Rbfox1 in conjunction with the specific increase of TrkB.T1 isoform expression also shows that the genes affected by Rbfox1 gain of function are surprisingly different from those influenced by Rbfox1 deletion. These findings not only identify TrkB as a major target of Rbfox1 pathophysiology but also suggest that gain or loss of function of Rbfox1 regulate different genetic landscapes.Brain-derived neurotrophic factor (BDNF) is a potent modulator of brain synaptic plasticity. Signaling defects caused by dysregulation of its Ntrk2 (TrkB) kinase (TrkB.FL) and truncated receptors (TrkB.T1) have been linked to the pathophysiology of several neurological and neurodegenerative disorders. We found that upregulation of Rbfox1, an RNA binding protein associated with intellectual disability, epilepsy and autism, increases selectively hippocampal TrkB.T1 isoform expression. Physiologically, increased Rbfox1 impairs BDNF-dependent LTP which can be rescued by genetically restoring TrkB.T1 levels. RNA-seq analysis of hippocampi with upregulation of  BDNF gene that converts a valine to methionine at codon 66   and truncated receptors lacking the kinase domain (TrkB.T1). Hypomorphic expression of the TrkB.FL causes hyperphagia-induced obesity due to reduced hypothalamic BDNF signaling, while genetic deletion of TrkB.T1 leads to increased anxiety related behavior associated with structural alterations in neurites of the amygdala  induction and is functionally essential for acute signaling cascades leading to LTP  before using BCA assay  for total protein quantification. Samples were then prepared for western blot using equal amounts of total protein before adding Laemmli sample buffer 2X  and loaded on 4–12% NuPAGE precast gels. Student t-test was applied for statistical significance assessment.Synaptosomes were obtained from the whole hippocampi of control animals  according to manufacturer’s instruction. cDNA was then generated using SuperScript III First-Strand Synthesis System . Real time PCR was performed using BioRad iTaq Universal SYBR-green Supermix (Cat.No. 172–5120) in a MX3000P (Agilent Technologies) apparatus with the following program: 95°C for 3 min; 95°C 10 s, 60°C 20 s for 40 cycles; 95°C 1 min and down to 55°C (gradient of 1°C) for 41 cycles (melting curve step). Delta Ct values were obtained using GAPDH as reference gene.Student t-test was applied for statistical significance assessment.Primers used:5’-AGCAATCGGGAGCATCTCT-3’TrkB common forward: 5’-CTGGCAGAGTCATCGTCGT-3’TrkB.FL reverse: 5’-TACCCATCCAGTGGGATCTT-3’TrkB.T1 reverse: 5’-TGCGACTTCAACAGCAACTC-3’GAPDH forward: 5’-ATGTAGGCCATGAGGTCCAC-3’GAPDH reverse: 5’- TGGCCCCAGTTCACTTGTAT-3’Rbfox1 forward: 5’- GCAGCCCTGAAGGTGTTGTA-3’Rbfox1 reverse: RNA immunoprecipitation was performed following the protocol from RNA was then extracted by adding an equal volume (150 µl) of buffer saturated phenol-chloroform pH 4.5 with isoamyl alcohol, vortexed and centrifuged at 20.000 g for 10 min. The aqueous upper part was carefully collected in a new tube before adding 150 µl of chloroform, vortexed and centrifuged again. The upper part was again transferred in a new tube and 50 µl of 5 M ammonium acetate, 15 µl of 7.5 M LiCl, 5 µl of 5 mg/ml glycogen and 1 ml of ice cold 100% ethanol were added before placing the samples at −80°C overnight to allow RNA precipitation. Samples were then centrifuged at 20.000 g/30 min at 4°C, washed once with 80% ethanol and centrifuged again. RNA pellets were finally resuspended in 20 µl of RNase-free water.RNA was polyadenylated using Poly(A) Tailing Kit  following the manufacturer instruction.cDNA was generated using SuperScript III First-Strand Synthesis System .RT-PCR was performed using BioRad iTaq Universal SYBR-green Supermix (Cat.No. 172–5120) in a MX3000P (Agilent Technologies) apparatus with the following program: 95°C for 3 min; 95°C 10 s, 60°C 20 s for 40 cycles; 95°C 1 min and down to 55°C (gradient of 1°C) for 41 cycles (melting curve step).RT-PCR products were run in agarose 2% gel.Primers used:5’-CGTGGTGGTGATTGCATCTG-3’TrkB common forward: 5’-CCATTGGAGATGTGGTGGA-3’TrkB.FL reverse: 5’-CAGTGGGATCTTATGAAACAAAACAA-3’TrkB.T1 reverse: 5’-TTTGAGCATGACTTACGTTTCG-3’TrkB.T1-upstream intron forward: 5’-CCCAGCCTTTGTCTTTCCTT-3’TrkB.T1-upstream intron reverse: 5’-CCGGAGTTACAAGAAGTGTGG-3’Camta1 forward: 5’-CTTGGTCCTGCTTTTTGGTC-3’Camta1 reverse: 5’-GAGCTGGATGATATGACGCTG-3’Sirt1 forward: 5’-CAGAGACGGCTGGAACTGTC-3’Sirt1 reverse: Nes-Cre animals with R26-Rbfox1flox/flox animals. Embryos at E18.5 stage were dissected and fast genotyped by using EZ Fast Tissue Tail PCR Genotypying Kit (EZ Bioresearch) in order to group Nes-Cre;R26-Rbfox1flox/+ hippocampi and R26-Rbfox1flox/+ hippocampi. RNA stability was assessed in hippocampal neurons at 4 DIV using Click-iT Nascent RNA Capture Kit  following manufacturer’s instruction. Briefly, 0.2 mM 5-ethynyl uridine (EU) ribonucleotide homolog was added to the neurons media to label new nascent RNA for 5 hr (pulse). After 5 hr, the media was replaced with normal media (without EU) for 9 hr (chase). Neurons were lysed at time 0 hr (right after 5 hr EU pulse) and at time 9 hr (after 9 hr of EU free media) and the RNA isolated using Qiagen RNeasy Mini kit (Cat.no 74104) according to manufacturer’s instruction. Biotin azide was then chemically bound to the EU containing RNA molecules and streptavidin magnetic beads were used to capture the newly synthesized pool of RNA. cDNA was generated by using SuperScript VILO cDNA synthesis kit .Primary hippocampal neurons for RNA stability studies were obtained by crossing Real time PCR was performed using BioRad iTaq Universal SYBR-green Supermix (Cat.No. 172–5120) in a MX3000P (Agilent Technologies) apparatus with the following program: 95°C for 3 min; 95°C 10 s, 60°C 20 s for 40 cycles; 95°C 1 min and down to 55°C (gradient of 1°C) for 41 cycles (melting curve step). Delta Ct values were obtained using β-Actin as reference gene.Student t-test was applied for statistical significance assessment.Primers used:5’-AGCAATCGGGAGCATCTCT-3’TrkB common forward: 5’-CTGGCAGAGTCATCGTCGT-3’TrkB.FL reverse: 5’-TACCCATCCAGTGGGATCTT-3’TrkB.T1 reverse: 5’-CCTGACACAGTGGTCATTGG-3’TrkC common forward: 5’-CTTGTCTTTGGTGGGGCTTA-3’TrkC.FL reverse: 5’-GACACATCCCCACTCTGGAC-3’TrkC.T1 reverse: 5’-TACCACAGGCATTGTGATGG-3’β-Actin forward: 5’-TCTCAGCTGTGGTGGTGAAG-3’β-Actin reverse: Nes-Cre) and N-cre-Fox1 mice  of about 3 months of age and in the C57BL/6 background. Deep-sequencing was performed on an Illumina HiSeq 4000 in paired-end mode with a read length of 150 base-pairs.RNA quality was assessed using an Agilent Bioanalyser. RNA integrity numbers (RIN) were observed to be greater than nine for all samples. Libraries were constructed from hippocampal total RNA using Illumina’s TruSeq Stranded Total RNA Kit (RS-122–2201). Three biological replicates were used for each of the two experimental groups: Ctrl mice , Preseq (version 2.0.3), Picard tools (version 1.119) and RSeQC (version 2.6.4). Reads were trimmed using Cutadapt (1.18) to remove adapter sequences, prior to mapping to the mm10 mouse reference genome using STAR (version 2.5.2b) in two-pass mode. Gene and transcript expression levels were quantified using RSEM (version 1.3.0) with gencode’s M16 mouse annotation. EBSeq (version 1.22.1) was used to test for differential isoform expression between experimental conditions. Group-based TPM filtering was applied to remove lowly expressed transcripts. Significant differentially expressed isoforms were identified with a false-discovery rate ≤0.05. The R package clusterProfiler (version 3.10.1) was used for gene ontology (GO) enrichment analysis of the significant differential expressed gene-isoforms identified by EBSeq. Significant over-represented biological process GO terms were identified with a q-value less than 0.05. The RNA-seq data generated for this study have been deposited in NCBI's Gene Expression Omnibus and is accessible through GEO Series accession number GSE136253.CLIP sequencing data   and the Flp-In T-Rex 293 Cell Line system  according to the manufacturer instructions. Rbfox1 expression was induced by Doxycycline  0.5 µg/ml. TrkB.T1 cDNA including the complete 3’UTR sequence (plasmid pLTM665) was transfected into the Rbfox1-Flp-In T-Rex 293 cells using X-tremeGENE 9 DNA Transfection Reagent .A Rbfox1 cDNA  was transiently transfected using X-tremeGENE 9 DNA Transfection Reagent .Neuro-2a (N2A) neuroblastoma cells  were transfected using TrkB.T1 cDNA including the complete 3’UTR sequence (plasmid pLTM665) in order to generate a stable N2A line expressing TrkB.T1 w/3’UTR. BDNF. All cell lines tested negative for mycoplasma.A HEK293 cell line with stable expression of TrkB.FL (HEK293-p618-2) was generated by transfecting HEK293 cells  with a TrkB.FL expression plasmid using X-tremeGENE 9 DNA Transfection Reagent  and by puromycin selection. TrkB.FL expressing HEK293-p618-2 were transiently transfected with TrkB.T1 expressing plasmid by using X-tremeGENE 9 DNA Transfection Reagent  and starved for 5 hr in serum free media (DMEM) before treatment with recombinant Coronal mouse brain slice for electrophysiological recording containing dorsal hippocampus was prepared according to Statistical significance was calculated using nonpaired two-tailed Student’s t-test. One-way ANOVA followed by Tukey’s multiple comparisons test was used for analysis of multiple groups . All dat In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Clive Bramham (Reviewer #1). Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.Thank you for submitting your work entitled "RbFox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels" for consideration by Summary:This study focuses upon the regulation of a truncated TrkB species, TrkB.T1, by the RNA binding protein RbFox1. There was agreement among the three reviewers that the regulation of TrkB.T1 by RbFox1 is of considerable interest. However, a number of reservations were raised. The major issues are the need for more evidence for the role of truncated TrkB in BDNF-dependent LTP; a resolution of how the levels of RbFox1 impact upon TrkB.T1 expression; and whether there is a non-neuronal contribution of Rbfox1. These are valid concerns that will require more experimentation in order to validate the major conclusions. Specific comments by the reviewers follow this letter. Based upon the common concerns and recommendations from the referees, we have therefore decided to decline the manuscript.Reviewer #1:The study provides important insights into the mechanism and functional regulation TrkB signaling via selective modulation in the expression of truncated TrkB.T1 receptors at synaptic sites. Regulation of TrkB.T1 is mediated by the RNA-binding protein RbFox1. In a gain-of-function model, RbFox1 stabilizes TrkB.T1 mRNA and increases expression of TrkB.T1 protein. Surprisingly, stabilization occurs through an unknown mechanism involving binding of RbFox1 to the intronic region of TrkB.T1 pre-mRNA, and not by binding to cis-acting elements in the 3'UTR. Functional studies in acute hippocampal slices show that transgenic overexpression of TrkB.T1 greatly inhibits LTP in the induced by rapid bath perfusion of BDNF. BDNF-LTP is partly restored in heterozygous transgenic mice, strongly supporting a role of TrkB.B1 in regulating TrkB function and plasticity in the adult hippocampus. The background for this work is the observation of enhanced TrkB.T1 expression in chromosome 16 trisomy mice. By identifying regulation of TrkB.T1 by RbFox1 and showing differences between gain and loss-of-function of the RNA binding-protein, the study opens new avenues for pre-clinical translational research.1) The effect on BDNF- LTP requires substantiation. Transgenic expression of TrkB.T1 modulates the response. To better evaluate the physiological role of TrkB.T1, BDNF-LTP should also be assessed in TrkB.T1 knockouts. TrkB inhibitors should be applied to confirm a TrkB-mediated response.2) Data on the binding sites and mechanism of RNA stabilization would improve the paper.Reviewer #2:This manuscript presents interesting data examining the consequences of RbFox1 upregulation as an approach to identify downstream genes regulated by RbFox1 which may have significance for brain disorders associated with RbFox1 mutation. The authors begin by following up on their prior Trisomy 16 work, to identify RbFox1 as a chromosome 16-encoded gene that could produce their previously observed upregulated expression of a truncated form of the TrkB receptor, TrkB.T1. Data is presented to show that viral overexpression of RbFox1 in hippocampal neurons produces upregulation of TrkB.T1 protein and mRNA without changing levels of full-length TrkB (TrkB.FL), and differential binding of RbFox1 to TrkB mRNAs is assessed. A mouse model with inducible RbFox1 expression is used to assess effects on gene expression by immunoblotting and RNA-seq and to probe BDNF-induced LTP. The authors compare their RbFox1 induced expression RNA-seq dataset with a published dataset from another group studying the effects of RbFox1 deletion  to support a main conclusion that genes affected by RbFox1 gain of function differ from genes affected by RbFox1 loss of function.Several aspects of the data make it difficult to definitively conclude that gain and loss of RbFox1 produce regulation of different genes:1) The comparisons of the results of RbFox1 overexpression and RbFox1KO in Figure 4B are hampered by the fact that there may be many differences in experimental design, mouse genetic background, sample preparation, sequencing, analysis and filters applied, etc., between the authors' dataset and data presented in Vuong et al., 2018. It's not clear that differences in seq data will all or even mostly be due to biological differences in the effects of Rbfox1 overexpression compared to Rbfox1 deletion. Insufficient details are provided in this manuscript to compare even major biological variables such as the age of mice used by the authors for the RNA-seq experiments.2) A proposed mechanism for the difference in genes regulated by gain or loss of RbFox1 expression is incompletely defined. The authors show that an RbFox1 heterozygote with reduced RbFox1 expression has an increase in RbFox2 expression relative to a control mouse, while RbFox1 gain of function does not alter RbFox2 levels. It is not clear whether RbFox2 is changed in the RbFox1 deletion context  though, or to what extent RbFox2 can interchange for RbFox1 binding sites and gene regulation.If RbFox1 differentially elevates levels of newly synthesized TrkB.T1 mRNA compared to TrkB.FL mRNA , then can the authors explain why this action of RbFox1 overexpression fails to produce any change in the level of TrkB.T1 protein  in the HEK293 dox-inducible cell line?Greater explanation is needed for the determination of reads as TrkB.T1 compared to TrkB.FL, especially given that TrkB.FL contains the sequence of TrkB.T1.In the BDNF-induced LTP experiments, it is not clear that the effect of 'inactivation' of one TrkB.T1 allele is specific to the context of RbFox1 upregulation. This manipulation should be symmetrically applied in the context of to the Ctrl mouse as well. The data presented are insufficient to conclude that RbFox1 upregulation leads to a deficit in the BDNF-induced LTP resulting from an increase in TrkB.T1 expression (as opposed to indicating possible distinct roles for both TrkB.T1 and RbFox1 upregulation in BDNF-induced LTP).Reviewer #3:In this manuscript, Tomassoni-Ardori et al. investigate the impact of Rbfox1 on the expression of different TrkB isoforms. The findings are somewhat interesting though the overall impact is somewhat limited by the many other recent manuscripts studying the function of this protein and the focus on Rbfox1 overexpression even though disease is associated with reduced functional Rbfox.However, the work does provide new insight into the potential role of Rbfox1 and the data demonstrating it binds and regulates TrkB.T1 is pretty conclusive, though there are a few shortcomings that should be addressed that are outlined below.1) In the second paragraph of the Results, the authors state that the observation that Rbfox1 specifically impacts TrkB.T1 mRNA without affecting full length suggests that it does not act by regulating splicing. I do not really understand this argument and think conducting experiments to rule out an effect on splicing  are warranted.2) It would be helpful to confirm that the upregulation of Rbfox1 in brains of lox-stop-lox/nestin-Cre animals is limited to neurons. Depending on what nestin-cre driver was used, it is possible that Rbfox1 is also upregulated in non-neuronal cells which could contribute to the gene expression changes observed.3) In order to demonstrate that TrkB.T1 was a key target of Rbfox1 despite the many other targets was to use a BDNF induced LTP paradigm. As responses in this setup would be expected to be dominated by BDNF/TrkB signaling, any regulator of TRkB levels would be expected to have strong phenotype here and dominate over the impact of other genes. Perhaps another complimentary functional experiment, if possible would be helpful here.4) Related to the above, it could be interesting to determine whether any of the other RNA-seq hits were TrekB dependent, this could be another opportunity to support a key role for Rbfox1 dependent regulation of TrkB.T1 in downstream phenotypes.5) The authors comment on potential differences between mouse and human, and it might be helpful to confirm that Rbfox1 similarly regulates TrkB.T1 in human neurons and determine whether knockdown of Rbfox1 is effective in that system.[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. A summary of the study and the reviewers' comments are included below.We are pleased to inform you that your article, "RbFox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels", has been accepted for publication in The BDNF TrkB receptor is responsible for synaptic plasticity, obesity and learning and memory. In addition to the full length TrkB tyrosine kinase receptor, a truncated form of TrkB (TrkB.T1) exists in the central nervous system. However the regulation of TrkB.T1 receptors is not well understood. In this study, the RNA binding protein, RBFox1, was found to upregulate the expression of TrkB.T1, which resulted in a deficit in BDNF-dependent LTP. This paper provides a new mechanism to explain the effects of BDNF upon synaptic plasticity.The revised manuscript has been evaluated by two prior reviewers. Both reviewers found the revised manuscript adequately addressed the previous criticisms and questions with additional experiments and analysis. They concluded the revision is suitable for publication. One remaining question was whether the regulation of truncated TrkB occurred through RNA stability or alternatively by a splicing event. this issue can be dealt with in the future, but it would be useful to acknowledge it in the text of the paper.Reviewer #1:In this manuscript, Tessarollo and colleagues identify that RbFox1 regulates levels of TrkB.T1, a TrkB isoform that regulates activity of TrkB.FL. The then go on to show that upregulation RbFox1 impacts LTP specifically through regulating TrkB/T1 levels.I was a reviewer of the last version of this manuscript, and the changes made by the authors have adequately addressed many of the concerns raised with the original version. The revised manuscript represents a more cohesive story with a logical flow that was harder to see in the previous version.There are still a few issues with the manuscript that should be addressed that are outlined below, but in general the revised manuscript is greatly improved and should be considered for publication.1) Despite additional experiments conducted by the authors, the mechanism by which RbFox1 specifically regulates TrkB.T1 is still a mystery. In the eyes of this reviewer, the data does not necessarily confirm an RNA stability mechanism and could be due to regulation of splicing to favor the T1 UTR. Altered splicing to include the T1 UTR could result in more T1 mRNA after RbFox overexpression in the pulse chase experiment  and is consistent with the observation that the 3'UTR does not increase mRNA stability . Additional experiments to look at splicing directly and/or experiments looking at stability using full mRNAs for TrkB.T1 and TrkB.FL would be ideal, but depending on the views of other reviewers it could also be acceptable to note that in the text that either mechanism is possible.2) The authors have done an excellent job demonstrating that (A) the LTP deficits following RbFox1 upregulation are Trkb.T1 dependent and (B) that RbFox2 is at least semi-redundant with RbFox1. Therefore, the gene expression data shown by the authors is not likely to underlie any of the core phenotypes of interest in the study. Because of this, it may be best to put less emphasis on this data through removing/rewording the significance statement for example.Reviewer #2:eLife.The authors have done a thorough revision of their manuscript, which is much improved from the prior submission. I believe that the manuscript is suitable for While the mechanism of action by which RbFox1 overexpression upregulates truncated TrkB.T1 remains unclear, the authors now show that this action is also achieved by RbFox2 but not RbFox3.It should be made clear within the manuscript text and figure legend whether the experiments in Figure 3—figure supplement 1 utilize a construct containing only the TrkB.T1 3'UTR, or the entire cDNA transcript inclusive of the 3'UTR.The authors have made gains in further understanding the relationship of RbFox1, TrkB.T1, and BDNF-dependent LTP. They have added data that TrkB.T1 deficient cultured hippocampal neurons display an increased response to BDNF. The manuscript also now includes data showing that conditional removal of one TrkB.T1 receptor allele in animals with up-regulated RbFox1 is sufficient to rescue hippocampal BDNF dependent LTP, suggesting that TrkB is a relevant target of RbFox1 in BDNF-dependent LTP.This would be a more appropriate conclusion than the more broad: 'genetic reduction of the TrkB.T1 isoform in animals with up-regulated RbFox1 is sufficient to restore hippocampal BDNF dependent LTP, suggesting that TrkB is a major target of RbFox1 pathological dysregulation' particularly since the author's observe a broad variety of gene dysregulation in their RNA-seq experiments.Statistical analysis and description of experimental design are improved. Analysis of RNA-seq data is improved for consistency across datasets being compared, although it remains unclear what biological variables exist between the authors' dataset and the previously published comparison dataset. [Editors’ note: the author responses to the first round of peer review follow.]Summary:This study focuses upon the regulation of a truncated TrkB species, TrkB.T1, by the RNA binding protein RbFox1. There was agreement among the three reviewers that the regulation of TrkB.T1 by RbFox1 is of considerable interest. However, a number of reservations were raised. The major issues are the need for more evidence for the role of truncated TrkB in BDNF-dependent LTP; a resolution of how the levels of RbFox1 impact upon TrkB.T1 expression; and whether there is a non-neuronal contribution of Rbfox1. These are valid concerns that will require more experimentation in order to validate the major conclusions. Specific comments by the reviewers follow this letter. Based upon the common concerns and recommendations from the referees, we have therefore decided to decline the manuscript.Thank you very much for the constructive comments that were provided by the whole reviewing team to our previous submission. The comments are all valid and constructive and we felt the need to thoroughly address them through the use of new mouse models and experiments to strengthen and improve our manuscript. Specifically:loxP) to demonstrate a direct role of TrkB.T1 function in neurons , where RbFox1 is also expressed .1) We have generated new information on the role of TrkB.T1 in BDNF-induced LTP by providing mechanistic information showing that up or down-regulation of TrkB.T1 modulates TrkB.FL signaling , which has been shown to be the primary determinant of BDNF-induced LTP. In addition, we have performed BDNF-induced LTP experiments in a new triple transgenic mouse model  To further understand how the levels of RbFox1 impact TrkB.T1 expression we have investigated the specificity of this function within the RbFox1 family members. The surprising finding that only RbFox1 and RbFox2, but not RbFox3, regulate TrkB.T1 despite the fact that all Fox proteins bind to the same RNA sequence suggests that other proteins are required for this phenomena . This observation is important because it steers the RNA biology field toward the investigation of a new molecular mechanism of RbFox protein function. In addition we have further strengthened our finding that RbFox1 gain or loss of function affects different genetic pools by re-doing our bioinformatics analysis starting from raw data from all studies and by employing the same parameters and filtering .loxP mouse model to demonstrate a direct role of TrkB.T1 function in neurons .3) We have validated that RbFox1 function is strictly neuronal by performing a detailed characterization of hippocampal nestin-cre/R26-RbFox1 activity  and by performing BDNF- induced LTP experiments in a new transgenic nestin-cre; R26- Rbfox1; TrkB.T1Reviewer #1:The study provides important insights into the mechanism and functional regulation TrkB signaling via selective modulation in the expression of truncated TrkB.T1 receptors at synaptic sites. Regulation of TrkB.T1 is mediated by the RNA-binding protein RbFox1. In a gain-of-function model, RbFox1 stabilizes TrkB.T1 mRNA and increases expression of TrkB.T1 protein. Surprisingly, stabilization occurs through an unknown mechanisms involving binding of RbFox1 to the intronic region of TrkB.T1 pre-mRNA, and not by binding to cis-acting elements in the 3'UTR. Functional studies in acute hippocampal slices show that transgenic overexpression of TrkB.T1 greatly inhibits LTP in the induced by rapid bath perfusion of BDNF. BDNF-LTP is partly restored in heterozygous transgenic mice, strongly supporting a role of TrkB.B1 in regulating TrkB function and plasticity in the adult hippocampus. The background for this work is the observation of enhanced TrkB.T1 expression in chromosome 16 trisomy mice. By identifying regulation of TrkB.T1 by RbFox1 and showing differences between gain and loss-of-function of the RNA binding-protein, the study opens new avenues for pre-clinical translational research.1) The effect on BDNF- LTP requires substantiation. Transgenic expression of TrkB.T1 modulates the response. To better evaluate the physiological role of TrkB.T1, BDNF-LTP should also be assessed in TrkB.T1 knockouts. TrkB inhibitors should be applied to confirm a TrkB-mediated response.loxP, R-26-RbFox1 and nestin-cre allele to test rescue of the BDNF induced LTP in neurons. The strategy led to a rescue . Furthermore, to mechanistically address the role of TrkB.T1 modulation on TrkB.FL function, we have used an in vitro system and showed that increased TrkB.T1 levels reduced TrkB.FL and ERK phosphorylation levels . A modulatory role was also confirmed in pure TrkB.T1 KO primary hippocampal cultures where TrkB.FL signaling was increased .We do agree with the reviewer that more experiments to strengthen the role of TrkB.T1 modulation in LTP ware important. Experiments with Trk inhibitors have already been performed and have suggested a key role for TrkB.FL signaling in BDNF induced LTP . In addition, in our initial paper  where we evaluated LTP in TrkB.T1 KO mice we failed to detect any significant difference. Here, we have also reported that TrkB.T1 heterozygous mice do not show any defect in BDNF induced LTP . Since a key point of the paper is to evaluate whether neuronal TrkB.T1 upregulation (since RbFox1 is expressed only in neurons), disrupts BDNF-induced LTP we did test whether it is the TrkB.T1 up-regulation in neurons causing the impairment . Therefore, we have generated a triple transgenic mouse with a conditional TrkB.T12) Data on the binding sites and mechanism of RNA stabilization would improve the paper.This is a valid point. So far, the data that we have accumulated show1) by analysis of the iCLIP data, that RbFox1 associates along the TrkB transcripts in two different nuclear fractions of the mouse brain . Direct binding is also confirmed by RIP analysis in primary hippocampal neurons ; 2) mechanistically, RbFox1 stabilizes specifically TrkB.T1 transcripts  and 3) RbFox1 RNA-binding capability is required for regulation of TrkB.T1 . (we have clarified this concept in the third paragraph of the Discussion). Importantly, during the revision process, we have found that in primary hippocampal neurons RbFox2, but not RbFox3 can substitute for RbFox1 . The implications of this finding are important because they suggest that other proteins binding to RbFox1 and/or RbFox2 confer specificity of action since all RbFox members bind to the same sequence ((U)GCAUG). We have added this important point in the Discussion (fourth paragraph). We think that future IP and proteomic analysis to identify proteins associated with the different RbFox gene products should help determine the identity of these proteins.Reviewer #2:This manuscript presents interesting data examining the consequences of RbFox1 upregulation as an approach to identify downstream genes regulated by RbFox1 which may have significance for brain disorders associated with RbFox1 mutation. The authors begin by following up on their prior Trisomy 16 work, to identify RbFox1 as a chromosome 16-encoded gene that could produce their previously observed upregulated expression of a truncated form of the TrkB receptor, TrkB.T1. Data is presented to show that viral overexpression of RbFox1 in hippocampal neurons produces upregulation of TrkB.T1 protein and mRNA without changing levels of full-length TrkB (TrkB.FL), and differential binding of RbFox1 to TrkB mRNAs is assessed. A mouse model with inducible RbFox1 expression is used to assess effects on gene expression by immunoblotting and RNA-seq and to probe BDNF-induced LTP. The authors compare their RbFox1 induced expression RNA-seq dataset with a published dataset from another group studying the effects of RbFox1 deletion  to support a main conclusion that genes affected by RbFox1 gain of function differ from genes affected by RbFox1 loss of function.Several aspects of the data make it difficult to definitively conclude that gain and loss of RbFox1 produce regulation of different genes:1) The comparisons of the results of RbFox1 overexpression and RbFox1KO in Figure 4B are hampered by the fact that there may be many differences in experimental design, mouse genetic background, sample preparation, sequencing, analysis and filters applied, etc., between the authors' dataset and data presented in Vuong et al., 2018. It's not clear that differences in seq data will all or even mostly be due to biological differences in the effects of Rbfox1 overexpression compared to Rbfox1 deletion. Insufficient details are provided in this manuscript to compare even major biological variables such as the age of mice used by the authors for the RNA-seq experiments.The reviewer raises a very valid point. Therefore, we have completely redone the comparative analysis of RNA-seq data using raw data from all studies, using the same parameters, and by applying the same filtering during the analysis. In addition, we have analyzed the changes in isoform expression and used the same cut-off between studies. This analysis greatly increased the number of genetic changes caused by RbFox1 loss of function  yet only 42 (from 38 of previous submission) overlap between that genetic model and our gain-of-function study. We have also changed our analysis to report changes in pathways rather than individual genes  which we think is more relevant to understanding the implications on brain function.We apologize for not having included all the details about the mice used for the analysis. We have now amended the Materials and methods section  to indicate that mice were backcrossed into a pure C57BL/6 genetic background for at least 10 generations and the nestin-cre mice were from the same source (JAX) as reported in the study by Vuong et al., 2018. The age of the animals is also indicated. Lastly, we have added more details in the Materials and methods section for the RNA-seq analysis . While we cannot completely rule out differences between studies we feel that, if any, these should be minimal after the new analysis.2) A proposed mechanism for the difference in genes regulated by gain or loss of RbFox1 expression is incompletely defined. The authors show that an RbFox1 heterozygote with reduced RbFox1 expression has an increase in RbFox2 expression relative to a control mouse, while RbFox1 gain of function does not alter RbFox2 levels. It is not clear whether RbFox2 is changed in the RbFox1 deletion context  though, or to what extent RbFox2 can interchange for RbFox1 binding sites and gene regulation.As indicated in our response to point 2 of reviewer 1 we have now shown that RbFox2 is not only upregulated in RbFox1 heterozygous mice but can also functionally compensate for RbFox1 . As suggested by the reviewer, we have also checked whether RbFox2 is up-regulated in the context of RbFox1 deletion  and found that indeed it is. We have added the reference , and also reported the data in Supplementary file 2. In addition, RbFox1 and RbFox3 knockdown experiments in primary neurons also show a 2- fold increase in RbFox2 protein , as also previously reported in RbFox1-KO brain .Indeed, as also indicated in our response to reviewer 1, point 2, we have made some progress with the new data toward the understanding of what might regulate the stability of different transcripts in a overexpression context but we think that showing the biological significance and importance of this new mechanism should be the first critical step to prompt the RNA biology community to dissect the molecular basis of this important phenotype.If RbFox1 differentially elevates levels of newly synthesized TrkB.T1 mRNA compared to TrkB.FL mRNA , then can the authors explain why this action of RbFox1 overexpression fails to produce any change in the level of TrkB.T1 protein  in the HEK293 dox-inducible cell line?We apologize for not having been more clear in explaining the HEK293-dox inducible experiment and its significance. Since it has been reported that RbFox1 can stabilize mRNA species by binding to the 3’UTRs and antagonize specific miRNA activity , we have tested if RbFox1 could increase the stability of TrkB.T1 cDNA with the 3’UTR. Our data show that RbFox1 presence does not change the level of TrkB.T1 with the 3’UTR suggesting that it is the binding to the intronic regions of the TrkB.T1 that changes its level of expression. This is in agreement with our iCLIP analysis and RIP experiments that show Rbfox1 binding to the intronic regions of TrkB.T1 . We have now clarified the concept in the manuscript .Greater explanation is needed for the determination of reads as TrkB.T1 compared to TrkB.FL, especially given that TrkB.FL contains the sequence of TrkB.T1.The reviewer is correct that more details are needed for how we determined the reads of TrkB.T1 and TrkB.FL since there is sequence overlap between the two isoforms. Even if TrkB.FL and TrkB.T1 share the same extracellular region, the unique TrkB.T1 exon and long 3’UTR provides specificity for the determination of reads as TrkB.T1 compared to TrkB.FL. We have added more details in the Materials and methods section about the RNA-seq analysis including the use of “EBSeq (version 1.22.1) to test for differential isoform expression between experimental conditions. Group-based TPM filtering was applied to remove lowly expressed transcripts. Significant differentially expressed isoforms were identified with a false-discovery rate £0.05”.In the BDNF-induced LTP experiments, it is not clear that the effect of 'inactivation' of one TrkB.T1 allele is specific to the context of RbFox1 upregulation. This manipulation should be symmetrically applied in the context of to the Ctrl mouse as well. The data presented are insufficient to conclude that RbFox1 upregulation leads to a deficit in the BDNF-induced LTP resulting from an increase in TrkB.T1 expression (as opposed to indicating possible distinct roles for both TrkB.T1 and RbFox1 upregulation in BDNF-induced LTP).loxP, R-26-RbFox1 and nestin-cre alleles to test the rescue of the BDNF induced LTP in the same cellular context where RbFox1 is upregulated (neurons); see new Figure 8. All manipulations are now symmetrically applied. The N-cre-Fox1 mouse had the R-26-RbFox1 and nestin-cre alleles, and the control (Ctrl) had the nestin-cre only allele. As indicated above, we also went further to better dissect the role of the TrkB.T1-isoform in the context of BDNF/TrkB signaling by showing that increasing TrkB.T1 levels reduces BDNF- induced signaling .We thank the reviewer for raising this very important point that also helped in addressing point 1 of reviewer 1. Specifically, we have generated a triple transgenic mouse with a conditional TrkB.T1Reviewer #3:In this manuscript, Tomassoni-Ardori et al. investigate the impact of Rbfox1 on the expression of different TrkB isoforms. The findings are somewhat interesting though the overall impact is somewhat limited by the many other recent manuscripts studying the function of this protein and the focus on Rbfox1 overexpression even though disease is associated with reduced functional Rbfox.The reviewer is correct that most studies have focused on RbFox1 loss of function though it is now emerging that there are pathological situations in humans where RbFox1 (and even RbFox2) is found to be up-regulated . Thus, we feel that we are covering a new understudied and yet important area of research.However, the work does provide new insight into the potential role of Rbfox1 and the data demonstrating it binds and regulates TrkB.T1 is pretty conclusive, though there are a few shortcomings that should be addressed that are outlined below.1) In the second paragraph of the Results, the authors state that the observation that Rbfox1 specifically impacts TrkB.T1 mRNA without affecting full length suggests that it does not act by regulating splicing. I do not really understand this argument and think conducting experiments to rule out an effect on splicing  are warranted.We do understand the difficulty of this concept especially because RbFox1 is known as a regulator of splicing. The data that lead to our conclusion are the following: 1) in vivo and in vitro RbFox1 overexpression causes up-regulation of only TrkB.T1 and no changes in TrkB.FL. In situations of alternative splicing we should have seen TrkB.FL expression levels going down; 2) These data, i.e. change of only one isoform, is confirmed by the very sensitive RNA-seq analysis in N-cre-Fox1 mouse hippocampus. 3) Analysis of new nascent RNA showed that TrkB.T1 but not TrkB.FL RNA is more stable upon RbFox1 overexpression. We have now modified the Discussion to clarify these points (third paragraph).2) It would be helpful to confirm that the upregulation of Rbfox1 in brains of lox-stop-lox/nestin-Cre animals is limited to neurons. Depending on what nestin-cre driver was used, it is possible that Rbfox1 is also upregulated in non-neuronal cells which could contribute to the gene expression changes observed.We thank the reviewer for bringing up this very important point. As suggested, we have performed a very careful analysis of nestin-cre activity in the hippocampus. The characterization of cre-activity as well as of Fox1 expression and potential overlap with GFAP is reported in the new panels of Figure 1—figure supplement 2 and in the Results section (third paragraph).3) In order to demonstrate that TrkB.T1 was a key target of Rbfox1 despite the many other targets was to use a BDNF induced LTP paradigm. As responses in this setup would be expected to be dominated by BDNF/TrkB signaling, any regulator of TRkB levels would be expected to have strong phenotype here and dominate over the impact of other genes. Perhaps another complimentary functional experiment, if possible would be helpful here.The reviewer’s point is well taken and indeed there could be other genes that are regulated by RbFox1 and that could impact TrkB levels or signaling. To strengthen our observation and test the direct regulation of TrkB.T1 in BDNF-induced LTP, we have performed this experiment in the context of conditional TrkB.T1 deletion in neurons to clarify that the previous rescue effect was not caused by an indirect down-regulation of TrkB.T1 in glia . Moreover, we have performed a new complementary functional experiment to show that increasing or decreasing TrkB.T1 modulates TrkB.FL signaling, which is key to BDNF-induced LTP .4) Related to the above, it could be interesting to determine whether any of the other RNA-seq hits were TrekB dependent, this could be another opportunity to support a key role for Rbfox1 dependent regulation of TrkB.T1 in downstream phenotypes.We agree that it would be nice to know if there are some TrkB- dependent RNA-seq hits. However, we are afraid that it would be almost impossible to demonstrate a direct relationship between those hits and TrkB-induced LTP because there are so many genes/neuronal pathways that are dysregulated by Rb-Fox1. To convey this point, we have changed panel Figure 5A to indicate that many neuronal pathways are dysregulated. Yet, we hope that the reviewer will agree that it is remarkable that correcting only the expression of one isoform can rescue the BDNF-induced LTP.5) The authors comment on potential differences between mouse and human, and it might be helpful to confirm that Rbfox1 similarly regulates TrkB.T1 in human neurons and determine whether knockdown of Rbfox1 is effective in that system.Following the reviewer’s comment we have investigated the possibility of testing our hypothesis in human hippocampal neurons, which are available. However, National Institutes of Health (NIH) regulations do not allow such experiments since human hippocampal neurons are of fetal origin. We have also considered the possibility of differentiating human iPSCs or ES cells but, to our knowledge, there are, as yet, no good protocols specific to the differentiation of hippocampal neurons. In the future, it will be important to test RbFox1 in conjunction with TrkB levels in patients with specific neurodegenerative or neuropsychiatric disorders, studies that we think will be prompted by our report.[Editors' note: the author responses to the re-review follow.]We are pleased to inform you that your article, "RbFox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels", has been accepted for publication in eLife. A summary of the study and the reviewers' comments are included below.The BDNF TrkB receptor is responsible for synaptic plasticity, obesity and learning and memory. In addition to the full length TrkB tyrosine kinase receptor, a truncated form of TrkB (TrkB.T1) exists in the central nervous system. However the regulation of TrkB.T1 receptors is not well understood. In this study, the RNA binding protein, RBFox1, was found to upregulate the expression of TrkB.T1, which resulted in a deficit in BDNF-dependent LTP. This paper provides a new mechanism to explain the effects of BDNF upon synaptic plasticity.The revised manuscript has been evaluated by two prior reviewers. Both reviewers found the revised manuscript adequately addressed the previous criticisms and questions with additional experiments and analysis. They concluded the revision is suitable for publication. One remaining question was whether the regulation of truncated TrkB occurred through RNA stability or alternatively by a splicing event. this issue can be dealt with in the future, but it would be useful to acknowledge it in the text of the paper.We have included the modification requested by the reviewers and indicated that future experiments will be needed to address the mechanism by which TrkB.T1 is regulated. The paragraph with the requested modifications has been included in the Discussion.


Alternatively, MSCs were seeded onto PDMS substrates  coated with a single monomolecular layer of graphene and cultured in basal medium (10 days). The ensuing expressions of FAK-p397, integrin, ROCK1, F-actin, Smad p1/5, RUNX2, OCN and OPN were evaluated by Western blot (n = 3). As controls, MSCs were plated onto uncoated PDMS in the presence of mechanotransduction inhibitors . MSC-impregnated graphene scaffolds exhibited positive immunoexpression of bone-related markers (RUNX2 and OPN) without the assistance of osteogenic inducers. In vitro, regardless of the stiffness of the underlying PDMS substrate, MSCs seeded onto graphene-coated PDMS substrates demonstrated higher expressions of all tested osteogenic and integrin/FAK proteins tested compared to MSCs seeded onto PDMS alone. Hence, graphene promotes osteogenesis via the activation of the mechanosensitive integrin/FAK axis.Graphene is capable of promoting osteogenesis without chemical induction. Nevertheless, the underlying mechanism(s) remain largely unknown. The objectives here were: (i) to assess whether graphene scaffolds are capable of supporting osteogenesis in vivo and; (ii) to ascertain the participation of the integrin/FAK mechanotransduction axis during the osteogenic differentiation induced by graphene. MSC-impregnated graphene scaffolds ( Graphene consists of a single sheet of carbon atoms arranged into a tightly packed two-dimensional (2D) honeycomb lattice. Graphene sheets have proven quite amenable to molecular chemical vapor deposition functionalization as well as present high specific surface area and ultrahigh mechanical strength. Graphene can also be produced by (CVD) that is a scalable method to produce high quality graphene substrates in two and three-dimensions .The demonstrated ability of CVD-grown graphene to induce osteogenic differentiation in vitro has rendered this method as a promising platform for cell-based bone tissue engineering and regeneration ,6,7,8,9.Although graphene has been shown to induce osteogenic differentiation in vitro the intracellular events involved in this phenomenon remain largely unresolved. The physical characteristic of graphene  may facilitate cell anchorage and thereby, promote stem cell differentiation ,8. PreviStem cell lineage commitment is modulated by the elastic characteristics of their microenvironments . For insThe objectives of this study were: (i) to assess the ability of a graphene scaffold to promote osteogenic differentiation of human MSCs in vivo and; (ii) to test the hypothesis that the osteogenic differentiation of MSCs on graphene is mediated via the activation of the integrin-FAK axis.To evaluate whether graphene was capable of inducing osteogenesis in vivo we seeded MSCs onto a graphene scaffold (red arrows in H&E) before transplantation into the subcutaneous space of SCID mice for four weeks . The hisTo determine whether graphene promotes osteogenic differentiation via the actions of mechanosensitive pathways, we analyzed the expression of key proteins involved in the integrin-FAK axis in response to substrates of diverse mechanical properties. MSCs were seeded onto PDMS substrates with tuned elastic moduli (PDMS) or graphene-coated PDMS (Gp) in the presence or absence of inhibitors. The expression of the proteins was evaluated after 10 days in vitro.p < 0.05) at all concentrations used. However, at 10 nM proliferation was reduced by approximately 30% after seven days (The effective concentrations of inhibitors were ascertained by their ability to suppress proliferation. MSCs were administered different concentrations of echistatin (a Disintegrin), Y-27632 (p160ROCK inhibitor) or DMH1 (Activin receptor-like kinase 2 inhibitor) and their growth monitored for seven days. A decrease in cell proliferation of approximately 30% compared to the control was set as the threshold for effective inhibition. Echistatin significantly inhibited proliferation as early as 1 day , or not. After 10 days, MSCs grown on Gp presented higher expression levels of FAK-p397, as well as all downstream proteins recruited in this axis compared to those seeded on PDMS alone. Highest expressions were observed on graphene-coated substrates (Gp) regardless of the stiffness of the underlying PDMS substrate. The expression of all mechanotransductory-related proteins was decreased by the presence of Echistatin (10 nM), strongly implicating the integrin-FAK axis in the osteogenic differentiation triggered by graphene A,B. The Y27632 (50 μM) was used to confirm a downstream role of ROCK1 in the osteogenic differentiation induced by graphene. As previously, regardless of the stiffness of the underlying polymer, MSCs on graphene-coated PDMS exhibited higher expression levels of ROCK1 in conjunction with its downstream affiliated transforming growth factor β modulating protein, Smad 1/5, and bone-related proteins , whose expressions were attenuated by the administration of Y27632 A,B.Finally, we checked the expression levels of the selected proteins before and after inhibiting Smad p1/5 in response to treatment with DMH1 (50 µM). The expressions of Smad p1/5 and of the downstream bone-related proteins  were higher on Gp compared to all PDMS conditions tested. The presence of DMH1 suppressed the expression of all proteins confirming that the osteogenic differentiation on graphene is regulated by the activation of Smad p1/5 A. The quCVD-grown graphene has emerged as a promising platform with which to enhance osteogenic outcome ,6,8,9,23Evidence exists for integrin-mediated mechanical signaling regulating stem cell paracrine factor production and release . We inveThe expression of FAK-p397 was higher in all graphene-coated PDMS substrates (Gp) compared to PDMS alone . Similarvβ3 receptor in a nondissociable manner  × 100.MSC  without the assistance of the conventional cocktail of osteogenic inducers considered necessary in other platforms. Here, we extended these findings by showing that graphene scaffolds also promote the osteogenic differentiation of MSCs in vivo. Osteogenesis was corroborated with the positive expression of RUNX2 and OPN, classical markers for osteogenic differentiation. Complementary in vitro tests demonstrated that the osteogenic differentiation was mediated by the activation of the mechanosensitive integrin-FAK axis. These developmental attributes appear to be inherent to the mono-atomic graphene sheet as changing the elastic modulus of the underlying PDMS had little effect on their ability to induce osteogenesis. Our findings deepen the understanding of the effects of graphene on biological systems and broaden the potential avenues for the use of this single-atom thin material to promote osteogenesis without the delivery limitations of exogenous chemical inducers.


Impurities produced during the synthesis process of a material pose detrimental impacts upon the intrinsic properties and device performances of the as-obtained product. This effect is especially pronounced in graphene, where surface contamination has long been a critical, unresolved issue, given graphene’s two-dimensionality. Here we report the origins of surface contamination of graphene, which is primarily rooted in chemical vapour deposition production at elevated temperatures, rather than during transfer and storage. In turn, we demonstrate a design of Cu substrate architecture towards the scalable production of super-clean graphene (>99% clean regions). The readily available, super-clean graphene sheets contribute to an enhancement in the optical transparency and thermal conductivity, an exceptionally lower-level of electrical contact resistance and intrinsically hydrophilic nature. This work not only opens up frontiers for graphene growth but also provides exciting opportunities for the utilization of as-obtained super-clean graphene films for advanced applications. The intrinsic properties of graphene and the resulting device performance are hindered by the impurities produced during the synthesis process. Here, the authors elucidate the origin of contaminations in CVD-grown graphene and devise a strategy towards the scalable production of ultra-clean graphene with >99% clean regions and low contact resistance. The surface contamination has been intensively highlighted as a major hurdle in probing intrinsic properties of graphene6, and strongly hinders device performance and applications of graphene, such as surface chemistry10 ultrahigh speed electronics2, and transmission electron microscopy (TEM) support11, where clean surface is highly needed. Among the various methods for graphene synthesis, chemical vapour deposition (CVD) approach, especially on Cu substrate, holds great potentials in the scalable and cost-efficient production in a controllable fashion12. Despite recent advances in dictating the grain size and scalability14 in CVD approaches, the growth of clean graphene films by eliminating surface contamination remains a daunting challenge2. Although much attention has been paid to post-growth processing aiming for higher degree of cleanness, with an emphasis on the suppression of transfer-related impurities18 and airborne contaminants5, the variations in reported cleanness indicate that the dominant processes and intrinsic root of contamination still remain to be unravelled15.Surface contamination has long been a great challenge in the whole society of carbon materialsHerein, we prove that the contamination on graphene surface is primarily introduced during the high-temperature CVD growth. The growth of metre-scale, super-clean graphene with advanced performances is facilely realised through the continuous supply of Cu vapour, via an ingenious substrate design using alternating stacks of Cu foil and foam.19. The competition between the formation of sp2 crystalline carbon (graphitisation process) and amorphous carbon during a CVD reaction primarily determines the cleanness of the graphene surface, and this phenomenon has been discussed intensely in academic and industrial settings in relation to the preparation of synthetic graphite and diamond20. In the course of graphene growth, Cu would catalyse the decomposition of hydrocarbons as well as the graphitisation process. However, the catalytic activity of Cu would be limited gradually upon the increase of graphene coverage, presumably leading to the formation of amorphous carbon21.During the high-temperature catalytic growth of graphene on Cu, the graphene surface becomes simultaneously contaminated due to the generation of amorphous carbon, which has been widely reported to be stable in CVD conditions Fig. 19. The c22 clearly manifests the universal distribution of amorphous carbon contaminants  band signal  with improved lateral resolution and sensitivitynent D 130 cm−1 ba12C/13C isotope-labelling technique21 was employed  analysis verifies that the carbon source fuels the dual formation of graphene and amorphous carbon during CVD , super-clean graphene  residues . Therefore, it was possible to quantify the amount of PMMA residues using mass signature of isotope 2H , represents the quality of CVD-derived graphene with optical and electrical properties comparable with that obtained by mechanical exfoliation. The large-area transfer of clean and unclean samples onto transparent polyethylene terephthalate (PET) substrates was achieved by means of a thermal release tape methodart Fig. , inset. art Fig. .Fig. 4Op35. In our study, we found that commonly used contact metals  were preferentially adsorbed on the contaminated regions upon deposition  measurements)  Fig. , inset37ped Fig. , substanped Fig.  and that2/Si substrate ranges from 14,900 to 18,500 cm2 V-1 s-1 at room temperature and ~31,000 cm2·V-1·s-1 at 1.9 K . The exceptional quality of obtained graphene was further corroborated by the evolution of the quantum Hall effect with magnetic field in devices, where the prominent quantum Hall platform is visible at low magnetic field intensities  , followed by annealing in H2  for 1 h to eliminate the surface oxygen and contamination. Subsequently, CH4 was introduced to initiate the graphene growth for different duration. Note that, the molar ratio of CH4 and H2 was carefully controlled to produce large graphene single crystals. The flow rate of CH4 is ~0.1–1.0 sccm with pressure from 0.3 Pa to 2.8 Pa. After growth, the system was quickly cooled down to room temperature while still under the same flow. The 13C-isotope-labelled methane is purchased from the Sigma-Aldrich company (production number #490229) with 13C atom ratio of 99%. The 2H-PMMA is purchased from Polymer source company (production number #P100226-dsPMMA) with Mn = 820,000 and Mw = 1,500,100.The super-clean graphene was grown by the low-pressure CVD method. The Cu foil  after electrochemical polishing was placed below a Cu foam (840 g2:CH4 molar ratio). The domain size distribution of graphene grown using different H2:CH4 molar ratio is 22.5 ± 1.2 μm for H2:CH4 molar ratio of 200, 125 ± 14 μm for H2:CH4 molar ratio of 300, 350 ± 11 μm for H2:CH4 molar ratio of 400, 750 ± 80 μm for H2:CH4 molar ratio of 500, 1750 ± 152 μm for H2:CH4 molar ratio of 1000, 2500 ± 575 μm for H2:CH4 molar ratio of 2000, respectively.The domain size of graphene single crystals grown using the vertical stacking structure of Cu foil and Cu foam can be tuned from tens of micrometres to large than three millimetres by carefully controlling the carbon source supply  in scanning tunnelling microscopy (STM) mode. A long working distance objective  was used for both excitation and collection of the backscattered light from the sample. The exciting wavelength was 632.8 nm and the laser power on the sample was less than 0.5 mW to avoid sample degradation. All the TERS spectra were collected with an acquisition time of 1 s.3+ beam operating at 25 keV. The scanning area was 200 μm × 200 μm with an acquisition time of 40 s. Negative ion spectra were collected for each sample. The software used for peak analysis was SurfaceLab 6.0 from ION-ToF.Annealing experiments were performed in the chamber of the ToF-SIMS spectrometer  before characterisation. The samples were analysed at 25 °C and after annealing at 100 °C for 1 h. ToF-SIMS spectra were acquired at the annealing temperature using a BiRaman spectra were obtained with LabRAM HR-800 with 514nm laser and ×100 objective. Optical microscopy images were obtained with an Olympus BX51 microscopy. Optical transmittance spectra were collected by a Perkin-Elmer Lambda 950 UV-vis spectrophotometer.2 cubed Titan 60–300 electron microscope under 80 kV. Energy-dispersive X-ray (EDX) spectra, bright-field (BF) and high-angle annular dark-field (HAADF) images were acquired using scanning TEM (STEM) mode at 60 kV in a double corrected FEI Titan Themis G2 electron microscope.SEM images were obtained with SEM . The graphene on TEM grids was characterised by TEM . HRTEM imaging was performed on an aberration-corrected and monochromated G2/Si substrates with marks for alignments and then subjected to AFM (Veeco dimension 3100) imaging to determine whether they were flat without wrinkle. Next, each graphene sample was etched into a Hall bar geometry or graphene transistor array using a PMMA etching mask (PMMA 950 K A2 @ 4000 rpm) designed by electron-beam lithography (EBL) (Raith 150 2nd) and reactive-ion etching (RIE) with O2 (Trion Technology Minilock III). Finally, after using EBL to design a PMMA mask (PMMA 950 K A4 @ 4000 rpm), 10-nm Pd and 80-nm Au were deposited on the samples using an electron-beam evaporator (Kurte J. Lesker AXXIS) and then a standard metal lift-off technique. Electrical characterisation at room temperature was performed in a vacuum probe station (Lakeshore TTP-4) using a Keithly Semiconductor Characterisation System (Model 4200-SCS). Electrical-transport and magneto-transport measurements at low temperatures were performed using a lock-in amplifier (Stanford Research 830) at 17 Hz with a source current of 10–100 nA.The graphene samples were transferred onto SiOThe element analysis was performed by XPS . AFM characterisation of graphene on Cu was carried out on Bruker dimension icon microscopy using the Scanasystg mode. The water contact angles were measured on a Dataphysics OCA 20 contact-angle system at room temperature.Supplementary InformationPeer Review File


Previous studies attempted to characterize the subjects with sarcoidosis according to differences in sex, age, and the presence of specific organ involvement. However, significant interactions among these factors precluded a clear conclusion based on simple comparison. This study aimed to clarify the age- and sex-stratified prevalence of specific organ involvement and the heterogenous nature of sarcoidosis. Using the data of 9,965 patients who were newly registered into a database at the Ministry of Health, Labour and Welfare, Japan between 2002 and 2011, we evaluated the age- and sex-specific prevalence of the eye, lung, and skin involvement of sarcoidosis. We also attempted corresponding analysis considering multiple factors. As compared with several decades ago, the monophasic age distribution in men became biphasic, and the biphasic distribution in women, monophasic. The prevalence of pulmonary and cutaneous lesions was significantly associated with age, whereas the prevalence of ocular involvement showed a biphasic pattern. The prevalence of bilateral hilar lymphadenopathy was significantly higher, whereas the prevalence of diffuse lung shadow was significantly lower, in subjects with ocular involvement than those without ocular involvement. Corresponding analysis visually clarified the complex interactions among factors. Our results contribute to a better understanding of the heterogeneous features of sarcoidosis. Sarcoidosis is a systemic granulomatous disorder of unknown etiology, and involves multiple organs. The disease occurs worldwide and affects both sexes and all ethnicities and age groups.7. However, due to the highly heterogeneous nature of the disease, the simple categorization of patients into two groups has inherent limitations, and the complex interactions among factors always need to be considered. In this regard, the detailed subtyping of patients using a large-scale survey is necessary to overcome this problem and clarify the characteristics of sarcoidosis.To better understand the heterogeneity of this disease, most previous studies have attempted to compare differences among patients, such as sex, age, and organ involvementet al. conducted a survey on the epidemiology of histologically proven sarcoidosis that was newly diagnosed in 2004 8. They showed a historical change in several clinical features of the disease over 50 years, such as an increase in female predominance and ocular involvement, and a decrease in bilateral hilar lymphadenopathy (BHL). After 2012, this system has been gradually revised and is now only used for patients with severe sarcoidosis (such as those who need high doses of systemic corticosteroids). However, before 2012, this system covered all subjects with sarcoidosis regardless of its severity. Taking advantage of this system before 2012, in the present study, we re-conducted a large-scale survey of histologically proven sarcoidosis diagnosed during 10 years from 2002 to 2011. In particular, we aimed to clarify the age- and sex-specific prevalence of the ocular, pulmonary, and cutaneous involvement of the disease. We also attempted to perform corresponding analysis considering multiple factors to better understand the complex and multidimensional nature of the disease.In Japan, there is a digital registration system of sarcoidosis under the Ministry of Health, Labour and Welfare. Using this system, Morimoto 9.The present study was performed using questionnaires for newly diagnosed sarcoidosis patients established by the public health system. In Japan, the Ministry of Health, Labour and Welfare defines an “intractable” disease as one for which studies on a national scale are necessary as the cause of the disease is unidentified; its treatment is difficult; it is a chronic disease; and its associated medical costs are high. According to this definition, sarcoidosis has been designated as an intractable disease since 1974. Attending physicians are requested to provide the clinical information using a questionnaire. The questionnaires are collected, and consent is obtained with the agreement of the expert panel in each prefecture . Following the diagnosis of sarcoidosis, each prefecture and the government support the medical costs of treatment. All prefectures in Japan enter the clinical information into a central database at the Ministry of Health, Labour and Welfare. This was initially a paper-based registration system, but later moved to a digital system. Sarcoidosis was diagnosed histopathologically on the basis of the American Thoracic Society (ATS)/European Respiratory Society (ERS)/World Association of Sarcoidosis and Other Granulomatous Disorders (WASOG) statement10, which provided the anonymous data.After 2012, this system has been gradually revised and is now only used for patients with severe sarcoidosis (such as those who need high doses of systemic corticosteroids). However, before 2011, this system covered all subjects with sarcoidosis regardless of its severity. Thus, in the present study, newly diagnosed sarcoidosis patients who were digitally registered and whose clinical information was entered into the central database at the Ministry of Health, Labour and Welfare from 2002 to 2011, were evaluated. Japan has two separate registry systems, one for newly diagnosed patients and the other for follow-up after the initial diagnosis. Thus, there should be no concerns regarding the duplicate entry of patients in our survey. Patients with granulomas of known causes and local sarcoid reactions were excluded. Those with no histopathological findings were also excluded. The enrollment into this health system had been conducted under agreement with use of the data for epidemiological survey from all subjects. The study was approved by the Clinical Research Ethics Committee of Hokkaido University Hospital (011-0188). All methods were performed with permission from the Ministry of Health, Labour and Welfare (07061)8. In this study, questionnaires were analyzed, focusing on the age at diagnosis, sex, and the presence or absence of ocular, pulmonary, and cutaneous lesions. The questions on pulmonary involvement included two items: BHL and diffuse lung shadow (DLS). Information on the presence of cardiac sarcoidosis was not detailed in this questionnaire.Details of the questionnaire used in this study have been previously describedt-test. For all statistical analyses, P < 0.05 was considered statistically significant. To analyze the tendency of the prevalence of organ involvement by age, the Cochran-Armitage trend test was performed. To supplement the pre-existing knowledge on the nature and course of sarcoidosis, we also performed corresponding analysis using categorized data . The patients were stratified using a cut-off age of 40 years (<40 vs. ≥40 years), based on previous reports12.Statistical analyses were performed using JMP version 13 . Categorical variables were compared using the chi-square test, and continuous variables were compared using an unpaired The number of sarcoidosis patients whose clinical information was digitally entered into the central database at the Ministry of Health, Labour and Welfare from 2002 to 2011 is shown in Table The baseline characteristics of all the patients  in this study are shown in Table 8 which is one of the unique clinical features of sarcoidosis in Japanese patients.We evaluated the age- and sex-specific prevalence of eye, pulmonary (BHL and/or DLS), BHL, DLS, and cutaneous lesions Table . Figure We next compared the clinical characteristics of the patients between two groups categorized by age, sex, and the involvement of each organ Fig. . The eldThe prevalence of ocular involvement was similar with or without the pulmonary involvement (BHL and/or DLS). However, when the lung involvement was classified into the presence of BHL and DLS, the prevalence of BHL was significantly higher in subjects with ocular involvement (P < 0.001). In contrast, the prevalence of DLS was significantly lower in subjects with ocular involvement (P < 0.001). Following these contrasting associations of BHL and DLS with ocular involvement, we classified subjects into four groups according to the presence or absence of BHL and DLS, and then compared the prevalence of ocular involvement among the groups; (i) BHL (−), DLS (−) (n = 1306), (ii) BHL (+), DLS (−) n = 4136), (iii) BHL (−), DLS (+) (n = 825), (iv) BHL (+), DLS (+) (n = 3698) The prevalence of ocular involvement was significantly higher in subjects with BHL (+), DLS (−) than in those with BHL (−), DLS (−)  significantly reduced with age. Ocular involvement was significantly predominant in females, and showed a biphasic pattern with age. In addition, results from corresponding analysis clarified complex associations among age, sex, and the involvement of the three organs (four lesions).14, and this probably reflects the recent drastic aging of our population. In the previous studies, monophasic distribution in men and biphasic distribution in women had been observed in the Japanese population. However, the proportion of elderly patients has increased and, subsequently, the monophasic distribution in men has tended to become biphasic, and the biphasic distribution in women, monophasic  was higher than that in several epidemiological surveys conducted several decades ago in Japan3. Meanwhile, ocular involvement was predominant in women, which is consistent with a previous study conducted in the medical University of South Carolina17, but in contrast with another study conducted in the Louisiana State University1. This inconsistency might be because of the nature of ethnic and/or regional difference of the disease.To better understand the heterogeneous features of sarcoidosis, comparisons of the clinical features of the disease  abnormalities. While we have this information, it does not reflect the presence of cardiac involvement in sarcoidosis; thus, we did not use these data in the current study. Meanwhile, the prevalence of cardiac sarcoidosis, which fulfilled the diagnostic criteria according to the Japanese Ministry of Health and Welfare guideline23.In conclusion, our large-scale epidemiological survey using a digital registration system revealed the age- and sex-specific prevalence of sarcoidosis and their complex interactions in the Japanese population. The findings of this study contribute to a better understanding of the heterogeneous features of sarcoidosis and the management of those with the diseaseSupplementary Table


Graphene-γMnS exhibited excellent and tunable NLO performance, illustrating that NCs materials have potential applications in NLO devices. Graphene-γMnS were prepared by facile one-step hydrothermal method. Structures and properties of samples were explored by characterization, and nonlinear optical (NLO) enhancement of nanocomposites (NCs) was fully studied. Nanoparticles and NCs were tested at 532 nm by a Z-scan technique. With γMnS attached in G layers, NLO susceptibility of graphene-γMnS was greatly improved under single-pulse laser irradiation compared with G and γMnS. The nonlinearity enhanced was attributed to local field effect and charge transfer between γMnS and graphene layers. And NLO property enhancement was restricted by non-radiative defects in graphene-γMnS. Exploring the mechanism of nonlinearity enhancement was significant for fabrication of NLO devices. However, the optical nonlinearity decreased first and then increased with further increased addition of GO, because the dispersion of γMnS attached on graphene surface might make density of sp Recently, research on graphene and graphene-semiconductor are rapidly developing research fields . Graphen2O4 enhanced was due to extent of conjugation with G layers [2 could be attributed to the combination of NLO absorption mechanism and NLO scattering [2O4, graphene-Pt, and enhanced graphene-TiO2, electrochemical property of graphene-γMnS was also many times higher than that of γMnS nanocrystal and graphene [Single graphene has weak NLO absorption and refraction, which can not meet the demand for various optical devices . HoweverG layers . In caseG layers . NLO proattering . γMnS isattering . Like NLgraphene ,13,14. Hgraphene . It is nIn this study, graphene-γMnS was synthesized by facile one-step hydrothermal method, and mechanism of NLO enhancement was discussed. Nonlinearity of NCs was controlled by changing amount of GO added, and samples were tested at 532 nm by picosecond (PS) laser pulse. We want to explore whether graphene-γMnS had potential applications in optical communication, optical limiter, and all-optical switch.4 and 98% H2SO4. Then, a solution was mixed with H2O2 and the mixture was washed several times with deionized water to remove impurities. Finally, products were dried at 45 °C for 48 h in vacuum drying oven. Improved Hammer method increased generation efficiency and oxidation degree of GO. Furthermore, this method could effectively prevent generation of toxic gases which caused harm to human body. High efficiency, high quality, and no toxicity in GO synthesis were important for large-scale production of GO.GO was prepared by improved Hummer method . Firstly2·4H2O were added to GO suspension. After half-an-hour stirring, the solution was transferred to Teflon-lined stainless-steel autoclave and reacted at 170 °C for 6 h. The reaction temperature in our previous study was 190 °C and product was different [Graphene-γMnS was prepared by facile one-step hydrothermal method. The synthesis process of NCs was shown in ifferent . Then, pGraphene-γMnS were tested by X-ray diffraction , scanning electron microscope , and transmission electron microscopy . FRIR spectra and Raman spectra were obtained on Bruker Optics Vertex 70  and Renishaw inVia , respectively. Ultraviolet Visible absorption spectra were acquired on Ultraviolet Visible absorption instrument . The Z-scan patterns were received on picosecond laser  with a wavelength 532 nm and the pulse width 30 ps. 2 domains formed, indicating that γMnS could be attached in graphene [XRD patterns of graphene, GO, graphene-γMnS and γMnS were shown in graphene . For purgraphene . And chagraphene . Figure The morphology of GO and NCs were exposed to SEM in Further information about structure could be seen in TEM. −1, 1632 cm−1, 1571 cm−1, 1239 cm−1, 1065 cm−1 and 627 cm−1 corresponded to stretching vibration of O-H, C=O, C-C, C-OH, C-S, and Mn-S, respectively [−1, 1632 cm−1, 1571 cm−1 and 1239 cm−1. When graphene-γMnS were formed, characteristic peaks at 1571 cm−1, 1065 cm−1 and 627 cm−1 were exhibited. At the same time, the characteristic peaks at 3411 cm−1 weakened, and 1632 cm−1 and 1239 cm−1 disappeared, demonstrating that large sp2 domains had been formed and smaller sp2 fragments existed between larger sp2 domains [More information about graphene-γMnS, γMnS, graphene, and GO could be obtained by FTIR spectra. ectively ,20,21. W domains ,23. 2 site [The UV-is absoption spectra of GO, γMnS and graphene-γMnS could be observed in 2 site . And γMn2 site . Figure 2 site . There wα-MnS/rGO was investigated, and results showed that the nonlinearity of NCs was enhanced, but the mechanism of enhancement was unclear. In this study, NLO absorption and refraction of samples could be measured by Z-scan technique using a single Gaussian beam. The transmittance T was measured as a function of laser incident energy density. The Nd:YAG laser system used for excitation was 30 PS laser pulse at 532 nm producing a repetition rate of 10 Hz, and the beam waist radius was about 10.6 μm at the focal plane. CS2 is used to calibrate the Z-scan curve so that the center of the curve is at the center of the x-axis. Nonlinear material CS2 was used to calibrate Z-scan and measured data could ignored the samples’ absorption and scattering effects. A cuvette was mounted on mobile platform controlled by computer, which moved samples along the focal plane of Z-axis and 250 mm focal length lens. Absolute ethanol was as a solvent and samples’ concentration was 0.125 mg/mL. The input single pulse intensity of the focal plane was adjusted to 15.1 GW·cm−2.In our previous study, the NLO response of It could be seen in 0(z) was obtained by q0(z) = βI0Leff/(1 + z2/z02) [OA Z-scan transmittance T could be calculated as (1)T(z)= z2/z02) . β was aeff was effect length which could be obtained byL(3)) and real part (Reχ(3)) could be obtained by Imχ(3) = cn0λβ/480π and Reχ(3) = n0n2/3π, where n2 was a nonlinear refractive index calculated asImaginary part (ImχSo nonlinear susceptibilities of γMnS and NCs could be obtained as (3) of γMnS was 1.21 × 10−12 esu, χ(3) of graphene was 0.78 × 10−12 esu, and χ(3) of graphene-γMnS was 6.23 × 10−12 esu. These demonstrated that χ(3) of graphene-γMnS was about five times larger than that of pure nanoparticles and about eight times larger than that of graphene. The susceptibilities of graphene, γMnS, and graphene-γMnS sample could be calculated by the above formula. It could be seen in (3) could be calculated and it was much larger than the sum of χ(3) of two separate components. This was consistent with the measured results, indicating that local field effect was one of the significant reasons for NLO property enhanced of NCs materials [−2, NCs exhibited SA. As the intensity increased to 8.5 GW·cm−2, 9.4 GW·cm−2, 15.1 GW·cm−2, 17.9 GW·cm−2, 18.9 GW·cm−2 and 20.8 GW·cm−2, a valley appeared in the peak at the focus and was getting deeper with incident intensity increased, which meaned that there was CT between them in two components [pT and vT were normalized values of peak-to-1 and 1-to-valley of OA Z-scan curves, respectively, and p-v = Tp + TvT [p-vT increased from 9.4 GW·cm−2 to 20.8 GW·cm−2, indicating that CT through interface was in this intensity range. And CT could not be observed at intensity below this range. To further prove CT only could be obviously observed at high incident intensity, χ(3) of samples at 6.6 GW·cm−2 were shown in −12 esu at low incident intensity. Larger χ(3) of graphene-γMnS demonstrated that CT appeared between graphene and graphene-γMnS at high incident intensity and no obvious CT at low incident intensity. Fluorescence experiments could further prove that there was CT between donor-receptors. The data in aterials . Charge aterials . Figure mponents . The pea Tp + Tv . It coulD/IG ratio. Surface defects had an important influence on the nonlinearity of NCs. −1 and 1583 cm−1, respectively [D/IG ratio of NC was obviously larger than that of gaphene, indicating that NCs had more surface defects and nanoparticles were attached in G. For all NCs in D/IG ratio increased first and then decreased as the amount of graphene increased. The increase of the ratio indicated that there were fewer defects due to the densities of γMnS decreased on the surface of graphene, while the decrease of the ratio represented that NCs with little GO addition had more defects. This was consistent with the results of SEM and XRD. For graphene-γMnS, the average sp2 domain size or the effective in-plane correlation length of the sp2 domain size aL were given by this formula plaserλ was the wavelength of the test laser (532 nm) [Dn could be approximately calculated by formula 2 fragment density, which decreased first and then increased. Defects affected the local state of a small area sp2 domain and the large area sp2 cluster on graphene.The information of NCs surface structure could be obtained by Raman spectroscopy. The density of NCs surface defects could be obtained by Iectively . The ID/(532 nm) . The defλplaser4 . The res3 matrix to the sp2 domain and the sp2 cluster directly affected the CT between the graphene and the γMnS nanoparticles, while the sp3 matrix played an important role on the active site for the attachment of the γMnS nanoparticles. Because the local state of the small area sp2 domain and the large area sp2 cluster were related to the CT time and relaxation time, which influenced changed nonlinearity. More local states on graphene surface increased CT time, resulting in the reduction in nonlinearity of NCs. As the addition of GO increased, the nonlinearity of NCs decreases first. It could be seen from SEM, XRD and Raman patterns that the distribution state of γMnS on graphene surface was greatly affected by the amount of GO added. For S1, S2, and S3, γMnS nanoparticles were stacked on the GO surface with a low degree of dispersion, which resulted in a large number of smaller sp2 domains growing. These lead to percolation between the larger sp2 clusters via growth of smaller sp2 domains, which increased the spatial overlap of electron and hole wave functions, thereby reducing the oscillator strength of excitons [2 domain of the small segment and did not interconnect the larger sp2 clusters, which led to increase in electron-hole pairs. The overlap of electron and hole wave functions reduced, resulting in the nonlinearity enhanced of NCs.The transfer from the spexcitons . As amou2 domain, the large area sp2 cluster on graphene, and defects changed. Tunable NLO performance of NCs by adjusting defects was important for the fabrication of nonlinear optics such as optical switches, optical sensors, and so forth.In summary, γMnS and graphene-γMnS composites were synthesized by hydrothermal method. The structure, absorption and refraction of γMnS and graphene-γMnS composites were characterized. The NLO property were investigated by 532 nm pulsed laser with pulse duration of 30 ps. The NLO enhancement of NCs was due to included local field effects and charge transfer. However, results of theoretical calculations were deviated from experimental data, and NLO enhancement was within an order of magnitude due to non-radiative defects in graphene-γMnS. This article also provided a facile approach of changing addition of GO to obtain tunable NLO property of NCs. As the addition of GO increased, NLO characteristics of NCs decreased first and then increased. Structure of NCs made local state of the small area sp