There is an ongoing debate whether an impairment of visual attentional functions constitutes an additional or even an isolated deficit of developmental dyslexia (DD). Especially performance in tasks that require the processing of multiple visual elements in parallel has been reported to be impaired in DD. We review studies that used parameter-based assessment for identifying and quantifying impaired aspect(s) of visual attention that underlie this multi-element processing deficit in DD. These studies used the mathematical framework provided by the “theory of visual attention”  to deriv Visual attentional functions are currently discussed as being related to developmental dyslexia (DD), a disorder in written language acquisition, that cannot be explained by age, visual sensory problems, or inadequate reading instruction . Due to Especially performance in tasks that require the processing of multiple visual elements in parallel seems to be impaired in DD . This muOne influential theoretical concept focusses on general resource limitations. It is suggested that people with DD have a reduced visual attentional span, assessed as the amount of elements that can be reported from a briefly displayed array . CriticaAdditionally or alternatively, impaired attentional selectivity aspects, i.e., changes in spatial attention and/or inefficiency in filtering information could also account for deficient multi-element processing. Indeed, people with DD might not show a normal slight leftward attentional bias. It is known that patients with hemi-neglect following right parietal damage show a rightward attentional bias, which is shown, for example, in rightward deviations in line bisection tasks. Normal participants instead deviate slightly, but reliably toward the left when bisecting lines  and alsoIn this review, we explore the role of visual attention functions for impaired multi-element processing in DD. A critical methodological challenge is to identify and quantify the impaired function(s). Recently, a number of studies have used a parameter-based account of visual attention assessment . In thest0, (ii) by an object’s processing rate, which depends on the relative attentional weight it receives, and (iii) by the capacity of the VSTM store K . TVA provides parameters for characterizing the general processing efficiency of the information processing system , and for characterizing attentional selectivity .Within the TVA framework, the efficiency of visual selection performance of a given participant can be described on the basis of a set of mathematically independent, quantitative measures of attentional components , in which the visual perception threshold , the growth parameter reflects the rate at which the stimuli (objects) can be processed (parameter processing speed C: number of element/s), and the asymptote of the growth function indicates the maximum number of objects that can be represented within VSTM (parameter VSTM storage capacity K). Thus, estimating these parameters of interest here permits to further differentiate if a deficient visual span performance  and efficiency in prioritizing targets over distractors (parameter top–down control α). Parameter wλ is derived in report tasks that involve trials with presentation of stimuli in only one and trials with stimuli in both hemifields. Based on report individual accuracy differences in bilateral vs. unilateral displays, the TVA model produces estimates of attentional weights iw separately for the left (wL) and the right hemifield (wR), and wλ is then computed as wL/(wL + wR). Hence, a value of wλ = 0.5 indicates balanced weighting; values of wλ > 0.5 indicate a leftward and values of wλ < 0.5 a rightward bias. A slight normal “pseudo-neglect” is indicated by a value of wλ > 0.5, because weights for objects to the left of fixation are slightly higher than those for objects to the right. If participants with DD indeed show a reduced or absent pseudoneglect, this would be indicated by significantly higher λw values compared to control participants. Parameter α, representing efficiency of top–down attentional control, is estimated from the performance in partial-report tasks, where participants have to report target objects, only, which are prespecified , whilst ignoring distractors. The parameter indicates the relative attentional weights of distracters compared to targets (wD/wT). Impaired top–down control in participants with DD would be indicated by higher α-values compared to control participants, indicating relatively more attentional weight allocated to distracters. In sum, estimating these selectivity parameters permits the exploration of the potential contribution of lateralized deficits or inefficiency of top–down control to impairments found in DD.Furthermore, TVA-based assessment allows to individually estimate critical selectivity aspects of attention of interest here: spatial laterality . The speed reduction was replicated at group level, with similar degree in children and adults with DD , in line with findings from line bisection studies . Thus, the TVA-based analyses do not support the previously suggested assumption that multi-element processing deficits in DD result from an inability to prioritize relevant over irrelevant information (Concerning the second selectivity aspect assessed, the reviewed studies reviewed above indicate that top–down control is not impaired in DD (ormation .We summarized the results of studies that used TVA-based assessment of visual attentional parameters to examine a potential relevance of deficient visual attentional processing in DD. Taken together, in children and adults with DD  marked rThe 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.


Idiopathic pulmonary fibrosis (IPF) is a progressive disorder marked by relentless fibrosis and damage of the lung architecture. A growing body of evidence now suggests that IPF progresses as a result of aberrant epithelial-fibroblast crosstalk. Injured epithelia are a major source of growth factors such as PDGF which guide resident fibroblasts to injury sites.In this study, we utilized a novel co-culture system to investigate the effect of fibroblast phenotype on their response to epithelial injury. Fibroblasts from normal lungs (NHLF) responded to epithelial injury and populated the wound site forming a fibroblast plug/mechanical barrier which prevented epithelial wound closure. IPF fibroblasts were impaired in their response to epithelial injury. They also expressed reduced PDGFRα compared to NHLFs and were defective towards PDGF-AA mediated directional movement. Neutralization of PDGF-AA and pan-PDGF but not PDGF-BB reduced the injury response of NHLFs thereby preventing the formation of the mechanical barrier and promoting epithelial wound closure. Co-culture of epithelial cells with IPF fibroblasts led to marked increase in the levels of pro-fibrotic growth factors - bFGF and PDGF and significant depletion of anti-fibrotic HGF in the culture medium. Furthermore, IPF fibroblasts but not NHLFs induced a transient increase in mesenchymal marker expression in the wound lining epithelial cells. This was accompanied by increased migration and faster wound closure in co-cultures with IPF fibroblasts.Our data demonstrate that the IPF fibroblasts have an aberrant repair response to epithelial injury. Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease of poorly defined etiology and pathogenesis. Decline in lung function eventually results in respiratory failure and death within 3 to 5 years of diagnosis. Usual interstitial pneumonia (UIP), the histological presentation of IPF, is marked by patches of fibrotic tissue in areas of normal appearing lung . PresencDespite extensive research in understanding disease pathogenesis, IPF remains poorly defined. The hypothesis that is currently most widely accepted proposes that IPF is initiated by repeated micro-injuries to the alveolar epithelium that lead to aberrant and irreversible scarring.During normal repair the injured alveolar epithelium secretes growth factors, cytokines, and chemokines that are essential for epithelial cell migration and differentiation. They also act as chemo-attractants and mitogens for fibroblasts that migrate into the wound site  and are Several studies have reported differences between fibroblasts derived from healthy lung and IPF lung . AlteredCell-cell contact between epithelial cells and fibroblasts appears to be important in signaling cascades and critical for wound repair . In ordeThe extent of re-epithelialization was investigated by wound closure of PKH26-labeled A549 cells in mono-cultures or in co-cultures with NHLFs or IPF fibroblasts over a 96-h time-course Figure . EpithelNext we investigated how epithelial cells and fibroblasts contributed to epithelial repair. NHLFs and IPF fibroblasts responded differentially to epithelial wounding. While NHLFs sensed and migrated in to wound area progressively in response to epithelial injury, IPF fibroblasts appeared to be characterized by a reduced migratory phenotype. By 48 to 96 h, the entire wound area was populated with NHLFs in NHLF-A549 co-cultures leading to the formation of a fibroblast plug. The number of NHLFs populating the epithelial wound area remained significantly higher compared to IPF fibroblast-A549 co-cultures between 48 and 96 h. IPF fibroblasts failed to populate the scratch wound area and these co-cultures exhibited complete re-epithelialization of the wound by the A549 cells by 72 to 96 h Figures . PresencCell-cell communication via direct contact or through soluble factors may explain the differential behavior we observe. To investigate if this was the case, the levels of secreted growth factors were analyzed. There was no significant difference in the level of bFGF, HGF, transforming growth factor beta-1 (TGF-β1), and platelet-derived growth factor-AA (PDGF-AA) in conditioned medium (CM) from either NHLF or IPF fibroblast mono-cultures  cells is an obvious limitation of this model. However, given the difficulties in procuring and maintaining primary human ATII cells in their physiological state Fibroblasts and injured alveolar epithelium can be a rich source of growth factors . InteracInjured alveolar epithelium can be a major source of PDGF isoforms  and our The directional transmigration of NHLFs may be explained by the PDGF-AA gradient created by the injured epithelium. Despite the increased PDGF-AA concentration in IPF co-culture, IPF fibroblasts failed to populate epithelial wounds. In line with this, PDGFRα expression was found to be significantly reduced in all IPF fibroblasts. IPF fibroblasts also presented decreased proliferation to PDGF-AA compared to NHLFs. PDGF-AA only signals via the PDGFRα  and decrbFGF-mediated migration and proliferation and FGFR1 expression were not different between the two fibroblast phenotypes. However, as we found that the concentration of bFGF required to drive fibroblast migration was much higher compared to that needed to induce proliferation, it is possible that at physiological levels increased bFGF in IPF co-cultures could induce proliferation of sub-epithelial IPF fibroblasts without necessarily inducing their transmigration into epithelial wounds.Neutralization of PDGF-AA was sufficient to inhibit the recruitment of NHLFs into epithelial wounds. As IPF fibroblasts already show reduced PDGFRα level and subsequent reduced responsiveness to PDGF-AA, further neutralization of PDGF-AA did not impact their response to epithelial injury. In both A549 mono-cultures and IPF co-cultures PDGF-AA neutralization inhibited while in NHLF co-cultures promoted epithelial wound repair. The latter could be the result of reduced migration of normal fibroblasts preventing the formation of the mechanical fibroblast barrier. However, the former finding suggests that A549 might themselves be responsive to PDGF-AA. Indeed, a previous study has shown presence of PDGFRα on the adenocarcinoma A549 cells, that express receptors which are not normally expressed by alveolar epithelial cells . We alsoEpithelial-fibroblast crosstalk can lead to changes in secreted factor profiles which can alter growth rates/migration of epithelial cells . We did PDGF-AA, bFGF, and TGF-β1 have been implicated in the induction of mesenchymal marker expression in epithelial cells -43 whilein vitro[Induction of mesenchymal markers in epithelial cells has been correlated with more rapid migration and wound closure  and vimein vitro. Epithelin vitro,46. In oIt is well appreciated that cancer and metastasis are underlying complications in IPF ,47. FibrIn order to put the unexpected finding of reduced IPF fibroblast proliferation and migration in our co-culture model in a disease context, one could entertain the following hypothesis. In spite of excessive growth factor release following alveolar epithelial cell injury, non-responsive IPF fibroblasts fail to recruit to the wound site leading to insufficient wound contraction and possibly basement membrane damage. Sub-optimal HGF concentrations impair repair and possibly further compensatory increase in PDGF and bFGF concentration may lead to bFGF mediated proliferation and ECM secretion by synthetic, non-migratory IPF fibroblasts and the recruitment and activation of heterogenous fibroblast populations form other areas of the lung forming an activated myofibroblast pool at the injury site.Our results described here provide new evidence that the altered phenotype of the IPF fibroblast may play an important role in fibrogenesis and offer new insight into some of the potential mechanisms. Using a novel co-culture model of epithelial injury-repair we demonstrate that the IPF fibroblasts have an aberrant response to epithelial injury. Our data show that the abnormal epithelial-fibroblast cross talk results in the generation of a pro-fibrotic environment and impaired alveolar repair.2. Primary fibroblasts were used between passages 4 to 7.A549 cells were obtained from ATCC, primary human lung fibroblasts (NHLFs) from Promocell . IPF fibroblasts were obtained from surgical lung biopsies of patients and cultured as described previously . All celTrans-well inserts 24 mm in size with 8 μm pore size  were coated with collagen-I at on both sides. A total of 100,000 NHLFs or IPF fibroblasts were allowed to attach to inverted trans-wells for 2 to 3 h. Trans-wells were placed in six well plates with 3 mL complete medium in the lower chamber. The upper chamber of each trans-well was incubated with 2 mL of complete medium containing 500,000 A549 cells or medium only (in case of fibroblast only controls) for a further 4 to 5 h. A549 cells were labeled with the fluorescent dye PKH26 (Sigma). The medium in both chambers was replaced with starvation medium containing 0.05% FBS for 16 h. Three 0.4-0.5 mm parallel scratches were made in the A549 cell layer using a sterile pipette tip. Cells were washed and medium was replaced. For PDGF-AA, PDGF-BB, and Pan-PDGF neutralization, medium was supplemented with 0.5 μg/mL of neutralizing antibody or isotype control (R & D systems).Experiments were carried out in triplicates with NHLFs (n = 2-4) and IPF fibroblasts (n = 3-5). CM was stored at -80°C for up to 3 days before a single thaw and further analysis and trans-wells fixed with 5% neutral buffered formalin solution (Sigma) for immune-fluorescent staining.The bottom side of trans-well membranes was wiped clean using moist cotton buds to ensure that only cells on the epithelial side remained. Staining was carried out by incubating with primary antibodies: rabbit anti-e-cadherin ; mouse anti-α-SMA (Sigma); or mouse anti-vimentin  followed by incubation with DAPI/AlexaFluor anti-rabbit488 or anti-mouse647 secondary antibodies (Invitrogen). Stained trans-wells were analyzed using LEICA, TCS SP2 confocal microscope .After seeding fibroblast as described earlier followed by the 16-h starvation, medium in the top chamber was replaced with 2 mL starvation medium containing 20 ng/mL PDGF-AA or 20 ng/mL bFGF (R & D Systems) for directional migration or starvation medium for non-directional baseline migration.Wound closure was monitored in real time by visualizing the fluorescently labeled A549 cells using an inverted microscope (Olympus IX71) equipped with a Nikon camera. Digitized images of three representative areas for every trans-well were captured at 0 h, 24 h, 48 h, 72 h, and 96 h. Images were analyzed using Image-J software .To analyze immuno-fluorescently stained trans-wells, 40× images were taken at 10 random points along a centrally placed scratch. At time points where the field of view could not encompass both wound edges lining the denuded area (0 h and 24 h), images were taken to include one wound edge and the denuded area ensuring similar coverage of scratch for all trans-wells. The number of positive staining cells was counted and averaged for all fields. Data were expressed as the number of cells per High Power Field (HPF). Fibroblasts were identified as elongated cells expressing DAPI/α-SMA or DAPI/vimentin. Epithelial cells (A549) were identified as cells expressing E-cadherin/DAPI. A549 cells transiently expressing mesenchymal markers were identified by the presence of triple staining for e-cadherin/vimentin/DAPI.CM was analyzed for growth factors. bFGF, HGF, and TGFβ-1 were assessed by using single-plex assays from Meso Scale Discovery . Levels of (EGF), IGF-1, PDGF-AA, and PDGF-BB were assessed using Quantikine ELISA . All assays were performed as per manufacturer’s protocol. For ELISA, CM from four biological replicates were pooled and concentrated 10 times using Amicon Centrifugal Filtration Units . Growth factor concentrations shown in this work fell within the dynamic range of the assays used.A549 cells were plated at 5,000 cells per well in a 96-well assay plate, starved for 48 h, and then stimulated for a further 48 h with CM. Proliferation of fibroblasts in response to PDGF-AA (150 ng/mL) and bFGF (2.4 ng/mL) was assessed in a similar way. Proliferation was assessed using the DELFIA Assay System .A549 cells were allowed to reach 100% confluence followed by 24 h starvation. Scratch wounds of 0.3 to 0.4 mm were generated across the wells that were then replenished with either starvation medium or starvation medium containing PDGF-AA (20 ng/mL), bFGF (10 ng/mL). Images were acquired and analyzed as described earlier.Cells were lysed using RIPA buffer . Proteins were quantified using the Pearce BCA assay kit (VWR). After separation on NUPAGE gels, proteins were transferred to a PVDF membrane and probed overnight with primary antibodies against PDGFRα, PDGFRβ, FGFR1, and GAPDH at 4°C followed by appropriate secondary antibody incubation for 1 h at room temperature. All antibodies were purchased from Cell Signaling Technologies .P <0.05 were considered statistically significant.Results were expressed as means ± SEM. Statistical analysis was performed using a one-way ANOVA followed by Dunnett’s post-test. Values of bFGF: Basic fibroblast growth factor; CM: Conditioned medium; ECM: Extracellular matrix; EMT: Epithelial mesenchymal transformation; FBS: Fetal bovine serum; FGFR: Fibroblast growth factor receptor; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; HGF: Hepatocyte growth factor; IL: Interleukin; MCP: Monocyte chemotactic protein; NHLF: Normal human lung fibroblast; PDGF: Platelet-derived growth factor; PDGFR: Platelet-derived growth factor receptor; SMA: Smooth muscle actin; TGF: Transforming growth factor.The authors declare that they have no competing interests.SP carried out experimental work, contributed to experimental design, data analyses and interpretation, and to the writing of the manuscript. CMH contributed to data interpretation and the writing of the manuscript. GJ contributed to experimental design, data analysis and interpretation, and to the writing of the manuscript. All authors read and approved the final manuscript.Immunofluorescence staining to determine fibroblast invasion into epithelial wound. Fibroblasts were identified as α-SMA/DAPI positive cells, A549 were identified as e-cadherin/DAPI positive cells. Co-culture with normal fibroblasts (NHLF-1 and NHLF-2) prevented A549 migration and re-epithelialization and filled the wound area progressively. Fewer IPF fibroblasts  migrated into scratch wound and these co-cultures exhibited enhanced A549 migration and re-epithelialization. Green: e-cadherin, Red: α-SMA, Blue: DAPI.Click here for fileSecreted growth factor profile in CM of fibroblasts in monocultores. (A) bFGF concentration in conditioned medium 48 h or 72 h after serum starvation. (B) HGF concentration 48 h or 72 h. (C) PDGF-AA concentration 72 h. (D) TGF-β1 concentration 48 h and 72 h. Triplicate experiments with multiple donors.Click here for fileThe effect of pan-PDGF neutralization on epithelial wound closure and fibroblast invasion. (A) Immunofluorescence staining of the epithelial side of trans-wells . PDGF neutralization resulted in recruitment of fewer NHLFs into epithelial wounds. (B) pan-PDGF neutralization resulted in a significant increase in wound closure in NHLF co-cultures while it prevented wound closure in A549 mono-culture and A549-IPF co-culture. *P <0.05, **P <0.01 compared to IgG controls.Click here for file


C. elegans to mammals, in the regulation of protein clearance via activation of FOXO transcription factors. In response to proteotoxic insults, TDP-43 redistributes from the nucleus to the cytoplasm, promoting nuclear translocation of FOXOs and relieving an inhibition of FOXO activity in the nucleus. The interaction between TDP-43 and the FOXO pathway in mammalian cells is mediated by their competitive binding to 14-3-3 proteins. Consistent with FOXO-dependent protein quality control, TDP-43 regulates the levels of misfolded proteins. Therefore, TDP-43 mediates stress responses and couples the regulation of RNA metabolism and protein quality control in a FOXO-dependent manner. The results suggest that compromising the function of TDP-43 in regulating protein homeostasis may contribute to the pathogenesis of related neurodegenerative diseases.Protein homeostasis is critical for cell survival and functions during stress and is regulated at both RNA and protein levels. However, how the cell integrates RNA-processing programs with post-translational protein quality control systems is unknown. Transactive response DNA-binding protein (TARDBP/TDP-43) is an RNA-processing protein that is involved in the pathogenesis of major neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we report a conserved role for TDP-43, from  C. elegans to mammals. Under conditions of stress, TDP-43 translocates from the nucleus to the cytoplasm, competes with FOXO transcription factors for binding to 14-3-3 proteins, and releases FOXO for nuclear translocation and activation. These data are consistent with the ability of TDP-43 to regulate protein aggregation. Together the results provide important insight into the role of TDP-43 in stress responses and disease mechanisms. Since chronic stress is associated with neurodegenerative diseases, the TDP-43 switch could be kept in overdrive mode in these disorders, with its capacity to buffer further stress and maintain protein homeostasis being compromised. This mechanism also suggests that other RNA-processing proteins that exhibit similar stress-induced behavior may be coupled to other cellular pathways to provide coordinated reprogramming in stress responses.TDP-43 is linked to pathogenesis of major neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How TDP-43 contributes to the development of these degenerative diseases remains unsolved, and the full range of TDP-43 functions has yet to be established. In the present study, we explored a conversed function of TDP-43 in regulating protein homeostasis from  A defining feature of all living cells is the ability to adapt to stress stimuli. This adaptive response is particularly important for maintaining protein homeostasis, which is critical for cellular functions. The cell employs a variety of protein quality control mechanisms in an effort to maintain the integrity of the proteome, including those regulating protein synthesis and degradation. The regulation of protein synthesis occurs at multiple levels, including transcription C. elegans, TDP-1, was shown to negatively regulate proteotoxicity associated with protein misfolding, suggesting that the nematode protein plays a role in the regulation of protein homeostasis TAR-DNA binding protein (TDP-43) is an RNA-binding protein that has been suggested to play a major role in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) C. elegans DAF-16 and mediate expression of genes involved in longevity, stress resistance, and protein quality control Despite extensive study of the complex pathways responsible for cellular stress responses, how the cell coordinates these different adaptive programs is not yet completely understood. In particular, the exact mechanisms by which RNA processing coordinates with other aspects of stress responses to maintain protein homeostasis remain unclear. Here, we present a mechanistic pathway through which TDP-43 couples RNA processing with active protein quality control during stress. Our results show that TDP-43 regulates the activities of FOXO transcription factors, which are orthologous to C. elegans lacking its sole TDP-43 ortholog, TDP-1, lives longer than wild-type (WT) controls (C. elegans to humans tdp-1(ok803lf);daf-2(e1370lf), exhibited a longer lifespan than did daf-2(e1370lf) alone ;daf-16(mu86lf), completely abolished the longevity effect of tdp-1(ok803lf) alone  alone . There ant alone . These df) alone  [55], inf) alone .C. elegans lacking TDP-1, the mutant also shows enhanced clearance of misfolded proteins tdp-1(ok803lf) mutant reduced the aggregation of TDP-C25-YFP, a misfolded protein reporter expressed in C. elegans neurons (tdp-1(ok803lf);daf-16(mu86lf). The daf-16 mutant reversed the reduction of protein aggregation conferred by the tdp-1 mutant (Correlating with increased lifespan in  neurons . To test1 mutant . This datdp-1(ok803lf) mutants and WT controls. These DAF-16-regulated genes included stress-resistance genes such as the metallothioneins mtl-1 and mtl-2 as well as uncharacterized genes dao-4, dct-8, and dct-17. The results indicated that most of the tested DAF-16 target genes are significantly up-regulated in tdp-1(ok803lf) mutant C. elegans  mutants and WT controls indicates that there are more genes down-regulated than up-regulated in tdp-1(ok803lf) mutants , with the first three showing a high degree of structural and regulatory similarity C. elegans DAF-16 activity by loss of TDP-1, shRNA-mediated knockdown of endogenous TDP-43 in HEK293T cells significantly increased the FOXO transcriptional activity, as indicated by the increase in luciferase activity  (Pearson's correlation coefficient>0.9) (Since NaCl treatment induced the nucleocytoplasmic translocation and granule formation of ent>0.9) . The redent>0.9) . ChangesInterestingly, we found that NaCl-stress-induced patterns of TDP-43 translocation and sequestration varied in a concentration-dependent manner. When HEK293T cells were treated with 0.3 M NaCl, TDP-43 underwent cytoplasmic translocation but was recruited to a previously undescribed, smaller type of granules that did not co-localize with G3BP (Pearson's correlation coefficient <0.4) . We alsoNext we explored whether the translocation of TDP-43 is a reversible process. After a 3-h treatment with sorbitol or NaCl, the stressors were washed off, and the cells were kept in normal medium. After 24 h, TDP-43 was completely translocated back to the nucleus, although G3BP-positive stress granules remained in the cytoplasm . These rSince redistribution of proteins in the cell is often associated with post-translational modifications, we examined how the phosphorylation of TDP-43 correlates with the changes in its localization induced by hypertonic stress. We used a phospo-TDP-43 Ser409/410-specific antibody to detect the phosphorylated TDP-43 in HEK293T cells by immunofluorescence microscopy . In untrTo understand how TDP-43 could regulate the activity of FOXOs during stress responses, we investigated the protein interactions that link these two proteins. We co-transfected HEK293T cells with tagged versions of the TDP-43 and FOXO3a proteins and performed co-immunoprecipitation assays. Immunoprecipitation assays using TDP-43 as bait failed to pull down FOXO3a , indicat2O2 treatment, cytoplasmic translocation of TDP-43 and nuclear translocation of FOXOs both increased, and their exclusive spatial correlation was maintained.Next we investigated whether TDP-43 and FOXOs are linked in their localization changes during stress responses. To easily visualize the localizations of FOXO proteins, we utilized two U2OS cell lines that stably expressed GFP-FOXO1 or GFP-FOXO3a. Interestingly, we observed a strong mutual exclusion in the nucleocytoplasmic compartmentalization of TDP-43 and FOXO proteins . Under n2O2 treatment, which increases the cytoplasmic fraction of TDP-43, the expression of TDP-43 led to a pronounced shift in FOXO3a from the cytoplasm to the nucleus  and a nuclear marker (PARP1) confirmed a clean separation of cytoplasmic and nuclear fractions. The expression of TDP-43 alone, without stress, did not induce any detectable change in the fractionation of FOXO3a. However, with 1 mM H nucleus . These r2 stress . Thus TD2O2 treatment, 14-3-3σ was able to pull down either TDP-43 or FOXO3a when 14-3-3σ was co-transfected with either of the two proteins  or the nuclear export signal (ΔNES) on the interaction between14-3-3 and FOXO proteins using the same competitive co-immunoprecipitation assays described above. The ΔNLS and ΔNES mutations enhanced the relative enrichment of cytoplasmic and nuclear TDP-43, respectively . The comFOXOs have been reported to positively regulate protein quality control systems, including proteasomes and autophagy In the present study, we have described a conserved signaling pathway in which TDP-43 senses stress and regulates protein quality control. This pathway is mediated, at least in part, by the ability of TDP-43 to regulate FOXO transcription factors. Therefore, TDP-43, a known RNA binding protein, represents a link between protein quality control and RNA metabolism, indicating a novel layer of regulation of protein homeostasis imparted by RNA-processing proteins. We propose that TDP-43 mediates stress responses designed to maintain protein homeostasis by coordinating the attenuation of protein synthesis and the selective enhancement of protein quality control systems. First, the loss of TDP-43 from the nucleus and its localization to stress granules in response to cellular stress constitute an adaptive response to keep target mRNAs from active translation C. elegans  strain, a transgene DNA construct was generated by subcloning TDP-1 cDNA with a C-terminal YFP tag into a modified plasmid, pPD30_38 , with the promoter replaced with that of the snb-1 gene, as previous described snb-1::TDP-C25-YFP(iwIs22) strain was reported previously All C. elegans and mutant strains RB929 [tdp-1(ok803)], CF1038 [daf-16(mu86)], CB1370 [daf-2(e1370)], IW417 , and IW177  were cultured under standard conditions at 20°C. Newly laid embryos were synchronized within 2 h and transferred to fresh NGM plates, with 50 embryos per plate. These animals were transferred to new plates every day until they reached the post-reproductive stage and were allowed to age under normal culture conditions. Animals were checked daily and considered dead if they showed no response to probing with a platinum pick. The animals that crawled out of the plate, had vulval burst, or died with internally hatched larvae or “bags of worms” were censored. The day when embryos were synchronized was defined as the first day for lifespan analysis. The lifespan data were analyzed using Prism 5 software.The WT N2 C. elegans were cultured at 20°C until they grow to L4 larval stage. L4 larvae were individually transferred to new plates and cultured at 25°C. Every 24 hours, these animals were transferred to new plates at 25°C until they stopped producing eggs. After transferring, the eggs laid on the plates were counted. For locomotion measurement, L4 larvae grown at 20°C were subject to a thrashing assay. The animals were transferred into M9 buffer  and allowed to adapt to the buffer for 1 min. Then the rate of body bending or thrashes for the animals was measured, with a thrash being counted when both the head and the tail bend over 45 degrees.C. elegans, animals were harvested and total RNA was isolated using a phenol-chloroform extraction with TRIzol reagent (Life Technologies), followed by purification with an RNeasy mini kit (Qiagen). A two-step RT-PCR was employed to assess relative changes in transgenic transcripts using an iScript cDNA Synthesis Kit and an IQ SYBR Green kit (Bio-Rad). Fluorescence was measured on a real-time PCR cycler (Bio-Rad), and CT values were analyzed based on standard curves. The worm gdh-1 gene was used as a control. The sequences of all the primers used are listed in the To quantify the expression of specific genes in Sal site and Gateway vector (pDonor-221) are listed in the HEK293T cells were grown in Dulbecco's modified Eagle's medium supplemented with antibiotics and 10% fetal bovine serum. Transient transfection of HEK293T was carried out with Lipofectamine 2000 according to the manufacturer's instructions (Life Technologies). The primers for subcloning TDP-43 cDNA into pRK5-myc vector at BamHI/HindIII restriction sites. The sequence of shRNA oligonucleotides (i), (ii), and (iii) as well as a control shRNA-RFP-C-RS are listed in the 5 per well, then transfected with the shRNA constructs after 24 h. After 24 h, puromycin was applied at 3 µg/ml to select for positively transfected cells, and cells were harvested at 72 h post-transfection.To knock down the expression of TDP-43, we generated shRNA constructs targeting different regions of the TDP-43 transcript and containing a puromycin resistance gene. The TDP-43 shRNA constructs were cloned by inserting small hairpin oligonucleotides matching TDP-43 mRNA sequences into the pRFP-C-RS plasmid (Origene) using Renilla luciferase, was used as an internal control. Cells were plated in 24-well plates at a density of 1×105 cells per well, and after 24 h, cells were transfected with expression plasmids of FOXOs, pSV40-Renilla, pGL3-FHRE-luciferase, or TDP-43. At 48 h, luciferase activities were measured by using the Dual-Luciferase Reporter Assay System (Promega) on a Synergy H1 luminometer (Bio-Tek). The experimental firefly luciferase activity was normalized to the control Renilla luciferase activity to reflect the FOXO activities.A reporter construct, pGL3-FHRE-Luc was originally from M. Greenberg (Addgene Plasmid 1789), which expresses the firefly luciferase driven by a promoter containing three copies of forkhead responsive elements, was employed to measure FOXO transcription activity In addition to HEK293T cells, two U2OS stable cells lines expressing FOXO1 or FOXO3a were used (Thermofisher). Cells were plated on coverslips pre-treated with polyethylenimine , in 6-well plates. Cells were stressed with 0.4 M sorbitol  for 1 h or 0.2 M (or 0.3 M) NaCl for 3 h. Coverslips were washed twice with 1× PBS and then fixed with 4% paraformaldehyde (PFA) for 10 min at RT. After the PFA was washed, the cells were permeabilized with 0.1% Triton X-100 in 1× PBS for 10 min. After treatment with blocking buffer containing 2% BSA in PBS with 0.1% Triton X-100 for 30 min, the coverslips were incubated with primary antibody at 4°C overnight. The primary antibodies were diluted in blocking buffer; they were: polyclonal anti-TDP-43, 1∶200 ; monoclonal anti-G3BP, 1∶200 ; and monoclonal anti-phospho TDP-43 (Ser409/410), 1∶200 . The coverslips were then incubated with secondary antibody for 1 h at RT: goat anti-rabbit Alexa Fluor 488, 1∶1000 ; goat anti-mouse Alexa Fluor 555, 1∶1000 ; or goat anti-mouse Alexa Fluor 555, 1∶1000 . The coverslips were mounted in buffer with 2.5% DABCO, 100 mM Tris-HCl (pH 8.8), 50% glycerol, and 0.2 µg/ml DAPI. Images were collected with a Zeiss AxioObserver Z1 with an Apotome imaging system.Cells were lysed in buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.4 mM EDTA (pH 8.0), 1% NP-40, 0.05% sodium deoxycholate, and complete protease inhibitor cocktail (11836153001). Cell lysates were incubated with anti-HA antibody  overnight at 4°C before being centrifuged at 10,000× g for 10 min. Supernatant was transferred to clean tubes and incubated with protein G-Sepharose beads  for 1 h. After five washes with lysis buffer, the beads were resuspended in SDS sample buffer and boiled for 5 min before the eluted materials were subjected to standard western blot analysis: Protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked with 5% milk in 1× phosphate-buffered saline with 0.1% Tween 20 (PBST) and incubated with the following primary antibodies: anti-c-myc- horseradish peroxidase, 1∶5000 ; monoclonal anti-c-myc, clone 9E10, 1∶3000 ; monoclonal anti-Flag M2, clone M2, 1∶5000 ; polyclonal anti-GAPDH,1∶30,000 ; monoclonal anti-V5, 1∶3000 ; polyclonal anti-TARDBP, 1∶3000 , polyclonal anti-PARP, 1∶1000 , polyclonal anti-Caspase 3, 1∶1000 , and polyclonal anti-Cu/Zn SOD, 1∶3000 . The following secondary antibodies were used: goat anti-rabbit IgG (H+L)-HRP conjugate, 1∶3000 ; goat anti-mouse IgG (H+L)-HRP conjugate, 1∶3000 ; goat anti-rabbit IgG IRDye, 1∶40,000 ; and donkey anti-mouse IgG, 1∶40,000 . After incubating with secondary antibodies, the membranes were developed on films or the Odyssey image system (Li-COR).2, 0.1 mM EDTA, 0.5 mM DTT, 0.4% NP-40, 0.5 mM PMSF, and complete protease inhibitor cocktail, then incubated on ice for 5 min. Cell lysates were then centrifuged at 600× g at 4°C for 3 min. Supernatants were transferred to clean tubes and saved as the “cytoplasmic fraction.” The pellets were washed twice with cytoplasmic extraction buffer and centrifuged at 600× g at 4°C for 3 min. After washing, the pellets were resuspended in a nuclear extraction buffer containing 20 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 25% glycerol, 0.5 mM PMSF, 0.2 mM EDTA, 0.5 mM DTT, and complete protease inhibitor cocktail and vortexed at room temperature for 1 min. The resuspended samples were incubated on ice for 10 min, then vortexed for 1 min and centrifuged at 16,000× g for 10 min. The supernatants were transferred to clean tubes and saved as the “nuclear fraction.”To isolate cytoplasmic and nuclear fractions, cultured mammalian cells were harvested in 1× PBS, centrifuged at 200× g for 1 min, and washed twice with 1× PBS. The cell pellets were resuspended in a cytoplasmic extraction buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgClC. elegans were extracted in 200 µl of buffer containing 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA (pH 8.0), 0.5% NP-40, 50 µM iodoacetamine, and protease inhibitor  by using a Bioruptor ultrasonicator at 4°C for 5 min. The lysates were then transferred to an Airfuge ultracentrifuge (Beckman Coulter) and centrifuged at 25 psi  for 5 min.A biochemical assay was used to detect insoluble aggregated proteins according to a previously described protocol, with some modifications The supernatant was transferred to clean tubes and saved as the “soluble” fraction. The remaining pellet was again resuspended in extraction buffer, then sonicated for 5 min. This resuspended pellet was applied to the Airfuge and centrifuged at 25 psi for 5 min. The remaining pellet was transferred to 100 µl of resuspension buffer containing 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA (pH 8.0), 0.5% NP-40, 0.5% deoxycholic acid, and 2% SDS, and sonicated for 5 min. This fraction was considered the “insoluble” protein aggregate fraction.p values for comparing Kaplan-Merier survival curves between groups were calculated by the Log-rank test. p values of qPCR, egg-laying, locomotion and Luciferase assay data were calculated with the Student's t-test.Image J software was used to analyze the colocalization between TDP-43/pTDP-43 and G3BP, and the colocalization was measured by Pearson's correlative coefficient. Figure S1tdp-1(ok803lf), daf-2(e1370lf) and the tdp-1;daf-2 double mutant were subjected to assays quantifying egg-laying  (A) and locomotion  (B). Error bars represent SEM.Loss of TDP-1 improves egg-laying and locomotion defects in the absence of DAF-2. The WT control, (TIF)Click here for additional data file.Figure S2tdp-1-null C. elegans. (A) Loss of tdp-1 in C. elegans leads to more down-regulated genes than up-regulated genes. Transcriptional profile analysis of mutant C. elegans lacking tdp-1 as compared to wild-type N2 animals. There were 485 genes down-regulated (blue) and 227 genes up-regulated (red) when the threshold of -fold change (FC) was set at 1.5. (B) Quantitative RT-PCR validation of representative genes analyzed by the microarray assay (black dots) and the DAF-16 transcriptional targets (red dots).DAF-16 transcriptional targets are specifically up-regulated in (TIF)Click here for additional data file.Figure S3Renilla luciferase control. Cell lysates were subjected to dual luciferase assays, and the ratio of firefly to Renilla luciferase activity was used to indicate the FOXO transcriptional activity. n>3, p>0.05.The FHRE-Luc reporter is not affected by the transfection of TDP-43 alone. HEK293T Cells were co-transfected with Myc-TDP-43 or control Myc vector, the FHRE-Luc reporter, and the (TIF)Click here for additional data file.Figure S4The dynamic change in the TDP-43 recruitment to stress granules induced by 0.2M NaCl treatment. HEK293T cells were treated with 0.2 M NaCl and analyzed at different time points by immunofluorescence microscopy for distribution of the endogenous TDP-43 (red) and the stress granule marker G3BP (green). With 30 min of the treatment, TPD-43 is predominantly nuclear. At 1 h, TDP-43 gains a slight cytoplasmic distribution in a diffuse and small punctate pattern, while G3BP is localized to well-demarcated stress granules. At 2 h, cytoplasmic TDP-43 forms larger puncta and co-localizes with G3BP in stress granules. Scale bar: 5 µM.(TIF)Click here for additional data file.Figure S5The dynamic change in the TDP-43 recruitment to cytoplasmic granules induced by 0.3M NaCl treatment. HEK293T cells were treated with 0.2M NaCl and analyzed at different time points by immunofluorescence microscopy for distribution of the endogenous TDP-43 (red) and the stress granule marker G3BP (green). At 1 h, TDP-43 has a somewhat diffuse and minor punctate distribution in the cytoplasm, and fewer and smaller G3BP-positive stress granules are present when compared to the 0.2M NaCl treatment. At 2 h, the TDP-43 puncta remain small and do not co-localize with G3BP-positive stress granules. Scale bar: 5 µM.(TIF)Click here for additional data file.Figure S6The hypotonic stress-induced translocation of TDP-43 from the nucleus to the cytoplasm is reversible. HEK293T cells were treated with 0.4 M sorbitol, 0.2 M NaCl, or 0.3 M NaCl. After 3 h of treatment, the stressors were removed, and the cells were cultured for another 24 h. Immunofluorescent staining was performed for endogenous TDP-43 (red) and the stress granule marker G3BP (green). TDP-43 is translocated back to nucleus, although G3BP-positive stress granules remain. Scale bar: 5 µM.(TIF)Click here for additional data file.Figure S7TDP-43 does not physically interact with FOXO proteins. HEK293T cells were co-transfected with Myc-TDP-43 and either WT Flag-FOXO3a (A) or Flag-FOXO3aAAA, which is a constitutively active form of FOXO3a (B). Co-immunoprecipitation was performed using anti-Myc (TDP-43) antibody for the pull-down, and the precipitates were analyzed by western blotting using anti-Myc-TDP-43 and anti-Flag-FOXO antibodies. No interactions between TDP-43 and the FOXO proteins were detected.(TIF)Click here for additional data file.Figure S8Differential cellular distribution of TDP-43 WT and mutants lacking the nuclear localization signal (ΔNLS) or the nuclear export signal (ΔNES). (A) Schematic diagrams of TDP-43 proteins with ΔNLS or ΔNES mutations. The other domains of TDP-43, including the RNA recognition domains (RRM) and the glycine-rich domain, are shown. (B) TDP-43 ΔNLS or ΔNES mutant proteins have enriched cytoplasmic or nuclear localization, respectively. HEK293T cells were transfected with the V5-TDP-43 WT, ΔNLS, or ΔNES construct. Fractionation assays were performed as described in (TIF)Click here for additional data file.Table S1Primers and oligonucleotides in materials and methods.(DOCX)Click here for additional data file.


The adsorption energy between NH4 and graphene increased stage by stage which showed NH4 was a strong donor. The calculation suggested that H2O molecule could help a quick desorption of NH4 from graphene by converting NH4 to NH3 or (NH3)n(H2O)m groups, which was consistent with the experimental results. This study demonstrates that the ammonia gas produced by a bubble system of ammonia water is mainly ammonium groups of NH3 and NH4, and the NH4 moleculars are ideal candidates for the molecular doping of graphene while the interaction between graphene and the NH3 moleculars is weak.The gas sensor based on pristine graphene with conductance type was studied theoretically and experimentally. The time response of conductance measurements showed a quickly and largely increased conductivity when the sensor was exposed to ammonia gas produced by a bubble system of ammonia water. However, the desorption process in vacuum took more than 1 h which indicated that there was a larger number of transferred carriers and a strong adsorption force between ammonia and graphene. The desorption time could be greatly shortened down to about 2 min by adding the flow of water-vapor-enriched air at the beginning of the recovery stage which had been confirmed as a rapid and high-efficiency desorption process. Moreover, the optimum geometries, adsorption energies, and the charge transfer number of the composite systems were studied with first-principle calculations. However, the theoretical results showed that the adsorption energy between NH Furthermore, ammonia detection has good prospects in medicine diagnose. For instance, measurements of exhaled ammonia may differentiate between viral and bacterial infections in lung diseases to justify the use of antibiotics , and ammifficult . Hugo E nterface . Lakshmagraphene . Howevergraphene .4 and graphene is much stronger than that of ammonia groups NH3. We take the view that hydrogen atom plays an important role in the adsorption between NH3 and graphene, and we find that H2O helps NH4 to take off one hydrogen atom and achieves a rapid desorption from graphene sheets.These explanations revealed the truth from some degree, but some of these explanations are still not theoretically grounded or lack experimental verifications. Therefore, it is hard to treat them as a fully complete conclusion. In this work, we studied the adsorption and desorption of ammonia groups on graphene theoretically and experimentally. Our study demonstrates that the interaction between ammonia groups NH2 (300 nm) layer working as the sensors’ bottom gate through a dry transfer procedure [N) in a graphene film was determined with micro-Raman scattering using the shape of the 2D Raman band at ~2700 cm−1 and the intensity of the Raman-active G-band scattering at ~1585 cm−1 by a LabRAM XploRA with 532-nm excitation and low power which results was confirmed as a single-layer graphene. Then, the Cr(5 nm)/Au(100 nm) electrodes, used as the source and the drain electrodes of graphene-FET with channel width of 10 μm . A sensing test cycle typically consists of three consecutive steps that include exposures of the device to (i) vacuum environment to record a base value of the sensor conductance, (ii) target gas to register a sensing signal, and (iii) vacuum environment for the sensor recovery. All the electrical measurements were carried out at room temperature. Five different sensors for the same process were measured which showed similar behaviors except the magnitude of conductance (the difference is less than 10 %) and each measurement was repeated for at least five times.A pristine graphene was first prepared by mechanical exfoliation (repeated peeling) of small mesas of highly oriented pyrolytic graphite, which was then transferred to highly p++ doped silicon wafer with a thermally formed SiOrocedure , 11. TheTheoretically, the density functional theory (DFT) calculations were performed with CASTEP ; the gen−1) was observed in graphene indicating that the quality of the graphene was maintained during the transfer, and the ratio of intensity G peak and 2D peaks was about 0.5, which was a typical value of single-layer graphene. The basic electrical properties of bottom-gated bilayer graphene FETs were first studied in the atmospheric environment. We found that the Dirac peak was about 60 V (VD = 60 V) . Prolonged vacuum treatment was capable of removing the adsorbed air molecules strongly. The total amount of effective charged impurities can be simply estimated by the amount of Dirac voltage shift, VD, using the following expression [The pristine graphene was prepared by mechanical exfoliation, and the Raman measurement was applied to study the layer numbers of graphene in room temperature under the atmosphere. In Fig. pression :1\documeCgs is the capacitance of the SiO2 between the graphene and the silicon substrate  and e is the unit charge. From Fig. 12/cm−2 in the air, while that is about 0.43 × 1012/cm−2 in the vacuum, with the corresponding Dirac voltage point decreased from 60 to 6 V. Moreover, for each trace in Fig. Kh and Ke) from which one can extract the mobilities for holes (electrons) according to [where rding to :2\documeC is a constant related to electrode geometry structure and dielectric material of the device, K is the slope of linear region , and μ is the mobility (μh for hole and μe for electron). From the above expression, the mobilities of the hole and electron were almost same and which value was about 2000 cm2(V•S)−1.where 2O is an acceptor (hole doping) molecule [2O was turned off and evacuated within about 5 s, the conductance quickly decreased toward its baseline in 1 min, which illustrates that the force between graphene and water molecules is relatively weak [The response of graphene gas sensor to high-purity argon (higher than 99.99 %) was first measured and there was no obvious change in conductance. Then, the time response of the sensor’s conductance to a mixture gas (the relative humidity is ~85 %) by diverting dry argon air through a water bubbler bottle was measured, and the results were shown in Fig. molecule . When thThe conductance of the gas sensor increased ~22.5 % when exposed to 5 ppm ammonia, shown in Fig. 3 molecules should physisorb to the film without significant change of the band structure, and only a slight charge transfer of 0.03 − 0.04 e per adsorbed NH3 molecule was predicted to occur [Ea) means stronger binding between molecule and graphene. The Ea between the gas molecule and graphene is defined asTheoretical calculations on isolated defect-free graphene predicted that NHto occur . It seemto occur , 19. In to occur . The conEsystem is the total energy of the optimized equilibrium configuration of the graphene and the adatom; Egraphene is the total energy of the isolated graphene; Emolecule is the total energy of the corresponding adsorption molecules in its ground state; n is the number of gas molecules in the system.where 2O molecule starting from the O atom, the H-O bonds pointing parallel to the graphene surface (P) [2O-graphene group for different H2O positions with sheet-adsorbate distances of 3.6 Å  as well as that in H2O-graphene group, and the charge transfer, however, was solely determined by the orientation of the NH3 molecule. There was a slight charge transfer of 0.02 e from the molecule to the graphene surface in the U orientation and a extremely slight charge transfer (0.01 e) from graphene to NH3 molecule in the D orientation, and the bottom atom of the physisorbed molecule was located 3.4–3.6 Å  , and two6 Å Fig. b, and onsmall 31–5 meV as 3 molecules on graphene [3 are not good candidates for effective molecular doping of graphene. Obviously, there are other chemical groups that physisorb more strongly on graphene sheets and shift the Fermi energy inside the valence or conduction bands. Bradley et al. found that vacuum-degassed SWNT-FETs were insensitive to NH3; they suggested that to detect NH3 gas in the FET response, one should dissolve the NH3 in a H2O monolayer which forms on the nanotube FETs under ambient lab conditions. They proposed that the NH3 molecule in this environment could become a cation with the charge compensated from the SWNTs, i.e., acting as an n-dopant [3 molecules and hydrogen adatoms to generate chemical species that have stronger interactions with the graphene layer. Specifically, an isolated H impurity on graphene can be captured by an NH3 molecule in an exothermic reaction that releases 1.00 eV of energy [These results were in agreement with those already reported in previous studies on the binding energies of physisorbed NHgraphene , 6. All n-dopant . In factn-dopant , 22, plaf energy .4 species on a defect-free graphene layer was calculated in different graphene positions and a special orientation [4 hydrogen atoms pointing toward the graphene sheets, while the other two hydrogen species are on the other side of the ammonium group, see Fig. 4 molecule were adsorbed on graphene in a stronger manner, and the interaction between dangling bonds of graphene and NH4 was very strong and partial electron transfer occurs from graphene. The adsorption energy was 528–644 meV which was much larger than that of NH3 and H2O adsorbed in graphene sheets, and a huge charge transfer (0.19–0.54 e) from NH4 to graphene proved that physisorbed NH4 acted as a stronger donor for the basal plane of graphene, and the bottom atom of the NH4 was located 2.19 Å  for all of the systems are calculated in Fig. 3 molecules and graphene from first-principle calculations. The electronic structure and electrical conductivity of the intrinsic graphene have limited changes due to the adsorption of NH3 molecules. Moreover, this study found that the ammonium (NH4) had strong interactions with graphene, forming a strong bond that introduces a large amount of shallow donor states into the system. When adding water molecules into the desorption process, the whole desorption process was greatly accelerated. We considered that ammonium molecules and water molecules generated the ammonia-water cluster and found that the cluster has weak adsorption with graphene sheets by calculation. The results are in accordance with the experiment. In a word, this study demonstrated that the ammonia gas produced by a bubble system of ammonia water were mainly molecular groups of NH3 and NH4, and the NH4 moleculars are ideal candidates for the molecular doping of graphene. However, the NH3 moleculars have weak interaction with graphene.In conclusion, the adsorptions of ammonia on graphene were investigated with experiment and first-principle calculation. The study demonstrates that the remarkable variation of the electrical conductivity is induced by the ammonia adsorption, and that the graphene gas sensor possesses an excellent characteristic of high sensitivity for ammonia gas detection. It was found experimentally that ammonia molecules produced by a bubble system of ammonia water were strongly adsorbed onto the graphene sheets; however, ammonia molecules were only weakly adsorbed onto the intrinsic graphene with small binding energy value and large distance between the NH


By merging bottom-up and top-down strategies we tailor graphene's electronic properties within nanometer accuracy, which opens up the possibility to design optical and plasmonic circuitries at will. In a first step, graphene electronic properties are macroscopically modified exploiting the periodic potential generated by the self assembly of metal cluster superlattices on a graphene/Ir(111) surface. We then demonstrate that individual metal clusters can be selectively removed by a STM tip with perfect reproducibility and that the structures so created are stable even at room temperature. This enables one to nanopattern circuits down to the 2.5 nm only limited by the periodicity of the Moiré-pattern, i.e., by the distance between neighbouring clusters, and different electronic and optical properties should prevail in the covered and uncovered regions. The method can be carried out on micro-meter-sized regions with clusters of different materials permitting to tune the strength of the periodic potential. Two main routes are usually followed to modify graphene's electronic and optical properties. On the one hand, bottom up approaches have proven to be efficient to change the overall electronic structure of graphene, enabling for example, the gap opening at the Fermi energy124578911121315131719π-bands with the characteristic linear dispersion and Fermi velocity of free standing graphene are only modified by the appearance of a small gap less than 100 meV227Let us first outline the bottom-up approach12452112325Additionally, this Moiré pattern formed by the graphene monolayer and the Ir(111) substrate can be used as a template for networks of monodisperse clusters of transition metals25The experimental bottom-up procedure is the following. We first grow a graphene monolayer on an Ir(111) substrate by chemical vapor deposition (CVD) of ethylene in UHV environments with the Ir(111) substrate held at 1050°C. Then, by evaporationg W or Ir from high purity filaments, we subsequently cover it with a hexagonal array of metal clusters with 2.5 nm periodicity (see methods for details on the sample preparation).‘graphene’ regions, i.e., ones covered with clusters and uncovered ones, with supposedly different electronic properties. It is noteworthy that our method can be used with clusters of different metallic elements, see We will now turn to the above mentioned top-down approach where STM appears as an ideal technique to tackle local manipulation on such samples due to its ability to modify and pattern 2D samples with ultimate resolution3031The procedure we have developed to engineer graphene nanostuctures consists in selectively removing single metallic clusters on top of graphene by gently approaching the STM tip towards them, as schematized in During the patterning process, the tip resolution is very robust and we usually observe almost no changes in our resolution after each cluster removal see . It furt25The possibility of picking up or manipulating individual clusters formed on the Moiré-pattern was previously mentioned25To this end, we have performed a careful study of the probability of removing a cluster as a function of both the tip-sample approaching distance and the bias voltage applied to the sample during the whole process, see 0G = 2e2/h, e: electron charge; h: Planck's constant), indicating that the extraction of a metal cluster involves the formation of an atomic size contact. Similar G ≈ 0G values have been reported for contacts between an atomically sharp Au tip and graphene regions strongly bonded with a metal substrate2 to sp3, in a similar way as reported for graphene regions underneath metal clusters on the graphene/Ir(111) systemTo get more insight into the physical processes involved in the cluster extraction, we recorded the current during the vertical displacement of the STM tip (I-Z curves), see for example inset of 35363736372 STM image of a graphene region uniformly covered by metallic clusters is shown in Let us now address several key points to infer the actual potential of our method to architecture functional graphene nanostructures, in particular, size limits, stability and quality. The range of applicability is obviously limited by the size of the nanostructures that can be created. We can build nanostructures from the 2.5 nm limit given by the Moiré pattern distance to the few micrometers one which is given by the typical STM scanning range; the possibility of growing single Moiré patterns domains extends over several micrometersFinally, we want to comment on the state of the graphene layer after the removal of the clusters. We aim to use pristine graphene on Ir(111) domains as one of our building blocks, thus, we need our cluster extraction method to produce perfectly clean graphene regions. To this end, we show in The full potential and applicability of our nanostructures is realized if the covered and uncovered regions display different electronic properties which has only been demonstrated for the homogeneous systemsπ→π* transitions between the valence and conduction band, so-called interband plasmonsE ≤ 0.4 eV should be forbidden in the covered, but allowed in the uncovered regions and can thus be localized to small graphene areas by removing the upper Ir-clusters, see L posses normal modes corresponding to the wave number q = nπ/L of the interband plasmon (q ≈ 0.03(0.05)Å−1 and energy E ≈ 0.25(0.375) eVL ≈ 10.5(6.2) nm for n = 1 or L ≈ 21(12.5) nm for n = 2. These length scales are well within the reach of our technique  system, plasmonic excitations have been measured by electron energy loss spectroscopyR can be obtained, see R ≤ 7 nm. In this regime, the uncovered area could resemble a quantum bit with qubit states “zero exciton” or “one exciton”. The excitonic states can further arbitrarily be connected by conventional wave function overlap or via Förster energy transfer which is mediated by the Coulomb interaction between the excitonic states, see A second and exciting new feature is given by the possibility to confine charged carriers, i.e., electrons as well as holes, within arbitrary geometrical regions due to locally modifying the electronic gap. One could hence design graphene quantum dots or nanowires of arbitrary size and form limited only by the cluster size of 2.5 nm which has to be contrasted with graphene nanostructures obtained by electron beam lithography and subsequent etching which have typical dimensions L = 20–100 nm. Using the effective-mass-approximation and thus the standard Dirac Hamiltonian with a variable mass profile, the discrete spectrum of a circular quantum dot as function of the radius sp2- to sp3-bonding on the covered regionsThe feasibility of the above proposals crucially depends on the impact that the Iridium substrate and the clusters on top have on the electronic properties of the graphene layer. Even though from ARPES experiments the band structure hardly seems to be affected beyond the gap of Δ ≈ 0.1 eV and ≈ 0.4 eV, respectively, the graphene Dirac cone has been reported to hybridize near the Fermi level with the S1 surface state of Ir(111)To conclude, we have presented a perfectly reproducible nanopatterning technique for graphene that combines bottom-up with top-down approaches. The precision is related to the periodicity of the Moiré-pattern that is formed by the graphene layer with the underlying substrate. Presupposing locally distinct electronic gaps in the covered and uncovered regions, new devices could be tailored with nano precision and we propose a novel platform for plasmonics relying on inter- rather than on intraband transitions. Also single graphene quantum dots/wires could be designed at will and arranged to arbitrary circuitries. Determining the optical gap and relaxation properties of mass-confined Dirac electrons via optical near-field scanning spectroscopy, emission spectroscopy or even transmission spectroscopy by chemically reduce the thickness of the sample would provide new insight on the role of the Ir(111) substrate on the excitonic decay rate. Finally, we note that the electronic spectrum drastically changes in the presence of a magnetic field due to the appearance of the zeroth Landau level not present in conventional semiconductor quantum dots which could be observable via Terahertz magneto-Raman spectroscopy.The STM experiments were performed with a home-built variable temperature instrument44+ sputtering at 850°C. The growth of graphene on the clean Ir(111) surface was performed by chemical vapor deposition (CVD) of ethylene (3 × 10−7 Torr during 1 min) in UHV environments with the Ir(111) substrate held at 1050°C. Under such conditions, small areas of the Ir(111) substrate remained intentionally uncovered by graphene, which allowed us to estimate the coverage of W or Ir used for the cluster formation. W and Ir were evaporated from high purity filaments composed of each corresponding material. An accurate calibration of the deposition rate as a function of the filament temperature, measured by an infrared pyrometer, was performed by means of STM images acquired on areas of bare -uncovered by graphene- Ir(111).Ir(111) surfaces were cleaned by 1 keV ArThe experimental data (plasmonic excitations in graphene on Ir(111) and Pt(111)) was obtained from the original publications and fitted to the theoretical predictions using the least-square method. The electronic properties of graphene were modeled using the standard effective-mass-approximation. Exciton lifetimes and hopping amplitudes were estimated via Fermi's Golden Rule.A.J.M.G. carried out the experiments supported by I.B. and J.M.G.R. A.J.M.G. and J.M.G.R. designed the research helped by I.B. A.J.M.G., I.B and J.M.G.R. analyzed the data. T.S. performed the main calculations with the collaboration of A.G.R. I.B. wrote the manuscript with the help of T.S. All authors contributed to the scientific discussion and revised the manuscript.Supplementary Material for the article Towards scalable nano-engineering of grapheneNano-lithographing Graphene with 2.5nm precisionNano-lithographing Ir on graphene with 2.5nm precisionNano-lithographing W on graphene with 2.5nm precision


When Hubel  referred Our interest in layer 1 (L1) of cortex sprang from the knowledge that it was the final destination of basal ganglia output. In spite of the fact that the whole cortex is represented in striatum and movement is certainly not its only function, here we review thalamic output to motor cortex because of our long-standing interest in movement and the basal ganglia. Similarly L1 is not only present in motor cortex but invests the complete cortex. In theorizing about basal ganglia and their influence on the “crowning enigma” it is as important to remember that movement is only the most obvious output and the easiest outcome to measure from both the dark basements of the brain and its crowing glory. With its plastic spines and input from so many parts of the environment L1 might be the best place to provide the necessary context for movements, or for perceptions, both of which, like all decisions of the cortex, need information from many sources including historical experience that can be coded in the pattern of spines.L1 is recognized throughout the entire cerebral cortex. Cajal  describeFrom work performed in mice and rat neocortex  three types of neurons in L1 are described as non-pyramidal GABAergic: Cajal-Retzius, elongated neurogliaform and single bouquet. Cajal-Retzius are the earliest born  containing axons reach any given square millimeter of rat L1  and midline rhomboid nucleus  is considered the area where afferents from globus pallidus , substantia nigra reticulata (SNR) and deep cerebellar nuclei form terminal fields in separate nuclei of the ventral thalamus: ventrolateral (VL), ventral anterior (VA) and ventromedial (VM). According to Scheibel and Scheibel  the bestTerminal sites of afferent axons to MT are conserved across species  in relation to basal ganglia output indicated that increases in GPi(EP) and SNR activity resulted in decreased VM activity  and inhibitory GABAThe presence of a cooperative integration between the distal dendritic tuft and the axosomatic compartment of pyramidal cells has been studied following co-activation of both compartments. It has been observed for example, that axosomatic action potentials can be generated by the coincidence of back propagation of action potentials and the effect of distal dendritic excitatory potentials  or the hyperpolarization-activated current (Ih) plus the effects of specific activation of neurotransmitter receptors are important mechanisms for microcircuit control. Kv channels in the distal apical dendrite of layer 5 pyramidal neurons control the interaction between subthreshold tuft excitatory input and trunk spike generation  and at low density in the somatic region with two different effects: an inhibitory action in the tuft that controls initiation and propagation of dendritic spikes, and an excitatory action in the soma that decreases the threshold for action potentials  on L1 is also important in the control of tuft currents and local interneurons. For example, α-adrenergic agents decrease Other neurotransmitters acting on L1 are also relevant for cortical function. For example, ascending ACh axons extend preferential terminations in L1 ) and caudolateral VA/VL receives GLU terminals from cerebellum. VM and rostromedial VA/VL project to L1 and caudolateral VA/VL to layer 2/3.Apart from cerebellar and basal ganglia influences relayed to motor cortex via MT, indirect cerebellar inputs reach basal ganglia through midline/intralaminar outputs.Plastic related changes in spines of pyramidal dendritic tufts make learning-related structural changes a likely substrate for learning in L1.What is the role of the large synaptic terminals from SNR to VM described as “detonator drives” that typically favor neurotransmitter spillover and volume transmission?What is the function of the synaptic contacts made in basal ganglia from MT and midline/intralaminar axons as they travel to motor cortex? Which neurons are being contacted?What is the function of the electrically coupled elongated neurogliaform interneurons?Is 5HT released in L1 involved in learning-induced dendritic changes as are other neurotransmitters released in the area?The excitatory input of L1 as studied in many cortical areas regulates the active regenerative electrical activity of dendrites of pyramidal cells of layer 2/3 and 5. This excitatory top down control on the dendritic tuft is crucial for integration and further generation of axosomatic discharge.Recent combined electrophysiological and behavioral observations indicate that L1 can be considered as a driver of pyramidal neurons with important behavioral consequences such as attention, expectation, anticipation, and action command.We would like to consider projections from MT and midline/intralaminar nuclei to L1 as a regulatory entity of pyramidal cell excitability in motor cortex. These projections can provide the necessary binding input to trigger or suppress final patterns of activity that would ultimately generate appropriate behavioral responses. Motor thalamus has already been labeled a “super integrator” (Bosch-Bouju et al., A source of concern when dealing with MT and midline/intralaminar afferents to L1 is the extent of their projection as observed in other neocortical areas. It is hard to conceive the point-to-point modulation suggested by some electrophysiological results e.g., Jiang et al. . PerhapsChanges in dendritic spines induced by motor activity and learning observed in dendritic tufts in L1 underline the functional rewiring observed as changes in spine morphology and neuronal dynamics (Yuste and Bonhoeffer, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


By conducting BLAST searches of the databases, we screened 22 introns (37–65 nt) with conserved lengths and sequences among closely related species. We then provide experimental and bioinformatic evidence for the splicing of 15 introns, of which 12 introns were remarkably G-rich and 9 introns contained completely inefficient splice sites and/or branch sites. These unorthodox characteristics of ultra-short introns suggest that there are unknown splicing mechanisms that differ from the well-established mechanism.According to the length distribution of human introns, there is a large population of short introns with a threshold of 65 nucleotides (nt) and a peak at 85 nt. Using human genome and transcriptome databases, we investigated the introns shorter than 66 nt, termed ultra-short introns, the identities of which are scarcely known. Here, we provide for the first time a list of  Pre-mRNA splicing is an essential process in eukaryotic gene expression. In higher vertebrates, however, the length of the target introns that need to be recognized vary markedly, from <50 nucleotides (nt) to >500,000 nt with degenerated splicing signals, or 5′ and 3′ splice sites at the ends of the introns. Interestingly, there are two statistical modes in the length distribution of eukaryotic introns; in humans, there is a narrow distribution of short introns with a peak at ~90 nt and a broad distribution of long introns with a peak at ~2000 nt ,2,3.in vivo and in vitro. According to this system, the essential splicing sequences in pre-mRNA, namely the 5′ splice site, the branch-site sequence, and the 3′ splice site, are simultaneously bound by the splicing factors U1 snRNP, U2 snRNP, and U2AF65/U2AF35, respectively, leading to early ATP-dependent formation of the spliceosomal A complex , which are very efficiently spliced iewed in ,5). Eleciewed in ). Nevertin vitro . Therefobona fide tiny human introns that are actually spliced. There are currently no objective- or evidence-based rules to determine the minimum intron length in the gene annotation procedure. We could list 22 evolutionarily conserved introns that are shorter than the 66-nt threshold, which we termed ultra-short introns. These ultra-short introns have not been characterized elsewhere. We then confirmed 15 introns with mRNA-Seq data, database annotations, and experimental evidence obtained by reverse transcription–polymerase chain reaction (RT–PCR) analysis. We demonstrated that nine ultra-short introns are actually spliced out, a process that is often required to escape from nonsense-mediated mRNA decay (NMD), suggesting that splicing of these ultra-short introns is a prerequisite for the expression of the host genes.To address this question, it is essential to identify and verify Using sequence data deposited in a human annotated transcriptome database (H-InvDB), we plotted the distribution of intron lengths . All of The authenticity of introns in the ultra-short range has never been investigated. Here, we validated the candidate introns in the ultra-short range to make a list of genuine ultra-short introns.To select unequivocal introns in the ultra-short range, we performed three screening steps in 4528 tentative intronic sequences retrieved from H-InvDB. Using a human mRNA/cDNA sequence database, we first eliminated the transcripts that were erroneously mapped in H-InvDB with insertions/deletions to the genomic sequence. From the resulting 4027 candidate introns, conserved introns were selected by two kinds of BLAST searches: one targeted a genomic database using flanking exons of the candidate introns as queries, and the other targeted a transcriptome database using concatenated flanking exons as queries. Only candidate introns for which the results of both BLAST searches contained the same species with high similarity scores were selected. As a result, we obtained 1253 short introns (37–85 nt), of which 23 were in the ultra-short range (37–65 nt). By removing one ultra-short intron, which was a partial transcript, 22 candidate introns were finally determined as conserved ultra-short introns .The authenticities of the 22 candidate ultra-short introns were further validated based on three criteria: (i) The presence of individually sequenced data in the International Nucleotide Sequence Database Collaboration (INSDC) databases (DDBJ/EMBL/GenBank); (ii) Verification with read sequences in the published mRNA-Seq data; and (iii) Our experimental verification by RT–PCR analysis of endogenous transcripts. Finally, we identified 15 ultra-short introns that fulfilled at least one of these three criteria (“SN” in (i) We inspected the original transcript data submitted to the INSDC database to judge whether the transcripts were cloned and sequenced individually at the specific interests of the investigators, or whether they were automatically sequenced in a high-throughput sequencing project. We found that eight introns were individually sequenced  We confirmed whether the 22 ultra-short introns are spliced out by searching the publicly available RNA-Seq database of healthy human tissues—A total of 20 samples from 18 tissues; 16 tissues from BodyMap 2.0 project see  and an aSpliced” in in vitro [(iii) We performed RT–PCR experiments to examine whether the selected ultra-short introns are actually spliced out by endogenous splicing in human tissues. These experiments demonstrated that nine ultra-short introns were spliced out from the endogenous transcripts  were located within the untranslated region (UTR) while the other 20 ultra-short introns were located within the coding sequences CDS; . Of thes and SN16GGGGCTGGG) in 43-nt  and 49-nt  introns that functions as an intronic splicing enhancer (ISE) [We found that the G-content in conserved human introns gradually increases from the short range 85 nt) to the ultra-short range ≤65 nt; , which i5 nt to tThe essential core splicing signals  in the listed ultra-short introns were evaluated using the SROOGLE Web tool see . AlthougUsing the SROOGLE tool, we also searched for various exonic splicing enhancer (ESE) sequences in upstream and downstream of the ultra-short introns, which would potentially underpin nearby suboptimal, or weak, splice sites . TheThe evident steric hindrance of the ultra-short introns together with the unorthodox sequence features suggests the presence of multiple splicing mechanisms that are distinct from the authentic spliceosome-dependent mechanism of action . Herevia nonfunctional splice sites and branch site indicates that the splicing reaction occurs without either U1 or U2 snRNP. U1 snRNP-independent splicing was reported to occur under conditions of enriched SR proteins [Drosophila spliceosome with a shorter intron (62 vs. 147 nt as the control) have a smaller head domain containing the early “A” complex, suggesting that the complex with a shorter intron consists of fewer splicing factors [(i) Splicing of ultra-short intron proteins ,23 or inproteins ,25. This factors .trans-acting factor that recognizes a particular ISE. The 5′ splice site activation mechanism in which the G-triplet interacts with U1 snRNP via non-canonical base pairing with the U1 snRNA [in vivo splicing studies in HeLa cells showed partial splicing depression of G-rich 49-nt  and 43-nt  introns by disruption of U1 snRNA (unpublished data). The non-canonical U1 snRNA binding in these two introns (SN5 and SN3) remains to be analyzed.(ii) The G-rich ISEs, instead of containing authentic splice site and branch-site sequences, could be critical for the recognition of ultra-short introns for splicing. We have demonstrated that a specific ISE, CAGGGGCTGGG, is essential to splice out two ultra-short introns (SN3 and SN5) , and we U1 snRNA  might bein vitro [(iii) A curious but important question is whether the G-rich ultra-short intron, which lacks an authentic branch site, is excised as a canonical lariat structure or not. We previously showed that a 56-nt intron  containing effective 5′ and 3′ splice sites and a branch site  was exciin vitro . Howeverhttp://hinv.jp/) [http://www.insdc.org/). Therefore, we used all of the human mRNA/cDNA sequences in the INSDC, except for computationally generated transcripts (pHITs and eHITs).An integrated database of human genes and transcripts, H-InvDB Web server  , was useftp://ftp.ncbi.nih.gov/genomes/H_sapiens/ARCHIVE/BUILD.36.3/Assembled_chromosomes/). Candidate intron sequences lacking a terminal GT and AG  were eliminated.The sequences of the candidate introns were extracted from the human genome reference sequence , we performed three screening steps using 4528 tentative introns retrieved from H-InvDB. The first BLAST searches against the human transcriptome were queried by exonic sequences derived from genome coordinates of H-InvDB annotation. We could accordingly eliminate the H-InvDB transcripts that were erroneously mapped to genomic sequences with insertion/deletion generated by over-fitting to the GT–AG rule in the automated H-InvDB annotation process. This process yielded 4027 candidate introns, which were then screened by two kinds of BLAST searches: (i) NCBI Transcript Reference Sequences were queried for concatenation of both flanking exons, and (ii) Genomic Reference Sequences were queried for introns with both flanking exon sequences. These procedures were applied to each intron with the following thresholds: Length of obtained sequence >55; Bit score >99; E-value ≤1×10−19; Identity ≥96%.To select unequivocal introns in the short range  were first checked against the source of the transcript sequences described in the INSDC database.http://www.ensembl. info/blog/2011/05/24/human-bodymap-2-0-data-from-illumina/). This dataset contains mRNA-Seq data from 16 human tissues, including adrenal, adipose, brain, breast, colon, heart, kidney, liver, lung, lymph, ovary, prostate, skeletal muscle, testes, thyroid, and white blood cells. We also investigated the sequence read archive databases (http://www.insdc.org/), which contain mRNA sequences derived from healthy human tissues: Brain (SRP002274), pancreatic islet (SRP010483), gastric tissue (SRP012016), and white blood cells (ERP000177). We generated index FASTA files for exonic fragments with defined lengths across each splice junction of the 22 ultra-short introns using ERANGE (getsplicefa.py program) [To gather evidence for the splicing of the 22 selected introns, we examined the following mRNA-Seq datasets. The BodyMap (2.0) project was searched via the Ensembl browser (program) , and theprogram) .We experimentally confirmed the splicing of these 22 introns using RT–PCR assays targeting endogenous gene transcripts. Total RNA extracted from human tissues  was reverse transcribed with PrimeScript reverse transcriptase (Takara Bio) and oligo-dT primers. To detect the spliced products, the reverse-transcribed cDNAs were amplified by PCR with specific primers , and collected annotated information for each intron and the hosted gene  through H-InvDB (http://www.ensembl.org/index.html). We finally identified the encoded proteins of the host genes using the curated results of H-Inv proteins, which were categorized based on the similarity between the predicted amino-acid sequence and the known sequences (http://hinv.jp/hinv/help/help_proteins.html).The frequency of the HITs spliced at the exact splice junctions corresponding to the ultra-short introns is represented as the ratio of the number of HITs spliced at the ultra-short introns to the number of all aligned HITs across the ultra-short intron region. We also checked for the presence of ultra-short introns in the Ensembl transcript database (http://sroogle.tau.ac.il) [The essential core splicing signals  of the 22 selected ultra-short introns were analyzed using SROOGLE Web server (u.ac.il) . This inu.ac.il) .


Vascular smooth muscular cell (VSMC) involve in vessel tone regulation and endothelial cell function. It is known and critical player in pathological conditions including atherosclerosis. VSMC is dynamic structure and response to different stimuli. MiRNAs molecules are important regulatory mechanism to promote/suppress VSMC proliferation and phenotype switch.Aim: We hypothesized that patients with acute myocardial infarction have altered VSMC transcriptome affecting cell phenotype and proliferation.® Human Genome U133 Plus 2.0 Array. The differently expressed mRNA/miRNA data were validated on independent cohort of patients with RT-PCR method assay.Methods: Aortic wall tissue obtained during coronary artery bypass surgery underwent laser dissection of VSMC. Frozen aortic sections were stained with Arcturus LCM kit to identify cells of interest follow by cell collection and tRNA extraction. The transcriptome profile will be performed using Affymetrix GeneChipResult: We identify nine miRNA differentially expressed in patients with acute myocardial infarction, with four of them related to VSMC proliferation and differentiation. The most prominent down regulated miRNA was known regulator of VSMC phenotype miR-143. Among up regulated miRNA, miR-486-5p and miR-29a-3p reported before as a potent regulator of VSMC function. Pairwise analysis with mRNA data from the same patients reveal reciprocal and statistically significant changes in the phenotype and proliferation related mRNA.Conclusion: In patients with acute myocardial infarction we observe remote phenotype and proliferation shift in the aortic wall VSMC. Switch of contractile phenotype and proliferation alteration might play compensatory role in patient with unstable plaque and myocardial infarction.


Gas permeability data are presented for mixed matrix membranes (MMMs) of few-layer graphene in the polymer of intrinsic microporosity PIM-1, and the results compared with previously reported data for two other nanofillers in PIM-1: multiwalled carbon nanotubes functionalized with poly(ethylene glycol) (f-MWCNTs) and fused silica. For few-layer graphene, a significant enhancement in permeability is observed at very low graphene content (0.05 vol.%), which may be attributed to the effect of the nanofiller on the packing of the polymer chains. At higher graphene content permeability decreases, as expected for the addition of an impermeable filler. Other nanofillers, reported in the literature, also give rise to enhancements in permeability, but at substantially higher loadings, the highest measured permeabilities being at 1 vol.% for f-MWCNTs and 24 vol.% for fused silica. These results are consistent with the hypothesis that packing of the polymer chains is influenced by the curvature of the nanofiller surface at the nanoscale, with an increasingly pronounced effect on moving from a more-or-less spherical nanoparticle morphology (fused silica) to a cylindrical morphology (f-MWCNT) to a planar morphology (graphene). While the permeability of a high-free-volume polymer such as PIM-1 decreases over time through physical ageing, for the PIM-1/graphene MMMs a significant permeability enhancement was retained after eight months storage. High-free-volume polymers, such as polymers of intrinsic microporosity (PIMs), have attracted attention for their high gas permeabilities [a) [et al. [et al. [Membranes are currently used for a variety of industrial gas separations, including nitrogen generation from air and the removal of COural gas . Howeverbilities . Howeverbilities ,5. The abilities , can givties [a) –9, the a [et al. ,11 for m [et al.  for a fuPMMM of a MMM containing a volume fraction ϕF of impermeable, spherical filler particles within a polymer of permeability PP, is given by equation (??).2 [Simple models of MMMs predict that the addition of an impermeable nanofiller to a permeable matrix will reduce permeability compared with the unfilled material. For example, the Maxwell model  predictsnau & He  reportednau & He , MgO [15 & He [2 , in varib). The randomly twisted shape makes it difficult for molecules to pack, and because there are no single bonds in the backbone about which rotation can occur, they cannot undergo the types of conformational change that enable conventional polymers to rearrange and fill space.The permeability of a glassy polymer is linked to the amount and distribution of free volume within the material. Glasses are in a non-equilibrium state, and many factors may influence the way the molecules pack together and, thus, the free-volume distribution. High-free-volume glassy polymers have high permeabilities as a result of frustrated packing of the constituent macromolecules. In a PIM, this is a consequence of the molecular structure of the polymer chain, which is composed of fused-ring sequences interrupted by sites of contortion such as spiro-centres b. The rac–e).The presence of a nanofiller surface restricts the conformational freedom of polymer chains in its vicinity, which may further frustrate the ability of the chains to pack together, thus increasing free volume and enhancing permeability. It is a reasonable hypothesis that the effect of a nanofiller will depend not only on its available surface area but also on the curvature of the surface at the nanoscale. Here we compare an essentially planar filler (graphene), with a cylindrical nanofiller (f-MWCNTs), and a filler composed of more-or-less spherical nanoparticles (fumed silica) c–e.ca 10 regularly stacked layers is generally regarded as graphitic. For the present work, few-layer graphene was considered a suitable planar nanofiller for comparison with multiwalled carbon nanotubes.The term ‘graphene’ may be applied to a wide range of materials. A classification framework for graphene-based materials has been suggested  and a re2.(a)′,6,6′-Tetrahydroxy-3,3,3′,3′-tetramethyl-1,1′-spirobisindane  was dissolved in methanol and re-precipitated from dichloromethane before use. Anhydrous K2CO3  was dried in an oven at 110°C overnight before use. Anhydrous dimethylacetamide (DMAc), toluene and methanol (MeOH), were purchased from Sigma-Aldrich and used as received. Natural graphite was purchased from NGS Naturgraphite GmbH.Tetrafluoroterephthalonitrile  was purified by sublimation; it was heated to around 150°C and the pure product collected without vacuum. 5,5(b)3 min−1 and the injection volume was 100 μl. Polystyrene standards of known molar mass were used for calibration.Gel permeation chromatography (GPC) measurements were carried out using a Viscotek GPC max VE 2001 instrument with two PL mixed B columns and a refractive index detector. Tetrahydrofuran was used as solvent at a flow rate of 1 cm1H nuclear magnetic resonance (NMR) spectroscopy was carried out using a Bruker 400 MHz spectrometer. For NMR sample preparation, PIM-1 (≈5 mg) was dissolved in deuterochloroform  and transferred into a 5 mm NMR tube.Ultraviolet–visible (UV–Vis) spectroscopy was carried out using a Cary 60 UV–Vis spectrophotometer at room temperature.(c)et al. [2CO3 (0.03 mol), DMAc (20 ml) and toluene (10 ml) were added to a round-bottomed flask equipped with a mechanical stirrer, nitrogen inlet and Dean–Stark trap. The mixture was refluxed at 160°C for 40 min, then the viscous solution was added to methanol to precipitate the product. The yellow product was dissolved in chloroform and reprecipitated from methanol. Further purification was carried out by refluxing the precipitate in deionized water overnight, then it was dried overnight in a vacuum oven at 110°C. GPC: Mn=43000, Mw=170000, Mw/Mn=4.0. 1H NMR : δ6.81 , 6.42 , 2.33 , 2.17 , 1.36 , 1.31 . IR : 3000–2800, 2238, 1443, 1262, 1107, 1009, 751. Elemental analysis, calculated for C29H20N2O4 (wt.%): C, 75.64; H, 4.38; N, 6.08. Found: C, 71.99; H, 4.17; N, 6.02.PIM-1 was synthesized by a method based on that of Du et al. . TFTPN ((d)Mn, and only a modest decrease in weight-average molar mass, Mw. To form the membrane with the highest graphene content (sample KA1-7-12(3)), a 5 ml portion of the stock solution was transferred into a 7.8 cm diameter flat-bottomed glass petri dish, and the solvent was allowed to evaporate over 3 days. For preparation of membranes with lower graphene contents, portions of the stock PIM-1/graphene solution were mixed with portions of a pure PIM-1 solution of concentration 35 mg ml−1. For samples KA-1-7(1), KA-1-7(2) and KA-1-7(3), 2 ml of stock solution were mixed with 2, 5 and 10 ml, respectively, of PIM-1 solution. For samples KA-1-7(5) and KA-1-7(6), 4 ml of stock solution were mixed with 20 and 40 ml, respectively, of PIM-1 solution. A 7.8 cm petri dish was used for samples KA-1-7(1) and KA-1-7(6), and a 6.8 cm dish for other samples.A stock PIM-1/graphene dispersion was prepared as follows: natural graphite (600 mg) and PIM-1 (1.4 g) were added to chloroform (100 ml). The solution was sonicated for 84 h  and then centrifuged twice at 6000 r.p.m. for 20 min to remove graphitic particles. Although prolonged sonication of a polymer solution might be expected to cause cleavage of polymer chains, control experiments with PIM-1 solutions showed little effect on number-average molar mass, (e)CG, was determined by UV–Vis spectroscopy. The absorption was measured at 660 nm and CG evaluated using a value of α=3620 ml mg−1 m−1 for the absorption coefficient, as determined by the Coleman group [−1 graphene, as well as 14 mg ml−1 PIM-1. The graphene contents of membranes used for permeation measurements were then calculated from the known dilutions. For calculation of the percentage graphene by volume, the graphene was assumed to have the same density as graphite (2.2 g cm−3).For determination of the graphene content, membrane KA-1-7(5) (0.23 g) was redissolved in 23 ml of chloroform. The graphene concentration of the solution, an group . This ga(f)Single gas permeation measurements were carried out in a fixed volume/pressure increase apparatus  at 25°C at a feed pressure of 1 bar, as described previously ,23.3.(a)Self-supported PIM-1/graphene MMMs were obtained by slow evaporation of the solvent from PIM-1/graphene dispersions in chloroform. A stock PIM-1/graphene dispersion was prepared by ultrasound-assisted exfoliation of natural graphite into a PIM-1 solution. Dilution of the stock dispersion with fresh PIM-1 solution enabled dispersions of various concentrations to be obtained, and hence MMMs with various graphene contents as indicated in 2 > H2 > He ≈ O2 > CH4 > N2. In the simplest model of membrane permeation, the solution-diffusion model, permeability coefficient P is the product of a diffusion coefficient D and a solubility or sorption coefficient S .3.1Theed PIM-1 .A significant enhancement in gas permeability is observed at low graphene content. This is discussed further and compared with the effect of other nanofillers below.(b)et al. [figure 2a. It can be clearly seen that while the Maxwell model predicts a decrease in gas permeability, the experimental results show a significant increase in the effective permeability of the MMM with increasing filler loading over the range 7–24 vol.%. Filler loadings above 24 vol.% were not investigated; it is difficult to achieve homogeneous dispersions at higher loadings. The nanoparticles used in that study were reported to have particle diameters in the range 11.1–13.3 nm, although there was evidence of larger aggregates in the MMMs. Filler density and Brunauer–Emmett–Teller (BET) surface area were reported as 2.2 g cm−3 and 205−245 m2 g−1, respectively.Data of Ahn et al.  for methet al. [figure 2b. The MWCNTs used for functionalization (FutureCarbon GmbH) were reported to have 8–12 walls and diameter in the range 12–15 nm. The BET surface area was 250 m2 g−1, similar to the fused silica described above. For the plot in figure 2b, a density of 2.1 g cm−3 was assumed in order to calculate vol.% filler; in practice, the density may vary depending on the diameter and number of walls [ca 1 vol.% filler, and then decrease.Permeability data from Khan et al.  for methof walls . It can b. For calculation of vol.% filler, the density of bulk graphite (2.2 g cm−3) was assumed. A similar pattern is seen with graphene as for f-MWCNTs, with an initial increase followed by a decrease with increasing filler content. While PIM-1 itself can exhibit substantial differences in permeability depending on sample history, it is noteworthy that broadly similar results were obtained by three different laboratories working with three different fillers, the CO2 permeability for methanol-treated PIM-1 being in the range 5000–6200 barrer, and the highest CO2 permeability achieved for these MMMs being about double, in the range 12 200–13 400 barrer. The significant difference between the fillers is the filler loading at which the greatest permeability enhancement is seen, which increases from ca 0.05 vol.% for few-layer graphene, to ca 1 vol.% for f-MWCNTs, to ca 24 vol.% for fused silica.Permeability data for methanol-treated PIM-1/graphene MMMs from the present work are included in figure 22 g−1 , but it(c)et al. [ca eight months storage under ambient conditions  to a cylindrical morphology (f-MWCNT) to a planar morphology (graphene).For this series of samples, the addition of just 0.05 vol.% few-layer graphene to PIM-1 gave a substantial enhancement in gas permeability; for CO