Plastic electronic waste (E-waste) is constantly growing around the world owing to the rapid increase in industrialization, urbanization, and population. The current annual production rate of E-waste is 3–4% in the world and is expected to increase to 55 million tons per year by 2025. To reduce the detrimental impact on the environment and save natural resources, one of the best solutions is to incorporate waste plastic in the construction industry to produce green concrete. This study examines the use of manufactured plastic coarse aggregate (PCA) obtained from E-waste as a partial replacement of natural coarse aggregate (NCA) in concrete. Six types of concrete mix with 10%, 20%, 30%, 40%, and 50% substitution of NCA (by volume) with PCA are prepared and tested. This study investigates the effect of manufactured PCA on the fresh and hardened characteristics of concrete. The properties of recycled plastic aggregate concrete (RPAC) studied include workability, fresh density, dry density, compressive strength (CS), splitting tensile strength (STS), flexural strength (FS), sorptivity coefficient, abrasion resistance, ultrasonic pulse velocity (UPV), and alternate wetting and drying (W–D). The results indicate that the CS, STS, and FS of RPAC declined in the range of 9.9–52.7%, 7.8–47.5%, and 11–39.4%, respectively, for substitution ratios of 10–50%. However, the results also indicate that the incorporation of PCA (10–50%) improved the workability and durability characteristics of concrete. A significant decrement in the sorptivity coefficient, abrasion loss, and UPV value was observed with an increasing amount of PCA. Furthermore, RPAC containing different percentages of PCA revealed better results against alternate W–D cycles with respect to ordinary concrete. Concrete is the most broadly used construction material in the building industry . It is aNowadays, the use of modern electronic appliances turns out to be an important part of our daily life. Due to the technological innovation, upgrade and advancement of technological products, the rate of obsolescence of electronic equipment has also increased, thus making E-waste an emerging waste in the world. Compared to other waste products, the degradation process of E-waste is comparatively more challenging, thereby significantly damaging the environment. It was reported that in 2013, the production of plastic products reached around 299 million tons . The EnvWhile investigating the strength properties of E-waste plastic concrete, Needhidasan et al.  studied Several researchers investigated the effect of different additives in E-waste incorporated concrete. Prasanna et al.  investigAlongside the strength characteristics, durability is an important aspect of structural concrete. To examine the durability characteristics of plastic concrete, durability tests were also carried out ,36, whicA brief assessment of the current literature indicates that the several types of research carried out on plastic concrete have focused on aspects of non-manufactured E-waste aggregates such as cleaning, sorting, and grinding or shredding the E-waste. The literature review suggests that a comprehensive study associated with the behavior of recycled plastic aggregate concrete (RPAC) containing manufactured plastic coarse aggregate (PCA) is missing. This study focused on utilizing the manufactured PCA through the proper heating procedure  of shapeOrdinary tap water is used in concrete mixes for hydration purposes, having a pH value range between 6.5 and 7, as per ASTM C1602/C1602M-12 standard . OrdinarThe E-waste utilized in this research work belongs to the family of acrylonitrile butadiene styrene (ABS) plastic, which consists of polycarbonate and acrylonitrile butadiene styrene. In previous works, ABS plastic was studied and its composition and other features were documented . The plaM25-grade concrete having a mix ratio of 1:2.14:3.08 (cement: fine aggregate: coarse aggregate) was prepared in this work . The maxThe experimental program of the present study, along with the number of specimens tested and standards used, is listed in 3 were prepared and tested after a 28-day curing period to determine their bend strength.The mechanical characteristics of concrete containing plastic aggregate were determined based on CS, STS, and FS tests. To determine the CS and STS, cylindrical samples of standard dimension (150 mm in diameter × 300 mm in height) after a 28-day curing period were tested as per ASTM C39/C39M-12 and ASTM C496/C496M-17 ,45, resp1/2 is the coefficient of sorptivity, and W1 denotes the dry weight of specimen (in grams), W2 represents the specimen’s weight after 4 hours ingression of water through the capillary rise, The durability properties of RPAC were examined based on the abrasion resistance, alternate wetting and drying (W–D), sorptivity coefficient, and UPV test. The sorptivity coefficient was determined as per ASTM C1585-04  and the The abrasion resistance of RPAC was measured as per ASTM C131/C131-20 standard . CylindrTo examine the influence of alternate wetting and drying (W–D) on the CS of plastic concrete, cubic specimens of 100 mm in size were prepared. Samples were subjected to 25 and 50 alternate W–D cycles after a curing period of 28 days. The duration of one complete cycle was 48 h (two days), in which the specimens were kept in water for the first 24 h, and then for the next 24 h, the samples were taken out from the water and then exposed to the air in the lab to dry ,49. The To determine the consistency, quality, and uniformity of concrete specimens, the UPV test was carried out . CylindrWorkability measures the ability of freshly mixed concrete to flow without segregation. It is a vital property of concrete that ensures proper handling and controls concrete’s strength and durability. The workability of RPAC was examined via slump test and 3, 2495 kg/m3, 2429.7 kg/m3, 2347.7 kg/m3, and 2272 kg/m3, respectively, at 10%, 20%, 30%, 40%, and 50% substitution level. With respect to the control mix, the maximum and minimum reduction in fresh density was 13.6% and 1.10%, respectively, at 50% and 10% replacement of NCA with PCA. In the literature, there is agreement that the density of concrete containing PCA decreases because of the low specific gravity of plastic aggregates with respect to mineral aggregates. A similar pattern of decrease in fresh density is well reflected in previously published works [The density of concrete depends upon the unit weight of concrete’s ingredients and their mix proportions. ed works . In the Compressive strength (CS) is an essential property of concrete that measures its ability to carry loads and is considered as one of the essential parameters in reinforced concrete design . The CS The flexural strength (FS) test, also known as transverse rupture strength, measures the ability of concrete to counter bending loads and was carried out as per ASTM C78/C78M-18  standardAbrasion resistance measures the ability of the concrete surface to counter wearing forces. Concrete abrasion allows the concrete to degrade over time, reducing its toughness by rendering it vulnerable to weathering. The poor resistance of concrete to abrasion makes it more vulnerable to deterioration, which is a common phenomenon. When a concrete structure is subjected to floating ice, a rapid movement of hard and pointed objects, friction, grinding behavior, or other factors, the surface of concrete slowly deteriorates thereby influencing the durability. This test was conducted as per the ASTM C-131  standardAlternative wetting and drying (W–D) is a durability test conducted to determine the ability of concrete to counter weathering when exposed to W–D conditions such as sea tidal waves. As a result of the alternative W–D of concrete, the stresses are induced in the concrete structure, which cause crack formation. Due to this phenomenon, the reinforcement is subjected to environmental factors such as moisture, air, etc., therefore leading to a decline in the durability characteristics of a structure. In this research work, the 28 days cured concrete specimens of concrete specimens of 100 mm in size were subjected to 25 and 50 alternate W–D cycles. The duration of one complete cycle (two days) means exposing the specimens to wetness for one day and then allowing them to dry for the next day.1/2) for all the concrete mixes containing plastic aggregates for 28 days of curing has a lesser value than the control mix. This reduction in sorptivity values with reference to the control mix is owing to the zero water absorption capacity PCA [Sorptivity measures the capacity of porous media to absorb water through capillary action. It provides a good measure of the durability of concrete as there are many chemicals that can penetrate the microstructure of concrete from soils and water by capillary action. The sorptivity coefficient is evaluated by determining the rise in the specimen’s weight as a result of the absorption of water through capillary rise with regard to time. The quantity of water absorbed by the samples comprising various proportions of PCA  through capillary rise per unit area is shown in city PCA ,32. WithThe UPV test was performed to analyze the uniformity, consistency, and quality of the concrete. It also evaluates the imperfections and compactness, i.e., cracks and voids inside a concrete sample. The UPV value has a direct relationship with concrete density, i.e., the denser the concrete, the higher will be its UPV value, and vice versa. The concrete specimen is considered to be of good quality if its UPV value lies in the range of 3.66–4.57 km/s  . Figure The incorporation of PCA in concrete significantly increased the workability (16.4–141% for 10% to 50% replacement ratios) due to the zero-water absorption of plastic aggregate. Moreover, the incorporation of PCA in concrete decreased the fresh and dry density of concrete composites for a maximum of 13.6% and 18.2%, respectively, reported at the 50% replacement level of NCA with PCA.The CS, STS and FS of RPAC were considerably reduced by increasing the percentage of PCA. The maximum percentage reduction in CS, STS and FS was achieved as 52.7%, 47.5% and 39.4%, respectively, for 50% replacement.The appreciable reduction (41.7%) in sorptivity value was observed for the maximum replacement of PCA considered in the study. The UPV values also remained within the required range of quality concrete after incorporating the full replacement of PCA. This suggests enhanced durability against the penetration of chemicals into the concrete.The incorporation of PCA in concrete improved the concrete abrasion resistance. Abrasion resistance increased in the range of 32–51% for different substitution ratios. This can be ascribed to the higher abrasion resistance and toughness of plastic aggregate with respect to natural aggregate. Moreover, loss in CS was also reduced after exposure to alternate wetting and drying cycles with respect to the control mix.The manufactured E-waste plastic aggregate can be used to substitute NCA in concrete by volume (10–50%); however, 30% substitution is recommended because up to this level, the characteristic strength of M25 concrete was obtained. Furthermore, the substitution level of 40% and 50% is recommended to be utilized in nonstructural lightweight elements. Additionally, the use of plastic aggregate is recommended to be used in marine conditions owing to its non-absorption capacity, which resists the ingress of hazardous chemicals such as chloride and sulphate, etc. The conventional steel and fiber-reinforced polymer (FRP) rebars in PCA-incorporated concrete are expected to perform better under an alkaline environment; however, more insights regarding the durability of FRPs in PCA-incorporated concrete shall be investigated first from the relevant literature ,60. In aThis study examined the effect of PCA on the fresh and hardened properties of concrete, including workability, fresh and dry density, CS, STS and FS. Furthermore, durability properties of RPAC such as abrasion resistance, alternate wetting and drying, sorptivity coefficient, and UPV were also explored. In the current study, PCA was used to replace NCA at replacement levels of 10%, 20%, 30%, 40% and 50%. Based on the findings, the following conclusions can be drawn:


MET can be observed in cases of focal gene copy number gain, such as MET-driven amp, or with a gain of chromosome 7, such as aneuploidy. Several studies have shown that only high-level focal MET amp (MET/CEP7 ratio ≥5) is oncogenic, with such tumors responding to targeted therapy. However, there are few reports on how to distinguish between focal amplification and aneuploidy using next-generation sequencing (NGS). A total of 1025 patients with advanced solid tumors  were tested with a non-invasive comprehensive cfDNA NGS panel (Guardant360) from July 2014 to June 2019. Since bioinformatics upgrades of Guardant360 were undergoing in September 2018, focal MET amp was determined by our independent algorithm using the cohorts tested before September 2018 (291 patients), and validation was performed in the remaining cohort (734 patients). MET alterations  associated with aberrant signaling were found in 110 patients (10.7%) among nine different cancer types, most commonly in non-small cell  and small cell  lung cancers, gastroesophageal cancer , and prostate adenocarcinoma . Among 291 patients tested before September 2018, 37 (12.7%) had MET alts. Among these, 24 (64.9%) had amps, 5 (13.5%) had exon 14 skipping, and 13 (35.1%) had single nucleotide variants (SNVs). Co-alterations, such as amp + SNVs, were found in four samples (10.8%). Among 24 MET amps, 29.2% (7/24) were focal according to our algorithm. MET copy number was significantly higher with focal amp compared to non-focal amp . In 734 patients tested after September 2018, our definition of focal MET amp was detected in 4.2% (31/734). Overall, focal amplification based on our algorithm was 3.7% (=38/1025). This study describes an approach to distinguish focal and non-focal MET amplification using comprehensive genomic profiling of cfDNA in advanced cancer patients. Focal MET amp accounted for ~30% of all MET amp, which was found in 3.7% of patients with diverse cancers and was associated with a higher plasma copy number. Clinical studies are warranted to assess the clinical utility of targeted therapies for tumors with focal MET amplification detected by NGS of cfDNA.Amplification (amp) of  MET is a proto-oncogene that encodes MET, and activating MET mutations have been reported in diverse carcinomas [MET, also known as c-MET, is a receptor tyrosine kinase that regulates many physiological processes, including proliferation, scattering, morphogenesis, and survival. The binding of its ligand, hepatocyte growth factor (HGF), to the MET receptor leads to the activation of several downstream signaling pathways, including RAS-ERK/MAPK, PI3K-AKT, or PLCgamma-PKC . MET is rcinomas . MET alterations , such as MET amplification (amp), exon 14 skipping, and single nucleotide variants (SNVs), can be oncogenic drivers. Furthermore, activating MET alts may also be acquired as a mechanism of resistance to tyrosine kinase inhibitors. MET exon 14 skipping has been described as a driver mutation in non-small cell lung cancer (NSCLC) [MET exon 14 skipping [MET amp has also been described as oncogenic, and studies using fluorescence in situ hybridization (FISH) have found MET amp in up to ~5% of patients with NSCLC or gastric adenocarcinoma [MET amp did not respond to therapy with a c-MET inhibitor [ (NSCLC) , and appnhibitor . MET amp can occur from focal gene copy number gain of the MET gene alone , co-amplification with adjacent genes in chromosome 7q such as CDK6 and BRAF , or gain of chromosome 7q copy number . Several studies have shown that only high-level focal MET amp (MET/CEP7 ratio ≥5), but not non-focal or lower levels of focal MET amp, are oncogenic and respond to targeted therapy [MET amp have been reported in the absence of other sensitizing MET alts, such as exon 14 skipping, particularly among those with higher MET to chromosome 7 ratios. Therefore, it is critical to distinguish between focal amplification and non-focal amplification. A typical companion diagnostic test interrogates only the target gene but may not include adjacent genes; therefore, in such cases, the distinction between MET focal amplification and non-focal amplification is not feasible. There are few reports on how to distinguish between focal amplification and aneuploidy using next-generation sequencing (NGS). therapy . MET inhMET alterations associated with aberrant MET signaling in 1025 patients with advanced solid tumors by using a comprehensive cfDNA NGS panel. We examined the pattern of focal and non-focal MET amps and established a way to investigate the prevalence of this potentially targetable alteration on NGS panels.NGS of cell-free plasma DNA (cfDNA) from patients with advanced cancers is a validated, non-invasive technique that can be used for comprehensive genomic profiling (CGP) of tumor-derived DNA. It can detect gene mutations with a relatively good correlation with tissue sequencing. In this study, we analyzed We reviewed the results of CGP from plasma of 1025 mostly pre-treated patients with advanced solid tumors. CGP of cfDNA was performed as a routine clinical practice between July 2014 and June 2019 from patients seen at institutes in Asia, Middle East, and Africa. Results from patients participating in prospective clinical trials were excluded. De-identified results were shared by Guardant Health Japan, Corp. Ethics review committee approval was granted by the institutional review board at Tokyo Medical and Dental University (G2020-021). ®, a comprehensive cfDNA NGS panel performed at Guardant Health, Inc. , a Clinical Laboratory Improvement Amendment (CLIA)-certified and College of American Pathologists (CAP)-accredited clinical laboratory. This assay interrogates single nucleotide substitutions, indels, amplification, and gene fusions in selected genes. During the study period, several versions of the assay were employed. In each version, MET amplification, MET single nucleotide substitutions, MET exon 14 skipping, and amplifications of BRAF, CDK6, and EGFR were included. Prior to September 2018, Guardant360 reported MET amplification regardless of focality; after bioinformatics upgrades, only focal MET amplification was reported. Therefore, for purposes of the present analysis, unfiltered Guardant360 amplification results for MET, BRAF, CDK6, and EGFR were provided for all samples. Copy number variants were scored based on the baseline copy number pooled in previous CGP data at Guardant Health, Inc. The result of amplification was reported as observed copy number. All 1025 patients were tested with Guardant360EGFR in 7p11.2, CDK6 in 7q21.2, MET in 7q31.2, and BRAF in 7q34. . . .In this study, additional analysis to distinguish focal and non-focal amplification was performed at an independent academic institution  using an original algorithm not used in the standard Guardant360 workflow. We divided the whole (1025 patients) into two cohorts; the cohorts tested before September 2018 (291 patients) was used to define focal amplification, and validation was performed in the remaining cohort (734 patients). Using the former cohort, we determined the algorithm to describe focal amplification as follows.(a)MET copy number ≥2.2.(b)MET gene is amplified without co-amplification of CDK6 and BRAF. Co-amplification status was defined as “increased together” when the copy number of other gene (CDK6 or BRAF) ≥2.2, and the difference with MET amplification is within +/−0.5.(c)MET amplification that satisfies both (a) and (b) is defined as focal.p-value). Statistical analysis was performed using EZR software (version 1.5.4 or later) and JMP software (version 14.2). For the validation cohort, positive percent agreement, negative percent agreement, positive predictive value, and negative predictive value were assessed using the updated Guardant360 bioinformatics pipeline results as the gold standard.Patient characteristics were summarized using descriptive statistics. Medians and respective 95% confidence intervals and range were calculated whenever possible. T-test or Mann–Whitney U test was used for numerical data, and Fisher’s exact test or chi-square test was used for categorical data. We set the accepted level of significance at 0.05 (n = 510), non-colorectal and non-gastroesophageal gastrointestinal cancers (“other GI cancers”) , breast cancer , and colorectal cancer . There was no statistically significant imbalance of diagnosis between the MET alteration and MET non-alteration groups . The cohort included 482 men (47.0%) and 543 women (53.0%). The most common cancer types were NSCLC , exon 14 skipping, and non-synonymous single nucleotide variants (SNVs). In total, MET alts were detected by Guardant360 in 110 of 1025 patients (10.7%), which was similar to a previous report [s report . MET alts were commonly found in SCLC 33.3% (3/9), gastroesophageal cancer 19.4% (7/36), prostate cancer 15.6% (5/32), and NSCLC 12.2% (62/510) ((62/510) . MET alts were found more frequently in men than in women  (MET mutations tended to be more common in prostate cancer (15.6%) than in breast (8.7%) and gynecological cancer (6.5%), although the number of cases of each was small. When analyzed by cancer type, there were significantly more MET alts in males than in females with non-colorectal and other GI cancers (p = 0.00478). However, no gender differences were observed for the frequency of MET alterations in other cancer types. 0.00234) . This maMET alts, according to Guardant360. Among these, 24 (64.9%) had amps, 5 (13.5%) had exon 14 skipping, and 13 (35.1%) had SNVs. Co-alterations, such as amplification + SNVs, were found in four samples (10.8%)  had  (10.8%) .MET amp, we found several patterns of gene amplification on chromosome 7  were focal, and 17 (70.8%) were non-focal  had co-amplification of MET and EGFR. In non-focal MET amplification, 82.4% of cases had co-amplification of MET, CDK6, and BRAF, suggesting aneuploidy. Other cases had co-amplifications of MET with either CDK6 or BRAF.Among the 24 samples with mosome 7 . Focal aon-focal . In the MET amplifications. MET copy number was significantly higher with focal amp compared to non-focal amp . . EGFR copy number was not different between the focal MET amp and non-focal MET amp groups . This illustrated that a higher copy number in MET and low copy numbers in CDK6 and BRAF, but not EGFR, were associated with focal MET amplification. We examined the features of focal and non-focal .00304).  In the cMET amplification. Among 10 patients with MET amplification, four had focal amplification, and six had non-focal amplification. The proportion of focal amplification in total MET amplification in the NSCLC cohort was 40%. The proportions of MET focal amplifications in gastrointestinal, breast, and other cancers were 25%, 50%, and 0%, respectively. Focal amplification was found in 7 out of 291 samples (2.4%), and the proportion of focal amplification among MET amp was 7 out of 24 samples (29.2%). However, the number of patients who had MET focal amplification was too small to elucidate any meaningful statistical conclusion. Further investigation is warranted in a larger patient cohort. Next, we investigated the proportions of focal vs. non-focal amplifications among different cancer types . In the EGFR driver mutations between focal and non-focal MET amps in NSCLC patients. Among 17 NSCLC patients with MET alts, 10 had MET amp. Among them, four had focal MET amp, and six had non-focal. Co-occurrence of EGFR driver mutations was found in five MET amp patients, two with a focal amp. EGFR T790M was also present in both patients with focal MET amp and one patient with a non-focal amp. This is suggestive of acquired resistance to third-generation EGFR TKI [Next, we examined whether there were differences in the coexistence of MET amplification in 30 patients (4.1%). The positive percent agreement , negative percent agreement , positive predictive value , and negative predictive value  were 83.3% (25/30), 98.7% (471/477), 80.6% (25/31), and 98.9% (471/476), respectively. Using our algorithm in the complete study cohort , focal amplification of MET was found in 38 patients (3.7%).Lastly, we compared our focal amplification definition to the amplification determined by a CLIA  certified laboratory at Guardant Health, Inc using an independent cohort.  of patients with diverse cancers and accounted for only ~30% of all MET amps. This distinction has clinical importance. Focal MET amplification is likely a driver alteration and, therefore, a therapeutic target. Non-focal amplification or aneuploidy is unlikely to be a driver alteration and, therefore, not an ideal therapeutic target.We describe a method for determining focal MET amp has been defined using the ratio of MET to CEP7 by FISH, and MET amps were found in up to ~5% of patients with NSCLC or gastric cancer [MET amp was as follows: “the MET: CEP7 signal ratio was ≥2.0 or when this ratio was <2.0, but there were >20 copies of MET signals in more than 10% of the tumor nuclei counted,” MET amp was detected in 2.5% of patients with advanced solid tumors in different cancer types. However, there was no significant difference in sensitivity to MET inhibitors between the MET amp group and the not amplified group [MET amp was proposed as a threshold, and a correlation with the therapeutic effect was seen in patients whose MET/CEP was 5 or more [MET-only amplification, without aneuploidy of chromosome 7, to enhance the therapeutic effect of MET inhibitors. Our study showed that a higher MET copy number in plasma was more likely to be due to focal amplification. In previous studies using tumor tissue,  or more ,7,8. ThiMET but also other adjacent genes on the same chromosome when designing companion diagnostic tests for MET amplification. Currently, a typical biomarker for amplification investigates only the target gene and does not include adjacent genes. However, this study illustrated that focal MET amplification was observed in only approximately 30% of “MET amplification,” and the rest were likely aneuploidy of chromosome 7. This distinction is critical for selecting a patient population in which MET amp drives cancer and may therefore be a target population for treatment with MET inhibitors. The idea of considering adjacent genes to identify focal gene amplification might be critical not only for MET but also for FGFR1 and FGFR2, for which there are emerging targeted therapeutics. Furthermore, this result indicates that it is necessary to interrogate not only BRAF, CDK6, and EGFR) in this study and defined aneuploidy as having CDK6 and/or BRAF amplified to the same degree. We defined the copy number of CDK6 or BRAF “increasing together” as within ±0.5 of the MET copy number, but the condition of less than 0.5 may not have been necessary because even if the copy number of CDK6 and BRAF is much higher (>0.5) than that of MET, it is still considered aneuploidy when all of them are high. For example, if our algorithm had excluded this rule, the NPA, PPV, and NPV would have been improved to 99.4% (from 98.7%), 89.3% (from 80.6%), and 99.0% (from 98.9%), respectively . Another point is whether or not to include EGFR in the definition. Unlike MET, CDK6, and BRAF genes on 7q, EGFR on 7p often behave independently, with apparently different values of copy number. Therefore, we removed EGFR from the definition of aneuploidy in this study. Further research is needed to validate this definition and its correlation with the clinical utility of MET targeting agents. Some of the MET tyrosine kinase inhibitors have been suggested or expected to have effects on MET amplification. For example, tepotinib and capmatinib, which are both approved by the FDA for NSCLC with MET exon14 skipping, have shown positive results for MET amplification. Improved anti activity of tepotinib plus gefitinib in patients with EGFR-mutant NSCLC and MET amplification was suggested [MET amplification was observed [EGFR mutation-positive lung cancers with MET amplification [EGFR mutations and MET mutations and amplifications [However, the algorithm needs to be optimized. We found several patterns of co-amplification of three genes on chromosome 7  using FISH and MET/CEP7 ratio in EGFR mutated NSCLC patients [MET high group , MET amplification  was observed only in 11.5%, and associated with suboptimal response to EGFR TKI, suggesting that MET amplification was another driver alteration, but it exists in limited population in MET high group. Roper and colleagues studied special and temporal heterogeneity of MET amplification in EGFR-positive NSCLC patients who received osimertinib [MET amplification and polysomy were observed. Therefore, it is important to carefully interpret MET copy number gain result from the tissue-based assay. It may be ideal to use liquid biopsy to capture heterogeneity in the clinical setting, rather than multiple biopsied used in this study. Our study showed the feasibility of capturing MET focal amplification using liquid biopsy.Recently, Lai and colleagues described patients . Among Mmertinib . In postMET alt dataset, we found NSCLC cases with EGFR driver and T790M with focal MET amp, suggesting resistance to EGFR TKI. It has been reported that NSCLC with secondary resistance to EGFR inhibitors tends to show MET amp [MET focal and non-focal amplification using FISH assays, the current golden standard to measure MET amplification. However, our de-identified data did not come with FISH results. Further study is warranted to validate the current proposed MET focal amplification definition by FISH assays in future studies.A major limitation of this study is the small sample size and the lack of complete clinical history, including treatment and response data. For example, in the  MET amp ,18. HoweMET amplification using unfiltered data from comprehensive genomic profiling of cfDNA in advanced cancer patients. Focal MET amp accounted for ~30% of all MET amps, was found in 3.7% of patients with diverse cancers, and was associated with a higher plasma copy number. Clinical studies are warranted to assess the clinical utility of targeted therapies for tumors with focal MET amplification detected by NGS of cfDNA.This study describes an approach to distinguish focal and non-focal


The high rate of infections among patients admitted to critical care units (CCUs) is associated with high rate of antibiotic consumption, especially broad-spectrum antibiotics. This study is to describe the antibiotics use in CCUs in primary and secondary hospitals in Vietnam, a setting with high burden of antibiotic resistance.This was a 7-day observational study in 51 CCUs in hospitals from 5 provinces in Vietnam from March to July 2019. Patients aged ≥ 18 years admitted to the participating CCUs was enrolled consecutively. We collected data on patient's demographics, initial diagnosis and antibiotic therapy within the first 24 hours. Antibiotic therapy was classified by the Anatomical Therapeutic Chemical (ATC) Index and the 2019 WHO Access, Watch, Reserve (AWaRe) groups.Out of 1747 enrolled patients, empirical antibiotic treatments were initiated in 1112 (63.6%) patients. The most frequently prescribed antibiotics were cefotaxime (22.3%), levofloxacin (19%) and ceftazidime (10.8%). Antibiotics were given in 31.5% of patients without diagnosis of infection. Watch and/or Reserve group antibiotic were given in 87.3% of patients and associated with patient's age  and the presence of SIRS on admission .We observed a high frequency use and a substantial variation in patterns of empirical antibiotic use in the CCUs in Vietnam. It highlights the importance of continuous monitoring antibiotic consumption in CCUs. We searched in the Pubmed between August 1, 2016 and August 1, 2021 with the terms “AWaRe classification” or “AWaRe”, “antibiotic” or “antimicrobials” and “Vietnam”. Only two studies evaluated the antibiotic purchases in community and in hospitals using the WHO Access, Watch, Reserve (AWaRe) classification. No study of has been performed specifically to evaluate the empiric antibiotic treatments in patients admitted to the critical care units (CCUs) in Vietnam up to the time of our study.This study is the first to explore the empirical antibiotic treatments in critical care units in Vietnam where the prevalence of antimicrobial resistance is high. Furthermore, we suggest that the AWaRe classification is a simple indicator for antibiotic use in the CCUs and could be used to monitor or evaluate empirical antibiotic use for different clinical diagnosis and syndrome.Our study showed that it is feasible to monitor the patterns of antibiotic use in CCUs using the AWaRe classification. Compliance with guidelines on antibiotic therapy should be further evaluated in stewardship programmes.Alt-text: Unlabelled box,,Severe acute respiratory infection (SARI) and sepsis are leading causes of mortality worldwide with more than 336 million episodes of SARI in 2106 and more than 19 million people with sepsis annually.,With a high burden of infectious diseases, critical care units had the highest consumption of antibiotics with an estimate of 71% of patients receiving any antibiotic.,WHO first released a global report on the consumption of antibiotics from 65 countries and areas in 2016 and the most updated report was release on 2018.E. coli increased from 64% to 71%, the resistance to carbapenem in K. pneumonia increased from 23% to 24% and the proportion of methicillin-resistant Staphylococcus aureus (MRSA) increased from 46% to 73%.Vietnam has one of the highest potential of antibiotic drug resistance in Asia.Viet Nam is composed of 63 provinces, including five centrally governed cities . Administrative divisions of Viet Nam is consisted with province, district, and commune and each level has health care facilities according to their capacity , in addition to national hospital in the central cities. This is the layer of reference system, e.g. a district hospital refer to a provincial hospital, and the national hospital. In this manuscript, ‘primary and secondary CCUs’ refers to CCUs in district or provincial hospitals.We conducted a cross-sectional study in 2 centrally governed cities (Hanoi and Can Tho) and 3 provinces  in 5 ecological regions in Vietnam from March to July 2019. In each province, we invited all CCUs in primary and secondary hospitals to participate in a 7-day prospective, observational cohort study in patients presented to critical care units. All patients admitted at the CCUs in selected hospitals for 7 days from the study initiation were included in this study. We collected the information of demographics, diagnosis, antibiotic prescriptions and data derived severity scores within 24 hours of admission and the outcomes at 7 days after the admission. Data was extracted from the medical charts. Doctors were not informed about the contents of the analysis and they managed their patients as they would normally.The initial diagnosis on admission to the CCUs were defined by the International Statistical Classification of Diseases and Related Health Problems 10th Revision (ICD-10)- WHO Version 2019. The diagnosis was made by the treating doctors in both ICD code and free text descriptions as a routine practise. All diagnosis were further cleaned and verified by two study doctors (VQD and TTD) by comparison of the consistency between ICD-10 coding and free-text data of diagnosis and severity across the patients and study sites. Discrepancies between the code and free texts were resolved by consensus by two study doctors. Patients were further defined as having severe acute respiratory infections (SARI) if they had a registered ICD-10 diagnosis code of J00-J06 (acute upper respiratory infections), J09-J18 (influenza and pneumonia) and J20-J22 (other acute respiratory infections)We used the 2019 WHO AWaRe classification of antibiotics to describe the empirical antibiotic therapy in patients presenting to the CCUs.Systemic inflammatory response syndrome (SIRS) was defined in patients with at least two of the following criteria within 48 hours of admission to CCUs: body temperature >38.0°C or <36.0°C, tachycardia >90 beats/minute, tachypnoea >20 breaths/minute, leucocytosis >1,200/mm3, <4,000/mm3 or bandemia ≥10%.The quick Sequential related Organ Failure Assessment (qSOFA) score was used to assess the severity of organ dysfunction in all patients. It consists of three components, assigning one point for each: respiratory rate ≥22 breaths per min, systolic blood pressure ≤100 mmHg, and Glasgow Coma Score (GCS) <15.Data were entered in Epidata  and analysed using IBM SPSS Statistics for Windows, Version 27.0. Armonk, NY: IBM Corp. Standard descriptive statistics were calculated for categorical (in percentage) and continuous variables . Differences between CCUs in the primary and secondary hospitals were analysed with Pearson chi-square test or Fisher's exact test for categorical variables when appropriated.Logistic regression was used to identify variables that predict abilities of choosing Watch and Reserve group antibiotics. A previous study in Vietnam have shown that doctors tended to choose a broader spectum for empirical treatment when the patients had more severe illness, older age and medical comorbidities.Eligible patients and/or their relatives were verbally informed about the study. The institutional review board (IRB) in the Hanoi Medical University approved the study (59/GCN-DDNCYSH-DHYHN). The IRB approved a waiver of consent based on the minimal risk to the participants.The authors did not receive any funds for conducting this study. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.We enrolled consecutively a total of 1747 adult patients admitted to 51 CCUs, including 36/51 (70.6%) CCUs in primary and 15/51 (29.4%) CCUs secondary hospitals in 5 provinces in Vietnam for a study period of 7 days in each study CCUs from March 2019 to July 2019. The demographics of patients was showed in Overall, 63.7% (1112/1747) patients received at least one antibiotic within 24 hours of admission. The overall rate of receiving antibiotics were 63.6% (1112/1747) . The rate of antibiotic use in patients with SIRS and any diagnosis of infections were 76.0% (835/1098), 91.9% (828/901) respectively. At the day 7 after CCU admission, 34.0% patient remained hospitalised while 60.2% patients were discharged to home. The overall 7th day mortality rate was 4.7% (82/1747) among patients admitted to CCUs for all causes and was 3.7% (29/794) among patients with SARI. Among patients receiving at least one antibiotic, the proportion of patients receiving any microbiological culture  was 12.9% (144/1112) . The rate of pathogen identification in primary and secondary hospitals were 16.1% (5/31) and 42/121 (34.7%), respectively. No susceptibility results were further collected.rd generation cephalosporins, fluoroquinolones and penicillins with beta lactamase inhibitors (There were 31.5% (267/846) patients without diagnosis of infection in the medical notes received any antibiotic, while 6.8% (62/901) patients with diagnosis of any bacterial infection received no antibiotic within 48 hours of admission. There was a total of 72 different antibiotics used over all CCUs and  represenhibitors . Table 3The proportion of any Access group, Watch group, Reserve group and non-recommended antibiotics were 24.1% (268/1112), 87.3% (971/1112), 0.54% (6/1112) and 5.0% (56/1112), respectively . All 6 pAmong the 1112 patients who received any antibiotics, 647 (37.0%) were treated with mono therapy, 444 (25.4%) received dual therapy and 21 (1.2%) received triple therapy . The ratIndependent factors associated with using Watch and/or Reserve groups were patient's age  and SIRS  .Table 5LTo the best of our knowledge, our study represents the first effort to describe the initially empirical antibiotic therapy in CCUs in Vietnam, a country with a high burden of antibiotic drug resistant.Our study was completed 5 months before the COVID-19 that was reported in Vietnam in January 2020In Vietnamese guideline on antibiotic use in 2015, antibiotics recommended for moderate pneumonia were amoxicillin plus clarithromycin or benzylpenicillin plus clarithromycin or β-Lactam  or ampicillin-sulbactam plus macrolide or fluroquinolone. For severe pneumonia, it was recommended to startIn a previous study, the percentages of antibiotic purchased in Access, Watch and Reverse groups in health care facilities in Viet Nam, were 47.2%, 52.4% and 0.1% respectively.Our study had some limitations. Firstly, we did not collect data to distinguish between antibiotic treatments and perioperative antibiotic prophylaxis. However, we considered the proportion of prescription for preoperative antibiotic prophylaxis was small because of the low rate of surgery within 24 hours of admission in our study participants (27/1747 or 1.5%). Secondly, because of the short-period observational study design, there may be some bias in evaluation of causes of admission. Additionally, the majority of initial diagnosis of infection were clinically made, partly related to the lack of rapid diagnostics whilst the empirical antibiotic prescribing decisions were influenced by doctors’ experiences and by level of hospitals. It makes the interpretation of empirical antibiotic choice difficult and must be related to the current burden of antibiotic resistant pathogens in community and in a particular CCU. Thirdly, due to the limitations of study design, data collection and low frequency of microbiological culture, we were unable to evaluate the necessity and appropriateness of antibiotic treatment in our study patients. Further studies are required to evaluate the compliance with antimicrobial treatment guidelines for empirical antibiotic selection and rational antibiotic use in relation to diagnosis and microbiological findings in Vietnam.In conclusion, there was an over prescription of broad spectrum antibiotic and high frequency of antibiotic combination for all causes of admission to CCUs in primary and secondary hospitals in Vietnam. It is crucial to implement the surveillance of antibiotic use in CCUs and establish a protocol the empirical antibiotic treatment to improve overall SARI patients’ outcome.VQD, SO supervised the project implementation, including designing the study and analysing the data. VQD, TTD reviewed and classified diagnosis on admission. SO, VQH, KBG and VQD collected data and monitor the study. VQD, TTD has consolidated and draughted the first report. All authors involved to the acquisition and interpretation of data. All authors contributed to and approved the final report.We declare no competing interest.


Peripheral blood mononuclear cells were used for comprehensive flow cytometric analysis.Clinical and pathological findings were compared in patients with EGPA and IgG4/IgG ratio in 61.5% patients. However, actual IgG4 levels, as well as the degree of the infiltration of IgG4-positive plasma cells, were still higher in patients with IgG4-RD than patients with EGPA. Examination by ACR/EULAR classification criteria showed only 13.6% of the EGPA patients met entry criteria, while all of them met the exclusion criteria. In regard to the immunophenotyping, EGPA patients had increases in activated CD4 and CD8 T cells compared with the healthy controls. However, no such similar changes occurred in IgG4-RD patients. On the other hand, both the EGPA and IgG4-RD patient groups had correlated increased plasmablasts and Tfh. These results indicate the presence of two axes: namely, the activation of T cells and that of B cells. Both axes are present in EGPA, but the T cell activation axis was not observed in IgG4-RD.An elevation of the IgG4 level was found in all EGPA cases, with the accompanying pathological findings of lymphocytic infiltration and fibrosis observed in 30.8% patients, and the elevation of IgG4 as well as pathological IgG4 infiltration are not specific. Meanwhile, EGPA and IgG4-RD differ in immunological phenotypes, indicating the possible importance of the predominant activation of T cells in the development of vasculitis.The elevation of serum IgG EGPA and microscopic polyangiitis (MPAs) show a common vasculitis that affects the small blood vessels, and EGPA and IgG4-related disease (RD) have common features including a history of allergic disease, elevated serum IgE, and eosinophilia. In addition, the increase of serum IgG4 in EGPA patients and its subsequent decrease on treatment have already been made known.Elevation of IgG4 in both serum and tissues are not highly specific to patients with IgG4-RD. Both activated CD4 T cells and activated CD8 T cells  and plasmablasts and follicular helper T cells  are elevated in EGPA patients, while only the B cell activation axis is seen in patients with IgG4-RD. Specifically, the T cell activation axis is not seen in patients with IgG4-RD.T cell activation is important for the development of the pathology of vasculitis, and the lack of a T cell activation axis would explain the clinical differences between EGPA and IgG4-RD. Additionally, these results increase the basic scientific knowledge concerning the clinical efficacy of B cell targeted therapy against IgG4-RD.et al.Eosinophilic granulomatosis with polyangiitis (EGPA) is one of the antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) diseases that presents, along with eosinophilia, with an allergic predisposition, with eosinophil infiltration in tissues, and with clinical presentations of vasculitis such as purpura and peripheral neuropathy.There are several clinical similarities between EGPA and IgG4-RD. For example, a history of allergies appears in 30% and 50% of the patients with EGPA and IgG4-RD respectively.This was conducted as a multicentre study. We enrolled in this study, patients who were both untreated and newly diagnosed with EGPA, IgG4-RD, between March 2013 and March 2018 from four facilities . In addition, newly diagnosed microscopic polyangiitis (MPA) patients were also enrolled as a control vasculitis group for the immunophenotyping portion of this study. Patient diagnoses were made by at least three doctors who are specialists in the field of Rheumatology. As a result, all subjects fulfilled either the classification criteria or diagnostic criteria.Comprehensive diagnostic criteria for IgG4-RD10.1136/rmdopen-2021-002086.supp2Supplementary dataThe laboratory tests included serum IgG, IgG4 and ANCA in addition to a general comprehensive laboratory test. Whole-body CT scan was perfomed in each of the patients to investigate specific organ involvement.4-positive plasma cell infiltration was counted, and ration of IgG4/IgG were calculated. The pathological assessment was done by two certified pathologists.All biopsy or resected samples were embedded in paraffin and stained with H&E and Masson-Trichrome (MT) in both diseases. Lymphoplasmacytic infiltration and eosinophilic infiltration were evaluated by H&E staining. Typical fibrosis of IgG4RD including storiform fibrosis and bird’s eye pattern fibrosis (kidney) was assessed by MT staining. Antibodies against IgG and IgG4 were used for immunohistochemical staining. IgGPeripheral blood immunophenotyping was performed by comprehensive eight-colour flow cytometric analysis, proposed by the National Institutes of Health/Federation of Clinical Immunology Societies as a Human Immunology Project, with some necessary modifications for detecting Tfh cells.Continuous data were expressed as the mean±SD, and categorical data expressed as the number (%). Baseline clinical characteristics and the proportion of immune cell subsets between groups were compared using the Mann-Whitney U test. The optimal cut-off value to distinguish two diseases was calculated using receiver operator characteristic (ROC) curve analysis. For easy exploration and visualisation of immunophenotyping data, we used principal component analysis (PCA) to statistically aggregate items, reducing the number of observed variables into a smaller number of principal components (PC) and reducing the dimensionality of the immunophenotyping data. PCA was performed as described before.Twenty-two EGPA patients and 20 IgG4-RD patients were enrolled in this study. The mean age was comparable, but the proportion of woman was dominant in the EGPA group . GlandulThe results of the laboratory tests are shown in +/IgG+ cells were higher in patients with IgG4-RD , eosinophil granulocyte counts , and C reactive protein (CRP)  showed best area under the ROC curve (AUC). Serological findings above these cut-off values thus are indicative of EGPA and likewise if below, indicate IgG4-RD. On the other hand, the cut-off value for IgG4 (1071 mg/dL) showed relatively narrow AUC (+/IgG+ cells > 40% and >10 IgG4 positive plasma cells/High-power field (HPF). If we calculated the cut-off value of the ratio of IgG4+/IgG+ cells and the number of IgG4 positive plasma cells/HPF for the differential diagnosis between EGPA and IgG4-RD, we found that a higher concentration of IgG4+ cells (ratio of IgG4+/IgG+ cells > 79% and >28 IgG4 positive plasma cells/HPF) were needed  the diagnosis of IgG4-related lung disease and IgG4-related kidney disease need to be more careful made since the lung and kidneys are favoured sites of AAV; (2) an increased level of serum IgG4 (>135 mg/dL) is not specific to IgG4-RD; however, higher IgG4 values (>1100 mg/dL) could increase the specificity towards the diagnosis; and (3) both diseases show eosinophilic infiltration and IgG4 positive plasma cells infiltration at sites of organ involvement. On the other hand, we also could clearly see the differences. The clinical and radiological features of mass-forming lesions are specific findings of IgG4-RD. A substantially increased level of white blood cell counts and serum CRP are not seen in IgG4-RD. In addition, morphological findings such as storiform fibrosis and obliterative phlebitis are specific pathologically.The results of immunological phenotyping further solidified this finding. Specifically, the Tfh-plasmablast axis is elevated in IgG4-RD to form its pathology, whereas in EGPA the T-cell activation axis, mainly that of Th1, was elevated independently along with the elevation of the Tfh-plasmablast axis. In MPA, a control disease, only T-cell activation was observed. This result, although it must be interpreted with caution, suggests that T-cell activation is important for the development of the pathology of vasculitis. The balance between the factors required for the activation of T cells and those required for the differentiation and activation of B cells could be responsible for the difference in IgG4 levels in IgG4-RD and EGPA, as well as the degree of local IgG4 infiltration.+ cytotoxic T lymphocytesThe limitation of this study is the relatively small number of cases analysed since we only enrolled untreated and newly diagnosed patients. In addition, the measurements of serum cytokines , which are important for testing the hypothesis mentioned above, were below the range of the limit of detection in several patients. Th2 and CD4Taken together, this study reaffirmed the importance of the clinical findings of mass formation in IgG4-RD and showed that the elevation of serum IgG4 as well as the pathological IgG4 infiltration are not specific only to IgG4-RD. Meanwhile, EGPA and IgG4-RD differed in immunological phenotypes, indicating a possible importance of the predominant activation of T cells for the development of vasculitis. The balance between the factors required for the activation of T cells and those required for the differentiation and activation of B cells could be responsible for the differences in IgG4-RD and EGPA.


Geraniol-poor Type C roses also showed higher cytokinins (in buds) and abscisic acid , and significantly shorter floral lifespan compared with geraniol-rich Type A and B roses. We conclude that geraniol enrichment, intensification of petal colour, and lower potential for H2O2-related oxidative damage characterise and likely contribute to longer floral lifespan in monoterpene-rich wild roses.Wild roses store and emit a large array of fragrant monoterpenes from their petals. Maximisation of fragrance coincides with floral maturation in many angiosperms, which enhances pollination efficiency, reduces floral predation, and improves plant fitness. We hypothesized that petal monoterpenes serve additional lifelong functions such as limiting metabolic damage from reactive oxygen species (ROS), and altering isoprenoid hormonal abundance to increase floral lifespan. Petal monoterpenes were quantified at three floral life-stages  in 57 rose species and 16 subspecies originating from Asia, America, and Europe, and relationships among monoterpene richness, petal colour, ROS, hormones, and floral lifespan were analysed within a phylogenetic context. Three distinct types of petal monoterpene profiles, revealing significant developmental and functional differences, were identified: Type A, species where monoterpene abundance peaked in open mature flowers depleting thereafter; Type B, where monoterpenes peaked in senescing flowers increasing from bud stage, and a rare Type C (8 species) where monoterpenes depleted from bud stage to senescence. Cyclic monoterpenes peaked during early floral development, whereas acyclic monoterpenes  peaked during floral maturation in Type A and B roses. Early-diverging roses were geraniol-poor (often Type C) and white-petalled. Lifetime changes in hydrogen peroxide (HThe online version of this article (10.1007/s00442-020-04710-z) contains supplementary material, which is available to authorized users. Floral fragrance in roses is largely made of volatile monoterpenes, isoprenoid alcohols, and their oxidised and glycosylated (non-volatile) derivatives. Monoterpenes are stored and emitted mostly from rose petal epidermal follicles , a non-radical reservoir of free radical ROS, was quantified with the expectation that H2O2 accumulation in plant tissues indicated senescence induction, while H2O2 depletion suggested delaying of senescence , Europe , and America (N = 8), and two with unknown origin. Sampled roses were diploid (N =27), tetraploid (N =14), and penta- and hexaploid (N = 3 each), and the remaining were of unknown ploidy. Most roses were single-whorled  and some were bi-whorled . Each rose species was represented by one healthy and fertile individual plant in the rose garden, implying that all species were autogamous or geitonogamous to different degrees. Each individual species acted as one biological replicate for comparative trait analyses (classified post factum). The empirical limitation of using just one individual per species meant that any of our results could not account for the potential diversity and plasticity in monoterpene profiles within species. However, by hosting a global collection of roses in a common garden, we were able to compare profiles among biological species under the same environmental conditions. Petal samples were harvested on mostly sunny days from 5 to 10 pseudoreplicate flowers per species  at three time-points in the floral life cycle: (1) unopened buds with visible petals; (2) fully open mature flowers; and (3) senescent, wilting petals, prior to abscission. New flowers were sampled at each stage. Collected petals were frozen in liquid nitrogen and stored at − 80 °C until further analyses.Petals from 73 wild roses  were harvested from plants maintained at the Gianfranco & Carla Fineschi Rose Garden in Cavriglia, Italy . These wild roses bloomed from April to June and were sourced from three geographic regions of origin, viz., Asia (−1 (purge flow at 50 mL min−1), and the column flow was 1.2 mL min−1. Volatile analytes were separated heating the column with the following sequence: 40 °C (1 min), increasing at 5 °C min−1 to 200 °C (6 min), and then to 260 °C at 10 °C min−1. Monoterpene standard mixtures were also prepared using high-purity components (Sigma-Aldrich). Standards were run separately and used for comparing ionisation patterns, retention times, and for preparing standard curves. Seventeen monoterpene species found in rose petals were quantified on petal fresh weight basis and classified into the following groups based on their chemical structure: (i) cyclic monoterpenes , (ii) acyclic monoterpene . These are also termed “geraniols” hereafter, and species were also classified on the basis of geraniols (G) content: high ; medium ; or low ; and (iii) other acyclic monoterpenes . Non-volatile monoterpene conjugates were not quantified.Monoterpenes were extracted by grinding 500 mg of frozen petals (pooled from > 3 flowers per stage) in 1 mL of heptane in and immediately transferred to an air-tight head-space sampler vial and incubated at 40 °C in a sonicator, stirred overnight. The extract was centrifuged at 4000 rpm for 5 min. 200 μL of the supernatant was collected. Volatiles were identified and quantified using gas chromatography–mass spectrometry . A small fraction of the extract was injected in splitless mode to the GC inlet at 260 °C fitted with an HP-INNOWax column (50 m × 200 μm × 0.4 μm). Ultrapure Helium  was supplied at the pressure of 33.6 Psi, at a total flow rate of 15 mL minR.sicula, R.nutkana, R.multibracteata, and R.acicularis. The sample size was 6–10 flowers per species for floral size, and 6–25 flowers per species for floral lifespan. Size was represented by floral circumference measured in situ as well as calculated from photographs (using ImageJ software v1.51 w). Floral lifespan, from unopened bud stage to petal abscission, was monitored during two flowering seasons. Individual flower buds per species were tagged at early unopened bud stage and floral lifespan was calculated as the time (in days) required for individual flowers to senesce and die .Species were categorised based on petal colour–pale pink (mixed white), pink (dark pink and purple), white, and yellow. Monoterpene profile types, geraniol abundance, and petal colour were linked to the phylogeny of 39 rose species, whose relatedness were discerned from published phylogenies of wild roses . Petal H2O2 was quantified by generating a standard curve with known amounts of pure H2O2. The quantification was done in a subset of 14 rose species representing Types A, B, and C monoterpene profiles based on tissue availability (see results). Each profile type included different petal colours and geraniol abundances (G10 to G1000). The 14 species were Rosa foliolosa, R. gallica, and R. nutkana , R. xanthina , R. arvensis , and R. bracteata, all Type A (N = 6); R. longicuspis , R. chinensis mutabilis and R. canina , and R. laevigata , all Type B, (N = 4); R. villosa , R. foetida , R. banksiae, and R. soulieana , all Type C (N = 4).Sixty mg of petals were ground in liquid nitrogen with 5% polyvinyl pyrrolidone (PVP), and the ground tissue was suspended in 2 mL of 1 N perchloric acid. The extract was cold centrifuged for 15 min. Five hundred μL of the supernatant were aliquoted and an equal volume of acidic phosphate buffer (pH 5) was added. Fifty μL were treated with ascorbate oxidase for 15 min at room temperature to remove ascorbates . A reaction mixture comprising 3-dimethylamino benzoic acid (DMAB), and 3-methyl-2-bonzothiazolinone hydrazine (MTAB), and phosphate buffer was placed in plate-microcuvettes. The neutralised extract and horse-radish peroxidase were added to the reaction mixture. Absorbance was monitored continuously at 590 nm, where an absorption peak is known for the deeply purple coloured product of the oxidative coupling between MTAB and DMAB in the presence of HAbscisic acid (ABA) was extracted from 100 mg fresh weight of petals per sample (tissues previously frozen in liquid nitrogen and stored at − 80 °C) in distilled water overnight at 4 °C (water: tissue ratio = 10:1 v/w). ABA was determined by an indirect enzyme-linked immunosorbent assay (ELISA) using DBPA1 monoclonal antibody, raised against S (+)-ABA , R. nutkana, and R. bracteata , all Type A; R. longicuspis  R. chinensis mutabilis , and R. laevigata , all Type B; R. villosa (pink G100), R. foetida , and R. banksiae , all Type C.Cytokinins (CKs) were extracted in acidified aqueous methanol, purified by two solid-phase extraction (SPE) steps and subsequently measured with LC–MS/MS . Outliers were identified and excluded from further analysis using Grubb’s test. Equality of variances among life-stages and among geographic groups was tested using Levene’s test (α = 0.05). Significance of differences among means was tested using a General Linear Model (multifactor nested ANOVA for repeated-measures design). Geographic region , monoterpene profile type (2 dof), life-stage (2 dof), and ploidy (4 dof) were the main factors. Differences in means among types, regions, and stages were analysed using Tukey’s test (α = 0.05) with conservative letter coding whenever possible. If variances were unequal, the Games–Howell test was applied. Significance of regressions (linear) among monoterpene abundance, extractable H2O2, and phytohormone content were tested within and among 3 geographic groups , 3 life-stages , and 3 monoterpene profile types . The consistency (trueness) of match between monoterpene profiles and their geographic group was tested by Discriminant Analysis, a technique where two or more groups, clusters, populations are known a priori, to which one or more new observations are matched. All statistical tests were conducted using Minitab statistics software v18 .The significance of normal distribution of total monoterpene content in petals was tested  using the Shapiro–Wilk (SW) test and they were found to be normally distributed   and specifically poorer of geraniol compared with Type A and B  Fig. a. Type Cd B Fig. a. Both gd B Fig. a, b. Thed B Fig. a. Levelsd B Fig. b. EU Typd B Fig. b. LinaloSpecies with white petals contained the least levels of monoterpenes, while yellow and pale-pink petals contained intermediate levels of monoterpenes and pink petals contained the highest levels of monoterpenes Fig. a. FlowerR. webbiana, R. rugosa) were closely related to pink, geraniol-rich (G1000) American Type A roses. All American roses were pink petalled and geraniol-rich. Type C condition was rare, typically associated with white-petalled and geraniol-poor (G10) roses  and R. foetida , which are among early-diverging roses . Early-diverging roses also included R. bracteata , and R. laevigata , which were all geraniol-poor  and Asian roses , compared with European roses (0.5). Assembled phylogenies placed American and European roses in separate monophyletic clusters and Asian roses formed different groups  and extractable Hges Fig. a. Unopenges Fig. b. ABA leges Fig. b. Buds hges Fig. c. NearlyLifetime petal monoterpene profiles in wild roses reveal three types of species Fig. , viz., tR. bracteata, R. laevigata, R. bella, R. rubus, and R. sempervirens) can be viewed as monoterpene-poor Type C species. Monoterpene abundance in senescent Type A roses decreases likely because of a hormonal intervention similar to that in Petunia flowers, where fertilised gynoecium releases ethylene to down-regulate petal volatile synthesis  can be a derived trait in many angiosperm families 2O2 pools, indicating lower potential for ROS-induced damage and better floral health.Abundance of acyclic monoterpenes (geraniols) is maximised at floral maturity in many wild roses and geraniol enrichment is associated with significantly smaller H(ii)Species that maximise monoterpene level in unopened buds (Type C) are rare, always geraniol-poor, small, white-petalled, and short-lived compared with common, typically geraniol-rich, pink petalled Type A and B roses. The geraniol-poor condition characterises most of the early-diverging and phylogenetically isolated species with short-lived flowers, supporting the notion that the last common ancestor of all modern species roses had monoterpene-poor, white-petalled, and short-lived flowers.(iii)Qualitative and quantitative petal monoterpene profiles can act as metabolic markers for floral longevity in rose breeding.In summary, our observations in wild roses show the following:Supplementary material 1 (DOCX 309 kb)Below is the link to the electronic supplementary material.


The potential to use waste glass, including cathode ray tube (CRT) glass, for making new products or as an admixture to existing ones is being intensively investigated. This kind of research intensified particularly in the period after CRT TV sets and computer monitors were replaced in the market by the advanced technology of thin film transistor (TFT) and liquid crystal display (LCD) screens. Cathode ray tube glass represents a considerable part of electronic waste (e-waste). E-waste globally increases at a far higher rate than other solid waste materials. There is a possibility to recycle cathode ray tube glass and use it in the construction industry. This paper shows the test results of physical and mechanical properties of blocks and paving flags. The reference specimen was made with quartz sand, while the other product employed a combination of quartz sand and ground panel cathode ray tube glass. The glass was ground to the fraction 0.25/1.00 mm, which corresponds to quartz sand fineness. The following tests were performed: shape and dimensions, resistance to freeze/thaw and de-icing salts, water absorption, splitting tensile strength and tensile strength by bending. Special attention was paid to the tests of Böhme wear resistance, slip resistance of the top surface of CRT products using a pendulum, radioactivity and leaching. The texture of the experimental concrete products was observed by SEM (scanning electron microscopy) and analyzed. The results obtained by experimental testing unequivocally show that CRT glass can successfully be used for making concrete blocks and paving flags. Considering the “service life”, i.e., the duration of the cathode ray tube screens, and the fact that they can still be found in households, it is clear that a considerable quantity of these devices is stored in landfills. Precise data on the amounts of generated waste CRT glass are not readily available. According to the data shared by the WEEE Forum, the amount of CRT displays generated in the Europe (30 countries) has been declining since 2009, and it is estimated that in 2020, this amount was around 250 kt [Computer and TV screens with cathode ray tubes (CRTs) have not been sold in Europe since 2011, when, through technological advancements, they were replaced by new TFT  and density is decreased. It is also concluded that few studies are based on concrete precast elements made with CRT glass. The subject of this paper is the production of concrete paving blocks and flags in which 50% of quartz in the finish–visible layer has been replaced by panel recycled cathode ray tube glass. The review of literature did not reveal any paper discussing this topic, which provides a certain originality to our paper. The main goal set for this research is proving the potential use of panel CRT recycled glass as a partial replacement for natural aggregate in the finish layer of concrete blocks and paving flags, which is a possible solution for disposal. We focus on the analysis of properties that relate the most to the finish layer of the paving elements. Within the experimental research, physical and mechanical properties as well as the durability of concrete are tested: shape and dimensions, splitting tensile strength, tensile strength by bending, water absorption, resistance to freeze/thaw and de-icing salts, wear resistance (Böhme test), slip resistance (SRT pendulum test), radioactivity and leaching. For the purpose of observing the concrete microstructure, SEM and EDS analyses were performed in order to acquire the knowledge of the concrete surface.For the production of concrete blocks, cement CRH Serbia CEM I 52.5R was used, which meets all the quality requirements of SRPS EN 197-1:2013. Physico-mechanical cement characteristics are presented in 3O4. The chemical composition of cathode ray tube glass is presented in The glass used in the experiment was panel CRT glass obtained by recycling old TV sets and other electronic devices. Large shards of glass a were grEDS spectroscopy of the ground CRT glass  determinTwo concrete mix designs were developed, and their compositions are shown in The production process of concrete blocks and flags is fully automated and computerized. Depending on the dimensions, the product is categorized as a block or flag ;Resistance to freeze/thaw and de-icing salts ;Water absorption ;Splitting tensile strength ;Tensile strength by bending ;Wear resistance—Böhme test ;Slip resistance—SRT pendulum ;Radioactivity testing;Leaching test; andSEM concrete analysis.The following tests were performed on the concrete blocks:2. The testing was performed in the Climatic Cabinet Controls 10-D1428/A with the time temperature cycle prescribed by the standard EN 1338, annex D. The mean values of the test results are provided in The testing principle of freeze/thaw resistance and de-icing salts is based on the procedure in which the prepared samples are submitted to 28 freeze/thaw cycles while the surface is covered with a 3% NaCl solution. The scaled off material was collected and weighed, and the result is expressed in kilograms per square meter. The number of tested samples was 6 of E concrete and 6 of WG concrete. The tested surface area of each sample was 22,500 mmTotal water absorption was tested on 10 samples of E concrete and WG concrete according to the standard EN 1338, annex E. After conditioning the specimen to (20 ± 5) °C, it was soaked to constant mass and then oven-dried to constant mass. The loss in mass is expressed as a percentage of the mass of the dry specimen. The mean values of the test results are provided in Splitting tensile strength tests were conducted on concrete blocks having 210 × 115 × 80 mm dimensions according to the EN 1338 standard, annex F. The upper and lower surfaces of the block were ground to smoothness. The tests were conducted on a digital hydraulic press UTEST UTC—5600 a. There Tensile strength by bending tests were conducted on concrete flags having 390 × 165 × 80 mm dimensions according to the EN 1339 standard, annex F. The flags were ground at their support points to achieve as satisfactory contact as possible. The load increments were such that failure was achieved after 45 s ± 15 s from the start of the load application b.3/mm2 is provided in Measuring of abrasion according to the Böhme test was performed on 6 cube-shaped samples with 71 mm sides on the Böhme Machine Controls 48-D5272. The samples were placed on the Böhme disc abrader, the test track on which standard abrasive is strewn, and the disc was rotated and the specimens were subjected to an abrasive load of (294 ± 3) N for 16 cycles. The mean loss in specimen volume ΔV after 16 cycles of testing in mmThe measurement of USRV on the specimen was made using the pendulum friction test equipment to evaluate the frictional properties of the specimen on the upper face. The pendulum friction test equipment  incorporates a spring-loaded slider made of a standard rubber attached to the end of the pendulum. Upon swinging the pendulum, the frictional force between the slider and test surface was measured by the reduction in length of the swing using a calibrated scale.−1). In building materials, the contents of the following radionuclide elements are determined: Ra—226, Th—232 (or Ra—228) and K—40. Based on the obtained values, the gamma index is calculated according to the equation:Ra is the concentration of radium (226Ra) in Bq/kg;CTh is the concentration of thorium (232Th) in Bq/kg; andCK is the concentration of potassium (40K) in Bq/kg.CIn order to confirm of the possibility of using concrete blocks and flags made with recycled glass, the radioactivity of the concrete in question was checked. The reference level for outdoor irradiation in an enclosed space originating from natural gamma emitters from building materials, excluding the outdoor irradiation in the open air, is E = 1 mSv a year, where E is the effective dose and the unit is Sievert , reference concrete (3 samples) and 3 samples of WG concrete. Samples of reference concrete and concrete with recycled glass (WG) were ground to pass the sieve with a mesh size of 1 mm without residue, and they were used as such in the tests. Gamma spectrometric analysis was performed using the HPGe gamma spectrometer Canberra DSA—2000 . An HPGeIn order to determine the impact of the addition of CRT glass on the environment, the leaching test was performed. This test examines the impact of potentially hazardous chemical compounds present in the finishing layer of concrete on the quality of ground waters, and determines if there is a potential risk for human health. The eluates for the reference concrete and WG concrete were obtained according to the standard EN 1744-3 . The cheThe SEM analysis was conducted for the purpose of assessing the texture of the surface of the finish layer of reference concrete and of concrete with a modified finish layer.The test results for the concrete paving blocks and flags are shown in 3 of concrete (basic + finishing layer) was 74 kg. The absence of a change in density is explained by the similar values of densities of quartz sand (2.65 g/cm3) and panel CRT glass (2.84 g/cm3), and of their low content in 1 m3 of concrete. After 28 cycles of simultaneous action of frost and defrosting salt, there was no damage to the concrete surface , neither in the reference series  of concentrated glass amounts to 1.076 (>1). According to the “Regulations on the limit values of radionuclide for drinking water, various food items, building materials and other commodities”, it is necessary to calculate the level of an effective dose, which is used to assess the application and conditions of use of materials in the construction industry. In the cases of the reference concrete and the concrete with recycled glass, the gamma index is uniform, amounting to 0.295 and 0.359, respectively. The measured values are considerably lower than the limits, so the presence of recycled panel cathode ray tube glass does not have an adverse effect on the environment and human health in terms of increased radioactivity emitted by CRT glass.3 WG concrete (basic + finishing layer), there is around 74 kg of panel CRT glass aggregate. For such a concrete mixture, the cathode ray tube glass has a mass share of around 3.4%. The obtained results of the chemical analysis of the eluate of WG concrete unequivocally indicate that it presents no danger to the environment and human health.The results of the leaching test are shown in The texture of the reference concrete is compact and homogeneous a. The teThe replacement of 50% of quartz sand with panel recycled glass in the finishing layer of concrete paving blocks and flags does not cause a change in bulk density, nor does it impair the durability of the product in terms of simultaneous action of frost and de-icing salt. Additionally, it does not increase water absorption or reduce tensile splitting strength and tensile strength by bending below the values prescribed by standards EN 1338 and EN 1339. Furthermore, the presence of recycled glass slightly reduced the slip resistance of concrete blocks, while the resistance to wear by Böhme grinding was significantly improved. It is our opinion that in future research, it would be interesting to perform a deeper SEM analysis which would include the interstitial transit zone (ITZ) between the aggregate grains and the cement matrix.The test of radioactivity has unequivocally shown that the use of recycled cathode glass in concrete does not increase the radiation dose above the limit that could adversely affect the environment and human health.All concentration values of potentially hazardous chemical compounds in eluates obtained by the leaching test are far less than 1 mg/kg, which is far below the limit values prescribed by regulations . The cheRecycled cathode glass can be successfully used for the production of concrete blocks and paving flags, representing part of the solution of the problem of existing landfills and a contribution to sustainable construction. The possible use of waste CRT glass presented in this paper is considerable, considering that the closed loop of recycling, which comprises the use of old CRT screens for the production of new ones, is not an option any longer.2. If for the production of the finish layer, panel CRT aggregate is used to replace 50% of quartz sand mass, which amounts to 10 kg/1 m2 (as in this paper), then the entire production would require 12,000 tons of glass. Even if it is acknowledged that not all concrete elements have two layers, it can be concluded that the entire collected amount of panel CRT glass can be consumed in this way. The cost of quartz sand in Serbia is around 5 euro cents per 1 kg. The costs of collecting old CRT devices, the separation of panel and funnel glass and crushing is 30 euro cents per 1 kg, which is around six times higher than the quartz sand cost. However, the cost of the reuse, recycling and utilization of CRT screens as a raw material amounts to 73 euro cents per 1 kg [Panel glass constitutes around two-thirds of one CRT screen, or around 10 kg . The surOne of the problems that must be solved in the future is forming legislative regulations and standards which would coordinate the classification of CRT glass and recycling and waste disposal solutions.


In the modern construction industry, lightweight aggregate concrete (LWAC) is often used to produce load-bearing structural members. LWAC can be up to 40% lighter by volume than normal strength concrete. However, the lack of adequate numerical models often limits the practical application of innovative building materials such as lightweight concrete in real projects. The present study conducted a comparative numerical deformation analysis of a full-scale bridge deck slab and girder. Using the physical model proposed by the authors and the finite element software ATENA, the deformations of full-scale lightweight and traditional reinforced concrete elements under the short-term effects of permanent and variable loads were compared. Depending on the safety and serviceability limit requirements, it was found that the amount of longitudinal reinforcement in lightweight reinforced concrete elements could be reduced compared with that in standard reinforced concrete elements with the same parameters. The results of the numerical analysis showed that the deformation analysis model proposed by the authors could serve as an alternative tool for the design of lightweight concrete flexural members with the selection of optimum geometric and reinforcement parameters limited by the stiffness condition. In the modern construction industry, lightweight aggregate concrete (LWAC), which can be up to 40% lighter than standard strength concrete by volume, is often used in the production of load-bearing structures in addition to standard strength concrete. For lightweight structural concrete, natural or artificial lightweight aggregates are used in addition to heavy aggregates  . ArtificIn addition to these advantages, lightweight concrete has better durability properties  than staA large number of analytical and numerical techniques for the serviceability analysis of traditional RC elements have been proposed and presented to the engineering community ,15,16. HThe authors recently proposed a constitutive model for assessing flexural stiffening of reinforced LWAC beams subjected to short-term loading . FurtherIt should be noted that the lack of adequate numerical models often limits the practical application of innovative building materials such as lightweight concrete in real projects . This isctσ = 0.55lctf, where lctf is the average tensile strength of LWAC calculated according to the Eurocode 2 (EC2) standard .The descending branch of the diagram is approximated by the following relationship:ε2 is calculated as follows:Strain ε3, corresponding to zero stress. This strain is calculated as follows:The length of the descending branch is defined by the maximal strain, Nonlinear numerical analysis was performed using the finite element software ATENA . In most cases, two-dimensional finite element models of RC elements were created by employing constitutive models of compressive and tensile concrete and the reinforcement. The behaviour of the reinforcement was represented by an elastic–plastic model corresponding to the yield strength of steel and the modulus of elasticity. A linear elastic diagram was used to model the compressive concrete. Justification is that the stress–strain relation in compression for LWAC adopted in EC2 is very close to linear. Plastic deformations occur only at high levels of compressive stresses. The difference in deflection estimates at service load when linear and nonlinear diagrams of compressive concrete were assumed are marginal. The proposed constitutive model  was used3, compressive strength of 31 MPa, and tensile strength of 2.29 MPa.A comparative numerical analysis was carried out for three full-scale experimental panels published by Vakhshouri  to evaluserM) does not exceed 2.5%. The analysis of the obtained results shows that the authors’ proposed model can be considered an alternative tool for analysing the real stress–strain state of lightweight RC.A comparison of the numerical and experimental bending moment–curvature diagrams is shown in 3, and the density of the lightweight concrete was 1800 kg/m3. The characteristic permanent load was calculated by considering the slab’s self-weight (dead load) and deck load .This section describes the comparative numerical deformation analysis of a full-scale bridge deck slab and girder under short-term loading. The behaviours of full-scale lightweight and standard RC structural elements under the short-term effects of permanent and variable loading were compared using the proposed physical model and the finite element program ATENA. Depending on the safety and serviceability limit requirements, it was determined that the amount of longitudinal reinforcement could be reduced in lightweight RC elements compared with standard RC elements with the same parameters. The subject of this study is a free-supported standard and lightweight concrete bridge deck slab and girder designed according to the EC2 standard . The slaR = 0.3%) and 5∅16 mm (ρR = 0.4%) diameter bars, respectively. The behaviour of the LWAC slabs with a reduced reinforcement intensity of 6∅12 mm (ρR = 0.27%) and 8∅12 mm (ρR = 0.36%) was also investigated to reveal the advantages of lightweight concrete and the reduced permanent load. The shrinkage deformations of concrete before loading were calculated as −1 × 10–4 and –1.14 × 10–4 for normal and lightweight concrete, respectively. The free shrinkage strain was assessed according to EC2 [st = 7 days—age of concrete at the end of curing, t0 = 28 days—concrete age at loading, RH = 75%—relative humidity, CEM 42.5 N—cement type. Numerical modelling was performed using the finite element program ATENA by applying the principles presented in C30/37 concrete was used for the normal RC elements, and LC30/33 lightweight concrete with the same compressive strength was used for the lightweight concrete. An S500 longitudinal reinforcement was used to reinforce both elements. Transverse reinforcements were not utilised. The normal and lightweight concrete slabs were designed to ensure mechanical resistance in flexure and shear for the designated initial parameters. According to the calculated safety limit state, normal RC slabs subjected to variable loads of 2.5 kPa and 5.0 kPa were reinforced with 5∅14 mm  and reinforced with S500 reinforcing bars. The girder was designed to withstand a design bending moment of ρ = 1800 kg/m3 was used for the lightweight concrete beams. The characteristic bending moment caused by permanent loading due to the lower self-weight of the girder decreased from gkM = 459 kNm to gkM = 389 kNm. The behaviour of the NWC and LWAC beams with the same reinforcement intensity was compared in the first stage for the lightweight and standard concrete elements. Taking into account the reduced permanent load, the behaviour of the LWAC beam in the middle zone  is slightly higher than that of the standard RC slab (Δqk = 2.2 mm). In general, the deflection values for both elements are small. Slightly different trends are obtained for slabs with a reinforcement percentage of 0.40%  to 3.1 mm, which is acceptable from a practical perspective. Thus, the amount of longitudinal reinforcement can be reduced by 12% by designing an LWAC bridge deck slab subjected to a variable pedestrian load of 2.5 kPa. A similar trend is observed for the slab subjected to a load of 5 kPa (gkM) and total load (EkM), the higher the efficiency of using LWAC in load-bearing structures will be, which leads to a more significant reduction in the amount of longitudinal reinforcement. The values of these ratios for the deck slabs are listed in The results of the analysis of the LWAC slabs with a reduced reinforcement percentage are shown in of 5 kPa b. The reρR = 2.33%), the numerical deformation analyses of elements having the same parameters using the LWAC and NWC physical models yield approximately the same calculation results. The slight differences in the curves for the LWAC and NWC girders are related to the different moduli of elasticity of these materials. The LWAC and NWC results are also in good agreement because the effect of tensile concrete between the cracks on the stiffness in highly reinforced elements is insignificant. Owing to this factor and the low ratio of permanent to total load  did not exceed 2.5%.The adequacy of the tension stiffening model for LWAC recently proposed by the authors was verified by comparing theoretical and experimental results for three full-scale lightweight concrete slabs. The comparative analysis showed that satisfactory prediction results were obtained at all loading stages preceding the yielding of reinforcement. The maximum discrepancy between the experimental and theoretical results at service load  compared with traditional RC elements with the same parameters.The effectiveness of the application of lightweight concrete increases with the ratio of the moments caused by the permanent and total loads. However, the reduced stiffness of the elements may require consideration of deflection limitations. These values are regulated by various design standards and depend on the purpose of the structure.Rρ > 2%), the effect of the tensile concrete between cracks on the stiffness is insignificant. In this case, the reduction in the concrete density does not have a significant structural effect.Lightweight concrete is less efficient than standard concrete, with a decrease in the ratio of permanent to total moments and an increase in the reinforcement percentage. In heavily reinforced elements (The results of the numerical analysis show that the deformation analysis model proposed by the authors can serve as an alternative tool for the design of lightweight concrete flexural members with a selection of the optimum geometric and reinforcement parameters, which are limited by the stiffness condition. The proposed model can be applied to the analysis of real lightweight concrete elements by implementing the performance-based design concept provided in modern design standards.