The pressure to use sustainable materials and adopt practices reducing the carbon footprint of the construction industry has risen. Such materials include recycled concrete aggregates (RCA) made from waste concrete. However, concrete made with RCA often presents poor fresh and hardened properties along with a decrease in its durability performance, especially when using its fine fraction . Most studies involving FRCA use direct replacement methods (DRM) to proportion concrete although other techniques are available such as the Equivalent Volume (EV) and Particle Packing Models (PPMs); yet their impact on the durability performance, especially its performance against freezing and thawing (F/T), remains unknown. This work, therefore, appraises the F/T resistance of FRCA mixtures proportioned through various mix proportioning techniques , produced with distinct crushing processes . The results show that the mix design technique has a significant influence on the FRCA mixture’s F/T resistance where PPM-proportioned mixtures demonstrate the best overall performance, exceeding the specified requirements while DRM-proportioned mixtures failed F/T resistance requirements. Moreover, the crushing process plays an important role in the recycled mixtures’ cracking behavior under F/T cycles, where less processing leads to fewer cracks while remaining the most sustainable option overall.  Canada has set its target of reaching the so-called net-zero by 2050 [2 emissions [Tackling climate change is among the top priorities worldwide, with emphasis on reducing CO by 2050 . There imissions ,4,5. Themissions ,7,8,9.RCA is produced by crushing returned or demolished concrete into aggregate-sized fractions. Generally, the coarse RCA fraction is used in new concrete construction, often limited to non-structural applications due to concerns over the material’s variability, quality and presence of impurities , whereas the fine RCA (FRCA) fraction is rejected, being considered as a by-product or waste ,5. Much of the research on RCA implicitly treats the material as being homogeneous and directly replaces certain proportions of natural aggregate by RCA through partial or full direct replacement methods (DRM) in the concrete mixture without further consideration of its multi-phase nature, especially for FRCA. Indeed, the multi-phase nature of FRCA, consisting of residual cement paste (RCP) adhered to residual sand (RS) and/or fractured coarse original virgin aggregate (OVA), influences the performance of recycled concrete mixtures, often resulting in inferior mechanical and durability properties. Moreover, given the high variability of RCA, accurately estimating and/or predicting the fresh and hardened state performance of recycled mixtures designed through the DRM method remains a challenge ,11,12,13Concrete waste is crushed then sieved to obtain certain particle sizes. Fine recycled concrete aggregates (FRCA) are normally considered as a by-product of coarse RCA (CRCA) production. Consequently, CRCA is composed of mainly original virgin aggregate (OVA) and residual mortar (RM); the latter can even represent up to 60% of the CRCA volume . MeanwhiPhysical properties of the FRCA particles are influenced by several factors such as the amount of RCP found in FRCA particles that can be influenced by the type and quality of the OVA and RS , the RCP’s quality  and the FRCA crushing process , leading to important variability among the final product ,21. MoreFRCA is generally considered a very low-quality material due to its high amount of RM, resulting in low specific gravity, high porosity and absorption, the latter of which corresponds to the increased water demand of the FRCA concrete. Some studies were performed to quantify the RM of FRCA ,23,24; hDespite all recent advancements in recycled concrete using FRCA, when compared to conventional concrete (CC), recycled concrete mixtures designed through conventional methods using partial over 30%) or full FRCA replacement were observed to often yield lower performance in the fresh  and hardened  states along with durability % or full,39,40,41Direct replacement methods (DRM), either partial/full or by weight/volume, are one of the earliest attempts to mixture proportion concrete using FRCA. Studies show that a replacement ratio of less than 30% provides optimal results ,42,43,44The Equivalent Volume (EV)  is the mq) along with the largest (LD) and smallest (SD) particle diameter present in the system. The coefficient of distribution is normally adopted based on the fresh state requirements of the mixture. Distribution coefficients ranging between 0.20 and 0.23 are often selected for self-consolidating mixtures, while values between 0.26 and 0.28 are normally targeted for vibrated and/or pumped concrete. It is worth noting that 0.37 is the coefficient of distribution that yields the highest packing density and thus lowest porosity to the granular system as per [CPFT is the cumulative (volume) percent finer than PD and PD is the particle diameter.Particle packing models (PPMs) are advanced mix design techniques used to improve the performance of concrete mixtures in both fresh and hardened states while optimizing the granular system, resulting in an improved packing density and reduced porosity ,49,50,51A convenient evaluation of the inner quality of concrete mixtures may be performed using non-destructive techniques (NDT), such as ultrasonic pulse velocity (UPV) and surface electrical resistivity (ER). Such techniques were verified to be suitable to appraise the inner quality of CC mixtures subjected to aggressive/harsh environments  along wiMoreover, regardless of the mixture proportioning technique used, RCA concrete mixtures generally present lower stiffness when compared to CC ,58,59, lFreeze-thaw (F/T) resistance of CC is required in Canada’s harsh environments and is linked to various factors such as the aggregate features, cement paste porosity and free water availability in the system. These parameters may be even more critical for recycled concrete since its higher porosity may contribute to an increase in the material’s absorption and permeability ,64. Some2 for comparative purposes. Moreover, the extended version of the DRI  can be used to evaluate the crack propagation and distribution within a CC [The Damage Rating Index (DRI) is a microscopic tool developed to assess the damage and its extent in CC ,68,69,70hin a CC ,68 as wehin a CC ,73,74.Nevertheless, the durability performance of concrete made of FRCA, namely its freezing and thawing resistance, while using several distinct mixture proportioning techniques  and crushing processes  remains mostly unknown. Moreover, techniques such as the SDT and DRI are novel test procedures used to evaluate the inner quality and or damage degree of concrete and have never been used to evaluate concrete made of FRCA before.Currently, there is a lack of studies on the durability and long-term performance of RCA, especially FRCA, which hinders its use in the industry. Moreover, the impact of distinct mix design procedures on the durability performance of recycled concrete mixtures made of FRCA is mostly unknown. Thus, this work aims to evaluate the durability related properties, namely the resistance against freezing and thawing (F/T) cycles, of concrete made of 100% FRCA replacement proportioned through various mix proportion techniques , produced with distinct crushing processes  vs. finely ground (FG)) and derived from conventional concrete mixtures incorporating natural and manufactured sand. By using non-destructive techniques (NDT), the compressive strength and the Stiffness Damage Test (SDT), the FRCA concrete mixtures’ inner quality and flaws were appraised before being subjected to F/T cycles as per ASTM C666 . FurtherTwo-hundred 35 MPa CC cylinders  were fabricated according to ASTM C39  while vaFollowing the CC production, the specimens were de-molded after 24 h and moist cured (100% RH and 20 °C) for 28 days before being processed to produce the FRCA. Further characterization of the residual cement paste (RCP) content was performed for each type of FRCA through the soluble silica sub-procedure according to ASTM C1084  and C114In order to produce the FRCA concrete mixtures, the same General Use-GU Portland cement and coarse limestone aggregate  were selected for use. Their physical properties were determined as shown in Three distinct FRCA concrete mixtures proportioned with distinct techniques  and containing 100% replacement of the fine aggregate by FRCA were designed . MoreoveTwenty-seven cylindrical (100 mm × 200 mm) specimens were produced for the CC and FRCA concrete mixtures. Likewise, three 75 mm × 75 mm × 300 mm prisms per concrete mixture  were fabricated for the accelerated freeze-thaw tests as per ASTM C666 , de-moldNon-destructive techniques  and dynamic modulus of elasticity through ultrasonic pulse velocity test (UPV)) and compressive strength tests were performed on three specimens from each concrete mixture at 28 days. Furthermore, the stress–strain relationship of selected mixtures before being subjected to F/T was evaluated at 28 days through the use of the Stiffness Damage Test (SDT) as per Sanchez et al. ,62. For All recycled mixtures fabricated were tested against F/T cycles through the use of the accelerated laboratory test procedure as per ASTM C666 . The procP; n is the fundamental transverse frequency (Hz) at 0 cycles and n1 is the fundamental frequency after “c” cycles of F/T. It is worth noting that the test was performed up until 300 cycles or until the sample reached a 40% decrease in the initial relative dynamic modulus of elasticity value.The relative dynamic modulus of elasticity of each sample was calculated based on the fundamental transverse frequency measurements, as shown in Equation (2):cL (%) is the length change after “c” cycles of F/T; l1 and l2 are the comparator length readings at 0 and “c” cycles, respectively, and gL is the effective gauge inner length measured between the gauge studs. As per ASTM C 666 [The length changes were measured according to ASTM C490  and calcTM C 666 , an expaDF) shown in Equation (4):DF is the durability factor of the specimen (0–100%), which corresponds to the reduction in dynamic ME over freeze-thaw cycles; NP (%) the dynamic ME after the Nth F/T cycle; N refers to the number of cycles at which the dynamic ME reaches the specified minimum value before discontinuing the test or the maximum number of cycles for the analysis ; and M is the specified number of cycles at which the exposure is to be terminated .Finally, the overall performance of the mixtures against F/T action was evaluated based on the durability factor  was used to assess the F/T damage in FRCA concrete mixtures. Concrete prisms subjected to 300 cycles of F/T were sawn in half longitudinally using a masonry saw equipped with a diamond blade followed by successive grinding and polishing  until a flat reflective surface was achieved. A grid of 1 cm × 1 cm squares was drawn on this surface in which distinct types of cracks , open crack in the aggregate (OCA), cracks in the residual and new cement paste ) were counted using a stereomicroscope at 16× magnification. Those cracks were then weighted using the weighting factors proposed by  and norme counts ,68.The inner quality of the concrete before being subjected to freezing and thawing (F/T) cycles was evaluated through non-destructive techniques (NDT), hardened state properties and the stress–strain relationship. The influence of the different mix designs used to proportion the FRCA mixtures was also evaluated using the SDT before being subjected to F/T to appraise the inner quality and flaws  of the recycled mixtures. The SDI, an output parameter obtained through the SDT, represents the amount of energy required to close inner flaws, while the PDI represents the plastic deformation of concrete subjected to loading. Due to testing limitations, only the natural sand (NS) was selected for evaluation along with the CF crushing procedure since the compressive strengths for PPM-proportioned mixtures made with CF–FRCA were closer to the targeted 35 MPa  compared to the FG–FRCA. Moreover, the PPM-proportioned FRCA concrete mixtures were compared by the crushing procedure due to the significant difference observed through the NDT and compressive strength results. The SDT appraises three important parameters SDI, PDI and static modulus of elasticity, which are presented in After being subjected to the F/T cycles, the mass loss of the concrete specimens was measured. The average changes in length for all FRCA concrete mixtures subjected to F/T cycles are displayed in The average residual dynamic modulus of elasticity results for all FRCA mixtures are illustrated in The durability factors for each FRCA mixture were calculated following the F/T cycles using Equation (4) and are presented in The Damage Rating Index (DRI) was used to complement the above results obtained and better understand the cracking behavior of recycled mixtures using FRCA. 2) is higher than other mixtures , its number of cracks in the cement paste and its proportions  are lower than the EV-proportioned mixtures, which range from 236 to 257 counts/cm2 and 60 to 70% for the crusher’s fines (CF) and finely ground (FG) FRCA, respectively. Otherwise, a lower number of cracks in the cement paste is observed in the PPM-proportioned mixtures at 91 and 231 counts/cm2 for the CF and FG FRCA, respectively.Overall, more cracks are observed in the new cement paste  for all FRCA concrete mixtures, while very few cracks in the residual cement paste  were observed. Although the total number of cracks in the DRM-proportioned mixture  and crushed using two distinct processes  to evaluate their resistance to freezing and thawing further. The electrical resistivity and dynamic modulus of elasticity  provide insight into the inner quality  and flaws  of the concrete before being subjected to freezing and thawing cycles. As presented in The same trend is observed through the compressive strength; however, the influence of the crushing procedure is apparent for the PPM-proportioned mixtures. The fully ground FRCA produced significantly higher results at 62 MPa and 65 MPa for the natural and manufactured sand, respectively, although a 35 MPa concrete was targeted, yet not achieved, for the mixtures proportioned through the EV and DRM . Nevertheless, PPM-proportioned FRCA mixtures, especially the fully ground FRCA, present better results when compared to EV- and DRM-proportioned mixtures, thus producing FRCA concrete with a better overall inner quality . Besides the lower amount of residual cement paste of the fully ground FRCA, the improvement of the FRCA–PPM–FG mixture may also be explained due to the content of the inert filler. Since the volume of filler added in PPM mixtures was equivalent to the volume of the residual cement paste of FRCA, FRCA–PPM–FG mixtures resulted in higher cement content and lower filler content than FRCA–PPM–CF mixtures. This slightly higher cement content might have improved the bond between the FRCA particles, and the new cement paste is fully ground mixtures due to the natural self-healing mechanism developed by the hydration of Portland cement, a phenomenon widely reported in the literature. Despite the results obtained through non-destructive techniques (NDTs) and the compressive strength, the EV-proportioned mixtures displayed the lowest performance  out of all mixture proportions used for FRCA concrete. As such, the SDIs of the DRM-proportioned mixtures were found to be similar to those of the PPM-proportioned mixtures, both of which resemble sound concrete. However, the same constituents were used in all mixtures, yet at different proportions, which were captured by the SDT through the SDI and PDI . Althoug3) when compared to DRM mixtures (524–551 kg/m3). Hence, the overall stiffness of the concrete may be governed by the higher number of interfaces found within the FRCA concrete. On the other hand, the PPM-proportioned mixtures contain the most FRCA (879–915 kg/m3), thus comprised of an even higher number of interfaces; however, all particles were more efficiently distributed/packed in a system with lower porosity. Since the residual cement paste was accounted for in EV-proportioned mixtures and disregarded in DRM, the difference in its performance may be justified by the higher FRCA content present in EV mixtures  and the crushing procedure  did not show any trends. Similar to the non-destructive techniques and compressive strength results, the DRM-proportioned FRCA concrete showed the poorest performance in mass loss, length change and residual modulus of elasticity and exceeded ASTM C666  limits wIn order to obtain a more thorough understanding of the resistance to freezing and thawing of the various concrete mixtures made of FRCA, a thorough microscopic analysis through the Damage Rating Index (DRI) method was performed. Consequently, the DRM-proportioned mixture evaluated in the microscopic analysis showed a significant number of open cracks within the aggregates, usually associated with distress as opposed to closed cracks in the aggregates , and proNoticeably, a similar trend is observed for the PPM- and EV-proportioned mixtures where fully ground FRCA was more susceptible to cracking than the crusher’s fines FRCA. Before being subjected to freezing and thawing, the fully ground FRCA exhibited better results than the crusher’s fines. A fully ground FRCA is subjected to a more intensive crushing procedure, hence the higher removal of residual cement paste, exposure of residual sand and original virgin aggregate (OVA) and potentially the introduction of a higher number of microcracks; however, cracks produced by freezing and thawing were uncommonly observed within the FRCA and mostly present within the new cement paste. The crushing procedure, therefore, influenced the cracking behavior of the FRCA concrete such that the formation of new interfaces may govern this behavior where residual to new mortar interfaces show more resistance to cracking than residual sand/OVA to new mortar interfaces due to the difference between the stiffness of the components . The litThe PPM-proportioned mixtures showed the highest inner quality before being subjected to freezing and thawing, followed by the EV and DRM-proportioned mixtures. The influence of the type of residual sand was not as significant as the crushing procedure, indicating that FRCA subjected to a more rigorous crushing sequence presented a better inner quality;The EV and PPM mixture proportioning techniques produced a concrete made of FRCA having adequate freezing and thawing resistance, while the DRM-proportioned mixtures were not considered resistant to freezing and thawing. The PPM mixtures showed the best performance followed by EV then DRM-proportioned FRCA concrete mixtures while no apparent trend was observed between the type of residual sand and crushing procedure;The overall durability factor of all FRCA mixtures subjected to freeze-thaw cycles was observed to have considerable variation between the mix design methods. A higher durability factor was observed for PPM mixtures, followed by EV then DRM. This demonstrates that the mix design procedure adopted to design FRCA concrete is more important than the material’s quality; PPM and EV-proportioning techniques being capable of reducing the variability presented while using the DRM;The DRI captured the differences in the damage propagation of FRCA concrete subjected to freezing and thawing. The highest level of damage was observed in DRM-proportioned mixtures, whereas the EV- and PPM-proportioned mixtures showed less damage. Despite showing a better performance before being subjected to freezing and thawing, the fully ground FRCA concrete presented more damage compared to the crusher’s fines FRCA. The crushing procedure significantly influences the mechanical properties, inner quality and crack generation and propagation in FRCA concrete. Further research is therefore required to understand the cracking behavior of RCA  concerning its multi-phase nature.Three types of mix proportioning techniques  were used in this work and assessed by evaluating the inner quality and the resistance to freezing and thawing of FRCA concrete designed with 100% replacement. The main findings can be highlighted hereafter:The freezing and thawing resistance of concrete made of FRCA was significantly improved when using mixture proportioning techniques different than the direct replacement method, which failed to comply with the specified requirements. Although it is the most used mix design technique in practice, it is evident that direct replacement methods not only show the poorest performance overall but also require a much higher cement content resulting in an unsustainable approach. Furthermore, it is clear that the inner quality of FRCA manufactured through multiple series of crushing  is enhanced when compared to only two series ; However, when the proportion was properly mixed, recycled concrete mixtures made of both FG and CF particles displayed quite suitable results. Therefore, it is recommended that the CF method be adopted since it requires less energy and thus provides a more sustainable material with less generation of waste.


Hypoxic microenvironment is a hallmark of solid tumors, especially glioblastoma. The strong reliance of glioma-propagating cells (GPCs) on hypoxia-induced survival advantages is potentially exploitable for drug development.Methods: To identify key signaling pathways for hypoxia adaptation by patient-derived GPCs, we performed a kinase inhibitor profiling by screening 188 small molecule inhibitors against 130 different kinases in normoxia and hypoxia. Potential kinase candidates were prioritized for in vitro and in vivo investigations using a ranking algorithm that integrated information from the kinome connectivity network and estimated patients' survival based on expression status.Results: Hypoxic drug screen highlighted extensive modifications of kinomic landscape and a crucial functionality of c-MET-PI3K. c-MET inhibitors diminished phosphorylation of c-MET and PI3K in GPCs subjected to hypoxia, suggesting its role in the hypoxic adaptation of GPCs. Mechanistically, the inhibition of c-MET and PI3K impaired antioxidant defense, leading to oxidative catastrophe and apoptosis. Repurposed c-MET inhibitors PF04217903 and tivantinib exhibited hypoxic-dependent drug synergism with temozolomide, resulting in reduced tumor load and growth of GPC xenografts. Detailed analysis of bulk and single-cell glioblastoma transcriptomes associates the cellular subpopulation over-expressing c-MET with inflamed, hypoxic, metastatic, and stem-like phenotypes.Conclusions: Thus, our “bench to bedside (the use of patient-derived GPCs and xenografts for basic research) and back ” analysis unravels the novel therapeutic indications of c-MET and PI3K/Akt inhibitors for the treatment of glioblastoma, and potentially other cancers, in the hypoxic tumor microenvironment. Hypoxia, a phenomenon depicted by low oxygen bioavailability, is a hallmark of the tumor microenvironment (TME). In solid tumors, hypoxic regions often arise because of rapid tumor cell proliferation, which outgrows the oxygen perfusion and nutrient supply from normal vasculature Glioblastoma (GBM) is a devastating form of brain cancer with a dismal median survival duration, a high level of resistance to current therapy, and common recurrence after treatment. The median survival rate of GBM is 15 months after diagnosis, with fewer than 7% of the patients surviving longer than five years Protein kinases participate in many signaling pathways, including those involved in cell proliferation, growth, metabolism, apoptosis, and differentiation. Protein kinases are attractive drug targets for malignancy due to their distinct selectivity, pro-oncogenic properties, and a wide selection of readily available kinase inhibitors. Over 50 kinase inhibitors are approved for clinical use by the US Food and Drug Administration, and many others are in clinical development. Molecular crosstalk between HIFs and protein kinases is indispensable for cancer cells' response to hypoxia. While aberrant kinase activities are common in many cancers, hypoxia can further disrupt cellular kinome network to increase the robustness and resilience of oncogenic pathways. HIFs have been shown to activate SAPK, MAPK, and PI3K signaling pathways In this study, we employed a “bench-to-bedside and back” approach to identify key signaling conduits exploited by GPCs in hypoxia and reveal opportunities for repurposing kinase inhibitors, currently at different phases of clinical trials. The use of patient-derived GPCs and xenografts in our kinase inhibitor screen and functional assay facilitated the “bench to bedside” analysis, while the “back again” section involved cross-validation with glioblastoma transcriptomes from public repositories. The full range investigative strategy unveiled new insights into the therapeutic prospect of targeting c-MET/PI3K in tumor hypoxia.Table S1. All cell lines were maintained in a humidified 37 °C incubator containing 5% CO2. Hypoxia was achieved by reducing O2 concentration to 1% in a hypoxic chamber.All GPC cell lines, NNI-11, NNI-24, and NNI-31, used in this study were derived from patients' tumors and obtained from the National Neuroscience Institute, Singapore. GBM tumor specimens and associated cell lines from the National Neuroscience Institute (NNI) were obtained with informed consent and de-identified in accordance with the SingHealth Centralised Institutional Review Board A. NNI-11 and NNI-24 were cultured in DMEM  with 10% fetal bovine serum (FBS) (Hyclone). The NNI-11, NNI-24, and NNI-31 were cultured in a serum-free growth medium as reported previously Suppl. File 1. The cells were treated with kinase inhibitors in normoxic or hypoxic conditions for 72 hours. For cell lines maintained in 10% FBS, they were fixed with 2% formaldehyde and stained with Syto60 fluorescent nucleic acid stain  to determine the drug cytotoxicity. The area of the stained cell colonies, which represents surviving and proliferative cells, was measured using ColonyArea Kinase inhibitors were obtained from SYNkinase  and Target Molecule Corp. . The kinase inhibitors, concentrations, and drug targets used in the study are described in Drug targets of the inhibitors which showed enhanced cytotoxicity in hypoxia were visualized and analyzed with Ingenuity Pathway Analysis (IPA)  and KinMap Suppl. File 1.For the ranking analysis, we used an unweighted scoring algorithm similar to Kinase Addiction Ranker Dose-response curves of selected c-MET and PI3K inhibitors, TMZ , and combined therapy of c-MET inhibitors with TMZ were assessed using Syto60 stain 72-hr post-treatment in normoxia or hypoxia. Drug-drug interaction between c-MET inhibitors and TMZ was examined using the Chou-Talalay method based on the fraction affected (Fa) values and combination index (CI), where CI < 1, CI = 1, and CI > 1 represent synergism, additive effect, antagonism, respectively GPC cells were transfected with siRNA targeting human c-MET from Trifecta RNAi kit  using Lipofectamine 2000 (Invitrogen) for 24 h. Scrambled siRNA was used as a negative control. Transfected cells were incubated at normoxia or hypoxia for 48 h followed by clonogenic assay. To examine the off-target effects of the inhibitors, the viability of c-MET-knockdown GPCs treated with c-MET inhibitors at normoxia and hypoxia for 72 h were examined.Table S2.Total RNA was extracted, and qPCR was performed as previously described ®CLx Infrared Imaging System (LI-COR Biosciences) and quantified using ImageJ. The effect of tivantinib on α-tubulin polymerization was examined with immunoblotting as described previously Cells were lysed using ice-cold M-PER Mammalian Protein Extraction Reagent (Thermo Scientific) containing Halt Protease Inhibitor Cocktail (Thermo Scientific) and Halt Phosphatase Inhibitor Cocktail (Thermo Scientific). Far-infrared immunoblotting was performed. Protein bands were visualized with the OdysseyTreated cells were stained with CellROX Deep Red Reagent . The cells were trypsinized, and fluorescence signals were detected at 0, 12, 24, and 48 h post-treatment with BD Accuri C6 Cytometer .Apoptotic cells were stained with propidium iodide (Sigma-Aldrich) and Annexin V-FITC  and detected using BD Accuri C6 Cytometer. Live and dead cells were distinguished by staining with propidium iodide and Hoechst 33342 (Invitrogen) and imaged using Ju LI Stage fluorescent microscope .6 NNI-24 cells were resuspended in Matrigel  and culture medium and implanted into the dorsal flank of 8-week old male NSG mice. Mice were maintained in a facility with a 12 h dark/light cycle and ad libitum access to chow diet and water. The tumor volume was measured weekly using a digital caliper and calculated using the following formula: (width2 x length)/2. When tumor volume reached approximately 100 mm3, the mice were randomized to six groups, which were given one-time intratumoral injection of saline (vehicle control), TMZ (25 mg/kg), PF042178903 (1 mg/kg), TMZ (25 mg/kg) + PF04217903 (1 mg/kg), Tivantinib (50 mg/kg), or TMZ (1 mg/kg) + Tivantinib (50 mg/kg), respectively. For histopathology and intratumoral cytokine profiling assays, the tumor xenografts were harvested at day three post-injection. Tumor weight was determined at week three post-injection. All procedures were performed according to the Nanyang Technological University's Institutional Animal Care and Use Committee guidelines .2×10Tissues were processed, sectioned, and stained as previously described with minor modifications TM Human 11-Plex Cytokine kit  according to the manufacturer's instruction.Cytokine profiling of the cell lysates, culture media, and tumor lysates was performed with LUNARISRNAseq data of clinical GBM tumors (n = 37 tumors) from different anatomic structures  were obtained from IVY Glioblastoma Atlas Project e.g., tumor volume over time) were analyzed using a mixed-model ANOVA. Dependent variables with two main factors  were analysed with a two-way ANOVA. Pairwise comparisons were performed with Sidak correction. Other variables were analyzed with one-way ANOVA, followed by post hoc Tukey's test. P-values < 0.05 indicate statistical significance.Dependent variables with repeated measures (2). The compounds screened included 39 U.S. FDA-approved drugs, 45 compounds which are in active clinical development , 42 compounds that were dropped from the clinical pipeline, 61 compounds in preclinical testing, and 1 compound withdrawn from the market (Suppl. File 1). The resultant cell viability data were subjected to unbiased hierarchy clustering analysis and presented in a heatmap .A total of 188 small-molecule compounds were screened against 130 different kinases using a cell-based viability assay. This functional assay directly measures GPCs, NNI-11, and NNI-24, cultured under normoxia and hypoxia (1% O). Thirty-six inhibitors (19.1%) were cytotoxic in normoxia, while 29 inhibitors (15.4%) were cytotoxic in hypoxia. Astoundingly, only 5 out of the 188 kinases inhibitors (2.7%) were cytotoxic to GPCs in normoxia and hypoxia, of which KX2-391  and Staurosporine ] were more hypoxic-selective whilst AT-7519 HCl  and BMS387032 HCl ] were more normoxic-selective. Lenvatinib  showed comparable cytotoxicity in both conditions. Clearly, the profound changes of GPCs' drug response and the low number of effective kinase inhibitors indicate the extensive modifications of cellular kinome activities under hypoxia.An inhibitor is considered cytotoxic when it reduced GPCs' viability by 10% relative to DMSO treatment. Most inhibitors , including the vehicle, DMSO, were non-cytotoxic at the tested concentration in both normoxic and hypoxic environments . The cytotoxicity effects of the hypoxic-selective inhibitors exhibited a dose-dependent trend and were comparable between GPCs grown at different conditions. The results suggest that the hypoxic selectivity is unaffected by the stemness of the tumor cells. Furthermore, they also highlight a dominant role for hypoxia in the TME on GPCs' response to drugs. The drug targets of hypoxic-selective inhibitors were then mapped out and amounted to 49 protein kinases . Certain kinases, notably PI3K/Akt, Src, c-MET, VEGFR1/2/3, and FLT3 were over-represented by multiple inhibitors, implying that these targets are likely to be true positives and key mediators of tumor hypoxic adaptation. We also analyzed the protein kinases with the IPA data repository to unravel the kinome network, differentially altered by hypoxia in GPCs . The interaction map revealed that Src and PI3K/Akt pathways are highly intertwined with other kinases, forming signaling hubs in the GPC's response to hypoxic stress. Several upstream receptor tyrosine kinases, namely c-MET, VEGFR1, AXL, FGFR4, and RET, interacted directly with the signaling hubs and HIF-1α, further supporting the pivotal roles of Src and PI3K/Akt signaling in hypoxia response. To rank and shortlist kinase candidates using information from the connectivity network, we employed a ranking algorithm similar to Kinase Addiction Ranker Figure ). The sum of scores from each category was used to rank the protein kinases . c-MET emerged as the top-ranked kinase. Src and PI3K/Akt, identified as signaling hubs in tumor hypoxic response, were also among the highest-ranking kinases. Importantly, c-MET, Src, PI3K/Akt share mutual interaction and with HIF-1α, clearly highlighting that the triad could be a targetable signaling conduit to undermine hypoxic adaptation by GPCs.The kinase inhibitor screen revealed 31 hypoxic-selective inhibitors whose molecular targets could be crucial for hypoxic response and survival in GPCs. To study if the hypoxic-selectivity of these inhibitors is affected by stemness and culture conditions, we performed another screen using GPCs maintained in either 10% FBS or serum-free conditions . The cytotoxicity effects of c-MET inhibitors were markedly increased under hypoxia. The hypoxic selectivity of crizotinib, foretinib, and tivantinib was consistent in NNI-24 and NNI-11. In line with the findings from c-MET inhibitors, the knockdown of c-MET also conferred hypoxic-selective cytotoxicity. Altogether, these observations indicate that the attenuation of c-MET compromises the survival of GPCs in a hypoxic environment.Although most kinase inhibitors have been developed against a specific kinase target, many inhibitors often inhibit multiple kinases in key signaling pathways and exhibit off-target effects. To mitigate such limitations, we examined the dose-response of five structurally different c-MET inhibitors, namely cabozantinib, crizotinib, foretinib, PF04217903, and tivantinib, as well as knockdown of c-MET on the viability of GPCs in normoxic and hypoxic conditions . Likewise, hypoxia also enhanced phosphorylation of c-MET following HIF-1α upregulation . While c-MET inhibitors had no impact on HGF expression, the phosphorylation of c-MET was effectively diminished by crizotinib, foretinib, and tivantinib, leading to the inactivation of c-MET signaling. To assess the off-target effects of these inhibitors, we treated c-MET-knockdown GPCs with the inhibitors . Crizotinib and foretinib did not induce further cytotoxicity, indicating that their hypoxic selectivity is attributable to c-MET inhibition. Surprisingly, tivantinib induced an additional 10-20% viability reduction to c-MET-knockdown GPCs in a non-hypoxic-selective manner, suggesting that the off-target of tivantinib has significant cytotoxicity. Tivantinib has been known to inhibit tubulin polymerization ), which suggests a disruption of α-tubulin assembly. The finding may explain the discrepancy in drug efficacy between tivantinib and other c-MET inhibitors.To understand how hypoxia affects c-MET signaling in GPCs, we first examined the expression of hepatocyte growth factor (HGF), the natural ligand of c-MET. We observed a significant increase in the expression of HGF in GPCs under hypoxia than in normoxia . However, Src phospho-activation was unaffected by hypoxia and c-MET inhibitors, even though Src inhibitors like KX2-391, bosutinib, and dasatinib did exert hypoxic-dependent cytotoxicity dose-dependently, especially in NNI-24 . Hence, the activation of Src may not be strictly c-MET-dependent in tumor hypoxia of GPCs. In contrast, the protein expression and phosphorylation of Akt were elevated under hypoxia and diminished by c-MET inhibitors . Exposure to hypoxia also increased the number of dead GPC cells treated with PI3K inhibitors like GDC-0941, ZSTK474, and PIK75 in a dose-dependent manner . Additionally, the hypoxic selectivity of c-MET and PI3K inhibitors was again validated in another patient-derived cell line, NNI-31 tumoroids, cultured in a serum-free environment. As anticipated, tivantinib (c-MET) and PIK75 (PI3K) inhibitors triggered apoptosis in hypoxic GPCs even at a low concentration . We also examined the activity of Ras-Raf-MEK-ERK pathway, another key signal transducer of c-MET, and found that it was suppressed in hypoxia. Expectedly, the treatment of c-MET inhibitors neither affected the expression nor phosphorylation status of B-Raf and MEK1/2 . The results indicate that MEK-ERK activation may serve as an early responder to hypoxia but has limited functionality in prolonged hypoxia The kinase-kinase interaction map suggests that Src and PI3K/Akt are the key downstream signaling hubs of c-MET activation . Cellular response to oxidative stress invokes the upregulation of Nrf2-regulated antioxidant genes like NQO1, PRDX1, SOD2, GPX1, and GPX4 . Treatment with c-MET inhibitors increased the high-ROS GPC population in hypoxia. In both GPCs, the population with high ROS levels peaked at 24 h. In NNI-11, the population plateaued from 24 to 48 h, but in NNI-24, such population declined at 48 h, likely because of oxidative stress-induced cell death . Clearly, the inhibition of c-MET by small molecule inhibitors further exacerbated ROS accumulation, outstripping the antioxidant capacity of the cells. Such a buildup of intracellular ROS likely due to impaired Nrf2-mediated gene regulation, as demonstrated by the persisted suppression of Nrf2-regulated antioxidant genes upon c-MET inhibitor treatment . Moreover, the supplementation of a strong antioxidant, N-acetyl cysteine, dose-dependently rescued GPCs from the cytotoxic effect of c-MET inhibitors in hypoxia, but not normoxia . The results further reinforce the involvement of the antioxidant mechanism in hypoxic adaptation. In summary, c-MET/PI3K/Akt signaling pathway is vital for GPCs to alleviate oxidative stress in hypoxic TME.Hypoxia alters intracellular reactive oxygen species (ROS) balance, a prosurvival signal in GPC self-renewal, and proliferation Figure ), while TMZ alone did not exhibit any hypoxic selectivity . Based on the fraction affected (Fa)-combination index (CI) plots computed using Compusyn software, most c-MET inhibitors synergized favorably with TMZ under hypoxia, but not in normoxia . Among the five c-MET inhibitors, PF04217903 and tivantinib exhibited the most notable drug synergism with TMZ under hypoxic conditions .TMZ is the current standard-of-care chemotherapeutic drug for newly-diagnosed GBM patients in vivo. As expected, co-treatment of TMZ with either PF04217903 or tivantinib effectively attenuated the growth of NNI-24 tumor xenograft and reduced tumor load . We also performed immunofluorescence staining of HIF-1α and cleaved caspase-3 to distinguish the extent of apoptotic death in the hypoxic and non-hypoxic regions of tumor xenografts . The immunofluorescence images revealed that hypoxic areas were pervasive in GPC xenografts. Vehicle-treated xenografts had minimal apoptosis. TMZ induced cellular apoptosis largely restricted to HIF-a negative regions, i.e., non-hypoxic.Next, we investigated the drug synergism in vivo results showed that inhibition of c-MET fortified TMZ cytotoxicity in the hypoxic environment, significantly mitigating tumor xenograft growth.Interestingly, the c-MET inhibitors PF04217903 and tivantinib induced activation of caspase-3 in the hypoxic regions. As a result, apoptosis was notable in both hypoxic and non-hypoxic areas of the tumors with co-administration of TMZ with c-MET inhibitor. Thus, our in vivo results. We found 843 upregulated and 2206 downregulated genes (3049 DEGs), of which the former were mapped to 178 transcription factors, and subsequently, 78 protein kinases were mapped to the hypoxic regions of GBM . Twenty-two out of the 78 enriched kinases intersect with the hypoxic-selective kinases from our screen, especially the highest-ranking targets, i.e., AURKA, SRC, RPS6KA3, etc. . The activation of c-MET and its downstream kinase targets are anticipated  based on Ivy GBM Atlas Project and Ingenuity Knowledge Base repositories. Interestingly, proinflammatory mediators and pathways are highly enriched in the hypoxic zone . Cytokine profiling also confirmed the increased expression and secretion of proinflammatory cytokines in cells exposed to hypoxia and tumor xenografts treated with and without TMZ treatment, whereas the treatment with c-MET inhibitors significantly resolved the proinflammatory response . Single-cell transcriptomes of GBM tumors from Darmanis et al. (2017) revealed that c-MET overexpression was only present in a subpopulation of the neoplastic GBM cells that were more hypoxic, inflamed, stem-like, and metastatic . This highly invasive subpopulation could be more vulnerable to the adjuvant therapy with c-MET inhibitors in clinical settings.Our above findings suggest intratumoral heterogeneity with respect to hypoxia-selective drug susceptibility. Due to the paucity of fresh GBM biopsies, we leveraged on the RNA-seq data of clinical GBM tumors from Ivy GBM Atlas Project Hypoxic TME is a hallmark of many cancers. As normal tissues are generally unaffected by hypoxia, exploiting this distinct feature of TME may lead to highly specific anti-tumor treatments. Tumor hypoxia is a pivotal modifier of tumor chemosensitivity, contributing to treatment failure Hypoxia modulates the activity of a series of kinase inhibitors on GPCs, with only a handful of small molecule inhibitors retaining their cytotoxicity irrespective of the oxygen environment Combining the kinase inhibitor screen, network analysis, and an unweighted scoring algorithm that includes clinical data, we identify c-MET-PI3K pathway as an essential and targetable signaling hub that is vital for GPCs' survival in hypoxic TME. Our interrogation of bulk and single-cell transcriptomes from clinical GBM specimens also supports the involvement of c-MET signaling in invasive tumor behaviors. The blockade of c-MET signaling, on the other hand, successfully weakened GPCs, resulting in increased apoptosis. Co-administration of c-MET inhibitors and TMZ demonstrated favorable drug synergism that significantly attenuated GPC tumor xenografts' growth. These findings confirm that the c-MET signaling network can serve as a potential oncogenic target to exploit hypoxic TME in GBM.In another study, abolishing HGF/c-MET axis with antisense transgenes also substantiated GBM response to radiotherapy in a synergistic manner Targeting c-MET activity with small molecule inhibitors adversely disrupts tumor proliferation, neovascularization, and distal metastasis across different cancer types, including gastric, breast, hepatic, pancreatic, and lung carcinomas, suggesting that HGF/c-MET axis is a crucial survival expedient for tumor cells in hypoxic TME Our findings are established on a drug screen that used IC50 values derived from cell-free assays and optimized to detect each inhibitor's differential effectiveness between normoxia and hypoxia. As the reported IC50 values for every inhibitor span across a considerable range (nM to µM), the resultant cytotoxicity read-outs should not be used to compare the efficacy between different inhibitors directly. Furthermore, the relationship between small-molecule inhibitors and their drug targets is a complex many-to-many association, rather than a simple one-to-one association. An inhibitor can have multiple targets, and different inhibitors share one or multiple common targets. The drug concentration adds another layer of complexity as target specificity is dose-dependent. A high throughput inhibitor screen poses practical challenges to eliminate the off-targets of every inhibitor. Hence, in our screening informatics strategy, all known targets of the hypoxic selective inhibitors were included in the subsequent analyses. The ranking algorithm also helps to sieve the information and extract the true targets based on network analysis and clinical data prediction. True targets that were underrepresented by our drug panel will be missed out in our approach. However, high-ranking kinases from our approach are experimentally shown to be hypoxic selective with supports from biological mechanisms and clinical prediction, and hence are highly likely to be true targets that are worth further investigation.Another limitation is associated with the use of flank implantation of patient-derived tumor cells in mice, which is unable to fully recapitulate the central nervous system environment. However, to our knowledge, none of the five c-MET inhibitors used in our study has been reported to cross blood-brain barrier (BBB) effectively, thus limiting our selection to subcutaneous xenograft models. Drug delivery across the BBB remains one of the most challenging fields for pharmaceutical and biotechnological products. Our findings can spur the development of c-MET inhibitors with desirable BBB-penetrating effect to further validate our results.Our observations of the changing kinomic landscape and kinase inhibitor responses strongly advocate an overhaul of cancer drug development by considering tumor hypoxia during preclinical settings. Using network analysis, unweighted scoring algorithm, and transcriptomes of clinical tumor specimens, we further enhance the predictive potential of the kinase inhibitor screen to assist in prioritizing potential candidates. Our results encourage drug repurposing of c-MET and PI3K/Akt inhibitors to improve GBM therapy. While GPCs were used in the present study, it is also noteworthy that tumor hypoxia occurs ubiquitously in many solid tumors, entailing the generalizability of our results to other cancers.Supplementary file legends, figures and tables.Click here for additional data file.Supplementary file 1.Click here for additional data file.Supplementary file 2.Click here for additional data file.Supplementary file 3.Click here for additional data file.


The present study mainly examined the effects of the volumetric concentration of nanobubbles (ultrafine bubbles) on the intensity of sonoluminescence (SL). The addition of nanobubbles at high acoustic amplitude enhanced the SL intensity for various bubble concentrations in comparison with that in pure water. This probably means that the resulting high amplitude is over the Blake threshold, and accordingly nanobubbles expand to some extent, leading to higher SL intensity. Therefore, nanobubbles have the potential to provide nucleation sites for sonochemistry. The influence of bubble size on the intensity of SL was also evaluated. The present study mainly deals with measurement of the SL intensity at different concentrations of nanobubbles and evaluates incremental increases in the intensity by nanobubbles compared with that in their absence. Recently, the authors reported preliminary assessments of the SL intensity at different concentrations of nanobubbles The present study examined the intensity of SL for various concentrations of nanobubbles as a function of acoustic amplitude, as well as the intensity in the absence of nanobubbles, while varying a greater number of parameters than in our previous work 2Nanobubbles were prepared in pure water  using a bubble generator apparatus (YBM Faby-10) that uses the impact of bubble cavitation and the shear force imparted by vortex flow, as described in a previous publication The size of nanobubbles was measured with a size distribution measurement apparatus (Shimadzu SALD-7500X10). The principle of size measurement using this apparatus has been described elsewhere The SL intensity-measurement procedure is described in 36 /mL is close to zero in As for the temporal stability of the bubble size, the authors confirmed at least that the deviation of the average bubble size of nanobubble water without dilution but prepared in the same way as the present method stayed within 4% after 17 min passed, which was longer than the time required for each of the present measurements.2 kPa, there is little difference in SL intensity with and without nanobubbles. This is probably because at relatively low amplitude it is hard for nanobubbles to pulsate enough to emit SL by overcoming the restriction due to surface tension of the surrounding liquid. Therefore, an effect of adding nanobubbles on SL intensity did not occur at relatively low amplitude. However, when the sound pressure becomes 5 × 102 kPa or more and the quenching phenomenon appears, the SL intensity with nanobubbles is higher than that without nanobubbles. The existence of nanobubbles has an effect of inhibiting the quenching since large-sized bubbles are expelled from the region of high-pressure amplitude while submicron-sized bubbles could stay there to cause pulsation leading to the emission of SL. In order to inhibit the quenching of SL, Hatanaka et al. 2 kPa. To find the difference clearly, the ratio of the SL intensity in the presence of nanobubbles against that in their absence at each pressure amplitude was calculated and the average over the range of relatively high amplitude of more than 6 × 102 kPa was obtained for different concentrations of nanobubbles. According to the results of numerical calculations reported in the literature 2 kPa compared to the value obtained with a lower amplitude of 1.75 × 102 kPa when producing nanobubbles at a relatively low ultrasonic frequency of 20 kHz. This is because the higher amplitude could be over the Blake threshold 2 kPa or more.The difference in the intensity between the absence and presence of nanobubbles was observed at the amplitude of more than 6 × 102 kPa or more in sound pressure amplitude compared with the intensity in the absence of nanobubbles. An influence of the concentration of nanobubbles on the relationship between sound pressure amplitude and SL intensity was investigated. 4Through the measurement of bubble size distribution and SL intensity under different conditions of bubble concentration and acoustic amplitude, it was clarified that the addition of nanobubbles at high acoustic amplitude enhanced the SL intensity for various bubble concentrations in comparison with that in pure water, and the increased bubble size provided higher SL intensity. These results might come from the gaseous nature of nanobubbles. The significance of the results was that the existence of nanobubbles under sonication could inhibit sonoluminescence quenching at high amplitude and provide cavitation nucleation sites useful for yield enhancement of sonochemical reaction. Such effects by nanobubbles could not be obtained at low amplitude because of the restriction due to surface tension by the surrounding liquid.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


End concrete cover separation is one of the most common failure modes for RC beams strengthened with external FRP reinforcement. The premature failure mode significantly restricts the application of FRP materials and could incur serious safety problems. In this paper, an innovative stress field-based analytical approach is proposed to assess the failure strength of end concrete cover separation and the conventional plane-section analysis is extended to evaluate the corresponding carrying capacity of FRP-strengthened RC beams. First, the dowel action of reinforcement and the induced concrete splitting, reflecting the interaction between concrete, steel and FRP, are considered in establishing the geometrical relationships of stress field for cracked concrete block. Then, the cracking angle and innovative failure criterion, considering the arrangement of steel and FRP reinforcement and cracking status of concrete and its softening effect, are derived to predict the occurrence of concrete cover separation and related mixed modes of debonding failure. Subsequently, an extended sectional analytical approach, in which the components of effective tensile strain of FRP resulted from flexural and shear actions are both considered, is presented to evaluate the carrying capacity of strengthened beams. Finally, the proposed calculational model is effectively validated by experimental results available in the literature. Due to the pronounced advantages, such as high strength, light weight, electromagnetic transparency, non-corrosive, and nonconductive properties, externally bonded (EB) fiber-reinforced polymer (FRP) and near-surface-mounted (NSM) FRP have become the prevailing techniques over the last three decades for flexural strengthening of existing reinforced concrete (RC) members ,2,3,4. EConcrete cover separation at the end of FRP reinforcement has been found to be the common failure mode in the retrofitting techniques using EB and NSM FRP methods and has obtained increasing research attention ,11,12,13Many analytical models and numerical techniques have been also presented to evaluate the failure strength of concrete cover separation ,43,44,45In addition, the fracture moment Consequently, the resultant force tion (5) ,30,31,32Equation (4) illustrates that the effective tensile strain of FRP reinforcement figured out by concrete tooth model is highly sensitive to crack spacing; therefore, crack spacing is the most critical factor to predict the failure strength of concrete cover separation. Consequently, the modified concrete tooth models by refining the calculation of crack spacing were subsequently presented ,32. AccoConcrete tooth model and the other mentioned analytical models have been so widely employed due to its simplicity and convenience in analysis and design. However, the great simplifications render the pronounced drawbacks in accuracy and application scopes. Thus, the more sophisticated numerical techniques have been used to improve the accuracy of prediction of concrete cover separation. Hawileh et al.  presenteGreat efforts on experiments and on analytical and numerical approaches have been made to investigate the concrete cover separation of RC members strengthened by external FRP reinforcement , whereas a full understanding of this failure mode is still lacked, and the precise and reliable calculation approach is still in demand. In this paper, a novel stress field-based analytical approach is presented to evaluate the failure strength of concrete cover separation induced at the end of external FRP reinforcement and the corresponding carrying capacity of FRP-strengthened RC beams that failed in concrete cover separation. First, dowel action of steel and FRP reinforcement and the induced concrete splitting, which reflect the interaction between concrete, steel and FRP reinforcement and are commonly ignored in existing investigations, are considered to be the critical factors to result in concrete cover separation and properly incorporated into the establishment of fundamental geometrical relationships of stress field for cracked concrete block. Then the influential factors such as transverse strain and splitting crack of concrete interacted with surrounding steel and FRP reinforcement are comprehensively taken account for the calculation of effective compressive strength of concrete in stress field. Accordingly, the cracking angle to identify the stress field, and the innovative failure criterion are derived to predict the occurrence of concrete cover separation and the related mixed modes of debonding failure; the arrangement details of steel and FRP reinforcement and the cracking status of surrounding concrete and its softening effect are properly considered in the derivation. Subsequently, the indispensable shear component in the effective tensile strain of FRP is taken into account by a simplified stress field approach to evaluate the carrying capacity of strengthened RC beams that failed in end concrete cover separation. Finally, the proposed calculational model is effectively validated by the experimental results available in the literature. Before the establishment of analytical model, it is fundamental to understand the mechanical state and failure mechanism of separation of concrete cover at FRP reinforcement end. According to many experimental investigations ,34,46,47More specifically, if the applied shear stress exceeds the bond strength between FRP and concrete, the crack creates at the FRP/concrete interface, which is known as interfacial debonding ,6,7,21. If FRP reinforcement is not attached, the horizontal splitting cracks can be also generated because the bond action between steel reinforcement and concrete ,49,50 anThis study mainly focuses on the end concrete cover separation of FRP-strengthened RC beams. Thus, the dominant dowel action of steel and FRP reinforcement and the induced concrete splitting would be considered, and the failure mechanism of concrete cover separation generated at the maximum moment region and the relevant bond action of steel reinforcement is not further discussed.Concrete cover between internal steel reinforcement and external FRP reinforcement is divided into discrete blocks by inclined separation cracks along the strengthened RC beam. Based on the conventional assumptions and simplifications, the stress field approach schematically shown in To derive the failure criterion and failure strength of end concrete cover separation, only the mechanical state of cracked concrete block at the end of FRP reinforcement needs to be appraised. Consequently, the fundamental hypotheses about mechanical and geometrical conditions are made to satisfy the requirements of stress field theory for concrete ,63,64,65Two adjacent inclined separation cracks are defined as the boundaries of concrete block. The tensile stress along the inclined cracks reaches the effective tensile strength of concrete, and the shear stress along the inclined cracks is ignored ,50,54.The first inclined separation crack straightly extends from the end of FRP reinforcement to the gravity center of the (outer) steel reinforcement; without considering crack spacing, the adjacent inclined and straight separation crack starts at the end of projection of the first inclined crack  steel reinforcement to the first inclined crack. The propagation length depends on the splitting degree and is assumed to be larger than the length of the upside of concrete block in this study.The dowel action is simplified as uniform load distributed on surrounding concrete block ,54,55,56Considering the plasticity and stress redistribution of concrete, the assumption and simplification of uniformly distributed tensile stress along inclined cracks and dowel action would generate the conservatively safe evaluation of failure strength of concrete cover separation, which is specifically validated and discussed in To establish the geometrical relationships of stress field for cracked concrete block, the mechanical state should be identified. First, the uniformly distributed tensile stress along the inclined separation cracks is defined as with 0.8 ,54,55,56Subsequently, it should be pointed out that the dowel actions of both steel and FRP reinforcement contribute to concrete cover separation according to experimental investigations . Howeverorcement  and thenIn this study, the distributed tensile stress on top surface of concrete block is ignored due to the development of splitting crack, and the dowel action Moreover, there exists shear stress acted on the top surface of concrete block surrounded by cracked concrete and steel bars, as schematically shown in The stress state of concrete block concerned in calculations is schematically in Replacing Defining the parameters β.Accordingly, Equation (10) can be further simplified into Equation (13) explicitly representing the relationship between cracking angle In addition, the relationship between cracking angle A parameter Consequently, through combining Equations (13) and (14), the relationship between geometrical parameter With the geometrical parameter rsection  can be dFurthermore, based on the geometrical relationship demonstrated in One of the critical failure conditions of concrete cover separation is concrete cracking along the inclined separation cracks at the end of FRP reinforcement. Thus, the transverse stress of micro concrete struts  adjacention (21) ,68:(21)εRelationship between the principal compressive strain ion (22) ,69:(22)εPlease note that the principal compressive strain concrete ,69. The ion (23) :(23)fcp0The effective compressive strength concrete ,63,64,65concrete ,63,64,65Consequently, the effective compressive strength of concrete in stress field is calculated by Equation (24):fficient ; ηε is tfficient ,63,64,65fficient ,77,78,79Equation (24) indicated that the generation and development of splitting crack would significantly reduce the effective compressive strength of concrete and affect the failure strength of concrete cover separation discussed as follows. To establish the failure criterion of concrete cover separation, stress state of a local part of micro concrete strut adjacent to the first inclined separation crack and external FRP reinforcement is shown in The principal tensile stress Case 1: Generally, the principal compressive stress concrete ,64. AccoIn contrast to most formulations of debonding failure strength, characterized by the individual material properties and geometries of concrete and external FRP reinforcement, the derived strength model additionally incorporates the influential factors such as interaction between concrete, steel and FRP reinforcement, cracking status of surrounding concrete and its softening effect, and arrangement details of internal steel reinforcement and external FRP reinforcement. Moreover, as the Mohr’s circle for stress of micro concrete in  devices ,81 are wOn the other hand, to ensure the occurrence of concrete cover separation, the maximum local shear stress orcement ,83, whiceriments ,10,16,47concrete ,83 in EqAnother possible mixed mode of cover separation and sliding failure is schematically shown in th 37.2° ,71. HencIn addition, according to the geometrical relationships of Mohr’s circle of stress shown in Based on Equation (28) and concrete ,63,64,65Case 2: By contrast, Case 2 does commonly occur due to the extremely low principal compressive stress of So far, the mechanical state of micro concrete strut adjacent to the first inclined separation crack shown in Select a cracking angle Specify the geometrical parameter Calculate the cracking strain of tensile concrete Select a splitting cracking width of Figure out the plasticity of concrete coefficient Check the correctness of principal compressive stress If Step 6 is false, adjust the value of If Step 6 is true, calculate the cracking angle If the computed cracking angle Check the computed cracking angle If Step 10 is false, repeat steps 1–8 and 10; andIf Step 10 is true, obtain the desired cracking angle Please note that under the condition of satisfying the requirements of failure strength listed in step 6 and the lower bound of splitting crack width of 0.015 Consequently, an analysis flowchart to estimate cracking angle As the cracking angle Referring to the aforementioned analytical approaches of local shear stress, the average shear stress For the scarcely occurring Case 2 with the average tensile stress With the estimated average shear stress The effective tensile strain of FRP corresponding to the occurrence of concrete cover separation is evaluated by Equation (31):It should be pointed out that the effective tensile strain of FRP corresponding to concrete cover separation is not identical to the one at the maximum bending moment section that failed in flexure, which is assumed to be sufficient and not the critical factor in this study. The location of end concrete cover separation renders the huge difficulties in precise evaluation of carrying capacity of RC beams strengthened with FRP reinforcement. Hence, most studies merely paid attention to assessing the effective tensile strain of FRP corresponding to concrete cover separation ,30,31,38For the intermediate crack-induced concrete cover separation, as shown in By contrast with the conventional sectional analysis performed at the critical section with the maximum bending moment, the presented analytical approach focuses on the section where the concrete cover is completely separated, namely the end of the first inclined separation crack . On the ion (32) ,84:(32)εSubsequently, the analyses of flexural and shear behavior of strengthened RC beams are respectively performed to specify the flexural strain Flexural strain The resultant force concrete , represeAccording to the common assumption that the plane section remains plane, the depth of compressive region Similarly, the compressive strain Consequently, the bending moment The shear force Considering the combination of steel and FRP longitudinal reinforcement, the calculational formulation based on the modified compression field theory (MCFT) ,62 and tThe development of angle of the MCFT ,62 and tthe MCFT , whereasthe MCFT ,18 reporry (CFT) , was modry (CFT) ,18 to es section ,18,87.Moreover, based on the solution of concrete plasticity , the uppTo facilitate calculation, the inclination angle of Considering crack spacing, the starting point of the second inclined separation crack, where the effective tensile strain of FRP at concrete cover separation is derived according to stress field approach, may be not coincident with the studied beam section, as the points A and A’ illustrated in The modification coefficient and (57) ,88,89,90h−c)bw/3 ,88,89,90Subsequently, the detailed process to assess the carrying capacity of strengthened RC beams that failed in concrete cover separation is illustrated as follows.Select a flexural strain Select a curvature Figure out the depth of compressive region Specify the reinforcement stresses of Compute the resultant force If If Compute the strain of FRP reinforcement If If Correspondingly, an analysis flowchart to assess the carrying capacity of strengthened RC beams is illustrated in To validate the proposed approach, the strengthened RC beams that failed in concrete cover separation with the desirable data in the available literature ,22,23,28With the proposed analytical model, the calculation of cracking angles of concrete blocks was first performed. The calculational results (Subsequently, incorporating the effective tensile strain of FRP reinforcement derived from the calculated cracking angles of concrete blocks, carrying capacities of the strengthened RC beams are assessed. In calculations, the effect of amplification coefficient of average crack spacing The statistical results in ferences ,92. SincA novel analytical approach based on concrete stress field was proposed to predict end concrete cover separation in RC beams strengthened with external FRP reinforcement. First, with the introduction of dowel action of steel and FRP reinforcement and the induced concrete splitting, which are the critical factors to reflect the interaction between concrete, steel and FRP reinforcement, the geometrical relationships of stress field for concrete were established through proper simplifications to configuration and mechanical state of cracked concrete block. Then, to assess the cracking angle and the correlated failure strength of concrete cover separation, the effective compressive strength of concrete in stress field was finely identified by incorporating the influential but prone to be neglected factors such as transverse strain, cracking status of surrounding concrete, and arrangement of internal steel reinforcement and external FRP reinforcement. Subsequently, an extended plane-section analytical approach, in which the components of effective tensile strain of FRP induced due to flexural and shear actions are both comprehensively considered according to the detailed location of concrete cover separation, is proposed to evaluate the carrying capacity of strengthened RC beams in a simple process. Finally, an excellent agreement between the predictions and experimental results was obtained to validate the proposed analytical approach; furthermore, the discussions and suggestions about the parameters concerned in the approach were proposed.The detailed conclusions of this study can be drawn as follows:By contrast with the conventional analytical models of concrete cover separation, merely focusing on the local response of concrete around FRP reinforcement, the proposed analytical approach based on stress field could comprehensively and properly consider the influence of the interaction between concrete, steel and FRP reinforcement, cracking status of surrounding concrete and its softening effect, and arrangement details of internal steel reinforcement and external FRP reinforcement on concrete cover separation; it is suited for practical design and analysis, and can be extended for external reinforcement with other composite materials.Dowel action of steel and FRP reinforcement, and the induced concrete splitting are the critical factors to establish the geometrical relationships of stress field for cracked concrete block and to derive the failure strength of concrete cover separation, and cannot be neglected.The assumption of the lower bound of splitting crack width of 0.015 The shear component in the effective tensile strain of FRP reinforcement cannot be ignored and was efficiently considered in the analysis of carrying capacity of strengthened RC beams that failed in end concrete cover separation.Crack spacing has a great effect on the assessment of carrying capacity of strengthened RC beams and, consequently, the coefficient incorporating the amplification factor of average crack spacing The proposed analytical approach obtained the satisfactory and conservatively safe prediction of the experimental results; by contrast, the commonly used concrete tooth model overestimated the failure strength of concrete cover separation, and generated unsafe prediction of carrying capacity of strengthened RC beams.