The comparison between 1-methyl-2-pyrrolidinone and N,N-dimethylformamide as dispersant solvents, and between sonication and homogenization as dispersion techniques, proves N,N-dimethylformamide and homogenization as the most effective choice. The functionalization of graphene nanosheets and reduced graphene oxide is confirmed using different techniques. Among them, energy-dispersive X-ray spectroscopy allows to map the pyrrolidine ring of the azomethine ylide on the surface of functionalized graphene, while micro-Raman spectroscopy detects new features arising from the functionalization, which are described in agreement with the power spectrum obtained from ab initio molecular dynamics simulation. Moreover, X-ray photoemission spectroscopy of functionalized graphene allows the quantitative elemental analysis and the estimation of the surface coverage, showing a higher degree of functionalization for reduced graphene oxide. This more reactive behavior originates from the localization of partial charges on its surface due to the presence of oxygen defects, as shown by the simulation of the electrostatic features. Functionalization of graphene using 1,3-dipolar cycloaddition is shown to be a significant step towards the controlled synthesis of graphene-based complex structures and devices at the nanoscale.Organic functionalization of graphene is successfully performed  via 1,3-dipolar cycloaddition of azomethine ylide is shown to be a significant step towards a controlled synthesis of graphene-based advanced nanoscale devices with engineered functionalities.Organic functionalization of graphene nanosheets and rGO  It is composed of carbon atoms packed in a honeycomb lattice.6 Because of its unique one atom-thick structure, it combines outstanding properties such as superior mechanical strength,7 excellent flexibility,8,9 high transparency,10 exceptionally high thermal and electrical conductivities as well as amazing electronic properties.11–19Since Novoselov and Geim isolated graphene for the first time in 2004,20 its outstanding properties are also a limitation. Due to the absence of a band-gap, graphene's use as an active element in electronic devices and sensors encounters many limitations and, therefore, some sort of engineering of graphene's structure is required.21 It has been suggested that a powerful method to overcome these impediments is provided by an efficient surface functionalization with suitable materials. Functionalization of graphene offers the possibility to finely tune the system's physical and chemical properties, resulting in a synergistic combination of the individual features of each component. However, while graphene's high specific surface area of 2630 m2 g−1 ,49,50 reaction with residual functional groups on graphene,51 or modification of graphene's unsaturated structure.52–57 In contrast, the non-covalent chemical modification is often realized by intermolecular interactions such as van der Waals forces,58 electrostatic interactions,59 or π–π stacking interaction.60–62Generally, graphene functionalization can be achieved through either covalent bonds or non-covalent interactions.2 carbon atoms of the graphene lattice, forming a covalent bond.63–67 While this mechanism leads to an abundant and quick functionalization, it is hard to control. Therefore, a more selective and controlled method has been shown as very promising. 1,3-Dipolar cycloaddition  of azomethine ylide has been investigated for the chemical modification of carbon nanotubes, fullerenes, and other carbon nanostructures,68–73 thanks to the availability of a variety of organic derivatives by selecting the appropriate precursors, with interesting applications in different fields like biotechnology, sensors at the nanoscale, and solar energy conversion.74–76 Recently, the graft of azomethine ylide on graphene via 1,3-DC has been reported after the successful production of graphene sheets directly from graphite dispersed in organic solvents.77 That was a pioneering work, but lacking a systematic and wide investigation on solvent effects, computational investigation, XPS analysis, or specific Raman signature of the functionalization. In a similar way, the selective binding between the amino groups involved in the 1,3-DC on dispersed graphene and gold nanorods has been obtained.78 However, a detailed description of graphene functionalized with azomethine ylide is still missing, as well as a deeper investigation on functionalization efficiency and signature.In particular, the covalent functionalization of graphene sheets using organic functional groups has been explored as a pivotal step towards the formation of graphene composites at the nanoscale. A commonly diffuse approach is the use of diazonium salts, often done through electrochemical or heating processes. Upon surface reduction of the diazonium compound, a highly reactive free radical is produced, which attacks the spN,N-dimethylformamide (DMF) are used as dispersant solvents, for their excellent efficiency in dispersing graphene as well as facilitating the in situ production and the grafting of the ylide onto graphene. Energy-dispersive X-ray spectroscopy (EDX) and electron energy loss spectroscopy (EELS) confirm the organic functionalization of graphene and verify its homogeneity on the entire surface. While these techniques have already been applied to analogous systems, a detailed characterization of functionalized graphene with Raman spectroscopy is presented here for the first time. Raman spectroscopy, performed on both the pristine and functionalized samples, allowed the detection of the characteristic Raman signature of graphene together with new distinctive peaks from the ylide. Moreover, X-ray photoemission spectroscopy (XPS) is used to estimate the coverage and the efficiency of the functionalization process, assessing the elemental composition. An ab initio density-functional theory (DFT) molecular dynamics simulation illustrates how the presence of epoxy groups in the rGO surface induces a local inhomogeneity of the partial charges of graphene's structure, promoting the 1,3-DC of azomethine ylide and showing how defects  could be exploited to acquire control on the degree of the functionalization. Finally, the simulated power spectrum provides a precise idea of the Raman signature of functionalized graphene, in agreement with the experimental data.While the techniques of production and chemical modification of graphene-like systems advance, the choice of the organic solvent suitable for the dispersion, together with the detailed understanding of the system, becomes fundamental in order to obtain an efficient functionalization. Here we present a detailed investigation on 1,3-DC of azomethine ylide on both graphene nanosheets (GNS) and reduced graphene oxide (rGO), including solvent effects. We compare sonication and homogenization as dispersion techniques, while 1-methyl-2-pyrrolidinone (NMP) and 22.1N,N-dimethylformamide , ethanol , N-methylglycine (98%), and 3,4-dihydroxybenzaldehyde (97%) were purchased from Sigma-Aldrich/Merck, Germany, while sodium carbonate  was purchased from Carlo Erba, Italy.Wet-jet milling exfoliated graphene powder was provided by BeDimensional Srl, Italy. Reduced graphene oxide (rGO) in flake form was purchased from Graphenea, Spain. 1-Methyl-2-pyrrolidinone , 2.2Graphene powder (35 mg) was dispersed in NMP or DMF (140 mL). For the dynamic light scattering (DLS) measurements, homogenization (OV5 Homogenizer from VELP Scientifica) was performed in time steps of 10 minutes, up to 1 hour, at 30 000 rpm. Sonication (SONOPLUS Ultrasonic HD 2070 from Bandelin) was performed in different time steps, from 15 minutes to 2 hours, at 60 W. For the functionalization procedure, homogenization was performed for 30 minutes at 30 000 rpm. Reduced graphene oxide powder (10 mg) was dispersed in DMF (40 mL). Homogenization was then performed for 30 minutes at 30 000 rpm.2.3N-methylglycine  were added to the graphene dispersion  in NMP or DMF, similarly for rGO dispersed in DMF . Sodium carbonate  was added in order to lower the acidity of the environment. This both helps the formation of the 1,3-dipolar compound and prevents the loss of the carboxyl group of the azomethine ylide. The reaction mixture was heated at 150 °C for 120 h, under magnetic stirring, with successive additions of the reagents every 24 h. In order to limit secondary reactions from the oxidation of the solvent at high temperature, an inert atmosphere (N2) was kept during the functionalization reaction. The resulting mixture was then thoroughly washed with clean solvent .To perform the organic functionalization, 3,4-dihydroxybenzaldehyde  and an excess of 2.479 respectively. Raman spectroscopy was carried out with a Renishaw InVia system, equipped with a confocal microscope, a 532 nm excitation laser and a 1800 line per mm grating . All spectra were measured with the following parameters: excitation laser power 500 μW, acquisition time for each spectrum 20 s, two acquisitions per spectrum, with a 100× objective . At least 3 different samples were measured, both for pristine and functionalized graphene, and they all showed the same features of the ones here reported as representative results. The samples were prepared by dropcasting (2 μL) onto clean silica substrate and dried under vacuum for several days. STEM-EDX/EELS measurements were carried out on a Zeiss Libra 120 microscope operating at an accelerating voltage of 120 kV, equipped with a thermionic LaB6 source and an in-column Omega filter for energy-filtered imaging and EELS analysis. EELS spectra were collected by integrating the EELS signal on the TEM CCD (a TRS 2k x 2k CCD camera binned 2 by 2) and by selecting the measured area using the entrance slit aperture of the Omega filter. The samples were prepared by washing the dispersant solvent with ethanol several times and collected with carbon film-supported copper grids. XPS core level emission spectra were acquired using a Surface Science Instrument SSX-100-301 spectrometer operating an Al Kα source (hν = 1486.5 eV), achieving an overall energy resolution of 0.9 eV. The collected spectra were fitted using the XPSPEAK software, subtracting a Shirley-type background80 and fitting the peaks using mixed Gaussian and Lorentzian components. The samples were prepared by repeated dropcasting onto clean highly doped silicon substrates (with native oxide), and dried under vacuum between several drops and at the end of the deposition process.DLS measurements were performed with a Malvern Zetasizer Nano ZS (equipped with a 633 nm HeNe laser), using quartz cuvettes (1 mL). Samples were equilibrated to 25 °C for 30 seconds before measuring and the data were acquired with a scattering angle of 12.8°. Values for NMP viscosity and refractive index at 25 °C are 1.6660 cP and 1.468, while for DMF are 0.8020 cP and 1.428 (data from the supplier). Graphene refractive index and absorption at 25 °C are 2.704 and 0.023,2.581,82 program at the DFT level of theory using the Perdew–Burke–Ernzerhof (PBE) exchange and correlation functional.83 Second-generation dispersion corrections (D2)84 were used to take into account a proper description of van der Waals interactions. Goedecker–Teter–Hutter (GTH) pseudopotentials,85 together with double-zeta quality basis sets (DZVP), were used. The Gaussian-and-Plane-Waves (GPW) method as implemented in CP2K86 was used; the energy cutoff for the auxiliary plane-wave basis was set to 340 Ry cutoff. The wavefunction convergence criterion was set to 10−6 hartree. Geometry optimization of the systems was performed by using Broyden–Fletcher–Goldfarb–Shanno (BFGS) algorithm by setting a root mean square (RMS) value of 10−4 hartree per bohr for the force and 10−4 bohr for the geometry as convergence criteria. The systems were treated as periodic. Reduced graphene oxide was modeled starting from the hexagonal graphene supercell (a = 1.98 nm) consisting of 164 carbon atoms. The Restrained Electrostatic Potential (RESP) charges87 for periodic systems were evaluated by using REPEAT methods88 as implemented in CP2K.89 After structural minimization, the functionalized system was subject to free molecular dynamics (MD). MD simulations were performed in the NVT ensemble (T = 300 K) employing a canonical-sampling-through-velocity-escalating (CSVR) thermostat90 with a time constant of 200 fs. A single integration time step of 0.4 fs was used. MD simulations were carried out for 12 ps. Vibrational spectra were obtained by calculating the Fourier transform of the atoms velocity auto-correlation function (VACF) taken from the last 10 ps of the simulated trajectory of the system.91All calculations were performed with the CP2K33.192 were dispersed in NMP and DMF in order to obtain a stable dispersion (∼0.2 mg mL−1), as shown in Fig. S1 in the ESI.93 since they minimize the interfacial tension between solvent and graphene. Moreover, both NMP and DMF are commonly used in 1,3-dipolar cycloadditions for their ability to favor the reaction.77,78,94,95 This is fundamental during the functionalization process, which is described in the following sections. In order to determine the best dispersant solvent and the most efficient dispersion technique, DLS measurements were performed on pristine GNS. Comparing the average lateral dimension of the dispersed nanosheets, we noticed a reduction in size from 850 nm to 600 nm (using sonication) or 400 nm (using homogenization) in NMP, in contrast with a decrease in size from 950 nm to 850 nm (using sonication) or 600 nm (using homogenization) in DMF , with respect to DMF (γ = 37.1 mJ m−2).96Exfoliated GNS produced by wet-jet milling (as described elsewhere)97 Recently, also homogenization, which consists in the shear mixing in suitable stabilizing liquids, has been demonstrated to achieve excellent exfoliation of graphite, even in a more scalable way.98 Here, we observed that in both NMP and DMF, homogenization allows a faster dispersion of GNS. For example, sonication in NMP for 60 minutes allows a decrease of the average lateral dimension from 850 nm to 600 nm, while the same result was obtained after only 30 minutes of homogenization. Therefore, homogenization results to be the advisable dispersion procedure, and the subsequent experiments were performed after dispersion via homogenization.Sonication has been widely utilized to induce exfoliation of bulk materials through growth and collapse of micro-bubbles due to pressure fluctuations in liquids.3.2N-methylglycine and 3,4-dihydroxybenzaldehyde to the GNS dispersion, as schematically represented in −1) and only via homogenization, while the subsequential functionalization procedure remained the same.Functionalized GNS were prepared by adding 3.3 PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C, as shown by the uniform distribution of the N signal in the EDX map  images were acquired in order to identify the GNS  of the 2D band is related to the number of layers of graphene sheets.99 In particular, a single curve with a FWHM of about 24 cm−1 /I(D′) reaches a maximum value of ∼13 for sp3 defects, decreases to ∼7 for vacancy-like defects, and has a minimum of ∼3.5 for boundary-like defects.102 Here, I(D)/I(D′) is 4.89 in case of pristine GNS in NMP, and 4.02 in case of pristine GNS in DMF, confirming the boundary-like nature of the defects in pristine GNS. Finally, the weak G* band is visible, here centered at 2463 cm−1, which arises from an intervalley process involving an in-plane transverse optical phonon and one longitudinal acoustic phonon .104Both pristine and functionalized GNS were dropcasted onto clean silica substrates, and the solvent was removed under vacuum see , in ordem−1 ref.  can be u105,106 The ratio I(D)/I(G) passes from an initial value of 1.02 for pristine GNS in NMP to a final value of 0.20 for functionalized GNS in NMP. Similarly I(D)/I(G) decreases from 1.01 to 0.68 for GNS in DMF. The decrease of I(D)/I(G) can be explained considering that the azomethine ylides are grafting onto graphene's most favorable bonding sites, which are in the defected areas of the lattice, possibly leading to a local structural relaxation and a decrease in the Raman intensity of the defects. Considering that the GNS lateral size is comparable with the Raman laser spot (∼1 μm), the initial high intensity of the D peak before the functionalization also derives from the high density of edge defects, which are passivated by the presence of the azomethine ylides after the functionalization, leading to a decrease of the intensity of the D peak. Observing the 2D peak, it is noticeable that its intensity remains almost constant, with I(2D)/I(G) slightly changing from 0.53 to 0.51 for GNS in NMP and from 0.49 to 0.47 for GNS in DMF. This behavior of the 2D peak confirms that the long-range order of the graphene lattice is maintained, and that we are not introducing some strong disorder towards amorphous graphene. A further sign of the successful functionalization of GNS is the rise of a new broad peak centered at 1548 cm−1 for GNS in NMP and at 1545 cm−1 for GNS in DMF. This band, called D′′ and usually seen in the range 1500–1550 cm−1, is thought to be related to either the amorphous phase 107 or to interstitial defects associated with the functionalization with small molecules.108–110 As aforementioned, the constant intensity of the 2D peak suggests that we are not inducing an amorphous phase, while the functionalization with azomethine ylide would explain creation of interstitial defects. Moreover, the peak centered at 1717 cm−1 for GNS in NMP and at 1716 cm−1 for GNS in DMF is usually assigned to the presence of the CO functional group,111 and would reasonably arise from the carboxyl group of the azomethine ylide. Finally, a set of four new peaks appears, which are labeled here as 1, 2, 3, and 4, centered respectively at around 1902 cm−1, 2057 cm−1, 2190 cm−1, and 2338 cm−1 for GNS in NMP and at 1895 cm−1, 2072 cm−1, 2201 cm−1, and 2343 cm−1 for GNS in DMF. These peaks are positioned in a region which is usually silent in Raman spectroscopy, and remarkably their positions do not significantly change with the change of the solvent /I(G) passes from 1.39 to 1.17 after the 1,3-DC of azomethine ylide. Likewise, I(D′)/I(G) decreases from 0.44 to 0.29. Furthermore, the intensity of the D′′ peak increases, with I(D′′)/I(G) moving from 0.29 before to 0.67 after the 1,3-DC, indicating the functionalization due to the presence of azomethine ylides. It is interesting to notice that also the intensity of the Raman signal from the CO group increases. As shown in −1, peak 2 at 2071 cm−1, peak 3 at 2204 cm−1, and peak 4 at 2341 cm−1. In the region of higher Raman shift, shown in −1 appears. This peak can be attributed to the O–H stretching118 of the carboxyl or the catechol groups in the azomethine ylide, and to our knowledge this is the first time it is seen in functionalized graphene. The Raman spectrum of functionalized rGO shows new and interesting features. As already seen for functionalized GNS, the intensity of the D peak decreases  or the substrate (same position for GNS and rGO), but arise from the functionalization with the azomethine ylide.3.6113 Functionalized rGO was modeled by adding the azomethine ylide in the central area of the rGO structure, followed by structural minimization. Functionalized rGO appears mildly corrugate, with a final morphology depending on the position of the epoxy groups and the azomethine ylide.In order to deepen our understanding of the functionalized graphene after 1,3-DC of azomethine ylide, models for pristine and functionalized rGO were built. Pristine rGO was modeled by positioning 10 oxygen atoms on top of randomly chosen carbon atoms, followed by structural minimization (as described in detail in Section 2). The total amount of oxygen was 8% in weight, in agreement with similar rGO models in literature.87 The RESP-derived partial charges, mapping the electrostatic features of the system, highlight the localization of the charges in the pristine rGO induced by the presence of the epoxy groups. The highest positive/negative charge values are, in fact, localized in the sp3 carbon atoms linked to the epoxy groups  derived partial atomic charges.rges see , demonstO in the carboxyl group of the azomethine ylide is visible, centered at 1730 cm−1. Moreover, there is a set of four vibrational regions  cm−1, (2000 ± 100) cm−1, (2100 ± 100) cm−1, and (2400 ± 100) cm−1 (the errors are related both to the simulation and to the extension of the regions 1′–4′). These values are consistent with the Raman features which appear in the intermediate region of both functionalized GNS and rGO. The corresponding first-order Raman bands are not visible in the experimental Raman spectra, being covered by the broad shoulder from the silicon substrate around 920–1050 cm−1  simulation at 300 K. During the MD simulation, the lattice did not undergo a large conformational rearrangement. The vibrational density of states has been obtained as the Fourier transform of the velocity autocorrelation function. The power spectrum was then decomposed and analyzed by computing the power spectra of the autocorrelation function of appropriate groups of atomic coordinates  and to nitrogen (C–N) are found at 286.1 eV.120 Finally, the contributions from CO and O–CO are seen at 287.8 eV and 290.8 eV, respectively.121Pristine and functionalized GNS and rGO were dropcasted onto silica substrates, and the solvent was removed under vacuum. This procedure was repeated several times in order to achieve a homogeneous coverage of the entire surface of the substrate, allowing measurements without any signal arising from the substrate. X-ray photoelectron spectroscopy provides a powerful and direct method for the determination of the surface elemental composition of a material. This information is crucial in order to quantify the efficiency of the functionalization, as well as the atomic abundance. In this study, the investigation was focused on the C 1s, N 1s, and O 1s core levels, which are related to the graphene substrate and the functionalizing material. Performing the deconvolution of the C 1s core level photoemission spectra of functionalized graphene (both GNS and rGO), we can identify five different components . The mai2  bonded to Na , and disappears after annealing at 180 °C.122 From the intensities of the C 1s, N 1s, and O 1s spectra, we can calculate the elemental abundances after the 1,3-DC of azomethine ylide, which are presented in Further information can be derived from the N 1s core level photoemission spectra of functionalized graphene . All spe4via 1,3-dipolar cycloaddition of azomethine ylide. For the first time, a comparison of the efficiency of this reaction in different dispersant solvents is provided. Even if the dispersion capability of NMP for graphene is slightly higher, the reaction proceeds more efficiently in DMF, allowing a greater organic functionalization of graphene in the liquid phase. To begin with, the functionalization was confirmed by detecting the presence of nitrogen in functionalized graphene with XPS, EDX, and EELS measurements. New Raman features arising from the functionalization with azomethine ylide were detected both in functionalized GNS and in functionalized rGO, and were assigned with the help of DFT simulations of the vibrational power spectrum of functionalized graphene. Thanks to the local inhomogeneity of the partial charges, due to the presence of oxygen functional groups in the pristine structure, a higher functionalization was achieved on rGO. This validates the interest in further exploring the possibility to control the position and the nature of defects, towards a precise building up of specific nanostructures, by exploring the design of the organic functionalization. Further chemical modifications of the functionalized graphene could be implemented by activating the carboxyl group of the azomethine ylide, with N-hydroxysuccinimmide for example, which would allow the binding of linker molecules and additional functional groups. Moreover, the efficiency of 1,3-DC of azomethine ylide suggests its use in future organic functionalization of higher quality graphene systems, like exfoliated flakes or epitaxial monolayer samples, where the controlled use of defect engineering would allow the realization of tailored devices starting from a performing substrate, in terms of transport properties. These results open the route for a wider range of applications, such as electrochemical devices for gas sensing or storage, enzymatic essays, catalysis, or protein interaction.In this work we have successfully functionalized graphene There are no conflicts to declare.NA-003-D1NA00335F-s001


To examine the effects of LUBAC-mediated linear ubiquitination on TDP-43 proteinopathies, we performed cell biological analyses using full-length and truncated forms of the ALS-associated Ala315→Thr (A315T) mutant of TDP-43 in Neuro2a cells. The truncated A315T mutants of TDP-43, which lack a nuclear localization signal, efficiently generated cytoplasmic aggregates that were colocalized with multiple ubiquitin chains such as M1-, Lys(K)48-, and K63-chains. Genetic ablation of HOIP or treatment with a LUBAC inhibitor, HOIPIN-8, suppressed the cytoplasmic aggregation of A315T mutants of TDP-43. Moreover, the enhanced TNF-α-mediated NF-κB activity by truncated TDP-43 mutants was eliminated in the presence of HOIPIN-8. These results suggest that multiple ubiquitinations of TDP-43 including M1-ubiquitin affect protein aggregation and inflammatory responses in vitro, and therefore, LUBAC inhibition ameliorates TDP-43 proteinopathy. TAR DNA-binding protein 43 (TDP-43) is a predominant component of inclusions in the brains and spines of patients with amyotrophic lateral sclerosis (ALS). The progressive accumulation of inclusions leads to proteinopathy in neurons. We have previously shown that Met1(M1)-linked linear ubiquitin, which is specifically generated by the linear ubiquitin chain assembly complex (LUBAC), is colocalized with TDP-43 inclusions in neurons from  TARDBP encodes TAR DNA-binding protein 43 (TDP-43), which is a key component of the insoluble and ubiquitinated cytoplasmic inclusions in the brain and spinal cord of ALS and frontotemporal dementia (FTD) patients -3-oxo-propenyl}-4-(1-methyl-1H-pyraol-4-yl)-benzoic acid sodium salt) was synthesized as described  vector (Promega), and the pRL-TK Renilla Luciferase control reporter vector (Promega) using Lipofectamine 2000. The medium was replaced at 4 h-post transfection. At 24 h after transfection, the cells were lysed and the luciferase activity was measured with a GloMax 20/20 luminometer (Promega) using the Dual-Luciferase Reporter Assay System (Promega). Neuro2a cells  Neuro2a cells by the CRISPR/Cas9 technique, resulting in the elimination of HOIP and reduced the HOIL-1L and SHARPIN levels , SOD1, FUS, C9ORF72, ATXN2, OPTN, VCP, UBQLN2, UBQLN4, SQSTM1, TBK1, and so on, but the mechanism of ALS onset has remained elusive. TDP-43-containing cytoplasmic inclusions are reportedly detected in 97% of ALS and 45% of FTD cases [282-360 showed that the region has a unique double-spiral-shaped fold, and is oligomerized with a prion-like filamentous structure [OPTN-associated fALS and sALS patients, and in tau neurofibrillary tangles from Alzheimer’s disease patients [ALS is a fatal neurodegenerative disease. Analyses of fALS have revealed the causative genes of ALS such as TD cases . Therefotructure . Ubiquittructure ,30. TDP-tructure ,32. Impotructure . Furthertructure . We detepatients ,22,23. Apatients . These rHoip or treatment with the LUBAC inhibitor HOIPIN-8 drastically reduced the insoluble aggregates of truncated TDP-43 , oligomerization, and aggregate formation. In this study, we showed that cytoplasmic aggregates of ectopically expressed, truncated ALS-associated TDP-43 mutants in Neuro2a cells contained M1-, K48-, and K63-ubiquitins in vitro, and the MG-132-mediated inhibition of proteasome activity enhanced the ubiquitination of TDP-43 . These rd TDP-43 . These rd TDP-43 . We furt247-414-GFP and A315T247-414-GFP, indicating that the production of the C-terminal fragment generally contributed to the aggregate formation, regardless of the single amino acid mutation of A315T. However, the human TDP-43 A315T mutation is a known etiology of ALS [Currently, a glutamic neurotransmission inhibitor, riluzole, and an antioxidant drug, edaravone, are approved for ALS treatment . Althougy of ALS ,41, and y of ALS ,43. Furt


Streptococcus mutans. They also inhibit periodontal pathogens that are responsible for periodontitis and root canal infection. Graphene-fluorine promotes enamel and dentin mineralization. These materials were also broadly studied in regenerative dental research, such as dental hard and soft tissue regeneration, as well as periodontal tissue and bone regeneration. Graphene oxide-based materials, such as graphene oxide-fibroin, were reported as promising in tissue engineering for their biocompatibility, bioactivity, and ability to enhance cell proliferation properties in periodontal ligament stem cells. Laboratory research showed that graphene can be used exclusively or by incorporating it into existing dental materials. The success of laboratory studies can translate the application of graphene into clinical use.Graphene is a single-layer two-dimensional carbon-based nanomaterial. It presents as a thin and strong material that has attracted many researchers’ attention. This study provides a concise review of the potential application of graphene materials in caries and periodontal disease management. Pristine or functionalized graphene and its derivatives exhibit favorable physicochemical, mechanical, and morphological properties applicable to biomedical applications. They can be activated and functionalized with metal and metal nanoparticles, polymers, and other small molecules to exhibit multi-differentiation activities, antimicrobial activities, and biocompatibility. They were investigated in preventive dentistry and regenerative dentistry. Graphene materials such as graphene oxide inhibit cariogenic microbes such as  Graphene is a two-dimensional mono-atomic sp2 hybridized carbon-based nanomaterial known as the thinnest and strongest element in existence . Of all Streptococcus mutans, which generates organic acids, thus reducing the oral pH level and leading to demineralization of the dental hard tissue surface [Porphyromonas gingivalis and Fusobacterium nucleatum are responsible for gingivitis and periodontitis, respectively [Dental caries is the chronic local damage of dental hard tissue  that acidic byproducts of bacterial metabolism of dietary carbohydrates often cause , and per surface . On the ectively . Severalectively .Generally, humans cannot reproduce or regenerate or regrow teeth or tooth tissues. Although oral and soft tissue can be repaired, the regeneration or repair of hard tissues  is inadequate or sometimes impossible. Interestingly, in addition to biomaterials sciences, preventive and regenerative dentistry is also advancing well. Preventive and regenerative dentistry research mostly focuses on preventing dental caries and periodontal disease. Simultaneously, they are focused on restoring lost tooth tissue because of caries and or periodontal diseases. These days, the high incidence of periodontal diseases is a major concern . ResearcRegardless of the challenges ahead, the latest advances in nanotechnology have played a biomimetic role and have shown tremendous potential in dental hard and soft tissue regeneration. Various nanomaterials are being added continuously and have produced many clinical benefits in dentistry using tissue engineering properties, which include: the advanced treatments of caries and periodontal diseases, bone regeneration, feasible biological tooth repair after caries, and is probably advancing towards regrowing entire lost teeth ,33. At tgraphene oxide, reduced graphene oxide, graphene, graphone, flurographene, graphyne, graphdiyne, doped graphene, and graphene quantum dots were well reported [Graphene is an excellent nanomaterial; however, in some cases, graphene may not adhere to the specific properties required for certain applications. Therefore, the functionalization of graphene comes to light in current research with improved properties known as graphene derivatives . Studiesreported ,37,38. Areported . In our Graphene oxide is the oxidized graphene-based sheets with several oxygen-functional groups , which provide the covalent or noncovalent combination of graphene oxide with nanoparticles and nano-biomolecules. Therefore, graphene oxide properties can be tuned by changing its molecular structure by functionalization of its oxygen functional group ,41. MoreGraphene oxide is a promising carrier for biomolecules and drugs. It can enhance biomaterials’ bioactivity, sustainability, and mechanical performance . At the Interestingly, all these graphene derivatives can be activated and functionalized with metal and metal nanoparticles, polymers, drugs, and other small molecules. They can be used in various research fields including regeneration, drug delivery, gene delivery, protein delivery, nanoparticles release, cell and tumor imaging, physiochemical properties development of other materials, multimodal bioimaging, and cancer therapy. This review includes studies published in the English language on graphene and graphene derivatives and their functionalized composites used in caries and periodontal disease management. Abstracts, editorials, letters, and literature reviews were excluded. The EMBASE, Web of Science, Google Scholar, and PubMed databases were systematically searched. In the search, the keywords consisting of “graphene”, “caries,” “demineralization”, “remineralization”, “periodontal”, “regeneration”, “tissue engineering” and “bone regeneration” were employed. These keywords would cover information about graphene and its derivatives in caries and periodontal disease management. After reading all the manuscripts, the obtained articles were selected to describe in the terms of caries management and periodontal disease management.Streptococcus mutans is the main cariogenic pathogen. They produce insoluble extracellular polysaccharides, which facilitate bacterial growth and the formation of cariogenic biofilms. This is why most of the research focuses on developing biomaterials to inhibit Streptococcus mutans [Dental caries is a highly prevalent disease. Cariogenic biofilms are mainly responsible for dental caries. Caries initiates with the chemical dissolution of dental hard tissue by the acid produced through dietary carbohydrate metabolism of bacteria that adhered (as biofilm) to the tooth surface. A prolonged stagnation of biofilm enhances enamel and dentin desolation and progresses to cavity formation on the tooth surface ,51. Thess mutans .Remineralization of demineralized caries also can stop caries progression. It is said that maximum mineralization in the human body is seen in teeth by continuous demineralization and remineralization throughout life with varying amounts to maintain tooth integrity . It breaAlthough caries risk assessment and remineralization of initial lesions have controversy, diverse advanced research and nanotechnology have developed risk-specific biomaterials or board functional nano-biomaterials and opened the doors to caries prevention . GraphenStreptococcus mutans, reduce dental hard tissue demineralization, or facilitate remineralization [Although graphene and its derivatives can inhibit cariogenic bacteria, most of them are studied together with antimicrobial metals or non-metal or polymer nanoparticles, such as copper, silver, zinc, peptides, and polymer nanoparticles, to improve the antibacterial properties or facilitate the sustainable release of incorporated nanoparticles ,69,70,71lization ,72,73,74Streptococcus mutans [Streptococcus mutans [Streptococcus mutans without any significant cytotoxicity [Streptococcus mutans biofilm [Streptococcus mutans growth significantly [Streptococcus mutans biofilm. There are also reports of suppressing acid production and glucan formation, which are responsible for caries and biofilm formation [Graphene nanosheet was reported as very effective against s mutans . Simultas mutans ,61. Substoxicity ,57,64,65 biofilm . Moreoveficantly . In addiormation .Streptococcus mutans [Streptococcus mutans [Streptococcus mutans growth [Staphylococcus aureus and Streptococcus mutans [Streptococcus mutans was significantly reduced [In other studies, amino-functionalized graphene oxide was reported with potential against cariogenic bacteria s mutans ,70. At ts mutans . Some ots growth ,74. Anots mutans . Some ot reduced ,75.Streptococcus mutans [Although graphene oxide and antisense vicR could significantly inhibit biofilm and extracellular polysaccharide production alone, graphene oxide–polyethyleneimine–antisense vicR was reported as superior in inhibiting extracellular polysaccharide regulation, virulence-associated gene expression, and biofilm formation. Therefore, the study suggested that graphene oxide–polyethyleneimine–antisense vicR ribonucleic acid could be a highly potent agent for caries prevention . On the s mutans . Interess mutans .By overseeing all these studies, it can be hypothesized that either pristine or functional nanocomposites of graphene and its derivatives could potentially be used against cariogenic bacteria. However, the established mechanism of antibacterial activities of graphene derivatives is still to be explored. Several antibacterial mechanisms have been described to demonstrate graphene and its derivatives in inhibiting cariogenic microbe and their biofilm. Physical damage, membrane stress, oxidative stress, and electron transfer were well considered . TherefoStreptococcus mutans acidic by-product, demineralization initiates dental caries. At the same time, the counteraction of remineralization protects teeth from decay. Graphene can facilitate remineralization. In a study, graphene-fluorine was reported to enhance the remineralization of white spot lesions [As a consequence of the  lesions . At the  lesions .In several studies, graphene oxide conjugated bioactive glass was reported to significantly increase the anti-demineralization effect, microhardness, shear bond strength, and adhesive remnant index with no or low cytotoxicity ,80. GrapIn one study, reduced graphene oxide-silver was found to reduce enamel surface roughness and mineral loss, thus reducing the lesion depth . MoreovePeriodontal diseases are highly prevalent among other dental diseases next to dental caries . PeriodoPorphyromonas gingivalis is known as the primary pathogen for periodontal diseases. Most research is targeted to develop novel biomaterials or drugs to inhibit Porphyromonas gingivalis [Streptococci mutans, Fusobacterium nucleatum, and Porphyromonas gingivalis) and reported to have a high inhibitory effect on Porphyromonas gingivalis [ngivalis . In recengivalis .Porphyromonas gingivalis and Enterococcus faecalis and reported to have enhanced antibacterial effects [Porphyromonas gingivalis, Fusobacterium nucleatum, and Candida albicans. They applied an oxidative stress-induced antimicrobial mechanism by measuring the reactive oxygen species (ROS) generation and were reported to exhibit antimicrobial effects against these oral pathogens [Staphylococcus aureus and Escherichia coli [Another study investigated graphene silver polymethyl methacrylate on  effects . At the athogens . Anotherhia coli .Porphyromonas gingivalis and Fusobacterium nucleatum. The study showed that graphene oxide nanosheets could penetrate and destroy the cell wall and membrane, and initiate plasma leakage, resulting in cell death [Porphyromonas gingivalis [Graphene oxide nanosheets were demonstrated as effective against ll death . Zinc oxll death ,102. Anongivalis .Streptococcus mutans, Fusobacterium nucleatum, and Porphyromonas gingivalis and their biofilms [Streptococcus sanguinis, Fusobacterium nucleatum, and Porphyromonas gingivalis [Staphylococcus aureus and Escherichia coli [Candida albicans biofilm [Conversely, the use of graphene oxide followed by brushing was reported to be effective in eliminating biofilms . In one ngivalis . Moreovehia coli . On the  biofilm .Porphyromonas gingivalis biofilm formation. Similarly, graphene oxide quantum dots and curcumin composite was also reported to inhibit polymicrobial biofilm formation, including periodontal pathogens such as Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia,Prevotella nigrescens, Escherichia coli, and Staphylococcus aureus [Studies showed that carbon quantum dots can inhibit s aureus ,109.Observing all this research, it can be assumed that graphene and its derivatives could effectively prevent periodontal disease by inhibiting periodontal pathogens and their biofilms; however, further investigations are necessary to investigate these in vitro results in clinical settings.Despite the prevention or reduction of periodontal disease, the reproduction of lost periodontal tissue is highly expected, thus regenerative periodontal therapy is a developing research interest. Several techniques, including stem cells, scaffolds, biomaterials, or a combination were applied in various studies ,111,112.Graphene oxide-silk fibroin was reported as an excellent candidate for tissue engineering. In a study, a composite of graphene oxide-silk fibroin was investigated for cell adhesion, proliferation, viability, and mesenchymal phenotype expression of periodontal stem cells, and was reported to have high potential in the therapeutic capacity of this composite for regenerative dental applications . On the In a study, minocycline hydrochloride conjugated graphene oxide was investigated in a peri-implantitis model and reported a very negligible bone loss in the minocycline hydrochloride-graphene oxide-titanium groups compared to that of titanium and minocycline hydrochloride-conjugated titanium group or the graphene oxide-titanium group. Moreover, there were almost no neutrophils found in graphene oxide-titanium and minocycline hydrochloride-graphene oxide-titanium groups, but a deposit of osteocyte cells was observed. Thus, suggested minocycline hydrochloride-conjugated graphene oxide could be a good therapeutic coating for preventing peri-implantitis .Another study investigated the bioactivity of human periodontal ligament stem cells on graphene oxide-coated titanium substrate, which demonstrated: a higher cell proliferation rate and alkaline phosphatase activity; upregulated osteogenesis-related genes such as alkaline phosphatase, bone sialoprotein, collagen type I, and osteocalcin; a higher runt-related transcription factor 2 expression than that of on the control sodium titanium substrate . SimultaIn addition, one study showed that a graphene oxide scaffold was biocompatible and capable to form new bone and periodontal tissue . In a seAt the same time, reduced graphene oxide has also shown potential for stem cell proliferation and is suggested for use in regenerative dentistry ,124. OneGraphene oxide and reduced graphene oxide have been included in a porous titanate scaffold on titanium implants to develop a drug delivery system for dexamethasone, the study of which showed that both dexamethasone-graphene oxide-titanium and dexamethasone-reduced graphene oxide-titanium increases alkaline phosphate activity in the bone marrow mesenchymal stem cells. They exhibit osteogenic genes, osteopontin and osteocalcin expression, and enhanced osteogenic activity . MoreoveReduced graphene oxide-coated hydroxyapatite also showed an osteogenic differentiation of human mesenchymal stem cells. Therefore, they suggested applying this composite for orthopedic and dental regeneration . GraphenThere have been tremendous advancements in tissue regenerative research using graphene derivatives; however, to date, no clinical trial is reported. More in vitro, in vivo, animal trials, and ultimately a human trial should be performed to validate the optimal result for applying these novel materials in clinical settings.Periodontal bone loss usually occurs due to several factors, including tooth extraction, trauma, infection, systemic or local alterations in the host response, malignancy, or multifactorial causes . Bone suStudies have well reported that graphene can promote the osteogenic differentiation of several cells, including osteogenic cells and stem cells , and have suggested applying this in different forms  . A studyThe chitosan-incorporated graphene oxide-chitosan-hydroxyapatite coating on titanium substrates was reported to show improved in vitro and in vivo osseointegration . MoreoveGraphene oxide-coated titanium substrates, loaded with bone morphogenetic protein-2, exhibited osteogenic differentiation of human bone marrow-derived mesenchymal stem cells, new bone formation, and sustained drug delivery without interfering with drug structure and bioactivity . In addiSimilarly, reduced graphene oxide also has the potential as a surface coating on titanium implants. To show the osteogenic differentiation of reduced graphene oxide, a study used collagen type I conjugated reduced graphene oxide and found collagen–graphene oxide/reduced graphene oxide can activate the differentiation of human bone marrow stem cells into osteoblasts . ComparaAnother study showed that multipass caliber rolled titanium alloy coated with dexamethasone-loaded reduced graphene oxide enhanced osteogenic cell growth and osteoblastic differentiation of MC3T3-E1 cells. In addition, a reduced graphene oxide-coated titanium implant was implanted in an artificial bone block and reported to be stable . On the In a study, reduced graphene oxide and pulsed electromagnetic fields were combinedly used to evaluate osteogenesis and the neurogenesis of mechanical stem cells. The investigation showed that the conjugates enhanced osteogenic, neurogenic, and adipogenic differentiation of human alveolar bone marrow stem cells. They suggested using this conjugate for stem cell and tissue engineering .In conclusion, in vitro studies demonstrated that graphene and its derivatives have antimicrobial and remineralizing properties for the management of dental caries and periodontal disease. Therefore, advanced research is essential to translate their application to clinical dentistry.


Cryogenic electron microscopy (cryo-EM) single particle analysis has become one of the major techniques used to study high-resolution 3D structures of biological macromolecules. Specimens are generally prepared in a thin layer of vitrified ice using a holey carbon grid. However, the sample quality using this type of grid is not always ideal for high-resolution imaging even when the specimens in the test tube behave ideally. Various problems occur during a vitrification procedure, including poor/nonuniform distribution of particles, preferred orientation of particles, specimen denaturation/degradation, high background from thick ice, and beam-induced motion, which have become important bottlenecks in high-resolution structural studies using cryo-EM in many projects. In recent years, grids with support films made of graphene and its derivatives have been developed to efficiently solve these problems. Here, the various advantages of graphene grids over conventional holey carbon film grids, functionalization of graphene support films, production methods of graphene grids, and origins of pristine graphene contamination are reviewed and discussed. The successful application of direct electron detection devices  and wellHowever, through cryo-EM studies, researchers have found that the conventional cryo-EM procedure does not always work and that there are obstacles in the specimen preparation procedure. The conventional plunge freezing procedure for cryo-EM sample preparation developed by Dr. Jacques Dubochet in the early 1980s  is stillIn recent years, many efforts have been applied to developing various methods and techniques to solve the problems that can occur during cryo-EM specimen vitrification. One method is modifying the surface of the holey carbon support foil by manipulating glow discharging protocols  or treatAnother type of technique is to coat the holey carbon/metal support foil of the grid with a continuous film, such as ultrathin amorphous carbon film , lipid mIn this review, various advantages of graphene grids are discussed by comparing them with conventional holey carbon grids. In addition, to form an overall outline of the current development of graphene grids for cryo-EM, the recent progress in graphene film functionalization and the preparation of high-yield and clean graphene grids are discussed. If not specified, the phrase “graphene grid” represents both grids coated with graphene oxide film and grids coated with continuous pristine graphene film.The successful exfoliation of 2D crystal monolayer graphene films was realized by Andre Geim and Konstantin Novoselov in 2004 . Since iHowever, unmodified graphene was not widely used, due to contaminant-induced hydrophobicity and degradation of image quality, until 2014, when Graphene oxide (GO) was also introduced as a substrate material for cryo-EM experiments since it is naturally hydrophilic, nearly electron transparent, and easy to synthesize . HoweverVitrified particles in a hole may not look like what we expect. The ideal model of cryo-EM samples shows evenly distributed particles with random orientations, and the vitreous ice is uniformly thin. Owing to the interaction of particles with the air–water interface, support film, and neighboring particles, different situations can occur .Next, the vitrified ice thickness in the hole affects the final resolution that we can achieve. Thick ice increases the background and therefore reduces the image contrast. In addition, the problems of defocus gradient  and highThus, finding suitable areas that have both thin ice and a high density of evenly distributed particles in samples prepared on holey carbon grids remains challenging. In most cases, protein particles are not at the same Z-height when they are vitrified using a conventional holey carbon film grid , resultiIn some cases, when using a holey carbon film grid, the nonspecific interaction between protein particles and carbon film is nonnegligible, resulting in few particles found in the holes, while more particles are adsorbed on the carbon film. To obtain more particles in the holes, a high concentration of specimen would be necessary. However, using GO grids, Many cryo-EM research groups have found that the quality of vitrified samples deteriorates in comparison with that of negative-stained samples. This phenomenon was later explained by the interaction of protein particles with the air–water interface.When the protein particles are confined to the thin layer of the solution on the grid, the particles can diffuse and approach the air–water interface quickly. It was estimated that there were more than 1,000 collisions per second with the air–water interface in the thin ice (≤100 nm), which gives sufficient opportunity for adsorption of particles in preferential orientation . Using tTo minimize the effect of the air–water interface, many approaches, including adding the surfactants OG , amphipoIn addition to our recent development of HFBI film–coated grids , the emeWhen irradiated using an electron beam, the particles embedded in vitreous ice move, and this type of motion is called beam-induced movement (BIM). The BIM process involves two phases, including an initial rapid “burst” phase and a following slower phase . The burThe BIM effect can cause blurring of images and limit the achievable resolution of cryo-EM. Regarding obtaining a 3-Å resolution reconstruction, the elimination of the BIM effect can significantly reduce the number of particles needed to reach the same resolution by ∼30-fold . Owing tThe pristine graphene film–coated grids showed a unique advantage in reducing the BIM effect due to their high mechanical strength and electrical conductivity. We note that The preferred orientation of particles is another common limiting factor in cryo-EM single particle analysis and can be induced by interaction between the protein particles and the air–water interface, the support film, or the neighboring particles. A preferred orientation causes a biased distribution of angular projections and yields an anisotropic resolution in the final reconstruction ; this isTo address the issue of air–water interface–induced preferred orientation, in addition to the above approaches of minimizing the interaction between the particles and the air–water interface, methods for altering the properties of the supporting film or the air–water interface, including plasma treatment in the presence of N-amylamine , the addTo address the existence of a preferred orientation, These problems of the tilt data collection strategy can be mitigated using graphene grids. First, a much thinner and uniform vitrified ice can be prepared, thus minimizing the concerns about the increase in the thickness of the ice for tilted specimens. Second, since most protein particles are adsorbed onto the layer of the graphene–water interface and sitting on the same plane, the focus gradient can be more accurately estimated, and CTF estimation can be performed more precisely. Third, more importantly, with their high electric conductivity and mechanical stiffness, graphene grids can effectively reduce BIM even under the condition of specimen tilting. Hence, a tilt data collection strategy using graphene grids would be a better solution for coping with preferred orientation problems in high-resolution structural studies ; the worIn particular, graphene grids can prevent the contact of protein particles with the air–water interface but cannot solve the preferred orientation problem due to the interaction between the particles and the graphene–water interface. The tilt data collection strategy with graphene grids would be necessary for many specimens. Of late, we developed a new type of grid based on a 2D crystal HFBI film that can adsorb protein particles by means of electrostatic interaction to protect particles from the air–water interface and play a role similar to that of a graphene grid, with minimal background, and to help form thin enough ice .Monolayer graphene is an ideal support for cryo-EM studies in comparison with holey carbon films due to the above advantages. To address the new potential for preferred orientation from the interaction between protein particles and the graphene–water interface and to increase the affinity of the graphene support to the specific molecules, there are many developments involving functionalizing graphene to regulate the interaction between the support and the protein particles.The first approach is functionalizing a graphene film using plasma treatment . NaydenoThe second approach is to oxidize graphene to GO and then chemically modify GO . Wang etThe third approach is to use reduced GO for high-resolution imaging . CompareThe fourth approach is similar to a previously developed affinity grid used for cryo-SPIM (cryosolid phase immune electron microscopy) . The affWith various advantages of graphene grids for cryo-EM study, many efforts to make reproducible and high-yield production of high-quality graphene grids have been performed in recent years. The nonuniform and low coverage as well as the surface contamination need to be efficiently solved during the production of graphene grids.Naturally hydrophilic GO is easy to obtain at low cost and has already been applied in cryo-EM studies . However3, is used to remove the unwanted copper support of the graphene monolayer , an extra support layer is needed when the graphene surface is isolated from the metal substrate. The PMMA layer is still the most widely used material for graphene transfer . HoweverThe structure of apoferritin has been resolved at atomic resolution with the development of new hardware, including cold field emission guns, monochromators, aberration correctors, and the latest generation camera coupled to a new energy filter , which iMonolayer graphene grids, with minimal background, have become a promising approach to solve these problems and offer the opportunity to reveal near-atomic structures of proteins with a small molecular weight (<100 kDa), low concentration, and even a transient intermediate state. For the samples that can be solved to a medium-high resolution using a holey carbon grid, the use of a graphene grid can significantly reduce the beam-induced motion, prepare uniform and thinner ice, and thereby increase the possibility of higher resolution.As of now, most graphene grids for cryo-EM studies are prepared by researchers themselves and thus often are associated with poor reproducibility, a lower coverage rate, cleanliness problems, etc. With the development of more scalable and robust methods for fabricating high-quality and ultraclean graphene grids, further commercialization will be possible, and thus, monolayer graphene grids as well as different functionalization treatments will become widely used in the cryo-EM community and make cryo-EM sample preparation more successful and reproducible.


Tripartite motif (TRIM) family genes get involved in the pathogenesis and development of various biological processes; however, the prognostic value of TRIM genes for idiopathic pulmonary fibrosis (IPF) needs to be explored.We acquired gene expression based on bronchoalveolar lavage (BAL) cells and clinical data of three independent IPF cohorts in the GSE70866 dataset from the Gene expression omnibus (GEO) database. Differentially expressed TRIM genes (DETGs) between IPF patients and healthy donors were identified and used to establish a risk signature by univariate and multivariate Cox regression analysis in the training cohort. The risk signature was further validated in other IPF cohorts, and compared with previously published signatures. Moreover, we performed functional enrichment analysis to explore the potential mechanisms. Eventually, the quantitative real time PCR was conducted to validate the expressions of the key genes in BAL from 12 IPF patients and 12 non-IPF controls from our institution.P < 0.05). A multiple stepwise Cox regression analysis was performed to construct a 4-TRIM-gene prognostic signature. We categorized IPF patients into one low-risk group and the other high-risk group as per the average risk value of the TRIM prognostic signature in the training and validation cohorts. The IPF individuals in the low-risk group demonstrated an obvious OS advantage compared with the high-risk one (P < 0.01). The time-dependent receiver operating characteristic approach facilitated the verification of the predictive value of the TRIM prognostic signature in the training and validation cohorts, compared with other published signatures. A further investigation of immune cells and IPF survival displayed that higher proportion of resting memory CD4+ T cells and resting mast cells harbored OS advantage over lower proportion, however lower proportion of neutrophils, activated dendritic cells and activated NK cells indicated worse prognosis.We identified 4 DETGs including TRIM7, MEFV, TRIM45 and TRIM47 significantly associated with overall survival (OS) of IPF patients (The TRIM family genes are significant for the prognosis of IPF and our signature could serve as a robust model to predict OS. Idiopathic pulmonary fibrosis (IPF) is a chronic and lethal interstitial lung disease, characterized by excessive extracellular matrix protein deposition and irreversible lung damage, resulting in progressive ventilation dysfunction, hypoxemia, respiratory failure and death , 2. The Alterations in genetic and molecular information obtained from lung tissues, peripheral blood mononuclear cells, bronchoalveolar lavage (BAL) cells, were found to be associated with disease severity and mortality of IPF patients . RecentlIn this study, we systematically compared TRIM gene expressions of BAL cells between normal individuals and IPF cases, and differentially expressed TRIM genes (DETGs) associated with IPF prognosis were used to construct a prognostic signature in a training cohort and further validated by other two independent cohorts. The potential mechanism was further investigated between low-risk and high-risk groups based on the prognostic signature.https://www.ncbi.nlm.nih.gov/geo/), which included a cohort of 62 IPF patients from Freiburg, Germany and a cohort of 50 IPF patients from Siena, Italy based on GPL14550 platform (Agilent-028004 SurePrint G3 Human GE 8 × 60 K Microarray), and another cohort consisting of 64 patients from Leuven, Belgium based on GPL17077 platform (Agilent-039494 SurePrint G3 Human GE v2 8 × 60 K Microarray)  > 0 and a false discovery rate (FDR) < 0.2 . VolcanoP < 0.05. OS-related DETGs were visualized on forest maps and applied to establish prognostic signature by stepwise multivariate Cox regression analysis. In the training cohort, the signature for prediction was to multiply the expression level of each selected prognostic gene by its corresponding relative regression coefficient weight as follows:To construct a reliable prognostic signature based on the DETGs, the Freiburg cohort was used as the training cohort to screen for hub genes, and the other two independent cohorts (Siena and Leuven cohorts) were used for validation. Univariate Cox regression analysis was used to select DETGs associated with overall survival (OS) of IPF patients at the threshold of k groups . Receivek groups , 16. ConUnivariate and multivariate Cox regression analyses were applied to determine independent clinical factors, including age, gender, GAP and risk-score. Through “rms” and “survival” packages in R, a prognostic nomogram was established to evaluate the probability of OS in IPF patients in the training cohort.P < 0.05, and |normalized enrichment score (NES) |> 1 [Gene set enrichment analysis (GSEA) version 4.1.0 was used to explore potential molecular mechanisms between patients in low- and high-risk groups underlying our constructed signature . The KyoES) |> 1 .Relative proportions of 22 given kinds of immunocytes in IPF samples were assessed by applying the CIBERSORT deconvolution algorithm , 20. Thr−ΔΔCt strategy, normalizing with GAPDH. The primer sequences used for qRT-PCR in this study are listed in Table We obtained BAL from 12 IPF patients and 12 non-IPF controls who underwent bronchoscopy in the First Affiliated Hospital of Chongqing Medical University. Written informed consent was obtained from all the patients, and the research was approved by the Ethical Committee of our institution. The BAL samples were immediately isolated and 1 ml Trizol to was added to the cell samples,then snap-frozen in liquid nitrogen and preserved in a − 80 °C freezer. Total RNA was isolated from frozen samples using the TRUEscript RT MasterMix  according to manufacturer’s instructions. Then total RNA was reverse-transcribed into complementary DNA using the TransScript First-Strand cDNA Synthesis kit . Quantitative real-time PCR (qRT-PCR) was then performed using the SYBR Green QPCR Mix . The data was calculated through the 2P < 0.05 was statistically significant.R software (version 4.1.2) packages were used to analyze GEO data. The qRT-PCR data was analyzed using unpaired Student’s t-test and visualized by using GraphPad Prism 9.4.1 . Among all the TRIM family members, 21 DETGs were obtained between 62 IPF patients and 20 healthy donors from the Freiburg cohort, as demonstrated in the heat map  × TRIM7 + (− 0.304) × MEFV + (− 0.409) × TRIM45 + (0.553) × TRIM47 (Table 1 × TRIM7P < 0.01)  Fig. A, with A01) Fig. B. Intere01) Fig. A, with A01) Fig. B. In con01) Fig. A, the Si01) Fig. B and the01) Fig. C.Fig. 5TP = 0.02), risk score  were significant independent risk factors , resting mast cells (P < 0.01), neutrophils (P < 0.05), activated dendritic cells (P < 0.05) and activated natural killer (NK) cells (P < 0.05) were significantly different between low- and high-risk groups  is a fatal condition, the most lethal form of interstitial pneumonia characterized with irreversible fibrosis in pulmonary parenchyma with unspecific etiology, leading to uncontrollable infection, respiratory failure and even death . IncreasA multidimensional prognostic staging system GAP model, consisting of gender (G), age (A), and 2 lung physiology (P) variables including forced vital capacity (FVC) and diffusion capacity of the lung for carbon monoxide (DLCO), was firstly constructed and validated in 2012, with good predictive and discriminative performance in the prognosis of IPF . A modifAnalysis of BAL is a useful procedure for differential diagnosis of interstitial lung diseases (ILDs) and for identification of granulomatous lung diseases. Recent studies have identified potentially useful peripheral and BAL biomarkers, including chemokines, cytokines, genes and proteins , 30. ConIn this study, we systematically evaluated the mRNA expression levels of TRIM family members in IPF and healthy samples. Four of DETGs were associated with OS of IPF Patients, which suggested that TRIM genes participated in IPF and harbored a potential predictive value of these genes. The four DETGs , were used to establish a predictive signature. TRIM7, also known as glycogenin-interacting protein 1 (GNIP1), was found to exert protective or detrimental effect in various cancers by different molecular mechanisms –37. MEFVInterestingly, members of the TRIM family as E3 ubiquitin ligases were found to critically regulate innate immunity and antiviral response . StudiesTo our knowledge, this study is the first one to investigate one specific gene family signature in IPF, which was more convenient to conduct gene analysis. Considering the significant roles of TRIM family members in various disease processes, our signature consists of only four TRIM genes, which were more concise than other models and have comparable predictive values with other models. However, there are still some limitations in this study. Firstly, the value of signature needs more validation in daily clinical work. Moreover, the signature was constructed to predict prognosis, and not able to predict a patient’s response to medical treatment.The TRIM family genes are significant for the prognosis of IPF and our signature could serve as a concise and robust model to predict OS.


We have studied whether growth factors, cytokines, hormones, neurotransmitters, and local hormones (autacoids) promote the proliferation of hepatic parenchymal cells  using in vitro primary cultured hepatocytes. The indicators used for this purpose include changes in DNA synthesis activity, nuclear number, cell number, cell cycle, and gene expression. In addition, the intracellular signaling pathways from the plasma membrane receptors to the nucleus have been examined in detail for representative growth-promoting factors that have been found to promote DNA synthesis and cell proliferation of hepatocytes. In examining intracellular signaling pathways, the effects of specific inhibitors of presumed signaling factors involved have been pharmacologically confirmed, and the phosphorylation activities of the signaling factors  have been evaluated. As a result, it has been found that there are many factors that promote the proliferation of hepatocytes , prostaglandin (PG)), and serotonin (5-HT)), while there are very few factors  that inhibit the effects of growth-promoting factors. We have also found that 5-HT and GH promote the proliferation of hepatocytes via different autocrine factors . Using primary cultured hepatocytes, it will be possible to further study the molecular and cellular aspects of liver regeneration. The liver is the central organ of metabolism and is responsible for the homeostasis of the body by metabolizing carbohydrates, proteins, lipids, and other substances. Furthermore, detoxification and regeneration are characteristic functions of the liver. These abilities are thought to have been acquired during the long evolutionary process to protect animals from catastrophic damage to liver tissues caused by food toxins  .The mechanisms of liver regeneration have been studied for many years, and numerous reports have been published. There are also excellent review articles integrating those results ,3,4,5,6.Therefore, we used a simpler in vitro primary cultured hepatocyte system to examine the actions of individual hepatocyte growth factors (and candidate substances) and their intracellular signaling pathways. Primary cultured hepatocytes retain metabolic activity comparable to that in vivo and express many kinds of receptors, each of which is known to be responsive to agonists ,7. It isIn this review, an in vivo model of liver regeneration is briefly described. In addition, based on our results, the factors that promote or inhibit the proliferation of hepatocytes are classified into their bioactive characteristics, and their respective intracellular signaling pathways are briefly summarized. Then, we will discuss the physiological significance of how each of these factors may play a role in liver regeneration in vivo.0 → G1 → S → G2 → M → G0 phase), which stops when the organ returns to its original size. During the post-PHx, the blood levels of the intracellular enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increase rapidly due to the destruction of the liver tissue , tumor necrosis factor-α (TNF-α), interleukin (IL)-6, serotonin (5-HT), and bile acid levels in the blood also increase after PHx . TherefoAlthough the aforementioned in vivo study of liver regeneration using live animal models has its advantages, there are limitations when studying the intracellular signaling mechanisms in detail. The main reason is that the liver tissue is composed of several different cell groups , and the mediators released by each cell affect each other in liver regeneration. Therefore, in the early 1970s, a method was developed to isolate, purify, and culture hepatocytes by using collagenase to perfuse the liver. This technique made it possible to study the intracellular signaling pathways for the proliferation of hepatocytes. As a result, it is now believed that liver regeneration does not initiate and progress by a single humoral factor, but rather by the interaction of a number of growth-promoting factors that exert a vigorous regenerative capacity.The hepatocytes are isolated from normal livers by the two-step in situ collagenase perfusion technique devised by Seglen to facilitate disaggregation of the adult rat liver . The hepAfter 3 h of incubation, the culture medium is replaced with a serum-free, defined medium, and the incubation is continued for an arbitrary period of time (0–21 h). Under the serum-free, defined culture conditions, the various effects of growth regulators on hepatocyte proliferation can be observed when different types of growth factors (and candidate substances) and signal-transducing factor inhibitors are added alone or in combination .0) and have stopped proliferation. However, in our culture system, the cell density at the time of cell seeding is relatively low, and the concentration of dexamethasone is reduced without adding insulin or EGF, which are considered essential for cell survival. By doing so, we have found that the effects of individual growth factors on hepatocyte proliferation can be observed much earlier than before.Under normal physiological conditions, hepatocytes are in the quiescent phase of the cell cycle  increases significantly after about 3–4 h A,B.We also found that there are many factors that promote hepatocyte proliferation, but there are few factors that can strongly inhibit the effects of these hepatocyte growth-promoting factors . Recentl2, 5-HT, and GH, which indirectly promote hepatocyte proliferation by secreting autocrine factors. In contrast, some factors, such as transforming growth factor-β1 (TGF-β1) and glucocorticoids, strongly suppress mitogen-induced hepatocyte proliferation . The physical properties, receptors, intracellular signaling pathways, and bioactive characteristics of representative hepatocyte growth-promoting and growth-inhibitory factors are briefly summarized below , and IGF-I. Co-mitogens, by themselves, do not promote hepatocyte proliferation, but, when used in combination with mitogens, the co-mitogens enhance the activity of the mitogens, and these include adrenergic α- and β-agonists and glucagon. In addition, there are many indirect mitogens, such as TNF-α, IL-1β, prostaglandin (PG) Eed below .EGF is a protein with a molecular weight of approximately 6 kDa, and it has three disulfide bonds. EGF binds to the EGF receptor (EGFR) and activates intracellular signaling pathways by phosphorylating the receptor tyrosine kinase (RTK); the EGF receptor is phosphorylated in vivo 30–60 min after PHx . IL. IL24]. TNF-α is a protein consisting of 157 amino acids and is a potent proinflammatory cytokine similar to IL-1 and IL-6. TNF-α promotes the proliferation of hepatocytes via TNF-α type 1 receptors . The majWe investigated the proliferative effects of TNF-α on hepatocytes in culture. The results showed that TNF-α alone promotes DNA synthesis and the proliferation of hepatocytes and that its effects are mediated via its autocrine secretion of TGF-α from the hepatocytes  25]..25].2. The physiological process involves constitutive COX-1 expression, while the inflammatory process involves inducible COX-2 expression. Biosynthesized PG products exhibit a variety of physiological effects, but inactivation is relatively rapid. PGs act on cells of the immune system to enhance inflammatory responses  by promoting the release of IL-1 and TNF-α. PGI2 is produced by vascular endothelial cells, regulates blood flow, and inhibits platelet aggregation. It has been reported that PGs stimulate hepatocyte DNA synthesis in culture [Prostaglandins (PGs) are low-molecular-weight local hormones. The receptors for PGs include EP, IP, and TP types, all of which are G protein-coupled. PGs are synthesized under the influence of cyclooxygenase (COX) from arachidonic acid and excised from the plasma membrane by phospholipase A culture .2 and PGI2, which promote DNA synthesis and the proliferation of hepatocytes. As a result, we found that both PGE2 and PGI2 indirectly promote DNA synthesis and the proliferation of hepatocytes via the secretion of the autocrine factor TGF-α from the hepatocytes  cells in the small intestinal epithelium and acts as a local hormone. 5-HT is stored in platelets and released upon stimulation and is involved in blood coagulation by promoting platelet aggregate formation (hemostatic thrombus). 5-HT has been reported to be involved in the promotion of liver regeneration [5-HT is a low-molecular-weight substance with receptor subtypes designated 5-HTneration . That is2B/Gq/phosphoinositide-specific phospholipase C (PLC)/Ca2+ pathway. We then found that the secreted TGF-α directly promotes DNA synthesis and hepatocyte proliferation  priming, 2) progression, and (3) termination. It has been recognized that liver regeneration begins early in each of these phases due to the interplay of numerous regulatory factors [ priming, progress2 receptors enhance the proliferative effects of EGF and HGF separately. Then, it is conceivable that transported endocrine mediators, either locally or somewhat distantly via the bloodstream, serve as signals to enhance the proliferation of hepatocytes at an early stage in vivo  to phagocytose and remove necrotic tissues. In addition, an early hemostatic response occurs at the site of the vascular injury. In other words, platelets adhere and release stored mediators  to promote the aggregation reaction. The platelets also release their own growth factors, such as HGF and PDGF. PGs and 5-HT, which are involved in inflammation and hemostasis, have been shown to act as indirect growth-promoting factors mediated by autocrine factors in vitro  17,26,2,226,27. Moreover, we also found that the branched-chain amino acid leucine and vitamin C stimulate hepatocyte proliferation via an autocrine mechanism ,30. HoweTGF-β1 strongly inhibits the hepatocyte proliferation-promoting effects of EGF, HGF, and TGF-α in culture ,32. In vRecently, the molecular mechanisms of action of various factors that regulate hepatocyte proliferation in vitro have been gradually elucidated. Based on these results, the in vivo mechanisms of liver regeneration are also becoming better understood. By integrating the results of these basic biological studies, we hope to extract useful insights that can be appropriately applied to several clinical issues  .


To study the protective effect and mechanism of icaritin (ICT) in a SH-SY5Y cells with virus-loaded TAR DNA-binding domain protein 43(TDP-43) by examining the effect of ICT on the expression of autophagy-related proteins in TDP-43-infected SH-SY5Y cells.A TDP-43-induced neuronal cell injury model was established by transfecting well-growing SH-SY5Y cells with virus loaded with the TDP-43 gene. The changes in cell viability were detected by the CCK-8 method. After successful transfection, the establishment of the model was verified by real-time quantitative PCR (qPCR) and Western blot methods. After the cells were subjected to drug intervention with ICT, the changes in the expression levels of TDP-43, cleaved Caspase-3, LC3 II/I, Beclin-1 and p62 were detected by Western blotting.After ICT intervention, it was found that compared with that of the TDP-43 group, the cell viability of the TDP-43+ICT group increased, the expression level of TDP-43 decreased, and the expression levels of the apoptotic protein cleaved Caspase-3, autophagy protein Beclin-1, and LC3-II/I decreased, while the expression level of the autophagy protein p62 increased.ICT has a protective effect on the SH-SY5Y cell injury model transfected with TDP-43. This protective effect may be related to reducing the protein expression of TDP-43 and inhibiting autophagy. TAR DNA-binding domain protein 43 (TDP-43) is a highly conserved rib nucleoprotein body consisting of 414 amino acids located on chromosome 1p36, which contains six exons. TDP-43 plays multiple roles in regulating gene expression at the transcriptional and translational levels . When exIcaritin (ICT) is a flavonoid compound and is a metabolite of icariin (ICA), both of which are the active ingredients in the extract of epimedium . The molTherefore, combined with the previous results of this research group, TDP-43-transfected SH-SY5Y cells were the research focus of this study and we used ICT for drug intervention in SH-SY5Y cells carrying the viral TDP-43 protein. Then, we initially explored whether ICT has a protective effect on nerve cell damage caused by TDP-43 transfection. At the same time, it was clear that it exerts a protective effect through the autophagy pathway, which provides a certain basis for the treatment of TDP-43 proteinopathy and drug development.2 culture flasks and cultured in a 5% CO2 incubator at 37 °C. The medium was replaced every 24–48 h and was composed of DMEM-F12, 15% fetal bovine serum, and 1% penicillin. When it grew to occupy 80–90% of the bottom of the bottle, the cells were passaged. The packaged lentivirus was transfected into cells, 200 µL of virus-containing medium was added to each well of a 96-well plate, and 1 ×104 cells were inoculated on the plate at an MOI of 0–60. The cell morphology was observed after 12 h of culture and the best MOI was selected.SH-SY5Y human neuroblastoma cells donated by the Collection Center of Wuhan University were inoculated into 25 cm4/mL with culture medium, and the suspension was seeded in a 96-well plate with 200 µL per well. After culturing for 1 day, the medium was changed, 10 µL of CCK-8 solution was added to each well, and the cells were incubated in a 37 °C incubator for 2 h. Then, a Synergy HTX microplate reader was used to measure the absorbance at 450 nm and calculate the cell viability.The CCK-8 assay was used to detect cell viability. The cells were grown under good conditions, the cell concentration was diluted to 5 ×10First, RNA was extracted, 200 µL of TransZol Up was added to the successfully transfected cells, 40 µL of chloroform was added and shaken for 30 s after the precipitate disappeared, and the mixture was allowed to stand. Then, 500 µL of CB9 and 500 µL of WB9 were added to the tube and discarded by centrifugation. The supernatant was finally added to 50 µL of RNase-free water, placed in an RNase-free tube and centrifuged to extract RNA. At a constant temperature of 65 °C, the mixing solution consisting of the primers shown in The cells were placed on ice, RIPA lysis buffer was added, and the cells were lysed on ice for 30 min. After centrifugation, the supernatant was taken as the protein sample. Then, the BCA method was used to determine the protein concentration. A standard curve was drawn at 562 nm on the microplate reader, and the blank of the standard curve was used as a control to determine the protein content of the sample. The protein was placed in boiling water at 100 °C and heated for 5 min to denature the protein. The separation gel and concentrated gel were selected according to the molecular weight of the target protein. Each 12% SDS–PAGE gel was added to 25 µg of total protein for separation. The concentrated gel electrophoresis voltage was set to 90 V, and electrophoresis was performed for approximately 30 min. After the same level, the separation gel electrophoresis voltage was set to 120 V, electrophoresis was performed for approximately 90 min, the electrophoresis was stopped when the sample band reached the bottom layer of the separation gel, and then transmembrane the protein. The nitrocellulose (NC) membrane was sealed with 5% skimmed milk powder for 1 h and washed with TBST three times after the sealing was completed, each time for 10 min. The NC membrane was incubated with the primary antibody overnight. After washing the membrane, the NC membrane was incubated with HRP-anti-rabbit antibody for 2 h. A color developing agent was added to the film and exposed in a fully automatic chemiluminescence gel imager, and then protein detection was performed after exposure.P < 0.05 was considered statistically significant.All statistical analyses were performed with IBM SPSS Statistics 22.0. If the data met normality, the mean ± SEM was used. The difference between the means of more than two groups was analyzed by one-way analysis of variance (ANOVA). If there were two variables in the statistical plot, two-way ANOVA was performed. When ANOVA showed significant differences, pairwise comparisons of means were made by Bonferroni’s post-hoc t test with correction. A value of Transfection was carried out with different concentrations of virus MOI. When the MOI was 30, the cell dendrites were clear, the growth state was good, and the transfection efficiency was high. When the MOI was 10 and 20, the cell state was good, but the transfection efficiency was low; when the MOI was 40, the transfection in the staining efficiency was good, but the cell status was less than when the MOI was 30, and the amount of cell death was greater than when the MOI was 30; the cell status was poor, the amount of cell death was greater and the transfection efficiency was low when the MOI was 50 and 60. Therefore, in this experiment MOI 30 was selected for transfection, as shown in After the cell model was established, ICT was added at different concentrations, including 0.0, 0.1, 1.0, and 10.0 µmol/L, to the TDP-43 group, and ICT was added for 12 h, 24 h, and 48 h to detect the cell viability of each group. The results showed that the cell viability of each group was the highest when the ICT concentration was 1.0 µmol/L and the incubation time was 48 h, as shown in The expression levels of LC3-II/I, cleaved Caspase-3, Beclin 1 and TDP-43 in the TDP-43 group were significantly higher than those in the Con group, and the expression levels of LC3-II/I, cleaved Caspase-3, Beclin 1 and TDP-43 in the TDP-43+ICT group after 48 h of ICT treatment were clearly decreased than those in the TDP-43 group. At the same time, the expression of p62 in the TDP-43 group was lower than that in the Con group, and the expression of TDP-43+ICT after 48 h of ICT was significantly higher than that in the TDP-43 group, as shown in TDP-43 is a member of the nuclear heterogeneous ribonucleoprotein family and includes two domains of RNA recognition motifs located on both sides of the N-terminal and C-terminal regions. The C-terminus affects the binding of heterogeneous and homologous proteins, while the N-terminus is mainly involved in the formation of homodimers . TDP-43 Autophagy is a regulated self-degradation process in cells that is highly conserved in many species, such as mammals and yeast. Autophagy avoids the accumulation of misfolded or aggregated proteins, which is essential to maintain the normal function of the central nervous system. The autophagy process includes the process of initiating autophagy after receiving the signal, forming autophagosomes, autophagolysosomes formed by the combination of lysosomes and autophagosomes, degrading autophagolysosomes into small molecules and reusing decomposition products . The ini1−42 contents (Previous research by our group showed that ICT can inhibit β-amyloid precursor protein cleavage enzyme 1 (BACE 1) to block the production of amyloid precursor protein (APP) to reduce the secretion of Aβ . We founcontents . Our grocontents .Moreover, studies have shown that ICA can act on diseases through autophagy and other pathways . TherefoTherefore, in this study, after transfecting SH-SY5Y cells with a virus carrying TDP-43, the cell morphology shrank, axons and dendrites were shortened or even disappeared, and cell death occurred. At the same time, the mRNA and protein expression of TDP-43 was measured. A significant increase in cell viability measured by CCK-8 decreased, and the cells in this state could better simulate the damage caused by TDP-43 in SH-SY5Y cells. Therefore, this experiment selected a virus carrying TDP-43 to transfect SH-SY5Y cells. Cell-based methods were used to establish cell models for research. Based on related literature and preliminary experiments, it was determined that the duration of virus transfection has different effects on the cells. Therefore, in this experiment, the cell viability of SH-SY5Y cell models after different transfection times was measured, and it was found that the cell viability was 12 h after transfection. The cell morphology changes were viewed under the microscope. At the same time, the cell viability was restored after ICT was added, and the cell morphology was partially restored to normal. Considering that TDP-43 has a greater impact on cytotoxicity, in this experiment the virus that carried TDP-43 was selected. SH-SY5Y cells were stained for 12 h as the experimental conditions for establishing cell models.After the model was successfully established, according to the experimental results of this research group and previous experiments, the cell viability was the best when the SH-SY5Y cell model transfected with TDP-43 was treated with ICT at a concentration of 1 µmol/L for 48 h. After ICT drug intervention for 48 h, the cell morphology and growth status were significantly improved compared to before the drug was added. CCK8 assays showed that SH-SY5Y cell viability increased significantly after TDP-43 transfection compared with before the drug was added. Western blot results showed that after drug intervention, the expression of TDP-43 in the TDP-43+ICT group was lower than that in the TDP-43 group. At the same time, the expression of the apoptosis protein cleaved Caspase-3, autophagy protein Beclin-1, and LC3-II/I decreased after adding ICT, and the expression of the autophagy protein p62 increased, suggesting that ICT may affect the protein expression of cleaved Caspase-3, Beclin-1, LC3-II/I and p62, and it also has a protective effect on the autophagy pathway in the injury model of TDP-43-transfected SH-SY5Y cells.in vivo level and explore its protective mechanism to provide new ideas for the development of novel drugs for the treatment of TDP-43 proteinopathy.In summary, this study used TDP-43-carrying virus to transfect SH-SY5Y cells and establish a TDP-43 transfection cell injury model. After ICT intervention, it was found that ICT reduced the autophagy pathway Beclin-1 and LC3-II/I protein expression by reducing cleaved Caspase-3 and increasing the expression level of p62 protein, thereby protecting the TDP-43-induced cell injury model. However, the current research on ICT is still in the preliminary stage, and the specific mechanism of its protective effect on the TDP-43-induced cell injury model is still unclear. This study explores the protective effect of ICT at the cellular level. In a follow-up study, our group will further verify its protective effect at the ICT has a protective impact on the SH-SY5Y cell injury model transfected with TDP-43. This protective effect may be relevant to regulating TDP-43, inhibiting the expression of cleaved Caspase-3, Beclin-1 and LC3-II/I and promoting the expression of p62 protein.10.7717/peerj.13703/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.13703/supp-2Supplemental Information 2Click here for additional data file.