Transmission electron microscopy (TEM) photograph and X-ray photoelectron spectroscopy (XPS) analysis of nanoporous graphene nanosheets indicated that the structure of nano-pore was irregular and the oxygen-containing groups in the surface were limited. More importantly, the as-prepared MMMs presented better separation performance than that of pristine EC membrane due to simultaneous enhancement of C3H6 permeability and ideal selectivity. The ideal selectivity of the MMMs with 1.125 wt‰ nanoporous graphene content for C3H6/C3H8 increased from 3.45 to 10.42 and the permeability of C3H6 increased from 57.9 Barrer to 89.95 Barrer as compared with the pristine membrane. The presumed facilitated mechanism was that the high specific surface area of nanoporous graphene in polymer matrix increased the length of the tortuous pathway formed by nanopores for the gas diffusion as compared with the pristine graphene nanosheets, and generated a rigidified interface between the EC chains and fillers, thus enhanced the diffusivity selectivity. Therefore, it is expected that nanoporous graphene would be effective material for the C3H6/C3H8 separation.Nanopore containing graphene nanosheets were synthesized by graphene oxide and a reducing agent using a facile hydrothermal treatment in sodium hydroxide media. The as-prepared nanoporous graphene was incorporated into ethyl cellulose (EC) to prepare the mixed matrix membranes (MMMs) for C Currently, the separation is mainly achieved by fractional distillation at cryogenic temperature343H6/C3H8 system using nanofillers, and most nanofillers are spherical shape like silica materials and C60, or square zeolitic like imidazolate framework ZIF-8 13143H6/C3H8 was 6.12 and the permeability of C3H6 was 0.098 Barrer with a nanocomposite membrane that contains 30 wt% silica particles under 2 bar feed absolute pressure and 35 °C temperature. Koros et al.3H6/C3H8, and a permeability of 56.2 Barrer for C3H6 with 48.0 wt% ZIF-8 loading, which were 150% and 258% higher than the pure 6FDA-DAM membrane respectively for selectivity and permeability.In recent years, a few studies have been reported about employing mixed matrix platform to the Cet al.26As mentioned above, most available literatures focused on the spherical and square shape nanofillers to enhance the gas separation performance. It is inevitable to generate the ‘sieve-in-a-cage morphology’ between these nanofiller materials and polymer interface due to the low aspect ratio of the spherical and square shape nanofillers and the weak interfacial adhesion. Such voids decrease the selectivity of the MMMs, even the permeability is increased71719202122233H6 diffusion coefficient. Furthermore, massive oxygen functional groups on the surface of graphene nanosheets generate better interfacial bonding strength with EC polymer28293H6/C3H8 separation performance, light reduction graphene oxide (L-rGO) containing oxygen-containing groups such as carboxyl, hydroxyl group and reduction graphene oxide (rGO) were synthesized and incorporated into EC polymer to prepare MMMs for C3H6/C3H8 permeation. The morphology and microstructure of the as-prepared graphene material were confirmed, and the physical properties of MMMs in terms of microstructure, crystallization, tensile property and thermal stability  is selected in this study as the polymer matrix based on its comparatively large free volume and relatively low glass transition temperatures, which might provide high gas permeability and a superb Clity see  were invated see .4 is inadequate for the chemical reduction of the vast oxygen groups on the GO nanosheets. However, intensive deoxygenation processes can be completed with abundant NaBH4 reduction and NaOH hydrothermal treatment, as shown in Different methods of treatment on GO nanosheets may result various morphologies, and these changes can be directly observed through TEM characterization. 2 regions in the graphene were gradually weakened and resulted in a defected surface. After that, these defected sites increasingly expanded and finally generated nanopores in the intensive hydrothermal treatment process. Therefore, an increased ID/IG intensity ratio gradually emerges from light chemical reduction (L-rGO) to chemical reduction (rGO), and then hydrothermal treatment (nanoporous rGO). Combined with the results of TEM images, it can be concluded that nanoporous rGO nanosheets would contain large amount of nanopores as compared with L-rGO and rGO nanosheets.Variation on the morphologies of graphene nanosheets can also be indirectly measured by the Raman spectra. As observed in 32−1 wavenumber are ascribed to the C–O, C=C and O–H groups in the L-rGO with a relative higher intensity in comparison with the rGO and nanoporous rGO nanosheets34−1 in rGO and nanoporous rGO results from the greatly decrease of the oxygen functional groups, indicating the completion of chemical reduction process.The micro-structure of graphene nanosheets were also characterized by FTIR spectroscopy. As shown in In order to further confirm the difference of micro-structure of L-rGO, rGO and nanoporous rGO nanosheets, XPS measurement was conducted . As showXRD is performed to investigate the effects of graphene nanosheets on the EC polymer chains. As shown in 3840The cross-section morphologies of three MMMs are presented in 424445The TEM image of nanoporous rGO MMMs solution further verifies the nanoscale morphology of nanoporous rGO nanosheets. As observed in 3H6 permeability, C3H6/C3H8 ideal selectivity gradually increase with the increase of graphene nanosheets content from 0 to 1.125 wt‰, while C3H8 permeability decreases with the content of graphene nanosheets increase from 0 to 0.375 wt‰ and then remain unchanged after 0.0375 wt‰ of graphene nanosheets loading. However, when the loading of the graphene nanosheets in the EC polymer matrix increases to 1.5 wt‰, both C3H6 permeability and C3H6/C3H8 ideal selectivity decrease, indicating that the graphene nanosheets are theoretically impermeable to all atoms and molecules, and greater loading in the MMMs could cause the agglomeration of the graphene nanosheets, resulting in the decrease of the effective surface area4748493H6 permeability of 66.05, 76.34 and 89.95 Barrer, respectively. Meanwhile, it is clearly showed that the ideal selectivity of pure EC polymer membrane is 3.45 and the permeability of C3H6 and C3H8 is 57.9 and 16.78 Barrer, which is consistence with the related literature3H6 permeability of 1.55-fold and C3H6/C3H8 ideal selectivity of 3.02-fold confirm the fact that the nanopores blended into the MMMs enhance the C3H6 permeability as compared with the pure EC membrane gas permeation performance. However, the blending of graphene nanosheets into the EC polymer matrix hinders the C3H8 permeability. Among three kinds of MMMs, nanoporous graphene MMMs have C3H6/C3H8 ideal selectivity and C3H6 permeability advantages over that of L-rGO and rGO MMMs, which indicates that oxygen-containing functional groups decrease the C3H6 permeability. A reasonable explanation of the gas separation mechanism is proposed as shown in In order to investigate the structure of graphene nanosheets and their loading content on the membrane permeation performances, three kinds of MMMs are fabricated with different nanofiller loading. 3H6/C3H8 diffusivity selectivity of L-rGO MMMs, rGO MMMs and nanoporous rGO MMMs are higher than that of pure EC membrane. It is notable that the nanoporous rGO MMMs has the highest C3H6/C3H8 diffusivity selectivity among L-rGO MMMs and rGO MMMs. The lack of nanopores in the nanosheets hinders the further enhancement of C3H6 diffusivity coefficient, even L-rGO nanosheets and rGO nanosheets generate a rigid interface with EC polymer chains and increase the tortuous pathway of the gas diffusion to enhance the diffusivity selectivity. We can conclude that the nanopores in the nanosheets further increase the tortuous pathway of the C3H6 diffusion with less resistance but restrain the diffusion of C3H8 molecules.To further explore the effect of nanopore in the graphene nanosheets on the gas permeation performance, the diffusivity coefficient (D) and solubility coefficient (S) of the membranes with the optimal 1.125 wt‰ of nanofiller loading are compared in 3H6/C3H8 solubility selectivity of L-rGO MMMs, rGO MMMs and nanoporous rGO MMMs with the optimal 1.125 wt‰ of nanofiller loading. The solubility of C3H6 and C3H8 in the polymer membranes depends on their relative condensability, characterized by critical temperature, the interaction between polymer and gases, the fraction and amount of free volumes in glassy polymers. The critical temperatures of C3H6 and C3H8 are in the following order: C3H6(364.76 K) < C3H8(369.8 K). It is believed that higher condensability has the higher solubility of gas in the polymer matrix5253543H6 exhibits greater solubility than C3H8. This is thanks to the high electron cloud on the double bond with high polarity and flat structure, resulting in more affinity of C3H6 to generate interaction with polymer, and thus enhancing the C3H6 solubility. Moreover, the incorporation of graphene nanosheets increases the density of polar groups such as -COOH and -OH in the MMMs. As a result, it creates polar spaces between the interface of nanofillers and polymer. This formation of polar spaces would result in the enhancement in the solubility coefficient of condensable gases3H6/C3H8 molecule to enhance the ideal selectivity of MMMs, due to the existence of nanopores and the formation of rigid interface.The diffusivity selectivity increases by 5.24-fold in comparison with 2.66-fold for L-rGO MMMs and 2.93-fold for rGO MMMs, although the loading of nanoporous graphene nanosheets in the polymer matrix cannot efficiently increase the solubility selectivity of the gases due to the decrease of the free volume of the glassy polymer. The incorporation of nanoporous graphene nanosheets into the EC polymer matrix mainly affects the diffusivity selectivity of C3H6/C3H8 permeation. The permselectivity of nanoporous rGO MMMs exhibits a significant increase with the simultaneous enhancement of diffusivity selectivity and solubility selectivity, especially the 5.24-fold increase of the diffusivity selectivity. This result indicates that nanoporous rGO nanosheets are superior in terms of enhancing the diffusivity selectivity of C3H6 gas molecule. The ideal selectivity for C3H6/C3H8 and the permeability coefficient for C3H6 exhibit a significant increase of 3.02-fold and 1.55-fold in comparison with the pristine EC membrane, respectively. This study shows that nanoporous rGO nanosheets can be effective nanofiller in the polymer matrix to enhance the C3H6/C3H8 permeability.In summary, nanoporous rGO nanosheets were successfully fabricated by sodium hydroxide using a facile hydrothermal treatment. The morphology and micro-structure of the nanoporous rGO material were confirmed by TEM and XPS measurement. Nanoporous rGO nanosheets were incorporated into the EC polymer matrix to prepare the MMMs for the C4), sodium hydroxide (NaOH), sodium dodecylbenzene sulfonates (SDBS), hydrochloric acid (HCl 36~38%), acetone and all other types of reactants were bought from Shanghai Chemical Co., Ltd. (China). The propylene (purity > 99.5%) and propane (purity > 99.9%) were purchased from KODI Gas Chemical Industry Co. Ltd. .EC  was purchased from Shanghai Reagent Corporation (China). Graphite oxide was obtained from Nanjing XFNANO Materials Tech Co. Ltd. (China). Sodium borohydride .In this case, GO colloidal solution (0.1 g in 150 mL deionized water) was obtained using a mild ultrasonic exfoliation method for 2 h. The above colloidal solution was centrifuged to remove the impurities under 8000 r min4 mass ratio of 1:2 at 80 °C for 0.5 h, and then the above L-rGO was collected by filtration. Subsequently, 10 mL SDBS (0.086 mol L−1) and 20 mL NaOH (12.5 mol L−1) were successively added dropwise to form a 60 mL solution. After stirred for 0.5 h, the reaction solution was decanted into a 100 mL Teflon-lined stainless steel autoclave and conducted the hydrothermal treatment at 180 °C for 3 h. Eventually, the reaction solution was cooled to ambient temperature, and the resulting solution was consecutively filtered and washed with sufficient deionized water (five times) and ethanol, and the nanoporous rGO nanosheets were placed in ethanol at a given concentration (0.012 g in 15 mL ethanol).The nanoporous rGO nanosheets were synthesized with the treatment of sodium hydroxide under hydrothermal condition. Firstly, GO colloidal solution (0.1 g in 150 mL deionized water) obtained from an mild ultrasonic exfoliation method for 2 h was chemically reduced to L-rGO using the GO to NaBHA series of different concentration of L-rGO, rGO and nanoporous rGO ethanol solutions were prepared via sonication for 1 h. Dried EC (in a vacuum situation at 40 °C for 24 h) was dissolved in methylbenzene to form a solution with the concentration of 19.7 wt%. Under a nitrogen atmosphere and at ambient temperature, the above dispensed graphene ethanol solutions were then added dropwise to the EC solutions with stirring, The resulting casting solution had a mass ratio of graphene to EC of 0.375, 0.75, 1.125 and 1.5 wt‰ respectively. After 24 h dissolution, the casting solutions were filtered with stainless steel filter, and still degassing for 24 h. The above casting solution was then cast onto the polyimide (PI) ultrafiltration supported membrane using a micrometer film applicator under RH 40% at 298 K. The final membranes were subsequently preserved for gas separation tests. All membrane thicknesses were approximately 15–20 μm.How to cite this article: Yuan, B. et al. Propylene/propane permeation properties of ethyl cellulose (EC) mixed matrix membranes fabricated by incorporation of nanoporous graphene nanosheets. Sci. Rep.6, 28509; doi: 10.1038/srep28509 (2016).


Neuroinflammation is a striking hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Previous studies have shown the contribution of glial cells such as astrocytes in TDP-43-linked ALS. However, the role of microglia in TDP-43-mediated motor neuron degeneration remains poorly understood. In this study, we show that depletion of TDP-43 in microglia, but not in astrocytes, strikingly upregulates cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production through the activation of MAPK/ERK signaling and initiates neurotoxicity. Moreover, we find that administration of celecoxib, a specific COX-2 inhibitor, greatly diminishes the neurotoxicity triggered by TDP-43-depleted microglia. Taken together, our results reveal a previously unrecognized non-cell-autonomous mechanism in TDP-43-mediated neurodegeneration, identifying COX-2-PGE2 as the molecular events of microglia- but not astrocyte-initiated neurotoxicity and identifying celecoxib as a novel potential therapy for TDP-43-linked ALS and possibly other types of ALS. Familial ALS cases are associated with mutations in genes such as Cu/Zn superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TARDBP) and, most recently discovered, C9orf72. Currently, most available information obtained from ALS research is based on the study of SOD1, but new studies focusing on TARDBP and C9orf72 have come to the forefront of ALS research.2 The discovery of the central role of the protein TDP-43, encoded by TARDBP, in ALS was a breakthrough in ALS research.5 Although pathogenic mutations of TDP-43 are genetically rare, abnormal TDP-43 function is thought to be associated with the majority of ALS cases.1 TDP-43 was identified as a key component of the ubiquitin-positive inclusions in most ALS patients and also in other neurodegenerative diseases such as frontotemporal lobar degeneration,7 Alzheimer's disease (AD)9 and Parkinson's disease (PD).11 TDP-43 is a multifunctional RNA binding protein, and loss-of-function of TDP-43 has been increasingly recognized as a key contributor in TDP-43-mediated pathogenesis.14Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the degeneration of motor neurons in the brain and spinal cord.16 Although numerous studies have focused on the intrinsic properties of motor neurons in ALS, a large amount of evidence showed that glial cells, such as astrocytes and microglia, could have critical roles in SOD1-mediated motor neuron degeneration and ALS progression,22 indicating the importance of non-cell-autonomous toxicity in SOD1-mediated ALS pathogenesis.Neuroinflammation, a striking and common hallmark involved in many neurodegenerative diseases, including ALS, is characterized by extensive activation of glial cells including microglia, astrocytes and oligodendrocytes.29 The role of COX-2 neurotoxicity in ALS and other neurodegenerative disorders has been well explored.32 One of the key downstream products of COX-2, prostaglandin E2 (PGE2), can directly mediate COX-2 neurotoxicity both in vitro and in vivo.37 The levels of COX-2 expression and PGE2 production are controlled by multiple cell signaling pathways, including the mitogen-activated protein kinase (MAPK)/ERK pathway,40 and they have been found to be increased in neurodegenerative diseases including AD, PD and ALS.46 Importantly, COX-2 inhibitors such as celecoxib exhibited significant neuroprotective effects and prolonged survival or delayed disease onset in a SOD1-ALS transgenic mouse model through the downregulation of PGE2 release.28Very interestingly, a vital insight of neuroinflammation research in ALS was generated by the evidence that both the mRNA and protein levels of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) are upregulated in both transgenic mouse models and in human postmortem brain and spinal cord.51 Although the contribution of glial cells to TDP-43-mediated motor neuron degeneration is now well supported, this model does not fully suggest an astrocyte-based non-cell autonomous mechanism. For example, recent studies have shown that TDP-43-mutant astrocytes do not affect the survival of motor neurons,51 indicating a previously unrecognized non-cell autonomous TDP-43 proteinopathy that associates with cell types other than astrocytes.Most recent studies have tried to elucidate the role of glial cells in neurotoxicity using TDP-43-ALS models, which are considered to be helpful for better understanding the disease mechanisms.Given that the role of glial cell types other than astrocytes in TDP-43-mediated neuroinflammation is still not fully understood, we aim to compare the contribution of microglia and astrocytes to neurotoxicity in a TDP-43 loss-of-function model. Here, we show that TDP-43 has a dominant role in promoting COX-2-PGE2 production through the MAPK/ERK pathway in primary cultured microglia, but not in primary cultured astrocytes. Our study suggests that overproduction of PGE2 in microglia is a novel molecular mechanism underlying neurotoxicity in TDP-43-linked ALS. Moreover, our data identify celecoxib as a new potential effective treatment of TDP-43-linked ALS and possibly other types of ALS.The goal of this study is to better understand the role of TDP-43 in neuroinflammation, a key feature of ALS. To gain insight into the roles of microglia and astrocytes in TDP-43-mediated neurotoxicity, we knocked down TDP-43 in primary cultured microglia and astrocytes using siRNA against TDP-43, and we then used biochemical approaches to detect the expression levels of COX-2 and iNOS, two markers of neuroinflammation. RNAi-mediated depletion of TDP-43 in microglia resulted in a greatly increased protein level of COX-2 with or without LPS treatment, whereas iNOS protein level was unchanged . In contTo characterize the molecular mechanism underlying the upregulation of COX-2 in TDP-43-deficient microglia, we examined the changes in cell signaling pathways accompanied by upregulation of COX-2 expression in TDP-43-deficient microglia. Given that the classical MAPK signaling pathway could regulate COX-2 expression and is significantly associated with various neurodegenerative diseases, we hypothesized that TDP-43 might be involved in the regulation of MAPKs. To test this possibility, we detected the total and phosphorylated levels of MEK, ERK, JNK and p38 in TDP-43-deficient microglia. As shown in β inhibitor) and SB203580 (p38 inhibitor), had no effect on TDP-43-mediated COX-2 expression  , and theWe next investigated whether the COX-2 inhibitor celecoxib could alleviate neuronal death driven by TDP-43-depleted microglia. We performed the conditioned medium assay as described in Although initially, most studies on ALS focused on the selective loss of motor neurons themselves, increasing efforts to understand the role of glial cells in disease pathogenesis have come to the forefront of the field. The present study reveals microglia-mediated COX-2-PGE2 production as the molecular determinant of TDP-43-associated neurotoxicity, emphasizing the important contribution of microglia to non-cell-autonomous motor neuron degeneration in TDP-43-linked ALS .51 On the basis of this finding, it is possible that other types of cells surrounding motor neurons are involved in the initial toxic effect of motor neurons. Consistent with this notion, microglia have recently been suggested to have a role in ALS initiation,52 but the contribution of microglia to TDP-43-linked ALS is still not fully understood. In this study, we show that loss of TDP-43 in microglia strikingly triggers the increase of COX-2 , indicating that signaling molecules other than NF-κB are required for this regulation. Relatedly, our results show that knockdown of TDP-43 in microglia resulted in the activation of AP-1 , anti-COX-2 , anti-GAPDH , anti-phospho-MEK (S217/221) , anti-MEK , anti-phospho-ERK (Santa Cruz Biotechnology), anti-ERK (Santa Cruz Biotechnology), anti-phospho-JNK (Epitomics), anti-JNK (Santa Cruz Biotechnology), anti-phospho-p38 (Epitomics), anti-p38 (Santa Cruz Biotechnology), anti-phospho-c-Raf (S338) , anti-c-JUN , anti-phospho-Glycogen Synthase (S641) (p-GS) (Epitomics) and anti-MAP2 (Santa Cruz Biotechnology). The secondary antibodies used included horseradish peroxidase-conjugated sheep anti-mouse and anti-rabbit antibodies . The antibody-bound proteins were visualized using an ECL detection kit  or Alexa Fluor 488 (green) donkey anti-mouse IgG .The anti-TDP-43 antibody was described previously,μM), BAY (Beyotime) (25 μM), SP600125 (10 μM), SB216763  (10 μM), SB203580 (10 μM), celecoxib (Sigma) (50 μM), or PBS (Gibco) or LPS (Beyotime) (0.1 μg/ml). The following sequences were used for siRNA targeting mouse TDP-43 (si-TDP-43): 5′-GGATCTGAAAGACTATTTC-3′ siRNA targeting rat TDP-43 (si-TDP-43): 5′-CCAATGCTGAACCTAAGCA-3′. For cell viability analysis, the cells were stained with PI and visualized using fluorescence microscopy. The dead cells with PI-positive staining were counted and the percentage of viability of cells was quantified.Mouse motor neuron cell line (NSC-34 cells) were cultured in Dulbecco's modified Eagle's medium (DMEM)  containing 10% fetal bovine serum (FBS) (Gibco) with penicillin (100 mg/ml) and streptomycin (100 mg/ml). Mouse microglial cell line (BV2 cells) were cultured in similar culture medium, but using 10% heat-inactivated FBS instead. For RNAi experiments, cells were transfected with siRNA against TDP-43 using the RNAiMAX (Invitrogen) transfection reagent according to the manufacturer's instructions. Subsequently, cells were treated for 24 h with DMSO  or U0126   for 15 min at 37 °C, and then the cells were dissociated with a plastic pipette. Subsequently, cells isolated from cortex tissue were plated on poly-D-lysine-coated 24-well plates  and cultured at 37 °C, 5% CO2. Mixed glial cells were cultured in DMEM/F12 with 10% heat-inactivated FBS and penicillin (100 mg/ml) and streptomycin (100 mg/ml), and the culture medium were replaced every 3 days. After 14 days, microglia were separated from mixed glial cells by shaking at 150 rpm for 2 h at 37 °C. Primary cultured astrocytes were similarly dissociated from the cortex of new born ICR mice and cultured in DMEM with 10% FBS and penicillin (100 mg/ml) and streptomycin (100 mg/ml). Cortical neurons were similarly dissociated from the cortex of ICR mouse embryos at embryonic day 17 (E17), and motor neurons were similarly dissociated from the spinal cord of ICR mouse embryos at E17 as previously described.68 In brief, the dissociated neurons were cultured in neurobasal medium (Gibco) with 10% FBS, 1 × B27 (Gibco) and glutamine  for 12 h. Subsequently, culture medium were change to neurobasal medium with 1 × B27 and glutamine (0.05 mg/ml). After 5 days, the neurons were subjected to experiments. The purity of motor neurons were shown in Primary cultured microglia were prepared as described elsewhere.Cells were harvested and lysed in cell lysis buffer (50 mM Tris-HCl (pH 7.6) with protease inhibitor cocktail , 150 mM NaCl, 0.5% sodium deoxycholate, and 1% Nonidet P-40). Then, the proteins were separated by 10% or 12% SDS-PAGE (polyacrylamide gel electrophoresis) and transferred onto polyvinylidene difluoride membranes .β-actin: 5′-GACCTGACTGACTACCTC-3′ and 5′-GACAGCGAGGCCAGGATG-3′, mouse iNOS: 5′-TCCCAGCCTGCCCCTTCAAT-3′ and 5′-CGGATCTCTCTCCTCCTGGG-3′, and mouse COX-2: 5′-CAGGCTGAACTTCGAAAC A-3′ and 5′-GCTCACGAGGCCACTGATACCTA-3′.Total RNA from BV2 cells was extracted with TRIzol Reagent (Invitrogen); subsequently, the RNA was reverse-transcribed into cDNA using PrimeScript RT Master Mix . Real-time PCR analysis was performed using SYBR Green Real-Time PCR Master Mix (Takara) using a CFX96 Real-Time System  and the following primers: mouse μl of 400 μl medium were measured with PGE2 ELISA kits  according to the manufacturer's instructions.Primary cultured microglia and BV2 cells were transfected with siRNA against TDP-43. After transfection, the cells were treated with U0126 or celecoxib for 24 h, and then the levels of PGE2 in 50 μg/ml of puromycin. After 5 days, survived BV2 cells were selected and the BV2 stable cell line expressing AP-1 reporter construct was generated. Subsequently, the BV2 cells stably expressing AP-1 constructs were transfected with siRNA against TDP-43. After 48 h, the cells were treated with U0126 for 24 h, and then the cells were harvested. Finally, the AP-1 promoter activity was measured using the luciferase assay kit  according to the manufacturer's instructions.BV2 cells were plated on 24-well plate and incubated for 16 h at 37 °C. Then the cells were transfected with Cignal lentiviral AP-1 Reporter (luc)  according to the manufacturer's instructions for 24 h. Subsequently, the culture medium were replaced with fresh medium. After another 24 h, the cells were cultured in culture medium supplement with 2.5


The films are grown under industrially safe conditions, i.e., the flammable gases (H2 and CH4) are diluted to well below their lower explosive limit. The high quality, spatial uniformity, and low density of domain boundaries are demonstrated by charge carrier mobility measurements, scanning electron microscope, electron diffraction study, and Raman mapping. The hole mobility reaches as high as ~5,700 cm2 V−1 s−1 in ambient conditions. The growth process of such high-quality graphene with a low H2 concentration and short growth times widens the possibility of industrial mass production.We demonstrate the growth of continuous monolayer graphene films with millimeter-sized domains on Cu foils under intrinsically safe, atmospheric pressure growth conditions, suitable for application in roll-to-roll reactors. Previous attempts to grow large domains in graphene have been limited to isolated graphene single crystals rather than as part of an industrially useable continuous film. With both appropriate pre-treatment of the Cu and optimization of the CH Due to its outstanding properties676913141517181920152223262728292 at typical growth conditions is a particular problem (the explosive limit of H2 is 4–75% (volume concentration) at room temperature in air). Although there are several reports on graphene growth using no H213322222934353637382V−1s−1, which is not qualified for “electronic-grade” applications2-free growth removes a useful second parameter for control over graphene quality, coverage, and domain size. Thus, the growth of graphene with a high charge carrier mobility is still a challenge under industrially safe CVD conditions.In addition, the safety issue of the CVD process is usually ignored, despite its importance in industrial production. Further economic development would desire the growth at large scale by roll-to-roll reactors, preferentially under intrinsically safe and atmospheric pressure conditions. The flammability of H2 (2%) in 80 min. The average domain size reaches ~1 mm, which is the largest achieved to date for continuous graphene. The hole mobility reaches as high as 5,700 cm2V−1s−1, the highest value among the reported mobilities using similarly low concentrations of H2. We achieve this by combining electropolishing and non-reductive annealing of the Cu foils with optimizing the CH4 concentration. This work brings the current growth method of graphene a large step towards scalable and safe production.In this paper, we demonstrate the growth of continuous monolayer graphene with millimeter-sized domains using diluted H4 and H2 that are diluted to well below their low explosive limit (LEL). 4, respectively. The as-grown samples are baked in air (at 200 °C for 1 min) to visualize the graphene-covered regions. In 60 min, the graphene partly covers the Cu substrate with ~70% coverage. Some isolated domains reach as large as ~1.5 mm , transmission electron microscope (TEM), OM, and Raman spectroscopy.2V−1s−1 (mean ± standard deviation) based on the Drude model12 cm−2. As a reference, we also measure a graphene sample grown by the typical low pressure CVD using H2 annealing as well as a commercial graphene sample. The mobility and carrier concentration of the reference samples are  and  respectively. Compared to the reference samples and the reported values for continuous monolayer graphene, the mobility of our graphene is notably higher than all other results using similarly low concentrations of H2 . The intensity ratio of the 2D peak to the G peak (I2D/IG) is ~2. The 2D peak is of Lorentzian shape with one single component and the full width at half maximum (FWHM) is ~26 cm−1. These are the fingerprints of high quality monolayer grapheneThe SEM image in 2 area. The I2D/IG, ID/IG, and FWHM of the 2D peak reaches 2.3  ±  0.2  drops from 320–100 nm over a 50 μm range as shown in We now discuss the essential factors for synthesizing the continuous graphene films while still achieving millimeter-sized domains under 2% HThe effect of electropolishing on graphene nucleation is shown in 2 diluted in Ar), as can be seen in 2  which is found to drastically reduce the nucleation density, by around 30-fold, compared with annealing in reductive gas  are electropolished in a home-built cell. Two pieces of 10  ×  10 cm2 (2%), and 0.20–0.80 sccm CH4 (50–200 ppm) for 10–80 min, followed by cooling down to room temperature under the same Ar and H2 flow (without CH4). The gas pressure is maintained at 1 atm.The polished Cu foil is loaded into a 2-inch quartz tube furnace, which is then heated up to 1030 °C at ~20 °C/min under 1 atm with 400 sccm (standard cubic centimeter per minute) Ar. The Ar flow rate is then increased to 4,000 sccm to anneal the Cu for 5 min. Graphene growth is carried out by flowing 3,920 sccm Ar, 80 sccm HThe post-growth graphene on Cu foils are baked at 200 °C for 1 min in air on a hot plate to oxidize the Cu areas that are not covered by graphene. The colour contrast between the graphene-covered area and exposed area can visualize individual domains2(300 nm)/Si wafer using a poly(methyl methacrylate) support layer and 0.05 M aqueous solution of (NH4)2S2O8 as Cu etchant2 (300 nm) /Si wafers with 50 × 50 μmFor charge carrier mobility measurement, the graphene film is transferred onto a SiO2(300 nm)/Si wafer. For electron diffraction study, the graphene film is transferred onto a 3 mm-diameter holey carbon copper grid (Agar Scientific No. AGS147).The samples are characterized by optical microscope (Nikon ECLIPSE LV150N), scanning electron microscope , transmission electron microscope , Raman spectroscopy , and optical profilometer (Wyko NT1100). For Raman characterization, the graphene film is transferred onto a SiOHow to cite this article: Wu, X. et al. Growth of Continuous Monolayer Graphene with Millimeter-sized Domains Using Industrially Safe Conditions. Sci. Rep.6, 21152; doi: 10.1038/srep21152 (2016).


Graphene nanostructures are attracting a great deal of interest because of newly emerging properties originating from quantum confinement effects. We report on using interferometric lithography to fabricate uniform, chip-scale, semiconducting graphene nanomesh (GNM) with sub-10 nm neck widths . This approach is based on fast, low-cost, and high-yield lithographic technologies and demonstrates the feasibility of cost-effective development of large-scale semiconducting graphene sheets and devices. The GNM is estimated to have a room temperature energy bandgap of ~30 meV. Raman studies showed that the G band of the GNM experiences a blue shift and broadening compared to pristine graphene, a change which was attributed to quantum confinement and localization effects. A single-layer GNM field effect transistor exhibited promising drive current of ~3.9 μA/μm and ON/OFF current ratios of ~35 at room temperature. The ON/OFF current ratio of the GNM-device displayed distinct temperature dependence with about 24-fold enhancement at 77 K. Graphene has risen as a fascinating two-dimensional 2D) material for its high carrier mobilityD materia57911121314151617181920222021252729The fabrication of graphene nanomesh for practical applications demands efficient nanopatterning technologies to inexpensively produce nanoscale periodic modulations over large-areas. To our knowledge, a reliable technology with a wafer-scale fabrication capability for graphene nanomesh is still a challenge. E-beam lithography is a highly versatile technique to fabricate graphene nanostructures. However, it is not an economically viable approach when large-scale production is required342) plasma treatment to fabricate GNMs with sub-10 nm neck widths and high uniformity over a 1 × 1 cm2 area. Our approach is facile, inexpensive, and high throughput; furthermore it is based on well-established processing steps, which have been extensively characterized for decades. Finally our approach is compatible with the integrated circuits (IC) manufacturing technologyWe demonstrate the use of interferometric lithography (IL)3839404142432/p+ silicon substrate . The corresponding SEM images are shown in 2 plasma etch.The narrow neck regions of the GNM strongly affect the charge transport through the structure2 wafer. The AFM profile studies on a set of samples with different etching durations  of 32 cm−1. For the unpatterned graphene, only a low-intensity or no D band was observed. In contrast, for GNMs with varying neck widths, we observe an increase in the relative intensity of the D band (centered at 1352 cm−1) as the mesh neck width decreases (ID/IG ratio of the unpatterned graphene and GNMs with different neck widths are shown in −1) and blue shifted by 11 cm−1 compared to the G band dominating the spectrum of unpatterned graphene. This trend may be attributed to quantum confinement or localization effects resulting from edge disorders such as variable edge roughness in GNM50512 plasma under a condition that is known to form oxygen-containing functional groups in graphite−1 by a maximum shift of 17 cm−1 with respect to the unpatterned graphene. Also, the measured FWHM of the G bands at the four locations are within a range of 42–47 cm−1, indicating that, on average, the G band of the GNM is 10 cm−1 broader than that of the unpatterned graphene. The blue shift and broadening of the G band for the narrowest GNM is likely due to the lateral quantum confinement and localization effects and/or chemical doping by functional groups on the GNM edges.In order to study the physical and electronic characteristics of the processed samples, GNMs with varying neck widths were characterized using Raman spectroscopy (see Methods). The Raman spectrum acquired from unpatterned graphene  as a function of gate voltage (Vg) at a constant source-drain voltage (Vds) are shown in Vds = −1 V. This is due mainly to a large GNM conduction channel filled with high density graphene ribbons capable of driving high currents. Importantly, at Vds = −100 mV, although the ON-state current density is reduced to ~2.5 μA/μm, the ON/OFF current ratio has increased up to ~35. The modulation of the ON/OFF current ratio by varying the source-drain voltage is a characteristic of the GNM as it has not been reported for pristine graphene devicesds indicates the semiconducting nature of the GNM36Id−Vg transfer curves , where kB is Boltzmann constant and T is the absolute temperaturekBT << Eg) was not sufficient to tunnel through the gap or hop through edge states and therefore the OFF-current state drops markedly. This temperature dependence is in strong contrast to that observed in unpatterned graphene FETs, where the pristine graphene is semi-metallic with a zero bandgap, and the temperature dependence of the transfer curves was negligible  by the experimentally derived equation Eg ~ α/w, where α is between 0.3 and 1.5 with unit [nm eV]The effective mobility of holes in the SL-GNM produced by IL are estimated to be ~80 cmEg = 30 meV for a GNM with an average neck width of ~10 nm. As shown in 21Using the measured ON/OFF current ratio at various temperature and inverting cm2 areas. The fabrication of GNM using IL is scalable and can be performed using standard top-down fabrication methods. The fabricated GNM exhibited a semiconducting behavior with an estimated bandgap ~30 meV. GNMs with various neck widths in the range of 50-10 nm have been fabricated on a macroscopic scale with an unprecedented level of control. Raman studies have shown a significant broadening and blue shift of the G band for the narrowest GNM. Such blue shift is attributed to quantum confinement effects. The GNM devices have been fabricated and they have shown drive currents ~3.9 μA/μm, ON/OFF current ratios ~35 at room temperature and ~215 at 77 K, and mobilities suitable for field- effective devices operating at room temperature. The availability of direct bandgap GNMs over large areas provides exciting opportunities for graphene-based photodetectorsIn summary, we have proposed and demonstrated a simple yet effective approach to produce highly uniform graphene nanomesh structures with sub-10 nm neck width across 1 × 1 2/+p Si substrate with 95% uniformity were prepared from Graphene Supermarket Inc. The GNM-FETs were fabricated using standard photolithography  solution (6:1 volume ratio of 40% NH4F in water to 49% HF in water), a 100 nm-thick Au film was sputtered onto the Si substrate. Then, source and drain electrodes were deposited using photolithography followed by electron-beam evaporation of Ti/Au (5 nm/95 nm). The channel length (L) of the transistor varies from 2–14 μm and the channel width (W) is 2 mm, providing maximum channel area of 28 × 103 μm2. Next, interferometric lithography was performed on the fabricated FET. A negative tone-resist (NR7-500PY) with nominal thickness 500 nm was spun on the sample, followed by two IL exposures using a 355-nm tripled YAG laser source at an incidence angle θ = 30° with a 90° rotation of the sample. The laser exposure time was 10 seconds, with pulse rate of 60 Hz and pulse energy of 100 mJ. After a hard bake at 110° for 60 seconds, the sample was developed in an MF-321 developer for 30 seconds and subsequently a 2D hole array pattern was made in the PR. Optimized O2 plasma etching  was performed for 80 seconds to reduce the PR linewidth and remove graphene in selected areas. The PR/GNM lateral etch rate was 0.8 nm/sec and the vertical etch is estimated to be 1.8 nm/sec. This etching recipe resulted in significant reduction in the PR width, while the PR thickness was only reduced by ~30 percent. Once the etching process is complete, the PR is removed by sample soak in acetone to obtain the final GNM. The samples were annealed at 300 °C in forming gas environment  for 30 min to remove/reduce the polymer resist from the etching mask, and other types of impurities on the mesh surface.High-quality SL CVD graphene wafers on 285 nm SiOhography . The hea−5 Torr) to eliminate/reduce the hole doping effects due to absorption of oxygen and water molecules at the graphene surface. The probe station was equipped with a Scientific Instruments M9700 temperature controller, and the measurements were carried out with a Keithley 2400 source measure unit (SMU) and Keithley 238 high-current SMU. For comparison purposes, the GNM can be approximated as a mesoscopic honeycomb network of GNR in which the ribbons are interconnected between larger graphene islands. Therefore, the electron and hole mobilities of the GNM were estimated using the formula μ = (gL/VdsCoxW), where g is the transconductance , where ε0 is the vacuum permittivity, εr and d are the dielectric constant and thickness of the SiO2 dielectric layer. It should be noted that in order to study the GNM mobility more accurately, the fringe effect at the ribbon edges of the GNMs and also the exact capacitance of the transistor must be considered, which requires a simulation model based on finite element analysis. SEM imaging was performed on a JEOL- 6500 field-emission microscope. The Raman experiment was carried out by focusing a 532 nm excitation light (output of laser diode) through a dry objective with numerical aperture 0.95. Prior to the Raman studies, the samples were annealed in forming gas environment  to remove polymer resist from the etching mask, and other types of impurities existed on the mesh surface. The scattered light is collected using the same objective and is directed to a CCD spectrometer . The AFM (Neaspec GmbH Inc.) was operated in tapping mode with resonance frequency of cantilever ~250 kHz and tapping amplitude ~50 nm. An ultra-sharp AFM tip (Hi’Res-C14/Cr-Au) with a spike radius of 1 nm was used. Statistical analysis on the acquired AFM images was carried out in AutoCAD software with careful tip corrections.The electronic properties of mesh-patterned graphene devices were characterized by a JANIS ST-500 micro-manipulated probe station under vacuum .


Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the loss of motor neuron-like cells. Mutations in the RNA- and DNA-binding proteins, fused in sarcoma (FUS) and transactive response DNA-binding protein 43 kDa (TDP-43), are responsible for 5–10% of familial and 1% of sporadic ALS cases. Importantly, aggregation of misfolded FUS or TDP-43 is also characteristic of several neurodegenerative disorders in addition to ALS, including frontotemporal lobar degeneration. Moreover, splicing deregulation of FUS and TDP-43 target genes as well as mitochondrial abnormalities are associated with disease-causing FUS and TDP-43 mutants. While progress has been made to understand the functions of these proteins, the exact mechanisms by which FUS and TDP-43 cause ALS remain unknown. Recently, we discovered that, in addition to being up-regulated in spinal cords of ALS patients, the novel protein oxidative resistance 1 (Oxr1) protects neurons from oxidative stress-induced apoptosis. To further understand the function of Oxr1, we present here the first interaction study of the protein. We show that Oxr1 binds to Fus and Tdp-43 and that certain ALS-associated mutations in Fus and Tdp-43 affect their Oxr1-binding properties. We further demonstrate that increasing Oxr1 levels in cells expressing specific Fus and Tdp-43 mutants improves the three main cellular features associated with ALS: cytoplasmic mis-localization and aggregation, splicing changes of a mitochondrial gene and mitochondrial defects. Taken together, these findings suggest that OXR1 may have therapeutic benefits for the treatment of ALS and related neurodegenerative disorders with TDP-43 pathology. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the loss of motor neuron-like cells, which leads to progressive muscle atrophy and death within 1–5 years. Genetic mutations have been found to cause both sporadic (sALS) and familial ALS (fALS) cases; in addition to mutations in superoxide dismutase 1 (SOD1) and C9ORF72, fused in sarcoma (FUS) and transactive response DNA-binding protein 43 kDa (TDP-43) are responsible for ∼5–10% of fALS cases and ∼1% of sALS cases . FUS andin vitro and disease severity in ALS patients (N-methyltransferase 1 (PRMT1) and direct interaction with transportin regulate FUS shuttling between the nucleus and cytoplasm  is up-regulated in the spinal cord of end-stage ALS patients as well as in a pre-symptomatic mouse model of ALS . Mice landitions ; our finnditions . Here, win vitro and in vivo  and at 120 min for Oxr1-C (∼11-fold)   compared with Hin silico pathway analysis on the Oxr1 binding partners. We identified EIF2 and mTOR, two pathways involved in the oxidative stress response . Interestingly, co-immunoprecipitation showed that the interaction between Oxr1-C and Fus or Tdp-43 was altered by a specific subset of ALS-associated mutations  and transactive response DNA-binding protein 43 kDa (Tdp-43) as Oxr1 binding partners. Given that FUS and TDP-43 mutations are associated with ALS and that OXR1 may be neuroprotective in this disease ,15,51,61ons Fig. B. In par2O2-induced stress, wild-type and Fus and Tdp-43 mutants form cytoplasmic aggregates that preferentially localize to stress granules, as labelled by the marker cytotoxic granule-associated RNA-binding protein (TIA-1)   ,34–42.FOXR1 isoforms by ∼87.9% results in increased percentage of cells with Fus- and Tdp-43-positive cytoplasmic inclusions . Taken together, these findings suggest that interaction between Oxr1 and Fus or Tdp-43 is important for proper cellular localization of Fus and Tdp-43 under oxidative stress, and over-expression of Oxr1 reduces mis-localization of certain ALS-Fus and Tdp-43 mutants.We next examined whether modulating Oxr1 levels could alter the cellular localization of wild-type or ALS-linked Fus and Tdp-43 mutants under oxidative stress. Interestingly, shRNA knockdown of all endogenous We next investigated mechanisms by which Oxr1 regulates Fus and Tdp-43 recruitment to stress granules. Both the proteasome and autophagy pathways are implicated in the clearance of Fus and Tdp-43 cytoplasmic inclusions –68. MoreSupplementary Material, Fig. S2A). Similarly, we confirmed that Fus binds to Prmt1 . However, methylation of over-expressed Fus is not affected by AMI-1 treatment . Furthermore, binding of over-expressed Oxr1-C with over-expressed Fus and Tdp-43 is not dependent on Prmt1 activity . However, we observed significantly increased number of cells with Fus-positive cytoplasmic aggregates after AMI-1 treatment, confirming that the presence of Prmt1 and Fus methylation is required for Fus localization to the nucleus . However, when cells were treated with arsenite to induce oxidative stress, we observed a slight but significant decrease in the ratio of exon exclusion to inclusion of Mtfr1 only in cells transfected with Tdp-43 M337V . In addition, a significant reduction in the total number of mitochondria was observed in cells over-expressing Tdp-43 M337V both in non-treated and treated conditions . We then tested if over-expression of Oxr1-C combined with Tdp-43 M337V restores normal mitochondrial morphology. In untreated cells, over-expression of Oxr1-C significantly increases mitochondria size to the level of cells transfected with empty vectors, and although mitochondria number is increased in cells co-transfected with Oxr1-C, it does not return to control levels .dynamics ,92, thusdynamics ,93. SimiSupplementary Material, Fig. S4C). Importantly, we found that over-expression of Oxr1-C with Tdp-43 M337V restores mitochondrial respiration to levels of empty vectors-transfected cells . Taken together, these data suggest that Oxr1-C improves certain aspects of mitochondrial dysfunction in ALS-associated Tdp-43 M337V mutant cells.We next tested whether the changes in mitochondrial morphology associated with the Tdp-43 M337V mutant affected mitochondrial function by analysing mitochondrial respiration. NSC-34 cells were transfected with various ALS-Tdp-43 mutants, and mitochondrial oxygen consumption was measured. Of all the Tdp-43 mutants tested in non-treated and oxidative stress conditions, only cells over-expressing Tdp-43 M337V show a significant decrease in mitochondrial respiration under non-treated conditions , as well as the EIF2 signalling; these pathways are well established as essential for the regulation of the stress response and cell survival  regulates the innate immune response and tolerance to infection  and Eef1a1, two nuclear transport proteins, were identified as Oxr1-C binding partners. Transportin is essential for the translocation of proteins through the nuclear pore complex  and Eef1in silico analysis of Oxr1 binding partners identified RNA-post-transcriptional modification as a downstream function regulated by Oxr1-C, and given the known function of Fus and Tdp-43 as splicing regulators, we assessed the splicing of genes that modulate mitochondrial morphology and function. Interestingly, we found that co-expression of Oxr1-C with Tdp-43 M337V rescued the splicing changes of a mitochondrial fission gene, Mtfr1, associated with Tdp-43 M337V specifically under oxidative stress, which suggests a function for Oxr1 in mitochondrial fission and replication. A recent study demonstrated that co-expression of the mitochondrial fusion protein mitofusin 2 (Mfn2) with Tdp-43 M337V mutant reduced the Tdp-43 M337V-induced mitochondrial morphological and functional abnormalities . NSC-34 cells were transfected using Magnetofection (Oz Biosciences) as per manufacturer's instructions. One day after transfection, cells were treated with H2O2 (Sigma) or sodium arsenite (Sigma) for dose and duration indicated in text. For proteasome inhibition experiments, cells were treated with 10 µm MG-132  for 4 h before subsequent oxidative stress treatments. For inhibition of autophagy-lysosomal degradation or arginine methylation, cells were treated with 1 µm Concanamycin-A (Santa Cruz) or 100 µm AMI-1 (Sigma), respectively, for 12 h before subsequent oxidative stress treatments.Negroid cervix epitheloid carcinoma (HeLa), Neuro2a (N2a) and mouse motor neuron-like cell line (NSC-34) were grown in DMEM supplemented with 10% FBS, 2 m2O2 treatment, cells were washed twice with cold PBS and lysed with lysis buffer  complemented with a cocktail of protease inhibitors (Roche). Lysates were cleared by centrifugation  and 50 μl of pre-equilibrated EZview Red Anti-HA (Sigma) was added to the lysates and incubated overnight at 4°C. Immunoprecipitated proteins were then washed five times with lysis buffer. Proteins were resuspended in NuPAGE LDS sample buffer (Life Technologies) and boiled for 5 min. Samples were run through the stacking gel of a NuPAGE gel (Life Technologies) and the entire band was cut and sent for trypsin digestion and analysis by LC-MSMS sequencing identification . The list of protein targets were analysed using Ingenuity software. The P-values calculated by the software are a measure of the likelihood that the association between the set of proteins identified in the experiment and a given process or pathway is due to random chance. The P-value for a given function is calculated by considering the number of functional analysis molecules that participate in that function and the total number of molecules that are known to be associated with that function in the Ingenuity Knowledge Base.To study interactors of Oxr1 (both full length and short isoforms), N2a cells were transfected with the HA-tagged Oxr1 full length or short isoform in pCX vector. Cells were transfected for 24 h. After HFor immunoprecipitation of HA, MYC and FLAG-tagged proteins to confirm binding targets, cleared protein extracts were incubated overnight at 4°C with EZview Red Anti-HA, MYC or FLAG Affinity Gel beads (Sigma), with no pre-clearing step. Immunoprecipitated proteins were washed three times with lysis buffer and resuspended in 20 μl of sample Laemmli buffer (Biorad) and protein extracts were run on a 4–12% or 10% pre-cast gels . Western blots were probed with the primary antibodies described below and with secondary antibody anti-HRP rabbit or mouse (Life Technologies) using ELC (Amersham) and Clean Blot IP detection reagents (ThermoScientific) with an ImageQuant LAS4000 (Amersham) or a Compact X4 (Xograph Imaging system).Primary antibodies used were as follows: mouse anti-FLAG ; rabbit anti-FUS ; rabbit anti-HA ; mouse anti-mono and dimethyl arginine ; mouse anti-Myc ; rabbit anti-Oxr1 HeLa, N2a and NSC-34 cells were rinsed with PBS, fixed with 4% paraformaldehyde for 15 min, and incubated with 0.1% Triton 100X for 20 min at room temperature. Primary antibodies against TIA-1 , COX4 , DCP1A , FLAG ; FUS ; rabbit HA ; Myc  and TDP-43  were incubated from 1 h to overnight at room temperature or 4°C depending on antibody. After three washes with PBS, coverslips were incubated with Alexa anti-mouse, rabbit, rat, or goat secondary antibodies  for 1 h or overnight. Coverslips were subsequently washed and mounted in DAPI medium (VectorLabs). Images were captured by Axioplan-2 Imaging fluorescent microscope (Carl Zeiss) or TCS SP5II confocal microscope (Leica Microsystems CMS GmbH).Gapdh was used as control gene (Table RNA was extracted using TRIzol reagent (Invitrogen) followed by purification and DNAse I-treatment on a mini column (Qiagen) as per the manufacturer's instructions. RNA was reverse transcribed using reverse transcriptase (Roche) and cDNA was used in a quantitative RT–PCR reaction using SYBR Green PCR master mix (Applied Biosystems) and a Step One real-time PCR machine (Applied Biosystems). ne Table .Table 2RNA was extracted from transfected NSC-34 cells using a mini column (Qiagen) as per the manufacturer's instructions and subjected to DNAse I treatment (Qiagen). cDNA was generated using reverse transcriptase and oligo dT primers (ThermoScientific). To quantify splicing, cDNA was used in a PCR reaction with Red Taq enzyme (Sigma) and run on a 2% agarose gel and visualized using an Alphalmager 3400. The forward and reverse primers for each reaction were located in exons situated on either sides of an exon indicated in bracket on the figures  plate reader . The oxygen probe signal was measured using a time-resolved mode at excitation and emission wavelengths of 380 and 650 nm, respectively. The slope was calculated as the slope of the linear part of the oxygen consumption curve and the percentage was calculated using values for cells transfected with empty vectors as 100%.Oxygen consumption assay was carried out as per the manufacturer's protocol  with minor changes. NSC-34 cells were plated at ∼3.3 × 102: 10-infinity; circularity: 0.00–1.00). Thirty to 40 cells per condition were analysed.To quantify COX IV-stained mitochondria in NSC-34 cells, confocal images (1024 × 1024 pixels) were captured using a Leica TCS SP5II confocal microscope  with a LAS AF software. Images were then exported and processed with NIH ImageJ FIJI software. The entire range of grey values (0–255) was covered by contrast optimization. Then, binary images of mitochondria were generated. The number of mitochondria per cell and the average area of a mitochondrion were subsequently evaluated by particle analysing tool ; the European Research Council (PAROSIN to P.L.O.) and the Clarendon and US–UK Fulbright Scholarships (to K.X.L.). Funding to pay the Open Access publication charges for this article was provided by the University of Oxford RCUK Open Access Block Grant.This work was supported by the


Three distinct transport regimes for increasing dopant concentration could be identified. First the agglomeration of dopants into clusters provides a route to increase the graphene conductivity through formation of ordered scatterers. As the cluster grows, the charge transfer efficiency between graphene and additional dopants decreases due to emerging polarization effects. Finally, large dopant clusters hinder the carrier motion and cause percolative transport that leads to an unexpected change of the Hall effect. The presented results help identifying the range of beneficial doping density and guide the choice of suitable dopants for graphene’s future applications.Graphene’s low intrinsic carrier concentration necessitates extrinsic doping to enhance its conductivity and improve its performance for application as electrodes or transparent conductors. Despite this importance limited knowledge of the doping process at application-relevant conditions exists. Employing  Graphene, a two-dimensional carbon system, is considered an enabling material in many fields. One of the earliest commercial uses could be as a transparent conducting film for touch screens or display applications or as electrodes in energy storage solutionsEnhanced performance of these devices requires the reduction of graphene’s electrical resistance. The most common approach for this task is to increase graphene’s intrinsically low carrier density through extrinsic dopingDespite significant efforts, the enhancement in carrier concentration by doping is limited and with it the sheet resistance reduction3in-situ study of the effect of doping on the transport properties of graphene. A measurement system was employed that combines sheet resistance, Hall effect, and Raman measurements  and its correlation with carrier transport.We here present an urements  which alThree distinct transport regimes could be identified for these dopants: At low dopant concentration, the agglomeration of dopants into clusters provides a route to increase the graphene conductivity through formation of ordered scatterers. As these clusters grow, the charge transfer efficiency from dopants to graphene decreases due to developing electric fields. Finally, at high dopant concentration, large dopant clusters hinder the carrier motion and cause percolative transport. These findings reveal the importance of the dopant morphology as factors benefitting and limiting the carrier transport in two-dimensional materials.4 at a pressure of 100 mTorr. Thus grown graphene was then transferred onto SiO2 samplesGraphene was synthesized by chemical vapor deposition on Cu foil following previous reportsThe samples were attached to a home-made measurement setup : A LabVi3, and HNO3.Three different dopants were investigated, i.e. UV generated ozone, AuClOzone was generated through dissociation of air by UV irradiation. For this purpose, a light source with emission at 185 nm and 254 nm was positioned in the vicinity of the sample .3 doping was achieved by placing the sample holder upside-down onto a vial of 0.1M aqueous AuCl3 solution. After the measurement was started, the vial was heated to 80 °C to initiate evaporation of the solution. Condensation of the solution on the sample resulted in a continuous increase of AuCl3 coverage with time. Continuous HNO3 doping was achieved with the same approach using a 0.25M solution of HNO3 in water.Continuous AuCl3 and AuCl3 vapor exposure  in the late 1950sWe now turn to the high doping concentration regime. EFM imaging in this range reveals that clusters have extended far enough to merge with neighboring clusters inset of . In thisρH is a robust measure of carrier density even in the presence of disorder, such as grain boundaries, polycrystallinity or random defectsHall effect measurements, that are generally regarded as the gold standard in the measurement of carrier concentration and mobilityThe dependence of Hall effect measurements on the properties of both phases allows us to extract the characteristics of adsorbate-covered graphene. In percolative systems the Hall resistance scales with the sheet resistance and the Hall resistances of the constituting phasesOne way of accomplishing this goal is to control the position of the adsorbate clusters. We employed microsphere lithographyWithout the effects of percolation influencing the Hall effect measurements, we can see that the mobility decreases continuously as more and more dopants adsorb and clusters grow throughout the experiment. The carrier density, however, reaches saturation as the clusters extend beyond a critical size which corroborates our explanation of the importance of dopant morphology on the limits of doping.Clustering of adsorbates is necessary to cause doping-induced enhancement of the graphene conductivity.Electric fields that develop in growing clusters limit the maximum achievable doping. The achievable limit is only weakly dependent on the nature of the adsorbate.The adsorbate phase is almost insulating and its main role is to provide carriers to the pristine region without taking part in the carrier transport significantly.The presented results reveal several important characteristics of the doping process.Concluding from these observations we identify the dopant cluster dimension as the most important factor for graphene doping. To achieve high mobility, dopants should be concentrated in few large clusters. However, efficient charge transfer from adsorbates to graphene requires many small clusters.3 and HNO3 seem to produce more dopant clusters of smaller dimensions which decrease the sublinearity but enhance the charge transfer and maximum charge density. These results are confirmed by AFM imaging of AuCl3 clusters at different exposures .


Graphene research and in particular the topic of chemical functionalization of graphene has exploded in the last decade. The main aim is to increase the solubility and thereby enhance the processability of the material, which is otherwise insoluble and inapplicable for technological applications when stacked in the form of graphite. To this end, initially, graphite was oxidized under harsh conditions to yield exfoliated graphene oxide sheets that are soluble in aqueous media and amenable to chemical modifications due to the presence of carboxylic acid groups at the edges of the lattice. However, it was obvious that the high-defect framework of graphene oxide cannot be readily utilized in applications that are governed by charge-transfer processes, for example, in solar cells. Alternatively, exfoliated graphene has been applied toward the realization of some donor–acceptor hybrid materials with photo- and/or electro-active components. The main body of research regarding obtaining donor–acceptor hybrid materials based on graphene to facilitate charge-transfer phenomena, which is reviewed here, concerns the incorporation of porphyrins and phthalocyanines onto graphene sheets. Through illustrative schemes, the preparation and most importantly the photophysical properties of such graphene-based ensembles will be described. Important parameters, such as the generation of the charge-separated state upon photoexcitation of the organic electron donor, the lifetimes of the charge-separation and charge-recombination as well as the incident-photon-to-current efficiency value for some donor–acceptor graphene-based hybrids, will be discussed. Therefore the properties of the resulting rGO significantly deviate from those of pristine graphene. Hence, this particular approach in not suitable for applications in which the novel electronic properties of graphene are of primary importance. Alternatively, wet exfoliation of graphite followed by functionalization is a more efficient strategy. Although functionalization of exfoliated graphene can be achieved by either covalent anchoring of organic molecules onto the graphene lattice  1,3-Dipolar cycloaddition of in situ generated azomethine ylides to introduce fused pyrrolidine rings into the skeleton of graphene . Azometh[1 + 2] Cycloaddition of malonate derivatives, the so-called Bingel reaction, yielding cyclopropane rings on graphene . This ve2-carbon lattice of graphene;Addition of in situ generated aryl diazonium salts, proceeding via the release of dinitrogen . The reaAddition of azides forming aziridine adducts onto graphene –43. The 2-hybridized carbon atoms at 1585 cm−1 and the 2D band at a higher frequency of around 2725 cm−1. In addition, functionalized graphene sheets exhibit a new band, the so-called disorder D-band at around 1350 cm−1, due to the presence of sp3-hybridized carbon atoms, present at defects and/or anchor sites of functional groups. Furthermore, the 2D band in functionalized graphene shifts to lower frequencies and narrows compared to that of graphite. Thermal gravimetric analysis gives information about the degree of functionalization in modified graphene sheets because the covalent attachment of the organic moieties can be thermally eliminated, while intact graphite is thermally stable under inert atmosphere. Moreover, microscopy techniques such as TEM and AFM offer significant insight into the morphology of graphene and especially the number of layers in a particular sample. Overall, based on such complementary spectroscopic, microscopy and thermal techniques graphene-based hybrid materials can be qualitatively monitored, while at the same time information about the dimensions and purity is obtained.Raman spectroscopy is an extremely useful tool for characterizing graphene-based materials. Pristine graphite exhibits two characteristic Raman modes, namely the G-band, due to the presence of spWith regard to energy conversion schemes, the challenge for constructing robust and functional architectures made of graphene and photo- and/or electro-active components is met by considering and applying to a large extent the aforementioned functionalization strategies. In this respect, a few covalently linked graphene sheets with organic electron-donor moieties to facilitate photo-induced electron-transfer processes have been synthesized. Briefly, photo-induced excitation of the organic electron donor in the graphene-based hybrid materials results on the formation of the singlet-excited state of the organic component. Then, charge-separation takes place and the efficiency of the whole process is governed by how fast or slow the recombination of charges occurs. A schematic description of such a process, which sometimes may be quite complex involving triplet states as derived upon intersystem crossing, is presented in 2. Typically, a suspension of the graphene-based hybrid material in THF is transferred to a cuvette, then, two OTEs cut from conducting glass are inserted, and a DC electric field is applied. Then, the graphene-based hybrid material from the suspension is driven to the surface of the positive electrode, and a robust thin-film  is deposited within a short period of time. The photocurrent action spectrum of the OTE/SnO2/graphene-based hybrid material electrode is evaluated by examining the wavelength dependence of the incident-photon-to-current conversion efficiency (IPCE). The IPCE values are calculated by normalizing the photocurrent densities by energy and intensity of the incident light according to the following equation:Usually, the photoelectrochemical measurements of graphene-based hybrid materials with photoactive organic electron donors are carried out in a three-compartment cell using a potentiostat, employing a saturated calomel reference electrode, a working electrode and a Pt wire gauze counter electrode. This configuration allows for photocurrent measurements under electrochemical bias. Electrophoretic deposition is applied to fabricate films of the graphene-based hybrid material onto an optically transparent electrode (OTE) covered with nanostructured SnOi is the photocurrent density (A·cm−2), Win is the incident light intensity (W·cm−2), and λ is the excitation wavelength (nm).where •−–(TPP)•+ was shown to have an energy gap of 0.87 eV, while the IPCE was calculated to be 1.3% . Th. Th39]. 2(py)Cl, results in the formation of another novel nanohybrid material (2(py)Cl–graphene hybrid material highlights the importance of the covalent binding and the chemistry chosen for the particular functionalization methodology for the enhancement of photo-induced electron transfer. It is noteworthy that triazole rings constructed by “click” reaction of an alkyne-functionalized graphene sheet (or chromophore) and an azide-functionalized chromophore (or graphene sheet) were also shown to amplify the charge transfer [Covalently functionalized few-layer graphene with the bipyridine ruthenium complex -4-pyridyl-ruthenium(II)chloride, Ru(bpy)material . A slow transfer .An overview of common photophysical properties for some of the discussed graphene-based nanohybrids is given in To summarize, recent advances in the synthesis of graphene sheets that are covalently functionalized with photo- and/or electro-active moieties for the formation of novel hybrid materials were reviewed. Although in early stages oxidation was the method of choice for the functionalizing graphene, the introduction of numerous lattice defects that significantly alter the electronic properties of graphene, required the development of new routes for obtaining graphene sheets that preserve to a large extent the high conductivity of the material. Therefore, graphene-based hybrid materials are formed by exfoliating graphite and a subsequent chemical functionalization. Such graphene-based donor–acceptor hybrid materials were found to possess interesting optical and photophysical properties. Charge-transfer phenomena mainly from the photoexcited organic chromophore units to the graphene layers were observed. Although the potential of these materials to be applied in solar and photoelectrochemical cells is great, there is surely a need for the preparation and the evaluation of more graphene-based materials in order to fully understand and describe the electronic interaction processes of these systems.Partial financial support from GSRT/NSRF 2007-2013 through action “ARISTEIA II” project FUNGRAPH (3150) “Functionalization of graphene with multichromophoric arrays of photoactive units for energy conversion” is acknowledged.


Cree births in Quebec are characterized by the highest reported prevalence of macrosomia (~35%) in the world. It is unclear whether Cree births are at greater elevated risk of perinatal and infant mortality than other First Nations relative to non-Aboriginal births in Quebec, and if macrosomia may be related.This was a population-based retrospective birth cohort study using the linked birth-infant death database for singleton births to mothers from Cree , other First Nations  and non-Aboriginal  communities in Quebec, 1996–2010. Community type was ascertained by residential postal code and municipality name. The primary outcomes were perinatal and infant mortality.th percentile) was substantially more frequent in Cree (38.0%) and other First Nations (21.9%) vs non-Aboriginal (9.4%) communities. Comparing Cree and other First Nations vs non-Aboriginal communities, perinatal mortality rates were 1.52  and 1.34  times higher, and infant mortality rates 2.27  and 1.49  times higher, respectively. The risk elevations in perinatal and infant death in Cree communities attenuated after adjusting for maternal characteristics , but became greater after further adjustment for birth weight .Macrosomia (birth weight for gestational age >90Cree communities had greater risk elevations in perinatal and infant mortality than other First Nations relative to non-Aboriginal communities in Quebec. High prevalence of macrosomia did not explain the elevated risk of perinatal and infant mortality in Cree communities. Mothers from Cree and other First Nations communities were much younger than mothers from non-Aboriginal communities. The proportion of mothers less than 20 years of age was over 4 times higher for births in Cree (19.3%) or other First Nations (20.2%) than non-Aboriginal (3.5%) communities. Mothers in Cree or other First Nations communities had much lower educational attainment. The proportion of mothers who had not completed high school was over 4 times higher for mothers in Cree (45.4%) or other First Nations (47.2%) than in non-Aboriginal (10.8%) communities, while the proportion of mothers with college or higher education was lower for mothers in Cree (11.5%) than in other First Nations (18.2%) or non-Aboriginal (57.9%) communities. The proportion of single, divorced or widowed mothers was substantially higher in Cree (21.6%) or other First Nations (34.1%) than in non-Aboriginal (9.1%) communities. Births to multiparous women were more common in Cree (70.1%) or other First Nations (66.1%) than non-Aboriginal (53.8%) communities. Birth weights were significantly higher for births in Cree (mean = 3772.5 g) and other First Nations (mean = 3549.5 g) vs. non-Aboriginal (mean = 3377.4 g) communities (p<0.0001).Maternal characteristics differed significantly between births in Cree, other First Nations and non-Aboriginal communities (Table 2). Preterm birth rates were identical in Cree and other First Nations communities, and both were roughly 1.1 times as high as in non-Aboriginal communities. Compared to births in non-Aboriginal communities, HBW and LGA occurred 3.21  to 4.06  times as frequently in Cree communities, and 1.94  to 2.34  times as frequently in other First Nations communities, while LBW  and 0.79 ) and SGA  and 0.50 ) birth rates were substantially lower in Cree and other First Nations vs. non-Aboriginal communities. Perinatal and infant mortality rates were 1.52  and 2.27  times as high for Cree , and 1.34  and 1.49  times as high for other First Nations  compared to non-Aboriginal  births. Relative to non-Aboriginal births, the absolute risk differences (increases) in perinatal death and infant death were 3.5  per 1000 and 5.1  per 1000 for Cree births, and 2.3  per 1000 and 2.0  per 1000 for other First Nations births, respectively. Macrosomic birth, perinatal and infant mortality rates were the highest in Cree communities. Infant death occurred about 50% more frequently in Cree vs. other First Nations communities. Large risk elevations were observed in postneonatal mortality in both Cree ) and other First Nations ) communities. The relative disparities in perinatal and infant mortality rates comparing Cree or other First Nations to non-Aboriginal communities fluctuated and showed no apparent trends between 1996–2000 and 2006–2010 (data not shown). Analyses of cause-specific infant mortality rates revealed significant risk elevations for infant death due to SIDS, infections and external causes in Cree communities , 19.32  and 4.70  times, respectively), and due to SIDS and external causes in other First Nations communities  and 3.88  times, respectively).The risks of preterm birth, infant macrosomia, perinatal and infant death were all significantly higher in Cree or other First Nations than non-Aboriginal communities (th-90th percentile) infants, there were no significant differences in perinatal mortality  and infant mortality  rates for births in Cree communities. In contrast, there were higher perinatal mortality rates , but no significant difference in infant mortality rates  comparing macrosomic vs weight appropriate for gestational age infants in non-Aboriginal communities.Comparing macrosomic vs birth weight appropriate for gestational age , year of birth, infant sex and gestational age attenuated the risk disparities (as indicated by the RRs) in perinatal and infant death comparing Cree or other First Nations to non-Aboriginal communities. However, significantly elevated risks remained in infant death ) especially postneonatal death ) comparing Cree to non-Aboriginal communities, and in postneonatal death ) comparing other First Nations to non-Aboriginal communities. Further adjustment for year of birth, infant sex and gestational age accentuated the risk disparities (as indicated by the adjusted RRs) in perinatal and infant death comparing births in Cree or other First Nations to non-Aboriginal communities. Additional adjustment for birth weight for gestational age further widened the risk disparities. The risk elevations in perinatal and infant mortality were much greater for births in Cree communities than in other First Nations communities relative to non-Aboriginal communities.The crude and adjusted RRs of perinatal and infant mortality comparing First Nations and other First Nations vs. non-Aboriginal populations from log-binomial regression models are shown in  showed similar patterns as those observed in the comparisons of the whole study birth cohort (Table 2). Significant risk elevations were observed in perinatal, infant and postneonatal mortality in both Cree , 2.47  and 5.61  times, respectively) and other First Nations , 1.62  and 3.58  times, respectively) relative to non-Aboriginal communities, and the risk elevations were greater in Cree than other First Nations relative to non-Aboriginal communities.The comparisons of birth outcomes and infant mortality in Cree, other First Nations and non-Aboriginal communities in rural areas We found that both Cree and other First Nations communities are at significantly elevated risks of perinatal and infant mortality relative to non-Aboriginal communities, but substantially greater risk elevations were observed in Cree than other First Nations communities. The high prevalence of macrosomia in Cree communities did not contribute to the high perinatal and infant mortality rates in Cree communities. The adjustment for birth weight small or large for gestational age did not diminish the risk disparities.1–12 and in First Nations and Inuit populations in Quebec using mother tongue or place of residence to identify First Nations and Inuit births [Elevated risks of perinatal and infant death have been reported in Aboriginal vs. non-Aboriginal populations even in developed countries,t births –5. Cree t births ,14. The The observed perinatal and infant mortality rates in Cree communities were not only substantially higher than the rates in non-Aboriginal communities, but also disappointingly higher than in other First Nations communities  in Quebec. This highlights the need for attention to improving perinatal and infant health in Cree communities. The largest mortality risk disparity was observed in postneonatal mortality comparing Cree to non-Aboriginal communities. Postneonatal mortality rate may reflect the impacts of socioeconomic conditions and infant care ,26. AborAn extremely large risk elevation was observed for infant death due to infections 19.32 times), and large risk elevations were observed for infant death due to SIDS (4.07 times) and injuries/external causes (4.70 times) in Cree relative to non-Aboriginal communities in Quebec. These risk elevations were much greater in Cree than other First Nations relative to non-Aboriginal communities, and deserve further studies. Such “preventable” infant deaths may be reduced through improvements in infant immunization coverage, promotion of safe infant sleep environment  ,30, and  times, aOur study confirmed the high prevalence of infant macrosomia in Cree communities ,14. In cThe main strength is the large population-based birth cohort. The main limitation is that some non-Aboriginal women living in First Nations communities would have been misclassified as Cree or other First Nations. However, such misclassifications should be small . Moreover, such misclassifications would only tend to bias the risk differences in perinatal and infant mortality rates in First Nations vs. non-Aboriginal communities towards the null. Another limitation is that the study could not address the causes of high birth weight such as maternal obesity and diabetes. However, these may be considered upstream risk factors that might affect perinatal and infant mortality risks through affecting fetal growth. Further studies are required to evaluate the contributions of these and other unmeasured risk factors to the elevated risks of perinatal and infant death in Aboriginal communities.Cree communities were at greater elevated risk of perinatal and infant mortality than other First Nations relative to non-Aboriginal communities in Quebec. The very high prevalence of macrosomia did not contribute to the substantially higher risk of perinatal and infant mortality in Cree relative to non-Aboriginal communities. There is strong need for interventions to improve perinatal and infant health in First Nations especially Cree communities in Quebec.S1 STROBE Checklist(DOC)Click here for additional data file.S1 Clinical Studies Checklist(DOCX)Click here for additional data file.


Frontotemporal lobar degeneration (FTLD) is one of the leading causes of dementia after Alzheimer’s disease. A high-ranking candidate to become a diagnostic marker for a major pathological subtype of FTLD is the transactive response DNA binding protein of 43 kDa (TDP-43). The main objective is to elucidate which antibodies are specific for pathological TDP-43, with special interest in its modified isoforms. Indeed, TDP-43 has been shown to be hyperphosphorylated and truncated in disease. A secondary objective is to review existing immunoassays that quantify TDP-43 in biofluids. A systematic review of literature was performed by searching PubMed and Web of Science using predefined keywords. Of considered research papers the methods section was reviewed to select publications that enabled us to answer our learning objective. After quality assessment, antibody characteristics and related outcomes were extracted. We identified a series of well-characterized antibodies based on a scoring system that assessed the ability of each antibody to detect TDP-43 pathology. A selection of 29 unique antibodies was made comprising 10 high-ranking antibodies which were reported multiple times to detect TDP-43 pathology in both immunostaining and immunoblotting experiments and 19 additional antibodies which detected TDP-43 pathology but were only scored once. This systematic review provides an overview of antibodies that are reported to detect pathological TDP-43. These antibodies can be used in future studies of TDP-43 proteinopathies. Additionally, selected antibodies hold the potential to be used in the development of novel immunoassays for the quantification of TDP-43 in biofluids, as a possible biomarker for FTLD-TDP.The online version of this article (doi:10.1186/s40478-015-0195-1) contains supplementary material, which is available to authorized users. MAPT) and progranulin genes (GRN) and a repeat expansion in C9orf72 [Frontotemporal lobar degeneration (FTLD) is the primary cause of early onset dementia after Alzheimer’s disease (AD) . Worldwi C9orf72 -13. Mole C9orf72 . The maj C9orf72 ,16. Unde C9orf72 . In FTLD C9orf72 ,19. Furt C9orf72 . Notewor C9orf72 ,21. A co C9orf72 .GRN mutation-related FTLD, a subgroup of FTLD-TDP [While mutations in a specific gene induce an associated molecular pathology, no strict relationship exists between clinical FTLD subtype and underlying proteinopathy ,23. IndeFTLD-TDP ,26. A hiFTLD-TDP . The TDPFTLD-TDP ,28. PathFTLD-TDP ,29-32. RFTLD-TDP -35 whileFTLD-TDP .Figure 1The primary objective of this systematic review is to identify which antibodies have previously been described to detect TDP-43 pathology. These antibodies are expected to be suitable for defining the characteristic profile of pathological TDP-43 in human brain and biofluids, using immunostaining and immunoblotting. Additionally, antibodies specific for TDP-43 pathology hold potential to be used in immunoassays for quantification of TDP-43 and its pathological isoforms in biofluids. In this regard, a secondary objective is to review existing immunoassays with reference to specific antibody pairs.A systematic review of existing literature was performed by searching PubMed and Web of Science using the predefined keywords “TDP 43” and variations like “transactive response DNA binding protein” or “TAR DNA binding protein” in publication title, and keywords indicating the use of antibodies  in title or abstract for PubMed and as topic for Web of Science. Only original research papers published between 06/10/2006  and 31/C. elegans or Drosophila as model organism were excluded. From relevant papers following data was extracted and tabulated: antibody characteristics , used immunoassays, assessed target , relevant immunoassay outcomes  and study strengths and limitations. For completeness of the systematic review, antibodies were characterized thoroughly by consulting available published data, manufacturer websites and corresponding authors. This allowed identification of epitopes at amino acid (AA) level [Papers that did not report the use of an antibody directed at TDP-43 (e.g. genetics based) were excluded. Because the aim was to assess specificity of antibodies with applicability to human disease, papers using yeast, A) level ,38 and iWhen provided with enough information to identify the exact antibody , published results were scored for each paper that used the same antibody both in immunostaining and immunoblotting experiments. The scoring is a basic “Yes/No” system indicating if an antibody reacts with pathological TDP-43 in the reported setup or not. For staining, a positive score signifies visualization of any form of pathological inclusions, while for blotting a positive score responds to the presence of high molecular weight smears and/or distinct C-terminal fragments ,40. A neFrom tabulated scores, a primary selection of antibodies was listed based on having a positive score on one or both immunoassays multiple times. A secondary selection was made from antibodies that scored positive on one or both immunoassays only once.The initial search resulted in a total of 671 publications, including 490 found in both databases. After selection of relevant English research papers 409 publications remained. These publications report the use of 833 antibodies (approximately 100 unique) from which the characteristics were extracted. Relevant immunoassay outcomes were scored for each well-characterized antibody . In 132 of these reports both immunostaining and immunoblotting were performed with the same antibody, leaving a total of 38 different antibodies for further assessment.The primary selection, representing antibodies capable of detecting TDP-43 pathology with both immunoassays, includes 9 unique antibodies reported 62 times with a double positive score and 33 times with a single positive score. One additional antibody only has multiple reports with a single positive score Table . The secA detailed look at the 10 highest-ranking antibodies  to quantify TDP-43. A total of 10 publications are available, describing 12 assays -50. HoweApart from aforementioned publications using ELISAs, only three other reports were found where TDP-43 is detected in biofluids, by means of an immunoblot -53.This review was undertaken to identify the most suitable antibodies for future research of TDP-43, while evaluating ELISAs already used for its quantification. As much as 29 antibodies met the selection criteria and are thus reported to detect TDP-43 pathology. In contrast, only five different ELISAs have been published and their antibody combinations leave room for improvement.The majority of antibodies in the final selection are characterized by an epitope in the C-terminal region, and many of them are specific for phosphorylated TDP-43. These findings are expected, since C-terminal truncation and phosphorylation are hallmarks of aggregated TDP-43 in disease ,30,32. HAnother reason for false positive and false negative results is the fact that antibodies are always used in a very specific setup. For example, antibodies used for studying exogenous expressed C-terminal fragments are not necessarily successful in detecting pathological TDP-43 in patients. Conversely, when antibodies are used to study patients without suspected TDP-43 pathology it is understandable that they will indeed not detect pathology. These remarks are supported by the fact that many antibodies in the primary selection do not consistently have a double positive score, and some are even reported with a double negative score. Finally, there is the potential occurrence of biased results, since three antibodies are considered “standard” antibodies for studying TDP-43 proteinopathies, and are thus widely used. These antibodies are 10782 (ProteinTech), 2E2-D3 (available from different manufacturers) and TIP-PTD-P01&-P02 (Cosmo Bio) ,55. ThisA final point of discussion when selecting antibodies to study TDP-43 is the use of commercially available antibodies or the generation of antibodies in-house. Despite favorable antigenicity profiling of TDP-43, only a few well-characterized antibodies within the systematic search are available from companies  TDP-43. To utilize TDP-43 as an effective biomarker applicable for human TDP-43 proteinopathies, it is very likely that all isoforms of TDP-43 will need to be quantified separately. From this point of view, the ELISA reported by Kwong et al.  is alreaIn the context of this systematic review it is necessary to comment on the recent publication by Feneberg et al. describing a limited role for TDP-43 as a diagnostic tool . The maiA final remark, which is also supported by Feneberg et al. , is thatIn order to make sure selected antibodies can be used in all future studies of TDP-43 proteinopathies, regardless of preferred immunoassay, we decided to score only those antibodies used for both immunostaining and immunoblotting in the same publication. As this might be a methodological limitation of the review process, we performed the same analysis on the entire dataset which resulted in a comparable selection, including all 10 highest-ranking antibodies. Another possible limitation is the use of a scoring system to generalize descriptive results reported with different antibodies, under different experimental conditions. However, using a scoring system is indispensable in order to compare antibodies across studies, and all immunoassay outcomes were independently scored by two researchers to ensure reliable categorization.This systematic review provides an overview of antibodies reported to detect TDP-43 pathology. These antibodies can be used in animal models and patient studies of TDP-43 proteinopathies, such as FTLD. Thus, researchers studying these disorders can choose highly sensitive and specific antibodies most suitable for their objective based on this review. Antibodies from the primary selection have consistently proven their functionality in detecting pathological TDP-43 in both immunostaining and immunoblotting. Antibodies from the secondary selection have the potential to detect TDP-43, but validation of their results is necessary before conclusions can be made regarding their future use. Additionally, high-ranking antibodies are leading candidates when selecting an antibody pair in future development of novel quantitative immunoassays for TDP-43 in biofluids or exosome preparations, as a possible biomarker for FTLD-TDP.