The main aim of this study was to investigate the improved autogenous healing of concrete caused by a crystalline admixture in combination with textile reinforced concrete (TRC). This phenomenon  has not yet been described by any independent study, and not at all in relation to TRC. The results of the study confirmed that the interaction between TRC and the crystalline admixture’s self-healing ability is advantageous and usable. The application of crystalline admixture could ensure the long-term entirety of the TCR element, where microcracks could occur. This allows for the creation of advantageous, thin (achieved by TRC) and waterproof  concrete structures. Moreover, this does not depend on temperature in the range of 4–30 °C . However, the interaction of both materials has its limits; the cracks must not be too wide (max. 0.1 mm), otherwise they will not heal. On the other hand, the advantage is that it does not matter what type of cement is used (CEM I and CEM II showed the same results), and the composition of the newly formed crystals in the cracks corresponds to the composition of the C-S-H gel, so it can be assumed that secondary hydration of the Portland cement occurred in the crack area. The problem of crack permeability in concrete is crucial for the assessment of any structure where there is a requirement for water impermeability. The concrete structures of buildings almost always have some cracks, due to uneven settlement or for other reasons. In the case where the substructure of the building is designed with waterproof concrete (as protection against ground moisture), it is necessary to ensure that water does not penetrate through the cracks into the building. In the place of projected joints in concrete, this is usually achieved by using special elements based on plastic, rubber, or bentonite strips. The problem is if the crack occurs out of the place of a projected joint. This happens very often . In suchThere some products exist to increase the autogenous healing ability of concrete. One of them is a crystalline admixture which causes secondary hydration in concrete. These additives are primarily intended to create a waterproof structure inside low-strength concrete. One side effect should also be a significant increase in the autogenous healing of concrete (according to the manufacturers’ claims), but the problem is that this assumption has never been proven by any independent tests.2, and a subsequent formation of disilicate and polysilicate anions. It is probable that the cumulative process is accompanied by the formation of 3Ca·2SiO2·3H2O, together with the creation of 3CaO·Al2O3·Ca(OH)2·12H2O [The crystalline admixture is a dry material (powder) consisting mainly of Portland cement, and additionally containing fine quartz sand and other inorganic chemicals. The chemical recipes are subject to the protection of company patents in all manufacturers in the US, Canada, and the EU . The crystalline admixture’s waterproofing effect in concrete is achieved by the reaction of various chemical components contained in the solution when combined within the concrete matrix ,2,3. The)2·12H2O . The phy)2·12H2O ,6,7,8,9.)2·12H2O ,15,16,17)2·12H2O ,19,20,21)2·12H2O ,23,24,25The results of the above mentioned studies were very convincing; they confirm the waterproof and durability effect of crystalline admixture in concrete. Several studies have also confirmed the importance of curing of fresh concrete with crystalline admixture ,27,28. In addition to the aforementioned studies, there have been some other publications focused on the crystalline technology, which include problems with curing , fiber cLikewise, the authors of this article have written several papers dealing with selected problems in the field of crystalline waterproofing systems: crystalline admixtures and their effect on selected properties of concrete , descripThe latest knowledge shows that organic compounds can also be theoretically used for improving self-healing ability, but with unreliable results. Some kinds of bacteria and fungi can, under very specific conditions, improve the self-healing ability of concrete, but this is difficult to use in practice .Based on previous studies and experience from practice, it is obvious that crystalline admixtures improve the self-healing ability of concrete. However, this phenomenon has not yet been described by any independent study, and not at all in relation to textile reinforced concrete (TRC). Therefore, the aim of this study was to describe this process in detail, with a focus on the use in TRC. The topic of the research was inspired by a question from the British company, Mott MacDonald, addressed to a co-author (J. Pazderka) by Dr. Noushin Khosravi. The replacing of part of the conventional secondary structural reinforcement by crystalline admixture was the main question. The need to ensure an acceptable level of crack development in concrete while reducing the structural reinforcement led us to the idea of using TRC. A detailed description of the interaction between TRC and the crystalline admixture’s self-healing ability could create new uses for this composite. First, preliminary tests on cubes were created, after that specific boundary conditions  applied to the TRC specimens were realized.First, the basic parameters of the improved self-healing ability  were tested. Pure concrete was used for this initial phase of testing.The experiment started in November 2015 and finished in June 2018. The aim of this research was to load these cracked samples with water pressure to determine their waterproofness, in terms of the crystalline admixture’s influence on the sealing of cracks. A water pressure test, using a modified methodology according to , was use3 [All specimens were standard concrete test cubes 150 × 150 × 150 mm3 . The speVarious methods of crack creation were considered. The creation of a realistic crack (if possible) in laboratory conditions is difficult, but it was necessary for the planned experiments. If the crack in the specimen does not resemble a real crack, then the experiment is valueless. Therefore, one of the objectives was to find a method for creating a realistic crack, with a predefined width. It was found (during the development of the methodology) that it was possible to make a realistic crack, but without a predefined width, or it was possible to make an unrealistic crack, with a predefined width. The first possibility was chosen. As a result, a method based on polypropylene fibers added into the concrete mixture was chosen for making realistic cracks in the test cubes. The cracks were made using a machine for testing the tensile splitting of concrete. Thanks to the presence of fibers, the cracks were created, but the concrete specimen was not destroyed. This method allows creating specimens with cracks, but the width of the cracks could not be controlled in advance. The crack width was measured by an Electrometer 900 optical microscope. Width of the cracks was in the range of 0.05–0.8 mm.Concrete specimens were cured under polyethylene foil for 48 h after the concrete embedment and, consequently, the formwork was stripped, and they were placed in special tanks full of water to stop autogenous shrinkage . The craAn important side objective of the experiment was to qualify the environment for saving the specimens (in relation to the maximum efficiency of the secondary hydration process in cracks). The aim was to determine whether air is necessary to cause chemical reaction or not. The basic presupposition was that ideal conditions for the growth of crystals are a specific combination of moist and dry environment. At first, all cubes were held in tanks under the water level. Then, 45 days after embedment, the specimens were placed in tanks with a humid environment  a, withouSubsequently, the cubes were placed in boxes , where the level of water was maintained at 20–30 mm from the bottom b. This cThe main experiment was divided into three phases . The firThe main aim of the experiment was to find out, if the crystalline admixture was able to help overgrow a large amount of the microcracks, whose formation is typical for thin structures made of TRC. This ability is important for the extension of the life of structures made from TRC.The temperature and air humidity of the environment were measured using a thermometer and hygrometer based on the Arduino UNO platform. Measurements lasted for the whole time of the experiment duration.3 (the first phase) and 50 × 50 × 10 mm3 (the second phase and the third phase). The composition of all test specimens in all phases was the same . The water–cement ratio was 0.4, this was caused by using fine aggregates and the polypropylene fabric.The dimensions of the test specimens were 100 × 100 × 10 mmthe same . For allIn the first phase, 9 test specimens were produced, in the second phase, 56 test specimens were produced, and in the third phase, 6 test specimens were produced containing a crystalline admixture (Xypex). The same quantity of reference specimens was produced as well.The test specimens were cured for two weeks, during which they were covered with a polyethylene foil to prevent the evaporation of moisture into the environment. During the curing, the temperature and humidity of the surrounding environment were monitored continuously by the Arduino UNO platform . Cracks were made by applying a bending load. The maximum crack width was about 0.1 mm, and the measurement of all crack widths was performed with a DigiMicro Profi II digital microscope , .In the first phase, the test specimens were placed in an environment with different boundary conditions .The crack development was monitored for the next 4 weeks, with control imaging every 7 days using the DigiMicro Profi II microscope . For specimens placed in direct contact with water, evaporation was allowed to the surrounding environment with ordinary relative humidity (40–80%). For specimens placed in the environment with a 100% relative humidity, a decrease in humidity values can be observed at regular intervals. This reduction was caused by regular monitoring of the test specimens, which were removed from their environment for a short time for the purpose of photographic documentation.The aim of the second phase was to determine the effect of curing on the activation of the crystalline admixture in concrete. Initial imaging was performed using the Digi Micro Profi II microscope at 50× magnification, and the specimens were placed in different environments based on a predetermined experimental scheme . The numThe test specimens were divided into sets of four test specimens. The first two sets (test specimens 1–8) were placed immediately in the environment where the water level was maintained throughout the experiment so that the upper surface of the specimens was in contact with air . The remThe last phase was focused on comparing the effectiveness of the crystalline admixture depending on the type of used cement. Two types of cement were tested, CEM I  in the p3 were produced, i.e., a total of two sets of test specimens of three pieces each for each type of cement, and the corresponding number of reference specimens, were made.A total of 12 test specimens with dimensions of 50 × 50 × 10 mmThe water pressure test to determine the depth of water penetration was performed in three different time periods for cracked and uncracked specimens. The first test was performed 90 days after embedment, the second was performed 190 days after embedment, and the third was performed 945 days after embedment. . For eacThe water pressure test for cracked test specimens was based on the  methodolIn all tested specimens, there was a crystal growth of white macroscopic crystals in the cracks. Material analysis  of the cThe scientific software, Fáze (Version 5), developed by K. Forstová under the guidance of assoc. prof. J. Němeček at CTU in Prague, was used for the quantification of changes due to the crystal growth. The main advantage of the program is its ability to recognize pores and cracks in the concrete structure. In order to compare individual areas of interest, an initial measurement was taken as a reference, and the area of cracks was marked 100%; i.e., no cracks are healed. Any subsequent crystal growth then led to a reduction in the crack area, and thus to a percentage loss .A Digi Micro Profi II microscope and a Canon PowerShot digital camera were used for the monitoring of the test specimens during the whole experiment in all phases .The measurement of the crack area during the first phase using the Fáze software  was performed at the beginning, before placement in individual experimental environments, and then at each test specimen inspection, i.e., 7, 14, 21, and 28 days after the start of the experiment. The values for each test specimen were then averaged and compared with each other .The averaged values of each test specimen were subsequently plotted in a graph for comparison . The meaFor specimens that had not been in contact with water, there were no, or only minor, changes, both for Xypex and reference specimens. Crystal overgrowth could be observed in specimens that had been placed in contact with water. For specimens with the Xypex Admix additive, a decrease of approximately up to 33% in the original crack surface was measured. For the reference specimens placed in the water environment, this decrease was about 53%. The results of the measurements also showed that the temperature was almost irrelevant for the secondary hydration process (in the room temperature range), the most important parameter for crack healing was partial contact with water, allowing water to rise through the specimen, and its subsequent evaporation into the environment .The measured values gained during the second phase  were aveThe obtained data confirmed the crystal growth in both sets of test specimens, with the crystalline admixture and reference specimens. In the reference test specimens, this overgrowth was smaller than in the specimens with the additive .In the case of the test specimens, which had been placed below the water surface before being placed in evaporating tanks, cracks were already overgrowing with crystals in this environment, and this continued even after their placement in evaporating tanks. For the specimens placed in evaporating tanks from the aquatic environment in the initial stages, the crystal growth in cracks was faster than for the specimens placed there in later stages, however, for the specimens placed in evaporating tanks in later stages, the resulting crystal growth in cracks was greater; it had largely already occurred in the aquatic environment.For the test specimens placed in a standard environment  before being placed in the evaporating tanks, the cracking showed a similar trend as for specimens 1–8, which were placed in evaporating tanks immediately after the end of curing.The results of the third phase show that the growth of crystals in the cracks was not dependent on the used cement type. The resulting crack area for specimens with the Xypex additive was around 30% in both cases. The resulting size of the crack area of reference specimens was in both cases around 59% . As a reThe sealed cracks were studied using an optical microscope . Images The overall results of the performed tests showed that the interaction between TRC and the crystalline admixture’s self-healing ability brings benefits. One of the objectives of the study was to approximate the methodology of the experiment as close to practice as possible . For this reason, some of the experiments were conceived as long-term. Loaded TCR often exhibits a net of smaller microcracks, of which the dislocation is determined by the used fabric, and the difference with ordinary concrete. Whereas the self-healing ability of the studied admixture is limited with respect to crack depth, this application gives a suitable solution to reaching long-term entirety of the TRC element. The result is a combination of the advantages of a thin structure  and maintaining the waterproofness of the concrete . One of the acquired findings of the study was, that this does not depend on temperature in the range of 4–30 °C . However, the interaction of both materials has its limits; the cracks must not be too wide (max. 0.1 mm), otherwise they will not heal. This was the main finding of the preliminary tests. However, the advantage is that it does not matter what type of cement is used (CEM I and CEM II showed the same results), and the composition of the newly formed crystals in the cracks corresponds to the composition of the C-S-H gel, so it can be assumed that the secondary hydration of Portland cement occurred in the crack area. This was confirmed by material analysis performed during the preliminary tests. Results acquired in the main test confirmed the initial assumption that the crystalline admixture improves the self-healing ability of TRC, as well as in the case of concrete with conventional reinforcement. Overgrowth of the cracks was more significant in the test specimens with crystalline admixture. The main output of the experimental program, which may be important for practical applications, is a detailed description of the kinetics of the self-treatment process under different conditions. Based on the presented results, it is possible to competently estimate the healing of cracks under specific conditions, which can be set artificially or may occur during construction. Artificial creation of conditions is much more complicated, but it compensates for disadvantages resulting from the presence of cracks in the structure.


Salmonella enterica and an efficient detection method. During induction with multi-stress conditions, nutrition starvation antagonizes with low-level acidity. Besides, high-level acidity was considered as an inhibitor for VBNC induction. Four inducers including nutrition starvation, salt stress, low-level acidity, and low temperature were concluded for a VBNC state. In addition, the keynote conditions for S. enterica entering a VBNC state included (i) nutrient-rich acidic environment, (ii) oligotrophic low-acidity environment, and (iii) oligotrophic refrigerated environment. Based on the keynote conditions, the environmental conditions of high acidity (1.0% v/v acetate) with low temperature (−20°C) could successfully eliminate the formation of S. enterica VBNC cells in flour food. In addition, combining with propidium monoazide pretreatment, PCR technology was applied to detect S. enterica VBNC cells. The sensitivity of the PMA–PCR technology was 105 CFU/ml in an artificially simulated food system. The results derived from this study might aid in the detection and control of VBNC state S. enterica in flour food products.The processing and storage conditions of flour food inevitably pose environmental stress, which promote bacteria to enter a viable but non-culturable (VBNC) state. The existence of VBNC cells causes false-negative detection in traditional culture-based detection methods, resulting in food quality and safety issues. This study aimed at investigating the influence factors including nutrition, acid, salt, and temperature for the entry into a VBNC state of  In addi or food . In the ormation . Besidesum cells . Moreoveum cells ,b. There in food . Althoug in food .S. enterica VBNC cells could be resuscitated by favorable conditions, the recovery of VBNC cells from different environmental stresses requires different methods, such as temperature upshift  treatment with nucleic acid amplification technologies has shown to be capable of rapidly detecting VBNC bacteria . The overnight bacterial culture (∼108 CFU/ml) was washed three times and resuspended by sterile saline. Besides, aliquots of these bacterial suspensions were separated into 1.5 ml tubes (∼20) to avoid the effect of repeated freeze–thaw. The viability of bacterial cells was characterized by the colony counting method, and the VBNC cells were determined by LIVE/DEAD BacLight® kit  with fluorescence microscope after the culturable colonies no longer form on an agar medium. The culturable and viable cell enumerations were preformed every 3 days.According to the food environmental condition, a total of three factors were selected as a single variable including nutrient, salt, and acid . The desS. enterica-specific gene invA was selected as the target gene, and corresponding primers were designed (The designed . The sel8 CFU/ml) was diluted 10 fold by adding 9 ml of sterile flour solutions to prepare the artificially contaminated food samples with different concentrations (107–10). Before the extraction of bacterial DNA, artificially contaminated samples were pretreated according to the following steps: (1) 1 ml of the flour solution was centrifuged for 10 min at 1,000 rpm to remove the macroparticles, then the supernatant was centrifuged again at 12,000 rpm for 10 min to collect precipitations. (2) The precipitations were resuspended in 500 μl of sterile saline and then mixed with 125 μl of ethyl acetate for 2 min to remove impurities such as oil and fat. After centrifugation at 12,000 rpm for 10 min, the precipitations were washed once with 500 μl of TE buffer and twice with 500 μl of sterile saline.Twenty-five grams of crystal cake powder was added to 225 ml of sterile saline and autoclaved. The 1-ml overnight culture  dye was added to the flour samples until its concentration reached 5 μg/ml. After incubation at room temperature for 10 min in the dark, the samples were exposed to a 650 W halogen lamp with a distance of 15 cm for 5 min, which inactivated unbinding PMA molecules rather than PMA–DNA molecules. All the dyeing process was performed in an ice bath to prevent DNA damage. Subsequently, the DNA extraction and PCR detection of PMA-treated cells were performed.The range of S. enterica cells were induced to a VBNC state by low-temperature storage , suggesting that nutrition may be a stimulated bacterial stress response mechanism to resist acetate. The culturable number in the other groups  showed a decreasing trend to reach 0  might contribute to inhibit bacterial growth.According to four external environmental factors , the orthogonal array was designed to induce 6 groups . Non-cul reach 0 . The acc reach 0 . The celS. enterica was as follows: acidity > nutrients > salt. With the reduction in acidity, the supplement of certain nutrients promoted S. enterica to resist environmental stress.In summary, under higher than 0.7% v/v of acetate, the order of environmental conditions in affecting the survival of S. enterica were no longer culturable in eight groups , the viable cells were still captured by fluorescence microscopy  in which bacteria lost culturability within a short-term storage (∼3 days) (data not shown), the formation of a VBNC state might require S. enterica to suffer by long-term induction of sublethal environmental stress and low temperature.Although croscopy . These rS. enterica VBNC state could be induced by lactic acid or peracetic acid . Other previous studies had described similar results and showed that temperature upshift and growth factors  were required for resuscitation  nutrient-rich acidic environment, (ii) oligotrophic low-acidity environment, and (iii) oligotrophic refrigerated environment.Salmonella cells to 0.7 or 1.0% acetic acid for 7 min usually caused 90% cells with inactivation and 99% of culturable cells with injury. In agreement with a related study, cell viability was evaluated based on cultivability, neglecting the generation of Salmonella VBNC cells induced by acetate in vitro (S. enterica to efflux protons (H+) by active transport maintaining intracellular pH homeostasis .Acetate had been utilized as an antimicrobial chemical for many years in food production. As previously described by in vitro . Encounteostasis . TherefoS. enterica could not be cultured after 3 days of storage at low temperature (4° and −20°C) by adding 0.7 or 1.0% v/v acetic acid (S. enterica was unable to enter into a VBNC state. When supplied with sufficient nutrients (≥50%), the culturable cells still existed under 0.7% v/v acetic acid after 3 days of storage, indicating that acid tolerance of S. enterica was improved by available nutrients, which might ultimately lead to generation of VBNC cells. However, when the concentration of acetate reached 1.0% v/v, no culturable cells could be found after 3 days of storage, whether provided with nutrients or not. As expected, the activity test results showed that all the bacterial cells were dead, indicating that the formation of a VBNC state of S. enterica could be controlled by adding 1.0% v/v acetate, even when supplied with rich nutrients. Interestingly, only group 7 had showed that all the bacteria were inviable with decreasing temperature under high-level acidity stress (1.0% v/v acetate). These results were consistent with those previously described by S. typhimurium at different growth temperatures and found that the reduction of low temperatures markedly decreased the acid resistance and increased the growth pH boundary of S. typhimurium. In summary, during the processing of flour foods, 1.0% v/v acetic acid could be used to clean the processing equipment, which can effectively eliminate the pollution of S. enterica and its VBNC cells.Under oligotrophic conditions, tic acid . The bacE. coli VBNC state, which was promoted by nutrient starvation, but stress removal will facilitate the disaggregation of the proteins by the DnaK–ClpB cochaperone system  combined with different concentrations  of nutrients. After 3 days of storage, the culturable number and viability of S. enterica in different conditions are shown in Salmonella could still survive after 3 days of storage at 4°C via a supplement of sufficient nutrients (100%)  . AlthougNC state . TherefoFoodborne pathogenic and spoilage bacteria had been previously shown to enter into a VBNC state in a food system under a freezing environment. Therefore, the results of this study might provide a theoretical basis for the control and reduction of foodborne bacterial VBNC cells.S. enterica using the constructed PMA–PCR technology to detect the VBNC status in the crystal cake food system is 105 CFU/ml, which is consistent with the PMA–PCR results in the pure induction system (data not shown). Compared to the detection limit of PCR for culturable S. enterica in food systems, the detection limit of PMA–PCR has no significant changes, suggesting that the PCR method by the PMA dye is non-effective. Therefore, the PMA–PCR method can be better applied to detect viable bacteria (culturable and non-culturable) in a food sample, preventing false-negative detection of a culture-based method on VBNC cells.In order to eliminate the interference of dead bacterial DNA, samples were subjected to PMA treatment before PCR amplification. The detection limit of S. enterica and an efficient detection method were investigated. The order of environmental conditions in effecting the cultivability of S. enterica was as follows: acidity > nutrients > salt. Four inducers for the VBNC state including nutrition starvation, salt stress, low-level acidity, and low temperature were concluded. However, during induction with multistress conditions, nutrition starvation antagonizes with low-level acidity. Besides, high-level acidity was considered as an inhibitor for a VBNC state formation. Therefore, the keynote conditions for S. enterica entering the VBNC state were concluded as (i) nutrient-rich acidic environment, (ii) oligotrophic low-acidity environment, and (iii) oligotrophic refrigerated environment. Thus, using an environment condition of high acidity (1.0% v/v acetate) with low temperature (−20°C), the formation of S. enterica VBNC state was eliminated in flour food. Combined with PMA pretreatment, the PCR technology could be applied to detect viable S. enterica cells (culturable and VBNC) removing the interference of dead cells. The detection limit of the PMA–PCR technology was 105 CFU/ml in an artificially simulated food system. In conclusion, this study identified specific environmental stresses to control, and applied a stable PMA–PCR method to detect, an S. enterica VBNC state, providing a theoretical basis for the control and reduction of foodborne bacterial VBNC cells.In this study, the influence factors including nutrition, acid, salt, and temperature for the entry into a VBNC state of All datasets presented in this study are included in the article/supplementary material.JL and KW conceived of the study and participated in its design and coordination. JF and TH performed the experimental work. CB and LC analyzed the data. YL and JL prepared and revised this manuscript. All authors reviewed and approved the final manuscript.YL was employed by the company Guangdong Zhongqing Font Biochemical Science and Technology Co. Ltd.The remaining 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.


TH), DOPA decarboxylase (DDC), solute carrier family 6 member 3 (SLC6A3) and DA beta-hydroxylase (DBH), with six important phenotypes of heroin dependence.Heroin dependence is a complex disease with multiple phenotypes. Classification of heroin users into more homogeneous subgroups on the basis of these phenotypes could help to identify the involved genetic factors and precise treatments. This study aimed to identify the association between genetic polymorphisms of DA synthesis and metabolism genes, including tyrosine hydroxylase  were genotyped by SNaPshot. Associations between SNPs with six phenotypes were mainly assessed by binary logistic regression. Generalized multifactor dimensionality reduction was used to analyze the gene-by-gene and gene-by-environment interactions.DBH rs1611114 TT genotype had a protective effect on memory impairment after heroin dependence . We also found that the income-rs12666409-rs129915-rs1611114 interaction yielded the highest testing balance accuracy and cross-validation consistency for memory change after heroin dependence.We found that Our results suggest that the memory change after heroin dependence was a result of a combination of genetics and environment. This finding could lead to a better understanding of heroin dependence and further improve personalized treatment. Drug dependence is a chronic relapsing brain disease that results from a combination of genetic and environmental factors , and theDA system modulates motivation, cognition and reward in the context of dependence –14. ExtrSLC6A3 rs27072 was associated with an early-onset age of nicotine dependence [TH rs6356 was significantly lower in the late-onset Parkinson’s disease group [DBH rs5320 smoked fewer cigarettes per day [DDC rs921451-rs3735273-rs1451371-rs2060762, which was significantly associated with nicotine dependence after correction for multiple testing [The A allele of pendence . The frese group . Male evSLC6A3 , TH , DDC (rs11575553 and rs12666409) and DBH  with these phenotypes. We performed survival analysis for DTFUD. Finally, we analyzed the gene-by-gene and gene-by-environment interactions in these significant phenotypes.We selected six phenotypes: memory change after heroin dependence, age of onset for heroin use, euphoria associated with first heroin use, DTFUD, daily heroin dose and daily methadone dose. We identified 14 potential functional single nucleotide polymorphisms (SNPs) by bioinformatics. We then analyzed the associations between the SNPs located in A total of 801 heroin-addicted individuals (mean age 39.30 ± 8.291 years) participated in our study. All of these participants were Chinese Han population. They were all unrelated individuals that were neither first-degree relatives, second-degree relatives nor third-degree relatives. This study included 709 males and 92 females. These participants were recruited from Lantian Compulsory Isolated and Detoxification Center, Xi’an Methadone Maintenance Treatment (MMT) Center and Xin’an Central Hospital Drug Rehabilitation Center. Heroin dependence was evaluated according to the Diagnostic and Statistical Manual of Mental Disorders fifth edition (DSM-V) criteria and a urine test. A structured interview was performed to assist with the diagnosis and to ask questions regarding (1) basic information: age, gender, nationality, education level, employment status, individual income, marital status and family atmosphere; (2) heroin use: the age of onset for heroin use, DTFUD, the daily dose of heroin used, reasons for first use of heroin, the administration routes of heroin, the euphoria associated with the first heroin use and the euphoria associated with heroin use after dependence; (3) individual responses after heroin use: body weight, sleep quality, memory, appetite and sexual desire; (4) methadone maintenance therapy or withdrawal symptoms: the number of withdrawal and relapse for heroin , the daily methadone dose used, and the methadone maintenance treatment duration. The detailed content of the questionnaire was shown in Supplementary file Written informed consent was obtained from all the study participants. The Ethical Committee of Xi’an Mental Health Center approved our study. We followed the approved guidelines to perform the protocol.SLC6A3 , 4 SNPs in TH , 2 SNPs in DDC (rs11575553 and rs12666409) and 4 SNPs in DBH  were selected. Some important information of these SNPs was summarized in Table https://snpinfo.niehs.nih.gov/snpinfo/snpfunc.html). Detailed effects of these SNPs were shown in Table S1 of Supplementary file SNP selection criteria were as follows: 1) located in the promoter, intron-exon border, exon, the 3’near region and untranslated regions (UTRs) of gene; 2) the minor allele frequency (MAF) greater than 0.05 based on dbSNP Han Chinese Beijing (HCB) and HapMap databases; (3) each SNP had three genotypes. Then 5 SNPs in  located  the minoThe genomic DNA was extracted by E.Z.N.A™ Blood DNA Midi Kit . Then, we used SNaPshot technology to genotype these SNPs. These specific experimental steps can be found in our previous research .Hierarchical data on phenotype variables were used to identify subtypes, and associations between these subtypes with SNPs were first assessed by Pearson’s chi-square test or Fisher’s exact test. Then, these associations were assessed again by binary logistic regression, and the basic information  were set as covariates.We assessed the potential correlation of DTFUD with these genotypes using Kaplan–Meier survival analysis. Survival curves were used to estimate the probability that individuals had not experienced dependence over a period of time following their first heroin use. The DTFUD was regarded as survival time in months. The origin of the first use was specified as the time of first heroin use, and the outcome of interest was the first occurrence of dependence. We used three methods  to compare the survival curves, and these methods give varying weight to different phases of follow-up time.http://sourceforge.net/projects/gmdr/) to analyze the gene-by-gene and gene-by-environment interactions. This method permits adjustment for discrete and quantitative covariates and is applicable to both dichotomous and continuous phenotypes in various population-based study designs [We used generalized multifactor dimensionality reduction . Consequently the loci should be considered significant at P < 0.003. All statistical analyses were performed using SPSS 20.0 software .research  P valuesFor the phenotype of memory, a total of 586 subjects (73.2%) that memory impaired were assigned to the “reduction” group, and 215 subjects (26.8%) that memory no changed were assigned to the “no difference” group.DBH rs1611114 TT genotype was significantly lower in the reduction group  and later-onset  groups accordingly.The age of onset for heroin use was converted to binary using median (28 years old), the sample was divided into earlier-onset  that have obvious feelings of euphoria were assigned to the “strong euphoria” group, and 628 subjects (78.4%) that have minimal level of happiness, no euphoria or unclear were assigned to the “week euphoria” group.TH rs10770140  and a low methadone dose group  based on the median daily methadone dose (45 mg). For the phenotype of heroin dose, the sample was divided into a high heroin dose group  and a low heroin dose group  based on the median daily heroin dose (0.5 g). No SNP was associated with the daily dose of methadone or heroin use  and a short duration group  based on the median of DTFUD (1 month). No SNP was associated with DTFUD in either Pearson’s chi-square test , income and rs3842727 have interaction. In three-way model (income-rs129915-rs1611114), income, rs129915 and rs1611114 have interaction. In four-way model (income-rs12666409-rs129915-rs1611114), income, rs12666409, rs129915 and rs1611114 have interaction. Among these significant interactions, the CV Consistency and Testing Bal. Acc in the four-way model (income-rs12666409-rs129915-rs1611114) were the most significant, this model was the best model (Table These sequencing data of SNPs were listed in Supplementary file Heroin dependence is a heterogeneous disease, and its clinical features and therapeutic outcomes vary substantially among patients. Regardless of linkage analysis or associative analysis, the results are obviously affected by disease grouping within samples, which finally result in poor stability. Features that correlated with dependence could be used as indicators to categorize heroin dependent patients into subgroups with uniform disease features.DBH rs1611114 was significantly associated with memory change after dependence. SNP rs1611114 was located in the 5’near region of DBH and was predicted to be located in the transcription factor binding site (Table S8 of Supplementary file DBH − 1021C/T variant accounted for 35–52% of the variation in plasma DBH activity, and the T allele predicted very low DBH activity [TH rs3842727 exhibited interactions in the memory change phenotype. Income, DBH rs129915, rs1611114 exhibited interactions in this phenotype. Rs1611114, DDC rs1266640, DBH rs129915 and income also exhibited interactions in this phenotype. These results showed that the memory change phenotype was a result of a combination of genetics and environment. TH converts L-tyrosine to L-DOPA, DDC catalyzes the decarboxylation of L-DOPA to generate DA, and then DBH catalyses the oxidative hydroxylation of DA to norepinephrine. SNPs in these genes may interact with each other in regulating the concentration of DA, and then affected the memory change after heroin dependence. Research showed that the higher-income group had greater memory capacity [In this study, we demonstrated that activity . Studiesactivity , 26. Hipactivity . DBH alsactivity . These scapacity . Lower icapacity . TherefoTH rs10770140 and rs10770141 were promoter variants that differentially affected the transcriptional activity of TH [TH Val-81-Met polymorphism was found to be associated with early-onset alcoholism [P value of this article was not corrected.ty of TH  (Table Sty of TH , 33. Howcoholism , as the SLC6A3 rs27072 may be related to the age of onset for tobacco or alcohol [SLC6A3 rs27072 was related to the age of onset for heroin use. We proposed three hypotheses to explain the difference. First, our study excluded alcohol and nicotine dependent subjects. Second, the proportion of our sample that use heroin before 18 years old was relatively small (Fig. S2 of Supplementary file Some studies have found that  alcohol –37. In p alcohol . HoweverDBH rs1611114 in memory change of heroin dependence. This phenotype was a result of a combination of genetics and environment. These findings were valuable for obtaining a better understanding of heroin dependence and improving personalized treatment.In conclusion, our results supported the important role of Additional file 1:. Supplementary file 1: Questionnaire.Additional file 2: Supplementary file 2: Table S1 The functional effects of all selected SNPs. Table S2 Results of association analysis between other SNPs and phenotype of memory. Table S3 Results of association analysis between other SNPs and age of onset for heroin use. Table S4 Results of association analysis between other SNPs and phenotype of euphoria. Table S5 Results of association analysis between SNPs and phenotype of methadone dosage. Table S6 Results of association analysis between SNPs and phenotype of heroin dosage. Table S7 Results of association analysis between SNPs and phenotype of DTFUD. Table S8 Transcription factor binding sites of rs1611114. Table S9 Transcription factor binding sites of rs10770140. Table S10 Transcription factor binding sites of rs10770141.Additional file 3:. Supplementary file 3: Figure. S1. Kaplan–Meier survival analysis of DTFUD. Figure. S2. Frequency distribution of age of onset for heroin use.Additional file 4:. Supplementary file 4: Sequence data.


Upon interactions with its specific ligand hepatocyte growth factor (HGF), the c-Met signal is relayed to series of downstream pathways, exerting essential biological roles. Dysregulation of the HGF-c-Met signaling pathway has been implicated in the onset, progression and metastasis of various cancers, making the HGF-c-Met axis a promising therapeutic target. Both c-Met and HGF undergo glycosylation, which appears to be biologically relevant to their function and structural integrity. Different types of glycoconjugates in the local cellular environment can also regulate HGF/c-Met signaling by distinct mechanisms. However, detailed knowledge pertaining to the glycosylation machinery of the HGF-c-Met axis as well as its potential applications in oncology research is yet to be established. This mini review highlights the significance of the HGF-c-Met signaling pathway in physiological and pathological context, and discusses the molecular mechanisms by which affect the glycosylation of the HGF-c-Met axis. Owing to the crucial role played by glycosylation in the regulation of HGF/c-Met activity, better understanding of this less exploited field may contribute to the development of novel therapeutics targeting glycoepitopes. Since the discovery of c-Met and its high-affinity cognate ligand HGF in 1980s , decadesGlycosylation is a common type of protein post-translational modification, playing a critical role in determining protein function and undergoing profound changes in cancer . It is n494 and Val495, generating mature HGF in a heterogeneous two-chain form  domain  domain composed of the full-length α-subunit connected by a disulfide bridge to the N-terminal portion of β-subunit, a small plexin, semaphoring and integrin (PSI) domain and four immunoglobulin-like regions in plexins and transcription factors (IPT) domains. The intracellular moiety of c-Met comprises three segments: a juxtamembrane segment (JM)  domain . Active ent (JM) , a tyros docking .N-terminal hairpin loop and the first K1 domain that engages with the IPT3 and IPT4 domains of c-Met, and a low-affinity site in the SPH domain of the HGF β-chain that interacts with the SEMA domain of c-Met  within its extracellular domain (O-glycosylation site in the extracellular domain of c-Met (O-glycosylated in the α-chain of HGF (Thr476)  ; Table 2(Thr476) . HoweverAll vertebrate cells are coated with glycans, which are in the hub of a great variety of biological activities, including cell-cell crosstalk, signal transduction and host-microbe interactions . GlycansO- and N-linkages. In N-linked glycosylation, a 14-monosaccharide glycan block (Glc3Man9GlcNAc2) is covalently linked to the asparagine residue of the aglycone part of the protein, usually involving a consensus peptide sequence Asn-X-Ser/Thr (where X stands for any amino acid except proline)  or a GlcNAc moiety is added to the hydroxyl group of a serine or threonine residue in the backbone of proteins  . The newex types . In O-glproteins . In conte moiety . The N- O-glycan truncation, and N- and O-linked glycan branching  chains are covalently attached. HSPGs are one of the most highly negatively charged biopolymers occurred naturally, collaborating with other ECM components to facilitate the ECM remodeling and structural integrity . The var to date , 2013. P to date .3 and GM2 downregulate the trans-phosphorylation of c-Met, which in turn impairs the recruitment of Grb2 as well as activation of downstream pathways  than that in the culture medium of CAFs from normal skin fibroblasts. Blockade of HGF highly reduces H-CAFs proliferation, indicating that HGF is a promising target of antitumor therapies. Under pathological conditions, overexpression of c-Met at both mRNA and protein levels has also been shown in several solid tumor progression  treatment impedes proper cleavage and phosphorylation of c-Met in GTL-16 cells (in vitro. Limited xenograft tumor growth in vivo with Fut8 KO HepG2 is also observed. To further explore the role of Fut8 in carcinogenesis, Fut8-/- mice with DEN/PB treatment are used to induce HCC. Compared to Fut8+/+ mice, Fut8-/- mice show limited liver carcinogenesis and liver regeneration (RTKs, including c-Met, have been considered as potential therapeutic targets in numerous cancers, such as HCC , colon c16 cells , and lea16 cells  and atte16 cells . α2,6-hy16 cells . In addi16 cells . Knockouneration , indicatO-linked glycosylation is another potential post-translational modification of c-Met. O-glycosylation site within the extracellular domain. Furthermore, C1GALT1, a critical mucin-type O-glycosyltransferase localized in the Golgi apparatus (O-glycan modification of c-Met by enhancing c-Met dimerization and activation following HGF stimulation in vivo and in vitro (Early studies indicated that pparatus , is respin vitro . siRNA-min vitro . Howeverin vitro .1a, mediate the activation of c-Met (Glycoconjugates including HSPGs and gangliosides in the local cellular environment also play important roles in regulation of HGF/c-Met activity . HSPGs aof c-Met . NeverthN-linked. Disruption of N-linked glycosylation appears to downregulate the expression level of several RTKs, including c-Met, and subsequently suppress the downstream signaling pathways. However, though multidisciplinary approaches have been applied to evaluate and develop effective therapeutics targeting the c-Met/HGF axis, the glycosylation machinery regulating the c-Met/HGF axis is less explored.Upon HGF-induced activation, aberrant c-Met activation facilitates enhanced tumor cell growth, angiogenesis and invasion in cancer, which is overall associated with poorer survival, suggesting that the c-Met/HGF axis is a promising therapeutic target against malignancies. Extracelluar domains of both c-Met and HGF are heavily glycosylated, with the primary glycosylation mode being Due to the fact that glycosylation also plays key physiological roles under normal circumstances, a question may arise that is it a practical target in cancer therapy? According to currently available data and also due to the non-template-driven nature of glycans, it is likely that glycosylation-related inhibitors targeting the c-Met/HGF axis, irrespective of their nature and source, i.e., independently from being glycolsyltransferase inhibitors or nucleoside antibiotics, will be likely used in combination with other therapeutics in the treatment.Another major challenge in the glycobiology field is to decipher the structural complexity of glycans. Glycomics, compared to genomics and proteomics, has lagged far behind, partly due to analytical difficulties arising from the diverse and heterogenous profile of glycans. Recent advance in analytical techniques has led to detailed characterizations of the glycoproteome of cancer cells. Given the specific glycan information of HGF and c-Met still remains elusive, the acquisition of those relevant knowledge would provide potent opportunities for scouting novel biomarkers and drugs targeting the c-Met/HGF axis in the foreseeable future.HL, LW, and XY proposed the study. XH and FT performed the research and wrote the first draft. All authors contributed to the interpretation of the study and to further drafts.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.


Background: Over the past decade, the political movement called ‘Revolución Ciudadana’ implemented a variety of policies and interventions (P&I) in Ecuador to improve higher education and strengthen local research capacity. We refer specifically to the ‘Mandato 14’ and the Higher Education Law  launched in 2008 and 2010, respectively.Objective: To assess the impact of these P&I (Mandato 14/LOES) on the production of health sciences-related articles (HSRA), and the relationship of these HSRA with the country’s health priorities.Methods: A Scopus search was performed to retrieve HSRA published from 1999 to 2017. Bivariate analysis was used to assess variation between the period I (1999–2008) and period II (2009–2017). Further, we examined the association between the top 10 causes of mortality and the total HSRA output.Results: The final study sample consisted of 2784 articles. After 2008, Ecuadorian production of HSRA increased steadily from 671 to 2133 publications (p<.001). Overall (1999–2017), the most common study design was cross-sectional (32.3%), the primary research focus was in the clinical-surgical area (49.3%), and the academic institutions were the primary drivers of scientific production during period II . Further, we found a decrease in the production of randomized controlled trials . Only 9% of research production involved the primary causes of mortality, and the proportion has remained unchanged over time .Conclusions: Ecuadorian HSRA output increased significantly after 2008. This larger volume of scientific output could be the result to the Mandato 14/LOES implemented in the last decade. However, a low percentage of HSRA are dedicated to addressing the country’s health priorities. Proper planning, execution and monitoring of national health research agendas would reduce the mismatch between health burden and the HSRA output in Ecuador and other low-and middle-income countries. The advancement of the practice of modern medicine is based on the production of biomedical research. As such, enormous resources are invested every year to execute this complex enterprise. For example, in 2010, ~ US$240 billion was invested in health research and development (R&D) globally .Over recent decades, despite political turmoil and economic crisis, research in the Latin America and the Caribbean (LAC) region has advanced considerably due to widespread efforts to improve higher education and research capacity . SeveralLikewise, Ecuador, an Andean country located in northwestern South America, implemented several policies and interventions (P&I) in the healthcare and higher education sectors under President Rafael Correa´s administration 2007–2017) that were aimed at strengthening local research capacities and improving population health –2017 tha. The MAIThis is a bibliometric study used the Scopus database to identify HSRA published in Ecuador during the period 1999 to 2017. This citation index database was chosen because of (i) higher coverage of journals than PubMed and Web of Science (WoS) ; (ii) thWe restricted our searching to articles and review documents published in any language. We searched in Scopus using the following equation:ecuador) AND PUBYEAR > 1998 AND PUBYEAR < 2018 AND  OR LIMIT-TO  OR LIMIT-TO  OR LIMIT-TO  OR LIMIT-TO  OR LIMIT-TO  OR LIMIT-TO  OR LIMIT-TO  OR LIMIT-TO  OR LIMIT-TO  OR LIMIT-TO ) AND  OR LIMIT-TO ).AFFILCOUNTRY  related to the health sciences, (ii) reporting studies conducted/executed in Ecuador, (iii) published during the period of 1999 to 2017, and (iv) containing all the pre-specified study variables. We excluded studies of non-human subjects, duplicate papers, and references for documents for which it was not possible to obtain a full article.We developed and tested one data extraction form to collect and organize additional data about (i) ‘institutional affiliation’; (ii) ‘type of study design’; (iii) ‘research focus’ in basic science, public health, clinical/surgical, and translational research ; and (iv) relationship with the top 10 causes of mortality in Ecuador, categorized as two standardized rankings (Supplementary Revolución Ciudadana initiative in the sector started in 2007 with the declaration of ‘A State of Emergency for Education’ [Descriptive statistics were used to summarize the baseline characteristics of the study publications included in the study. Continuous variables were reported as median and interquartile range (IQR) due to skewness and categorical variables were reported as frequencies and percentages. We defined a pre-policy intervention period before 2008. The rationale to set this cut-off point was that the first ucation’ . ResultsOur search strategy identified 5041 HSRA in Scopus. After exclusion of duplicates  and publications that did not satisfy the inclusion criteria , a total of 2748 publications were retrieved for the final review and analysis .Figure 1The total HSRA production increased by ~215% between period I (1999–2008) and period II (2009–2017), from 671 to 2133 publications (Supplementary Table 4). Overall, there has been a steady increase in the number of HSRA publications in Ecuador across the 18 years analyzed , and durRevolución Ciudadana, public and private academic institutions took the lead of the production of HSRA publications and showed the largest increase in percent change, by 466% (Supplementary Table 5).Overall (1999 to 2017), private universities were the main actor with the highest number of HSRA publications (24.5%); public university and other institutions ranked second (18.8%), followed by the association between the university and hospital institutions (12.6%) . HoweverAcross the 18 years analyzed, clinical and surgical topics were the primary focus of research (49.3%) for Ecuadorian healthcare researchers . This fiThe proportion of HSRA publications related to the top 10 causes of mortality increased from 8.2% in the period I to 9.3% in the period II, but this increment did not reach statistical significance between the analyzed periods (p = 0.37) . DespiteWe found 60 HSRA (2009–2017) publications that mentioned the Prometeo program either in the authorship affiliation or in the acknowledgements section. However, a further analysis of these publications showed that although were conducted locally, 13 of the 60 publications addressed topics unrelated to Ecuador or used data from other countries (Supplementary Table 7). Of the remaining Prometeo HSRA publications (n = 43) the largest proportion (44.7%) came from the Central University of Ecuador . 23.4% of these publications were produced in collaboration with Japan, followed by Venezuela (19.1%) .Revoluación Ciudadana´s P&I on the production of HSRA publications in Ecuador from 2007 to 2017. The primary findings are as follows. First, the P&I (M14 and LOES) implemented in the Ecuadorian higher education system may have resulted in an increased volume of HSRA publications. Second, academic institutions were the primary drivers of HSRA publications in the country. Third, most of the HSRA output consists of observational studies (55.6%). Fourth, the primary research focus is in the clinical-surgical area. Fifth, a low percentage of the overall HRSA production was dedicated to addressing the country’s health priorities, specifically, the primary causes of mortality.These results highlight the potential impact of the Ecuador significantly increased its production of HSRA publications across the entire period, and especially over the past decade, from 671 to 2133 publications. This growth could be the result of M14 and LOES, which catalyzed improvements in the qualification and accreditation processes of local higher education institutions (HEI), increased the investment of substantial funds for the development of a critical mass of scientists, provided for more government funding of research projects, and enhanced international collaboration  , review , and ecological  studies. Methodologically speaking, these study designs are useful for establishing preliminary evidence of a causal relationship. In contrast, there were few studies of a higher level of evidence to inform health policy-making such as cohort, RCTs and meta-analysis studies: 3.5% (n = 99), 2.9% (n = 83) and 0.1% (n = 4), respectively. We were surprised to find a decreasing trend for cohort and RCTs studies, from 4.8% to 3.2% and 6.7% to 1.8%, respectively (p < .001). This shift could be explained by the fact that analytical studies demand more financial, time, and logistical resources or by a lack of skilled personnel, or by the pressure exerted by the LOES institutional ranking. Ecuadorian researchers may have chosen to perform quick, relatively easy, and inexpensive studies in order to satisfy the new institutional ranking mechanism. Other factors that could explain these findings are ethical and regulatory system obstacles . In EcuaOf the 2784 HSRA included in this study, 1372 (49.3%) of them were related to topics in the clinical/surgical area compared to other areas such as public health . Further, only 252 (9%) HSRA of the overall production addressed the top 10 causes of mortality in the country, and this proportion remained unchanged across both analyzed periods. This mismatch between the HSRA output and disease burden has been previously reported elsewhere ,22–24 anOur investigation has several strengths. We included lab-based studies in our analysis and thus were able to assess certain diseases more likely to be addressed by these types of studies, such as cancer. Potential limitations should also be considered. Although we used a comprehensive database (Scopus) to retrieve Ecuadorian HSRA, otherwise eligible publications not indexed in Scopus  may have been omitted. However, due to the extensive period analyzed, we anticipated that the effect of any missed publications would be minimal. We did not measure agreement among reviewers; thus, the possibility of misclassification cannot be ruled out. This study did not assess other dimensions or indices to identify and evaluate the impact of research such as morbidity or disability-adjusted life years (DALYs) , researcRevolución Ciudadana movement, Ecuador benefitted from the largest increase in revenues in contemporary history because of boom in oil, its most important export, and a concomitant growth in GDP, which grew from US$51 billion in 2007 to US$94.47 billion in 2013 [Revolución Ciudadana were not a constraint, as is commonly reported in Low-and Middle-Income Countries (LMICs) [During the decade of the  in 2013 . This in in 2013 ,33. Thus (LMICs) ,34. The  (LMICs) . Second, (LMICs) . Third,  (LMICs) . Fourth, (LMICs) . Fifth,  (LMICs) .Revolución Ciudadana’s P&I (M14 and LOES) that were implemented in the past decade. Most of the health sciences research output consisted of observational studies and the primary research focus was in the clinical-surgical area. Despite these advances, a low percentage of HRSA have been dedicated to addressing the country’s health priorities, and the proportion has remained unchanged over time. Proper planning, execution, and monitoring of national health research agendas would reduce the mismatch between health burden and the HSRA output in Ecuador and other LMICs.In summary, our study showed that Ecuadorian HSRA increased significantly after 2008. This larger volume of scientific output could be the result of the Click here for additional data file.