Here we present a facile technique for the large-scale production of few-layer graphene flakes. The as-sonicated, supernatant, and sediment of the graphene product were respectively sprayed onto different types of silicon wafers. It was found that all devices exhibited current rectification properties, and the supernatant graphene devices have the best performance. Schottky junctions formed between graphene flakes and silicon n-type substrates exhibit good photovoltaic conversion efficiency while graphene/p-Si devices have poor light harvesting capability. Tremend cm2/Vs). A monol cm2/Vs), which m cm2/Vs). Graphen cm2/Vs).J-V characteristics of graphene/silicon interfaces has not been studied.Previous use of graphene in organic solar cell applications was mainly confined as flexible transparent electrodes to replace transparent indium tin oxide or fluorine-doped tin oxide for collecting charge carriers. RecentlIn this work we present a facile technique for the large-scale production of few-layer graphene flakes. Next, as-sonicated (So), supernatant (Su) and sediment (Se) of the graphene product were respectively sprayed onto n-/p-type silicon wafers. The current rectification properties of the formed Schottky junctions between different graphene sources and silicon substrates were compared.3)3·9H2O and Co(NO3)2·6H2O were dissolved in ethanol under agitation. Subsequently, the MgO powder with a surface area of 130 m2/g was mixed the solution and followed by drying at 60°C overnight. The catalyst was obtained by calcinating the resulting mixture in air at 500°C for 2 h. Graphene sheets can grow on the catalyst system from pyrolysis of acetylene at 1,000°C with the argon flow as carrier gas. The mixture product can be collected after 30 min of reaction and cooled under argon flow for about 10 min. Impurities like catalystsupport MgO and Fe-Co metal particles can be removed by washing the mixture product with hydrochloric acid under sonication. The purified graphene sheets can be obtained after filtration and washing.The graphene flakes were synthesized through chemical vapor deposition of acetylene on a MgO-supported Fe-Co bimetallic catalyst (Fe-Co/MgO with a stoichiometric composition of 2.5:2.5:95 wt%). The catN-methyl-2-pyrrolidone  under sonication. The G-NMP solution can be directly utilized to make G/n-Si devices. Part of the G-NMP solution was centrifuged; the supernatant (Su) and the sediment (Se) were taken out respectively and used for the fabrication of G/n-Si devices. The three different solutions were sprayed respectively on n-type silicon wafer , p-type silicon substrate  and glass substrates (for reference) by airbrushing. A silicon wafer with a window of pre-deposited insulating layer and a glass substrate were placed on a heating platform side by side (so that the resulting graphene flake films on Si and glass substrates should have the same thickness) and heated up to 150°C in order to evaporate the NMP solvent from the coatings. The schematic diagram of graphene-on-silicon Schottky device was displayed in FigurePurified graphene flakes were first dispersed in pure e NMP, 0. mg/mL unKα radiation line and general area detector diffraction system were used as an excitation source and detector, respectively. The experiments were carried out in Bragg-Brentano geometry.To characterize the morphological properties of graphene nanosheets, several techniques such as microscopy and X-ray diffraction (XRD) were utilized. Atomic force microscopy (AFM) images were obtained on Veeco Dimension 3100 AFM system . Scanning electron microscopy (SEM) images were obtained using a JEOL 7000 F high-resolution scanning electron microscope . This microscope has a resolution of 1.2 nm at an accelerating voltage of 15 kV and a working distance of 10 mm. The final products were mounted on aluminum pins with double-sided carbon tape and their corresponding SEM images were obtained. Elemental analysis was performed with Genesis energy dispersive spectrometer system . The X-ray powder diffraction profiles of graphene sheets were recorded in θ-2θ mode on Bruker D8 Discovery diffraction system . The monochromatic Cu J-V) characteristics were investigated in the dark and under illumination using a solar simulator at air mass coefficient 1.5 (approximately 100 mW/cm2) inside a glove box in a nitrogen environment. The illumination was on the graphene flake side. The devices were irradiated in an area of 1 × 1 cm2 and data were recorded using a Keithley 2400 source meter .To understand how a different graphene source affect the G/n-Si Schottky junction properties, current density-voltage , G band  and the 2D band . The D band arises due to the breathing modes of sp2 atoms in rings and its intensity is usually associated with defects in the carbon-based material[G band is often referred to the E2g mode at the Brillouin zone center due to the bond stretching of sp2 atoms in both ring and chains[D band is the second order mode and its shape as well as its position is used to identify a single layer from bilayer and few (less than five) layer graphene[As shown Figurematerial, in the material. The G bd chains. The 2D graphene. Figured-spacing) between two subsequent graphene sheets can be calculated from the (002) diffraction peak position[B cos θ), where λ is the x-ray wavelength, B is the full width at half maximum of the diffraction peak, and θ is the Bragg angle)[d-spacing and the size of crystallite, the graphene sheets in this work were estimated to have in average of about four layers.The crystallinity and number of layers in the graphene nanosheets can be analyzed by XRD technique. The XRD profile of the graphene sheets grown by radio frequency catalytic chemical vapor deposition method was shown in Figureposition. The widg angle). Based oThe top view morphologies of a typical G/Si device made of supernatant were displayed in SEM and AFM images Figure. Both SE3 at ±1 V. As seen from FigureJ-V curve. The Shockley ideal diode equation gives the J-V characteristic of an ideal diode in either forward or reverse bias (or no bias). The equation is as follows:The typical dark current–voltage characteristic of G/n-Si heterojunction device is displayed in the top inset of FigureJ is the diode current, JS is the reverse bias saturation current , VD is the voltage across the diode, VT is the thermal voltage , and n is the ideality factor, also known as the quality factor, or sometimes emission coefficient. The ideality factor n varies from 1 to 2 depending on the fabrication process and the semiconductor material and, in many cases, is assumed to be approximately equal to 1. As shown in Figuren(Su) > n(So) > n(Se) . The saturation current density Js can be expressed with thermionic emission model[2)where on model in the fA is the Richardson constant, which is 112 A/(cm2K2)for n-Si and 32 A/(cm2K2) for p-Si substrates[eϕSB is the zero-bias Schottky barrier height. By combining equations 1 and 2 and fitting the J-V curves of the devices in dark, the Schottky barriers are estimated to be 0.52 to 0.67 eV on average for the graphene/n-Si devices and 0.61 to 0.73 eV for the graphene/p-Si devices at 300 K, which is consistent with the higher current densities observed in n-Si devices versus p-Si devices. These values are comparable to the values of the bilayer graphene-on-silicon devices[where bstrates, and eϕS devices, but con devices, and the devices.Usually graphene flakes, generally known to exhibit weak p-type conductivity, and difon/ioff ratio of >102, suggests that the electron–hole pairs could be efficiently generated and separated in the G/Si solar cells, which helps facilitate the Schottky junctions to harvest solar light more effectively. The schematic energy band diagrams of G/n-Si and G/p-Si were displayed in FigureTo investigate the underlying physics of the G/Si Schottky junction solar cells, photoresponse characteristics of both G/n-Si and G/p-Si devices were shown in Figure2 or nitric acid in G/n-Si devices[Although the photovoltaic conversion efficiency of the G/Si solar cells is relatively low, the photovoltaic performance could be improved by doping graphene with nitrogen in G/p-Si configuration, which could be attributed to the enhanced n-type conductivity of the graphene film at higher N doping concentration, or treating graphene with a strong acid such as SOCl devices. AdditioWe developed chemical vapor deposition approach to the synthesis of graphene at large scale and low cost, which makes the wide applications of graphene possible. The graphene coating on n-Si wafer forms Schottky junction with rectification behavior. The fabrication of Schottky junctions has the merits of low cost and simplicity. By comparing different graphene sources, it was found that the supernatant graphene is the most desirable material for the fabrication of G/n-Si junction with excellent rectifying capability and good photovoltaic conversion efficiency. Although G/p-Si also exhibit rectification behavior, they demonstrate poor photovoltaics conversion efficiency due to the weak p-type conductivity of our graphene flakes.The authors declare that they have no competing interests.MM and ZL contribute equally in this work. MM carried out the experiments and measurements; ZL offered idea, performed data analysis and drafted the manuscript. JC and CT participated in the discussion. All authors read and approved the final manuscript.MM is Ph.D. candidate connected with the Department of Physics and Astronomy, University of Arkansas at Little Rock. ZL is a visiting professor at the Department of Physics and Astronomy, University of Arkansas at Little Rock, JC and TC are the professors at Department of Physics and Astronomy, University of Arkansas at Little Rock.


Before the deposition, optical microscopy and Raman spectroscopy were utilized to identify the number of the graphene layers. After the deposition, scanning electron microscopy was used to observe the morphologies of the particles. Systematic analysis revealed that the average sizes of the nanoparticles increased with the number of graphene layers. The density of nanoparticles decreased as the number of graphene layers increased, revealing a large variation in the surface diffusion strength of nanoparticles on the different substrates. The mechanisms of formation of these layer-dependent morphologies of silver on n-layer graphene are related to the surface free energy and surface diffusion of the n-layer graphene. The effect of the substrate such as SiO2/Si was investigated by fabricating suspended graphene, and the size and density were similar to those of supported graphene. Based on a comparison of the results, the different morphologies of the silver nanoparticles on different graphene layers were theorized to be caused only by the variation of the diffusion barriers with the number of layers of graphene.The distributions of sizes of silver nanoparticles that were deposited on monolayer, bilayer, and trilayer graphene films were observed. Deposition was carried out by thermal evaporation and the graphene films, placed on SiO It, therefore, has various unique electrical and optical properties of interest to scientists and technologists[2 and Al2O3 with special thickness between graphene and the substrate are typically used to make graphene optically visible[A single atomic layer of graphene is the thinnest spologists-3. Graph visible-7. The e visible. Raman a visible-14. Rece visible-20. The  visible,22.In this work, layers of graphene were fabricated in a sample by micromechanical cleavage. The number of layers of graphene was determined by micro-Raman spectroscopy and optical microscopy. After silver nanoparticles were deposited on the sample using a thermal deposition system, the distribution and sizes of the particles on flakes with different numbers of layers were systematically analyzed. To analyze the effect of the substrate, suspended graphene was fabricated, and the size and density thereof were found to be similar to those of supported graphene. The different results for the mono-, bi-, and tri-layer graphene are theorized to be caused only by the variation among the diffusion barriers of the various graphene layers, which provides a method of determining the number of graphene layers and provides information that can be utilized to elucidate the interaction between a graphene flake and its substrate.2 over a Si substrate. Graphene flakes that contained different numbers of graphene layers were distributed on different areas on the same sample. The variation in the color contrast with the number of graphene layers is observed under an optical microscope, as presented in FigureA graphene flake was fabricated by micromechanical cleavage with scotch tape. It was capped with 300-nm-thick layer of SiO2/Si substrate and the monolayer, bilayer, and trilayer graphene flakes were obtained, and shown on right-hand sides of Figure2/Si substrate.Silver nanoparticles were deposited on graphene flakes at a rate of 0.5 nm/min using a thermal deposition system to a thickness of 5 nm at a fixed temperature of the deposition system of 300 K. To elucidate the surface diffusion, the sample after deposition was maintained at 373 K in a vacuum for 1 h. To study the surfaces of the graphene flakes, scanning electron microscopy (SEM) images of the distribution of nanoparticles on the SiO2/Si substrate, monolayer, bilayer, and trilayer graphene. The sizes of the nanoparticles on the monolayer graphene flake are distributed in the range of 0 to 50 nm, whereas those of the nanoparticles on the trilayer graphene flake were distributed in the range of 10 to 70 nm. Whereas the sizes of the nanoparticles on the SiO2/Si substrate were distributed in the range of 0 to 50 nm, the majority of them were in the range of 10 to 30 nm.To investigate the density and size of the nanoparticles, average size and density were determined by histogram analysis. The histograms, on the left-hand sides of Figure2/Si substrate without any graphene flake. The nanoparticles on the trilayer graphene are largest, with a mean size of approximately 36 nm; as the number of graphene layers decreased, the average size fell to 32, 25, and 18 nm. The density of nanoparticles on the trilayer graphene was also the lowest, at around 3.1 × 1014/m2. Reducing the number of graphene layers increased the density from 3.1 × 1014 to 9.1 × 1014/m2. The density of nanoparticles decreased as the number of graphene layers increased, revealing a large variation in the surface diffusion strength of nanoparticles on the different substrates. The mechanisms of formation of these layer-dependent morphologies of silver on n-layer graphene are related to the surface free energy and surface diffusion of the n-layer graphene. Different morphologies of silver on the differently layered graphene are obtained when silver particles are deposited on the graphene surfaces because of the differences of surface free energy. The variation in surface free energy with number of layers is responsible for the variation in the morphologies of silver on the differently layered graphene. The particles are randomly deposited on the graphene surface, and surface diffusion causes randomly arranged particles to combine to form relative large islands. The surface diffusion coefficient D ∝ exp(−En/kT) indicates the interaction between particles and the substrate surface, where En and k denote the diffusion barrier and the Boltzmann constant, respectively. The density of the particles on the substrate (N) can be expressed as N ∝ (1/D)1/3 for isotropic surface diffusion. The diffusion barrier specifies the interaction between particles and the substrate surface. The difference between the diffusion barriers of the substrate and monolayer can be calculated as ΔE = E0 − E1 = 3kT ln(N0/N1) = 61.7 mev by combining the above equations. The difference between the barriers of the substrates with other numbers of layers can be similarly obtained, and they are 39.1 meV (E1-E2) between the first and second layers and 3.1 meV (E2-E3) between the second and third. The difference between the diffusion barriers of silver decreased as the number of graphene layers increased. According to one review[2/Si substrate and different graphene layers, which the most is trilayer, is in the range of 9 × 1013 to 1.25 × 1013/m2, and the difference between diffusion barriers of different layers is in the range 500 to 291 meV. The area density of the silver nanoparticles herein is approximately ten times this value, indicating that silver nanoparticles can be more easily prevented from moving along the graphene surface, revealing a stronger interaction between silver and graphene than between gold and graphene.In Figuree review, the are2/Si is responsible for the variation in morphology, suspended graphene was fabricated by mechanical exfoliation of the graphene flakes onto an oxidized silicon wafer. First, ordered squares with areas of 6 μm2 were defined by photolithography on an oxidized silicon wafer with an oxide thickness of 300 nm. Reactive ion etching was then used to etch the squares to a depth of 150 nm. Micromechanical cleavage of HOPG with scotch tape was then used to deposit the suspended graphene flakes over the indents. The deposition conditions and silver nanoparticles were the same for both types of graphene. Figure2 does not affect the distribution of silver nanoparticles on the surface of graphene.To determine whether the substrate effect of SiO2, suspended graphene was also fabricated. The size and density of suspended graphene were found to be similar to those of the supported graphene. According to these results, only variations in the interactions between n-layer graphene and the silver nanoparticles were responsible for the variation in their distribution.In this work, the distribution of sizes of silver nanoparticles that were deposited on graphene films with different numbers of atomic layers of graphene was investigated. A systematic analysis revealed that the average size of the nanoparticles increased, and the area density and the difference between diffusion barriers of the nanoparticles decreased as the number of graphene layers increased. To analyze the effect of a substrate such as SiOThe authors declare that they have no competing interests.C-WH carried on the experimental parts: the acquisition, analysis, and interpretation of data. C-WH was also involved in drafting the manuscript. H-YL and C-HH took the analysis and interpretation of data. They also had been involved in revising the manuscript. R-JS and W-HW  prepared the graphene samples using micromechanical method and obtained the OM images. C-YL has made substantial contributions to the conception and design of the study, and in the critical revision of the manuscript for important intellectual content. H-CC, the corresponding author, had made substantial contributions to conception and design, and had been involved in drafting the manuscript and revising it critically for important intellectual content. All authors read and approved the final manuscript.C-WH received his BS degree in Electrical Engineering from the National University of Kaohsiung, Kaohsiung, Taiwan, in 2008. He studied his MS degree in 2008 and Ph.D. degree directly in 2009. Currently, he is a Ph.D. candidate in the Department of Photonics, National Cheng Kung University, Tainan, Taiwan. He focuses on the property of graphene and surface plasmon resonance of nanoparticles. H-YL received her doctoral degree from the Institute of Biomedical Engineering, National Cheng Kung University in 2010. Her current research interest focuses on the SERS properties of graphene and possible sensing application. C-HH received his doctoral degree from the Institute of Electro-Optical Science and Engineering, National Cheng Kung University in 2009. His current research interest focuses on the Raman and SERS properties of graphene incorporating with metal nanoparticles.


In natural conversation, the minimal gaps and overlaps of the turns at talk indicate an accurate regulation of the timings of the turn-taking system. Here we studied how the turn-taking affects the gaze of a non-involved viewer of a two-person conversation. The subjects were presented with a video of a conversation while their eye gaze was tracked with an infrared camera. As a control, the video was presented without sound and the sound with still image of the speakers. Turns at talk directed the gaze behaviour of the viewers; the gaze followed, rather than predicted, the speakership change around the turn transition. Both visual and auditory cues presented alone also induced gaze shifts towards the speaking person, although significantly less and later than when the cues of both modalities were available. These results show that the organization of turn-taking has a strong influence on the gaze patterns of even non-involved viewers of the conversation, and that visual and auditory cues are in part redundant in guiding the viewers’ gaze. Behind the apparent ease of conversation, whether a formal or a more casual one, lies a tight organization of speaking turns According to the organization of turn-taking, one party talks at a time, and the change of the turn is allowed in transition-relevance places Gaze, as an important nonverbal gesture, has a role in coordinating conversation and turn-taking as well as in informing about the person’s target of attention. The speaker’s gaze direction may signal that the turn is approaching completion: the speaker makes eye contact with the listener around turn exchange, whereas the eye contact is less common when the speaker is still continuing the turn In addition to face-to-face conversation, gaze coordination occurs in many other situations involving social interaction. For example when subjects are listening to a previously recorded description of the same TV scene they are viewing, their eye movements follow the eye movements of the speaker with an average delay of 2 s In the present study we showed a group of volunteers a short video of a real conversation to find out how people follow a two-party conversation with their gaze. As a control, the same video was presented without sound and the same soundtrack with a still frame captured from the original video. To be able to assess the effects of turn transitions and the turn-transition cues to followers of a natural conversation, we developed quantitative methods for analysing gaze behaviour of a group of subjects. This experimental setup with passive viewing of a conversation presented from video enabled us to expose each subject to an identical natural conversation, to evaluate the effect of the verbal and nonverbal cues separately, and to focus our analysis on any conversational events of interest to assess the similarity of the viewers’ gaze behaviour. Although our subjects were non-involved viewers of the conversation, their gaze was expected to be informative of the most attention-capturing aspects of the conversation.We hypothesized that accumulation of the syntactic, pragmatic and prosodic cues of turn transition would prompt a shift of gaze even in a viewer of the conversation. We further expected to see synchronous gaze behaviour between subjects, directed by the organization of turn-taking. We also studied the timing of the gaze patterns with respect to turn transitions.Nineteen Finnish-speaking young adults  with normal or corrected-to-normal vision volunteered in the main experiment. Nine subjects participated in the two control experiments, video with no sound  or still image with sound .The subjects gave their written informed consent prior to the experiment after the course of the study had been explained to them. The study had prior approval by the Ethics Committee of the Hospital District of Helsinki and Uusimaa.(i) the viewer would be guaranteed an access to the facial and posture orientation of the participants of the conversation and (ii) to the topic talked about, (iii) the speaker should change rather rapidly and one participant should not hold the turn for long, and (iv) the scene should be clean of any other possibly interesting items.In the main experiment, we presented an audiovisual stimulus: a 5.5-min video clip of two women  having a conversation. As a control, (i) the same video was shown without sound (later referred as ‘video only’) and (ii) the same soundtrack was played with only a still frame captured from the original video (later referred as ‘sound only’). The criteria for choosing the original video clip were that The video was presented with ClearView 2.7.1 software  on a grey background, and its size was 20 deg×16 deg (16 deg×12 deg for 4/19 subjects because of different display resolution settings) when viewed from the distance of 60 cm. The sound was presented through stereo loudspeakers, located on both sides of the screen 3 P2: mt. Mh >Kuinka paljon< siel oli ihmisiä.Mt. Mh >How many<people were there.[1∶17∶3]The example illustrates syntactic (S), pragmatic (Pra), and prosodic (Pro) cues for turn transition. Syntactic cues mark places where the utterance could be syntactically complete notwithstanding the preceding context. Pragmatic cues mark places where the utterance can be understood as complete in its conversational context. Prosodic cues refer to places where the intonation of the utterance sounds as if the speaker is finishing his/her turn of talk In addition to the turn-transition cues, routinely annotated in conversation analysis, we also defined the ends and the starts of speech, followed or preceded by a minimum of ∼0.5 s of silence. We defined ‘fast turn transition’ (fTT) as an event type in which the speaker change (from the end of one speaker’s speech to the start of the other’s) occurred in less than 300 ms and without overlap of the two speakers; altogether 41%  and ClearView 2.7.1 software, with a sampling frequency of 50 Hz. The eye tracker was calibrated before the experiment by asking the subjects to fixate at five points that covered the screen. The tracking was done binocularly and based on video-oculography by dark-pupil–corneal-reflection method; the infrared camera and the infrared light sources were integrated into the computer screen.As the faces of the conversation participants in the stimulus video stayed relatively still during the whole recording, positions of two regions of interest (ROIs), one ROI for each face, were defined as stationary rectangles see .The gaze data were further analysed in Matlab 7 (MathWorks). Gaze direction changes were defined as gaze moving from one ROI to the other in less than 70 ms, later referred as ‘ROI change’. This limit was chosen because a 10-deg saccade lasts for about 40 ms For the analysis of the group-level effects, we computed the mean ROI change rate over all subjects separately for all different conversational events, in 200-ms windows across a 5-s (from –2 to 3 s) epoch centered on each event; the same analysis was made for the main experiment and for the two control conditions. For comparison, the baseline level of ROI change rate was estimated in the same manner around “events” placed equidistantly once every 6 s throughout the video.The effect of visual and auditory cues to the subjects’ responses to turn transitions was assessed by comparing the cumulative distributions of ROI changes around fTTs in the three experimental conditions with Kolmogorov-Smirnov test in Matlab.p<10–9; see The data of p  = 0.5, between the two ROIs. The colour codes refer to p-values  for obtaining the observed sample from such an underlying distribution. We noted that for 68% of the total time at least 14 out of the 19 subjects viewed the same ROI.The statistical significance of the similarity vs. randomness of the subjects’ gaze behaviour is indicated with coloured dots along the x  = 0 line of The effects of various turn completion cues and of other events in the conversation were assessed by examining the gaze-direction changes around each cue. Our goal was to understand, by tracking the eye gaze, how uninvolved viewers follow a conversation between two other persons. The gaze patterns across our 19 subjects showed similarity that differed statistically significantly from random viewing. Turns at talk directed the subjects’ gaze so that the current speaker received most of the gazes. The time spent looking at the current speaker was on average 74%, which closely resembles the behaviour of participants in a two-party conversation in which the listener looks at the speaker on average 75% of the time The fTTs induced gaze-target changes also in the control experiments when either only the verbal (sound only) or only the nonverbal (video only) cues of turn transition were present. In human speech, speech-sound envelope and mouth opening are highly correlated, and they are both modulated at 2–7 Hz Although we found–as we had expected–that the stronger the cue for turn transition, the more gaze-direction changes it elicits, very similar behaviour was seen also in the video-only condition, in which the subjects could not hear the verbal cues. Thus this result had to arise from the strong temporal correlation of turn transitions and the different turn-transition cues. In addition, because the cue strength is inversely related to the temporal distance to turn transition, it is obvious that the stronger is the cue for turn transition, the more its effects are contaminated by the effects of the close-by turn transition. Consequently, we had to abandon any further attempts to analyse the effects of different verbal cues of turn transition on the viewer’s gaze behaviour. Our results suggest a need for further research on visual cueing–by means of participants’ gaze as well as by body posture and kinesics–of turn transition, and its coordination with the verbal cueing.We did not observe anticipation of turn transition in the viewers’ gaze behaviour; instead, most of the gaze-target changes occurred about 0.5 s after fast turn transition, and about 0.3 s after start of speech. This lag equals the lag of a saccade towards a simple auditory target Our experimental setup is in line with the currently dominant tradition of social neuroscience, where the effects of social stimuli to an uninvolved viewer are assessed. However, this “spectator stance” has been criticized see e.g. , as it n


Perhaps the most controversy is regarding the function of RNAs found in introns of annotated transcripts, where most of the reads that map outside of exons are usually found. However, it has been reported that the levels of RNA in introns are minor relative to those of the corresponding exons, and that changes in the levels of intronic RNAs correlate tightly with that of adjacent exons. This would suggest that RNAs produced from the vast expanse of intronic space are just pieces of pre-mRNAs or excised introns We present data that challenges the notion that intronic RNAs are mere by-standers in the cell. By performing a highly quantitative RNAseq analysis of transcriptome changes during an inflammation time course, we show that intronic RNAs have a number of features that would be expected from functional, standalone RNA species. We show that there are thousands of introns in the mouse genome that generate RNAs whose overall abundance, which changes throughout the inflammation timecourse, and other properties suggest that they function in yet unknown ways.So far, the focus of non-coding RNA discovery has shied away from intronic regions as those were believed to simply encode parts of pre-mRNAs. Results presented here suggest a very different situation – the sequences encoded in the introns appear to harbor a yet unexplored reservoir of novel, functional RNAs. As such, they should not be ignored in surveys of functional transcripts or other genomic studies. Mammalian cells require molecular machineries with sufficient complexity and diversity to acquire, process, and distribute vast amounts of information. The unique features of non-coding RNAs could facilitate key steps in the information processing activities of hundreds of regulatory pathways, suggesting a role for them as the major informational “currency” of the cell,2. The lHowever, the prevalent belief is that these RNAs simply represent pre-mRNAs en route to splicing or spliced-out introns en route to degradation. Moreover, it has been suggested that the levels of RNAs in the intronic regions are consistently lower than those in exons and that the levels of intronic and exonic RNAs highly correlate – all coTo gain insight into the functional significance of non-exonic RNA, we asked whether introns could indeed harbor functional transcripts in the cell whose features and physiological behavior differed from that of pre-mRNAs or spliced out introns. In this report, we present the results of a global investigation of intronic transcription during the mammalian inflammation cascade. The cascade sets in motion a series of intricate responses to the perturbation of invading pathogens, using many interlocking feedback loops to determine the precise nature and extent of the challenge, and carefully regulating the corresponding pathways to resolution. Thus, it is expected to require a significant intracellular information exchange, where we and others expect non-coding RNAs to function-15 and aMice were treated with lipo-polysaccharide (LPS) by inhalation, followed by isolation of RNA from lung at 3, 6, 12, 24 and 48 hours post-treatment. In total 42 animals were studied: 7 animals at each time-point and a group of control, un-treated animals. We have chosen the single-molecule sequencing (SMS) platform for RNA analysis due to its superior performance and reproducibility for quantification of RNA expression, in part due to the fact that it does not depend on PCR amplification and ligation for the library preparation. Total RTotal RNA from each sample was treated with DNAse, subjected to a RiboMinus procedure to deplete ribosomal RNAs and sequenced by SMS. The basic statistics reflecting the distribution of mapped reads in annotated exons, introns and intergenic regions are shown in TableConsistent with our previously published-results, 63.6–64It is worth noting that in our analysis intronic RNAs refer to any RNAs encoded by genomic regions annotated as introns, including excised introns, alternative isoforms of exons, as well as standalone sense or antisense transcripts transcribed from their own promoters that overlap intronic regions. The first clue that intronic RNAs should not be disregarded came from the observation that the signal from intronic RNA was not evenly spread among genes: rather, annotated genes differed significantly in the amounts of intronic RNAs Figure. On one As the next step, we investigated whether levels of intronic RNAs in general mirrored those of the exons of their associated transcripts, or whether the two were relatively independent from each other. Some controversy in this regard exists in the literature. The report by van Bakel et. al. found a We then calculated Spearman rank correlation between RNAseq densities of each intron and the corresponding exons throughout the time course by comparing 42 exon-intron densities for each animal in each time point for each intron-exon pair (Methods). The histogram of the resulting correlation coefficients for 220,645 exon-intron pairs from this analysis is shown in FigureWe then asked how well different introns of the same transcript correlate with each other. We used two metrics – correlation of different introns of the same transcript with each other throughout the time course, and the range of differences of the intronic RNAseq signal within the same transcript. We expected that if an intron indeed harbored independently regulated transcripts, these transcripts would not correlate with the levels of either exons or other introns from the same locus. The correlations between an intron N and all other introns in a given locus were split into bins according to the correlation that this intron had with the corresponding exons as shown in FigureIndeed, introns that correlated well with their corresponding exons also had higher correlations with the other introns, as evidenced by the upward trend of the minimal correlation box plots in the right portion of the FigureWe then asked how introns of the same transcript might differ in terms of abundance with their corresponding RNAs. For each transcript with 2 or more introns analyzed in FigureThe median ratio of intronic densities to their corresponding exonic densities was found to be 11.95% across all samples in the “Total Intron-Exon Pair-Wise Dataset” (Methods). However, as mentioned above we also observed heterogeneity in the level of intronic RNA signal, even among different introns of the same gene. Therefore, we asked how many individual introns have at least comparable or higher read densities than that of their corresponding exons in at least one animal at any time point. We calculated the maximum ratio of intron/exon densities for each intron that could be found in any of the 42 animals at any timepoint. The distribution of these ratios is shown in FigureWe were intrigued by the introns whose densities are higher than that of exons in this analysis and investigated this further. The introns were stratified based on the maximum ratio of intron/exon density. In the >10 fold category, represented by 96 introns, most (~90%) contained annotated non-coding RNAs, with half of the introns containing snoRNAs found only in the Ensembl database Table. While tAlso as expected, once we lowered the maximum intron/exon density ratio to include the introns with still significant, but lower ratio of 2–10 and 1–2 fold, the situation reversed. More than 90% of introns in these much more numerous categories did not have any annotated RNA . The remaining 7 seem to represent novel RNAs with no BLAST hits to any sequence of known function and 4 examples are shown in FigureThese two examples shown in FigureAccurate and precise regulation of a biological molecule separates functional behavior from noise. Therefore, we asked whether the steady-state levels of intronic RNAs could be as tightly regulated as those of exons. We reasoned that the level of control exerted over the steady-state level of a transcript would manifest itself in the variation of its levels measured among different genetically-identical individuals maintained in the same conditions. Levels of tightly regulated RNAs would vary less than those of RNAs under little regulatory control. Our system is well-suited for measurement of such variation – we have profiled 7 animals of the same gender and similar age per time point from a genetically-homogeneous strain of mice using SMS, which generates highly reproducible results, a key tTo accomplish this, we calculated the coefficient of variation (CV) of each individual intron and the corresponding exons among 7 animals for each time point, resulting in 1,231,535 intron-exon combinations (Methods). While the much lower intronic density precludes a direct comparison between the CV’s of the introns and exons (Methods), we asked how many introns had the same or lower CV than the corresponding exons at any time point. Surprisingly, 40,948 out of 98,190 introns used in this analysis satisfied this criterion, despite the fact that the RNAseq median densities within these introns were 20.78% of that of the corresponding exons. In other words, the levels of 41.7% of introns have the same or lower animal-to-animal variation within the same time point of LPS treatment (or control) as the corresponding exons, even though their relative levels are on average ~5 fold lower, suggesting an extraordinary level of biological control of steady state expression of intronic RNA.As a next step, we interrogated the entire genome  for the presence of RNAs that change during different stages of inflammation irrespective of the presence of annotation. Determining un-annotated RNAs presents a unique challenge because their sequences are not known and the read length is shorter than the sequence of the entire RNA molecule. Therefore, we have chosen an unbiased approach to identify such regions, based on splitting the genome into a series of non-overlapping bins of different sizes, and then counting the number of SMS reads in each bin, in each sample, and then comparing to the control. We have previously reported this approach with theAn example of a known non-coding RNA detected with this method, the primary transcript for miRNA mir-21, whose transcription increases during inflammation, is showTableOverall, thousands of such examples exist: just at the 3 hr time point, we found 3,890 annotated transcripts corresponding to 1,556 loci that had at least one intronic down-regulated bin and 6,165/2,472 transcripts/loci that had at least one intronic up-regulated bin with no accompanying changes in exons of these genes  RNA regions were first identified based on profiling of histone modifications associated with elongating RNA Pol II in mouse and thenWe thus investigated the distribution of our informative reads and DE bins among the mouse linc RNA regions published by Guttman et al. In total, we could remap 1,666 out of the original 1,673 regions to mm9. We realized that 530 of those regions originally found in the intergenic space now map to or overlap UCSC Genes as exemplified by FigureIt has been reported that cells depleted for the components of RNA degradation machinery, such as the exosome, have higher levels of some non-coding RNAs,38. It hThe discovery of pervasive transcription of the mammalian genome,32,41-43Here, we challenge this general notion and provide evidence that many intronic RNAs can display features consistent with function: their levels of expression and their biological variation during a physiological time course, or among different individuals of the same strain, can often occur with a magnitude similar to that of exons. Furthermore, levels of intronic RNAs often do not depend on levels of their exonic counterparts. Based on these criteria, many introns produce RNA molecules whose fates in the cell are different from that of exonic RNAs, in response to the important biological stimulus of LPS induced inflammation. The combination of these and other parameters as shown in TableWhile these arguments do not tell us how intronic RNAs function, they suggest that they should not be ignored or automatically assigned to the category of annotated pre-mRNAs as suggested by van Bakel et al.,9. On thThis knowledge has an immediate application in molecular diagnostics as intronic RNAs can apparently provide additional information about a biological state that cannot be obtained just by the analysis of exons alone. As a matter of fact, microarray-based analysis of specific biological systems have indeed shown that non-exonic RNAs, including intronic RNAs, not only can be used as diagnostic markers,46, theyThis begs the question of what potential functions could be carried out by intronic RNAs. We think that there are at least two likely possibilities: precursors to smaller functional RNAs, and RNA scaffolds. The general theme of production of smaller RNAs from larger precursors is now recognized in the field. For exaWhile the fact of pervasive transcription is firmly established now as mentioned above, as history shows, it can fade somewhat with time, with focus instead shifting to separately developed lists of long non-coding RNAs detected in specific experiments or filtered by certain properties that hint at functionality,34,52-55This study presents a highly quantitative, comprehensive and unbiased RNAseq dataset showing that most of the RNAs that change during inflammation correspond to the non-coding, predominately intronic, regions of the genome. These non-coding RNAs would not have been detected if only the exons of protein-coding mRNAs or relatively few non-coding RNAs from existing lists were considered as most of the genomic regions they are derived from are not annotated. In general, large numbers of intronic RNAs have properties comparable to those of stand-alone functional RNA species, suggesting that they are more than just discardable parts of pre-mRNAs. In summary, these results argue for a global change in thinking away from one where intronic RNAs are automatically relegated into the pre-mRNA category. Rather, the community should analyze RNAs from these regions with the same interest and rigor as it does mRNAs or linc RNAs.Female Balb/c mice, approx. 6–8 weeks old  were used in this study as a well-established model of LPS-induced inflammation. All animals were weighed and randomized prior to their first challenge: in consideration of their weight they were distributed evenly to groups of eight animals each. For the induction of respiratory inflammation, all animals were exposed to a defined LPS aerosol, except the negative control group which was exposed to clean air only. The aerosols were generated using an Aeroneb nebulizer. The LPS inhalation was done using aerosolized lipopolysaccharide (deposited dose approx. 20 ng LPS) with 0.021% LPS solution for an inhalation period of 10 min on three consecutive days. After the final challenge a necropsy was performed at the following time points: 0 , 3, 6, 12, 24 and 48 hours.Animals were treated using the vehicle 7 days before first inhalative challenge and daily 1 hour before challenge to LPS or clean air (the control group). Animals from all groups were sacrificed painlessly with an overdose of pentobarbital sodium (Narcoren®) 0 (control) and 3, 6, 12, 24 and 48 hours after LPS-challenge. Seven animals were assessed per time point. The inflammatory status in lungs was analysed including the numbers of macrophages/monocytes, neutrophils, eosinophils and lymphocytes by counting a total number of 300 cells per cytospot.Whole lungs were excised, cut into small pieces (max 0.5 cm diameter) and transferred in a sufficient volume of RNAlater (Ambion) and frozen in liquid nitrogen. The tissue samples were stored at −70°C until RNA isolation.Total RNA from tissue samples and cell lines was extracted using TRIzol® reagent  using manufacturer’s protocol. The sample was homogenized while suspended within an appropriate volume (10x sample volume) of reagent. After a brief incubation at room temperature, the samples were centrifuged to remove insoluble material and the supernatant transferred to a fresh tube. Choloroform (0.2x TRIzol volume) was then added to the contents of the new tube and vigorously vortexed. After a few minutes of incubation at room temperature samples were then centrifuged for 15 minutes at 12000 x g at 4°C. This results in the formation of three distinct phases. The topmost aqueous phase, which contains RNA, was then carefully transferred to a new tube. The addition of isopropyl alcohol (0.5X TRIzol volume) to the samples followed by a 10 minute room temperature incubation and a subsequent 10 minute centrifugation at 12000 x g at 4°C precipitated the RNA into a gel-like pellet at the bottom of the tube. After the removal of the supernatant, the pellet was washed twice in 1 ml of ice-cold 75% ethanol. The resultant pellet of RNA was then allowed to dry for about 5–10 minutes and finally resuspended in DEPC-treated water. The quality and the quantity of the resulting RNA was then measured using spectrophotometric techniques on a NanoDrop instrument (Thermo Scientific).Total RNA was first DNase treated to remove any residual DNA. Approximately 40 μg of total RNA , 2 μl Rnase-out  in a total volume of 200ul) is incubated for 30 minutes at 37°C. Samples are then cleaned using the RNeasy MinElute cleanup kit  following manufacturer’s protocol. In brief, 700 μl Buffer RLT and 500 μl 100% Ethanol are added to the sample which is then added to a MinElute spin column. The columns are washed with 500ul Buffer RPE followed by 500 μl 80% Ethanol. After an additional 2 minute centrifugation to remove any residual ethanol the sample is eluted in 14ul DEPC treated water. Quality of RNA was then assessed using an Agilent 2100 Bioanalyzer and their RNA 6000 Nano Kit  using manufacturer's protocol and the sample was quantified using a Nanodrop as per above. Samples were depleted for rRNA through the use of the RiboMinus Eukaryote Kit for RNA-Seq  following the manufacturer's protocol. The RNA was then converted into cDNA, and subjected to SMS as previously described[All genomic coordinates listed throughout this work correspond to the mm9 version of the mouse genome.We extracted intronic intervals from the knownGene.txt file downloaded from the UCSC genome browser site, based on the mm9 version of the mouse genome and last modified 30-May-2011 00:13. We then removed parts of introns that overlapped exons annotated in the same knownGene.txt file, also see Additional fileTo calculate intronic densities, we counted number of reads that fall within each intronic interval  to generate the “Total Intron-Exon Per Time-Point Dataset”. One complication that we faced was that the CV of a transcript level among animals depends on the expression level of a transcript, with more abundant transcripts having less variation – as would be expected, since more abundant transcripts could be measured more reliably than the less abundant ones. Indeed, the Spearman rank correlation between average RNA signal density and the corresponding CV was −0.58, indicating that transcripts with lower abundance have higher CV. Since introns had a tendency to have lower abundance than exons, their variation would be higher due to the intrinsically higher variation of detection of lower-abundant RNAs. To avoid intronic transcripts with low levels of expression in this experiment, we sorted the “Total Intron-Exon Per Time-Point Dataset” by average intronic density and selected the top half of data points for analysis where average exonic density was positive . The median CV for introns and exons in the resulting dataset were 35.37% and 18.97% respectively and the median density of an intron being 18.16% of the corresponding exons.t-test was used to estimate the fold change significance across 7 animals and p-value < 0.001 cutoff was applied. If one group consists only of zero values, then artificially 0.5 read is added to one animal and 0.5 read is subtracted from another animal of the group before expression density and Student score calculation.Optimal detection of regions of transcription requires the use of nested bins of different sizes. For example, exons of protein-coding mRNAs that have a median size on the order of 100 bp in the mouse genome would be expected to be optimally detected with a smaller size bin on the order of 100 bp. On the other hand, relatively low abundance longer transcripts would be detected best with a bin of longer size that has a chance to capture more SMS reads representing such transcripts. Therefore, genome sequence was split into non-overlapping bins of variable size 100, 200, 500 and 1000 bp and expression density of each bin for each animal for each time point was calculated. Up-regulated and down-regulated bins  on each time point comparing to control (which is zero time point) were identified. One-tailed paired Student’s Bins of different sizes were then merged together. Whenever a bin of smaller size overlapped a bin of a larger size, a bin of the smaller size was chosen as it is more likely to represent the accurate bounds of the detected transcribed element. The power of a more precise bin approach is illustrated by detection of a specific isoform of Adora 3 consisting of only two exons out of a total of 9 known for this locus and upregulated at the 3 hour time point  of annotated UCSC Genes were selected from list of up-regulated and down-regulated on 3 hrs after LPS bins described above. Up-regulated or down-regulated bins where the corresponding gene had respectively at least one up-regulated or down-regulated exon at 3 hrs after LPS  were filtered out from the list.1,383 snoRNAs annotated by the Ensembl and not UCSC Genes database were selected to assess enrichment of functional non-coding RNAs presented in TablewhereN - number of unique Introns in the tested dataset,M - number of unique Introns from tested dataset passing the threshold,n - number of unique Introns from N overlapping snoRNAs,m - number of unique Introns from M overlapping snoRNAs.First strand synthesis was performed using Superscript III  following the manufacturer’s protocols with 200 ng Total DNase-free RNA (see above) as a template. See the Additional fileAuthors declare no competing interests.DS, TE and PK performed the statistical and bioinformatics analyses; MT and ZY performed sample preparation and SMS sequencing; CW and SUI provided theoretical insights; GstL, BS and TAM designed and performed the inflammation time course study; PK and GstL provided the theoretical framework and guidance for the intronic RNA analysis and wrote the manuscript. All authors read and approved the final manuscript.Figure S1. A scheme of the strategy to partition intronic coordinates in the cases of overlapping transcripts. Boxes –exons, lines – introns. Regions 1–4 were used to calculate the average read density of the corresponding introns.Click here for fileTable S1. Correlation of specified introns of Prkca and Slc24a3 with their corresponding exons.Click here for fileTable S2. Properties of individual mouse introns.Click here for fileTable S3. Details of RT-PCR analysis in selected mouse introns.Click here for fileFigure S2. An example of DE bins specifically detecting a specific up-regulated isoform of Adora3 locus.Click here for fileFigure S3. Examples of DE bins detecting regions around annotated miRNAs, found both in an intron  and an interegenic region .Click here for file


In vivo knockdown of GAP-43 in olivary neurons, leads to the atrophy of their CFs and a reduction in the ability to sprout toward surrounding denervated PCs. These findings demonstrate that GAP-43 is essential for promoting denervation-induced sprouting and maintaining normal CF architecture.Structural plasticity occurs physiologically or after brain damage to adapt or re-establish proper synaptic connections. This capacity depends on several intrinsic and extrinsic determinants that differ between neuron types. We reviewed the significant endogenous regenerative potential of the neurons of the inferior olive (IO) in the adult rodent brain and the structural remodeling of the terminal arbor of their axons, the climbing fiber (CF), under various experimental conditions, focusing on the growth-associated protein GAP-43. CFs undergo remarkable collateral sprouting in the presence of denervated Purkinje cells (PCs) that are available for new innervation. In addition, severed olivo-cerebellar axons regenerate across the white matter through a graft of embryonic Schwann cells. In contrast, CFs undergo a regressive modification when their target is deleted.  Structural plasticity is limited in the central nervous system (CNS) of adult mammals, constituting a significant impediment to recovery from injuries such as those caused by trauma, stroke, and neurodegenerative and demyelinating diseases Duffau, . NeverthThe cerebellar climbing fiber (CF), the terminal arbor of the olivo-cerebellar axons, has provided the first example, in the mammalian CNS, of individually observed fibers undergoing sprouting after brain injury . CFs undergo lesion-induced sprouting, activity-dependent remodeling, expansion of their area of innervation in response to an enlarged target territory, and regressive modifications after elimination of their target  respond dramatically to axonal injury. The resection of olivo-cerebellar axons leads to the regression of the remaining stump and the death of many axotomized neurons in the IO during the first few weeks after injury  are linked to agenesis of the corpus callosum and severe mental retardation , a model of human muscular dystrophy, in which degeneration-regeneration events in muscle fibers are accompanied by remodeling of intramuscular terminal nerve fibers , mossy fibers grow ectopically to their normal target layer, innervating the distal L1CAM and NCAM are expressed at constitutively high levels in the IO (Horinouchi et al., In addition to maintaining of CF structure, GAP-43 might also govern the organization of the presynaptic terminal and, consequently, neurotransmitter release. When GAP-43 is silenced, CF varicosities undergo alteration in morphology, becoming rounder and larger compared with control varicosities (Grasselli et al., Several studies have also established the involvement of GAP-43 in neurotransmitter release and synaptic plasticity (Dekker et al., Thus, increasing evidence suggest that GAP-43 has a double role in mature CFs in sustaining both injury-induced sprouting and the maintaining their structure under normal conditions, possibly by mediating cytoskeletal reorganization that is triggered by cell adhesion molecules and CFs interactions with their target. In addition GAP-43 appears to regulate the organization of CF presynaptic terminal and neurotransmitter release.Emerging technologies, such as 2-photon microscopy and laser axotomy, will allow us to monitor cells during injury and repair in live mammalian brains and induce microscopic lesions, enabling us to determine the sequence of structural remodeling events that occur in single fibers after axotomy (Holtmaat and Svoboda, 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.


Previous studies suggest that the responsiveness of TrkB receptor to BDNF is developmentally regulated in rats. Antidepressant drugs (AD) have been shown to increase TrkB signalling in the adult rodent brain, and recent findings implicate a BDNF-independent mechanism behind this phenomenon. When administered during early postnatal life, ADs produce long-lasting biochemical and behavioural alterations that are observed in adult animals.We have here examined the responsiveness of brain TrkB receptors to BDNF and ADs during early postnatal life of mouse, measured as autophosphorylation of TrkB (pTrkB).ex vivo. Basal p-TrkB status in the brain of BDNF deficient mice was significantly reduced only during early postnatal period. Enhancing cAMP (cyclic adenosine monophosphate) signalling failed to facilitate TrkB responsiveness to BDNF. Reduced responsiveness of TrkB to BDNF was not produced by the developmental increase in the expression of dominant-negative truncated TrkB.T1 because this reduction was similarly observed in the brain microslices of trkB.T1−/− mice. Moreover, postnatal AD administration produced long-lasting behavioural alterations observable in adult mice, but the responses were different when mice were treated during the time when ADs did not (P4-9) or did (P16-21) activate TrkB.We found that ADs fail to induce TrkB signalling before postnatal day 12 (P12) after which an adult response of TrkB to ADs was observed. Interestingly, there was a temporally inverse correlation between the appearance of the responsiveness of TrkB to systemic ADs and the marked developmental reduction of BDNF-induced TrkB in brain microslices We have found that ADs induce the activation of TrkB only in mice older than 2 weeks and that responsiveness of brain microslices to BDNF is reduced during the same time period. Exposure to ADs before and after the age when ADs activate TrkB produces differential long-term behavioural responses in adult mice. Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are critical regulators of neuronal survival, differentiation, axonal and dendritic growth, and synapse formation BDNF and TrkB play a central role in the mechanism of action of antidepressant drugs (AD) Previous finding suggests that the responsiveness of TrkB to BDNF is regulated during development; BDNF readily activates TrkB in early postnatal rat brain microslices, but this effect of BDNF is strongly blunted after the second week postnatally bdnf and trkB mRNA expression in mice Exposure to ADs during early life has been shown to produce long-lasting behavioural changes in rodents that are evident even in adulthood. Specifically, depressive and anxiety-like, motor and sexual behaviours are modified in adulthood ex vivo BDNF at different stages of mouse postnatal development. Our results suggest the intriguing hypothesis that during the postnatal development, there is a shift in TrkB responsiveness from a receptor that is readily activated by BDNF but refractory to ADs to a TrkB clearly phosphorylated by ADs but only weakly activated by BDNF. This shift may play a role in the development of certain adult emotional phenotypes.We have here studied the phosphorylation response of TrkB to systemic ADs and We have previously shown that a single intraperitoneal (i.p.) injection of AD induces phosphorylation of TrkB within an hour in the prefrontal cortex (PFC) and hippocampus (HC) of adult rodent brain Our results showed that, along postnatal period, TrkB response to ADs is age-dependently regulated. In the PFC, acute IMI treatment of mice between P5 and P11 failed to increase TrkB phosphorylation at the PLCγ1 site (pY816) . Furthermore, as already reported in adult brain data not shown).We have previously shown that ADs specifically induce phosphorylation of TrkB at the PLCγ1 site (Y816) and the phosphorylation of CREB (cAMP response element binding protein), the downstream target of this pathway, whereas no increase in phosphorylation levels is observed at the Shc site (pY515) or its downstream target Akt et alFigure S2).Knüsel et aldata not shown).The studies by Knüsel The developmentally reduced ability of BDNF to induce TrkB phosphorylation in the mouse brain microslices is probably not due to reduced penetrance of BDNF into mature brain tissue since a highly related neurotrophin, NGF (nerve growth factor), readily induced TrkA phosphorylation (Y674/5) in hippocampal microslices obtained from P24 mice  Bdnf+/− mice  and/or serotonin (5-HT) that subsequently can activate Gs –linked postsynaptic receptors. Consequently, we next tested whether acute treatment with IMI might restore the responsiveness of TrkB to BDNF ex vivo in adult hippocampus. As expected, IMI treatment increased the pTrkB levels in the hippocampus but no further phosphorylation was observed when BDNF was applied .Cyclic adenosine monophosphate (cAMP) signalling has been shown to facilitate or “gate” BDNF-induced TrkB signalling in mature hippocampal neurons ex vivo assay in brain microslices obtained from adult mice pre-treated with fluoxetine (FLX) via drinking water (0.08 mg/ml) for 21 days Long-term systemic treatment with antidepressant fluoxetine has been shown to reopen developmental-like plasticity in the adult rodent brain in vitro cell-free kinase assay to examine whether there are any structural modifications in the TrkB receptor protein itself that might prevent BDNF from binding to and activating TrkB in the mature brain tissue. When the adult PFC and HC homogenates were subjected to the kinase assay in the presence of adenosine triphosphate , a clear increase in TrkB phosphorylation in response to BDNF was observed (ex vivo assay (.We used an data not shown), suggesting that long-lasting alterations of the levels of BDNF or TrkB do not explain the behavioural phenotype observed in adult animals.Exposure of rodents to ADs during postnatal life has been shown to elicit alterations in emotional behaviour that are evident in adulthood, long after the cessation of the treatment Table S1 and post hoc test showed that mice exposed to L-PS CLO show a significantly increased Time in the light .When we tested behaviour of adult animals at 3 months of age, we found that animals exposed to CLO during E-PS or L-PS, long before the behavioural testing, showed differential alterations in exploratory locomotion and anxiety-related behaviour that correlated with the age of AD treatment. Specifically, exposure to CLO treatment produced strong long-term behavioural effects in the Light-Dark test when the drug was given during the L-PS, but not when it was administered during the E-PS (Table S1 and post hoc test showed a reduced latency to feed in mice treated with CLO in the E-PS when compared to control mice at the same age (p<0.05) (Table S1 and Table S1), but the post hoc test did not show any difference among controls and CLO treated animals. No differences were found between the groups for weight loss before the test for postnatal age and treatment (Table S1).In the novelty-suppressed feeding (NSF) test, only the administration of CLO during the E-PS significantly influenced adult behaviour. In this paradigm, food-deprived mice are presented a food pellet in a novel environment and the latency to approach the food is measured ex vivo BDNF stimulation in late postnatal stage and adulthood. AD exposure before or after this developmental switch produces differential long-lasting effects on motor and anxiety-like behaviours in adult animals. Although an inverse temporal correlation between the appearance of the responsiveness of TrkB to systemic ADs and the robust reduction of BDNF-induced TrkB phosphorylation in ex vivo microslices cannot be taken as any evidence of causality, it is tempting to speculate that the same maturation processes that bring about the AD responsiveness, lead to developmental changes that restrict the effects of BDNF on TrkB.In the present study, we observed an age-dependent switch from a TrkB receptor readily activated by BDNF but not by ADs during early postnatal stage to a receptor activated by ADs but less sensitive to direct Pharmacologically, ADs such as the drugs used in the present study bind to and inhibit the monoamine reuptake pumps which lead to enhanced monoamine receptor signalling. While monoaminergic neurons are among the first neurons to be generated during early embryonic development, full maturation of these cells extends beyond postnatal life in rodents Ser133 is induced by a variety of signalling pathways, including PKA (protein kinase A), Ca2+-calmodulin kinase II and Mitogen-activated protein kinase pathways AD-induced TrkB phosphorylation during late postnatal period takes place specifically at the Y816 (binding site of PLCγ1), but not at the Y515 (binding site of Shc), which is consistent to our previous findings in adult brain et alWe have here confirmed and extended the unexpected observations previously reported by Knüsel trkB.T1−/− mice. Another possible explanation could be that the ability of BDNF to induce TrkB activation in the mature brain depends on additional gating mechanisms such as cAMP or adenosine i signalling, might facilitate or restore the ability of BDNF to activate TrkB, but this did not turn out to be the case. Moreover, direct up-regulation of [cAMP]i signalling in the ex vivo assay failed to facilitate BDNF-induced TrkB phosphorylation in mature microslices. It is possible that functional maturation of neuronal networks and closure of sensitive periods may modify the responsiveness of TrkB to BDNF (and ADs). Chronic treatment with AD fluoxetine has been shown to reopen developmental-like plasticity in the adult rodent cortex ex vivo BDNF in the adult mouse brain. However, the cell-free TrkB kinase assay suggests that there does not appear to be any structural modifications in the TrkB protein itself that would prevent the receptor from binding to and being activated by BDNF.It was previously suggested that the developmentally regulated increase in the truncated TrkB.T1 receptor that can act as dominant-negative partner for full-length TrkB, might explain the specific reduction of the responsiveness of TrkB to BDNF. We ruled out this possibility, since hippocampal TrkB receptors responded similarly to BDNF and ADs in the tissue samples prepared from WT and ex vivo assay after early postnatal life remains unclear. Although recent evidence suggest that lack of BDNF has only minor effects on the survival and structure of cortical neurons in the CNS in vivo infusion of BDNF into the adult brain increases TrkB phosphorylation, albeit only at relatively high concentrations via TrkB bdnf+/− mice and in mice over-expressing an inhibitory form of trkB (TrkB.T1) in brain The reason for and significance of the apparently complete loss of TrkB responsiveness to BDNF , 2, the brains quickly removed and bilateral hippocampus and prefrontal cortex were dissected out on a dish cooled on dry-ice. Samples were homogenized in a NP++ buffer  and 2 mM Na3VO4). After incubation on ice for 15 min, samples were centrifuged  and the supernatant collected for further analysis.Dams with their litters were housed individually. For postnatal acute antidepressant treatments, age-matched litters (P5-21) were randomly assigned to receive i.p. injection of saline (SAL) , imipramine (IMI) , or clomipramine  and following indicated lag-time (30–60 min) hippocampus and medial prefrontal cortex were collected as described in Figure S4).Dams with their litters were housed individually. The litters were randomly assigned to 4 groups: saline injected (SAL) , and clomipramine injected (CLO)  Mice were weaned on P22 and males were housed together (4–6 mice/cage) until the behavioural experiments. For biochemical analyses the animals were killed after 2 weeks of the last behavioural test and their tissues were collected and processed as described before.For the acute AD treatments, adult (∼P90) mice received a single i.p. injection of imipramine  or SAL and were killed 30 minutes after. For chronic AD treatment, adult (∼P90) mice had free access to either tap water or to 0.08 mg/ml solution of fluoxetine  for 21 days ex vivo BDNF stimulation assay was performed according to Knüsel et alThe et al2, 0.05% BSA, 2% DMSO with or without 100 µM ATP and/or 50 ng/mL BDNF. The reaction was initiated adding 40 µg of NP++ lyzed protein extract to the mix and the incubation was allowed to proceed for 10 min at 37°C. For a subset of samples, the reaction was quenched by adding an equal volume of 4× Laemmli sample buffer and proteins were separated by SDS-PAGE and TrkB phosphorylation status analyzed with western blotting as described below. Rest of the samples we immediately transferred to Trk antibody containing ELISA (enzyme-linked immunosorbent assay) plates, 3% BSA/PBS-T (+2 mM Na3VO4) added ad 200 µl, and phospho-TrkB ELISA proceeded as previously described Assays were performed according to the procedures described by Angeles Y816 , anti-p-TrkA/BY490/Y515 , MA, USA), anti-p-TrkA/BY674-5/Y705-6 , anti-TrkBout , anti-p-CREBS133 , anti-CREB , CA, USA), anti-p-AktThr308 , anti-AKT , anti-p-PLCγ1Y783  or anti-Trk (#sc-11 (rabbit), 1∶1000, SCB). Further the membranes were washed with TBS/0.01% Tween® (TBST) and incubated with horseradish peroxidase conjugated secondary antibodies . After subsequent TBST washes, secondary antibodies were visualized using electrochemiluminescence kits (Amersham Biosciences) followed by an exposure to Fuji LAS-3000 Camera  for ECL detection.Sample protein concentrations were measured using the Lowry Method (Bio-Rad DC protein assay). Lectin precipitation was carried out essentially as described Battery of behavioural tests Testing was performed for 10 min in an acrylic cage (28.5×28.5×20 cm)  divided into two equal sized compartments: one part with transparent walls, open topped and brightly illuminated (∼450 lx by a 40 W light bulb fixed 55 cm above the floor), the other part made from black plastic (passing infrared light) and covered by a lid. The two compartments were separated by a partition containing an opening (7×5 cm) in its centre at floor level. The animal was placed in the centre of the light compartment facing away from the opening, and the latency to enter dark area, time spent in the compartments, total distance travelled, immobility time and the number of entries to dark were measured over 10 min. In addition, rearing time was also calculated. Testing apparatus was thoroughly cleaned before each animal using 70% ethanol.ad libitum. On testing day each animal was removed from its home cage and placed in a holding cage for 30 min before the test and then put in one corner of the arena. The latency to beginning of a feeding episode was measured (maximum over a period of 5 min). Immediately, after starting of feeding the mouse was removed from the arena and placed with the food pellet in its home cage and allowed to feed ad libitum over a period of 5 min. The amount of food consumed was quantified by weighting the pellet.Novelty-Suppressed Feeding: The Novelty-Suppressed Feeding (NSF) was performed as previously described post hoc test. The criterion for significance was set to p<0.05.Immunoblot bands were quantified using NIH ImageJ. All the data are expressed as mean ± SEM (Standard Error of Mean) and as percentage of control. Statistical analyses were performed using GraphPad Prism 4.0 for Windows . For comparison between two groups Two-way Student's t-test was used. Two-way ANOVA or two-way ANOVA for repeated measures was used to reveal main effect and interaction between the factors followed by Bonferroni Figure S1Effect of imipramine on the phosphorylations of TrkB Shc binding site and Akt during development. (a) Phosphorylation of TrkB Shc binding site (Y515) after acute imipramine treatment  in prefrontal cortex (PFC) and hippocampus (HC). (b) Phosphorylation of Akt (Thr308) after acute imipramine treatment  in prefrontal cortex (PFC) and hippocampus (HC). Phospho-protein values are normalized against corresponding total protein levels. (TIF)Click here for additional data file.Figure S2Acute BDNF stimulations does not regulate total TrkB protein levels in brain microslices. Representative blots showing the levels of full-length TrkB after control or BDNF stimulation in cortical (PFC) or hippocampal (HC) microslices prepare from P5-P60 old mice.(TIF)Click here for additional data file.Figure S3Imipramine and fluoxetine does not directly induce TrkB phosphorylation in hippocampal microslices ex vivo. P20 hippocampal microslices were incubate with vehicle or different concentrations of imipramine  (a) or fluoxetine  (b) for 15 min at 37°C and TrkB phosphorylation (Y705/6) analyzed with western blotting. n = 3 per group.(TIF)Click here for additional data file.Figure S4The effect of postnatal clomipramine on weight gain of mouse. A daily dose of clomipramine  or saline during early  or late  postnatal period produced no changes on weight gain during (P4-21) (a) or after the treatments (P28-120) (b). n = 10–15 per group.(TIF)Click here for additional data file.Table S1Postnatal treatments with clomipramine lead to long-lasting and distinct behaviours depending on early exposure period. The effect of early  and late  postnatal clomipramine treatment  on behaviour in light-dark box test and novelty suppressed feeding test in adult (P90) animals. Two-Way ANOVA followed with Bonferroni post hoc test was performed for statistical analysis; *P<0.05, ** P<0.01. n = 10–15 per group.(DOCX)Click here for additional data file.


New paradigms have been recently proposed in the pathogenesis of both chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), evidencing surprising similarities between these deadly diseases, despite their obvious clinical, radiological and pathologic differences. There is growing evidence supporting a "double hit" pathogenic model where in both COPD and IPF the cumulative action of an accelerated senescence of pulmonary parenchyma (determined by either telomere dysfunction and/or a variety of genetic predisposing factors), and the noxious activity of cigarette smoke-induced oxidative damage are able to severely compromise the regenerative potential of two pulmonary precursor cell compartments . The consequent divergent derangement of signalling pathways involved in lung tissue renewal (mainly Wnt and Notch), can eventually lead to the distinct abnormal tissue remodelling and functional impairment that characterise the alveolar parenchyma in these diseases . Chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) are two severe multifactorial pulmonary disorders characterised by quite distinct clinical and pathological features. COPD is characterised by a poorly reversible and progressive airflow limitation that is determined by the concurrence of airways inflammation and emphysema (from now on both included in the acronym COPD),2, whereNevertheless, a number of similarities can be recognised between the two disorders. Firstly, both COPD and IPF are chronic and progressive diseases of elderly people , that severely affect the lung function, and both are related to long term inhalation of external noxious agents (mainly tobacco smoking),4,6,7. SDespite the great deal of research, effective treatments are lacking for both COPD and IPF. This can be a consequence, at least in part, of the limited understanding of their pathogenesis, despite the overwhelming plethora of studies and theories proposed so far. Interestingly, for both diseases a gradual shift from "inflammatory-based" pathogenic theories to more complex approaches occurred in recent years ,13. In tWAF1 and p16INK4a )[The most striking new information linking the pathogenesis of IPF and COPD relates to their proposed inclusion within the category of diseases with alveolar senescence and lung "premature aging" -33. The 16INK4a ),25,34,3516INK4a ),30. In a16INK4a ).Although abnormalities directly affecting telomerase genes have not been demonstrated in COPD, telomere decreased length has been demonstrated in either lung cells or peripheral leukocytes in COPD patients, compared with control subjects ,24-27. TThe pathogenic role of genetic abnormalities in IPF is much more evident than in COPD, and familial IPF has been recently included within the category of genetic diseases with "telomere dysfunction" together with acquired aplastic anaemia and dyskeratosis congenita -44. ThesFamilial IPF cases have been linked not only to mutations of telomerase genes, but also to mutations in surfactant proteins coding genes (surfactant protein-C and A2)-54. ChroThus different mechanisms can be responsible of the progressive loss of pneumocyte precursors in IPF, including accelerated senescence, surfactant abnormalities and endoplasmic reticulum stress, all potentially causing precursor cell exhaustion and abnormal alveolar re-epithelialisation -59. The In COPD a large amount of data suggest that parenchymal remodelling and progressive dilation of alveolar spaces is related to decreased and/or deregulated production of extracellular matrix proteins, in particular elastin, with the eventual impaired capability to sustain connective and epithelial tissue repair. Connective tissue insufficiency can be either caused by genetic defects as observed in alpha-1 anti-trypsin deficiency , or it may be ascribed to ill defined causes inducing mesenchymal precursor cell senescence and progressive decrease of matrix protein production, as suggested in this review. Accordingly, senescence related markers in COPD are mainly demonstrable in mesenchymal cells  , and a vAll this taken into account, it is possible to hypothesise that in the pathogenesis of both COPD and IPF the main driving abnormality is the precocious senescence of pulmonary parenchyma.But how can be reconciled this proposed similarity between the basic pathogenic mechanisms underlying COPD and IPF with the obvious diversity of their clinical and pathological presentations? A possible explanation can be searched in the diversity of the genetic alterations, including genetic/epigenetic inheritance or predisposing gene polymorphisms, compromising the renewal capacity of different target cells -71. As pHuman and experimental studies provide strong evidence that molecular networks regulating parenchymal lung tissue renewal are perturbed in both COPD and IPF, although in different ways. Among these networks, particular relevance has been focused on the interplay between Notch and Wnt, two signalling pathways playing critical roles in epithelial and mesenchymal precursor cell maintenance and differentiation -78.In IPF the senescent phenotype seems to mainly affect the epithelial precursors of alveolar tissue (type-II pneumocytes), thus preventing a correct epithelial renewal at anatomical sites where mechanical stress and alveolar damage are expected to be maximal  ,79. Pneuwaf1 and p53 [But how can senescent pneumocytes abnormally trigger these pathways in IPF? Signals provided by the milieu of damaged alveolar cells can trigger a variety of reparative mechanisms, including the recruitment and stimulation of endogenous and exogenous progenitors , and it  and p53 ,97. Smal and p53 , express and p53 ,99. Bron and p53 ,56,99. I and p53 . Further and p53 ,100. Int and p53 .Interestingly, the same signalling pathways involved in IPF (Wnt- and Notch), seem to have a relevant role also in COPD, but in the opposite way. In fact, both Wnt- and Notch- appear as significantly inhibited in COPD, rather than activated as observed in IPF ,102,103,The observed inhibition of Wnt-signalling in COPD may be ascribed to a variety of concurrent causes, including the smoking-related up-regulation of extracellular Wnt antagonists (e.g. secreted frizzled-related proteins) . InteresAll these data taken together, it is possible to hypothesise that in COPD the abnormal senescence of mesenchymal precursors can cause both an impaired production of extracellular matrix proteins (e.g. elastin), as well as a derangement of the interplay between signalling pathways that regulate alveologenesis (in particular Wnt and Notch). These abnormalities are likely sufficient to cause the progressive weakening of the scaffold interstitial structures sustaining the pulmonary parenchyma, with eventual alveolar dilatation and emphysema.Interestingly, the here proposed pathogenic scheme, centred on alveolar senescence and mesenchymal precursor cell insufficiency, can in part reconcile some controversial issues regarding the significance and role of inflammation and autoimmunity in the development of airway disease occurring in COPD ,109-115.In summary, in both COPD and IPF a common pathogenic scheme can be traced where an accelerated cellular senescence determined by the "two hits" paradigm (genetic predisposition to cell senescence with the concurrence of tobacco smoke), determines an impaired regeneration of the lung parenchyma after damage. The divergence in two horns in this model is provided by the affected precursor cells , the relevance of genetic background, as well as by the basic signalling pathways involved in the development of either emphysema or fibrosis Figure . Both meThe complexity of this network is difficult to be completely deciphered in both IPF and COPD, since beyond precursor cell senescence, as here described, other genetic predisposing factors and molecular mechanisms are likely involved in both diseases, including micro-RNA regulation -133. IntThese evolving concepts open new options to better understand the pathogenesis of both IPF and COPD, as far as the involvement of both parenchymal and small airway components are concerned ,143, andThe authors declare that they have no competing interests.All Authors participated in the design of the review and helped to draft the manuscript. All authors read and approved the final manuscript.