Limestone is widely used in the construction industry to produce Portland limestone cement (PLC) concrete. Systematic evaluations of hydration kinetics, compressive strength development, and carbonation resistance are crucial for the rational use of limestone. This study presents a hydration-based model for evaluating the influences of limestone on the strength and carbonation of concrete. First, the hydration model analyzes the dilution effect and the nucleation effect of limestone during the hydration of cement. The degree of cement hydration is calculated by considering concrete mixing proportions, binder properties, and curing conditions. Second, by using the gel–space ratio, the compressive strength of PLC concrete is evaluated. The interactions among water-to-binder ratio, limestone replacement ratio, and strength development are highlighted. Third, the carbonate material contents and porosity are calculated from the hydration model and are used as input parameters for the carbonation model. By considering concrete microstructures and environmental conditions, the carbon dioxide diffusivity and carbonation depth of PLC concrete are evaluated. The proposed model has been determined to be valid for concrete with various water-to-binder ratios, limestone contents, and curing periods. With increasing water-to-binder ratios or decreasing cure periods, the carbonation depth of concrete increases. When the curing period increases from one day to three days, the concrete carbonation depth decreases by approximately 25%. While the curing period increases from three days to 28 days, the concrete carbonation depth decreases by approximately 25%–30%. This is because the rate of cement hydration at early ages (before three days) is much quicker than that at late ages (28 days). Hence, early age curing is effective for reducing the carbonation depth of concrete.The experimental results for the carbonation of PLC concrete from references ,7 were uParrot  investigThe carbonation resistance of PLC concrete depends on a trade-off between some competing effects. First, the limestone replacement increases the degree of cement hydration. This factor increases the carbonation resistance of PLC concrete. Second, limestone replacement reduces the cement content in concrete. This factor decreases the carbonation resistance of PLC concrete. The increasing and deceasing factors contribute to the carbonation resistance of concrete with different weights. The carbonation resistance of PLC concrete relates to increasing and deceasing factors.2 diffusivity on local relative humidity requires additional examination. Second, the change in carbonation depth may also be related to the changes in the pore solution and the composition of the cement hydrates with limestone present (and reacting) in the cement paste. This point was not considered.In this study, the effects of carbonatable material contents, porosity, and environmental conditions on concrete carbonation are considered. However, the carbonation model also shows some limitations. First, the drying of concrete during carbonation tests was not considered . The depThis study presents a systematic study on hydration, strength development, and carbonation of PLC concrete. The conclusions of this study are summarized as follows:First, a kinetic hydration model is proposed for cement-limestone blends. The dilution effect and physical effect from limestone powder replacements are taken in account. The dilution effect is considered by the effective W/C ratio. The physical effect is considered by the limestone nucleation effect indicator. The input parameters of the hydration model are concrete mixing proportions, compound compositions, Blaine surface area of cement and limestone, and concrete curing temperatures. The values of the reaction coefficients of the hydration model can be determined using the input parameters. Furthermore, the time-dependent degree of hydration can be calculated.Second, the compressive strength of PLC concrete is evaluated using the gel–space ratio. Limestone replacement can increase the degree of cement hydration. For concrete with low water-to-binder ratios, the improvements related to the degree of cement hydration are more obvious. At late ages, for concrete with higher water-to-binder ratios, limestone replacements impair compressive strength, while, for concrete with lower water-to-binder ratios, strength reduction due to limestone replacements is marginal. Regarding compressive strength, the use of limestone in concrete with low water-to-binder ratios is a more rational option.2 diffusivity and carbonation depth of PLC concrete are evaluated by considering concrete material properties and environmental conditions. With increasing water-to-binder ratios and limestone content or reductions in curing period, the carbonation depth of concrete increases. The carbonation resistance of PLC concrete is related to both increasing factors (limestone replacements can increase the reaction degree of cement) and deceasing factors (limestone replacements decrease cement contents in mixing proportions).Third, the calculation results from the hydration model, such as carbonatable material contents and porosity of concrete, are used as input parameters for the carbonation model. CO


Follow-up time ranged from 3 to 15 months after their prenatal test result. The reporting of susceptibility loci was initially ‘shocking’ for five parents while the other seven felt ‘worried’. Ten out of 12 participants indicated they would like to be informed about the susceptibility locus again, two were unsure. Most had no enduring worries. Participants unanimously indicated that pregnant couples should have an individualized pre-test choice about susceptibility loci (non)disclosure. We observed no negative psychological impact with the prenatal diagnosis and disclosure of SL on participants. A key factor in mitigating parental anxiety with SL disclosure appears to be post-test genetic counseling. Our report confirms that pregnant women and their partners prefer an individualized choice regarding the scope of prenatal testing.Genomic microarray may detect susceptibility loci (SL) for neurodevelopmental disorders such as autism and epilepsy, with a yet unquantifiable risk for the fetus. The prenatal disclosure of susceptibility loci is a topic of much debate. Many health care professionals fear that reporting susceptibility loci may put a psychological burden on pregnant couples. It is our policy to disclose prenatal susceptibility loci as we recognize them as actionable for prospective parents. The aim of this report was to evaluate the psychological impact of disclosing a prenatal diagnosis of susceptibility loci. The psychological impact of disclosing susceptibility loci was evaluated in the first patients who received such results. Eight out of 15 women who had a susceptibility locus disclosed and four of their partners consented to share their experiences through a telephonic evaluation ( Genomic microarray may detect more copy number variants (CNVs) that cause clinically relevant abnormalities and generates results faster than conventional karyotyping (CK) , rapid diagnostics and more adequate care may be mobilized when parents have the knowledge of the SL   received pre-test counselling by a senior obstetrician and received a patient information leaflet which specified that ‘all pathogenic results will be reported’. Pregnant couples were informed about array testing. The occasional occurrence of unexpected findings was discussed. These could either be pathogenic CNVs not related to the prior indication for invasive testing or susceptibility loci (SL), Patients received no detailed information regarding SL. Unexpected findings were discussed, but there was no strong emphasis on SL as a category of outcomes of invasive prenatal testing. SNP array testing was performed as a first-tier diagnostic test as described before  from the postnatal literature were available.An expert ultrasound examination was offered if the SL was associated with structural abnormalities.We offered targeted parental SNP array in all cases because knowing whether an SL was inherited aided in evaluating the clinical implications of the SL within the family.The couples were informed about the possibility for early postnatal intervention programs (www.mee.nl), and the option to terminate the pregnancy was discussed.The pregnant women and their partners were offered support from a medical psychologist specialized in prenatal care.When a SL was diagnosed pregnant couples were contacted directly by a clinical geneticist informing them that there was no causative chromosomal abnormality found, but a deviant finding that may require special attention. They were invited for extensive post-test counseling available the next day. For extensive information about our counseling methods and pregnancy management, see Govaerts et al. (manuscript in preparation), in short:In this report we describe the experiences pregnant couples had when a susceptibility locus was found after invasive genetic testing, in the absence of ultrasound anomalies. Between July 2012 and December 2013, 14 couples received a prenatal diagnosis of a SL, and all of them were contacted. A clinical geneticist contacted them and asked whether they were willing to share their experiences by phone in order to assess the impact of disclosing a prenatal susceptibility locus. This interview was part of aftercare in order to learn about the long term psychological impact of SL disclosure in pregnancy. All patients proceeding with invasive prenatal testing, signed consent for further follow up during pregnancy and after delivery. Eight women and four of their partners agreed to share their experiences, see Fig. The follow-up interview period between invasive PND and the contact ranged between 3 and 18 months. The mean time before the follow-up interview was 10 months after disclosure. Three participants were still pregnant at the time of follow-up.Consenting participants were approached for a follow-up interview by phone, using semi-structured questions (mean duration: 30 min). Women and their partners were interviewed individually. In Table Qualitative analysis was performed on the answers of all participants. A posteriori, three independent judges  categorized the answers to the open-ended questions  Field .Qualitative analysis of the interviews showed that 7 out of 12 participants said they were ‘worried’, while the other 5 participants said ‘it was real shock’ to hear about the SL. We provide quotes to the answers by category. Participants marked with an asterisk (*) were still pregnant during follow-up.‘It startled me, you just don’t want to hear that about your baby. But I thought that our baby would develop normally, maybe a bit slower than others, but healthy in the end.’ ‘It was unpleasant, because we thought everything would be fine. Thus far, it appears as such.’ ‘We were a little shocked at first, but we understood that there was only a very small chance that something could really be wrong. So we were not too worried.’ ‘We were a bit upset the first days. When we had an advanced ultrasound, everything looked normal, that was a relief to us.’ ‘It came very unexpectedly, I was a little overwhelmed, but I wasn’t really, really alarmed.’ ‘At first we were not too alarmed, because the baby did not have Down syndrome. But we felt the SL diagnosis was slightly worrisome, because we did not know what we could expect at all.’ ‘I was upset, because they could not tell me exactly how high the risk of developing the clinical features was. I just sat there stared at the geneticist and asked what it was, and if it was dangerous.’ Quotes of parents who were worried (7 out of 12)‘To us, it was very unclear at first. We heard something was wrong and it came as a shock, I was nervous. When we had an appointment with the geneticist to talk about it, we understood that the risk was quite low. I thought; ‘we’ll have to wait and see’, but my husband was really worried. There was a picture of a patient with the same deletion, clearly showing something was wrong. This was very upsetting to us. We didn’t really know what to do with the provided information. I wasn’t expecting it and did not think about the possibility of this kind of outcome when we engaged in prenatal diagnosis, only about the possibility of a trisomy. Maybe our older daughter has this deletion too, but she is a healthy, normal girl.’ ’That was a real shock. It was quite upsetting. We thought; What is going to happen next? It was not a very nice time.’ ‘We were startled, it was quite something. But we were informed of the possibility of such results.’ ‘Unpleasant. It came as a shock. We did an amniocentesis hoping to hear that everything is alright, and then this susceptibility locus came as a test result. I was very emotional.’ ‘That was a real shock. It was not clear what was wrong, that made me worry a lot. The more I thought about it, the more worried I became. I had a lot of questions. I kind of panicked. Luckily, we had an appointment with the geneticist the next day. After that, I felt calmer.’ Quotes of parents who were shocked (5 out of 12)What was it like for you when you were told about the SL that was found? What was your first reaction?‘I don’t think about it too much now.’ ‘I don’t think about it anymore. I think I just have a normal, healthy son.’ ‘I don’t look back on it. I gave birth to a healthy son. ‘‘I like to think that nothing is wrong. At the moment, I don’t see any reason to think there is. ‘‘During pregnancy I was worried about other physical abnormalities. But now that I gave birth, I am not worried anymore.’ ‘I don’t think about the SL anymore. I think I coped with the information quite well.’ ‘Most abnormalities were excluded with expert ultrasound examinations. We are only unsure of other neurodevelopmental symptoms like behavioural problems. But we think everything will be fine.’ ‘After the expert ultrasound examinations we felt reassured. The SL does not have to mean anything.’ ‘I think the chance of expression of the SL is very small. And if it will express itself, I think it will be mild and actionable.‘‘We will have to wait and see. I think it is nothing very severe, since I carry it myself and do not have any symptoms. I am not too worried anymore.’ ‘I think it might be something very mild. My wife has it too. Maybe we will not even notice it. ‘‘I do not see anything out of the ordinary regarding my daughter at this point.’ Quotes of parents that do not think about it often anymore (11 out of 12)‘It is something that you keep carrying with you. If she behaves weirdly, then I immediately think that this behaviour is related to the SL. I also do not like the fact that she already had a medical file before she even was born.‘Quote of a parent that experienced a stigma (1 out of 12)‘I want to know as much as possible. That is the reason I chose for invasive prenatal diagnosis in the first place.’ ‘Yes, even though it was distressful when we first heard about the susceptibility locus. But if something might be wrong with your child, you want to know about it.’ ‘If I could choose, than I would like to know.’ ‘Absolutely.’ ‘Yes, I think so, because I prefer to know as much as possible. ‘‘Personally, I want to know everything, but I have an academic degree. I can imagine that this kind of information might be very confusing for people with a lower educational level.’ ‘Of course. Especially with regards to my advanced maternal age.’ Yes (10 out of 12 parents)‘If I would get pregnant again, I might not want to know. But in this pregnancy, I would not want to have missed this information.’ ‘It depends if it really matters. It did give us a lot of stress, because we thought it was something very severe at first. But I would be very curious in the future (next pregnancy). A friend of mine, who had children at a young age, did not have any genetic information about her children at all. But her son has a neurodevelopmental disorder and she did not know about it in advance. It can be useful, because you know where it might come from.‘Hesitant (2 out of 12 parents)just have the normal worries any parent has’.Worries about the health and development of the child ranged from 2 to 7 on a 10-point scale, see Table Since it has been suggested that disclosure of SL may raise emotional harm, we evaluated the psychological impact of prenatal SL disclosure on pregnant couples. Women and their partners initially felt worried and shocked. Most parents indicated that the SL was not what they had expected from invasive PND, however some recalled being informed on such possibility during the pre-test counselling. Previous research showed that pregnant women are hardly ever ready for receiving abnormal prenatal test results, even if they are well informed , all born children had no congenital anomalies or dysmorphic features, but were still too young to be examined for neurological development. None of the fetuses had ultrasound anomalies. It is yet unknown whether these children will develop neurodevelopmental symptoms in the future. Most parents did not have lingering worries regarding the SL, except for one woman who experienced a stigma regarding her seemingly normal and healthy born daughter. She told that each time her daughter behaved aberrant, she immediately feared it might be caused by the SL. This is something that is also encountered in other studies on abnormal prenatal diagnoses, even if the child has seemingly normal appearance (Bernhardt et al. Nearly all parents indicated they would want to be informed of SL again if offered a choice. Parents who said they preferred to know about SL, said they did so because they could quickly mobilise adequate care if needed. For instance, if developmental problems would occur, they could have access to early interventions for i.e. autism. These findings are congruent with our earlier study in which we found that a vast majority of pregnant couples, when offered a choice during pretest genetic counseling, opted for SL disclosure (van der Steen et al. This small study showed that in our setting, there was no long-term psychological burden for pregnant couples whose fetus was diagnosed with a susceptibility locus. A key factor in mitigating parental anxiety with SL disclosure appears to be post-test genetic counseling. This paper confirms that parents highly appreciate an individualized choice on the scope of prenatal testing. We believe that if genomic microarray testing is offered, a chance of the detection of results like susceptibility loci should be routinely mentioned during pre-test counselling. An opt-out possibility may be sufficient to support the reproductive autonomy of pregnant couples.


Brassica chinensis L.) to tetracycline, cephalexin, and sulfamethoxazole at 50% minimum inhibitory concentration (MIC) levels and MIC levels, respectively, was conducted to explore plant growth, antibiotic uptake, and the development of antibiotic resistance in endophytic systems. The three antibiotics promoted pakchoi growth at 50% MIC values. Target antibiotics at concentrations ranging from 6.9 to 48.1 µg·kg−1 were detected in the treated vegetables. Additionally, the rates of antibiotic-resistant endophytic bacteria to total cultivable endophytic bacteria significantly increased as the antibiotics accumulated in the plants. The detection and quantification of ARGs indicated that four types, tetX, blaCTX-M, and sul1 and sul2, which correspond to tetracycline, cephalexin, and sulfamethoxazole resistance, respectively, were present in the pakchoi endophytic system and increased with the antibiotic concentrations. The results highlight a potential risk of the development and spread of antibiotic resistance in vegetable endophytic systems.Antibiotic contamination in agroecosystems may cause serious problems, such as the proliferation of various antibiotic resistant bacteria and the spreading of antibiotic resistance genes (ARGs) in the environment or even to human beings. However, it is unclear whether environmental antibiotics, antibiotic resistant bacteria, and ARGs can directly enter into, or occur in, the endophytic systems of plants exposed to pollutants. In this study, a hydroponic experiment exposing pakchoi ( Antibiotic pollutants and their environmental impacts have become a mounting concern owing to their broad usage and persistence in the environment. A large range of veterinary and human antibiotics have been detected in soil, animal manure, sediment, municipal or industry wastewater, surface water, groundwater, and drinking water samples ,3,4,5,6.Once antibiotics are released into agricultural lands, crops are exposed to them due to their persistence, and the level of exposure depends on the physicochemical properties of the compounds, sorption potential, and environmental conditions ,17. EvenTo date, the majority of research on the impact of antibiotic contamination in plants has focused on evaluating the toxicity of antibiotics to plants or detecting the ability of antibiotics to accumulate in plants. Limited knowledge is available regarding the potential effects of antibiotic stress on the development and spread of antibiotic resistance, including antibiotic-resistant bacteria and antibiotic-resistant genes (ARGs), in plant endophytic systems. There is a diverse range of endophytic bacteria, which includes pathogens, mutualists, and commensals that grow within the roots, vasculature, and aerial tissues of plants . RecentlBrassica chinensis L.), a frequently consumed vegetable in China, was selected and planted in a hydroponic system and exposed to different antibiotics. Then, the antibiotic uptake and its effects on plant growth and the presence of AREB and ARGs in the endophytic system were investigated and evaluated. The findings will facilitate a more accurate assessment of the potential risks of antibiotic contamination to food quality and environmental health.To assess possible consequences, pakchoi , an enzyme in the folic acid synthesis pathway. Two genes, sul1 and sul2, mediated by transposons and plasmids, and expressing DHPS highly resistant to sulfonamide, have been found [Tetracycline , cephalexin , and sulfamethoxazole  were purchased from Dr. Ehrenstorfer GmbH  and selected to represent the different classes of antibiotics  based on their frequent usage in the local livestock farms in Xinxiang City, China . Tetracyon genes . β-lactaon genes . Sulfonaen found .Methanol, acetonitrile, formic acid, and acetone of HPLC grade were purchased from Fisher Scientific . Other chemicals were of analytical grade and obtained from Yaohua Chemical Reagent Factory . Ultrapure water was supplied using a Millipore Milli-Q system . Oasis HLB cartridges  purchased from Waters  were used for the extraction and purification of the target antibiotics. Individual stock standards were prepared by dissolving antibiotics separately in methanol and were stored at −20 °C in brown vials.B. chinensis L.) were used for this study. TC, CPL, and SMX were separately added into the hydroponic solution of the test system at two concentrations: 50% of minimum inhibitory concentration (MIC) of each antibiotic, and the MIC of each antibiotic [A hydroponic experiment with antibiotic treatments was performed in an experimental greenhouse in the College of Life Sciences, Henan Normal University, China, during the autumn of 2016. Seeds of pakchoi ; (3) CPL-treated ; and (4) SMX-treated . Each treatment was designed with three replicates, and a total of 21 hydroponic tanks were used in the present study. Additionally, each tank had nine cylindrical holes (4 cm depth and 3 cm diameter) in its cover containing sponges (3 × 3 × 2.5 cm) in individual cylinders as a rooting medium. Five uniform seeds were planted per hole and irrigated with half-strength Hoagland solution every 2 days until the roots of the seedlings were immersed in the solution. Finally, only one or two strong seedlings were selected to leave in each hole, and they were grown directly in nutrient solution.Prior to testing, the seeds were surface-sterilized in 0.1% sodium hypochlorite solution for 10 min and then rinsed with sterile deionized water . Seeds oDuring the experiment the room conditions were maintained at 25 ± 2 °C in daylight and 18 ± 2 °C at night, with a relative humidity between 65% and 70%. Each planter was equipped with an electric aeration pump and was aerated for 2 h every day. Because of evapotranspiration from the vessels, lost water was supplemented with fresh nutrient solution without the addition of extra antibiotics. Pakchoi was harvested after 55 days of cultivation. Then, the sponges attached to the vegetables were trimmed off. The plants were rinsed first with tap water and then with deionized water and dried on adsorbent paper. The growth parameters and abundance of endophytic bacteria were measured immediately. Antibiotic analyses and DNA extraction from endophytic bacteria were completed within two weeks of sampling.0 indicates the measurement of the control treatment and Mt indicates the measurement of the antibiotic treatment.All plants were harvested at the end of the test, and then, plant heights, root lengths, and fresh biomasses of 10 plants from each treatment were measured and recorded. The growth inhibition rate was calculated using following formula:Samples of the edible pakchoi portions (stem and leaf) were selected and freeze-dried for 24 h until moisture was no longer present . Then, t−3. Each 100 μL of diluted suspension was spread on meat-peptone agar and on corresponding antibiotic-containing agars  for cultivation at 28 °C for 3 days. Each sample was replicated three times. The colony-forming units (CFUs) of TCEB and AREB  were enumerated.To isolate endophytic bacteria, the edible portions of fresh pakchoi were immersed in 3% hydrogen peroxide for 30 min, followed by rinses with sterile deionized water (3 min × 3 times). Then, they were immersed in 70% ethanol for 1 min and rinsed as before . FinallyThe surface-sterilized edible pakchoi portions were cut into pieces and ground with liquid nitrogen before extraction under sterile conditions. Total DNA was extracted using PowerPlant DNA Isolation Kit  following the manufacturer’s instructions . The contetA, tetC, tetG, tetK, tetL, tetM, tetO, tetQ, tetT, tetW, tetB/P, and tetX), 5 sulfonamides-resistant genes , and 6 β-lactams-resistant genes . PCR detection assays were performed as previously described [PCR detection assays were used to screen for the presence or absence of 23 types of ARGs in the antibiotic-treated samples, including 12 tetracyclines-resistant genes (escribed . Primers2 > 0.99)  using a LightCycler real-time PCR system  with SYBR Green I. Details of the primers are listed in  > 0.99) . The 20 p < 0.05) between treatments.Ten plant samples from each treatment were used to test the effect of antibiotics on plant growth. For other analyses, three repetitions, each of which was the mixture of different plant parts from six plants, were performed. The mean values and standard deviations (SDs) of all data were calculated using Microsoft Office Excel 2016 . Statistical analyses were performed using the software SPSS 21.0 . Duncan’s multiple comparisons were used to determine the significant differences . When the exposure dose was increased to MIC levels, this growth-promoting effect was maintained in CPL-treated plants. However, the TC- and SMX-treated plants were significantly inhibited (p < 0.01), as indicated by the growth parameters being less than those of the controls. According to the detection of growth inhibition rates . These changes in AREB abundance resulted in dramatic increases in the ratios of AREB to TCEB from 0.23%, 3.77%, and 1.26% of TC, CPL, and SMX resistance, respectively, in the controls, to 0.79–1.23% , one β-lactamase gene (blaCTX-M), and two sul genes (sul1 and sul2) responsible for TC, CPL, and SMX resistance, respectively, were present in the corresponding antibiotic-treated samples. Thus, further quantification using qPCR was conducted to monitor their responses to different treatment doses. To minimize the differences in background bacterial abundances and DNA extraction efficiency, 16S rRNA gene was also quantified and the absolute numbers of the above four quantified ARGs were normalized to that of the ambient 16S rDNA  [tetX gene isolated from patients are still resistant to tigecycline [tetX gene in edible pakchoi should be highly concerning. The other ARGs, sul1, sul2, and blaCTX-M, are widely present in various environmental media [sul1 gene is normally found linked to other resistance genes in the Tn21 type integron, while sul2 is usually located on small plasmids of the IncQ family [Among the four detected ARGs, the presence of acteria) ,53,54. Hecycline . Therefoal media ,57,58, aQ family . qPCR shPrevious studies have demonstrated the antibiotic uptake  and the The present study investigated the growth of pakchoi and the antibiotic resistance in its endophytic system under TC, CPL, and SMX exposure. Pakchoi was shown to absorb antibiotics from the hydroponic culture environment. The absorption was selective toward different antibiotics, and the absorption amount was related to the antibiotic concentration. The accumulated antibiotics in the plant influenced the growth of the plant and increased the levels of AREB and ARGs, even at sub-inhibitory doses, which should be noted due to considerations surrounding the possible transfer of ARGs through the food chain.


How do meaningful events that naturally occur within a culture, such as a national holiday, shape evaluative judgments of objects related to the holiday? We focused on evaluations of red roses and gift chocolates, which are everyday objects, but also iconic of Valentine’s Day in the U.S. We reasoned that if cultural events shape evaluations, then roses and chocolates would be evaluated differently near Valentine’s Day. Using a large and diverse U.S. sample, we found that as Valentine’s Day neared, evaluations of roses and chocolates (but not a comparison object) were evaluated more positively. Increases in positivity of roses and chocolates covaried with their increased cultural relevance, as quantified by the volume of web search queries involving these terms. These findings provide a demonstration of  For somWhether Valentine’s Day is loved or loathed, roses and chocolates are pervasive cues around the Valentine’s Day season. In the U.S., there may be nothing more iconic of Valentine’s Day than a gift of red roses and a box of chocolates . Each yeOur main question is: Do evaluations of roses and chocolates change as Valentine’s Day approaches, and are shifts in evaluations related to shifts in the cultural salience of roses and chocolates? This conjecture is plausible given that evaluative judgments are assumed to be constructed at the moment of the evaluation rather than reflecting trait-like attitudes . AccordiTo illustrate, when evaluating roses, a person brings to mind the mental representation of roses. This may include their physical features, related experiences . We examined the extent to which daily web search queries for “roses” and “chocolates” predict increases in the positivity of roses and chocolates as Valentine’s Day neared.N was specified at the start of data collection and GCS stopped data collection when the specified N was achieved. The rate at which GCS collects data depends on a number of factors not disclosed to the researcher. Because we had no a priori predictions regarding how evaluations change following Valentine’s Day, we focused on data collected during the time period from 2/3/15 to 2/14/15.Respondents were recruited using Google Consumer Surveys (GCS). We recruited respondents in two waves of data collection. Wave 1 began on 2/3/15. Wave 2 began on 2/12/15. For both waves, the desired Table 1. This study was carried out in accordance with the recommendations of Cornell University’s Institutional Review Board. The committee granted the project Exemption from IRB review because it involved survey procedures, no identifying information was requested, and the content of all measures fell under minimal risk.Our sample consisted of 14,793 respondents. We collected information about respondents’ relationship status and divorce history and other demographic information  was inferred by GCS. However, inferred demographic data are not available for all respondents. Detailed information about the sample composition is outlined in d > 0.2 and r > 0.1). Additionally, although the diverse nature of our sample’s characteristics as well as the diverse settings in which they completed the survey , increase the generalizability of our conclusions, it also requires highly precise estimates to detect the effect given these other extraneous factors. We conducted a post hoc power analysis using G∗Power (3.1) software , and groups set to 8 (2 waves × 4 relationship status groups); statistical power (1-β) of the present study to detect a small effect , a Google Inc. product available at a cost at Individuals who are using the Internet and would like to access web content that would normally be behind a pay-wall  are prompted to complete a short survey in exchange for free access to the web content  to 7 (very much). Respondents also responded to the following open-ended prompt: “When you see this image, write down all the words, and/or phrases that come to mind.” We found support that red roses and gift chocolates are more strongly associated with love and Valentine’s Day, compared to the online dating product .We assumed that roses and gift chocolates are attitude objects that also symbolize love, more so than an online dating product, which is associated with relationships but not necessarily love. In a separate supplemental study, we obtained empirical support for these assumptions. Specifically, we presented a different sample (n = 2859) of respondents in wave 2, for whom it was presented first, to check if presenting the status question earlier in the survey affected respondents’ evaluations. Analysis of wave 2 data showed that order of the relationship status question did not affect responses on any of the three evaluation questions  and will not be discussed further. The divorce status question was always asked last.The order in which the set of three evaluation questions and the set of two attachment questions were presented was counterbalanced across participants. The relationship status question was presented last, except for a subset  to 5 (extremely) .Respondents were presented with an image of roses, gift chocolates, and an online dating product  and asked “Which of the following describes your CURRENT relationship status?” and provided the following options: “Single; NOT interested in dating”; “Single; Interested in meeting someone”; “Dating more than one person”; “Dating only one person”; Partnership/Married; and “Other.” We also asked respondents whether they have ever been divorced .We asked respondents: “www.google.com/trends/ that has been used in past research to quantify changes over time in Internet-based information seeking . We used this categorical variable (referred to simply as wave) in all analyses. We also tested our hypothesis using a continuous measure of days until Valentine’s Day by subtracting the specific date in February from 14 .We coded temporal proximity to Valentine’s Day into one of two groups: We performed three repeated measures ANOVAs to test each pairwise comparison . Attitude object  was the within-person factor and wave (wave 1 vs. wave 2) was the between-person factor. We repeated all analyses to examine whether age or gender moderated the effects. In addition, we repeated all our primary tests to statistically control for respondents’ gender and age by including them in the models as covariates . The results of these secondary analyses were highly similar to those reported here .n = 121) for relationship status. Additionally, we excluded respondents who self-identified as “dating more than one person,” because this group had a considerably smaller sample size (n = 374) than the other four groups (ns > 1977). This left a sample size of 14,298 for these analyses. We repeated the three repeated measures ANOVAs described above, with relationship status entered as a categorical variable along with its interaction with wave. We performed all pairwise comparisons using Tukey’s honestly significant difference (HSD) test.Additionally, because past research has found that people’s responses to Valentine’s Day vary depending on their relationship status , we testp = 0.017.Because we tested our full model with three repeated measures ANOVAs (one for each pairwise), we applied a Bonferroni correction and adjusted the criterion for each test to df. To test for significant differences in the magnitude of two dependent correlations , we used Steiger’s Z  assumption was violated, we report adjusted ’s Z ZH; .We repeated all the analyses described above with experimental factors  as covariates . The conclusions do not vary when covariates are included in the analyses.We first assessed descriptive statistics to provide empirical validation of our methodological approach .F = 14649, p < 0.001, M = 3.56, SD = 1.36) were evaluated more positively than roses , and roses in turn were evaluated more positively than the online dating product . Evaluations of roses, chocolates, and the online dating product were all positively correlated with one another. But, notably, the correlation between roses and chocolates  was significantly greater than (p < 0.001) the correlation between roses and the online dating product , and also significantly greater than (p < 0.001) the correlation between chocolates and the online dating product . This is consistent with our assumption that roses and chocolates are more similar to one another, than the online dating product.Evaluation judgments significantly differed across attitude objects  and chocolates , but more negative evaluations of the online dating product .Women, compared to men, expressed stronger positive evaluations of roses .To provide a strong empirical test, we compared temporal changes in evaluations of roses, and of chocolates, to temporal changes in evaluations of the online dating product. As shown in ps < 0.007), but not for the online dating product (ps > 0.121). For example, roses were evaluated more positively Friday, February 13th, the day immediately before Valentine’s Day (wave 2) compared to the Friday, February 6th, 1 week earlier (wave 1).In 2015, Valentine’s Day landed on a Saturday. In our data, evaluations varied meaningfully by the day of the week, with more positive evaluations as the weekend approached compared to earlier in the week  = 11.44, p < 0.001, Interestingly, neither respondent gender nor relationship status moderated the effect of proximity to Valentine’s Day on evaluations of roses and chocolates  and “chocolates”  increased as Valentine’s Day approached, peaked on Valentine’s Day, and decreased following Valentine’s Day. The volume of search queries for “online dating”  did not show the same clear pattern. In fact, in 2015, the frequency of searching for “online dating” peaked the day after Valentine’s Day.An examination of the results obtained from Google Trends data supports our intuitions that the salience of roses and chocolates increases in relevance near Valentine’s Day. As shown in Do these trends in U.S. web search activity in the time period between February 3rd and February 14th of 2015 reflect cyclical, seasonal patterns that occur annually in the U.S. around Valentine’s Day, or are these trends unique to 2015? To address this question, we conducted auxiliary analyses using all available data from Google Trends . In every single year between 2004 and 2016, we found a prominent peak in search frequencies for “roses” and “chocolates.” Moreover, the volume of search queries for “online dating” did not show the same cyclical seasonal pattern. This lack of seasonal web activity for “online dating” further supports the assumption that online dating is not necessarily associated with love and Valentine’s Day. Moreover, the trends data for “online dating” also offers a comparison, showing that it is not the case that people are simply conducting more searches around Valentine’s Day or conducting more searches related to relationships.r(10) = 0.81, SE = 0.38, p = 0.004; 95%CI = ], and chocolates ], but not for the online dating product ]. These patterns provide evidence that not only do evaluations of roses and chocolates become more positive as Valentine’s Day approaches, but that their rate of increase in positivity maps on to the rate of increase in people’s searching patterns for these objects .Importantly, we predicted that the increased positivity in evaluations observed in our sample should covary with population indicators of web search frequencies for “roses” and “chocolates.” Indeed, the search frequency of “roses” and “chocolates” on a given day predicted their respective mean evaluations in our sample on that day. To quantify this association, we used day  as the unit of analysis. The zero-order correlations between sample-level mean evaluations and population-level Google search term frequency were statistically significant for roses [at the moment, shaped by a variety of chronic and temporary influences  remind respondents of Valentine’s Day; instead, respondents simply evaluated images of roses and chocolates, but did so at a time when Valentine’s Day was near or far. Thus, this is a demonstration of naturally occurring cultural priming, which is starkly different from experimentally manipulated standard priming methods used to activate conceptual knowledge . These findings add to the rich literature on the intersection of culture and social cognition. Whereas past work has focused primarily on differences between cultures . Null findings are typically difficult to interpret. However, given the high statistical power (>0.99) of our study to detect even a small effect, the absence of differences is informative.Indeed, the finding that individuals regardless of their relationship statuses and gender showed similar increases in positivity of roses and chocolates as Valentine’s Day approached is noteworthy. The meaning of Valentine’s Day depends on one’s personal, social, and relational goals. To the extent that Valentine’s Day serves as a celebration and validation of one’s relationship, it should activate positive thoughts particularly for those in a relationship . But, throses—love and chocolates—love, not associations that reflect idiosyncratic experiences , web searches for “roses” and “chocolates” show demonstrable peaks during the Valentine’s Day season. This was not the case for “online dating.” Moreover, further supporting the idea that people possess roses—love and chocolates—love associations, there are seasonal peaks of web searches for the term “love” too . Finally, our supplemental study shows that objects more strongly associated with the concept love, are in fact evaluated more positively. Taken together, a conceptual priming account provides the most parsimonious explanation for how cultural events associated with a national holiday affect evaluative judgments.The entirety of the results, including the supplemental study and historical analyses of all existing Google Trends data, support this account. First, the supplemental study shows that roses and chocolates are symbols of love. In the language of conceptual priming, people possess decrease liking for positive attitude objects  increase the activation of culturally relevant concepts in the minds of members living within a given culture, and (2) how they lead to increases in positivity.naturally occurring cultural priming. Contrary to the Stein’s famous quote, rose is not a rose is not a rose is not a rose.Our study shows that cultural events provide a backdrop that shape evaluative judgments—the first demonstration of VZ and JT came up with the hypotheses, study design, and materials for the main study. VZ and GP designed the study and developed the materials for the supplemental study. VZ analyzed the data for the main study, and VZ and GP analyzed the data for the supplemental study. GP generated the historical analysis of the Google Trends Data. VZ, GP, and JT wrote the ms.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pten expression is one of the major mechanisms leading to the aberrant activation of the AKT/mTOR signaling. Here, we show that concomitant downregulation of Pten and upregulation of c-Met occurs in a subset of human HCC, mainly characterized by poor prognosis. Using CRISPR-based gene editing in combination with hydrodynamic injection, Pten was deleted in a subset of mouse hepatocytes (sgPten). We found that loss of Pten synergizes with overexpression of c-Met to promote HCC development in mice (sgPten/c-Met). At the molecular level, sgPten/c-Met liver tumor tissues display increased AKT and mTOR signaling. Using Rictor conditional knockout mice, we demonstrate that sgPten/c-Met-driven HCC development strictly depends on an intact mTORC2 complex. Our findings therefore support the critical role of mTORC2 in hepatocarcinogenesis. sgPten/c-Met mouse model represents a novel valuable system that can be used for the development of targeted therapy against this deadly malignancy.Hepatocellular carcinoma (HCC) is a deadly malignancy with limited treatment options. Activation of the AKT/mTOR cascade is one of the most frequent events along hepatocarcinogenesis. mTOR is a serine/threonine kinase and presents in two distinct complexes: mTORC1 and mTORC2. While mTORC1 has been extensively studied in HCC, the functional contribution of mTORC2 during hepatocarcinogenesis has not been well characterized, especially  However, patients with advanced HCC only experience ~3 months of benefits from Sorafenib or Regorafenib treatment.3 Consequently, it is imperative to elucidate the molecular pathogenesis of HCC in order to develop innovative therapies against this malignancy.Hepatocellular carcinoma (HCC) is one of the major causes of morbidity and mortality worldwide, especially in less developed countries.6 By activation of AKT and other downstream effectors, the PI3K pathway regulates a broad spectrum of processes essential for cancer, including cell survival, proliferation, growth, metabolism and angiogenesis.8 The PI3K pathway can be activated by genetic alterations in PIK3CA, TSC1/2, LKB1 and Pten, or by the activation of upstream inducers such as IGF and HGF/c-Met signaling. This complex signaling network has been shown to play a critical role in hepatocarcinogenesis.11 In normal tissues, the PI3K/AKT pathway is negatively regulated by the tumor suppressor phosphatase and tensin homolog (Pten).8 Expression of Pten is reduced in about half of all HCC tumors, leading to constitutive activation of the PI3K/AKT pathway.13It is well established that the Phosphoinositide-3-Kinase (PI3K)/v-AKT Murine Thymoma Viral Oncogene Homolog 1 (AKT) pathway is frequently dysregulated in cancer.16 c-Met is often overexpressed in human HCC samples and considered to be a therapeutic target in this disease.18The c-Met proto-oncogene encodes the receptor for hepatocyte growth factor (HGF). HGF-induced c-Met activation drives an intricate cascade of molecular events, involving multiple downstream targets, such as the mitogen-activated protein kinase (MAPK) and PI3K pathways. The c-Met signaling has been shown to promote tumor invasion and metastasis by sustaining cell proliferation, survival, migration and angiogenesis.20 mTORC1 functions via regulating cellular growth and metabolism, and it is highly sensitive to Rapamycin. The major downstream targets of mTORC1 include p70 ribosomal S6 kinase (p70S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). p70S6K phosphorylates PRS6, leading to increased glycolysis and lipogenesis. 4E-BP1 functions together with eukaryotic translation initiation factor 4E (eIF4E) to regulate CAP-dependent translation. Unlike mTORC1, how mTORC2 is regulated and its functional contribution to tumorigenesis remain poorly understood.20 AGC kinases, which include AKT, SGK and PKC-α, are considered to be the major substrates of mTORC2, and in turn regulate cell cycle progression, cell survival, and metabolism. In human HCC, AKT has been found to be activated in ~50% of tumor specimens, and is associated with aggressive tumor growth and poor prognosis.22 Our recent and other studies demonstrate that an intact mTORC2 is required for the activation of AKT in vitro and in vivo.23 Furthermore, chromosomal gain of Rictor, the unique and essential subunit of mTORC2, is found in ~25% of human HCCs.13 These data indicate the activation mTORC2 in HCCs. However, the precise mechanisms whereby mTORC2 modulates hepatocarcinogenesis remains poorly understood.The serine/threonine kinase mTOR is one of the major downstream effectors of PI3K signaling. mTOR acts as part of two distinct multiprotein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2).24 As loss of Pten expression is a major mechanism leading to activated PI3K/AKT signaling, we investigated whether ablation of Pten synergizes with c-Met to promote HCC development in vivo. We test the hypothesis by using CRISPR-based technology to delete Pten (sgPten) while co-expressing c-Met using sleeping beauty transposon system and hydrodynamic transfection (sgPten/c-Met). Using sgPten/c-Met induced HCC as preclinical model and conditional Rictor KO mice, we demonstrated the critical role of mTORC2 in hepatocarcinogenesis.We recently showed the oncogenic cooperation between PI3K/AKT and c-Met pathways along liver carcinogenesis.n=25) and longer survival/better prognosis , characterized by <3 and >3 years’ survival following partial liver resection, respectively. The clinicopathological features of liver cancer patients are summarized in A collection of formalin-fixed, paraffin-embedded HCC samples was used in the present study. Fifty frozen HCC and corresponding non-tumorous surrounding livers from the same collection were used. Tumors were divided in HCC with shorter survival/poorer prognosis  for in vivo experiments.The constructs used for mouse injection, including PX330-sgPten, pT3-EF1α-c-met, pT3-EF1α, pT3-EF1α-Cre and pCMV/sleeping beauty (SB) transposase, have been described previously.Rictorfl/fl mice29 from the Jackson Laboratory . Hydrodynamic injection was performed as described previously.30 In brief, the plasmids encoding the genes of interest along with SB transposase in a ratio of 25:1 were diluted in 2 ml saline (0.9% NaCl), filtered through 0.22 μm filter, and injected into the lateral tail vein of the mice in 5–7 s. For the tumorigenesis models, 20 μg sgPten, 20 μg c-Met with 1.6 μg SB plasmid were delivered into FVB/N mouse liver (n=14). To determine the requirement of mTORC2 in sgPten/c-Met-driven liver carcinogenesis, high dose of Cre (60 μg) or pT3-EF1α (60 μg) was mixed with sgPten (20 μg), c-Met (20 μg) and SB (4 μg) and injected into Rictorfl/fl mice . Mice were housed, fed and monitored in accordance with protocols approved by the Committee for Animal Research at the University of California, San Francisco.Wild-type FVB/N mice were obtained from Charles River Laboratories  and the 31 Immunohistochemistry (IHC) was performed as previously described.24 The primary antibodies against c-Met , p-AKTS473 , Pten , fatty acid synthase , acetyl-CoA carboxylase , p-ERK , and Ki67  were used in the present investigation.Liver specimens were fixed in 4% paraformaldehyde and embedded in paraffin. Liver histopathologic analysis on mouse lesions was assessed by two experienced liver pathologists (SR and FD) in accordance with the criteria described in detail previously.24 Equal loading was assessed by GAPDH and/or β-actin. The antibodies used are as follows: Pten , c-Met , p-Met , p-AKTS473 , p-AKT308 , total-AKT , p-c-RafS259 , p-GSK3β , p-PKC , p-PRAS40 , p-mTOR , p-4EBP1 , p-RPS6 , p-ERK1/2 , total-ERK1/2 , HK1 , HK2 , PKM1 , PKM2 , GAPDH , β-Actin .Frozen mouse liver specimens were homogenized in Mammalian Protein Extraction Reagent  containing the Complete Protease Inhibitor Cocktail and sonicated. Protein concentrations were determined with the Bio-Rad Protein Assay Kit  using bovine serum albumin as standard. Supernatant was boiled in Laemmli sample buffer for western blot analysis as previously described.P–values <0.05 were considered as statistically significant.Differences between two groups were analyzed with unpaired t test using Prism 6 Software . n=50). For this purpose, we examined the protein expression patterns of c-Met and Pten in a collection of human HCC specimens by immunohistochemistry. We found that c-Met levels were upregulated, when compared with non-tumorous surrounding counterparts, in 22 of 50 (44%) of the HCC specimens .28 Long-term overexpression of c-Met alone by hydrodynamically injecting pT3-EF1α-c-Met and pCMV/SB into mice led to liver dysplasia, but not tumor development (not shown), in accordance with previous findings.33 Subsequently, we co-injected mice with sgPten, pT3-EF1α-c-Met as well as pCMV/SB (sgPten/c-Met). We found that in sgPten/c-Met mice liver tumors developed as early as ~9 weeks post injection (n=14). Upon dissection, numerous tumors could be found throughout the liver, leading to increased liver weight and liver/body ratio  or activated mutant forms of PIK3CA cooperate with c-Met to induce liver cancer.uman HCC .12, 13 Fpecimens  and 2. Wnjection . By 11–1njection , n=14. Udy ratio .32 No cholangiocellular lesions were detected in the sgPten/c-Met mouse liver. At the cellular level, loss of Pten expression could be easily visualized in sgPten/c-Met HCC lesions  could be found ~9 weeks post injection in the livers of sgPten/c-Met mice. At later time points, liver parenchyma was occupied by confluent HCAs and HCCs . Tumor l lesions . Overexp lesions . Further lesions .de novo lipgenesis, were also detected in sgPten/c-Met tumor cells  and AKT substrates . Increased p-mTOR, p-PKC, phosphorylated 4E binding protein 1 (p-4EBP1) and ribosomal protein S6 (p-RPS6) were also detected, underscoring the activation of the mTOR cascade in sgPten/c-Met mice. It is well known that aberrant metabolism is one of the hallmarks of cancer; and increased glycolysis and lipogenesis are the major metabolic events downstream of mTOR. Consistently, we found high expression of hexokinase 1 (HK1), hexokinase 2 (HK2) and pyruvate kinase 1 and 2, muscle isoform (PKM1 and PKM2), major enzymes involved in glycolysis, in sgPten/c-Met liver tumor lesions . Furtheror cells . PreviouIn summary, our study indicates that loss of Pten synergizes with overexpression of c-Met to induce HCC formation in mice. Tumor cells showed high levels of AKT/mTOR and Ras/MAPK cascades as well as elevated glycolysis and lipogenesis, which are often induced in human HCC samples.35 Increased p-AKT expression in sgPten/c-Met HCC lesions supports the activation of mTORC2 in tumor cells. Thus, sgPten/c-Met mice provide us with an excellent in vivo model to investigate the functional role of mTORC2 in hepatocarcinogenesis. For this purpose, we inactivated the mTORC2 pathway using conditional Rictor knockout mice (Rictorfl/fl mice).29 Specifically, we hydrodynamically injected sgPten, c-Met and Cre plasmids into Rictorfl/fl mice , thus allowing the simultaneous expression of sgPten and c-Met oncogenes, while deleting Rictor in a subset of mouse hepatocytes. As a control, sgPten, c-Met and pT3-EF1α (empty vector) were co-injected into Rictorfl/fl mice   .Rictorfl/fl mice, similar to that observed in sgPten/c-Met injected FVB/N mice  hepatocytes failed to transform into tumor cells; thus, they remained as normal hepatocytes and did not expand. Indeed, individual Pten(−) hepatocytes could be readily found scattered throughout the liver in sgPten/c-Met/Cre Rictorfl/fl mice .39 As mixed HCC and CCA tumors are rather rare entities in humans, it was mandatory to generate a pure HCC model depleted of Pten to further study the molecular signaling triggered by Pten loss in hepatocarcinogenesis.The mTOR cascade is a major signaling pathway regulating cell growth and metabolism. Previous studies have demonstrated the critical role of mTOR in HCC.in vivo modeling of liver tumorigenesis.30 However, the approach is especially successful in inducing liver tumors by the overexpression of oncogenes.30 In order to generate liver tumors with deletion of a given tumor suppressor gene using hydrodynamic transfection, the only feasible approach is to apply shRNA-based gene silencing. However, the shRNA sequence is highly toxic to hepatocytes , and only miR-30-based shRNA can be used in vivo.40 Furthermore, shRNA leads to partial gene silencing, not complete knockout of the target gene. Thus, shRNA-based gene silencing may not be sufficient to induce a phenotype. CRISPR-mediated gene editing is a powerful technology that allows the complete elimination of a target gene in cells.42 Recently, Xue et al.28 demonstrated that combining CRISPR-mediated gene silencing and hydrodynamic injection may be a reliable and efficient way to knockout a target gene in the liver. Specifically, the study showed that hydrodynamic injection of plasmids encoding CRISPR-based gene knockout for Pten and TP53 (sgPten and sgTP53) led to the formation of CCA in mice.28 In the present study, we found that CRISPR-based gene knockout of a tumor suppressor can be combined with SB-mediated somatic integration of an oncogene to induce liver tumor development in vivo. Specifically, we demonstrated that loss of Pten (using sgPten) synergized with overexpression of c-Met to promote HCC formation. The resulting tumors showed loss of Pten and high expression of c-Met, as well as their downstream effectors, including activation of the AKT/mTOR and Ras/MAPK cascades. Thus, our present investigation supports the combination of hydrodynamic transfection with CRISPR mediated gene editing for modeling liver tumor in vivo. It is important to note that unlike liver specific Pten knockout mice, which develop both HCC and CCA, only HCC lesions (but no CCA) were found in sgPten/c-Met mice. Therefore, sgPten/c-Met mice represent an excellent model to study the consequences of Pten loss during HCC development.Hydrodynamic transfection, which combines hydrodynamic tail vein injection with SB-mediated somatic integration, has become an increasingly popular method for 43 In HCC, the investigations on mTORC2 are still very limited. Activated AKT is found in ~50% of human HCCs,21 and AKT activation requires an intact mTORC2.23 In addition, previous studies showed that chromosomal gains at Rictor locus and Rictor overexpression are present in a subset of human HCC samples and associated with early tumor recurrence.23 Interestingly, our most recent analysis using the TCGA data set suggests that mSin1, another unique mTORC2 component, is upregulated in human HCC samples , further supporting the role of mTORC2 in human liver cancer. Obviously, further experiments are required to elucidate the mechanisms whereby mTORC2 is activated in human HCC. In this study, using sgPten/c-Met mouse HCC model in combination with Rictor conditional KO mice, we demonstrate that mTORC2 is required for sgPten/c-Met-driven hepatocarcinogenesis. The results provide solid in vivo evidence for the important role of mTORC2 along hepatocarcinogenesis. Mechanistically, we showed that loss of mTORC2 inhibited AKT activation induced by sgPten/c-Met co-expression. mTORC2 regulates multiple AGC kinases; among them, AKT proteins are considered to be the major mediators.20 In the liver, AKT1 and AKT2 are the only isoforms expressed. Studies have shown that both AKT1 and AKT2 contribute to liver regeneration after partial hepatectomy,44 and may play distinct roles in liver tumorigenesis.45 Further experiments are necessary to identify the kinases downstream of mTORC2 that are required for sgPten/c-Met induced HCC formation in mice.While mTORC1 has been extensively studied in tumorigenesis, including HCC, the functional contribution of mTORC2 in cancer initiation and progression remains poorly characterized.46 It is important to note that the first generation of mTOR inhibitors, the Rapamycin homologs , including Everolimus, are able only to partially inhibit mTOR. Specifically, Everolimus inhibits the S6K/RPS6 axis downstream of mTORC1, but it does not affect the 4EBP1/eIF4E branch of mTORC1 nor it inhibits mTORC2. As a consequence, it is not surprising that clinical studies demonstrated very limited efficacy of Everolimus for cancer treatment. In HCC, Everolimus treatment failed to improve overall patients’ survival.47 The second generation mTOR inhibitors include pan-mTOR and PI3K/mTOR inhibitors. These new inhibitors can suppress both mTORC1 and mTORC2, leading to increased anti-tumor efficacy. Currently, there are only limited studies on the efficacy of second generation mTOR inhibitors for HCC treatment.48 Importantly, the sgPten/c-Met murine HCC model shows high levels of mTORC1 and mTORC2 activities, and our investigation demonstrates its dependence by mTORC2. Therefore, sgPten/c-Met mice are an excellent preclinical model for testing the therapeutic efficacy of the new class of mTOR inhibitors in HCC.HCC is a lethal malignancy worldwide, and a better understanding of the signaling pathways involved in its molecular pathogenesis is required for the development of effective treatments. Conventional chemotherapy has proven to be ineffective to treat HCC patients. Thus, the development of novel targeted therapies is the alternative approach. Mouse models are critical tools to evaluate the therapeutic potential of these new drugs. As mTOR cascade has been shown to be a key regulator of tumorigenesis, mTOR inhibitors have been developed and tested clinically in various tumor types, including HCC.


A novel, more affordable, and widely applicable quantitative polymerase chain reaction (qPCR) protocol analyzing the IgG4/IgG RNA ratio in blood also achieves excellent diagnostic accuracy (n = 125). Moreover, this qPCR test performed better than serum IgG4 levels in sensitivity (94% vs. 86%) and specificity (99% vs. 73%) and correlates with treatment response (n = 20). Conclusions: IgG4+ BCR clones and IgG4/IgG RNA ratio markedly improve delineation, early diagnosis, and monitoring of IgG4‐RD of the biliary tree and pancreas. Immunoglobulin G4 (IgG4)‐related disease (IgG4‐RD) of the biliary tree and pancreas is difficult to distinguish from sclerosing cholangitis and biliary/pancreatic malignancies (CA). An accurate noninvasive test for diagnosis and monitoring of disease activity is lacking. We demonstrate that dominant IgG4 AIPautoimmune pancreatitisANOVAanalysis of varianceAUCarea under the curveBCRB‐cell receptorCCAcholangiocarcinomaCAbiliary/pancreatic malignanciescDNAcomplementary DNACPchronic pancreatitisHISORthistology, imaging, serology, other organ involvement, response to treatmentIACIgG4‐associated cholangitisIgG4immunoglobulin G4IgG4‐RDIgG4‐related diseaseIQRinterquartile rangeNGSnext‐generation sequencingPSCprimary sclerosing cholangitisqPCRquantitative polymerase chain reactionROCreceiver operating characteristicSSCsecondary sclerosing cholangitissIgG4serum IgG4ULNupper limit of normalImmunoglobulin G4 (IgG4)‐related disease (IgG4‐RD) is a multiorgan inflammatory disease that frequently affects the biliary tree  and pancreas .+ B‐cell receptor (BCR) clones in peripheral blood of 6 patients with active IAC, but not in healthy or disease controls, using next‐generation sequencing (NGS).+ clones within the BCR repertoire. Of note, the same clones were encountered in the inflamed tissue.Recently, we identified dominant IgG4+ B cells/high‐power field in combination with marked improvement of pancreatic morphology/biliary strictures and biochemical parameters upon 4 weeks of corticosteroid treatment (group C).Consecutive patients meeting the HISORt diagnostic criteria for IACsIgG4 levels  were measured in 122 patients by automated nephelometry in the local laboratory (using BN ProsPec Siemens in Amsterdam and BNII Siemens in Oxford). For 3 patients, a sIgG4 level was only measured after start of treatment, not at time of RNA collection.Peripheral blood was collected in PAXGene Blood RNA tubes  at the Academic Medical Center in Amsterdam and in Tempus Blood RNA tubes  at the John Radcliffe Hospital in Oxford and handled according to manufacturer's instructions. Simultaneously or at least within 1 week, serum was collected for IgG4 measurement. Processing of blood RNA tubes was performed by a trained technician masked for the origin of the samples or outcome of the sIgG4 levels. In a similar fashion, the person performing the data analysis (M.D.) was blinded. Complementary DNA (cDNA) was synthesized from 1,000 ng of total RNA input using Superscript III RT .heavy sequences were analyzed. NGS visualizes expanded BCR clones as a deviation in the repertoire because the BCR sequence they carry is determined more frequently than low abundant BCR sequences and thus account for a relatively large proportion of all the sequences found. Moreover, it is not unlikely that plasmacytoid cells are identified in peripheral blood; these cells produce increased amounts of BCR messenger RNA, producing a comparable deviation in the repertoire as expanded B cells. For clarity, we will use the term “dominant clone” to denote these clones.The linear amplification protocol has been extensively described earlier.Supporting Fig. S1). Additionally, the following rules were applied: (1) two different reverse primers were designed, one for all possible IgG subtypes and one specific for the IgG4 subtype, and (2) the two reverse primers were designed on virtually the same position to allow comparable conditions. After optimization, this resulted in one universal forward primer (5′‐GCTGCCTGGTCAAGGACTAC‐3′), one generic IgG reverse primer (5′‐TCTTGTCCACCTTGGTGTTG‐3′), and one specific IgG4 reverse primer (5′‐CTACGTTGCAGGTGTAGGTCTTC‐3′). Duplicate qPCR reactions were performed in a 10‐μL total volume in the presence of 10 pmol of the forward and reverse primers, cDNA from 50 ng RNA input, and 5 μL of SensiFAST SYBR Lo‐ROX reagent  for 40 cycles , using the LightCycler 480 system . Both for IgG and IgG4, the starting concentrations in the sample were calculated using LinRegPCR software+ RNA molecules that were IgG4+. Thus, the more expanded IgG4+ clones were present in blood, the higher the percentage IgG4 RNA message of total IgG RNA message.IgG and IgG4 primers for qPCR were designed to specifically amplify sequences that encode the constant region of the heavy chain of the receptor and according to common standards for primer design , Kruskal‐Wallis test of ranks, or χ2 test, where appropriate. A receiver operating characteristic (ROC) curve was used to determine cut‐off values for diagnosis of IgG4‐RD. Diagnostic accuracy was expressed in terms of sensitivity and specificity rates with 95% confidence intervals. Graphpad Prism  and PASW Statistics software  were used to perform statistical analyses. Two‐sided P values of <0.05 were considered statistically significant. This study was performed in agreement with the Standards for Reporting of Diagnostic Accuracy.Following criteria for (non)parametric analysis, values are expressed as mean ± SD or median and interquartile range (IQR). Differences between groups were analyzed using Student Supporting Tables S1 and S2.A total of 50 IgG4‐RD patients meeting the HISORt criteria for IAC and/or AIP were prospectively included at two departments of Gastroenterology and Hepatology in Amsterdam and Oxford between December 2011 and December 2015. In total, 15 patients were included in HISORt group A, 13 in HISORt group B, and 22 in HISORt group C. Forty‐eight patients with PSC, 27 with CA, and 10 patients with CP served as disease controls. Patient characteristics are described in + BCR clones within the IgG+ repertoire of IgG4‐RD patients  revealed multiple dominant IgG4nts Fig. A comparents Fig. B. This iG4+ Fig. C, a greaCRs Fig. D, and a ols Fig. E, confirSupporting Fig. S2). The cutoff for the qPCR test was determined at 5% of all IgG+ RNA molecules being IgG4+ using an ROC curve. Subsequently, the cut‐off value was validated in additional cohorts from two clinical sites . The overall sensitivity and specificity of all cohorts combined was 94.0% and 98.7%, respectively .In order to distinguish IgG4‐RD patients from disease controls on a widely available platform, a qPCR protocol was developed that estimates the fraction of total IgG RNA that encodes IgG4 (qPCR test). Using this qPCR test in blood samples of a Dutch discovery cohort—consisting of 15 IgG4‐RD patients, 7 PSC patients, and 8 CA patients , respectively. Because sIgG4 is part of the diagnostic criteria for IgG4‐RD (HISORt groups B and C), we performed a subanalysis on the different diagnostic subgroups. This showed that the sensitivity of sIgG4 in patients in whom sIgG4 was not part of the diagnostic criteria was lower , whereas the qPCR test performed equally in all three groups .Comparing the findings of the qPCR test to a well‐used marker, sIgG4, the latter showed a sensitivity and specificity for sIgG4 of 85.7% and 73.3% (χP < 0.0001). All patients responded well and showed clinical improvement with resolution of symptoms, together with a decrease of biochemical markers .Subanalysis of follow‐up samples in 20 Dutch IAC/AIP patients 4 and 8 weeks after start of corticosteroid therapy revealed an IgG4 RNA decrease from 20.3% ± 13.4% of total IgG RNA (mean ± SD) before treatment to 5.8% ± 2.2% after 4 weeks and 1.9% ± 0.8% after 8 weeks , in line with other cohorts. This cannot only be explained by the fact that a definite diagnosis of IgG4‐RD in patients with normal sIgG4 needs histology, but it is also known that sIgG4 levels are lower in IgG4‐RD patients after resection for a presumed malignancy given that at least part of the cells producing the sIgG4 are removed. In disease control groups with PSC and CA, however, sIgG4 levels, but not IgG4 RNA, were elevated, contributing to low specificity of sIgG4 levels. This highlights again the usefulness of this qPCR test as a simple and accurate test for diagnosis and monitoring in an otherwise difficult‐to‐diagnose disorder.The observation that a quarter of the IgG+ clones among the top 10 of IgG+ clones. One of the 3 patients was diagnosed with CCA 32 months after fulfilling the HISORt criteria for IgG4‐RD. This emphasizes the need for even more accurate diagnostic criteria than the HISORt criteria and the potential of the presented qPCR test.Three IgG4‐RD patients with elevated sIgG4 had a qPCR score below the threshold of 5%, and repertoire analysis performed in 2 patients showed no dominant IgG4+ B cells on liver biopsy, a phenomenon that is regularly observed in liver explants of PSC patients with a severe disease course.Although most PSC patients with elevated sIgG4 values had normal IgG4/IgG RNA ratio, their qPCR scores were, on average, higher than in PSC patients with normal sIgG4. It has been suggested that PSC patients with elevated sIgG4 levels have worse clinical outcome in terms of disease severity and time to transplantation compared to PSC patients with normal levels.+ BCR clones are supported by a recent study confirming that plasmablasts are present in blood of IgG4‐RD patients.Supporting Table S2) supports our view of specific immune responses underlying IgG4‐RD and may explain the advanced age and male predominance of IgG4‐RD patients.Both our previous and current findings on IgG4+ BCR clones in IgG4‐RD has enabled us to develop an affordable qPCR test, which may allow accurate diagnosis of IgG4‐related disease of the biliary tree and pancreas.In conclusion, identification of dominant IgG4Author names in bold designate shared co‐first authorship.onlinelibrary.wiley.com/doi/10.1002/hep.28568/suppinfo.Additional Supporting Information may be found at Supporting InformationClick here for additional data file.


This paper presents the residual mechanical properties of concrete made with crushed bricks and clay roof tile aggregates after exposure to high temperatures. One referent mixture and eight mixtures with different percentages of replacement of natural aggregate by crushed bricks and roof tiles are experimentally tested. The properties of the concrete were measured before and after exposure to 200, 400, 600 and 800 °C. In order to evaluate the basic residual mechanical properties of concrete with crushed bricks and roof tiles after exposure to high temperatures, ultrasonic pulse velocity is used as a non-destructive test method and the results are compared with those of a destructive method for validation. The mixture with the highest percentage of replacement of natural aggregate by crushed brick and roof tile aggregate has the best physical, mechanical, and thermal properties for application of such concrete in precast concrete elements exposed to high temperatures. Aggregate production is economically and socially important as well: the value of natural raw materials produced annually amounts to approximately 35 billion euros, and the aggregate production industry employs around 250,000 people. The estimated demolition waste material in the EU member states amounts to approximately 180 million tons per annum, which is 1.3 kg a day per capita. There are significant differences in produced construction waste quantities within the European Union; for instance, Germany and the Netherlands produce about 1.9 kg per capita daily, whereas Sweden, Greece and Ireland produce less than 0.5 kg [The production of mineral raw materials in the European Union member states is very dynamic because it is fairly dependent on the realization of and investment in projects approved for a defined period of time . AggregaMore than 80% of the total construction obtained by waste in certain European countries, such as The Netherlands, Belgium and Denmark, is recycled . In thisCrushed clay bricks and roof tiles are among the best aggregates, for use in concrete are known have to resistance at high temperatures, and concrete with such aggregates performs much better than similar concretes containing granite aggregate . Clay br20 [f3 for natural fine aggregate, f10 for crushed and mixed fine aggregates, and f1,5 for the coarse fraction. Based on the listed limits, as shown in Crushed brick (CB), crushed roof tile (CT) and dolomite/limestone  have been used in this study as aggregates for concrete mixtures. There was no need for additional processing of dolomite because it already was separated into fractions. The bricks and roof tiles were crushed at the brick and roof tile factories and delivered to the concrete-mixing location. Since both CB and CT represent waste products in the process of making new bricks and roof tiles, no additional processing of these materials was required. If CB and CT are to be used as aggregates for concrete, it is necessary to prove that they are adequate replacements for conventional NA. The CB and CT were sieved into 0–4 mm, 4–8 mm and 8–16 mm fractions. Based on the granulometric composition, particle size groups were determined as shown in 20 . Table 120 . The larThe coarse aggregate abrasion test (Los Angeles–LA) was conducted in accordance with the standard HRN EN 1097-2/A1  and the Aggregate freezing and thaw resistance were tested in accordance with standard HRN EN 1367-2 , by whicThe grading curves of CB, CT and NA in further tests are shown in CB and CT can be used as full or partial replacement for NA (in this study dolomite aggregates).Special attention must be paid to the concrete composition with regard to the water quantity required and the percentage of replacement of NA by brick and roof tile waste, depending on concrete requirements.The mixture compositions are shown in Portland cement CEM I 42.5 N was used throughout the preliminary experimental test phase. Its characteristic mechanical, physical and chemical properties are in compliance with standard HRN EN 197-1 . PolycarConcrete was mixed in a laboratory mixer of 70 L maximum volume. Each of the nine compositions was mixed three times in order to determine the repeatability of the test results. Concrete was consolidated on a vibrating table with 150 Hz frequency.After the concretes  were mixAs expected, the densities of mixtures containing CB and CB aggregates are lower than the density of the reference mixture with NA.The consistency of all nine mixtures is in accordance with the recommendations of the standard HRN EN 206-1 .The air content is lower in the RM mixture (2.53%) than in the mixtures with CB and CT (4% to 5.5%), which was expected due to the more irregular particle shape of coarse aggregates.By comparing the mixtures in Concrete mixtures were cast in molds and the samples were covered with a plastic sheet; they were then left in molds for 24 h. After they were demolded, the concrete samples were placed in a water tank in a digitally controlled temperature environment of 20 °C until they were tested. The density of CB and CT aggregate concrete is lower than that of the reference mixture, as was expected. Any further increase of the CB and CT percentage may lead to the production of lightweight concretes.The flexural strength of mixtures with CB and CT aggregates is on average 9% lower than that of the NA concrete mixture.The gas permeability of concrete was measured on cylinder-shaped samples, 50 mm in diameter and 50 mm in length. All tests were conducted on three samples and etc.) would result in elements with a higher drying shrinkage contribution at identical strain when compared to concrete with NA. However, in short elements such as floor blocks, the modulus of elasticity plays no significant role, and the use of CB and CT in resisting and semi-resisting floor blocks may therefore be considered possible.According to By comparison with the ultrasonic speed velocity results listed in The drying shrinkage of concrete, as shown in Samples for testing mixture properties after exposure to high temperatures were made using identical mixture compositions, and the samples were cured in an identical manner up until 28 days of age compared to samples tested at room temperature. After 28 days, before their placement in an electric furnace at 105 °C, the samples were kept at room temperature for seven days in order to avoid the emergence of cracks due to the sudden drying at 105 °C. The drying procedure was carried out until the samples were dried completely. In order to be able to design concrete structures as well as to assess the safety with regard to thermal events or after a real accident according to , it is nThe heating and cooling regimes of samples exposed to temperatures of 200 °C and 800 °C are shown in Concrete, like the majority of materials, changes mass under the influence of temperature. The weight was measured before and after high temperature exposure on digital scales with a precision of ±0.1 g.Mass losses after heating are shown in The concrete mass loss after heating was calculated using Equation (1)  in hardened cement paste. According to the results shown in After exposure of concrete to a temperature of 600 °C, the loss of compressive strength becomes more significant: for RM, it is 29%, and for CB and CT concrete, it is 13% to 25%. The samples with the highest percentages of CB and CT aggregate (BM36) show the lowest compressive strength loss at 600 °C, as expected. Crushed bricks and roof tile aggregates concrete enable a better passage of gas through concrete.3 deteriorates, releasing CO2 as gas from the concrete. If the gas exit route is blocked, the resulting pressure causes microcracking and deterioration of the concrete structure. Since the brick and roof tile production process includes a basic limitation of the clay chemical composition, which is a maximum percentage of calcium-carbonate of 4%. CB and CT aggregates of concrete will presumably have a significantly better integration and higher remaining strength after high temperature exposure when compared to dolomite aggregate concrete, which contains around 30% calcium-carbonate. After exposure to high temperatures, the decarbonization of dolomite results in porous dolomite lime with a large specific surface area. The dolomite lime increases its volume under the influence of the earlier described reaction and gas leaves the concrete.At temperatures higher than 800 °C, decarbonization of limestone occurs and the aggregate containing CaCOAll samples exposed to 1000 °C show a low residual compressive strengths, e.g., RM 5%, BM36 11% and BM50 7%. The samples were tested immediately after cooling to room temperature, a day after they were heated. All samples that remained for seven days at room temperature, and humidity conditions deteriorated and fractures occurred as a result of dolomite expansion, as described in the previous section.CB and CT aggregates are sharper and coarse particles, which may result in a better bond between the aggregate particles and cement paste, subsequently resulting in better mechanical properties of concrete. The presented results indicate that CB and CT are better at tolerating high temperatures when compared to dolomite, due to the lower percentage of CaO in the chemical composition and the geometrical properties of its particles, as is evident in the percentage of the remaining compressive strengths illustrated in The modulus of elasticity of concrete decreases after exposure to high temperatures, as is shown in Considering that floor blocks will not be exposed to outside environmental conditions (exposure class X0), it is presumed that the permeability is the property with most pronounced with regard to durability. Since methods of laboratory testing of gas permeability have not been standardized yet but have been described and recommended by Technical Committee RILEM 116-PCD within the European organization RILEM , the tesThe review of possible applications for crushed clay bricks (CB) and roof tiles (CT) waste as partial substitution for natural aggregates (NA) in concrete identified the research area of this paper. The identification of research needs leads to the in depth research and proof of the possibility for using concrete with CB and CT as aggregates for the production of precast concrete elements.The first step in the process of determining the properties of concrete with CB and CT as aggregates comprised experimental tests of CB and CT, which proved that such material may be used as a partial or full substitute for NA, depending on the required concrete properties.Basic concrete properties were determined in order to optimize concrete with CB and CT as aggregates.The densities of concrete mixtures with crushed brick and roof tiles (mixtures BM) are 11% to 14% lower than those of concrete with natural aggregate (mixture RM).Experimental testing on BM and RM samples in the hardened state at room temperature showed that samples with CB and CT as aggregates compared to the reference samples RM had on average: a 27% lower compressive strength, a 16% lower flexural strength, a 19% lower hardened-state density, a 50% lower modulus of elasticity, a 20% lower ultrasonic pulse velocity, a 18% higher air permeability, and 28% less drying shrinkage.It was concluded that special emphasis must be placed on using such concrete for horizontal and vertical structural elements (beams and columns) due to the lower modulus of elasticity and the possibility of larger deformations occurring.The BM36 mixture with the highest percentage of replacement of NA with CB and CT aggregates has the best physical, mechanical, and thermal properties with regard to the application of precast concrete elements exposed to high temperatures.In general, mixtures with CB and CT as aggregates have better physical and mechanical properties than regular concrete after exposure to high temperatures.The conducted laboratory experimental tests on mixtures BM with CB and CT aggregates after exposure to temperatures of 20, 200, 600, 800 and 1000 °C, and the comparison of the results with RM, lead to the conclusions:It is important to mention that this research is not applicable for crushed bricks and tiles in general , but for waste products of the process of making new bricks and roof tiles only. Furthermore, in order to make conclusions about the applicability of concrete with CB and CT aggregates in precast concrete elements in general (regardless of temperature to which the concrete is exposed), further research will be carried out to address the durability properties of the concrete.