Staphylococcus aureus resistance, and whether the possible restriction is associated with changes in antibiotic mutant prevention concentrations (MPCs), the enrichment of daptomycin- and gentamicin-resistant mutants was studied by simulating 5-day single and combined treatments in an in vitro dynamic model. The MPCs of the antibiotics in the combination were determined at concentration ratios equal to the ratios of 24 h areas, under the concentration–time curve (AUCs) of the antibiotics, as simulated in pharmacodynamic experiments. The MPCs of both daptomycin and gentamicin decreased in the presence of each other; this led to an increase in the time when antibiotic concentrations were above the MPC (T>MPC). The increases in T>MPCs were concurrent with increases of the anti-mutant effects of the combined antibiotics. When anti-mutant effects of the antibiotics in single and combined treatments were plotted against the T>MPCs, significant sigmoid relationships were obtained. These findings suggest that (1) daptomycin–gentamicin combinations prevent the development of S. aureus resistance to each antibiotic; (2) the anti-mutant effects of antibiotic combinations can be predicted using MPCs determined at pharmacokinetic-based antibiotic concentration ratios; (3) T>MPC is a reliable predictor of the anti-mutant efficacy of antibiotic combinations.To explore whether combined treatments with daptomycin and gentamicin can prevent the development of  Staphylococcus aureus [S. aureus strains, the development of daptomycin resistance was restricted in the presence of gentamicin [Emerging trends in antimicrobial resistance (AMR) presuppose the need to search for effective tools to improve treatment outcomes. One such tool is the use of combined therapy, with two or more antimicrobial agents, to mitigate the effects of AMR. Daptomycin is widely used to treat gram-positive infections including endocarditis and bacteremia, especially those caused by s aureus ,2,3,4. Hs aureus ,6,7,8 his aureus ,10; howeS. aureus strains [S. aureus cells when staphylococci were exposed to stepwise-increasing concentrations of daptomycin, alone or in combination with gentamicin [S. aureus susceptibility to both drugs noted in respective mono-treatments. However, in other studies [Anti-mutant activity of the daptomycin–gentamicin combination was investigated in several in vitro studies, conducted in both static ,12 and d strains . Howeverntamicin , the com studies ,15, onlyS. aureus. The observed enhancement was attributed to lengthening the time within the dosing interval, when antibiotic concentrations exceeded the mutant prevention concentration (MPC) (T>MPC), as a result of lowering the MPCs of each antibacterial in the presence of the other. The MPCs of linezolid and daptomycin used in this combination were determined at concentration ratios equal to the ratio of the 24 h area under the concentration–time curve (AUC) of linezolid to the AUC of daptomycin simulated in the pharmacokinetic experiments. This allowed determination of the antibiotic MPCs used in the combination studies at pharmacokinetic-based concentration ratios.A previous in vitro pharmacodynamic study of the combination of daptomycin with linezolid  revealedS. aureus mutants resistant to both drugs, and if such restriction is associated with changes in antibiotic MPCs, the enrichment of daptomycin- and gentamicin-resistant mutants was studied by simulating 5-day single and combined treatments in an in vitro dynamic model. The exposures of daptomycin were simulated to provide subtherapeutic doses of the lipopeptide and to allow higher probability of daptomycin-resistant S. aureus mutant growth. In contrast, gentamicin-pharmacokinetics were simulated at therapeutic exposures, according to previous in vitro studies that reported high intensity of growth of gentamicin-resistant mutants, even at high-dose aminoglycoside regimens [To explore whether combined treatments with daptomycin and gentamicin can prevent the enrichment of regimens ,15,17. Aregimens , the MPCMPCs were assessed for daptomycin and gentamicin alone and in combination . IncreasS. aureus mutants resistant to 2 × MIC of daptomycin and gentamicin are shown in S. aureus mutants resistant to 2 × MIC of antibiotic emerged after 24 h from the beginning of the experiment. At high daptomycin exposure (D100) the amplification of antibiotic-resistant cells was less pronounced, and occurred 72 h after beginning the experiment . At regimen D30+G160, the growth of gentamicin-resistant mutants was completely restricted. Similar trends, showing enhanced anti-mutant efficacy of daptomycin–gentamicin combinations, were observed when bacterial growth was analyzed on agar plates with 4 × MIC of daptomycin and gentamicin ) in the presence of gentamicin was consistent with increases in T>MPC levels (M(D)s declined from 122 (regimen D30) to 28 (log CFU/mL) × h (regimen D30+G65), along with the lengthening of T>MPC from 0% to 12%, respectively. Changes in the anti-mutant effect of gentamicin (expressed as AUBCM(G)) under the influence of daptomycin are shown in T>MPCs. For example, when AUBCM(G) decreased from 530 (regimen G65) to 425 (log CFU/mL) × h (regimen D30+G65), T>MPCs increased from 7% to 24%.Enhancement of anti-mutant effects of daptomycin  of daptomycin (AUBCM(D)) and gentamicin (AUBCM(G)), respectively. The present study demonstrates the protective effects of lengthening the times above MPC for daptomycin and gentamicin against the emergence of resistant S. aureus mutants. Similar conclusions, regarding the key role of increased T>MPCs of antibiotic combinations in enhancing their anti-mutant effects, were reported in our studies of combinations of daptomycin with linezolid [In the current study, the anti-mutant potential of daptomycin–gentamicin combinations against trations ,15,17. Afampicin , daptomyfampicin , linezolfampicin . In the inezolid  and lineinezolid .S. aureus observed in mono- and combined treatments and their respective T>MPCs, AUBCMs were plotted against T>MPCs achieved in both single and combined treatments (merged data for each antibiotic) (T>MPC relationships with AUBCM(D) or AUBCM(G)  were found. This indirectly suggests that MPCs determined at pharmacokinetic-based concentration ratios are predictive of the anti-mutant effectiveness of antibiotic combinations. The sigmoid shape of the relationships between resistance and T>MPC has been reported previously with fluoroquinolone [To explore the relationships between daptomycin- and gentamicin-resistant ibiotic) . Reasonauinolone ,21,22,23uinolone ,25 monotS. aureus strains [The anti-mutant efficacy of daptomycin–gentamicin combinations has been described previously in a study using an in vitro pharmacodynamic model with simulated endocardial vegetations . Gentami strains . Our fin strains ,15.S. aureus strain was used. This limits the potential clinical relevance of our findings and suggests the need for further investigation of the anti-mutant potential of daptomycin–gentamicin combinations with a larger number of S. aureus strains. In addition, the clinical relevance of the current study is limited as we used subtherapeutic daptomycin doses. To improve the clinical significance of our research future simulation of therapeutic doses of daptomycin against S. aureus strains with decreased lipopeptide susceptibility should be considered. Another limitation is that the current study was carried out in in vitro conditions; this did not allow considerations of any effects of the immune system and/or protein binding, which might influence the anti-mutant efficacy of the antibiotics.It is worth noting that in the current study only one daptomycin-susceptible S. aureus 293 was used in the study; it was susceptible to both daptomycin and gentamicin with MICs of 0.5 and 0.25 mg/L, respectively.Daptomycin powder was purchased from Acros Organics ; gentamicin sulfate was purchased from PhytoTechnology Laboratories . Clinical isolate Antibiotic MPCs, alone and in the presence of each other, were determined as described elsewhere . Each exBoth single and combined treatments mimicked subtherapeutic dosing regimens of daptomycin, with respective AUCs of 30 mg × h/L  and 100 mg × h/L (regimen D100), and therapeutic dosing regimens of gentamicin, 5 and 7 mg/kg once daily, with respective AUCs of 65 mg × h/L (regimen G65) and 160 mg × h/L (regimen G160) calculated using peak serum gentamicin concentrations reported in human studies  ,27. CombAll pharmacodynamic experiments with once-daily dosing of daptomycin or gentamicin used alone or in combination were conducted for five consecutive days. The simulated antibiotic half-lives were as follows: 9 h for daptomycin  and 3 h 2+/L (CSMHB), because daptomycin antimicrobial activity is influenced by the presence of Ca2+ [To simulate mono-treatments with daptomycin and gentamicin a previously described dynamic model  was used of Ca2+ . One flaIn combined treatments simultaneous mono-exponential elimination of daptomycin and gentamicin was simulated; the model was modified according to the Blaser and Zinner principle . Briefly8 colony-forming units (CFU)/mL. Then antibiotics were administered into the central unit of the model. The duration of each experiment was 120 h.The operation procedure used in the pharmacodynamic experiments was as described elsewhere . AntibioS. aureus 293 in the pharmacodynamic experiments, the central unit of the model was multiply sampled throughout the observation period (120 h). The samples were serially diluted, if necessary, plated on Mueller–Hinton agar (MHA) with 2×, 4× and 8 × MIC of daptomycin or gentamicin, and incubated for up to 72 h at 37 °C. The viable counts were screened visually for growth. The lower limit of detection was 10 CFU/mL .To monitor the time courses of antibiotic-resistant subpopulations of S. aureus mutants resistant to 2 × MIC of antibiotic, the area under the bacterial mutant concentration–time curve (AUBCM) [M(D); for staphylococci resistant to gentamicin it was designed as AUBCM(G).To characterize the time courses of  (AUBCM)  was calcM(D)s and AUBCM(G)s, determined in simulated mono- and combined treatments (merged data), were plotted against T>MPC and fitted by the sigmoid function:M(D) or AUBCM(G), x is T>MPC, Y0 and a are the minimal and maximal values of the AUBCM, respectively, x0 is x corresponding to a/2, and b is a parameter reflecting sigmoidicity.The AUBCAll calculations were performed using SigmaPlot 12 software .S. aureus mutants; (2) the inhibition of resistant S. aureus mutants using daptomycin–gentamicin combinations can be predicted by MPCs determined at pharmacokinetic-based antibiotic concentration ratios, and (3) T>MPC is a reliable predictor of the anti-mutant efficacy of antibiotic combinations as studied using in vitro dynamic models.The present study suggests that (1) daptomycin–gentamicin combination restricts the development of daptomycin- and gentamicin-resistant


This study was conducted to investigate the effect of ground granulated blast furnace slag on the structural performance of precast concrete beams, evaluating the flexural, shear and bonding performance by using the replacement ratio of the ground granulated blast furnace slag as a variable. The design strength of the concrete was set at 45 MPa in consideration of the characteristics of precast concrete products, and the replacement ratio of the ground granulated blast furnace slag to replace cement was 30 to 70%. The experimental results showed that all specimens had similar behavioral characteristics regardless of the replacement ratio of the ground granulated blast furnace slag. Comparison of the prediction results obtained by ACI 318-19 and EC 2 showed that the mean flexural strength and shear strength were higher than 1.19 and 1.43, respectively, and the mean bond strength was 1.57, satisfying the required performance. Therefore, the experimental results showed that in using the ground granulated blast furnace slag as an admixture for precast concrete, the cement replacement ratio may be increased up to 70% without causing any problems in securing the structural performance. Summarizing the results of the present study, a ground granulated blast furnace slag replacement ratio of 50% or lower may be reasonably applied. Since the development of Portland cement in the early 19th century, concrete has been applied to architectural and civil engineering structures as a key structural material because of its simple manufacturing method and excellent durability. And with the advances of the construction industry, many developments and studies have been made to secure the required quality and performance. Various approaches have been proposed to improve concrete quality and performance. In particular, the recycling of industrial byproducts to secure a cement replacement can provide both environmental and economic advantages.Steel slag, a byproduct from the steel industry, is largely divided according to the production process into blast furnace slag and steel furnace slag. Blast furnace slag, generated from the process of producing pig iron from a blast furnace using iron ore, coke and limestone as raw materials, is further divided into water-cooled slag and air-cooled slag according to the cooling method. The production of blast furnace slag is dependent on the iron-making process, and about 300 kg of blast furnace slag is generally generated when 1 ton of molten steel is produced. Water-cooled slag accounts for about 70–80% of generated blast furnace slag; the rest is air-cooled slag. Blast furnace slag has a chemical composition similar to that of conventional construction materials such as cement and natural aggregate, and its stability allows for recycling into various forms, including cement replacement, admixture, aggregate and bricks ,3,4,5. AAccording to the results of previous studies, Majhi et al., 2020) [020 [2], As the demand for iron is increasing, the generation of steel slag is increasing every year. Although most steel slag is recycled at present, it is necessary to find high value-added applications to utilize it more positively according to advances of the construction industry. Gradually breaking free from the traditional labor-intensive methods of the construction industry, new construction methods and structural systems are drawing attention to make site management easier and minimize on-site work . PrecastHigh-strength concrete is used to increase the durability of precast concrete products, and the application of ground granulated blast furnace slag may effectively improve the workability and chemical resistance of high-strength concrete. In addition, steam curing for expression of the initial strength may resolve the limitation of the replacement ratio of ground granulated blast furnace slag and the problems related to curing, and thus it may allow for extensive recycling. However, research on the structural performance of precast concrete members using ground granulated blast furnace slag is still loose ends.Therefore, the present study was conducted to investigate the effect of ground granulated blast furnace slag on the structural performance of precast concrete beams in terms of flexural, shear and bonding strength by using the replacement ratio of the ground granulated blast furnace slag as a variable. The experimental results were compared with predictions by ACI 318-19 and EC 2 as well as with results provided in previous reports. Based on this comparison, an appropriate replacement ratio of ground granulated blast furnace slag was investigated to secure structural stability of precast concrete beam members manufactured using ground granulated blast furnace slag. And it is expected that the results of this study can be used as basic data to review the appropriate replacement ratio of ground granulated blast furnace slag in the manufacture of precast concrete members.Considering the properties of existing precast concrete products, the concrete was designed with a design strength of 45 MPa by varying the replacement ratio of ground granulated blast furnace slag, as shown in To investigate the compressive strength of the concrete, circular concrete cylinders of Φ100 × 200 mm were prepared when the specimens were placed, and the cylinders were cured under the same conditions as the specimens. The compressive strength of the concrete was tested on the days of experiment for each specimen series; the test results are shown in To evaluate the structural performance of the precast concrete beam members depending on the replacement ratio of ground granulated blast furnace slag, specimens of the F, S and B series, 5 for each series, were fabricated, as shown in a/d) was designed to be 4.0, and thus the clear span was 2900 mm long. The F series had a tensile reinforcement ratio of 1.94%, and shear reinforcements were arranged in the experimental interval in a gap of 100 mm to prevent shear failure. The S series specimens were 2000 mm long and had a cross-sectional area of 200 × 350 mm. To induce shear failure, no shear reinforcements were arranged in the S series specimens. The B series specimens were 2300 mm long and had a cross-sectional area of 250 × 400 mm. To prevent flexural failure from preceding bond failure, two-fold longitudinal reinforcements were arranged. In addition, shear reinforcements were densely arranged with gaps of 50 mm to prevent shear failure. As shown in As shown in Loading to the specimens was performed using a universal testing machine (UTM)  in the shape of a simple beam, as shown in The deflection of the beam by loading was measured by installing a linear variable differential transducer (LVDT)  at the central bottom of the loading point where the maximum deformation of the specimens was generated. In addition, strain gauges for concrete were attached to positions 10 mm and 30 mm away from the compressive edge to measure the strain of the concrete at the loading point. As shown in Flexural cracks of the F series specimens were generated at loads of 24.3 to 32.3 kN; the deflection was 0.46 to 1.04 mm. The tensile reinforcements reached yield strain at 216.8 to 241.5 kN, and the displacement was 12.5 to 14.1 mm. The load and deflection at the maximum stress were 276.2 to 286.7 kN and 29.7 to 36.0 mm, respectively. In the cases of the F series, the stress of all the specimens, except the F50 specimen, significantly decreased (about 10% or more) after the maximum stress. This may be because of the crushing of the concrete that happened at the compressive edges.Here, As shown in c/d) depending on the replacement ratio of ground granulated blast furnace slag. The neutral axis depth in the experimental results was calculated using the strain measured by the strain gauges attached to the tensile reinforcement and the compressive edge concrete according to the Bernoulli-Euler hypothesis, as shown in Here, As shown in Ductility refers to the load resistance capability while maintaining the deformation of the member without a significant decrease of the stress; it is represented by a curvature ductility factor  (0.25 or lower for C50 at ultimate limit state). As shown in As shown in yP–0.6yP zone, corresponding to the service load state, was less than 0.3 mm, which satisfied the allowable crack width constraints provided by ACI 318-19 [I 318-19  and EC 2I 318-19 . The groAs shown in Here, As shown in The bond stress-slip relationships and the bond strength of the B series specimens are shown in Here, As shown in As shown in Comparison of the bond strength between the bottom reinforcements and the top reinforcements of the B series specimens showed that the mean bond strength of the bottom reinforcements was higher in all the B series specimens than those of the top reinforcements, at ratios of about 1.03 to 1.25, except for the B30 specimen. The consolidation effect of compacting during concrete placement may have increased the bond strength at the bottom reinforcements. In addition, the bond strengths in consideration of the compressive strength of the concrete (The bond strength obtained from the experiment with the B series specimens was evaluated in comparison with the prediction results from ACI 318-19 , Fujii-MThe bond strength was compared between the inner longitudinal reinforcements and the corner longitudinal reinforcements. The mean experimental/analytical ratios of the ACI 318-19, Fujii-Morita and Maeda formulas were 1.06 to 1.32 and their coefficient of variation were 7.3 to 9.6% for the inner longitudinal reinforcements; these values were 1.46 to 1.81 and 10.9 to 15.7% for the corner longitudinal reinforcements. This showed that predictions were better for inner longitudinal reinforcements than for corner longitudinal reinforcements. The difference may be because the prediction formulas did not consider the constraint by the transverse reinforcement. In addition, according to the positions of the longitudinal reinforcements, ACI 318-19  evaluateThis study was conducted to apply ground granulated blast furnace slag to precast concrete members to find an appropriate replacement ratio. The flexural, shear and bond performances of precast concrete beams were evaluated using the replacement ratio of ground granulated blast furnace slag as a variable. Although it may vary depending on the chemical composition of ground granulated blast furnace slag, the following conclusions could be obtained in this study.The flexural strength of the F series specimens was predicted by ACI 318-19 and EC 2. The mean ratio of the yield moment of the experimental results to that of the prediction results was 0.99, and the mean ratios of the ultimate moment were 1.21 and 1.19, respectively. This showed that the experimental results were predicted conservatively;The mean shear strengths of the S series specimens, measured experimentally, were 1.43 times and 1.71 times higher than the shear strength required by the ACI 318-19 and EC 2 standards, regardless of the replacement ratio of ground granulated blast furnace slag. However, in consideration of the concrete compressive strength, the shear strength of the S series specimens decreased as the replacement ratio of ground granulated blast furnace slag increased. Therefore, consideration is needed to determine the appropriate replacement ratio that can secure the required shear performance;The bond strengths of the B series specimens, measured experimentally, were 1.26 to 1.57 times higher than the values predicted by the formulas of ACI 318-19, Fujii-Morita and Maeda, regardless of the replacement ratio of ground granulated blast furnace slag. Therefore, the required bond strength was sufficiently satisfied. The formula proposed by Maeda predicted the experimental results at a mean experimental/analytical ratio of 1.26 with a coefficient of variation of 8.8%, indicating that the prediction was better than those obtained using formulas according to ACI 318-19 and Fujii-Morita;In consideration of the experimental results, in using the ground granulated blast furnace slag as an admixture for precast concrete, the cement replacement ratio may be increased up to 70% without causing any problems in securing the structural performance. However, in order to ensure sufficient safety of precast concrete members, it is considered reasonable to apply the replacement ratio of ground granulated blast furnace slag to 50% or less.


The MET receptor is a critical member of cancer-associated RTKs and plays an important role in different biological activities, including differentiation, migration, and cell proliferation. In this study, novel MET inhibitors were introduced and applied on esophageal squamous carcinoma cell line KYSE-30, and the level of proliferation and migration, as well as the activated form of MET receptor protein were assessed in the examined cells. The human KYSE-30 cell line was cultured according to ATCC recommendations. The mRNA level of the MET gene was measured in the examined cell line using the quantitative RT-PCR assay. Cytotoxicity evaluation test was performed at different concentrations of heterocyclic anti-MET compounds . Finally, the capability of these compounds in MET receptor inhibition was evaluated using the migration assay and Western blot. All experiments were performed in triplicate and repeated three times with similar results. p ≤ 0.05) by all the above-mentioned compounds. Moreover, the majority of compounds significantly prevented the cell migration (p ≤ 0.05) and inhibited MET autophosphorylation. Interestingly, the level of phosphorylated MET was significantly correlated with KYSE-30 cell migration. Cell growth and proliferation were significantly inhibited (The obtained data introduced and confirmed the biological activities of the mentioned novel compounds in KYSE-30 cells and proposed that the therapeutic inhibition of MET with these compounds may be a powerful approach for inhibiting cancer cell migration and proliferation although some structural optimizations are needed to improve their inhibitory functions.  Despite recent advances in medical and surgical treatments, including new surgical methods, preoperative chemo-irradiation, and postoperative chemotherapy, the patients’ five-year survival rate remains low (15-25%) due to the poor prognosis of the disease[2]. Therefore, specific prognostic and diagnostic biomarkers are required for either the targeted therapy of the disease or improvement of the patients’ survival. ESCC is the sixth leading cause of cancer deaths globally, developmental abnormalities[5], and tumorigenesis[6]. MET protein was first discovered in a human osteogenic sarcoma cell line as a part of a hybrid gene generated by the fusion of translocated promoter region from chromosome 1q25 and MET . Cells with the epithelial origin normally express MET[9] although endothelial cells[10], adipose tissues[11], hepatocytes, hematopoietic cells[12], melanocytes, and neonatal cardiomyocytes express MET receptors, as well[13]. MET receptor activation occurs through the binding of its cognate ligand to the extracellular domain, triggering kinase activity, and phosphorylation of two tyrosine residues (i.e. Y1234 and Y1235) in the cytoplasmic domain of the receptor. Docking sites for intracellular adaptor proteins are phosphorylated residues that can modulate the cell survival, differentiation, and proliferation through the activation of downstream components involved in different cell signaling cascades, including the MAPK, PI3K-AKT, and STAT signaling pathways[16-19]. The irregular activation of MET kinase happens by a variety of mechanisms such as the ligand-mediated HGF, autocrine or paracrine stimulation, gene mutation, and amplification, as well as cross-talk with other receptors[20]. The malfunction of MET kinase has been reported in several malignancies, comprising thyroid, renal cell, colorectal, ovary, breast, pancreas, prostate, gastric, and liver cancers, along with ESCC[16]. RTKs serve as essential transmembrane receptors and rely on signals from the extracellular environment into the cells. Their inhibition may cause the deregulation of cell signaling pathways and lead to metabolic defects[21] since it promotes proliferation, invasion, migration, and tumor cell metastasis[22]. Current clinical drugs against the HGF/MET pathway include SMIs and monoclonal antibodies. The SMIs can block MET kinases through binding to its ATP-binding pocket and can suppress the MET-dependent biological activities of the cell[24]. SMIs are considered as one of the most emerging molecular therapeutic strategies against cancer cells, and extensive efforts have focused on designing, synthesis, and biological evaluation of these molecules against specific kinases[25]. The present study was designed to evaluate the impact of MET phosphorylation on the biological behavior of the ESCC cell line KYSE-30 through introducing and investigating the efficacy of some novel heterocyclic inhibitor molecules against MET kinase activity.Pathological MET activation has been suggested as a therapeutic target for cancer treatmentCompounds[26]. Drug-likeliness was examined by applying Lipinski’s rule of five for all compounds[27]. DMSO was used to make a stock solution of the compounds because of better solubility, and then DMSO toxicity was assessed by MTT assay. In this study, eight compounds  were selected for experiments. The nuclear magnetic resonance and mass spectrometry were employed to confirm the chemical structure of compounds[26].In this study, we used six novel heterocyclic compounds, which were designed and synthesized by Saadatmandzadeh and co-workers, the Department of Chemistry, Ferdowsi University, Mashhad, Iran. The structural features of compounds are summarized in Cell culture2 at 37 °C[30].For experiments, human esophageal squamous carcinoma cell line KYSE-30 was chosen due to its invasive nature and obtained from the National Cell Bank, Pasteur Institute of Iran (Tehran). The cells were cultured in the RPMI -1640 medium  supplemented with 10% fetal bovine serum  and 100 U/mL-100 mg/mL of penicillin-streptomycin (PAA) in a humidified atmosphere with 5% CORNA extraction, cDNA synthesis, and RT-PCR6 cells were lysed by adding 750 μL of the lysis buffer and 150 μL of chloroform and then centrifuged at full speed for 12 minutes. All steps were performed according to the manufacturer’s instructions. The purity and quantity of total RNA were measured by a NanoDrop spectrophotometer . Moreover, the integrity of the RNA was evaluated by the electrophoresis on 1% agarose gel, and 28S and 18S ribosomal RNA bands were observed accordingly. After the treatment of the extracted RNA by deoxyribonuclease I, cDNA was synthesized using the Thermo Fisher Scientific (USA) kit based on the manufacturer’s protocol. Next, cDNA was subjected to PCR amplification in a 20-µL final reaction volume, including 1 µL of cDNA, 0.5 µL of each primer (20 mM), 6 µL of dH2O, and 12 µL of Taq DNA Polymerase Master Mix RED . The glyceraldehydes 3-phosphate dehydrogenase gene was used as the internal control, and the target gene (MET) evaluated by using a speciﬁc primer set, including sense 5’TTGGATAGGCTTGTAAGTGCCC3’ and antisense 5’TACTGCACTTGTCGGCATGAA3’. Amplifications were conducted in 45 cycles as the initial denaturation step at 95 °C for 10 minutes, the denaturation step at 94 °C for 15 seconds, the annealing step at 60 °C for 30 seconds, extension at 72 °C for 30 seconds, and the final extension at 72 °C for 5 minutes. Eventually, PCR products were detected on 2% agarose gel electrophoresis.The total RNA was extracted from the KYSE-30 cell line using the total RNA extraction kit . Approximately 2 × 10Cell proliferation assay4 KYSE-30 cells per well were applied and treated with the increasing concentration (ranging from 1.95 to 16000 µM/µL) of the compounds in 96-well plates for 48 hours at 37 °C[31]. The cell viability was assessed by Resazurin reagent (Sigma-Aldrich). The fluorescent intensity of resorufin, as the reduced form of resazurin, was measured in viable cells by a plate reader . In addition, the IC50 value for each compound was calculated by Graph Pad Prism . The experiment was performed in triplicates, and the mean of three independent assays was measured as the final IC50 value.Nearly 5 × 10In vitro wound healing assay6 and incubated at 37 °C for 24 hours. Next, to create a cell-free zone, confluent cells were scraped with a sterile 100-μL pipette tip and after scraping, the cellular debris was removed by washing three times with PBS. Cells were then incubated with the IC50 concentrations of the compounds for 24 hours. The process of cell migration was followed by using an inverted microscope and the images were obtained immediately after scraping (0 hour) and at different time points (e.g. 24 and 72 hours). The experiments were performed in triplicate. The area of the scratches in different images was measured using Image J software . To quantify the migration ability of the cultured cells, the cell-free areas of the scratches at different times were calculated as a percentage of the initial scratched area at 0 hour in each group of drugs. The experimental data were plotted to describe the percentage of the cell-free area versus time points[32].The effects of heterocyclic compounds on the migration of KYSE-30 cells were examined using the scratch assay. KYSE-30 cells were seeded onto six-well plates at a density of 1 × 10Western blot 6 cells/well and allowed to attach overnight. The cells were treated with the IC50 concentrations of the compounds as indicated above. The cultured cells were washed with PBS, lysed with a 70-µL ice-cold radio-immunoprecipitation assay buffer  and treated with a protease inhibitor (Sigma-Aldrich). Then cell lysates were centrifuged at 13,000 × rpm at 4 °C for 10 minutes, and the supernatants were analyzed for the presence of target protein. For immunoblotting analysis, identical amounts of extracted proteins from each sample were subjected to electrophoresis on 10% SDS poly-acrylamide gel electrophoresis, and the proteins were transferred to polyvinylidene ﬂuoride membrane. Next, the blot was incubated in the blocking buffer containing 2% bovine serum albumin at room temperature for 2 hours. Then polyvinylidene ﬂuoride membranes were probed overnight with 1:500 dilution of primary monoclonal rabbit antibody against phospho-Y1234/1235 MET  at 4 °C, followed by incubation with anti-rabbit immunoglobulin G peroxidase  at room temperature for 1 hour. Using sc-47778 , blotting was performed for β-actin as a control for protein loading. Finally, the rabbit peroxidase-conjugated secondary antibody was applied, and antibody-bounded proteins were visualized using the enhanced chemiluminescence Western blot analysis system . Twenty-five microgram of protein samples were separated on a 10% SDS acrylamide gel  at 150 V for 1 hour, and the proteins were transferred to a nitrocellulose membrane . After blocking in 5% fat-free milk, the membrane was treated with the diluted primary antibody (1:800) at 4 °C overnight and then with the diluted secondary antibody (1:3000) at room temperature for 1 hour. Subsequently, immunoblot membranes were visualized by an enhanced chemiluminescence reaction using the detection kit . The Western blotting assay was repeated at least three times on every sample with similar results. Finally, the relative intensity of immunoreactive protein bands was quantified by Image J software[33].KYSE-30 cells were seeded onto six-well plates at a density of 1 × 10Statistical analysisp ≤ 0.05 was regarded as statistically signiﬁcant. The statistical analysis was conducted using SPSS software  and GraphPad Prism . Data were presented as the mean ± SD from three independent experiments. The scratch assay data were statistically compared using the one-way ANOVA test to find differences in the migration of KYSE-30 cells between different treatment groups. A Important structural features of novel compoundsLipinski’s rule of five was applied on all the compounds, as reported in Cell proliferation 50 values in p ≤ 0.05). IC50 values for different compounds were determined as 75 µM (D8), 162 µM (D7), 167 µM (D1), 307 µM (D6), 556 µM (D2), and 674 µM (D6). In addition, similar results were found regarding the proliferative potential related to three independent experiments after treatment, and all results were expressed as the mean ± SD of three replicate wells. The inhibitory effect of compounds on MET kinase activity is shown as curves in 50 value among all compounds. The IC50 concentration had a rising trend in D7, D1, D6, D2, and D5. Eventually, the cell growth and proliferation were significantly inhibited by all these compounds (p ≤ 0.05), the details of which are provided in The expression of MET mRNA was confirmed in the KYSE-30 cell line by RT-PCR. The cytotoxic effects of all synthesized compounds, including D1, D2, D5, D6, D7, and D8, were assessed on KYSE-30 cell proliferation. The results were expressed as ICWound healing assay 50 value compared with D5 and D6 , the related data of which are illustrated in The wound healing assay was performed to analyze the effect of the applied compounds on the migration of the KYSE-30 cell line. All the compounds (except for D8) significantly decreased the KYSE-30 cell migration . Among tEffects of the compounds on MET auto-phosphorylation50 values for compounds, corresponding to the MET auto-phosphorylation, are summarized in Table 3. Five out of six novel applied MET SMIs, including D1, D2, D5, D6, and D7 (except for D8), efﬁciently inhibited MET autophosphorylation at tyrosines 1234/1235 in the KYSE-30 cell line. However, D8 demonstrated no significant MET inhibitory effect on KYSE-30 cells  of MET and decreased the cell proliferation and survival. IC30 cells . Correlation between MET autophosphorylation and KYS-30 cell migration50 values of the applied compounds), a linkage was found between the level of MET phosphorylation and cell migration. It was revealed that ESCC cell migration was significantly inhibited by these SMIs in the examined cell line. The inhibition of MET RTK phosphorylation reduced the cell migration ability of KYSE-30 cells. A low level of MET phosphorylation, confirmed by the Western blot, decreased the migration potential of KYSE-30 cells though a high level of MET phosphorylation (a strong band in the related Western blot test) was significantly correlated with a large distance of KYSE-30 cell migration (p = 0.006). The obtained data are depicted in Based on the combined results of Western blot analysis (which detected the phosphorylated form of MET protein) and wound healing assay  can block the HGF/MET pathway, specific agents against the MET/HGF signaling are not well-defined yet. MET overexpression has been reported in aggressive tumors such as gastric, renal, esophageal, and lung malignancies. Therefore, MET kinase inhibitors are expected to provide a clinical beneﬁt in related patients. Our results suggested that an effective dose of these compounds can be used as therapeutic agents. Except for D8, all heterocyclic compounds, which were used in this study, inhibited not only cell migration but also the autophosphorylation of the MET receptor in KYSE-30 cells. The D8 compound with a functional amino group (-NH2) had the lowest IC50 concentration (IC50 = 75.29 μM) among the other compounds of interest. Our results showed that D8 significantly (p = 0.05) inhibited cell growth in its IC50 concentration. It may imply that D8 could effectively reduce cell metabolism, division, and proliferation although its MET inhibitory effect was low. D1, D2, D5, D6, and D7 had the IC50 concentrations of 167 μM (p = 0.04), 556 μM (p = 0.03), 674 μM p = 0.006), 307 μM (p = 0.02), and 162 μM (p = 0.017), respectively. Based on the results, other compounds, including D1, D2, D5, D6, and D7, significantly decreased cell proliferation and growth, as well as the migration of KYSE-30. Furthermore, these compounds at an IC50 concentration reduced the autophosphorylation of Tyr1234 and Tyr1235 in the MET receptor, confirming their specificity for the MET receptor. Indeed, it is believed that compounds should have the highest efficiency with the lowest concentration in order to impose the minority of chemical changes and side effects in vivo. Since MET inhibition is important in drug selection, our compounds need to be structurally optimized to reach the MET receptor specificity. RTKs play a crucial role in different cellular processes, such as cell metabolism, diﬀerentiation, proliferation, and survival. 58 RTKs have been identified for humans and are classiﬁed into 20 subfamilies. The same molecular architecture is observed in all RTKs consisting of an extracellular ligand-binding domain and a cytoplasmic tyrosine kinase domain, which the latter is a key player in most cellular activities[41]. The obtained findings may confirm the role of MET in the migration of other malignancies. It has been shown that shikonin suppresses MET-mediated EMT and inhibits invasion and migration in human lung cancer cells[41]. MET mainly contributes to the invasion and migration of hepatocarcinoma cells, and its down-regulation is suggested as a therapeutic modality for the disease in experimental models[42]. Furthermore, the blocking of the HGF-MET pathway and angiogenesis by NK4 in malignant pleural mesothelioma significantly attenuates invasion and migration capacities[43]. Yang et al.[44] have displayed that the crosstalk between FoxM1 and MET/AKT promotes EMT and plays a role in the invasiveness and migration of tongue squamous cell carcinoma. It has been frequently represented that microRNAs inhibit many types of cancer cell invasion and migration such as cervical[45], breast[46], non-small cell lung[47], gastric[48], ovarian, melanoma[49], and osteosarcoma malignancies[50] by targeting MET. It has also been revealed that the in vitro downregulation of MET phosphorylation at tyrosine residues by the silencing of 6-phosphogluconate dehydrogenase leads to the significant inhibition of cells. Consequently, 6-phosphogluconate dehydrogenase seems to be required for effective MET signaling and migration[51]. The oncogenic role of MET in ESCC and its role in the migration have been reported, as well. Some HGF/MET inhibitors are currently used in the clinic, including crizotinib (suppresses the migration and invasion of the human NCI-H441 lung carcinoma cell), MK-2461 (a potent ATP-competitive inhibitor significantly reduces migration and tubulogenesis), and foretinib . All these chemical compounds bind to the ATP-binding pocket of MET and block its activity, leading to the suppression of a variety of MET-dependent biological activities. In line with these reports, the results of the current study also revealed that the expression level of phosphorylated MET is significantly decreased by treatment with these novel compounds. Therefore, MET can be introduced as a potential therapeutic target for ESCC therapy. Although detailed investigations are required to identify MET function in ESCC. To the best of our knowledge, the presented compounds in this study (the D group) and their MET inhibitory potential have not been reported yet. Based on the results, ESCC cell migration was significantly inhibited by D series of SMIs in the examined cell line. Considering that the level of MET phosphorylation was determined using the Western blot, and cell migration was also measured at different levels of MET phosphorylation states (by using our MET inhibitor compounds), we can combine the results and correlate the level of MET phosphorylation and migration in the examined cells. Our results showed that a weak intensity of the Western blot band, which describes a decreased MET autophosphorylation state, was correlated with the reduced distances of cell migration. In other words, MET phosphorylation leads to an increase in cellular migration in ESCC and lung cancerIn general, this study introduced some novel and potent MET inhibitor compounds with excellent kinase inhibition activity and effective potential for preventing the cell growth and migration. As far as we know, the present study is the first one that focused on the impact of phosphorylated MET on KYSE-30 cell migration ability, as well as the structure and function of novel compounds as new MET inhibitors for ESCC. The evidence may introduce such small inhibitors as potential therapeutic agents for repressing MET activity and inhibiting cancer cell proliferation and migration.


Pervious concrete is an eco-efficient concrete but has problems regarding its mechanical performance and permeability balance. This research investigated the feasibility of using a combination of styrene–butadiene rubber (SBR) and acrylate polymer to improve the toughness of pervious concrete while keeping its permeability. Single-sized aggregate and no sand were considered in the concrete mixture. Acrylate polymers with different solid content, PH, density, and viscosity were emulsion copolymerized with an SBR polymer. Eleven scenarios with different mix proportions and 220 specimens for compressive strength, flexural strength, flexural stiffness, impact resistance, and fracture toughness tests were selected to evaluate the effects of the copolymer on the toughness of copolymer-modified pervious concrete (CMPC). The studies showed that (1) the influence trend of the copolymers generally varied according to different mechanical indexes; (2) XG–6001 acrylate polymer mainly and comprehensively enhanced the toughness of the CMPC; (3) it was difficult to increase the enhancing property of the XG–6001 acrylate polymer with the growth of its mix proportion; (4) the zero-sand pervious concrete with 90% SBR and 10% XG–6001 acrylate emulsion copolymerization proved to have relatively high toughness. The proposed CMPC holds promising application value in sustainability traffic road construction. Pervious concrete has recently regained popularity as it is considered as an eco-efficient concrete in the sustainable development of transportation infrastructure . Normal Polymers are added to the pervious concrete mixes, either in the form of an aqueous emulsion or in a dispersed form, in order to improve its mechanical behavior ,6. The pHowever, Sofi et al.  reportedToughness is a measure of the energy absorption capacity of reinforced concrete and represents the concrete’s ability to resist fracture when subjected to static or dynamic load ,26. A seThe aim of this study is to demonstrate the feasibility of using a combination of SBR and acrylate polymer to improve the toughness of CMPC with zero sand by means of a laboratory testing program and comprehensively statistical manners.Single-sized pervious concrete has less shrinkage, high permeability, and noise elimination. This property was observed in our team’s earlier research, and a new, high mechanical performance porous cement pavement was further composed by enhancing its bonding paste with zero sand ,37,38. IPortland cement with a strength grade of 42.5  was adopted. Its chemical compositions are presented in Coarse aggregates of the pervious concrete were crushed limestone (mined in Chongqing) ranging from 4.75 to 9.5 mm in size, in which the nominal maximum aggregate size was 10 mm. After washing, the aggregates were oven dried at 120 °C. The clean and dry aggregates are shown in In this study, the copolymer latex was prepared by emulsion copolymerization of SBR and acrylate polymer . The thrThe basic mix proportions of the pervious concrete mixture used in this study were water, cement, and aggregate in a ratio of 0.41:1:4.7 by weight according to our team’s previous work ,40,41. CIn this research, CMPC’s toughness is presented with compressive strength, flexural strength, flexural stiffness, impact resistance, and fracture toughness, so that the cube specimens, beam specimens, and cylindrical specimens were prepared for the corresponding test. Each test was carried out in 11 scenarios of the additive level for copolymer latex. The number of specimens for each scenario was six for the compressive strength test, three for the flexural strength test, three for the flexural stiffness test, five for the impact resistance test, and three for the fracture toughness test. The total number of specimens was 220. The specimens’ sizes and numbers are detailed in b is the width of the specimen (100 mm), and h is the height of the specimen (100 mm).Compressive strength test and flexural strength test: The compressive strength and flexural strength were tested by following the Chinese Standard GB/T 50081 . The cubFlexural stiffness test: The flexural stiffness revealed the stiffness measured in bend for characterizing the property of concrete pavement on orthotropic steel decks or subgrade . The fleh is the height of the specimen (100 mm), and b is the width of the specimen (100 mm).The flexural stiffness of the specimens was tested at 28 days. Its test procedure was roughly similar to the flexural strength test; the only difference was the loading manner. The loading process was undertaken for five cycles as shown in 1N denotes the first cracking number of blows, and N2 is the failure number of blows. The impact energy is calculated as follows:W is the absorbed impact energy by specimen, N is the number of blows, m is the drop weight (3 kg), g is the gravity acceleration (9.8 m/s2), and h is the drop distance (0.69 m).Impact resistance test: Impact resistance represents the CMPC’s energy absorption capability under repeated impact load. The impact resistance was tested at 28 days by using the Repeated Blows Drop Weight Impact (RBDWI) test according to American standard ACI 544-2R . The bloFracture toughness test: Pervious concrete’s fracture toughness is a measure of its ability to resist the unstable macroscopic crack growth. The most problematic concrete property is its response to the tensile load as the tensile strength; thus, the fracture mode I among all the fracture mechanics parameters was studied in this research . The fraA is the area of the fracture ligament, The fracture energy,  follows :(4)GF=w03/2) was calculated as follows [H is the height of the beam, and The fracture toughness  follows ,48:(5)KIThe compressive strength test results were determined and multiplied by the size conversion factor 0.95 according to the Chinese Standard GB/T 50081-2019 . Figure However, it could be seen that the CMPC with respect to SP3 failed to grow in terms of compressive strength (11.5 MPa in average); it decreased by roughly 13% compared with the non-polymerized pervious concrete. Above all, it could be observed that the polymer SBR, copolymer SP1, and copolymer SP2 could promote the compressive strength of the pervious concrete even though their enhancement effect on compressive strength was equal.The flexural strength test results were taken and multiplied by the size conversion factor 0.85 according to Chinese Standard GB/T 50081-2019  as well.In terms of 0.5F and Δε during the flexural stiffness test are presented in 0.5F. For example, the maximum 0.5F in relation to SP1_30% did not induce a maximum Δε. The SP2_10% gave rise to a maximum Δε but not a maximum 0.5F. Furthermore, the flexural stiffness test data were taken according to the Chinese Standard JTG 3420 [The JTG 3420  and are N1 existed in the corresponding material. In The impact resistance test results were taken according to American standard ACI 544-2R , and theN1, and the failure number of blows, N2. The absorbed impact energy of the specimen accompanied with the first cracking is denoted as W1, and the further absorbed impact energy during the period from the first cracking to the specimens’ failure is denoted as ΔW, where N1, the average of which was 629 for SBR, 872 for SP1, 1318 for SP2, and 164 for SP3. It is apparent that with the exception to SP3, the copolymer additive significantly increased the first cracking number compared to the SBR. Interestingly, N1 decreased with an increase in the mix proportion of P1 or P2. This implies that small quantities of P1 or P2 additive can easily enhance the pervious concrete’s impact energy absorption capability.As shown in N1 and N2 were observed in 1 trend as shown in In addition, only slight differences between 1 and ΔW, copolymer SP2 could mainly increase the CMPC’s impact resistance; its proper mix proportion was 10%.Generally, it can be seen from The fracture toughness test results were taken according to Chinese Standard DLT5332-2005  and the FG, the SBR additive increased the test data from 737.9 to 1220.5 N·m. However, more than the SBR additive, the average value for the SP1 additive increased by 67.1%; for SP2, it increased by 40.7%, and for SP3, it increased by 17.2%. This indicated that the SP1 contributes greatly to the fracture energy absorption, and SP1_30% is the best performer, while SP2_10% is a close follower.ICK, a slight increase from 347.6 to 360.3 kN/m3/2 was observed when the water was replaced by SBR latex. In contrast, significant increases were exhibited while the additives changed to copolymers. The average value for SP1 additive increased by 66.7%; for SP2, it increased by 78.2%; and for SP3, it increased by 59.4%. From the results, it is evident that when it comes to fracture toughness, SP2 plays an important role, and SP2_10% is the best performer, with the close followers being SP1_30% and SP3_10%.From the perspective of FG and ICK grew with an increase in the P1 additive, while the P2 additive exerted opposite effects. This implies that a little mix of P2 can significantly increase pervious concrete’s ability to resist unstable macroscopic crack growth.In addition, another trend was observed: the From the previous section’s study, the compressive strength, flexural strength, ultimate strain, flexural stiffness, ultimate absorbed impact energy, fracture energy, and fracture toughness were selected as the toughness discriminating criteria. It is clear that the copolymer largely influences the CMPC’s toughness. However, it is difficult to determine which type of copolymer and which mix proportion of P1 or P2 is appropriate. Therefore, the following paragraphs aimed to identify the best performer that greatly increased the CMPC’s toughness. Data normalization was adopted to scale down the selected criteria to a range between 0 and 1. Then, the comprehensive effect of copolymers on CMPC could be determined by accumulating the normalized criteria value.D is the original value of the test data,The data’s normalization process is illustrated in the following equation:It is necessary to take the reciprocal of the flexural stiffness before normalizing it, as the flexural stiffness is inversely proportional to flexural toughness. The comprehensive effect of copolymers on CMPC’s toughness is illustrated in The reason for most of the copolymer additives in this study increasing the mechanical properties can be explained as follows: the plain cement paste coating the aggregate has high fluidity compared to the copolymerized cement paste during the specimen cast molding period. Considering the relatively high polymer-to-cement ratio in this study and the copolymer’s potential ability of enhancing the adhesion of cement paste, the copolymerized cement paste was more likely adhere to the aggregate surface, while the plain cement paste tended to flow to the lower place. This caused a strong bonding between the aggregates of the CMPC while a weak bonding existed in the plain pervious concrete; thus, the relative excellent mechanical properties of the CMPC was induced.Another point that needs to be mentioned is that in previous studies ,14, the In addition, according to the Chinese standard JTG D40-2011 , the conThe SBR polymer was proved to be non-toxic in the literature ; therefoThis study investigated the toughness of SBR acrylate polymer-modified pervious concrete with single-sized aggregates and no sand. In addition, the optimal acrylate type and its mix proportion for the CMPC without sand were proposed. The following conclusions were drawn from the results:(1) The combination of SBR and acrylate polymer largely increases the toughness of pervious concrete without sand, indicating that the reduction of the sand mixture is feasible while maintaining the pervious concrete’s mechanical performance.(2) The toughness of CMPC is largely affected by the type and mix proportion of acrylate polymer. Furthermore, the influence trend varies according to different mechanical indexes. The normalization process of test data, which scales the data with different dimensions down to the range from zero to one, is applicable to comprehensively evaluate the effect of a copolymer on the toughness of CMPC.(3) XG–6001 acrylate polymer mainly and comprehensively enhanced the toughness of the CMPC in contrast with other acrylate polymers. It was difficult to improve the enhancing property of the XG–6001 acrylate polymer with the growth of its mix proportion.(4) The zero-sand pervious concrete with 90% SBR and 10% XG–6001 acrylate emulsion copolymerization exhibits relatively high toughness compared with previous studies in the literature, demonstrating its ability to retain stability, durability, and cracking resistance ability, and proving its strong application potential in sustainability traffic road construction.In this study, the polymer-to-cement ratio was relatively high, and the testing program and statistical evaluation applied only investigated the toughness of the CMPC in a common environment; thus, the further study will seek to optimize the polymer-to-cement ratio and pay attention to the performance of CMPC in special conditions, such as freeze–thaw and erosion. Enhancing the skid resistance for the CMPC while retaining its high toughness property is an area of further study as well. Micro-surfacing technology is a potential way to promote the skid resistance of the CMPC in the field in terms of enriching its micro-texture based on its macro-texture. The authors will investigate the probable specific micro-surfacing manners or more effective methods to raise the traffic safety of CMPC pavement in further researches.


Immunoglobulin G4 (IgG4) cholangiopathy is used to describe regional biliary involvement in a systemic fibro-inflammatory and infiltrative disease, IgG4-related disease. Occasionally, it tends to present with localized disease leading to an extensive workup to rule out malignancy and infections, especially since IgG4-related disease is an uncommon entity. Herein, we present a case of a 68-year-old male who presented with pruritus and steatorrhea with imaging studies revealing a biliary hilar mass concerning for malignancy. Subsequent extensive testing was inconclusive of malignancy and finally noted to have elevated IgG4 levels as part of a broader workup. The patient was started on prednisone with the resulting improvement in his symptoms and the imaging findings. Immunoglobulin G4 (IgG4)-related disease is a systemic disease characterized by diffuse lymphoplasmacytic infiltration of IgG4+ plasma cells along with dense storiform fibrosis of the tissues. It generally affects the pancreas with a type 1 autoimmune pancreatitis and the hepatobiliary tree but has the potential to involve any organ in the body. When affecting the biliary system, it tends to be reminiscent of primary sclerosing cholangitis. Rarely, it presents with obstructive jaundice and weight loss as a hilar mass highly suspicious for cholangiocarcinoma or Klatskin tumor. In such scenarios, diagnosis is generally delayed as it requires a high index of suspicion. IgG4 levels are rarely sent as initial tests for evaluation of a hilar mass but ultimately help to confirm the diagnosis in the right clinical setting. Here, we present a case of IgG4 cholangiopathy presenting as a biliary hilar mass concerning for malignancy.A 68-year-old male with a history of hepatitis C and remote pancreatitis visualized on prior endoscopic retrograde cholangiopancreatography (ERCP) presumed to be from alcohol use presented with a two-month history of progressive pruritus, steatorrhea, fatigue, and a 20-pound weight loss. Physical examination was notable for scleral icterus, jaundice, and skin abrasions from pruritus. Abdominal examination was benign. Laboratory data revealed obstructive jaundice with a total bilirubin of 8.3 mg/dL, alanine aminotransferase (ALT) of 188 U/L, aspartate aminotransferase (AST) of 78 U/L, and alkaline phosphatase of 596 U/L. Cancer antigen 19‐9 (CA 19-9) was elevated to 53.91 U/ml.Computed tomography (CT) of the abdomen/pelvis and magnetic resonance cholangiopancreatography (MRCP) showed mild to moderate intrahepatic biliary ductal dilatation with a 4.5 cm x 2.5 cm hilar lesion favoring Klatskin tumor, Bismuth‐Corlette type IV Figures , 2. ERCPHe underwent repeat ERCP with endoscopic ultrasound (EUS)-guided biopsy. On EUS, the pancreas was noted to be atrophic and have scattered calcifications, confirming the prior diagnosis of pancreatitis; the pancreatic duct was irregular with mild dilation in the body. Two plastic stents were placed in the right and left intrahepatic ducts during endoscopic retrograde cholangiopancreatography (ERPC). Cholangioscopy showed a malignant-appearing lesion, and repeat biopsies showed atypical appearing cells. Oncology was consulted due to high concerns for a malignancy. Positron emission tomography (PET) scan when obtained showed an avid tumor and lymph nodes in the biliary hilum and diffuse mediastinal and retroperitoneal lymphadenopathy (LAD), which was atypical for hilar cholangiocarcinoma. Hence, differential diagnoses were broadened.IgG4 level was elevated at 312 mg/dl. In light of unrevealing workup for malignancy, elevated IgG4 levels, cholangitis, and the history of chronic pancreatitis, the patient was started on a trial of steroids for suspected IgG4 cholangiopathy. Prednisone 60 mg daily was given for four weeks for induction followed by an eight-week taper, then stopped. While on prednisone, his symptoms significantly improved confirming the clinical diagnosis. Bilirubin normalized one week after placement of the stents during ERCP. Furthermore, his liver enzymes normalized, and there was a significant downtrend of IgG4 levels (111 mg/dl). The stents were removed after the completion of steroid induction, and liver function test (LFTs) remained normal. Repeat EUS revealed shrinkage of the hilar mass. PET scan also showed decreased lymph node activity, and no vascular involvement was seen. The patient had a recurrence of symptoms when taken off steroids and was later successfully transitioned to azathioprine in the outpatient setting. Currently, two years out, he continues to do well on intermittent prednisone 10 mg with complete resolution of the 4-cm perihilar mass on MR imaging.IgG4-related disease is a systemic disease characterized by diffuse lymphoplasmacytic infiltration of IgG4+ plasma cells along with dense storiform fibrosis of the tissues. It generally affects the pancreas with a type 1 autoimmune pancreatitis and the hepatobiliary tree but has the potential to involve any organ in the body including salivary glands, lymphadenopathy, and kidneys. IgG4 cholangiopathy, also referred to as IgG4-sclerosing cholangitis (IgG4-SC), is an IgG4-related disease with the involvement of the biliary system and is generally seen with biliary strictures and associated cholangitis. The concomitant presence of autoimmune pancreatitis should raise concerns for IgG4-related disease [Common presenting symptoms are non-specific and include obstructive jaundice, weight loss, or new-onset diabetes ,3. The mThe most characteristic feature of IgG4-SC is its dramatic response to steroids and can act as a surrogate confirmatory test . Our patIgG4 cholangiopathy is the involvement of IgG4-related disease in the biliary system. As the clinical features and diagnostic evaluation between IgG4 cholangiopathy and cholangiocarcinoma overlap, it can be difficult to differentiate between the two, especially when patients present with isolated perihilar masses without any pancreatic involvement. Ultimately, a high index of suspicion prompting evaluation of IgG4 levels supported by critical pathology review is required to make a definitive diagnosis, guide treatment, and reduce the risk of patient morbidity and mortality.


This work aimed to estimate the knowledge and practice of general dentists in the governorate of Manouba regarding the management of patients at risk of infective endocarditis.  A survey involving private sector general dentists in the governorate of Manouba was performed. It contained 21 questions, and it was addressed to 111 dentists. Only 82 dentists responded. To carry out the descriptive study, we used the SPSS software version 21.0.  Our results proved the lack of knowledge among dentists in the governorate of Manouba with regard to the management of patients at risk of infective endocarditis. An overestimation of the risk and an overprescription of antibiotic were found in order to ensure an over-protection for the patients as well as the dentist. In fact, 85.4% of these dentists prescribed antibiotic prophylaxis for the two groups of patients (high risk and moderate risk). Only 9.8% followed the right modality of antibiotic prophylaxis prescription; 4.9% of the dentists prescribed antibiotic only 1 hour before the act and 4.9% of them prescribed antibiotic 1 hour before the act and continued the treatment in case of the presence of an infectious site.  A discrepancy towards an over-estimation of risk and overprescription of antibiotic was found between the recommendations and real practice. Similar studies in the other governorates of Tunisia are recommended in order to better understand the problem. Infective endocarditis (IE) is an uncommon but potentially devastating disease with an estimated annual prevalence of 11–50 cases per million population . In a reSince the publication of the American Heart Association (AHA) in 1990, it has been conventionally considered appropriate to prevent IE by antibiotic prophylaxis (AP) before procedures believed to cause bacteremia –5. HowevRecently, a meticulous analysis of epidemiological data by Dayer et al. reported a significant increase of incidence of IE in England (0.11 cases per 10 million people per month) that appeared to correspond with the NICE recommendations to cease AP . NICE guIn our study, we aim to assess the attitude and practice of dentists regarding patients at risk of IE and their way to provide dental care in these clinical situations, through a questionnaire.A cross-sectional survey was conducted to determine antibiotic prescription modalities among 129 private dentists for general practice in Tunisia (in the governorate of Manouba). The list of dentists practicing in this governorate was provided by the Dental Council, following a prior request. The survey took place over a period of 5 months.Our study included general practitioners registered with the Dental Council practicing in the governorate of Manouba. This study does not relate to students with pending thesis, residents registered with the Dental Council, and dental specialists or having had specialized training or practicing in public health.p < 0.05 for a significant difference between the variables.A self-administered structured questionnaire was developed and tested among a sample of 20 interns and residents, who were interviewed to gain feedback on the overall acceptability of the questionnaire in terms of length and language clarity (no tests were carried out in order to measure the acceptability of the questionnaire). The final version of the questionnaire was written by a resident. It consisted of 3 sections. The first section solicited general demographics . The second section consisted of an assessment of knowledge about different heart diseases and the third section consisted of antibiotic prescription modalities in patients at risk of infective endocarditis. The questionnaire was distributed to the 129 dentists in the sample, as a written document; multiple responses for the same question were allowed. To carry out the descriptive study, we used the SPSS software version 21.0 . Normality was tested by the KS test for age and length of practice. The chi-squared test was used during the bivariate analytical study when comparing the percentages of qualitative variables with a threshold In the present study, out of 129 dentists, 82 responded to the questionnaire and the response rate was 73.8%. The responses were analyzed as percent scores.Mean age of the 82 dentists is 39 ± 11 years, the minimum being 27 years and the maximum 65 years. It is made up of 38 (46.3%) women and 44 (53.7%) men; the sex ratio is 1.15. The average length of practice is 11 ± 9.5 years, the minimum being 1 year and the maximum 35 years.Most of them  studied at faculty of dental medicine of Monastir and the 10 (12.2%) others outside the country .The assessment of the risk of IE in different types of heart diseases , by our sample, is shown in Tables 1Evaluation of antibiotic prescribing patterns is depicted in Certain dental procedures are associated with bacteremia although the magnitude will vary . The numThe use of probiotic was proposed to modify the oral environment in recent study. This showed promising results in oral microbiota control for both adults  and chilThe cumulative exposure to bacteremia is significantly greater from everyday procedures such as tooth brushing and chewing, when compared to dental procedures , 11, 16.Through our study, we tried to assess the estimation of the risk of IE by our respondents. Cyanotic congenital heart diseases were underestimated as a high risk of IE. Most of heart diseases with moderate risk or risk free of IE were overestimated. Our results emphasize the lack of knowledge of the infectious risks relating to the various heart diseases. Our results are similar to those found on survey among dentists in Hyderabad City, India, showing a relatively low level of knowledge of the new guidelines .Lack of medical information regarding heart disease even with the definitions provided during the interview.General practitioners prefer to overestimate than underestimate. The vital prognosis in a private practice constitutes a phobia pushing the general practitioner to overestimate and therefore to protect himself.This can be explained by the following:In vivo, PA are believed to act by interfering with 3 of the major stages in the pathogenesis of IE: bacteremia (by reducing the numbers of microorganisms in blood), adherence , and multiplication of the microorganisms on heart valves (by interfering with the metabolic activity of the microorganism) . HoweverFor dental acts justifying an antibiotic prophylaxis, there is an incorrect contraindication by these dentists. This is due to the responsibility and the fear felt towards this category of patients: 23.2% contraindicated endodontic treatment in single/double rooted teeth, 20.7% contraindicated single-visit endodontic treatment in teeth with vital pulp and rubber dam in place, and 14.6% contraindicated tooth extraction.For dental procedures that do not justify antibiotic prophylaxis, we note that there is an over-prescription of ATB, particularly for local or locoregional anesthesia in uninfected tissue (42.7%), bloodless scaling (37.8%), and conservative supra-gingival treatment (17.1%).For contraindicated dental acts, we note that there is a remarkable lack of knowledge of these acts and a remarkable over-prescription of ATB: 18.3% do not contraindicate intraligamentary anesthesia with no AP whilst 45.1% indicate AP for this act. 72% of respondents still proceed to root separation with periodontal disease with AP. 40.2% do not contraindicate periapical surgery, with AP. Endodontic retreatment (28%) and implant placement (30.7%) are indicated with AP too.In the present study, we tried to assess the attitude of dentists regarding patients at risk of IE induced by dental procedures justifying antibiotic prophylaxis and contraindicated acts in patients at risk of IE. In this regard, we have adopted the classification of ANSM 2011. We were able to draw the following conclusions:These conclusions present some disagreement with the new views of experts in 2017 who were part of French Society of Cardiology (ESC), French Society of Stomatology, Maxillofacial Surgery and Oral Surgery (SFSCMFCO), French Society of Periodontology and Oral Implantology (SFPIO), and French Society of Endodontics (ESE). These experts consider that endodontic treatments and implants are no longer systematically contraindicated [Savarrio et al. conducted a study for 30 patients receiving non-surgical root canal treatment, a detectable bacteremia was present in 30% of the patients. In a review of 53 cases of IE following dental procedures, 7 were attributed to previous RCT. Duval et al. concluded that bacteremia result from previous infectious lesions rather than dental care . In all Farrington showed that incidence of bacteremia after pulpotomy is 4%. But if there is no certainty that a bacteremia is induced during RCT, then it would seem unlikely that a pulpotomy procedure should cause microorganisms to enter bloodstream , 23.Roberts stated that the placement of matrix band and wedge resulted in bacteremia comparable with that encountered following dental extraction, thus providing evidence that these procedures should be covered by AP . In 2004The evidence-base for the efficacy of antibiotic prophylaxis in preventing IE is weak and views on the risk-benefit analysis have shifted in recent years, with moves to reduce the utilization of antibiotic prophylaxis , 6, 25. In the present study, we noted that 85.4% of respondents prescribe antibiotic prophylaxis for both groups of patients (at high risk and at moderate risk). This over-prevention of IEs is also reflected in the prescription modalities: Only 1/3 of our sample follows the reference modalities: either only 1 hour before the act (4.9%) or 1 hour before the act and continue the prescription of antibiotics in the presence of an associated infectious focus (24.4%).The 2007 guidelines stated that AP should be administered in a single dose 1h before the procedure , 6, 11. As for the doses indicated, in our study, the percentages are very close. 54.9% prescribe 2g of amoxicillin and 45.1% 3 g of amoxicillin in the absence of penicillin allergy. In attempt to reduce the adverse gastrointestinal effects of high dose, the AHA has revised its recommended oral dose of Amoxicillin from 3 to 2 g . HoweverIn the event of Penicillin allergy, 58% of the respondents prescribe Clindamycin. This molecule is not available on the Tunisian market which leads us to underline the non-application of their theoretical knowledge in the field. Other molecules are prescribed such as Clarithromycin, Rovamycin, Pyostacin, Spiramycin, Birodogyl, and Azithromycin. Apart from Azithromycin and Clarithromycin which can be used according to the AHA 2007 as AP in a single dose 500 mg 1 hour before the act, the other molecules do not have the marketing authorization as AP , 28. CliBacterial resistance to antibiotic could be involved in the inefficacy of the prophylaxis , 32. PasPerform an appropriate technique under ideal aseptic conditionsBe performed by a specialist who is trained to perform these types of treatment under ideal conditionsPerform a CBCT to assess difficulty and feasibility in one sessionPrescribe a flash ATBEnsure that working length is respected and that the risk of propelling infected debris beyond the apex is lowProvide recapitulation and adequate irrigation to reduce bacteremiaFor infected teeth in patients at risk of IE, endodontic treatment should be the treatment of choice because extraction has been associated with greater bacteremia provided:The size of the population studied does not allow us to represent all practitioners, but it gives an idea of the therapeutic attitudes among general practitioners. This sample is insufficient to carry out an analytical study, which makes our study a descriptive one.Our findings revealed a confusion in the indicated and contraindicated acts and showed a gap between recommendations and clinical practice. There are deficiencies in knowledge regarding prescribing antibiotic and appropriate antibiotic prophylaxis.This study showed the importance of improving knowledge about patients at risk of infective endocarditis and procedures requiring antibiotic prophylaxis.


Altmetric indicators allow exploring and profiling individuals who discuss and share scientific literature in social media. But it is still a challenge to identify and characterize communities based on the research topics in which they are interested as social and geographic proximity also influence interactions. This paper proposes a new method which profiles social media users based on their interest on research topics using altmetric data. Social media users are clustered based on the topics related to the research publications they share in social media. This allows removing linkages which respond to social or personal proximity and identifying disconnected users who may have similar research interests. We test this method for users tweeting publications from the fields of Information Science & Library Science, and Microbiology. We conclude by discussing the potential application of this method and how it can assist information professionals, policy managers and academics to understand and identify the main actors discussing research literature in social media. Research literature is increasingly mentioned, shared and discussed on social media. This represents a substantial challenge as well as an opportunity to anyone trying to study the interactions that take place in the digital environment  has given rise to the development of altmetric data aggregators that provide data on a variety of social media sources. These include Altmetric.com, CrossRef Event Data, or Plum Analytics, among others. Despite the evident advantage of offering unique data access points, they do have limitations. Zahedi and Costas  systemicFor a variety of reasons, Twitter is the social media platform that has received most attention since the earliest days of altmetric studies. In part, this is because it is the public forum with the second-highest figures for coverage of scientific literature mentions after Mendeley  network. Any such network is based on an asymmetrical matrix in which rows and columns are composed of different entities. Recently, Hellsten et al.  suggesteTo introduce a novel methodological proposal by which actors in a given network can be grouped on the basis of their cognitive interests thus, to some extent, removing social relationships that could potentially blur the boundaries between communities.To test our methodical approach in a specific case study: Twitter mentions of scientific literature in the field of Information Science & Library Science.To replicate this approach in a different field—Microbiology—to observe potential inconsistencies in the methodology and discuss differences between the two case studies.In the present paper we build on our literature review to better refine methods by which communities with common scientific interests can be identified on social media. We test our methodological proposal using Twitter mentions to scientific papers in two research fields: Information Science & Library Science and Microbiology.Our study closely follows recent work in which a genuine effort has been made to conceptually define and then build a framework in which methodological solutions in the field of altmetrics can be expanded. For instance, Costas et al.  recentlyThe present study builds on previous work which analyzed differences in interests of topic by social media platform  was conducted using Gephi 0.9.2 visualization software .The data needed to reproduce our analyses are available at We downloaded publication data for two research fields: Information Science & Library Science and Microbiology. We used the former as a case study to test our methodological approach. We then replicated the method in the latter field to compare results and analyze discrepancies in different contexts.Information Science & Library Science: 14,475 publications were mentioned by at least one altmetric source, giving a total of 167,110 mentions from Altmetric.com. Some 151,505 of these (90.66%) were Twitter mentions of 13,458 (92.97%) publications.Microbiology: 192,836 publications were mentioned by at least one altmetric source, giving 1,876,599 mentions from Altmetric.com. Some 1,599,315 of these (85.22%) were Twitter mentions of 173,406 (89.92%) publications.On 17 July 2019 we retrieved all records indexed in the Web of Science (WoS) InCites database (excluding the Emerging Sources Citation Index) published between 2012 and 2018 in the WoS categories of Information Science & Library Science ; and in Biotechnology and Applied Biochemistry  and Microbiology —these two represent a combined total of 413,910 publications, henceforth referred to as ‘Microbology’. From Altmetric.com’s Altmetric Explorer portal, we extracted all social media mentions of these records by using their DOIs as our search item. Information Science & Library Science has 35,695 publications with DOI (42.21%), and Microbiology has 366,449 (88.53%). Table Our purpose here is to map only those actors who are genuinely involved in Twitter discussions. Retweets have been excluded as they could potentially distort results: they correspond to the platform’s social function and do not necessarily indicate participation in scientific debate  and in Microbiology to 699,507 (43.74%).Simultaneously, we extracted author keywords of publications mentioned using terms included in the WoS Author Keywords. These are widely used in bibliometrics and have been previously applied in altmetrics  with 44,421 keywords, of which 20,027 are unique, and 35,411 Twitter mentions ; and in Microbiology, our final dataset constituted 101,206 publications (59.16%) with 540,227 keywords, of which 163,674 are unique, and 328,110 Twitter mentions (49.91%).We now describe our methodological proposal to identify communities of interest. This approach can be divided into three distinct phases.Firstly, we construct a co-word network (semantic map) from the author keywords of publications tweeted in the field. The network is constructed regardless of the number of mentions received and is solely based on the co-occurrence of keywords in scientific publications. It is pruned to remove the weakest co-occurrences, less frequent keywords, and isolated components. Due to the different network sizes and edges’ weights (number of times than two keywords co-occur) in the two areas, the established minimums are not the same for both. This map enables us to identify research areas in the field. To do so, we use a social network community detection method. The chosen is the Louvain community detection algorithm . This network is also pruned to remove the weakest relations also following a heuristic strategy, which means that there is no a standard value, but different tests are carried out for this purpose, and reduced to its main component. A community detection is applied to it, using the Louvain community detection algorithm and following the same criteria as in the semantic map. The resulting communities are reflected by areas. To generate the socio-semantic network, each social actor is assigned to its topic, generating a second grouping of social actors, whose quality is calculated by the modularity value. Figure We identified a total of 13,243 Twitter users mentioning 8452 scientific publications of which 92.65% were articles and 3.42% reviews. Twitter users mention a mean 2.23 publications (SD ± 8.79) and 10.59 keywords (SD ± 32.32).Social Media: a community consisting of 5511 Twitter accounts, disseminating 2870 publications in 11,684 tweets and sharing 225 keywords. It includes publications related to social media use, the ethics of their use, their use by young people, and the application of big data techniques in social media analysis.Bibliometrics: a community consisting of 4989 Twitter accounts, disseminating 2229 publications in 11,984 tweets and sharing 175 keywords. This community includes publications related to bibliometrics and altmetrics analysis and covers issues relating to open science and science policy.Libraries: a community consisting of 2854 Twitter accounts, disseminating 1658 publications in 6297 tweets and sharing 141 keywords. This community includes publications relating to general, academic or specialized libraries, their evaluation, and the analysis and training of users.Information retrieval: a community consisting of 3522 Twitter accounts, disseminating 1486 publications in 7651 tweets and sharing 118 keywords. This community includes publications relating to information storage and retrieval, its application in electronic health records, the use of ontologies and classification systems and their interoperability.The author keywords co-occurrence network is composed of 20,025 nodes and 100,604 edges. It is reduced to 659 nodes and 1315 edges by removing edges with less than 3 co-occurrences and getting its main component. Figure Figure Some 1107 Twitter users combine mentions to topics related with social media and bibliometrics (9.25%). In fact, 44.22% of those who discuss topics related to bibliometrics also discuss topics related to social media. This figure falls slightly when combined with information retrieval (39.61%) and drops further when combined with libraries (19.87%). Finally, one singular cluster is that consisting of 366 actors (3.35%) who mention all four topics.Figure Figure We replicated our approach in a larger field—Microbiology—to see how it would work in a different context. We identified 48,109 Twitter users mentioning 101,206 scientific publications of which 86.52% were articles, 11.03% reviews, and 1.88% editorial material. Twitter users mentioned a mean 5.93 publications (SD ± 63.65) and 25.27 keywords (SD ± 197.84).The author keywords co-occurrence network is composed of 163,650 nodes and 1,173,938 edges. It is reduced to 2309 nodes and 7559 edges by removing keywords with less than 50 occurrences, edges with less than 5 co-occurrences and getting its main component. Figure Omics and Phylogenic Classification: a community consisting of 26,654 Twitter accounts, disseminating 35,450 publications through 143,604 tweets and sharing 591 keywords. It included publications covering studies of genetic material, bacterial microorganisms and biodiversity.Immunology and Viral Diseases: a community consisting of 18,695 Twitter accounts, disseminating 23,499 publications through 85,030 tweets and sharing 490 keywords. It is related to viral diseases, their diagnosis, novel treatments, and vaccines.Bioengineering: a community consisting of 11,523 Twitter accounts, disseminating 17,625 publications through 47,743 tweets and sharing keywords. It includes publications in biotechnology, metabolic engineering, and synthetic biology.Bacteria: a community consisting of 19,077 Twitter accounts, disseminating 33,805 publications through 111,915 tweets and sharing 660 keywords. It includes publications related to diseases of bacterial origin, epidemiology, and outbreaks of infectious diseases.Stem Cell Development: a community consisting of 7206 Twitter accounts, disseminating 11,208 publications through 31,081 tweets and sharing 223 keywords. It includes publications on regenerative medicine, gene therapy, and cancer treatment.Tick transmitted diseases: a community consisting of 1048 Twitter accounts, disseminating 1044 publications through 4477 tweets and sharing 30 keywords. It includes publications relating to tick and flea transmitted diseases.When assigning Twitter users to each of these six topic groups Fig. , we founIn the present study we propose a methodological approach to the identification of social media communities on the basis of common scientific interests. It enables us to link social media users on the basis of the keywords of the publications they mention and then group users by topic. We first applied this to Twitter users who mention publications in the fields of Information Science & Library Science. We then tested its feasibility by replicating the study in the field of Microbiology. Our proposal responds to the need for new efforts in social network analysis  rather than objects (i.e. publications), we minimize potential relationships derived from social relations between actors rather than from common research interests (e.g. colleagues from the same institution).This methodology has the potential of being applied in other scenarios from the ones proposed here. Other social media platforms could be considered, as well as other types of contents shared through social media. Some of the many and varied contexts in which it can be applied are political participation and political engagement (Halpern et al.,