This study was conducted to determine the effects of methionine(Met) supplementation on productive and reproductive performance, immuneresponse and antioxidant status in breeder quails reared under heat stress(HS). A total of 125 breeder quails were divided into five groups. One groupwas kept in an environmentally controlled room at 22  Several techniques are available toalleviate the adverse effect of HS on poultry performance. Because of thehigh costs associated with cooling structures that house animals, solutionshave been aimed primarily at dietary manipulation .Nutritional strategies have included the following: adjusting feeding timesor temporary feed withdrawal , dietary energy andprotein levels manipulation , diet supplementation with vitaminC , Se or Cr , probiotics and prebiotics , essential oils and water supplementation with electrolytes .Recently, the connection between HS and oxidative stress has received muchinterest . Oxidative stress is the presence ofreactive species in excess of the available antioxidant capacity animalcells. The effects of HS are possibly due to acceleration in the rate ofreactive oxygen species (ROS) formation and/or an increase in ROS reactivity. The defenses of organisms against ROS may be mediated bynonenzymatic or enzymatic antioxidants, mainly represented by the enzymessuperoxide dismutase (SOD), catalase (CAT) and the glutathione (GSH) defensesystem . Antioxidant mechanisms are more oftenattributed to vitamins  than to amino acids.However, sulfur amino acids (SAAs) play a major role in the antioxidantsystems of the cell in different ways.Methionine (Met) is a methyl group donor and a precursor of cysteine,cystathionine, homocysteine, S-adenosylhomocysteine and glutathione . Diets with Met deficiency result in an increase in heatproduction , followed by an increase in adverse effectsof HS when the environmental temperature is high . Balancing the amino acid composition in the diet with Metsupplementation improves production performance through pathways ofpolyamine metabolism  and glutathionine(derived from methionine) and may reduce the damage from oxidative stress.Also, the Met requirement determined by NRC (1994) is for laying quails andthis may be insufficient for supporting the reproductive performance andhatchability in breeder quails . It has been shown that dietary supplementation of Met above NRC(1994) recommendation enhanced cellular immunity in birds . Therefore, dietary supplementation of breeder diets with Met abovethe NRC recommendations may increase the transmission of antibodies tooffspring in breeders.There is very limited information in the literature on the use of Met inbreeder quails. Therefore, the aim of the present study was to determinewhether dietary supplementation with Met could reduce the adverse effect ofHS on the performance, antioxidant status and immune response, as well asthe transfer rate of maternal IgY to offspring (no report in the literature)in heat-stressed breeder quails.2All experimental protocols adhered to the guidelines of, and were approvedby, the Animal Ethics Committee of the University of Kurdistan .2.1A total of 200 Japanese quails at 45 d of age were obtained from a localsupplier. The obvious runts and birds with extreme weight were eliminated.Overall, 125 Japanese quails at 45 d of age were distributedin a completely randomized design, consisting of five treatments, fivereplicates and five  birds per replicate. Aperiod of 20 d was provided for the birds to adapt to the basal diets, cagesand a thermoneutral environmental temperature  requirements. The birds had unlimited access to water and feedthroughout the entire experiment period.2.2Egg production, feed intake, average egg weight , feed conversion ratio (grams of feedper grams of eggs) and eggshell, yolk and albumen percentage were evaluated. Eggproduction was determined by daily egg collection, which was recorded on onespreadsheet per replicate. At 2, 4, 6 and 8 weeks of assay, all eggsproduced in a single day were collected for quality evaluation .Average albumen weight per replicate was determined to be the differencebetween average egg weight and average yolk weight plus eggshell weight perreplicate. In order to determine eggshell weight, eggs were cracked, and theshells were washed and dried at room temperature for 48 h prior to beingweighed on an analytical balance. Shell thickness was measured by amicrometer as an average of three points . The Haugh unit wascalculated according to Brant et al. (1951), (100 log (Approximately 250 eggs (50 eggs per treatment) were collected over a3 d period in week 8 and stored at 15 Unhatched eggs were analyzed for embryo mortality causes, which wereclassified as early, middle and late death. In order to determine offspringserum IgG (five blood samples per treatment), all 1 d old chicks werehatched from each replicate, bled by cardiac puncture, and blood wascollected. The serum was then separated by centrifugation at 2.32.3.1IgY was isolated from egg yolks using the Polson et al. (1980) method.Briefly, an equal volume of buffer  was added to yolk and stirred. Solid polyethylene glycol 6000(Sigma-Aldrich) was added to a concentration of 3.5 %, stirred untildissolved, and the resultant protein precipitate was pelleted bycentrifugation at The total IgG levels in the maternal serum, IgY in the egg yolks and day-oldchick's serum IgG were determined using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditionsaccording to Laemmli (1970). The resultant from the IgY precipitation stepswas dissolved in the sample buffer with 2 % 2-mercaptoethanol and run on a5 % stacking gel and 10 % separating gel. Samples and standards wereloaded on the gel at a concentration of 20 2.3.2The cellular immune response was assessed by a cutaneous basophilichypersensitivity test using phytohaemagglutinin (PHA). At day 30 and day 45of the experiment, one quail from each replicate was selected, and the toethickness of both the left and right feet at the third and fourth interdigitalspaces was measured by micrometer. Immediately following the measurements,0.05 mL of phosphate-buffered saline (PBS) and 0.05 mL PHA  antibodytiter estimation. A suspension of sheep red blood cell (SRBC) as described in the literature. SOD activity was measured by the xanthineoxidase method, which monitors the inhibition of nitro blue tetrazoliumreduction and the change of absorbance at 560 nm . CATactivity was measured following the decrease in absorbance at 240 nm due tohydrogen peroxide decomposition . GPx activity was measured at412 nm by quantifying the rate of oxidation of reduced glutathione tooxidized glutathione . The thiobarbital method  was used to determine the MDA level with a wavelength of532 nmto determine absorbance.Plasma levels of triglycerides, total cholesterol, low-density lipoprotein(LDL)-cholesterol and high-density lipoprotein (HDL)-cholesterol wereanalyzed using a spectrophotometer  per theinstructions on the corresponding reagent kit .2.5The obtained data were submitted to analysis of variance using the generallinear model (GLM) procedure of the SAS statistical package . Significant difference among means of treatments was detected byDuncan's multiple range test procedures. The differences were consideredsignificant at 33.1The effects of HS on breeder quails were observed as a decrease in egg weight(EW), egg production (EP) and daily feed intake (DFI) in the HS group ascompared to the thermoneutral (TN) conditions group (Table 2). Metsupplementation increased EW, EP and DFI compared to the basal group exposedto HS. The feed conversion ratio (FCR) was significantly .The reduced EP and EW could be attributed in part to the decrease in feedintake and to an impairment in the utilization of nutrients. Heat stresscould also negatively impact nutrient utilization in poultry via changes inthe intestinal morphology, where different intestinal segments  exhibit lesions that vary in their degree, as well asdifferences in their relative weight, villus height, villus surface area,crypt depth , immunoglobulin A-secretingcell area and epithelial cell area  and by decreasingthe activity of digestive enzymes .When the ambient temperature exceeds the thermoneutral zone for birds, feedintake is decreased in order to minimize the production of metabolic heat, which adversely affects productive performance.To minimize these adverse effects of HS, many practical approaches have beensuggested to enhance the thermotolerance of birds in order to supportproductivity . In our study, we investigated Metas a way to protect quails against HS. Diet supplementation with 1.15, 1.30and 1.45 times the Met requirements restored the impairment in DFI, EP, EW andegg quality in quails submitted to HS. The reduced EP and egg quality inheat-exposed quails might be due to the reduction in feed intake and animpairment in the utilization of nutrients. In addition, dietsupplementation with Met improves productive performance through pathways ofpolyamine metabolism . Glutathioninederived from Met may also reduce damage from oxidative stress. Therefore,the TSAA requirement would be higher under hyper-thermoneutral conditionsthan under thermoneutral conditions. The inclusion of a high level of Met(1.30 and 1.45 times the Met requirement) improved productive performance inthe HS group compared to the TN group. Dietary supplementation with Met forbreeder quails exposed to HS significantly improved the egg parametersincluding egg weight and HU scores to levels similar to those of quailsreared under TN conditions. Several investigators also reported significantincreases in egg production with higher Met The internal quality of eggs can be evaluated particularly by HU. Birdsexposed to HS and fed the basal diet had the lowest HU score compared toother HS groups. Thus, it can be deduced that Met concentrations of 1.15,1.30 and 1.45 times the NRC (1994) recommendations in breeder quail dietsunder a heat-stressed environment were sufficient to improve the quality ofalbumen as evidenced by HU scores to a level similar to that of quailsreared under TN conditions.Heat stress can affect the reproductive function of poultry in differentways. Heat-stressed birds had depressed egg production due to imponderablesin the calcium–estrogen relationship, so we can infer that HS decreasesalbumin consistency, yolk size and calcium deposits in the egg shell. Thedepression in the ovarian blood flow is a possible mechanism for thereduction in ovarian function as a differential ovarian blood flow patternwas detected in laying hens subjected to HS . Dietarysupplementation with Met in breeder quails exposed to HS significantlyimproved the egg parameters including egg weight, eggshell thickness,albumen percentage and yolk percentage. Consequently, Met could eliminatethe negative effects of HS with regard to hatchability.The results of this study showed that the embryonic mortality of breederquails decreased by dietary supplementation with Met in the HS group. Tolbaet al. (2015) reported that Met supplementation in the diet of quails canimprove the infertility egg ratio, early embryonic mortality and laterembryonic mortality. In this context, the reduction in mortality in Metgroup quails may also be attributed to improved immunity as discussed below.In addition to impairing performance, HS also affects the immune response inbirds. In the current study, Met improved the maternal serum IgG, egg yolkIgY and offspring serum IgG in comparison to the basal groupexposed to thermal stress. The levels of supplementation can also stronglyaffect immunomodulation. In the present study, higher levels of Met fed toheat-stressed quails resulted in higher levels of maternal serum IgG, eggyolk IgY and offspring serum IgG compared to similar parameters in TN quails.The highest antibody transmission rate from breeder quails to offspring alsobelonged to quails that received 1.45 times the Met requirement. Thepercentage of IgY transfer can be expressed as the percentage of the dam'splasma IgY levels circulating in the blood of 1 d old chicks (approximately33.60 %). In agreement with our results, Loeken and Roth (1983) reportedthat the amount of IgY transported is independent of egg size and known to beproportional to the maternal serum IgY concentration.In this study, the humoral and cell-mediated immune responses were suppressedunder HS conditions. It is well known that environmentally stressed poultrygenerally have a depressed humoral immune response . Inagreement with the present study, Met supplementation optimized the responseto PHA as well as total antibody response to SRBC as a The heterophil-to-lymphocyte ratio has been used as a sensitive indicator ofstress, including HS, in poultry . An increased Heat stress could also induce oxidative stress resulting in an imbalance inantioxidant status in birds . Under HS conditions, as thebird's body attempts to maintain its thermal homeostasis, increased levelsof ROS occur. Consequently, tissues and cellspossess defense mechanisms to detoxify ROS by radical scavengers such asSOD, CAT and GPx . The results of our study are inagreement with data available in the literature. In the present study, theactivities of SOD, CAT and GPx were lower and MDA concentration in serum washigher in quails exposed to HS compared to the TN group. Dietary Metsupplementation increased the serum and liver activity of antioxidantenzymes  and reduced the concentrations of MDA, anindirect parameter of lipid peroxidation and overproduction of ROS, inheat-stressed quails. Inclusion of the higher level (1.45 times) of Met in thediet resulted in higher levels of antioxidant enzymes and lower MDA than inthe TN group. Synthesizing antioxidant enzymes such as SOD and GPx is animportant regulation with regard to animal responses to stress conditions.Glutathione is a tripeptide synthetized from glutamate, glycine andcysteine. Cysteine can be synthetized from homocysteine from the precursorMet ; therefore, when dietary Met is available inadequate amounts, larger quantities can be directed towards cysteinesynthesis via the transsulfuration pathway.With regard to HDL and LDL plasma levels, the present study showed apositive trend for an increase in HDL and a decrease in LDL in supplementedMet  recommended)vs. the HS group fed a basal diet. This indicates the beneficial andprotective effects of these methyl donors on bird health in particular andon productive performance in general. In addition, administration of Met tothe diet restored plasma levels of HDL-cholesterol, LDL-cholesterol, totalcholesterol and triglycerides near to those of the TN group.Possible mechanisms for the lipid-lowering effects of Met might be relatedto its antioxidant properties. In our study, Met supplementation reducedserum peroxide content. The maintenance of the normal structure oflipoprotein receptors is necessary for their function, improving thecellular uptake of serum lipids from the blood. Reactive oxygen speciesproduced during oxidative stress react with lipoproteins to produceoxidation states, decreasing the cellular uptake of lipids from the blood. Thus, theantioxidant action of Met might contribute to elevated cellular lipiduptake, resulting in a decrease in serum cholesterol levels.5Breeder quails subjected to the stress of a high thermal environmentexhibited deteriorated productive performance, reproduction, egg quality,antioxidant status and immune response. Dietary supplementation with either1.15, 1.30 and 1.45 times the Met requirements (as NRC recommendation) inbreeder quails reared under HS conditions can alleviate the adverse effectsof HS and improve the performance, reproduction, antioxidant status andimmunity, as well as maternal antibody transmission. In addition,administration of Met to the diet restored most parameters nearer to the TNgroup. In some cases, supplementation of the diet with 1.45 times the Metrequirement resulted in better responses compared to quails under TNconditions. Therefore, it was concluded that Met in higher levels ofrecommended NRC (1994) could be used as a feed additive in breeder quailsfor the alleviation of the adverse effects of HS.


TM 3M, St. Paul, MN, USA) and a glass containing resin-based composite , which contains fluoride. Class V preparations were placed on 22 premolars; the gingival margin was below the cementoenamel junction and the occlusal margin was placed above the cemento-enamel junction. Ten teeth were randomly selected to be restored with Z100 while the other 10 were restored with Tetric EvoCeram Bulk Fill. Both groups were restored following manufacturer’s instructions. All teeth had an acid resistant varnish placed within one millimeter of the preparation margins. Both groups were placed in artificial caries challenge solution (pH 4.4). At the end of the 4 days; 100 µm buccolingual sections were obtained for each tooth; these were photographed under polarized light microscopy and the demineralized areas adjacent to the restorations were measured and quantified. The mean (±S.D.) area (µm2) of demineralization from the occlusal margin (enamel) and dentin margin were: Z100 2781.889 ± 1045.213; 3960.455 ± 705.964 and for Tetric EvoCeram Bulk Fill 1541.545 ± 1167.027; 3027.600 ± 512.078. Student’s t-test indicated that there was significantly less enamel and dentin demineralization adjacent to Tetric EvoCeram Bulk Fill compared to Z100; there was significantly less demineralization in enamel compared to dentin in both Tetric EvoCeral Bulk Fill and Z100. Tetric EvoCeram Bulk Fill performed better inhibiting demineralization at restoration margins when compared to Z100 and provided better demineralization inhibition in enamel than cementum/dentin.Recurrent caries is still considered the main reason restorations need to be replaced. There are different materials available now that promise to reduce the possibility of recurrent caries by releasing fluoride and inhibiting restoration marginal caries. The purpose of this in vitro study was to evaluate the demineralization inhibition potential of a non-fluoride-releasing resin (Z100 Dental caries is still an ongoing problem not only for adults but also for children. Approximately 59 percent of children between the ages of 12 and 19 years old have experienced dental caries .There are different materials available now that promise to reduce the possibility of recurrent caries by releasing fluoride and inhibiting restoration marginal caries . These mTM 3M ESPE, St. Paul, MN, USA) and a glass containing resin-based composite  which contains fluoride. The purpose of this in vitro study was to evaluate the demineralization inhibition potential of non-fluoride-releasing resin  Pediatric Dentistry Clinic. Currently, UTHSCSA has a standing Institutional Review Board (IRB) acceptance of using human teeth for studies without individual IRB review for each study because extracted teeth were accumulated with no identifying features. The teeth were stored in deionized water until the initiation of the experiment to avoid dehydration. A class V preparation was placed on the buccal surface of each tooth with the occlusal margin in enamel and the gingival margin in dentin/cementum, which provided a restoration where caries inhibition in enamel and dentin would be easily evaluated on sections through the tooth. . An acid+2, 2.2 mM PO4−3, 50 mM acetic acid [After the teeth were restored, they were placed in an artificial caries solution (pH 4.4) for four days . The arttic acid . At the tic acid . A computic acid . 2) of demineralization from the occlusal margin (enamel) for Z100 was 2781.889 ± 1045.213 and for Tetric EvoCeram Bulk Fill was 1541.545 ± 1167.027 (2) of demineralization from the gingival margin (dentin/cementum) for Z100 was 3960.455 ± 705.964 and for Tetric EvoCeram Bulk Fill was 3027.600 ± 512.078 (p = 0.023). When comparing dentin demineralization, a Student’s t-test indicated there was significantly less dentin demineralization adjacent to Tetric EvoCeram Bulk Fill, compared to Z100 (p = 0.003) and when comparing demineralization adjacent to Tetric EvoCeram Bulk Fill, there was significantly less demineralization in enamel compared to dentin (p = 0.001). Lastly, and when comparing demineralization adjacent to Z100, there was significantly less demineralization in enamel compared to dentin (p = 0.008).The mean (±S.D.) area (µm1167.027 . The mea 512.078 . A StudeThis in vitro study intended to examine the inhibition of demineralization at the restoration margins of two different restorative products. In this study, Tetric EvoCeram Bulk Fill performed better than Z100 inhibiting adjacent tooth demineralization in both enamel and dentin . The TetAlthough the effects fluoride has in caries prevention are widely known, the study of bioactive materials and their inhibition of adjacent tooth demineralization, has not received attention. The study of demineralization inhibition with fluoride containing materials started in the 1990s and has continued until the present day. Donly and Grandgenett  studied 2 nanoparticles into resins have shown similar fluoride release rates to those of resin-modified glass ionomers [Different studies have shown the ability fluoride containing restorations have in preventing wall lesions and in some cases create inhibition zones ,12. The ionomers . NanoparBioactive materials are not new, they have been around since 2010. With the continuous improvement of restorative materials, better clinical long-term results can be expected. We need to keep in mind that caries is an active process and for it to start and advance, there must be an imbalance between demineralization and remineralization . TherefoTetric EvoCeram Bulk Fill performed better inhibiting demineralization at the restoration margins when compared to Z100 and provided better demineralization inhibition in enamel than in cementum/dentin.


This may even be more crucial when the member is subjected to cyclic loading. Although monotonic bond tests are available in the literature, only limited experimental studies exist on bond characteristics under cyclic loading conditions. In order to fill this gap and propose preliminary design recommendations, 10 specimens of 10-mm-diameter ribbed CFRP rebars embedded in specially designed high strength concrete (f’c = 70 MPa) blocks were subjected to monotonic and cyclic pullout tests. The experimental results showed that cyclically loaded CFRP rebars had less bond strength than those companion specimens loaded monotonically.Composite reinforcing bars (rebars) that are used in concrete members with high performance (strength and durability) properties could have beneficial effects on the behavior of these members. This is especially vital when a building is constructed in an aggressive environment, for instance a corrosive environment. Although tension capacity/weight (or volume) ratios in composite rebars (carbon fiber reinforced polymer (CFRP), glass fiber reinforced polymer (GFRP),  Reinforcements made of carbon fiber are widely used as retrofitting materials to increase the capacity of existing buildings . Recent Recently conducted experimental studies by various researchers showed similar behavioral properties of carbon rebars in concrete sections when compared to steel rebars. These studies also revealed that required embedment lengths of these rebars should be taken into consideration correctly and carefully. Unlike steel rebars, less information is available in the literature on the bond behavior of carbon rebars in normal and high strength concrete members, particularly for cyclic loading conditions.In the past decades researchers have carried out numerous experimental studies and proposed bond models to describe the behavior of steel rebars in terms of their embedment and anchorage lengths. Alsiwat and Saatcioglu  formulatSimilar to steel rebars, composite rebars also do not uniformly transfer forces to concrete . Steel rThis experimental study mainly focuses on comparing the bond behavior of carbon fiber reinforced polymer (CFRP) rebars in a high strength concrete block under monotonic and cyclic loading conditions. In addition to the loading procedure, various embedment lengths were also taken as test parameters. Obtained results showed that the sequence of loading has a significant impact on the behavior of CFRP reinforced concrete members.All rebars had the same diameter (d) of 10 mm and were placed concentrically in the concrete blocks. According to the predetermined specimen sizes, 10 plywood molds have been manufactured with 350 mm × 350 mm × 300 mm dimensions. In this research, the concrete cover was kept constant and taken as two times the diameter of the rebar to conform to the ACI 440  guidelinUnconfined specimens were designed and constructed. As stated in the preceding section, the concrete cover was kept constant in order to observe the effect of 2 d cover spacing as per the ACI 440 guideline. It was thought that carbon rebars would be more appropriate to use in high strength concrete members . As showd = 10 mm diameter and a 79 mm2 nominal cross-sectional area can resist 110 kN tension force under 1% elongation [Sika CarboDur BC10 deformed rebars were selected in this research, as shown in ongation .2. Measured rupture strengths for the specimens were 146.9, 104.4 and 126.6 kN, respectively. By using the average rupture value and also the experimentally obtained strength and modulus of elasticity values, it was found out that the fracture strain was slightly over 1.0%, which was different than the catalogue value.Three CFRP rebar specimens were tested at the Materials Laboratory of Bogazici University . AccordiAll specimens were tested at the Structures Laboratory of Bogazici University. The existing loading frame, which was used previously for steel rebar tests, was modified according to the requirements of this research. The test setup was equipped with special steel mounting parts to provide a reliable connection between the rebar and hydraulic jack (200 kN capacity with ± 200 mm of stroke). A special conical steel jaw attachment was designed to carry 250 kN of load without any slipping occurrences. The steel jaw assembly is the most crucial part of the experimental setup.In order to obtain proper and accurate force distribution between the rebar and surrounding concrete, compatible strain gauges were chosen for the CFRP rebar surfaces and attached to various locations before concrete pouring.Although CFRP rebars exhibit high tensile strengths in the longitudinal direction, they are weak in the transverse direction because of their orthotropic material properties. Without using any protecting cover for the rebar, the jaw would crush the rebar laterally, leading to erroneous results. For this reason, to prevent any material crushing, the tested rebars were surrounded with a 250-mm-long steel pipe sleeve with a diameter slightly larger than the rebars. The spacing between the rebar and steel sleeve was filled with a high strength epoxy having more than 3 MPa of bonding capacity to the steel.The schematic arrangement of the test setup is shown in As opposed to other proposed test setups  that produce compression on the concrete surface, the test setup developed in this work allows us to measure the bond capacity of CFRP rebars with the pure tension area in a concentric or eccentric loading configuration. Also, various embedment lengths from 5 d to 30 d with different concrete covers can be tested. Note that many test setups allow a 5 d embedment length. It is believed that the real behavior of the bond mechanism can be better achieved with the testing method developed in this work.The specimens were tested monotonically and cyclically. In cyclic tests, only specimens with 25 d, 20 d and 15 d embedment depths were tested using the force-controlled test method with 5 kN of force increments and three repetitions. Displacement-controlled cyclic testing was performed for other specimens. Displacements were increased by 0.01 mm at a time.One Linear Variable Differential Transformer (LVDT) (100 mm) was connected to the rebar with a manacle to measure the relative slip c. AnotheIn order to monitor the strain distribution inside the concrete block on rebar surfaces, three BFLA-5 type strain gauges  were attached on the rebar. Strain gauge measurements were taken at the beginning of the bonded area (SG1), in the middle of the bonded area (SG2) and at the end of the bonded area (SG3) a.Expected failure modes are explained in this section by comparing the experimental and expected failure modes. If an adequate embedment length is provided in an unconfined concrete, the pullout force could exceed the tension capacity of the concrete and a shear failure would occur. Splitting failure occurs when the surrounding concrete cannot resist the circumferential  tensile stresses. Splitting generally occurs on the surface of the member as flexural cracks start forming and the rebar is stressed and elongated towards failure with a continuous longitudinal crack along the rebar. Splitting cracks generally occur on the concrete surface  and propagate longitudinally and transversely as the rebar is axially stressed.In general, two types of splitting modes were expected prior to testing: concrete rupture and V-notch failures. Concrete rupture is most likely to occur when the force exceeds the tension capacity of the unconfined concrete specimen c. As shoThe pullout mechanism may be defined in three different failure modes: rib crushing, failure of the surface and concrete crushing. Rib crushing occurs in raised types of ribs such as in the steel rebar. If the load exceeds the capacity of the rib, it starts bending or yielding. The CFRP rebars used in this work have indented ribs. Therefore, rib crushing is not an expected failure mode in the tests. However, failure of the surface or concrete crushing may occur according to the ratio of the rib height to the rib gap.When the ribs are high and spaced too closely, the shear stress may govern the behavior and the rebar would be pulled out a. As shole embedded in a concrete block. The force in the rebar is resisted by an average bond stress u acting on the surface of the rebar. Equilibrium of forces can be written as follows;Af,bar is the cross-section area of one rebar, db is the rebar diameter and ff is the stress developed in the rebar at the end of the embedment length.C is the cover to the center of the rebar, u is the average bond stress acting on the surface of the GFRP rebar, bd is the diameter of the reinforcing bar, fc’ is the specified compressive strength of the concrete and le is the embedded length of the reinforcing bar.The development length equation for steel reinforcing bars found in ACI 318-08  is basedExperimentally obtained test results are summarized in Expected bond capacities given in Under monotonic testing, maximum axial loads of 28.3 and 86.4 kN were obtained for 5 d and 25 d specimens, respectively. As for cyclic (not reversed) loading, 16.2 and 70.0 kN load values were reached for 5 d and 25 d specimens, respectively. Developed bond stresses corresponding to these load values were 18.0 and 11.0 MPa for monotonic loading, and 10.3 and 8.9 MPa for cyclic loading. The experimental bond stresses could be significantly reduced when cyclic loads are applied. This results in reduced capacities for the specimens under cyclic loading. Reductions in short- and long-development-length specimens are 40% and 20%~25%, respectively. Experimental results showed that the attained minimum and maximum loads of 16.2 and 86.4 kN, respectively, were far below the maximum capacity (104.4 ~146.9 kN) of the deformed CFRP bars used.versus slip (in mm) graphs are plotted and illustrated in Bond stress (in MPa) Specimens S21, S22, S23 and S26 failed from the splitting mode while specimens S24, S25, S27, S28, S29 and S30 failed from the pullout failure mode. It was observed that splitting failure occurred under relatively high bond strengths, showing that the tensile strength of unconfined concrete controlled the behavior at this load level. For the splitting-type failures, loud sounds were heard, whereas for the pullout (or slip) failure no sound was heard. Since the concrete blocks were not confined, the splitting failures in some tests were quite brittle and caused complete wide cracks throughout the concrete block.The general shapes of the behavioral curves obtained from this study were similar to the curves given in the existing literature. Typical failure modes for splitting and pullout failures are given in Achillides and Pilakoutas  tested Ci.e., >2 d) is more suitable for relatively deeper embedment lengths when high strength concrete is used.In general, rebars with 5 d and 10 d embedment lengths failed due to pullout of the bar while rebars with 15 d, 20 d and 25 d embedment lengths failed due to splitting of the concrete. These failures were more brittle since the tension capacity of the unconfined concrete block was exceeded. Therefore, a thicker cover concrete  at early displacement levels for both cyclic and monotonic loading.Cyclic loading reduced the bond capacity of the specimens. Reductions in short- and long-development-length specimens are within the range of 40% and 20%~25%, respectively.For tested specimens, the bond strength values changed between 10~18 MPa under monotonic loading and 9~14 MPa under cyclic loading. These results showed that obtained monotonic loading capacities were considerably higher than those obtained by the previous research.Measured bond stresses are generally higher than the calculated bond capacities according to ACI 440 . For theMonotonic and cyclic experimental bond behavior of CFRP rebars in high strength (68.8 MPa) concrete was investigated. Various embedment lengths were studied. All (10) specimens had a constant concrete cover of 2 d. All monotonically-loaded specimens were loaded under a displacement-controlled testing procedure as well as the 5 d and 10 d embedded cyclic loading specimens. Other cyclic loaded specimens  were tested under the force-controlled testing procedure. The following conclusions could be drawn from this experimental work:


IgG4-related disease is characterized by lymphoplasmacytic inflammation and fibrosis, often leading to mass-forming lesions in different organs. When IgG4-related disease affects the bile ducts, it is called IgG4-related sclerosing cholangitis. A 74-year-old male complained of dysphagia and abdominal pain. Endoscopic retrograde cholangiography and magnetic resonance cholangiography revealed bile duct changes suspicious of a bile duct carcinoma or cholangitis. Liver biopsy showed storiform fibrosis, lymphoplasmacytic infiltration, obliterative phlebitis, and a portal-based inflammatory nodule with expansion of a portal tract. Hot spots revealed 339 IgG4-positive cells per high power field (HPF) and an IgG4/IgG ratio of 72%. Eight months earlier, an inguinal lymph node had been removed, showing expanded interfollicular zones and increased plasma cells. Hot spots revealed 593 IgG4-positive cells and an IgG4/IgG ratio of 92%. The serum IgG4 of the patient was elevated nearly 10 times upper limit of normal. The diagnosis of IgG4-related sclerosing cholangitis associated with IgG4-related lymphadenopathy was made. There was good response to treatment with prednisolone and azathioprine. The differentiation of IgG4-related sclerosing cholangitis from primary sclerosing cholangitis and bile duct carcinoma is often difficult. Liver biopsy only rarely contributes to this setting, but we describe and report in detail a case where liver biopsy showed a portal-based inflammatory nodule with the characteristic features of this disease. IgG4-related disease (IgG4-RD) has a broad spectrum of clinical manifestations, depending on which organ systems are involved. This disorder is characterized by fibro-inflammatory activity that may present as mass-forming lesions. The condition can affect one or multiple organs at the same time and is associated with elevated serum IgG4 in most cases –4. The dWhen IgG4-RD affects the bile ducts, it goes under the term IgG4-related sclerosing cholangitis (IgG4-SC). In some cases the differential diagnosis of IgG4-SC is complicated by its high degree of clinical and radiologic resemblance to primary sclerosing cholangitis (PSC) and bile duct carcinoma . The curA 74-year-old male complained of dysphagia and abdominal pain lasting for two months. Esophagogastroduodenoscopy revealed esophageal candida infection that was treated with nystatin, a drug that has not been reported as causing hepatic injury. He had a history of arterial hypertension, prostatic hypertrophy, yet unspecified myopathy causing walking-disabilities for 40 years, and diabetes mellitus type 2 diagnosed four years prior to debut of the gastrointestinal symptoms. For the past 55 years he had smoked 20 cigarettes a day and had an alcohol consumption of 21 units (252 g) a week. Four and a half and one and a half years earlier, he had undergone surgical removal of a malignant melanoma Clark's level 2 from his left cheek and a basal cell carcinoma from his back. Two months earlier, a benign inguinal lymph node as well as nine colonic hyperplastic polyps had been removed. The lymph node was detected by physical examination as part of the follow-up program for malignant melanoma and was also seen at positron-emission tomography (PET).μmol/L [5-25 μmol/L], and alkaline phosphatase (AP) 500 U/L [35-105 U/L)]. The amylase was not elevated. The elevation of CRP was due to a urinary tract infection, and E. coli was isolated from the peripheral blood. He was treated with antibiotics, whereafter the CRP normalized and the hemoglobin almost normalized (7.4 mmol/L). After two failed attempts of endoscopic retrograde cholangiopancreaticography (ERCP), a magnetic resonance cholangiopancreaticography (MRCP) revealed a normal main pancreatic duct, intrahepatic cholestasis, and bile duct changes suspicious of a bile duct carcinoma Bismuth-Corlette type IV in the hilar region, involving both the right and left hepatic bile ducts. By magnetic resonance imaging (MRI), no hepatic tumors or pseudotumors but a benign cyst were observed  showed that the CBD had a diameter of 11 mm, intrahepatic cholestasis with stenosis at the hepatic duct bifurcation, a liver cyst located to segment 8, a right-sided renal tumor classified as Bosniak 3, pancreatic calcifications, and a presumed benign cyst located to the pancreatic neck. Serological tests revealed C-reactive protein (CRP) 79 mg/L [< 6 mg/L], hemoglobin 6.6 mmol/L , elevated alanine aminotransferase (ALT) 164 U/L [10-70 U/L], bilirubin 252 observed .μmol/L, and AP 343 U/L. Serum cancer associated antigen 19-9 (CA 19-9) was strongly elevated to 3003 kU/L [0-37 kU/L] but dropped to 165 kU/L after stent placement. Hereafter, either a bile duct carcinoma or PSC was suspected. Bile duct brush cytology revealed inflammation and atypical cells. Additional blood tests showed strongly elevated IgG4 , elevated IgG , negative cytoplasmic and perinuclear neutrophil cytoplasmic antibodies (c-ANCA and p-ANCA), and positive IgM rheumatoid factor. Serologic markers concerning viral hepatitis as well as anti-smooth-muscle antibodies, anti-liver-kidney microsome type 1 (LKM1) antibodies, anti-mitochondrial antibodies (AMA), glomerular basement membrane antibodies (GBM), liver cytosol specific antibody type 1 (anti-LC1), and anti-nuclear antibodies were all negative. He had a normal glomerular filtration rate (GFR).One month later, an ERCP showed multiple strictures of the small intrahepatic bile ducts in several liver segments, dilation of the CBD (12 mm), and a stenosis of the bifurcation . Unfortu2) were found, corresponding to an IgG4/IgG ratio of 72%  were found, corresponding to an IgG4/IgG ratio of 92%  type 1 or other manifestations of IgG4-RD , 17. CliIgG4-related lymphadenopathy is both an “under- and overdiagnosed entity”, as mentioned by Cheuk et al. . Five diOur patient was diagnosed with malignant melanoma and basal cell carcinoma 4.5 and 1.5 years prior to the development of IgG4-RD. It is at present not clear whether IgG4-RD may be a form of paraneoplastic syndrome in patients with malignancies. A few follow-up studies indicate an increased risk for the development of malignancies in IgG4-RD, including patients with type 1 AIP and IgG4-SC –31. HoweThe patient presented here had unspecified myopathy and noticed a marked improvement related to treatment of IgG4-RD with steroids. It is well known that inflammatory myopathy responsive to steroids can develop in association with autoimmune diseases such as systemic sclerosis, systemic lupus erythematosus, Sjögren's syndrome, and rheumatoid arthritis, a condition termed overlap myositis . Hence, In conclusion, we present a case of a male with IgG4-related sclerosing cholangitis involving the small intrahepatic as well as the hilar bile ducts, initially suspected to represent bile duct cancer, diagnosed with liver biopsy, and associated with IgG4-related lymphadenopathy.


In this data article, the experimental data of the density, water absorption and compressive strength of lateritic earth moist concrete are presented. 10% and 20% of fine aggregate were replaced with laterite. Potable water at 0.3 water/cement ratio was used and ordinary Portland cement used as the binder. Concrete cubes ( Specifications TableValue of the data•The influence of laterite in the density, water absorption and compressive strength of earth moist concrete is presented here.•The data gives a basis on ways to utilize earth moist concrete in construction.•The research data could be helpful in further researches on earth moist concrete.1The dataset presented in this article are experimental results of the water absorption and compressive strengths of grade 20 earth moist concrete cubes, fine aggregate was partially replaced with laterite. The tests were carried out at Landmark University Concrete laboratory and the test set up for the compressive strength was shown in 2The concrete aggregate materials were all sourced for in Omuaran, Kwara state, Nigeria. Earth moist concrete with 0.3 water/cement ratio was mixed at 0%, 10% and 20% laterite replacement for the fine aggregate. Concrete cubes (


MET amplification with more than 20 copies. As expected, MET overexpression led to its constitutive activation and upregulated signaling towards the MAPK, PI3K/AKT, STAT3 and NF-κB pathways. Since the usual feature of MET-amplified cell lines is the "MET addiction" for their cell proliferation, the effect of the highly selective ATP competitive MET inhibitor PHA665752 was analyzed. While PHA665752 strongly inhibited the MAPK pathway, it did not reduce cell proliferation in TTA1 cells (IC50 = 4100 nM). This resistance to PHA665752 of the TTA1 cell line was demonstrated to be related to EGFR-MET functional cross-talk and PI3K/AKT and NF-κB signaling. Nevertheless, PHA665752 suppressed the anchorage-independent growth capacity of the TTA1 cell line and reduced cell migration and invasion in a transwell assay. The role of activated MET in these neoplastic properties of the TTA1 cells was also proved with si-MET-RNA targeting. Thus, this work highlights the TTA1 cell line as the first model of MET amplification in an ATC cell line, which leads to MET constitutive activation and underlies its neoplastic properties. Besides being a useful model for MET inhibitors screening, the TTA1 cell line also supports the argument for searching for MET amplification in ATC, as it could have therapeutic implications.In thyroid cancers, MET receptor overexpression has been associated with higher risk of metastatic progression. In this study, it was shown that the anaplastic thyroid cancer (ATC)-derived TTA1 cell line overexpressed MET. By using FISH and relative quantification by qPCR, it was demonstrated that this overexpression resulted from a  PIK3CA, PTEN or CTNNB1 mutations affect the PI3K/AKT and WNT-βcatenin pathways [EGFR, PDGFRA, PDGFRB, VEFGR, KIT and MET [Follicular cells-derived thyroid cancers are the most frequent endocrine tumors with an increasing incidence worldwide , 2. Thyrpathways . Gene am and MET .MET is the trans-membrane tyrosine kinase identified as the high affinity receptor for hepatocyte growth factor (HGF). The binding of HGF and activation of the tyrosine kinase domain provide multiple docking sites for SH2 molecules through autophosphorylation of Tyr1349 and Tyr1356. These molecules act as intracellular transducers for PI3K-AKT, RAS-MAPK and STAT3 pathways by which MET activation promotes different cellular responses, such as proliferation, cell survival, cell scattering/migration and morphogenesis , 12. DerMET gene in the TTA1 ATC-derived cell line was described. It was postulated that MET overexpression and constitutive activation of downstream signaling pathways could have a role in neoplastic properties of this cell line. By the use of a specific pharmacological inhibitor, PHA665752, and si-RNA mediated MET downregulation, it was demonstrated that the activation of the MET-dependent signaling pathways in the TTA1 cell line contributes to neoplastic properties by sustaining anchorage-independent cell growth, cell motility and invasiveness rather than to proliferation and apoptosis protection.In this study, among a panel of 11 human thyroid cancer cell lines, the amplification and overexpression of the The expression of MET mRNA was analyzed in eleven thyroid cancer cell lines, including 3 PTC cell lines  and 8 ATC cell lines . With the exception of the HTh74 and TTA1 cell lines, all of them bear an identified driver genomic alteration  leading to a constitutive activation of the MAPK pathway. As shown in Figure MET amplification [MET copy number in the TTA1 cells in comparison to low MET-expressing cells was analyzed. FISH experiments demonstrated that TTA1 cells possessed a high copy number of the MET gene, compared to three thyroid carcinoma cell lines , which expressed low levels of MET -mediated MET downregulation, or a pharmacological MET inactivation. As shown in the Figures ut 70 nM . And the Figures . TTA1 ce Figures . While M Figures . Cell cy Figures . And cle Figures .We then explored the molecular basis of the PHA665752 resistance of the TTA1 cells with the hypothesis that it could be mediated by the PI3K/AKT, STAT3 pathways and/or NF-κB pathways that were not totally inhibited by PHA665752-induced MET inactivation. Since functional cross-talk of MET was described in various cancers with other RTKs , 18, we Lapatinib, an inhibitor of the EGFR family members, did not affect TTA1 cells viability. The Pi3K inhibitor LY294002 alone slightly reduced TTA1 cell growth, thus indicating implication of the PI3K/AKT pathway in TTA1 cell proliferation. The STAT3 inhibitor LLL12 had no inhibitory effect. Interestingly, lapatinib synergized with PHA665752 to reduce cell growth by about half , compared to only 3 in control PTC without distant metastasis. In the 39 PTC with distant metastasis, 36% harboured MET mutation, as opposed to 20% of the 20 control PTC, thus indicating a strong association of MET exon 2 or 14 mutations with tumor progression. Nevertheless, only one MET mutation was detected in the recent large scale genomic analysis of 507 non aggressive PTC [MET were described in different types of cancers. In thyroid cancer the first description was the TFG-MET fusion, found in one papillary thyroid cancer after the depth analysis of the Cancer Genome Atlas (TCGA) RNA-seq data [The tight regulation of the HGF-MET signaling that occurs in normal tissues is lost through multiple ways in several cancers. Missense activating point mutations of  cancers . Most of cancers . In foll cancers . Another cancers . Howeverd in PTC . Interessive PTC . In aggrsive PTC , and no sive PTC . Gene fuseq data .MET gene was demonstrated as resulting from the upregulation of hypoxia inducible factor (HIF-1), probably through hypoxia [MET gene amplification is the most frequent genetic alteration that leads to MET activation in non-small cell lung cancer, colorectal and gastric cancers with poor prognosis [MET amplification has been found to be significant in a series of ATC (5 out of 42 anaplastic carcinomas (11.9%) with 3.5 to 8.7 copy number gains) [MET amplification in an ATC-derived cell line with a biological impact related to neoplastic properties.The MET pathway oncogenic activation can also be achieved by the MET overexpression through a HGF-independent dimerization. In PTC, increased transcription of the  hypoxia . The und hypoxia . MET genrognosis . In aggrrognosis . MET ampr gains) . Our stuMET amplification contributes to the tumoral properties of the TTA1 cells, an RNA interference-based strategy and a pharmacological approach with PHA665752, a MET inhibitor with a high selectivity (Ki 4 nM) for the catalytic domain of the kinase, were used [MET-amplified cancer cells are highly sensitive to MET inhibitors [MET amplification (fold>8), 50 nM of PHA665752 were enough to inhibit cell growth [MET-amplified cell lines were sensitive to 200 nM PHA665752 or presented an IC50 between 20 to 100 nM, while cell lines without MET amplification were resistant to the drug [MET amplification defines their “MET addiction”, which is related to the constitutive activation of this receptor, because of its ligand-independent dimerization when overexpressed at the cell surface. In the TTA1 cells, it was demonstrated that MET overexpression was also associated with its constitutive activation leading to the stimulation of downstream signaling pathways. However, the high level of amplification with more than 20 copies of MET was not associated with a high sensitivity of TTA1 cells to MET inhibition by PHA665752. A lack of interaction between PHA665752 and its target could not be asserted, since the drug undoubtedly produced its expected inhibitory effects on MET receptor activation and the downstream signaling effectors ERK1/2 and AKT.In order to know whether ere used . It is ghibitors , 33. In l growth . In othethe drug , 33. TheIn cancers, MET activation is commonly implicated in both cell proliferation and invasiveness . HoweverThen, we used the TTA1 cells to investigate the molecular basis of the resistance to MET inhibitors, which frequently occurs with MET targeting therapies. We demonstrated that TTA1 cell proliferation is dependent on the cooperation between MET and EGFR, which act in cross-talk to activate downstream signaling pathways, such as the PI3K/AKT and NF-κB pathways. These are two pathways that we demonstrated as being necessary for TTA1 cell proliferation. Nevertheless, the hypothesis that other RTKs-mediated PI3K and NF-κB activation could also drive cell proliferation in the TTA1 cells cannot be excluded. Functional cross-talk of MET with other RTKs, such as EGFR, HER2, HER3, RET or IGF1R, was identified in various cancers as mediating inhibitors resistance , 37–40. MET amplification was demonstrated as a central mechanism to BRAF inhibition resistance in a murine model of ATC [In the TTA1 cells, it was further demonstrated that MET activation contributes to their neoplastic properties such as the anchorage-independent growth and migration and invasion capacities, thus corroborating the numerous histological analyses demonstrating the significant correlation between MET overexpression and a higher risk of metastasis in thyroid cancers , 43. In l of ATC . In othel of ATC , 50. TheALK-rearranged ATC [https://clinicaltrials.gov/ct2/show/NCT02034981). Cabozantinib (XL184), targeting VEGFR1/2, MET, RET, KIT and FLT3, has been assessed in several cell line models and has been approved by the FDA for the treatment of progressive medullary thyroid cancer [https://clinicaltrials.gov/ct2/show/NCT02041260). Analyses of the genetic alterations in follicular thyroid cancers were essentially focused on mutations with sequencing analysis, and gene amplifications were not extensively investigated. This work argues for the importance of further investigation into MET amplification in these cancers, as it could have therapeutic implications for patients who are refractory to standard therapy.Several MET targeting-MKI and specific MET kinase inhibitors have entered clinical trials in various cancers with MET alterations . In thisnged ATC , and entd cancer . In a phd cancer . A phased cancer . A phaseThe human hepatocellular carcinoma cell line HEPG2 was kindly provided by C. Perret . The human anaplastic thyroid cancer cell lines HTh74, TTA1, ACT1, CAL62, C643, SW1736, HTh104 and the human thyroid papillary cancer cell lines TPC1 and KTC1 were kindly provided by J. Fagin . These cell lines have been previously authenticated . The humThe selective ATP competitive MET inhibitor PHA665752 was purchased from Sigma-Aldrich, and the EGFRs inhibitor lapatinib, the PI3K inhibitor LY294002 and the STAT3 inhibitor LLL12 were purchased from Selleckchem. The cabozantinib and crizotinib RTKs inhibitors were kindly provided by B. Blanchet . The inhibitors were diluted in dimethylsulfoxide (DMSO).The following antibodies were from Santa Cruz Biotechnology, Inc. : anti-MET (sc-10), anti-P-ERK1/2 (sc-7383), and anti-GAPDH. The anti-P-MET (Tyr1234/1235)(D26), anti-ERK 1/2, anti-P-AKT (Ser473), anti-P-AKT (Thr308), anti-AKT, anti-P-STAT3 (D3A7), anti-STAT3, anti-P-EGFR (Tyr1068), anti-EGFR antibodies and anti-cleaved caspase-3 (Asp15) were from Cell Signaling Technology, and the anti-actin antibody was from Sigma-Aldrich.Total cell protein lysates were extracted using Tris-NP40 buffer (20 mM TrisHCl (pH 7.5) 1 mM EDTA, 150 mM NaCl, NP40 1%) containing protease and phosphatase inhibitors (Roche). Protein concentration was determined using the Bradford reagent (BioRad). Proteins were separated by 7–12% SDS-PAGE and transferred on nitrocellulose membranes . After blocking with 5% non-fat milk or 5% bovine serum albumin, proteins were immunoblotted overnight with primary antibodies followed by incubation with HRP-conjugated secondary antibodies. Immunostaining was revealed by chemiluminescence  and visualized using the ImageQuant LAS4000 imaging system .Electrophoretic mobility shift assay (EMSA) was carried for analysis of NF-κB activation, as previously described . Briefly-∆∆CT method, with PPIA as the reference gene. Oligonucleotide sequences are detailed in RNA extraction was performed with RNeasy™ Mini Kit (Qiagen) according to manufacturer’s instructions. RNA were retrotranscribed in cDNA by using the high capacity cDNA reverse transcription kit . Quantification of mRNA was performed by RT-qPCR SYBR Green in capillaries  or in 96-microwell plates . Values of mRNA were calculated by relative quantification using the 2®Green probes in 96-microwell plates in Light Cycler 480 (Roche). Oligonucleotide sequences are detailed in DNA was extracted with QIAGEN™ QIAamp™ DNA Mini Kit and QIAamp Mini spin columns according to manufacturer’s instruction. Nucleic acids quantification was made by spectrophotometry . Cell line DNA quantification was performed by real time PCR using SYBRMET copy number was calculated using COL6A5 as the reference gene, located at 3q22.1. This gene has been chosen after verification of the stability of the ratio ALB/COL6A5 for all cell lines. The results of relative quantification was normalized with normal healthy human DNA containing 2 copies of the MET gene, one sample serving as calibrator, the other serving as control.Relative quantification of Light™ SPEC MET/CEN7 Dual color probe . The control probe with orange dye targets the centromere of chromosome 7 (D7Z1), serves to locate the chromosome of interest and the specific probe with green dye targets area genomic locus including the MET gene located at 7q31.2 (D7S486-D7S2347). Six metaphases and 6 nuclei were analyzed for each sample.FISH analysis was performed on metaphase spreads obtained according to standard protocols using Zyto50 values were estimated by non-linear logistic regression model by using the Graphprism software.Cells were seeded in 96-microwell plates and viability was assessed by using either the MTT (Thiazollyl Blue Tetrazolium Bromide) assay, as described by Buffet and coll. , or the Cell cycle was analyzed by flow cytometry as previously described . ApoptosSmall interfering (si) RNAs for c-MET , RelA (si-RelA:5′- GGAUUGAGGAGAAACGUAA-3′) and RelB (si-RelB: 5′-GACTGCACCGACGGCATCT-3′) were purchased from Eurofins Genomics. Cells were transfected with Lipofectamine 2000 or Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer’s protocol. Cells were either seeded in 96-well plates for cell viability assay or in 6-well plates for harvesting at day 2 for qRT-PCR analysis.The anchorage independent growth was analysed through a soft agar colony formation assay, as described previously . ColonieCell migration and invasion were assessed in Transwell polycarbonate membrane inserts (Corning) as previously described . For thet-test (GraphPad software on line). P values < 0.05 were considered significant with the following degrees: *p < 0.05; **p < 0.01, ***p < 0.001.Statistical significance was assessed using the Student’s


This cross-sectional study examines the association of law enforcement seizures of heroin, fentanyl, and carfentanil with opioid overdose deaths in Ohio from 2014 to 2017. The Centers for Disease Control and Prevention5 has endorsed using law enforcement drug seizures as a proxy indicator for the illicit drug supply, and a joint investigation between the Centers for Disease Control and Prevention and the state of Ohio6 demonstrated concurrent increases in fentanyl overdose deaths and fentanyl seizures. Less known, however, is whether there are associations between law enforcement drug seizures and drug overdose deaths.The United States continues to experience an unprecedented overdose crisis. Fentanyl overdose deaths increased 525% from 3105 in 2013 to 19 413 in 2016 and then increased 45.2% from 19 413 in 2016 to 28 466 in 2017, with fentanyl overdose deaths outpacing deaths from both prescription opioids and heroin in 2016 and 2017.6 Each record contained a list of drugs detected by qualitative testing via gas chromatography–mass spectrometry, the confiscation date and county, and the weight in grams. We first constructed monthly statewide counts for seizures that contained heroin, fentanyl, carfentanil, cocaine, and methamphetamine and then calculated the percentage of seizures of heroin, cocaine without heroin, and methamphetamine without heroin that also contained fentanyl or carfentanil. Overdose death data were obtained from the Ohio Department of Health from January 2014 to December 2017 and were rereviewed and recoded for fentanyl or fentanyl analogue detection. Overdose deaths were those for which the cause of death, according to International Statistical Classification of Diseases and Related Health Problems, Tenth Revision codes, was unintentional (X40-X44), suicide (X60-X64), homicide (X85), or undetermined intent (Y10-Y14). We coded overdose deaths as opioid-involved if International Statistical Classification of Diseases and Related Health Problems, Tenth Revision codes T.40.0, T.40.1, T40.2, T40.3, T40.4, or T40.6 were indicated, or if the Ohio Department of Health’s rereview included a separate flag for fentanyl or fentanyl analogues. We summed opioid-involved deaths to yield a monthly, statewide count.Ohio’s Bureau of Criminal Investigation provided drug seizure information associated with state and local drug cases from the state’s 88 counties that were tested by 3 public laboratories from 2014 to 2017.P < .05 was considered statistically significant.Drug seizures are presented for heroin (29 917 seizures), cocaine (24 462 seizures), and methamphetamine (20 957 seizures) by year from 2014 to 2017, including the percentage of fentanyl or carfentanil in each drug category. Percentages of fentanyl or carfentanil are presented for a subsample with nonmissing weights by 3 weight strata , with mean, median, and 99th percentile weights provided for seizures greater than 30 g. A Mantel-Haenszel test compared the distribution of fentanyl or carfentanil across weight categories over the 4-year period. A multivariate, generalized, autoregressive, conditional-heteroskedasticity model estimated the association between drug seizures and drug overdose deaths. This cross-sectional study received a full review and was approved by the institutional review board at the Ohio Department of Health. All data were administrative records and the study did not require an informed consent process. All analyses were completed using Stata MP statistical software version 15.1 (StataCorp). Additional model details are included in the eAppendix in the 2 = 3528; P < .001). Among 18 276 cocaine seizures with weights available in 2017 not containing heroin, 7.1% contained fentanyl or carfentanil, all of which were less than 1 g. Among 558 cocaine and 753 methamphetamine seizures weighing more than 30 g, less than 0.5% contained fentanyl or carfentanil , with a median age of 40 years . The mean (SD) monthly number of opioid-involved overdose deaths was 260 (78). Of the 29 917 seizures of heroin identified, 23 175 (77.5%) included weights in grams. The percentage of these containing fentanyl or carfentanil increased from 3.4% in 2014 to 48.6% in 2017. Most of the increase involved seizures weighing less than 30 g. By 2017, 52.0% of heroin seizures less than 1 g and 11.2% of heroin seizures greater than 30 g contained fentanyl or carfentanil. Overall, changes in the distribution of the percentage of heroin seizures containing fentanyl or carfentanil over time and across weight categories were statistically significant (χfentanil .P < .001). Carfentanil seizures were the only other significant covariate, with a coefficient of 0.34 .Increases in opioid overdose deaths were associated with increases in fentanyl or carfentanil seizures from 2014 until mid-2017 . Heroin By integrating overdose mortality data from Ohio’s Vital Statistics System with state crime laboratory data from Ohio’s Bureau of Criminal Investigation, we demonstrate a significant association between law enforcement drug seizures and overdose deaths in Ohio from 2014 to 2017. To our knowledge, this is the first study to offer an empirical basis for using crime laboratory data as a viable indicator of opioid overdose deaths. This analysis is limited by the administrative character of crime laboratory data, which, like other institutional data systems , are subject to internal measurement error. Because quantitative testing was not performed by Ohio’s Bureau of Criminal Investigation, the analysis could not identify percentage amounts of fentanyl and carfentanil in heroin, cocaine, and methamphetamine seizures and, thus, was unable to determine whether fentanyl adulteration was intentional or the result of unintentional trace contamination. The absence of drug seizures from private laboratories limits a comprehensive portrait across Ohio; however, the strength of our findings and large sample size suggest that our conclusions would not change materially by including samples from private laboratories. Our analyses were limited to Ohio and may not generalize beyond this state.These findings underscore the importance of partnerships between public health and public safety to address the opioid overdose epidemic. Active data sharing between law enforcement and public health agencies can facilitate timely, actionable data to identify fentanyl hot spots and coordinate rapid responses that could limit overdose mortality.


Tursiops truncatus) and a short-beaked common dolphin (Delphinus delphis), considered members of ‘vulnerable’ and ‘endangered’ subpopulations in the Mediterranean, respectively, by the International Union of Conservation of Nature is described in this paper. The birth of the hybrid was registered in the Bay of Algeciras (southern Spain) in August 2016, and the animal has been tracked on frequent trips aboard dolphin-watching platforms. This unique occurrence is the result of an apparent ongoing interaction (10 years) between a female bottlenose dolphin and common dolphins. The calf has a robust body with length similar to Tursiops, while its lateral striping and coloration are typical of Delphinus. It displays the common dolphin’s ‘criss-cross’ pattern. However, the thoracic patch is lighter than in D. delphis and its dorsal area is light grey, with a ‘V’ shape under the dorsal fin. This paper also provides a comprehensive mini-review of hybridizations of T. truncatus with other species.A case of intergeneric hybridization in the wild between a female bottlenose dolphin ( Dephinus delphis) which, since 2003, are considered ‘Endangered’ in the Mediterranean Sea according to the Red List criteria by the International Union of Conservation of Nature (IUCN) [Tursiops truncatus), a species which is also considered as a ‘Vulnerable’ Mediterranean subpopulation by the IUCN. Striped dolphins  are occasionally detected (‘Vulnerable’ in the Mediterranean by IUCN) mixing with common dolphin, but the groups are mainly formed by mothers, calves and immature juveniles.The Bay of Algeciras, located in the south of Spain , hosts ae (IUCN)  and alsoe (IUCN) . Also, iD. delphis, S. coeruleoalba and T. truncatus, included in the clade Delphininae [T. truncatus populations from the Black Sea (eastern Mediterranean) and Scotland (north-eastern Atlantic Ocean) [S. coeruleoalba and D. delphis species [The three species involved in this study phininae , share tphininae , 7. Furtphininae . T. trunc Ocean)  showed ic Ocean) . Further species –16.Common and bottlenose dolphins overlap ranges in temperate and tropical waters , althougThe bottlenose dolphin was identified among the common dolphins due to morphologic differences in size and coloration: she was bigger than the common dolphins, with a robust body, a falcate dorsal fin, of light grey coloration to darker grey dorsally, and showed a light blaze marking on her sides. A well marked demarcation at the end of the melon, convex flippers and a short and stubby beak  were alsBefore 2016, Billie was detected three times assisting common dolphin births, leading newborns to the surface and offering alloparental care for only a few minutes after labour and always accompanied by other common dolphins. Allomaternal care often occurs among bottlenose dolphins , 20 and s and curled flukes, produced by the neonatal posture in the uterus during the gestation period [On 11 August 2016, Billie was observed raising and pushing to the surface a neonate . Visiblen period . No otheCetaceans exhibit surprising karyotype uniformity, suggesting that they have a higher potential to produce hybrid offspring than do other mammals –27. HoweMarine mammal hybridizations are very difficult to detect in comparison to other taxonomic groups of animals and plants, although several examples in captivity and in the wild have been reported –35. InteThe study was carried out on board opportunistic dolphin-watching platforms (14 m and 12 m in length), offering trips of 90 min with consistent daily itineraries from August 2016 until May 2017. Once the group of interest (the one including the mother/hybrid pair) was detected, standardized data were gathered (weather permitting), such as date, time, GPS position, structure and group size. Group composition data were collected using a combination of sampling methods. Individual-following protocols , 38, focTissue sampling by means of biopsy dart was not attempted as it was considered invasive and inappropriate due to the immaturity of the calf . It is wData regarding the group composition of the species involved were collected over 25 weeks (11 August 2016 to 29 May 2017). Sightings were classified according to 19 descriptors. A box plot analysis , was applied to analyse the behaviour of the mother/hybrid pair, comparing the composition and the frequency they were found in mixed-species groups, separated or alone.This study was carried out in strict accordance with the cetacean protocol included in the Marine Regulations, 2014 and has been approved by the Committee on the Ethics of Animal Experiments of the Ministry for Education, Heritage, Environment, Energy and Climate Change of Gibraltar.D. delphis)  and in only six sightings (1 h 53 min) was the pair sighted alone, distanced from the common dolphins (minimum 500 m between groups). The pair were detected together less than 10 m apart in 112 sightings (99.1%); 1 occasion (0.83%) was Billie  separated from the hybrid by 100 m, both of them accompanied by common dolphins. Sea surface temperature (SST) during these observations was an average of 19.35°C (66.83°F) with minimum of 14°C (57.2°F) and maximum of 26°C (78.8°F). From 2 June 2017 until the end of the year, the hybrid was not sighted again, leading to the reasonable suspicion of death.After the first sighting on 11 August 2016, re-sighting took place on 17 August 2016, after which they were seen on an almost daily basis mixing with ‘nursery groups’ of common dolphins (delphis) . Data weThe female bottlenose dolphin was observed showing continuous epimeletic and nurturant behaviour towards the newborn, offering care and protection and exhibiting near-body contact for the first three months of observation. The two main swimming positions for calves and their mothers are defined as ‘echelon position’  and ‘infant position’  . The hybCalves often show rubbing behaviour with their mothers, with particular focus on her head region . Body-coThe mother/hybrid pair showed normal breathing and developmental patterns and close swimming positions until 4 September 2016, when at 24 days the neonate was observed breaching and swimming in echelon position alongside an adult common dolphin . At the D. delphis) and bottlenose dolphin (T. truncatus)  , and chaTursiops spp. Neonates are 1.1 m, while in common dolphins 0.8 m. The presumed hybrid was less than half the size of the female bottlenose at birth (approximately 1.25 m), and remained in echelon position close to her most of the time, characteristics consistent with a newborn. By the middle of November, the animal was just over half the length of Billie, and therefore considered an infant or calf [According to Whitehead and Mann , Tursiop or calf , 38, 40. or calf , 40, 51.D. delphis, although in this case it was light brown in colour from the anterior insertion of the flipper to the lower jaw and gape  has only been observed once in the wild [T. truncatus and several other species including Delphinus capensis [T.truncatus, and the calf didn’t survive either. In 2018, Gridley, reported multiple intra-generic matings between T. truncatus and Tursiops aduncus producing a health F1 hybrid, which survived to adulthood and also produced back-crossed hybrid offspring [On the other hand, hybridization events in Delphinidae in captivity have been reported multiple times, and  species , but thithe wild , 57. In capensis , 58, whiffspring .S. coeruleoalba and Delphinus have a closer phylogenetic relationship, being more closely related to each other than to T. truncatus. Furthermore, the greatest number of interactions between the three species cited have been observed between D. delphis and S. coeruleoalba [D. delphis and S. coeruleoalba coexist in sympatry in three different areas of the Mediterranean, including the Alborán sea [S. coeruleoalba displayed more opportunistic trophic habits compared with D. delphis’ in the north of Spain (Bay of Biscay) [T. truncatus and D. delphis [D. delphis feeding in the water column or near the surface and T. truncatus focusing on bottom prey’ [According to morphological , 52 and uleoalba –81. In forán sea –81. In a Biscay) . ‘Fissio delphis , 83, dep delphis , 84. A n delphis  in such om prey’ . This haD. delphis with S. coeruleoalba was expected to be higher than with T. truncatus. However, interactions between D. delphis and T. truncatus [T. truncatus and their potentiality to produce hybrids with up to ten different genera of dephinids (D. delphis. This is corroborated in S. coeruleoalba calves.Accordingly, the probability of hybridization of runcatus , 86 are runcatus , ‘the coruncatus , 82, 86 ephinids . All facDespite the uniqueness of this hybridization, DNA samples from the hybrid were not obtained. Considerations were that the rare, but extremely dangerous experiences during cetacean sampling , 88, theThe Bay of Algeciras is a heavily anthropised area, but it serves as a feeding, nursing and breeding ground for cetaceans, including both common and bottlenose dolphins and future hybrids. Enforcement of the cetacean observation protocols and the introduction of an environmental education plan to minimise the impacts on cetaceans in the Bay of Algeciras are vital. In this regard, conservation measures have already been proposed for this hotspot area for cetaceans facing detrimental threats .


Cracks in concrete structures can be indicators of important damage and may significantly affect durability. Their timely identification can be used to ensure structural safety and guide on-time maintenance operations. Structural health monitoring solutions, such as strain gauges and fiber optics systems, have been proposed for the automatic monitoring of such cracks. However, these solutions become economically difficult to deploy when the surface under investigation is very large. This paper proposes to leverage a novel sensing skin for monitoring cracks in concrete structures. This sensing skin is constituted of a flexible electronic termed soft elastomeric capacitor, which detects a change in strain through changes in measured capacitance. The SEC is a low-cost, durable, and robust sensing technology that has previously been studied for the monitoring of fatigue cracks in steel components. In this study, the sensing skin is introduced and preliminary validation results on a small-scale reinforced concrete beam are presented. The technology is verified on a full-scale post-tensioned concrete beam. Results show that the sensing skin is capable of detecting, localizing, and quantifying cracks that formed in both the reinforced and post-tensioned concrete specimens. Cracks that manifest in concrete structures can be caused by a combination of poor construction practices, deleterious chemical reactions such as corrosion and alkali-aggregate reactions, construction overloads, cyclic freezing and thawing damage . Cracks Various evaluation techniques can be leveraged during an inspection to determine the location and extent of cracking, and to evaluate the general condition of the concrete. These methods include visual inspections and nondestructive evaluation techniques such as impact-echo , ultrasoA solution is the implementation of automated monitoring solutions, also known as structural health monitoring (SHM). Conventional SHM approaches to crack monitoring include resistive strain gauges , vibratiNovel surface strain sensing technologies, or sensing skins, with excellent durability, sensitivity, and cost-effectiveness for geometrically large systems, have gained popularity in the research community as an organic step beyond FOS. These include strain sensing sheets based on large area electronics and integrated circuits ,15,16, eThis study aims at extending previous research efforts on the SECs to the monitoring of cracks in concrete. In particular, the performance of SECs at localizing and assessing flexural crack development in concrete infrastructures through strain measurements is evaluated on a small-scale and a full-scale concrete beam using a network of strip-shaped SECs. The remainder of the paper is organized as follows. This section provides a background on the SEC technology, including its fabrication process and electromechanical model, and presents validation results on a small-scale concrete beam.C) can be derived from the parallel plate capacitor equation:d and length l  is transduced into a measurable change in the SEC’s capacitance. An SEC is presented in Equation  can be sm × 5 mm b. The neThe electromechanical response of an end-bonded SEC  maximum relative change in capacitance, taken as the peak-to-peak amplitude in signal for each load step; (2) residual relative change in capacitance, taken as the difference between the maximum reading during a given load step and the capacitance left after unloading; and (3) average relative change in capacitance, taken as the average change in capacitance over a loading step. This paper presented the study of a novel sensing skin for the detection, localization, and quantification of cracks in concrete. The sensing skin, constituted from an array of soft elastomeric capacitors (SECs), is an inexpensive, durable, and robust sensing solution that can be leveraged to measure strain over large surfaces. The strain measurement values are collected in the form of discrete point values among the network. The spatio–temporal comparison of strains enables the detection, localization, and quantification of cracks.The sensing skin was first introduced and validation results preliminarily conducted on small-scale reinforced concrete beams were presented. Initial characterization of a free-standing SEC led to a gauge factor that was used to map electrical signal to strain and thus, cracks. Time series measurements from the SECs and visual observations from crack growth were in agreement. The sensing capability was further studied by deploying a sensor network of 20 SECs onto the surface of a full-scale post-tensioned concrete beam. A bending test with a loading and unloading sequence was conducted until structural failure, and data from the SECs and visual observation of cracks collected. Results demonstrated that data collected from the distributed SEC network correlated with crack-induced damage. The extraction of time series features, among which the maximum relative change in capacitance at each loading step, showed good agreement with the observed normalized crack length.Overall, the SEC network showed capable of detecting, localizing, and quantifying cracks in concrete. The application of dense networks of SECs could provide a cost-effective monitoring solution for real-time, long-term crack monitoring on civil structures. Future research will include the influence of the gauge length on the accuracy of SEC’s final configuration, bonding sensitivity between the concrete and sensing materials, optimal dense sensor network design for multi-crack detection, and crack initialization characterization algorithms.


There was wide aberrant RON and MET expression in the cancer tissues. In 227 pancreatic cancer samples, 33% had RON overexpression, 41% had MET overexpression, and 15.4% had RON and MET co-overexpression. RON and MET expression were highly correlated. RON and MET expression levels were significantly related to OS. Patients with RON and MET co-overexpression had poorer OS. BMS777607 and PHA665752 inhibited pancreatic cancer cell viability and migration, and promoted apoptosis by inhibiting RON and MET phosphorylation and further inhibiting the downstream signaling pathways in vitro. They also inhibited tumor growth and further inhibited phosphorylated (phosphor)-RON and phospho-MET expression in the mouse xenograft models in vivo effectively. INCB28060, which inhibits the MET signaling pathway alone, was not effective. RON and MET can be important indicators of prognosis in pancreatic cancer. Tyrosine kinase inhibitors targeting RON and MET in pancreatic cancer are a novel and potential approach for pancreatic cancer therapy.RON (recepteur d'origine nantais) and MET (hepatocyte growth factor receptor) are tyrosine kinase receptors. Various cancers have aberrant RON and MET expression and activation, which contribute to cancer cell proliferation, invasiveness, and metastasis. Here, we explored RON and MET expression in pancreatic cancer and their relationship with overall survival (OS) time, and evaluated their significance as therapeutic targets of tyrosine kinase inhibitors in pancreatic cancer. We enrolled 227 patients with pancreatic cancer in the study. RON and MET expression was analyzed by immunohistochemical staining. Four human pancreatic cancer cell lines expressing variable levels of RON or MET and four MET superfamily inhibitors  were used. The effect of the four tyrosine kinase inhibitors on cell viability, migration, and apoptosis were determined using cell viability, scratch wound healing, and Caspase-Glo 3/7 assays. Cellular signaling was analyzed by immunoprecipitation and western blotting. The therapeutic efficacy of the tyrosine kinase inhibitors was determined with mouse xenograft pancreatic cancer models  Cancer currently poses a serious threat to human health. Advances in precision medicine have allowed many common cancers to be detected early and treated effectively, reducing mortality. As a leading cancer, pancreatic cancer has very high mortality. The 1-year survival rate of pancreatic cancer is <20%. Due to the lack of early diagnosis and effective treatment, the death rate from pancreatic cancer will become the second leading cause of cancer-related death in the next 20 years . TherefoRON  is a member of the MET proto-oncogene family, which also includes another member, MET  , 5. MET Here, we used a panel of pancreatic cancer cell lines expressing different levels of RON and MET as the model. The cell lines have previously been used to determine the role of RON and MET in regulating pancreatic cancer tumorigenic activity . Here, wWe explored RON and MET expression in pancreatic cancer and their relationship with survival time to investigate whether they can be used as a new prognostic marker in pancreatic cancer. Then, we explored the signaling pathways of aberrant RON and MET expression in pancreatic cancer, and evaluated the significance of RON and MET as therapeutic targets of TKIs targeting RON and/or MET in pancreatic cancer, further providing new means and therapy for treating pancreatic cancer in the future.Human pancreatic cancer cell lines expressing variable levels of RON and MET were selected as the TKI targeting model. The BxPC3 ], AsPC1 ], and Panc1 [KRAS [mut]] cell lines were from American Type Cell Culture  and had been authenticated in 2010 with cytogenesis. The L3.6p1 cell line was provided by G.E. Gallick  . Flow cyWe analyzed 227 patients who had been pathologically diagnosed with pancreatic cancer with or without liver metastases between January 2010 and June 2015 at the Affiliated Hospital, Zhejiang University School of Medicine. All patients underwent pathological biopsy for pancreatic cancer surgery. The clinical parameters included patient demographics, tumor-node-metastasis (TNM) stage, tumor differentiation, tumor size, and treatment modality. Furthermore, 20 patients with benign pancreatic or liver disease, such as pancreatitis and cysts, and hepatolithiasis, were enrolled as a control group. All tissues were fixed in 10% buffered formalin and embedded in paraffin. This study was approved by the Ethics Committee of the First Affiliated Hospital (reference numbers: 2017427-1), Zhejiang University School of Medicine.In human pancreatic cancer tissue, IHC staining was carried out using Zt/f2 (5 μg/mL) as the primary antibody for RON and rabbit anti-MET mAb  for MET, followed by EnVision System reagents  as previously described . The neg4 cells per well in a 96-well plate in triplicate) were incubated in a 5% CO2 atmosphere at 37°C. Cell viability at 24, 48, and 72 h after TKI treatment (0–15 μM) was determined using Cell Counting Kit-8 . BxPC3 cell apoptosis following TKI treatment (0–10 μM) was measured using an ApoLive-Glo Multiplex Assay kit  to detect caspase-3/7 activity in the cells according to the manufacturer's instructions.Pancreatic cancer cells (1 × 105 cells) were seeded in 6-well plates and allowed to grow until 100% confluent. A scratch was made in the plate using a P200 pipette tip after the inhibitors  had been added. According to the CCK-8 experimental results at 24 h  with 2 nM MSP (RON activation) and 2 nM HGF (MET activation), followed by TKIs  at 37°C for 60 min  were mixed with anti-phospho-tyrosine PY-100 (1:100) coupled to protein G Sepharose beads. Proteins were separated in 8% SDS-PAGE under reduced conditions. Phospho-RON or phospho-MET was detected by western blotting using R5029 or ab51067.The DuoSet IC ELISA was used for measuring human phospho-RON in cell lysates according to the manufacturer's instructions. Capture antibody  was coated in a 96-well microplate and incubated overnight at room temperature. Then, cell lysates (30 μg/100 μL/well) were added and incubated for 2 h at room temperature, followed by the addition of diluted anti-phospho-tyrosine PY-100–horseradish peroxidase (HRP) to each well. After adding substrate solution, the absorbance of each well was measured using a microplate reader (Bio-Tek ELx800) at 450 nm wavelength. The phospho-RON levels in the cell lysates were evaluated based on the optical density value.6 BxPC3, AsPC1, L3.6p1, or Panc1 cells in the subcutaneous space of the right flank as previously described (n = 4 per group). Treatment began when all tumors had a mean volume of ~100 mm3. BMS777607, INCB28060, PHA665752, or Tivantinib was administered by gavage at 25, 5, 25, and 20 mg/kg daily per mouse, respectively, and continued for 14 days. Control mice were injected with vehicle (DMSO in PBS). Tumor volume and mouse body weights were recorded every 4 days. The volume (V) of the subcutaneous tumors was calculated as follows: V = (length × width2)/2. The animals were euthanized if the tumors became necrotic or ulcerated through the skin or when tumor volumes were >2,000 mm3 or if the mice bred for >60 days after becoming tumor-burdened. The tumors were harvested for the subsequent experiments.All mouse experiments were approved by the institutional animal care committee (reference numbers: 2017400-1). Female athymic nude mice  were acclimated to the animal housing facility for at least 1 week before the study. Then, the mice were injected with 5 × 10escribed , 38. Thet-test. Results are shown as the mean ± SD. P < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS  and GraphPad 7. The relationship between RON and MET expression and clinicopathological characteristics was compared using the chi-square test. Overall survival (OS) was calculated from the diagnosis of pancreatic cancer until death or the date of the last follow-up. Survival data were analyzed by the Kaplan–Meier method and log rank test. The independent prognostic factors of survival were identified using Cox proportional hazard model analysis. The significance of the experimental and control groups was analyzed using one-way analysis of variance (ANOVA) or the two independent samples A total of 227 patients (156 men and 71 women) with pancreatic cancer were enrolled in the study. All patients were followed up until December 2018, when only 10 patients were confirmed to be still alive. The median age at tumorectomy was 63 years . All patients were diagnosed with infiltrating ductal adenocarcinoma. p < 0.05) and tumor size (p < 0.05), respectively. RON expression level was highly correlated with MET expression (p < 0.01). However, RON and MET expression was not associated with patient age at tumorectomy (<65 vs. ≥65 years), gender, lymph node metastasis, tumor-node-metastasis (TNM) stage, or chemotherapy treatment.RON and MET staining was detected in the cell membrane and cytoplasm in cancerous and non-cancerous cells in patients with pancreatic cancer . The panp < 0.01). Patients with RON overexpression had poorer OS compared with patients with weak/moderate or negative RON expression (p < 0.01) (p < 0.01) (p < 0.01) (p < 0.01), tumor size (p < 0.01), lymph node metastasis (p < 0.05), distant metastasis (p < 0.01), TNM stage (p < 0.01), and chemotherapy treatment (p < 0.05).The role of RON and MET expression in OS was assessed using Kaplan–Meier analysis and the log-rank test . At the  < 0.01) . The cor < 0.01) . Patient < 0.01) . As novep < 0.01) than patients with RON or MET negative expression. Patients with RON expression had much higher RR  than the RON-negative control. The same result was obtained for MET expression . In addition, distant metastasis (p < 0.01), age (p < 0.05), tumor size (p < 0.01), and chemotherapy (p < 0.01) remained independent prognostic factors of poor OS  in BxPC3 cells treated with 1.5 μM PHA665752, 1.5 μM BMS777607, and 0.15 μM tivantinib, with 57.49, 59.12, and 55.07% open area, respectively, compared to the scrambled control vector set with 10.07% open area. AsPC1 cells yielded similar results to that of the BxPC3 cells. Wound healing was also reduced (p < 0.001) in L3.6p1 cells treated with 1.5 μM PHA665752 and 1.5 μM BMS777607, with 28.98 and 32.07% open area, respectively, compared to the 8.26% of the empty vector. In Panc1 cells, only Tivantinib reduced cell migration significantly compared to scrambled control at 24 h (p < 0.01). Compared with the other three inhibitors, INCB28060 had no inhibitory effect on pancreatic cancer cell migration after 24 h  for 60 min at 37°C, and then phospho-RON and phospho-MET expression were analyzed by western blotting. MSP and HGF significantly stimulated RON and MET phosphorylation as compared to the control. BMS777607 and PHA665752 significantly silenced phospho-RON and phospho-MET expression. Moreover, inhibiting phospho-RON and phospho-MET expression significantly diminished both AKT and ERK1/2 phosphorylation, but had no effect on their protein expression. INCB28060 only inhibited phospho-MET expression, and had no effect on the downstream signaling pathway. Therefore, RON and MET signaling is critical in AKT and ERK1/2 activation, and BMS-777607 and PHA665752 might inhibit RON- and MET-mediated tumorigenesis .P < 0.01) compared to the MSP-alone group. These results corroborated BMS777607 and PHA665752 significantly decreasing phospho-RON and phospho-MET expression  into the mammary fat pad caused tumor growth in a time-dependent manner. The pancreatic cancer cells were oncogenic in the nude mice and rapidly caused tumor formation and growth in vivo. Treatment of the mice bearing pancreatic cancer xenografts (~100 mm3) was initiated with repeated injection of 20 mg/kg/day Tivantinib, 5 mg/kg/day INCB28060, 25 mg/kg/day BMS777607, or 25 mg/kg/day PHA665752, with a total of 14 doses for 14 days. In BxPC3 xenografts, Tivantinib, PHA665752, and BMS777607 inhibited BxPC3 cell–mediated tumor growth in a time-dependent manner. Significantly reduced (p < 0.0001) tumor volume was observed after Tivantinib, BMS777607 and PHA665752 treatment at day 20. At day 27, the mice in the control and INCB28060 groups were euthanized when tumor volumes exceeded 2,000 mm3. Compared to the control group, the PHA665752-, BMS777607-, and Tivantinib-treated mice had 52.32, 70.36, and 64.0% reduced tumor growth, respectively . Then, at day 37, 43, and 47, mice in the PHA665752, Tivantinib, and BMS777607 groups were euthanized when tumor volumes exceeded 2,000 mm3. Compared with the control, BMS777607, Tivantinib, and PHA665752 extended the life of the mice by 20, 16, and 10 days, respectively .Inoculating pancreatic cancer cells (5 × 10p < 0.01)  . Compare < 0.01) . And thep < 0.01) was 35.67 and 54.97% in the BMS777607 and Tivantinib groups, respectively (p < 0.01)  . During Analysis of the lysate proteins from BxPC3 cell xenograft tumors confirmed that phospho-RON and phospho-MET expression was significantly diminished in the BMS777607-treated groups. The PHA665752-treated groups had significantly diminished phospho-RON expression and slightly diminished phospho-MET expression. Moreover, inhibiting phospho-RON and phospho-MET expression significantly diminished both AKT and ERK1/2 phosphorylation, but had no effect on their protein expression. In the INCB28060-treated groups, inhibiting only phospho-MET expression had no effect on the downstream signaling pathway .Pancreatic cancer is a digestive system malignancy of global health concern. Angiogenesis, the presence of highly resistant cancer stem cells, and dysregulation of the cell cycle and apoptosis are thought to be critical in pancreatic cancer chemoresistance . Due to in vivo  to explore the function of RON and MET in pancreatic cancer cells and whether RON and MET could serve as new targets for future treatment of pancreatic cancer. BMS777607 and PHA665752, both targeting RON and MET, could inhibit pancreatic cancer cell viability and migration and induce apoptosis effectively , 3. Theyignaling . Some reignaling . Furtherignaling . Here, tIn addition, many molecular targets have been developed in pancreatic cancer. Erlotinib and gefitinib, TKIs in phase III studies, block EGFR  selectively. Trastuzumab and cetuximab are mAbs against the HER2/neu (erb-b2 receptor tyrosine kinase 2) receptor and EGFR, respectively . MoreoveRON and MET belong to a family of tyrosine kinase receptors and have similar structures. Many researchers have investigated the possible cross-talk between RON and MET , 19, 47.Moreover, the positive correlation of RON expression with MET has been reported in prostate cancer, and patients with RON and MET co-expression had the lowest 10-years disease-free survival in node-negative breast cancer , 49. Othin vitro and in vivo experiments. In the future, we will evaluate the potential use of these TKIs targeting RON and MET in combination with gemcitabine. Moreover, further understanding of the cross-talk of these receptors may present more possibilities for treating pancreatic cancer.In the present study, we compared the inhibitory effects of four TKIs on human pancreatic cancer cell lines expressing variable levels of RON and MET. We demonstrate clearly that RON and MET can be new therapeutic targets in pancreatic cancer. RON is more meaningful as a new future therapeutic target than MET. However, RON and MET may interact with many other RTKs. In human cancer, the RTKs RON, MET, and EGFR are frequently co-expressed and promote resistance to targeted therapeutics . TKIs tain vivo and vitro experiment. Increased RON and MET expression by pancreatic cancer cells is a suitable target for anti-RON and anti-MET drugs in future cancer therapy. These findings also provide support for the use of TKIs targeting RON and RON/MET as a novel and potential approach for pancreatic cancer therapy.In conclusion, RON and MET are widely expressed in pancreatic cancer tissues. RON and MET expression are highly correlated with OS in pancreatic cancer. RON and MET may be involved in the malignant process of pancreatic cancer, and can serve as a biomarker for evaluating the prognosis of patients with pancreatic cancer. There is a complex cross-talk between RON and MET. We believe that the MSP–RON signaling pathway, but not the HGF–MET signaling pathway, may be the dominant mechanism in pancreatic cancer. TKIs targeting RON and MET have a better inhibitory effect on pancreatic cancer cell The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.The Ethics Committee of The First Affiliated Hospital, Zhejiang University School of Medicine, approved the present study (reference numbers: 2017427-1 and 2017400-1).We have obtained consent to publish this paper from all the participants.in vitro cellular experiments and data analysis were performed by C-YH, T-HW, T-MT, and Y-ZL. The animal experiments were carried out by C-YH, T-MT, and Y-ZL. The manuscript was drafted by H-PY, M-HW, and C-YH. All authors read and approved the manuscript.Study conceptualization and supervision was carried out by H-PY, X-MX, and M-HW. Sample collection, resources, IHC, and/or clinical characterization were conducted by H-PY, C-YH, X-MX, BH, Z-GW, YQ, and T-HW. The 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.