We exploit graphene’s flexibility and simulatemechanical tuning of a finite size PnC. Under electrostatic pressureup to 30 kPa, we observe an upshift in frequency of the entire phononicsystem by ∼350%. At the same time, the defect mode stays withinthe bandgap and remains localized, suggesting a high-quality, dynamicallytunable mechanical system.In the field of phononics, periodic patterning controls vibrationsand thereby the flow of heat and sound in matter. Bandgaps arisingin such phononic crystals (PnCs) realize low-dissipation vibrationalmodes and enable applications toward mechanical qubits, efficientwaveguides, and state-of-the-art sensing. Here, we combine phononicsand two-dimensional materials and explore tuning of PnCs via appliedmechanical pressure. To this end, we fabricate the thinnest possiblePnC from monolayer graphene and simulate its vibrational properties.We find a bandgap in the megahertz regime within which we localizea defect mode with a small effective mass of 0.72 ag = 0.002 m Specifically, we expect the entire acousticband structure of such a PnC to be highly tunable by applying mechanicalpressure. Nevertheless, tunability of 2D phononic systems as wellas localized defect modes in them have not been studied yet.Recently, PnCs made from two-dimensional (2D) materials have beenconsidered.40 The applied pressure smears outthe phononic bandgap as the out-of-plane displacement breaks the symmetryand causes perturbations of the artificial lattice, yet the mode shapeof the defect mode remains highly localized. Overall, we can tunethe resonance frequency of the defect mode by more than 350% and accessnew regimes of strain engineering.Here, we investigate mechanical tunability in a realistic graphenePnC. We fabricate a suspended micron-sized monolayer graphene PnCvia focused helium ion beam milling (FIB) and characterize it spectroscopically.We then use experimentally established parameters to calculate thephononic band structure of the resulting PnC. We find a phononic bandgapfrom 48.8 to 56.5 MHz inside of which we localize a defect mode withan effective mass of 0.72 ag. Finally, we computationally investigatethe mechanical tunability of the PnC under pressure induced by a localelectrostatic gate.2/(V s)41 and large breaking strength >10%38 perfectly fulfils these requirements. By using large area CVD graphene,we can fabricate many devices on a single chip. Finally, the deviceneeds to host a large enough number of unit cells with sufficientperiodicity to form a well-defined PnC. While this task is simplein thick SiN, it is much more challenging for fragile freestandingmonolayer graphene. To overcome this, we choose a much smaller unitcell compared to typical SiN-PnCs (∼100 μm size) anduse helium FIB-milling to pattern the PnC.42 This direct lithography allows one to pattern graphene down to 10nm features,43 while causing little damage.45 A patterned prototype monolayer graphene PnC is shown in a =350 nm, hole diameter d = 105 nm) around a centralregion. Within its 10 μm diameter, the two-dimensional PnC containsmore than 30 unit cells. The honeycomb lattice inspired by Tsaturaynet al.15 exhibits a robust bandgap46 while retaining a relativelylarge fraction of material to ensure a stable device. Additional PnCwith various patterning sizes are shown in Figures S1–S3.Our device design of a tunable,two-dimensional PnC consists of the following key elements. First,the PnC material must be freestanding to allow out-of-plane displacement.Second, it is necessary to use an electrically conductive material.In that case, an electrostatic gate electrode can be used to applypressure and to induce tension as the membrane is pulled toward thegate. Third, the material needs to be flexible to allow large mechanicaltunability with small pressures. Monolayer graphene with its highcarrier mobility >200 000 cm47 Such tension redistribution should affect the vibrational propertiesof our PnC. To this end, we fabricate another prototype device .Next, we map the tension within the produced structures using Ramanspectroscopy. We expect tension hot spots in the thin ribbons andrelaxation in the centers of the hexagons.e device 1B with l17 However, due to the size limits of suspended graphene, our devicesare smaller than typical SiN-PnCs (mm size)17 and containfewer unit cells. Furthermore, we want to apply pressure to the entiresystem and investigate localized modes in the bandgap. Therefore,we also simulate a more realistic system of finite size (“finitemodel”). For both models, we use the honeycomb lattice withfeasible parameters and account for tension redistribution upon fabrication. This model is well-accepted and fast.rication1D,E. We 51 and therefore do not hinder radiation shielding. The bandgap originatesfrom Bragg scattering, with each hole acting as a scatterer for out-of-planeoscillations. Upon negative interference conditions, directional Braggbandgaps open at the high symmetry points. Where these gaps overlap,radiation shielding becomes possible, as wave propagation is isotropicallyforbidden.1 The bandgap position dependsreciprocally on a. With our fabrication schema, wecan tailor the bandgap center from 350 to 26 MHz by varying a from 0.175 to 2 μm . Overall, the simulations in the infinite modelsuggest the possibility of a large quasi-bandgap, which we will nextuse to control phonons.By applying periodic boundary conditions to the unitcell 2A, wecaa, d/a, and T0 as before) and employ fixedboundary conditions along the PnC’s perimeter. We choose acircular device as such a geometry allows uniform suspension and minimizesedge effects. In the center of the 30.6 μm device, we createa 1.9 μm hexagonal defect,15 as sketchedin 52 and the central defect area is large enough to measureresonances interferometrically.54 Next, we simulate thefirst 1500 eigenfrequencies and the corresponding spatial mode shape.In f versus mode number N for the PnC (blue) and compare it to an unpatterned graphene membraneas reference (green). The graph for the PnC shows signs of a bandgap,as we observe an initial flattening of the curve followed by a suddenincrease. This region of reduced mode density coincides exactly withthe bandgap from our infinite model (blue area) and stands in contrastto the unpatterned membrane for which the frequencies gradually increasewith mode number. The second indication of the bandgap is evidentwhen we examine the effective mass of the modes2D is the areal density of graphene and z (zmax) is the (maximum) vibration amplitude in z-direction. For the fundamental mode we obtain meff = 80.9 ag = 0.252 mphysical,which roughly matches the literature value for the mode shape of auniform, circular membrane of meff = 0.269 mphysical.55 We observe a pronounced drop of meff in the bandgap region . Inthis model, we consider a finite number of unit cells of the honeycomblattice . As most modes in the bandgap, this modeis localized at the edges of the PnC in the real space. However, onemode at frequency 49.9 MHz is localized at the central defect . Nevertheless,we estimate the top and bottom of the bandgap, Supporting Information).Having demonstrated the overall tunability of our system, we nowsimulate the effect of electrostatic pressure on the phononic systemand the defect mode in a realistic device. To do so, we switch tothe finite model and apply pressure in negative pressure 4C. In thpressure 4C. We atpressure 4C, and rpressure S6E. Neve33Next, we investigate tunability of the defect mode. Upon applying30 kPa pressure to a device with an initial tension of 0.01 N/m, theresonance frequency of the defect mode upshifts from 49.9 to 217.5MHz .54 This modehas a nonzero net displacement and can be directly actuated via electrostaticdrive. It will be straightforward to distinguish the defect mode fromother modes by its localization in the center of the device and itslikely increased quality factor. Indeed, the quality factor is definedby Q = 2πEstored/Ediss, where Ediss is the dissipated energy per oscillation including alldissipation mechanisms and Estored isthe mode’s total energy. As the mode shape shows zero displacementnear the clamping points, we expect strongly suppressed bending lossesand thus enhanced Q’s. Additionally, the phononicshield hinders radiation losses into the substrate, which become especiallyimportant at higher frequencies.16 Whilebending and radiation losses may play a secondary role among the mechanismslowering Q in graphene resonators, our experimentsnevertheless should determine the contribution of these mechanisms.Finally, by applying pressure we increase the stiffness of the resonator.This increases the energy stored in the system17 and supposedly further enhances the quality factor. Thedemonstrated level of strain control in our system invites futurestudies on dissipation dilution via strain engineering following thework of Ghadimi et al.17We now discuss experimental signatures of this system. The spatialfeatures of the extended modes in our device 3E,H are 39 and transferred CVD grapheneis often covered by fabrication residues, so using thin exfoliatedgraphene multilayers could be a solution for which we expect to findexperimental signatures. The increased uniformity in multilayer graphenecomes along with a decreased tunability, yet we anticipate more than100% relative tuning for up to ∼35 layers . For our graphene PnC, we do not expect to reach Q’s comparable to SiN. Nevertheless, we estimate meff of our defect mode to be at least 8 ordersof magnitude lower than in other 2D-SiN-PnCs.15 This immensely increases the measurement rate of quantum statesΓmeas ∝ 1/meff and decreases thermomechanical noise.15 The frequencies in our system are controlled by simply adjustinga gate voltage, and we expect the tuning to take place on time scalescomparable to regular graphene resonators and therefore achieve tuningbandwidths >15 kHz.57We also note that our results can be easily extended to the entirefamily of two-dimensional materials. Currently, it is challengingfor us to experimentally achieve sufficient uniformity in the graphenemembrane in order to generate a spatially uniform bandgap and localizethe defect mode. Monolayer graphene is rather sensitive to surfacecorrugationsIn summary, we have fabricated and simulated a tunable PnC madefrom monolayer graphene. For an experimentally informed honeycomblattice structure, we find a robust vibrational bandgap in the megahertzrange. The bandgap persists for a finite-size system, and we use itto localize a defect mode and shield it from its surroundings. Thisdefect mode shows a very small effective mass of 0.724 ag, ordersof magnitude smaller compared to traditional PnCs. As our centralresult, we demonstrate a frequency upshift of the defect mode as wellas the entire phononic system by more than 350% by applying an experimentallyfeasible pressure of 30 kPa. While the bandgap smears out due to out-of-planedisplacement perturbing the lattice, the defect mode stays withinthe bandgap and remains highly localized. We suggest experimentalsignatures of the defect mode allowing its differentiation from othermodes in the system. Overall, our design of a 2D-material-based PnCadds a new knob to dynamically and rapidly tune frequencies in a broadrange of phononic applications. Our results invite future experimentsas our approach allows adjustable coupling of a PnC to external systemsand may lead to better understanding of the dissipation mechanismsin graphene.Supporting Information Section I provides a detailedprocess description.The pattering of the CVD grown graphene membraneswas carried out in a He-ion microscope (Orion Nanofab). Supporting Information Section IV.Raman mapping was performed on a Horiba XploraRaman spectrometer using a 100× (NA 0.9) objective and 532 nmexcitation. Spectra were acquired with a laser power of 0.5 mW andan integration time of 3 s. Tension (via strain) values were derivedfrom the 2D-mode position following standard procedures, see E2D = 1.0 TPa,38 Poisson’s ratio of v = 0.15, thickness of h = 0.335 nm, and a densityof 3. The initial tension T0 = 0.01 N/m thus corresponds to an initial strain: Supporting Information, Sections II and III.For the finite element modeling we use COMSOL Multiphysics (Version5.5) and assume the following material parameters for monolayer graphene:Young’s modulus


Moreover, the theory of graphene’s adsorption of gases is explained. The results show that gas sensing characteristics such as response/recovery time and response are related to the target gas, gas concentration, test temperature, and so on. In the stability test, the difference between the maximum resistance and the minimum resistance of the device is 1 ohm without ammonia, the change is less than 1% of its initial resistance, and the repeatability is up to 98.58%. Therefore, the sensor prepared with high quality single-layer graphene has good repeatability and stability for ammonia detection.The ammonia sensing properties of single-layer graphene synthesized by chemical vapor deposition (CVD) were studied. The Au interdigitated electrode (IDE) was prepared by microelectromechanical systems (MEMS) technology, and then, the single-layer graphene was transferred to the IDE by wet transfer technology. Raman spectroscopy was used to monitor the quality of graphene films transferred to SiO Studies have shown that people working with ammonia for a long time are often poisoned , and expritation ,3. In ad2−, O2−, and O−, thus achieving gas detection [2 hybrid orbitals. The carrier mobility of single-layer graphene at room temperature is about 15,000 cm2/(V·s) [According to the sensors category, commonly used ammonia detectors can be divided into infrared ammonia detectors, semiconductor ammonia detectors, and electrochemical ammonia detectors . Resistaetection ,10; on tetection ,12. Theretection . This ism2/(V·s) ,15, withm2/(V·s) ,17. In am2/(V·s) . Recentlm2/(V·s) .3 response [Many graphene-based sensors have been developed through doping, chemical modification, and other methods to detect NHresponse ,20,21. Aresponse ,23. The response .Based on the literature mentioned above, a single-layer graphene film was selected as the ammonia gas sensor’s sensitive material in this study. The single-layer graphene was transferred to the interdigitated electrode (IDE) prepared by the microelectromechanical systems (MEMS) process through wet transfer technology ,26. The The IDE is used as the sensor’s resistance. It has a planar structure, in which metallic conductors are placed in a comb-like arrangement. It is reported that the IDE can effectively reduce the test resistance of the gas sensing film, which is beneficial for testing the performance of the thin film’s gas sensing . It has 2 insulator layer, metal leads deposition, metal lift-off technology, and so on.The IDE was prepared using a standard microelectromechanical systems (MEMS) process, as shown in 2 film of 2000 Å thickness was deposited on the cleaned silicon wafer by plasma-enhanced chemical vapor deposition (PECVD), used as the insulating layer between metal electrode and silicon wafer, and the next process was lithography. Before photolithography, in order to increase the adhesion between the photoresist and the insulation layer, it was necessary to carry out Hexamethyldisiloxane (HMDS) steps on the wafer in an environment of 130 °C, and then, spin the positive photoresist AZ6130 on the silicon wafer at a speed of 3000 rad/min. The exposure technology adopted contact UV exposure and then, placed the wafer into the developer (DI water: AZ400K = 4:1), keeping for 30 s to form the electrode pattern. Before metal modification, oxygen plasma was used for surface activation treatment to increase the cleanliness and surface activation of silicon wafers. The parameters were as follows: power of 200 W, time of 2 min, and the carrier gas was oxygen. Furthermore, Ti 200 Å/Au 2000 Å metal layer was deposited by EXPLORED , and the parameters were as follows: vacuum degree of 5 × 10−6 torr, power of 500 W, and Ar with a flow rate of 14 sccm (standard cubic centimeter per minute). Upon the metal layer, the photoresist was dissolved in acetone solution, which is called lift-off technology. Then, the IDE cells were formed by ultraviolet laser fine processing equipment FPC03 . Finally, the monolayer graphene was transferred to the IDE by wet transfer technology.Firstly, the silicon wafers were cleaned using a standard inorganic cleaning process. The silicon wafers were cleaned in a strong acid mixture of sulfuric acid . Next, the silicon wafers were rinsed with de-ionized (DI) water and blow-dried with nitrogen. The external contamination on the surface of the silicon wafer was removed in the above cleaning process. Then, a SiOThe single-layer graphene used in this experiment was grown on a copper substrate by chemical vapor deposition technology, and PMMA was used as a protective layer, as shown in 2 substrates were characterized by Scanning Electron Microscopy (SEM) and Raman spectroscopy. −1) and 2D band (2692 cm−1) [−1, for the G peak is 26.79 cm−1, and the residual doping is 90.72 meV [As shown in 92 cm−1) , and the0.72 meV . Figure 0.72 meV ,33. Simu3 was performed in a photoelectric comprehensive test platform (CGS-MT). It is a closed device with a volume of 2000 cm3, the experimental device is shown in 3 was maintained for 10 min. This is a complete test cycle. In order to ensure there were no accidental experimental errors, three tests were performed in each group of experiments. In the experiment, the change of the device’s resistance was tested under different ammonia concentrations and different temperatures. We can also directly monitor device resistance changes under different conditions through external software tests in real-time.Detection of the NH3NHR is the resistance of the sensor when exposed to ammonia gas, and the airR is the resistance of sensor when exposed to air [The gas sensing properties of graphene were studied by measuring the resistance when they passed through gases in different environments. The sensor response was defined as the ratio of the change in ammonia resistance to in air, and was calculated by the following relation. Here, the d to air .S(%)=RN3 is a reducing gas, when exposed to NH3, the NH3 molecules come into contact with the single-layer graphene films and the electrons would be transferred from the NH3 molecules to the surface of graphene [3 supply, the NH3 molecules adsorbed on the graphene surface will be separated from graphene [3 desorption to the electron molecules, there will be no electron transfer on the graphene surface, and the resistance value will finally return to the initial state.The theory of graphene’s adsorption of gases is explained by the first principle, which is essentially the transferring of electrons between graphene and gas molecules . As NH3 graphene ,35,36,37graphene . Finallygraphene ,35,36,37As we know, the graphene has a non-ignorable resistance response to the humidity ,40. In t3 concentration of 100 ppm. From 3 gas. As shown in 3 for 5 min and the sensitivity of the sensor recovered to its baseline after 15 min. Meanwhile, with the reaction process’ occurrence, the sensor’s sensitivity was improved, but the reaction rate gradually decreased continuously. In the initial reaction stage, the change of sensor relative resistance is the largest. In the process of the reaction of 1–5 min, the response was 3.26%, and in response to the 5–10 min reaction stage, the resistance changes by only 1.37%. This can be attributed to the fact that the graphene surface is constant when exposed to NH3 and many ammonia molecules adsorbed to the graphene membrane surface. At the same time, electrons and holes can be recombined on the graphene membrane surface, causing the concentration of holes on the surface of the graphene to drop, causing the increase in sensor resistance. However, because the graphene surface adsorption of gas molecules is limited, as the reaction progresses, many gas molecules are adsorbed on the surface, the effective adsorption surface area decreases, and the response rate is slowed down. In the last, it kept a state of dynamic balance. After closing the intake channel, the dynamic balance was instantly broken, and the adsorbed gas molecules on the surface of the graphene film gradually decreased. At the same time, the surface electron migration rate attenuated, resulting in a decrease in resistivity, and finally, kept the stability.3 gas concentration is, the higher the sensor’s response speed and sensitivity will be. This is because NH3 gas with a higher concentration has more molecules per unit volume. When the intake channel is opened, more molecules are absorbed onto the surface of the graphene film. A large number of electrons provided by ammonia molecules compound with holes on the graphene surface, resulting in a decrease in hole concentration on the graphene surface. The decrease in cavity concentration leads to the decrease in the film surface’s conductivity, which is finally manifested as the increase in sensor resistance. In addition, we performed a series of tests at different gas concentrations. The experiment was carried out at room temperature, and the whole test process only changed the gas concentration. As shown in 3, it causes the NH3 molecules to move violently at high temperatures. Therefore, the influence of temperature on the performance of the sensor is studied. In this paper, the corresponding tests are carried out at different temperatures. The motion of molecules is related to temperature. For NHRepeatability and stability are important parameters to characterize a sensor. Stability is the ability of a device to maintain the same performance over time. In this experiment, three IDEs processed in the same batch were selected for testing, and their test results were very similar. A graphical representation of the stability test results is shown in In this paper, a resistive graphene-based gas sensor with good repeatability and stability has been prepared, which is different from previous studies focusing on improving sensitivity and selectivity. Single-layer graphene was transferred to an IDE by wet transfer technology. Raman spectral analysis showed that monolayer graphene had fewer defects and stable performance. The experimental results show that the sensor prepared by this method has a good response to ammonia gas, and the response has a stable relationship with the ambient temperature and gas concentration when the ambient humidity is relatively stable. Through repeated tests, it is found that the stability error of the sensor is within 1%, and the repeatability reaches 98.58%. In the future, we will study the feasibility of graphene sensors for environmental monitoring and perform surface functionalization of single-layer graphene layers, based on which the sensor’s sensitivity will be improved.


FOXC2 are predominately associated with lymphedema. Herein, we demonstrate a key role for related factor FOXC1, in addition to FOXC2, in regulating cytoskeletal activity in lymphatic valves. FOXC1 is induced by laminar, but not oscillatory, shear and inducible, endothelial-specific deletion impaired postnatal lymphatic valve maturation in mice. However, deletion of Foxc2 induced valve degeneration, which is exacerbated in Foxc1; Foxc2 mutants. FOXC1 knockdown (KD) in human lymphatic endothelial cells increased focal adhesions and actin stress fibers whereas FOXC2-KD increased focal adherens and disrupted cell junctions, mediated by increased ROCK activation. ROCK inhibition rescued cytoskeletal or junctional integrity changes induced by inactivation of FOXC1 and FOXC2 invitro and vivo respectively, but only ameliorated valve degeneration in Foxc2 mutants. These results identify both FOXC1 and FOXC2 as mediators of mechanotransduction in the postnatal lymphatic vasculature and posit cytoskeletal signaling as a therapeutic target in lymphatic pathologies.Mutations in the transcription factor  The lymphatic vasculature has a critical role in maintaining tissue homeostasis by returning interstitial fluid to the venous circulation, absorbing lipids from the digestive tract, and providing a network for immune surveillance and response . MutatioFOXC1 have primarily been dominantly associated with eye anterior segment defects, cerebellar malformation, and cerebral small vessel disease. In contrast, mutations in FOXC2 have been dominantly associated with lymphedema-distichiasis syndrome characterized by failure of lymph drainage in limbs, venous valve failure, and the growth of an extra set of eyelashes (FOXC2 mutations (FOXC1 and FOXC2 are closely related members of the forkhead box (FOX) transcription factor family with nearly identical DNA binding domains, similar expression patterns in mesenchymal tissues during development, and essential roles in cardiovascular developmental processes . Mutatioyelashes . Work fryelashes . Additioyelashes . Furtherutations . HoweverFoxc1 postnatally impairs valve maturation, while Foxc2 deletion impairs maturation and induces valve degeneration, as previously described  in vitro, which also improves LEC barrier integrity in vivo, while valve degeneration is partially rescued in only Foxc2 mutants. Finally, via generation of transgenic mice that express Foxc2 within the Foxc1 locus, we show Foxc2 is capable of functionally substituting for Foxc1 in lymphatic development and maturation. Together, our data show a complementary role for FOXC1 in addition to FOXC2 as key mediators of mechanotransduction in the postnatal lymphatic valves and implicate new mechanistic targets for therapeutics in the treatment of lymphatic-associated diseases.Here, we report an essential role for FOXC1 during lymphatic valve maturation and maintenance. Detailed comparison of FOXC1 and FOXC2 expression and roles in lymphatic valves suggests some overlap with a broader importance for FOXC2, but more subtle, key contribution for FOXC1. In mice, endothelial cell (EC)-specific deletion of escribed . HoweverOur group previously reported that FOXC1 and FOXC2 expression co-localizes with PROX1 in lymphatic valve-forming cells at E17 and later at P3 . HoweverFoxc1 during murine embryonic development impairs lymphatic valve maturation (flFoxc1 mice (ERT2Chd5-Cre mice (Foxc1 mutant (EC-Foxc1-KO) mice. Tamoxifen was administered from P1-P5 to induce Cre-mediated recombination and we confirmed deletion of Foxc1 via qPCR analysis of isolated CD31-positive cells from hearts of P6 individuals and by immunostaining of the mesenteric lymphatic vasculature with antibodies against FOXC1, FOXC2, and VEGFR-3  protein ligand fibronectin-EIIIA to regulate the formation of the ECM core of valve leaflets  mice and administered tamoxifen postnatally. PROX1 immunostaining showed no significant differences in total valve number of LEC-Foxc1-KO mice  mice and administered tamoxifen from P1-P5 with inactivation confirmed by qPCR in ECs isolated from P6 hearts and by immunostaining of the mesenteric lymphatic vasculature with antibodies against FOXC1, FOXC2, and VEGFR-3  mice and administered tamoxifen from P1 to P5 with inactivation in compound mutant individuals verified by qPCR and immunostaining of the mesenteric lymphatic vasculature with antibodies against FOXC1, FOXC2, and VEGFR-3  developed severe chylous ascites  compared to controls   , indicatFOXC1 knockdown in LECs impairs cell adhesion, we slightly modified our protocol: LECs were first seeded on fibronectin-coated surfaces, then transfected with scramble control, FOXC1, FOXC2, or combined FOXC1/FOXC2 (50%:50%) siRNAs and kept under static conditions for 2 days. Given that FOXC2 inactivation and exposure to OSS in LECs results in increased actomyosin contractility  suite of tools (https://ecrbrowser.dcode.org) -phosphorylated myosin light chain (pMLC) signaling pathway has been well established as a regulator of cytoskeletal contractility mechanisms . FurtherDKO mice . Recent DKO mice . Our RNADKO mice  revealed embryos . Further embryos  were notpression . Becausepression  and our of tools  was usedof tools  in activof tools . Then, wode.org)  tool to PRICKLE1 , ARHGAP2ARHGAP23  loci. ToPRICKLE1 , ARHGAP2P23 loci . We founin ECR-1 . Additio ECR-1–2 . Finally23 locus .ICAM1 that was not predicted to bind FOX transcription factors by in silico analysis  allele, in which the Foxc1 coding region has been replaced with the cDNA coding (from the start codon to the stop codon) for Foxc2    and FoxCrostomes . Althougnditions . TherefoPecam1 or Sdc4 and LEC-specific deletion of Cdh5 in vivo resulted in impaired valve morphogenesis phenotypes and poor orientation and elongation of LECs with the direction of flow in mesentery collecting vessels  for Foxc2 by using restriction enzyme digestion and PCR cloning. To facilitate the elimination of the selectable marker gene rNeo in mice, we introduced the ACN cassette flanked by two flox sites  mice , c2/+Foxc1 , and c2/c2Foxc1  individuals.SalI-linearized targeting vector (100 μg) was then electroporated into TL1 ES cells (129S6) as described . The cel/+) mice  were theGenotyping of mice for use in analysis was performed by Transnetyx Inc  using real-time PCR.Dissection, immunostaining, and imaging of the lymphatic vasculature in mesentery tissue from 4 week old C57Bl6 mice for whole-mounts was performed as previously described . BrieflyThe lymphatic collecting vessel vasculature of neonates was analyzed by whole mount immunostaining of mesentery tissue harvested from pups at the indicated time. Briefly, mesentery tissue was dissected from the intestinal tract, laid out in plastic dish and left until it was firmly attached. Following fixation with 2% PFA in PBS, tissues were washed with PBS, then permeabilized and blocked in PBS solution containing 0.5% BSA, 5% serum, 0.3% Triton X-100, and 0.1% Sodium Azide. Tissues permeabilized/incubated with blocking buffer were then incubated with primary antibodies listed in Imaging was performed using a Zeiss AxioVision fluorescence microscope and Zeiss Axiovision software, Zeiss LSM-510 Meta, LSM-800 and LSM 880 confocal microscopes and Zeiss Zen Blue acquisition software, or using a Nikon A1 Confocal Laser Microscope System NIS-Elements software. Images were processed with Imaris and Adobe Photoshop software. Imaris colocalization function was used to produce pictures showing p-MLC2 , vinculiPpia) or 18S was used as an internal standard for mRNA expression. Primer sequences are provided in Hearts from neonatal mice were digested in collagenase Type I solution (2 mg/mL) for 40 min at 37°C with gentle agitation. Cells were then filtered through a 70 μm cell strainer and the pellet was resuspended in Buffer 1 . The cell suspension was then incubated with magnetic Dynabeads (Invitrogen) pre-coated with CD31 antibody to isolate the endothelial cell population. After several washes with Buffer 1, RNA was extracted from endothelial cells using RNA STAT solution (Tel-Test) followed by phenol-chloroform treatment. Extracted RNA was subjected to DNAse I treatment and concentration was determined using a NanoDrop machine (Thermo Scientific). cDNA was synthesized using an iScript reverse transcriptase kit (Bio-Rad) according to the manufacturer’s instructions. Triplicates of cDNA samples for qPCR analysis were performed using a Fast qPCR machine (Applied Biosystems), Fast SYBR reaction mix (Applied Biosystems), and gene specific primer sets. Peptidylprolyl isomerase A  coated with 40 µg/ml human fibronectin, cultured for 24 hr and then subjected to LSS (4 dyn/cm2), OSS (4 dyn/cm2 and flow direction change every 4 s) using Ibidi Pump system, or kept in static conditions for an additional 24 hr prior to fixation, immunostaining, and mounting using Ibidi Mounting Medium (ibidi GmbH).Flow experiments with cultured LECs were performed as described previously . Brieflyhttps://genome.ucsc.edu/ENCODE/)(2012). Putative sites in the human genome were then searched against the mm10 mouse genome using the ECR Browser (https://ecrbrowser.dcode.org) (Putative FOX-binding sites were determined first by using the Hypergeometric Optimization of Motif EnRichment (HOMER)  suite ofode.org)  and rVishttps://www.promocell.com/product/human-dermal-lymphatic-endothelial-cells-hdlec/) were cultured and used according to the manufacturer's protocol. The cells were cross-linked with 1% formaldehyde, followed by sonication. The sheared chromatin was immunoprecipitated with dynabeads  conjugated with anti-FOXC2 antibody , anti-FOXC1 antibodies , or control IgG . DNA extraction and PCR were performed as previously described  from juvenile foreskin  (escribed  with priPRICKLE1 ECR-1.>hg19 chr12:42876176–42876523TGTTGGCTCCTGAACAACACTCCTCTTTGTGAAACTTACAGCACCCATTTGAAACAAGTTTCCAGAGAAAACACTTCAAGAAAGTGTTTGAGAAGTCACAAGACTCGAGTTGTAAAAACAAATTCCACACATAGCCTGGCTTATAAGGACACAGACTAACACCACACAAGGCGGTGCTCTAATGAGCCCATTATTTTCCATAATGGGGGATGCAGATATTTTCTCAAAATCGTGTTCTCCTCAGTCTTCTATTGATTTTTTGGATTTCTATTTTCAACAGTGGCCCGAGGAAACGGCAGCCAGACTTGACTCCAATGTACACACAGACTCAGGTTTCGCCCCGTCACCPRICKLE1 ECR-2.>hg19 chr12:42878066–42878454GAGCACCTACCGCCGCCCGCCCGCTCCATTCTCCCGAGCCCAGTGAGTGAAGCCGCTAAGATGCAAATACCCTAGGACGCTTATGTAAACTTCCCCCTCCCGCAGGTGCACGCGCGGGCCACGAAACGCTGGGAGAATATGAAAGGCCACCTCTTAAAGAAATCATCTCCACTCTGCCCATAACAATGATGTCAGCAAATGGCACATTTAAGCAAGTTCTCACTTAGAAGGGCTCATTAGCATATGAATTCTCTTAGGACTTTCCCTGCATTTCGGAGTGATTCCTACTGCTTAGCGCAGGAGATTTATTTTTATCAGTAAATAACAGCAAAGAAAAGGAGCCAGGTCACGCGATGTACTAACTCAAGTACACTACTGAGAGTTTTTACPRICKLE1 ECR-3.>hg19 chr12:42879624–42879943AGTTACTTGTAAATACGTTTTGTTATATTTTCAACAGGTACTGTCATGGTTTATTACCCATTGTAAGCGTTTTTAGTGATGAAGCAGTGTGGAGACAGTTGCAAGCTTCCTACAAAGGTTCAATCTCAATGAAACAGACTTTAGTCTGGTCTGAAAGGGGTTGTTATTGTCAAGGGTGAACTTATGCAGAATGGAGAGCAAGGCCCCCAACCAGCATCCTTTTGTTTCAGCCAGGTGGAATATTCATGCTTGCAGATCACACTTTAGGGGCCAGTTGAGAAAGGAAGCCCAAAAATTCACAGGGCTTGGTTCCCTCGCTCPRICKLE1 ECR-4 and ECR-5.>hg19 chr12:42981201–42982292TACATTAGCCATGACTTATTAAACTTGGAGATTTTCAAGTTCATCAGCAAGTGTGCAAACCCTTAACTGTGGGTAACATCCATTTATTTGTAGCACCTTTCGGTTTTAATATGTAGAGCACATGCATTGTTAACCTCTAAATCCTTTGT(ATAAACA)TTTCTGGAAGAGCTGGTAAAATATCTCCTTCTGTGTTTCCTGACTCGCCAGTTGATGGCATTTAGAAACCCTCTGGTACCAGCAGGTGCTGTATTTTGCTTTCTTCAGGCTCCAGCTGGGCTACAATGACAGATTCCTGTCCCAGGCCAAGCCTAGCCACCAAGGCTAGGACCACATTGGAGGCAAACTGAACCAGGCTCCACCAGACAGCAAGATGAATGGCTGCTGTTTAAGTTTAAAATCCCCTGGTGGGA(GTAAATATTGTTCCAGAGAAAAGCCTTGACAAATA)CTGCGTCATCCTTACAGAACTGTCTTGATTAAAGCAGAATCTTTGGATTAAGTTGATGCTCAATTCAAAATGTATCTATCTTGCTGTCATGGGATTTTTTTTTGTTTTTTTTTTCCTTTCTAGAGTCTGAAAACAGACATAATGTTGGGGACGGTCAAACAAGGCTGCCGGCTCCCAAGGGGCTAGAGTCCACTCCTGATAATAGAAGGCGGCTGAACACTGACACTTCACTGAGGATAATGGAGACAGCAAAGGCTTAGTGGGAAAGGGCCAGTTGTCACCTAAGTGACAGGCAACAGCTGAGCTCACACATCTGGAGCCGGACTACGGCAAACATTAGCAACCCTCACCAGTCTACACCTTGGGCCTGTCTGAAAAGACAGATGGAAGTTCCCTCTACTCCTAAAGTACATTAAAAAATGTCTGATGGTGAACCACATCAATTATATAACATCAACTGCAGGCACAGCCTTCCAAAGTACTGATTAAGACGAGGCAGTAGACAACACTGTATGCATGAACAGATACAAGATACCATTTCAGTGATTTGTCATTCATAAAACTTATCCTAAAAGACACATATACATGCATCCATTTGATAGCACAAATGCATGTTAACTCTGCAGGAGAGGCAGATTTTTACATGTPRICKLE1 ECR-6.>hg19 chr12:42982328–42982743CCTTTAGGCTGGTTCCCGTGGTGTGTTTGCCTTGTTCATGGTCTCAGTTCTGCCGCTGATACCCTTTTAAAAATCAGCAACCAAACGCGTTCGGCTTGTGATCCTGAACCCCCTTAGGCAAGCTGGAACTAAGCGTGATGCAGCCGTCCTCCCTCTCTCCCAACCCCCAACCTCGTTCTTCAGCCTCCTGAAGACAATCTGTGAACAATTTTCCCAAAGTCCCAAGAATAACACAGCACTGCCAATAGTCACTGGCGATGCCGTTTGTTTTTCTTAGAGGGTAATGAAAATTTAACAGCTTTCTGCTGCATCCTGAGTCCCGCTCCTAATAACTATTAACATGCCTAGTTTCTTCAACTTTTTCTACCTCAAGAGAGGAAGACGCTCCCATTTTTTCCCTATATCTGTGCTACATARHGAP21 ECR-1 and ECR-2.>hg19 chr10:25009076–25009514CTGTCATTGTTAATAAAAGCCAAGTGTGCAACAAACTGGAAATACTGCTTGCTAGCAAGGACAAGATGTGTCTAAATTCTTGGTTTCAGGACATCTCTTAAATGACCAAAAAAAAAAAAAAAAATCAAATAACATTAGTTCTT(GCAAACAGAATGCAAATACAATGCTAATTAAAGTATTCACAAGACAATGACAAATA)AGGCTTCAGGACCACAATACATATTATTATGTAACTGCAATACACATTAAGCAATCACCAGGTTGCAGGTAGGCCTTCCAAAAGGAGTTATTATGGTTTACCGTGATCAGAGGATTGTGGTGTTCCACTTAATCATGCTTTTGCCTGCAAGCAGGTGTTTACAGATGTCAAAAAGTAAAACACTGATTCTAAATGTAAACCTACAGTTCTGCCTAATAAATTGTACAGTAATAGCACACARHGAP21 ECR-3.>hg19 chr10:25017295–25017667CTATATTAATAAAAACTACAAGAAAGCTTTATACACTAAATCTAGGCAAGACATTTATGAAGATGAGAACTGTATCCTTAAAAGGTAAGTGTTGGCTTTGCTAATGACATAATATTGTTTTGGTGAACCAATATCAAGGGAAAAAATGTCAAAGCCAAAAATAGAGGCAAAGTATCCCAGCCCCTGGTGTGGAAGGCCATTCTATGATAATCTATGAATGATTTCTACTCTGAATATGTTAACAGAAGCTGGCACATCTGAGAAGCACAAGTGTTTGCTAGTGAATCCACAAATGAAATTTGCAATTTGGGTTGAAGCCTTTGCAAAACTACGTTAAGACCAATAGCCCTCAGAAGAGTAAGGGGTTTTGTTTARHGAP23 ECR-1.>hg19 chr17:36585363–36585767TTCTAGACACAGGCCCAGGACCCCGGGCTCTGCCGGCGAGGCTGCCCTCCCCTCTGCCCTCTCCGACCGGCTGTGGGTGGGTCAGAGCGCGGGGTGCCAGGGGCATTACTCAGCGCTGGGCTGCTCTGCCTGGGTTCTTTCATCTGCCAGCTGCTGAGGCTGGGGAGGGGCCAGCAGGGGCCTCCCAGCCCCATCCCCCCATCAGGGCCATTCCCTTACCTCTGAGCCTGGCTGCCCGCCCTGCAGGAGCCCCCCAGCAGGCCTCCCTGCTCCTAAGTTGAAGGGTTGAACACTGTCAGGCCAACAGTTTCCCTGAGCTCGGAAAAGAAATTCCCCGGGGTCCAGGTTGAGGTCAAGGCCAGGGCTGAGGCCTGTTCCTCTTTAGACAGGGCTGAAAGACTTGGGTo identify lymphatic valves, mesentery tissue was stained with PROX1 antibody and areas of high expression were quantified as mature (visible leaflets) or immature (no visible leaflets) on four lymphatic collecting vessels per individual. Total number of valves were determined and percent of mature valves was normalized to the total counted per individual. For assessment of apoptosis in lymphatic collecting vessels, mesentery tissue was immunostained with PROX1 and active caspase-3 antibody and the percentage of PROX1/caspase 3-positive LECs was quantified from 20X high-power fields generated from confocal z-stacks using Fiji software to determine the total number of LECs per field using thresholded PROX1 immunostaining. Quantification was completed from three biological replicates.in vitro quantifications, relative nuclear intensity levels were measured using Fiji software. Nuclei were considered as regions of interest from thresholded Hoechst staining pictures. FOXC1 or FOXC2 intensity was then measured in each nuclei using RawIntDen function. For quantification of F-actin and p-MLC2 area per cell, LECs were fist manually segmented and defined as individual regions of interest, then F-actin (or p-MLC2) staining was thresholded using similar parameters for all pictures and F-actin+ (or p-MLC2+) area was measured per each region of interest. Quantification was completed from three independent experiments.For in vivo experiments was performed using GraphPad Prism v5 or v8. P values were obtained by performing a 2-tailed Student’s t test or one-way ANOVA and Tukey’s test. Data are presented as mean ± standard deviation of representative experiments from at least three biological replicates. For quantification of apoptosis in LECs of compound Foxc1; Foxc2 mutant mice, the ROUT method with Q set to 1% was used to identify outliers in the data set that were then excluded from statistical analysis using student’s t-test. P values less than 0.05 were considered statistically significant. Statistical analysis for in vitro experiments was performed using GraphPad Prism v8 to perform mixed-effects analysis and calculation of correlation coefficients. P values less than 0.05 were considered statistically significant for mixed-effects analysis.Statistical analysis for All procedures and animal studies were approved by Northwestern University’s IACUC or by the Animal Ethics Committee of Vaud, Switzerland. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This study provides new insight into the mechanically regulated activation of, and distinct roles for, FOXC1 and FOXC2 in lymphatic vessel valve development. The identification of unique roles for these closely related transcription factors is intriguing and will pave the way to a deeper understanding of the transcriptional mechanisms regulating their distinct activities. The study also implicates FOXC1 as a gene that may potentially underlie primary lymphoedema.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, including Natasha L Harvey as the Reviewing Editor and Reviewer #2, and the evaluation has been overseen by Didier Stainier as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Mark Kahn (Reviewer #3).Thank you for submitting your article "Shear stimulation of FOXC1 and FOXC2 differentially regulates cytoskeletal activity during lymphatic valve maturation" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Here, Norden, Sabine and colleagues dissect the roles of transcription factors FOXC1 and FOXC2 in lymphatic vessel valve development, revealing that FOXC1 primarily controls focal adhesions in lymphatic endothelial cells, while FOXC2 primarily controls adherens junction integrity. The work builds on previous studies by the authors that have demonstrated (1) the importance of FOXC2 and shear stress for lymphatic vessel valve development and , (2) important roles for both FOXC1 and FOXC2 in embryonic lymphangiogenesis via regulation of RAS/ERK signalling . Aspects of this current study overlap with some of the authors' prior work and this should be clearly articulated in the manuscript where relevant. Distinctions and extensions of the work should also be clearly articulated. There are key novel and interesting findings described in this manuscript that address important questions regarding the distinct mechanisms by which FOXC1 and FOXC2 activity are regulated, together with the mechanisms via which these transcription factors regulate cytoskeletal architecture.Essential revisions:Foxc1 seems modest, and is characterized by a larger proportion of immature valves in mesenteric lymphatic vessels. It thus becomes important to know how the authors define immature valves. It is not obvious why those shown in Figure 2E, F are defined as immature, and not representing valves that are imaged from a different (90°) angle compared to valves that show the typical v-shape. Staining of the valve leaflets would demonstrate this more clearly. The inclusion of staining for markers of valve maturation such as α5-laminin and/or α9-integrin would help to demonstrate this point. To more convincingly demonstrate that Foxc1 modulates the Foxc2 KO phenotype, side-by-side comparison of Foxc2 single and Foxc1;Foxc2 double mutants should be shown. Of note, deletion of Foxc2 seems to be incomplete . Considering that the authors show that Foxc2 can compensate for Foxc1 function, Foxc1 deletion in combination with the incomplete Foxc2 deletion may just decrease total Foxc signaling sufficiently but not necessarily indicate distinct functions of the two genes as proposed. The question is whether Foxc1 deletion would affect the phenotype of Foxc2 mutants if Foxc2 is efficiently deleted. Why is Foxc1 staining apparent on the cell membrane and present in the Foxc1 mutant in Figure 3—figure supplement 1?1) The phenotype caused by early postnatal deletion of Foxc1; Foxc2 mutants resulting in increased apoptosis in lymphatic collecting vessels' requires more supporting evidence. We see one image  aiming to support the claim that cell elongation and junctional integrity is impaired but it is not clear how representative this image is. It is surprising that such a high number of apoptotic cells is detected in control vessels, drawing into question the specificity of the caspase-3 staining, and whether the signal is indeed detected in the LECs. No evidence is provided for cell elongation defects leading to increased apoptosis. This statement should also be demonstrated: "Of note, apoptotic bodies were more frequently observed at branched areas of the lymphatic collecting vessels potentially indicating areas of valve degeneration." The authors should expand on the cellular mechanisms underlying the presumed rupture of lymphatic vessels and accumulation of chylous ascites in EC-Foxc1;Foxc2-DKO pups. Ultrastructural analyses could be informative here.2) The conclusion 'Cell elongation and junctional integrity is markedly impaired in compound EC- specific Foxc1;Foxc2 double mutants. Figure 3—figure supplement 3D shows instead extremely high PROX1 levels, along with 'discontinuous junctions', making it difficult to judge if this is a representative image. The junctional phenotype presented in Figure 3—figure supplement 3D is indeed dramatic , but the phenotype is seemingly different in Figure 3—figure supplement 2D where the phenotype is not obvious at all. 'Discontinuous junctions' in the Foxc1;Foxc2 double mutants should be demonstrated more convincingly.3) Inconsistent quality of staining between different experiments and even panels in the same figure (as if staining and imaging is done at different time points and not using littermate mice) make assessment of the phenotypes difficult, especially when a single high magnification image is provided as a supporting evidence. For example – Figure 3—figure supplement 2 VE-cadherin staining, Figure 2E-F CD31 staining. Figure 3 and Figure 3—figure supplement 3H show low PROX1 levels overall in Foxc1 and Foxc2 have distinct functions in regulating the actin cytoskeleton, it is not clear why Foxc2 is capable of functionally substituting for Foxc1 in vivo.4) The authors should provide further evidence to demonstrate how relevant the described in vivo phenotypes are to those described in vitro. If 5) Previous studies have demonstrated that FOXC2 is regulated by oscillatory shear forces in LECs, and that such forces likely play a key role in valve development. In Figure 4 the authors use in vitro flow studies to conclude that FOXC1 expression is regulated primarily by laminar shear while FOXC2 primarily by oscillatory shear. However, the endothelial cells shown in Figure 4A do not appear aligned with flow as would be expected for laminar shear and the authors fail to identify a clear mechanism that would account for such a distinct regulation. This theory appears to rely primarily on antibody detection of nuclear FOXC1 which is not as robustly detected as FOXC2. In vivo studies, especially Figure 10, suggest that these transcription factors are functionally redundant at the protein level as replacement of FOXC1 with FOXC2 does not impair valve maturation. Much of the data shown are carefully analyzed, but these findings and their connection to where FOXC1 is best detected in vivo in the valve tip seems like an over-reach. More specific mechanistic data identifying how such remarkable distinctions in shear regulation are achieved should be provided.Foxc2-KO compared to mRNA? Foxc2 mRNA appears to be reduced by approximately 50% compared to control EC Foxc2 levels. Does this level of reduction in EC-Foxc2-KO animals explain the partial penetrance of the phenotype of chylous ascites?6) Figure 1—figure supplement 1: What is the level of FOXC2 protein present in LEC of EC-Foxc2-KO compared to EC-Foxc1;Foxc2-DKO mice.7) Figure 3—figure supplement 2: A lower power image depicting the broader profile of α9-integrin expression throughout multiple valves would enable the reader to determine how α9-integrin deposition is affected more globally in EC-Foxc1;Foxc2-DKO LEC? It would be informative to provide more detail as to why these three genes were selected for further analysis. Were they the only genes involved in RhoA/ROCK signalling that were changed in expression? It would also be informative to know whether RAS/ERK signalling is affected in mutant LEC at this stage of development, given the authors' previous work which demonstrated regulation of this key pathway by FOXC1 and FOXC2 in embryonic LEC.8) Figure 6: How do the changes in Prickle1, Arhgap21 and Arhgap23 levels compare to other gene expression changes in LEC-9) Figure 6D: Additional controls are required in which ChIP of both FOXC1 antibodies at a negative control region of the genome (e.g. an enhancer region demonstrated or predicted not to bind FOXC1) is assessed. This should demonstrate the selectivity of FOXC1 antibodies in terms of their specific versus non-specific/background binding to DNA and may help to explain the distinct results observed with each antibody in different regions of the genome.Foxc1c2/c2 mice. What is the remaining signal in FOXC1 stained c2/c2Foxc1 tissue?10) Figure 10D-F: It is not clear from the images presented that a call can be made regarding FOXC2 levels being modestly increased in  Essential revisions:1) The phenotype caused by early postnatal deletion of Foxc1 seems modest, and is characterized by a larger proportion of immature valves in mesenteric lymphatic vessels. It thus becomes important to know how the authors define immature valves. It is not obvious why those shown in Figure 2E, F are defined as immature, and not representing valves that are imaged from a different (90 degrees) angle compared to valves that show the typical v-shape. Staining of the valve leaflets would demonstrate this more clearly. The inclusion of staining for markers of valve maturation such as α5-laminin and/or α9-integrin would help to demonstrate this point. To more convincingly demonstrate that Foxc1 modulates the Foxc2 KO phenotype, side-by-side comparison of Foxc2 single and Foxc1;Foxc2 double mutants should be shown. Of note, deletion of Foxc2 seems to be incomplete . Considering that the authors show that Foxc2 can compensate for Foxc1 function, Foxc1 deletion in combination with the incomplete Foxc2 deletion may just decrease total Foxc signaling sufficiently but not necessarily indicate distinct functions of the two genes as proposed. The question is whether Foxc1 deletion would affect the phenotype of Foxc2 mutants if Foxc2 is efficiently deleted. Why is Foxc1 staining apparent on the cell membrane and present in the Foxc1 mutant in Figure 3—figure supplement 1?Foxc1, Foxc2, and compound Foxc1; Foxc2 mutant mice. To address the first part of this comment, we have included new representative images of α9-integrin/VE-Cadherin immunostaining of lymphatic valve regions side-by-side with PROX1/CD31 immunostained lymphatic valves in Figure 2. Inset images of only the α9-integrin channel more clearly show the presence of mature, intraluminal bi-leaflet valve structures in Control and EC-Foxc1-KO mice as well as valve regions that are considered immature that are not characterized by a distinct intraluminal leaflet structure by α9-integrin immunostaining. In EC-Foxc2-KO mice, we have observed that overall, α9-integrin expression is reduced compared to littermate control mice. However, there are regions characterized by the presence of intraluminal leaflets, as depicted by PROX1, CD31, and VE-Cadherin immunostaining, that are shortened in length. We describe these observations in the subsection “FOXC1 and FOXC2 are required for postnatal lymphatic valve maturation and maintenance”.The authors thank the reviewers for this comment and careful assessment of our described phenotypes in our Foxc2-KO mice and EC-Foxc1; Foxc2-DKO mice. Figure 2H and N depict representative images of a mesentery collecting vessel immunostained with PROX1/CD31 and Figure 2R and T depict 10X power images of collecting vessels immunostained with PROX1 to show the difference in phenotypes more clearly.To address the second part of this comment, we have also updated Figure 2 to include representative images showing side-by-side comparison of EC-Foxc1-KO, EC-Foxc2-KO, EC-Foxc1; Foxc2-DKO mice and respective littermate controls and included representative images in Figure 2—figure supplement 1. In Figure 2—figure supplement 1G, we show that FOXC2 expression is markedly reduced in EC-Foxc2-KO mice and FOXC1 expression is reduced compared to littermate controls, likely as a result of perturbed flow contributing to the reduced induction of FOXC1 expression. In contrast, Figure 2—figure supplement 1E shows strong reduction of FOXC1 expression but no discernible difference in FOXC2 expression. Thus, the maintenance of FOXC2 expression in EC-Foxc1-KO mice may in part explain the differences in phenotype severity observed in Foxc2 mutant mice compared to Foxc1 mutant mice. We have revised our Discussion section (second paragraph) to note this important difference. The difference between our qPCR data, depicted in Figure 2—figure supplement 1B, and our immunostaining evidence is likely attributable to the fact that CD31-positive cells were isolated from cardiac tissue to use for qPCR analysis. It is likely that FOXC2 expression levels are different between lymphatic endothelium in mesentery collecting vessels and the cardiac vasculature. Thus, this may explain the differences in deletion efficiency. The repeated immunostaining of collecting vessels with FOXC1, FOXC2, and VEGFR-3 antibodies also now shows clear expression of FOXC1 in the nucleus of both lymphatic endothelial cells and smooth muscle cells of arterioles. As shown in Figure 2—figure supplement 1E, G, and I, there is no membrane signal detected. We suspect that there may be some cross-reactivity with our secondary antibodies used for the immunostaining combination of FOXC1 and VE-Cadherin, thus warranting our use of VEGFR-3 antibody to address this issue and verify the reduction of FOXC1 expression in the endothelium of collecting vessels.To address the third part of this comment, we performed immunostaining of FOXC1, FOXC2, and VEGFR-3 in mesentery collecting vessels of EC-2) The conclusion 'Cell elongation and junctional integrity is markedly impaired in compound EC- specific Foxc1; Foxc2 mutants resulting in increased apoptosis in lymphatic collecting vessels' requires more supporting evidence. We see one image  aiming to support the claim that cell elongation and junctional integrity is impaired but it is not clear how representative this image is. It is surprising that such a high number of apoptotic cells is detected in control vessels, drawing into question the specificity of the caspase-3 staining, and whether the signal is indeed detected in the LECs. No evidence is provided for cell elongation defects leading to increased apoptosis. This statement should also be demonstrated: "Of note, apoptotic bodies were more frequently observed at branched areas of the lymphatic collecting vessels potentially indicating areas of valve degeneration." The authors should expand on the cellular mechanisms underlying the presumed rupture of lymphatic vessels and accumulation of chylous ascites in EC-Foxc1;Foxc2-DKO pups. Ultrastructural analyses could be informative here.Foxc1; Foxc2 mutants and is accompanied by increased apoptosis throughout lymphatic collecting vessels’ as the previous statement was not accurate. Our group previously reported that valves of LEC-Foxc2-KO mutant mice were characterized by increased apoptosis and apoptotic cells were often arranged in doublets with symmetrically organized PROX1-high apoptotic bodies, suggesting that apoptosis may occur in dividing cells. Additional evidence identified that half of the dying cells in LEC-Foxc2-KO mice were PROX1/Ki67-positive, demonstrating that valves of LEC-Foxc2-KO mice were characterized by abnormal activation of cell proliferation and increased cell death. This phenotype was associated with improper activation of TAZ signaling in the absence of FOXC2 . We have included a summary of this finding in the subsection “Cell elongation and junctional integrity is markedly impaired in compound EC-specific Foxc1; Foxc2 mutants and is accompanied by increased apoptosis throughout lymphatic collecting vessels”. We then sought to characterize junctional integrity and apoptosis in compound Foxc1; Foxc2 mutant mice to assess if there was a synergistic effect of inactivation of both Foxc1 and Foxc2 in the lymphatic endothelium.We thank the reviewers for this helpful comment and careful critique of our data. We have revised our conclusion to more accurately state, ‘Cell elongation and junctional integrity is markedly impaired in compound EC-specific Foxc1; Foxc2 mutant littermates that show apoptotic bodies are in closer proximity to branched regions.To further address this reviewer comment, we carefully re-evaluated our caspase-3 immunostaining and repeated analysis to characterize the percent of PROX1/cleaved caspase-3 positive lymphatic endothelial cells in 20X high-power field images. Figure 2—figure supplement 4I-K has been updated to include new representative images and quantitative analysis. In addition to representative images of maximum intensity projections of PROX1/cleaved caspase-3 immunostained collecting vessels, we also show panels of z-sections from selected regions in higher magnification where PROX1/cleaved caspase-3 positive cells were identified. We also now show images from two compound 3) Inconsistent quality of staining between different experiments and even panels in the same figure (as if staining and imaging is done at different time points and not using littermate mice) make assessment of the phenotypes difficult, especially when a single high magnification image is provided as a supporting evidence. For example – Figure 3—figure supplement 2 VE-cadherin staining, Figure 2E-F CD31 staining. Figure 3 and Figure 3—figure supplement 3H show low PROX1 levels overall in Foxc1;Foxc2 double mutants. Figure 3—figure supplement 3D shows instead extremely high PROX1 levels, along with 'discontinuous junctions', making it difficult to judge if this is a representative image. The junctional phenotype presented in Figure 3—figure supplement 3D is indeed dramatic , but the phenotype is seemingly different in Figure 3—figure supplement 2D where the phenotype is not obvious at all. 'Discontinuous junctions' in the Foxc1;Foxc2 double mutants should be demonstrated more convincingly.Foxc1; Foxc2-DKO mice more clearly depict that collecting vessels in these mice are characterized not only by discontinuous junctions, but the increased presence of overlapping junctions as well. We have also generated videos of 3D reconstructions of the collecting vessels depicted in Figure 2—figure supplement 4A-H (Videos 1 – 4), characterizing this observation.We thank the reviewers for this helpful comment and careful critique of our representative images. To address the first part of the reviewer comment, we have repeated immunostaining of tissues and re-evaluated the post-processing of our representative images for several figures to address issues with inconsistencies. This includes new images for PROX1/CD31 immunostaining in Figure 2C, E, I, and K and new images for PROX1/VE-Cadherin immunostaining in Figure 2—figure supplement 4A-H that now show consistent PROX1 expression levels with representative images in Figure 2. Additionally, our new representative images for Figure 2—figure supplement 3E-H and Figure 2—figure supplement 4A-H show consistent VE-Cadherin expression levels among different tissue samples. Furthermore, our new images of VE-Cadherin immunostaining in EC-4) The authors should provide further evidence to demonstrate how relevant the described in vivo phenotypes are to those described in vitro. If Foxc1 and Foxc2 have distinct functions in regulating the actin cytoskeleton, it is not clear why Foxc2 is capable of functionally substituting for Foxc1 in vivo.Foxc2-KO and EC-Foxc1; Foxc2-DKO mutants in Figure 2—figure supplement 4A-H that shows differences in the presence of discontinuous junctions in collecting vessels of EC-Foxc2-KO mice, but both discontinuous and overlapping junctions present in collecting vessels of EC-Foxc1; Foxc2-DKO mice. This new data recapitulates our in vitro evidence depicting the same observations in FOXC2-KD and FOXC1; FOXC2-KD cultured lymphatic endothelial cells as represented in Figure 4—figure supplement 1, thus providing additional support for the relevancy of our in vivo and in vitro observations. In regard to the second part of the reviewer comment, although FOXC1 and FOXC2 are functionally similar, as characterized by their similar DNA-binding capacity, their transcriptional targets are likely to be different under oscillatory or laminar shear stress. For example, our group previously reported that loss of FOXC2 leads to distinct phenotypes in lymphatic endothelial cells under oscillatory shear stress and laminar shear stress . Thus, as FOXC1 is induced only by laminar shear, but FOXC2 is induced by both laminar and oscillatory shear, their downstream targets are likely different within the collecting vessels of lymphatic endothelial cells. We have also updated the fourth paragraph of the Discussion section to elaborate on these differences.We have now included new representative images of VE-Cadherin immunostaining in EC-5) Previous studies have demonstrated that FOXC2 is regulated by oscillatory shear forces in LECs, and that such forces likely play a key role in valve development. In Figure 4 the authors use in vitro flow studies to conclude that FOXC1 expression is regulated primarily by laminar shear while FOXC2 primarily by oscillatory shear. However, the endothelial cells shown in Figure 4A do not appear aligned with flow as would be expected for laminar shear and the authors fail to identify a clear mechanism that would account for such a distinct regulation. This theory appears to rely primarily on antibody detection of nuclear FOXC1 which is not as robustly detected as FOXC2. In vivo studies, especially Figure 10, suggest that these transcription factors are functionally redundant at the protein level as replacement of FOXC1 with FOXC2 does not impair valve maturation. Much of the data shown are carefully analyzed, but these findings and their connection to where FOXC1 is best detected in vivo in the valve tip seems like an over-reach. More specific mechanistic data identifying how such remarkable distinctions in shear regulation are achieved should be provided.As indicated in the Materials and methods section, the flow experiment presented in Figure 3  was run for only 24 hours (subsection “Cell transfection and immunostaining”). This shorter time – 24-hour flow instead of 48-hour flow presented in our previous publications  – is not sufficient to drive an important cell elongation in the direction of flow. In In addition, we also provide the reviewer with the quantification of length:width ratio for static, LSS and OSS conditions presented in Figure 3, which shows that there is a significant elongation of cells under LSS in average . To clarThe observation that FOXC1 and FOXC2 expression are already different in LECs after only 24 hours under laminar shear stress is interesting as it suggests that FOXC1 upregulation by laminar flow is an early response of LECs to directional flow. How expression levels of FOXC1 and FOXC2 are distinctly regulated by the directionality of flow is a very interesting question that addresses the more general one of how LECs sense flow direction and are able to respond differently to LSS and OSS. Answering such a question would require further investigation and, in our opinion, is beyond the scope of this paper in which the central message is that each factor is differentially regulated by flow and plays slightly distinct but complementary roles in regulating the cell cytoskeleton in response to shear stress.6) Figure 1—figure supplement 1: What is the level of FOXC2 protein present in LEC of EC-Foxc2-KO compared to mRNA? Foxc2 mRNA appears to be reduced by approximately 50% compared to control EC Foxc2 levels. Does this level of reduction in EC-Foxc2-KO animals explain the partial penetrance of the phenotype of chylous ascites?Foxc2-KO mice compared to littermate controls. We also suggested that the differences observed in reduction of mRNA levels compared to what we observe in regard to protein expression may be attributable to the different vascular beds analyzed. The difference in the penetrance of the phenotype of chylous ascites in the current study may be attributable to both the timeline of experimental analysis as well as the differences in Cre-drivers used. In our group’s previous study, tamoxifen was administered to ERT2; Foxc2fl/flProx1-Cre mice beginning at P4 with approximately 50% of mutants developing chylous ascites 4 days after tamoxifen administration and nearly all mutants presenting chylous ascites 6-8 days after tamoxifen administration . In the current study, tamoxifen administration started at P1 and we primarily investigated tissues at P6. We believe it is likely that our ERT2; Foxc2fl/flCdh5-Cre mice would present chylous ascites at later time points.As previously described in our response to reviewer comment #1, Figure 2—figure supplement 1F and G show a marked reduction of FOXC2 protein within collecting vessels of EC-7) Figure 3—figure supplement 2: A lower power image depicting the broader profile of α9-integrin expression throughout multiple valves would enable the reader to determine how α9-integrin deposition is affected more globally in EC-Foxc2-KO compared to EC-Foxc1;Foxc2-DKO mice.Foxc2-KO mice at P6, but the collecting vessels of EC-Foxc1; Foxc2-DKO mice are absent of α9-integrin-positive valve regions.We thank the reviewers for this suggestion. Figure 2—figure supplement 3A-D now depicts 10X low power images of α9-integrin/VE-Cadherin immunostaining to show that a few, α9-integrin-positive degenerating valve regions are detected in collecting vessels of EC-8) Figure 6: How do the changes in Prickle1, Arhgap21 and Arhgap23 levels compare to other gene expression changes in LEC-Foxc1;Foxc2-DKO LEC? It would be informative to provide more detail as to why these three genes were selected for further analysis. Were they the only genes involved in RhoA/ROCK signalling that were changed in expression? It would also be informative to know whether RAS/ERK signalling is affected in mutant LEC at this stage of development, given the authors' previous work which demonstrated regulation of this key pathway by FOXC1 and FOXC2 in embryonic LEC.Foxc1; Foxc2-DKO mice and littermate controls in panel K. Figure 5—figure supplement 1K now shows the changes in expression for RhoA, Rock1, and Rock2, and several GTPase activating proteins associated with regulation of RhoA signaling and endothelial barrier function and lumen maintenance . Here, we see that there is a modest, yet significant increase in Arhgap18 expression and a significant reduction of Arghap20, although its expression was generally lower compared to other GAPs. Because of the previously reported evidence of the formation of a physical complex between Prickle1 and Arhgap21/23  and our observation that all three genes were significantly downregulated in our RNA-seq analysis, we focused our investigation on these putative downstream targets. In regard to changes in RAS/ERK signaling, we attempted to perform whole-mount immunostaining of pERK using the same antibody utilized in our group’s previous study  but we were not successful in identifying a positive signal in control or mutant lymphatic valves at P6, testing the antibody at various concentrations. While we are interested in characterizing whether RAS-ERK signaling may be affected in our mutants during postnatal development in other lymphatic vascular beds, the authors believe that this is not of focus for the mechanism described in this study and is beyond its scope.We agree with the reviewers that providing additional details regarding the choice to focus on Prickle1, Arhgap21, and Arhgap23 would be helpful. We have now updated Figure 5—figure supplement 1 in the revised manuscript to include additional data from our RNA-seq analysis for LECs isolated from the dorsal skin of embryonic LEC-9) Figure 6D: Additional controls are required in which ChIP of both FOXC1 antibodies at a negative control region of the genome (e.g. an enhancer region demonstrated or predicted not to bind FOXC1) is assessed. This should demonstrate the selectivity of FOXC1 antibodies in terms of their specific versus non-specific/background binding to DNA and may help to explain the distinct results observed with each antibody in different regions of the genome.ICAM1 promoter that is not predicted to bind to FOX transcription factors. Panel J then shows data from three separate experiments showing no detection of band signals from ChIP with our FOXC1 or FOXC2 antibodies. Within the text of the manuscript, we have also included relevant literature that has identified variations in immunohistochemistry related work for antibodies recognizing the same immunogen peptide that are dependent on the source of the manufacturer. This information is included in the second paragraph of the subsection “FOXC1 and FOXC2 regulate LEC expression of negative RhoA signaling regulators PRICKLE1, ARHGAP21, and ARHGAP23”.We thank the reviewers for this important suggestion. We have now included Figure 5—figure supplement 1I and J to directly address this comment. Panel I depicts the location of primers used to amplify a transcriptionally active region of the c2/c2 mice. What is the remaining signal in FOXC1 stained Foxc1c2/c2 tissue?10) Figure 10D-F: It is not clear from the images presented that a call can be made regarding FOXC2 levels being modestly increased in Foxc1c2/c2Foxc1 mice, we have also included new representative images of FOXC1/VEGFR-3 immunostaining in Figure 9D and E. As previously stated in our reply to reviewer comment #1, we suspect that the remaining signal in the FOXC1 channel of Figure 9H is non-specific and may be related to a cross-reactivity issue of secondary antibodies. However, we believe that Figures 9F-H clearly depict a reduction in nuclear FOXC1 expression as expected.We agree with the reviewers that it is likely not accurate to describe that FOXC2 levels are modestly increased. Thus, this statement has now been removed from the revised manuscript. To verify that FOXC1 protein expression is absent in collecting vessels of


It is proven that the simulation results are in good agreement with the experimental phenomena.In this paper, the Voronoimosaic model and the cohesive element method were used to simulate crack propagation in the microstructure of alumina/graphene composite ceramic tool materials. The effects of graphene characteristic size and volume content on the crack propagation behavior of microstructure model of alumina/graphene composite ceramics under different interfacial bonding strength were studied. When the phase interface is weak, the average energy release rate is the highest as the short diameter of graphene is 10–50 nm and the long diameter is 1600–2000 nm. When the phase interface is strong, the average energy release rate is the highest as the short diameter of graphene is 50–100 nm and the long diameter is 800–1200 nm. When the volume content of graphene is 0.50 vol.%, the average energy release rate reaches the maximum. When the velocity load is 0.005 m s Ceramic materials have attracted attention because of their high hardness and wear resistance ,2,3, and2O3 matrix to obtain Al2O3/WC/TiC/graphene composite ceramic tool materials, whose optimal indentation fracture toughness, Vickers hardness, and flexural strength reached 9.42 MPa.m0.5, 24.64 GPa, and 646.31 MPa, respectively. Ahmad et al. [Graphene has a high specific surface area and can be closely combined with ceramic matrix, which can improve the fracture toughness of ceramic materials ,11,12. Id et al.  used nanNagaraj et al.  found thThe finite element method (FEM) is a numerical analysis method based on variational and interpolation principles ,26,27. TIn previous studies, the microstructure model of alumina/graphene composite ceramics was established by the inserting cohesion units, and the influence of interfacial bonding strength between alumina and graphene on the crack propagation behavior was studied. Based on previous studies, this paper studies the effects of graphene particle size, graphene content, and velocity load on the crack propagation path, and compares them with the experimental results.2O3 ceramic tool material, the microstructure of graphene was characterized by ellipse. It is necessary to design the size of the long diameter and short diameter of the ellipse, and then characterize the graphene microstructure by randomly generating the corresponding size of the ellipse in the same 10 μm × 10 μm square using Python scripting language. The microstructure model of graphene is shown in The overall size of the Voronoi mesh model established in this paper is 10 μm × 10 μm, the number of grains is about 160, and the average grain diameter is about 0.8 μm. On the basis of obtaining the Voronoi mesh to characterize the microstructure of single-phase AlIn our previous work, the influence of interfacial bonding strength on the crack propagation path of AG composite ceramics was studied. The results show that the toughening effect is best when the cohesion parameters of alumina grain boundary and intragranular are taken as the cohesion parameters of alumina/graphene phase interface . Therefoε represents the strain rate. V represents the velocity load, which is applied to the upper and lower sides of the model. H represents the half of the total height in the model. Relevant research [3 S−1 and 1 × 105 S−1. For this model, the speed range of the strain rate is between 0.005 m s−1 and 0.05 m s−1. However, the smaller the value of velocity load, the longer the time needed for crack propagation simulation calculation [The average energy release rate is used to characterize the fracture toughness of composite ceramics . In thisresearch  shows thculation . TherefoThe effects of the short diameter and long diameter of graphene on the crack propagation path were studied, respectively. The specific data are shown in The long diameter of graphene is set as 800–1200 nm, and the short diameters are set as 10–50 nm, 50–100 nm, 100–150 nm, and 150–200 nm, respectively. The simulation results of the crack propagation are shown in When the phase interface is weak, the smaller the short diameter of graphene, the more obvious the toughening effect. When the short diameter of graphene is 10–50 nm, the resistance to crack is the strongest.The simulation results of the crack propagation path are shown in When the phase interface is weak, the smaller the short diameter of graphene, the more obvious the toughening effect. When the phase interface is strong, the toughening effect is relatively good when the short diameter graphene is 50–100 nm. According to the above analysis, the short diameter sizes of graphene are determined to be 10–50 nm and 50–100 nm.When the short diameter is 10–50 nm or 50–100 nm, and the long diameter is 800–1200 nm, 1200–1600 nm, 1600–2000 nm, and 2000–2400 nm, respectively, the crack propagation at different interface strengths is simulated.The simulation results of crack propagation paths with different long diameters are shown in The simulation results of crack propagation paths with different long diameters are shown in The effect of graphene volume content on the crack propagation path of AG composites was simulated under different microstructures .The simulation results of crack propagation at weak interface are shown in The simulation results of crack propagation of each microscopic model under the strong interface are shown in −1, the crack begins to form at about 2 μs. With the increase of velocity load, the time of crack generation is gradually advanced. When the velocity load is 0.005 m s−1 and 0.01 m s−1, the crack propagation path is a single crack propagation and a few secondary cracks, and the curve at this stage is relatively stable. In general, the velocity load of 0.005 m s−1 in this study can ensure the convergence of simulation calculation.The law of the energy dissipation under different velocity loads is shown in According to the above research results, with the increase of graphene volume content, the average energy release rate of materials showed a trend of increasing at first and then decreasing. When the volume content of graphene is 0.50 vol.%, the average energy release rate reaches the maximum. This is different from that reported by Meng et al.  that the2O3 near graphene is smaller than that far away from graphene. The grain size of Al2O3 is about 0.8 μm, which is consistent with the model established in this paper.The graphene sheets in the alumina/graphene composite ceramic cutting tool materials reported by Meng et al.  of our rThrough the simulation and analysis, the toughening modes such as crack deflection, graphene fracture, and crack bridging are observed in the composite ceramic tool material. As shown in In this paper, the Voronoimosaic model and the cohesive element method were used to simulate crack propagation in the microstructure of alumina/graphene composite ceramic tool materials. The effects of graphene characteristic size, graphene content, and loading speed were studied. The conclusions are as follows:(1) When the phase interface is weak, the average energy release rate is the highest when the short diameter of graphene is 10–50 nm and the long diameter is 1600–2000 nm. When the phase interface is strong, the average energy release rate is the highest when the short diameter of graphene is 50–100 nm and the long diameter is 800–1200 nm.(2) With the increase of graphene volume content, the average energy release rate increased first and then decreased. The average energy release is the largest when the content of graphene is 0.50 vol.%.−1. It is proven that the simulation results are in good agreement with the experimental results.(3) The results show that the convergence of the simulation calculation can be ensured when the velocity load is 0.005 m s


The electrodes of MoS2/graphene in superlattice configuration show improved catalytic activity for H2 and O2 evolution with smaller overpotential of +0.34 V for the overall water splitting when compared with analogous MoS2/graphene heterojunction with random stacking.Evidence by selected area diffraction patterns shows the successful preparation of large area (cm × cm) MoS I2 and Pt ,6. MoS2 on (HER) ,8,9 and,2 as electrocatalyst, it has been shown that the assembly of this chalcogenide with graphene improves its performance as electrodes [2 to the external circuit. For this reason, there has been considerable interest in developing different procedures for the preparation of MoS2/graphene heterojunctions to be used in electrolysis [Regarding the use of MoSectrodes ,11,12. Gtrolysis ,14,15 2 strongly interacting with graphene has been recently reported. The process is based on the pyrolysis of (NH4)2MoS4 and chitosan as precursors of MoS2 and N-doped defective graphene, respectively [In this context, a procedure consisting in the one-step formation of MoSectively . One of ectively ,18. Alsoectively .2/graphene heterojunction in superlattice configuration, particularly when considering the well-known activity of MoS2 for important reactions, such as HER, OER, and oxygen reduction reaction (ORR) processes [Since both boron nitride and graphene are two-dimensional (2D) materials with very similar lattice parameters, the term superlattice refers to the coincidence of the hexagons of boron nitride overlapping those of graphene. Fundamental studies have shown that superlattice configuration of 2D materials heterojunctions can give an assembly with tunable electrical conductivity in contrast to the random heterojunctions of the two materials ,21. Thisrocesses .2/graphene heterojunction in superlattice configuration. It will be shown that this fl-MoS2/graphene material (fl meaning few layers) exhibits much lower onset potential for HER and OER than an analogous electrode prepared with random MoS2/G heterojunction, illustrating the advantages of lattice matching to improve the electrochemical performance.This manuscript reports a novel procedure for the preparation of large area (cm × cm) films of MoSMolybdenum sulfide exfoliation was carried out while using polystyrene as an exfoliating agent (ALDRICH). The polystyrene was dissolved in dichloromethane at a concentration of 3 mg/mL and powdered Molybdenum sulfide was mixed at a concentration of 1.5 mg/mL. The sample was sonicated while using a Sonic tip (FisherbrandTM Model 705 at 50% 700W for 5 ho per sample with a on/off sequence consisting in 1 s off and 1 s pulse and an ice bath was used to prevent solvent evaporation). After sonication, the dispersion was centrifuged at 1500 rpm for 45 min ).2 quartz substrate to prepare the films : 4000 rpm, 30 s). The pyrolysis of polystyrene treatment was performed using an electric oven and using the following heating program: heating at 5 °C/min at 900 °C for 2 h.The supernatant was preconcentrated and deposited on quartz films by rotation coating on 2 × 2 cm2  at a concentration of 1.5 mg/mL and commercial graphene with an area of 700 m2  at a concentration of 7.5 mg/mL were added to a 30 mg/mL polystyrene solution  in dichloromethane and the suspension submitted to ultrasounds using a Sonic tip at 700 W for 5 h. After sonication, the dispersion was centrifuged at 1500 rpm for 46 min. and the supernatant used to prepare films of randomly configured MoS2/G heterojunction following the same procedure indicated above.For the preparation of films with random configuration, commercial MoS2). Voltammetric experiments were carried out with a potentiostatic device CH 920c , using H2SO4 1.0 M saturated with air as an electrolyte. The working electrodes were the MoS2/fl-G films that were deposited on graphite. The graphite was deposited on the quartz films using the automatic carbon coater  with carbon evaporation supplied BALTEC-CEA035).Electrochemical measurements were made in a conventional three electrode electrochemical cell while using a Pt disc pseudo-reference electrode and a Pt wire auxiliary electrode that accompanies the graphene modified glassy carbon (GCE) working electrode  or few layers structure of MoS2 (25 cm−1). In the present case, the difference between the A1g and E2g vibration modes of MoS2 was 24 cm−1, which agrees with the case of few layers MoS2. This configuration of MoS2 as few layers platelets is also in accordance with the previously commented AFM measurements of MoS2 particles that were supported on graphene.2/graphene  heterojunction, as stated in the introduction. Aimed at this purpose, an additional sample of r-MoS2/G was prepared by introducing commercial preformed graphene during the process of exfoliation of bulk commercially available MoS2 (Aldrich). 2/G sample used for the comparison, including a set of TEM images and EDS analysis with the corresponding elemental mapping. (2 and graphene not overlapped and clearly distinguishable for each 2D material, as expected for samples lacking the superlattice configuration (The main purpose of this study is to determine the possibility of preparing large area films of superlattice MoSmapping.  Importanguration , frame d2/G electrode and a reference electrode. The quartz plates used for preparation of electrodes were previously modified by subliming carbon before MoS2/G film preparation, thus improving the electrical contacts, in order to increase the electrical conductivity of MoS2/G electrodes. Prior controls with sublimed carbon quartz plates lacking MoS2/G electrocatalyst did not exhibit any peak in cyclic voltammetry. Experiments were carried out for air-saturated, 1.0 M aqueous H2SO4 solution as electrolyte. 2/G and r-MoS2/G under these conditions n. As it can be seen there, for s-MoS2/G a cathodic peak at −0.8 V vs. Ag/AgCl corresponding to ORR is recorded before a step peak corresponding to HER with onset being determined by extrapolation of the current density plot at −0.70 V. In the anodic region, the s-MoS2/G electrode exhibits the expected OER process with an onset potential of 0.87 V. For comparison, 2 electrodes together with the new data herein obtained for s-MoS2/G.Electrochemical measurements were performed with a conventional three-electrode single cell with Pt as auxiliary electrode accompanying the MoS2 crystals using polystyrene as promoter, followed by film casting and pyrolysis is a suitable procedure for obtaining few layers MoS2 deposited on few layers defective graphene in superlattice configuration. The procedure allows for obtaining large area films that are suitable for electrochemical characterization. By comparing the performance of two MoS2/G electrodes, one with lattice matching and another with random configuration in the heterojunction, it has been observed that the electrocatalytic activity of MoS2/G improves significantly in the superlattice configuration. This probably reflects the favourable orbital overlapping and electron migration in the MoS2/G heterojunction when the two lattices match one on top of the other. The present results show the far-reaching potential of superlattice assembly of 2D heterojunctions for application in electrocatalysis.In summary, it has been shown that the exfoliation of bulk MoS


In this work, the synthesis of graphene-like nanosheets (GNS) by an electrochemical exfoliation method, their microstructural characterization and their performance as fillers in a ceramic matrix composite have been assessed. To fabricate the composites, 3 mol % yttria tetragonal zirconia (3YTZP) powders with 1 vol % GNS were processed by planetary ball milling in tert-butanol to enhance the GNS distribution throughout the matrix, and densified by spark plasma sintering (SPS). According to a thorough Raman analysis and SEM observations, the electrochemically exfoliated GNS possessed less than 10 graphene layers and a lateral size lower than 1 μm. However, they contained amorphous carbon and vacancy-like defects. In contrast the GNS in the sintered composite exhibited enhanced quality with a lower number of defects, and they were wavy, semi-transparent and with very low thickness. The obtained nanocomposite was fully dense with a homogeneous distribution of GNS into the matrix. The Vickers hardness of the nanocomposite showed similar values to those of a monolithic 3YTZP ceramic sintered in the same conditions, and to the reported ones for a 3YTZP composite with the same content of commercial graphene nanosheets. Since the first isolation of single-layer graphene in 2004 by the mechanical exfoliation of graphite —the “Scotch tape” method —its uniqIn the last decade, graphene has mainly been synthesized using two different approaches: bottom-up, in which graphene is grown from small molecular carbon precursors, and top-down, in which graphene is exfoliated from graphite as parent material . Among tElectrochemical exfoliation has had a strong impact on the development of techniques to obtain graphene because it provides an economical, simple and fast way to produce it. It is easily reproducible because it can be performed under environmental conditions, and toxicity-free components that can be easily removed after the process are used. In addition, the use of graphite as starting material, together with the good results obtained from this process, reduce the cost of producing graphene, resulting in an efficient and affordable method for the scientific community . MoreoveAmong the different applications of graphene, its use as a filler in composite materials has awakened the interest of the scientific community in the last years, owing to the relevant properties that these nanostructures impart to most materials ,16. In tMost of the published studies about graphene-ceramic composites generally use commercially acquired graphene nanosheets. Although promising results in terms of enhancement of mechanical and electrical properties have been reported for composites with cost-effective graphene nanoplatelets prepared using advanced powder processing techniques ,23, the In this work, the synthesis of graphene-like nanosheets has been assessed by means of a simple, cost-effective and fast electrochemical exfoliation technique, using graphite as parent material. After a detailed characterization of the as-synthesized nanosheets by Raman spectroscopy and electron microscopy observations, they were incorporated as filler in a 3YTZP matrix nanocomposite. Powders with 1 vol % GNS were processed by planetary ball milling in tert-butanol to enhance the GNS distribution throughout the matrix, and densified by spark plasma sintering (SPS). The quality and level of defects of the GNS in the composite were assessed by Raman spectroscopy. The microstructure and hardness of the obtained nanocomposite was analyzed and compared to the reported ones for 3YTZP composites prepared with commercial nanosheets.The graphene-like nanosheets were obtained by the electrochemical exfoliation method , using aAfter the exfoliation process, the suspensions were washed with DI water and isopropyl alcohol by vacuum filtration using 200 nm pore filter alumina membranes  and centrifuged  at 8500 r.p.m. for 15 min to remove graphite aggregates. The suspensions were frozen with liquid nitrogen and freeze-dried for 48 h at −80 °C in order to avoid re-agglomeration of the obtained nanosheets during drying .−1) Raman spectra were fitted to a sum of five functions: two Gaussian and three pseudo-Voigt functions. In the second-order spectra (from 2250 to 3300 cm−1) three Lorentz and three pseudo-Voigt functions were used. The fits were carried out using the OriginLab software .Raman spectroscopy and high-resolution scanning electron microscopy  were used to characterize the number of layers, morphology and size distribution of the as-synthesized GNS. To that end, a few droplets of GNS suspension in isopropyl alcohol were deposited on a glass slide for Raman spectroscopy or on a Cu transmission grid with C coating for HRSEM inspection after drying. At least 10 Raman spectra were acquired on the electrochemically exfoliated GNS using a dispersive microscope Raman Horiba Jobin Yvon LabRam HR800 (ICMS), with a green laser He-Ne (532.1 nm) at 20 mW. The first-order , which were previously annealed at 850 °C for 30 min in air. Planetary ball milling  was used to homogenize the powders in a 10 w/w% tert-butanol (t-BuOH)/water mixture at 700 r.p.m. for 15 min. A 45 mL zirconia jar and seven 15 mm diameter zirconia balls were used. After drying on a rotary evaporator, the composite powders were homogenized in an agatha mortar and spark plasma sintered at 1250 °C for 5 min, with an applied pressure of 75 MPa and heating and cooling ramps of 300 and 50 °C/min, respectively . A sheet of graphite paper was placed between the powders and the die/punches to both ensure their electrical, mechanical and thermal contact and also for an easy removal. The temperature was continually monitored by means of an optical pyrometer focused on the side of the graphite die. Cylindrical samples with 10 mm diameter and 2 mm thickness were obtained. The surface graphite paper from the SPS molding system was manually eliminated by grinding.3 and 2.2 g/cm3, respectively. Scanning electron microscopy (SEM) using backscattered electrons (BSE) for imaging  was used to analyze the dispersion of the GNS in the ceramic matrix. This microscope has two in-lens detectors which allow obtaining high resolution images at short work distances. Polished in-plane (i.p.) and cross-section (c.s.) surfaces were analyzed to account for the existence of any structural anisotropy on the composite. The grain size of the ceramic matrix was estimated from SEM images acquired on polished c.s. surfaces previously annealed in air for 15 min at 1150 °C. The planar equivalent diameter, d = 2(area/π)1/2, namely the diameter corresponding to a circle with the same area as the measured grain, was taken as a measure of the grain size, averaging 200 to 300 grains, according to UNE-EN ISO 13383-1:2016 standard. The software packages ImageJ and OriginLab were used to determine the relevant parameters. The fracture surface of the composite was also examined by HRSEM .To account for possible structural modifications of the graphene-like nanosheets after the composite powder processing and sintering, at least ten Raman spectra were acquired on the obtained powders after planetary ball milling, and on the fracture surface of the sintered composite. The first- and second-order Raman spectra were fitted to the functions described in V (GPa) = 1854.4 P/D2, where P is the applied load in N and D the average diagonal of the imprint in μm.The hardness of the nanocomposite was estimated from standard Vickers micro-indentations  performed on the mirror polished i.p. and c.s. surfaces. These two orientations were evaluated to account for any possible anisotropy effects. Ten indentations were performed on each surface with 1.96 N applied load during 10 s. The hardness values were calculated following the equation: HTypical HRSEM micrographs (acquired in Secondary Electron Image mode) for the electrochemically exfoliated nanosheets are shown in −1. The G and 2D bands are always found in pristine graphene. The G band is due to the doubly degenerate zone center E2g mode and the 2D band is the second order of zone-boundary phonons −1. These in 1 [4 ,27,35 or in 1 [4 ,30,36 deed as T2 , D3 [34,as T2 [3  or D’’ [as T2 [3  or other3 and D’ bands are not described when analyzing the Raman spectra of graphene-based nanomaterials because they are very weak peaks. Nevertheless, when these bands present a remarkable intensity, they appear to overlap with the D and G peaks. This makes the deconvolution of the first-order spectrum (from 1000 to 2000 cm−1) essential for the correct interpretation of the Raman spectrum, as it is suggested by different authors [Usually, the D‘’, D authors ,35,38,393) and three pseudo-Voigt  functions for the first-order spectra, and three Lorentz (2D) and three pseudo-Voigt  functions for the second-order spectra.D/IG and ID’/IG ratios . The existence of a pronounced D3 band , in agr−1, which had not been observed in the spectrum of the as-exfoliated GNS (−1) and monoclinic  phases of the zirconia matrix [−1 and a broad band in the range ~700–1100 cm−1 were found. These bands can be attributed to the presence of a low percentage of an alumino-silicate (AS) glass [−1—to the fittings.The Raman spectra of the composite powder after planetary ball milling and of the sintered ceramic composite are presented in ated GNS a. In ordated GNS a revealia matrix , sharp pS) glass  that cou3) and four pseudo-Voigt  functions for the first-order spectra, and three Lorentz (2D) and three pseudo-Voigt  functions for the second-order spectra. 3 band is found, pointing to a lower amount of amorphous carbon in the GNS after sintering, in agreement with published results that the ID3/IG ratio decreases as the crystallinity increases [A decrease of the defect-related D and D’ peaks intensity, along with an increase of the intensity of the 2D band, is observed for the GNS in the sintered composite, in comparison with the as-exfoliated GNS . Also, ancreases . All of ncreases ,31,32,39Another parameter that can give information about defects in graphene is the band width for D, G, D’ and 2D bands, as their widths increase with a growing number of defects . MartinsThe HRSEM images of the fracture surface of the nanocomposite, shown in The Vickers hardness of the nanocomposite, evaluated on i.p. and c.s. surfaces, revealed no mechanical anisotropy, as similar hardness values were obtained for both surfaces . This maGraphene-like nanosheets (GNS) with a number of graphene layers lower than 10, containing amorphous carbon and vacancy-like defects, and with a lateral size lower than 1 μm were successfully synthesized using a simple, cost-effective and fast electrochemical exfoliation method. The incorporation of 1 vol % GNS to a 3YTZP matrix resulted in a composite material with a homogeneous distribution of GNS into the matrix, despite the high tendency to agglomerate presented by the as-exfoliated GNS. The selected processing method—planetary ball milling of the composite powders and spark plasma sintering (SPS)—produced a fully dense nanocomposite containing GNS with significantly enhanced quality, as revealed by Raman spectroscopy. The GNS were wavy, semi-transparent and with very thin thickness. The microstructural anisotropy—preferential alignment of the GNS in the direction perpendicular to the pressing axis during SPS—revealed by SEM observations was not reflected on the hardness values of the nanocomposite, which were isotropic. The hardness of the composite studied here was very similar to the reported one for a 3YTZP composite with the same content of commercial graphene nanosheets.