This review article summarizes the published investigations on the preclinical studies of three novel IPF drugs, namely pirfenidone, nintedanib, and N-acetylcysteine (NAC). In addition, the study protocols, differences, and the main findings in the recent clinical trials of these pharmacological treatments are reviewed. The strategy for drug development and the timeline from the discovery to the clinical use have been very different in these regimens. Pirfenidone was discovered in 1976 but only recently received approval in most countries, and even now its exact mechanism of action is unknown. On the contrary, nintedanib (BIBF1120) was identified in large drug screening tests as a very specific inhibitor of certain tyrosine kinases, but no published data on preclinical tests existed until 2014. NAC, a mucolytic drug with an antioxidant mechanism of action was claimed to possess distinct antifibrotic properties in several experimental models but proved to be ineffective in a recent randomized placebo-controlled trial. At present, no curative treatment is available for IPF. A better understanding of the molecular mechanisms of IPF as well as relevant preclinical tests including animal models and  The positive results emerging from animal studies and phase I trials stimulated the initiation of several clinical trials in idiopathic pulmonary fibrosis (IPF) in 1990s and 2000s; however, most of them produced only disappointing results when they reached the randomized phase III studies . In contAlthough these trials mark a novel era in the clinical treatment of IPF, they also highlight the need for researcher-driven studies on drug efficacy as well as necessity of conduction comparative studies of different regimens of the patients representing various phenotypes of IPF. It would be crucial to clarify the experimental foundations of these molecules and their mechanisms of action to create a foundation for future basic and clinical studies. Since the cost of drug development is so enormous, it would be most advantageous if clinical researchers in the near future could develop markers of therapeutic effect, that is, not only for monitoring changes in disease severity but also as possible indicators for treatment cessation.in vitro and in vivo, but there was still a lack of firm conclusive evidence to support its clinical use in the treatment of IPF.in vitro on human lung fibroblasts cells have revealed that pirfenidone exerts many effects, that is, a decrease in fibroblast proliferation, reduction of TFG-β stimulated reactions, lowered levels of a myofibroblast marker alpha smooth muscle actin (α-SMA), and reduced expression of heat shock protein 47 (HSP47)  fluid. Pirfenidone has been shown to prevent the accumulation of hydroxyproline, procollagen I and III, inflammatory cells and transforming growth factor-beta (TGF-β) in BAL, and/or lung tissue –18. Late (HSP47) , 25. A cThe timeline of pirfenidone from the drug development and preclinical studies to the worldwide acceptance as the first drug of choice of IPF is shown in not CDK4 , several vascular endothelial growth factor receptors , and fibroblast growth factor receptor 1 (FGFR-1) but not CDK4 . As a ponot CDK4 , gynecolnot CDK4 , and brenot CDK4 . Its effnot CDK4 . Howevernot CDK4 , but it not CDK4 . Irrespein vitro. Only afin vitro  and nintedanib (Inpulsis) are shown in For the first time in the history of the clinical research of the pharmaceutical treatment on IPF, the primary endpoints of several studies have revealed positive results. Even though the reduction of FVC decline by 30% did not exceed the hopes of clinicians and patients, these results are still promising since they raise the possibility of a prolonged survival for IPF patients. The three recently published studies, namely Ascend, Inpulsis, and Panther clearly showed that randomized and placebo-controlled multicenter trials are the most appropriate way to reveal clinical benefits of pharmacological treatment of IPF. The results of all these abovementioned studies will affect daily clinical practice not only through positive results of pirfenidone and nintedanib but also with the negative results obtained with NAC.In view of the long history of preclinical and clinical investigations of NAC and pirfenidone, it can be concluded that it requires a long time to develop a drug from its discovery till its clinical application. On the contrary, the time period to the clinical use of nintedanib has been markedly shorter. Thus, there are some elements that need to be considered when future clinical trials are evaluated. First, it seems that interim analyses have sometimes broken study liability. In the first randomized pirfenidone trial , the decin vivo studies have indicated that NAC can increase the levels of glutathione in BAL fluid of patients with IPF  reveal surprisingly similar antifibrotic mechanism of actions, namely decreased amounts of histological fibrosis, decreased tissue levels of collagen and/or hydroxyproline, and decreased levels of TGF-β and α-SMA. Each drug has, in addition, displayed several specific antifibrotic properties. A glutathione deficiency in epithelial lining fluid of the patients with IPF was observed several decades ago  but the with IPF . In addiwith IPF , 50. Thewith IPF .Careful evaluation of the design of the future clinical studies will be necessary, as IPF is such a rare disease. The results of recent clinical trials raise many questions, for example, will it be possible to perform clinical trials in the future with sufficient numbers of patients on trials investigating a novel drug versus pirfenidone, and moreover, will placebo-controlled trials still be approved by the authorities? How about the patients that do not fulfill the current IPF criteria or are at a very early or an advanced stage of the disease? It would probably be useful if future clinical trials could be performed using uniform inclusion criteria and have similar endpoints to allow data pooling and comparison between studies. In addition, more specific tools for the diagnostics of acute exacerbations would be helpful for providing more diverse endpoints. Most importantly, even though some important progress has been made, clinical studies must be continued, and novel drugs will still need to be investigated in a RCT setting. Furthermore, the patients with other types of fibrotic interstitial lung diseases are still lacking clinical research advances.Although major progress has been achieved in the clinical research into IPF, a curative therapy for this severe lung disease is still lacking, which emphasizes the need for all kinds of research, not simply clinical trials in IPF. Before one can develop novel pharmacological therapies to cure IPF, we will need innovative approaches, including basic and translational techniques, and most importantly, relevant animal models as well as protocols using human cells. If it were possible to provide respiratory physicians new tools for the treatment of the IPF patients, this in turn could advance further clinical research, for example, identifying those patients that will benefit most from the new therapeutic agents.


Angiogenesis is a pre-requisite for the growth and the metastatic dissemination of solid tumors. Therefore, interfering with the angiogenic process has long been proposed as a therapeutic strategy to restrain cancer progression . In thisin vitro studies on genetically manipulated endothelial cells revealed that L1 is necessary and sufficient for cell proliferation, migration and tube formation. Unexpectedly, L1 also exerted a regulatory function on tumor vessel architecture and function. L1-expressing tumor vessels showed the typical alterations of cancer vasculature, with poor pericyte coverage and basement membrane deposition, loss of endothelial polarity and enhanced permeability. In contrast, L1-deficient vasculature exhibited an almost “normal” phenotype, as shown by pericyte coverage, production of a continuous basement membrane and endothelial cell polarity. This resulted in the abrogation of tumor vessel permeability, indicating that L1- dependent regulation of vessel architecture has functional implications [We have recently reported a series of observations that point to the protein L1 as a candidate target for such strategies. L1  is a cell surface molecule that belongs to the immunoglobulin superfamily of cell adhesion molecules, and has been extensively characterized as an important player in the development and plasticity of the nervous system . Furtherications .This set of results, together with a series of observations on genetically manipulated endothelial cells in culture , pointedBesides shedding light on novel mechanisms causally linked to the dysregulated architecture and function of cancer vessels, our data suggested that interfering with the function of vascular L1 might represent an innovative therapeutic option. Indeed, we observed that treating tumor-bearing mice with L1-neutralizing antibodies recapitulated the genetic inactivation of endothelial L1, with decreased tumor growth and angiogenesis accompanied by vascular normalization . Future Antiangiogenic drugs are commonly used in combination with conventional chemotherapeutics or targeted therapies and it can be anticipated that combined strategies will also represent the best option for L1- based treatments. Besides the obvious expectation of an additive effect between the cytotoxicity towards neoplastic cells and prevention of tumor neovascularization, it is tempting to speculate that the vascular normalization promoted by L1 inactivation, by restoring a more uniform blood perfusion of the tumor tissue, might promote a better distribution of the anti-neoplastic drugs, thus enhancing the therapeutic response. Indeed, despite this remains a controversial issue, the hypothesis that vascular normalization improves the clinical efficacy of chemotherapy is supported by a growing body of evidence .Thus, L1 is emerging both as a key player in the orchestration of vascular pathophysiology associated to cancer development and as a promising target for innovative therapeutic strategies targeting tumor vessels. Future preclinical studies will give further insights into the feasibility and the optimal applications of L1-based antitumor treatments.


Self-splicing introns are present in the mitochondria of members of most eukaryotic lineages. They are divided into Group I and Group II introns, according to their secondary structure and splicing mechanism. Being rare in animals, self-splicing introns were only described in a few sponges, cnidarians, placozoans and one annelid species. In sponges, three types of mitochondrial Group I introns were previously described in two demosponge families  and in the homoscleromorph family Plakinidae. These three introns differ in their insertion site, secondary structure and in the sequence of the LAGLIDADG gene they encode. Notably, no group II introns have been previously described in sponges.Agelas oroides , Cymbaxinellapverrucosa  and Axinella polypoides . We show, for the first time, that sponges can also harbour Group II introns in their COI gene, whose presence in animals’ mitochondria has so far been described in only two phyla, Placozoa and Annelida. Surprisingly, two different Group II introns were discovered in the COI gene of C. verrucosa. Phylogenetic analysis indicates that the Group II introns present in C. verrucosa are related to red algae (Rhodophyta) introns.We report here the presence of mitochondrial introns in the cytochrome oxidase subunit 1 (COI) gene of three additional sponge species from three different families: The differences found among intron secondary structures and the phylogenetic inferences support the hypothesis that the introns originated from independent horizontal gene transfer events. Our results thus suggest that self-splicing introns are more diverse in the mitochondrial genome of sponges than previously anticipated.The online version of this article (doi:10.1186/s12862-015-0556-1) contains supplementary material, which is available to authorized users. Nephtys) [Mitochondrial introns are mobile genetic elements that form self-splicing RNA molecules. They are divided into Group I and Group II introns depending on their secondary structure and splicing mechanism . Unlike Nephtys) . Within Nephtys) , 15, 18.Plakinastrella onkodes [P. onkodes, which does not encode any ORF [In sponges, mitochondrial introns have been found to be present in three species of the Plakinidae family (Homoscleromorpha) , in four onkodes , which c any ORF . Interes any ORF .Agelas oroides ; 2- Axinella polypoides Schmidt, 1862; and 3- Axinella verrucosa . It is worth noting that the genus Axinella is not monophyletic. Indeed the two Axinella species belong to different families, Axinellidae (A. polypoides) and Hymerhabdiidae (A. verrucosa), while A. oroides belongs to Agelasidae [Cymbaxinellapverrucosa is thus used to identify A. verrucosa. We also describe, for the first time, the presence of Group II introns in sponges. Indeed, the sponge C. verrucosa was found to harbour in its COI gene two Group II introns in addition to one Group I intron. Our results suggest that self-splicing introns are more diverse in the mitochondrial genome of sponges than previously anticipated.We show here the presence of Group I mitochondrial introns in the COI gene of three additional Demosponge species: 1- elasidae –21. To aelasidae  will be A. oroides , A. polypoides  and C. verrucosa . The products were sequenced using Sanger sequencing  against the non-redundant protein database of the National Center for Biotechnology Information (NCBI) (accessed May 2015), revealed that all three introns 723 include a LAGLIDADG ORF of 948-1,176 bp, which is mainly encoded in the loop of the P8 element. However, no significant similarity  to known proteins was found when conducting Blastp searches using the ORF present in introns 870 of A. oroides and A. polypoides as query. The ORFs detected in the P8 element of introns 870 of these two species were shorter  than the LAGLIDADG ORF of other sponge introns 870 (888 bp in Tetilla radiata). A DNA alignment of the ORFs and LAGLIDADG CDSs identified in sponge introns 870 revealed the presence of numerous deletions and frameshift mutations in A. oroides and A. polypoides ORFs  against the non-redundant protein database of NCBI (accessed May 2015) indicated that both introns 966 and 1141 encode a putative RT ORF (of 2199-2220 bp), which is mainly encoded in domain IV.The introns 966 and 1141 present in the rons see . Specifirons see  and 6 fooup IIA1 . Blastp oup IIA1  searchesC. verrucosa were added to the RT dataset of Zimmerly et al. [C. verrucosa intron 1141, which clusters together with Rhodophyta and Stramenopiles sequences. Interestingly, a careful inspection of all introns within this clade indicated that they are all inserted at orthologous positions in the COI sequence, although for several sequences, the intron insertion site is incorrectly annotated in NCBI. The RT sequence of C. verrucosa intron 966 forms another clade with the RT sequence of Placozoan BZ49 COI intron 6 and Rhodophyta sequences. Again, we verified that all species within this clade share the same intron insertion site in their COI sequence. Two additional metazoan branches consist of: 1- the Nephtys RT sequence; and 2- the NC008151 Trichoplax adhaerens COI intron 2 RT and the NC008834 Placozoan BZ2423 COI intron 2 RT. These last two sequences appear to be related to Viridiplantae sequences.To identify the origin of the putative RT ORFs encoded within introns 966 and 1141, the new RT sequences of y et al. , togetheOur results show that introns in sponges are not limited to the families Tetillidae, Aplysinidae, and Plakinidae, but are also present in Agelasidae, Axinellidae, and Hymerhabdiidae. We also show that not only Group I introns but also Group II introns are present in sponges. The new data allow us to discuss the putative origin of the mitochondrial introns.Introns can be transmitted either horizontally or vertically. The results presented here reinforce the idea that both Group I and Group II mitochondrial introns are horizontally transmitted in sponges , 13, 15,Tetilla radiata , Plakina crypta and Plakina trilopha  are structurally more similar to one another than to the introns 870 of A. polypoides and A. oroides Tetilla rInterestingly, not all introns were found to encode a functional HEG. Goddard and Burt proposed a cyclical model of intron gain and loss , which eC. verrucosa and A. polypoides appear to be in stage 1; intron 723 of A. oroides is in stage 2; while introns 870 of A. polypoides and A. oroides are in stage 3 and 4, respectively.Emblem et al. , distingA. oroides and C. verrucosa are in different stages of their intron life cycle and show very different secondary structures and rates of evolution, as evident from the difference in branch length in the LAGLIDADG tree  inserted at position 387 in COI [The huge increase in sponge sequence data, leads to novel insights regarding the evolution of the mitochondrial genomes of sponges e.g., , 38, 39), 3938, 37 in COI . Since fA. oroides (voucher TAU Po.25598), C. verrucosa (voucher TAU Po.25600) and A. polypoides (voucher TAU Po.25597) preserved in 95 % ethanol, were obtained from the Steinhard Museum of Natural History  [Amplification of the COI gene was performed in two steps. Detailed information concerning the PCR conditions are outlined in Additional file TTGG-3′)  and Cox1TTGG-3′) .C. verrucosa the primer Cox_Calc_R1 showed non-specific annealing to position ~600 bp of the COI sequence. The complete sequence of C. verrucosa was therefore obtained in three fragments. First, a shorter fragment embracing the 5′ region of the COI fragment was obtained by performing a re-amplification of the Cox_Calc_D1-Cox_Calc_R1 product with the primers LCO1490 and COX550PoeR (5′- CATAGTWATMGCCCCNGCTAATAC-3′). Based on the sequence of the Cox1D1-Cox550PoeR fragment and the sequence of A. polypoides, a forward primer, CoxAxiD2, was designed (5′-ATWAGATTAGAATTATCKGCTCC-3′). A middle fragment was then obtained by performing a re-amplification of the Cox_Calc_D1-Cox_Calc_R1 fragment with the CoxAxiD2-Cox1R1 primers. New primers Verrucosa_D1 (5′- GTTATTGTCTCTGCCAGTTTTAGCCGGG-3′) and Averr2014Da (5′-TGCCAGAAGTTTACATTTTAATATTGCC-3′) were then designed based on this middle region. The 3′ end of the sequence (including the Group II introns) was obtained with a different set of amplification. The first PCR was performed applying only the forward primer Verrucosa_D1 on a C. verrucosa genomic extract. This procedure was aimed to enrich the template in mitochondrial copies. A re-amplification was then performed using Averr2014Da and Cox_Calc_R1.For A. oroides, Sequencing of the COI gene was complicated by the presence of a secondary structure. To facilitate sequencing, PCR fragments of A. oroides were cloned into the pJET #K1231 vector (Thermo Scientific). The new sequences were deposited in the EMBL-EBI European Nucleotide Archive under accession numbers LN868208- LN868210.Sequencing of the COI genes was performed using primers specifically designed for each sample. In the case of Amphimedon queenslandica complete COI. Specifically, in A. oroides and A. polypoides sequences, two introns were inserted after positions 723 and 870, when using the sequence of Amphimedon queenslandica as reference. Similarly, three introns were identified in the sequence of C. verrucosa, inserted after positions 723, 966 and 1141, also when using the sequence of A. queenslandica as reference.The position of each intron was determined by performing a manual alignment of the sequence obtained with the complete COI sequence of closely-related species. Following Szitenberg et al.  introns http://unafold.rna.albany.edu/?q=mfold) [The secondary structure of Group I introns was determined following Rot et al. . First, q=mfold)  was thenq=mfold) , and usiq=mfold)  and Toorq=mfold) . The Mfoq=mfold)  was usedq=mfold)  were conS. singularis was not yet available in NCBI while preparing this manuscript and could not be included in our phylogenetic analyses. Since few complete mitochondrial genomes were available from species closely-related to A. polypoides and C. verrucosa, Blastn 2.2.32+ searches [Dragmacidon reticulatum, HQ379410 Axinella infundibuliformis (for A. polypoides) and KJ008097 Cymbaxinellap sp.GL 2014 (for C. verrucosa) were thus added to the dataset.To confirm the phylogenetic position of the obtained COI sequences all complete mitochondrial genome sequences of demosponges and homoscleromorphs available in NCBI (accessed March 2015) were downloaded, and the complete COI CDSs were extracted from these sequences. All sponge COI CDSs known to harbour an intron –15 were searches  were conwww.geneious.com). Positions with more than 50 % of missing data were excluded from the alignment. The final alignment is available as supplementary material  and Bayesian criteria. ML trees were built with RAxML version 8.0.26. The dataset was partitioned based on codon positions and each position was assumed to evolve under an independent GTR + Gamma model of sequence evolution. The ML tree was identified using 100 starting trees. Support values were computed using 100 slow bootstrap replicates. The Bayesian tree was reconstructed with MrBayes v3.2.2 using the same partitions and model as in the ML analysis. Two independent runs, each with four chains, were sampled every 500 generations. Each chain was run for 100,000,000 generations. Posterior probabilities (PP) were computed after excluding the first 25 % of the trees (burnin). Convergence of the run was assessed by verifying that the average standard deviation was below 0.01 before the burnin threshold, and that the potential scale reduction factors of the parameters were equal to 1. The homoscleromoph sequences were used as root for the demosponge tree and To reconstruct the phylogenetic origin of the LAGLIDADG ORF encoded within Group I introns, the dataset of Szitenberg et al.  was downLAGLIDADG sequences were aligned using MAFFT v7.017 under the L-ins-I algorithm. Positions with more than 50 % of missing data were excluded from the alignment. The alignment is available as supplementary material  were added. Blastx 2.2.32+ and Blastp 2.2.32+ searches were then conducted with default settings against the non-redundant protein database of NCBI (accessed May 2015) to identify sequences with an E-value = 0.0 and at least 45 % identity to the metazoan RTs. These sequences were added to the dataset. RT sequences were aligned with MAFFT v7.017 under the L-ins-I algorithm. Positions with more than 50 % missing data were excluded from the alignment. The alignment is available as supplementary material (Additional file To reconstruct the phylogenetic origin of the RT ORF encoded within the Group II introns of s et al.  was folls et al.  was downPhylogenetic tree reconstructions were performed using RaxML 8.0.26 and MrBayes v3.2.2 as described above for the LAGLIDADG analysis. The best model of evolution was also found to be VT + I + G + F using ProtTest 3.4 . The onlSequences were deposited in the EMBL-EBI European Nucleotide Archive under accession numbers LN868208- LN868210. Sequence alignments used in the phylogenetic analysis are included as (Additional files


Where and Which questions, which can be answered with gestures and/or words. After extracting all adult Where and Which questions and child answers from longitudinal videotaped sessions, we examined the timing from the end of each question to the start of the response, and compared the timing for gestures and words. Child responses could take the form of a gesture or word(s); the latter could be words repeated from the adult question or new words retrieved by the child. Or responses could be complex: a gesture + word repeat, gesture + new word, or word repeat + new word. Gestures were the fastest overall, followed successively by word-repeats, then new-word responses. This ordering, with gestures ahead of words, suggests that the child knows what to answer but needs more time to retrieve any relevant words. In short, word retrieval and articulation appear to be bottlenecks in the timing of responses: both add to the planning required in answering a question.When young children answer questions, they do so more slowly than adults and appear to have difficulty finding the appropriate words. Because children leave gaps before they respond, it is possible that they could answer faster with gestures than with words. In this study, we compare gestural and verbal responses from one child between the ages of 1;4 and 3;5, to adult  When adults answer questions, their answers are surprisingly fast, regardless of the language involved. The median gap between the end of a yes/no question, for example, and the start of the answer, is about 200 ms for speakers of English, with a range over other languages from 0 ms to nearly 400 ms . This clYoung children take much longer to answer questions than adults do , apparenTo date, studies of how children answer questions have focussed on verbal answers, where children either repeat one or more words from the adult question in their answer, or construct a verbal answer from scratch. This suggests that one factor in answering a question is how much word production children have to plan in order to come up with an answer. When they repeat a word from the adult’s question as the answer, they need not retrieve anything from memory, so the cost should be minimal. But when they have to retrieve one or more appropriate words, and the relevant construction, from memory before they begin to articulate their answer, the cost should be much greater.Where question, or to select an alternative in answer to a Which question, would seem to require less planning and articulation than producing an utterance, where children must first decide which words to repeat of those already said, say, or else search for and retrieve one or more appropriate words and then articulate those in their response.What happens, though, when children , have the option of answering the question with a gesture, either alone or in combination with speech? Once children have decided on an answer, to extend hand and arm to designate a place in answer to a Children start to produce manual gestures fairly early to indicate interest in or desire for something in their immediate environment. They start to point (interest: ‘look’) and reach (desire: ‘I want’) from as early as 10–12 months. These two gesture-types have been considered to be proto-speech acts for asserting and requesting . The vasChildren demonstrate comprehension as well as production of POINTS from around 12 months on e.g., . When adBy 1;6, children appear to use POINTS differentially: to elicit a term for something, they simply point and look, and adults then typically offer a label for that referent. But when children combine a point with vocalization or a word, adults are significantly more likely to treat that combination as a request and to give the child the object targeted see .where question with a POINT to the relevant location. In this study, we compare gestural and verbal responses to adult Where and Which questions addressed to one child between the ages 1;4 and 3;5.When young children answer questions, they may substitute an indicating gesture for speech, for example answering a To make this comparison, we examined the relative timing of any gesture onset in an answer, compared to the timing of any vocal or verbal onset in the answer. Answers could take several forms: (a) they could be simple, containing just a single response-element: a gesture, a babble , or a word—either a word repeated from the adult question or a new verbal choice made by the child; or (b) they could be complex, consisting of two elements: a gesture + babble, gesture + repeat, gesture + new-word(s), or repeat + new-word(s).Since gestural responses do not require any lexical retrieval, we expect that children should be able to produce a gestural response as soon as they have understood the question. Producing a new verbal response should be more complex and therefore take more time: the child must retrieve any relevant word(s), plan the answer, and then articulate it. So any measurable difference between gestural and verbal response times would provide a preliminary indication of question-comprehension time (how long it takes to issue a gesture) and hence how much time is required to produce a verbal response.what to answer but needs additional time to retrieve relevant words. The conceptual cost of identifying a relevant answer is the same for gestural and verbal responses. But for verbal responses, lexical retrieval is one bottleneck in the timing of an answer, followed by planning the response and articulation , this would be evidence that the child knows culation , 2006. WFor this study, we drew on the corpus for one child, Alex, from the Providence Corpus of American English , in the Where and Which questions can be answered with either a gestural or verbal response, or both. We extracted all Where and Which questions, and measured the onset times of all responses provided by the child. We identified a total of 502 Where and Which questions in the 35 videos selected, and then identified all the responses given. The adult (mother) in each case treated the child’s responses as answers to the question just asked. Moreover, the majority of these responses explicitly provided relevant semantic information: a target-place in response to Where questions, and a chosen alternative in response to Which questions. The one response-type we were unable to assess was vocal babble on its own: these babbles were consistent in form but, to us, uninterpretable. His mother, however, treated these too as appropriate responses.In order to analyze the time it took for the child to answer his mother’s questions, we compared gestural and verbal responses. Gestural responses offer a non-verbal form of response that avoids the need to find appropriate words, while verbal responses require finding and producing an appropriate answer. gesture: manual gesture (G) to the target object’s location, (ii) speech: babble (B), repeat of a term from the adult question (R), new verbal response (V), and (iii) location: on camera or off camera (O). We also coded as ‘no response’ (N) any questions the child failed to answer, questions where the child was impeded from responding , and questions asked when the child was already manipulating, or speaking about, the relevant object.We coded all the child responses using the following categories: (i) Where or Which), (v) the adult’s actual question, and (vi) the content of the child’s response. Next, we used a Python script to extract 12 s of audio/video for each of the 502 questions we had identified, beginning 2 s before each question onset. Each video clip was imported into ELAN and paired with a synchronized transcription file. The onsets and endings of all questions, and of all answers  were transcribed into question, gestural, and verbal tiers, respectively. Adult Where and Which questions received 235 (47%) unimpeded gestural and/or verbal responses from the child.We collected metadata for each question/response pair, including: (i) age of child, (ii) video file, (iii) timestamp for that question in the video session (HH:MM:SS), (iv) question type . Gesture onsets were marked immediately prior to the beginning of movement (the preparation of the gesture), and duration of the stroke was also measured where possible. In the nine instances where a question was repeated multiple times, we used our discretion in determining which instance the child was attending to. Lastly, we excluded outlier responses that were two SDs or more from the relevant mean  from our statistical analyses.Where and Which questions was rechecked by an independent coder. This coder and the second author agreed on the timing measurements, within 200 ms  88% of the time, with high inter-rater reliability . When the timing window was expanded to within 400 ms, the agreement rate rose only very slightly, to 91%.The timing of 10% of Alex’s responses to Where questions received 137 responses, and Which questions 84 responses. ‘Single’, simple, responses followed 145 (66%), and ‘double’, complex, responses followed 76 (34%). In the double responses, we measured the onset timing for each element separately (76 × 2). The single or simple responses consisted of one element in the response. There were 14 gestures, 13 babbles, 56 repeats, and 62 new-word responses , as shown in Table 3. Double or complex responses consisted of those where the child combined a gesture with a vocalization or word, or combined a repeat from the adult’s question with some added verbal material. These combinations were ordered, with 10 gesture + babble responses, 7 gesture + repeated word, 39 gesture + new-word, and 2 repeated word + new-word responses, along with another 18 other complex answers where the second element seemed to be produced for added clarification rather than being an integral part of the initial response. In these cases, Alex produced an indicating gesture after saying a repeated or new word. In seven instances, he babbled and then, after a pause, pointed; in five more, he repeated a word from the question, and only then pointed; and in another five, he produced a new word and then pointed. In each instance, Alex appeared to be trying to clarify his answer by adding the gesture afterward . And in one, he produced a one-word answer, paused, and then repeated a word from the adult question. We therefore excluded the timing for these from our overall computations of the relative timing of the two parts in double responses.Of these 221 questions included in our statistical analyses, Table 2.In all, we measured 297 response tokens . The overall mean response times, from the end of the question to the onset of the child’s answer, are summarized in F = 9.355, p < 0.0001], with babbles and repeats both produced significantly faster than new-word responses (Table 2). His gestures were faster than both repeated word responses , and new-word responses . And his repeated word responses in turn were significantly faster than his new-word responses . Finally, within his complex double responses (Table 4), his gestures were significantly faster than the verbal responses they were paired with, where the gesture onset preceded the word onset .Overall, Alex’s gestures were produced faster than his babble responses. His babbles, repeats of adult words, and new-word responses differed significantly overall .The single gesture responses consistently indicated the location in response to r = 0.085, n.s.), but when Alex constructed his answer with his own words, he produced a larger number of more complex (longer) utterances as he got older . We also looked, within his single responses, at how long he took to produce 1–, 2–, and 3+ word answers. On average, longer answers took longer to produce for both repeats and new-word responses, as shown in Table 5. However, there was effectively no correlation between length-of-response and timing here, largely because both repeats  and new words  displayed wide variance in timing with production of utterances of the same length. This is attributable to extraneous factors such as how well the child was actually attending when the adult issued the question, whether he could remember immediately where something was, and his basis for deciding which alternative to choose.Does planning a longer verbal response take longer? We looked at the correlations with age for Alex’s responses (a) where he repeated one vs. two vs. three or more words from the adult question, and (b) where he produced one vs. two vs. three or more words retrieved from memory. In the first case, where he had just heard the word(s) he repeated in the adult’s question, there was no change with age, whether he repeated 1, 2, or 3+ words (Table 5). As Casillas et al. (under review) noted, young children do get faster at answering yes/no questions as they get older. However, children’s increasing speed in answering Wh-questions is often obscured by their ongoing acquisition of different Wh-words, and the added planning needed to answer Wh-questions, as children master the meaning of each Wh–type in turn. Of Where and Which, Where is typically acquired first . However, we did not have enough data to detect any changes in speed for Alex’s answers to these Wh- questions over time, as he got older.As children get older, they get better at planning and better at articulating words and sequences of words, so they can produce longer answers. But they should also gradually speed up, perhaps doing so more readily when they repeat words from the adult question than when they construct an entirely new answer themselves. This would initially yield the different means for responses where Alex repeated one or more words from the adult question, compared to where he constructed a new answer with words retrieved from memory . But responding with an utterance requires that children be able to retrieve any pertinent words and, if necessary, combine two or more words in a syntactic construction, for subsequent articulation.But in order to answer questions, children need to be able to retrieve the right words. This in turn depends on their having already made the appropriate mapping as they linked forms and (preliminary) word meanings so they could recognize those words from others—for comprehension. This first step is essential for children trying to retrieve the relevant or most appropriate word(s) for production. Question-answering depends on both comprehension and production, with comprehension of the question followed up by (a) the idea of a possible answer, and (b) its instantiation as a gesture, as an utterance, or as some combination of the two , even if these fail to match the adult targets, adults can generally understand what the children intend. But children continually monitor and fine-tune their production, eventually matching the conventional forms produced in the speech community around them. One way to characterize their articulatory development is in the form of two ‘rules’ for early word production, Be consistent and Be precise  to their repertoire, and learning how to answer each question type. Yet they clearly know, by age two if not earlier, that they are expected to answer as soon as they can. At this point, they begin to produce floor-holders like um, or start an utterance and keep repeating the first word  until they have their full answer ready (When people ask questions, they expect to hear answers in the next turn, and the median gap between question and answer for d 200 ms . But at er ready .Turn-taking is a skill fundamental to language use: it is critical for coordinating with others, whether to exchange greetings, answer questions, exchange information, collaborate in all sorts of activities, or co-construct a story. While some form of turn-taking, with attention to gaze, for example, first emerges during infancy, it is only once children produce recognizable words that they begin to participate in conversational exchanges, and begin trying to observe the adult’s ‘No gap, no overlap’ pattern of taking turns.Indeed, turn-taking is central to all conversational interaction: it relies on gaze and gesture as well as on the child’s utterances. Examining different aspects of turn-taking and, in particular, how children answer different question-types, allows us to take a closer look at how children make use of what they know about both language and interaction so far . They noThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We reported a simple and effective way of fabricating one-dimensional (1D) graphene oxide nanoscrolls (GONS) from graphene oxide (GO) sheets through shock cooling by liquid nitrogen. The corresponding mechanism of rolling was proposed. One possibility is the formation of ice crystals during the shock cooling process in liquid nitrogen to be the driving force. The other might be due to the uneven stress of the sheets inside or outside ice during the lyophilization. After reducing, graphene nanoscrolls (GNS) exhibited good structural stability, high specific surface area, and high specific capacitance. The capacitance properties were investigated by cyclic voltammetry, galvanostatic charge-discharge, and electrical impedance spectroscopy. A specific capacity of 156 F/g for the GNS at the current density of 1.0 A/g was obtained comparing with the specific capacity of 108 F/g for graphene sheets. Those results indicated that GNS-based rolling structure could be a kind of promising electrode material for supercapacitors and batteries. Graphene was one-atom-thick carbon material with remarkable electronic, mechanical, and thermal properties ,2. It caCurrently, only two of the predicted structures  tubular structure, CNS were formed by rolling up of flat graphite thin nanoplates , attractIn this work, we reported the graphene oxide nanoscrolls (GONS) of a distinct form of 1D tubular graphene architecture, which fabricated by shock cooling of aqueous graphene oxide (GO) dispersion by liquid nitrogen. The resulting stable and curly 1D structure was found to be high specific surface area and high specific capacitance. As a consequence, a high reversible capacity, good thermal stability and good rate capability, and excellent cyclic stability were achieved.4, NaNO2, NaOH, concentrated H2SO4, concentrated HCl, and H2O2 (30%) were all analytical grade and purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd . All aqueous solutions were prepared with ultrapure water (18 MΩ).Graphite powder (400 mesh) was obtained from Beijing Chemical Reagents . KMnO2SO4 (98%) was stirred in an ice bath, and 5.8 g KMnO4 was slowly added with stirring for 0.5 h. The solution is heated to 30°C for 2 h, 40 mL of deionizer water is added slowly, the reaction is heated to 90°C for 30 min, then, 80 mL of deionizer water is added. When the temperature is cooled to 60°C, 10 mL H2O2 (30%) is added to give an orange yellow solution. Two hundred milliliters of 5% HCl solution is added, decanted the supernatant, and centrifuged with deionizer water to pH 4 to 6, and the mixture solution is further dialyzed in a dialysis bag in 2 days, and low density large graphene oxide is obtained.GO is prepared by the modified hummers method . BrieflyGO was produced by the modified Hummers method through acid oxidation of flake graphite. The primary graphite was suspended in water under sonication for 1 h, which followed by centrifugation at 4,000 rpm for 30 min and dispersed in the water. The resulting yellow mixture was cooled to room temperature. Graphene oxide was dispersed in water to form graphene oxide dispersion  in a 100-mL beaker which was rapidly cooled by surrounding with liquid nitrogen. The water was removed by freeze-drying machine. The remaining GONS (0.24 g) was dispersed in water (150 mL) and reduced by sodium nitrite (0.34 g) in a 250-mL round-bottom flask, which was then heated to 90°C for 2 h, the water was removed, and the residue was washed by water to obtain GNS.−1 in N2. Raman spectra were characterized by a JY Horiba Raman  under 514-nm laser for the accumulation intensity of three-time scan.Scanning electron microscope (SEM) images were obtained by a high-resolution scanning electron microscope  at 3.0 kV. Transmission electron microscopy (TEM) experiments were performed on a high-resolution transmission electron microscope  equipped with selected area electron diffraction (SAED). X-ray diffraction (XRD) was recorded on a Rigaku D/Max-RB diffractometer  with CuKα radiation at 40 kV and 120 mA. Thermogravimetric analysis (TGA) was carried out using a thermogravimetric analyzer  from 30°C to 600°C at a heating rate of 10°C min5 to 0.05 Hz at 10-mV amplitude referring to open-circuit potential (OCP). Cycling stability of the materials was performed on galvanostatic charge/discharge curves at current density of 1 A/g after 2,000 cycles.Capacitance performances of GNS materials were tested by cyclic voltammetry (CV), galvanostatic charge/discharge, and electrochemical impedance spectroscopy . All of the electrochemical experiments were carried out in a three-electrode system. The fabrication of working electrodes was carried out as follows. GNS (4.2 mg) were mixed with carbon black and poly (tetrafluoroethylene) at a mass ratio of 85:10:5 in several drops of ethanol. The working electrode was formed after drying. The potential range of CV examination was −0.2 to 1.0 V, and that of galvanostatic charge/discharge tests was 0 to 1.0 V. Electrochemical impedance spectroscopy (EIS) tests were performed in the frequency range of 10The transformation from 2D graphene sheet into the curly tubular 1D structure could be understood with the aid of the illustrations in Figure In this study, we attributed one possibility of the rolling of 2D graphene oxide sheets to the rapid formation of ice crystals, which caused by shock cooling with liquid nitrogen. As water starts to form ice crystals in this experimental procedure, GO sheets in the suspension were rejected from the moving solidification front  and forcWhile Xu et al. suggested that the rolling was formed during the sublimation of ice . During 2NH2 water solution as shown in Figure Figure Figure 2 carbon structure. Its intensity was known to increase as the number of defects increases [−1) and D band  was observed with a large intensity ratio ID/IG at 1.09. And G band of GONS  and D band  were observed with an intensity ratio ID/IG at 0.917. Raman spectroscopy confirmed the quality of GO and GONS, the relative intensity of the disorder D band, and the crystalline G band (ID/IG) for as-produced GO and GONS were only around 0.179. This indicated not only the high quality of as-prepared GONS but also the differences were possibly related to the increased π-π stacking and the scrolled structure in GO.Raman spectra of GO and GONS were recorded using the 514-nm excitation line  as the electrolyte .−1. Figure −1. It was noted that the integrated area in the CV of GNS was larger than that of the rGO materials at the same scan rate, suggesting its higher specific capacitance. Additionally, we found that the area surrounded by CV curves form GNS electrode was apparently bigger than that of the rGO electrode at the same scan rate, implying the higher specific capacitance of GNS electrode, which indicated a remarkable contribution of rolling graphene oxide in raising the capacitance of graphene. The GNS presented well-symmetric and rectangular CV curves with obvious redox characteristic peaks indicated a low resistance of GNS electrodes to mass transfer and good charge propagation of ions at the interfaces between the electrolyte and the GNS material [−1 in the rGO electrode [Figure material . We founmaterial . For rGOlectrode .Figure 8Figure Figure Z′) was a combined resistance of the ionic resistance of the electrolyte, the intrinsic resistance of the substrate, and the contact resistance at the active material/current collector interface. The semicircle in the high-medium frequency range corresponds to the Faradaic charge transfer resistance, and in the low-frequency range, it corresponds to the capacitor’s diffusive resistance of the electrolyte in the electrode pores and the ion diffusion in the host material [Nyquist impedance plots of the GNS and rGO electrodes are shown in Figure material . The Nyqmaterial . Thus, aImportantly, the GNS exhibited good rate capability. A specific capacitance of 156 F/g can be achieved at 1 A/g, and the specific capacitance could remain at high level of 148 F/g with a good retention above 93% (Figure In our work, one-dimensional GONS were fabricated by rolling GO with assistance in liquid nitrogen by a simple and effective way. The mechanism of formation was proposed, and the formation of ice crystals during the shock cooling step in liquid nitrogen were believed to be the driving force to form such unique tubular structures. GNS exhibited good structural stability, high specific surface area, and high specific capacitance after reducing GONS. Comparing with the specific capacity of 108 F/g for graphene sheets, a remarkable capacity of 156 F/g is obtained at the current density of 1.0 A/g, which was graphene sheets transformed to the unique rolling structure of GNS. Those encouraging results indicated that GONS based on the rolling structure of graphene sheets were a kind of promising material energy storage field.


Linear arrangement of repeat unit 5–8–5 defect along zigzag-direction of graphene), DS (a Slope angle between repeat unit 5–8–5 defect and zigzag direction of graphene) and DZ (Zigzag-like 5–8–5 defects) defects in the zigzag direction become larger than those in the pristine graphene in the same direction. A maximum increase of 11.8% of Young’s modulus is obtained. Furthermore, the brittle cracking mechanism is proposed for the graphene with 5–8–5 defects. The present work may provide insights in controlling the mechanical properties by preparing defects in the graphene, and give a full picture for the applications of graphene with defects in flexible electronics and nanodevices.Understanding the effect of defects on mechanical responses and failure behaviors of a graphene membrane is important for its applications. As examples, in this paper, a family of graphene with various 5–8–5 defects are designed and their mechanical responses are investigated by employing molecular dynamics simulations. The dependence of fracture strength and strain as well as Young’s moduli on the nearest neighbor distance and defect types is examined. By introducing the 5–8–5 defects into graphene, the fracture strength and strain become smaller. However, the Young’s moduli of DL ( Graphene, a two-dimensional 2D) material with a single atomic carbon layer array in honeycomb lattice, has attracted widespread interest due to its novel electronic propertiesD materia5678910et al.et al.et al.161819et al.ab initio calculations. By employing nonequilibrium molecular dynamics simulations, divacancy was reconstructed by Liang et al.et al.et al.et al.et al.An important class of graphene defect, 5–8–5 defect, has been studied both experimentally and theoretically. Through electron bean method, graphene sheets with 5–8–5, 5–8–4–8–5 defects were prepared by Robertson In this paper, a family of the graphene with 5–8–5 defects are designed and prepared. Then, systematically detailed studies on the mechanical responses of graphene with various 5–8–5 defects are examined. The dependence of ultimate strength, ultimate strain and Young’s modulus of graphene with defects on the nearest neighbor distance between defects and defect types is determined. A possible brittle cracking mechanism was proposed: i.e. the failure of graphene with 5–8–5 defects can initiate from either the bond shared by the 5–8 rings or the bond shared by 6–8 rings, depending on the defect arrangement as well as the loading orientation.Linear arrangement of repeat unit 5–8–5 defect along zigzag-direction of graphene), DS (a Slope angle between repeat unit 5–8–5 defect and zigzag direction of graphene), DZ (Zigzag-like 5–8–5 defects), DY (A repeat unit of Y-like 5–8–5 defects), DT (A repeat unit of Triangular 5–8–5 defects), DQ (a repeat unit of Quadrangular 5–8–5 defects) and DH . The size of the graphene is approximately 20 × 20 (nm)2. The adaptive intermolecular reactive bond order potential (AIREBO)28Graphene with six different kinds of 5–8–5 defects are prepared and presented in −1 and the increment every 1 ps.Simulations were performed by employing Larger-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS)The stress on each carbon atom is calculated via the following virial stress equation33α and β are the indices of stress tensors along the Cartesian coordinate axis, i and j are the atomic positions, Ωα, vα and mα are the atomic volume, velocity and mass, respectively. rj and f i are the distance and force. In addition, the thickness 3.4 Å is employed to compute the atomic stress in graphene sheet3036In s  at s = 54.6 Å is 6% higher than that when s = 8.4 Å. The d1 (1.384 Å) at s = 54.6 Å is 2% lower than that when s = 8.4 Å. Our results of the average bond lengths are in excellent agreement with the experiment data and DFT results for the two 5–8–5 defects1, d2, d3, d4 and d5 are 1.33–1.45 Å, 1.30–1.49 Å, 1.53–1.67 Å, 1.43–1.46 Å, and 1.31–1.53 Å, respectively. θ as a function of the nearest neighbor distance (s) for the graphene with DL defect. The average angle θ changes monotonically with the increase of s. θ and s can be fitted by a linear function, that is, θ = ks + b. Using the least-squares technique, the fitting parameters are obtained, that is, θ = 0.04115 * s + 107.7 (see  d5 > d1 . For exa07.7 see .s for each structure) are presented in The stress-strain curves of pristine graphene and graphene with various 5–8–5 defects (the shortest Based on s) of graphene with various defects. As s increases, the fracture strength and strain in the zigzag direction first increase and then tend to constants. The fracture strength and strain of DH in the armchair direction first increase and then tend to constants as s increases while those of DL, DS, DZ, DT, and DY are not sensitive to the nearest neighbor distance s. For a selected graphene or graphene with defects, the fracture strength and fracture strain of zigzag oriented are larger than those of armchair oriented, while the Young’s moduli in the armchair direction are much higher than those in zigzag direction.s increases. Comparing with Young’s modulus of pristine graphene, the Young’s moduli of defects DT and DY in the zigzag direction become smaller with small s, and become larger with large s . The maximum Young’s modulus of 863 GPa is obtained, which is 11.8% higher than that of pristine graphene. The average Young’s moduli are larger than those of graphene containing one-GB36From 29ge s see . For insTo identify the failure mechanisms of graphene with defects, conformations of graphene with defects after the fracture occurs are shown in s = 46.2 Å are illustrated in 39414240In order to present the fracture process of graphene sheet with defects, the atomic stress distributions for DS, DY and DH defects with the nearest neighbor distance z axis  on the nearest neighbor distance and defect types was investigated. The fracture strength and strain in the zigzag direction first increase and then tend to constants with the nearest neighbor distance increases. For a selected graphene or graphene with defects, the fracture strength and fracture strain of zigzag oriented are larger than those of armchair oriented. The Young’s moduli in the armchair direction are much higher than those in zigzag direction. The fracture strength and fracture strain become smaller by introducing 5–8–5 defects in graphene. Comparing with pristine graphene, the Young’s moduli of graphene with defects in the armchair direction become smaller, whereas the Young’s moduli of DL, DS, and DZ defects in the zigzag direction become larger. The brittle cracking mechanism is proposed for the graphene with 5–8–5 defects, and the first breaking bond is either shared by 5–8 rings or by 6–8 rings.How to cite this article: Wang, S. et al. Large-Scale Molecular Simulations on the Mechanical Response and Failure Behavior of a defective Graphene: Cases of 5-8-5 Defects. Sci. Rep.5, 14957; doi: 10.1038/srep14957 (2015).


This work paves the way for enhancing light modulation in graphene plasmonics by employing noble metal plasmonic structures.Subwavelength metallic slit arrays have been shown to exhibit extraordinary optical transmission, whereby tunnelling surface plasmonic waves constructively interfere to create large forward light propagation. The intricate balancing needed for this interference to occur allows for resonant transmission to be highly sensitive to changes in the environment. Here we demonstrate that extraordinary optical transmission resonance can be coupled to electrostatically tunable graphene plasmonic ribbons to create electrostatic modulation of mid-infrared light. Absorption in graphene plasmonic ribbons situated inside metallic slits can efficiently block the coupling channel for resonant transmission, leading to a suppression of transmission. Full-wave simulations predict a transmission modulation of 95.7% via this mechanism. Experimental measurements reveal a modulation efficiency of 28.6% in transmission at 1,397 cm Graphene-plasmon optical modulators with broadly tunable operating frequencies are sought for photonic applications. Here, Kim et al. demonstrate tunable mid-infrared transmission that utilizes resonant absorption in graphene ribbons to modulate the extraordinary optical transmission effect in metallic slit arrays. Significant efforts have been made in the past 5 years to create graphene plasmon-based optical modulators that function from THz to mid-infrared frequencies. These devices have exploited the unique plasmon dispersion relation of graphene, which exhibits optical modes with high-confinement factors12345791011121314151617181920211213231819202526−1, which is 2.67 times higher than that measured for an equivalent GPRs-only structure on the same supporting dielectric structure.In this paper, we report an approach to use graphene plasmonic modes for light modulation in a transmission geometry that satisfies the above conditions—high modulation efficiency, at carrier densities accessible with electrostatic gating. Our modulator design is based on a triple resonant structure, where the plasmonic resonances in GPR are matched to a dielectric substrate Fabry–Perot resonance, and also to the optical resonances in a subwavelength metallic slit array, which is designed to exhibit extraordinary optical transmission (EOT) in the mid-infrared. In full-wave simulations, the proposed structure shows 95.7% modulation efficiency in transmission. We measured a mid-infrared transmission modulation efficiency of 28.6% at 1,397 cmx membranes, as shown in 2829303132x membrane with a DC conducting indium tin oxide /a-Si (60 nm) contact on the bottom side of the membrane, as shown in x thickness. The calculations were performed at λ−1=1,340 cm−1, which is the resonant frequency of 50 nm GPRs for EF=−0.465 eV, and a graphene carrier mobility (μh) of 15,000 cm2 V−1 s−1 was assumed for the graphene sheetEF denotes that the graphene is hole doped. As shown in x thickness (txSiN), and leads to variation in plasmonic absorption by GPRs. In contrast to GPR reflection modulators that achieve maximum absorption at tsub=λ/4nsub . Considering this structure as a Fabry–Perot cavity, the maximal absorption point corresponds to a transmission resonance in the forward direction  for a 2 μm layer of SiNx, as shown in 1314λ−1=1,340 cm−1, which occurs not for maximal doping level but when the plasmonic resonance in GPRs and substrate resonance are matched, as shown To achieve electronically tunable transmission, the substrate must be transparent at the operating frequency, yet also allow for electrostatic gating. In addition, the substrate will support Fabry–Perot-type resonances, which can lead to constructive or destructive interference effects that modify the electric field intensities on the top surface and thus the absorption in the GPRs. To satisfy these constraints, we use a SiNsub refs , 20, thealso see . This efλ−1=1,340 cm−1. After combining the GPRs and the subwavelength metallic slit array, the 0th order transmittance (T) as a function of frequency and EF is shown in EF=−0.465 eV), there is a strong dip in the transmission spectrum at the crossing between two resonant modes, as shown in EF=−0.465 eV), both the metallic surface plasmon modes on the bottom metal surface and inside the subwavelength metallic slits are diminished significantly (ηT) is defined by 1−T/Tmax, where T is transmittance as a function of EF, and Tmax corresponds to the transmission spectrum for the graphene Fermi level position that maximizes the transmitted intensity at the resonant frequency. In this simulation for GPRs, Tmax occurs at EF=−0.310 eV, where there is sufficient doping to prevent graphene inter-band absorption, but insufficient carrier density to support plasmonic modes in the GPRs. μh=15,000 cm2 V−1 s−1), the coupled structure shows a moderate improvement over the bare GPRs, with the theoretical maximum modulation efficiency increasing from ηT=74.5–95.7%. As the graphene carrier mobility is lowered, the overall modulation efficiency decreases in both devices. However, the relative benefits of the GPRs–EOT structure are enhanced with lower graphene carrier mobility, as shown in μh=3,000 cm2 V−1 s−1 the modulation efficiency of the bare GPRs is 50.6%, while the GPRs-EOT structure achieves 85.9% efficiency, and for μh=1,000 cm2 V−1 s−1 these values change to 27.3% and 65.2%, respectively. Thus the GPRs-EOT structure can exhibit large transmission modulation and is more robust against ribbon disorder compared with bare GPR-based devices.In the proposed coupled structure, GPRs are located inside subwavelength metallic slits to modulate the coupling between the surface plasmon modes on the top and bottom metal surfaces. Four parameters dictate the subwavelength metallic slit array design: the metallic material, metal thickness, slit width, and period in transverse direction, as shown in ficantly  because ficantly . To evalEF values far above or far below −0.465 eV, resonant absorption of the GPRs is far away from the transmission resonance of the subwavelength metallic slit array, and the two effects effectively behave independently. As EF approaches −0.465 eV, however, the plasmonic resonance in GPRs creates large deviations in the local dielectric function near the energy of the EOT mode. Specifically, at energies just below or above the plasmonic resonance in GPRs, the effective permittivity is increased or decreased, respectively. This allows the subwavelength metallic slit array to support two distinct modes, even though its geometry selects for only one wave vector. That is, a longer wavelength mode exists which experiences a larger permittivity, and a shorter wavelength mode exists which experiences a smaller permittivity. This creates the splitting, or anti-crossing behaviour between the graphene plasmonic resonant mode and the EOT resonant mode, as observed in Modulation can be interpreted qualitatively using an effective medium theory that considers plasmonic modes in the subwavelength metallic slit array coupled to deeply subwavelength GPRs. There is significant spatial overlap between the plasmonic modes in GPRs and the EOT modes of the subwavelength metallic slit array, such that plasmonic resonances in GPRs can alter the local dielectric environment experienced by EOT modes when the two frequencies approach one another. For Q-factor of the GPRs decreases, and this anti-crossing behavior is lostμh≤1,000 cm2 V−1 s−1), the GPRs–EOT modulator still displays relatively high efficiencies, but the modulation is achieved by absorption in the GPRs, where the optical modes decay too rapidly to interact strongly with the EOT structure. Notably, the modulation enhancement of the GPRs–EOT device over the bare GPRs device becomes more significant in this damping-dominant regime. Therefore we suggest that coupling between the two resonant modes is the dominant mechanism for light modulation for high carrier mobility graphene , while the GPRs with low carrier mobility graphene simply damp the EOT mode.As shown in 35x membranes. First, a transparent back gate electrode composed of 4 nm of ITO and 60 nm of a-Si was sputtered onto the bottom side of the SiNx membrane. Since this back electrode is directly connected to the Si frame of the SiNx membrane, we can apply a gating voltage through the Si frame1017λ−1=1,403 cm−1 with undoped graphene in a mid-infrared transmission measurement. Transmission measurements were performed using a Fourier transform infrared (FTIR) microscope with a polarizer to eliminate the transverse electric component from the incoming light.To demonstrate the modulator performance, we fabricated bare GPRs and GPRs–EOT on 2 μm SiNEF=−0.294 eV for bare GPRs device and EF=−0.353 eV for GPRs–EOT device, corresponding to graphene Fermi levels that exhibit maximum transmittance at the EOT resonance frequency. The resulting gate-dependent modulation efficiencies in transmission are shown in λ−1=1,403 cm−1 where no plasmons exist in the GPRs because of low doping, as shown in EF reaches −0.542 eV, which corresponds to the crossing point between the EOT resonance and the plasmonic resonance in GPRs. λ−1=1,397 cm−1, the GPRs–EOT device shows maximal modulation efficiency of 28.6% with EF=−0.542 eV, while the bare GPRs device has a maximum modulation efficiency of only 10.7% at the same EF.−1) in our measurements. The resulting calculated transmission spectra are shown in −1). Moreover, the angular spread of incident light lowers the maximum theoretical transmittance of the extraordinary optical transmission structure from 42.1 to 9.32%.The experimental measurements shown in 2 V−1 s−1 is in good agreement with the graphene plasmon resonance linewidth, which indicates that damping was a dominant factor in the light modulation for this experiment. Simulations with scaling factors of 0.633 for the bare GPRs device and 0.734 for the GPRs–EOT device are able to fit the measurement results. With these fitting parameters, the expected modulation efficiencies with purely normally incident light are 11.4% for the bare GPRs device and 36.0% for the GPRs–EOT device.In addition to the broad light incidence angular distribution, some imperfections in fabrication could degrade the modulation efficiency, including PMMA residue on graphene, carrier density variation in the graphene created by localized charges, a variation in the width of the GPRs resulting from lithography, edge states of the GPRs induced by the etching process, and some resonators that are electronically isolated. These imperfections result in broadening of the graphene plasmon resonance linewidth and a lower modulation efficiency compared with simulations. To account for the broad incident angular distribution and imperfections in fabrication, we tuned graphene carrier mobility and employed a scaling factor as fitting parameters to explain the linewidth and modulation efficiency see . FittingIn contrast to the decreased transmission seen here due to plasmonic absorption in the GPRs, it has been reported that transmission can increase via absorption, described as absorption-induced transparency373838In summary, GPRs coupled to subwavelength metallic slit array that exhibit extraordinary optical transmission enable strong transmission modulation at mid-infrared frequencies. Light absorption in GPRs efficiently suppresses the extraordinary optical transmission resonance resulting in high modulation efficiency. Simulations indicate a transmission modulation efficiency of 95.7%. This modulation occurs with small changes in the graphene Fermi level position, within ranges that are accessible with electrostatic gating methods. Experimental mid-infrared transmission measurements of a fabricated device demonstrate that the proposed device exhibits 2.67 times higher modulation efficiency than that of a bare GPRs device. The experimental modulation efficiency could be enhanced by use of graphene with higher carrier mobility and by transmission measurements using a parallel rather than a convergent beam illumination configuration since extraordinary optical transmission resonances are quite angle-sensitive. The results illustrate the potential for coupling graphene resonances and conventional noble metal plasmon resonances to achieve transmission-type light modulation, which may be useful in, for example, actively tunable amplitude modulated infrared metasurfaces and real-time hologram systems.x was measured using mid-infrared ellipsometry (t=0.3 nm) having a permittivity of σ is calculated by random phase approximation20The frequency-dependent dielectric function of SiNpsometry . For ITO2 flow rate of 50 sccm and CH4 flow rate of 1 sccm . The PMMA/graphene layer was then rinsed in DI water and transferred onto the substrate. Finally, the PMMA layer was removed with acetone. The quality of the CVD-grown graphene was characterized by Raman spectroscopy, where a negligible D-band was observed—indicating a low defect density—and a 2D to G ratio of ∼2 confirmed the monolayer nature of the graphene sheet  at 1,000 °C for 1 hour with Hccm refs , 45. Aftne sheet . The grane sheet , a simplx membrane  by RF sputtering at room temperature. The RF sputtering powers for the ITO and the a-Si were 48 W and 150 W, respectively. Both GPRs and the subwavelength metallic slit array were patterned by 100 keV electron beam lithography using a PMMA resist. The GPRs were then cut using reactive ion etching with oxygen at 80 W for 15 s, with the patterned PMMA serving as a soft etch mask. The metallic layer was deposited by electron beam evaporation of Ti (3 nm) followed by Au (80 nm).The DC conducting contact (ITO 4 nm/a-Si 60 nm) was deposited on the bottom side of 2 μm SiNThe authors declare that the data supporting the findings of this study are available within the article and its How to cite this article: Kim, S. et al. Electronically tunable extraordinary optical transmission in graphene plasmonic ribbons coupled to subwavelength metallic slit arrays. Nat. Commun. 7:12323 doi: 10.1038/ncomms12323 (2016).Supplementary Figures 1-6, Supplementary Notes 1-3 and Supplementary References.


We proposed a ferroelectric domain controlled graphene based surface plasmon polariton modulator. Ferroelectricity-induced electronic and optical property tuning of graphene by domain in lithium niobate was theoretically investigated considering both interband and intraband contributions of surface conductivity. With the corrected Sellmeier equation of lithium niobate, the propagation of transverse magnetic mode surface plasmon polaritons in an air/graphene/lithium niobate structure was studied when monolayer graphene was tuned by down polarization direction ferroelectric domain with different polarization levels. The length of the ferroelectric domain was optimized to be 90 nm for a wavelength of 5.0 μm with signal extinction per unit 14.7 dB/μm, modulation depth 474.1 dB/μm and figure of merit 32.5. This work may promote the study of highly efficient modulators and other ultra-compact nonvolatile electronic and photonic devices in which two-dimensional materials and ferroelectric materials are combined. The most thriving research field in which graphene is being studied is plasmonics, which describes the interaction between electron oscillations and electromagnetic field348et al. researched a graphene growth and transfer method on periodically poled lithium niobate (PPLN)17p-n junctions in graphene makes ferroelectric materials potential alternatives to gate electrodes. Single-layer molybdenum disulfide  on PPLN also has been studied for the convenience of ferroelectric polarization pattern fabrication without need of lithography21222324As a two-dimensional 2D) electron system with a monolayer of carbon atoms arrayed in a honeycomb lattice, graphene possesses a linear dispersion relation, and the extraordinary properties of the chiral fermions make it a promising platform for electronic and photonic applicationsD electroK points) at the corners of the 2D hexagonal Brillouin zone is depicted in μc, i.e., Fermi energy level EF. With random-phase approximation (RPA) under the self-consistent-field linear response theory, the surface conductivity of graphene can be derived from the Kubo formula consisting of both interband and intraband transitionsThe band structure (or energy dispersion) of monolayer graphene with six Dirac cones , the real part of the surface conductivity decreases with increasing carrier concentration, while the imaginary part increases with carrier concentration and the valley (corresponding to 2ħω/μc) shifts from the MWIR range to the near-infrared (NIR) wavelength range. Generally, the interband contribution dominates from the visible to the NIR range for slightly doped graphene, while the intraband process plays a more significant role in the mid-IR, and far-IR regions, including the terahertz(THz) regions1σV = σS/Δ),where  see 2627, contact see 2627. As showεr = 2.5 is the background relative permittivity. The in-plane component is shown in 27where Δ = 0.34 nm is thickness of monolayer graphene, The anisotropic refractive indices of CLN can be obtained from the three-oscillator Sellmeier equation,c-axis  is perpendicular to the graphene/CLN interface, i.e., the ordinary and extraordinary refractive indices  is negative293536σ) <0, the wavelength shifts from the infrared to visible region when the chemical potential increases, which is calculated using the RPA model, i.e., into the high loss region where the SPP propagation length is quite small, as shown in 93738The transverse magnetic (TM) and transverse electric (TE) modes are supported in conventional materials that are used for optical fiber, waveguide, and other optical devices3132434εair and εCLN are permittivities of air and CLN, respectively. From λ(TM)SPP) is much shorter than that in conventional noble metals, whose λ(TM)SPP is usually hundreds of nanometers even though at visible rangeδ(TM)SPP) SPP)  makes thμc of approximately 0.001 eV, which corresponds to a very small carrier concentration of approximately 1 × 108 cm−2. As the absorption of SPP in a graphene modulator depends strongly on the chemical potential, a depolarized ferroelectric domain of CLN could be used to tune the graphene into a quasi-neutral state7 V/m and the SPP propagates at the interface, which is similar to the conventional insulator/metal/insulator (IMI) structure. Although the energy flow gets into the graphene structure on the depolarized ferroelectric domain, it is almost perfectly absorbed. In addition, the SPP will still propagate into the depolarized region and there is a short extinction length of SPP, which can be observed from Poff and Pon  is also an vital factor for consideration. In this modulator, we observe that when the domain length varies from 250 nm to 30 nm, the modulation depth increases from 235.9 dB/μm to 825.3 dB/μm  is the most common method to grow graphene and then graphene can be transferred onto a ferroelectric surface. Consequently, the surface chemical reconstruction, O-derived defects, and temperature-dependent polarization will change the amount of ferroelectric surface-bound charge394849505152532In addition, other ferroelectric domain structures in different shapes225657In conclusion, in this paper, we have proposed a graphene modulator controlled by the ferroelectric domain in LN. Based on the ferroelectricity-induced tuning of the electronic and optical properties of graphene, we can use domains with different ferroelectric polarization levels to realize propagation control of SPPs in the air/graphene/CLN structure. Furthermore, the domain length can be optimized by considering the signal extinction per unit length, modulation depth, normalized power ratio, and FoM. For the typical wavelength of 5.0 μm in the MWIR range, we obtained an optimized value of 90 nm. In addition, other ferroelectric materials, such as lead zirconate titanate and barium titanate, can be utilized in similar structures fabricated using 2D and ferroelectric materials. This work can promote the study of highly efficient modulators and other nonvolatile electronic and photonic devices for future on-chip applications.The simulation was performed using the commercial finite element method (FEM) software “COMSOL Multiphysics” and we used “Boundary Mode Analysis” module to obtain the TM mode in the air/graphene/CLN structure. The heights of the two-dimensional structure simulation of the three types of materials were 300 nm, 0.34 nm and 300 nm, respectively, and the length was 1200 nm for each material. To obtain accurate results, the graphene structure was vertically meshed into five layers having an interval length of 1 nm. The left boundary was set as the input port and the right one was the output port. Then, the remaining boundaries on the exterior were set to be “Scattering Boundary Condition”. Two “Perfect Matched Layers” (PMLs) were added above and below this structure to eliminate the stray electromagnetic waves.How to cite this article: Wang, H. et al. Graphene Based Surface Plasmon Polariton Modulator Controlled by Ferroelectric Domains in Lithium Niobate. Sci. Rep.5, 18258; doi: 10.1038/srep18258 (2015).


Intriguing reaction mechanism of catalyzed CO oxidation is revealed. These studies suggest a new class of graphene-based catalysts and pave the way for future applications of graphene in solid-state catalysis.Graphene-based solid-state catalysis represents a new direction in applications of graphene and has attracted a lot of interests recently. However, the difficulty in fine control and large-scale production of previously proposed graphene catalysts greatly limits their industrial applications. Here we present a novel way to enhance the catalytic activity of graphene, which is highly efficient yet easy to fabricate and control. By first-principles calculations, we show that when the underlying metal substrate is doped with impurities, the catalytic activity of the supported graphene can be drastically enhanced. Graphene supported on a Fe/Ni(111) surface is chosen as a model catalyst, and the chemical reaction of CO oxidation is used to probe the catalytic activity of graphene. When the underlying Fe/Ni(111) substrate is impurity free, the graphene is catalytically inactive. When a Zn atom is doped into the substrate, the catalytic activity of the supported graphene is greatly enhanced, and the reaction barrier of the catalyzed CO oxidation is reduced to less than 0.5  Graphene has attracted enormous interests due to its unusual electronic properties originating from the unique honey-comb lattice123567891011121314157891112Here through first-principles calculations we report a novel way of activating graphene for catalyzed CO oxidation. We show that when supported on a metal substrate that is doped with an impurity metal atom, graphene can be activated to be an excellent catalyst for the chemical reaction of CO oxidation. The observed high catalytic activity of graphene originates from the significant changes of local chemical properties of graphene right on top of the impurity due to charge transfer. Unlike previously suggested catalysts5678Ni (111) is regarded as an excellent metal substrate for graphene due to the very small lattice mismatch between them. Graphene supported on Ni (111) has been extensively studied both experimentally and theoretically17181919eV per carbon pair. All of these results agree very well with previous studiesThe atomic model of graphene on Fe/Ni (111) is shown in 2 and CO molecules. According to our calculations, both O2 and CO do not bind to graphene on impurity-free Fe/Ni (111), which is actually expected because of the chemical inertness of graphene. We then investigate the effects of substrate doping on properties of supported graphene. Compared with doping in graphene, the substrate doping we considered here has an obvious advantage that it does not ‘damage’ graphene and therefore is easier to control. We choose Zn as a dopant in current study because Zn is cost effective and has been widely used as dopant in various kinds of applications. One Zn atom is doped in the top Fe layer as shown in 2 does bind to graphene at places around the C atom that forms chemical bond with the impurity Zn atom.The chemical reactivity of the supported graphene is tested by the adsorption of O2 on graphene that is supported on Zn doped Fe/Ni (111). The adsorption energy of O2 in this case is calculated to be around 0.42 eV. The O-O bond of the O2 molecule is significantly elongated to 1.48 Å (compared with 1.24 Å in gas phase). We also tested other inequivalent adsorption sites near the Zn impurity  simulation of the system with adsorbed O2 (2) and two C-O bonds (between graphene of O2) as functions of MD steps are shown in 2 on graphene after the impurity doping is stable at room temperature. To understand the interaction between the supported graphene and the O2 molecule, we plot the isosurface of charge redistribution caused by O2 adsorption in * orbital of O2, causing the O-O bond elongated. The adsorption induced change of O2 electronic structures is further illustrated in 2 molecule before and after adsorption is plotted. Before adsorption, the O2 molecule is magnetic and the 2π* orbital of spin-down channel is unoccupied. After adsorption on graphene, three are electrons transferred to the originally empty 2π*, pulling the orbital below the Fermi energy and eliminating the magnetic moment of the molecule. This picture is consistent with the Bader charge analysis that nearly one electron is transferred to O2 after adsorption.We would like to point out here that since the system under study is big, the numerical error in total energy calculations could be big also so that the calculation of the adsorption energy of Oorbed O2  at room 2 implies that the molecule is activated for CO oxidation. Since CO does not bind to graphene, there are no CO poisoning problems and we only need to consider the Eley-Rideal (ER) mechanism of the reaction for which the CO molecule approaches the O2 from vacuum. As aforementioned, the reaction barrier is determined by c-NEB method. In 2→CO2+O and the second step: CO+O→CO2). Reaction barriers of both steps are low (<0.5 eV), indicating high catalytic activity of the supported graphene. In the first step of the reaction, the barrier of 0.47 eV originates from the breaking of the O-O bond and also one C-O bond between the O2 and graphene. To get some rough ideas for how much these two bond breaking processes contribute to the barrier, we performed c-NEB calculations for the breaking of O-O bond on graphene (without the presence of the CO molecule). The results of calculations are shown in eV of energy is needed to break the O-O bond on graphene surface. After one O atom of O2 is taken away in the first step of reaction, the binding between the left-over O atom and the graphene becomes stronger, which can be seen from the fact that in this case the C-O bond is shorten from 1.50 Å to 1.35 Å. The shorter C-O bond length means more energy is needed to break the bond. The reaction barrier of the second step of the reaction is estimated to be 0.40 eV  surface, graphene is chemically inert. We show that after doping an impurity Zn atom in the underlying Fe layer, graphene is activated and become an excellent catalyst for CO oxidation. The reaction barriers of the two-step chemical reaction of CO oxidation catalyzed by graphene supported on Zn doped Fe/Ni (111) are estimated to be low (<0.5 eV). Unlike the previously proposed graphene- supported or covered catalysts, the catalysis center here is the graphene itself. Since CO does not bind to graphene, the CO poisoning is not an issue for the proposed catalyst here. Also, considering the cost-effective materials  we used here, the proposed method of activating graphene may have important applications in the future design of graphene-based catalysis. We believe that the observed substrate-impurity induced high catalytic activity of graphene is not limited to the specific system discussed in this paper. Our studies pave a way for a new class of graphene-based solid-state catalysts.In summary, via eV and 3 × 3 × 3 sampling of the Brillouin zone are used. The convergence criterion for structural relaxations is set to 0.01 eV/Å.The first-principles calculations are performed with spin-polarized density functional theory (DFT) by utilizing Vienna ab-initio Simulation Package (VASP)23How to cite this article: Guo, N. et al. Greatly Enhancing Catalytic Activity of Graphene by Doping the Underlying Metal Substrate. Sci. Rep.5, 12058; doi: 10.1038/srep12058 (2015).


Ubiqitinated and TDP-43 immunoreactive cytoplasmic aggregates are hallmark features of ALS molecular pathology. Since clinically most ALS begins focally and advances contiguously, it is important to characterize their distribution. Our objective was to determine the extent and distribution of TDP-43 immunoreactive aggregates in the lower motor neuron columns as a function of disease onset, and to correlate ubiquitinated with TDP-43 aggregates in the lumbar region. We examined TDP-43 cytoplasmic aggregates at four separate neuraxis levels – hypoglossal nucleus and cervical, thoracic, and lumbar anterior horns – in five controls and 20 sporadic ALS nervous systems from patients whose disease began in various sites, i.e. five bulbar, five arm, five trunk, and five leg onsets. We correlated ubiquitinated to TDP-43 aggregates on adjacent histological sections for the lumbar regions. We found that TDP-43 cytoplasmic aggregates are seen in about 8% of motor neurons but there is marked variability between nervous systems, ranging from 0.4% to 20.6%. The aggregates are uniformly distributed within individual nervous systems. There is no obvious correlation between site of disease onset and rate of spread. Almost all ubiquitinated aggregates correlate to TDP-43 aggregates. Thus, TDP-43 immunoreactive cytoplasmic aggregates have a low overall average frequency that does not correlate with either disease course or clinical spread and is the prime ubiquitinated protein. TARDBP gene that encodes TDP-43 have been identified in 3% of FALS and 1–5% of SALS  neuropathology ,3. TDP-4 of SALS , indicat of SALS ,11.ALS has a focal onset and an orderly contiguous outward spread that creates pathological changes in the nervous system often related to the onset point, at least in terms of neuron counts . Since qn = 5) to obtain semi-quantitative analysis rather than to reflect typical clinical presentations. Accordingly, there were four groups of five based on location of the onset of clinical disease during life – leg, trunk, arm, and bulbar. From each nervous system we studied two sets of paired adjacent, formalin-fixed, paraffin-embedded, 6-μm-thick histological sections from each of the four major neuraxis levels – hypoglossal nucleus and lumbar, thoracic, and cervical anterior horns – for a total of 16 sections from each patient. The two sets of paired adjacent sections were separated by 150–250 μm within each neuraxis level for greater anatomic sampling.We studied 20 sporadic ALS and five controls nervous systems in our ALS tissue repository. ALS nervous systems were from patients who met modified El Escorial criteria for probable or definite sporadic ALS . The nerM Tris buffer (pH 7.6), blocking was performed for 5 min using a 0.1 M Tris buffer/2% fetal bovine serum (FBS). Both the TDP-43 primary antibody  and ubiquitin primary antibody  were diluted in Tris/FBS solution and applied to their respective sections. The slides were then incubated in a 4°C humidified chamber for 16–20 h. Another round of washing with Tris buffer and blocking in the 0.1 M Tris buffer/2% FBS solution was performed prior to secondary antibody application. The secondary antibody  in 1:1000 dilution ratio in Tris/FBS buffer, was then applied to both sections and incubated at room temperature for 1 h. After another round of washing in the Tris buffer and blocking in 0.1 M Tris buffer/2% FBS, a Vectastain Elite ABC kit (VECTOR® PK-6200) was used on the slides. The slides were rinsed and washed in Tris buffer, and liquid DAB substrate kit (Zymed no. 00-2014) was applied to the slides and incubated at room temperature for 5 min and then washed with distilled water. Slides were counterstained with hematoxylin, dehydrated, cleared, and mounted.We performed immunohistochemistry on the paired adjacent sections, one for TDP-43 and one for ubiquitin. The slides were deparaffinized and immersed in Quench endogenous peroxidase for 30 min. Antigen retrieval was performed by applying unmasking solution  to the slides and then placing them in a Pascal pressure cooker. After washing the slides in 0.1 Digital images were taken on a Leica DM 2500 microscope using Spot Advanced software. The procedure was designed to capture the entirety of both anterior horns and hypoglossal nuclei, with maximum resolution. To ensure consistency across the samples, a specific technique was followed during digital microscopy, which varied slightly by neuraxis level due to anatomical differences. A transverse line centered through the central canal divided the anterior and posterior horns. Each anterior horn was then divided into grids that tiled the region of interest and each tile was imaged. The lumbar, cervical, and thoracic sections were all imaged at 400X magnification with grid sizes of 9, 6, and 4 images, respectively; the differences in numbers of images were due to the differences in sizes of the respective anterior horns. The hypoglossal nuclei in the medulla were imaged at 200X magnification and only one picture was needed per motor neuron region. A total of 3554 images at 5.46 pixels/micrometer  resolution was obtained.All images were de-identified and given to five separate observers working in ALS histology for counting. The observers received an identical training tutorial instructing them on the counting methodology. This consisted of counting the total number of neurons and the number of neurons showing TDP-43 abnormalities within each image. TDP-43 abnormalities were divided into three categories based on histological morphology – dense round inclusions, skeins, and overlaps of skeins and inclusions. Quantitative assessments of hypoglossal nuclei in the medulla sections were sometimes difficult due to anatomical variation as a function of the exact rostral-caudal level . After cFor this part of the study, we looked at the lumbar level images. We identified motor neurons with ubiquitin-positive immunostaining and then searched for corresponding TDP-43 immunoreactivity in the paired adjacent section.www.r-project.org).We sought correlation between site of onset and distribution of pathology with a repeated measures ANOVA using between-subject factors. The analysis was carried out in R version 2.9.2 (TDP-43 immunoreactive cytoplasmic aggregates were seen in all 20 ALS nervous systems . ThirteeTDP-43 immunoreactive cytoplasmic abnormalities were seen in both healthy appearing neurons in ALS nervous systems and in smaller atrophic appearing neurons. In absolute counts, there was an average of 5–6 abnormal neurons per neuraxis level with a range of 0–20. Of the 5345 ALS nervous system neurons that were counted, there was a total of 16 skeins, 154 dense round inclusions, and 278 overlaps of skeins and dense round inclusions . A totalAt each neuraxis level, there was marked inter-but not intra-nervous system variability of frequency of TDP-43 aggregates . All fouDefinite 1:1 correlation between ubiquitin and TDP-43 immunoreactive cytoplasmic aggregates was seen in 106 of the 113 motor neurons . While TWe found relatively low frequency and highly variable degrees of TDP-43 immunoreactive cytoplasmic aggregates over the rostral-caudal expanse of the lower motor neuron columns between different SALS nervous systems, but fairly uniform distribution between neuraxis levels within individual nervous systems. Our average of 8.3% of neurons with TDP-43 aggregates is consistent with a recent quantitative study by Giordana et al.  who founThe progression of appearance of TDP-43 abnormalities from solubility to insolubility, from nucleus to cytoplasm, and from puncta to skeins to dense round inclusions is unclear. Sanelli et al.  proposedInterestingly, there seems to be a lack of obvious correlation between the presence or absence of TDP-43 immunoreactive cytoplasmic aggregates and the morphological state of the neuron; pathological aggregation coexists in otherwise healthy appearing neurons and often does not seem to affect or correlate with its morphological status. Also interesting is that TDP-43 immunoreactive cytoplasmic aggregates were seen in small, atrophic neurons as well as large otherwise healthier appearing neurons at the high microscopy magnifications that we employed in the study. By combining high power microscopy and immunohistochemical markers, we were able to identify that small, atrophic cells were indeed motor neurons. Morphometric studies of ALS have failed to show cell shrinkage or left-shift of frequency histograms of neuron size and it is generally believed that neuronal loss is due to apoptosis, a presumed but not proven explanation ,18. HoweAn important observation was that the locations of TDP-43 cytoplasmic aggregates were uniformly distributed throughout the nervous system and did not show any graded correlation with respect to clinical disease onset. Lack of correlation might be partly explained by high background noise: there is marked variation among normal nervous systems; our sample sizes were small; and our patients ranged in age, gender, and disease course as well as site of onset. A correlation might have existed earlier in the disease course but by the time of post mortem observation, the nervous system is so oversaturated by the degeneration that gradations are undetectable. However, it is also possible that the TDP-43 aggregates themselves are of less importance in disease pathogenesis than altered protein function, an idea supported by recent studies by Giordana et al.  and WegoA robust correlation was seen between ubiquitin and TDP-43 immunoreactive cytoplasmic aggregates, consistent with the belief that TDP-43 is the prime ubiquitinated protein. Recent studies by Sanelli et al. , Sumi et