The production of an innovative, high-performance graphene-based polymer nanocomposite using cost-effective techniques was pursued in this study. Well-dispersed and uniformly distributed graphene platelets within a polymer matrix, with strong interfacial bonding between the platelets and the matrix, provided an optimal nanocomposite system for industrial interest. This study reports on the reinforcement of high molecular weight multimodal-high-density polyethylene reinforced by a microwave-induced plasma graphene, using melt intercalation. The tailored process included designing a suitable screw configuration, paired with coordinating extruder conditions and blending techniques. This enabled the polymer to sufficiently degrade, predominantly through thermomechanical-degradation, as well as thermo-oxidative degradation, which subsequently created a suitable medium for the graphene sheets to disperse readily and distribute evenly within the polymer matrix. Different microscopy techniques were employed to prove the effectiveness. This was then qualitatively assessed by Raman spectroscopy, X-ray diffraction, rheology, mechanical testing, density measurements, thermal expansion, and thermogravimetric analysis, confirming both the originality as well as the effectiveness of the process.  Multimodal high-density polyethylene (HDPE) is an engineered thermoplastic semi-crystalline polymer, which is widely used in automotive, films, pressure pipes and fittings, bottles, tubes, and cables jacketing ,2,3,4,5.It is therefore the topic of this study to introduce a more cost-effective, optimal way of fabricating a nanocomposite of high molecular weight multimodal-HDPE matrix, reinforced with a bottom-up graphene. These polymers are indeed widely used in a long-term application in an extreme environment, which includes hydrostatic, thermal, and environmental stresses ,21,23. H3 functional group on either the surface, or at the edge of the graphene sheets. Accordingly, a better stress transfer can potentially be achieved through the strong interfacial bonding created between graphene platelets and the polymer matrix. Achieving a thorough dispersion and distribution of graphene within the multimodal-HDPE, by melt intercalation, via co-rotating intermeshing twin-screw extruder, has never yet been reported according to the authors’ knowledge. The results of this research provide greater insight into different melt intercalation factors, affecting the multimodal HDPE-graphene nanocomposite performance and criterion for effectively producing the next generation of black multimodal-polyethylene compounds for use in high-pressure pipes, automotive, and energy cable applications [In the present work, we attempted to degrade the polymer to a sufficient level, through thermo-oxidative, as well as thermo-mechanical degradation during the melt extrusion process. This created a compatible medium for the graphene to disperse and distribute thoroughly within the polymer matrix. The polymer is consequently able to interact physically or chemically with the residual oxygen functional groups at the graphene surface which contains almost 5% oxygens, or through the short molecules introduced by thermo-mechanical degradation, with defective spications ,20,21,233 were used in this study. The multimodal high-density polyethylene matrix used in the present study was engineered specifically for nanocomposite applications. According to the production process technology, the melt flow rate (MFR) of the polymer gives an indication that the split MFR ratio between the reactors is significantly high [3 at 20 °C. Unstabilized high-density polyethylene powders, produced with Ziegler Natta catalyst via proprietary Borstar process, , with a melt flow rate of about 7.5 g/10 min 190 °C, 21.6 kg), Mw = 280 kg/mole, Mn = 8.49 kg/mole, Mw/Mn = 33, and a density of 950 kg/m1.6 kg, MThe graphene-based multimodal-HDPE nanocomposites (PE-g) were prepared via melt intercalation using a Coperion ZSK 18 twin extruder, with a screw diameter of 18 mm and a barrel length of 720 mm (L/D = 40). The screw rotation speed (rpm) was 600 min-1, barrel temperature profile was in the range of 170-240 °C see , and feeA schematic of a modular twin-extrusion screw configuration used in the present study is given in Since graphene has the ability to shield the polymer from heating and becoming completely molten by enhancing the thermal stability in the feeding and melting zones ,31,32,33Optical microscopy analyses were conducted on ZIESS Axio scope.A1 HAL 100/HBO 100, operated with an AxioCam MRc 5 camera, and AxioVision software. Film samples were sectioned to a thickness of 15 μm, using a fully automated rotary microtome Leica RM2265 . Transmission electron microscopy (TEM) was performed using a Hitachi HT7700, at an accelerating voltage of 120 kV. Film samples were cryo-sectioned to a thickness of ~80 nm at -125 °C, using a Leica EM UC7/FC7 Cryo-Ultra-microtome. Density measurement was performed with an analytical balance, equipped with a density measurement kit , following an ASTM D792-method B, based on the Archimedes’ principle where the weight of the sample immersed in an n-dodecane fluid decreases by an amount equal to the displacement of the liquid weight. f) was measured by calculating the deflection of the beam at the mid-span (δ), following the equation εf=6 δ t/L2, where t is the thickness of the beam specimen and L is the span between the supports [Raman measurements were carried out using Renishaw inVia confocal Raman microscope with 633 nm and 532 nm lasers. With the exception of the deformation test, all the Raman data was collected using He-Ne ion laser with a wavelength of 633 nm . However, an Nd-YAG laser with a wavelength of 532 nm  was used to evaluate the stress-transfer along the interfacial surface between the polymer matrix and reinforcement. The dog-bone specimens, having been prepared for tensile testing, were deformed in a three-point bending rig. The strain (εX-ray diffraction (XRD) measurements were performed using a Bruker D8-Advance diffractometer equipped with Cu Kα1 radiation (λ = 1.54060 Å). The diffraction patterns were recorded with a step size of 0.15087°, and dwell time of 5s. A scanning electron microscope (SEM) was utilized to assess the cryofractured cross-section surface of the uncoated polymers, using an FEI QUANT 250 FEG SEM, at an accelerating voltage of 2 kV. Samples were notched prior to being submerged in liquid nitrogen for ten minutes. Tensile properties of the nanocomposites such as tensile modulus, stress and strain at both yield and break, as well as other aspects of the tensile stress-strain curve were studied on the stamped ISO 527-2 dog-bone specimens, type 1B. This was achieved using die punch equipment  on the compression molded samples. These were measured with the Zwick/Roell Universal Testing Machine (UTM)–Z050, using a load cell of 2.5 kN, grip-to-grip separation of 115 mm, a gauge length of 50 mm, and a cross-head speed of 50 mm/min. A contact type extensometer  was used to measure the strain of the specimen. The results were based on a minimum of 6 specimens. z in an ambient gas atmosphere (50 mL/min), using a wedge-shaped quartz probe. The dimension of the samples was 10 × 3.4 × 0.5 mm3. The same equipment was employed for studying the coefficient of thermal expansion (CTE) via a flat-tipped standard expansion probe in an ambient gas atmosphere (50 mL/min). Flat samples with dimensions of 6 × 6 × 4 mm3 were heated at 3 °C/min from room temperature to 100 °C. They were then held for 10 min, then cooled to 0 °C at the same rates of 3 °C/min, then held for 10 min, and subsequently heated at 3 °C/min to 120 °C, under a constant load of 0.05 N. The displacement was reset to zero at the start of the last sequence where the measurement started. Q400 Thermomechanical analyzer (TMA) was employed to determine the bending properties of the nanocomposites with dynamic mode through the 3-point bending test. Samples were cooled to -150 °C at the cooling rate of 3 °C/min for 10 min, and subsequently heated to 150 °C at the heating rate of 3 °C/min. The modulate force was 0.01 N at a frequency of 1 HG’ is the storage shear modulus, G” is the loss shear modulus, ω is the angular frequency, and Simple qualitative characterization related to an indirect measurement of molecular weight and processability of the polymer was studied upon the melt-flow rate (MFR). Pellets of 3-8 g were charged into a cylinder at 190 °C under the load of 21.6 kg, which was achieved using the Melt Indexer MI-4 manufactured by GÖETTFERT Werkstoff–Prüfmaschinen GmbH , following ISO 1133 procedure B. The rheological behavior of the samples was studied using stress-controlled rotational rheometer, an Anton Paar Physica MCR 301 with CTD450 heating unit, at 190 °C under a nitrogen atmosphere. The compression molded sample, weighing 1.5 g, 25 mm in diameter, and 1.5 mm thick was conditioned at 40 °C for 48 h. The sample was then placed onto a 25 mm parallel plate fixture and trimmed to a thickness of 1.2 mm by slowly lowering the upper plate. Dynamic frequency sweep was conducted from 500 to 0.0154 rad/s at 5% strain. The reason for starting from the maximum frequency was to avoid sample degradation under high temperature and low angular speed. The polydispersity index (PDI) was measured as follows [Thermogravimetric analysis (TGA) was carried out with Q500 TGA  with a heating rate of 10 °C /min from room temperature to 1000 °C in a nitrogen atmosphere. High-resolution (Hi-Res)-dynamic mode was performed with a sensitivity of 2.00 and a resolution of 4 °C. A twin-screw extrusion system with a modular screw configuration was utilized in this study to fabricate a polymer nanocomposite with well-dispersed and uniformly distributed graphene flakes. The screw configuration was optimized after several trials, starting with a screw design employed for compounding such polymers with a high-volume fraction of nanoparticles (carbon black) or nanoclays . A combination of different elements was utilized and arranged according to mixing requirements and material properties to be attained ,30,31,322) was around 0.5 µm2 and 0.0063, respectively. Graphene monolayers are transparent under an optical microscope, opacity of 2.3 ± 0.1%, while the optical loss become greater in the wrinkled and overlapped samples [2, with maximum particle size of around 4.7 µm2, and a %area fraction of 79.4 , while the breathing modes of the sp2 carbon atoms in a hexagon ring gives rise to the D-band [K point by double resonance, whereas the overtone of the D’ and D-bands gives rise to 2D’ and 2D-bands [I2D/IG), and Lorentzian fittings of the 2D peak provide good correlation with the number of layers of graphene in a flake sample [−1. Z. Lin et al. [−1, 1582.5 cm−1, and 2658 cm−1, respectively. The 2D-band of the nanocomposite shown in −1 to 2658 cm−1, fitted by four Lorentzians, with an overall FWHM decreased from 65.52 cm−1 to ~53 cm−1. Four fitted Lorentzians, each with a FWHM of ~ 24 cm−1, most likely arose from the asymmetry between the valence and conduction bands present in the bilayer graphene [I2D/IG) from 0.98 for graphene to 1.55 for PE-g-1%, reveals the reduction of the graphene layers [Overlay Raman spectra of the neat multimodal-HDPE, graphene powder, and PE-g-1% samples, are shown in ectively ,44,45. Te D-band ,44,45. Te D-band ,44,45. I2D-bands ,44,45. T2D-bands ,44,45. Te sample . In Figun et al.  in fact graphene . Besidese layers . Overallρc) of the graphene/multimodal-HDPE (PE-g) and carbon black/multimodal-HDPE (PE-CB) nanocomposites at nanofiller loadings of 0.1, 0.5, 1, 2, and 5 wt.%. The PE-CB sample is a commercial grade, produced based on the same polymer matrix, but reinforced by a carbon black with a density of 1700-1900 kg·m−3. A monolayer graphene is made up of covalently-bonded sp2-hybridised carbon atoms, densely packed in a honeycomb lattice [−3 [ρm = 950 kg·m−3) increased linearly with the addition of the nanofillers, i.e., densities of both nanocomposites increased by the same amount. The slope values are calculated at 4.003 for PE-g and 4.093 for PE-CB, suggesting that the graphene platelets were homogenously dispersed and distributed throughout the polymer matrix. An increase in the nanocomposite density is attributed to the high density of the reinforcements (ρr) employed to reinforce the polymer matrix, according to the equation of the form (ρc = 1/(Wr/ρr) + (Wm/ρm)), where Wr and Wm are the weight fractions of reinforcement and matrix, respectively [ lattice ,22. Thertice [−3 . On the tice [−3 . Each prtice [−3 . AccordiThe interfacial adhesion strength between graphene sheets and a polymer matrix can be explored through the microscopic examination of cryofractured cross-sectional surfaces. Shown in These flakes are well dispersed and evenly distributed within the multimodal-HDPE matrix, which may have formed a continually interconnected network structure throughout the matrix ,52,53,54−1 and 28 cm−1, respectively. Surprisingly, the 9% strain is around the yield point as can be seen in the stress-strain curve shown in The interfacial adhesion strength between the polymer matrix and graphene platelets was further investigated by the stress-induced Raman band shifts ,58,59,60G’) and loss (G”) moduli, as well as their frequency dependence [pendence ,61. CG) and crossover frequency point (Cω) have decreased from 0.044 MPa·s and 4.5 rad/s for the neat multimodal-HDPE to 0.035 MPa·s and 1.2 rad/s for PE-g-1%, respectively. The shift of Cω to lower region indicates that the nanocomposite exhibited higher average molecular mass and/or the entangled molecules were induced by the three-dimensional network of graphene platelets within the matrix [C shifting to lower values after the reinforcement indicates that the polymer exhibited broader molecular weight distribution, which is evident from the increase in the polydispersity index (PDI) by 18.4%, i.e. the larger the PDI, the broader the molecular weight distribution [cG upon the addition of the reinforcement possibly arose also from exposing the neat polymer to high temperature, under a combination of high shear and elongation forces, for a prolonged period of time. In At a high-shear rate, both materials exhibited thinning behavior, which resulted in a decrease of extensional viscosity. However, the incorporation of 1 wt.% graphene increased the melt viscosity of the nanocomposite, though the relative increase gradually lessened at high-shear rate. The presence of graphene has considerably increased the pseudoplasticity at a low shear rate region. At the angular frequency (ω) of 0.0154 rad/s, the complex viscosity increased from 0.13 MPa·s for neat multimodal-HDPE to 0.24 MPa·s for the PE-g-1%. Furthermore, the loss and storage moduli of the neat polymer increased by a value of 92% and 77% with 1 wt.% loading of graphene, respectively. The greater amount of storage and loss moduli of PE-g-1% suggests that the formation of a strong interfacial bonding between the polymer matrix and the high-modulus graphene reduced the loss tangent, the nanocomposite accordingly became more elastic ,61,62,63e matrix . Howeverribution . The shiThe synergistic effect advantages of graphene are further investigated by thermal expansion and thermogravimetric analyses (TGA). As shown from the TGA thermograms in −6 °C−1 over the temperature range of 30–103 °C, as shown in The coefficient of thermal expansion (CTE) of the PE-g-1% was calculated to be 0.55 × 10A high-performance novel graphene-based multimodal polyethylene nanocomposite was produced directly without any particular treatment to the composite constituents. Different characterization techniques such as TEM, SEM, optical microscopes, Raman spectroscopy, Rheology, density measurements, TGA, and CTE were employed in order to demonstrate the novelty of the fabrication method. Microscopic analysis showed that graphene platelets were homogenously dispersed and distributed within the polymer matrix. The electronic and optical microscopies showed that the graphene was dispersed with an average size of less than 450 nm, and distributed with a %area fraction of only 0.0063. The adhesion strength between the graphene sheets and polymer matrix examined by microscopic examination of the cryofractured surfaces of the nanocomposite, mechanical testing and stress-induced Raman band shifts, revealed a strong interfacial bonding attained through thermo-mechanical and thermo-oxidative degradation to a controlled level during the melt extrusion process. The cryofractured surface of the nanocomposite became rough with fibrils almost entirely absent. Deflection of the nanocomposite led the characteristic 2D and G Raman peaks of graphene to shift significantly towards high wavenumbers. This was confirmed further by the mechanical testing where the storage modulus of the polyethylene reinforced with 1 wt.% of graphene increased up to 118% at room temperature.The thermal performance of the nanocomposite was investigated via rheology testing, TGA and thermal expansion analysis. Loading a polymer with 1 wt.% graphene has resulted in a significant shift of the crossover frequency point and crossover modulus point to the lower regions. The homogenous dispersion and distribution of graphene platelets within the polymer matrix, as well as the strong adhesion bonding, led to the formation of an interconnected 3D network in the polymer which accordingly restricted the movement and expansion of the polymer chains movements. This has resulted in a significant increase in the onset degradation temperature by more 31 °C as a consequence of a thermal barrier or synergetic effects induced by graphene. The nanocomposites exhibited almost zero thermal expansion below 100 °C. The results of this study change the idea of there being difficulty in using melt extrusion for producing well dispersed and disturbed graphene-based hydrophobic polymer nanocomposites. Furthermore, considering there is no need to chemically treat graphene or polymer, or in fact change anything in the existing plants, such a result can significantly attract the industry.


The spectral dependence measured using the Z-scan technique is corroborated by a density matrix quantum theory formulation to extract a n2,eff ∝ λ2 dependence. The temporal evolution obtained using the time-resolved Z-scan measurement shows the nonlinearity peaking at zero delay time and relaxing on a time-scale of carrier relaxation. The dependence of the n2,eff on pulse duration is obtained by expanding the input pulse using a prism-pair set-up. Our results provide an avenue for controllable tunability of the nonlinear response in graphene, which is limited in silicon photonics.Graphene’s giant nonlinear optical response along with its integrability has made it a vaunted material for on-chip photonics. Despite a multitude of studies confirming its strong nonlinearity, there is a lack of reports examining the fundamental processes that govern the response. Addressing this gap in knowledge we analyse the role of experimental parameters by systematically measuring the near-infrared spectral dependence, the sub-picosecond temporal evolution and pulse-width dependence of the effective Kerr coefficient ( As a scale-invariant host of chiral carriers3, graphene exhibits a universal optical response in absorption, with a single sheet capable of absorbing ~2.3% of normally incident light. Its reduced dimensionality together with the peculiar Dirac type dynamics of the quasiparticles enable graphene to have a relatively strong nonlinear optical response in the presence of intense electromagnetic illuminations5. Intrigued by its potential applications in photonics and optoelectronics several theoretical and experimental studies of the nonlinear optical properties of graphene have been carried out22. Due to the centrosymmetric crystalline structure of graphene, even-order nonlinearities are forbidden, and the first nonlinear contribution is a third order effect. In particular, the Kerr type nonlinearity is dominantly attributable to the third order nonlinear interactions in graphene. The refractive index, n, considering the first nonlinear term is intensity dependent and is given by the expression n = n0 + n2I, where n0 is the linear refractive index, n2 is the Kerr coefficient, and I is incident intensity. This Kerr-type nonlinearity in graphene has been reported to be large, confirmed by studies characterising frequency mixing7, harmonic generation8, self-phase modulation10, and self-refraction15.The Dirac band structure of graphene18 of the nonlinear refractive index 14. A point of contention while comparing these results are the varying spectral and temporal properties of the excitation source, sample preparation techniques, and substrate material. The measurements are performed with the excitation wavelength ranging from the visible to the mid-infrared, while the pulse duration ranges from hundred femtoseconds to tens of picoseconds, under different sample preparation methods24. It has been recognised that the n2 depends on the wavelength, pulse duration and power in semiconductor systems25. Particularly in graphene, the n2 expected to become larger with longer wavelengths4. This trend has been observed and reported thereafter in the near15 and mid-infrared12 range, while spectral dependence in other regions remains experimentally unexplored. It has also been shown in graphene oxide, that the nonlinear parameters of the system change when excited with nanosecond and picosecond pulses26. The decrease of n2 with increasing incident intensity due to saturation effects has been observed and reported previously in several studies27. Raman studies have shown p-type doping occurs when chemical vapour deposition (CVD) graphene is transferred onto quartz24. Considering these factors, it is more apt to consider the measured nonlinear parameters in conjunction with the experimental conditions. This makes it imperative to understand the dependence of these factors to gain a fundamental understanding of the governing processes.The experimental studies probing the Kerr-nonlinearity in graphene have shown variation in neff2,) of graphene in hundreds of femtosecond regime using our pump-probe integrated Z-scan (PPZS) set-up. We refer to the nonlinear refractive index (n2) as neff2, because we find that the nonlinearity in graphene comprises of the conventional Kerr-type nonlinearity and a component stemming from graphene’s unique structure. In addition to that, our set-up allowed for temporal measurements, unravelling the evolution of the nonlinearity in a pump-probe style measurement to reveal an ultrafast nonlinearity. Thus, this study provides a foundation for the development of predictable models imperative for device design, allowing for large controllable tunability of the nonlinear response of graphene.In this study we report a systematic analysis of the near-infrared spectral dependence, the sub-picosecond temporal evolution and pulse-width dependence of the effective Kerr coefficient  configuration, while the Open Aperture (OA) configuration captures the effect of absorption. This technique was modified by Wang et al.29 to extract the temporal evolution through the integration of a secondary time delayed beam to obtain the time-resolved Z-scan measurement. The PPZS set-up is schematically illustrated and further elucidated upon in the Methods and Materials section. The set-up can be operated in multiple modes of measurement. In the single-beam mode, the set-up is a standard Z-scan measurement. In dual-beam mode, with the addition of a lock-in amplifier, chopper and cross-polarisation filtering, the set-up can be used to perform a standard pump-probe measurement for the extraction of relaxation time constants. When used in conjunction with the Z-scan components, the dual-beam mode is used for the time-resolved Z-scan measurements.Originally introduced by Sheik-Bahae 2) were characterised by Raman spectroscopy, shown in Fig. −1, 2661 cm−1 and 1328 cm−1, respectively. The relative intensities and widths of the G and 2D peaks confirm that the sample is single layer. We also perform a temporal correlation measurement on the sample, shown in Fig. τ1 relaxation time constant of about ~113  fs. The methodologies and results are further discussed in the Methods and Materials section.In order to verify the quality and monolayer nature of the samples, the CVD fabricated graphene on quartz (GoQ) samples . Since the measurement is sensitive to the beam quality, the beam is characterised using a knife edge beam profiler for quality and stability over the period of the measurement. To ensure that the Z-scan modulation observed in the far-field is originating from graphene, a bare quartz substrate is measured at 900 nm at high on-axis irradiance to show no effect  as the correlated pulses are of similar pulse durations. This uncertainty is accounted for in our theoretical model and is illustrated by the shaded (yellow) region in Fig. neff2, value that is lower as compared to longer wavelengths, with approximately the same on-axis irradiance. Our calculation suggests a quadratic dependence of neff2, on λ which agrees with the experimental results. It is noted that neff2, exhibits negligible dependence on the Fermi level for a low-doped graphene monolayer. In our case neff2, is dominated by contributions from interband transitions and since the λ2 dependence is a direct consequence of the linear band diagram around the Dirac point, the most relevant transition occur at the zero detuning region where ω = 2|k|vF. Detuning is defined as ω is the frequency of the excitation photon and k. The phenomenological relaxation coefficients, γ1/2, account for the collective broadening effects for the population and coherence decay, respectively. The theoretical derivation is succinctly provided in the Methods and Materials section. Under our theory, the electromagnetic coupling for normal illumination is defined by E is the electric field and the unit vector The cumulative results of the spectral dependence are presented in Fig. μ ≪ ℏω), absorption bleaching due to optically induced Pauli-blocking plays the leading role. This assumption is supported by observation of the absorption drops upon high optical irradiance, refer to the OA trace in Fig. steady state population, ω is then given by JNL is the microscopic origin of the intensity dependence of the refractive index so that n2 ∝ |JNL|/|E|3. For a small enough γ2, the Kerr-type nonlinear induced current is given by:gs and gv are the spin and valley degeneracy factors, respectively, D = 1/4π2 is the density of states, and β~π is a dimensionless quantity that arises from angular integration around the Dirac cone. Due to low doping, k2 term appearing in Eq. k = 0 circle the Bloch wave-number k is linearly proportional to the frequency which in turn yields λ2 dependence of the Kerr nonlinear coefficient.The nonlinear optical absorption has multiple origins namely bleaching effects culminating in saturation, and two photon absorption. Since the graphene sample used in our experiment is low-doped  Z-scan measurement is performed to locate the peak and valley positions of the sample. In order to locate the zero-delay position of the probe, the sample (GoQ) is placed at the peak position and the probe is scanned until a cross-correlation signal of the pump and probe pulses is obtained, shown in the inset of Fig. τ1, while at longer time scales, τ2, ΔTpv shows no discernible variation. The symmetric shape of the figure leads us to believe that in this measurement we are simply observing the probe following the pump signals rather than unravelling the nonlinear phenomena hidden at shorter time scales. This is also supported by the fact that the pulse duration of the pump and probe is similar to the measured temporal cross-correlation extracted in Fig. TP−V or the induced phase shift ΔΦ seen in Fig. 2) at the centre peak of the pulse. Therefore, according to neff2, for the peak power is extracted to be 1.12 × 10−8 cm2/W. As the intensity decreases moving away from the peak, the neff2, increasing in accordance with saturation. This measurement clearly shows that on the time scale greater than the heating and cooling times, the probe follows the pump and Kerr nonlinearity is tunable and controllable over this time scale. In this context, the heating and cooling times refers to the time-scale of carrier excitation to higher states and subsequent carrier relaxation to the ground state, respectively30. The modulation is understood through the relation n = n0 + Δn, where Δn = neff2,I, and the Δn parameter is modified as the neff2, varies nonlinearly on the intensity prior to complete saturation5.The temporal evolution of the nonlinearity is shown in Fig. n term, by changing the pulse properties, the switch can be controlled. However, there is saturation that takes place at relatively high intensities, leading to a deviation between the pump power within the pulse and the observed Δn.For the purposes of all-optical switching the on/off time of the nonlinearity is controlled by the pulse-duration and power of pulse. If the pulse-duration is longer than the effective relaxation times of the nonlinearity, then the evolution of the observed nonlinearity simply follows the Gaussian power distribution of the pulse. In this way through modulation of the Δτ1. Pulse stretching is achieved using a dispersion-based prism-pair apparatus, where the pulse can be temporally expanded by varying the separation distance of prisms31. A schematic of the set-up is shown in Fig. It is well understood that the relative timescales of the excitation pulse and the system response times determine the induced dynamics in the system. To investigate the effects of pulse duration on nonlinear refraction, we stretch the ~100 fs pulse upto ~475  fs at 900 nm to observe the effects in the long pulse regime; longer than the duration of the nonlinearity or relaxation time, neff2, on the excitation pulse duration, with neff2, becoming larger with increasing pulse-width. This trend was theoretically speculated by Vermeulen et al.10 and shown here experimentally. The neff2, ranges from 1.02 × 10−8 to 1.7 × 10−8 cm2/W in this pulse duration regime with a minimum between 200–300 fs. The decay constant τ1~113 fs measured in Fig. p- and n-doping making the time constants long and shorter, respectively32. This data trend coincides with previously reported Z-scan studies on graphene performed with picosecond excitation where the neff2, is larger than what is reported in the femtosecond regime. In addition to this, a similar comparative analysis performed on carbon disulphide (CS2), reference material used for calibration of Z-scan measurements, revealed a similar dependence of the neff2, on pulse duration33. In a recent publication10, this pulse-width dependence of neff2, is theoretically derived for a regime when the effective decay constant is larger than the pulse duration in Z-scan measurements. The interplay of relative carrier heating and cooling times is said to induce a nonlinear response that may not originate only from the conventional electronic Kerr-type nonlinearity but also from what they refer to as saturable photoexcited-carrier refraction (SPCR). As mentioned previously, the nonlinearity originates from the carrier population difference. Given a pulse of an arbitrary pulse duration that impinges on the system, the maximum excited carrier density is reached at the peak of the pulse. If the pulse duration is shorter than the system’s relaxation constant i.e., τ1~113 fs, then the maximum carrier density is reached on the time-scale of the pulse duration, the carriers then remain in the excited state even after the pulse passes, subsequently relaxing on a time-scale of τ1. However, if the pulse duration is longer than τ1 then carriers continuously undergo excitation and relaxation as the pulse passes with the carrier population difference continuously evolving, and therefore the optical nonlinearity becomes dependent on the duration of the pulse. The saturability in graphene deviates from saturability in other 2D materials due to the presence of its unique gapless band structure which facilitates spontaneous saturation near the Dirac point even when there is no field23. Therefore, it is apt that we refer to the Kerr-type nonlinearity characterised using the Z-scan method as neff2, due to the presence of this contributing mechanism.The results show a clear dependence of neff2, on the spectral and temporal properties of the pulse, has allowed us to gain a more fundamental understanding of the parameters governing the observed nonlinear optical effect in graphene. This sheds significant light on the widespread debate in the field regarding the large variation in the neff2, value for graphene due to varying experimental conditions and sample preparation techniques. The dependence of neff2, on the exciting wavelength revealed a quadratic  relationship observed experimentally and confirmed theoretically. The quantum theory developed can be used to calculate the nonlinear response under varying the relaxation coefficients and Fermi energy, and the results can be extended to other spectral regions. The time-resolved Z-scan measurement revealed that the heating and cooling dynamics within our graphene sample are simply too fast to be probed with ~100 fs pulses using this method. However, this method reveals a practical application in all-optical ultrafast switching of the nonlinearity, completely controlled by the pulse-duration and power of the impinging laser pulse.The systematic measurement and analysis of the dependence of neff2, and the laser pulse-duration in the hundreds of femtosecond regime, with neff2, growing larger with longer pulse-duration. Our results also coincide with the trend that is observed over several papers14 performing the measurement with different pulse durations, further reconciling our measurements, theory and experiments. In our measurements we used a prism-pair set-up to expand the pulses which is highly sensitive to beam alignment, posing a challenge if the pulse duration needs to be changed dynamically. Alternatively, transmission diffraction gratings can provide large pulse expansion ratios with high efficiencies and easier alignment34. Using the diffraction gratings a larger range of pulse durations can be scanned and a decisive relationship between neff2, and pulse duration can be established.Through the pulse-duration dependent measurement, we were able to confirm the theoretically predicted relationship between neff2, remains positive. The sign of neff2, has been a constant point of disagreement in literature, with the sign alternating from positive to negative depending on the method of characterisation employed or the conditions of the experiment. Theoretical studies have revealed that neff2, is very sensitive to the Fermi energy of the sample22. Fermi energy modulation can be practically achieved with ease by electrically gating graphene samples in contrast to changing the spectral and temporal properties of the pulse to achieve the same tuning. Therefore, it becomes absolutely critical to perform an experimental characterisation of the dependence of neff2, on Fermi energy. By scanning this parameter and observing the nonlinear response, it is possible to observe if or when the change in sign takes place, which would resolve the supposedly contradictory reports. With this study we have gained critical fundamental understanding of the underlying processes governing the nonlinear optical phenomena and the effect of experimental parameters which can be used to largely tune the effect.Throughout all our experiments, the value for λ/2 waveplate (λ/2), polarising beam-splitter (P-BS) and polariser (Pol1) before the sample. An analyser polariser (Pol2) placed after the sample is rotated to achieve extinction of the pump beam.The experimental set-up for this measurement is illustrated in Fig. x is the Rayleigh length (zR) normalised position (z/zR) and ΔΦ0 is the nonlinearity induced phase shift. When a pulsed source is used with pulse-width comparable to the duration of the nonlineairty, the nonlinear refractive index is extracted via k0 is the wave vector, I0 is the on-axis irradiance and Leff is the effective length of the sample35. In our analysis the value for ΔΦ0 is extracted using a nonlinear least-squares curve fitting routine in MATLAB. The routine takes in the aforementioned function along with a starting value for ΔΦ0, and a vector constraining the upper and lower bound for the value. The routine then works to find a minimum for the sum of squares of the input function in an iterative manner, with the allowed number of iterations being easily modified. In our measurements the value for ΔΦ0 falls in the range of , which was the upper and lower bound input, while a starting point of 0 with 5000 iterations generally allowed for convergence.The normalised CA transmittance is fitted to the equation: 2) were characterised by Raman spectroscopy, shown in Fig. −1, 1576 cm−1 and 2661 cm−1 respectively. The D band signifies disorder in the graphene lattice and is typically weak in high quality graphene, as seen in our sample measurement. A point to note as it pertains to the D band is that it is a resonant band and has very weak modes underlying it. The shape and position of this band can vary significantly depending on the excitation laser wavelength. The G band is a signature of an in-plane vibrational mode stemming from the stretching of the C-C bond, characteristic to all sp2 carbon systems. The 2D band is the second overtone of the D band and is the result of a two-phonon lattice vibrational process. It is always strong in graphene and does not represent defects regardless of the presence of the D band. The peak at ~2460  cm−1 has been referred to as the G* band and is attributed to a double resonance36. The peak appearing at ~2325  cm−1 may arise from additional defects or resonances. The relative intensities of the 2D and G peaks confirm that the sample is single layer.In order to verify the quality of the samples, graphene on quartz (GoQ) samples , to obtain a τ1 relaxation time constant of about ~113 fs.We also perform a temporal correlation measurement on the sample, shown in Fig. τ, k′} = {t, k − δk(t)}, where δk obeys The nonlinear response coefficients have been calculated via Semiconductor Bloch Equations (SBEs). In the continuous excitation regime, the optical response can be estimated based on the dynamics of (i) the microscopic population difference 23.Since the crystalline structure of graphene is centrosymmetric, even order nonlinearities are prohibited and therefore the first nonlinear term is the third order. We describe the effect of this third order nonlinearity by considering three complex fields with the time dependence of The conductivity tensors for the six distinct photon processes contributing to the third order optical effect in graphene are presented in the supplemental document. The leading term (purely interband term) is:dgr is the equivalent thickness of graphene which is typically around d ≈ 0.3 nm3 and ε0 is the free space permittivity.In the case of graphene, the definition of the nonlinear bulk susceptibility is ambiguous due to the arbitrariness in the definition of the thickness of the two-dimensional structure. The Kerr coefficient is defined through the intensity dependent refractive index i.e. n = n0 + (n2 − ik2)I where I = 2ε0Re{n0}c|E|2 (c is the speed of light). The complex nonlinear coefficient n2−ik2 is related to the bulk susceptibility χ(3) as3:The equivalent third order bulk susceptibility of graphene is related to the third order surface dynamic conductivity via:n0 are both normalized to the thickness dgr, the Kerr coefficient is less sensitive to a particular selection for the thickness of the 2D material. The estimated values for the Kerr coefficient of graphene and the expected spectral dependence are presented in supporting information document.Since bulk susceptibility and the linear refractive index Supporting Information - Experimental Characterization of Ultrafast, Tunable and Broadband Optical Kerr Nonlinearity in Graphene


Topographic corrugations, such as wrinkles, are known to introduce diverse physical phenomena that can significantly modify the electrical, optical and chemical properties of two-dimensional materials. This range of assets can be expanded even further when the crystal lattices of the walls of the wrinkle are aligned and form a superlattice, thereby creating a high aspect ratio analogue of a twisted bilayer or multilayer – the so-called twisted wrinkle. Here we present an experimental proof that such twisted wrinkles exist in graphene monolayers on the scale of several micrometres. Combining atomic force microscopy and Raman spectral mapping using a wide range of visible excitation energies, we show that the wrinkles are extremely narrow and their Raman spectra exhibit all the characteristic features of twisted bilayer or multilayer graphene. In light of a recent breakthrough – the superconductivity of a magic-angle graphene bilayer, the collapsed wrinkles represent naturally occurring systems with tuneable collective regimes. These phenomena can be observed, because the layer orientation differs from the perfectly oriented AA or AB stacking, and a superlattice structure is formed2. At specific orientations (magic angles), the graphene bilayer exhibits ultra-flat bands near charge neutrality, which upon doping results in zero-resistance states with a critical temperature Tc up to 1.7 K1.A twist between the individual layers of a graphene bilayer enables the observation of new phenomena, such as superconductivity or Mott insulating states, which are unknown for single layer graphene3, folding a graphene monolayer using an atomic force microscope (AFM)5 or even directly by growing twisted bilayer graphene using CVD6. Twisted bilayer graphene with the misorientation angle in a suitable range can be conveniently identified using Raman spectroscopy, as superlattice-activated Raman processes give rise to two new Raman modes: RTO and RLO8, where the subscripts refer to the respective phonon branches. The RTO and RLO modes originate from the phonons with a wave vector q in the interior from the Brillouin zone. The activated wave vector is dependent on the actual misfit angle θ; therefore, the misfit angle can be determined from the measured Raman shift of the RTO and RLO modes. The same Raman features have been found also in systems with more than two twisted graphene layers10. On the other hand, the reports on twisted bi- or multilayer graphene systems have been so far limited to planar structures, with the notable exception of graphene nanoscrolls11.Twisted bilayers can be created by stacking exfoliated or chemical vapour deposition (CVD)-grown graphene monolayers12. It was theoretically shown that if the wrinkle height is sufficiently large, the wrinkle can collapse13 in such a way that the distance between the walls of the wrinkle becomes small enough to effectively form a bilayer17. If the height of such a wrinkle does not reach approximately 8 nm18, the collapsed wrinkle can remain standing. However, after exceeding the critical height, the wrinkle bends towards the surface and is termed ‘folded collapsed wrinkle’14. In such a case, depending on the number of folds, the resulting structure will be composed of 2n + 1 layers (n > 0). Thus, in some cases, it can be expected that both walls of the graphene wrinkle will be oriented in such a way that they form a collapsed wrinkle consisting of a twisted bilayer, if standing, which we call a ‘twisted bilayer wrinkle’, or twisted multilayer wrinkle, if folded. That said, the appearance of twisted wrinkles may not be as rare as could be expected17. Wrinkles are common to transferred CVD-grown graphene and usually form during the cooling phase as a result of a thermal expansion coefficient mismatch between the metal catalyst and graphene19. These out-of-plane corrugations usually manifest as long and narrow wrinkles spaced by several micrometres (depending on the particular graphene growth and transfer procedures)13. The adhesion of the graphene is high on the standard substrate (Si/SiO2)20, and thus the compressive stress is partially retained at the interface, and only a part of the layer is relaxed into the wrinkles. However, if such a substrate presents a large number of instabilities (such as height topography corrugations/features), then the probability of the appearance of wrinkles will be higher22.Out-of-plane corrugations are ubiquitous in graphene due to their low bending stiffness, resulting in various shapes and dimensions: from (sub)nanometre-sized, low aspect ratio ripples to long, high aspect ratio wrinkles13, optical23 and chemical properties24 of corrugated graphene compared to flat graphene25. Furthermore, exotic states (such as snake states) are expected to be observable in these thin wrinkles26. Wrinkled graphene also exhibits interesting mechanical properties, e.g., an increased bending stiffness of standing collapsed wrinkles27 or an improved interfacial shear stress transfer at the graphene/polymer interface when the graphene wrinkles are fully supported by the polymer28.The selective formation of wrinkles with a specific geometry has a promising impact on future applications, as the morphology of graphene itself is a factor which plays an important role in its actual band structure. Graphene’s effective gauge field follows the local curvature of the topographical networks of rugae and can change locally the electronic29. From a fundamental point of view, the twisted wrinkles represent a perfect model system for the observation of various collective phenomena, such as superconductivity or Mott insulating regimes.The prospect of combining the electronic, optical and chemical properties of wrinkles and twisted bilayer graphene is very appealing from the perspective of technical realisation, as both outward sides of such a vertical graphene bilayer are easily accessible for creating contacts or for chemical functionalisation. The twisted bilayer and multilayer wrinkles thus have interesting applications in optoelectronics due to their high aspect ratio (length/width) and relative ease of patterning, e.g., by modifying the protocol presented in ref.TO and RLO modes, and the excitation energy dependence of the giant enhancement of the G mode.In the present letter, we demonstrate that the ‘twisted wrinkles’ do indeed exist, proving them via the synergy of atomic force microscopy (AFM) and Raman micro-spectroscopy studies. The AFM measurements showed topographic features characteristic of both narrow standing wrinkles and broad folded ones. Indisputable evidence of the presence of twisted wrinkles was generated by the combination of Raman spectroscopic mapping and Raman spectroscopy co-localised with AFM using a range of visible excitation energies which exhibit the characteristic properties of twisted bilayer graphene, such as the R30. These wrinkles typically exhibit a fine structure, such as shown in Fig. 15. Furthermore, Fig. ca. 3-μm long and 200-nm wide, ended with a perpendicular crack; then, the wrinkle continued only as a 1-μm long, narrow, high wrinkle , labelled as region ‘1’. Another crack perpendicular to the wrinkle propagation direction again caused a change in its morphology, and the wrinkle propagated further to the right as a broad wrinkle (~500 nm width) with a higher narrow profile superimposed at its bottom edge (region ‘2’).Monolayer graphene transferred to the nanopillar arrays showed a large number of wrinkles that could be easily located optically Fig.  thanks td, is only 0.34 nm for the AB-stacked bilayer graphene. Even though it has been shown recently that the equilibrium interlayer distance is dependent on the misfit angle and can be as large as 0.6 nm31, it is impossible to resolve the real width of the wrinkle in AFM topography even with a very sharp tip. High resolution surface potential measurement can provide clues to distinguish standing and folded collapsed wrinkles on a flat support due to doping effects from the substrate18, however, the very same effect could make the interpretation of Raman spectra more difficult, see below.The analysis of the selected cross-sections of the thin wrinkle Fig.  demonstr32. Moreover, the FWHM of the 2D mode is larger in comparison with the 2D mode in the reference spectrum; and, finally, the features of a twisted bilayer, the RLO and RTO modes, appear  interaction of the two layers when they form a bilayer, as observed previously on a large set of twisted bilayers with downshift of ~1–3 cm−1 6. We can conclude that the narrow wrinkle indeed exhibits the properties of a twisted bilayer (multilayer) and can henceforth be termed ‘twisted bilayer (multilayer) wrinkle’.To clarify the nature of the observed wrinkles Fig. , we measTO and RLO modes.The spatial dependence of the Raman spectra of the twisted wrinkle was studied in detail using a laser energy of 2.33 eV ), where a is the graphene lattice parameter34. Hence it is obvious that even the height of the standing collapsed wrinkle allows for the creation of a superlattice.The misfit angle VHS) and the resonance window width (γ) can be obtained from formula32:ℏωG is the energy of the G phonon, and M is a constant that encompasses the product of the matrix elements for electron-photon and electron-phonon interactions. The inset in Fig. EVHS was approximately 1.64 eV, γ was 0.14 eV and M was 0.20 eV. EVHS can then be related to the mismatch rotation angle θ using Elmax = E0|sin(3θ)|32, where E0 = 3.9 eV. When Elmax = 1.64 eV, we find that θ is approximately 14°. This agrees well with the value of θ derived from the position of the RTO mode. We also noted that the giant enhancement of the G mode correlating with the appearance and shifts of the RTO and RLO modes rules out the interpretation of the increased G intensity as a simple addition of graphene layers in the collapsed wrinkle, as proposed previously by Zhu et al.13. If this were the case, the G band intensity would be roughly multiplied by a factor of three  and not by a factor of ~20 or more  inside the laser spot with radius r, centred at  is given by the following equation36:Finally, to determine whether the G band enhancement originated from standing or folded collapsed twisted wrinkles, we estimated the relative contribution of such a wrinkle to the spectrum in the Gaussian-shaped Raman laser spot. The intensity, 15 and corresponding 100-fold G band intensity enhancement37. The laser spot was then divided to 7 × 7 nm2 squares, and the Gaussian intensity calculated for each square through Eq. SLG)) for a standing collapsed wrinkle of ~2.5. No changes in 2D band intensity would be visible in such a case. To achieve the highest G band enhancement observed in this work (50x), the width of the wrinkle running through the middle of the spot must be at least ~200 nm if singly folded according to the calculation above. The observed wrinkle morphologies as well as G band enhancement span the whole range between the borderline cases above. Indeed, the wrinkles with the narrowest AFM profile, i.e., most likely the standing collapsed ones, largely invisible in optical micrographs, show only a minor G band enhancement  of 7 nment Fig. ; while t2 pillar array, where the wrinkle walls were aligned with respect to each other. The mutual alignment of the walls creates a graphene superlattice structure, which results in the appearance of the RLO and RTO modes and a laser excitation dependence of the enhancement of the G mode. Furthermore, we found that the mismatch rotation angle slightly varied along the wrinkle owing to the alignment of the wrinkle with respect to the orientation of the graphene domain(s). Both standing and folded wrinkles can be found and discriminated based on their width and G band enhancement factors.In conclusion, we have demonstrated the existence of twisted bi- or multilayer wrinkles on CVD-grown graphene transferred to a Si/SiO39 of these materials, by the deposition of 2D materials on predefined substrates21, or by deposition of electrodes with a small gap on top of the graphene layer40. Furthermore, the interaction between the sidewalls of the wrinkle can be controlled by temperature. The compressive stress, caused by the change of temperature and the negative thermal expansion coefficient of graphene, results in a progressive closing of the wrinkle walls and thus decrease of the interlayer distance17. The possibility of fine tuning the level of interaction between two misoriented layers by changing d has been shown recently41. Finally, the natural superlattices of the wrinkled graphene are promising candidates for facile experimental realisation of high-purity magic angle bilayers, which exhibit tuneable collective states at low temperatures.The existence of these twisted wrinkles is important as it presents the opportunity to control the optical and electronic properties of wrinkles in graphene and other 2D materials. For the creation of these well-defined wrinkles, no complex lithographic processes are needed. The formation of wrinkles with a well-defined length/width ratio can be controlled simply via uniaxial compression42. In brief, the polycrystalline copper foil was heated to 1000 °C and annealed for 20 min under a flow of 50 standard cubic centimetres per minute (sccm) H2. The copper foil was exposed to 30 sccm CH4 and 50 sccm H2 for 20 min, whereafter the copper foil was cooled to room temperature. The as-grown graphene was subsequently transferred onto a Si/SiO2 nanopillar array, with a pillar-to-pillar spacing of 240 nm and a pillar top diameter of 54 nm21, using nitrocellulose (NC), and residual NC was removed from the graphene by annealing the sample at 180 °C for 30 min in air. Further details are given in the Supporting Information  laser excitation, a laser power of approximately 1 mW, a grating of 600 lines/mm and lateral steps of 50 nm in both directions. The laser was focused on the sample with a 100x objective.+–Kr+  lasers. The spectrometer was interfaced to a microscope (Olympus) equipped with a piezo stage, and a line scan was measured through the wrinkle with a 50-nm step size. The intensity response of the CCD detector was calibrated with a tungsten halogen light source  for each excitation energy. The laser was focused on the sample using a 100x objective, and for all wavelengths, a laser power of approximately 1 mW was used.The excitation energy-dependent Raman spectra were acquired with a LabRam HR spectrometer (Horiba Jobin–Yvon) using He–Ne [1.96 eV (633 nm)] or Ar0 = 70 kHz, nominal tip radius = 2 nm), and the captured topography was processed using the Gwyddion software. Moreover, the co-localised AFM–Raman measurement was performed using the Horiba-AIST-NT Omegascope system (with a LabRAM HR Evolution Raman spectrometer), first capturing the AFM image with the sharp RFESP-300  probe by Bruker, followed by the recording of the high-resolution Raman map of the same area using the 532-nm laser excitation wavelength.AFM was measured in the tapping mode with a Dimension Icon microscope (Bruker Inc.). Images were captured using Bruker SCANASYST-AIR probes (k = 0.4 N/m, fSupplementary Information


An important component of change of direction speed is the ability to decelerate. Objective methods to examine this quality have been rarely reported in the literature. The aim of this study was to investigate the within- and between-session reliability (intraclass correlation coefficients (ICC), coefficient of variation (CV), standard error of measurement (SEM) and smallest detectable difference (SDD)) of using a laser Doppler device (LAVEG—LAser VElocity Guard) to quantify deceleration ability in 20 amateur rugby union players. Each player performed one familiarisation and two experimental sessions (seven days apart) consisting of three maximal 15 m sprints from a standing start, with an immediate deceleration to a complete stop upon hearing an audible cue at the 15 m mark. Deceleration was evaluated by determining the distance required to decelerate to 75%, 50%, 25% and 0% (‘stopping distance’) of the velocity achieved at 15 m of the maximal sprint. Within-session relative reliability was moderate to good (ICC = 0.64–0.83) with borderline acceptable variation (CVs = 10.51%–16.71%) across all variables. Between-session reliability reported good to excellent relative reliability (ICC = 0.79–0.93) with acceptable absolute reliability, particularly for stopping distance . The assessment shows promise as a method to quantify deceleration ability in athletes. In courerformed . Whereasectively . Furtherectively  and rugbectively  has beentch play .Despite the importance of deceleration in sport, until recently there has been limited attention in the literature to evaluate this quality. Biomechanical studies have examined deceleration as a precursor to a change of direction manoeuvre, identifying the presence of distinct kinematic and kinetic differences between the ‘final’ plant step and steps preceding the change of direction manoeuvre ,7. FurthChange of direction speed or ability is defined as the ‘the ability to decelerate, reverse or change movement direction and accelerate again and is considered pre-planned’ . This quThe first reported method for assessing an athlete’s ability to decelerate from a sprint was suggested by Plisk . Plisk’s2 = 0.961) and stopping distance (R2 = 0.851), which could then be used to set realistic conditions for acceleration–deceleration drills. The use of laser Doppler devices provides an avenue to objectively quantify deceleration capabilities. However, no research has yet evaluated the reliability of previously used deceleration tests [More recently researchers have used laser Doppler technology (LAVEG— LAser VElocity Guard) to quantify stopping distances . This ston tests ,13,14 anTwenty male amateur rugby union players  who were free from injury were recruited to take part in the present study. All subjects trained and played rugby union regularly (minimum once per week) for at least one year preceding the experiment. The aims and details of the study and what participation in the study would involve were explained to each subject before completion of the written informed consent forms. Institutional  ethical approval (ethics number: HST1617-24) was granted before subject recruitment began.The reliability and sensitivity of the LAVEG  deceleration test was assessed using a repeated-measures design. Three measurements were taken on two separate occasions, seven days apart. Subjects were asked to perform three maximal, 15 m sprints from a standing start, followed by an immediate deceleration to a complete stop. This distance was chosen because it is a common, short sprint distance in rugby union [Subjects attended the testing field on three separate occasions. The first session ensured that each subject completed a familiarisation session in which the procedure was conducted before data were recorded. This was necessary due to prior pilot work, whereby a definitive learning effect was observed from session one to session two. Height was recorded to the nearest 0.01 m using a stadiometer  and body mass was measured to the nearest 0.1 kg using digital scales . Prior to each testing session, the LAVEG was placed 2 m behind the start of the 15 m sprint track and a zero-point was calibrated at the start of the 15 m sprint . At eachEach testing session included a familiarisation task of three submaximal sprint and stops of increasing intensity (50% maximum intensity/ 70% maximum intensity/ 90% maximum intensity). Subjects were then asked to perform three maximal 15 m sprints. Upon hearing the audible cue at the 15 m mark they were instructed to decelerate and stop as quickly as possible and remain in the stop position for a minimum of 3 s. Two minutes’ rest was instructed between each trial to allow a full recovery. Measures of instantaneous velocity were recorded by the LAVEG. The same investigator (J.A.) performed all measurements with the LAVEG throughout both testing sessions. During the manual operation of the LAVEG within each trial the investigator focused the crosshair of the viewing lens on the lower torso  throughout each trial.The testing process was then repeated seven days later, at the same time of day (±1 h) and under similar environmental conditions to control for circadian variation and environmental influences on performance . Each su−1) was recorded and quartiles  of this velocity were calculated for each trial. Distance relative to the 15 m mark to the point where each subject in each trial reached 75%, 50%, 25% and 0% (stopping distance) of velocity at 15 m was recorded from the “distance/velocity” trace on the LAVEG software .The LAVEG measured the positional information of each participant at 100 Hz. This information was then used to produce an instantaneous velocity profile for each subject. The data were smoothed using a 51-point moving average at intervals of 0.5 s to remove excess noise, and then extrapolated from the LAVEG software to Microsoft Excel. Velocity at 15 m (m·sPOOLED × √1 − ICC), smallest detectable difference (SDD = 1.96 × √2 × SEM) and coefficient of variation (SD/mean × 100), were determined for within session  and between sessions. ICCs were considered excellent if ICC > 0.8 [Data were collected over two sessions and statistically analysed using SPSS software . Descriptive statistics  were reported for both sessions. The data were explored for normality using a Shapiro–Wilks test. Bland–Altman plots were used to inspect for the presence of heteroscedasticity of data between sessions. Intraclass correlation coefficient’s (two-way mixed effect model with absolute agreement) were determined for within- and between-session data. Standard error of measurement (SEM = SDCC > 0.8  with minCC > 0.8 . SEM andp > 0.05) were observed between trials during session one and two for velocity at 15 m, and distances to 75%, 50%, 25% and 0% (stopping distance) of 15 m velocity. Within-session reliability was shown to be moderate to good across each testing session, with complete stopping distance (distance at 0% of 15 m velocity) shown to have excellent reliability (>0.8 ICC) in both testing sessions. Within-session CVs met minimal acceptable variation of <15% with the exception of distance to 75% and 50% of 15 m velocity in session two (The descriptive data for both sessions are reported in sion two .p > 0.05) were observed between sessions for velocity at 15 m, and distances to 75%, 50%, 25% and 0% (stopping distance) of 15 m velocity. ICCs demonstrated excellent reliability (ICC ≥ 0.79) between sessions for all variables, whilst between-session CVs met minimal acceptable variation of <15% with the exception of distance to 75% of velocity at 15 m  to determine deceleration ability from a maximal 15 m sprint. Stopping distance (0% of velocity at 15 m) demonstrated excellent within- and between-session relative reliability. Furthermore, between-session reliability was excellent for distances to achieve specific percentage of velocity decrement (ICC ≥ 0.79), confirming the study hypotheses. Within-session reliability was moderate, but good for overall stopping distance (ICC = 0.64–0.83). Reasonable between-session absolute reliability was reported; suggesting a moderate sensitivity for stopping distance (distance to achieve 0% of 15 m velocity) with relative SDD of 9.11% for between sessions. Thus, further work is needed to perhaps improve the absolute reliability further to establish a greater level of sensitivity to monitor changes in athletes as a result of interventions.−1; 30 m: +0.06 ± 0.13 m·s−1; 50 m: +0.08 ± 0.15 m·s−1). To the authors knowledge, this is the first study to use and examine the reliability of the LAVEG to assess deceleration ability. The results reveal that stopping distance from a 15 m maximal sprint has good to excellent within- and between-session reliability  and acceptable between-session absolute reliability ; SDD = 0.64 (9.11%)). Furthermore, reporting distances to specific percentages of velocity at the end of the 15 m sprint provided good relative reliability (ICC = 0.79–0.93) and acceptable absolute reliability  between sessions, but much lower absolute within-session reliability ; suggesting that overall stopping distance is the most reliable metric to quantify deceleration ability.Despite a lot of attention to methods to evaluate both change of direction speed and agility, little attention has been paid to develop methods in order to evaluate deceleration ability. Previous suggestions have ranged from counting the number of steps to stop from sprints of various intensity , and stoAlthough for most variables reported in the study the variation between trials met an acceptable level <15%) with the exception of distance to achieve 75% and 50% of velocity at 15 m in session two. In general, a large variation in all four measures was evident within each session (CV = 10.51%–16.71%) and could be largely explained by the movement variation associated with the task and the intra-operator reliability of using the LAVEG. The ability to decelerate is a complex skill that involves integration of several muscular systems, control of multiple large joints and extremely high forces across multiple planes of movement when performed maximally , as in t% with thThe LAVEG requires manual operation by the tester to track a single point on the athlete’s torso  during trials. Thus, intra-operator consistency could also account for some of the variation between trials. Further work should be carried out to explore typical inter-operator agreement  to isolate the impact of operator influence on reliability when using this protocol.−1; session 2: 6.81 ± 0.5 m·s−1 vs. velocity at 15 m session 1: 5.36 ± 0.72 m·s−1; session 2: 5.41 ± 0.66 m·s−1). Although an audible bleep was used in this study by virtue of the athletes running through a timing gate as a command to begin deceleration, athletes may still ‘anticipate’ the deceleration prior to the audible bleep as they approach the timing cells. Utilising acceleration–deceleration efforts [Only one previous study  has used efforts  may avoi efforts  and expl efforts ,13. ThisThe findings of the present study suggest that using laser devices such as the LAVEG is a reliable method to quantify stopping distance from a 15 m sprint task and offers a solution to quantify deceleration ability in team sport athletes. The results further show that ‘stopping distance’ has a greater level of relative and absolute reliability than determining deceleration distances to certain percentage velocity decrements and, thus, should be the sole metric to determine deceleration ability. Based on the reported data, changes in stopping distance of >0.64 m (9.11%) identify meaningful changes in response to training or competition for amateur rugby players. Future research is needed to explore the use of alternative versions of the test  and compare the agreement of measuring ‘stopping distance’ from the LAVEG against video methods previously used.


In this study, a ternary nanocomposite comprising graphene oxide and carbon loaded with zero‐valent iron nanoparticles was developed as a promising nanoadsorbent, especially for polyphenols available in food industry by‐products. The fabricated nanoadsorbents were characterized in terms of structural, morphological, and chemical attributes. Zero‐valent iron nanoparticles (nZVI) were produced by a modified method leading to the formation of nanoparticles below 50 nm. Also, active carbon was transformed to a needle‐like shape instead of its native shape so that the active surface area was drastically increased which favors the higher adsorption process. Moreover, the space between graphene oxide sheets was enhanced by ultrasonication so that more active carbon and nZVIs could be oriented between these sheets. Finally, the FTIR and Raman data demonstrated the formation of O‐H stretching groups and a D/G value of 0.85 corresponding to the maintenance of a desired structure of the graphene oxide sheets, respectively. To summarize, the developed nanocomposites can be employed as a promising tool for the adsorbance of food and beverage industry by‐products, especially polyphenols. The aforementioned features make AC a promising material for the adsorption of several grafting agents, such as phenolic compounds , ferrous sulfate (FeSO4.7H2O), potassium permanganate (KMnO4), sodium nitrate (NaNO3), hydrogen peroxide H2O2 (30 wt%), polyvinylpyrrolidone (PVP), sulfuric acid , hydrochloric acid , and ethanol (absolute) were of all analytical grade and prepared from Merck Company. Milli‐Q water was implemented in the entire production stages.Graphite, active carbon, NaBH2.24.7 H2O) 0.05 M and sodium borohydrate 0.2 M. Initially, 0.05 M of ferrous sulfate (FeSO4.7 H2O) was placed in a three‐necked flask which had an inlet for N2 gas and an outlet for H2 from the middle outlet of the flask. NaBH4 (0.2 M) was suddenly added to the middle of the flask with short intervals (5–15 s) and as the extra content of the hydrogen gas is released during the interaction, the solution was stirred at 400 rpm. After 1 hr, the black‐colored solution was centrifuged and rinsed for three times with double‐distilled water and alcohol in the ratio of 1:1 and centrifuged again. Finally, the yield was dried by N2 gas and kept in a vacuum desiccator.nZVIs were prepared from the mixture of ferrous sulfate . Graphene oxide in deionized water was dispersed in an ultrasonic bath during 3 hr and added to active carbon powder. In the next step, PVP 0.1716 g) and ferrous sulfate were added to the solution under nitrogen gas and stirred for 3 hr. Sodium borohydrate (2.5 g/50 ml) was added dropwise into the solution, and the stirring process continued for 5 hr. According to Equation  was employed to detect the FTIR spectra of the samples. Accordingly, KBr pellets and samples were dried in a vacuum oven at 353 K for 12 hr to reduce the impact of moisture on the spectrum, and the FTIR spectrum was scanned in transition mode from 400 to 4,000 cm−1 range by a Bruker Optics model Senterra, employing 532‐nm wavelength incident laser light and 20 mW power. The high throughput of this instrument permitted the use of very low energy densities at the sample.Raman spectra were obtained in the 600–3,500 cm2.6Field emission scanning electron microscopy  operating at 15 kV was implemented to view the morphology of the nanocomposite plus energy‐dispersive X‐ray spectra (EDS) as well as elemental mapping.2.7The surface and height profile of samples were analyzed by atomic force microscopy  after placing a drop of the sample onto a surface of mica in tapping mode.33.13.1.1The generated ZVI nanoparticles were examined by FE‐SEM Figure a and b. 3.1.2Active carbon was analyzed using the FE‐SEM device, and results are shown in Figure 3O4/activated carbon nanocomposites for the treatment of wastewater, the developed nanocomposites represented rod‐shaped active carbons with a high surface area, and the Fe3O4 particles were dispersed uniformly on the active carbon surface which approves the results of this study. Moreover, Liu et al.  as shown in Figure 300‐G600), and an aggregated cluster form of nZVI was observed, and graphene sheets were not exposed, whereas in the conditions that active carbon had a greater concentration than graphene , the structure of graphene oxide and the rod‐shaped active carbon was clearly visible. However, in the samples which had lower extent of graphene oxide, more active carbon and nZVI structures were detected due to the considerable spaces between the graphene oxide sheets. By comparing the FE‐SEM images of the treatments at equal concentrations of graphene oxide and active carbon , an intermediate architecture between the mentioned arrangements was formed. Accordingly, Li, Pan, Nie, Liu, and Sun , the observed ordered structure is homologous to the SEM image. The AFM image of graphene oxide  stretching vibration  and C‐O   stretching that was absent in graphite and detected in GO structure  Accordingly, active carbon plus iron nanoparticles were clearly observed as graphene oxide level was reduced. When GO concentration was higher than active carbon, nZVI was observed in the form of clusters and GO sheets. Also, active carbon was not observable as shown in Figure 300‐G600) which is in line with the relevant AFM image illustrating the ordered and intact nZVIs (Figure As seen in the FE‐SEM images, the amount of zero‐valent iron should be reduced with respect to the reduction rate in graphene oxide. Also, in samples containing higher levels of active carbon in comparison to graphene oxide, it is expected that the iron level is between the two other samples, while the iron level was significantly increased. Furthermore, graphene oxide sheets and the rod shape of active carbon was visible in FE‐SEM image (Figure s Figure d. On theIt seems necessary to analyze the composite structure in order to optimize the availability level of the iron nanoparticles. As seen in Figure Furthermore, the element mapping of C, Fe, and O is shown in Figure 4600‐G300 had the lowest GO content plus a higher nZVI content due to higher chance of the deposition of nZVI on the available surfaces. According to the studied structural and chemical attributes of the fabricated nanocomposite, it is suggested that this nanostructure can be employed as a robust compound for the nanoadsorption of different compounds, especially polyphenols found in the by‐products of food industry waste.nZVI has been employed to adsorb different types of compounds and pollutants. Active carbon and graphene oxide have also been used for adsorption and purification purposes. In this study, a combination of these materials was implemented to synthesize a nanocomposite/nanoadsorbent, and the structural along with the chemical characteristics of the fabricated nanocomposites was explored. Our results demonstrated that the preparation method leads to the formation of needle‐shaped active carbon bodies which enhances the available surface area for reactions. Moreover, with the help of ultrasonication, the space between graphene oxide sheets was enhanced which in turn accommodates more active carbon and nZVIs that can be used for adsorption processes. Also, the concentration of graphene oxide had an increasing effect on the deposition process and leads to the formation of homogeneous particle sizes. By investigating the structural properties via FTIR and Raman spectra, the formation of O‐H stretching groups plus the D and G bands showed that the structure of graphene oxide sheets maintained their stable structure. Overall, among the entire samples, sample CAll authors declare that there is no conflict of interest.There was no human or animal testing in this study.


This work suggests potential application of the PANI NF/graphene cofiller system for DLP-type 3D printing.Three-dimensional printing based on the digital light processing (DLP) method offers solution processability, fast printing time, and high-quality printing through selective light curing of photopolymers. This research relates to a method of dispersing polyaniline nanofibers (PANI NFs) and graphene sheets in a polyacrylate resin solution for optimizing the conductive solution suitable for DLP-type 3D printing. Dispersion and morphology of the samples with different filler contents were investigated by field emission scanning electron microscope (FE-SEM) and optical microscope (OM) analyses. The polyacrylate composite solution employing the PANI NFs and graphene was printed well with various shapes and sizes through the 3D printing of DLP technology. In addition, the electrical properties of the printed sculptures have been investigated using a 4-point probe measurement system. The printed sculpture containing the PANI NFs and graphene sheets exhibited electrical conductivity (4.00 × 10 Of the various technologies that can contribute to the Fourth Industrial Revolution, 3D printing is becoming a future-oriented manufacturing process because it can produce customized products of desired design, shape and size. In particular, 3D printing is considered to be an advanced polymer processing technology, since various polymers including polyurethane (PU), polyacrylate, polylactide (PLA), polystyrene (PS), acrylonitrile butadiene styrene (ABS) resin, and polyvinyl chloride (PVC) are used as raw materials ,2.Methods for 3D printing are divided into several categories, such as material extrusion, digital light processing (DLP), stereolithography (SLA), powder bed fusion, material jetting, binder jetting, powder jet fusion, and so forth. Material extrusion is a 3D printing process in which molten polymers are selectively dispensed through the nozzle . MedicalIf the printed material is electrically conductive, it will be possible to expand the application range of polymers for 3D printing and additive manufacturing. Various efforts have been made to fabricate 3D conductive structures using the DLP method ,6. In adConducting polymers, such as polyaniline (PANI), poly (PEDOT), polypyrrole (PPy), and polythiophene (PT), have a conjugated system structure and are therefore defined as polymeric materials capable of exhibiting electrical conductivity after appropriate doping ,9. TheseIn this work, PANI NFs and graphene sheets were introduced into a polyacrylate resin having excellent processability and applicability, and DLP-type 3D printing using the conductive polyacrylate solution was discussed. Field emission scanning electron microscope (FE-SEM) and optical microscope (OM) images were used to investigate the presence and dispersion of conductive fillers. The role of PANI NF/graphene cofiller dispersed in the polyacrylate resin was confirmed by measuring the surface resistance and electrical conductivity of 3D printed sculptures. The printed sculpture containing the optimal amount of PANI NF/graphene cofiller exhibited high printability and improved electrical properties, suggesting that the PANI NF/graphene cofiller system can provide a unique approach to design DLP-type 3D printing.−2 mol of aniline was added to 40 mL of 1M aqueous HCl solution and stirred for about 30 min. After that, 5.3 × 10−3 mol of APS was added and reacted at room temperature for 3 h to obtain emerald-colored PANI precipitates. The prepared PANI precipitates were washed with water, ethanol and acetone solvent.Aniline (99%) and ammonium persulfate  were obtained from Sigma-Aldrich . Graphene paste was obtained from MExplorer Co., Ltd. . The graphene sheet has a density of 25 mg/mL, and the average thickness and lateral size of the graphene sheet are approximately <5 nm and 2–3 μm, respectively. The polyacrylate resin solution  was obtained from Carima . Hydrochloric acid , ethanol (95%), and acetone (99%) were purchased from Daejung Chemical & Metals Co., Ltd. . The PANI NFs used in this experiment were prepared by a chemical oxidation polymerization method which was optimized from our previous work . 1.1 × 1Preparative conditions of polyacrylate solutions are shown in The 3D printer used in this experiment was a DLP-type printing system . The resin solution containing different amounts of conductive fillers was 3D printed and evaluated for printability. The maximum printable concentration of PANI NF was 5 wt % and no lamination occurred when the content of PANI NF exceeded 5 wt %. In the case of graphene, 3D printing could be achieved at a content of less than 2 wt % by weight. Based on the microscope images and the results of printability, the weight ratios of PANI NF to graphene controlled in this work were 1.0:0.06 (PG1), 1.0:0.12 (PG2), and 1.0:0.25 (PG3), respectively.ρ = (ln2/πt)(I/V), where ρ is the static resistivity, R is the surface resistance, and t is the thickness of the sample . Ele. Ele8]. e matrix ,16,17.6 times. In addition, when a proper amount (1.2 wt % with respect to polyacrylate resin solution) of graphene sheet was introduced, an additional electrical conductivity improvement of about 40 times occurred. The results mean that even after 3D printing is complete, the conductive fillers provide conductive regions within the polyacrylate resin. The results also have shown that the conductive 3D printing solution prepared by our work can be reprocessed as conductive sculptures with various designs and sizes. The sheet resistance and electrical conductivity values obtained from the results are enough to be used as conventional conductive resins for antistatic property. It is expected that the progress of DLP-type 3D printing technology using a PANI NF/graphene cofiller system and its expansion of application fields, such as electronic and biomedical devices, will be further accelerated.In this study, DLP-type 3D printing of polyacrylate composites employing PANI NF and graphene dispersion was investigated. The presences of PANI NFs and graphene were clarified using FE-SEM and OM images, and the optimal contents of PANI and graphene were 5 wt % and 1.2 wt %, respectively. The prepared NFs were highly dispersible with the polyacrylate resin, resulting in a high electrical conductivity value of about 10


The mechanism of age‐related decline in the angiogenic potential of the myocardium is not yet fully understood. Our previous report revealed that the aging of cardiac microvascular endothelial cells (CMECs) led to changes in their expression of receptor Trk isoforms: among the three isoforms , only the truncated TrkB‐T1 isoform continued to be expressed in aged CMECs, which led to decreased migration of CMECs in aging hearts. Thus far, how BDNF induces signalling through the truncated TrkB‐T1 isoform in aged CMECs remains unclear. Here, we first demonstrated that aged CMECs utilize BDNF–TrkB‐T1 signalling to recruit Willin as a downstream effector to further activate the Hippo pathway, which then promotes migration. These findings suggest that the aging process shifts the phenotype of aged CMECs that express TrkB‐T1 receptors by transducing BDNF signals via the BDNF–TrkB‐T1–Willin–Hippo pathway and that this change might be an important mechanism and therapeutic target of the dysfunctional cardiac angiogenesis observed in aged hearts. Age‐related decline in the angiogenic potential of the myocardium is common among the elderly. Furthermore, the angiogenic factor‐mediated therapeutic effect, which is potent in young individuals, is less effective in older individuals. Thus, achieving functional regeneration following MI in the elderly remains a significant challenge  play a central role in cardiac angiogenesis following MI due to their potent ability to initiate angiogenesis and to stimulate the secretion of multi‐angiogenic factors, such as vascular endothelial growth factor (VEGF) and platelet‐derived growth factor (PDGF), which act as autocrine and paracrine factors  and western blot. As 293 T cells did not express TrkB‐T1 receptor . In addition, most of the green fluorescence was located in the cell membranes in the TrkB‐T1‐VC155‐ and Willin‐VN173‐cotransfected group . Furthermore, a competitive BiFC strategy using TrkB‐T1‐FLAG‐tag and Willin‐HA‐tag as competitors for TrkB‐T1‐VC155 and Willin‐VN173, respectively, also confirmed that the fluorescence density decreased significantly when the TrkB‐T1‐FLAG‐tag expression vector or the Willin‐HA‐tag expression vector was transfected in the TrkB‐T1‐VC155‐ or Willin‐VN173‐transfected group compared with the TrkB‐T1‐VC155‐ and Willin‐VN173‐cotransfected group . This indicated that the intracellular domain of TrkB‐T1 interacts with Willin much more strongly than TrkB‐FL dose. The critical amino acids for the interaction between the intracellular domain of TrkB‐T1 and Willin were further screened by homology modelling combined with bioinformatics analysis. The homology modelling analysis predicted that the intracellular domain loop of TrkB‐T1 was included in structural pocket of the FERM domain of Willin . We engineered TrkB‐T1 and Willin fusions to VC155 and VN173 of EGFP, which are derivatives of green fluorescent protein produced when VC155‐TrkB‐T1 and VN173‐Willin interact with each other . In addition, knockdown of Willin using si‐Willin‐2095 siRNA, which was confirmed to effectively downregulate Willin , abrogated the lengthening of the migrating distance of BDNF in TrkB‐T1‐EGFP‐positive cells . This suggested that BDNF–TrkB‐T1 recruited Willin to facilitate the activity of the pseudopod. Next, we further confirmed this effect in old CMECs. We first confirmed that si‐Willin‐751 best knocked down Willin expression in old CMECs . We also demonstrated that BDNF (160 ng/ml) lengthened the migration distance of the pseudopod of CMECs compared with the BSA control . In addition, knockdown of Willin using si‐Willin‐751 in old CMECs abrogated the effect of BDNF of increasing the migrating distance . Furthermore, under the same dose of BDNF treatment (160 ng/ml), the mean migration distance of old CMECs was similar to that of TrkB‐T1‐transfected 293 T cells . The percentage of polarized cells was 81.03% ± 0.03% at 5 min and reached 90.08 ± 0.03% at 30 min after BDNF treatment. However, the percentages of polarized cells in the BSA‐treated groups at parallel time points were similar to each other . In addition, the mean diameter of stress fibres in the BDNF‐treated group was significantly larger than that of the BSA‐treated group 30 min after BDNF treatment . Together, these results suggest that the BDNF–TrkB‐T1–Willin pathway increased the polarity of actin and the polymerization of stress fibres in BDNF‐treated old CMECs.The polarity of actin and the polymerization of stress fibres are crucial for initiating mobility potential. Therefore, we further tested whether the BDNF–TrkB‐T1–Willin pathway is able to increase the polarity of actin and the polymerization of stress fibres. We performed a time‐lapse experiment combined with 2.4p < 0.05), while the differences in the migration distances after treatment with BDNF in the anti‐TrkB‐T1‐pretreated group, the si‐Willin‐751‐transfected group and the anti‐TrkB‐T1+si‐Willin‐751‐transfected group were not statistically significant compared with the respective BSA and DMEM control groups . The results revealed that the BDNF–TrkB‐T1–Willin pathway promoted the migration of old CMECs, and the increased migration effect in old CMECs after BDNF treatment was attributed to binding with TrkB‐T1 receptors, which led to the recruitment of the downstream effector, Willin.The above evidence clearly demonstrated that the BDNF–TrkB‐T1–Willin pathway promoted the migration potential of old CMECs. We therefore applied an in vitro scratch wound healing assay to investigate whether the BDNF–TrkB‐T1–Willin pathway is able to promote the migration of old CMECs. We confirmed that the migration distance of old CMECs in the BDNF‐treated group was significantly longer than that of the controls . si‐Willin‐751 transfection in old CMECs knocked down Willin, and under si‐Willin‐751 knockdown and BDNF treatment, MST1, MST2, LATS1, LATS2 and Yap were upregulated compared with the non‐Willin‐751 + non‐BDNF‐treated group . Importantly, BDNF promoted the phosphorylation of MST1/2 and LATS1/2 and the expression of Willin . These results suggest that in old CMECs, BDNF treatment upregulated and activated key effectors of the Hippo pathway via the BDNF–TrkB‐T1–Willin pathway. Furthermore, the results also suggest that as the downstream effectors of Willin, the expression levels of MST1, MST2, LATS1, LATS2 and Yap sensed the downregulation of Willin after si‐Willin‐751 treatment and subsequently upregulated themselves as a feedback response to maintain the pathway activity in vivo. In addition, Willin, MST1, MST2, LATS1, LATS2 and Yap were expressed in both young and old CMECs. At the mRNA level, the differences in the expression levels of Willin, MST1, MST2, LATS2 and Yap between young CMECs and old CMECs were not statistically significant (p > 0.05), while the expression of LATS1 in young CMECs was significantly higher than that in old CMECs . However, the western blot results confirmed that the differences in expression of Willin, MST1, LATS1 and Yap between young and Old CMECs were not statistically significant at the protein level . This suggests that old CMECs only expressing TrkB‐T1 might be a key reason why old CMECs recruit Willin to activate the Hippo pathway to transduce the BDNF signal to promote their migration.Willin is an upstream component of the Hippo signal pathway; therefore, Hippo signal molecules  . However, knockdown of Willin expression by si‐Willin‐751 delayed the dephosphorylation of Yap. The phosphorylation level of Yap in the si‐Willin‐751‐treated old CMECs was still higher at 15 min after BDNF treatment compared with the same time point of BDNF‐treated old CMECs , which suggested that the BDNF–TrkB‐T1–Willin pathway promoted the dephosphorylation of Yap in old CMECs. The Yap localization between the cytoplasm and nucleus under BDNF‐ and non‐BDNF‐treated conditions was further investigated in old CMECs. Compared with the non‐BDNF treatment condition, the level of phosphorylated Yap in the cytoplasm was decreased from 5 min to 60 min after BDNF treatment . However, when Willin was knocked down by si‐Willin‐751 in old CMECs, BDNF treatment induced a progressive increase in the level of phosphorylated Yap in the cytoplasm . In parallel, the level of dephosphorylated Yap in the nucleus of old CMECs was increased from 5 min to 60 min after BDNF treatment . Furthermore, when Willin was knocked down by si‐Willin‐751 in old CMECs, BDNF treatment for 60 min caused a decrease in the level of dephosphorylated Yap in the nucleus . These results reveal that in old CMECs, BDNF treatment for approximately 60 min induced a decrease in the level of Yap phosphorylation in the cytoplasm and an increase in the level of Yap dephosphorylation in the nucleus, while knocking down Willin expression in old CMECs abrogated the BDNF‐mediated decrease in Yap phosphorylation in the cytoplasm and increased Yap dephosphorylation in the nucleus.As the dephosphorylation of Yap and its translocation into nuclei are a hallmark of activation of the Hippo pathway  and Willin (Prey A) revealed that TrkB‐T1 recruits Willin as a downstream effector via its intracellular domain of 11 amino acids of the C‐terminus. Furthermore, the results of homology modelling analysis further revealed that the 11 C‐terminal amino acids of TrkB‐T1 (intracellular domain) are the binding domain between TrkB‐T1 and the FERM domain of Willin. All these data clearly show that TrkB‐T1 receptor is able to recruit Willin via its specific 11 C‐terminal amino acids to interact with the FERM domain of Willin, but this does not happen for TrkB‐FL, which does not have the relevant domain. In support of this finding, the FERM domain of Willin is sufficient to activate the Hippo pathway via MST1/2 and to antagonize Yap‐induced phenotypes in mammalian cells , the mean migration distance of old CMECs was similar to that of TrkB‐T1‐transfected 293 T cells  and in peripheral nerves, epithelial layers, skin, the placenta, the uterus and the cervix . Furthermore, Willin was an upstream effector of MST1, MST2, LATS1, LATS2 and Yap, and a negative‐feedback relationship existed to regulate the interactions between Willin and these Hippo pathway key effectors in vivo. As the downstream effectors of Willin, the expression levels of MST1, MST2, LATS1, LATS2 and Yap sensed the downregulation of Willin to upregulate their own expression levels as a feedback response to maintain the pathway activity in vivo.Willin/FRMD6 was first identified via a yeast two‐hybrid screen of a rat sciatic nerve library using the neuronal transmembrane protein neurofascin as bait (Gunnmoore et al., The dephosphorylation of Yap and its translocation into the nucleus are hallmarks of activation of the Hippo pathway (Zeng & Hong, We previously reported that the BDNF–TrkB pathway is able to promote the migration of young CMECs via activation of PI3K/Akt. This effect is mainly attributed to the activation of TrkB‐FL receptor as BDNF treatment significantly increases the expression of TrkB‐FL but not TrkB‐T1, though both receptor isoforms are expressed in young CMECs (Cao et al., The findings of the present study suggest a novel mechanism of the BDNF–TrkB‐T1 pathway in aged CMECs. Thus, aged CMECs utilize the TrkB‐T1 receptor to transduce BDNF signals. The BDNF–TrkB‐T1 pathway recruits Willin as a downstream effector to further upregulate key elements of the Hippo pathway. This promotes the dephosphorylation of Yap and facilitates the translocation of dephosphorylated Yap into the nucleus, which activates the Hippo pathway to promote the migration of old CMECs Figure . To our 4A detailed method is available in Supporting Information Data 4.1For details, see Supporting Information Data 4.2For details, see Supporting Information Data 4.3For details, see Supporting Information Data 4.4For details, see Supporting Information Data 4.5For details, see Supporting Information Data 4.6For details, see Supporting Information Data 4.7For details, see Supporting Information Data 4.8For details, see Supporting Information Data 4.9For details, see Supporting Information Data 4.10For details, see Supporting Information Data 4.11For details, see Supporting Information Data 4.12For details, see Supporting Information Data 4.13For details, see Supporting Information Data 4.14For details, see Supporting Information Data 4.15For details, see Supporting Information Data Z.W. and Y.C. performed most of the experiments and analysed the data; X.C. and G.X. finished the yeast two‐hybrid screening; Z.Y., H.Z. and X.Q. contributed to the discussion; X.Z., X.S. Y.C., L.L. and N.Z. performed animal preparation and data collection; and D.C. conceived and designed this work and wrote the manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Trypanosoma brucei is a complex system that is not found in humans, so there is interest in targeting this system for drug development. This system uses hundreds of small non-coding guide RNAs (gRNAs) to modify the mitochondrial mRNA transcriptome. This process occurs in holo-editosomes that assemble several macromolecular trans factors around mRNA including the RNA-free RNA editing core complex (RECC) and auxiliary ribonucleoprotein (RNP) complexes. Yet, the regulatory mechanisms of editing remain obscure. The enzymatic accessory RNP complex, termed the REH2C, includes mRNA substrates and products, the multi-domain 240 kDa RNA Editing Helicase 2 (REH2) and an intriguing 8-zinc finger protein termed REH2-Associated Factor 1 (H2F1). Both of these proteins are essential in editing. REH2 is a member of the DExH/RHA subfamily of RNA helicases with a conserved C-terminus that includes a regulatory OB-fold domain. In trypanosomes, H2F1 recruits REH2 to the editing apparatus, and H2F1 downregulation causes REH2 fragmentation. Our systematic mutagenesis dissected determinants in REH2 and H2F1 for the assembly of REH2C, the stability of REH2, and the RNA-mediated association of REH2C with other editing trans factors. We identified functional OB-fold amino acids in eukaryotic DExH/RHA helicases that are conserved in REH2 and that impact the assembly and interactions of REH2C. H2F1 upregulation stabilized REH2 in vivo. Mutation of the core cysteines or basic amino acids in individual zinc fingers affected the stabilizing property of H2F1 but not its interactions with other examined editing components. This result suggests that most, if not all, fingers may contribute to REH2 stabilization. Finally, a recombinant REH2 (240 kDa) established that the full-length protein is a bona fide RNA helicase with ATP-dependent unwinding activity. REH2 is the only DExH/RHA-type helicase in kinetoplastid holo-editosomes.Uridylate insertion/deletion RNA editing in  Trypanosoma brucei are early-branching eukaryotes that require extensive insertion and deletion of uridylates in most mitochondrial mRNAs (reviewed in  but the precise control mechanisms in editing remain unclear. However, the helicase REH2C subcomplex may play an important regulatory role. REH2 is the largest characterized member of the DExH/RNA helicase A (RHA) subfamily of proteins . These pin flies . Recombiin vitro . Howeverproteins . A genetize REH2 .H2F1 that affect the binding of these proteins with each other, the stability of REH2, and RNA-mediated stable or transient association of the REH2C subcomplex with other editing components in vivo. About 20 different protein variants were examined in vivo. Comparisons of the REH2 with other eukaryotic DExH/RHA helicases identified conserved OB fold residues that affect the assembly and interactions of REH2C and are also important in distant processes, namely mRNA splicing in yeast, and the assembly of the dosage compensation complex in fly. H2F1 upregulation stabilized the large REH2 polypeptide in vivo. However, mutation of individual zinc fingers compromised the stabilizing property of H2F1 but not its association with REH2 or other examined components of the editing apparatus. Most if not all fingers may contribute to REH2 stabilization. Finally, we used a recombinant protein construct to establish that the isolated full-length REH2 (240 kDa) is a bona fide RNA helicase enzyme with ATP-dependent unwinding activity. REH2C is most likely an enzymatic editing RNP in kinetoplastid holo-editosomes.The current study identified features in REH2 and Previous studies revealed that the native REH2C exhibits RNA-mediated interactions with at least two variants of RESC –5. TheseH2F1 and GAP1 proteins, of REH2C and GRBC, respectively. We found that ectopic REH2 was able to reconstitute interactions of the native REH2 protein in trypanosomes  , where ddCq = Cq test IP–Cq mock IP, as in . This scores the relative difference between the reference and target Cq values. The current plots of input extract used 18S rRNA as reference . Sequence alignment of Prp43p, REH2, and other helicases was done using the CDD tool in NCBI . Shorter bars indicate a smaller differential versus 18s rRNA in the sample. For example, ND7 is relatively abundant compared to other transcripts in the sample . The WT construct is induced or not (+/-). All mutants are induced. All end-point amplicons were examined in gels to confirm that they were single products during linear amplification. Abbreviation of the RNA names is as in Independent biological replicates (two independent cultures used in (TIFF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.