The strengths and mechanical characteristics of concrete play a key role in the safety levels for the recovery and reuse of existing RC buildings and civil engineering works. This is one of the main focuses of the current research trend. To this aim, the characteristics of concrete must be investigated: the characterization of the concrete and its in situ conditions play a key role. For these reasons, many studies on in situ and laboratory test methods and procedures have been carried out over the last two decades. In the past few years, non-destructive investigation methods have been considered reliable and used in many engineering applications, also for RC constructions. More recent codes and guidelines identify destructive test methods as a reference for practice application. However, non-destructive investigation methods can be used though exclusively in combination with destructive tests to support them. In this study, a significant database is considered to assess the reliability of the relationship between destructive and non-destructive methods for in situ concrete in existing RC constructions. The results of the analyses are used to verify the effectiveness of the methods and prediction models and suggest more effective test procedures. It can be stated that many of the existing empirical methods (based on pre-established correlations) are unable to provide a reliable evaluation of the compressive concrete strength and its variability. In practical applications, non-destructive methods often lead to unsatisfactory results for the existing reinforced concrete constructions. Finally, based on the results, some first operational indications are provided for practical investigations and future possible codes and guideline improvements. One of the main focuses of the current research trend is the recovery and reuse of existing RC buildings and civil engineering works. They are publicly and privately owned and are often severely degraded. Consequently, they are often underused and in many cases unused. The topic is particularly important in Europe, in which post-war reconstruction activities (after 1945) led to the construction of a very high number of RC buildings and civil engineering works. Currently, these buildings are very old and require assessment and retrofitting interventions.For example, in Italy, the RC construction heritage is among the oldest in Europe and requires substantial and extraordinary maintenance as well as functional, structural, and seismic retrofitting. The criticalities are amplified by the recent seismic classifications. As an example, in However, the seismic performance assessment of existing buildings and civil engineering works is a very complex and difficult task. In recent years, the research and professional activities and the effects of earthquakes  have higThe increasing need for the use and rehabilitation of existing RC buildings and civil engineering works requires improving the assessment and investigation procedures, particularly for the execution and processing of the test results of the materials. The assessment and seismic retrofitting of the existing RC buildings has been dealt with in specific codes in many countries since the nineties  from whiIn the current Italian code, the part dedicated to the existing constructions is reported in chapter 8 which contains the general principles and the main issues of existing constructions; even though significant options and choices are left up to the professional’s practice. More quantitative details are reported in the guidelines  which adThe latest Italian code  has intrThe purpose of this study is to identify the critical issues in current investigation procedures and possible improvements. Similar studies have been carried out on these topics in the past. However, no alternative and corrective methods useful for the applications were clearly identified.In particular, in this study, the operational issues of DTs and NDTs for concrete in existing RC construction, as well as the reliable interpretation of the results, are investigated.A contribution of the study is the database considered. The database was obtained from laboratory investigations and professional practice activities. Thanks to the database, the reliability of the relationship between destructive and non-destructive methods for in situ concrete inthe existing RC constructionsis considered and assessed.As reported in the following sections, the investigation procedures for existing RC construction based on NDTs and the commonly used NDT–DT correlations highlighted significant criticalities, particularly in their ability to investigate and estimate the characteristics of the concrete. Such methods, which are often developed in laboratories and validated for new concrete, are often not suitable for investigating the existing RC constructions. Based on the reported experimental campaigns, some possible improvements are consequently defined from the obtained results.To define the method followed in the study and before describing the materials, it is necessary to highlight the most recent and relevant literature, although a complete review is not among the objectives of the study. For the mechanical characterization of concrete , the codes and guidelines consider DTs and NDTs. For existing RC construction, NDTs ,11,19,20In practical applications, one of the greatest difficulties is to identify the homogeneous areas of the structure in terms of concrete properties, and typically, NDT methods are used for this purpose . HoweverRN) and ultrasonic velocity (V). The approachwas born in the 1970s and initially, its application was not based on direct correlation with destructive testing. In addition, with particular reference to the current application on existing structures, its use was significantly different. It was mainly dedicated to new concrete  is still open and every possible contribution is still important.The SonReb method is undoubtedly the most common approach. SonReb procedures arebased on rebound number . In theHowever, it must be emphasized that the most commonly used form in the last 30 years has been the exponential one where the relationship between the strength of the concrete and the NDTs is based on the form (1):a, b and c are derived for each study with different experiments .In this work, it was not considered necessary to report further details and indicate the various expressions and their comparisons. On the other hand, considering Italian data similar to what is contained herein, a SonReb reliability study  was receMore recently, some interesting applications have been made regarding a new emerging application area for artificial neural networks (ANNs) in civil engineering . Some ANIt must be noted about the validation of the SonReb methodthat many forms were defined on laboratory-prepared samples, many even using cubic specimens ,47,48,49To this aim, the time reference could be the European code  and therGlobally, the classic SonReb method seems inconsistent with the knowledge objectives of existing constructions. It is not consistent with the Italian code provision ,11. FurtFor the above reasons, the evaluation of the mechanical characteristics of concrete  must be based mainly  on DTs. NDTs must be considered only as a  support for the evaluation of the homogeneity of the characteristics.Above all, the NDTs must never be used individually but only and always in conjunction with the DTs.In fact, NDTs are not satisfactory methods for the estimation of the mechanical characteristics and, in particular, the strength of concrete.These application restrictions are expressly provided for in the Italian code ,11.Consequently, the NDTs should not be used a priori for the rough determination of the characteristics of the concrete and neither for the a priori identification of homogeneous areas.Using them in this way could be misleading.Nevertheless, DTs too present many uncertainties; they are linked to: (i) reliability and accuracy of the methods, (ii) characteristics and variability of the strength of the concrete in the structures, and (iii) execution of extraction tests. Conversely, some experimental programs on reinforced concrete elements showed the high within-member, within construction variability of in situ concrete strength ,38,47,49In the practice application, design of investigation campaigns and results analyses play a key role insubsequent assessments. They are affected bythe limited number of experimental observations and thus the obtained results (in terms of concrete characteristics) could not be statistically representative ofthe entire construction or its part. Consequently, many more studies (as this study) are needed for the validation of NDTs, and any new contribution could be useful.In the database, the samples are cylindrical samples extracted from existing RC structures by core drilling.Moreover, some considerations regarding the usefulness of in situ NDTs are reported. The characteristics of the in situ tests are compared with the provisions of the codes in force at the time of the design and construction of the structures. It is to be noted that without this reference, the interpretation of the results of the tests would be ill-conditioned. In order to not make the study too long, further analyses of the database will be conducted in subsequent studies. As an example, the variability in the individual structures due to position, type of element, number of samples extracted, size of the structure, etc. has not been analyzed in depth. Similarly, no issues relating to the construction methods, management and degradation of the structures were developed, which also have a significant influence on the results of the tests.The database is extremely interesting. It is derived from experimental and professional activities that were conducted or coordinated at the University of Basilicata and it considers activities carried out by other test centers and by professionals. This heterogeneous origin can be considered a valuable characteristic of the database which was created aftermany years of activities and research. The latest data were obtained in 2021. The database relates to hundreds of RC constructions (buildings and civil engineering works) located in the south-central Italian territory, over 7 regions. The RC buildings and civil engineering works were designed and used for public  and private  use and were built from the 1950s. The database wascollected during several years of experimental programs and during several programs of seismic vulnerability assessments of public and private RC constructions. In these activities, the codes and guidelines for DTs and NDTs were used. Columns, walls and beams are the structural elements tested; their choice and the location of the tests were defined by the engineers, based on the construction characteristics considered.Subset DB_1: 2010 samples with compression tests (Subset DB_2: 1039 samples with Specific Weight (SW).Subset DB_3: 1175 samples with Subset DB_4: 202 samples with compression tests and ultrasonic velocity tests, The database contains DT and NDT results. In consideration of the main objective of this study, only the NDT cases were excluded, thus the following subsets were identified.The difference between DB_1 and DB_3 reflects the operational difficulty of carrying out NDTs in a widespread and reliable way on existing constructions. For example, in civil engineering works the execution of reliable NDTs (in particular ultrasound) is often prevented by the construction type, shape, size, and current state of the works under investigation, for example, see the work in Lastly, a limited subset of samples (DB_4.1) was considered based on the results of some laboratory and in situ investigations. DB_4.1 reports the data relating to the compression tests and direct velocity values, both performed in situ  and in the laboratory . The database is constantly improved and updated and possesses the characteristics shown in the following figures and tables.The database can be considered reliable in terms of credibility and adequacy. Major errors were removed (outliers and the like). Primarily, the quality of the database can be considered consistent with the main objectives of the study. The database is based on measurements that were conducted in certified laboratories considering the same standard code.Below, each database is investigated individually. Following the main objective of the study, the main results are reported but thanks to the significant value of the database, other studies and applications will be possible in the future beyond the objectives of this work.c,coref). The first analysis shows the distribution in compressive strength classes of different design and construction periods. The analysis of the c,coref values indicates the variability given by the period under consideration. The mean values show an anomaly in the age group <1961  is possible for a subset of 1039 samples.The specific weight depends on the water/cement ratio, aggregate/cement ratio, type of aggregate, original manufacturing procedures, and age.It can be considered to be representative of the global concrete quality: high porosity is generally associated with a lower strength of the structural elements. Therefore, SW could provide indications on performance and durability (which can affect the retrofitting strategies).In particular, the analyses look for the correlation between SW and compressive strength. Moreover, high porosity can affect the result of the compression test, as shown in c,coref relationship is analyzed. Some SW values seem to be of critical value for compressive strength and, in some cases (on single buildings), it is possible to identify a clear correlation between SW and compressive strength. However, contrary to expectations, there is no clear and generalizable relationship between SW and compressive strength, as is reported in Furthermore, the SW—c,coref, RN (rebound index), V (direct ultrasonic velocity test) are simultaneously available. Cases where ultrasonic velocity was evaluated through surface measurements were excluded. To identify a potential relationship between DTs and NDTs, the analysis of the results of this subset plays a key role. However, the analyses generally show a poor or no correlation between the compressive strength and the considered NDTs. This result is particularly clear for the hammer tests (rebound number). The variability of the in situ compressive strength can be much greater than that shown by the NDTs  in which the NDTs .Moreover, the relationship between NDT variability and DT variability for each construction was analyzed. A total of 80 buildings were analyzed and the results are shown in a and b are defined based on the DT and NDT results. In this way, it is also possible to consider a lower number of in situ tests, increasing the reliability of the tests and thereby reducing costs.The method can be effectively applied with the correction reported in the next paragraph.For NDTs, considering the methods described in the previous paragraphs (in particular the combined SonReb method), it is believed that it is more reliable to use only the results of the ultrasonic tests to build a calibrated relationship between DTs and NDTs. This result is consistent with other recent studies (for example ) which, c,coreV, c,insituV, c,coref) were obtained from an experiment conducted in the laboratory of the University of Basilicata [c,coreV, c,insituV, c,coref)were obtained. All these values are subsequently compared and Subset DB_4 reports the ultrasonic velocity as NDTs. DB_4 was defined considering only a subset of samples with a direct velocity measurement both on the structural elements (in situ) and on the single extracted samples (in the same points) before the compression test . In totasilicata  on fourbAs expected, in c,coreV-c,coref and c,insitu-fc,coreV in In c,coreV-c,coref are reported; whereas in c,insituV-c,coref are reported; the best fit of the data is reported. In the first case, the internal variability of the ultrasonic method is very limited.In c,coref,c,coreVseems able to correctly estimate the c,coref values and follow its evolution. The effects of age and compressive stress, also referred to in several studies, seem less evident in the c,coreV-c,coref relationship whereas for the c,insituV-c,coref relationship, age also has a clear influence on the deterioration of the concrete and subsequent reduction of the reliability of the correlation.It is heavily influenced by the material and by the context  and it sc,coreV and c,insituV. This proposal is also consistent with other studies ..Vc,core In the literature and previous studies, methods based on the NDT–DT relationship are considered reliable and are considered for applications also on existing RC constructions. As a matter of fact, as reported in the previous sections, in the last few years, for existing RC construction the investigation procedures based on NDTs and the commonly used NDT–DT correlations have shown significant critical issues, particularly in their ability to investigate and estimate the characteristics of the concrete. Such methods, which are often developed in laboratories and validated for new concrete, are often not suitable for investigating the existing RC constructions.On the contrary, this study shows a significant degree of uncertainty, operational difficulties and potential errors in interpreting the results; they are based on experimental experiences on existing RC constructions. Based on the reported experimental campaigns, the critical issues in current NDT–DT relationships are clearly shown and some possible improvements are consequently defined from the obtained results . For thiIn the last few years, the characteristics of concrete have been investigated using empirical expressions in order to evaluate the in situ compressive strength using NDTs, namely the rebound hammer test and ultrasonic pulse velocity test. In other studies, the variability of the most common NDT (rebound hammer test and ultrasonic pulse velocity test) was analyzed ,13,52,53The present study reported data and procedures, based on a database of real data. A new contribution from the study is really the database. The database was obtained from laboratory investigations and professional practice activities. On the contrary, several interesting and recent studies are developed in laboratories and validated only for new concrete where the results do not take into account the quality of the concrete in existing reinforced concrete constructions and its low strength and degradation problems.However, based on the results of this study, it can be stated that many of the existing empirical methods (based on pre-established correlations) are unable to provide a reliable evaluation of the compressive concrete strength and its variability.In particular, the results obtained show variability in compressive concrete strength but highlighted the non-variability of the Rebound Number (RN) and its non-correlation with concrete compressive strength. Ultrasonic velocity shows an extremely limited variability although it can be increased by relating the in situ ultrasonic velocity and the ultrasonic velocity samples.The rebound hammer test is not representative of the compressive concrete strength and is also misleading. It must be excluded. The classic SonReb method should not be used;Ultrasonic velocity tests can be used only if suitably calibrated with Ultrasonic velocity tests on the extracted concrete before the compression tests;Using current methods and procedures, NDTs cannot be used a priori to identify homogeneous areas.In conclusion, based on this work, it can be highlighted that:Lastly, this study contains the experimental data useful as a reference both for practical professional activities and for the comparison and validation results used in research activities .–An increase in the amount of experimental data available;–The improvement of data collection procedures and standardization of analysis procedures;–The updated indication in the codes and guidelines.This study allows for further developments due to:In the next step of the study, new procedures and methods will be developed based on the reported results, in particular, following the results of


Chromodomain helicase DNA binding protein (CHD) family plays critical roles in regulating gene transcription. The family is linked to cancer disease, but the family member’s role in tumorigenesis remains largely unknown. Here, we report that CHD6 is highly expressed in colorectal cancer (CRC). CHD6 knockdown inhibited cancer cell proliferation, migration, invasion, and tumorigenesis. Consistently, Villin-specific Chd6 knockout in mice attenuates cancer formation in AOM/DSS model. We found that aberrant EGF signals promoted the stability of CHD6 by diminishing ubiquitin-mediated degradation. EGF signal inhibits GSK3β activity, which in turn prevents phosphodegron formation of CHD6, thereby hindering E3 ligase FBXW7-mediated CHD6 ubiquitination and degradation. CHD6’s chromatin remodeler activity engages in binding Wnt signaling transcription factor TCF4 to facilitate the transcriptional expression of TMEM65, a mitochondrial inner membrane protein involved in ATP production and mitochondrial dynamics. In addition, Wnt signaling is also an upstream regulator of CHD6. CHD6 promoter contains TCF4 and β-catenin binding site, and CHD6 can be transcriptionally activated by Wnt ligand to facilitate TMEM65 transcription. Thus CHD6-TMEM65 axis can be regulated by both EGF and Wnt signaling pathways through two different mechanisms. We further illustrate that CHD6-TMEM65 axis is deregulated in cancer and that co-administration of Wnt inhibitor LGK974 and the anti-EGFR monoclonal antibody cetuximab largely restricted the growth of patient-derived xenografts of CRC. Targeting CHD6-TMEM65 axis may be effective for cancer intervention. CRC is highly aggressive and is the third leading malignance in the world population, causing near 900,000 death per year, and its incidence has been a health care challenge2. Despite treatment progress has been made, CRC continues to be one of the deadliest cancer types with different molecular phenotypes/strong resistance to therapies3 and very high mortality rate2. Thus, there is an urgent need to identify more molecular biomarkers for CRC. Further, it is critical to explore the molecular mechanisms underlying CRC progression, which may help provide new therapeutic targets.Colorectal cancer (CRC) is a genetic disease with accumulated genetic alterations during the progression and invasion4. CHD proteins contain nine members and can be classified into three subfamilies on the basis of their functional domain similarity4. CHD activities are critical for a wide spectrum of cellular functions, including transcriptional regulation, cell growth, cell death, development, virus infection5, autophagy6, DNA damage response7, and genome integrity4. Deregulations of CHD in various human cancers are emerging, indicating that chromatin dynamics is critical during tumorigenesis. Subfamily III includes CHD6, CHD7, CHD8, and CHD9. These four enzymes are similar in their constituent domains, but they have non-redundant roles in the cell functions. The mechanisms behind their distinct and non-overlapping activities are unclear. Low CHD7 expression leads to enhanced survival of pancreatic cancer patients, suggesting that CHD7 promotes oncogenesis8. CHD8 is critical in recruiting E2F1 to the promoter of cyclin E2 for transcriptional activation9. The roles of CHD subfamily III proteins, including CHD6 and CHD9, in cancer remain to be characterized. CHD9 mutations have been found in gastric and colorectal cancers10. Functional analysis of the large CHD6 protein (2715 aa) has been hindered by its large size.Chromodomain helicase DNA-binding protein (CHD) family is composed of several members. This group of enzymes belongs to the SNF2 superfamily of ATP-dependent chromatin remodelers11. FBXW7 mutations are observed in cancers16. FBXW7 is a transcriptional target of tumor suppressor p5318. FBXW7 binds target proteins and facilitates their ubiquitination and degradation19. Most of the FBXW7 target proteins are oncoproteins, including c-MYC21, Cyclin E22, c-JUN24, mTOR25, Notch26, Aurora B27, FOXO4, and MCL-132. FBXW7 is a tumor suppressor19 as manifested in many types of cancers with FBXW7 mutation34. The functional activity of FBXW7 is important in hindering cancer growth. Nonetheless, many FBXW7 targets involved in tumorigenesis remain to be characterized.FBXW7 is an important component of the SCF (SKP1–CUL1–F-box protein) ubiquitin E3 ligase complexCHD6 gene transcription during tumorigenesis, and illustrate a role of CHD6 in regulating downstream target gene TMEM65 through Wnt signaling and TCF4 transcriptional activation. CHD6 collaborates with TCF4 to positively regulate TMEM65 gene expression, thereby impacting mitochondrial homeostasis and promoting cancer growth and metastasis. Our understanding of the biological role of CHD6 in regulating TMEM65-mediated mitochondrial dynamics and metastasis of cancer reveals therapeutic strategies for cancer intervention and treatment.Here, we show that CHD6 is overexpressed in CRC. We have characterized the upstream regulators of the CHD6 during tumorigenesis, including EGF, glycogen synthase kinase-3β (GSK3β), FBXW7α (hereafter abbreviated as FBXW7), and Wnt3a. Moreover, we identify CHD6 downstream target TMEM65, which is a mitochondrial protein overexpressed in many cancers and is involved in regulating mitochondrial dynamics. Our results shed light on the EGF-GSK3β axis in preventing FBXW7-mediated CHD6 degradation and the Wnt-β-catenin axis in promoting the CHD6 was highly amplified in CRC  level was higher in CRC adenocarcinoma tissues than in normal tissues  staining of a panel of 104 CRC and 76 corresponding normal tissue specimens (tissue microarray (TMA)) showed that CHD6 protein expression is significantly higher in CRC than in adjacent normal tissue (66 of 76 paired samples (86.8%)) ) Fig. . Kaplan–%)) Fig. . CollectCHD6 knockdown (KD) in DLD1, HCT116, and SW620 cells and validated the KD efficiency by western blot 35 growth, G1-S progression, cell survival, oxygen consumption rate (OCR), ATP production, and mitochondrial mass   showed a lower tumor burden  mice and inducible villin-specific conditional Chd6-knockout  mice  staining at three weeks after TAM injection ) and Chd6fl/fl;Villin-CreERT + TAM mice ) were injected with Azoxymethane (AOM), followed by Dextran Sodium Sulfate (DSS) treatment . The ten most significantly enriched cancer-related pathways commonly associated with high-CHD6 group in both datasets were selected and plotted  using Ingenuity Pathway Analysis (IPA) software  or degron is present in CHD6 Fig. . Indeed,To confirm that FBXW7 targeted the specific binding motif (LPTPXXT) or phosphodegron for CHD6 degradation, we constructed the CHD6 T2127A/T2131A mutant within (2125 LPTPXXT 2131) motif. The results showed that FBXW7 accelerated the turnover rate of CHD6 WT but not the CHD6 T2127A/T2131A mutant Fig. . Again, Given that FBXW7 recognizes phosphorylated degron of target proteins for degradation, and the FBXW7 binding motif on CHD6 is a GSK3β-recognized motif (S/TXXXS/T) and phosphorylation site Fig. , we hypoCHD6 construct for microarray analysis. The differentially expressed genes were shown in the volcano plot between the control (no Dox) and CHD6 KD cells (Dox)  Fig. . The toplls Fig. . Intereslls Fig. . To inve KD Fig. . Second,PCR Fig. . Third, CHD6 KD inhibits OCR and ATP production , a protein involved in heme synthesis ) ) Fig. . Again, X)) Fig. , and its KD Fig. , suggest KD Fig. . Co-IP r KD Fig. , suggestCHD6 KD leads to decreased TMEM65 (a PPOX interacting protein) and decreased oxidative phosphorylation and ATP production.Given that mitochondrial protein TMEM65 impacts mitochondrial functions and interacts with PPOX, this observation raises an interesting issue regarding the association of TMEM65 with other mitochondrial proteins in the complex. Our gel filtration studies indicate that TMEM65 are present in complexes with different mitochondrial proteins, such as ATP5a, Mt-CO2, NDUFA8, ATP5b, and PPOX, reiterating its role in regulating mitochondrial homeostasis    with or without ectopic expression of TMEM65 , compromised expression of TMEM65, and reduced PPOX expression. Importantly, TMEM65 overexpression reversed, at least in part, these impacts from CHD6 KD  and showed that they can downregulate mRNA expression of TMEM65  were established, and the expression levels of CHD6 were characterized. Two PDXs contain high CHD6, while the other two PDXs contain low CHD6  was more efficient in hindering tumor growth than Cetuximab or the LGK-974 inhibitor alone in high CHD6-expressing PDX (case no: 241808) as revealed by the reduced tumor volume, diminished TMEM65 staining, increased cleaved caspase-3 staining, and decreased Ki67 staining Fig. . TogetheTMEM65 gene expression through Wnt and TCF4 signaling. Our data shed light on CHD6 upstream regulatory circuit and reveal how EGF/Wnt oncogenic signal promotes CHD6’s downstream activity toward TMEM65 pathway to cause deregulation of mitochondrial dynamics and subsequently promote cancer metastasis and tumorigenesis  among the group III subfamily members (CHD6–9), so far these members share non-redundant functional roles in the cell. Other than CHD6, roles of other members in CRC development required further investigation. Importantly, as indicated in our analysis  of CHD6 on FBXW7 substrate binding site from CRC patients. However, the significance and functional impact of this mutant have not been elucidated. We demonstrated that this cancer-derived CHD6 mutant is resistant to FBXW7-mediated degradation, suggesting that its continual stability/activity is critical in mediating tumorigenesis.sis Fig. , CHD6 is45. The deletion leads to cerebellar defects and ataxia. So far, no tissue-specific Chd6-knockout mouse is employed to investigate its role in cancer. CHD6’s impact on human disease has been documented in a chromosomal translocation t   event between CHD6 (located at 20q12) and the basic helix-loop-helix transcription factor 4 gene (located at chromosome 18), which resulted in mental retardation syndrome46. However, CHD6’s biological role remains an enigma. We set up a Chd6 CKO mice by using the CRISPR-Cas9 genome-editing system to understand the genetic contribution of Chd6 during the development of tumorigenesis. Villin-specific knockout of Chd6 using Cre-loxP system led to slower formation of cancer upon the treatment of AOM/DSS, suggesting the critical role of Chd6 in promoting the growth of colon cancer. Our CRISPR-mediated knockout of Chd6 can be utilized in other types of cancer studies if using proper Cre expressing system, such as liver or prostate cancer. It would be a useful tool to understand the roles of CHD6 in other cancers.CHD6 Δexon12 (to delete the ATPase domain) mice have been characterized47. FBXW7 is a tumor suppressor48 protein through recognizing and degrading several oncoproteins, including FOXO4, cyclin E, c-JUN, Aurora B, and MYC32. It is reasonable to have a tumor suppressor E3 ligase FBXW7, which is frequently mutated in colon cancer, to degrade a potential oncogenic protein CHD6. Our study adds a new member to the target list of FBXW7 E3 ligase.EGF/AKT signaling is highly active in many types of cancers, and it becomes a critical therapeutic target for anti-cancer drugs. Although AKT activation is frequently observed in advanced clinical stages in many types of cancers, the consequence or target of aberrant activation of AKT remains not fully understood. GSK3β is inhibited through AKT-dependent phosphorylation. Our studies demonstrate that GSK3β mediates CHD6 phosphorylation, which destabilizes CHD6 through enhancing FBXW7-mediated CHD6 ubiquitination. Therefore, EGF/AKT activation can increase the stability of CHD6 to drive the activity of downstream signals. The fact that FBXW7 mediates CHD6 degradation is reminiscent of the regulatory mechanism of many FBXW7 targets. For examples, FBXW7 binds to SREBP phosphorylated by GSK3β. The binding leads to SREBP degradation50, but little is known about its regulation or other biological functions. Noticeably, TMEM65 is overexpressed in cancer, which is consistent with the role of its upstream regulator CHD6 as CHD6 is also highly expressed in CRC.TMEM65 is localized at the mitochondrial inner membraneCHD6 KD leads to upregulation of p-Drp1, which in turn causes fragmented mitochondria (fission), while TMEM65 expression can reverse this impact caused by CHD6 KD. That is, TMEM65 can downregulate p-Drp1 to manifest mitochondrial elongation (fusion). Determining how CHD6/TMEM65 can downregulate p-Drp1 warrants further investigation. Fused mitochondria seem to promote increased oxidative metabolism, ATP production, and reduced ROS. These observations mediated by CHD6/TMEM65 expression are consistent with the role of regulated mitochondrial dynamics in cancer metastasis and tumorigenesis52. It is obvious that CHD6/TMEM65 expression has a role of increasing ATP production in mitochondria (bioenergetics) and another role of resisting apoptosis. It is then important to point out that both aspects are regulated by mitochondria53 and are critical in cancer development.Mitochondria are very dynamic organelles. They constantly undergo fission and fusion to impact the shape, size, number of mitochondria, subcellular transport, mitochondrial quality control (mitophagy), and programmed cell death (apoptosis) in the cell. The balance of fission and fusion events manifested mitochondrial dynamics, which is regulated by a number of mitochondria-shaping proteins such as Drp1. Drp1 (DNM1L) has a role in mitochondrial fission via mediating membrane fission through its oligomerization and interacting with membrane-associated tubular structures. We showed that TMEM65 can downregulate p-Drp1, suggesting that TMEM65 may have a role in antagonizing mitochondrial fission. Activated fission and/or inhibited fusion results in fragmented mitochondria, whereas active fusion and/or inhibited fission leads to mitochondrial elongation. Indeed, 54. It is conceivable that CHD6/TMEM65 deregulation may have a pathological impact on these diseases. For example, defects in TMEM65 lead to a mitochondrial disorder manifested as a complex encephalomyopathic phenotype. Further, TMEM65 interacts with connexin 43 (Cx43)55, and its KD affects the protein level of Cx4355 to regulate heart function. It remains to be determined whether CHD6 affects the expression of Cx43.In addition, mitochondrial deregulation results in a variety of diseases56, thereby boosting oxidative phosphorylation activity. This phenomenon is consistent with the role of CHD6/TMEM65 in increasing OCR and ATP production.We observed that TMEM65 overexpression leads to more cristae in mitochondria compared to controls. This increase of cristae number could result in higher levels of respiratory chain proteins and the ATP synthase44. We showed that CHD6 engages in the Wnt signaling pathway and modulates TMEM65 expression through its binding to TMEM65 promoter in a TCF4-collaborating manner. Interestingly, CHD6 is positively regulated by Wnt signaling as its expression is upregulated by TCF4 activity and Wnt ligand. Given that EGF can also stabilize CHD6, it is then conceivable that CHD6 participates in both EGF and Wnt signaling pathways, which have major impacts in promoting the development of CRC  plus Wnt signaling (Wnt inhibitor) might have a better synergistic effect in treating CHD6-high CRC. Indeed, Cetuximab plus Wnt inhibitor LGK-974 as a combination treatment strategy for CHD6-high, Ras/Raf WT CRC PDX studies demonstrated the feasibility  and Chd6flox/flox;Villin-CreERT (6 mice). For tumor induction, 6-week-old mice were injected with 10 mg/kg AOM (Sigma) intraperitoneally (i.p.) one time at the beginning of the experiment (day 0). On day 5, 2% DSS  was given in drinking water for 7 days followed by regular drinking water for 2 weeks. This cycle (7 days DSS plus 14 days water) was repeated twice with 1.5% DSS. Mice were sacrificed at 80 days after AOM injection. To induce Chd6 knockout, mice were administered with 50 mg/kg tamoxifen  for 5 days 2 weeks before AOM injection.Two groups of mice were confirmed by genotyping: 58. Briefly, 5-week-old female BALB/c nude mice were purchased from GemPharmatech Co., Ltd. 1 × 106 HCT116 cells, containing stably tetracycline-inducible shCHD6 construct, were subcutaneously injected into the right flank. When tumor volumes reached 50–80 mm3, all the tumor-bearing mice were randomly divided into two groups of 6 animals per group. Animals were either i.p. injected with doxycycline  or vehicle every three days. Tumor widths and lengths were measured twice per week using a caliper. Tumor volumes were calculated by the formula: V (mm3) = (width2 × length)/2.Xenograft cancer models were established as previously described6 HCT116 cells, containing stably tetracycline-inducible shCHD6 with or without TMEM65 overexpression, were injected into the spleen of BALB/c nude mice under general anesthesia. The mice in CHD6 KD group were i.p. injected with 50 mg/kg doxycycline every three days. On day 30, mice were sacrificed by CO2 inhalation.1 × 1059. Basically, four KRAS/BRAF WT PDX lines were selected. Patient-derived tumor tissues were cut into small fragments (3 mm3) and subcutaneously implanted into immunocompromised mice . For single drug treatment, when tumor volume reached ~50 mm3, mice were randomly allocated to two groups and were i.p. injected with either cetuximab  or vehicle once a week. For combination treatment, when tumor volume reached ~50 mm3, mice were randomly allocated to the following treatment groups: (1) vehicle control; (2) cetuximab ; (3) LGK974 , (4) cetuximab and LGK974 using doses described above. Administration of cetuximab was the same as addressed above. LGK974 was administered to mice via i.p. injection every day. Mouse weights and tumor volumes were measured every three days during the experiments.PDX experiments were performed as previously describedHCT116 cells and mouse colon tissues were fixed with fixation solution . After 24 h, samples were washed 3 times with 0.1 M cacodylate buffer and post-fixed with 1% osmium tetroxide (Ted Pella Inc) for 2 h at room temperature. Then the samples were dehydrated in a graded ethanol series and embedded into SPI-Pon 812 . The embedding models were polymerized at 60 °C for 2 days. Ultrathin sections (60–80 nm) were cut with a Leica Ultracut microtome (Leica UC7) and stained with 2% uranyl acetate and 2.6% lead citrate. TEM imaging was performed on a Hitachi TEM system (HT7800). The lengths of mitochondria in TEM images were measured using ImageJ.61. In brief, fresh CRC tumor tissues were washed with cold PBS containing penicillin-streptomycin, and cut into 3–5 mm fragments. Pieces were digested with EDTA (5 mM) on ice for 60 min with mixing. After being digested into clumps of cells, the sample was mixed with Matrigel and seeded into a 24-well plate. After Matrigel polymerization (10 min at 37 °C), 500 μL/well advanced DMEM/F12 medium containing 10 mM HEPES, 100 U/mL penicillin/streptomycin, 2 mM GlutaMAX, 1× B27, 1× N2 (Life Technologies), 10 nM gastrin I (Biogems), 500 ng/mL R-spondin1 (Peprotech), 10 μM SB202190 (Sigma), 10 μM Y-27632 (Abmole), 50 ng/mL recombinant EGF, 500 nM A83-01 (Biogems), 100 ng/mL recombinant Noggin (Peprotech), 10 mM nicotinamide (Sigma), 1 mM N-acetylcysteine (Sigma) was added to each well containing organoids. On the second day, the organoids were transduced with shCTL or shCHD6 lentivirus using polybrene (10 μg/mL) .Human CRC organoid was cultured as described previously2. HCT116, SW620, and HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. DLD-1 cells were cultured in RPMI 1640 medium (RPMI) with 10% FBS. LipofectamineTM 2000 (Invitrogen) and polyethylenimine  were used for cell transfection in this study following the manufacturer’s standard protocol.All colorectal cell lines and HEK293T cells used in this study were obtained from ATCC, confirmed to be mycoplasma-free, and incubated in humidified incubator at 37 °C with 5% (vol/vol) COCHD6, TMEM65, TCF4, and FBXW7 were amplified by PCR. These cDNAs were cloned into a pCMV5 vector to generate fusion proteins with N-terminal Flag or Myc tag. CHD6 cDNA was also subcloned into pEGFP-N1 vector (Addgene) to produce a fusion protein with a GFP tag at the C-terminus of CHD6. TMEM65-Flag was also re-cloned into a lentiviral vector pWPI. CHD6 mutant was generated by using a Fast Mutagenesis Kit V2 (Vazyme) according to the manufacturer’s instructions. The resulting plasmids were verified by sequencing.The cDNAs for CHD6 KD, lentiviral shRNA constructs were purchased from (GeneCopoeia). We screened 6 hairpins targeting human CHD6 transcripts and found two independent sequences that reduced protein levels by > 80% . For the doxycycline-inducible CHD6 KD, the shRNAs targeting CHD6 were inserted into Tet-pLKO-puro vector (Addgene). Tet-pLKO-shCHD6-1 (CHD6 shRNA #1: 5′-CATTCCAGCAATCATAGTTAA-3′) was designed to target 3′UTR of CHD6. The shRNA sequence in Tet-pLKO-shCHD6-2 is the same as shCHD6-31 (5′-GCACAGAAGATCAAGCGATTT-3′). For the doxycycline-inducible FBXW7 KD, the shRNA sequence (5′-TGATACATCAATCCGTGTTTG-3′) was inserted into Tet-pLKO-puro vector. For TMEM65 KD, the sequence (5′-TCCAGGTTAGGCCTGTCAATT-3′) was inserted into pLKO.1 puro vector.For 7, and were co-transfected with 10 μg shRNA lentiviral plasmid, 5 μg psPAX2 and 5 μg pMD2.G using polyethylenimine . The supernatant containing lentivirus was collected at 48 and 72 h after transfection and were filtered through Millex-GP Filter Unit . HCT116, DLD-1, and SW620 cells were infected twice with filtered viral supernatant containing 10 μg/mL polybrene .To prepare lentivirus, HEK293T cells were seeded into a 10 cm dish at a density of 1 × 10Cells were washed twice with cold PBS and lysed in RIPA buffer . Sample proteins were separated by SDS-PAGE and transferred onto PVDF membranes. For protein turnover assay, cells were treated with 60 μg/mL cycloheximide  for the indicated times before collection. The following antibodies were used in the immunoblotting and immunoprecipitation experiments: CHD6 , CHD6 , TMEM65 , p-AKT (Ser473) , AKT , Vinculin , FBXW7 , COX IV , PPOX , VDAC1 , mtTFA , p-Drp1 (Ser616) , Drp1 , Parkin , GAPDH , Flag-tag , HA-tag , Myc-tag , β-Catenin , TCF4 , GSK3β .2 agarose beads at 4 °C overnight.Cells were lysed in IP lysis buffer . Lysates were incubated either with CHD6 antibody or IgG at 4 °C overnight. Protein A/G agarose beads  were added into each sample and then incubated at 4 °C for 3 h. Samples were washed 4 times using cold IP lysis buffer and boiled with 2× SDS loading buffer. For Flag-tag immunoprecipitation, cell lysate supernatants were incubated with Flag-MFrozen sections were blocked in 2% bovine serum albumin for 1 h at room temperature. After blocking, sections were incubated with CHD6  and TMEM65  antibodies overnight at 4 °C. The samples were subsequently incubated with Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies. Nuclei were stained with DAPI (Invitrogen). Fluorescence signals were imaged using fluorescence microscope (Leica).2HPO4/NaH2PO4). Cell lysates were incubated with Ni-NTA agarose (Invitrogen) at room temperature for 4 h and washed with Wash Buffer . Eluted proteins were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies.For CHD6 ubiquitination assay, cells were transfected with the indicated plasmids for 48 h. MG132  was added to the culture media for 6 h. Then cells were collected and lysed in Buffer A , CHD6 , TMEM65 , COX IV , PPOX , p-Drp1(Ser616) , OPA1  and cleaved Caspase-3 (Asp175)  antibodies were used.Immunohistochemistry was performed following a standard protocol. Paraffin-embedded sections were dewaxed in xylene and rehydrated in gradient ethanol. Antigen retrieval was performed using 0.01 M sodium citrate in a high-pressure cooker. After cooling down, sections were incubated in 3% HTotal RNA was extracted using TRIzol Reagent  according to the manufacturer’s protocol, and reverse-transcribed into cDNA using ReverTra Ace® qPCR RT Master Mix (TOYOBO). The expression of target genes was detected using the 2× SYBR Green qPCR Master Mix  by a LightCycler® 480 II (Roche).4 cells per well) and cultured at 37 °C and 5% CO2 overnight. Sensor cartridge was hydrated in calibrant and incubated in a 37 °C, non-CO2 incubator. For the XF Cell Mito Stress Test Kit , assay medium was prepared by supplementing XF Base Medium  with 10 mM glucose , 1 mM pyruvate, and 2 mM glutamine. Cells were washed with assay medium twice, and incubated at 37 °C, non-CO2 incubator for 45 min. Oligomycin, FCCP and Rotenone/antimycin A were each resuspended in assay medium and added to the microplate. The OCR was determined by a Seahorse XF24 analyzer .Prior to assay, cells were seeded on XF24 microplate (5 × 106 cells/mL) was diluted to 9 volumes of boiling TE buffer  and was incubated at 100 °C for 2 min. After incubation, samples were centrifuged at 1000× g for 1 min, and 50 μL supernatant of each sample was transferred to a well of 96-well microplate. ATP level was detected by adding 50 μL luciferase to each sample, and the bioluminescent signal was measured with a Microplate Reader .The total ATP was detected using ATP Bioluminescence Assay Kit CLS II . 100 μL of cell suspension (1 × 102O + 2 H+). ECAR data indicates the total proton Efflux Rate, and the mitochondrial Proton Efflux Rate can be calculated according to the ECAR that is changed by adding rotenone and antimycin A. The mitoATP Production Rate is calculated according to Equation: mitoATP Production Rate (pmol ATP/min) = OCRATP (pmol O2/min) × 2 (pmol O/pmol O2) × P/O (pmol ATP/pmol O). OCRATP can be calculated as the OCR that is inhibited by adding oligomycin (ATP synthase inhibitor). Assay medium was prepared by supplementing XF Medium-phenol free, pH 7.4  with 10 mM glucose , 1 mM pyruvate , 2 mM glutamine . Oligomycin, Rotenone, and antimycin A were prepared prior to assay.For the determination of glycolytic ATP production rate (glycoATP Production Rate) and mitochondrial ATP production rate (mitoATP Production Rate), Agilent Seahorse XF Real-Time ATP Rate Assay Kit (103592-100) was used according to the manufacturer’s protocol. The glycoATP production rate is equivalent to Glycolytic Proton Efflux Rate  and stored in a closed box at 4 °C overnight. On the second day, 500 μL of 2 M oxalic acid (heat for dissolution) was added and mixed with the stored samples. 500 μL of the mixture was heated at 98 °C for 30 min, and the remaining samples were kept at room temperature. All prepared mixtures were centrifuged at 16,000× g for 2 min. 200 μL of mixture from each sample was transferred to a black microtiter plate and the fluorescence  was measured in a fluorescence microplate reader . The fluorescence of corresponding unboiled samples was used to correct for background fluorescence.Heme content measurement assay was performed as described previouslyL-Wnt3a-expressing cells were seeded into 10 cm dish, and cultured in DMEM supplemented with 10% FBS. After 4 days, cell culture supernatant was collected, filtered, and kept at 4 °C. The cells were cultured for another 3 days for a second supernatant collection. The second supernatant was combined with the first collection and stored at −80 °C.64. Briefly, control and CHD6 KD HCT116 cells were cross-linked using 1% formaldehyde for 20 min at room temperature. Reactions were quenched by treating with 0.125 M glycine for 5 min at room temperature. Then cells were washed with cold PBS and collected in 1 mL ChIP lysis buffer . Chromatin fragmentation was performed using a Diagenode BioruptorPico sonicator . Clear chromatin extracts were incubated with 20 μL Magna ChIP™ Protein A + G Magnetic Beads  and 2 µg antibody  or IgG overnight at 4 °C. Immunoprecipitated chromatin was washed with low-salt buffer  twice, washed with high-salt buffer  twice, and then washed twice with LiCl buffer  and once with TE buffer (pH 8.0). Sample beads were resuspended in 50 μL elution buffer  with 5 μL proteinase K (20 mg/mL) at 65 °C overnight. DNA was purified using PCR purification Kit . Eluted DNA was used for qPCR.ChIP was performed as previously described58. Briefly, CHD6-transfected HEK293T cells were lysed in lysis buffer . The cleared supernatants of each cell lysates were run through a size-exclusion column Superose 6 (GE), and proteins were eluted by PBS  in GE AKTA avant150 chromatography system at a flow rate of 0.4 mL/min. A total of 40 fractions (0.3 mL/fraction) were collected. Collected protein fractions were resolved with SDS-PAGE, followed by immunoblotting with the indicated antibodies.Fractionation of cell lysates by size-exclusion chromatography was performed as previously described with minor modificationCHD6 KD (dox-inducible CHD6 KD). HG-U133 Plus 2.0 GeneChips (Affymetrix) was used to examine the gene expression profiles according to the manufacturer’s instructions. Analysis of microarray data was performed in R programming environment (http://cran.r-project.org), and the Bioconductor (http://bioconductor.org) R packages simpleaffy (http://bioinformatics.picr.man.ac.uk/simpleaffy) and EnhancedVolcano (https://github.com/kevinblighe/EnhancedVolcano) were used for analyzing Affymetrix data and generating volcano plot. TCGA expression and mutation data were downloaded from cBioportal66. The CRC datasets  were downloaded from the Gene Expression Omnibus (GEO), the Oncomine, The Cancer Genome Atlas and The Human Protein Atlas databases. For the CRC study, tumors were stratified with median CHD6 expression. The datasets of GSE31595 and GSE2109 were annotated by GSEA (Broad Institute) on KEGG and Cancer Hallmark Pathways databases. The GEO2R web application (https://www.ncbi.nlm.nih.gov/geo/geo2r/) was used to computerize the differentially expressed genes  by comparing the gene expression profiles of CHD6-high and -low CRC (GES2109 and GES14333). The resulting gene list was submitted for pathway analysis using the ‘Core Analysis’ function in the IPA .RNA was extracted from HCT116 cells with or without CHD6 mRNA level was detected by RT-qPCR. The human TMAs were purchased from Outdo Biotech . The TMAs contained 180 human colon tissue cores, including 76 paired specimens of CRC and corresponding normal tissues and 28 identified CRC specimens. The immunostained slides were scanned using Slide Scanning System SQS-1000 (TEKSQRAY). TMA images were analyzed with HALO image analysis software (Indica Labs).All cancer patient samples were collected with the patients’ written informed consent and approval from the Institutional Review Board of the Sixth Affiliated Hospital of Sun Yat-sen University. For CHD6 expression level analysis, 18 paired CRC and adjacent normal colon tissues were collected from the Department of Surgery at the Sixth Affiliated Hospital of Sun Yat-sen University. Total RNA was isolated, and t-test (GraphPad Prism 6). Multiple group comparisons were performed by one-way ANOVA followed by Tukey test  or Dunnett test . The statistical significances about cell proliferation and tumor growth in different groups were calculated by two-way ANOVA. Survival analysis was performed using SPSS 25 by Kaplan–Meier survival curve and verified by log-rank test. The Pearson correlation was calculated using a package written in the R language (http://cran.r-project.org). χ2 test was used to examine the relationship between groups. Cox proportional hazards regression for univariate and multivariate analyses were employed to evaluate which clinicopathologic factors had prognostic values. The P values and hazard ratios are shown and P < 0.05 was defined as statistically significant.The differences between two groups were measured by Student’s Supplementary information


The use of structural lightweight concrete in the construction industry is on the rise in the last few decades mainly because of the higher strength per unit density, as it reduces the total deal load of the structural elements as compared with normal strength concrete. In addition, the environmental concerns of the concrete industry have gained supreme importance in recent times, demanding vital and effectual steps. In this regard, the current study was carried out to formulate an alternative approach for producing a sustainable lightweight structural concrete. The study followed two stages: initially, the selection of optimized manmade plastic aggregates based on trial concrete mixes, and finally, to gauge the physical-, mechanical- and durability related properties of the concretes integrating optimized manmade aggregate series at different replacement fractions. As a result of the first phase: two aggregate series out of eight were selected based on the compressive strength and durability properties of their concretes. In the next stage, all the properties for the optimized aggregate concrete were analyzed in terms of compressive strength. It was noted that the physical, mechanical and chloride penetration resistances have generally displayed a decreasing trend, with an increase in the manmade plastic aggregate replacement fractions as compared with reference lightweight concrete. However, the two aggregates, i.e., 70% DS-30% LLDPE and 50% QF-50% PET at the replacement fractions of 25% and 100%, were found to be the best two contenders that fulfilled the criteria for structural lightweight concrete, i.e., ASTMC330/C330M-14, and were proposed for structural lightweight purposes with low and relatively high strength and chloride resistance-based durability requirements, respectively. In addition, the brittleness ratios and structural efficiency parameters for the concretes of the 70% DS-30% LLDPE and 50% QF-50% PET also supplemented the aforementioned findings. Overall, this study presents a sustainable approach for the effective utilization of plastic waste for producing structural lightweight concrete. Concrete construction has become one of the popular techniques all over the world, with its applications varying from a small single-family house to multi-story structures, long-span bridges and other mega-infrastructures such as dams, etc. The main reason for its popularity is the availability of raw materials for its components and well-established casting and curing procedures, along with its superior mechanical and durability properties against different environmental conditions compared with other available construction materials. However, with the advent of new and lightweight construction materials, one of the highlighted factors for normal concrete is the lower strength per unit density, which increases the size of structural elements.Lightweight concrete has several intrinsic advantages such as lessening the total weight of a structure by a reduction in the structural elements’ sizes, which subsequently affects the usage of the two most expensive ingredients in reinforced concrete, i.e., steel reinforcement and cement. In addition, the lightweight concrete has remarkable sound and heat penetration resistance because of very low thermal conduction, mainly because of the presence of air voids as compared with conventional concrete. The density of normal concrete is mainly influenced by the density of the aggregates used in the concrete mix design. Therefore, one the suitable alternatives to producing lightweight concrete is to use lightweight aggregates to lower the density of the resulting concrete, as the aggregates occupy the maximum volume in the concrete. However, the incorporation of lightweight aggregates can negatively affect the physical, mechanical and durability properties of lightweight concrete, especially if the target is to use the resulting concrete for structural purposes.In the past, a few researchers have tried to produce structurally lightweight concrete by using different materials as a replacement of normal coarse aggregates, i.e., expanded polystyrene (EPS) lightweight aggregate, organic lightweight aggregate, solid waste or recycled by-products and expanded clay aggregates ,2,3,4. AIn addition, a few efforts have been made in the past to use plastic waste for producing lightweight concrete ,14,15,16The reduction in fresh density was observed for the replacement of coarse aggregate with expanded polystyrene . The dryIt was also reported that the recycling process, the type of plastic and properties of synthetic aggregates manufactured using plastic significantly affected the properties of the resulting lightweight concrete . AdditioIn addition, the environmental concerns of the concrete industry have become of utmost importance in recent times and need urgent and efficient steps to lower the carbon footprints of the construction industry. Thus, in light of the above, the current experimental study was planned to formulate a sustainable green lightweight concrete for structural applications complying with ASTM C330/C330M-14 . It was In the second stage, a detailed investigation was carried out for the concrete mixes prepared by the selected aggregates at various fractions, i.e., 25%, 50%, 75% and 100%, to examine the possibility of using the selected aggregate for manufacturing a concrete fulfilling ASTM C330/C330M-14  criteriaIn the first stage of the research study, the trail mixes of concretes were prepared by keeping the majority of the parameters (such as water-to-cement ratio) constant, except the type of coarse aggregate. A total of ten concrete mixes were prepared using eight different types of manmade plastic aggregates and two control mixes having the lightweight aggregates (readily available in the market) and normal weight aggregates, as shown in The optimized manmade plastic aggregate series was selected based on the performance of concrete mixes with regard to compressive strength, durability indicator, i.e., chloride ion penetration, and availability of the raw material. As seen from the results in For the second stage of the current study, the two optimized manmade plastic aggregates (MM1 and MM2) were used as coarse aggregate at different replacement ratios of 25%, 50%, 75% and 100%. In addition, for comparison purposes, a commercial lightweight (Lytag aggregates) and a normal-weight coarse aggregate were also used to prepare reference concrete mixes (LCC and NCC). 3 and 1132 kg/m3, respectively. The water absorption was found to be 2.75% and 0.95% for MM1A and MM2A, respectively. For the detailed investigation, ten concrete mixes were prepared with a constant water-to-cement ratio of 0.5, as shown in Ordinary Portland cement (Type I) having a specific gravity of 3.15 was utilized in the current study for the casting of concrete mixes. The dry unit weight for MM1A and MM2A was 750 kg/mThe dry density, compressive strength, flexural strength, splitting tensile strength, modulus of elasticity, abrasion resistance, thermal conductivity and resistance to chloride ion tests were conducted on all the concrete types. The test setup for the mechanical testing is shown in The following section presents the results of major physical-, mechanical- and durability-related properties for the concrete mixes prepared by using the two optimized manmade plastic aggregates at different replacement ratios. It should be noted here that the results for all the properties are expressed in terms of the compressive strength, as it is one of the most important parameters for concrete in general.2 value of 0.3078 was obtained for the best-fit equation, which also represented a greater divergence in the current study in comparison with other studies.The results for the dry density of MM1C and MM2C in relation to cube and cylinder compressive strength are presented in 2 values of 0.663 (solid Line) and 0.724 (dotted line) for the power- and linear-based regression analyses. Overall, based on the results of density and cylinder compressive strength, the MM1A and MM2D qualified to be used as a structural lightweight concrete, as per the ASTMC330/C330M-14 [The cylinder compressive strength and density relation of the current study was also compared with the existing studies ,41,42,43C330M-14  criteriaThe trend of the abrasion resistance with respect to compressive strength for all concrete series is shown in Overall, it was observed that the two series selected based on the density and compressive strength requirements, i.e., MM1A and MM2D, show almost the same values based on the results of abrasion and cylinder compressive strength, even with the different trend followed by their respective concrete series.In this section, the author presents the relationship between the compressive strength and drying shrinkage of the concretes at various replacement percentages of manmade plastic aggregates. In addition, different plastic types were used for two plastic aggregates; therefore, to quantify the effect, the author performed the regression analysis and tried to formulate and equation based on the current experiment results. The trend of the drying shrinkage with respect to compressive strength for all concrete series is shown in It was observed that the addition of a plastic-based aggregate for MM1C and MM2C reduced the compressive strength, with a rise in the drying shrinkage. However, the increment in MM1C was found to be higher as compared with MM2C. This behavior of a rise in drying shrinkage in concrete containing manmade plastic aggregate was expected, as the primary factor influencing it is the amount of water loss, which in the case of the aforementioned concrete will just be from the matrix alone, as the plastic aggregates will not contribute in this phenomenon. The equation to predict the drying shrinkage for manmade plastic aggregate concrete in terms of compressive strength is also proposed in The relation between the tensile strength and compression of normal concrete is well documented in the codes, however, the effect of incorporating the manmade plastic aggregates in tensile strength in relation to compressive strength is quantified in this section. The author measured the compressive strength of the concretes by using the cylindrical and cube specimens. In the past, both cube and cylindrical compressive strength has been used by researchers; therefore, the author presented the relation between tensile and the compressive strength measured by two differently shaped specimens to eliminate the shape effect of specimens, keeping the main variable parameter as the plastic replacement. The trend of the tensile strength with respect to cube and cylinder compressive strength for all concrete series is shown in 2 values of 0.8521 (Cube-Tensile) and 0.7456 (Cylinder-Tensile). Furthermore, the equations proposed in From Overall, based on the results of tensile strength, density and cylinder compressive strength, MM1A and MM2D qualified to be used as a structural lightweight concrete as per the ASTMC330/C330M-14  criteriaThe trend of the bending strength with respect to cube and cylinder compressive strength for all concrete series is shown in 2 values of 0.8123 (cube-bending) and 0.7126 (cylinder-bending). Furthermore, the equations proposed in It was observed that irrespective of the aggregate type, the decrease in compressive strength was accompanied by a decrease in the bending strength at increasing percentage replacement of aggregate series. However, the decrease ratio between the compressive and bending strength of concrete for each replacement level of aggregate was found to be different. The maximum decrease for MM1C and MM2C was found to be 44% and 26%, respectively, with reference to control mixes. From In the current study, chloride penetration resistance was used as an indicator to access the durability and microstructure of the concrete containing different percentages of plastic aggregates. The trend of the chloride penetration resistance with respect to cylinder compressive strength for all concrete series is shown in It was observed that irrespective of the concrete type, the higher the concrete strength, the higher the amount of charge passed; however, the slopes of both the concretes (MM1C and MM2C) were different. It was observed that at lower strength values, MM1C showed higher values of charge passed as compared with MM2C, while the difference seemed to narrow down with increasing values of compressive strength. Decreases of about 18% and 73% in the amount of charge passed were observed for MM1CD and MM2CD, respectively, as compared with LCC. All the concrete series except for MM2CD showed values of charge being passed through concrete greater than 2000 coulombs, which refer to the classification of the concrete permeability ranging from medium to high, as per ASTM C1202 . TherefoIn the preceding sections, the physical-, mechanical- and durability-related properties of concrete containing two different types of manmade plastic aggregates at various fractions are presented. The primary aim was to determine a suitable candidate for sustainable structural lightweight concrete. As plastic was used in the manmade aggregate, it was therefore expected that the concrete brittleness behavior would be improved because of the ductile nature of plastic. In this regard, the brittleness ratio, as defined by Hucka and Das (1974) , i.e., tIt was observed that MM1C concrete series show an inverse relationship between the percentage of MM1A and brittleness ratio, i.e., as the amount of MM1 aggregates surged, the concrete became less brittle as compared with the reference lightweight concrete. On the other hand, MM2C showed a marginal increment in the brittleness ratio with the addition of MM2A aggregate type as compared with LCC. This difference is primarily because of the difference in the type of plastic and filler material being used for the manufacturing of both types of aggregates. It was noticed that MM1C can be used in applications with low strength and high ductility, while MM2C can be targeted for relatively higher strength with moderate ductility requirements.In addition, another important factor which is normally discussed for structural lightweight concrete is the structural efficiency. The structural efficiency is defined as the ratio between the cylindrical compressive strength and density of the given concrete as per ACI 213R-14 (2014) . The strIt was observed that for MM1C series, structural efficiency dropped with the increasing percentage of manmade plastic aggregates. The decrease was mainly because of the decrease in the compressive strength values. On the other hand, MM2C showed marginal decreases for 50% and 75% replacements of manmade plastic aggregates, and it was found to increase at the 100% replacement level, almost equivalent to that of the reference lightweight concrete. Therefore, from the structural efficiency aspect, MM2CD also showed its capacity to replace the commercially available lightweight aggregate. From the above discussion, it can also be deduced that replacing the commercially available lightweight aggregate, or natural lightweight aggregates, with the produced aggregates can effectively lower the carbon footprints of concrete and, in parallel, will help the utilization of plastic waste, as the aggregates occupy 70% of the concrete volume, and therefore a larger quantity can be replaced.This study aimed to formulate a sustainable green lightweight concrete for structural applications complying with ASTM C330/C330M-14 . It was Out of eight manmade plastic aggregates having different plastic and filler types, two, i.e., the first made by 70% DS and 30% LLDPE (MM1) and the second made by 50% QF and 50% PET (MM2), were selected as the optimized aggregate series based on the performance of concrete mixes with regard to compressive strength, durability indicator, i.e., chloride ion penetration, and availability of the raw material.The results of the physical properties analyzed in terms of compressive strength give rise to the best two contenders, i.e., MM1 and MM2 at 25% and 100% replacement fractions, respectively. On the other hand, the mechanical properties generally displayed a decreasing trend with increase in the manmade plastic aggregate replacement fractions; however, MM1 and MM2 at 25% and 100% replacement fractions, respectively, fulfilled the criteria for structural lightweight concrete, i.e., ASTMC330/C330M-14 .From the results of chloride ion penetration resistance, it was found that the aggregates made by 70% DS and 30% LLDPE were suitable for the projects having relatively low contact with chloride ions, while the aggregates made by 50% QF and 50% PET can be used for structural lightweight concrete applications irrespective of chloride levels. The brittleness ratio and structural efficiency also supported the use of MM1CA and MM2CD for structural lightweight concrete applications.Finally, two aggregates, i.e., 70% DS-30% LLDPE and 50% QF-50% PET at the replacement fractions of 25% and 100%, were proposed for structural lightweight purposes with low and relatively high strength and chloride-based durability requirements, respectively. As a whole, the current study provides an alternative approach to target the sustainable ways for producing lightweight structural concrete by the utilization of plastic waste.Therefore, this study was carried in two stages, i.e., the selection of optimized manmade plastic aggregates based on trial concrete mixes and the evaluation of the possibility of using the optimized aggregate series for structural lightweight construction applications. In this regard, the physical-, mechanical- and durability-related properties of the concretes integrating optimized manmade aggregate series were assessed, keeping compressive strength as the primary datum. The following conclusions are drawn: