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


Long INterspersed Element-1 (LINE-1 or L1) is a retrotransposable element that has shaped the evolution of mammalian genomes. There is increasing evidence that transcriptionally active L1 could have been co-opted through evolution to play various roles including X-inactivation, homologous recombination and gene regulation. Here, we compare putatively active L1 distributions in the mouse with human. L1 density is higher in the mouse except for the Y-chromosome. L1 density is the highest in X-chromosome, implying an X-inactivation role. L1 is more common outside genes (intergenic) except for the Y-chromosome in both species. The structure of mouse L1 is distinguished from human L1 by the presence of a 200 bp repeat in the 5′ UTR of the former. We found that mouse intragenic L1 has significantly higher repeat copy numbers than intergenic L1, suggesting that this is important for control of L1 expression. Furthermore, a significant association between the presence of intragenic L1s and down-regulated genes in early embryogenesis was found in both species. In conclusion, the distribution of L1 in the mouse genome points to biological roles of L1 in mouse similar to human. The Long INterspersed Element-1 (LINE-1 or L1) is a retrotransposable element, which constitutes 18–20% of mammalian genomes Although L1s are abundant in human and mouse genomes, most of them do not have retrotranposition activity owing to truncations in 5′ regions, rearrangements, or mutations cis-regulatory element in embryogenesis), cell differentiation and DNA repair Until recently L1s were thought to be selfish DNA elements in which their only function was to replicate Mouse and human L1s share similar ORFs but differ markedly in 5′ UTR sequence, which may be responsible for differences in transcriptional activities between the two species In order to further explore other possible functions of L1 in mammals, this paper presents an in-depth comparative study between human and mouse L1s. First, the distribution of L1s within and outside of gene bodies  were mapped between the two species. Intragenic and intergenic L1s were compared in terms of conservation of L1 structural features in mouse instead of performing straightforward L1 sequence comparison. Finally, statistical tests were performed of intragenic L1 association with gene expression profiles during the early stages of human and mouse embryogenesis.Alu).Mouse and human L1 information were downloaded from the L1Base, which is a public database containing L1 elements residing in human and mouse reference genomes Homo sapiens reference sequence (Refseq) build 36.3 and Mus musculus mouse Refseq build 35. The intragenic L1 group , and GF . Human L1s can be grouped into two major subfamilies according to their sequences in the 3′ end of ORF2 We hypothesized that intragenic mouse L1s should more conserved than intergenic, as was found earlier for human t-test for non-categorical characteristics, were conducted to test the null hypothesis that for a given feature of L1, there should not be much different between intragenic and intergenic L1s. Statistical tests were conducted separately on both mouse and human L1s. In mouse, there are 42 categorical functional characteristics and 11 non-categorical characteristics of L1s (groups related to the host genome (intragenic/intergenic) and condition, e.g., conserved, CpG islands, and L1 functional feature. Since age of L1s may confound the relative contributions of young and old elements to the intragenic/intergenic regions, we adopted Mantel-Haenszel (MH) chi-square testing model Two statistical tests, namely chi-square test for categorical characteristics and Student's s of L1s . In humas of L1s . For chit-tests with unequal variances For non-categorical (quantitative) features, unpaired Student's t-test t-statistics were calculated from the average and standard deviation of differences between paired samples of each developmental stage and the one-cell stage. Genes with p-values less than 0.05 were considered as differentially expressed. Chi-square analysis was then performed to test if genes containing L1 sequences are associated with up regulation with respect to the one-cell stage. The 2×2 contingency tables were constructed with rows of number of genes with L1 present and L1 absent, and columns of number of up-regulated genes and the rest. Similar 2×2 contingency tables were also constructed for testing L1 association with down-regulated genes in which columns were constructed as down-regulated genes and the rest. Chi-square tests were performed for both human and mouse between each pair of time-points using CU-DREAM (http://pioneer.netserv.chula.ac.th/~achatcha/cu-dream/) To test the hypothesis that intragenic L1s regulate genes in other physiological cellular processes such as embryogenesis in mammalian species, we analyzed publicly available microarray data from different stages of preimplantation embryonic development, namely one-cell, two-cell, four-cell, eight-cell, sixteen-cell, morula and blastocyst stages , where the higher number represents more significant value. From these tests, it was found that intragenic L1s are significantly more conserved in mouse as well as human. In mouse intragenic L1s, conserved features are distributed along the structure of L1 except for the 5′ UTR. Only one functional feature, the SA-154 acceptor splice site on antisense mouse L1 sequences, is poorly conserved among intragenic mouse L1. There are three conserved features in ORF1, six in ORF2 and one in the 3′ UTR. Unlike mouse intragenic L1s, the 5′ UTR of human intragenic L1 contains two conserved features. There are three and nine conserved features in the ORF1 and ORF2 of human intragenic L1, respectively. For both mouse and human, intragenic L1s have significantly higher intactness score and GC contents than that of intergenic L1s. For human L1s, ORF1 and ORF2 codon adaptation indexes (CAI) are significantly higher for intragenic L1s, whereas for mouse intragenic L1s, the monomer features, namely mean number of monomer repeats and monomer splice sites, are significantly greater. The complete listing of functional features and their statistical values are in Previous study showed that intragenic human L1s are more conserved than intergenic ones t-tests  and 3. TL1s are expressed in early embryogenesis In this study, we tested for association of L1 location and sequence with respect to genes and L1 functions in two mammal species. Four main observations were made. First, L1 density is greater in mouse than human, including L1s within genes. Second, intergenic L1s density is greater in autosome and X-chromosome but less in Y-chromosome. Third, mouse intragenic L1s are less conserved than human and contain significantly more monomer repeats than that of intergenic ones. Finally, mouse and human intragenic L1s are associated with down-regulation of gene expression during early embryogenesis.On the X-chromosome, L1 density is higher than all other chromosomes combined in mouse and human. This is consistent with the role of L1s in X-inactivation activity, where L1s are thought to act as boosters of X-inactivation chromosome spreading from a center of inactivation Intragenic L1s are more conserved than intergenic L1 for the mouse and human. Interestingly, mouse intragenic L1s are overall less conserved than human. The lower conservation of mouse L1 is particularly marked in the 5′ UTR, in which variation in monomer repeats was shown previously to control L1 promoter activity The greater conservation and possible activity of intragenic L1 in mouse is suggestive of function. We investigated whether intragenic L1 might play a role in gene regulation in early embryogenesis. Significant associations were found for down regulated genes with intragenic L1 and down regulation of the genes, starting from the 2-cell to the morula stage in mouse, whereas associations were significant for 8-cell to blastocyst in human. The different “L1 associated with down regulation” (LaD) profiles align well with the varying zygotic activations and the levels of global hypomethylation among mammals Kcnq1 was reported to be a paternally imprinted gene that is down regulated during embryogenesis development PDE3A) gene functions in the cGMP-PKG signaling pathway PDE3A must be inhibited to allow expression of other important genes during physiological development. Hence, under the global hypomethylation state during zygotic activity, intragenic L1 may be expressed which down-regulates these genes, perhaps by the same AGO2-dependent mechanism as described in cancer cells Although the timing of zygotic activation differs between mouse and human, intragenic L1 appears to be important for controlling gene expression in both species. Among the orthologous genes obtained from intersecting the mouse and human LaD gene sets , two genTokudaia osimensis, an XO species Although these tests are suggestive for possible function of intragenic L1s in mouse and human such as X-inactivation and embryogenesis, there are alternative explanations that do not require L1s to have functions. For example, some rodent species thought to lack potentially mobile L1 still have X-inactivation We reanalyzed both mouse and human L1 data from L1base. Statistical analyses showed that mouse and human L1s are distributed similarly over their host genomes with greater density of intergenic L1s in X and autosomal chromosomes but greater density of intragenic L1s in Y chromosome. Intragenic L1s are more conserved than intergenic, and mouse intragenic L1 are more likely to be transcriptionally active owing to higher monomer repeat copy number in the 5′ UTR. Furthermore, mouse and human intragenic L1s could play a role in gene regulation during early embryogenesis as they are associated with genes down regulated during zygotic activation. Therefore, distributions of L1 in other mammalian species need to be studied to fully comprehend the functional repertoire of L1.Table S1List of mouse L1 characteristics.(PDF)Click here for additional data file.Table S2List of human L1 characteristics.(PDF)Click here for additional data file.Table S3Molecular functions of 14 orthologous down-regulated genes during early embryogenesis in human and mouse.(PDF)Click here for additional data file.


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


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


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


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


Sporadic inclusion body myositis (sIBM) is the most frequently acquired myopathy in patients over 50 years of age. It is imperative that neurologists and rheumatologists recognize this disorder which may, through clinical and pathological similarities, mimic other myopathies, especially polymyositis. Whereas polymyositis responds to immunosuppressant drug therapy, sIBM responds poorly, if at all. Controversy reigns as to whether sIBM is primarily an inflammatory or a degenerative myopathy, the distinction being vitally important in terms of directing research for effective specific therapies. We review here the pros and the cons for the respective hypotheses. A possible scenario, which our experience leads us to favour, is that sIBM may start with inflammation within muscle. The rush of leukocytes attracted by chemokines and cytokines may induce fibre injury and HLA-I overexpression. If the protein degradation systems are overloaded , amyloid and other protein deposits may appear within muscle fibres, reinforcing the myopathic process in a vicious circle. Sporadic inclusion body myositis (sIBM) is the most frequent acquired progressive myopathy presenting over 50 years of age in Western populations , albeit n = 200) and non-IBM patients with other neuromuscular disorders (n = 171) [There is a highly characteristic pattern of limb muscle involvement with selective weakness of finger flexion Fig. a, b and n = 171) . They arn = 171) , 82. Furn = 171) .Fig. 1ClUntil recently, the diagnostic gold standard was considered to be certain pathological criteria. These criteria  include Furthermore, it has been noted frequently that the above-mentioned canonical pathological features may be missing in patients with typical clinical features of the condition (leading to a sensitivity of only 11 % ), particFinally, Pestronk  proposedThese diagnostic difficulties relate to the fact that the physiology of sIBM is not yet fully understood and that its proposed mechanisms are still debated . In shorIncluded in the canonical pathological criteria, one of the main features of this disease is the abnormal accumulation of ubiquitinated proteinaceous congophilic inclusions within some muscle fibres. This was first described in 1991 by Mendell et al.  who studBefore considering further what might be called the ‘amyloid hypothesis’, it must be noted that not all investigators have concluded that the accumulation of proteins associated with neurodegeneration is of specific significance. For example, using a proteomic approach, Parker et al.  did not Nevertheless, defenders of the theory of β-amyloid-mediated sIBM myofiber injury have tried to understand the physiology of the amyloid deposits by studying protein degradation pathways Fig. . One of The second pathway of protein degradation is autophagy Fig. . A portiConcomitantly p62, a shuttle protein transporting poly-ubiquitinated proteins for both the proteasomal and lysosomal degradation pathways accumulates within muscle fibres into aggregates that are positive for phosphorylated tau . This fepresenilin-1 gene under the dependence of a muscle specific promotor, showed increased Aβ42 levels in skeletal muscle and exacerbation of inclusion body myositis-like pathology and motor deficits [+ T cells by MHC-I molecules) [Thus, in sIBM, accumulation of aggregates seems to be related to proteasome and autophagy dysfunctions, but with what pathophysiological consequences? Various mouse models have attempted to provide further insight. A double transgenic mouse, overexpressing APP and the deficits . Moreovedeficits . Specifideficits , 66. It lecules) . Intereslecules) . It is llecules) . This silecules) . Here aglecules) . Furtherlecules) . Knowinglecules) , this in+ T cells, Fig. + T cells  which may promote lymphocyte activation by providing secondary signals to T cells in addition to T cell receptor stimulation by antigen, and elicit cytotoxic function markers such as perforin [+ T cells can be expanded ex vivo from muscle of polymyositis and sIBM patients. These clones show cytotoxic activity against autologous myotubes in vitro [+ cells in both peripheral blood and muscle inflammatory infiltrates of sIBM patients  that displayed normal suppressor function [On the other hand, immuno-regulatory second signals such as programmed death ligand 1  are also triggered, presumably as a negative feedback loop. PD-L1 is expressed in polymyositis, dermatomyositis and sIBM muscle fibres but not in normal muscle. Staining is predominantly localized (in muscle fibres as well as mononuclear cells) to areas of strong inflammation . Further+) are frequently observed  areDespite the clear evidence of global immune activation in sIBM, the question remains as to whether sIBM is primarily an inflammatory or a degenerative myopathy. If amyloid is the consequence of a primary immune reaction, including secretion of cytokines that increase MHC-I expression to such an extent that protein degradation capabilities are overloaded , then tap = 0.007, RMI p = 0.004) and on the sIBM weakness composite index (p = 0.04), than those who were not treated. The first stage of disease progression, towards handicap in walking, also occurred more rapidly among patients receiving immunosuppressive treatments . We therefore concluded that immunosuppressive treatments do not ameliorate, and may even aggravate, the natural course of sIBM [The main argument comes from the lack of significant beneficial clinical effect from conventional immunosuppressant drug therapy. Eight prospective double-blind studies have shown that conventional immunosuppressants (corticosteroids or methotrexate) or immunomodulatory regimens  have minimal or no benefit for sIBM patients , 84, 100 of sIBM . We haven = 6), rheumatoid arthritis (n = 4) and Sjögren syndrome (n = 4), with 3 patients with multiple autoimmune disorders [p < 0.001), and among antibodies against extractable nuclear antigens (in order of their frequency), anti-SSA/Ro-52 or Ro-60, SSB/La, RNP-A or C, and PM-Scl75 were statistically more frequently observed in sIBM patients [sIBM has been frequently described in association with other autoimmune disorders , 24, 53.isorders . The preisorders . For inspatients .Recently, apparently sIBM-specific autoantibodies have been described. An antibody recognizing a 43 kD antigenic target was found in the serum of 13 out of 25 sIBM patients but not in 40 controls  . The antEvidence for a sIBM-prone immunogenetic background comes from observations of a strong association with prominent alleles of HLA class I or II  , 53, 81,From a pathological point of view, Pruitt et al.  have shoFinally, and in contrast to the observations noted above concerning the outcome of prospective, double-blind, studies of immunosuppressive therapies in sIBM, two clinical trials, which included only a small number of patients, suggested that massive immunosuppression can lead to strength stabilization. The first compared six patients treated with anti-T lymphocyte globulin and methotrexate, with five patients treated with methotrexate only. The latter showed a significant loss of strength after 12 months but the anti-T lymphocyte globulin group remained stable . In the N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) gene mutation (leading to quadriceps-sparing myopathy). GNE encodes for a key enzyme in sialic acid biosynthesis and the myopathy is associated with the formation of autophagic vacuoles [GNE patients and correlated with pro-inflammatory markers (such as IL-6), which remained expressed at levels comparable to control muscles [GNE do not show features of inflammation [As indicated above, sIBM is accompanied by degenerative protein accumulations, and deficient protein degradation pathways  and one accumulated protein candidate might be HLA class I . This sivacuoles . But thivacuoles , except vacuoles , 54. Nev muscles . Furtherammation . Experimammation . In a moammation . Hence, In a sIBM transgenic mouse model, that is marked by enhanced levels of Aβ1-42 in skeletal muscle  , the lonIn muscle biopsies of sIBM patients, compared to other forms of myositis, Schmidt et al.  found thAs we have seen previously, various pro-inflammatory cytokines are secreted in the milieu of sIBM inflammatory infiltrates, notably TWEAK . TWEAK ip = 0.007 and 0.04). These correlations between the degree of inflammation and signs of mitochondrial dysfunction strongly suggest a causative link between them. Furthermore, evidence of mitochondrial dysfunction is also highly correlated with fibre atrophy (p < 0.0001) [Moreover, Rigyel et al.  have sho 0.0001) . The aut 0.0001) .From the various arguments summarized above, a possible scenario for the pathogenesis of sIBM, and one that we favour, is that it may start with inflammation within muscle  with thp = 0.04), suggesting that rimmed vacuoles may be a later feature of the disease. Moreover, no differences in disease characteristic or progression were observed between patients with or without vacuoles [From a clinico-pathological standpoint, Chahin and Engel  showed tvacuoles .The rush of leukocytes, attracted by chemokines and cytokines, may induce fibre injury, mitochondrial dysfunction and HLA class I overexpression through, presumably, components of pro-inflammatory cell stress mechanisms such as nitric oxide production . If the


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


Several clinical trials have shown that immune treatment focus on programmed death-1 and programmed death-ligand 1 (PD-L1) yields a good clinical efficacy in advanced non-small cell lung cancer (NSCLC). We investigated whether the PD-L1 expression was related to clinicopathologic and molecular characteristics in patients with surgically resected NSCLC.EGFR, KRAS, NRAS, PIK3CA, BRAF, HER2 mutations and ALK, ROS1, RET fusion genes. The PD-L1 expression was evaluated by immunohistochemistry and staining of 5 % or more was scored as positive expression. Survival analysis was evaluated using the Kaplan–Meier method. Multivariate regression was performed using the Cox proportional hazards model.Between December 2008 and 2013, formalin-fixed, paraffin-embedded samples were obtained from patients with lung adenocarcinoma at Zhejiang Cancer Hospital. RT-PCR was used to analyze EGFR mutation , followed by EML4–ALK rearrangement , KRAS , HER2 , ROS1 rearrangement , PIK3CA , RET rearrangement , BRAF , and NRAS mutations . Twenty-four (6.2 %) patients carried coexisting mutations. PD-L1 expression was detected in 48.3 % (186/385) of all the patients. PD-L1 positive patients more frequently carried coexisting mutations , followed by single-gene  and pan-negative mutations . PD-L1 expression decreased disease-free survival (DFS) in univariate analysis (P = 0.014). Multivariate analysis revealed that PD-L1 expression was not an independent risk factor for poor DFS and overall survival (OS) .Mutations were detected in 76.6 % of the 385 patients tested: PD-L1 overexpression is more frequently observed in oncogene-mediated lung adenocarcinoma, especially with coexisting mutation subtypes. PD-L1 expression is not a prognostic factor in surgically resected lung adenocarcinoma patients. Lung cancer is the leading cause of cancer-related death in China . The staBlockade of immune checkpoints in cancer with monoclonal antibodies has recently emerged as a promising approach to the treatment of solid tumors. Programmed death 1 (PD1), which belongs to the CD28 family of proteins, is a T cell surface receptor that regulates T cell activation and proliferation. Its ligand, programmed death-ligand 1 (PD-L1), is frequently expressed in many types of carcinomas –14. ReceThis study focused on patients with completely resected lung adenocarcinoma and evaluated the association of PD-L1 expression with clinicopathologic parameters and driver genes, as well as its prognosis value in Chinese patients.The Ethics Committee of Zhejiang Cancer Hospital approved this study and written informed consent was obtained from each participant.A total of 385 adenocarcinoma patients underwent resection between December 2008 and 2013 in Zhejiang Cancer Hospital. Histological typing was determined according to the 2004 World Health Organization classification . Tumor-nProteintech Group Inc., Chicago, IL, USA, Catalog number: 66248-1-Ig) was 1:100 in Dako antibody diluent; slides were incubated with this antibody overnight at 4 °C. Then, the slides were incubated with Ventana Omni Mapanti-rabbit secondary antibody for 60 min. AVentana Chromo MapKit was used for antibody detection, and then the slides were counterstained with hematoxylin. Next, the slides were dehydrated and cover slipped as per normal laboratory protocol. Two independent pathologists (Wei Wu and Guoping Cheng) assessed the expressions.Immunohistochemical (IHC) staining of PD-L1 expression was performed on 4-6 μm thick formalin-fiated, paraffi-embedded tissue. The concentration of rabbit primary antibody that reacts to PD-L1 The concentration of rabbit primary antibody that reacts to PD-L1 (PD-L1 immunostaining results were classified into two groups based on the degree and intensity of staining: (1) negative, when staining was absent or detected in <5 % of the cells; and (2) positive, when membranous staining was present in ≥5 % of the cells.We used another antibody  to confirmed the PD-L1 expression in 102 patients. The PD-L1 immunostaining criterion is same with the former antibody.Genomic DNA or RNA was extracted from tumor tissues according to standard protocols . Briefly, the isolated RNA samples were used for reverse transcription into cDNA using Revert Aid First Strand cDNA Synthesis Kit . Either genomic DNA or cDNA was used for PCR amplification and sequencing. EGFR, HER2, KRAS, NRAS, BRAF, and PIK3CA were PCR amplified using genomic DNA. Cycle sequencing of the purified PCR products was carried out with PCR primers using the commercially available ADx Mutation Detection Kits .The ALK, ROS1, and RET fusion mRNA was detected by PCR with fusion gene detection kit . In brief, total RNA was extracted with QiagenRNeasy FFPE Kit. The mRNA was reverse-transcribed to cDNA at 42 °C for 1 h. β-actin was used as the internal control. The RT-PCR conditions were as follows: an initial denaturation at 95 °C for 5 min, followed by 95 °C for 25 s, 64 °C for 20 s, and 72 °C for 20 s to ensure the specificity; and 31 cycles at 93 °C for 25 s, 60 °C for 35 s, 72 °C for 20 s were performed for data collection and sensitivity analysis. All of the positive genes including mutations or fusions were confirmed with Sanger sequencing. All the experiments were performed according to the user manual as described previously .P < 0.05 was considered statistically significant. The median follow-up was 54 months (6.5–65) and the last follow-up date was July 31, 2015.The Chi squared test was used to evaluate the relationships between different driver genes and PD-L1 expression. Survival curves of pathologically confirmed samples were calculated using the Kaplan–Meier method until death or last follow-up. Multivariate analysis was performed using the Cox regression model. Statistical analysis was performed with the SPSS 18 software . Patients’ clinical profile is presented in Table EGFR, KRAS, NRAS, PIK3CA, BRAF and HER2 mutations and ALK, ROS1, RET fusion genes. This analysis included EGFR mutations , followed by EML4–ALK rearrangements , KRAS , HER2 , ROS1 , PIK3CA , RET , BRAF , and NRAS , and 24 coexisting mutations (6.2 %). All the nine genes were negative in 90 patients, defined as pan-negative. The details of coexisting mutations are listed in Table All the patients were analyzed for P < 0.001).The PD-L1 membrane expression was detected in 186 of the 385 lung adenocarcinoma patients (48.3 %) Figs. . The relP = 0.337).Another antibody  was used in 102 patients to detect the PD-L1 expression. The same trend of PD-L1 expression difference existed in patients with different gene abnormality. The PD-L1 positive patitnets was more frequently carried coexisting mutations , followed by single-gene positive  and pan-negative mutations (P = 0.014)   and PD-L1 expression negative were significantly risk factors for tumor recurrence or metastasis Figs. , 4, whilIn multivariate analysis, only early stage suggested lower risk for DFS, while PD-L1 expression was not correlated with recurrence or metastasis. Early stage was an independent and favorable prognostic factor for OS Table .Table 5MThis study shows that PD-L1 is overexpressed in 48.3 % (186/385) of lung adenocarcinoma patients and this overexpression is more frequently seen in patients with coexisting mutations, but less frequently in patients with pan-negative genes. The PD-L1 overexpression is not a prognostic factor for overall survival. To the best of our knowledge, this is the first study with the largest number of patients correlating the nine common driver genes in lung adenocarcinoma and PD-L1 expression.PD-L1 expression and driver genes [PD-L1 positive status was significantly associated with EGFR mutations . Mu et al. observed no significant correlation between PD-L1 expression and EGFR/ALK status in stage I NSCLC patients [PD-L1 expression and EGFR status in lung adenocarcinoma [EGFR mutations or ALK rearrangements, and other driver genes were not well investigated. Last but not least, racial differences may play an important role in the controversial results.Several studies have reported the association between er genes , 24. Theer genes  observedpatients . Similararcinoma . TherefoIn the present study, PD-L1 overexpression was more frequent in patients with coexisting mutations than in pan-negative patients. One explanation is that the genetic differences affected epigenetics, which may alter the expression of tumor-associated self-antigens, which in turn, affected tumor antigenicity. Increased number of driver genes reflects a higher level of neoantigens, which alters the immune microenvironment and increases the PD-L1 expression .Because of heterogeneity of tumors, the efficacy of chemotherapy or molecular targeted treatment is relatively limited, combination treatment with different anti-cancer mechanisms drugs hold much potential in this area. Previous studies demonstrated that EGFR and ALK genes could induce PD-L1 expression to facilitate evasion of the host anti-tumour immune response, suggesting an active role for these genes in remodelling the immune microenvironment , 28. In The role of PD-L1 in predicting the prognosis of NSCLC was controversial in previous studies . Some stOur study limitations are as follows. One major limitation was its retrospective nature. Second, only 24 patients with coexisting mutations were included, and the small sample size may influence the results of the current study. Third, different antibodies were used in different anti-PD-1 or PD-L1 therapies in clinical trials currently. The choice of antibody and the threshold for positivity might influence the results of different studies. Only one antibody and 5 % threshold were used in the present study. Different anti-PD-L1 antibodies may need to be validated in the same sample in future studies.In conclusion, we demonstrated the expression of PD-L1 in over 48 % of lung adenocarcinoma patients and the expression was associated with coexisting driver genes. PD-L1 expression is not associated with overall survival in patients with completely resected NSCLC.


Here, we report a patient with sarcoidosis who developed edematous erythema and interstitial lung disease. At the initial visit, clinically amyopathic dermatomyositis (CADM) with rapidly progressive interstitial lung disease (RP-ILD) was suspected because he had progressive dyspnea but no muscle weakness. The presence of anti-CADM-140/MDA5 autoantibodies was immediately assessed to facilitate a precise diagnosis, with negative results. Thereafter, skin and transbronchial lung biopsies revealed noncaseating granuloma with Langhans giant cells in both specimens, leading to a diagnosis of sarcoidosis. In this case, clinical features of skin and lung were unable to distinguish DM (including CADM) from sarcoidosis, but the lack of anti-CADM-140/MDA5 antibody was useful for differentiating CADM with RP-ILD mimicking sarcoidosis from bona fide sarcoidosis. Sarcoidosis is a disorder of unknown etiology affecting multiple organs and is characterized by the formation of granulomatous lesions. It has many clinical manifestations including skin and pulmonary symptoms. In light of reported cases of sarcoidosis coexisting with dermatomyositis (DM) –6, it isμL, red blood cell count of 517 × 106/μL, hemoglobin 17.0 g/dL, and platelets at 22.0 × 104/μL. Although serum AST was 56 IU/L, ALT was 57 IU/L, LDH was 260 IU/L, creatine kinase (CK) was 34 IU/L, and aldolase was 6.1 IU/mL, within thenormal range. Serum C-reactive proteins, KL-6 and SP-D, were elevated to 1.05 mg/dL, 1758 U/mL, and 279 ng/mL, respectively. Rheumatoid factor, antinuclear autoantibodies, and anti-Jo-1 antibodies were absent. Bacterial culture examination of sputum and thetuberculin skin test were negative. SpO2 (in room air) at rest was decreased to 92%. Chest radiography and CT indicated diffuse fine nodular or reticular shadow in both lung fields that suggested interstitial lung disease (ILD) (5 cells/mL with increased lymphocytes (49%) and an elevated CD4/CD8 ratio (8.40). TBLB and skin biopsy from the area of anterior chest erythema revealed infiltration of lymphocytes and epithelioid cells into the tissues and formation of noncaseating granuloma with Langhans giant cells. No caseous necrosis or malignant cells were seen. This is consistent with the pathological findings of sarcoidosis  revealed mediastinal lymphadenopathy, emphysematous change, and granular/nodular shadow on both lung fields. Blood chemistry revealed an elevation of serum LDH and serum KL-6. Because his respiratory symptoms were progressive, he was referred to our university hospital for further examination and treatment of the skin and respiratory symptoms. At the first visit, he had dyspnea on exertion and facial erythema as well as erythema on his anterior chest and back . He was se (ILD) . Electrocoidosis . GalliumThis is a case report of sarcoidosis in a patient with lung and skin manifestations mimicking CADM and RP-ILD. Our case highlights the difficulty of diagnosing patients who manifest clinical features indistinguishable from DM (including CADM), particularly skin and pulmonary symptoms. Symptoms of both diseases sometimes closely resemble each other and it is difficult to distinguish them. Moreover, patients with both DM and sarcoidosis have been reported –6, althoInitially, this patient was suspected of having CADM and RP-ILD due to edematous erythema of the face and trunk and progressive dyspnea on exertion. RP-ILD accompanied by DM, especially CADM, is a life-threatening complication and it is well recognized that respiratory symptoms in these patients often progress rapidly and resist treatment even if intensive immunosuppressive therapy is given. However, it is also known that early intervention with high-dose corticosteroids and immunosuppressive agents can rescue patients from this life-threatening condition. On the other hand, sarcoidosis therapy usually does not require the type of intensive treatment that is necessary for DM and RP-ILD. In this regard, it would be important to differentiate sarcoidosis from DM in the early stages as quickly as possible. Anti-CADM-140/MDA5 antibody is a recently discovered myositis-specific autoantibody . The preIn summary, we report a sarcoidosis patient who developed edematous erythema and ILD mimicking CADM and RP-ILD. In this case, measurement of anti-CADM-140/MDA5 antibody was useful to distinguish sarcoidosis from CADM with RP-ILD. Successful treatment with PSL alone achieved complete remission of the sarcoidosis. This case emphasizes the importance of being aware of other conditions mimicking DM and RP-ILD, as well as the usefulness of measurement of anti-CADM-140/MDA5 antibody.