Exposure to AO promotes the formation of a silicon-rich coating layer on the surface of the laminate, which in turn reduces roughness and increases stiffness, as evidenced by measurements of flexural properties and spectral data after exposure.The preparation of ultra-thin CFRP laminates, which incorporate a cycloaliphatic epoxy resin reinforced with polyhedral oligomeric silsesquioxane (POSS) reagent nanofiller, using out-of-autoclave procedure is reported. The influence of the amount of POSS within the laminate on the mechanical properties and surface roughness of the laminates is analysed before and after exposure to atomic oxygen (AO) to simulate the effects of low Earth orbit (LEO). The addition of 5 wt% POSS to the base epoxy leads to an increase in both flexural strength and modulus, but these values begin to fall as the POSS content rises, possibly due to issues with agglomeration. The addition of POSS offers improved resistance against AO degradation with the laminates containing 20 wt% POSS demonstrating the lowest erosion yield (1.67 × 10 The adoption of carbon fibre reinforced plastics (CFRP) is increasingly widespread in different engineering sectors such as aerospace, wind turbines, construction, and automotive, due to the high strength-to-mass ratio that they offer . ConsequOwing to their multi-functionality, deployable structures have become more effective in the application of booms, antennas, parabolic reflectors, mirrors, solar sails, masts, and deployable solar arrays . ExampleHowever, a significant concern for composites design is the cost of the manufacturing process, the sourcing of raw materials, and the labour cost associated with the layup. Current costs of production for CFRP-based materials for aerospace applications are typically in the range of £6.38/kg, some twenty times higher than the cost of steel (£0.30/kg) or four times higher than the cost of aluminium £1.36/kg) [.36/kg 1. It may Atomic oxygen (AO) is typically found in LEO at 160 to 2000 km, the most populated altitude range above the Earth. AO is formed due to the photodissociation of diatomic oxygen molecules from the Earth’s residual atmosphere into high energy radicals by the action of UV radiation from the Sun. AO is the predominant cause of erosion in satellite structures in LEO  and poly1:5 empirical formula, where R may be a hydrogen atom, an organic residue, or a functional group, and which have a nanoscopic diameter of approximately 1–3 nm [g), reduction in moisture absorption, and mechanical response. In this work, an octafunctional POSS reagent (EP0409 glycidyl POSS) was selected with glycidyl ether (epoxy) groups at the apices. A diagram of the structural formula of glycidyl POSS is shown in The use of polyhedral oligomeric silsesquioxane (POSS) reagents is increasingly considered as a substitute to silicon-based protective coatings. POSS molecules are cage-like structures with a RSiOy 1–3 nm . These ny 1–3 nm . The POSy 1–3 nm ,20,21 hag values and high thermal stability  cured with 4,4′-methylenebis and 4,4′-methylenebis(2-isopropyl-6-methylaniline)). Nevertheless, these materials with a high aromatic content are extremely susceptible to degradation when exposed to VUV radiation in the LEO environment. In contrast, the chemical structure of cycloaliphatic epoxy resins incorporates fully saturated carbocyclic rings which offer exceptional ultraviolet blocking effects for VUV stability. While cycloaliphatic epoxies do exist for use in space applications , these are more expensive than the more commonly used diglycidylether- or tetraglycidylamine-based monomers. The ECC monomer is a low viscosity material (400 mPas at 25 °C) but is very brittle when cured, thus requiring toughening for engineering use. The cycloaliphatic epoxy resin system featured in this work has previously been studied by Suliga [CFRP structures often incorporate aerospace-qualified epoxy resins for spacecraft structures due to their high Ty Suliga  and is ry Suliga , but theTM H manufactured by Dupont was used as a commercial baseline comparison with the laminates.The CY184 epoxy resin (EEW 144–172 g/eq) and Aradur 2954 cycloaliphatic amine curing agent were purchased from Huntsman Advanced Materials ; the EP0409 glycidyl POSS was purchased from Hybrid Plastics , and the MTM441 was supplied as prepreg by Solvay Group . All samples were used as received without further purification. Moreover, for the development of the ultra-Sthin laminates, a carbon fibre braid provided by A&P Technology Inc.  was used, as well as unidirectional carbon fibre in tape presentation provided by Oxeon  . KaptonTBoth laminates were laid up in a similar fashion, using identical fibre orientations/ply distribution, but with the important distinction that while the MTM44-1 was supplied as a prepreg and could be laid up directly, the cycloaliphatic epoxy system was formed from liquid components and required pre-polymerisation to achieve a low molecular weight, free-standing, integral film.The method applied to develop ultra-thin laminates was based on the use of a thin metallic plate used to wrap a carbon fibre braid sleeve around to achieve samples of approximate dimensions 80 mm × 100 mm. The carbon fibre braid sleeve was covered by a sheet of resin prepreg and cut into two separate layers so that each layer had a MTM44-1 prepreg sheet on only one side. Several tapes of unidirectional (UD) fibres equal to the same area as that of the carbon fibre braid were attached next to each other. Both sides of this UD layer were stacked with two prepreg resin sheets , left.All the layers were stacked carefully by peeling the backing paper of the prepreg sheets and placing them in the correct order with the braid fibres being placed perpendicular to the UD fibres.1), Aradur 2954 (2), EP0409 Glycidyl POSS (3)) were mixed to form a liquid resin (see The constituents of the resin system (CY 184 (x2y3z) is used where the subscripts denote the content of each component. Initial attempts were made to achieve a film based on a prepreg system similar to MTM44-1 but, owing to the fluidity of the system, it was proposed to use MTM44-1 to set the internal layup configuration and apply the designed resin system only to the two external layers, as these will be directly in contact with a LEO radiation environment. Therefore, a similar layup configuration was followed as displayed in Samples with different POSS contents  were considered in this study and the convention (1The baseline MTM44-1 resin system was processed using an out-of-autoclave (OOA) moulding procedure by curing the samples inside an oven with a low-pressure vacuum bag . Being a3/min. The glass transition temperatures were determined by calculating the midpoint of the curve inflexion during cooling and rescan experiments.Differential scanning calorimetry (DSC) experiments were performed using a TA DSC Q2000. Hermetically sealed Tzero aluminium pans were used, with sample masses of 9.1 ± 0.2 mg. Samples were equilibrated at 30 °C and then heated to 300 °C at a heating rate of 10 °C/min, unless otherwise stated, with the sample cell kept under a constant nitrogen flow of 50 cm−1 at room temperature and co-added to produce the final spectrum.Surface spectroscopy was measured using Fourier-transform infrared (FTIR) spectroscopy in the Attenuated Total Reflectance (ATR) mode on a Perkin Elmer Fourier-transform infrared (FTIR) spectrometer. A total of 15 scans were performed per sample over a spectral range of 650–4000 cmSurface topography of the cured polymer and laminate samples was characterised using an Alicona Infinite Focus instrument and Automation Manager surface analysis and imaging software. Surface roughness distribution data and 3D images of the sample surfaces were obtained for scanned areas of 2 mm × 2 mm using a 5× objective. The average surface roughness was calculated from a minimum of 1640 measurement points.2 flow of 0.3 NL/min to simulate space conditions. Each sample from each batch was weighed before exposure and then after increments of exposure to AO, the mass losses were determined.The AO exposure test was carried out using a JLS Designs Plasmatherm 550–570, radio frequency plasma generator . Four samples from each specimen were placed within the chamber, on an aluminium panel and the following parameters were applied: frequency of 150 W, with a constant pressure of 100 Pa and a constant OTM H polyimide sample using Equation (1) [F is the AO fluence (atom/cm2), ΔkM is the mass loss of KaptonTM H film (g), kρ is the density of KaptonTM H (g/cm3), kA is the area of KaptonTM H film (cm2), and kE is the erosion yield of KaptonTM H (cm3/atom), which is a constant (3 × 1024 cm3/atom) measured in the LEO environment [yE is the erosion yield of tested sample (cm3/atom), ΔM is the mass loss of tested sample (g), ρ is the density of tested sample (g/cm3), A is the sample surface area (cm2) and F is the AO fluence (atom/cm2) calculated using Equation (1).AO fluence was calculated based on the mass loss of a reference Kaptontion (1) :(1)F=∆Mironment . Once th21 atom/cm2 for one year of exposure [The samples were assigned to six different batches, one sample of each specimen  and exposed to different time intervals  to analyse a total of 12 months of exposure in space conditions. The cumulative AO fluence received in each exposure run and its equivalent duration in orbit is shown in exposure .E is the effective flexural modulus (GPa), P is the force (N), L is the support span (mm), δ is the deflection of the centreline of the specimen at the middle of the support span (mm), b is the width of specimen tested (mm), and t is the thickness of beam tested (mm).Each sample was cut into two or three specimens 52 mm × 12.5 mm) both in the direction of the UD plies and perpendicular to them. Three-point bend tests were conducted using a Shimazu mechanical test machine with a 1 kN load cell, following ASTM D7264 standard . Zweben .5 mm botEI is the flexural rigidity (Nmm2), P is the force (N), L is the support span (mm), and δ is the deflection of the centreline of the specimen at the middle of the support span (mm).However, the magnitude of flexural modulus is affected by the thickness of the laminate sample and, as this parameter displayed some variation (0.46–0.55 mm), the flexural rigidity  was used1)/amine (2) blend was not available as a prepreg and an alternative means of introducing the matrix into the lay-up needed to be found. Initial experiments involved mixing the reactants thoroughly by means of an ultrasonic bath followed by spreading the resultant resin system blend over a solid surface coated with a Frekote release agent using a doctor blade tool to ensure the desired constant thickness of 100 µm. The resultant thin resin film would then be left to dry before it could be retrieved as a self-sustaining film, which could be peeled from the solid surface and transferred directly to the laminate layup. The free-standing film was found to have a cured Tg of 71 °C using DSC.Unlike the commercial MTM44-1 system, the cycloaliphatic epoxy (3) and the spectra are presented as a function of POSS content . It was noted that the compaction plate used to apply pressure to the laminate samples during the oven vacuum bag cure did not completely cover them. This effect may have caused a differential in the force to which the samples were subjected and, combined with any spring-back behaviour associated with other residual stresses arising from the cure process, may have resulted in the slight curvature observed following cure. FTIR spectral analysis of the epoxy blends was performed to confirm the incorporation of the POSS reagent ( content . Selectene cages . As the 5025030) and both surfaces of the sample containing 5 wt% POSS (14324335) were slightly smoother than the remaining samples. When the four samples were being manufactured in batches, film samples were stored in a fridge to arrest polymerisation. However, it is known [Minimising the surface roughness of the laminates is a key step towards optimising their performance, as the probability of a damaging reaction with energetic photons and AO present in the LEO environment increases with the surface area of the exposed material . Additiois known  that eveThis might explain the slight differences in cure state and why one side of the 0% POSS samples appeared to have a significantly flatter surface with fewer imperfections and the 5% POSS resin had smoother surfaces than the other two POSS-integrated samples. Laminates incorporating resins with a higher viscosity are also more susceptible to the formation of voids and resin accumulations on the surface as it is more difficult for the resin to penetrate the carbon fibre layers and fill any voids below the surface . GlycidyThe bottom surface of the laminate sample containing 0% POSS displayed a significantly smoother surface compared to those of the nanocomposites . AdditioThe surface roughness distribution of the scanned surfaces also exhibits significantly different amplitudes for the different samples, e.g., as the two surfaces of the 0% POSS sample have the same amount of POSS and the resin systems of the other two surfaces were spread over the carbon fibres at the same time, these data therefore appear to indicate that the two theories which were previously discussed for the dissimilarities in the amount of surface imperfections combined could justify the differences in the surface uniformity of the samples. While determining the surface topography of the samples, it was found that the depth of the surface imperfections for the blends containing 10 wt% and 20 wt% POSS was too large for the Alicona instrument to obtain an accurate representation of the surfaces, with these being represented as gaps in the surface by the program. Consequently, it was only possible to obtain reliable data for the surface roughness of the epoxy samples containing 0 wt% and 5 wt% POSS.11 direction, parallel to the UD fibres or the E22 direction, perpendicular to them.The tangent modulus data obtained for the virgin, cured ultra-thin laminates using 3-point bend tests are shown in The data used to obtain the flexural modulus of each sample are displayed in 11 and E22 values. The POSS cages have a higher modulus than the polymer chains and these rigid structures can form bonds with the organic molecular chains of the epoxy resin matrix and restrict their movement, thus improving their flexural properties [Initially, the stiffest of the matrices is the unmodified base epoxy and, at low concentrations of 5%, the addition of the POSS nanofiller results in an increase in the flexural modulus of the specimens followed by a decrease in this property as the POSS concentration is further increased; this is true for both the Eoperties . It is ioperties . Consequ11 axis, which is aligned with the UD fibres, indicating that the flexural properties of the laminates are more dependent on the orientation of the UD fibres than that of the braid fibres. A study presented by Swanson et al. [For all the samples, the average tangent modulus is significantly larger along the En et al.  found thEach sample was weighed before and after every exposure to keep track of any mass changes during exposure and these data are shown in TM H) was included to provide a means of calibrating the response, since the behaviour of this material is well documented, and this was used to calculate the AO fluence. The data show that the base amine-cured cycloaliphatic epoxy, containing no POSS, (15025030) lost 4.1 % of its mass over the same 12-months of simulated exposure, while increasing amounts of POSS reduced the amounts of mass lost, with the blend containing 20 wt% POSS displaying the lowest mass loss (2.5 %) after the equivalent of 12 months of AO exposure.A polyimide witness sample (KaptonTwo kinds of AO are involved in the degradation mechanisms: the ground state (3P) and electronically excited (1D) form . The groy) was also carried out, but the trends are hard to discern , but for completeness the data are supplied in the 5025030) appears to display an erosion yield (Ey) that fluctuates  around Ey = 4 × 10-24 cm3/atom over the equivalent of 12 months of LEO exposure. This compares well with Ey = 3.9 × 10-24 cm3/atom [20 atom/cm2, while the latter figure was recorded using a stronger plasma/VUV source. Similarly, Banks et al. [-24 cm3/atom for an aromatic epoxy EP. The blends containing POSS all initially showed similar behaviour: the erosion yields were initially high and proportionate to the POSS content, but the Ey values appear to rise again, and this is particularly marked in the case of the 14824835 blend, perhaps indicating further damage to the surface. More detailed work is necessary to explore this.Determination of the erosion yields . According to data acquired in-orbit during NASA’s Materials International Space Station Experiments (MISSE) programme [The result of AO exposure on the mechanical  properties of the laminates is shown in rogramme , the res5025030), becoming significantly stiffer as a result of crosslinking and AO reaction to form a silica surface layer. Having assessed the bulk properties of the laminates, more detailed analysis was performed on the surfaces of the laminates to confirm the mechanism responsible for the increases in flexural properties.However, as the AO fluence grows, then the stiffness of the laminates varies, so that after a 12-month period of simulated exposure, the laminates display very similar behaviour  proved challenging for the Alicona microscope, leading to the display of small gaps in the surface data  has a more uniform surface than the other samples, with more granular surfaces observed for the nanocomposites. The flexural properties of the laminates were found to improve slightly from 0% to 5% POSS concentration before decreasing steadily after agglomerations of the additive are formed. After the laminates were exposed to AO, the formation of a protective layer was observed, especially in those containing 10 wt% and 20 wt% POSS. Microscopic and topographical analysis reveals an increasingly uniform protective layer following exposure. The presence of POSS reduces the erosion yield, compared with the unmodified cycloaliphatic epoxy, with the erosion yield falling proportionately as the amount of silicon is increased. The samples containing POSS had superior flexibility properties when compared to laminates containing the commercial epoxy MTM44-1; the highest flexural modulus and strength was recorded for the laminate containing 5 wt% POSS. The challenge is to achieve a suitable manufacturing method capable of incorporating sufficient POSS content (some 5–10 wt% in this case) without compromising the processing characteristics or the final mechanical properties. In this case, the laminates were produced on a relatively small scale, so lab-based methods were employed to obtain the films, whereas the logical step would be to concentrate on optimising the preparation of the films to produce homogeneous dispersions of POSS; this aspect is now receiving our attention.


This paper presents an analytical prediction coupled with numerical simulations of a split Hopkinson pressure bar (SHPB) that could be used during further experiments to measure the dynamic compression strength of concrete. The current study combines experimental, modeling and numerical results, permitting an inverse method by which to validate measurements. An analytical prediction is conducted to determine the waves propagation present in SHPB using a one-dimensional theory and assuming a strain rate dependence of the material strength. This method can be used by designers of new SPHB experimental setups to predict compressive strength or strain rates reached during tests, or to check the consistencies of predicted results. Numerical simulation results obtained using LS-DYNA finite element software are also presented in this paper, and are used to compare the predictions with the analytical results. This work focuses on an SPHB setup that can accurately identify the strain rate sensitivities of concrete or brittle materials. Protection of buildings and structures in emergency situations is a key issue that needs to be addressed . Prevent−1). The setup used was based on the Kolsky bar setup [A description of dynamic material behavior is generally difficult to assess in the case of brittle materials. Different experimental techniques must be coupled in order to cover such a wide range of strain rates, as shown in ar setup ,8. The e−1), and this comprised a projectile and a concrete specimen sandwiched between two long elastic bars termed the input and output bars  ,24, the In Equation (1), ain rate ,24, and y (1991) . The comAdditionally, experimental data have also been reported to define the strain rate sensitivity of concrete under compression , and datTo understand the real experimental measurements for the dynamic compression of concrete , a new analytical technique should be used in comparison with compression of the metals . The newZ σIThe strain and stress values depend on the material properties of the input bar and of the projectile and its initial impact velocity Finally, the reflected wave The average strain rate and strain induced to the concrete specimen can be calculated as follows:The set of Equations (1)–(7) were solved considering the geometry of the bars, the projectile, the specimen and the initial impact velocity of the projectile. The values −1) (Equations (6) and (7), respectively).Based on the previous analytical description, the material parameters and the geometry of the setup and specimen a. As repBoth trends shown in The following section concerns designing the experimental setup, and it addresses what to change in order to obtain the maximum possible strain rate from the experiments. −1), while the maximum strain rate is about 714 (s−1) for a radius of 43 mm (2.15 times larger than the concrete specimen radius) (−1) if the specimen length decreases from 50 to 30 mm (−1). Concerning the analytical approach, the dynamic strength of concrete according to CEB was assumed to estimate the transmitted and reflected wave intensities. For a strain rate equal to 1166 (s−1), the CDIF is equal to 5.3 (strength 159 MPa). However, the size cannot decrease continuously for concrete, since the material behavior must be representative and the aggregate sizes must be considered. Thus, the specimen must be designed considering the representative elementary volume (REV) [It is well known that increasing the bar radius and assuming a constant specimen radius (20 mm in this work) increases the strain rate, as shown in  radius) . Howeverto 30 mm . By coupme (REV) ,29.The above calculations and analysis should be used to design the setup geometry and its configuration in order to obtain the correct and expected stress levels or strain rates during experiments. It can be useful to test specific concrete types such as ultra-high strength concrete (UHSC) with a static compressive strength close to 100 MPa. The proposed analytical approximation can be used with success to predict the effects of a strong strain rate sensitivity on the material behaviors of concrete or other brittle materials in SHPB testing.The following numerical simulations present how it is possible to describe the dynamic compressive strength and strain rate of concrete during dynamic failure using the SHPB technique. Previously neglected effects (dispersion of the wave and loading time) were revealed in the results of a full three-dimensional model. The simulation corresponding to the process of elastic wave propagation and related to the SHPB technique was performed using an explicit integration scheme in LS-DYNA . All parIn addition, the continuous-damage surface cap model was used to simulate the concrete behavior and its properties at high strain rates. The parameters were obtained by calibrating a typical concrete; namely, C30 grade. A detailed description of the model is reported in ,33,34. IBased on the previous numerical solutions, experiments were mimicked and the same values were measured as the three elastic waves propagating along the two elastic bars. Before using the previous analysis (Equation (2-a–c)), the key point was to demonstrate that force equilibrium was reached during dynamic loading. In the following curves, 30 m/s was reported for impact velocity and 0.05 m was reported for length of the specimen in regards to the three waves after the shifting time to zero, Once the force equilibrium has been reached and achieved a conservation of energy and quantity of movement, the theory of elastic waves may be used to described the material behavior of concrete under dynamic loading. Even if the transmitted wave seems large, the strain applied stays relatively small, as shown in The material behavior , which wConcerning all assumptions described in The main parameters allowing the strain rate sensitivity of concrete under dynamic compression to be described are d Nc see . The usuectively ,33. Usinectively ,25. Thusectively ,26,27 thThe real strain rate sensitivity presented in  Nc=0.58 . The CEBThe numerical simulations of the dynamic compression of concrete were analyzed in detail for two initial impact velocities: 10 and 30 m/s. The results of the numerical simulations were compared with the CEB , which wTo avoid plastic deformation of the bars and to have pure elastic wave propagation, the yield stress of a bar was assumed equal to 250 MPa see a. SimulaThe analysis showed that material model parameter identification should be conducted using the geometry and complexity of the whole SPHB experimental setup  in cases of brittle materials with a high strain rate sensitivity.The pathological influence of finite element discretization (mesh size) is an important problem during concrete simulation due to the softening effect (decreasing of the stress with strain) ,38,39. TFor the calculations presented in this article, the authors used a material model with a fracture damage energy coupled to visco-plasticity (see Equations (A1)–(A9) in ectively ,33. The The mesh size sensitivity of the numerical results is acceptable  to assume that the results were not mesh size dependent. Based on these curves the behavior (stress-strain and strain-rate curves) for both cases was also similar. The model combined with the analyzed material model behavior was correct, and a mesh size dependency was not observed.The stress-strain curve may vary for different kinds of concrete. The slope of the curve in the unloading part is crucial. It is important to know whether the curve can be measured using the SPHB technique. The main parameter measured up to now has been strength. However, the addition of some components as aggregates or fiber reinforcements in the concrete can change other mechanical properties. A parametric study was conducted to prove that potential changes in the softening curve shape of a material can be defined and observed during dynamic testing. The analysis was conducted by considering the changes of the two material parameters fc and B . During B, the material starts to become more brittle.The results corresponding to all previously described examples are reported in All effects are reported in An analytical solution to predicting stress and strain wave intensities was presented. This could be used to simplify the design process of SHPB and check consistency in further experimental test results.The effect of the initial impact velocity of the projectile on the strength and strain rate reached in the specimen was determined for different bar diameters.A method to calibrate the material model for concrete including strain rate sensitivity was presented. A numerical simulation was used to find a correct value of the parameters that define the strain rate sensitivity. As discussed, the original parameters have very low values of dynamic strength for compressed concrete in comparison with the analytical solution.The presented analysis proved that the solution was not sensitive to mesh size. The important aspect is that possible changes in fracture energy during compression or the shape of the softening (descending) part of the curve can be identified using this experimental technique.This work assumes that the concrete specimen is in equilibrium during the simulations, and that the friction coefficient has limited influence on the final results.The dynamic behavior of concrete during impact or a blast is very often analyzed using numerical simulations. During these kinds of loadings, high strain rates are reached and observed in the material. To predict the material’s behavior and dynamic strength, very precise tests and dynamic measurements are necessary, as has been discussed in this paper. Experimental results are then used to calibrate the concrete material model parameters. If the initial boundary value problem is used to simulate the dynamic behavior of the structure with the correct material model, its prediction will agree with the experimental observations. The following main concepts and results are presented in detail in this article:For each case considered in this work, the previous dynamic experimental results recommended by the CEB as CDIF were compared with the numerical results. This work may be used to summarize the design process of SHPB for concrete to reach a certain strain rate. Extension of the analysis to other classes of concrete (concrete C30 was assumed) or glass is possible using the same procedure. These new materials  can also be tested using the setup described herein. However, a limitation is imposed not to exceed the yield stress of the Hopkinson bars.


Stachel sequence. However, the low affinity of p15 limits its utility for ADGRG2 characterization. In the current study, we used alanine scanning mutagenesis to examine the critical residues responsible for p15-induced ADGRG2 activity. We next designed systematic strategies to optimize the peptide agonist of ADGRG2, using natural and unnatural amino acid substitutions. We obtained an optimized ADGRG2 Stachel peptide T1V/F3Phe(4-Me) (VPM-p15) that activated ADGRG2 with significantly improved (>2 orders of magnitude) affinity. We then characterized the residues in ADGRG2 that were important for ADGRG2 activation in response to VPM-p15 engagement, finding that the toggle switch W6.53 and residues of the ECL2 region of ADGRG2 are key determinants for VPM-p15 interactions and VPM-p15-induced Gs or arrestin signaling. Our study not only provides a useful tool to investigate the function of ADGRG2 but also offers new insights to guide further optimization of Stachel peptides to activate adhesion GPCR members.The adhesion GPCR ADGRG2, also known as GPR64, is a critical regulator of male fertility that maintains ion/pH homeostasis and CFTR coupling. The molecular basis of ADGRG2 function is poorly understood, in part because no endogenous ligands for ADGRG2 have been reported, thus limiting the tools available to interrogate ADGRG2 activity. It has been shown that ADGRG2 can be activated by a peptide, termed p15, derived from its own N-terminal region known as the  The adhesion G protein–coupled receptors (aGPCRs) are a family of 33 receptors in humans that are involved in many diverse biological processes, including brain functions, immune responses, and fertility , 9, 10. Stachel (German for stinger), within the N-terminal region of an aGPCR member, which induces receptor activation through its interaction with the 7TM domain. The tethered Stachel sequence located at the CTF plays an important role in aGPCR activation in response to different physiological stimulations, such as ligand binding, mechanostress, or removing of the NTF  (Stachel peptide p15 (sequence: TSFGILLDLSRTSLP) derived from the Stachel sequence (A) (Adhesion G protein–coupled receptor G2(ADGRG2), also known as G protein–coupled receptor 64 (GPR64) or human epididymal gene product 6 (HE6), has attracted substantial attention for its specific expression and essential functions in the male reproductive system . Recent DGRG2-β) . In our sequence , to elicsequence A  . p15 doeStachel sequence–derived synthetic peptides have a significantly low affinity to the 7TM  (VPM-p15) that activated ADGRG2 with significantly improved affinity. Furthermore, we characterized the residues in ADGRG2 that are important for ADGRG2 activation in response to VPM-p15 engagement.In contrast to other peptidergic GPCRs,  the 7TM , 26. Ford ADGRG2 . The lowStachel peptide p15 on ADGRG2. We generated an ADGRG2 mutant in which the entire ECD (including the entire GPS motif) was removed (ADGRG2-ΔGPS-β). Then, we measured the concentration-dependent p15 activities toward ADGRG2-ΔGPS-β and full-length ADGRG2(ADGRG2-FL) receptor. Increasing the amount of p15 led to a significant increase in cAMP levels, which was determined by the GloSensor assay (A). p15 also elicited a rise in the intracellular calcium level, which was determined using a CalfluxVTN Ca2+ assay. This change could be inhibited by the Gq/11 selective inhibitor YM-254890, suggesting that p15 induced the calcium influx via a Gq-dependent pathway (B). Of interest, we found through the bioluminescence resonance energy transfer (BRET) assay that p15 promoted the recruitment of β-arrestin1 and β-arrestin2 to ADGRG2, respectively (C–D). It is notable that p15 induced significantly more recruitment of β-arrestin1 or β-arrestin2 through the truncated mutant compared with the wildtype ADGRG2. Moreover, the ADGRG2-β (CTF fragment) shows a significantly higher constitutive activity for arrestin recruitment compared with that of the wildtype ADGRG2 (E–F). Because the Stachel sequence of ADGRG2-β could have more chance to access to the 7TM domain compared with that of the Stachel sequence in the ADGRG2 full length, these results indicate that the arrestin recruitment to ADGRG2 might be strengthened by the activation of ADGRG2 via Stachel sequence interaction. Taken together, these data demonstrate that the Stachel peptide p15 activates multiple signaling pathways downstream of ADGRG2.First, it is necessary to confirm the activation of or assay A. p15 al pathway B. Of intectively , C–D. Ite ADGRG2 , E–F. BeStachel peptides of ADGRG2 , p15 induced the strongest ADGRG2 activation (B). To find out which amino acid (aa) residues of p15 are required for receptor activation, we performed a systematic alanine scanning. The results showed that mutations of T1A and T12A enhanced p15 activity. Conversely, mutations of any residues from the middle of LLDL at positions ranging from +6 to +9, F3A, or R11A all greatly impaired p15 activity .We next optimized p15 by amino acid substitution, according to the following strategies: (1) diversifying the noncritical residues identified in the alanine scanning to amino acids with different properties; (2) changing critical amino acids into natural or unnatural amino acids with different structures but similar properties ; (3) enhancing the hydrophobicity of the C terminus of the  peptide A.Figure B). Next, we changed the Ser(S) at +10 position to A, V, F, L; Thr(T) at +12 position to A, V, F, L to diversify the residue properties at these positions following strategy 2. We also changed +15 position to Tyr(Me) or P15, following strategy 3. However, these p15 mutants did not exhibit enhanced signal transduction capacities compared with the wildtype p15 (D). Finally, according to strategy 2, we mutated the Phe(F) at +3 position to unnatural amino acids Tyr(Me), Phe(4-CN), Phe(4F), Phe(4-Me), F3, F3, Phe(4-NO2). Interestingly, the F3Tyr(Me), F3Phe(4-Me), and F3 mutants showed higher signal transduction activities (C). According to the results of the above studies, concentration–response curves with p15 mutants T1V, F3Tyr(Me), F3Phe(4-Me), and F3 showed a lower EC50 value than wildtype p15 in the same second messenger assay (E). To further enhance the potency of the p15 mutants, we performed combined mutations of p15 for those who showed improvement at different p15 positions. The double mutants T1V/F3 and T1V/F3Phe(4-Me) (VPM-p15) showed a significant lower EC50 than other mutants, and the effect of the mutant VPM-p15 on EC50 was most profound, with an EC50 of 1.41 ± 0.16 μM and an approximately 170-fold increase compared with the wildtype p15 peptide  at +1 position to F, W, H, R, K, D, E, V, respectively. Following strategy 1, the substitution of the polar T to a hydrophobic V at +1 position in T1V peptide induced about three times more cAMP accumulation compared with the wildtype p15, as determined by the GloSensor assay B. Next, type p15 D. Finalltivities C. Accorder assay E. To fur2+ assay and to recruit β-arrestin by BRET assay. Concentration–response curves showed that VPM-p15 enhanced the abilities of ADGRG2 to activate both Gs and Gq , in both ADGRG2-FL, ADGRG2-ΔGPS-β overexpressed HEK293 cells (A–D). Subsequently, VPM-p15 promoted the recruitment of β-arrestin1 or β-arrestin2 to the ADGRG2 truncated mutant ADGRG2-ΔGPS-β, ADGRG2-β, and ADGRG2-FL . Therefore, the optimized VPM-p15 induced both G protein and arrestin activations downstream of ADGRG2.ADGRG2 has been shown to maintain the fluid reabsorption through coupling to both Gq and β-arrestin1 . We ther93 cells , A–D. Su-FL E–H, , A–B. ThStachel sequences of the region p15 are highly conserved among different species (A). Because adhesion GPCRs are most homologous to class B GPCRs, we therefore used the PTH1R (a class B GPCR) and a Swiss-model software to generate an ADGRG2 three-dimensional coordinate model (A). According to this model, we then selected ADGRG2 mutations in extracellular loops 1, 2, 3, and transmembrane helices and then mutated them to alanine by QuikChange (A–B and A). We transfected HEK293 cells with ADGRG2-ΔGPS-β WT or its corresponding mutants and detected the expression of receptors on the cell surface by cell-surface ELISA (B). We adjusted the amount of transfected plasmid and used the mutants without significant alterations of receptor-surface expression for further delineation of the ADGRG2-VPM-p15 engagement.To provide a foundation for further optimization of the ligand that activates ADGRG2, we then investigated the molecular mechanism of the engagement of VPM-p15 with ADGRG2. Through sequence alignment, we found that ADGRG2  species A. Becauste model A. Accord A–B and A. We trace ELISA B. We adjThe binding capacities of ADGRG2 WT or its mutants for VPM-p15 were determined by BRET assay, which has been used in previous studies , 28, 29.ECL2 and I758ECL2, as well as the mutations of W8246.53 in TM6 , almostD and ECL2, I758ECL2, and W8246.53) abolished intracellular cAMP accumulation, whereas F829 and F838 enhanced cAMP accumulation, and N759 ECL2, S760 ECL2, F8266.55, F8286.57, V836ECL3, I8447.53, and F8457.54 might reduce cAMP accumulation. The other residues, including F7645.35, Y7655.36, V7685.39, and F7725.43 may regulate receptor dynamics, which are important for transducing the ligand–binding signal to downstream coupled G proteins . These results indicate that G protein and arrestin activity are mediated by at least several common residues located in the ADGRG2 7TM region.Both p15 and VPM-p15 were able to induce β-arrestin recruitment Stachel peptides varies between 7 and 18 amino acids . Consistently, a recent study reported that ΔNTF and P622 mutants of ADGRG2 interacted with β-arrestin1 and β-arrestin2, which induced constitutive internalization of ADGRG2 in steady states . Similaf ADGRG2 , 24, 25.f ADGRG2 , 35, 36.f ADGRG2 , E–F. CoStachel peptides is that they require large amounts to elicit significant activation levels in vitro with EC50 values ranging from ∼80 to 400 μM in general  diversifying noncritical residues to natural or unnatural amino acids with different properties and (2) changing critical amino acids into natural or unnatural amino acids with similar properties with different structures  and Dual-Luciferase Reporter Assay System (E1960) were purchased from Promega. Tetramethylbenzidine was purchased from Thermo Fisher. Coelenterazine 400a (FP-BB839B) was purchased from Interchim. Gq/11 selective inhibitor YM-254890 (AG-CN2-0509-M001) was purchased from Adipogen. All other reagents or chemicals were purchased from Sigma-Aldrich unless otherwise specified.Wildtype ADGRG2 full-length (ADGRG2-FL) was cloned from mouse total cDNA libraries using the following primers: forward, ATTCTCGAGGATGCTTTTCTCTGGTGGG; reverse, ATTGAATTCCATTTGCTCGATAAAGTG, and then ADGRG2-FL, ADGRG2 C-terminal truncation mutant (ADGRG2-β), and ADGRG2-ΔGPS-β (the entire ECD of ADGRG2 was deleted) were subcloned into the pcDNA3.1 expression vector. The ADGRG2-ΔGPS-β mutants  were generated using a QuikChange Mutagenesis Kit (Stratagene). All mutations were verified by DNA sequencing. All primers are listed in Stachel peptides were synthesized using standard Fmoc-chemistry on an automated peptide synthesizer MultiPep (Intavis AG). Final side chain deprotection and cleavage from the solid support was achieved by using TFA, water, and thioanisole (95:2.5:2.5vol%). Peptides were subsequently purified to >95% purity by preparative RP-HPLC (Shimadzu LC-8). Peptides were suspended in dimethyl sulfoxide and diluted to ≤4% dimethyl sulfoxide (vol/vol) in experiments.ADGRG2 Human embryonic kidney 293 (HEK293) cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, penicillin (100 IU/ml), and streptomycin (100 μg/ml) as described , 38. For2-independent medium in each well for 2 h. The cAMP signal was examined using a luminescence counter (Mithras LB 940).The Glosensor cAMP assay was performed as described , 39, 40.2+ assays, HEK293 cells in 24-well dishes were cotransfected with plasmids encoding ADGRG2 or its mutants, CalfluxVTN constructs were used at a 6:1 ratio. Empty vector pcDNA3.1(+) was used to normalize the total amount of transfected plasmid DNA. The transfection and CalfluxVTN Ca2+ assays were performed as previously reported  under a Thermo plate reader. The BRET signal was calculated as the ratio of emission of YFP (527 nm) to Rluc (370–480 nm).The ADGRG2-YFP and β-arrestin1-Rluc or β-arrestin2-Rluc plasmids were transiently transfected into HEK293 cells. Forty-eight hours after transfection, the cells were washed with PBS, detached with PBS and 5 mM EDTA, and resuspended in PBS with 0.1% (w/v) glucose at room temperature. The cells were then distributed (80 μg of protein per well) in a 96-well microplate  and incubated in the presence or absence of ADGRG2 50 values of ADGRG2 WT and its mutants for binding VPM-p15 were calculated by GraphPad, and data were normalized by EC50 of WT.In the detection of binding capacities of ADGRG2 WT or its mutants for VPM-p15, HEK293 cells were transfected with Rluc-ADGRG2-ΔGPS-β or mutants. Transfected cells were stimulated by increasing the concentration of FITC-conjugated VPM-p15. Binding capacities were determined by BRET, ECAfter transfection for 24 h, HEK293 cells were cultured at 37 °C in 24-well plates pretreated with polylysine. After another 24-h incubation, the cells were washed with Tris Buffer Saline Tween20 (TBST) once and fixed with 4% paraformaldehyde at room temperature for 10 min. Then the cells were washed three times with TBST and sealed with 5% bovine serum albumin solution for 1 h at room temperature. After three washes with TBST, the cells were incubated with primary antibody overnight at 4 °C. After another three washes with TBST, secondary antibody was added, and the cells were incubated for 1 h at room temperature. Tetramethylbenzidine was added after washing three times with TBST. After 5 to 20 min, the reaction was stopped with 0.25 M HCl solution until the color of the solution changed appropriately to blue. Then the reaction solution was transferred to a 96-well plate, and the absorbance at OD450 was measured with a microplate reader.p < 0.05. The sequence alignments were performed using T-coffee.All the data are presented as the mean ± SEM from at least three independent experiments. Statistical comparisons were performed using an ANOVA with GraphPad Prism 6.0 . Significant differences were accepted at The structure presented in this paper has been deposited in the Protein Data Bank with the following code: 6NBI. All remaining data are contained within the article.The authors declare no conflicts of interest in regards to this manuscript.


The aim of this research was to develop new electrospun membranes (EMs) based on polycaprolactone (PCL) with or without metronidazole (MET)/nano-hydroxyapatite (nHAP) content. New nHAP with a mean diameter of 34 nm in length was synthesized. X-ray diffraction (XRD) and attenuated total reflectance Fourier transform infrared spectroscopy (FTIR-ATR) were used for structural characterization of precursors and EMs. The highest mechanical properties  were found for the PCL membranes, and these properties decreased for the other samples in the following order: 95% PCL + 5% nHAP > 80% PCL + 20% MET > 75% PCL + 5% nHAP + 20% MET. The stiffness increased with the addition of 5 wt.% nHAP. The SEM images of EMs showed randomly oriented bead-free fibers that generated a porous structure with interconnected macropores. The fiber diameter showed values between 2 and 16 µm. The fiber diameter increased with the addition of nHAP filler and decreased when MET was added. New EMs with nHAP and MET could be promising materials for guided bone regeneration or tissue engineering. The evolution of medicine nowadays requires a change in the treatment methods and biomaterials used. Certainly, there is a requirement for biomaterials that can demonstrate complex functions and allow a proper host–material interaction. This shift leads to the development of a new generation of biomaterials , charactin vitro bioactivity [The common administration of antibacterial agents like mouthwashes shows poor results in disease control because a small amount of the drugs reach the periodontal pocket and the drugs are constantly flushed from the periodontal pocket, due to a very high fluid-clearance rate (40 replacements of the fluid/hour in a 5 mm pocket) . Cliniciactivity . PCL briactivity . The feaactivity . Using tThe aim and novelty of this research was the synthesis of new nHAP using two dispersing agents and the use of the new nHAP for the development of a new EM based on PCL with or without MET/nHAP content. The new nHAP was characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD) and attenuated total reflectance Fourier transform infrared spectroscopy (FTIR-ATR). All precursors and EMs were structurally characterized by XRD and FTIR-ATR. For mechanical characterization, the force at maximum load, Young’s modulus, the stiffness and the tensile strength of EMs were investigated. SEM investigation was used to analyze the topography of EMs.The null hypotheses tested in the article were as follows: (1) the composition does not affect the structure and the topography of new EMs; (2) the composition does not influence the mechanical properties of EMs.−1, MET, dichloromethane (DCM), dimethylformamide (DMF), poly (PVA), diammonium hydrogen phosphate ((NH4)2HPO4), calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) and ammonium hydroxide solution (NH4OH) were acquired from Sigma-Aldrich GmbH, Steinheim, Germany. Darvan 821A was purchased from R. T. Vanderbilt, Norwalk, CT, USA. Darvan 821A is an ammonium polyacrylate mainly used as a dispersing agent to avoid the formation of particle aggregates of nHAP in homogeneous and highly concentrated nHAP colloidal suspension. Deionized water was used in all experiments. The resistivity of absolute pure water was 18.2 MΩ·cm at 25 °C or electrical conductivity 0.055 µS/cm. All commercial materials were used without further purification.PCL with molecular weight (M.W.) 80,000 g·mol4OH solution. Calcium nitrate solution was mixed with ammonium hydrogen phosphate solution in conformity with the standard stoichiometry for pure HAP at a Ca/P ratio of 1.67. Moreover, nHAP was prepared according to the following reaction:nHAP was synthesized using a wet chemical method from calcium nitrate and ammonium hydrogen phosphate from Ca and P precursors. Two solutions of 1200 mL were prepared in two beaker glasses (3000 mL) by separately mixing calcium nitrate and ammonium hydrogen phosphate with double distilled water by strong stirring in an aqueous solution at ambient temperature. A solution dispersing agent of 0.2 vol.% of 1:1 Darvan 821A/PVA was added to both solutions. Each aqueous solution had the pH adjusted to 10.5 by using 25% NH4)2HPO4 was added dropwise into Ca(NO3)2 solution at 70 °C with strong stirring, and a pH of 10.5 was maintained by using 25% NH4OH solution. The suspension was observed under stirring for 12 h. Then, the formed precipitate was filtrated and washed three times with double distilled water and anhydrous ethanol. Finally, the nHAP particles were lyophilized  and dried in an oven at 80 °C for 12 h. After that, the powder was crushed and kept at 300 °C in a furnace for 6 h to obtain a fine nHAP dry powder.The solution of  spectroscopy was carried out in an attenuated total reflection mode (ATR). The spectra were recorded on FTIR spectrophotometer  equipped with an ATR attachment with a horizontal ZnSe crystal. The spectra were scanned in the mid-IR range from 400 to 4000 cmThe XRD measurements were run at room temperature using a Shimadzu 6000 XRD diffractometer , with a monochromator of graphite for the Cu-Kα radiation. The source power was operated at a voltage of 40 kV and current 30 mA, and the scan speed was 2°/min. The PDF2 reference library was used for qualitative identification of the crystalline phases. The average of the mean crystallite size of the synthesized nHAP was determined by using the Debye–Scherrer formula given by Equation (2) :(2)D=λ·KTEM  was used at 80 kV high voltage and 20× magnification to investigate the size and morphology of nHAP. Morphology of the EMs was investigated by scanning electron microscopy  using high vacuum, 15 kW, 1000× magnification and 10.7–13.9 mm working distance. The diameter of nHAP from the TEM image and the fiber diameter from SEM image were evaluated using Image J software . After collecting the data, the mean diameter and standard deviation (SD) were calculated and the graphs were generated.n = 5) under dry conditions using a mechanical testing machine  using a loading rate of 1 mm/min−1. The force at maximum load, the Young’s modulus, the stiffness and the tensile strength were evaluated. The Young’s modulus of the samples was measured from the slope of the elastic portion (linear) of the stress–strain curve. The stiffness (S) measures the resistance offered by an elastic body to deformation and was calculated using Equation (3):F is the force on the body and δ is the displacement produced by the force along the same degree of freedom.The developed EMs with a rectangular shape  were tested for tensile strength (TS) was measured using Equation (4):TS is the tensile strength (MPa), F is the force on the cross-section of the sample at final tension (N) and A is the nominal cross-sectional area of the sample (mm2).The tensile strength  by one-way analysis of variance (ANOVA) and by Tukey’s test with the level of significance set at 0.05 in order to determine the significant differences between the mean values of the tested EMs.The average crystallite of nHAP from XRD measurement a showed TEM investigation  of cryst−1, shows the positions of the absorbance peaks more clearly. Thus, −1, C–H (aromatic) stretching at 3099 cm−1, C–NO2 and N–O stretching at 1535 cm−1, CH2 scissoring at 1471 cm−1, C–OH and C–O stretching at 1047 cm−1 and C–NO2 and C–N stretching at 825 cm−1 [−1 and C–N stretching at 825 cm−1, did not show any displacements after it was introduced in the polymer (PCL) membrane. It can be said that no chemical bond was formed between drug and the polymer (PCL) matrix. Some characteristic peaks for MET, such as CH2 at 1471 cm−1 and C–OH and C–O at 1047 cm−1, were found to overlap with the PCL peaks in the drug-containing EMs.The FTIR spectra of the different precursors  and EMs  are show825 cm−1 ,24,25. Ip < 0.05) when evaluated with one-way ANOVA statistical analysis. The highest values for mechanical properties  (p > 0.5). There was one exception: the stiffness increased when 5 wt.% nHAP was added to the PCL matrix (p > 0.5). The addition of MET or 5% nHAP-20% MET to PCL decreased the stiffness.The results of mechanical properties showed statistically significant differences (trength) a–d were p < 0.5) when compared with the PCL membrane. This behavior could be explained by the decrease in PCL percentage, because this is the only compound that contributed to the curing and polymer matrix construction. The stiffness (or rigidity) is the resistance of an elastic body to deflection or deformation by an applied force. Young’s modulus is a property of a material that indicates the ability to resist mechanical deformation [in vitro and in vivo tests are required in the future in order to see the advantages of nHAP or MET addition.The addition of MET to the PCL membrane caused a greater reduction in mechanical properties than the addition of nHAP. This could be explained by the difference in mechanical properties between nHAP and MET. When nHAP and MET were added together, the new EMs showed the lowest mechanical properties . All mecormation . The incormation . Caballéormation  investigormation . When nHormation . From thin vitro and in vivo for the clinical application of these EMs.By using two dispersing agents, namely Darvan 821A and PVA, we have confirmed advantages in obtaining new nHAP particles. The imaging and characterization of nHAP particles by TEM, XRD and FTIR confirmed the presence of nHAP (mean diameter of 34 nm) obtained by new synthesis. XRD and FTIR showed the presence of PCL, nHAP and MET in newly obtained EMs without chemical bonds between them. The investigated mechanical properties  decreased with the addition of nHAP and MET. The stiffness increased with the addition of 5 wt.% nHAP. The lowest mechanical properties were registered for the EM with nHAP and MET. The SEM images confirmed that all EMs showed a fibrous and porous structure. The greatest diameter of the fiber was obtained when 5 wt.% nHAP was added, and the diameter decreased when the filler was replaced with MET. Therefore, all null hypotheses for the newly obtained EMs are rejected. New experimental EMs with nHAP and MET could be promising materials for guided bone regeneration or other tissue engineering applications. These results encourage us to pursue further investigation


This study presents a flow pump technique usually used for evaluating the permeability of soils, which was, for first time, applied to measure the water permeability of concrete. Additionally, a new easy-to-apply method to determine permeability is proposed, based on a modification of Valenta’s formula. In the calculations, the apparent air content of concrete mixes was taken into account. An additional purpose of the conducted research was to determine the influence of a new generation of polycarboxylate superplasticizer and chemically active admixtures on the permeability, compressive strength, and other properties of concrete. The following four types of concrete were tested: concrete without admixtures, concrete with an admixture to increase the compressive strength, concrete with a superplasticizer, and concrete containing two admixtures simultaneously. The results showed that the proposed method allows to obtain reliable measurements within a very short period of time. The obtained results confirmed that new method may be very useful in engineering practice, particularly in terms of the watertightness of hydrotechnical concretes and the properties of the concretes used in bridge construction, underground parts of office buildings, or sealed tanks. Concrete is one of the major construction materials widely used for infrastructure and building construction across the world. The durability of concrete structures is primarily influenced by the movement of both gaseous and liquid substances via the system of pores, which can possibly result in the deterioration of the quality of concrete ,2,3. TheThe water permeability of permeable and highly permeable concretes can be assessed easily by direct methods. However, the measurement of water permeability has become more complicated as the quality of concrete has improved. Laboratory permeability tests for modern dense concrete or high-performance concrete may meet some difficulties in keeping the steady state flow through the concrete sample, and there is a high risk of leaking around the sample if the surface is not sealed. For concrete with a high density, it is not possible to obtain a measurable quantity of water flow, even over a long period of time .3, while the resolution of the pressure measurement is 1 kPa on display and 0.1 kPa via software.One method that can be adapted to test the permeability of concrete is the flow pump technique used in the study of low-permeability materials ,17,18,19However, when testing a large volume of weakly permeable concrete, the limitation associated with obtaining a flow rate through the whole sample in this method is a serious problem. The flow pump technique in adjustment to concrete testing consists of maintaining a constant level of hydraulic head and controlling the flow rate under a given pressure. However, when testing a large volume of weakly permeable concrete, the limitation of this method is obtaining a flow rate through the whole sample. This approach makes it possible to reproduce the test conditions that are used in measuring the depth of water penetration under pressure in accordance with EN 12390-8 . TherefoThe four different types of concretes used in this work were made with Portland cement (CEM I 32.5 R) complying with the European Standard EN 197-1 , a fossiSeven cubic samples with sides of 150 mm were prepared for each set of tests on concrete: four for the compressive strength tests and three for the water penetration depth tests. To determine the permeable porosity, six cylindrical samples with a diameter of 45 mm and a height of 40 mm were cast from the same batches of concretes. Additionally, four cylindrical samples with a diameter of 100 mm and a height of 100 mm for each set of tests on concrete were prepared for the flow pump tests at a constant hydraulic head.All specimens were soaked in water at room temperature after preparation for 10 days. Then, the samples were stored in a controlled chamber at a relative humidity (RH) of 95% and a temperature of 20 °C until 28 days of age. This conditioning of the samples was necessary due to the requirements of the admixture (A) and the delay of cement grain hydration by the superplasticizer (B). The goal was to achieve a high degree of hydration of the polyether side chains of the superplasticizer.In the case of the concrete mixes, their bulk density was tested in accordance with EN 12350-6  and appaEach type of concrete was tested for its water permeability in terms of water penetration depth. These tests were performed on three samples for each type of concrete. The testing procedure for measuring the water penetration depth was carried out in accordance with EN 12390-8 , where tIt is believed that in non-saturated medium , the water penetration under external hydraulic pressure is associated with sorptivity and unsteady flow ,8,35,36.k is the penetration coefficient identified with the permeability coefficient (m/s), d is the water penetration depth at time t (m), α is the permeable porosity, t is the test time (s), and H is the hydrostatic head causing the water percolation (m).According to the modified Valenta equation cited by Neville  and RamaC is the ratio of the avgd to dmax. The average depth of penetration avgd is calculated by first measuring the area Aw and maximum width wmax of the wetted region for the two split sections using CAD software [avgd is taken as the average of the Aw divided by wmax for each half.Hedegaard and Hansen  expressesoftware . Then, dAn increase in sample weight indicates that the samples were still hydrated or saturated throughout the test. The changes that occur in the samples throughout the test can be quantified by checking the measured inflow volumes and accurately measuring the sample weight before and after the test. Changes in the mass of samples during the test make it possible to determine the porosity, that is, the volume of pores that are filled with water, expressed as a percentage or fraction of the volume of concrete.These samples were characterized by a much smaller mass and it was possible to apply an electronic balance with a resolution of 0.01 g, thereby significantly increasing the accuracy of the measurements. These results are shown in The flow pump technique developed by Olsen et al.  was espeA number of constant head permeability tests were carried out according to ASTM D2434 [The experimental setup shown in −15 m/s. To avoid any lateral leakage and to ensure uniaxial flow, the circumference of the cylindrical specimen was covered with a rubber membrane that was sealed to the pedestal and upper cap with O-rings. The hydraulic head was applied using a volume–pressure controller. The driving pressure used in this study was 0.5 MPa. It has been found that the presence of compressive stress below a certain threshold value decreases the permeability, but when the applied stress exceeds this threshold, a significant increase in the permeability occurs [The permeability cell is a type of triaxial cell originally developed to test the permeability of fine-grained soil or rock. The cell is able to withstand the confining pressures (up to 1.0 MPa) necessary for measuring flow in materials with permeability as low as 10y occurs ,45,46. TThe flow rate was determined from the volume of water flowing during an imposed specific time phase. The change in flow rate was recorded regularly, with the frequency of measurements depending on the type of material and the time elapsed since the beginning of the test. The readings were taken several times a day (collected during a given period). The volume of the water that passed through the sample in unit time for the test pressure was then calculated and expressed as the water permeability. When steady state conditions had been achieved at the test pressure, the inflow corresponding to this could be used to calculate the permeability coefficient. The initial and final masses of the samples were measured with a resolution of 0.01 g to determine their relative contents of pores filled with water.Amriou and Bencheikh  noted thk is permeability coefficient (m/s), Q is the volumetric flow rate (m3/s), L is the length of the flow path (m), F is a cross-section of the sample exposed to water flow (m2), and ΔH is the drop in hydraulic potential difference (m).The permeability coefficient k of the samples was determined using Darcy’s experimental law   are presented in Water permeability is generally determined by accumulated flow and a permeability coefficient. The curves of the total flow or permeation were plotted on the basis of the change in the flow rate. −13 ÷ 10−14 m/s, the initializing times were approximately 140–150 h. Kameche et al. [The water permeability of saturated concrete is seldom the prevailing conveying mechanism in practical applications . If non-e et al.  reportedThe transient rise and transient decay phases of the presented tests in low-permeability concretes took a shorter period of time. This does not mean that when the stable permeation stage is reached in a shorter amount of time, the material is more permeated. Instead, it indicates that, before the tests began, the concretes also had a very high degree of cement hydration. The fluid–cement matrix interactions during measurements can significantly affect the permeability. The interactions of the water–cement matrix could be reduced in this method because of the continuing hydration of mature cement paste in a relatively short time required to carry out the measurement. Nokken and Hootoon  noticed Different effects of the applied admixtures were also observed, as shown in In order to compare the results obtained from this method with those obtained from the depth penetration method, the coefficient of permeability was evaluated as an average value of 71 and 73 h. In The coefficient of permeability decreased with time. The reduction in the permeability coefficient value over time was not due to the continuous hydration of the cement. During a long testing period, even small temperature variations cause appreciable fluctuations in the response of the rate flow versus time because the high accuracy measurements of flow water volume. These fluctuations are caused by the thermal expansion and contraction of both the equipment and water for the permeability test . In FiguA summary of the results for each concrete is given in In the proposed direct method of determining concrete permeability, the average permeability coefficient values in a given unit of time ~72 h) were assumed. The results of the tests were affected by solar gains in the room and a diurnal temperature variation on the outside. Therefore, visible cyclic scattering of points in time is visible in 2 h were It can be seen from The results from the flow pump indicate significantly lower permeability values, up to an order of magnitude lower than the calculated values based on the penetration depth .ε is the apparent air content in concrete mix.The coefficients of permeability for all of the concretes changed in a similar way, although they concerned materials of different porosities and compressive strengths. Generally, the values were different by approximately an order of magnitude, but even more than two orders of magnitude if correction was applied taking into account the average to maximum penetration depth ratio. The values obtained on the basis of the modified Valenta Equation (1) by incorporating the apparent air content are interesting, as this approach can easily be applied in practice. A better agreement with the reference data in this case was found using the following formula:The favorable agreement affords confidence in the validity of the developed method. The average fitting error of the data calculated from Equation (4) compared to the reference values was only 59%.Two different methods for evaluating the permeation property of tested concretes were applied: The flow pump technique and the depth of penetration method. The experimental results confirmed that the depth penetration test and the flow pump test at a constant hydraulic head result in similar values of the coefficient of permeability when similar samples are tested under the same conditions.For the first time, a novel direct method of measuring the in-flow of cement-based materials was applied based on the application of a constant head and measuring an extremely low flow rate. The flow pump technique is better suited to automatic data acquisition and is more reliable and sensitive than conventional methods. The results for the cumulative water flow versus time were plotted to determine flow at steady state. Next, the steady flow rate was determined and the coefficient of the water permeability of concrete was calculated using Darcy’s law.The fundamental advantages are that the flow pump method is very fast, highly accurate, and simple enough to apply. The greatest dynamics of the changes in the flow of water under constant pressure were observed during the first day. The second day was a transitional stage. Next, there was a very small decrease in the permeability coefficient corresponding to the quasi steady state conditions. The tests performed confirmed that the three days of measurements guarantee the determination of the permeability coefficient in weakly permeable concrete. It can be expected that in the case of more permeable concretes, a shorter time would be needed to reach steady state conditions. The results obtained by the water penetration depth method were adequate for such materials. Moreover, a novel indirect method of determining concrete permeability based on the modified Valenta equation used in the depth of water penetration tests was proposed. These results confirmed that the proposed methods can be used for evaluating the water permeability of concrete. Based on the experimental data provided, the presented methods appear to have merit in determining water permeability and are capable of properly and reliably identifying the influence of the properties of concrete on permeability.This paper also presented experimental results concerning the influence of a new generation of polycarboxylate superplasticizers and a chemically active admixture to increase the compressive strength and to improve the other properties of concrete, but it was not a main goal of this research.To summarize, it should be emphasized that the proposed flow pump method of testing the water permeability of concrete has not been previously used in scientific research and has the advantage that it can be successfully used in practice. The tests were carried out on concrete samples prepared in the laboratory, but the flow pump technique is suitable for testing concrete from structures. It is common knowledge that concrete made according to the same recipe in situ (in engineering constructions) shows different quality characteristics from that of concrete cured in a laboratory. A lot of work on this subject has been carried out already, in which field research plays an important role. In order to determine the permeability of concrete in a structure, it is sufficient to take core samples of a small height (approximately 100–150 mm) and a small diameter of 100 mm or more at designated points. Shallow boreholes with a small diameter will disturb the structure to a minimal extent, and concrete losses can be easily filled in. Drilling can be done in sections without reinforcement. However, it is also possible to test samples containing horizontal reinforcement . The proposed precise and fast way of measuring water permeability (at least 24 h) provides a new perspective on concrete testing in structures compared to the known indirect methods such as water absorption, which are very time-consuming.


Statistical learning (SL) is essential in enabling humans to extract probabilistic regularities from the world. The ability to accomplish ultimate learning performance with training  has been known to be dissociated with performance improvement per amount of learning time . Here, we quantified the potential and efficiency of SL separately through mathematical modeling and scrutinized how they were affected by various executive functions. Our results showed that a high potential of SL was associated with poor inhibition and good visuo-spatial working memory, whereas high efficiency of SL was closely related to good inhibition and good set-shifting. We unveiled the distinct characteristics of SL in relation to potential and efficiency and their interaction with executive functions. It is a critical process in our daily lives based on the fact that, through SL, learners grasp probabilistic regularities to predict upcoming events and prepare appropriate actions effectively. It has been examined across tasks using various types of stimuli, including visual stimuli8, tactile stimuli9, non-linguistic sounds10, auditory syllables11, and action segmentation12. SL has been scrutinized by an alternating serial reaction time (ASRT) task in which odd-numbered visual stimuli generate a fixed motor sequence and even-numbered visual stimuli establish random sequences such that participants learn the motor sequence implicitly16.Statistical learning (SL) is an implicit mechanism that requires learners to extract probabilistic regularities from the environment18, is known to be related to the frontal lobe of the brain that has been intertwined with higher-order cognitive functions, particularly executive functions21. For example, the effect of SL was negatively correlated with a composite score that is an average value of normalized scores of several neuropsychological tests to assess executive functions19. An electroencephalography (EEG) study found a dynamic human brain network, showing that a functional connectivity of frontal areas was negatively correlated with individuals’ SL performance15. Even though these studies posit a close relationship between SL and the executive functions, there is still a limited understanding of SL. Executive functions include various cognitive processes such as response inhibition, set-shifting, and working memory25. From these processes, which one is the most pertinent to SL remains unclear. Considering that executive functions are integral to high-level cognitive processes in humans, it is worth scrutinizing each individual process of executive functions in relation to SL.The effect of SL, being defined as the difference in learning performance between probable targets  and improbable targets 26. When discussing learning mechanisms, we have to consider two distinct features, that is, the potential of learning and the efficiency of learning. The potential of learning is the ability to accomplish an ultimate learning performance given that learners are provided with all the optimal conditions during the progression of learning27, and the efficiency of learning is the performance improvement per amount of learning time28. Interestingly, these two features have been known to be dissociated from each other26. For example, even though older adults required more time for motor skill acquisition compared to younger adults, their final performance was comparable to that of younger adults30. This indicates that, even though the older and younger adults needed different learning times (implying different learning efficiency) to achieve a comparable level of performance, their final levels of performance were similar . Potential and efficiency are of great importance in learning. However, no study has conducted an investigation with respect to these two features, particularly in SL. Therefore, in the present study we distinguished the efficiency of learning from the potential of learning in SL and examined their relations to executive functions, hoping to unveil the contribution of several processes of executive functions  to both the potential and efficiency of SL.Learning is not a single entity but is underpinned by several mechanisms32. Mathematical modeling, by providing precise quantification, describes assumptions about how observed data is generated and developed33 such that it helps better understanding and clarification of research questions or theories than qualitative descriptions34. In this approach, a model comparison is crucial to select the best model to describe empirical data and to predict its possible changes more precisely35. Researchers have used mathematical modeling in describing the benefits from practice in learning and found the exponential function36 to be the standard equation to describe and predict the improvement in learning performances40. In the present study, we focused on SL and aimed to find the best model to reflect SL performances by testing goodness-of-fit of the exponential function in comparison with power or linear functions (as control models)44. According to a formula of the exponential function, a learning rate decreases and eventually stays consistent even with increasing practice36. Using this function, we estimated the potential and efficiency of SL and investigated the dynamic changes in SL performance  of cognition in a simplified fashion by converting problems or ideas that should be identified in the experiment into mathematical representations, using mathematical formulationsOut of several executive functions, we aimed to elucidate which of them had the most influence on the potential and efficiency of SL. To this end, we first examined the effect of SL in an ASRT task through the difference in learning performance between probable targets and improbable targets. The ASRT task had three different conditions: pattern type with a high probability condition (Pattern-High), random type with a high probability condition (Random-High), and random type with a low probability condition (Random-Low) Fig. . BecauseParticipants showed high accuracy  and fast reaction times (RTs) . These results indicate that participants successfully performed the ASRT task.45 with two independent variables of conditions  and learning time , and included the interaction between the conditions and learning time for the prediction of SL effects in behavioral performances . The results are shown in the Table P = 0.008, 95% confidence interval (CI95%) = 0.8 to 5.0], while a marginal influence was observed for accuracy .We hypothesized that an increase in performance differences between Random-High and Random-Low occurs through the progression of SL, and thus an interaction between conditions  and learning time  predicts the effects of SL. We used a multiple linear regressionentclass1pt{minima46. Using maximum likelihood estimation (MLE)48, we fitted an exponential model 49, and Bayesian information criterion  using objective measures (i.e. accuracy and RTs). However, through mathematical modeling we were able to investigate the change of SL over time using the estimated learning curve in the exponential model , which are known to assess several executive functions (see Supplementary method). In Session 2, participants performed an alternating serial reaction time (ASRT) task16. Several tools such as E-Prime 3.0113, MATLAB114, and Psytoolkit116 (a web-based environment) were used for running tests. In our ASRT task, no feedback was given to participants regarding their task performance. The ASRT task was composed of 36 blocks, which were alternated with rests Unbeknown to the participants, we alternated between two kinds of main trials: pattern trials and random trials Fig. a. In the117. For example, 3r2, 2r4, 4r1, or 1r3 were pattern type triplets because they had two pattern trials  which were shown regularly in the triplet and only one random trial  between the pattern trials. However, r3r, r2r, r4r, or r1r triplets were random type triplets, because they included two random trials  and only one pattern trial  in the middle. With respect to the probability, some triplets  and probability (High vs. Low): Pattern-High, Random-High, and Random-Low Fig. b. As forExact probability of occurrence of the triplets was calculated as follows. The pattern type and random type were shown in the same proportion of 1:1. In the random type, Random-High and Random-Low were shown in the proportion of 1:3. Thus, the probabilities of occurrence of the Pattern-High, Random-High, and Random-Low were 50%, 12.5%, and 37.5%, respectively . Therefore, the third trial is the critical element in categorizing the triplets as either high probability or low probability, and thus accuracy and RTs only from the third trials were considered for the analysis.Notably, a triplet is made of three sequentially presented trials, and a probability of the triplet is determined by the third trial. For example, Fig. 118. Our main interest was to identify the dynamic changes of SL performances over the course of learning time. To do this, first we confirmed if participants successfully achieved SL during the ASRT task with a simple model. The first factor was an effect of probabilistic structure in RTs. We examined this effect by comparing the performances of the high probability and low probability in the same type . Since the Random-High and Random-Low have the same type (Random) with different probability (High vs. Low), this factor made it possible to solely examine the pure effects of SL. The second factor was the effect of learning time, which was represented by the block order showing how much learning time had passed. We investigated these two factors (PROBABILITY and BLOCK) independently through the multiple linear regression model45 in Eq. , PROBABILITY = High, Low, INTERACTION = BLOCK Here, we supposed that the coefficient of INTERACTION in the model represents the effect of SL over the course of learning time, since the slope of BLOCK would differ between the conditions (Random-High vs. Random-Low) as participants progress in learning.Likewise, the effect of type (Pattern vs. Random) was investigated in the same probability (High) by comparing the performances of Pattern-High and Random-High. The block order was used to measure the effect of learning time. These two factors were considered in the following multiple linear regression model to see the effect of learning type on RTs Eq. .3\documeBLOCK = 1, 2, …, 36 (block order), TYPE = Pattern, Random, INTERACTION = BLOCK × TYPE.For all the analyses we used only RTs of correct responses, and the block order was centered for a better fit.15 or not. We calculated the SL scores in every block to investigate the dynamic changes of SL over the course of learning time (block order). Specifically, since an individual’s overall speed of RT could affect the individual SL scores, we adjusted the SL score in each block divided by the mean RT of its corresponding block. We subsequently tested a first-order exponential model 48. To select a model to best explain our SL scores, we used two criteria of goodness-of-fit: the corrected Akaike information criterion (49 and the Bayesian information criterion (50. Because we did not have many numbers of data point (36 blocks) and participants (40 participants), we used a corrected term (119. The equation of To scrutinize the effect of SL together with time, we investigated the learning curve in SL. To this end, we defined SL scores as the following: absolute values of difference in RTs between Random-High and Random-Low. This indicated whether participants learned the statistical probabilities of the triplets52. Specifically, when we compared the 120. The We compared the three models following the scales of Table 453. In the step response, the saturation level of estimated values—41. In our model, 53. In principle, arbitrary large and small 58. Here, we used the 122 for bound constrained minimization and the initial parameter values of one.The exponential function turned out to be the best fit for the SL scores compared to other models. This function is described as follows: 123 and two-sided p value between each individual’s neuropsychological test scores and the two parameters—We calculated Kendall's tau coefficientSupplementary Information.


Supplemental Digital Content is available in the text Type A aortic dissection (TAAD) is a life-threatening disorder yet it is hard to diagnose. The dissection might extend to the coronary artery causing ST-segment elevation myocardial infarction (STEMI). Physicians might not recognize this particularly early in its presentation and patients proceed to receive the primary percutaneous coronary intervention. We present such a case and found that the marked pressure difference between the radial and ascending aortae could be a useful clue for diagnosing the aortic dissection-related myocardial infarction.A 58-year-old male was presented to our emergency department for the complaint of left side chest pain that lasted for an hour with concomitant hypotension. STEMI was diagnosed at that time.The emergent primary percutaneous intervention was performed. When the diagnostic catheter was advanced to the ascending aorta, the systolic aorta pressure became 20 mm Hg higher than radial systolic pressure. Due to the abnormally large pressure differential between the peripheral radial artery and central ascending aorta, TAAD was suspected.After angiography and computer tomography confirmed the diagnosis of TAAD, the patient was sent for emergent surgery.The patient was died because of extensive dissection and shock.We present such a case and found that the marked pressure difference between the radial and ascending aortae during catheterization could be a useful clue for diagnosing the aortic dissection-related myocardial infarction. This clue had hinted our speedy examination of the occluded coronary artery and dissection flap, and led to an early and accurate diagnosis. It is therefore important to diagnose as early as possible the STEMI that occurs secondary to TAAD. To shorten the “door-to-balloon” time, the required image examinations are often not done soon enough to diagnose the primary disorder. More frequently, TAAD is misdiagnosed as STEMI and treatment procedure taken is either the primary percutaneous coronary intervention (PCI) or thrombolytic therapy. TAAD dissection might occlude the ostium of coronary artery which slows down the procedure of PCI in searching the ostium of coronary artery. A prolonged procedure of PCI might hold making the correct diagnosis of TAAD and subsequently delaying the proper surgical therapy. Here, we present a case with the patient initially presented with STEMI and shock, and who received primary PCI thereafter. During the advancement of the diagnostic catheter, we found a marked pressure difference between the ascending aorta and the radial artery. This finding gave a hint for a possible TAAD. We then performed a manual aortogram which revealed the presence of an intimal flap, confirming the diagnosis of TAAD.Stanford type A aortic dissection (TAAD) is a life-threatening disease but is hard to diagnose. It has high mortality (18%–58%), and the mortality rate increases with time lapsed after symptom onset.2Written informed consent was obtained from the patient's wife for publication of this case report and accompanying images.3A 58-year-old male with hypertension without regular medical control was presented to our emergency department for the complaint of left side chest pain that lasted for an hour with concomitant hypotension. He had no cold sweating, radiating pain, or claudication. The 12-lead electrocardiogram suggested inferior wall and right ventricle STEMI, including ST elevation in leads III, and aVF, lead I with reciprocal ST depression, and right side V4R elevation . Due to the abnormally large pressure differential between the peripheral radial artery and central ascending aorta, TAAD was suspected. After a short trial that failed to locate ostium of the right coronary artery, we withdrew the diagnostic catheter from the ascending aorta back to the innominate artery and there, we found the maximal pressure damping site around the junction of aorta arch and innominate artery  is extremely rare. Since TAAD has a high mortality rate (estimated 1%–2% per hour increase immediately following symptom onsets in untreated patients), physicians should be aware of the possible diagnosis of TAAD in STEMI patients. Although careful physical examination and accurate history taking, such as tearing chest pain and radiating to back, pulse or/and blood pressure differentials, may help in the differential diagnosis of TAAD from true STEMI, it is still challenging as symptoms are mostly similar between these two. Moreover, the typical clinical presentation as well as radiographic findings might not always be present. On top of these, for STEMI patients, the required image studies (such as transthoracic echocardiography or computed tomography) may not be indicated initially, because the imaging procedure may cause delay and prolong the “door-to-balloon” time. Therefore, some STEMI patients with TAAD might proceed to receive primary PCI, which might not readily detect TAAD. It is, therefore, crucial to increase the awareness of cardiologists during primary PCI regarding clues of TAAD related STEMI.Only 2.5% of patients with TAAD has initial presentation as STEMI. If the catheter were placed in the false lumen, greater resistance will be experienced. However, if the catheter were instead placed in the true lumen, the resistance will appear low or even be free of resistance. Nowadays, the radial rather than the femoral approach is used for primary PCI and the chance of driving the catheter into the true lumen is higher. For this reason, the diagnosis of TAAD is even more difficult to make. In our case, there is no resistance while advancing the catheter. It was only the marked pressure difference between the radial artery and the ascending aorta that had hinted us the possibility of TAAD. We withdrew the catheter to locate the site with the greatest pressure drop and found it near the junction between the aorta arch and innominate artery. The aortography, conducted with manual injection of contrast medium, showed a dissection flap over the ascending aorta, thus confirming the diagnosis of TAAD. The false lumen of TAAD could compromise the true lumen of major branches (such as innominate artery) making the pressure drop. Therefore, the pressure differential we found could be a useful and time-saving clue of TTAD related STEMI. In addition to the pressure changes observed during PCI, other phenomena also occur and are helpful. For example, following the injection of contrast medium into the aorta, the following might be observed:In a study by Wang et al, increased resistance in PCI during physical advancement of the diagnostic catheter was felt in 60% of cases with STEMI secondary to TAAD.(1)a discrepancy in diameters between the aortic root and the ascending aorta (100% occurrence),(2)presence of intimal flap in the ascending aorta (75%), and(3)the disappearance of the coronary artery ostium (40%). While these clues may not present themselves simultaneously in any individual patient, physicians should be cautious of these signs to save time in searching the occluded ostium of coronary artery and to lower the risk of further damages to the dissected vessel. The sooner the diagnosis of TAAD is made, the better is the survival chance.All of the above findings are important diagnostic auxiliary clues for TAAD related STEMI.Catheter directed a large blood pressure difference between the radial and ascending aorta is a helpful clue for the diagnosis of TAAD related STEMI.Data curation: Yu-Po Hung.Formal analysis: Tzu-Hsiang Lin, Szu-Ling Chang.Writing – original draft: Chi-Yao Huang.Writing – review and editing: Wen-Lieng Lee, Chih Hung Lai.Chih Hung Lai orcid: 0000-0003-1409-4979.