To access the properties of theoretical graphene, it is crucial to manufacture layers with a defect-free structure. The imperfections of the structure are the cause of deterioration in both electrical and mechanical properties. Among the most commonly occurring crystalline defects, there are grain boundaries and overlapping zones. Hence, perfect graphene shall be monocrystalline, which is difficult and expensive to obtain. An alternative to monocrystalline structure is a quasi-monocrystalline graphene with low angle-type boundaries without the local overlapping of neighboring flakes. The purpose of this work was to identify factors that directly affect the structure of graphene grown on a surface of a liquid metal. In the article the growth of graphene on a liquid copper is presented. Nucleating graphene flakes are able to move with three degrees of freedom creating low-angle type boundaries when they attach to one another. The structure of graphene grown with the use of this method is almost free of overlapping zones. In addition, the article presents the influence of impurities on the amount of crystallization nuclei formed, and thus the possibility to order the structure, creating a quasi-monocrystalline layer. HighlightsMetallurgical graphene is grown on a liquid metal surfaceGraphene grain boundaries obtained in the research are of low-angle typeThe lower the nucleation rate, the more ordered the quasi-monocrystalline structureA large number of impurities prevents the formation of an ordered  structureImpurities initiate heterogeneous nucleation of graphene nucleiGraphene is one of the carbon derivatives with sp2 hybridization. In graphene, carbon atoms are arranged in a honeycomb structure, which provides the material with its outstanding properties, such as chemical stability, optical transparency, enormous mechanical strength and electrical characteristics . Graphen2 to even 1 m2 and bigger) is a much safer form of graphene, since it is highly unlikely to be inhaled. Processing large-area graphene, namely transferring it to desired substrates, requires the usage of metal etchants (wet transfer) or electrolyte solutions (hydrogen delamination) [However, as a nanomaterial, especially in the powder form, graphene can cause a serious health hazard through pulmonary, oral or dermal exposure, where inhalation causes the greatest risk ,7,8,9. Wination) , that agFor the commercial implementation of graphene, a scalable, repeatable and efficient production technique must be developed . ProductAnother approach is to slow down the nucleation rate, to achieve a limited number of graphene nuclei. That will allow the formation of larger single crystals before neighboring islands merge ,26,27. IThe graphene synthesis on liquid metal takes advantage of the quasi-atomically smooth surface of the liquid, which prevents the negative influence of defects and grain boundaries present in solids . The graLiquid metals have demonstrated a great potential to synthesize large-area, uniform and almost defect-free graphene sheets. Liquid can play the role of a matrix for carbon deposition in a CVD process, or a solvent for carbon atoms to dissolve in, from which the graphene precipitates out further in the process in the process the graphene precipitates out . Using aSeveral liquid metal substrates have been reported suitable for graphene growth, including copper, gallium and silver ,37,38,39In this report, an investigation of graphene growth on liquid copper via precipitation, and how this growth is affected by solid impurities, is presented. Liquid copper was chosen because the liquid is perfectly smooth at the sub-micrometer level with no surface defects, which is an issue in the case of solid substrates. Graphene grown on a solid metal substrate reproduces the surface defects of the growth substrate. In addition, the liquid surface allows the formed nuclei to move and rotate, which creates the possibility of creating an ordered structure, as demonstrated by the article. Another raised issue was the dependence of graphene nuclei mobility on their number and density. The aim of this research was to obtain a monolayer of graphene with low-angle type boundaries in which properties are closer to the theoretical ones, as it was described by Kula et al. .Graphene was grown on a liquid copper surface inside a molybdenum crucible. As mentioned before, copper can spread only over several metals, among which nickel it the most suitable. Since it has better wettability than molybdenum, the nickel layer was deposited on the crucible. A nickel layer with a thickness of 0.2 µm was applied on a molybdenum crucible by the Radio Frequency Physical Vapor Deposition (RF PVD) method. Before putting on the nickel coating, the surface of the samples was subjected to argon plasma etching with a pressure of 2 Pa, auto-polarization potential of −800 V and a time of 10 min. Plasma etching was applied to remove any impurities from the crucible surface. Then, the coating was applied using DC pulse magnetron sputtering  with a power of 1.0 kW, a pressure of 0.5 Pa, a potential of −50 V and a time of 30 min. The dimensions of the crucible were 53 mm in diameter and 5 mm in height. Then, a copper disc  with a 52 mm diameter and a 1.0 mm thickness was put into the crucible. The copper purity was 99.9995%.The graphene synthesis was conducted in a vacuum furnace . After inserting the samples, the furnace was pumped to a pressure of 10 Pa. The next step of the process consisted of heating the substrate to the temperature of 1100 °C under argon with a 3% hydrogen atmosphere. The pressure was maintained at 10 kPa. The growth substrate was kept at these conditions for 10 min. Eventually, the system was cooled down to 1050 °C with a cooling rate of 0.5 °C/min in the same atmosphere. The source of carbon was the mixture of acetylene, ethylene and hydrogen in a mass flow proportion of 2:2:1, respectively. The gas mixture was simultaneously added into the reaction chamber for 10 min with a flow rate of 5 L/min during the initial thermal stage of 1100 °C. The synthesis was conducted according to the patent .A Hitachi scanning electron microscope , working in secondary electron (SE) mode at 5 kV accelerating voltage, was used for the qualitative morphology analysis of the metallurgical graphene layers.Raman spectroscopy  with an excited wavelength of 532 nm was used to characterize the graphene.A graphene layer was separated from the growth substrate using the electrochemical method and transferred into a copper mesh. Quality measurements of the obtained graphene were conducted with a transmission electron microscope (TEM). For this purpose, an HR TEM  was used with an atomic resolution microscope) with a maximum accelerating voltage of 200 kV.The most common structural defects are graphene grain boundaries ,47,48,49By measuring the misorientation angles, we proved that in the case of high-strength metallurgical graphene, there are low-angle grain boundaries at the misorientation angle below 10°. This phenomenon was a consequence of the growth mechanism on a liquid phase. Graphene islands appearing on a liquid surface have three degrees of freedom: they are able to move and rotate in the XY plane, which is shown in The process purity is one of the main factors which determines the number of appearing nuclei and their sizes. Each foreign particle on the growth surface acts as an active site, favoring the heterogenous graphene nucleation starting with those particles. It is clearly visible that the particle is in the center of the forming graphene flakes. The same impurity particle can be an active site for another graphene nuclei. Based on the EDS spectrum , the parA transmission electron microscope (TEM) was used for grain boundary examination. Measurement was conducted for graphene grown on a clean surface. This study, as shown in A low-angle boundary is formed on the contact surface of two grains with a misorientation angle smaller than a few degrees. −1), G (1580 cm−1) and 2D (2690 cm−1). Among others, it is possible to evaluate the number of layers and structural defects by the analysis of the peak area, position, intensity and intensity ratios of spectrum peaks. In the spectrum analysis, the relative intensities of individual peak values are important, not the absolute counts’ values. The D peak intensity and ID/IG intensities ratio increases with the increase in the number of graphene layer defects [Additionally, Raman spectroscopy was used for the further investigation of the prepared samples. The analysis of the graphene Raman spectra requires three main peaks to take into consideration: D (1350 cm defects ,61. Figu2D/IG ratio is 1.52, proving the monolayer character of the sample. No additional peaks are visible in the spectra. On the other hand, the spectrum of locally overlapped flakes represented in D/IG ratio equal to 0.37, shows significant defects in the graphene structure. A more defective structure is also supported by the presence of the D′ peak, which results from disorders in the graphene layer and appears around 1620 cm−1 [2D/IG ratio is equal to 1.16, which could even represent a monolayer, but the shape of the 2D peak is more characteristic of a multilayer structure. This can be explained by the local overlapping of the graphene flakes.The Raman spectrum of the sample with aligned graphene flakes shown a, is typGraphene growth on a liquid metal surface was examined and presented in this paper. The study shows that the obtained graphene grains were of low-angle type due to their ability to move on the surface of the substrate leading to a controlled flakes arrangement. The study also indicates that a lower number of graphene nuclei is favorable for the formation of a monolayer without overlapping zones. The fewer the nuclei, the more possibilities for movement and rotation, as well as a higher probability of perfect alignment. The increasing number of nuclei may result in mutual interference between flakes, which will immobilize them and therefore, prevent them from creating a continuous sheet. The clean and smooth surface of liquid allows graphene flakes to rearrange and lean towards each other creating a quasi-monocrystalline structure. However, when the surface of the growth substrate is contaminated with foreign particles, a perfect alignment is impossible due to the heterogeneous nucleation of graphene on those particles. This leads to a higher nucleation rate, the immobilization of occurring graphene flakes and the growth of additional layers. In order to obtain a monolayer graphene, a fundamental issue is to keep the substrate and process purity at the highest level.


After the injection of TDP-43 PFFs, the elevated levels of pTDP-43 were present in several pyramidal tract-associated regions of Atg5+/- mice. Additionally, the occurrence of spontaneous potentials detected by electromyography demonstrates evidence of lower motor neuron dysfunction in M1-TDP-43 PFFs-injected Atg5+/- mice, and prolonged central motor conduction time detected by motor evoked potentials provides evidence of upper motor neuron dysfunction in C7-TDP-43 PFFs-injected Atg5+/- mice. These results show that injection of TDP-43 PFFs into the M1 or C7 of Atg5+/- mice induces the spreading of pathological TDP-43 along corticospinal tract axons in both an anterograde and retrograde manner. Importantly, TDP-43 PFFs-injected Atg5+/- mice also display ALS-like motor dysfunction. Taken together, our findings provide direct evidence that TDP-43 PFFs-injected Atg5+/- mice exhibited ALS-like neuropathology and motor phenotypes, suggesting that autophagy deficiency promotes the formation and spreading of pathological TDP-43 in vivo.Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease, characterized by phosphorylated TDP-43 (pTDP-43)-positive inclusions in neurons and glial cells. However, the pathogenic mechanism that underlies ALS remains largely unknown. To investigate the effects of autophagy deficiency in the formation and spreading of pathological TDP-43 along corticospinal tract axons, TDP-43 preformed fibrils (PFFs) were prepared and unilaterally injected into the fifth layer of the left primary motor cortex (M1) or the left anterior horn of the seventh cervical spinal cord segment (C7) of Atg5 After the injection of TDP-43 PFFs, the elevated levels of pTDP-43 were present in several pyramidal tract-associated regions of Atg5+/- mice. In addition, the occurrence of spontaneous potentials detected by electromyography (EMG) demonstrates evidence of lower motor neuron (LMN) dysfunction in M1-TDP-43 PFFs-injected Atg5+/- mice, and prolonged central motor conduction time (CMCT) detected by motor evoked potentials (MEPs) suggests upper motor neuron (UMN) lesion in C7-TDP-43 PFFs-injected Atg5+/- mice. Moreover, behavioral analysis indicates that both M1- and C7-TDP-43 PFFs-injected Atg5+/- mice developed an ALS-like syndrome in a time-dependent manner.In our present study, TDP-43 preformed fibrils (PFFs) were prepared and unilaterally injected into the fifth layer of the left primary motor cortex (M1) or the left anterior horn of the seventh cervical spinal cord segment (C7) of Atg5+/-) and C57BL/6 mice weighing 18-20 g were used. Animals were kept under standard laboratory conditions with free access to standard laboratory food and water . All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals. The protocols were approved by the Institutional Ethics Committees of the Zhengzhou University.Eight-week-old male B6: 129-Atg5<tm1Nmz> (RBRC02231) . Unilateral injections were made into the fifth layer of the left M1 or left anterior horn of C7 at the following coordinates: M1: anteroposterior, 0.7 mm; mediolateral, 1.5 mm; dorsoventral, 1.4 mm; and C7: mediolateral, 0.8 mm; dorsoventral, 1.2 mm. Each injection site received 5 µl of TDP-43 PFFs or PBS at a rate of 0.2 µl/minute using a Hamilton Syringe .One hundred and twenty Atg5Date and cause of death were recorded for each mouse. For ethical reasons, animals were closely monitored and sacrificed as moribund prior to actual death using criteria for severe illness. To determine the duration of survival reliably and humanely, the moribund state, defined as the inability of mice to right themselves 30 seconds after being placed on their side, was used. The moribund mice were scored as “dead” and were euthanized.After euthanasia and perfusion, the brain and spinal cord from perfused animals were fixed in 30% sucrose solution containing 4% paraformaldehyde. Tissues were embedded in paraffin blocks, cut into 4 µm using a Rotary Microtome , and mounted on glass slides. Sections of spinal cord and brain tissues were then deparaffinized and rehydrated in a graded ethanol series. Immunohistochemistry was performed as previously described Frozen spinal cord or brain samples were thawed on ice and sonicated in 5× v/w RIPA buffer  containing protease inhibitor cocktails (Thermo Fisher Scientific). Samples were centrifuged for 30 minutes at 4 °C, 100,000 g. The supernatant was taken as RIPA-soluble fractions. The pellet was sonicated with 5× v/w RIPA buffer as above. Then, the pellet was sonicated in 2× v/w urea buffer  and centrifuged for 30 minutes at 22 °C, 100,000 g. This supernatant was collected as the RIPA-insoluble/urea-soluble fractions. Samples were analyzed by 10% SDS-PAGE with polyvinylidene fluoride (PVDF) membranes. Following transfer to the PVDF membranes, the immunoblot was sequentially probed with antibodies that recognize pTDP-43  or LC3A/B . The densities of the proteins were normalized to GAPDH.°C. A standard EMG apparatus  was applied. To determine UMN and LMN deficits, we collected motor unit action potentials (MUAPs) and spontaneous activities including fibrillation potentials, positive sharp waves and fasciculation potentials. A sweep speed of 10 ms/div, a gain of 100 uv/div and a band pass filter with low and high cut-off frequency of 20 and 10,000 Hz were performed. The mice were electrically grounded through a disposable needle electrode implanted subcutaneously at the abdominal wall. Next, we used concentric needles  to detect the following muscles: bilateral biceps brachii, bilateral T10 paraspinal muscles, bilateral tibialis anterior, and bilateral gastrocnemius. Spontaneous activities were recorded for 120 seconds and MUAPs were recorded at different sites of each muscle until 20 MUAPs were obtained.Mice were anesthetized with isoflurane and placed in the prone position on a thermostatically controlled warming plate to maintain body temperature at 37 Cortical MEP (cMEP) and spinal MEP (sMEP) were obtained using two monopolar needle electrodes. For cMEP, the cathode was positioned at the midline of the interaural line through the scalp; the anode was positioned 4-5 mm lateral and anterior to the cathode. For sMEP, two monopolar needle electrodes were inserted into C7, close to the emergence of the spinal nerve root. cMEP and sMEP were recorded with the active needle electrode inserted into the muscle of the forelimb footpad and reference needle electrode inserted under the skin of the second digit. The onset of the first, generally negative, deflection was taken as the MEP latency. The MEP with the shortest latency was considered as the final evaluation criteria of MEP latency, the peak-to-peak amplitude of the motor response was used for the mean MEP amplitude. The CMCT is the propagation time between motor cortex and spinal cord, which is the difference between the cMEP latency and the sMEP latency.Biopsied biceps brachii were dissected out from the forelimb of an anesthetized animal and immersed immediately in isopentane cooled in liquid nitrogen. Serial frozen sections were cut at 10 μm thickness and stained by hematoxylin and eosin (H&E). All steps were performed according to the standard procedures To evaluate behavioral performance, 15 mice from each group were weighed weekly and underwent a battery of behavioral tests every two weeks starting from the second month post-surgery. Baseline performance was measured before surgery. The experimenter was blinded to the treatment group for all behavioral tests.Rotarod test was performed using the rotarod apparatus . The test began by placing the mice on the rod rotating at 30 rpm. The latency to fall off the rotarod within the maximum time (180 seconds) was recorded as a measurement of the competence of their motor function. Three trials were performed per day with a 15-minute inter-trial interval. The mean latency to fall off the rotarod was calculated for the data analysis.The hanging wire test was performed to evaluate general muscle strength. Each mouse was placed on a horizontally positioned screen with many 1 cm × 1 cm grids. The period until the mouse left the screen was recorded, with a maximum time of 180 seconds. Three trials were performed per day, and the mean latency was recorded for the data analysis.To obtain footprints, paws of the mice were painted with two different non-toxic paints (fore-paws in red and hind-paws in green). The mice were then allowed to walk along a restricted cardboard tunnel  into an enclosed box and a sheet of white paper was placed on the floor of the tunnel, and one set of footprints were collected for each mouse. Three steps from the middle portion of each run were measured for the following parameters (cm): (1) stride length (front and hind legs). (2) The front- and hind-base width. The mean value of each set of values was used for the data analysis.2 value to test for significance. For all statistical tests, significance was taken as *p < 0.05, **p < 0.01, ***p < 0.001.The statistical analyses were performed using SPSS 24.0 . Behavioral and electrophysiological data of mice were presented as mean ± standard deviation (SD), and two-way ANOVA was employed for comparison of multiple groups. Disease onset and survival statistics were performed by Kaplan-Meier survival curves and the data were analyzed using the log-rank test, generating a χFigure ) into the fifth layer of the left M1 or the left anterior horn of C7 of Atg5+/- mice . Diagrams of estimated conduction pathway for pathological TDP-43 spreading were shown in Figure . Firstly, pTDP-43 immunoactivity was detected at the injection sites using an anti-pTDP-43 (phosphorylated at Ser409/Ser410) antibody. As early as 2 months post-injection (mpi), pTDP-43-immunoreactive (pTDP-43-ir) pathology was detected in the neurons and glial cells in the second, third and fifth layers of the M1 around the injection region of M1-TDP-43 PFFs-injected Atg5+/- mice ; however, the staining was not observed on the contralateral side . In the cervical enlargement of C7-TDP-43 PFFs-injected Atg5+/- mice, pTDP-43-ir pathology was found in the neurons of the left spinal cord anterior horn adjacent to the injection sites at 2 mpi . No pTDP-43-ir pathology was detected in the age-matched PBS-injected Atg5+/- mice . Thus, these data indicated that TDP-43 PFFs could induce the formation of pTDP-43 pathology in vivo. These data supported that the injection of TDP-43 PFFs into the brain or spinal cord induce pTDP-43-ir pathology deposits in situ.To investigate whether pathological TDP-43 could spread along corticospinal tract axons, we injected TDP-43 PFFs , medulla oblongata , and cervical enlargement  of M1-TDP-43 PFFs-injected Atg5+/- mice. In the cortex, neuronal and glial cytoplasmic pTDP-43-ir pathology mainly deposited in the fifth layer of the left M1 , while no pTDP-43 pathology was observed on the contralateral side . In the CA1 region of the hippocampus, the positive neuronal and glial cytoplasmic pTDP-43 staining was more intense on the left side  than the contralateral side . In medulla oblongata, the positive neuronal and glial cytoplasmic pTDP-43 pathology was detected in both sides of hypoglossal nucleus  and the pTDP-43-ir nerve fibers in the left corticospinal tract , and the pTDP-43 staining was stronger in the left side of the hypoglossal nucleus , however, the pTDP-43-ir staining was not detected in the contralateral side of the corticospinal tract . Furthermore, neuronal and glial cytoplasmic pTDP-43 was deposited in the right side of anterior horn of the cervical enlargement . However, no pTDP-43 pathology was detected in both sides of M1-PBS-injected Atg5+/- mice .At about 6 mpi, pTDP-43-ir staining was detected in the hippocampus , CA1 region of hippocampus , and the fifth layer of the M1  compared to the left side  at about 5 mpi. The pTDP-43-ir nerve fibers were detected in the left corticospinal tract at about 5 mpi . As with M1-PBS-injected Atg5+/- mice, no pTDP-43 pathology was detected in both sides of C7-PBS-injected Atg5+/- mice . In addition, we also injected TDP-43 PFFs into the C7 of C57BL/6 mice. However, the formation of TDP-43-ir neuronal and glial cytoplasmic pathology in the fifth layer of the M1 was not detected until 18 months . Taken together, these data suggest that the injection of TDP-43 PFFs into motor cortex or ventricornu of Atg5+/- mice induces the spreading of TDP-43 pathology along corticospinal tract axons anterogradely and retrogradely in vivo.In C7-TDP-43 PFFs-injected Atg5+/- mice, the results showed that LC3A/B-II/LC3A/B-I levels were significantly decreased in these regions of adult Atg5+/- mice compared with age-matched C57BL/6 mice . To further demonstrate whether there was an increase of pTDP-43 pathology in TDP-43 PFFs-injected Atg5+/- mice, immunoblotting analysis was also performed in the abovementioned regions of M1-TDP-43 PFFs- and M1-PBS-injected Atg5+/- mice at 6 mpi. In the soluble fractions, no significant difference of pTDP-43 levels was found between M1-TDP-43 PFFs- and M1-PBS-injected Atg5+/- mice . Nevertheless, in the insoluble fractions, pTDP-43 levels were increased in all examined M1-TDP-43 PFFs-injected Atg5+/- mice, while it was faintly detected in age-matched PBS-injected Atg5+/- mice . The same results were also found in C7-TDP-43 PFFs-injected Atg5+/- mice at 5 mpi (data not shown). These results support that the injection of TDP-43 PFFs into M1 or C7 of Atg5+/- mice could induce the spreading of pathological TDP-43 along corticospinal tract axons anterogradely and retrogradely.To evaluate whether autophagy deficiency can affect the aggregation of pathological TDP-43, we first detected LC3 expression levels in M1, hippocampus, medulla oblongata, and cervical enlargement of C57BL/6 and Atg5+/- mice exhibited any neurophysiological abnormality. Spontaneous activity, motor unit potential recruitment, and MUAPs were used to quantify LMN dysfunction. MEPs were used to evaluate potential impairment of the corticospinal tract. Firstly, needle EMGs were recorded from the biceps brachialis, T10 paraspinals, tibialis anterior, and gastrocnemius muscles. In M1-TDP-43 PFFs-injected mice, abnormal spontaneous activity including fibrillation potentials , fasciculation potentials , and positive sharp waves  were detected on the right side of biceps brachialis, T10 paraspinals, tibialis anterior, and gastrocnemius muscles as early as 2 mpi. At 5 mpi, abnormal spontaneous activities were found in both side of biceps brachialis, T10 paraspinals, tibialis anterior, and gastrocnemius muscles, and there was no significantly difference between the two sides . Significantly, the abnormal spontaneous activities were detected in the right biceps brachialis of all the M1-TDP-43 PFFs-injected mice, however, the M1-PBS-injected Atg5+/- mice only showed mild abnormal spontaneous activity in right biceps brachialis muscles at 7 mpi . The frequency of abnormal spontaneous activity was developed in a time-dependent manner as shown in Figure . Secondly, we collected 20 MUAPs in each mouse for the motor unit measurement. The findings showed a significantly increase in the mean amplitude and duration of MUAPs at 2, 5, and 7 mpi in M1-TDP-43 PFFs-injected Atg5+/- mice, compared to age-matched M1-PBS-injected Atg5+/- mice . Furthermore, we performed a biopsy in both sides of the biceps brachialis in M1-TDP-43 PFFs-injected Atg5+/- mice at 7 mpi. H&E staining showed muscle atrophy, round muscle fibers, and muscle fibers with central nuclei in the right biceps brachialis Figure K, while no morphological changes were found in the left biceps brachialis . Taken together, these findings suggest LMN dysfunction in M1-TDP-43 PFFs-injected Atg5+/- mice.Besides the morphological evidence of TDP-43 PFFs-induced pathology, we further investigated whether the M1- and C7-TDP-43 PFFs-injected Atg5+/- mice to quantify UMN impairment . At about 2 mpi, CMCT was significantly prolonged in the injection side of C7-TDP-43 PFFs-injected Atg5+/- mice compared to age-matched C7-PBS-injected Atg5+/- mice , and the CMCT increased in a time-dependent manner  in C7-TDP-43 PFFs-injected Atg5+/- mice. Moreover, cMEP amplitude decreased in C7-TDP-43 PFFs-injected Atg5+/- mice compared to age-matched C7-PBS-injected Atg5+/- mice  at about 2 mpi, and this decline became more significant over time . At the end stage, responses to cortical stimulation were absent in C7-TDP-43 PFFs-injected Atg5+/- mice, indicating devastating damage of the corticospinal tract. Taken together, the quantitative analyses of CMCT and cMEP amplitude indicated UMN dysfunction in C7-TDP-43 PFFs-injected Atg5+/- mice.Furthermore, we performed cMEP and sMEP in C7-TDP-43 PFFs-injected Atg5+/- mice exhibit ALS-like symptoms. First, the lifespan of TDP-43 PFFs-injected Atg5+/- mice was significantly shorter compared with PBS-injected controls, and the Kaplan-Meier curve showed overall survival in Figure . To assess the motor function of TDP-43 PFFs-injected Atg5+/- mice, we performed a series of behavioral tests. At approximately 8 weeks post-injection, C7-TDP-43 PFFs-injected Atg5+/-mice gradually lost weight  and performed poorly on the rotarod test  and hanging wire test , revealing a significant deficit in muscle strength, motor coordination, and balance. In addition, C7-TDP-43 PFFs-injected Atg5+/-mice showed significantly shorter stride length and wider base width compared to PBS-injected controls in the footprint test . As for M1-TDP-43 PFFs-injected Atg5+/-mice, they began to lose weight at around 10 weeks post-injection , and they also showed deficient motor performance in the rotarod test and hanging wire test compared to age-matched PBS-injected Atg5+/-mice . However, the severity of motor disability was much higher in C7-TDP-43 PFFs-injected Atg5+/-mice compared to age-matched C7-TDP-43 PFFs-injected Atg5+/-mice . Moreover, in the footprint test, M1-TDP-43 PFFs-injected Atg5+/-mice also showed significantly shorter stride length and wider base width similar to those found in C7-TDP-43 PFFs-injected Atg5+/-mice . In conclusion, these changes suggest that the M1- and C7-TDP-43 PFFs-injected Atg5+/- mice exhibited ALS-like symptoms, including short survival, weight loss, abnormal gait, and motor dysfunction.We further investigated whether M1- and C7-TDP-43 PFFs-injected Atg5in vivo, we injected TDP-43 PFFs into the cortex or spinal cord anterior horn of Atg5+/- mice. The results show that unilateral injection of TDP-43 PFFs into the fifth layer of the left M1 or the left anterior horn of C7 of Atg5+/- mice could induce the formation and spreading of pathological TDP-43 along the corticospinal tract. Moreover, we also found the presence of TDP-43 pathology in our animal models at CA1 region of hippocampus and hypoglossal nucleus in M1-TDP-43 PFFs-injected Atg5+/- mice at 6 mpi and in C7-TDP-43 PFFs-injected Atg5+/- mice at 5 mpi. This supports the hypothesis that TDP-43 pathology could spread via axonal pathways To investigate whether inoculation of TDP-43 PFFs can transmit pathological TDP-43 along corticospinal tract axons in vivo recapitulates ALS-like pathology. Furthermore, M1-TDP-43 PFFs-injected Atg5+/- mice showed abnormal spontaneous activity in both sides of biceps brachialis, T10 paraspinals, tibialis anterior, and gastrocnemius muscles at around 5 mpi, and C7-TDP-43 PFFs-injected Atg5+/- mice showed prolonged CMCT and decreased cMEP amplitude at around 2 mpi, which recapitulated the electrophysiologic changes found in ALS patients. On the other hand, these results also reflect the anterograde and retrograde spreading of pathological TDP-43.It mimics the TDP-43 pathology in patients with ALS. These data indicate that TDP-43 seeding in vitro evidence has suggested that autophagy induction improves TDP-43 metabolism and clearance and prevents death in cultured neurons and astrocytes expressing mutant TDP-43 Drosophila models for TDP-43 proteinopathy, evidence shows that inducing autophagy effectively diminishes TDP-43 aggregation and finally rescues behavior deficits +/- mice, and pTDP-43 levels were significantly increased in M1-TDP-43 PFFs-injected Atg5+/- mice compared with age-matched PBS-injected Atg5+/- mice. It indicated that autophagy deficiency may promotes the formation and spreading of pathological TDP-43 in vivo. Furthermore, our results showed that TDP-43 PFFs-injected Atg5+/- mice exhibits ALS-like neuropathology and motor phenotypes.It is known that autophagy is a vital degradation mechanism of dysfunctional organelles and large protein aggregates +/- mice to SOD1 G93A transgenic mice in further study, thus making our results more convincing.As we all know, SOD1 G93A transgenic mouse is a commonly used animal model for ALS. Previous studies have found increased autophagy in SOD1 G93A mice in vivo. These data suggest that promoting autophagy and inhibiting cell-to-cell transmission of pathological TDP-43 may have therapeutic potential in the treatment of ALS.In conclusion, we provided the evidence that TDP-43 PFFs could induce the transmission of pathological TDP-43 along corticospinal tract axons in both an anterograde and retrograde manner


These results provide the first quantitative description of the density of defects and transport properties, which plays an important role for potential applications.In recent years, graphene oxide has been considered as a soluble precursor of graphene for electronic applications. However, the performance lags behind that of graphene due to lattice defects. Here, the relation between the density of defects in the range of 0.2 % and 1.5 % and the transport properties is quantitatively studied. Therefore, the related flakes of monolayers of graphene were prepared from oxo‐functionalized graphene (oxo‐G). The morphologic structure of oxo‐G was imaged by atomic force microscopy (AFM) and scanning tunneling microscopy (STM). Field‐effect mobility values were determined to range between 0.3 cm The effect of defects: The room‐temperature mobility values of single‐layer graphene derived from oxo‐graphene with densities of defects between 0.2 % and 1.5 % are presented. The fundamental findings can explain the generally diverging results reported for reduced graphene oxide used in applications. In addition, we investigated the effect of structural defects on the electrical properties of monolayer oxo‐G by two‐probe measurements under ambient conditions. Our results quantify the correspondence relationship between structural defects and transport capacities in high‐defect single‐layer graphene materials.0 %  and graphene with various densities of defects  obtained by wet‐chemical preparation and reduction with hydroiodic acid (HI).0 % is determined to about 0.5 nm with lateral dimensions of 10–20 μm. Contaminants are visible at the edge of the G0 % sheet, which stems from the used tape during the exfoliation and transfer processes. In contrast, the roughness of GD is with 1.0 nm twice as high as for G0 % due to bitopic oxo‐groups at the carbon basal plane and possible adsorbates  was used. Comparing the atomically‐resolved honeycomb structure for defect‐free highly ordered pyrolytic graphite (HOPG) in Figure D reveals distinguishable topographical features with the appearance of islands and rows at bright spots, as depicted in Figure As shown in Figure 0 %, there are two distinct peaks, the G band (at 1570 cm−1), associated with the in‐plane stretching vibration of C−C bonds, and the 2D band (at 2670 cm−1), activated by a double‐resonant Raman scattering.−1 is noticed, probably evolving from grain boundaries or other lattice imperfections.−1) and D′ band (1620 cm−1). In addition, the broadening of all bands is observed with increasing density of defects. The full‐width‐at‐half‐maximum  of the 2D peak (Γ2D) increases from approximately 24 cm−1 in the single‐layer G0 % to about 178 cm−1 in the single‐layer G1.5 %. In Table 0 %, G0.2 %, G0.4 %, G0.5 %, G0.9 % and G1.5 %.The density of defects in single layers of graphene can be quantified by Raman spectroscopy. Figure  band at 570 cm−1,ID/IG is used for determining the density of defects in the GD samples. In the case of single‐layer G0 %, it contains an extremely low density of defects, which belongs to the low‐defect density regime according to the Raman model introduced by Lucchese, Cançado and Ferrari et al., with a ratio of ID/IG=0.2±0.07 corresponding to about 24 000 C atoms within the intact graphene lattice. According to Equation The intensity ratio of NC corresponds to intact carbon atoms and Acell=0.2462×sin (60°)=0.05239 nm2, the average distance between defects LD is about 25 nm. The related defect density (nD) is about 5.1×1010 cm−2, using nD (cm−2)=1014/(πLD2).D samples relate to the high‐defect density regime. In this regime, the ID/IG ratio increases with an increase of LD, based on the relation of ID/IG∝LD∝NC.ID/IG ratio decreases from 4.5 to 1.0 and the LD values gradually decrease from 3.5 nm to 1.3 nm, respectively. The related nD increases from about 2.6×1012 cm−2 to 1.9×1013 cm−2. As depicted in Figure ID/IG ratio versus Γ2D. With increasing the density of defects, the Γ2D values increase, which is consistent with our discussion above.in which the 0 % flakes were mechanically exfoliated from a bulk graphite and transferred to a heavily p‐doped Si substrate with a 300 nm thick SiO2 layer (Si/SiO2),Γ2D value of 23.6±2.6 and I2D/IG>4 was determined by Raman spectroscopy to prove the single‐layer nature of the produced flake , served as source and drain electrodes, were deposited onto single‐layer graphene channel materials by using e‐beam lithography and evaporation processes. Avoiding any thermal decomposition of chemically‐derived GD samples, no annealing process was performed for all prepared devices after the lift‐off process.Field‐effect transistors were fabricated by using the obtained monolayer graphene samples as conducting channels Figure  a. The mElectrical transport measurements were performed at ambient conditions in a two‐terminal configuration. The performance of transistors relies on the properties of channel materials, gate dielectrics, electrodes and test conditions. Therefore, to reliably compare electrical performances for the obtained monolayer graphene samples, all transistors were prepared with parallel electrodes, the same manufacturing processes and test conditions.IDS–VDS) are determined and visualized in the insets of Figure 0 % device in Figure IDS–VBG) with asymmetric Dirac voltage  located at +20 V. The observed p‐doping behavior was probably attributed to the heavily p‐doped Si/SiO2 substrate, impurity doping as a result of exfoliation and transfer processes or polar adsorbates like water or oxygen acting as charge traps between substrate and the graphene surface. Furthermore, a hysteretic behavior between forward and reverse sweeps are observed. For GD transistors  and distance between defects (LD), respectively. It is found that the mobilities follow, within experimental uncertainties, a nonlinear relationship with LD and NC, because the defect‐free area of graphene increases over‐proportionally with increasing LD.in which LD≤3 nm) in oxo‐G were identified by Raman spectroscopy. The isolation of these domains limits the charge transport in reduced oxo‐G. Therefore, the mobility values of charge carriers of graphene with densities of defects between 0.2 % and 1.5 %, change by three orders of magnitude, from 0.3 cm2 V−1 s−1 and 33.2 cm2 V−1s−1. More generally, the mobility of charge carriers varies by orders of magnitude, although it looks like that the density of defects varies only a little. The fundamental findings reported here can explain the generally diverging results often reported for reduced graphene oxide used in applications.In summary, we have studied the room‐temperature electrical properties of single‐layer graphene derived from oxo‐G containing defect densities varying from 0.2 % to 1.5 %. The defects give rise to a heterogeneous topographical morphology of oxo‐G. The isolated graphene domains (−10 mbar). All transport measurements were performed under ambient conditions by a two‐probe station and two source‐measurement units (Keithley 2450).AFM characterization was performed by using a JPK NanoWizard 4 Atomic Force Microscope in tapping mode at room temperature. Raman characterization was carried out with a Horiba Explorer spectrometer with a 532 nm laser for excitation under air conditions. Scanning tunneling microscopy (STM) was conducted by using Omicron‐STM1 microscope under ultra‐high vacuum . Reduction was performed by vapor of hydriodic acid (HI) and trifluoroacetic acid (TFA)  at 80 °C (10 min). Subsequently, the surface of GD was cleaned with doubly distilled water (Carl Roth) to remove iodine species. The density of defects of individual flakes was determined by Raman spectroscopy (Horiba Explorer spectrometer with a 532 nm laser for excitation under air conditions). Subsequently, flakes with defined density of defects were selected for FET device fabrication.The defective G0 % and GD flakes, respectively.Standard electron beam lithography procedure (Raith PIONEER TWO) was used to define and expose the geometry of metal contacts. Subsequently, a 5 nm/70 nm Cr/Au layer was deposited with thermal evaporation (Kurt J. Lesker NANO 36) and lifted off in acetone to make electrode contact to monolayer GThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


The study of processing characteristic and property optimization of ultra-high-molecular-weight polyethylene (UHMWPE) pipe is increasingly performed, mainly focusing on difficulties in the melting process and poor thermal-mechanical properties after forming, which have limited the wider engineering application of UHMWPE pipe. In this study, organo-montmorillonite (OMMT)-modified UHMWPE pipe with good thermal-mechanical properties was prepared by screw extrusion molding. First, high-density polyethylene was subjected to fluidity modification so that the screw extrusion molding of UHMWPE pipe was feasible. Then, OMMT-modified UHMWPE pipes under different addition amounts of OMMT were innovatively prepared by extrusion. Furthermore, the effects of the addition amounts of the compatibilizer HDPE-g-MAH and the silane coupling agent γ- propyl trimethoxy silane (KH560) on the thermal properties of OMMT-modified UHMWPE pipe were investigated for the first time. Compared with those of pure UHMWPE pipe, the Vicat softening temperature (from 128 to 135.2 °C), thermal deformation temperature (from 84.4 to 133.1 °C), bending strength (from 27.3 to 39.8 MPa), and tensile strength (from 20.8 to 25.1 MPa) of OMMT-modified UHMWPE pipe were greatly increased. OMMT-modified UHMWPE pipe with good thermal-mechanical properties was able to be prepared by extrusion for the first time. The compatibilizer method of HDPE-g-MAH was slightly more effective than the coupling agent method of KH560. Ultra-high-molecular-weight polyethylene (UHMWPE) is a polymer that has been used in pipelines because of its good physicochemical and tribological properties ,2. NowadCompression molding without flow, which features simple operation and low cost, can overcome the disadvantage of the high viscosity of UHMWPE ,6,7,8,9.Screw extrusion molding, which features continuous production, can overcome the disadvantage of the low production efficiency of compression molding . By the However, the fluidity modification of UHMWPE will inevitably induce a degree of decrease in thermal and mechanical properties. Thus, the strengthening modification of UHMWPE is necessary for good thermal-mechanical properties. Strengthening modification methods of UHMWPE mainly include physical modification ,21,22, cIn this study, the fluidity modification of UHMWPE was performed so that the screw extrusion molding of UHMWPE pipe was feasible. Then, with different addition amounts of organo-montmorillonite (OMMT), OMMT-modified UHMWPE pipes were innovatively prepared in a single screw extruder. In addition, the effects of the addition amounts of HDPE-g-MAH and KH560 on the thermal properties of OMMT-modified UHMWPE pipe were investigated for the first time. With the management of thermal and mechanical properties, the Vicat softening temperature, thermal deformation temperature, bending strength, and tensile strength of OMMT-modified UHMWPE pipe were greatly increased. OMMT-modified UHMWPE pipe with good thermal-mechanical properties could be prepared by extrusion for the first time.UHMWPE (granule/GUR4152) was supplied by Ticona Co., Ltd., Frankfurt, Germany. OMMT (powder/I.44P), used as filler, was provided by Nanocor Co., Ltd., Arlington Heights, IL, USA. HDPE (powder/5070), used for the fluidity modification of UHMWPE, was provided by Beijing Yanshan Petrochemical High-Tech Co., Ltd., Beijing, China. HDPE 5070 was chosen because the melt flow index (MFI) of this HDPE is high (6.1~8.0). HDPE-g-MAH (powder/ME5506), used as a compatibilizer, was obtained from Nantong Rizhisheng New Polymer Material Technology Co., Ltd., Nantong, China. A silane coupling agent (liquid/KH560), used to improve the interaction between UHMWPE/HDPE and OMMT, was supplied by Hangzhou Jiexika Chemical Co., Ltd., Hangzhou, China. The chosen silane should have both an inorganic reactive group and an organic reactive group so that it can enhance the interaction between the filler OMMT (inorganic phase) and the matrix UHMWPE/HDPE (organic phase). Ethanol (liquid) was supplied by Dongguan Jintai Chemical Technology Co., Ltd., Dongguan, China.A single screw extruder (with a screw diameter of 45 mm), used for ultra-high-molecular-weight material, was developed by Beijing University of Chemical Technology, China. A high-speed mixer (SHR-25A) was provided by Zhangjiagang Yongli Mechanical Co., Ltd., Zhangjiagang, China. An electric constant temperature drying oven (SFG-02.400) was provided by Hengfeng Medical Device Co., Ltd., Huangshi, China.The preparation of OMMT-modified UHMWPE pipe samples is summarized as follows:Step 1. The mixtures of materials were prepared with a compatibilizer method and a coupling agent method.(a)For the compatibilizer method, the OMMT was kept in a drying oven at a constant temperature of 110 °C for four hours. Then, UHMWPE, HDPE, OMMT, HDPE-g-MAH, and other additives were added into a mixer for homogeneous mixing.(b)For the coupling agent method, a solution of KH560 and ethyl alcohol was prepared with a volume ratio of 1:10. Then, the solution was premixed uniformly with OMMT, and the mixture was kept in a drying oven at a constant temperature of 110 °C for four hours. Lastly, UHMWPE, HDPE, OMMT, KH560, and other additives were added into a mixer for homogeneous mixing.The mixer has two levels of revolving speed: 725 and 1440 rpm. At room temperature, the mixer works at 725 rpm for five minutes and then at 1440 rpm for another five minutes.Step 2. The mixtures were processed into pipe samples see  in a sin3 for the Vicat softening temperature and 80 × 10 × 4 mm3 for the thermal deformation temperature. The pressure 0.45 MPa was used to test the thermal deformation temperature. The sample bars were obtained from pipe samples by cutting and flattening according to China national standards (the same below).A tester of Vicat softening temperature and thermal deformation temperature  was used to test the Vicat softening temperature and thermal deformation temperature of OMMT-modified UHMWPE pipe according to China national standard GB/T1633-2000. The lengths, widths, and thicknesses of the sample bars were 10 × 10 × 4 mm3.A Shore D durometer  was used to test the Shore hardness of OMMT-modified UHMWPE pipe according to China national standard GB/T2411-2008. The length, width, and thickness of the sample bar were 50 × 50 × 4 mmSEM  was used to analyze the microstructures of OMMT-modified UHMWPE pipe. Cooled with liquid nitrogen, the sample was sprayed with gold after a brittle fracture.DSC  was used to measure the melting enthalpy, melting point, and crystallinity of OMMT-modified UHMWPE pipe. Under the protection of nitrogen gas, each sample of 5 milligrams was heated from room temperature to 200 °C at a rate of 10 °C/min and then cooled to room temperature.3 for the bending strength and 120 × 10 × 4 mm3 for the tensile strength.A universal material testing machine  was used to test the bending strength and tensile strength of OMMT-modified UHMWPE pipe according to China national standards GB/T9341-2000 and GB/T1040-2006. The lengths, widths, and thicknesses of the sample bars were 80 × 10 × 4 mmAn X-ray diffractometer  was used to measure the diffraction angle at the diffraction peak of OMMT-modified UHMWPE pipe. The diffraction test was performed under CuKα-ray radiation with a tube voltage of 40 kV, a tube current of 50 mA, and a scanning speed of 5°/min.Firstly, the thermal and mechanical properties of pure UHMWPE pipe were tested and are shown in In order to study the effect of the addition amounts of the filler OMMT on the thermal and mechanical properties of UHMWPE pipe, OMMT-modified UHMWPE pipes were prepared using different addition amounts of OMMT. The other additives in wOMMT ≤ 8%. When the nano-filler OMMT is introduced, it disperses in the matrix of UHMWPE/HDPE. With an increasing amount of OMMT, more polymer macromolecules should be adsorbed on the surface of the nano-filler OMMT. As a result, UHMWPE/HDPE entanglements will be strengthened. The strengthened entanglements will enhance the intermolecular forces and the rigidity of the OMMT-modified UHMWPE. According to its testing principle, the Vicat softening temperature can be regarded as the hardness of materials under heating conditions, i.e., the rigidity of materials. Therefore, the Vicat softening temperature increases with an increasing amount of OMMT for wOMMT ≤ 8%. When the OMMT-modified UHMWPE is heated, OMMT around the molecular chains of UHMWPE/HDPE plays the role of a physical cross-link, which limits the thermal motion of UHMWPE/HDPE molecular chains. Therefore, the thermal deformation temperature increases with increasing amounts of OMMT for wOMMT ≤ 8%. Moreover, with the filling of OMMT, the OMMT-modified UHMWPE is denser in molecular chain distribution, more complex in space structure, and more difficult to invade than the matrix UHMWPE/HDPE. Therefore, the Shore hardness increases with increasing amounts of OMMT for wOMMT ≤ 8%.As shown in wOMMT = 8%, and then decrease. Therefore, a reasonable addition amount of OMMT is 8%. At wOMMT = 8%, the Vicat softening temperature, thermal deformation temperature, and Shore hardness are 135.2 °C, 133.1 °C, and 90 HD, respectively.In wOMMT = 5%, 8%, and 10% were compared, as shown in wOMMT = 5% and 8%, while aggregation of OMMT is found at wOMMT = 10%. At wOMMT = 8%, OMMT disperses uniformly in the matrix UHMWPE/HDPE without aggregation; however, excessive amounts of OMMT give rise to aggregation, which can be a material defect and weaken the thermal and mechanical properties of OMMT-modified UHMWPE. In wOMMT > 8%.To study the deterioration of the Vicat softening temperature, thermal deformation temperature, and Shore hardness, SEM micrographs of the fracture surfaces at wOMMT = 5%, 8%, and 10%. The melting enthalpy (ΔH) and melting point (Tm) of the OMMT-modified UHMWPE, which can be obtained from the DSC curves, are also shown on the left and right sides in H and the melting enthalpy of fully crystallized UHMWPE (ΔH0) known, the crystallinity (Xc) of the OMMT-modified UHMWPE can be obtained by the following equation and as shown in In addition, wOMMT = 8%, and then decreases with increasing amounts of OMMT. For wOMMT ≤ 8%, the addition of OMMT gives rise to the heterogeneous nucleation crystallization of UHMWPE/HDPE, which increases the crystallinity and the melting point of OMMT-modified UHMWPE; however, for wOMMT > 8%, an excessive amount of OMMT results in aggregation, which will weaken the effect of the heterogeneous nucleation crystallization of UHMWPE/HDPE. It is also found that the bending and tensile strengths of the OMMT-modified UHMWPE have the same trend as the crystallinity  were compared, as shown in wOMMT = 8%) occur at 2θ = 3.314 and 2.538 deg, respectively. Then, the layer distances can be obtained by the Bragg equation:wOMMT = 8%) are 2.663 and 3.477 nm, respectively.To better understand the changes in the bending and tensile strengths, small-angle X-ray diffraction (XRD) curves of OMMT and OMMT-modified UHMWPE (wOMMT = 8%) moves to a smaller angle, which indicates that the molecular chains of UHMWPE/HDPE enter the lamellar structure of OMMT. With more molecular chains of UHMWPE/HDPE entering the lamellar structure of OMMT, the OMMT-lamellar-coated molecule chains of UHMWPE/HDPE are able to sustain larger bending loads. In addition, under tension loads, the molecular chains of UHMWPE/HDPE in the lamellar structure of OMMT extend along the lamellar structure, which gives rise to a uniform distribution of tensile stress. As a result, both the bending and tensile strengths of the OMMT-modified UHMWPE increase with an increasing amount of OMMT for wOMMT ≤ 8%. However, for wOMMT > 8%, an excessive amount of OMMT induces aggregation, which will be a material defect and weaken the effect of OMMT-lamellar-coated molecular chains.Compared with that of OMMT, the diffraction peak for the crystal (001) of the OMMT-modified UHMWPE  than by using KH560 (wKH560/wOMMT = 1%).A further comparison between wHDPE-g-MAH = 3%) and using KH560 (wKH560/wOMMT = 1%), small-angle XRD curves of the corresponding OMMT-modified UHMWPE were compared, as shown in θ = 2.538 and 2.584 deg, respectively. The layer distances of OMMT for the corresponding OMMT-modified UHMWPE can be obtained by Equation (2), and the calculated layer distances are 3.477 and 3.415 nm, respectively. A longer layer distance by using HDPE-g-MAH than by using KH560 indicates that more molecular chains of UHMWPE/HDPE enter the lamellar structure of OMMT. The distance between molecular chains is shorter in the lamellar structure than outside the lamellar structure. With more molecular chains in the lamellar structure, the intermolecular force becomes stronger. Thus, the strong intermolecular force limits the thermal motion of molecular chains of UHMWPE/HDPE and increases the thermal deformation temperature and Vicat softening temperature.To better understand the differences in thermal properties between using HDPE-g-MAH , thermal deformation temperature (from 84.4 to 133.1 °C), bending strength (from 27.3 to 39.8 MPa), and tensile strength (from 20.8 to 25.1 MPa) of OMMT-modified UHMWPE pipe were greatly increased. Our results showed that(1)screw extrusion molding of UHMWPE was feasible by the modification of UHMWPE;(2)wKH560/wOMMT = 1% for the coupling agent method;for the process parameters in this study, the reasonable addition amounts of OMMT and HDPE-g-MAH were 8% and 3%, respectively, for the compatibilizer method, and the reasonable addition amount of KH560 was (3)with the management of thermal-mechanical properties, OMMT-modified UHMWPE pipe with good thermal-mechanical properties was able to be prepared by extrusion for the first time; and(4)the compatibilizer method of HDPE-g-MAH was slightly more effective than the coupling agent method of KH560.


We have previously observed that tropomyosin receptor kinase B (TrkB) induces breast cancer metastasis by activating both the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) and phosphatidylinositol-3-Kinase (PI3K)/AKT signaling pathways and inhibiting runt-related transcription factor 3 (RUNX3) and kelch-like ECH-associated protein 1 (KEAP1). These studies indicated that TrkB expression is crucial to the pathogenesis of breast cancer. However, how TrkB regulates bone morphogenetic protein (BMP) signaling and tumor suppression is largely unknown. Herein, we report that TrkB is a key regulator of BMP-mediated tumor suppression. TrkB enhances the metastatic potential of cancer cells by promoting cell anchorage-independent growth, migration, and suppressing BMP-2-mediated growth inhibition. TrkB inhibits the BMP-mediated activation of SMAD family member 1 (SMAD1) by promoting the formation of the TrkB/BMP type II receptor complex and suppresses RUNX3 by depleting BMP receptor I (BMPRI) expression. In addition, the knockdown of TrkB restored the tumor-inhibitory effect of BMP-2 via the activation of SMAD1. Moreover, the TrkB kinase activity was required for its effect on BMP signaling. Our study identified a unique role of TrkB in the regulation of BMP-mediated growth inhibition and BMP-2-induced RUNX3 expression. The bone morphogenetic protein (BMP) family, a member of the transforming growth factor-β (TGF-β) superfamily, consists of over 20 multifunctional growth factors with a broad range of regulatory functions, including embryonic development, apoptosis, chemotaxis, proliferation, and differentiation ,2,3,4. ASimilar to the functions of TGF-β in the cancer microenvironments, BMPs, and their receptors play a dual tumor-suppressive and -promoting role in cancer. For example, in an analysis of large patient cohorts, Gremlin 1, a BMP-antagonist, was found to be significantly increased in the tumor tissues of breast cancer patients . In anotBrain-derived neurotrophic factor (BDNF), a neurotrophin, interacts with and activates tropomyosin receptor type B (TrkB) and plays a key role in the survival, differentiation, and maintenance of neurons . Recent To understand the correlation between TrkB and BMP signaling, we examined whether TrkB expression can modulate the transcription activity of a luciferase gene driven by the BMP response element (BRE). RIE-1, HeLa, and NMuMG cells were transiently-transfected with TrkB, which reduced BRE-luciferase activity relative to that of the control . Thus, TAs expected, the BMP-2-mediated transcriptional activity was reduced by TrkB overexpression in both TrkB-overexpressing cell lines . In normTo further substantiate the role that BMP signaling plays in regulating tumor invasion and the function of TrkB, we knocked down TrkB expression in MDA-MB-231 and Hs578T cells, which have high TrkB expression . We founOur results suggest that TrkB suppresses BMP-mediated tumor inhibition by regulating the processes upstream of SMAD1. We speculate that the mechanism underlying this process might be associated with TrkB-BMP receptor interactions. We found that TrkB directly interacted with BMPRII, but not with BMPRI A,B. In aMoreover, when the cytoplasmic and N-terminal regions of BMPRII, which included the extracellular and transmembrane domains, were deleted, the binding affinity for TrkB remained unchanged. However, when an area between amino acids 203 and 350, which corresponded to the tyrosine kinase domain of BMPRII, was removed, this abolished its binding affinity E. Thus, We assessed the effects of a TrkB kinase inhibitor on BMP-mediated tumor inhibition in RIE-1 and RIE-1-TrkB cells. The inhibition of TrkB kinase activity with K252a significantly increased BRE transcriptional activity in RIE-1-TrkB cells upon treatment with BMP-2, whereas no change to BRE transcription was observed in RIE-1 cells, with or without K252a treatment A. SimilaSimilarly, in MDA-MB-231 and Hs578T breast cancer cells, which expressed endogenous TrkB, the BRE transcriptional activity increased in response to K252a and BMP-2 treatments C,D. ConsTo assess the effects of TrkB activation on BMP-associated transcriptional activity, we generated RIE-1 TrkB kinase-dead (KD) cell lines using vectors that expressed a kinase-dead mutant of TrkB (K588M). As expected, both RIE-1-TrkB and HeLa-TrkB cells exhibited reduced reporter activity in response to BMP-2 treatment, whereas in cells that expressed the TrkB KD, BMP-2 treatment increased reporter activity A,B. In aWe were also interested in the possibility that TrkB KD and BMPRII form a complex. Wild-type TrkB was strongly associated with BMPRII; however, the interaction between BMPRII and the TrkB KD mutant was significantly reduced with or without BMP-2 treatment. Furthermore, BMPRII phosphorylation following BMP-2 treatment was observed only in TrkB wild-type cells D. BecausBMP-2 induces the expression of the tumor suppressor RUNX3 and consequently decreases c-Myc expression when RUNX3 binds to the c-Myc promoter. Moreover, in comparison to the constitutively-active BMPRIA (CA-ALK3), the transfection of a dominant-negative version of BMPRI (DN-ALK3), in the presence of BMPRII, does not suppress c-Myc transcription activity , indicatOur results show that BMPRI-BMPRII complex formation was significantly suppressed in the presence of TrkB E,F. UndeOur results suggest that TrkB expression might enhance the tumorigenic potential of the tumor by countering BMP-2-mediated growth inhibition. Based on the above findings, we evaluated whether TrkB overexpression inhibited RIE-1 cell metastasis. To metastasize to distant organs, cancer cells must overcome anoikis as part of cancer progression . We firsWe have previously shown that TrkB induces tumorigenesis and metastasis via the induction of the JAK2/STAT3 and PI3K/AKT pathways. In this study, we showed that TrkB modulates BMP-mediated the growth inhibition of cancer cells. BMP-2 treatment induced SMAD1 phosphorylation and downstream transcription in RIE-1 and HeLa cells, which were significantly reduced by TrkB. In contrast, the loss of TrkB in highly metastatic breast cancer cell lines (MDA-MB-231 and Hs578T) restored BMP signaling. Moreover, growth inhibition induced by BMP-2 treatment was rescued when TrkB was also knocked down in these cells. Our results indicate that the TrkB-mediated suppression of BMP activities is necessary for the induction of tumorigenicity and cancer metastasis, which also supports results from our previous study. We have previously demonstrated that TrkB expression is dramatically increased in triple-negative breast cancers (TNBCs) relative to other subtypes and induces the growth and metastasis of breast cancer cells in vivo ,25. In aFurthermore, when the TrkB+ CSCs were eradicated, TNBC recurrence was prevented, which increased patient survival. In our study, TrkB overexpression in RIE-1 cells markedly increased anchorage-independent growth, migration, and mammosphere formation. These results suggest that TrkB may regulate cancer progression and recurrence by acquiring CSC traits.We also investigated whether TrkB can regulate the expression of BMP antagonists to inhibit BMP signaling. We found that TrkB significantly upregulated the expression of Gremlin 1 (a BMP antagonist). A previous study demonstrated that BMP signaling prevents TGF-β-mediated CSC transition in breast cancer and TGF-β-induced epithelial-to-mesenchymal transition (EMT) in renal fibrosis. In a mesenchymal subpopulation (MSP) of the human breast mammary epithelia, BMP antagonists (Chordin-like 2 and Gremlin 1) were significantly increased, whereas BMP ligands (BMP-2 and BMP-4) were markedly decreased. Additionally, BMP-4 treatment inhibited the capacity of HMLE, an MSP that had arisen from immortalized human mammary epithelial cells (MECs), to migrate and form mammospheres. The twist is overexpressed in HMLE cells ,29. MoreOur studies (both past and present) showed that TrkB represses RUNX3 expression by activating the PI3K/AKT signaling pathway and inhibiting the canonical BMP signaling. Introducing BMPRI in the presence of TrkB upregulated RUNX3 expression, which is further enhanced by the addition of BMP-2. Furthermore, upon transfection with BMPRI, RUNX3 expression is increased in MDA-MB-231 TrkB-shRNA but not in MDA-MB-231 control cells, and this increase is further enhanced by BMP-2 treatment. The activity of RUNX3, a tumor suppressor, is inhibited in various cancers by mislocalization, hypermethylation, or loss of heterozygosity ,33,34. RAdditionally, BMP signaling plays a role in the Hippo signaling pathway by inducing the phosphorylation of YAP (inactivated form) and stimulating the Ras association domain family  to inhibit tumor development and progression. BMP-2 treatment activates the Hippo kinase cascade, which includes mammalian Ste20-like kinase 1 (MST1), MOB kinase activator 1 (MOB1), and YAP (inactivation), and this action is tumor suppressive. BMP-2 also induces BMPRII expression, apoptosis by inhibiting the nuclear translocation of YAP, and RASSF1-MST1 complex formation by disrupting AKT-RASSF1 interaction . AnotherTo date, the link between TrkB and BMP signaling is unknown, and our study is the first to report this association. We have identified a unique role for TrkB in the regulation of BMP and RUNX3-mediated growth inhibition of cancer cells.RIE-1, HeLa, NMuMG, 293T, and human highly metastatic cancer cells (Hs578T and MDA-MB-231) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). BMP-2 from PeproTech (500-P195BT) used at the final concentration of 50 ng/mL for the indicated time. The protein kinase inhibitor K252a was purchased from Abcam.RIE-1, HeLa, and NMuMG cells were infected with a V5-TrkB using a pLenti6.3/V5-TOPO TA Cloning Kit  to generate TrkB overexpression cells  and the Proteins extracted from human breast normal and tumor samples obtained as previously described ,41.We performed Western blotting, immunoprecipitation, and immunofluorescence analysis, as previously described . phospho3 cells/well seeded into 6-well cell culture plates. For the wound healing assay, 1 × 106 cells/well seeded into 6-well cell culture plates. For the invasion assay, 1 × 104 cells seeded into 24-well BD Matrigel invasion chambers with 8-µm pores . For anoikis assay, RIE-1 and RIE-1-TrkB cells were seeded into an Ultra-Low Cluster plate  at 1 × 105 cells per well in a six-well plate and photographed at 7 days. For mammosphere assay, 1 × 103 cells/well were seeded into 96-well ultra-low adhesion plates in DMEM medium.All the assays were performed as previously described ,43,44. F4 cells were transfected with a BRE-Luciferase reporter plasmid using Lipofectamine 2000 . The cell lysates collected 48 hr after transfection, and the luciferase activities were measured using the Enhanced Luciferase Assay Kit .3 × 10Total RNA was isolated using RNeasy Mini Kits , and RT-PCR analysis was performed using a One-Step RT-PCR kit  according to the manufacturer’s instructions.For quantitative RT-PCR, reverse transcription performed with the Superscript Ⅳ First-strand synthesis system  and amplification performed with SYBR Green Mix I , and All PCR analyses were conducted in triplicate using the 7900HT Fast Real-Time PCR System . The primer sequences used to amplify the investigated genes listed in 5 cells/well in 0.5 mL culture medium. After waiting 4 hr for surface attachment, we incubated the cells in the presence or absence of porcine BMP-2 (5 ng/mL) for 24 hr. The cells were then pulse-labeled with 0.5 µCi [3H]-thymidine for 2 hr, fixed with 1 mL methanol/acetic acid, 3:1 (vol/vol), for 1 hr at 25 °C, washed twice with 2 mL 80% methanol, incubated at 37 °C with 0.2 mg/mL trypsin for 30 min and then solubilized with 0.5 mL 1% sodium dodecyl sulfate (SDS). Incorporated activity levels were measured with a scintillation counter.Actively growing asynchronous cells were plated in 24-well plates at a density of 0.5 × 10t-test (two-tailed). Differences were considered statistically significant at p < 0.001.Data expressed as the means ± SEM. Statistical analyses of the data conducted via the Student’s TrkB promotes the formation of TrkB/BMPRII complex by interacting between the TrkB kinase domain and then TrkB suppresses activation of SMAD1. Eventually, TrkB suppresses BMP and RUNX3-mediated growth inhibition of cancer cells.


In this study, graphene was synthesized on the Si(100) substrates via the use of direct microwave plasma-enhanced chemical vapor deposition (PECVD). Protective enclosures were applied to prevent excessive plasma etching of the growing graphene. The properties of synthesized graphene were investigated using Raman scattering spectroscopy and atomic force microscopy. Synthesis time, methane and hydrogen gas flow ratio, temperature, and plasma power effects were considered. The synthesized graphene exhibited n-type self-doping due to the charge transfer from Si(100). The presence of compressive stress was revealed in the synthesized graphene. It was presumed that induction of thermal stress took place during the synthesis process due to the large lattice mismatch between the growing graphene and the substrate. Importantly, it was demonstrated that continuous horizontal graphene layers can be directly grown on the Si(100) substrates if appropriate configuration of the protective enclosure is used in the microwave PECVD process. Graphene is a monolayer or several layers of hexagonally shaped carbon atoms . This 2DA complicated graphene transfer process is one of the main limitations preventing the broader use of graphene in semiconductor device technology. In this instance, graphene is grown on the copper of nickel catalytic foils ; followe2, Al2O3, MnO2, HfO2, and TiO2 films 2D/IG rat highest  at the sature in . A decreorted in ,51, simiThus, the different effects of the gas flow ratio, plasma power, and temperature on the graphene structure were found in various studies. The discrepancy of the results reported by other authors was more considerable than the results reported in the present study. The graphene structure’s dependence on the technological synthesis process conditions found in the present research can be explained by considering the main graphene growth-related physical and chemical processes. Notably, the decrease in the number of graphene layers with increasing process time can be explained by the hydrogen etching prevailing over the growth of additional graphene layers as it was reported in . 2D/IG ratio with plasma power ubstrate , and SiOtrate [2 . Thus, otrate [2 ,69.In conclusion, the transfer-less and catalyst-less synthesis of graphene on Si(100) substrates via a combination of direct microwave plasma-enhanced chemical vapor deposition and protective enclosures was performed.4/H2 gas flow ratio, temperature, and plasma power on graphene structure revealed that the most significant technological parameter used in the present study was methane and hydrogen gas flow ratio. Plasma power effects were the least pronounced. It seems that if a temperature is sufficiently high for graphene synthesis, the crucial process is a competition between plasma etching by hydrogen and carbon-containing active species influx towards the surface. If hydrogen flow is too high and/or methane flow is too low, etching prevails against growth and no graphene is formed. Afterwards, the number of graphene layers increases with carbon species flow and/or with decreased hydrogen flow. Hydrogen species density is a much more critical etching factor than increased plasma power. The thermally stimulated desorption of carbon atoms is important, while the formation of the plasma-induced radiation defects has less influence on graphene growth and defect density. A study of the effect of the CHA study of the enclosure effects revealed no top hole size effects for investigated enclosures. The absence of top holes in the middle of the enclosure reduced the plasma effect on the growing graphene and decreased defect density. The graphene was successfully synthesized using just a rectangular steel sheet folded in two places as a simplified protective enclosure, considering these results. Graphene was grown at a temperature of 700 °C using a protective sheath with such a simple structure. Analysis of the positions of 2D and G peaks revealed unintentional n-type doping of the graphene. It was explained by charge transfer from the Si(100) substrate to the graphene. The presence of compressive stress was found in graphene. It was supposed that the large lattice mismatch between the growing graphene and the silicon induced thermal stress.An atomic force microscopy study confirmed the growth of continuous horizontal graphene layers.


Nanoscale photothermal effects enable important applications in cancer therapy, imaging and catalysis. These effects also induce substantial changes in the optical response experienced by the probing light, thus suggesting their application in all-optical modulation. Here, we demonstrate the ability of graphene, thin metal films, and graphene/metal hybrid systems to undergo photothermal optical modulation with depths as large as >70% over a wide spectral range extending from the visible to the terahertz frequency domains. We envision the use of ultrafast pump laser pulses to raise the electron temperature of graphene during a picosecond timescale in which its mid-infrared plasmon resonances undergo dramatic shifts and broadenings, while visible and near-infrared plasmons in the neighboring metal films are severely attenuated by the presence of hot graphene electrons. Our study opens a promising avenue toward the active photothermal manipulation of the optical response in atomically thin materials with potential applications in ultrafast light modulation. A new breed of ultrafast light modulators that operate across the visible to terahertz regions may be possible by harnessing photothermal control of plasmons in graphene and thin-metal films. Theoretical analysis performed by Eduardo Dias and coworkers from the ICFO research institute in Barcelona, Spain suggests that ultrafast optical excitation of thin gold/silver-graphene structures can significantly raise the electron temperature in the carbon layer and as a result dramatically modify the plasmonic response. Calculations indicate that strong changes (>70%) in the optical reflection of such structures are possible, with a strength that increases with the fluence of the excitation pulses. Importantly, the fluence is below the damage threshold of the materials. The photothermal modulation approach could have useful applications ranging from all-optical light modulation to optical sensing. These important applications have been extensively investigated in conducting materials due to the substantial enhancement produced in the strength of nanoscale photothermal processes as a result of the excitation of their collective electron oscillations, known as plasmons12. Among plasmonic materials, graphene offers additional appealing properties, such as large field confinement and enhancement15, remarkably low optical losses16 and the ability to tune its plasmons electrically27, which have generated expectations for applications in optical modulation31, light detection33, and sensing36. Unfortunately, plasmons in graphene have been observed only in the mid-infrared (mid-IR) spectral range, far from the technologically appealing visible and near-infrared (vis-NIR) spectral regime. Photothermal excitation has been proposed as a way to reach the vis-NIR plasmonic regime37, but experimental attempts in this direction have only been limited to the mid-IR39. As an alternative approach to extrapolate the appealing plasmonic properties of graphene to vis-NIR, both monolayer noble metals40 and hybrid systems formed by graphene in close proximity to few-nanometer metal films41 have been predicted to display relatively low losses and a large electrical tunability in the vis-NIR domain. Progress in this direction has been recently made with the experimental demonstration of plasmons in laterally patterned sub-2-nm crystalline silver42 and thicker amorphous gold43 films. In particular, epitaxially grown silver films serve as a novel platform for ultracompact nanophotonic devices that can further benefit from the comparatively high quality factor of plasmons in defect-free crystalline silver samples42.Heat generation driven by light absorption in nanostructures has proven useful for photothermal therapy44. Importantly, due to the small electronic heat capacities of graphene45 and ultrathin noble metals46 compared with their total heat capacities, the lattice remains close to ambient temperature for optical pump-pulse fluences as high as ~10 J/m2, which is the upper limit of the range typically used to investigate thin-film dynamics through pump-probe spectroscopy48. In combination with the strong temperature dependence of the graphene optical conductivity, the transmission and reflection of a light probe can undergo radical variations produced by heating49, while intense photothermal effects should be expected due to the exceptionally small electronic heat capacity and generally weak electron-phonon coupling in this material45. Metals present a weaker thermal dependence, although the effects of intense pumping have also been observed to affect the probe as heat propagates away from the pumped region48. Consequently, graphene, thin metal films and hybrid systems comprising these materials hold strong potential to achieve large photothermal modulation over a broad spectral range extending from the visible to the THz region. Despite the vast range of applications that such modulation could open, the ultrafast photothermal response of these systems has been poorly explored.Under ultrafast light irradiation, the absorbed energy is first deposited on conduction electrons, which remain at an elevated temperature for ~1 ps before transferring a substantial fraction of heat to the atomic latticeIn this paper, we theoretically investigate the thermal control of plasmons assisted by light absorption in graphene, thin metals and graphene/metal hybrid films. Based on a realistic description of the temperature-dependent optical properties of these materials, combined with the light-electron-lattice heat flow under ultrafast optical pumping, we predict a remarkable thermally driven attenuation of the plasmon strength in mid-IR graphene plasmons and vis-NIR metal plasmons of hybrid systems. This attenuation is directly revealed by plotting the reflection coefficient of planar structures. We further propose a practical scheme to exploit this dependence, consisting of laterally patterning the metal so that propagating external light can directly couple to the noted plasmons. Our results predict a large modulation of the light reflection produced by optical pumping using attainable intensities below the damage threshold level. Graphene plays an important role in these structures because in its absence the photothermal response is much weaker; we relate this result to the small heat capacity of the carbon layer. Our findings open a promising route towards ultrafast photothermal light modulation over a broad spectral range extending from vis-NIR to far-IR.We illustrate the main concept explored in this paper in Fig. 37. This effect is thus an ideal ingredient for the pump-probe scenario envisioned in Fig. EF  for realistic levels of doping, and the same is true for noble metals. A femtosecond optical pump can then raise the electron temperature Te to 1000′s K, while heat transfer to the lattice, which takes place over a picosecond timescale44, does not substantially move the lattice temperature T0. In fact, we estimate that for fluences of the order of 1–5 mJ/m2, which roughly correspond to ultrafast pulses of ~100 fs with peak intensities of 0.1–0.5 GW/cm2, the electron temperature in graphene increases up to ~5000 K52, whereas electrons in the metal stay within a few 100′s K, as shown in Fig. 53. Therefore, in what follows, unless otherwise stated, we assume Graphene displays stronger photothermal effects than noble metals see Fig.  due to iTe through the chemical potential μ, the Drude weight μD and the inelastic scattering rate 1/τ, which enter its optical conductivity σ, as shown in Eq. . In contrast, the plasmon frequencies in noble metals exhibit a very mild dependence on the electron temperature (see Methods).The optical response of graphene is highly dependent on see Fig. . The nonTe, as shown in Fig. EF because the phase space for electron-phonon interactions is enlarged. Similar trends of the temperature dependence of the scattering rates are found in noble metals, as shown in Fig. The inelastic scattering rate of graphene also depends strongly on Te, as calculated using methods discussed in previous studies54. In all cases considered in Fig. Te as a result of the increase in Re{σ}. For the mid-IR graphene plasmons considered in Fig. Te rises , while thermal smearing of Dirac fermions shifts down the chemical potential μ for the Te values under consideration and diffuses the quenching within an energy interval of roughly ±kBTe  around 2μ .In Fig. see Fig. . We note57) or additional elements . Recently, an alternative route to excite graphene plasmons has been proposed49, where two interfering optical pump beams create a periodic spatial temperature profile in graphene to assist the coupling of a probe beam to plasmons. The characteristic diffusion time of the temperature profile created by the optical pump is of the order of a few hundred femtoseconds58, which should allow a probed optical pulse to excite plasmons, as considered in this study. Here, we further demonstrate that not only graphene plasmons but also metal plasmons or even subwavelength acoustic plasmons can be excited using this approach. We assume a periodic pattern of electron temperature Te created by the optical grating pump only inside the graphene sheet with a modulated temperature range extending from 300 to 8000 K. Due to the strong temperature dependence of the graphene conductivity σ  in 1-nm-thick (four atomic layers) gold gratings, which is a smaller value than in hybrid structures, despite the large fluences under consideration compared with those needed to heat graphene electrons to the values of Te used in Fig. We finally explore in Fig. see Fig.  in SI, w37, but their applicability has been limited to the mid-IR regime. We have shown that the addition of a noble metal film in close proximity to graphene enables similar levels of modulation >70% to be reached over a wider spectral range extending down to the visible domain. More precisely, optical pumping can bring graphene electrons to a high temperature, while the large heat capacity of the metal prevents similar heating inside it; then, high-energy metal-like plasmons are strongly affected by the opening of additional loss channels ; additionally, lower-frequency acoustic plasmons supported in these hybrid structures also become more lossy. A practical way to exploit these effects consists in laterally patterning the metal , thus breaking the energy-momentum mismatch that otherwise prevents light coupling into the plasmons of planar films. We envision applications in ultrafast optical modulation, which could be potentially pushed to the single-photon level through plasmon blockade in sufficiently small graphene nanostructures60. Spectrally resolved optical sensing could also be carried out by exploiting the temperature-dependent plasmon shifts reported here, which would enable a continuous sweep of the probe frequency as the system cools down over a picosecond timescale. Although the achieved thermally-induced spectral shifts can be limited by the presence of a finite diffusion time in the temperature profile, the amplitudes of the associated changes in the reflectivity provide an alternative sizeable signature for optical sensing in the ~300–3000 K temperature range.In summary, the remarkably small electronic heat capacity of atomically thin systems, such as graphene and few-atomic-layer noble metal films, allows us to elevate their electron temperature via ultrafast optical pumping in such a way that their optical responses are dramatically modified with moderate pump intensities below the damage threshold. This effect, which constitutes the basis for ultrafast thermo-optical modulation, is particularly strong in graphene due to the large photothermal response of this material, in which the conical electronic band structure causes large changes in the optical behavior for moderate amounts of deposited electronic heat. The photothermal modulation of the frequency and strength of graphene plasmons has been extensively studied in the past62We use the local random-phase approximation to calculate the optical conductivity of graphene asτ is a phenomenological inelastic scattering lifetime, Te, energy E and chemical potential μ, kB is the Boltzmann constant and 63where EF is the Fermi energy defined at zero temperature. The graphene conductivity has two contributions corresponding to the electronic intraband (first term in Eq. (μD) and interband . However, at higher electron temperatures, the chemical potential becomes increasingly smaller than EF  transit EF Fig. , allowinτ depends on both electron and lattice temperatures. For clean graphene samples , the inelastic scattering rate is dominated by electron-phonon scattering. Following previous studies65, the energy-dependent scattering rate associated with the b-type phonon is given to first order byIn general, the inelastic scattering rate 1/kk′ is the scattering angle between incoming and outgoing wave vectors k and k′, l = 1) and hole (l = −1) energies in the conical band dispersion regime, vF ≈ 106 ms–1 is the Fermi velocity andwhere θb-phonon scattering probability. Here, q = k′−k, b-type phonon that can be readily evaluated using Eqs. (16–21) and Table IV of ref. 64. Then, the total DC inelastic scattering rate is given byis the electron-where Te-dependent electronic heat as63The specific heat capacity of graphene Fig.  is calcuwhereis a thermal coefficient.59. In addition, the Drude model is in agreement with recent observations of plasmons supported by few-atomic-layer crystalline silver films42. We therefore use the Drude model and write the metal permittivity as48For ultrathin metal films, a recent study shows that the Drude response works extremely well compared with the random-phase approximation incorporating film electron wave functions and atomic-plane corrugationε∞ is a background accounting for the polarization of interband transitions, the plasmon frequency B  and68where mentclass2pt{minimmentclass2pt{minimγ0 is found by matching the total inelastic scattering rate γm to the experimental results at ambient temperature T0 = 300 K . The rightmost approximate expression in Eq. (γm in Fig. Te dependence of the plasmon resonance frequency of metallic nanostructures is mostly controlled by that of the classical metal plasma frequency ωp, which is robust against the electron temperature range considered in this work (see Fig. where n in Eq.  is validsee Fig.  in SI.Supplementary Information