We describe an autosomal dominant, multi‐generational, amyotrophic lateral sclerosis (ALS) pedigree in which disease co‐segregates with a heterozygous p.Y374X nonsense mutation within TDP‐43. Mislocalization of TDP‐43 and formation of insoluble TDP‐43‐positive neuronal cytoplasmic inclusions is the hallmark pathology in >95% of ALS patients. Neuropathological examination of the single case for which CNS tissue was available indicated typical TDP‐43 pathology within lower motor neurons, but classical TDP‐43‐positive inclusions were absent from motor cortex. The mutated allele is transcribed and translated in patient fibroblasts and motor cortex tissue, but overall TDP‐43 protein expression is reduced compared to wild‐type controls. Despite absence of TDP‐43‐positive inclusions we confirmed deficient TDP‐43 splicing function within motor cortex tissue. Furthermore, urea fractionation and mass spectrometry of motor cortex tissue carrying the mutation revealed atypical TDP‐43 protein species but not typical C‐terminal fragments. We conclude that the p.Y374X mutation underpins a monogenic, fully penetrant form of ALS. Reduced expression of TDP‐43 combined with atypical TDP‐43 protein species and absent C‐terminal fragments extends the molecular phenotypes associated with TDP‐43 mutations and with ALS more broadly. Future work will need to include the findings from this pedigree in dissecting the mechanisms of TDP‐43‐mediated toxicity. In this study Cooper‐Knock et al identified an autosomal dominant ALS pedigree associated with p.Y374X‐TARDBP. In patient tissue the authors confirmed reduced TDP‐43 protein expression, abnormal TDP‐43 fragmentation and deficient TDP‐43‐mediated splicing. This work extends the phenotypes associated with TDP‐43 mutations. The C:N‐terminal peptide ratio was calculated by dividing non‐log absolute abundances of the light peptides. Truncation peptides include one cleavage site independent of the enzyme used for digestion (giving a nonspecific digestion pattern), which suggests that cleavage has occurred endogenously prior to digestion.TDP‐43 protein fragmentation in the urea insoluble protein fraction from motor cortex tissue was quantified by immunoblot and mass spectrometry (MS) as previously described . In brie2.8g for 10 min. RNA was extracted from 250 μl lysate, with the remainder used for immunoblotting.Motor cortex tissue (~0.1 g) was lysed in 400 μl RNase free RIPA lysis buffer  using a Precellys evolution homogeniser and 8 × 1.4 mm Zirconium Oxide beads at 5500 rpm for 2 × 30 s, 10‐min incubation on ice and another round of homogenisation. Lysates were collected after centrifugation at 17,000 Protein extract concentrations were determined using a Bradford Reagent (Bio‐Rad), resolved by SDS‐PAGE electroblotted onto nitrocellulose membrane and probed using the relevant primary antibody. STMN2 (1:1000 dilution)  and TDP‐43 (C terminus) (1:1000 dilution)  were detected with horseradish peroxidase (HRP)‐conjugated rabbit secondary antibody (Promega), while α‐tubulin, clone DM1A (1:2000)  and TDP‐43 (1:3000)  were detected with HRP‐conjugated mouse secondary antibody (Promega). Immunoblots were visualised using a Licor Odyssey Fc and densitometry analysis performed using Image Studio Lite (Version 5.2.5).2.9g, 10 min, 4°C. The upper phase was collected and the RNA was precipitated overnight at −20°C with equal volume isopropanol and 1 μl glycogen  and subsequently pelleted at 17,000g, 20 min, 4°C. RNA pellets were washed with 70% DEPC ethanol and resuspended in DEPC water. All RNA samples were treated with DNaseI (Roche) and quantified using a Nanodrop (NanoDropTechnologies). Following quantification, 2 μg RNA was converted to cDNA using BioScript Reverse Transcriptase (Bioline). Human snRNA U1 ; human STMN2 , and human UNC13A  primers were used in this study. qRT‐PCR reactions were performed in duplicate using the Brilliant III Ultra‐Fast SYBR Green QPCR Master Mix (Agilent Technologies) on a C1000 TouchTM thermos Cycler using the CFX96TM Real‐Time System (Bio‐Rad) using an initial denaturation step, 45 cycles of amplification  prior to recording melting curves. qRT‐PCR data were analysed using CFX Manager software (Version 3.1) (Bio‐Rad) and quantification performed using the comparative CT method (35) and GraphPad Prism (Version 9).RNA was extracted from motor cortex using PureZOL (Bio‐Rad). Briefly, one fifth the volume of chloroform was added and tubes were vigorously shaken for 15 s followed by 10 min incubation at RT and centrifugation at 12,00033.1A Y374X truncating mutation in TDP‐43 c.1119_1120delTT) was identified in the present study. This mutation was detected in three affected kindred members Figure . In addidelTT was3.2The index pedigree is shown in Figure 3.3C9ORF72 [Neuropathological features were most prominent in lower motor neurons. There was loss of lower motor neurons that was most marked in the anterior horns of the spinal cord Figure  with BunC9ORF72 .3.4). To confirm expression of the mutated allele we analysed protein lysates from cultured fibroblasts, isolated from three members of the kindred, and motor cortex tissue, isolated from one member. The truncated protein was expressed  . Y374X‐TMotor cortex tissue from a single Y374X‐TDP‐43 ALS patient was fractionated to enrich for pathological insoluble species Figure  althoughMS‐PRM enabled antibody‐free quantification of TDP‐43 fragments and confirmed the findings of the immunoblot analysis. Heavy isotope‐labelled peptides corresponding to the N‐ and C‐terminal ends of the TDP‐43 protein Figure  were syn3.6POLDIP3/SKAR gene is altered by loss of TDP‐43 function resulting in exclusion of exon 3, and so RT‐PCR for this gene was performed [t‐test p = 0.2165, Figure Our analysis suggests that the quantity of total TDP‐43 protein is reduced in the presence of the Y374X mutation Figure  and thererformed , 27. The) and protein .We also utilised an indirect measure of TDP‐43 splicing function which has been linked directly to ALS pathogenesis. Recently, reports have connected reduced nuclear TDP‐43 content to missplicing of STMN2 and UNC13A and neurotoxicity in ALS , 10. Mis4Here, we have presented data from a multi‐generational ALS kindred with a Y374X truncating mutation within TDP‐43. This is the first time this atypical mutation has been shown to co‐segregate with ALS in a single pedigree, which represents good evidence for monogenic pathogenicity. The age of onset varied significantly in our pedigree which may explain the observation of this mutation previously in an apparently sporadic ALS case .Whilst dysfunction of TDP‐43 is undoubtedly associated with ALS  and gainThe discovery of TDP‐43 CTF  and coulIt should be noted that we tested TDP‐43 fragmentation in motor cortex tissue for consistency with our previous work . HoweverC9ORF72‐FTLD neurodegeneration can be associated with RNA foci and dipeptide‐repeat protein inclusions without TDP‐43 pathology [A caveat in our study of TDP‐43 fragmentation function is that the analysis is based on a single Y374X TDP‐43 ALS patient compared to up to five neurologically normal controls. In addition we focused on the motor cortex which was devoid of classical TDP‐43 pathology in this case. However, we note that there is precedence in the literature for disease‐associated neuronal dysfunction in the absence of classical TDP‐43 pathology. For example, TDP‐43 pathology is relatively more frequent in the spinal cord compared to the motor cortex even when there is clearly clinical evidence of upper motor neuron involvement . In C9ORathology . Finallyathology .Of significant interest is that the major domain identified to be abnormally phosphorylated in ALS cases, S409/410, is absent from the Y374X truncated protein , 35. HowIn conclusion we have demonstrated that the Y374X TDP‐43 truncating mutation is very likely to be a cause of autosomal dominant ALS. Moreover this mutation is associated with typical TDP‐43 pathology at a macromolecular scale in the spinal cord but also with atypical insoluble TDP‐43 protein fragments and reduced TDP‐43 protein function. These data extend the spectrum of observations associated with TDP‐43 pathology in ALS and will inform future work aimed at ameliorating neurotoxicity associated with this protein.J.C.K. C.H., P.J.S, R.J.M, J.R.H, and J.K. conceived and designed the study. J.C.K., E.F., J.R.H, M.S., S.P.A., T.M., T.S., L.C., G.M.H., C.H., J.K., and S.B.W, were responsible for data acquisition and data analysis. J.C.K., E.F., M.R.T., K.T., O.A., J.R.H, T.H.J., M.S., T.M., S.P.A., T.S., L.C., G.M.H., C.H., J.K. S.B.W, and P.J.S. were responsible for the interpretation of the findings. T.H.J., J.C.K., E.F., M.S., C.H., J.R.H., R.J.M., and P.J.S. drafted the manuscript with assistance from all authors. All authors meet the four ICMJE authorship criteria, and were responsible for revising the manuscript, approving the final version for publication, and for accuracy and integrity of the work.Emily Feneberg, Kevin Talbot, Martin R. Turner, and Olaf Ansorge have a patent filed for the TDP‐43 sequences described in this work (Patent application no. 2004863.3). Other authors delare no conflict of interest.Table S1 Sequenced genes with no identified ALS‐associated mutation in members of the Y374X‐TDP‐43 ALS pedigree.Click here for additional data file.


We integrate these graphene-coated glass rovings into a vacuum-infused epoxy–glass fabric composite and also demonstrate the potential use of as prepared graphene-based composites for de-icing applications.Graphene-based de-icing composites are of great interest due to incredible thermal, electrical and mechanical properties of graphene. Moreover, current technologies possess a number of challenges such as expensive, high power consumption, limited life time and adding extra weight to the composites. Here, we report a scalable process of making highly conductive graphene-based glass fibre rovings for de-icing applications. We also use a scalable process of making graphene-based conductive ink by microfluidic exfoliation technique. The glass fibre roving is then coated with graphene-based conductive inks using a dip-dry-cure technique which could potentially be scaled up into an industrial manufacturing unit. The graphene-coated glass roving demonstrates lower electrical resistances (∼1.7 Ω cm We report a scalable process of making highly conductive graphene-based glass fibre rovings and their integration into a vacuum infused epoxy–glass fabric composite. We then demonstrate the potential use of as prepared composites for de-icing applications. Whereas ice accumulation on a wind turbine can cause mechanical and electrical failures; results in up to 50% power losses.10 Several thermal, chemical and mechanical methods8–11 have been developed for eliminating the surface ice such as chemical fluid systems,9,11 pneumatic boots,12 electro-impulsive,13 hot air system,11 ultrasound system,7,14 microwave technology15 and electro-thermal de-icing system.2 Among these, electro-thermal de-icing technique is considered to be the most effective and energy efficient due to its ability to control the temperature and heat dissipation by Joule heating.16Recent years have seen significant interest in developing smart de-icing systems due to problems associated with the ice formation in many industrial applications such as aircrafts,17 However, ice accumulation reduces the advantages that the composite brings to the structure. The electro-thermal system is identified as one of the most promising de-icing systems for polymer composites, as it does not cause delamination and damage to composite structure.9,11 However, the application of the electro-thermal system within composites is limited by the poor thermal conductivity and high thermal sensitivity of polymeric materials.18,19 Many studies20–22 have reported uses of conductive polymers, metals, CNT and carbon black to make conductive polymer composites; however they still suffer from poor thermal and electrical conductivity, and higher energy consumption. Therefore, it is desirable to use a conductive material that can provide excellent electro-thermal properties as well as can achieve desired temperature without compromising existing mechanical and thermal properties of composites.Currently, fibre reinforced polymer composites are increasingly popular in aerospace, automobile and civil engineering industries due to their higher strength and lower weight.2 carbon allotropes, has been focus of mass investigations in recent years due to its large surface area,23 record thermal conductivity,24 excellent mechanical strength,25 and superior electronic mobility.26 It has demonstrated much higher thermal conductivity ∼5000 W mK−1 than CNT (∼3000 W mK−1) and amorphous carbon (∼1 W mK−1).27 Previous study has demonstrated the use of graphene-based inks to produce highly conductive electrical conductor with lower resistance.28 Moreover, a small amount of graphene would be sufficient to form a percolative electric network due to higher aspect ratio and surface area;29 thus reduce overall weight of the composite. Recent studies have highlighted using of Graphene Nanoribbon (GNR),2,30 perfluorododecylated GNR31 and Graphene Nanoplatelets (GNP)32 for de-icing applications. However, there are number of problems still exist with these techniques such as complicated and expensive manufacturing route for GNR, not suitable for industrial applications and material waste. Moreover, strong van der Waals forces between graphene fillers may cause re-stacking in the polymer matrix, which may result in defective composites.33 Moreover, increased GNR concentration modifies the resin viscosity and increases the brittleness of composites.34Graphene, a single atom thick two-dimensional closely packed honeycomb lattice of spHere we report a scalable manufacturing route for next generation graphene-based de-icing composites. We utilise microfluidic exfoliation technique to synthesize graphene-based ink in a scalable quantity and use a dip-dry-cure coating technique to make highly conductive graphene-based glass fibre rovings. These graphene-coated glass rovings are characterised both electrically and thermally; then integrated into a composite structure. We demonstrate the use of as prepared composite for de-icing applications using Joule heating phenomena.2.2.1The natural flake graphite  was kindly supplied by Graphexel Limited, UK. Sodium deoxycholate (SDC) powder was purchased from Sigma Aldrich, UK. The glass fibre roving  was purchased from AGY (USA). 290 gsm (grams per square meter) plain woven glass fabrics, EL2 Epoxy Laminating Resin and AT30 Epoxy Hardener were purchased from Easy Composites, UK.2.228,35 Briefly, 50 g graphite powder and 10 g SDC are added into a glass bottle and mixed with 500 ml deionised (DI) water. This mixture is sonicated for 30 minutes using an ultrasound bath to allow homogenous dispersion and added into an input reservoir of a Microfluidizer . The dispersion is slowly passed through ‘Z-type’ microfluidic channels of ∼200 μm and ∼87 μm diameter with diamond construction at high pressure (∼200 MPa). This allows the exfoliation of graphite to few-layer graphene (FLG) at 100 ml min−1 flow under high shear rate [∼108 s−1] with a 100% exfoliation yield. The exfoliated dispersion is then passed through a cooling channel surrounded by cold water (∼25 °C) to prevent over-heating of the dispersion and collected. This process is repeated 20 times to produce FLG (MF flakes) which is then used as a conductive ink for glass roving coating.We use microfluidization technique to exfoliate graphene in a scalable quantity following previously reported methods.2.3We use a simple dip-dry-cure coating technique to coat glass fibre roving with graphene-based ink. The graphene-based ink is added into a 100 ml cylinder flask. Glass rovings are cut to 25 cm and dipped into graphene dispersion for 5 seconds. The coated glass fibre rovings are then passed through eyelets to squeeze extra ink out of coated glass tows and to maintain coating evenness. We then use a Mathis Laboratory dryer  to dry (at 100 °C) and cure coated glass fibres. We also use various curing temperatures , curing times (5 min to 30 min) and a number of coating cycles to optimise coating conditions.2.4We use vacuum resin infusion process and room temperature thermoset EL2 epoxy resin to manufacture de-icing composites that contains graphene-based (coated) glass rovings. Briefly, 6 layers of glass fabric (dimension: 10 × 10 cm) are laid on a pre-cleaned and pre-coated (with a release agent) metal plate. The graphene-based glass rovings are then inserted after 3 layers of glass fabric and connected to wires for electrical and thermal characterisation. The sample is sealed by a plastic bag and vacuumed pressed using a pump. EL2 Epoxy Laminating Resin and AT30 Epoxy Hardener are degassed separately for 30 minutes and mixed together immediately before we use. The resin with hardener is then flown over layered glass fabrics at a constant flow rate using a vacuum pump, which enables the impregnation of glass fabrics with resin. The resin infused preform is then cured at room temperature for 24 hours to make graphene-based glass composites for de-icing application.2.52 (290 nm oxide on plain silicon). The images and Raman spectra of MF flakes are taken at 10 different locations on the drop-casted sample to assess the flake size, flake thickness and flake types. We use an optical microscope to measure the flake size and Philip XL30 Field Emission Gun (FEG) Scanning Electron Microscope (SEM) to assess the surface topography of the untreated and coated glass rovings. A Dimension Icon (Bruker) Atomic Force Microscopy (AFM) is used to determine the flake thickness. A Renishaw Raman System equipped with 633 nm laser is used to collect Raman spectra of MF flakes, untreated and coated glass rovings. The surface of MF flakes, untreated and coated glass rovings is characterised using a Kratos Axis X-ray Photoelectron Spectroscopy (XPS) system. The thermal decomposition of graphite, SDC and MF flakes are evaluated using a TA instrument, TGA Q5000. The resistances of graphene-coated glass rovings per unit length are measured using a two probe multi-meter . The average resistance is calculated from 5 measurements at different positions along the length of graphene-based (coated) glass fibre rovings.The graphene-based dispersion containing MF flakes is diluted 1000 times and then drop-casted on Si/SiO2.6The electro-thermal behaviour of coated glass fibre rovings is measured using a thermal camera  and a power supply unit  which supplies direct current at different voltages from 0–30 V. The graphene-based glass fibre rovings are clamped on a wooden plate using two metals clamps at 1, 5, 15 and 20 cm distances. A multi-meter is also used to measure the current. The thermal camera is installed on a tripod and connected to the computer. The thermal images obtained by the camera is shown on the computer through a software . The change in temperature of the coated glass is observed and recorded at various volts and currents. Thermal images of coated glass at various volts and currents are also captured.3.3.1d < 100 μm) at high pressure (up to 209 MPa),35 which generates liquid velocities of 400 m s−1 and several order of magnitudes higher shear rates (>108 s−1)36 than conventional rotor-based or other homogenisers. It is used primarily for particle size reduction,37 nano-emulsion of immiscible liquids,38 for disrupting or lysing cells39–41 and de-agglomeration and dispersion of carbon nanotubes and graphene nano-platelets (GNP) into polymers.41,42 Recent studies have highlighted using microfluidization technique to produce graphene,28,35 graphene quantum dots36 and two-dimensional (2D) boron nitride nano-sheets.43 It is a simple and environmental friendly technique that can produce graphene-based ink with a 100% exfoliation yield.28 Moreover, this process could be scaled up to produce hundreds of kilos graphene-based inks, which would suit perfectly for industrial applications such as manufacturing of graphene-based multi-functional composites.We use microfluidization technique to exfoliate few layers graphene (MF flakes) from graphite into a water-based dispersion. Microfluidizer can pass fluids through micro-channels  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C bond in aromatic rings (∼284.6 eV),  of starting graphite above 750 °C; whereas MF flakes start to decompose at lower temperature (∼5.5 wt% up to 750 °C), may be due to the lower thermal stability of smaller MF flakes.44 As expected, SDC suffers significant decomposition around 400 °C.We use Graphexel 2736 grade graphite (average flake size < 50 μm) supplied by Graphexel, UK. 3.2For electro-thermal de-icing application, it is desirable to produce highly conductive and uniform glass-fibre roving using a scalable process. We use a very simple dip-dry-cure technique that could potentially be scaled-up for industrial applications. As illustrated in 45 However, after a certain temperature, further increase in temperature results in slight increase in the electrical resistance of graphene-based glass fibre roving. This optimum temperature point for lowest resistance varies with the curing time, as higher curing time provides lowest resistance at lower optimum curing temp and vice versa. We use 220 °C for 5 minutes as optimum curing condition to enable rapid drying and curing in a continuous manufacturing process. 2,46 Moreover, the contact resistance between graphene flakes are reduced with the increase of coating cycles. The lowest resistance of coated glass-fibre roving with graphene-based ink is obtained as ∼1.7 Ω cm−1 with 15 coating cycles.−1, ∼1582 cm−1 and ∼2700 cm−1, corresponding to D, G and 2D peaks, respectively. The D peak is caused by the defects and disorders in the hexagonal graphitic layers, while G peak attributed to an E2g mode of graphite due to the vibration of sp2-bonded carbon atoms in a two dimensional hexagonal lattice.47 These Raman spectra are similar to the spectra of a typical liquid-phase exfoliated graphene or graphitic materials.28,35,48 The wide scan XPS spectra of control glass-fibre reveal the presence of C1s, O1s, Si2s and Si2p and provide the evidence of some silane coating on the fibre surface, Raman spectra of control glass roving shows smooth featureless background of uncoated fibre, which is masked by strong fluorescence, C (284.6 eV) and C–O (286.4 eV), C, which is similar to graphene or graphite.49The high resolution C1s spectrum of control glass-fibre can be fitted into two main components: C–C/CSEM images provide further evidence of the presence of graphene flakes, covering the glass fibre, 3.3We use four different lengths  of graphene-based (coated) glass fibre rovings for electro-thermal characterisation and five samples for each length. We apply various voltages across the entire sample, and measure the current generated and the change in the temperature of graphene-based rovings (due to Joule heating) with voltages and the power consumed per unit length, I–V curves for various length of graphene-based glass roving show the linear relationship between the applied voltages and current passes through conductive rovings, p = IV/L), the curves for the samples of different length collapse on the same straight line indicating that the major dissipation mechanism is the thermal radiation and convection rather than thermal conductivity through the contacts. Moreover, thermal images show uniform heat distribution along the length of conductive graphene-based glass rovings . Moreover synthesis of GNR is based on complex, time consuming and expensive process of unzipping CNTs.50 Furthermore, GNR was mixed with epoxy resin which may alter the rheology and uniformity of epoxy/hardener mixture and limit the concentration of conductive materials.51 In contrast, we use a scalable process for manufacturing graphene-based glass roving. Moreover, the coating of individual glass roving with graphene-based ink would enable insertion of coated warp or weft glass roving into the fabric or composite structure as required. This would potentially reduce the material waste and provide much better control and flexibility to remove various amount of ice from different parts of the structure.In order to demonstrate a potential de-icing application of graphene-based glass rovings, we manufacture a glass–epoxy composite using a vacuum resin infusion process, where five graphene-based glass rovings are inserted in the middle of 6 glass fabrics layers (ESI, Fig. S4a and bFinally, we demonstrate the de-icing capability of our graphene-based glass/epoxy composites by dipping it into an ice-bucket, 4.−1 and efficient heating to a desired temperature at lower power consumption. We also demonstrate the use of this graphene-based glass rovings for manufacturing glass/epoxy composite for de-icing applications. The de-icing test shows efficient removal ice from graphene-based composite. We believe, the obtained results from this study would be an important step towards realising graphene-based next generation lighter, stronger and cost-effective smart de-icing composite for aerospace and other applications.We report a scalable process of manufacturing graphene-based surface heating for de-icing applications. The graphene-based glass roving demonstrates low resistance, ∼1.7 Ω cmThere are no conflicts to declare.RA-008-C8RA02567C-s001


Fractional quantum Hall effect (FQHE) appeared at 1.7 K after encapsulation by h-BN, yielding ultra-high mobility of ~280,000 cm2 V−1 s−1. Integrated wafer-scale graphene thermal emitters exhibited significant broadband emission in near-infrared (NIR) spectrum. Overall, the proposed methodology is promising for future integration of wafer-scale 2D materials in advanced electronics and optoelectronics.The integration of large-scale two-dimensional (2D) materials onto semiconductor wafers is highly desirable for advanced electronic devices, but challenges such as transfer-related crack, contamination, wrinkle and doping remain. Here, we developed a generic method by gradient surface energy modulation, leading to a reliable adhesion and release of graphene onto target wafers. The as-obtained wafer-scale graphene exhibited a damage-free, clean, and ultra-flat surface with negligible doping, resulting in uniform sheet resistance with only ~6% deviation. The as-transferred graphene on SiO Defect-free integration of 2D materials onto semiconductor wafers is desired to implement heterogeneous electronic devices. Here, the authors report a method to transfer high-quality graphene on target wafers via gradient surface energy modulation, leading to improved structural and electronic properties. Note that, an essential prerequisite is transferring 2D materials from their growth substrates to industrial wafers. Yet, a significant gap still exists in transfer method, which frustrates the recent progress in wafer-scale single-crystal growth of 2D materials10. Conventionally, wet transfer methods commonly use poly(methyl methacrylate) (PMMA) film as a transfer medium to support 2D materials when separated from the growth substrate and scooped from the liquid surface to a target substrate12. For graphene, the wet transfer methods may significantly degrade its properties due to introduced cracks, wrinkles, polymer contaminations, and water doping by water adsorptions on the graphene surface18. These factors act as extra scattering centers to limit carrier mobility and decrease device performance. To overcome these issues, previous attempts have shown that the optimization of PMMA and replacement of PMMA with small molecules or other polymers would facilitate clean graphene transfer27, conformal contact with the target substrate may reduce the formation of cracks and wrinkles31, and the development of dry transfer methods may diminish water doping by preventing the submersion of target substrate in liquids39. However, no method has so far entirely solved these issues, and most approaches are not compatible with high-volume semiconductor technology at the wafer level41.The integration of two-dimensional (2D) materials into current silicon technology may embed high mobility, dangling band-free interface, atomic-scale channel size into practical electronic and optoelectronic devices43. In this case, the higher surface energy of a target substrate, the better it serves as the thin film ‘acceptor’ due to the better wetting and larger adhesion strength at interface44. Thus, the surface energies of transfer medium and target substrates should be engineered to ensure reliable adhesion and release45, critical features for securing the wafer-scale 2D materials integration46. As a result, the gradient surface energy (GSE) modulation approach conduced to the integration of 4-inch single-crystal ultra-flat graphene onto silicon wafers. The transferred graphene wafer maintained its flatness, exhibiting intact and clean surfaces with negligible water doping. Consequently, the resulting wafer-scale graphene illustrated a uniform sheet resistance of only ~6% deviation over a 4-inch area. The as-transferred graphene on SiO2/Si exhibited outstanding electrical performances with smaller Dirac points and much higher carrier mobility  at room temperature when compared to conventional wet transfer (~2000 cm2 V−1 s−1). The quantum Hall effect (QHE) was also observed at room temperature in graphene transferred on SiO2/Si, and fractional quantum Hall effect (FQHE) was recorded at 1.7 K in transferred graphene encapsulated by h-BN with high mobility reaching ~280,000 cm2 V−1 s−1. Furthermore, the integrated thermal emitter arrays fabricated on a 4-inch graphene/silicon wafer showed significant emission with a broad spectrum in NIR region.Here, we designed a multi-functional tri-layer transfer medium with gradient surface energy distribution, according to the thin-film adhesion theory that transfer of thin film from one layer to another layer is mainly dominated by the difference in surface energy of each layerTo minimize the adverse effects of grain boundaries and wrinkles on the charge carrier mobility, single-crystal ultra-flat graphene films were grown on 4-inch Cu111)/sapphire wafers and the details can be found in Methods. A multi-functional tri-layer transfer medium was designed to support the wafer-scale graphene during transfer  and A (the adhesive), respectively; and γAB is the interface energy between phase A and B. The fracture strength σf of the interface is related to λAB by:Km is a function of the mechanical properties. Considering that γAB ≪γA, the fracture strength σf is proportional to the surface energy ratio of the adherend to the adhesive (γB /γA).Most importantly, the surface energy gradually decreased from the destination SiOyer Fig. b, c, who2/Si (γ1) was much larger than that of graphene/borneol (γ2), the complete wetting and reliable adhesion of graphene to the SiO2/Si wafer was facilitated  that is close to PMMA (γ3) ensured the damage-free release of wafer-scale graphene onto SiO2/Si wafer  allowed successful integration of the 4-inch single-crystal graphene onto the SiO 22 Fig. , owing t 22 Fig. . In gene 22 Fig. –14. In t2/Si using the GSE strategy , whose standard deviation was only ~6% over the 4-inch wafer , resulting from the uneven distribution of cracks and contaminations introduced during the transfer21  and 2D band (ω2D) were extracted from the Raman spectra, and the correlation maps were plotted in Fig. ωG = 1582 cm-1, ω2D = 2677 cm-1), corresponding to neither doping nor strain47. The PMMA-transferred graphene films are often deeply p-doped when using wet transfer methods due to water doping at the interface21, leading to changes in the Fermi level of graphene and declined carrier mobility. As shown in Fig. 2/Si wafer experienced deeply p-doping. By comparison, GSE-transferred graphene on SiO2/Si wafer nearly experienced no p-doping and strain, thereby close to the intrinsic graphene  of transferred graphene were gathered in Fig. Γ2D of GSE-transferred graphene (~30 cm-1) was smaller than that of PMMA-transferred graphene (~40 cm-1), indicating GSE-transferred graphene with little random strain fluctuation and potentially high charge carrier mobility49.To examine the doping and strain level of GSE-transferred graphene, the peak positions of G band  was an order of magnitude higher than that of GSE-transferred graphene, showing a relatively low μ of 1930 cm2 V−1 s−1. Accordingly, the average hole mobility of GSE-transferred graphene reached ~6000 cm2 V−1 s−1 . In addition, the mobility of wet-transferred graphene by only using PMMA/borneol as the transfer medium is ~3950 cm2 V−1 s−1, much lower than that of GSE-transferred graphene, which indicate the water-adsorption-induced doping will significantly degrade the electrical properties of graphene  to measure the field-effect carrier mobility of graphene on SiOs−1 Fig. , comparas−1 Fig. , a value2/Si extracted from the Hall effect measurement was 9500 cm2 V−1 s−1 at room temperature. By measuring the Hall resistance (Rxy) and magnetoresistance (Rxx) at different temperatures  and Hall (Rxy) magnetoresistance measurements at 1.7 K with a fixed magnetic field (B = 8.5 T), quantized Hall platform and magnetoresistance zeros were observed at all possible integer fillings of n = 0 and n = 1 Landau Levels (LLs) h/e2 with minimum Rxx at fractional filling factors υ = 2/3 and 4/3 in n = 0 LL, as well as υ = 7/3, 8/3, 10/3, and 11/3 in n = 1 LL were observed, confirming the fractional quantum Hall effect (FQHE) feature. The back gate (Vg) dependent resistant measurements at the different magnetic fields on h-BN-encapsulated transferred graphene Hall-bar device were then performed to resolve the broken Landau level degeneracy and FQHE in the Landau fan diagram  flake was employed to pick up and encapsulate the transferred graphene from SiOate Fig.  to fabriate Fig. . The Halene Fig. . Accordiene Fig. , the extLs) Fig. . Furtherram Fig. . The obs55. As shown in Fig. I through graphene thermal emitter device with narrow constrictions led to enhancements in the Joule heating, as well as localized light emission of graphene at the middle of the constriction56. As shown in Fig. Graphene-based silicon-chip blackbody emitters in the near-infrared region, including telecommunication wavelength hold promise in applications in on-Si-chip, small footprint, and high-speed emitters of highly integrated optoelectronics and silicon photonics2O3 layer was deposited on the graphene before voltage application  camera under vacuum at power density P = 3.0 kW cm−2 , allowing the observation of quantum Hall effect (QHE) at room temperature. Fractional quantum Hall effect (FQHE) also appeared at 1.7 K in the transferred graphene encapsulated by h-BN, with mobility reaching ~280,000 cm2 V−1 s−1. Furthermore, integrated thermal emitter arrays with the 4-inch graphene/silicon wafer illustrated significant emission with a broad spectrum in NIR region. In sum, the proposed methodology can be used as a universal approach for the integration of other intrinsic 2D materials, such as h-BN and 2D MoS2 on the wafer level, paving the way for the development of integrated high-performance electronics and optoelectronics.The transferred graphene enhanced the electrical performance due to the negligible doping level and much fewer scattering centers when compared to conventional PMMA-transferred graphene. The Hall-bar devices fabricated with graphene on SiOFurthermore, our GSE transfer method was successfully reproduced by an independent research group (as shown in the Peer Review file), demonstrating the robust reproducibility of GSE method in the transfer of large-area 2D materials.Ra < 0.2 nm) using a sputtering system (ULVAC QAM-4W). The deposition rate was 0.3 nm s-1 with a power of 300 W at 8 × 10−4 Torr, and a ~500-nm-thick Cu film was obtained on sapphire after 30 min. After that, the Cu/sapphire was then annealed at 1000 °C with 2000 sccm Ar and 10 sccm H2 for 2 h at atmospheric pressure in a homemade annealing furnace to obtain Cu(111) single crystal.The Cu(111) film was deposited on a single-crystal sapphire  and 40 sccm H2 was introduced for graphene growth. Fully covered graphene was obtained after ~2 h, and CH4 gas flow was turned off while the sample cooled to room temperature.The 4-inch Cu(111)/sapphire was heated to 1000 °C with 2000 sccm Ar at atmospheric pressure, then 40 sccm CH59. The heating and annealing processes were carried out under 500 sccm H2 and 100 sccm Ar.Commercially available polycrystalline Cu foils were placed in a homemade annealing furnace equipped with a 6-inch quartz tube. Three heating zones were asynchronously heated up from room temperature to target temperatures  in 40 min and kept for 1 h, which leads to the formation of a temperature gradient through the Cu foils (about 2 °C/cm) and promotes the anomalous Cu grain growth2 for 30 min. Then the growth of graphene was initialed by the introduction of 1 sccm CH4. Fully covered graphene was produced after 1-h growth. Finally, the system was cooled down to room temperature.The rough graphene was grown on Cu(111) foil using a low-pressure CVD system. Firstly, The Cu(111) foil was heated to 1000 °C with 500 sccm Ar, followed by annealing with 500 sccm H4S2O8 . After washing with deionized water to remove residual etchant, the PMMA/Borneol/graphene film was attached to the Si substrates, and the PDMS  was laminated on the surface of PMMA after the graphene was dried using the commercial laminator . The composite film of PDMS/PMMA/Borneol/Graphene was detached from Si substrate in water because the water will intercalate into the interface of graphene and Si substrate due to the hydrophilic surface of SiO2/Si , and the PDMS was exfoliated from the PMMA at 180 °C in 5 min. To further enhance the interaction of graphene and substrate, we baked the graphene at 180 °C for 3 h before removing PMMA and borneol with the vapor of hot acetone , leaving the monolayer graphene on target substrate.A step-by-step protocol is available as a Supplementary Protocol in The detachment of graphene from Cu(111)/sapphire can also be achieved by electrochemical bubbling delamination when PDMS/PMMA/borneol was used as a composite support layer, which reduced the time of transfer of graphene and preserved the Cu(111)/sapphire wafer.4S2O8. After washing with deionized water to remove residual etchant, the PMMA/graphene film was attached to the SiO2/Si substrates at the surface of water. To further enhance the interaction of graphene and SiO2/Si, the PMMA/graphene/SiO2/Si was baked at 180 °C for 3 h before removing PMMA with the vapor of hot acetone, leaving the monolayer graphene on SiO2/Si.First, a layer of PMMA was spin-coated on graphene/Cu(111)/sapphire or graphene/Cu(111) foil at 1000 rpm for 1 min, and baked at 170 °C for 3 min. After that, PMMA/graphene film was detached from growth substrate by etching the Cu film in an aqueous solution of 1 mol/L (NH)Ead, short chain of PMMA (C22H38O8) terminated with -CH3 without introducing additional polarity of the polymer was used in our first-principles calculations. Both borneol and PMMA short chain were placed on the surface of graphene with a ~3.3 A distance, measured from the center plane of molecule to the plane of graphene. The adsorption energy is defined as:To evaluate the adsorption energies of borneol and PMMA on graphene surface a = 24.56 Å, b = 24.56 Å, c = 21.70 Å. The 11 × 11 supercell of graphene was chosen for all the calculations. The PBE with the optB86b-vdW correction functional was used to determine the total energies for each structure, taking into account the Van der Waals interaction between polymer and graphene.All the structures were optimized using the density functional theory (DFT) implemented in VASP. To keep consistency, all the periodic cells are of the same size with 2/Si with marks for alignment. Electron-beam lithography and plasma etching with air (Diener Pico) were employed to pattern graphene into a Hall-bar geometry. After a PMMA mask (PMMA 950 K A4 @ 4000 rpm) was patterned by EBL, Pd/Au (5/40 nm) electrodes were deposited by thermal evaporation , followed by a standard lift-off technique. Limited by the stage size of electron beam lithography, we fabricated 42 graphene Hall-bar devices on 10 slices from the same 4-inch GSE-transferred graphene wafer and 18 graphene Hall-bar devices on 5 slices from another 4-inch PMMA-transferred graphene wafer. The area of each slice is 1 × 1 cm2.Hall-bar devices were fabricated on the graphene/SiOElectrical transport at room temperature was determined using a vacuum-probe station (Lakeshore CRX-VF) with a semiconductor characterization system . Electrical transport at low temperature and magneto-transport data were acquired using a low temperature and strong magnetic electronic measurement system . Device resistance was measured using a lock-in amplifier (Stanford Research 830) with an AC driving current of 0.1–1 μA.2O3 layer was grown by atomic layer deposition method. The Raman spectroscopy of graphene were measured by a confocal microRaman spectroscope  with a solid-state laser at 532 nm. The Raman signals were dispersed by a grating of 1800 lines/mm ensuring a high spectral resolution of ~1.0 cm−1. The spectrometer was calibrated by a quartz tungsten lamb at temperature of 3200 K before measurement. The thermal emission spectra were recorded using a spectrometer and a liquid nitrogen cooled Si CCD with a 50× objective lens. The radiation signal was acquired using an infrared camera. All measurements were carried out at room temperature and in vacuum.The arrays of graphene thermal emitters were fabricated by UV lithography machine, and AlOptical microscopy was conducted on a Nikon Olympus LV100ND. Raman spectra of transferred graphene were collected on a Horiba LabRAM HR Evolution Raman system using a 532 nm laser with a laser spot size 1 μm, and a 100x objective and 600 lines/mm grating were used to collect the Raman signal.The contact angle images of tested liquids on different surfaces were obtained using Biolin THETA optical tensiometer, the volume of the droplet on surface was controlled at about 4 μL.SEM images were obtained on an FEI Quattro S field-emission scanning electron microscope using a 5 kV acceleration voltage. The aberration-corrected STEM images of graphene were performed using a Nion U-HERMS200 microscope at 60 kV.The AFM morphology images were collected on a Brucker Dimension Icon using the ScanAsyst mode.The sheet resistances of transferred graphene on 4-inch wafers were collected by CDE ResMap 178 four-probe resistance tester.Supplementary InformationPeer Review FileSupplementary Movie 1Supplementary Movie 2


N = 55) during a face-to-face interaction. We used functional-near infrared spectroscopy hyperscanning to measure dyads' brain activity and microcoded their turn-taking. We also measured infants’ inter-hemispheric connectivity as an index for brain maturity and later vocabulary size and attachment security as developmental outcomes potentially linked to turn-taking. The results showed that more frequent turn-taking was related to interpersonal neural synchrony, but the strength of the relation decreased over the course of the proto-conversation. Importantly, turn-taking was positively associated with infant brain maturity and later vocabulary size, but not with later attachment security. Taken together, these findings shed light on mechanisms facilitating preverbal turn-taking and stress the importance of emerging turn-taking for child brain and language development.Even before infants utter their first words, they engage in highly coordinated vocal exchanges with their caregivers. During these so-called proto-conversations, caregiver–infant dyads use a presumably universal communication structure—turn-taking, which has been linked to favourable developmental outcomes. However, little is known about potential mechanisms involved in early turn-taking. Previous research pointed to interpersonal synchronization of brain activity between adults and preschool-aged children during turn-taking. Here, we assessed caregivers and infants at 4–6 months of age (This article is part of a discussion meeting issue ‘Face2face: advancing the science of social interaction’. Across In early human ontogeny, infants as young as eight weeks of age actively engage in turn-taking in vocal interactions with their caregivers . These e (a)As turn-taking depends on precise temporal coordination, one key candidate underlying mechanism is the mutual entrainment and synchronization of endogenous neural oscillators in the brains of the conversation partners . AccordiImportantly, we know little about when the relation between interpersonal neural synchrony and turn-taking emerges during development. Gaining insights into the functional role of interpersonal neural synchrony is important as it could pave the way for interventions promoting neural synchrony and its related benefits ,24. Furt (b)As infants continue to partake in early proto-conversations, including turn-taking, they become more competent at turn-taking and increasingly initiate turn-taking sequences . We suggEarly turn-taking has been associated with developmental benefits in language processing and acquisition ,29. For Next to infants’ individual development, turn-taking in mother–infant proto-conversations is assumed to be one fundamental feature of interpersonal connectedness ,22 and t. 2 (a)N = 81) were excluded from the neural synchrony analysis due to early termination of the experiment (n = 15), not codable video/audio (n = 5), and bad functional near-infrared spectroscopy (fNIRS) signal quality (n = 6). Subsequent sample sizes were further decreased because parents either did not return the language development questionnaire (n = 27) or did not participate in the home visits to assess attachment (n = 23). Mothers were, on average, 33.35 years old , of European White origin, and highly educated (76% of mothers graduated with a university degree). Participants were recruited from the database of the Department of Developmental Psychology (Wiener Kinderstudien). Most of the registered families were recruited at Vienna General Hospital, and their children were added to the database after their parents gave their written consent. Mothers and infants received a small present for their participation. The local Ethics Committee approved the study (reference no. 00352).Fifty-five mother–infant dyads  were included in the final sample. Infants were all born healthy and full-term (after the 37th gestation week and with a birth weight of more than 2.500 g). Twenty-six additional dyads of the original sample Mothers and infants participated in three conditions: distal joint watching, proximal joint watching and free play. In the joint watching conditions, the infant was seated in a highchair  or in the mother's lap  and watched a calm aquarium video for 90 s. The order of distal and proximal joint watching conditions was counterbalanced. In the free play condition, mother and infant were instructed to freely play face-to-face, as they would at home, but without toys and without singing for five minutes. The free play condition always followed the two watching conditions. Only interpersonal synchrony data from the free play condition are reported here  (i)We used two NIRSport 8-8  devices in the tandem setting and simultaneously recorded concentration changes in oxy-haemoglobin (HbO) and deoxy-haemoglobin (HbR) in mother and infant. The 8 × 2 probe sets were attached to an electroencephalography cap with a 10–20 configuration . The probe sets over the left and right inferior frontal gyrus (IFG) surrounded F7 and F8, whereas the probes on the medial prefrontal area (mPFC) surrounded FP1 and FP2. These regions of interest were based on previous work involving adult–child interactions ,39. In eμMol) in HbO and HbR. HbR analyses are reported in the supplements.fNIRS measurements were processed using MATLAB-based functions derived from Homer 2 . Raw datWe assessed the relation between the fNIRS time series in each caregiver and infant using (Morlet) wavelet transform coherence (WTC) as a function of frequency and time . WTC is Using WTC, we assessed infants' intra-personal inter-hemispheric functional connectivity between homologous channels in the passive distal watching condition (as an age-appropriate resting phase). The frequency band of interest was determined to be 0.063 Hz–0.167 Hz (corresponding to 6–16 s), according to the potential infant HRF function , by visu (ii)κ = 0.98, infant vocalizations: κ = 0.78).To assess vocal turn-taking in the free play condition , tra (iii)The ELFRA-2  is a par (iv)r = 0.75, thus, an averaged AQS score was used in subsequent analyses. These AQS scores ranged between −1.0 and +1.0, with higher scores representing higher attachment security of the observed dyad.We used the Attachment Q-Sort (AQS ) to obse (v)All statistical analyses were calculated in RStudio . We used generalized linear mixed models (GLMM), generalized linear models (GLM), and linear models (LM) to analyse the hypothesized associations between turn-taking, interpersonal neural synchrony, inter-hemispheric functional connectivity, infant's language development and attachment.post-hoc analyses (emmeans) were applied to contrast factors. Multiple comparisons were corrected by using Tukey's honest significant difference. All continuous predictor variables were z-standardized, and distributions of residuals were visually inspected for each model. Models were estimated using maximum likelihood. Model fit was compared using a Chi-square test  of a GLMM with interval (1–5), the number of bidirectional turn-taking, response latencies, and region of interest (ROI: IFG versus lPFC versus mPFC) as fixed factors. We included the interaction between intervals, number of turns, and ROI. ROI was inserted as random slopes and dyads as random intercepts. Random slopes for interval and turn-taking did not lead to model convergence and were thus excluded. To further examine significant main and interaction effects, tio test ).Hypothesis 2: Turn-taking and infants' inter-hemispheric connectivity. We calculated two separate GLM including inter-hemispheric connectivity values as the response variable (assuming a Gaussian distribution and logit link). The first GLM tested infant response latencies, and number of infant-to-mother turns as the main effects. The second GLM tested the effects of mother-to-infant and infant-to-mother overlaps. Further turn-taking variables are considered in analyses reported in the supplements.Hypothesis 3: Infants’ expressive language abilities and turn-taking. Infants' vocabulary at 24 months assessed by the ELFRA questionnaire was entered as the response variable (assuming a Gaussian distribution) of three LM. One included the main effects of dyadic turn-taking . Another one included the main effects of infant turn-taking variables . The third LM included mother turn-taking variables (number of turns and response latency).z-transformed, assuming a Gaussian distribution) of three LM. One included the main effects of dyadic turn-taking . Another one included the main effects of infant turn-taking variables . The third LM included mother turn-taking variables (number of turns and response latency).Hypothesis 4: Infants’ attachment and turn-taking. Infants' attachment security score at 12 months of age assessed by the AQS was entered as the response variable , which yielded 1-ß = 0.856. We, thus, deemed the sample size to be appropriate for this set of analyses.The ery rate ). We alsery rate ) in relaery rate , we assu. 3 (a)N = 55) throughout the free play condition are included in electronic supplementary material, table S1. Number of bidirectional turn-taking and response latencies were highly correlated . Neither frequency of turn-taking nor overlaps from mother to infant or infant to mother were significantly related, p > 0.189.Descriptive statistics for the turn-taking patterns of mother–child dyads ( (b)n = 55) provided a significantly better fit to the data than the null model . The comparison of the full model with the reduced models revealed that the three-way interaction turns*intervals*ROI  and the main effect ROI  were statistically significant. These effects indicate that interpersonal neural synchrony was different between brain regions and that this difference was modulated by the frequency of turn-taking and that their slopes were different over time. Neither the two-way interactions turn-taking*intervals, intervals*ROI, turn-taking*ROI nor the simple effects of turn-taking, intervals and response latencies reached significance (p > 0.139). To understand the three-way interaction, we conducted three separate GLMM to test the interaction between turns and intervals in relation to interpersonal neural synchrony in each ROI. We corrected p-values  for multiple comparisons. The GLMM for the inferior frontal gyrus and lateral prefrontal cortex revealed no significant main effects and interaction effects (p > 0.270). Only the GLMM for the medial prefrontal cortex revealed a significant main effect of turns , a significant main effect of interval , and a significant interaction between turns and intervals . This significant set of effects shows that more frequent turns were related to higher neural synchrony, specifically in the medial prefrontal cortex. Neural synchrony between mother and infant also increased over time. But more specifically, more frequent turns were associated with higher neural synchrony in the medial prefrontal cortex in the first intervals .Firstly, we analysed whether more frequent turn-taking during a proto-conversation was related to mother–infant neural synchrony in frontal regions. The GLMM ( (c)n = 54) output displayed that the frequency of infant-to-mother turns was significantly associated with infants’ inter-hemispheric connectivity , as depicted in p = 0.701). Moreover, infants' and mother's frequency of overlaps were not significantly associated with inter-hemispheric connectivity (p > 0.255). Taken together, more infant-initiated turns in the proto-conversation were associated with higher inter-hemispheric connectivity in infants' brains.Next, we tested infants’ inter-hemispheric connectivity as a neural correlate to infant-initiated (infant-to-mother) turns and infants' (mother-to-infant) response latencies. The first model ( (d)n = 28). The first LM assessed the relation between infants’ vocabulary and dyadic turn-taking patterns and revealed a significant correlation between the number of turns of the dyad and infant vocabulary . More frequent turn-taking was related to infants' larger vocabulary at 24 months (a). Next, we split the dyadic turns into uni-directional turns and ran an additional regression analysis. The result showed that the number of mother-to-infant turn-takings explained more variance in infants’ vocabulary than infant-to-mother turn-taking . The third LM assessed the relation between infants' vocabulary and mother-to-infant overlap frequency and found a significant negative relation . Fewer mother-to-infant overlaps were associated with infants’ larger vocabulary (b). Infants' vocabulary was not significantly related to infant-to-mother overlaps (p = 0.794).We tested the relation between infants' vocabulary at 24 months and dyadic as well as individual turn-taking patterns during the mother–infant interaction at 4–6 months p-corrected > 0.122. In an exploratory analysis, we dichotomized infants’ attachment scores into securely and insecurely attached infants . The te. 4In the present study, we examined turn-taking patterns in mother–infant proto-conversations in relation to interpersonal neural synchrony and developmental outcome measures: infants' inter-hemispheric connectivity, expressive language abilities, and attachment. The more turn-taking mother and infant showed the more they displayed interpersonal neural synchrony during the proto-conversation. The relation was stronger in the initial phase of the interaction and decreased over time. Next, infants’ number of turns was related to higher inter-hemispheric connectivity in infants’ frontal brain regions. In addition, the number of turns in general, but especially infants' turns, was related to a larger vocabulary at 24 months of age. However, mother–infant turn-taking was not related to the infant's attachment security. The results underscore the role of interpersonal neural synchrony in early turn-taking as well as brain maturation and language development as potential outcomes related to coordinated proto-conversation in early caregiver–infant interactions.As predicted, we observed a positive relation between the frequency of turns and neural synchronization between mothers and their infants in a proto-conversation. This association was limited to the medial prefrontal cortex among our regions of interest. Medial prefrontal regions have been implicated in caregiver–infant synchrony, especially in face-to-face exchanges . This isOur hypothesis that neural synchrony might be a shared underlying mechanism for turn-taking across earlier and later human development can therefore neither be fully confirmed nor dismissed. By contrast to the current results, neural synchrony increased over time in mothers and their preschool-aged children in association with more frequent turn-taking in a previous study . There aNext, we examined the relationship between mother–infant turn-taking and infant maturity at the neural level, measured by inter-hemispheric connectivity. Consistent with our hypothesis, we found that more frequent infant-to-mother turn-taking, but not mother-to-infant response latencies, was positively related to brain maturation. These results support previous findings that infants' individual differences in inter-hemispheric connectivity can be identified and related to individual emotional and sensorimotor differences ,28. InteEven though early social interactions are maintained to be a core component of children's language development , few stuNext to supporting the development of infants' language skills, conversational turn-taking is suggested to help build connections between people ,22. PrevOur results highlight the importance of preverbal turn-taking between caregivers and infants in early development. Turn-taking arises together with interpersonal neural synchronization in the medial prefrontal cortex, a potential biomarker of mutual engagement and prediction. Moreover, turn-taking in proto-conversations is related to infants’ brain maturation and language development. Our results also indicate that the range of turn-taking qualities in the present sample might comprise ‘good enough’ communication patterns to facilitate the bond between healthy and (mostly) securely attached infants and their caregivers . TherefoTaken together, our results show that mother–infant turn-taking, similar to later in development , relies


Sporobolus anglicus (previously known as Spartina anglica), a widely distributed saltmarsh plant, is colonized by DMS-degrading microorganisms.Saltmarshes are hotspots of organosulfur compound cycling due to production of dimethylsulfoniopropionate (DMSP) by benthic microorganisms, macroalgae, and saltmarsh vegetation. Degradation of DMSP is a source of dimethylsulfide (DMS), an important precursor for formation of secondary organic aerosol. Microorganisms degrading DMS play a role in controlling the amount of DMS available for emission into the atmosphere. Previous work has implicated sediment microbial populations as a major sink for DMS. Here, we show that 13C-DMS stable isotope probing, microbial community diversity and functional genetic potential in phyllosphere and rhizosphere samples was assessed by high-throughput sequencing of 16S rRNA gene amplicons, cloning and sequencing of methanethiol oxidase genes, and by metagenomic analysis of phyllosphere microbial communities.Dimethylsulfide degradation potential was assessed by gas chromatography and 13C-DMS stable isotope probing and included populations related to Methylophaga and other Piscirickettsiaceae in rhizosphere samples. DMS-degraders in the phyllosphere included Xanthomonadaceae and Halothiobacillaceae. The diversity in sediment samples of the methanethiol oxidase (mtoX) gene, a marker for metabolism of methanethiol during DMS and DMSP degradation, was similar to previously detected saltmarsh mtoX, including those of Methylophaga and Methylococcaeae. Phyllosphere mtoX genes were distinct from sediment mtoX and did not include close relatives of cultivated bacteria. Microbial diversity in the phyllosphere of S. anglicus was distinct compared to those of model plants such as rice, soybean, clover and Arabidopsis and showed a dominance of Gammaproteobacteria rather than Alphaproteobacteria.The DMS degradation potential of microbial communities recovered from phyllosphere and rhizosphere samples was similar. Active DMS-degraders were identified by Sporobolus anglicus suggest that DMS cycling in saltmarshes is more complex than previously recognised and calls for a more detailed assessment of how aboveground activities affect fluxes of DMS.The potential for microbial DMS degradation in the phyllosphere and rhizosphere of  Dimethylsulfide (DMS) is the most abundant biologically formed sulfur compound emitted to the atmosphere and performs a crucial role in the global sulfur cycle . As an i−1 , while trimethylamine monooxygenase has been shown to co-oxidise DMS to DMSO (Thiobacillus thioparus (Hyphomicrobium EG (mtoX) has been identified from the DMS-degrading bacterium Hyphomicrobium sp. VS . Overall diversity in the phyllosphere of S.anglicus was assessed by high-throughput metagenomic sequencing.We assessed DMSP contents of Sporobolus anglicus plants including phyllosphere and rhizosphere were sampled from the Stiffkey saltmarsh (52°57′55.6″N and 0°55′24.0″E) in Norfolk (United Kingdom) in June and October 2012, in February, June, and October 2013 and in August 2014. Between 3 and 6 S. anglicus plants were collected from several subsites within the lower saltmarsh. These subsites were located at a maximum distance of 25 m to each other. Phyllosphere and rhizosphere samples of S. anglicus were used in triplicates. For the above ground parts (phyllosphere), leaf wash, leaf and stem samples were used, whereas below ground parts (rhizosphere) included root samples including endorhizosphere and rhizoplane, and rhizosphere sediment (ectorhizosphere), respectively. Leaf wash samples were prepared according to S. anglicus plant samples, in October 2013, bulk sediment of S. anglicus plants was sampled to characterise the sediment type, particle size, pH, total N, total C, organic matter, and water extractable anions . In August 2014, S. anglicus phyllosphere samples were collected for metagenomics. Plants used for determination of microbial community composition and in incubation studies were generally from areas characterized by a sandy loam sediment  and rhizosphere (roots and sediment) samples were measured as DMS following alkaline hydrolysis as reported and described previously  in samplS. anglicus tissue samples, sediment samples, and stable isotope probing experiments using the FastDNA™ Spin Kit for Soil  following the manufacturer’s instructions. For S. anglicus tissue and sediment samples, 0.5 g of sample was used. Leaf wash samples were collected on 0.22 μm filters as described above. Filters were placed in the lysis tubes and the subsequent steps performed according to the manufacturer’s instructions. DNA for the S. anglicus phyllosphere metagenomes was extracted from triplicate S. anglicus phyllosphere samples according to S. anglicus phyllosphere sample for the extraction of metagenomic DNA.DNA was extracted from all 12C2]-DMS and [13C2]-DMS incubations, and metagenomes. Amplicon samples were sequenced on the Illumina MiSeq platform, metagenomic DNA was sequenced on the HiSeq platform. Library preparation for 16S rRNA amplicon HTS was carried out according to S. anglicus phyllosphere metagenomic sequencing, multiple displacement amplification was carried out using the Illustra GenomiPhi V2 DNA amplification kit  prior to sequencing. Library preparation and paired-end sequencing with a 20 pmol spike-in of PhiX (Illumina) was carried out by the Genomics facility of the School of Life Sciences at the University of Warwick.High-throughput amplicon sequencing was applied for taxonomic classification (16S rRNA gene) of non-enriched phyllosphere and rhizosphere tissue samples, sediment samples, “light” and “heavy” [S. anglicus was carried out according to S. anglicus (leaf wash) was studied in February and June 2013. Leaf wash samples were prepared as described above. All SIP incubations were carried out in triplicates. Approximately 2 g rhizosphere sediment or 1–2 g leaf wash sample was incubated with either [13C2]-DMS or [12C2]-DMS in a 125 ml serum vial sealed with a butyl rubber stopper. Overall, 100 μmol carbon g−1 sample in form of either [13C2]-DMS or [12C2]-DMS was provided over three pulses. Substrate utilization was monitored using gas chromatography as described above. Samples were harvested and processed immediately after DMS was not detectable anymore.DNA-stable isotope probing (DNA-SIP) of DMS-assimilating microorganisms in phyllosphere and rhizosphere of 13C2]-DMS and [12C2]-DMS incubations active DMS-assimilating populations were identified. Fractions were initially analysed by DGGE and subsequently subjected to 16S rRNA amplicon high-throughput sequencing. In addition, individual DGGE bands were cut out and sequenced according to DNA-SIP fractions were used as template for the amplification of bacterial 16S rRNA genes using the primers 341F-GC and 907R . By compmtoX) encoding the methanethiol oxidase was assessed in leaf, root, and rhizosphere sediment samples from June 2012 and in root and rhizosphere sediment samples from October 2012 and February 2013. Functional gene PCRs were performed in a 50 μl reaction volume containing 1 × buffer including MgCl2, 0.8 mM dNTPs (0.2 mM each dNTP), 0.4 μM forward (MtoX41Fmodv2_inos) and reverse (MTOX346Rmod) primer  and BSA  were added to enhance strand separation in GC-rich regions and to help stabilise the enzyme, respectively. PCRs were carried out in a T-100 thermocycler . PCR cycling regime and subsequent cloning of PCR products was carried out according to The presence and diversity of the gene  primer , 2 U TaqRaw fastq reads from 16S rRNA gene amplicon HTS were joined and demultiplexed using QIIME . QualitymtoX sequencing data was carried out using BLASTx , DMSP cleavage , DMS oxidation , methanethiol degradation (mtoX), and genes involved in the downstream metabolism of DMSP, DMS, and methanethiol degradation products, such as genes involved in one carbon metabolism either via the tetrahydrofolate-dependent pathway  or the tetrahydromethanopterin-dependent pathway  and genes involved in sulfur oxidation . E-values are indicated in Metagenomics-Rapid Annotation using Subsystem Technology (MG-RAST) was used for analysis of taxonomic and functional assignment of the metagenomic reads . MG-RASTSporobolus anglicus phyllosphere and rhizosphere samples have been submitted to the Sequence Read Archive (SRA) under the accession number PRJNA670606. Raw read data from the 16S rRNA under dimethylsulfide DNA stable isotope probing have been submitted under the accession number PRJNA670609. Read data of the three Sporobolus anglicus metagenomes have been submitted to the Sequence Read Archive (SRA) under the accession numbers ERX675820, ERX675821, and ERX675822. DNA sequences of methanethiol oxidase genes (mtoX) have been deposited in Genbank under accession numbers MW245658–MW245712.16S rRNA raw read data from S. anglicus tissue samples confirmed that plants sampled for this study had high DMSP concentrations and showed differences in DMSP concentration between plant tissues and sampling sites. The highest DMSP concentrations were detected in S. anglicus leaf, stem, and root tissue samples (−1 fresh weight (FW). DMSP was not detectable or only present at very low concentrations in leaf wash and rhizosphere sediment samples. Variability in the DMSP concentration was observed, not only between different seasons and months, but also between individual plants and their tissues , and particle size distribution , whereas the highest DMSP concentrations in the S. anglicus plants growing in the sandy loam were observed in the stems  in these samples typically took between 4 and 13 days, except for samples from October 2012 where the degradation of the DMS took between 13 and 25 days in leaf wash, root and rhizosphere sediment samples (−1 sediment d−1 (data not shown).The hypothesis that  samples . Degrada samples . The rhiS. anglicus phyllosphere and rhizosphere samples from June and October 2012 and February, June and October 2013. The bacterial community composition of S. anglicus . Also, Deltaproteobacteria showed a high abundance in the rhizosphere  effect size (LEfSe) method  confirmeS. anglicus, multidimensional scaling (MDS) was performed. Plots displaying the 16S rRNA gene-based bacterial community composition at the genus level of all phyllosphere and rhizosphere samples were calculated, showing data points with superimposed codes representing plant part, sample tissue, DMSP content, pH, sampling month, and temperature , Rhodobacteraceae , and Psychrobacter  within the phyllosphere, and Chromatiales (14.04%), Desulfobulbaceae  and Desulfobacteraceae  in the rhizosphere. These analyses corresponded well to the results obtained by the LEfSe analysis.Operational taxonomic units primarily providing the discrimination between the two observed sample clusters  from February and June 2013. DNA was extracted from phyllosphere and rhizosphere SIP incubations with 13C-labelled DMS (and control incubations with 12C-DMS) after degradation of approximately 100 μmol carbon g−1 sample. Initial evaluation of SIP fractions by DGGE analysis of bacterial 16S rRNA amplicons indicated successful labelling (data not shown). Bacterial 16S rRNA amplicon high-throughput sequencing was done for the “heavy” and “light” 12C- and 13C-labelled fractions of each experiment. A total of 6,636,546 quality-filtered, chimera-checked sequences were obtained from 48 samples and assigned to 1,115 distinct OTUs at 97% identity . Taxonomic profiling showed distinct community profiles in “heavy” and “light” DNA fractions of phyllosphere and rhizosphere SIP samples  and other (unassigned) Piscirickettsiacaea (13C-heavy DNA fractions from the phyllosphere were enriched in Gammaproteobacteria [including Methylophaga and other (unassigned) Piscirickettsiacaea] but also highlighted a range of other genera as DMS degraders  oxidizes inorganic sulfur compounds such as thiosulfate, S and sulfides and hydrogen sulfide to sulfate , as well as taxa not previously shown to degrade DMS, but which are shown here as having the potential to contribute to DMS degradation in the phyllosphere of S. anglicus.Thus, SIP identified several candidate DMS-degrading populations including some that were previously linked to DMS cycling (Methylophaga (family Piscirickettsiacaea), Halothiobacillus , or Rhodanobacter (family Rhodanobacteraceae) were not detected in the 16S rRNA gene sequencing data of the same unenriched S. anglicus samples, suggesting that these genera were present at a low abundance. Also, the families Halothiobacillaceae, Rhodanobacteraceae, Xanthomonadaceae, and Alicyclobacillaceae were not detectable in the 16S rRNA gene sequencing data of the same unenriched S. anglicus samples. However, the family Piscirickettsiacaea (family of Methylophaga) could be detected in S. anglicus unenriched samples with relative abundances ranging from 0.1% in some leaf wash samples to 39.2% in root samples and 37.6% in rhizosphere sediment samples (data not shown) highlighting these as potentially abundant DMS degrading bacteria in association with S. anglicus especially below ground.At the genus level, mtoX gene encoding the methanethiol monooxygenase was used as a functional marker gene to detect methanethiol-degrading bacteria  and rhizosphere samples (marked in red and brown) clustered with mtoX clones previously reported from surface sediment of the same saltmarsh  was found in 0.1–0.2% of the metagenomic reads of the S. anglicus phyllosphere.Direct metagenomic analysis based on sequencing of phyllosphere community DNA of three  library . Similarabundant . The micArabidopsis thaliana, clover, soybean, and rice showed major differences in community composition between those plants and S. anglicus likely reflecting the differences in habitat (see A comparison of the taxonomic diversity to phyllosphere metagenomes of itat see .dddP and dddD), DMS oxidation , and methanethiol degradation (Methanethiol oxidase—mtoX), which were all present in the investigated metagenomes  were Gammaproteobacteria and Alphaproteobacteria . The abundance of the gene encoding the DMS monooxygenase (dmoA) was higher in the Sporobolus anglicus phyllosphere metagenomes compared to the DMS dehydrogenase (ddh), suggesting that DMS is more likely to be degraded to methanethiol and used as a carbon and sulfur source via its subsequent degradation to formaldehyde and hydrogen sulfide via the methanethiol oxidase (mtoX) than the oxidation to DMSO via the DMS dehydrogenase , e.g., mch (Methenyl-H4MPT-cyclohydrolase), fhcA (Formyltransferase/−hydrolase), foldD (Methylene-H4F-dehydrogenase/cyclohydrolase), mtdA (Methylene-H4F dehydrogenase), and purU  in C1 and DMS cycling.The phylogenetic affiliation of the organisms encoding these genes was inferred by blast searches of the relevant genes detected in the metagenomic contigs against the NCBI nr database . This shrogenase . DMS is bacteria . AlthougS. anglicus contains DMSP is not a novel finding, it was important in the context of our analyses to assess the environmental and seasonal factors that could affect the DMSP content of S. anglicus sampled in a natural saltmarsh. The DMSP concentrations that we measured were similar to those reported previously (S. anglicus (Sporobolus plants as shown in previous studies with greenhouse or laboratory grown Sporobolus. Here we show that plants sampled from locations with different sediment properties (sand or sandy loam) had different tissue DMSP concentrations. As expected, a higher total N and total C content was shown for the sandy loam in this study  but also between plants suggesting a patchier distribution of these potential DMS degraders seasonally and spatially, which may indicate a lower overall abundance of these bacteria.Active DMS degraders in the rhizosphere were predominantly  strains  or findieriments . InteresmtoX, in unenriched microbial communities demonstrated that MT-degrading microorganisms are closely associated with S. anglicus. Sequences of mtoX clustering closely with Methylophaga are likely to originate from uncultivated Piscirickettsiacaea shown to be involved in DMS degradation by SIP. The finding of an mtoX clade that was only detected in the phyllosphere suggests that some of these Gammaproteobacteria may have a habitat preference, but a better taxonomic placement of these sequences would require more detailed analysis of neighbouring genes in their genomic context or resolving the corresponding genomes as metagenome assembled genomes.The detection of the gene encoding the methanethiol oxidase, S. anglicus-associated microorganisms degrading DMS and related sulfur compounds provided new insights. Although it is challenging to integrate these different datasets seamlessly, the following conclusions can be drawn. Based on SIP, the potential for DMS cycling by Piscirickettsiaceae is clear, but other DMS degraders outside this family were also identified in phyllosphere samples. SIP incubations may favour fast-growing organisms, which potentially outcompete slower growing ones that may be more dominant in situ. Therefore identification by SIP is not necessarily a reflection of their dominance or contribution to the process. This issue could be addressed in future studies under substrate-limiting conditions, ideally using small-scale chemostat experiments.The application of different approaches to characterise Piscirickettsiaceae were present at up to 39% read abundance in some unenriched rhizosphere samples clearly demonstrating that these bacteria can be abundant members of the community. They are thus likely relevant for cycling of DMS (and other C1 compounds) in situ. This was further supported by finding several hits to C1 cycling genes in the metagenomes which were affiliated with Methylophaga and related organisms and mtoX amplicons affiliated with this group. Other candidate DMS degraders may be less abundant in situ, and the lack of genes of sulfur and C1 cycling matching such taxa may be due to a lack of genomes of representative DMS-degrading strains in the sequence databases. This can be illustrated using Methylophaga as an example. DMS metabolism has only been reported for three Methylophaga species and only in genomes of two of these, M. thiooxydans and M. sulfidovorans, have genes involved in DMS metabolism been identified  can be found at: EK and HS conceived the research, analyzed the data, and wrote the manuscript. EK carried out the experimental research. AM supported the data analysis. All authors contributed to the article and approved the submitted version.This study was supported with funding from the Natural Environment Research Council .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


X substrate is presented as temperature sensors by semiconductor technology and its electrical properties were measured. The results show that the temperature coefficient of the resistance value is 2.07 × 10−3 in the temperature range of 300–450 K and 2.39 × 10−3 in the temperature range of 450–575 K. From room temperature to high temperature, the “metal” characteristics are presented, and the higher TCR obtained at higher temperature interval is described and analyzed by combining Boltzmann transport equation and thermal expansion theory. These investigations provide further insight into the temperature characteristics of graphene.Graphene membranes act as temperature sensors in nanoelectromechanical devices due to their excellent thermal and high-temperature resistance properties. Experimentally, reports on the sensing performance of graphene mainly focus on the temperature interval under 400 K. To explore the sensing performance of graphene temperature sensors at higher temperature intervals, micro-fabricated single-layer graphene on a SiN Compared with conventional temperature sensors, micro-temperature sensors have attracted research attention because of their miniaturization , intelliaterials ,10. Grapaterials , but theaterials . For thi2/Si substrate, SiN membrane and a suspended architecture [X/Si substrate to explore the temperature characteristics of graphene in a wider temperature range was fabricated in this study.The temperature characteristics of graphene-based sensitive elements such as reduced graphene oxide (rGO) ,14 and gitecture . TherefoIn this article, graphene prepared by chemical vapor deposition (CVD) was developed to a thermal resistance nanoelectromechanical graphene temperature sensor by using standard semiconductor processes such as transferring and sputtering. The temperature characteristics of graphene were tested in the temperature range of 300–575 K, and its temperature characteristics were explained by combining Boltzmann theory and thermal expansion theory. Experimental support for the development of temperature sensors with graphene as a sensitive element in a higher range will be provided in this study. Simultaneously, the previously proposed model was improved from the perspective of thermal expansion of the substrate, and the concept that the tensile strain caused by thermal expansion of the substrate intensifies the “metallic” property of graphene was explained. In the temperature interval studied here, the increase of the positive temperature coefficient (PTC) could be described as the superposition of the dominant scattering mechanism of graphene itself and the tensile strain of the external substrate.X/Si substrate ) pre-deposited with metal electrodes [2 plasma to etch the back graphene of the sample, and 40% FeCl3 was used to etch the Cu substrate supporting the graphene for about 6 h. After that, the PMMA/graphene sample was transferred to the target substrate and heated on a hot plate at 85 °C for 30 min. The PMMA layer was dissolved in acetone (CP) solution at 50 °C for 10 min, and then the target was washed with alcohol (EA) solution and deionized (DI) water. Finally, The graphene was patterned by using lithography and O2 plasma etching. The parameters of O2 plasma etching are as follows: power, 60 W; gas flow, 30 sccm; and etching time 3 min.Graphene was grown on copper substrate by the chemical vapor deposition (CVD) method . A wet tectrodes ,24. The The structure of the graphene temperature sensor is shown in After the graphene temperature sensor device was fabricated, the temperature characteristic of the graphene temperature sensor was tested by using a high-temperature sealed tube and a high-temperature furnace combined with a Wheatstone bridge. 0 was equal to the initial resistance of the sensor at room temperature with a humidity of 45% to 50%. The voltage at both ends of the bridge changed linearly with the change of resistance. The output voltage signal was amplified by an operational amplifier, sampled by a low-pass filter, and finally converted to the corresponding resistance value.To accurately measure the change in resistance during the heating process, the device resistance was measured using a Wheatstone bridge (see illustration). The bridge was equilibrated down at room temperature, where R−1 and 2700 cm−1, respectively, and the intensity ratio of the 2-D peak to the G peak (I2D/IG) is 3.21, which indicates that the transferred graphene is a monolayer film [−1, which indicated that the graphene film was of high quality [To characterize the number of layers and defects of graphene after transferring, samples were subjected to Raman scattering  . As showyer film . No D peThe electrical and thermal conductivity of graphene would be weakened because of the presence of cracks, folds, and organic residues on the graphene surface ,29,30,31tol(T) ≠ ρPH(T) +ρother(T). ρtol(T) represented the temperature characteristic that graphene exhibits as resistance varies with temperature. ρPH(T) represented the temperature characteristic exhibited by phonon scattering, and ρother(T) represented all the scattering mechanisms except phonon scattering. This statement explains the non-linear experimental result in the experiment. The humidity test of the device was carried out, and the results are shown in R0 represents the resistance value at initial temperature T0 and R is the resistance value at temperature T.The temperature coefficient of resistance (TCR) is one of the performance indicators of a temperature sensor. To characterize the performance of the sensor, the TCR can be calculated according to Equation (1). As could be visible in 1 = (5.52425 − 4.2155)/(4.2155·(450 − 300)) = 2.07 × 10−3. The TCR at 450–575 K temperature range can be calculated as follows: TCR2 = (7.13572 − 5.52425)/(5.52425·(575 − 450)) = 2.39 × 10−3.The TCR at 300–450 K temperature can be calculated as follows: TCRX expands with increasing temperature. As a result, the resistance of graphene gradually increases during tensile strain, which indirectly aggravates the “metal” characteristics of graphene, presenting a higher temperature coefficient.After calculation, the TCR of the high-temperature interval is greater than that of the lower-temperature interval. One possible reason is that during the heating process, graphene is subjected to tensile strain from the substrate because SiNA graphene temperature sensor with a temperature range from 300 K to 573 K was fabricated by using standard semiconductor integration technology. Scanning electron microscopy, optical microscopy, Raman analysis revealed that graphene is a single layer of defect-free structure with a clean surface. An electrical performance test using a wheatstone bridge revealed that temperature sensor resistance increased with the increase of temperature, and the reason is that the phonon scattering dominates the presence of the characteristics of “metal” in the temperature range. Simultaneously, the TCR of the high-temperature interval was higher, a possible reason for enhancing this characteristic was explained. In brief, the temperature characteristic of graphene in the range of 300 to 574 K was investigated, and a wide range, good linearity, and high sensitivity graphene temperature sensor has been fabricated, which meets the needs of industrial production and other fields for accurate temperature control at high temperatures.