Our aim was to investigate the expression of c-Met in advanced esophageal squamous cell carcinoma in four phase II trials who had tumor tissues from archival in our center and analyze the correlations between c-Met expression and clinical features.Ninety patients with advanced ESCC who were admitted to the phase II clinical trials in the Department of Gastrointestinal Oncology, Peking University Cancer Hospital and Institute from March 2007 to March 2014 were finally eligible for present study and the corresponding tissues and clinical data were collected. The expression of c-Met in the tissue samples was detected by immunohistochemistry (IHC). c-Met overexpression was defined as ≥ the median value of H-score. Kaplan-Meier and Cox multivariate regression were conducted to evaluate the relationship between c-Met expression and ESCC survival.vs 422 d, P = 0.011). In the group treated with chemotherapy combined with anti-EGFR drugs, patients with lowexpression of c-Met had a better OS than those with overexpression of c-Met .The overexpression of c-Met is 43.3% in advanced ESCC. There was no statistical difference between c-Met expression and clinical features except sex and tumor location. Survival analysis documented that the overexpression of c-Met predicted a worse prognosis (OS: 253 d c-Met may be an independent prognostic factor in advanced ESCC. The overexpression of c-Met may predict a worse efficacy of anti-EGFR therapy. Esophageal carcinoma is one of the most common gastrointestinal cancers, the incidence of which ranks the fifth among all of the malignant cancers in China . More thRecently, a meta-analysis focusing on c-Met overexpression and the prognosis of gastric cancer including 14 studies  indicateTherefore, in this study, we detected c-Met expression in advanced ESCC patients who involved in the phase II clinical trials in our center. We investigated the expression levels of c-Met in advanced ESCC and estimated the relationship between c-Met expression and clinical features.Tumor samples were retrospectively identified from patients with ESCC who were enrolled in four phase II clinical trials between 2007 and 2014 at the Peking University Cancer Hospital in Beijing, China. Four phase II clinical trials for first-line treatment of ESCC were involved: Paclitaxel and Cisplatin in Patients With Advanced Squamous-Cell Carcinoma of the Esophagus ; IrinoteHistological confirmed stage IV according to AJCC 6.0 or recurrent ESCC.Patients have not received any palliative chemotherapy or radiotherapy before.Adjuvant therapies should be received more than 6 months before recurrence.Inclusion criterion:Adjuvant therapies are received within 6 months before recurrence.Patients who have other primary carcinomas except ESCC.Histology confirmed mixed tumor.Exclusion criterion:Anti-total c-Met (SP44) rabbit monoclonal primary antibodies were purchased from Ventana Company .Tissue samples for diagnostic purposes were obtained with the consent of each patient. All tumor specimens were fixed in 10% buffered formalin, embedded in paraffin and then made into continuous 4 μm tissue sections for IHC examination.All patients signed written informed consent for their information to be used for study. Study approval was obtained from independent ethics committees at Peking University Cancer Hospital. The study was undertaken in accordance with the ethical standards of the World Medical Association Declaration of Helsinki.IHC evaluation was performed using anti-total c-Met(SP44) rabbit monoclonal primary antibody. The staining was carried out according to the manufactures’ protocol on the BenchMark XT platform from Ventana utilizing the ultraView detection kit. The c-Met staining intensities were evaluated by two pathologists who were blinded to the diagnosis of individual patients. c-Met was localized primarily in the cytoplasm and membrane. Specifically, intensity was scored according to a four-tier systems: 0, no staining; 1+, weak; 2+, moderate; and 3+, strong.H-score assessment was based on staining intensity(0–3) and the percentage of positive cells (0-100%). Each individual intensity level was multiplied by the percentage of cells and all values were added to obtain the final IHC score, ranging from 0 to 300. The final score was calculated from the scores of assessment at membranous and cytoplasmic expression.The other evaluation of c-Met was performed according to MetMab IHC defined scoring criteria  in the clinical trial of NCT01456325, a MetMab phase III trial in advanced NSCLC .To our knowledge, there were no validated scoring systems for interpretation of c-Met staining intensity. Here, positive results were judged by two systems of criterion.P values of <0.05 were considered significant. All analysis were performed using SPSS 19.0 software .A number of prognostic factors between different c-Met expression groups were examined in univariate analysis. These factors included: (1) sex: male and female; (2) baseline age, dichotomized as ≤59 years vs. >59 years; (3) tumor location; (4) presence of distant metastasis; and (5) presence of lymph node invasion. Comparison of categorical variables was performed with the chi-square test or the Fisher’s exact test. Initial analysis involved a univariate investigation of relationship between c-Met overexpression and OS time using Kaplan-Meier survival curves. To identify independent biomarkers, multivariate analyses adjusted for the baseline variables were performed using a Cox regression model for OS. After screening all the patients’ medical record, one hundred and ten cases were detected for c-Met IHC Figure . Due to H-score of c-Met IHC ranged from 0 to 270, with the median value of 20, which was chosen as the cutoff point for separating c-Met over-expression tumors from c-Met low-expression tumor. Of the 90 patients, 51 cases had an H-score ≤ 20, considered as IHC low-expression, and 39 cases had an H-score > 20, considered as IHC over-expression. No statistically significant difference of c-Met expression was found between different groups of sex, age, tumor location, tumor differentiation, lymph node invasion and distant metastasis . Patients with tumor located in upper thoracic (475d) and middle thoracic (422d) had a better prognosis than those in lower thoracic (258d) (P = 0.005).The relationship between OS and clinical features of ESCC patients was analyzed through log-rank and chi-square test. The results showed that no statistical significance of OS was found in different groups of age, stage, distant metastasis, tumor differentiation. However, OS was significantly different by sex and tumor location. Female patients had a better prognosis than male patients . The median OS was 333d (95% CI 252d-482d). The OS was closely related to the expression of c-Met in ESCC tissues. As shown in Figure vs 422d, P = 0.011). In the multivariate Cox regression model adjusted for baseline variants, there was still a statistical significance between OS and c-Met expression .Progress-free survival(PFS) was not significantly different between the two c-Met expression groups . Similar results were obtained in c-Met low-expression group . In TP plus nimotuzumab group, the OS of patients with c-Met low-expression was significantly better than those with c-Met over-expression . In TP alone group, no statistical significance was found   plus nimotuzumab, 55 patients received chemotherapy of TP alone, and 10 patients received other chemotherapies. In c-Met overexpression group, OS of patients who received TP plus nimotuzumab was not different from that of patients who received TP alone (232d P = 0.289).The other evaluation of c-Met was performed according to MetMab IHC defined scoring criterion. According to this criterion, 16 patients (17.8%) had c-Met over-expression and 74 patients (82.2%) had c-Met low-expression. There was no significant relevance between c-Met expression and different groups of age, sex, tumor location, tumor differentiation, distant metastasis. The patients with c-Met low-expression had a trend of better prognosis with no statistical significance (c-Met expression has been reported in a number of human primary tumors, including gastric, breast, colorectal, liver and renal cancer. c-Met plays an important role in tumor development and metastasis . As the Our study detected c-Met expression in 90 ESCC patients and analyzed the relationship between c-Met expression and clinical features and prognosis. According to H-score assessment, 43.3% of patients had c-Met overexpression, similar to other reports in China . The mosOne study reported that an increased expression of c-Met was seen along the metaplasia-adenocarcinoma sequence and patients with esophageal adenocarcinoma with c-Met positive tumors showed lower 6-month survival rates after surgical resection than those with c-Met negative tumors . There wIn our study, patients with c-Met lowexpression had a better OS than those with c-Met overexpression both in TP plus nimotuzumab group and in TP group. In c-Met overexpression group, patients treated with TP plus nimotuzumab had a worse OS than those treated with TP alone. On the contrary, in c-Met lowexpression group, patients treated with TP plus nimotuzumab had a better OS than those treated with TP alone. This suggests that patients with c-Met overexpression may not be suitable for anti-EGFR treatment. There has been no study on anti-EGFR treatment and c-Met expression before. However, a study finds out that in lung cancer, amplification of c-Met causes gefitinib resistance by driving ERBB3-dependent activation of PI3K, a pathway thought to be specific to EGFR/ERBB family receptors . RecentlThere were some limitations in this study. First, this study was conducted in a relatively small sample size. Secondly, due to a retrospective study, only cancer tissues before treatment were detected. It would be better that cancer tissues after treatment, lymph nodes, distant metastasis, and normal tissues adjacent to cancer could be detected for c-Met IHC and c-Met gene amplification. On the other hand, information of patients treated with different therapies was not well matched. Thus, our findings should be validated in subsequent prospective studies in future before clinical application.In short, c-Met plays an important role in development of ESCC and may have an effect on anti-EGFR therapy, which needs further studies.Patients with c-Met over-expression had a poorer prognosis than those with c-Met low-expression, suggesting that c-Met may be an independent prognostic biomarker in ESCC. Patients with c-Met over-expression treated with nimotuzumab had a shorter OS, indicating c-Met might be a predictor of efficacy of anti-EGFR therapy.


Test results showed that as SCM ratio (RSCM) increased, the strength gain ratio at an early age relative to the 28-day strength tended to decrease, whereas that at a long-term age increased up to RSCM of 0.8, beyond which it decreased. In addition, the beneficial effect of SCMs on the freezing-and-thawing and chloride resistances of the concrete decreased at RSCM of 0.9. Hence, it is recommended that RSCM needs to be restricted to less than 0.8–0.85 in order to obtain a consistent positive influence on the compressive strength and durability of SCM concrete.The objective of this study is to examine the compressive strength and durability of very high-volume SCM concrete. The prepared 36 concrete specimens were classified into two groups according to their designed 28-day compressive strength. For the high-volume SCM, the FA level was fixed at a weight ratio of 0.4 and the GGBS level varied between the weight ratio of 0.3 and 0.5, which resulted in 70–90% replacement of OPC. To enhance the compressive strength of very high-volume SCM concrete at an early age, the unit water content was controlled to be less than 150 kg/m However, since the late 1990s the concrete industries have exerted considerable effort and made investments to minimize the use of OPC, partly because of serious worldwide issue to reduce greenhouse gas emissions. It is generally estimated that the production of one ton of OPC consumes approximately 2.8 tons of raw materials such as limestone and coal and that it releases about 0.7–0.95 tons of carbon dioxide  and ground granulated blast-furnace slag (GGBS) as SCMs has gradually increased in the construction industry because of their environmentally beneficial recycling effect and remarkably low CO2 inventory [The use of high-volume supplementary cementitious materials (SCMs) as partial replacement for OPC in concrete has become increasingly attractive for the development of sustainable construction materials with low COnventory . Furthernventory –8. (1) Tnventory . On the nventory , 11 thatMalhotra et al. –12 did pRF) was fixed at 0.4, whereas that of GGBS (RG) varied between 0.3 and 0.5; as a result, 70–90% of the OPC was replaced with FA and GGBS. Concrete mixes with RF of 0.25 and RG of 0.15 were also prepared as control specimens in each group. To achieve good strength gain, especially at an early age, the unit water content was controlled to be less than 150 kg/m3, and a polycarboxylate-based water-reducing agent was added after being specially modified through the adjustment of the amount of polyethylene glycol alkyl ether and the addition of an amine. Simple equations to predict strength development of the very-high-volume SCM concrete samples are proposed based on the nonlinear multiple regression analysis of the measured results. Four very-high-volume SCM specimens, together with the companion control mixes, were selected in order to examine their durability under the following environments: repeated freezing and thawing, chloride penetration, and sulfate attack.The objective of the present study is to examine the practical feasibility of producing very-high-volume SCM concrete (incorporating FA and GGBS) with relatively good strength gain at an early age. A total of 36 high-volume SCM concrete mixes with different mixture proportions were prepared according to designed concrete compressive strengths of 24 MPa (Group I) and 30 MPa (Group II). As a partial replacement for OPC, the weight ratio of FA (2)-to-aluminum oxide (Al2O3) ratio by mass of 1.91, which belongs to Class F of ASTM C618 [2-to-Al2O3 ratio by mass of 2.29, which is very similar to that of OPC. The basicity of GGBS calculated from the chemical composition was 1.94. The specific gravity and specific surface area, respectively, were 3.15 and 3466 cm2/g for OPC, 2.23 and 3720 cm2/g for FA, and 2.91 and 4497 cm2/g for GGBS.OPC (ASTM Type I) was partially replaced with commercially available FA and GGBS powders, which produces a ternary-type binder. The chemical compositions of these materials were determined by X-ray fluorescence (XRF) analysis and the results are given in STM C618 . The LOISTM C618  had highLocally available natural sand with a maximum particle size of 5 mm and crushed granite with a maximum particle size of 25 mm were used for fine aggregates and coarse aggregates, respectively. The specific gravity and water absorption were 2.61 and 1.16%, respectively, for fine aggregate and 2.62 and 1.78% for coarse aggregate, as given in 2+ and OH− ions from the mineral compositions of the cement. From previous tests [To maintain good workability of the concrete at lower unit water content, the molecular structure of a polycarboxylate-based water-reducing agent was specially modified as follows. (1) The degree of polymerization of the main chain in the acryl acid-type polycarboxylate polymer was reduced by a factor of 10 see . (2) Theus tests , the optfcu) of 24 MPa (Group I) and 30 MPa (Group II). The selected test parameters in each group were as follows. (1) Two levels of the water content (W) were used, 140 kg/m3 and 150 kg/m3. (2) The unit binder content (B) for each water content level was varied as 310 kg/m3, 330 kg/m3, and 350 kg/m3 for Group I and 370 kg/m3, 390 kg/m3, and 410 kg/m3 for Group II; as a result, the water-to-binder ratios (W/B) for the Group I mixes were calculated to be 45.2%, 42.4%, and 40.0%, respectively, for W of 140 kg/m3 and 48.4%, 45.5%, and 42.9% for W of 150 kg/m3, while those in the Group II mixes were 37.8%, 35.9%, and 34.2% for W of 140 kg/m3 and 40.5%, 38.5%, and 36.6% for W of 150 kg/m3. (3) SCM level (RSCM) as a partial replacement for OPC was varied as 0.7, 0.8, and 0.9. At each RSCM, RF was fixed to be 0.4, whereas RG varied as 0.3, 0.4, and 0.5. The addition of FA as a partial replacement of OPC is favorable to the reduction of hydration heat of concrete but unfavorable to the strength development of concrete at an early age. Considering this fact, the present study selected RF to be 0.4. The volumetric fine aggregate-to-total aggregate ratio (S/a) was designed to be 48% for W of 140 kg/m3 and 46% for W of 150 kg/m3. For comparison, a control mix with a typical RSCM (RF of 0.25 and RG of 0.15) was also prepared for each group. Considering the demand increase trend on the use of SCM, the typical SCM concrete was selected for the control mix, instead of OPC concrete. From the practical mixture proportions of ready-mixed concrete batches, the unit water and binder contents determined for the control mixes were 184 kg/m3 and 342 kg/m3, respectively, for Group I and 165 kg/m3 and 400 kg/m3 for Group II. The targeted air content and initial slump of all concrete mixes were 4.5 ± 1.5% and 210 ± 25 mm, respectively. To meet the designed initial air content (Ac) and slump (Si), an air entraining agent and the specially modified polycarboxylate-based high-range water-reducing agent were added, as given in fcu of 24 MPa produced from the following mixture proportions: W of 140 kg/m3, B of 310 kg/m3, and RSCM of 0.7 (RF of 0.4 and RG of 0.3). Concrete specimens denoted by I-C and II-C indicate the control concrete with a typical RSCM value in each group.For easy recognition of test parameters, the concrete specimens were notated sequentially using the targeted compressive strength, water content, binder content, and SCM level as a partial replacement for OPC. For example, specimen I-140-310-0.7 indicates a concrete with 3 capacity. The initial slump (Si) and air content (Ac) of fresh concrete were measured in accordance with the ASTM C143 and C231 provisions, respectively [All concrete specimens were mixed using a twin forced mixing-type mixer with 0.35 mectively . All spe2 solution (4 g/L), an external electrical potential was applied axially across the specimen, forcing the chloride ions outside to migrate into the specimen. The catholyte solution was a 10% NaCl solution, whereas the anolyte solution was a 0.3 N NaOH solution. The penetration depth, measured from the visible white silver chloride precipitation at saturation ages of 28 and 91 days, was then converted into the chloride migration coefficient according to the procedure specified in NT Build 492. The sulfate resistance of the concrete was evaluated from the variations of compressive strength of the specimens saturated in a curing tank containing 5% sulfuric acid solution for 28 days.The compressive strength of the concrete was measured using cylindrical specimens of 100 mm in diameter and 200 mm high at ages of 3, 7, 28, 56, and 91 days in accordance with ASTM C39 . The durRSP) and air entraining agent (RA) to the total binder by weight used to achieve the target Si and Ac are given in RA was required for the very-high-volume SCM concrete mixes than for the companion control mixes, regardless of W and RSCM values. The value of RA was between 0.028% and 0.042% for Group I mixes and between 0.032% and 0.045% for Group II mixes, indicating that Ac of fresh concrete without the air-entraining agent was commonly lower in Group II mixes than in Group I mixes. To achieve the target compressive strength, a greater amount of binder was needed for the Group II mixes than for the Group I mixes at the same water content. This implies that increasing B at the same water content is accompanied by a decrease in the number of macrocapillaries and artificial air pores [RSP added to meet the targeted Si was slightly higher for the Group II mixes than for the Group I mixes. This is attributed to the fact that W/B of the Group II mixes was lower than that of the Group I mixes. On the other hand, the value of RSP tended to be independent of RSCM, indicating that the GGBS content has little influence on the workability of concrete [The ratios of the modified polycarboxylate-based water-reducing agent (ir pores . The speconcrete .W of 140 kg/m3 met the targeted 28-day compressive strength (fcu), as given in W of 150 kg/m3 failed to achieve fcu, in particular, for the concrete with RSCM of 0.9, and for the Group I concrete with W/B of 48.4% and the Group II concrete with W/B of 40.5%. As expected, the measured 28-day compressive strength (fc′) decreased with increasing W/B and RSCM. The ratio of fc′ of the very-high-volume SCM concretes relative to that of the control concrete is shown in RSCM, indicating that the rate of the decrease was greater for Group II mixes than that for Group I mixes. All concrete mixes with RSCM more than 0.8 developed lower fc′ than the control concrete. Furthermore, fc′ of the concrete with W of 150 kg/m3 was commonly lower by approximately 10% than that of the control concrete with W of 140 kg/m3, even at the same W/B, indicating that fc′ of very-high-volume SCM concrete is somewhat affected by W. Overall, to obtain a value of fc′ equivalent to that of a conventional concrete with a typical RSCM, very-high-volume SCM concrete should have W/B < 40% and RSCM = 0.7.Most concrete mixes with fc′ is taken to be inversely proportional to W/B and Ac [fc′ of concrete with various SCMs based on a nonlinear multiple regression (NLMR) analysis using an extensive amount of test data collected from the available literature. In the database for the regression analysis, the primarily ranges of the main parameters are as follows: W/B = 0.25–0.6, RF = 0.1–0.4, and RG = 0.2–0.4. The number of ternary-type-binders using OPC, FA, and GGBS in the database is small and RSCM is mostly within 0.5. Overall, the following equation, proposed by Yang, is thought to be suitable for concrete with a typical RSCM not exceeding 0.5:f0 (=10 MPa) is the reference value for the 28-day compressive strength of concrete and RS is the silica fume level as a partial replacement for OPC.In general,  and Ac . Conside and Ac  proposedfc′ of high-volume SCM concrete is somewhat sensitive to W, though sensitivity depends on the type and level of SCMs. Furthermore, to obtain the same fc′ of OPC concrete or concrete with a typical SCM level, a lower W/B is required for high-volume SCM concrete as compared to OPC concrete or typical SCM concrete. Considering these experimental observations, (fc′ of high-volume SCM concrete (see W0 (=100 kg/m3) is the reference value for the unit water content.vations,  was modirete see . Considefc′ than the Yang's model. The mean and standard deviation of the ratios between the experimental results and the predicted results are 0.89 and 0.103, respectively, for the Yang's model and 0.99 and 0.062 for the current model. This indicates that the Yang's model based on concrete mixes with typical SCM levels is likely to overestimate the 28-day compressive strength of high-volume SCM concrete.Comparisons of the measured 28-day compressive strength and predictions obtained from the Yang's model  and the W on the strength development rate. As RSCM increased, the strength gain ratio at an early age relative to the 28-day strength tended to decrease, whereas that at a long-term age increased up to RSCM of 0.8, beyond which it decreased somewhat. A slightly higher ratio at an early age and a slightly lower ratio at a long-term age were observed for Group II mixes as compared to Group I mixes, indicating that the strength development rate is affected by W/B. Relative to the 28-day strength of high-volume SCM concrete, the strength gain ratio at an age of 3 days ranged between 0.2 and 0.28 for Group I mixes and between 0.27 and 0.33 for Group II mixes, whereas that at age of 91 days ranged between 1.33 and 1.46 for Group I mixes and between 1.27 and 1.43 for Group II mixes. As compared with the predictions from the ACI equation, those values are lower by approximately 27–50% at 3 days and higher by approximately 14–31% at 91 days. This indicates that, by the ACI 209 equation, the compressive strength of very-high-volume SCM concrete is likely to be slightly overestimated at an early age or, conversely, underestimated at a long-term age. Although the specially modified polycarboxylate-based water-reducing agent was added to enhance the early strength of very high-volume SCM concrete, a strength gain lower than that found using the ACI 209 equation was measured at the ages of 3 and 7 days. However, it can be estimated that these low gains at an early age are not detrimental because the early strength gain of concrete with typical RSCM is frequently found to be 10–40% lower than that of OPC concrete or the values predicted using the ACI 209 equation [The typical compressive strength development rate of high-volume SCM concrete is shown in fc′(t) is the compressive strength according to age t (in days). The strength development rate at early and long-term ages is determined by the variation of the constants A1 and B1. In general, a lower value of A1 leads to a higher compressive strength gain at an early age. For OPC concrete cured by air drying, it is recommended that the values of A1 and B1 are 4.0 and 0.85, respectively. However, these values need to be modified for very-high-volume SCM concrete in order to minimize the error observed in R2) of more than 0.93, as listed in W/B, RG, and RF, the constant A1 tends to increase, whereas B1 decreases. The determined values of the constants appear to be more significantly affected by RG than by RF, whereas they are independent of W. Based on regression analysis using these influencing parameters, the two constants A1 and B1 in (The ACI 209 provision  empiricafc′(t)=tAd B1 in :(4)A1=1fc′ in (γm), standard deviation (γs), and coefficient of variation (γv) of the ratios between the experimental and predicted results are also given in the same figure. Compressive strengths at different ages predicted using  are  are fc′Ed) recorded every 30 cycles of freezing-and-thawing is shown in Ed above 98% throughout the 300 freezing-and-thawing cycles. The high-volume concrete with RSCM of 0.8 showed the same behavior as the control mixes. For the very-high-volume concrete with RSCM of 0.9, the value of Ed remained at 98% until the 210th freezing-and-thawing cycle, beyond which it gradually decreased to 90% until the end of the tests (300 cycles). This indicates that the freezing-and-thawing resistance of the selected high-volume SCM concrete mixes is comparable to that of the control mixes with the typical RSCM.The relative dynamic modulus of elasticity (Dnssm) of concrete specimens at ages of 28 and 91 days, which were calculated from the measured chloride penetration depth, in accordance with the procedure specified in NT Build 492 [Dnssm than that with designed strength of 24 MPa (Group I). Furthermore, the value of Dnssm tended to decrease with increasing age. The ratios of Dnssm values between ages of 91 and 28 days were calculated to be 0.81 and 0.61 for the I-C and II-C control specimens, respectively, and 0.44 and 0.33 for the I-140-330-0.8 and II-140-390-0.8 specimens, indicating that the decrease of Dnssm with age is higher in the very-high-volume SCM concrete than in the control concrete. At an age of 28 days, a slightly higher Dnssm was calculated for the very-high-volume SCM concrete than for the control concrete, regardless of concrete compressive strength. However, at an age of 91 days, Dnssm tended to decrease with increasing RSCM up to 0.8, beyond which it started to increase.uild 492 . As expefc′ owing to the saturation in sulfuric acid solution was between 30 and 32% for the control concrete, whereas it decreased to 8–20% for the very-high-volume SCM concrete; in other words, the ratio of fc′ after saturation in a sulfuric acid solution for 28 days relative to the concrete cured at room temperature was measured to be 69%, 81%, and 92% for specimens I-C, I-140-330-0.8, and I-140-350-0.9, respectively. This indicates that the deterioration of fc′ owing to sulfate attack decreased with increasing RSCM. This trend was similarly observed in terms of the damage to the specimens; that is, the presence of damaged chips and flaws decreased with increasing RSCM. Hence, it can be proposed that the very-high-volume SCM concrete has superior sulfate resistance as compared to conventional concrete.The variation of compressive strength of concrete measured from specimens saturated in 5% sulfuric acid solution for 28 days is shown in Dnssm in concrete mixes with RG varying from 0 to 0.85 recorded at an age of 91 days decreases with increasing RG. However, the addition of SCM exceeding a certain limit would result in decreased impermeability, as demonstrated in freezing-and-thawing and chloride resistances. Hence, RSCM needs to be restricted to less than 0.8–0.85 in order to maintain a positive influence on the durability of concrete.The beneficial effect of SCMs on the durability of concrete can be explained by improvement in both the impermeability and diffusion taking place in water-filled pores or by capillary suction. Gruyaert et al.  showed tW of 150 kg/m3 was commonly lower than that of the companion concrete with W of 140 kg/m3 by approximately 10%, even at the same W/B, showing that fc′ of high-volume SCM concrete is somewhat sensitive to W.The compressive strength of the concrete with fc′ equivalent to that of conventional concrete with typical RSCM, W/B and RSCM in very-high-volume SCM concrete need to be restricted to less than 40% and to 0.7, respectively.To achieve a value of RSCM increased, the strength gain ratio at an early age relative to the 28-day strength tended to decrease, whereas that at a long-term age increased until reaching RSCM of 0.8, beyond which it decreased somewhat.As Unlike the ACI 209 equation which overestimates the early strength of high-volume SCM concrete and underestimates the strength at a long-term age, the proposed equations describe well the compressive strength development of very-high-volume SCM concrete; the mean and standard deviations of the ratios between the experimental and predicted results were 1.0 and 0.082, respectively.RSCM. However, the beneficial effect of SCMs on the freezing-and-thawing and chloride resistances of concrete decreased at RSCM of 0.9.In general, the freezing-and-thawing, chloride, and sulfate resistances of the high-volume SCM concrete mixes were comparable to those of the control mixes with the typical The present investigation needs to be further extended to examine the carbonation resistance and inelastic deformation of very-high-volume SCM concrete in order to improve the compressive strength. From the experimental observations on the compressive strength and durability in the current study, the following conclusions may be drawn.


IgG4-TIN is the most common pattern of renal involvement in IgG4-related disease. There are several proposed diagnostic criteria of IgG4-TIN recently. Two of them proposed by the Mayo Clinic and JSN are predominant. However, histopathological criteria of the number of IgG4+ plasma cells and several histological features are still under discussion due to low amount of tissue in renal biopsy specimens and low frequency of this kind of specimens. We aimed to screen IgG4-TIN on archived renal biopsy samples and evaluated the application of two proposed diagnostic criteria.We selected 480 interstitial inflammation samples for light and electron microscopy and immunohistochemistry of CD138, IgG and IgG4 test. The Mayo Clinic proposed criteria diagnosed high-probability IgG4-TIN and JSN criteria confirmed IgG4-TIN.Twelve high-probability IgG4-TIN were screened by histology, imaging, serology and other organ involvement according to the Mayo Clinic proposed criteria. The previous principal pathological diagnoses were IgAN (n=4), CreGN (n=4), tubulointerstitial nephritis (n=3) and LN (n=1). Three cases showed storiform fibrosis and a bird’s eye pattern. The distribution of IgG4+ plasma cells was focal, multifocal or diffuse, with a mixed mild, moderate or strong stainingpattern. Their treatment and clinical outcomes varied depending on different levels of proteinuria, serum creatinine, eGFR and original glomerular disease presentation. Therefore, we applied strict histological criteria of storiform fibrosis and evenly distributed IgG4+ plasma cells by JSN to confirm typical IgG4-TIN. Two cases were finally diagnosed as real IgG4-TIN. One was previously diagnosed as idiopathic interstitial nephritis with rapid response to corticosteroid therapy. The other was CreGN with immune complex deposits, which had poor outcome and long-term hemodialysis.IgG4-TIN might present concurrently with glomerular disease. The proposed criteria by the Mayo Clinic is flexible, sensitive, and superior in the identification of early-stage or atypical IgG4-TIN, with enhanced risk of misdiagnosis as compared to the proposed criteria by JSN, which is stricter, more specific, and might overlook early-stage or atypical IgG4-TIN. We propose a new set of criteria to improve pathologist-derived diagnosis. IgG4-RD is recognized as a systemic autoimmune disease that is characterized by significant lymphoplasmacytic infiltration of IgG4 positive plasma cells, with obliterative phlebitis and storiform fibrosis leading to organ swelling or nodular lesions –3. It waPatients with sufficient acute or chronic interstitial inflammation  by light microscopy were enrolled in this study from April 2008 through December 2013, irrespective of the presence or absence of glomerular disease. Altogether, 480 patients who were first admitted as renal injury without any remarkable medical history were studied following approval by the ethical committees of Hangzhou Hospital of Traditional Chinese Medicine.HE-stained slides were reviewed by two pathologists. Cases were selected by lightmicroscopy as having an average plasma cell count of more than 5 plasma cells in at least 3 HPF fields . SectionHigh-probability IgG4-TIN cases were screened according to the criteria proposed by the Mayo Clinic as shown in Table The histological criterion proposed by JSN was much stricter as shown in Table Statistical analysis was performed with SPSS software version 17.0. Normally distributed data were expressed as mean ± SD. The clinical characteristics between two groups were compared by Student’s t test. Categorical variables of histological features between two groups were expressed as percentages and were compared by Chi-square test or Fisher’s exact test. Correlations were assessed by Spearman’s rank correlation to estimate the relationship of the clinical characteristics associated with IgG4 positive cells and the stage of interstitial fibrosis. P-values less than 0.05 were considered statistically significant.The procedure of screening cases is shown in Fig. According to the proposed criteria of the Mayo Clinic , twelve Originally, nine cases were diagnosed with glomerular diseases and three cases were diagnosed with TIN pathologically. Both CreGN and IgAN were predominant glomerular diseases among them. The others were idiopathic TIN, drug-induced TIN and LN Table . Most ofIn the high-probability IgG4-TIN group, all 12 cases presented with substantial histological differences, including the stage of fibrosis, the number of IgG4 positive cells and their distribution. Their treatment and clinical outcomes were absolutely different, and were dependent, at least in part, on proteinuria, serum creatinine, eGFR and presence of original glomerular disease. Therefore, we applied strict morphological criterion by JSN to confirm the diagnosis of IgG4-TIN and found three cases that met it. Two cases (case 5 and case 11) met all criteria of histology, imaging, serology and other organ involvement. The left case (case 10) met all criteria but the imaging criterion.. The histological feature of this case, such as storiform fibrosis, was mild and distributed focally. Moreover, IgG4 positive cells were unevenly distributed and moderately stained. Therefore, we checked through the finding of more references, in which it was recommended to identify the presence of dense, diffuse infiltrates of IgG4 positive cells >50/HPF, which were highly specific –24. MoreThe epidemiology and etiology of IgG4-TIN remain unclear. It has been hypothesized that autoimmune disorders and allergic reactions might be the heterogeneous causes –28. HoweThe characteristic histological features such as storiform fibrosis and a bird’s-eye pattern were shown in more than 90 % of IgG4-TIN cases by JSN , 32. By The clinical and pathological evidence of unconfirmed IgG4-TIN remain understudy. It’s unknown whether IgG4-TIN is concurrent with or earlier than glomerular lesions, or the second injury of glomerular diseases. In previous studies, some pauci-immune CreGN, DN, LN and MN met the criteria of IgG4 > 10 per HPF, but they were not real IgG4-TIN , 17. TheIgG4-TIN is easily missed without IgG4 test at the time of renal biopsy. The essential nature of increased IgG4 plasma cells is unclear and it will exist in various kidney diseases meanwhile. Strict histological criteria are helpful, and sometimes necessary in differentiation of IgG4-TIN.


MET gene copy number (CN) has been considered to be a potential biomarker to predict the response to MET-targeted therapies in various cancers. However, the current standard methods to determine MET CN are SNP 6.0 in the genomic DNA of cancer cell lines and fluorescence in situ hybridization (FISH) in tumor models, respectively, which are costly and require advanced technical skills and result in relatively subjective judgments. Therefore, we employed a novel method, droplet digital PCR (ddPCR), to determine the MET gene copy number with high accuracy and precision.The analysis of MET gene CN and MET/CEN-7 ratio determined by SNP 6.0 and FISH, respectively.The genomic DNA of cancer cell lines or tumor models were tested and compared with the MET CN detected by ddPCR and SNP 6.0 is strong (Pearson correlation = 0.867). In tumor models, the MET CN detected by ddPCR was significantly different between the MET gene amplification and non-amplification groups according to FISH . Given that MET gene amplification is defined as MET CN >5.5 by ddPCR, the concordance rate between ddPCR and FISH was 98.0%, and Cohen's kappa coefficient was 0.760 .In cell lines, the linear association of the MET gene copy number with high precision and accuracy as compared with the results from SNP 6.0 and FISH in cancer cell lines and tumor samples, respectively.The results demonstrated that the ddPCR method has the potential to quantify the  MET gene amplification has been reported to be correlated with poor prognosis in patients with GC [MET gene copy number prior to MET-targeted therapy. In routine studies, SNP 6.0 and FISH are the standard methods to evaluate the MET copy number of cancer cell lines in vitro and tumor samples in vivo, respectively. However, these methods have many limitations, including the advanced technical skills required high costs and the need for experienced experts to analyze the tumor samples, which results in variable results between labs. For example, FISH analysis is usually performed on formalin-fixed, paraffin-embedded (FFPE) tissues, which requires a number of complex processes, such as fixation and immunohistological staining, and may cause genomic DNA damage and fracture. These issues increase the probability of false negative results because of the low quality and quantity of DNA. Therefore, the detection of gene amplifications is challenging because of the low sensitivity of these approaches.In normal physiological functions, MET is expressed in cells of epithelial origin, where it plays an essential role in cell growth and homeostasis . However with GC –9 and maMET copy number without need for standard curves. In a typical digital PCR, the sample is randomly distributed into discrete partitions such that some contain no nucleic acid template and others contain one or more template copies. The partitions are PCR amplified to end point and then read using a droplet reader to determine the fraction of positive partitions based on fluorescence amplitude, from which the absolute concentration of the target or reference DNA is estimated statistically by modeling as a Poisson distribution. Therefore, ddPCR is an end-point measurement that enables to quantify nucleic acids without the need for standard curves, external calibrators and endogenous controls [The droplet digital PCR (ddPCR) is a method that can absolutely quantify the controls .MET copy number in cancer cell lines and tumor samples and compared our results with those obtained using SNP 6.0 and FISH for the same samples.In this study, we sought to employ ddPCR assays to absolutely quantify the 2 and 95% humidity. Genomic DNA was extracted from the cancer cell lines with the TIANampGenomic DNA Kit  according to the manufacturer’s instructions. One hundred and fifty-six FFPE of patient-derived xenograft (PDX) models (including 116 GC and 39 HCC models) were obtained from the Shanghai LIDE Biotech Company. For the PDX model samples, genomic DNA was extracted using the DNeasy Blood and Tissue Kit  according to the manufacturer’s protocol. The DNA concentration was determined with a NanoDrop spectrophotometer (Thermo), and digested with the HaeⅢ enzyme  at 37°C for 1 h. The digested DNA sample was diluted 10-fold with autoclaved Millipore H2O and stored in a -20°C freezer.Eight human GC and thirty-eight HCC cell lines were purchased from five organizations: the American Type Culture Collection (ATCC), Japanese Collection of Research Bioresources (JCRB), Korean Cell Line Bank (KCLB), Shanghai Institutes of Biological Sciences, CAS (SIBS), and Zhongshan Hospital Fudan University (ZHFU) see . The celMET copy number was calculated as the ratio of the concentrations of MET and AP3B1 and multiplied by two. Each data for MET CN using ddPCR represents two or three merged technical replicate wells with no template control (NTC) as a negative control.The TaqMan PCR reaction mixture was assembled in a final volume of 20 μL with 2x Supermix, 20x primers and 20x probes and 20 ng of the genomic DNA as the template. Each reaction mixture was then loaded into a DG8 cartridge (Bio-Rad) with 70 μL of droplet generation oil to generate a droplet. The droplets from each well were then transferred into a 96-well PCR plate. The plates were heat-sealed and then thermally cycled under the following conditions: 95°C for 10 min (one cycle); 40 PCR cycles of 95°C for 15 seconds and 60°C for 1 min; and a hold at 4°C. After PCR, the plates were placed on a QX200 droplet reader (Bio-Rad) that analyzed the droplets of each well of the plate and quantified the target DNA. The PCR data were analyzed using QuantaSoft version 1.7.4.0917 (Bio-Rad) to determine the copy number variation (CNV). The 20x reference assay for AP3B1 included primers targeting the centromere loci on Chromosome 5 and a probe labeled with a HEX fluorescent signal (Bio-Rad). The 20 x CNV PCR assay for MET included primers targeting the region from intron 20-exon 21 on Chromosome 7, and the probe was labeled with a FAM fluorescent signal . Each well was replicated for all of the samples. The http://www.broadinstitute.org/ccle/home), and the raw data for the other 11 cell lines were collected using the Affymetrix Genome-Wide Human SNP Array 6.0 platform, including BEL7402, HCCC-810, NOZ, OCUG-1, OZ, QGY7701, QGY7703, SMMC7721, SNU354, SNU368, and SNU739. All of the raw data were processed using PICNIC software and presented as MET copies.Among the 46 cell lines, the SNP 6.0 raw data for 35 of the cell lines were downloaded from the CCLE project . The CEN-7 centromere probe was used as a reference control, according to the manufacturer’s instructions. The formalin-fixed, paraffin-embedded specimens (cell pellets) were sectioned and then subjected to deparaffinization and rehydration. H&E staining was performed first. The heat pretreatment was performed in pretreatment solution in a microwave oven for 3 min and 50 sec, cooled to RT over 15 min, and then washed. The sections were digested in RTU pepsin for 6 min at 37°C (the time can be adjusted for different section thicknesses). After washing, the sections were completely dehydrated. The sections containing the probe were denatured at 66°C for 10 min and then hybridized at 45°C for 1–2 h. After stringent washing, the sections were dehydrated, mounted in medium with DAPI, and stored in the dark at 4°C for 15 min before reading. A fluorescence microscope and appropriate filters were used to scan and identify the relevant tumor area. The signals of the red MET gene and green CEN-7 gene were counted in 20 tumor nuclei in a minimum of two areas to determine the MET/CEN-7 ratio. The FISH scoring was defined as follows: ratio ≤2.0: MET amplification was not observed; ratio >2.0: MET amplification was observed. If the ratio was borderline (1.8–2.2), an additional 20 nuclei were counted, and the ratios were recalculated.MET CN were combined for the statistical analyses. Pearson’s correlations and linear regression were used to evaluate the relationship between ddPCR and SNP 6.0 or FISH. To compare the differences in MET CN analyzed by ddPCR between the gene amplification and non-amplification groups analyzed by FISH, t-tests were used. The consistency of gene amplification based on ddPCR and FISH was evaluated according to the concordance rate and Cohen's kappa coefficient. The analyses were performed with SAS 9.4 software.The valid HCC and GC measurements for MET copy number detection, and the results of the ddPCR assay were compared to those of SNP 6.0 to determine whether ddPCR can replace the standard molecular biology techniques. The results were shown in MET copy numbers of cell lines in vitro using an Affymetrix microarray. Next, we used ddPCR to test the MET copy number in the same samples by normalizing to the AP3B2 reference gene. We observed that the cell lines with high MET copy numbers according to SNP 6.0 also had high ddPCR measurements. The linear association for MET copy number measurements between ddPCR and SNP 6.0 is strong based on Pearson’s correlation . Moreover, the slope and intercept of MET CN by SNP 6.0 to ddPCR in the linear regression were significant (P <0.001) with a value equal to 1.996 (standard errors of estimates = 0.177) and -4.159 (standard errors of estimates = 0.752), respectively  compared to the MET non-amplification group  analyzed by FISH using a t-test with unequal variance (P = 0.044) . Furthermore, using linear regression, the slope and intercept of the ratio of FISH to MET CN by ddPCR were significant (P < 0.001), with a value equal to 2.723 (standard errors of estimates = 0.176) and -0.803 (standard errors of estimates = 0.272), respectively  . The linectively . GeneralMET CN >5.5 by ddPCR had the highest concordance rate (98%) and Cohen's kappa coefficient (κ = 0.760) . Notably, among the 155 valid PDX samples, although most of samples had the consistency of MET gene amplification between ddPCR and FISH, only 3 PDX models  that were positive for MET CN by ddPCR did not show a detectable FISH ratio >2.0  and hepatocellular carcinoma (HCC), which results in dysregulation of MET signaling and is associated with clinical prognosis and poor outcome [MET gene amplification as a potential biomarker to estimate patients with cancers who will benefit from the treatment with selective MET inhibitors or antibodies. So far, there has been no standardized method to validate MET gene amplification or MET copy number.Human genomes exhibit segmental copy number variation (CNV) at thousands of loci . MET gen outcome . The abe outcome –9 have dMET copy number in cancer cell lines in vitro and tumor samples in vivo, respectively. However, a number of issues can complicate the detection of gene amplification. First, these methods are very costly and require advanced technical skills, and thus, informatics technicians or experienced pathologists must analyze the data and make subjective judgments. Second, in vivo tumor samples, such as FFPE tissues, are always tested by FISH and use long fragment probes, resulting in variations in the gene copy number detection depending on the probes targeting different gene loci. Thus, we sought to identify a method with simple technical requirements to more accurately detect the MET copy number.SNP 6.0 and FISH are the methods that are conventionally used to evaluate MET copy number or assess gene amplification from cancer samples consisting of cell lines or FFPE tumors. The results showed that ddPCR could determine the MET status in tumor samples. We found there was a positive correlation between the MET copy numbers detected by ddPCR and SNP 6.0 according to Pearson’s correlations . Although microarray technologies are valuable approaches for CNV determination, they have limited dynamic range and are expensive for high-throughput screening in population studies [MET amplification in 155 FFPE PDX tumors, ddPCR can reliably reflect the MET amplification status compared to FISH, with a 98% concordance rate. Moreover, there is significant difference of MET CNV detected by ddPCR between FISH-positive and FISH-negative groups, providing a highly significant proof of principle.In this study, we evaluated the feasibility of using a novel method of ddPCR to quantify the  studies . Because studies . Based oMET copy number values were high (CN >5.5) in ddPCR did not show gene amplification by FISH (MET/CEN-7 ratio >2.0). The reason for the discordance of the MET amplification between ddPCR and FISH may be the underestimation of MET amplification by FISH or overestimation of MET amplification by digital PCR. On the one hand, the presence of MET gene amplification could be masked because of characteristic of incomplete hybridization of long fragment probes used in FISH, resulting in non-specific identification of MET amplification. In particular, the variation in the MET amplification could be ascribed to the probe targeting different genomic loci detected by FISH. On the other hand, the fixation process used for the FFPE tissues could cause DNA fragmentation, damage or fusion, thus leading to a false-negative result. Additionally, considering that loss of chromosome frequently occurred in many cancers can result in genomic chromosomal instability [MET:AP3B1 in ddPCR, rather than high MET copy number.Notably, although in most of 155 FFPE PDX tumors, status of MET detected by ddPCR were comparable to those detected by FISH, three FFPE tumors whose tability –16, it iMET CN of another PDX sample (GAPF101) is 0.00022 with no successful CN call by ddPCR, extremely lower than MET/CEN-7 ratio of 1 by FISH. It implies that the deletion or damage of MET might occur in partial region of MET which were targeted by different probes employed in ddPCR and FISH, thus resulting in the difference of MET status between FISH and ddPCR. It will be intriguing to explore the reasons for the discordance in the future research.Of note, MET copy number in cancer samples either in vitro or in vivo, without the need for standard curves or endogenous control.Otherwise, the analysis of tumor samples by FISH requires complex experimental technology and may depend on the subjective judgment of pathologists. The results can vary between different pathologists based on their experiences. In this study, we show that ddPCR is a quantitative method that can absolutely quantify the MET copy number in cancer cell lines or FFPE DNA and highly correlated with SNP 6.0 or FISH, respectively. MET gene amplification has been described to be associated with tumorigenesis and metastatic progression and is regarded as a biomarker to predict benefit from MET-targeted therapy in various clinical studies [Overall, these data suggest that ddPCR has the potential to detect the  studies –9. Our fS1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


AbstractMulloidichthysflavolineatusflavicaudus Fernandez-Silva & Randall is described from the Red Sea and Arabian Sea, including Socotra and Gulf of Oman. It is characterized by a yellow caudal fin, 25–28 gill rakers, and 37–38 lateral-line scales and it is differentiated from nominal subspecies Mulloidichthysflavolineatusflavolineatus by 1.7% sequence divergence at the mitochondrial cytochrome b gene. The morphometric examination of specimens of Mulloidichthysflavolineatusflavolineatus revealed variation in head length, eye diameter, and barbel length, in western direction from the Hawaiian Islands, South Pacific, Micronesia, and the East Indies to the Indian Ocean. The population of Mulloidichthysflavolineatusflavicaudussubsp. n. in the Gulf of Aqaba differs from that of the remaining Red Sea by shorter barbels, smaller eyes, shorter head, and shorter pelvic fins. We present a list of 26 endemic fishes from the Gulf of Aqaba and discuss the probable basis for the endemism in the light of the geological history of this region.The number of goatfish species has increased recently, thanks in part to the application of molecular approaches to the taxonomy of a family with conservative morphology and widespread intraspecific color variation. A new subspecies PageBreak Mulloidichthysflavolineatus was described by Mulloidichthysflavolineatus from nearby Réunion, but it was later considered invalid by him , which also has a yellow caudal fin  gene  gene .PageBreakPageBreakPageBreakPageBreakMulloidichthysflavolineatus have mixed with a school of Mulloidichthysayliffe. Mulloidichthys of the Western Indian Ocean. It mimics and often schools with the snapper Lutjanuskasmira. It is an amazing example of parallel evolution with Mulloidichthysmimicus Randall & Guézé, 1980 of the Marquesas Islands and Line Islands in the Central Pacific, which closely mimics the stripe pattern of Lutjanuskasmira and forms aggregations with it.The caudal fin continues to be yellow from the Red Sea into the Gulf of Aden and Socotra, as shown by Fig. Mulloidichthysflavolineatus in Oman and Maldives include many individuals with yellowish caudal fin mixed with a few gray-tailed and yellow-tailed fish ; the California Academy of Sciences, San Francisco, CA, U.S.A. (CAS); the Museum of the Hebrew University of Jerusalem, Israel (HUJ); the Senckenberg Museum, Frankfurt, Germany (SMF); and the National Museum of Natural HistoryU.S. (NMNH). These were the primary sources of goatfish specimens examined in this study.Type specimens were deposited at the Lateral-line counts begin with the first pored scale completely posterior to the upper end of the gill opening and end at the base of the caudal fin . Counts of gill rakers were made on the first gill arch; they include all rudiments.standard length (SL), measured from the median anterior point of the upper lip to the base of the caudal fin ; body depth is taken vertically from the base of the first dorsal-fin spine where it emerges from the body ; body width is the maximum PageBreakwidth measured just posterior to the gill openings; head length (HL) from the front of the upper lip to the posterior end of the opercular membrane, and snout length from the same anterior point to the nearest fleshy edge of the orbit; orbit diameter is the greatest fleshy diameter, and interorbital width the least fleshy width; upper-jaw length is taken from the front of the upper lip to the end of the maxilla; barbel length is the maximum straight length; caudal-peduncle depth is the least depth, and caudal-peduncle length the horizontal distance between verticals at the rear base of the anal fin and the caudal-fin base; length of fin spines and rays of the dorsal and anal fins are measured from where they emerge from the body to their tip; caudal-fin length is the horizontal length from the posterior end of the hypural plate to a vertical at the tip of the longest ray; caudal concavity is the horizontal distance between verticals at the tips of the shortest and longest rays; pectoral-fin length is measured from the base of the uppermost ray; pelvic-fin length is measured from the base of the pelvic spine to the tip of the longest soft ray. Proportional measurements in the text are rounded to the nearest 0.05.Lengths of specimens are given as Mulloidichthysflavolineatusflavolineatus and Mulloidichthysflavolineatusflavicaudus subsp. n. were applied in the diagnoses and comparisons: the number of gill rakers, lateral-line scale counts, barbel length, eye diameter and head length. We also compared the length of the pectoral and pelvic fins, but these did not show differences between Mulloidichthysflavolineatusflavolineatus and Mulloidichthysflavolineatusflavicaudus subsp. n.Only meristic characters and measurements that vary between Because goatfishes present allometric changes in body form  during oMulloidichthysflavolineatus we obtained cytb sequences from 217 specimens sampled at nineteen sites throughout the Red Sea, the Arabian Sea, the Indian Ocean and the Pacific Ocean. To elucidate phylogenetic relationships we sequenced an additional fragment of the mitochondrial genome, the ATP synthetase 8 and ATP synthetase 6 (ATPase-8 and ATPase-6) regions, from individuals representative of the cytb diversity. We also sequenced an individual of Mulloidichthysvanicolensis and one of Mulloidichthyspfluegeri to use as outgroups. Briefly, DNA was extracted from fin clips and Polymerase Chain Reactions (PCR) were carried out using the primers L8331 (5'-AAA GCR TYR GCC TTT TAA GC-3') and H9236 (5'-GTT AGT GGT CAK GGG CTT GGR TC-3')  per 7.5 µL of PCR product, at 37 °C for 15 min, followed by deactivation at 85 °C for 15 min. We cleaned all PCR products using ExoSAP  and then sequenced them in the forward direction  using a genetic analyzer ABI 3130XL  at the Hawai‘i Institute of Marine Biology EPSCoR Sequencing Facility. The ATPase-8 and ATPase-6 sequences were aligned, edited, and trimmed to a common length using GENEIOUS PRO vers. 4.8.4  . We carrs. 4.8.4 , and thes. 1.8.0 , based os. 1.8.0 . We alsos. 1.8.0  and the axml-bb/ , respecthttp://dx.doi.org/10.5061/dryad.f54m5The data underpinning the analysis reported in this paper are deposited in the Dryad Data Repository at Taxon classificationAnimaliaPerciformesMullidaeFernandez-Silva & Randallsubsp. n.http://zoobank.org/779C9D55-B037-4548-A717-F5C33BC1ACD5Yellowtail GoatfishMulloidesflavolineatus : Mulloidichthysflavolineatus : SMF 35486 [ex SMF 24824], 142 mm SL, Red Sea, Sudan, Sanganeb Atoll , April 1991, coll. F. Krupp, V. Neumann & T. Paulus.SMF 24818, 6: 106–125 mm SL, Red Sea, Sudan, Sanganeb Atoll , April 1991, coll. F. Krupp, V. Neumann & T. Paulus; USNM 221124, 181 mm SL, Red Sea, Egypt, off “ancient site” of Koseir along reef in cove ca. 5 km north of Koseir town , 8 Jan 1965, coll. H. A. Fehlmann; HUJ 9985, 3: 73–93.5 mm SL, Red Sea, Gulf of Suez, El Kura, a few km south of Dahab ; CAS 237352, 4: 107–147 mm SL, Red Sea, Saudi Arabia, Thuwal, inner Fesar , 18 April 2014, coll. P. Saenz-Agudelo; BPBM 41246 [ex CAS 237352], 102 mm SL, same data as preceding.Gulf of Suez: HUJ 5635, 107 mm SL, A-Tur. Gulf of Aqaba: BPBM 19843, 4: 114–122 mm SL, Nuweiba; CAS 58876, 252 mm SL, Eilat; CAS 206715, 133 mm SL, Eilat; CAS 206726, 198 mm SL, Eilat; CAS 206736, 167 mm SL, Eilat; HUJ 5905, 2: 128–144 mm SL, Eilat; HUJ 20216, 4: 107–110 mm SL (poor condition), Eilat; HUJ 8315, 169 mm SL, Ras Muhammed; HUJ 8543, 108 mm SL, Ras Muhammed; HUJ 8658, 2: 164–235 mm SL, Nabq; HUJ 8645, 3: 159–177 mm SL, Sanafir Island; HUJ 11663, 109 mm SL, Eilat; HUJ 8642, 243 mm SL, Nabek. Sudan: SMF 24821, 203 mm SL, Sanganeb Atoll; SMF 24823, 13: 97.5–161 mm SL, Sanganeb Atoll.SL; head moderately compressed, the length 3.0–3.3 in SL; snout long, slightly blunt. Barbels usually not reaching a vertical at posterior margin of preopercle, their length 4.1-5.0 in SL. Eye diameter 10.3–13.5 in SL. Pectoral-fin rays 16–18. Gill-raker counts 25–28 ; lateral-line scales 37–38. Caudal fin yellowish to yellow. .Body elongate, the depth at first dorsal-fin origin 4.1–4.5 in SL in Table SL for the holotype and in parentheses for selected paratypes (n=7), except where indicated.Meristics are provided in Tables PageBreakBody elongate, its depth at first dorsal-fin origin 4.1 (4.2–4.5), and maximum width 6.7 (6.5–7.3), head length 3.2 , snout length 6.9 (6.8–7.7), orbit diameter 13.0 , barbel length 4.5 , caudal-peduncle length 4.8 (4.6–5.1), caudal-peduncle depth 11.6 (11.4–12.0), pelvic-fin length 4.9 (4.7–5.3), pectoral-fin length 4.8 (4.7–5.1), longest anal ray 7.5 (7.2–7.7), longest dorsal spine 4.8 (4.6–5.1), longest dorsal ray 7.2 (7.2–7.8).SL; jaws with small conical teeth, in two rows with teeth more irregularly placed between both rows; no teeth on the vomer and palatines; anterior nostril small, elliptical, two-thirds eye diameter in front of eye; posterior nostril small, elliptical, at dorsoanterior corner of orbit; opercular spine flat, at mid-eye height.Mouth small, maxilla not reaching a vertical at front of orbit, upper-jaw length 12.3 (12.2–13.9) in PageBreakScales very finely ctenoid; head fully scaled; scales on the base of caudal fin, other fins without scales; dorsal fin behind the vertical at fourth lateral line scale, origin of second dorsal above 18th (17th in some paratypes) scale. Pored scales on lateral line with many branching tubules.PageBreakPageBreakPageBreakColor in life silvery white to yellowish, slightly darker over lateral line; margin of each scale on upper half of body darker than scale. Yellow stripe on side of body at level of eye, from posterior margin of orbit to caudal-fin base, bordered by a narrow whitish stripe (stripe sometimes slightly blue); the stripe usually containing a black spot above posterior part of pectoral fins , sometimes faint due to fading, stripe anterior to spot occasionally indistinct; barbels white; dorsal fins usually transparent, sometimes first dorsal fin with yellowish tinge; pectoral, anal, and pelvic fins whitish, translucent; caudal fin yellowish or yellow. Color when fresh often pink and all fins yellow. Uniformly creamy white in preservative.Mulloidichthysflavolineatusflavicaudus subsp. n. is named in reference to the yellow color of the caudal fin, in contrast to the whitish gray color of the caudal fin of Mulloidichthysflavolineatusflavolineatus.Mulloidichthysflavolineatusflavicaudus subsp. n. is restricted to the NW Indian Ocean biogeographic province, where it ranges from various locations in the Red Sea (including the Gulf of Aqaba), the Gulf of Tadjoura, the Gulf of Aden, and Socotra  and shorter head (3.0–3.6) (Tables The population of ) Tables  and 3. IMulloidichthysflavolineatusflavicaudus subsp. n. differs from its nominal subspecies Mulloidichthysflavolineatusflavolineatus in having 25–28  gill-raker counts , usually 37–38 lateral-line scales (37–40 in Mulloidichthysflavolineatusflavolineatus) and a yellow caudal fin (white to light gray in Mulloidichthysflavolineatusflavolineatus). Also, the eyes are smaller in Mulloidichthysflavolineatusflavicaudus subsp. n. (10.3–13.5 in SL) than in Mulloidichthysflavolineatusflavolineatus (9.8–15.6 in SL).Taxon classificationAnimaliaPerciformesMullidaeYellowstripe GoatfishMullusflavolineatus Lacepède, 1801: 384, 406 .MulloidichthysflavolineatusPageBreak(Christmas Island);  : RandallBPBM 20135, 162 mm SL, Indian Ocean, Mauritius, East Coast, Oyster Bay , 1 November 1973, coll. J.E. Randall.Hawaiian Islands: BPBM 28726, 83 mm SL, Kona Coast, South Kohala; BPBM 4087, 288 mm SL, Laysan; BPBM 4086, 180 mm SL, Laysan; BPBM 4088, 2: 139–230 mm SL, Lisiansky; BPBM 25457, 130 mm SL, O‘ahu, Wai‘anae coast; BPBM 25674, 175 mm SL, O‘ahu; BPBM 1749, 183 mm SL, O‘ahu; BPBM 1750, 173 mm SL, O‘ahu; BPBM 15308, 152 mm SL, Midway Atoll; BPBM 25517, 119 mm SL, Midway Atoll; USNM 147073, 158 mm SL, Midway Atoll. Johnston Atoll: BPBM 4090, 85 mm SL; BPBM 4091, 93 mm SL; BPBM 7520, 166 mm SL; Philippines: USNM 327877, 107 mm SL, Sorsogon, Gubat Bay; USNM 405724, 209 mm SL, W Luzon, Port Matalvi; USNM 147062, 222 mm SL, Mindoro, Varadero Bay; USNM 147066, 169 mm SL, W Luzon, Zambales; USNM 322272, 3: 138-155 mm SL, Babuyan, Maybag Island; USNM 147069, 2: 171–176 mm SL, Batangas, Maricaban; USNM 84231, 128 mm SL, Mindanao, Zamboanga; USNM 84232, 139 mm SL, Mindanao, Zamboanga; USNM 147070, 164 mm SL, Palawan, Candaraman; USNM 147072, 145 mm SL, Tulayan Island, Jolo; USNM 145294, 2: 98–100 mm SL; USNM 147065, 231 mm SL, Sulu, Siasi Island; USNM 147076, 135 mm SL, Sulu, Simaluc Island, Tawi Tawi. Indonesia: USNM 147067, 221 mm SL, Moluccas, Bouru Island; USNM 147064, 3: 195–205 mm SL, Moluccas, Makian I; USNM 405723, 200 mm SL, Moluccas, Makian Island; USNM 267514, 2: 102–126 mm SL, Mentawai Islands, Pulau Siburu; USNM 267503, 2: 155–156 mm SL, Mentawai Islands, Pulau Siburu; USNM 147058, 203 mm SL, Sulawesi, Talisse Island; USNM 87989, 255 mm SL, Sumatra, Poeloe Toekus; USNM 75887, 250 mm SL, Borneo, Tandjoeng, Setebah. Cocos-Keeling: SU 35630, 200 mm SL, Cocos-Keeling I. Papua New Guinea: USNM 267499, 203 mm SL, Trobriand Kuia Islands; USNM 267515, 2: 116–120 SL, New Britain, Rabaul. Solomon Islands: USNM 382371, 205 mm SL, Santa Cruz Islands. Micronesia: BPBM 77, 8: 75–230 mm SL, Guam; BPBM 4089, 3: 209–220 mm SL, Wake Island; BPBM 24628, 12: 79–160 mm SL, Chuuk, Puluwat Atoll. Japan: BPBM 7086, 2: 88–108 mm SL, Minami-Tori Shima; BPBM 7087, 210 mm SL, Minami-Tori Shima. South Pacific: BPBM 27868, 5: 81–119 mm SL, Samoan Islands, Tutuila Island; BPBM 27906, 3: 84–107 mm SL, Samoan Islands, Tutuila Island; BPBM 15299, 16: 81–159 mm SL, Phoenix Islands, Orona Atoll; BPBM 12937, 165 mm SL, Rapa; BPBM 2136, 198 mm SL, Marquesas Islands, Nuku Hiva. Western Indian Ocean: USNM 229036, 9: 129–192 mm SL, Chagos Archipelago, Salomon Atoll; CAS 237312, 2: 137–144 mm SL, Maldives, Faafu Atoll; BPBM 34673, 2: 107–115 mm SL, Maldives, N Malé Atoll; CAS 35383, 2: 85.5–142 mm SL, Maldives, Malé Atoll; BPBM 41252, 2: 120–135 mm SL, Mauritius, Oyster Bay.PageBreakSL; head moderately compressed, the length 2.9–3.8 in SL; snout long, slightly blunt anteriorly. Barbels usually not reaching a vertical at posterior margin of preopercle, their length 3.7–6.0 in SL. Eye diameter 10.1–15.6 in SL. Pectoral-fin rays 16–18. Gill-raker counts 27–29 (rarely 26 or 30); lateral-line scales 37–40 . Caudal fin varying from usually white or light gray to occasionally yellowish or yellow.Body elongate, the depth at first dorsal-fin origin 4.0–4.6 in Silvery white to yellowish, slightly darker over lateral line, margins of each scale on upper half of body darker than scale. Yellow stripe on side of body at level of eye, beginning from posterior margin of orbit and ending at caudal-fin base, bordered by two whitish narrow stripes (sometimes slightly blue); the stripe usually containing a black spot above posterior part of pectoral fins , sometimes faint due to fading, stripe anterior to spot occasionally indistinct; barbels white; dorsal fins usually transparent, sometimes first dorsal fin with yellowish tinge; pectoral, anal, and pelvic fins whitish, translucent; caudal fin varying from usually white or light gray to occasionally yellowish or yellow. Sometimes body color pattern of broad irregular red-brown bars, especially at night. When fresh, body color can turn pink and all fins yellow. Uniformly creamy white in preservative.Mulloidichthysflavolineatusflavolineatus is wide-ranging from East Africa north to the Maldives and Chagos Archipelago and east to the Hawaiian, Marquesas and Pitcairn Islands, north to the Ryukyu and Bonin Islands and south to Lord Howe Island, New Caledonia and Rapa Island  and individuals in the rest of the Indian Ocean and the Pacific Ocean  and five from the Pacific (Hawai‘i and Okinawa). Phylogenetic reconstructions based on Bayesian inference  and Mulloidichthysmartinicus  less than 350,000 years ago (unpublished results).Notably, the age of split of the Mulloidichthysflavolineatusflavicaudus subsp. n. from the Gulf of Aqaba have consistently smaller eyes, longer head, and longer barbels than fish from the Red Sea proper  than in the rest of the Red Sea (4.40 in SL). However, both populations extensively share cytb haplotypes and the analyses of haplotype frequencies do not support genetic differentiation, although this comparison is based on mitochondrial markers only   . The Gully Table . Moreovely Table . Therefoly Table .Mulloidichthysflavolineatus (PageBreakJohnston Atoll) from the remainder of the Indo-Pacific. Mulloidichthysflavolineatus have shorter barbels than those in the Indian Ocean, but he includes the Hawaiian Islands with the rest of the Pacific in this study. We found the Hawaiian population has shorter barbels, shorter head, smaller eyes, higher gill-raker PageBreakcounts, and higher lateral-line scale counts than all other populations examined, and that there is a range of variation as we move from Hawai‘i to other islands of Oceania, the West Indies, the Western Indian Ocean, and the Red Sea , Upeneusdavidaromi, Upeneusniebuhri Guézé, 1976, and Upeneuspori Ben-Tuvia & Golani, 1989, but inclusion of the latter to the Red Sea endemics is a mistake, as this species is also reported from Oman, Madagascar and South Africa.


Pneumocystis jiroveci pneumonia. A review of the etiology, pathophysiology, and treatment of methemoglobinemia is presented.We report a case of methemoglobinemia with significant hemoglobin desaturation in a young female with AIDS who was being treated for  It is caused by a number of medications and toxins and can quickly degrade oxygen transport sufficiently enough to cause or aggravate severe tissue hypoxemia. Its hallmark features are hemoglobin desaturation out of proportion to blood partial pressure of oxygen and “chocolate brown” blood. Definitive diagnosis can be made quickly and easily with co-oximetry, but must be suspected, as co-oximetry is not routinely performed in patients. It is important to recognize, as correction of the pathologic hemoglobin redox state with methylene blue is simple, rapid, effective, and lifesaving. We present a case of methemoglobinemia secondary to primaquine, which was successfully treated with methylene blue and discontinuation of the drug.Methemoglobinemia is the presence of a significant amount of oxidized iron (Fe4 count = 26/mm3) was admitted to a local hospital for cough, fever, and respiratory distress. Initial blood cultures grew gram-positive cocci in clusters, and endocarditis was suspected. Vancomycin was started, but a transthoracic echocardiogram was nondiagnostic, and the patient was transferred to our institution for further evaluation.A 32-year-old female with a history of intravenous drug abuse and AIDS . On day 7, she developed moderate digital and perioral cyanosis and an increasing lactic acid level (7.3 mmol/L). ABG showed pH 7.42, pCO2 33, with a saturation of only 86% despite a pO2 of 165. Co-oximetry revealed a carboxyhemoglobin level of 1.6% and a methemoglobin level of 20.1%. She received methylene blue 100 mg IV and was switched from primaquin to intravenous pentamidine. Methemoglobin did not recur. She ultimately died from complications of multiorgan system failure (MOSF) on hospital day 12.She was started on empiric gatifloxacin, primaquin, clindamycin, and prednisone for community-acquired pneumonia, with coverage for 3+ in the heme moiety of Hgb. Normally, met-Hgb is produced at low levels by oxidative stress in the blood at a rate of 3% per day [+3 is rapidly and efficiently reduced to normal (Fe+2) via the cytochrome b5 reductase pathway  that resists reduction. Due to the chronic nature of the inherited type, compensatory mechanisms such as increased red blood cell mass and cardiac output have time to develop, and thus patients are characteristically cyanotic but asymptomatic , 5.  TabAcquired met-Hgb results from exposure to drugs, pollutants, and toxins, which are also listed in 2-Hgb affinity within the affected multimer, left shifting the Hgb dissociation curve. This further decreases oxygen delivery by decreasing oxygen offloading in peripheral tissues [Mild cyanosis is characteristic of methemoglobinemia. Only about 1.5 g/dL (10–15%) of met-Hgb is needed to produce detectable cyanosis, and up to 70% met-Hgb can be relatively well tolerated if the amount of Hgb is adequate, and there has been enough time to develop compensatory mechanisms. Met-Hgb levels of 25–50% can cause headache, confusion, and chest pain . More pr tissues , 20. Man2 saturation will be decreased from that predicted by the PaO2 and a normal oxygen-Hgb dissociation curve. A rule of thumb is that the saturation, as measured by pulse-oximetry, will drop by one half of the met-Hgb concentration between 3 and 20%. Thus, a patient with 98% saturated arterial hemoglobin who develops a met-Hgb level of 10% can be expected to have pulse oximetry of 93% [Pulse oximetry, especially in combination with arterial blood gas analysis, is useful in the diagnosis of methemoglobinemia. The absorption spectrum of met-Hgb overlaps with that of oxyhemoglobin at 660 and 910 nm, the wavelengths used in most pulse oximeters , and so y of 93% . With hiy of 93% , 23. Defy of 93% , 24. Initial treatment consists of discontinuation of the offending drug. Methylene blue, a dye that acts to transport an electron from NADPH to hemoglobin, may be administered at a rate of 1-2 mg/kg for 5 minutes to hasten met-Hgb reduction, although in theory other antioxidants such as vitamin C , tocopherol, or N-acetyl cysteine may also be beneficial, and have been utilized in occupational and congenital diseases , 19. Of Methylene blue 1.5 mg/kg was administered intravenously, causing the patient's pulse oximetry to increase to 93% after 1-2 minutes and with immediate resolution of her cyanosis. Repeat co-oximetry demonstrated complete resolution of the met-Hgb. She was switched from primaquin-clindamycin to IV pentamidine to complete her therapy for PCP. While her met-Hgb never recurred, she did not survive her hospitalization. Supportive care was withdrawn after the development of severe MOSF. We suspect that methemoglobinemia may have played a role in her progression to MOSF, by converting a borderline hypoxemia into a profound deficiency in tissue oxygen delivery during her critical illness. 2) and measured Hgb saturation, especially when pulse oximetry reads about 85%. Definitive diagnosis is made by sending an arterial blood sample for co-oximetry. Treatment is removal of the offending drug, with methylene blue administration if tissue hypoxia is critical.Many drugs routinely used in the ICU can cause methemoglobinemia, adding to deficient oxygen delivery in this vulnerable patient population. A high index of suspicion should be kept for methemoglobinemia in ICU patients. Clues to the presence of met-Hgb are cyanosis, chocolate brown blood, and dissociation between expected (from paO


Thus, our study aimed to determine the biocompatibility and stability of the matrix containing structurally stabilized epidermal growth factor (S-EGF) and basic fibroblast growth factor (S-bFGF).Diabetes mellitus is a disease lack of insulin, which has severely delayed and impaired wound healing capacity. In the previous studies, various types of scaffolds and growth factors were used in impaired wound healing. However, there were several limitations to use them such as short half-life of growth factors in vitro. Also, these matrixes had longer releasing period that result in enhancement of half-life. Finally, when these matrixes were applied on the wound of diabetic mice, there were no inflammatory responses, in vivo. Thus, our results demonstrate that these matrixes are biologically safe and biocompatible as wound-dressing material.We stabilized EGF and bFGF that are structurally more stable than existing EGF and bFGF. We developed biocompatible matrix using S-EGF, S-bFGF, and hyaluronate– collagen dressing (HCD) matrix. The developed matrix, S-EGF and S-bFGF loaded on HCD matrix, had no cytotoxicity, Our stabilized EGF and bFGF was more stable than existing EGF and bFGF and the HCD matrix had the capacity to efficiently deliver growth factors. Thus, the S-EGF and S-bFGF loaded on HCD matrix had improved stability. Therefore, these matrixes may be suitable for impaired wound healing, resulting in application of clinical treatment.The online version of this article (doi:10.1186/s40824-016-0056-4) contains supplementary material, which is available to authorized users. The wound healing process consists of inflammation, contraction, neoangiogenesis, extracellular matrix deposition, granulation tissue synthesis, re-epithelialization and remodeling. These stages are related in various cellular and molecular signals  and whenin vivo [Recently, several studies have reported that the growth factors, as therapeutical agents, are widely used in impaired wound healing , 4. Platin vivo .in vivo. Indeed, the inadequate experimental conditions that did not continuously release growth factors from scaffolds and lose their activity readily when they are loaded onto various types of scaffolds [Although these growth factors have therapeutic effects in diabetic wound healing, they have been limited to use as therapeutic agents due to the short half-life of growth factors, caffolds , 14–17. There have been variety types of scaffolds such as collagen sponge , photo-cin vitro. Furthermore, we investigated in vivo stability of modified growth factors-loaded HCD matrix, via application on the diabetic mice.The hyaluronate- collagen dressing (HCD) matrix has been already used as wound-dressing materials because of advantages such as haemostatic effect, reduction of pain, maintenance of moisturizing condition, and feasible to exchange the wound dressing material because the surface of the HCD matrix does not adhere to wound. For these reason, we considered that HCD matrix is suitable for base material. So, we have assessed the biocompatibility and stability of modified growth factor-loaded HCD matrix, 2 and aliquot them into the mold for the lyophilization , Collagen  and Pluronic F68  are base materials for production of hyaluronate- collagen dressing (HCD) matrix. For the production of HCD matrix, we first dissolved the 0.1 % of collagen in refined water (pH3 ~ 4) and raised pH to 7 ~ 8 then dissolved 0.8 % of HA. The 0.1 % of F68 was added to help blending collagen and HA then evenly mixed them with homogenizer. Subsequently, we added stabilized growth factors with concentrations of 0.1, 0.3, 1, and 2.5ug/cm2 and medium was replenished every two days. Each of cells were seeded at density of 1X105cells/well on 96 well plates and incubated for 24 h. We classified the groups as negative control (no treatment), positive control  [2. Then thyiazolyl blue tetrazolium bormide  was added and incubated for 3 h. At the indicated time, DMSO was added and absorbance was read at 570 nm.L929 cells  were cultured in Dulbecco’s Modified Eagle’s Medium-high glucose  supplemented with 10 % FBS and 1 % penicillin/streptomycin (P/S). The NIH/3T3 fibroblast cells (Sigma-Aldrich) and Balb/3T3 fibroblast cells (Sigma-Aldrich) were cultured in DMEM (Gibco) supplemented with 0.5 % bovine calf serum (BCS) and 1 % P/S. These cells were incubated at 37 °C incubator with 5 % CO10993-5) , S-EGF, 5cells/well on 6well plate. After 24 h of cultivation, medium were removed and 1.5 % agar  diluted in distilled water was overlaid on the cells. After solidification of agar, 0.01 % neutral red vital dye (Sigma-Aldrich) was treated on medium and placed in dark 37 °C incubator for 90 min. The sterilized paper discs (6 mm) containing HA, were laid on agar and incubated in dark for 24 h. After incubation, cytotoxicity was rated with decolorized zone around the paper discs [L929 cells were seeded 4X102 of S-EGF and S-bFGF (diameter of 10 mm) was immersed in 10 ml of 0.1 % BSA (Sigma-Aldrich) dissolved in PBS and incubated in shaking incubator at 37 °C with 15 rpm for 7 days. Thereafter, to determine the amount of growth factors, these mixtures were diluted to 1/1000 and analyzed by ELISA kit . Also, to confirm concentration of released S-EGF and S-bFGF from HCD matrix, we installed matrices (diameter of 30 mm) in the Franz Cell and incubated in shaking incubator at 37 °C with 50 rpm. Then, collected samples on day 1, 3, 7, 10, 14, 21 and confirmed quantity of S-EGF and S-bFGF with ELISA kit (R&D system).Each of disk-shaped HCD matrix containing 1 μg/cmICR mice  were purchased from Orient Bio  and housed in wire cage at 20–22 °C at a relative humidity of 40–50 %. We used type1 diabetic mice, induced by intraperitoneal (I.P.) injection of streptozotocin (STZ)  dissolved in 0.05 M citrate buffer (pH4.5). One week after induction of diabetes, blood glucose levels were measured using OneTouch Select meter . The diabetic phenotype in animals was confirmed by blood glucose levels over 300 mg/dL and maintained for three weeks. The animal experiments were carried out in accordance with guidelines set by the Department of Laboratory Animal Resources, Yonsei University College of Medicine and Seoul, Korea (Permit number = 2015-0190).2, HCD+S-EGF 1 μg/cm2, HCD+S-EGF 2.5 μg/cm2, HCD+S-bFGF 0.3 μg/cm2, HCD+S-bFGF 1 μg/cm2, HCD+S-bFGF 2.5 μg/cm2. After application of matrix on wound site, Vaseline gauze  and Neo dressing  were placed on matrix for the minimal dehydration of wound sites. In each groups, twelve animals were used.STZ-induced type1 diabetic mice  were anesthetized with I.P. injection of Zoletile (30 mg/kg body weight) and Rumpon (10 mg/kg body weight). The hair on the back of mouse was shaved and subsequently wiped with 70 % ethanol. A 10 mm diameter full-thickness of skin wounds were created on the backs of STZ-induced diabetic mice and fixed with a silicone ring to prevent wound contraction. The groups are classified as Defect control, HCD only, HCD+S-EGF 0.3 μg/cmp < 0.05, **p < 0.01 were considered statistically significant.Statistical analysis was performed via Student’s T-TEST and ANOVA. The data was expressed as the mean ± standard deviation (SD). Values of *First, we performed cytotoxicity assay for the evaluation of the biological stability of stabilized epidermal growth factor (S-EGF) and basic fibroblast growth factor (S-bFGF). All concentrations of S-EGF and S-bFGF have no significance compared to negative control, indicating that S-EGF and S-bFGF do not retain toxicity Fig. . The resFor biocompatibility of HCD matrix, we evaluated cytotoxicity of three base materials, respectively. The collagen and F68 had more than 90 % of cell survival rate, whereas more than 1 % of HA showed reduction of survival rate Fig. . Since tWe evaluated the final concentration of S-EGF and S-bFGF that are loaded on HCD matrix. As a result, the concentrations were 89.44 ± 4.6 pg/ml and 85.27 ± 2.07 pg/ml, respectively Fig. . When weAdditionally, we determined the biological stability of S-EGF and S-bFGF loaded on HCD matrix. Similar to HCD group as a negative control, each concentration of S-EGF and S-bFGF loaded on HCD matrix showed more than 90 % of cell survival rate at all concentrations. These results define the biological safety of our S-EGF Fig.  and S-bFin vivo. These matrixes had no inflammatory effect at day 7, indicating that the S-EGF and S-bFGF loaded on HCD matrix is negligent as wound-dressing material. In addition, the S-EGF and S-bFGF loaded on HCD matrix showed slight acceleration of wound healing when compared to defect control and HCD matrix only group (Fig. Next, we assessed the stability of S-EGF and S-bFGF loaded on HCD matrix oup Fig. . As showoup Fig. . Thus, tin vitro. Indeed, this matrix was biologically safe, in vivo. Although, the further studies toward S-EGF and S-bFGF loaded on HCD matrix are needed to evaluate the effectiveness, in vivo, S-EGF and S-bFGF loaded on HCD matrix is expected to be valuable wound-dressing material in impaired wound healing.Taken together, our results demonstrated that S-EGF and S-bFGF are more stabilized than existing EGF and bFGF after modification, resulting in longer half-life and releasing period of growth factors from HCD matrix, in vitro. Furthermore, when these matrixes were applied on the wound of diabetic mice, the inflammatory response was not occurred, indicating that this matrix is biocompatible. According to these results, the S-EGF and S-bFGF loaded on HCD matrix may overcome the disadvantages of established scaffolds in wound healing study. Taken together, the S-EGF and S-bFGF loaded on HCD matrix may contribute to the application in impaired wound healing because of the biological safety.For impaired wound healing, we developed the S-EGF and S-bFGF loaded on HCD matrix, which has no effects on cytotoxicity and proliferation. Also, we confirmed stability and releasing period of S-EGF and S-bFGF loaded on HCD matrix. As a result, these matrixes showed more stabilized and longer releasing period of growth factors than existing EGF and bFGF,