The undesirable properties of conventional recycled fine aggregate (RFA) often limit its application in the construction industry. To overcome this challenge, a method for preparing completely recycled fine aggregate (CRFA), which crushes all concrete waste only into fine aggregate, was proposed. The obtained CRFA had high apparent density, and its water absorption was lower than that of the conventional RFA. To take advantage of the CRFA, this paper introduced the modified packing density method for the CRFA concrete mix design. The modified packing density method took account of the powder with a particle size of smaller than 75 μm in the CRFA and balanced both the void ratio and the specific surface area of the aggregate system. Concrete (grade C55) was prepared using the CRFA to validate the feasibility of the proposed method. The unit price of the prepared CRFA concrete was around 12.7% lower than that of the natural aggregate concrete. Additionally, the proposed procedure for the concrete mixture design could recycle all concrete waste into the new concrete and replace all the natural fine aggregate in the concrete mixture. With plenty of urbanization happening over the world, the construction industry gradually becomes the major consumer of the natural resources in the world. Thus, many developing countries face a shortage of natural fine aggregate and the difficulty of dealing with concrete waste. According to the statistics, the global aggregate production has increased from 21 to 40 billion tons from 2007 to 2014 . CDW is Since concrete is made of natural coarse aggregate, natural fine aggregate, and cementitious material, recycled concrete waste is usually classified as recycled aggregate (RA), i.e., recycled coarse aggregate (RCA) or recycled fine aggregate (RFA), and recycled powder. Recycled aggregate consists of original aggregate and the adhered mortar. The porosity of the adhered mortar distinguishes recycled aggregate from the natural aggregate and causes the recycled aggregate to have lower density and higher water absorption. Hence, the quality of recycled aggregate mostly depends on the quality and amount of the adhered mortar .In the conventional recycling procedure, larger recycled aggregate particles are separated from the crushed concrete and used as RCA, and the smaller ones are used as RFA. For this procedure, some researches ,8,9 haveIn order to further enhance the properties of RFA and increase the utilization rate of concrete waste, Yang et al.  introducIn the conventional procedure, a natural coarse aggregate that accounts for 40% of concrete waste is often sieved into RCA instead of RFA, which raises the percentage of mortar in RFA. In contrast, CRFA could make use of this part of natural coarse aggregate since it crushes all concrete waste into fine aggregate. Regarding the crushed products of natural coarse aggregate, the ratio of mortar in CRFA is lower than that in conventional RFA, and the apparent density and porosity of CRFA are better than those of conventional RFA . The advFor CRFA concrete mix design, the packing density method should be an acceptable approach. It minimizes the void ratio of the aggregate system and the consumption of the cement paste by maximizing the bulk density of the blended aggregate ,23. The Since there is not a suitable mix design method available for the CRFA concrete currently, it is difficult to apply the CRFA in the construction industry. In order to solve this problem, this work proposed a modified packing density method, which can not only take the feature of the CRFA into account but also provide a balance between the low void ratio and the low specific surface area. In the rest of the paper, the preparation process of the CRFA and the modified packing density method for the CRFA concrete mix design are introduced in Due to the size limitation of the pilot plant, the steps to remove the impurities of concrete waste and crush them into concrete blocks smaller than 200 mm are not included in the preparation process. The pilot plant preparation process of the CRFA can be divided into two main stages.In the first stage, the concrete waste is crushed by PE250 × 400 jaw crusher . The size of the obtained aggregate can be controlled by adjusting the outlet size of the jaw crusher. All the concrete waste blocks smaller than 200 mm are crushed into blocks smaller than 50 mm.The second stage is the cyclic sand making. The blocks smaller than 50 mm are crushed repeatedly until all the aggregate has an average particle size of less than 4.75 mm. The obtained aggregate with an average particle size of smaller than 4.75 mm is called the CRFA. The equipment used in this cyclic process is composed of a feeder machine, a conveyor belt, a vibrating screen, and the PCX8040 sand making machine . During the procedure, the whole powder produced is retained in the CRFA. The equipment is displayed in When the conventional packing density method is applied to the concrete mix design, it designs the aggregate mixture with the highest density, i.e., maximum packing . AlthougAfter the proportion of the aggregate mixture is obtained, the water-to-cement (W/C) ratio can be determined based on the strength requirement. The volume of the cement paste is equal to the void content of the combined aggregate. In order to coat the aggregate particles and obtain a workable concrete mixture, the paste content should be higher than the void content . The ratAs the water absorption of the CRFA is higher than that of natural aggregate, additional water is required to improve the workability of the CRFA concrete. However, adding too much water will reduce the concrete strength since the CRFA cannot retain the additional water strongly, and part of the absorbed water may flow into the concrete mixture and influence the W/C ratio. In order to take account of this effect, the saturation factor (SF) is introduced herein to evaluate the degree of the saturation of the CRFA; the recommended range of SF is 0–60% . The CRFFor further reducing the cement content and the MFF of the CRFA concrete, a three-stage optimization is conducted. In the first stage, the EF is optimized, and the minimum value satisfying the workability requirement is regarded as the optimal EF. In the second stage, the cement paste is replaced with the CRFA of the same volume to further lower the paste content. The ratio between the paste content after and before the replacement is called the cement paste index (CPI). The slump test is conducted to determine the minimum CPI for workable concrete mixtures. In the third stage, the CRFA is replaced by a natural coarse aggregate of the same volume for decreasing the specific surface area of the aggregate mixture. The minimum MFF satisfying the concrete workability requirement is regarded as the optimal MFF. After the three-stage optimization, the obtained concrete mixture proportion has a low cement paste content, and the obtained aggregate mixture has both a low void content and a low specific surface area.Step 1. Preparing the materialsThe procedure for the proposed CRFA concrete mixture design is outlined in Step 2. Determining the proportion of coarse aggregate of different sizesThe CRFA and coarse aggregate are dried, and their properties  are measured.Step 3. Determining MFFThe proportion that maximizes the compacted bulk density of the coarse aggregate mixture is selected as the proportion of the coarse aggregate.Step 4. Calculating the aggregate contentThe CRFA is added into the coarse aggregate mixture obtained in the previous step. Then, the MFF above which the increment in the bulk density is not remarkable is selected as the initial MFF.3 of concrete can be calculated by Equations (1) and (2):3 of concrete (kg);i in 1 m3 of concrete (kg);3);i.Step 5. Selecting the W/C ratio for the target strength gradeThe aggregate content for 1 m3 of concrete (kg);3 of concrete (kg);3);3);3);i (kg/m3);3);Step 6. Selecting SF and calculating the additional water contentThe W/C ratio of the CRFA concrete is the same as that of the natural aggregate concrete. The content of the water and the cement can be calculated by Equations (3) and (4), respectively:3 of concrete (kg);Step 7. Determining the water reducer contentAn SF is selected based on the W/C ratio. The large SF values are used for the concrete with a low W/C ratio, and the small values are used for the concrete with a high W/C ratio. For example, an SF of 10% is used for the W/C ratio of 0.50, and an SF of 60% is used for the W/C ratio of 0.33. The content of the additional water can be calculated by Equation (6):3 of concrete (kg);The water reducer content (Step 8. Optimizing EFIn the optimization stage (steps 8–10), the content of the water reducer can be adjusted to achieve the best fresh performance of the concrete.Step 9. Optimizing CPIThe workability test is conducted on the concrete with different excess factors, and the minimum value satisfying the workability requirement is regarded as the optimal EF. For different excess factors, the amount of water and cement needs to be recalculated by Equations (3), (4), and (7).3 of concrete in step 9 (kg);3 of concrete in step 9 (kg).Step 10. Optimizing MFFThe workability test is conducted on the concrete with different cement paste indices, and the minimum value satisfying the workability requirement is regarded as the optimal CPI. For different cement paste indices, the content of the water, the CRFA, and the cement need to be recalculated by Equation (4) and Equations (6)–(9). 3 of concrete in step 10 (kg);i in 1 m3 of concrete in step 10 (kg);The workability test is conducted on the concrete with different mass fractions of the fine aggregate, and the minimum value satisfying the concrete workability requirement is regarded as the optimal MFF. For different MFFs, the content of the water, the CRFA, and the coarse aggregate need to be recalculated by Equations (6), (7), (10), and (11). Step 11. Mixture proportion adjustmentThe obtained optimal proportion is checked for meeting the strength requirement. If the strength requirement is not satisfied, we go back to step 5 and reselect the W/C ratio and SF.3 of concrete is calculated by:3 of concrete at the optimal proportion (kg);i in 1 m3 of concrete at the optimal proportion (kg);3 of concrete at the optimal proportion (kg);3 of concrete at the optimal proportion (kg);3 of concrete at the optimal proportion (kg);3).The concrete mixture proportion is adjusted by the concrete apparent density. The adjusted material content of 1 mThe properties of P.II 42.5R Portland cement used in this work are listed in The concrete waste (strength grade: C40) used for preparing the CRFA was obtained from the blocks of the hollow slabs and beams of a highway viaduct. The coarse aggregate and the fine aggregate of the concrete waste were the crushed granite stone and the river sand, respectively.Two types of natural coarse aggregate (crushed stone) with a particle size of 10 and 20 mm were used. The properties of the crushed stones are presented in HG7 polycarboxylate water reducer at a solid content of 20% was employed as the admixture.Eight physical properties of the CRFA, namely, particle gradation, fineness module, the content of fine powder, crush index, apparent density, saturated-surface-dry water absorption, loose bulk density, and loose void volume, were tested by the methods suggested in standard GB/T 25176-2010 “Recycled Fine Aggregate for Concrete and Mortar”  and stanThe particle appearance of the CRFA of five different particle sizes of 1.18–2.36 mm, 0.60–1.18 mm, 0.30–0.60 mm, 0.15–0.30 mm, and 0–0.15 mm was also analyzed using Olympus, SZX10 stereomicroscope . The powder particles smaller than 0.15 mm were sieved by a 0.080 mm standard cement negative pressure screen. After that, the dried powder particles smaller than 0.080 mm were divided into two samples. One sample was gilded by Cressington 108 Sputter Coater ), and microcosmic analysis was conducted on it by ZEISS EVO-18 Premium scanning electron microscope . The particle distribution of the other sample was measured by using the Horiba LA-950 laser particle size analyzer .For each concrete mixture proportion, the initial slump, the slump after 1 h, the initial slump-flow, and the slump-flow after 1 h were tested by the methods recommended in GB/T 50080-2016, “Standard for The Test Method of Performance on Ordinary Fresh Concrete” . Cubic sThe appearance and particle grading of the CRFA produced by the procedure described in The CRFA powder with a particle size of smaller than 0.080 mm was characterized using laser particle size analysis. The particle size distribution of the powder is presented in The mixture of the CRFA concrete (grade C55) was designed by following the procedure described in 3) is obtained. Thus, the optimal proportion of the coarse aggregate is 20% of crushed aggregate 1 plus 80% of crushed aggregate 2.All the aggregate was dried according to step 1. In step 2, two types of a coarse aggregate of different sizes, namely, crushed aggregate 1 and crushed aggregate 2, were mixed at different proportions by mass. Six different mass fractions of crushed aggregate 1, i.e., 0, 10, 15, 20, 25, and 30%, were tested here. The compacted bulk density of the coarse aggregate with six different proportions is listed in 3; therefore, the initial MFF is 40%. The primary mix proportion of aggregate is 40% of the CRFA, 12% of crushed aggregate 1, and 48% of crushed aggregate 2. Mixed at this proportion, the aggregate mixture has both a low void content and a low specific surface area. As the compacted bulk density of 1 m3 of the aggregate mixture at this proportion is 1835 kg/m3, the content of the CRFA, crushed aggregate 1, and crushed aggregate 2 in 1 m3 of the concrete is equal to 734, 220, and 881 kg/m3, respectively.Following step 3, the MFF was set at 0, 36, 38, 40, 42, 44, 46, 48, and 50%. The compacted bulk density of the aggregate mixture obtained from different mass fractions of the fine aggregate is presented in 3, as determined by some pre-experiments. In the trial test, an excess factor of 1.05 just met the workability requirement, so step 8 is not reported herein. The initially calculated mixture proportion obtained from steps 1–8 is presented in A W/C ratio of 0.333 and an SF of 60% were selected in step 5 and step 6. The initial content of the water reducer in step 7 was set at 7.4 kg/mIn step 10, the CRFA is replaced with the coarse aggregate of the same volume. The mass fraction of the CRFA varies from 41.2 to 37.2%, with increments of 2%. The calculated mixture proportions and the corresponding performance of the concrete samples in step 10 are presented in Compared with the initial proportion, i.e., R11, the concrete mixed in proportion R22 has better workability and higher compressive strength. The 28-day compressive strength of the concrete mixed in proportion R22 is 64.0 MPa, which is larger than 55 + 1.654 × 5 MPa and satisfies the requirement of concrete grade C55. Furthermore, the final optimum MFF of 39.2% is smaller than the MFF of 44% measured by the conventional dry packing density method. This phenomenon resembles the wet packing density method; in fact, the MFF calculated by the wet density method is around 5% smaller than the one measured by the dry density method . Therefo3, so proportion R22 should be modified by using the apparent density to measure the material consumption for 1 m3 of the CRFA concrete. The apparent density of the concrete mixed in proportion R22 is 2304 kg/m3, and the final mix proportion for 1 m3 of the CRFA concrete is presented in The amount of concrete obtained by using proportion R22 shown above is often larger than 1 m3 of the CRFA concrete can be evaluated by the material prices in Guangzhou, China, as presented in 3. The price of the natural fine aggregate concrete can be evaluated approximately by using the river sand of the same mass in the proportion; thus, the unit price of the river sand concrete is calculated as 502.78 RMB Yuan/m3. It can be concluded that using the CRFA is economically feasible and can reduce the concrete unit price by around 12.7%, which is attractive in the construction industry.The cost of 1 mIn order to improve the properties of conventional recycled fine aggregate, the CRFA is introduced in this work. Instead of crushing part of concrete waste into recycled coarse aggregate, the technique used for producing the CRFA crushes all concrete waste completely into fine aggregate. Since the crushed natural coarse aggregate in concrete waste constitutes about 40% of the CRFA, the proportion of the mortar is lower in the CRFA than in conventional recycled fine aggregate, which increases the apparent density of the CRFA and decreases the water absorption of the saturated-surface-dry CRFA.For taking advantage of the CRFA, a procedure for the concrete mix design based on the modified dry packing density method is proposed. Different from the conventional packing density method, the modified approach chooses the mass fraction of the fine aggregate above which the increment in the packing density is not noticeable. The modified approach can take account of the fine powder in the CRFA and provide a balance between the low void content and the low specific surface area. Additionally, the modified approach can also be suitable for other fine aggregates with a high powder content.To validate the proposed procedure for the CRFA concrete mixture, concrete grade C55 was prepared. The obtained CRFA concrete can reach the strength requirement of concrete grade C55 and offers suitable fresh performance and lower cement content. The optimum mass fraction of the fine aggregate calculated herein is 4.8% smaller than the MFF figured out by the conventional packing density method. Moreover, the CRFA concrete is well economical, and its unit price is around 12.7% lower than the natural aggregate concrete. Furthermore, by applying the proposed procedure, all concrete waste can be reused in the new concrete, and natural fine aggregate can be 100% replaced by the CRFA.


R (oxytetracycline) and TylanR (tylosin tartrate). These effects of antibiotic treatments suggest a role of microbiota in the interaction between the fat body and brain that is important for honeybee behavioral development.Recurrent honeybee losses make it critical to understand the impact of human interventions, such as antibiotic use in apiculture. Antibiotics are used to prevent or treat bacterial infections in colonies. However, little is known about their effects on honeybee development. We studied the effect of two commercial beekeeping antibiotics on the bee physiology and behavior throughout development. Our results show that antibiotic treatments have an effect on amount of lipids and rate of behavioral development. Lipid amount in treated bees was higher than those not treated. Also, the timing of antibiotic treatment had distinct effects for the age of onset of behaviors, starting with cleaning, then nursing and lastly foraging. Bees treated during larva-pupa stages demonstrated an accelerated behavioral development and loss of lipids, while bees treated from larva to adulthood had a delay in behavioral development and loss of lipids. The effects were shared across the two antibiotics tested, TerramycinThis paper has an associated First Person interview with the first author of the article. Summary: Antibiotic treatments in bee colonies cause an increase or decrease in rate of behavioral development and lipid stores of workers, affecting fat body–interaction, likely through effects on microbiota. Apis mellifera; Linnaeus) is a generalist pollinator and their colonies are managed and used globally in agriculture for the pollination of many crops and fruit grown in the open fields such as such as apples, almonds, blueberries, and cranberries . Bees treated with antibiotics during pupa stage exhibit a higher lipid content at 1 day of age. However, bees that were removed from antibiotic treatment after immature development (+/−), expressed a lower lipid profile than those in antibiotic treatment during all developmental stages (+/+). Bees that were placed in treatment only during adult development (−/+) had a similar lipid profile to (+/+) after being introduced to an antibiotic treated colony (on days 7 and 14 of age). There is no significant difference in the lipid amount between oxytetracycline and tylosin tartrate . See There is a trend of lipid change by age . Colonies were determined to not be an influence factor in the rate of behavioral development, generalized linear mixed model (GLMM) analysis showed that colony effect explained very little of the variation (0.002%) for each of the worker counts.Behavioral development was examined by recording job performance at different ages of bees in the four treatment groups . There w31.67, P≤0.0001). However, bees treated with antibiotics throughout their entire development (+/+), had a delayed rate of development . These individuals maintained a higher fat reserve for the duration of the experiment and the transition to stable lipid loss was delayed or did not occur  had a faster rate of development, shifting from hive jobs to field jobs quicker  lack the necessary bacteria (or numbers of bacteria) to have a steady metabolic rate. Similar to +/+, when −/+ are introduced to an antibiotic colony, they might lose that important bacteria at being exposed to antibiotics with no fast way of regaining them, and the effects are shown in their rate of behavioral development.Additionally, antibiotic effect on development seems to depend on the timing of treatment. This timing could relate to a rescue effect of microbiota due to nest-mate microbiota. Studies have shown that newly emerged workers gain their microbiota through nest-mate interactions . Once beOur findings may have other general implications for chemicals that bees encounter in their environment. For instance, the herbicide glyphosate also alters microbiota beneficial for honeybee growth and defence against pathogens . The intn=12) colonies of A. mellifera were selected for the experiment. The experiment was conducted during the summer seasons of 2013  and 2014  with different hives. The apiary is located at the University of Puerto Rico's Agricultural Experimental Station in Gurabo, PR, (18°15′26.6″N 65°59′11.5″W). Colonies were screened and health assessed. Colonies that had received previous treatment, or were sickly or weak were excluded. Colonies were paired based on hive population and composition. Selected control colonies were treated with powdered sugar, the vehicle of antibiotic. Experimental colonies were treated with the commercial oxytetracycline, Terramycin , or with tylosin tartrate, Tylan , following the recommended commercial dose of the powdered antibiotic.A total of twelve (n=12) in a general cross-fostering design  and trial 2, treatment with Tylosin tartrate . Colonies were paired based on composition and randomly assigned a treatment; control or antibiotic. Initial treatment was performed in standard (40.6 cm W×50.4 cm L×24.4 cm D) one-story wooden hive boxes with eight frames. After 3 weeks of treatment, brood frames were collected from each pair and placed in an incubator  at 33°C for 24 h. From each colony approximately 500 emerging bees were marked on the thorax with numbered color tags  to indicate source colony and treatment  and the antibiotic colonies (n=10) for each of the trials, prior to cross fostering. Also, an additional 100 bees from each colony were marked with paint of different colors in the thorax to indicate source colony and treatment and were cross fostered. For example, 100 bees from C1 and 100 bees from A1 were marked with paint, 50 from C1 stayed in C1 while the other 50 were cross fostered with A1, and in the same manner 50 bees from A1 were cross fostered with C1, to be later collected for the fat content analysis. This was performed for each pair of colonies.Additionally, for further fat content analysis, twenty newly emerged bees (not numbered tagged) were collected at random from the control . Bees collected from the −/− and +/+ groups at 1 and 7 days of age were washed in 70% ethanol  and had their digestive tracts removed. The digestive tracts were homogenized by vortex mixer in 10 mL of nutrient broth medium  followed by dilution plating on nutrient agar (Himedia) and incubated at 35°C under aerobic conditions for 48 h . Individual bacterial colonies were counted and multiplied by the degree of dilution to obtain CFU of the original sample (We examined the efficacy of antibiotics by measuring colony-forming units (CFU) . Of note, 1-day-old bees were collected only from the −/− and +/+ groups. This is due to bees being newly emerged and thus not cross-fostered at this age. Age of collection was selected to match onset of behaviors as described by Since bees present a lipid profile that follows worker tasks and age stages , previouCollected bees were dissected by separating the abdomen from the thorax. The entire digestive tract, along with the sting apparatus and any wax scales observed on the outside were removed from the abdomen.To measure lipid content of bees by age and treatment, we used the total body fat extraction method as described by To examine differences in rate of behavior development, the four treatment groups were followed and recorded in glass-walled observation hives with eight frames, in two daily scans of 2 h each (at 10:00 and at 14:00 h) until the beginning of foraging activities. We quantified cleaning behaviors (removing debris from honeycomb cells), brood cell visits (nursing), and foraging behavior during daily behavioral scan samples . Age at To measure rate of development, we focused on earliest performance of behaviors by focal group of individuals. Bees were divided by age group, representative of the age cohorts by task: cleaning, nursing and foraging, should be performed or start by : 3–6, 7–U-test to determine whether the CFU count was significantly different between −/− and +/+ at 1 and 7 days of age.We performed a Shapiro-Wilk test to determine if the distribution of the CFU counts was significantly different than normal. After confirming the data was not normal, we performed a Mann–Whitney Since trial 1 (oxytetracycline) and trial 2 (tylosin tartrate) were conducted at two different summer seasons and with different colonies, data from both antibiotics were analyzed as independent factors. We performed a Shapiro-Wilk test to determine if the distribution of lipid content was significantly different than a normal distribution. After confirming the data did not showed a normal distribution, we performed a square-root transformation to ensure our data followed a normal distribution. After the assumption of normality was met, we performed a three-way ANOVA with type of treatment, age and antibiotic as independent factors to determine their interaction with lipid content. A Tukey test was used as a post-hoc analysis.We determined that the behavior count data was over dispersed, therefore we used negative-binomial generalized-linear mixed model (GLMM) to determine if job count intercept and slope varied by hive. We determined that colony effects explained very little of the variation (only a 0.002%) for each of the worker counts. Therefore, we used a negative-binomial generalized-linear model (GLM) to examine the number of workers performing each task using type of treatment, age and type of antibiotic as independent factors.https://doi.org/10.5061/dryad.gf1vhhmn2) was analyzed using the statistical program R (Data generated (rogram R  v. 3.5.2


Meanwhile, 5-Aza-dC treatment decreased DNA methylation and upregulated the expression of GABRD in the NAc, whereas MET had the opposite effect. Our results also reveal that 5-Aza-dC might alter the methylation landscape of the GABRD gene by directly repressing DNMT1 and DNMT3A expression. Furthermore, reinstatement of heroin-seeking behavior was significantly inhibited by directly overexpressing GABRD and remarkably reinforced by GABRD gene silencing in the NAc. Collectively, these results suggest that targeting the GABRD gene and its methylation might represent a novel pharmacological strategy for treating heroin addiction and relapse.Epigenetic modifications such as DNA methylation play important roles in regulating gene expression and may mediate neuroplasticity and lead to drug-induced aberrant behaviors. Although several brain regions and neurobiological mechanisms have been suggested to be involved in these processes, there is remarkably little known about the effects of DNA methylation on heroin-seeking behavior. Using a Sprague-Dawley rat model, we show that heroin self-administration resulted in gamma-aminobutyric acid type A receptor subunit delta (GABRD) gene hypomethylation, which was associated with transcriptional upregulation of GABRD in the nucleus accumbens (NAc). Systemic  Heroin addiction is a chronic and relapsing brain disorder characterized by compulsive drug-seeking and the negative emotional state of withdrawal. Relapse to drug-seeking after abstinence has long been a challenge in the treatment of heroin addiction. Heroin addiction is characterized by specific behavioral alterations, indicating long-lasting alterations in gene and protein expression within specific reward-related brain regions. Accumulating evidence has demonstrated that epigenetic mechanisms such as DNA methylation regulate drug-induced gene expression profiles and enduring behavioral phenotypes . Recent de novo” methyltransferases that establish new methylation patterns (DNMT3A and DNMT3B) , polo-like kinase 2 (PLK2) and protein-tyrosine phosphatase non-receptor type 5 (PTPN5) (DNA methyltransferases (DNMTs) are responsible for adding methyl groups to cytosine-guanine dinucleotides (CpGs) in the genome . In mamm DNMT3B) . CpG isl DNMT3B) . Conside DNMT3B)  found th (PTPN5) . Changes (PTPN5) . Additio (PTPN5) . Interes (PTPN5) . Previou (PTPN5) . Xu et aγ-aminobutyric acid (GABA) A receptors (GABAARs) are the crucial inhibitory neurotransmitter receptors in the central nervous system. As a class of transmembrane ligand-gated chloride channels, GABAARs consist of five subunits from the 19 known different subunit isoforms, α1–6, β1–3, γ1–3, δ, ε, θ, π, and ρ1–3 . The chaIn the present study, we combine global whole-genome and candidate gene approaches to investigate the effect of DNA methylation on heroin-seeking behavior by analysing DNA methylation profiles and the correlated gene expression changes in the NAc of rats. Our research also explores whether DNA methylation regulates GABRD gene transcription in the NAc after repeated heroin treatment, and whether the pharmacological enhancement or inhibition of DNMTs alters heroin self-administration and the reinstatement of heroin-seeking behavior in rats. Finally, we investigate whether overexpression or down-regulation of GABRD in the NAc of rats significantly alters the associated cues-induced or heroin priming reinstatement after extinction of heroin self-administration.https://www.ncbi.nlm.nih.gov/sra/PRJNA673675). On average, more than 331,106,942 sequence reads were obtained per sample, the differentially methylated regions (DMRs) were identified between the three groups. The methylKit and eDMR R package was used for comprehensive DMRs analysis, the eDMR can optimize regional methylation analysis based on bimodal normal distribution and weighted cost function. A comparative analysis revealed that there were 38,177 DMRs between the heroin self-administration and the yoked-saline group, and 23,129 DMRs existed between the yoked-heroin and the yoked-saline group. As shown in p < 0.05, p < 0.05). Besides, methylation level of the GABRP gene  was shown to be increased in the heroin-self-administration compared with that in the yoked-saline group (p < 0.05)  .r > 0.50, p < 0.05). Therefore, the average percentage value for 1–5 CpG sites were used for subsequent analysis. The average GABRD methylation levels were significantly reduced in heroin self-administration group as compared to those in the yoked-saline group .To further confirm the results, pyrosequencing was performed. Significant correlations among five CpG sites in GABRD were observed ; besides, there were significant correlation between CG3 and CG4 . In total, the average methylation levels of GABRP were obviously increased in heroin self-administration and yoked-heroin groups compared with that in the yoked-saline group . The multiple comparisons illustrated that GABRD mRNA expression in the heroin self-administration group increased compared with that in the yoked-saline and yoked-heroin groups  = 0.056, p = 0.946]. These results revealed a significant negative correlation between GABRD gene methylation and mRNA expression in the NAc.The relative mRNA expression of GABRD and GABRP were determined by RT-qPCR; one-way ANOVA showed that GABRD expression was significantly different in the three groups . The post hoc comparisons showed fluorescence ratios in pGL3-promoter-GABRD which contains the GABRD segment that included five CpG sites were significantly higher than that in basal reporter plasmids (pGL3-promoter vector). These results revealed that the candidate fragment of GABRD had somehow enhancer-like activity  = 4.175, p = 0.071]  and interaction (MET and heroin)  on GABRD gene methylation; however, no effect of heroin on methylation was observed . Thereafter, post hoc multiple comparisons were performed, and the results showed that the GABRD gene exhibited an overall increase in DNA methylation in the Her + MET group, and there were significant differences between Her + MET vs. Her + Sal (p = 0.003), Her + MET vs. Con + MET (p = 0.034), and Her + MET vs. Con + Sal (p = 0.047) groups  = 4.87, p = 0.045], CpG3 , and CpG4 . Meanwhile, significant interaction of MET treatment and heroin effects at CpG3  and CpG5  were detected. Conversely, contrary to the methylation levels, mRNA expression level in the Her + MET group was obviously lower than that in the Her + Sal group , heroin , and interaction (heroin and 5-Aza-dC)  on active responses during heroin reinstatement. Furthermore, the post hoc analysis showed that heroin priming reinstatement was prevented by pre-treatment with 5-Aza-dC   .r > 0.743, p < 0.001). Two-way ANOVA showed a significant main effect of 5-Aza-dC  and heroin  on the average methylation levels of these five CpG sites. Meanwhile, there was significant effect of 5-Aza-dC at the CpG1 , CpG2 , CpG4 , and CpG5  sites. Simultaneously, significant effects of heroin at CpG1 , CpG2 , CpG3 , CpG4 , and CpG5  were detected. The significant effect of interaction was only indicated at CpG3 . Furthermore, the multiple comparisons revealed that average methylation levels of the GABRD gene were significantly decreased after treatment with 5-Aza-dC  = 6.10, p = 0.029 and F = 9.79, p = 0.009] for main effect of 5-Aza-dC, respectively; F = 5.50, p = 0.037 and F = 5.40, p = 0.038 for main effect of heroin self-administration, respectively. However, no interaction effect 5-Aza-dC and heroin self-administration was observed , but DNMT3B expression remained unaffected . Post hoc multiple comparisons showed that DNMT1 and DNMT3A levels decreased in the heroin self-administration group after treatment with 5-Aza-dC in the NAc .Pyrosequencing was used to investigate the effect of 5-Aza-dC on DNA methylation. The results revealed significant correlations among the five GABRD CpG sites (4.64E−4] . Additio4.64E−4] . Two-way(s.c.): 1,428.71 ± 151.36 vs. 1,585.29 ± 147.07, p = 0.42; 5-Aza-dC vs. aCSF(intra-NAc): 1,640.86 ± 204.93 vs. 1,456.29 ± 173.56, p = 0.28].As shown in F = 0.083, p = 0.78, F = 8.83, p = 0.01, p = 0.038, We investigated whether GABRD overexpression or RNAi in the NAc of rats would cause changes in heroin reinstatement. Our results showed that over-expression of GABRD in the NAc had no effect on cues-induced reinstatement of heroin-seeking behavior  (1.4 ± 0.1 mm thickness), and serial 1 mm brain sections were cut . The NAcGenomic DNA was extracted from the NAc using the QIAamp DNA Mini kit . DNA was sheared and then purified using the QIAquick PCR purification kit, and libraries were constructed using the Illumina library preparation kit. Thereafter, the QIAquick PCR purification kit was used to purify the libraries, and the EpiTect bisulfite kit  was suitable for bisulfite conversion. Bisulfite converted libraries were amplified using PfuTurbo HotStart DNA polymerase , size selected (200–300 bp), quantified using qPCR, and run through the Illumina HiSeq 2000 platform as previously described . The dowDouble luciferase reporter assay was carried out to verify the enhancer activity of the target sequence and examine the effect of methylation on its activity. Reporter gene vectors (pGL3-promoter) carrying GABRD DNA fragments (GABRD: chr5:172809066–172809129) were constructed as follows. The pGL3-promoter was digested with NheI/XhoI, and the recombinant plasmids were transformed into TOP10 competent cells. The correct clones were subjected to sequencing, and the plasmid was extracted using TIANprep Mini Plasmid Kit  after verification by sequencing. The constructed plasmids were then methylated using the CpG methyltransferase M.SssI Kit  to establish the methylated groups. Thereafter, the EpiJET DNA Methylation Analysis Kit was used to perform the methylation test. HEK293T cells were cultured in Dulbecco's modified Eagle medium  with 10% fetal bovine serum  and 1% penicillin/streptomycin . HEK293T cells were stably co-transfected with recombinant plasmid or methylated plasmids using the Lipofectamine 2000 Transfection Reagent  after the cells reached 70% confluence. The reporter assay was carried out using the Dual-Luciferase Reporter Assay system  two days after co-transfection according to the manufacturer's protocol. The recombinant plasmids were divided into 3 groups: i) pGL3-promoter (Pro), ii) pGL3-promoter-GABRD (Pro-GABRD), iii) Me-pGL3-promoter-GABRD (Me-Pro-GABRD).GABRD and GABR pi (GABRP) PCR and sequencing primers were designed using the PyroMark Assay Design 2.0 software. The sequences of the forward primers are shown in ® 480 system (Roche Applied Science), and the 2−ΔCq method was used to analyze the relative changes in mRNA expression levels.Total RNA was extracted and reverse-transcripted into single-strand cDNA according to the method described previously . RT-PCR For protein analysis, NAc samples were homogenized in 300 μl ice-cold lysis buffer (SDS: PMSF = 100: 1) and then centrifuged at 12,000 rpm for 20 min at 4°C. Supernatants were collected, and the total protein concentration was measured using the bicinchoninic acid protein assay reagent kit . The protein solutions were mixed with loading buffer, boiled at 100°C for 5 min, and then separated by 10% SDS-polyacrylamide gel electrophoresis and electroblotted onto a polyvinylidene difluoride membrane . Then the membrane was blocked with 5% skimmed milk for 1 h at 37°C. Subsequently, the membrane was incubated overnight at 4 °C with the appropriate antibodies—anti-GABRD , anti-GAPDH , anti-DNMT1 , anti-DNMT3A , and anti-DNMT3B . Thereafter, membranes were washed three times with PBST  (10 min each) and incubated with secondary antibody (Goat anti-mouse IgG (H + L) − HRP, 1:10,000, Jackson ImmunoResearch) for 2 h at 37 °C and then washed five times with PBST (5 min each). Protein bands were detected using a chemiluminescence detection system . Protein expression intensities were expressed as ratios relative to that of GAPDH based on gray-scale analysis.p < 0.05 was considered indicative of a statistically significant difference. Gene methylation levels were analyzed using Pearson's regression analysis and one-way or two-way analysis of variance (ANOVA) followed by Student–Newman–Keuls post hoc comparisons. One-way and two-way ANOVA was used to analyze mRNA or protein expression, with treatment and heroin self-administration as the independent variables. Effects of MET or 5-Aza-dC on the nose-poke responses and infusions during heroin self-administration and reinstatement were analyzed using two-way repeated-measures ANOVA with treatment group and time as factors. Effect of MET and 5-Aza-dC treatment on locomotion activity, and Effect of GABRD overexpression or RNAi on protein expression level of GABRD were compared using Student t-test.Statistical analysis was performed using SPSS 16.0  and GraphPad Prism 5.0 . All data are expressed as the means ± standard deviations (SDs); https://www.ncbi.nlm.nih.gov/sra/PRJNA673675.Our Illumina sequencing data to SRA database had been successfully processed and will be released on 2024-10-01, Accession to cite for these SRA data: PRJNA67367. Our SRA records will be accessible with the following link after the indicated release date: The animal study was reviewed and approved by Laboratory of Behavioral Neuroscience, Animal Care and Use Committee of the Ningbo Institute of Microcirculation and Henbane .HL designed the study; QH, WZ, and HL wrote the manuscript; WX, ZL, JL, and WC performed the experiments; HZ, ML, DZ, ZX, and DF analyzed the data.This work was supported by the National Key R&D Program of China (Grant No. 2017YFC1310400), The Natural Science Foundation of Zhejiang (Grant No. LY18H090008), The Natural Science Foundation of China (Grant No. 81671321), and The Ningbo Science and Technology Project .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The effect of mill-rejected granular cement (MRGC) on enabling concrete to autogenously heal its cracks was investigated. The crack-healing efficiency of concrete containing 5%, 10%, 15%, and 20% wt. of MRGC as a replacement for natural fine aggregate was investigated at the age of 28 days. Concrete specimens were induced with artificial cracks and placed in water or air at 20 ± 2 °C to cure and heal the cracks for an additional 28 days. Compressive, flexural, and tensile strengths and water permeability tests were carried out to evaluate crack-healing by evaluating the strength to regain and the reduction in water permeability of concrete. For the air-cured specimens, the gain in compressive strength was between 45% and 79%, the flexural strength was between 74% and 87%, and the tensile strength was between 75% and 84% of the reference specimens for the MRGC content was between 0% and 20%, respectively. For the water-cured specimens, the gain in compressive strength was between 54% and 92%, the flexural strength was between 76% and 94%, the tensile strength was between 83% and 96% of the reference specimens for the MRGC content between 0% and 20%. The water permeability coefficients of the concrete specimens cured in water after cracking decreased by one order of magnitude, while those of the specimens cured in the air increased by the same order of magnitude. The crack-healing efficiency of concrete could be enhanced by increasing the MRGC content of concrete and hydration water. Cement concrete is a man-made composite rock composed of coarse aggregate, fine aggregate, and cement paste. The conventional cement concrete referred to as concrete uses ordinary Portland cement (OPC) paste to bind the aggregates and fill the voids between them. Therefore, cement paste plays a vital role in the properties and performance of concrete. When OPC is mixed with water, a chemical reaction (hydration) takes place and gives off various products. Calcium silicate hydrate (C-S-H) is a predominant cement hydration product responsible for the cementitious property of cement paste. Generally, the strength of concrete could be defined as the bond strength between the C-S-H gel and the aggregates.Concrete is the most common construction material worldwide. According to Meyers , the gloConcrete, even though it has many advantages, has some limitations that negatively affect its performance. For example, the fact that concrete being strong in compression but weak in tension makes concrete more susceptible to cracking under minimal stress intensities. Concrete cracks form when the tensile stress exceeds the tensile strength of the concrete. The tensile stress in concrete can be induced by internal or external factors such as shrinkage, expansive chemical reactions, temperature variation, freeze-thaw cycles, poor construction practice, external loads, etc. ,3. Crack3), C-S-H, crystallization of calcium hydroxide (Ca(OH)2, and sediments. When water containing dissolved atmospheric carbon dioxide enters concrete cracks, the carbonation reaction takes place between the carbon dioxide and calcium hydroxide present near the cracked surfaces. CaCO3 produced from the carbonation reaction may fill the crack space to prevent further ingression of water. The crystallization of newly formed calcium hydroxide on the cracked surfaces could also play a role in narrowing down the crack widths by filling up the crack space. Crack space can also be filled with sediments present in the water entering the cracks. Unhydrated cement particles present in the vicinity of the cracked surfaces’ hydrate in the water entering the cracks produce C-S-H gel, which could also fill the crack space and bind the cracked surfaces [Several researchers have introduced some techniques for the maintenance and repair of cracks in concrete structures ,8,9,10. surfaces ,30,31.One of the autogenous self-healing mechanisms discussed above is the crack filling and cracked surface binding effect of the hydration of the unhydrated cement particles. However, the effectiveness of this mechanism depends on the amount of the unhydrated cement particles present during crack formation. The amount of unhydrated cement particles present in cement paste depends on several factors such as moisture, curing mechanism, age, etc. . HoweverThis paper presents the work done to investigate the use of coarse cement grains as crack-healing agents in concrete. Coarse cement grains, classified as mill-rejected granular cement (MRGC), were obtained from cement industries. MRGC is coarse cement that did not meet the required fineness and did not separate to be reprocessed. The main objective of this research work is to evaluate the effect of MRGC and its amount on the self-healing performance of concrete. Concrete mixes containing different amounts of MRGC as a partial replacement for fine aggregate were tested. Artificial cracks were induced on the specimens and allowed to heal the cracks. The crack-healing performance of the concrete specimens was evaluated based on the percentage regain in strength and impermeability after the specimens were allowed to heal the induced cracks.Concrete specimens were prepared using locally available materials with the partial replacement of MRGC for a natural fine aggregate. The materials used in this study are presented below. Ordinary Portland cement (OPC) obtained from Ethio Cement Factory  was used in this study. The OPC used in this study complies with CEM I Portland cement as in BS EN 197-1:2011 . The phyMill-rejected granular cement (MRGC) was also obtained from the same source asthe OPC. MRGC, as the name indicates, is a portion of cement rejected by the cement processing because it has a grain size coarser than the specified 75 μm sieve size. The chemical compositions of the MRGC was the same as that of OPC . The graGrain size analysis was performed on the MRGC according to the BS EN 933-1 [3,2.70, and 1.26%, respectively.Natural sand with 4.75 mm in maximum size, obtained from Gibe riverbed located about 180 km southwest of Addis Ababa, Ethiopia, was used for concrete mix. Grainsize analysis of the sand was performed, according to the BS EN 933-1 standard procedure ), specifCrushed stone with a 20-mm maximum size, obtained from a quarry on the outskirts of Addis Ababa, Ethiopia, was used for concrete mix. Grain size analysis of the coarse aggregate was performed in accordance with BS EN 933-1standard procedure . AbrasioThe physical properties and grain size distribution  indicateThe presented study was carried out by preparing concrete mixes containing different percentages of MRGC as a partial replacement for sand. At the age of 28 days, the specimens were cracked using controlled compressive, tensile, or flexural stresses. When cracks visible to the naked eye started to appear, the loading stopped. The stress levels causing the initial visible cracks were recorded. Some of the cracked specimens were placed in air at room temperature and others in water at room temperature for 28 days to heal their cracks. After 28 days, the specimens were tested to determine the regain in compression, tensile, and flexural strengths. The change in permeability after crack-healing was also used to evaluate the crack-healing efficiency of concrete containing MRGC.Crack healing in the concrete was assessed based on the evaluation of the regain in the compressive, tensile, and flexural strengths and a drop in permeability values obtained when the specimens were tested after the crack healing period. The results obtained after the healing period were compared with the performance of the uncracked reference specimens to determine the percentage regain in strength or drop in permeability due to crack healing.The concrete mix was designed with a targeted compressive strength of 25 MPa in accordance with BS EN 206-1  and BS 8The concrete mix was performed by adding half of the coarse aggregate, sand, MRGC, and the remaining half of the coarse aggregate, consecutively, into a 300-L capacity tilt drum mixer. After the mixer was run for 30 s, about half of the water was added within the next 15 s. The mixer was run for a total of 3 min and stopped for 10 min. After adding the cement and run the mixer for 30 s, the remaining water was added within the next 30 s and the mixing was continued for 2 min afterward. The mixing and sampling of fresh concrete were carried out in accordance with the BS 1881-125  standardThe slump test was carried out on each concrete mix in accordance with BS EN 12350-2 . The sluConcrete specimens were cast and compacted using a vibration table to ensure good compaction. The surface of each specimen was leveled with a trowel and sealed off with a plastic cover to avoid moisture loss. After the specimens were air-dried at a temperature of 20 ± 3 °C for 24 h, they were placed in a water bath with a temperature of 20 ± 2 °C for 28 days. During the testing times, the specimens were removed from the water bath and wiped off with a damp cloth to remove any excess water from the surface.Concrete specimens prepared for each type of test were 100 mm × 100 mm × 500 mm prisms for a four-point bending test, 150-mm cubes for a compressive strength test, 100-mm diameter and 200-mm long cylinders for an indirect tensile strength test, and 200-mm diameter and 100-mm thick discs for a water permeability test. Three specimens were prepared for each concrete mix shown in To investigate the effect of MRCG and its percentage replacement of the natural sand on enabling concrete to autogenously heal its cracks, artificial cracks were introduced into the specimens. At the age of 28 days, fine cracks were induced into the specimens using displacement controlled compressive stress, split tensile stress, and flexural stress. The loading was stopped when cracks visible to the naked eye started to emerge, and the cracking load and the corresponding displacement were recorded. The cracked specimens were unloaded, placed in air or water at room temperature to heal the cracks, and tested 28 days after the initial cracking.The concrete cube specimens were compressed under a displacement control mode using a standard compression machine. The cubes were loaded until cracks started to appear on the surface of the specimen. The loading of the specimens was stopped when the cracks appeared, and the final displacement was taken as a control. The crack-inducing compressive stress was determined by dividing the cracking load by the cross-sectional area of the specimen. Six replicates of concrete specimens were cracked for each concrete mix, and three of them were immersed in a water bath while the remaining three were placed outside to cure in the air until they were tested.To create artificial cracks using the indirect tensile stress, the cylindrical specimens were loaded diametrically across the circular cross-section until cracking occurred. The loading stopped after the formation of the cracks, and the peak load corresponding to the crack formation was recorded along with the displacement of the load.Four-point bending was another method used to create artificial cracks in the concrete specimens. The prismatic specimens were placed on two parallel supports, and the load was applied on them through two parallel pins placed at the third points of the specimens. The bending load was applied to the specimens until the first visible flexural crack forms. To prevent total collapse and to control the crack width, the beams were reinforced with two #6 standard reinforcing bars, as shown in The effect of MRCG on healing concrete cracks was evaluated based on strength and permeability test results. The specimens induced with artificial cracks, after being submerged in water at a temperature of 20 ± 3 °C for a specified period, were tested to investigate if there was any strength to regain in the specimens due to autogenous healing of the cracks. The crack healing efficiency of MRGC was evaluated by comparing the initial cracking strength and the reloading strength after crack-healing as percentages of the 28-day strength . CompresCompressive strength tests were carried out on the initially cracked and water-cured and air-cured cubic specimens 28 days after cracking. The test was performed in accordance with BS EN 12390-3:2019  standardSplitting tensile strength tests were carried out on the initially cracked and water-cured and air-cured cubic specimens 28 days after cracking. The test was performed in accordance with BS EN 12390-6:2019  standardFour-point bending strength tests were carried out on the initially cracked and water-cured and air-dried cubic specimens 28 days after cracking. The test was performed in accordance with BS EN 12390-5:2019  standardWater permeability of concrete was also one of the methods used to evaluate the effect of MRGC on enabling concrete to autogenously heal its cracks. The crack healing efficiency was evaluated by comparing the permeability values of the specimens measured before cracking, after cracking, and the crack-healing period. After the specimens were cracked at 28 days, they were cured in the air or in water until the testing day of 28 days. The constant head permeability test was carried out on concrete discs that are 200 mm in diameter and 100 mm in thickness using the UTC 1080 apparatus, following a BS EN 12390-8  standardk (m/s) was calculated using the following equation.V = volume of water (m3), L= length of the concrete specimen (m), t = elapsed time (s), A = area of the specimen (m2), and h = applied pressure head (m).The coefficient of permeability The results indicate that the workability of concrete decreases with the increase in MRGC content. This is in agreement with the fact that the workability of concrete decreases with an increase in the content of a fine aggregate. Therefore, the amount of mixing water or a superplasticizer should be increased with an increase in the MRGC content of concrete to improve its workability.The first cycle compressive strength, which is the compressive stress required to induce cracks, and the compressive strength regained due to crack healing after the specimens were cured in air and in water are presented in It was observed that the percentage regained in compressive strength by the control mix (C0) after the crack-healing period was 45.23% and 54.53% for the air-cured and water-cured specimens, respectively. The percentage regained in the compressive strength by the water-cured specimen was 9.3% more than that of the air-cured specimen. Similarly, the percentage regained in compressive strengths by the other mixes for the air-cured and water-cured, respectively, were 60.25% and 75.33% for C5, 66.15% and 81.51% for C10, 74.64% and 90.18% for C15, and 79.72% and 92.44% for C20. The strength regained by the water-cured specimen exceeded that of being air-cured by 15.08%, 15.36%, 15.54%, and 12.72% for C5, C10, C15, and C20, respectively. The percentage regained in compressive strength, relative to C0 mix, was 15.02% for C5, 20.92% for C10, 29.41% for C15, and 34.49% for C20 mixes when cured in air and 20.80% for C5, 26.98% for C10, 35.65% for C15, and 37.91% for C20 mixes when cured in water.The results indicated how compressive strength was regained for all mixes. However, the regain in strength values in the water-cured specimens were higher than those in the air-cured specimens. Furthermore, the regain in compressive strength was observed to increase with an increase in the percentage replacement of MRGC. The regain in compressive strength could indicate that crack-healing activities took place in the specimens. The increase in the strength regain with the content of MRGC could be related to an increase in the availability of unhydrated cement grains, which hydrate to produce new products such as C-S-H and CH that fill and seal the cracks ,46. SincThe first cycle flexural strength, the flexural stress required to induce cracks, and the regain in flexural strength due to crack healing are presented in Similar to the results observed for the compressive strength, the percentage regained in flexural strength of the five mixes, for the air-cured and water-cured, respectively, were 73.84% and 75.87% for C0, 76.49% and 80.07% for C5, 82.58% and 87.78% for C10, 84.78% and 91.98% for C15, and 86.99% and 94.29% for C20. The flexural strength regained by the water-cured specimens exceeded those of air-cured specimens by 2.03%, 3.58%, 5.20%, 7.20%, and 7.30% for C0, C5, C10, C15, and C20, respectively. The percentage regained in flexural strength, relative to the C0 mix, was 2.65% for C5, 8.75% for C10, 10.95% for C15, and 13.15% for C20 mixes when cured in air and 4.20% for C5, 11.91% for C10, 16.10% for C15, and 18.42% for C20 mixes when cured in water.The results of the flexural strength agree with those of compressive strength such that a general regain in strength was observed in all mixes. However, the regain in strength values of the water-cured specimens were higher than those of the air-cured specimens. The regain in flexural strength also increased with the increase in the content of MRGC. This could be due to the healing of the induced cracks. The increase in the strength regained with the content of MRGC could be related to an increase in the presence of extra unhydrated cement grains, which hydrate and produce additional cement paste responsible for healing the cracks ,46. SincThe first cycle splitting tensile strength, the splitting tensile stress required to induce cracks, and the splitting tensile strength regained due to crack healing after the specimens were cured in air and in water are presented in The trend in the percentage regained in tensile strength was also similar to those of compressive and flexural strengths. The percentage regained in the tensile strength of the five mixes, for the air-cured and water-cured, respectively, were 75.19% and 83.20% for C0, 77.04% and 88.15% for C5, 80.23% and 91.16% for C10, 81.74% and 93.91% for C15, and 84.07% and 96.16% for C20. The tensile strength regained by the water-cured specimens exceeded those of air-cured specimens by 8.01%, 11.11%, 10.93%, 12.17%, and 12.09% for C0, C5, C10, C15, and C20, respectively. The percentage regained in flexural strength, relative to C0 mix, was 1.85% for C5, 5.04% for C10, 6.55% for C15, and 8.88% for C20 mixes when cured in air and 4.94% for C5, 7.96% for C10, 10.71% for C15, and 12.96% for C20 mixes when cured in water.The results for the tensile strength regained conform with those of flexural and compressive strengths in which a general strength regained was observed in all mixes. However, as in the case of the regain in compressive and flexural strengths, the tensile strength values of the water-cured specimens regained were higher than those of the air-cured specimens. The tensile strength regained also increased with the increase in the content of MRGC, which could be attributed to the crack-healing effect of MRGC due to hydration ,46.The water permeability coefficient k of the concrete specimens before cracking, after cracking, and after the specimens were allowed to heal the induced cracks for 28 days are presented in The permeability  of the uncracked specimens as a percentage of the permeability of the control C0 were 38.36% for C5, −33.64% for C10, −42.83% for C15, and −59.94% for C20. The drop in the permeability of the air-cured and water-cured specimens, respectively, as a percentage of the permeability of the cracked specimens were 87.67% and 97.06% for C0, 94.24% and 99.08% for C5, 96.86% and 99.41% for C10, 98.61% and 99.85% for C15, and 98.72% and 99.85% for C20.From the above results, it can be noted that the water permeability of the concrete specimens decreased with the increase of MRGC content for the uncracked specimens and after the crack-healing period. This could be attributed to an increase in the presence of extra unhydrated cement grains ,46, whicThe regain in compressive, flexural, and tensile strengths and the drop in water permeability of concrete after specimens were allowed to heal the induced cracks indicate the occurrence of autogenous crack-healing activities in the concrete.The use of mill-rejected granular cement as a partial replacement for a fine aggregate could enable concrete to heal its cracks autogenously. The crack-healing efficiency of concrete could be increased by increasing the MRGC content of the concrete in the presence of sufficient water for hydration.The crack-healing effect of MRGC could be attributed to its hydration at a later age. MRGC particles are coarser than OPC particles, which enables the former to hydrate slowly. During the initial hydration, most of the finer OPC particles hydrate while hydration of the MRGC particles is limited to their surface. This could preserve unhydrated cement particles that can be used by concrete to heal cracks forming during its service life autogenously. Therefore, the use of MRGC could help concrete autogenously heal the cracks during its lifetime due to the hydration of the MRGC with the water entering through cracks.The mechanical properties of concrete were partially recovered due to the autogenous self-healing capability of concrete with the help of MRGC. The average regained in the mechanical properties of the water-cured specimens was about 90% of the reference strength (first cycle strength).The coefficient of permeability of concrete could be reduced by increasing the MRGC content of concrete in the presence of sufficient moisture.Based on the trend of the observed results, a further increase in the percentage replacement of MRGC for fine aggregates could improve the mechanical properties and crack-healing efficiency of concrete. However, MRGC is cement by nature and has to be evaluated from an economic viewpoint as well.The following conclusions could be made based on the results from the experimental study on the crack-healing effect of mill-rejected granular cement when used as a partial replacement for a fine aggregate.


High meat quality is one of the demands made by consumers. Therefore, studies have concluded that meat quality can be improved through some feed additives to broiler diets. Other studies showed that broilers meat quality improved by adding some nutrients to the maternal diet, so this study was conducted to evaluate the paternal effects of dietary 0.1% methionine on meat quality and carcass traits of offspring. Our results suggested that paternal dietary supplementation with 0.1% methionine enhances offspring carcass eviscerated yield, thigh muscles yield and reduces abdominal fat, also meat shear force, drip loss, pH-value, and color. The expression of genes associated with meat quality support these results.p < 0.05), the relative eviscerated yield and relative thigh muscle yield were higher in MET group (p < 0.05); but the relative abdominal fat was lower (p < 0.05). In thigh and breast muscles, a positive response of pH24 h value, shear force (g) and drip loss (%) were observed in MET group (p < 0.05). The lightness (L) and redness (a) were increased (p < 0.05) in breast muscles of MET group, while only the redness (a*24 h) and yellowness (b*24 h) were increased (p < 0.05) in thigh muscles of MET group. The gender has a significant (p < 0.05) effect on carcass traits and muscle redness (a*), where these traits improved in males, and no interaction between treatments and gender were observed for these results. The expression levels of PRKAG2 and PRDX4 supported the changes in muscle pH, with these up-regulated in thigh and breast muscles of MET group, the PPP1R3A gene supported the changes in pH value being down-regulated (p < 0.01) in these same muscles. The BCO1 gene expression was consistent with the changes in meat color and was up-regulated (p < 0.01) in thigh muscles of MET group, consistent with the changes in b* color values. Finally, it was concluded that the supplementation of 0.1% Met to rooster diets could improve carcass characteristics and meat quality of progeny.The effects that maternal dietary methionine have on progeny have been reported on broilers. However, the paternal effects are not known, so the current study was conducted to explore the influences of paternal dietary methionine (Met) have on progeny carcass traits, meat quality, and related gene expressions. A total of 192 hens and 24 roosters from Ross parent stock at 36 weeks of age were selected. From week 37 to 46, the roosters were allocated to two groups with three replicates of 4 cocks each, , and methionine group . The results revealed that, although the heavier live body weight in progeny at day 49 of control group compared to MET group ( Continuous genetic selection, has resulted in fast growing broilers that reach market weight by six weeks of age with high meat yield . The selFor corn-based broiler diets, methionine (Met) is considered the first limiting specific amino acid for synthesis of protein and the source of S-adenosylmethionine, the donor of methyl group for DNA methylation ,8,9. A mThere are studies indicating that the dietary Met improves productive performance  and enhaRoosters have an essential role in the broiler breeding system, where they constitute 50% of the genetic make-up of progeny and the flock fertility . MethionThe use of animal in this research was approved by the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences . A total Ross 192 hens and 24 roosters at 36 weeks of age obtained from Hebei Feilong Poultry Co., Ltd., . The roosters were randomly split into two groups (control group and methionine group) with three replicates of four roosters each. The control group was fed a basal diet with 0.28% endogenous Met, whereas the MET group were fed a diet with 0.38% Met, formulated as shown in At day 49, the progeny broilers  were randomly selected, weighed, and slaughtered by cutting the jugular vein after fasted for 12 h. The eviscerated yield was measured as a live body weight percentage. The percentages of thigh and breast muscle, and abdominal fat weight were measured as an eviscerated carcass weight percentage. Samples of muscles (thigh and breast) were quickly snap-frozen in liquid nitrogen, then stored at −80 °C for subsequent analysis.45 min, L*24 h, a*45 min, a*24 h, b*45 min, b*24 h) were assessed at 45 min and 24 h after slaughter using a spectrophotometer (Konica Minolta CR 410) by the three-dimensional CIELAB color space system [The breast and thigh muscles were stored at 4 °C after stripping. The meat color of thigh and breast muscles such as lightness (L), redness (a), and yellowness (b)  using the QuantStuio 7 Flex RealTime PCR System . The amplification protocol was as follows: 95 °C for 3 min, followed by 40 cycles of 95 °C for 3 s and annealing temperature for 34 s, as mentioned previously [−△△CT method [All procedures of isolation, quantification, and reverse transcription of total RNA from muscles were conducted as reported earlier . Brieflyeviously . The datp < 0.05. The lower cases (a/b) represent significant differences within the main effects of treatments while upper cases (A/B) represent significant differences within the main effects of gender.The present experiment was designed based on a completely randomized design. The general linear model by two-way ANOVA, 2 × 2 and, T-test for (shear force (g), drip loss (%) and gene expression data) were applied to analyze the data using SPSS 26 (IBM). The model of two-way ANOVA included the main effects of treatments (control or MET), the gender  and interactions between treatments and gender. The means were compared by using Tukey’s range test to determine significant differences among means, differences were considered significant at a value of p < 0.05), the eviscerated weight was not different between the two groups. The higher relative eviscerated yield and relative thigh muscle % were observed in progeny from the MET group . The relative abdominal fat was lower in MET group (p < 0.01) and no differences in relative breast muscles % between the progeny. The carcass characteristics were significantly increased (p < 0.01) in males, while no significant interaction between treatments and gender was observed.The results of progeny carcass traits are shown in -value and color are presented in p < 0.05), while the redness (a*24 h) and yellowness (b*24 h) in thigh muscles of MET group were higher (p < 0.05). A higher pH24 h in breast and thigh muscles was observed (p < 0.01) in the MET group, and no differences in other indicators. Males displayed higher meat redness (a*) p < 0.01). The male’s thigh and breast muscles drip loss (%) and shear force (g) as presented in p < 0.01 and p < 0.05, respectively) in MET group. There were no diet × gender interaction for any of the meat quality measures.The results of breast and thigh muscles pHPRKAG2 and PRDX4 genes positively related to pH-value were up-regulated significantly (p < 0.01) in breast and thigh muscles of MET group. In contrast, the expression of PPP1R3A pH-value negatively related gene down-regulated significantly (p < 0.01), consistent with pH-value. The expression levels of BCO1 related to meat color, was up-regulated in the MET group thigh muscle and, consistent with the b*-value.To verify the effect of methionine level on progeny’s meat quality traits, we analyzed the expression of four genes associated with meat quality. The relative gene expression of breast and thigh muscles of male progeny are presented in Results from the current study demonstrate that paternal 0.1% Met dietary supplementation, positively affects the relative eviscerated carcass yield and relative thigh muscle yield in the progeny, while the live body weight was negatively affected. These findings are in accordance with previous research ,36,37, w24h values, as reported in former studies [The results regarding meat quality indicated that the coated Met supplementation has a significant positive effect. The pH value has a vital effect on meat quality, influencing the attributes responsible for processing suitability, nutritional properties, and shelf life of meat . Met sup studies ,45,46. T studies ,45. Drip studies ,46,47. M studies ,46. Ther studies ,48.PRDX4 and PRKAG2 in the muscles of Met group were significantly up-regulated, while the PPP1R3A was significantly down-regulated, all changes consistent with the meat pH-values. The PRDX4 gene has an essential function in regulating the pH-value of chicken breast muscles by catalyzing hydrogen peroxide reduction and positively correlated with the pH-value [PRKAG2 affects glycogen metabolism in muscle by activating the AMP signaling pathway, and is inversely related to muscle glycogen metabolism that, is responsible for lower pH in muscles [PPP1R3A [To verify the meat quality results, we determined the gene expression rates of four genes associated with meat quality. The results revealed that the expression of pH-value ,50. PRKA muscles . The gly[PPP1R3A .BCO1 expression is up-regulated in thigh muscles of MET group and is consistent with increased yellowness (b*), because BCO1 expression is consistent with the cumulation of carotenoids affecting the meat yellow color by changing the yellow pigments lutein and zeaxanthin content [In our experiment, the  content ,54.A better progeny performance is achieved when they come from roosters with higher semen quality . EpigeneIn conclusion, paternal dietary supplementation with 0.1% methionine could improve progeny carcass traits including increased eviscerated carcass and thigh muscle yields and reducing abdominal fat content, also meat quality characteristics such as drip loss, shear force, pH value, and color. Further research is required to explore the mechanism(s) of transgenerational effects of paternal nutritional supplementation on progeny.


Neoadjuvant therapy improves long-term survival by providing locoregional disease control and reducing the risk of long-term recurrence. The most important prognostic factor of survival after neoadjuvant therapy followed by surgery is the burden of lymph node involvement. Several trials have demonstrated improved long-term survival with neoadjuvant therapy; however, there are little data evaluating adenocarcinoma and squamous cell carcinoma (SCC) and difference in outcomes for similar pathological stage with and without neoadjuvant treatment. This studyThese results corroborate what has been found in other recent studiesThis study reinforces the understanding that post-neoadjuvant stage influences prognosis. While some may advocate ‘complete’ restaging prior to progressing to surgery, induction therapy is known to potentially impact on the reliability of staging modalities. It may also be important to consider the post-neoadjuvant stage when deciding on the merit of adjuvant treatment. There has been some suggestion that adjuvant treatment may confer some benefit in node-positive patients with adenocarcinoma who received neoadjuvant chemoradiotherapy, and it already forms part of the standard of treatment in patients receiving MAGIC protocol chemotherapy.


Introduction: Students in the Frontiers in Human Aging course at UCLA participate in service-learning (SL) with older adults. In 2020, completion of SL coincided with the outbreak of the novel coronavirus disease (COVID-19) pandemic. We evaluated the impact of SL on student attitudes on aging and community service in the context of the pandemic. Methods: Students were assigned to senior residential and daycare programs for 18-20 hours of SL. A retrospective pretest-posttest survey asked about attitudes and interests before and after SL and how the COVID-19 pandemic affected these perceptions; 73 (of 103) students responded. Mean differences before and after SL were tested and differences were assessed within groups reporting COVID-19 effects. Results: SL improved students’ attitudes and ability to engage with older adults, knowledge about aging concepts, interest in future work with older adults, attitudes on community service, social well-being and feelings of usefulness (p<0.001). There was no significant change in overall anxiety about aging (p=0.1), however, students showed increased anxiety about losing independence and finances when older (p<0.05). At least 50% of students reported that the COVID-19 pandemic increased their awareness of needs of older adults (81.9%) and decreased connection to their peers (50.7%); the impact of SL remained unchanged by these effects. Conclusion: Despite the overall benefits of SL, increased anxiety about aspects of aging suggests the need to address these concerns. While the COVID-19 pandemic did not seem to affect the impact of SL, this event did seem to influence perceptions about aging and social integration.


Background and objectives: Infections caused by resistant bacteria are a growing public health problem that is linked to many different causes, among them the antibiotics’ incorrect use plays an important role. According to the World Health Organization (WHO) the most dangerous behaviors are the early interruption of antibiotic therapy and the use of molecules without appropriate prescription. The authors conducted a systematic review to assess if antibiotic prescription with different regimens is connected to the onset of bacterial resistance. Methods: The authors performed an electronic and manual literature search on four databases  from their inception to 15 June 2019. The date of the last search was 27 November 2019. Any article comparing cultural or genic analysis of resistance in patients that took antibiotics with at least two different regimens was included. No language restrictions were applied. Risk of bias for randomized controlled trials (RCTs) was assessed using the Cochrane collaboration’s tool whereas case-control and cohort studies were evaluated through the Newcastle–Ottawa scale. Results: The initial search resulted in a total of 1744 titles. After careful evaluation of all results, only three studies satisfied the outcome of the present review. From the qualitative analysis of data, it emerges that even if antibiotics are administered for a shorter period than the conventional one the species that inhabit the oral cavity can adapt quickly and express genes of antibiotic resistance. Additional evidence from this analysis is that not only does the proportion of resistant bacteria increase in the oral cavity, but also in more distant districts such as the intestine. Conclusions: Despite the great number of studies retrieved by electronic databases only few studies investigated the target of this review. The reason for this evidence is that it is not ethical to investigate and compare different antibiotic regimens, shorter or longer than the appropriate one. This evidence is applicable both to prophylactic administrations and to those aimed at treating infections. Besides this, the WHO affirms that, in the absence of infective complications, the prescription of antibiotic after every type of surgical intervention cannot be admitted and that studies dealing with antibiotic regimens that do not comply with drug’s pharmacodynamics characteristics cannot be ethically admitted. PROSPERO acknowledgement of receipt [149149]. Bacterial resistance is the capability of the bacteria to multiply in the presence of drug concentrations that are mostly inhibitors of the same species or equal to the maximum achievable concentration during the therapeutic use. The rising of resistant bacteria is a growing public health problem that is mainly linked to antibiotics’ incorrect use. One of the connected problems consists of the increasing complexity of medical treatment and the growing amount of costs that is estimated as $30 billion annually .Other important issues are the over or incorrect prescription, patients not finishing antibiotic treatments, the overuse in livestock and fish farming, and the lack of hygiene and poor sanitation and infection control in hospitals and clinics . There aPrevious studies have already demonstrated that patients’ noncompliance towards antibiotic prescription is an emerging issue and that it is mainly linked to misunderstanding of the treatment ,4,5,6.Evidence has been already highlighted, in form of a call to action, against the indiscriminate use of antibiotics for animal agriculture and fish farming  and regaStrategies to cope with increasing bacterial resistance include accurate knowledge of the microbial component that feeds infections ,10 and tIn medicine and dentistry, the prescription of antibiotics in recent decades has been the subject of debate since many authors presented results of studies in which a reduction of prescription duration resulted in substantial equal effects in terms of infection resolution and postoperative adverse effects onset ,15,16,17When the electronic and the manual search were completed 1948 titles matched the inclusion criteria. The elimination of duplicate results led to 1744 titles of which 1730 were removed analyzing their title and abstract. According to this process three randomized controlled trials (RCTs) were selected and included in this review .After full-text evaluation, five studies were excluded because they did not give a quantitative analysis of bacterial resistance and six studies were excluded because they did not compare different antibiotic regimens.The three studies were randomized controlled trials carried out in different countries: France, Sweden, and Brazil.Two trials were conducted at University dental clinics ,19 whereHelicobacter pylori infection, chronic periodontitis, or whose treatment plan included at least one tooth extraction belonged to the test group. In the test group patients were prescribed an antibiotic for a determined period; in the control group patients were prescribed alternatively another antibiotic, the same antibiotic for a shorter amount of time, or placebo. Patients were excluded if they matched at least one of the following exclusion criteria: heart or renal failure, cancer, pregnant, nursing, any antibiotic molecule taken in the previous 45 days, history of allergic reaction to the tested antibiotic.All studies included adult patients (age range: 18–81 years) ,20. PatiChardin et al. enrolled patients recruited from emergency dental consultations of three university hospitals in case they had an absolute indication for tooth extraction . Patients were divided into two groups: the first group was given 1 g amoxicillin twice daily orally therapy for 3 days with a subsequent 4-day placebo period. The second group was given 1 g amoxicillin twice daily orally therapy for 7 days. The administration of amoxicillin started on day 0 (participant’s inclusion in the study). The oral surgery procedure was performed 2 days after the beginning of the antibiotic treatment (day 2); the follow-up period lasted for 7 days after tooth extraction (until reaching day 9). The authors also planned an additional follow-up period of around 1 month (until reaching day 30). Researchers evaluated the bacterial susceptibility to amoxicillin in each study group; such evaluation was performed analyzing the proportion of streptococci with reduced susceptibility out of their total number .In the RCT of Feres et al., the authors evaluated the antibiotic resistance in subgingival plaque samples of patients affected by chronic periodontitis whose diagnosis had been made more than 5 years earlier. Such samples were taken in different time frames: baseline, at 3, 7, and 14 days during antibiotic administration and at 3, 7, 14, and 90 days post antibiotic therapy. All patients belonging to the test group were enrolled after a baseline-monitoring visit and before receiving a full mouth SRP under local anesthesia and instructions about proper home care procedures; in that occasion the authors took the first sample. Ten patients belonging to the test group were given orally metronidazole  and 10 orally amoxicillin . The antibiotic therapy started at the baseline-monitoring visit. At the end of the study period (day 90), researchers took a further sample from each patient, and everyone was given full mouth maintenance SRP. All samples were taken using sterile curettes from the distal aspects of six posterior teeth with pocket depth of at least 5 mm .Helicobacter pylori infection by the rapid urease test and whose diagnosis had been confirmed by culture, histology, or PCR at least a year earlier. For the treatment of this condition 14 patients received 20 mg of omeprazole capsules  and 1 g amoxicillin capsules  twice daily for 14 days (Group 1). Fourteen patients received omeprazole capsules 20 mg along with placebo  capsules twice a day for the same period (Group 2). Eight men and six women composed the group 1; among them seven patients were affected by peptic ulcer disease and seven patients by dyspepsia without active ulceration. Patients belonging to group 2 included seven men and seven women; among them eight patients were affected by peptic ulcer disease and six by dyspepsia without active ulceration. No statistically significant differences were retrieved between the two groups regarding age, sex, or endoscopic diagnosis. Patients returned to the endoscopy ward on treatment day 10 and 28 days after treatment (day 42) for endoscopy and bacteriological sampling. Oral samplings were collected before gastroscopy with the aim of avoiding contamination with the gastric microflora ) OR (short course antibiotic AND long course antibiotic)) AND  OR antibiotic resistance). The abovementioned search strategy was the one used for Medline; the same strategy was, afterwards, adapted for the other databases. Gray literature was also screened questioning Google Scholar and considering the first 100 listed hits along with a supplementary manual search through Journal of Clinical Periodontology, Journal of Periodontology, Clinical Oral Implants Research, International Journal of Oral Implantology, International Journal of Oral and Maxillofacial Surgery, and International Journal of Oral and Maxillofacial Surgery. The manual search aimed at finding appropriate articles published up to November 2019. Lastly, all included articles were further analyzed checking their references with the aim of retrieving other relevant publications and contacting corresponding authors by e-mail for recovering unpublished articles or raw data.Two reviewers (GM and LM) conducted the screening process independently and in duplicate. The screening process consisted of the evaluation of titles and abstracts of the retrieved studies according to the inclusion criteria. Subsequently, the same reviewers performed the assessment of the full-text articles. In case of disagreement the author supervisor (RP) was consulted.The authors extracted the data with the help of data extraction forms specially designed for this purpose. In case of articles where the title and abstract information were not detailed enough in order to make a final decision, the reviewers screened the full-text article. The primary outcome was the development of antibiotic resistance in single bacterial species or genus evaluated through cultural or metagenomic analysis. Summary measures were given as difference in means.The reviewers independently extracted data regarding antibiotic resistance through a structured form.The Cochrane Collaboration’s Risk of Bias tool was used for assessing risk of bias of all included RCTs and the Newcastle–Ottawa quality assessment scale for case control studies for all included CCTs, with the aim of defining their methodological quality.The evaluation of the selected publications identified three studies ,19,20. AThe present review showed that different antibiotic regimens are not directly related to an increase in bacterial resistance against these molecules. This evidence, however, must be read in the light of a propensity to experiment with antibiotic doses other than those indicated by the manufacturers. This attitude has already been stigmatized by WHO which has also highlighted the ethical problems connected to it. Indeed, the pharmacokinetic and pharmacodynamics characteristics of the prescribed antibiotic molecules must be correctly known and considered when deciding to prescribe a therapy.