated from the homogenized mouse epididymal fat tissue with the QIAzol Lysis reagent  and purified using the RNeasy Lipid Tissue Mini kit  according to the manufacturer’s instructions.In each group, we chose one mouse whose body weight was closest to the group’s average body weight. Microarray analysis was conducted by Biomatrix Laboratory . The quantity and quality of the extracted total RNAs were assessed by an ND-1000 spectrophotometer  and a 2100 Bioanalyzer , respectively. The total RNAs were then reverse transcribed into fluorescent cRNAs labeled with Cy3 using the Low Input Quick Amp Labeling Kit . The labeled RNAs were hybridized onto a SurePrint G3 Mouse GE 8×60K Microarray using the Gene Expression Hybridization Kit . The fluorescent signals were scanned with an Agilent Microarray Scanner and analyzed using the Feature Extraction software . Each spot’s raw data was normalized by subtracting with the blank spots’ mean background signal. The relative gene expression levels were calculated by comparing the signal intensity of the valid spots from the microarray experiments using the GeneSpring GX v11.5.1 software package .http://david.abcc.ncifcrf.gov/) for comprehensive functional annotation. In this study, we performed GO analysis in the DAVID database to identify statistically significant data (p < 0.05) using the genes with a significant change in expression, i.e., more than 2.0- or less than 0.5-fold change compared to the Co-Co condition, from microarray analysis data .Additionally, GSEA was also performed to obtain functional annotation and interpretation using all the gene expression data from the microarray analysis. Based on the size of the set, a normal enrichment score was evaluated for each gene set; FDR and nominal Total RNA was reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit  and the thermal cycler . Quantitative real-time PCR was performed using the PowerUp SYBR Green Master Mix  in accordance with the manufacturer’s instructions. Quantitative analyses were performed using the CFX96 Real-Time System . The primer sequences for each of the genes analyzed by quantitative real-time PCR are shown in p < 0.05 was considered statistically significant.All data are expressed as mean ± SEM. The differences between groups were analyzed statistically using one-way analysis of variance. Post hoc analysis was performed using Fisher’s Least Significant Difference test when significant main effects were observed. Differences in the running distance between both exercising groups were statistically analyzed using the Student’s t-test. For all tests, p < 0.001), and the Ex-Co group, at 42.5 ± 0.6 g (p < 0.001), than that in the Co-Co group, at 53.2 ± 2.5 g. There was no significant difference in body weights between the Ex-Ex and Ex-Co groups. At week 20, the body weights were significantly lower in the Ex-Ex group, at 46.7 ± 1.3 g, than the Co-Co group, at 62.9 ± 3.1 g (p < 0.001), and the Ex-Co group, at 58.2 ± 2.4 g (p < 0.01). The Ex-Co group’s body weight was reverted to the Co-Co group’s body weight, and no significant difference was found between the groups. Total distance run by the mice was 1,777 ± 142 km, or 12.7 ± 1.0 km/day and 780 ± 55 km, or 11.1 ± 0.8 km/day in the Ex-Ex and Ex-Co group, respectively (p < 0.001).The change in the mice’s body weight over time was measured (p < 0.01 for both comparisons); no significant difference was found between the Co-Co and Ex-Co group. The weight of the Ex-Ex group’s retroperitoneal fat-pads was also significantly lower than those of the Co-Co and Ex-Co groups (p < 0.001 for both comparisons); no significant difference was observed between the Co-Co and Ex-Co group. We also calculated the sum of these fat-pad weights  × 2 + retroperitoneal fat-pad weight (g) × 2). The sum of these fat-pad weights of the Ex-Ex group was significantly lower than that of the Co-Co and Ex-Co group (p < 0.001 for both comparisons), but no significant difference was found between the Co-Co and Ex-Co group.The weights of the epididymal or retroperitoneal fat-pads or their sum were measured . The weiadiponectin and leptin, and the major glucose transporter in adipose tissue, Glut4, were investigated (adiponectin mRNA expression was significantly higher than that of the Co-Co and Ex-Co groups (p < 0.05 for both comparisons), but no significant difference was found between the Co-Co and Ex-Co group. On the other hand, the Ex-Ex group’s leptin mRNA expression was lower compared to those of the Co-Co and groups (p < 0.05); but no significant difference was found between the Co-Co and Ex-Co group. The Glut4 mRNA expression of the Ex–Ex group was significantly higher than that of the Co-Co, and Ex-Co groups , but no significant difference was found between the Co-Co and Ex-Co group.The changes in the mRNA expression of adipokine-related genes, stigated . The Ex-p53, p21, and p16, were investigated , but there was no significant difference between the Ex-Ex and Ex-Co groups. On the other hand, the Ex-Ex group’s p21 mRNA expression was significantly lower than that of the Co-Co group (p < 0.01). The Ex-Co group’s p21 mRNA expression showed the tendency to revert to the Co-Co group condition, and therefore, there was a significant difference between the Ex-Ex and Ex-Co groups (p < 0.05).The changes in mRNA expression of senescence-related genes, such as stigated . The p53TNF-α, IL-1β, and IL-6, were examined (TNF-α mRNA expression of the Ex-Ex group was significantly lower than that of the Co-Co group (p < 0.001). The TNF-α mRNA expression of the Ex-Co group, showed the tendency to revert to the Co-Co group condition (p = 0.051 between the Ex-Ex and Ex-Co groups and p = 0.079 between the Co-Co and Ex-Co groups). The IL-6 mRNA expression of the Ex-Ex and Ex-Co group was significantly lower than that of the Co-Co group , but there was no significant difference between the Ex-Ex and Ex-Co group. The IL-1β mRNA expressions of the Ex-Ex and Ex-Co groups showed the tendency to decrease compared to the Co-Co group, but no significant difference between the three groups.The changes in mRNA expression of the genes associated with SASP, i.e., examined . The TNFThis study was designed to clarify the effects of exercise on the expression of senescence-related genes in the visceral adipose tissue. This study also aimed to study the presence of a maintenance period of this effect after exercise was stopped.p53, p16, and IL-6 in response to exercise. The effect of exercise also appeared long-lasting, as the reduced level of the genes was maintenance 10-week after exercise was stopped. These mice had reverted to the normal condition as almost the same as the sedentary control mice in body weight, abdominal fat-pad weight, and gene expressions related to adipokine and glucose metabolism. This study is the first to report that exercise ameliorates the expression of senescence-related genes in adipose tissue and that this effect is maintained for a period after stopping exercise.This study found a significant decrease in the expression of the senescence and SASP- related genes, i.e., the mRNA level of p53, p21, and IL-6 in visceral adipose tissues using diet-induced obese mice. Recently, many articles have reported that the expression and activation of the senescence-related marker genes were elevated in obese adipose tissue. The p53 and p21 expression in adipose tissue were increased in young, 12-week-old, obese ob/ob mice [Ay mice aged 20 weeks [ob/ob or diet-induced obese mice and rats, aged 8 weeks to 10 months, were reported in several articles [In this study, a 20-week voluntary wheel running exercise was shown to decrease body weight and visceral adipose tissue significantly compared to the sedentary control; the mRNA expressions of the senescence-related genes were significantly decreased concomitantly. These results are consistent with the findings of Schafer et al. [20 weeks . Similararticles ,10,11,25articles .Ay/+ and B6.V Lep ob/ob, and high-fat diet-induced obese mice, M1-polarized proinflammatory macrophages and inflammatory cytokines, such as TNF-α, IL-6, and iNOS, were increased in the perigonadal adipose tissue [KKay mice has severe oxidative stress and reactive oxygen species (ROS), such as an increased level of H2O2 production, TBARS, and NADPH oxidase subunits, and a decrease in Cu-, Zn-SOD, GPx, and Catalase [It is well known that obese adipose tissue has inflammation and oxidative stress. In genetically obese mice, B6.Cg e tissue . The adiCatalase . HypoxiaCatalase .ob/ob and high-fat diet-induced obese mice.Wu et al.  reportedp16 mRNA seems to be one of the most sensitive and reliable senescence-related genes [p16 mRNA expression was increased substantially in various tissues compared to p21 mRNA expression. Several articles reported that the elimination of p16-positive cells from several tissues alleviated aging-related diseases and promoted longevity [According to previous articles, the ed genes ,33,34,35ed genes  reportedongevity ,33,34,35p53, p21, p16, and IL-6 mRNA expressions between mice under sedentary control conditions and mice under a wireless wheel running exercise regimen. Although they did not show details of this running device, the daily and total running distance of their mice were almost half of the mice in the Ex-Ex group in our study. Furthermore, the mice in the exercise group in their study did not show any significant decrease in the weight of the visceral fat-pads. In our experiment, the mice in the exercise groups showed robust decreases in the weight of their visceral adipose fat-pads compared to the sedentary control mice  gene in adipose tissues. These data suggest, in our study, the continued decrease in the expression of p16 and other senescence-related genes for a period after the termination of exercise might be induced by DNA methylation induced by exercise in our study.However, the detailed mechanisms underlying these exercise or dietary restriction-induced legacy effect or metabolic memory have not been studied in these papers. Ceriello  discussep16 mRNA expression, in the Ex-Co group tended to revert to the level in the sedentary control group. Hence, the most important thing about exercise is to continue it for as long as possible.We think that further investigations are needed to determine the duration of this exercise-induced legacy effect and whether the changes in gene expression could lead to protein synthesis and the body’s systemic function in the near future. Lastly, almost all the parameters, except We investigated whether the exercise could change the expression of senescence-related genes in the visceral adipose tissue of normal young mice and whether the effects of exercise could be maintained for 10 weeks after the termination of exercise. We found that exercise could lessen the expression of senescence-related genes in murine visceral adipose tissue, and these effects might be maintained for a while after stopping exercise. The mechanisms of exercise-induced legacy effects should be investigated further.


Cadherins play an important role in tissue homeostasis, as they are responsible for cell-cell adhesion during embryogenesis, tissue morphogenesis, differentiation and carcinogenesis. Cadherins are inseparably connected with catenins, forming cadherin-catenin complexes, which are crucial for cell-to-cell adherence. Any dysfunction or destabilization of cadherin-catenin complex may result in tumor progression. Epithelial mesenchymal transition (EMT) is a mechanism in which epithelial cadherin (E-cadherin) expression is lost during tumor progression. However, during tumorigenesis, many processes take place, and downregulation of E-cadherin, nuclear β-catenin and p120 catenin (p120) signaling are among the most critical. Additional signaling pathways, such as Receptor tyrosine kinase (RTK), Rho GTPases, phosphoinositide 3-kinase (PI3K) and Hippo affect cadherin cell-cell adhesion and also contribute to tumor progression and metastasis. Many signaling pathways may be activated during tumorigenesis; thus, cadherin-targeting drugs seem to limit the progression of malignant tumor. This review discusses the role of cadherins in selected signaling mechanisms involved in tumor growth. The clinical importance of cadherin will be discussed in cases of human and animal cancers. Cadherins are transmembrane glycoproteins responsible for cell-cell adhesion and maintenance of normal tissue architecture. In addition, cadherins have been found not only between the tumor cells but also in body fluids (mainly in blood) ,2,3. TheThe structure of cadherin is well known and has been extensively reviewed by others ,3,4,5. BThe role of cadherins in the process of cancer development has been studied widely over the last decades ,10,11,12The purpose of this review is to discuss the role of cadherins in the tumor growth and its clinical importance.Cadherins preferentially bind to catenin proteins that are found in three subtypes, α-catenins, β-catenins, and p120ctn family catenins. A juxtamembrane region of cadherins, which is proximal to the cell membrane, binds to p120ctns, while the distal region called catenin-binding domain binds to β-catenins. Monomeric forms of α-catenins associate with the cadherin-catenin complex through β-catenins, while homodimeric forms of α-catenins do not bind β-catenins, but F-actin . The conCadherins, together with catenins, interact through the AJs with actin cytoskeleton and regulate cellular processes during embryonic development as well as during neoplastic transformation and progression. In cancer, any disturbances in cell-cell and cell-matrix adhesion are related to tumor progression and allow cancer cells to become more motile, degrade the extracellular matrix, enter the blood vessels and form distant metastases ,30. Fz, LRP6, Axin2, Naked, Dkk1, and Rspo, resulting in positive or negative regulation by TCF/β-catenin [The signaling activation takes place at the site of cadherin-catenin interaction. Thus, the catenin-binding domain of cadherin is crucial in cadherin function and it plays an important role in maintaining epithelial integrity. For example, phosphorylation of either E-cadherin or β-catenin affects β-catenin binding to cadherin, while phosphorylation of β-catenin may also lead to α-catenin binding to the cadherin-β-catenin complex . During -catenin ,35,36,37Furthermore, p120-catenin, which interacts with the cadherins through the juxtamembrane domain is regulated by tyrosine kinases and modulate cadherin intracellular trafficking, stability, adhesive capacity and motility . p120 caMany studies have shown the role of β-catenin as a proto-oncogene in human cancer ,31. MutaEpithelial-mesenchymal transition (EMT) is a pivotal process of morphogenesis whereby epithelial cells acquire the mesenchymal phenotype. EMT is known as the phenomenon of loss of E-cadherin expression during cancer progression . EMT occκB activity, which leads to inflammation-associated carcinogenesis [In carcinomas, EMT can be initiated and promoted by many oncogenic signaling pathways, including hypoxia and signals of the tumor microenvironment . Subsequogenesis . There iogenesis . A recenogenesis . The othogenesis . HoweverCadherin switching is primarily the result of transcriptional regulation of cadherin expression through several factors, such as SNAI1, SLUG, TWIST, ZEB1 and ZEB2 ,62. ThesThe protein p120 catenin is another important factor that plays a role in the regulation of downstream signaling and cell behavior upon cadherin switch ,71. p120In humans, the loss of E-cadherin expression during EMT is associated with tumor development. However, decreased expression of E-cadherin has also been observed in canines with more aggressive mast cell tumors (Grade 3) . SimilarCadherin expression has been widely studied in human cancers as well as in animal models ,10,21. EP-cadherin is expressed in myoepithelial cells, and its overexpression in high-grade tumors is correlated with tumor aggressiveness and a poor prognosis in humans . SimilarN-cadherin (the product of the CDH2 gene) influences the nervous system, brain, heart, skeletal muscles, blood vessels and hematopoietic microenvironment function ,90,91. NThe most frequently evaluated cadherin is E-cadherin . This adIn dogs, reduction in E-cadherin expression, like humans, is associated with increased tumor size, high histological and invasion grades, lymph node metastasis and a high mitotic index . AnotherUnderstanding how cadherins influence the cell behavior can be used to design possible therapeutic interventions to regulate its activity and prevent tumor cell growth, invasion and metastasis ,114. Solanum nigrum Linn.), which stimulates E-cadherin expression and reduce vimetin expression  leading to the suppression of EMT. At the same time α-solanine downregulates matrix metalloproteinase (MMP) expression, which plays a key role in metastatic process; therefore, it is has a strong anti-carcinogenic activity [A good example is α-solanine . In human breast, hepatocellular and prostate cancer hypermethylation of the CDH1 promoter has been observed ,125. It Cadherins play an important role in tissue homeostasis and dysfunction. Destabilization of cadherin-catenin complex may result in tumor progression. Cadherins show varied biological functions. The loss of E-cadherin expression during EMT in humans is associated with tumor development and worse prognosis. For these reasons, many studies have shown that E-cadherin can act as tumor suppressor; however, more recently, a role in the cancer progression has been also described.Many oncogenic signaling pathways modify cadherin cell-cell adhesion. The most commonly described are cyclin kinase inhibitor p27-mediated signaling, MAPK, Ras, Rac1 signaling, PI3K/AKT signaling, as well as Hippo signaling. Abnormal cadherins expression is connected to metastasis, angiogenesis, adhesion and invasion.Higher expression of N-cadherins is related to tumor aggressiveness, cancer metastasis, apoptosis and angiogenesis in many human and animal cancers.Overexpression of P-cadherin is also usually a bad prognostic factor, as it is related to tumor progression and invasion and shorter overall survival.Overall, these adhesion molecules are novel promising targets in cancer treatment, but they may also be useful in predicting a patient’s prognosis in human and veterinary medicine.