Background: This study aimed to discover the association between parameters of event-related potentials (ERPs) and categorization of images of visual scenes, both emotionally-charged and neutral, in carriers of different genotypes of the COMT, HTR2A, BDNF genes.Methods: Electroencephalogram (EEG) and ERPs were recorded at 128 leads, with two ear referents. Images of different visual scenes were presented to the study participants sequentially on a monitor screen. The participants’ task was to examine these images and indicate what emotions  they elicit. Comparison of event-related potentials was carried out using unpaired Student t-test in EEGLAB toolbox.Results: COMT. A stronger reaction, as reflected in the amplitude of the ERPs, in participants with the recessive homozygous Met/Met genotype was observed on latency around 200 ms to the stimuli, assessed as positive. Carriers of dominant homozygous Val/Val genotype had higher amplitude of 200 ms peak when assessed scene images as either neutral or negative in comparison to other genotypes. Participant with the Val/Met heterozygous genotype had higher amplitude of ERP that Met/Met group on same latency when assessed stimuli as negative.HTR2A. Significant increase in negativity in the parietal-occipital regions revealed in the range of 350-420 ms in participants with the recessive homozygous A/A genotype when choosing any type of assessment, compared to carriers of the heterozygous genotype A/G and the dominant homozygous G/G genotype.BDNF. Participants with Val/Val genotype categorized the visual images more thoroughly, as reflected in greater activation of the parietal-occipital zones and higher amplitude on ERP peak on 190 ms (negative assessment) and 160 ms  then Val/Met carriers.Conclusions: TheCOMT, HTR2A, BDNF gene different genotypes are associated with the process of categorizing emotionally charged and neutral visual scenes, and this relationship is reflected in the ERP parameters. Studying hereditary factors that determine specifics of the categorization of visual images are very important for opening up new opportunities for individualizing educational experiences, i.e. those that could take into account individual visual characteristics of students. Specifically, emotions that actualize these visual images can play an essential role in the process of categorizing visual scenes. As it was shown in our previous work, particular features of image categorization may vary dependent on genetic differences among carriers at the level of genes that control the functioning of brain neurotransmitter systems  and categorization of images of visual scenes, both emotionally-charged and neutral, in carriers of different genotypes ofCOMT) gene encodes the production of the catechol-O-methyltransferase enzyme that regulates the inactivation of catecholamines . The COMT enzyme is involved in the metabolic degradation of the dopamine neurotransmitter in brain regions such as the prefrontal cortex, amygdala and striatum. Research have shown that the Val158Met polymorphism ofCOMT gene is associated with the activity of the brain stem, amygdala, basal ganglia and prefrontal regions, and also is involved in the modulation of neural substrate structures responsible for processing negative and positive emotions , in which dopamine content is typically high (The catechol-O-methyltransferase (5-HTR2A receptor gene and the brain neurotrophic factor gene (BDNF). The genotypes for these genes (as well as for theCOMT gene) could be represented in a homozygous dominant form, homozygous recessive and heterozygous forms. These genotypes are associated with different sensitivity of neuron receptors to the presence of neurotransmitters in the synaptic cleft; in the case of theHTR2A gene, the level of survival of dopaminergic neurons, while in the case of theBDNF gene, the nutrition of serotonergic neurons , in which guanine can be replaced by adenine (G196A). Such a substitution causes the amino acid valine to be replaced by methionine in codon 66, and the alleles of this gene are called Val and Met. The Met allele interferes with the intracellular traffic ofBDNF, reducing the secretion of neurotrophic brain factor, which is associated with the transition from plasticity to stability in neural networks (BDNF (BDNF) and high  cortical plasticity, as well as in individuals with different genotypes for theHTR2A serotonin receptor gene  and approved ethically by the Academic Council of the Academy of Psychology and Pedagogy of SFU, on 22 February, 2017. Before the start, each participant was informed about the goal of the research, procedure, experimental conditions, and safety of their personal data. Oral consent was obtained from each participant before continuing with the study.This study involved 73 participants, of both sexes, average age 23±5 years . The study was conducted from March 24, 2017 to May 25, 2017 at the Laboratory of Psychophysiology and Experimental Psychology, Department of Psychophysiology and Clinical Psychology, Academy of Psychology and Education Sciences, Southern Federal University . The participants were students of Rostov universities, including the Southern Federal University (SFU). SFU students received bonus points in their academic disciplines for their participation in the study.DNA analysis was carried out as follows: after two hours of dry hunger, buccal epithelium was collected from the inner surface of the participant’s cheeks by two sterile cotton probes. Then, probes with biological material were immersed in a transport medium and sent to the «Biologicheskie reshenija i tehnologii» , where DNA was extracted from clinical material using the «Proba-NK» reagent kit . Thermocycler CFX96 «Touch»  was used for real-time polymerase chain reaction (PCR).For analysis, we used genomic DNA preparations obtained from biological samples using DNA extraction kits with the removal of PCR inhibitors and the presence of at least 100 genomic copies in 1 μl. During the genetic examination, the following DNA sections were analyzed: COMT catechol-O-methyltransferase gene . Possible genotypes: Val/Val, Val/Met, Met/Met. Val158Met alleles are codominant.–BDNF brain neurotrophic factor gene . Possible genotypes: Val/Val, Val/Met, Met/Met.–HTR2A serotonin receptor gene ). Possible genotypes: G/G, G/A, A/A.–Genetic analysis showed that carriers of dominant (GG), heterozygous (GA), and minor (AA) genotypes of Tr2 polymorphism of the second type of serotonin receptor gene have similar genotypes for Tr3 polymorphism . From now on, we will use the designation of genotypes by Tr2 polymorphism (A/G), implying the presence of a carrier of a similar genotype by Tr3 polymorphism.The genotyping resulted in the following sample composition: COMT gene, 25 participants  were carriers of Val/Val genotype, 41 – Val/Met  and 7 – Met/Met  genotypes; HTR2A gene, 19 participants were carriers of A/A genotype (26%), 29 – A/G  and 25 – G/G ; BDNF gene, 46 participants were carriers of Val/Val genotype (63%), 26 – Val/Met  and 1 – Met/Met .Extended data.The stimuli database (2) was on the monitor all the time of trials. Images were presented in center of screen with duration of 500 ms. The interstimuli interval was 500–1500 ms. Stimuli pictures were presented in random order regardless of their emotional valence. Participants were asked to examine the images and indicate emotion they elicit – by pressing the corresponding key on the keyboard: “1”– negative, “2” – neutral, and “3” – positive emotion. The next image appeared on the screen only after the subject evaluated the previous one and the interval between stimuli was completed. Additionally, participants were instructed to blink only when stimulus ended.Participants were seated in good lit room at viewing distance of 100 cm in front of the monitor. A grey background  recording was carried out using Neurovision-136 equipment manufactured by «MKS» (Russia), with a discretization frequency of 1000 Hz. ERPs were recorded at 128 leads, using «5–10» system, and with two ear referents. Bandpass filter of 0.5-50 Hz was applied.COMT, BDNF, andHTR2A genes, e.g. inCOMT group ERP compared as Val/Val vs Val/Met vs Met/Met carriers when they chose negative, neutral or positive answer, etc. All visualization was conducted in EEGLAB.The frequencies of emotionally different responses were processed using the REdaS Package for R, version 0.9.3., in order to calculate confidence intervals for averaged response rate (α = 0.05) . The ERPCOMT, BDNF andHTR2A genes polymorphisms corresponded to the Hardy-Weinberg distribution. The estimation of the distribution of genotype frequencies for all loci, as well as the positions of the loci, are presented below inThe results of estimating the frequency distribution of alleles and genotypes of theBehavioral results. The calculation of confidence intervals for assessing relative frequencies of differential responses to emotiogenic stimuli resulted in the following outcomes for participants withCOMT genotypes. Participants with different genotypes of theCOMT gene selected negative responses to the stimuli with approximately equal average frequency, as illustrated inParticipants with the Met/Met genotype reliably responded with the neutral reaction 10% more often than participants with Val/Met and 15% more often than Val/Val genotype carriers. Also, the same Met/Met subgroup of participants gave on average 10% and 13% fewer positive answers, respectively.ERP data analysis. The analysis of ERPs in response to images of different emotional modality showed the following. When respondents viewed the presented image as emotionally negative, the amplitude of the ERP in the parietal and occipital regions of the brain (range 150 – 250 ms) in the subgroup with the Met/Met genotype was significantly lower  than in the Val/Met subgroup  and Val/Val (p <0.001 t = -3.59) subgroups of participants . Also, iThe topography of the potentials distribution also shows that the main changes in brain activity associated with the selection of neutral responses appear in the parieto-occipital regions of the cortex, as is the case with the selection of negative responses . NeverthCOMT gene, the amplitude of ERP wave in the range from 199 to 210 ms was significantly higher than in the Val/Met subgroup  and Val/Val (p <0.05 t = -0.28 ) subgroup  and G/G (p <0.022 t = -2.42) genotypes. At the same time, the ERP in subgroups of participants with the A/G and G/G genotypes, when compared with each other, did not have any significant differences , and their amplitudes differed very slightly  and G/G (p < 0.008 t = -2.81) subgroups. The ERP of the A/G and G/G subgroups remained positive, and when comparing between these two subgroups, the corresponding potentials did not show significant differences  . ExampleThe topography of the distribution of ERPs for the latency of 400 ms associated with a neutral response showed a picture similar to the situation of choosing a negative response, namely spatially uniform and higher positivity in the parietal-occipital and posterior temporal regions in subgroups with the genotypes A/G and G/G, compared with the increasing negativity in the A/A subgroup in the same brain regions .When the visual stimuli were evaluated as emotionally positive, the amplitude of the corresponding ERPs in the latency from 370 to 405 ms in participants with the A/A genotype showed increasing negativity, significantly different from that in subgroups with the A/G  and G/G  genotypes. Comparison between the A/G and G/G subgroups revealed no significant differences , in terms of the amplitude of the indicated latency . It is uThe topography of the distribution of ERP for the latency of 400 ms associated with positive responses showed a picture similar to the two previous situations. The parieto-occipital and posterior temporal regions showed higher positive amplitude of event-related potentials in subgroups with the A/G and G/G genotypes compared to the A/A subgroup, which had the increased negativity in these brain regions .HTR2A gene.Presented below are the amplitudes of ERPs at the Oz lead that exemplify selection of different response options when evaluating stimuli by respondents with different genotypes of theBDNF gene as only these subgroups were sufficiently represented in the sample. Participants with the indicated genotypes on average selected negative and positive responses to the presented stimuli with an approximately equal frequency, as illustrated inThe calculation of confidence intervals for assessing the frequency of responses different in their emotional valence was carried out only for the Val/Val and Val/Met subgroups of theBDNF subgroup was also performed only for participants with the Val/Val and Val/Met genotypes. When the presented images were evaluated negatively emotionally charged, the amplitude of the ERPs in the parietal-occipital areas within in the 180-195 ms latency for the Val/Val subgroup was significantly higher  than for the Val/Met subgroup  than for the subgroup with the Val/Met genotype   . ExampleBDNF gene , Val158Met, is involved in the catecholamine system associated with the perception of emotions. Dopamine in areas, such as the prefrontal cortex, tonsil and striatum, modulates brain activity in response to aversive stimuli and regulates the processing of emotions. For example, it was found that theCOMT Val158Met gene genotype is associated with the success of the detection of emotions, and subsequent perception of the emotions’ valence , potentially associated with the distortion of the effectiveness of post-transcriptional processes, is considered in the medical literature to be a risk factor for cognitive pathologies. A polymorphism rs6311, located in close proximity to the promoter region of the gene, is associated with changes in the expression of theHTR2A gene (The receptor for 5-hydroxytripamine (serotonin) 2A (5-HTR2A) is a key receptor involved in monoaminergic regulation of the basic biological functions of the body and of human behavior. A polymorphic version of theHTR2A gene are associated with individual differences in the manifestation of empathy and in experiencing happiness. Brain structures responsible for providing a mental model of empathizing with other people, include the medial prefrontal cortex, as well as the temporoparietal region. It was also found that participants with the dominant homozygous and heterozygous GG genotypes of theHTR2A gene experienced more pronounced feelings of happiness and greater activation of a part of the mentalization network, than people with the minor homozygous AA genotype.The MRI data obtained in the work of .In our present work, when participants evaluated emotional valence presented to them via visual stimuli, there was no statistically significant difference in the frequency of assessing these stimuli as neutral, positive or negative in those with different genotypes of theHTR2A gene, there was a statistically significant increase in negativity in the parieto-occipital regions (the time range of 350–420 ms) in comparison with participants who carried genotypes A/G and G/G of theHTR2A gene. This latter finding can be interpreted in support of the fact that decision-making in evaluating visual stimuli in participants with the A/A genotype of theHTR2A gene occurs more intensively (compared with subjects with the A/G and G/G genotypes), regardless of what response regarding emotional valence of a particular stimulus was selected by the participants.Analysis of the ERPs recorded while assessing emotional valence of presented visual stimuli found that in participants with the A/A genotype of theExisting studies that feature data from the post-mortem analysis of the brain of mentally healthy people found that carriers of different alleles of the T102C polymorphic marker differ in the amount of 5HTR2A mRNA and synthesized on its basis protein product, where the carriers of the A/A genotype showed a lower level of the gene expression .The interactions between genes and the environment lead to changes in the brain, depending on emotions- and behavior-transforming experiences. A positive interaction with the environment through new experiences and physical activity can engage the neuroplasticity mechanisms to improve brain functioning. Adult neurogenesis and brain neurotrophic factor (BDNF) serve as mediators of neuroplasticity , more carefully process the details of the visual stimuli, which manifests itself as an increase in activation of the parietal-occipital zones , as it is reflected in their responses to the images estimated to be emotionally negative or neutral.The obtained data can be interpreted as indicating that individuals with high cortical plasticity . In those with the dominant homozygous Val/Val genotype of theCOMT gene, a higher amplitude of the ERPs was observed for the stimuli assessed as either neutral or negative . The same tendency, but to a lesser extent, was detected for the study participants with the Val/Met heterozygous genotype of theCOMT gene.A stronger reaction, as reflected in the amplitude of the ERPs, in participants with the recessive homozygous Met/Met genotype of theHTR2A gene .There were no statistically significant differences in the frequency of evaluating visually presented stimuli as either emotionally neutral, positive, or negative, as reflected in responses of the participants with different genotypes of theHTR2A gene, compared to participants with the heterozygous genotype A/G and the dominant homozygous G/G genotype of theHTR2A gene.Analysis of the ERPs associated with participants’ evaluation of presented visual stimuli as emotionally negative, neutral, or positive revealed a statistically significant increase in negativity in the parietal-occipital regions in the range of 350–420 ms in participants with the recessive homozygous A/A genotype of theBDNF gene) chose neutral responses significantly more often than participants with reduced cortical plasticity .When the study participants evaluated emotional valence of visually presented stimuli, those with high cortical plasticity , processed the visual images more thoroughly, as reflected in greater activation of the parietal-occipital zones , which is shown to be typical for people with the homozygous Val/Val genotype when they evaluate emotional valence of visual information.Participants with high cortical plasticity .Data is made available under the terms of the I don't have any comments.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:behavioral geneticsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The authors took into account the corrections proposed by me and made addition to the text of the article. I think the article is worthy of indexing. Good luck!Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Psychophysiology, experimental psychology, general psychologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The work is devoted to the actual problem of reflection of evoked potentials in the parameters of the process of evaluating visual scenes as causing positive, negative or neutral emotions in respondents. The analysis was carried out taking into account the representation of different genotypes in the study participants for the COMT, BDNF and HTR2A genes.Describe in detail the characteristics of the study sample .It is desirable to describe the database of stimulus images  that was used in the work – what types of visual images were used. The work is characterized by novelty, relevance and from a scientific point of view is quite interesting.  I recommend indexing it. Comments:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Psychophysiology, experimental psychology, general psychologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thank you for your report! 1.  Correction was made in the text (see "Participant") 2.  Correction was made in the text (see "Procedure" section) In the Introduction  it is stated “… in carriers of polymorphisms of COMT, HTR2A, BDNF genes.” From a genetics point of view, this sentence is incorrect, since everyone has genetic polymorphism, the authors should rephrase this sentence.In the Introduction  it is stated “As a result, carriers of the Met allele have a thicker cortical layer in these particular areas.” Although it is indicated later, what SNP the authors selected for the analysis, its designation , and functional significance have to be reported in the Introduction section. In addition, it is well documented that Val158Met alleles are codominant. Please, indicate this information also.There is a mistake in the Introduction  “In the 5HTR2A gene, a 1438A/G polymorphism located in the promoter region is possible…”. Please report “-1438A/G”, since this SNP is located in the promoter gene region. Although, mention the data regarding on association of HTR2A allele with reduced/enhanced expression.In Materials and Methods  the authors have to transliterate or to present unique designations for the Russian phrases ≪Биологические решения и технологии≫,ООО “НекстБио”, ≪Терцик≫, ДНК-Технология, Литех, МКС.In Materials and Methods it is indicated that the authors conducted real-time PCR using “Tertsik” DNA-analyzer; however, this PCR machine is not used for Real-time PCR. The authors should report the correct DNA Analyzer and the appropriate determination of genotyping. It remains unclear, why the authors did used GelDoc UV-transilluminator for TaqMan-based fluorescent PCR. They have to use Real-time DNA Analyzer.In Materials and Methods Section please provide information regarding to % of women, mean age, since sex differences may modulate recognition of emotions.In Materials and Methods Section  the authors use the term “mutation” for the studied SNPs instead of polymorphism or SNP.In Materials and Methods Section  the authors should indicate genotype frequency of the observed genotypes.Statistical analysis should be based on the appropriate statistical criterion. Since the authors deal with quantitative data (the amplitude of the ERP), the use of unpaired Student t-test requires the testing for the normality distribution and the assumption of the homogeneity of variance. Assuming small sample size (N = 73), the normality of distribution is unlikely. Please, provide required normality of distribution testing or in the case of its failure, use non-parametric statistical tests. In addition, operations with genetic data  require the check for the Hardy-Weinberg disequilibrium. Please, report this information.I suggest to include BDNF Met/Met-genotype together with Met/Val-genotype into one group  for the statistical analysis.The study sample is rather small (N = 73), therefore I suggest to indicate a preliminary character of conducted study and to mention this issue in Limitations.The text has to be checked for the sequence of tenses. The article can be indexed after all corrections .The present article is devoted to the analysis of differences in ERP parameters depending on genetic differences  during facial recognition. The idea is interesting, however, some issues occur:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:behavioral geneticsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Thank you for your report!Question 1-8: Corrections made in text of the article according to report.Question 9: Hardy-Weinberg equilibrium verification results were added in the "Procedure" section. There are table 1 with frequency distribution and text with description for it.  Concerning about ERP statistics and normality distribution. Usually in articles with ERP method,such a question is not raised, since the technique itself, due to the number of trials (recomended more >25) and a subsuqent averaging, assumes a normal distribution of event-related potentials. For example, in our case the quantity of numeriacal data is very lagre  Therefore, researchers are fairly free to use parametric methods without checking data for normality. This is why a T-test was used. However, a table of Shapiro Wilk normality test results for latencies at which differences were found are attached.            Type of response Shapiro-Wilk test statistics and p-value on latency (ms)    200             300            400                  Negative W = 0,97    W = 0,96    W = 0,98 p = 0,16      p = 0,07     p = 0,41                   Neutral W = 0,98   W = 0,97    W = 0,97 p = 0,36     p = 0,17     p = 0,26                  Positive W = 0,97   W = 0,99    W = 0,99 p = 0,11     p = 0.95      p = 0,98 As seen in example - all data in this latency for 73 participants and 3 responses type are normaly distributed (p>0.05)Question 10: Since there was only one Met\Met genotype carriers, his\here results were excluded from research. There is a note about this in the text, in the begging of section "Results / BDNF".Question 11: "Limitations" section was added in end of the article. Two majors limitations were described in the section, and futher direction of research was added.Question 12: Corrections made in text of the article.


Small amounts of glycidylisobutyl polyhedral oligomericsilsesquioxane (G-POSS) (up to 10 phr) were added into a immiscible polyamide12 (PA12)/polyolefin elastomer (POE) blend (70 wt%/30 wt%) by simple melt mixing. The effects of the G-POSS on phase morphology and mechanical properties were investigated by FE-SEM, tensile testing, Izod impact test and dynamic mechanical analysis. FE-SEM analysis revealed that domain size of the dispersed POE phase in matrix PA12 is decreased significantly by adding the G-POSS, indicating a compatibilization effect of the G-POSS for the immiscible PA12/POE blend. The PA12/POE blend compatibilized with POSS showed simultaneous enhancement in mechanical properties including tensile modulus, strength and toughness. Further, thermally triggered shape memory effect was observed in this compatibilized blend. Polymer blending is an efficient and simple way to develop new polymeric materials possessing desirable physical properties and specific functionalities for various end-use applications. Most of the polymer blends are immiscible and need proper compatibilization in order to obtain proper phase separated morphologies which determine the performance properties. The compatibilization induces finer phase morphology by improving interfacial adhesion between the component polymers in the blend, which promotes their synergistic combination ,2.Blends of polyamides (PA) with polyolefins (PO) are important class of polymer blends because of their balanced properties of strength, toughness and moisture resistance. For the compatibizations of the immiscible PA/PO blends, functionalized polyolefins, such as maleic anhydride (MA)-grafted polyolefins, have been investigated ,5,6,7,8.8O12) with additional organic functional groups covalently bonded to each vertex Si on the cage. Due to the organic functional groups, the POSS cage is naturally compatible with polymers, and can be chemically bonded to polymer chain [Among various inorganic nanofillers, polyhedral oligomeric silsesquioxane (POSS) is unique which features a well-defined nanosized Si–O cage structure  is one of the important engineering plastics with a broad range of applications including oil resistant tubes for automotive, cables, food packaging films and powder coatings for metals. For the improvement in toughness of the PA12, blending it with various rubbers such as styrene/ethylene-butylene/styrene block (SEBS) rubber , epoxidi2 or –COOH) of PA12 during melt mixing and van der Waals interaction of isobutyl groups with POE, respectively. The compatibilization and reinforcing effects of the G-POSS for the PA12/POE (70 wt%/30 wt%) blend were investigated by observation of phase morphology and mechanical properties. A thermally-triggered shape memory effect of this blend is also reported.Hence, this work employed glycidylisobutyl POSS (G-POSS) having a cage structure with one glycidyl and seven isobutyl groups at each corner of the cage  as a comPA12 (Rilsan AESNO TL) was purchased from Arkema . POE  was obtained from Dow Chemical Co., USA. Glycidylisobutyl POSS  was purchased from Hybrid Plastics Co .PA12/POE (70 wt%/30 wt%) blends with G-POSS content of up to 10 part per hundred (phr) of the polymers were prepared by melt mixing at 200 °C in a Haake internal mixer equipped with a cam rotor  at a rotor speed of 60 rpm. PA12 was first melted for 1 min followed by addition of POE and mixed for another 1 min, then desired amount of G-POSS was loaded and mixed into the mixture and mixing continued for 10 min till the mixing torque was stabilized. The obtained PA12/POE/G-POSS mixture was formed into a sheet by compression molding at 200 °C in an electrically heated press  for the property measurements.Phase morphologies of the blend were investigated by a field emission-scanning electron microscope  at an accelerating voltage of 15 kV. To avoid ductile deformation during fracture, the samples were chilled in liquid nitrogen before breaking to initiate a brittle fracture. The cryogenically fractured surface was sputter-coated with platinum prior to SEM observation.Tensile properties were measured at 25 °C using a universal testing machine  at a crosshead speed of 50 mm/min with an initial gauge length of 20 mm. At least five specimens were used for the test. Elastic moduli were obtained from the initial slope of the stress–strain curves (up to about 1% strain). Tensile toughness of each sample was obtained by calculating the area of the stress–strain curves.Notched Izod impact strength was measured using a sample with a thickness of 4 mm and width of 10 mm at 25 °C using an impact tester  according to ISO 180. The test was conducted using 10 specimens, and the average values are presented here.−1 from −100 to 200 °C.Dynamic storage modulus and tan δ as a function of temperature were determined using a dynamic mechanical analyzer  under a cyclic tensile strain with an amplitude of 10% at a frequency of 1 Hz. The temperature increased at a heating rate of 2 °C minTg and well below Tm of the blend) followed by cooling the deformed shape to 0 °C was heated to 70 °C. The shape fixing ratio (Rf) and shape recovery ratio (Rr) of samples were determined by following equations, respectively.m is an initial strain imposed onto the sample (50% in this case), εu is a strain measured upon cooling the deformed sample at 0 °C and εp is a strain measured upon heating the temporarily deformed shape at 70 °C.In order to evaluate thermally-triggered shape memory behavior of the samples, temporarily-fixed sample obtained by uniaxial deformation of dogbone-shaped specimen with a thickness of ca. 1 mm to 50% at 70 °C  and high-speed sintering (HSS) for precise manufacturing of parts with complicated shape ,39. FurtThis study demonstrated that a POSS having both a glycidyl group and several alkyl groups on its cage can act as a compatibilizer for an immiscible PA12/POE (70 wt%/30 wt%) blend system. The incorporation of a small amount of G-POSS reduced the dispersed POE domain size in PA12 matrix of the blend. The combination of the decreased rubber particle size and the presence of rigid POSS nanoparticles in the blend led to interesting simultaneous improvement in modulus and toughness. Moreover, the PA12/POE blend embedded with POSS represented excellent thermally triggered shape memory effect. The PA12 blends with good mechanical properties and shape memory effects may have diverse applications such as smart packaging films, sensors, fast deployable and actuating devices. Further studies on the use of this material for modern processing technologies will be performed.


This process is essential for resolving polycistronic pre-mRNAs produced by eukaryotic operons into monocistronic transcripts. SL trans-splicing and operons may have independently evolved multiple times throughout Eukarya, yet our understanding of these phenomena is limited to only a few well-characterised organisms, most notably C. elegans and trypanosomes. The primary barrier to systematic discovery and characterisation of SL trans-splicing and operons is the lack of computational tools for exploiting the surge of transcriptomic and genomic resources for a wide range of eukaryotes.Spliced leader (SL) bona fide SL RNA molecules. SLOPPR identifies RNA-Seq reads that contain a given 5′ SL sequence, quantifies genome-wide SL trans-splicing events and predicts operons via distinct patterns of SL trans-splicing events across adjacent genes. We tested both pipelines with organisms known to carry out SL trans-splicing and organise their genes into operons, and demonstrate that (1) SLIDR correctly detects expected SLs and often discovers novel SL variants; (2) SLOPPR correctly identifies functionally specialised SLs, correctly predicts known operons and detects plausible novel operons.Here we present two novel pipelines that automate the discovery of SLs and the prediction of operons in eukaryotic genomes from RNA-Seq data. SLIDR assembles putative SLs from 5′ read tails present after read alignment to a reference genome or transcriptome, which are then verified by interrogating corresponding SL RNA genes for sequence motifs expected in trans-splicing and operons throughout Eukarya and improve gene discovery and annotation for a wide range of eukaryotic genomes. Both pipelines are implemented in Bash and R and are built upon readily available software commonly installed on most bioinformatics servers. Biological insight can be gleaned even from sparse, low-coverage datasets, implying that an untapped wealth of information can be retrieved from existing RNA-Seq datasets as well as from novel full-isoform sequencing protocols as they become more widely available.SLIDR and SLOPPR are flexible tools that will accelerate research into the evolutionary dynamics of SL The online version contains supplementary material available at 10.1186/s12859-021-04009-7. Thise genome  and has e genome , 4, 5. Snd fungi –9, thougnd fungi .trans-splicing is the processing of polycistronic pre-mRNAs generated from eukaryotic operons [trans-splicing machinery coordinates cleavage of polycistronic pre-mRNA and provides the essential cap to the resulting un-capped monocistronic pre-mRNAs [C. elegans, which possesses two types of SL [One clear biological function of SL  operons . In cont operons . The trare-mRNAs , 13. Thies of SL : SL1, whes of SL –14.Schistosoma mansoni and the tunicates Ciona intestinalis and Oikopleura dioica each possess only a single SL, which is used to resolve polycistronic mRNAs but is also added to monocistronic transcripts [Spadella cephaloptera and the cnidarian Hydra vulgaris splice a diverse set of SLs to both monocistronic and polycistronic transcripts [trans-splicing an obligatory component of gene expression [trans-splicing for all nuclear mRNAs, but only a subset of genes are organised as operons [trans-splicing also occurs in many other organisms including rotifers, copepods, amphipods, ctenophores, cryptomonads and hexactinellid sponges, operons and polycistronic mRNAs have not been reported in these groups [The same SL2-type specialisation of some SLs for resolving downstream genes in operons exists in many other nematodes –20 but inscripts –23. Siminscripts , 25. Rempression . In cont operons , 28. Alte groups , 30.trans-splicing machinery and its role in facilitating polycistronic gene expression and broader RNA processing. One barrier in dissecting the evolutionary history of these phenomena is the difficulty in systematically discovering novel SLs. Identifying the full SL repertoire of an organism would traditionally require laborious low-throughput cloning-based Sanger sequencing of the 5′ ends of mRNAs . Hi. Hitranss  after alignment to a genome or transcriptome reference . Unlike em loops , 38. Thesplicing , 39. ForCiona intestinalis [Hydra vulgaris [RNA-Seq reads are first aligned to the genome or transcriptome reference using HISAT2  or BOWTIstinalis  or 46 bpvulgaris . Tails fvulgaris .The extracted tails are then dereplicated, 3′-aligned and clustered at 100% sequence identity using VSEARCH . Each reSm binding  sites, and secondary structure stem loops are predicted using RNAFOLD from the ViennaRNA package [Sm motif 20–60 bp downstream of the splice donor site [trans-splice acceptor site of the gene annotation.SLOPPR is designed as a genome annotation tool that predicts operons from genome-wide distributions of SL SL-quant . The opeSL-quant , 22, 51,gle gene , 52, pavtrans-spliced 5′ SL tail, all reads are first aligned end-to-end to the genome reference using HISAT2 [RNA-Seq reads containing SLs are identified using a three-step strategy . Since sg HISAT2  and unalg HISAT2 . The 5′ g HISAT2 . Finallyg HISAT2 . Likewistrans-splicing process means that SLs must only be present at the first exon of a gene, i.e. the 5′ end. We can thus identify problematic gene annotations that consist of fused operonic genes via internal exons that receive SL reads [The nature of the SL SL reads , 52. SLOSL reads , unique SL reads  and SL rtrans-splicing events allows for identifying the distinct trans-splicing patterns of SL2-type SLs expected from their specialisation for resolving downstream operonic genes. If SL2-type SLs are not known, K-means clustering and linear discriminant analysis are used to assign SLs to one of two synthetic clusters assumed to correspond to SL1-type and SL2-type SLs  based on the background gene counts . The nortrans-splicing , the SL2:SL1 CPM ratio is computed and summarised across all genes that receive both SL types. The operon prediction algorithm is based on finding tracts of adjacent genes with SL2-bias, which are designated as downstream operonic genes  and non-operonic genes. In consequence, by manually specifying SL1/SL2-type SLs, SL2:SL1 ratio cut-off, upstream gene SL2-type bias and intercistronic distance cut-off, a large gamut of relationships between SLs and operonic genes can be explored, even in situations where no subfunctionalisation of SLs for operon resolution exists, for example in kinetoplastids or tunicates [Finally, intercistronic distances among the predicted operonic genes are computed and compared to genome-wide intergenic distances to diagnose tight physical clustering of operonic genes Fig. e. These unicates –23.C. elegans, Ciona intestinalis, Hydra vulgaris and Schistosoma mansoni, and using the same reference genomes and RNASeq datasets that were used in benchmarking SLFinder [trans-spliced (SL trans-splicing rate) and whether any novel SL candidates were identified.We first compared the accuracy of SLIDR against that of SLFinder , focusinSLFinder . All RNASLFinder , trimminC. elegans . SLIDR also identified all eleven SL2 sequence variants from as few as 28 reads and detected 18 SL RNA genes, which correspond to all 18 functional sls-2 genes and correctly omit the sls-2.19 pseudogene  despite very high genome alignment rates of 93–95%. The SL sequence contained extra 5′ nucleotides supported by the minority of reads (5′-taaggcATTCTATTTGAATAAG-3′). All but one of the eleven SL RNA genes identified by SLIDR were on chromosome NC_020175.2 (one was on NC_020166.2), and all were part of a 264 bp repeat unit that contains functional SL copies in the genome . In compSm motif, which identified the same SL at identical coverage but yielded > 50 additional SL RNA genes (likely pseudogenes)  is 24 bp long and is part of an 80 bp SL RNA [Sm binding sites differ between SL-A (5′-GAUUUUCGG-3′) and all other SLs (5′-AAUUUUGA-3′ or 5′-AAUUUUCG-3′) [Sm binding site motifs and allow for detecting both the short and long SLs.The cnidarian p SL RNA , whereasp SL RNA . The Sm UUCG-3′) . We ran SLIDR detected the full SL-B1 sequence from 865,003 reads spliced to 18,768 genes and identified two SL RNA genes. SLIDR also detected two 5′ truncated versions of SL-D (encoded by two SL RNA genes) and two potential novel SL variants at much lower coverage  SL with an unusually long Sm binding site (5′-AGUUUUCUUUGG-3′) and a total SL RNA length of 90 bp [trans-splicing by this SL [Sm binding site.The platyhelminth of 90 bp . The tra this SL . SLIDR wtrans-spliced, which exceeds the 2,459 genes previously identified from large-scale RNA-Seq data  supported by 41,243 reads, and two 5′-truncated SL variants supported by only 3 reads each  but faling rate .SLFinder assembled a single hook that missed the two 5′ A nucleotides and included three unspecific 3′ nucleotides Table . All 132C. elegans and three other eukaryotes, we then examined SLIDR’s performance (using the same criteria as above) in a range of other nematodes with well-characterised SL and SL RNA repertoires: Caenorhabditis briggsae, Pristionchus pacificus, Meloidogyne hapla, Trichinella spiralis and Trichuris muris. We also tested how SLIDR performs with a transcriptome reference instead of a genome. In this situation, SLIDR cannot confirm splice acceptor sites because these are not expected to be present in a transcriptome; however, if the transcriptome contains SL RNAs it will still be possible to find splice donor and Sm binding sites. We demonstrate this in Prionchulus punctatus using a draft de novo transcriptome assembly and compare the results against C. elegans using a well-curated transcriptome.Having validated SLIDR in As above, all RNA-Seq data underwent quality-trimming and SLIDR required the default GT/AG splice donor/acceptor sites. The results demonstrate that SLIDR detects all known SLs in all cases and often discovers novel SL variants Table . We alsoC. briggsae is a close relative of C. elegans that possesses a similar SL repertoire and shows considerable synteny of operons [Cbr-SL1 is encoded by a repetitive gene cluster , of which four are shared with C. elegans [trans-spliced [C. elegans [ operons , 18. Cbr copies; ) linked  elegans . Only 37 elegans , 56, thotrans-splicing events [Cbr-SL1  and Cbr-SL3 (17 reads spliced to 17 genes)  and five out of six full-length SL2-type SLs (Cbr-SL13 was absent) supported by up to 20,317 reads and spliced to up to 1,968 genes  and all six SL2-type SLs   and four SL2-type (Ppa-SL2) SLs, which are encoded by 187 and 16 genes respectively [Ppa-SL1a- and Ppa-SL2a-enriched RNA-Seq, about 90% of 23,693 expressed genes are SL trans-spliced [Sm binding motifs [ectively . Based o-spliced . We ran -spliced  to examig motifs .Ppa-SL1a  and little evidence of Ppa-SL2a (40 reads spliced to 16 genes). The SL1-enriched library yielded Ppa-SL1a at double the coverage  and Ppa-SL2a/m at low coverage (at most 140 reads), consistent with SL1-enrichment. However, the SL2-enriched library contained predominantly Ppa-SL1a  despite increased coverage of Ppa-SL2a , suggesting that the SL2-enrichment may not have been as effective as suggested by qPCR control experiments [Ppa-SL1 variants, all four Ppa-SL2 variants and six novel SL variants . SLIDR detected all but one of the same known SL1 and SL2 variants as before, with similar coverage and numbers of SL Ppa-SL1a and Ppa-SL2a variants [trans-splicing rate can, in part, be explained by SLIDR’s reliance on a small fraction of RNA-Seq reads that contain sufficient evidence of an SL tail at their 5′ end. Any underrepresentation of SL tails in the original libraries [trans-splicing rate estimates [trans-spliced transcripts, the SL trans-splicing rate would be overestimated. Since this is impossible to test bioinformatically with the available data, more work is required to verify the extent of SL trans-splicing in this organism.These results highlight a rich diversity of known and previously unreported SL1-type and SL2-type SLs beyond the canonical variants , 19. Theibraries . ConversM. hapla possesses the canonical C. elegans SL1 and four additional variants, all of which are trans-spliced to a minority of only 10% of 14,420 genes [Medicago truncatula. Since reads from unstranded single-end libraries originate from the 5′ end of the transcript only 50% of the time, usable coverage is effectively halved. SLIDR was run with parameters -S ’.{30,80}AT{4,6}G’ -R 90 to allow for larger variation in Sm binding motif location and a longer SL RNA.The plant-root knot nematode 20 genes . We obta20 genes . These dtrans-splicing rate of at least 40%, which suggests that the previously reported 10% is an underestimate [trans-spliced genes  are SL2-type SLs specialised for resolving downstream genes in operons [Sm binding motif to the splice donor site [The parasite 48 genes  and are 48 genes . Three o operons . We down operons . SLIDR wnor site .trans-splicing rate of at least 38%  are SL2-type SLs [trans-splicing in this organism is unknown [Sm binding motif to the splice donor site [13 genes , 67. Thrtype SLs . The gen unknown . We downnor site .SLIDR detected all 13 known SLs from 15-15,046 reads spliced to 17-3,122 genes  but only discovered two (Ppu-SL1 and Ppu-SL3) out of six known SLs, supported by only 327/353 reads and spliced to 122/63 genes respectively  and three stranded 2 × 150 bp RNA-Seq libraries. SLIDR detected SL1, all eleven SL2 variants, and all 28 functional SL RNA genes, consistent with the results obtained with a genome reference  and identified how many predicted operons matched reference operons (specificity) and how many reference operons were predicted (sensitivity). SLOPPR correctly identified SL1- and SL2-type SL subfunctionalisation in all species and identified up to 96% of known operons . We quantified SL trans-splicing events by the canonical SL1 sequence and 11 SL2 variants as supplied by the SL-quant pipeline [Up to 20% of genes in s of SL2  or are es of SL2 . We desipipeline .trans-splicing at 52% of genes, which falls short of the expected SL trans-splicing rate of 70–84% [trans-spliced, only 1% were strictly SL2 trans-spliced and 15% were trans-spliced by both SL1 and SL2  that were defined based on tight gene clusters that receive SL2 [eive SL2 . We decieive SL2 . We suppeive SL2 .trans-splicing rate of 27% and predicted 631 operons comprising 1,346 genes with a median intercistronic distance of 333 bp , of which 37 (5%) were among the 48 (77% detected) reference operons  than they did between SL1 and SL2-type SLs, which caused SLOPPR to cluster the SLs by library instead of SL type  showed no evidence of SL2-enrichment (0.98% of genes) and comparable SL1 levels to the non-enriched control library (8.2% of genes). These results echo the SLIDR results using the same libraries (see above) and would suggest a far lower SL trans-splicing rate than 90% [trans-splicing rate, only 117 operons were predicted, of which 99 (84%) matched the 6,909 operon-like gene clusters and 67 (57%) matched the 2,219 plausible reference operons [SLOPPR detected SLs at only 20% instead of 90% of genes, even when including the non-enriched library (SRR1182510). The SL1-enriched library (SRR1542610) supported SL1 and SL2 than 90% . Due to  SRR154260 showed trans-splicing at 28% of genes and predicting at most 205 operons, of which 102 (50%) matched the 2,219 reference operons , we finally aimed to illustrate that SLOPPR is also able to infer operons in organisms that lack such specialisation. Here we demonstrate this ability in two tunicates, bona fide operonic genes but will also contain all monocistronic genes that receive the SL. Therefore, these initial candidate operonic genes must be filtered by intercistronic distance to partition out true operonic genes. SLOPPR can be configured to either use a user-supplied cut-off if the expected intercistronic distances are known, or to bisect the distribution of intercistronic distances empirically into two groups using K-means clustering and retaining those genes with short distances. By exploring parameter combinations, specificity and sensitivity in partitioning out operons can be optimised  and trans-splicing sites [The tunicate ematodes , 42. Usiematodes , 42. Theematodes , 42, sim elegans . The genng sites .http://ghost.zool.kyoto-u.ac.jp/download_kh.html). We used the same 13 RNA-Seq libraries from bioproject PRJNA376667 that performed best in SLIDR  with the fraction of reads annotated as C. intestinalis, suggesting that the libraries are contaminated with other organisms  and KH operon annotations  from the Ghost database  but a distinct set of genes had very low intercistronic distances, likely representing true operons . SLOPPR predicted only 856 operons with a median intercistronic distance of 1 bp (inferred cut-off: 68 bp). Of these operons, 823 (96%) matched reference operons, indicating high specificity, but only 62% of the reference operons were detected  matched reference operons and represented 83% of reference operons  matched reference operons and 90% of the reference operons were detected . This yielded 1,128 operons, indicating that only 44 operons had intercistronic regions , and operon annotations from the Genoscope Oikopleura Genome Browser (https://wwwdev.genoscope.cns.fr/oikopleura/). We used four unstranded 2 × 90 bp libraries from bioproject PRJNA269316 and 16 stranded 2 × 100 bp libraries from bioproject PRJDB5668, representing various life stages. Similarly to C. intestinalis, we observed poor and highly variable background alignment rates (17–69%) related to poor taxonomic purity  and genome annotations from OikoBase   Fig. . Re-runnbp) Fig. . We thusbp) Fig. .trans-splicing at upstream genes (-u) across the same three analysis runs and obtained much more stringent sets of 111–165 operons of which 106–143 (87–95%) matched reference operons : LinuxProgramming language: BASH, Rdata.table, glmpca, ggdendro, MASS, ggplot2, parallel, reshape2Other requirements: CUTADAPT (tested v2.3), GFFREAD (tested v0.11.4), HISAT2 (tested v2.1.0), BOWTIE2 (tested v2.3.5), SAMTOOLS (tested v1.9), BEDTOOLS (tested v2.28.0), SEQTK (tested v1.3), VSEARCH (tested v2.15.1), BLASTN (tested v2.9.0), SUBREAD (tested v1.6.2), ViennaRNA (tested v2.4.14), R (tested v3.6.0), R packages License: Creative Commons Attribution License (CC BY 4.0)Any restrictions to use by non-academics: NoneAdditional file 1:Table S1. Detailed results from all SLIDR runs.Additional file 2:Table S2. Detailed results from all SLOPPR runs.Additional file 3: GFF3 operon annotations generated by SLOPPR.


Breast milk contains human milk oligosaccharides (HMOs), including sialyllactose (SL). SL is composed of sialic acid and lactose, and is divided into 3′-SL and 6′-SL according to the binding position. SL has immunoprotective effects against bacteria and viruses, and acts as a probiotic in the gastrointestinal tract. In this study, we developed a bioanalytical method for simultaneous analysis of 3′-SL and 6′-SL in liver and kidney tissues of Yucatan minipigs using liquid chromatography–tandem mass spectrometry (LC-MS/MS) under conditions optimized in our previous study. LC-MS/MS was performed using a hydrophilic interaction liquid chromatography (HILIC) column  with a mobile phase consisting of 10 mM ammonium acetate in water (pH 4.5) and acetonitrile with gradient elution at a flow rate of 0.3 mL/min. A surrogate matrix method using water was applied for analysis of endogenous SL. The developed method was validated with regard to selectivity, linearity, precision, accuracy, the matrix effect, recovery, parallelism, dilution integrity, carryover, and stability according to the US Food and Drug Administration guidelines. We performed a tissue distribution study of minipigs, and analyzed liver and kidney tissues using the developed method to determine the tissue distribution of 3′-SL and 6′-SL. The tissue concentrations of 3′-SL and 6′-SL were readily measurable, suggesting that the method would be useful for evaluating the tissue distributions of these compounds in minipigs. N-tetraose (DSLNT), and sialyllacto-N-tetraose (SLNT). 3′-SL and 6′-SL are endogenous compounds within the body, and the level of 3′-SL is maintained during lactation, but 6′-SL level decreases gradually in breast milk over time ) of the nominal concentration.A calibration curve was generated for the surrogate matrix at concentrations of 20, 40, 500, 1000, 5000, 10,000, and 20,000 ng/g. Linear regression for 3′-SL and 6′-SL was performed with a weighting factor of 1/xAccording to the white paper of Wakamatsu et al. , the metn = 6). The inter-day results for 3′-SL and 6′-SL ranged from −5.3% to 7.5% for accuracy and 2.9% to 10.0% for precision (n = 18).In liver tissue, the intra-day results for 3′-SL and 6′-SL ranged from −2.8% to 2.7% for accuracy and 0.5% to 7.2% for precision (n = 6). The inter-day results for 3′-SL and 6′-SL ranged from −4.7% to 7.5% for accuracy and 3.0% to 10.0% for precision (n = 18).In kidney tissue, the intra-day results for 3′-SL and 6′-SL ranged from −5.3% to 6.0% for accuracy and 1.0% to 7.2% for precision  ranged from 4.7% to 10.1% for 3′-SL, 6.9% to 27.8% for 6′-SL, and 2.0% to 7.9% for internal standard. The results of the recovery test of kidney tissue were as follows. The mean recovery ranged from 90.3% to 123.6% for 3′-SL, 91.2% to 115.7% for 6′-SL, and 95.1% to 96.2% for internal standard. Precision (n = 6) ranged from 4.2% to 4.9% for 3′-SL, 2.9% to 14.9% for 6′-SL, and 3.6% to 4.2% for internal standard.Recovery was assessed at the three QC sample concentration levels . The results of the recovery test of liver tissue were as follows. The mean recovery ranged from 60.0% to 84.8% for 3′-SL, 59.2% to 85.8% for 6′-SL, and 76.1% to 94.3% for internal standard. Precision . This was thought to be due to the background concentration of SL in the authentic matrix being higher than the spiked concentration of LQC. The recovery data are shown in n = 6) ranged from 3.6% to 12.4% for 3′-SL and 4.2% to 15.7% for 6′-SL.The matrix effect was evaluated using six individual authentic samples at the LQC, MQC, and HQC concentration levels of 60, 1500, and 15,000 ng/g, respectively. The results of the matrix effect test of liver tissue were as follows. The mean values of the internal standard-normalized matrix factor for six individual samples ranged from 0.952 to 1.15 for 3′-SL and 1.06 to 1.12 for 6′-SL. Precision (n = 6) ranged from 0.4% to 7.3% for 3′-SL and 1.3% to 20.9% for 6′-SL.The results of the matrix effect test of kidney tissue are as follows. The mean values of internal standard-normalized matrix factor for six individual samples ranged from 0.853 to 0.993 for 3′-SL and 0.889 to 0.996 for 6′-SL. Precision  and kidney (20.9%). As in the recovery evaluation, this was thought to be because variation in the background concentration of SL in the authentic matrix affects that of the LQC. The results for the matrix effect are shown in y = 0.000119x + 0.0386 and y = 0.0000474x + 0.00556 in the liver, and y = 0.000114x + 0.0756 and y = 0.0000416x + 0.00245 in the kidney, respectively. The background concentration in the authentic matrix was 324 ng/g for 3′-SL and 117 ng/g for 6′-SL in the liver, and 66.3 ng/g for 3′-SL and 58.9 ng/g for 6′-SL in the kidney.The background concentration in the authentic matrix was calculated from the x-intercept of the calibration curve constructed with addition of internal standard to the authentic matrix. The calibration curve equations for 3′-SL and 6′-SL were n = 6) values for 3′-SL and 6′-SL ranged from 0.4% to 5.6% for the liver and 0.7% to 7.9% for the kidney. The results of the parallelism tests are shown in The three QC samples  in the authentic matrix were analyzed with six replicates and substituted into the calibration curve of the surrogate matrix. Accuracy and precision were assessed by comparing the measured concentrations with the nominal concentrations added to the background concentration in the authentic matrix. The accuracy (RE) of 3′-SL and 6′-SL ranged from −7.2% to −4.6% for the liver and −14.8% to −6.6% for the kidney. Precision  followed by the blank sample in the surrogate matrix. No peaks were observed at the retention time of 3′-SL and internal standard in the blank sample. The peak area in the blank sample was 2.1% of that in the LLOQ sample at the retention time of 6′-SL. These results satisfied the acceptance criterion, i.e., that the peak area at the retention time of SL should be <20% of that of the LLOQ sample.n = 6) was 0.6% for 3′-SL and 1.5% for 6′-SL in the liver, and 0.5% for 3′-SL and 1.2% for 6′-SL in the kidney. These results satisfied the ± 15% acceptance criterion, suggesting that samples with concentrations exceeding the ULOQ may be diluted prior to the analysis.If the concentration exceeded the ULOQ, it would be necessary to dilute the sample with blank matrix. Dilution DQCs above the ULOQ were prepared in six replicates in the blank tissue homogenate at a concentration of 100,000 ng/g. Dilution integrity was evaluated based on the accuracy and precision of the samples diluted 10 times with the surrogate matrix (water). The accuracy (RE) was −8.0% for 3′-SL and 12.0% for 6′-SL in the liver, and −9.0% for 3′-SL and 14.0% for 6′-SL in the kidney. Precision  for 4 h using samples with long-term stability, i.e., those stored at −80 °C for 34 days. All QC samples of 3′-SL and 6′-SL showed stability values of −7.6% to 3.7% for liver and −10.1% to −6.0% for kidney. The post-preparative stability (RE) of samples after 21 h of storage at 10 °C in an autosampler tray ranged from –3.1% to 8.7% for liver QC samples and −10.2% to 4.7% for kidney QC samples of 3′-SL and 6′-SL. To investigate freeze/thaw stability, QC samples were frozen at −80 °C and thawed at room temperature over four cycles. The stability of all QC samples of 3′-SL and 6′-SL ranged from −6.7% to 7.6% for the liver and −9.3% to −3.2% for the kidney. All stability results satisfied the acceptance criterion of ±15% of the nominal concentration. A tissue distribution study was conducted involving intravenous injection of minipigs with 3′-SL and 6′-SL at a dose of 12.5 mg/kg. Kidney, liver, and blood samples were collected. The tissue concentrations and pharmacokinetic parameters are shown in The results indicate 3′-SL and 6′-SL were rapidly distributed to the kidney and liver tissue following intravenous gavage. In addition, the T/P ratio of the kidney was 1.0–1.3 fold and that of the liver was 0.12–0.14 fold, indicating that 3′-SL and 6′-SL were shown to be primarily eliminated by the kidney via urine excretion. The exposure of tissues to 6′-SL was approximately 20% higher than that of 3′-SL, with a 6′-SL concentration of 3030 ng/g in the liver and 27,000 ng/g in the kidney. The 3′-SL levels were 2550 ng/g in the liver and 22,300 ng/g in the kidney. However, the difference between 3′-SL and 6′-SL was not found to be based on tissue distribution.These results provide useful information for further studies on the tissue distribution of 3′-SL and 6′-SL. Additionally, the main excretion route requires elucidation by monitoring the amount of 3′-SL and 6′-SL in bile or urine over time.13C3]3′-sialyl[3 -13Cglc] lactose sodium salt, was purchased from Omicron Biomedicals, Inc. .Standards of 3′-SL (purity 98.55%) and 6′-SL (purity 98.75%) were provided by GeneChem Inc. . Methanol and acetonitrile (HPLC grade) were purchased from Burdick & Jackson . Acetic acid (HPLC grade) was purchased from Sigma-Aldrich . Ammonium acetate (HPLC grade) was purchased from Fluka . Deionized water was obtained using a Millipore Elix and Milli-Q Biocel system . An internal standard, 3′-Sialyl[3-13Cglc] lactose sodium salt, and the stock solution was prepared by dissolving in water at a concentration of 0.1 mg/mL. Water was used as a surrogate matrix for minipig tissue to prepare the calibration curve and low quality control (LQC). Samples for the calibration curve were prepared by adding 5 μL of suitable working solution to 95 μL of the surrogate matrix, with final concentrations of 20, 40, 500, 1000, 5000, 10,000, and 20,000 ng/g for both 3′-SL and 6′-SL. LQC samples (60 ng/g) were prepared using the surrogate matrix, and middle quality control  and high quality control  samples were prepared using homogenized minipig tissues.Standard stock solutions of 3′-SL and 6′-SL were prepared separately by dissolving each compound in water at a concentration of 1 mg/mL. Working solutions for 3′-SL and 6′-SL were prepared by serially diluting the stock solutions with water. The internal standard was prepared using a stable isotope-substituted sialyllactose [1,2,3-g for 10 min at 10 °C. The supernatant (400 μL) was transferred to fresh polypropylene tubes and evaporated at 45 °C for 90 min using a speed vacuum . The residue was reconstituted with 100 μL of a mixture of 10 mM ammonium acetate in water (pH 4.5) and acetonitrile , and vortexed for 10 min. An aliquot of 2 μL was injected into the LC-MS/MS system.Approximately 1 g of minced liver or kidney tissue was weighed and transferred into a polypropylene tube, after which 9 mL of water was added. The mixture was thoroughly homogenized . Aliquots of 40 μL of the homogenized minipig tissue samples were placed in polypropylene tubes with 40 μL of internal standard solution (1000 ng/mL in water) and 500 μL of methanol. The mixture was then vortexed for 10 min and centrifuged at 16,363 × The method developed here was validated in terms of selectivity, linearity, precision, accuracy, the matrix effect, recovery, parallelism, dilution integrity, carryover, and stability according to the FDA guidelines .The selectivity of an analytical method is usually evaluated by comparing the chromatograms of the blank samples from individual sources and standard fortified samples to determine the presence of interference at the retention time of the analyte. However, as SL is an endogenous compound, selectivity was evaluated in a different way. Two qualification ion transitions were established, and the peak area ratios between the qualified and quantified ion transitions of SL were calculated. The ratios of peak area between the HQC sample in the surrogate matrix and the authentic liver and kidney tissue samples were compared and evaluated for equivalence, as described previously . The 3′-To prepare a calibration curve, samples were made at seven concentrations in the surrogate matrix and consisted of double blank , zero blank , 20, 40, 500, 1000, 5000, 10,000, and 20,000 ng/g. The calibration curve was acceptable when the concentration of each calibrator was within ±15% (±20% for the lower limit of quantification (LLOQ)) of the nominal concentration, and when at least 75% of the calibrator, including the LLOQ and upper limit of quantification (ULOQ), met the acceptance criteria.The intra-day and inter-day accuracy and precision were tested at the LLOQ (20 ng/g), and for three QC concentrations . Accuracy and precision are expressed as the relative error  and coefficient of variation , respectively. According to the FDA guidelines, accuracy and precision should be within ±15% (±20% for LLOQ) and <15% (20% for LLOQ).The intra-day test was performed with six replicates at each QC level within 1 day. The inter-day test was performed by repeating the intra-day test for three days. LLOQ and LQC samples were prepared using a surrogate matrix. For the MQC and HQC samples, the background concentration of SL was determined by analyzing the homogenized blank minipig tissue sample, and the background concentration was subtracted from the measured concentrations . The accRelative error (%) = (Measured conc. of QC − Background conc.)/Nominal conc. × 100.A recovery test was performed on the three QC samples  with six replicates per level. Recovery was evaluated by comparing the peak area of a blank sample fortified with a known amount of analyte after sample preparation and the peak area of the sample fortified with the analyte before extraction. The final peak area was calculated by subtracting the peak area of background SL in the authentic matrix. The precision  of recovery should be within 15%.The matrix effect refers to the enhancement or suppression of a chromatography signal by interference or co-eluting compounds in the matrix. In this study, the matrix effect was evaluated at 60, 1500, and 15,000 ng/g by an internal standard-normalized matrix factor, which was calculated as the matrix factor of the analyte divided by that of the internal standard. The matrix factor was calculated as the peak area of six individual blank samples fortified with a known analyte after sample extraction divided by that of standard solution. The peak area of analyte in the authentic matrix was calculated by subtracting the peak area of background SL. The precision  of the internal standard-normalized matrix factor among six individual samples at each QC should be within 15%.Parallelism shows that the calibration curve in the authentic matrix is parallel to that in the surrogate matrix. The FDA guidelines specify that parallelism should be evaluated for endogenous compounds . In prevThe carryover test was performed to evaluate whether the previous sample affects the response of the next sample. Carryover was assessed by analyzing the ULOQ sample followed by a blank sample in the surrogate matrix. In the blank sample, the peak area at the retention time of SL should be <20% of that of the LLOQ sample.If the determined concentration exceeds ULOQ , the sample should be diluted. In cases with dilution integrity, the diluted sample can be quantified accurately. Six dilution quality control (DQC) samples of 100,000 ng/g were prepared in the homogenized blank tissues and then diluted 10-fold with surrogate matrix. The diluted samples were evaluated for accuracy and precision by substituting them into the calibration curve in surrogate matrix.The biological samples were evaluated for benchtop, freeze/thaw, post-preparative, and long-term stability. The stability tests were performed by measuring LQC and HQC samples with three replicates. The background concentration of SL in the blank authentic tissues was set to the nominal LQC concentration in the stability test. As the background concentration of SL in the authentic matrix is similar to or higher than the LQC (60 ng/g), the deviation of the background concentration of SL markedly affects the accuracy of LQC. The background concentration of the blank samples was determined on initiation of the stability test. The HQC samples were prepared by adding a standard solution to the authentic matrix, and the results were calculated by subtracting the endogenous concentration in the blank matrix. Benchtop and long-term stability were evaluated simultaneously with the benchtop stability test carried out at room temperature (approximately 24 °C) for 4 h using samples with long-term stability (stored at −80 °C for 34 days). The post-preparative stability was assessed at 10 °C on an autosampler tray for 21 h. The freeze/thaw stability was evaluated by four cycles of freezing at −80 °C and thawing at room temperature.The tissue distribution of 3′-SL and 6′-SL in Yucatan minipigs was assessed following intravenous administration at a dose of 12.5 mg/kg. The animal experiments were performed in compliance with the Animal Welfare Act and Guide for the Care and Use of Laboratory Animals of Institute for Laboratory Animal Research (ILAR). The animals were kept in a single cage at constant room temperature (23 ± 3 °C) and 30–70% humidity under a regular 12-h light/12-h dark cycle. Animals were provided with approximately 25 g/kg/day food and water was freely available.v/w) in an Ultra-Turrax apparatus . After blood collection into K2-EDTA tubes, blood samples were gently mixed and placed on crushed wet-ice/Kryorack . Following centrifugation at 20,740 × g for 3 min at 4 °C for separation of plasma, the plasma was transferred into labeled polypropylene tubes.Two minipigs were sacrificed at 2 h after administration; liver and kidney tissues, and blood from the jugular vein, were then collected. Each tissue was immediately removed and washed with normal saline, blotted dry with a paper towel, cut into pieces with scissors, and individually homogenized with three volumes of normal saline .The results are presented as the mean ± SD. Statistical analyses were performed using Microsoft Excel 2016  or Phoenix WinNonlin Professional .A very rapid and highly sensitive LC-MS/MS method, for the separation and quantification of 3′-SL and 6′-SL in the liver and kidney tissue of minipigs, was developed and validated in terms of selectivity, linearity, accuracy, precision, matrix effects, recovery, dilution integrity, carryover, and stability. This analytical method, which was developed using the surrogate matrix method (because SL is an endogenous compound) was applied successfully to minipig tissue samples in a tissue distribution study. The developed method was useful for evaluating the tissue distribution of 3′-SL and 6′-SL in the minipig model.The results showed that 3′-SL and 6′-SL were distributed rapidly to the kidney and liver tissue after intravenous gavage. In addition, 3′-SL and 6′-SL were shown to be mainly eliminated via the kidney with urine excretion. These results provided useful information for further studies for elucidation of main excretion route by monitoring the amount of 3′-SL and 6′-SL.


Lightweight aggregate concrete manufactured by solid waste or recycled by-products is a burgeoning topic in construction and building materials. It has significant merits in mitigating the negative impact on the environment during the manufacturing of Portland cement and reduces the consumption of natural resources. In this review article, the agricultural and industrial wastes and by-products, which were used as cementitious materials and artificial lightweight aggregate concrete, are summarized. Besides, the mechanical properties, durability, and a few advanced microstructure characterization methods were reviewed as well. This review also provides a look to the future research trends that may help address the challenges or further enhance the environmental benefits of lightweight aggregate concrete manufactured with solid waste and recycled by-products. The low density LWAC can be used as non-structural applications, while the high density LWAC can be used as structural applications. The mediate density LWAC can be used in both structural and non-structural applications depending on the field requirements [Light weight aggregate concrete (LWAC) has been widely applied in constructions with the merits of lightweight, heat preservation, fire resistance, low shrinkage, and creep resistance under normal conditions . The mosIt was reported that  the LWACThe United States is one of the earliest countries that applied the LWAC. Since 1913, the shale concrete has been successfully prepared and implemented in building bridges (Lightweight-Aggregate-History). In Asia, Japan began to produce and use LWAC in 1955 and then it has been widely used for building urban roads, bridges, railways, and marine constructions . China bIt is no doubt that the production of high-strength LWAC has significant benefits in modern constructions. However, for the production of high-strength LWAC, the high-quality Portland cement, and artificial lightweight aggregates are the key factors that govern the final properties of LWAC. As well-known, the production of high-quality Portland cement is an energy-intensive process, and the manufacture of LWA also requires a large amount of energy consumption , which pThe primary purpose of developing green high-performance light-weight aggregate concrete is to reduce the carbon footprint. It was reported that the LWAC can be prepared by using recycled materials as aggregate  and ceme2 emissions [Portland cement, which provides good mechanical properties, high economic value, and excellent durability, has been widely used as the cementitious binder in modern constructions . Howevermissions . Accordimissions  and othemissions  are the In order to reduce the energy consumption and improve the properties, special type cement can be used to partially or entirely replace Portland cement in light-weight aggregate concrete, especially for the aggressive environment, such as marine area, and sulfate or chloride attack. With exposure to these environments, calcium aluminate (CA) cement, calcium sulfoaluminate (CSA) cement, and supersulfate (SS) cement have been applied to manufacture durable LWAC with exposure to aggressive environment.2 with forms of tricalcium silicate (C3S) and dicalcium silicate (C2S), the main oxides of CAC are CaO and Al2O3 in the form of monocalciumaluminate (CA) instead of C2S and C3S. Although the CA cement has an outstanding performance, the high price (four to five times higher than the Portland cement) has significantly confined its wide applications, except some extreme cases, such as high temperature or acid attacking. It was claimed that the high-temperature resistance of CA cement lightweight aggregate concrete is better than the normal Portland concrete. This study compares the high-temperature resistance of CA/expanded clay aggregate LWAC and OPC/natural aggregate concrete. The results showed that after exposure to 1000 °C, the residual mechanical properties of the CA/expanded clay aggregate LWAC is better than the OPC/natural aggregate concrete [CA has significant merits compared to Portland cement, including the rapid hardening, high temperature and temperature change resistance, chemical attack resistance, biogenic corrosion resistance, impact resistance, and abrasion resistance. Unlike the Portland cement, in which the main oxides are CaO and SiOconcrete .Apart from CA cement, calcium sulfoaluminate (CSA) cement generally has a higher strength in the early and late stages compared to OPC ,18. CSA-e (C4AF) . The ye’quations , which cquations ,22,23,24The excellent corrosion resistance of CSA results from the formation of a dense pore structure according to these reactions. A previous study has evaluated the interactions between the OPC, CSA in LWAC . In thisBecause of the low heat of hydration and the excellent corrosion resistance with exposure to aggressive environments, supersulfate cement intrigues broad interests in recent years. Mixed with blast furnace slag, calcium sulfate, and alkaline activators, supersulfate cement showed its great potential as a promising binder to prepare durable concrete with exposure to the aggressive environment . The effOne of the most impressive merits of the supersulfate cement LWAC is its environmental resistance with exposure to chloride or sulfate attacking. Grounds investigated the resistance of decomposed ettringite to sulfate solutions when supersulfate cement samples are exposed to sulfate . The preAlthough the CA, CSA, and SS cement are considered to have application prospects because of its low carbon dioxide emissions and low energy requirements, it is worth noting that the challenges from academic and practical perspectives are mainly the high manufacturing cost and the lack of understanding the phase formation and hydration process of the mineral contents, and the unpredictable long-term durability.It has been widely recognized that Portland cement production not only consumes tremendous energy but also contributes to significant greenhouse gas emissions. As a result, the search for sustainable and low-carbon cementitious materials to replace Portland cement is burgeoning in recent years. The use of supplementary cementitious materials (SCMs) in normal concrete constructions has been investigated for several decades and showed a few promising benefits to enhance the mechanical properties and durability of concrete materials. However, the effects of the SCMs on the properties and durability of LWAC have yet been systematically investigated, especially by using the solid recycling wastes as SCMs or low-carbon cementitious materials.In order to study the effect of different SCMs on the carbonization resistance of LWAC, Bogas  selected2 emissions can also be significantly reduced by this substitution. It is worth mentioning that the slag had no obvious effect on the bond strength of concrete of the LWAC [Ground granulated blast slag (GGBS) as SCMs can partially replace cement, which can improve the workability of concrete by 30% compared with OPC concrete . It is mthe LWAC .In addition to these conventional SCMs, a few other cementitious materials use for LWAC comes into our views. Lynn  was concBui et al.  studied The application of SCMs to ordinary concrete has obtained relatively good application rules and application values. However, the use of these reused SCMs for lightweight aggregate concrete and special concrete under extreme environments is still in the experimental stage. The practical ways and effects need long exploration, especially insufficient research on its durability. An extensive systematic analysis on SSA in concrete and concrete-related products showed that this material had the potential for some different forms, such as lightweight aggregate production and fine aggregate ,39,40,41Reusing waste materials from agricultural and industrial wastes to prepare light-weight aggregates is an effective way to maximize the use of natural resources and reduce energy consumption . In geneAgricultural residues are extensively discarded or landfilled as solid wastes. Their utilization efficiency is quite low so that this waste accounts for a severe environmental problem. Recycling utilization of these wastes, such as oil palm shells, coconut shells, and corn cob, as lightweight aggregates to manufacture LWAC has brought broad interest in recent years.Oil palm shell (OPS), as a kind of by-product of oil palm-based agriculture, has the potential to serve as the lightweight aggregate of concrete. The amount of waste OPS is huge, and the most majority of OPS is from many Asian countries such as Malaysia, Indonesia, and Thailand, as shown in Shafigh  used theIf the dry density was considered, the control OPS concrete was about 20.8% lighter than the normal-weight concrete. Using OPBC sand instead of ordinary sand will further reduce the density of the concrete samples. When the proportion of the OPBC sand were 12.5%, 25%, 37.5%, and 50%, the densities were 21.7%, 22.2%, 26.2%, and 27.4% lighter than ordinary concrete, respectively, shown in In addition to OPS, coconut shell (CS) is also a large amount of agricultural waste in the coconut industry. It can be used as coarse aggregates or as a potential building material in the construction industry, which is helpful to solve the environmental problem of using recycling solid waste . It was 2 with the combination of Al2O3. This means that the corn cob ash can be used as a cementitious material. Corn cob as a lightweight aggregate for producing lightweight concrete in the non-structural applications was first researched by Pinto, and they measured the density, compressive strength, and thermal insulation properties of the corn cob LWAC. The results were compared with a non-structural lightweight concrete made with a lightweight artificial aggregate, namely, the expanded clay. The results pointed out that, although the compressive strength of the corn cob lightweight concrete is lower than the expanded clay lightweight concrete, the thermal conduction coefficient showed its advantages as non-structural applications [Apart from OPS, corn cob, an agricultural solid waste from maize and corn was also widely incorporated with cementitious powder to prepare lightweight concrete. Previous studies ,55 have ications .The cork industry generates large quantities of waste, which has been studied for the application in the construction industry. In terms of grain density, the density and absorption of the loose body are comparable to that of expanded perlite, vermiculite, and other artificial light aggregates. Compared with expanded perlite or vermiculite, the use of cork particles as lightweight aggregate can reduce energy consumption, consume renewable resources, and be more cost-effective. Besides, cork has a high absorption value and can be used as a lightweight aggregate for internal curing of concrete. As for other physical properties, the advantages of cork as a lightweight aggregate are verified .Besides these materials, Ozturk et al.  revealedAlthough the reuse of agricultural waste as an aggregate of lightweight concrete can achieve the acceptable performance of LWAC, its property still has room for improvement. Many industrial wastes include drill cutting, and steel cutting has been widely applied as aggregates.Drill cuttings consist of a fine mix of rock particles produced by drilling for oil and gas during either exploration or production, and they will be contaminated with drilling fluids and contain high concentrations of chloride salts . This liIn addition to drilling cuttings, waste plastic is another recycled material as aggregates. Colangelo proposed polyolefin waste aggregate (PWA) refined from recycled plastic materials as LWA instead of natural aggregate in the manufacture of LWAC . It can Steel cutting is another under-utilized resource. El-Sayed  did reseIt was claimed that if the raw materials were appropriately selected, the LWAC would show its distinctive advantage compared to the NWC . HoweverTo improve the mechanical properties of LWAC, reducing the water-binder ratio is a widely used method. However, when the coarse aggregate is replaced by the lightweight aggregate, the volume of the aggregates will be significantly increased, and the water-binder ratio will be considerably reduced. This brings a negative impact on the workability of the LWAC. Pierce et al.  used cruAlthough many industrial and agricultural wastes have been recycled and reused for manufacturing lightweight aggregates, or as supplementary cementitious materials to replace Portland cement, their utilization efficiency is still quite low. As a result, how to high-effectively reuse these wastes remains a significant challenge. Furthermore, in the actual production process, using industrial waste to manufacture aggregate often needs additional processing, which inevitably consumes energy and causes secondary pollution. Exploring environmentally friendly and cost-effective approaches are imperative to develop green LWAC.Concrete composite can be considered as a two-phase material, i.e., the aggregates embedded in the cementitious binder phase. According to the properties of aggregate and mortar, concrete can be divided into two categories. In the first type of concrete, including NWAC and some LWAC, the relatively strong aggregates are embedded in the relatively weak cementitious binder matrix. The short-term strength mainly depends on the water-cement ratio and the cementitious binder. In the second type of concrete, the relatively weak aggregates are embedded in the relatively strong cementitious binder matrix; hence, the influence of the aggregate on the strength of the concrete must be considered. By comparing the relationship between the mortar strength and the concrete strength, the influence of the aggregate on the overall compressive strength of concrete can be evaluated, as shown in Yap  aimed toThe mechanical properties of the LWAC made of oil palm shell (OPS) as the lightweight aggregates had been tested by Teo . The resAfter the investigation of the OPS, another study claimed that using palm oil clinker (POC) to replace the OPS as coarse aggregates has a positive impact on the mechanical properties of the LWAC . In thisThe application of granulated cork with bark (GCB) as lightweight aggregate to prepare concrete has been investigated most recently . In this3, as aggregates. The mechanical testing results demonstrated that, with using the recycled concrete as lightweight aggregates, the compressive, splitting tensile strength, and the modulus of elasticity have increased 14%, 32%, and 22%, respectively. It was claimed that the recycled aggregates obtained from the non-structural lightweight concrete can satisfy the requirements and be used in structural LWAC.Apart from the agricultural wastes, many types of industrial wastes, including recycled concrete, recycled clay bricks, fly ash, and various types of sludge, were also used as lightweight aggregates to prepare LWAC. Bogas  investigThe demolished waste clay bricks were used as lightweight aggregates to prepare LWAC . The dem2, Al2O3, Fe2O3, CaO, MgO, K2O, and Na2O are about 46%, 18%, 9%, 5%, 1%, 2%, and 2%, respectively. The 28 days compressive strength and splitting tensile strength can reach 56 MPa and 3.0 MPa, respectively, and the measured modulus of elasticity is about 19 MPa. An interesting phenomenon being observed in this study was the 7 d compressive strength had reached 91% of the 28 d compressive strength. This was the reason that the loading transfer mechanism between the hydrated cement paste and the lightweight aggregates. The microstructure analysis of the ITZ revealed that the cement paste had penetrated into the lightweight aggregates, which reinforced the interfaces between the aggregates and the cement paste.Except using the recycled construction materials as lightweight aggregates, using industrial wastes to prepare lightweight aggregates is another important branch of the LWAC manufacturing method. Lo  prepared2 (55%), Fe2O3 (14.3%), Al2O3 (20.6%), K2O (2.7%), CaO (2.7%), MgO (1.4%), and balance (3.5%). This study investigated the synergistic impact of the aggregates and cememtitious binder. The sludge ceramsite was 100% used as the coarse aggregates and the cement was partially replaced by GGBS. It was found that, although using the SC along with RA and GGBS had significantly reduced the compressive strength, if solely using SC as coarse aggregate, the compressive strength of the LWAC can reach about 13.9 MPa. The elastic modulus results of all samples were in the range of 6.5–9.5 MPa. Unlike the statement from Zhao’s study [Sludge ceramsite (SC), recycled aggregates (RA), and ground granulated blast-furnace slag (GGBS) were used as coarse aggregates, fine aggregates, and supplementary cememtitious materials to prepare the LWAC . The mai’s study , the micFrom compression and crack development perspectives, microcracks often occurred at the interface between the binder and the aggregate. As the stress increases, the cracks in the vicinity of aggregates gradually expanded, and the cracks in the mortar interlocked each other until the failure occurred, as demonstrated in It is not surprising that the mechanical properties of the LWAC is relatively lower than NWC, and the strengths of the LWAC decrease with increasing content of LWA ,79,80,81Shrinkage is one of the primary reasons that account for the cracks. The early age cracks of cement-based materials will lead to premature degradation of concrete structure, so as to shorten its service life. To reduce shrinkage and improve resistance to shrinkage cracking performance of cement-based materials, a variety of methods were adopted, including (1) expansive-based cement/admixture which compensate the shrinkage through the expansion-induced stress ,87; (2) Internal curing, also known as self-maintenance curing, refers to a curing method that relies on pre-absorbent materials to release water to maintain sufficient moisture inside the concrete under adiabatic conditions, it was developed to eliminate self-desiccation and reduce autogenous shrinkage . In receThe self-shrinkage reduction mechanism of the lightweight aggregates is primarily from the gradually releasing of the internal water. During the hydration process, a humidity gradient is formed between the internal curing agent and the cementitious material, and the capillary pressure difference will be generated so that the moisture in the LWA will be released to compensate for the loss of humidity .The effects of LWA performance and mixing parameters on the auto-shrinkage performance have been studied previously. Harmful effects of internal curing on the early strength had been reported in most cases . HoweverEarly age crack that emerged on cementitious materials caused by the autogenous shrinkage plays a significant role in determining the strength of concrete materials, especially for LWAC. Therefore, one of the best ways to reduce early age cracks in the cement matrix is the incorporation of SCMs. Meddah investigated the effect of SF on autogenous shrinkage of LWAC. The results showed that the greater the autogenous shrinkage, the greater the induced internal tensile stress, and the appropriate SCMs can increase the volume stability . Bentz  establisThe shrinkage reducing agent is composed of polyether or polyalcohol organic matter or their derivatives . The mecShrinkage reducing admixture (SRA) is a water-soluble organic chemical with low viscosity, whose main function is to reduce the surface tension of the solvent . At the As for the research of lightweight aggregate concrete, most of them were focused on the aggregate types , mechaniThe carbonation of concrete is a diffusion process of carbon dioxide through the porous microstructure of concrete. The ingressed carbon dioxide reacts with the calcium hydroxide and forms calcium carbonate, which reduces the pH value of the pore solution as low as 11, and thus results in the corrosion of the steel reinforcement in concrete . As wellAlthough LWAC exhibits excellent performance, its carbonization mechanism is still lacking to the best of our knowledge. Newman  pointed RS) to the total aggregate is another factor that determined the carbonation resistance. It was claimed that the increase of the VRS can slightly escalate the carbonation of the LWAC. The main contribution of this study is that the potential mechanisms of the impacts were pointed out. It was believed that the high w/b ratio will lead to the high porosity, which increase the potential carbonation degree of the LWAC, and the increase of VRS will result in a high specific surface area of the waste clay brick, which enhance the pozzolanic reaction between the Ca(OH)2 and the clay bricks. This will dwindle the amount of the Ca(OH)2 in cementitious binder and thus mitigate the carbonation problem of the LWAC. This mechanism is worthy of further systematically investigation.Zhao recycled the waste clay bricks as lightweight aggregates for the LWAC preparation . The wasApart from the w/b ratio, the mineral admixture is another important factor that determines the carbonation resistance of the LWAC. Gao  investigIn the physical and chemical deterioration mechanism of concrete, chloride ion and/or other corrosive ions diffusion in concrete is the most noteworthy phenomenon. The microstructure of concrete is the main factor controlling this process, which is related to water transport and ion diffusion in concrete . The porGüneyisi et al.  used silThe water saturation condition of the LWA is the main factor affecting the freeze resistance of LWAC. Zhu  carried In Tang’s study , lightwem is the magnitude as defined in seismology; m is equivalent to the log scale of the amplitude “a” of the AE signal. N is the number of signals with a magnitude greater than m, and the coefficient b is the negative slope of the logN–m plot.Acoustic emission (AE) is a relatively new method that can be used to explore the mechanism of explosive spalling of LWAC at high temperature ,120. Theb  varies with the type of damage. At the initial stage, a large number of low-amplitude acoustic emission signals were generated. Later, fewer signals were produced, while the amplitude was high. This means that the value of b decreases gradually as the sample approaches the impending failure. This is at the heart of the so-called b-value analysis used to assess damage [Specifically, the coefficient index s damage . By moniMeans of neutron radiography (MNR) can be used for the visualization and quantification of liquid water transport . The higNon-destructive techniques (NDTs) play an essential role in the quality control of new buildings and the evaluation of the service condition of existing concrete structures ,128. TheThe research work in recent years provides a framework for further research on the improvement of the mechanical properties of LWAC produced by waste and recycled by-products. The physical properties of LWAC, mixing ratio design method, freshness, and hardening properties of LWAC were discussed.It was found that many solid waste materials, from both industrial and agricultural by-products, can be used as supplementary cementitious materials and lightweight aggregate materials to prepare green lightweight aggregate concrete. Several main industrial by-products, including silica fume, fly ash, lime powder, ground granulated blast slag, and various sludge, have been investigated as replacement of Portland cement. The results indicated that, provided with proper design and manufacturing process, these industrial by-products can be used as cementitious materials to prepare green lightweight aggregate concrete.Apart from the supplementary cementitious materials, it was found that, many types of agricultural by-products, including oil palm shell, oil palm-boiler clinker, coconut shell, corn cob, cork, and tobacco wastes, and several industrial wastes, including drill cuttings, waste plastic, recycled clay bricks, various sludge, and steel cutting can be used as lightweight aggregates for manufacturing green lightweight concrete. The mechanical properties testing results shows that the lightweight aggregate concrete manufactured from the above mentioned agricultural and industrial by-products can meet the field application requirements. Although the durability testing of the lightweight aggregate concrete is quite limited, several studies have shown that the durability of the lightweight aggregate concrete is strictly correlated to the porosity and the saturation degree of the lightweight aggregates.(1)The chemical reaction mechanisms between the recycled aggregates and cementitious binders should be investigated systematically. So far, the chemical bonding between the aggregates and the cementitious binders remains unclear. Further studies should focus on the chemical analysis of the aggregates, cementitious binder, and the interfacial area to elucidate and characterize the chemical reactions of the various recycled lightweight aggregates and the cementitious binders.(2)For the lightweight aggregate concrete in the construction, prewetting of the lightweight aggregate is an appropriate way to reduce water absorption and improve the workability. The interface wettability between the lightweight aggregate and the cementitious pastes needs to be investigated. The impact of the saturation degree of various recycled aggregates on the workability, mechanical properties, and even durability should be further investigated.(3)The porosity of the aggregates is another critical factor that determines the final properties of lightweight aggregate concrete. So far, the relationship between the porosity of the recycled lightweight aggregates and the final properties of the lightweight aggregate concrete is still lacking, especially when the chemical compositions of the lightweight aggregates were different.(4)At present, researches on lightweight aggregate concrete mainly focus on high strength, which is an inevitable trend of future development. However, the durability challenge and the segregation resistance in engineering applications are worthy of this concern.Although previous studies have demonstrated that the green lightweight aggregate concrete made of solid waste materials have a great potential to be widely used in practical applications, they are still facing several critical challenges need to be further investigated: