Genetic diversity was measured by estimating Nei’s population genetic distance using 15 reproducible bands from random amplified polymorphic DNA (RAPD) genotyping. Results: Principal component analysis (PCA) of the secoiridoid concentrations clustered samples according to cultivar. Clustering based on genetic distances is not concordant with phenolic clustering. A cultivar effect was also demonstrated in the association between the concentration of individual phenols with DPPH reducing activity. Conclusions: Taken together, the study shows that the olive oil phenolic content defines “cultivar-specific phenolic profiles” and that environmental factors other than agronomic conditions contribute more to phenotype variance than genetics.Background: The phenolic fraction of extra virgin olive oil (EVOO) has disease preventive and health-promoting properties which are supported by numerous studies. As such, EVOO is defined as a functional food. The aim of the present study was to characterize the phenolic profile of olive oil from cultivars farmed in the Ionian Islands  and to investigate the association of phenols to antioxidant activity, which is central to its functionality. Furthermore, the study investigates whether multivariate analyses on the concentration of individual biophenolic compounds and genetic population diversity could classify the olive oil samples based on their geographic origin. Methods: Phenols were determined in 103 samples from different Ionian Island tree populations by  As s31P-NMR (nuclear magnetic resonance) and 1H-NMR spectroscopy [Numerous factors affect the phenolic concentration of extra virgin olive oil, such as olive cultivar and fruit maturation, agronomic and environmental factors, processes used for olive oil extraction, packaging conditions, storage conditions, and shelf-life ,26,27,28troscopy ,30; and Consequently, there has been an increase in the implementation of multivariate statistical methods on multiple biochemical and biophysical numerical data to identify which characteristics could serve as quality and composition identity “markers” between EVOOs from cultivars of different origin. In a previous study by our group, it was shown that discrimination of olive oil by geographical origin was possible by implementing multivariate analyses on the volatile compound profiles of olive oils extracted from the Ntopia cultivars from four different Ionian Islands . The aimOlea europaea during the early harvest period (October) of the year 2017. Each of the 103 samples corresponds to one extraction from multiple immature, healthy, green, and firm fruits (monocultivar EVOO) from 35 Lefkada Asprolia, 22 Zakynthos Ntopia, 26 Kefalonia Ntopia (13 old and 13 recent trees), and 20 Kerkyra Lianolia trees , Lefkada , Kefalonia , and Zakynthos  . Tree ager trees . Region,er trees .Fruit samples were stored in open containers at 4 °C and transferred within 24 h to the laboratory. Only mechanical methods were used to obtain olive oil in the laboratory. In brief, olives were crushed, pitted, ground, and malaxed for 30 min, as previously described . Caution3 and 550 μL of the solution was transferred to a 5 mm NMR tube.A quantity of 5.0 g of olive oil was mixed with 20 mL cyclohexane and 25 mL acetonitrile. The mixture was homogenized by a vortex mixer for 1 min and centrifuged for 5 min at 4000 rpm. 25 mL of the acetonitrile phase was collected and mixed with the internal standard consisting of 1.0 mL of a syringaldehyde solution (0.5 mg/mL) in acetonitrile, and evaporated under reduced pressure using a rotary evaporator . The residue was dissolved in 750 μL CDCl1H NMR spectra were recorded at 400 MHz using a Bruker 400 NMR spectrometer. Typically, 50 scans were collected into 32 K data points over a spectral width of 0–16 ppm with a relaxation delay of 1 s and an acquisition time of 1.7 s. Prior to Fourier transformation (FT), an exponential weighting factor corresponding to a line broadening of 0.3 Hz was applied. The spectra were phased, corrected, and integrated automatically using TOPSPIN. When necessary, accurate integration was performed manually for the peaks of interest. Standard solutions and calibration curves were prepared as previously described in Dimantakos et al. [s et al. . Raw valF, A1, A2, t1 and t2):The antioxidant activity of the olive oil samples was determined via measurement of their capacity to reduce the free radical DPPH , as adapted recently ,34. Thusf is the final DPPH absorbance value which is expected to be attained after an “infinite” amount of time which allows for expression of the total antioxidant activity of the olive oil sample, A1 and A2 are the amplitudes of each of the two exponentials, while t1 and t2 are their corresponding time constants. Experiments were repeated three times in a Shimadzu UV 2100 UV-VIS spectrophotometer at room temperature. DPPH and Trolox were obtained from Sigma-Aldrich,  and ethyl acetate was purchased from Merck .In Equation (1), I(t) is the DPPH absorbance at 517 nm at time t, IRaw values are presented in 2O and sample concentration and purity were determined in a UV spectrophotometer by measuring absorption at 260 nm and calculating the 260/280 and 260/230 ratios. The samples were stored at −20 °C until further processing.DNA was extracted by ethanol precipitation, as previously described . BrieflyO. europaea cultivar genome, 5 decamer oligonucleotide sequences were selected from the Eurofins Genomics 10mer RAPD kits v.02.10.2014 . Selection was based on their ability to amplify polymorphic alleles in O. europaea, as previously reported [http://www.gelanalyzer.com/?i=1 (accessed on 15 June 2019)).To screen for polymorphic alleles across the reported  (Supplem0.5 and Nei D (Nei genetic Distance) = −Ln(I), where Jx and Jy are the sum of the squared allele x and allele y frequencies, respectively, across populations, and Jxy is the sum of the product of the squared allele frequencies across populations. All calculations were performed by implementing the allele frequency calculation functions of the GenAlEx v6.51b2 excel add-in for haploid binarized genotypes [p-values are calculated through two types of tests: multiscale bootstrap resampling which calculates the AU (Approximately Unbiased) p-value and a p-value returned by normal bootstrapping. The p-value of a cluster is a value between 0 and 1, indicating the strength of cluster support by the data.The magnitude of genetic similarity between the cultivar varieties (populations) was estimated by calculating Nei’s genetic distance estimators . Of the enotypes . The binenotypes , where pp < 0.05. A principal components analysis (PCA) was implemented to identify which variables, i.e., individual phenolic compounds, contribute the most to the variance between cultivars, i.e., which combinations of variables can discriminate the cultivar varieties, and also identify putative correlations between the variables (redundancy in discriminating between the cultivar varieties). PCA was run on scaled data (standard deviation = 1 and mean = 0) by invoking the prcomp R v4.0.3 (2020-10-10) built-in function. Functions of the factoextra v1.0.6 and the ggplot2 v3.3.3 [Differences in the composition of the phenolic compounds between cultivars were tested with a one-way analysis of variance (ANOVA) and Tukey’s post hoc test for multiple comparisons. Statistical significance was set at 2 v3.3.3 , R v4.0.p = 0.05 [Pearson’s r was calculated to test the effect of the concentration of individual phenolic compounds on antioxidant activity per cultivar group. For all statistical tests the level of significance was set at p = 0.05 .The analyzed olive oil samples were produced from monocultivar extractions, where each cultivar represents a distinct geographical region with the exception of Kefalonia _Ntopia, which is divided into two subgroups (populations) based on tree age . The forThe reverse trend was obtained for the rest of the secoiridoid derivatives under investigation where the Asprolia _Lefkada variety had the highest mean concentration values a. Trend Based on the above, and since in the present study the harvest, ripening stage, and olive oil extraction conditions were maintained as identical for the different island cultivars, we attributed the observed phenolic compound concentration trends to a “variety/cultivar effect”. To further investigate the cultivar effect on the concentration of individual phenols, we performed one-way ANOVA with Tukey’s tests for pairwise comparisons. The significantly different tests per secoiridoid derivative and antioxidant activity are summarized in We also observed that based on the pairwise tests two groups of phenolic substances could be formed. The first group is formed by oleomissional and ligstroside aglycon (number of significant tests = 4) and the second is formed by oleokoronal and oleuropein aglycon (number of significant tests = 5) . In specAltogether, our observations are in line with the proposed cultivar differences described by Diamantakos et al. , where tPhenolic compounds act as antioxidants though radical scavenging, hydrogen atom transfer, and metal-chelation. Antioxidant activity has been shown to differ between olive oils produced from different cultivars . Based oTaken together, our results indicate that differences in the phenolic content are more likely to allow cultivar classification than the antioxidant activity alone.x-axis on the PCA biplot  explains 70.1% and is driven by the difference between Lefkada_Asprolia and Kerkyra_Lianolia, illustrated as the most distant clusters on the A biplot a. The fiup means a indicatup means b. Intereup means b. TherefThe same analytical approach has been applied in Italian cultivars by Losito et al. , where tSince the multivariate analysis demonstrates that cultivars can be distinguished based on the concentration of intermediates in the biochemical transformation towards oleocanthal and oleacein, we next investigated the effect of genetics on cultivar phenotypic diversity. To do so, we analyzed a subset of the dataset comprising 61 samples with complete genetic and biochemical data.p values  in the phenolic compound concentrations in EVOOs from those trees. In addition, our results also indicate that the concentration of olive oil phenolics is a phenotype that is greatly influenced by the environment, decreasing the proportion of variation explained by genetics (heritability). Moreover, quantitative traits are complex and therefore controlled by interactions between multiple genes and the environment. In the livestock breeding sector, field and herd management is significantly optimized to reduce environmental variation and improve the estimation of heritability. The advent of innovative high-throughput genotyping technologies such as genotyping-by-sequencing has greatly improved the identification of genetic markers that associate with specific traits . These t infancy ,48,49 anThe present study represents the first investigation of the phenolic content in olive oils produced from Ionian Island cultivars under tightly controlled agronomic conditions. Multivariate analyses showed that the olive oil phenolic content defines “cultivar-specific phenolic profiles” and can be further investigated as a classifier by expanding the repertoire of measured secoiridoids. Interestingly, antioxidant activity was not stably correlated with a specific phenolic compound across cultivars, likely reflecting the effect of the cultivar on the complexity of the biochemical mechanisms mediating free radical DPPH reduction. This finding has implications for the development of methods to identify EVOOs with high antioxidant activity so that its functionality can be stably preserved. Finally, in accordance with previous reports, RAPD genotype-based cultivar classification did not reflect phenolic profile sample clustering. This highlights the need to develop novel and high-throughput genotyping platforms and to optimize agronomic practices to reduce environmental variation. This will allow quantitative trait association mapping and, ultimately, marker assisted EVOO classification.


The current study was designed to investigate the effect and underlying mechanism of warm acupuncture combined with bone marrow mesenchymal stem cells (BMSC) transplantation on cartilage tissue injury in rabbit knee osteoarthritis (KOA). In the study, 50 rabbits were randomly divided into 5 groups: blank group, KOA group, warm acupuncture group, BMSCs group, and warm acupuncture combined with BMSCs group. After warm acupuncture combined with BMSCs, the Modified Lequesne MG knee joint assessment scale was used to evaluate the degree of knee joint behavior, the Taiping Peng method generally observed the histomorphology changes of KOA rabbit cartilage, and hematoxylin-eosin staining, safranin O green staining, and toluidine blue staining were conducted to evaluate the extent of cartilage tissue pathology. Furthermore, transmission electron microscopy and TUNEL staining were used to observe cell apoptosis, and immunohistochemistry and qPCR analysis were used to detect the expression of apoptosis-related proteins and mRNA. Results showed that administration of warm acupuncture combined with BMSCs recovered the joint function and significantly decreased Lequesne MG score. The degree of cartilage tissue pathological damage has been improved, cartilage ultrastructure degeneration has recovered, peripheral blood vessels have mild edema, blood supply has gradually recovered, and even small amounts of red blood cells have appeared. In addition, warm acupuncture combined with BMSCs treatment suppressed chondrocyte apoptosis in rabbits with knee osteoarthritis by reduced TUNEL-positive chondrocytes and simultaneously reversed the mRNA expression of Bax, Bcl-2, and Caspase-3. These results indicate that warm acupuncture combined with BMSCs transplantation has a potential protective effect on rabbit KOA, which may be mediated by inhibiting chondrocyte apoptosis. Knee osteoarthritis (KOA) is a disease that causes articular cartilage tissue damage and joint dysfunction due to abnormal apoptosis of the chondrocyte . With thThe articular cartilage regeneration ability of the knee joint is weak and cannot be repaired by itself after pathological changes occur. If not treated in time, the degree of articular cartilage damage will gradually worsen, and joint function may even be lost. Excessive apoptosis of chondrocytes is an important reason for the degeneration of articular cartilage into osteoarthritis –6. UnderBone marrow mesenchymal stem cells (BMSCs) have the characteristics of stromal cells and secrete a variety of cytokines –12. The In the past few decades, traditional Chinese medicine has been widely used in the clinical treatment of KOA due to its diverse treatment methods, positive curative effects, few side effects, and low cost. Warm acupuncture as a commonly used clinical treatment of KOA, mainly used in the treatment of early and middle KOA, has the advantages of simple operation, few side effects, and positive short-term and long-term effects . In our On the basis of the above, it can be hypothesized that warm acupuncture combined with BMSCs might exert protective properties on KOA. To verify this hypothesis, this experiment investigates the potential protective effect of warm acupuncture combined with BMSCs transplantation on KOA cartilage tissue and proposes that this protective effect may explore new clinical treatment methods for KOA by inhibiting cell apoptosis.A selection of 50 six-month-old New Zealand rabbits  weighing 2.5–3 kg was provided from the Experimental Animal Center of Ningxia Medical University (certificate number: SCXK Ningxia 2015-0001). The animals were maintained under effectively fixed conditions of temperature (22–24°C), humidity (40–70%), photoperiod (12 h/12 h light/dark cycle), and food and water available ad libitum. The experiment was carried out in accordance with the current guidelines for the care of laboratory animals in Ningxia Medical University. All researchers who performed animal testing were blind.Rabbit bone marrow mesenchymal stem cells (RBXMX-01001) were purchased from Saiye ; fetal bovine serum (FBS) (10270-106) was purchased from Gibco ; DMEM (SH30022.01B) was purchased from HyClone (USA); the TUNEL assay kit (11684795910) was purchased from Roche (Switzerland); 0.25% trypsin-EDTA (C0201), penicillin-streptomycin liquid (C0222), L-glutamine (C0235), and DAPI staining solution (C1006) were purchased from Beyotime Biotechnology ; anti-Bax (ab270742), anti-Bcl-2 (ab250782), and anti-Caspase-3 (ab32351) antibodies were purchased from Abcam ; 10% goat serum (G9023) was purchased from Sigma-Aldrich ; the hematoxylin-eosin staining kit (G1120), modified safranin O-fast green FCF cartilage stain kit (G1371), toluidine blue O cartilage stain solution (G2543), EDTA decalcifying solution (E1171), and paraformaldehyde 4% (E1110) were purchased from Solarbio Science & Technology Co., Ltd. ; RNAprep Pure Tissue Kit (DP431) was purchased from Tiangen Biotech ; and RT-Master Mix (639505) was purchased from Takara Bio .The experimental protocol used is illustrated in μg/mL streptomycin were added. BMSCs were stored in a 37°C saturated humidity 5% CO2 incubator. BMSCs were identified and cultured to maturity, and all available bone marrow mesenchymal stem cells were collected for injection therapy [Rabbit BMSCs were purchased from Cyagen Biotechnology Limited Company , and all procedures were approved by the Ethics Committee of the General Hospital of Ningxia Medical University . Briefly, stem cells are isolated from the internal thread cryogenic vials and cultured in Dulbecco's modified Eagle's medium (DMEM), and 10% fetal bovine serum, 1 × insulin-transferrin-selenium, 2 mm L-glutamine, 100 U/mL penicillin, and 100  therapy ; the res6/mL, the cell suspension was separated into a 1 mL syringe for reserve [Normal saline (NS), trypsin, and complete medium were preheated to 37°C during the preliminary experiment. The third-generation BMSCs cultured in vitro were digested with trypsin and observed under an inverted microscope. When the cells retracted to a round shape, the wall of the culture bottle was tapped to make the cells fall off. The cell suspension was collected, followed by centrifugation at 1000 r/min for 3 min, and the supernatant was suspended with NS. After adjusting the cell concentration to 1 × 10 reserve , 24.The rabbit model of cartilage degeneration in KOA was reproduced by plaster immobilization in the right hind limb extension position for 6 weeks , 26, and  (I) Local pain stimulus response   (II) Gait change   (III) The range of motion of the joint   (IV) Degree of joint swelling Rabbits in each group were assessed by the Lequesne MG scale scoring system on the second day after successful modeling and the first day after the end of treatment to determine the changes in knee joint status and behavior . The scoμm sections made with a microtome . The sections were stained with hematoxylin and eosin (HE) staining, safranin O green FCF staining, and toluidine blue staining, and transmission electron microscopy (TEM) analysis was carried out.The cartilage was fixed with 4% ice-cold paraformaldehyde for 2 days and then immersed in EDTA decalcification solution for 4 weeks after removal. The cartilage pieces were dehydrated and embedded in paraffin, successive 5 The paraffin slices were dried at 70°C for 2 hours, deparaffinized, rehydrated, and stained with hematoxylin and eosin (HE), the histopathological changes of cartilage tissue were observed visually under a light microscope  (×400), and then photographs were acquired. Injuries to the articular cartilage were assessed by the Mankin score . The ManThe paraffin slices were dried at 70°C for 2 hours, deparaffinized, and rehydrated. Then slices were stained with fresh Weigert dye solution, differentiated 15 seconds with acid ethanol solution, and dyed with solid-green dye solution. The slices were washed with distilled water and soaked with safranin O dye solution. After washing with an acetic acid solution, tissue samples were deparaffinized and rehydrated by using xylene substitute and graded alcohols . The injThe paraffin slices were dried at 70°C for 2 hours, deparaffinized, and rehydrated by using a xylene substitute and graded alcohols. For toluidine blue staining, samples were immersed in toluidine blue O stain for 30 minutes and then washed thrice with distilled water for 2 minutes. After differentiating in acetone, the injured sites were visually observed through a light microscope  (×100) and images were acquired [μm semithin sections were stained with 0.5% toluidine blue. After toluidine blue staining to orientate the injury site, fragments were cut by an ultramicrotome into 75 nm thick slices. The ultrathin slices were obtained on colloid-coated copper grids and double-stained with 0.4% uranyl acetate and 2% lead acetate [The cartilage blocks (5 mm × 5 mm × 2 mm) in the right knee were collected and fixed with 2% glutaraldehyde for 2 h at 4°C and washed thrice with 0.1 M phosphate buffer every 2 hours. The samples were then postfixed with 1% cold osmium tetroxide for 2 hours and rinsed twice with 0.1 M cacodylate buffer every 15 min. Then, they were dehydrated and embedded in epoxy resin. 0.5  acetate . SubsequChondrocytic apoptosis was detected with the terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick end labeling (TUNEL) using a cell death detection kit . In short, dewaxing was performed to slices in addition to a rehydration process followed by permeation with proteinase K for 15 minutes. Then, the DNA fragmentation of apoptotic chondrocyte bound the terminal deoxynucleotidyl transferase (TdT) enzyme in a reaction buffer. Slides were mounted with DAPI  for nuclear staining after rinsing with PBS for 10 minutes at room temperature . In 6 diThe fresh paraffin slices were dried at 70°C for 2 hours, deparaffinized, and rehydrated by using xylene substitute and graded alcohols. Then slices were placed in 3% hydrogen peroxide solution for 10 minutes. After rinsing with PBS three times, 10 mol/L urea was added to the slices at 37°C for 30 minutes. After irrelevant antigens were blocked using 10% goat serum for 30 minutes at room temperature, the primary antibodies  were incubated at 4°C overnight. The surface of cartilage slices was evenly covered with DAB, stained with hematoxylin, and soaked in 1% hydrochloric acid alcohol after rinsing with distilled water for 5 min. Tissue samples were deparaffinized and rehydrated by using xylene substitute and graded alcohols . The imaμL) was prepared using a KAPA SYBR Fast qPCR Kit  according to the manufacturer's protocol. Specific primers were used at final concentrations of 100 nM. Amplification was performed using a QuantStudio 5 Real-Time PCR Systems thermal cycler . The cycling profile included one cycle of 95°C for 5 min, followed by 40 cycles of 95°C for 3 s and 60°C for 30 s, with a final stage of melting curve analysis. GAPDH RNA expression was also measured and used as the internal sample control. The value of each gene expression was calculated using the 2∆∆Ct method and normalized to GAPDH levels [KOA-associated gene expression was measured using qPCR. The cartilage was homogenized in RNAprep Pure Tissue Kit , and total RNA was extracted according to the manufacturer's protocol. Then, 500 ng of total RNA was subjected to reverse transcription using RT-Master Mix  according to the manufacturer's protocol to generate complementary DNA (cDNA). The cDNA was serially diluted 10-fold with sterilized distilled water and qPCR was performed to detect the expression of cartilage-associated marker genes. The qPCR reaction mixture . However, the increase in the Lequesne MG score was reduced significantly (p < 0.01) in the warm acupuncture combined with the BMSCs group compared to that in the KOA group. In addition, the effect of the warm acupuncture combined with BMSCs is better than that of warm acupuncture group and BMSCs group separately (p < 0.05 and p < 0.01).KOA rabbits showed a higher behavior score than the blank group (p < 0.05); Compared with the KOA group, the Mankin scores of the three intervention groups were significantly lower (p < 0.05), and the Mankin score of the combined treatment group was significantly lower than that of the treatment group separately (p < 0.05).In the blank group, articular cartilage structure was good, the cells were uniformly distributed, the cells were evenly distributed, there was single column arrangement, and extracellular matrix staining was uniform. There were cracks in the rough surface of cartilage, the number of cartilage cells was obviously reduced, the distribution was sparse, shallow cells experienced pressured deformation, the hierarchical structure was disturbed, and the cracks were visible locally through the radiation layer. However, the cartilage structure was normal, without cracks, the tidal line structure was basically intact, the chondrocytes were arranged in a relatively regular manner, there was no clustering phenomenon, and the cartilage matrix staining was normal in the warm acupuncture combined with BMSCs group . TherefoChondroitin sulfate and keratin sulfate in the cartilage matrix were acidic and red after reacting with slightly alkaline safflower O. Collagen fibers in the superficial layer of cartilage and subchondral bone contain a large number of alkaline amino acids, which are green when combined with acid fixation. In the blank group, the cartilage structure was intact, the layers were clear, the cells were arranged in a single row, the cartilage matrix was red, and the subchondral bone was green -A. In thThe cartilage tissue in the blank group was clearly arranged, the structure was intact, the number of chondrocytes was normal, and the toluidine blue staining was uniform . MeanwhiTransmission electron microscopy was used to observe the changes of apoptotic body. As demonstrated in p < 0.01).In the same way, the results of TUNEL staining are similar, as shown in p < 0.01 and p < 0.01; Figures p < 0.01, The immunohistochemical analysis result indicated that the KOA group exhibited upregulated expression of Bax and Caspase-3 and downregulated expression of Bcl-2 compared with the blank group. The warm acupuncture combined with BMSCs can significantly inhibit the increase in the expression of Bax and Caspase-3 and reduce the expression of Bcl-2 . At the The main finding of the present study is that warm acupuncture combined with BMSCs can markedly improve the impairments of knee joint behavior and decrease the extent of cartilage tissue pathology relative to that of the rabbits subjected to KOA. Moreover, our results exhibited that warm acupuncture combined with BMSCs intervention can decrease the formation of apoptosis body and reversed the expression of apoptosis-related proteins Bax, Bcl-2, and Caspase-3 induced by KOA. These results suggest that warm acupuncture combined with BMSCs offers potential protective effects against KOA rabbits, and their protective effects may be related to the inhibiting chondrocyte apoptosis.The Lequesne MG index reflects the severity of KOA . The resSome evidence suggested that apoptosis plays a vital role in the pathogenesis of KOA , 40. TheSome studies have shown that the level of Bcl-2 decreased and those Bax and Caspase-3 increased after KOA; that is, apoptosis was activated , 43–45. In conclusion, this study is the first to report that warm acupuncture combined with BMSCs transplantation can effectively inhibit KOA cartilage tissue injury in rabbits. The effect is obviously better than that of simple warm acupuncture and simple BMSCs, confirming the synergistic effect. Also, warm acupuncture combined with BMSCs transplantation may reduce injury to KOA rabbit cartilage tissue by reducing the expressions of Bax and Caspase-3, raising Bcl-2 levels, and inhibiting cartilage apoptosis. Therefore, it can be speculated that the warm acupuncture combined with BMSCs may inhibit chondrocyte apoptosis on KOA rabbits, delaying KOA cartilage tissue injury, which promoted chondrocytes survival. However, the experiments in this study have several limitations, particularly on the mechanism of warm acupuncture combined with BMSCs to treat KOA, as well as lack of studies exploring the primary mechanism of protection. More cartilage effects and various relevant cell survival mechanisms of warm acupuncture combined with BMSCs on KOA need to be further explored. At present, the targeted differentiation mechanism and influencing factors of BMSCs are not clear. Further studies are needed to determine how to improve the directed differentiation rate of BMSCs. In addition, the current experiment is still some initial animal experiment, and advanced animal and clinical studies are needed. Long-term large-scale randomized clinical trials are needed to improve the interpretation, so as to determine the level of efficacy in future studies. Although there are still many questions about the application of BMSCs which need to be further studied, BMSCs should not be ignored as the direction of future cartilage repair. It is believed that, with the deepening of the research and the promotion of the application, the tissue-engineered cartilage will be better applied in clinic.


To investigate the postoperative course of patients who explanted a diffractive bifocal intraocular lens (IOL) due to waxy vision and implanted with an extended depth of focus IOL.® implantation because of dissatisfaction due to waxy vision at the Takabatake West Eye Clinic between January 2018 and November 2019. The indication criteria for this surgery were patients with uncorrected distance visual acuity of 0.05 logMAR or better, without eye diseases that may affect visual function, and no dissatisfactions about photic phenomena. We investigated patient demographics, uncorrected and corrected visual acuity, manifest refraction, contrast sensitivity, subjective symptoms, time to IOL explantation, explanted IOL type, and spectacle independence.This study evaluated 29 eyes of 25 patients who underwent diffractive bifocal IOL explantation followed by TECNIS SymfonyThe time to the IOL exchange after the initial IOL implantation was 55.3 ± 50.4 days (range: 14–196 days). The logMAR corrected distance visual acuity before and after IOL exchange were −0.13 ± 0.06 and −0.14 ± 0.06, respectively (p = 0.273). After IOL exchange surgery, the area under log contrast sensitivity function increased significantly from 1.07 ± 0.12 to 1.21 ± 0.12 (p < 0.001), and the waxy vision symptoms improved. The spectacle independence rate at the last visit was 88.0%.For patients who complain of waxy vision despite good visual acuity after diffractive bifocal IOL implantation, exchange to extended depth of focus IOL was considered one of the useful surgical options. In recent years, patients have higher expectations from cataract surgery, and many patients desire independence from spectacles as a surgical outcome. After multifocal intraocular lens (IOL) implantation, good distance and near visual acuities can be obtained, and so high patient satisfaction is achieved by reducing dependence on spectacles and improving quality of life , 2. WhilⓇ  an EDOF IOL which maintains contrast sensitivity close to that of monofocal IOLs by suppressing light loss and correcting chromatic aberration using a unique diffraction technology [Diffractive bifocal IOLs distribute incident light into distant and near images and lead to concerns that the contrast sensitivity will decrease due to light loss , 8–10. Achnology . Since tThis study aimed to investigate the postoperative course of patients who had their diffractive bifocal IOLs explanted due to waxy vision and underwent EDOF IOL implantation.® (including toric type) implantation because of dissatisfaction to waxy vision at the Takabatake West Eye Clinic between January 2018 and November 2019. The indication criteria for this surgery were as follows: (1) uncorrected distance visual acuity (UDVA) of 0.05 logMAR or better, (2) area under the log contrast sensitivity function (AULCSF) of 0.80 or better, (3) absence of eye diseases that may affect visual function, and (4) no dissatisfactions regarding photic phenomena . All patients who met the indication criteria were given the option of surgery to replace with another IOL to improve contrast sensitivity, or follow-up without surgery. After sufficient informed consent, the treatment was chosen by the patient himself.This retrospective study was approved by the institutional review board of Yamaguchi University and adheres to the tenets of the Declaration of Helsinki. All participants actively agreed to participate in the study and were given the choice to opt-out, in case they wished to. This study evaluated 29 eyes of 25 patients who underwent diffractive bifocal IOL explantation followed by TECNIS SymfonyAll the surgeries were performed by the same experienced surgeon (RT). The primary cataract surgery used a standard phacoemulsification technique under local anesthesia. A 2.4–3.0 mm clear corneal incision was made at the steepest corneal meridian, depending on the power and axis of the corneal astigmatism. Toric type IOLs were selected with 1.5 D or more for with-the-rule and 1.0 D or more for against-the-rule and oblique astigmatism. The corneal incision’s position and IOL axis were aligned using the VERION image guided system .® was implanted in the capsular bag using an injector. No intraoperative complications were observed.The IOL exchange surgery used the same corneal incision of the previous cataract surgery. The corneal incision was opened, a viscoelastic material was injected, and the IOL was released from the capsular bag. The IOL optics were cut in half with scissors for easy explantation through the small wound and TECNIS SymfonyPatient demographics and clinical outcomes, including the visual acuity (VA), contrast sensitivity, subjective symptoms, time to IOL exchange, explanted IOL type, implanted IOL type, and postoperative spectacle independence, were investigated based on medical records. Waxy vision was defined as being equivalent to blurred vision, regardless of the sighting distance after spectacle correction.®, the uncorrected and distance-corrected intermediate VAs  were measured at 70 cm. In some patients, the UNVA and DCNVA at 40 cm were also measured.The uncorrected and corrected distance VAs  were measured at 5 m before and after IOL exchange surgery. The uncorrected and distance-corrected near VAs  were measured at 40 cm before IOL explantation. One month after implantation of TECNIS SymfonyThe contrast sensitivity was measured using the CSV-1000 instrument  under scotopic conditions with best spectacle correction. From the contrast sensitivity, the AULCSF was determined as described previously .All the statistical analyses were performed using R version 3.6.3 . The decimal VA values were converted to the logarithm of the minimum angle of resolution (logMAR) for analysis. The means ± standard deviations were used to describe the distributions of continuous variables, and percentages were used to describe the distributions of categorical variables. Univariate analysis was performed with the Wilcoxon signed-rank test. The tests of statistical significance were two-tailed, and p < 0.05 were considered statistically significant.We analyzed the data for 29 eyes of 25 patients . ® Multifocal ZLB00  in 23 eyes, AcrySof® IQ ReSTOR® +3.0 D  in 2 eyes, and AcrySof® IQ ReSTOR® +3.0 D TORIC  in 4 eyes. Diffractive bifocal IOLs were implanted in both eyes in 7 cases, of which monocular exchange was in 3 cases and binocular exchange was in 4 cases. If there was a difference in subjective symptoms between the eyes, the eye with more severe symptoms first underwent IOL exchange, and if there was no difference, the eye was IOL exchanged with the dominant eye.The time to the IOL exchange after the initial IOL implantation was 55.3 ± 50.4 days (range: 14–196 days). Explanted IOLs were TECNIS®-implanted patients with the intended emmetropia or -1.0 D mild myopia targets. The purpose of using spectacles was for near vision in 2 cases and for distance vision in 1 case, and they were only used occasionally when necessary. As for subjective symptoms, disappearance of waxy vision was observed in 23 patients. Mild monocular waxy vision symptoms remained in 2 patients, but the symptoms were less than before IOL exchange surgery and spectacle independence was achieved, so they did not want any further intervention.The spectacle independence rate was 88.0% at the last visit. Three patients using spectacles were non-monovision binocular TECNIS SymfonyEven if the indications for multifocal IOL transplantation are carefully considered before surgery and good VA is obtained after surgery, some patients may not be satisfied with the surgical outcomes. In this study, patients who complained of waxy vision despite good UDVA after implantation of diffractive bifocal IOLs were exchanged for EDOF IOL and showed improvement in contrast sensitivity and subjective symptoms. Replacement with EDOF IOL, which provides comparable contrast sensitivity to that of monofocal IOL , 11, was® is one of the EDOF IOLs that was able to suppress the decrease in the contrast sensitivity due to its proprietary diffractive echelette design and achromatic technology [Kamiya et al.  conductechnology , 11, 13.Blurred vision is the main cause of dissatisfaction in patients with multifocal IOLs. Previous reports examining cases of dissatisfaction after implantation of multifocal IOLs reported that approximately 95% of the causes of dissatisfaction were blurred vision . Most ofThere are concerns about the risk of complications when exchanging IOLs , 18–20. Hybrid monovision with monofocal IOL in the dominant eye and a multifocal IOL in the nondominant contralateral eye may reduce the waxy vision caused by bilateral multifocal IOL implantation , 22. SevThe spectacle independence rate after multifocal IOL implantation averaged 80.1% in 63 studies , 26. ConFactors causing dissatisfaction after multifocal IOL implantation are not only restricted to waxy vision but also to poor VA and photic phenomena, and cases have been reported in which exchange monofocal IOLs were required –7. Altho® or common to different types of EDOF IOLs, since there was no control group. Additionally, the number of patients analyzed was small, and the subjective symptoms were confirmed by interview, and a questionnaire survey on satisfaction was not conducted. However, since no patient wanted further surgical intervention after IOL exchange, we believe that patient satisfaction was obtained.There are several limitations of this study. First, this was a retrospective study; a randomized controlled study with exchanges to monofocal IOLs may provide further information confirming the validity of these results. Second, it is not possible to assess whether the results of this study are specific to the TECNIS SymfonyIn conclusion, our study suggests that IOL exchange is a surgical option for dissatisfied patients with waxy vision in diffractive bifocal IOL-implanted eyes. Although monofocal IOLs are generally used as IOLs to be replaced, this study suggested that replacement with EDOF IOL is also effective. Further studies with more subjects using questionnaires on overall patient satisfaction, comparisons with replacement to monofocal IOLs, and verification of different types of EDOF IOLs are required to confirm these findings.S1 Table(XLSX)Click here for additional data file.


In this study, silver nanoclusters as prooxidant biosensor were eco‒friendly synthesized using chicken egg white protein without any chemical reducing agents for measuring copper(II)-induced prooxidant activities of catechin, epicatechin, epigallocatechin gallate, resveratrol, gallic acid, chlorogenic acid, and rutin. The prooxidant activities were evaluated via measuring the absorption at 450 nm wavelength of the Cu(I)‒neocuproine chelate formed by extraction of protein-bound Cu(I) with neocuproine reagent. Accuracy was determined by evaluating recovery values of wine, grape and apple samples and the obtained values were between 97.2%‒98.9%. Intra-day precision and inter-day reproducibility experiments were studied with three different experiments in a day and three different days respectively. The obtained relative standard deviation values were 0.96% and 1.91%. The detection limit of the biosensor was found as 0.2 µM. The total prooxidant activities of fresh apple and grape fruits, apple and grape juices, and red wine were determined and the results obtained were compared with the findings of the carbonyl assay. In this study, a cheap, easily applicable, sensitive, and reproducible biosensor was developed. It was seen that it could be used in the measurement of the prooxidant activity of different food samples and give an idea about diet, healthy life, and nutrition. Copper is a vital and essential micro-bioelement for organism which required for the function of over 30 proteins, including ceruloplasmin, cytochrome c oxidase, tyrosinase, and superoxide dismutase. Also it was found mainly in the Cu(II) form in the biological systems, since in the presence of oxygen via Fenton‒type reaction Cu(I) is oxidized to Cu(II). The most reactive species, hydroxyl radicals release as a result of this reaction and induce “site-specific” oxidative damage to biological macromolecules . The prooxidant activity is the capacity of reducing transition metal ions to their lower oxidation states by antioxidants, exciting the production of reactive species. Therefore, it is important to understand whether it is beneficial or harmful to health, depending on the amount of these compounds. As secondary metabolites derived in plants, phenolic exhibit various physiological properties such as antimicrobial, antiinflammatory, antioxidant, and antiallergenic [3]. Despite their antioxidative properties are well known, they may act as a prooxidant depending on some conditions such as high concentration, high pH, in the presence of redox‒active metal ion and biological material in the medium [4]. Furthermore, it has been noticed that prooxidant activity increases with increasing the number of free‒OH substitutions on the molecule structure [5]. In addition, since these compounds are used as food additives, it is very important to examine their antioxidant or prooxidant behavior depending on the conditions in which they are found.4 as chemical reducing agent [7‒10] and there are rare biological reduction synthesis methods for AgNC . Metal NCs were able to interact with each other and aggregate irreversibly to reduce their surface energy without stabilization [13]. In this context, metal NCs are biologically synthesized via the reduction of metal ions by the suitable reducing and stabilizing agents such as protein, peptide or nucleic acid where the thiol, carboxyl, and amine groups of biological molecules may be effective to stabilize NCs . The NCs are able to act as sensitive probes with their optical response is highly dependent on the interaction with the organic scaffold. The application fields of different types of silver nanoclusters (AgNCs) have included iron [10], mercury  or biothiols [16] detection, and biological imaging [17].In recent years, noble metal nanoclusters (NCs) have received extraordinary attention due to their many properties such as easy synthesis, subnanometer size, photostability, and biocompatibility [6]. Unfortunately, the most of silver nanocluster (AgNC) synthesises found in the literature contain NaBHMeasuring prooxidant activity is a developing research area, required to be interested in with more work. Also it is needed to develop cheap, easily applicable, sensitive, and reproducible sensors. Existing assays in the literature are expensive especially in detecting radicals by electron spin resonance (ESR) spectroscopy, laborious, limited with respect to application area, and cause positive errors in results because of interferences, likewise in protein carbonyl assay. In our previous studies, we have developed new methods to overcome these disadvantages in the literature with the needs of this research area [18‒22].In this study, chicken egg white protein directed silver nanocluster (CEW‒AgNC) biosensor, where the protein was used as both reducing and protecting agent, was prepared to detect prooxidant activity of phenolic compounds including in fruits, fruit juices, and red wine. In the method, it is thought that cuprous ions formed as a result of reduction of cupric ions by phenolic compounds are bound to thiol groups of protein in CEW‒AgNC surface. The protein bound cuprous ion which is the indicator of prooxidant activity, was determined colorimetrically by forming a Cu(I)‒neocuproine (Nc) complex with Nc reagent. Total prooxidant activities (TPAs) of fresh fruits, commercial fruit juices, and red wine were examined and statistically compared with the carbonyl assay. 4) and neocuproine (Nc) from Aldrich ; gallic acid (GA), silver nitrate (AgNO3), sodium dihydrogen phosphate dihydrate (NaH2PO4.2H2O), ethanol (EtOH), and 2,4‒dinitrophenylhydrazine (DNPH) from Sigma‒Aldrich ; ethylenediaminetetraacetic acid (EDTA) disodium salt and copper(II) sulfate from Fluka ; disodium hydrogen phosphate (Na2HPO4), hydrochloric acid (HCl) and sodium hydroxide (NaOH) from Riedel‒de Haën . Fresh apple, apple juice, fresh grape, grape juice, and red wine were purchased from a local market. The following chemicals/reagents were supplied from the indicated sources: catechin (CAT), resveratrol (RES), epigallocatechin gallate (EGCG), epicatechin (ECAT), chlorogenic acid (CLA), and rutin (RT) from Sigma ; tetrachloroauric acid (HAuClNeocuproine (7.5 mM) and the stock standard antioxidant (10.0 mM) solutions were prepared in EtOH and diluted daily for experiments. Copper (2.0 mM), pH 7.4 phosphate buffer (0.5 M), and EDTA (0.1 M) solutions were prepared in distilled water. DNPH reagent was prepared in 0.2 N HCl solution. Two grams of fresh apple and grape were extracted using the microwave-assisted extraction technique as described in our previous study after all samples were washed with water and cut into small pieces with a plastic knife [20]. Fruit juices and wine samples were diluted with distilled water and the proposed assay was directly applied.‒1) was added to AgNO3 solution  and vortexed for 2 min before the addiction of NaOH solution . The final mixture was incubated in a 37 °C shaken water bath for 20 h. After this time, the color changed from white to yellow, and the synthesized CEW−AgNCs were stored in the refrigerator at 4 °C before use.Silver nanoclusters were prepared after slight modifications as described in our previous study [20]. Briefly, protein solution  solution, (x) mL of standard antioxidant or sample solution, and (1−x) mL of distilled water were added to a test tube. After the incubation for 15 min at room temperature, 0.5 mL EDTA and 1 mL Nc solutions were added and incubated for 20 min. At the end of this time the absorbance values were recorded at 450 nm against reagent blank including all solutions except the analyte. First incubation period was required to reduce cupric to cuprous by phenolic compounds. Second incubation period was required to remove free cupric ions remaining in the solution, and to break the bond of protein−bound (Cu) to obtain Cu(I)−Nc chelate with neocuproine reagent. The calibration equations were calculated by using the graphs plotted between concentration versus absorbance for each compound and the molar absorption coefficients were figured out from the slope of the calibration line concerned.The carbonyl assay is based on the measurement of the absorbance of the dinitrophenylhydrazone (DNP) compound formed as a result of the reaction of the 2,4‒DNPH reagent with the carbonyl groups released as a result of protein oxidation . This assay was applied with slight modification similarly to Akyuz’s study [19], CEW−AgNC solution was incubated with phosphate buffer, Cu(II), standard antioxidant or sample, and DNPH solutions for 30 min at room temperature. After this time, absorbance measurements were recorded at 370 nm against reagent blank.F−test was used to compare the precisions of both methods.All experiments were performed in triplicate. Excel software (Microsoft Office 2016) was used for calculating the mean and the standard error of the mean {mean ± standard deviation (SD)}. The S‒terminal residues of protein in the silver nanoclusters inducing site‒specific protein damage via reactive oxygen species (ROS) formation. The prooxidant activities of phenolic compounds were indirectly determined by measuring protein bound‒Cu(I) known as a marker of prooxidant activity, utilizing ethanolic Nc reagent to obtain Cu(I)‒Nc chelate. This study was the green synthesized method using chicken egg white proteins that was carried out with slight changes in the synthesis method of our previous study [20] and applied for the first time to measure the copper-induced prooxidant activities of phenolic antioxidants as prooxidant biosensor. A novel CEW−AgNC prooxidant biosensor was proposed to measure copper catalyzed prooxidant activities of CAT, ECAT, EGCG, RES, GA, CLA, and RT as standard phenolic compounds, fresh fruits, commercial fruit juices, and red wine. The developed assay involved the reduction of cupric ions by the phenolic antioxidants and binding of cuprous ions to 3 solution  was studied and the best results were obtained at 2.25 mM AgNO3 concentration. Although, a 6:1 ratio between nucleic acid and silver concentrations was mentioned in DNA‒stabilized AgNC synthesis in the literature , this ratio was not suitable for those prepared with protein. Finally, pH values of synthesized CEW‒AgNC solutions were evaluated between 11‒13 and the highest absorbance values were obtained at pH 13.For optimizing synthesis conditions, firstly concentration of phosphate buffer solution that was used for preparing protein solution before the synthesis of CEW‒AgNC was studied. Within this experiments, 0.1, 0.2, and 0.5 M phosphate buffer solutions were used for preparing protein solution which was used for synthesis of NC, and maximum absorbances of synthesized NC solutions were achieved with 0.2 M phosphate buffer. Secondly, concentration of AgNOThe developed assay involved two incubation periods, that the first incubation was required for the reduction of cupric ion, and binding to the protein thiol in the CEW‒AgNC . The second incubation was required to eliminate excess Cu(II) with EDTA and separating the bond between copper and protein using neocuproine reagent to obtain Cu(I)‒Nc chelate . To optimize these periods, time intervals from 1 to 60 min was applied to epicatechin (ECAT), gallic acid (GA), and rutin (RT) compounds at equal concentrations (40 µM) with triple experiments. As can be seen in Figures 1A and B, 15 and 20 min for first and second incubation periods respectively were found more appropriate for the three tested compounds.Absorption spectra of epicatechin between 10‒50 μM concentrations with respect to the proposed biosensor and absorption spectra of synthesized CEW‒AgNC solution were given in Figure 2. Validation parameters of the proposed biosensor were studied and the results obtained were summarized in Table 1. Using the slope of the calibration line of the epicatechin standard and standard deviation of the blank, limit of detection (LOD) and limit of quantification (LOQ) values were calculated. According to the calculations, the LOD and LOQ values which were the lowest values in the literature, were found to be 0.2 and 0.7 μM ECAT equivalent, respectively. CEW‒AgNC biosensor was found more sensitive than CEW‒AuNC biosensor, which was reported that AgNCs were more reactive than their gold analogs . Accuracy of the proposed biosensor was determined by evaluating percent recovery values of wine, grape, and apple samples that spiked at three different concentrations 10, 20, 30 μM ECAT standard. The obtained recovery values were 98.4, 97.2, 98.9 for wine; 98.6; 98.2; 97.9 for grape; and 98.1; 98.7; 98.9 for apple, respectively. Intra-day precision was studied with three different experiments in a day and inter-day reproducibility was studied at three different days by determining epicatechin standard at 20 μM concentration level. The obtained relative standard deviation values were 0.96 and 1.91, respectively. All experiments were repeated three times.F‒test at 95% confidence level and the TPAs of epicatechin and red wine (10 fold diluted) were expressed as mg ECAT L−1 equivalents. There was no significant difference at 95% confidence level between both methods with respect to the F‒test data presented in Table 2.The statistical comparison of CEW‒AgNC biosensor and carbonyl assay were performed by appling the r = 0.999. The order of molar absorption coefficients of the studied compounds by using the CEW‒AgNC was as follows: ECAT > RT > CAT > RES > EGCG > CLA > GA. Calibration equation, correlation coefficient (r) and linear range (µM) values of some natural antioxidant standards were given at Table 3. As shown in the Table 3, excellent linearity was obtained between absorbance and concentration with respect to the proposed biosensor for all compounds. All correlation coefficient values were found above o‒dihydroxy structure in the B ring included the highest scavenging activities. They also reported that under physiological conditions flavonoids which had the half peak reduction potencial values below <0.06 mV, underwent redox cycles and were thus might be reduce transition metals. Eghbaliferiz and Iranshahi [27] stated that RES exhibit prooxidant activity in the presence of copper. In the study of Ahmad et al. [28], resveratrol caused mutations to the plasmid DNA. They treated the DNA samples with increasing concentrations of RES (10‒200 µM) in the presence and absence of copper ions and said that resveratrol was act as prooxidant at higher concentrations. Zhen et al. [29] studied the prooxidant activity of some hydroxycinnamic acid compounds, measuring DNA damage in the presence of Cu(II) ions. They found that the prooxidant activity of the compounds bearing o‒dihydroxyl group such as CLA were displayed much higher than the others which had no functional groups. Simić et al. [30] examined the electrochemical oxidation of some phenolic compounds with cyclic voltammetric method. They reported that the compounds with low anodic potential (Epa) (<0.45 V) demonstrated antioxidant activity, whereas the compounds with high Epa values (>0.45 V) acted as prooxidants. In their study, RT behaved as an antioxidant with the 0.23 V Epa values. Yang et al. [31] investigated the DNA damage caused by quercetin, rutin, p‒coumaric acid and their derivatives with 300 µm concentration. In their study, it was found that rutin had higher prooxidant activity than phenolic acids. Lambert and Elias [32] reported that green tea catechins such as CAT, ECAT, and EGCG, might be responsible for the induction of apoptosis in tumor cells, although they were known as antioxidant compounds. Furthermore, in the study of Zhou and Elias [33] it was mentioned that EGCG showed higher prooxidant activity with low pH of solution medium and they detected lower prooxidant activity with the higher pH values. This information was supported with the iron(III)-induced prooxidant activity assay worked in pH 5.5 and found the highest prooxidant activity value of EGCG in the literature, instead of the other assays worked in physiological pH [22].The molar absorptivity of ECAT was found higher than CAT and other phenolic acids and this result was supported similarly with one of our previous works [19]. On the other hand, in the study of Kondakçı et al. [18], the order of prooxidant activity of tea catechins was found similar as ECAT > CAT > EGCG with respect to the molar absorption coefficients. Also it was supported with the literature as the prooxidant activity of RES was lower than catechins and higher than phenolic acids [20]. Rice‒Evans et al. [26] stated that the polyphenols with the Comparison of the proposed method with previously published methods in terms of limit of detection (LOD) values as μM ECAT equivalent was shown in Table 4. It can be seen that the lowest LOD value in the literature was achieved with the proposed biosensor.The primary components of the samples were as follows: chalcones, flavanols, flavonols, and condensed tannins in apple and apple juice [36]; anthocyanins, flavanols, flavonols, phenolic acids in grape, and grape juice [37]; anthocyanidins, flavanols, flavonols, and especially resveratrol in red wine , respectively. TPA values of fresh apple and grape, commercial apple and grape juices, and red wine were determined as mM epicatechin equivalent and compared with those of carbonyl assay as reference method. As you can see in Figure 3 the results were compatible with each other.In the study of Rice‒Evans et al. [26], it was indicated that the amount of epicatechin was approximately 55 times higher than resveratrol in red wine. Since red wine contains strong prooxidant compounds such as catechin derivatives and resveratrol, higher TPA was determined than apple and grape samples. TPA of the apple containing phenolic acid derivatives was found to be low due to relatively low prooxidant ability of related compounds. In the evaluating Figure 3, the lowest TPA value was found in fresh fruits and so it can be concluded that consuming fresh fruit is healthier than consuming fabricated fruit juices. In this context, in order to be protected from various diseases and to live healthy, all fabricated products and prepackaged foods should be refrained and taken care to consume fruits and vegetables in the most natural form as much as possible.S‒terminal residues on the CEW‒AgNC surface inducing site‒specific protein damage via ROS formation. Protein bound‒Cu(I), as a marker of prooxidant activity, was precisely determined via CEW‒AgNC biosensor with the lowest LOD value (0.2 µM) in the literature using neocuproine (Nc) reagent. Accuracy was evaluated with recovery values obtained up to 100% that spiked at three different concentrations of ECAT standard. Precision and reproducibility studied by determining ECAT standard as intra-day and inter-day experiments were found 0.96% and 1.91%, respectively. The TPA values of fresh fruits, commercial fruit juices, and red wine were calculated and the results were statistically compared with those of carbonyl assay. The most important advantage of using nanoclusters in medicine especially in cancer diagnosis and treatment is their ability to penetrate to the kidney tissue because of their ultrasmall size and to easily dissipate from the body to reduce toxicity in vivo unlike nanoparticles [38]. In addition, considering the results of the proposed method, it was thought to give important ideas in terms of healthy life and nutrition with the controlling of oxidative stability of fabricated products and additives that had extended shelf life.Phenolic bioactive compounds known as health-beneficial antioxidants are able to exhibit prooxidant behaviour causing to increase oxidative process exponentially depending on high concentration, high pH, presence of high free transition metal ion and biological material in the medium. Thus, the determination of prooxidant activity of phenolics has gained importance as it may guide to diets and the treatment of various diseases. There are rare prooxidant biosensors in the literature to determine the prooxidant activities of natural antioxidants through protein damage. The aim of this study was to develop a novel, low cost, easily applicable, fast, sensitive, and reproducible biosensor determining the prooxidant activity in contrast to the methods in the literature, which were expensive, time-consuming, and require qualified personnel like ESR. In this study, spectrophotometric silver nanocluster based prooxidant biosensor, prepared by using chicken egg white protein was synthesized in the principle with green chemistry and applied as a prooxidant biosensor for the first time to detect copper catalyzed prooxidant activity of phenolic antioxidants abundant in foods, beverages, cereals. Protein was utilized as both reducing and stabilizing agent in the silver nanocluster synthesis procedure. In the assay, after phenolic antioxidants reduced cupric ions, cuprous ions were bound to the protein The corresponding author is responsible for all contributions.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.


This study aimed to evaluate the indications of laparoscopic repeat liver resection (LRLR) for recurrent hepatocellular carcinoma from the viewpoint of its difficulty.One hundred and one patients who underwent LRLR and 59 patients who underwent open repeat liver resection (ORLR) were included. The difficulty was classified according to the preoperative predictive factors for difficult LRLR, including an open approach during previous liver resection, history of two or more previous liver resections, history of previous major liver resection, tumor near the resected site of the previous liver resection, and intermediate or high difficulty with the difficulty scoring system. We compared the surgical outcomes between the LRLR and ORLR groups based on the difficulty class .In the low‐ or intermediate difficiulty class, intraoperative blood loss and the proportion of patients with postoperative complications were significantly lower in LRLR than in ORLR, and the duration of the postoperative hospital stay was significantly shorter in LRLR than in ORLR. In the high difficiulty class, total operative time and operative time before starting hepatic parenchymal resection were significantly longer in LRLR than in ORLR, and there were no significant differences in other surgical outcomes between the two groups.LRLR is recommended for patients in the low or intermediate difficulty class. However, LRLR does not have an advantage with longer operative time for patients in the high difficulty class compared with ORLR. Laparoscopic repeat liver resection (LRLR) is recommended for patients in our suggested low or intermediate difficulty class. On the other hands, LRLR does not have an advantage with longer operative time for patients in the high difficulty class, compared with open repeat liver resection. To eliminate operative bias, the patients who underwent repeat liver resection that involved segmentectomy or extensive surgery, noncurative resection, concomitant resection of other organs , resection of multiple lesions, and resection of the caudate lobe (segment 1) were excluded from this study. A total of 160 patients who underwent repeat partial liver resection were included in the study (101 underwent LRLR and 59 underwent ORLR). The median age of the subjects was 70  years, and 134 subjects were male. Of the 160 patients, 76 were seropositive for anti‐hepatitis C virus antibody and 42 were seropositive for hepatitis B surface antigen. Eleven patients had alcoholic hepatitis, and five had nonalcoholic steatohepatitis.This study conformed to the ethical guidelines of the Declaration of Helsinki and was retrospective in nature, and we obtained approval from the Ethics Committees of Osaka City University (No. 3166) and Osaka City General Hospital (No. 1910076). All participants provided written informed consent.2.2The preoperative risk factors for unfavorable surgical outcomes after LRLR were selected based on previous studiesIn our previous study,2.3U‐test was used to compare continuous variables. Categorical variables were summarized as numbers and percentages and compared between groups using Fisher's exact test or the chi‐squared test, as appropriate. P < .05 was considered statistically significant. Statistical analyses were performed using JMP v. 11 .The Mann–Whitney 33.1Surgical outcomes of each difficulty class (low or intermediate and high difficulty class) after LRLR are described in Table 3.2Patients’ backgrounds are described in Table 3.3A significantly longer total operative time, less intraoperative blood loss, and shorter postoperative hospital stay were observed in the LRLR group than in the ORLR group. There was no difference in the operative time before starting liver parenchymal resection and the proportion of patients with postoperative complications between the groups Table .3.4P < .0001), and the duration of postoperative hospital stay was significantly shorter in the LRLR group than in the ORLR group (P < .0001). Moreover, the proportion of patients with postoperative complications was also significantly lower in the LRLR group than in the ORLR group (P = .014). In the LRLR group, one patient had bile leakage. In the ORLR group, two patients had bile leakage, one had intractable ascites, and two had pleural effusion.In the low or intermediate difficulty class (patients with 0 to 3 preoperative predictive factors), there were no significant differences in the total operative time and operative time before starting hepatic parenchymal resection Table . Intraop3.5P = .0009 and P = .0017, respectively; Table In the high difficulty class (patients with 4 or 5 preoperative predictive factors), total operative time and operative time before starting hepatic parenchymal resection were significantly longer in the LRLR group than in the ORLR group , tumor near the resected site of the previous liver resection, and intermediate or high difficulty in the DSS was useful in making the decision for the indication of LRLR. LRLR is recommended for patients in the low or intermediate difficulty class (0 to 3 predictive factors). In contrast, presently LRLR cannot be recommended for patients in the high difficulty class (4 or 5 preoperative predictive factors) because LRLR has a longer operative time without any benefits in other surgical outcomes for such patients, compared with ORLR.Conflict of Interest: None.Funding Information: This work was supported by the Health, Labour and Welfare Policy Research Grants from the Ministry of Health, Labour and Welfare of Japan (Policy Research for Hepatitis Measures [H30‐Kansei‐Shitei‐003]).Author Contributions: Study design: Masahiko Kinoshita, Akishige Kanazawa, and Shoji Kubo designed the study. Acquisition of data: Masahiko Kinoshita, Akishige Kanazawa, Shogo Tanaka, Shigekazu Takemura, Ryosuke Amano, Kenjiro Kimura, Hiroji Shinkawa, Go Ohira, Kohei Nishio. Data analysis: Masahiko Kinoshita, Akishige Kanazawa, and Shoji Kubo. Manuscript drafted by Masahiko Kinoshita, Akishige Kanazawa, and Shoji Kubo. All the authors reviewed the manuscript.Table S1Click here for additional data file.Table S2Click here for additional data file.


Pulmonary thromboembolism (PTE) is a potentially life-threatening condition with high morbidity and mortality, and computed tomographic pulmonary angiography (CTPA) is an important diagnostic tool for patients in whom PTE is suspected; however, intraoperative PTE is very difficult to diagnose and often has a rapid clinical course. We experienced a case of intraoperative PTE with persistent tachycardia refractory to conventional treatments despite negative preoperative CTPA findings.A 53-year-old man with a pelvic bone fracture who had been on bed rest for 10 days underwent open reduction and internal fixation under general anesthesia. He remained tachycardic (heart rate of 120 beats/min) despite treatments with fluid resuscitation, analgesics, and beta-blockers.Preoperative CTPA, computed tomography (CT) venography, and transthoracic echocardiography showed no signs of deep vein thrombosis and PTE. However, the levels of D-dimer were elevated. After the start of the surgery, tachycardia (heart rate between 100 and 110 beats/min) could not be treated with fluid resuscitation. Systolic blood pressure was maintained between 90 and 100 mm Hg using continuous infusion of phenylephrine. Ninety minutes after the surgery, systolic and diastolic blood pressures suddenly dropped from 100/60 to 30/15 mm Hg with a decrease in end-tidal carbon dioxide concentration from 29 to 13 mm Hg and development of atrial fibrillation. Arterial blood gas analysis revealed hypercapnia. Under the suspicion of PTE, cardiopulmonary resuscitation (CPR) was immediately initiated. Three CPR cycles raised the blood pressure back to 90/50 mm Hg with sinus tachycardia (115 beats/min). Transesophageal echocardiography showed right ventricular dysfunction and paradoxical septal motion. However, emboli were not found. Postoperative chest CT revealed massive PTE in both pulmonary arteries.Immediately, surgical embolectomy was performed uneventfully.The patient was discharged from the hospital 1 month later without any complications.The patient with moderate risk for PTE  should be closely monitored and managed intraoperatively even if preoperative CTPA findings are negative. The development of PTE needs to be expected if tachycardia is refractory to conventional treatments. In addition, the incidence of PTE was the highest (0.78%) in patients undergoing pelvic open reduction and internal fixation among 11,313 adult trauma patients on thromboprophylaxis therapy. Therefore, more attention should be paid to the development of PTE in patients with a pelvic bone fracture.Pulmonary thromboembolism (PTE) is a rare but potentially life-threatening condition associated with high morbidity and mortality. However, intraoperative PTE is very difficult to diagnose and often has a rapid clinical course. Furthermore, the results of preoperative tests do not take effect until the intraoperative period because the physiological milieu changes under both the anesthetic and surgical conditions. Nevertheless, there are limited predictive factors for PTE and no available guidelines for its screening. The obstruction of the pulmonary arteries (the mechanism of PTE) is often accompanied by nonspecific hemodynamic changes, such as normal blood pressure compensated for by tachycardia. Catecholamine-driven tachycardia in combination with right ventricular dilatation is an important compensatory mechanism to maintain cardiac output. However, normal blood pressure with tachycardia may not always indicate PTE because other confounding factors affect intraoperative hemodynamics, such as underlying cardiovascular or autonomic diseases, anesthetics, bleeding, fluid shifting, etc. Therefore, it would be reasonable to suspect the occurrence of PTE in patients at risk for PTE if tachycardia persists even after all the candidate causes other than PTE are ruled out. In addition, given the fact that the development of shock and the severity of hemodynamic compromise are important predictive and prognostic factors for mortality, close monitoring of vital signs to identify and halt the progress of PTE is mandatory.Perioperatively (before and after surgery), computed tomographic pulmonary angiography (CTPA) and D-dimer test are the mainstay for diagnosing PTE in patients with suspected PTE in the absence of hemodynamic instability.Herein, we report intraoperative development of PTE preceded by tachycardia refractory to conventional treatments in a bedridden patient owing to a pelvic bone fracture with no evidence of deep vein thrombosis (DVT) and PTE on preoperative CTPA and computed tomography (CT) venography.2A 53-year-old man , a heavy alcoholic, was admitted to a local medical center under the diagnosis of right acetabular and iliac wing fracture after falling from a tree. The patient was bedridden under conservative treatment. Three days later, he presented with abdominal pain and tenderness and was then transferred to our hospital for further evaluation and management. The systolic/diastolic blood pressure, heart rate on electrocardiogram, and body temperature were 160/94 mm Hg, 120 beats/min, and 37.5°C, respectively. Under the suspicion of dehydration and fracture-induced pain, fluid resuscitation was initiated with Hartmann solution and analgesics were administered. Nonetheless, the heart rate did not decrease. Hence, bisoprolol (5 mg per os) was administered once daily. Thiamine, haloperidol, and quetiapine were administered to treat alcohol withdrawal delirium. Every time the patient became responsive to verbal stimuli, he kept reporting abdominal pain. However, no pulmonary symptoms were present. Anteroposterior chest radiograph and transthoracic echocardiography (TTE) were normal. Abdominal CT revealed retroperitoneal and right iliacus hematoma. However, no injury was found in major vessels or organs. The laboratory findings showed increased levels of D-dimer (26.64 μg/mL), the normal range of which was < 0.1 μg/mL. Because he was immobilized after the injury with elevated levels of D-dimer, combined CT venography and CTPA was performed 7 days after the injury to rule out DVT and PTE; however, the results were negative.2) were 142/92 mm Hg, 100 beats/min, and 100%, respectively. Anesthesia was induced with bolus administration of 130 mg propofol and continuous infusion of remifentanil at 0.1 μg/kg/min. Endotracheal intubation was facilitated with 50 mg rocuronium. The ventilator was set in the volume-controlled mode to deliver a tidal volume of 6 to 8 mL/kg at a respiratory rate of 10 to 12 breaths/min. The tidal volume and respiratory rate were adjusted to maintain end-tidal carbon dioxide concentration (EtCO2) between 33 and 35 mm Hg. Anesthesia was maintained with sevoflurane (1.5–2.0 vol%) in oxygen/air mixture (inspired oxygen saturation of 50%) to ensure bispectral index values between 40 and 60. The infusion rate of remifentanil (0.1–0.2 μg/kg/min) was adjusted to maintain blood pressure and heart rate within 20% of the baseline values. The right radial artery was catheterized for real-time monitoring of arterial blood pressure and arterial blood sampling. The right internal jugular vein was catheterized for real-time monitoring of central venous pressure, bolus administration of drugs, and continuous infusion of drugs. The results of arterial blood gas analysis after anesthesia induction were as follows: pH, 7.468; partial pressure of arterial carbon dioxide (PaCO2), 37.2 mm Hg; partial pressure of arterial oxygen (PaO2), 193.5 mm Hg; bicarbonate (HCO3−), 26.3 mmol/L; base excess, 2.6 mmol/L; hemoglobin, 8.2 g/dL.Three days later (10 days after the injury), he presented to the operating room for open reduction and internal fixation of the pelvic bone fracture. Before anesthesia induction, the systolic/diastolic blood pressure, heart rate, and peripheral oxygen saturation  ensued. Under the impression of PTE, 3 cycles of cardiopulmonary resuscitation (CPR) were intermittently performed for 25 minutes until spontaneous circulation [systolic/diastolic blood pressure of 80–90/50 mm Hg and sinus tachycardia (115 beats/min)] was achieved . At the end of the last CPR cycle, the infusion rate of the fluid was reduced to 3 to 5 mL/kg/min, and a probe for transesophageal echocardiography (TEE) was inserted into the esophagus. TEE showed right ventricular dysfunction with paradoxical septal motion. However, no embolus was found in the main, right, and left pulmonary arteries. Intraoperatively, 500 mL of 6% hydroxyethyl starch, 1250 mL of packed red blood cells, and 450 mL of fresh frozen plasma were administered in total. The amount of blood loss and urine output was 700 and 100 mL, respectively.At 10 minutes before the surgery, 100 μg phenylephrine was bolus administered twice at a 5-minute interval to ensure a systolic blood pressure of > 100 mm Hg. At the beginning of the surgery, phenylephrine was continuously infused at a rate of 0.2 μg/kg/min owing to the transient effects of its bolus administration. To compensate for intraoperative blood loss and preoperative dehydration, 6% hydroxyethyl starch, packed red blood cells, and fresh frozen plasma were administered at a rate of 8 to 10 mL/kg/min, and systolic blood pressure and heart rate were maintained between 90 and 100 mm Hg and between 100 and 110 beats/min, respectively. Ninety minutes after the surgery, systolic/diastolic arterial blood pressure and EtCO2 and central venous pressure were elevated from 20 to 31 mm Hg and reduced from 30 to 4 mm Hg, respectively, after weaning from cardiopulmonary bypass. Intraoperative TEE and postoperative TTE showed normalized right ventricular function and septal motion. Anticoagulation therapy with warfarin was commenced from the third postoperative day. The inferior vena cava filter was also placed. One month after the surgery, CTPA and CT venography revealed no DVT and PTE. The patient was then discharged from the hospital without any complications. The patient provided informed consent for the publication of this case.Chest CT performed immediately after the surgery showed emboli in both the pulmonary arteries Fig. . The pat3 and Wells score were 10 and 3, respectively, indicating an intermediate and moderate probability of having PTE (27.5–28.5% and 36.1–37.3%), respectively. However, anticoagulation therapy or intermittent pneumatic compression for the prevention of DVT or PTE was not performed because of the risk of bleeding and ischemic events distal to the pelvic bone fracture. Alternatively, CTPA and CT venography were performed to detect any PTE or DVT. Because of its non-invasiveness, availability, and high sensitivity (83%) and specificity (96%), CTPA is widely used in patients at high risk for PTE.,13 CTPA combined with CT venography can diagnose DVT and PTE at the same time without using additional radiocontrast and exposure to excessive radiation. As the negative predictive value of CTPA was 89% in patients with a moderate probability (Wells score of 3) based on the results of a previous study, we believed that the negative findings of CTPA and CT venography almost excluded the preoperative development of PTE and DVT in our patient. Accordingly, preoperative echocardiography showed no evidence of PTE . As trauma produces fibrin, the usefulness of the levels of D-dimer is limited for the diagnosis of PTE in this case.Our patient was immobilized because of a pelvic bone fracture for 10 days before undergoing surgery under general anesthesia. The heart rate was mostly>100 beats/min, and the D-dimer test was positive. On the basis of the above information, the revised Geneva score Accordingly, sustained tachycardia and normal blood pressure were observed in clinical settings. In addition, the Wells score and revised Geneva score that were developed to predict the probability of PTE have heart rate  as one of the predictive variables.,9 Particularly, the heart rate of ≥95 beats/min has the highest score among the variables included in the revised Geneva score. In our case, until the development of hemodynamic instability attributed to PTE, preoperative and intraoperative heart rates remained > 100 beats/min. Preoperative blood pressure was normal, and intraoperative blood pressure was maintained normal with continuous phenylephrine infusion. Unfortunately, all the perioperative efforts to control the rapid heart rate  were unsuccessful. Therefore, it was reasonable to assume that tachycardia was caused by PTE after excluding the candidate causes of tachycardia. However, the evidence of preoperative PTE and DVT was nearly absent on CTPA and CT venography findings. In addition, hypotension after anesthesia induction was easily treated with phenylephrine, and no other significant changes in vital signs were observed until the development of PTE during the surgery. Particularly, general anesthesia and surgery were additional causes of tachycardia . Moreover, symptoms related to PTE were masked by general anesthesia. Therefore, it was very challenging to predict the intraoperative development of PTE in our patient.Tachycardia occurs frequently in the early stage of PTE. Acute large or multiple PTE increases pulmonary vascular resistance, resulting in an increase in right ventricular afterload. The increased afterload causes right ventricular dysfunction and dilatation that displace the interventricular septum towards the left leading to a reduction in cardiac output and systemic arterial blood pressure, which is compensated for by tachycardia.–21 It is also helpful in the differential diagnosis of the causes of intraoperative hemodynamic instability, such as hypovolemia, pericardial tamponade, and severe hypokinesia in the left ventricular wall. In a report published in 2004, TEE performed by cardiac anesthesiologists could detect visible emboli immediately before surgery in only 26% of patients undergoing emergent pulmonary embolectomy for severe PTE. However, with advanced technology and increased levels of expertise, a recent study in 2014 reported that the evidence of emboli and the right ventricular strain was found in 87% and 92% of patients with intraoperative massive PTE, respectively. Although delayed, TEE monitoring was commenced after the occurrence of hemodynamic instability, and we found right ventricular dysfunction that indicated the development of PTE. However, pulmonary emboli could not be visualized presumably because of the lack of attending anesthesiologist's proficiency.Thus, the intraoperative use of TEE was not indicated for a prompt and accurate diagnosis of PTE in our case. However, it is very regrettable that we did not use TEE after anesthesia induction because PTE actually occurred in our patient. TEE is widely used to confirm the presence of pulmonary emboli and right ventricular dysfunction during surgery without affecting surgical performance. Hence, they were not used in our patient. As anticoagulation therapy is indicated for patients with normal blood pressure and right ventricular function, the therapy was not provided to our patient. The presence of hemodynamic instability indicated systemic thrombolysis or surgical embolectomy, and surgical embolectomy was performed because the patient had absolute contraindications to systemic thrombolysis (major trauma or surgery in previous 3 weeks and active bleeding).At the beginning of CPR, we infused norepinephrine to increase arterial blood pressure and right ventricular contractility via strong α1 and modest β1 adrenergic effects, respectively. Another beneficial effect of norepinephrine is the restoration of coronary perfusion pressure gradients to the right ventricular subendocardium. In addition, fluid overloading was avoided to reduce right ventricular preload and systolic wall stress that worsen myocardial ischemia and ventricular interdependence. Although systemic administration of vasodilators, such as nitroglycerin or nitroprusside that release nitric oxide, can decrease pulmonary arterial and venous resistance, they may also worsen hypotension and systemic hypoperfusion because their action is not specific to the pulmonary vasculature.On the basis of our experience, negative CTPA and CT venography findings do not guarantee that intraoperative PTE does not occur in a patient with a moderate probability of having PTE. Although TEE was not used after anesthesia induction in our case, we strongly recommend the intraoperative use of TEE to investigate the causes of hemodynamic instability (including PTE) for patients with a moderate or high probability of having PTE. As tachycardia is an important predictive factor for PTE, anesthesiologists should consider the probability of intraoperative PTE occurrence if tachycardia is refractory to conventional treatments.In conclusion, a comprehensive understanding of the risk factors and clinical features of PTE and monitoring and maintaining hemodynamics are mandatory for managing patients at risk of PTE.Conceptualization: Jong Hae Kim, Jung A Lim.Data curation: Hyungseop Lim, Hyun Mi Kim, Jung A Lim.Writing – original draft: Jong Hae Kim, Hyungseop Lim, Jung A Lim.Writing – review & editing: Jong Hae Kim, Jung A Lim.


The aim of this study was to evaluate the effect of ageing and feeding strategies on the calpain protease system and meat quality traits in Braford steers.longissimus thoracis et lumborum (LTL) steaks aged for 2, 7, 14, and 21 days.Thirty Braford steers were employed; 15 animals were supplemented with corn silage during finishing and 15 were kept only on pasture. Meat quality traits and calpain system protein activity were evaluated in Aged meat showed higher pH and calcium content, while Warner Bratzler shear force (WBSF) decreased to day 21. No interaction between ageing and diet was seen for quality traits. Steers finished with corn silage showed higher values of water holding capacity, WBSF and free calcium, and lower values of pH and cooking loss. Calpain and calpastatin activities decreased with ageing. Finishing steers on pasture produced higher values of calpains and lower values of calpastatin activities. The higher values of calpain 1 activity were observed in muscles aged 2 days from pasture finished animals, and the lower activity of the inhibitor in the 21 days aged samples of the same group.These results suggest a diet by ageing interaction in calpains and calpastatin and this interaction impact in Warner Bratzler Shear Force in Braford LTL muscle. Bos indicus and Bos taurus breeds [Bos indicus genetic influence were tougher than steaks from Bos taurus cattle [post mortem strategies such as ageing could be effective for improving tenderness [In Argentina, beef cattle production systems have been based traditionally on pasture in temperate regions. As a result of the expansion of agriculture, beef cattle production was displaced to tropical and subtropical regions, and British breeds were replaced by other genotypes, such as composites between s breeds . Numerous cattle ,3. Nevernderness .post mortem proteolysis and meat tenderization [post mortem ageing  on meat tenderness and calpains (1 and 2) and calpastatin activity in beef longissimus thoracis et lumborum (LTL) muscle of Braford steers finished on pasture or with silage supplementation.One of the most important proteolytic systems involved in meat tenderization is the calpain system . In skelrization ,8. Howevrization . Giusti rization  suggesterization , demonstad libitum pasture and supplemented with corn silage  (Suppl) whereas the other 15 steers were fed ad libitum similar pasture without corn supplementation (Contr). Animals on both diets had free access to water. Steers, with 26±2 months of age in average, were slaughtered according to standard commercial procedures on the same day after mixing groups to avoid peri-slaughter effects. Carcasses were weighed for hot carcass weight (HCW) determination and were deboned following a 2-day cooling period at 1°C to 5°C. Samples from LTL muscle were vacuum-packed , and stored at 2°C±1°C in darkness for the corresponding ageing time ; subsequently they were frozen at −18°C until analysis. Before meat quality measurements, samples were thawed at 4°C±1°C overnight.The present study was conducted with animals from a commercial breeding herd in Santiago del Estero, in northwest Argentina (coordinates 27°17′34.3″S - 62°15′14.1″W). Animal handling and experimental procedures were in accordance with the Handbook of Procedures for Animal Welfare of the National Service of Animal Health of Argentina . As was described by Coria et al , thirty L*  and the chromatic coordinates a*  and b* . Measurements were carried out using a Minolta CR-400 colorimeter . The instrumental conditions were artificial D65 illuminant, 8 mm port size and 2-degree standard angle observer. Determinations were carried out on 2.5 cm thick steaks. Each sample was allowed to bloom for 45 min at room temperature prior to the first measurement, and six scans of each steak were averaged for statistical analysis. Then, muscle pH was measured using a Testo 205 pH meter . Each measurement was performed in triplicate, taking the mean values as the assay result.Meat colour was assessed using a Commission Internationale d’Eclairage Lab System, which provides values for colour components: The water holding capacity (WHC) was determined in duplicate following the filter paper press methodology described by Coria et al . The WHCThe cooking loss (CL) was determined only once by measuring the sample weight before and after heat treatment. Post cooking measurements were done after 20 min cooling at room temperature. Steaks were subjected to heat treatment with dry heat cooking . The CL was reported as the percentage of weight loss with respect to the initial weight of the sample.Instrumental tenderness was measured using the Warner-Bratzler test, assessing the resistance to a shear force in cooked meat. After cooking, samples were cooled down to 4°C overnight in refrigerator. Then, cores of 1.3 cm diameter were obtained parallel to the muscle fiber orientation and sheared once across the middle (perpendicular to the fibers) using a 1.016 mm Warner Bratzler blade (WB) probe in a TA.XT Plus Texture Analyzer . The test was performed with a crosshead speed of 2.0 mm/s (pre-test), 2.0 mm/s (test), 10.0 mm/s (post-test). The highest peak in Newton (maximum shear force) was measured in 7 to 10 cores per sample.Muscles samples (12 g) held at −80°C were removed from frozen storage and kept at room temperature for 10 min before they were finely diced, and then kept on ice again for 20 min before they were centrifuged at 20,000×g during 20 min at 4°C. An aliquot of the supernatant was mixed with 4 M KCl. Finally, calcium concentration was determined in triplicate using the Ca-Color AA kit  following the manufacturer’s instructions. Absorbance was read at 570 nm in a NanoDrop 2000c UV-Vis spectrophotometer .post mortem ageing [Meat texture is determined by collagen and is improved with prolonged m ageing . TherefoActivity was measured in frozen samples using the method described by Coria et al . BrieflyCalpain and calpastatin activity was determined using the casein zymography. Clear bands indicating calpain activity were quantified comparing the density of each band with the reference standard of each gel. Before statistical analysis, calpain band densities were calculated as a percentage of a standard included in each gel. The standard was a sample of pasture-finished steers taken randomly in the same experiment.Data were checked for normality using Infostat Software . Meat trThere was no significant interaction (p>0.05) between the main effects of all analyzed variables. Therefore, each effect is discussed individually.The effect of the different ageing time on meat quality is shown in L*) and yellowness (b*), even though no differences were observed in redness (a*). These results are in accordance with a study published by Stanišić et al [L* and b* during the ageing of longissimus dorsi muscle. Higher values observed in L* parameter during ageing could be explained by the protein degradation that occurs during ageing, that leads to the weakening of protein structures, resulting in a greater scattering of light, thus increasing the lightness of meat [b* parameters were described in Brangus steers finished on pasture, in Friesian x Simmental bulls raised in semi-intensive systems, and in Angus cows and bulls finished on pasture with 14 days of ageing [b* parameter increased during ageing and explained this difference by changes in the metmyoglobin amount due to meat surface oxidation. Marino et al [Significant effect of ageing was observed in colour parameters lightness . These results were consistent with previous publications, although the rate of decline varies. Papaleo Mazzucco et al [Warner-Bratzler shear force values decreased linearly with ageing time until day 21. The percentage changes were in average of 13.8%, 31.6%, and 42.4% after 7, 14, and 21 days, respectively, with respect to the initial value  with the weakest ability to bind water [The WHC and CL values remained constant during ageing time. These characteristics are pH-dependent . Post mond water . The WHCnd water . In thislongissimus muscle at 10 and 14 d of ageing were 0.64 and 0.97 mM, respectively. Furthermore, Senaratne [post mortem ageing could facilitate activation of calpains with subsequent proteolysis of muscle proteins resulting in an improvement on meat tenderness.Calcium concentrations were between 0.64 mM and 0.93 mM and increased linearly during ageing. Likewise, Parrish et al  reportedenaratne  documentsemimembranous and longissimus dorsi muscles of bulls evaluated at 5 and 12 days of ageing [post mortem, which is the minimal toughness that can be reached by meat after prolonged storage [In the present study, no differences were observed in between 2 and 21 days of ageing . Similarly, in other studies, the total collagen content did not differ in f ageing ,26. Tota storage . As expeFeeding strategy is the management factor which is most actively used in relation to improvement and/or control of animal performance, nutritional value, eating and technological quality. In agreement with Teira et al , corn-fipost mortem proteolysis like the calpain/calpastatin system can also affect CL values [No difference was found in WHC between feeding strategies. Similarly, in other studies, feeding strategy did not produce differences in WHC ,33. NeveL values . ExplainL values , pastureCooking loos and WHC values were higher than those obtained in other studies performed in Braford steers ,37. Nevepost mortem creating an influx of calcium into the skeletal muscle causing a decrease in calpain levels, or potentially an increase in calpastatin activity. Likewise, in the present study corn silage supplementation offered could produce the mentioned effect by increasing polyunsaturated fatty acids of sarcoplasmic reticulum membrane, making it more susceptible to oxidation and thereby causing early release of calcium [post rigor produce delayed and/or reduced proteolysis in beef, explaining the differences observed in the present study between feeding strategies in calpains and calpastatin activities and in WBSF values.As can be seen in  calcium . Althoug calcium  showed tThe WBSF of beef from the corn-supplemented group were higher than for beef from the pasture-only group. The higher pH values in grass-fed steers compared to grain-fed steers, influences the activity of endogenous enzyme systems at slaughter which, in turn may affect myofibril fragmentation and thus affects ultimate meat tenderness. According to Destefanis et al  classifiIn agreement with another study that has assessed the influence of diet on meat quality and collagen properties, in the present study the collagen content did not differ between the feeding groups . These aThere was significant interaction between ageing and feeding effect on calpain-1 and calpastatin activity. The higher values of calpain 1 activity were observed in muscles aged 2 days from pasture finished animals, and the lower activity of the inhibitor in the 21 days aged samples of the same group .post mortem ageing in beef [post mortem or until 5 days of ageing [Most literature indicates that calpain-1 activity is diminished by 14 or 21 days of  in beef . In the  in beef . Additiof ageing ,47. Difff ageing , calpastf ageing .pre rigor muscle environment is critical in determining the behaviour of myofibrillar proteins and their subsequent impact on meat quality attributes such as tenderness and colour. According to Dransfield [post mortem, depend on pH and have an important influence on tenderness. Muscle pH and temperature also interact continuously during rigor development as they impact on both physical shortening and proteolytic enzyme activity [In our study, finishing steers with corn silage produced lower levels of proteases and higher levels of the inhibitor. In previous research, Volpelli et al.  suggesteansfield , the effactivity . As prevpost mortem meat tenderness development during ageing [post rigor could result in higher calpastatin activity, and may have the potential to influence calpain activity and tenderness during ageing.Several studies present compelling evidence that the calpain system plays a primary role in g ageing ,6. Therepost mortem ageing in LTL muscle in Braford steers had a significant effect on meat quality, specifically in tenderness. The comprehensive understanding of factors is highly relevant to the development of effective ageing strategies to maximize the positive impacts of ageing. In this sense, the results obtained suggest that ageing produces significant differences up to 21 days. Furthermore, both proteases and their inhibitor activity remained until 21 days of ageing, contributing to the degradation process of muscle fibers and tenderization of meat. A feeding strategy by ageing interaction on calpain system proteins was observed. The higher values of calpain 1 activity were observed in muscles aged 2 days from pasture finished animals, and the lower activity of the inhibitor in the 21 days aged samples of the same group. Likewise, finishing steers on pasture produced lower free calcium concentration and calpastatin activity, and therefore more tender meat than corn silage supplementation. The tougher meat obtained could be due to inactivation of calpain by excessive calpastatin activity, stressing its role on meat tenderness.Feeding strategies and