To transform the conventional process of concrete production to a more sustainable process, the replacement of high energy-consumptive PC with new binders such as fly ash and alkali-activated slag (AAS) from available industrial by-products has been recognized as an alternative. This paper investigates the effect of curing conditions and steel fiber inclusion on the compressive and flexural performance of AAS concrete with a specified compressive strength of 40 MPa to evaluate the feasibility of AAS concrete as an alternative to normal concrete for CO2 emission reduction in the concrete industry. Their performances are compared with reference concrete produced using OPC. The eco-efficiency of AAS use for concrete production was also evaluated by binder intensity and CO2 intensity based on the test results and literature data. Test results show that it is possible to produce AAS concrete with compressive and flexural performances comparable to conventional concrete. Wet-curing and steel fiber inclusion improve the mechanical performance of AAS concrete. Also, the utilization of AAS as a sustainable binder can lead to significant CO2 emissions reduction and resources and energy conservation in the concrete industry.Conventional concrete production that uses ordinary Portland cement (OPC) as a binder seems unsustainable due to its high energy consumption, natural resource exhaustion and huge carbon dioxide (CO In order to address this, alkali-activated slag (AAS) has been considered as an alternative binder to cement, as well as a way of reusing available industrial by-products. Damineli et al. 4− in the pore solution.The compressive strength values of AAS concrete were significantly increased by fiber inclusion whereas OPC concrete shows no noticeable change in the compressive strength. It can be noted that lateral confinement by steel fiber is improved due to the smaller particle size of GGBS compared to OPC. Furthermore, the increase ratio in compressive strength for AAS concrete with wet-curing is higher than that with dry-curing. This indicates that wet-curing enhances the bonding properties between fiber and mortar phase for AAS based binders. f), first crack deflection (δf), ultimate strength (ff), and deflection at the peak load (δf) are given. As shown in flexural responses in A flexural strength-deflection curve itself may be quite sensitive in distinguishing amongst different fiber inclusions, and the curve roughly indicates differences in the toughness performance of concrete with different fiber volume fractions. 5 (3δ), I10 (5.5δ), I20 (10.5δ), I30 (15.5δ), and I50 (25.5δ). For the residual strength factors, R5,10, R10,20, R20,30 and R30,50 are also specified in the ASTM. In this paper, for the flexural toughness indices and residual strength factors, I100 (50δ), I200 (100δ), R50,100, and R100,200 were added to consider overall flexural response including the deflection at peak load of test specimens. For the R50,100 and R100,200 added, the factors were reduced as per the following equations:ASTM C1018  is the m50,100 and R100,200 which are toughness indices added in this paper, the difference of indices between AAS and OPC specimens was widened. This means that the AAS binder is more efficient for bonding in the interfacial zone between steel fibers and the matrix phase than OPC binder during post-cracking behavior.2 emissions of concrete. 2 evaluation of the OPC and the AAS concrete, respectively. As listed in the tables, the CO2 emission of the AAS concrete is 15%~24% of that of the OPC concrete because the AAS based binder has about 97% lower CO2 emission than the OPC based binder. The CO2 emissions of concretes considering fiber inclusion are compared in Korea LCI Database Information Network  and Japabi) and CO2 intensity (ci) were proposed by Damineli BL et al. [2 intensity permits an estimation of the mix design’s global warming potential. The indices are calculated as:b is the total consumption of binder materials (kg/m3), c is the total CO2 (kg/m3) emitted to produce, and p is the compressive strength (MPa) at 28 days.To compare environmental efficiency, a binder intensity . AAS concrete has lower binder intensity compared to OPC concrete even though it was reported that Ca(OH)2-based AA GGBS concrete requires a greater amount of binder in order to obtain the same compressive strength as OPC concrete [The binder intensity with variables and the relationship between the binder intensity and compressive strength are presented in 2 intensity with variables and the relationship between the CO2 intensity and compressive strength. As estimated in 2 intensity of AAS concrete is about 20%–25% of that of OPC concrete. In view of the curing method, as shown in 2 intensity by 40%–50% compared to dry-curing because electric equipment is needed to maintain the water at a constant temperature for curing. The best-fit curves in previous research [2 intensity in this study and are presented in 2 intensity for OPC is around the best-fit curve. However, there is little difference between the curves because Yang et al. [2 emitted in production and transport as c. In this study, CO2 emitted for transport was omitted because the CO2 emission is influenced by the transportation distance between the plant and construction sites. In this study, CO2 emitted in production only (cradle to gate) was considered to calculate CO2 intensity (ci), and the best-fit curves for AAS-wet and -dry lie under the curve proposed by Yang et al. [bi and ci) were evaluated. The following observations and conclusions can be made and drawn on the basis of the compressive test results and intensity estimation in this study.AAS based binders may provide similar compressive performance with a lower fiber content due to better fiber dispersion and enhanced bond characteristics in the steel fiber–matrix transition zone. Furthermore, the wet-curing method is very helpful for the performance enhancement of AAS with steel fibers.It can be inferred that AAS binder can enhance performance, especially the compressive strength of concrete, and can reduce the total amount of binder necessary to achieve the performance required.Based on the results of the ASTM toughness index and residual strength factor, it is concluded that AAS binder is more efficient for bonding in the interfacial zone between steel fibers and matrix phase than OPC binder in post-cracking behavior. This is because the internal stress distribution in the tension zone would be enhanced due to the formation of the dense and uniform transition zone in the matrix.2 intensity. It indicates that more economic and eco-efficient fiber reinforced composites with AAS can be produced. In the future, additional studies on the mechanical properties of AAS concrete under fatigue and creep are needed.The significant effects of fiber inclusion and wet-curing on the eco-efficiency of AAS were confirmed on the basis of the binder intensity and COIn this study, AAS was used as an alternative binder to OPC in order to reduce environmental impact of concrete. To estimate eco-efficiency of AAS based binder, compressive tests were conducted and the effects of fiber inclusion and wet-curing on the two indicators (


Higher c-Met expression was associated with higher FNG (4 vs. 3) in primary tumors . c-Met expression was numerically greater in PD-L1+ vs. PD-L1- tumors. Higher c-Met expression in metastatic sites compared to primary tumors suggests that testing for biomarkers of response to c-Met inhibitors should be conducted in metastases. While higher c-Met expression in PD-L1+ tumors requires further investigation, it supports exploring these targets in combination clinical trials.In preclinical models, c-Met promotes survival of renal cancer cells through the regulation of programmed death-ligand 1 (PD-L1). However, this relationship in human clear cell renal cell carcinoma (ccRCC) is not well characterized. We evaluated c-Met expression in ccRCC patients using paired primary and metastatic samples and assessed the association with PD-L1 expression and other clinical features. Areas with predominant and highest Fuhrman nuclear grade (FNG) were selected. c-Met expression was evaluated by IHC using an anti-Met monoclonal antibody (MET4 Ab) and calculated by a combined score : intensity of c-Met staining (0–3) x % of positive cells (0–100). PD-L1 expression in tumor cells was previously assessed by IHC and PD-L1+ was defined as PD-L1 > 0% positive cells. Our cohort consisted of 45 pairs of primary and metastatic ccRCC samples. Overall, c-Met expression was higher in metastatic sites compared to primary sites (average c-Met CS: 55 vs. 28,  The e repair , 5. In Re repair . In the e repair .C-Met and the tyrosine-kinase AXL can be upregulated in RCC and have been shown to play a possible role in the development of resistance to VEGFR inhibitors making these pathways rational targets for therapeutic trials –13. CaboThe HGF/c-Met pathway has also been implicated in attenuating inflammatory responses, which suggests potential for immunomodulation with inhibition of this pathway , 18. PreIn this context, and given the known tumoral heterogeneity in this disease , we aimen = 14), bone (n = 4), lymph nodes (n = 10), soft tissues (n = 5), adrenal gland (n = 6), pleura (n = 3), brain (n = 1), thyroid (n = 1), and others (n = 10). Although most primary tumors had only one corresponding metastasis, 10 cases (22%) had two or more metastatic lesions that could be retrieved, which resulted in a total of 54 metastases that were analyzed.We identified 45 patients with both primary and metastatic lesions available for analysis. Patient characteristics at the time of primary surgery are summarized in Table p = 0.0003, respectively)  and patients with T-stage T3-T4 appear to have higher c-Met expressions compared to those with Tx-T2  (Table c-Met expression (average c-Met combined score (CS) > 0) was detected in 34 of 45 (75.6%) primary ccRCC samples and 41 of 54 (75.9%) metastases. Overall, c-Met expression levels were higher in metastatic sites compared to paired primary tissues : 55  vs. 28 , ) Figure  and 1B. p = 0.34, respectively) and metastatic sites , although these differences were not statistically significant  displayed PD-L1 positivity in the primary tumor and 9 cases (20%) were PD-L1 positive in paired metastases, including 7 cases (16%) with PD-L1 positivity in both primary tumors and metastases. PD-L1 positive tumors had numerically greater c-Met expression than PD-L1 negative tumors in both primary  compared to those with low c-Met expression : 6.1 (5.3–NA) vs. 9.3 (2.56–NA) months). Similarly, those with high primary c-Met expression had numerically worse overall survival (OS) compared to those with low c-Met expression (median OS (95% CI): 18.1 (12.1–NA) vs. 34.6 (11.4–NA) months).Among eight patients who received systemic therapy in between surgical resection of their primary and metastatic sites, 6 patients were treated with VEGF-TT. Thirty-five patients had no systemic therapy in between primary and metastases resection. Patients treated with VEGF-TT in between surgical resection of primary tumor and metastases appeared to have greater c-Met expression in their metastatic sites compared to those who had no systemic treatment in between surgeries . In contrast, c-Met expression in primary tumors was similar in patients who received VEGF-TT before resection of metastases and those who received no treatment .c-MET proto-oncogene in tumor development and aggressiveness [We demonstrate that in our cohort of ccRCC samples, c-Met expression is significantly higher in corresponding metastatic sites compared to paired primary tissues. Given that the treatment landscape for mRCC has expanded to include small-molecule inhibition of VEGFR, MET, and AXL pathways –16, our siveness .et al. evaluated the prognostic significance of Met molecular status using tissue microarrays (TMAs) of primary tumor and corresponding normal renal tissue samples, and found higher Met expression and copy number associated with dedifferentiation and higher tumor extent. However, 84% of their samples were low-grade (FNG 1 or 2) and only 2 samples overall were FNG 4. Furthermore, they did not evaluate paired metastatic samples compared to the primary RCC tissues [et al. investigated Met expression in matched metastatic and primary ccRCC and found no significant difference between the two sites. Their analysis showed a low level of correlation of Met expressions between the primary and distant tissue (correlation coefficient R = 0.5), and their study was limited by a smaller sample size (n = 31) [While c-Met has been shown to be a key player in the development and progression of a variety of solid tumors, it has therapeutic, and potentially diagnostic, relevance in RCC. As a consequence of hypoxia and inactivation of the Von-Hippel-Lindau tumor suppressor gene (VHL), MET and AXL are upregulated , 9. Thes tissues . Shuch e(n = 31) . Given tet al., which identified c-Met and PD-L1 to be significantly upregulated and co-localized in renal cancer tissues [et al. show that PD-L1 positivity was significantly associated with increased c-Met expression [et al. found that in 62 sunitnib-treated mRCC patients with high c-Met expression, 85.5% of these also had PD-L1 overexpression (defined as 2–3 staining intensity) [et al. highlight the role of HGF/c-Met pathway as a potential regulator of inflammation and autoimmunity, particularly with immune-related systemic diseases. For example, HGF treatment can increase the levels of PD-L1 expression by dendritic cells and CTLA-4 molecules expressed on T cells, thereby inducing immune tolerance. HGF also markedly alters the antigen-presenting function and differentiation of dendritic cells and interferes with pro-inflammatory NF-κB signaling, thereby inhibiting an inflammatory response [An interesting finding in our study is that PD-L1 positive tumors were noted to exhibit numerically higher c-Met expression than PD-L1 negative tumors, in both primary and metastatic sites Table , 3 A-D.  tissues . In a clpression . Furthertensity) . Collectresponse . In the response . With thIn a descriptive analysis, we observed that patients in our cohort who were treated with VEGF-TT and had high c-Met expression had numerically worse clinical outcomes, in terms of reduced TTF and OS, compared to those patients treated with VEGF-TT who had low c-Met expression. While our small sample size limited formal statistical testing, our findings are consistent with other data showing that high c-Met expression portends worse clinical prognosis , 14, 15.In this analysis of paired primary and metastatic samples, we used a validated anti-MET monoclonal antibody  and all tumor slides underwent independent pathological review and were scored by an expert genitourinary pathologist (SS). FNG was assessed using established criteria . NeverthIn conclusion, we demonstrate that c-Met expression is higher in metastatic sites compared to paired primary tissues in patients with ccRCC. Given the expanding role and ongoing clinical trials of c-Met inhibition in mRCC, our results suggest that the accurate assessment of c-Met expression as a biomarker for these agents should be conducted in metastatic sites. Although the observation of higher c-Met expression in PD-L1+ tumors requires further investigation, it supports the growing rationale for exploring these targets in combination therapeutic trials.A cohort of primary ccRCC tumors and the corresponding metastatic sites from 53 patients, who had undergone surgical tumor resections, were identified from two institutions: Brigham and Women’s Hospital and Beth Israel Deaconess Medical Center. Formalin-fixed paraffin-embedded (FFPE) tissue blocks from primary tumor and corresponding regional lymph node or distant metastases were retrieved. For each nephrectomy or metastasectomy specimen, all hematoxylin and eosin-stained slides containing tumor were reviewed by an expert genitourinary pathologist (SS). Fuhrman nuclear grade (FNG) was assessed using established criteria . For eacc-Met expression was evaluated by immunohistochemistry (IHC) using an anti-MET monoclonal antibody  at 1:250 dilution. Out of 53 pairs, 4 cases were excluded for insufficient tumor cells and 4 cases were excluded for failing to meet quality control (QC) for c-Met staining, resulting in 45 pairs that met the QC for IHC reporting. Therefore, the analytic cohort included 45 primary tumors and 54 metastatic sites corresponding to the 45 primary tumors. c-Met expression was measured using a combined score  and calculated by: the intensity of c-Met staining (range 0–3) multiplied by the percentage (%) of positive cells (range 0–100).PD-L1 expression was previously evaluated by IHC and quantified using an H-score, as reported by Callea et al . Specifiwww.r-project.org), and a p-value (two-sided) of < 0.05 was considered statistically significant when appropriate.Patient and tumor characteristics at the time of primary surgery were summarized descriptively. When several samples were available within one primary or multiple metastatic sites, an average of c-Met combined score (average c-Met CS) were calculated. Similarly, the PD-L1 expressions according to tumor sites were dichotomized as PD-L1+ (> 0% positive cells) and PD-L1- (= 0% positive cells) in the analysis. The comparisons between paired primary and metastatic samples used Wilcoxon signed-rank test for c-Met expression status (average c-Met CS). The associations of the average c-Met CS with PD-L1 expression (+/–), as well as clinicopathological features of FNG and tumor stage (T-stage) according to the primary and metastatic sites were assessed using the Wilcoxon rank-sum test. In an exploratory analysis of patients treated with first-line VEGF-TT, time to treatment failure (TTF) and overall survival (OS) were estimated using the Kaplan Meier method, according to high c-Met expression levels (defined as c-Met CS ≥ median) versus low (c-Met CS < median). Descriptive statistics were also used to summarize c-Met expression by disease sites (primary vs. metastasis) in patients who had VEGF-TT in between sampling time points compared to those without treatment prior to sampling. All statistical computations were performed using R 13.3.1 (


Formation of cracks impairs the durability of concrete elements. Corrosion inducing substances, such as chlorides, can enter the matrix through these cracks and cause steel reinforcement corrosion and concrete degradation. Self-repair of concrete cracks is an innovative technique which has been studied extensively during the past decade and which may help to increase the sustainability of concrete. However, the experiments conducted until now did not allow for an assessment of the service life extension possible with self-healing concrete in comparison with traditional (cracked) concrete. In this research, a service life prediction of self-healing concrete was done based on input from chloride diffusion tests. Self-healing of cracks with encapsulated polyurethane precursor formed a partial barrier against immediate ingress of chlorides through the cracks. Application of self-healing concrete was able to reduce the chloride concentration in a cracked zone by 75% or more. As a result, service life of steel reinforced self-healing concrete slabs in marine environments could amount to 60–94 years as opposed to only seven years for ordinary (cracked) concrete. Subsequent life cycle assessment calculations indicated important environmental benefits (56%–75%) for the ten CML-IA  baseline impact indicators which are mainly induced by the achievable service life extension. As such, it is logical that the construction sector is encouraged to develop innovative construction materials and techniques that are more durable and environmentally friendly. Since concrete production contributes 5% of the annual anthropogenic global COs effect . While cThe inspiration to develop self-healing materials comes from biological systems, which do have the natural ability to heal after being wounded. In fact, also in the case of concrete, some help from nature is obtained as concrete in itself possesses natural self-healing properties, i.e., autogenous healing of cracks. Due to ongoing hydration of clinker minerals or carbonation of calcium hydroxide, cracks may heal after some time . HoweverSome research studies even go one step further and really modify concrete elements in order to obtain autonomous repair of cracks. This is obtained through the use of embedded capsules ,19,20 or2 to 104 if encapsulated polyurethane is provided [From the aforementioned mechanisms, encapsulation of polyurethane by brittle glass capsules is a promising approach. By means of acoustic emission analysis the rupture of the capsules upon crack formation has been proven  and compprovided . By neutprovided ,32. Moreprovided . Autonomprovided ,34. In tThe evaluation of the environmental sustainability of a material should always be done in an objective way. Since the end of the 1960s, a step-by-step methodology has been gradually developed to achieve this goal . NowadayNatural chloride diffusion tests on uncracked concrete as well as accelerated chloride diffusion tests on (un)cracked and self-healing concrete with two different encapsulated PU-based healing agents were done to obtain the necessary experimental input to a probabilistic prediction model based on Fick’s second law of diffusion and to estimate the time to chloride-induced steel depassivation of the embedded reinforcing steel. Subsequently, life cycle assessment calculations were done in the commonly used and well-accepted SimaPro 8 software cf. ISO 14040-14044. The calculations were performed for steel reinforced concrete slabs made with ordinary (cracked) and the self-healing concrete and compared with each other.This study is only a part of the ISHECO project . As described in the ISHECO project outline, the service life extension is to be assessed with probabilistic prediction models that are commonly used in engineering model codes but also by means of dedicated and more accurate numerical modeling approaches. This paper only focuses on the first approach. The second modeling approach and a subsequent fine-tuning with the first approach are still under investigation and will be published at a later stage.It has been proven that concrete where a certain amount of cement is replaced by fly ash has a higher resistance to chloride penetration, which is beneficial for the durability ,46,47,48Some of the concrete samples contained cylindrical borosilicate glass capsules  filled with a one component polyurethane-based healing agent. Filling of the capsules was done by sealing one end with a methyl methacrylate (MMA) based glue, subsequently injecting the polyurethane precursor into the capsules and finally sealing the other end with the MMA based glue. Upon the occurrence of a crack in the concrete matrix, the capsules easily break and release their content into the crack. Two different polyurethane (PU) precursors were used for this research. The first healing agent is a non-commercial PU which was developed in the framework of another project . Its viscosity at 25 °C is 6700 mPas. It essentially consists of a polyether polyol and methylene diphenyl diisocyanate (MDI). For application in self-healing concrete, this PU precursor reacts with the moisture in the concrete matrix and hardens into a solid flexible foam inside the crack within 48 h. The polyurethane is denoted as PU_HV (High Viscosity). The second healing agent is a commercially available PU precursor with an approximate viscosity of 200 mPas at 25 °C. It is also a MDI and polyether polyol based prepolymer. In addition, it contains inert hydrophobic compounds that control the viscosity and rheological behavior. The viscosity of this second healing agent is more than 30 times less than the viscosity of the first one. Therefore, this product will be named PU_LV (Low Viscosity). For crack healing in concrete, this PU precursor flows into the crack and cures to a closed-cell polymer upon contact with moisture in the concrete matrix.The viscosity of the healing agent is an important parameter for self-healing concrete. The viscosity should not be too high, in order to be able to flow out of the capsules and fill the crack. On the other hand, when the viscosity is too low it may leak out of the crack or be absorbed by the surrounding matrix . AccordiFor the natural chloride diffusion tests on uncracked concrete, a series of cylindrical concrete samples  were drilled from cubes (side: 150 mm) that were cured at 20 °C and 95% relative humidity until the age of 21 days and then partially coated with an epoxy coating on all sides except for the test surface to allow for a unidirectional chloride ingress during exposure.For the accelerated chloride diffusion tests, cylindrical concrete specimens with a height of 50 mm and a diameter of 100 mm were made using plastic molds cut from a PVC tube with an inner diameter of 100 mm. Cracks were introduced into the specimens in an artificial way by introducing thin brass plates with a thickness of 300 µm into the fresh concrete upon casting . The plaAfter the preparation of the molds, the concrete was cast and the specimens were stored in an air-conditioned room at a temperature of 20 °C and a relative humidity of more than 95% for 24 h. Subsequently, the specimens were demolded and stored in the same environment to cure until the age of 28 days. After the curing period, the plates were removed from all samples creating standardized cracks with a predefined width, depth and length of 0.3 mm, 25 mm and 60 mm, respectively. For the specimens with the self-healing properties the capsules broke due to the removal of the plates. In this way, the healing mechanism was triggered and the healing agent was released in the crack.Since the exposure surface containing the artificially induced crack was a troweled surface with a certain roughness, it was mechanically flattened by cutting off a layer of approximately 1 mm. In this way, a perfectly flat test surface was obtained. This was also done for the uncracked specimens. For the specimens with self-healing properties, the flattening of the test surface occurred after the PU was given the time to harden completely for 48 h. All samples used for the accelerated chloride diffusion tests were also partially coated to ensure unidirectional chloride ingress. All different test series that were used in this research are listed in − concentration in the North Sea. After 77, 139, 192, 262 and 311 days of exposure each time three cylinders were removed from the solution. Subsequently, 10 concrete powders were collected from each cylinder by grinding material in 2 mm layers parallel to the exposure surface. Determination of the total chloride concentration per ground layer of powder consisted of an acid-soluble extraction in a nitric acid solution followed by a potentiometric titration against silver nitrate cf. Mu [To determine the proper mix specific input parameters for service life prediction of the uncracked concrete, the partially coated cylindrical specimens taken from cubes were stored in an aqueous 33 g/L NaCl solution at 20 °C after 28 days. This concentration corresponds with the normal Cle cf. Mu .2 solution. An aqueous NaCl solution with a concentration of 165 g NaCl per dm3 solution was prepared for the accelerated test. All specimens were completely immersed in this solution and kept at a constant temperature of 20 °C. At exposure times of 49 and 133 days, three specimens of each test series were removed from the exposure solution. The chloride profiles of the specimens at the respective exposure times were determined by grinding off material in layers of 2 mm parallel to the exposed surface. A diamond coated drilling head with a diameter of 16 mm was used to grind away the material in a zone of 16 mm × 78 mm around the crack he crack , similarAfter the collection of the powders of all the ground concrete layers, the powders were dried in an oven at 105 °C for a minimum of 7 days. Subsequently, the total chloride content of each powder was determined in the same way as described in 0, the initial chloride concentration (m%/binder); Cs, the constant surface concentration (m%/binder); and erf(.), the error function and Dapp, the apparent diffusion coefficient (m2/s). It should be noted that Dapp counts as a time-averaged value over the entire exposure period and not an instantaneous value. Values for Cs and Dapp were estimated from Equation (1) by applying non-linear regression analysis on the experimental chloride profiles. The first layer (0–2 mm) of the chloride profiles was omitted in this regression analysis.Under the assumption that the diffusion coefficient and surface chloride concentration remain constant in time, the chloride ingress can be estimated using the well-known formula suggested by Collepardi et al.  which isapp does change with exposure time and therefore, this time dependency should be included in the prediction models. According to the same authors, the following updated formula should be in agreement with this requirement for the continuous exposure condition (Equation (2)):0, the instantaneous diffusion coefficient (m2/s)) at reference time t0 which is usually 28 days (=0.0767 years) and n, the ageing exponent (−). In this equation there are three unknown variables  that need to be optimized using the least-of-squares method. According to Visser et al. [s, D0 and n at once. Thus, also in this research the very same approach as proposed by Visser et al. [s, D0 and n using Equation (2).However, Visser et al.  showed tr et al.  it is imr et al.  was adopThe probabilistic limit state function (Equation (3)) used for estimating the time to chloride-induced steel depassivation looks almost identical to Equation (2) of Visser et al. .(3)Ccrie has been included to account for the fact that the temperature of the aqueous exposure solution during the laboratory diffusion tests differs from the actual seawater temperature in practice. In accordance with fib Bulletin 34 [e represents the typical Arrhenius function (Equation (4)).The only difference is that an environmental transfer variable kletin 34 , variablVariable d (Equation (3)) corresponds with the concrete cover d on top of the reinforcing steel. Eurocode 2 prescribes a minimum concrete cover of 50 mm for construction class S6 which corresponds with a design service life of 100 years in exposure class XS2. Despite the fact that this concrete cover is strictly specified, the actual value is subject to variation due to the unavoidable small errors that may occur during the construction phase of a steel reinforced concrete element or structure. Therefore, it is a stochastic variable. As such, a standard deviation of 8 mm on the minimum cover was taken into consideration. The latter value is seen as appropriate for concrete without particular execution requirements . The procrit in Equation (3), counts as the decisive criterion in the limit state function. Once the chloride concentration at the location of the steel rebar reaches this critical value, onset of active corrosion can start. In this case study, this event is seen as the end of service life. In [The critical chloride concentration, represented by Clife. In  it has blife. In , this vaf) as a function of time were calculated for Equation (3) using the First Order Reliability Method (FORM) available in the probabilistic Comrel software. Cf. fib Bulletin 34 [f ≤ 0.10) to qualify for use in exposure class XS2. 0, Cs and D0 is discussed more in detail in Reliability indices (β) and probabilities of failure . As such, the extra material needed to repair the slab as soon as steel corrosion would be at risk was accounted for. For a slab repair, an extra concrete volume representing the 50 mm cover on top of the rebars plus the thickness of these rebars was taken into account. For the self-healing concrete slabs and its repairs the presence of one layer of 4150 capsules filled with PU precursor in the tensile zone of the slab was considered.The goal of this LCA was a quantification of the environmental impact reduction that could be achieved by using the proposed self-healing concrete instead of a more traditional 15% fly ash concrete in exposure class XS2. To do this in an adequate manner, the LCA study takes into account the difference in service life between traditional (cracked) concrete and the same concrete with self-healing properties. Therefore, a steel reinforced concrete slab with a design service life of 100 years and a variable load of 5 kN/mFor almost all concrete constituents, the life cycle inventory (LCI) data were collected from the renowned Ecoinvent database  Table 4Table 4.3 of concrete, including all internal underlying sub-processes .For the allocation of impacts related to the industrial by-product fly ash, the economic allocation coefficient as proposed by  was applAs indicated in 2 eq), ozone depletion , human toxicity , freshwater aquatic ecotoxicity , marine aquatic ecotoxicity , terrestrial ecotoxicity , photochemical ozone creation , acidification  and eutrophication .CML-IA was used as impact method. It is an update of the CML 2 baseline 2000 method which was released by the Center of Environmental Science (CML) of Leiden University in 2013. It gives an eco-profile with ten baseline impact indicators regarding abiotic depletion , global warming  the chloride profile reaches a stable value at a depth of around 14 mm. For the last exposure time (311 d) this event is only observed at a depth of around 18 mm onwards. Based on these results, it is quite evident that at a certain moment, the zone in direct contact with an embedded rebar will reach the critical chloride concentration that causes breakdown of the protective passivation layer on top of the rebar.This seems to be the case for all five exposure times . As such, the values for Cectively . For thiNote that the experimental data corresponding with the first ground layer (0–2 mm) of all five profiles clearly deviate from the fitted profiles. On the one hand, this is not surprising because the first layer was never considered in the fitting process  for all (un)cracked and healed specimens at the two exposure periods are shown in The graphs in At an exposure time of 133 days, the difference in chloride content between cracked and uncracked concrete was less pronounced than for 49 days of exposure. This is because the ingress of chlorides at later times was dominated by diffusion. Since there were already a lot of chlorides present in the concrete near the crack surface due to early capillary absorption, the diffusion of chlorides through the crack is slowed down compared to uncracked concrete. It was found that the maximum increase in chloride content between 49 and 133 days of exposure for the cracked concrete was 14%, while for uncracked concrete an increase of 27% (depth 0–2 mm) to 104% (depth of 14–20 mm) was found. However, the chloride content at an exposure time of 133 days at a depth of 18–20 mm was still 10 times higher due to the presence of a crack. Consequently, the presence of a 300 µm crack has a big influence on the chloride penetration in concrete, especially at larger depths below the exposed surface and thus possible locations of the steel reinforcement.i) was defined according to Van den Heede et al. [When going from the top layers deeper inside the concrete, the chloride concentration drops very fast for the healed specimens. This can clearly be seen from the steep slopes of the chloride profiles of the healed specimens at depths from 0 to 6 mm below the exposed surface. From a depth of 6 mm onwards, healing of cracks decreases the chloride content to a large extent compared to unhealed cracks. The hardened polyurethane inside the cracks clearly forms a partial barrier against immediate ingress of chlorides through the cracks. In order to make clear conclusions about the performance of the self-healing mechanism, a self-healing efficiency at every depth i below the exposed surface ) this would mean that there is a negative SHE in these first layers. This was denoted as not applicable (N/A). For all other layers in the profile, the SHE (%) is given above the curves in The SHE gives an idea about the general performance of the self-healing mechanism regarding the durability in chloride environments. However, it might be interesting to have a look at the individual results as well. In the case of crack healing with the high viscosity polyurethane (PU_HV) at an exposure time of 49 days, there is not much variability between the different healed specimens, especially not in the layers from 12 to 20 mm. All healed specimens showed more or less the same healing performance, but none of them behaved as uncracked concrete. At 133 days of exposure, the mean healing efficiency of PU_HV was even higher than at 49 days, but the variability between the different specimens was also higher. One of the healed specimens behaved as almost uncracked with a SHE at the deepest ground layer (18–20 mm) of 97%. For the other two specimens in this series, the SHE at this depth was 74% and 58%.For crack healing with the low viscosity polyurethane (PU_LV) similar results were found at both exposure times. Two out of three specimens were almost perfectly healed, behaving very similar to uncracked concrete. One specimen however performed much worse than the others. This can be seen clearly in s and Dapp were determined using Equation (1). However, the model for chloride ingress based on Fick’s second law of diffusion (Equation (1)) was developed based on sound, uncracked concrete. C0 in Equation (1) is the initial chloride concentration in the concrete, which reaches a stable value far enough away from the surface exposed to chlorides. In the case of cracked  concrete, the experimental chloride profiles in the cracked zones also have a tendency towards a stable value at a depth larger than 14 mm below the exposed surface  respectively with their standard deviation σ. The surface chloride concentration of all series, except CR, increased due to a longer exposure time. This is logical, since there is more ingress of chlorides with time. For the specimens with healed cracks larger values of Cs were found than for (un)cracked samples. The reason for the high surface chloride content in the crack zone of the healed samples is already explained in app is always approximately reduced by half compared to 49 days of exposure. The presence of a  crack thus has a limited influence on the development of the diffusion coefficient over time. At both exposure times, Dapp is largest for uncracked concrete and has the smallest value for healed concrete . The fact that the diffusion coefficient of cracked concrete is lower than the one of uncracked concrete is mainly because chlorides do not only enter the concrete through diffusion from the exposed surface. The chloride solution can easily enter into the crack and there is also ingress of chlorides perpendicular to the crack faces. This causes the concentration gradient of chlorides in the concrete to be different and will consequently have an effect on the apparent diffusion coefficient. In the case of self-healing concrete there is much less immediate ingress of chlorides deep into the crack, since this is (partly) blocked by the hardened polyurethane inside the crack. Therefore, the fitted curves of the healed specimens look similar to the ones for uncracked specimens  was made. First, the ratio of Dapp (or Cs) for healed concrete and uncracked concrete was calculated. This ratio was then multiplied by the realistic value of the instantaneous diffusion coefficient (and surface chloride concentration) of uncracked concrete found with the natural diffusion test . The same was done to make an estimation of C0 for self-healing concrete as input for the service life prediction model . For ordinary cracked concrete, these ratios did not need to be calculated since that service life prediction was simply done by assuming all model input for the uncracked concrete in combination with a reduced concrete cover of only 25 mm to account for the presence of the crack.For a service life prediction of self-healing concrete, input of the surface chloride concentration and diffusion coefficient is required. However, the values of Dtest see . These v0_UNCR, Cs_UNCR, and D0_UNCR to C0_PU_HV/LV, Cs_PU_HV/LV, and D0_PU_HV/LV per exposure time  and 94 years (PU_LV) resulting from the accelerated diffusion tests with a 49 d exposure period is still a lot more than the seven years that was calculated for the cracked concrete without presence of an embedded healing mechanism . The samtime see  was too With a design service life set at 100 years, steel reinforced concrete elements, like for instance the slab that was considered in this research, would need to be repaired only once in course of the predefined timespan when using either one of the two studied self-healing concrete types . When usWhen assessing the ten CML-IA baseline impact indicators for the considered functional unit , a high sustainability potential seems to be present for the self-healing concrete with encapsulated PU precursor in comparison with ordinary (cracked) concrete . ADP  was 18 times higher due to the presence of a crack at 49 days of exposure and 10 times higher at 133 days of exposure.In the case of concrete where the crack was healed by encapsulated polyurethane, the accelerated chloride diffusion tests showed that the hardened polyurethane inside the cracks formed a partial barrier against immediate ingress of chlorides through the cracks. Perfect healing of the crack, so that the concrete would behave as uncracked, was never possible. However, from a depth of 6 mm onwards, the self-healing efficiency for both types of polyurethane at the two tested exposure times was always 75% or higher. This reduction of chloride concentration in the zone around the crack will have important benefits for the durability of reinforced concrete since a much lower amount of chlorides will reach the steel reinforcement through the cracks.The autonomous healing technique used for this research was manual and of course it should still be extended to a real healing technique for construction practice. In previous research, it was already proven that the proposed healing mechanism works to heal realistic cracks ,34. For Probabilistic service life prediction to estimate the time to chloride-induced steel depassivation showed that steel-reinforced self-healing concrete slabs containing one layer of 4150 PU-filled capsules in the tensile zone would only require repair works  after 60–94 years. For ordinary cracked concrete slabs, repair would already be needed after seven years. In other words, a substantial service life extension seems indeed possible by embedding encapsulated PU precursor as autonomous healing mechanism in concrete. Based on the currently available prediction outcome, no preference could be formulated for the high or low viscosity PU precursor as encapsulated healing agent. It should also be noted that the now obtained service life information still needs to be verified and fine-tuned with more accurate numerical modeling of the chloride ingress in (un)cracked and self-healing concrete. This specific aspect is at this moment still under investigation and will be dealt with in a next stage of the ISHECO project.Life cycle assessment calculations in SimaPro 8 for the same steel reinforced concrete slabs with inclusion of the required number of repairs with a 100-year lifespan—the functional unit of this LCA—mark substantial reductions in environmental impact for all ten CML-IA baseline impact indicators (56%–75%) when self-healing concrete would be used instead of ordinary (cracked) concrete. The difference in repair frequency (1 vs. 14) mainly explains this. The overall impact of the presence of encapsulated PU precursor turned out to be negligible.


The paper presents the results of tests on the effect of the low calcium fly ash (LCFA) addition, in the amounts of: 0% (LCFA-00), 20% (LCFA-20) and 30% (LCFA-30) by weight of cement, on fracture processes in structural concretes. In the course of the experiments, compressive strength of concrete and fracture toughness for: I (tensile), II (in-plane shear) and III (anti-plane shear) models of cracking were measured. The tests determined the effect of age of concretes modified with LCFA on the analyzed parameters. The experiments were carried out after: 3, 7, 28, 90, 180 and 365 days of curing. Fracture toughness of concretes was determined in terms of the critical stress intensity factors:  The world’s current Portland cement production contributes about 1.35 billion tons of carbon dioxide CO2 to the greenhouse gases in the earth’s atmosphere [The principles of sustained development and ecology are essential in the design of concrete compositions. However, it is well known that the basic binder in the composition of plain concrete, i.e., Ordinary Portland Cement (OPC), is not an environmentally friendly material. Cement manufacturing is one of the leading energy consuming and heavy pollutant processes which is accountable for CO2 released during cement clinkering is around 0.7 to 0.9 tons per ton of OPC, meaning that the cement industry generates around 7% of total CO2 emissions worldwide [2 is China [2/tons of cement and 1.1 tons CO2/tons of clinker, while in 2008, respectively 0.631 tons CO2/tons of cement and 0.865 tons CO2/tons of clinker [It is estimated that the COis China . Neverthis China . As far  clinker .2 emission as well as the usage of waste materials from other industries. The structures made of sustainable concrete are environmentally sustainable and are constructed in such a way that the total impact on the environment during their full life cycle, including service life, is reduced to a minimum. In this context, a responsible, sustainable construction should be energy efficient and made of environmentally friendly materials. Concrete used in such structures must meet the requirements for strength and durability and its components should be obtained, produced, and used in an environmentally friendly manner [Therefore, concrete as the most popular building material has been included in the sustainable building program. It is connected with the fact that for many years the cement industry has been trying to follow the sustainable development strategy, whose main purpose is the reduction of environmental pollutions, primarily the reduction of COy manner ,7,8,9,10y manner  health, y manner . Moreovey manner  the needy manner  along wiIn this regard, for over several dozen years, the development of a new generation of concrete composites, i.e., environmentally friendly concrete composites, was followed by a significant increase of the production of concrete mixtures containing supplementary cementitious materials (SCMs), of different classes and types , often hUndoubtedly, one of the most commonly used mineral additives in the form of FA, is LCFA which is a porous waste by-product from coal combustion in thermal power plants. LCFA is formed by a multi-stage pyrolysis of hard coal, thoroughly described in .As LCFA is a by-product obtained in the process of hard coal combustion, there are hundreds of millions tons of industrial waste produced annually across the World. LCFA is an abundant commodity in many countries using steam coal for power production. In some countries all the LCFA is utilized whereas in some others only a low percentage is used. LCFA utilization amounts to 39% in the USA and 47% in Europe, whereas the global average is estimated to be close to 25%, which is approximately 200 million tons per annum [Nowadays, there is much research into the ability of LCFA to enhance: Construction, mining and terrain management. LCFA can be utilized in products such as: Building materials, zeolite synthesis, wood substitute products, soil stabilization, road base/embankments and ground consolidation, land reclamation, and soil amendments in agricultural applications, e.g., . A comprThe issues connected with the substitution of cement binders with LCFA are important not only from the scientific point of view, but also in terms of applicability, from the economic point of view. Although, the governments of many countries had a long discussion on the perspectives and strategies of the implementation of diversified energy systems, the prognosis concerning the energy production from coal as the main source stays stable. On the basis of , coal usConcrete composites with the addition of LCFA can certainly be included in the sustainable and environmentally friendly concrete category. Nowadays, in the cement and concrete industry, LCFA is used for production both in plain concretes as well as in novel composites, e.g., as an addition to nano concrete . As the The overall objective of the conducted scientific analyses was a description of fracture processes in concretes with the LCFA additive. Concretes with these additives have been used in the industry for approximately 80 years, and in the past two decades their use in the composites with cement matrix has significantly increased. Therefore, it is obvious that in the course of such a long period of time the effect of the LCFA additive on the basic physical and mechanical characteristics of concrete could be thoroughly explored. However, a closer look at the literature devoted to this area of science suffices to reveal a scarcity of complete data related to the analysis of initiation and propagation of microcracks and cracks developing in result of the impact of complex stress states on concrete.2 and Al2O3) calcium hydroxide. These reactions lead to the increase of C-S-H phase in concrete, which has a significant effect on the properties of concrete. The introduction of LCFA to the composition of cement causes changes in the phase composition and the microstructure of cement paste which is important for the development of mechanical parameters of concrete. Typically, composites with greater diversification of phases are characterized by higher sensitivity to the formation of local stress concentrations, which may imply the presence of damages and microracks in these locations. Such defects occur mainly in the areas of adjoining structures; under the influence of increasing external loads, the development, accumulation and, consequently, the premature material destruction occurs.LCFA is a pozzolanic material that demonstrates binding capacity through active LCFA ingredients  structural stresses, amongst others . Full knIn order to carry out an in-depth analysis of cracking processes occurring in concrete structures, i.e., crack initiation and its growth, the engineers, most frequently, use non-destructive methods, e.g., . NumericThe reduction of concrete strength results from the initial structural defects existing inside material , mainly Seeing high degree of heterogeneity of concrete composites one can state that, even in the case of the ideal external load, local interactions between the grains and pores cause the occurrence of all three models of cracking.mode I, opening or tensile mode,mode II, sliding or in-plane shear (pure shear) mode,mode III, tearing or anti-plane shear mode.On the basis of the above results, it can be concluded that a thorough analysis of crack initiation and propagation requires the knowledge of all fracture mechanics parameters for modes: I, II and III, i.e., c, e.g., ,42,43,44The tests of fracture toughness in complex stress states are associated with the description of the present critical state in the element, which can be written as the following Relationship (1):With the known values of critical stress intensity factors: tion (2) . The reaUnfortunately, up till now, the question of LCFA influence on fracture toughness of concrete composites was not investigated thoroughly. In the assessment of fracture toughness of concretes containing LCFA the available results of experiments concern mainly the first mode fracture and seldom the second one. Fracture toughness under mode I—articles ,48,49,50s [KIIIc ,52,53 anIIIc [Wf . The comIIIc [Wf , devotedhe paper , assesseAn additional problem is the fact that all previous papers usually refer to the study of the fracture toughness of concrete containing significantly different amounts of LCFA (from 20 to 55%) regularly after 28  days of their curing.This paper provides new testing methods and novel experimental results concerning generalized fracture toughness in the investigated concretes. The studies present original research techniques, e.g., in order to determine the fracture toughness In particular, the paper contains a comparative analysis of the generalized fracture toughness f curing . Due to f curing . HoweverSo far, the analysis of the effect of curing time in low- and high volume LCFA concrete aimed at the evaluation of important properties, e.g., Mechanical properties, corrosion resistance, water absorption, water permeability, and frost resistance or microstructure and crack propagation. A list of papers describing these studies are summarized in In the present study—having in mind the reason of working on this subject—we examine changes in concrete compressive strength, All experiments were planned for two compositions of concrete mixture, with varying percentage of LCFA additive often used in the cement industry. The choice of the target range was determined by the fact that in the tests  and 30% (LCFA-30) of the weight of cement. The results of the experiments were compared to the values obtained for the reference concrete (LCFA-00), which was a composite made with the use of cement CEM I. For each of the three composites, all experimental tests were conducted in six time periods, with the age of concrete: 3, 7, 28, 90, 180 and 365 days.analysis of physicochemical properties of LCFA,evaluation of pozzolanic activity of LCFA.In the range of LCFA testing, the scientific objectives were:experimental determination of the impact of LCFA additive on the critical stress intensity factor of concrete under: I, II and III mode fracture, between the third and 365th day of curing,calculation of particular concretes with generalized fracture toughness In the range of fracture toughness, the scientific objectives were:An advanced diagnostic equipment was used during the experiments. For the evaluation of fracture toughness, three separate test stands were organized see . On the The concrete mixtures were prepared at the laboratory of the Faculty of Civil Engineering and Architecture, Lublin University of Technology . The following materials were used: OPC, LCFA, sand, gravel, plasticizer and water.3, respectively. The fineness specific surface of OPC and LCFA used was 328 and 364 m2/kg, respectively. The volume change of OPC used was 0.56 mm with an initial and final setting time of 207 and 298 min, respectively.In this research, both OPC and LCFA were used as a binder, OPC having the compressive strength equal to 23.3 MPa on the second day and 50.0 MPa after 28 days of curing. The LCFA used, in different percentage, to replace cement is a product of energetic combustion of hard coal in the local thermal-electric power station. Bulk density of both materials was determined by pycnometric method, whereas the specific surface area by Blaine method. The specific density of OPC and LCFA used was 3.11 and 2.14 kg/mThe characteristics of typical grains, microstructures, and the dominant morphologies of OPC and LCFA particles can be found in ,49,51, w3S—60.69, C2S—15.82, C3A—9.24, C4AF—7.28, CaSO4 (gypsum)—5.10. X-ray diffraction (XRD) analysis allowed for the identification of the crystalline phases of LCFA [2), mullite (Al6Si2O13), magnetite (Fe3O4), and hematite (Fe2O3) [The mineralogical composition of OPC was analyzed by the Bogue method. These studies showed that, in OPC tested, there are (in %) the following phases: C of LCFA  and show (Fe2O3) .2 + Al2O3 + Fe2O3, 0.65% of SO3 and 3.2% of Loss of Ignition (LOI), meeting the requirement of ASTM C618 standard.We evaluated the chemical composition of OPC and of LCFA by X-ray fluorescence (XRF). Three aspects of natural radioactivity of LCFA, and of concrete containing it, were considered: Concentration of radioactive elements , radioactivity coefficients ion dose . The stuion dose .w/b ratio 0.4.Concrete mixes were designed using natural pit sand 0–2 mm a, natura3 and the dosing range of 0.1–1.0% of mass of cement. The plasticizer is used in an amount of 0.6% of mass of the binder.A calcium lignosulfonate-based plasticizer used in this study had density of 1.16 g/cmThe composition of prepared concrete mixes is shown in 6 cubic specimens (150 mm) for testing the compressive strength—6 beams (80 × 150 × 700 mm) with one initial crack for testing fracture toughness under mode I fracture—6 cubic specimens (150 mm) with two initial cracks for testing fracture toughness under mode II fracture—6 cylindrical specimens with a diameter of 150 mm and a height of 300 mm having an initial circumferential notch, for testing fracture toughness under mode III fracture—Assortment of specimens for compressive strength tests and fracture toughness tests of concrete, for each of the mixtures in all time periods, was as follows:3 prisms of the mortar. The water to solid ratio in mortars is 0.5. According to the EN 450-1 standard, the acceptable level of pozzolanic activity is attained when the 28 day compressive strength of the LCFA and cement mortar constitutes 75% of the value for the control sample and after 90 days, 85% respectively.The applicability of the LCFA depends mainly upon its pozzolanic activity. To assess the pozzolanic activity of the used LCFA we selected a physical method, according to the EN 450-1 standard. EN 450-1 defines as a standard pozzolanic activity index of the LCFA the Strength Activity Index which is the ratio (in %) of the compressive strength of mortar containing 75 wt % of cement CEM I 42.5R, 25 wt % of the LCFA and cement mortar without addition. According to EN 450-1 standard the pozzolanic activity index of the LCFA is determined after 28 and 90 days of hydration. The compressive strength of cement is measured according to the procedure described in EN 196-1 standard, using six 40 × 40 × 160 mmDue to that compressive strength tests and fracture toughness tests were conducted in 6 time periods between 3 and 365 days, pozzolanic activity index was also determined at the same time intervals, i.e., after: 3, 7, 28, 90, 180 and 365 days.We performed compressive tests  for uniaxial compression strengths with maximum load of 3000 kN while controlling the loading rate between 0.5 MPa/s and 0.8 MPa/s. The compressive strengths were tested with application of cubic specimens see  accordind = 150 mm, b = 80 mm, L = 700 mm, S = 600 mm, a0 = 50 mm —Crack mouth opening displacement (CMOD),Load (f).Load—Displacement  :(3)KIcS=Tests method to evaluate concrete fracture toughness used see . The tarused see a and speused see b.F)—Time (t),Load (f),Load—Displacement , proposed by Watkins [B, b, a—according to The tests were performed with the use of the MTS 810 hydraulic press a, like i Watkins .(4)KIIcof curve ; B, b, aFor tests being in accordance with III model of cracking (anti-plane shear), a special innovative device was designed and manufactured . This deThe device for the fracture toughness test at the mode III fracture consisted of the: cylindrical specimens with a diameter of 150 mm and a height of 300 mm having an initial circumferential notch of 2 mm thickness b, steel To mount the specimens in the grips of the torsional testing machine, two types of circular steel plates with drilled holes were designed and manufactured. During the forming process, the bottom plates with 15 mm thickness were anchored in the specimens at their top and the bottom through 6 bolts M12/65. Then, top plates with 10 mm thickness were bolted to these plates through 6 bolts M12/20. These plates hold the specimen directly in the grips of the torsion testing machine from the top and bottom through M28/70 bolts. More details of the device preparation were described in , whereast),Angle of rotation (T)—Time,Torque  .r, radius of the specimen KIIIng to Tc ; r, radispecimen ; ωc, cring to Tc .With the known values of Strength Activity Index  indicateAs a result of the study it can be concluded that the pozzolanic activity of the LCFA meets the requirements of EN 450-1 standard. Strength Activity Indices were at 28 days, 92.13%, and after 90 days, 111.84%, which means that they exceed significantly the minimum values specified in the requirements of the EN-450-1 standard.The compressive strength data  for: 3, 7, 28, 90, 180 and 365 days of age of sustainable concrete are plotted in The graph of relative changes After comparison the relative changes of generalized fracture toughness in time , one canAccording to the results, the LCFA used in the study is characterized by high pozzolanic activity . The resThe analysis of fracture processes in these concretes was primarily based on the results of the macroscopic fracture toughness, designated for three modes. This allowed for the estimation of generalized fracture toughness The parameter values of fracture mechanics depend on the age of concretes during the conducted tests. It was observed that the LCFA additive in the amount of 30% of mass of cement reduces the the LCFA , after aWhen comparing the effect of LCFA on the generalized fracture toughness , and comIt is possible to make sustainable concrete with the addition of low calcium fly ash with high compressive strength and generalized fracture toughness.The low calcium fly ash additive in the amount of 20 and 30% of mass of cement significantly affects the change of generalized fracture toughness in tension, shear and torsion.The values of the critical stress intensity factor The low calcium fly ash additive in the amount of up to 30% of mass of cement drastically reduced the compressive strength and the generalized fracture toughness at an early age.20% low calcium fly ash additive ensures high compressive strength and generalized fracture toughness in matured concretes.Concretes with 30% low calcium fly ash additive are characterized by the highest dynamic increase of the parameters After 180 and 365 days, generalized fracture toughness of LCFA-30 concrete is higher in comparison to the values obtained for reference concrete.Results of the generalized fracture toughness and the compressive strength are convergent qualitatively.Effect of curing time on the compressive strength and the generalized fracture toughness of concretes with low calcium fly ash additive is directly related to the pozzolanic activity of this ashes.On the basis of the comprehensive fracture toughness tests, in which a portion of the binder was replaced with active pozzolanic low calcium fly ash, it can be concluded that:Sustainable concretes with the low calcium fly ash additive are used in building structures, both residential and industrial. Range of composites, made on the basis of the binders modified with this waste is very rich. Such concretes are used in the construction industry: Concrete, reinforced concrete and prestressed; in structures: monolithic, prefabricated, composite and prestressed.In the case of concretes, which are scheduled for commissioning after 28 days, or later, the preferred solution is to use composites modified with the 20% LCFA additive.The use of concretes with 30% LCFA additive in typical structures is possible, however, it should be taken into account that their strength and fracture toughness can be reduced for up to six months, compared to plain concretes.It is not advisable to construct concrete structures, with the low calcium fly ash additive, if their commissioning is planned before the 28th day after the placing of concrete mixture.It is not recommended to use composites with the low calcium fly ash additive in the prefabricated elements, in which the interoperational transport strength would be less than a week.In any case, it is not advisable to use concretes with 20% and 30% low calcium fly ash additive, if they are subjected to any load in less than three days from the preparation of concrete mixture.On the basis of the obtained results, several practical tips and recommendations on the use of sustainable concretes, with the low calcium fly ash, in concrete industry can be suggested:


Gallbladder cancer (GBC) is a malignant cancer with very poor prognosis. Although promyelocytic leukemia zinc-finger protein (PLZF) was reported to be deregulated in numerous cancers and also relevant to clinical prognosis, its role in GBC progression has been little known. In this study, we found PLZF expression was decreased in GBC, correlating to advanced TNM stage, distant metastasis, and shorter overall survival. Moreover, ectopic PLZF expression in GBC cells (NOZ and GBC-SD) significantly reduced the cell proliferation, migration, and invasion. Consistently, overexpression of PLZF in xenograft mice model could suppress tumor growth and liver metastasis. Mechanical investigations verified PLZF could regulate the expression of cell cycle arrest-associated gene p21 and epithelial–mesenchymal transition (EMT)-related genes (E-cadherin and N-cadherin) in GBC cell lines. Importantly, PLZF remarkably increased the mRNA transcription of interferon-induced protein with tetratricopeptide repeat 2 (IFIT2) by increasing STAT1 protein level, a known factor involved in tumor progression. Furthermore, ablation of IFIT2 in PLZF overexpression cells abrogated the tumor-suppressive function of PLZF, at least partially, leading to impaired tumor growth and EMT program. These studies indicated PLZF inhibited the proliferation and metastasis via regulation of IFIT2. In conclusion, our study demonstrated PLZF could be a promising tumor biomarker for GBC, and also be a potential therapeutic target. Due to easier local infiltration and distant metastasis, GBC has an extremely poor prognosis with the median survival of 9.2 months and the 5–year survival rate of 5%4. Given the lack of specific tumor marker and effective therapeutic targets for GBC, novel insight into the GBC progression contributes to the identification of potential targets for early diagnosis and therapy.Gallbladder cancer (GBC) is a highly lethal and the most common biliary tract cancer, ranking the sixth leading cause of cancer-related death of digestive system6. PLZF is involved in diverse cellular processes, particularly in stem cells self-renewal or differentiation, and immune cells development7. Nevertheless, the role of PLZF appeared to be controversial in tumor progression. Several studies showed PLZF was able to reduce cell growth and survival in numerous cancers including melanoma, malignant mesothelioma, prostate cancer, and non-small cell lung cancer cells via c-myc suppression or poly ADP-ribose polymerase (PARP) and Mcl-1 expression increase13. Importantly, low expression of cytoplasmic PLZF strongly correlated with high tumor grade, lymph node metastasis and indicated a short overall survival (OS) time in non-small cell lung cancer14. Meanwhile, Hur et al. claimed a tumor-promoting effect of PLZF by repressing the p53 pathway15. In thyroid carcinoma, high cytoplasmic expression of PLZF was found to be involved in capsular invasion and lymph node metastasis16. However, the role of PLZF in GBC has remained to be elucidated.One protein of Kruppel-like zinc-finger proteins family, promyelocytic leukemia zinc-finger protein (PLZF), also known as ZBTB16, was first discovered in acute promyelocytic leukemia as a fusion protein with the retinoic acid receptor α17. It could constitute complexes with itself or with two other related human ISGs, IFIT1 and IFIT3. In addition, IFIT2 has been considered as a tumor suppressor in many tumors to promote cellular apoptosis, suppress tumor proliferation, and metastasis18.Interferon-induced protein with tetratricopeptide repeat 2 (IFIT2) is a member of IFN-stimulated genes (ISGs), which are induced after the treatment of type I or III IFNs and in vivo that PLZF served as a potent tumor suppressor for decreased tumor growth and liver metastasis of GBC. Moreover, IFIT2 expression has been markedly increased following PLZF overexpression, and was demonstrated to be required for the tumor inhibition of PLZF in GBC cells. Therefore, our study demonstrated PLZF reduced GBC progression by IFIT2-dependent p21 increase and suppression of tumor epithelial–mesenchymal transition (EMT). Also, we found PLZF promoted the transcription of IFIT2 by increasing STAT1 protein level. Hence, our study provided a valuable biomarker for prognosis and a potential therapeutic target for GBC.In the present study, we provided evidences in vitroFormalin-fixed, paraffin-embedded (FFPE) tumor samples with histologically confirmed GBC were obtained from 80 patients who had GBC surgical resection and postoperative adjuvant chemotherapy at the Department of Pathology  from January 2004 to February 2015. Twenty FFPE gallbladder samples were obtained from gallbladder stone patients. Matched fresh primary GBC samples and relevant non-tumorous tissues were obtained from 15 patients among the 80 GBC patients. All the fresh specimens were routinely snap-frozen in liquid nitrogen. The paraffin-embedded samples for immunohistochemistry (IHC) were evaluated by two certified pathologists at the Department of Pathology. Follow-up data and medical records were collected from the hospital electronic medical records. And the acquisition of patient specimens and medical data got the permission from Ethical Committee of Renji Hospital, Shang Hai Jiao Tong University School of Medicine and the patients or their relatives.The human embryonic kidney 293T cells and human GBC cell line GBC-SD were purchased from the Chinese Academy of Life Sciences . Another GBC cell line NOZ was kindly gifted by Xinhua hospital .2.GBC-SD and 293T cells were maintained in RPMI-1640 medium . NOZ cells were maintained in Willian’s E medium. All the cells were maintained in the medium supplemented with 1% antibiotics and 10% fetal bovine serum (GIBCO) at 37° C with 5% COHuman PLZF expression plasmid was constructed by insertion of the coding sequence (CDS) of PLZF into pCDH-CMV plasmid . STAT1 plasmid was kindly gifted by Hao Jia from Department of Biochemistry and Molecular Cell Biology, Shanghai Jiaotong University School of Medicine. The PLZF short hairpin RNA (shRNA) was constructed by Shanghai GenePharma Medical Biotechnology Company. The IFIT2 shRNA was constructed by Shanghai GenePharma Medical Biotechnology Company. PCDH-flag-PLZF sense: 5′-TGCTCTAGAGCAATGGATTACAAGGATGACGACGATAAGGATCTGACAAAAATGGGCAT GATC-3′; PCDH-flag-PLZF antisense: 5′-CCGGAATTCCGGTCACACATAGCACAGGTAGAGGTACG-3′. The sense sequence of IFIT2 shRNA was: 5′-CCAAATCCTTCATGTAATA-3′. The sequence of the IFIT2 shRNA was the following: 5′-TGCTGTTGACAGTGAGCGCGCCAAATCCTTCATGTAATATTAGTGAAGCCACAGATGTAATATTACATGAAGGATTTGGCTTGCCTACTG CCTCGGA-3′. Recombinant lentiviruses were produced using HEK-293T cells and the viruses were harvested and used to infect human GBC cells with 4 μg/ml polybrene (Sigma). Then, the stable expression cells were selected by 2.5 μg/ml puromycin. Mock were these empty vector stably transfected cells and NC were these negative control plasmid stably transfected cells.Total RNA was isolated from cell lines using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Complementary DNAs were synthesized using Reverse Transcriptase kit. The gene expression levels were detected by using SYBR Premix Ex Taq  and performed real-time PCR on the ABI Prism 7500 system . Data were normalized to internal control β-actin.The primers were PLZF-forward: 5′-TGCGGCTGAGAATGCATTA-3′; PLZF-reverse: 5′-ACACAGCAGACAGAAGACGG-3′. IFIT2-forward: 5′-CTCAAAGGGCAAAACGAGGC-3′; IFIT2-reverse: 5′-CCAGGCATAGTTTCCCCAGG-3′. β-Actin-forward: 5′-GGACTTCGAGCAAGAGATGG-3′; β-actin-reverse: 5′-AGCACTGTGTTGGCGTACAG-3′. All the primers above were provided by Biosune Biotech. The experiments were done in triplicates.Plasmids encoding STAT1 and PLZF-Flag proteins were transiently transfected into HEK-293T cells, and 48 h after transfection, cells were lysed in IP lysis buffer with protease inhibitor cocktail and phenylmethanesulfonyl fluoride (PMSF). Co-IP assays were performed with Flag beeds . For normal western blot, total protein was extracted from GBC cells using RIPA lysis buffer supplemented with 1% PMSF and proteinase inhibitor cocktail. Bicinchoninic acid (BCA) assay was used to measure the protein concentration. Equal amounts of protein were loaded on a 8% sodium salt -polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to NC membranes . Then, the blots were blocked in 5% skimmed milk with 0.1% Tween 20 for 1 h at room temperature followed by incubating at 4° C overnight with primary antibodies. The membranes were washed with tris-buffered saline and tween (TBST) and then incubated with secondary antibody at room temperature for 2 h. The blots were detected by ECL chemiluminescence kit (Millipore). The PLZF antibody was obtained from Santa Cruz Biotechnology . β-Actin antibody from Abclonal Biotech was used as loading control. Other antibodies were purchased from Proteintech group .The plasmid containing the IFIT2 promoter region was kindly gifted by Hao Jia from Department of Biochemistry and Molecular Cell Biology, Shanghai Jiaotong University School of Medicine. The IFIT2 promoter and PLZF plasmid were co-transfected into NOZ cells. After 24-h transfection, cells were harvested and the luciferase activities were measured by the Dual-Luciferase Reporter Assay System (Promega). The values of luciferase assay were expressed after normalization to Renilla luciferase activity. Each transfection was carried out in triplicates.4 PLZF overexpression cells were seeded to 24-well plate covered with sterile coverslips. The cells were incubated for another 12 h. Then, the primary antibody and matched secondary antibody were adopted to stain PLZF and IFIT2. In all, 4, 6-diamidino-2-phenylindole (DAPI) was used to stain cell nuclei. All assays were performed in triplicates.We also performed immunofluorescence (IF) staining to detect the function of PLZF on IFIT2. In all, 2 × 102. After 2-week growth, the cells were fixed and stained with Coomassie Brilliant Blue for 30 min. The number of clones was counted and all the assays were repeated three times.Cell Counting Kit-8 (CCK-8) and plate colony formation assays were performed to detect the cell proliferation capacity. For CCK-8 assay, 2000 cells with 100 μl medium were seeded into each well of 96-well plates. Considering of the evaporation, periphery wells of 96-well plates did not seed cells. After every 24 h, 10 μl CCK-8 was added to the medium for 1 h at 37° C and then we measured the absorbance of the plates at 450 nm. For the plate colony formation assay, 600 cells with 2.5 ml medium were seeded into each well of six-well plates and then maintained at 37° C with 5% CO4 cells with 100 μl serum-free medium were seeded into the upper chamber. In the lower chamber, 700 μl medium with 10% fetal bovine serum was added. After 16 h, the migrated cells were fixed with 4% paraformaldehyde and stained with Coomassie Brilliant Blue. The cells were stained with Coomassie Brilliant Blue. Then, the cells were counted in six random fields of each chamber. For invasion assay, 8 × 104 cells were seeded into the upper chamber with Matrigel (BD)-coated membrane for 48 h. All experiments were carried out in triplicates.Transwell assay was used to assess the migration and invasion abilities of the GBC cells. Twenty-four-well transwell chambers with 8 μm pore size polycarbonate membrane  were used in this assay. For migration assay, 4 × 10Specimens fixed in 10 % buffered formalin were made into paraffin-embedded sections. Sections were deparaffinized and rehydrated by dimethylbenzene and ethanol. The antigen activity of specimens was retrieved by 0.01 M sodium citrate buffer (pH 6.0). Later, the sections were kept in 3% hydrogen peroxide for 20 min to erase endogenous peroxidase. Then, the sides were blocked with goat serum and incubated with primary and secondary antibody. 3, 3'-diaminobenzidine (DAB) system was applied in the positive staining. The final results were assessed independently by two pathologists who knew nothing about the group design. The scoring of the staining was based on the intensity and proportion of the positive staining. The staining intensity was classified into four groups ; and for the staining area .6 NOZ cells were injected subcutaneously into the right lower regions of 4-week-old male nude mice. Tumor length (L) and width (W) were measured every 3 days with vernier caliper. The tumor volume (V) was calculated by the following formula: V = L × W2 × 0.519. After 5 weeks, all the mice were sacrificed and the subcutaneous xenografts were excised. To further verify the metastatic capability of PLZF in vivo, 100 μl PBS containing 5 × 106 NOZ cells were injected subcutaneously into the nude mice. After 7 weeks from the injection, the mice were sacrificed and the livers were excised and made into sections for hematoxylin and eosin (H&E) staining and IHC staining. All the animal experiments were approved by the Institutional Review Board of the Renji Hospital of Shanghai Jiao Tong University.To investigate the proliferation effect of PLZF in vivo, 100 μl phosphate-buffered saline (PBS) containing 1 × 10t-test. Clinicopathological data were analyzed using Pearson’s Χ2 test. Kaplan–Meier method and log-rank test were performed to analyze the survival probabilities. Univariate and multivariate analysis were analyzed using Cox proportional hazard regression model. All of the statistical analyses were performed with SPSS 19.0 software. P-values <0.05 were considered statistically significant.All data were presented as the mean ± s.d. The variance of each experimental group was determined by an unpaired two-tailed Student’s P = 0.012) and distant metastasis (P = 0.008).In 15 pairs of GBC tissue samples, the mRNA expression levels of PLZF were detected to be lower in GBC tissues compared with that in adjacent non-neoplastic tissues Fig. . Then, wP = 0.005). Moreover, tumor size , TNM stage , lymph node metastasis , and distant metastasis  were also risk factors for OS. Besides, multivariate analysis using Cox model, including tumor size, TNM stage, lymph node metastasis, distant metastasis, and PLZF expression showed that only PLZF was an independent prognostic factor for OS in GBC patients  to detect the expression changes of genes correlating with cell proliferation and apoptosis, and found p21 was significantly upregulated both in overexpressing PLZF NOZ and GBC-SD cells  transfection in control group and PLZF-overexpressing NOZ cells, followed by examining GBC cell proliferation and migration. Figures Given that we demonstrated the tumor repressive role of PLZF in vitro, we next determined whether PLZF suppressed GBC cell growth and metastasis in vivo. GBC cells with stable expression of PLZF or control vector were subcutaneously implanted into nude mice. The tumor volumes were measured every 3 days. After 5 weeks, the mice were sacrificed and xenograft tumors were removed and analyzed. As shown in Fig. 21. But the mechanism of tumor proliferation and metastasis is still poorly understood. Cumulative evidences have shown PLZF was downregulated in numerous cancers and relevant with the clinical prognosis. Moreover, the functions of PLZF in cancers remained to be ambiguous and open to interpretations of either a tumor suppressor or tumor promoter as reported previously7. However, little is known about the role of PLZF in GBC progression. In this study, the mRNA transcripts of PLZF were significantly downregulated in GBC tissues in contrast to the adjacent counterparts. Consistently, the protein levels of PLZF were reduced in many GBC tissues. Moreover, the decreased PLZF was demonstrated to be associated with tumor size, TNM stage, lymph node metastasis, and distant metastasis. Multivariate analysis using Cox’s regression suggested that PLZF was an independent prognostic factor for OS in GBC patients. In agreement with its role in clinical samples, PLZF was further found to suppress GBC cell growth and metastatic activity in vitro, supporting PLZF being a potential tumor suppressor in GBC.GBC is a malignant cancer with very poor prognosis because of its high potential to metastasize in a short time22. Lots of studies have shown p21 has low expression in GBC tissues and absence of p21 expression independently predicts poor outcome24. Moreover, silence of p21 has been proved to promote GBC cells (NOZ and GBC-SD) proliferation significantly25. It is involved in cell proliferation inhibition in both p53-dependent and -independent way26. A previous study showed PLZF could interact with some corepressors and bind to p21 promoter to repress its transcription15. But our results showed opposite phonotypes. This could be due to the fact that the cells we used were different from those studied or the regulation of PLZF might be context dependent.In this study, we found PLZF overexpression could suppress cell proliferation and tumor growth. Furthermore, we investigated the genes involved in tumor growth and found PLZF could increase p21 mRNA level, but no impact on the apoptotic genes like p53, BCL-2, BCL-XL, and BAX. p21 is a well-studied gene required for cell cycle arrest. Reduced p21 expression was considered as an early event in gallbladder carcinogenesis30. EMT gets cells to obtain strong capability of motility and spreading, leading to metastasis to the distant regions32. The underlying molecular mechanism of PLZF in cancer metastasis remained little understood. Only one study in melanoma demonstrated PLZF suppressed migration and invasion partially through integrin β3 and MMP99. We examined the expression levels of EMT markers for possible mechanism investigations, which showed PLZF overexpression decreased N-cadherin expression but enhanced E-cadherin level, whereas vimentin and fibronectin were not altered, suggesting PLZF could selectively induce mesenchymal markers for initiating EMT program.EMT is regarded as a potent driver conferring cells with metastatic features, including lower expression of epithelial markers like E-cadherin and higher expression of mesenchymal markers like N-cadherin, vimentin, and Fibronectin35. Growing studies have verified ISGs are involved in multiple processes including apoptosis, angiogenesis, and metastasis37. To validate this prediction, qRT-PCR analysis was used and showed that PLZF overexpression enhanced the expression of a subset of ISGs included IFIT2 in particular. This observation was also confirmed by WB and IF analysis, indicating PLZF could regulate IFIT2 mRNA transcription. Dual-luciferase reporter assays were then performed to further verify PLZF effect on IFIT2 expression regulation. The results showed PLZF increased IFIT2 in a dose-dependent manner. IHC staining of PLZF and IFIT2 in GBC tissues also confirmed a strong correlation. Accumulating evidences have established IFIT2 as a tumor suppressor in various tumor types. IFIT2 promotes apoptosis through the intrinsic apoptosis mechanism by regulating antiapoptotic and proapoptotic factors41. Moreover, IFIT2 was reported to suppress the migration and metastasis by regulating atypical protein kinase C, leading to EMT and cell migration42. We therefore verified whether IFIT2 was required for the function of PLZF. We introduced IFIT2 small interfering RNA into PLZF overexpression cells and found the expression of p21 and E-cadherin was decreased, but N-cadherin was increased in PLZF overexpression cells with IFIT2 silencing. Moreover, downregulation IFIT2 expression could rescue the inhibitory effects of proliferation, migration, and invasion in PLZF overexpression cells. IFNs exert their biological effects by activating STATs expression, which translocate into the nuclear and bind ISGs to transactivate their activation43. STAT1 is the central mediator of ISGs’ promotion44. Notably, we showed that ectopic PLZF expression could enhance STAT1 and pSTAT1 after IFNγ treatment, indicating that PLZF may function as a suppressor of STAT1/IFIT2 pathway. Moreover, our results showed PLZF could interact with STAT1 protein and increase its protein level by an unknow mechanism. Previous studies have demonstrated that STAT1 is mainly degraded by ubiquitination and proteasome pathway45. So PLZF might inhibit STAT1 degradation in that way. Collectively, these results suggested PLZF might function concomitantly with IFIT2 for decreased GBC cell proliferation, migration and invasion.To clarify the underlying mechanism of PLZF, we performed chip–chip data analysis using online database and found a link between PLZF and ISGs. Through high-affinity binding with cell surface receptors, IFN molecules can activate the Janus kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway, resulting in induction of thousands of ISGs at the transcript level and in vivo. PLZF might be a potential tumor biomarker and a critical therapeutic target for GBC.Above all, we revealed that PLZF was remarkably reduced in GBC tissues and low PLZF expression was correlated with advanced TNM stage, distant metastasis, and short overall survival. Furthermore, we found PLZF overexpression inhibited tumor growth and metastasis by activating STAT1/IFIT2 pathway in GBC cells and nude mice. To our knowledge, we are the first to identify the tumor-suppressor role of PLZF in GBC both in vitroSupplement 1Supplement 2Supplementary Figure Legends


C-Met is a receptor tyrosine kinase with multiple functions throughout embryonic development, organogenesis and wound healing and is expressed in various epithelia. The ligand of c-Met is Hepatocyte Growth Factor (HGF) which is secreted among others by mesenchymal stroma/stem (MSC) cells.Physiological c-Met functions are centred around processes that underly cellular motility and invasive growth. Aberrant c-Met expression and activity is observed in numerous cancers and makes major contributions to cell malignancy. Importantly, HGF/c-Met signaling is crucial in the context of communication between cancer cells and the the tumor stroma.Here, we review recent findings on roles of dysregulated c-Met in urogenital tumors such as cancers of the urinary bladder, prostate, and ovary. We put emphasis on novel aspects of cancer-associated c-Met expression regulation on both, HGF-dependent and HGF-independent non-canonical mechanisms. Moreover, this review focusses on c-Met-triggered signalling with potential relevance for urogenital oncogenesis, and on strategies to specifically inhibit c-Met activity. It is e1234 and Tyr1235 within the catalytic loops [1003 in the juxtamembrane domain and Tyr1349/Tyr1356 within the C-terminal region , src-homology-3- (SH3-) domain-containing proteins and phospho-tyrosine-binding proteins (PTB) Fig. . These aActivated c-Met in normal cells – predominantly in stromal cells including MSC - plays a major role in organogenesis and wound healing whereas in tumor cells, HGF-activated c-Met contributes to invasive and metastatic behavior , 27. VarIn prostate cancer, it was shown that androgen deprivation is connected with a more aggressive phenotype and leads to an increased c-Met expression. In parallel, the androgen receptor, which is known as a negative regulator of c-Met, is downregulated –39. HencA recent report shows that c-Met upregulation in castration-resistant prostate cancer tissue is associated with a different distribution pattern compared to biopsies from naïve prostate cancer tissue. Interestingly, a c-Met fragment lacking the c-terminal part of full-length membranous c-Met was found to accumulate in the nuclei of tumor cells preferentially in castration-resistant prostate cancer . These rElevated c-Met expression in tumor cells also leads to enhanced c-Met activity within the tumor tissue, since profound HGF expression within the tumor stroma activates c-Met on cancer cells in a paracrine manner. HGF expression was shown to be clearly increased in urogenital cancers such as urinary bladder, prostate and ovarian cancer –45. In pIt is now well established that receptor tyrosine kinases can also act via signaling pathways other than the Ras/Raf cascade. “Canonical signaling” is currently used as a term for the signaling route extending from ligand binding to the cell surface exodomains of receptors resulting in dimerization/oligomerisation and receptor trans-/ autophosphorylation with subsequent intracellular signal release. Recently, “non-canonical signaling” has emerged as a concept to describe the specific generation of defined intracellular tyrosine kinase receptor fragments which have different functions . The forFour cleavage products of c-Met with potential functions in signaling have as yet been described. The extracellular N-terminal fragment (Met-NTF) can be eliminated from the full-length receptor by metalloproteinases such as ADAM-10 and ADAM-17. Subsequent processing of the membrane-proximal C-terminal fragment (Met-CTF) by presenilin-dependent, regulated, intramembrane proteolysis (PS-RIP) results in an intracellular c-Met fragment (Met-ICD) which becomes subject to proteasomal degradation –62. It iBy shedding the Met-NTF, the resulting fragments Met-NTF and-CTF can also be lysosomally degraded. Ancot et al. published a model describing the fate and subcellular location of the receptor-derived breakdown products post processing of the full-length cell surface receptor by PS-RIP and its proteasomal degradation upon removal of Met-NTF. However, if c-Met is membrane-bound and internalized, it can also undergo intracellular proteolytic cleavage. This process gives rise to Met-NTF and Met-CTF, which can subsequently be degraded lysosomally. Thus, fragment Met-CTF escapes processing by PS-RIP when the cleavage takes place intracellularly . Both shExperimental overexpression of a segment comprising only the intracellular segment of c-Met devoid of the extracellular domain in NIH3 Ts fibroblasts showed an elevated oncogenic potential with enhanced proliferation and invasiveness compared to control fibroblasts . Interesc-Met cleavage products are functional in the control of cell death and survival . The recCleavage of c-Met yields the intracellular p40Met fragment, a membrane-bound p100Met fragment and, in addition, a peptide ten amino acid residues in length designated M10 and corresponding to C-terminal end of the receptor protein , 75. TheThe ratio of full-length c-Met and caspase-3 appears to have an influence on the extent of apoptosis in hepatocytes: High abundance of c-Met can suppress caspase-3 activity and, thus, suppress apoptosis. Under conditions of a strong apoptotic stimulus, however, this inhibitory effect of c-Met on caspase-3 is overcome and cell death is no longer blocked .Remarkably, caspase-3 is also active in non-apoptotic melanoma and glioblastoma cells as well as in invasive bladder cancer promoting tumor progression –79. MukaAnalysis of Met-overexpressing tumor samples from patients with non-small cell lung cancer demonstrated the existence of all Met fragments suggesting a variety of cellular conditions within lung tumors . HoweverInterestingly, not only c-Met can contribute to tumor progression in a ligand-independent manner. Tate et al. indicated a role for c-Met ligand HGF in prostate cancer in the absence of its receptor . HGF indThe identification of prominent c-Met expression in certain urogenital tumors suggested that the HGF/SF – c-Met axis may serve as an attractive target to be included into chemotherapeutic regimens. Different approaches have been taken towards molecularly targeting c-Met and HGF such as (i) competitive interference with HGF/c-Met interaction, (ii) inhibition of the tyrosine kinase activity of c-Met, or (iii) blocking of activated c-Met downstream signaling mediators Fig. . AccordiUsing an ovarian cancer mouse model, the orally available c-Met inhibitor PF-2341066 was successfully applied to reduce cell proliferation, adhesion and invasion as well as to induce apoptosis . This smAnother c-Met inhibitor, Crizotinib (PF-02341066), represents a designed tyrosine kinase inhibitor based on the core structure of the c-Met-inhibitor PHA 665752 in which some functional groups of the originating molecule (PHA 665752) were altered. As a result Crizotinib has stronger affinity for the ATP-binding pocket of the kinase domain within the c-Met receptor. Consequently, binding of ATP to the c-Met kinase domain and subsequent tyrosine phosphorylation of the receptor and signal mediators are blocked , 90. Mor50: 0.4 nM) and vascular epithelial growth factor receptor-2 (VEGF-R2/KDR) (IC50: 0.9 nM) [50: 3,6 nM), PDGFRβ (IC50: 9.6 nM), RON (IC50: 3 nM), c-KIT , FLT-3  und TIE-2  and low affinities to FGFR1 (IC50: 660 nM), und EGFR  [The orally administrable tyrosine kinase inhibitor Foretinib  exhibits a certain specificity and high affinity binding for both c-Met (IC 0.9 nM) . This dr 2,9 μM) –95. An aForetinib has also been demonstrated to block MAP kinase signaling and cell cycle progression during in vitro studies, substantiating this drug as a multi-target tyrosine kinase inhibitor . PromisiPotential effects of Foretinib as a multi-target tyrosine kinase inhibitor besides its specificity for c-Met are also focused on inhibition of VEGF-R2 signaling as an important pathway during angiogenesis and subsequent neo-vascularization. Here, Foretinib exhibits properties which primarily target the tumor microenvironment, thereby displaying dual anti-tumorigenic strategies. Promising effects of Foretinib in targeting VEGF-R2 signaling were achieved by an approximately 10-fold reduction in tumor size of tumors of the rare and aggressive small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), following tumor initiation by the two model cell lines SCCOHT-1 and BIN-67, respectively. MSC within the tumor microenvironment were shown to contribute to this outcome , 99. Sup1349. Tumor treatment with DCC-2701 was able to efficiently reduce the tumor burden in vivo by inhibition of c-Met phosphorylation and c-Met-mediated signaling, for cell growth and migration [The tumor microenvironment plays an pivotal role for therapeutic approaches –105. Theigration .In summary, the abundance of c-Met expression and intracellular signaling in urogenital cancers may provide a selective molecular target for tumor therapeutic interventions. Moreover, multi-target c-Met tyrosine kinase inhibitors with the property to simultaneously affect cancer cells and the tumor microenvironment represent efficient and focused drugs to target urogenital cancers. In this context, it is important to note that cancer growth and metastasis of c-Met-positive urogenital cancers involves further stimuli besides HGF-binding and subsequent c-Met signaling since no anti-tumor effect was detectable in an ovarian cancer clinical trial after using a humanized IgG2 antibody directed against HGF (AMG-102), even though AMG-102 prevents HGF binding to c-Met and subsequent c-Met activation . It is l