The complete thermal decomposition loss of PMMA film was effectively avoided by introducing a copper clad layer. After the first thermal transformation process, flat, clean, and high-quality amorphous carbon films were obtained. Next, the in situ obtained amorphous carbon layer underwent a second copper sputtering and thermal transformation process, which resulted in the formation of a final, large-sized, and highly uniform transfer-free multilayer graphene film on the surface of the dielectric substrate. Multi-scale characterization results show that the specimens underwent different microstructural evolution processes based on different mechanisms during the two thermal transformations. The two-step thermal transformation method is compatible with the current semiconductor process and introduces a low-cost and structurally controllable polymeric carbon source into the production of transfer-free graphene. The catalytic protection of the copper layer provides a new direction for accelerating the application of graphene in the field of direct integration of semiconductor devices.Direct in situ growth of graphene on dielectric substrates is a reliable method for overcoming the challenges of complex physical transfer operations, graphene performance degradation, and compatibility with graphene-based semiconductor devices. A transfer-free graphene synthesis based on a controllable and low-cost polymeric carbon source is a promising approach for achieving this process. In this paper, we report a two-step thermal transformation method for the copper-assisted synthesis of transfer-free multilayer graphene. Firstly, we obtained high-quality polymethyl methacrylate (PMMA) film on a 300 nm SiO Graphene’s atomic-level thickness and excellent charge carrier properties have made it a highly sought-after material in the field of semiconductor device integration ,2. In thCompared with gaseous carbon sources , which hExisting studies on the preparation of transfer-free graphene from polymeric carbon sources mainly focus on nickel as a catalyst, which is a kind of magnetic metal and difficult to use in semiconductor processes. Copper, however, has lower carbon solubilization ability  and is iIn this study, PMMA was used as a representative carbon source to investigate its chemical composition evolution during the two-step thermal transformation process for the growth of transfer-free graphene films. The protective and catalytic effects of a metallic copper layer were examined, and the microscopic transformation process from PMMA to amorphous carbon to graphene was elucidated in detail. The role and surface structure evolution of metallic copper films in the thermal transformation of PMMA were also systematically studied. This research provides important insights for ensuring the successful transformation of PMMA into transfer-free graphene films and offers suggestions for further improving the quality of such films based on polymeric carbon sources. The findings of this work can help to enhance the compatibility of graphene film preparation technology with semiconductor processes and promote the application of graphene films in the integrated fabrication of semiconductor devices.−4 Torr) at 100 °C for 5 min to remove water molecule adsorption.The dielectric substrate used in this experiment was a 4-inch, 300 nm thermal silicon oxide wafer prepared by the Advanced Electronic Materials and Devices School Platform of Shanghai Jiao Tong University . The specimens were cut into small slices of approximately 1 cm × 1 cm by a diamond cutting knife. To ensure high-quality spin-coating of the polymer solution, hydrophilic treatment was required due to the hydrophobic nature of the wafers in their natural state. The silicon oxide slices were ultrasonically washed in acetone, deionized water, and ethanol for 15 min each, and the liquid residues were blown off using dry nitrogen before each washing procedure. UV treatment was used to improve the wettability of the polymer solution on the silicon oxide slices and the optimized exposure time is ~30 min. PMMA solution with a mass fraction of 0.5 % was then uniformly spin-coated on the treated silicon oxide slices using a homogenizer, with spinning velocity and time of ~6000 rpm and ~60 s, respectively. The Cu films with a thickness of 200 nm were sputtered using a magnetron sputtering system. The thermal transformation experiments were carried out in a tubular furnace with hydrogen gas protection (~100 sccm). The steady-state temperature and holding time were controlled in the range of 400–950 °C and 10–60 min, respectively, and the specific parameters can be seen in the corresponding figure. After the first thermal transformation, the copper film on the specimen’s surface was removed by nitrogen blowing a. For th2. The surface roughness of the specimen was measured using the MFD-300 atomic force microscope manufactured by Oxford Instruments, with a longitudinal resolution of 0.02 nm and a test range of 20 × 20 μm2. The microtopography and EDS spectrum of the sample were analyzed using the RISE-MAGNA Raman image-scanning electron microscope , with an accelerating voltage of 20 kV, beam current of 200 pA, and optimal focal length of 5 mm. The Raman spectrum and optical photographs of the sample were obtained using the Renishaw inVia Qontor with a laser power of 60 mW, spot size of 0.5 μm, and an acquisition wavenumber range of 1200–2800 cm−1, with the surface mapping sweep step size set to 0.5 μm. Chemical bonding information of the heat-transformed products was analyzed by the Mini Flex 600 XPS spectrometer produced by Rigaku , using monochromaticized Al Kα as the X-ray source, with a test power of 150 W, scanning step size of 1000.0 meV, and residence time of 100 ms. The full spectrum scanning range was 0–1200 eV, while the C 1 s and the Cu 2p3/2 spectrum scanning ranges were 273–296 eV and 924–968 eV, respectively. For electrical performance characterization, a four-probe resistor meter produced by Valley Polymerization Technology Co., Ltd.  was used. The test voltage and current were set to 1.0 V and 10 μA, respectively, with the probe spacing set to 1.0 mm. The number of repeated measurements of single-point resistance was set to 10 times. The ToF-SIMS depth profiles were captured using ION-TOF GmbH  under a chamber pressure of below 5 × 10−9 mbar to analyze the content of Si, O, Cu, and C in each layer of the specimen before and after annealing. For high mass resolution analysis, the spectra mode with pulsed 30 keV Bi3+ (~0.48 pA pulsed current) ion beam was applied. The focused analysis area was 150 × 150 μm2, with 2 keV Cs ion beam sputtering at the same time, 400 × 400 μm2 sputter raster, and noninterlaced mode.Thermogravimetric analysis of PMMA solid particles was carried out using the TGA 8000 thermal analyzer produced by PerkinElmer . The temperature range for analysis was from 25 °C to 700 °C with a heating rate of 10 °C/min, and the gas flow rate was ~40 mL/min in both air and NThe thermogravimetric analysis (TGA) of PMMA particles a reveals−1 and 1580 cm−1, respectively. These characteristic peaks correspond to the defect D peak and graphite-type G peak of the amorphous carbon film. The evolution of the characteristic peaks of amorphous carbon film with thermal transformation temperature indicates that the amorphous carbon film only begins to grow at about 400 °C, and no corresponding characteristic peaks appear below this temperature. The temperature is almost the same as the complete thermal decomposition temperature of PMMA particles in After the first thermal transformation, the morphology of the specimens with the spin-coated PMMA film changed significantly in comparison to the specimens without spin-coated PMMA g. The coAccording to the idea of the first thermal transformation of PMMA film, we carried out a second copper sputtering treatment on the obtained transfer-free amorphous carbon film and proceeded with a series of thermal transformation experiments again. The final morphology of these samples is shown in The copper layer on the surface of the sample undergoes a “dewetting” phenomenon in the high-temperature environment, but it remains intact below the temperature of 850 °C and 60 min. The EDS mapping of the copper film in −1). The appearance of these characteristic peaks confirms the in situ transformation of amorphous carbon film into graphene, and the intensity ratio of 2D peak to G peak determines the multilayer structure of the transfer-free graphene film. The intensity of the 2D peak in the Raman spectrum also indicates that longer thermal transformation time results in graphene film with better crystallization quality. Evaluating the uniformity of the intensity ratio of Raman 2D peaks to G peaks within a certain area of graphene film is another effective way to evaluate the surface uniformity of graphene. 2D/IG result of the red dashed line in 2D/IG ratio of the graphene film obtained in this process is around 0.16, demonstrating that the graphene film has good uniformity. The macroscopic topography photo of the silicon oxide slice after copper layer etching, shown in 2-type structure, which is also the typical structure in graphene film. The peak position at 285.40 eV mainly corresponds to the carbon sp3 bonding structure, which shows that there are some defects in the multilayer graphene film and is also one reason for the apparently defective D peak in the Raman spectrum in 3/2 region  can also provide us with more structural details of the transfer-free graphene films. Meanwhile, by analyzing the bonding type of chemical elements, we can also determine the impurity binding of the graphene film and evaluate the overall quality of the graphene film. XPS results of in situ growth of multilayer graphene films on silicon oxide substrate are shown in 2 region e testifi3 binding bonds in XPS C 1 s spectrum.Since the multilayer graphene film obtained in this project is in situ grown on the silicon oxide substrates, the traditional physical transfer process is avoided, and the electrical four-probe test is particularly convenient. The electrical results show that the surface resistance of the transfer-free multilayer graphene film is below 30 kΩ/□, which is similar to the nitrogen-doped graphene film prepared in the reference . Though 3+, H2, and H2O through the thermal decomposition process, and then gaseous carbon radicals will deposit and stitch on the surface of copper foil to grow graphene films. It is apparent that the polymer carbon source PMMA will undergo gas phase decomposition under high-temperature pyrolysis conditions, resulting in the loss of mass. Therefore, during the first thermal decomposition process in our project, the copper layer on the surface of the specimen loses its original support due to the loss of gaseous products of the lower PMMA layer, and then obvious wrinkles and bulges occur, and the copper layer is easily blown away by gas. It should be mentioned that, based on the structural characterization of the obtained products on the silicon oxide substrate after blowing away the copper layer, it is found that due to the physical barrier of the surface copper layer, the PMMA layer does not undergo complete and thermal pyrolysis with no solid residues, like in the free space, but generates amorphous carbon film on the substrate through amorphous transformation (see By comparing the first and second thermal transformation results, we can clearly see that the copper layer plays significantly different roles in the two thermal transformation processes, and both of these roles are indispensable parts of the synthesis of transfer-free multilayer graphene film. Reference  investigUnlike the PMMA pyrolysis process, the amorphous carbon film does not experience gaseous phase transformation during the second thermal transformation, so the surface copper layer remains intact after the thermal annealing. In order to deeply study the specific changes of each element in this process, the content distributions of Si, O, Cu, and C elements before and after thermal transformation were analyzed by ToF-SIMS, and the results are shown in γ is the interfacial energy, t is the graphene thickness at the interface, and ΔtransG is the graphene formation energy per unit volume with copper catalyzed, respectively [2, which indicates that it is more thermodynamically favorable to synthesize graphene film at the interface. Therefore, under the protection and catalyzation of the copper layer, graphitization transformation occurs, and the graphene multilayer film is ultimately generated (Simplified thermodynamic calculations show that the energy difference before and after the second thermal transformation can be expressed as ectively ,40. The In summary, this study investigated the two-step thermal transformation process of PMMA polymer to obtain transfer-free graphene film on dielectric substrates, utilizing film spin coating, copper layer sputtering, and wet etching techniques. The microstructural evolution of the PMMA film layer was examined, providing valuable insights for in situ graphene film synthesis. The copper layer plays a crucial role in the physical protection and catalytic crystallization processes, and precise control of its structural parameters is important for obtaining high-quality transfer-free graphene film. The two-step thermal transformation process successively yields clean, flat transfer-free amorphous carbon film as low as 400 °C and multilayer graphene film at 850 °C, which is more economical and energy-saving than the nickel-catalyzed method. The quality of these films can be optimized by controlling the initial spin coating and the thermal annealing parameters. Our study deepens the understanding of copper-assisted polymer transformation for graphene growth, without the need for post-transfer procedures. This approach has potential in compatible semiconductor processes and can offer more choices for graphene-based functional device manufacturing.


We study the processes of dynamical diffraction of the plane X-ray waves on the graphene film/SiC substrate system in the case of the Bragg diffraction geometry. The statistical dynamical theory of X-ray diffraction in imperfect crystals is applied to the case of real quasi-two-dimensional systems. The necessity of the taking into account of the variability of the lattice parameter of multilayer graphene, as well as the influence of thickness on the thermal Debye–Waller factor at the calculation of the complex structural factors and Fourier components of polarizability, is demonstrated. It is shown that the change of the structural characteristics of the 3-layer graphene/substrate system, as well as its strained state, leads to a significant change in the diffraction profiles, which makes it possible to determine the characteristics by the X-ray diffraction method. The development of modern physical materials science is inseparably linked with the determination of structural characteristics of materials. This is due to the material structure mainly determines their most important physical and exploitation properties. At the same time, the main characteristics of materials  as a rule strongly depend on their defect structure. Therefore, the problem of creation of the materials with predetermined properties is closely related to the improvement of already known and the development of new methods of characterization of the material structure.3 and SiC substrate annealing6 are effective methods for large-scale graphene production. Since the properties of graphene depend, in particular, on its structural parameters, it is important to carry out an accurate quantitative characterization of the structure of the graphene film on the substrate.Among other perspective materials, graphene has unique mechanical, electrical properties and can be used in many areas. At the same time, the properties of the graphene film depend significantly on the method of its preparation. In particular, chemical vapor deposition (CVD)11. The effectiveness of any diffraction method is largely determined by the analytical expressions that provide an adequate description of the scattering pattern. Both the kinematical approximation and the dynamical theory are used in the X-ray characterization of crystalline materials.For structural characterization of thin films, multilayer systems, superlattices, etc. nondestructive diffraction methods are widely usedThe kinematical description gives a simple expression for the scattering amplitude, which, in fact, is the Fourier transform of the electron density of the crystal. However, the kinematical approximation is violated for crystals whose dimensions are comparable with the extinction length. In this case, dynamical effects (multiple scattering effects), namely the interaction between diffracted and transmitted waves, together with refraction and absorption, can become significant. At the same time, partially dynamical effects can be manifested even for ‘thin’ crystals, so their rigorous description requires the use of dynamical scattering theory.12 and optical potential method15 to describe the Borrmann effect were developed.The dynamical scattering theory attracted an attention of researchers and got a development almost immediately and intensively after the discovery of the radiation diffraction on crystal structures. However, only since 1960s, when methods for growing almost perfect single crystals had developed, a rapid growth of a series of studies related to the dynamical diffraction was additionally motivated and stimulated. Particularly, the dynamical theories based on the Ewald–Bethe–Laue approach17 for the case of incident spherical wave diffracted on the mosaic crystal. This theory was repeatedly verified experimentally .Then, the Takagi–Taupin equations became the fundamental base of a statistical dynamical diffraction theory proposed in Refs.19). Just that approach was used to calculate the distributions of X-ray intensity scattered by imperfect crystals24.The case of incident plane waves was considered for describing the angular distributions of the diffracted X-ray  systems, including the firstly know 2D material—graphene, which contributes to the motivation of the study. The characterization of quasi-monolayer structures by the methods of dynamical diffractometry has the specific and certain features. The scope of this article deals with extension of the statistical dynamical theory of X-ray diffraction in imperfect crystals to the case of realistic quasi-monolayer systems with inhomogeneous strain fields and microdefects.Thus, the most studies and therefore obtained results describe mainly the bulk crystals. There is still a lack in the studying the quasi-two-dimensional (242:We consider the diffraction of the plane X-ray waves in the case of the Bragg diffraction geometry (the case of reflected radiation). According to the optical potential method, the polarizability of the crystal is represented as a complex quantityAccordingly, the Fourier component of polarizability can be represented as follows:Hr and χHi are also complex quantities proportional to the corresponding structural factors43:re λ2/πvc, re is classical electron radius, λ is wavelength of X-ray radiation, vc = , h, k, l are Miller indices. The summation in  of the atoms in the unit cell, fr and fi are the real and imaginary parts of the atomic form factor:f0 and ∆f are the atomic form factors at absolute zero temperature and the dispersion correction respectively, and exp(−M) is the thermal Debye–Waller factor. The atomic photoelectric absorption cross section has the form45:46, Ma is atomic mass, NA is Avogadro constant, C = 1 or cos(2θB) is polarization factor, respectively, for σ- and π-polarization,In the case of a non-centrosymmetrical crystal, the Fourier components χ47:ai, bi, ci are tabulated parameters, S = sinθB/λ. Dispersion corrections ∆f were taken from work46.An interpolation formula was used to calculate the atomic form factors48:Hp is the planar component of the scattering vector and Hz is its z-component. The root-mean-square displacements of lattice atoms 44.The exponent of thermal Debye–Waller factor in expression  was calc49, which were obtained within the framework of the generalized dynamical theory of scattering in imperfect single crystals with randomly distributed defects39.As mentioned, one of the effective methods of preparation graphene is its growth by annealing the SiC substrate. The analytical expressions for the intensity of diffracted X-ray radiation on the graphene film/SiC substrate system were used the recurrence relations between the coherent components of the amplitude reflection coefficients of adjacent layersM layers and a substrate, in the case of the Bragg diffraction geometry is described as follows:The coherent component of the reflectivity of a multilayer crystal system, which consists of RM(∆θ) is calculated using the recurrence relation between the amplitude reflection coefficients of two systems consisting of M and (M − 1) layers49:j = 1, …, M, R0 ≡ r0, ∆θ is angular deviation of the crystal from the Bragg angle. rj and tj are the amplitude coefficients of reflection and transmission of the jth layer, respectively:j indicates the connection of the corresponding value with the jth layer, b = γ0/|γH| is the parameter of the diffraction asymmetry, γ0 and γH are the direction cosines of the wavevectors of the incident and diffracted plane waves, respectively, H is the reciprocal-lattice vector, K = 2π/λ, dj is the thickness of jth layer, δ = 1, 2. The accommodations of the strong Bragg wavevectors in the jth layer, yj in formulas  due to the sample rotation and by the deviations ∆Hj of the reciprocal-lattice vector Hj due to the average strain caused by defects or chemical composition of jth layer:j is the angular deviation of the jth layer from the substrate orientation due to strain, ψ is the angle between the crystal surface and the reflective planes. The parallel and normal strain components in the jth layer are described as follows:R is a tension parameter (0 ≤ R ≤ 1) that represents or can be associated with a strain strength or percentage, D = 2c13/c33 is distortion factor, a0, c0 and aj, cj are lattice parameters for the substrate and layer, respectively, l0 and lj are the components of Miller indices for substrate and layer, respectively.The normalized angular deviation formulas  and 15)yj in forAt first, using formulas –8), the, the8), 4, the parameter β was used for to describe of the structure of graphene layers. This parameter characterizes an occupancy of graphene layers . Thereby the used model has two fitting parameters for each layer: interlayer spacing and occupancy of graphene layer. In our calculation, we consider graphene layers as the completely occupied (β = 1).In the articleIt should be noted, that we characterize a quasi-two-dimensional object, which is such according to its physical properties. However, here it is assumed that in the plane, parallel to the surface of the system, all layers  are homogeneous. It is due to this that the diffraction problem can be considered as one-dimensional along the axis, which is perpendicular to the surface of the system, which is inhomogeneous in this direction.44. It should be noted that the use of root-mean-square displacements for films of small thicknesses  corresponding to a bulk sample would lead to significant errors in the calculation of the thermal Debye–Waller factor and the Fourier components of the crystal polarizability and, as a result, to errors at the calculation of the reflection curves (RCs). This fact is illustrated Fig. Formula  takes inentclass1pt{minima51. So, as initial values of lattice parameters were used lattice parameters of graphite, namely a = 0.246 nm and c = 0.6708 nm. The Fourier components of the polarizability were calculated for these lattice parameters.Figure Figure 4. It can be seen that the results of the calculations of this work and the experimental data are in good agreement to each other.For comparison, Fig. 52. This layer is not yet graphene, because it is partially connected to the substrate due to the presence of sp3-hybridized regions. According to Ref.53, the interplanar distance between the buffer layer and the SiC substrate is d = 0.216 nm.It should be noted, that one of the stages of creating multilayer graphene films on a SiC substrate is the formation of a buffer layer Figure D materials known as ‘straintronics’58 and ‘twistronics’61. Figure a0 in formulas (a0cos30°. Poisson’s ratios for multilayer graphene νi = 0.15 (in-plane) and νo = − 0.09 (out-of-plane) were taken from the work62.One of the remarkable properties of graphene—known as a strongest material ever tested—is its high flexibility along with strength. Such unique mechanical properties caused the development of the relatively new research direction in the physics of graphene and related 2formulas  and 20)D materiaIt can be seen that the change in the strain state of the 3-layer graphene/substrate system leads to a change in the shape of RCs.In this article, the statistical dynamical theory of X-ray diffraction of the plane X-ray waves in imperfect crystals is applied to the case of real quasi-two-dimensional systems. The case of the Bragg diffraction geometry is considered. The necessity of the taking into account of the variability of the lattice parameter of multilayer graphene, as well as the influence of thickness on the thermal Debye–Waller factor at the calculation of the complex structural factors and Fourier components of polarizability, is demonstrated. It is shown that the change of the structural characteristics of the 3-layer graphene/substrate system, as well as its strained state, leads to a significant change in the diffraction profiles, which makes it possible to determine the characteristics by the X-ray diffraction method.D materials. Particularly, such the sensitivity can contribute to the overcoming the challenges dealing with detecting so-called magic angles in the twisted 2D material offering a method to modify its electronic properties.The X-ray diffraction characteristics are sensitive to the structural strains independently on their types . Therefore, the XRD method can act as a powerful or at least additional tool for detecting of any responses in the structural changes occurring in graphene and other currently discovered 2The proposed dynamical approach, as an additional tool, allows describing correctly the diffraction profiles from such structures in the region of the Bragg peak from the substrate and its immediate vicinity. Due to this, we can expect to improve the reliability of the diffraction characterization and possibly obtain additional accurate information about the structure of the system, because the proposed method allows us to take into account the influence of the substrate and its defects on the formation of the total diffraction pattern.


With the escalating demand for electrochemical energy storage, commercial lithium-ion and metal battery systems have been increasingly developed. As an indispensable component of batteries, the separator plays a crucial role in determining their electrochemical performance. Conventional polymer separators have been extensively investigated over the past few decades. Nevertheless, their inadequate mechanical strength, deficient thermal stability, and constrained porosity constitute serious impediments to the development of electric vehicle power batteries and the progress of energy storage devices. Advanced graphene-based materials have emerged as an adaptable solution to these challenges, owing to their exceptional electrical conductivity, large specific surface area, and outstanding mechanical properties. Incorporating advanced graphene-based materials into the separator of lithium-ion and metal batteries has been identified as an effective strategy to overcome the aforementioned issues and enhance the specific capacity, cycle stability, and safety of batteries. This review paper provides an overview of the preparation of advanced graphene-based materials and their applications in lithium-ion, lithium-metal, and lithium-sulfur batteries. It systematically elaborates on the advantages of advanced graphene-based materials as novel separator materials and outlines future research directions in this field. Given the escalating concerns surrounding pollution and climate change as a result of the extensive utilization of non-renewable resources, it has become crucial to prioritize the development of energy storage technologies that are sustainable, efficient, and environmentally friendly. With the decreasing cost of renewable energy sources, such as wind and solar power, rechargeable batteries have emerged as a superior option to traditional mechanical and hydraulic energy conservation due to their higher energy density and accessibility. In particular, lithium-ion batteries have gained immense popularity in various industries, including the automotive, electronic, and aerospace sectors, owing to their remarkable operating voltage, substantial energy density, minor memory effect, and eco-friendliness. Additionally, lithium-metal and -sulfur batteries, which boast higher theoretical specific capacity and lower costs, are widely regarded as one of the most promising candidates for next-generation energy storage systems.In recent decades, the separator has developed significantly as a crucial component of lithium-ion/metal/sulfur batteries through extensive research efforts. It has been established that the separator must exhibit desirable insulation and mechanical strength to prevent short-circuiting of electrodes and the appropriate pore size and porosity to facilitate the uniform transmission of lithium ions. Furthermore, excellent thermal stability, adequate wettability, and electrolyte retention are critical to enhancing battery capacity and performance. Hence, selecting a separator with comprehensive performance is essential to improve battery performance ,2,3,4. C2 hybridized hexatomic carbon ring of graphene. Meanwhile, the controllable surface functional groups enable more specific properties to be achieved, for instance, the effective catalysis action on redox reactions and the adsorption effect of Li+, which significantly improves energy density and cycle stability of batteries while expediting the Li+ transfer and electrochemical reaction kinetics . A. A4 as tobtained B 17]. G. G4 as tynthesis . In the ynthesis . For the[3/FeCl3 C 22]..4 as theDespite the progress achieved by single metal substrates, as previously mentioned, the great challenge for them is controlling the layer number and uniformity of graphene. Hence, the preparation of graphene by CVD on alloy substrates has rapidly developed due to the advantage of multiple metal templates. Ruoff reported the CVD growth of graphene on “90–10” Cu-Ni alloy, where a higher temperature, longer deposition time, and slower cooling rate helped to increase graphene coverage on Cu-Ni alloy foil D 23]. W. W23]. W4  flakes to obtain large amounts of graphene. However, pore size cannot be precisely controlled in pyrolysis. To address this issue, the addition of in situ pore-building additives at high temperatures has been explored as a competitive strategy. For instance, Zhang et al. developed a simple method to produce a fluorescent graphene layer by pyrolyzing amide salt . Xu et a2(OH)2CO3 with polymethyl methacrylate (PMMA) and subsequently induced PMMA to yield few-layer graphene . G. G68]. G3O4 particles with graphite particles and intense ultrasonic waves to enhance the exfoliation of graphite . . + trans0 cycles C 105]. . + transCompared with the previously discussed basic lithium-ion batteries, lithium-metal batteries have garnered significant attention from researchers for their exceptional energy density and high voltage. However, the formation of metal dendrites during the electrode process can react with the electrolyte, leading to a rapid decay in battery cycle life, a substantial reduction in coulomb efficiency and energy density, as well as deformation and melting of the separator due to the puncturing and thermal effects of lithium dendrites. These phenomena can ultimately cause irreversible capacity loss, internal short circuits, and even explosions ,107,108.−2 . . +, whic mA g−1) . Gong etcapacity .4 (LFP) full cell with PDA/Gr-CMC as the separator revealed a specific capacity close to 100% of the initial discharge capacity after 500 cycles . . 4 (LFP) mA cm−2 . 2N3 as the transition metal nitride was also investigated . . −1 (equof 82.7% . Peng et mA g−1) . Zhou ety of 98% .3BO3/NH2CN for GO reduction to obtain B-doped and N-doped graphene and coated them on the separator, respectively, for comparison. The battery with a nitrogen-doped graphene composite separator maintained a specific capacity of 430 mAh g−1 after 400 cycles at 0.2 C, delivering better cycling stability . . −1 at 00 cycles . Zuo et ectively . Li et a0 cycles . Zhou etectively . Zhang eeparator . Lei et 0 cycles . The aboGraphene-based materials for separator modification in lithium-ion/metal/sulfur batteries have recently received vast attempts. In this review, we provided a comprehensive summary of the recent advancements in the preparation of graphene-based materials using techniques such as CVD, pyrolysis, and others. We also surveyed their applications as functionalized separator materials in next-generation lithium-ion/metal/sulfur batteries in + transfer channels while buffering the volume change of electrodes. Specifically, for lithium-ion batteries, the high wettability and abundant functional groups lead to improved Li+ transfer kinetics, superior energy density, and enhanced rate/cycle performance. For lithium-metal batteries, the graphene-based interlayer mitigates local heat accumulation around irregular lithium deposition and inhibits further dendrite growth. Additionally, the outstanding mechanical strength of these materials effectively prevents separator puncturing. Finally, in lithium-sulfur batteries, the graphene-based interlayer can function as an effective sulfur adsorption agent and capture network, as well as an activator for dead sulfur.Generally, functionalized separators utilizing porous graphene-based materials exhibit significant enhancements in conductivity, wettability, and thermal stability due to their large surface area, exceptional chemical stability, excellent mechanical properties, and controllable oxygen-containing surface functional groups. These materials also provide numerous active sites for redox reactions and LiHowever, there are still great challenges for the industrial production of separators assembled with a graphene-based interlayer. Regarding preparation, it is quite challenging to make sufficient integration of low cost, minimal pollution, high yield, and high quality. While micromechanical cleavage can produce graphene sheets of just a few atomic layers with high accuracy, its low production efficiency restricts its application to laboratory settings. CVD is a commonly employed method to produce high-quality single-layer graphene material, but issues, such as the service life of templates and pollution from the etching process, need to be considered for large-scale industrial production. Pyrolysis with SFA encounters difficulties in completely removing the residual SFA, while SFA-free pyrolysis requires even more stringent production conditions. Liquid-phase exfoliation is a low-cost method for preparing large-scale few-layer graphene, but it is challenging to control the layer number and avoid restacking of the graphene layer during extended production times.+ transfer kinetics and the interaction between the graphene-based interlayer and electrolytes/electrodes. Additionally, new composite strategies, rational structure design schemes, and improved film formation and coating methods need to be developed. For example, in situ preparation of an integrated graphene-based interlayer and substrate material may lead to more uniform modification compared to simply coating graphene-based material onto commercial separators, as most studies do at present. Afterward, the self-shutdown function when overheating and shortcuts were rarely mentioned in the present works about separators functionalized with graphene-based materials, which may be a desirable scheme for improved safety. Other potential strategies that are worth further investigation include self-supporting graphene-based interlayers without substrates and the use of graphene-modified separators in gel/solid electrolytes for higher performance in next-generation energy storage devices.In terms of application in separator modification, further research is needed to understand the mechanism of electron/Li


Despite the strong evidence linking the transactive response DNA-binding protein 43 (TDP-43) aggregation to the pathogenesis of frontotemporal lobar degeneration with TDP-43, amyotrophic lateral sclerosis and several neurodegenerative diseases, our knowledge of the sequence and structural determinants of its aggregation and neurotoxicity remains incomplete. Herein, we present a new method for producing recombinant full-length TDP-43 filaments that exhibit sequence and morphological features similar to those of brain-derived TDP-43 filaments. We show that TDP-43 filaments contain a β-sheet-rich helical amyloid core that is fully buried by the flanking structured domains of the protein. We demonstrate that the proteolytic cleavage of TDP-43 filaments and exposure of this amyloid core are necessary for propagating TDP-43 pathology and enhancing the seeding of brain-derived TDP-43 aggregates. Only TDP-43 filaments with exposed amyloid core efficiently seeded the aggregation of endogenous TDP-43 in cells. These findings suggest that inhibiting the enzymes mediating cleavage of TDP-43 aggregates represents a viable disease-modifying strategy to slow the progression of amyotrophic lateral sclerosis and other TDP-43 proteinopathies. Reconstitution of TDP-43 filaments that exhibit sequence and morphological features similar to those found in the brain helps to uncover a new mechanism for the formation and propagation of pathology in amyotrophic lateral sclerosis and other neurodegenerative diseases. The pathological hallmark of these diseases is the presence of proteinaceous cytoplasmic inclusions in degenerating neurons. TDP-43 is the primary component of the cytoplasmic inclusions in ~97% of ALS and ~45% of FTLD  cases4.Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two of the most common debilitating neurodegenerative diseases (NDs) in people aged between 45 and 65 years6. Furthermore, TDP-43 aggregates of distinct properties have been isolated from ALS and FTLD-TDP brains and are increasingly commonly found in the brains of patients suffering from other NDs8. Therefore, understanding the molecular, structural and cellular determinants of TDP-43 aggregation is essential to unravel its role in NDs and developing novel strategies to diagnose, prevent, treat or slow the progression of these devastating disorders.Increasing evidence suggests that cellular changes leading to TDP-43 mislocalization and aggregation in the cytoplasm resulting in the gain of toxic functions that drive neurodegeneration in ALS and FTLD-TDP9. The NTD of TDP-43 acquires a novel ubiquitin-like fold when produced as an isolated domain10. This NTD forms functional but non-pathological homo-oligomers under physiological conditions and is resistant to cellular stress11. The extent to which the structure of the NTD and the mechanism of its assembly in isolation affects the misfolding and aggregation of the FL TDP-43 remains unknown.Full-length TDP-43 (FL TDP-43) consists of 414 amino acids with two RNA recognition motifs (RRM1 (105–169) and RRM2 (193–253)) flanked by an N-terminal domain (NTD (1–80)) and C-terminal domain (CTD (277–414)). The folded domains of RRMs bind specifically to the UG-repeats of RNA and the TG-repeats of DNA and play an important role in TDP-43 function in transcription regulation13. It is intrinsically disordered, contains a long stretch of sequences enriched with glutamine/asparagine repeats and glycine residues, which are predicted to be highly amyloidogenic15, and harbors several mutations associated with familial and sporadic ALS/FTLD-TDP17. Misfolded and aggregated forms of FL TDP-43 or its C-terminal fragments (CTFs) are commonly found in the brains of ALS/FTLD patients , outside the amyloid core, influence these mechanisms in the context of the FL TDP-43 protein.Previous studies showed that not all TDP-43 inclusions in ALS/FTLD-TDP cases possess the classical characteristics of amyloid fibrils23. Altogether, our in vitro and in-cell-seeding studies suggest a new working model for TDP-43 pathology formation, where post-filament formation PTMs, especially N-terminal cleavages, could play critical roles in regulating TDP-43 fibril uptake, seeding activity and pathology spreading.In our work, we describe a new method for generating highly ordered filaments derived from native monomeric FL TDP-43 in vitro and devoid of oligomers, amorphous aggregates or monomers. We demonstrate that an amyloid fibril core is buried within these TDP-43 filaments and masked by the flanking ordered domains of TDP-43. Using a limited proteolysis protocol, we were able to unmask the amyloid core, and show that it corresponds to a sequence that is virtually identical to that of the amyloid core of human brain-derived pathological TDP-43 filaments (differs by only three amino acid residues)6-SUMO protein tag  showed that some fibril populations possess apparent periodicity with varying helical twists appearing to have left-handed helical twists  studies showed that the FL TDP-43 filaments are unbranched and several µm-long with a mean diameter of 20.7 nm (±3.3 nm) that bear morphological similarity to the TDP-43 filaments isolated from patient sources Fig. . FL TDP-sts Fig. ,m. The ssts Fig. .Our observations led us to propose a model whereby TDP-43 aggregation, driven by the CTD, results in the formation of amyloid filaments that are heavily decorated by the structured domain of the protein (NTD and RRMs), which mask the amyloid core of the filaments, thus rendering the amyloid core inaccessible to amyloid-specific dyes such as ThS/ThT and CR. To test our hypothesis, we analyzed these filaments with cryo-EM Fig. . The cryNext, we developed a method to unmask the amyloid core of the FL TDP-43 filaments by removing the globular domains that decorate the TDP-43 filaments by limited proteolysis followed by EM analysis. Treatment with trypsin resulted in a reduction of their diameter from 22 to 15 nm after 60 min Fig. . However23. Consistent with these findings, immunogold labeling followed by EM analysis showed that only antibodies that recognize the epitopes present on the NTD but not on the CTD exhibited extensive decoration of gold particles on the surface of TDP-43 filaments  has the propensity to form fibrils on its own. For this, we expressed and purified recombinant CP following the same strategy described for FL TDP-43 protein , we used CP monomers as a substrate to assess the seeding activity of the amyloid core using the ThT assay  by immunocytochemistry (ICC) using the p409–410 antibody  due to the inclusion of mutations within the bipartite nuclear localization signalody Fig. . The traody Fig. . Howeverody Fig. , arrows.ody Fig. . No signody Fig. . SupplemIn mouse primary neurons transduction with fluorescently labeled CP fibrils and PK-unmasked TDP-43 filaments, we detected the aggregation of endogenous TDP-43 protein  by immunoblot using TMEM106B-specific antibody. As shown in Extended Data Fig. 35, TMEM106B fragments were only present in samples from individuals >60 years old , whereas TDP-43 filaments were detected by immunogold labeling in all participant cases  is almost identical to the amyloid core sequence we identified by limited proteolysis and mass spectrometry (279–360). Despite their similar sequence, the recombinant peptide 279–360 exhibited a very distinct fold with major differences in secondary structures , in comparison with the core amyloid structure of brain-derived TDP-43 filaments or TDP-43 fibrils produced in vitro from the entire CTD domain or shorter CTD-derived peptide fragments  could be consistently and efficiently detected and/or immunolabeled with antibodies against extreme C-terminal phosphorylation sites  suggests that the CTD of many of these filaments remains intact40.Although increasing evidence supports that seeding-directed misfolding and nucleation of TDP-43 aggregation and cell-to-cell propagation as key processes driving TDP-43 pathology spreading in the brain40. Nonaka et al. also showed that PK treatment does not alter the cellular seeding activity of aggregates isolated from diseased brains (FTLD-TDP) or ALS, suggesting that brain-derived aggregates are highly enriched with CTFs with a possibly exposed PK-resistant amyloid core40. These findings have significant implications for the mechanisms of TDP-43 pathology formation and point to targeting the proteolysis of TDP-43 monomers and filaments as viable approaches for developing novel therapies to treat TDP-43 proteinopathies based on preventing TDP-43 aggregation, seeding activity, pathology formation and spreading. Therefore, identification of the enzymes that mediate the proteolytic cleavage of TDP-43 filaments and aggregates represents an important first step that could pave the way for novel therapies to slow the spreading of pathology and progression of ALS and other TDP-43 proteinopathies. Interestingly, previous studies from our laboratory showed that post-fibrillization cleavage of the highly negatively charged C terminus of αSyn could be essential for efficiently packing αSyn fibrils within Lewy bodies (LBs)42. This is consistent with the C-terminally truncated αSyn fibrils localizing primarily in the core of the LBs43. Furthermore, we recently showed that O-linked-N-acetylglucosaminylation modified αSyn fibrils or post-fibrillization nitrated αSyn fibrils exhibit diminished seeding activity in neurons and in vivo45. These findings indicate that the PTMs found in pathological aggregates associated with NDs not only are markers of pathology but also serve as key regulators of their processing and pathogenic properties.These observations, combined with previous findings and our in vitro and cell-seeding results Figs.  and 7, lOur data also suggest that the sequestration of TDP-43 within the filaments could result in a loss of protein function Fig. . However48.Altogether, our findings contribute to advancing our understanding of the role of TDP-43 aggregation in the pathogenesis of TDP proteinopathies and provide new insights to guide the development of new therapies and diagnostics. For example, our work suggests that upregulating PTMs within the amyloid core sequence and inhibiting the proteolysis of TDP-43 aggregates represent viable strategies to inhibit TDP-43 aggregation and seeding and pathology spreading, respectively. The methodologies and assays we describe should also open new avenues for future studies to: (1) elucidate the sequence, molecular and structural determinants of TDP-43 aggregation, pathology formation, and spreading; (2) develop more robust tools  and methods to detect and capture the diversity of TDP-43 pathology in the brain; and (3) develop diagnostic methods based on the detection of differences in the structural properties of native TDP-43 aggregates isolated from human brains. Finally, our work calls for investigating the role of post-fibrillization proteolytic cleavage of TDP-43 in mediating the seeding of other protein pathologies, for example, tau, amyloid-β and αSyn, which increasing studies show to co-occur with TDP-43 pathology6-Sumo-TDP-43) into a pD451-SR vector. ER2566 Escherichia coli (E6901S) competent cells were purchased from New England BioLabs. Isopropyl-ß-d-thiogalactopyranoside was ordered from Applichem . ThT was purchased from Sigma. ThS was purchased from Fluka. Phenyl methane sulfonyl fluoride (PMSF) was purchased from Axonlab (A0999.0005). Trifluoroacetic acid (TFA) was purchased from Sigma. The primary antibodies such as rabbit polyclonal full-length TDP-43 antibody (18280-1-AP), rabbit N-terminal-specific TDP-43 antibody (10782-2-AP) and rabbit C-terminal-specific TDP-43 antibody (12892-1-AP) were purchased from Proteintech and others included C-terminal anti-TDP-43 antibody  and p409–410 49. The secondary goat anti-rabbit labeled with Alexa Fluor 680 was purchased from Invitrogen. Goat anti-Rabbit IgG (H&L) was purchased from Aurion (810.011). The PageRuler prestained protein ladder (26617), SeeBlue Plus2 prestained protein standard (LC5925) and the SnakeSkin dialysis tubing (68700) with a molecular-weight cutoff of 7 kDa were purchased from Thermo Scientific.TDP-43 plasmid constructs used in this study were purchased from ATUM by the insertion of DNA coding for fusion protein  and incubated at 30 °C for 20 h with shaking at 180 r.p.m. Bacterial overexpression was initiated by mixing the pre-culture medium with 3 l LB medium to obtain the OD600 of 0.05 and continued to grow at 30 °C until the OD600 reached between 0.4 and 0.5. Cells were then induced with 0.4 mM IPTG, growth was continued overnight at 15 °C and pelleted by centrifugation at 4,160g for 10 min at 4 °C.The respective TDP-43 plasmids were transformed into 6-Sumo-TDP-43 was started immediately by resuspending cells in 80 ml lysis buffer , pH 8.0) with the addition of 0.3 mM PMSF and two protease inhibitor tablets (Roche). Cell lysis was carried out on the ice by sonication  using a Vibra cell VCX130 from Sonics. Centrifugation  was followed to separate the supernatant from the crude cell lysate. Next, 12 μl of Benzonase Nuclease HC (Novagen) was added to the supernatant and incubated at room temperature for 30 min with mild stirring. Following that, the supernatant was filtered through 0.45-μm syringe filter membranes and loaded onto a 20-ml Ni-NTA affinity column  pre-equilibrated with five column volumes of buffer A. Weakly bound nonspecific proteins were washed off from the column by passing of ten column volumes of buffer A at a flow rate of 2 ml min−1. Imidazole gradient with the buffer B , pH 8.0) from 5% to 100% was carried out to elute His6-Sumo-TDP-43. Elution fractions were analyzed to identify the fractions containing high-purity His6-Sumo-TDP-43 using SDS–PAGE and analytical C8 reversed-phase ultra-high performance liquid chromatography (UPLC).Purification of His6-Sumo-TDP-43 were pooled, added with ubiquitin-like-specific protease 1 , and incubated overnight at 4 °C to cleave the native full-length TDP-43 (FL TDP-43) from the His6-Sumo fusion. Cleavage was monitored using UPLC and SDS–PAGE analysis. Subsequently, a cleaved sample was filtered through 0.45 μm syringe filter membranes and the buffer was exchanged with reverse-IMAC buffer A , pH 7.0) using HiPrepTM 26/10 desalting column.Fractions identified for high-purity His6-tag proteins by passing the Ulp-1 treated sample into a 5 mL Ni-NTA affinity column  pre-equilibrated with 5 column volumes of reverse-IMAC buffer A. One step imidazole gradient was performed with buffer B , pH 7.0). Native TDP-43, lacking the His6 tag, elutes in the wash fractions. His6 tag proteins bound to the column eluted at 100% buffer B concentration. Collected fractions were analyzed using UPLC and SDS–PAGE. Protein concentration was determined by BCA assay . The molecular weight of His6-Sumo-TDP-43 and native FL TDP-43 was measured using LC–MS-LTQ ion trap mass spectrometer (Thermo Scientific) having the C3 poroshell 300SB 1.0 × 75-mm 5-μm column from Agilent . All obtained LC–MS spectra were deconvoluted with MagTran software version 1.03b from Amgen. Theoretical molecular weights of His6-SUMO-TDP-43 and full-length TDP-43 are 57775.2 Da and 44608.4 Da, respectively.Using reverse-IMAC, native FL TDP-43 was purified from the rest of the His6-Sumo-TDP-43 (279–360) CP were pooled, added with Ulp-1  and incubated overnight at 4 °C to cleave the TDP-43 (279–360) CP from the His6-Sumo fusion. Cleavage was monitored using UPLC and mass spectrometry analysis. Subsequently, a cleaved sample was filtered through 0.45-μm syringe filter membranes and injected into a reverse-phase HPLC C18  column, and the peptide was eluted using buffer A, 0.1% TFA in water, and buffer B, 0.1% TFA in acetonitrile. The purity of the elution from HPLC was analyzed by UPLC and electrospray ionization (ESI)–MS. Fractions containing highly pure TDP-43 (279–360) CP were pooled, snap frozen and lyophilized.Fractions identified for Hisg for 30 min at 4 °C  using a SnakeSkin dialysis tubing with a molecular-weight cutoff of 7,000 Da (Thermo Fisher). Following overnight dialysis, the fibrillization of TDP-43 was performed in FFB for 5 d at room temperature. This resulted in the aggregation of TDP-43 from the oligomeric prefibrillar form to mature TDP-43 filaments over the total incubation period. After 5 d, TDP-43 fibrils were isolated from coexisting oligomeric species by centrifugation of the incubation buffer at 25,000 °C Fig. . The pel−1 and digital integration time, 2 s. The spectrum of each sample is the average of ten repeats followed by a binomial approximation and plotted as the mean residue molar ellipticity.CD spectra of TDP-43 samples, loaded in a quartz cuvette with 1-mm path length, were collected using a Jasco J-815 CD spectrophotometer operated at 20 °C within the range of 200–250 nm. Data acquirements used the following parameters: data pitch, 0.2 nm; bandwidth, 1 nm; scanning speed, 50 nm min−1 with four times zero filling.Spectra were recorded on a Tensor 27 Fourier transform infrared spectroscopy spectrometer (Bruker) equipped with a BIO-ATR II cell and a photovoltaic LN-MCT detector cooled with liquid nitrogen. Around 30 µl of the sample was placed onto the zinc selenide (ZnSe) crystal of the BIO-ATR II cell. Spectra represent averages of 128 scans at room temperature, using an aperture of 4 mm and an instrument resolution of 4 cmSamples for SDS–PAGE were mixed with 2× Laemmli buffer and loaded onto 15% polyacrylamide gels. The gel was run at 180 V for 1 h in a running buffer (Tris-glycine-SDS running buffer), followed by staining with a solution containing 20% (vol/vol) isopropanol, 10% acetic acid (vol/vol) and 0.05% (wt/vol) Coomassie brilliant blue R and destaining with boiling distilled water.−1 and αSyn fibrils at 150 µg ml−1 were mixed with 20 µM ThT in 30 mM Tris and 100 mM NaCl at pH 7.4. All fluorescence spectra obtained were an accumulation of three curves, recorded using FluoroMax3 spectrometer (Horiba) at room temperature using an excitation wavelength of 450 nm with a slit width adjusted to 1 nm and emission wavelength from 460 to 650 nm with a slit width at 5 nm. For ThS fluorescence, samples were mixed with 20 mM ThS and measured using the same settings as described above.Samples for ThT fluorescence assay were prepared with pelleted TDP-43 filaments at the concentration of 250 µg mlFL TDP-43 filaments at 5 µM or αSyn fibrils at a concentration of 5 µM in 30 mM Tris and 100 mM NaCl at pH 7.4 was added with 20 µM CR and incubated for 5 min at room temperature. Following this, CR absorbance spectra were collected at 400–600 nm using a Cary 100 Bio UV–vis spectrophotometer  at room temperature. The stock solution of CR was prepared in 30 mM Tris and 100 mM NaCl at pH 7.4 and 1 mM concentration and filtered using a 0.45-µm filter.Following electrophoresis, the proteins in the gel were transferred onto nitrocellulose membranes  using a semidry transfer system (Bio-Rad) under a constant current of 0.6 A at 20 V for 1 h. The membranes were then blocked for 1 h at room temperature in a 1:3 ratio of Odyssey blocking buffer (Li-COR Biosciences):PBS. The membranes were then probed with the respective TDP-43 (full-length) antibody (18280-1-AP) overnight at 4 °C. After three washes with PBS buffer containing 0.1% (vol/vol) Tween 20 (Fluka), the membranes were incubated with secondary goat anti-rabbit antibody conjugated to Alexa 680 . The membranes were then washed three times with PBS buffer containing 0.01% (vol/vol) Tween 20, once with PBS buffer, and scanned on a Li-COR scanner at 700 nm.Before the application of the sample, Formvar and carbon-coated 200 mesh containing copper EM grids (Electron Microscopy Sciences) underwent a glow discharge for 30 s at 20 mA using a PELCO easiGlow Glow Discharge Cleaning System (TED PELLA). Subsequently, 5 μl of the sample was placed onto the EM grids and waited for a minute. Then, samples in the grids were carefully blotted out using filter paper and air-dried for 30 s. Following that, the grids were washed three times with ultrapure water and 0.7% (wt/vol) uranyl formate solution, respectively. Grids were examined using a Tecnai Spirit BioTWIN electron microscope. The microscope was equipped with a LaB6 gun, operated at an acceleration voltage of 80 kV, and images were captured using a 4k × 4k charge-coupled device camera (FEI Eagle). The crossover distance and the width of the helical filaments were measured manually on the EM images using ImageJ software. For each filament, the average crossover distance and width were calculated and plotted in the graphs using OriginPro software. A total of 21 helical FL TDP-43 filaments were evaluated from the acquired EM images.For immunogold labeling of filaments, 5 μl of TDP-43 filaments were pipetted onto glow-discharged Formvar/carbon-coated grids, blotted off of the excess samples after 2 min, and washed two times with autoclaved distilled water. The grids were then blocked with 0.1% bovine serum albumin  in PBS (Dulbecco) for 5 min. Following that, grids were incubated in a water-saturated chamber for 2 h with primary antibodies of anti-TDP-43 specific for N terminus and C terminus (Proteintech) diluted at 1:100 in 1% BSA in PBS. Later, the samples were washed four times with PBS drops. The grids were then incubated with 10-nm gold-conjugated anti-rabbit antibody (Aurion) diluted at 1:25 in PBS, for an hour in a water-saturated chamber. After rinsing three times (each rinsing is for 5 s) and washing for four times (each washing is for 2 min incubation) with PBS drops, the samples in the grids were cross-linked using 1% glutaraldehyde in PBS for 5 min. Finally, the grids were washed with ten drops of autoclaved distilled water with incubation of 1 min at each drop. Grids were then negatively stained with three drops of 0.7% (wt/vol) uranyl formate solution. Immunogold-labeled grids were examined at transmission EM as described above.−1 was added with 5 μl of PK  solution or 5 μl of trypsin  solution prepared in Tris calcium buffer  incubated at 37 °C. At 0, 5, 15, 30 and 60 min, 150 μl from the reaction mixture (PK and trypsin) was taken out, and we added 2.5 μl of 300 mM PMSF prepared in dimethylsulfoxide  to inhibit the proteolysis. Samples at these different time points were further used for transmission EM and dot blot analysis.A reaction mixture consisting of 750 μl of TDP-43 filaments at the concentration of 250 μg mln = 3) on the nitrocellulose membranes  packed using a 96-well dot blot apparatus  connected under vacuum conditions. After the adsorption of samples, the membrane was washed twice with PBS-T  while still packed in the dot blot apparatus. Following that, the membrane was blocked in a 1:3 ratio of Odyssey blocking buffer (Li-COR Biosciences):PBS-T. After an hour of incubation at room temperature, the membrane was treated with respective TDP-43 N terminus and C terminus (Proteintech) overnight at 4 °C. After three washes with PBS-T, the membrane was incubated with secondary goat anti-mouse or anti-rabbit antibodies conjugated to Alexa Fluor 680 . The membrane was then washed three times with PBS-T, once with PBS buffer and scanned on a Li-COR scanner at 700 nm. The intensities of antibody staining were quantified using the densitometry analysis tool from ImageJ (Fiji) software. The intensity of undigested TDP-43 fibrils at time 0 was used as standard and set to 100%.Around 20 μl of protease-treated and untreated TDP-43 fibrils at different time points (see above) was loaded in replicates  solution prepared in Tris calcium buffer  incubated at 37 °C for 30 min. Following inhibition with 5 mM PMSF, samples were ultracentrifuged (Beckman Coulter) for 45 min at 200,000g. After the centrifugation, the supernatant was removed and the remaining pellet was washed twice with 30 mM Tris, and 100 mM NaCl pH 7.4 to remove the soluble species after cleavage. To the pellet, 100 μl of a 1:1 TFA/HFIP solution was added and incubated at room temperature for 30 min under mild shaking conditions for the disaggregation of the core. The TFA/HFIP solution was removed using a gentle nitrogen stream, and the sample tubes were flash-frozen in liquid nitrogen and lyophilized. The lyophilized material was dissolved in 30–40 μl of 2% acetonitrile/0.1% formic acid for subsequent LC–MS/MS analysis.In total, 300 μl of TDP-43 filaments at 250 μg ml2, pH 7.4), adjusted to a concentration of 250 μg ml−1 and added 2 μl of PK . Following the incubation for 30 min at 37 °C, 5 mM PMSF was added and subsequently used for LC–MS/MS analysis.For the MS/MS analysis of non-fibrillar TDP-43 following the PK treatment, non-fibrillar TDP-43 (purified fresh from reverse-IMAC purification) was buffer exchanged to Tris calcium buffer  and 0.1% formic acid in 98% acetonitrile (mobile phase B). The mass spectrometer operated in a data-dependent MS/MS mode with a full scan range of 400–1,600 m/z and a resolving power of 60,000 m/z. The five most intense ions detected from the MS1 survey scan were selected for fragmentation by collisional induced dissociation (CID) at 35% normalized collision energy over 30 ms reaction and with dynamic exclusion enabled for 40 s, to minimize repeated selection of the same parent ion. An isolation width of 2 m/z was used for parent ion selection.In total, 15 μl of samples were analyzed using ESI in positive ion mode (3.5 kV ionization voltage) on an LTQ Orbitrap XL (Thermo Fisher), equipped with an Accela Pump HPLC and a CTC ThermoPAL autosampler (Thermo). Peptides were separated on a 4.6 mm inner diameter × 75 mm length column, packed with 3.5 μm, 100 Å Symmetry C18 (Waters). LC–MS/MS was performed using a 25-min gradient at a flow rate of 150 μl min50. Peptides were identified with the X! Tandem search engine as an internal tool of OpenMS platform51, using TDP-43 as a sequence database. An accuracy of 10 ppm and 0.5 Da was used for parent and fragment ions assignment, respectively. The search was carried out with no specificity and with the maximum missed cleavages admitted. Oxidation on methionine residues was selected as a variable modification. The most representative peptide spectrum matches of the identification list were selected for ion chromatogram extraction and mass deconvolution.After data acquisition, raw data were converted into peak list (mzML) using ProteoWizard toolsAround 10 μl of samples containing FL TDP-43 filaments or PK-unmasked FL TDP-43 filaments in 30 mM Tris buffer pH 7.4 with 100 mM NaCl were mixed with 10 μM ThT and were pipetted into a laboratory-made flow cell on a glass slide, sealed using nail polish and incubated overnight upside down at room temperature in a dark chamber. Samples were visualized on a Nikon Eclipse Ti microscope by mounting on a Plan Apo ×100/1.45 oil TIRFM objective (Nikon) in TIRF mode. ThT in the samples was excited with 405 nm laser and emission was collected using an excitation 455/10-nm and emission 485/30-nm bandpass filter cube (TIRF). Fluorescence images were captured using an ANDOR camera.Following the protein concentration estimation using BCA assay, 200 μl of samples containing FL TDP-43 filaments or PK-unmasked TDP-43 filaments (at two different amounts of 20 μg and 40 μg for each) in 30 mM Tris buffer at pH 7.4 with 100 mM NaCl was taken in different microcentrifuge tubes. To each of which, 10 μM ThT was added. For every sample, a triplicate of 60 μl was pipetted into a 384-well plate . ThT fluorescence was measured using a 440-nm excitation filter, and a 480-nm emission filter set at 37 °C. The final ThT fluorescence values shown are the average of three independent recorded triplicates and error bars show the s.d. of the triplicate measurements.All samples used for AFM measurements were incubated at room temperature on freshly cleaved and positively functionalized mica discs. Functionalization was done by a 10-μl droplet of 0.5% (vol/vol) APTES (3-aminopropyl) triethoxysilane deposition on a freshly cleaved mica surface. After 1 min of incubation, APTES was flushed three times with 1 ml of milli-Q water and the surface was dried by a gentle stream of nitrogen gas. Ten microliters of each sample diluted in PBS buffer was incubated at room temperature for 10 min on freshly functionalized mica. After 10 min, the sample was rinsed with 1 ml of milli-Q water and the mica surface was dried by a gentle nitrogen stream.52. All images were obtained using the NX10 Atomic Force Microscope (Park Systems) in non-contact amplitude modulation mode in ambient conditions. Commercially available silicon ultra-sharp tips  with a nominal tip radius of 2 nm and nominal resonance frequency of 330 kHz were used. Close to the surface, tip–sample interaction forces cause a reduction in the amplitude of the oscillation and a phase shift lag or advance. The phase shift signal contains information regarding the specific nature of the tip–sample interaction dissipative forces. Mechanical interaction between the tip and the sample can introduce imaging artifacts or damage the TDP-43 filaments, altering in both cases the recorded features. To control these interactions, phase images were recorded simultaneously with height images. Height images were used for further analysis of a sample and phase images were used as a control of tip–sample interactions. To consistently compare the topographical features, only images obtained with a comparable phase shift  were used in further analysis. All images were recorded at 1,024 × 1,024 pixels for 4 × 4 mm2 of sample area and were subsequently flattened with XEI software (Park System) with the same guidelines to keep consistency between different samples. Images were flattened with 1st regression order first by plane and then line by line until images reached a flat baseline on their profile. During this process, all structures were masked from software calculations to avoid any modification of their height.AFM measurements and image processing were performed as described52. ‘DNA trace’ allows a connection of the highest points along the fibril axis within a given cross-sectional area on the AFM image. A constant step for tracing equal to 4 nm was kept for all analyzed images, which corresponds to 2 pixels on 4 × 4 mm2 images. The average height of a single filament was obtained by calculating the mean height of all traced and connected points along the filament axis. To avoid the artificial height due to an increase in the calculations of the mean, areas of crossing between filaments were excluded. For filaments with a length exceeding the area of an image, only the longest part was traced. In this case, only one representative part of filament per structure was taken to avoid a false count of measured structures. Histograms with the distribution of filament height were created using the OriginPro software (OriginLab).For the height analysis of the filaments, home-build software ‘DNA trace’ was usedLyophilized TDP-43 (279–360) CPs in microcentrifuge tubes were weighed in a balance (Sartorius) and added a few microliters of dry DMSO  was added to the film of the CP, followed by the addition of FFB to give a final concentration of CP at 100 μM. A stock solution of ThT at 500 μM was also prepared. For the ThT-based aggregation assay, 400 μl of respective samples were prepared to have the final composition of 10 μM of CP, 10 μM of ThT, and respective percentages (% wt/vol) of different seeds (see below for the preparation of each seed). For every sample, a triplicate of 120 μl each were pipetted into a polybase black 96-well plate with an optical bottom (Corning). Aggregation kinetics were carried out in a FLUOstar Omega plate reader (BMG Labtech) by measuring the ThT fluorescence as a function of time (min) using a 440-nm excitation filter, and a 480-nm emission filter set at 25 °C. ThT fluorescence was monitored online by obtaining the data points once every 300 s, and before every measurement, the plate was shaken at 100 r.p.m. for 5 s. The aggregation curves of every sample shown are the average of three independent recorded triplicates and error bars show the s.d. of the triplicate measurements.Lyophilized TDP-43 (279–360) CP was added with dry DMSO (2.5% vol/vol) and FFB to have the final concentration of 50 μM in 400 μl. Following 3 d of incubation at room temperature under no shaking conditions, the presence of fibrils was confirmed by EM analysis. Seeds were prepared by sonication of the fully grown core fibrils at 400 ml using a probe sonicator  for 1 s on/1 s off cycle for 5 s at 40% amplitude followed by one more time of sonication for 1 s on/1 s off cycle for 5 s at 20% amplitude.Following the preparation of filaments, seeds were prepared by sonication using a probe sonicator  for 1 s on/1 s off cycle for 5 s at 40% amplitude followed by one more sonication for 1 s on/1 s off cycle for 5 s at 20% amplitude.−1 was added with 2 μl of PK  solution prepared in Tris calcium buffer  incubated at 37 °C for 30 min. Following inhibition with 5 mM PMSF, samples were ultracentrifuged (Beckman Coulter) for 45 min at 100,000g. After centrifugation, the supernatant was removed and the obtained pellet was washed twice with 30 mM Tris, and 100 mM NaCl at pH 7.4 to remove the soluble species after cleavage. To the pellet, 150 μl of 30 mM Tris and 100 mM NaCl at pH 7.4 was added and resuspended very well by vertexing. Seeds were prepared by sonication using a probe sonicator  for 1 s on/1 s off cycle for 5 s at 40% amplitude followed by one more sonication for 1 s on/1 s off cycle for 5 s at 20% amplitude.In total, 300 μl of TDP-43 filaments at 250 μg ml33. Human postmortem brains were obtained from the University of Pennsylvania, CNDR Brain Bank53. All necessary written informed consent forms were obtained from the participants or their next of kin in accordance with University of Pennsylvania Institutional Review Board guidelines and confirmed at the time of death. For each brain-derived extract, 10 µl of each sample  was used to prepare the PK-treated and PK-untreated seeds . Then, 20 μl of brain extract was added with 80 μl of FFB and split into two 50-μl aliquots. To one 50-μl aliquot, 0.5 μl of PK  was added with solution prepared in Tris calcium buffer . To the other aliquot, 0.5 μl of FFB was added and incubated at 37 °C for 15 min (–PK). Both samples were incubated at 37 °C for 15 min, followed by the addition of 1 μl of 300 mM PMSF. Finally, samples were water bath sonicated for 30 s and added to 350 μl of TDP-43 CP aggregation assay reaction mixture having the final 10 μM CP concentration and monitored for ThT-based aggregation kinetics as mentioned before.Sarkosyl-insoluble TDP-43 protein from the frozen frontal cortex of FTLD-TDP participants and control cases was prepared as described previously33 diluted at a 1:40 ratio in 1% BSA in PBS. Later, the samples were washed four times with PBS drops. The grids were then incubated with 10-nm gold-conjugated anti-rabbit antibody (Aurion) diluted 1:20 in PBS, for 1 h in a water-saturated chamber. After rinsing three times (each rinsing is for 5 s) and washing for four times (each washing was for a 2-min incubation) with PBS drops, the samples in the grids were cross-linked using 1% glutaraldehyde in PBS for 5 min. Finally, the grids were washed with ten drops of autoclaved distilled water with incubation of 1 min at each drop. Grids were then negatively stained with three drops of 0.7% (wt/vol) uranyl formate solution. Immunogold-labeled grids were examined at transmission EM as described above.Sarkosyl-insoluble brain extracts were diluted  with PBS (Dulbecco). For the immunogold labeling, 4 μl of diluted brain extracts was pipetted onto a glow-discharged Formvar/carbon-coated grids, blotted off of the excess samples after 2 min and washed two times with autoclaved distilled water. The grids were then blocked with 0.1% BSA (Aurion) in PBS (Dulbecco) for 5 min. Following that, grids were incubated in a water-saturated chamber for 2 h with rabbit polyclonal primary TDP-43 antibody 35. Blots were scanned using an Odyssey infrared system (ODY-2816 Imager) and the images were analyzed with the Image Studio software (LI-COR Biotechnology).In total, 5 µl of sarkosyl-insoluble fraction was resolved on 12% Bis-Tris gels  using MOPS-SDS as a running buffer. Proteins were transferred onto 0.45-μm nitrocellulose membranes and blocked with Rockland blocking buffer (MB-070). Membranes were immunoblotted overnight at 4 °C with the rabbit polyclonal antibody TMEM239 at a 1:2,000 dilution in blocking buffer−/Å2) for each exposure, and defocus range of −0.9 to 2.0 microns. In total, 3,171 movies were stored in gain-normalized tiff format.Fibrils from the TDP-43 CP (279–360) were screened with a negative-staining (NS) transmission EM for fibril concentration and morphology. Aliquots of optimized fibril samples were applied onto gold Ultrafoil 1.2/1.3 grids and plunge frozen in liquid ethane with a Vitrobot Mark IV (Thermo Fisher Scientific: TFS). Frozen cryo-EM grids were imaged on a TFS 300 kV Titan Krios at the NeCEN facility. Images were collected at the NeCEN facility on a K3 electron counting direct detection camera (Gatan) in counting mode  using the EPU 2.6.1 (Thermo Fisher Scientific) at a magnification of ×80,000  and a total dose of 40 electrons per square angstrom . One hundred micrographs were manually inspected to select a representative set of nonoverlapping straight filaments using the e2helixboxer.py from EMAN2 (ref. 56). Dose-weighted averages were denoised with JANNI and subjected to the semi-automated filaments tracing with crYOLO (v1.7.5)57. Filament start-end coordinates in STAR format were imported into RELIONv3.1, and helical segments were extracted using a box size of 256 pixels (271 Å) and 10% inter-box distance. A total of ~106 segments were extracted and subjected to multiple rounds of the reference-free 2D classification to select only segments with clear 4.86-Å β-strand separation along the fibril axis, measured from the 2D class-average layer line profile. The helical twist was determined by measuring a half-pitch distance from 2D class-average images of 1,024 pixels extracted and scaled to 256 pixels segments. Initial 3D reference structure was generated with relion_helix_inimodel2d from set of representative 2D class averages and estimated helical parameters. The best segments were subjected to subsequent 3D classifications with the generated 3D initial reference. Several rounds of 3D classification with T = 25 and ‘ignore CTFns unit first peak’ option resulted in 8,104 and 5,795 segments contributed to the first (one protofilament) and second (two protofilaments) conformations of TDP-43 CP fibrils.A total of 3,171 movies were imported into RELION (v3.1)59. Finally, the resulting 3D reconstructions were post-processed with RELION, and the global resolution was measured, 4.1 Å for the first conformation and 3.7 Å for the second conformation. Post-processed maps were symmetrized with refined helical parameters for both conformations. A second conformation with 3.7-Å resolution and resolved large side chains were used for model building. Both were assumed to have a left-handed twist, according to the handedness of previously reconstructed TDP-43 filaments.The best segments of both conformations were subjected to additional rounds of 3D classification with a single class and local optimization of helical twist and rise, resulting in well-separated β-strands and resolved large side chains. The resulting 3D classes were subjected to 3D refinement iterated with particle polishing and CTFn refinement60. Large side chains of 334 TRP, 316 PHE, 313 PHE, and a few others were used as a guide. Both N−C and C–N orientations were built, and the model with the best fit of side chains was used for subsequent refinement. An initial model was used to build five helical symmetry-related copies with MolRep from CCPEM61, which were subjected to real-space refinement with NCS restraints in PHENIX62. After several rounds of refinement, side chains were manually inspected and adjusted for the energy-favored geometry in COOT. The final model was validated using MolProbity63 and Protein Data Bank (PDB) validation service from wwPDB64. Accurate details of the data acquisition and final statistics of the refined model are given in the Supplementary Table The CP atomic model was subsequently built de novo into post-processed and symmetrized maps with COOTd-lysine-coated coverslips  and transduced after 24 h with 0.25 µg of recombinant TDP-43 monomers, CP monomers, FL TDP-43 filaments, CP fibrils or PK-unmasked TDP-43 filaments. Briefly, samples were diluted with dPBS and mixed with single-use tubes of BioPORTER as a protein delivery reagent  as previously described34. Protein–Bioporter complexes were added to the cells and incubated for 4 h. Cells were placed back on fresh medium in presence of doxycycline (1.0 μg ml−1) and cultured for 3 additional days (3 d.p.t.).iGFP-NLSm cells (clone 6.B7) were plated on poly-34. Briefly, after blocking, cells were incubated with the monoclonal antibody phospho-specific p409–410 antibody overnight at 4 °C. After three washes with dPBS, cells were incubated with appropriate secondary antibodies in a blocking buffer for 1 h at room temperature in the dark. Coverslips were mounted with Fluoromount G containing DAPI (Thermo Scientific).iGFP-NLSm were fixed with 4% paraformaldehyde (PFA) in PBS and permeabilized with 0.1% Triton X-100 for 15 min. When indicated, to remove cytoplasmic soluble proteins and quantify the formation of TDP-43 pathology, cells were fixed in 4% PFA containing 1% Triton X-100 for 15 min at room temperature as previously described65. The number of DAPI-positive nuclei (no. DAPI/µm) was quantified using HALO.To quantify the extent of phospho-positive aggregates, whole coverslips were scanned and images were digitalized using a Lamina Multilabel Slide Scanner (Perkin Elmer). The images were processed using the Indica Labs HALO software, and the percentage area occupied by p409–410 immunopositive staining was quantified automatically using HALOd-lysine-coated  Quad Ibidi dishes with glass coverslips . Next, 2.5 µg of Atto 488-labeled FL TDP-43 filaments, PK-unmasked TDP-43 filaments and CP fibrils were diluted with dPBS, sonicated and mixed with single-use tubes of BioPORTERTM as a protein delivery reagent . Protein–Bioporter complexes were added to the cells and after 4 h of incubation the transduction mix was removed and OptiMEM containing 100 μM of trypan blue was added to the cells to quench the extracellular labeled material. The culture dish was secured inside a moist chamber maintained at 37 °C in a humidified atmosphere containing 5% CO2 and cells were imaged at ×40 in a high-resolution Leica DMI6000B microscope using the Leica LASX software.Live cell-imaging uptake analysis was performed in QBI-293 cells plated in poly-g for 1 h at 4 °C. The supernatants were collected and stored for later SDS–PAGE analysis. The sedimented pellets were resuspended in PBS. This wash step was repeated (three times) until the dye in excess was removed. The samples collected during the repeated centrifugations steps were loaded onto an SDS–PAGE gel, and labeling on the pelleted samples was confirmed by scanning the gel using Typhoon FLA 7000  with excitation and emission wavelengths of 400 and 505 nm, respectively. Alexa Fluor 488-labeled filaments/fibrils were then fragmented via sonication using a probe sonicator  for 1 s on/1 s off cycle for 5 s at 40% amplitude followed by one more time of sonication for 1 s on/1 s off cycle for 5 s at 20% amplitude. Alexa Fluor 488-labeled filaments/fibrils were then assessed by EM analysis and SDS–PAGE/fluorescence scanning.Different forms of TDP-43 aggregates including the FL filaments, PK-treated TDP-43 filaments and CP fibrils were taken at a concentration of 10–20 μM in a final volume of 300 μl of PBS and the pH was adjusted to 8.5. One equivalent of Alexa Fluor 488 maleimide was added and incubated at 4 °C overnight. The labeled fibrils/filaments were then ultracentrifuged at 100,00066. In brief, the cortex was isolated and cleaned in HBSS and was dissociated enzymatically (papain) at 37 °C for 30 min followed by gentle trituration in triturating media . Cortical neurons were then resuspended in adhesion medium  plated on poly-d-lysine-coated glass coverslips on 24-well plates. After 3 h, adhesion medium was replaced with neuronal growth medium  to quench fluorescence from fibrils present outside the neuron. Cells were then fixed using 4% PFA for 15 min at room temperature and then co-stained together with anti-TDP-43  and MAP2 antibody (1:500 dilution).Primary cortical neurons were prepared from postnatal day 0 pups of wild-type mice  and cultured as previously describedSCR_014980) or GraphPad Prism 9.1.1. The statistical tests were performed using ANOVA followed by Tukey’s multiple-comparisons test. Sample sizes (n) are indicated in the figure legends. The corresponding results of the statistics and the exact P values are provided in the figures legends of the respective experiments. All experiments using the recombinantly produced FL TDP-43, its variants and CP were carried out independently at least three times. No statistical methods were used to predetermine sample sizes but our sample sizes are similar to those reported in previous publications68. All data distribution was assumed to be normal, but this was not formally tested. No data were excluded from the analyses. Data collection and analysis were not randomized and were not performed blind to the conditions of the experiments.The statistical analyses were carried out using KaleidaGraph (RRID: Further information on research design is available in the Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41593-023-01341-4.Supplementary InformationSupplementary Figs. 1–11, Supplementary Tables 1–6, Supplementary references and source data of gels and immunoblots for Supplementary Figs. 1, 2 and 3Reporting SummarySupplementary Data 1Source data for Supplementary Figs. 5 and 6


Sarcoidosis is a systemic granulomatous disease of unknown etiology with the potential to involve many organs of the body. Less than 1% of patients with sarcoidosis have GI manifestations. Here, we report a case of GI tract sarcoidosis that presented with severe symptomatic anemia.A 51-year-old female with a history of pulmonary and liver sarcoidosis presented to the emergency room with a one-week history of chest pain and shortness of breath. A physical exam was significant for conjunctival pallor. On admission, her hemoglobin was 6.9 g/dL. Her iron studies showed anemia of chronic disease. There was no evidence of recurrent pulmonary sarcoidosis on the CT scan of the chest. Transthoracic echo showed no abnormal wall motion movements. A nuclear stress test was negative for perfusion defects. She underwent esophagogastroduodenoscopy (EGD) and colonoscopy to further evaluate potential sources of active GI tract blood loss. Biopsies of gastric mucosa and small bowel revealed non-caseating granulomas. Immunohistochemical stains for acid-fast bacilli and fungus were negative. Random biopsies of erythematous mucosa from the colonoscopy were unremarkable. The patient’s history of pulmonary and liver sarcoidosis along with non-caseating granulomas found in the gastric mucosa and small bowel suggest GI tract sarcoidosis manifestations. She was started on corticosteroids with complete resolution of symptoms in five months.Clinical presentation varies widely based on the specific organ involvement, as well as the underlying pathophysiology of the organ damage. The pathogenesis of sarcoidosis is poorly understood and attributable to both genetic and environmental factors. Overall, the treatment of sarcoidosis is not standardized. It is primarily driven by the effect of sarcoidosis on the patient’s symptoms and quality of life. However, symptomatic sarcoidosis usually responds well to corticosteroids. We believe that clinicians should maintain a high level of vigilance for patients with a known history of sarcoidosis and new symptoms, as these might signal sarcoid involvement of a new organ and help guide the diagnostic and treatment process. Sarcoidosis is a systemic granulomatous disease of unknown etiology with the potential to involve many organs of the body. GI tract involvement is estimated to occur in less than 1% of patients with sarcoidosis . Here, wA 51-year-old female with a past medical history of hypertension, type 2 diabetes, end-stage kidney disease on peritoneal dialysis, and iron deficiency presented to the emergency room with a one-week history of chest pain and shortness of breath. The chest pain was described as stabbing pain and numbness sensation under her left breast with associated shortness of breath. Two days prior, she started feeling dizzy with mild abdominal cramping. She denied dysuria, hematuria, hematemesis, hematochezia, or melena. Her medications included amlodipine 10 mg QD, metoprolol 25 mg BID, insulin glargine 10 units HS, and insulin lispro 5 units with meals.The patient had similar left-sided chest pain eight years ago. CT scan then revealed a 16 x 13 mm left upper lobe nodule. PET scan showed an increase in metabolic activity as well as a hypermetabolic liver lesion concerning for metastasis. She then underwent a left thoracotomy that demonstrated the lung nodule to be a mediastinal lymph node. The biopsy was diagnostic for non-caseating granulomas and suspicious for sarcoidosis. Fungal and acid-fast bacilli cultures performed were negative. Serology for antinuclear antibody, anti-neutrophilic cytoplasmic antibody, and Histoplasma titers were within normal limits. At that time, she was treated with prednisone which improved her symptoms with residual chest pain.Upon admission, her physical exam was remarkable for conjunctival pallor and positive digital rectal exam. Labs showed hemoglobin of 6.9 gm/dL. Iron studies showed iron 83 mcg/dL 37-145 mcg/mL), total iron binding capacity 188 mcg/dL (251-346 mcg/mL), and ferritin 554.0 ng/mL (13.0-150.0 ng/mL) suggesting anemia of chronic disease. Troponins trended 143-167-163 ng/mL and were elevated at baseline in the setting of her ESRD. Creatinine was 4.9 mg/dL which was her baseline. EKG was normal with no ST or T wave changes compared to prior. There was no evidence of recurrent pulmonary sarcoidosis on the CT scan of the chest. Transthoracic echo showed no abnormal wall motion movements and grade 1 diastolic dysfunction. The nuclear stress test was negative for perfusion defects. She underwent esophagogastroduodenoscopy (EGD) and colonoscopy to further evaluate potential sources of active GI tract blood loss. Biopsies of gastric mucosa and small bowel revealed non-caseating granulomas (Figure 45 mcg/mLInvolvement of the GI tract in sarcoidosis is extremely rare. The largest published literature on this topic analyzed 305 cases of GI involvement . GI invoClinical presentation varies widely based on the specific organ involvement, as well as the underlying pathophysiology of the organ damage . The patDysphagia is the main presenting symptom of the esophageal sarcoidosis. Its nature has been attributed to multiple mechanisms, such as mucosal or muscular layer infiltration and involvement of the myenteric plexus or extrinsic compression -6. With Histoplasma capsulatum enteritis [Regardless of the organ involvement, sarcoidosis of the GI tract may present as anemia  such as nteritis ,14. Inventeritis . GI sarcnteritis . Granulonteritis . In addiAsymptomatic and non-progressive disease requires no treatment, as a majority of patients undergo spontaneous remission. However, in general, symptomatic extrapulmonary sarcoidosis such as GI tract sarcoidosis is usually treated with 20-40 mg of daily prednisone as the initial dose. Then, the corticosteroid should be tapered to the lowest effective dose. Patients usually respond well to corticosteroids and are treated for at least one year . AdditioDespite 305 cases of GI sarcoidosis reported, this clinical entity still remains a rarity. Anemia related to GI sarcoidosis has previously been described; however, there was no case reported, where it would have such a severe presentation as in our patient. We believe that clinicians should maintain a high level of vigilance for patients with a known history of sarcoidosis and new symptoms, as these might signal sarcoid involvement of a new organ and help guide the diagnostic and treatment process.


The application of graphene-based materials in medicine has led to significant technological breakthroughs. The remarkable properties of these carbon materials and their potential for functionalization with various molecules and compounds make them highly attractive for numerous medical applications. To enhance their functionality and applicability, extensive research has been conducted on surface modification of graphene (GN) and its derivatives, including modifications with antimicrobials, metals, polymers, and natural compounds. This review aims to discuss recent and relevant studies related to advancements in the formulation of graphene composites, addressing their antimicrobial and/or antibiofilm properties and evaluating their biocompatibility, with a primary focus on their biomedical applications. It was concluded that GN surface modification, particularly with compounds intrinsically active against bacteria , has resulted in biomaterials with improved antimicrobial performance. Furthermore, the association of GN materials with non-natural polymers provides composites with increased biocompatibility when interfaced with human tissues, although with slightly lower antimicrobial efficacy. However, it is crucial to highlight that while modified GN materials hold huge potential, their widespread use in the medical field is still undergoing research and development. Comprehensive studies on safety, long-term effects, and stability are essential before their adoption in real-world medical scenarios. In recent years, graphene materials have attracted significant interest due to their remarkable properties and various applications. Graphene (GN) is a two-dimensional carbon allotrope composed of a single layer of carbon atoms arranged in a hexagonal lattice . This stGraphene derivatives can be generated by introducing oxygen-containing functional groups to the GN structure or reducing its oxide form, obtaining graphene oxide (GO) or reduced graphene oxide (rGO) , respectWithin the medical field, there have been notable technological advances in the application of GN and its derivatives in drug/gene delivery, biosensing, bioimaging , wound hTo date, different mechanisms have been proposed to explain the antimicrobial activity of GN and its derivatives . GN’s shThe antimicrobial potential of GN is related not only to its structural properties  but also to the surface modification, the nature of the targeted microorganism (Gram-positive or Gram-negative bacteria), and the environment where GN and microbial cells interact .Smaller GN particles with a higher surface-to-volume ratio can interact more effectively with microbial cells and affect their membrane integrity ,30. In aStudies have revealed that GN and its derivatives, such as GO and rGO, can inactivate both Gram-positive and Gram-negative bacteria in either planktonic or sessile states . HoweverWhen using GN materials for medical applications, it is crucial to comprehensively evaluate their biocompatibility to ensure that they are safe for use in real scenarios, such as medical devices or wound dressings. Numerous studies have been conducted and the results are mostly promising ,42,43,44Despite the plentiful range of GN-based materials developed in recent years, most are still far from practical biomedical applications. As a result, further investigation is necessary to understand the functionality, applicability, and safety of these carbon materials. Hence, this review aims to critically discuss recent progress in the formulation of graphene composites, assessing their antimicrobial and/or antibiofilm activities while also evaluating their biocompatibility, with a focus on biomedical applications. Research involving GN and its derivatives modified with antimicrobials, metals, polymers, or natural compounds is addressed . By summOne desirable feature of graphene is its capacity to bind to a variety of molecules, including antimicrobial agents, peptides, and biocides. This capability not only broadens the range of potential applications of GN materials but also has the potential to enhance their antimicrobial activity and biocompatibility . Table 12) surfaces for potential application in medical devices. The results demonstrated that Dox-modified GO/TiO2 surfaces reduced the viability of adhered bacteria by 90% compared to the 40% reduction observed with GO/TiO2 surfaces. This suggests that Dox exhibited a synergistic effect with the GO material, efficiently inhibiting bacterial adhesion. This antibiotic acts by inhibiting bacterial protein synthesis , i, i67], iRegarding biocompatibility, human cells exposed to GN-based composites maintained their viability and proliferation capability ,69. In gOwing to their biodegradability, renewability, and biocompatibility, there has been growing interest in composites that incorporate natural compounds. In recent years, several natural compounds, including vivianite , usnic aAmong the studies presented, natural compounds were mainly associated with graphene oxide.S. aureus and Staphylococcus epidermidis by up to 3 Log in a dose-dependent manner (E. coli and S. aureus while preserving the viability of osteoblast cells [2+) and calcium (Ca2+) ions released from the composite may have contributed to potentiating its antimicrobial performance. Moreover, hydroxyapatite is well known for its exceptional biocompatibility. Croitoru et al. [S. aureus culturability by 1 and 3 Log, respectively, and E. coli culturability by 5 Log. These GO-based materials showed biocompatible behavior at lower concentrations. Pandit et al.  conjugatt manner . Furtherst cells . The feru et al.  modifiedOverall, natural compounds, particularly quercetin and juglone, enhanced the antimicrobial activity and biocompatibility of GN-based materials intended for medical purposes. This review comprehensively summarizes and discusses recently developed graphene-based materials, with a focus on their antimicrobial properties and biocompatibility, while exploring potential medical applications such as tissue engineering, medical devices, and wound healing. Biocompatible and antibacterial nanomaterials are in high demand for a variety of medical applications. The surface modification of GN and its derivatives  with antimicrobials , metals or metal oxides, polymers, and natural compounds has enhanced the functionality and applicability of these materials, resulting in improved antimicrobial performance and increased biocompatibility towards human tissues. The combination of graphene materials with agents that possess intrinsic antimicrobial properties, such as antimicrobial peptides, metals (silver or copper), or chitosan, enhances the effectiveness of GN materials in inactivating bacteria, especially Gram-positive bacteria, because of their less complex membrane structure. Conversely, although promising for a wide range of applications, the use of non-natural polymers for GN surface modification results in composites with lower antimicrobial activity than those obtained through the modifications mentioned above. However, GN–polymer composites exhibit superior biocompatibility compared to antimicrobial or metal-based GN composites. In the latter case, adverse effects on human cells are highly dependent on the type of metal used and the methodology employed for their production.Despite the significant progress made in these biomaterials, few in vivo studies have validated their effectiveness and applicability, which may hinder their translation into real medical scenarios. Additionally, studies addressing the long-term effectiveness, biocompatibility, and stability of these composites are lacking. Therefore, further research is needed to introduce these promising biomaterials in the medical field.