The influence of multi-pass cold drawing on the evolution of microstructure, texture, and properties of Cu matrix composite, reinforced by in situ grown graphene, has been systematically investigated. Under continuous and severe plastic deformation, the grains in the composite were continuously refined to nanoscale. In addition, graphene in the composite could be gradually refined, exfoliated, and redispersed. Interestingly, dynamic recrystallization of the composite was formed after 80% drawing reduction and its formation mechanism was discussed. The texture of the as-drawn composite comprised a mixture of fiber textures with dominated <111> and minor <100> orientation after 99.7% severe drawing reduction. The tensile properties and electrical conductivity of the as-drawn composites were also investigated. This work provides a better guideline on the plastic deformation behavior of the advanced graphene/metal nanocomposite. Metal matrix composites reinforced by graphene (Gr/MMCs) have recently attracted considerable attention and researchers strive to improve its structural or functional properties ,2,3,4,5.As a result, the plastic deformation of composites is the next step. In addition, it is crucial to the realization of the transformation from blank materials to semi-manufactured products. Commonly, several plastic deformation methods, such as extrusion , rollingHerein, graphene/Cu composite wires were prepared by in situ grown graphene on Cu powders, spark plasma sintering (SPS), hot-extrusion, and multi-pass cold drawing. During the process of cold drawing, grains in the composite could be continuously refined to nanoscale and the homogeneity of microstructure was improved. Moreover, graphene in the composite was found to be gradually refined, exfoliated, and redispersed. Interestingly, after severe plastic deformation, the recrystallization of composites was formed. The underlying deformation mechanism was discussed. As a result, the composites showed an improvement of mechanical properties and electrical conductivity after cold drawing. This work provides a deep understanding of the plastic deformation process of Gr/MMCs and can be a guide for the preparation of Gr/MMCs with high performance.2 (17 mL/min)/Ar (83 mL/min) mixture. Thereafter, in situ grown graphene on Cu composite powders could be achieved.Commercial flake Cu powders (purity of 99.9%) with a diameter of 30~50 μm and solid molecular naphthol ) were used for the raw Cu matrix and carbon source, respectively. The typical growth process of graphene, which was systematically studied in our recent investigation , can be The as-prepared composite powders were first sintered using SPS  . Then, a rod (Φ9.5 × 40 mm) was cut from the as-sintered composite. Subsequently, the rod was extruded at 750 °C (extrusion ratio of 7.4:1). The as-extruded composite (diameter of 3.5 mm) was drawn by the multi-pass process at room temperature and the reduction of each single pass was less than 10%. Finally, the as-drawn composites with different diameters were obtained. The area reduction is calculated as the drawing reduction, and 3 (5 wt%) to remove Cu. The composites for TEM analysis were prepared by several steps of mechanical grinding to obtain a foil (thickness of 50~60 μm), and ion thinning technique. The C content of graphene in Cu matrix is tested by carbon and sulfur analyzer . The room temperature tensile properties of block composites were studied by an Instron-1186 tensile testing machine. In addition, the tensile speed, gauge length, and gauge width were 0.5 mm/min, 15 and 2 mm, respectively. The tensile properties of as-drawn composites (length of 20 mm) were measured on a tensile machine  with a speed of 0.5 mm/min. The resistance of the samples was tested on a low resistance instrument based on the four-wire measurement method . The electron backscatter diffraction  of the composites was carried to investigate the microstructure of the composite. The EBSD system is HKL Nordlys , and the data processing software is Channel5. The samples for EBSD investigation were prepared by the cross section ion polishing instrument . For SEM and EBSD test, the observation positions of as-extruded and as-drawn composites were investigated on the transverse direction–normal direction (TD–ND) plane.The microstructure was carried on scanning electron microscopy  and transmission electron microscope . Prior to the SEM investigation of individual graphene in graphene/Cu composite powders, the composite powders were first etched by HNO−1), G band (1603 cm−1), and 2D band (2300~3000 cm−1) of the graphene. The ID/IG (intensity ratio of D to G band) of the as-grown graphene is calculated as 0.89, indicating a low defect density of the graphene [In graphene . All of W) and length (dL) of the grains are counted as 1.88 and 3.9 μm, respectively. Nevertheless, The dispersibility of graphene is one of the most challenging difficulties for the preparation of composite. Nevertheless, this problem can be easily solved by the graphene in situ growth method. Therefore, the as-obtained composites can be a model material for the study of plastic deformation mechanism. As shown in Typical SEM-BSE images of the as-drawn composites are shown in EBSD was used to study the microstructure evolution of the composites during the drawing process, including the evolution of grain size, grain boundaries (GBs), recrystallization, microstrain, and texture. To investigate the characteristics of GBs in the composites, the type and misorentation angle distribution of GBs are presented in Furthermore, Since the composites are drawn at room temperature, how does the mechanism of the dynamic recrystallization form? The underlying reasons are discussed as follows: (i) Initially, for the matrix, the GBs in the initial as-extruded composite are dominated by HAGBs see c. The reDuring the drawing deformation of FCC metals, the <100> and <111> orientation will parallel to the axis direction, and form a mixture of <100> and <111> fiber textures. Among them, the <111> fiber texture is considered to be a stable texture, while the <100> fiber texture is a transitional texture. In addition, the <100> fiber texture is mainly attributed to the recrystallization during deformation ,52. As s6 S/m when the drawing reduction is 99.7%, indicating that the as-drawn composite still has good electrical conductivity. There are complicated factors affecting the EC of the as-drawn composites. First, the increasing number of interfaces resulting from the refinement of grain size and graphene lead to the intensification of electron scattering [attering , which wattering . TherefoIn this work, the cold drawing behavior of in situ grown graphene/Cu nanocomposite, including the evolution of microstructure, graphene, texture, and properties, have been systematically investigated. Graphene sheets were homogeneously distributed in Cu matrix during the cold drawing process. Furthermore, the refinement and exfoliation of graphene could be clearly found with the increasing drawing reductions. The grain of the composite could be continuously refined to nanoscale and the homogeneity of microstructure was improved after severe drawing deformation. In addition, recrystallization of composites after 80% reduction can be formed, which was affected by a synergistic effect of matrix and graphene. The final fiber texture of the as-drawn composite was a mixture of fiber textures with dominated <111> and minor <100> fiber textures. After cold drawing, the composites presented an enhancement of tensile strength and showed excellent electrical conductivity.


The Covid-19 pandemic has had a deep impact on the lives of the entire world population, inducing a participated societal debate. As in other contexts, the debate has been the subject of several d/misinformation campaigns; in a quite unprecedented fashion, however, the presence of false information has seriously put at risk the public health. In this sense, detecting the presence of malicious narratives and identifying the kinds of users that are more prone to spread them represent the first step to limit the persistence of the former ones. In the present paper we analyse the semantic network observed on Twitter during the first Italian lockdown (induced by the hashtags contained in approximately 1.5 millions tweets published between the 23rd of March 2020 and the 23rd of April 2020) and study the extent to which various discursive communities are exposed to d/misinformation arguments. As observed in other studies, the recovered discursive communities largely overlap with traditional political parties, even if the debated topics concern different facets of the management of the pandemic. Although the themes directly related to d/misinformation are a minority of those discussed within our semantic networks, their popularity is unevenly distributed among the various discursive communities. The Covid-19 pandemic has had a deep impact on nearly every human activity; as such, it is not surprising that it has generated a widespread online debate which, in turn, attracted the interest of scholars from different disciplines . Due to super-spreaders.Indeed, the already prolific research on the diffusion of d/misinformation –18 was eThe comparison of several platforms is also the research target of : Gab, FaAcross the entire literature overview, a limited attention is paid to identify the narratives shared by the various users. In this sense, the contribution of , 20 is tnot encoded into the constraints ,,NEMtropyIn the present manuscript, we analysed the narratives emerged on Twitter during the Covid-19 epidemic in Italy. Our data set covered a period of one month (from the 23rd of March 2020 to the 23rd of April 2020) and included approximately 1.5 millions of tweets. As observed in previous studies , even inNEMtropy and described in [In the present paper, we have refined the partition in communities obtained in  by usingribed in , insteadWe represent it as a bipartite network of users and hashtags, where a link connects a user to an hashtag if the former one has used the latter one at least once during the observation period. Then, using a bipartite entropy-based null-model  as a benmediated events stimulate the discussions on Twitter more in DX and FI community: by looking at the temporal evolution of the communities of the semantic network, we were able to identify these events, as TV-appearances of politicians.As observed in , 20, theConfirming other previous studies , the comEven if the literature is limited to a few studies, it has been shown that often non trivial correlations between offline and online behaviours occur –71. In t


Poorly soluble environmental antigens, including carbon pollutants, are thought to play a role in the incidence of human sarcoidosis, a chronic inflammatory granulomatous disease of unknown causation. Currently, engineered carbon products such as multiwall carbon nanotubes (MWCNT) are manufactured commercially and have been shown to elicit acute and chronic inflammatory responses in experimental animals, including the production of granulomas or fibrosis. Several years ago, we hypothesized that constructing an experimental model of chronic granulomatosis resembling that associated with sarcoidosis might be achieved by oropharyngeal instillation of MWCNT into mice. This review summarizes the results of our efforts to define mechanisms of granuloma formation and identify potential therapeutic targets for sarcoidosis. Evidence is presented linking findings from the murine MWCNT granuloma model to sarcoidosis pathophysiology. As our goal was to determine what pulmonary inflammatory pathways might be involved, we utilized mice of knock-out (KO) backgrounds which corresponded to deficiencies noted in sarcoidosis patients. A primary example of this approach was to study mice with a myeloid-specific knock-out of the lipid-regulated transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ) which is strikingly depressed in sarcoidosis. Among the major findings associated with PPARγ KO mice compared to wild-type were: (1) exacerbation of granulomatous and fibrotic histopathology in response to MWCNT; (2) elevation of inflammatory mediators; and (3) pulmonary retention of a potentially antigenic ESAT-6 peptide co-instilled with MWCNT. In line with these data, we also observed that activation of PPARγ in wild-type mice by the PPARγ-specific ligand, rosiglitazone, significantly reduced both pulmonary granuloma and inflammatory mediator production. Similarly, recognition of a deficiency of ATP-binding cassette (ABC) lipid transporter ABCG1 in sarcoidosis led us to study MWCNT instillation in myeloid-specific ABCG1 KO mice. As anticipated, ABCG1 deficiency was associated with larger granulomas and increased levels of inflammatory mediators. Finally, a transcriptional survey of alveolar macrophages from MWCNT-instilled wild-type mice and human sarcoidosis patients revealed several common themes. One of the most prominent mediators identified in both human and mouse transcriptomic analyses was MMP12. Studies with MMP12 KO mice revealed similar acute reactions to those in wild-type but at chronic time points where wild-type maintained granulomatous disease, resolution occurred with MMP12 KO mice suggesting MMP12 is necessary for granuloma progression. In conclusion, these studies suggest that the MWCNT granuloma model has relevance to human sarcoidosis study, particularly with respect to immune-specific pathways. Sarcoidosis. Currently, no single definitive cause has been identified for sarcoidosis, an inflammatory granulomatous disease reported to have an incidence of 33.4 cases/year per 100,000 patients in the United States . SarcoidThe rationale for the construction and study of a carbon nanotube-induced granuloma model of sarcoidosis was based upon numerous epidemiologic studies linking sarcoidosis incidence with environmental carbon exposures. Among the risk factors associated with sarcoidosis are exposures to wood-burning fireplaces or stoves and the occupation of firefighting ,6. SarcoThe goal of our studies was to establish a chronic in vivo model of granulomatous inflammation mimicking sarcoid pathophysiology. In our model, MWCNT are administered via oropharyngeal instillation as a bolus into wild-type (C57/Bl6) mice. Initial dosage ranged from 25 to 100 ug with 100 ug providing consistent granuloma formation maintained up to 90 days after instillation . When coThe transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), is part of a subfamily of nuclear receptors with ligand-inducible activity . PPARγ iInterferon-gamma is a hallmark cytokine of sarcoidosis ,31,32. PIn order to further define the role of PPARγ in granulomatous disease, we utilized macrophage-specific PPARγ KO mice in an attempt to better elucidate pathways associated with sarcoidosis. The most striking feature in PPARγ KO mice 60 days after oropharyngeal instillation of MWCNT was the presence of extensive granuloma formation within the lungs as assessed by histopathology and granuloma scoring indices compared to wild-type mice . FurtherIn a more recent study, we examined the effects of ligand-mediated activation of PPARγ in MWCNT-instilled wild-type mice . ResultsAs fibrosis is present in some 20% of sarcoidosis patients and is a leading cause of mortality , we inveInitial experiments carried out in wild-type mice produced evidence that 60 days after concurrent instillation of the ESAT-6 peptide with MWCNT, both granulomatous involvement and pulmonary fibrosis had significantly increased compared to MWCNT instillation alone . InteresDue to the differences in wild-type and PPARγ KO responses to ESAT-6 inclusion in the MWCNT model, pulmonary retention of ESAT-6 peptide in lung tissues was examined by mass spectrometry. Surprisingly, results indicated a lack of persistence of ESAT-6 in wild-type lungs at 60 days post instillation whereas ESAT-6 remained detectable in 60-day PPARγ KO lungs . Such fiOur previous MWCNT instillation studies had noted elevated numbers of CD3+ T lymphocytes infiltrating pulmonary granulomatous areas in both wild-type and PPARγ KO mice ,27. Our Alveolar macrophage ATP-binding cassette (ABC) transporters are critical to surfactant clearance and pulmonary lipid regulation ,49. In hBased on these findings, a further study was carried out to determine the effects of ABC deficiencies in the MWCNT model . InteresFurther studies of the MWCNT model in both wild-type and PPARγ KO mice revealed another regulatory pathway for ABC transporter control—that of microRNA 33 (miR-33) . Mir-33 To assess the possibility of similar pathway mechanisms in granulomatous inflammation, murine BAL samples from MWCNT-instilled and sham control animals were simultaneously analyzed with human BAL samples from sarcoidosis patients and healthy controls. Transcriptional profiles from the mouse model were compared to those from human samples to identify overlapping molecular programs . To bettQuantitative PCR was used to validate four network genes identified as commonly upregulated in both the murine MWCNT model and in human sarcoidosis. Data confirmed elevated gene expression of MMP12, signal transducer and activator of transcription 4 (STAT4), cathepsin K (CTSK), and lymphocyte protein tyrosine kinase (LCK) . These cOur gene array studies on commonalities between the human sarcoidosis and murine MWCNT granuloma model showed that MMP12 was highly expressed in both species . This suInitial studies of the MWCNT model noted a consistent elevation of the metalloproteinase, MMP12, in both granulomatous tissues and BAL cells of MWCNT-instilled mice , which iA brief comparison of pulmonary mediator findings in the murine MWCNT model and in sarcoidosis patients indicates striking similarities between groups . Among tAs noted in a recent review of in vitro/in vivo sarcoidosis models, more innovative approaches are needed to explore the pathogenesis and pathophysiology of sarcoidosis . A focus


Low frequency perturbations in the chemical potential will consequently lead to variation in resistance, giving rise to 1/f noise. Such current noise amounting to 3, may originate, for example, from charge traps, localized carrier states at the edges, or fluctuations in the dielectric constant of the gate insulator, leading to involved gate voltage dependence of 1/f noise12. Part of the 1/f noise originates from charge carrier mobility fluctuations which may arise a.o. due to substrate roughness, ripples in graphene, gas adsorbates, or coupling to phonons15. In h-BN encapsulated graphene samples, a weak gate modulation in the current fluctuation is seen with peak at Dirac point and saturation at higher charge density17. This reduction in noise is inherent to reduced charge density fluctuations in h-BN encapsulated devices19. In such samples, contact resistance is prone to low-frequency fluctuations10. Recently, it has been observed that current crowding21 plays an important role in 1/f noise at graphene-metal contacts22.The characteristic density of states at Fermi level with linear energy dependence in monolayer graphene23, the 1/f noise mechanisms are expected to be the same as in non-superconducting junctions. However, if the fluctuations are conveyed into fluctuations in the pair correlation length, then even stronger 1/f noise may appear in the critical current 24. In short ballistic SGS junctions fabricated using h-BN encapsulation, the critical current has been demonstrated to grow with the number of transmission channels28. This strong tunability of ballistic SGS junctions makes them as a unique platform for realizing quantum circuits with long-lived coherent states. Microwave spectroscopy on short ballistic SGS Josephson junctions has demonstrated resilience of these junctions against strong magnetic fields, which further enhances their promise for hybrid quantum circuits31. It has been known for quite some time that the low frequency fluctuations are detrimental for the coherent operation of superconducting qubits32. Despite the importance of critical current fluctuations in SGS junctions for their coherence properties and, for instance, for the sensitivity of novel graphene bolometers34, detailed study of low frequency In proximity-induced superconducting graphene junctions (SGS)f noise in graphene devices are basically models that utilize the same phenomenological approaches developed for semiconductor devices37. Correlation between mobility and carrier number density (n) fluctuations38 is expected to take place in graphene according to several models and it has indeed been observed in graphene experiments12. In graphene, short and long range scatterers have distinct roles, which can produce quite different charge density dependencies for 1/f noise in different samples8. The majority of scatterers may also reside in charge traps, which naturally leads to correlation between mobility fluctuations and carrier number39. In small devices, charge noise acting on the gate-sensitive channel resistance of graphene has also been employed to account accurately for low frequency current noise5. In Ref.40, charge traps have been considered in the context of SGS junctions and 5, the central factor is the derivative of the conductance with respect to chemical potential, which gives distinct gate dependence for the variance Theoretical models for 1/f noise in the magnitude of supercurrent induced by superconducting proximity effect in graphene. We employ microwave frequency reflection measurements at f dependence for 7, we find a local noise minimum in the normalized critical current noise In this work we use h-BN encapsulated graphene samples with NbTiN contacts to address 1/41. The metallic leads of the sample were reactively sputtered from NbTi target in Our experiments employed encapsulated graphene Josephson junctions fabricated on strongly doped silicon with 285 nm of thermally grown oxide. The doped silicon acted as a back gate giving an areal capacitance of mentclass2pt{minimp-doping for our sample at the charge carrier density level of l is related to mobility 42. By taking the contact resistance approximately equal to the resistance at gate bias far away from CNP, we estimate the mean free path l with our channel length The main frame of Fig. mentclass2pt{minimmentclass2pt{minimT = 10 mK base temperature. The electrical connections were via bias-T components  by having the necessary reference impedances inside the cryostat and by connecting them via a microwave switch and equivalent cabling to the measurement system. The calibration, however, did not fully account for the employed bond wires, which leaves a small uncertainty of The sample was excited using an rf-carrier signal at voltage levels from 0.045 For the critical behavior of a Josephson junction, it makes a difference whether the rf-carrier voltage or current biases the sample. In the voltage bias regime, there is a critical voltage for the carrier amplitude that depends on the frequency according to the ac Josephson relation L that is in series with a termination resistance C on the order of 2 pF due to 2-cm-long cabling before the 50 L and the reflection phase L and obtain the Josephson inductance as f noise magnitudes.We model our sample as an inductance entclass1pt{minimaentclass1pt{minimaInitially, the samples were characterized around mentclass2pt{minimf noise dependence above 0.2 Hz. At 1.4 f noise components, but the high frequency regime is dominated by the equivalent system noise voltage Figure f part of the noise. Noise of the form f noise, on the other hand, is quite universal and can be employed as a general characteristics for evaluation of the sample quality. At high bias, the separation of the 1/f component by fitting is straightforward as the noise can be fitted directly by a ; for a tunnel junction (TJ) i.e.According to theory, the inverse Jospehson inductance is given by entclass1pt{minimaR at microwave frequencies. When 16. Local modulation of the chemical potential close to the charge neutrality point will create in-homogeneous electron-hole charge puddles across the graphene sample. Variation of “percolating paths” among these charge puddles creates finite fluctuations of local resistance, giving rise to enhanced normalized current fluctuations around the Dirac point. Further away from the Dirac point, the sample resistance becomes dominated by the electrode-graphene interface and the contact-induced 1/f noise becomes dominant.The measured noise in normal state was converted to resistance fluctuations using Eq.  by repla44. The influence of thermally activated phase slips on the measured noise is illustrated in the inset of the Fig. T above 400 mK. At temperatures i.e. normal resistive mode where, eventually, the voltage fluctuations are governed by the resistance noise.In the superconducting state, Josephson inductance fluctuations are dominated either by critical current fluctuations or by phase variation across the junction. At finite voltage, phase variation will be caused by phase diffusion due to phase slips, excited either thermally or by quantum tunneling45. However, as our sample has good contacts, charging energy is not a relevant junction characteristics for it. According to Ref.46, in the limit of large scaled conductance G is the conductance of the sample. From this relation, the effective charging energy at the CNP is on the order of 1 neV which is negligible compared to Josephson energy Phase variation and inductance noise may also be induced by charge noise in quantum Josephson junctions in which charging energy becomes comparable to Josephson energyIn Fig. Even though the difference between S and N case in Fig. entclass1pt{miniman| to enhanced fluctuations due to variation in the proximity induced energy gap in the sample.Near the Dirac point, the normal state resistance noise 47, they showed that the AB relation becomes 49, the latter term can vary substantially with charge density. The inverse proximity effect depends on the balance between the effective volume of the contact region (where the charge density is constant due to strong screening of the superconductor) and the charge density of the tuned 2D-material section. The effective contact length D is the 2D diffusion constant and 47. The reduced proximity gap is given by 11.Small contacts on S-2D-S systems lead to strong modification of supercurrents. For example in the work of Kopnin et al.et al. who developed a chemical potential fluctuation model based on charge traps40. Qualitatively, our data for the superconducting state is in agreement with this theoretical model as the noise grows away from the Dirac point. With charge traps, however, the amount of noise should be related with the derivative The influence of charge traps in superconducting graphene junctions has been investigated theoretically by Pellegrino We have studied low-frequency fluctuations in Josephson inductance in h-BN encapsulated, monolayer graphene SGS junctions using microwave reflectometry at frequencies of


Cell adhesion molecule L1 regulates multiple cell functions, and L1 deficiency is linked to several neural diseases. Recently, we have identified methyl CpG binding protein 2 (MeCP2) as a potential binding partner of the intracellular L1 domain. By ELISA we show here that L1’s intracellular domain binds directly to MeCP2 via the sequence motif KDET. Proximity ligation assay with cultured cerebellar and cortical neurons suggests a close association between L1 and MeCP2 in nuclei of neurons. Immunoprecipitation using MeCP2 antibodies and nuclear mouse brain extracts indicates that MeCP2 interacts with an L1 fragment of ~55 kDa (L1−55). Proximity ligation assay indicates that metalloproteases, β-site of amyloid precursor protein cleaving enzyme (BACE1) and ɣ-secretase, are involved in the generation of L1−55. Reduction in MeCP2 expression by siRNA decreases L1-dependent neurite outgrowth from cultured cortical neurons as well as the migration of L1-expressing HEK293 cells. Moreover, L1 siRNA, MeCP2 siRNA, or a cell-penetrating KDET-containing L1 peptide leads to reduced levels of myocyte enhancer factor 2C (Mef2c) mRNA and protein in cortical neurons, suggesting that the MeCP2/L1 interaction regulates Mef2c expression. Altogether, the present findings indicate that the interaction of the novel fragment L1−55 with MeCP2 affects L1-dependent functions, such as neurite outgrowth and neuronal migration. The cell adhesion molecule L1 not only plays crucial roles during the development of the nervous system, but it also regulates important functions in the adult nervous system under physiological and pathological conditions . In hImportant physiological processes such as neuronal differentiation, migration and survival, axon outgrowth and fasciculation, synaptogenesis, myelination, synaptic plasticity, learning and memory, as well as regeneration after injury are all abnormal in the absence of L1, leading to morphological and functional changes as well as behavioral disturbances ,20,21,22First indications suggesting a functional interdependence of L1 and MeCP2 were gained by studying reprogrammed human stem cells from a Rett syndrome individual: reduced L1 expression as well as reduced neurite outgrowth and neuronal cell survival were observed, and expression of wild-type MeCP2 in these cells resulted in increased L1 levels, neurite outgrowth, and neuronal cell survival . MoreoveFollowing up on these observations, we have recently found that MeCP2 in a nuclear mouse brain extract associates with immobilized recombinant intracellular L1 domain (L1-ICD) . This reAmong the MeCP2-deficient mouse lines investigated so far, several show a similar abnormal phenotype, the most remarkable of which are a staggering gait, other disturbances of motor coordination and hypoactivity, as well as changes in anxiety, learning capabilities, and social behavior ,32. MorpSince mutations in L1, absences of wild-type L1 or wild-type MeCP2, as well as increased expression of MeCP2 by duplication of the MeCP2 gene lead to similar phenotypes, it is deemed likely that a balanced interaction of L1 with MeCP2 is essential for normal development of the nervous system.In the present study, we have identified an L1 fragment of approximately 55 kDa (L1−55) comprising the intracellular domain as a novel binding partner of MeCP2. We also present evidence that this interaction occurs in the cytoplasm and nucleus of cultured cerebellar and cortical neurons and that MeCP2 contributes to regulating L1-dependent functions, such as neurite outgrowth and neuronal migration.Because of the functional importance of MeCP2 and L1, we further investigated their interactions. As a first step, we performed immunoprecipitation with L1 and MeCP2 antibodies using nuclear brain fractions from wild-type and L1-deficient mice. By Western blot analysis, a ~80 kDa MeCP2-immunopositive band was detected in the L1 immunoprecipitate from wild-type mice, whereas this band was not detectable in the L1 immunoprecipitate from L1-deficient mice and in control immunoprecipitates with a non-immune mouse antibody a. WesterBy ELISA, a concentration-dependent and saturable binding of recombinant L1-ICD to substrate-coated recombinant MeCP2 was observed, whereas the recombinant intracellular domain of the close homolog of L1 (CHL1-ICD) did not bind to MeCP2 d. A concTo determine the site(s) in L1-ICD that bind(s) to MeCP2, five peptides which cover the total L1-ICD sequence were assayed in a competition ELISA for their capacity to inhibit the binding of L1-ICD to MeCP2. Peptides comprising L1-ICD amino acids 1–35 (P1), 23–57 (P2), 14–29 (Pb), 25–43 (Pc), and 19–32 (Ps) reduced the binding of L1-ICD to MeCP2, whereas peptides comprising amino acids 1–17 (Pa), 55–73 (P3), and 70–89 (P4) did not affect the binding f. The peTo analyze whether MeCP2 and L1 interact also in a cellular context, proximity ligation assay was performed with cultured wild-type and L1-deficient cerebellar neurons. Using a mouse L1-ICD antibody and a rabbit MeCP2 antibody, positive signals were observed as red puncta in the cytoplasm and nuclei of wild-type cerebellar neurons but not in L1-deficient neurons a, indicaTo further support the notion that L1 interacts with MeCP2 in the nucleus, images were taken from z-axis stacks. In several cells, most z-axis stacks showed only red puncta, whereas several stacks displayed purple puncta due to the overlap between the blue nuclear DAPI stain and the red signal of the proximity ligation assay, which are indicative of a nuclear interaction of L1 and MeCP2 d, supporMeCP2/L1-positive puncta were also observed in cortical neurons e. HoweveRecently, we identified a ~55 kDa L1 fragment (L1−55) interacting with heterochromatin protein 1 isoforms α, β, and γ via the KDET motif, and we proposed that the generation of this fragment involves the metalloprotease ADAM-10 and/or -17, BACE1, and γ-secretase . Since iL1−55 was detectable in MeCP2 immunoprecipitates but not in control immunoprecipitates from nuclear extracts from cortical neurons c. Levelsn = 3; p < 0.001, one-way ANOVA with Bonferroni’s multiple comparison test) in L1-expressing cells after transfection with MeCP2 siRNA relative to L1-expressing cells transfected with control siRNA  in neurons after transfection with MeCP2 siRNA relative to the levels in neurons transfected with control siRNA , Wnt family member 7A (Wnt7a), and Hes family BHLH transcription factor 5 (Hes5), since comparisons of transcriptome data from the cerebellum of L1-deficient mice  and MeCPInterestingly, after the treatment of neurons with L1 antibody 557, Mef2c mRNA levels were not altered when compared to mock-treated neurons, but they were reduced by L1 siRNA and MeCP2 siRNAs to a similar extent, as seen in siRNA-transfected unstimulated neurons a.The reduction of Mef2c mRNA levels by MeCP2 and L1 siRNAs coincided with reduced Mef2c protein levels b,c. ThesTo further support this notion, we used the cell-penetrating tat-L1/KDET peptide to disturb the KDET motif-mediated interaction of L1 with MeCP2 and analyzed the effect of this peptide on Mef2c gene expression. In the presence of the peptide, the Mef2c mRNA levels were reduced in non-stimulated and L1 antibody 557-stimulated cortical neurons a. SimilaIn summary, these results indicate that the L1/MeCP2 interaction is required for regulation of Mef2c expression.After having identified MeCP2 as a potential binding partner of L1-ICD , we veriMeCP2 not only binds to DNA sequences containing methylated CpG dinucleotides but also to unmethylated DNA, thereby functioning both as a transcriptional repressor and an activator. Moreover, MeCP2 also contributes to the organization of the chromatin structure and is involved in splicing mechanisms ,54,55,56The functional interplay of L1 and MeCP2 and the interaction of L1 and MeCP2 with other interaction partners may affect L1- and MeCP2-mediated cellular functions. It is noteworthy in this respect that MeCP2 interacts with HP1γ and that the MeCP2/HP1γ interaction contributes to the reorganization of heterochromatin during myogenesis ,59. InteWe expect that the interaction of L1−55 with MeCP2 can also affect other MeCP2- and L1-dependent nuclear functions, such as mRNA processing, splicing, chromatin folding, and DNA repair. The L1−55/MeCP2 interaction might also affect the interplay of other L1 fragments with their nuclear binding partners, such as topoisomerase I . AblatioL1-deficient mice have been described . Mice weMouse monoclonal L1 antibody C-2  against the intracellular L1 domain was obtained from Santa Cruz Biotechnology . Rat monoclonal function-triggering L1 antibody 557 has been described . The rab® HD Transfection Reagent (Cat #E2311) was obtained from Promega .Recombinant full-length human MeCP2 (amino acids 1–486) with GST-tag (#H00004204-P01) was obtained from Abnova . Recombinant full-length human MeCP2 with a Flag-tag was obtained from Genway . Synthetic L1 peptides P1 (CFIKRSKGGKYSVKDKEDTQVDSEARPMKDETFGE), P2 (RPMKDETFGEYRSLESDNEEKAFGSSQPSLNGDIK), P3 (GDIKPLGSDDSLADYGGSVD), P4 (CSVDVQFNEDGSFIGQYSGK), P5 (CSGKKEKEAAGGNDSSGATSPINPAVALE), Pa (FIKRSKGGKYSVKDKEDTQ), Pb (EDTQVDSEARPMKDET), Pc (KDETFGEYRSLESDNEEK), Ps (DSEARPMKDETFGE), and tat-L1/KDET (YGRKKRRQRRRDSEARPMKDETFGE) comprising the cell-penetrating HIV tat sequence YGRKKRRQRRR thiazin-7a(7H)-yl]-4-fluorophenyl]-5-fluoro-2-pyridinecarboxamide  (Cay21599-1), (2S)-N-[acetyl]-L-alanyl-2-phenyl]glycine 1,1-dimethylethyl ester  (Cay13197-5), (2R)-N4-hydroxy-N1-[(1S)-1-(1H-indol-3-ylmethyl)-2-(methylamino)-2-oxoethyl]-2-(2-methylpropyl)-butanediamide  (Cay14533-1), and aprotinin (CAS 9087-70-1) (Cay14716-10) were obtained from Biomol . 4′,6-diamidino-2-phenylindole (DAPI) was obtained from Thermo Fisher Scientific .g for 10 min. The pellet was overlaid with 20% iodixanol (OptiPrep™ (60% iodixanol); PROGEN Biotechnik, Heidelberg, Germany) in 20 mM Tris/HCl, pH 7.4, homogenized by applying 10 strokes and centrifuged at 10,000× g for 40 min. The pellet was resuspended in homogenization buffer and centrifuged at 3000× g for 5 min. This pellet was resuspended in 200 µL Nuclear Extraction Buffer , vortexed at the highest speed for 15 s, incubated for 30 min, and centrifuged at 5000× g for 5 min. The supernatant was used as a nuclear protein extract.Protocol A: Brains of adult mice were mechanically disrupted in a homogenization buffer  per 50 mL; 3 mL per brain) by using a Potter-Elvehjem tissue homogenizer with a Teflon pestle  and by applying 20–25 strokes. This and subsequent steps were carried out at 4 °C. A total of 15 min after homogenization, NP-40 was added to a final concentration of 0.2%, and the samples were vortexed at the highest speed for 10 s and centrifuged at 3000× 2, 2 mM CaCl2, 1 mM phenylmethylsulfonyl fluoride (PMSF), pH 7.4) per brain using a Potter-Elvehjem. The homogenate was centrifuged at 200× g for 15 min at 4 °C. The pellet containing the nuclei was gently resuspended in 5 volumes lysis buffer (homogenization buffer containing 0.1% NP40) and loaded onto a discontinuous sucrose gradient, which consisted of 0.8, 1.0, and 1.2 M sucrose in isotonic buffer . After centrifugation for 2 h at 82,705× g at 4 °C, the pellet was resuspended in 1.5 mL homogenization buffer and centrifuged for 20 min at 1000× g and 4 °C. The pellet was resuspended in 25 µL Roeder C buffer  per brain and incubated for 30 min at 4 °C, and it was vortexed and homogenized by pipetting up and down. The homogenate was then centrifuged at 100,000× g at 4 °C for 30 min, and the supernatant was collected as nuclear extract.Protocol B: Brains of juvenile mice (younger than 20 days old) were homogenized in 2 mL (per brain) ice-cold homogenization buffer  was used. Cells were harvested in a Cytoplasmic Extraction Buffer (CEB) using a cell scraper. The NEB fraction was used as nuclear extract for immunoprecipitation.2+ and Mg2+ (PBS), and the samples were incubated overnight at 4 °C with gentle rotation. After the addition of 20 µL Protein G-coupled magnetic bead suspension and incubation of the samples for 2 h at room temperature with gentle rotation, the beads were isolated using a magnet and washed four times with PBS. The beads were suspended in 15 µL SDS sample buffer, boiled for 5 min at 95 °C, and separated using a magnet. The resulting supernatant was subjected to Western blot analysis.For co-immunoprecipitation, 200–500 µg of nuclear proteins was incubated with 5 µg of L1 antibody C-2, MeCP2 antibody, or non-immune anti-mouse or anti-rabbit antibodies. The incubation volume was adjusted to 250 µL using phosphate-buffered saline containing CaFor ELISA, 25 µL of 5 µg/mL recombinant GST-tagged MeCP2 was incubated overnight at 4 °C in 384-well microtiter plates with a high binding surface . All of the following steps were performed at room temperature. Wells were washed with PBS, treated with blocking solution  for 2 h, washed again with PBS containing 0.005% Tween 20 (PBST), and incubated with increasing concentrations of recombinant His-tagged intracellular L1 domain (L1-ICD) or CHL1 domain (CHL1-ICD) as ligands for 1 h under gentle agitation. For competition ELISA, 2.5 µM L1-ICD were preincubated for 1 h without or with a 5-fold molar excess of L1 peptides. The mixtures were then incubated with substrate-coated recombinant Flag-tagged MeCP2 (25 µL of 10 µg/mL was used for coating). After washing three times with PBST, an L1 antibody C-2 (1:200) or a CHL1 antibody C-18 (1:200) in blocking solution was applied for 1 h followed by three washes with PBST and incubation with horseradish peroxidase-coupled anti-mouse or anti-goat secondary antibodies (diluted 1:2000 in blocking solution) for 1 h. Wells were washed again with PBST, and 1 mg/mL ortho-phenylenediamine dihydrochloride (Thermo Fisher Scientific) was used for the detection of bound L1-ICD and CHL1-ICD. The reaction was terminated by the addition of 25 µL 2.5 M sulphuric acid. Absorbance was measured at 492 nm with an ELISA reader .2 biosensor surface  was used for substrate coating. Binding was determined by measuring the peak wavelength shift of reflected light using the BIND Technology (SRU Biosystems) as described [In label-free binding assays, L1-ICD and CHL1-ICD were used as soluble binding partners, and a 384-well plate with a TiOescribed .2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with L-glutamine, 4.5 mg/mL glucose, 10% fetal calf serum (DMEM/FCS), and 100 µg/mL penicillin/streptomycin. At a confluency of 50–70%, the medium was replaced with DMEM/FCS without penicillin/streptomycin, and the cells were transfected with 1 µg plasmid containing L1 cDNA, 7 µL 10 µM siRNA, and 3 µL FuGENE transfection reagent in 100 µL DMEM. After 16 h, the medium was replaced with DMEM/FCS containing penicillin/streptomycin. After further incubation for 8 h, the cell layer was subjected to scratch injury using a 50 µL pipette tip. After 24 h, cells were fixed in 4% formaldehyde for 20 min at room temperature. The gap size was measured with an Axiovert microscope with the AxioVision 4.6 imaging system .HEK293TN cells  were plated in 6-well plates  and maintained at 37 °C in a 5% COg for 15 min. Cells were then diluted in Neurobasal A medium (Thermo Fisher Scientific) supplemented with 2 mM L-glutamine (Invitrogen), 4 nM L-thyroxine (Sigma-Aldrich), 1 mg/mL BSA (Sigma-Aldrich), 12.5 μg/mL insulin (Sigma-Aldrich), 30 nM sodium selenite (Sigma-Aldrich), 100 μg/mL transferrin , 0.1 mg/mL streptomycin, and 100 U/mL penicillin (Invitrogen). For the proximity ligation assay, cells were seeded onto poly-L-lysine-coated 12 mm coverslips in a 24-well plate at a density of 2.5 × 105 cells per well. For the determination of protein levels, cells were seeded at a density of 2 × 106 cells per well of a 6-well plate coated with poly-L-lysine, and for neurite outgrowth, cells were seeded at a density of 5 × 104 cells per well of a 48-well plate coated with poly-L-lysine (Sigma-Aldrich).Cerebellar neurons were prepared from cerebella of 6- to 8-day-old mice. Cerebella were incubated with 10 mg/mL trypsin and 0.5 mg/mL DNase I (Sigma-Aldrich) in Hanks’ balanced salt solution (HBSS) for 15 min at 37 °C, washed with HBSS, mechanically dissociated and centrifuged at 100× 5 cells per well. For the determination of protein expression or neurite outgrowth, cells were seeded at a density of 2 × 106 cells per well of a 6-well plate coated with poly-L-lysine or at a density of 5 × 104 cells per well of a 48-well plate coated with poly-L-lysine (Sigma-Aldrich), respectively.For the culturing of cortical neurons, cerebral cortices were dissected from 15.5- to 16.5-day-old embryos and incubated in 0.025% trypsin (Sigma-Aldrich) in HBSS at 37 °C for 30 min. The cortices were then incubated in HBSS containing 1% BSA (Sigma-Aldrich) and 1% trypsin inhibitor  at 37 °C for 5 min. After washing in HBSS, the tissue was mechanically dissociated, and the dissociated cells were cultured in Neurobasal medium (Thermo Fisher Scientific) supplemented with 1% B-27 (Thermo Fisher Scientific), 2 mM L-glutamine (Thermo Fisher Scientific), 100 U/mL penicillin (Thermo Fisher Scientific), and 100 μg/mL streptomycin (Thermo Fisher Scientific). For the proximity ligation assay, cells were seeded onto poly-L-lysine-coated 12 mm coverslips in a 24-well plate at a density of 2.5 × 106 cells per well of a 6-well plate coated with poly-L-lysine, and cells from three wells were used per condition. For experiments with protease inhibitors, neurons were treated 2 h after seeding with 10 µM DAPT, GM6001 or LY2886721, or with 1 µM aprotinin for 24 h. Treatment of cerebellar and cortical neurons with 50 µg/mL tat-L1/KDET peptide was performed 30 min after seeding.For L1 function triggering, neurons were treated with 50 µg/mL L1 antibody 557 for 2 h at 37 °C. For immunoprecipitation, cells were seeded at a density of 3 × 10After seeding, cortical neurons were maintained for 2 h before transfection with 400 ng plasmid containing L1 cDNA, 2 µL 10 µM siRNA, and 1 µL FuGENE transfection reagent per well. L1 antibody 557 (12.5 µg 557 per well) was added to the cultures 24 h after transfection, and 48 h after transfection, the cells were washed gently with pre-warmed culture medium, fixed in 2.5% glutaraldehyde, and stained with 1% toluidine blue and 1% methylene blue in 1% sodium tetraborate for 1 h at room temperature. Neurite outgrowth was analyzed by measuring the total length of neurites in an Axiovert microscope with the AxioVision 4.6 imaging system .Cultures were fixed for 15 min at room temperature in 4% formaldehyde, washed with PBS and subjected to proximity ligation assay using Duolink PLA products (Sigma-Aldrich) according to the manufacturer’s protocol  with minor modifications. In brief, cells were blocked using Duolink Blocking solution supplemented with 0.5% Triton X-100 and incubated for 24 h at 4 °C with L1 antibody C-2 and MeCP2 antibody diluted 1:10 in Duolink Antibody Diluent. Cells were washed two times using Duolink Wash Buffer A and incubated with a mixture of secondary antibodies conjugated with oligonucleotides (Duolink Anti-Mouse PLA Probe MINUS and Duolink Anti-Rabbit PLA Probe PLUS). The proximity ligation reaction was then performed according to the manufacturer’s protocol using the Duolink In Situ Detection Reagent RED. Thereafter, the coverslips were incubated in 5 µg DAPI/mL PBS for 15 min, washed twice with PBS, and mounted in Immuno-Mount (Thermo Fisher Scientific). A total of 10 images per condition were taken using an Olympus F1000 confocal microscope and analyzed using ImageJ software . The number of red puncta and the number of DAPI-stained nuclei were determined using ImageJ, and the number of red puncta per image was divided by the number of nuclei per image. The average values of red puncta per cell (=nuclei) were determined in 10 images per condition, and 200–500 cells per condition were analyzed.® II reverse transcriptase (Thermo Fisher Scientific) was used. qPCR was performed in triplicates using reverse transcribed mRNA, the 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific), the qPCR kit SYBR® Green I, ROX , and primers for the determination of the mRNA levels of BDNF , Wnt7a , Hes5 , A2bp1 , Mef2c , and the reference gene actin . The SDS 2.4 software (Thermo Fisher Scientific) was used for analysis of the qPCR data. The mRNA levels of BDNF, Wnt7a, Hes5, A2bp1, and Mef2c relative to the mRNA levels of the reference gene actin were calculated. Data of relative gene expression  were used for statistical analysis.For reverse transcription, oligoT18 primer and SuperScriptTests are indicated in the legends.


In this study, the electrical properties of graphene–polypyrrole (graphene-PPy) nanocomposites were thoroughly investigated. A numerical model, based on the Simmons and McCullough equations, in conjunction with the Monte Carlo simulation approach, was developed and used to analyze the effects of the thickness of the PPy, aspect ratio diameter of graphene nanorods, and graphene intrinsic conductivity on the transport of electrons in graphene–PPy–graphene regions. The tunneling resistance is a critical factor determining the transport of electrons in composite devices. The junction capacitance of the composite was predicted. A composite with a large insulation thickness led to a poor electrochemical electrode. The dependence of the electrical conductivity of the composite on the volume fraction of the filler was studied. The results of the developed model are consistent with the percolation theory and measurement results reported in literature. The formulations presented in this study can be used for optimization, prediction, and design of polymer composite electrical properties. Uniform dispersion of nanofillers in a polymer matrix improves the electrical conductivity, thermal conductivity, mechanical properties, and chemical stability of the composite ,2. CarboComposites with graphene are more efficient than other carbon material composites owing to its astounding electrical conductivity, flexibility, simple synthesis methods, and super-electrochemical action when it is composited with conducting polymers ,10,11. Ts-like shape of the percolation curve for polymer composites. As shown in By considering the experimental behaviors of polymer composites, the transition of the composite from insulator to semiconductor/conductor can be mathematically described by the percolation theory with respect to the volume fraction of the filler ,20,21. TTo mitigate the challenges in quantifying the various factors determining the electrical conductivity and other properties of graphene–PPy, the Monte Carlo simulation approach (MCSA) is employed in this study. Fang et al.  predicteMoreover, to predict the electrical properties of polymer composites, their tunneling and intrinsic resistances must be evaluated. The resistivity of single-walled carbon nanotubes (CNTs) varies between ×10−2 Ωm , while tm [106 Ω . The tot0 ±300 Ω . The tun0 ±300 Ω ,32,33.Therefore, for graphene nanocomposites, maximum contact resistance, the effect of polymer thickness on the capacitance behavior of the composite, and the contribution of the intrinsic resistance of fillers to the overall electrical conductivities of the resulting composites, are important parameters which must be evaluated. These aforementioned parameters and others are investigated in this study. In addition, a prediction based on the simulations of the contact resistance of PPy on graphene is presented. The developed model can be used to quantify the electrochemical behavior of the polymer composite.In this study, a Monte-Carlo is used in the computation of electrical conductivity model developed to investigate the associated percolation in graphene–PPy. The model incorporated a reported contact model  and McCuThe analysis of the variation in the conductivity level of a polymer with included two-dimensional materials is complex. A simulation approach that can produce a random distribution of fillers in polymers is essential for the modeling of the electrical conductivities of polymer composites. A statistical computation based on the generation of random parameters to obtain numerical results is referred to as the Monte Carlo model. The computational steps employed in this study involve the generation of a random graphene network distribution using the MCSA, modeling of the sheet-to-sheet resistance of the graphene in the composite, inclusion of the filler intrinsic resistance, and application of the rules of mixtures to quantify the electrical conductivity of the composite.A three-dimensional percolation network was created by a Monte Carlo model to predict the threshold of the composite. We developed a cubic representative volume element (RVE) of the graphene-PPy composite and obtained a conductivity equation. It was assumed that the electrical resistance of the graphene–PPy composite can be modeled by the resistance of the graphene percolation networks and electrical conductivity contribution of the polymer in the RVE. As shown in hs of γg ,38.The dimensions of the cuboid RVE are twork is ,40:(1)(γion (4)) . The filTwo types of resistance determine the electrical conductivities of nanocomposites ,40, i.e.According to Yu et al. , the tunequation . The curn [J, is ,26:(5)J=r limits , λ1 and The electronic charge,  film is :(9)V= qCAccording to Equation (10), the tunneling resistance is estimated to be The intrinsic resistance of the filler was modeled by considering the law of resistance:The effective electrical conductivity of the graphene dispersed in the PPy is obtained by the McCullough equation , based oThe summary of the parameters used in the simulation are given in In this section, we discuss the (i) effect of the polymer insulation thin film on the tunneling resistance; (ii) relationship between the intrinsic resistance and tunneling resistance; (iii) composite capacitance; (iv) validation of the model with experimental measurements; (v) tunneling resistance and electrical conductivity; (vi) effect of the aspect ratio on the composite electrical conductivity; and effect of the graphene electrical conductivity on the composite.For the determination of tunneling resistance, the graphene height of rectangular potential barrier is set to 4.6 eV , the PPy105 , and the−12 C/Vm . Figure −12 C/Vm . Figure −12 C/Vm , Yu et a−12 C/Vm , and Sim−12 C/Vm . In thisg et al.  and Yu eg et al. .In addition, the specific capacitances of the graphene–PPy composites with different insulation thicknesses of 0.1–3 nm and graphene diameters of 500–800 nm are presented in 3 S/m, the aspect ratio was varied by employing a graphene diameter of 500–700 nm. The results indicate that the aspect ratio had a considerable influence on the formation of the conducting path of the composite. As shown in The influence of the aspect ratio The effect of the graphene intrinsic conductivity was also investigated, as shown in −107 S/m ,51. The −107 S/m ,46,52.The contribution of the tunneling resistance to the electrical conductivity of the graphene–PPy composite was analyzed by considering the insulation thickness in the range of 0.1–1 nm, intrinsic PPy conductivity of 10 S/m, volume fraction of approximately 0.04, graphene diameter of 500 nm, and graphene conductivities of Furthermore, in this study, the developed model was validated by reported measurements. Kim et al.  carried  105 S/m , respect 105 S/m  were 5 μTo further confirm the robustness of the model, the results of a set of experimental electrical conductivity measurements of the graphene-polystyrene (graphene–PS) composite (10 data points), carried out by Stankovich et al. , were coIn summary of this section, an excellent electrically conductive material such as graphene has been shown to be suitable for the enhancement of the electrical properties of polymer (polypyrrole). However, the performances of this composite are largely dependent on the thickness of the polymer. The tunneling of electrons between the sheets of the conductor is a function of the thickness of the polymer film. From the modeling results, the effects of filler diameter and matrix thickness has been shown to have varying impact in the tunneling resistance of the composite. For example, a low equivalent resistance is required to obtain a high conductive and high capacitive supercapacitor . Therefot (3 eV) ; these cThis study considered the use of the Simmons and McCullough equations, in conjunction with the MCSA, to calculate the contact resistance in the graphene–PPy composite and electrical conductivities of its constituents. The results of the developed model agreed well with the results of previous measurements. The barrier junction of the polymer composite strongly depended on the filler diameter and polymer thickness. With the increase in insulation thickness, the intrinsic conductivity of the filler had a negligible effect on the effective conductivity of the composite. In addition, the capacitive behavior of the composite showed that the graphene–PPy is a good electrochemical composite, owing to the large diameter of the graphene and very small thickness of the PPy. The influences of aspect ratio, graphene intrinsic conductivity, and volume fraction on the electrical conductivity of the graphene–PPy composite were investigated. The consistency of the model results with those of measurements and previous studies shows its suitability for the theoretical evaluation of polymer composite electrical properties. Further experimental and theoretical studies on the feasibility of using graphene–PPy composites for solar cell and battery applications are required.


Tc) of Nb. Maximum modulation depths of 57.3% and 97.61% are achieved for EIT and group delay at the THz transmission window, respectively. A comparison is carried out between the Nb-graphene-Nb coupled SRR-based devices with those of Au-graphene-Au SRRs, and significant enhancements of the THz transmission, group delay, and EIT responses are observed when Nb is in the quantum mechanical phase. Such hybrid devices with their reasonably large and tunable slow light bandwidth pave the way for the realization of active optoelectronic modulators, filters, phase shifters, and slow light devices for applications in chip-scale future communication and computation systems.Metamaterial photonic integrated circuits with arrays of hybrid graphene–superconductor coupled split-ring resonators (SRR) capable of modulating and slowing down terahertz (THz) light are introduced and proposed. The hybrid device’s optical responses, such as electromagnetic-induced transparency (EIT) and group delay, can be modulated in several ways. First, it is modulated electrically by changing the conductivity and carrier concentrations in graphene. Alternatively, the optical response can be modified by acting on the device temperature sensitivity by switching Nb from a lossy normal phase to a low-loss quantum mechanical phase below the transition temperature ( Metallic superconductors are macroscopic quantum systems and gain their electromagnetic properties from pairs of electrons, Cooper pairs . Due to The surfaces of hybrid materials can be engineered to achieve electromagnetic response at will. Such engineered materials, metamaterials, develop their exotic electromagnetic properties from the geometries of the engineered unit cells  through the interactions of artificial atoms and photons . For exastatic’ superconducting THz metamaterials was reported where the authors observe tuning response with intense THz light [An integrated optoelectronic device with the capability of continuous tuning and controlling of the group velocity of light is therefore of interest in the microwave, millimeter wave, terahertz (THz), and far infrared regime, which make possible, for example, the realization of (i) tunable optical phase shifter, (ii) time-delay lines  as toolsHz light ,35, tempHz light ,37,38,39Hz light . MetamatHz light ,42.active’ slow light optoelectronic device operating at cryogenic temperatures. The advantages of our proposed devices include: (i) Nb as a metallic superconductor obtains quantum mechanics phases at cryogenic temperatures to offer active modulation and tuning of THz response in wide temperature and frequency ranges. Such sensitive temperature sensing, as a result of the temperature-dependent superfluid density of the Nb superconductor, cannot be achieved with any normal or noble metal-based metamaterial devices. (ii) The current most promising platform for a quantum computer is based on superconducting processors operating at cryogenic temperatures. Therefore, it is necessary to develop tunable slow light devices, sensors, detectors, and modulators operating at low temperatures. (iii) The combination of graphene as a 2D material with their tunable carrier concentration with voltage offers an additional knob to control light in such novel integrated circuits at cryogenic temperatures.In this work, a single layer of graphene is integrated with superconducting Nb plasmonic SRRs to realize a voltage and temperature tunable ‘Tc = 9.2 K) of Nb and (ii) electrically by changing the conductivity and carrier concentrations in graphene.The architecture of the proposed hybrid graphene–superconductor photonic integrated circuit is schematically shown in R = 8 μm, and bias line width of Wb = 4 μm. The unit cell periodicity is set to be (Py = 114.4 µm) × (Px = 76 µm). The single unit cell is repeated into arrays to ease light coupling. The thickness of Nb is considered to be 100 nm. 2. The THz transmission of bare SiO2/Si substrate is used as a reference. In order to design and model the device architecture, the RF module of COMSOL Multiphysics V5.5 is used. SiO2 and Si are considered dielectrics with ε = 3.9 and 11.56, respectively [τ = 15 fs is the scattering time, and ω is the angular frequency [σ0 is mentioned in the paper. Graphene is simulated as a 2D layer with surface current boundary conditions. The surface current of graphene ectively . In the ectively . At DC, vely [σ0 . The AC requency ,46. The requency ,48. The response ,49,50. T×6.5 THz . Here, εT = 4.5 K, far below the transition temperature Tc of Nb. The THz transmissions of three sets of SRRs are shown in fb) and 0.58 THz (labeled fd), respectively. The bottom resonator’s longer side is directly excited by the incoming THz E-field. Therefore, the excited net dipole moments in the longer arm result in a resonance with a broader full width at half maximum bandwidth (FWHM), as the radiation damping is SRR structure size-dependent. The total length of the bottom resonator is also slightly larger than the top resonator resulting in resonance at a lower frequency.We first consider the THz response of three sets of static superconducting SRRs in the absence of graphene, at x-direction, see fd. On the other hand, the larger SRR (bottom) acts as a superradiant (or bright) resonator with resonance frequency fb. However, the destructive interference between two subradiant and superradiant resonances leads to two dips as hybridized modes of “bonding” and “anti-bonding” and one peak as an asymmetrically coupled mode [f− and anti-bonding f+ resonance frequencies are shown in the lower panel of f− bonding mode, while they are in the same direction for f+ anti-bonding mode. The suppressed currents due to strong asymmetric coupling and destructive interference between a subradiant mode from the top SRR and a superradiant mode from the bottom SRR lead to induced electromagnetic transparency (EIT) in the coupled SRRs [The electric field distribution in the coupled resonators shows the strength of the resonators to incident THz radiation polarized along the led mode , as showled SRRs .Now, we discuss the results for the active, electrically tunable device based on the integration of graphene with Nb SRR arrays in a superconducting circuit. The preliminary graphene characterization of the continuous patch was carried out at room temperature and is shown in σ0 = 0.3 mS (close to Dirac point), σ0 = 0.9 mS (corresponding to gate voltage VBG = 0 V), and highest σ0 = 2.5 mS conductivity values shown in dashed red, purple, and green in f10.3mS and a single resonator LSP circuit with sub-radiant resonance f12.5mS. f -mode by 41 GHz when the graphene conductivity changes from σ0 = 0 mS, a static resonator circuitry with no graphene in the dark resonator’s gap, to σ0 = 2.5 mS where the graphene conductivity is set to highest value (f12.5mS mode shown in dashed green). The electric field distribution of this mode shows the damping of the resonance at the dark element . The bias line exhibits resonance at ties see a. The elties see  show no Tc and (ii) when Nb is in the resistive normal state above Tc. The THz transmission spectra as a function of frequency for different graphene conductivities between σ0 = 0 mS and σ0 = 2.5 mS at T = 4.5 K < TcNb and at T = 10 K > TcNb are shown in T = 4.5 K, and T = 10 K. A clear enhancement of THz transmission, superradiant and subradiant modes, and frequency tuning can be seen when the device is in superconducting quantum phase state at T = 4.5 K. Quality factor of first (f1) and second (f2) resonances for the temperatures T = 4.5 K and T = 10 K are shown in σ0 = 1.8 mS, where the second resonance completely damps, and only one single resonance remains.To obtain further insight into the voltage-controlled active hybrid graphene–superconductor photonic integrated circuit demonstrated in this paper, we compare the detailed results for (i) when Nb is in superconducting quantum phase state below T = 4.5 K are sharper, and hence, the device is more sensitive to incident THz light at the superconducting state than that at T = 10 K, where Nb obtains a normal metallic phase. The change of the transmission minimum at the first dip between graphene conductivity σ0 = 0 and σ0 = 2.5 mS for T = 4.5 K is 0.16, where it is 0.01 at T = 10 K. The variation for the second dip at T = 4.5 K between σ0 = 0 and σ0 = 1.7 mS is 0.3, which reduces to 0.27 for T = 10 K. The field concentration in the gap is more enhanced at T = 4.5 K. As a result of more interaction of electric field in the gap with graphene, the change in transmission at T = 4.5 K is larger than that at T = 10 K. This suggests that our hybrid superconducting THz devices are suitable for THz sensing at wide temperature ranges below the Tc of Nb.The resonances at T= 4.5 K)/Au, (b) Nb (at T = 10 K)/Au, (c) Au/Nb (at T = 4.5 K), (d) Au/Nb (at T = 10 K), and (e) Au/Au, respectively. One can clearly find the role of superconductivity. Looking at T = 4.5 K < TcNb. The strong difference between the THz responses of the Nb superconductor and Au resonators is originates from the difference in ohmic (nonradiative) losses of the resonators. The nonradiative losses depend on the resistance of the device material building blocks. The lower nonradiative losses of Nb at the superconducting state result in exciting sharper resonances [We also compare the effect of the superconducting quantum phase on the THz response of such active devices with those of devices in the normal phase based on different materials such as gold . Figure sonances ,56.Rs = 100 mΩ between f = 100 GHz and 600 GHz, at T = 5 K [The THz surface resistance of Nb is small, a value around  T = 5 K . ReplaciRLC electrical circuit equivalent of the SRR. In the absence of coupling, each single SRR can be considered as an antenna with a frequency-dependent impedance. The electric circuit in R1, an inductance L1, and a capacitance C1, represents the bright resonator. The upper loop represents the dark resonator with R2, L2, and C2 circuit elements. In the presence of coupling, due to the electric field of charges in close proximity between two bright and dark SRRs, a parallel coupling capacitor Cc can connect the two circuit loops. In the circuit, R1 and R2 illustrate the losses in each SRR; L1 and L2 represent the stored magnetic energy due to induced current in each SRR; C1 and C2 illustrate the energy stored in the finger of each bright and dark SRR due to accumulated charges. RG accounts for the extra induced (resistive) losses as a result of the integration of graphene with Nb in the finger of the dark SRR. V1 and V2 in the circuit represent the electromotive force on the electrons due to the incident THz radiation in the bright and dark SRRs, respectively. We assume that the value of V1 is 0.65V2 as the bright SRR provides a larger coupling with the incident THz transmission. The inductance of each resonator Li is defined as the ratio of magnetic field flow divided by current J density, as z-component of the magnetic field Hz and induced current density J for each SRR are obtained from the COMSOL simulation. After determining the inductance of each SRR, the capacitance is calculated from the equation We use an equivalent lumped element circuit model to further investigate the resonance response and frequency tuning of the coupled resonators as a function of graphene conductivity.  ωr=1LiC ,57.RG as a function of graphene conductivity σ0 is determined from the equation A free fitting parameter as =1RiLiCi , where tectively . The obtRw) is calculated. RG changes from 2.5 Ω for σ0 = 0.3 mS to 25 Ω for σ0 = 2.5 mS. The current induced in the bright and dark SRRs  will flow through the bottom and top loops. The voltages are related to currents through Kirchhoff’s law  at tion see a. Next, nces see . The EITω is the angular frequency. Group delay is calculated relative to the bare substrate reference. T = 4.5 K < cT and (b) T = 10 K > cT. Maximum negative group delay dips are observed at around 0.46 THz and 0.66 THz at both temperatures; however, the group delay value and shape observed at T = 4.5 K < Tc, where Nb is in the superconducting quantum phase, is much larger and sharper. The positive group delay between these frequencies indicates the slow light effect through the hybrid device. It can be seen that the delay time is quite loss-sensitive and is much larger when Nb is in the superconducting quantum phase at T = 4.5 K. A maximum positive group delay of 2.86 ps at 0.627 THz is observed when there is no graphene present in the Nb gap in the dark resonators . The maximum group delay is in the order of other superconducting metamaterials [T = 4.5 K and T = 10 K. Our results indicate that the proposed hybrid photonic integrated circuit based on graphene-Nb SRR can be used as an efficient and tunable THz slow light modulator device.Here, we demonstrate how to actively control slow light in hybrid graphene–superconductor resonators integrated with superconducting circuits. The group delay aterials ,60. In caterials ,61,62. Oσ0 = 1.6 mS for T = 4.5 K. As the graphene conductivity increases, the EIT transmission has an obvious decline that results in the MD growing. When σ0 becomes larger than 1.6 mS at T = 4.5 K, the EIT peak shows no more positive group delay. Positive group delay corresponds to the slowness of slow light devices. In addition, active modulation of slow light can be realized by graphene conductivity. The modulation depth of group delay is determined by σ0 = 1.6 mS for T = 4.5 K. Applying a voltage on the device pads can significantly change the amplitude of the EIT peak, rather than the resonance frequencies, as is clear from shown in  increaseT = 4.5 K)/Au, (b) Nb (at T = 10 K)/Au, (c) Au/Nb (at T = 4.5 K), (d) Au/Nb (at T = 10 K), and (e) Au/Au. One can clearly find the role of the superconducting quantum phase state. The strongest EIT response and slow light effect are observed when both coupled resonators are based on Nb and when the Nb is set at T = 4.5 K < TcNb. This is because at this temperature, the THz surface resistance of Nb is much smaller. Replacing Nb in the bright resonator with Au will reduce the group delay, but this reduction is considerable when Nb in the dark resonator is replaced by Au. The device response will significantly weaken when the SRR structure is built from Au, which retains large surface resistance at T = 4.5 K.T = 4.5 K)/Au, 2-Nb (at T = 10 K)/Au, 3-Au/Nb (at T = 4.5 K), 4-Au/Nb (at T = 10 K), 5-Au/Au, 6-Nb (at T = 4.5 K)/Nb (at T = 4.5 K), 7-Nb (at T = 10 K)/Nb (at T = 10 K), at σ0 =0 mS and σ0 = 0.9 mS, are calculated and presented in T = 4.5 K, has the largest DBP and is superior to the other superconducting metamaterials that has been reported [The maximum group delay, EIT window bandwidth, and maximum delay-bandwidth product (DBP) for different hybrid circuits, including device number 1-Nb (at reported . LikewisWe have proposed a voltage and temperature-controlled photonic integrated circuitry by the integration of graphene with an array of Nb subwavelength split-ring resonators in a superconducting circuit, and have reported the first demonstration of a hybrid graphene–superconductor THz metamaterial slow light device. Additionally, we have shown that an equivalent circuit model is a useful tool for active hybrid device optimization. Furthermore, we have demonstrated electromagnetic induced transparency, subradiant and superradiant resonances in such a novel class of hybrid photonic integrated circuits. The quantitative analysis has shown that the modulation depth of EIT transmission amplitude and slow light group delay can be realized via voltage and temperature. The maximum values of 57.3% and 97.61% have been obtained for the modulation depth of EIT and group delay with voltage. A comparison of Nb SRR-based devices with Au SRRs have shown the reduction of the maximum delay-bandwidth product from 0.46 to 0.04. Our devices, with their large and tunable slow light bandwidth, can pave the way for the realization of active optoelectronic modulators for applications in future quantum communication and computation systems.


Dear Editor,1. Recently, we investigated the roles of Med23 in VSMC proliferation and differentiation as well as injury-induced neointima formation by generating and examining the smooth muscle specific Med23-knockout (Med23sm−/−) mice. Our results showed that Med23 regulates the balance of VSMC growth and differentiation in mouse aorta. The Med23sm−/− mice showed enlarged lumen and impaired contractility of aorta. RNA profiling of the aorta samples revealed that gene expression programs switching between proliferation and differentiation upon the presence or absence of Med23. More importantly, we found that Med23 deficiency prevented neointima formation after vascular injuries, through repressing VSMC proliferation. Collectively, our data demonstrate that Med23 has positive effects on VSMC plasticity and plays a novel pathological role in vascular injury induced neointima formation through promoting the proliferation and growth of VSMCs, thus providing a novel mechanism understanding the VSMC plasticity and related vascular diseases.Vascular smooth muscle cells (VSMCs) that line the arteries and veins, are able to increase their proliferation rate greatly following vessel wall injury and in atherogenesis. Changes in VSMC proliferation rate and differentiation state have been proposed as required procedures of injury repair. The regulation of the balance between proliferation and differentiation is of key importance in vascular biology. Our previous works have shown that Mediator MED23 subunit restricts smooth muscle cell lineage development and promotes growth related gene expression1. Nevertheless, the roles of Med23 in regulating smooth muscle development and function have not been examined in vivo in mice. Since homogenous knockout of Med23 is embryonic lethal2, we generated smooth muscle specific Med23-knockout (Med23sm−/−) mice to investigate the in vivo function of Med23. We crossed conditional Med23-knockout mice (Med23fl/fl)3 with the aortic smooth muscle actin (Acta2)-Cre expressing line4 to generate the Med23sm−/− mutant mice. Western blot analysis of tissue samples from Med23sm−/− mice showed specific and efficient Med23 ablation in stomach and aorta, the walls of which are lined with smooth muscle, but not in kidney, spleen, liver, fat tissue or lung  ratio 9, SMCs migrate from tonica media to intima, and proliferate further there. Although the proliferation of VSMC plays a central role in injury repair, excess proliferation of VSMC may lead to arterial injury induced neointima formation. In order to investigate the cellular mechanism behind the reduced neointima formation in Med23sm−/− mice after arterial injury, we examined cell proliferation and migration of VSMCs isolated from the aorta , Rhobtb1 (Rho-related BTB domain containing 1) and musculoskeletal gene Mustn1 were among the down-regulated genes in Med23sm−/− samples, in addition to Egr1. While the expression of muscle-related genes Myl1, Tbx20, Myoz2 and brain derived neurotrophic factor (Bdnf) were up-regulated in Med23sm−/− samples  and phosphorylated Akt (p-Akt)  Fig. . Therefoles Fig. . On the Fbb Fig. . Upon PDles Fig. . These rMed23 is important for the function of smooth muscle in vivo, through maintaining the balance of SMC growth and differentiation. Moreover, Med23 is involved in neointima formation induced by arterial injury, which may improve our understanding on vascular diseases.Taken together, our results suggest that Supplementary Information


Retinopathy of prematurity (ROP) is one of the main causes of blindness in children worldwide. Brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin-related kinase B (TrkB), play critical protective roles in the development and function of neurons and vasculature. Lack of BDNF expression results in increased endothelial cell apoptosis and reduced endothelial cell-cell contact. Premature babies who develop ROP tend to have lower serum BDNF levels. BDNF expression is also significantly lower in mouse retinas following exposure to hyperoxia compared to those reared in room air. Specifically, BDNF promotes angiogenic tube formation of endothelial cells (EC), and it is considered an EC survival factor required for stabilization of intramyocardial vessels. We hypothesized that the activation of TrkB receptor protects retinal vasculature in the mice during oxygen-induced ischemic retinopathy (OIR), a model of ROP. To test this hypothesis, we treated neonatal mice with 7,8-dihydroxyflavone (DHF) (5 mg/kg body weight), a TrkB receptor agonist. We examined its potential protective effects on retinal vessel obliteration and neovascularization, two hallmarks of ROP and OIR. We found that retinas from DHF treated postnatal day 8 (P8) and P12 mice have similar levels of vessel obliteration as retinas from age-matched control mice subjected to OIR. Similarly, DHF showed no significant effect on mitigation of retinal neovascularization during OIR in P17 mice. Collectively, our studies demonstrate that the TrkB receptor agonist DHF provides no significant protective effects during OIR. Retinopathy of prematurity (ROP) is a major cause of visual impairment and blindness that affects a significant proportion of children born prematurely in the USA . The devBrain-derived neurotrophic factor (BDNF) activity, through binding to its receptor, tropomyosin receptor kinase B (TrkB), plays important roles in the development and function of various types of neurons , 13. BDNBDNF/TrkB signaling pathway also plays critical roles during vascular development. Lack of BDNF expression increased endothelial cell apoptosis and reduced endothelial cell-cell contact. In contrast, overexpression of BDNF resulted in higher vascular density . In vitrad libitum access to standard rodent chow and water. The day of the birth was considered postnatal day zero (P0). At desired termination time points animals were euthanatized using carbon dioxide.Experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the University of Wisconsin School of Medicine and Public Health (IACUC assurance number: D16-00239). Adult C57BL/6J mice were obtained from the Jackson Laboratory and were bred in our mouse colony at UW-Madison. Their litters were used for the studies described below. Mice were housed and allowed Oxygen-induced ischemic retinopathy (OIR) was induced in C57BL/6J mice by exposing P7 pups with their mothers to 75 ± 0.5% oxygen in an air-tight incubator for a period of 5 days. The temperature of the incubator was maintained at 23 ± 2°C, and oxygen was continuously monitored with a PROOX model 110 oxygen controller . At the end of the 5-day period, animals were gradually introduced to room air over a period of 5–6 h before they were transferred into their regular housing area. Animals were kept under room air conditions until desired time-points were reached. Naïve C57BL/6J mice of same age were used as controls. At the end of each experiment, the animals were exposed to CO2 and death was confirmed before eyes were enucleated. For consistency and rigor, the number of pups per litter used for OIR experiments was kept small (5–7 pups). In addition, the pups’ body weight was monitored to make sure all the pups are the same size. For treatment experiments the pups were randomly divided into two groups, the treatment and control groups. Similar conditions were used for age-matched room air studies.7,8-dihydroxyflavone  was used in these studies. The stock solution (10 mg/ml) was prepared in dimethyl sulfoxide (DMSO). The working solution was prepared in 1X PBS (3.3% DMSO in 1XPBS), as previously described by others , 29. DMSPreparation of wholemount retina for assessing vessel obliteration and neovascularization at different time points was done as we described previously –32. BrieAGCCGCAAAGAAGTTCCA; Bdnf_R, GCAACC GAAGTATGAAATAACCA; TrkB_F, ACGAGTATGGGAAGGATG; TrkB_R, TTCAGGG TAATTTGGGTTTG; TrkB-Variant1_F, GATGTCCCGAGATGTGTA; TrkB-Variant1_R, GGTGGTGAATTTCCTGTA; TrkB-Variant2_F, CATCTGTGGTGGGATTCT; TrkB-Variant2-R, CCAGTGGGATCTTATGAAAC. Expression of each transcript was assessed in triplicate reactions. Relative expression of targeted transcript was normalized to the housekeeping gene RpL13A using the quantitative 2−ΔΔCT method [RNA isolation, cDNA preparation and quantitative PCR (qPCR) were performed as we previously described . BrieflyT method .Room-air and OIR mice (P17 or P18) were injected intraperitoneally with DHF (5 mg/Kg) or control (0 hour) and retinas were harvested after 2, 4 or 6 hours. The mouse retinas (2 retinas per sample) were harvested and homogenized in 300 μL of cold lysis buffer  with proteases and phosphatase inhibitor . The total protein concentrations were measured using the Pierce BCA protein assay kit . Thirty microgram (30 μg) of retinal lysates were separated by electrophoresis on 4–20% gradient gels  and transferred to nitrocellulose membranes . The membranes were incubated with anti-TrkB , anti-phosphorylated (Y705)-TrkB , anti-phosphorylated (Y816)-TrkB  and anti-β actin  antibodies. The membranes were washed with TBST  and incubated with appropriate HRP-conjugated secondary antibodies  as recommended by the supplier. Secondary antibodies were diluted as the following: Rabbit-HRP 1:4000 for TrkB, Rabbit-HRP 1:2500 for p-TrkB (Y705 and Y816), and Mouse-HRP 1:5000 for beta actin (mouse-HRP). Following incubation, the blots were washed with TBST and developed using enhanced chemiluminescence reagents . The intensities of protein bands, relative to loading control, were quantified using ImageJ software and plotted using GraphPad Prism version 8 .Statistical analysis was performed as we previously described , using GWe assessed mRNA expression levels of Bdnf, and Ntrk2 in retinas from P7, P12, P15 and P17 mice kept at room-air . ExpressTo determine whether the intraperitoneal (IP) delivery of DHF can reach the retina and activate TrkB, we treated room-air mice with DHF or vehicle and determined the retinal levels of TrkB phosphorylation by Western blot analysis. DHF treatment was shown by others to promptly increase phosphorylation of TrkB as early as 10 minutes and phosphorylation steadily decrease after 8 hours and is nearly undetectable after 16 hours in retinal ganglion cells . TherefoNeonatal mice were treated daily with either DHF or vehicle solution starting from one day before exposure to hyperoxia at P7 until one day before returning to room air at P12  according to OIR method. Retinas from P12 mice were wholemount immunostained with Isolectin B4 for visualizing blood vessels. Vascular obliteration was quantified by measuring the avascular area in the center of the retina around the optic nerve. Most of the vessel obliteration occurs within the first 24 h after exposing P7 mice to 75% oxygen . To examTo determine whether DHF can favorably affect the retinal vasculature during OIR phase II, neonatal mice were exposed to hyperoxia for 5 days (OIR Phase I) and then kept at room air for additional 5 days (OIR phase II) before retinas were immunostained with anti-collagen IV antibody and analyzed; collagen IV is a basement membrane protein that is specifically localized in the retina on the outside of blood vessels. Daily treatment with DHF or vehicle started on P6 and continued until P16, one day before collecting the eyes at P17. We next determined whether treating animals only during OIR phase II (P12-P16) would have a different outcome. Intraretinal revascularization activity results in complete vascularization of the avascular area in the central retina by postnatal day 25 (P25). Also, all neovascularization tufts regress by P25 , 41. To We assessed mRNA expression levels of Bdnf, total TrkB, TrkB.FL and TrkB.T1 in retinas from OIR-P12, OIR-P15, OIR-P17 and OIR-P28 mice . ExpressExpression of total TrkB showed an upregulation trend with age and significantly peaked at OIR-P17 before it showed significant downregulation at OIR-P28 as compared to OIR-P17 . ExpressTo determine whether DHF can activate TrkB in OIR mice, we treated animals with DHF or vehicle and determined the retinal levels of TrkB phosphorylation by Western blot analysis. We assessed TrkB phosphorylation levels in retinas from OIR animals after 4 and 6 hours of injection with DHF or vehicle; we decided to examine the level of phosphorylation 6 hours after DHF treatment instead of the 2 hours used in room-air conditions because the later time point failed to show any significant difference in phosphorylation after DHF treatment. Phosphorylation levels of TrkB at tyrosine residues Y816 and Y705 were assessed using phospho-specific antibodies. DHF treatment of OIR mice did not show a significant increase in phosphorylation level of TrkB at either residue  but rathRetinal vascular development and homeostasis are strictly controlled by oxygen and nutrients needs of the neural retina. Interruption of normal oxygen level during active angiogenesis in premature babies can result in the development of ROP , 41. TheAlthough the expression of Bdnf mRNA in OIR-P12 retina was comparable with that of room air-P12, the mRNA expression of Bdnf was downregulated in retinas from OIR-P15 and remained low in OIR-P17 retinas compared with room-air-P15 and room air-P17 retinas. Similarly, Hellgren and colleages reported similar downregulation of Bdnf mRNA expression in retinas from mice exposed to OIR conditions as compared with retinas from mice reared in room air . This isThe expression data of the Ntrk2 gene showed the level of TrkB.T1 variant to be predominant as compared with TrkB.FL variant. While TrkB.T1 variant is still an important Bdnf receptor, it lacks the catalytic tyrosine kinase domain, which makes it behave as a dominant-negative receptor by binding excess Bdnf, which could impede Bdnf-Trkb.FL signaling . This maLack of DHF protective effect against hyperoxia-induced vessel obliteration and ischemia-mediated neovascularization during OIR could be attributed to several factors which deserve further investigation in future experiments. Hypoxia-induced transcription factors (HIFs) mediate cellular responses to varying oxygen levels by inducing the expression of genes critical for vascular development and homeostasis, such as VEGF –47. AfteOur data here showing lack of a protective impact of DHF during OIR is consistent with conflicting reports in the literature. Although DHF is reported to work as BDNF mimic by activating TrkB and when used, it has neuroprotective effects on relevant neurological diseases –22, otheIn conclusion, to the best of our knowledge, this is the first report that examines the protective effect of DHF on retinal vasculature integrity during OIR. Our findings showed that treating animals with DHF does not provide significant protection against hyperoxia-induced vascular obliteration and ischemia-mediated neovascularization and revascularization. Plausible reasons for these observations are that high oxygen level could result, directly and/or indirectly, in decreased total TrkB protein levels, its activation and signaling capacity that are subject of future investigation. Thus, our studies suggest that DHF does not have the same kind of protective effect on the retinal vasculature as reported on neurons.S1 File(PDF)Click here for additional data file.


OBJECTIVES/GOALS: Penn instituted a Professional Development Core (PDC) to complement existing CTS education programs. Sessions were designed to advance participant knowledge and skills in key competency areas including communication, expectation setting, implicit bias and organizational structure, self-efficacy and resilience in order to enhance abilities to successfully execute career and research goals. METHODS/STUDY POPULATION: The PDC enrolled 4 cohorts totaling 87 trainees and scholars from 2016-2019. This included 35% predoctoral trainees , 39% postdoctoral trainees , and 26% junior faculty . The two-year curriculum consists of 14 lunchtime sessions held bimonthly during academic terms. Session structures include a variety of interactive presentations, activities and facilitated discussions as well as reading material, assessment tools, and case studies. Facilitators include topic experts in academia, entrepreneurship, communications, and professional and personal development. The program is evaluated qualitatively through student satisfaction surveys after each session. RESULTS/ANTICIPATED RESULTS: Of the 2018-2019 participants, 90.8% rated the overall quality of PDC sessions as Very Good (56.05%) or Outstanding (34.75%). DISCUSSION/SIGNIFICANCE OF IMPACT: Feedback indicates that the group benefited from combining predoctoral and postdoctoral trainees, although not all content was immediately relevant to early stage trainees. Trainees appreciated the opportunity to engage with experts in disciplines typically considered outside of traditional science but critical to CTS career success. The flexibility of the curriculum allowed for inclusion of timely topics, newer suites of sessions focus on the multiple dimensions of valuing your science.