The legacy of the use and misuse of antibiotics in recent decades has left us with a global public health crisis: antibiotic-resistant bacteria are on the rise, making it harder to treat infections. At the same time, evolution of antibiotic resistance is probably the best-documented case of contemporary evolution. To date, research on antibiotic resistance has largely ignored the complexity of interactions that bacteria engage in. However, in natural populations, bacteria interact with other species; for example, competition and grazing are import interactions influencing bacterial population dynamics. Furthermore, antibiotic leakage to natural environments can radically alter bacterial communities. Overall, we argue that eco-evolutionary feedback loops in microbial communities can be modified by residual antibiotics and evolution of antibiotic resistance. The aim of this review is to connect some of the well-established key concepts in evolutionary biology and recent advances in the study of eco-evolutionary dynamics to research on antibiotic resistance. We also identify some key knowledge gaps related to eco-evolutionary dynamics of antibiotic resistance, and review some of the recent technical advantages in molecular microbiology that offer new opportunities for tackling these questions. Finally, we argue that using the full potential of evolutionary theory and active communication across the different fields is needed for solving this global crisis more efficiently.This article is part of the themed issue ‘Human influences on evolution, and the ecological and societal consequences'. The rise of antimicrobial resistance in human and animal pathogens poses a serious threat to human health and food production, respectively, as traditionally employed antibiotics are becoming ineffective in the face of rapidly evolving bacteria populations . The incEscherichia coli is an obvious example of such a species [Bacteria are prevalent across all habitats, with the total number of bacterial species estimated to exceed one million . However species ,11. Solv species . Althoug species , even in species –17. Henc species , and an The emergence and spread of antimicrobial resistance has become an active area of research in recent years . Conside2.Antibiotic resistance is a global health crisis with multiple dimensions. Generating effective solutions to this crisis requires active collaboration and communication among scientists representing different disciplines. Unifying the language that we use and having a clearly defined terminology among physicians, microbiologists, evolutionary biologists and environmental scientists—among others—is a necessity so that we can proceed in solving the problem of antibiotic resistance in the most efficient manner. Below we describe some of the key terminology we use, and advocate their use as defined here across disciplines.(a)et al. [Although the increase in resistance of human pathogens to antimicrobial agents is one of the best-documented examples of contemporary evolution in action, the actual word ‘evolution’ is rarely used in the papers describing this research . As demoet al. , the woret al. . To avoi(b)The potential for eco-evolutionary feedback loops in determining the interaction between ecological and evolutionary dynamics has been increasingly recognized in recent years –27. Whil(c)conjugation, DNA is transferred in cell contact. Transformation does not require cell-to-cell contact but short fragments of naked DNA are taken up by naturally transformable bacteria. Transduction involves transfer of DNA via bacteriophages. Essentially, these mechanisms allow extremely rapid evolution since with HGT bacteria can change their genetic make-up practically instantly regarding a trait that has dramatic effect on fitness in the presence of antibiotics. While HGT is the main mechanism by which bacteria acquire antibiotic resistance, another key mechanism is chromosomal mutations. The main difference between these mechanisms is how fast resistance adaptation occurs in bacteria.HGT is the process where bacterial cells can exchange genetic material. Antibiotic resistance genes are often carried on MGEs  that act as vectors transferring genetic information between bacteria and even between species boundaries. Three main mechanisms for HGT are conjugation, transformation and transduction. In (d)Most often the aim of antibiotic usage is to kill the bacteria by using high enough concentrations of the given antibiotic. The key concept here is the minimal inhibiting concentration (MIC), which is the concentration that inhibits visible bacterial growth . However(e)The correlation of therapeutic antibiotic use and antibiotic resistance level in human populations is well documented ,34. AlthThe problem of human-mediated selection for antibiotic resistance in the environment can go well beyond the threats to human health and food production. Antibiotic use shapes the microbial community associated not only with humans and human-linked animals but in the environment by enrichment of antibiotic-resistant bacteria at the expense of antibiotic-sensitive bacteria. The possible effects of this large-scale event for the processes driven by microbes are yet to be seen.3.In this section, we outline how an understanding of eco-evolutionary dynamics could help in tackling the problem of antibiotic resistance and what are the key gaps in knowledge related to this.(a)The essence of the antibiotic resistance problem is that bacterial populations react to selection pressures caused by antibiotics by evolving resistance against antibiotics. This phenomenon is well documented in the case of high, therapeutic antibiotic concentrations, and several studies have also demonstrated how resistance evolves in the presence of sub-MICs ,42. The Overall, in natural environments sub-MIC antibiotics are one probable reason for enrichment of antibiotic-resistant bacteria even when the concentrations are extremely low. In addition, in the environment, there may be sub-MICs of different antibiotics which can cause synergistic selection pressures. However, to date, the empirical evidence for how MICs of antibiotics act as a selective agent in natural bacterial communities is still restricted. Obtaining reliable estimates can be challenging, since, for example, antibiotic concentrations in soil can be very patchily distributed and bulk estimates do not provide a comprehensive picture. We, however, argue that there is a pressing need for studies that combine sampling  and surveys of antibiotic concentrations and resistance genes in natural environments with controlled experiments using realistic levels of antibiotics to mimic selection pressures that bacterial communities are faced with in nature.(b)In natural settings, bacteria harbouring antibiotic resistance genes are part of complex communities where they interact with other species. However, at the moment, we are largely missing a population-level perspective on what processes constrain and drive antibiotic resistance in species-rich communities when complex species interactions are present. For example, in the case of spread of antibiotic resistance genes via HGT, microbial communities can be seen as gene-sharing networks where species composition and diversity can be a major factor determining how resistance genes spread . In the In addition to bacteria interacting with other bacteria, key interactions in microbial communities are those between bacteria and their natural enemies such as protozoan predators and the interactions between bacteria and their viral parasites (bacteriophages). For example, in natural microbial communities, protozoans form the most abundant and diverse group of predators feeding on bacteria. The most pronounced effect of protozoan predators and phage parasites is the mortality that they cause in bacterial populations. There is some experimental evidence demonstrating that protozoan predation, phage parasitism and maintenance of antibiotic resistance genes can be linked. One study shows that the plasmid was better maintained when the protist predator was present , while a(c)Development of molecular techniques has been crucial for increasing our understanding of the evolution of antibiotic resistance in microbial communities. Importantly, they are not subject to the culture bias caused by the fact that only a small minority of bacterial species can be cultivated with the current methodology . Metagen4.A large amount of research has been conducted to better understand the emergence of antibiotic resistance, which is recognized as a serious and global problem. However, most of the research has not taken species interactions into account although in natural populations bacteria are embedded in species communities characterized by diverse interactions. Also, antibiotic leakage to natural environments has the potential to radically alter resistance evolution and microbial community dynamics and structure. With this review, we want to (i) highlight the potential importance of species interactions in the evolution of antibiotic resistance; (ii) discuss how low environmental antibiotic concentrations could affect the evolution of antibiotic resistance and indirectly trophic interactions; and (iii) unify some of the key terminology between evolutionary biologists and researchers in biomedical fields working on antibiotic resistance problem. Establishing direct links between the fundamental axes of eco-evolutionary dynamics and species interactions offers an exciting future venue of research and is needed to tackle the problem of antibiotic resistance.


The pore structure of concrete can also be affected by the formation of an oriented structure of water in concrete materials; (ii) incorporation of fly ash makes a difference for the performance of concrete submersed in solutions as the total porosity and the pore connectivity can be lower. Especially when the dosage of fly ash is up to 30%, the pores with the diameter of larger than 100 nm show significant decrease. It demonstrates that the pore properties are improved by fly ash, which enhances the resistance against the calcium leaching; (iii) chlorides have a significant impact on microstructure of concrete materials because of the chemical interactions between the chlorides and cement hydrates.In this paper, the pore structure characterization of concrete exposed to deionised water and 5% NaCl solution was evaluated using mercury intrusion porosity (MIP), scanning electron microscopy (SEM) and X-ray diffraction (XRD). The effects of calcium leaching, fly ash incorporation, and chloride ions on the evolution of pore structure characteristics were investigated. The results demonstrate that: (i) in ordinary concrete without any fly ash, the leaching effect of the cement products is more evident than the cement hydration effect. From the experimental data, Ca(OH) Pore structure is one of the most important parameters, which determines the properties of cement-based materials. Pore structure is generally characterized by total porosity and pore size distribution (PSD). Previous researches have indicated that chloride ions, originating from deicing salts or sea water, can affect the pore structure characterization of the cement materials ,2, as th2 and C–S–H may be dissolved into water when concrete is exposed to marine environment [Fly ash, a by-product from thermal power plants and metallurgical industrials, has found common practice in civil engineering, in response to the increasing requirement of energy across the world. Many researchers have studied the macroscopic properties  of fly ash as well as the micro-mechanisms like transport properties ,14,15. Pironment ,21,22. TEven though numerous investigations regarding the performance of pore structure have been carried out ,24,25,26D is the pore diameter (μm), γ is the surface tension of mercury (mN/m), θ is the contact angle between mercury and the concrete (°) and P is the applied pressure (Mpa). The surface tension of mercury applied here is 480 mN/m, and the contact angle between mercury and concrete used is 117°.Mercury intrusion porosimetry (MIP) has been widely used to measure the porosity and pore size distribution of cement-based materials for years ,32,33. TThrough Barrett–Joyner–Hanlenda (BJH) interpretation , the porAs can be seen from the data in et al. [2 and a progressive decalcification of C–S–H with the use of a 50% concentrate solution of ammonium nitrate, and established a model of both strength decreasing and porosity increasing during the leaching process. As the dissolution of Ca(OH)2 increases to 25%, the micro pore structure of concrete changes dramatically, from a dense mixture  to an incompact mixture (high pore volume: 69 × 10−2 mL and large pore size: l00 nm).Furthermore, the result of et al.  investig2 are recorded as 34°, 47°, 51° and 64°, respectively. Based on the results of characteristic broad peak, as compared to the neat concrete of 0 day immersion time, the peak size of Ca(OH)2 of 180 days pure water submersion is evidently lower, which indicates the depletion of Ca(OH)2. The density and the pore structure of concrete is thought to be changed undesirably, along with the ongoing consumption of Ca(OH)2. With the increasing the immersion time, the pore structure of the concrete deteriorates.In this experiment, the results of X-ray diffraction data of OPC at the immersion ages of 0 and 180 days are presented in 2) and water generates hydration products similar to those of Portland cement,  i.e., calcium silicate hydrates (C–S–H), a rigid gel composed of extremely small particles with a layer structure [2 as well as the decalcification of C–S–H gels. In this regard, the leaching effect may deteriorate the pore structure of concrete. However, as can be seen from the 2 and the subsequent decalcification of C–S–H gels. It should be noted that the porosity of concrete can be lower if the initial Ca(OH)2 content in concrete is lower. Consequently, the concrete with a certain amount of fly ash added reflects better leaching resistance than the ordinary concrete. It is therefore to say that the fly ash concrete has a good resistance against calcium leaching, which was also confirmed by previous surveys that have tested the influence of fly ash intrusion on the permeability of concrete. Kunhe Fand et al. [2+ content in pore solution is lower than that of tap water, which indicates that the dissolution resisting characteristic of the roller-compacted concrete with low cement content and high fly ash content is strong.tructure . This retructure . The inid et al.  has repoAs a porous media, cement and concrete materials adopt a pore structure with broad pore size distribution from nanometer to micrometer scales . General4+ and Al3+. Therefore, secondary hydration is therefore to take place to form calcium silicate hydrate (Cao·SO2·H2O). Therefore, the pore system shows less connectivity, which will lead to very low transport rates.After the chloride ingress, some chlorides can be attached to the pore walls or cement products and react with them, which is called chloride binding ,48,49. T3A). The reaction product is known as calcium chloroaluminate hydrate, Friedel’s salts, 3CaO·Al2O3·CaCl2·10H2O [4AF, also reacts with chloride originating the equivalent ferric salt, 3CaO·Fe2O3·CaCl2·10H2O, but the reaction takes place at a slower rate [2 and form the CaCl2 complex, which makes the concrete more compact. As soon as the reaction begins, the binding between the chloride ion and cement product can modify the shape of concrete microstructure. Midgley et al. [− source to study the pore size distribution of cement paste under chloride ingress. It was found that the intrusion of chloride ion leads to more fine pores and less big pores. Jensen et al. [et al. [The chemically bound chlorides in the cementitious materials at the beginning react with the main ordinary Portland cement component, tricalcium aluminate 3. Coarse aggregate that originated from quarry of AnTuo Mountain, Shenzhen, was used. With the unit weight of 2700 kg/m3, the maximum and the minimum particle size of coarse aggregate are 20 mm and 5 mm, respectively. Sodium chloride that has purity above 99% was used to perform chloride bath for the specimens. Distilled water was used in concrete production in order to eliminate the influence of other unwelcome ions in water.In experimental studies, the P.O 42.5 Portland cement which is produced by Starfish Onoda cement limited company of Shenzhen was used. Type I fly ash (FA) was obtained from a power station in Ma Bay of Shenzhen. The chemical compositions of cement and fly ash are given in Water/binder ratio (W/B) was kept constant as 0.47, concrete without any fly ash was taken as the control mixture (Ordinary Portland Cement), while the remaining mixtures had two fly ash replacements with the proportion of 15% and 30%, respectively. The physical characterizations and mix proportions of all materials are summarized in All specimens with a size of 100 mm × 100 mm × 100 mm were prepared and cured for 28 days under continuous air curing conditions till the submersion testing. The specimen was at first cut into several blocks by a sawing machine. Then, the blocks were cut into fragments with a diameter of not more than 10 mm and with a height of not more than 30 mm, using a small diamond saw. After clearing away the stones and sands at the surface area, the specimens were submersed in deionised water and 5% (by weight of solution) NaCl solution, respectively. The frequency of renewal of the NaCl solution is around one month. The submersion age was set as 90 and 180 days.The specimens were immersed in absolute ethyl alcohol for more than a week in order to stop the cement hydration process. Afterwards, the specimens were put into an oven to be dried under the temperature of 60 °C. In this research, the pore size distribution of concrete was examined by Micromeritics AutoPore IV 9500  capable of generating pressure range from subambient to 33,000 pounds. Values of intrusion volume were measured at stepwise increasing pressures equilibrating at each pressure step . Thus, tPowder X-ray diffraction (XRD) was employed for calcium hydroxide identification. Scanning Electron Microscopy (SEM) was performed in microscopes. Prior to imaging, the concrete specimens were first crushed into small pieces. These pieces were then placed in a vacuum under 50 °C until constant weight, and coated with a thin gold layer.2 is obvious. The formation of an oriented structure of water in concrete exposed to water saturation as well bears the responsibility for destruction of the pore structure of concrete.As to the ordinary Portland concrete, the deteriorated effect of calcium leaching is found to be the dominant parameter governing the evolution of pore structure in spite of the growth of solid hydration products. During the process of immersion, the leaching phenomenon of Ca(OH)2 and dissolution of C–S–H gel.The concrete with a certain amount of fly ash added reflects higher calcium leaching resistance degree than the ordinary Portland concrete, which is closely linked to the double effects of fly-ash substitution of concrete: micro-filler effect and pozzolanic reaction. Especially when the dosage of fly ash is up to 30%, the pores that are larger than 100 nm significantly decline. The pore system of fly ash concrete shows less connectivity, which can resist the leaching of Ca(OH)The chloride ions can have a significant influence on the microstructure of cement materials. The pore size of concrete under chloride saturation has experienced a huge reduction compared to the deionised water immersion. The SEM images shows that the concrete under chloride saturation has a more smooth surface and denser structure in contrast with the deionised water immersion.This study discusses an experimental program carried out to investigate the pore structure characterization of cement paste exposed to deionised water and chlorides. The following conclusions can be drawn according to the results of this study:


Antimicrobial resistance is a public health problem of global importance. In Singapore, much focus has been given to antibiotic usage patterns in hospital settings. Data on antibiotic use in primary care is lacking. We describe antibiotic usage patterns and assess factors contributing to antibiotic usage among adults presenting with acute febrile illness (AFI) in primary care settings in Singapore.We analyzed data from the Early Dengue infection and outcome study. Adults with AFI presenting at 5 Singapore polyclinics were included. We used multivariable logistic regression to assess demographic, clinical and laboratory factors associated with antibiotic usage among adults with AFI.Between December 2007 and February 2013, 1884 adult AFI patients were enrolled. Overall, 16% of adult AFI patients reported antibiotic use. We observed a rise in the use of over-the-counter medications in late 2009 and a decrease in antibiotic use during 2010, possibly related to the outbreak of pandemic influenza A H1N1 virus. After adjusting for age, gender, polyclinic and year of enrolment, the following factors were associated with higher odds of antibiotic use: living in landed property (compared to public housing) ; body mass index (BMI) <18.5 ; elevated white blood cell (WBC) count ; and persistence of initial symptoms at 2–3 days follow-up with OR (95% CI) for categories of 1, 2, 3, and ≥4 persisting symptoms being 2.00 (1.38–2.92), 2.67 (1.80–3.97), 4.26 (2.73–6.64), and 2.79 (1.84–4.24) respectively.Our study provides insights on antibiotic usage among adult patients presenting to primary care clinics with febrile illness, and suggests that high socio-economic status, and risk factors of a severe illness, that is, low BMI and persistence of initial symptoms, are associated with higher antibiotic use. Further work to understand trends of antibiotic usage in both private and public primary care clinics, and factors that influence patient expectation and physician prescribing of antibiotics is important. Antimicrobial resistance (AMR) is a public health problem of global importance. The recent O’Neill review on AMR, commissioned by the government of the United Kingdom, suggests that the death toll from antimicrobial-resistant infections could exceed 10 million annually by 2050, surpassing mortality from cancer . Thus, iWe analyzed demographic, clinical and epidemiological data from adults with acute febrile illness (AFI) enrolled in the Early DENgue infection and outcome (EDEN) study . The aimχ2 statistics and p-values.We described the frequency of antibiotic usage among AFI patients, and examined the use of over-the-counter (OTC) medications  and antibiotics over time. Discrete and continuous variables were summarized using mean  or median and  as appropriate; categorical variables were summarized using frequencies and percentages. We investigated associations between antibiotic use among AFI patients and demographic characteristics, clinical symptoms and laboratory parameters using p-value <0.2 in the univariable analysis were considered for inclusion in multivariable analysis. We included age group, gender, recruiting polyclinic, and year of enrolment as covariates in all models. We added explanatory variables in a forward step-wise procedure, using the likelihood ratio (LR) test to assess the contribution of each variable to the model. We retained variables with a LR test p-value <0.05 and summarized the association between each variable and antibiotic usage with odds ratios (ORs) and 95% confidence intervals (CIs). Statistical analysis was performed using Stata 12 (Stata Corp).We used multivariable logistic regression to identify factors independently associated with antibiotic use. In a sub-group analysis, we further examined these factors among patients meeting the WHO definition for ILI. Variables with We analyzed data from 1884 adult AFI patients enrolled in the EDEN study between December 2007 and February 2013. The mean age of patients was 36.7 years , 1235 (66%) were male, and 958 (51%) were Chinese. Most patients (74.3%) lived in public housing. Average body temperature at initial presentation was 38.2 °C, median duration of illness was 5 days , and 759 (40%) patients satisfied the WHO definition of ILI. Less than 5% of AFI patients were known to have diabetes; other comorbidities  were uncommon (<1%)  patients used antibiotics, ranging from 5 to 31% across 5 polyclinics. OTC medication was used by 56% of patients, and included use of analgesics , cough medicines  and cold remedies . Figure 6% patienAmong AFI patients, 94% reported two or more symptoms at the initial presentation. Headache (69%), muscle pain (64%), and loss of appetite (54%) were the three most commonly reported symptoms. Vomiting, diarrhea, swollen lymph nodes, rashes, and bleeding were each reported by fewer than 10% of patients Fig. .Fig. 2Sy2 , and elevated WBC count . Persistence of symptoms at 2–3 days from initial presentation was also associated with increased odds of antibiotic use; adjusted OR (95% CI) for categories of 1, 2, 3, and ≥4 persisting symptoms were 2.00 (1.38–2.92), 2.67 (1.80–3.97), 4.26 (2.73–6.64), and 2.79 (1.84–4.24) respectively ; having a body mass index (BMI) below 18.5 kg/mIn a sub-group analysis of patients who met the WHO definition of ILI, after adjusting for age, gender, recruiting polyclinic, and year of enrolment, an elevated WBC count at initial presentation , and persistence of symptoms at 2–3 days from initial presentation  were associated with higher odds of antibiotic usage  are likely to be related to higher socioeconomic status . A possiHaving low BMI and persisting symptoms at 2–3 days from the initial presentation were associated with a higher odds of antibiotic usage. Prescribing clinicians may perceive these as risk factors for more severe or longer-lasting illness, or secondary infection –20, and Fever patients with elevated WBC count at initial presentation were more likely to use antibiotics. A similar association was also observed in the subgroup of patients with ILI. A high WBC count might indicate a bacterial infection, and thus the likelihood of antibiotic prescription. Studies from Switzerland and China found that more antibiotic use among fever patients was assoiated with higher WBC count , 23.Our data indicate that antibiotic use decreased markedly in late 2009 and 2010, during which time the use of OTC medications increased. This coincided with the period following the epidemic of influenza A H1N1, which was introduced into Singapore in May 2009 and peaked in the latter part of 2009 and early 2010 . The redThe EDEN study was primarily designed to assess Dengue virus infection patterns, and antibiotic use was self-reported by the participants. Data on prescription, type, dosage and timing of antibiotic use were not available. Certain respiratory symptoms, such as cough, were not systematically recorded, and may have led to an underestimation of ILI.Among fever patients in Singapore, approximately 80% of primary care consultations take place at private institutions . The EDEOur study provides insights on antibiotic usage among adult patients presenting to primary care clinics with febrile illness, and suggests that high socio-economic status, and risk factors of a severe illness, that is, low BMI and persistence of initial symptoms, are associated with higher antibiotic use. Further work to understand trends of antibiotic use, both in the public and private sectors, as well as factors that influence patient expectation and physician prescribing of antibiotics is important, in order to identify opportunities to rationalize antibiotic use and develop policies for optimal antibiotic use in primary care.


Innovative work behavior has been one of the essential attribute of high performing firms, and the roles of entrepreneurial orientation and self-leadership have been important for promoting innovative work behavior. This study advances research on innovative work behavior by examining the mediating role of self-leadership in the relationship between perceived entrepreneurial orientation and innovative work behavior. Structural equation modelling is employed to analyze data from a survey of 404 employees in banking sector. The results of reliability measures and confirmatory factor analysis strongly support the scale of the study. The results from an empirical survey study in the deposit banks reveal that participants’ perceptions about high levels of entrepreneurial orientation have a positive impact on innovative work behavior. The results also provide support for the full mediating role of self-leadership in the relationship between participants’ perceptions of entrepreneurial orientation and innovative work behavior. Additionally, this study provides some implications for practitioners in the banking sector to facilitate innovative work behavior through entrepreneurial orientation and self- leadership. In recent years, the service sector has played an important role in promoting the growth of economy in many countries  is the presence of organizational-level entrepreneurship  has attracted the expanded attention of practitioners and scholars. SL behaviors involves the influence individuals exert over themselves to achieve the self-motivation and self-direction needed to behave in desirable ways Manz . SL is dThe ability to behave innovatively in the workplace is becoming more and more significant for continuous innovation in today’s business organizations , as of June 2014, the number of deposit banks  operating in Turkey was 26 of which 11 privately-owned, 3 state-owned and 12 foreign deposit banks. The dataset for this study was compiled from 17 banks among these deposit banks: 8 privately-owned, 3 state-owned and 6 foreign deposit banks. These banks have higher number of branch offices and employees than the banks in other groups and unreached deposit banks. In addition, the banks in the sample represent the general classification of deposit banks in Turkish banking system. The participants of the study were randomly selected from the positions of associate, manager and senior manager within these banks who were knowledgeable about the key constructs of the study. Multiple participants were also selected from each bank. Participants in this study answered the questionnaire in a voluntary manner and were informed of the aim of the survey. Participants were also assured of the anonymity and the confidentiality of their answers. Questionnaires were administered to 461 employees and a total of 404 (88 % respond rate) were usable. The demographic characteristics of the respondents are represented in Table All items in the questionnaire were measured on a five-point Likert scale ranging from ‘strongly disagree’ to ‘strongly agree’. The questionnaire was originally developed in English, and then underwent a back-translation procedure . The RSLQ consisted of 35 items and was developed by Houghton and Neck  and baseTo measure the IWB, the scale of De Jong and Den Hartog  was usedTo control the existence of confounding variables from demographic characteristics on the relationship between the predictors and outcome variables, position  and gender  were applied as covariates suggested by prior research Janssen . Positio2/df  =  1.71, RMSEA  = .042, CFI  =  .923, RMR = .04, SRMR  =  .05).Confirmatory factor analysis was performed to assess the validity of the multi-item measurement scale. According to Hair et al. , comparaTable The data for this study were collected using the self-report questionnaire that may lead common method bias or variance. According to Podsakoff et al. , severalTable  = .273, p = .761; F = 1.476,  p = .230; F = .692, p = .501, respectively).Independent-samples t-tests were conducted to compare EO, SL and IWB for gender differences. These tests indicated that female (mean ± SD: 4 ± .49) are more likely than male (mean ± SD: 3.8 ± .53) to use general SL strategies  and differences between means were non-significant for EO and IWB . Additionally, ANOVA tests were conducted to control for mean difference due to the position of participants. ANOVA showed no differences between positions regarding scores of EO, SL and IWB .This study applies SEM to verify the hypotheses, and utilizes AMOS software to obtain the empirical results by means of the method of maximum likelihood estimation (MLE). The first step in SEM is to assess the overall model fit with some fit indices , mediator (SL) and outcome (IWB) simultaneously, since SEM has the advantages of correcting for unreliability of measures  = 202.368, p < .001. Fit indices also indicated that full mediation model provided a good fit to data: χ2/df  =  1.705, RMR = .042, RMSEA = .042, SRMR = .0538, IFI = .924, CFI = .923, as did the partial mediation model: χ2/df  =  1.706, RMR = .042, RMSEA = .042, SRMR = .0537, IFI = .924, CFI = .923. However, the alternative model of partial mediation effect of SL did not provide a better fit to data than the full mediation model and the fit indices remained almost unchanged: χ2 difference (df = 1) = .036, n.s. Thus, the rule of parsimony indicates the fully mediated model is the preferred model . Furthermore, a resampling method known as bootstrapping was used to test the significance of the mediational effect of SL in the relation between perceived EO and IWB. The bootstrap method is considered a more rigorous approach and has greater statistical power than a majority of other procedures  facilitate empowerment, thinking ‘outside of the box’, coping with uncertainty and/or complexity associated with the innovation process without fear of punishment or failure, and proactively participating in the innovation processes, thereby exhibiting IWB Amo . In agreThe results of the study indicate that SL operated as an intervening variable between EO and IWB. As hypothesized, SL fully mediates the relationship between EO and IWB. The result shows that the effect of EO on IWB is increased by developing SL skills. This result makes an important contribution to the literature. EO holds a prominent position by providing an environment to develop innovations in organizations. Taking steps only in boosting EO to enhance IWB is not enough; it is essential to place consideration on developing SL as well. The results of this study support that SL enhances IWB. It is also found that individuals who have strong SL are more likely to have high innovative behavior than individuals who have weak SL. Hence, SL notably helps individuals to develop IWB by providing individuals with self-management, self-motivation and self-influence on their own thoughts and/or behaviors. The development of innovative behaviors in organizations becomes easier with the SL skills that include the processes of self-influence and self-management, therefore organizations that seek out to facilitate IWB, need to recognize the importance of individuals’ SL skills.De Jong and Den Hartog  claim thThe results of this study indicate that there is a significant difference between the SL scores of men and women, which is in agreement with the findings of Norris . The womThe results of this study also have managerial implications. Due to the importance of the banking sector in developing countries, there is a need for banking managers to become efficient in managing innovative behaviors in order to support the constantly changing needs of customers and the rapidly changing market. If banking managers are interested in giving employees a sense of control over themselves, and building a sense of fostering innovativeness, proactiveness and taking risk, they can manage employees’ innovative behavior more effectively, and accordingly maximize employees’ IWB. The findings of this study also suggest critical implications in terms of both selecting and training employees and managers within banks. Banks should consider implementing SL selection standards and actively provide training programs in order to develop SL skills among employees and managers, which in turn promotes motivation to exhibit IWB.The results of the study should be considered in light of several limitations. Self-reported data from a single source may pose potential problems such as CMV. However, as discussed in the “


Improper antibiotic use is one of the main drivers of bacterial resistance to antibiotics, increasing infectious diseases morbidity and mortality and raising costs of healthcare. The level of antibiotic consumption has been shown to vary according to socioeconomic determinants (SED) such as income and access to education. In many Latin American countries, antibiotics could be easily purchased without a medical prescription in private pharmacies before enforcement of restrictions on over-the-counter (OTC) sales in recent years. Brazil issued a law abolishing OTC sales in October 2010. This study seeks to find SED of antibiotic consumption in the Brazilian state of São Paulo (SSP) and to estimate the impact of the 2010 law.Data on all oral antibiotic sales having occurred in the private sector in SSP from 2008 to 2012 were pooled into the 645 municipalities of SSP. Linear regression was performed to estimate consumption levels that would have occurred in 2011 and 2012 if no law regulating OTC sales had been issued in 2010. These values were compared to actual observed levels, estimating the effect of this law. Linear regression was performed to find association of antibiotic consumption levels and of a greater effect of the law with municipality level data on SED obtained from a nationwide census.Oral antibiotic consumption in SSP rose from 8.44 defined daily doses per 1,000 inhabitants per day (DID) in 2008 to 9.95 in 2010, and fell to 8.06 DID in 2012. Determinants of a higher consumption were higher human development index, percentage of urban population, density of private health establishments, life expectancy and percentage of females; lower illiteracy levels and lower percentage of population between 5 and 15 years old. A higher percentage of females was associated with a stronger effect of the law.SSP had similar antibiotic consumption levels as the whole country of Brazil, and they were effectively reduced by the policy. Ternhag . (2014)  reported. (2014) ,36 Perceet al. (2015) [Higher density of private health establishments and lower density of SUS health establishments were associated with antibiotic consumption. These results are in the expected direction, considering that patients that attended the public sector already needed prescriptions before 2010 and that private doctors may be more eager to meet their patients’ demand for an antibiotic.  Density . (2015)  found noDemographic structure of the population has been widely analysed to date as a possible predictor of antibiotic consumption, with many different results being achieved. People under 5 and over 75 years old were prescribed more antibiotics in Sweden ; while tet al. (2010), Filippini et al. (2006) and Marra et al. (2010) have all found that antibiotic consumption was higher in areas of higher physician density. [Even though the policy effect was greater in municipalities with previously higher consumption, it did not seem to be much related with the SED analysed—only the percentage of females in the population presented a good association in the multivariable model, with very little correlation with the data. This policy was previously described to be more effective in areas with better SES within Brazil . It is ndensity. –11Campylobacter infection, keeping consistency with previous studies [Additionally, a more objective measure of incidence of infectious diseases would have been incidence of a specific disease such as  studies . The varEuropean Union countries monitor their use of antibiotics through the European Surveillance of Antimicrobial Consumption project, which aims to produce international data on antibiotic consumption using information from sales and reimbursement data, encompassing all antimicrobials being consumed in the country  in order to guide possible public health policies.  LAC do nA limitation associated with this study is that its results cannot be extrapolated to the individual level. However, for the main purposes of this study and for the main implications for policy, the municipality level may be the most important. It is also important to remind that this study did not aim to investigate causation—it is not clear which of the associated factors, if any, were truly causative. Measurement error of the outcome may have occurred if pharmaceutical volume coverage differs across meso-regions/municipalities. Moreover, not all antibiotics are consumed in the municipality where the sale has occurred, and not all antibiotics are consumed within the month of its purchase. These factors may have increased misclassification of the municipality or month of consumption. Some variables may not be a good predictor at the municipality level—for instance, GDP per capita, as, at a much greater extent than at country level, not all income generated in a municipality goes to its own population. The addition of several variables in the multivariable models may have increased the amount of uncertainty and caused an over-adjustment of intermediate variables as some of them possibly mediated the effect of others. The use of other medication group as a control would have improved the analysis and dismissed possible secular trends. Finally, the large differences in municipality sizes may have caused the standard errors of this study to have been under-estimated.As in most other LAC where OTC sales have recently been restricted, the policy has changed the trend of increase and effectively reduced antibiotic consumption in SSP. Antibiotic consumption continued to decline in the second year after the policy was issued, and in 2012 it had lower levels than in 2008. SSP had similar levels of consumption to those yielded by literature for the whole country of Brazil. ,20 SED o


It has only recently become evident that a number of HGF-regulated functions in inflammatory processes and immune responses are imparted via DCs. However, the mechanisms by which Met signaling in DCs conveys its immunoregulatory effects have not yet been fully understood. In this review, we focus on the current knowledge of Met signaling in DCs with particular attention on the morphogenic and motogenic activities. Met signaling was shown to promote DC mobility by regulating matrix metalloproteinase activities and adhesion. This is a striking resemblance to the role of Met in regulating a cell fate program during embryonic development, wound healing, and in tumor invasion known as epithelial–mesenchymal transition (EMT). Hence, we propose the concept that an EMT program is executed by Met signaling in LCs.Langerhans cells (LCs), the epidermal dendritic cell (DC) subset, express the transmembrane tyrosine kinase receptor Met also known as hepatocyte growth factor (HGF) receptor. HGF is the exclusive ligand of Met and upon binding executes mitogenic, morphogenic, and motogenic activities to various cells. HGF exerts anti-inflammatory activities  Howein vivo . The indnd liver –11. In and liver , 12. Thend liver , 13, 14.A corresponding aberrant activation of the EMT program by Met signaling during tumorgenesis results in invasive growth and metastasis of tumor cells , 13, 14.Biologically active HGF is a disulfide-linked heterodimeric molecule composed of a 69 kDa α-chain and a 34 kDa β-chain that is derived from an inactive single-chain precursor  . Pro-HGFAll biological functions of HGF are exerted by binding to its unique receptor Met. Like its ligand, Met is synthesized as an immature single-chain precursor that is cleaved by intracellular endoproteinases to form the mature membrane-bound disulfide-linked α-β heterodimer Figure . Met shaMet interacts with other cell surface receptor that can modulate Met signaling in ligand-dependent and -independent manner. Remarkably, this includes a number of surface receptors involved in regulation of cellular motility and migration, such as integrin α6β4 , plexin-Beyond the well-recognized role of Met signaling in epithelial cells and tumor development, early reports already provided evidence for a role in hematopoietic cells , 43, andConstitutive Met expression is limited to hematopoietic progenitor cells and their antigen-presenting progenies, including B cells, monocytes/macrophages, and DCs , 44. HowFor the Met ligand HGF, it has been shown that it—frequently in synergy with other growth factors—can support erythropoiesis, thrombopoiesis, and myelopoiesis and development of Met-expressing thymocytes , 43. FibIn response to infection or tissue injury the production of HGF is further stimulated by pro-inflammatory cytokines, including IL-1α, IL-1β, TNF-α, and IL-6 , 61. By via direct impact on DC function   , 59, 69.s)   , and it nd TROP2 –97, and nd TROP2 –100. Remnd TROP2 , 102. Thnd TROP2 .via HGF/Met in immune cells could concurrently stimulate potential tumor cells toward invasive growth. The knowledge of the HGF/Met signaling mechanisms in DCs is still in its infancy and must be extended to (i) develop save Met-based therapies in the future and (ii) corroborate the concept that a Met-driven execution of an EMT program in DCs is indeed a generic mechanism.In summary, Met signaling in skin resident DCs including LCs appears to be a critical determinant for maintaining normal immune function and as an important constituent that interlaces tissue regenerative functions with the appropriate immune responses that must be accomplished after tissue injury, infection, or inflammation. Other studies suggest a protective role of HGF/Met signaling against autoimmunity by directing DCs toward a tolerogenic phenotype. This and a number of further activities of HGF/Met signaling on other immune cells suggest the HGF/Met pathway as a potential target for treatment of inflammatory and autoimmune disorders, including skin diseases and transplantation , 104. DuAll authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Monitoring the incidence and intensity of catastrophic health expenditure, as well as the impoverishing effects of out of pocket costs to access healthcare, is a key part of benchmarking Kenya’s progress towards reducing the financial burden that households experience when accessing healthcare.n =33,675). We undertook health equity analysis to estimate the incidence and intensity of catastrophic expenditure. Households were considered to have incurred catastrophic expenditures if their annual out of-pocket health expenditures exceeded 40% of their annual non-food expenditure. We assessed the impoverishing effects of out of pocket payments using the Kenya national poverty line. We distinguished between direct payments for healthcare such as payments for consultation, medicines, medical procedures, and total healthcare expenditure that includes direct healthcare payments and the cost of transportation to and from health facilities. We used logistic regression analysis to explore the factors associated with the incidence of catastrophic expenditures.The study relies on data from the nationally-representative Kenya Household Expenditure and Utilization Survey conducted in 2013 (When only direct payments to healthcare providers were considered, the incidence of catastrophic expenditures was 4.52%. When transport costs are included, the incidence of catastrophic expenditure increased to 6.58%. 453,470 Kenyans are pushed into poverty annually as a result of direct payments for healthcare. When the cost of transport is included, that number increases by more than one third to 619,541. Unemployment of the household head, presence of an elderly person, a person with a chronic ailment, a large household size, lower household social-economic status, and residence in marginalized regions of the country are significantly associated with increased odds of incurring catastrophic expenditures.Kenyan policy makers should prioritize extending pre-payment mechanisms to more vulnerable groups, specifically the poor, the elderly, those suffering from chronic ailments and those living in marginalized regions of the country. The range of services covered under these mechanisms should also be extended such that the proportion of direct costs paid to access care is reduced. Policy makers should also prioritize reducing supply side bottlenecks such as availability of healthcare facilities in close proximity to the population, especially in rural and marginalized areas, and improvements in quality of care. For the poor and the vulnerable, initiatives to cover the cost of transport to and from a health facility, such as transport vouchers could also be explored. There is increasing commitment by low and middle income countries (LMICs) to achieve universal health coverage (UHC) , the goaKenya’s health sector is financed by a mix of public, private, and donor resources. Between 2009 and 2013, donor financing reduced from 34.5 to 25.6%, while financing from public sources increased from 28.8 to 33.5%  as well as all levels of private healthcare facilities are still paid for through OOP payments. Second, health insurance coverage in Kenya remains low even though it has increased from 10 to 17.1% between 2007 and 2013 , Kenya aOOP payments deter some Kenyans from seeking care and cause others to become impoverished as a result of paying for care. A previous study by Chuma and colleagues estimated that 14.8% of households experienced catastrophic healthcare expenditure in 2007 [Our analysis contributes to the policy dialogue around UHC in Kenya and as well as the broader literature on catastrophic health spending in three ways. First, by using recent data, it provides policy makers with information to take stock of progress (or lack thereof) on improving financial risk protection among the population, and provides a benchmark for the future. Second, unlike the analysis by Chuma and colleagues, and most analyses that only consider catastrophe and impoverishment due to direct payments made to healthcare providers, we also consider the impact of transport costs borne by users to access healthcare services. This is based on the recognition that transport costs are often quite significant when compared to direct payments to healthcare providers . Third, Kenya is a lower middle income country in East Africa with a per capita Gross Domestic Product (GDP) of USD 1245. It has an estimated population of 44.86 million , 45.9% oThis analysis is based on the 2013 KHHEUS . The KHHOf the individuals sampled, 19.3% reported illness in the four weeks preceding the survey . Of thesThere is no consensus on the threshold for catastrophic health payments. In this analysis, households were considered to have incurred catastrophic expenditures if their total out of-pocket health costs in a year exceeded 40% of their annual non-food expenditure (referred to as capacity to pay) . We chosThe catastrophic head count (HC), which represents the incidence of catastrophe, refers to the proportion of households that incurred catastrophic health payments and is estimated as follows :1\documeWhere N is the sample size while E is an indicator that assumes a value of 1 if the OOP payments of a household i exceed the defined catastrophic expenditure threshold, and 0 if it does not. While catastrophic head count estimates the fraction of households that incur catastrophic costs, it does not give information on the extent of catastrophe (by how much a households OOP payment exceeds the catastrophic threshold). The catastrophic overshoot (O) provides this information. Catastrophic overshoot refers to the average degree by which OOP payments, as a proportion of total expenditure, exceeds the catastrophic payment threshold (Z). Catastrophic overshoot (O) is estimated as follows :2\documei represents the OOP payments of household i, Xi represents the household non-food consumption expenditure of household i, and Z represents the catastrophic threshold. Subsequently, the average catastrophic overshoot can be estimated as follows [Where T follows :3\documeThe intensity of catastrophic expenditure is computed by averaging the catastrophic overshoot over all households that exceed the catastrophic threshold. This measure, referred to as the mean positive overshoot (MPO), is computed as follows:E(h) is computed as follows [The distributional profile of the incidence and intensity of catastrophic payments was assessed by computing concentration indices. The concentration index is derived by computing the covariance between the fractional rank of the individual sorted by socio-economic status and the variable of interest . The ind follows :5\documeWhere:n is the number of individuals in a given population, λi denotes the socioeconomic rank of the individual ranging from the richest (λi = 1) to the poorest (λi = n), hi is the level of the variable of interest for individual i, bh is the upper bound of the variable of interest, and ah is the lower bound of the variable of interest. Erreygers concentration index satisfies the four criteria argued by Erreygers to be essential [transfer: A small transfer of the variable of interest from a richer to a poorer individual is translated into a pro-poor change in the concentration index, 2) mirror: The concentration index of the presence of the variable of interest, and the concentration index of the shortfall of the variable of interest should be mirror images of each other, 3) level independence: An equal increment of the variable of interest for all individuals does not affect the concentration index, 4) Cardinal invariance: A linear transformation of the variable of interest does not affect the value of the index.And, ssential , 31 nameTo assess the impact of OOP  payments on poverty estimates, we first specified a poverty line by adopting the Kenya national poverty line of consumption expenditure of Kenya shillings 1562 (USD 15.62) for rural areas and Kenya shillings 2913 (USD 29.13) for urban areas per individual per month . We thenWe developed a logistic regression model to examine the association of the incidence of catastrophic expenditures and a range of household level and county level covariates. We developed two models, one with catastrophic expenditure due to healthcare costs as the dependent variable, and a second one with catastrophic expenditure due to healthcare and transport costs as the dependent variable.Covariates were selected based on findings from literature on determinants of catastrophic expenditures , 37–40. Table Table Direct OOP spending for outpatient services were the greatest driver (64.68%) of total OOP health spending , while payments for medicines comprised the highest proportion (44.57%) of direct healthcare payments (outpatient and inpatient OOP) Fig. .Figure Table The mean positive overshoot was 3.75% when only direct OOP paid to healthcare providers are considered and 5.73% if both direct OOP costs paid to healthcare providers and transport costs are considered. The mean positive overshoot represents the extent to which OOP payments exceed the defined catastrophic threshold (40% of non-food budget share). The concentration indices are negative for both catastrophic expenditures and overshoot, showing that both the incidence and intensity of catastrophe are concentrated among the poor.Figure Table th quintile are the ones that are pushed into poverty by OOP payments to access healthcare.To graphically illustrate the impoverishing effects of OOP payments, Fig. Table Households had increased odds of incurring catastrophic expenditures due to healthcare costs if their household head was older. For instance, households whose heads were over 55 years of age had a 72% increased odds incurring catastrophic expenditures due to direct healthcare costs compared to households whose heads were below 25 years of age . A household with an unemployed head had 75% more odds of incurring catastrophic expenditure due to direct healthcare costs compared to a household with an employed head . Low social economic status also increased the odds of a household incurring catastrophic expenditure. For example, households in the poorest quintile had 5.61 times more odds of incurring catastrophic expenditure due to direct healthcare costs compared to the households in the richest quintile . Other variables that increased the odds of incurring catastrophic costs due to direct healthcare costs include household with a larger number of household members , households with a household member with a chronic disease  or an elderly household member , and households that were located in a marginalized county  (Table This study presents a detailed analysis of catastrophic health spending in Kenya using the most recent nationally representative household survey. Direct comparison of our findings with those from other settings is limited by differences in methodological choices such as how health expenditure was measured and choice of the threshold for catastrophic health expenditures. This not-withstanding, our findings appear to be comparable to those reported elsewhere in the Sub-Saharan Africa region , 43. ForWhen compared to similar analysis in Kenya from previous periods, our results show that the country is moving in the right direction. Compared to 2007, when 14.8% of Kenyan households incurred catastrophic expenditures due to direct costs of healthcare . In the Further, the fact that the level of catastrophic expenditure due to direct healthcare costs is still high reinforces the observation made in other countries that user fee removal is not enough. For example studies from Uganda, Bukina Faso and Zambia reported that user fee removal did not reduce, and sometimes even increased OOP payments to access healthcare services –46. In tOur results highlight the financial burden that paying for transportation to healthcare facilities poses for Kenyan households and especially the poor. It is worth noting that assessments of catastrophic health spending in LMICs typically focus on direct healthcare payments, and customarily ignore other indirect costs such as the cost of transportation to the health facility. The seeming vulnerability among the poor is not only due to the poor having lower incomes, but also because a significant proportion of the poor live in rural and marginalized regions of the country, where access to healthcare facilities is limited because facilities are few and far between. Our study thus adds to the growing evidence that transport costs comprise a significant proportion of OOP healthcare costs. For example, findings from Zambia report that transport costs comprised 73.00% of OOP costs incurred access healthcare services in Zambia . TranspoOur findings also reinforces evidence from other settings that outpatient care and costs of medicines are the greatest cost drivers of direct OOP costs paid to healthcare facilities , 51. ThiOur study offers some insight into who amongst the Kenyan population are most vulnerable to catastrophic expenditures. Households that are larger, poorer, have an unemployed head, have a member with a chronic ailment such as diabetes of hypertension, have an elderly member, or live in marginalized regions of the country have an increased odds of incurring catastrophic expenditures. These findings are consistent with evidence from other settings on the determinants of the incidence of catastrophic health expenditures. For example, the presence of a household member with a chronic illness, or the unemployment of the household head were found to increase the odds of a household incurring catastrophic health expenditures in Nepal , China , Kenya , and GhaOur findings on the influence of health insurance on health spending add to the mixed findings from the literature on this topic. While some studies have found that having health insurance reduces the odds of incurring catastrophic health expenditures , 56, 57,Our analysis has a number of implications for policy in Kenya and similar settings. First, the design of health financing mechanisms should prioritize the cost drivers of OOP spending. For example, social health insurance schemes, such as the NHIF in Kenya, should explicitly include essential medicines in their benefit packages. Further, in addition to policies to remove user fees paid directly to health facilities, policy makers should explore policies to reduce the burden of transport costs especially among the poor, and the vulnerable. For example, while the government of Kenya has implemented a free maternity healthcare programme and introduced a health insurance subsidy for the poor programme , they shSecond, policies aimed at providing financial risk protection should prioritize vulnerable groups in the population. For instance, interventions for prevalent chronic diseases such as diabetes and hypertension should be included in benefit packages of UHC schemes. Further, in extending coverage, special priority should be given to the poor, the elderly and those living in rural and marginalized regions of the country. While the Kenyan government has introduced an insurance subsidy programme for the poor and the elderly, these remain pilots funded by donors and therefore have very limited coverage . The KenThird, efforts at reducing financial barriers to access can only succeed if accompanied by efforts to remove supply side bottlenecks. Specifically, the Kenyan government should invest in increasing geographical access to healthcare facilities among the population, especially those in the rural and marginalized regions of the country. The NHIF should also scale up the number of facilities it contracts to provide healthcare services to its members, and specifically focusing on increasing the numbers of primary healthcare facilities, and the network of contracted facilities in rural and marginalized areas. The Kenyan government should also prioritize implementing measures to improve the quality of care in health facilities. The government should strengthen the supply chain and availability of essential medicines and supplies in public healthcare facilities, while the NHIF should strengthen its purchasing function to ensure that essential medicines and supplies are available in both the public and private facilities that it contracts to provide services for its members.The KHHEUS was conducted in 2013, which makes it outdated. However, KHHEUS is the only data source that provides detailed information on health consumption patterns at the household-level in Kenya, and the 2013 round is the most recent round. Further, as has been observed by others, surveys such as the KHHEUS rely on self-reported data on healthcare use, which is susceptible recall bias .KHHEUS 2013 offers us the most recent estimate of catastrophic health spending in Kenya. While there is an observed decline in both the incidence of catastrophic health spending and its impoverishing effects, the levels are still unacceptably high especially among the poor. There are signs that recent government of Kenya reforms, specifically the user fee removal policy might have contributed to increased financial risk protection among the poor. The fact however that CHE persist in the Kenyan population means that the Kenyan government has to do more to not only ensure that prepayment mechanisms are scaled up and adhered to, but that supply side bottlenecks such as quality of care and geographical access to facilities are improved. It is also clear from our analysis that transport costs comprise a significant portion of OOP and should be given policy priority by policy makers, especially among the poor.


The main strategy to reduce the environmental impact of the concrete industry is to reuse the waste materials. This research has considered the combination of cement replacement by industrial by-products, and natural coarse aggregate substitution by recycled aggregate. The aim is to evaluate the behavior of concretes with a reduced impact on the environment by replacing a 50% of cement by industrial by-products  and a 100% of natural coarse aggregate by recycled aggregate. The concretes prepared according to these considerations have been tested in terms of mechanical strengths and the protection offered against steel reinforcement corrosion under carbonation attack and chloride-contaminated environments. The proposed concrete combinations reduced the mechanical performance of concretes in terms of elastic modulus, compressive strength, and flexural strength. In addition, an increase in open porosity due to the presence of recycled aggregate was observed, which is coherent with the changes observed in mechanical tests. Regarding corrosion tests, no significant differences were observed in the case of the resistance of these types of concretes under a natural chloride attack. In the case of carbonation attack, although all concretes did not stand the highly aggressive conditions, those concretes with cement replacement behaved worse than Portland cement concretes. The environmental impact associated to this activity is high due to the extraction of raw materials in quarries, the energy consumption related to cement production and the carbon dioxide emissions released in the fabrication and transportation processes –3. Howevetc. The success of these by-products was absolute, and nowadays it is very usual to use them. Nevertheless, other pozzolans have been successfully implemented in cement products, although their use has not been so extended or many times they have never been applied to real construction. As examples of these minor pozzolans, the following can be listed: rice husk ash [etc. The light usage of these minor pozzolans on the concrete manufacture can be related to the small quantity of them that are produced respect to the total cement demand, the heterogeneity of these materials composition and/or the need of laborious pre-treatments in some cases.One of the main strategies to reduce the environmental impact of the concrete industry is the reuse of waste materials. The first approach consisted on replacing variable amounts of cement by industrial by-products with pozzolanic activity. Under this strategy, a number of supplementary cementing materials were tested and developed, such as silica fume, fly ash, ground-granulated blast furnace slag, husk ash , sewage husk ash , metakaohusk ash , other mhusk ash , red sluhusk ash , spent chusk ash , calcinehusk ash , etc. ThAnother way to reduce the contamination derived from the concrete industry is the reuse of recycled aggregate to replace a quantity of the traditional natural aggregate. In this case, environmental considerations can be solidly argued, since neither economical nor technological advantages are easily deduced –13, althi.e., its proportion and composition. In this way, the main problems arise when recycled aggregates come from demolished buildings due to the presence of bricks and tiles [et al. [et al. [The conclusions of another review by Hansen  and Amernd tiles . López-G [et al.  conclude [et al. ,20. Alth [et al. , it has  [et al. . In that [et al. –25, even [et al. . In orde [et al.  indicateThis research has considered the combination of both strategies: cement replacement by industrial by-products and natural coarse aggregate substitution by recycled aggregate. Regarding to the binder, a combination of two pozzolanic materials with complementary properties is proposed in order to increase the level of cement replacement without decreasing the concrete’s performance. Fly ash and spent catalytic cracking catalyst have been selected since the first one has shown low short-term reactivity , whereasRegarding to the environmental impact of this work, the total amount of wastes affected by this research is huge. About 160,000 tons per year of spent catalyst are produced, and this quantity is increasing annually at a 5% rate . For flyThe EU has only recently introduced recycling targets for construction and demolition waste. A 70% recycling target was introduced in the new EU Waste Framework Directive 2008/98/EC to be achieved by 2020. It includes only recycling of non-hazardous construction and demolition waste and excludes soil and stone. The total recycling rates vary significantly among the countries, but in general, the rate is quite reasonable (>50%) for 11 countries. Five countries have very high recycling percentages (>70%), already in excess of the 2020 target. Six countries have recycling rates between 50% and 70%, one country between 30% and 50% and six countries below 30%. The most significant policy measures to promote the recycling of construction and demolition wastes have been landfill ban or taxes and source separation mandates . In ordeTherefore, it has to be admitted that a huge effort has been made by the scientific community to study and understand how recycled concrete aggregate can be implemented in structural concrete. Nevertheless, the impact of these actions on the corrosion performance of steel reinforcement has to be assessed. The aim of this research is to evaluate the behavior of concretes with a reduced impact on the environment, with a 50% of cement replacement by industrial by-products and a 100% of natural coarse aggregate replacement by recycled aggregate. The concretes prepared according these considerations have been tested in terms of protection offered against steel reinforcement corrosion under carbonation attack and chloride-contaminated environments. In addition, some mechanical tests at 28 days have been performed in order to show their main characteristics at a standard age.2.2.1.et al. [et al.  observedet al. , so the et al. [et al. [et al. [et al.  observed [et al.  register [et al. . Kou et  [et al.  observedi.e., keeping the coarse aggregate mass constant instead of the volume fraction, in spite of the density difference between RA and NA, produces a higher volumetric proportion of coarse aggregate in RA concrete, resulting in a lower density for them. In this sense, this fact will not only affect mechanical properties of recycled aggregate concrete but also aspects related to durability, as those studied in the second part of this work. In addition it can be observed that the use of the alternative binder slightly reduces the concrete porosity due to the pozzolanic activity of fly ash and spent catalyst.i.e., FA. Thus, at 28 days, the contribution of this part of the binder is not expected to be significant enough, although a positive evolution of strength should be observed in the long term [Summing up, both changes applied to control concrete (aggregate and binder) reduce the mechanical performance of studied concrete. The causes of these facts could be attributed several factors. In the case of the aggregate, lower RA density leads to a decrease of RA concretes density, which can be directly related to their strength losses. Besides, coarse aggregate substitution has been made in mass basis, so taking into account the lower RA density, the binder to coarse aggregate ratio is lower for RA concretes, and then a lower strength is also expected. Therefore, in volumetric terms, in this research, RA concretes have a higher proportion of a less strong component, which joint to high replacement level, leads to a poor mechanical behaviour. In the case of the binder, there is a high substitution degree (50%) and most part of it consists of a pozzolan with slow reaction rate, ong term , since pong term . Additioong term , and tak2.2.Icorr) and corrosion potential (Ecorr) evolution of steel reinforcement embedded in different concretes which contain increasing levels of chloride added during the mixing. This chloride addition was tested not to simulated real service conditions but to evaluate the chloride threshold admitted by each matrix (concrete) to stand an acceptable corrosion level. All corrosion graphs present a boundary region between 0.1 and 0.2 μA/cm2 that separates the inactive corrosion rate zone from the active corrosion rate zone. Values under 0.1 μA/cm2 are considered negligible in terms of durability, while values over 0.2 μA/cm2 produce a significant reduction in the service life of the structural element due to the corrosion process [ process .Icorr) and corrosion potential (Ecorr) evolution of steel reinforcement embedded in NA-OPC concrete with different chloride concentrations. It can be observed that chloride threshold for NA-OPC concrete lies between 1% and 2% of chloride, since Icorr values for 1% lays in the boundary region and 2% slightly exceeds 0.2 μA/cm2 almost constantly. Ecorr values are coherent since the higher the Icorr value, the lower the Ecorr value.Icorr) and corrosion potential (Ecorr) evolution of steel reinforcement embedded in RA-OPC concrete with different chloride concentrations. In this case, the behaviour exhibited by this concrete is almost the same than NA-OPC concrete, i.e., chloride threshold for RA-OPC concrete lies between 1% and 2% of chloride.Icorr) and corrosion potential (Ecorr) evolution of steel reinforcement embedded in NA-AB concrete with different chloride concentrations. The performance of this concrete is better than NA-OPC concrete. In this case the chloride threshold for NA-AB concrete is placed over 2% of chlorides since a clear depassivation of steel is not observed for 2% of chlorides. This behaviour has been previously attributed to higher formation of Friedel’s salt due to a higher Al2O3 concentration given by FA and SFCC [Icorr values registered in NA-AB to control concrete, it can be observed a general reduction for all chloride concentrations.and SFCC ,49,50. MIcorr) and corrosion potential (Ecorr) evolution of steel reinforcement embedded in RA-AB concrete with different chloride concentrations. In this case, the general behaviour is similar to NA-OPC and RA-OPC, establishing a chloride threshold between 1% and 2% of chlorides, although it is clearer now that 2% of chlorides are in the active corrosion zone. It seems that the benefits produced by the use of AB, which traditionally exhibits better resistance to chloride ingress [ ingress  is compe ingress . However ingress .Icorr) and corrosion potential (Ecorr) evolution of steel reinforcement embedded in all concrete types when they are exposed to an external chloride attack. This experiment is divided in three stages. The first one is the curing period, to evaluate if steel reinforcement has achieved a passive state. It can be observed that all specimens were correctly passivated before they were subjected to sea water attack. During the partial immersion in sea water solution (second stage), steels of all specimens experienced an increase in Icorr values and a consequent decrease in Ecorr. However a clear disruption of the passive state was not observed for any specimen, so all of them stood the aggressive condition for the 80 days that they were in the sea water solution. The results indicate that the chloride concentration provided by the sea water solution was not high enough to produce a change in the steel surface in that period of time. The changes observed in Icorr and Ecorr reflected that certain amount of chloride reached the steel surface. A reduction of the partial pressure of oxygen on the steel surface, as a consequence of the immersion, is also deduced. The third stage aims to evaluate the damage on the steel passive film. The increase in the oxygen concentration available to develop the corrosion process while the depassivating agent has actuated usually shows if a significant damage on the steel conditions has been done. In this case, it can be observed that all specimens returned in low corrosion rate values and initial corrosion potentials, so it can be concluded that all concretes protected steel reinforcements under this type of attack.Icorr) and corrosion potential (Ecorr) evolution of steel reinforcement embedded in all concrete types when they are exposed to a carbonation attack. This experiment is also divided in three stages similar to sea water attack. The first one is the curing period, to evaluate if steel reinforcement has achieved a passive state, and again, this starting condition was correctly attained. Second stage consisted in exposing the steel reinforced specimens to a 100% CO2 atmosphere with a 65% RH. In this environment, all specimens showed an abrupt increase in Icorr at the same time that a decrease in Ecorr values are registered. This indicated that carbonation front reached steel surface and general corrosion of steels started. Among different concretes, no significant differences in the time lapse between the initiation of the attack and the increase of the corrosion rate were observed. Therefore, similar carbonation resistance of all concretes could be deduced, although the high CO2 concentration used does not allow to effectively discriminate this property. In the last stage, almost no changes were monitored in Icorr and Ecorr values, so an effective depassivation was achieve for all specimens. In addition, it can be observed that AB specimens registered higher Icorr values, indicating worse general performance against this type of aggressive environment. This result is somehow expected since AB’s alkaline reserve, in case of existing, should be significantly lower than OPC concretes’ [ncretes’ , due to ncretes’  which isncretes’ . Focusinncretes’ .3.3.1.Portland cement type used was CEM I 52.5 R, designated according to the European Standards . Fly ashFour types of concrete were prepared in order to evaluate the effect of the recycled aggregate and supplementary cementing materials, according to the mix proportions shown in The mixing was prepared on a rotatory concrete mixer and the procedure was the following: firstly, the coarse aggregate was placed in the mixer with the plasticizer with one third of total water ; secondly, fine aggregate and binder, and finally the resting amount of water. The fresh concrete was mixed for 10 min.Six prismatic and six cylindrical specimens of each concrete type were casted. Three prismatic specimens of 100 mm × 100 mm × 400 mm dimensions were used for determining flexural strength and the rest of them for measuring dynamic elastic modulus. Three cylindrical specimens of Ø150 × 300 mm dimensions were used for compressive strength and the rest of them for determining static elastic modulus tests. Both prismatic and cylindrical specimens were cured in moist room  until tested .2. After the 50 days curing period in humid chamber , corrosion test specimens were partially immersed in NaCl 0.5 M solution (simulating seawater) or carbonated in a chamber with 100% CO2 and 65% RH. The curing period was long enough to ensure a steady passive state prior to aggressive conditions attack. Alternatively, different corrosion specimens were prepared adding different amounts of chlorides during the mixing, ranging from 0% to 5% respect to binder mass.In addition, corrosion specimens similar to those reported in previous works ,60 were 3.2.At 28 days, three cylindrical specimens of each concrete were tested to determine the compressive strength according to the standard test method ASTM C 39/C 39 M .k = 2.10) and Catman v.5.0 analysis software. A press machine of 3500 kN capacity was used for both compressive strength and elastic properties tests. Longitudinal and transverse strain values were obtained for each loading cycle up to a maximum value when the applied load is equal to 40% of the sample ultimate load, as specified by the standard test method ASTM C 469 [In order to determine the elastic properties of the cylindrical concrete specimens, under longitudinal compressive stress, the Ohmic Extensometry technique was used on the rest of cylindrical specimens. An HBM Spider 8–600 Hz equipment was used jointly with HBM strain gauges  test device of working frequency range 1 Hz to 100 kHz, with EMAT vibrator and piezoelectric receiver was used to perform the test. The resonant frequencies were determined following the forced resonance method, in which, the specimen is placed in a special device with three support points on its base and it is forced to vibrate by an electro-mechanical driving unit. At least 10 readings of the resonance frequency were made in each sample. The dynamic Young’s modulus of elasticity is calculated from the fundamental longitudinal frequency, mass, and dimensions of the specimen tested. The values obtained by this method are, in general, greater than the static modulus of elasticity obtained on cylindrical specimens.3.3.Wdry) was determined. Then samples were water saturated according to ASTM 1202-97 [Wsub). Finally, the surface of the samples was dried and saturated weight (Wsat) was determined. Open porosity was calculated by the Three samples of each type of concrete were dried at 50 °C in a furnace until constant mass was reached, and dry weight ( 1202-97 . After c3.4.Rp) [Rp is the slope of the polarization curve around the corrosion potential (Ecorr): Rp = ΔE/ΔI when ΔE→0. The Rp value is related to Icorr by means of a constant, denominated B by Stern [The electrochemical technique used to assess the corrosion rate was the Polarization Resistance (Rp) ,65,66. TB is related to Tafel’s constants βa and βc by the The constant B value of 26 mV, the maximum Icorr error factor is 2. For the case of steel embedded in concrete, a value of 26 mV was found [B = 52 mV is more appropriate for passive steel. The electrode polarization has been made from Ecorr − 10 mV to Ecorr + 10 mV at a scan rate of 0.5 mV/s. Rp has been calculated as the slope of the lineal fitting of the polarization curve.It was stated by Stern  that wheas found  for the Rp and corrosion potential were periodically measured during the time of the experiment. Corrosion rate measurements were made by a scanning potentiostat model EG&G 362 from Princeton Applied Research. The reported results are the average of three identical bars. Although it may be controversial to average Ecorr values in the presence of passive/pitting/active transitions no significant scatter between replicate electrodes was found. In order to validate the electrochemical Icorr values obtained through Rp measurements, they were compared by means of Faraday’s law to the gravimetric losses obtained in the same rebars.4.The following conclusions can be drawn from this research:The use of total natural coarse aggregate replacement by recycled aggregate and 50% of OPC substitution by 15% of SFCC and 35% of FA reduced the mechanical performance at early ages of concretes in terms of elastic modulus, compressive strength, and flexural strength;The loss of properties may be attributed more specifically to recycled aggregate than the use of alternative binder;An increase in open porosity was observed which is coherent with the changes observed in mechanical tests when recycled aggregate was incorporated;Regarding the influence on the performance against most usual corrosion processes, no significant differences were observed in the case of the resistance of these types of concretes under a natural chloride attack;In the case of carbonation attack, although all concretes did not stand the highly aggressive conditions, those concretes with cement replacement showed higher corrosion values than OPC concretes.In order to improve the understanding of these materials in terms of its durability, a deeper study of the microstructure evolution should be addressed. These studies may include long term mechanical performance, evolution of porosity, permeability and pozzolanic reaction. In addition, determination of carbonation and chloride ingress kinetics would be valuable to perfectly characterize corrosion protection offered by the proposed materials.


Patients with idiopathic multicentric Castleman's disease were significantly younger than those with IgG4-related disease (p<0.001). Splenomegaly was observed in only idiopathic multicentric Castleman's disease (p=0.002), while pancreatitis and sialo-dacryoadenitis were restricted to IgG4-related disease (both p<0.001). Serum IgG4 concentrations were commonly elevated at >135 mg/dL in both groups (p=0.270). However, the IgG4/IgG ratio in IgG4-related disease was significantly higher than that in Castleman's disease (p<0.001). Histologically, sheet-like plasmacytosis was highly characteristic of idiopathic multicentric Castleman's disease (p<0.001), while plasmacytic infiltration in IgG4-related disease was always associated with intervening lymphocytes. Similar to laboratory findings, the IgG4/IgG-positive plasma cell ratio, but not the IgG4-positive cell count, was significantly higher in IgG4-related disease (p=0.002). Amyloid-like hyalinized fibrosis was found in 6/8 lung biopsies (75%) of Castleman's disease. The over-expression of IL-6 mRNA was not confirmed in tissue samples of Castleman's disease by either in situ hybridization or quantitative real-time PCR. In conclusion, useful data for a differential diagnosis appear to be age, affected organs, the serum IgG4/IgG ratio, sheet-like plasmacytosis in biopsies, and the IgG4/IgG-positive cell ratio on immunostaining. Since IL-6 was not over-expressed in tissue of idiopathic multicentric Castleman's disease, IL-6 may be produced outside the affected organs, and circulating IL-6 may lead to lymphoplasmacytosis at nodal and extranodal sites.The present study aimed to compare clinicopathologic features between idiopathic multicentric Castleman's disease (n=22) and IgG4-related disease (n=26). Histology was analyzed using lymph node and lung biopsies. The expression of IL-6 mRNA in tissue was also examined by  Castleman's disease is a heterogenous entity typically presenting lymphadenopathy with characteristic histopathologic changes . It is cInternational consensus criteria were recently proposed for iMCD . They arIgG4-related disease (IgG4-RD) is a systemic inflammatory disorder histologically characterized by plasma cell-rich, sclerosing inflammation potentially affecting various organs including the pancreas, salivary glands, lacrimal glands, bile duct, lungs, lymph nodes, kidneys, retroperitoneum, and aorta –20. Sincin situ hybridization and real-time PCR in order to elucidate the cellular origin of IL-6 production in iMCD and establish whether tissue examinations for IL-6 mRNA expression contribute to the diagnosis.In the present study, we compared clinicopathologic features between iMCD and IgG4-RD. Since IgG4 is a biomarker for IgG4-RD, IgG4 elevations in tissue and blood were examined in both conditions using immunohistochemistry and serological tests, respectively. The expression of IL-6 mRNA in tissue samples was also analyzed using This study was approved by the Ethics Committee of the Kobe University Graduate School of Medicine. A search of the pathology database at our institutes and affiliated hospitals identified 22 patients with iMCD. All tissue samples taken in iMCD cases were biopsies from either the lymph nodes or lungs. Therefore, 26 cases of IgG4-RD, in which lymph node or lung biopsies were available, were selected for comparison from our institutional database. Cases of iMCD were diagnosed during 16 years from January 2002 to December 2016, while the total number of IgG4-RD cases diagnosed at our institutes in the same period was more than 250 with a part of the cohort reported in a previous study . Among tin situ hybridization for immunoglobulin light chains .Since this study started before the diagnostic criteria for iMCD were proposed , no stanThe diagnosis of IgG4-RD was established based on the international consensus statement for IgG4-RD . All patTable Systemic symptoms, such as fever, weight loss, and fatigue were observed in only iMCD cases 8/22, 36%), one of which also had pleural effusion and ascites. As shown in Table 2, 36%, oIn 3 patients with iMCD, lymphadenopathy was not obvious on imaging, and, thus, did not meet the proposed diagnostic criteria for iMCD . Their cOf the 22 patients with iMCD, 6 (27%) received corticosteroid monotherapy, but showed only partial responses. Additional tacrolimus was given to one patient, but remission was not obtained. Of 12 (55%) patients who were treated with tocilizumab, 10 achieved clinical remission, while another case continued to have general malaise. The remaining patient initially treated with corticosteroid also responded to tocilizumab; however, the IL-6 blockage therapy was stopped due to adverse effects  listed in the major diagnostic criteria for iMCD was compared between iMCD and IgG4-RD Table . The twoIn immunohistochemistry, IgG4-positive plasma cell counts did not differ between the two conditions, with IgG4-positive plasma cells >50 cells/hpf being commonly observed in both Figure  and 2E. Both conditions showed extensive inflammatory extension along intrapulmonary connective tissue, including bronchovascular bundles, the alveolar interstitium, interlobular septa, and pleura Figures  and 4A. Similar to the lymph nodes, the lungs involved in iMCD often showed sheet-like plasmacytosis corresponding to grade 3 in the iMCD diagnostic criteria for lymph node biopsies  Figure . PlasmacSimilar to lymph node biopsies, no significant difference was observed in IgG4-positive plasma cell counts between the two conditions, while the IgG4/IgG-positive plasma cell ratio was significantly higher in IgG4-RD (p=0.002) Figures . ElevateIn situ hybridization was performed on all lymph node and lung biopsies of iMCD in order to elucidate which cells express IL-6 in iMCD. A single lung specimen was found to have a small focus showing positive signals in the endothelial cells of small vessels , other lung diseases (n=5), and normal lungs (n=5) were used as controls. The lung disease controls consisted of one case each of bone marrow transplantation-associated organizing pneumonia, rheumatoid arthritis-related interstitial pneumonia, aspergillosis, bronchopneumonia, and extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue. Normal lung tissue was obtained from unremarkable background parenchyma in bullectomy specimens.Since IL-6 expression was not obvious on In lymph node biopsies, no significant difference was observed in IL-6 expression values among iMCD, IgG4-RD, and disease controls .Several previous studies examined potential cellular sources of IL-6 production in unicentric and multicentric Castleman's disease using hemistry –32. In t6 in MCD –32. More6 in MCD . HoweverIn conclusion, although iMCD and IgG4-RD are both plasma cell-rich, multi-organ disorders, discriminating between the two will be possible, particularly based on age, affected organs, laboratory data , and microscopic findings . Serum IgG4 elevations and IgG4-positive cell counts appeared to be less useful than expected. The present results also suggested that IL-6 is not produced at sites affected by iMCD. IL-6 may be produced elsewhere, and circulating IL-6 may lead to lymphoplasmacytosis in the lymph nodes, lungs, and other organs.Clinical records were reviewed for age, sex, affected organs, and laboratory data including IgG, IgG4, CRP, and hemoglobin. IgG4 concentrations were not measured at the time of the diagnosis in 6 cases of iMCD. Serum IL-6 concentrations were available in 20 cases of iMCD. Affected organs were determined by imaging at the time of the diagnosis. Lymph node involvement was defined as the presence of enlarged lymph nodes (>1 cm in the short-axis diameter) according to the diagnostic criteria for iMCD .Tissue specimens were fixed in 10% buffered formalin and embedded in paraffin. All samples were stained with hematoxylin and eosin (H&E), and lung biopsies underwent additional staining with Elastica van-Gieson (EVG). In lymph node biopsies, 5 individual histologic parameters described in the diagnostic criteria for iMCD were examined and graded into 0 to 3, with grades 2 and 3 being considered to be significant . SamplesLymph nodes of iMCD were classified into three categories based on histopathologic changes according to the iMCD international diagnostic criteria . Cases iAll cases were immunostained for IgG and IgG4 using a Ventana Benchmark XT  according to the manufacturers’ protocols. Deparaffinized sections were heat-treated and incubated with primary antibodies. The antibodies used were as follows: IgG  and IgG4 . The numbers of plasma cells positive for IgG or IgG4 were counted at three hot spots and calculated as the average per hpf. In endoscopic small lung biopsy specimens, positive cells were counted at the single highest spot.In situ hybridization was conducted manually using RNAscope®  in order to detect intracytoplasmic mRNA coding IL-6 according to the manufacturer's protocol. Briefly, 4-μm-thick sections of formalin-fixed paraffin-embedded tissue were baked and deparaffinized. After peroxidase blocking, antigen retrieval, and a protease treatment, slides were hybridized with the pre-designed probe specific for IL-6 mRNA (Advanced Cell Diagnostics). Signals were amplified by sequential hybridization and then visualized with Fast Red. Slides were counterstained with hematoxylin. Lung cancer tissue was used as a positive control.The expression of IL-6 mRNA in lymph node or lung biopsies was compared using real-time PCR among iMCD, IgG4-RD, and controls. Since the quality of extracted RNA was low or unstained slides were not available in some cases, this experiment was conducted on 26 lymph node samples  and 22 lung specimens .RNA was extracted from formalin-fixed paraffin-embedded tissue using the RNeasy FFPE Kit  following the manufacturer's instructions. Affected areas were selected for RNA extraction under a microscope. RNA content was assessed by the NanoDrop 2000c spectrophotometer . cDNA was synthetized using the Super Script IV First-Stand Synthesis system (Invitrogen). Real-time PCR was performed for quantitative analyses, according to the standard protocol using a StepOnePlus Real-time PCR System . Specific primers and probes for IL-6 (Hs00174131_m1) and β-actin (Hs01060665_g1) were obtained from Applied Biosystems. PCR was performed using the TaqMan Gene Expression Master Mix (Applied Biosystems) with 1 μl of cDNA in a 20-μl final reaction mixture. The mRNA expression levels of IL-6 were normalized to those of β-actin. Each experiment was performed in duplicate, and the mean was adopted for the analysis.Statistical analyses were performed using the Wilcoxon rank-sum test or Fisher's exact test for the comparison of clinicopathologic features between the two groups. In the analysis of real-time PCR, the Kruskal-Wallis test was applied to compare expression values among three or four groups, and if any significant difference was suggested, the Steel-Dwass test was conducted as a post-hoc analysis between the two relevant groups. A p-value less than 0.05 was considered to be significant. The software used for statistical analyses was JMP® 13 .