Epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinases ErbB family and it is found to be overexpressed in gastric cancer. However, the mechanism of the regulation of the EGFR expression is still unknown. We used the Sequenom EpiTYPER assay to detect the methylation status of the EGFR promoter in normal and tumour tissues of 30 patients with gastric cancer. We also carried out quantitative real time PCR (qPCR) to detect the expression level of EGFR in our 30 patients. Notably, increased methylation level at EGFR promoter was found in tumour tissues than the corresponding adjacent noncancerous. In both Region I DMR and Region II DMR detected in our study, tumor tissues were significantly hypermethylated . Region I_⊿CpG_2 was also found to be associated with the presence of distant metastasis (P = 0.0323). Furthermore, the results showed a strongly significant association between the relative EGFR expression and the EGFR methylation changes in both Region I and Region II . Our findings help to indicate the hypermethylation at EGFR promoter in gastric cancer and it could be a potential epigenetic biomarker for gastric cancer status and progression. Gastric cancer is the third most common malignant tumor and the second most frequent cause of cancer death worldwideEGFR belongs to the family of receptor tyrosine kinases (RTK) ErbB, which consisting of HER1/EGFR/ErbB1, HER2/Neu/ErbB2, HER3/ErbB3 and HER4/ErbB4EGFR expression in cancer cells is tightly controlled, but the mechanism of the regulation of the EGFR expression is not fully studied. Epigenetic regulation is a biological mechanism by which gene expression is modulated through DNA methylation and histone modifications. DNA methylation is among the best studied epigenetic modifications and the methylation of cytosine at CpG dinucleotides is an important regulatory modification throughout the genomeIn our study, we used the Sequenom EpiTYPER assay to study the relationship between the methylation changes of the EGFR promoter and gastric cancer as well as its clinical characteristics such as histology differentiation, histologic grading, infiltration, TNM stage, and distant metastasis. We also carried out quantitative real time PCR (qPCR) to detect the expression level of EGFR to see the relationship between the EGFR methylation changes and the relative EGFR expression. We aimed to investigate whether methylation status of the EGFR promoter correlates with malignancy and patient outcome in gastric cancer.We analyzed 30 pairs of tissues (gastric cancer tissues and corresponding noncancerous tissues) from surgically removed primary gastric cancer in Ruijin Hospital of Shanghai Jiaotong University School of Medicine between July 2011 and May 2013. All participants were Han Chinese in origin and were examined histopathologically to confirm the diagnosis. All patients  were at initial presentation and had no radiotherapy or chemotherapy history before surgery. Control tissues were the corresponding non-cancerous mucosa from the stomach of cancer patients, and excised beyond 5–7 cm from neoplastic lesions. The tissue samples were immediately frozen in liquid nitrogen and stored at −80 °C until analysis. A standard informed consent was established and all the participants signed the consent. The study protocol was approved by the ethics committee of the Shanghai Institute for Biological Sciences, and the methods were carried out in accordance with the approved guidelines.Genomic DNA was isolated from 25 ug tissue samples using the QIAamp DNA Mini Kit following the manufacturer’s protocol  and a Thermo NanoDrop2000  was used to detect 260/280 nm UV absorbance ratio and concentration. Bisulfite conversion of DNA was carried out using the Epitect Bisulfite Kit .http://www.epidesigner.com) were as follows: Region I-F: 5′- aggaagagagGGGTAGTGAGTAGATTTGTGTTTGTT-3′, Region I-R:5′- cagtaatacgactcactatagggagaaggctATATCCCACTACCCCTATAACTCCC-3′; Region II-F:5′- aggaagagagGGAGTTGGGTGTTTTTATTTTAGATG-3′, Region II-R:5′- cagtaatacgactcactatagggagaaggctTACAAACCCAACCTATATCCAAATC-3′. Polymerase chain reaction (PCR) amplification was performed using a 5 ul reaction mixture and followed by SAP cleanup and T Cleavage. 20 ul H2O and 6 mg of Clean Resin  were added to the T Cleavage transcription products to remove bilvalent cation adducts. The samples were then transferred to a SpectroCHIP® array and sequenced on a MassARRAY analyzer . The amplicon comprised 26 CpG sites (8 of Region I and 18 of Region II ) were located in: Human Genome 19 assembly – chr7: 55,085,467-55,085,911 and chr7: 55,086,061-55,086,416 (GRCh37/hg19), Region I_CpG_1: 55,085,495; Region I_CpG_2: 55,085,520; Region I_CpG_3: 55,085,538; Region I_CpG_4: 55,085,661; Region I_CpG_5: 55,085,708; Region I_CpG_6: 55,085,860; Region I_CpG_7: 55,085,862; Region I_CpG_8: 55,085,886; Region II_CpG_1: 55,086,091; Region II_CpG_2: 55,086,122; Region II_CpG_3: 55,086,142; Region II_CpG_4: 55,086,146; Region II_CpG_5: 55,086,162; Region II_CpG_6: 55,086,164; Region II_CpG_7: 55,086,166; Region II_CpG_8: 55,086,172; Region II_CpG_9: 55,086,175; Region II_CpG_10: 55,086,210; Region II_CpG_11: 55,086,221; Region II_CpG_12: 55,086,248; Region II_CpG_13: 55,086,269; Region II_CpG_14: 55,086,272; Region II_CpG_15: 55,086,285; Region II_CpG_16: 55,086,288; Region II_CpG_17: 55,086,304; Region II_CpG_18: 55,086,391. The data for each CpG site or aggregates of multiple CpG sites were analysed using EpiTyper Software . And 10 DNA samples from 5 patients were randomly selected to be replicated in this study on the same bisulfite-converted sample, and yielded a highly consistent result (R2 = 0.95). Experiment on two independent bisulfate-converted samples of 5 patients was also yielded a highly consistent result (R2 = 0.92).Quantitative methylation analysis of DNA was performed using MassARRAY EpiTYPER assays . Two regions of the EGFR promoter were detected in our study . PrimersRNA was extracted from fresh, frozen tissue using RNA isolation reagents following the manufacturer’s protocol . cDNA synthesis was performed using SuperScript® III First-Strand Synthesis Kits according to the manufacturer’s protocol .TM 7 Thermal Cycler . The primers F: 5′-AGGCACGAGTAACAAGCTCAC-3′ and R: 5′-ATGAGGACATAACCAGCCACC-3′ were designed for the EGFR with 177 bp products. The primers F: 5′-GGAGCGAGATCCCTCCAAAAT-3′ and R: 5′-GGCTGTTGTCATACTTCTCATGG-3′ were designed for the GAPDH with 197 bp products. The PCR reactions included 2 × SYBR Green SuperReal PreMix Plus , 10 nM forward and reverse primer and 5 ul 80-times diluted cDNA template. Cycling conditions for all primer pairs were 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. All samples were performed in duplicate to capture intra-assay variability, and multivariate samples were randomly chosen to test the reproducibility of the assay. All of the samples were successfully detected and showed a coefficient of variation (CV)<1%. The expression of GAPDH was used to normalize that of the EGFR gene, and the expression level of EGFR was expressed as 2−⊿⊿Ct, where ⊿Ct = Ct (EGFR)-Ct (GAPDH), ⊿⊿Ct =⊿Ct (Tumour)−⊿Ct . The relative expression level was 1 in control tissue, while it was the value of 2−⊿⊿Ct in tumour tissue.Real-time Quantitative PCR was performed on an ABI VIIAhttp://www.r-project.org/). Student paired t-tests were used for the comparison of EGFR expression and methylation changes between primary tumors and adjacent noncancerous tissues. We also used the Wilcoxon signed-rank test to do the comparisons of paired tissues. The association of methylation changes as well as EGFR expression and clinical parameters were detected using linear regression analysis. Spearman correlation test was also used for the correlation analysis of participant characteristics and the methylation differences in DMR I and DMR II. A P value<0.05 was considered significant in these analyses.The statistical analyses were performed using the R program  patients, which is consistent with the established knowledge of EGFR overexpression in ~30% of GC patientsCombined the data of EGFR methylation and expression, we found the EGFR expression was significantly associated with the EGFR methylation status in both Region I  is expressed in primary breast tumors despite tumor-specific promoter methylationet al. found that hypermethylation of a specific CpG in prostate cancer cells resulted in transcriptional upregulation of 15-LO-1et al. explained the mechanism of genomic imprinting mediated by insulators as is present at the H19/Igf2 locus32Loeb, D. M. We also found that the methylation levels in Region I_⊿CpG_2 of the EGFR promoter were associated with the presence of distant metastasis, representing an unfavourable prognostic factor. Thus suggested that evaluation of high methylation levels of EGFR may be useful in identifying high-risk gastric cancer patients who eligible for multimodal treatments.In summary, we found hypermethylation at the EGFR promoter in gastric cancer, which could be one of the mechanisms for high expression level of EGFR in gastric cancer. Consequently the methylation levels of EGFR could be considered as a potential epigenetic biomarker for gastric cancer status and progression.L.H. and Y.L. supervised the experiment. X.W. and H.Z. designed the experimental protocol and carried out the experiment. Y.L., X.W., H.Z., J.Y., M.K., F.L., T.W. J.D. and Y.T. analyzed and discussed the experimental results. Finally, X.W. and Y.L. wrote the manuscript. All authors reviewed the manuscript.How to cite this article: Weng, X. et al. Hypermethylated Epidermal growth factor receptor (EGFR) promoter is associated with gastric cancer. Sci. Rep.5, 10154; doi: 10.1038/srep10154 (2015).


We propose to improve the prognostic assessment after radical prostatectomy (RP) by dividing high-risk prostate cancer (hrPCa) (according to the d’Amico classification) into subgroups combining 1, 2 or 3 criteria of aggressiveness  > 7).Data from 4795 hrPCa patients who underwent RP in two French university hospitals from 1991 to 2013 were analyzed. Subgroups were formed to determine whether an increasing number  of criteria of tumor aggressiveness was associated with poorer oncological results and early biochemical recurrence (BCR) (PSA > 0.2 ng/ml). These results were compared using Fisher’s exact test and BCR was compared according to the Kaplan-Meier method.p < 0.0001). However, no predominant negative criterion was found.Eight hundred fifteen patients were treated by RP for hrPCa (8 %). Four hundred eleven patients (79.5 %) presented 1 RF (Risk Factor), 93 (18.0 %) 2 RF and 13 (2.5 %) 3 RF. Lymph node invasion and positive margin rates were 12.4 and 44.1 %, respectively. The prognostic sub-stratification based on these 3 factors was significantly predictive for adverse pathologic features and for oncologic outcomes. BCR free survival was respectively 56.4, 27.06 and 18.46 % for 1RF, 2RF and 3RF  contains supplementary material, which is available to authorized users. Prostate cancer (PCa) is the most common form of malignant cancer in Europe and, the second leading of death attributable of cancer , 2. DespIn high-risk prostate cancer patients, the best course of treatment is often unclear, and the oncological outcomes appear heterogeneous among series and treatment options. Even though several treatment options, including RP, RT, and androgen deprivation therapy (ADT) alone or in combination, are available but the recurrence rate remains high regardless of the type of treatment . RecentlThus, large multicentric series have reported that a significant percentage (about 30 %) of PCa preoperatively defined as high risk, was organ-confined and favourable in RP specimens –13. ThesThe aim of this study was to define subgroups combining 1, 2 or 3 criteria of aggressiveness  > 7) among surgically treated hrPCa and to define their risk of progression.After institutional review board approval , we retrospectively examined data from 815 consecutive patients who underwent radical prostatectomy and bilateral extended pelvic nodes dissection (in 98.1 % cases) for clinical high-risk prostate cancer in D’Amico risk classification  between 1990 and 2013 in two French academic centers . Surgical procedures were performed by 7 different senior surgeons, who used standardized techniques  and applied the same anatomic template during pelvic lymph node dissection, as previously described . We exclThe clinical stage was assigned according to the 2002 TNM staging system, prostate biopsy cores were obtained with transrectal ultrasound guidance, using a >10-core biopsy protocol, and pretreatment PSA was measured before digital rectal examination. Genitourinary pathologists assessed the biopsy and pathologic gradings according to the Gleason gradings system before 2005 and the modified ISUP Gleason score after 2005. The pT stage was graded according to the 2002 AJCC staging system for PCa.Biochemical recurrence (BCR) was defined as a PSA value 0.2 ng/ml after RP, confirmed by at least two consecutive measurements.Data were summarized by frequency and percentage for categorical variables, and by median and range for continuous variables. Comparisons between groups were performed using the Mann–Whitney rank sum test for continuous variables and Chi square or Fisher’s exact test for categorical variables.Biochemical Recurrence Free Survival was calculated from the date of the surgery to the date of the diagnosis of the biochemical recurrence. BCR was estimated using Kaplan-Meier method and univariate analysis was performed using the log rank test. Logistic regression models were built to determine the independent predictive value of PSA, Gleason score and clinical stage at diagnosis.p < 0.05. Stata 13.0 software  was used for all statistical analysis.All statistical tests were two sided, and differences were considered statistically significant when p < 0.001). The proportions of the clinical stage were different between the 3 groups: cT1c-cT2b: 91.2 % (n = 375), 35.5 % (n = 33) and 0 %; cT2c-cT3: 8.8 % (n = 36), 64.5 % (n = 60) and 100 % (n = 13)) respectively for 1RF, 2RF and 3RF (p < 0.0001). Furthermore, the proportions of Gleason sum >7 found into prostate biopsy was greater in population with less RF (43.6 % (n = 179), 62.4 % (n = 58) and 100 % (n = 13) respectively for 1RF, 2RF and 3RF (p < 0.0001). In overall population, the pT stage was pT2 in 29 % of patients, pT3a in 37.9 %, pT3b in 32.9 %, and pT4 in 0.2 %. Positive surgical margin was reported in 44.1 % of cases (n = 228) and increased within the risk sub-stratification: 40.6, 58.1, and 53.8 % for 1RF, 2RF and 3RF respectively (p < 0.0007)). Lymph node metastasis was noted in 12.4 % of patients: 9.2 % (n = 37), 22.6 % (n = 21) and 38.5 % (n = 5) for 1RF, 2RF and 3RF respectively (p < 0.0001). The number of nodes removed was exactly the same for the 3 groups of patients (median 10 LN (1–39). The majority of the patients had only one criterion of aggressiveness (79.5 %) : radiotherapy in 1.5 % of cases, androgen deprivation therapy in 2.1 % of cases, and RT combined with ADT in 1.7 % of cases. Salvage treatments  were salvage radiotherapy in 18 % of cases, androgen deprivation therapy in 11.8 %, RT combined with ADT in 0.8 % and chemotherapy in 4.5 %.Overall, median follow-up was 25.2 months. The 2-year and 5-year RFS rate was 55.21  and 41.67  respectively.p < 0.0001) , 27.06 %  and 18.46 %  for 1RF, 2RF and 3RF .Figure In this current study, we demonstrated the different outcomes in high risk PCa according to the number of preoperative risk factors identified: the increasing number of RF was correlated with poorer BCR-free survival.Indeed, at present, urologists face the dilemma of deciding which treatment is best adapted for hrPCa patients: ADT, radiotherapy, RP or a multimodality approach. When asked, most physicians commonly say they prefer ADT associated with radiotherapy . NeverthPreoperatively, the identification of high-risk PCa can be based on at least three well-defined predictors of the extent of the disease and the post treatment outcome as defined by d’Amico et al. . Others The first population identified, characterized by 1 risk factor and benefited the most from surgery because of favorable cancer control with good BCR-free survival rates. Similarly, prior studies have attempted to better stratify patients with hrPCa based on the number of risks factors , 16. JonConversely, the risk of recurrence appears to be very high in patients with high-grade disease and who still have at least 2 risk factors despite radical surgery along with a great deal of adjuvant and salvage treatment strategies. Multimodal treatment strategies are probably warranted in those cases. Prospective trials on the timing of adjuvant or salvage ADT and RT will hopefully provide some answers.In our study, the PSM rate was around forty percent and more then 70 % had pT3 disease, but only 1.5 % of the patients received adjuvant radiotherapy (ART). On the other hand, salvage treatments (35.2 %) were often proposed. Most of recommendation proposed RT after RP in cases of extracapsular extension, GS > 7, PSM because of a high risk of local recurrence. Currently, the question about the time of treatment is always open: immediate adjuvant RT or salvage RT after biological monitoring. 3 studies showed a benefit of immediate ART  in comparison with no adjuvant treatment. But the time of treatment is still discussed and we should wait the results of AFU-GETUG 17 trial. Some recent studies showed a better functional recovery if RT is not performed immediately after RP. For these two reasons, urologists in this study preferred salvage RT.Finally, we have demonstrated that men with only one high risk factor had a better BCR-free survival rate than men with two or more.Therefore, we identified a subgroup, for which RP led to favorable outcomes, corresponding to the ideal candidate for RP in high risk PCa. Recently, Fossati et al. showed an increased diagnosis of localized and less extensive high-grade prostate cancer was observed over the last two decades. In this context, patients with high-risk disease selected for radical prostatectomy had better cancer control over time .The nomogram published by Briganti et al. has led to selection improvement for patients candidates for RP as a primary treatment for high risk PCa . This prOur study is not without certain limitations. Firstly, though the study period frame was long, it featured (ranging from 1990 to 2013) and with only 523 patients, sub group analysis based on years of surgery was not statistically achievable. Secondly, the changes over time in pathology assessment (Gleason score grading), patient selection and operative techniques such as nerve-sparing procedures impacting the margin status, presented unavoidable biases. No centralized pathology was available between the two centres. Finally, because of the lack of key data, the final analysis only included 523 of the 814 patients identified.In multivariate analysis, Gleason score and number of RF was not an independent factor as showed in other studies , 18. We Finally, it is surgery (RP) itself that provides the most accurate knowledge of the pathologic tumour and to adapt adjuvant therapies based on the risk of recurrence (pT stage and lymph node invasion).Thus, the use of such a sub-stratification prognostic approach should improve the accuracy of predicting pathologic and oncologic results, thus optimizing the selection of patients for whom primary cancer control is possible. However, even when optimal predictions are made, those high-risk patients showing adverse pathologic outcomes should still be considered as candidates for a multimodal, combined approach. In these cases, cancer control after surgery should be optimized by either adjuvant RT, HT, or a combination of both treatments.Finally, oncologic results after RP are heterogenous within the hrPCa risk group. Sub-stratification based on three well-defined criteria leads a better identification of the most aggressive cancers. In contrast, the presence of a single RF seems to be the most appropriate circumstance for RP. Such results may help urologists in scheduling post-operative monitoring and management.ADT, androgen deprivation therapy; BCR, biochemical recurrence; GS, Gleason score; hrPCa, high risk prostate cancer; HT, hormo; LNI, Lymph node involvement; PCa, Prostate cancer; PSA, prostate specific antigen; RF, risk factor; RP, radical prostatectomy; RT, radiation therapy; SC, specimen confined


Acropora yongei and Pocillopora damicornis from the southernmost geographical limit of these species at Salmon Bay, Rottnest Island (32°S) in Western Australia. Changes in buoyant weight were normalised to colony surface areas as determined from both X-ray computed tomography and geometric estimation. Extension rates for A. yongei averaged 51 ± 4 mm y−1 and were comparable to rates reported for Acroporid coral at other tropical and high latitude locations. Mean rates of calcification for both A. yongei and P. damicornis in winter were comparable to both the preceding and following summers despite a mean seasonal temperature range of ∼6 °C (18.2°–24.3 °C) and more than two-fold changes in the intensity of downwelling light. Seasonal calcification rates for A. yongei (1.31–2.02 mg CaCO3 cm−2 d−1) and P. damicornis (0.34–0.90 mg CaCO3 cm−2 d−1) at Salmon Bay, Rottnest Island were comparable to rates from similar taxa in more tropical environments; however, they appeared to decline sharply once summer temperatures exceeded 23 °C. A coral bleaching event observed in December 2013 provided further evidence of how coral at Rottnest Island are still vulnerable to the deleterious effects of episodic warming despite its high latitude location. Thus, while corals at Rottnest Island can sustain robust year-round rates of coral growth, even over cool winter temperatures of 18°–19 °C, there may be limits on the extent that such environments can provide refuge against the longer term impacts of anthropogenic climate change.To assess the viability of high latitude environments as coral refugia, we report measurements of seasonal changes in seawater parameters  together with calcification rates for two coral species,  Declining rates of coral growth have stimulated debate over the future of coral reefs and the mechanisms or strategies that may be available for hermatypic corals to endure in the face of Earth’s rapidly changing climate. Recent studies have suggested that range expansions to ‘high latitude refugia’  could offer one such pathway for survival from increasing seawater temperatures . Already−2 d−1 and Ωar > 3.5; Temperature is generally thought to be the primary environmental driver controlling the geographic distribution of coral, rates of coral calcification and reef formation worldwide ; howeverAcropora spp. coral community during the Last Interglacial (∼125 ka ago) when ocean temperatures were 1° to 2 °C higher than present day (−1) in two of the coral species at Rottnest Island , nor any corresponding measurements of the environmental conditions under which these coral have been growing. Thus, we do not know the degree to which these coral species have acclimatized to the conditions at Rottnest Island or to what extent this habitat can support the continued growth and accumulation of hermatypic coral over the long term.Rottnest Island  off the coast of Perth in Western Australia (WA) hosts a range of temperate to sub-tropical habitats due to the influence of the perennial Leeuwin Current, which transports warm, oligotrophic water poleward along the WA coastline . These wsent day . To dateA. yongei and P. damicornis at Rottnest Island respond to seasonally varying changes in light, temperature and seawater chemistry over two summers and one winter. We then compare our results to the seasonal growth patterns of similar coral taxa living in other high and low latitude reef environments to better determine the suitability of this high latitude environment as coral refugia, thereby supporting the continued growth of hermatypic coral given anticipated trends in climate-driven ocean warming. This study will provide important baseline calcification rate data for high latitude hermatypic branching corals at the edge of their geographical distributions in WA. We initially hypothesized (1) that both A. yongei and P. damicornis at Rottnest Island would exhibit a growth ‘hiatus’ or decrease in rates of calcification during the winter months r months ; both asThe Rottnest Island Authority (RIA) provided approval and permits for this research (#2012/164683 and 2014/227143). The Government of Western Australia Department of Parks and Wildlife (DPaW) provided research permits and license to take fauna for scientific purposes (#SF009096 and #SF009705) and the Government of Western Australia Department of Fisheries provided research permits and exemption from the Fish Resources Management Act 1994 (#2202 and #2410).A. yongei coral are currently reported to occur . Coral and filamentous turf algae co-inhabit this inner reef environment exhibiting mixed assemblages typical of other near-shore habitats at high-latitude  for Rottnest Island was measured from December 2013 through July of 2014 using light loggers  calibrated using a high precision LiCor 192A cosine PAR sensor that was factory calibrated. Additional in-water measurements of light were made on the 24th January 2014 and on the 30th July 2014 to estimate depth-dependent light attenuation coefficients (dk) in summer and winter, respectively. During each field sampling trip, discrete samples of water were collected from the study site using a 2.2-L Van Dorn bottle and measurements of pH on the total hydrogen ion concentration scale (pHT) were made using a Schott Handylab pH 12 m (±0.03) using both seawater (‘Tris’) and NBS buffers and calibrated against a certified reference pH buffer provided by Andrew Dickson at the Scripps Institute of Oceanography (Batch #123). A SeaFET ocean pH sensor  was also deployed at a depth of 2.5 m in Salmon Bay for 5 days in January 2014  and 12 days in July 2014  to measure diurnal changes in pHT (±0.03). Additional seawater samples were collected and filtered with 0.7-µm nominal glass fibre filters  and taken back to UWA for the analysis of total alkalinity (TA) and dissolved inorganic nutrients. TA (±5 µeq kg−1) was measured using a modified version of the spectrophotometric approach developed by pCO2), and aragonite saturation state (Ωar) were calculated from measured pHT, TA, and temperature using the CO2SYS program . Daily down-welling planar photo-synthetically active radiation  within Salmon Bay, Rottnest Island in December 2012. Samples were buoyantly weighed, epoxied onto acrylic plates (n = 5 of A. yongei and n = 3 of P. damicornis) and could not be included in the study, thus resulting in a final sample size of n = 5 for A. yongei and n = 6 for P. damicornis at the beginning of the experiment . To increase our overall sample number, additional coral samples were deployed in February 2013 (n = 11 of A. yongei and n = 3 of P. damicornis) all of which survived, giving a total sample size of n = 16 for A. yongei and n = 9 for P. damicornis. In November 2013, we deployed even more samples of each coral species to investigate potential size effects on rates of calcification for both species (n = 6 of A. yongei and n = 4 of P. damicornis); however, data from these colonies were used solely to examine relationships between calcification and colony size  calibrated against two different rectanguloid sections of coral skeleton. Reconstructions of the scans were performed by NRecon software and surface area analyses were performed in CTAn software . To prevent sacrificing any living sample colonies growing on tiles during the course of the experiment, the surface area of each colony was calculated using regressions of total surface area versus dry skeletal weight created from a range of separately collected colonies of P. damicornis and A. yongei whose individual masses spanned a very large range from 2–370 g . Due to size restraints, the X-ray CT scanner was only used to analyze small colonies less than 30 g (<15 cm); therefore, for larger colonies, we estimated total colony surface area using geometric estimation and then corrected these estimates based on regressions of X-ray CT surface area against geometric estimation surface area . Rates of calcification for each coral colony were then calculated based on the change in dry skeletal mass, the total surface area estimated from the initial and final mass, and the number of days between buoyant weighing.Changes in skeletal mass were calculated from the change in buoyant weight normalized to total bioactive surface area measured using both geometric estimation  and X-raA. yongei colonies randomly selected from across the eastern part of Salmon Bay to assess how growth (extension) rates for A. yongei varied across the total study area. Each branch was tagged 2 cm from the growing tip using coloured plastic cable ties  with a Bonferroni post-hoc test was performed to test for significant differences in rates of calcification and rates of linear extension across time. After finding no significant effect of colony location on linear extension rate , colony data were pooled. A priori analyses further indicated that rates of calcification at the two locations within Salmon Bay  did not significantly differ ; therefore, these data were also pooled.Seasonal changes in TA, pHA. yongei: 1.67 vs. 1.60 mg CaCO3 cm−2 d−1; P. damicornis: 0.66 vs. 0.70 CaCO3 cm−2 d−1) and we found that there were no significant differences between the two deployments ; therefore, these data were pooled. Changes in seasonally averaged calcification rates and linear extension rates were also tested using a one-way ANOVA. For all data analyses, measurements from November through April were treated as ‘summer’ and measurements from May through October as ‘winter’ based on our temperature records . Before conducting rANOVA tests, the dependent variables were tested for sphericity (Mauchly’s test). When calcification rate data for A. yongei and P. damicornis deviated from the assumptions of sphericity, we adjusted the degrees of freedom using the Greenhouse-Geisser adjustment with an epsilon of 0.35 and 0.43, respectively  than in winter  in Salmon Bay measured at the depth of the tripod (1.5 m) was very low in both seasons (0.053 m−1 during summer vs. 0.075 m−1 during winter), corresponding to a seasonal range in light reaching the benthos of 15–48 mol m−2 d−1. Light levels were ∼80% of surface values at the tripod site (1.5 m) and ∼70% at the brick site (2.5 m). Thus, light levels these two deployment locations generally differed by only ∼10%.Weekly average water temperatures were found to range from 18.2 °C (July 2013) to 24.3 °C (February 2013) giving a seasonal temperature range of ∼6 °C . These ft test, t(26) = − 1.19, p > 0.05, T ranged from 7.9–8.19, on average summer pHT was only 0.03 pH units higher than winter  = 1.04, p > 0.05, T over diurnal cycles indicate daily average values that were close to equilibrium with the atmosphere  = 2.54, p < 0.05, −3, t test, t(12) = 1.12, p > 0.05, −3; t test, t(13) = − 6.22, p < 0.05, −3,, t-test, t(13) = − 4.12, p < 0.05, TA did not differ between seasons  = 3.2 cm2, R2 = 0.98; P. damicornis: y = 0.8 × + 5.0, RMSE = 4.8 cm2, R2 = 0.95, A. yongei: RMSE = 1.1, R2 = 0.99, y = 4.1x0.97; P. damicornis: RMSE = 1.0, y = 2.1x1.20, R2 = 0.99, A. yongei  = 7.0, p < 0.05) and P. damicornis  = 3.9, p < 0.05). Post-hoc tests for A. yongei showed that the significant changes over time were attributed to the abrupt calcification rate declines of 25% during February 2014, which was followed by consistently low rates throughout autumn 2014; both time points were found to be significantly different from five other previous time points  = 1.4, p > 0.05). Similarly, average rates of calcification for P. damicornis did not significantly differ between seasons  = 3.0, p > 0.05).Mean rates of calcification over the entire study ranged from 1.31–2.02 mg CaCOmicornis  and 8B. P. damicornis showed a negative linear relationship with seasonal changes in temperature  or P. damicornis .Calcification rates for −1, mean of 51 ± 4 mm y−1, A. yongei coral at Rottnest Island, similar to those reported more than 20 years ago  = 4.4, p < 0.05); however, post hoc tests revealed that this was a result of the significantly higher rates of extension in the month of April 2013 , which was significantly higher than four of the other time points . Average rates of linear extension for A. yongei did not significantly differ between seasons  = 1.7, p > 0.05). No significant correlation was found between mean rates of linear extension and rates of calcification .Relatively high linear extension rates  reef flat were visibly bleached  using state-of-the-art X-ray CT scanning  and P. damicornis (0.34–0.90 mg CaCO3 cm−2 d−1) at Salmon Bay, Rottnest Island were comparable to coral from other high latitude sites such as Porites astreoides (1.45–2.19 mg CaCO3 cm−2 d−1) and Diploria strigosa (0.68–1.78 mg CaCO3 cm−2 d−1) in Bermuda at 32°N  calcified at rates between 0.8–1.4 mg CaCO3 cm−2 d−1; however, they used geometric estimation to calculate surface area, which we found overestimated the true surface area by ∼20%. Thus, the rates of A. pulchra calcification that they reported at Ningaloo Reef are more likely to be the range of 1.0–1.7 mg CaCO3 cm−2 d−1; values that are nonetheless comparable to the annual calcification rates that we measured for A. yongei at Rottnest Island, ∼1,000 km south of Ningaloo Reef. The calcification rates for P. damicornis at Salmon Bay, Rottnest Island were also similar to rates measured during the same years by 3 cm−2 d−1). P. damicornis using the more accurate method of X-ray CT. Thus, both the summer and winter calcification rates at Salmon Bay, Rottnest Island are comparable to rates for similar coral taxa in more tropical environments despite the much lower temperatures for this high latitude environment . We recognise, however that a lack of visible bleaching does not preclude the existence of thermal stress  in Salmon Bay, Rottnest Island, measured using a hand-held GPS on snorkel in December 2013.Maps showing the distribution of Click here for additional data file.10.7717/peerj.781/supp-2Figure S2Acropora yongei, and (B) bleached Pocillopora damicornis on the shallow reef at Rottnest Island in December 2013.Photographs showing (A) bleached Click here for additional data file.10.7717/peerj.781/supp-3Figure S3T in Salmon Bay, Rottnest Island taken during (A) the 19th–25th of January 2014 and (B) the 18th–30th of July 2014. The solid red lines denote the diurnal averages of 8.03 for January and 8.07 for July. The dashed black lines represent the hypothetical pH in equilibrium with the atmosphere of 8.044 for January and ∼8.048 for July at in situ temperatures (pCO2 = 395 µatm).Continuous measurements of pHClick here for additional data file.


A refractive index sensor composed of two straight metal-insulator-metal waveguides and a ring resonator is presented. One end of each straight waveguide is sealed and the other end acts as port. The transmission spectrum and magnetic field distribution of this sensor structure are simulated using finite-difference time-domain method (FDTD). The results show that an asymmetric line shape is observed in the transmission spectrum, and that the transmission spectrum shows a filter-like behavior. The quality factor and sensitivity are taken to characterize its sensing performance and filter properties. How structural parameters affect the sensing performance and filter properties is also studied. Surface plasmon polaritons (SPPs) are charge density waves, formed by the interaction between incident photons and free electrons in a metal surface, that travel along the metal–dielectric interface . Since SSPP waveguides, including insulator-metal-insulator (IMI) waveguides , metal-iHere, a MIM waveguide-coupled ring resonator structure is discussed. In this study, to realize the Fano resonance, we etched one chip repeatedly, rather than multiple chips with different parameters, to adjust the coupling between the straight waveguides and ring resonator. The filter properties and the refractive index sensitivity are simulated by finite-difference time-domain (FDTD) methods. In addition, the effects of structural parameters on the transmission spectra are investigated.S is the dipole source, r is the radius of the ring resonator, d is the coupling distance between the waveguide and the ring resonator, and l is the distance between the sealed end of the output waveguide and the center of the ring resonator. To ensure that only the fundamental transverse magnetic mode is supported in the MIM waveguide, the widths w of the waveguides and ring resonator are fixed at 50 nm 1/2, where εin and εm are the dielectric constants of the insulator and metal, respectively. The wave vector in free space is given by k0 = 2π/λ0, and κ can be determined with Equation (2). Thus, the real part of the effective index Re (effn) of a MIM waveguide can be expressed as Re(effn) = mε + (κ/κ0)1/2. The effective wavelength sppλ of the SPPs can be obtained with the equation spp = λλ0/Re(neff). Output energy flow outP is the integral of the x-component of the Poynting vector at the interface of the output port. The transmittance is given by T = Pout/Pin, where Pin is the input energy flow. We use the quality factor Q = 0λ/FWHM to evaluate the filter properties of the MIM waveguide-coupled ring resonator, where 0λ is the resonance wavelength and FWHM is the full width at half maximum of the resonance peak.In Equation (2), d = 6 nm, r = 300 nm, and l = 0 nm. There are resonances at peak I (λ0 = 0.887 μm) and peak II (λ1 = 0.675 μm). The FWHM of peak I is 0.034 μm, and that of peak II is 0.028 μm. Propagation of SPPs in the MIM waveguide-coupled ring resonator is shown in Hz| distributions are calculated. Hz| field distributions of the MIM waveguide-coupled ring resonator structure at 0.675 µm, 0.887 µm, 0.611 µm, and 0.772 µm, respectively. Standing wave modes are formed in the ring. The |Hz| field distributions of the pass band correspond to the resonance peaks at 0.675 and 0.887 µm, respectively, while, the stop band distributions correspond to resonance peaks at 0.611 and 0.772 µm, respectively. In addition, standing waves help to explain the steady-state |Hz| distributions in the ring.To characterize the SPP resonances in the ring, the steady-state magnetic field |L = m(λspp/2), where m is a positive integer. For λ = 0.675 µm, Re (effn) of the MIM waveguide is 1.4367 and the corresponding effective SPP wavelength sppλ is 0.470 µm. Therefore, m = 2sppL/λ = 8, which agrees well with the numerical results in λ = 0.887 µm, Re (effn) = 1.41, sppλ = 0.629 µm, and m = 6. For λ = 0.611 µm, Re(effn) = 1.4529, sppλ = 0.421 µm, and m = 9. Finally, for λ = 0.772 µm, Re(effn) = 1.4211, sppλ = 0.543 µm, and m = 7. Overall, the resonant wavelengths in L = m (λspp/2). SPPs cannot be coupled into the output waveguide when m is an even number. The calculations based on the standing wave condition are consistent with the steady-state distributions of the normalized magnetic field |Hz| simulated with the FDTD method.The standing wave condition is r affects the transmission of the MIM waveguide-coupled ring resonator, it was increased from 270 nm to 300 nm in 10-nm increments with d = 6 nm and l = 0 nm. r = 270 nm, 280 nm, 290 nm, and 300 nm. The transmittances of the I and II resonance peaks are approximately 0.7 and 0.5, respectively. With increasing r, the resonance peaks exhibit red shifts. When r > 290 nm, a new resonance peak emerges at shorter wavelength. Q factor on r. At increased r, both peaks exhibit red shifts and the Q factor increases. The coupling length between the straight waveguides and the ring resonator increases with increasing r, leading to a stronger coupling coefficient. In addition, the longer resonance length increases Q and the SPP filter has higher resolution at higher Q.To investigate how the radius d over the range 6–20 nm in 2 nm increments, with r = 300 nm and l = 0 nm. Both resonance peaks I and II exhibit slight blue shifts with increasing d, as shown in Q factors as a function of d. The transmittance decreases with increasing d; however, the Q factor increases with increasing d. A smaller coupling coefficient (weaker coupling strength) will result in a decreased transmittance, a narrower FWHM, and a higher Q factor. Some of the SPPs that fail to form resonances in the ring dissipate because of ohmic heat loss. Accordingly, the noise will decrease, leading to an increased Q factor.l = 80 nm, and values of w, r, and d that are the same as those in l = 0 nm, 40 nm, and 80 nm with fixed r = 300 nm and d = 10 nm. Resonance peak I splits into two resonance peaks when l ≥ 40 nm. The splitting increases with increasing l, revealing Fano resonances.Q factor of resonance peak I at 0.887 µm as a function of l. The Q factor increases markedly as l increases from 20 nm to 80 nm. When l = 80 nm, the asymmetric line shape of peak I clearly reveals the Fano resonance and the smaller FWHM entails a larger Q factor.n. The transmission spectra with n = 1.01, 1.03, and 1.05 for l = 80, r = 280 nm, and d = 10 nm are plotted in n. The value δ = Δλ/Δn quantitatively characterizes the refractive index sensitivity of the Fano and Lorentz peaks. As shown in δ for the peaks I, II, III, and IV are 868.4, 921, 605.3, and 300, respectively. The Fano peaks are thus more sensitive to n variations than the Lorentzian peaks.To simulate the refractive index sensitivity of the Fano resonance, the MIM waveguide-coupled ring resonators were filled with dielectrics having various refractive indices r increases. The transmittance decreases and the Q factor increases with increasing d, and the resonance peaks exhibit slight blue shifts. Fano resonance occurs when the sealed end of the output waveguide is moved from the ring center to the left. It is generated by the interference between the broadband resonance of the ring resonator and narrow-band resonance of the sealed end of the output waveguide. The Fano resonances exhibit a higher refractive-index sensitivity relative to that of the Lorentzian resonances. This study thus provides a pathway based on MIM waveguide-coupled resonators for designing sensors that detect refractive index changes. In addition, electromagnetic inverse scattering has revealed that neural networks can be used to verify ring resonator models because of their higher computational accuracy and optimization [The transmission properties of a MIM waveguide-coupled ring resonator are simulated by FDTD methods. The transmission spectra display red shifts when the ring radius mization ,24,25.


Paraconiothyrium variabile in both non-assisted and hydroxybenzotriazole-aided form against six azo dyes.Enzymatic elimination of synthetic dyes, one of the most environmentally hazardous chemicals, has gained a great interest during the two last decades. The present study was performed to evaluate the decolorization and detoxification potential of the purified laccase of Km of 0.49 mM and Vmax of 189 mmol/min/mg. Evaluation the toxic effect of laccase-treated dye sample based on the growth inhibition of standard bacterial strains revealed decrease in toxicity of all applied dyes after treatment by laccase.The obtained results showed that Acid Orange 67, Disperse Yellow 79, Basic Yellow 28, Basic Red 18, Direct Yellow 107, and Direct Black 166 were decolorized up to 65.3, 53.3, 46.7, 40.7, 34, and 26.2 %, respectively, after 1 h treatment with laccase (0.5 U/mL). Addition of HBT up to 5 mM enhanced decolorization percent of all the investigated dyes. The results of kinetic study introduced the monoazo dye of Acid Orange 67 as the most suitable substrate for laccase with P. variabile laccase as biocatalyst efficiently decreased the toxicity of all studied synthetic azo dyes.Application of the  Synthetic dyes are being increasingly applied in the textile, paper, cosmetics, pharmaceutical, leather dyeing, color photography, and food industries –4. Azo dTrametes versicolor for decolorization of four dyes including Red FN-2BL, Red BWS, Remazol Blue RR and Blue 4BL was reported by Mendoza et al.  was then utilized for determination of growth inhibition percent (GI%), where OD600S and OD600C are OD600 of sample and OD600 of control, respectively.A modified microtoxicity assay procedure using six standard bacterial strain  with Dunnett’s T3 post hoc test were employed using the SPSS software .Three replicates of each above mentioned experiment was performed and mean ± standard deviation of each value was reported. In order to calculate the statistical significance  between mean values the independent sample As presented in the time course of decolorization of six synthetic azo dyes Fig. , laccaseP. varibile. In the study of Zeng et al. [T. trogii SYBC-LZ didn’t decolorize Acid Red 1 (a monoazo dye) and Reactive Black 5 (a diazo dye) while the highest decolorization percent (80.4 %) achieved for the anthraquinonic dye of Remazol Brilliant Blue R (RBBR). Grassi et al. [T. trogii while more than 80 % of anthraquinonic dye of RBBR and Indigo Carmine was eliminated at the same time.Application of laccases and laccase mediated system (LMS) to decolorize mutagenic and carcinogenic synthetic colorants has been received great interest during two last decades , 35. Litg et al.  it was f percent 0.4 % achThe results of pH alteration on decolorization of investigated dyes were illustrated in Fig. p-value < 0.05) by elevating of temperature to 70 °C which was in agreement with the acquired results of Ashrafi et al. [Decolorization percent of all studied dyes was gradually increased by enhancing the reaction temperature and maximum decolorization of Disperse Yellow 79 (60 %), Acid Orange 67 (71.3 %), and Basic Yellow 28 58.4 %) was observed at the temperature of 45 °C Fig. . Basic R8.4 % wasi et al. .p-value < 0.05) after increasing of laccase activity from 0.5 U/mL to 1 U/mL. Maalej-Kammoun et al. [As represented in Fig. n et al.  determinAs represented in Fig. T. trogii’s laccase. They introduced HBT as the best mediator for maximum decolorization [Treatment of hazardous organic pollutants and synthetic dyes using laccase-mediated systems, especially in the case of laccases with low redox potential and recalcitrant pollutants, has been well documented . For exarization . Laccaserization , 40. Howrization . So, litrization , 40.Km value belonged to the monoazo dye of Acid Orange 67 (0.49 mM) and triazo dye of Direct Black 166 (4.25 mM), respectively. Unsuitability of Direct Black 166 as a laccase substrate is evident from the high value of the calculated Km of this colorant (4.25 mM) and its related maximum velocity (33 mmol/min/mg). The resistance of azo dyes to laccase-catalyzed oxidation compared to other classes of colorants has been well documented. Grassi et al. [T. trogii B6J among tested synthetic dyes. Generally, presence of electron withdrawing groups such as –SO3H, −SO2NH2 together with the high molecular weight decreases the ability of laccases for elimination of azo dyes [B. subtilis was able to decolorize Direct Blue 1 (a diazo dye), Reactive Black 5 (a diazo dye), and Sudan Orange G (a monoazo dye) by 88, 85 and 98 %, respectively at alkaline pH and in the absence of redox mediators [Based on the kinetic parameters of the laccase toward studied dyes Tabale 1) the lowest and the highest i et al.  reported the lowege 67 0.4 mM and tThe obtained results of energetic assessment for laccase-mediated decolorization Table  indicateM. luteus, S. aureus, B. subtilis, E. coli, P. aeruginosa, and S. typhi by 68, 56, 61, 51, 43, and 47 %, respectively, when exposed to each bacterial strain for 10 h. On the other hand, the monoazoic dye of Disperse Yellow 79 represented GI% of 30, 23, 27, 21, 14, and 18 % for M. luteus, S. aureus, B. subtilis, E. coli, P. aeruginosa, and S. typhi, respectively  after physicochemical or biological treatment many biological assays such as inhibition the growth of bacterial, yeast, and mammalian cell lines as well as phytotoxicity evaluations on plant seeds were developed , 41, 42.n et al.  reporteds et al.  found loP. variabile for decolorization of 6 azo dyes in the presence and absence of HBT as the laccase mediator was evaluated. All the synthetic dyes were decolorized using non-assisted laccase. In addition, decolorization percent of all applied colorants improved in the presence of HBT (up to 5 mM) as laccase mediator. Evaluation the toxicity of laccase-treated dye samples assisted by growth inhibition percentage (GI%) of standard bacterial strains revealed significant decrease in the toxicity of all studied dyes after treatment by laccase.The potential activity of the purified laccase of


POU1F1  gene with backfat thickness (mm), carcass weight (kg), pH, and color values  in crossbred pigs (Landrace x Yorkshire x Duroc). Frequency of the AA genotype indel was at the highest level (66.67%). Frequency of A allele (0.81) was higher than that of b allele (0.19). This population followed Hardy-Weinberg equilibrium. Carcass weights and a* values of the three genotypes were all significantly different (p < 0.05), respectively. However, backfat thickness, L*, b*, visual color, and pH of the three genotypes were not significantly different (p > 0.05). Visual color was negatively correlated with L* (r = -0.521) and b* (r = -0.390) values, L* value was correlated with b* (r = 0.419) value, and a* value was positively correlated with b* (r = 0.612) value. These results indicate that the POU1F1 gene affected carcass weight and meat redness.This study was carried out to investigate the association of  As of 2012, the annual average per capita meat consumption in Korea reached 40.6 kg, with pork accounting for the highest average consumption at 19.0 kg . In KorePOU1F1  gene is expressed in the pituitary gland of pigs. It is associated with growth regulation as well as secretion of growth hormones, prolactin, and thyroprotein β-subunit  as well as carcass traits  [POU1F1 gene is related to pig growth, and the genotype frequencies of intron 1 vary among breeds. Their analysis of 15 breeds, including 11 breeds of Chinese traditional varieties and four traditional breeds, confirmed 100% frequency of the BB genotype in Chinese traditional varieties of Tibetan, Lingao, Rongchang, Songliao Black, and Min. Frequencies of the BB genotype were high in the Meishan, Erhualian, Fenjing, and Leping Spotted breeds, although the highest genotypes were found to be AA type, AB type, and BB type in the Landrace, Pietrain, and Duroc breeds, respectively. Further, A allele was shown to be present at higher frequencies than B allele in all four breeds [POU1F1 gene intron region 3 and meat quality characteristics of the carcass [POU1F1 genotype [POU1F1 gene intron region 1 in crossbreeds of the three varieties.The -subunit , 7. POU1of meat) –12. Song carcass  as well POU1F1 genotype frequency in 168 crossbred pigs that were raised domestically in order to obtain basic data on meat quality assessment for the establishment of a high-quality pork production system by analyzing meat color and carcass characteristics.Therefore, this study estimated Standard laboratory animals used in this study included 168 crossbred pigs, with a live weight of approximately 110 ± 5 kg. The breeds were Landrace, Yorkshire, and Duroc (LYD or YLD) and were raised in Gyeonggi-do, Chungcheong-do, and Jeolla-do on Nong-Hyup animal feed, which meets the specifications of the National Research Council (NRC).To determine carcass grades and extract DNA, we transported animals to a slaughterhouse in Chungnam. After securing loin portions as laboratory samples, carcass grades were surveyed, and the meat was frozen and transported to the laboratory. The muscle tissues were then thawed at room temperature (-25°C), and an appropriate amount was purified using a QIAamp Mini kit  following the manufacturer’s protocol. Purified genomic DNA was dissolved in distilled water or TE buffer  and collected by centrifugation. The amount of genomic DNA was confirmed by performing electrophoresis, followed by storage at -25°C until polymerase chain reaction (PCR) analysis.POU1F1 gene are shown in Table POU1F1 gene. For the normal genotype (AA type), an amplified product of approximately 1,091 bp in size was obtained, whereas BB type amplified product was 778 bp. The synthesized primers were diluted to a final concentration of 10 pmol.The primers used for amplification of the 2, 1 mM of each dNTP, 10 pM of primers, and 2.5 U of Taq. PCR conditions were as follows: pre-denaturation at 94°C for 5 min, followed by 35 cycles of denaturation for 30 s at 94°C, annealing for 1 min at 55°C, and extension for 1 min at 72°C. PCR was completed with a final extension for 5 min at 72°C. The gene was amplified and analyzed using 1.5% agarose gel electrophoresis with TAE  buffer. The quality of DNA was checked by adding 0.1 mg of ethidium bromide to 1 μL of electrophoresis buffer in the gel and the electrophoresis medium, followed by electrophoresis for approximately 20 min at 100 V. A 100-bp ladder plus  size marker was used as reference for the electrophoretic analysis, and images were obtained under UV illumination.For the PCR mixture, prime Taq DNA polymerase from GeNet Biosystem  was used. Each PCR mixture was 20 μL and consisted of 1× buffer , 1.5 mM MgCl* (lightness), a* (redness), and b* (yellowness) values on the cut surface of the fillet were measured using a color meter , and the standard meat color board at the time was Y = 92.40, X = 0.3136, and y = 0.3196. The visual color based on the meat color reference standard suggested by the National Pork Producers Council (NPPC) was directly compared for assessment based on seven steps, ranging from a light color score of 1 to a dark color score of 7. The pH of muscle was measured using a glass electrode pH meter  after mixing 5 g of sample with 20 mL of distilled water, followed by homogenization for 1 min at 8,000 rpm using a homogenizer .Backfat thickness, carcass weight, meat color, and pH were surveyed as carcass characteristics. For backfat thickness, grading data from the slaughterhouse were used, whereas weight after slaughtering was measured and used as the carcass weight of each pork sample . For meat color, Hunter LPOU1F1 intron 1 were analyzed using the SAS package . For intergroup genetic equilibrium, significance was tested using the χ2-test. After pigs were slaughtered, backfat thickness (mm), carcass weight (kg), meat color, and pH were determined to calculate the mean and standard deviations of each genotype, and correlation among carcass characteristics was calculated. For comparison of differences among groups, significance was tested at a 5% level by using Duncan’s multiple-range tests.The gene and genotypes of POU1F1 intron 1 are shown in Figure FOU1F1 genotype of Korean beef [The results of PCR analysis of ean beef .Figure 1POU1F1 intron 1 SNP genotype and allele frequencies are shown in Table The results of the analysis of the *, a*, and b*values, meat color , and pH, which are the main carcass characteristics associated with the POU1F1 SNP genotypes AA, AB, and BB, are shown in Table The results of the analysis of backfat thickness, carcass weight, Hunter L* (lightness) and b* (yellowness) values, which were within the ranges of 37.94–39.54 and 9.78–10.90, respectively (p > 0.05). However, the POU1F1 intron 1 gene was found to affect color expression since Hunter a* value (redness) was the highest in AB type pork (p > 0.05). In the meat color  assessment using the standard meat color board, the AA, AB, and BB genotypes showed values of 4.05, 4.63, and 5.00, respectively, with BB type showing a higher level of redness than AA type. In terms of pH, average frequency was 5.69 for the AB and BB genotypes, although no significant differences were observed between them. This result is similar to the findings of Yu et al. [No significant differences were observed in terms of meat color based on Hunter Lu et al. , who rep* value and relatively high a* and b* values in the crossbreeds. Results for pH values were within the range of 5.75–5.49, which is almost the same as that reported by Kim et al. [In terms of average carcass characteristics, Oh et al.  obtainedm et al.  and Oh em et al. , whereasm et al.  reported*  and b*  values, whereas a low positive correlation was observed for backfat thickness, meat color (r = 0.12), and redness (r = 0.23). The L* and b*  values showed a low positive (+) correlation, whereas a* and b*  values showed a very high positive correlation.The results of the correlation analysis of carcass characteristics are shown in Table * value), redness , and yellow (b*) values were very similar to those reported previously [These results are similar to those of Kim et al. , who repeviously .POU1F1 intron region 1 are related to carcass weight, meat color, as well as growth. Further extensive study on the relationship between pig growth and changes in POU1F1 gene expression in relation to carcass characteristics is necessary in order to identify quantitative traits that might possibly be related to pig growth or meat quality of pork.The results of this study suggest that changes in the POU1F1 genotypes of 168 domestically bred pig carcasses are as follows. Analysis of POU1F1 intron 1 genotypes showed that the AA genotype has the highest frequency (66.7%), whereas frequencies of the AB and BB genotypes were 28.6% and 4.76%, respectively. The A allele showed a very high expression frequency (0.81), whereas B allele was estimated to have a low frequency (0.19). A χ2-test demonstrated that this allelic frequency was not in Hardy-Weinberg equilibrium. Backfat thickness and pH were not significantly different according to the genotype, but carcass weight was significantly higher in AB (92.00 kg) and BB (92.33 kg) types than in AA type . Further, Hunter a* value was higher in AB type than in BB type (p < 0.05). In addition, meat color  showed a negative correlation with L* (lightness) and b* (yellowness) values. The results of this study showed that changes in the POU1F1 gene influence carcass weight, backfat thickness, and meat color. Therefore, extensive investigation of the relationship between carcass characteristics and changes in the POU1F1 gene is necessary to provide a reliable index for meat quality improvement.The results of the analyses of carcass characteristics according to


Background and Aims. Endoscopic fundoplication is an emerging technique for the treatment of gastroesophageal reflux disease (GERD). The aim of this study is to determine the ideal position of the staples in relation to gastroesophageal junction (GEJ). Methods. Ten endoscopic fundoplication procedures were performed in each group using fresh ex vivo porcine stomachs: Group A: 2 staples each at 3 cm above the GEJ and 180° apart; Group B: 2 staples at 3 cm and 90° apart; Group C: 2 staples at 4 cm and 180° apart; Group D: 3 staples at 3 cm with 90° between each staple . After the procedure, the stomach was gradually filled with water. Gastric yield pressure (GYP) was determined by detection of reflux of the water in esophagus or by rupture of staples. Results. Mean increase of GYPs (±SD) after the procedure was as follows: Group A: 16.9 ± 8.7; Group B: 8.1 ± 7.9; Group C: 12.2 ± 9.4; Group D: 22.7 ± 13.3. GYP in Group A and Group D was higher than Group B . Conclusions. We recommend the placement of 3 staples at 3 cm distance from the GEJ, which resulted in the highest increase of GYP. Gastroesophageal reflux disease (GERD) is a problematic condition, which develops when reflux of gastric contents causes clinical symptoms and complications . Proton ™; Medigus, Omer, Israel), a combined video- and ultrasound-guided transoral surgical stapler, has been cleared by the US Food and Drug Administration and CE marked for use in the European Union for endoscopic fundoplication for the treatment of GERD in patients who require and respond to PPI. Patients with any of following conditions are contraindicated for MUSE: (1) hiatal hernia > 3 cm, (2) failure to reduce hernia with positive end-expiratory pressure up to 10 cm H2O, (3) stricture or varices in the esophagus, (4) body mass index (kg/m2) < 21 or > 35, and (5) nonresponders to PPI therapy. Several studies showed favorable short-term and long-term efficacy of endoscopic fundoplication using MUSE [Recently, the Medigus Ultrasonic Surgical Endostapler  is a validated bench model using ex vivo porcine stomachs, which has been used effectively for testing various medical devices –12. The Endoscopic fundoplication can be performed with the MUSE. The MUSE system consists of an endostapler, staple cartridge, monitor, and console unit. The distal tip of MUSE contains an anvil for stapling, a miniature video camera, and an ultrasound transducer. The insertion length of the MUSE is indicated on the shaft of the device. After the staple cartridge is loaded, the operator types the insertion length at the level of the GEJ into the console unit. Then, the monitor shows the location of the stapling position, which is programmed to be at 3 cm above GEJ. The distal shaft of the MUSE can be retroflexed to 270°, which lifts the proximal part of fundus and enables the stapling of stomach to distal esophagus. The MUSE is disposable, but stapling can be performed several times by replacing the staple cartridges during the case. Each time the stapler is fired, also called “stapling,” it delivers five closely spaced staples.This prospective randomized experimental study has been exempted from institutional review because no human or animal subjects were involved. The inanimate models were obtained from a commercial food supplier. Four different stapling techniques were compared by varying the distance of stapling location from the GEJ and the angle between the staples in the horizontal plane: Group A: 2 staples each at 3 cm distance, angle 180 degrees; Group B: 2 staples at 3 cm, angle 90 degrees; Group C: 2 staples at 4 cm, angle 180 degrees; Group D: 3 staples at 3 cm, 90 degrees between each staple quintuplet . Randomization was done by a computer-generated randomization list. A paper, which disclosed the procedure group, was sealed in an opaque envelope. Before the procedure, horizontal length of the stomach was measured . After eEfficacy of the procedure was assessed by gastric yield pressure (GYP) and gastric yield volume (GYV) , 14. To p value < 0.05 was considered statistically significant.Statistical analyses were conducted using SPSS 18.0 software . Continuous variables were documented as mean and standard deviation or interquartile range (IQR). Increase of GYP was compared between the four groups. For this statistical analysis, we used the repeated measures ANOVA. GYV between before and after the procedure were compared in each group using Wilcoxon signed-rank test. Categorical variables were compared using Chi-square test and Fisher exact test. A p = 0.03, p < 0.01, resp.).A total of 40 endoscopic fundoplication procedures, 10 procedures per each group, were successfully performed. The shape of the stomachs used in the study were consistent with no difference in average specimen diameter or length between each group. The length of the specimen (axis from GEJ to pylorus) in each group was as follows: Group A: 25.0 ± 1.9 cm; Group B: 26.0 ± 3.2 cm; Group C: 25.5 ± 2.4 cm; Group D: 26.0 ± 3.1 cm. A Hill grade I valve, defined as the close approximation of the cardia to the shaft of the endoscope, was created after the endoscopic fundoplication procedures in all specimens . The prop = 0.03; Group B versus Group D, p = 0.01). Incidents of burst pressure being lower than GYP occurred in 6 cases, 4 cases, 5 cases, and 7 cases of each group, respectively. Total rupture rate was 55%. The rupture rate did not differ between all groups.Baseline GYP before the procedure was 0 mmHg in all groups. GYP after the procedure was as follows: Group A: 16.9 mmHg ; Group B: 8.1 mmHg ; Group C: 12.2 mmHg ; Group D: 22.7 mmHg . Pressure increase of Group A and Group D was higher than Group B .Mean GYV before the procedure was as follows: Group A: 274.0 mL ; Group B: 272.0 mL (IQR 172.5–327.5); Group C: 319.0 mL ; Group D: 316.0 mL . Mean GYV after the procedure is as follows: Group A: 2299.0 mL ; Group B: 1663.0 mL (IQR 862.5–2565.0); Group C: 1857.0 mL ; Group D: 2639.0 mL . In all groups, the GYV was significantly larger than before the procedure .


Despite the large number of published papers analyzing the prognostic role of Ki-67 in NSCLC, it is still not considered an established factor for routine use in clinical practice. The present meta-analysis summarizes and analyses the associations between Ki-67 expression and clinical outcome in NSCLC patients.PubMed, Cochrane, and Embase databases were searched systematically using identical search strategies. The impacts of Ki-67 expression on survival in patients with NSCLC and NSCLC subtypes were evaluated. Furthermore, the association between Ki-67 expression and the clinicopathological features of NSCLC were evaluated.P < 0.001) and disease-free survival  in NSCLC patients. Analysis of the different subgroups of NSCLC suggested that the negative association between high Ki-67 expression and OS and DFS in Asian NSCLC patients was stronger than that in non-Asian NSCLC patients, particularly in early-stage (Stage I-II) adenocarcinoma (ADC) patients. Additionally, while high expression was more common in males, smokers, and those with poorer differentiation, there was no correlation between high Ki-67 expression and age or lymph node status. Importantly, significant correlations between high Ki-67 expression and clinicopathological features  were seen only in Asian NSCLC patients.In total, 32 studies from 30 articles met the inclusion criteria, involving 5600 patients. Meta-analysis results suggested that high Ki-67 expression was negatively associated with overall survival  contains supplementary material, which is available to authorized users. Lung cancer (LC) is often fatal and is very common worldwide. It has been reported that the overall 5-year survival rate of lung cancer patients was ~16 %, and that it was < 70 % even in patients diagnosed at stage I [was associated with a poorer overall survival (hazard ratio (HR) 1.56, 95 % confidence interval (CI) 1.30–1.87), it did not evaluate the association between Ki-67 expression and disease-free survival. Most importantly, because of the limited number of studies and patients included, it did not examine high Ki-67 expression in Asian patients [Ki-67 is expressed in proliferating cells and has been used in clinical practice as an index to evaluate proliferative activity in NSCLC and other cancers , 3. Morepatients . Thus, aIn this study, we performed a meta-analysis to explore the relationship between Ki-67 expression and its prognostic value in NSCLC. Associations between Ki-67 expression and the clinicopathological features of NSCLC, including age, gender, smoking status, lymph node status, and tumor differentiation, were also evaluated.The protocol, including the objective of our analysis, criteria for study inclusion/exclusion, assessment of study quality, primary outcome, and statistical methods, was in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (“PRISMA”) statement , Ki-67 (‘Ki-67’ or ‘Ki67’ or ‘MIB-1’ or ‘MIB 1’ or ‘proliferative index’), prognosis, survival, and outcome, in all possible combinations. Using these parameters, we filtered out all the eligible articles and looked through their reference lists for additional studies. The systematic literature search was undertaken independently by two reviewers (SW and ZW) and ended in November 2014. Disagreements were determined through consensus with a third reviewer (CL). Authors of the eligible studies were contacted for additional data relevant to this meta-analysis, as necessary.Inclusion criteria for the primary studies were 1) inclusion of patients with a distinct NSCLC diagnosis by pathology, 2) measurement of Ki-67 expression using immunohistochemistry (IHC) in primary NSCLC tissue, 3) investigation of the relationship between Ki-67 expression and overall survival (OS) or disease-free survival (DFS) in patients with NSCLC and availability of valid survival data either provided directly or that could be calculated indirectly, and 4) publication in the English language. When authors had several publications or reported on the same patient population, only the most recent or complete study was included.Exclusion criteria for the primary studies were 1) an overlap among articles or duplicate data; 2) the use of animals or cell lines; 3) insufficient data availability for estimating HR and 95 % CI, such as typical of abstracts, editorials, letters, conferences data, expert opinions, reviews, and case reports; 4) investigation of the relationship between Ki-67 and NSCLC using methods other than IHC; 5) inclusion of patients who underwent chemotherapy or radiotherapy interventions; and 6) a study sample comprising fewer than 20 patients.Two investigators (SW and WZ) conducted the data extractions independently . Any disFor study quality control, we used the Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) and extracted 18 items .To identify the prognostic effect of Ki-67 expression, the overall HR and 95 % CI were evaluated for elevated Ki-67 expression. The combined ORs and HRs were initially estimated graphically using forest plots. Subgroup analyses were then conducted when the risk (OR or HR) was significant  [n = 1) [n = 1) [n = 1) [We identified 2046 potentially relevant articles through the search strategy described in Methods. As shown in Fig. n = 4) –19, use  [n = 1) , or prov [n = 1) . Additio [n = 1) , 24. Thu [n = 1) –9, 23–47As demonstrated in Table All studies included information on disease stage, and the proportion of stages I + II was 67.9 %. IHC was the only technique used to detect Ki-67 expression, using various antibodies and cut-off values , and 2503 (44.70 %) tissue samples had ‘high’ Ki-67 expression Table .n = 6) or from Kaplan–Meier survival curves (n = 7), as described by Tierney [Of the 32 studies, 19 provided HR and 95 % CI values directly, whereas in the other 13 studies, they were calculated from available data  and 14 involved non-Asian patients (n = 2287). The overall HR and 95 % CI for NSCLC patients was 1.59 , with significant heterogeneity  with significant heterogeneity .Of the 28 studies investigating the association between Ki-67 expression and OS, 14 involved Asian patients  with heterogeneity , but no association with shorter OS was observed in patients in stages III–IV .Next, subgroups including TNM stage  and type of NSCLC (10 studies for ADC and two for non–ADC) were analyzed. The analyses indicated that high Ki–67 expression was associated with a shorter OS in stage I, stages I–II, and stages I–III patients . However, the association was not significant in the non-ADC group . Additionally, only Asian patients (vs. non-Asian patients) and the early-stage group (stages I–II vs. advanced stage) in the ADC group demonstrated significant associations between high Ki–67 expression and shorter OS. The combined HRs were 3.01, 95 % CI 1.96–4.02, P < 0.001 and 3.30, 95 % CI 1.37–7.96, P = 0.008, respectively. Non-Asian ADC patients and ADC patients at advanced stages of the disease showed no significant association between high Ki–67 expression and OS .Another subgroup analysis (ADC vs. non–ADC) demonstrated that the ADC group showed a significant association between high Ki–67 expression and shorter OS . The pooled HRs and 95 % CIs were as follows: 1.80 (95 % CI 1.20–2.70) vs. 1.53 (95 % CI 1.28–1.84) for a cut–off value of 10 %, 1.57 (95 % CI 1.27–1.95) vs. 1.60 (95 % CI 1.22–2.08) for a cut–off value of 25 %, and 1.56 (95 % CI 1.30–1.86) vs. 1.72 (95 % CI 1.27–2.33) for a cut–off value of 50 % with significant heterogeneities  showed the highest risk, when compared with stages I–II or I–III, with the following combined HRs and 95 % CIs: HR 4.31, 95 % CI 2.37–7.84, P < 0.001; HR 1.51, 95 % CI 1.02–2.23, P = 0.038; and HR 2.02, 95 % CI 0.97–4.20, P < 0.06, respectively.The pooled HR and 95 % CI for DFS provided in eight studies was 2.21, 95 % CI 1.43–3.43, 2-4) and poorer differentiation grade (moderate or poor); the combined ORs and 95 % CIs were as follows: OR 1.89, 95 % CI 1.53–2.33, P < 0.001; OR 2.20, 95 % CI 1.72–2.82, P < 0.000; OR 1.88, 95 % CI 1.60–2.22, P < 0.001; OR 1.46, 95 % CI 1.13–1.88, P = 0.004; and OR 1.47, 95 % CI 1.15–1.88, P = 0.002, respectively. Moreover, significant associations between Ki–67 and gender , being a non-ADC patient, higher tumor stage, and poorer differentiation were seen only in Asian NSCLC patients. The combined ORs and 95 % CIs were as follows: OR 2.18, 95 % CI 1.67–2.81, P < 0.001; OR 2.22, 95 % CI 1.82–2.70; OR 1.47, 95 % CI 1.12–1.94, P = 0.006; and OR 1.50, 95 % CI 1.15–1.94, P = 0.002, respectively ; the combined ORs and 95 % CIs were OR 1.08, 95 % CI 0.85–1.37, P = 0.553 and OR 1.01, 95 % CI 0.83–1.22, P = 0.927, respectively (Table There was no significant association between Ki–67 expression and age (>60 vs. < 60) or lymph node status N1– vs. N0; P < 0.001 and HR 2.74, 95 % CI 1.25–4.22, P < 0.001, respectively) after the ‘trim and fill’ method was used , but it showed seemingly significant publication bias for OS after assessing the funnel plot , consistent with a previous meta-analysis, published in 2004 [P < 0.001). Sensitivity analysis suggested that the association between high Ki-67 expression and NSCLC prognosis was stable and unchanged after removing any one study. Also, the results of the current meta-analysis show that high Ki-67 expression was more common in males , smokers , those in later tumor stages , or those with poorer differentiation , which has been linked to more aggressive tumors. Overall, the results of the current meta-analysis suggest that increased Ki-67 expression exerts a significantly adverse effect on the prognosis of NSCLC patients. To our knowledge, this study is the most comprehensive and detailed meta-analysis to evaluate the association between Ki-67 expression and survival in NSCLC patients.Meta-analytic techniques using non-randomized controlled trials (NRCTs) may be useful in certain clinical settings where the number or the sample size of the RCTs is insufficient . The resNSCLC is a malignancy displaying substantial heterogeneity, and the clinical and biological characteristics of the different subtypes of NSCLC vary substantially . In thisP = 711). Therefore, the alteration of Ki-67 expression may contribute to the differences in the tumor biology observed between Asian and non-Asian patients with NSCLC. Although, future validation and investigations are needed, these data may provide new insights into biological aggressiveness of NSCLC in Asian versus in non-Asian patients.It was reported that Asian ethnicity is a favorable prognostic factor for OS in NSCLC and is independent of smoking status , 53. Howet al. [et al. introduced 50 % as the cut-off value for defining Ki-67 expression in SQCC [Heterogeneity was significant in this meta-analysis, and it could not be ruled out by using a random-effects model or multiple subgroup analyses. For reasons of homogeneity, we analyzed only the studies dealing with NSCLC histology and restricted the analysis to the histological subtypes or tumor stages for which we had sufficient numbers of studies. Furthermore, the technique(s) used to identify the expression of Ki-67 can be a potential source of bias. The use of different antibodies (anti-Ki67 mAb or anti-MIB-1 mAb) and a protocol to count the number of cells stained by these antibodies without a received standard antibody concentration may yield variation among the studies. Moreover, the cut-off value used to define a tumor with ‘positive’ Ki-67 staining is often arbitrary and varies according to the investigator, from a low percentage to more than 50 %. Martin et al.  introduc in SQCC . In thisIt is important to note that the current study encountered difficulties, similar as most meta-analysis. First, it was based on summary data rather than data from individual patients. Therefore, multivariate analyses for confounding factors such as histological subtypes, gender and smoking status were not performed. A meta-regression model that adjusted for those factors that were found to be correlated with high K-67 levels was also not performed, too. Second, our search was limited to published studies and excluded unpublished trials or results in abstract form, which may have led to publication bias. Third, the unequal number of studies from Asian countries, with data derived mainly from Japan, China, and Korea may have also been a source of bias. Hence, our analysis may reflect outcomes from East Asia rather than from Asia in general.There are several advantages to this study. First, large numbers (32 studies and 5600 patients) were analyzed, whereas only 17 studies and 1863 NSCLC patients were considered in the meta-analysis published in 2004. Second, important clinical parameters including age, gender, tumor stage, histology, and race/ethnicity were included in the analysis. The association between high Ki-67 expression and DFS was also investigated. However, as several limitations still exist, the results need to be interpreted cautiously. First, the number of included studies and included NSCLC patients were relatively small. Moreover, heterogeneity was inevitable among the groups due to the impossibility of matching patient characteristics across all studies. This may have weakened the results to some extent. Second, publication bias was unavoidable for clinical evidence, because the relevant data were extracted from non-randomized controlled trials. Third, reports in languages other than English were excluded; therefore, potential language bias may have been present. Lastly, some data for OS were extracted from survival curves or other available data rather than provided directly. Although the method used for extrapolation of HRs and 95 % CIs is widely accepted and we did not identify any major differences in current study , in Asians, and in ADC patients is a poor prognostic indicator for survival outcome. Further adequately designed prospective studies with standardization of the immunohistochemistry technique, especially standardization of the cut-off threshold value, need to be conducted to confirm these results.


Furthermore, although raspberry ketone (RK), RD derivative, also increased intracellular ROS in B16F10 cells, increase in ROS was suppressed by disodium dihydrogen ethylenediaminetetraacetate dehydrate (EDTA). The amounts of increased ROS with RK in HaCaT cells without melanocyte were further increased by tyrosinase. Therefore, tyrosinase, a metalloprotein having copper, was speculated to be one of causative agents allowing phenol compounds to work as a prooxidant. Hydroxyl radical was generated by adding a mixture of tyrosinase and H2O2 to RD, and the amount of the radical was further increased by UVB, indicating that RD cytotoxicity was caused by intracellularly increased ROS, which possibly related to phenol induced prooxidants.Specific phenol compounds including rhododendrol (RD), a skin-brightening ingredient in cosmetics, are reported to induce leukoderma, inducing a social problem, and the elucidation of mechanism of leukoderma is strongly demanded. This study investigated the relationship among the cytotoxicities of six phenol compounds on B16F10 melanoma cells and HaCaT keratinocytes and generated reactive oxygen species (ROS). As a result, the cytotoxicity of RD on B16F10 cells was higher than that on HaCaT cells, and RD significantly increased intracellular ROS and hydrogen peroxide (H In 2013, leukoderma has been reported to appear on the skin of women who use particular skin-brightening cosmetic products in Japan. The damage has been found widely not only in Japan but also in Asia, inducing a major social problem. More than 5,000 people have leukoderma in Japan at August 2013. Since skin-brightening cosmetic products containing a specific phenol compound, called rhododendrol (4-(4-hydroxyphenyl)-2-butanol (RD)) , are rep2O2), which induces Fenton reaction [Melanocytes produce melanin actively as their characteristic features, and the cells have an indispensable enzyme, tyrosinase . Tyrosinreaction , 9.On the other hand, phenolic compounds including RD are used as building blocks for preparing various pharmaceutical compounds, cosmetic ingredients, and food additives because of their special characters in oxidation-reduction (redox) reactions. However, some phenolic compounds are known to show specific cytotoxicities to melanocytes. For instance, raspberry ketone (4-(4-hydroxyphenyl)-2-butanone (RK)) , which i in vitro. Actually, the authors have already confirmed that oxidation reactions promoted with iron proteins such as hemoglobin enhance the toxicity in ultraviolet- (UV-) ray-irradiation induced inflamed cutaneous tissues by comparing generated ROS quantitatively with electron spin resonance (ESR) spectroscopy [There are a few studies that directly detect the generation of reactive oxygen species (ROS) generated by metalloproteintroscopy .Recently, the cytotoxicity of RD in melanocyte has been reported to be suppressed by phenylthiourea, a copper chelating agent, and the knockdown of tyrosinase . This re2O2, which is known to induce directly the Fenton reaction in B16F10 cells. Hydroxyl radical (OH∙) was directly measured by ESR for confirming that the Fenton reaction was induced by H2O2 and copper with tyrosinase. Furthermore, the oxidation pathway of RD to catechol derivatives by tyrosinase was also investigated by high performance liquid chromatography (HPLC) analysis.This study, therefore, investigated the relationships among generated ROS and the cytotoxicities of RD in B16F10 melanoma cells and HaCaT keratinocytes having no melanin and compared the results to those of RK, MEHQ, p-hydroxyphenylacetamide (pHPA) , 2-p-hy, and p-h2O2 were purchased from Wako Pure Chemical Industries . RD and EDTA were purchased from Nacalai Tesque . MEHQ was purchased from Tokyo Chemical Industry , respectively. Tyrosinase from mushroom, neutral red, and Triton X-100 reduced were purchased from Sigma-Aldrich , and 2′,7′-dichlorofluorescin diacetate (DCFDA) was purchased from EMD Millipore . Hoechst33342 was purchased from Life Technologies . 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was purchased from Labotec (Tokyo). Dimethyl sulfoxide (DMSO) was purchased from Pierce Biotechnology . Ferrous sulfate was purchased from United States Pharmacopeial Convention . Dulbecco's modified Eagle medium (DMEM) was purchased from Nissui Pharmaceutical (Tokyo). Fetal bovine serum (FBS) was purchased from Biological Industries Israel .RK, pHPA, 2pHP-IA, pHPE, and H2 condition. HaCaT cells were cultured in DMEM containing 5% FBS at 37°C at a 5%-CO2 condition.B16F10 cells were cultured in DMEM containing 10% FBS at 37°C at a 5%-COB16F10 and HaCaT cells were cultured in DMEM containing 5.0 mM RD, RK, MEHQ, pHPA, 2pHP-IA, pHPE, or 10.0 mM MEHQ and pHPA individually, and for measuring the survival rates, the medium was replaced with DMEM containing 0.165 mg/mL neutral red. After being cultured for 2 h, the cells were washed with phosphate buffered saline (PBS), and neutral red incorporated in the cells was extracted with NR extraction solution, which was a mixture of methanol, acetic acid, and water at a ratio of 50 : 1 : 49. The absorbance of neutral red in a well was measured at 550 nm with a reference absorbance of 650 nm by a microplate reader (VMax Kinetic ELISA Microplate Reader) . The cell survival rates were expressed as percent values of those of the cells incubated without test compounds described above.μM DCFDA, and 4.0 μM Hoechst33342 for 30 min. After being incubated for 30 min, the cells were washed with HBSS and observed with a fluorescence microscope (EVOS FLoid Cell Imaging Station) .B16F10 cells were cultured in Hanks' balanced salt solution (HBSS) containing 1.0 mM RK, 20.0 μM DCFDA for 30 min, washed with HBSS, and solubilized with 0.5% Triton X-100 reduced. The fluorescence intensity of the medium containing the solubilized cells was measured at an exciting wavelength of 485 nm and an emission wavelength of 530 nm with a fluorescence microplate reader (Gemini EM fluorescence microplate reader) (Molecular Devices). The intracellular ROS level was expressed as a relative value, which was calculated from fluorescence intensity per 1.0 μg protein of the control cells incubated without test samples.B16F10 and HaCaT cells were cultured in HBSS containing 1.0 mM RD, RK, MEHQ, pHPA, 2pHP-IA, or pHPE and 20.0 μM DCFDA for 30 min. B16F10 cells were cultured in HBSS containing 0.25, 0.5, or 1.0 mM EDTA with 1.0 mM RK and 20.0 μM DCFDA for 30 min. The fluorescence intensity of the medium was measured by the same method described above. The intracellular ROS level was expressed as a relative value, which was calculated from fluorescence intensity per 1.0 μg protein of the control cells incubated without tyrosinase, EDTA, and RK.The effects of tyrosinase and EDTA on change in intracellular ROS by exposure to RK were investigated by the following procedure. HaCaT cells were cultured in HBSS containing 50.0, 75.0, or 100.0 units/mL tyrosinase with 1.0 mM RK and 20.0 2O2 was measured with Amplite fluorimetric hydrogen peroxide assay kit “Red Fluorescence” . The amount of H2O2 was expressed as a relative value, which was calculated from fluorescence intensity per 1.0 μg protein of the control cells incubated without test samples such as RD and RK.After being cultured in HBSS containing 5.0 mM RD or RK for 30 min, B16F10 cells were washed with HBSS and solubilized with 0.5% Triton X-100 reduced, and the intracellular amount of HμL, and a quartz cell (Labotec) with a home-made cover glass (40 × 5 × 0.5 mm). As an ESR standard marker, manganese oxide (MnO) powder  was used. As a spin-trap agent, 5,5-dimethyl-1-pyrroline-N-oxide  and 10 w/w% DMSO solution were used. The following test samples were solved in 0.1 M PBS (pH 7.5). RD (100 μM) was added to 100 units/mL tyrosinase. DMPO (20 μL) was added to the sample solutions (20 μL) immediately, and the radical signals were measured by ESR. UVB (290~340 nm) irradiation was introduced to samples at a dose of 200~1000 J/m2 by a USHIO Optical Modulex . To identify observed peaks, signals were analyzed by specialized ESR analysis software  , which was installed in the ESR device, and allowed to determine g-value and calculated hfcc from the distance between peaks. To identify ESR signals, the g-value and hfcc of the measurable peaks were compared with the peak values of the standard OH∙. The standard OH∙ was generated by the reaction of ferrous sulfate solution (100 nM) and H2O2 (30 mM).A JES-FA200 ESR spectrometry  was used for ESR signal measurement. ESR spectrometry conditions used for estimating radical species with adequate spin-trap reagents were as follows: microwave frequency, 9414.499 ± 5.000 MHz; microwave power, 4.00 mW; field center, 335.32 ± 0.5 mT; sweep width, ±5.00 mT; modulation frequency, 100.00 kHz; sweep time, 0.5~5 min; amplitude, 1.500~2.500; and time constant, 0.03~0.5 s, at room temperature. This study used an ESR universal cavity (ES-UCX2: TE011-mode cavity) (JEOL) with X-band microwave units (8.750–9.650 GHz), an aqueous sample cell  with a sample volume of 20~100 μM RD was added to 100.0 units/mL tyrosinase, the sample mixture was incubated for 0, 1.5, and 30 min at 37°C and was injected into an HPLC instrument. Sample (50.0 μL) was separated on an octadecylsilane (ODS) reverse-phase column (Shodex C18P-4E column)  at 40°C. The mobile phase, which was a mixture of acetonitrile and 0.03 M KH2PO4 at a ratio of 60 : 40, was used at a flow rate of 0.7 mL/min. The HPLC system consisted of a SCL-10A system controller, a LC-10AD pump, a SPD-10A UV/VIS detector, a CTO-10AC column oven, and a SIL-10A injector . The separated peaks were monitored at 300 nm with the detector.After 100.0 Protein concentration was determined with bicinchoninic acid- (BCA-) protein assay kit (Pierce Biotechnology). t-test, and the probability less than 0.05 (P < 0.05) was considered statistically significant. Correlations were analyzed by calculating Pearson correlation coefficients (r).Comparison between two groups was performed by Student'sAfter B16F10 cells were treated with 1.0 mM RK, which is made by replacing the hydroxyl group of RD with ketone group, for 30 min, the generation of ROS was clearly confirmed in the cells . Since tTyrosinase was investigated to be involved in the increase in intracellular ROS level. As a result, 50.0, 75.0, and 100.0 units/mL tyrosinase were confirmed to further promote increase in the intracellular ROS level in HaCaT cells treated with 1.0 mM RK .EDTA, a metal chelating agent, was investigated to decrease ROS generated in B16F10 cells with RK intracellularly. As a result, 0.5 and 1.0 mM EDTA were confirmed to reduce the intracellular ROS level increased with 1.0 mM RK .2O2 level in the cells was measured. As a result, 5.0 mM RD and RK were found to increase the H2O2 level significantly  by their g-values and hfcc values in the measurable peaks in ESR. ∙ spectra. The samples were irradiated with UVB at 200, 500, and 1000 J/m2, and the peak intensity of OH∙ spectra of a sample mixture of tyrosinase, H2O2, and RD was significantly increased at a dose of 1000 J/m2. Further, the peak intensity of OH∙ spectra was found to increase with UVB irradiation dose-dependently.After RD was added to a sample mixture of tyrosinase and Hd by ESR . The heid by ESR  and furtd by ESR . These fμM RD was added to 100.0 units/mL tyrosinase, the sample mixture was incubated for 1.5 or 30 min at 37°C and was analyzed by HPLC. The separated peaks were monitored at 310 nm. Although the sample gave a peak derived from RD at a retention time (RT) of 5.8 mm , resulting in the possible appearance of cytotoxicity eventually by the prooxidant effects of RD and RK. Since (1) 50.0~100.0 units/mL tyrosinase further increased the intracellular amount of ROS, which had been increased by 1.0 mM RK, in HaCaT cells by 144.2~262.5% significantly , and (2)h as OH∙ –34. In th as OH∙ . Reactioh as OH∙ . Since tly 11.0% , RD alsoendently , and theThe substrates of tyrosinase are L-tyrosine, L-dopa, and 5,6-dihydroxyindole (DHI) . L-tyros2O2 and dopa to tyrosinase solution [2O2 is reported to be generated in oxidization process from phenol derivative to quinone derivative by copper [2O2 [2O2 is now well-known to be produced in the generating process of quinone by the oxidization of phenolic hydroxyl group of anthrahydroquinone on industrial scale process, which is called anthraquinone oxidation (AO) process [2O2 was generated in the oxidization process of RD in this study (∙), through the conjugation of allylic C-H bonds with the π-bond  [∙ as indicated in the following: 2O2 induces the generation of OH∙ [π-allylic conjugation in phenolic compounds suggested that the conjugated system extends to the side chain of phenolic compounds, and electrons were able to move between the terminal oxygen atom of the side chain and the benzene ring of phenolic compounds. The distance between the terminal oxygen atom of the side chain and the benzene ring of RD or RK was speculated to be comparable to that of L-tyrosine, indicating that the moving distance of electrons in RD or RK might be the same as that in L-tyrosine, relating to a melanocytes-specific cytotoxicity induced by RD or RK.The production of superoxide is enhanced by adding Hsolution . Moreovey copper . 2,3-Dihper [2O2 .H2O2 is  process . The resis study , and RD-ugation) , 41. Theugation) , 28–30, n of OH∙ , 28–30, RD is approved to be used as a skin-brightening ingredient in quasidrug category in Japan. Therefore, many safety evaluation tests for RD have been carried out, and the safety of RD is ensured at the same safety level as that of medicine. However, RD has induced a strong adverse effect, leukoderma, in 2013. There is a question why RD has passed many safety tests. This study confirmed that RD induced a melanocytes-specific cytotoxicity and increased an intracellular ROS level in B16F10 cells. Although three-dimensional cultured skin models are used in safety evaluation tests, there are no safety evaluation tests using melanocytes. Therefore, for improving the safety of quasidrug or cosmetic ingredient, safety evaluation tests, which investigate whether a test compound shows a melanocytes-specific cytotoxicity and increases an intracellular ROS level in melanocytes, are suggested to be necessary. The authors hope that this study's results could contribute to the establishments of these safety evaluation tests.2O2 level in B16F10 cells. Since the structure of RD is similar to that of L-tyrosine, RD is thought to react with copper in tyrosinase. Moreover, the results of reverse-phase HPLC analysis suggested that RD was oxidized to a quinone derivative through catechol and semiquinone derivatives by tyrosinase. Therefore, H2O2 was speculated to be generated in the oxidization process to quinone derivative from RD in B16F10 cells. Furthermore, ESR spectroscopy in this study confirmed that OH∙, which is known to show a high cytotoxicity, was generated by the reaction of tyrosinase and H2O2, indicating that reaction of copper in tyrosinase and H2O2 might induce Fenton reaction simultaneously. This study proposed that the RD-induced melanocytes-specific cytotoxicity could be caused possibly by OH∙ generated by the reaction of copper in tyrosinase and H2O2, and this reaction might be one of the mechanisms for explaining RD-induced leukoderma.RD increased the intracellular H