Acid whey is the main co-product resulting from the production of fresh cheeses and Greek-type yogurts. It generally goes through a spray-drying process prior to valorization, but it needs to be deacidified (lactic acid recovery) and demineralized beforehand to obtain a powder of quality with all the preserved compounds of interest such as lactose and proteins. Electrodialysis (ED) is a process actually used for acid whey treatment, but scaling formation at the surface of the ion-exchange membrane is still a major problem. In this work, a combination of two new avenues of ED treatment has been studied. First, the integration of a nanofiltration (NF) membrane in an ED conventional stack was compared to a classical ED stack with an anion-exchange membrane in a standard current condition. Secondly, both configurations were tested in the overlimiting current condition to study the impact of electroconvective vortices on process efficiency. The combined effects of the NF membrane and overlimiting current condition led to a higher lactic acid recovery rate of acid whey (40%), while the conventional ED stack in the overlimiting current condition led to a higher demineralization . Those effects were related to the conductivity, pH, global resistance, and energy consumption of each treatment that are influenced by water splitting phenomenon, which was decreased in the overlimiting condition. Dairy production generates effluents with variable composition depending on the final product. Acid whey is generated from the manufacture of Greek-type yoghurts, fresh cheeses (cream or cottage cheeses), and caseins resulting from milk acidification. In the past few years, the demand for those dairy products has continued to increase, leading to the production of significant volumes of acid whey. Considered as the most contaminated waste generated in dairy production because of its high organic content ,2, this Many processes and experimental conditions have been tested to realize this critical preliminarily step such as nanofiltration (NF) , ion-exc+/Zn2+ and Na+/Mg2+ systems and demonstrated that an NF membrane can increase the limited current density in ED. In the case of acid whey demineralization and the deacidification process, these membranes could allow a better mass transfer due to their larger pore size and their surface charge, particularly for lactic acid. However, the use of nanofiltration membrane in an ED cell for the deacidification of acid whey has never been reported in the literature.In addition, the use of charged filtration membranes stacked in an ED cell was demonstrated to be effective for demineralization of solutions. Hence, in 2011, Bazinet and Moalic  were theIn this context, the main goal of this study was to explore the use of NF membrane in the replacement of anion-exchange membrane (AEM) stacked in the ED cell as well a combination of the overlimiting current condition in order to potentially optimize lactic acid recovery and the demineralization of acid whey and prevent the formation of scaling.The acid whey samples were obtained from the Parmalat Canada  dairy processing plant and transported at 4 °C until they reached the laboratory. They were aliquoted, stored at −30 °C, and thawed at 4 °C before each experiment. Their composition, as described in 2 was used for the experimentations in this work. This cell included a dimensionally stable electrode (DSA-O2) as the anode and a stainless steel electrode as the cathode. The first membrane configuration “CACAC”  with an effective membrane surface area of 100 cm “CACAC” a, a conv “CACAC” b, the AE2S04 20.0 g/L  was used as the electrode rinsing solution. First, 500 mL of whey and NaCl solutions circulated in two different loops to create two deacidification/demineralization units  was used as the recovery solution and Na2SO4 were circulated in the ED stack . In parallel, the limiting current density (LCD) of the ED stack was determined by the Cowan and Brown method . BrieflyED stack  as for tED stack . Since tED stack , the con2) and was applied during 1 h with no temperature control and at flow rates of 400 mL/min in whey and NaCl compartments. A volume of 10 mL of samples was collected every 15 min during the process. They were frozen at −30 °C until further analyses such as lactic acid, lactose, and mineral contents. To complete this protocol, both configurations were carried out in an overlimiting current condition at 3.0 A  during 30 min with no temperature control and the same flow rates as in the underlimiting conditions. In this study, the temperature was deliberately not controlled. Indeed, since the overlimiting current would increase the temperature, by controlling the temperature increase, its potential effect on the molecules’ transfer and energy efficiency would have not been taken into account. Furthermore, in a context of sustainability, controlling the temperature increase during ED at the overlimiting temperature would use lots of energy, and the potential energy saving related to the increase in temperature would be lost. So, to test both processes in real conditions and to be more close to the reality, the temperature was consequently not controlled. The ED duration was fixed to 30 min since during preliminary tests, it was observed, as expected, that the demineralization was faster and did not need to reach 1 h as for the underlimiting conditions. A volume of 10 mL of samples was also collected at 0, 5, 10, 20 and 30 min. Then, they were frozen until further analyses. All ED combinations of current conditions and configurations were repeated three times.This underlimiting current condition corresponds to a constant current of 0.7 A (current density of 7 mA/cmCurrent–voltage (CV) curves as well as chronopotentiograms (ChP) were determined according to Villeneuve et al.  and MikhThe pH of both compartments (NaCl and whey) was measured using a Symphony pHmeter  equipped with a temperature probe to compensate for temperature changes.2SO4 (180 µL H2SO4/L) solution as the mobile phase and at a flow rate of 0.4 mL/min. The column temperature was kept constant at 40 °C. Samples were centrifuged during 5 min at 5000 rpm  and filtered  before injection (15 µL). Each sample was analyzed during a 36 min run time. Lactose anhydrous (PHR1025) and L-(+)-lactic acid (L1750) from Sigma-Aldrich  were used as external standards.Lactic acid and lactose concentrations in collected samples during ED were determined with high-performance liquid chromatography (HPLC) with a Waters chromatograph  equipped with a Hitachi differential refractometer detector L-7490 , a 600E controller, a column oven, and a thermostated 717 Plus autosampler. An ICSep ICE-ION-300 column  was used with 8.5 mM of Hi in ppm) and the end (Cf in ppm) of ED treatments according to Equation (1).The deacidification or lactic acid recovery rate (DaR) related to each treatment was calculated by considering the acid lactic concentration detected in the samples collected at the beginning (Ci in mS/cm) and the end (Cf in mS/cm) of each treatment.Acid whey and NaCl conductivities were measured with an YSI conductivity meter  equipped with an immersion probe . Then, the demineralization/mineralization rate based on the conductivity measurement was calculated with the same equation than for the deacidification rate (Equation (1)), considering the conductivity of whey and NaCl at the beginning (Ci in ppm) and at the end (Cf in ppm) of each treatment.As described by Dufton et al. , the samThe voltage (U) and current intensity (I) directly obtained from the two power supplies  were used to calculate the global system resistance (R) according to Ohm’s law (U = R·I).The energy consumption (in Wh) was calculated according to Equation (2) where I is the current intensity (in A), U(t) tis he voltage (in V) as a function of time, and t is the duration of the ED treatment (min.)+ migrated from whey, since it is the ion having the higher migration rate [Energy consumption for demineralization was calculated according to the concentration of Kion rate .p < 0.05 .Data were subjected to one-way and two-way, and three-way analyses of variance (ANOVA). A Tukey multiple comparison test was performed to compare treatments together at a probability level of 2, respectively. These results were in accordance with Villeneuve et al. [CV curves were analyzed for both AEM and NF membranes. The experimental values of LCD were determined by the intersection of the extrapolated tangential lines of the Ohmic and plateau regions . For thee et al.  for the 2 for the determination of the transition time, before the limiting current begins, for AEM, since no limiting current was observed for the NF membrane. The resistance, thickness of the boundary layer, and transition time were 44.4 ± 5.4 Ω, 284 ± 26 μm, and 11.1 ± 0.3 s, respectively These results were in accordance with those obtained by Villeneuve et al. [e et al.  for the P < 0.001 for both). Indeed, for the CNfCNfC configuration, the pH was stable at 4.5 during the treatments in the underlimiting current condition, while it increased from 4.47 ± 0.03 to 4.66 ± 0.09 in the overlimiting one (P < 0.001) (P < 0.001); in the underlimiting current condition, it decreased from 4.46 ± 0.14 to 4.03 ± 0.16, while the decrease in the overlimiting condition was more pronounced with a final value of 2.83 ± 0.08 (P = 0.242), but a general trend of decrease in pH was observed. Only the membrane configuration has an impact on pH evolution for each treatment (P < 0.001). Indeed, both CACAC underlimiting and overlimiting current conditions showed an increase in pH from 5.58 ± 0.49 to 7.37 ± 0.67 and 7.86 ± 1.87 to 11.53 ± 0.06. The opposite trend was observed for CNfCNf, since the pH decreased for both current conditions, until the end of the process, from 5.74 ± 0.09 to 5.03 ± 0.21 and from 5.66 ± 0.58 to 4.58 ± 0.15 in underlimiting and overlimiting conditions, respectively.The pH evolution in the whey compartment was influenced by the current condition applied and the membrane configuration tested (< 0.001) a. For th3 ± 0.08 a. For th3 ± 0.08 b, no staAs previously mentioned in the literature for the CACAC configuration ,27, the P = 0.595 for configuration, P = 0.516 for current, P = 0.314 for double interaction configuration/current). In contrary, considering the NaCl compartment, it was confirmed that lactic acid migrated from the whey compartment to the NaCl compartment and was influenced by the double interaction between the membrane configuration and current condition (P < 0.001). Furthermore, in the underlimiting current condition, the CACAC configuration led to a greater migration of lactic acid anion while its migration was higher in the overlimiting condition for the CNfCNfC configuration. Indeed, this organic compound was present mostly in a form of lactate (around 75%) at the pH of raw acid whey (4.45 ± 0.02). Consequently, it was able to migrate through the positively charged membranes (AEM and NF), whatever the configuration.As shown in P = 0.027). As shown in P = 0.020) between the two current conditions. Indeed, after only 30 min, the deacidification rate obtained in the overlimiting condition (40.00% versus 26.03%) had practically doubled compared to the underlimiting condition after 60 min of treatment. Consequently, the replacement of AEMs by NFs in an ED stack with the application of an overlimiting current condition could lead to a higher deacidification rate.These results of lactate migration were confirmed by the deacidification rates calculated for each treatment. However, only the current condition had an impact on the global deacidification rate  ,37. Theycid whey .Recently, Talebi et al. used the combination of diafiltration (Dia)-NF and ED for the removal of lactic acid in separated steps. The use of Dia-NF prior to ED resulted in a 71% removal of lactic acid, compared to 36% with NF only prior to ED. In the present study, 40.00 ± 4.33% were obtained with CNfCNf configuration in the overlimiting current in only one step . Indeed,The concentration of lactose remained constant in the whey compartment during the whole process for each treatment condition (results not presented here). No lactose was found in the NaCl compartment whatever the configuration used or the current condition applied. These results showed that CACAC and CNfCNfC membrane configurations allowed the deacidification of acid whey while preserving its compounds of interest such as lactose. Moreover, as reported by Chandrapala et al. (2016) , assuminP < 0.001 for both whey and NaCl compartments) and the current condition applied (P < 0.001 for both whey and NaCl compartments) had an impact on the evolution of conductivity during treatments (P = 0.044 and P < 0.001 respectively for whey and NaCl compartments) (P = 0.001).The configuration (rtments) . In the rtments) . Moreovertments) a was higrtments) b. For dertments) a and higrtments) . Those r+, Ca2+…) migrated through the CEM, while negatively charged species  migrated through the AEM or NF membrane. By leaving the whey compartment, those species contributed to the decrease in its conductivity, which led to an increase in the demineralization rate and to a simultaneous increase of the conductivity in the NaCl compartment  was obtained in 30 min in the overlimiting current . These rP = 0.042). Indeed, the current applied in the overlimiting condition increased the migration rates obtained for the CACAC (P < 0.001) and CNfCNfC (P = 0.0003) configurations. However, the configuration had no impact on the demineralization rates obtained in the underlimiting current condition (P = 0.093), since it only had an impact while the overlimiting current condition was applied (P < 0.001). The same double interaction was observed for sodium migration (P < 0.001), but it has opposite origins. For this ion, the membrane configuration influenced drastically the migration rates whatever the current condition applied (P < 0.001 for the underlimiting and overlimiting currents). The current condition had only a statistical impact on the CACAC membrane configuration (P < 0.001), and it did not occur on the CNfCNfC membrane configuration (P = 0.259) because of the large standard deviations obtained for the three repetitions carried out on CNfCNfC in the overlimiting condition. These results for sodium were also explained by the CV curve, which indicated that the NF membrane allowed its migration. For potassium ion, its migration was influenced by the distinct effect of the configuration and current condition (P = 0.207). Each result was different depending on the membrane configuration used (P = 0.001) and the current applied (P = 0.003). The same trend was observed for magnesium and phosphorus , since configuration (P = 0.005 and P < 0.001 respectively) and current (P < 0.001 and P = 0.007 respectively) alone had separate effects (P = 0.186 and P = 0.776 for double interaction respectively) on their migration.More generally, whatever the ED configuration or condition of current applied, the potassium was the ion having the highest migration rate, and it can be due to its high electrophoretic mobility and concentration . As alrexOyPzn− were anions having lower migration through the AEM and NF membrane compared to cation migration through the CEM in all the conditions tested. This fact is due to the relatively low electrophoretic mobility of these anions compared to the monovalent chloride and other organic anions. Moreover, the phosphorous-containing ionic migration is hampered by competitive anions having higher concentration in the whey solution . Furthermore, as shown in Regarding the phosphorous-containing ion migration, one can observe that HP < 0.001). Indeed, the application of the overlimiting condition increased the cell resistance in comparison with the underlimiting one, whatever the configuration. However, in the underlimiting condition, the overall system resistance of CNfCNfC remained stable all along the treatment, while there was an increase in the resistance of CACAC . A larger amount of energy was necessary to deacidify acid whey when using the CACAC configuration compared to the CNfCNfC configuration. Indeed, a conventional stack needed around 68% more energy than the stack with the NF membrane. This energy cost was not justified, since this configuration led to a lower deacidification rate, as presented in There was no statistical difference in energy consumption for the deacidification step with both membrane configurations when the underlimiting current condition was applied , leadingAs reported by Chen et al. , a stand+) were observed for both configurations. However the overlimiting condition increased by 7–9 times the energy consumption compared to the underlimiting condition, and these results are not in accordance with Dufton et al., since the use of pulsed electric fields in their study allowed a decrease in energy consumption compared to the constant current in the underlimiting condition [Concerning the demineralization step, energy consumption, as presented in ondition . Additioondition  reportedP < 0.001), meaning that the difference of potential applied had a greater impact on minerals than on lactate ions. However, even if the CACAC configuration needed more energy in the overlimiting current condition, it led to greater demineralization rates than all the other tested conditions. As for the CNfCNfC membrane configuration, it required the same amount of energy than the CACAC, but for half the demineralization rate obtained. Consequently, even if this configuration was promising for deacidification, it was less interesting for demineralization.In addition, it seemed that the demineralization of acid whey consumed more energy than for deacidification. Indeed, in the underlimiting condition, the energy consumption had practically doubled for both membrane configurations to demineralize acid whey  while it had a more significant impact on demineralization with a CACAC configuration (87%). However, these two optimal conditions needed more energy than treatments performed with the underlimiting current condition. Their conductivity, pH, and global resistance were also more influenced by the water splitting phenomena that occurred while the limiting current was reached. Furthermore, it appeared that the selectivity for sodium was drastically decreased or suppressed in the overlimiting condition for the NF membrane, while no change was observed for other cations.Further experiments also have to be carried out on the CNfCNfC membrane configuration to study the generation of EVs and their development on both AEM and NF membrane types. In addition, applying a pulsed electric field in both underlimiting and overlimiting current conditions could be interesting in order to compare their impact on acid whey treatment efficiency and to potentially enhance the demineralization and deacidification processes.


Alcohol consumption by pregnant women may produce neurological abnormalities that affect cognitive processes in children and are together defined as fetal alcohol spectrum disorders (FASDs). However, the molecular underpinnings are still poorly defined. In our earlier studies, we found that ethanol exposure of postnatal day 7 (P7) mice significantly induced widespread neurodegeneration mediated via endocannabinoids (eCBs)/cannabinoid receptor type 1 (CB1R). In the current study, we examined changes in the β-catenin protein levels that are involved in the regulation of neuronal function including neuronal death and survival. We found that moderate- and high-dose postnatal ethanol exposure (PEE) significantly reduced active-β-catenin (ABC) (non-phosphorylated form) protein levels in the hippocampus (HP) and neocortex (NC). In addition, we found that moderate- and high-dose PEE significantly increased the phosphorylated-β-catenin (p-β-catenin)/ABC ratios in the HP and NC. Antagonism/null mutation of CB1R before PEE to inhibit CC3 production mitigated the loss of ABC protein levels. Collectively, these findings demonstrated that the CB1R/β-catenin signaling mechanism causes neurodegeneration in neonatal mouse brains following PEE. Alcohol exposure during pregnancy can cause abnormal fetal development and functional anomalies to multiple brain regions that result in a broad spectrum of neurobehavioral and cognitive deficits termed fetal alcohol spectrum disorders (FASD) ,2. The pThe eCB signaling system, containing endocannabinoids, cannabinoid receptors type 1 and 2, and the enzymes involved in their metabolism ,24, is aMale and female C57BL/6J and CB1R heterozygous mice were housed in typical (12 h light/12 h dark cycle) laboratory conditions. Mice were allowed to ad libitum access to food and water. CB1R heterozygous mice were backcrossed on the C57BL/6J background for over 10 generations. We generated CB1R wild-type (WT) and knockout (KO) mice from a described CB1R heterozygous   breedingIn the present study, we used the PEE paradigm, in which acute ethanol treatment induces widespread neurodegeneration in many brain regions, including the hippocampus and cortex, without causing any lethality . Half ofMice were perfused with a solution containing 4% paraformaldehyde and 4% sucrose in 0.05 M cacodylate buffer (pH 7.2) at 8 h after the first dose of saline or ethanol administration. This time point was shown to exhibit maximum caspase-3 activation (in one or more brain regions) in previous studies ,46,47. Tg for 1 min, and the supernatant was aspirated and stored at −80 °C until use. The nuclear pellet was then resuspended in a nuclear extraction reagent (NER)  [g for 10 min (4 °C). The supernatant was collected in prechilled tubes and stored at −80 °C for further studies. The samples were prepared in a sample buffer as previously described by our laboratory [At 4 to 24 h after the first saline or ethanol injection, the HP and NC tissues were subjected to homogenization using buffer  containing freshly added 1% protease inhibitor mixture . The HP and NC homogenates were handled as described previously ,49. The GA, USA) . The nucboratory ,51. In aboratory ,51. The post hoc test. A p < 0.05 cutoff was used to represent statistical significance in all the comparisons. Prism software  was used to perform the statistical analyses.The experiments were performed using an equal number of animals per treatment. All the data are shown as the mean ± SEM. A statistical analysis of the data was performed by either a one-way analysis of variance ANOVA or a two-way ANOVA with Bonferroni’s The P7 mice were administered a moderate  or high  dose of ethanol at 0 h and again at 2 h. The BELs were determined at 3 and 9 h after first dose ethanol treatment. Consistent with an earlier finding , we obse3, 28 = 26; high-dose: F3, 28 = 32, p < 0.05) and NC  at the 4–24 h (after the first ethanol administration) time points compared to the levels in the saline group (0 h) (one-way ANOVA with Bonferroni’s post hoc test).Both moderate- A and hig3, 28 = 26, p < 0.05) and NC  at 8 and 24 h  at 8 and 24 h and in the NC  at 4 and 8 h but not at 24 h  and NC  at the 4–24 h (after the first ethanol administration) time points compared to the levels in the saline group (0 h) (one-way ANOVA with Bonferroni’s post hoc test).Both moderate- Aand highp < 0.05) in the HP  and NC . Additionally, high-dose PEE in the CB1R KO mice failed to induce the loss of ABC in the HP  and NC  (one-way ANOVA with Bonferroni’s post hoc tests).Because pharmacological blockade or genetic ablation of CB1R blocks PEE to induce neurodegeneration without affecting ethanol metabolism in P7 mice ,25,32,54β-Catenin is involved in controlling many of the cellular functions of the developing CNS and has been shown to orchestrate neuronal differentiation, neuron death/survival, axonal elongation, synapse formation, and plasticity, neurotrophin transcription, neurogenesis, and regeneration ,57,58,59Numerous studies report alterations in β-catenin in response to ethanol insult; nonetheless, most of these studies used chronic ethanol exposure and, therefore, may manifest cellular adaptation in reaction to ethanol challenge. Chronic ethanol exposure significantly suppressed β-catenin signaling and the expression of Wnt effectors in bone ,62,63. IWe and others showed earlier that PEE causes significant activation of caspase-3 in neonatal mice ,20,54, aA previous study in non-neuronal cells showed that activation of CB1R by exogenous agonists increased the phosphorylation of β-catenin, followed by loss of cytosolic and nuclear β-catenin . β-catenThese findings raise the query as to how PEE abolished β-catenin activity. Protein degradation is the most studied mechanism for β-catenin regulation in the brain and other tissues. The β-catenin complex is phosphorylated by a destruction complex consisting of Axin, adenomatous polyposis coli, and glycogen synthase kinase-3, causing proteasomal degradation. Wnt activation causes this destruction complex to dissociate, leading to the stabilization of β-catenin ,72. Moretranscriptional repressor element-1 (RE-1) silencing transcription factor (REST) and CoREST support several repressive histone-modifying activities [Growing studies suggest that actin and actin-binding proteins impact gene expression at various levels ,80. For tivities ,82,83 wiIn summary, the outcome of this study suggests that CB1R can regulate neurodegeneration during a sensitive period of brain development. The findings also indicated that PEE-induced neurodegeneration is mediated through the inactivation of β-catenin. Overall, our results revealed the participation of CB1R signaling in the postnatal ethanol-induced inactivation of β-catenin transcriptional activity in the developing brain. The crosstalk of the CB1R signaling and β-catenin transcriptional activity pathways suggests new potential strategies for therapy.


The versatile properties of a nanopaper consisting of a porous network of multi-walled carbon nanotubes were applied to enhance the mechanical and electrical properties of a thermosetting epoxy polymer. The embedded nanopaper proved useful both in the monitoring of the curing process of the epoxy resin by the self-regulating Joule heating and in the supervising of tensile deformations of the composite by detecting changes in its electrical resistance. When heated by Joule heating above its glass transition temperature, the embedded carbon nanotube nanopaper accelerated not only the modelling of the composites into various shapes, but also the shape recovery process, wherein the stress in the nanopaper was released and the shape of the composite reverted to its original configuration. Lastly, in comparison with its respective epoxy adhesive, the internally heated electro-conductive carbon nanotube nanopaper/epoxy composite not only substantially shortened curing time while retaining comparable strength of the adhesive bonding of the steel surfaces, but also enabled a release of such bonds by repeated application of DC current. We recently used a nanopaper made of multi-walled carbon nanotubes (MWCNTs) to monitor infiltration of epoxy resin through a glass fiber textile and study the curing process and deformation of a glass fiber/epoxy composite . The embThe idea of curing of carbon allotrope/epoxy composites by means of self-heating has proven successful in several composite arrangements. For instance, carbon fiber/epoxy composites can be cured by Joule heating. Such cured composites compare favorably with oven-cured ones, thanks to the lower energy consumption of the Joule heating process . SimilarIt has recently been shown that polypropylene with an embedded carbon fiber veil can be molded into a composite product by Joule heating, with properties comparable to that prepared by an oven heating process . SimilarDifferent embedded reinforcing conductive structures in polymer matrices increase strength and add electrical properties to such polymer composites. These properties may be used for forming shapes of industrial products and parts or for an electrical-driven actuation, among others. The recent progress in the investigation of shape memory polymer composites with embedded carbon black, carbon nanotubes (CNTs), carbon nanofibers (CNFs), and graphene is reviewed in . The carThe above examples show possible versatile uses of the various carbon allotrope structures embedded in epoxy polymers or in similar polymers. However, we think that the use of the carbon nanotube nanopaper embedded in the epoxy in various technological applications is still insufficient. In our previous paper , we descPurified MWCNTs produced by chemical vapor deposition of acetylene were supplied by Sun Nanotech Co. Ltd., Jiangxi, China. According to the supplier, the nanotube diameter was 10–30 nm, length 1–10 μm, purity ~ 90%, and volume resistivity 0.12 Ω cm. Further details about the nanotubes and results of the transmission electron microscope (TEM) analysis can be found in our previous papers ,21.2, frequency 24 kHz,  for 15 min at 50% power of the apparatus, 50% pulse mode, and temperature about 50 °C. NaOH aqueous solution was added to adjust pH to 10. The aqueous dispersion of MWCNTs  was prepared by sonication using the Dr Hielscher GmbH UP400St apparatus  from pristine nanotubes, the nanotubes were deposited on a porous polyurethane electrospun non-woven membrane by vacuum filtration. Two hundred and fifty mL of the homogenized MWCNT dispersion was filtered through a funnel 90 mm in diameter. Then, the resulting disk-shaped filtration cake was washed in situ several times with deionized water (at 65 °C) and afterwards with methanol. The filtering membrane was peeled off and dried between two filtration papers for 24 h at room temperature. The measured electrical conductivity of the MWCNT nanopaper was S = 11.97 S/cm.Tg, which was determined by differential scanning calorimetry (DSC) analysis , were 18.4, 35.1 and 58.1 °C for the component ratios of 100:100, 100:75, and 100:50, respectively. The chosen mixing epoxy component ratio of 100:75 enabled manipulation and shape forming of experimental epoxy samples above Tg (35.1 °C) manually. The curing time was 48 h at room temperature. The exothermic heat of the curing was determined by the DSC analysis as −275 J/g and peak temperature 140 °C at a heating rate 10 °C/min. The cured epoxy was elastic and flexible (without any damage at the bending over radius of 70 mm); its ultimate tensile strain was 3.5% and hardness 66 Shore D. The two-component epoxy resin Epox G 200  is a transparent epoxy casting system with adjustable hardness and extended processing time. According to the supplier, the ratio of the epoxy components (A/B-hard/elastic) 100:50 yields a hard epoxy of hardness 79 Shore D, while the component ratio of 100:100 yields a flexible one (44 Shore A). The glass transition temperatures The samples of MWCNT nanopaper/epoxy composite for the tests were fabricated in steps, which included at first the gluing of Cu strip electrodes to the opposite sides of a nanopaper plate. Then, the plate was put on a polytetrafluorethylene (PTFE) foil, filled with epoxy resin, and its surface covered by the PTFE foil again. To avoid wrinkling in the course of curing by inner Joule heating at a temperature around 100 °C for 6 min, the plate was kept under a pressure load of 300 kPa. The measured electrical conductivity of the resulting composite was S = 3.16 S/cm. The glass transition temperature of the epoxy matrix with the embedded nanopaper, determined as peak temperature, was 53.9 °C.Tg and thus retained the imposed shape. Thereafter, the voltage was reapplied, and the deformed composite strip straightened against gravity, regaining its original flat shape.To illustrate the shape recovery of the bent strips of the MWCNTnanopaper/epoxy composite induced by Joule heating, two electrodes were attached lengthwise to the opposite sides of the strip . The electrodes were made of Cuprexit (a thin copper layer supported by a 0.22 mm thick fiberglass plate). The composite strip was stapled to the conductive copper layer of the Cuprexit electrodes at 60 °C, to which in turn were soldered conductive wires. Subsequently, the composite strip was warmed up by Joule heating to about 60 °C and molded by hand alongside an edge to form a right-angled shape. The composite strip was held in that shape by hand until the material cooled to below its Tg and thus retained the imposed shape. The straight portion of the partially coiled composite strip was attached by double-sided tape to a vertical side of a box and the coiled portion was left protruding above. Next, the misshapen composite strip was reheated in an oven to 60 °C and its coiled portion straightened against gravity, regaining its original flat shape.An identical composite strip was warmed in an oven to 60 °C and a part of the strip was reeled onto a glass rod. It was then held by hand in that shape until the material cooled below its 3/min at pressure of 101.325 kPa  was set for all experiments. A platinum crucible was used to hold the sample, which weighted about 4 mg. The temperature was increased at a rate of 20 °C/min within the range from ambient room temperature to 1200 °C.Thermogravimetric analysis (TGA) of the epoxy and MWCNT nanopaper/epoxy composite samples was carried out using the thermogravimeter SETARAM SETSYS Evolution 1200 . The samples were examined under inert atmosphere of helium , which minimized the oxidation-dependent weight fraction loss of carbon nanotubes. Gas flow of 30 cmThe structure of the MWCNT nanopaper and the cross-section of the nanopaper/epoxy composite were analyzed by a scanning electron microscope (SEM) . The surface temperature of the specimens was measured by means of a thermal camera , which was able to create a detailed temperature pattern. Sensor resistance was measured lengthwise by the two-point technique using the Multiplex datalogger 34980A , which stored the readouts once per second. The DC power supply Metex AX 502  was used to power the Joule heating of composites. Tensile tests were carried out using the Testometric M350-5CT system .The temperature progression induced by the imposed electric power on the MWCNT nanopaper and the dependence of the reached maximal temperature on the electric power are shown in The process of epoxy resin curing is shown in gT as peaks by the DSC was 35.1 °C for the former and 53.9 °C for the latter. The peak shift to the higher temperature was similar to the effect of the decreasing of the ratio between the resin components A:B in favor of component A. The peak shift was not due to creation of the second phase of epoxy as the immobilized rigid amorphous fracture near the MWCNT surface ). The prepolymer was prepared with sufficient excess of epichlorohydrin so the termination was by free oxirane rings capable to react chemically with amine groups. Component B (Trimethylolpropane tris ether [CAS: 39423-51-3]) was a hardener or a curing agent containing three polymeric chains of poly(propylene glycol) terminated by amine groups. The poly(propylene glycol) chains themselves are highly movable, capable to rotate around a single chemical bond. The chains contain oxygen, which has a relatively low rotational barrier, and therefore the conformation of the chains changes easily under, e.g., an applied mechanical stress. The reaction of components A and B leads to the formation of a three-dimensional polymer network, resulting in a final epoxy matrix. At an excess of component B at the 100:100 ratio, some poly(propylene glycol) branches remain unbounded, by a lack of oxirane rings with a high molecular mobility potential, and the epoxy is elastic. With a lack of component B at the 100:50 ratio, the proportion of functional groups for crosslinking is more equimolecular. The epoxy contains less free unbounded poly(propylene glycol) branches, resulting in resistance against conformation changes, stiffness, and a higher  surface , increas surface . It was  surface . 2, while the other ones were assigned to the respective oxygenated functional groups C–O (286.2 eV), C=O (287.1 eV), O–C=O (288.6–289 eV), and C1s–π–π* (291.1–291.5 eV). X-ray photoelectron spectroscopy (XPS) signals were measured to obtain information on functional groups attached to the nanotube surfaces . The sig3 denoted the measured apparent density of the nanopaper and 3 the measured average density of nanotubes, which was close to the theoretical value of 1.8 g/cm3 [A TGA analysis was performed on the MWCNT nanopaper, the MWCNT nanopaper/epoxy composite, and the cured epoxy to determine the weight fraction of MWCNTs, .8 g/cm3 .A tensile test of the MWCNT nanopaper, the MWCNT nanopaper/epoxy composite, and the cured epoxy was carried out to determine their fracture tensile strength and strain. According to the test results in A detailed view of the structure of an individual nanotube consisting of about 15 rolled sheets of graphene obtained by means of TEM is shown in Tg of 53.9 °C. At this temperature, the composites went from being rigid and glassy to rubbery and flexible, and thus highly formable. When a bending force was applied, the strips deformed easily to the chosen shapes, which they held after cooling below Tg, despite the deformed embedded MWCNT nanopaper  to the forming temperature of 70 °C, which was above their glass transition temperature anopaper . Tg to the transformation temperature of the shape memory, shape recovery stress in the embedded MWCNT nanopaper was released due to the epoxy matrix softening. Apparently, the embedded nanotubes were compressed on the inside surface of the bent strip and stretched on the outside. When the nanotubes were released and were able to move within the epoxy matrix, the deformed arrangement of the stretched portion and compressed portion of the nanopaper recovered to its initial arrangement. Moreover, the strip’s Joule heating through inter-nanotube contacts was more efficient than heating in an oven. Consequently, when Joule heating was applied to the bent composite strip attached to a box, the time of the strip shape’s unbending reached about 20 s  on which a few drops of the epoxy were deposited; the MWCNT nanopaper was attached, a few drops of the epoxy were added on top of the nanopaper surface, and then the second steel strip was overlaid. The overlapped strips were under pressure load of 300 kPa heated by electric power of 6 W to a temperature of about 160 °C for 5 min  could be released by force of 120 N or about 12 kg of load under gravity, which can be readily exerted by human hand.Rapid heating of an embedded nanopaper may facilitate polymerization of the epoxy components of the composite and thus aid its adhesive properties. To test this surmise, we used two zinc-coated steel strips  reached The MWCNT nanopaper/epoxy composite could not only be easily formed, but also retained its shape memory. Namely, when Joule heating was reapplied to a molded MWCNT nanopaper/epoxy composite, it reverted to its original shape. Such a process was in an order of magnitude shorter when Joule heating was applied than when an oven was used. Thus, the shape recovery time as well as processability, light weight, and low manufacturing costs may be a highly desirable combination.Facile forming and shape memory apart, another advantageous property of the MWCNT nanopaper/epoxy composite is its adhesiveness. Albeit no substantial difference in the strength of the bonding was observed when the composite was compared with a respective conventional epoxy adhesive, the time period necessary for curing and thus establishing a firm connection between the surfaces was substantially shorter in the nanomaterial, thanks to the possibility of application of Joule heating. In addition, repeated application of the DC current released these bonds, which suggests that materials based on the MWCNT nanopaper/epoxy composites might be used e.g., to form electrically controllable seams.We combined a multi-walled carbon nanotube free-standing nanopaper and an epoxy resin into a reinforced thermosetting polymer matrix composite and demonstrated its versatile use in technological processes with advanced processing parameters, thus saving time and electrical energy. In our earlier papers ,21,24, tg decreased its adhesive stress and consequent release was done readily by human hand. The list of possible applications of the MWCNT nanopaper/epoxy composite and modulation of its characteristics by Joule heating, as presented in An innovative finding was self-regulation of the epoxy curing temperature . After t


Glucocorticoid hormones are vital; their accurate operation is a necessity at all ages and in all life situations. Glucocorticoids regulate diverse physiological processes and they use many signaling pathways to fulfill their effect. As the operation of these hormones affects many organs, the excess of glucocorticoids is actually detrimental to the whole human body. The endogenous glucocorticoid excess is a relatively rare condition, but a significant proportion of adult people uses glucocorticoid medication for the treatment of chronic illnesses, therefore they are exposed to the side effects of long-term glucocorticoid treatment. Our review summarizes the adverse effects of glucocorticoid excess affecting bones, adipose tissue, brain and skin, focusing on those effects which involve the Wnt/β-catenin pathway. Glucocorticoids (GCs) are pleiotropic hormones with an overall impact on different processes: cell proliferation and survival, growth, reproduction, metabolism and cardiovascular function essential for life. They are also responsible for the proper operation of the immune system and central nervous system ,2,3. GluEndogenous glucocorticoid excess, Cushing’s syndrome (CS), is a relatively rare disease, with an annual incidence of 0.7–2.4 cases per million population . GlucocoGlucocorticoid signaling engages many signaling pathways to exert its pleiotropic effect in the human body. The most studied pathways include the GH/IGF-1 axis, TGFβ-SMAD signaling, MAPK signaling, PI3/Akt signaling, Wnt-signaling and BMP signaling. All these at various levels can be responsible for signs observed in patients suffering from GC excess.The Wnt/β-catenin pathway is an ancient signaling cascade that controls numerous cell processes  during embryonic development and adult homeostasis . Its derWnt proteins are a family of small signaling glycoproteins, which bind to a member of the Frizzled (Fz) receptor family. In the absence of Wnt-ligand, β-catenin is constantly degraded by a destruction complex composed of Axin and Adenomatous polyposis coli (APC) which admit the phosphorylation of β-catenin by glycogen synthase kinase 3β (GSK-3β) and casein kinase 1 (CK1). Phosphorylated β-catenin is subjected to proteosomal degradation, resulting in low free cytoplasmic and nucleic β-catenin levels in the absence of Wnt-ligand binding. The binding of Wnt to the Fz receptor requires low-density lipoprotein receptor-related-protein 5 or 6 (LRP 5/6) as coreceptors. The formation of the Wnt-Fz-LRP 5/6 complex leads to the membrane recruitment of the Axin complex by Dishevelled proteins (Dvl), Axin being bound to a cytoplasmic tail of LRP 5/6. In the absence of the degrading complex, β-catenin escapes from phosphorylation and consequential ubiquitination. This leads to an increase in free β-catenin levels which form complexes in the nucleus with the DNA-bounded T cell factor/lymphoid enhancer factor (TCF/LEF) and activate Wnt target gene expression. Up to now,10 families of Wnt-pathway agonists were identified [The common occurrence of adrenal adenomas in patients with familial adenomatous polyposis highlighted the role of the Wnt/β-catenin pathway in adrenal tumorigenesis ,15. WithIn another study, genetic alterations were found in 5 of 33 adrenal adenomas (15%). Of these, three were cortisol-secreting, one aldosterone-producing and one nonfunctioning adenoma . In two Homeostasis of the adult skeleton depends on the balance between bone-forming and bone-resorbing processes and relies on the complex communication between osteoblasts, osteocytes and osteoclasts.Bone-forming osteoblasts are derived from mesenchymal stem cells (MSCs) located in the bone marrow. Master transcriptional regulator Runx controls the commitment of MSCs toward osteogenesis and the following differentiation processes . PreosteThe communication between bone cells is conducted partly through the production of cytokines. Osteoblasts release M-CSF, which binding to its cognate receptor on preosteoclasts, provides signals essential for the survival and proliferation of osteoclast precursors . The recGlucocorticoid-induced osteoporosis (GIO), the most common cause of secondary osteoporosis ,28, is cGC excess rapidly induces the apoptosis of osteoblastic cells as it was demonstrated on different cell lines and in patients with CS or receiving GC treatment. Sclerostin treatment of human osteoblastic cells initiates apoptosis through a caspase-activated mechanism . sFRP-1 Glucocorticoids are detrimental to osteo-anabolic processes through their effects on oxidative stress levels ,35. The Glucocorticoid excess enhances the expression of Wnt-inhibitors. Supraphysiological levels of glucocorticoids enhance sFRP-1 expression in primary mesenchymal cell cultures in vitro and in osteoblasts and bone marrow cells in vivo . The expWnt-pathway inhibition influences cell proliferation and maturation of cells from osteoblastic lineage. Dkk-1 expression in rat calvaria cells was associated with inhibited osteoblast differentiation and with the lack of mineralized nodules; parallelly, increased adipocyte differentiation could be detected in vitro . In Yao’The loss of osteocytes by GC-induced apoptosis damages the osteocyte-canalicular network and the mechanosensory ability of bone, which, in turn, decreases the capacity of osteocytes to regulate osteoclasts’ function. In mechanosensory osteocytes, the capacity to produce RANKL is much higher than that in osteoblasts . Mice laIntact Wnt-signaling exerts an indirect negative role on osteoclastogenesis: in pluripotent mesenchymal cells, and stromal cells Wnt3a upregulates OPG expression , therebyThe role of sFRP-1 in the regulation of osteoclasts now seems to be bidirectional: first, sFRP-1 inhibits osteoclast formation by preventing the fusion of osteoblast precursors , secondlRecently, Albers et al. demonstrated a direct effect of the Wnt-signaling pathway on osteoclastogenesis and bone resorption beside the indirect effects mediated by osteocytes . They foIn summary, we can conclude that the Wnt/β-catenin pathway is essential in normal bone homeostasis, and the harmful effects of GC excess are closely related to the inadequate operation of this pathway.Adipogenesis is a well-orchestrated process during which MSC-derived preadipocytes develop into mature adipocytes. This process involves the interplay of several transcription factors, from which PPARγ functions as a master regulator .Wnt10b gene detected in families with early-onset obesity represent additional evidence for the strong relation between PPAR- and Wnt-signaling [There is plenty of evidence for strong communication between adipose tissue and bones, based (partly) on the interaction between PPAR- and Wnt-signaling pathways. Sustained activation of the Wnt/β-catenin pathway by expression of Wnt10b and inhibiting expression of PPARγ-associated adipogenic genes, in murine preadipocytes, maintains preadipocytes in an undifferentiated state . Inverseignaling .Clinically, the strong but opposite-sign communication between adipogenesis and osteoblastogenesis appears most expressively in patients with Cushing’s syndrome presenting with osteoporosis, osteopenia and increased bone fragility associated with central obesity ,53. GC eGCs change the characteristics of adipose tissue differently in VAT and SAT, as it was demonstrated in methylprednisolone-treated rats . An incrAdipose tissue produces Wnt-inhibitors belonging to the sFRP family. Gene expression of all sFRPs (sFRP1-5) was analyzed in subcutaneous and visceral adipose tissues of obese and non-obese women . In SAT,Adipocytes secrete a variety of signaling proteins  and proinflammatory cytokines which are responsible for the auto-, para- and endocrine effects of adipose tissue. Adipose tissue-derived cytokines  were proved to affect the function of osteoblasts and/or osteoclasts ,59 and nLeptin is one of the first discovered adipokines and nowadays it is considered as the prototype of all signaling molecules produced by the adipose tissue. It is responsive to extensive communication between adipose tissue and brain, pancreas and reproductive organs . The relData from the last two decades have provided sufficient evidence for the role of Wnt-signaling in different processes of neurogenesis. Recently, Guo summarized these results and concluded that the Wnt/β-catenin signaling pathway plays a context-dependent role in oligodendrogenesis, oligodendrocyte differentiation and myelination. In addition, the effects associated with the Wnt/β-catenin pathway depend on the developmental stage of CNS, oligodendrocyte and CNS microenvironment .Detrimental effects of increased GC levels on brain structure, neurological function and emotional life have been demonstrated in numerous studies. GC excess negatively affects memory retrieval, cognitive functions and behavior and they have significant effects on brain structure ,67.Of brain regions, the hippocampus seems to be the most vulnerable to the neurotoxic effect of GCs ,68,69. MClinically, brain development and affective problems of preadolescent children with or without fetal exposure to synthetic GCs were evaluated by Davis et al. . A signiAccumulating evidence suggests that long-lasting effects of GCs on the brain are regulated through epigenetic mechanisms. Rat embryonic neural stem cells exposed to dexamethasone underwent heritable alterations; GC effects induced in parent cells were detectable in daughter cells which were never exposed directly to GCs . MethylaExtensive genome-wide alterations in promoter methylation, typically the hypomethylation of the affected promoters, were detected in the hippocampi of fetal guinea pigs treated prenatally with GCs . The samInhibitory effects of dexamethasone on the neural precursor cells and the role of the Wnt/β-catenin pathway in this process were described by Boku et al. . The negThe negative effects of GCs were demonstrated on human neural progenitor cells (hNPCs) responsible for the major cell types  of the brain. Treating hNPCs with dexamethasone caused a marked decrease in proliferation and neuronal differentiation of hNPCs while increased glial cell formation. Dexamethasone induced the upregulation of the Wnt-signaling inhibitor Dkk-1 which effect could be inhibited by adding a GR antagonist, mifepristone. The final conclusions were that Dkk-1 was the primary target for GR in hNPCs and the regulation of hNPCs by dexamethasone and Dkk-1 was mediated through the canonical Wnt-signaling pathway .High levels of GCs induced the generation of reactive oxygen species (ROS) in primary hippocampal and cortical cell cultures . AccordiSkin, besides its primary function of protecting the body, is an important part of immunological processes and extraadrenal steroidogenesis . RecentlKeratinocytes, one of the main cell types of the epidermis, synthesize cholesterol, the precursor of all steroids . EpidermRelated to Cushing’s syndrome, a part of the signs and symptoms are relatively common  and makes CS difficult to diagnose. Some of the skin-related manifestations  are between those signs which best discriminate Cushing’s syndrome from other diseases, while other skin-related signs  are very common in patients with CS  .GCs have profound effects on the different layers of the skin. In the epidermis, the decreased size of keratinocytes, the thinning of the epidermis and the diminution of the lamellar lipid bilayer in the stratum corneum are well-known histopathological features of GC excess . The molIn the dermis, GCs lead to a decreased number of fibroblasts with the rearrangement of the dermal fibrosis network . The antThere are only little data about the effect of GC excess on hypodermis, but we have to note the confusion in the literature related to the definition of skin-associated adipocyte layers. Traditionally, the thick layer of adipocytes underlining the reticular dermis has been called hypodermis or subcutis and it was considered that all skin-associated adipose tissues represent one depot . The preRelated to the effects of GC excess on skin compartments, it seems plausible that skin atrophy involves all skin layers, including dermal white adipose tissue. An indirect proof for this assumption was presented very recently by Baida et al. . They stEpidermal appendages are also influenced by GC excess. Based on the data of 481 patients published by the European Registry on Cushing’s syndrome, hair loss was present in 31% of patients . Female Ectopic expression of Wnt-inhibitor Dkk1 in epidermis drives to the lack of epidermal appendages: hair, whiskers, teeth and mammary gland . EpithelAs the canonical Wnt-signaling plays an important role in skin cells’ proliferation and differentiation, its impact on wound healing is self-evident. Wnt10a-deficient mice, besides their phenotype including alopecia and follicle growth failure, showed delayed wound healing compared to their wild-type littermates . De novoIn non-healing edges of venous ulcers, components of the Wnt-pathway were found to be deregulated: phosphorylated β-catenin was found mainly in the cytoplasm and nuclear; while in healthy skin specimens, β-catenin was present mostly at the membrane of the basal layer . Jozic eWe have to conclude that, related to GC excess-induced skin alterations, the sole role of the Wnt/β-catenin pathway has not yet proven in many processes, but its function obviously contributes to these phenotypes. Future research can clarify the bidirectional link between the Wnt-pathway and GC-induced skin lesions.Balanced operation of the Wnt/β-catenin pathway is indispensable for homeostatic processes in many tissues, including bones, brain, adipose tissue and skin .. HoweveSelective inhibition of sFRP-1 was also proposed as a potential tool to stimulate Wnt-signaling, thereby osteo-anabolic processes in bone ,108. In Wnt/β-catenin signaling can be stimulated by physical exercise. In a study conducted among women affected by abdominal obesity, osteoblast cell cultures were exposed to sera of these women at an initial timepoint and 4, 6 and 12 months after they attended a physical training program combined with a hypocaloric diet . ProteinA similar study was carried out among breast cancer survivors, where serum levels of Dkk-1 and sFRP-1 were followed before and after a 12-week long physical exercise training program. The conception of this study was based on the results of former studies showing that elevated serum Dkk-1 levels correlated with poor prognosis in certain types of cancer . SignifiAnother evidence for the beneficial effects of physical exercises on Wnt-pathway was the mechanosensitive expression of sclerostin (specific product of osteocytes). Mechanical unloading of wild-type mice caused downregulation of Wnt/ß-catenin signaling with the upregulation of the sclerostin levels . In SostThe beneficial effects of physical exercise were also highlighted by Chen, who recently reviewed the paradox of chronic stress and chronic exercise, both known to increase basal cortisol levels. Their metanalysis underlined that solely increased basal cortisol levels are not necessarily detrimental to the human body and their work suggested that other factors are also needed for the harmful effects of glucocorticoid excess . RelatedRelated to this, Noguchi et al. proved that the detrimental effects of the glucocorticoid excess on bone could be prevented by oxygen ultra-fine bubbles saline injection . In predSummarizing these results, it can be assumed that the restorative effects of physical exercise on Wnt/ß-catenin pathway can be linked partly to an enhanced oxygen supply, and inversely, deleterious effects of GC excess on Wnt-pathway are more pronounced when hypoxic conditions are present and probably the severity of the damaging effects is related to the degree of hypoxia.The relationship between GC excess and severe comorbidities is well-known: fractures occur in 30%–50% of the patients subjected to long-term hypercortisolism ; the rat


Study of filamentous-actin (F-actin) subsequently showed that SEMA3F-mediated retention is associated with F-actin disassembly. In conclusion, SEMA3F signaling actively regulates neutrophil retention within the injured tissues with consequences for neutrophil clearance and inflammation resolution.Neutrophilic inflammation is central to disease pathogenesis, for example, in chronic obstructive pulmonary disease, yet the mechanisms that retain neutrophils within tissues remain poorly understood. With emerging evidence that axon guidance factors can regulate myeloid recruitment and that neutrophils can regulate expression of a class 3 semaphorin, SEMA3F, we investigated the role of SEMA3F in inflammatory cell retention within inflamed tissues. We observed that neutrophils upregulate SEMA3F in response to proinflammatory mediators and following neutrophil recruitment to the inflamed lung. In both zebrafish tail injury and murine acute lung injury models of neutrophilic inflammation, overexpression of SEMA3F delayed inflammation resolution with slower neutrophil migratory speeds and retention of neutrophils within the tissues. Conversely, constitutive loss of  Effective host responses to injury and infection require both rapid recruitment of neutrophils into tissues and timely inflammation resolution. Research efforts have focused separately on the initiation and resolution phases of inflammation and, in terms of resolution, on how neutrophil survival responses determine the duration and extent of the inflammatory response . Less foDirected neutrophil migration, essential for neutrophil recruitment to the injury site and an effective innate immune response, is critically dependent upon polarization of the cell. Cell polarity is tightly regulated by phosphoinositide 3-kinase (PI3K) signaling products, with PI3K activation at the leading edge linked to microtubule assembly and activation of key regulators of the actin cytoskeleton, including Rho and Rac –4. TheseSema3f, a member of the semaphorin family of secreted and transmembrane axon guidance molecules , a disease characterized by neutrophilic inflammation. In tissue sections of surgically resected COPD lung, we identified staining for SEMA3F, and the coreceptor neuropilin 2 (NRP2) (+ myeloid cell populations (https://doi.org/10.1172/JCI130834DS1). Equivalent levels of secreted SEMA3F were found between these treatment groups (We have previously established that the trophils . This re2 (NRP2) , with NRulations . Ex vivot groups . In parat groups . Ex vivot groups . Naive at groups .nrp2 coreceptor (http://www.sanger.ac.uk/science/tools/basicz). We therefore tested the effects of genetic ablation of sema3f by morpholino-modified (MO-modified) antisense oligonucleotide knockdown of both zebrafish homologs of human SEMA3F (sema3fa and sema3fb)  refs. . While kruitment , disruptr injury . Furtherobserved . Importal counts .mpx:GFP background, in which stop codons were introduced into the sema3fa and sema3fb genes using genome editing with transcription activator–like effector nucleases (TALENs)  . Followigenotype , confirmgenotype . Inflamm sema3fb , recapit sema3fb . Photocotogether .Sema3f transcript and SEMA3F protein over an extended time course (Nrp2 at both mRNA and protein levels (Sema3f was the key determinant for neutrophil egress from the lung. We therefore crossed Mrp8 Cre with fl/flSema3f lines to generate a transgenic mouse with Mrp8-driven neutrophil-specific knockdown of Sema3f (fl/flMrp8Cre+/–Sema3f) (fl/flMrp8Cre+/–Sema3f (KO) mice exhibited both a greater influx and more rapid rate of clearance of these cells  IgM levels between 24 and 48 hours in the Sema3f-deficient mice  . Followise cells  than Sement mice . Importaent mice .Sema3f regulated airway neutrophil counts in the face of preserved apoptosis rates, we examined peripheral blood  was instilled into the trachea of mice and total cell and neutrophil differential counts were performed at 48 and 72 hours . More neE. coli) or yeast (Zymosan) by neutrophils  . To furttrophils . Surpristrophils  while prtrophils .sema3fa and sema3fb RNA overexpression resulted in a reduction in overall F-actin levels  are well established, the expression of molecules that function to retain neutrophils within the injury site represents a mechanism through which the longevity of the inflammatory response may be regulated.To date, effective therapeutic strategies that target persistent neutrophilic inflammation remain an important unmet clinical need. In this work, we identify a mechanism by which inflammatory cells can deliver a neutrophil-specific retention signal to the injury site in the form of a secreted axon guidance molecule, SEMA3F. Initial phenotyping revealed, both acutely in a murine LPS model of lung injury and chronically in lung sections from patients with COPD, that recruited myeloid cells express the class 3 semaphorin SEMA3F and its high-affinity coreceptor NRP2. With evidence that CD66b–/–Sema3e murine model  , and recne model . Our worWithin axons, actin rearrangement is observed following activation of semaphorin signalling pathways. In our experiments, we also observed that SEMA3F promotes neutrophil rounding in association with F-actin disassembly and persistence within the airspaces. Taken together, these data suggest a mechanism by which neutrophil expression of SEMA3F selectively regulates neutrophil retention within the inflammatory site as a consequence of promoting F-actin disassembly and suppressed neutrophil migratory speeds. Future work to explore the mechanisms by which neutrophil SEMA3F expression regulates neutrophil retention within the tissue and whether this is a cell autonomous mechanism or dependent on the presence of other immune cell populations  will be of interest. Importantly, this occurs in the face of preserved leading edge PI3kinase activity, preserved directional migration, and critically preserved neutrophil effector functions. This is in keeping with SEMA3F acting downstream of the PH domain proteins, with F-actin disassembly and aberrant F-actin distribution, and turnover selectively affecting neutrophil retention in the face of preserved antimicrobial effector function. Thus we propose that SEMA3F acts as a break in neutrophil movement among the different vascular, interstitium, and airspace compartments within the lung.Sema3f loss within the neutrophil compartment on effective antimicrobial responses was explored in vivo. Importantly, neutrophils deficient in Sema3f were able to mount an antimicrobial response that was sufficient to control bacterial numbers where animals were exposed to a bacteremic model of streptococcal pneumonia. With a dissociation between the consequences of Sema3f loss on neutrophil migratory speed versus respiratory burst generation and bacterial clearance, an important future direction of this work will be to dissect the downstream mechanisms by which neutrophils retain key effector functions in the face of aberrant F-actin turnover and distribution.Finally, when considering the therapeutic utility of targeting SEMA3F to promote inflammation resolution, the consequence of In summary, our work aims to elucidate how neutrophils respond to signals within an inflamed microenvironment that induce them to persist. We identify SEMA3F as a neutrophil retention signal with effects conserved across a number of different species . These observations have important implications for inflammation resolution and tissue injury and are of interest given both the current lack of effective therapeutic strategies for treating neutrophil inflammation and the potential to selectively target the neutrophil retention response.2O. Serial sections were stained with anti-Ly6G clone 1A8  and the following polyclonal antibodies: anti-SEMA3F , NRP1 , NRP2 , or an isotype control following deparaffinization. Lung sections from patients with COPD undergoing resection for suspected lung tumor were stained with anti-SEMA3F, NRP2, or isotype control, and visualized with DAB or stained with anti-CD66b , anti-NRP2 , and DAPI  with TSA plus system amplification  and autofluorescence quenching with TrueView .Murine paraffin-embedded blocks were prepared from lungs fixed via the trachea with 10% buffered formalin at 20 cm H6 cells/mL in RPMI 1640 supplemented with 10% FCS and 1% penicillin/streptomycin at 37°C in a humidified incubator with 5% supplemental CO2 in the presence or absence of LPS from Escherichia coli , IL-1b , TNFa , or LTB4  for 4 to 12 hours.Human peripheral blood neutrophils were isolated from whole blood using dextran sedimentation and discontinuous plasma-Percoll gradients. Cells were cultured 5 × 10PLUS iced water bath (Diagenode Europe SA) and added to lysis buffer. Following SDS-PAGE protein separation , transfer of proteins to a PVDF membrane was performed using a Bio-Rad Mini Trans-Blot cell at 100 V for 90 minutes. Intercept (PBS) blocking buffer (Li-Cor) was used to block blots and dilute primary and secondary antibodies. Primary antibodies were directed against SEMA3F  and NRP2 , and P38  was used as a loading control. Blots used for the detection of SEMA3F were incubated with horseradish peroxidase–conjugated (HRP-conjugated) anti-rabbit , and blots used for the detection of NRP2 were incubated with IRDye800CW-conjugated anti-rabbit . Signals were detected using an Odyssey FC Imaging System (Li-Cor) and protein expression was quantified by scanning densitometry and normalized to the expression of P38. Enzyme-linked immunosorbent assay (ELISA) was performed according to the manufacturer’s protocol to quantify SEMA3F levels (Cloud-Clone Corporation) in supernatants of human blood neutrophils following 4 hours culture ex vivo with inflammatory stimuli, IgM levels (Abcam) in BAL fluid from mice either after 24 or 48 hours after nebulization with LPS , or after 14 hours post-intratracheal instillation of 107S. pneumoniae D39 type 2 strain. Flow cytometry was performed to analyze surface expression of L selectin and CD11b by using anti–L selectin [PE] (BD Pharmingen) and anti-CD11b [PE] (BD Pharmingen), respectively.Lysates were prepared using sonication in a BioruptorSema3f) and the endogenous control assay (actb) (Applied Biosystems).Human peripheral blood neutrophils were isolated as described above, with murine peripheral blood neutrophils isolated from WT C57BL6 mice by negative magnetic selection  and inflammatory neutrophils were recovered from BAL fluid following challenge with 1 mg nebulized LPS. RNA was extracted using the mirVana total RNA isolation protocol (Ambion). Samples were treated with DNAse (Ambion) and random hexamer cDNA was synthesized by reverse transcription. TaqMan commercially available primer probe sets were obtained from Applied Biosystems for the target assay  after red blood cell lysis (BioLegend). Total viable neutrophil counts were obtained after blocking unspecific staining with Fc block (BioLegend) and using live/dead aqua dye (Applied Biosystems) in combination with the following antibodies (BioLegend): anti-CD45 [AF700] (clone 30-F11), anti-CD11b [BV450] (clone M1/70), and anti-Ly6G [PE] (1A8). For total lung counts or after infection, lung digest was performed with Collagenase V (Sigma-Aldrich), Collagenase D (Roche), DNAse I (Roche), and Dispase (Gibco). Following red cell lysis (Sigma-Aldrich), viable cell counts were performed using live/dead aqua dye (Applied Biosystems). Immune cell populations were identified after Fc block (BioLegend) using the following markers : anti-Siglec F [PE-CF594] (clone S17007L ), anti-CD11b [BV450] (clone M1/70), anti-Ly6C [FITC] (clone HK1.4), anti-Ly6G [PE] (clone 1A8), anti-MHC II [PerCP-Cy5.5] (clone C068C2), anti-CD11c [PE-Cy7] (clone N418), anti-CD64 [APC] (clone X54-5/7.1), and anti-CD45 [AF700] (clone 30-F11). BAL fluid was harvested from mice after LPS or after infection and total nucleated cell counts were obtained either with hemocytometer or NucleoCounter (Chemometec). Total viable counts for the different immune populations were obtained by using either cytospins or the same markers used for lung tissue. Flow cytometry was performed to analyze NRP2 surface expression by using anti-NRP2 [APC] .Murine venipuncture was performed directly following humane killing and full blood counts were conducted using the Coulter counter method by the clinical pathology service at the Royal Dick Veterinary School, Edinburgh, Scotland. For 7 serotype 2, S. pneumoniae left lung was homogenized in sterile conditions by using a tissue homogenizer (Bullet Blender) and lysing tubes (Precellys). Ten-fold serial dilutions were plated into blood agar plates, grown overnight at 37°C and 5% CO2, and CFU counts determined.Fourteen hours after infection with 10Tg(mpx:GFP)i114 , Tg(mpx:GAL4)sh267;Tg(UAS:kaede)s1999t, Tg(lyz:PHAkt-EGFP)i277, Tg(mpx:LifeActRuby)sh429, and macrophage-specific fluorescent zebrafish line Tg(fms:GAL4.VP16)i186;Tg(UAS:nfsB.mCherry)c624. Transcription activator–like effector nuclease (TALEN) technology was used to induce frameshift mutations in sema3fa and sema3fb genes. Fertilized eggs were injected with RNAs coding for heterodimers of a left and right subunit containing a C-terminal Fok1 nuclease domain and an N-terminal site-specific DNA binding domain, specifically targeting sema3fa or sema3fb using the tool on http://talent.cac.cornell.edu/node/add/talen-old, with TALE repeats designed to flank the Mwol (sema3a) and BsII (sema3b) restriction endonuclease restriction site of exon 8 as previously described  and microinjected into one-cell-stage embryos. All MOs were from Genetools. One-cell-stage embryos were microinjected with pre-mRNA translation blocking MO to sema3fa  containing full-length zebrafish  sema3fa  or splicy RT-PCR . The conInflammatory responses were elicited in zebrafish embryos by tail transection as previously described . NeutropTg(lyz:PHAkt-EGFP)i277 larvae were injured 3 days after fertilization (dpf), mounted in 1% low melting point agarose (Sigma-Aldrich) containing 0.017% tricaine, and imaged on an UltraVIEWVoX spinning disk confocal microscope (Perkin Elmer) with an inverted Olympus IX81 microscope. Neutrophils in the region between the site of injury and the posterior blood island were individually imaged in the 488 nm laser line with 20 Z slices. Using Image J software, a transecting line was drawn from the trailing edge to the leading edge and a fluorescence intensity plot generated. Polarity index was calculated by (log10a/b) × ([a + b]/c), where a represents the mean fluorescence intensity of the trailing edge of the cell, b represents the mean fluorescence intensity of the leading edge of the cell, and c represents the mean fluorescence intensity of the whole cell (Mrp8Cre) targeted Sema3f deletions to neutrophil populations. fl/flSema3f mice were generated via the Medical Research Council (MRC) International Mouse Phenotyping Consortium  with animals backcrossed to a C57BL/6 background  and ketamine (76 mg/kg), and then instilled with 50 μL PBS or 1 μM SEMA3F via the trachea, followed by recovery with 1 mg/kg atipamezole.Z-stacks were acquired and projected using Imaris  for analysis. For fixed slices, image stitching was performed using Zen black or blue software (Carl Zeiss) and represented an area of 1 mm × 1 mm of tissue, imaged at 1 time point/position. Image analysis was performed using IMARIS V 9.1 software  low melting point agarose gel (Life Technologies). Lungs were dissected en bloc, washed in PBS, and placed in either methanol-free 4% formaldehyde solution in PBS  for 2 hours (fixed lungs) or RPMI media (live lung culture), and 300 μm vibratome sections were generated . Fixed lE. coli O55:B5 (Sigma-Aldrich) (MOI 1:1) for 30 minutes at 37°C. Cells were washed with ice-cold PBS, resuspended in FACS buffer, and analyzed by flow cytometry . Data analysis was performed with FlowJo software .Neutrophil chemotaxis to fMLF (0–100 nM) over 60 minutes was measured in the presence or absence of recombinant SEMA3F protein (10–100 nM) (R&D Systems) using 5-μm filter NeuroProbe ChemoTx microplates. For microfluidic chip assays, whole blood was applied to a stable LTB4 gradient (100 nM) and migration assessed using real-time light microscopy . To dete6/mL) were cultured with recombinant SEMA3F protein  (R&D Systems) for 1 hour before addition of 5 μM 2′7′-dichlorofluorescein diacetate (DCF) for 30 minutes. Cells were then stimulated with 100 nM fMLF for 30 minutes and FL1 geometric mean fluorescence was determined by flow cytometry.Human neutrophils  and fMLF (100 nM). Neutrophils were fixed, permeabilized, stained with DAPI, and Alexa Fluor 488–conjugated phalloidin. Images were obtained using an Operetta High-Content Imaging System (Perkin Elmer) or LSM880 Airyscan Fast (Zeiss) and analysis of F-actin was performed by Harmony analysis software (Perkin Elmer).t tests were used to compare 2 groups. One-way and 2-way ANOVA with Bonferroni’s, Dunnett’s, or Sidak’s post hoc test were used to compare different groups if the data followed a normal distribution and if the samples analyzed had the same genetic background. P values smaller than 0.05 were considered significant. The number of fish, mice, and donors used are listed in the figure legends.The data are expressed as mean ± SEM. Graph and curves were made using GraphPad Prism software versions 7 and 8. Mann-Whitney, unpaired, and paired Student’s The Role of inflammation in Human Immunity, AMREC reference 15-HV-013. This study was approved by the The Blood Resource Management Committee, Centre for Inflammation Research, Queen’s Medical Research Institute, Little France, Edinburgh; the Academic and Clinical Central Office for Research and Development (ACCORD) medical research ethics committee, AMREC . Human lung tissue was obtained by biopsy through the Edinburgh Tissue Bank, which was reviewed and approved by the Edinburgh Tissue Bank ethics committee and AMREC. The NHS Lothian Bio-Resources provided lung tissue from nontumor regions of lung following biopsy used for the immunohistochemistry in this project (application number SR451). All animal experiments were conducted in accordance with the Home Office Animals (Scientific Procedures) Act of 1986 with local ethical approval.All participants gave written informed consent in accordance with the Declaration of Helsinki principles. Healthy human blood donation was obtained under the University of Edinburgh ethics protocol of the project entitled, TP, SE, MASG, LR, PC, ASM, DH, XLRI, LMC, JMM, FEE, TM, ERW, FM, AZ, JLC, and CL performed the research. TP, SE, MASG, LR, FVE, and PME interpreted the data. TP, SAR, AJWF, MKBW, and SRW designed the research and wrote the manuscript.


Chronic kidney disease (CKD) has a high prevalence worldwide. Renal fibrosis is the common pathological feature in various types of CKD. However, the underlying mechanisms are not determined. Here, we adopted different CKD mouse models and cultured human proximal tubular cell line (HKC‐8) to examine the expression of C‐X‐C motif chemokine receptor 4 (CXCR4) and β‐catenin signalling, as well as their relationship in renal fibrosis. In CKD mice and humans with a variety of nephropathies, CXCR4 was dramatically up‐regulated in tubules, with a concomitant activation of β‐catenin. CXCR4 expression level was positively correlated with the expression of β‐catenin target MMP‐7. AMD3100, a CXCR4 receptor blocker, and gene knockdown of CXCR4 significantly inhibited the activation of JAK/STAT and β‐catenin signalling, protected against tubular injury and renal fibrosis. CXCR4‐induced renal fibrosis was inhibited by treatment with ICG‐001, an inhibitor of β‐catenin signalling. In HKC‐8 cells, overexpression of CXCR4 induced activation of β‐catenin and deteriorated cell injury. These effects were inhibited by ICG‐001. Stromal cell–derived factor (SDF)‐1α, the ligand of CXCR4, stimulated the activation of JAK2/STAT3 and JAK3/STAT6 signalling in HKC‐8 cells. Overexpression of STAT3 or STAT6 decreased the abundance of GSK3β mRNA. Silencing of STAT3 or STAT6 significantly blocked SDF‐1α‐induced activation of β‐catenin and fibrotic lesions. These results uncover a novel mechanistic linkage between CXCR4 and β‐catenin activation in renal fibrosis in association with JAK/STAT/GSK3β pathway. Our studies also suggest that targeted inhibition of CXCR4 may provide better therapeutic effects on renal fibrosis by inhibiting multiple downstream signalling cascades. The results indicate CXCR4 plays a crucial role in mediating renal tubular cell injury and is associated with activation of β‐catenin. Furthermore, JAK/STAT/GSK3β pathway mediates SDF‐1α/CXCR4‐induced activation of β‐catenin. These findings suggest that CXCR4 mediates renal fibrosis through activating JAK/STAT/GSK3β/β‐catenin pathway.22.1Male C57BL/6 mice, weighing 22‐24 g, were purchased from Southern Medical University Animal Center  and housed in a standard environment on a regular light/dark cycle with free access to water and chow. For the unilateral ureteral obstruction (UUO) model, male C57BL/6 mice were performed double‐ligating of the left ureter using 4‐0 silk after a midline abdomen incision. Sham‐operated mice had their ureters exposed and manipulated but not ligated. Mice were killed 7 and 14 days after UUO. The ischaemia‐reperfusion injury (IRI) model was established by bilateral renal pedicles clipped for 32 minutes using microaneurysm clamps. During the ischaemic period, body temperature was maintained between 37°C and 38°C using a temperature‐controlled heating system. After removal of the clamps, reperfusion of the kidneys was visually confirmed. Mice were killed 7 and 10 days after IRI. For the unilateral IRI (UIRI) model, male C57BL/6 mice were subjected to unilateral renal IRI by an established protocol, as described previously.2.2Human proximal tubular epithelial cells (HKC‐8) were provided by Dr L. Racusen . Cell culture was carried out according to the procedures described previously.2.3Protein expression levels were analysed by Western blot analysis as described previously.2.4Paraffin‐embedded mouse kidney sections (4 µm thickness) were prepared by a routine procedure. Periodic acid‐Schiff (PAS), Masson trichrome staining and Sirius red staining  were performed by a standard protocol or according to the manufacturer's instruction. Immunohistochemical staining was performed using routine protocol. Primary antibodies used were as follows: CXCR4 , β‐catenin , collagen I , α‐SMA , E‐cadherin , fibronectin  and active β‐catenin , anti‐p‐GSK3β (ser 9) , p‐STAT3 or p‐STAT6  and p‐JAK2 .2.5Frozen kidney sections (3 µm) were fixed with 4% paraformalin for 15 minutes at room temperature. HKC‐8 cells cultured on coverslips were fixed with cold methanol:acetone (1:1) for 10 minutes at −20°C. The slides were blocked with normal donkey serum and incubated with primary antibodies as follows: anti‐fibronectin , anti‐β‐catenin antibody , anti‐STAT3 antibody , anti‐STAT6 antibody  and anti‐E‐cadherin . After washing, the slides were incubated with Cy3‐ or Cy2‐conjugated donkey anti‐mouse or donkey anti‐rabbit IgG . Nuclei were stained with DAPI (Sigma‐Aldrich) according to the manufacturer's instruction. Images were taken by confocal microscopy  or Olympus DP80 microscope with EMCCD camera.2.6Total RNA was prepared using TRIzol RNA isolation system  according to the manufacturer's instruction. The first strand of complementary DNA was synthesized using 1 μg of RNA in 20 μL of reaction buffer using AMV‐RT and random primers at 42°C for 60 minutes. PCR amplification was performed using a HotStarTaq Master Mix kit . The sequences of the primer pairs are shown in Table 2.7Determination of Scr and BUN was analysed by an automatic chemistry analyzer . The level of Scr or BUN was expressed as mg/dL or mmol/L, respectively.2.8P < .05 was considered significant.All data examined were expressed as mean ± SEM. Statistical analysis of the data was carried out using SPSS 19.0 . Comparison between groups was made using one‐way ANOVA followed by Student‐Newman‐Keuls test or Dunnett's T3 procedure. 33.1To identify the role of CXCR4 in renal fibrosis, we first examined the expression of CXCR4 and CXCL12 (SDF‐1α), the ligand of CXCR4, in various CKD models. The protein expression of CXCL12 (SDF‐1α) was elevated time‐dependently in UUO and IRI mice Figure . Consist3.2To investigate the potential role of CXCR4 in renal fibrosis, we injected mice with AMD3100,3.3We then examined the correlation between CXCR4 and β‐catenin. First, we assessed the expression of GSK3β, a serine/threonine protein kinase that mediates β‐catenin degradation and its phosphorylation at Ser9 suggests the deactivation of GSK3β.3.4To further confirm the role of CXCR4 in renal fibrotic lesions, we carried out the mouse model of unilateral ischaemia‐reperfusion injury (UIRI). Groups of mice were subjected to UIRI and administered AMD3100 at the dose of 1 mg/kg/d  was assessed. As shown in Figure 3.6To further establish the role of CXCR4 in β‐catenin signalling, mice were delivered a flag‐tagged CXCR4 expression plasmid (pFlag‐CXCR4) by a hydrodynamic‐based gene therapy3.7To further figure out the important role of β‐catenin activation in CXCR4‐induced tubular injury, we performed analysis in a human proximal tubular cell line (HKC‐8). First, HKC‐8 cells were transfected with CXCR4 expression plasmid (pFlag‐CXCR4) or treated with SDF‐1α, the ligand of CXCR4. As shown in Figure 3.8It was previously reported that the cytoplasmic domain of CXCR4 protein involves in JAK2 and STAT3 phosphorylation.3.9To further testify the modulation of JAK/STAT pathway by CXCR4, we next analysed the activity of JAK2/STAT3 and JAK3/STAT6 in UUO and UIRI mice administered AMD3100. The detailed experimental designs were presented in Figures 3.10To further prove the role of CXCR4 in renal fibrosis, we knocked down renal CXCR4 expression in vivo. The mice were injected with shRNA vector encoding the interference sequence of CXCR4 (pLVX‐shCXCR4) through a hydrodynamic‐based gene delivery approach. The detailed experimental design was shown in Figure 4More than 10% of the global populations are suffering from CKD. Unfortunately, a large portion of them would inevitably progress into the end‐stage renal disease (ESRD).Renal fibrosis is initially characterized by the excessive matrix deposition in the interstitial area. With the progression of disease, the massive collagen deposition in the interstitial area could invade nearby nephron, the basic structural and functional unit of the kidney, and spread quickly. In humans with a variety of nephropathies and experimental CKD models, it is often seen that tubular cells may undergo cell injury in different ways with the secretion of fibrosis‐related molecules, especially in the late stage of disease.C‐X‐C motif chemokine receptor (CXCR) family is a member of seven‐transmembrane receptors, which exerts chemoattractant effects on leucocytes and other cell types in controlling cell activation and guiding cell movements under basal and inflammatory conditions.β‐catenin is the downstream effector of Wnt signalling in tubular cell senescence,We first assessed the expression of CXCR4 and β‐catenin in experimental mouse models. The results show that CXCR4 and β‐catenin were both up‐regulated predominantly in injured tubular cells in a time‐dependent manner. The results of staining on sequential sections further claim the co‐localization of CXCR4 and β‐catenin in tubular cells. Furthermore, the expression of CXCR4 has a positive correlation to the expression of MMP‐7 and triggers signal amplification of MMP‐7, the downstream target of β‐catenin Figure . We alsoWe next performed the analysis in experimental mouse models and in cultured tubular cells. In UUO and UIRI mice, blockade of CXCR4 by AMD3100 significantly inhibited the activation of p‐GSK3β/β‐catenin signalling, thereby protected against renal fibrotic lesions and preserved kidney functions Figures , 3, 4, 5One of the novel findings in the present study is that the transcription factors STAT3 and STAT6 could regulate GSK3β/β‐catenin signalling in renal tubular cells Figure .This obsThe authors declared no competing interests.Yahong Liu, QijianFeng, Jinhua Miao, Qinyu Wu, Shan Zhou and WeiweiShen conducted the experiments and prepared the materials involved in this study. Lili Zhou conceived this study and its design and coordination. Lili Zhou, Fan FanHou, Youhua Liu, YanqiuFeng, Jinhua Miao and WeiweiShen contributed to the analysis and interpretation of the data. Lili Zhou and Yahong Liu drafted the manuscript. All authors read and approved the final manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file.