The phonon-mediated both in-plane and out-of-plane heat transfer is clarified for this prospective heterobilayer. This study furthermore explored the impact of various interatomic potentials on the thermal conductivity of the heterobilayer. These findings are useful in explaining the heat conduction at the interfaces in graphene/2D-SiC vdWH and may provide a guideline for efficient design and regulation of their thermal characteristics.Graphene based van der Waals heterostructures (vdWHs) have gained substantial interest recently due to their unique electrical and optical characteristics as well as unprecedented opportunities to explore new physics and revolutionary design of nanodevices. However, the heat conduction performance of these vdWHs holds a crucial role in deciding their functional efficiency. In-plane and out-of-plane thermal conduction phenomena in graphene/2D-SiC vdWHs were studied using reverse non-equilibrium molecular dynamics simulations and the transient pump-probe technique, respectively. At room temperature, we determined an in-plane thermal conductivity of ~ 1452 W/m-K for an infinite length graphene/2D-SiC vdWH, which is superior to any graphene based vdWHs reported yet. The out-of-plane thermal resistance of graphene → 2D-SiC and 2D-SiC → graphene was estimated to be 2.71 × 10 However, the zero intrinsic bandgap restrains graphene applications to transistor and logic devices. Thus, extensive efforts were made in the last few years to obtain a tunable bandgap along with intrinsic good thermal conductivity. The hybridization of graphene with finite-bandgap semiconductors would solve this limitation and give rise to new potential applications. In this context, van der Waals heterostructures (vdWHs) made from various two dimensional (2D) materials in a single stack attracted great attention due to their unparalleled platforms for exploring new physics, a novel design of devices as well as exotic electrical and optical properties8. Several graphene-based vdWHs including graphene/h-BN, graphene/MoS2, graphene/MoSe2, graphene/stanene, graphene/silicene, graphene/phosphorene, graphene/germanene with exciting electronic and optical properties have been explored16.The Joule heating generated by electrical current is a key challenge in nanoelectronic and optoelectronic devices, which results in a high power density and spatial heat localization. Such concentrated heat, if not effectively eliminated, could accumulate in thermal hot spots, reduce the overall electronic current density, and lead to significant material failure. Graphene has been suggested as superb material for heat management in nanoelectronic devices because of its exceptional thermal conductivity19. On the other hand, a quasi-2D SiC flake has been synthesized by Lin et al.20 recently via the thermochemical reaction between Si powder and exfoliated graphene at high temperature. A thin atomic structure with hexagonally bonded SiC nanograins has also been fabricated by Susi et al.21. Moreover, 2D-SiC offers a wide direct bandgap with exotic thermal, electrical, optical, and mechanical properties27. Extraordinary tunable electronic properties have also been demonstrated using 2D-SiC as a promising substrate in stanene/2D-SiC and bismuthene/2D-SiC vdWHs29. The epitaxial growth of good-quality graphene from SiC and the latest productive efforts to synthesize 2D-SiC greatly inspired the development of graphene/2D-SiC vdWHs as well. Using 2D-SiC as a compatible substrate a wide band-gap of ~ 30 meV along with a well-preserved Dirac cone and tunable electronic properties has been reported for graphene/2D-SiC vdWHs very recently30, which could be promising for nanoelectronic and optoelectronic applications. It is intriguing to know whether this heterostructure may also succeed to retain the exotic thermal conductivity of graphene. Understanding the thermal behavior of these materials is thus of fundamental importance in the evolution of high-performance nanoelectronic devices.Although graphene based vdWHs offer remarkable possibilities in band-gap engineering and improve electronic and optical properties, nevertheless, the choice of a compatible substrate is an important prerequisite to experimentally synthesize these vdWHs. Recent literature revealed that silicon carbide (SiC) is the most appealing substrate for the epitaxial synthesis of graphene31. Thermal properties of the stacked structures can be very dissimilar from its constituents33; they not only dependent on the choice of 2D materials used for its construction but are also considerably modified by the orientation of the two lattices. Although in-plane lateral thermal conductivity of graphene and 2D-SiC is well known, nevertheless, the mechanism of cross-plane thermal transport in graphene/2D-SiC vdWHs requires deep knowledge before they can effectively compete in real-world technology. The phonon-mediated both intra and interlayer heat transfer needs to be answered to clarify the detailed heat dissipation phenomena of this prospective heterobilayer. However, despite the promising electronic features in graphene/2D-SiC, there is no study on the thermal transport behavior of this heterobilayer.Prior investigations demonstrated that the interface thermal conductivity in graphene related carbon systems is very low due to the substrate effects, which is the main constraint for efficient thermal transport and heat dissipationIn this work, we investigated thoroughly the in-plane and cross-plane heat conduction in graphene/2D-SiC vdWHs by means of molecular dynamics (MD) simulations. The lateral thermal conductivity was calculated via the reverse non-equilibrium molecular dynamics (RNEMD) technique and the cross-plane thermal conductivity was estimated utilizing the transient pump-probe (TPP) technique. To clarify the heat dissipation phenomena between graphene and 2D-SiC layers, phonon properties of the graphene/2D-SiC heterobilayer as well as individual graphene and 2D-SiC were estimated utilizing the velocity correlation of atoms. The calculated in-plane thermal conductivity for the graphene/2D-SiC heterobilayer is outperformed comparable to any graphene based heterostructures reported yet. The thermal resistance at the interface in both the graphene to 2D-SiC and 2D-SiC to graphene directions demonstrated that the heterobilayer has no thermal rectification effect. This study provides a profound intuition into the heat management for nanoelectronics based on the graphene/2D-SiC heterobilayer.ε, r and σ denote the energy, the distance between two atoms, and the atomic length parameters, respectively. χ signifies the interface interaction parameter that can be adjusted to modulate the coupling strength. These parameters were considered as 34. The interface interaction parameter was fixed as χ = 1 for default interaction strength. For all vdW interactions, we set the cutoff distance of the LJ potential as 10 Å. The initial spacing between graphene and the 2D-SiC sheet was set at 3.4 Å, which can be further modified automatically under the relaxation of the structure. To remove the edge effects, periodic boundary conditions were used in both in-plane (x and y) and out-of-plane (z) directions. The atomic interaction with the system image was eliminated by setting a high vacuum distance as 10 nm in the out of-plane direction. We set a time step of 0.5 fs in all MD simulations. All the calculations were conducted by means of Large Scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) package35. We employed the optimized Tersoff potentials implemented by Lindsay et al.36 and Albe et al.37 to interpret the interactions between C–C within the graphene structure and Si–C within the 2D-SiC sheet, respectively. These potentials were successfully implemented to predict the thermal behavior of graphene and 2D-SiC precisely.We employed 12–6 Lenard–Jones (LJ) potential to model the vdW interaction between graphene and the 2D-SiC layer which can be represented as:38 were employed to compute the in-plane thermal conductivity of the graphene/2D-SiC heterobilayers. The RNEMD technique is a straightforward approach where the thermal conductivity is determined by applying a heat flux as the input and calculating the temperature gradient as the output according to the Fourier's law:,dT/dx, and x direction, respectively. A hot and cold slab both of widths 20 Å were placed in the heterobilayer structure at the L/4 and 3L/4 positions, respectively, where L denotes the heterobilayer length in the x direction. To establish the temperature difference between hot and cold regions, the velocity rescaling method for thermal energy modulation of the atoms was implemented. Thus, the heat flux flowing through the material can be expressed as Δε denotes the disparity in thermal energy between the hot and cold atoms in every time step M (M is classified as exchange frequency of heat energy). t is the MD time step, A = wh, the cross-sectional area of the system. The factor 1⁄2 was incorporated in the equation because of the system’s periodicity which means that heat energy will flow in both directions. The temperature gradient was calculated by dividing the whole sheet into N slabs along the computational direction (x) as illustrated in Fig. s’ can be calculated by using,s’, the Boltzmann constant, mass and velocity of atom ‘a’ in the slab, respectively.Throughout the analysis, the RNEMD simulationsmentclass2pt{minim5 time steps to adapt the heterobilayer to the preferred temperature and pressure. To achieve the target temperature, the velocity-verlet integration approach was employed. We then applied NVE MD simulations for 105 time steps to raise the initial temperature to the desired temperature. To stabilize the temperature and pressure as well as to preserve the volume and energy of the heterobilayer, 2 × 105 time steps of NPT and 3 × 105 time steps of NVE MD simulations, respectively, were conducted. Before applying the heat energy, we again placed m, ν. Besides, to check the stability of the system temperature, we also record the system temperature as a function of time. If the system temperature remains constant over time, reflecting the equilibrated state of the system at that temperature. Finally, after imposing the heat energy, the system was run another We conducted our simulation in two steps: structure relaxation and heat-imposing steps. At relaxation step, the system was first set to an equilibrium state at the target temperature. Following energy minimization of the structures via Conjugated Gradient Algorithm, isothermal-isobaric (NPT) ensemble was conducted for 1032. This method was successfully used to study the out-of-plane thermal transport of different 2D heterostructures40. After equilibrating the system at designated temperatures, a 50 fs ultrafast thermal impulse was applied to the graphene layer. The graphene temperature rises rapidly during the ultrafast thermal impulse of 50 fs, while the 2D-SiC temperature is set at 300 K according to the TPP technique. After 50 fs, the graphene temperature started rapidly decreasing, and the 2D-SiC temperature rises gradually as the ambient environment is a vacuum. The heat energy will dissipate by means of interlayer heat exchange before a new thermal equilibrium is achieved by the graphene/2D-SiC bilayer. The total energy of graphene and the changes of temperature in each layer were noted at each time step. The variation of energy will obey the equation below:Et, t, and AR denote the total energy, time and contact area, respectively. The temperature in the heat applied layer and the unimpeded layer is indicated by Tgraphene and T2D-SiC, respectively. With the least squares approach we used an integral formula to calculate the optimal R value as40:Et and this integral, in which the slope is /R. The out-of-plane thermal resistance R at the interface can be found on the basis of the contact area AR. We also analyzed the thermal rectification effect on R. The heat transport from graphene to 2D-SiC is denoted as graphene → 2D-SiC, and the heat transport from 2D-SiC to graphene is denoted as 2D-SiC → graphene, respectively. To obtain noise-free data points, the collected data points are averaged at every 100-time steps. To eliminate the statistical errors, we conducted three separate simulations with specific initial conditions for both in-plane and cross-plane thermal conductivity calculations. Through combining each simulation, the final values were determined using the standard deviations displayed as an error bar.To obtain the out-of-plane thermal transport, we calculated the interfacial thermal resistance using the TPP technique30. It is therefore of great general concern to probe the thermal behavior of this heterostructure and to know whether the unique thermal conductivity of graphene is still preserved. The RNEMD simulation was used to probe the in-plane thermal behavior of this appealing heterobilayer. Before applying the RNEMD simulation, the steady-state condition of the system was confirmed by estimating the atomic velocities of the graphene and 2D-SiC layers at thermal equilibrium. Figure SiC is considered as one of the most important substrates to synthesize and use with graphene for technological applications. Moreover, 2D-SiC has been shown to enable graphene to retain the exotic electronic features, as well as to introduce a direct bandgap creation in the heterobilayerT + ∆T and T − ∆T, respectively, where a ∆T of 50 K is considered. The temperature profile of the heterobilayer having the area of 100 × 10.8 nm2 (length × width) at the steady-state condition is shown in Fig. 44 similar phenomena for various heterobilayers as well as 2D material systems. The thermal conductivity was calculated by taking the temperature gradient, which was obtained from linear fitting. Temperature profiles adjacent to the hot and cool regimes were exempt from the linear fitting process due to the incessant interchange of energy in the heat sinks, where phonon scattering causes an increase in nonlinearity. The temperature gradient ∇T was determined using only the linear region as shown by the blue square bracket in Fig. ∇T for graphene, 2D-SiC and the heterobilayer are 0.081762 K/nm, 0.096669 K/nm and 0.087131 K/nm, respectively.To characterize the thermal conductivity, the temperatures of hot slab and cold slab are monitored as 2 graphene  could be used for measuring thermal conductivity in all types of 2D materials. Accordingly, both dG and d2D-SiC are taken to be 3.35 Å. In this context, the overall thickness of the heterobilayer (dG/2D-SiC) is 6.7 Å. We considered both of these thicknesses in this study, which we denoted as vdW thickness (6.85 Å) and unified thickness (6.7 Å).Usually, the size of the system has a substantial impact on the calculated thermal conductivity in MD simulations. The heterobilayer structure with lengths 22.16, 42.63, 85.25, 170.1, 255.15, 336.80, 428.65, and 555.28 nm were modeled to study the size effects. The widths of all the structures were the same, with the value of 10.8 nm. Moreover, the thickness of the system largely affects the in-plane thermal conductivity. The height of a monolayer is generally considered as the thickness of that particular 2D material45 as:Figure les Fig.  of the g3N by Gao et al.51 stated that the overall thermal conductivities are the cumulative result of various phonons with different mean free paths (MFPs). The MFP of phonons change by the scattering of various phonons, which largely relies on the system size as well. When the length of the system increases, phonons with longer MFP take part in the heat conduction, which causes the increase of the in-plane thermal conductivity. If the length of the heterobilayer is lower than the phonon MFP, the phonons will be transported ballistically and the thermal conductivity will rise almost linearly with length. If the length of the system is higher than the MFP of phonon, the phonons will be transported through the system diffusively, which slows down the increasing rate of the thermal conductivity.Moreover, with the increased length of the heterobilayer, the thermal conductivity rises monotonously and eventually converges. The thermal conductivities of the heterobilayer, graphene and 2D-SiC rise monotonously from 95.46 to 1065.71 W/m-K, 178.12 to 1780.80 W/m-K and 55.76 to 408.9 W/m-K, respectively, with an increased length of 22.16 to 555.28 nm. It is perceived that the thermal conductivity of heterobilayer lies between graphene and 2D-SiC. An earlier study on graphene and C52. If all phonon modes are considered to have the same average group velocity and MFP similar to a single phonon mode, the thermal conductivity for infinite length can be predicted as:L signify the infinite-length thermal conductivity, the effective MFP of the phonon, and the system length, respectively. Figure 1/k and 1/L. By performing a linear fitting on the RNEMD results, the infinite-length thermal conductivities of graphene, 2D-SiC and the heterobilayer are 2239.49 W/m-K, 430.19 W/m-K, and 1451.59 W/m-K, respectively. The calculated thermal conductivity for the graphene/2D-SiC heterobilayer outperforms compared to any graphene based heterostructures predicted in the literature, such as graphene/phosphorene, graphene/h-BN, graphene/MoS2, graphene/MoSe2, and graphene/silicene46. Moreover, to elucidate the interlayer interactions, we have estimated the 54, however, they are much higher than the individual supported graphene (2239.49 W/m-K) and 2D-SiC (430.19 W/m-K) in the heterobilayer structure. This suggests that the in-plane thermal conduction decreases in both graphene and 2D-SiC layers due to interlayer interactions. The predicted lower thermal conductivity in graphene is due to the lessening of the flexural (ZA) phonons in graphene/2D-SiC vdWHs. As described by Seol et al.55, the main heat carrier in the free-standing graphene is the ZA phonon, which can be scattered and leaked when attached with a substrate. The enhanced phonon scattering between the interlayers reduces the ZA phonons. In free-standing single-layer graphene, in-plane (LA and TA) phonons and out-of-plane phonons are well decoupled. Consequently, there is no chance of enhancing phonon scattering in the out-of-plane direction. In contrast, when graphene stacked with a certain material, then in-plane and out-of-plane phonons are well coupled with supported layer phonons, and different parameters of graphene such as translation and rotation, as well as the supporting layer are also altered. As a consequence, phonon modes in the graphene/2D-SiC heterostructure will also be modified, and phonon scattering through the interlayer will be increased, which will minimize the presence of flexural phonons as well as lateral (LA and TA) phonons. A similar anomaly was also perceived in graphene/C3N43, graphene/stanene41 and graphene/MoS245 structures.The infinite-length thermal conductivity was also calculated via the kinetic theory and Matthiessen's rule by adapting the MD outputs with diverse system sizes60 used several distinct potential models to evaluate the thermal conductivity of monolayer graphene and its ribbon. Amid these potential models, the Tersoff63 model is a conventional potential built for demonstrating the energetics of covalent structures with classical inter-atomic potentials. Along with the Tersoff potential, the reactive empirical bond order (REBO) potential was also applied to estimate the thermal conductivity of single-64 and multi-layer66 graphene. Even though the Tersoff, and extended REBO such as AIREBO potential are often used in carbon-based systems, recently the reactive force field (ReaxFF) potential68 has drawn great attraction for better performance in doped-graphene, functional graphene oxide, and graphite/polymer nanocomposite interfacial thermal resistance70. Nonetheless, the improved model of the Tersoff potential, referred to as the opt-Tersoff suggested by Albe et al.37 was established mainly for Si, C, and SiC structures and demonstrated that the thermal and phonon characteristics of such materials can be well replicated due to their precise calculation of the energy and interatomic forces. We used both the modified and initial Tersoff potentials to study the impact of parameterization on the thermal conductivity of graphene/2D-SiC heterobilayers. A comparison of the thermal conductivity estimated by the original and optimized Tersoff potentials is shown in Fig. 37 precisely generates the phonon dispersion using the analytical model that is confirmed by the first-principles calculations. Thus, the obtained thermal conductivity from the optimized Tersoff potential is more precise due to the enhanced acoustic phonon branches and decreased phonon–phonon scattering intensity along with the best fitting of phonon group velocities at BZ center.In order to determine the thermal conductivity by MD simulations, interatomic potentials are the essential parameter that adequately describes the atomic interactions. Both intralayer and interlayer interactions play a pivotal role in obtaining the thermal conductivity of multilayer graphene and related heterobilayers. Previous studies71. Quite different phonon dispersion, phonon density of states (PDOS), and scattering effects are observed in 2D materials compared to their bulk structure, leading to different thermal conductivity with different configurations72. With an intension to quantitatively explain the thermal transport process, the PDOS of the heterobilayer is explored. We calculated the PDOS using the Fourier transform of the velocity autocorrelation of atoms73. The velocity autocorrelation function (VACF) is formulated as:ith atom of materials α at time t and time 0 is viα(t) and viα(0), respectively. The angle brackets represent the ensemble averaging. The PDOS was estimated using the following expression74:F(ω), ω, and π∫ Zα(t)cos(ωt)dt. The calculated VACF for the graphene, Si and C atoms are illustrated in Fig. Phonon plays a major role in the heat conduction in 2D materials rather than the electron conduction75, recent investigations have presented otherwise. Seol et al.55 reported by evaluating the heat transport of supported graphene on amorphous SiO2 that the flexural acoustic phonon (ZA) may contribute to the thermal conductivity of suspended graphene as 77% and 86% at 300 K and 100 K, respectively, because of the large mean scattering time of ZA phonons as well as high specific heat. Lindsay et al.76 also demonstrated that the ZA phonon makes a dominant contribution to graphene thermal conductivity. Besides, as Fig. To show the variations in phonon frequency in graphene and the 2D-SiC layer, the total and decomposed PDOS for in-plane and out-of-plane directions of freestanding samples were calculated. Figure 77 the flexural acoustic or ZA phonon contributes insignificantly due to its low group velocity and wide Grüneisen parameter78. On the other hand, Lindsay et al.76 noticed that most of the heat in graphene is carried by the ZA phonons. At low temperature, the ZA phonon causes a ~ T1.5 behavior, whereas the LA and TA phonons induce a ~ T2 behavior of the thermal conductivity. The ZA phonon contributes greatly to the thermal conductivity of compound 2D materials, including 2D-GaN and 2D-ZnO. Moreover, all of these low-frequency phonon states engage in the heat conduction of 2D-SiC24. Thus, the thermal transport in the graphene/2D-SiC heterobilayer is predicted to result from the contribution of all low-frequency phonon modes. The participation of theses modes also increases with increasing sheet length. As a consequence, the thermal conductivity of the graphene/2D-SiC heterobilayer may be increased with increasing sheet length.Figure x, y, and z directions are used here to eradicate the size effect. During the thermal relaxation, the temperature evolutions in the graphene/2D-SiC heterobilayer with a size of 100 × 10 nm2 are illustrated in Fig. 12 W/m2 was imposed on the graphene layer for 50 fs at T = 300 K. The temperature of graphene −7 km2/W, similar to other vdW heterobilayers55. An outline of the thermal resistance of typical 2D material interfaces is given in Table R between graphene and 2D-SiC is greater than graphene/silicene, graphene/h-BN and graphene/MoS2 heterobilayer.Although 2D materials exhibit an outstanding in-plane thermal conductivity, the weak vdW interaction across the interface constrains the out-of-plane heat conduction. To elucidate the out-of-plane thermal energy exchange, we evaluated the thermal resistance at the interface of the heterobilayer. Periodic boundary conditions in the R along the graphene → 2D-SiC and 2D-SiC → graphene directions to expose the thermal modification behavior in the graphene/2D-SiC heterostructure. Moreover, we take different lengths of 5.10, 10.2, 22.42, 42.10, 62.31, 85.55, 100, 121.1 and 142.2 nm with a fixed width of 10.8 nm to explore the impact of system area on R. Figure R at T = 300 K. Firstly, it is found that the measured R is almost the same in both the graphene → 2D-SiC and 2D-SiC → graphene directions for a fixed heterobilayer area, which demonstrates the absence of the thermal rectification effect in the heterostructure. As a reference, with a system size of 2, −7 km2/W and 2.65 × 10−7 km2/W, respectively, revealing only 2.21% difference. Moreover, the value of R was observed to upsurge monotonically with growing system size. As the system area enlarges, R rises initially, and then the rate of increase slows down from 454 nm2. And finally, R remains almost constant at 2/W from area 1080 nm2. The maximum R increases to 70.4% for increasing system area from 50 to 1420 nm2.The thermal conduction at the interface between two different solids strongly depends on the direction of the heat current especially for dissimilar materials, which is known as a thermal rectification characteristic. We also calculated the R with system size, we calculated the ZA phonon of the heterobilayer for various system sizes because the ZA phonon dominantly contributes to the out-of-plane thermal conduction in graphene related materials. Figure R increases rapidly up to area 454 nm2, slows down from area 454 nm2 and then stays almost constant from area 1080 nm2. This indicates that the R is strongly size-dependent. In order to better illustrate and analyze the size-dependent phenomena of R, we also quantify the overlap factor S for various heterobilayer areas. The S mainly quantifies the amount of phonon overlapping between the interlayer as well as indicating the interlayer energy exchange efficiency. We quantify the S between the graphene out-of-plane (ZA) phonon modes and 2D-SiC in-plane (LA and TA) phonon modes as we apply an ultrafast heat impulse from graphene to 2D-SiC. The S between out-of-plane and in-plane PDOS is calculated using the following relation as79S rises gradually to 850 nm2, and from 1080 nm2 it is almost constant. This implies the overlapping of phonons, as well as the energy exchange efficiency between the interlayer, increases up to 850 nm2 and appears to slightly decrease the scattering of phonons between the interlayer. As a result, the overall thermal resistance of the interface slows down from 454 to 850 nm2 areas. On the other hand, from 1080 nm2 the overlapping of phonons as well as the energy exchange capacity is almost constant, which means that from 1080 nm2 phonon scattering between the interlayer is almost constant. Consequently, the R remains almost constant from 1080 nm2 because it relies explicitly on the phonon scattering between the interlayer.To clarify the variation of 2, graphene/MoSe2, graphene/stanene, silicene/graphene, graphene/C3N, as well as individual 2D-SiC, phosphorene, hexagonal boron nitride (h-BN), MoS2 and MoSe2 of the same system size. The in-plane thermal conductivity of graphene, 2D-SiC and heterobilayer at infinite length were determined as 2239.49 W/m-K, 430.19 W/m-K and 1451.59 W/m-K, respectively. The total PDOS revealed that the active phonon frequencies in 2D-SiC are only 0–35 THz with a low phonon density in the high-frequency region, strictly bounding the lateral thermal conductivity to a lower value than graphene. With a system size of 2, we estimated that out-plane thermal resistance of graphene → 2D-SiC as 2.71 × 10−7 km2/W and 2D-SiC → graphene as 2.65 × 10−7 km2/W at 300 K, respectively, indicating the absence of thermal rectification in the heterobilayer. Out-of-plane thermal resistance was found to expand monotonically with growing system size. The maximum R increases to 70.4% for increase of the system area from 50 to 1420 nm2. We found that the increase of the ZA phonon peak with the length caused the monotonic increase of the out-of-plane thermal resistance with system size. The superior thermal conduction, along with good electrical properties, could make the graphene/2D-SiC heterobilayer appealing as a high-performance thermal interface material or electronic material for next generation nanoelectronics.The lateral and cross-plane thermal conductivity of the graphene/2D-SiC heterobilayer were systematically studied using the RNEMD simulation and TPP method, respectively. The original and optimized Tersoff potentials were used to illustrate their effect on the graphene/2D-SiC heterobilayer thermal conductivity. The optimized Tersoff potential provides better thermal conductivity estimation than the original Tersoff potential due to the correctly parameterized interatomic forces. A high thermal conductivity of ~ 452 W/m-K was obtained for the 100 nm length and 10.8 nm width heterobilayer at room temperature using the optimized Tersoff potential, superior to most other graphene based vdWHs such as graphene/MoS


Cryptoendolithic communities are almost the sole life form in the ice-free areas of the Antarctic desert, encompassing among the most extreme-tolerant organisms known on Earth that still assure ecosystems functioning, regulating nutrient and biogeochemical cycles under conditions accounted as incompatible with active life. If high-throughput sequencing based studies are unravelling prokaryotic and eukaryotic diversity, they are not yet characterized in terms of stress adaptations and responses, despite their paramount ecological importance. In this study, we compared the responses of Antarctic endolithic communities, with special focus on fungi, both under dry conditions , and after reanimation by wetting, light, and optimal temperature (15 °C). We found that several metabolites were differently expressed in reanimated opposite sun exposed communities, suggesting a critical role in their success. In particular, the saccharopine pathway was up-regulated in the north surface, while the spermine/spermidine pathway was significantly down-regulated in the shaded exposed communities. The carnitine-dependent pathway is up-regulated in south-exposed reanimated samples, indicating the preferential involvement of the B-oxidation for the functioning of TCA cycle. The role of these metabolites in the performance of the communities is discussed herein. Endolithic microbial communities are found inside rocks in the coldest or hottest drylands worldwide, from Atacama Desert in Chile to McMurdo Dry Valleys in Antarctica, where conditions of high solar radiation, drastic temperature fluctuations, water deficit, prolonged periods of desiccation, oligotrophy, and high salinity levels prevent the settlement of microbial epilithic patinas ,2,3.4·2H2O) to anhydrite (CaSO4).Scientific interest in endolithic microorganisms rose after their discovery in the McMurdo Dry Valleys, continental Antarctica, one of the harshest environments on Earth and accounted as a Terrestrial Martian analogue. Cryptoendoliths, due to their low complexity and diversity compared to other microbial consortia such as soil and biological crusts communities, are in fact excellent models to explore biotic and abiotic drivers of diversity, explore microbial communities responses under stress conditions, giving clues into the limit of life on Earth, and also for exploring the possibility for life elsewhere in the Solar System . The landscape of the McMurdo Dry Valleys is mostly composed of ice-free exposed rocks and oligotrophic mineral soils ,5. They Endolithic communities are the main, if not the sole, life-forms representing the main standing biomass in the McMurdo Dry Valleys, occupying approximately 4% of sandstone boulders , up to 3Trebouxia spp.  [Among endolithic microorganisms, fungi are pivotal in these microbial consortia and are organized as follows. Lichen-forming fungi  are the most abundant ,23, and occales)  that areoccales) . Yeasts occales) . Yeasts Recent studies are elucidating the biodiversity, structure, and composition of Antarctic cryptoendolithic communities ,32,33,34Microbial community metabolomics is recently providing a new route to establish stress-response and survival strategies in environmental samples ,41. A reIn the present work, we evaluated the responses of differently stressed communities after reanimation by wetting, light, and temperature above freezing point, providing critical insights on metabolic processes under the extreme aridity and oligotrophy.Three sandstone rocks colonized by cryptoendolithic communities were collected at Finger Mt.  and incubated for 96 hrs at 15 °C in an incubator (Panasonic MIR-254) equipped with a white light lamp  and after metabolic machinery reactivation, samples have been exposed to wetting  of 35ms with 3.8 kV spray voltage, 40 sheath gas, and 25 auxiliary gas. The system was operated in positive ion mode. Calibration was performed before each analysis against positive or negative ion mode calibration mixes  to ensure error of the intact mass within the sub ppm range.Twenty μL of extracted supernatant samples was injected into an ultra-high-performance liquid chromatography (UHPLC) system  and run on a positive mode: samples were loaded on to a Reprosil C18 column  for metabolite separation. Chromatographic separations were made at a column temperature of 30 °C and a flow rate of 0.2 mL/min. For positive ion mode (+) MS analyses, a 0–100% linear gradient of solvent A  to B  was employed over 20 min, returning to 100% A in 2 min and holding solvent A for a 1-min post time hold. Acetonitrile, formic acid, and HPLC-grade water and standards  were purchased from Sigma Aldrich. The UHPLC system was coupled online with a Q Exactive mass spectrometer (Thermo) scanning in full MS mode (2 μscans) at resolution of 70,000 in the 67 to 1000 http://metlin.scripps.edu), which incorporates tandem mass spectral data from model compounds. Raw data sets were uploaded to XCMS Online, and a single group job was created to analyze the difference between the communities under dry conditions and after metabolic machinery reactivation in northern sun-exposed rocks. The raw data files are then processed for peak detection, retention-time correction, chromatogram alignment, metabolite feature metadata, and statistical evaluation using the predefined workflow settings for Orbitrap, and metabolite identification is facilitated through METLIN standard database matching and KEGG pathway database. XCMS extracted metabolomic features with statistically significant expression changes among the two groups to produce a list of raw differentially expressed features based on p-values  and then perform pathway analyses directly from their raw metabolomic data. The output can be visualized through Pathway Cloud Plot. We then analyzed in detail only pathways with metabolites with p value ≤ 0.001.Raw files of metabolomics data from technical and biological replicates of south and north exposed samples both dry and reanimated samples were analyzed using XCMS Online, a freely accessible metabolite database called METLIN   compare the effect of reanimation  on metabolism and response of Antarctic endolithic communities with those under dry conditions  and (ii) reveal which metabolic pathways, with a special focus on fungi as main component of these communities, play a critical role for reactivation.We found that a quite diverse activity of microbial communities in these two conditions, resulting in a different magnitude of change to the overall functioning, can be discerned by comparing biochemical pathways in (i) North dry samples (NDS) vs. North reanimated samples (NRS), (ii) South dry samples (SDS) vs. South reanimated samples (SRS), and (iii) NRS vs. SRS.Aspergillus fumigatus and a few yeasts such as Saccharomyces cerevisiae, Yarrowia lipolytica, Schizosaccharomyces pombe, and Rhodotorula glutinis. Members of Rhodotorula genus were also retrieved from halite nodules from samples collected in the Atacama desert; in this work, Aspergillaceae, Sporidiobolaceae, and Sordariaceae families were the most highly represented in these shotgun metagenome sequences [One of the main differences found concerned the NDS vs. NRS comparison, where the “saccharopine” and “spermine and spermidine” pathways resulted in strongly up-regulated in reactivated samples. In fungi, lysine is synthesised through the “saccharopine pathway”; this pathway also occurs in higher plants and animals, but instead of functioning in lysine synthesis, it works in the catabolic reaction, leading to lysine degradation . The up-equences .Additionally, enzymes involved in the fungal AAA pathway are unique to lysine synthesis; in addition, several saccharopine pathway intermediates are incorporated into secondary metabolites  that areColletotrichum truncatum was inhibited [Rhizoctonia solani, Fusarium oxysporum, and Cochliobolus carbonum [On the contrary, the performance of microbial endolithic communities subjected to abiotic stress (dormant state) requires a concerted orchestration of the multiple cellular and metabolic remodeling processes. We found, indeed, that the lysine synthesis was strongly inhibited by water deficit, contrary to what we observed in NRS. Drought, indeed, induced substantial shifts in NRS, resulting in an up-regulation of the “spermine and spermidine” biosynthesis, showing the important role of metabolites involved in this pathway in protecting microbial activity in drought periods. Spermine and spermidine are polyamines that are essential metabolites present in all living organisms, and they act in different ways in the cell functions in which they are involved, from growth to development and differentiation and protection of DNA from reactive oxygen species (ROS). During the last decades, fungi have contributed to the understanding of polyamine metabolism; a few years ago, the use of specific inhibitors and the isolation of mutants have allowed the manipulation of the pathway providing information on its regulation . The polnhibited . The samcarbonum . Additiocarbonum ,55,56 reNε-trimethyl-lysine biosynthetic pathway that has been fully characterized in fungi including Neurospora crassa. Genetic studies in the yeast Candida albicans led to the identification of the aldolase that catalyses the second step of the carnitine biosynthesis pathway, suggesting that fungi utilize this pathway to produce carnitine for the β-oxidation [When comparing dry and reanimated samples collected in the southern exposed surfaces, we reported an alteration of “L-carnitine biosynthesis pathway” in SRS. Carnitine biosynthesis led to the endogenous production of L-carnitine, a molecule that is essential for energy metabolism that is necessary when rocks are reanimated and their metabolic machinery is reactivated; conversely, in a dormant state, the microorganisms decrease their energetic costs. The carnitine biosynthesis pathway is highly conserved among many eukaryotes and some prokaryotes, although it acts in diverse physiological processes. In this work, we reconstructed the L-carnitine pathway from the precursor xidation . Indeed,xidation . In addixidation ,60. Theyxidation .Finally, we compared the reanimation processes between northern and southern exposed communities. Recently, it has been reported that abundance and composition of metabolites presented a clear pattern of correlation across opposite exposed surfaces of the same mountain, revealing significant changes in metabolic profiles following sunlight deprivation . OverallIn the present study, we found that the endolithic communities exposed directly to the sun (northern surface) use canonical pathways for energy production compared to the shady samples, suggesting that the latter may preferentially use alternative mechanisms, more appropriate to keep metabolism active under more unfavorable conditions.North vs. South reanimated samples, the TCA cycle appears significantly perturbed. As discussed above, SRS use the carnitine-dependent pathway, where the carnitine acetyltransferases exchange the CoA group of acetyl-CoA for carnitine, thereby forming acetyl-carnitine, which can be transported in peroxisome for the B-oxidation. Then, the products of the peroxisomal b-oxidation (shortened fatty acids and acetyl units) were transported to the mitochondrial TCA for ATP generation that are essential for growth and survival [By comparing responses of survival  and thatsurvival , may stoBased on these results, we may hypothesize that the two opposite sun-exposed communities activate different metabolic pathways to restore their vitality and that the south-exposed ones, being more stressed, prefer peroxisomal oxidation (through biosynthesis of carnitine) and TCA cycle as the main pathways for producing energy.Cryptoendolithic microbial ecosystems have unique properties as a model system for studies of microbial ecology in both hot and cold deserts. In this study, we provided critical insights on how Antarctic endolithic communities respond to stresses, maintaining biological activities under harshest conditions that are typically incompatible with active life. We also identified metabolites that might be expressed in response to optimal conditions (after reanimation), characterizing the response dynamics of these communities to changing environmental conditions. Our results show that endolithic microorganisms, in particular fungi, exist in dormant states but rapidly reactivate the saccharopine and polyamines biosynthesis pathways that are tightly correlated with the growth, development, or reproduction of the organisms and energy metabolism and may play an important role when the communities face environmental changes in its living habitat.Although further research on a larger number of samples is necessary to attribute shifts in particular microbially driven endolithic processes to the observed changes in functional and genetic diversity under different conditions, these findings will be useful in modelling predicting important pathways that have evolved to support these life-forms on the extreme edge of Antarctic desert.These results may be also applied to microbial endolithic ecosystems in drylands worldwide, in an era of climate change and rapid desertification.


We propose a new categorization of turn-taking events which combines the criterion of speaker change with whether the original speaker inhales before producing the next talkspurt. We demonstrate that the latter criterion could be potentially used as a good proxy for pragmatic completeness of the previous utterance . We also present evidence that breath holds are used in reaction to incoming talk rather than as a turn-holding cue. In addition to analysing dimensions which are routinely omitted in studies of interactional functions of breathing , the present study also looks at patterns of breath holds in silent breathing and shows that breath holds are sometimes produced toward the beginning (and toward the top) of silent exhalations, potentially indicating an abandoned intention to take the turn. We claim that the breathing signal can thus be successfully used for uncovering  The importance of breathing for production of speech needs little justification. It is, after all, the intricate coordinative patterns of the respiratory system that are the main driving force behind much of speech production as well as other vocal communicative behaviors. However, in spite of its importance, breathing has been generally overlooked in speech science. This claim can be easily verified by even a cursory look at standard phonetics textbooks with their focus firmly placed on articulatory phenomena and relatively little attention paid to the glottal and the subglottal systems. Supraglottal aspects of speech production enjoy a similar position of dominance when it comes to studies of communicative aspects of vocalizations in spontaneous conversation. While it is true that the field has enjoyed an increased interest in recent years, the contribution of the respiratory system to signaling speakers' communicative intentions is still far from clear.pause interruptions, which coincide with turn-holding silences in interlocutor's speech , which focused primarily on properties of pre-speech inhalations, we investigate both inhalatory and exhalatory segments, as well as instances of respiratory holds. We also extend existing accounts by describing respiratory patterns found in overlapping speech. In addition, we present evidence that the respiratory signal can be used to identify turn-taking events which are otherwise obscured by the commonly used silence-based classification of conversational floor state  rather than eliciting natural turn-taking behavior. In addition, the conversations they investigated were relatively short (about 4 min) and involved subjects who were immobilized by means of shoulder straps, footrests, and hands held clasped on the lap.As indicated above, studies of respiratory mechanisms employed in production of spontaneous speech are rare. Even less frequent are studies of respiratory patterns underlying management and coordination of turn-taking. For instance, while Winkworth et al.  characteBy contrast, the first description of respiratory turn-taking cues proper was done by McFarland . In thissmooth transitions occurring after complete turns, (2) competitive interruptions, in which the incoming speaker successfully grabs the floor from the interlocutor, and (3) competitive butting-ins, in which the incoming speaker fails to interrupt the interlocutorFigure 4 suggests that inhalation durations in turn-taking are comparable to those in listening  in Dutch, Torreira et al. , data acquisition choices (one or two belts) and the aspects of the respiratory signal selected for analysis. These differences notwithstanding, the studies provide evidence for systematic variation in breathing in the vicinity of turn-taking events, with the shortening of the inhalation in turn-holding being the most robust cue found. By contrast, breathing patterns related to claiming or releasing the turn were less consistent. Even though the effects were generally weak, the breathing signal was demonstrated to improve prediction of turn-taking in technical systems.In order to characterize turn-taking events, they first have to be identified as such. Several approaches to the classification of turn-taking events have been used in the past. The speaking turn, as introduced in the seminal work by Sacks et al. , is defispeaker change. This technique goes back to the method of interactional chronography, introduced by Norwine and Murphy  speaker changes  to minimize distortions in the respiratory signal. Speech was recorded using directional close-talking condenser microphones (Sennheiser HSP 4) to reduce the amount of cross-talk.isovolume manoeuvre  method  was calculated as the interval between the 5th and 95th percentiles of all peak and trough values, and resting expiratory level (REL) was estimated dynamically as the median value of troughs within a 60-s window. In addition, breath holds were identified using the method proposed by Noto et al.  were excluded from the analysis. Also excluded were all intervals in which edges of the speech segments coincided with an inhalation N = 1, 801).For the purpose of the present study, we further classified the WSS, BSS, and BSO intervals depending on whether the following utterance by the original speaker was preceded by an inhalation (+INH) or directly followed the exhalatory segment (-INH), see 2 s), slope (in SV per second), speech lag , duration . Also excluded were inhalations and exhalations with extreme values of slope . In this work, we operationalized backchannels as talkspurts shorter than 1 s. This criterion, proposed by Heldner et al. , should be low compared to the probability of a speaker change not accompanied by a hold, P(speaker change|no hold). Conversely, if they are used in response to interlocutor's interruption, the opposite should be true. We find that the probability of a speaker change is much higher (0.74) when a silence coincides with a respiratory hold than when it does not (0.38), suggesting that holds are produced predominantly reactively, in response to incoming speech.In order to verify to what extent breath holds are used proactively and to what extent they are produced to ward off (or wait out) an interlocutor's interruption, we calculated conditional probabilities of speaker change depending on whether or not a respiratory hold is present. If respiratory holds were used as an effective turn-holding signal, the probability of a speaker change in their presence, Distributions of exhalation duration, amplitude, and slope as well as onset and offset levels in the six interval types are plotted in The distributions of exhalatory duration show a similar grouping with [−INH] intervals being generally longer than their [+INH] counterparts. In addition, several effects previously attested in the literature are also apparent in that plot. For instance, as observed in several studies  significantly improved fit of the resulting model (indicated with likelihood ratio test) and were included in the final model, summarized in R2 for this model equals 0.18; if the duration and onset level are also included in the modelR2 values of 0.7–0.9 for linear functions  and shorter inhalations, which are followed much more quickly by speech. Again there is no substantial difference between BSO and TT intervals, and only slightly steeper slopes in intervals involving overlap, BSO+INH and TT(O), than in those coinciding with silence, BSS+INH and TT(S).The contribution of the inhalatory features to prediction of interval type was assessed again using multinomial logistic regression. Similar to exhalatory features, only slope was included in the model due to high collinearity between slope, amplitude, and duration. Given that speech lag was defined only for a subset of intervals, we fitted two separate models. First, we fitted a model which included slope and offset level using all five interval types. Next, for WSS+INH, TT(S), and TT(O) intervals, we fitted a model which additionally included speech lag as a predictor. The results of these models are summarized in R2 of this model equalled 0.06. When duration was added as a predictor, this value changed only slightly to 0.07.In the first model , fitted R2 of this model equalled 0.21, compared to R2 of 0.14 for a model with slope and offset level as the only predictors. This relatively high R2 value compared to the model in R2 = 0.22).The second model , which oIn the present section we turn to the problem of identifying abandoned speech initiations. To that end we identified breath holds during silent breathing . In Of more interest to our present goal is the smaller concentration of respiratory holds toward the beginning (and the top) of the exhalation. This indicates that speakers sometimes hold their breath right after an inhalation. This behavior is illustrated in 2 s) and inhalation amplitude (as a fraction of the speaker's respiratory range) in silent cycles split depending on whether or not they coincide with a hold, as well as in turn-taking cycles accompanied by silence, TT(S). Overall, the TT(S) category is characterized by both increased inhalation duration and amplitude, with the values for the Hold class placed in between the other two. These tendencies are also reflected in the results of the multinomial logistic regression in R2 values. Even though both inhalation amplitude and slope significantly improve the model fit, McFadden's pseudo-R2 is quite low (0.03) indicating the predictive power of these features for discrimination between the three categories is limited.In In this paper, we have aimed to provide an overview of respiratory patterns related to several floor management strategies. The basic analytical categories used in the present work were formulated in terms of between- and within-speaker silences and overlaps  is also the expected endpoint for pragmatically complete breath groups.At the same time, the BSO-INH and BSS-INH intervals were characterized by the longest exhalations of all the analyzed categories. Given that these intervals, alongside BSO+INH, also had the highest likelihood of coinciding with a respiratory hold, the results are consistent with the idea that these cases involve turn-competition, whereby the previous speaker is trying to keep the turn.P(speaker change|hold) = 0.74] than in its absence [P(speaker change|no hold) = 0.38]. This suggests that respiratory holds do not function as a proactive turn-holding resource, as proposed by Local and Kelly  and BSO+INH in comparison to TT(S) and BSS+INH, the differences were practically nonexistent. In other words, incoming speakers' breathing behavior is not substantially altered by presence of overlapping speech, possibly indicating that the latter is not a reliable indicator of turn competition.In addition to respiratory patterns in the vicinity of speech, we also investigated occurrences of breath holds in silent cycles with a view to identifying abandoned attempts at taking a turn. Indeed, recent evidence  for getting closer to competitive/non-competitive speaker changes without the need for an explicit analysis of interlocutors' communicative intentions. Fifth, using these categories, we were able to shed light on To the best of our knowledge, this is the first study which attempted to identify unrealized turn-taking intentions using a fully automatic method. By comparing breathing features of hidden event candidates with their overt counterparts  we hope to have shown that inclusion of additional data streams can be helpful for this task and we are looking forward to seeing other multimodal data  being used for this purpose. At the same time, even though this method provides support to the hypothesized character of the identified events, an independent qualitative investigation of their pragmatic function is certainly warranted, including the question as to whether the identified patterns are actually used by conversation partners for turn-management. Furthermore, analysing patterns of respiratory holds in the vicinity of overlapping speech might be useful for solving the thorny problem of discriminating between collaborative and competitive overlaps , using a hyperscanning paradigm including several conversational partners in parallel. The results will allow evaluating and augmenting the neural evidence for speech planning in conversation  can be found at: Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.MW and MH: conception and design of the work, acquisition, analysis, and interpretation of the data, drafting the work, and final approval of the version to be published. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rauvolfia serpentina (L.) Benth. ex Kurz. However, to a much larger extent, the investigated conversion yielded vinorine ‐epoxide, a new metabolite with an epoxide functional group that is rare for indole alkaloids. The described work represents a successful example of combinatorial biosynthesis towards an increase in biodiversity of natural metabolites. Moreover, characterisation of the products of the in vitro and in planta transformation of potential pharmaceuticals with human CYPs might be indicative of the route of their conversion in the human organism.Human drug‐metabolizing cytochrome P450 monooxygenases (CYPs) have enormous substrate promiscuity; this makes them promising tools for the expansion of natural product diversity. Here, we used CYP3A4 for the targeted diversification of a plant biosynthetic route leading to monoterpenoid indole alkaloids. In silico, in vitro and in planta studies proved that CYP3A4 was able to convert the indole alkaloid vinorine into vomilenine, the former being one of the central intermediates in the ajmaline pathway in the medicinal plant  Alternative oxidation: We used CYP3A4 for the targeted diversification of plant monoterpenoid indole alkaloids. In vitro and in planta studies proved that the cytochrome mainly converted vinorine into vinorine ‐epoxide, a new metabolite containing a rare for indole alkaloid epoxide group. A minor route led to the formation of the natural constituent vomilenine. Cytochrome P450 monooxygenases (CYPs) are versatile enzyme catalysts, capable of performing a variety of reactions, most of which are oxidation reactions.Rauvolfia serpentina (L.) Benth. ex Kurz., at least seven different CYP enzymes are involved.Catharanthus roseus, they perform very distinguished reactions, like regio‐ and stereospecific hydroxylation of 7‐deoxyloganic acid to form loganic acid (CYP72A224), decyclization of loganin (CYP72A1) or the multi‐step oxidation of nepetalactol (CYP76A26) R. serpentinaS,20R)‐19,20‐dihydroperaksines, the latter of which feature a different structural scaffold.Biosynthetic pathways leading to the formation of complex plant specialized metabolites very often encompass numerous CYP enzymes. The importance of these catalysts for a given plant's metabolism is underscored by the number of genes corresponding to this enzyme family, ranging from around 150 to more than 400, depending on the plant species.The indolenine alkaloid vinorine, one of the central intermediates in the pathway, was recently shown to facilitate regeneration and to promote recovery of motor function after nerve crush injury via up‐regulation of nerve growth factor protein levels and extracellular signal‐regulated kinase activation.R. serpentina. We assayed whether this CYP can provide a more specific vinorine hydroxylating activity excluding formation of the side product perakine or, alternatively, produce recently unknown metabolites. Since previous work on human CYP2D6 transiently expressed in Nicotiana benthamiana did not show any vinorine conversion,To investigate regio‐ and stereospecificity of an alternative CYP enzyme for the oxidation of vinorine, we aimed at probing a human drug‐metabolizing CYP for its suitability to substitute the endogenous VH from To evaluate whether vinorine could be a possible substrate for CYP3A4, we performed docking studies using the MOE 2019.01 software (S1.6). One hundred docking positions of vinorine to the active site were generated in proximity to the haem group of the CYP. Out of 20 best‐scored alternative docking positions, the second best‐scored featured a small distance between the double bond of vinorine and the hydroperoxo‐ferric complex in the active centre. To check the stability of the arrangement, a final energy minimization with the fixed backbone atoms was performed. The resulting tertiary enzyme structure with the docked vinorine is displayed in Figure R. serpentina , as described earlier,The postulated docking arrangement is mainly recognised by the hydrophobic amino acid residues F215, F108 and F304 and accompanied by the formation of a hydrogen bond of the NH group of the indene moiety with S119. With this docking pose, the C19−C20 double bond of the substrate is in very close proximity (3.03 Å) to the oxygen atom of the ferric hydroperoxo intermediate, which allows effective generation of the epoxide  in vitro assays with baculosome preparations of recombinant CYP3A4 (S1.2) and b) in planta transient system expressing CYP3A4m/z [M+H]+ 351) and a minor peak at 9.4 min  as compared to the controls, together with a distorted peak characteristic of an indole alkaloid with an aldehyde functional group . After the addition of sodium borohydride, the distorted peak disappeared and an alkaloid with tR=8.5 min and m/z [M+H]+ 295 was registered as a major component of the reaction mixture , detected in both control and experimental samples  after borohydride reduction, derived from perakine – the contaminant of the vinorine sample.A trace amount of raucaffrinoline  with m/z [M+H]+ 351  shifted upfield to δ 3.06 ppm , as did the signals of the protons at C15, C18 and C21, adjacent to the vinyl moiety in vinorine. This indicates the dislocation of the deshielding effect of the 19–20 double bond in the novel compound. The data of a CLIP‐COSY experiment displayed coupling of the H19 proton with the H18 methyl protons only and that of the H21 protons between themselves, suggesting a quaternary carbon at C20, which was subsequently confirmed by 13C NMR experiments ‐epoxide led to the conclusion about the ‐configuration of the novel alkaloid . The distance between the H18 protons and H16 in this configuration (≤3 Å) was appropriate for the induction of a clear NOE signal. In the possible alternative ‐configuration , the distance between the mentioned protons increased to around 4 Å, while the distance between the H18 protons and the axial H14 (δ 2.33 ppm) was reduced to less than 3 Å. However, no through‐space interaction was detected between H18 and H14. Thus, oxidation of vinorine by CYP3A4 resulted in the formation of the novel product, vinorine ‐epoxide, containing an epoxide moiety, which is a rare feature for indole alkaloids.To determine the stereochemistry of CYP3A4‐catalysed epoxidation, the data of the NOESY experiment were analysed . For H19, clear NOE interaction was observed only with the H18 protons of the methyl group; additionally, we registered strong signals between H18 protons and H15 as well as H18 and H16 . Interpretation of these data using 3D models of vinorine  was identified as vomilenine (2 % yield) upon comparison with the authentic reference sample . Selective extraction of the m/z 351 ion chromatogram did not allow detection of vomilenine in the control samples . Inefficient conversion  precluded structure elucidation of the corresponding alkaloids, but the similarity of their UV spectra to that of vomilenine  indicated that the oxidation occurred outside of the UV‐absorbing chromophore.Incubation of vomilenine with the CYP3A4 baculosome preparation did not result in its rearrangement into perakine , as described for the plant CYP82S18.N. benthamiana with subsequent administration of vinorine. This resulted in the formation of vinorine ‐epoxide, as confirmed by HPLC‐ESI‐MS analysis and comparison with the authentic reference sample . No other product of vinorine CYP3A4 oxidation was detected. The yield of the product was ∼4 % of the extracted alkaloids (as determined by HPLC and HPLC‐MS), probably due to the high activity of plant background enzymes competing with CYP3A4 for the substrate. As in the baculosome assays, the major component of both experimental and control plant samples was the product of vinorine deacetylation, vellosimine ‐epoxide, a new metabolite, which was not previously found in nature or synthesized, containing an epoxide functional group that is rare for indole alkaloids. An alternative, minor route of vinorine hydroxylation by CYP3A4 led to the formation of vomilenine, which is the natural constituent of the plant indole alkaloid network. The described work represents a successful example of the realization of the combinatorial biosynthesis concept. This implies various combinations of heterologous enzymes and substrates in a different metabolic environment, to ultimately increase the biodiversity of natural metabolites. In addition, the characterisation of the products of the either in vitro or in planta transformation of compounds with potential pharmaceutical use with human CYPs might be indicative of the way of their conversion in the human organism.In conclusion, we utilized CYP3A4 for the targeted diversification of the biosynthetic route of monoterpenoid indole alkaloids. In vitro and in planta studies proved that the human CYP mainly converted vinorine into vinorine  should be addressed to the authors.SupplementaryClick here for additional data file.


Zebrafish IL-4–like cytokines promote type 2 and suppress type 1 immunity.Zebrafish IL-10 is potently anti-inflammatory and essential for gill homeostasis. Danio rerio) and, together with an existing IL-10 mutant line, characterized the consequences of loss of function of these cytokines. We demonstrate that IL-4/13A and IL-4/13B are required for the maintenance of a Th2-like phenotype in the gills and the suppression of type 1 immune responses. As in mammals, IL-10 appears to have a more striking anti-inflammatory function than IL-4–like cytokines and is essential for gill homeostasis. Thus, both IL-4/13 and IL-10 paralogs in zebrafish exhibit aspects of conserved function with their mammalian counterparts.Mucosal surfaces such as fish gills interface between the organism and the external environment and as such are major sites of foreign Ag encounter. In the gills, the balance between inflammatory responses to waterborne pathogens and regulatory responses toward commensal microbes is critical for effective barrier function and overall fish health. In mammals, IL-4 and IL-13 in concert with IL-10 are essential for balancing immune responses to pathogens and suppressing inflammation. Although considerable progress has been made in the field of fish immunology in recent years, whether the fish counterparts of these key mammalian cytokines perform similar roles is still an open question. In this study, we have generated IL-4/13A and IL-4/13B mutant zebrafish ( In mammals, IL-4 and IL-13 are structurally and functionally related cytokines that participate in several physiological processes but are mainly known for stimulating type 2 immune responses . These r+ Th2 cells, basophils, eosinophils, mast cells, and group 2 innate lymphoid cells (ILCs) but also by granulocytes associated with allergic responses, including basophils, eosinophils, and mast cells , namely il4/13a and il4/13b  were maintained under standard conditions (∼28°C under a 14-h light/10-h dark cycle) within the Biological Services Unit (The University of Manchester) and the Central Biomedical Services . Embryos were collected and raised in egg water  up to 5 d postfertilization (dpf) and then transferred to the main aquarium system. Younger fry were fed powder food and rotifers, whereas older fry and adults were fed powder food and brine shrimp. All food was supplied by Zebrafish Management fish food. e46/e46il10 mutant zebrafish were a kind gift from S. Johnston (University of Sheffield) and were generated by the Sanger Institute through the Zebrafish Mutation Project (Zebrafish ( Project . All regil4/13a or il4/13b zebrafish genes using the Harvard chopchop program (https://chopchop.rc.fas.harvard.edu). The il4/13a target site was 5′-GGGTTTTACGTTGAAAGGCA-3′, and the il4/13b target site was 5′-GAAATCATCCAGAGTGTGAA-3′ and were incorporated into a gRNA template for transcription using PCR. The forward primer contained the target gene specific sequence followed by a constant sequence that overlaps with the remaining sequence of Streptococcus pyogenes chimeric gRNA DNA template , whereas the reverse primer was complementary to the gRNA DNA template (see Guide RNAs (gRNAs) were designed to target the first exon of either S. pyogenes nls-zCas9-nls mRNA synthesized using a mMESSAGE mMACHINE SP6 Kit (Life Technologies) from a pCS2 construct , 100 pg/nl H2B-mCerulean3 tracer mRNA similarly generated from a pCS2 construct, and 0.05% (w/v) phenol red to allow visualization of injections. Embryos were injected at the one-cell stage and screened for fluorescence at 24 h postfertilization to identify positively injected fish, which were then raised to adulthood.Injection solutions included 30 pg/nl of either gRNA, 250 pg/nl of codon-optimized il4/13a and il4/13b zebrafish mutant lines, genomic DNA was amplified by PCR using gene-specific primers flanking the targeted site (see Il4/13a+/− and il4/13b+/− heterozygous fish were in crossed to generate homozygous mutants for both il4/13a (il4/13a−/−) and il4/13b (il4/13b−/−) in the F3 generation. To generate il4/13a;b−/− double mutants, il4/13a−/− and il4/13b−/− homozygous fish were intercrossed and their progeny in crossed. il4/13a;b−/− double mutants were in crossed to maintain the line.To identify modified alleles in the F1 progeny for the establishment of n = 9–10) were fixed in 4% (w/v) paraformaldehyde in PBS at 4°C overnight. Fish were then washed in 0.1% (v/v) Tween 20 PBS solution and dehydrated through an ascending series of methanol. Digoxigenin-labeled RNA antisense probe for zebrafish mpx was kindly supplied by S. Renshaw (University of Sheffield), and whole mount in situ hybridization was performed as described in (mpx+ cells was performed using ImageJ software.Three-day-old embryos (ribed in . Embryosn > 3) using the RNeasy Mini Kit (QIAGEN) and reverse transcribed using the ProtoScript II First Strand cDNA Synthesis Kit (New England BioLabs) with Oligo (dT) primers, according to the manufacturer’s instructions. Quantitative PCR (qPCR) was performed using SYBR Green JumpStart Taq ReadyMix (Sigma), ROX Reference Dye, and cDNA (diluted 1:2). All samples were run in triplicate on 96-well PCR plates (BioLabs). Negative controls were run without cDNA. Data were analyzed by the ΔCt method using bactin for normalization. The primers used for qPCR are listed in RNA was isolated from homogenized larvae and/or adult gills (n = 3), −/−il4/13a (n = 4), −/−il4/13b (n = 4), −/−il4/13a;b (n = 4), and e46/e46il10 (n = 6) as well as wildtype and e46/e46il10 animals following 8-h resiquimod (R848) challenge (n = 3) and fixed in 4% (w/v) paraformaldehyde overnight at 4°C. The next day, the samples were washed in PBS and decalcified in 0.25 M EDTA solution for 3 d at room temperature. After decalcification, heads were washed with tap water and placed in 70% (v/v) EtOH. All specimens were dehydrated, and paraffin wax embedded. Sections (5 μm) were cut on a Leica rotary microtome RM2235 and mounted on slides . Sections (n = 3) were deparaffinized, rehydrated, and stained with H&E. Staining and coverslipping was performed on a Leica workstation operated by the Histology Facility at The University of Manchester. Slides were scanned on a slide scanner (Pannoramic P250 Flash III), and images were processed using 3DHISTECH software. Gill tissue damage was quantified using a score adapted from Mitchell et al. . Sequence adapters were removed, and reads were quality trimmed using Trimmomatic_0.36 (http://ftp.ensembl.org/pub/current_gtf/danio_rerio/Danio_rerio.GRCz10.84.gtf.gz) using STAR_2.5.3 . Normalization, principal component analysis (PCA), and differential expression were identified using DESeq2_1.16.1  and downregulated (log2 fold change < −1) genes in −/−il4/13a, −/−il4/13b, and −/−il4/13a;b gills.Gills were dissected from 6-mo-old fish; RNA was isolated and subjected to sequencing analysis. Prior to performing RNA sequencing (RNA-seq), the integrity of RNA samples was assessed using a 2200 TapeStation (Agilent Technologies), according to the manufacturer’s instructions. Total RNA was submitted to the Genomic Technologies Core Facility of the University of Manchester, and libraries were generated using the TruSeq Stranded mRNA assay (Illumina), according to the manufacturer’s protocol. Adapter indices were used to multiplex libraries, which were pooled prior to cluster generation using a cBot instrument. The loaded flow cell was then paired-end sequenced  on an Illumina HiSeq 4000 instrument. Finally, the output data were demultiplexed  and BCL-to-Fastq conversion performed using Illumina’s bcl2fastq software (version 2.17.1.14). Unmapped paired-end sequences were tested by FastQC . Gene ontology analysis and pathway analysis were performed with Enrich (https://software.broadinstitute.org/morpheus). xCell analysis was used to perform cell type enrichment analysis from gene expression data for 64 immune cell types (https://xcell.ucsf.edu).Cluster analysis was performed using k-means clustering (Manhattan distance) using maxdView software. Each cluster was then hierarchically clustered. Clustering was performed on the means of each sample group (log 2) that had been h Enrich  using huh Enrich . SeveralR848 gill challenge was performed as previously described  using 5 p values <0.05  were considered statistically significant. Throughout, error bars represent SEM.Statistical analysis was performed using GraphPad Prism version 8 (GraphPad Software). The il4-like genes, namely il4/13a and il4/13b, have been described  and an il4/13b allele carrying a 7-bp deletion were isolated (il4/13a (il4/13a−/−) and il4/13b (il4/13b−/−) were found to be viable, and they were intercrossed to generate il4/13a;b−/− double mutants. These were also found to be viable and fertile.In zebrafish, two similar escribed . To geneisolated . Homozygil4/13a mRNA in −/−il4/13a embryos was observed (il4/13b mRNA (1.6-fold) was detected in il4/13a−/− embryos , supporting the inflammatory phenotype. A similar increase of mpx was observed in il4/13a−/− larvae (3-fold) but not in il4/13b−/− larvae  and tnfα (40-fold) mRNA in the gills of il4/13a;b−/− double mutants (Genes belonging to cluster 4 were found highly upregulated in gills from response . Accordiresponse . Moreove mutants . To furt mutants , suggestil4/13a and il4/13b on zebrafish gill homeostasis, histology was performed. No significant morphological changes were observed in the gills of −/−il4/13a, −/−il4/13b, or −/−il4/13a;b fish compared with wildtype (e46/e46il10 mutants lacking functional IL-10, which have also been reported to shift toward type 1 immune responses (To further evaluate the effects of wildtype , suggestesponses . A thickesponses , 4B, indil10 in gill homeostasis, RNA-seq was performed on RNA extracted from the gills of e46/e46il10 mutant animals. Again, PCA confirmed a distinct expression profile for e46/e46il10 mutant animals (e46/e46il10 mutant animals. Moreover, a depletion of Th1 and Th2 cells but an enrichment of regulatory T cells and Th17 was also predicted (il1β, tnfα, and il6 mRNA encoding inflammatory cytokines but an increase of ifng1–2 mRNA was detected in e46/e46il10 gills compared with wildtype (To further investigate the role of  animals . Gene on animals . We also animals . To inveredicted . To valiwildtype .−/−il4/13a, and −/−il4/13b animals after 1 h as shown by significantly enhanced levels of il1β, il6, tnfα, and ifng1–2 mRNA encoding inflammatory cytokines in R848-treated fish compared with untreated controls (il1β, il6, and tnfα in both wildtype and mutant fish compared with the 1-h timepoint. ifng1–2 displayed a different kinetics, being significantly upregulated in both wildtype and mutant fish still at 8 h (We next sought to investigate the importance of IL-4/13a and IL-4/13b in modulating an inflammatory response in the gills to an exogenous source of irritant. To induce gill inflammation, we used an intervention we have recently established, whereby a solution of R848, a synthetic compound that mimics viral ssRNA and interacts with TLR7/8 in mammals , is applcontrols . Howeverl at 8 h . Again, −/−il4/13a;b double mutants for 1 and 8 h. Significantly enhanced levels of il1β, il6, tnfα, and ifng1–2 mRNA were observed in the gills of wildtype and −/−il4/13a;b animals after 1 h. Moreover, levels of il6 were significantly higher in −/−il4/13a;b compared with wildtype (il1β, il6, and tnfα were reduced almost to baseline in both wildtype and double mutants. However, ifng1–2 levels were still elevated and again significantly higher in the gills of −/−il4/13a;b double mutants compared with wildtype (Next, we evaluated the response to R848 in the absence of both IL-4/13a and IL-4/13b. R848 challenge was performed on the gills of wildtype . At 8 h wildtype , suggeste46/e46il10 fish were challenged with R848 for 1 and 8 h, as described before. Significant upregulation of il1β, il6, tnfα, and ifng1–2 mRNA were observed in the gills of stimulated wildtype and e46/e46il10 fish compared with untreated controls after 1 h. However, no difference was observed in the response between wildtype and mutant fish (il1β, il6, and tnfα transcripts were still significantly upregulated in gills from e46/e46il10 animals, and ifng1–2 transcript levels were even more pronounced (il10. In keeping with the exaggerated inflammatory response at 8 h posttreatment, thickening of gill filaments and lamellae were observed in gills from R848-treated e46/e46il10 animals but not wildtype animals (il10 gene induced an exaggerated inflammatory response in the gills following R848 stimulation.Loss of IL-10 resulted in marked alteration of gill morphology in the steady state as shown by the thickening of the gill arch and filaments as well as lamellar epithelial changes , 4B and ant fish , suggestonounced , indicat animals , 6C. AllIn recent years, the field of fish immunology has progressed considerably with many molecules and cell components of both innate and adaptive immune systems having been identified , 22, 58.il4/13a and il4/13b and characterized the consequences of loss of function of these genes under both steady state and inflammatory conditions. Under controlled laboratory conditions, zebrafish lacking IL-4/13A and IL-4/13B were viable, fertile, and did not show any overt phenotype, indicating that such cytokines are not essential for survival and development in zebrafish. Similar findings are observed in IL-4−/− and IL-13−/− mice that are healthy and display no phenotypic abnormalities (In this study, we have used the zebrafish as a model organism to shed light on fish malities .il4/13a and il4/13b induced a proinflammatory state in zebrafish larvae, indicating the role for IL-4/13A and IL-4/13B in suppressing inflammation. An anti-inflammatory role for fish type 2 cytokines has been previously demonstrated in trout in which the treatment of head kidney cells with rIL-4/13A and rIL-4/13B2 lead to the downregulation of proinflammatory cytokines (mpx in −/−il4/13a;b double mutants, supporting the inflammatory phenotype. The increase in mpx transcripts in the −/−il4/13a single mutants indicates that il4/13a may have a potential neutrophil-specific effect albeit one that does not drive a corresponding change in neutrophil number. Effects on mpx transcript were not observed in the −/−il4/13b mutant, suggesting a divergent function for these paralogs in neutrophils in zebrafish. In mammals, neutrophils express IL-4 type I receptor (−/−;mpx:EGFPil4/13a;b zebrafish line would be valuable to address neutrophil behavior in the absence of functional IL-4/13A and IL-4/13B.We found that disruption of both ytokines , resemblytokines . Neutropytokines . We founreceptor , 62, andreceptor , 64 to rreceptor . To date+ Th2-like lymphocytes resident in the zebrafish gill mucosa were a source of il4/13b expression but not il4/13a that was detected in gill tissue (il4/13a following helminth Ag exposure, resembling mammalian ILC2 cells (il4/13a. To shed light on the function of IL-4/13A and IL-4/13B in zebrafish gills, transcriptome analysis was performed in mutants for these cytokines. We found an enrichment of genes implicated in Th1 differentiation and IFN-γ signaling in the gills of il4/13a;b−/− double mutants, further supported by qPCR analysis. These data suggest that IL-4/13A and IL-4/13B are jointly required for the maintenance of a Th2-like phenotype in zebrafish gills, and their loss leads to an imbalance toward type 1 immunity. Evidence for IL-4/13 cytokines regulating type 2 immunity was previously reported in fish. In zebrafish, the interaction of IL-4/13A with IL-4Rα on B cell surfaces promoted B cell proliferation and Ab production (il4/13 paralogs was observed in the gills of Atlantic salmon following parasite infection (Previously, we demonstrated that CD4-1l tissue . Recent C2 cells . In mammC2 cells . ILCs mioduction ; in trouoduction , and uprnfection .−/−il4/13a;b double mutants. In mammals, basophils and eosinophils represent innate sources of IL-4 and IL-13, and their infiltration into tissues is one of the hallmarks of type 2 immune responses (−/−il4/13a;b double mutants is somehow connected, perhaps reflecting a divergent effect of IL-4/13 on the recruitment of these cell populations to gills. This is unanticipated from mammals in which basophils and eosinophils infiltrate tissues coordinately (The transcriptome data predicted a reduction in basophils but an increase in eosinophils in the gills of esponses . Basophiesponses , and a cesponses . It mighdinately .tnfα and il6 transcripts in the gills of −/−il4/13a;b double mutants indicate an inflammatory phenotype. Recent studies have identified subsets of polarized macrophages in zebrafish, describing a proinflammatory M1-like phenotype and anti-inflammatory M2-like phenotype, resembling the situation found in mammals (−/−il4/13a;b double mutants might also contribute to the induction of M1 macrophages, as previously demonstrated (The increase in the fraction of M1 macrophages as well as enhanced levels of  mammals . Our dat mammals –78. The nstrated , and to e46/e46il10 fish and found that IL-10 might be fundamental in maintaining homeostasis in the gills. Transcriptome analysis revealed evidence of smoldering inflammation in the gills of e46/e46il10 fish. In mammals, a lack of IL-10 leads to the development of spontaneous inflammation in the skin, intestine, and lungs (Despite the induction of proinflammatory signals, loss of IL-4/13A and IL-4/13B did not affect tissue morphology, suggesting that there might be further regulatory mechanisms in place to avoid the disruption of homeostasis in the gills. IL-10 is known to control mammalian immune responses to avoid inflammation and maintain tissue homeostasis. We examined the gills of nd lungs . In thisRecent work has described the use of R848 in inducing inflammation and cytokine response in zebrafish gills , highligWe used R848 to induce inflammation in the gills and found that loss of both IL-4/13A and IL-4/13B led to an enhanced response in the gills of double mutants, which appeared to be type 1 driven, in agreement with transcriptome data in the steady state. In mammals, IL-4 and IL-13 act together to ensure a successful Th2 inflammatory response, and a Th1 phenotype with increased expression of IFN-γ is observed in IL-4/IL-13–deficient mice . We infeMycobacterium marinum model (IL-10 appeared to be fundamental in the maintenance of gill homeostasis, and its loss enhanced the inflammatory response to R848 in the gills, supporting a potent anti-inflammatory function for this cytokine. An anti-inflammatory role for IL-10 has been previously shown in carp  and in zum model . For the−/−il4/13a, −/−il4/13b, and −/−il4/13a;b zebrafish mutants would be valuable models to investigate the function of il4/13 paralogs in the context of infection and disease. This would further our knowledge of fish immunology and provide comparative models to answer outstanding questions in mammals.Previous studies indicate that fish IL-4/13 paralogs might have divergent functions, with IL-4/13A providing a basal level of type 2 immunity and IL-4/13B required for specific T cell–mediated immunity . Our finData Supplement


Demand for the fabrication of high-performance, transparent electronic devices with improved electronic and mechanical properties is significantly increasing for various applications. In this context, it is essential to develop highly transparent and conductive electrodes for the realization of such devices. To this end, in this work, a chemical vapor deposition (CVD)-grown graphene was transferred to both glass and polyethylene terephthalate (PET) substrates that had been pre-coated with an indium tin oxide (ITO) layer and then subsequently patterned by using a laser-ablation method for a low-cost, simple, and high-throughput process. A comparison of the results of the laser ablation of such a graphene/ITO double layer with those of the ITO single-layered films reveals that a larger amount of effective thermal energy of the laser used is transferred in the lateral direction along the graphene upper layer in the graphene/ITO double-layered structure, attributable to the high thermal conductivity of graphene. The transferred thermal energy is expected to melt and evaporate the lower ITO layer at a relatively lower threshold energy of laser ablation. The transient analysis of the temperature profiles indicates that the graphene layers can act as both an effective thermal diffuser and converter for the planar heat transfer. Raman spectroscopy was used to investigate the graphite peak on the ITO layer where the graphene upper layer was selectively removed because of the incomplete heating and removal process for the ITO layer by the laterally transferred effective thermal energy of the laser beam. Our approach could have broad implications for designing highly transparent and conductive electrodes as well as a new way of nanoscale patterning for other optoelectronic-device applications using laser-ablation methods. For the fabrication of transparent electronic devices, such as touchscreen panels (TSPs) and flat-panel displays (FPDs), it is essential to develop transparent conductive electrodes (TCEs) with high transmittance and low resistivity. Although indium tin oxide (ITO) has been mainly applied as a typical material for TCEs in electronic devices because of its high figure of merit and good compatibility with conventional semiconductor-processing technology ,2,3, the2 electronic hybridization configuration [Graphene has been widely attempted to be used as one of the promising candidates for TCEs in various optical and electronic device applications owing to its high transmittance and high flexibility ,13,14. Tguration ,16,17. OIn this study, to realize a higher conductance of a TCE in an electronic device, a chemical vapor deposition (CVD)-grown graphene layer was transferred to indium tin oxide (ITO)-film-coated glass or polyethylene terephthalate (PET) substrates, and graphene on an ITO (graphene/ITO) double layer was suggested as a new TCE material. However, it is very difficult to produce fine electrode patterns because of the difference in etch selectivity between graphene and ITO layers. In the case of the wet etching process for the removal of the ITO layer, it is indeed challenging to maintain graphene-electrode patterns without damage. The electron-beam-lithography and plasma-etching techniques have been reported as some patterning methods for graphene ,19,20. O2) at a flow rate of 60 sccm, a few-layered graphene film was grown with methane (CH4) feedstock gas at a flow rate of 5 sccm. Subsequently, polymethyl methacrylate (PMMA) was spin-coated over the CVD-grown graphene layer on the Cu foil; then, the whole PMMA/graphene/Cu foil structure with Cu foil downside was carefully floated on ammonium persulfate 2S2O8, Sigma-Aldrich Korea Ltd., Seoul, Korea)) solution to etch off the Cu foil [A-few layered graphene films were produced in a CVD furnace and transferred to soda-lime-glass and PET substrates ,28,29, o Cu foil ,31, foll Cu foil , despite4) laser system  was used for the direct patterning of the prepared graphene/ITO-layered films  and 0.27637 , respectively. Those of the graphene/ITO/PET substrate were 0.18134  and 0.56833 , respectively. There is a tendency that in the same substrate, the lateral etch rate of graphene is higher than that of the ITO layer. This could be because of the difference in the thermal conductivity as well as film thickness between the two layers. For the same material, for example, the graphene layer with PET shows a higher value with increasing σ compared to that with a glass substrate. This result would be possibly due to the lower thermal conductivity [The difference in the laser-driven etched spot patterns between the graphene upper and ITO lower layers can be explained as a result of the higher thermal conductivity of graphene than that of the ITO layer . Therefouctivity ,40 as weuctivity . The theuctivity . The bou∂T∂z|ITO . κ is thComsol Multiphysics [The transient temperature distribution confirms the different characteristics of thermal-energy transport on graphene/ITO double and ITO single layers, enabled by the laser irradiation for 10 ns . The anaiphysics . First, iphysics  to 533 Kiphysics ) of the iphysics .−1. For the region 1, the three characteristic bands, D-, G-, and 2D-band, indicative of the existence of a graphene layer were found [−1) of weak intensity and both G- (~1580 cm−1) and 2D-band peaks (~2700 cm−1) on the graphene layer were observed. When the Raman spectra were acquired along the edge of a laser-etched spot, where the graphene upper layer was etched selectively but the ITO lower layer remained (region 2), very small traces of both the D- and G-band peaks were observed, but no 2D-band peak was detected, indicating that most of the graphene upper layer had been removed as a result of laser ablation. Compared to the Raman spectra of a center region of the laser-etched spot, on which no graphene-related peaks were observed (region 3), some of the residual amorphous carbon on the ITO lower layer was found, which is expected to have resulted from the incomplete heating and removal process for the ITO lower layer by the laterally transferred effective thermal energy of the laser beam.To confirm the removal of the graphene/ITO double layer after laser ablation, Raman spectroscopy was performed to examine a laser-etched spot on the graphene/ITO double layer a, where re found ,55,56,57In conclusion, we experimentally showed the laser-ablation-assisted patterning of graphene/ITO double layers for future TCE applications. To elucidate the underlying mechanism for the difference between ITO single and graphene/ITO double layers during the laser-ablation process, we also conducted a theoretical analysis of transient temperature spatial distribution, revealing thermal-energy transport on graphene/ITO double and ITO single layers. From the analysis, we demonstrated a role of effective thermal-energy transfer during laser ablation, indicating that the graphene layers can act as both an effective thermal diffuser and converter for the planar heat transfer. The analysis of Raman spectroscopy also supported this laser-ablation mechanism of the selective etching of graphene layers due to the effective thermal-energy transfer. Our study may provide insight into designing optimal electrode patterning using graphene layers, which would not only enable better electrode formation with high transparency and conductance for TCE applications but would also serve as a new way of nanoscale patterning for other optoelectronic-device applications using laser-ablation methods. Future work will be carried out towards the further reduction of the size of the pattern width as well as the development of complex shapes of pattern design.