To elucidate the mechanism of the Ex-upregulated NO system in the kidney, we examined the additive effect of Ex and tempol on the renal NO system in SHR and WKY. Our data showed that, in SHR, both Ex and tempol increase the levels of H2O2 and nitrate/nitrite (NOx) in plasma and urine. We also observed an increased renal NOS activity and upregulated expression levels of eNOS and nNOS with decreased NADPH oxidase activity. The effects of the combination of Ex and tempol on these variables were cumulate in SHR. On the other hand, we found that Ex increases these variables with increased renal NADPH oxidase activity, but tempol did not change these variables or affect the Ex-induced upregulation in the activity and expression of NOS in WKY. The SOD activity in the kidney and aorta was activated by tempol only in SHR, but not in WKY; whereas Ex increased SOD activity only in the aorta in both SHR and WKY. These results indicate that Ex-induced endogenous H2O2 produced in the blood vessel and other organs outside of the kidney may be carried to the kidney by blood flow and stimulates the NO system in the kidney.Both exercise training (Ex) and superoxide dismutase (SOD) mimetic tempol have antihypertensive and renal protective effects in rodent models of several hypertensions. We recently reported that Ex increases nitric oxide (NO) production and the expression levels of endothelial and neuronal NO synthase (eNOS and nNOS) in the kidney and aorta of the spontaneously hypertensive rats (SHR) and normotensive Wistar–Kyoto rats (WKY). We also found that endogenous hydrogen peroxide (H Exercise training (Ex) reduces the systemic blood pressure in human  and the Nitric oxide (NO) is a vasodilatory factor that can be synthesized by three isoforms of NO synthase (NOS): endothelial, neuronal, and inducible NOS , and it controls the systemic blood pressure and peripheral hemodynamics . NO has 2−) is generated by NADPH oxidase [2O2) by superoxide dismutase (SOD). Reactive oxygen species (ROS) and oxidative stress are elevated in SHR [2O2 administered intravenously increases the levels of eNOS and nNOS in the kidney and aorta of WKY [2O2 could mediate the expression of NOS in the kidney and vessels of SHR. H2O2 may increase NOS expression and activity by improving the rate of gene transcription and mRNA processing and stability [Superoxide anion  and eNOS expression in the mouse aorta , and the of mice . Conside of mice  and H2O2ice [2O2  increaseFive-week-old male SHR/Izm and WKY/Izm were obtained from SLC . These rats were housed in the animal care facility at the Tohoku University School of Medicine under controlled temperature (24°C) and a 12 h light-dark cycle. All rats had free access to standard laboratory chow and water. All protocols involving rats were reviewed and received prior approval by the Animal Welfare Committee at the Tohoku University School of Medicine.n = 10 in each group): control, Ex, tempol-treated (Tmp), and an Ex + Tmp. The treadmill running  was performed with a rat treadmill  to the Ex and Ex + Tmp groups, and tempol (1 mmol/l) was given to the Tmp and Ex + Tmp groups through drinking water for 8 weeks.SHR and WKY were randomly divided into four groups (The systolic blood pressure (SBP) was monitored by the tail-cuff method . The rats were placed metabolic cages  individually on the day before the final experimental day, and urine samples were collected on ice over a period of 24 h. On the final experimental day, the rats were anesthetized with pentobarbital sodium , and blood samples were collected by decapitation. These samples were centrifuged for 5 minutes at 1,500 rpm and separated from the sediments and stored at −80°C.2O2 was measured using an Amplex Red Hydrogen Peroxide/Peroxidase Assay kit  [Creatinine and urea nitrogen were measured by a standard autoanalysis technique, . HOR, USA) . NitrateOR, USA)  using th2− generation by the lucigenin-enhanced chemiluminescence method [μmol/g protein/unit time for the NOS activity.The preparation of tissue samples including the renal cortex (CO), outer medulla (OM), inner medulla (IM), and aorta was described previously . The proe method , 27 and e method . The NO β-actin.The level of NOS was examined by Western blot, as described previously , 35. TheP < 0.05 was considered as statistical significance. All statistical analysis data were performed using the SPSS 19 software .Data are presented as the means ± SEM. The comparison between the WKY control and SHR control used the one-way ANOVA. In WKY or SHR, compared with the control group, Ex, Tmp, and Tmp + Ex were all equivalent to the single factor intervention group, so one-way ANOVA was used. Subsequently, for the comparison between Ex, Tmp, and Tmp + Ex groups, data analysis was performed by Fisher's probability least significant difference (LSD) test for multiple comparisons after one-way ANOVA. In all analyses, a two-tailed 2O2 and NOx were significantly higher in the control SHR group than in the control WKY group (P < 0.01), but plasma creatinine and urea nitrogen and creatinine clearance were not significantly different between these control groups. In SHR, Ex and tempol significantly decreased the SBP, and there is an additive effect of the combination of Ex and tempol on the SBP. Ex significantly decreased the plasma creatinine and increased creatinine clearance (Ccr), which reflects the glomerular filtration rate (GFR), and tempol tends to increase Ccr (P=0.062). Other variables were not different among the four groups. In contrast to SHR, neither Ex nor tempol affected SBP or plasma variables in WKY.The SBP, DBP, body weight, kidney weight, and plasma variables in the four groups of SHR and WKY are shown in Tables 12O2 in plasma and urine  compared to controls. Notably, the combination of Ex and tempol further increased H2O2 in plasma and urine by 50% and 62% (P < 0.01) compared to the control group, and the differences between the Ex + Tmp and the Ex and Tmp only groups reached statistical significance  (2O2 by 36% in urine (P < 0.05) compared to controls, but Tmp did not significantly change it. The combination of Ex and Tmp increased H2O2 by 35% in urine (P < 0.05) compared to controls, but the plasma and urinary H2O2 levels were not significantly different between the Ex + Tmp group and Ex only group  . In WKY,ly group .P < 0.01; tempol: by 18% and 26% P < 0.01); the combination of Ex and Tmp further increased the NOx in plasma and urine to 38% and 47% compared to the controls (P < 0.01), and the NOx levels in the plasma and urinary were significantly higher in the Ex + Tmp group compared to the Ex and tempol groups (P < 0.01) (P < 0.01), but they were not significantly different in the Ex + Tmp group compared to the Ex group  . In WKY,Ex group .In SHR, Ex and Tmp significantly decreased the activity of NADPH oxidase in the CO, OM, and aorta. An additive effect of the combination of Ex and Tmp on the NADPH oxidase activity in these tissues was also found . In WKY,In SHR, Ex significantly increased SOD activity in the IM and aorta, but not in the CO and OM; Tmp significantly increased SOD activity in the kidney sections and aorta; there is an additive effect of the combination of Ex and Tmp on the SOD activity in the IM and aorta . In WKY,In SHR, Ex and Tmp significantly increased NOS activity in the kidney sections and aorta; an additive effect on NOS activity was observed for Ex and Tmp in the kidney sections and aorta . In WKY,The protein levels of eNOS and nNOS in the kidney sections and aorta were also examined. As shown in As shown in 2O2 and NOx levels and renal NOS expression with decreased NADPH oxidase activity in SHR, but with increased NADPH oxidase activity in WKY. We also observed an additive effect of Tmp and Ex on these variables in SHR, but not in WKY.Although Ex increased the NOS activity and protein levels of eNOS and nNOS in the aorta and kidney of SHR and WKY, the mechanism of the Ex-upregulated NO system in the kidney has not yet been discovered. In the present study, we found that Ex increases concomitantly plasma and urinary H2O2 and NOx levels in SHR, and endogenous H2O2 upregulates NO production [2O2 and NOx in SHR, but not in WKY. Since the effects of Ex and tempol are similar, we think that Ex-induced NO production may also be related to the endogenous H2O2 in the plasma and urine of SHR.In our previous study, we found that Tmp increases the plasma and urinary Hoduction . The pre2− in SHR may be dependent on elevated NADPH oxidase activity, and tempol increases the H2O2 level and decreases NADPH oxidase activity through the activation of SOD in SHR [2O2 [2O2 and downregulation of NADPH oxidase activity might depend on increased SOD activity in SHR. Furthermore, we showed an additive effect of the combination of Ex and tempol on H2O2, NADPH oxidase, and SOD activities in the aorta of SHR. In addition, the previous study has reported that the catalase and glutathione peroxidase activities were not significantly increased in SHR [2O2 could not be upregulated. Thus, in the present study, we also reported upregulation of H2O2 not only in plasma but also in urine of SHR.We have shown that upregulation of OD in SHR . It has SHR [2O2 , so we pSHR [2O2 . These dd in SHR . Subsequ2O2 is more significant in urine than in plasma of WKY. Taken together, Ex-increased plasma H2O2 may be carried to the kidney with blood flow and causes the significant increase in urine of WKY. In addition, the degree of Ex-induced NADPH oxidase activity in the kidney and aorta of WKY was still lower than that in the SHR control, and SOD activity of WKY control is two more times more than of the SHR control. We found that tempol had no effect on SOD activity not only in the WKY control group but also in the Ex-induced WKY group.It has been shown that Ex has no effect on the plasma and urinary TBARS levels in WKY . In the 2O2 increased the protein level of NOS in the kidney of WKY [2O2 increased eNOS phosphorylation in rats' aortas [2O2 levels in SHR. Furthermore, the Ex-increased NO system in the kidney and aorta of WKY is also accompanied with the increase in H2O2. Thus, endogenous H2O2 may be a mediator of Ex-induced NOS activity and NOS expression in the kidney and aorta [2O2 and the NO system in SHR (data not shown) could be explained by the sufficiency of low dose of tempol on suppressing the oxidative stress in combination with Ex in SHR.We previously reported that both Ex  and tempy of WKY , and exo' aortas . A key fnd aorta , 31. The2− and SOD activity. Taken together, Ex-increased endogenous H2O2 in the plasma and urine might be produced in the blood vessel and other organs including the skeletal muscle, but not the kidney. The endogenous H2O2 was produced by Ex-increased SOD activity and NADPH oxidase activity in the aorta and would be then carried to the kidney with blood flow and causes the increase of the renal NOS activity and expression in SHR and WKY. Further studies are needed to identify the nonrenal H2O2-mediated mechanism of Ex-induced NOS activity and expression in the kidney.Fukai et al. reported that Ex-upregulated the expression levels of vascular eNOS and extracellular SOD in wild-type mice . In the + reabsorption, tubuloglomerular feedback (TGF), and sympathetic nerve activity [Ex increased NOS activity and expression in the kidney and aorta, which in turn improved numerous physiological effects of NO in SHR, including vasodilatory responses  and variactivity , 11, 42.activity , Ccr, glactivity . It is aactivity , and theactivity . The renactivity . Whether2O2 has the effect on vasodilation. H2O2 has been shown to be a major component of the endothelium-derived hyperpolarizing factor (EDHF) in several vascular beds in multiple species [2O2 could also cause interprotein disulfide bond in protein kinase G (PKG) I-α, which activates the kinase independent of the NO-cyclic guanosine monophosphate (cGMP) pathway and coupled to vasodilation [2O2-induced vasorelaxation in mice mesenteries [2O2 on vasorelaxation. However, other studies have yielded conflicting results that H2O2 revealed vasoconstrictor effects: endothelium-dependent contraction [It is widely accepted in recent years that not only NO but also H species , 46, and species . H2O2 codilation . Moreoveenteries . Consisttraction  or vasomtraction , 52 effe2O2 production in the blood vessel and other organs including the skeletal muscle but not the kidney.In conclusion, the present study indicates that the combination of Ex and Tempol offers additive effects on the NOS activity and levels of eNOS and nNOS in the kidney with decreased renal NADPH oxidase activity and increased arterial SOD activity in SHR. We also demonstrate that the Ex increases the renal NO system with increased renal NADPH oxidase activity and arterial SOD activity in WKY. These results suggest that the Ex-upregulated NO system in the kidney is mediated through the increased endogenous H


The static mechanical, dynamic thermal-mechanical properties and thermostability of as-prepared g-C3N4/epoxy nanocomposites were significantly ameliorated compared with that of the pure epoxy matrix. The tensile modulus and flexural modulus of g-C3N4/epoxy nanocomposites increased by 31.81% and 28.28%, respectively. Meanwhile, the tensile and flexural strength was also improved by 16.02% and 12.67%, respectively. The g-C3N4/epoxy nanocomposites exhibited an increased storage modulus and glass transition temperature. The markedly improved mechanical and viscoelasticity properties were attributed to the stronger interfacial interaction caused by enlarged contact area and increased chemical bonding, and enhanced mechanical interlocking on the interface. The loss factor of epoxy nanocomposites also raised by 40% due to the comprehensive effect of frication caused by the relative slip between nanosheets, micro-constrained layer damping structure and the reversible cycle of breakage and re-established of the hydrogen bond. Meanwhile, the 10% weightlessness temperature , semi weightlessness temperature  of g-C3N4/epoxy nanocomposites have increased by about 15 °C and 14 °C, respectively.In this study, graphitic carbon nitride (g-C Epoxy resin as an excellent matrix material was used widely in the field of aerospace, automobile and architecture. However, some properties of epoxy resin are far from satisfactory at present ,2,3,4. T3N4) as a polymeric semiconductor with graphite-like structure has attracted extensive attention of researchers all over the world owing to its low-cost and good chemical stability. C3N4 is a carbon-nitrogen compound that has been discovered [3N4) is the most stable one at room temperature and atmospheric pressure. Allotropes of other structural forms are stable only at high temperatures, which are hard to prepare and difficult to study. Wang et. al. [3N4 could be used as a metal-free photocatalyst to decompose water to produce hydrogen under visible light, which has triggered an upsurge in the research of carbon nitride in the field of photocatalysis. The 2D monolayer of g-C3N4 exhibited a considerable specific surface area and abundant residual functional groups (−NH2 or −NH). These groups can act as active sites for the formation of strong interface bonding or grafting other functional groups to improve its compatibility with polymer matrices. All these characteristics confirmed that the enormous potential of g-C3N4 as an ideal reinforcement for the high-performance composites with excellent mechanical properties. Shi et al. [3N4 nanosheets on thermal stability and mechanical properties of sodium alginate films. The initial thermal degradation temperature increased by 29.1 °C and the tensile modulus remarkably increased from 60 to 3540 MPa after adding 6 wt.% g-C3N4 into the matrix. Furthermore, Shi et al. [3N4/polypropylene nanocomposite in the following research. The initial and half thermal degradation temperature was raised by 14.6 °C and 27.7 °C, respectively. The storage modulus was also increased from 2445 MPa to 2783 MPa. Sareshkeh et al. [3N4 reinforced epoxy-vinyl resin. The presence of g-C3N4 increased to the tensile strength and Izod-impact strength by 49.2% and 25.37%, respectively, and promoted the Tg (glass transition temperature) and IDT  by 6.46% and 12.05%, respectively. The interlaminar shear strength and interfacial shear strength of composite laminates were increased from 51.84 to 72.09 MPa and 44.62 to 73.41 MPa, respectively. Although the remarkable progress in g-C3N4 reinforced composites have been achieved, the potential of g-C3N4 in the design and fabrication of high-performance and functional composites still remain to be further explored and utilized.Recently, graphitic carbon nitride  was purchased from Aladdin . Epoxy resin (HT-723A) and curing agent (HT-723B) were supplied by Wells Advanced Materials Co., Ltd. .3N4 and epoxy nanocomposites is shown in 3N4 were prepared through the thermal decomposition of urea. In a typical synthesis process  was added into epoxy resin and mixed by a high-speed shear dispersion homogenizer at 10,000 rpm for 20 min, and then the curing agent was mixed into the above suspension at a mass ratio of 30:100. The mixture was casted into a steel mold for curing after defoaming treatment. A typical curing procedure was as follows: 35 °C for 30 min, 50 °C for 120 min and then 120 °C for 180 min. According to the content of g-C3N4 in an epoxy matrix, the as-prepared g-C3N4/epoxy nanocomposites were labeled as EP-CN0.0/0.5/1.0/1.5/3.0/4.0/5.0, respectively.The preparation of epoxy nanocomposites is also shown in 3N4, epoxy and nanocomposites were characterized by X-ray diffraction . The surface functional groups of g-C3N4 were investigated by flourier transform infrared spectroscopy . The morphologies of g-C3N4 was observed using transmission electron microscopy  and scanning electron microscope . The specific surface area (SSA) and pore size distribution of g-C3N4 were tested by nitrogen physisorption apparatus .The crystal structure of g-C3N4/epoxy nanocomposite was measured using a universal testing machine  with a high-precision load cell with self-identification function (TEDS). According to the tensile standard of ASTM D638, a dumbbell-shaped specimen was selected, with a total length of 165 mm, a width of 13 mm at the middle, 19 mm at both ends, a gauge length of 50 mm and a thickness of 3.2 mm. The standard of ASTM D790 was applied for the flexural test. At least five valid data were obtained from seven samples in each group. The fracture surfaces of the tensile test samples were investigated by SEM.The static mechanical properties  of g-CDynamic thermal mechanical behavior was investigated by a dynamic thermomechanical analyzer  at a fixed frequency of 1 Hz and a temperature range from 25 to 180 °C with a heating rate of 3 °C/min. Three points bending mode was chosen and the samples were machined with dimensions of 60 mm × 10 mm. The linear viscoelastic region was probed by amplitude scanning and the amplitude of 15 μm was determined. The loss factor was also measured by DMA at the mode of temperature step multi frequency scanning in a temperature range of 25–35 °C with multiple excitation frequencies. Considering to expand the test range as much as possible without resonance, these frequencies, 1/5/10/15/20/25/30 Hz, were chosen.Thermogravimetry (TG) analysis of nanocomposites was carried out on a DSC/TGA simultaneous thermal analyzer  from 50 to 750 °C at a heating rate of 10 °C/min.3N4, as shown in 3N4 [Two typical diffraction peaks can be observed in the XRD spectra of g-Cn in 3N4 ,32.3N4 is shown in 3N4 with the tri-s-triazine building units is confirmed. The absorption peak at 812 cm−1 is attributed to the out-of-plane bending vibration characteristic of heptazine rings and the characteristic mode of the triazine units of g-C3N4 [−1 is assigned to the C≡N triple bonds [3N4 prepared by thermal condensation polymerization. The absorption peaks in the range of 1650–890 cm−1 represent the typical stretching vibration modes of heptazine-derived repeating units and connected units of C−N(−C)−C (full condensation) or C−NH−C  [−1 is attributed to the stretching mode of the O−H bond and the vibration mode of the N−H bond [The FTIR analysis of g-Cf g-C3N4 . The weale bonds . Due to nsation) . The broN−H bond .3N4 powder was investigated by TEM and SEM, as shown in 3N4 nanosheets were obtained in our experiment.The microscopic morphology of as-obtained g-C3N4 is a perfect planar lattice structure formed only by the sp2 hybridization of C and N atoms. However, a certain degree of curling deformation may occur in the plane of g-C3N4 due to the repulsion between N atoms [3N4 were synthesized with the pyrolysis of urea and a large amount of ammonia and steam were released, which contributed to the expansion of the stacked-layer and promoted the graphitization of g-C3N4, thus making the thickness of the single-layer thinner and promoting the formation of nano-lamellar porous morphology. The N2 adsorption isotherms and pore size distribution of g-C3N4 are shown in 3N4 has a considerable specific surface area (97 m2/g) and pore volume (0.45 cm3/g). In addition, the pore size and distribution analysis confirm that micropores and mesopores are both existed on the g-C3N4 surface and micropores have a dominant position on pore structure of g-C3N4.The ideal g-C N atoms  and stre N atoms ,39. The 3N4/epoxy nanocomposites are shown in 3N4 centered at 26.6° appears in the XRD spectra of epoxy nanocomposites when g-C3N4 loading is no less than 3 wt.% which indicates that the larger loading of g-C3N4 (≥3 wt.%) led to incomplete exfoliation in our experiments. Compared with graphene, the dispersion of g-C3N4 in epoxy resin has certain advantages in terms of surface functional groups. The layers of g-C3N4 are bound by a hydrogen bond and Van der Waals force. In the dispersion process, epoxy resin permeated into the interlayer and the nanosheets were fully encapsulated by epoxy under shear stress. As shown in 2 or −NH) on g-C3N4 can react with the epoxy groups of the matrices, which impels the intercalation of epoxy into the interlamination and then promotes the dispersion of g-C3N4 in epoxy matrix.The state of fillers in the cured matrix greatly affects the properties of the nanocomposites. The XRD technique is a crucial method for determining the exfoliation degree of graphene in the studies of graphene reinforced polymer composites and the disappearance of stack peaks always indicates a uniformly dispersion . The XRD3N4/epoxy nanocomposites are depicted in 3N4. The maximum growth of the tensile modulus  was obtained from EP-CN4.0. Meanwhile, the flexural modulus of g-C3N4/epoxy nanocomposites also exhibits an increasing trend (raised from 2628 ± 48 to 3361 ± 37 MPa) with the increased loading of g-C3N4. EP-CN5.0 achieves the strongest value of flexural modulus (3361 ± 37 MPa) with an increased rate of 28.28%. The markedly enhanced tensile and flexural modulus of nanocomposites can be attributed to the good interfacial bonding between g-C3N4 nanosheets and epoxy. As displayed in 2 or −NH) on g-C3N4 contributes to the formation of stronger interfacial interaction between g-C3N4 and epoxy. In addition, the curly structure and the large specific surface area of the g-C3N4 nanosheet also enhance the mechanical interlocking effect with the matrix.The tensile and flexural properties of pure epoxy and g-C3N4 originated from the good interfacial adhesion between g-C3N4 and the epoxy matrix [3N4, thus resulting in more consumption of the energy and toughening of the matrix. The quantity of deviated cracks depends on the number of particles the crack encountered and this point can be confirmed by the refinement of the surface grain reflected in 3N4 nanosheets existed in a state of island-like with good interface condition in the matrix. The agglomerates appeared with further increasing of g-C3N4 and the agglomeration and homogeneous distribution of g-C3N4 were coexistent in the matrix of EP-CN3.0 and the “islands” are marked by the white dotted circle in 3N4 is completely infiltrated by epoxy and the laminated structure of g-C3N4 sheets are clearly exhibited on the surface. Although there are some gaps (marked by white arrows), the agglomerate is still completely infiltrated by the matrix, exhibiting good bonding with the matrix (marked by black arrows). Nevertheless, as the increased loading promotes the formation of more strong interfacial interaction, it also leads to poor dispersion of the g-C3N4. The existence of excessive g-C3N4 results in the formation of large-size agglomerates and the reduction of crack deviation efficiency. The agglomerates with layered structure dramatically reduce the specific surface area and wettability of g-C3N4 thus the mechanical properties of nanocomposites decrease. On the whole, the enhancement of g-C3N4 is comparable to that of graphene [3N4 would be required to achieve the same enhancement effect.The increasing of the tensile strength (16.02%) and flexural strength (12.67%) of nanocomposites are not obvious enough as a modulus, and a considerable number of particle enhancement nanocomposites presented similar results. The fracture morphology of epoxy nanocomposites after the tensile test was investigated using SEM. The fracture section of epoxy is a relatively smooth surface with wave morphology a while ty matrix . The dirgraphene , but a l3N4/epoxy nanocomposites are shown in 3N4/epoxy nanocomposites exhibit an increased storage modulus at the glassy state region due to the reinforcing effect of the g-C3N4 nanosheets, compared to pure epoxy. The storage modulus of EP-CN5.0 is 2892 MPa at 30 °C 1 Hz while that of epoxy is 2754 MPa. The effect of loading on the enhancement is similar to that of static bending test, but the storage modulus of nanocomposites decreases significantly with the increase of temperature, which is mainly determined by the matrix material used in our experiment [3N4. Tg is associated with the fundamental changes in polymer chain dynamics, thermal stability and many other critical applications [g [g of EP-CN1.0 and EP-CN1.5 both increases about 8 °C in comparison to that of the epoxy matrix. It is also worth noting that when the loading is more than 3 wt.%, as the loading increases, the enhancing effect on Tg weakens. It can be seen from g of the other several kinds of addition configuration have all been improved to varying degrees which is attributed to the restriction effect of strong attractive interactions formed by the reaction between residual functional groups (−NH2 or −NH) of g-C3N4 and epoxy group on the motion of epoxy molecular chain. In addition, the functional groups carried by g-C3N4 remedy the disproportionality of local functional group and played a role in repairing local grids, as shown in g value of EP-CN5.0 with an excessive amount of g-C3N4 is 3 °C below that of pure epoxy due to the deterioration of dispersion, reduction of interfacial area and formation of fillers-rich or poor regions [The storage modulus plots for pure epoxy and g-Cperiment . Figure ications ,43. Genetions [g . Nano-sc regions .3N4 loading and the maximum value is obtained at 3 wt.% loading. The loss factor of EP-CN3.0 under the excitation frequency of 1 Hz at 30 °C increased from 0.035 to 0.049. It is generally believed that the energy dissipation originated from interfacial friction is the main reason for the enhancement of damping properties of nanocomposites. However, the strong interfacial interaction between g-C3N4 and matrix means the critical interfacial shear strength could be relatively high and the interfacial slip is not easy to occur. Nevertheless, the loss factor of nanocomposites has been significantly improved, which indicates the existence of other forms of energy dissipation. Moreover, it is possible that the increased loss factors are related to the multiscale laminated structure of g-C3N4. The multiscale nanoparticles exhibited both fully dispersed and insufficiently dispersed states in the matrix. In the state of fully dispersed, most of the particles existed in the form of non-oriented single nanosheet, while in the state of insufficiently dispersed the existence of stacked structures in the matrix can be divided into three situations, as shown in 3N4. Therefore, in addition to interfacial friction, the reversible cycle of breakage and re-established of hydrogen bond  and semi weightlessness temperature  are generally regarded as an index of the thermal stability. As displayed in initial and Thalf of g-C3N4/epoxy nanocomposites increased by about 15 °C and 14 °C, respectively. This is possibly due to the addition of g-C3N4 has a hindrance to the decomposition of epoxy resin. Similar results were found in graphene and nano-clay reinforced nanocomposites [3N4 delayed the permeation of oxygen and the escape of volatile degradation products and also char formation [The effect of g-Cmposites . The so-ormation .3N4 reinforced epoxy nanocomposites were prepared and characterized. In our experiment, when the loading was less than 3 wt.%, g-C3N4 can obtain excellent dispersion in epoxy. Results indicated that the static mechanical, dynamic thermal-mechanical and thermal stability properties of g-C3N4/epoxy nanocomposites had been significantly improved. Compared to neat epoxy, the tensile modulus and flexural modulus of g-C3N4 reinforced epoxy matrix increased by 31.81% with a loading of 4 wt.% and 28.28% with a loading of 5 wt.%, respectively. However, the maximum values of tensile and flexural strengths were achieved at smaller additions (1–1.5 wt.%). In terms of the magnitude of the enhancement, the enhancement effect of g-C3N4 on strength was relatively weak. The loss factor of nanocomposites (3 wt.%) under the excitation frequency of 1 Hz at 30 °C increased from 0.035 to 0.049. Meanwhile, the Tg of nanocomposites (1 wt.% and 1.5 wt.%) increased about 8 °C in comparison to that of epoxy matrix and Tinitial and Thalf increased by about 15 °C and 14 °C, respectively. The structural characteristics of graphite-type carbon nitride, such as multi-scale lamination structure, large specific surface area and naturally carried amino groups, played an excellent role in promoting the dispersion of nanoparticles and strengthening epoxy matrix. The g-C3N4 as a novel filler for fabricating epoxy nanocomposites is not only feasible but also excellent. The strategy developed in this study could provide a new avenue for the fabrication of high-performance composites. In the follow-up study, we will do more in-depth research on the effect of g-C3N4 on the friction properties of the epoxy matrix, the effect of the existing form of staking structure in the matrix on the properties of nanocomposites, and the relationship between energy dissipation of hydrogen bond in the interlayer and temperature.Here, the g-C


Primary cilia act as crucial regulators of embryo development and tissue homeostasis. They are instrumental for modulation of several signaling pathways, including Hedgehog, WNT, and TGF-β. However, gaps exist in our understanding of how cilia formation and function is regulated. Recent work has implicated WNT/β-catenin signaling pathway in the regulation of ciliogenesis, yet the results are conflicting. One model suggests that WNT/β-catenin signaling negatively regulates cilia formation, possibly via effects on cell cycle. In contrast, second model proposes a positive role of WNT/β-catenin signaling on cilia formation, mediated by the re-arrangement of centriolar satellites in response to phosphorylation of the key component of WNT/β-catenin pathway, β-catenin. To clarify these discrepancies, we investigated possible regulation of primary cilia by the WNT/β-catenin pathway in cell lines  commonly used to study ciliogenesis. We used WNT3a to activate or LGK974 to block the pathway, and examined initiation of ciliogenesis, cilium length, and percentage of ciliated cells. We show that the treatment by WNT3a has no- or lesser inhibitory effect on cilia formation. Importantly, the inhibition of secretion of endogenous WNT ligands using LGK974 blocks WNT signaling but does not affect ciliogenesis. Finally, using knock-out cells for key WNT pathway components, namely DVL1/2/3, LRP5/6, or AXIN1/2 we show that neither activation nor deactivation of the WNT/β-catenin pathway affects the process of ciliogenesis. These results suggest that WNT/β-catenin-mediated signaling is not generally required for efficient cilia formation. In fact, activation of the WNT/β-catenin pathway in some systems seems to moderately suppress ciliogenesis. Primary cilia are tubulin-based rod-shaped organelles on the surface of most mammalian cells. They play a fundamental role in embryo development and tissue homeostasis. Importantly, defects in primary cilia structure and function lead to variety of developmental disorders collectively called ciliopathies . MoreoveCilium formation is organized by the mother centriole (MC)-derived basal body, the older centriole of the pair that makes up the centrosome. While centrosome is best known as microtubule organizing center coordinating mitosis, primary cilium formation is tightly connected with G1/G0 phase . The groWNT signaling pathways are developmentally important signaling routes regulating cell differentiation, migration, and proliferation and their activity controls shaping of the embryo . WNT sigWhilst the connections between primary cilia and hedgehog signaling are well documented , the relTo make the matters even more puzzling, two opposing models have recently emerged regarding possible function of WNT/β-catenin pathway in cilia formation. Activation of the WNT/β-catenin pathway in neural progenitors of the developing cerebral cortex was reported to hamper cilia formation in mice , arguing®  supplemented by 10% FBS and 1% Penicillin/Streptomycin. Where indicated, RPE-1 cells were starved by serum free medium, NIH3T3 cells were starved by 0.1% FBS containing medium, and HEK293 cells were starved by serum free medium for 24 h. Cells were seeded at 50,000/well (RPE-1 and NIH3T3) or 120000/well (HEK293) of 24 well plate. Treatments by small molecules were done for indicated times: LGK974 (0.4 μM)  for 72 h  supplemented by 10% FBS , 1% Penicillin/Streptomycin  and 1% L-glutamine , HEK293 T-Rex  and NIH3T3 cells were grown in DMEM Glutamaxn = 3. Statistical analyses by students t-test or one-way ANOVA were performed using Graphpad Prism, P < 0.05 (∗), P < 0.01 (∗∗), P < 0.001 (∗∗∗), and P < 0.0001 (****).Western blot was performed as previously described . AntibodoC methanol, washed 3× by PBS, blocked (2% BSA in PBS with 0.01% NaN3), 3× washed by PBS, incubated with primary antibodies for 1 h, 3× washed by PBS, incubated with secondary antibodies  for 2 h in dark, washed 3× by PBS, incubated 5 min with DAPI, 2× washed by PBS and mounted to glycergel (DAKO #C0563). Microscopy analysis was done using Zeiss AxioImager.Z2 with Hamamatsu ORCA Flash 4.0 camera, 63× Apo oil immersion objective, and ZEN Blue 2.6 acquisition SW (Zeiss). Image stacks acquired using Zeiss AxioImager.Z2 were projected as maximal intensity images by using ImageJ distribution FIJI , P < 0.01 (∗∗), P < 0.001 (∗∗∗), and P < 0.0001 (****). Results are presented as mean plus SEM. Primary antibodies used: Arl13b , γ-tubulin , CP110 , and TTBK2 .RPE-1, NIH3T3 and HEK293 cells were seeded on glass coverslips, treated as indicated, washed by PBS and fixed for 10 min in −20ion FIJI . Where aommended . For theTransfection and dual luciferase assay of HEK293 WT and KO cells was carried out as previously described . In brieUsed guide RNAs were following: LRP5 gRNA gagcgggccgacaagactag, LRP6 gRNA ttgccttagatccttcaagt, AXIN1 gRNA cgaacttctgaggctccacg, and AXIN2 gRNA tccttattgggcgatcaaga. gRNAs were cloned into pSpCas9 (BB)-2A-GFP (PX458)  or pU6-(BbsI)_CBh-Cas9-T2A-mCherry  plasmids. Following transfection by Lipofectamine 2000 (Thermo Fisher Scientific) the transfected cells were FACS sorted [FACSAria Fusion (BD Biosciences)] and clonally expanded. Genotyping of LRP5 KO and AXIN2 KO mutants was done following genomic DNA isolation  by PCR using DreamTaq DNA Polymerase (Thermo Fisher Scientific). Used primers: LRP5 forward: gttcggtctgacgcagtaca, LRP5 reversed: aggatggcctcaatgactgt, AXIN2 forward: cagtgccaggggaagaag, and AXIN2 reversed: gtcttggtggcaggcttc. PCR products were cut by BfaI  in case of LRP5 KO and Hpy188III  for AXIN2 KO screening, respectively. Successful disruption of individual ORFs was confirmed by sequencing, First, we tested if primary ciliogenesis can be modulated by activation of WNT/β-catenin pathway in RPE-1 by recombinant WNT3a. Experiment outline is schematized . We initHaving found WNT3a-activated WNT/β-catenin signaling is not sufficient to promote cilia formation, we tested a possibility that steady state WNT signaling is required for effective ciliogenesis. WNT/β-catenin pathway is intensively studied as a driver of oncogenic growth, thus there are currently available various small molecules that inhibit WNT ligand secretion. To this end, we used a Porcupine inhibitor LGK974 to block the secretion of endogenous WNT ligands and in turn block the steady state WNT signaling . As a poTo corroborate our findings, we established a panel of HEK293 cells devoid of critical components of WNT signaling pathways. To specifically block the course of WNT/β-catenin pathway we used LRP5/6 double knock out HEK293 cells, to block the course of any WNT signaling pathway we used DVL1/2/3 triple knock out HEK293 cells  and to oFirst, we have verified successful disruption of LRP5 gene by sequencing , 3A, andHaving characterized our model system, we examined cilia formation in those cells. Consistently with previous work, HEK293 cells form cilia less frequently than RPE-1 or NIH3T3 cells . We wereRegulation of ciliogenesis is a complex process involving multiple factors directly or indirectly influencing cilia initiation and elongation. The regulators of cilium formation encompass a wide range of molecules such as components of centrioles, regulators of vesicular trafficking, intraflagellar transport proteins, membrane proteins, and components of cytoskeleton .WNT3a is considered a prototypical “canonical” WNT ligand that activates WNT/β-catenin pathway . MoreovePrincipally, there are several important methodological differences between our work and the previous results  which maOur data show that while WNT3a consistently activates the WNT/β-catenin pathway, it has no or minor negative effects on ciliogenesis. Elevated β-catenin levels following APC ablation have been related to reduced ciliogenesis and cell cycle defects in the developing cortex in mice . Indeed,In sum, we found no evidence that endogenous WNT/β-catenin signaling, while ablated either pharmacologically in RPE-1 or NIH3T3 by LGK974, or genetically by removal of LRP5/6 in HEK293, is required for primary cilia to form. Our findings presented in this article challenge some of the published evidence and argue against positive role of WNT3a or WNT/β-catenin pathway in ciliogenesis or cilia length regulation.The original contributions presented in the study are included in the article/OB designed and performed the experiments, and wrote and edited the manuscript. PP and AK performed selection and verification of CRISPR edited HEK293 cell lines. VB edited the manuscript. LC designed the experiments, and wrote and edited the manuscript. All authors contributed to the article and approved the submitted version.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.


The article presents a study on the adhesion of thermoplastic films to a room temperature-hardening epoxy resin, which deals with an important question on sensor integration into fibre composites. By means of a morphological box, a test specimen is developed, which allows to test strength values for the adhesion of thermoplastic films to epoxy resin. Polyimide (PI), which is typically used as a carrier material for flexible sensors, is compared with the thermoplastics polyetherimide (PEI), polyethersulfone (PES) and polyamide 6 (PA6). To evaluate the spatial formation of the interface, images taken with a light microscope, fluorescence microscope and electron microscope and an energy-dispersive X-ray spectroscopy (EDX) analysis are presented. The images show that during the curing process of the epoxy resin the initially expected pronounced interphase does not form. In this respect, it is surprising that PEI achieves such a high adhesion strength even without extended interphase formation, that the failure of the test specimen occurs in the epoxy resin region at a tensile stress of 70 MPa and not at the interface between epoxy and PEI, as might initially be assumed. It is also surprising that PES exhibits the lowest adhesion strength of 5 MPa to room temperature-hardening epoxy resin, although in previous investigations it was often used as a soluble toughness modifier for epoxy resins. The tensile adhesion strength of PI to epoxy resin was found at 27 MPa and the tensile adhesion strength of PA6 to epoxy resin was found at 13 MPa. For sensor integration, the findings mean that flexible sensors on PEI substrates promise a low tendency to delaminate even in the room temperature-hardening epoxy resin used, while the other materials tested indicate an increased tendency to delaminate. The combination of thermoplastic films with an epoxy resin matrix can be useful for a wide range of applications. On the one hand, the thermoplastic film can be applied for toughness modification to increase the crack propagation resistance of fiber-composite components . On the The findings presented in this article originate from a research project in which film sensors based on a suitable thermoplastic material film are integrated into a fiber-composite. In the sensor integration, there can be different measured variables, the present research pursues the goal of integrating the sensors into the fiber-composite for cure monitoring. The monitoring of the entire curing process enables a precise determination of the degree of crosslinking, allowing to obtain the best achievable mechanical properties of the epoxy resin ,8,9. ThiThere is no unified model for predicting the quality of adhesion, but rather a variety of theories, each of which takes different mechanisms into account . In the The literature reports the following mechanism for interphase formation. At the beginning of the interphase formation, the monomers of the epoxy resin diffuse into the thermoplastic, forming a thermoplastic region that is in a gel-like state . IndividWhile the fracture toughness of the bond between epoxy resin and thermoplastic is a parameter frequently investigated in the literature, there are few studies dealing with the strength of the interface between flexible carrier films for integrated sensors and epoxy resin. The few articles dealing with the determination of strength values almost exclusively use interlaminar shear specimens. This article presents tensile strength values for the interfaces between different thermoplastics and epoxy resin and investigates the relation to the interface formation.The epoxy resin used consists of 100 weight parts of the resin component RIMR 426 together with 26 weight parts of the hardener component RIMH 435. Both components come from Hexion . The resin is a mixture of the diglycidyl ether of bisphenol A, and the diglycidyl ether of bisphenol F and 1,4-bisbutane. The hardener component is a mixture of the aminic hardeners 3-aminomethyl-3,5,5-trimethylcyclohexylamine, trimethylhexane-1,6-diamine, polyoxypropylenediamine and m-phenylenebis(methylamine). The epoxy resin is suitable for curing at room temperature and was processed at room temperature in the tests.The following thermoplastic films were used: The PA6 film used is a 25 A test specimen was developed to examine the strength of the bond between the thermoplastic films and the epoxy resin. Therefore different concepts were elaborated in a morphological box. In the preliminary phase of the concept design, only the film materials and the epoxy resin system had been determined as well as the requirement to produce the test specimens from liquid resin by a casting process without fiber material. The sub-functions for the mechanical testing-method are divided into the stress type occurring in the interphase and the load type introduced into the specimen by the testing machine. Yet, there are no test standards for the analysis of the mechanical properties of this type of polymer combination. Therefore, testing standards for adhesion in composites, in particular adhesive bonding of plastics, have been considered. In this case, an adaption is possible due to acting adhesive forces between the epoxy-rich and thermoplastic-rich phase. The specimen geometry is characterized by the type of joint which describes the structural arrangement of the contact zone between the thermoplastic film and the epoxy. Regarding the specimen manufacturing the casting mold and its number of components, the casting process and the arrangement of the thermoplastic film are considered.Subsequently, the part solutions for the selected part-functions are sought and presented in the morphological box, see t for the specimen height and b for the specimen width.For the testing of the specimens an Instron  5567 universal testing machine with a load cell up to 10 In addition, various imaging techniques were used to investigate the interphase. For this purpose, grinding patterns were created by first embedding cross-sections in acrylic resin, then grinding and polishing them in different polishing steps with the finest grain size of 0.25 For fluorescence microscopy, the excitation wavelength ranges between 375 and 435 In order to examine the specimens under the SEM subsequently, the surface of the ground specimens for microscopic investigation is sputtered with gold for 180 s so that it is not charged by the electron beam. The accelerator voltage of the SEM is 15 er range . Due to t-test. At least five specimens were tested for each configuration. The data show that in the case of PES, PA6 and PI the interface between thermoplastic and epoxy resin does not reach the tensile strength of the reference samples. The significantly weakest bond is the interphase between the PES film and the epoxy resin. In the case of PEI, the interphase reaches the strength of the reference samples and in the bar chart, it can be seen that the measured strength values of the reference samples and the PEI-epoxy interface do not differ significantly. As the fractures occurred in the surrounding epoxy resin in four of five specimens with integrated PEI-films, the measured strength value only represents a lower estimate of the real strength of the interface.The comparison of typical tensile curves of different test specimens shows an arc in the stress-traverse path curve of the reference specimens before failure, see The subsequent microscopic investigation was intended to find a relation between interphase formation and the strength value obtained in the tensile test. While the contrast in light microscope images is created by the optical transmission of thermoplastic materials, fluorescence microscopy uses the physical property of fluorescence in different frequency ranges. Due to their solubility in epoxy resin, an interphase was initially expected for the thermoplastics PEI and PES and the sharp limit recognizable in the light microscope images  was doubThe fluorescence microscope images of the different thermoplastic films are shown in μm , 113.5 tIn order to be able to resolve even very small structures, images were taken with an electron microscope in addition to those taken with a light microscope and the fluorescence microscope. The electron microscope also offers the option of examining the interphase with energy dispersive X-ray (EDX) analysis. of 10 kV . The widThe chemical composition of the epoxy resin suggests no sulfur content in the epoxy resin. The origin of the low constant sulfur content far away from the integrated thermoplastic film has not yet been definitely clarified. In the EDX investigations of the other thermoplastic films in The three microscopic methods used to investigate the interface between the epoxy resin and the used thermoplastic films do not show any evidence of extensive interphase formation in the two to three-digit micrometer range, as has been reported so far ,18. The Due to the mediating effect of EDX, this method is restricted to a few microns. The extent of this averaging could be more accurately predicted by Monte Carlo simulations as used in . On the By comparing the experimental methodology with the literature, the deviating observations can be explained. Teuwen et al. worked with a mixture of the epoxy resins TGMAP and DGEBF with the hardener DDS and observed the dissolution necessary for interphase formation only above 120 i et al.  found den length . The mean length  on the sThe observations of the interface zone become particularly interesting when compared with the results of the mechanical experiments. Although PEI has not formed an extended interphase with the epoxy resin, the adhesion of both materials is so strong that the fracture of the test specimens takes place in the surrounding epoxy resin and not in the boundary zone. The stated strength is therefore only a lower estimate of the strength of the PEI-epoxy resin interface. The formation of a pronounced interphase, therefore, does not seem to be the necessary condition for high strength of the interface. At the same time, chemical compatibility (measured by solubility) does not seem to be a sufficient condition for high strength of the interface, since for PES a higher solubility in epoxy resin than PEI is reported by Vandi . Since eVarious requirements have to be met before sensors can be integrated into load-bearing fiber composites. The sensors must withstand the harsh conditions during the manufacturing process, satisfy the electrical boundary conditions imposed by the surrounding composite and must not impair the mechanical properties of the composite in order not to weaken the main load-bearing function. Mechanical properties include resistance to crack propagation, often measured by the critical energy release rate, and delamination tendency, which can be measured by the mechanical strength of the interface. This article shows that one way to measure the strength of the contact surface is to examine specimens consisting of a film embedded in epoxy resin.The strength of the interface is an important requirement for evaluating possible substrate materials for sensor production. The presented investigations show that, of the thermoplastic films considered, only PEI is suitable as a sensor substrate, since an increased tendency to delamination of the composites with integrated sensors can almost be excluded due to the strong bonding. Especially PES, which is promising from the point of view of solubility, showed the weakest bond to the room-curing epoxy resin in the tests, which leads to a decreased delamination resistance in a load-bearing fiber composite. An open question for future investigations is the influence of the temperature during the curing reaction on the dissolution of the PES film and therefore on the strength of the contact zone.The microscopic examinations were initially meant to show the relation between interphase formation and high interfacial strength. However, no extensive interphase can be detected. A closer look at the used techniques reveals that they are not suitable for the investigation of interphases in the sub-micron range, in the case of EDX, light microscopy and fluorescence microscopy because of the resolution and in the case of electron microscopy because of the contrast. Light microscopy and fluorescence microscopy are particularly suitable for the detection of interphases in the two to three-digit micrometer range due to the high contrast. Although electron microscopy has low contrast, it could possibly exploit its resolution advantage better in combination with a suitable etching process. EDX analysis is completely unsuitable for the observation of concentration gradients on submicron scales due to its mediating effect. In addition, because of the similar atomic composition of PEI, PI and the epoxy resin used here, these materials cannot be distinguished by EDX analysis.From the entirety of the investigations carried out here on the PEI-epoxy interface, it can be concluded that the formation of an extended interphase is not a necessary condition for high bond strength. On the other hand, high chemical compatibility is not a sufficient condition for high bond strength, as can be seen from the example of PES.


Epoxy-based adhesives are widely used for repairing or jointing the metal sheets in the industry. Because of their superior mechanical properties, the metallic nanoparticles can be selected as the additive of the epoxy adhesive. The strength of the Cu nanoparticles (CuNPs) can be expected to improve the mechanical properties of neat epoxy. In this study, CuNPs were added at various weight ratios, such as 1, 2, 5, 10, 15, and 20% into the epoxy resin adhesive. Tensile tests of the dog-bone specimens and the lap-shear tensile tests of the single lap joints were performed for obtaining the mechanical properties. In order to investigate the failure mechanisms, the fractured surfaces of the tensile test samples and adhesively joined sheets were imaged by using a Scanning Electron Microscope. The thermal properties of the adhesives were obtained by using Thermo Gravimetric Analysis and Differential Thermal Analysis. The mechanical and thermal properties of epoxy resin adhesive were improved by adding the CuNPs. The best adding ratios of CuNPs into epoxy were obtained by both mechanical and thermally point of views. As a result of this study, 15 wt% the ratio of Cu nanoparticle adding into the epoxy-based adhesive is suitable for improving the mechanical properties. On the other hand, 20% is the proper Cu nanoparticle adding ratio for the thermal properties improving. Bonding by an adhesive is widely used in many areas of the industry, from the aerospace to medical applications6. Because of their superior physical, chemical, electrical and mechanical properties, these types of joints are preferred by many appliers from the engineers to physicians8. Many adhesive materials are using for these types of bondings, such as polymers, metals, etc. One of them is the epoxy resin adhesive. Many researches have been published about the epoxy and micro or nano additives of it12. One of the application areas of the epoxy-based adhesive is to repair or patch the parts13. For example, the outer surface of an airplane body can be damaged by the impact of an object while it flies at high speed. To repair these types of parts, a proper adhesive that is compatible with the outer surface material of the airplane is needed14. These types of applications can also be met in many industrial practices. Adhesive joints are expected to behave like having similar mechanical properties of the main material. For this reason, the mechanical properties of epoxy adhesives must be improved by adding some additives when applied on to metallic surfaces15. The mixture of the epoxy resin and its curing agent exhibits as a brittle behavior after the mixture cured. But some applications of these types of adhesives are expected to have ductile behavior, especially the metal–metal joints17. The mechanical properties of the epoxy resin adhesive can be improved by some additive materials20. Both the size and type of additives can be enhanced the superior properties to matrix23. Macro, micro, or nano-sized particles have different properties and effects from their bulk form24. For example, nano-sized metallic particles have better mechanical properties26. The mechanical or physical property of the epoxy resin adhesive can be improved by adding several metallic nanoparticles. The higher weight ratio of the additives can be affected negatively to composites because of the difficulty of these additives dispersed the homogeneously into the matrix. Also, the higher additive ratios can be responsible for the lower adhesion between the matrix and the additive.Bonding of the parts made by similar and/or dissimilar materials is widely used as an industrial processes17. When repairing some electronic parts by using epoxy-based adhesives, they can be conserved from the adverse effects of higher temperatures of soldering or welding27. Adhesive materials can consist of several number of components. Similar to composite materials, these materials can be mentioned in two parts, the matrix, and the additives. Selecting of the matrix and additives is related to the adherent material surfaces. Epoxy-based adhesives can be used on many adherent materials such as polymers, wood, and metals, etc. Some particles can be used as the additive of the epoxy-based adhesives30. CuNPs are used in many areas as alloyed or alone instead of their bulk or micro-sized form. Because of the ductile behavior of the Cu, the ductility of the epoxy can be enhanced by adding the CuNPs.Adhesive joints can be used at various temperatures. Durability and strength at extreme temperatures have always been a significant limitation of adhesives31. For this reason, the nano-sized particles were selected as additive of adhesives. The expected usage area of this adhesive is the repair or manufacturing of a machine or airplane parts. Lap-Shear and tensile tests, Thermo Gravimetric Analysis (TGA), Differential Thermal Analysis (DTA), and Scanning Electron Microscopy (SEM) imaging were performed for obtaining the adhesives and joint’s properties.This study is about the application of CuNPs into the epoxy-based adhesive for industrial bonding applications. Al  2024-T3 sheets are jointed by using the epoxy-based, and CuNPs added adhesive. CuNPs content was selected as 1, 2, 5, 10, 15 and 20% in weight ratios. Al sheets used in this study are widely used in industry, especially in the aircraft industry. By adding the CuNPs, the adhesive is expected to improve its mechanical properties. However, the mechanical properties can be affected negatively for higher weight ratios by adding CuNPs. This negative effect can also be about micro or higher sized powders3, and the viscosities are 600–900/50–100 mPa s at 25 °C and the gelation time of the epoxy-hardener system is approximately 1 h. The epoxy resin and curing agent were mixed 100/40 in weight ratio as recommended by the manufacturer. This ratio can express in terms of the stoichiometric ratios as 71.4–28.6%. Al alloy adherent sheets purchased from TAI (Turkish Aircraft Industry in Turkey) were used as adherent material in a 1.6 × 25 × 101.6 (in mm) dimensions. CuNPs used in this study were purchased from Genbiotek Biosystem Laboratory Company in Turkey. The shape and size of the CuNPs are shown in Fig. Epoxy resin (MGS® L285 Bisphenol A (BPA) Epoxide value/100 g is 0.59–0.65) and its curing agent (MGS® H285) is the type of cyclo-aliphatic amine at 70–90% and polyoxyl alkyl amine mixture at 10–30% produced by Hexion (in Spain) and purchased from Dost Kimya (in Turkey) was used in this study. The densities of the epoxy resin/hardener are about 1,180–1,230/940–970 kg/m32. As recommended by the manufacturer, the mixture temperature was limited between the + 10 and + 50 °C in an ice bath. Then the mixture was stayed at vacuum for 10 min at 0.02 MPa to avoid the air bulbs. At last, the hardener was added in the mixture at 100/40 weight ratio as advised by the manufacturer and mixed mechanically for 10 min. Mixtures were stayed for 5 min in a vacuum at 0.05 MPa for removing the bubbles. Produced adhesive material was used for preparing both the dog-bone tensile test specimens and the Single Lap Joint (SLJ) tensile test specimens.In this study, seven types of epoxy-based adhesives were prepared, and the contents of the manufactured adhesives are presented in Table Tensile test samples were poured in an open mold. The open steel mold was prepared and dimensioned according to ASTM D638-10, and the mold cavity was calculated. Lap joint tests were performed according to ASTM D1002-10. The cavity between the two adherents (0.2 × 12.7 × 25.4 in mm) was calculated. The adhesive amount was adjusted that all cavities could be filled.33. The mechanical properties of the adhesive joints were obtained in accordance with the ASTM F2255 standard, and the dog-bone tensile test specimens were prepared in accordance with the ASTM D638-10 for obtaining the mechanical properties of adhesive materials. For reliability of the results, five specimens that have the same contents were tested. Tensile tests were performed on these specimens for obtaining the mechanical properties of the adhesives by using the Shimadzu AGS X series (made in Japan) tensile test machine at room conditions (25 °C and 60% relative humidity). Both the axial and the transversal displacements were measured by using Epsilon 3,560 model bi-axial extensometer during the tensile tests.Various methods can be applied to producing the bulk form of specimensf34 that the total energy of the materials carried out. These values were calculated and presented in Table The tensile test results are presented in Fig. The accuracy of the results of strength tests of adhesive bonds depends on the conditions under which the bonding process is carried out. For this reason, the Single-Lap-Joint tensile tests in accordance with ASTM D1002-10 were performed for obtaining the adherents’ bonding properties by using Shimadzu AGS X tensile test machine at a constant crosshead speed of 1 mm/min. The metal pads at grip areas of the samples were placed for eliminating the bending effect during the tensile test. Following ASTM D3933-98, chemically prepared adherents having a bondline length of 12.7 mm were bonded together, as shown in Fig. t = 0.2 mm) was provided by using the shims of proper thickness at grip areas of the tensile tests as seen in Fig. τ) and shear strains (γ) were calculated by using Eqs. , bondline length (12.7 mm), measured elongation at the jointed region (mm), thickness (0.2 mm) of the joint and the tensile load (N) respectively. The multiply w.L is the area of the shear at the adhesive joint.The thickness of the SLJ (ing Eqs.  and 2),,t = 0.2 Shear strength of the joint values calculated by Eq.  and giveFor investigating the type of failure after tensile tests, both the adhesive’s and adherents’ (Al sheets’) ruptured surfaces were imaged by Zeiss (LEO) 1430VP model Scanning Electron Microscope (SEM) made in Germany. The effect of Cu content on the failure mechanisms for some of the fractured tensile tests and SLJ tensile test specimens and can be seen in Figs. Thermal stability and the decomposition properties of the adhesives were obtained by using the Thermo Gravimetric Analysis (TGA) and the Differential Thermal Analysis (DTA) techniques. TGA results are plotted in Fig. M), resultant melting Enthalpy (ΔH), and Glass Transition Temperatures (TG) of the samples are presented in Table Almost all of the specimens exhibit the endothermic reaction between the temperature range at 225–345 °C. The area under the curve of this temperature range (in terms of heat flow versus time) is named the resultant melting enthalpy value of the specimen. Melting Temperature  is one of the thermal properties of a polymeric material. This value limits the using temperature of an adhesive39. The maximum TG value (67.1 °C) was obtained for the NE sample while the minimum (56.23 °C) for the 20CuE sample (see Table G by adding the particles) were obtained at a previous study for adding the Nano-silica particles38. Another thermal property is the decomposition temperature of a polymeric material. Decomposition temperature can be named the limit of the thermal stability of a material. The thermal stability of a material can be observed by TGA results. It can be seen in Fig. Thermal properties of the adhesive materials were examined by TGA and DTA techniques. Glass transition temperature  temperatures in almost all samples. The reason for this can be CuNPs that were not as effective as at the beginning of the tests.Another thermal property of an adhesive is the melting energy, and it was obtained by using DTA results. All samples exhibited the endothermic reaction between 225 and 345 °C interval, as seen in Fig. 40 on the effect of silver nanoparticles decorated with boron nitride nanoplates incorporated into the epoxy adhesive on thermal stability, thermal stability increased. These findings are consistent with the results obtained in this study, and shown in the TGA graphics. Dynamic mechanical analyzes were also performed in the same study and it has been shown that it provides optimum mechanical properties for 20% of the particle additive. In this study, this rate was found to be 10 wt% for CuNPs additive. The electrical and mechanical properties of the epoxy adhesive were enhanced by adding MWCNT and Ag nanoparticles41. In that study, the epoxy adhesive that consisted of 0.5 wt% MWCNT and 0.5 wt% Ag presented the higher mechanical properties than the neat epoxy.No studies on epoxy-based copper nanoparticle adhesives discussed in this study have been found. In a studyThis study was carried out to investigate the effect of copper nanoparticle addition on the properties of epoxy adhesives. Epoxy resin is hardened by adding the curing agent. After curing, these types of adhesives exhibit brittle behavior. But also these type of adhesives are used to repair or joint of the metallic components. For this purpose, a ductile property is required from the epoxy-based adhesive. Because of this, CuNPs were added into the epoxy resin adhesive as various weight ratios such as 1, 2, 5, 10, 15, and 20 wt%.While preparing the samples of this study, neat epoxy was used as a pure form by an ultrasonic mixer, but the composite samples needed to be mixed with CuNP. After the mixing, the samples were kept into the vacuum environment to avoiding the occurred air bubbles while mixing by the ultrasonic mixer. This process could not thoroughly remove the air bubbles in the composite samples. But the neat epoxy samples had not any air bubble. For this reason, some of the mechanical properties obtained by obtained the tensile tests are not values as high as expected. This negative situation could be avoided by mixing the neat epoxy sample by the ultrasonic mixer. But this application was meaningless for the user of the adhesive.By inspection of the SEM images given, one can see the CuNP were distributed almost homogeneously. But there are small and few agglomerated regions that are also seen in the SEM image. For the more mixing time, the more homogeneous mixture can be reached. In that case, the mixing process may be harmful to epoxy or nanoparticles because of the temperature rising and the mechanical effects of ultrasonic waves.Modulus of Elasticity of the Neat epoxy was decreased by adding the CuNP. It can be concluded from this result that the ductility of the epoxy-based adhesive was enhanced.The strength of the adhesive and strength of the joint is the different characteristics of an adhesive material. Maximum tensile strength (96.34 MPa) of the bulk specimen was obtained for 15 wt% Cu nanoparticle added specimen, but the maximum joint strength (15.52 MPa) was obtained for 10% Cu nanoparticle added specimen. After the SLJ tests, almost all of the failures occurred by adhesively (fracture between the adhesive and adherent). This can be commented as because the perfect integration between the adhesive and adherents can not be reached, or the strength of the composite adhesive is higher than the linking between the adhesive and adherents’ surfaces.These different results can be related to the surface condition of the adherent materials. It can also be concluded that the nanoparticles affected adhesion between the adhesive and adherents surfaces’. The bulk specimen property is the determiner only for the adhesive material. By the point of view of mechanical properties, the 15 wt% the ratio of Cu nanoparticle can be mentioned about the optimum value for the adhesive, but if we use this material as an adhesive 10% weight ratio must be selected. For the higher particle ratios, the interface between the adhesive and adherent gets smaller. Because of this effect, the 10 wt% Cu nanoparticle ratio can be advised as the proper for Al–Al joints.The modification mechanism of the mechanical properties can be commented as: CuNP have resisted to deformation of epoxy. The deformation of the neat epoxy tensile test sample is higher than almost the nanoparticle added ones. But adding CuNP have affected negatively on adhesion between the adherent surfaces and adhesive.Another essential property of an adhesive is the thermal stability and the glass transition temperature. Especially for the metallic adherents, the adhesive must conserve its properties during the application temperature range. For the higher Cu nanoparticle ratios into the epoxy resin adhesive, the thermal stability of the adhesive decreased. The reason for this can be imagined as the absorbed heat by the metallic nanoparticles in epoxy at the beginning of the thermal analysis, and then this CuNP behave as a heat source in the mixture. If heat transfer continues, the epoxy matrix reaches over the decomposition temperature. In order to conserve the thermal stability of epoxy, the maximum Cu nanoparticle content must be selected as 20 wt%.


The EX-PRESS glaucoma filtration device appears to be an effective addition to our options to treat refractory glaucoma. The possibility to create a sclerostomy without tissue excision provides a safe and reliable outflow pathway for aqueous that is standard in size, reducing much of the variability associated with a surgical procedure. Prospective randomized studies comparing EX-PRESS implantation with trabeculectomy show encouraging results. However, complications usually encountered in filtration surgery have been reported, and EX-PRESS implantation can also lead to specific device-related complications. This article reviews the most common complications associated with this procedure. The EX-PRESS glaucoma filtration device  has gained increasing recognition as an alternative to trabeculectomy. The shunt consists in a non-valved biocompatible stainless steel device 2.64–2.96 mm long with a 400 micron external lumen diameter and options of a 50 micron or 200 micron internal lumen diameter. The device connects the anterior chamber to the intrascleral space to divert the aqueous humor to the subconjunctival space, with aqueous shunted from the anterior chamber to beneath a partial thickness scleral flap. Mitomycin C (MMC) use at a concentration of 0.2–0.4 mg/mL during the procedure is recommended to prevent scarring. MMC can be applied to tissues using surgical sponges, while an alternative method involves the irrigation of MMC directly in the subtenon space; the different MMC application route may lead to changes in surgical outcomes .The shunt is available in different versions with an inner lumen of 50 μm  and 200 μm (P-200). Originally, the EX-PRESS shunt was implanted under the conjunctiva, but this unguarded technique resulted in several complications such as hypotony and extrusion and erosion of the implant ,4,5. To Similarly to all filtration surgeries, failure caused by episcleral and subconjunctival fibrosis remains a challenging problem. Although success criteria definition may differ between studies, many comparisons between EX-PRESS and standard trabeculectomy showed similar failure rates ,11,12,13A recent meta-analysis based on four randomized controlled trials of 292 eyes confirmed that EX-PRESS implantation and trabeculectomy have similar efficacy in intraocular pressure (IOP)-lowering, medication reduction, vision recovery, and qualified operative success rates. EX-PRESS implantation is associated with higher rates of complete operative success and fewer hyphemia cases when compared to trabeculectomy .In our series of 248 consecutive patients with uncontrolled glaucoma who underwent EX-PRESS implantation over a period of 9 years (with a maximum follow-up of 7 years), we found a complete success rate decline from 83% at 1 year to 57% at 5 years [Another recent study evaluating Japanese patients found that advanced age, higher postoperative IOP, pseudo-exfoliation glaucoma, and simultaneous cataract surgery negatively influence the volume of the filtration bleb 2 years after surgery . It mustFailure of EX-PRESS shunts implanted under a scleral flap is normally a consequence of episcleral or bleb scarring, despite the use of anti-metabolites. Growth of fibrotic tissue between the external flange of the device and the inner portion of the scleral flap has been shown in vivo  and in eThe first option to consider in case of failure is a bleb needling or revision with antifibrotic agents, which have been proven to be a safe and efficacious procedure in eyes with an EX-PRESS shunt . AnotherIn case of definitive failure, the EX-PRESS shunt does not need to be removed to proceed to additional glaucoma surgery. Conventional tube surgery can be performed avoiding the area of the failed bleb.Most studies show no significant difference in intraoperative or postoperative complications, but some do note a lower incidence of postoperative hypotony and choroidal effusion with use of the EX-PRESS ,11,20,21This finding might be related to the smaller and standard outflow pathway created by insertion of the shunt. However, postoperative hypotony has been reported after surgery, with a transient decrease in the anterior chamber depth, probably due to a transient overfiltration through the EX-PRESS implant .Nonetheless, management of postoperative hypotony can be achieved in the same manner as in the classic filtration procedures.A significant reduction in the axial length, with an important hyperopic refractive shift, was recently observed in a highly myopic patient 18 months following hypotonia after glaucoma filtering surgery with EX-PRESS .EX-PRESS shunt malpositionThe EX-PRESS shunt is designed with a flange that fixes the implant to the sclera and prevents it from penetrating the anterior chamber. It also has a small spur-like extension to prevent movement extrusion.To avoid shunt movement after the insertion, it is essential to perform the pre-incision with the correct needle size. A 25-gauge needle track for the newer P series shunts is required, because of the slightly squared off shape. EX-PRESS glaucoma shunt dislocation into the anterior chamber has been described by Teng et al. twenty-one days after the procedure with no evidence of corneal decompensation . MoreoveThe original technique of implantation under a conjunctival flap was associated with high rates of implant dislocation and subsequent erosion .Traverso et al. showed, in a series of 26 eyes of 25 patients using the sub-conjunctival technique, two cases (7.7%) of device rotation (one treated by reposition) and three cases (11.5%) of conjunctival erosion at 2 and 3 years . ErosionTo remove the device, after an accurate dissection of the overlying conjunctiva and identification of the scleral flap, any fibrotic tissue should be dissected to allow for lifting of the flap. Once the shaft is fully exposed, the device should be gently lifted and rotated to dislodge the distal spur and allow removal. Creating a wider incision for removal is not needed to perform a safe extraction of the shunt.If the integrity of the scleral flap allows a tight closure, a tissue punch can be used to further enlarge the original entry site and convert to traditional trabeculectomy. If the scleral flap has eroded significantly, a patch graft should be used to close the opening and secured tightly to prevent filtration through this site.The EX-PRESS glaucoma filtration device distal portion contains an accessory orifice which serves as an alternative route in case of obstruction of the main central lumen. In a case series of 345 EX-PRESS shunt implantations, Kanner et al. reported that the most common device-related complication was shunt obstruction  . Only onIt has already been shown that the application of MMC may play a role in the pathogenesis of bullous keratopathy after filtrating surgery ,34. TherIn the first study that has compared endothelial changes after trabeculectomy, EX-PRESS shunt and Ahmed valve implantation, Casini et al. did not find significant modifications either at 1 month (0.2% decrease) or at 3 months (0.3% decrease), suggesting that EX-PRESS might represent the treatment of choice in patients with significant low corneal endothelial cell density before surgery or other risk factors for corneal damage . The lowRecent studies evaluating longer follow-ups demonstrate a statistically significant reduction of the corneal endothelium cell density, which was found by Ishida et al. 24 months after EX-PRESS implantation , compareOn the other hand, Omatsu et al. found no effects on the corneal endothelial cells until at least 24 months after the initial EX-PRESS surgery, supporting the benefit of using this procedure in patients who have a lower corneal endothelial cell density prior to the surgery .The physiopathological mechanism underlying the damage to the corneal endothelial cells following EX-PRESS implantation remains unknown, with recent data suggesting that it might be different from that of corneal endothelial cell loss after trabeculectomy. The corneal superior area, where the EX-PRESS device was inserted, was found to have significantly more severe corneal endothelial cellular density reduction than the inferior area 2 years after surgery, whereas no area dependency was observed in patients who underwent trabeculectomy .Possible reported causes of endothelial cell loss after EX-PRESS are MMC , foreign body reaction or immune reactions due to the stainless material used for the tube, changed flow of the aqueous humor around the tip of the tube, and mechanical ablation between the corneal endothelium and the tube.To reduce the risk of a postoperative contact between the tube and the endothelium, the insertion of the device should be performed while the anterior chamber is inflated by a continuous infusion or secured with viscoelastic. The needle entry point is at the grey line (junction between sclera and cornea) to avoid corneal stroma. Tojo et al. reported a case of partial decompensation following filtering surgery with EX-PRESS of the corneal endothelium adjacent to the filtering bleb 9 months after the surgery. Specular microscopy also revealed a marked reduction in the endothelial cell density at the center of the cornea. However, the authors underlined that the device was erroneously inserted from the cornea, not from the trabecular meshwork .Corneal surgery  may be taken into consideration in cases of severe endothelial cell loss, and patients presenting lower endothelial cell count should be carefully evaluated and informed before EX-PRESS implant surgery.Trabeculectomy bleb-associated infections are a serious and vision-threatening complication whose incidence varies among different studies; however, there is cumulative evidence that the incidence of bleb-associated infections has reduced over time . The ColWhile evaluating longer follow-ups, a paper on 460 eyes which underwent trabeculectomy reported cumulative probabilities of blebitis and bleb-associated endophthalmitis of 2% and 5%, respectively, during the 20 postoperative years .Little has been published regarding the incidence of blebitis and endophthalmitis after EX-PRESS implantation. Few cases of bleb-related endophthalmitis following EX-PRESS implantation have been reported ,47, inclThe aim of every glaucoma surgeon is to minimize complications while maximizing outcomes. The EX-PRESS glaucoma filtration device appears to be an effective addition to our surgical options to lower IOP in patients with refractory glaucoma. The possibility to create a standard sclerostomy without tissue excision provides a safe outflow pathway for aqueous with a standard size, reducing much of the variability associated. Prospective randomized studies comparing EX-PRESS implantation with standard trabeculectomy show encouraging results. However, the usual complications encountered in filtration surgery have been shown, and the implant of the EX-PRESS can also lead to specific device-related complications. The correct positioning of the implant is crucial for preventing these complications.


The Wnt/β-catenin signaling pathway plays a crucial role in early embryonic development. Wnt/β-catenin signaling is a major regulator of cell proliferation and keeps embryonic stem cells (ESCs) in the pluripotent state. Dysregulation of Wnt signaling in the early developmental stages causes several hereditary diseases that lead to embryonic abnormalities. Several other signaling molecules are directly or indirectly activated in response to Wnt/β-catenin stimulation. The crosstalk of these signaling factors either synergizes or opposes the transcriptional activation of β-catenin/Tcf4-mediated target gene expression. Recently, the crosstalk between the peroxisome proliferator-activated receptor delta (PPARδ), which belongs to the steroid superfamily, and Wnt/β-catenin signaling has been reported to take place during several aspects of embryonic development. However, numerous questions need to be answered regarding the function and regulation of PPARδ in coordination with the Wnt/β-catenin pathway. Here, we have summarized the functional activation of the PPARδ in co-ordination with the Wnt/β-catenin pathway during the regulation of several aspects of embryonic development, stem cell regulation and maintenance, as well as during the progression of several metabolic disorders. The mammalian pre-implantation period is one of the critical and unique phases during early embryonic developmental processes. A transition from single-cell zygote to the blastocysts stage involves a series of crucial events that lead to the initiation of cell lineage specification and differentiation into the inner cell mass (ICM) and trophectoderm (TE) ,2. TheseThe canonical Wnt pathway is called a β-catenin dependent pathway. It depends upon the subsequent accumulation of the activated β-catenin level in the cytoplasm. In the presence of the canonical Wnt ligand, a multi-protein complex (Wnt/Fzd/Lrp6) is formed at the membrane that triggers the downstream signaling. This complex directs the β-catenin destruction complex Gsk3/APC/Axin) to the membrane and leads to the stabilization of β-catenin levels in the cytosol /APC/Axin. The accA spatially defined and well-controlled Wnt signaling pathway orchestrates normal embryonic development. This process is initiated at fertilization and is continued until the formation of a complete organism ,15. To rThe early developmental processes are governed by the regulated processes of cellular proliferation, pluripotency maintenance, cell migration, and differentiation ,7,10. InThe role of Wnt signaling in governing pre-implantation development is somewhat controversial. However, β-catenin was detected from the 2-cell stage to the blastocyst stage of mouse embryos, suggesting that Wnt/β-catenin signaling has a functional role in driving the early processes of embryogenesis . The devThe development of placenta and maintenance of its integrity is another crucial aspect of normal embryonic development and successful pregnancy outcome. Numerous reports highlighted the critical involvement of the Wnt/β-catenin signaling pathway as well as the significance of PPARδ functions during trophoblast differentiation and placentation ,30,31. T-/- in mice inhibited trophoblast differentiation towards the giant cells that substantially impact placental development [Placenta originate from the outermost trophectoderm layer of a blastocyst that differentiated into several trophoblast cell types ,32. Diffelopment . Deficieelopment . Placentelopment ,34. Knocelopment . Likewiselopment ,36. PPARelopment . PPARδ ielopment ,6,35, liStem cells are characterized by self-renewal by maintaining the pluripotency and the proliferation potential of the progenitor cells . This phWnt/β-catenin signaling represents a major pathway upon which various signals converge to influence metabolism and cancer progression . ImbalanSo far, PPARδ has been majorly implicated in lipid catabolism in adipose and muscle tissue by enhancing fatty acid oxidation (FAO). This molecular cross-talk between Wnt signaling and PPARδ has been implicated in many metabolic syndromes, such as obesity, diabetes and the development of cancer ,46,50. TWnt signaling has a broad range of roles in the control of early embryonic developmental processes. Wnt/β-catenin signaling is remarkably known for the regulation of cell proliferation and differentiation events during early embryogenesis and stem cell maintenance. This multitude effect of Wnt signaling may involve crosstalk with other signaling components. The above model diagram  highligh