C be a nonempty closed convex subset of a real Hilbert space f is Lipschitz continuous and strongly monotone, which essentially improves the relevant result in (Luo and Yang in Optim. Lett. 8:1261–1272, C is very complex and the projection operator Let  C of Let C be a nonempty closed convex subset of Let mentclass2pt{minimely, see , 42–46, I is the identity operator on λ is an arbitrary positive constant.It is easy to verify that A class of variant variational inequalities is the inverse variational inequality 34])Ifis a one-to-one correspondence, thenis a solution ofif and only ifis a solution ofAs for the existence and uniqueness of solutions for Lipschitz continuous and strongly monotone inverse variational inequalities, Luo et al.  proved t are  are 6) aThe main iterative algorithms to approximate the inverse variational inequalities  are projThere is also a lot of research on the properties of the inverse variational inequalities. We refer the reader to the papers , 35, 37,Although inverse variational inequalities have a wide range of applications, they have not received enough attention. For example, some fundamental problems, including the existence and uniqueness of solutions, still need further study.f is Lipschitz continuous and strongly monotone. This means that conditions  are  are \doc(i)→ denotes strong convergence;(ii)⇀ denotes weak convergence;(iii)ω-limit set of In this section, we list some concepts and tools that will be used in the proofs of the main results. In the sequel, we use the notations: The next inequality is trivial but in common use.f is said to be (i)monotone if(ii)η-strongly monotone if there exists a constant (iii)L-Lipschitz continuous if there exists a constant (iv)nonexpansive if(v)firmly nonexpansive ifLet It is well known that For the projection operator  andis a real sequence such that(i)(ii)orThenAssume thatis a sequence of nonnegative real numbers such thatwhereis a sequence inis a sequence of nonnegative real numbers, andandare two sequences insuch that(i)(ii)(iii)impliesfor any subsequenceThenRecall that a function u ifu, if it has at least one subgradient at u. The set of subgradients of φ at u is called the subdifferential of φ at u, which is denoted by φ at u. A function φ is called subdifferentiable, if it is subdifferentiable at every φ is differentiable, then its gradient and subgradient coincide.Recall that an element w-lsc) at u if Recall that a function In this section, with the help of Lemma Firstly, applying Lemma LetCbe a nonempty closed convex subset of a real Hilbert spaceand letbe a Lipschitz continuous and strongly monotone operator. Then the variational inequalityhas a unique solution. Furthermore, ifisL-Lipschitz continuous andη-strongly monotone, then for anyis a strict contraction and the sequencegenerated by the gradient projection methodconverges strongly to the unique solution ofwhere the initial guesscan be selected inarbitrarily.Secondly, we show the following two facts.Ifis Lipschitz continuous and strongly monotone, thenis a bijection and thusis a single-valued mapping.f is L-Lipschitz continuous and η-strongly monotone with T. Using Lemma T is a strict contraction and hence T has only one fixed point. Consequently, the equation In order to complete the proof, it suffices to verify that, for any IfisL-Lipschitz continuous andη-strongly monotone, thenisLipschitz continuous andstrongly monotone.f, we haveFor any f is L-Lipschitz continuous, we obtainOn the other hand, noting that LetCbe a nonempty closed convex subset of a real Hilbert spaceand letbe a Lipschitz continuous and strongly monotone operator. Then the inverse variational inequalityhas a unique solution.From Lemma In Theorem not need –6). So,. So,\docC be a nonempty closed convex subset of a real Hilbert space L-Lipschitz continuous and η-strongly monotone operator. Using Theorem Let μ and α such that Set Take For the current SetCalculateWe now establish the strong convergence of Algorithm LetCbe a nonempty closed convex subset of a real Hilbert spaceand letbe anL-Lipschitz continuous andη-strongly monotone operator. Then the two sequencesandgenerated by Algorithm converge strongly to the unique solutionofand the unique solutionofrespectively.First of all, for each Secondly, using Lemma ave from  that16\L̃-Lipschitz continuous, we have from \documentC is very simple and the projection operator Algorithm ukushima  suggesteukushima  and the C be the level set of a convex function, i.e.,c is weakly lower semi-continuous, subdifferentiable on ∂c is a bounded operator . It is worth noting that the subdifferential operator is bounded for a convex function defined on a finite dimensional Hilbert space Take For the current SetCalculateNext theorem establishes the strong convergence of Algorithm LetCbe given by (and letbe anL-Lipschitz continuous andη-strongly monotone operator. Assume thatis weakly lower semi-continuous and subdifferentiable onand∂cis a bounded operator. Suppose that the sequencesatisfies (i) asand (ii) Then the two sequencesandgenerated by Algorithm converge strongly to the unique solutionofand the unique solutionofrespectively.given by , and letM a positive constant, which represents different values in different places. Firstly, we verify that For convenience, we denote by equality  and the milar to , we also∂c is bounded on bounded sets, it follows that there exists a constant w-lsc of c and , a, a\docummilar to , we haved. Using , (28), L0. Using  and the Noting that her with  leads toNext we estimate the convergence rate of Algorithm u and the radius one \document∈C. Thus  follows ∈C. Thus  and 40)\documentBy Theorem Finally we are in a position to estimate the convergence rate of Algorithm Assume that the conditions in Theorem hold and the conditionis satisfied. Then, in the ergodic sense, Algorithm has theconvergence rate ifwithandand has theconvergence rate ifFor des from  and 41)\documenthen from  and 42)\documentThe conclusion can be similarly proved for In this section, in order to show the practicability and effectiveness of Algorithm L, η, α, μ, τ, and τ̃ as in Sect. nth step iteration.In the following two examples, we denote by It is worth noting that for the following two examples, condition  is not sf is 3-Lipschitz continuous and 1-strongly monotone. Hence, Let n and equals 3.3541803 when Except the first steps, the error The numerical results generated by implementing Algorithm f is Let From Fig. In this paper, we give an existence-uniqueness theorem for the inverse variational inequalities, whose conditions are weaker than those of Luo et al. . Based o


Damage in the bonding interface is a major factor that leads to the degradation of macroscopic performance of reinforced concrete (RC) structure because the damage generally results in the debonding or slipping between reinforcement and concrete. Based on hierarchical mesh methodology, a multi-scale finite element (FE) model consisting of coarse aggregate, mortar and steel rebar was established to analyze the failure process of RC structure in this paper. In order to develop the mesoscopic FE model, Monte-Carlo method was used to randomly generate the size and position of coarse aggregates; a criterion of mesh reconstruction was proposed to separate the macroscopic mesh into the mesoscopic mesh and the mesh of transitional zone; the damage constitutive relation model for concrete presenting significant difference of its tensional and compressive properties was adopted to control the damage evolution in concrete when loading; the birth-death element method was used to adaptively reform the multi-scale FE model, and finally macroscopic performance degradation of RC structure was evaluated reasonably. A example of standard RC specimen under unaxial load was performed to verify both the accuracy and efficiency of the developed FE model in analyzing failure mode of RC specimen under unaxial tension and compression. By using the developed multiscale FE model, the destruction process of a four-point bending RC beam was analyzed. The simulation results coincide well with the test results from another literature. From the perspective of mechanics, the degradation of bonding ability in the interface between concrete and steel rebar when loading is caused by the shear stress due to the difference of their material properties. When the shear stress reaches to a critical value, the relative slip between concrete and steel rebar along the anchorage direction is generated in the bonding interface. If the slippage is small, these two materials still can collaborate; while the debonding is generated once the slippage exceeds a certain critical value. Main interactions between concrete and reinforcement in the process of debonding include pullout effect –2, tensiAt the present, research methods for the interfacial bonding performance of the RC structure mainly include the pull-out test –10 and tThe finite element method is a supplement of experimental research and has been used widely because of its convenience in research on reinforced concrete bond behavior. At present, there are two strategies to deal with the interaction between concrete and reinforcement: 1) (continuous mode) an average stress-strain relationship of concrete and reinforcement is developed to modify the constitutive relationship of concrete or reinforcement , 16–17; From the perspective of material damage, the bonding failure of reinforced concrete is a multi-scale process that mesoscopic damage accumulates and then induces macroscopic rupture . SpecifiThe above models and methods focus on the macro mechanical performance of bonding interface of reinforced concrete. They don’t consider the micro/meso-structure of bonding interface, which means that they aren’t able to describe condition and transformation of damage in a microscopic or a mesoscopic level. However, concrete material is a highly heterogeneous material comprised of coarse aggregate, mortar and interfacial transition zone (ITZ) between them . Random In order to overcome the shortcomings of traditional research methods, this paper developed a multiscale simulation method to research debonding process of reinforced concrete. This method presents real meso-structural morphology and random non-uniformities of RC material. First, a multi-scale finite element model based on adaptive mesh refinement algorithm was established, in which the mesoscopic damage evolution in the bonding interface was simulated. Second, the feasibility and accuracy of the developed FE model was verified by test results of concrete specimen under uniaxial tension and compression. Last but not least, the multi-scale model of a four-point bending RC beam was developed to analyze the macroscopic performance degradation and mesoscopic damage evolution process, as well as the influence of mesoscopic damage evolution on macroscopic performance degradation.In order to study the interfacial debonding or slipping of reinforced concrete caused by mesoscopic damage, it is necessary to research the failure process in a mesoscopic level. However, the large volume of concrete structure makes the computation of mesoscopic model inefficient. Therefore, a multi-scale finite element model of the RC structure is developed in this paper, which can simulate the deterioration of the macroscopic mechanical performance of the structure and the mesoscopic damage evolution on the bonding interface at the same time. There are mainly three steps to develop the multi-scale model: 1) developing a mesoscopic model of concrete material with random coarse aggregates, 2) realizing multi-scale modeling of the RC structure by adaptive mesh encryption technology and 3) calculating the damage of each element in every analysis step to simulate damage evolution in the bonding interface.The mesoscopic modeling of bonding interface includes the following steps: 1) determining the location and size of random coarse aggregates, 2) modeling and meshing the bonding interface (2D model) in commercial software (ANASYS in this paper), and 3) assigning constitutive relationships to the mesoscopic elements.At the mesoscopic level, steel could be regarded as a homogeneous material, and thus the model only needs to consider the surface shape of steel rebar. However, concrete is heterogeneous and consist of two components, mortar and coarse aggregate. Therefore, the established mesoscopic model should describe both the surface shape of the steel rebar and the distribution of coarse aggregates in the mortar. The shape and the location of steel rebar could be determined by the design parameters. Meanwhile, according to the gradation or the ratio of concrete, the number, size and position of the coarse aggregates could be generated by Monte Carlo simulation.ri and rj are the radii of the ith and jth aggregate, and k is the amplification factor.The first step in Monte-Carlo process is to determine the maximum size of the grains, and then the coarse aggregates are generated one by one based on random centroid position and diameter within the maximum size range. The coarse aggregate isn’t allowed to locate near the generated coarse aggregates and steel rebar. When total area of the aggregates whose diameters are within the current size range meets the requirement, the coarse aggregates within the next size range are generated sequentially until all the coarse aggregates are generated. The order of the generation process is from the maximum size range to the minimum size range. The schematic diagram of random aggregate generation process is shown in The mix ratio of concrete chosen in this paper is that the coarse aggregate accounts for about 45% of the total volume of concrete, including big grains whose diameters range from 20mm to 40mm and small grains whose diameters range from 5mm to 20mm . The masD is used for the mortar and coarse aggregates.Linear elastic constitutive relationship is used to describe the mechanical property of steel, and the constitutive relationship with the damage variable Es is the elastic module of the steel, which is 210GPa [Steel is modeled with linear constitutive relation, and the stress-strain equation is as follows:s 210GPa  in the dm and a refer to the mortar and coarse aggregates respectively. The constitutive equation is as follows:Em and Ea are the initial elastic modulus of mortar and coarse aggregates respectively. Based on the uniaxial compression test [Em is 22.2GPa and Ea is 75GPa. Ec, the elastic modulus of the concrete, is taken as 31.5GPaThe constitutive relationships of mortar and coarse aggregates are shown in ion test  and the Dm and Da respectively. The damage resulted from the previous loading increment step is introduced into the elastic modulus of the later loading increment step to simulate the damage evolution of the mesoscopic RC structure. DR refers to the damage zone where Dm>0 or Da>0. The mesoscopic damage evolution is characterized by both the extension of damage area and the increase of damage value of mesoscopic mortar elements. The mesoscopic damage evolution conditions will lead to the macroscopic performance deterioration, including the area of damage zone, the direction of damage propagation, the extension rate, etc.The damage variables of mortar and coarse aggregates are denoted as Di under uniaxial tension is expressed as follows:εi−t0 is the tensile strain threshold for damage evolution and εi−tu is the tensile strain limit. The residual tensile strength coefficient λi−t is defined by the relationship: fi−tr = fi−tλi−t, where fi−t is the uniaxial tensile strength and fi−tr is the residual tensile strength after damage initiation.The damage variable Di under uniaxial compression is expressed as follows:εi−c0 is the compressive strain threshold and εi−cu is the compressive strain limit. The residual compressive strength coefficient λi−c is defined by the relationship: fi−cr = fi−cλi−c, where fi−c is the uniaxial compressive strength and fi−cr is the residual compressive strength after damage initiation.The damage variable Because mortar and coarse aggregate are brittle materials, their mechanical properties are similar to that of concrete. Therefore, the strength criterions of mortar and coarse aggregates are also similar to that of concrete under biaxial stress, as shown in σ1>σ2>0, regarding σ1 as the principal stress, Dm and Da could be calculated by Eq , the damage equations of mortar and coarse aggregate under biaxial stress could be obtained from the Eq  and Eq . When σ1ed by Eq . When σ2ed by Eq . When σ1ed by Eq . When k ed by Eq .εi−t0 under uniaxial tension. According to the proportioning scheme of common concrete [Em is 22.2GPa, the corresponding fm−t is 4MPa, and the tensile strain threshold of mortar εm−t0, which represents the ratio of fm−t to Em, is equal to 0.00018. εm−tu is two times as εm−t0 and equals 0.00036. Because the mortar is more brittle under uniaxial tension than under biaxial tension, λm−t could be taken as 0.3. When Ea is 22.2GPa, the corresponding fa−t is 15MPa. The tensile strain threshold of coarse aggregate εa−t0, which represents the ratio of fa−t to Ea, is equal to 0.002. εa−tu is two times as εa−t0 and equals 0.004. Because coarse aggregate is also more brittle under uniaxial tension than under biaxial tension, λa−t could be taken as 0.3.According to the characteristics 2), the maximum tensile strain criterion could be used to determine the tensile strain threshold concrete , when Emεi−c0 under uniaxial compression, which could be written as follows:φi is the friction angle. According to the proportioning scheme of common concrete [Em is 22.2GPa, the corresponding fm−c is 40MPa. The compressive strain threshold of mortar εm−cu is taken as 0.00036, which is ten times of εm−tu. When subjected to uniaxial compression, mortar is not as brittle as subjected to uniaxial tension. Therefore, λm−c and φm could be taken as 0.7 and 30°respectively. When Ea is 75GPa, the corresponding fa−c is 150MPa. The compressive strain threshold of coarse aggregate εa−cu can be taken as 0.004, which is ten times of εa−tu. By the same token, because coarse aggregate is not so brittle under uniaxial compression, λa−c and φa can be taken as 0.7 and 30° respectively.According to the characteristic 3), Mohr-Coulomb criterion could be used to determine the strain threshold concrete , when EmIn order to improve the computational efficiency for large RC structures, refined mesh with small element size is used for the vulnerable parts, and meanwhile coarse mesh with large element size is used for the other parts of the model, i.e., a multi-scale modeling method is applied for large RC structure.The established multi-scale model based on adaptive mesh encryption technology  is shownn increments, and every displacement increment is 0.005mm. The damage state of the element is judged twice in one increment.The flow chart of damage evolution simulation is shown in εc−t0, fm−c and φc in Eq  and the deflection of mid-span is shown in It could be seen that the drawing force increases linearly with the deflection in the initial stage. When reaching 70% of the peak value, the drawing force starts to increase nonlinearly. After reaching the peak value, the drawing force shows a downward trend.M of the portion subjected to pure bending and the mean curvature φ of the beam axis. The relationship between the overall effective stiffness and mid-span deflection is shown in The overall effective stiffness It could be seen that the curve in f is the mid-span deflection, M is the nominal bending moment, B is the initial overall stiffness and D is defined as the macroscopic damage variable of the beam, which could be denoted by Dro−Bmac as:Assume that the mid-span deflection caused by the nominal bending moment in the damaged beam is equal to that caused by an effective bending moment in the undamaged beam, i.e.B, Dmacro−B is equal to zero, which means that there is no damage in the beam. When Dmacro−B is equal to 1 and the beam has been damaged completely. When 0<Dmacro−B<1, the damage is evolving gradually. The relationship between Dmacro−B and mid-span deflection f is shown in When The trend of macroscopic damage evolution is opposite to that of the deterioration process of global effective stiffness, which could be divided into four stages: 1) Plateau period, the macroscopic damage is 0; 2) Concave growth period, the macroscopic damage suddenly increases to 0.1 and then tends to be steady; 3) Steady growth period, macroscopic damage increased steadily to 0.6; 4) Convex growth period, the macroscopic damage rapidly increases to 1, and the failure of the beam occurs.The deterioration process of bending performance of RC beam in section 4.1 is due to the mesoscopic damage evolution. By analyzing the mesoscopic damage states of the beam at different stages of the overall effective stiffness curve, the relationship between mesoscopic damage evolution and macroscopic performance degradation is studied.Presumably, concave descending of the overall stiffness may be due to the failure in pure bending zone of RC beam, while the plateau descending of the overall stiffness is probably due to continuous extension of the damaged zone along the axial of reinforcement.Further research is conducted for the influence of mesoscopic damage evolution on the overall effective stiffness.The following conclusions are obtained in this paper:A multi-scale finite element model for RC structure is established based on the adaptive mesh encryption method, in which the interfacial mesoscopic damage evolution is simulated. At the mesoscopic level, the material is considered as a three-phase heterogeneous material consisting of mortar, coarse aggregates and steel rebar. The distribution of coarse aggregates in the mortar was determined by random Monte-Carlo method. The mesoscopic damage is categorized into tensile damage and compressive-shear damage, of which the damage thresholds are defined by the maximum tensile strain criterion and Mohr-Coulomb criterion respectively. Compared with the traditional simulation method, the proposed multi-scale modeling method considering mesoscopic damage evolution is more suitable in the simulation of the deterioration of macroscopic performance of the actual RC structure.The stress-strain curves and failure modes of concrete under uniaxial tension and uniaxial compression are obtained by the proposed simulation method considering the mesoscopic damage in the bonding interface. The proposed algorithm is verified by comparison with the results given by specification and literature.The multi-scale algorithm developed in this paper is proved to be effective in the simulation of the failure process of a four-point bending RC beam, and the influence of mesoscopic damage of bonding interface on the macroscopic performance deterioration of the RC beam is obtained. Macroscopic performance deterioration and the mesoscopic damage evolution could be divided into four stages. In the first stage, the tensile force increases linearly with the deflection of the beam. In the second stage, the damaged zone appears below the reinforcement in the pure bending portion of the beam, and the tensile force increases nonlinearly. In the third stage, the damaged zone in the pure bending portion extends towards the compression zone, and meanwhile the damaged zone above the reinforcement in the bending-shear portion extends along the steel rebar. The overall effective stiffness of the beam reduces by 50%, and the drawing force reaches the peak value when the damaged zone extends to half of the bending-shear portion. In the fourth stage, the compression-shear damage in the upper bonding interface above the support blocks the coalescence of damaged zone along the steel rebar, and meantime drawing force starts to decrease. The results show that when the bonding strength is relatively low, the failure of the bending-shear portion would be caused by coalescence of the damaged zone along the axial direction, which leads to the significant reduction of macroscopic bending performance of the RC structure.Numerical techniques that replace crack defects with the degradation of material stiffness have been investigated for simulating the fracture behavior of concrete indirectly, owing to the methods’ good compatibility with FEM. One such representative method for concrete material is the damage model, which enables the simulation of 2D or 3D crack propagation behavior including the debonding and slip between concrete and reinforcement. However, concrete is regarded as a homogenous material in most research in this field. For example, in ref –37, cracThe multi-scale numerical simulation method can effectively simulate the mechanical properties of RC structure. This study is only a limited part of it. The following work can be carried out in the future:This paper focuses only on reinforced concrete beams. In order to study the evolution of mesoscopic damage and its influence on the response of the whole structure, it is necessary to further develop the adaptive mesh encryption technology and conduct multi-scale modeling for the complete structure.The model established in this paper is a two-dimensional model. It will be more accurate to study the microscopic damage with three-dimensional solid model. But there are many difficulties, such as: the development of effective three-dimensional aggregate production and placement algorithm because the existed method to judge overlapping of 3D aggregate is inefficient; the development of three-dimensional adaptive algorithms for large scale structures.In fact, the damage accumulation and the extension of damaged zone in the interface zone are closely related to the loading rate and the loading mode. Therefore, it is necessary to study on the meso-damage evolution of the interface zone under the action of earthquake or fatigue load.S1 FileThe data are size and distribution of coarse aggregates. They are generated by Monte Carlo simulation in Matlab software and then are inputted into ANASYS software to produce the mesoscopic model of RC beam in section 4.(TXT)Click here for additional data file.S2 FileThe data are initial data to draw (XLSX)Click here for additional data file.


He was found inappropriate at street via police officers about 12 h after last seen by the family, and according to his family, he addicted. He referred to the clinic, but he was very intoxication and also died the deceased was transferred to the Legal Medicine Organization of Shiraz for a medico-legal autopsy. The autopsy was performed six hours after the body found. Due to the identification performed autopsy for the deceased and viscera specimen  and urine sample sent to the toxicology laboratory, also performed amphetamine test addition of toxicology routine tests . There were no violent wounds found on the body or injection sites. An autopsy from the deceased revealed skin congestion and bleeding and congestion of the conjunctiva and pulmonary edema and congestion observed in both lungs. In the stomach, there were multiple condoms content of white powder . No otheAutopsy performed within gastrointestinal, tube-shaped packages were found in the stomach. Overall, 97 complete packages and some empty plastic open and scattered that is not recoverable were found in the stomach and totally, the weight of them was 1.095 grand one of them for detection of content sent to the toxicology laboratory. Sample of the creamy powder obtained in the package of tube-shape from the autopsy analyzed via high-performance liquid chromatography (HPLC), and finally, heroin and noscapine detected. The cause of death in this victim was a heroin overdose due to rupturing one heroin package in the stomach.In certain cases, an enormous amount of drug released when a packet, heroin well absorbed from the gastrointestinal tract. The autopsy findings, in this case, revealed cyanosis and pulmonary edema and congestion. Heroin and its metabolites were detected in the gastric contents. The estimated quantity of heroin that leaked into the stomach was 3.5 gm.Heroin well absorbed from the gastrointestinal tract. Symptoms and signs of opiate toxicity include nausea, vomiting, constipation, depression of consciousness, respiratory depression, coma, and death –3. Tissu


In this work, we report for the first time on the influence of polyhedral oligomericsilsesquioxanes (POSS) on the structure and properties of liquid crystalline polyurethane (LCPU). LCPU/POSS hybrids were synthesized via a two-step method. In the first step, 4,4′-methylenephenyl diisocyanate (MDI) and polytetramethylene ether glycol (PTMG) reacted with functionalized trisilanolphenyl POSS (TSP-POSS) bearing three hydroxyl groups. In the second step, the growing chain was extended with 4,4′-bis(hydroxyhexoxy)biphenyl (BHHBP). FTIR measurements confirmed the chemical bonding between the POSS and LCPU matrix and showed the influence of the silsesquioxane modification on the intensity of hydrogen bonds. The DSC and POM techniques confirmed the formation of liquid crystalline phases. The incorporation of silsesquixanes into the LC matrix leads to higher melting and isotropization temperatures along with the broadening phase transition effect. Scanning electron microscopy showed a good distribution of POSS moieties, both in the bulk and on the surface of the liquid crystalline PU matrix, whereby wide-angle X-ray diffraction (WAXD) patterns revealed halos from both the liquid crystalline and unmodified polyurethane matrix. The stress at the breaking points for LCPU/POSS hybrids containing 50% and 60% of elastic segments is greater than the stress at the breaking point of the reference material (LCPU), what is due to good dispersion of POSS in less elastic matrix. Thermal properties of the LCPU/POSS materials obtained, determined by TGA, revealed that the char residue increased with the amount of POSS for 40% of elastic segments materials. The synthesis and modification of thermotropic liquid crystalline polymers by applying different types of substrates and additives has been widely studied over the last decade . One of N,N-dimethylformamide (DMF) added only to decrease the viscosity and dissolve the chain extender. As a reactive nanofiller, trisilanolphenyl POSS (TSP-POSS) was chosen, due to its structure containing aromatic rings on the corners of the POSS cage; and it was hypothesized that such a molecule may have an influence on the formation of hydrogen bonds by causing steric hindrance. The single cage diameter of such nanoparticles is ca. 1–3 nm [In our work, we used 4,4′-methylenephenyl diisocyanate (MDI) and polytetramethylene ether glycol (PTMG), and—as a chain extender—BHHBP to form side-chain polyurethanes with liquid crystalline properties. A low molar mass of the polyol (Mw = 650) was selected for better LC properties in the PU matrix . The mes. 1–3 nm . We inve−1 OH, 3029 cm−1 Ar–H, 2923 cm−1 asymmetrical CH2, 2805 cm−1 symmetrical CH2, 1605 cm−1 and 1496 cm−1 Ar, 1440 cm−1 O–CH2, 1270 cm−1 Calif-OH, 1240 cm−1 Calif–O–Caromat, 820 cm−1 Caromat–H—The chemical structure of BHHBP was confirmed by the FT-IR (ATR) spectra. The following characteristic bands were observed: 3260 cmBHHBP creates strong birefringent liquid crystalline textures. Mosaic textures, characteristic for highly-ordered smectic phases, were observed. In the cooling segment 125–120 °C, the smectic phase (Sm2) exhibited mosaic textures. A slight change of the textures appeared in the temperature range 100–90 °C. According to the literature , dendricIn the heating mode, two endothermic phase transitions, characterized by enthalpy changes of 52 and 102 J/g, were observed. The first peak is associated with crystalline-to-smectic phase transition and the second one represents a smectic-to-isotropic change. Moreover, in the cooling mode, two transitions occurred with exothermic enthalpy changes of 104 and 45 J/g. The BHHBP smectic phase appears in both the heating and cooling segments, which confirms the enantiotropic properties—The LCPU/POSS hybrids obtained were characterized by the means of IR analysis—−1 correspond to the stretching vibrations of hydrogen bonded NH groups. As the absorption bands at 3500 cm−1, associated with the stretching vibrations of the free NH groups, did not occur, it may be concluded that all the NH groups participate in the formation of hydrogen bonds. The bands at 2950 cm−1 and 2850 cm−1 correspond to the asymmetric and symmetric stretching vibrations of CH groups, respectively. The POSS load does not affect the aforementioned bands significantly. The bands at 1720–1660 cm−1 are due to the stretching vibrations of urethane carbonyl groups. The bands in the wavenumber range of 1150–1030 cm−1 are associated with the asymmetric stretching vibrations of ether linkages C–O–C that are present in the soft segments derived from the polyol component. The bands observed at 1100 cm−1 are correlated with the stretching vibrations of free ether groups in the soft segment, while the bands at 1040 cm−1 correspond to the ether groups that participate in the formation of hydrogen bonds. Those bands are overlapped with the bands associated with the stretching vibrations of the Si–O–Si groups present in the silsesquioxane cage. The stretching vibrations of Si–O bonds are also represented by the bands observed in the wavenumber range of 610–590 cm−1. As shown in −1 arise from the stretching vibrations of Si–C bonds that are formed between a silicon atom and methyl group in the POSS cage. The highest intensity of this band is observable for the material with the highest POSS content. The bands observed in the spectral range 1260–1230 cm−1 correspond to C=N stretching vibrations. Those bands overlap with the bands associated with the stretching vibrations of CH2OH groups (derived from polyol component and chain extender) that are usually observed at 1250 cm−1. The bands at a wavenumber of 2250 cm−1, originating from the stretching vibrations of isocyanate groups, are absent, which confirms that the isocyanate substrate completely reacted.The bands at 3300 cm−1 correspond to the stretching vibrations of the ordered hydrogen bonded carbonyl groups present in the urethane linkages, while the bands at 1725 cm−1 are associated with the stretching vibrations of the free carbonyl groups [−1 corresponds to the vibration of the disordered carbonyl groups that are hydrogen bonded in the soft phase, which also indicates incomplete phase separation .In the heating mode, all the samples undergo a melting process at different temperature ranges depending on the soft/hard segment ratio and the POSS load. The PU with 40% of elastic segments showed the highest range of thermal transition due to the highest amount of the mesogenic chain extender .  for those samples where the effect of liquid crystalline transition can overlap the effect of crystallization. The influence of POSS on liquid crystalline properties highly depends on hard/soft segments ratio. For 40% and 60% elastic segments, the most appropriate load of POSS is 2 wt%. For equal amounts of segments, the highest value of POSS gives better liquid crystalline properties. All the effects shown in the DSC thermograms can be combined with the FTIR results of the hydrogen bonds formation after the incorporation of POSS to the PU structure. The lower intensity of hydrogen bonds with carbonyl groups may have an impact on liquid crystalline phase’s formation, leading to a higher range of phase transitions.A polarized optical microscope was used to determine the extent of birefringence of the liquid crystalline nanocomposites with respect to temperature. It was observed that the liquid crystalline phase was dispersed uniformly as a polydomain texture and its fraction was decreasing continuously with an increase of temperature—At the first stage, the samples were heated up to 190 °C, above the isotropization and flow temperatures of the elastomers under investigation. The shapes of the materials changed and the recovery of the optical anisotropy of the samples to a large extent ceased after cooling the samples to room temperature. In the second stage, the elastomers were subjected to lower temperatures and it was observed that by avoiding the flow temperatures of the samples, the reversibility of the optical anisotropy was enhanced. The isotropization temperatures determined based on the microscopic measurements were comparable to the end temperatures of the peaks in the DSC heating cycle thermograms. The flow temperatures (Tflow) were determined as temperatures at which the initial stages of the liquefaction of the samples occurred, indicated by substantial smoothing of the surface and the edges of the samples, followed by changes of the shape identified on the microphotographs. The results are summarized in The temperatures at which change of the shape and liquefaction were observed are shown in TGA and DTGA results are presented in WAXD measurements were conducted after annealing the materials at 130 °C for 24 h in order to achieve the liquid crystalline phase transition temperature .The detailed patterns were obtained using the curve-fitting technique based on the Gaussian and Lorentzian assumptions. According to the literature , there aWith the objective to examine the influence of POSS molecules on the mechanical properties of a liquid crystalline matrix, stress at break was measured—The reinforcement properties of TSP-POSS were observed for the samples with 50% and 60% elastic segments. Such an effect is a result of good dispersion of POSS in a less elastic matrix. According to SEM microphotographs , the disIn order to investigate the POSS distribution in the LCPU matrix, SEM microphotographs were taken on the surface and in the bulk of the materials after fracturing—It was observed that POSS nanoparticles are well dispersed on both the surfaces and in the interiors of the samples. Some POSS crystallites were found for the material containing 40% elastic segments, which may be a result of higher rigidity disturbing the homogenous distribution of nanoparticles in the polymer matrix.N,N-dimethylformamide, 4,4′-methylenephenyldiisocyanate, and 1-butanol were acquired from Sigma Aldrich. MDI was stored in an inert gas atmosphere in a freezer. TSP-POSS was produced by Hybrid Plastics and stored at a low temperature. PTMG with a molecular mass of 650 and 1,4-butandiol were dried under vacuum for 24 h at 80 °C.4,4′-dihydroxybiphenyl (BHBP) and 6-chloro-1-hexanol (97%) were purchased in Alfa Aesar and used as received. Anhydrous ethanol, sodium hydroxide, anhydrous 4,4′-bis(hydroxyhexoxy)biphenyl (BHHBP) was synthesized based on the procedure described by Lee et al.  with som−1. The reaction was terminated by adding a mesogenic chain extender  dissolved in anhydrous DMF at 80 °C. All of the syntheses lasted for ca. 75 min. Finally, the reaction mixture was poured into a steel mould and dried for 24 h at 120 °C.Three different types of LCPUR matrices were prepared via a two-step prepolymer method, containing 40%, 50%, or 60% elastic segments. Each PU system was modified with 2, 4, and 6 wt% of TSP-POSS. The reaction path was such that MDI diisocyanate was first melted at 60 °C and then 10 mL of anhydrous DMF was added. In the next step, the polyol and POSS were added to the solution, which was then stirred vigorously, and heated up to 80 °C—−1 from 4000 to 400 cm−1, with an average of 16 scans.Infrared, spectroscopic Fourier transform measurements were performed on a Nicolet iS5 spectrometer  equipped with a diamond crystal attenuated total reflectance unit (ATR). Spectra were measured with a resolution of 8 cmFor DSC measurements, Mettler–Toledo DSC  was applied. The DSC measurements were performed under a heating/cooling rate of 10 K/min, with two heating and two cooling segments in the temperature range 20–220 °C. All the measurement were done under a nitrogen atmosphere using an intracooler as a cooling system.POM observations were performed using a polarized microscope DM2700P  equipped with a heating stage . Samples of submillimeter dimensions were cut and placed on a microscopic cover glass, and heated at a heating range of 10 K/min.A Bruker D Phaser diffractometer  was used for WAXD investigations in reflection mode. A standard CuKα anode with the wavelength λ = 1.54184 Å was applied.Measurements were performed using a TA Instruments TGA 550 Discovery , operating in dynamic mode at a heating rate of 10 K/min from 40 to 600 °C in an atmosphere of nitrogen. The sample mass was ca. 3.5 mg, measured in open Pt crucibles.Stress at break tests were performed based on the ISO 527-2 type 5A and ISO 37 type 2 standards. Samples were prepared in a form of thin films in a shape of “paddles.” Measurements were conducted using a Brookfield CT3 texture analyzer .Scanning electron microscopy (SEM) micrographs were taken using a JEOL InTouchScope JSM-6010LV  microscope with energy-dispersive X-ray analysis (EDS) capabilities, operated at 10 kV accelerating voltage.New organic—inorganic hybrid materials based on liquid crystalline polyurethanes, modified by polyhedral oligomeric silsequioxanes, were synthesized using BHHBP, which exhibits smectic liquid crystalline phases as a chain extender. The effect of POSS on liquid crystalline properties was investigated by the DSC technique and it was found that incorporation of rigid silsesquioxanes moieties with aromatic rings results in a higher phase transition temperature and a broader range of LC phenomena. The best mesomorphic properties were displayed by polyurethane with 40% elastic segments, which could lead to conclusions that this ratio of soft and rigid segments–liquid crystalline phase formation is favored. Higher POSS loads cause a separation of two DSC peaks into three, with a lower enthalpy change effect that can be explained by the existence of two mesophases of different thermodynamic stabilities in the LCPU. FTIR measurements in the absorption range of carbonyl groups showed lower intensities for the peaks responsible for hydrogen bonds, which might be a result of incomplete phase separation due to steric hindrance of the aromatic POSS structure. Incorporation of TSP–POSS does not lead to the formation of agglomerates, which was proven by scanning electron microscopy and WAXD measurements. X-ray diffraction patterns showed a shift of the main amorphous halo to lower theta angles as a result of the crosslinking properties of TSP–POSS. The nanoadditive in low amounts facilitates the nucleation and formation of higher ordered crystalline structures, as evidenced by the value of the degree of crystallinity of PU (40% elastic segments) containing 2% of silsesquioxane. Stress at break measurements revealed that the reinforcement effects of TSP–POSS were observable for polyurethane materials containing 50% and 60% elastic segments, in which a fine distribution of nanoparticles was observed by the SEM technique. On the other hand, incorporation of POSS moieties in PU matrix with 40% elastic segments lead to a lower stress at break due to an increase of the degree of crystallinity. Thermogravimetric analysis results show that the char residue increases with the amount of POSS for 40% elastic segments materials; this observation can indicate a material’s reduced flammability through the formation of a protective layer on the sample’s surface, which limits oxidation processes during burning.


The JNK expression was higher in tumor tissue compared with the corresponding non-tumor tissue sample in 17.4% patients (p < 0.01). The inhibition of aberrant c-MET expression in human CC cell lines was achieved by blocking the phosphorylation of c-MET with Tivantinib. Notable losses in cell viability and colony-forming capability were detected (p < 0.01). Synergistic activation of the JNK/c-jun pathway was demonstrated after Tivantinib treatment. Knockdown of the JNK by siRNA or competitive binding of c-MET receptor by stimulation with HGF-antagonized anti-tumor effects of Tivantinib was observed. Our data suggest that inhibition of c-MET could be a possible alternative approach for the treatment of human CC, for which Tivantinib may an effective inhibitor. The synergistic activation of the JNK/c-jun pathway contributed to the elevated apoptosis in CC cells via treatment with Tivantinib.Clinical treatment options for human cholangiocarcinoma (CC) are limited. c-MET, a high-affinity receptor for hepatocyte growth factor (HGF), is deregulated in many cancers. Its role in cholangiocarcinogenesis remains unclear. In current study, 23 corresponding tumor- and non-tumor tissues, taken from patients with intrahepatic (iCC) and perihilar cholangiocarcinoma (pCC), who underwent liver resection, were analyzed. The relationship of clinicopathological features and c-MET, as well as c-jun N-terminal kinase (JNK) was evaluated. The anti-tumor effects of Tivantinib, a small-molecule inhibitor with potent activity against the c-MET kinase, was investigated in three human CC cell lines, namely HUCC-T1, TFK-1, and EGI-1. In comparison with the results obtained in non-tumor tissue samples, c-MET was overexpressed in 91.3 % of tumor tissues ( Most patients are diagnosed at an advanced stage, for which radical surgical resection is not feasible. The combination of Gemcitabine and Cisplatin is the only first-line palliative treatment for those patients and has limited benefits8.Untreated cholangiocarcinoma (CC) is one of most invasive malignancies with high mortality16. c-MET activates multiple downstream signaling pathways such as the phosphtidyl inositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, the mitogen activated protein kinase (MAPK) pathway, and the STAT pathway, and is also involved in cell proliferation, differentiation, survival, mortality, and movement19. The aberrant expression of c-MET was recently considered as a potential target and biomarker in malignant tumors22. Although overexpression of c-MET has been described in patients with CC and in a mouse xenograft CC model, the precise function of c-MET signaling in cholangiocarcinogenesis still remains unclear24. The aim of this study was to explore the expression of c-MET in corresponding non-tumor and tumor tissues from CC patients, and its relationship with numerous clinicopathological factors. Tivantinib, a small-molecule kinase inhibitor with potent activity against c-MET, was investigated as an alternative therapeutic approach for CC in vitro.The pro-tumorigenic function of c-MET, a high-affinity receptor of the hepatocyte growth factor (HGF), has a critical role in many solid tumors, including human CCs26. After three washes in phosphate-buffered saline (PBS), the tissue sections were incubated with 100 μL of secondary antibody in a dark, humid chamber at room temperature for 1 h. Finally, 100 μL of 4′,6-diamidino-2-phenylindole (DAPI) solution  was introduced into each tissue area for 10 min before being counterstained with Mayer’s hematoxylin for 10 s, dehydrated in ethanol, and mounted. Immunohistochemistry was examined using a Zeiss Axiovert 40 CFl microscope. This study was carried out with the patients’ informed consent and approval from the local ethics committee. The approval number is 159/2002 and followed the guidelines stated in the Declaration of Helsinki.Twenty-three corresponding tumor- and non-tumor tissues were collected from patients with intrahepatic (iCC) and perihilar CC (pCC), who underwent liver resection. Clinicopathological characteristics of the patients are shown in Table 2 at 37 °C. Tivantinib , Apitolisib , and Refametinib  were dissolved in 100% dimethyl sulfoxideand stored at −20 °C. Recombinant human HGF was purchased from Sigma-Aldrich, Munich, Germany.HUCC-T1, TFK-1, and EGI-1 , German Collection of Microorganisms and Cell Cultures ) were used for in vitro experiments. All cell lines were cultivated in RPMI-1640 medium supplemented with 10 % fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin in 5 % CO3/well) and were treated with increasing concentrations of Tivantinib or a combination of Apitolisib and Refametinib. To investigate the effect of Tivantinib or the combined treatments on cell viability at different concentrations, the cells were kept in culture for 1, 2, 3, and 6 days. At the time points 1, 2, and 3 days, 20 μL of MTT -2,5-diphenyltetrazolium bromide, 5 mg/mL, Sigma-Aldrich, Munich, Germany) solution was added to each well and incubated for another 4 h, after which the medium was carefully discarded. Formazan (MTT metabolic product) was resuspended in 200 μL of 2-propanol . The plate was placed on a shaker at a speed of 550 r.c.f. for 30 min. Then, the optical density was read at 570 nm using a Biochrom Anthos 2010 microplate reader . The cell viability after 6 days of treatment with Tivantinib was evaluated using a resazurin-based PrestoBlue reagent . As metabolically active cells are capable of reducing the PrestoBlue reagent, the colorimetric changes can be used as an indicator to quantify cell viability in cultures. Cell viability was measured according to the manufacturer’s instructions. Briefly, the PrestoBlue solution (22 µL) was added into each well after 2 h of incubation for the 6-day treatment condition, plates were then placed back into the incubator for a further 2 h of incubation, after which absorbance was measured at 570 nm for excitation and 600 nm for emission.Cells were seeded onto 96-well plates at various cell densities to avoid overgrowth . The number of colonies, defined as > 50 cells/colony, were counted. Triplicate wells were set up for each condition under investigation.5 to 2 × 105) were seeded in six-well plates and treated after being left to incubate overnight. Fluorescence-activated cell sorting  was performed to detect the sub-G1 cell fraction. This was done to determine apoptosis and the respective phases of the cell cycle after propidium iodide (PI) staining as previously described27. In addition, apoptosis was assessed morphologically using Hoechst 33342 staining and fluorescence microscopy.Cells  was used to dilute the primary antibodies and as a negative control. After five washes in PBS, the cells were incubated with the fluorochrome-conjugated secondary antibody at 37 °C for 45 min before being washed again with PBS. Nuclear staining was achieved with DAPI . Finally, cover slides were mounted in a fluorescence mounting medium obtained from Dako.® SAPK/JNK siRNA II  and SignalSilence® c-jun siRNA II . HuCC-T1 and EGI-1 cell lines were transfected with siRNA using Lipofectamine® 2000 Transfection Reagent  as per the supplier’s protocol. Transfection efficiency was evaluated after 24, 48, and 72 h using western blotting. The apoptotic effect of Tivantinib on human CC lines after siRNA transfection was detected using MTT assay and FACS.For c-jun N-terminal protein kinase (JNK) and c-jun knockdown in human CC cell lines, the following pre-designed and pre-validated small interfering RNAs (siRNAs) were purchased from Cell Signaling Technology: SignalSilenceCells were lysed using RIPA buffer  for 10 min on ice, followed by centrifugation at 16,110 × g for 15 min at 4 °C. The supernatant was collected and the protein concentration was determined using a BCA™ Protein Assay Kit . Whole cell extracts (20 μg) were heated with LDS sample buffer  at 70 °C for 10 min, separated using SDS-polyacrylamide gel electrophoresisin 4–12 % Bis-Tris gel , and transferred to the nitrocellulose membrane . After blocking for 1 h, the membrane was incubated overnight with primary antibodies at 4 °C. Subsequently, the membrane was probed using horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The bands were visualized using a West PICO chemiluminescent substrate  and photographed using an image acquisition system .The following antibodies were used: Actin, p-Met, c-MET, BCL-XL, FADD, p-Bad, bim, cleaved caspase-8, cleaved caspase-3, Phospho-p44/42 MAPK (Erk1/2), p44/42 MAPK (Erk1/2), p-c-jun, p-AKT, AKT, p-Raf, c-Raf Phospho-MEK1/2, MEK1/2 ; caspase-8, JNK1, and p-JNK ; CK19, c-jun, and p-c-jun ; c-MET neutralizing antibody ; and the secondary antibodies for goat anti-rabbit and goat anti-mouse .50, t-test, and one-way analysis of variance were calculated using IBM SPSS Statistics 24.0. The figures were plotted using GraphPad Prism 6.0. The values obtained are the mean (± SEM) of three replicates. Statistical difference was considered significant when p ≤ 0.05.The ICp < 0.01). In each TNM stage or tumor histological grade, there were more patients who had a greater expression of c-MET in the tumor tissue than in the non-tumor tissue , TFK-1(200.2 nM), and EGI-1(151.5 nM)  Fig. . This manM) Fig. . The celnM) Fig. .Fig. 2Bl29. Induction of apoptosis was observable at Tivantinib concentrations of 500 and 5000 nM. In addition, cells showed apoptotic features, such as DNA fragments, after 72 h of incubation during DAPI staining  (Table p > 0.05).High expression of JNK1 was detected in tumor tissue samples compared to corresponding non-tumor tissue in 17.4% of patients 4/23, 1) Table . There w3, 1 Tabl35. In the present study, c-MET overexpression was found in CC tumor tissues compared with non-tumor tissues. It is hypothesized that c-MET might be a promising therapeutic target in CC treatment.c-MET overexpression is well reported in multiple carcinomas22. In the present study, Tivantinib, a non-selective Met inhibitor that stabilizes its non-phosphorylated inactive conformation, was used to inhibit the c-MET expression in vitro37. However, there are conflicting clinical trial results. In some cases, Tivantinib failed two phase III studies involving second-line treatment of Met-high, advanced, hepatocellular carcinoma (METIV-HCC/JET-HCC), despite its success during phase II studies. The reasons for this might be multifactorial and discussion centers around trial design, inadequacies in the selection of patients according to tumor c-MET status, unclear definition for primary c-MET expression as biomarker for treatment, the inevitable upregulation of c-MET, which had been exposed during first-line treatments such as Sorafenib, and differences in implementation of Tivantinib during studies42. A recent systematic review and meta-analysis on the efficacy and safety of Tivantinib for the treatment of solid tumors concluded that Tivantinib prolonged the progression-free survival, but not the overall survival in patients43. This was similar to the results of a phase III study involving epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer44. So far, there is limited information about Met inhibition in CC and no evidence on the efficacy of Tivantinib in CC.A robust pipeline of high-quality inhibitors targeting different aspects of c-MET activation is currently being investigated in phase I, II, and III clinical trials across multiple tumor types. Preliminary data demonstrate promising clinical activity of these agents, along with an acceptable toxicity profile45.The study demonstrated that targeting c-MET using Tivantinib led to a dose- and time-dependent decrease in the growth, viability, and colony formation in human CC cell lines. Compared with TFK-1, a cell line with low baseline levels of c-MET expression, HuCC-T1 and EGI-1, both of which had relatively high baseline levels of c-MET expression, were found to be more sensitive to Tivantinib. This indicated that Tivantinib inhibited the activation of c-MET signaling and is also involved in the progression of CC, as it effectively inhibits both the full and the phosphorylated form of c-MET. Further, based on recent evidence on its apoptosis-inducing ability, Tivantinib was shown to cause cleavage of Procaspase-3 and -8 followed by the FADD. Cleavage of Caspase-8 was a hallmark of extrinsic apoptotic pathway activation, whereas cleavage of Caspase-3 represented the intrinsic apoptosis involved19. Different downstream targets of the c-MET signaling pathway were analyzed to investigate Tivantinib’s pro-apoptotic mechanisms. This is in contrast to previous studies with c-MET inhibitors48 in which high expression of p-AKT, AKT, p-raf, c-raf, p-ERK, and ERK after 24 h of exposure to Tivantinib was observed and confirmed in CC cell lines, the pancreatic cancer cell line Capan-1, and the hepatocellular carcinoma cell line Huh7. However, in previous studies with c-MET inhibitors, combination with the Pi3k inhibitor Apitolisib or the MEK inhibitor Refametinib did not affect the anti-tumor efficacy of Tivantinib. This indicated that activation of c-MET downstream pathways, the Pi3k/Akt/mTOR pathway, and the MAPK/ERK pathway seemed to have a marginal role in triggering apoptosis. As both the intrinsic and extrinsic apoptotic pathways were activated in CC cells during Tivantinib treatment, cell apoptosis related to pathways were assessed. Data indicated that Tivantinib activated the JNK/c-jun pathway and induced CC cells apoptosis. The data on JNK was also confirmed by blocking c-MET receptor with neutralizing antibody. Apoptosis was reduced via a decrease in JNK through siRNA transfection or competitive binding of the c-MET receptor by HGF, but not via the silencing of c-jun. The JNK/c-jun pathway could be activated using multiple factors and c-jun activation could be independent or dependent on this pathway50. Present results indicated that the JNK/c-jun pathway might be involved in the synergistic apoptotic effect exhibited by Tivantinib; among others, it was JNK, rather than its downstream protein c-jun, which had a role in Tivantinib-induced apoptosis. Tivantinib might antagonize the effect of c-MET down-mediated expression of JNK to proceed the apoptotic pathway, the effect of c-jun might not be key role during this process. This was in agreement with previous studies in which c-jun was not essential for apoptosis52. In contrast, the JNK is a subfamily of the MAPK superfamily, which specifically phosphorylates the transcription factor c-Jun on its N-terminal transactivation domain at two serine residues Ser63 and Ser7353. JNK is a key regulator in many cellular events. Apoptosis mediated by the JNK/c-jun pathway was influenced by the cell type, the duration of stimulation, or its upstream and downstream signal pathways. The JNK/c-jun pathway could activate or inhibit apoptosis54. The present study confirmed that when exposed to Tivantinib, activated JNK was responsible for caspase-3 activation, followed by the downstream proteins p-c jun, bim, and p-Bad.c-MET engagement participates in multiple transduction pathways41. Here, it was demonstrated that Tivantinib also activated JNK and its pathway. JNK, in addition to MET overexpression, could be a potential predictor of Tivantinib efficacy in CC. Therefore, the expression of JNK1 in non-tumor and the corresponding tumor region from patients with CC was detected. There is evidence that JNKs, especially JNK1, had a critical role in death receptor-initiated extrinsic and mitochondrial intrinsic apoptotic pathways57. Based on this, it could be hypothesized that CC arising from epithelial origin may already lose the ability to quickly renew epithelial through JNK-mediated cell-programmed death due to tumorous immortality and heterogenesis. In this study, JNK1 was overexpressed in non-tumor tissues compared with the corresponding tumor tissues. There was no difference in the expression of JNK regarding each TNM stage and tumor histological grade. This illustrated that JNK, similar to c-MET, may participate in each developmental stage of cholangiocarcinogenesis and could be a possible alternative predictor for Tivantinib treatment. However, to prove this hypothesis, additional research is needed to define the optimal selection strategy for patients based on the JNK status combined with/without c-MET expression for Tivantinib treatment in CC. Moreover, further investigations are also warranted to demonstrate the relationships in the JNK and c-MET pathway, as well as other possible pathways involved in regulating cell apoptosis in Tivantinib treatment.In previous clinical trials, high c-MET expression was described as an incidental biomarker of tumor sensitivity to the anti-tumor activity of TivantinibIn summary, inhibition of c-MET could be a possible alternative approach for the treatment of human CC, for which Tivantinib may an effective inhibitor. The synergistic activation of the JNK/c-jun pathway contributed to elevated apoptosis in CC cells through the treatment with Tivantinib. The data presented indicate great therapeutic potential for CC treatment with c-MET inhibition.Supplemental Figure 1Supplemental Figure 2Supplemental Figure 3


IntroductionThe emergence and continuous spread of drug resistant bacteria has become one of the leading health concerns globally. Persistent failure to develop and/or discover new antibiotics along with irrational use of existing antibiotics is associated with rise in antibiotic resistance. There is poor understanding of antibiotics usage and their preciousness among the masses which result in careless utilization and hence, the emerging antibiotic resistance. The aim of this study is to evaluate the knowledge and attitude of Pakistanis towards antimicrobial resistance (AMR).MethodsThis observational cross-sectional survey was designed in the form of an online pro forma circulated in January 2019. It was a self-structured pro forma which included age, gender, and 10 questions - five to assess the knowledge and five to assess the attitude towards AMR. Each question was to be responded with a “Yes” or a “No.” For knowledge assessing questions “do not know” was also an option. Data was entered and analysed using SPSS version 22 .ResultsOf 1,132 participants, 837 (73.9%) thought that it was alright to stop antibiotics course whenever they felt better, 505 (44.6%) thought that frequent and unnecessary use of antibiotics actually decrease their effectiveness, and 208 (18.4%) participants thought it was correct to take antibiotics for cold and influenza. There were 157 (13.9%) participants who had not followed the duration of treatment as given in their doctor’s prescription, 49 (4.4%) who had changed their antibiotic dose without doctor consultation, 467 (41.3%) who had reused leftover antibiotics from their previous prescription, 700 (61.8%) who had suggested it to their doctors to prescribe them antibiotics and 378 (33.4%) participants who had purchased antibiotics without any prescription in the last one year.ConclusionPakistani individuals are not as knowledgeable about antibiotic resistance as severe the issue is in this region. Their attitude towards utilization of antibiotics is not very promising. It becomes essential to initiate antibiotic stewardship programs and educate the masses regarding efficacious and safe use of antibiotics in this region. Antibioibiotics . Howeveribiotics .Another major reason for antibiotic resistance is overuse and misuse. Although, medical literature has always strongly advised cautious use of antibiotics, they still remain highly over-prescribed all across the globe. Evidence from various studies has stated that treatment indications, agent choice, and antibiotic therapy duration were not medically correct in 30%-50% of the cases , 5. Irrard of all oral antibiotics used globally are taken without prescriptions and are used for incorrect indications such as tuberculosis, malaria, and for childhood viral infections [Patients and the general population contribute to another important factor leading to antibiotic resistance - self-medication . Around fections .In a study conducted with physicians, it was seen that 20% of their antibiotic prescriptions were influenced by their patients' choice and demand . This isThis observational cross-sectional survey was designed in the form of an online pro forma circulated via social media in January 2019. It was a self-structured pro forma which included age, gender, and 10 questions - five to assess knowledge and five to assess attitude. Each question was to be responded with a “Yes” or a “No.” For knowledge assessing questions “do not know” was also an option. Since it was a self-structured pro forma, its internal reliability was first calculated through a pilot survey (n = 20). Data was entered and analysed using SPSS version 22 . Internal reliability deduced via Cronbach alpha was 0.79. Frequency and percentages were calculated for categorical variables including questions for assessing knowledge and attitude. Mean and standard deviation (SD) were calculated for continuous variable of age.One thousand one hundred and thirty-two pro formas were completed. There were 612 (54.06%) men and 520 (45.94%) women. The mean age of all participants was 35.84 ± 11.09 years.The responses to knowledge about antibiotics resistance are shown in Figure The attitude of the participants towards antibiotics resistance was assessed by five questions as shown in Figure Irresponsible and inappropriate prescriptions of antibiotics are among the major reasons contributing to the rising antimicrobial resistance. Almost one-third of the participants in this study took antibiotics without prescription. A study published in 2016 stated that over the counter sales of antibiotics is one of the major reasons for higher prevalence of resistance in the developing countries . InapproThe public health sector in Pakistan has recognized antibiotic resistance as its major health concern. In 2017, National Antimicrobial Resistance Action Plan was created for Pakistan. Its major strategic priority is “Development and implementation of a national awareness raising and behavioral change strategy on antimicrobial resistance [The urgent need for implementing antibiotic stewardship programs in Pakistan has been highlighted previously . HoweverPakistani individuals are not as knowledgeable about antibiotic resistance as severe the issue is in this region. Their attitude towards utilization of antibiotics is not very promising. The rapidly rising trend in antibiotic resistance is becoming a public health concern in Pakistan. It has become essential to initiate antibiotic stewardship programs and educate the masses regarding efficacious and safe use of antibiotics in this region.


Objectives:Understanding how phenotypic traits vary has been a longstanding goal of evolutionary biologists. When examining antibiotic-resistance in bacteria, it is generally understood that the minimum inhibitory concentration (MIC) has minimal variation specific to each bacterial strain-antibiotic combination. However, there is a less studied resistance trait, the mutant prevention concentration (MPC), which measures the MIC of the most resistant sub-population. Whether and how MPC varies has been poorly understood. Here, we ask a simple, yet important question: How much does the MPC vary, within a single strain-antibiotic association? Using a Staphylococcus species and five antibiotics from five different antibiotic classes—ciprofloxacin, doxycycline, gentamicin, nitrofurantoin, and oxacillin—we examined the frequency of resistance for a wide range of concentrations per antibiotic, and measured the repeatability of the MPC, the lowest amount of antibiotic that would ensure no surviving cells in a 1010 population of bacteria.Results: We found a wide variation within the MPC and distributions that were rarely normal. When antibiotic resistance evolved, the distribution of the MPC changed, with all distributions becoming wider and some multi-modal.Conclusion: Unlike the MIC, there is high variability in the MPC for a given bacterial strain-antibiotic combination. The increase in antibiotic-resistant bacteria is globally an urgent public health issue . The min10 cells . The MPC is defined as the MIC of the least-susceptible, single-step mutant . This is10 cells . This co10 cells . The con10 cells .While the MIC for each bacterial antibiotic-strain pair is typically considered a single value with high repeatability , it is uStaphylococcus aureus , was our ancestral strain and grown overnight in Luria Broth (LB) media , and then frozen with 25% glycerol at -80°C. Several hundred aliquots were made from the master tube and also kept frozen with 25% glycerol at -80°C. S. epidermidis (ATCC 14990) was evolved to each of five antibiotics: ciprofloxacin, doxycycline, gentamicin, nitrofurantoin, and oxacillin. We obtained and purified one independent spontaneously resistant mutant for each antibiotic, resulting in five resistant strains. For all resistant strains collected, we confirmed resistance by streak-purifying colonies onto agar plates containing antibiotic concentrations above the known MIC. For all experiments described here, we used freshly thawed aliquots of the ancestral strain and the resistant strains. Each replicate experiment required one aliquot. Strains were grown (aerated) in LB media for approximately 8 h at 37°C to a density of roughly 109 cells per ml and serially diluted to approximately 105 cells per mL for MIC determination on agar plates.A master tube of We used five antibiotics: Ciprofloxacin hydrochloride (CPR) , Doxycycline hyclate (DOX) (Sigma-Aldrich D9891), Gentamycin sulfate salt (GEN) (Sigma-Aldrich G1264), Nitrofurantoin (NTR) (Sigma-Aldrich N7878), and Oxacillin sodium salt (OX) (Sigma-Aldrich 28221). Ciprofloxacin, a synthetic second-generation fluoroquinolone, inhibits DNA synthesis by inhibiting bacterial enzymes DNA gyrase and topoisomerase, which are involved in the unwinding and supercoiling of DNA during DNA replication . Doxycyc3–104 cells were inoculated in each well with 100 μl LB and allowed to grow for 22 h, shaken at 220 revolutions per minute (rpm) and incubated at 37°C (Tecan Infinite M200 PRO Multimode Microplate Reader). The liquid MIC estimate was determined by the lowest antibiotic concentration observed to inhibit growth by at least 95%, compared to the positive control. We also included negative controls on each 96 well-plate to validate no contamination of media.MIC estimates in liquid culture were determined using microtiter plates with serial and equidistant dilutions of antibiotics. Approximately 105 cells, resulting in a CFU population that has a limited probability of spontaneous mutation . We inoculated each plate using 10mutation . These cmutation , which i10 cells on LB plates containing dilutions of antibiotic . We resuspended and combined all bacterial pellets in 7.5 mL of the original supernatant to give 1010 cells. We used liquid MIC estimates to plan the incremental concentrations used in MPC experiments. We performed two preliminary MPC experiments with concentrations ranging from 1 × liquid MIC estimate to 64 × liquid MIC estimate, increasing by a factor of two. We repeated MPC experiments 20 times, with three replicates per antibiotic concentration. To measure MPC, we plated at least 1010 bacterial cells on agar plates and spread the inoculum via the Copacabana method  and Kolmogorov-Smirnov test (p < 0.001)). The ancestral strains did have a mix of distributions; doxycycline and nitrofurantoin both failed to reject the null hypothesis of a Shapiro-Wilk test (p > 0.05). We also demonstrate using a two-sample Kolmogorov–Smirnov test, that the MPC distributions change as resistance evolves. In all direct comparisons of ancestral and resistant strains (with the same antibiotic) the distributions of the MPC values are different (p < 0.001).The distribution of most MPCs do not appear normal . All of We also found that the MSW changed when resistance is evolved . There iOur results show a range of MPCs in replicate experiments, indicating a large role for stochasticity and limited repeatability for this trait. In this study, the MPC trait is not easily predictable. This variation in MPCs is in contrast to MICs, which are generally predictable for each bacterial strain-antibiotic combination within a particular laboratory setting. For example, although variation in the MIC among different labs has been shown as a result of variations in strains as well as assay variations, individual studies within labs show consistency in the determination of the MIC . Thus, wThe change in the MPC is large enough to account for the change of distribution and variation within the resistant strain as there is little to no overlap in the inter-quartile range (IQR). This supports the idea that although the MPC distribution is large and somewhat unpredictable, we can be confident that the MPC of a resistant strain is higher than an ancestral strain.Our results here suggest two potentially relevant clinical notes. First, it has been proposed that if clinicians target MPCs, there can be no resistant bacteria left in a population within an individual patient . While tin vitro do not match the resistance mechanisms that would be found in a clinical setting (There has been some contention as to the utility of MPCs when the resistance mechanisms evaluated  setting . In this setting . The dis setting .10 cells, an inoculum size similar to the number of bacterial cells found in naturally-occurring bacterial infections (It is known that the MIC fluctuates with inoculum size, with smaller inocula leading to lower MIC estimates . Even whfections . TestingLuria-Delbruck fluctuations, defined as fluctuations in the frequency of spontaneous mutations in microbial populations , may affIn summary, we find that even in highly controlled laboratory environments, MPCs vary widely, not only from differences in strain and antibiotic, but from replicates with the same strain and same antibiotic. Several other factors may also affect MPC variation, such as CFU concentrations, mutation type, and inocula size and in the future, these factors should be investigated. Understanding how and why the MPC varies can allow us to lay the foundations for more comprehensive dosing strategies that take into consideration the presence and elimination of single-step resistant mutants. From a clinical perspective, caution should be taken when determining how reliable certain therapeutic treatments will be in terms of completely eliminating resistant mutants. From an evolutionary perspective, we show the significant role of stochasticity in bacteria evolving antibiotic resistance.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.PY conceived of the presented idea and planned the experiments. CG, NL, CH, PK, AB, AV, LS, GB, MW, EC, LF, and JN carried out the experiments. NL analyzed the data. CG, NL, and PY discussed and contributed to the interpretation of results. CG, NL, CH, MW, and PY contributed to the final version of the manuscript. PY supervised the project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Neoadjuvant chemoradiation or chemotherapy has improved the treatment efficacy of patients with resectable, borderline resectable, and locally advanced pancreatic ductal adenocarcinoma (PDAC). Due to the optimal regimen remains inconclusive, we aimed to compare these treatments in terms of margin negative (R0) resection rate and overall survival (OS) with Bayesian analysis.We reviewed literature titles and abstracts comparing three treatment strategies  in PubMed, Embase, Cochrane Library, the American Society of Clinical Oncology and ClinicalTrials.gov database from 2009 to 2018 to estimate relative odds ratios (ORs) for margin negative (R0) resection rate and hazard ratios (HRs) for overall survival (OS) in all include trials.p < 0.001) and there was no significant difference in R0 resection rate . However, in the network meta-analysis from all enrolled clinical trials, neoadjuvant chemoradiation showed significantly higher R0 resection rate over upfront surgery , whereas neoadjuvant chemotherapy did not provide better efficacy in R0 resection over upfront surgery . For R0 resection rate, neoadjuvant chemoradiation has the highest probability of ranking one compared with neoadjuvant chemotherapy or upfront surgery (79% vs 21% vs 0%). For OS, neoadjuvant chemotherapy has the highest probability of ranking one compared with neoadjuvant chemoradiation or upfront surgery (98% vs 0% vs 2%). Neoadjuvant chemotherapy was associated with higher rates of postoperative complications (rank worst: 84%), followed by neoadjuvant chemoradiotherapy (13%) and upfront surgery (3%).A total of 14 literatures with 1056 patients were enrolled in this Bayesian analysis. In the pairwise meta-analysis from limited head-to-head studies, compared with neoadjuvant chemotherapy, neoadjuvant chemoradiation showed superior OS significantly (HR 0.8, 95% CI 0.60–0.99, Different neoadjuvant treatment was selected based on various purposes, whether increasing R0 resection rate or not. Future clinical trials comparing neoadjuvant chemoradiation with neoadjuvant chemotherapy are warranted to confirm our results. Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers in the world . PrognosSince 2009, local pancreatic ductal adenocarcinoma has been broadly classified into three categories: resectable, borderline resectable and locally advanced disease based on vascular involvement assessed by preoperative imaging in the expert consensus , 5. In gIt is becoming more obvious that patients with BRPC, who are at a higher risk for R1 resection, are potentially in need of neoadjuvant therapy with the goal of improving overall survival (OS) , 8. AccoThe studies mentioned above addressed the effectiveness and safety of neoadjuvant therapies involving neoadjuvant chemotherapy, or chemoradiation compared with upfront surgery in patients with local PDAC. However, they did not focus on the advantage between neoadjuvant chemoradiation and neoadjuvant chemotherapy, and there were very limited data available for head to head research comparing the main neoadjuvant regimens. Furthermore, to the best of our knowledge, no literature has reported a comprehensive comparison of three methods (neoadjuvant chemoradiation vs neoadjuvant chemotherapy vs upfront surgery). This study compared these methods for the treatment of resectable, borderline resectable, and locally advanced PDAC comprehensively by applying a network meta-analysis, with an expectation to provide some reference for selecting appropriate treatment.The Bayesian analysis was conducted and reported in accordance with the PRISMA  guidelines. We searched PubMed, Embase (Ovid), Cochrane Library, the American Society of Clinical Oncology and ClinicalTrials.gov database using “neoadjuvant therapies of pancreatic ductal adenocarcinoma” as part of the titles and abstracts with English restrictions. A time-frame from January 1, 2009, which was the date of introducing local anatomic subcategories of pancreatic ductal adenocarcinoma, i.e., RPC, BRPC and LAPC , 5, to DReferences were included if: 1. local PDAC; 2. compare two of the three treatment strategies  with each other; 3. report enough information to calculate hazard ratios (HRs); 4. unrestricted age, gender, performance status (PS), ethnicity and country.The references were excluded according to the following criteria: 1. neoadjuvant targeted therapy; 2. neoadjuvant chemoradiation and chemotherapy mixed; 3. posters and abstracts; 4. single-arm studies.The search strategy in strict accordance with Population Intervention Comparison Outcomes Study (PICOS) design framework included the following domains of Medical Subject Heading (MeSH) terms: ‘Pancreatic Neoplasms’ and ‘Neoadjuvant Therapy’; MeSH and Subheadings were combined with ‘AND’ or ‘OR’.First, the titles and abstracts of articles were screened. Review articles, case series, case reports, guidelines and conference abstracts were excluded from our study; and then full-text articles that met the inclusion criteria were thoroughly reviewed. Two investigators  reviewed the full manuscripts independently and extracted information including patient characteristics, period and type of study, treatment protocols, the sample size and outcomes  into the electronic database. Disagreements in study and data selection among investigators were resolved by discussion and consensus. The quality and risk of bias of randomized controlled trials (RCTs) were assessed by using Cochrane Collaboration’s tool , and theP < 0.05 was considered as significant level. Heterogeneity was assessed with the I2 statistic. I2 values less than 25% and greater than 50% were regarded as indicating low and high heterogeneity, respectively [http://www.getdata-graph-digitizer.com). We applied the traditional pairwise meta-analysis between direct comparisons with Stata13 . The network meta-analysis was conducted with GeMTC version 0.14.3 (http://drugis.org/software/addis1/gemtc) and WinBUGS version1.4.3  by fixed-effect models. For R0 resection rate, we used GeMTC for network meta-analysis. Parameters for the GeMTC software were selected as: number of chains, 4; tuning iterations, 20,000; simulation iterations, 50,000; thinning interval, 10; inference samples, 10,000; and variance scaling factor, 2.5. Besides, we chose 5000 burn-ins and a thinning interval of 1 for each chain for HRs. The consistency model would be used when there was no significant inconsistency; otherwise, the inconsistency model was applied. We assessed the convergence of the model using the potential scale reduction factor (PSRF) of the Brooks–Gelman–Rubin method [The outcomes we analysed were median OS and R0 resection rate. R0 was defined as margin negative if tumour cells were present > 1 mm from the any surface. R1 was defined as margin positive if tumour cells were present within 1 mm from the any surface , 15. Resectively . When HRectively , and Kapn method ; PSRF clWe identified 1089 studies from the title and abstract review to start with  were in the range of 1.00 to 1.01, so the model was proven convergent and stable; the consistency model was adopted. According to the established network, based on the consistency model, neoadjuvant chemoradiation showed significantly higher R0 resection rate over upfront surgery , whereas neoadjuvant chemotherapy did not provide better efficacy in R0 resection over upfront surgery  Fig. a. ComparThe rank probabilities, calculated by the network consistency model, ranked the probabilities of rank order of treatment regimen overall outcomes evaluated . Regimens with a higher value in the histogram were associated with higher probabilities for better treatment outcomes. The probability distribution of each regimen which was ranked at each of the possible positions was showed by the histogram Fig. a-b. For The comparison of postoperative complications among different treatment strategies was carried out. None of the five studies provided data on postoperative complications. The rankings of the three competitive treatment strategies were summarised in terms of postoperative complications Fig. . RegimenOur study compared neoadjuvant chemoradiation, neoadjuvant chemotherapy and upfront surgery for local PDAC including both R0 resection rate and OS by using pairwise meta-analysis and network meta-analysis. In our pairwise meta-analysis, there was no significant difference between neoadjuvant chemoradiation and neoadjuvant chemotherapy in R0 resection rate, with only three studies reporting the data. However, in this network analysis, our results suggested that neoadjuvant chemoradiation provided R0 resection rate advantage over upfront surgery in indirect evidence and had the highest probability of ranking one compared with neoadjuvant chemotherapy or upfront surgery. In general, neoadjuvant chemoradiation might be the most popular choice of treatment for local PDAC, especially for BRPC and LAPC.In our study, neoadjuvant chemoradiation provided an OS advantage over neoadjuvant chemotherapy suggested by the pairwise meta-analysis, but not that of network meta-analysis likely because the enrolled patients could not be subgrouped, such as RPC, BRPC and LAPC. Zhan et al. proved that neoadjuvant therapy would be beneficial for patients with BRPC and LAPC in the meta-analysis of prospective studies, but neoadjuvant chemoradiation did not achieve a better prognosis than neoadjuvant chemotherapy alone (median OS: 16.7 months vs 16.8 months) . As moreInterestingly, Schorn et al. addressed that neoadjuvant therapy reduced the risk of local recurrence  but not the risk of distant metastasis  in BRPC and LAPC in the meta-analysis of observational studies . As the Our study also had several limitations. First, although there was no obvious inconsistency and severe risk of bias detected, our study only enrolled three RCTs with a relatively small number of participants that might weaken the evidence of our meta-analysis. Second, given the dissimilar disease of participants with RPC, BRPC and LAPC, further subgroup analysis was not conducted. Lastly, different (neo) adjuvant chemotherapy regimens might had an effect on the OS.In conclusion, according to the comprehensive evaluation suggested by Bayesian analysis, neoadjuvant chemoradiation provided better R0 resection rate advantage compared with neoadjuvant chemotherapy and upfront surgery, and neoadjuvant chemotherapy improved OS for local PDAC. Selection of different neoadjuvant therapy will reply on treatment purpose of whether need increase R0 resection rate or not. Additional head to head clinical data that further define the long-term efficacy of neoadjuvant chemoradiation and chemotherapy are needed to confirm our results.


We report a case of eosinophilic granulomatosis with polyangiitis (EGPA) complicated with a IgG4 related disease like symptoms presenting as eyelid swellings. In the present case, the serum level of IgG4 and the ratio of IgG4 to IgG were generally increased by the disease course of EGPA. Considering the course of clinical symptoms, there is a possibility that orbital manifestations were one of the clinical features during the disease course of EGPA while the histological features of right eyelid tissue and other ocular manifestations were consistent with the diagnosis of IgG4 related disease. Eosinophilic granulomatosis with polyangiitis (EGPA) is characterized by severe asthma, blood and tissue eosinophilia, and antineutrophilic-cytoplasm antibody (ANCA) targeting neutrophil myeloperoxidase in only a subset of patients. EGPA is classified among small-vessel vasculitides associated with ANCAs and the hypereosinophilic syndrome and the pathological findings are consisting of necrotizing vasculitis, eosinophilic infiltration, and extravascular granuloma formation. IgG4 related disease (IgG4-RD) is a fibro-inflammatory condition characterized by sclerosing lesions in extranodal sites, lymphadenopathy, and elevated serum IgG4 . Histopaβ2 agonist, theophylline, leukotriene receptor antagonist, and anti-IgE monoclonal antibody. However, he often suffered from asthma attacks. One year later, laboratory data revealed hypereosinophilia (blood eosinophil count was 1584 per μL) and serum concentration of myeloperoxidase anti-neutrophil antibody (MPO-ANCA) was elevated at the level of 102 U per mL. For treatment of asthma symptom, he was treated with oral prednisone 30 mg per day from June 2012. We observed an improvement of the asthma control with a rapid decrease of serum concentration of MPO-ANCA at the level of 15.9 U per mL on October 2012. The prednisolone was tapered, and he was on 10 mg per day of prednisolone from May 2013. On August 2013, the patient presented the discomfort of bilateral eyelids and papillary swelling of upper eyelids was observed . A skin biopsy was performed and histology showed perivascular eosinophilic infiltration and deposition of multinucleated giant cells and degenerated eosinophils. The symptom of both legs was caused to the peripheral nerve involvement by mononeuritis multiplex. He was diagnosed as EGPA and treated with steroid pulse therapy. After treatment, the symptom was improved, and blood eosinophil count was decreased to 330 per μL. In this case, serum IgG and IgG4 levels were measured from the first visit  had any ophthalmologic manifestations at diagnosis and 2 (0.7%) had orbital inflammatory disease . In theiIn a recent report, the possibility of interaction between IgG4-RD and allergic disorders has been advocated , 10. In


The spinning of concrete is a process in which concrete mixture is moulded and compacted under the action of the centrifugal force arising during the fast rotational motion of the mould around its longitudinal axis. As a result of the spinning of the liquid concrete mixture, an element annular in cross section, characterised by an inhomogeneous layered wall structure, is produced. The heavier constituents tend towards the cross-section wall’s outer side, while the lighter components tend towards its inner side. The way in which the particular constituents are distributed in the element’s cross section is of key importance for the macro properties of the manufactured product. This paper presents procedures for investigating spun concrete and interpreting the results of such investigations, which make it possible to characterise the microstructure of the concrete. Three investigative methods were used to assess the distribution of the constituents in the cross section of the element: micro-computed tomography (µCT), 2D imaging  and nanoindentation. A procedure for interpreting and analysing the results is proposed. The procedure enables one to quantitatively characterise the following features of the microstructure of spun concrete: the mechanical parameters of the mortar, the aggregate content, the pore content, the cement paste content, the aggregate grading and the size (dimensions) of the pores. Special attention is devoted to the determination of the variation of the analysed quantities in the cross section of the element. The result of the application of the investigative procedures is presented for an exemplary spun concrete element. The proposed procedures constitute a valuable tool for evaluating the process of manufacturing spun concrete elements. The main aim of this study was to assess the capabilities of three different investigative methods, namely nanoindentation, micro-computed tomography (µCT) and 2D optical scanning, as applied to evaluate selected properties of the microstructure of spun concrete. In particular, it was attempted to use these methods to describe the internal microstructure of concrete, e.g. local strength parameters, pore space morphology, spatial distribution of aggregate and cement paste. The performance of utilised methods was demonstrated on the example of spun concrete samples. This type of concrete has an internal structure different from the commonly used cast in place concrete. Due to the production process, the spun concrete is characterised by a layered structure across the wall of the annular cross section. Therefore, the parameters describing the internal microstructure vary within the cross-section. Proposed methodology for determining aforementioned parameters can be a practical tool helpful, for example, in the development of the optimal technology for the production of spun concrete elements.The spinning of concrete is a process in which a concrete mixture is shaped and compacted under the action of a normal  force arising during the fast (500–700 rpm) rotational motion of the mould around its longitudinal axis. Because of its peculiarities, this method can be used to produce exclusively hollow elements. As a result of spinning, the concrete mixture introduced into the mould consolidates and retains the acquired shape as the process continues. The moulding pressure produced by spinning is not uniform across the layer being consolidated. The moulding pressure on the inner layer of the product wall is close to zero, while, on its outer side, it reaches a maximum value. A graph of the pressure across the product wall can be presented in the form of a triangle.The generated normal pressure is initially taken by the water present between the particles of the concrete mixture. As a result, the hydrostatic pressure increases and under this pressure the water begins to displace  from one layer to another. At the same time, the concrete mixture consolidates. The displacing water takes the finest concrete mixture constituents (mainly cement) with it. In the course of this process, the cohesion of the mixture increases and the mechanical bond between the mixture constituents acquires some strength and in certain conditions a state of equilibrium is reached, whereby the filtration stops. Since at this moment the moulding pressure is fully transferred to the solid particles of the already considerably compacted concrete mixture, the ultimate effect of the compaction will depend on the composition of the concrete mixture (to a large extent on the aggregate grading).Under such a distribution of pressure during spinning, water is not carried away from the cross-section wall thickness equally . Consequently, the mixture layers closer to the mould axis begin to consolidate only after the limit density of the outer layer is reached while the moulding pressure increases. This means that, unlike in the case of vibrated concrete, the structure of spun concrete across the element wall is inhomogeneous and layered. The structure is characterised by the fact that the heavier constituents (large particles) tend towards the cross section’s outer side, while the lighter components (cement paste) tend towards its inner side. As a result, the outer layer can become highly compact and after the concrete sets it can acquire high strength and resistance to chemical and mechanical impacts. The inner layer will consist of highly consolidated cohesive cement paste and after setting it can become highly impervious and resistant to the impact of flowing water. According to Ref. , the 2–4The deformation and strength characteristics of the layered structure of spun concrete have been the subject of only a few studies. Mainly macroscopic investigations of spun concrete are carried out. Marquardt was one of the first researchers  who descAchverdov, investigating the cross section of a spun concrete pipe, found  that theDilger, Ghali and Krishna Mohan Rao, when investigating spun concrete poles ,4, found3), plasticiser amount (0–2%), rotational speed (400–700 rpm) and spinning time (5–11 min) on the structure of spun concrete. They conducted tests on 230 mm high annular spun concrete samples with the inside diameter of 45–60 mm. The test results confirm the conclusions drawn in Ref. )The investigative methodology comprising nanoindentation, micro-computer tomography (µCT) and two-dimensional optical scanning seems to be useful for evaluating selected features of the internal microstructure of spun concrete. The methodology allows one to gain an insight into the interior of concrete and it yields qualitatively and quantitatively valuable results. The developed procedures for investigating the internal structure of spun concrete can be successfully used, e.g., to evaluate the process of manufacturing spun concrete elements, by verifying the porosity of the produced material, the fractionation of the aggregate, and the cement content, taking into account the variation of the parameters across the wall.Taken together, the main contribution of the work to the field is the development of a procedure for the quantitative description of selected microstructure parameters. It should be emphasised that special attention was paid to the evaluation of the variability of these parameters in the cross section of the element. In the future perspective, the developed procedure will serve as a base for upcoming studies planned by the authors. For instance, the procedure allows for a comparative assessment of elements manufactured under different technological regimes. Thus, the methodology proposed in the article may prove to be a useful tool in the optimisation of manufacturing process concerning, i.e. selection of time and speed of mould spinning. Moreover, the procedure can be utilised in the analysis of the spun concrete microstructure changes along the traction pole, for which the diameter decreases towards its top.