It is demonstrated that the increasing number of graphene layers with CVD graphene plays a key role in reproducible strain gauge application since defects of individual layers may become less important in the current path. This may lead to a more stable response and, thus, resulting in a lower scattering.. Further results demonstrate the piezoresistive effect in a network consisting of liquid exfoliated graphene nanoplatelets (GNP), which result in even higher strain sensitivity and reproducibility. A model-assisted approach provides the main parameters to find an optimum of sensitivity and reproducibility of GNP films. The fabricated GNP strain gauges show a minimal deviation in PRE effect with a GF of approximately 5.6 and predict a linear electromechanical behaviour up to 1% strain. Spray deposition is used to develop a low-cost and scalable manufacturing process for GNP strain gauges. In this context, the challenge of reproducible and reliable manufacturing and operating must be overcome. The developed sensors exhibit strain gauges by considering the significant importance of reproducible sensor performances and open the path for graphene strain gauges for potential usages in science and industry.The two-dimensional material graphene promises a broad variety of sensing activities. Based on its low weight and high versatility, the sensor density can significantly be increased on a structure, which can improve reliability and reduce fluctuation in damage detection strategies such as structural health monitoring (SHM). Moreover; it initializes the basis of structure–sensor fusion towards self-sensing structures. Strain gauges are extensively used sensors in scientific and industrial applications. In this work, sensing in small strain fields (from −0.1% up to 0.1%) with regard to structural dynamics of a mechanical structure is presented with sensitivities comparable to bulk materials by measuring the inherent piezoresistive effect of graphene grown by chemical vapor deposition (CVD) with a very high aspect ratio of approximately 4.86 × 10 The two-dimensional 2D) material graphene has exceptional intrinsic properties, including a very high mobility , extraorD materiaMany recent experimental and numerical investigations have been devoted to the piezoresistive effect (PRE) and to achieve high gauge factors (GF) with graphene strain gauges ,14,15. A4) is used which dissociates on the Cu substrate into carbon atoms forming the graphene lattice. Graphene islands with different lattice orientations are created all over the substrate which expand and grow together [CVD can be used to grow mono- or multiple-layers of graphene on a catalytic transition metal substrate like copper (Cu)  or nicketogether . A graintogether ,35) or ttogether . The grotogether  between together  up to mitogether . These ttogether .Polyimide (PI) is a common substrate used for metallic strain gauges and other flexible electrical devices. The material is a flexible polymer which has a maximal elongation of 20% and a minimal bending radius of 0.3 mm as well as high heat resistance and temperature stability over a range of −269 °C to 400 °C . The graPolymethyl methacrylate (PMMA) (E-beam resist ARP 679.02 from Allresist) is spin-coated onto mono- and multi-layer graphene on Cu for 20 s at a rotational speed of 1200 rpm, followed by a hot bake step to evaporate the solvent. The Cu foil is placed in a sodium persulfate solution to etch the Cu. The PMMA-graphene stack is lifted out of the liquid using the PI substrate with a thickness of 50 µm. The graphene layer is blow dried using nitrogen gas and to promote the adhesion to the substrate. In terms of mechanically stacked bi-layer graphene the fabrication steps are modified. The PMMA-graphene stack is lifted out with a second mono-layer of graphene on Cu. A thermal treatment in an oven at 150–180 °C removes the liquid and brings the two layers in close contact . The Cu 2 Cu sheet [The mono-layer graphene used for the devices was grown in-house in a Moorfield NanoCVD rapid thermal processing cold wall reactor system on a 2 × 2 cmCu sheet . The str−1 and corresponds to defects within the sp2 hybridized honeycomb structure of graphene [−1 [−1, the number of graphene layers can be determined [Raman spectroscopy is the most commonly used method to qualitatively analyze graphene . Propertgraphene . The nexhene [−1 . By calctermined . A specttermined ,43. The Raman spectrum of the used mono-layer as well as multi-layer graphene for the strain gauge application show the characteristic spectrum with a very low D peak intensity which is negligible in comparison to the other peaks, indicating high quality graphene with few defects see . The 2D/Apart from the described bottom up manufacturing approach in To utilize exfoliated GNP in dispersion, drop casting , vacuum After spray deposition, ready-made GNP strain gauges on a 50 µm thick polyimide foil were fabricated. The main fabrication steps are described in The aqueous dispersion used for the strain gauge devices (purchased from Thomas Swan) are shear exfoliated GNP with a sheet resistance of approximately 20 Ω/□ for a film of 25 µm thickness . The conThe conductance behavior of connected clusters in a randomly ordered network may be described by the percolation theory . This apAfter formation of the percolated network, the working principle of the PRE in a network can be generally explained by the tunneling effect , which cSimplifying the GNP to circular rigid bodies, the motion of the particles can be estimated. For a low-voltage range the tunneling resistivity is given by Simmon’s equation  as: (1)ρe is the elementary charge, m is the mass of an electron and h is the Planck constant. The tunneling distance d is assumed to be the smallest possible distance between the nanoparticles by considering the lower limit of van der Waals distance between adjacent graphene sheets [Here, e sheets . λ is thA laminated morphology of the GNP coating can be observed in the scanning electron microscope (SEM) image in The resistance change can be measured by a Wheatstone bridge circuit (WBC) with a high resolution for small strain fields. The GF was calculated by considering the offset in a quarter bridge of the WBC after application on a mechanical structure. The strain gauge sensor was attached to the structure via an adhesive layer. A simple cantilever beam setup (length 300 mm and width 30 mm) is a low-cost device for sensitivity tests with a well-defined strain field. For data acquisition the amplifier module Quantum MX840B of HBM is used which provided an internal transducer for the WBC. The relative change in the resistance ΔR/R of a conductor consists of two terms: a change due to the alteration in the conductor geometry by strain ε and transverse contraction ν, and an alteration in electrical resistivity ρ due to changes in electron mobility, which is expressed by the PRE Δρ/ρ:GF is a general measure for the sensitivity of the sensor and quantifies the relative change of the resistance R due to the applied strain ε on graphene byThe Mono-, bi- and multi-layer CVD graphene as well as GNP strain gauges were investigated in a quasi-static load test with a strain field from −0.1% up to 0.1%, where their sensitivities were quantified. The results presented in 8, which is the highest measured so far. The reliability and reproducibility are the key points in the sensor application. Therefore, the sensitivity for mono-, bi- and multi-layer graphene were investigated of a rectangular shaped CVD graphene sensor with an aspect ratio of approximately 4.86 × 10Using the cantilever setup and applying compression and tension loads, a strain field from around −0.1% up to 0.1% can be partially detected by the graphene sensors. In case of mono- and bi-layer graphene the tensile loads were detectable while the compressive loads are unreliable in two of three attempts. The scattering in both cases is higher in comparison to the multi-layer graphene. The GF of the bi-layer graphene are the most strongly scattered. Nevertheless, it can be seen that only the multi-layered graphene layer provided appropriate measuring signals for all applied sensors. Moreover, the multi-layer graphene shows a contrasting behavior in comparison to the mono-layer sensors. By increasing the strain, a more sensitive electromechanical behavior to a compressive load than to a tensile load is observed see .F. Investigations of Huang et al. with nanoindentation of graphene layer with a width of 1.5–4 μm show that the normalized resistance change is related to the changes in the Fermi-velocity [In a CVD graphene based strain sensor, a large number of mechanisms of action occur which cause the piezoresistive effect (PRE). The intrinsic PRE of a graphene membrane is different to the network of GNP and shall be considered in velocity . Smith evelocity . The provelocity . Investivelocity . 8) of the fabricated CVD graphene strain gauges, a higher scattering of the GF was observed in the bi-layer in comparison to the mono- and multi-layer strain gauges. In this context, the presence of defects can play a pre-dominant role as they can induce significant changes in the drift mobility, which may result in a higher variation of the GF [In our investigations with a very high aspect ratio  and a low potential barrier, the electromechanical behavior of the spray deposited strain gauge is verified. As a consequence, uncertainties in fabrication of spray coatings  can be overcome by a low potential barrier. Thus, our fabricated GNP strain sensors are mainly focused on enhanced reproducibility and scalability for large quantities with a low variation on the sensing behaviour for the initial state. The long-term durability shall be considered in fatigue tests for future studies.In the context of sensor development, we have investigated the inherent piezoresistive effect of individual CVD graphene layers as well as the electromechanical network behavior of GNP by considering the requirement of reproducibility, which is one of the key points in sensor applications. Using a simple cantilever, we were able to characterize the PRE in a well-defined strain field. In case of CVD graphene, we achieved a higher reproducibility by increasing the number of layer. Thus, defects have less influence on the electromechanical behavior, since defects of individual layers may become less important in the current path. This led to a more stable response and, thus, resulting in a lower scattering of the electromechanical behavior. Minimal deviation of the GF over a batch of fabricated GNP strain gauges is achieved far beyond the percolation threshold for densely packed networks. These experimental measurements were confirmed in a model-assisted approach for the used GNP dispersion. Further investigations may deal with the conflict of objectives between sensitivity and reproducibility.This work provides the path for development of strain gauges based on graphene. Very thin and transparent strain sensitive sensors may be developed for application scenarios such as wearable sensors on human skins or microelectromechanical systems (MEMS). The implementation of integrated GNP coatings by spray deposition allows a large area sensing system and creates new options for smart structures with sprayable sensors. Future work will be extended from quasi-static to dynamic load tests by considering the environmental cross-sensitivities.


Drosophila melanogaster. Overexpression of HSP67Bc, the functional ortholog of human HSPB8, suppressed the eye degeneration caused by the cytoplasmic accumulation of a TDP-43 variant with a mutation in the nuclear localization signal (TDP-43-NLS). TDP-43-NLS accumulation in retinal cells was counteracted by HSP67Bc overexpression. According with this finding, downregulation of HSP67Bc increased eye degeneration, an effect that is consistent with the accumulation of high molecular weight TDP-43 species and ubiquitinated proteins. Moreover, we report a novel Drosophila model expressing TDP-35, and show that while TDP-43 and TDP-25 expression in the fly eyes causes a mild degeneration, TDP-35 expression leads to severe neurodegeneration as revealed by pupae lethality; the latter effect could be rescued by HSP67Bc overexpression. Collectively, our data demonstrate that HSPB8 upregulation mitigates TDP-43 fragment mediated toxicity, in mammalian neuronal cells and flies.Aggregation of TAR-DNA-binding protein 43 (TDP-43) and of its fragments TDP-25 and TDP-35 occurs in amyotrophic lateral sclerosis (ALS). TDP-25 and TDP-35 act as seeds for TDP-43 aggregation, altering its function and exerting toxicity. Thus, inhibition of TDP-25 and TDP-35 aggregation and promotion of their degradation may protect against cellular damage. Upregulation of HSPB8 is one possible approach for this purpose, since this chaperone promotes the clearance of an ALS associated fragments of TDP-43 and is upregulated in the surviving motor neurones of transgenic ALS mice and human patients. We report that overexpression of HSPB8 in immortalized motor neurones decreased the accumulation of TDP-25 and TDP-35 and that protection against mislocalized/truncated TDP-43 was observed for HSPB8 in  Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neurone disease, which leads to progressive muscle weakness and eventual respiratory failure. ALS is a complex disease that develops in familial (fALS) forms, associated with specific gene mutations in 5–10% of the cases and in sporadic (sALS) forms in the remaining 90% of patients .TDP-43 is a predominantly nuclear protein that belongs to the family of heterogeneous nuclear ribonucleoproteins (hnRNPs) and functions in RNA processing . TDP-In addition, experiments in several different cellular models have shown that both TDP-25 and TDP-35 form detergent-insoluble ubiquitin-positive cytoplasmic inclusions. These inclusions sequester endogenous TDP-43 (11) and correlate with reduced TDP-43 levels in the nucleus . These TIndependently of which specific form is more aggregation-prone or toxic, since inclusions formed by truncated TDP-25 and TDP-35 cleavage fragments are associated with enhanced cellular toxicity, all approaches aimed at decreasing their aggregation rate and facilitating their disposal may represent successful strategies to counteract motor neurone degeneration in ALS. One way of achieving this objective could be to boost the protein quality control (PQC) system, which survey protein folding/aggregation and assist protein clearance. Indeed, we have already demonstrated that upregulation of the small heat shock protein HSPB8  , might rDrosophila melanogaster (Dm). We find that overexpression of HSPB8 and its Dm functional ortholog HSP67Bc , we found that, while GFP was distributed in a diffuse manner, GFP-TDP-43 was confined to the nucleus in the vast majority of motor neurones, while both GFP-TDP-35 and GFP-TDP-25 were observed to be mislocalized to the cytoplasm . Cells tThe aggregation propensity of TDP-25 versus TDP-43 and TDP-35 might depend not only on the intrinsic properties of these different species but also on the ability of the cell to clear them. If differences in the turnover of the cleaved TDP-43 fragments exist, their clearance should be differentially affected when the degradative systems are pharmacologically impaired. Therefore, we analysed how inhibition of the proteasome and of autophagy impacts on the intracellular levels of the different GFP-TDP variants. In this assay, we use the NP40 fractionation method, which, according to our results, is the most suitable method to analyse the accumulation of GFP-TDP aggregates. Inhibition of autophagy or of the proteasome system mildly increased the levels of the NP40-soluble monomeric forms of GFP-TDP-43 and GFP-TDP-35, while leaving monomeric NP40-soluble GFP-TDP-25 unaffected . In contThe observation that HSPB8 expression is enhanced by the expression of both GFP-TDP-35 and GFP-TDP-25 fragments ,16, founTo probe further, the importance of HSPB8 for the degradation of GFP-TDP fragments we performed silencing experiments in inducible stably-transfected GFP-TDPs-NSC34 cell lines that expressed comparable levels of GFP-TDPs mRNA (data not shown). In these cells, HSPB8 silencing resulted in a significant increased accumulation of both GFP-TDP-35 and GFP-TDP-25 NP40-soluble species retained by cellulose acetate membranes . ConsistAs the GFP tag has the potential to influence the stability, distribution and/or aggregation properties of truncated TDP-43, we repeated our experiments using a FLAG tag instead of GFP; the smaller size of the former is likely to reduce any perturbation effects. We therefore produced N-terminal FLAG-tagged TDP variants , and characterized their biochemical behaviour after transient overexpression in immortalized motor neurone NSC34 cells. Similarly to the GFP-tagged TDPs described above, 2xFLAG-TDP-43 was found to be confined to the nucleus with very few cells forming aggregates (7.14 ± 1.36% of cells), while both 2xFLAG-TDP-35 and 2xFLAG-TDP-25 were mislocalized into the cytoplasm . HoweverSupplementary Material, Fig. S1). In combination, these results further suggest that HSPB8 participates in the disposal of accumulating and/or mislocalized TDP43 variants independently on the type of tag used.We next evaluated the effects of overexpression of HSPB8 on the intracellular distribution and accumulation of the 2xFLAG-tagged TDP-43, TDP-35 and TDP-25. Similarly to the observation made using GFP-tagged TDP-43, TDP-35 and TDP-25, co-expression of HSPB8 decreased the accumulation of both soluble and insoluble species of all 2xFLAG-tagged TDP variants as measured by WB  and FRA Drosophila melanogsater (Dm) family of sHSPs comprises 11 members that have specific functions in the control of protein homeostasis  and co-expression of HSP67Bc with WT TDP-43 did not significantly affect the eye phenotype caused by WT TDP-43, nor its expression levels  have been shown to result in premature lethality in flies . This fin levels . By contn levels . Both thn levels , findingn levels .Figure Supplementary Material, S2B and S2C). Second, we co-expressed another member of the Drosophila sHSP family, CG14207, which promotes HSP70-dependent protein refolding, with WT and mutant TDP-43-NLS. However, in contrast to HSP67Bc, CG14207 was not able to induce the autophagy-mediated degradation of misfolded proteins, for example of expanded polyglutamine sequences . Taken together, our results suggest that the protective effects on eye degeneration and the reduction of TDP-43-NLS levels in the fly eyes that co-express HSP67Bc are due to its pro-degradative function.The decrease in the extent of eye degeneration and in the total protein levels of mutant TDP-43 NLS observed in flies that co-express HSP67Bc is likely to be due to its pro-degradative function. To explore this issue further, we first used the UAS-eGFP or the UAS-LacZ transgenes to control for the possibility that the differences observed in the absence and presence of HSP67Bc would be due to dilution effects of the GAL4 protein with an increased number of UAS transgenes. We observed no difference in the eye phenotype of flies expressing WT TDP-43 and the TDP-43-NLS mutant alone or with GFP or LacZ . Increased eye degeneration due to the downregulation HSP67Bc was found to correlate with a small but significant increase in the high molecular weight (MW) species typically formed on expression of mutant TDP-43-NLS ; these TDP-43-NLS high MW species accumulate further following depletion of HSP67Bc , signif S4A and . As withescribed ) and alsescribed . Interesescribed . Similar HSP67Bc . Finally HSP67Bc . TogetheTDP-43-NLS is an artificially generated mutant that mimics the toxicity due to cytoplasmic accumulation of TDP-43. To study further the protective role of HSP67Bc against TDP-43 mediated toxicity, we generated another fly model which expresses the truncated form TDP-35 observed in ALS patients. Flies expressing WT TDP-43 were used as a control, and to avoid any artefacts due to differences in transgene expression between TDP-43 or TDP-35, both constructs encoding for TDP-43 or TDP-35 were incorporated at the same genomic locus . FurtherA number of ALS-linked missense mutations have been identified in the gene encoding for TDP-43, including the M337V mutation . We therProtein misfolding and aggregation is a common feature of many neurodegenerative disorders, including ALS, FTLD-U, polyglutamine diseases such as Huntington Disease (HD), Parkinson disease (PD) and Alzheimer disease (AD) and decreasing protein aggregation has been shown to be protective in several cellular and animal models of such conditions ,35–40. PThe multi-functional RNA binding protein TDP-43 is emerging as a key pathogenic protein in several neurodegenerative diseases. Indeed, in almost all cases of sporadic and inherited forms of ALS and FTLD-U, protein aggregates have been shown to contain not only ubiquitin, but also TDP-43 11,51). In ALS and FTLD-U, several post-translational modifications of TDP-43 have been identified, which include hyperphosphorylation and ubiquitination, as well as caspase-mediated proteolysis. TDP-43 cleavage leads to the production of 25 kDa and 35 kDa N-terminally truncated fragments of TDP-43 (referred to as TDP-25 and TDP-35). Virtually all these modifications can contribute to the mislocalization of TDP-43 from the nucleus  to the cytosol (where it aggregates), thereby causing the loss of normal TDP-43 function for the modulation of splicing and transcription ,51. In A. In the in vivo. Since the mammalian and the Drosophila melanogaster (Dm) families have independently evolved, and as we had previously identified the functional ortholog of human HSPB8 in flies, called HSP67Bc . Interestingly, high MW species, as well as cytoplasmic aggregates, have been found in cellular models expressing the specific mutant TDP-43-NLS that we studied here in vivo ; these hS2 cells ,28. In autophagy ,28, did The protective effect of HSP67Bc was further reinforced using a fly model expressing truncated TDP-35. Again, while HSP67Bc had no detectable effect on WT TDP-43 levels and the observed eye phenotype, its overexpression was sufficient to abolish the lethality of pupae caused by the TDP-35 fragment. We have previously characterized the toxic effects caused by overexpression of TDP-43 and TDP-25 in fly eyes and observed that both proteins caused only a mild rough eye phenotype, with no major differences in their effects on fly eye morphology . When coFinally, overexpression of HSP67Bc also protected against the eye degeneration induced by the ALS-causing mutant M337V TDP-43, further generalizing its beneficial effects to different forms of mislocalized TDP-43.In summary, our data provide clear evidence that upregulation of HSPB8 could be an effective approach to combating those neurodegenerative diseases that are characterized by (motor) neurone loss and proteinaceous inclusions. Since aggregated TDP-43 and TDP-35 also contain RNA, and as their aggregation has an impact on RNA homeostasis, by sequestering specific TDP-43-bound RNAs , it is pDimethyl sulfoxide (DMSO), doxycycline and MG132 were obtained from Sigma-Aldrich , 3-Methyladenine (3-MA) was obtained from Selleckchem .pEGFP-TDP-43, pEGFP-TDP-35 and peGFP-TDP-25, coding, respectively, for the GFP-fused TDP-43 full length protein (GFP-TDP-43) and its C-terminal 35KDa (GFP-TDP-35) or 25 KDa (GFP-TDP-25) fragments, were kindly provided from Dr. L. Petrucelli . 2xFLAG-et al. 2012 . The fly stocks gmr-GAL4 (stock number 1104) and actin 5C-GAL4 (stock number 4414) were obtained from the Bloomington Drosophila Stock Centre. Transgenic flies expressing V5-HSP67Bc were constructed at Genetic Services Inc. , and the background strain w1118 of Genetic Services was used as a control . RNAi liTransfected cells were fixed 48 h after transfection with 4% paraformaldeyde and methanol and processed as previously described . To deteQuantification of the number of cells with GFP-TDPs or 2xFLAG-TDPs aggregates was performed on cells co-transfected with TDPs and pDSRed-monomer (used as a transfection control) by measuring the numbers of cells with TDP aggregates, normalized to the total number of transfected cells (pDSRed-monomer positive cells). At least 100 cells/field were counted, and three fields were analysed for each coverslip. The experiments were performed in triplicate and the data have been expressed in each case as mean ± sem.The effects of HSP67Bc overexpression or knockdown on eye degeneration in the gmr-GAL4-TDP43 WT and NLS mutant, as well as in the gmr-GAL4-TDP25 or gmr-GAL4-TDP-35 lines, were evaluated by taking light microscopic images of the eyes of 1-day old adult flies with a stereoscopic microscope model SZ40 (Olympus). For each genotype and condition more than 100 fly eyes were evaluated. For fly eye histology, heads of the appropriate genotype were collected and fixed for 2 h at room temperature in 4% paraformaldehyde in PBS. Samples were serially dehydrated in ethanol and processed as previously described .2, glycerol 3%, pH 7.4; Sigma-Aldrich) supplemented with complete EDTA-free (Sigma-Aldrich) and 1mM dithiothreitol . NP40 soluble and insoluble fractions were separated by centrifugation at 16,000 g for 15 min at 4 °C. The protein content of the soluble fraction was determined by the bicinchoninic acid method . WB analysis was performed on 12% SDS polyacrylamide gel, loading 15 μg of proteins for PBS and NP40-soluble extracts or an equal volume of the corresponding NP40-soluble fraction for NP40-insoluble extracts. Samples were electro-transferred to nitrocellulose membranes  using TransBlot Turbo Apparatus (Bio-Rad). Nitrocellulose membranes were processed as previously described  and homogenized using slight sonication. For NP40 extraction, the pellets were resuspended in lysis buffer  was used as a loading control and rabbit polyclonal ubiquitin antibody (DAKO) was used to detect ubiquitinated proteins in fly head extracts. Human TDP-43 in fly head extracts was detected using the TARDBP (41–7.1) antibody . The antibody against Dm-HSP67Bc has been previously described .n = 3).For transiently transfected NSC34 cells, 1.5 µg of the PBS or NP40-soluble protein extracts (quantified used the BCA assay as described above) were loaded onto 0.2 µm cellulose acetate membrane  and filtered through a Bio-Dot SF Microfiltration Apparatus (Bio-Rad). An equal volume of the corresponding NP40-soluble extract was loaded in the case of NP40-insoluble extracts. Slot-blots were probed as described for WB to detect GFP-TDPs or FLAG-TDPs retained insoluble species. For stably transfected GFP-TDPs-NSC34 cell lines, cells were harvested 72 h after induction with 1 μg/ml of doxycycline, and samples were prepared as described above for transiently transfected cells. 9 µg of the NP40-detergent soluble protein extracts were loaded onto cellulose acetate membranes and filtered. An equal volume of the corresponding NP40-soluble extract was loaded in the case of NP40-insoluble extracts. Slot-blots were probed with GFP antibody as described above for WB. Densitometric optical analysis of slot-blots was performed and represented as mean ± SEM (t-test. Values represent mean ± SEM of 3 replicates. Fly biochemical data and eye morphological comparisons between genotypes were analysed using ANOVA, followed by the Bonferroni post-hoc or the Student’s t-test. * = P > 0.05; ** = P > 0.01; *** = P > 0.001. Values represent mean ± SEM.Cellular biochemical data were analysed using Student’s


Eukaryotes and fungi were the least affected components in these subjects but archaea, except Methanobrevibacter were significantly decreased in them. The two dominant archaea viz. Methanobrevibacater and Methanosphaera followed opposite trends in abundance from NGTs to Known-DMs subjects. There was a substantial reduction in eubacteria, with a noticeable decrease in Bacteroidetes phylum (p = 0.098) and an increased abundance of fungi in New-DMs subjects. Likewise, opportunistic fungal pathogens such as Aspergillus, Candida were found to be enriched in New-DMs subjects. Analysis of eubacterial interaction network revealed disease-state specific patterns of ecological interactions, suggesting the distinct behavior of individual components of eubacteria in response to the disease. PERMANOVA test indicated that the eubacterial component was associated with diabetes-related risk factors like high triglyceride (p = 0.05), low HDL (p = 0.03), and waist-to-hip ratio (p = 0.02). Metagenomic imputation of eubacteria depict deficiencies of various essential functions such as carbohydrate metabolism, amino acid metabolism etc. in New-DMs subjects. Results presented here shows that in diabetes, microbial dysbiosis may not be just limited to eubacteria. Due to the inter-linked metabolic interactions among the eubacteria, archaea and eukarya in the gut, it may extend into other two domains leading to trans-domain dysbiosis in microbiota. Our results thus contribute to and expand the identification of biomarkers in diabetes.Diabetes in India has distinct genetic, nutritional, developmental and socio-economic aspects; owing to the fact that changes in gut microbiota are associated with diabetes, we employed semiconductor-based sequencing to characterize gut microbiota of diabetic subjects from this region. We suggest consolidated dysbiosis of eubacterial, archaeal and eukaryotic components in the gut microbiota of newly diagnosed (New-DMs) and long-standing diabetic subjects (Known-DMs) compared to healthy subjects (NGTs). Increased abundance of phylum Firmicutes ( The eubacterial assemblage associated with the human body together with other microbes like archaea, eukaryotes and fungi are referred to as “microbiota.” Trillions of these microbes that live in our distal gut are believed to be co-evolving with their hosts  too are disturbed due to the disease condition or vice-versa. In the present study, we investigated the composition of the intestinal microbiota of newly diagnosed (New-DMs) and long-standing diabetic subjects (Known-DMs) and compared it with normal glucose tolerant subjects (NGTs). We used Ion torrent PGM sequencing technology, to analyse eubacterial and archaeal 16S rRNA gene, 18S rRNA gene from eukaryotes and fungal ITS tagged amplicon from fecal samples.We studied 49 adults, who are parents of children in the Pune Children Study (PCS) conducted by Diabetes Unit of KEM Hospital Research Centre  ≥130 mmHg or diastolic blood pressure (DBP) ≥85 mmHg.Plasma glucose, cholesterol, HDL-cholesterol, and triglyceride concentrations were measured using standard enzymatic methods . Between-batch coefficients of variation for all these assays were <3% in the normal range. Plasma insulin was measured using Delfia technique . Overweight was defined as BMI ≥25 kg/mTotal community DNA was extracted from each fecal sample using QIAmp DNA Stool Mini kit  as per manufacturer's protocol. The PCR amplification and sequencing of resulting amplicons was performed as described earlier , all New-DMs (n = 14) and all Known-DMs (n = 16) were pooled by mixing equal quantities of concentration normalized PCR products. This way we obtained three pools for each group, NGTs, New-DMs and Known-DMs for archaeal 16S rRNA, eukaryotic 18S rRNA and fungal ITS1 genes. All the pooled samples were then sequenced using Ion Torrent PGM. Since fungal ITS amplicons varied in length, we fragmented 100 ng of it with Ion Shear Enzyme mix  for 20 min and 200 bp size fragments were selected before adapter ligation step  is a computational tool that uses marker gene data for prediction of functional composition of metagenome. Briefly, OTU abundance table was first normalized for 16S rRNA copy number against known gene copy number for each OTU. Functional predictions were categorized into KEGG pathways and an annotated table of predicted gene family counts (KOs) for each sample using predict metagenome option. Gene family table then categorized by function and further statistical analysis was performed in STAMP v2.0.1  on 7,300 Real-time PCR system from Applied Biosystems Inc. (USA). The primers and PCR conditions used for qPCR assays were described earlier  and ANOVA test is used to compare differences among the study groups. Different type of data generated through QIIME was imported and analyzed in ade4, vegan and ggplot2 packages within R software  and observed  species diversity indices were lowered in New-DMs and Known-DMs subjects  were significantly lowered, Firmicutes (p = 0.010) were significantly higher while Bacteroidetes (p = 0.098) showed decreased trend in abundance in New- and Known-DMs subjects  revealed the presence of 83 significantly different OTUs (p < 0.01) of which Prevotella copri, Faecalibacterium prausnitzii and Lachnospiraceae OTUs were enriched in NGTs whereas Lactobacillus ruminis OTUs were found enriched in Known-DMs  demonstrating enrichment of L. ruminis in these subjects. After the Random Forest analysis, top 20 OTUs were considered as highly discriminative among the three groups . However, notable compositional changes were observed in enterotypes of both New-DMs . Notably, the E2 (five subjects) in New-DMs and E3 (eight subjects) in Known-DMs were dominated by taxa that were being enriched in these subjects.We were able to stratify the gut microbial communities of NGTs, New-DMs as well as Known-DMs subjects into three distinct enterotypes (E) Figure . As obses Figure  and Knows Figure  comparedMethanobrevibacter  and associated decrease in Methanosphaera abundance from NGTs to New-DMs to Known-DMs subjects. From the three pools of Eukaryotic sequence data, we obtained 41,959 good quality sequences that clustered into 383 OTUs and could be assigned to four phyla: Chloroplastida, Metazoa, Stramenopiles, and Metamonada. Members of Stramenopile especially members of genus Blastocystis were found abundant in all groups. Fungi, particularly members belonging to Saccharomycetales were abundant in New-DMs compare to NGTs and Known-DMs. For fungal ITS data, we could obtain 106,185 reads that clustered into 871 OTUs belonging to phyla Ascomycota being most dominant followed by Basidiomycota and Zygomycota to be least dominant. From the Ascomycota group; Aspergillus and Emericella, the two alternative forms of the same fungus predominated most of the sequences ; which clustered into 65 OTUs belonging to Euryarchaeota and Thaumarchaeota phyla. The former being the most dominated phylum occupying more than 99% reads of all three groups. We noticed the gradual increase in p = 0.03), triglyceride (p = 0.05), and waist-hip ratio (p = 0.02) to be associated with OTU diversity across all samples (Supplementary Table p = 0.01), and in the case of New-DMs, oral glucose tolerance test (p = 0.05) to have an influence on distinct OTU diversity. Further, the covariance between genus abundance and clinical and anthropometric parameters were examined using co-inertia analysis  of these datasets, resulting in a relationship with RV coefficient = 0.219, P = 0.196 between the datasets , amino acid metabolism , vitamin B metabolism , glutathione metabolism and other functions  such as acetate and butyrate. These SCFAs are known to confer many health benefits; individuals lacking bacterial families producing SCFAs suffer from many diseases  and Methanosphaera stadtmanae are well adapted to the human gut environment, interestingly, the latter has acquired most of these adaptations through inter-domain lateral gene transfer . PERMANOVA is considered a powerful technique in detecting changes in community structure in response to environmental parameters  belonging to these butyrate-producing genera were found enriched in non-diabetic controls in diabetes associated metagenomic study (Qin et al., We used network analysis to capture specific ecological interactions among the eubacterial consortium in relation to diabetes status. Such interaction networks can predict the outcome of community alterations (Faust et al., Finally, with the bioinformatics tool PICRUSt (Langille et al., One of the strengths of our study is the comparison of gut microbiota of different grades of glucose intolerant subjects from a cohort which is has been followed for the past 20 years, this allowed a confident separation between newly diagnosed and known diabetic subjects. The participants are from the similar socioeconomic background and have a predominantly vegetarian diet. The age and gender distribution in the three groups were similar. One of the limitations of this study is that we were unable to describe sequential events in gut microbiota from healthy to diabetic state due to the cross-sectional design of this study. Another limitation of the study is the relatively small number of participants from one part of the country. Given the diversity in lifestyles, dietary habits, and social-economic status in the country, this study underscores a need for nationwide longitudinal studies. Our study is subject to inherent biases introduced by the use of high-throughput 16S rRNA gene amplicon sequencing. These include the region of 16S rRNA gene sequenced, set of primers used for gene amplification and use of sequence database for taxonomic assignments of the amplicon reads.In conclusion, our results add to the growing literature suggesting an association between gut microbiota and diabetes. Broad similarities between our results and literature reports suggest that our measurements are reliable and support consistent association across populations. Additionally, we have broadened the boundaries of diabetes associated gut microbiota by providing the consolidated description on eubacterial, archaeal, and eukaryotic dysbiosis in these subjects. Given the peculiarities of diabetes in Indians, these results suggest an important avenue be further explored for causality and possible interventions to prevent or modify the course of diabetes and related disorders. We anticipate the need for subsequent studies describing differences in gut microbial communities of diabetic patients from different populations and identification of relevant population specific biomarkers.YS, SG, and CY contributed to conception, design, and coordination of the study and to the critical revisions of the manuscript for important intellectual content. SJ and CY were involved in subject recruitment and sample collection. SB acquired and processed the fecal samples for 16S rRNA amplicon sequencing. SB performed detailed bioinformatics analysis. SB and MS performed archaeal, eukaryotic and fungal amplicon sequencing. SB prepared the first draft of the manuscript and contributed to the critical revisions of the manuscript for important intellectual content. SB and SJ undertook statistical analysis and interpretation of results. SJ contributed to the critical revisions of the manuscript for important intellectual content. All authors gave final approval of the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


SGs are studied in several diseases, including ALS and FTD, which involve the degeneration of motor and cortical neurons, respectively, and are now realized to be linked pathogenically by TDP-43, originally discovered as a component of ubiquitin-positive aggregates within patients’ neurons and some glial cells. So far, studies to undercover the role of TDP-43 in SGs have used primarily transformed cell lines, and thus rely on the extrapolation of the mechanisms to cell types affected in ALS/FTD, potentially masking cell specific effects. Here, we investigate SG dynamics in primary motor and cortical neurons as well as astrocytes. Our data suggest a cell and stress specificity and demonstrate a requirement for TDP-43 for efficient SG dynamics. In addition, based on our  TDP-43 is a highly conserved, ubiquitously expressed RNA-binding protein associated with the heterogeneous ribonucleoprotein (hnRNP) family. The RNA-binding ability of TDP-43 is conferred by two RNA recognition motifs (RRM1 and RRM2), while the C-terminal glycine-rich region mediates protein-protein interactions. In addition to known roles in alternative splicing and transcriptional regulation in the nucleus4, TDP-43 functions to stabilize and transport mRNA in the cytoplasm6. Additionally, TDP-43 is recruited to cytoplasmic RNA granules that are formed following exposure to various environmental stresses . These granules, termed stress granules (SGs), are membrane-less organelles that are believed to facilitate cell survival via the storage of non-essential mRNAs, translation factors and RNA-binding proteins during stress exposure9. SGs follow a linear dynamic featuring an initial nucleation/formation followed by assembly into larger structures, and eventual disassembly as the cell recovers. In transformed cell lines, depletion of TDP-43 has a negative impact on each of these steps11, indicating a key role for TDP-43 in the regulation of this essential cell survival mechanism.TAR DNA-binding protein 43 (TDP-43) was originally characterized as a transcriptional repressor of the HIV-1 genome via binding to the trans-activation response (TAR) element13. These diseases impact particular neuronal populations, with ALS being due to motor neuron degeneration and accompanying gliosis16 and FTD involving extensive degeneration of cortical neurons. Despite TDP-43-containing cytosolic inclusions in neurons and some glial cells being a pathological hallmark of ALS and FTD, the mechanism(s) by which TDP-43 contributes to neurodegeneration remains unclear. Furthermore, a persistent question in the field is whether TDP-43 evokes toxicity via the gain of an unknown cytosolic function or via the loss of some nuclear function. This outstanding question stems from the observation that neurons presenting cytoplasmic pathological inclusions usually also demonstrate a depletion of the nuclear pool of TDP-4317.The term “TDP-43 proteinopathies” first emerged with the discovery of ubiquitinated cytoplasmic inclusions of TDP-43 in the neurons of patients affected with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS)18, few studies have been conducted in cell types relevant to neurodegenerative disease19. Thus, we aimed to investigate SG dynamics and the impact of TDP-43 on these structures in primary neurons and glia. We observed distinct differences in SG morphology and dynamics between cortical and motor neurons, astrocytes, and fibroblasts. The data demonstrate that TDP-43 is required for optimal SG dynamics in primary neurons and glia exposed to oxidative stress. Moreover, the dependence on TDP-43 for SG formation was exacerbated in the context of hyperosmotic stress. Finally, neurons “aged” via prolonged culture times had impaired SG dynamics accompanied by decreased TDP-43 expression.Stress response mechanisms are heavily studied in neurodegenerative diseases and there has been a recent convergence on the involvement of SGs in ALS and FTD. Although it is appreciated that SG morphology and composition differ according to the stress stimuli and the cell type20; and astrocytes, glia cells implicated in ALS progression14. As a cell type unrelated to these disease conditions, we also cultured mouse embryonic fibroblasts. Given that the majority of published studies on SGs utilize sodium arsenite (SA) as the SG-provoking agent21, our first goal was to determine if SA can provoke SG formation in the primary cultures selected. SGs were labelled with an oligo(dT) probe since polyadenylated mRNA is obligatorily recruited into SGs following stress exposure regardless of cell type, whereas protein composition can vary between cell types, as demonstrated by the variable colocalization of known SG markers HuR22 . Finally, the most surprising result was that cortical neurons were more resistant to SG formation, and SGs persisted longer. Treatment with 0.5 mM SA for 30 min, the gold-standard in the field, induced SGs in only 15% of the neurons, whereas treatment for 60 min was required to initiate robust SG formation .  This experiment suggests that primary cortical neurons are refractory to a certain level of stress exposure. However, once formed, SGs were maintained until 210 min, much longer than in other cell types, at which point there was a precipitous disassembly of SGs. Taken together, these results indicate there are detectable differences in the timing of SG formation and disassembly amongst different cell types relevant to ALS/FTD.In transformed cell lines exposed to 0.5 mM SA, several small SGs assemble within minutes of exposure and then coalesce into a fewer number of larger puncta over a defined time window11 and accelerates SG resolution compared to control siRNA-treated cells10. Given the observations that different cell types have different kinetics of SG formation and disassembly, and our previous data that TDP-43 is central to SG dynamics in transformed cells, we questioned whether the involvement of TDP-43 also varies in cell types relevant to neurodegenerative diseases. In astrocytes in which nuclear TDP-43 expression was reduced to 39% of control levels els Fig. , we obseels Fig. . Howeverels Fig. . In contels Fig. , the reqels Fig. . Similar11. Thus, the effect of TDP-43 knockdown by siRNA on stress granule dynamics was assessed in astrocytes and neurons. To measure SG coalescence, as indicated by SG size and number per cell, cells were assessed at high magnification over two time points following SA exposure. As expected, in control siRNA-treated astrocytes exposed to SA, the number of SGs per cell significantly decreased over time, from a mean of 26 SGs per cell at 30 min to 14 SGs per cell at 90 min  while the size of individual SGs increased about 1.2 μm2 between 30 and 90 min . In contrast, in siTDP-43 treated astrocytes selected based on diminished TDP-43 labelling (as measured by pixel intensity), individual SG size and number per cell remained unchanged when TDP-43 levels were lowered . Similar results were obtained with a second independent siRNA for TDP-43  siControl-injected motor neurons exposed to SA demonstrated the expected increased size of individual SGs of ~1 μm2 and a decrease in the number of SGs per cell from 60 to 120 min as in the previous experiments . In contrast, SG size and the number of SGs per cell did not change in motor neurons depleted of TDP-43 upon exposure to SA . Similarly, SG coalescence was observed in cortical neurons expressing basal levels of TDP-43 following SA exposure , but not in TDP-43 siRNA-treated cortical neurons . Specifically, neurons undergo a reduction in the number of SGs per cell over time, and a significant  increase in individual SG size . These data were replicated with an independent TDP-43 siRNA  of D-sorbitol and exposures for up to 3 h. Surprisingly, SGs were not observed in cortical neurons exposed to up to 1 M D-sorbitol (data not shown), while astrocytes robustly formed SGs in response to 0.8 M sorbitol stress, consistent with a previous report19. Under these conditions, SGs were very small. Indeed, although the SGs were near the diffraction limit and thus not able to be reliably measured, we inferred that the observed cytoplasmic puncta were SGs based on comparison to unstressed siControl astrocytes, which displayed a relatively disperse (i.e. non-granular) cytoplasmic mRNA pattern  had smaller and fewer SGs compared to siControl treated astrocytes . These data establish the existence of cell type-specific responses to stress and further show that TDP-43 has variable relevance to SG dynamics in response to different stress conditions.It is appreciated that SG composition can vary according to stress exposureern Fig. . Despiteern Fig. . However26 that are driven by osmotic gradients, of which two are mainly expressed in the CNS28. Of these, Aquaporin 4 (AQP4) is primarily expressed by astrocytes31 and has a prominent role in brain homeostasis and neuronal survival. AQP4 expression is elevated in select brain regions of patients affected with neurodegenerative diseases such as prion disease32, multiple sclerosis27 and ALS26, as well as in rodent models of mutant SOD1-mediated ALS33. In this study, by immunoblot, we found that AQP4 protein levels were increased 2-fold in TDP-43-depleted astrocytes compared to control siRNA-treated astrocytes and was absent from MEFs, as expected . In attempt to determine if this increased expression results in more AQP4 channels at the plasma membrane, where it needs to be inserted to regulate osmotic balance, we performed surface labelling for AQP4 using non-permeabilized cells coupled with flow cytometric detection. Surprisingly, only 50% of TDP-43-depleted astrocytes had surface labelling for AQP4 relative to siControl astrocytes  and 56DIV , consistent with what we observed in 28DIV siControl-injected motor neurons  indicating effective SG coalescence. However, in aged motor neurons (56DIV), SG coalescence did not proceed; size and number were unchanged over time . The same defect in coalescence was observed in cortical neurons cultured for 21DIV (an established in vitro aging paradigm for these neurons34) while SG assembly proceeded as expected in 7DIV cultures  compromises normal SG dynamics and may be linked to TDP-43 levels.As ALS and FTD are age-related diseases and SGs are considered to be a central component of disease pathogenesis, we investigated the impact of ‘aging’ of cortical and motor neurons on SG dynamics by prolonged culture, as a means to model aging DIV Fig. . In 28DIres Fig. . Interesres Fig. . Thus, tEnvironmental stress is considered to play a role in the phenotype of several neurodegenerative diseases, and SGs are a first line mechanism for survival following stress exposure. While much has been done in transformed cells, the molecular events in the cell types implicated in these diseases are not well documented. We used a stress model of primary motor and cortical neurons, as well as astrocytes, to study SG dynamics in primary cells relevant to ALS/FTD neurodegeneration.11. Cortical neurons responded slowly to arsenite stress, with the majority of neurons displaying SGs only after 60 min of exposure. SG disassembly was noted after 300 min; this timeline of SG kinetics was twice as long in these neurons compared to astrocytes. While SG formation has been previously reported in primary neurons and glial cells35, this is the first study to detail the differences in SG assembly and disassembly kinetics in cell types relevant to neurodegenerative disease, namely neurons and astrocytes. Inclusions containing TDP-43 are characteristic of most cases of ALS and about half of all FTD cases. It has been proposed that SGs may serve to “seed” these inclusions36. One could speculate that the prolonged time required for SG assembly and disassembly may be relevant in this context and possibly contribute to neuronal vulnerability in these diseases. Indeed, persistent SGs due to delayed or failed disassembly have been reported where mutant forms of TDP-43 or other ALS-related genes have been transiently overexpressed in transformed cells37. In addition, SG disassembly is reportedly mediated by autophagy or chaperone-mediated protein degradation42, mechanisms which have been previously reported to become less efficient as neurons age44. That the kinetics of SG disassembly are protracted in neurons, coupled to the natural decline of these mechanisms intended to help dissipate SGs, may be highly relevant to the generation of the large cytoplasmic inclusions that characterize ALS/FTD, and are rich in RNA binding proteins beyond just TDP-43.We report differences in the morphology and kinetics of SGs formed in different cell types. Small SGs form quickly after arsenite stress and then rapidly disassemble in MEFs. In contrast, SGs formed in the majority of astrocytes within 30 min of arsenite treatment, followed by a plateau and subsequent decline at 150 min, indicative of SG disassembly similar to what we previously showed in SK-N-SH and HeLa cells11. We have previously shown that SG coalescence is relevant to SG interactions with processing bodies and the protection of polyadenylated mRNA following stress exposure45. Thus, we speculate that reduced nuclear TDP-43 levels would have negative consequences on these aspects of RNA granule homeostasis in neurons and astrocytes as well. This could, in turn, impact RNA homeostasis in general, possibly leading to a complex dysregulation of both transcript levels/stability and miRNA processing.Second, we demonstrate the importance of TDP-43 in SG assembly and disassembly in neurons and astrocytes. Specifically, reducing the levels of TDP-43 resulted in an acceleration of SG disassembly in astrocytes and cortical neurons. We attribute the more modest effect in cortical neurons to a less efficient depletion of TDP-43 levels. Interestingly, SG coalescence was impaired by TDP-43 depletion in all cell types examined and reaffirms the important role of TDP-43 in regulating this essential cell stress response mechanism previously documented in transformed cells46 as well as in patients27, and a loss of polarity of the protein has been reported46. TDP-43-mediated regulation of AQP4 expression and trafficking has not previously been reported and may represent a novel mechanism for further exploration given that astrocytes are known to play a contributing role to neurodegeneration48.In primary astrocytes, we discovered that TDP-43 is critically required for proper SG formation in hyperosmotic conditions. Specifically, following assembly, the SGs formed in TDP-43 depleted cells are smaller compared to control siRNA-treated astrocytes. We postulated that the glial-relevant water channel AQP4 may be impacted by TDP-43. Indeed, we observed that AQP4 was less efficiently integrated into the plasma membrane despite a robust TDP-43-dependent up-regulation. AQP4 has been previously reported to be up-regulated in mouse models expressing mutant SOD1in vitro model of aging. Our data indicate that although aged motor and cortical neurons form SGs in response to oxidative stress, they do not coalesce as expected. With the important caveat that aging cultures in vitro is not equivalent to in vivo aging, our findings suggest the possibility of a disturbance of RNA granule dynamics during normal aging. In addition, an unexpected observation was that TDP-43 protein levels were diminished in the aged cultures. Aging has long been considered as a risk factor for ALS and FTD20. Indeed, current models propose that these diseases arise due to “two-hits”, genetic susceptibility and a second trigger such as external stress or aging. Although we appreciate that these results are in vitro, it is tempting to speculate that aging could be a modifier of SG dynamics and thus may have relevance to the initiation and/or progression of age-related neurodegenerative diseases. Taken together, our data suggest that the cell and stress specificity in SGs dynamics and the impact of aging may be contributing factors to neuronal vulnerability in ALS and FTD cases with TDP-43 inclusions. A better understanding of the molecular mechanisms surrounding this stress response in a cell specific manner will be essential for future ALS/FTD therapeutic strategies aimed at modulating stress granule dynamics.Finally, we explored the impact of aging on SG formation in response to stress by subjecting neurons to long-term culture conditions, an established 49, from embryonic day 13 (E13) CD1 mouse embryos . After dissociation in trypsin, cells were plated at a density of ~450,000 per well in 12-well dishes (Greiner Bio-One) containing round glass 18-mm coverslips (Thermo Fisher Canada) coated with poly-D-lysine  plus Matrigel basement membrane matrix . Cultures were maintained in minimum essential medium enriched with 5 mg/ml glucose and supplemented with 1.3% horse serum (Invitrogen), 10 μg/ml bovine serum albumin, 26 ng/ml selenium, 20 μg/ml triiodothyronine, 10 μg/ml insulin, 32 μg/ml putrescine, 9.1 ng/ml hydrocortisone, 13 ng/ml progesterone (Sigma-Aldrich), 200 μg/ml apo-transferrin , and 10 ng/ml nerve growth factor (UBI Life Sciences). On days 4–6, cultures were treated with 1.4 μg/ml cytosine-β-D-arabinoside  to minimize growth of non-neuronal cells. The cultures were maintained at 37 °C in 5% CO2. Cultures were used at 21 or 56 days post-plating, as indicated in the figure legends. At this age in vitro, motor neurons are distinguishable from other neuronal types and glia in the culture because they develop and differentiate to resemble their counterparts in the intact spinal cord, both morphologically and by expression of biological markers. They have large cell bodies (>20 µm in diameter) and dendritic trees, and express choline acetyltransferase, glutamate receptors, and neurofilament proteins52.The use of animals and all procedures were performed according to the guidelines of the Canadian Council on Animal Care and were approved by the CRCHUM Institutional Committee for the Protection of Animals, and MNI/McGill / Animal Care Committee. Primary cultures of dissociated spinal cord along with dorsal root ganglia (DRG) were prepared as described53. Briefly, cerebral cortices were trypsinized , and then treated with trypsin inhibitor  and DNAse . Neurons were dissociated by trituration with a Pasteur pipette. Cells were then plated on glass coverslips coated with polylysine and laminin (Sigma Aldrich) and maintained in Neurobasal medium (Invitrogen) supplemented with 0.0025% Glutamax (Invitrogen) and 0.02% B27 (Invitrogen). Cultures were used five days after plating, or as indicated in the figure legend.Primary cortical neurons were prepared from E18.5 C57BL/6 mouse embryos , as previously publishedPrimary astrocytes were cultured from C57BL/6 pups (P0–P3) bred in house from stock obtained from Charles River Laboratories . Cortices were dissociated with 18 G and 21 G needles and cells were grown in Dulbecco’s modified Eagle medium , supplemented with 10% FBS, 1% glutamine and 1% Penicillin-Streptomycin (Sigma) on poly-L-ornithine (Sigma) coated plates and coverslips. Cells were used at confluence (~14 DIV).Primary mouse embryonic fibroblasts (MEFs) were prepared from E12.5 C57BL/6 embryos . Briefly, the entire body except the liver was treated with trypsin . The reaction was stopped with 10% FBS supplemented high Glucose DMEM and the cells were dissociated mechanically and plated in DMEM (Invitrogen) supplemented with 10% FBS, 1% glutamine and 1% Penicillin-Streptomycin (Sigma).Astrocytes and MEFs were transfected at 60–70% confluence with 40 pmol of custom siRNAs using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions and collected after 72 h. siRNA sequences used were: mouse TDP-43 #1, 5′-AAGCAAAGCCCAGACGAGCCUUUGA-3′; mouse TDP-43 #2 : 5′-GCAAUCUGGUAUAUGUUGUCAACUA-3′; and the negative control low GC siRNA . Cortical neurons were transfected at 7 DIV for 5 days with 5 pmol (per 18 mm coverslip) of mouse siTDP-43.50. Specifically, cultures were bathed in EMEM without sodium bicarbonate, titrated to pH 7.2. Solutions of Tris-EDTA (5 mM Tris and 0.5 mM EDTA), pH 7.2, containing 125 pmol of siRNA (negative control low GC and mouse TDP-43) plus 70 kDa dextran  were clarified by centrifugation at 11,000 × g for 15 min and microinjected into neuronal nuclei using glass microcapillaries (World Precision Instruments) coupled with a microinjector . After completion of the injection, cultures were returned to the incubator in culture medium plus 0.75% gentamicin .Motor neurons in mature spinal cord cultures do not transfect; thus, plasmids are expressed by intranuclear microinjection. The protocol was performed as previously publishedTo induce SGs, cells were treated with 0.5 mM SA (Sigma-Aldrich) for the indicated times at 37 °C. Recovery was initiated by changing the media. For osmotic stress, astrocytes were treated with 0.8M-1M D-Sorbitol (Sigma) for indicated times. Cover slips were collected and fixed with 1% paraformaldehyde (PFA) at 30, 60, 120, and 180 min after the start of the stress.Cells grown on coverslips were fixed in 1% PFA in PBS and then permeabilized with 0.1% Triton X-100 in 2X saline sodium citrate (SSC) for 15 mins, then washed with 1 M Tris, pH 8 for 5 mins. Coverslips were blocked with 0.0005% BSA, 10 mg/ml yeast RNA diluted in 2X SSC, washed with 1 M Tris, pH 8 for 5 mins, and incubated with hybridization buffer (1.3 ng FITC or Cy3 labelled oligo(dT) probe, 0.005% BSA, 1 mg/ml yeast RNA, 10% dextran sulfate, and 25% formamide, diluted in 2X SSC) for 1 h in a humid chamber. Coverslips were subsequently washed twice with 4X SSC for 5 min and once with 2X SSC for 5 mins Coverslips where incubated for 1 h with primary antibody: rabbit TDP-43 , and when indicated with SMI32R , GFAP , HuR , CAPRIN1 . After washing twice with 4X SSC for 5 min and once with 2X SSC for 5 min, labelling was visualized with fluorescently conjugated secondary antibodies against the desired species (Jackson ImmunoResearch). Coverslips were washed twice with 4X SSC, once with 2X SSC, and then mounted with ProLong Antifade reagent (Invitrogen). Images were collected using 40X (1.25 NA) and 63X oil (1.7 NA) objective lenses on a confocal microscope  equipped with LAS AF software (Leica) for acquisition. Total cellular fluorescence intensity of the oligo(dT) probe and TDP-43 was determined using Photoshop CS4 (Adobe). Discrimination of TDP-43 levels was based on measurements of TDP-43 pixel intensity in controls/at baseline in the accompanying experiment. Importantly, all images were acquired with the same settings.11. Briefly, to assess SG kinetics at different time points, SGs were identified by oligo(dT), and cells were scored as positive when they had at least two foci. A minimum of 70-100 cells were counted per time point, per condition and per experiment. For SG number and size, we used automatic recognition by ImageJ  using the following parameters: all SGs ranging from 0.2 to 15 µm2 in 10 randomly selected cells per condition in each experiment.SGs parameters were quantified using ImageJ, as previously published11, cells were collected by scraping in ice-cold PBS. Lysis was performed with RIPA buffer , incubated 10 min on ice, 10 min at room temperature then centrifuged 10 min at 13500 g. The collected supernatants were quantified with the BCA Protein Assay Kit . 20 µg of lysates were loaded for standard SDS-PAGE. The antibodies used were: rabbit anti-TDP-43 , rabbit anti-AQP4 , and mouse anti-Actin  as a loading control. Blots were visualized with peroxidase-conjugated secondary antibodies and ECL Western Blotting Substrate . Images were acquired using a range of acquisition times using the BIO-RAD ChemiDoc MP imaging system, and then exported as tifs via the Image Lab application without any manipulation. Chosen images were those just below overexposure (easily seen by the red pixels on the image). Densitometry was then performed with Adobe PhotoShop. No post-acquisition modifications were made.As we previously published54. Briefly, cells were detached and labelled for surface AQP4 with primary antibody (Proteintech 16473–1-AP) and secondary antibody (Jackson ImmunoResearch). Note, the recognized epitope is resistant to trypsin digestion. Non-specific background staining was assessed using an appropriate fluorochrome-matched secondary antibody. Cells were processed the same day for analysis on a BD LSR II flow cytometer and data was analyzed using FACSDiva (BD Bioscience).Extracellular staining of AQP4 was performed as previously describedt test as indicated in the figure legends, with statistical significance established at p < 0.05.Data were compared via two-tailed Student Supplementary information


Pregnancy requires balance between tolerance to the haploidentical fetus and the mother's ability to mount immune responses. There are parallels to this phenomenon that occur in metastatic cancer. We assessed soluble program death ligand‐1 soluble PD‐L1 (sPD‐L1) and galectin‐9 in the blood of pregnant women during gestation as these molecules are highly involved in immune suppression during cancer.sPD‐L1 and galectin‐9 concentrations by ELISA. Term placentas were collected in formalin and IHC was completed for PD‐L1 and galectin‐9 expression.Maternal blood was collected from 30 primigravida women at monthly intervals during pregnancy, delivery and 6‐week post‐partum. Blood was analyzed for sPD‐L1 (0.438 ng/mL) and galectin‐9 (1976 pg/mL) were elevated early in normal pregnancies compared to non‐pregnant controls . sPD‐L1 increased throughout gestation, whereas galectin‐9 remained elevated until parturition; both proteins returned to control levels post‐partum. Women carrying male fetuses had significantly higher galectin‐9 levels, but not sPD‐L1, than those carrying females . Trophoblast cells of the term placenta coexpress galectin‐9 and PD‐L1.Maternal blood levels of sPD‐L1 increased during pregnancy and may play a role in immune tolerance that is critical for the fetus.Immune‐regulatory molecules galectin‐9 and  PD‐L1 promotes T regulatory cell development and function and directly inhibits effector T‐cell differentiation, cytokine production, and target cell lysis.Galectin‐9 is another molecule with a role in inflammation, development, and malignancy that may have a similar role in pregnancy.2This study was approved by the Mayo Clinic Institutional Review Board. Ten milliliters of peripheral blood was collected from 30 consenting primigravida women with healthy, uncomplicated pregnancies. Collections were monthly starting at 8 weeks, and then 2‐hour post‐delivery and 6‐weeks post‐partum (n = 10 average time points per person). A section of the placenta was also collected at the time of delivery, fixed in formalin, and embedded in paraffin for immunohistochemistry. Blood was also collected from 15 non‐pregnant, reproductive age females undergoing blood donation at Mayo Clinic. Whole blood was processed to plasma and buffy coats were isolated using Ficoll‐Paque gradients . Plasma was stored at −80°C until use.To measure plasma levels of galectin‐9, DuoSet ELISA antibodies were utilized as per the manufacture's instruction . For sPD‐L1, a sandwich ELISA was developed using paired mouse monoclonal antibodies (H1A and B11) raised against the extracellular domain of human PD‐L1. The specificity of each antibody was validated by immunohistochemistry and flow cytometry. Both antibodies bind to the extracellular domain of PD‐L1 and to different sites on the PD‐L1 molecule. The assay is specific for PD‐L1 and does not exhibit cross‐reactivity to other B7‐H homologues , or third‐party recombinant protein P‐selectin (R&D Systems). Binding of H1A to PD‐L1 in the ELISA can be blocked by pre‐incubation of standards with antibody Figure  A,B. H1ATen term placentas were fixed in formalin and embedded in paraffin. Antibodies against galectin‐9  or PD‐L1  were utilized for staining, along with isotype controls. The galectin‐9 antibody was diluted 1:500 for immunohistochemistry while the PD‐L1 antibody was diluted 1:400 for staining.t test. Longitudinal data were measured by ANOVA, and compared to post‐partum concentrations. P‐values < .05 were considered significant.All samples were run in duplicate and averaged. Data are presented in means and standard error of the mean (SEM). Statistical significance was determined using a 2‐tailed student's 33.1All women included in our pregnant cohort were primigravida and ranged in age from 19 to 34 years with an average age of 28.4. They were non‐smokers and had no known infections, autoimmune disease or prior cancer diagnoses. Of this cohort, 15 women carried and delivered male offspring and 15 had females. This occurrence further permitted a direct comparison of infant sex on the levels of the factors assayed in this study. The control cohort was comprised of reproductive age, non‐pregnant women who were blood donors at Mayo Clinic. The non‐pregnant women in the control population ranged in age from 22 to 44 years, with an average age of 36.8 years. All patients in the control group had negative pregnancy tests at the time of blood collection, but pregnancy or other history was not collected for these subjects.3.2P = .45). However, by 15 weeks, sPD‐L1 levels were significantly higher than non‐pregnant controls . On the other hand, galectin‐9 levels were nearly 3 times non‐pregnant levels at 8 weeks of gestation . Both molecules had returned to control levels by 6‐week post‐partum . Table Elevations in both sPD‐L1    was originally cloned from a cDNA library of the human placenta and was predicted to function as an organ‐specific negative regulator of cellular immune responses.Studies of galectin biology have demonstrated numerous roles for this family of proteins in inflammation, infection, cancer, development, and signaling.We found that both galectin‐9 and sPD‐L1 levels increase with length of gestation. As the placenta highly expresses both immunomodulatory proteins, this increase may be due to the growth of the placenta through pregnancy; however, the placenta is not the only source of these proteins. Throughout the maturation process, dendritic cells release increasing amounts of sPD‐L1 which can initiate apoptosis of CD4 +  and CD8 +  T‐cells as a mechanism of immune homeostasis.If the role of sPD‐L1 and galectin‐9 during pregnancy is to modulate maternal immune tolerance to support fetal growth and development, it is important to consider their role in spontaneous miscarriage or recurrent pregnancy loss. Studies have demonstrated that mRNA expression of PD‐L1, but not PD‐1, is significantly decreased in decidual tissues from cases of recurrent miscarriage compared to controls.The sex of the fetus is another important variable which must be considered when studying fetal‐maternal interactions. Many pregnancy outcomes have been associated with the sex of fetus; for example, male fetuses have an increased incidence of preterm birth and gestational diabetes, whereas mothers carrying female fetuses more often present with hypertensive disorders.Our results support the relevance of the PD‐1/PD‐L1 and galectin‐9 pathway in fetal‐maternal tolerance and pregnancy‐related complications. Therefore, manipulation of these pathways may represent a novel approach to maintaining and enhancing maternal tolerance in pregnancy. Currently, although anti‐PD‐1 and anti‐PD‐L1 antibodies have become routine in oncology care, there are no tools to block the action of galectin‐9 for humans. Further studies are still needed to better understand the potential role of these molecules in pregnancy, especially in pregnancy loss. Continued collaborations between clinicians and scientists, especially those made up of multidisciplinary teams, will provide us with creative new ways to understand complicated biology and improve outcomes in obstetrics and oncology.H. Dong and S. Harrington are the inventors of U.S. patent application WO2015050663A1, and H. Dong and S. Markovic hold US patent 9302005 B2. The authors have no other relevant affiliations or financial involvement with any organization or entity having financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. Click here for additional data file.


This fact indicates that graphene predominantly exhibits dielectric-like behavior at visible and near-infrared wavelength, which can be attributed to the low carrier density in graphene compared with metals.Graphene has proven to be a promising conductive layer in fabricating optical plasmon resonators on insulator substrate using electron beam lithography and has the potential to construct electrically controlled active plasmon resonators. In this study, we investigate the effect of graphene on plasmon resonance using graphene and Au plasmon resonator system as a model at visible and near-infrared wavelength. Our experiment data show that the presence of graphene does not weaken and annihilate the plasmon resonance peaks, instead it predominantly makes the peaks redshift, which is similar to the behavior of depositing SiOThe online version of this article (doi:10.1186/s11671-016-1753-6) contains supplementary material, which is available to authorized users. Optical plasmon resonators are artificial nanostructures on sub-wavelength scale, with the ability to engineer electromagnetic space and control the propagation of electromagnetic wave , 2. In tGraphene, a two-dimensional plane of carbon atoms arranged in a honeycomb lattice, is a semimetal with zero energy band gap. A single-layer graphene has a transmission of 97.7% at visible and near-infrared region  and has 14/cm2 using highly efficient ion-gel dielectric [In addition to be a promising conductive layer in fabricating complex optical plasmon resonators, graphene also has the potential to construct electrically controlled active plasmon resonators at broadband wavelength , 19, 20 electric , which ielectric . At visielectric , 14, 20.2 film is deposited on the Au plasmon resonators. Since depositing SiO2 film or graphene on the Au plasmon resonators both exhibit the same effect, this indicates that graphene predominantly shows dielectric-like behavior at visible and NIR frequencies. The research is beneficial for understanding the role of graphene in Au plasmon resonators at visible and NIR frequency.In this work, we investigated the effect of graphene on plasmon resonator system at visible and near-infrared wavelength using Au plasmon resonators as a model system. The experiment data show that the plasmon resonance peaks exhibit clear redshift no matter whether graphene is placed below or above the Au plasmon resonators, and the plasmon resonance peaks’ shape experience little change. Frequency redshift also appears when a SiOi and width Wi  were all decreased by 16 nm in order to offset the increase caused by RIE. Figure Figure 2, and the spectra are collected from 20 × 20 μm2 in the sample center using a ×10 objective. The sample exhibits one absorption peak at about 715 nm under X-polarized light excitation (see black curve), but two plasmon resonance peaks occur at around 570 and 807 nm for Y-polarized light, respectively (see red curve). All of these absorption peaks are consistent with the numerical simulation results when only Au plasmon resonator array on quartz [Figure n quartz  is consi2 RIE. The RIE condition was appropriate to removing graphene while having little effect on Au nanostructure. Figure In order to reveal the effect of graphene on the optical spectra of the Au plasmon resonator array at visible and NIR wavelength, we compared the extinction spectra of sample with and without graphene. Graphene in the fabricated Au plasmon resonator array was removed using O2 RIE-treated sample. The transferred graphene covers the top surface of the Au plasmon resonator array as shown in Fig. 3. The black curve and red curve in Fig. To further confirm the effect of graphene on the spectra of the Au plasmon resonator array at visible and NIR frequencies, we transferred a single-layer CVD graphene onto O2 as dielectric film and investigated the extinction spectra of Au plasmon resonator array after depositing different thickness SiO2 film on it. Figure 2 film on the sample. We first removed the graphene on Au plasmon resonator array with RIE, then we used E-beam to evaporate 15 nm SiO2 film on the sample, and this process was repeated twice. In this way, sample S4 was covered with 15 nm SiO2 film and S5 with 30 nm. Figure 2, S4, and S5. The extinction spectra of both S4 and S5 have a redshift for X- and Y-excitation as the thickness of SiO2 increases. For the samples excited by X-polarized light, plasmon resonance peak shows redshift from 660 to 695 nm when the thickness of SiO2 increased from 0 to 15 nm. When the thickness of SiO2 increased from 15 to 30 nm, the redshift of plasmon resonance peak increased further to 725 nm, as shown in bottom panel of Fig. 2 thickness increases. Their behaviors are similar no matter whether graphene is located on or below the Au plasmon resonator array. And furthermore, the redshift caused by a single layer graphene is equal to that brought about by 30 nm SiO2. Therefore, graphene predominantly shows dielectric-like behavior in Au plasmon resonator array at visible and NIR frequencies, and its effect is equal to about 30 nm SiO2.To clearly show the behavior of Au plasmon resonator array after depositing dielectric film, we chose SiOm is the electron mass, n3D is the free electron number per unit volume, and e is the electric charge. We get q < <kF, where q is the wave vector, kF is the Fermi vector) satisfy [q is the wave vector, vF = 1.1 × 106 m/s is the Fermi velocity at SiO2 substrate [ћ is the reduced Plank constant, and ε is the dielectric constant. nGr is the free electron number per unit area. For a typical doping induced by SiO2 substrate [nGr ~ 1011/cm2, the plasmon oscillation frequency q = 0.1kF. Furthermore, free electron in gold is about 6 × 1022/cm3, much higher than that in graphene. Therefore, ω,Grp < <ωp,3D in the graphene and Au plasmon resonator system, and we can treat graphene as dielectric in engaging plasmon mode at visible and NIR frequency.The dielectric-like behavior of graphene at visible and NIR frequency can be attributed to the low carrier density in graphene compared with metals. In three dimension metal, the plasmon oscillation frequency at zero wave vector is satisfy .\documenubstrate , ћ is thubstrate , nGr ~ 2 film thickness in Fig. 2 film increase. For the resonance peak at about 660 nm, its shift rate increases to 2.2 nm/nm. For the resonance peak at 725 nm, its shift rate further increases to 2.9 nm/nm. This discovery provides convenience to design wavelength tunable plasmon resonance and make more sensitive sensor based on plasmon resonance.Finally, we presented the dependence of plasmon resonance peaks on SiO2 RIE, but we found the presence of graphene does not weaken and annihilate the plasmon resonance peaks, and it predominantly makes plasmon resonance peaks shift to the red end. Graphene shows dielectric-like behavior, which is similar to that of SiO2 film on Au plasmon resonator array, at visible and NIR frequencies. The research provides useful insight into the role of graphene in Au plasmon resonators and give some valuable guidance on designing graphene-based tunable plasmon resonators at visible and NIR frequency.In summary, we fabricated Au plasmon resonator array on quartz using graphene as discharge layer. The residual graphene on sample can be easily removed by O4) and hydrogen (H2). A protective polymethyl methacrylate (PMMA) film was applied to the deposited graphene layers, and the PMMA/graphene was transferred to the insulating substrate by dissolving the Cu foil using a 1 M FeCl3 solution. The transfer process was completed by attaching the PMMA/graphene to a quartz substrate followed by removal of the PMMA with acetone.Graphene was grown by chemical vapor deposition (CVD) on a 25-μm thick copper (Cu) foil  at 1000 °C using a mixture of methane  to methyl isobutyl ketone (MIBK) mixture for 90 s at room temperature followed by rinsing in IPA and drying by nitrogen flow. For the following process of pattern transfer, a metal layer was first deposited by an Anelva L-400EK e-beam evaporation system followed by a lift-off process using acetone at 40 °C. Graphene was removed using AVELVA reactive ion Etching (RIE) system, in which the O2 flow was calibrated in the following ways: flow rate was 40 sccm, pressure was 10 Pa, and RIE time was 15 s. SiO2 film was deposited using Anelva L-400EK e-beam evaporation system.The process of EBL exposure and development on quartz substrates includes the following procedures. The quartz substrate coated with graphene was spin coated with resist PMMA 950 K A2 (MicroChem Company) at 3000 rpm, giving a resist thickness of 80 nm. The resist was prebaked on a hot plate at 180 °C for 90 s. The e-beam exposure was performed using a JEOL JBX-6300FS system, operated at 100 kV with beam current as 30 pA, beam diameter of 3 nm, exposure dose of 1100 μC/cmThe optical spectra, including transmission and reflection, were measured using a Craic 308 PV microscopy spectrophotometer at room temperature and in the air.


In this paper, we present an innovative and ultra-fast process for the deposition of high-quality graphene on different metal foils and thin metal films. The graphene layer can be homogeneously deposited in only 30 s process time. Due to the weak adhesion to the substrate material, the monolayer graphene is easy to transfer using the established processes. For the production, we use magnetic filtered high-current arc evaporation (Φ-HCA) with a solid, graphitic carbon source. This ultra-fast growth process can pave the way towards a cost-effective graphene synthesis for the mass production e.g., in a roll-to-roll process, avoiding time consuming established processes. HoweverSo far, the most established method to fabricate large area monolayer graphene films is a CVD  or PE-CVD (Plasma enhanced CVD) process using carbon containing precursor gases like methane ,3,4,5. UIn contrast, arc evaporation as one of the most established physical vapor deposition methods (PVD) can produce a wide range of carbon nano-structures. Fullerenes, Nanotubes, and Carbon Black can be produced just by changing the deposition parameters, e.g., substrate temperature, gas composition, gas pressure, and plasma power ,9. Even If graphene should be used as a substitute for transparent conductive oxides (TCO) using the high transparency and excellent electrical conductivity or as a material for high frequency applications, a closed monolayer coating with low defects is necessary. These requirements cannot be fulfilled using the state-of-the-art arc evaporation processes as described above.2) on poly crystalline copper foils for 30 s. As far as we know, this is the first publication reporting the formation of low-defective closed graphene films using an arc evaporation method.Therefore, we here present an innovative approach, which allows the ultra-fast growth of homogeneous large-area monolayer graphene  of highly sp3-hybridized amorphous carbon films resulting in a predominantly sp2-hybridized graphite-like structure with a strongly reduced resistivity [In this paper, we used an arc evaporation tool which was originally developed to produce ultra-hard carbon films  with a high percentage of sp3 bonds ,14. In oistivity ,16.In contrast, in the process describe here, the carbon deposition and the thermal organization, resulting in a closed graphene film, occurs simultaneously. This deposition can be optimized for different substrate materials by several external parameters such as pressure and composition of background gas, temperature, impinging angle, and deposition rate. Using this technique, the deposition of transparent and conductive graphene-like carbon coatings on Insulators is also possible . The fundamental design of the arc coating tool used in the present study is shown in The evaporator works in a pulsed mode using 1.0 ms pulses with an arc current of up to 3000 A. For the current process only a small amount of carbon is necessary, corresponding to the atomically thin thickness of the graphene structure. Consequently, only 17 plasma pulses are necessary to form a closed graphene film. That fact makes the growth process extremely fast. After igniting the plasma discharge, the ions are firstly guided through the gas atmosphere in order to reduce their kinetic energy by collisions with the gas atoms. Additionally, hydrogen was used to reduce the crystal nucleus on the surface and enhance the growth of larger grains. The impinging angle of the carbon was changed in order to reduce the effective impact energy. The best conditions were obtained for an angle of incidence of 0° . The copper foil was firstly cleaned in acetone and isopropyl alcohol and subsequently treated by chemical polishing using a mixture of acetic acid, nitric acid, and sulfuric acid  for 5 miArc-Evaporation graphene was produced using the following process parameters: arc current 1500 A, magnetic filter current 200 A, pulse time 1 ms, pulse interval of 3 s, partial argon pressure 100 Pa, partial hydrogen pressure 20 Pa, substrate temperature 1020 °C. Before starting the coating process, the substrate was pre-heated for 20 min in 45 Pa hydrogen in order to increase the grain size of the copper foil and to remove the native oxide. After coating, the samples were cooled down for 10 min in argon/hydrogen atmosphere.For comparison, CVD graphene was produced on copper using the following parameters: argon flow 90 sccm, hydrogen flow 20 sccm, methane flow 20 sccm, overall working pressure 70 Pa coating time 30 min, substrate temperature 1000 °C. Before starting the coating process, the substrate was pre-heated for 30 min with 60 sccm argon in order to increase the grain size of the copper foil and to remove the native oxide. After coating, the samples were cooled down for 20 min in argon atmosphere.2 on Silicon) followed by drying in atmosphere and baking procedure . For removing the PMMA, the sample was washed in Acetone for 20 min and subsequently rinsed in Isopropyl alcohol and pure water.For transfer, an established electrolytic transfer process was used ,21: 1 M Raman measurements were conducted using a WiTec alpha 300 micro Raman microscope using 488 nm excitation laser wavelength with 30 mW with a lens magnification of 50×. The single spectra on one point were taken out from the large area scan with an integration time of 100 ms and one accumulation. Mappings were made with a lateral resolution of 0.66 µm per pixel with an integration time of 100 ms. For comparison for each of these mappings an average spectrum from every point inside the scanned area was created.−1, G-peak at 1581 cm−1, and 2D-peak at 2703 cm−1. The D-peak, originating from defects or grain boundaries which are breaking the translational symmetry of the Raman scattering process [As described above, the deposition process can be carefully optimized by changing the deposition parameters. The amount of carbon which is deposited on the substrate surface is determined by the number of plasma pulses. Consequently, this parameter is the most significant one to form a monolayer of graphene, because arc evaporation is not self-limiting. For the current study, we varied the number of plasma pulses from 10 to 35 with an arc current of 2.5 kA to identify the best conditions for monolayer graphene. After the deposition, we characterized the graphene using high-resolution Raman mapping. For all samples, we observed the typical Raman spectrum for graphene with the characteristic D-peak at 1350 cm process . For monAfter 10 plasma pulses, the graphene growth is still incomplete, resulting in a beginning nucleation of graphene domains and uncoated regions. This can be seen by a decreasing Raman I(2D)/I(G) ratio compared to 15 plasma pulses. After 15 pulses, we recognized a complete coverage of monolayer graphene. By increasing the number of plasma pulses, the Raman I(2D)/I(G) ratio decreases. Additionally, the 2D-peak position shifts to higher wave numbers, indicating the growth of multilayer graphene, as reported in . After 32. Because the graphene growth ultimately limits the production rate [In order to speed up the process, we additionally varied the time between two plasma pulses  from 1.5 to 5 s. The shorter the pulse interval can be chosen, the higher is the effective deposition rate. ion rate , it woulion rate .However, even by using an optimized pulse interval of 3 s, a non-neglectable D-peak can be seen, which is a sign of defective graphene.Consequently, for a further optimization of the growth process, we varied the plasma pulse current from 1.5 to 2.5 kA. The plasma pulse current determines the plasma density during the deposition. Hence, this parameter influences the deposition rate of the growing film and subsequently the number of defects and the grain size of the graphene layer. Using the previously described optimized parameters of the modified Φ-HCA process, it is possible to reproducibly deposit high quality graphene on poly-crystalline copper foils. −1. The D-band, originating from defects or grain boundaries which are breaking the translational symmetry of the Raman scattering process [The 2D band is roughly 4 times higher than the G peak (Graphite) which is a sign of single layer graphene with only few defects . Additio process , is almo2. For investigating the homogeneity of the graphene layer, large area Raman scans with high resolution were conducted. The Raman I(2D)/I(G) ratio is predominantly above 3 inside the graphene grains, which clearly shows the formation of high-quality single layer graphene. Variation in Raman I(2D)/I(G) intensity was created by the grain boundaries of the single layer graphene. Surprisingly, the graphene layer can be identified even in the microscopic image of the copper surface b, indicaFurthermore, 2 on Silicon wafer) using the established electro-chemical transfer [Subsequently, the graphene layer was transferred for further investigation on oxidized silicon  Φ-HCA-graphene, (b) CVD-graphene.To compare our Φ-HCA-graphene with graphene grown on copper foils using CVD, we additionally prepared samples by means of a standard CVD process . In contrast, the Arc-Evaporation-graphene is almost free of copper residuals and shows only few contaminations with the polymer film. Additionally, the Arc-Evaporation-graphene was much easier to transfer. It seems that the adhesion between the Arc-graphene and the copper is much weaker than in case of transferring standard CVD-graphene. This can be caused by a completely different growth process compared to CVD.The Raman I(2D)/I(G) reaches the value of 3 indicating the presence of monolayer graphene whereas the intensity of the D-band increases. The presence of D-band requires defects , which can be a result of the treatment during the transfer process . Hence, The Raman I(D)/I(G) ratio a as well2/Vs for one monolayer. The electrical properties can be further enhanced by using an optimized transfer process. However, these values reported here are already comparable to other established methods [Additionally, electrical properties of the transferred graphene on Silicon oxide were characterized by means of four tip measurements (van der Pauw arrangement) and Hall measurement. Due to some defects after the transfer process, our transferred graphene reaches a sheet resistance of 4 kΩ/square and a charge carrier mobility of 400 cm methods ,24.Overall, a monolayer graphene with only few defects can be grown very fast on multi crystalline copper foils using this new Φ-HCA process and subsequently transferred by means of electro-chemical methods.A new ultra-fast deposition process for graphene on copper foils is presented. A modified magnetic filtered vacuum arc system (Φ-HCA) with hydrogen containing argon as background gas and with heated and tilted substrates can form graphene with excellent homogeneity covering large areas. The filtered arc source supplies the surface only with that amount of material which is necessary for the reorganization of carbon atoms into graphene. Consequently, only 17 plasma pulses are necessary to form a closes graphene film. This makes the deposition process much faster than many established CVD processes.After transfer, the graphene shows almost negligible copper residuals and only few contaminations of the polymer transportation layer.To sum up, this fast and cost-effective process can pave the way towards an industrial roll-to-roll process and opens a broad field of application of this kind of carbon layers.