Concrete itself, and issues relating to the recycling and management of reinforced concrete waste, are highly relevant, especially when urban expansion is being achieved by increased building construction volumes. This research investigates concrete waste and its (re)usage possibilities and resolves several major issues related to the question of how natural materials can be replaced by compounds made from concrete waste, thereby saving natural resources. The experiment was carried out using concrete mixtures, which were combined with natural aggregates and crushed concrete waste (fraction 4/16). The resulting mix of concrete was achieved using natural aggregates, thus replacing natural aggregates with waste, which had partially and fully replaced bulky aggregates with crushed concrete waste. The main aim of the investigation was to investigate how aggregates made from crushed concrete waste impact the properties of concrete. The exothermic effect on the concrete mixture during the hardening process was investigated. Furthermore, a macrostructural analysis of hardened concrete was conducted using scanned sample images; the adhesion zone between newly formed concrete stone and aggregates derived from natural rock from crushed concrete waste was investigated. Using an electron microscope to observe aggregate from crushed concrete waste and the contact zone of hardened cement stone revealed that the aggregate from waste adheres poorly with hardened cement stone. Furthermore, both the mechanical properties of new, hardened concrete and determined resistance to frost indicators are weak. Concrete density and compression strength decreased , and absorption increased almost twofold due to aggregates derived from crushed concrete waste, since their cleavage strength indicator was twice as high, while water absorption was four times higher than that of natural aggregate. The results indicate that recycled concrete obtained from demolished buildings is environmentally sustainable and can be recommended for lower quality concrete for use in related engineering projects.  The world is intensively attempting to solve concrete waste recycling and re-usage problems. Concrete waste can be used as an aggregate in newly mixed concrete production . Many reCoarse aggregate is produced by crushing and sieving concrete waste. This aggregate consists of several types of particles : naturalIn the presence of such composition concrete aggregates, fresh concrete is of two types, according to mechanical properties. Aggregates, that is to say, rock, is characterized by a high compression strength (100–250 MPa), and lower mechanical strength aggregates . Due to this reason, some changes to fresh concrete parts can be seen when calculating concrete composition. In this fresh concrete, a higher amount of concrete is used than in concrete with aggregates from natural rocks. Additionally, a higher amount of water is used, since crushed concrete aggregates are characterized by a higher need for water, and to mix the mixture, a higher amount of concrete is used.Environmental performance and environmental life cycle assessment (LCA) of the manufacture of reused concrete, along with uncertainty of shear resistance models are the best tools according to related research carried out by leading scientists ,18,19,20The goal of this work is to not only research concrete, which is produced from crushed old concrete waste instead of coarse aggregate, and mechanical properties, but also the exploitation properties and structural indicators. By using coarse aggregate from crushed concrete waste for research, it is possible to determine the hardened concrete class and mechanical properties of ecological concrete.3, a particle density of 2.43 g/cm3, and the particle size is from 0.125 up to 4 mm. This aggregate fits LST EN 12620:2003+A1:2008 standard requirements.In this work, a binding material was used—Portland cement CEM II/A-LL 42.5 N—which corresponds to LST EN 197-1:2011/P:2013 standard requirements. For a fine aggregate, natural sand was used, which has a bulk density of 1.63 g/cmTwo types of coarse aggregates were used: natural gravel and crushed concrete waste. Crushed concrete waste aggregate was prepared from concrete waste brought from three different crushing sites. This concrete waste was not contaminated with any salts or chemicals since it was picked out in the building material waste site by hand. When wanting to use aggregates made from concrete waste in concrete production, it is important that they are close to the compression strength class of the produced concrete. Due to that, the crushed constructions have to be picked out and sorted beforehand. During raw material research, the strength of crushed concrete constructions was determined. Cubes of 100 mm × 100 mm × 100 mm were cut from brought waste and their compression strength was determined (from 45 to 65 MPa). Crushed concrete pieces were sieved through 16 mm and 4 mm meshes. The main characteristics of this aggregate are shown in All concrete compositions were picked according to raw material characteristics with the calculating experimental method by using the computer program “AGbetonas” (2003). This program was created using concrete standard recommendations. The mixed fresh concrete slump is 3 cm.In this work, research was carried out by changing the amount of concrete waste and all coarse aggregate amounts in concrete. Due to this, three fresh concrete mixtures were prepared, which were marked CW0, CW1, and CW2. Fresh concrete compositions are presented in According to the provided concrete compositions, 100 mm × 100 mm × 100 mm concrete cubes were formed. Their hardening was carried out according to EN 206-1 standard requirements.Exothermic process temperatures in the fresh concrete mixture were determined by a methodology created by the “Alcoa” firm . A fresh concrete sample weighing 1.5 kg was placed into 100 mm × 100 mm × 100 mm textolite vessels. After filling the vessel with mixture, the sample had a T-type thermocouple placed in it, which was then placed into a glass tube. The thermocouple was connected to a data-transmitting device and a personal computer. Instantly after the fresh concrete was mixed, the form was placed into a metal box and isolated with 50 mm thick polystyrene foam. Temperature rises done in an automatic method were constantly recorded into the computer without any interruptions.The concrete samples’ main properties were determined while upholding methodologies specified in normative documents: concrete sample density according to LST EN 12390-7:2019, concrete sample absorption according to LST EN 13369:2018, and sample compression strength according to LST EN 12390-3:2019. Samples were compressed with an “AUTOMAX 3000”  press, corresponding to LST EN 12390-4:2019.2, effective porosity WE, general active porosity WR, porous space reserve R, capillary mass stream speed in a vacuum perpendicular to freezing temperatures G1, and capillary mass stream speed in a vacuum in the direction of freezing G2. The sample predicted exploitation resistance to frost was calculated according to the sample structural characteristics, using predictive formulas according to [Using the methodology in ,25, concrding to . R—porous space reserve,%; D—conditional pore and capillary wall strength; G1—capillary mass stream speed in a vacuum in the direction of freezing; G2—capillary mass stream speed perpendicular to freezing direction; N—structural direction unevenness indicator; g1—capillary mass stream speed during normal conditions.The sample destruction start can be calculated using this formula:The scanned sample image was investigated with a video creation program. The image was scanned with 800 dpi image quality. For image creation, the Adobe Photoshop CS2 program was used, using different colours to illustrate different phases: pores, hardened cement rock together with fine aggregate, coarse aggregate from natural rock, and coarse aggregate from crushed concrete waste.2 (15 g/dm2), then the experiment ended.The sample resistance to frost was indicated according to technical conditions CEN/TS 12390-9:2006/P:2007. The experiment was carried out according to an alternative experiment method CF/CDF . Prepared concrete samples a were weThe contact zone between the aggregate from concrete waste and newly formed cement rock was investigated with a scanning electron microscope, SEM EVO LS 25, Zeiss Germany , using the navigational system . The imaThe dominant space was marked in the program, which could be located in any part of the sample. Then the microscope lens/diaphragm pushed into the marked spot. After finding the contact zone, the image was zoomed in as necessary, so that it was be possible to see and fixate the dominant space.To determine the aggregates, obtained from concrete crushing waste, the influence on fresh concrete hardening and the exothermic effect change during the Portland cement hydration duration was determined. Three fresh concrete samples have their compositions shown in max, since both mixture temperatures’ highest value was almost the same. Fresh concrete, marked as CW0, and only has natural coarse aggregates, had a Tmax of 31.28 °C, which was reached after 14 h 28 min. Meanwhile, after replacing part of the natural coarse aggregates with concrete waste , a Tmax of 31.24 °C was reached after 14 h 59 min. Mixture CW2 (15 h 34 min), which has coarse aggregates obtained by crushing concrete waste, had a Tmax of 30.50 °C, while its reaching period lengthened by 1 h 6 min when compared with fresh concrete without waste’s (CW0—14 h 28 min) temperature, for which only natural coarse aggregates were used. By replacing part of the coarse aggregates with concrete waste, it can be noted that concrete waste insignificantly affects the mixture temperature THardened concrete, made from researched mixture macrostructure analysis, was done by analyzing the sample cross-cut area and calculating each composite phase areas. In In After calculating each colored zone in Sample CW1 crosscut photos are shown in After calculating the different color areas, it was determined that white colored pores take up 0.81% , light gray colored cement rock takes up 45.21%, and dark gray and black colored coarse aggregates from natural rock and crushed concrete waste—53.98% .In Visually, it was noticed that half of the coarse aggregates during the crushing process are separated from the cement rock, while half the aggregates in the mixture are strongly stuck to the cement rock. Coarse aggregates in concrete are evenly spread through and they are all separated from one another with a mixed cement paste with fine aggregates. After calculating every colored zone area b it was After completing the concrete made by using concrete waste macrostructure analysis, that is, after completing the concrete sample cut research and calculating every composite phase occupied area, it can be stated that the mixtures with waste , evenly spread across the entire concrete mass. However, they encounter several problems: when mixing mixtures, air partially included into the fresh concrete, together with old concrete pieces, stay in there during compaction. Aggregates with cracks and fissures enter into the mixture. They are particles that appear during the crushing process when the hardened concrete strength, absorption, and other properties are negatively affected.While analyzing concrete microstructure, examinations of microscopic contact zones were done, which allowed to determine the closeness connection between the cement rock and aggregate. With SEM, concrete samples were investigated after hardening for 28 days .Microscopic research showed that the contact zone between the waste and newly formed cement rock depends on the aggregate origin. When using natural aggregates, it can be noted that they bond well with hardened newly formed cement rock. When fresh concrete uses aggregates from waste, then between it and the newly formed cement rock a crack appears, separating these main fresh concrete components, and due to this, the contact between them weakens. To avoid the weakening of these zones, it is necessary to mix in an admixture and an addition to fresh concrete .3 [The hardened concrete sample absorption after three days of submersion in water was determined by researching the physical and mechanical properties. The results are shown in 3 .Concrete sample compression strength after 7 and 28 days of hardening were determined .It is seen that by raising the waste amount in the mixture, concrete compression strength lowers, for the concrete sample CW1, compression strength was 91%, counting from the sample compression strength of CW0. While investigating the compression strength change, all aggregates were changed with concrete waste. It can be seen that by using waste, the compression strength fell by 18%, while compared with the sample compression strength when there is no concrete waste. A decrease in strength can be explained by a weakened connection between the aggregate from waste and newly formed cement rock.Using experiments and calculations, the formed sample resistance to frost was determined. Sample destruction when freezing and thawing was determined with experiments, while the predicted exploitation resistance to frost was determined with calculations. Predicted constructional concrete resistance to frost can be determined according to physical and structural characteristics . AccordiAccording to the research results , it can Predicted sample exploitation resistance to frost was calculated according to sample structural characteristics by the fixating sample destruction start. According to these indicators, it can be seen that the highest predicted exploitation resistance to frost is of the concrete samples, which are prepared by using natural aggregates, while using crushed waste this size was smaller.Effective sample porosity size shows how many pores and empty spaces the sample has that a chance to be filled with water during the the 3 day submerging process have. General sample porosity shows how many pores and empty spaces have a chance to be filled with water throughout a very long period . According to From 2 (15 g/dm2). The best results, according to this indicator, were obtained during the experiments of sample CW0, which used natural aggregates. Its mass loss after 56 freezing and thawing cycles was only 0.87 g/dm2. The sample surface had no visible big cracks; the contact zone between the cement rock and aggregate was undamaged. On some parts of the cement rock, only insignificant micro-cracks were visible.To effectively investigate concrete resistance to frost, it is necessary to determine not only structural concrete characteristics and to calculate the predicted exploitation resistance to frost, but to also carry out their comparison analysis using the resistance to frost values obtained during the experiment process. The determined sample resistance to frost results are shown in In the concrete samples, where part of the coarse aggregate was replaced by waste (CW1), and samples where used aggregates were only made of waste (CW2), mass loss was highest, however, they did not reach maximum values. In these samples, after 56 cycles, significant cracks appeared on the sample surface and contact zone between the aggregate and cement rock, which are marked in The appearance of cracks in the contact zone betweenthe aggregate and cement rock can be explained by a weakened connection between the aggregate from waste and newly formed cement rock.After comparing the results shown in The investigated concrete costs comparison is shown in After researching how concrete properties change when using crushed concrete waste instead of coarse natural aggregate for mixing, it can be determined what concrete products—as in, what concrete class products—could be produced. From the research results, it is clear that by changing the natural aggregate to concrete waste after hardening lowers the mechanical characteristics of the obtained concrete. However, not all concrete products can be produced from higher class concrete, similar to the fact that not all products need to have a high resistance to frost. For products which do not have a high resistance to frost and mechanical characteristic requirements, crushed concrete waste can be used perfectly fine. In this way, a high amount of waste can be used up.By using the standard research method to determine the main aggregate characteristics, it is evident that coarse aggregate obtained from concrete waste differs from aggregate made with natural rock. It was also noted that the concrete waste cleavage indicator and absorption can be significantly higher than natural aggregate.The research conducted using concrete samples, for which crushed concrete waste (4/16) was used as coarse aggregates, indicates that the properties of hardened concrete depend on the amount of concrete used in fresh concrete. The concrete density and compression strength decreased , and absorption increases  due to the aggregates derived from crushed concrete waste, since the cleavage indicator is two times higher, while water absorption is four times higher than in natural aggregate.Using a raster electron microscope to observe aggregates from crushed concrete waste and the contact zone of newly hardened cement rock, demonstrates that coarse aggregate from waste does not bond well to the newly formed cement rock, which impairs the mechanical property of concrete and causes a deterioration in its physical properties.Concrete includes coarse (4/16 mm) aggregates obtained from crushed concrete waste, resulting in a lower compression strength of 5–8%, and a lower density—8–18%. Notably, there is almost a 200% rise in the absorption rates, since the concrete structure and the use of fractional concrete waste properties change when compared with natural aggregates.Even though effective and general sample porosity increased after adding concrete waste, porous space reserve in these samples became smaller than 30%. This indicator is a strong influence when predicting the resistance to frost. Due to this, and after calculating all structural parameter influences, we determined that the predicted exploitation resistance to frost declined by 19%–28%.2 when investigating concrete samples with no concrete waste in their composition. By adding part of the concrete waste , the mass loss rose 4.7 times, whereas the mass loss rose 6.2 times when coarse concrete aggregates were replaced by crushed waste. After measuring the concrete sample resistance to frost with an experimental method, it was determined that the mass loss after 56 freezing and thawing cycles amounted to 0.87 g/dm


Escherichia coli is an important pathogenic indicator in drinking water. Viable but non-culturable (VBNC) E. coli induced by low level chlorination was found to have higher antibiotic tolerance. The emerging of VBNC bacteria in drinking water systems is posing challenges to the control of bio-safety. It is necessary to study the underlying mechanisms of VBNC state E. coli under actual residual chlorine condition of drinking water pipe network. In this study, we investigated the changes of morphology and gene expressions that might present such state. The results indicated that the size of VBNC E. coli was not remarkably changed or recovered culturability under favorable environmental conditions. Results from transcriptomic analysis revealed that the regulated genes related to fimbrial-like adhesin protein, putative periplasmic pilin chaperone, regulators of the transcriptional regulation, antibiotic resistance genes and stress-induced genes, rendering VBNC cells more tolerant to adverse environmental conditions. In total of 16 genes were significantly up-regulated under the VBNC state, including three genes encoding toxic protein , indicating that VBNC E. coil was still a threat to human. The work is of great relevance in the context of better understanding this poorly understood physiological state. However, several reports have mentioned that some waterborne pathogens could be induced into a viable but nonculturable (VBNC) state by chlorine or chloramine disinfectants10. VBNC bacteria cannot be detected by the conventional plate counting method which leads to an underestimation of the potential microbiological risks. Thus, the pathogenic bacteria in the VBNC state may pose a threat to public health.Bacteria are prime targets to remove from drinking water since waterborne pathogenic microorganisms can cause epidemic diseases. In many countries, chlorine at levels of 0.05–0.5 mg LEscherichia coli in the VBNC state persisted in the presence of certain antibiotics, e.g., 200 μg mL−1 ampicillin (Amp) and 5 μg mL−1 ofloxacin (Ofx) 11. In addition, Oliver et al. reported that Vibrio vulnificus could enter the VBNC state, protecting cells against a variety of potentially lethal environmental challenges 12. In our previous study, it was found that low doses of chlorine (0.5 mg L−1) disinfection effectively reduced the number of culturable E. coli and induced a VBNC state, after which the metabolic activity of the bacteria was reduced and persistence of E. coli in the presence of 9 typical antibiotics was enhanced13. Moreover, Moreno et al. also reported that low-level of residual chlorine in DWDS was capable of inducing VBNC state antibiotic-tolerant bacteria, which could be considered as an undetectable risk8. Chlorination, a vital step in controlling the biosafety of drinking water, was directly related to public and environmental health. Thus, it is necessary to thoroughly investigate the VBNC state induced by low concentrations of chlorine.It has been reported that VBNC bacteria exhibit an extraordinary tolerance to adverse conditions. Vibrio spp. and Pseudomonas aeruginosa in the VBNC state could continuously express known toxin and virulence genes15. To survive under unfavorable conditions, major metabolic pathways  could be altered to obtain energy and reduce the metabolic rate under the VBNC state16. Some studies focused on the gene expression of VBNC bacteria to explore their regulatory mechanisms19.In addition to the compromise of physical structure, chlorine has also been reported to impact the metabolic processes, efflux pumps, and pathogenicity of bacteria by regulating gene expression. Therefore, it is important to comprehensively investigate the antibiotic tolerance of VBNC bacteria at the morphological and genetic levels. Generally, quantitative reverse transcription PCR (RT-qPCR) is applied to quantitatively determine the expression levels of known target genes in viable cells. It was reported that human pathogenic 20. Conventional investigation through individual genes is inadequate for discovering the complete regulation and cross talk of these genes. Nowadays, the high-throughput transcriptome sequencing (RNA-seq) technique has been applied in constructing frameworks of gene expression which has markedly accelerated the capacity of analyses21. Several comparative transcriptome analyses focused on some important environmental bacteria, including Pseudomonas syringae (Fmicb)18, Rhodococcus biphenylivorans19 and Vibrio splendidus22. These studies highlighted a more comprehensive and data-intensive approach for better understanding of the mechanisms of bacterial stress responses. In DWDS, the inducer is chlorine, a chemical oxidant that may endow many different characteristics to the VBNC bacteria compared with physical inducers such as low temperature. Accordingly, it is worthwhile to analyze the adaptive responses of E. coli in the VBNC state induced by low-level chlorine via transcriptomic analysis.The regulation of gene expression involves complex and specific biological processes composed of a series of functionally related moleculesE. coli was selected as the test which is the indicator for pathogenic bacteria in the drinking water quality standards all over the world24. The bacteria were exposed to chlorine at the level comparable to residual chlorine in a DWDS to induce the VBNC state. The gene expression variation of the VBNC bacteria was assessed via transcriptome approaches. Then, the morphology was investigated by scanning electron microscope (SEM) and flow cytometry (FCM). It was expected that the special regulation behavior at low level chlorination circumstances could be revealed and deeper understanding of VBNC cells could be acquired.In this study, as a continuation of the previous work, −1, the concentration generally applied in DWDS2. In our previous study, it demonstrated that 105–6 cells mL−1 of bacteria entered the VBNC state at 0.5 mg L−1 residual chlorine in 6 h  method, a direct viable count (DVC) still presented observed growth in the range between 3- and 4-log cells mL−1. Significantly high numbers of HPC and DVC were found both in the biofilm and effluent of the model system25.Chlorination is vital for controlling bacterial regrowth in DWDS. It usually takes several hours to days to transport water from a treatment plant to household taps. Such residence time is sufficient for the induction and resuscitation of bacteria into/from the VBNC state. To induce a VBNC state, we chose chlorine at 0.5 mg LE. coli  was verified. Resuscitation experiments were performed with VBNC cells using liquid media. As shown in Fig. 3 CFUs mL−1). The same initial concentration of culturable E. coli was used to draw the growth curve as a control. We found that the growth curve of VBNC cells was significantly different from that of the conventional growth curve, suggesting the occurrence of resuscitation . In our results, approximately one in twelve (1/12) tubes displayed this recovery.VBNC microorganisms can be resuscitated under certain conditions. In this study, the resuscitation of VBNC ion Fig. . The the1 to 103 cells/mL in real drinking water. This means that residents in these cities will be exposed to up to 1013–15 VBNC (101–3 cells/mL = 1013–15 cells/million tons) cells per day, which might resuscitate under the appropriate conditions.This recovery was not high for the absolute percentage of bacteria. However, the risk of resuscitation will be even more serious considering the large water supply in large modern cities. Currently in China, many cities have a water supply capacity of over 1 million tons/day. The probability of VBNC cells is approximately 1026. Unlike the VBNC state induced by cold temperature, the bacteria sterilized by low chlorine concentrations showed no changes in cell morphology. However, we observed a number of damages on the cell membrane  of the chlorinated bacteria (red arrows), as shown Fig. et al. confirmed that bleach inhibits the biosynthesis of outer cell wall mycolic acids and induces oxidative damage in bactreria27. In the presence of 0.5 mg L−1 chlorine, some bacteria were relatively intact E. coli had enhanced permeability in the presence of selected chlorine concentrations (data not shown). These results were in accordance with the conclusions reached by Virto et al.28 and Tatsumi et al.29 who suggested that approximately a dose of 1 mg L−1 chlorine was linked to an increase in the membrane permeability of four types of bacteria including E. coli and neutrophils.The results of FCM corroborated the conclusion that some cells were SYTO 9 positive Fig. . FCM ana33, but few studies have investigated VBNC cells produced in municipal pipe networks. Their researches confirmed that VBNC cells existed in municipal pipe networks exhibited strong viability, and they could resuscitate25. Undoubtedly, more researches are needed to explore bacteria in a VBNC state, which may yield insight into strategies for the prevention and control of VBNC bacteria.Recently, several studies have focused on the induction conditions and properties of VBNC cellsE. coli have been constructed. The libraries of VBNC cells and culturable cells had 27.97 million and 29.82 million reads respectively, and these reads were aligned with the E. coli K12 genome sequence (NCBI GenBank accession No. NC_000913). After incubation with a low concentration of chlorine, the expression of 362 genes of E. coli was found to be changed significantly (expressed in the log2 (FoldChange) with base 2, hereinafter indicated as |log2 (FoldChange)|) (|log2 (FoldChange)| >1 and p < 0.05) | >5 indicated that these genes had extremely significant changes after exposure to chlorine and needed special attention. Detailed information of the 362 genes with significant differences was provided in supporting information Table etc.cDNA libraries derived from VBNC and culturable 05) Fig. , of whicIn the Venn diagram, the total expressed genes were plotted with two comparisons of transcriptional profiles Fig. . EstabliTo ascribe gene functions to differentially expression genes (DEGs), a total of 3194 DEGs were assigned to 3 parts: biological process, molecular function and cellular component. To determine the Gene Ontology (GO) function of the up- or downregulated genes, GO enrichment analysis was performed on the 2048 upregulated and 1777 downregulated genes. The 30 most enriched GO terms are presented in Fig. Additionally, the genes were mapped to the KEGG pathway. Among the mapped genes, the upregulated genes in the VBNC cells were mainly enriched in two pathways: ABC transporters and microbial metabolism in diverse environments. The enriched genes had lower Q values, indicating significant enrichment Fig. . In Fig.4. However, VBNC cells still have high level of gene expression, and the behavior of these genes has a greater contribution to the survival mechanisms and resuscitation of VBNC cells. The present study focused on highly expressed genes. Figure E. coli were related to 6 functional groups, including ABC transporter, biotin, curli, biofilm, nitrate reductase and others. ABC transporters, including ATP-binding components of amino acid ABC transporters, ATP-dependent high affinity P-Type ATPase metal ion transporters, saccharides ABC transportors, etc., were upregulated from 3.15 to 6.39 log2 (FoldChange), suggesting that some genes could be highly expressed in VBNC bacteria to survive35. With respect to the expression of other genes, some genes involved in biotin synthesis could encode proteins that produce some growth factors, increasing the survivability of bacteria. Enterobactin  is a high-affinity siderophore that acquires iron for microbial systems. Pathogenic bacteria can steal iron from other living organisms using this mechanism, even though the concentration of iron is kept extremely low due to the toxicity of free iron37. The existence of such levels of biotin not only increases the pathogenicity of pathogenic bacteria , but also sequesters the iron resources of other bacteria. Curli is composed of two subunits: CsgA and CsgB. CsgE prohibits the self-assembly of CsgA into amyloid fibers. CsgE acts directly on the secreted substrate CsgA to prevent premature subunit assembly38. The expression levels of the csgE gene were significantly higher in bacteria in the VBNC state (4.18 log2 (FoldChange), P < 0.05) compared to the control bacteria, and the formation of pili was reduced, leading to the reduced motility of bacteria39. The cells that cannot swim could reduce energy consumption and are more suitable for dormancy. Additionally, biofilm formation would be regulated through the high expression of yjcH  and yibE (YibE is involved in extracellular polysaccharide production and is a threonine-rich protein). Overexpression of the yjbEFGH operon alters colony morphology, leading to the production of novel extracellular polysaccharide genes that promote the production of extracellular polymer substances and enhance the mechanical strength of biofilms40. Due to the bacterial reduction of nitrate and generation of nitrite with high expression of the nar V, W, Y, Z genes, more amino acids and other nitrogen compounds would be formed in cells, enhancing the strength of bacterial cell walls42. In other words, these enhanced processes would increase the persistence and survivability of the cells. Furthermore, the VBNC cells showed partial resistance, such as the expression of resistant genes  that are homologues of vancomycin resistance genes43. The polymyxin b resistance protein (ArnA)44, TA systems and the porin protein (CirA) increase the transcription of ybdZ.A VBNC state is a state of dormancy. In the VBNC state, many genes are downregulated to reduce energy consumption4. Thus, the downregulation of gene expression in dormant bacteria seems to be a common conclusion. In this study, we also listed genes that were extremely downregulated (log2 (FoldChange) < −4)  and cold shock protein . Hydrogen metabolism in E. coli is tightly regulated by anaerobic conditions of growth produced. The genes hyaA, B, C, D, E, and F were downregulated, which could decrease the synthesis of active HYD1, and reduce energy metabolisms1. CspA and CspB have been shown to be cold shock-inducible in E. coli. The low expression levels of CspA and B in VBNC cells suggested that bacteria were reduced in response to low temperature stresss2. Other downregulated genes  involved in ATP-dependent transport reduced the related biological process energy consumptions3, 4. The results of downregulation gene expression were consistent with previous studies.Dormant bacteria improve their prospects of survival by reducing metabolic activity4) Table . Eleven 2 (FoldChange) >5) , 5 encoding stress-induced proteins , 3 encoding regulators  and 3 encoding efflux systems   which could be useful for regulating the metabolic levels. RpoS is the master regulator of the general stress response in E. coli. Rapidly growing cells contain little, if any, RpoS. In contrast, in order to response to many different stress conditions, cellular RpoS levels rapidly increased up to 20-fold. This regulation occurred at the levels of transcription and translation, and RpoS proteolysis occurred in various stress conditions  that differentially affected these levels of control47. RpoS is a typical upregulated gene47 which could lead to the inhibition of bacterial division and increase the survivability under high-level expression. In particular, the metal efflux system genes (cusC and F)49 and the multidrug efflux protein gene (garP)30 were extremely upregulated, which could increase metal- and antibiotic persistence.For further analysis, we selected genes that were expressed at extremely high levels (log5) Table . There wP) Table . These gE. coli cells could be induced into the VBNC state if water was treated with low concentrations (0.5 mg L−1) of chlorine and VBNC E. coli had the potential to resuscitate. Results of the SEM showed that: the size of VBNC bacteria was not markedly changed, and FCM examination showed that: VBNC cells’ permeability was enhanced. Transcriptomic analysis suggested that the enhancement of the efflux system and stress-induced function contributed to the strong environmental tolerance of the VBNC cells and VBNC E. coli was still a threat to human health.From the morphology and based on transcriptomic analysis, we investigated the regulation of gene expression of VBNC cells induced by a low chlorine level. E. coli CMCC4410315 was cultured with Luria-Bertani (LB) broth  at 37 °C, 180 rpm until logarithmic phase (12 h). Cells were harvested  and re-suspended in sterilized buffered saline and then diluted to a final concentration of 107–108 CFU mL−1. The cell number was counted by plate counting and disposable counting chambers . The bacteria were divided into two parts, one of which was used to induce the VBNC state, and the other for control. The control was culturable E. coli with the same treatment except chlorination.−1 was made by addition of disinfectant stock solution. To maintain stable chlorine level, chlorine was added every 2 hours. The concentration of free chlorine was determined using the DPD method . All cells were incubated at 25 °C, 180 rpm and sampled at 0 min, 15 min, 30 min, 1 h, 3 h, 6 h, 9 h, 12 h, 18 h and 24 h. Before sampling, 100 μL of 20% sodium thiosulfate  was added to inactivate residual chlorine.An initial disinfection concentration of 0.5 mg L4. When cells were exposed to chlorine for 6 hours or longer, the numbers of VBNC and viable cells were equal because no colonies were formed on the plates. Cells were considered to have entered VBNC state (105–6 cells mL−1) and have been ready for the next preparation step in this study  by centrifuging at 7800 rpm, 4 °C, 20 minutes and harvested for RNA extraction.In this study, culturable cells were identified by plate counting on NA agar. The number of viable cells was counted by flow cytometry. The number of VBNC cells was calculated as the difference between the numbers of viable and culturable cells. This is a classic characterization method that could count numbers of VBNC cellsE. coli. In each experiment, 4 mL of prepared VBNC state bacteria were separately dispensed into 50 mL centrifuge tubes. Each tube contained 36 mL sterilized LB broth medium and 4 mL bacterial culture, the ratio of VBNC E. coli to LB broth was 1:9. The initial concentration of VBNC cells was about 104 cells mL−1. All of these tubes were cultured at 37 °C, 180 rpm. 1 mL of sample was collected hourly and coated on nutrient agar (NA)  plate, and the resuscitation was evaluated by plate counting.Resuscitation experiments were conducted in LB broth medium with the prepared VBNC state 50, whereas 5-cyano-2, 3-ditolyl tetrazolium (CTC) was used for viable cells counting 51. SYBR Green  was added into 0.2 mL suspensions in 1.5 mL tubes . CTC was added to the same system at a final concentration of 2 mM . Samples were analyzed by FCM  within 2 h after resuspension, and Forward scatter (FSC), side scatter (SSC), fluorescence signals SYBR Green and CTC using 525 nm and 620 nm band pass filters were measured in logarithmic amplification mode. 5,000 cells for each sample were collected. Dyeing operations were all conducted in the dark.Cell viability was analyzed by flow cytometer (FCM) . SYBR Green was used for total cells counting 1. All these experiments were performed in triplicate. Cells were considered to be in VBNC state when none of the colonies was demonstrated on a NA plate (1 mL of sample was coated).For culturability analysis, each sample was incubated on NA at 28 °C for 24 h according to the national standards7–8 cells mL−1) were centrifuged at 12,000 × g for 1 min and re-suspended in sterilized buffered saline. The control in this paper was culturable E. coli with the same treatment except chlorination. RNA was extracted using EasyPure RNA Kit . RNA purity was examined using the micro-spectrophotometer Nano-100 . Qubit  was used to measure the RNA concentration. The experiments were repeated three times under the same conditions. The RNA which was extracted from biological duplication was used for RNA sequencing. In addition, Pearson correlation between samples was checked to ensure accuracy of the results of RNA-seq.Previously prepared control (4.1) and VBNC (4.2) bacteria solutions . Sequencing libraries were built using NEBNext® Ultra™ Directional RNA Library Prep Kit  following the manufacturer’s instructions. Briefly, the mRNA was fragmented and purified with poly-T oligo-attached magnetic beads . Then, first strand cDNA and second strand cDNA were synthesized. PCR was performed of universal PCR primers and Index (x) Primer. Finally, the Library preparations were sequenced on an Illumina Hiseq 2000 platform.53 was used to analyze the gene expression variations between culturable and VBNC cells. The reads numbers mapped to each gene was counted by HTSeq v0.6.1.FPKM (Expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced) method54.DESeq R package was used to analyze differential expression of culturbale and VBNC state. After multiple testing correction, the adjusted P-value less than 0.05 by DESeq was selected as a threshold of differential expression55.GOseq R package was used to GO enrichment analysis of differential expressed genes. GO terms with adjusted P-value < 0.05 were assigned as significantly enriched by differential expressed genes56.KEGG was used to understand functions of the biological system from molecular-level information. In this study, KOBAS software was adopted to analyze the statistical enrichment of differential expression genes in KEGG pathwaysFive typical genes were chosen for the expression levels via qRT-PCR to confirm the transcriptome results. The information of primers was listed in Table 25. Briefly, SYTO 9 and propidium iodide (PI) were added in 1.5 mL tubes at a final concentration of 0.5 μM and 10 μM, respectively . Samples were analyzed by FCM (the same operations as method 4.4), and the fluorescence was recorded for SYTO 9 and PI using 480/525 nm and 488/617 nm, respectively. The morphological structures were observed by SEM  at the 20,000 × magnification.Morphological characteristics of culturable cells and VBNC cells were investigated using the LIVE/DEAD BacLight bacterial viability kit Characterization and potential mechanisms of highly antibiotic tolerant VBNC Escherichia coli induced by low level chlorination.Dataset 1.


Pervious concrete is considered to be porous concrete because of its pore structure and excellent permeability. In general, larger porosity will increase the permeability coefficient, but will significantly decrease the compressive strength. The effects of water-cement ratio, fiber types, and fiber content on the permeability coefficient, porosity, compressive strength, and flexural strength were investigated. The pore tortuosity of the pervious concrete was determined by volumetric analysis and two-dimensional cross-sectional image analysis. The concept and calculation method of porosity tortuosity were further proposed. Results show that the permeability coefficient of the pervious concrete is the most suitable with a water-cement ratio of 0.30; the water permeability of the pervious concrete is influenced by fiber diameter. The permeability coefficient of pervious concrete with polypropylene thick fiber (PPTF) is greater than that with copper coated steel fiber (CCF) and the polypropylene fiber (PPF). The permeability coefficient is related to tortuosity and porosity, but when porosity is the same, the permeability coefficient may be different. Finally, general relations between the permeability coefficient and porosity tortuosity are constructed. Concrete is the most widely used material in civil infrastructure systems, and low strength concrete is usually used in earth rock dams, water-retaining walls, and other constructions ,2. It is3 [According to Marinkovi et al. ,6, waste3 . Because3 . UsuallyGenerally, compared with ordinary concrete, the strength and structural performance of pervious concrete is more variable because of the porosity . A studyFibers have the effect of delaying and hindering cracks, and they are widely used in cement-based composite materials  and resiPorosity is used to characterize pervious concrete, but not all porosity is effective for water to flow through. Some pores are closed and do not connected with others. In addition, there are small pores, like capillary pores, and they have no contribution to the transport of fluid. Nevertheless, these pores affect the strength and permeability coefficient of pervious concrete. A new method was proposed by Kia Alalea  to study2 = 0.99. This research can further promote the understanding and application of pervious concrete.The natural aggregate was replaced by recycled aggregate and recycled fine aggregate, and fiber and ultra-fine silicon powder (SP) were added in this study. The effects of fiber types, including polypropylene thick fiber (PPTF), copper coated steel fiber (CCF) and polypropylene fiber (PPF) with different percentages of the permeability, porosity, compressive strength, and flexural strength of the pervious concrete, are studied. In addition, three water-cement ratios of 0.25, 0.30, and 0.35 are evaluated. This study is an attempt to propose the concept and calculation method of tortuosity based on the characteristics of pore structure. Also, the second degree equation of the permeability coefficient, porosity, and tortuosity is fitted, where RAccording to GB175-2007 , PO42.5 As shown in Three types of fibers were considered in the test. Their photos and characteristics are shown in Generally speaking, according to ACI 522R 2010, the water-cement ratio is between 0.26 and 0.45 for pervious concrete and fine aggregates ranging from 0% to 15% . CCF andFirstly, the aggregate, cement, and mineral admixture were added to the mixer, and then 80% water was added and it was stirred for 30 s to wet the cement into a cement slurry bonded to the aggregate surface. In the next step, the remaining water and fiber were then added together and stirred for a further 120 s to allow the cement to fully saturate and mix well with the fiber.The cylinder specimens with a diameter of 100 mm and height of 50 mm were cast for the permeability coefficient and the porosity test; the cubic specimens with side length of 100 mm were cast for compressive strength test, and the prism specimens of 100 mm × 100 mm × 400 mm were cast for flexural strength test, in which the three specimens for each group were prepared.The vibrating table was used firstly to vibrate the concrete, and then a trowel was applied to smooth the casting surface. After curing for 24 h in a natural environment, the specimens were transferred to a curing room at 95% RH and 20 °C for 28 days under standard conditions.The constant head method is derived from the Japan Society of Concrete Engineering, and its principle of measuring the permeability coefficient is Darcy’s law. The experiment device of the constant head method is shown in Q in the time was measured again to calculate the permeability coefficient. The calculation of the permeability coefficient can be derived from the Darcy formula:k is the permeability coefficient, t is the time, Q is the amount of overflow water in the time, L is the thickness of the test specimen, A is the surface area of the cylinder, H is the water level difference, t is the time, and i is the hydraulic slope.The test specimen was sealed with a sealing material and placed on a bracket of the cylinder. After the water level difference was stabilized, the amount of overflow water P is the porosity, V is the volume of test specimen after drying, V1 is the volume reading of the test specimen immersed in water, and V2 is the volume reading after the bubbles are discharged.The test specimen was dried until it attained constant weight, and then we measured its apparent size. Next, the test specimen was immersed in a suitable container with water. Last, the container was shaken up and down to remove the air bubbles. The calculation formula for porosity is shown in Equation (4):As shown in Based on the methods and materials in the experiment, the main results of this test are shown in 3, the permeability coefficient and porosity gradually reduced with the rise of water-cement ratio. When PPTF was 3 kg/m3, with the increase of the water-cement ratio, the permeability coefficient first increased by 12.02%, and then it decreased. The porosity decreased gradually, and the decreasing was minor. When the PPTF was 4 kg/m3, the permeability coefficient fell off gradually, while the porosity had a different trend: the figure for it climbed before dropping dramatically. The water-cement ratio of 0.30 was higher than the water-cement ratio by 0.25, with the number at 23.53%.The effect of PPTF content on the water permeability of the pervious concrete at different water-cement ratios is shown in In general, the variation of porosity is slight while the range of permeability coefficients is pretty large. As the water-cement ratio raises, the permeability gradually reduces . In factIt can be seen from the 3, the porosity decreased rapidly. With the increase of PPF content, the porosity dropped after boosting. As for the permeability coefficient, there was an increase in the initial stage, but it suffered a reduction later and finally stabilized. Ali A. Aliabdo and H. Toutanji [As shown in Toutanji ,28 also Because the porosity of the recycled aggregate pervious concrete is mainly composed of connected porosity and semi-connected pores, as fiber content increases, PPTF and CCF occupy a part of the volume of the pervious concrete connected pores, resulting in a decrease in connected porosity. However, the adhesion between fiber and cement substrate is relatively poor. Under the action of pressure, water can easily enter the interface between fiber and substrate, causing the interfaces to communicate with each other, so the connected porosity has an increase. In general, fiber content is low, and the water permeability coefficient varies little with the change in fiber content.PPF has good dispersion in concrete, due to the incorporation of fibers, and the fluidity of the concrete is changed. As a result, the thickness distribution of the cement slurry between the aggregates is uneven, hence affecting the porosity of the recycled aggregate concrete. It can be known from physical properties of fiber that the diameter of PPF is the finest, CCF is the second, and the diameter of PPTF is the thickest. Therefore, it is apparent that the fiber diameter has a significant influence on the permeability of pervious concrete.3, the compressive strength increased from 18.90 MPa to 20.99 MPa, and finally, reached 19.96 MPa, with a decrease of 8.94%. When the fiber content was 2 kg/m3, the compressive strength climbed slowly as the water ratio increased. For instance, as the water-cement ratio increased from 0.25 to 0.35, the compressive strength rose approximately 5.61%. In general, the compressive strength changes insignificantly as the water-cement ratio varies. This result is consistent with the test results of Saeid Hesami [The effect of the water-cement ratio on the compressive strength is shown in d Hesami ,30.In the mix ratio, as the water-cement ratio increased, the amount of water increased and the amount of cement decreased. In the water-cement ratio of 0.25, the content of cement mortar was relatively low, and the aggregate could not be wrapped well, resulting in loose filling. Also, the hydration was insufficient, so the compressive strength of pervious concrete was eventually reduced.In the water-cement ratio of 0.30, it had a certain fluidity, which improved the strength and bonding ability between the aggregates and the concrete saturate. The hydration was more sufficient, and the strength was higher than that of the pervious concrete with a water-cement ratio of 0.25. In the water-cement ratio of 0.35, the fluidity of mixture was so large that it could easily lead to a slurry. The strength of concrete structure was increased, but the lifting range was small. It can also be seen from the influence of water-cement ratio on porosity in 3, the compressive strength was the highest at 21.43 MPa. After that, there was a slight decrease. Generally speaking, CCF and PPF all contributed to raising the compressive strength. This finding agrees with the test results of Ali A. Aliabdo et al. [As shown in o et al.  and Habuo et al. . When thPPF and PPTF have the same performance except for aspect ratio. However, the volume of PPF is significantly larger than that of PPTF, and the distribution is more random. CCF belongs to steel fiber, its compressive strength can reach 200 MPa, and it tends to go upward with the increase of fiber content. When the fiber content is too much, the dispersion is poor, and the interface between fiber and substrate is weak, which results in a decrease in the compressive strength of pervious concrete.The influence of fiber types and content on flexural strength as shown in Three types of fibers all have contributed to improving flexural strength . CCF andThe permeability coefficient is an indicator that reflects the permeability of the pervious concrete. When designing the mix ratio of pervious concrete, the design porosity can be controlled to ensure the permeability coefficient. However, due to differences in raw material properties and differences between the concrete forming process and the pouring quality, the actual porosity of pervious concrete cannot be guaranteed to be the same as the designed porosity . MoreoveAs shown in The percolation channels in pervious concrete are influenced by pore structure, mixture ratio, and the aggregate size. Ideal percolation channels are shown in h is the height of pervious concrete specimen (here 50 mm); d is the diameter of pervious concrete specimen (here 100 mm); α is the angle between the pervious concrete axis and the equivalent flow length. In the ideal state, the percolation channels of the permeable concrete are all connected pores with vertical distribution, h is the shortest percolation channels, and at this time α equals 0. In the extreme state, l′ means the longest percolation channels in extreme state, and at this time α equals In l and the tortuosity R can be calculated by Formulas (5) and (6):h is the height of pervious concrete specimen.The equivalent flow length k = 29.08x1 + 16.84x2 − 3.61x1x2 − 9.52, R2 = 0.99, where k is the permeability coefficient, x1 is the porosity, and x2 is the tortuosity. Based on the materials and measurement methods used in this test, the following conclusions can be drawn:1. In a water-cement ratio of 0.30, the permeability of the recycled pervious concrete with fiber is the most appropriate. In a ratio of 0.25, the hydration is insufficient, while in the water-cement ratio of 0.35, excess cement slurry is easy to deposit at the bottom and seals the bottom pore.2. For pervious concrete with different types of fibers, in the water-cement ratio of 0.30, the permeability coefficient of the recycled aggregate pervious concrete with PPTF is 7.64 mm/s, which is better than that of CCF and PPF at 5.74 mm/s and 6.01 mm/s, respectively.3, the permeability coefficient and strength have relatively balanced results. The flexural strength is the highest at 3.42 MPa, the compressive strength is 21.43 MPa, and the permeability coefficient is 7.64 mm/s.3. In this paper, when the amount of PPTF content is 3 kg/m4. The permeability coefficient of pervious concrete is linearly related to porosity, but the permeability coefficient may be different at the same porosity.5. Fiber can significantly improve the flexural strength of pervious concrete. The flexural strength of pervious concrete is 2.46 MPa. When PPTF, CCF, and PPF were added, the flexural strength increased by 32.05%, 35.14%, and 30.12%, respectively.2 = 0.99.6. The concept of tortuosity is proposed and a method for calculating tortuosity is provided. The equation of permeability coefficient, porosity, and tortuosity is fitted, where R


MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles  and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary. Abnormal stimulation of multiple signalling transduction pathways downstream of MET promotes many of the hallmarks and challenges associated with cancer. For example, it is through proto-oncogene tyrosine-protein kinase SRC that MET acts as an epithelial-mesenchymal transition (EMT) promoter7. Aberrant activation of MET can occur through several mechanisms. These include MET amplification and higher gene copy numbers, as well as MET dysregulation through MET mutations. These which increase MET stability and accumulation on the cell surface and, thus, a targetable alteration. MET stimulation induces phosphorylation of specific tyrosine residues which, in turn, activate multiple downstream signalling pathways.Mesenchymal–epithelial transition factor (MET) is a receptor tyrosine kinase oncogenic driver in some non-small cell lung cancers (NSCLC)MET amplification is an oncogenic driver9 and MET over-expression occurs in ~ 72% of NSCLC10. Companion diagnostics, particularly those that are minimally-invasive such as in the blood, are urgently needed for patient stratification and ease of selecting patients who have a chance of benefiting from MET inhibitors. Research, by our group and others, has strongly implicated extracellular vesicles (EVs) as minimally-invasive packages of biomarkers; if due consideration is given to appropriate pre-analytical sample preparation11. Many methods for EV separation from biological samples have been proposed. The most commonly used is ultracentrifugation, typically with density-gradients12. This method can result in quite pure EV preparations, but it is time-consuming, requires high-end equipment, and is not translatable to a routine clinical diagnostic setting. Some methods are simpler and less laborious, but purity of EVs is often compromised13. In the context of considering EVs as sources of biomarkers reporting on MET status of their cells of origin, here we evaluated a number of EV separation methods, that to the best of our knowledge have never been directly compared. We have then further investigated if EVs carry MET information representative of their cells of origin. Specifically, we have investigated the presence of the gene transcripts encoding for MET (MET mRNA), total MET protein, and activated/phosphorylated MET protein. The experimental design for this study is summarised in Fig. The MET pathway is an important actionable target and so several MET inhibitors have been developed. These drugs can offer substantial benefit for treatment of several cancers where previously very few treatment options were available. However, only sub-groups of patients with these cancers can hope to benefit from MET inhibitors. Specifically, in up to 7% of NSCLC, 6 cells) recorded by nanoparticle tracking analysis (NTA) was obtained by UC-DG method of EV separation; low speed centrifugation (LSC) produced the second highest quantities, followed by ExoQuick  analysed, the highest particle counts  was used to detect surface proteins on single EVs (objects). As with NTA results, by IFCM-across all cell lines-, total EV concentration was higher following EV separation by UC-DG, when compared to LSC and ExoQuick Fig. a. TetrasIrrespective of EV separation method, common EV markers were detected by immunoblots in all EV lysates Fig. , S3, altThe highest concentration of RNA (ng/μL) was observed in EV samples from UC-DG, followed by LSC, then ExoQuick Fig. . As expeBy RT-qPCR, MET was detected in EBC1, H596 and Hs746T cells and their released EVs following separation by UC-DG, ExoQuick, and LSC , measurement of total MET (Pan MET) and phospho-MET  for cell and EV lysates (following the three different methods of EV separation) were performed. These showed that although cell lysates had higher amounts of total MET and pMET (Tyr 1234/1235) compared to EVs, all EV isolates carried some MET and pMET . Thus, a precision medicine approach is necessary so as to not waste precious time of a patient’s life by administering an anti-cancer treatment from which that individual could not gain benefit; but would still endure its side-effects. Thus, the typical way of identifying those patients who would be candidates for such targeted anti-cancer treatment is to evaluate MET expression/over-expression and activation in tumour tissue samples. However, many tumours, e.g. those in the lungs, are not easily accessible in order to evaluate the solid tissue (e.g. by immunohistochemistry) for this purpose. Thus, if an entity released by the tumour cells into blood reflects the MET status of the tumour, this would have potential as a minimally-invasive means of predicting those individuals whose tumours are likely versus unlikely to respond to MET-targeted treatment.Indeed, EVs in blood are showing promise as carriers of minimally-invasive biomarkers in cancer. However, for each given clinical indication and drug/drug family the relevance of EVs as biomarkers must be established. Thus, here we must establishing if EVs could inform on the MET/pMET status of their cancer cells of origin; which method(s) would be most suitable for isolating EVs for this purpose; and if EV-MET/EV-pMET could be reliably, robustly and reproducibly evaluated in a way that is translatable to the clinical [such as enzyme-linked immunosorbent assay (ELISA)], rather than only via fundamental laboratory procedures (such as immunoblotting).14. Subsequently, however, the Reproducibility Project-intending to replicate this work-reported that they were unable to reliably detect pMET in exosomes15. Thus, no conclusive assumptions can be made as to whether or not MET and pMET are carried by EVs, from such opposing reports .Very few studies have reported on either MET or pMET in relation to EVs. In one study of melanoma, EVs were reported to increase the metastatic behaviour of primary tumours in pre-clinical models, by “educating” bone marrow progenitor cellsWe believe this to be the first study extensively evaluating and comparing methodologies for EV separation and characterising from cells (including both lung cancer cells and gastric cancer cells) of known MET status, to establish the optimal method for EV separation and also to determine how the resulting EVs reflect the MET and pMET status of their cells of origin.16. Of course, there are pros and cons for every method of EV separation18. In efforts to remove contaminating non-vesicular particles, ultracentrifugation on density-gradient has been shown to increase stringency, compared to other methods, by separating particles of different densities19. More recent published studies have also demonstrated that reasonably pure EV preparations, from plasma, can be separated by ultracentrifugation on iodixanol (OptiPrep) density-gradient (UC-DG). This method is proposed to produce higher yields of EVs than previously described separation techniques, without lipoprotein and albumin contamination20. Increasing numbers of commercially-available EV separation kits claim a quick and precise EV separation method, that could be used in the clinical setting. However, published studies suggest that commercial kits such as ExoQuick and total exosome separation, provide a higher extraction efficiency of general isolates than traditional UC, but that EV samples separated by UC are purer21.MISEV2018 guidelines highlight the importance of standardisation of sample collection, EV separation, and EV characterisation methods, to obtain meaningful data. This indicates that a large percentage of the heterogeneous EV population was lost. This may bias the outcome of a study i.e. it may give us the impression that the population consists of mainly small vesicles, but that may be because the rest of the vesicles have been lost by this process. Of course, we feel it is important to reiterate that no method for EV is ever likely to successfully collect all EVs.In this study, we have shown by NTA and IFCM that the highest yield of EVs were separated from CM using UC-DG, indicating that, of the three methods compared, UC-DG is superior for most comprehensive recovery of EV. NTA also analysis suggested that particles separated by ExoQuick are the smallest, with more heterogenous EV populations and particle sizes measured in EV samples separated by UC-DG. As expected, particles isolated by LSC were the largest (based on mode particle size). It has been suggested that ExoQuick may be most suitable where the population consists of mainly small EVs. However, it must also be considered that the yield of EVs was so much lower with ExoQuick than with the other methods evaluated20. TEM indicated that the majority of EVs separated by UC-DG were 40–140 nm in size. As predicted, and in line with NTA findings, some of the largest EVs, up to 200 nm, were separated by LSC. ExoQuick isolated were typically very small (~ 10–30 nm), with extensive background material. Previously studies have reported that ExoQuick isolates particles may be linked to the aggregation and precipitation of other elements in any given sample, but that are not EVs per se. This claim was supported by data showing that the amount of residual proteins was significantly higher following ExoQuick compared to UC22. In keeping with this, in our study, ExoQuick isolates had the lowest EV/particle/object numbers per million donors cells  and by imaging flow cytometry (IFCM)); had the lowest RNA yields; and yet contained the highest protein levels, suggesting the presence of non-EV material.EV characterisation by transmission electron microscopy (TEM) also showed that the apparent size and heterogeneity is dependent on the EV separation technique applied to each cell line’s CM. TEM images also suggest that the UC-DG method produces the purest EVs with the lowest levels of background materialIFCM was used to analyse surface EV protein markers on intact, individual EVs. By IFCM, consistent with the NTA data, total EV yield was found to be higher with UC-DG compared to LSC, and significantly higher when compared to ExoQuick. This was the situation with all three cell lines. All EV surface markers studied  were detected in samples to varying concentrations. Overall, the IFCM data confirms the NTA data that EV populations separated from CM from any given cell line -but using different methods of EV separation-differ in yield, with UC-DG separating a significantly higher amount of EVs.21. As MISEV2018 guidelines indicate, we must continue to analyse a number of EV markers to establish if we have isolated EVs, as no one marker is likely to be common to all cells types heterogeneous EV populations, isolated using different methods. The detection of GRP94, albeit typically at relatively low levels, supports the finding by others that GRP94 is not necessarily a negative marker for all EVs. Its detection here with some cell lines’ isolates, particularly with ExoQuick isolates and LSC isolates—and so a lesser extent, UC-DG isolates—also indicates  that some of our EV preparation are purer than others.Immunoblotting was used to analyse EV protein markers on the lysed heterogenous pool of EVs (and cells). Immunoblotting confirmed the presence of EV markers  but, again, with differences depending on the cell line and the EV separation methods; in concert with studies on other cell lines/EVsIt is noteworthy that different EV characterisation methodologies may be considered to give inconsistent data. However, because different types of analyses evaluate different aspects of EVs, it is typical that they do not produce exactly the same results. If that was so, some methodologies would be redundant. Thus, the intention of using a broad range of techniques for EV analysis is for added value and to get as comprehensive understanding of the EV population(s) in hand, as possible. This is a relatively new but expanding field, with no single type of analysis accepted for characterising of all aspects of EVs. There may never be. So, for example, considering CD63 and CD9 analysis by flow cytometry vs immunoblotting: for immunoblotting, lysed pools of EVs are studied; for IFCM, intact single EVs are studied. So, one method is evaluating single intact EVs, the other investigates the heterogeneous pool of EVs in a lysed state. Thus, the reason for using both techniques at this stage in EV research is to generate as complete an understanding as possible about the EVs. Similarly, for TEM vs NTA, these two complementary methodologies will not generate exactly the same results, as they are investigating EVs in different ways. NTA was used with the aim of estimating quantities and sizes all particles in the samples, whereas TEM was used to visually evaluate EV structure, size and sample background. So, although there is not full accordance, we believe that in a study that compares methodologies it is important to be as comprehensive with our characterisation as possible. It highlights the limitations of individual techniques and the need to continue using a range of techniques for fundamental characterisation of EVs.T values were compared, and all data was highly reproducible between repeats . Furthermore, MET mRNA was detected with all EV samples analysed, as well as in their cells of origin.With the objective of assessing how well EVs from each separation method represent the MET transcript status of their cells of origin, RT-qPCR and ELISAs were performed. Different yields of RNA were separated from EV samples obtained via different methods of EV isolation. However, for optimal use of RT and qPCR primers, a constant amount of RNA had to be used. This caused a bias towards the ExoQuick method of EV isolation. Specifically, there was less RNA available from ExoQuick isolates, but the same amount of RNA had to be used for RT-qPCR as for UC-DG and LSC. Due to the lack of reliable control RNA for EVs, quantification could not be performed. However, we can conclude that C23. However, our stock of these cells was purchased directly from ATCC for this study, so we are confident of their pedigree. Crucially, however, what was observed in the cells was also observed in their corresponding EVs.For the most part, EVs appear to reflect MET status of their cells of origin. As mentioned above, RT-qPCR showed the expression of MET in all cell types and their EVs, following EV separation using each of the three methods. The Hs746T results were as predicted, whereas the H596 results were unexpected, as these cells have been suggested not to harbour METTotal MET and phosphorylated MET proteins were also detected in all cell lines and all EVs, regardless of the EV separation method. However, the amounts detected in EVs were substantially less than in the cells. Overall, the highest levels of total MET and pMET were apparent in ExoQuick isolates, although these isolates were not rich in EVs . It can be considered that, as TEM results indicate the ExoQuick preparations carry most impurities, it is possible that this larger amount of total protein and, more specifically, total MET and pMET is due to the presence membrane fragments; rather than only intact EVs.Altogether, our data suggests that UC-DG, compared to ExoQuick and LSC, is the most efficient method to recover the highest yield of EVs from CM to produce the purest EV samples. Due to lengthy, labour-intensive protocols, UC-based methods are arguably not most suitable for diagnostic use in a clinical setting. However, they provide a valuable platform for quality EV separation in research. Our data suggests a potential use of commercially-available kits in the clinical setting, if pure EVs are not required. Overall, our studies showed that although, of course there was less MET mRNA, MET and p-MET proteins carried in EVs than in their cells of origin, the EV cargo reflected that observed for their cells of origin.Cell lines of differing MET status were included. EBC1  have MET amplification; NCI-H596 [H596] (ATCC) have mutated MET (i.e. an exon 14 skipping mutation); and Hs746T (ATCC), portrays both MET and mutated MET amplification (exon 14 skipping mutation). As recommended by the Suppliers of these cells, EBC1 were cultured in RPMI-1640 medium; H596 were cultured in RPMI-1640 medium; and Hs746T were cultured in DMEM). 10% (v/v) EV-depleted FBS was required for all lines. All experiments were done a minimum of 3 times, starting with a new vial of cells each time.6 cells/T175 cm2 culture flask in 30 mL of medium. EBC1 cells were seeded at 3.5 × 106 cells/T175 cm2 culture flask in 30 mL medium. Twenty hours later, medium was replaced and conditioned for 48 h. CM  was centrifuged at 750g for 10 min (twice) and 2800g for 10 min (twice) to remove cell debris. Cells were counted to calculate EV numbers/releasing cells.H596 and Hs746T cells were seeded at the density of 5 × 10Tangential flow filter  was used to concentrate CM, exactly as per manufacturer’s recommendation. Each 150 mL of conditioned media (CM) was concentrated to 3 mL (i.e. concentrated by a factor of 50) for each cell line, prior to isolation using UC-DG and ExoQuick, respectively. Fresh TFF columns were used for each cell line to avoid cross-contamination. Each TFF column was re-useable up to 20 times, as per manufacturer’s recommendation.g for 18 h at 4 °C . Seventeen fractions (1 mL each) were then collected (top to bottom). To calculate each of the 17 fraction’s density, the refractive index (RI) of each was measured using a Pal-Ri refractometer (Atago). The refractometer was zeroed/blanked using 300 μL of distilled water. Then a 300 μL aliquot of each UC-DG fraction was placed on the refractometer and read. Based on the measured RI, the following equation was used to calculate the density, as density = 3.3411 × RI–3.4584. EV/particle concentration in each fraction were also measured by nanoparticle tracking analysis . Samples within density fractions of 1.12–1.2 g/mL , were pooled, washed with sterile particle-free PBS and centrifuged at 120,000g at 4 °C  in order to pellet EVs and to remove OptiPrep. EVs were re-suspended in PBS or cell lysis buffer (50% in each).EVs, including small EVs, are expected to be isolated by UC-DG. OptiPrep was diluted with PBS to create density-gradient fractions of 5–40%. Density-gradient fractions (bottom-up) were layered over the sample and centrifuged at 120,000ExoQuick-TC kit  was used as per manufacturer’s recommendation. Specifically, 5 mL of CM must be used for EV isolation using the kit. Therefore, 2 mL of basic culture medium  was added to CM concentrates that were obtained following TFF (as described above) in order to bring the total volume of CM to 5 mL. No other changes were made to the process recommended by the manufacturer of ExoQuick-TC.24. Following the centrifugation at 750g for 10 min (twice) and 2800g for 10 min (twice) to remove cell debris, LSC of CM was performed using a 70Ti fixed-angle rotor at 10,000g for 1.5 h at 4 °C. Supernatant was discarded and the EV pellet was re-suspended in PBS or cell lysis buffer (50% in each).Large EVs are expected to be isolated by low speed centrifugation (LSC). Here, these EVs were isolated from CM by adapting a previously published protocolParticle size distribution was determined by NTA configured with a 488 nm laser and a high sensitivity scientific CMOS camera. Samples were diluted (1:20–1:100) in PBS  and analysed under constant flow conditions (flow rate = 50). Data was analysed using NTA 3.1.54 software.25. Briefly, 4 μL of sample in filtered PBS were fixed  with 4% glutaraldehyde on carbon-coated grids . Grids were washed 3 times (5 min) at room temperature (RT) with purified water. These were then contrasted with 2% phosphotungstic acid , washed 3 times, and examined by JEOL 1011 TEM .Samples were prepared by adapting a previously published protocolg for 10 min. at 4 °C. Soluble protein was measured by BCA assay . Representative details of protein quantification in Supplementary Fig. Cell pellets were lysed in ice-cold lysis buffer  with added protease inhibitor cocktail  and phosphatase inhibitor cocktail  on ice for 25 min. These were then centrifuged at 10,000g for 10 min. at 4 °C. Soluble protein was measured by BCA assay . Representative details of protein quantification in Supplementary Fig. EVs obtained by UC and LSC were lysed as for cells. ExoQuick isolates were lysed by mixing equal volumes of ExoQuick elute and lysis buffer. All samples were incubated on ice for 25 min, centrifuged at 10,00026, except that 5% BSA/PBS containing 0.1% Tween 20 (PBS-T) was used. Primary antibodies from Abcam were anti-CD63 ; anti-CD9 ; anti-syntenin-1 ; anti-actinin-4 ; and anti-GRP94 . Secondaries from Cell Signalling were anti-mouse  or anti-rabbit .Cell and EV protein (30 μg)—isolated as described above—was used for immunoblotting as we previously described27. EVs were analysed using an Amnis ImageStreamX Mark II Flow Cytometer with the following antibodies, all from Biolegend: anti-CD63-FITC ; CD9-PE ; CD81-PE-Cy7 ; HLA-DR-BV421  and ADAM10-APC 27. EV-free IFCM buffer, unstained EVs, single-stained controls and FMO (fluorescence-minus-one) controls were run in parallel. Data analysis used IDEAS v6.2 . EVs were gated as SSC-low vs fluorescence, then as non-detectable brightfield (fluorescence vs Raw Max Pixel Brightfield channel)  characterisation of EVs was adapted from a previously published protocolRNA from cells and EVs was isolated using PureLink RNA mini kit  following the manufacturer’s recommendation. Concentrations of cellular and EV RNA was measured using NanoDrop spectrophotometer and RNA purity was estimated using 260/280 ratio. The cellular and EV RNA yields are shown in Fig. Cell and EV protein samples (30 μg)—isolated as described above—were analysed using ELISA  for total MET and pMET (pY1234/1235). ELISAs were performed according to their manufacturer’s instructions..Unpaired two-tailed Student’s t-test was performed for comparisons between two groups. One-way ANOVA was used to compare more than two groups. Statistical analyses were performed using Graph Pad Prism 6 software. p < 0.05 was considered statistically significantSupplementary Information.


As is reported, an increasing body of evidence sheds light on the critical and underappreciated role of antibiotic tolerance in the disease burden of bacterial infections. Considering this tense situation, new therapeutic strategies are urgently required for combating antibiotic tolerance. Herein, we provide an insightful illustration to distinguish between antibiotic resistance and tolerance, and highlight its clinical significance and complexities of drug-tolerant bacteria. Then, we discuss the close relationship between antibiotic tolerance and bacterial metabolism. As such, a bacterial metabolism-based approach was proposed to counter antibiotic tolerance. These exogenous metabolites including amino acids, tricarboxylic acid cycle (TCA cycle) metabolites, and nucleotides effectively activate bacterial metabolism and convert the tolerant cells to sensitive cells, and eventually restore antibiotic efficacy. A better understanding of molecular mechanisms of antibiotic tolerance particularly  Staphylococcus aureus, and the identification of streptomycin in 1943 enables the effective control of tuberculosis by Mycobacterium tuberculosis. As such, these previous endeavors paved the way for the coming of the golden era of antibiotics. However, just as the saying goes, “Where there is oppression, there is resistance”, bacteria have evolved versatile means to antagonize antibiotic killing, such as antibiotic resistance -encoded phosphoethanolamine transferase catalyzes the addition of the cationic moiety to the phosphate groups of lipopolysaccharides  for a tolerant strain is remarkably higher than that in a susceptible strain -dependent transport to allow sufficient cell penetration , isocitrate lyase mediates broad antibiotic tolerance by remodeling Mtb’s TCA cycle, including increased glyoxylate shunt activity and suppressed TCA cycle , and a Gram-positive bacterium (Mycobacterium smegmatis) to aminoglycosides via promoting the transmembrane proton gradient (ΔpH), which in turn enhances PMF and stimulates the uptake of aminoglycosides  caused by the bacillus Mtb is a lengthy and arduous process (approximately six mouth), which majorly due to the presence of a small population of tolerant bacteria . Meanwhiof drugs . Apart functions . It has response . CollectP. aeruginosa was co-cultured with carbon source metabolites in different central metabolic pathways, there were significant differences in the bactericidal activity of tobramycin, implying that the sensitivity of P. aeruginosa to tobramycin was related to the disorder of central carbon metabolism. Consistently, lower TCA cycle metabolites such as fumarate, succinate, α-ketoglutarate, and pyruvate significantly sensitized stationary-phase cells to tobramycin. Mechanistic investigations showed that fumarate activated cellular respiration and generated more PMF by stimulating the TCA cycle as a tobramycin potentiator, which was consistent with the prior findings that aminoglycosides’ sensitivity is associated with PMF and drug internalization levels (2), thereby displaying a lower TCA cycle and decreased cellular respiration, which eventually lead to decreased antibiotic efficacy. In 2018, Su et al. found that glutamate promoted the inactivation of drug-resistant bacteria by kanamycin, and the underlying mechanism revealed a previously unknown prevalent pathway termed P cycle  and succinate dehydrogenase . Given those results, it is probable that pyrimidines may potentially serve as novel antibiotic adjuvants by promoting purine biosynthesis and altering the metabolic status of bacteria.In cells, nucleotides synthesis is made up of purine and pyrimidine synthesis pathway. On the basis of 5-phosphoribosyl pyrophosphate (PRPP), the no acids . Meanwhino acids . In 2019no acids . ConsistM. tuberculosis by inhibiting the dormancy pathway , but fructose was the only one that increased the efficacy of gentamicin against tolerant S. aureus (in vitro, with only a few studies that have tested the strategy of using metabolites as antibiotic adjuvants in vivo animal models, whereas in vivo trials are essential for the application of new antibiotic strategies to actual clinical treatment. Notably, the dynamic changes of metabolites in the physiological environment of resistant bacterial infections cannot be obtained by in vitro experiments alone (in vivo infection dynamics and to verify the therapeutic effect of antibiotics (in vivo efficacy of metabolites as novel antibiotic adjuvants.However, the current researches on improving antibiotic efficacy based on the bacterial metabolic network also have some limitations. First, although the metabolic state of bacteria is a uniform characteristic defining antibiotic efficacy across in various physiologic states, the same metabolites did not increase the sensitivity of each pathogen, it is important to note that these strategies must be tuned on a pathogen-by-pathogen basis to be successful. For example, the sensitivity of aminoglycoside to . aureus , suggestts alone . It is iibiotics . These sDespite the gaps in our search for new approaches to reverse antibiotic tolerance by metabolite-based approaches, these findings discussed in this review provide a variety of strategies and ideas for exploring novel therapeutic regimens for combating antibiotic tolerance, and in future studies, the complex metabolic networks of bacteria should be more fully utilized.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The thermal conductivity of concrete is a key factor for efficient energy consumption in concrete buildings because thermal conductivity plays a significant role in heat transfer through concrete walls. This study investigated the effects of replacing fine aggregates with coal bottom ash (CBA) and the influence of curing age on the thermal properties of high-strength concrete with a compressive strength exceeding 60 MPa. The different CBA aggregate contents included 25%, 50%, 75%, and 100%, and different curing ages included 28 and 56 days. For concrete containing CBA fine aggregate, the thermal and mechanical properties, including the unit weight, thermal conductivity, compressive strength, and ultrasonic velocity, were measured. The experimental results reveal that the unit weight and thermal conductivity of the CBA concrete were highly dependent on the CBA content. The unit weight, thermal conductivity, and compressive strength of the concrete decreased as the CBA content increased. Relationships between the thermal conductivity and the unit weight, thermal conductivity and compressive strength of the CBA concrete were proposed in the form of exponential functions. The equations proposed in this study provided predictions that were in good agreement with the test results. In addition, the test results show that there was an approximately linear relationship between the thermal conductivity and ultrasonic velocity of the CBA concrete. Currently, due to the increasing frequency of extremely hot weather conditions, efficient energy consumption is required in the construction field. In particular, there is an increasing demand for energy-efficient buildings, in which the internal temperature can be optimized ,2,3. OneRegarding concrete with low thermal conductivity, some experimental studies have been performed ,9,10,11.The demand for electricity is also an increasingly prevalent issue, and a thermal power plant is one of the methods to supply electricity. However, coal-fired thermal power plants create an enormous amount of bottom ash and fly ash ,15,16,17However, most previous studies investigated the mechanical properties of CBA concrete with a normal compressive strength of less than 40 MPa, which included CBA as an aggregate replacement. Moreover, few studies have assessed the thermal properties of high-strength CBA concrete. Therefore, to develop concrete with low thermal conductivity for energy efficiency, an experimental study must be performed to investigate the effects of the partial or total replacement of natural aggregates with CBA on the thermal properties of concrete.In this experimental study, the thermal and mechanical properties of high-strength CBA concrete with a target compressive strength exceeding 60 MPa were investigated. The CBA concrete included 0%, 25%, 50%, 75%, and 100% replacement of natural fine aggregates with CBA aggregates. The unit weight, thermal conductivity, compressive strength, and ultrasonic velocity of the produced concrete were measured. In addition, relationships between the thermal conductivity and the unit weight, compressive strength, and ultrasonic velocity for the CBA concrete were proposed.3, respectively, and the water absorption of the fine and coarse aggregates were 0.69 and 1.44, respectively.Both the fine and coarse aggregates used in this study were crushed materials. Due to the depletion of natural resources, crushed fine aggregates have been used favorably in Korea. The crushed fine aggregate is shown in 2, Al2O3, and Fe2O3, which had contents of 60.03%, 20.25%, and 9.80%, respectively, thereby comprising greater than 90% of the CBA.The CBA was collected from a thermal power plant company . The chemical components of the CBA used were determined through energy-dispersive spectroscopy , and the analysis results are shown in 3, respectively. On the other hand, the CBA water absorption was much higher than that of the crushed fine aggregate, which were 6.87% and 0.69%, respectively.The CBA aggregate was screened to remove particles greater than 5.0 mm and to retain particles greater than 0.15 mm. The CBA used in this study is shown in 3, which corresponded to 0.6% of the weight of the OPC, was used.Ordinary Portland cement (OPC) used in this study was type I in accordance with KS L 5201 . The spe3, respectively. A water-cement ratio of 0.3 was applied in all of the concrete mixtures.The mixing proportions of the control concrete and CBA concrete are provided in All of the concrete specimens were fabricated in a laboratory mixer. Cylindrical specimens with dimensions of 100 mm × 200 mm were fabricated to determine the unit weight, thermal conductivity, and compressive strength of the different mixtures. After casting the concrete, the concrete specimens were covered with plastic wrap and moist-cured for one day. Thereafter, the specimens were demolded at an age of 24 ± 1 h and then cured under submersed conditions at 23 ± 2 °C in a water tank until the ages of 28 and 56 days after the casting of concrete.The unit weight, compressive strength, thermal conductivity, and ultrasonic velocity of the CBA concrete were measured at curing ages of 28 and 56 days. Both end surfaces of the cylinders for compressive strength tests were ground before implementing each experiment.The unit weight (bulk density) of hardened CBA concrete was measured by using the cylindrical specimens after curing for 28 and 56 days, respectively. The unit weight was determined by dividing the mass of the cylindrical specimen by the volume of the specimen.The compressive strength of the cylindrical specimens was tested with a universal testing machine (UTM). Loading was applied under displacement control using a UTM with a capacity of 2000 kN. The mean values of three specimens were recorded to obtain the material properties of the concrete.There are several testing methods and their related devices for the measurement of the thermal conductivity of concrete. First, the thermal conductivity test can be carried out in accordance with ASTM D 5334-05 . The ASTThe transient plane source (TPS) method has been explained in detail by Gustafsson  and Log In this study, the measurement of the thermal conductivity was based on the TPS method. The thermal conductivity of the CBA specimens was measured using a TPS1500 testing device supplied from Hot Disk Ltd.  as shown in The sensor contained a nickel double spiral that applied a heating pulse to the specimen. The concrete specimen was controlled to satisfy thermal equilibrium before the measurements. After a thermal equilibrium time of at least 90 min under laboratory temperature conditions, measurements were made with an applied heating power of 0.3 W. Three measurements were taken for each specimen to ensure accurate test results.To investigate the workability of the fresh concrete, a slump test was performed. The slump test results are shown in 3, whereas the unit weight of the mixture containing 100% CBA fine aggregate decreased to as low as 2190.2 kg/m3. Specifically, the unit weights of the CBA concrete mixtures CBA25, CBA50, CBA75, and CBA100 were 2.1%, 3.2%, 5.3%, and 7.6% less than that of the control mixture, respectively. This decrease in the unit weight of the CBA concrete mixtures occurred because the unit weight of CBA fine aggregate was lower than that of crushed fine aggregate, as shown in The unit weights of the concrete specimens with different CBA replacement ratios are shown in 3, whereas the unit weight of mixture CBA100 decreased to as low as 2225.7 kg/m3. Specifically, the unit weights of the CBA concrete mixtures CBA25, CBA50, CBA75, and CBA100 were 2.0%, 3.7%, 5.8%, and 6.7% less than that of the control mixture, respectively.At a curing age of 56 days, the unit weight of the control mixture was 2386.5 kg/mThe thermal conductivities of the concrete specimens with different CBA contents are presented in A decrease in the thermal conductivity of the CBA concrete would increase the thermal insulation provided by the concrete and reduce the heating and cooling costs for buildings constructed from these materials. Therefore, the test result of the thermal conductivities for CBA concrete in this study implies that CBA could be utilized to fabricate high-strength concrete with low thermal conductivity for efficient energy consumption.The effect of curing age on the thermal conductivity of the CBA concrete specimens is also shown in The relationship between the thermal conductivity and the unit weight of the CBA concrete with two different curing ages is shown in ρ is the unit weight of the CBA concrete (kg/m3).Asadi et al.  proposedACI committee 213 R-03  proposedWongkeo  and ZhanThe equation from Asadi et al.  overestiThe test results of the compressive strength of the concrete specimens with different CBA contents are shown in 2 is transformed into CSH. These phenomena are why the concrete compressive strength increased with the increase in curing age.The relationship between the compressive strength and the unit weight of the CBA concrete under two different curing ages is presented in 3).The relationship between the compressive strength and the unit weight of the CBA concrete, based on the test results in this study and in previous studies ,38, is s3, whereas it overestimated the compressive strength values when the unit weight ranged from approximately 2300 to 2400 kg/m3.This equation underestimated the compressive strength values when the unit weight ranged from approximately 1700 to 1900 kg/mIn addition, the relationship between the thermal conductivity and the compressive strength is shown in Moreover, the relationship between the thermal conductivity and the compressive strength, which is based on the test results in this study and in previous studies ,38, is sOverall, the thermal conductivity test results in the figure had some deviations with various CBA concrete compressive strength values. Therefore, the equation overestimated the thermal conductivities when the measurements of compressive strength ranged from approximately 30 to 50 MPa, whereas it underestimated the thermal conductivities when the measurements of compressive strength ranged from approximately 60 to 80 MPa.An ultrasonic pulse velocity test was carried out to assess the material characterization of the CBA concrete. The measured quantity of this experiment was the travel time of the ultrasonic pulse between the transducers that were held on each surface of a concrete specimen, as shown in According to the ASTM C597-09 , the freThe relationship between the thermal conductivity and the ultrasonic velocity is shown in The unit weight of CBA concrete decreased as the replacement of CBA as fine aggregate increased. This decrease in the unit weight of the CBA concrete mixtures occurred because the CBA had a lower unit weight and a higher porosity than the crushed fine aggregate.The thermal conductivity of the CBA concrete was highly dependent on the CBA content. In addition, overall, the thermal conductivity of the CBA concrete increased as the curing age increased. When the curing age increased from 28 to 56 days, the thermal conductivity of the concrete increased by 3.1~6.5%.The relationship between the thermal conductivity and the unit weight of the CBA concrete was modeled with an exponential function. The results indicate that the equation proposed in this study provides predictions that are in good agreement with the test results.The compressive strength of the CBA concrete decreased as the CBA content in the concrete increased. In addition, an equation relating the thermal conductivity of the CBA concrete to the compressive strength was proposed. The equation overestimates the thermal conductivity of moderate-strength concrete (30~50 MPa), whereas it underestimates the thermal conductivity of high-strength concrete (60~80 MPa) because the test results used as a basis for the equation have some deviations.The ultrasonic velocity of the CBA concrete decreased as the amount of CBA fine aggregate in the concrete increased. Moreover, the test results show that there was an approximately linear relationship between the thermal conductivity and ultrasonic velocity of the CBA concrete.In this research, an experimental study was performed to investigate the thermal conductivity of CBA concrete with a compressive strength exceeding 60 MPa. The research findings are summarized as follows: