Hydrogels are characterised focusing on the contribution of the nanomaterial to the polymer network. Our results suggest that graphene is not a mere embedded nanomaterial within the hydrogels, rather it represents an intrinsic component of these networks, with a specific role in the emergence of these structures. Moreover, a hybrid hydrogel with a graphene concentration of only 0.2 mg mL−1 is used to support the growth of cultured brain cells and the development of synaptic activity, in view of exploiting these novel materials to engineer the neural interface of brain devices of the future. The main conclusion of this work is that graphene plays an important role in improving the biocompatibility of polyacrylamide hydrogels, allowing neuronal adhesion.In tissue engineering strategies, the design of scaffolds based on nanostructures is a subject undergoing intense research: nanomaterials may affect the scaffolds properties, including their ability to interact with cells favouring cell growth and improving tissue performance. Hydrogels are synthetic materials widely used to obtain realistic tissue constructs, as they resemble living tissues. Here, different hydrogels with varying content of graphene, are synthesised by  In general, 3D scaffolds have been designed to accomplish important features for implantable prostheses: biocompatibility, stability and homogeneity at the microscale, conductivity and controlled mechanical properties. Biocompatibility is in general defined as “the ability of a material to perform with an appropriate host response in a specific application”6, although new definitions, based also on cell function, are increasingly proposed7.Adapting the microenvironment of three-dimensional (3D) tissue-scaffolds to regulate neural cell adhesion or tissue growth is essential to design new therapies for neurological applications. In these developments, 3D systems are being highly investigated as they are showing, when compared 9. These nanomaterials represent biocompatible substrates, and they can be functionalised with polymers to modulate neuronal survival and growth10. Graphene has also attracted the attention of neuroscientists, demonstrating biocompatibility with neurons in 2D interfaces, being able to preserve the basal physiological level of neuronal activity13. Very interesting examples are related to 3D porous graphene structures14, which have been used as new scaffolds for neural stem cells in vitro. For example, Li and co-workers15 found that the graphene-foam not only supports cells growth, but also keeps them in an active proliferation state better than 2D graphene films.Recently, emerging carbon-based nanomaterials, with their unique physicochemical properties, have opened up additional and novel opportunities for neuronal tissue engineering developments. Among these, carbon nanotubes have been the most inspiring ones18. Hydrogels are the best candidates to play the role of extracellular matrix for 3D cell culture19, mainly due to their aqueous environment and their porous structure, which make it possible to link and maintain proteins in a functional state21. This fact allows improving cell culture development by anchoring, for example, growth factors to the polymer network, which makes these systems even more similar to the in vivo condition.Hydrogels - 3D polymer networks that possess high water contents - have been used to obtain more realistic tissue constructs because of their ability to mimic the nature of soft tissues23. In this direction, many efforts have been made to fabricate hybrid graphene-based hydrogels with enhanced properties, mainly using graphene oxide (GO)25, graphene oxide peroxide (GOP)26, or even reduced graphene (rGO) from GO28. However, most of these studies do not provide details about the role of the nanomaterial within the network, moreover GO sheets are insulating and its electronic properties differ substantially from those of graphene. As a matter of fact, there are almost no examples in literature of pristine graphene-based hydrogels29, which are mainly limited due to the difficulties to obtain pristine graphene aqueous solutions in a simple, scalable and cost efficient way.Hydrogels can be obtained from a wide range of materials; however, one of the main disadvantages of this kind of polymers is their low mechanical strength. Over the last years there has been a growing interest in reinforcing polymeric hydrogels with nanosized materials to improve and adequate their poor mechanical consistence. Remarkably, the nanomaterials embedded in a cross-linked polymer network not only make the system stiffer and tougher, but can provide novel features, adding synergistic benefits to the hybrid 3D materialset al.30 chemically modified a polysaccharide with pendant methacrylate groups, which acted as a physical exfoliant of graphite to produce multi-layer graphene flakes, followed by co-polymerization with methacrylic acid and a cross-linker. Toumia and co-workers31 dispersed graphite in an aqueous solution of a commercial amphiphilic surfactant containing aromatic moieties (PEG-PEPEMA) which was chemically grafted to a polyvinylalcohol (PVA) hydrogel network. Shim et al.32 probed the effects of different PVA and dextran polymers containing phenyl or pyrene units for the physical exfoliation of graphite in water and subsequent hydrogelation of the obtained suspensions with glutaraldehyde. Finally, Ahadian and co-workers developed a friendly aqueous route to graphite exfoliation down to few-layer graphene sheets by use of bovine serum albumin in water as exfoliant33, and used it to fabricate hydrogel nanocomposites made of methacrylated gelatin.With the latest advances in graphene research, one can find a handful of recent works that timely address this conceptual approach from top-down cleavage of graphite. Fan 2+ imaging experiments.Here, we present a new class of graphene-based hydrogels and we show their ability to support the growth of living primary neurons. The produced nanocomposites were compared to the hydrogels in the absence of nanomaterials and were fully characterised. The presence of graphene brought about a noticeable improvement in the properties of the hybrid materials and varying the graphene amounts allowed to study its role in the resulting features. Hippocampal neurons and astrocytes developed efficiently only in the graphene-doped hydrogels. In these graphene hybrids, the cultured neurons gave rise to active synaptic networks as observed by immunofluorescence microscopy and CaN,N′-methylenebisacrylamide (MBA), potassium peroxodisulfate (KPS) and melamine were purchased from Sigma Aldrich.Graphite powder was acquired from Bay Carbon Inc. (SP-1 reference). Acrylamide (Am), 34. In a typical experiment, a graphite/melamine mixture (1:3) (30 mg) was ball-milled at 100 rpm for 30 minutes using a Retsch PM100 planetary mill. The resulting solid mixture was dispersed in water (20 mL) to produce a dark suspension, which was then left to settle down for five days while some precipitate (mainly pure or poorly exfoliated graphite) segregated from the liquid. The liquid fraction with stable sheets in suspension was carefully extracted. Melamine was removed by washing the extracted liquid with hot water, leading to few-layer graphene sheets almost without defects. The obtained graphene dispersions were stable at room temperature for some weeks. The zeta potential of the dispersion was measured at 25 °C using Milli-Q water and CRISON buffer solution (pH = 7) by BIC 90Plus analyser.The exfoliation of graphite was carried out using a ball-milling procedure developed in our group−1) together with the MBA (0.2 mg mL−1). The solution was homogenised by stirring and mild sonication. Then, KPS was added  and polymerization took place at 75 °C for 1 hour. The resulting materials were immersed in water for at least four days, and the water was replaced every day in order to remove any unreacted monomer and initiator molecules.Pure polyacrylamide hydrogels were prepared using Am, MBA as cross-linker and KPS as initiator. Am was initially dissolved in ultrapure Milli-Q water (200 mg mL−1 and 2 mg mL−1).A similar process as the previously described for the preparation of AM was followed to synthesise the nanocomposite, adding in this case all the reactants to the aqueous graphene dispersion. Graphene water dispersions with different concentrations were used , which analysed 9 × 9 µm2 slices of sample and depth steps of 1 µm until reaching 10 µm. According to the dimensions of the probed volume and given that we registered 6200 spectra during the mapping, we end up with a pixel size of 0.13 µm3.The whole set of spectra obtained was fitted to the G-band region in three dimensions.Raman spectra were recorded with an InVia Renishaw microspectrometer equipped with a 532 nm point-based laser. Power density was kept in all cases below 1 mW/µm−1 (cell load = 100 N). The extent of the deformation was measured in terms of compression ratio which equals the ratio of the initial height of the sample to the final height of the sample. The extent of the deformation was also measured in terms of strain, which equals the ratio of the displacements of the samples. After holding the constant deformation, the deformed hydrogel was removed from the press. This behaviour was represented in the typical stress-strain curves from which it was possible to obtain the Young’s modulus. In this case that value was calculated between 15% and 20% of strain. Moreover, fatigue behaviour was studied using the aforementioned program in a dynamic way, reaching 100 compressive cycles with each sample. Creep experiments were conducted on hydrogels at ambient conditions with a Mecmesin Multitest 2.5-i dynamic mechanical analyser. Cylindrical disks of gels (×3) with a diameter of 1.5 cm and an initial thickness of 1.5 cm were molded. Hydrogel disks were uniaxially compressed between two plates. The deformed hydrogels were then held under constant applied load at 15% compressional strain for an extended period of time.Compressive tests were performed on hydrogels at 25 °C with a Mecmesin Multitest 2.5-i dynamic mechanical analyser. Cylindrical disks of gels (x6) with a diameter of 1.5 cm and an initial thickness of 1.5 cm were molded. Hydrogel disks were uniaxially compressed between two plates at the rate of 6 mm min37. All procedures were approved by the local veterinary authorities and the SISSA ethical committee, in accordance with the Italian law (decree 26/14) and the UE guidelines (2007/526/CE and 2010/63/EU). The use of animals was approved by the Italian Ministry of Health. All efforts were made to minimise suffering and to reduce the number of animal used. Hippocampi were isolated from the rest of the brain and upon enzymatic treatment and neurons were mechanically dissociated. Cells were plated on three different substrates: poly-L-ornithine-coated, pure polyacrylamide hydrogel (AM) and 0.2 mg mL−1 graphene hydrogels (AMG0.2). Before using for culturing, both hydrogels were sliced at 300–400 µm thickness and mounted on the glass coverslip  by a thin adhesive layer of Polydimethylsiloxane (PDMS)5. One hour prior to plating, hydrogels were treated with an air plasma-cleaner in order to facilitate cell adhesion and at the end sterilised with an UV lamp. Briefly, enzymatically dissociated hippocampal neurons37 were suspended in the culture medium consisting of MEM (Gibco), supplemented with glucose (35 mМ), Apo-Transferrin (1 mМ), HEPES (15 mМ), insulin (48 µМ), Biotin (3 µМ), Vitamin B12 (1 mМ) and Gentamicin (Gibco) (500 nМ), Fetal Bovin Serum . Cultures were incubated at 37 °C, in a humidified atmosphere with 5% CO2 in the same culture medium. Cultures were used for experiments after 8–10 days in vitro (DIV).Primary hippocampal cultures were prepared from postnatal (2–3 days) rats as previously reported2+ measurements, cultures were fixed in 4% formaldehyde  in PBS for 30 min, at room temperature. Cells were then permeabilised with Triton X-100 (1%) for 45 min, blocked with FBS (5%) in PBS (blocking buffer) for 45 min at room temperature and incubated with primary antibodies for 45 min. The primary antibodies used were: rabbit polyclonal anti-β-tubulin (1:250 dilution) and mouse monoclonal anti-GFAP (1: 500 dilution). After the primary incubation and PBS washes, cultures were incubated for 45 min with Alexa 594 goat anti-rabbit , Alexa 488 goat anti-mouse  and with DAPI  to stain the nuclei. Samples were mounted in Vectashield (Vector Laboratories) on 1 mm thick coverslips and imaged using a confocal microscope . Both 2D-polyornithine and hydrogels substrates were investigated at 20x magnification and serial confocal planes (z-stack) were acquired every 500 nm. The Z profile reconstructions of the images were performed offline using the image-processing package Fiji38.After Ca2+ measurements, cultures were loaded with cell permeable Ca2+ dye Oregon Green 488 BAPTA-1 AM (Molecular Probes); stock solution of the Ca2+ dye (4 mМ) was prepared in DMSO and cultures were incubated with a final concentration of 4 µМ for 30 min at 37 °C in the cell culture incubator. Samples were then placed in a recording chamber mounted on an inverted microscope (Nikon TE-200) where they were continuously superfused by a recording solution of the following composition (in mМ): 150 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose (pH adjusted to 7.4 with NaOH). The loaded cultures were observed with 40x objective (0.60 NA). Recordings were performed form visual fields (120 × 160 µm2) containing on average 6 ± 2 neurons. Prior to recording Ca2+ signals, we selected the cells by drawing regions of interest (ROIs) around cell bodies. Images were continuously acquired by a Till Photonics Till-Imago System, exciting the Ca2+ dye with a 488 nm wavelength light generated by a monochromator device equipped with integrated light source . Images were acquired continuously for 1800 s (200 ms exposure time) by a cooled slow-scan interline transfer camera  operating on 60 × 80 pixels binning mode. The imaging system was controlled by an integrating imaging software package  using a personal computer. In order to induce rhythmic bursts, bicuculline methiodide (a blocker of GABAA receptors) (20 µМ) was bath-applied after 10 minutes recording; at the end of each experiments, TTX (1 µМ)  was added to the recording solution to confirm the neuronal nature of the recorded signal. Recorded images were analysed off-line both with the Clampfit software  and Igor Pro Software . Intracellular Ca2+ signals were expressed as fractional amplitude increase (ΔF/F0 where F0 is the baseline fluorescence level and ΔF is the rise over baseline); we determined the onset time of neuronal activation by detecting those events in the fluorescent signal that exceed at least five times the standard deviation of the noise. We then computed the difference between consecutive onset times, to obtain the inter-event interval (IEI). All data are presented as mean ± standard deviation (SD) of the mean (n is the number of cells). Statistical significance for IEI was calculated with Student’s t-test .For CaAll data generated or analysed during this study are included in this published article (and its Supplementary Information files).34. Graphene dispersions in water were obtained and thoroughly characterised  and N,N′-methylenebisacrylamide (MBA) were chosen as the initiator and the cross-linker respectively. Figure Graphene-based hydrogels are formed by −1) were synthesised. Figure A chemically cross-linked polyacrylamide gel (AM) prepared without graphene and a series of hybrid graphene-polyacrylamide gels  and the results show structural changes in the hydrogels with graphene compared to the sample without nanomaterial  are used in similar proportions. Moreover, we can exclude a relevant role of the residual melamine in the hydrogel structure, because the amount of melamine in the final gels is negligible. Hydrogels can be chemically or physically crosslinkedial Fig. . Three wial Fig. . The fir46, are ascribed to the different stretching (ν) and bending (δ) vibrations of the polyacrylamide skeleton, being the most intense the methylene and C-N stretchings, and bending of amino groups. Upon acquisition of Raman spectra from the hybrid graphene hydrogels, the characteristic features of graphene are visible, mainly the disordered carbon (D-band), the sp2 tangential mode (G-band), and the second-order vibration mode (2D-band). Raman spectroscopy was also employed to reveal the state of dispersion of graphene within the hydrogel matrix, throughout three-dimensional mappings at a micrometer scale . The final swelling degree decreases when increasing graphene content in the hybrid hydrogels Fig. . The mos48. Compressive tests were conducted on all the samples. For practical reasons, we only show the compressive Young’s modulus (E) for samples AM, AMG0.2 and AMG2, but, although not shown, samples AMG0.05 and AMG0.1 presented identical values in the elastic moduli as AMG0.2. These values are in the range of other materials already used for 3D neuronal growth50. The Young’s modulus of the hybrid prepared in the presence of a dispersion of 2 mg mL−1 of graphene is considerably higher in comparison with the other two hydrogels. The fatigue behaviour was studied only for AM and AMG2 gels, in order to evaluate if the introduction of graphene could deteriorate the gel and affect the fatigue response. In this case, the compressive toughness of the materials was measured from the area of the stress-strain curve of non-stopped 100 compressive cycles. It is evident that the incorporation of graphene in the hydrogel increases the toughness of the material  to address its potential interference with neuronal growth. The final content of melamine in this hybrid hydrogel is only 5.4 · 10−4 wt%, which is not comparable to the amount of cross-linker used to form the nanocomposite sample (0.1 wt%). The intrinsic role of graphene would be thus revealed, with potential interest in further scalability and applications. We cultured cells in three different conditions: in traditional 2D polyornithine-layered substrate (control)4 and in the hydrogels, cross-linked in the presence (AMG0.2) or in absence (AM) of graphene. After 10 DIV we compared neuronal growth by immunofluorescence techniques and confocal microscopy. As shown in Fig. 4. Despite repeated trials (3 different culture series) hippocampal cells when seeded on AM hydrogels did not adhere to the structure and, even more, barely grew. We detected only occasionally few clusters of small β-tubulin III-positive cells, virtually deprived of outgrowing axons, as shown in Fig. 4. The zeta-stack reconstruction revealed that hippocampal cells were able to grow within the porous niches present on the surface of the graphene-based hydrogels, but they did not migrate deeper in the structure4. This is further sustained by the reconstruction of Fig. We tested the 3D polyacrylamide scaffold efficacy to support neuronal cell growth and activity and we compared one of the nanocomposite sample (AMG0.2) to its blank AM counterpart. Although different in the pore size ranges, both materials have pores within dimensions known to permit cell survival in culture4. At 8–10 DIV neurons are usually mature and active and display synaptic signaling spontaneously, traditionally mediated by glutamate and GABAA receptor mediated-synaptic pathways36. With our imaging set up we sampled representative regions of 120 × 160 μm2. Neurons were stained with the membrane permeable Ca2+ dye Oregon green 488 BAPTA-1 and simultaneously visualised within the sampled area. On average, 6 ± 2 fluorescent cells were detected in the recorded field. In Fig. 2+ activity usually appeared as spontaneous bursts of activity fully blocked by TTX (1 µM) applications . TTX is a well-known blocker of voltage-gated fast Na+ channels54, responsible for the generation of action potentials. In our recordings, spontaneous Ca2+ activity was detected in 36% of control cells visualised in each field  and, similarly, in 25% AMG0.2 cells  of 32 ± 7 s (n = 21 cells) that is similar to that measured in AMG0.2 cultures  application. This antagonist of synaptic inhibitory components is known to potentiate rhythmic activity patterns56. In fact, in both groups, in respect to spontaneous activity, the addition of bicuculline significantly decreased the IEI average values 4. In Fig. 2+ episodes brought about by bicuculline in active cells that, on average, display IEI of 22 ± 4 s in control (n = 21 cells) and, similarly, of 16 ± 6 s in AMG0.2 cultures , therefore indirectly affecting neuronal growth.The main result obtained from our neurobiological studies is that the presence of graphene inside the structure of the hydrogel is crucial for allowing cell adhesion and the formation of mature and active synaptic networks. In fact, only sporadic clusters of few neurons can be found in the blank hydrogel, in the absence of proper axon formation. Ultimately, neurons only grew in the scaffold synthesised by using a graphene dispersion in water with a concentration of just 0.2 mg mL4. This is further suggested by the observed similarity in the activity patterns when recorded by calcium imaging in the two culturing conditions4. However, in both networks, episodes of calcium activity, whose neuronal and synaptic nature was supported by TTX experiments, were reflecting spontaneous synaptic activity. Based on these results, we are carrying out further experiments to adjust the pore size dimensions and interconnectivity in the graphene-based hydrogels in order to improve the growth and migration of neurons deeper within the sample, favouring by this way 3D neuronal network connections.The neuronal networks grown in control and AMG0.2 display basically similar Z projection (≈20 µm thickness), thus in the graphene-hybrid hydrogels, neurons did not develop in a genuine 3D geometryWe have designed hybrid graphene hydrogels as new 3D scaffolds for neuronal growth. Acrylamide was used as main monomer for the formation of the polymer networks and the radical polymerization was conducted in aqueous graphene dispersions of different concentrations. The scaffolds were thoroughly characterised, and the compressive moduli were measured, being higher for the sample that contains a noticeable amount of graphene. Moreover, no significant fatigue behaviour was observed in the studied samples, even in the hydrogel with the highest amount of graphene. The most important result observed from the neuronal studies is that neuronal networks are only visualised in the hybrid graphene hydrogel, but not in the scaffold without graphene. The mechanical properties of the scaffold do not seem to be critical for neuronal growth in the presence of graphene and thus an intrinsic beneficial role of graphene is revealed.Supplementary info


Programmed death-ligand 1 (PD-L1) is an immunosuppressor that plays an important role in cancer treatments. Although majority of the studies demonstrated that PD-L1 expression was regulated by cellular intrinsic and extrinsic controls, and IFN-γ was a key molecule of extrinsic control, other studies imply that other cytokines play important roles in PD-L1 expression. In this study, we investigated the regulation of PD-L1 by chemokine signaling pathway in gastric cancer (GC) cells.Bioinformatics was used to explore the PD-L1-related genes in GC and propose a hypothesis. PD-L1 and CXCR3 expression were detected by western blot in SGC7901 and MKN74 cell lines. Meanwhile, PD-L1 and CXCR3 expressions were immunohistochemically assessed for their relevance. Moreover, PD-L1, pSTAT3 and pAkt were detected after treatment with CXCL9/10/11. Furthermore,PD-L1, pSTAT3 and pAkt were evaluated after blocking chemokine signaling in SGC7901 cells.vivo experiments. CXCR3 and PD-L1 were expressed in GC cell lines and tissues, and the expression of CXCR3 and PD-L1 was positively related. PD-L1 was upregulated after treatment with CXCL9/10/11, accompanied by activation of STAT3 and Akt. After blocking chemokine signaling, upregulation of PD-L1 and activation of STAT3 and Akt were diminished.Based on online database analysis, CXCL9/10/11-CXCR3 is proposed to upregulate PD-L1 expression by activating the STAT and PI3K-Akt pathways. This hypothesis was confirmed by in vitro and CXCL9/10/11-CXCR3 upregulated the expression of PD-L1 by activating the STAT and PI3K-Akt signaling pathways in GC cells. There was a significant positive correlation between the expression of PD-L1 and CXCR3 in gastric cancer patient tissues.The online version of this article (10.1186/s12885-018-4384-8) contains supplementary material, which is available to authorized users. Although the incidence of gastric cancer (GC) has greatly reduced in developed countries, it remains the third leading cause of cancer-related deaths worldwide , and oneCancer immunotherapy has shown major advancements during the past few years. Immune checkpoints such as cytotoxic T-lymphocyte-associated antigen CTLA-4) and programmed death-ligand 1 (PD-L1) can suppress the activity of T-lymphocytes, which could recognize and eliminate cancer antigens  and prog–7. In 20Majority of the recent studies have shown that PD-L1 expression is regulated by intrinsic and extrinsic mechanisms in cancer cells. Intrinsic immunologic tolerance associates carcinogenesis with PD-L1 expression, such as overexpression of ALK and Ki-67, activation of Kras, and inactivation of PTEN and Lkb1 –14. AddiIn the present study, bioinformatics was used to explore the PD-L1-related genes in GC and propose a hypothesis. In vitro experiments were conducted to confirm the hypothesis, which clarified that CXCL9/10/11-CXCR3 upregulated PD-L1 expression by activating the STAT and PI3K-Akt pathways in SGC7901 and MKN74 GC cell lines. These findings suggest a novel mechanism regulating the immune-evading factor PD-L1.https://gdc-portal.nci.nih.gov/). Another two mRNA expression profiles (GSE15459 and GSE62254) were downloaded from Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/). Above two datasets were acquired using Affymetrix Human Genome U133 Plus 2.0 Array. TCGA dataset included surgically resected 375 GC samples with complete data. Dataset GSE15459 included surgically resected 192 GC samples with complete data from the Singapore patient cohort [RNASEQV2 dataset was obtained from the Cancer Genome Atlas (TCGA)  < 0.25, the first 20 pathways were ranked as the “significant enrichment genes” according to the FDR value from low to high,if P = 0 and FDR = 0, the pathways should be included. The software R 3.2.3 was used to perform all the statistical analyses.The GSE62254, GSE15459 and TCGA-STAD-RNASEQV2 data were substituted into GSEA2.0.14 to analyze the effect of high and low expression level of CD274 on various pathways of biological pathways, and the reference gene set was set to the c2.cp.kegg.v5.1.symbols.gmt gene set in the MsigDB database of the GSEA website. The analysis was repeated 1000 times for each one according to the default weighted enrichment statistic method. In the GSEA, based on the condition of r > 0.6, genes were selected for further analysis. The database for annotation, visualization and integrated discovery (DAVID) was used to analyze list of genes derived from high-throughput genomic experiments. DAVID online tool [Based on TCGA dataset, the correlation coefficient r between CD274 and the expression of each gene was calculated by Spearman correlation analysis. Based on the condition of ine tool  for Geneine tool .CXCL9/10/11 were purchased from Pepro Tech (USA). The PD-L1 antibody was purchased from Cell Signalling Technology (USA). The CXCR3 antibody was purchased from ABCAM (USA). The CXCR3 antagonist AMG487 was purchased from Tocris (USA). The PI3K/AKT inhibitor LY294002 and the STAT3 inhibitor STATTIC were obtained from Sigma . The anti-STAT3, anti-pSTAT3, anti-Akt, anti-pAkt, anti-ERK, anti-pERK, and anti-GAPDH antibodies were obtained from Santa Cruz Biotechnology (USA).Human gastric cell lines SGC-7901 and MKN74 were obtained from the Type Culture Collection of the Chinese Academy of Sciences (China). Cells were grown in RPMI 1640  containing with 10% fetal bovine serum and 1% penicillin–streptomycin in a humidified atmosphere of 95% air and 5% CO2 at 37 °C .5 per well in 6-well plates and incubated overnight; Cells were treated with CXCL9/10/11 (100 ng/ml) for indicated times. Cells were lysed in lysis buffer. The method of Western blot was discussed in our previous study [Cells were seeded at 2 × 10us study .The siRNA sequences were from View solid biotechnology co., LTD . The AKT siRNA sequence was 50-CUCACAGCCCUGAAGUACUtt-30. The siRNAs were transfected with Lipofectamine 2000  per the manufacturer’s instructions.The files of 92 patients who underwent surgical resection of gastric cancer between 2006 and 2011 at the First Hospital of China Medical University, were included in our study. Patient information included age, gender, TNM stage, histopathologic type and metastases. All patients were not treated with any chemotherapy. This study was approved by the Human Ethics Review Committee of China Medical University; informed consent was obtained from all patients.Immunohistochemical staining was performed using a streptavidin-peroxidase procedure. CXCR3 and PD-L1 expression were investigated using antibodies against CXCR3  and PD-L1 . All other reagents were from Sigma. The specificity of all antibodies was confirmed by western blotting.PD-L1 and CXCR3 were detected by immunohistochemistry (IHC) as described previously. Staining was graded into four categories: 0 , 1+ (staining of 6–25%), 2+ (staining of 26–50%), 3+ (staining more than 50%). Tissues with scores of 2+, or 3+ were considered to be positive [P-value of 0.05 or less was considered to indicate a statistically significant difference. Spearman correlation analysis was used to determine the correlation of PD-L1 and CXCR3 expression.All statistical tests were performed using SPSS  17.0 computer software. Differences between two groups were evaluated by Student’s t-test. A Using the TCGA, GSE15459 and GSE62254 datasets, the pathways ranked as the “significant enrichment genes” were shown in Fig. A total of 81 genes that were closely related to PD-L1 were selected by Spearman correlation analysis , especially to I + II stage patients, but not correlated to gender, age, Lauren classification and lymph node metastasis of patients with gastric cancer. The expression of CXCR3 was correlated to gender (P = 0.025), especially to women patients, but not correlated to age, Lauren classification, TNM stage and lymph node metastasis , with statistical significance , gene mutations and cytokines. IFN-γ of the cytokine family is a key factor triggering PD-L1 induction in tumor cells. However, the specific mechanisms underlying PD-L1 expression remain largely unknown. Stimulation with IFN-γ, TLR ligands and LPS via MyD88, TRAF6, MEK, STAT1, NF-κB and PI3K-dependent pathways increased PD-L1 expression in different types of cells, such as inflammatory cells, fibroblasts and cancer cells –30. AddiCXCL9/MIG, CXCL10/IP10 and CXCL11/ITAC are members of the ELR-negative CXC chemokine subfamily, also known as IFN-γ-induced monocyte cytokines, IFN-γ-inducible protein 10 and IFN-γ-inducible T cell α chemokine –34. CXCRvivo experiments, which is an important factor of immune evasion in GC cells, and CXCL9/10/11-CXCR3 could regulate PD-L1 expression through STAT and PI3K-Akt signaling pathways in GC cells. Recently, molecular targeted therapy has created hope for advanced GC patients. Checkpoint blockers such as PD-L1 offer novel immunotherapy options for cancer patients. Based on our findings, CXCR3 could be a potential target in GC therapy. Further studies are required to confirm this conjecture.In summary, our findings suggest a regulatory mechanism of PD-L1 through data analysis, in vitro and Additional file 1:Table S1. The first 20 significantly enriched gene pathways in TCGA dataset. (XLS 18 kb)Additional file 2:Table S2. The first 23 significantly enriched gene pathways in GSE15459 dataset. (XLS 21 kb)Additional file 3:Table S3. The first 24 pathways significantly enriched gene pathways in GSE62254 dataset. (XLS 19 kb)Additional file 4:Table S4. 81 genes that had a close relationship to PD-L1 were selected for Gene functional classification after spearman correlation analysis. (XLS 25 kb)Additional file 5:Table S5. CXCL9/10/11 and CCL4 were grouped together after Gene functional classification in DAVID. (XLS 20 kb)


Glioblastoma Multiforme (GBM) is a lethal primary brain tumor with poor survival lifespan and dismal outcome. Surgical resection of GBM is greatly limited due to the biological significance of brain, giving rise to tumor relapse in GBM patients. Transactive response DNA binding protein-43 (TDP-43) is a DNA/RNA-binding protein known for causing neurodegenerative diseases through post-translational modification; but little is known about its involvement in cancer development. In this study, we found that nutrient deprivation in GBM cell lines elevated TDP-43 expression by a mechanism of evasion from ubiquitin-dependent proteolytic pathway, and subsequently activated the autophagy process. Exogenous overexpression of TDP-43 consistently activated autophagy and suppressed stress-induced apoptosis. The inhibition of autophagy in TDP-43-overexpressing cells effectively increased the apoptotic population under nutrition shortage. Furthermore, we demonstrated that HDAC6 was involved in the activation of autophagy in TDP-43-overexpressing GBM cell lines. The treatment with SAHA, a universal HDAC inhibitor, significantly reduced TDP-43-mediated anti-apoptotic effect. Additionally, the results of immunohistochemistry showed that TDP-43 and HDAC6 collaborated in GBM-tumor lesions and negatively correlated with the relapse-free survival of GBM patients. Taken together, our results suggest that the TDP-43-HDAC6 signaling axis functions as a stress responsive pathway in GBM tumorigenesis and combats nutrient deprivation stress via activating autophagy, while inhibition of HDAC6 overpowers the pathway and provides a novel therapeutic strategy against GBM. Glioblastoma (GBM), classified as Grade IV brain tumor by World Health Organization (WHO), is the most common and aggressive malignant primary brain tumor . GBM is TDP-43, also known as transactive response (TAR) DNA binding protein-43 (TARDBP), belongs to heterogeneous nuclear ribonucleoprotein (hnRNP) family and is a well-characterized RNA-binding protein (RBP). TDP-43 has been closely linked to the pathogenesis of neuronal degenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) upon post-translational modification , 6. BesiNutrient deprivation is often observed in solid tumors due to the rapid growth rate and ineffective blood supply in tumor tissue. Autophagy has been implicated as the key regulatory mechanism for cells to escape from cytotoxic stress. The role of autophagy in cancer therapy has been implicated to against cytotoxic, plenty of clinical trials have been launched targeting the inhibition of autophagy while the expression of autophagy-associated proteins, such as beclin-1 and LC3, are also connected with the poor prognosis of cancers –13. The In this study we investigated the mechanism of how TDP-43 promotes survival of the cells upon nutrition deprivation and contributes to the progress of GBM. We found that elevated expression of TDP-43 activates autophagy in stressful conditions and rescues cells from apoptotic process. Immunoprecipitation of TDP-43 revealed the increase of ubiquitination indicating the shortening of protein half-life of TDP-43 during starvation. Moreover, we discovered that HDAC6 acts as a downstream effector of TDP-43 in response to nutrient deficiency. In meanwhile, the inhibition of HDAC increases the apoptotic events even when TDP-43 is overexpressed. Concomitantly, we compared our findings to the clinical data and found that TDP-43 and HDAC6 are co-expressed and their high-expression reflected the better survival than that of others.p < 0.01), while TDP-43 also enhanced the formation of neurospheres of U87MG . To further assess the role of TDP-43 in tumorigenicity, we subcutaneously implanted the TDP-43-overexpressing cells into nude mice. In comparison to the empty vector control (Flag-control), the size of tumors was greatly increased in Flag-TDP-43 group , the TDP-43 was found to be involved in Warburg effect-associated pathways . In contrast, siTDP-43 enhanced the starvation-induced proportion of apoptotic cells in U87MG as compared to scrambled siRNA control . These data indicate that TDP-43 protects GBM cells from nutrient deprivation-induced cell death.Limited vascularisation caused by rapid tumor growth restricts local oxygen and nutrient supply and results in a stressful condition. However, the malignant neoplasms, including GBM, are able to survive this stress, although little is known about the detailed molecular mechanisms supporting such survival. Warburg effect is one of the most well-established mechanisms of tumor survival under nutrient shortage. In an s Figure . Nutriens Figure . In addis Figure . We alsos Figure . In meanp < 0.05). Western blotting further indicated that the endogenous autophagy marker LC3-II accumulated in nutrient-deprived U87MG cells with TDP-43 over-expression.  as a downstream effector of TDP-43 , we expep < 0.001; Figure hi/HDAC6hi. Collectively, we demonstrated that the co-elevated expression of TDP-43 and HDAC6 positively correlated with the high-grade glioma and GBM, suggesting the TDP-43/HDAC6 could be a potential marker of prognosis in GBM.Since our data suggested that TDP-43 and TDP-43/HDAC6 axis promotes GBM-related tumorigenesis, we therefore examined the expression patterns of TDP-43 and HDAC6 in GBM patient samples. First, we analyzed microarray dataset from TCGA Brain (Oncomine database) and found that expression levels of TDP-43 were significantly elevated in GBM specimens in comparison with the clinical white matter  Figure . SecondlATG7 mRNA that encodes an autophagy regulating factor, which is required to maintain autophagy system [Understanding of the molecular mechanisms of tumorigenesis in glioblastoma multiforme (GBM) is crucial for developing the effective therapeutic approaches and improving patient survival. In this study, we focused on expression of the nutrient deprivation-induced TAR DNA-binding protein TDP-43) and its downstream effector histone deacetylase-6 (HDAC6) to dissect their involvements in tumorigenesis of GBM. Our results indicate that TDP-43 expression is essential for tumor progression Figure  by promo and its y system . NotablyTDP-43 has been reported to be involved in repression of HIV transcription and regulation of alternative splicing of CFTR gene –27. BecaHistone deacetylases (HDACs) are involved in epigenetic modification through histone deacetylation. Eighteen HDACs were identified in mammals, among them class I HDAC family members that include HDAC1 and HDAC2, are localized in the nucleus and regulate some biological events in cancer cells, including apoptosis and proliferation. Unlike type I HDACs, HDAC6 is categorized as type IIB HDAC and is mostly located in the cytoplasm, where it is predominantly engaged in cytoskeletal regulation , 41. HDATo summarize our findings, we demonstrate that: (1) TDP-43 expression is required for tumor progression; (2) TDP-43 modulates autophagy in starved cells, which is in charge of tumor survival under nutrient deprivation; (3) increased stability sustains TDP-43 expression in starved cells; (4) HDAC6 expression mediates anti-apoptotic function of TDP-43. To our knowledge, this is the first demonstration that TDP-43 is involved in tumor progression via autophagy. These findings contribute to our understanding of the potential role of TPD-43 in cancer. In summary, TDP-43/HDAC6 functions as a stress responsive pathway in GBM tumorigenicity and promotes survival of nutrient-deprived GBM cells via activating autophagy Figure .The human GBM cell line, U87MG, and its derived TDP-43-overexpressed stable cell line were cultured in Dulbecco’s Modified Eagle’s Media  with 10 % fetal bovine serum , 150g/mL G418 , 100 units/mL penicillin, and 100 μg/mL streptomycin  under standard culture conditions . Subculturing was performed with 0.25% trypsin-EDTA . Media were replenished every two days.U87MG cells were seeded in 6-well plates  and were incubated for 24 hours. U87MG cells starved in nutrient deprivation condition for 6 days. After 6 days, U87MG cells were refreshed with fresh medium and cultured for 14 days. The cells were fixed with 9% formalin, and stained with 4% trypan blue (w/v) for 20 min. The stained cells were washed using PBS and counted.The cells were cultured in tumor sphere medium consisting of serum-free DMEM/F-12, N2 supplement, 10 ng/mL human recombinant bFGF, and 10 ng/mL EGF until sphere formation was observed after about 4 weeks of cultivation.5 cells was added, and the dishes were incubated at incubator for 4 weeks. Plates were stained with 0.005% (w/v) crystal violet for 1 hour and then a dissecting microscope was used to count the number of colonies.Each well (35 mm) of a six-well culture dish was coated with 1.5 ml bottom agar mixture  FCS, 0.6% (w/v) agar). After the bottom layer solidified, 1.5 ml top agar-medium mixture  FCS, 0.3% (w/v) agar) containing 1 × 10Protein samples were prepared with RIPA buffer  containing 1% protease inhibitor. Equal weights of total protein were separated by SDS/PAGE. After the proteins had been transferred onto a polyvinylidene difluoride membrane , the blots were incubated with blocking buffer (1 X PBS and 5% skim milk) for 1 hour at room temperature and then hybridized with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. The blots were obtained by X-ray film exposure, and the intensities were quantified by densitometric analysis . The primary antibodies used in this study were anti-Flag , TDP-43 , cleaved-caspase-3 , HDAC6  and beta-actin . LC3 (Novus NB100 2220).Total RNA was isolated from GBM cells using TRlzol  followed by phenol:chloroform purification and ethanol precipitation. Reverse transcription reactions were carried out using SuperScript III reverse transcriptase . Oligonucleotides were designed using Primer Express 2.0 . Oligonucleotide specificity was computer tested  by homology search with the human genome and later confirmed by dissociation curve analysis. The qRT-PCR was performed with power SYBR Green PCR Master Mix  according to manufacturer’s instruction. Signals were detected with 7900HT Fast Real-time PCR system . The expression level of each gene was normalized to endogenous beta-actin and experimental control through ΔΔCt method.The RNAi oligonucleotides targeting TDP-43 (siTDP-43) and the scramble sequence (scRNA) oligonucleotides were all purchased from Ambion. Transient transfection was carried out using INTERFERin siRNA transfection reagent  according to manufacturer’s instruction. Cell-based experiments were performed after 2-day incubation.Plasmid transfection was carried out using jetPEI DNA transfection reagent  according to manufacturer’s instructions.All procedures involving animals were performed in accordance with the institutional animal welfare guidelines of Taipei Veterans General Hospital. The GBM cell line U87-Flag and U87-TDP-43 were harvested, washed, resuspended in PBS and subcutaneously implanted into the dorsolateral side of the flank region of 8-week-old male BALB/c nude mice . After 14 days of subcutaneous inoculation tumor volume was measured by calipers.2O2. Sample were blocked in 5mg/ml BSA  for 30 mins before hybridizing with 1/100 diluted primary antibodies TDP-43 , cleaved-caspase-3 , HDAC6  overnight at 4°C. Signals were amplified by the TSA Biotin System  following the manufacturer’s instruction and the samples were counterstained with hematoxylin. The sections were examined under Olympus BX61 microscope , and three fields of view were randomly selected and photographed for evaluation. The relative staining index (rSI) represents the percentage of positive-expressed cells in the counting region.Tumor specimens from mice were fixed with 4% paraformaldehyde . Sections were deparaffinized and rehydrated, and subjected to antigen retrieval by boiling in 10 mmol/L (pH 6) citrate buffer  for 10 mins. Sections were cooled in PBS for 10 mins before treating with 3% Ht-test was used to compare the continuous variables between groups. The Kaplan-Meier procedure was used to calculate survival probability estimates. A log-rank test was used to compare the cumulative survival durations in different groups. The statistical significance level was set at 0.05 for all tests.The Statistical Package of Social Sciences software  was used for statistical analysis. An independent Student’s


Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron degenerative disease. ALS-affected motor neurons exhibit aberrant localization of a nuclear RNA binding protein, TDP-43, into cytoplasmic aggregates, which contributes to pathology via unclear mechanisms. Here, we demonstrate that TDP-43 turnover and toxicity depend in part upon the endocytosis pathway. TDP-43 inhibits endocytosis, and co-localizes strongly with endocytic proteins, including in ALS patient tissue. Impairing endocytosis increases TDP-43 toxicity, aggregation, and protein levels, whereas enhancing endocytosis reverses these phenotypes. Locomotor dysfunction in a TDP-43 ALS fly model is also exacerbated and suppressed by impairment and enhancement of endocytic function, respectively. Thus, endocytosis dysfunction may be an underlying cause of ALS pathology. Impaired turnover of TDP-43 by impaired autophagy or proteasomal function have been suggested to be the cause of TDP-43 accumulation, a hallmark of ALS. Here the authors demonstrate that endocytosis is also important for regulating TDP-43 turnover and toxicity. Most ALS cases are sporadic with only 5–10% of cases being inherited. No effective treatment for ALS currently exists.ALS is a neurodegenerative disease characterized by motor neuron loss, muscle weakness, paralysis, and eventually respiratory failure. Death typically occurs within 3–5 years post diagnosis2. Normally, TDP-43 is a nuclear protein with many roles in messenger RNA (mRNA) metabolism, but in ALS, TDP-43 re-localizes to cytoplasmic aggregates. Several studies suggest these TDP-43 aggregates induce disease partly via a toxic gain of function. However, the source of such toxicity, and possible loss of function (LOF) effects remain highly debated3. Regardless, there is significant interest in understanding how TDP-43 aggregates form, and in developing therapeutic strategies that prevent TDP-43 aggregation or facilitate TDP-43 aggregate clearance.A hallmark of ALS is the accumulation of cytoplasmic ubiquitinated protein aggregates in affected motor neurons, of which TAR DNA-binding protein 43 (TDP-43) is the primary component4. SGs are dynamic, membrane-less assemblies composed of non-translating mRNA–protein (mRNP) complexes5. Interestingly, many genes that are mutated in ALS include RNA binding proteins that also localize in SGs . Such mutations usually make these proteins more aggregation prone, which can lead to more rapid or persistent SG assembly7. Supporting a role of SGs in TDP-43 toxicity, preventing SG assembly either genetically or chemically has shown therapeutic benefit in models of TDP-43 toxicity9.TDP-43 aggregates may form due to TDP-43 concentration in cytoplasmic stress granules (SGs)11. Mutations in genes involved in either process such as UBQLN2, OPTN, p62, and VCP occur in some ALS patients6. Furthermore, induction of non-selective autophagy rescues TDP-43 toxicity in human iPSC-derived neurons, astrocytes, and a mouse frontotemporal lobar dementia model13.In contrast, other studies suggest that impaired turnover of TDP-43, either via defects in autophagy or proteasomal function, contributes to ALS pathology14, the mechanism of which is not yet fully understood. Evidence that granulophagy is selective includes a dependence on “adaptor”  and “receptor” 15 proteins dispensable for non-selective autophagy. Additionally, granulophagy is active only under specific stress conditions, or when cytoplasmic mRNA decay is blocked in yeast, which causes aberrant SG formation. Since TDP-43 localizes in SGs, we initially hypothesized that inducing granulophagy may help clear TPD-43 aggregates.Interestingly, SGs are targeted by an autophagic pathway called granulophagy16. For example, TDP-43 expression in yeast, which recapitulates TDP-43 aggregation, SG localization, and cellular toxicity, enabled the identification of Pbp1 as a modifier of TDP-43 toxicity. This lead to the discovery of Ataxin-2 (Pbp1 ortholog) in humans as an ALS risk factor17.For many neurodegenerative diseases featuring protein aggregates, yeast has helped identify relevant modifiers of disease aggregate toxicityHere, we discover that granulophagy and non-selective autophagy weakly affect TDP-43 turnover, and do not affect TDP-43 toxicity in yeast. Instead, TDP-43 turnover, aggregation, and toxicity strongly depend on endocytosis, both in yeast and human cell lines. TDP-43 inhibits endocytosis, and co-localizes with endocytic proteins in yeast, cell lines, and ALS frontal cortex tissue. Additionally, endocytic defects exacerbate motor neuron dysfunction in a TDP-43 ALS fly model. Importantly, enhancing endocytosis increases TDP-43 turnover, and reduces TDP-43 toxicity and motor neuron dysfunction. Our work implicates defective endocytosis function in ALS pathology.Given prior studies on TDP-43 turnover and our interest in granulophagy, we examined how non-selective autophagy, granulophagy, and the proteasome affected TDP-43 turnover in yeast. TDP-43 was expressed via a galactose-regulatable promoter. Galactose concentrations were optimized to limit overexpression and help identify mutants that exacerbate TDP-43 toxicity  promoted TDP-43 turnover by use of a steady-state YFP/GFP fragment assay, in which appearance of a YFP or GFP fragment indicates vacuolar turnover of YFP or GFP-fusion proteinsess Fig. . N-S stress Fig.  and TDP-ess Fig. . These d5. TDP-43 indeed localized in yeast SGs during stationary phase, when granulophagy is active14  treatment, which disassembles SG, caused loss of Pub1 but not TDP-43 foci  binding protein 1 (Pub1) foci. Pub1, and its mammalian ortholog TIA-1, are SG assembly proteins14. Next, we examined if TDP-43 turnover resembled that of poly(A) binding protein 1 (Pab1), which is a core SG protein that binds mRNA poly(A) tails and is a protein target of granulophagy14. Interestingly, following Dcp2 inactivation in a temperature sensitive dcp2-7 atg15Δ strain, which blocks mRNA decay and potently induces granulophagy14, Pab1-GFP accumulated in vacuoles, whereas TDP-43-YFP remained mostly in cytoplasmic foci  Vps34. Vps34 functions in two conserved PI3K complexes, “I” and “II”, which include Vps15 (a Vps34 regulatory S/T kinase), Vps30/Beclin 1, and either Atg14 (complex I-specific) or Vps38/UVRAG (complex II-specific). Yeast PI3K complex I promotes autophagy, whereas PI3K complex II promotes endocytosis and Golgi vacuole trafficking22. In mammalian cells, conflicting evidence exists as to whether PI3K complex II also aids autophagy besides promoting endocytosis24. Regardless, PI3K inhibition induces increased death of iPSC-derived motor neurons that express a TDP-43 ALS disease variant25. Examining PI3K mutants was thus of interest.Two non-autophagic vacuolar trafficking pathways that could promote TDP-43 turnover include endocytosis and Golgi vacuole traffickingvps34Δ and vps15Δ strains showed enhanced sensitivity to TDP-43 expression  thought to be core to PI3K complexes. As expected, re-introduction of Vps15, Vps34 and Vps38 expression plasmids into their respective null strain backgrounds suppressed TDP-43 toxicity  for Rab5 proteins (Vps21/Ypt52/Ypt53) in yeast. Lack of Rab5 function causes a strong block in endocytosis between early (EE) and late endosome (LE) formation; see ref. 28 for review of all Rab protein functions.We measured endocytosis rate using an FM4-64 assay29  or Vps38 (II subunit) in TDP-43 expressing yeast. Strikingly, TDP-43 toxicity was almost completely rescued by Vps38 overexpression, whereas Atg14 overexpression had no effect Fig. . In addiVRP1, IDB17, EDE1, SLA1, and APL3 (AP-2 subunit), showed enhanced TDP-43 toxicity  showed high sensitivity to TDP-43 expression . Notably, vps9Δ strains showed a strong sensitivity to TDP-43 expression, even accounting for modest sickness of the vps9Δ strain alone , two of which (Vps16 and 18) increase TDP-43 toxicity when absent , CORVET (vps3Δ), and HOPS-specific (vps41Δ) mutants was also increased , vps28Δ (ESCRTI), vps36Δ (ESCRT-II), and vps2Δ (ESCRTIII) strains . Nonetheless, we began by transfecting cells with various TDP-43-expressing constructs. These included WT TDP-43, a Q331K disease mutant allele, and aggregation-prone C-terminal fragments of TDP-43 (TDP-25 and 35). These are observed in ALS patient aggregates, and are associated with cellular toxicity37.We next examined if TDP-43 was affected by endocytic function in human cells. A complicating issue here is that in human cells, endocytosis and autophagic pathways typically converge prior to lysosome fusion11. However, TDP-43 foci co-localized more frequently with endogenous Rab5 foci , impaired endocytosis as assessed by a transferrin uptake assay Fig. . TDP-43 oci Fig. . This suoci Fig. . Multipl ER Fig. . Using s ER Fig. . In summ41. Notably, dynasore induced significant increases in TDP-43 foci in cells expressing TDP-43, Q331K, TDP-35, or TDP-25 proteins  were generated whose expression level approximated endogenous TDP-43 , reduces TDP-43 foci levels and toxicity.We next examined if inhibiting endocytosis impaired TDP-43 protein clearance. As in yeast, ectopically expressed TDP-43 protein levels (WT and Q331K) increased when endocytosis was inhibited by dynasore Fig. . EndogenWe next examined how enhancing endocytosis rates by overexpressing Rab5 affected TDP-43 levels, and again utilized Rab8 overexpression as a control. Strikingly, ectopic TDP-43 levels were strongly reduced with Rab5 overexpression Fig. , whereas40, we examined accumulation of lipidated LC3 (“LC3-II”), which is a proxy for autophagosome number and autophagy rate18. Relative to rapamycin treatment, which served as a control for autophagy induction, the effect of Rab5 overexpression was subtle , dynasore treatment, or a combination of both upon endogenous TDP-43 protein levels. Consistent with prior findings, Atg5 KD indeed caused TDP-43 accumulation, to a similar degree as dynasore treatment Fig. ; lane 4,ver Fig. . ThapsigDrosophila model that recapitulates key ALS features44. In this model, WT or disease-associated G298S TDP-43 is specifically expressed in motor neurons, and locomotor dysfunction is assessed by the time a larva takes to re-orientate itself after being turned onto its back. Both WT and G298S TDP-43 expression significantly increases larval turning times relative to controls  converts PIP2 phosphoinositiol to PI(3)P phosphoinositol48, which aids EE and autophagosome maturation; note PI3K also generates this. Second, ALS2 is a Rab5 GEF that is mutated in a form of familial ALS49. Third, knockdown of ESCRTI (Tsg101) and III (Vps24) proteins results in accumulation of TDP-43 in cytoplasmic foci50. Mutations in CHMP2B, another ESCRTIII protein, are also associated with rare forms of ALS51. Notably, as suggested in studies on ALS2 and the aforementioned ESCRT proteins52, endocytic dysfunction may also impair autophagy by blocking autophagosome fusion with the endocytic pathway. Fourth, recycling endosome defects that alter signaling at neuromuscular junctions have been observed in a TDP-43 fly model53. Finally, C9ORF72, the most commonly mutated gene in ALS6, harbors a DENN domain commonly found in Rab GEF proteins54, co-localizes with Rab5, and its depletion impairs endocytosis rate in neuroblastoma cells55. Conflicting evidence also exists as to whether C9orf72 promotes57 or inhibits58 autophagy via interactions with other Rab proteins and/or effects on mTOR signaling and transcription of autophagy/lysosomal genes. Regardless, our data and that of others argues that endocytic dysfunction may be a common mechanism underlying ALS caused by multiple different mutations.Other evidence suggests that endocytosis may be perturbed in ALS, and that this may also impact autophagy. First, 59, which could promote both endocytic entry and intercellular transmission. Alternatively, TDP-43 may disrupt the endocytic pathway such that a hybrid endocytic compartment is formed, which may permit TDP-43 internalization via a novel mechanism. Finally, TDP-43 could traffic on the exterior of endocytic vesicles and be incorporated into vacuoles/lysosomes by an alternative import mechanism.A key question is how TDP-43 traffics with the endocytic pathway, especially as other SG-localizing proteins . Additionally, although knowledge of TDP-43 interaction with specific cellular membranes is lacking, TDP-43 interacts in vitro with membranes via amino acids 311–319, which are critical for its neurotoxicity60. Membrane interactions also aid TDP-43 aggregation, particularly mutant forms60. This region can also damage membranes61, which could be related to TDP-43’s ability to inhibit endocytosis. Alternatively, TDP-43 aggregates may sequester and inhibit endocytic proteins. One possibility is Rab5, which is strongly concentrated into large TDP-35/25 aggregates when present at endogenous levels  and huntingtin protein aggregates can impair endocytosis function partly via Hsc70 chaperone sequestration26. TDP-43 aggregates may behave similarly, and may also translationally repress Hsc70 mRNA through a sequestration mechanism, which in turn affects synaptic vesicle cycling62. Determining how TDP-43 aggregates inhibit endocytosis, and if this defect is common to other neurodegenerative disease protein aggregates is a key future goal.Another key question is how TDP-43 might inhibit endocytosis. Our FM4-64 data indicate continued presence of FM4-64 at the plasma membrane even at late time points . In addition, ubiquitination of substrates is usually required for substrate incorporation into LEs via ESCRT protein function64. Thus, accumulation of ubiquitinated TDP-43 could indicate endocytic defects, rather than autophagic/proteasomal defects as is commonly assumed. Finally, TDP-43 aggregates do localize with autophagy-associated proteins such as Ubiquilin1, p62, Atg5, and LC365 as we too observed  medium supplemented with appropriate amino acids. Either 2% glucose or 0.25% galactose plus 1.75% sucrose as a carbon source to inactivate or drive GAL1-driven expression of TDP-43 was used unless otherwise stated. Transformation was performed by standard LiAc methods. Strains generated in this study were constructed by homologous recombination methods. Briefly, EUROSCARF antibiotic/biosynthetic cassettes were PCR amplified with 50 bp regions of homology flanking the start and stop codons of the gene to be deleted. These PCR products were transformed into yeast, which were then subject to appropriate selection and PCR conformation to identify yeast in which the gene in question was successfully replaced  or mid-log cells  were examined using a DeltaVision Elite microscope via a ×100 objective. All image analysis was done using Fiji software67. TDP-43 foci co-localization with different proteins was scored manually, in a blind manner, in a minimum of 50 cells (in which each cell may contain multiple foci). Cell and foci counts for all microscopy data (including human cell immunostaining) are listed in Supplementary Data The methods have been described previouslyTDP-43 expression in mid-log cells was halted by transcriptional shut off . Alternatively, general protein synthesis was halted by adding 0.2 mg/ml CHX to the yeast culture. In both cases, protein lysates were obtained via a standard NaOH method and examined via Western blot.Western blotting was performed via a standard protocol. All images shown are representative of >3 biological replicates. Primary antibodies used are listed in Supplementary Table 68. Briefly, cells were harvested at mid-log phase and kept on ice for 10 min. Then the cells were suspended in YPD medium containing 8 µM FM4-64 at 30 °C for indicated times. Vacuolar membrane intensity was quantified using Fiji.Yeast endocytosis rate assay was conducted as described previously29. Briefly, yeast cells were cultured to the mid-log phase. Cells was concentrated to 5–8 folds in a 1.5 ml tube. Suspend cells in YPD medium with 1 mg/ml Lucifer yellow and incubate for 30 min. Cells were then concentrated and washed three times in PBS with 10 mM sodium azide and 50 mM sodium fluoride. Images were then taken under the microscope.Lucifer yellow assay was performed as previous reportedHEK293A cells were cultured in DMEM medium  with 10% FBS  50 U/ml penicillin and 50 μg/ml streptomycin . HEK293A cells stably expressing TDP-43, TDP-43-Q331K, TDP-35, and TDP-25 with FLAG at the N-terminus and GFP at the C-terminus were constructed and selected with 1 μg/ml puromycin . SH-SY-5Y cells were cultured with 1:1 mixture of Eagle’s minimum essential medium  and F12 medium (Thermo Fisher Scientific 11330032), 10% FBS.oC after blocking with 5% goat serum. Cells were then incubated with Alexa fluor conjugated secondary antibody  for 1 h at room temperature. After three washes with TBS-T, cells were stained with DAPI  and imaged using a Deltavision Elite microscope with a ×40 objective. Antibodies used for the immunofluorescence were: HA , UVRAG , Rab5 , LC3 . All shown images are representative of >3 biological replicates.Cells were transfected with plasmids, cultured for 48 h in 8-well chamber slides , fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Trion X-100 for 5 min. Cells were incubated with indicated primary antibodies overnight at 4 68. Briefly, cells were harvested in homogenization buffer (HB)  and lysed using a dounce glass-glass homogenizer (B-pestle). Gradient columns  consisted of HB, 25, 35, and 42% sucrose from top to bottom. Gradients were ultracentrifuged at 21,000×g for 3 h at 4 oC. The interfaces between HB and 25% layers, and between the 25 and 35% layers were harvested, which correspond to the late and early endosome-containing fractions, respectively. All samples were examined by western blotting.Endosomes were fractionated as previously described69. Briefly, homogenization of cells was as described for the endosome fractionation assay described above. About 2 ml gradient columns  were generated with HB buffer and 5–30% OptiPrep with 2.5% increments. Gradients were ultracentrifuged at 100,000×g for 16 h. Each fraction  was harvested and examined by western blot using antibodies against appropriate organelle markers . Cell number was counted after 48 h growth.HEK293A cells expressing empty vector or different stably integrated versions of TDP-43 were cultured in puromycin containing medium for selection purposes. About 1 × 10The transferrin assay was performed in accordance with Thermo Fisher product guidelines. Briefly, cells were kept on ice for 10 min. Then cells were washed with live cell imaging solution (Thermo Fisher Scientific) and then incubated with 25 µg/ml fluorescent transferrin  at 37 °C for 15 min. Then cells were washed again with live cell imaging solution, imaged and quantified for intracellular fluorescence using Fiji.Drosophila stocks and crosses were kept on standard yeast/cornmeal/molasses food at 22 °C. Human TDP-43 variants with YFP C-terminal tags were generated as previously described50. Untagged TDP-43WT was obtained from J. Paul Taylor and untagged TDP-43G298S was obtained from Takeshi Iwatsubo. Gal4 drivers used included the eye-specific GMR Gal4 and the motor neuron driver D42 Gal4. For controls, w1118 was crossed with the appropriate Gal4 driver. To manipulate Rab5 y1w*;P[UASp YFP Rab5]O2, y1w*;P[UASp YFP Rab5]Pde80^8b, and yd2w1118;P{EPgy2}Rab5EY10619/CyO were used (Bloomington Drosophila Stock Center). To manipulate Rab7 w1118-;P[UAS Venus Rab7 WT]/CyO, y1w*-;P[UAS Venus Rab7 CA1]/CyO, y1w*-;P[UAS Venus Rab7 DN1]/CyO, and w1118 Sp/CyO; FRT82B Rab7KO/TM3 were used70. To manipulate Atg8a, y[1 w[1118]; P{w[ + mC] = UASp-GFP-mCherry-Atg8a}2 was used (Bloomington Drosophila Stock Center). To manipulate Rab8, Rab8[1 red[1 e[4/TM6B, Sb Tb was used (Bloomington Drosophila Stock Center).All oC, and performed as described previously50, using a Leica MZ6 microscope equipped with Olympus DP73 camera.Assays were conducted at 25 50. Briefly, third instar wandering larvae were placed and acclimated on a grape juice plate at room temperature. The crawling larvae were turned onto their backs , and the time that each larva took to re-orientate , and make a forward movement was recorded. Thirty larvae were used per genotype. Statistical significance was analyzed by Student’s t test.Assays were performed as previously described2O2 for 30 min. Tissue samples were then blocked with 5% goat serum for 1 h before incubation with primary antibody at 4 °C overnight. Antibody staining of TDP-43 (ProteinTech) and Rab5 (Abcam) was diluted to 1:200 and 1:100, respectively. After four TBS-T washes, samples were incubated with Alexa fluor conjugated secondary antibody at a 1:1000 dilution for 1 h at room temperature. After washing, immunostained tissue samples were mounted with ProLong Gold Antifade DAPI and imaged.Paraffin-embedded tissue microarray slides  were provided by the Southern Arizona VA Healthcare Agency and immunostained using a standard protocol from Abcam . Briefly, glass slide-adhered samples were dewaxed with xylene and fixed with successive 100, 100, 90, 80, 70% ethanol and distilled water for 10 min each. Antigen retrieval was accomplished by incubating samples in boiled sodium citrate buffer for 20 min. Endogenous peroxidases were quenched with 3% HThe data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2


The JAVELIN Solid Tumor clinical trials group recently reported the results of a multi-center open-label phase 1b study NCT01772004) evaluating avelumab in patients with progressive or platinum-resistant metastatic or recurrent non-small cell lung cancer (NSCLC) [004 evaluin vitro [in vitro.Among the five currently FDA-approved anti-PD-1/PD-L1 agents, avelumab is unique due to its unmodified fragment crystallizable (Fc) domain which may potentiate antibody-dependent cell-mediated cytotoxicity (ADCC) via interaction with CD16 (FcɣRIIIa) on natural killer (NK) cells . The impin vitro . Howeverin vitro . This strd line of therapy) and with other driver mutations . Multiple studies across multiple treatment modalities including immunotherapy have shown lower response rates with more advanced disease / later lines of therapy.With a median follow up of 8.8 months in the NSCLC dose-expansion cohort, the JAVELIN investigators enrolled 184 patients with stage IIIB or IV NSCLC with disease progression after prior platinum-based doublet therapy for metastatic disease across 58 academic centers . This stn = 22), and 1-year progression-free and overall survival rates were 18% and 36%, respectively. Ongoing follow-up has confirmed an additional 4 responses, raising the ORR to 14.1% (n = 26). Accounting for these factors, ORR was similar (12%-14.1% vs. 14%-20%) in this study compared with randomized prospective studies evaluating other PD-1 or PD-L1 inhibitors in the second-line advanced NSCLC setting [vs. 11.4%) [Objective response rate (ORR) was 12% . These findings are exploratory and hypothesis-generating at present but add to a growing compendium of literature attempting to clarify the appropriate use of PD-L1 expression as a predictive biomarker for patient selection. A preliminary exposure-response analysis of this cohort of NSCLC patients explored the relationship between PD-L1 expression and avelumab exposure (trough concentration after first dose) [To explore potential biomarkers of response to avelumab, clinical responses were stratified by PD-L1 expression. A proprietary assay for PD-L1 expression (anti-PD-L1 rabbit mAb clone 73-10) using a cutoff value of ≥1% on tumor cells showed a progression-free survival benefit for PD-L1 positive patients relative to PD-L1 negative counterparts (12.0 In consideration of this heterogenous, heavily pre-treated, PD-L1 non-selected advanced NSCLC patient population, avelumab demonstrated a toxicity profile and anti-tumor activity similar to other anti-PD-1 and PD-L1 mAbs .


Oxidative wet-chemical delamination of graphene from graphite is expected to become a scalable production method. However, the formation process of the intermediate stage-1 graphite sulfate by sulfuric acid intercalation and its subsequent oxidation are poorly understood and lattice defect formation must be avoided. Here, we demonstrate film formation of micrometer-sized graphene flakes with lattice defects down to 0.02% and visualize the carbon lattice by transmission electron microscopy at atomic resolution. Interestingly, we find that only well-ordered, highly crystalline graphite delaminates into oxo-functionalized graphene, whereas other graphite grades do not form a proper stage-1 intercalate and revert back to graphite upon hydrolysis. Ab initio molecular dynamics simulations show that ideal stacking and electronic oxidation of the graphite layers significantly reduce the friction of the moving sulfuric acid molecules, thereby facilitating intercalation. Furthermore, the evaluation of the stability of oxo-species in graphite sulfate supports an oxidation mechanism that obviates intercalation of the oxidant. Scalable graphene production from graphite via an intercalation-oxidation-reduction process is still hampered by low reproducibility and many lattice defects. Here, the authors show that reducing molecular friction by using highly crystalline graphite and mild oxidizing conditions is the key to high quality graphene. However, mainly high temperature processes are facilitated to produce graphene5. Up to now, it remains challenging to prepare the same quality of graphene wet-chemically utilizing chemical functionalization methods. The advantages of a wet-chemical process are its scalability and the possibility to introduce functional groups at the same time. Chemical modification can give new functions to graphene, which enable e.g., medical applications10.The outstanding electronic and mechanic properties of graphene and its chemical reactivity led to the development of graphene technology11. For improving the quality of the produced graphene, it is therefore highly desired to understand the underlying mechanisms in this process in more detail in order to be able to better control the individual synthesis steps. Key factors on the way to high-quality graphene are the proper formation of the intermediate graphite sulfate, a stage-1 graphite intercalation compound (GIC) with every layer of the graphite intercalated  of 0.3–0.05%14. This oxo-functionalized graphene was found to be suitable to establish a controlled chemistry of graphene where lattice defects play a minor role18. We term this product oxo-G1 (index = number of layers) in order to distinguish it from the known graphene oxide, which possesses an undefined amount of defects and consequently a disturbed order of the lattice.Conventional synthesis of graphene via graphite intercalation, oxidation, delamination into graphene oxide, and subsequent reduction 2)n has been reported, needs more clarification for improving the yield and the quality of the synthesized graphene21. The intercalation of graphite with sulfuric acid increases the layer distance to 7.98 Å24. This intercalation process is initiated by electronic oxidation of the graphite29. However, why the initial oxidation is needed and to what extend oxo-functional groups are introduced on the basal planes of the graphite scaffold at this stage, is currently unknown. Puzzling are also recent contradictory reports: while some groups observed the intercalation process to be reversible upon water addition24, we recently reported the formation of hydroxylated graphene under similar reaction conditions14. This contradiction unfolds the lack of our understanding of the overall graphene synthesis reaction.Nevertheless, how a proper stage-1 GIC is obtained that enables oxo-Gion Fig.  and the 1. Our oxo-G1 from the graphite with the highest crystallinity yields a graphene with an exceptionally low density of lattice defects down to 0.02% after reductive defunctionalization. Statistical Raman spectroscopy (SRS) and high-resolution transmission electron microscopy (HRTEM) prove the high lattice quality. Mechanistic ideas about the formation ability of different types of graphites to form stage-1 graphite sulfate are derived from ab initio molecular dynamics (MD) simulations, which provide an understanding why a high degree of crystallinity of graphite is mandatory. The simulations show that the friction of moving sulfuric acid molecules in the graphite intercalation compound is intimately coupled to the stacking and the electronic oxidation state of the graphite layers. Ideal AB stacking and initial oxidation significantly reduces friction and supports the spreading of the sulfuric acid molecules within the graphite. Oxo-species  are found to be unstable within the graphite intercalation compound below a formal oxidation state of about Here we reveal that crystallinity of the graphite is the decisive factor for obtaining a proper stage-1 GIC and subsequently oxo-G2SO4-GIC formation was described in the literature21, the origin of hindrance could not yet be elucidated.The formation of graphite sulfate was studied experimentally by immersing a high quality graphite crystal in a quartz glass cuvette with conc. sulfuric acid and ammonium persulfate as oxidant Fig. . The for30, and NG1 is the most crystalline graphite with 28% 3R-phase (ABC stacking) and crystallite size larger than 10 μm and 72% 2H-phase (AB stacking) with crystallite size of about 100 nm. NG2 also bears crystallinity with about equal amounts of 3R-phase (46%) and 2H-phase (54%) with however crystallite sizes of 100 nm and less. In contrast, TG bears no significant stacking order30. Raman spectroscopy of those graphite grades  according to the intensity ratio of the defect induced D peak and the G peak (ID/IG)32, which are for NG1 LD = 35 nm (ID/IG = 0.12), for NG2 LD = 36 nm (ID/IG = 0.11) and for TG LD = 19 nm (ID/IG = 0.38). The symmetrical shape of the 2D peak of TG also indicates the missing stacking order33.To shine more light on the intercalation and subsequent functionalization and graphene formation process three different grades of graphite were used: Sri Lankan natural graphite with 70–150 μm grain (NG1), natural graphite with 10 μm grain (NG2), and turbostratic graphite (TG). X-ray powder diffraction of those graphites was conducted in an earlier study−1 due to the high doping level. The 2D peak is almost absent in both NG-GICs as expected  of the D and G peak, respectively 11. However, few spectra of processed NG2 also exhibit characteristic spectral features of graphite  with lateral dimensions up to 10 μm is found for NG1, whereas for NG2 such flakes are only isolated from the supernatant and with lateral dimensions <3 μm. Apparently, mainly graphite-like particles are formed. In contrast, graphene was not found from TG. Consequently, we prepared dispersions of functionalized graphene from NG1 to investigate the potential use of this synthetic approach.Subsequent sonication of water purified samples of functionalized graphite leads to exfoliation and a deposition of flakes on Si / 300 nm SiO1 with an optical density of 0.5 could be obtained by iterative centrifugation and redispersion in a 1/1 mixture of H2O/MeOH assisted by ultrasonication  indicates a low degree of oxo-functionalization, which was estimated to θf = 0.04 in an earlier study14. Furthermore, graphene obtained after reduction with NaBH4 and stabilized by sodium cholate exhibits an absorption maximum at 266 nm, instead of 254 nm for oxo-G1 (θf = 0.5), indicating an improved quality of graphene . Moreover, the IG values of recorded Raman data let conclude that about 72% of the area are either single layer graphene (including areas of probed single layer graphene / substrate) with only 28% of few-layer graphene  technique. In Fig. 34. Although heteroatoms, such as nitrogen or oxygen, can’t be discriminated from carbon by contrast, the carbon framework of graphene is well resolved as it can be seen in Fig. 35. Although some areas of the carbon lattice are hidden by the adsorbed material, the periodic extension of the uncontaminated carbon framework gives evidence for a coherent hexagonal lattice.The interpretation of SRS results is further confirmed by HRTEM performed with atomic resolution after deposition of functionalized graphene on TEM grids by the LB technique. The carbon lattice was observed without additional cleaning or heating procedures, as we described in a recent HRTEM study36 and to probe how their mobility depends on the graphite stacking and the GIC oxidation state.The observation that micron-sized graphite grains with perfect crystalline stacking (NG1) are easier to intercalate and to oxidize than more defective graphite grades with smaller grains (NG2) and perturbed stacking (TG) is surprising. Intuitively one would expect that the reduced cohesion between the graphite layers and the increased layer separation in perturbed graphite makes it easier for the sulfuric acid molecules to enter the graphite lattice, widen the layer spacing and fill the available space. This suggests that for GIC formation and oxidation the initial ability of the sulfuric acid to penetrate the graphite lattice is less important, but the mobility of the sulfuric acid between extended graphite sheets is the more decisive factor. To explore this idea, a series of ab initio molecular dynamics (MD) simulations were performed in order to determine the interaction of confined sulfuric acid molecules with the graphite lattice22. The region between the graphite layers was filled with 8 sulfuric acid and one water molecules to reflect the composition of concentrated sulfuric acid (see Method section). Different stages of oxidation were obtained by adding one, two and three isolated pairs of hydroxyl groups to this unit cell, each in 1,2-trans configuration since this is the most stable arrangement of OH pairs , leaving behind a two-fold positively charged graphite layer see Fig. . Similar2SO4)2)n) of one positive charge per 24 carbon atoms19 and can be well understood from an analysis of the electronic structure  for our three configurations is shown in Fig. λ can be extracted from the plateau region, which is reached after about 0.4 ps. The comparison between the three simulations reveals that oxidation  shown in Fig. This result is corroborated by an analysis of the po was converted into a free energy surface via ∆A = −kBT In po from which a line scan  combined with large grain sizes as in NG1 efficiently reduces the corrugation of the free energy surface for the slip of the sulfuric acid molecules between the graphite sheets and supports successful stage-1 GIC formation. In contrast, any deviation  as in NG2 and TG leads to an increase in the free energy barrier and thus hinders the intercalation process.Finally, our observations on the stability of oxo-species in graphite sulfate suggest an alternative oxidation mechanism. Since oxo-species do not remain attached to the graphite sheets up to an oxidation state of ion Fig. . This prExperimental and theoretical results demonstrate that crystalline graphite with large grain size can act as a precursor for wet-chemically prepared graphene of high quality, as directly observed by HRTEM and quantified by statistical Raman spectroscopy. Friction of sulfuric acid confined between layers of graphite is significantly lowered with electronic oxidation, which initiates the intercalation process. Intercalation and oxidation reinforce each other: if intercalation is hindered by high friction, oxidation remains insufficient and the partial intercalation is reversible upon addition of water. Epoxides and hydroxyl groups are not stable at low oxidation levels, but become protonated and cleaved from the carbon lattice. Only at higher oxidation levels oxo-species remain attached to the graphite layers.Moreover, friction of confined sulfuric acid is lowest for AB-stacked graphene layers. Therefore, it is plausible that natural graphite, bearing grain boundaries and irregular stacking orders, is not an equally potent precursor for graphene production. Along the same line, turbostratic graphite is not forming a proper GIC (low oxidation degree) and thus reverts back to graphite upon water addition. Sulfuric acid, however, intercalates graphite of high crystallinity with large grains upon oxidation. The oxidation proceeds very likely outside the layer confinement by electron transfer, and charge compensation is accompanied by proton diffusion, leading to ers Fig. .The here presented route towards oxo-functionalized graphene and graphene on substrates or stabilized by surfactants in water can be expected to also lead to novel graphene derivatives with tailored functionality suitable for electronic or bio-sensing applications.42. UV–Vis spectroscopy was conducted on a Perkin Elmer—Lambda 1050.Graphite grades NG1 (grade 2012) and TG were obtained from Asbury Carbon. NG2 was purchased from Future Carbon. Large flake graphite was obtained from NGS Trading & Consulting GmbH. Sulfuric acid, hydroiodic acid 57% in water (HI), trifluoroacetic acid (TFA), and ammonium persulfate were obtained from Sigma-Aldrich® and used as received. Doubly distilled water was obtained from Carl Roth GmbH and used as received. Methanol was distilled in a solvent circulation apparatus before use. Centrifugation was accomplished by a VWR Micro 1814 table centrifuge. Sonication was performed using a Sonorex Digital P10 from Bandelin. LB films were prepared using a LB mini trough from KSV NIMA. AFM imaging was performed using a NT-MDT Solver Pro. Raman spectra were recorded with LabRAM HR Evolution from Horiba Scientific using an 532 nm laser line. Spectra were processed as reported elsewhereGraphite  was suspended in sulfuric acid . Over a period of three days ammonium persulfate  was added in small portions under stirring.g) and the supernatant was removed. The graphite sulfate was freezed by liquid nitrogen and doubly distilled water (1.5 ml) was added. The mixture was cooled for 16 h to 8 °C. The reaction mixture was purified by centrifugation and redispersion in water  until the supernatant was of neutral pH. Finally, the carbon material was taken up in 1/1 volume mixture of water/methanol. The dispersion was sonicated in a bath sonicator for 2 min. The oxo-G1 solution was obtained by centrifugation  and collecting the supernatant. Small particles were removed by centrifugation . Finally, larger particles were removed by centrifugation  leaving oxo-G1 in dispersion.Graphite sulfate (400 mg) was centrifuged  until the surface pressure reached 5 mN m−1. The substrate was then pulled up (2 mm min−1) to deposit flakes of oxo-G1 on Si/SiO2 (300 nm) substrates.The as-prepared oxo-G43, the Perdew-Burke-Ernzerhof PBE exchange-correlation functional44, Vanderbilt ultrasoft pseudopotentials45 and a plane wave basis set with kinetic energy cutoff of 30 Ry. To account for dispersion effects, the semi-empirical Grimme D2 correction was applied46. The MD simulations were carried out in the NVT ensemble using a Nose-Hoover thermostat set to 400 K in order to compensate for the known overstructuring of hydrogen-bonded liquids at room temperature in PBE47. An additional Nose-Hoover thermostat, a fictitious mass of 400 a.u. and a time step of 4 a.u. were used for the propagation of the electronic degrees of freedom. For obtaining converged results for the friction coefficient in the MD simulations, large supercells with lateral size of 24. The interlayer region was filled with 32 sulfuric acid and four water molecules for the neutral GIC simulations and eight hydrogen atoms were removed in the oxidized GIC(2SO4)2)n,21.The Car-Parrinello molecular dynamics simulations were carried out using the CPMD code48. The image acquisition time was 2 s and the dose rate for the nominally 300,000× magnified images (Fig. 6 e− nm−2 s−1. The diameter of irradiated area was approximately two times the camera width and the sample area imaged was changed after recording few micrographs with varying defocus; therefore, the maximum total dose per imaged area was about 108 e− nm−2, since the post-sample beam shutter was used. Vignettes were balanced using Gwyddion software.The TEM investigations were performed with a FEI Titan 80–300 transmission electron microscope fitted with a CEOS CETCOR third order spherical aberration corrector and a Gatan UltraScan 1000XP CCD camera. The microscope was operated at an electron acceleration voltage of 80 kV and the electron source extraction voltage was set to 2 kV reducing the primary electron energy distribution to 0.5 eV FWHMThe data that support the findings of this study are available from the corresponding authors upon request.Supplementary InformationPeer Review File