Calcium carbide residue (CCR) is the end-product of production of acetylene gas for the applications such as welding, lighting, ripening of fruits, and cutting of metals. Due to its high pH value, disposing of CCR as a landfill increases the alkalinity of the environment. Therefore, due to its high calcium content, CCR is mostly blended with other pozzolanic materials, together with activators as binders in the cement matrix. In this study, cement was partially substituted using CCR at 0%, 7.5%, 15%, 22.5% and 30% by weight replacement, and nano silica (NS) was utilized as an additive by weight of binder materials at 0%, 1%, 2%, 3% and 4%. The properties considered were the slump, the compressive strength, the flexural strength, the splitting tensile strength, the modulus of elasticity, and the water absorption capacity. The microstructural properties of the concrete were also examined through FESEM and XRD analysis. The results showed that both CCR and NS increase the concrete’s water demand, hence reducing its workability. Mixes containing up to 15% CCR only showed improved mechanical properties. The combination of CCR and NS significantly improved the mechanical properties and decreased the concrete’s water absorption through improved pozzolanic reactivity as verified by the FESEM and XRD results. Furthermore, the microstructure of the concrete was explored, and the pores were refined by the pozzolanic reaction products. The optimum mix combination was obtained by replacing 15% cement using CCR and the addition of 2% NS by weight of cementitious materials. Therefore, using a hybrid of CCR and NS in concrete will result in reduction of cement utilization in concrete, leading to improved environmental sustainability and economy. According to Andrew [2 emission comes from the cement industry, where approximately 1.25 tons of CO2 is generated from producing one ton of cement. In other reports by Andrew [2 emission, where 90% of the emission from industries comes from the cement factories [Infrastructural development is significantly improving globally as most countries are moving towards urbanization. This leads to a rapid surge in the use of concrete, being the most widely utilized construction material. This, therefore, leads to the higher demand and utilization of cement as it is the main constituent material for concrete and mortar production. However, cement is the most environmentally unfriendly and expensive constituent material in concrete. The world is now focusing more on environmental and natural sustainable materials to address the rapidly increasing infrastructural demand. This, therefore, gives researchers a challenge in improving and achieving sustainability in the building and construction industries. It was projected that global demand for concrete will reach 18 billion tons annually by 2050 [o Andrew , about 5y Andrew , cement actories 2) to generate C2H2 (acetylene gas) and Ca(OH)2 , as presented in Equation (1) [CCR is the end product of production of acetylene gas used for many applications such as welding, lighting, fruits ripening, and metals cutting. The production of CCR involves a reaction occurring chemically between water and calcium carbide (CaCtion (1) ,8,9.(1)2 as a hydration product. The Ca(OH)2 in the presence of SiO2 and Al2O3 from cement or other pozzolanic materials generates excess compounds (C-S-H and C-A-S-H), the main products for strength improvement in cementitious matrix [Due to its high alkalinity, utilization of CCR for other applications seems unsuitable and is typically disposed of as landfills, mostly in slurry form. Due to its high pH value, CCR raises the alkalinity of the disposal area. It also causes health threats as it flies as dust into the environment ,10,11. Ts matrix ,13. TherKhongpermgoson et al.  develope2 generated by the CCR during hydration with the SiO2 and Al2O3 from the pozzolanic material to produce excess hydration products for strength development. However, based on the findings from the literature, only a small replacement of CCR with other Pozzolanic material yielded good results. Therefore, Nano silica due to its very high SiO2 content, finer size and high reactivity [Based on the existing literature, different pozzolanic materials have been used in combination with CCR to enhance the reaction between the Ca(OH)activity , if used3A and C4AF phases, and consuming Ca(OH)2 to densify the concrete microstructure. Murthi et al. [Nanotechnology is continuously becoming more acceptable in the construction and building industries. It is used in the form of nanomaterials in concrete and mortar to enhance the durability and mechanical performance of the concrete. It also improves environmental and material sustainability through the decrease in the use of cement in concrete and achieving higher strength at early ages with less cement content. Furthermore, nanomaterials significantly activate and enhance the pozzolanic reactivity of SCMs such as fly ash, slag, and RHA in concrete ,20,21,22i et al.  reportedi et al.  investigi et al.  and Huani et al. , NS as a2 which is utilized for pozzolanic reaction in concrete. Hence, in this research work, the effect of CCR as a partial substitute to cement and NS as addition by binder weight on the mechanical properties of concrete was investigated.Based on the critical review of previous research work carried, very few or no available literature that studied the effect of partial replacement of cement with CCR and NS as additive in concrete. Therefore, there is a need to study the effect of the hybrid of CCR and NS as cementitious materials in concrete. This is due to the fact that according to the literature, both CCR and NS when used with other Pozzolanic materials in concrete, significantly improved concrete’s properties. NS activates the pozzolanic reactivity of the SCMs in concrete, while CCR produces excess Ca(OH)Ordinary Portland Cement Type 1, which satisfied the standard specifications of ASTM C150  and whicThe coarse aggregate used was crushed granite having a maximum size of 19 mm which was free from dust and impurities. The properties of the coarse aggregate as obtained reference to ASTM C127  were preA Superplasticizer of polycarboxylate-based group which satisfied the BS EN 934-2  specificThe control mix was designed using the guidelines outlined in ACI 211.1R , for a tTo investigate the combined effects of calcium carbide residue (CCR) and nano silica (NS) on the properties of the concrete, twenty-five mixes were developed as shown in Prior to casting the concrete, the aggregates were brought to saturated surface dry conditions to prevent absorption of the mixing water, which can affect the consistency of the mixes and decrease strength. Before mixing, both cement and CCR were ensured to be fully dried and free from lumps and agglomerate. The mixing and curing were done based on the guidelines in ASTM C192/C192M  using a The workability of the fresh concrete was determined immediately after mixing using the slump test method outlined in ASTM C143/C143M .The compressive strength test was conducted on each mix after 3, 7, and 28 days curing period using 100 mm cubes with reference to the methods outlined in BS EN 12390-3 . A 2000 The splitting tensile strength test was carried out based on specifications outlined in BS EN 12390-6 . CylindrFor the flexural strength test, beams having dimensions of 100 mm × 100 mm × 500 mm were prepared and tested in accordance with the procedure outlined in ASTM C293/C293M , i.e., bcE represents the modulus of elasticity; cσ represents the stress corresponding to 40% of maximum compressive force; σ1 represents the stress equivalent to the longitudinal strain of 0.00005, ε2 represents the longitudinal strain equivalent to σ2.The methods outlined in ASTM C469  were useThe water absorption test was conducted on all the mixes in accordance with the specifications of ASTM C642  using 10Field emission scanning electron microscope (FESEM) test was conducted on some selected mixes. Small fractures were extracted from the samples after 28 days of curing and allowed to dry completely, carrying out the FESEM test. Small pieces of the concrete were obtained after conducting the 28 days compressive strength testing. The concrete piece was cleaned and dried, and then coated with a thin gold film before placing it in the FESEM machine. During the test, morphology of the concrete in the form of high-resolution pictures for up to 10,000 magnifications were obtained. The FESM results were used to examine the effect of CCR and NS on the microstructure of the concrete, such as densification, interfacial transition zone, and traces of hydration products in the concrete matrix and pores. For the XRD analysis, few mixes were selected to study the effect of CCR and NS on the hydration products of the concrete. The test was carried out after a 28 day curing period, where the paste was extracted from the compressive strength test samples. The paste was then cleaned, dried, and grinded to a very fine powder. The XRD analysis was then conducted using an X’Pert3 MRD Materials Research X-ray Diffraction System. The schematic flow chart of the whole research work is presented in The workability of the concrete containing calcium carbide residue (CCR) as a partial substitute to cement and nano silica (NS) as addition by weight of binder was measured using slump test, and the result presented in 2, which further reacts with the little SiO2, and Al2O3 from the cement to form more hydration products such as C-S-H and C-A-S-H which are the major contributors for strength development and pore filling in concrete [2 produced at the early age of the hydration reaction, which hinders strength development. Li and Yi [2 kept diminishing; thereby the reduction in strength also lessens.The results of compressive strength for all the concrete mixes containing different percentages of CCR as supplementary cementitious material and NS by binder weight is presented in concrete ,49. Kartconcrete  also repconcrete , where tconcrete  also fouconcrete , also rei and Yi  also rep2 content and high reactivity of NS, thereby reacting with the excess Ca(OH)2 produced by the CCR during hydration reaction to make more hydration products such as C-S-H gels which densified the pores in the hardened microstructure and improves strength develop in the concrete. Furthermore, NS itself, due to its very fine particle sizes, had pore filling ability thereby densifying the microstructure of the concrete and increasing strength [2, thereby leading to lower strength development. Nazari and Riahi [NS addition to the concrete mixes containing CCR as SCM significantly improved the compressive strength all ages. Even for the mixes containing up to 22.5% CCR, NS improved the compressive strength to be greater than the control mix. The addition of 1% and 2% NS to mixes containing 22.5% CCR such as M22.5C1N and M2.5C2N improved their strength. At 3, 7, and 28 days, the strengths of M22.5C1N were higher than those of the control by 1.1%, 9.1%, and 7.2%, respectively. While the strengths of M2.5C2N were higher than for the control mix by 28.4%, 34.5%, and 16.6%, respectively. The reasons for the improvement in strength with the addition of NS was due to the higher SiOstrength ,53,54. Ond Riahi  also repnd Riahi  for NS i2, which reacts with the SiO2 and Al2O3 in cement to generate additional C-S-H. The C-S-H filled up the pores in the concrete matrix and refined it, densified the ITZ between the cement paste and aggregate, and hence enhanced the compressive strength and hence improved tensile strength. Abdulmatin et al. [2 as a by-product of hydration. This Ca(OH)2 leached out easily, therefore increasing porosity in the concrete’s microstructure and hence reducing the tensile strength.n et al.  also repn et al.  also rep2 (pozzolanicity) in NS reacting with the extra Ca(OH)2 generated by the CCR to make more C-S-H gels, which made the concrete microstructure denser by filling the pores, refining the ITZ between cement paste and aggregate, thereby improving bonding and consequently enhancing the splitting tensile strength. Furthermore, NS due to its pore filling ability from its finer size, also leads to densification of the microstructure and ITZ in the cement matrix, thereby increasing strengths.NS addition to CCR concrete improved its splitting tensile strength at 7 and 28 days. However, this increment is for up to use of 15% CCR in the concrete. For example, considering mixes containing 15% CCR, the splitting tensile strength of mixes M15C1N, M15C2N, M15C3N, and M15C4N were higher by 21.3%, 36%, 30.8% and 3.3%, respectively, at 7 days and 11.1%, 24%, 14.4%, and 4.9%, respectively, at 28 days compared to mix M15C0N. The splitting tensile strength of mixes M22.5C1N, M22.5C2N, and M22.5C3N were higher by 12.1%, 6.6%, and 20.7%, respectively, at 7 days and 14.0%, 5.2% and 18.3% at 28 days, respectively, when compared to M0C0N. This improvement resulted from the high SiOThe relationship between 7 and 28 days splitting tensile strength versus 7- and 28-days compressive strength of the concrete mixes containing CCR and NS was presented in 2, which reacts with some elements of the cement-like SiO2 and Al2O3 to generate extra C-S-H. The excess C-S-H further refined the microstructure and densified the paste-aggregate ITZ, which consequently leads to higher bending resistance. Adamu et al. [2 generated during hydration, which can be leached out easily, leaving pores in the hardened matrix. This will result in higher porosity in the matrix and consequently reduce the flexural strength.u et al.  also fou2 from NS and Ca(OH)2 from CCR [NS addition further enhanced the flexural strength of the mixes containing any percentage of CCR. For instance, the flexural strength of M22.5C1N, M22.5C2N, and M22.5C3N were greater than M0C0N by 4.6%, 12.3%, and 20%, respectively, at 7 days and 8.2%, 14.7%, and 18.7%, respectively, at 28 days. The addition of up to 2% NS to mixes containing 30% CCR improved its strength. M30C1N had higher flexural strength than M30C0N by 12% and 6.6% at 7 and 28 days, respectively, while M30C2N had higher values compared to M30C0N by 14.2% and 11.6% at 7 and 28 days, respectively. The increase in flexural strength resulted from the densification and enhancement of the ITZ and improving adhesion between aggregate and paste which significantly increases the bending resistance. This is due to the excess hydration products generated from the pozzolanic reaction between SiO2 from the CaO in CCR and some elements in cement-like SiO2 and Al2O3. This resulted in increasing in C-S-H contents, which densified the concrete microstructure and consequently increased the strength and stiffness, thereby enhancing the MoE of the mixes. However, higher CCR contents from 15% without any NS addition resulted in a decrease in MoE, as the MoE values of M22.5C0N and M30C0N were lower than for the control mix by about 21.4% and 41.5%, respectively. This decrease might be due to excess Ca(OH)2, which could not react with the elements from cement and thereby leached out easily, hence creating pores in the concrete microstructure. These pores caused a decline in strength and stiffness of the concrete and hence decreased its MoE. Contrary to these findings, Amnadnua et al. [a et al.  reported2) and CCR (high Ca(OH)2 from its high CaO), creating supplementary hydration products filling the porosity in the concrete and densified its microstructure thereby increasing strength and stiffness and consequently higher MoE [NS addition significantly improved the MoE of the concrete mixes containing CCR. Therefore, with NS as an additive, up to 22.5% CCR as SCM can be used to improve the stiffness and MoE of concrete. As shown in gher MoE ,54. Adamgher MoE  also rep2). The Power model as given in Eqn 3 can be compared to the models for normal concrete according to ACI 318 as given in Eqn 4 [CE is the Modulus of Elasticity in MPa, and CF is the compressive strength in MPa.The relationship between the compressive strength and MoE of the concrete is presented in in Eqn 4  and for in Eqn 4 , where iin Eqn 4  for concin Eqn 4 , a combi2 in the cement matrix, which increases porosity and water absorption.2 reacts with the hydration products from CCR and cement, such as crystalline Ca(OH)2, producing excess amorphous C-S-H, filling the nanopores in the cement matrix, producing a more homogenized and densified microstructure, thereby decreasing water absorption [The addition of NS to mixes containing CCR further decreased their water absorption values. For instance, mixes containing 7.5% CCR, the water absorption values of M7.5C1N, M7.5C2N, M7.5C3N and M7.5C4N were lower by 9.2%, 16.1%, 17.3%, and 2.3%, respectively, in comparison to mix M7.5C0N. Additionally, in comparison to the control mix, the water absorption of M22.5C1N, M22.5C2N, M22.5C3N and M22.5C4N were lower by 4.6%, 13.6%, 22%, and 7.7%, respectively. The decreases in water absorption might be attributed to the physical properties of NS were due to its very fine sizes, it could fill the pores and densify the cement matrix, hence decreasing the water absorption. Another reason can be due to the chemical properties of the NS, where its high SiOsorption ,54.2 and Ca(OH)2 produced during the hydration of the concrete mix containing CCR and NS. This reaction resulted in more C-S-H generation and consumption of Portlandite (Ca(OH)2), therefore a denser microstructure. Furthermore, it can be observed that the microstructure of M15C2N was the most densified, having the highest degree of pozzolanic reaction. Therefore, its microstructure was the most refined. This can be justified as mix M15C2N has the highest mechanical strengths and least water absorption value. From 2 (Portlandites) were found on the morphology of mix M22.5C0N. This is coming from the unreacted Ca(O)2 produced by the excess CCR during the hydration reaction which contains a high amount of CaO. In the absence of excess pozzolanic elements to react with the Ca(OH)2, the Ca(OH)2 easily leaked out, leaving pores inside the cement matrix as seen in the morphology of mix M22.5C0N. By comparing the morphology of mix M30C4N , SiO2 (represented as S), and CaCO3 (represented as K). The peak hydration product was the crystalline Ca(OH)2 occurring at about an angle (2-theta) of 28–30°. This is due to the CaO content in cement which reacts in the presence of water to give Portlandite as a by-product, with no supplementary pozzolanic material to consume it. Zhou et al. [2 rapidly decreased, where quartz (SiO2) had the highest hump peak occurring between angles (2-theta) 26–28°, as seen in 2 with the surplus Ca(OH)2 to produce additional C-S-H, amorphous in nature and hard to detect using XRD [2 and Ca(OH)2 were detected, having lower intensities compared to mixes containing high CCR with low NS, or low CCR with high NS. Therefore, there is a tendency that in mix M15C2N both SiO2 and Ca(OH)2 undergo a higher pozzolanic reaction and consume each other to generate more C-S-H gels. This can further be justified as mix M15C2N has the highest strengths values and lowest water absorption. Lastly, the XRD results for mix M30C1N 2 was not adequately consumed. Therefore, during XRD analysis, a high-intensity peak of the crystalline Ca(OH)2 was detected. During the XRD analysis, other peaks of elements such as mullite (Al6Si2O13), represented as M, were detected, which might occur from reaction between Al2O3 from cement and SiO2 from NS. Additionally, CaCO3 (represented as K) was also detected in some mixes, which might also be from the reaction of cement elements with the CCR elements or from limestone used in producing cement.The X-ray Diffractometer (XRD) analysis was carried out to examine the effect of CCR and NS on the hydration reaction and products of the concrete. Representative mixes containing both CCR and NS were selected, and the results of the XRD analysis were presented in u et al. , also resing XRD . Figure x M30C1N e have thCost analysis was carried out for all the concrete mixes. Concrete mix proportions in 2 is emitted to the environment [2 emission from cement is by utilizing SCM in concrete. This will lead to reduction in the amount of cement produced and consequently lower CO2 emissions. In this study, the effects of CCR as partial substitute to cement and NS as additive on the CO2 emission of concrete were studied. The equivalent CO2 emissions from cradle to get for each of the mixes were calculated using Equation (6) in conjunction with the data in i represents the mix number, n represents the total number of mixes, iχ represents the quantity of raw material i in kg/m3 and COi)−ø2.One of the major disadvantages of using cement in cementitious composites is due to the high amount of greenhouse gas emissions to the environment. According to some studies for one ton of cement produced, an equivalent of 1 ton COironment ,65,66. O2 emissions from cradle to gate for all the concrete mixes. In comparison to the control mix , the CO2 emissions of the concrete mixes decrease with increment in partial replacement of cement using CCR. This is due to the lower CO2 emission of the CCR compared to cement it replaced as shown in 2 emission of the concrete due to its very low CO2 emission as seen in 2 emission of the concrete mixes, which increases with the increment of NS addition.(1)The workability of concrete decreases with increment in partial substitution of cement using CCR, due to higher surface area and loss of ignition. NS addition to the concrete containing CCR further decreases the concrete’s workability, thereby increasing water demand during mixing due to its very fine sizes;(2)The strengths and modulus of elasticity of concrete increased with replacement of up to 15% cement using CCR. On the contrary, higher replacement levels of cement with CCR above 15% resulted in a decrease in the listed properties;(3)NS addition up to 3% by weight of binder further enhanced the mechanical strengths and modulus of elasticity of the concrete. This is due to enhanced pozzolanic reactions between quartz from NS with Portlandites from CCR generating excess calcium silicate hydrates responsible for strength development. However, addition of higher NS above 3% resulted in a decrease in the mechanical properties of the concrete, due to agglomeration and reduced consistency;(4)Both CCR and NS decrease the porosity of the concrete, resulting in a decrease in water absorption. However, this improvement was for the use of up to 22.5% CCR and up to 3% NS;(5)The microstructural morphology showed that the combination of NS and CCR in concrete densified its microstructure, refined its pores, and produced more hydration products such as C-S-H;(6)The optimum mix was obtained using a combination of 15% CCR with 2% NS, which gave the best performance in terms of mechanical strengths, elastic modulus, water absorption, and microstructural refinement;(7)2 emission of the concrete. NS on the other hand increases the concrete’s cost but does not have much effect on the CO2 emission;The use of CCR as cement replacement in concrete reduces the cost and CO(8)The developed concrete can be used for structural applications with a strength of more than 30 MPa where up to 30% cement can be replaced with CCR with the addition of NS.The effect of partial substitution of cement using CCR and addition of NS by weight of binder on the mechanical properties of concrete was investigated. The following conclusions were drawn:This study was limited to normal strength concrete with a target strength of 30 MPa at 28 days. The CCR was obtained from only one source, and likewise the NS. The maximum amount of CCR was limited to 30% as a partial replacement by weight of cement, and the NS was limited to 4% as additive by weight of cementitious materials. The research was also limited to investigating the effect of the hybrid of CCR and NS on the mechanical strength, water absorption, MoE, and microstructural evaluations. The developed concrete can be used for structural applications where up to 22.5% CCR can replace cement with up to 3% NS added. The developed concrete is limited to use for structural applications, with concrete design strength not exceeding 30 MPa at 28 days.


Accurate assessment of predictive biomarker expression is critical in patient selection in clinical trials or clinical practice. However, changes in biomarker expression may occur after treatment. The aim of the present study was to evaluate the effects of chemotherapy on MET expression in gastric cancer (GC).MET gene amplification was assessed by chromogenic in situ hybridization (CISH). The antitumor effect of MET targeted therapy was investigated in human gastric cancer cells in vitro and in vivo, and the underlying molecular mechanisms were analyzed by western blot.MET expression was examined immunohistochemically before and after treatment in 122 patients with unresectable or recurrent GC, and was evaluated according to H-score or the scoring criteria used in the MetMAb trial. in vitro and in vivo in MKN45 cells with MET and phosphorylated MET (pMET) high expressions via suppressing downstream PI3K/Akt and MAPK signaling pathways. Furthermore, combination therapy targeting both MET and HER2 demonstrated a synergistic antitumor activity.MET expression was associated with Lauren classification as well as tumor differentiation by either scoring system. MET amplification was not associated with clinical characteristics. Of the 71 patients who had paired pre- and post-treatment tumor tissues, 28 patients (39%) were initially positive for MET expression, and 43 (61%) were negative. Twenty-five patients (35%) showed significant changes in MET expression after treatment (P=0.007). Additionally, there was a concomitant overexpression of MET and HER2 in a subset of GC patients. MET inhibitor volitinib could significantly inhibit cell proliferation and xenograft growth MET expression is altered post chemotherapy and MET status should be evaluated in real-time. Both MET and pMET expressions might need to be considered for patients suitable for volitinib treatment. Gastric cancer (GC) is the third most common cancer in China, causing more than 373,000 deaths annually . BecauseMET gene amplification and expression have been associated with poor prognosis in GC , hepatocyte growth factor receptor (MET) has recently emerged as an important target in GC. Aberrant constitutive activation of the MET signaling pathway frequently occurs in multiple neoplasms, including GC . MET oveis in GC , 16.Several drugs have been developed to target the MET signaling axis, one of which is onartuzumab, a monovalent anti-MET antibody. Although onartuzumab activity was observed in early clinical trials, the MetGastric and the MetLung phase III studies on onartuzumab in gastric and lung cancer showed negative results, which were attributable to various reasons, including patient selection , 18. AnoThe present study evaluated the effects of treatment on MET expression using pre- and post-treatment paired samples and different immunohistochemistry (IHC) scoring criteria. The expression profile of MET and HER2 as well as the prognostic role of MET were also investigated. We also tried to explore the efficacy and mechanisms of a novel MET inhibitor volitinib both in GC cell lines and PDX model.Pre-treatment tumor samples from patients with unresectable/recurrent GC who were enrolled between January 2008 and August 2013 were collected after obtaining patient informed consent and ethics committee approval from Peking University Cancer Hospital (PUCH). The biopsy tumor samples were obtained by gastroscopy from the primary GC site. The tissues were fixed in formalin and then paraffin-embedded in blocks, which were prepared at the time of biopsy. Medical data including age, gender, clinical diagnose, previous treatments, tumur stage according to the TNM classification system, tumor grade, the histologic classification of based on Lauren’s criteria, HER2 test results, and survival follow-up information were collected from medical records. MET protein expression and gene amplification testing of all formalin-fixed paraffin-embedded (FFPE) tumor samples, and the testing data were collected.MET gene copy number in biopsy vs resection specimens, some biopsy (n=81) and resection (n=72) samples that were preserved in AstraZeneca laboratory were also detected and analyzed.To compare MET protein expression and vs cytoplasmic, were assessed and recorded.Four-micrometer-thick tumor sample sections were freshly cut from the archival tumor FFPE blocks for MET IHC analysis to evaluate MET protein expression levels. IHC detection of MET was performed using a rabbit monoclonal anti-total MET antibody  on an automatic immunostainer . A minimum of 100 tumor cells was examined for MET staining. Staining intensity  and prevalence of these intensities in tumor cells were evaluated. The tumours were scored according to the criteria used in the onartuzumab (MetMAb) phase II trial in GC, where MET-positive samples were defined as having ≥50% of tumor cells showing moderate or strong intensity MET staining . AnotherMET CISH analysis to assess MET gene copy number. MET gene copy number per cell was determined by CISH technique using both MET-specific and centromere 7 (CEP7)-specific probes following the manufacturer’s recommendations . A minimum of 50 non-overlapping cells with hybridization signals were examined for each sample. Mean MET gene and mean CEP7 copy number per cell and MET/CEP7 ratio were recorded. Tumors with MET/CEP7 ratios ≥ 2.0 (designated as “amplified”) and/or MET ≥ 4.0 copies (named “high polysomy”) were considered MET CISH-positive.Four-micrometer-thick tumor sections were freshly cut from archival tumor FFPE blocks for MET FISH analysis. MET FISH evaluation was performed on unstained FFPE tissue sections using a MET/CEP7 probe cocktail (Dako) according to the manufacturer’s instructions. A minimum of 50 non-overlapping cells with hybridization signals from each sample were examined under a fluorescence microscope . Tumors with MET/CEP7 ratios ≥ 2.0 (named “amplified”) and/or MET ≥ 5.0 copies (named “high polysomy”) were considered MET FISH-positive  rabbit monoclonal primary antibody . In cases of IHC 2+ staining, FISH was performed. Tumor specimens with a reported , HER2 po2 and cultured in RPMI 1640 medium  supplemented with 10% fetal bovine serum (Gibco-BRL) and 1% penicillin and streptomycin (Gibco-BRL). Volitinib was kindly provided by AstraZeneca Pharmaceuticals . Trastuzumab (Herceptin) was purchased from Roche Pharmaceutical Ltd. . Recombinant human HGF was purchased from R&D Systems.Human gastric cancer cell lines SNU-216, MGC803, MKN45, NCI-N87 and BGC823 were purchased from the cell bank of Peking Union Medical College . All cells were incubated at 37℃ in a humidified incubator with 5% COCells were seeded into 96-well plates at a density of 3,000~5,000 cells/well and allowed to adhere overnight. Cells were incubated with volitinib with 2-fold serial dilutions alone or the combination of trastuzumab in culture medium in the presence or absence of 20 ng/mL HGF for 72 hours. After treatment, cell proliferation was measured using a CCK8 kit  according to the manufacturer’s protocol. Absorbance was measured at 480 nm using spectrophotometry.Cells were harvested at 70~80% confluence and were lysed using a CytoBuster protein-extraction reagent , containing complete protease inhibitor and phosphatase inhibitor cocktail . The extracted protein concentrations were quantitated using a BCA protein-assay Kit . After protein electrophoresis and membrane transfer, the membranes were probed with primary antibodies overnight at 4°C and incubated with the secondary antibodies for 1 hour at room temperature. The signals were detected using the Clarity Western ECL substrate . The primary antibodies were as follows: anti-MET antibody (#8198), anti-pMET antibody (#5605), anti-AKT antibody (#9272), anti-pAKT antibody (#9271), anti-ERK antibody (#4695), anti-pERK antibody (#4370), anti-S6 antibody (#2217), anti-pS6 antibody (#4858) purchased from Cell Signaling Technology. Anti-β-actin antibody (Lot #014 M4759) was purchased from Sigma-Aldrich .3, mice were randomly assigned to vehicle control or treatment groups with at least five mice per group. Mice in treatment groups were given volitinib at a dose of 10mg/kg daily by oral gavage or trastuzumab at 20 mg/kg weekly by intraperitoneal injection, or in combination for three weeks. Tumors were measured with digital calipers twice a week, and tumor volumes were calculated by the formula: volume = length × (width)2/2. Tumor growth inhibition (TGI) was calculated using the following formula: TGI% = [1-(ΔT/ΔC)] × 100% . All animals were performed in accordance with the guidelines approved by independent ethics committee of Peking University Cancer Hospital.Two kinds of xenograft models were included in this study. Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) female mice with 6-week-old  were inoculated with 1×107 MKN45 cells suspended in PBS to establish MKN45-derived xenograft. Three gastric cancer patient-derived xenograft (PDX) models were obtained as describes as our previously published report . When tuin vitro and in vivo studies, differences between the groups were analyzed using one-way ANOVA or unpaired two-tailed t test. A two-sided P value < 0.05 was considered statistically significance.Statistical analysis was performed using the SPSS 18.0 software. Chi-square test or Fisher’s exact test were used to evaluate the association of clinical characteristics with MET expression and MET amplification. A Mann-Whitney test or Kruskal-Wallis test was used to analyze the relationship between MET H-scores and clinical characteristics. Overall survival (OS) was estimated and analyzed by a Kaplan-Meier curve and Cox regression model. The lo-rank test was used to detect differences in OS among various groups. For the A total of 122 patients were eligible in this study, of which tumor sections from 116 patients were assessed in terms of MET staining, but not for 6 patients due to insufficient number of tumor cells in the prepared sections. Of these 116 patients, 71 had paired pre- and post-chemotherapy samples. The schematic flow diagram of the analysis is shown in P<0.001) indicated an association between tumor grade (P<0.001) and tumor location (P=0.047) with MET expression. The rate of MET-positive cells was higher in well-differentiated and intestinal tumors, as well as tumors located in the antrum. H-score assessment indicated a median MET H-score of 130, which was chosen as the cut-off value for classifying MET-positive and MET-negative tumors. No significant differences in MET expression based on gender, age, and primary tumor site were detected. However, significant associations were observed between MET H-score and Lauren classification (P<0.001) and tumor grade (P<0.001).According to the MetMAb scoring criteria, 47% of pre-treatment samples (54/116) were positive. MET CISH analysis. The average MET gene copy number per cell was 2.4, and the range was from 2.0 to 11.02. Seven patients (14%) showed MET amplification. Of the 7 patients, 6 patients (85.7%) were MET IHC-positive, whereas 1 patient was MET IHC-negative. No significant association between MET amplification and gender, age, primary tumor site, Lauren classification, or tumor grade was detected, which would be further validated in future large samples.Only 49 patients in this study were eligible for vs resection specimens (vs 46.6%) or gene copy number (12.9% vs 14.3%) between the AstraZeneca laboratory and PUCH laboratory were detected. However, we observed a higher MET protein staining in biopsy specimens compared to resection ones from the AstraZeneca laboratory , as well as in the PUCH laboratory   were initially positive for MET expression and 43 (61%) were negative. There were 25 patients (35%) who showed changes in MET expression after treatment. vs 36%, P=0.03)  were HER2-positive and 78 (72%) were HER2-negative. HER2 and MET positivity were not mutually exclusive; there were 18 MET-positive patients in the 30 HER2 positive patients (60%). Notably, compared to the HER2-negative group, there were more MET-positive patients in the HER2-positive group (60% vs 374 days (309-439), P=0.274]. However, when an H score=200 was used to define MET positivity/negativity, we observed a worse OS in the MET-positive group than in the MET-negative group . In the multivariate Cox regression model adjusted for other variants, a statistically significant difference in OS between the MET-positive and MET-negative patients was also observed .We evaluated the association between pre-treatment MET expression and survival using various H-score values. Of the 116 patients whose tumor tissues were obtained prior to treatment, 113 had available survival data. The median follow-up time was 374 days. There was a numerical but not statistically significant difference in OS when the MET H-score cut-off was 130, as defined by the median H score [median OS, 319 days (95% CI: 226-412) in vivo and the possible mechanisms. As expected, MKN45 cell  was much more sensitive to volitinib than other cells  of 85.2%  high expressions compared to other gastric cancer cells  for the publication of any potentially identifiable images or data included in this article.JG and CZ conceived and designed the study. HL, TT and YS were involved in study performing, the data collection, data analysis, and writing of the manuscript. ZP, JW, and YL obtained the patient’s samples and assisted in performing experiments. ZL evaluated the results of experiments. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (grant numbers 82072728), and Sanming Project of Medicine in Shenzhen, China (SZSM201812062).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer CD declared a shared affiliation with one of the authors YS to the handling editor at the time of review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The overall five-year survival rate of patients with loco-regional advanced head and neck squamous cell carcinomas (HNSCC) is only around 40%. The curability of HNSCC with radiochemotherapy was previously found to be associated with clinical and biological parameters including tumor volume, hypoxia, epidermal growth factor receptor expression, and human papillomavirus infection status. Different signaling pathways, e.g., constitutively activated receptor tyrosine kinase signaling, increased DNA damage repair and intracellular defense against reactive oxygen species were identified as factors driving HNSCC progression and its resistance to therapy. c-MET was found to be hyperactivated in HNSCC and has been reported to drive tumor progression, therapy resistance, and metastatic spread. Here, we investigated the therapeutic potential of c-MET targeting strategies for HNSCC radiosensitization and discovered putative resistance mechanisms impeeding success of therapeutic intervention. This study highlights the importance of detailed knowledge about biological mechanisms and regulatory networks for future patient stratification and individualized treatment concepts.MET gene expression and protein phosphorylation with reduced disease-specific and progression-free survival. To investigate the role of c-MET-dependent radioresistance, c-MET-positive cells were purified from established HNSCC cell lines and a reduced radiosensitivity and enhanced sphere-forming potential, compared to the c-MET-depleted cell population, was found in two out of four analyzed cell lines pointing to regulatory heterogeneity. We showed that c-MET is dynamically regulated after irradiation in vitro and in vivo. Interestingly, no direct impact of c-MET on DNA damage repair was found. The therapeutic potential of eight c-MET targeting agents in combination with irradiation demonstrated variable response rates in six HNSCC cell lines. Amongst them, crizotinib, foretinib, and Pha665752 exhibited the strongest radiosensitizing effect. Kinase activity profiling showed an association of crizotinib resistance with compensatory PI3K/AKT and MAP kinase signaling. Overall, our results indicate that c-MET is conferring radioresistance in HNSCC through modulation of intracellular kinase signaling and stem-like features.The receptor tyrosine kinase c-MET activates intracellular signaling and induces cell proliferation, epithelial-to-mesenchymal-transition and migration. Within the present study, we validated the prognostic value of c-MET in patients with head and neck squamous cell carcinoma (HNSCC) treated with radio(chemo)therapy using the Cancer Genome Atlas database and found an association of increased  MET gene expression significantly correlates with reduced locoregional control, decreased overall survival and enhanced distant metastasis after post-operative radio(chemo)therapy, especially in patients with human papillomavirus (HPV)-negative HNSCC, while it does not have prognostic potential in patients treated with primary radio(chemo)therapy  and[n = 77) ,43 and c= 0.779) F. We use= 0.779) . In addi= 0.779) . A high = 0.779) . Additiosurvival . In addisurvival . ImportaIn summary, this data shows a central role of c-MET in pre-irradiated sublines with increased kinase activity not only for c-MET, but also for EGFR signaling. TCGA and HIPO subgroup analysis of patients that received radiotherapy implies the importance of c-MET and its downstream intracellular signaling for HNSCC progression during radiotherapy .MET expression is associated with locoregional control in HNSCC patients after postoperative, but not after primary radio(chemo)therapy [+ and c-MET− cell population, 1000–1500 sorted cells per well were plated under single cell conditions within matrigel in 96-well plates for 3D-colony formation assay and irradiated with increasing doses (2–8 Gy). The fraction of cells surviving irradiation was significantly higher for the c-METhigh population in SAS and UT-SCC-5 cell lines in comparison to c-METlow cells. However, we did not observe differences in FaDu and Cal33 cells indicative for a heterogenous c-MET dependency between the tested HNSCC cell lines  demonstrated that dysregulated )therapy ,35,44. W)therapy   repair, while SAS cells are HR deficient. To investigate how c-MET may determine tumor radiosensitivity in patients with HNSCC, we correlated the MET gene expression with occurrence of canonical pathways in silico within the TCGA dataset (n = 473). According to our functional analysis, we found that MET expression negatively correlates with genes regulating DNA damage repair while it is positively associated to genes involved in cell motility, metastasis, and epithelial-mesenchymal transition (EMT)  induced by ionizing radiation can be quantified using the yH2AX foci assay. We quantified initial yH2AX foci 30 min after irradiation with 4 Gy, which are indicative for immediate damages, and residual, unrepaired foci 24 h after irradiation. These foci counts were used to calculate DNA repair kinetics from isolated c-METanalysis . Howeveron (EMT) G. This idoes not H. Despitpatients I. The MEinvolved D. To bri− and c-MET+ populations of Cal33 and SAS cell line were cultured for one week and membrane c-MET expression was determined by flow cytometry. Cal33 cells are composed of 21.2% c-MET− and 78.8% c-MET+ cells (ratio = 1:3.7). After purification and culture, the proportion of c-MET− (69.7%) to c-MET+ (30.3%) within c-MET− cultures equilibrates back to a ratio of 2.3:1. About 30% of c-MET− cells re-gain c-MET expression. The c-MET+ culture resembles the proportion of the original culture (ratio = 1:4.5) with 18.3% c-MET− cells and 81.7% c-MET+ cells after seven days  within the TCGA dataset. Interestingly, PROM1, the gene encoding for CD133, negatively correlates with MET which is in line with our co-staining data. The other three tested biomarkers CD44, SLC3A2 (encodes for CD98 protein) and EGFR show a significantly positive correlation with MET in TCGA data, as shown above on protein level in HNSCC cell lines in our flow cytometry experiments  within the TCGA database that contains results for phosphorylated c-MET protein at tyrosine 1235 (Y1235) measured by reverse phase protein array, which is indicative of an activated kinase status. We found that higher level of phospho-c-MET is significantly associated with a decreased progression-free and disease-specific survival in patients with HNSCC . When patients are stratified according to radiotherapy treatment this correlation is not seen  (MET transcript and phospho-c-MET (Y1235) level. However, we found a significant association between high MET transcript levels and lower survival rates. Meaning, the lack of correlation between transcript and phospho-protein might be attributed to a lower number of cases within the proteomics dataset  F. These  dataset G. AdditiMET expression and c-MET cell surface protein abundance is altered in radioresistant and stem-like HNSCC cells. Moreover, we could show that c-MET is dynamically regulated upon irradiation and that a transient knock-down of the MET gene is increasing radiosensitivity only within a subset of investigated HNSCC cell lines. We hypothesize that this subset of HNSCC cells with c-MET dependency may be highly sensitive to chemical c-MET-specific targeting. Therefore, we screened eight clinically relevant c-MET inhibitors  in three different HNSCC cell lines  within a dose range of 1–100 µM for different time points  and in combination with 4 Gy irradiation using cell viability assay (CellTiterGlo) (The above-described data demonstrated that iterGlo) A. The deiterGlo) B. We valiterGlo) . ComparaiterGlo) . These riterGlo) .p < 0.05, fold change > 1.5) identified the neurotrophin, PI3K/AKT, and RAS signaling as key resistance mechanisms (Receptor tyrosine kinases require adenosine triphosphate (ATP) as source for subsequent phosphorylation of downstream targets. Crizotinib and Pha665752 are potent c-MET inhibitors through competitive binding within the ATP-binding pocket that prevent c-MET-induced downstream signal activation, e.g., of PI3K/AKT, MAPK, and STAT. To investigate the underlying molecular mechanisms determining crizotinib sensitivity, we analyzed the activity of different intracellular kinase cascades in the responder cell line Cal33 in comparison to the non-responder line SAS 4 h and 24 h after crizotinib and Pha665752 treatment using Western blot analysis. In Cal33 cells, we found that c-MET targeting slightly increase MET phosphorylation, followed by an induction of AKT, ERK1/2, and EGFR phosphorylation. These late effects may illustrate the activation of compensatory kinase pathways. Within SAS cells, c-MET was hyperphosphorylated initially and reduced after treatment with crizotinib and Pha665752 affecting downstream AKT and EGFR phosphorylation. Moreover, JNK phosphorylation was induced while ERK1/2 was not affected D. These chanisms . In summMET gene expression with poor overall survival while immunohistochemical staining demonstrated a significant increased association of c-MET protein expression with worse relapse-free survival and the presence of regional lymph node metastasis [MET gene expression is associated with unfavorable prognosis of HNSCC patients after radio(chemo)therapy [MET overexpression in 58%, MET amplification in 39%, MET protein phosphorylation in 30%, and HGF overexpression in 58% of the analyzed tumors of patients with HNSCC [MET gene. This indicates that acquired radioresistance goes along with altered c-MET signaling in HNSCC cells. To analyze effects of c-MET on cell-intrinsic radiosensitivity we prospectively purified c-METhigh and c-METlow cell populations, and found a significantly higher fraction of clones surviving irradiation within the c-METhigh population in comparison to c-METlow cells. However, we did not observe any difference of both populations in FaDu and Cal33 cells, indicating that the association of c-MET and radioresistance differs between the different HNSCC cell lines. Until now, it is not fully understood how c-MET is affecting radiosensitivity. A recently published study by Bensimon et al. applied quantitative phosphoproteomic technique and identified a novel c-MET phospho-site that bears as substrate for the DNA damage response (DDR) machinery including DNA-dependent protein kinase (DNA-PK), nuclear mitotic apparatus protein 1 (NUMA1) and checkpoint kinase 1 (CHEK1) in c-MET addicted cancer cell lines [high and c-METlow cells within the c-MET-dependent cell line SAS and UT-SCC-5 using the yH2AX assay. Surprisingly, we did not find any differences in initial and residual yH2AX foci reflecting DNA double strand breaks after irradiation with 4 Gy. This was validated within the TCGA data set, were we found that MET expression is positively associated with stem-like features and negatively with DNA repair mechanisms. To shed further light on this, we investigated the effect of irradiation on c-MET expression. Therefore, we analyzed the dynamic changes in protein expression after treatment of HNSCC cell lines with 4 Gy single dose and found an >20-fold enrichment in SAS cells within 2 days that equilibrates to baseline level within one week. Cal33 cells increase c-MET >6-fold until day 7 while c-MET level in FaDu cells even decline. This heterogeneous, cell line dependent response of c-MET to irradiation was proven with flow cytometry and immunofluorescence analysis and is fitting to the identified c-MET dependency influencing the cell-intrinsic radiosensitivity of SAS and UT-SCC-5 cells. These results are in-line with a previously published study that investigated the stem-like characteristics of c-METpos cells within primary HNSCC cultures using limiting dilution analysis in immunocompromised NOD/SCID mice [pos population compared to 1/27,093 for the c-METneg population. In addition, the authors determined the stem-cell frequency of other known CSC-markers and found with 1 tumorigenic cell within 497 cells (1/497) a higher stem-cell frequency in the ALDHhigh population than in the CD44+ population containing only 1 in 7364 [+ population using the sphere-formation assay and found that this population is enriched for sphere-forming cells compared to the c-MET− population in particular after irradiation. We cannot exclude that this effect is independent from c-MET, because co-expression analysis demonstrated that c-MET is highly co-expressed and co-regulated with other known CSC markers such as ALDH, CD98, and CD44. Therefore, we applied siRNA-mediated transient gene knock-down to specifically down-regulate MET expression that was validated on protein level. Despite we efficiently down-regulated c-MET protein level, there was no radiosensitizing effect within the investigated HNSCC cell lines while the purified c-METhigh population was resistant. Downstream signaling analysis demonstrated that even upon c-MET protein reduction the intracellular kinases are still active, which is a consequence of compensatory signaling pathways overcoming loss of intracellular c-MET signal transduction. When analyzing the proteomic TCGA dataset of patients with HNSCC we found that high level of phosphorylated c-MET, i.e., active c-MET is significantly associated with disease-specific survival. We found a discrepancy of MET gene expression, protein expression and kinase activity that was confirmed within the TCGA dataset. MET gene expression does not correlate with phospho-MET level. The phosphorylation of the intracellular domain of c-MET can activate the PI3K/AKT, RAS/MAPK, and STAT3 signaling pathway. This tight regulation is lost within HNSCC and abnormal, activated or overexpressed MET causes tumor progression, invasion and metastasis [MET over-expression and EGFR amplification that may impact on treatment efficiency [MET mutation R1004G as prognostic factor for the response to crizotinib treatment [The pleiotrophic role of the receptor tyrosine kinase (RTK) c-MET in cellular processes and its important role for cancer progression suggest that c-MET may be a promising target for anticancer therapy ,50,51,52tastasis . Moreove)therapy ,44,54. Sth HNSCC . These ath HNSCC ,32,36,57tastasis ,61,62,63ficiency . In combficiency ,67,68,69ficiency . These dficiency . This daficiency . EGFR maficiency ,66. The ficiency . Clinicareatment .MET gene expression and phosphorylation status. Overall, these results highlight the importance of comprehensive genomic profiling and biomarker-driven patient stratification to assess and predict the therapeutic potential of targeted therapies in the field of radiooncology.The key role of the receptor tyrosine kinase c-MET for cell survival and cancer progression in patients with HNSCC support c-MET as promising target for anticancer therapy. Within the present study we showed that c-MET plays a role for cellular plasticity and stemness in HNSCC, which are major determinants of tumor radioresistance, and provide support for the potential of c-MET targeting strategies for HNSCC radiosensitization. Mechanistic investigation of resistant HNSCC cells to c-MET targeting identified compensatory kinase pathways such as PI3K/AKT and RAS that overcome c-MET blockade and may prevent clinical efficiency in future application. Additional studies are needed to investigate the observed differences between the prognostic potential of


Tursiops truncatus) and reference values for 27 Indo-Pacific bottlenose dolphins (Tursiops aduncus), 13 beluga whales (Delphinapterus leucas), and 6 Pacific white-sided dolphins (Lagenorhynchus obliquidens) in zoos and aquariums accredited by the Alliance for Marine Mammal Parks and Aquariums and the Association of Zoos & Aquariums. Blood samples were collected as part of a larger study titled “Towards understanding the welfare of cetaceans in zoos and aquariums” . Two blood samples were collected following a standardized protocol, and two veterinarian examinations were conducted approximately six months apart between July to November 2018 and January to April 2019. Least square means, standard deviations, and 95% confidence intervals were calculated for hematology, serum, and plasma biochemical variables. Comparisons by age, gender, and month revealed statistically significant differences (p < 0.01) for several variables. Reference intervals and values were generated for samples tested at two laboratories for up to 56 hematologic, serum, and plasma biochemical variables. To apply these data, ZooPhysioTrak, an iOS mobile software application, was developed to provide a new resource for cetacean management. ZooPhysioTrak provides species-specific reference intervals and values based on user inputs of individual demographic and sample information. These data provide a baseline from which to compare hematological, serum, and plasma biochemical values in cetaceans in zoos and aquariums.This study reports comprehensive clinical pathology data for hematology, serum, and plasma biochemistry reference intervals for 174 apparently healthy common bottlenose dolphins ( Tursiops truncatus), Indo-Pacific bottlenose dolphins (Tursiops aduncus), beluga whales (Delphinapterus leucas), and Pacific white-sided dolphins (Lagenorhynchus obliquidens). Of these species, common bottlenose dolphins have been the focus of the most rigorous research in order to characterize hematological and serum reference intervals [Accurate hematological, serum, and plasma biochemical reference intervals and values developed from healthy cetaceans facilitate the determination of the health status of individuals , 2. Compntervals –7. Venn-ntervals , 9 and tSparse reports of hematological, serum, or plasma values exist for Indo-Pacific bottlenose dolphins , 11 and Several studies have reported on the variation in hematological, serum, and plasma biochemical values for beluga whales in zoos and aquariums , 13–16. Results of studies of cetaceans in zoos and aquariums are particularly valuable because blood samples can be obtained voluntarily via a trained fluke present and blood draw behavior and the animal’s health history is known. The objective of this study was to obtain samples from a large cross-section of the zoo and aquarium populations in order to derive robust reference intervals and values for more variables than previously described. To assist in overcoming importation and exportation restrictions, reference intervals were developed for samples sent to laboratories in the United States and Mexico for common bottlenose dolphins. We aimed to report values for the first time or expand the available hematological, serum, and plasma biochemical reference values for Indo-Pacific bottlenose dolphins, beluga whales, and Pacific white-sided dolphins.This study was authorized by the management at each participating zoo and aquarium and, where applicable, was reviewed and approved by research committees. In addition, the study protocol was reviewed and approved by the U.S. Navy Marine Mammal Program Institutional Animal Care and Use Committee #123–2017.Zoos and aquariums that were accredited in 2017 by the Alliance for Marine Mammal Parks and Aquariums and the Association of Zoos & Aquariums were eligible for participation in this study provided they cared for common bottlenose dolphins, Indo-Pacific bottlenose dolphins, Pacific white-sided dolphins, or beluga whales.e.g., adult female). For Indo-Pacific bottlenose dolphins, beluga whales, and Pacific white-sided dolphins, all available individuals trained to voluntarily provide biological samples were selected. Blood sample collection attempts were conducted with each dolphin approximately six months apart between July through November 2018 and January through April 2019. Time periods were selected to correspond with one peak visitor month and one non-peak visitor month at each facility.To select cetaceans to participate in the study, each location provided a list of demographic information of all available participants. For common bottlenose dolphins, stratified sampling was used to select participants by dividing potential subjects into groups by sex and age class . Age class by sex was weighted by their frequency in the group of available participants to generate a target number of participants that represented the demographic distribution across facilities. Then, participants were randomly selected within their respective category , samples were not collected if the dolphin declined to comply with the requested behavior on the sample collection date. In addition, blood samples were not analyzed if they did not meet the volume required for testing. Of the samples processed at the UNAM, 94.4% were obtained from dolphins living in professionally managed ocean habitats . Of the samples process at the AHDC, 94.9% were obtained from dolphins living in professionally managed zoo/aquarium habitats .A total of 43 facilities participated in the study by providing data from cetaceans who met the inclusion criteria. Blood samples were collected from 174 common bottlenose dolphins, 27 Indo-Pacific bottlenose dolphins, 13 beluga whales, and 6 Pacific white-sided dolphins. The Cornell University Animal Health Diagnostic Center (AHDC) laboratory analyzed 122 common bottlenose dolphins, 47 Indo-Pacific bottlenose dolphins, 21 beluga whale, and 6 Pacific white-sided dolphin samples originating from Bermuda, Hong Kong, Jamaica, Singapore, Spain, and the United States for 56 hematologic, serum, and plasma biochemical variables. The Universidad Nacional Autónoma de México (UNAM) laboratory analyzed 162 common bottlenose dolphin samples originating from Mexico for 55 hematologic and serum biochemical variables. Samples analyzed at different laboratories were not compared. As blood samples were collected through a voluntary fluke-presenting behavior  vacutainer, a 5 mL BD vacutainer with a serum separator, and a 2.7 mL BD sodium citrate vacutainer using a 21 G, 3/4” infusion set. Two blood smears were made from the EDTA vacutainer within one hour of collection. Slides were not fixed or stained. After collection, the serum tubes sat at room temperature for 45 minutes until clot formation. The tube was centrifuged at 112 x g for 5 minutes. The serum was aspirated using a pipette and transferred to a 4 mL cryovial. Plasma vacutainers were immediately centrifuged at 112 x g. Plasma was aspirated using a pipette and transferred to a 1.2 mL cryovial. All samples were stored in a refrigerator at or below 4° C until shipping the same day as collection. Blood samples were tested in real time as they were collected and were not stored frozen for batch analysis.Samples originating in Mexico were shipped refrigerated to the UNAM for analysis. Hematology testing was performed by hematology analyzer EXIGO Vet® and blood chemistry was performed by biochemistry analyzer Dirui CS-T240 model. Samples originating from all other countries were shipped to the AHDC for analysis. Hematology testing was performed by hematology analyzer ADVIA 2120 and blood chemistry was performed by automated wet chemistry analyzer Cobas C501. The fibrinogen assay (Clauss method) was performed using an automated clot detection instrument  with the manufacturer’s reagents, standards, and quality control materials . Hematological, serum, and plasma biochemical values included in the study were limited to those reported by each laboratory.i.e., veterinarians not at the participating facilities) with expertise in marine mammals. Variables included in the analysis were sex, age at the time of sampling, and month the sample was collected. The list of hematologic, serum biochemical, and plasma variables tested at AHDC and UNAM are listed in Veterinary examinations, cytology findings, and blood reports for all study participants were blinded and then evaluated by external veterinarians  to determine the health status of each individual. A dolphin was considered to be healthy if they had no known disease, had an absence of abnormal clinical signs, were not on any medications that could potentially affect blood values, and did not receive antimicrobial treatments within 14 days before or after sampling. Whales and dolphins that were administered eye drops, nutritional supplements, or contraceptives were maintained in the study. Pregnant females were excluded from the final analysis.2, and month  by use of a mixed model to control for repeated samples. Age2 was included in the model to account for non-linear relationships between age and the dependent variables. Samples from common bottlenose dolphins tested at AHDC and UNAM were analyzed separately and not compared. Significance was defined as p < 0.01. A full mixed model using a training set that included 70% of the data set was created. The data were randomly split by observation rather than by individual to include a variety of individuals from multiple locations, a range of ages, and sample months. If significant differences were identified in variables by sex, age, age2, or month, models were run with only significant variables and reference intervals or values were developed using the appropriate parameters. For example, if sex and month were significant predictors, then they were included as parameters in the reference interval computations.Analyses were completed using the software package SPSS 21 and Microsoft Excel. For common bottlenose dolphins, Indo-Pacific bottlenose dolphins, and beluga whales, mean clinicopathologic values were compared by sex, age, agePrediction equations were extracted from each 70% training model and tested on the remaining testing subset (30%) of data. These predicted values were compared to the actual values by assessing the output from the mixed models, means, and standard deviations of the predicted value relative to the observed value.2, or month, models were run with only significant variables and reference intervals or values were developed using the appropriate parameters. If no significant predictors were identified, reference intervals and values were established from values that lay within two standard deviations from the mean for common bottlenose dolphins, Indo-Pacific bottlenose dolphins, and beluga whales. Reference intervals were produced for common bottlenose dolphins. As the sample size was smaller than 40 individuals, reference values were produced for Indo-Pacific bottlenose dolphins, Pacific white-sided dolphins, and beluga whales.Reduced linear models were produced that excluded both animal ID and facility ID for application to future samples. If significant differences were identified in variables by sex, age, agei.e., 95% confidence intervals) were developed using the following function:crit = critical value, and n = sample size, Xo = score on the predictor, sdX = standard deviation of the predictor [For variables with significant predictors, reference intervals or values (redictor .k IVs and the second summation is over k(k-1)/2 pairs of IVs expressed as standard scores, zio = absolute value of the IV, βij = standardized partial regression coefficient [The standard error of the estimate for a single observation with multiple predictors was calculated as:fficient .Due to the small sample size for Pacific white-sided dolphins, non-parametric reference values were generated. Data were ranked in order of magnitude and values between the 10th and 90th percentiles were retained as the reference values .ZooPhysioTrak is a native iOS mobile application that is compatible with the iPad, iPhone, and iPod Touch. Native applications are well-integrated with the operating system and do not utilize a web server or live internet connection during operation. ZooPhysioTrak was written in the Swift programming language. Test versions of ZooPhysioTrak were distributed via TestFlight. In order to ensure accurate operation, the build process for ZooPhysioTrak included automated unit testing to confirm it was producing and displaying accurate results.Several tests were employed based on a walkthrough of the statistical analysis with example values. They ensured that the calculation was done correctly, produced a correct numerical result, showed a correct textual display of the range, and displayed a correct unit. Within the program’s database, variables with significant predictors also contained example inputs and expected outputs; these were also tested to confirm the calculation for that variable was correct. Miscellaneous tests ensured that the program data was internally consistent, and that the ordering of displayed values was as desired.r2 for the full mixed models are given in 2 indicating that the effect lessoned with age. Reticulocyte count and absolute reticulocyte count were significantly lower as the calendar year progressed . Values of lymphocytes were significantly higher for female dolphins and significantly decreased with age. MPV values significantly increased with age and were significantly lower early in the year. Sex, age, age2, and month were not significant predictors for the remaining variables.All prediction equations from full mixed models and reduced linear models were based on models created with the training data subset . Predicti.e., winter/late rainy season). Reticulocytes, monocytes, Pt/Fin, Ca/P ratio, potassium, sodium, strong ion ratio, and lipase were significantly lower near the end of the year. There were no significant differences in all other variables.For common bottlenose dolphin samples analyzed at the UNAM, 16 variables were significantly affected by either month or sex predictors in the mixed models. Female dolphins were more likely than male dolphins to have higher triglyceride values. Dolphins were significantly more likely to have higher hematocrit, RDWa, total protein, globulin, phosphorus, bicarbonate, and amylase values as the calendar year progressed  are presented in For application to future samples, the results of the linear regression  are provided in ZooPhysioTrak was designed to provide a feature that supplies hematological, serum, and plasma biochemical reference intervals and values for common bottlenose dolphins, Indo-Pacific bottlenose dolphins, beluga whales, and Pacific white-sided dolphins Figs  and 2. UThe application took a no-keyboard approach. All data is entered via switches, segmented controls, and rotating pickers. Only valid data choices are displayed. If an interval can not be calculated, a reason such as “need age” or “no data” is displayed as appropriate. ZooPhysioTrak is compatible for side-by-side work with other applications. Data is not linked to a database for exportation; however, variables are listed in the same formatting order as provided by the diagnostic laboratories for easy comparison.Reference intervals and values for hematological, serum, and plasma variables established from healthy individuals have been previously reported for a variety of cetacean species living in the wild and under professional care , 19–22. Blood samples are tested at in-house laboratories or through a variety of commercial diagnostic laboratories. Laboratory choice is often determined by the historical use of a specific laboratory, physical proximity to aid in rapid turnaround of results for ill animals, trust of results, and economic cost including both shipping and analysis. Samples tested at different laboratories should only be compared following appropriate statistical analysis of parallel samples because the variation in results between testing laboratories leads to differences in reference intervals or values . For exa2, and month of the year were all found to be important determinants of several blood variables in the present data as well.The present study expands upon these reports for common bottlenose dolphins, beluga whales, and Pacific white-sided dolphins. Previously undescribed hematological, serum, and plasma reference values for Indo-Pacific bottlenose dolphins were established. Sex, age, season, and nutrition affect various clinicopathologic variables in cetaceans , 19, 24.In wild common bottlenose dolphins, juveniles had higher TIBC concentrations than adult dolphins , 25. ThiAlkaline phosphatase decreased with age for common bottlenose dolphins tested at AHDC and for Indo-Pacific bottlenose dolphins. Alkaline phosphatase is associated with the osseous activity that occurs during early growth , 28. PriIn Indo-Pacific bottlenose dolphins and common bottlenose dolphin samples analyzed at AHDC, hematocrit decreased with age. These trends were consistent with previous reports of hematocrit and platelet counts in common bottlenose dolphins under professional care . It is iFor common bottlenose dolphin samples analyzed at the AHDC lab and Indo-Pacific bottlenose dolphins, creatinine significantly increased with age. These findings were consistent with previous reports of creatinine values increasing with age for common bottlenose dolphins under professional care , 24. SeaIn the present study, creatinine kinase significantly decreased with age in common bottlenose dolphin samples analyzed at the AHDC laboratory. Juvenile common bottlenose dolphins in other populations also had significantly higher creatinine kinase than adults , 25–26. Significant predictors for blood variables were different for the two groups of common bottlenose dolphin samples that were tested at different diagnostic laboratories. It is possible that these differences were due to inter-laboratory variation. Another potential reason for the difference in predictors is the habitat locations. The majority of samples sent to the UNAM lab were obtained from animals housed in professionally managed ocean habitats while the majority of samples sent to the AHDC lab were housed in professionally managed zoo/aquarium habitats. Seasonal changes affect many hematological and serum biochemical values of wild dolphins , 24. BluChanges in triglyceride values are associated with dietary changes, lipid metabolism, and potentially hormonal factors. Consistent with findings in wild common bottlenose dolphins , triglycIn common bottlenose dolphins, juveniles have higher mean concentrations of blood urea nitrogen than adults , 25. In The ZooPhysioTrak iOS application was developed in order to facilitate easy access to the reference intervals and values for the four species of cetacean. The application supplies a streamlined method of viewing reference intervals and values for an individual while taking into account significant predictors. ZooPhysioTrak delivers valuable information that can be used by animal care professionals to guide health assessments and decisions.This work allows the cetacean facility clinician and care providers to see the comparative health results of quality controlled, commonly used diagnostic tests usually run in many unrelated laboratories. Laboratory choice is often determined by the need for a rapid turnaround time with ill animals, trust of results, and economic considerations that include test costs and mailing expense. Data quality can be affected by machine type, technicians with varying levels of experience and skill, interpretation of cytology findings, and variations in quality control. This presents one limitation of the present study. While the samples were analyzed following standardized protocols at both testing laboratories, it is possible that there is intra-testing variability within the same lab because the samples were not tested as a single batch. However, this variability would fall within the range that would occur when using these values to compare results from future samples.Following current best practice, reference intervals and values from laboratories that are generated from healthy conspecifics can be used to complement robust individual reference intervals. Generally, voluntary blood samples can be frequently collected from cetaceans and used to build a database of an individual’s historic reference values. Both species and individual level reference intervals can be used in concert to inform health management decisions. Where questions of results develop for individuals, laboratories may provide a comparative bench mark with individual reference values alongside intermittent parallel samples for quality control. The list of standard health tests should be analyzed by reputable, experienced laboratories with the goal of setting in-house species and individual reference intervals over time. Importantly, a robust historical record of an individual’s blood values will allow professionals to take into account any potential variation that may exist as a result of the visitor high and low seasons. The reference ranges presented in ZooPhysioTrak include values from blood samples collected in one high and one low visitor season at each facility. The high and low visitor season varied in the month of the year that they occurred based on the geographic location. Future research should also examine if the high/low visitor seasons affect blood values.While the reference intervals and values presented here and in ZooPhysioTrak are for healthy individuals, sustained values in the higher or lower portions of the given ranges should be closely monitored. For example, serum iron may vary depending on age, gender, environment, diet, and nutritional status . GeneralContinued research is necessary to understand the effects of diet, seasonality, and water temperature on blood values. Diet has been suggested to affect several blood variables , 9, 36. 2, and month predictor variables were found to be important determinants of several blood variables in the present data. For common bottlenose dolphins, the results reported here were generated from apparently healthy dolphins residing in a large number of accredited zoos and aquariums resulting in the first reported reference intervals that are applicable across facilities. This is the first report of a hematological, serum, and plasma values and reference values for Indo-Pacific bottlenose dolphins. While blood variables for beluga whales have been previously reported, the sample collection and processing protocols given in ZooPhysioTrak and reported values provide a standardized starting point for comparing future values. The reference values provided for Pacific white-sided dolphins presented an updated and expanded suite of blood parameters. The reference intervals and values and ZooPhysioTrak supply veterinarians and animal care professionals with valuable information that can be utilized in providing cetaceans in zoos and aquariums optimal care. Future research should examine the effects that other variables  may have on blood values.The hematological, serum, and plasma reference intervals and values provide an important diagnostic tool for future health assessments for cetaceans in accredited zoos and aquariums. The data are representative of cetaceans in a variety of habitats and management experiences. Sex, age, ageS1 Data(XLSX)Click here for additional data file.S1 Fig(TIFF)Click here for additional data file.S1 TableList of blood variables included in the analysis from the Cornell University Animal Health Diagnostic Center.(DOCX)Click here for additional data file.S2 TableList of blood variables included in the analysis from the Universidad Nacional Autónoma de México.(DOCX)Click here for additional data file.


Validated biomarkers that more accurately predict prognosis and/or measure disease burden in patients with high-grade gliomas would help triage which treatment strategies are most promising for evaluation in Phase III multicenter trials. Multicenter trials to evaluate imaging biomarkers in this group face particular challenges; these trials have historically been slow to accrue and have not recently succeeded in validating new imaging biomarkers useful in treatment development. Due to variability in image acquisition protocols, scanner hardware, image analysis, and interpretive schemes, promising results obtained in single centers are poor predictors of success in the multicenter setting. Multicenter preliminary data to support further evaluation of imaging biomarkers is rarely available. The need for more efficient trial designs that bring multicenter data earlier into the process of biomarker development has become increasingly clear. In this presentation, the planning process within ECOG-ACRIN’s Brain Tumor Working Group for a platform multicenter trial called GABLE  designed to evaluate biomarkers for distinguishing pseudoprogression from true progression in patients with newly diagnosed GBM is described. In our planning process, it was determined that efficiencies can be gained from evaluating multiple biomarker types in parallel rather than serially; in the context of the proposed trial, not only conventional imaging biomarkers but plasma biomarkers and radiomic biomarkers can be evaluated simultaneously. Patient tolerance limits the feasibility of evaluating multiple non-standard-of-care imaging biomarkers in parallel. For this group of biomarkers, a “fast-switching” serial evaluation strategy using multiple interim analyses was developed to triage out biomarkers unlikely to succeed in identifying patient groups with clinically significant differences in median survival. For biomarker triage, an endpoint of event-free survival (events of either death or NANO progression) was proposed. Simulations were used to evaluate alpha and beta error using this evaluation strategy.


Background and Objectives: Facing neoadjuvant chemotherapy followed by surgery, neoadjuvant immunotherapy is an innovative concept in localized muscle-invasive bladder cancer. Herein, we performed a review of the available and ongoing evidence supporting immune checkpoint inhibitor (ICI) administration in the early stages of bladder cancer treatment. Materials and Methods: A literature search was performed on Medline and clinical trials databases, using the terms: “bladder cancer” OR “urothelial carcinoma”, AND “neoadjuvant immunotherapy” OR “preoperative immunotherapy”. We restricted our investigations to prospective clinical trials evaluating anti-PD-(L)1 and anti-CTLA-4 monoclonal antibodies. Data on efficacy, toxicity and potential biomarkers of response were retrieved. Results: The search identified 6 ICIs that were tested in the neoadjuvant setting for localized bladder cancer—4 anti-PD-(L)1 inhibitors  and 2 anti-CTLA-4 inhibitors (Ipilimumab and Tremelimumab). Most of the existing literature was based on single-arm phase 2 clinical trials that included from 23 to 143 patients. The pathological complete response rate (pCR) and pathological response rate (pRR) ranged from 31% to 46% and from 55.9% to 66%, respectively. Survival data were immature at this time. The safety profile was acceptable, with severe treatment-related adverse events ranging from 6% to 41%. Conclusions: The results of early phase trials are encouraging, and more investigations are needed to strengthen the rationale for immune checkpoint inhibitor administration in localized muscle-invasive bladder cancer. Bladder cancer accounts for 90% to 95% of urothelial carcinoma, with invasion of the muscle wall accounting for 30% . The curThe PD-1 pathway has an important role in local immunosuppression in the tumor microenvironment, but it can also have a role in the modulation of T cell priming. In recent years, immune checkpoint inhibitors (ICIs) targeting programmed cell death, regarding protein-1 (PD-1), programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4), have become another pillar of cancer treatment. Concerning urothelial carcinoma, they have shown durable responses in locally advanced and metastatic patients, at first in a second-line setting , and morp-values. In addition, as immunotherapy is a rapidly developing field, we examined abstracts from major oncology conferences until ASCO-GU 2021 (February 2021). We performed our literature search covering the period from 1 January 2010 to 1 March 2021. Three independent authors  performed the screening of titles, abstracts, and full-text sharing, and accepting the final list of publications that would be included.We conducted a comprehensive review, using a literature search for current data on perioperative ICIs, using the PubMed and ClinicalTrials.gov databases. The following terms were used: “bladder cancer” OR “urothelial carcinoma”, AND “neoadjuvant immunotherapy” OR “preoperative immunotherapy”. Only prospective studies evaluating ICIs in the neoadjuvant setting in localized bladder cancer were included. Studies were excluded if they involved non-muscle invasive bladder cancer (NMIBC), locally advanced, or metastatic MIBC. Other exclusion criteria were a lack of proven diagnosis, efficacy or safety, and the absence of key information, such as hazard ratios, confidence intervals and The search identified 6 ICIs that were tested in the neoadjuvant setting for localized bladder cancer: 4 anti-PD-(L)1  and 2 anti-CTLA-4 (Ipilimumab and Tremelimumab). Most of the existing literature was based on single-arm phase 2 clinical trials, as summarized in Oncological outcomes among the studies are displayed in ICIs have mainly been tested in the neoadjuvant setting in cisplatin-ineligible patients. Despite their demonstrated efficacy, ICIs come with a certain degree of potential toxicity. A wide range of side effects has been observed in the presented trials, with fatigue and skin rashes being the most common. Severe treatment-related adverse events  ranged from 6% to 41% among studies, thus leading to discontinuation or delaying immunotherapy administration in some cases. To our knowledge, no death was reported. The highest rates of adverse events were seen when ICIs were combined. Thus, the associations of Nivolumab + Ipilimumab and of Durvalumab + Tremelimumab reported 41% and 21% of severe AEs rates, respectively, while monotherapies claimed to have less than 10% of severe toxicity. Moreover, the association of several ICIs seems to emphasize side effects without increasing the pathological response rate. Overall, monotherapy with ICIs is usually well tolerated. In the recent PURE-01 trial, thyroid dysfunction was the most frequent all-grade adverse event (AE) (18%). Grade 3 AEs were observed in 6% of patients, and discontinuation of Pembrolizumab was necessary for one of the fifty enrolled patients . HoweverWith such a craze for PD-(L)1 and CTLA-4 monoclonal antibodies, there is a real need for selecting good responders to immunotherapy. All previous trials have analyzed a panel of biomarkers in order to distinguish those people who will benefit the most from ICIs. PD-L1 expression on surgical specimens was the most studied biomarker and has been positively correlated with response in the PURE-01 and NABUCCO trials, whereas MDACC did not find any statistical association. The tumor mutational burden was also assessed with a cut-off placed between 10 and 15 Mut/Mb and was found to be associated with pT0 response . First op = 0.06) and improved the rate of pCR, defined as pT0N0 . This study defined pCR as a surrogate for the efficacy for neoadjuvant chemotherapy since 85% of pT0 patients were free of disease at five years. Other trials conducted in this area were unable to demonstrate the benefit of neoadjuvant chemotherapy for different reasons . In the last decade, alternative regimens (GC) and schedules such as dose-dense MVAC (ddMVAC) have also been tested in two single-arm studies, showing similar pCR compared to conventional MVAC but within a much shorter period of time. More recently, in a randomized phase-III study, ddMVAC has demonstrated a higher local control rate  compared to neoadjuvant CG , with more severe asthenia and gastrointestinal side effects [Neoadjuvant cisplatin-based chemotherapy combinations for cT2-T4a N0 bladder cancer are the current standard of care strategy for fit patients, having demonstrated an overall survival benefit in phase III trials and in meta-analyses . The SWO effects . However effects . Importa effects . ConsequThe emerging data from the different trials exploring the role of ICIs in the neoadjuvant setting of UC are promising. The pCR rates observed might lead to a change in the neoadjuvant scenario and set new treatment standards. However, some questions remain unanswered. Firstly, the identification of patients who are most likely to benefit from ICI administration needs to be investigated. There are no molecular biomarkers that are properly validated for patients’ selection. The anti-tumor effect of immunotherapy does not correlate with the PD-L1 expression status, as PD-L1 low-expression tumors may also respond to ICIs. The neoadjuvant setting is an ideal translational research setting, due to the possibility of pre- and post-treatment evaluation. Secondly, we need mid- and long-term survival data to demonstrate the oncological benefits associated with ICI administration; unlike with neoadjuvant chemotherapy, we do not yet have evidence that pCR is a surrogate of overall survival. Moreover, the currently available data come from single-arm phase 2 trials; we need randomized controlled trials to assess the benefits associated with ICIs administration compared to neoadjuvant chemotherapy in all-comers, and also versus upfront surgery in cisplatin-ineligible patients. Thirdly, we need to determine the optimal setting between a neoadjuvant regimen or the combination of a neoadjuvant and maintenance post-surgery regimen, as in the first-line metastatic setting .Neoadjuvant cisplatin-based chemotherapy has been the standard of care for muscle-invasive bladder cancer for decades. With outstanding results in locally advanced and metastatic urothelial carcinoma, the place of ICIs is being moved forward to earlier stages of the disease. Several phase-2 trials have tested ICIs in localized bladder cancer in the neoadjuvant setting, with promising results and acceptable toxicity. Predictive biomarkers of response are yielded to improve the selection of patients for these new treatment strategies. In order to incorporate them in daily practice, ICIs need randomized controlled trial evidence and longer follow-ups to be implemented as a new standard of care in localized muscle-invasive bladder cancer.


A long-term maximum compressive strength was measured to be ~16 MPa for both the optimized SMPC and the control ordinary pervious concrete (Control PC). The phosphorus removal was favorable for both the optimized SMPC and the Control PC based on the dimensionless Freundlich parameter (1/n). Both the optimized SMPC and Control PC had a first-order phosphorus removal constant of ~0.03 h−1. The optimized SMPC had a slightly lower capacity of phosphorus removal than the Control PC based on the Freundlich constant, Kf (mg1−1/n kg−1 L1/n): 15.72 for the optimized SMPC vs. 16.63 for Control. This study demonstrates a cleaner production and application of off-spec FA-added, seawater-mixed pervious concrete to simultaneously attain water, waste, and concrete sustainability.A mix proportion of off-spec fly ash (FA)-added, seawater-mixed pervious concrete (SMPC) was optimized for compressive strength and permeability and then the optimized SMPC was tested for the rate and extent of aqueous phosphorus removal. An optimum mix proportion was obtained to attain the percentages (% wt.) of FA-to-binder at 15.0%, nano SiO Pervious concrete pavement is one of the most effective management practices for stormwater runoff control . It redu2 emissions to the atmosphere  is the hydroxide concentration in water (mol/L).The rate of aqueous phosphorus 6(OH)2 and Ca3(PO4)2 as the phosphorus removal mechanism as the study did not characterize the precipitates. It could have been attributed to other minerals present in seawater. A further investigation is needed to clarify this speculation.There was an increase in pH to ~11.0 from the initial 7.7 after 24 h of contact time during the kinetic experiment in the current study. However, it is premature to account the precipitation in the form of Caeq is the removed phosphorus amount at equilibrium (mg/kg), fK is the Freundlich constant (mg1−1/n kg−1 L1/n), eC is the aqueous phosphorus concentration at equilibrium (mg/L), and 1/n is a dimensionless parameter. The fK and 1/n for SMPC were found to be 15.72 and 0.4357, respectively and those for the Control PC were 16.63 and 0.4272, respectively. Based on the 1/n’s which were less than 1, it can be said that both SMPC and Control PC favored phosphorus removal. The fK indicates a removal capacity, but they are only comparable when the 1/n’s are the same because of the dependence of the unit of fK on 1/n. As the values of 1/n in this study were similar for SMPC and Control PC , it is construed that SMPC had a slightly reduced phosphorous removal ability than the Control PC, judged by its lower fK of 15.72, compared to 16.63 of the Control. These findings are in good agreement with Wu et al. [fK of 16.6 and a 1/n of 0.719, whereas light weight silica aggregates had a fK and 1/n of 17.4 and 0.720, respectively . On the other hand, Vazquez-Rivera et al. [fK of 2.48 and a 1/n of 1.7 from aqueous phosphorus removal with pervious concrete optimized with nano-iron and FA.An isotherm study was conducted at room temperature (20 ± 2 °C) for 72 h of contact time which was determined from the kinetic study. A non-linear removal phenomenon was observed a for botu et al.  who repoa et al.  reportedThis study demonstrated a cleaner production of pervious concrete with seawater as a mixing water and an off-spec FA as a partial cement replacement to simultaneously attain water, waste, and concrete sustainability;The permeability of SMPC specimens ranged from 1.98 to 8.87 mm/s with the maximum compressive strength of ~16 MPa, which was found to be practically acceptable;The optimized SMPC had a slightly greater compressive strength at the early ages than the Control PC;For both SMPC and Control PC, aqueous phosphorus concentrations were dramatically decreased by 90% after 72 h of contact time, indicating that SMPC showed a plausible potential to improve water quality as a green infrastructure to manage stormwater runoff;Future studies are warranted to elucidate the mechanisms of aqueous P removal by FA-added, seawater-mixed pervious concrete, to test other waters including intermediate or reject seawater from desalination processes, and to understand the effect and fate of the constituents of concern in seawater such as microplastics.Cement and water are key ingredients to produce cement-based infrastructure. However, the production of cement creates environmental damage in terms of carbon dioxide release and the use of freshwater for mixing cement-based materials is unfavorable considering the shortage of freshwater resources worldwide. FA has been used as a partial replacement of Portland cement in sustainable concrete production. However, those off-spec FAs that are not in compliance with standards such as ASTM are typically landfilled, which can potentially cause different environmental and health problems. Based on the findings, the following conclusions and recommendations can be made: