Although Hb did not affect the cytoplasmic accumulation of TDP-43, Hb attenuated TDP-43-induced toxicity by reducing oxidative stress and mitochondrial dysfunction. Moreover, a shortened lifespan and motility defects in TDP-43-expressing Drosophila were significantly mitigated by dietary treatment with hydroxocobalamin. Taken together, these findings suggest that oral intake of hydroxocobalamin may be a potential therapeutic intervention for TDP-43-associated proteinopathies.TAR DNA-binding protein 43 (TDP-43) is a member of an evolutionarily conserved family of heterogeneous nuclear ribonucleoproteins that modulate multiple steps in RNA metabolic processes. Cytoplasmic aggregation of TDP-43 in affected neurons is a pathological hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer’s disease (AD), and limbic predominant age-related TDP-43 encephalopathy (LATE). Mislocalized and accumulated TDP-43 in the cytoplasm induces mitochondrial dysfunction and reactive oxidative species (ROS) production. Here, we show that TDP-43- and rotenone-induced neurotoxicity in the human neuronal cell line SH-SY5Y were attenuated by hydroxocobalamin (Hb, vitamin B TDP-43 (TAR DNA-binding protein 43) is encoded by the TARDBP gene ; it pred2+ homeostasis [TDP-43 interacts with NF-κB, a key factor contributing to the inflammatory response, and activates NF-κB in microglia. Activated NF-κB induces the production of proinflammatory cytokines, such as IL-1β and TNF-α ,16. Furteostasis ,21. In aeostasis . Moreoveeostasis . In addieostasis . Prolongeostasis .A recent clinical study indicated that methylcobalamin (vitamin B12) is a slightly effective for patients with early-stage ALS. This study suggested that methylcobalamin does not affect all patients with ALS, but it can extend survival and reduce disease progression in patients with early-stage ALS . HoweverHydroxocobalamin (Hb) is a manufactured analog of the vitamin B12 . The cyanide ligand of vitamin B12 is replaced by hydroxide in Hb . There aDrosophila disease model, the short lifespan and climbing defects induced by TDP-43 expression were significantly rescued by hydroxocobalamin treatment. Taken together, these findings suggest that vitamin B12 may be a potential therapeutic target for TDP-43-associated proteinopathies.In this paper, we found that TDP-43-induced neurotoxicity was rescued by hydroxocobalamin treatment. We found that hydroxocobalamin protects cells by regulating mitochondrial dysfunction without affecting cytoplasmic accumulation of TDP-43 aggregates. In addition, in the The following reagents were purchased from the indicated companies: dimethyl sulfoxide , rotenone , mifepristone , MG-132 (MilliporeSigma), PERK inhibitor (MilliporeSigma) and hydroxocobalamin hydrochloride . The following antibodies were purchased from the indicated companies: TDP-43 , phospho-eIF2α , eIF2α , α-tubulin , COX-Ⅳ , cleaved caspase-3 , anti-mouse IgG , anti-rabbit IgG , phospho-PERK (Santa Cruz Biotechnology), and Lamin A/C (MilliporeSigma).The SH-SY5Y human neuroblastoma cell line  was maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% heat-inactivated fetal bovine serum  and 50 μg/mL penicillin–streptomycin (Gibco).g at 32 °C for 120 min and incubated overnight in a 37 °C CO2 incubator. The medium was changed to DMEM containing 10% FBS to avoid the toxicity of virus particles. Cells were harvested 2 or 3 days after infection.Third-generation lentiviral constructs, including pLenti-C-mGFP-P2A-Puro (vector) and human WT TDP-43, were used to generate lentiviruses as previously described . HEK293TA Cell Counting Kit-8  was used to measure cytotoxicity according to the manufacturer’s instructions. Briefly, SH-SY5Y cells were seeded in 96-well plates and treated with rotenone or Hb for 24 h, in which DMSO was used as a negative control. CCK-8 solution (10 µL) was added to each well and was incubated at 37 °C for 2 h. The absorbance at 450 nm was measured using a microplate reader . Cell viability was reported as a percentage of the control. All experiments were performed in triplicate.6 cells/well) were cultured with/without Hb pretreatment. Cells were then resuspended in 500 μL of binding buffer  and incubated with 5 μL of FITC-conjugated Annexin V according to the manufacturer’s instructions. Subsequently, the cells were incubated for 15 min in the dark at 37 °C. After adding 4 μL of propidium iodide (necrotic cells), flow cytometry was performed within 1 h using a MoFlo Astrios instrument .The numbers of necrotic and apoptotic cells were measured using an Annexin V-FITC apoptosis detection kit  according to the manufacturer’s instructions. Briefly, rotenone-treated SH-SY5Y cells  with freshly added protease and phosphatase inhibitor cocktail  by homogenizing cells. Next, an equal amount of protein lysates was separated on 4–12% Bis-Tris gels  or NuPAGE 3–8% Tris-Acetate gels (Thermo Fisher Scientific) and was transferred to a polyvinylidene difluoride  membrane. The membranes were blocked with TBS blocking buffer containing 5% skim milk (BD Biosciences) or 2% bovine serum albumin (BSA) for 1 h and incubated with primary antibodies for 12 h at 4 °C. Detection was carried out using an ECL-Plus kit . Samples from three independent experiments were used, and the relative expression levels were determined using a Fusion-FX instrument .g for 30 min at 4 °C. Supernatants containing the soluble fractions were harvested. The resting pellets were further extracted with cell lysis buffer containing 2% SDS , sonicated and then boiled at 95 °C for 5 min. The supernatant from this step was collected as an insoluble fraction.Proteins were fractioned according to their solubility by making some modifications to a protocol previously described. Cells were harvested, washed, and homogenized in lysis buffer containing protease and phosphatase inhibitor cocktails. The homogenate samples were centrifuged at 100,000× g for 5 min at 4 °C. Cytosolic supernatants were harvested and frozen. The pellet was then resuspended by vortexing in ice-cold NER buff-er, and the extracts were centrifuged at 16,000× g for 10 min at 4 °C. Supernatants containing the nuclear extract were collected.For subcellular fractionation, cells were lysed by NE-PER nuclear and cytosolic ex-traction reagents kit (Thermo Fisher Scientific) according to the manufacturer’s procedures. Briefly, nuclear and cytoplasmic fractions were obtained in ice-cold CER I and CER II buffer by centrifugation at 16,000× g for 5 min at 4 °C. Supernatants containing the cytosolic extract were harvested, and pellets were solubilized in ice-cold 2% CHAPS in TBS buffer. After vortexing, samples were centrifuged. Supernatants containing mitochondrial extracts were harvested. The extracts were mixed with 4× Bolt LDS Sample buffer and 10× Bolt Sample Reducing Agent buffer and then boiled at 95 °C for 5 min.The mitochondrial fraction was obtained using a Mitochondria Isolation Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Briefly, cell fractions were prepared in ice-cold mitochondrial A, B and C buffers through centrifugation at 12,000× For immunocytochemistry, cells were fixed with 4% paraformaldehyde for 30 min at room temperature. Cells were washed three times with PBS and then blocked with 5% BSA for 1 h at room temperature. For cell permeability, the blocking buffer was made of PBS-T . The primary antibody diluted in PBS-T containing 1% BSA was incubated with cells overnight at 4 °C. The next day, cells were then washed three times with PBS and incubated with an Alexa Fluor-conjugated secondary antibody for 1 h at room temperature. Then, samples were mounted and observed using a fluorescence microscope . Photomicrographs from three randomly chosen fields were obtained.4 cells per well, and all cells were then infected with GFP-tagged or GFP-tagged human WT TDP-43. The Hb (10 µM) or vehicle (DMSO) group was transferred to the IncuCyte ZOOM™ apparatus, and incubations continued over 72 h, with images collected every 3 h. All images were analyzed for confluence (%).Images were captured and analyzed to determine cell growth and proliferation using the IncuCyte ZOOM™ Live-Cell Imaging system  as previously described for monitoring the kinetics of proliferation and cytotoxicity of the cultured cells ,42. Incu5 cells/well in 200 μL of growth medium and incubated for 24 h at 37 °C with 5% CO2. After replacing the growth medium with 200 μL of XF assay medium supplemented with 5 mM glucose, 1 mM pyruvate and 4 mM glutamine, which was prewarmed at 37 °C, cells were degassed for 1 h before starting the assay procedure in a non-CO2 incubator. The OCR and ECAR were recorded at baseline, followed by sequential additions of 1 μM oligomycin, 2 μM FCCP, and 0.5 μM antimycin A plus 0.5 μM rotenone. Nonmitochondrial oxygen consumption (in the presence of antimycin A + rotenone) was subtracted from all OCR values, and outlier technical replicates outside the standard deviation of the mean were discarded for both ECAR and OCR. In SH-SY5Y cells, values were normalized to the mean protein level in each well measured using a BCA protein assay kit (Thermo Fisher Scientific).The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in TDP-43-transfected SH-SY5Y cells using a Seahorse XF96 flux analyzer . Cell lines were seeded in 24 wells of an XF 96-well cell culture microplate at a density of 1.5 × 10® PrimeScriptTM RT–PCR kit  according to the manufacturer’s instructions using an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems). Internal control was using 18S rRNA. The 2−ΔΔCt method was used to analyzed relative changes in gene expression [The TDP-43-overexpressed cells were isolated RNA using the TRIzol Plus RNA Purification Kit (Invitrogen) according to the manufacturer’s instructions and 100 ng of RNA were synthesized into cDNA templates at 37 °C for 120 min using a High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific). Quantitative RT–PCR was conducted using the one-step SYBRg for 3 min and resuspended in 500 μL of buffer containing 20 mM HEPES pH 7.4, 10 mM KCl, 2 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 1 mM DTT. The homogenates were incubated on ice for 20 min and centrifuged at 720× g for 5 min. Transfer supernatant (contain cytoplasm) into a fresh tube and keep on ice. Then, the supernatants were centrifuged at 10,000× g for 5 min for the isolation of cytoplasmic fraction. Finally, DNA was extracted from the supernatant or whole cell using the QIAamp DNA Blood Mini kit according to the manufacturer’s protocol  and 100 ng of DNA was used to quantitative RT-PCR. All primer sequences used in this study are found in Cells were collected at 600× Drosophila stocks were maintained on standard cornmeal agar media at 24 °C unless indicated otherwise. UAS-TDP-43 was described previously [eviously . All stoTDP-43-overexpressed adult male flies (0 to 1 d old) were divided and moved into individual vials contained RU-486 (Mifepristone) or EtOH only fly growing media (n > 100). Flies were transferred into new media every two-day, dead flies were recorded. Adult climbing ability was evaluated using a previously described method .p < 0.05 and are indicated as follows: * p < 0.05; ** p < 0.01; *** p < 0.001; and n.s., not significant.Data were analyzed using one-way ANOVA followed by a post hoc analysis as indicated, depending on the variables compared . Differences were considered significant when Rotenone is a natural pesticide that increases ROS production and ER stress by impairing the mitochondrial electron transport system ,46,47,48Next, we tried to determine whether Hb exerts a protective effect against other types of stress, such as proteotoxic stress. MG132, a potent ubiquitin proteasome system inhibitor, induces the death of SH-SY5Y cells. However, Hb treatment did not affect MG132-induced cytotoxicity in SH-SY5Y cells B.Previous studies showed that the inhibition of mitochondrial complex I activity induces ROS production, ATP depletion and the loss of the mitochondrial membrane potential ,51,52. MATF4/CHOP/GADD34, and upregulation of these genes plays an essential role in cell death [ATF4/CHOP/GADD34 transcripts in rotenone- or rotenone/Hb-cotreated SH-SY5Y cells using real-time PCR. We observed increased levels of the ATF4, CHOP, and GADD34 transcripts in cells exposed to rotenone, and their levels were significantly decreased by Hb treatment  . SH-SY5YWe performed immunoblot analyses to elucidate the effect of Hb on TDP-43 protein levels. The level of the exogenous TDP-43 protein was not affected by Hb treatment. Therefore, Hb does not appear to exert a direct effect on the TDP-43 protein level A. Next, Recent studies have reported that the inhibition of TDP-43 mitochondrial localization blocks mitochondrial dysfunction and TDP-43-induced neurotoxicity ,58. We pATF4, CHOP, and GADD34, were significantly increased in TDP-43-infected cells compared to the controls, and Hb treatment significantly reduced the expression levels of those genes , and ALS [2+ overload has been shown to contribute to necrosis or apoptosis by inducing mitochondrial dysfunction, abnormal generation of ROS, and ER stress [2+-sensitive dye, for 1 h to examine intracellular Ca2+ homeostasis in TDP-43-expressing cells. As shown in 2+ overload was observed in TDP-43-expressing neuronal cells and was significantly reduced by Hb treatment. A previous study suggested that chronic stress caused by the misfolded mutant TDP-43 protein induces inappropriate release of Ca2+ from the ER to the cytoplasm and eventual programmed necrosis [2+ concentrations in the cytoplasm drive TDP-43-mediated neurotoxicity. Taken together, these data indicate that Hb modulates cell survival by alleviating the mitochondrial dysfunction induced by TDP-43 in neuronal cells.Furthermore, we examined whether TDP-43 affected the mitochondrial membrane potential. GFP- and GFP-tagged human WT TDP-43-expressing cells were then analyzed using flow cytometry to quantify the fluorescence intensity of TMRE, a fluorescent dye that labels mitochondria in a membrane potential-dependent manner. The TMRE intensity was significantly lower in TDP-43-expressing cells than in controls. Moreover, Hb treatment significantly restored the mitochondrial membrane potential in TDP-43-expressing cells C. Consis and ALS ,67,68,69R stress . Subsequnecrosis . Thus, iA reduced mitochondrial membrane potential and increased mitochondrial permeability regulate the release of mitochondrial DNA (mtDNA) into the cytoplasm and activate the cyclic GMP-AMP synthase (cGAS) stimulator of the interferon genes (STING) pathway ,73. MoreThe cGAS-STING signaling pathway is known as a major mediator of inflammation in the environment of infection, cellular stress, and tissue damage . Next, wATF5, LonP1, HSP60, and HSPA9) genes in TDP-43-expressing neuronal cells. The transcription of mtUPR-related genes was upregulated by the expression of TDP-43. Subsequently, the increased expression of the LonP1, HSP60, and HSPA9 genes was reversed by Hb treatment. However, the expression of the ATF5 gene was not affected by Hb (In addition, the mtUPR is the first stress protective response initiated by mitochondrial damage, eventually mitigating damage by removing or repairing misfolded proteins . We perfed by Hb C. These Drosophila model of TDP-43 proteinopathies, which expresses human TDP-43 in the nervous system. In our previous studies, we established a Drosophila model of TDP-43 proteinopathies in vivo and showed that overexpression of TDP-43 led to neurodegeneration [We next investigated whether Hb inhibits TDP-43-induced toxicity in vivo using a neration ,77. We iVitamin B12 deficiency is associated with central nervous system lesions, including subacute combined degeneration of the spinal cord, suggesting that vitamin B12 plays an important role in the spinal cord and brain ,78. In a2+ signaling. Furthermore, mitochondria are known to be key centers mediating TDP-43 cytotoxicity in the development and progression of ALS [Mitochondria are important organelles participating in various cellular processes, including ROS generation, ATP synthesis, and intracellular Can of ALS ,85. NeurDrosophila models. First, we investigated mitochondrial dysfunction upon the overexpression of TDP-43 in the human neuroblastoma cell line SH-SY5Y and confirmed that Hb treatment significantly alleviated the loss of the mitochondrial membrane potential and intracellular Ca2+ imbalance caused by TDP-43 expression (In this study, we examined whether the neurotoxicity induced by TDP-43 was alleviated by Hb treatment. Hb treatment attenuated TDP-43-induced global mitochondrial dysfunction in mammalian neuronal cells and pression . Moreovepression .Drosophila disease model, the short lifespan promotion and climbing defects induced by TDP-43 expression were significantly rescued by Hb treatment. However, Hb did not affect the aggregation and mislocalization of TDP-43. Mitochondrial dysfunction, oxidative stress and UPS impairment are the major components of TDP-43-induced neurotoxicity [Cells have a mitochondrial quality control system called the mtUPR for the repair and recovery of damaged mitochondria. mtUPR activation in mitochondria promotes cell survival and organelle repair. Recent studies have reported the role of the mtUPR in AD, PD, and ALS ,86,87. Htoxicity ,22,76,84


The ability to precisely control moiré patterns in two-dimensional materials has enabled the realization of unprecedented physical phenomena including Mott insulators, unconventional superconductivity, and quantum emission. Along with the twist angle, the application of independent strain in each layer of stacked two-dimensional materials—termed heterostrain—has become a powerful means to manipulate the moiré potential landscapes. Recent experimental studies have demonstrated the possibility of continuously tuning the twist angle and the resulting physical properties. However, the dynamic control of heterostrain that allows the on-demand manipulation of moiré superlattices has yet to be experimentally realized. Here, by harnessing the weak interlayer van der Waals bonding in twisted bilayer graphene devices, we demonstrate the realization of dynamically tunable heterostrain of up to 1.3%. Polarization-resolved Raman spectroscopy confirmed the existence of substantial heterostrain by presenting triple G peaks arising from the independently strained graphene layers. Theoretical calculations revealed that the distorted moiré patterns via heterostrain can significantly alter the electronic structure of twisted bilayer graphene, allowing the emergence of multiple absorption peaks ranging from near-infrared to visible spectral ranges. Our experimental demonstration presents a new degree of freedom towards the dynamic modulation of moiré superlattices, holding the promise to unveil unprecedented physics and applications of stacked two-dimensional materials. For instance, twisted bilayer graphene (TBG) with a magic twist angle can flatten the electronic band structure near zero Fermi energy, which allows achieving Mott insulating behavior and unconventional superconductivity2. Besides, twisted transition metal dichalcogenide (TMD) heterobilayers have enabled trapping interlayer excitons in the potential wells created by moiré superlattices6.The formation of moiré superlattices in twisted layers of two-dimensional (2D) materials has recently become a crucial method to realize unprecedented photonic and electronic properties that are unattainable in the constituent layers13. Heterostrain is inequivalent strains on the two constituting layers of stacked 2D materials, which leads to the slippage of one layer over the other due to inequivalent lattice deformation18. Theory results have recently predicted that heterostrain can modulate the local atomic registry of moiré superlattices and enable intriguing properties such as flat moiré bands in TBG12, topologically protected helical modes in twisted TMD19, and excitonic instability in TBG13. The effect of heterostrain is expected to be far more significant than that of homostrain on altering moiré patterns7, which may enable discovering a new class of physical properties beyond what have thus far been observed in the absence of heterostrain13. For instance, heterostrain-enabled one-dimensional (1D) moiré potentials have shown linearly polarized photoluminescence emission with two orders of magnitude higher intensity20.Very recently, heterostrain in twisted layers of 2D materials has arisen as a powerful, new degree of freedom to tailor moiré patterns and the resulting physic properties of the stacked 2D materials22, which requires the precise control of the twist angle and heterostrain during device fabrications. Recent experimental studies have demonstrated the continuous tuning of the twist angle, which substantially alleviates the strict requirement for controlling the twist angle during the fabrication23. However, controlling heterostrain dynamically has yet to be experimentally realized, limiting the feasibility of heterostrain-enabled on-demand manipulation of moiré superlattices.Overall, the desired physical properties of moiré superlattices are very sensitive to the modulation of the moiré potential landscape16, which played a key role in achieving a large heterostrain of up to ~ 1.3%. The induced heterostrain in TBG devices was dynamically tunable by continuously changing the bending radius of the PET substrate. Raman spectroscopy provided unambiguous evidence for the generation of a substantial heterostrain by presenting triple G peaks that arise from both unstrained and strained graphene layers having a single degenerate mode of the G peak and two split G+ and G− peaks, respectively. Strong polarization dependence of the triple G peaks further confirmed the formation of heterostrain. We also verified the influence of the experimentally obtained heterostrain on the modulation of moiré superlattices and the electronic band structures of TBG through tight-binding simulations. The 1.3% heterostrain was predicted to allow achieving multiple absorption peaks including one in the near-infrared spectral range that is attributed to the optical transition between the two saddle points, which is forbidden in the absence of heterostrain.Here, we present a generalized method for dynamically tuning heterostrain in TBG to modify moiré superlattices. TBG devices were assembled on a flexible PMMA-coated PET substrate by mechanical stacking. A relatively large twist angle of 13.2° was intentionally used to minimize the interlayer friction force2) layer to allow convenient identification of monolayer graphene. The number of graphene layers was identified via various methods including Raman spectroscopy and optical contrast (see Supplementary Note 1 for more details on the sample identification). The crystal orientation was determined from the cleaved angles of exfoliated flakes24. The cleaved edge was aligned to the long side of a PMMA-coated PET substrate on a transfer stage.Figure 2/Si substrate  by −1/% is the strain coefficient. Table Figure mentclass2pt{minimmentclass2pt{minimmentclass2pt{minim−1 with the full width at half maximum (FWHM) of 16 cm−1  and G (green) peaks as shown in the bottom two panels of Fig. T peak remains unchanged while the G peak shifts by ~ 37 cm−1, which provides strong evidence for the development of substantial heterostrain in the TBG device. The measured peak positions of the three peaks, GT, G and G, at different bending radii are summarized in Fig. Interestingly, the substrate bending causes the single G peak to split into multiple peaks that are clearly discernable as shown in the bottom three panels. At a bending radius of 2 cm . In addition, heterostrain increases the number of pronounced DOS peaks (marked as red and green arrows). These new DOS peaks can be attributed to the strongly modified electronic band structures under the influence of heterostrain as shown in Fig. The changed electronic band structures in the presence of heterostrain result in the altered density of states (DOS) Fig. e, which . The calculated Re (30. Unstrained TBG (blue curve) shows strongly enhanced optical absorption  possesses multiple peaks enhanced over 2 We also calculated the real part (Re) of the dynamical conductivity, ces Fig. f. The sp6. Moreover, the dynamically tunable moiré superlattices enabled by heterostrain may achieve unprecedented physical properties. It is also worth noting that such heterostrain in TBG can also be achieved in an integrated strain engineering platform on a silicon substrate32.In summary, we demonstrated a strain engineering platform that enables the dynamic tuning of heterostrain in TBG. Raman spectroscopy measurements confirmed the existence of heterostrain by showing triple G peaks; the unstrained top graphene layer showed the G peak that is the same as in the pristine MLG whereas the strained bottom graphene layer presented the two split G peaks, which also showed strong polarization dependence. It was verified that the heterostrained TBG possesses distorted moiré superlattices, which strongly influence the electronic band structures and optical properties. Our heterostrain platform gives a new approach to tune the moiré materials continuously and accurately, thus may find important applications in tunable electronics and optoelectronics based on moiré-engineered graphene. This approach is very general and can be employed for modulating moiré superlattices in twisted transition metal dichalcogenide (TMD) heterostructures as well, which are expected to play important roles in engineering moiré excitons for a variety of applications including quantum emission2 substrate . Number of graphene layers was identified by analyzing optical contrast and Raman spectra. Long graphene strips were selected as bottom layers of TBG, while smaller monolayer flakes were selected as tops layers. Both the selected bottom and top layers has straight edges, such that crystal orientation could be identified from flake edges.Graphene flakes were prepared by mechanical exfoliation of Kish graphite  by scotch tape and deposited onto Si/SiOA 135-μm thick PET substrate was cut into 1 cm × 5 cm rectangles. PET substrates were cleaned by acetone, isopropanol alcohol (IPA) and deionized (DI) water, and then dried at 65 °C for 5 min. About 400 nm polymethyl methacrylate  was spin-coated onto PET at 4500 rpm for 90 s.Twisted bilayer graphene was mechanically stacked on PMMA-coated PET with a home-made transfer stage. First, long edge of bottom graphene was aligned along horizontal direction by rotating the sample stage. At the same time, PMMA-coated PET thin film was attached to a PDMS stamp of ~ 4 mm × 2 mm × 0.6 mm, which was adhered to a glass slide. Bending direction of PET rectangle was also aligned to horizontal direction, such that uniaxial strain will be applied along long side of bottom graphene. Then Glass/PDMS/PET/PMMA stack was used to pick up the bottom graphene layer. After baking at 180 for 2 min, bottom graphene is firmly adhered to PET substrate. Next top graphene was aligned 13.2° to horizontal, which was subsequently picked up by Glass/PDMS/PET/PMMA/Bottom graphene. Finally, detach PET substrate from glass slide and PET/PMMA/TBG was obtained. Cured PMMA can successfully transfer strain from PET to bottom layer of TBG during the bending process.Graphene G peaks were measured by Raman spectroscopy  with a 100× objective and 1800 g/mm grating. Laser wavelength was 532 nm. Before measurement, laser power was adjusted to low level to avoid heating effect. Lorentz fitting was applied to determine peak position and FWHM.Supplementary Information.


We investigate the Landau damping of acoustic phonons (We perform self-consistent analysis of the Boltzmann transport equation for momentum and energy in the hypersound regime i.e.,  LDOPW) is the loss of energy from the collective motion of plasma waves to individual particles. This causes plasmons to decay by exciting an electron below the Fermi level2. The mechanism of LDOPW has been observed in various systems, such as plasma oscillations (Langmuir waves) and accelerators3. In semiconductors, Landau damping of acoustic phonons (LDOAP) occurs in the hypersound regime during electron–phonon interactions5. This has been studied using Raman spectroscopy6, and high-resolution electron-energy-loss spectroscopy (HREELS)7.Landau damping of plasma waves 10, acoustomagnetoelectric effect (AME)13, acoustothermal effect (ATE)14 and acoustomagnetothermal effect (ATME)16. The mathematical relation between absorption coefficient 20 and confirmed experimentally in GaAs/AlGaAs22 and GaAs/LiNbO323 SSLs. Azizyan25 calculated the absorption coefficient in a quantized electric field, while Shmelev and Zung24 calculated the absorption coefficient and renormalization of the short-wave sound velocity. Cerenkov emission (CE) is one of the most commonly used methods for investigating acoustic effects29. When a Non-quantized electric field (ED) with a drift velocity 28.The interaction of acoustic phonons with charge carriers in bulk semiconductor materials causes an amplification of acoustic waves and was predicted by Tolpygo and Uritskii in 1956SAW) in the hypersound region is described as quantized lattice vibrations or surface phonons, which typically extend to 1013 Hz. SAW is generated by the deformation of the material caused by the intraband transitions of electrons under an applied field31. Theoretical and experimental studies of acoustic wave effects in graphene35, quantum wells (QWs)36, carbon nanotubes41, and rectangular quantum wires42 have been investigated in the megahertz (MHz), the gigahertz (GHz) and the terahertz (THz) regions. Thalmeier et al.43 observed Landau oscillations as a function of gate voltage in graphene. Zhang et al.44 obtained a strong absorption when the carrier density and the field were increased as a result of electrons colliding with the acoustic phonons under a drift electric field. Such interaction can also generate sound waves. Considering acoustic phonons as quantized sound waves of frequency (LDOCE). LDOCE occurs when the drift velocity is less than the speed of sound in a material. The absorption of the phonon energy is determined by the energy balance of the system. As the frequency of the acoustic phonon increases, the absorption also increases, until there is a resonance beyond which the absorption decreases. This phenomenon has been observed in several graphene-based AE experiments and has been used in the fabrication of sensing devices such as humidity sensors46, photodetectors47, and gas sensors48.In low-dimensional structures, the motion of surface acoustic wave  into GNRs creates a tunable multiband absorption effects, which opens an energy gap (N), the quantized wave vector B and the electron momentum. B varies with the width of the GNR, the lattice constant (Pi). To the best of our knowledge, there has been no theoretical investigation of LDOCE in GNR even though some experimental evidence of the phenomenon exists35. Poole et al.35 reported a nonlinear AE in GNR of width DC current. They observed a resonance between the measured current maxima, after which the current decreased. Liang et al.48, Zheng et al.49 and Okuda et al.50 reported similar behavior, where acoustic charge transportation was induced by SAW propagation in the graphene. Morgado et al.51 also reported negative Landau damping in bilayer graphene, where they measured a DC electric current induced by a static voltage across the graphene sheet. In this work, we study the nonlinear AE in SLG and GNR-500 with degenerate energy dispersion in the hypersound regime. We adopted the Boltzmann kinetic equation for the electron system interaction with the sound waves and calculate the absorption coefficient (fk), thus affecting those electrons whose velocities in the direction of sound propagation are close to the sound velocity. Herein, we theoretically examine the effect of DC fields on LDOCE in an SLG/GNR-500.The quantum Hall effect is observed when sound waves in a material are subjected to magnetic fieldsLDOAP, we analyzed the effect of electron–phonon interactions in a gate-controlled single SLG and GNR-500. An in-plane current I was applied along the x-direction of the graphene sheet, which was biased by the presence of the source-to-drain voltage 52, expressed as:k is occupied and the final electron state k′ is empty. fk is the unperturbed Fermi–Dirac distribution function. The factor A being the area of the material, the summation in Eq. . The absorption coefficient relates to the AE current via the Weinrich25 relation, as follows:Considering Thus, the acoustoelectric current Integrating and simplifying Eq.  yields6\58 indicates the contribution of an in-plane acoustic phonon. In Fig. 5 cm−1. The plot shows a nonlinear AE current AE current does not exhibit a simple linear dependence on THz, and then increased at higher frequencies. A similar trend was observed when the temperature was increased to 30K. However, at 50K and 70K, the increase in 34), where they measured the acoustoelectric current for several temperatures at various frequencies in the MHz region. However, in this study, the frequencies are in the THz region.From Eq. , \documementclass2pt{minimmentclass2pt{minimT) is shown as a 3D plot in Fig. T, the graph decreased to a minimum and then increased to a point and remained constant at higher temperatures, while the dependence of D = 0.9vs, 0.92vs and 0.94vs, which depicts a linear relationship. However, Γq decreases when vD increases.To further illustrate this, the simultaneous dependence of the mentclass2pt{minimmentclass2pt{minimAE current. Thus, Fig. 57), where the AE current varied linearly with the frequency. Figure 29. In Fig. 57, where acoustic-phonon frequencies above 10 THz were attained. The field E in the SLG can be calculated using L is the length from the source to the drain electrode in the graphene), the in-plane current n is the electron density) can be calculated.The Weinreich relation entclass1pt{minimaentclass1pt{minimaentclass1pt{minimaSLG into 54 with the energy dispersion given by53B is the quantized wave vector. By considering that the acoustic phonon and the electric field are directed along the GNR axis, GNR, the energy level degenerates. At low temperatures, when Patterning mentclass2pt{minimN is the width of the graphene, Pi is the sub-band index. The absorption reveals the characteristic feature of the acoustic phonon spectrum in the materials that occurs in the Terahertz frequency range. In addition to the parameters used in Fig. 54). The twin peaks occur as a result of electron transport in the dual-band formed in the GNR. In the first band, the electrons are initially absorbed until they encounter a gap, where they lose their energy. They then gains energy in the second band by absorbing the energy of the confined phonons. This occurs at low drift velocities of In Eq. , \documementclass2pt{minimThe first gap occurs at 35). In Figs. q is shown in Fig. 48, Okuda et al.50, and Morgado et al.51. In the Terahertz range, the simulated results of absorption in graphene obtained by Ullah et al.55 are in qualitatively agreement with Fig. Therefore, knowing, s in Eq. . Using tSLG can be calculated by using L is the length from the source to the drain electrode in the graphene), the in-plane current n is the electron density) can be calculated.The We have theoretically demonstrated the acoustoelectric current generation in graphene nanoribbon resulting from Landau damping of acoustic phonons. The AE current in a single layer of graphene was calculated when the temperature and the electric fields were applied. Graphene nanoribbon exhibited a larger acoustoelectric current than a single layer of graphene when a non-quantizing field was applied. The acoustoelectric current shifts as the drift velocity is varied. This makes acoustoelectric current in graphene nanoribbon tunable and a good acoustic wave filter for phonon spectroscopy.Supplementary Information.


We study the lattice configuration and electronic structure of a double moiré superlattice, which is composed of a graphene layer encapsulated by two other layers in a way such that the two hexagonal moiré patterns are arranged in a dodecagonal quasicrystalline configuration. We show that there are between 0 and 4 such configurations depending on the lattice mismatch between graphene and the encapsulating layer. We then reveal the resonant interaction, which is distinct from the conventional 2-, 3-, 4-wave mixing of moiré superlattices, that brings together and hybridizes twelve degenerate Bloch states of monolayer graphene. These states do not fully satisfy the dodecagonal quasicrystalline rotational symmetry due to the symmetry of the wave vectors involved. Instead, their wave functions exhibit trigonal quasicrystalline order, which lacks inversion symmetry, at the energies much closer to the charge neutrality point of graphene. The electronic structures of such systems—for example twisted bilayer graphene6, graphene on hexagonal boron nitride (hBN)13, and twisted bilayer transition metal dichalcogenides15 with small twist angles 3, mini Dirac points formation13, Hofstadter’s butterfly16, the emergence of superconductivity17, correlated phases18, and orbital magnetic moment19.When two or more two-dimensional atomic layers which do not share a common periodicity are overlaid, an additional periodicity in the form of moiré interference pattern emerges28. Owing to the momentum mismatch21, quasicrystalline twisted bilayer graphene exhibits the electronic structure of almost decoupled bilayer graphene at most energy ranges. Nevertheless, it also hosts unique electronic states which satisfy the 12-fold rotational symmetry28. Such quasicrystalline states arise from the resonant interaction between the states at specific wave vectors via the rotational symmetry of the quasicrystal as well as the translational symmetry of the constituent atomic layers28. The red and blue hexagons in Fig. 28. Thus, even transition metal dichalcogenides (TMDC) or square lattices can show the quasicrystalline states.A special case occurs when two hexagonal lattices are overlapped at mentclass2pt{minimmentclass2pt{minimmentclass2pt{minim30. The lattice mismatch between graphene and hBN results in a hexagonal moiré superlattice potential with a superlattice period 13. Recently, Leconte and Jung show that BN/G/BN at specific configurations can host two hexagonal moiré patterns overlaid at a twist angle of 31. However, the interaction mechanism responsible for such unique electronic states in BN/G/BN, as well as the actual electronic band structure, and whether the wave functions actually satisfy the symmetry of the quasicrystal have not yet been investigated.Recently, rapid progress has been made in stacking more than two incommensurate atomic layers and a number of studies have investigated the effects of multiple moiré superlattice potentials on the electronic structure. The most notable example among them is a double moiré system, which is composed of a graphene layer encapsulated by hBN layers (BN/G/BN)entclass1pt{minimaentclass1pt{minimaentclass1pt{minimamoiré patterns in double moiré superlattice are arranged in a dodecagonal (12-fold) quasicrystalline configuration. Then we reveal the resonant interactions that bring together and hybridize twelve degenerate Bloch states of monolayer graphene and show that such interactions reconstruct the band dispersion of pristine graphene at these wave vectors. Compared to the resonant states of quasicrystalline twisted bilayer graphene where the actual atomic lattices are arranged in a dodecagonal configuration28, the resonant states of BN/G/BN appear at the energies much closer to the charge neutrality point of graphene. However, their wave functions show the quasicrystalline order with a three-fold rotational symmetry rather than fully satisfying the 12-fold rotational symmetry of the double moiré pattern.Here, we investigate the conditions where the two hexagonal A and B) sublattices. Graphene has carbon atoms in both sublattices, while hBN has nitrogen atom on A site and boron atom on B site. The lattice constant of hBN, 32, is slightly larger than that of graphene, 33. Here, we do not consider the lattice relaxation between graphene and hBN35, since the effects of such relaxation on the electronic structures is an order of a few meV. Nevertheless, our effective theory that respects the lattice symmetry is able to properly describe both the gap at the primary Dirac point and the asymmetric gap opening at the two inequivalent mini Dirac points13, as well as the orbital magnetism of the structure19.We consider a trilayer system composed of graphene sandwiched by hBN. Both graphene and hBN are two-dimensional honeycomb lattice whose unit cell comprises of two ,38, 39 and graphene on hexagonal boron nitride13. In this work, we scaled 39. The effective potentials by hBN to graphene, 13 , there are more sets of states, with different wave numbers, that show the resonant interaction between the constituent monolayer states. However, the set in Fig. It should be noted that this is not the only resonant coupling in this system. As shown in previous work on quasicrystalline twisted bilayer graphene . For (2), on the other hand, we agree that the parameters used in this work might not be accurate. The difference in the interlayer interaction strength Finally, we discuss several factors that may affect the results in this work. (i) Effects of the theoretical model and parameters: In this work, we described the quasicrystalline resonant states by the effective model in the framework of a tight-binding Hamiltonian. Then, it is natural to ask how more accurate models, such as first-principle methods, affect the results. In our effective model, we describe the electronic states by using a few monolayer Bloch states which contribute significantly to the states. In first-principle methods, we get fully coupled wave functions of the states, and may analyze the contributions of each monolayer Bloch state by the projection of the wave functions to the Bloch states. The difference between the two models mainly comes from (1) the interaction between the resonant chain Eq.  and the hain Eq. , which ant}ψ Eq. , which aform Eq.  with sli28, the graphene encapsulated by TMDC layers will also exhibit the resonant states respecting the quasicrystalline rotational symmetry. The difference between the lattice symmetry of the hBN layer (honeycomb lattice) and the chalcogen layer  makes a phase difference between the A-to-A and A-to-B interaction in Eq. ((ii) Effects of using TMDC as encapsulating materials: We can get similar quasicrystalline resonant states in the system composed of a monolayer graphene encapsulated by two TMDC layers at suitable angle. Since the metal atom in a TMDC layer is far from the interface, the dominant interlayer interaction comes from the orbital hybridization between the t(q) Eq.  should b35. Thus, our systems, especially the structure with an angle of A-to-A and A-to-B in Eq. ((iii) Effects of lattice relaxation: Incommensurately stacked atomic layers tend to exhibit local lattice relaxation, especially when the moiré pattern is sufficiently large(iv) Tuning conductivity by gating: It is well-known that the conventional quasicrystals are poor conductors at the energies where the quasicrystalline states are dominant . And the van der Waals quasicrystals would be also likely poor conductors at the energies where the quasicrystalline states are dominant , as we can expect from the large band curvatures and the wave functions distributed selectively on a limited number of sites. Thus, if we tune the Fermi energies near the quasicrystalline states by gating, which is possible only when the states are within a feasible range from the charge neutrality point as in the current system, then we will be able to tune conductivity significantly by gating.moiré patterns are arranged in a dodecagonal quasicrystalline configuration. We first find the condition which gives a We investigated the lattice configuration and electronic structures of a double moiré superlattice of which two hexagonal atomic lattices are arranged in a dodecagonal configuration, the resonant states of double moiré superlattice lack the 12-fold rotational symmetry; they hexagonal quasicrystalline order at a specific point We then reveal the resonant interaction which brings together and hybridize twelve degenerate Bloch states of monolayer graphene as well as the band dispersion around the resonant states. Compared to the resonant states of quasicrystalline twisted bilayer graphene, of which


Larval zebrafish have transparent bodies that allow non-invasive visualization of whole cells of single spinal motor neurons, from somas to the neuromuscular synapses. This unique feature, combined with its amenability to genome editing, pharmacology, and optogenetics, enables functional analyses of ALS-associated proteins in the spinal motor neurons in vivo with subcellular resolution. Here, we review the zebrafish skeletal neuromuscular system and the optical methods used to study it. We then introduce a recently developed optogenetic zebrafish ALS model that uses light illumination to control oligomerization, phase transition and aggregation of the ALS-associated DNA/RNA-binding protein called TDP-43. Finally, we will discuss how this disease-in-a-fish ALS model can help solve key questions about ALS pathogenesis and lead to new ALS therapeutics.Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by progressive degeneration of motor neurons in the brain and spinal cord. Spinal motor neurons align along the spinal cord length within the vertebral column, and extend long axons to connect with skeletal muscles covering the body surface. Due to this anatomy, spinal motor neurons are among the most difficult cells to observe  TARDBP gene, in the degenerating motor neurons  is a fatal neurological disorder in which motor neurons in the brain and spinal cord are selectively degenerated, leading to progressive muscle weakness. Approximately 1–2 individuals per 100,000 are diagnosed with ALS each year, with motor symptoms typically appearing in mid-adulthood (average age 55) . About 1 neurons . Better TARDBP mostly occur in the IDR has led to the idea that pathological phase transitions of TDP-43 mediated by altered homotypic and heterotypic interactions through IDR contribute to ALS pathogenesis. Cytoplasmic aggregation of TDP-43 has been observed by overexpressing wild-type and mutant TDP-43 in cellular and animal models of ALS  that self-associates upon blue light exposure (PHR (PHR or a point mutant version of Cry2 (Cry2olig) exhibiting enhanced clustering (The optoDroplet approach, recently introduced by exposure  to inducure (PHR . This teure (PHR , and hasustering  was fuseustering . These oustering , suggestustering . The ligustering . This reustering .in vivo contexts (A key step toward understanding of motor neuron degeneration in ALS is to induce TDP-43 oligomerization and phase transition in motor neurons and evaluate their cellular outcome in contexts . In zebrcontexts , can be contexts , enablincontexts . opTDP-4contexts . Live imcontexts . The precontexts . This obcontexts . KnowledA major advantage of optogenetics is that it allows precise temporal control of a photo-responsive probe. In zebrafish larvae expressing opTDP-43 in most of the spinal motor neurons, the extension of the photostimulation duration from 3 h to 3 days resulted in the accumulation of cytoplasmic opTDP-43 aggregates in the motor neurons . The cytin vivo in motor neurons. The accumulation of cytoplasmic TDP-43 aggregates is a hallmark of degenerating neurons in ALS. However, how TDP-43 is localized in the nucleus under normal conditions and the mechanisms by which it gradually loses its normal state and forms cytoplasmic aggregates are not clear. Thus, it is tempting to speculate that light-dependent opTDP-43 phase transition is a fast-forward replay of the changes occurring in TDP-43 in ALS, especially as TDP-43 dynamics is currently anatomically inaccessible in human motor neurons. Uncovering intermediate stages of the opTDP-43 phase transition would provide mechanistic insights into TDP-43 cytotoxicity and potential therapeutic targets. The illumination time-dependent formation of cytoplasmic opTDP-43 aggregates suggests that oligomerization and phase separation of opTDP-43 is tunable and can be used to reveal the temporal sequence of downstream events driven by pathological TDP-43 phase transition in the spinal motor neurons. This opTDP-43 phase transition can be targeted with subcellular resolution to explore spatial sources of TDP-43 toxicity, such as the nucleus, axon, and pre-/post-synaptic terminals. The ability to observe diseased motor neurons in a transparent animal also allows us to directly investigate systemic responses of neighboring cell types, including interneurons, myofibers, glial cells, and immune cells, to aberrant TDP-43 transitions occurring in the motor neurons. Moreover, since the IDR of TDP-43 is classified as a prion-like domain and TDP-43 has a prion-like aggregation-seeding ability (The optoDroplet approach has explicitly demonstrated the causal relationship between TDP-43 phase transition and neurotoxicity  ability , potentiKA wrote the manuscript and generated the figures, with inputs from HH and KK. All authors approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The mean RALA town-wide assessment score across communities was 56.5 + 15.6 out of a possible 100 points. The mean RALA program and policy assessment score was 40.8 + 20.4 out of a possible 100 points. While grocery stores and schools are important for enhancing food and physical environments in rural areas, many opportunities exist for improvements to impact behaviors and address obesity.The built environment contributes to an individual’s health, and rural geographies face unique challenges for healthy eating and active living. The purpose of this descriptive study was to assess the nutrition and physical activity environments in rural communities with high obesity prevalence. One community within each of six high obesity prevalence counties in a rural Midwest state completed the Nutrition Environment Measures Survey for Stores (NEMS-S) and the Rural Active Living Assessment (RALA). Data were collected by trained community members and study staff. All communities had at least one grocery store and five had at least one convenience store. Grocery stores had higher mean total NEMS-S scores than convenience stores (26.6 vs. 6.0,  Obesity is a multifaceted public health issue and adult obesity continues to impact much of the United States (U.S.). Data from the National Health and Nutrition Examination Survey (NHANES) indicate that age-adjusted obesity prevalence among U.S. adults aged 20 years and older is 41.9% . AmericaEnvironmental features can hinder or support active living and healthy eating environments. The built environment is defined as manmade surroundings that include buildings, public resources, land use patterns, the transportation system, and design features . The buiThe built environment contributes to an individual’s health, and rural geographies face unique challenges for active living and healthy eating, including isolation, transportation distances, and lack of facilities ,15. UnheIn 2014, the Division of Nutrition, Physical Activity and Obesity (DNPAO) at the CDC funded several land-grant universities under the High Obesity Program (HOP) cooperative agreement to reduce obesity prevalence in high-obesity prevalence counties using a community-based, policy, systems, and environmental intervention approach . RecipieSix counties with an obesity prevalence >40% in a rural Midwest state (South Dakota) were identified as part of the CDC HOP funding aimed at improving the nutrition and physical activity environment to support healthy behaviors . One comhttps://nems-upenn.org/tools/, accessed on 29 November 2021). While higher scores are better, there are no cut points for what constitutes an ideal score on NEM-S; rather, the tool can be used to track changes pre/post intervention or examine associations between store environments and eating behaviors [To examine the nutrition environment of each community, the Nutrition Environment Measures Survey for Stores (NEMS-S)  was utilTo examine the physical activity environment of each community, the Rural Active Living Assessment (RALA)  was utilT-tests were used to identify differences in the mean scores for availability, price, and quality of food between grocery stores and convenience stores. Chi-squared tests were used to assess differences in the frequency of availability of healthful food items between grocery and convenience stores. T-tests were also used to compare overall RALA TWA and PPA scores. For all analyses, statistical significance was set at p ≤ 0.05. Data are presented as mean ± standard deviation.Analyses were conducted utilizing IBM© SPSS Statistics Version 24. Descriptive statistics were utilized to compile data on total scores and section scores for the NEMS-S and RALA tools. p < 0.001), as well as higher scores for availability , and quality  of healthful foods. Mean price scores did not vary between grocery stores and convenience stores (p = 0.07). Availability of healthful options in both grocery and convenience stores varied by food type (p < 0.05).All communities had at least one grocery store and five of six had at least one convenience store. Overall NEMS-S scores, in addition to sub-scores for availability, price, and quality for grocery stores and convenience stores by community are presented in ood type . HealthfIn terms of pricing of healthful options compared to regular options among the seven grocery stores, healthful options for 100% juice and whole-grain bread were priced lower than regular options in only 14.3% of stores see . HealthfThe mean RALA town-wide assessment score across communities was 56.5 ± 15.6, ranging from 30 to 74 out of a possible 100 points. Communities scored highest in school location and parks/playground sections, and lowest in the trails section. Recreational facilities and water activities fell in the middle range .The mean RALA program and policy assessment score was 40.8 ± 20.4 ranging from 3 to 56, out of a possible 100 points. Communities strongly supported physical activity through school-based policies and school programming. Town programming scored lower. Town policy held the lowest scoring subscale . Total TThe physical environment in which people live plays a role in shaping both diet and physical activity behaviors, thus ultimately impacting obesity ,24. The p < 0.001), it should be noted that these scores are on a scale of −9 to 54, and grocery stores received only 49% of total possible points. Therefore, although grocery stores are better nutrition environments in these rural areas, they are not necessarily ideal. These findings appear to be in line with others in rural areas. A study examining NEMS-S scores among counties in Montana with varying degrees of rurality based on rural–urban continuum (RUC) codes ranged from 28.7 in the most metro county (RUC code 3) to 21.8 in the most rural county (RUC code 9) with a mean among all counties surveyed of 23.8  [n = 1) to 8 (n = 1) to 9 (n = 4). For comparison, a study using the NEMS-S in urban Pennsylvania found supermarkets to have total scores approaching 40 points [Nutrition Environment. All communities had a minimum of one grocery store and all but one community had a minimum of one convenience store. Differences were observed, however, between the two types of retail environments in terms of availability  of healthy foods. While this was reflected in a higher total NEMS-S score for grocery stores vs. convenience stores  . In the 0 points . While p0 points ,30.As indicated by previous research, the healthfulness of individual’s diets is correlated with the availability of healthful food in stores within the same zip code , and rurPhysical Activity Environment. The RALA town-wide assessment and program and policy assessment scores across communities were 57 and 41 out of 100 possible points, respectively. In the present study, the town-wide assessment ranged from 30 to 74 and the program and policy section ranged from 3 to 56, highlighting wide variation in the amenities, programs, and policies that support physical activity among communities. These data are comparable with two other studies that have previously utilized the RALA to assess the physical activity environment in rural areas. Robinson et al.  assessedThe amenities within the community that emerged as strengths were linked to schools, such as school location within the community and parks and playground availability, as both sections scored ≥78%. The parks and playground section was the least variable (±20%), with all communities scoring a minimum of 15/25 (60%). These data highlight the key role schools play in supporting physical activity within small, rural communities through active transportation to and from school, and the parks and playgrounds associated with the school. The availability of recreational facilities and water activities was less common of an amenity, with the least common amenity section being trails. Only two of the communities had trails, scoring 16/20 with the other four communities earning zero points in this category. These lower scoring sections highlight the opportunity for improvement. While the availability for water activities and trails can be costly to amend, the availability of recreational facilities could be addressed through shared use agreements between the community and the school district to utilize gymnasiums outside of school hours for other recreational needs within the community.Schools had stronger policies and programs to support physical activity when compared with the community. Although the highest scoring section, the average score in the school policies section was 50%. Only one community earned all points in this category. Since there are national standards for school wellness policies, it was expected to see this section emerge as the highest scoring section but concerning to note the average was only 50%. School programs was the second highest section, highlighting the school as a strength within these rural communities.Similar to the present findings, school location, parks, and playground amenities were shown to be the highest scoring town-wide assessment sections, and school policy was shown to be the highest scoring program and policy assessment section when assessed in eight communities across Alabama and Mississippi , as wellThis study is not without limitations. While the NEMS-S is a validated tool, it captures information only about certain locations within communities , and other places where food is available  were not assessed, nor was the overall availability of food within the community. The RALA is also a validated tool, but it was completed by only one group of stakeholders within the community and could be biased to the information those individuals were aware of and willing to report. Therefore, it is unknown what information remained uncaptured in the assessment. Additionally, while NEMS-S data were collected by Extension professionals, RALA data were collected by community members, which may impact quality of data collection and reporting. Finally, funding limited the type and location of work possible; thus, there remains a small number of communities to assess, and the study is descriptive in nature. The findings presented are specific to rural communities and are unlikely to be generalized to other geographic locations.Conducting assessments on the nutrition and physical activity environments within rural communities is helpful in identifying existing supports that can be enhanced, and in identifying barriers and challenges to overcome. This facilitates interventions that are feasible and impactful for communities while optimizing the use of existing resources. In the rural communities examined in the present study, nutrition environments are enhanced by grocery stores—although price and overall availability of healthful food requires further improvement—and schools serve as an important physical activity hub. This information will be used for developing and implementing community-based, obesity prevention interventions, tailored for rural communities.


Polymer electrolyte fuel cells hold great promise for a range of applications but require advances in durability for widespread commercial uptake. Corrosion of the carbon support is one of the main degradation pathways; hence, corrosion-resilient graphene has been widely suggested as an alternative to traditional carbon black. However, the performance of bulk graphene-based electrodes is typically lower than that of commercial carbon black due to their stacking effects. This article reports a simple, scalable and non-destructive method through which the pore structure and platinum utilisation of graphene-based membrane electrode assemblies can be significantly improved. Urea is incorporated into the catalyst ink before deposition, and is then simply removed from the catalyst layer after spraying by submerging the electrode in water. This additive hinders graphene restacking and increases porosity, resulting in a significant increase in Pt utilisation and current density. This technique does not require harsh template etching and it represents a pathway to significantly improve graphene-based electrodes by introducing hierarchical porosity using scalable liquid processes. Polymer electrolyte fuel cells (PEFCs) are an electrochemical energy conversion technology that is key to helping decarbonise the transport, portable, and stationary power sectors ,2,3. How2 and H2) transport and water management [2 g−1 [Fuel cell CLs are typically 2–20 μm thick and consist of electrically-conducting carbon supports decorated with Pt-based nanoparticle catalysts. They utilise proton-conducting polymers (referred to as ionomer) as both binder and electrolyte, which in turn are in contact with the proton-conducting polymer membrane ,15. To mnagement ,17. Thusnagement ,20,21,22t [2 g−1 ,24. Hight [2 g−1 ,26. Carbt [2 g−1 ,27.2-bonded framework, which is significantly more resistant to oxidation than carbon black [2 g−1), in practical bulk systems, the ‘graphene’ used is rarely fully exfoliated. Graphene-based CLs are typically formed from dispersions of few-layer graphenes (FLGs), which have surface areas of only a few hundred metres squared per gram. Furthermore, 2D sheets are prone to restacking during deposition, which dramatically reduces their accessible surface area and Pt utilisation, due to the catalyst particles becoming trapped between the layers [2 diffusion highways as they are generally regarded as being key to low-tortuousity gas diffusion, particularly in thicker CLs [Graphene has been widely proposed as an attractive catalyst support material, mainly due to its spon black ,17,28,29e layers ,31,32. Te layers ,33,34,35e layers ,36. Micre layers  and mesoe layers ,24,36. Mcker CLs ,38. The cker CLs ,40,41,42cker CLs . One of While attempts have been made to manufacture porous graphene electrodes by novel techniques, such as freeze-drying, they typically use the less conductive and more defective variant of graphene, (reduced) graphene oxide, or they rely on a non-scalable manufacturing route ,31,44,453Ni aerogel as a CL, in which K2CO3 additives were found to induce changes in the CL morphology, modifying the porosity and improving mass transport.The most widely reported technique for mitigating the propensity of graphene inks to re-stack and form low-porosity CLs when deposited is to introduce a second electrically-conducting carbon support material to act as a spacer ,44,46,47In this study, a simple, industrially-scalable and non-damaging method was developed that improved the pore structure and Pt utilisation. This method uses urea as a sacrificial additive to the standard aqueous-based CL ink, which, when deposited, forms water-soluble organic crystals physically separating the FLG sheets during deposition. Urea was chosen as it is low-cost, environmentally benign, and widely available; and unlike salt additives, it was found to not destabilise graphene ink suspension. The urea can then be removed by simple soaking in water, leaving voids between the FLG particles. This process maintains the structure of the graphitic component while introducing meso- and macroscale porosity to the CL, dramatically increasing current density. Due to the mildness of the treatment, the benefits are obtained without damaging the graphene sheets or disrupting the continuous ionomer network.2 g−1), sodium metal , naphthalene (99%), dimethylacetamide , 4-bromothioanisole (97%), chloroplatinic acid hydrate , sodium borohydride (≥98.0%), and urea  were supplied by Sigma Aldrich . The acetone (99.8%) and propan-2-ol (99%) were supplied by VWR Ltd. . The Nafion® Dispersion  was obtained from DuPont Fluoroproducts . The carbon black (Vulcan XC72R) and Freudenberg H23C7 was supplied by Fuel Cell Stores . The argon (99.95%) was supplied by BOC gases .The graphene nanoplatelets  before use. All the other chemicals were used as supplied.The water was deionised using an Elga PureLab Chorus on site. The DMAc was dried over 4 Å molecular sieves  for 48 h before use. The graphene nanoplatelets were dried under vacuum  with a glass stirrer bar until all the sodium dissolved, leaving a deep green solution (ca. 4 h) of sodium naphthalide. Graphene nanoplatelets  were added and stirred (12 h) to form a black solution. 4-Bromoanisole  was added to the solution (12 h) before removing the mixture from the glovebox, filtering it over filter paper and washing it with water and acetone to produce a black powder of functionalised few-layered graphite .FLG Functionalisation. The procedure used was adapted from Clancy et al. . In an aPlatinum Nanoparticle Deposition. The chloroplatinic acid hydrate (1.08 g) and f-FLG (500 mg) were mixed with water (500 mL) and briefly bath-sonicated  to produce a metastable suspension. The mixture was vigorously stirred and sodium borohydride solution  was rapidly added and left to stir (16 h). The mixture was then filtered to produce Pt@FLG.Catalyst Ink Formulation. The graphene-based catalyst inks were prepared by mixing Pt@FLG , Nafion solution ., water (30 mL) and propan-2-ol (30 mL) in a 100 mL Mason Jar. The commercial Pt/C catalyst ink consisting of Pt/C , Nafion solution 11 wt% were supplied by DuPont Fluoroproducts ., water (30 mL) and propan-2-ol (30 mL) were likewise prepared in a 100 mL Mason Jar. The ratio between the ionomer and the carbon support was 0.8:1. In the sacrificial templated samples, urea 120 mg  was also added to the mixture. The mixture was bath-sonicated (30 min) and used immediately after dispersion.−1, with an offset serpentine spray pattern. A loading of 0.4 mgPt cm−2 was calculated from the mass change on the MEA due to spraying. The GDEs sprayed with urea additives in the ink were soaked face-down in a Petri dish filled with distilled water, after 24 h the GDE was removed from the water and dried. The graphene GDE was assembled together with the Gore Select membrane and a 0.4 mgPt cm−2 HyPlat GDE to act as an anode. The MEAs were hot-pressed at 150 °C and 360 psi. The cell assembly was sealed with a torque of 4 Nm applied to the 12 bolts of the MEA assembly.Membrane Electrode Assembly. The catalyst ink was sprayed directly onto the gas diffusion layer (GDL) H23C7 were suppliedFreudenberg  using an ultrasonic spray system . The bed of the spray coater was heated to 90 °C during spraying. The flow rate of the Exactacoat was set to 0.4 mL min2 with no back pressure and at 100% RH, 80 °C; 1.5/3 was used as the H2/O2 stoichiometric flow rate. The cell was conditioned by purging with Ar/Ar for 10 min, followed by Ar/H2 for 10 min, then air/H2 held at OCV for 5 min. The cells were held at 5 A for 1 h, followed by an Ar/H2 purge; 25 CV ‘cleaning’ scans were run between 0.06 and 1 V at 20 mV s−1. Cyclic voltammetry (CV) measurements were performed between 0.06 and 1 V at 20 mV s−1. ‘Fast’ polarisation curves were run at 0.05 V/pt and 10 sec/pt until the current density stabilised. ‘Slow’ polarisation curves were run for data collection purposes at 0.025 V/pt and 30 sec/pt.The fuel cell testing was performed on a Scribner 850e test system under air/H−1) and drop-cast on lacey carbon copper grids .TEM was performed on a JEOL 2100 with an accelerating voltage of 100 keV. The samples were briefly bath-sonicated in HPLC-grade ethanol  was run on a K-Alpha XPS system. The MEA samples were mounted on conductive carbon tape with the top of the GDE pointed upwards and a charge-compensating flood-gun applied to avoid charging effects.The CL microstructure was analysed by scanning electron microscopy (SEM). Small pieces of the GDEs were embedded in epoxy resin, then the GDE cross-sections were mechanically polished to achieve a mirror-like surface. The observations were performed using a ZEISS MERLIN  field emission gun scanning electron microscope (FEG-SEM) equipped with two X-ray energy dispersive spectrometry (EDS) detectors. The backscattered electron images and the platinum and fluorine EDS elemental maps were recorded at an accelerating voltage of 4 kV.The surface areas and pore size distribution were measured through the application of Brunauer, Emmett and Teller (BET) theory, on a Quantachrome Quadrasorb Evo  using nitrogen gas.The pre-exfoliated FLG nanoplatelets were further exfoliated in order to maximise the exposed surface area by using a previously established reduction methodology . The exfAlthough spraying the CL directly onto the electrolyte membrane has been reported to have improved fuel cell performance, in this work the ink was directly sprayed onto the microporous layer (MPL) of the gas diffusion layer (GDL) . This apThe elemental composition of the CLs were estimated via XPS. The standard CL formulation, composed of carbon black/graphene, Nafion and Pt, was found to possess a small nitrogen content, which was likely due to residual DMAc solvent. Urea contains 47 wt% nitrogen, so any significant change in the quantity of urea within the CL was identifiable from the nitrogen content. The nitrogen content in the CL after adding urea to the catalyst ink increased from 0.5 wt% to 2.3 wt%, decreasing to 0.9 wt% after washing , indicat2 transport. The addition of the urea thus significantly changed the pore structure of the catalyst layer, introducing meso- and macropores and improving accessibility to the active Pt NPs.Cross-sectional SEM images of the graphene-based CL without urea a,b revea2 diffusion limitations [2 mass transport resistances.itations . The MEA2 g−1 to 11.9 m2 g−1 due to the addition of urea at the fabrication stage  of cycles had to be performed before the power density stabilised. This instability was found to be significantly worse if the MEA was not soaked in water before assembly, requiring hundreds of polarisation curves to reach a stable performance. We suggest that the initial inability of the MEAs to reach a stable performance was due to the in situ removal of the excess urea from within the CL in the high-humidity environment. It was found that the initial performance reached equilibrium faster during high-humidity conditioning procedures.2, or low surface energy solvents; or direct sublimation, in order to allow a greater quantity of the sacrificial additive to be incorporated into the CL. Furthermore, different additives would be expected to have different morphological effects on the CL; moreover, combinations of different additives may allow more precise control over the PSD and porosity of CLs.This article reports a scalable, non-destructive and simply-implemented method for controlling the internal morphology and pore structure of the micro- to mesoscopic length scales of a graphene-based CL. The use of urea additives allowed the one-step fabrication of the CL, followed by a simple and environmentally friendly single-additive removal step. The addition of the urea to the CL ink mitigated the graphene sheets’ (re)stacking during spraying, creating a more porous morphology, increasing the efficiency of Pt utilisation and improving mass transport, leading to significantly higher current densities. The addition of the urea was only effective on graphene-based CLs; traditional Pt/C based on bulk carbon supports did not benefit from the addition of urea. Understanding how the sacrificial additive affects fuel cell performance, including the power density difference between graphene-based CLs and those based on Pt/C, is key to further improving nanomaterial-based electrodes. The technique reported here could be improved by modifying the process for the removal of the additive, as this step often leads to the mechanical breakdown of the CL. Other potentially scalable approaches to urea removal include the use of supercritical CO


Tendon is a major component of musculoskeletal system connecting the muscles to the bone. Tendon injuries are very common orthopedics problems leading to impeded motion. Up to now, there still lacks effective treatments for tendon diseases.Tendon stem/progenitor cells (TSPCs) were isolated from the patellar tendons of SD rats. The expression levels of genes were evaluated by quantitative RT-PCR. Immunohistochemistry staining was performed to confirm the presence of tendon markers in tendon tissues. Bioinformatics analysis of data acquired by RNA-seq was used to find out the differentially expressed genes. Rat patellar tendon injury model was used to evaluate the effect of U0126 on tendon injury healing. Biomechanical testing was applied to evaluate the mechanical properties of newly formed tendon tissues.In this study, we have shown that ERK inhibitor U0126 rather PD98059 could effectively increase the expression of tendon-related genes and promote the tenogenesis of TSPCs in vitro. To explore the underlying mechanisms, RNA sequencing was performed to identify the molecular difference between U0126-treated and control TSPCs. The result showed that GDF6 was significantly increased by U0126, which is an important factor of the TGFβ superfamily regulating tendon development and tenogenesis. In addition, NBM (nonwoven-based gelatin/polycaprolactone membrane) which mimics the native microenvironment of the tendon tissue was used as an acellular scaffold to carry U0126. The results demonstrated that when NBM was used in combination with U0126, tendon healing was significantly promoted with better histological staining outcomes and mechanical properties.Taken together, we have found U0126 promoted tenogenesis in TSPCs through activating GDF6, and NBM loaded with U0126 significantly promoted tendon defect healing, which provides a new treatment for tendon injury.The online version contains supplementary material available at 10.1186/s13287-021-02679-x. Tendon is a major component of the musculoskeletal system which connects the muscles to the bone. Injuries to tendon including tendinopathy and tendon rupture are very common orthopedics problems. Tendon disease is one of the most common diagnoses of people engaged in sports professions, accounting for 30% of the total number of injuries diagnosed . Althougβ (TGFβ) family of growth factors, GDF6/7, Scleraxis (Scx), Egr1, Mkx, etc. [ β-catenin/ERK signaling pathway, which could be partially rescued by U0126 [The arising of tissue engineering shows promising prospects for tendon tissue regeneration. Many studies have shown that mesenchymal stem cells (MSCs) or tendon stem/progenitor cells (TSPCs) have been successfully applied in tissue engineering to treat tendon injuries –6. MSCs kx, etc. . Howeverkx, etc. . The inhkx, etc. . In our by U0126 .ε-caprolactone (PCL) scaffolds fabricated by electrospinning have been widely used in soft tissue regeneration as well as tendon engineering. PCL is a low-cost aliphatic linear polyester characterized by its good biocompatibility and bioresorbablity. It has been approved by the U.S. FDA for clinical use [As we know, Type I collagen is the main functional component of the tendon. The other components are a small amount of proteoglycans, glycoproteins and minor collagens , 15. Theical use . Gelatinical use –21. Compical use . And it ical use . Using gical use . In the The NBM was generated according to the previously published protocol by Neo Modulus (Suzhou) Medical Sci-Tech Co., Ltd. . The surThe animal experiments were approved by the Animal Research Ethics Committee of Guangzhou University of Chinese Medicine (No. TCMF1-2019053). 10-week-old male SD rats or GFP-transgenic SD rats were used in this study. The details of TSPCs isolation and culture have been described previously . Briefly3 per well) were subcultured in a 96-well plate. After 24 h of incubation, the medium was changed into U0126 containing medium at different concentrations. Cells were incubated at 37 °C for 1 and 3 days. The cell proliferation was determined using methyl thiazolyl tetrazolium (MTT) reduction assay. After incubation, cells were treated with the MTT solution  for 4 h at 37 °C. The dark blue formazan crystals formed in intact cells were solubilized with 100 μL DMSO and the plate was shaken for 10 min. The absorbance at 570 nm was measured with a microplate reader.The cells (3 × 105 cell/well) and cultured until the cells reached confluence. Then the medium was changed to the tenogenic induction medium with ascorbic acid (25 μM) and CTGF (25 ng/ml) at 37 °C, 5% CO2. The medium was changed every 2 or 3 days. Two weeks later, the expressions of tendon-related markers were evaluated by quantitative real-time RT-PCR (qRT-PCR). At 2 weeks after tenogenic induction, the cells were stained with Sirius Red (0.1%). For quantification, Sirius Red was eluted with 0.1 N sodium hydroxide, and the optical density at 540 nm was determined using a spectrophotometer.The TSPCs were plated in 6-well plates . Real-time PCR was performed using the CFX96 Real-Time PCR Detection System . Amplification conditions were as follows: first at 95 °C for 5 min, and then 45 cycles of 95 °C for 15 s and 60 °C for 60 s. Primer sequences were listed in Additional file Total RNA was obtained from the TSPCs treated with or without U0126 using TRIzol Reagent . The quality and integrity of total RNA samples were assessed using a 2100 Bioanalyzer or a 2200 TapeStation (Agilent Technologies) according to the manufacturer’s instructions. The preparation of whole transcriptome libraries and deep sequencing were performed by the Sangon Biotech Corporation . The differential genes (log2ratio ≥ 1 or ≤ − 1) identified by RNA-seq have been uploaded in Additional file In order to demonstrate the formation of neo-tendon tissue in vivo, the cell sheets formed by TSPCs treated with or without U0126 were transplanted to the dorsal sites of nude mice. Briefly, a total of 8 mice were used; after anesthetized with ketamine and xylazine, an incision was made on the dorsum and a subcutaneous pocket was created to expose the posterior midline. The cell sheet was sutured to the posterior midline at both ends, there was tensile strength on the tendon graft with the mice movement. Six weeks later, the tissues were harvested and subjected to histological analysis.Forty SD male rats  were used in this study. A 2.0 mm wide tendon defect was created from the distal apex of the patella to the insertion of the tibia tuberosity with two stacked sharp blades according to our well-established protocol. U0126 was dissolved in DMSO and diluted with PBS. Then NBM scaffold was immersed in the U0126 solution overnight at 4 °C. The operated rats were divided into four groups: (a) Blank control group; (b) NBM-PBS group; (c) NBM-U0126 (100 mΜ) group; and (d) NBM-U0126 (200 mΜ) group. The NBM loaded with PBS and/or U0126 was placed in the tendon defect and sutured to the patellar bone and tibia tuberosity using Ethicon 6-0 suture. At 4 weeks after surgery, the animals were sacrificed by an overdose of ketamine and xylazine, and the patellar tendons were harvested for histological examination and biomechanical test.2) was calculated based on the ultimate load divided by the cross-sectional area at the breaking point measured by a high-resolution Vevo 770 animal ultrasound system  with images taken immediately prior to the biomechanical test. The Young’s modulus (N/mm2) was calculated from the linear slope of a stress strain curve.The procedure of biomechanical testing was described in a previous study . GeneralThe regenerated patellar tendon tissues were washed with PBS, fixed in buffered formalin, embedded in paraffin and sectioned for histological examination. Immunohistochemistry was done as described previously . Brieflyp < 0.05 was considered statistically significant. At least three sets of independent experiments were performed for each assay.All data were presented as mean ± SD. The statistical analysis was performed using one-way analysis of variance (one-way ANOVA). A value of Scx, Col1, Tnmd, Fmod and Mkx. While, PD98059 did not show an obvious promoting effect. The Sirius Red staining at 2 weeks after tenogenic induction also demonstrated that U0126 significantly promoted the tenogenesis of TSPCs  did not affect the cell viability of TSPCs  analysis revealed that many signaling pathways were enriched, the Top 30 of which were shown in Fig. GDF6, an important member of TGF-β signaling which has been well known as an indispensable factor for tenogenesis and tendon development, was significantly increased by U0126 which was confirmed by qRT-PCR and immunocytochemical analysis  in four-week-old male mice, which has direct functional consequences for the mechanical integrity of the tissue  [GDF6 has promoting effect on the tenogenic differentiation of BMSCs [U0126 has been used as a specific inhibitor of the ERK1/2 signaling pathway by selectively inhibiting MEK-1 and MEK-2 . It has  in vivo . And the in 2009 . They haperties) . Our preof BMSCs .The NBM, composed of electrospun gelatin and polycaprolactone nanofiber nonwovens, mimics the ECM of tissues. It was fabricated by electrospinning of in situ cross-linked GE nanofiber nonwovens and subsequent lamination with electrospun PCL nanofiber nonwovens by heating the layer assemblies above the melting temperature of PCL. It has been used to treat soft tissue defects . The preGDF6 expression. Most importantly, we also developed a novel combination of NBM and U0126 which could be used to promote tendon injury healing in a rat tendon defect model.In this study, we have demonstrated that U0126 could promote tenogenesis of TSPCs by activating Additional file 1. Fig. S1. Release kinetics of U0126 from NBM scaffold in vitro.Additional file 2. Table S1. Sequences of primers for real-time PCR.Additional file 3. Table S2. Differentially expressed genes (log2ratio ≥ 1 or ≤ -1) in U0126-treated TSPCs.


Various techniques such as scanning electron microscopy, UV-vis absorption spectra, tensile testing, Shore A hardness, surface resistance, thermal conductivity, and thermogravimetric analysis were conducted to characterize the microstructure and properties of the obtained composites. The results showed that the π–π stacking interfacial interaction between phenyl groups of SIS and graphene play an important role in the properties’ improvement, and the effect of interfacial interaction on the properties was revealed.Interfacial interaction is one of the most important factors in the construction of high-performance graphene-based elastomer composites. In this paper, graphene/poly (styrene- Polymer composites based on nanofillers such as fullerene, carbon nanotubes, nano-diamond, and layered silicates have attracted much attention in recent years, because of their prominent properties and numerous applications ,2,3. NowThe other important factor that influence the properties of graphene-based polymer composites is the interfacial interaction, which determines the stress dispersion and transport performance of the obtained nanocomposites ,12,13,14b-isoprene-b-styrene) (SIS) is a triblock copolymer belonging to the family of thermoplastic elastomers. The SIS composites with carbon nanotube or graphene have applications in photomechanical actuation [Styrene-based block copolymers are basically thermoplastic elastomers, with good performance for a wide range of applications, such as toys, packaging, adhesive, and medicinal materials . Poly  (Melt flow rate = 23 g/10 min) was purchased from Xingzhi New Materials Co. Ltd. . Chloroform was obtained from Sinopharm Chemical Reagent Co., Ltd.  without purification.Graphene was purchased from Suzhou TanFeng Graphene Tech Co., Ltd. . Poly (styrene-Graphene/SIS nanocomposites with different graphene concentrations (0~2.0 wt%) were prepared by a solution mixing method. In a typical procedure, the graphene was dispersed into 10 mL chloroform using a bath sonicator for 1 h. Then, 4 g of SIS was added into the dispersion with vigorous stirring for 2 h, and the mixture was sonicated for 1 h. Finally, the obtained mixture was poured into a polytetrafluoroethylene mold, the graphene/SIS films were obtained with an evaporation-induced self-assembly process, and the obtained films were dried at 60 °C to a constant weight.Scanning electron microscopy  was used to examine the morphology of the graphene sheets. UV-Vis absorption spectra were conducted with a S 3100 spectrophotometer . The tensile properties of graphene/SIS composites were measured by a universal testing machine ; at least five samples were tested to obtain average values. Measurements of Shore A hardness consisted of vertical immersion of the indenter into the composite surface with a Shore hardness tester . The surface resistance of the obtained composites was measured with an ultra-high resistance micro-current tester . The thermal conductivity of the composites was measured using thermal conductivity test equipment . Thermogravimeter (TG) analysis was performed with a TG-209-F3  under the nitrogen atmosphere at a heating rate of 10 °C/min from 30 to 650 °C.Scanning electron microscopy (SEM) of the graphene powder is given in Solution mixing has been found to be an effective method for the preparation of graphene-based polymer composites, as the polymer can be absorbed on the surface of graphene through interfacial interaction, resulting in a good dispersion state ,12. The Tensile testing was used to evaluate the mechanical properties of pure SIS and the graphene/SIS composites. The stress–strain behavior of all samples is presented in The investigation of Shore A hardness of SIS and corresponding graphene nanocomposites as a function of filler content are shown in According to the electrical percolation theory, graphene sheets provide percolated pathways for electron transfer, which imparts electric conductivity to the nanocomposites. However, the improvement efficiency of graphene sheets dramatically depends on the degree of sheets’ dispersion and interfacial interaction ,33. The Thermally conductive polymer composites are attracting considerable attention, especially in recent years because increasingly more powerful electronics are being developed . The theIncreased thermal stability is typical of polymer-layered nanocomposites, usually attributed to heat and mass barrier effects of layered nanocompounds, which delay heat and pyrolysis products’ diffusion. The thermal stability of graphene/SIS composites is presented in In summary, the graphene/SIS composites were prepared through solution mixing followed by an evaporation-induced self-assembly process, enhanced mechanical, electrical, thermal properties were obtained through π–π stacking interfacial interaction, and the effect of interfacial interaction on the properties was revealed. The results of UV-vis absorption spectra revealed that π–π stacking interaction between phenyl groups of SIS and graphene can be formed in the process of evaporation-induced self-assembly. The π–π stacking interaction can serve as a sacrificial bond to dissipate energy, leading to the effective dissipation of stress, and the tensile strength of the 0.5 wt% graphene/SIS composites is increased by 26.4% from 1.4 to 1.77 MPa. The π–π stacking interaction benefits from the formation of a conductive network; the surface resistivity of 2.0 wt% graphene/SIS composite decreased by four orders of magnitude. The high specific surface area and π–π stacking interaction provide more sites that can scatter phonons and damp the vibrational amplitude at the interface, inducing a higher thermal resistance. The π–π stacking interaction is benefits for the effective obstruction of low molecules from the degraded SIS and the shield function of the heat; thus, the obtained composites exhibit improved thermal stability in comparison to pure SIS.