Graphene has emerged as a promising material for optoelectronics due to its potential for ultrafast and broad-band photodetection. The photoresponse of graphene junctions is characterized by two competing photocurrent generation mechanisms: a conventional photovoltaic effect and a more dominant hot-carrier-assisted photothermoelectric (PTE) effect. The PTE effect is understood to rely on variations in the Seebeck coefficient through the graphene doping profile. A second PTE effect can occur across a homogeneous graphene channel in the presence of an electronic temperature gradient. Here, we study the latter effect facilitated by strongly localised plasmonic heating of graphene carriers in the presence of nanostructured electrical contacts resulting in electronic temperatures of the order of 2000 K. At certain conditions, the plasmon-induced PTE photocurrent contribution can be isolated. In this regime, the device effectively operates as a sensitive electronic thermometer and as such represents an enabling technology for development of hot carrier based plasmonic devices. The photoresponse of graphene-based photodetectors is dominated by photovoltaic and photothermoelectric effects. Here, the authors leverage strongly localised plasmonic heating of graphene carriers to detect a second photothermoelectric effect occurring across a homogeneous channel in the presence of an electronic temperature gradient. The hot carrier regime gives rise to a strong photothermoelectric (PTE) effect that can under certain circumstances dominate over the photovoltaic (PV) effect6. A strong PTE effect, which we will refer to as the PTE junction (PTE-j) effect, has been reported for graphene devices at the junction of monolayer and bilayer graphene4 at the junction between regions of graphene with different Fermi energies (EF), such as supported/suspended graphene interfaces6 and at graphene p–n junctions with buried split-gates2 and top-gate control5. Graphene–metal interfaces are another well-known example of junctions with different EF. In this case, the EF of graphene on top of or below a metal pad is shifted compared to the metal-free graphene channel due to the difference in work functions of the materials  and is driven by the difference in graphene’s Seebeck coefficients ((S1 – S2) = ΔS) either side of the junction  through 3. The dependence of the PTE-j current on ΔS results in multiple photocurrent sign reversals over a gate voltage sweep due to the nonmonotonic dependence of S1,2 on EF  thermoelectric (TE) experiments where a heater is an internal part of the device contacts14. The electronic temperature gradient across the channel may, in principle, be present when only one side of the metal–graphene–metal device is illuminated. However, studies of these graphene photodetectors to date have typically reported carrier temperature increases of just a few degrees Kelvin or less15 so that the contribution from VPTE-ch was negligible compared to VPV or VPTE-j.In this paper, we report an additional but distinct PTE contribution, where a global electronic temperature difference is established across the device channel , as shown in Fig. 2; however, at these scales the fabricated particles appear slightly elliptical, as evident from Fig. 2c18. In our study, we utilize the L resonance, which occurs near a wavelength of 700 nm. It should be noted that the results presented in this study are obtained for several devices. Please refer to Supplementary Discussion The geometry of our graphene photodetector is similar to that of typical metal–graphene–metal detectors previously reported2) over the device at illumination wavelength 740 nm, close to the NP L-resonance . As the channel length is substantially greater than the width of the laser beam, the separate photovoltage responses of each electrode can be clearly resolved  electrode , characteristic of any dipole antenna /et, where t is the oxide thickness and Vg and VD are gate voltage and the Dirac point position, respectively, we deduce an EF = ℏνF(πn)1/2 of 0.25 and 0.4 eV for the graphene over the gold electrode and SiO2, respectively. The higher p-doping in the graphene channel can be explained by SiO2-induced charge transfer19. The doping EF values are consistent with the results of Raman spectroscopy analysis, discussed in Supplementary Discussion Figure 2 channel, shown in Fig. T between the source and drain electrodes is established, generating a thermovoltage VTE = SΔT. The gate-dependent thermovoltage measured across the device directly reflects the variation of the Seebeck coefficient for the channel graphene1 and thus provides a characteristic footprint of the TE and PTE-ch effects. As shown in Fig. 20. The sign of the thermovoltage indicates the sign of the majority charge carrier and is expected to change from positive to negative, as the gate voltage crosses the Dirac point. However, the observed thermovoltage remains positive, indicating that the Dirac point is not reached for this range of gate bias values, consistent with the resistance measurements shown in Fig. It is important to understand the gate dependence of the conventional TE effect to directly compare to the PTE-ch effect, which has the same physical origin. Therefore, we analyse the gate-dependent resistance and TE properties of a representative two-terminal graphene test device with a 5 × 8 µmNext, we compare the overall trends observed for the plasmonic and nonplasmonic contacts to the TE response of the test device. It is evident that the gate-dependent photovoltage of the plasmonic contact demonstrates a quite similar behaviour to the thermovoltage Fig. . In partTC21, the voltage generated due to the PTE-ch effect for the plasmonic contact can be expressed asTH and T0 are the carrier temperatures at the plasmonic and nonplasmonic contacts, respectively. The hot carriers generated in the vicinity of the plasmonic contact diffuse into the graphene layer and create an electronic temperature gradient. The profile of the temperature gradient is discussed further in Supplementary Discussion T0 remains at Tbath ~ 300 K for a wide range of cooling lengths of 0.2–1 µm, which are typical values for graphene observed at room temperature23.The PTE-ch effect arises due to the carrier temperature difference between two contacts and thus provides an opportunity to analyse the plasmon-induced carrier temperature in graphene. Since the hot carrier population established in graphene after photoexcitation can be described by a thermal distribution with a well-defined hot carrier temperature 14, we integrate Eq.  results in greater saturation, which is clearly observed in the experimental results for both electrodes . In this experiment, we study the photovoltage as a function of the time delay Δdes Fig. . Notablydes Fig. .Fig. 5Pl30. Accordingly, we fit the experimental photovoltage data with two-sided bi-exponential decay to extract relaxation times, the results of which are shown in Fig. 30 where this observation was attributed to the accumulation of photocarriers in the high-field region leading to a longer relaxation time required.The width of the dip observed for the overlap condition corresponds to the carrier relaxation time required for the graphene device to return to equilibrium. The photocarrier dynamics in graphene are well described by a bi-exponential decay with a short sub-picosecond (fast) component immediately after the temporal overlap of the pump/probe pulse followed by a longer picosecond (slow) componentτr = 0.85 ± 0.13 ps  graphene31, the estimated diffusion length is of ~700 nm substantially less than the channel length of 5.5 μm, further supporting our assumed value for T0. As discussed earlier, of particular interest is the flat-band condition occurring at Vg ~ 25 V, where the PV and PTE-j effects are cancelled. At this gate bias, we determine k ≈ 63 nV K−2 , the hot carrier temperature can reach ~200 K over the initial bath temperature of Tbath ≈ 10 K32. In stark contrast, in our experiments, under same illumination conditions, plasmonic amplification results in carrier temperatures as high as ~2200 K. High electronic temperatures aided by plasmonic enhancement sets out the potential for hot carrier-driven photochemistry applications. Moreover, as a sensitive electron (or hole) thermometer, the mechanism and device geometry we demonstrate here can be utilized for optimization of hot carrier generation for the growing field of plasmonic-driven hot carrier applications.To show that plasmon-induced carrier heating is indeed efficient, we compare our results to typical carrier temperatures observed for graphene p–n junctions, where the PTE-j effect is dominant−1 with a device resistance of 1 kOhm. Nevertheless, this is about an order of magnitude lower than the best-reported responsivity to date (6.1 mA W−1) for two-terminal metal–graphene photodetectors using high-mobility and low-defect exfoliated graphene33. The mobility of our devices is measured to be in the range of 700–1000 cm2 V−1 s−1. Notably, significant improvement could be achieved by encapsulation with exfoliated or CVD-grown hBN layers36, which typically results in the material with mobility in the range of 25,000–100,000 cm2 V−1 s−1 and higher Seebeck coefficients by a factor of 237. Consequently, the responsivity of our device geometry could be significantly improved.In terms of the detectivity of our devices, in the presence of plasmonic nanostructures, the device responsivity at the resonance wavelength is strongly enhanced, achieving a ~5 times improvement over conventional devices, corresponding to 0.125 mA WIn summary, we have demonstrated that plasmonic nanostructures can be utilized as part of an asymmetric contact arrangement in a photodetector device to generate a strong electronic temperature gradient across the graphene channel and thus drastically enhance a PTE current generated across the device channel. This effect is distinct from the typical PTE effect generally observed at graphene p–n junctions. By studying the gate-dependent photoresponse, we have identified a regime where this plasmon-induced PTE contribution can be isolated and where our device can operate as an effective electron temperature thermometer. Ultrafast photocurrent studies have shown the device response time of ~2 ps. Consistent with our proposed plasmon-enabled PTE mechanism, we have estimated the local plasmon-induced electronic temperatures to be ~2200 K on picosecond time scales. The demonstrated device geometry can be utilized as a sensitive electron thermometer and also has the potential to contribute to the development of free space compact and ultrafast photodetectors for a range of wavelengths due to plasmon-enabled spectral tunability.2-plasma treatment at 100 W during 2 min. The exposed Cu foil was then etched in an ammonium persulphate solution (15 g L−1) followed by a thorough rinse in deionized water. The floating PMMA/graphene stack was scooped out using the target substrate. After drying, the sample was annealed at 180 °C for 30 min. PMMA was removed in acetone followed by isopropanol alcohol rinse. The graphene was patterned using photolithography process followed by O2-plasma treatment at 50 W for 30 s. The presence of high-quality single-layer material was confirmed by Raman spectroscopy and atomic force microscopy (Supplementary Discussion The photodetectors were fabricated on a highly conductive Si wafer (p-type) to avoid photogating effects due to absorption in Si. A 90-nm-thick layer of thermal oxide was used as a back gating dielectric. Photolithography technique was used to fabricate microscopic contacts  followed by electron beam lithography used to define nanocontacts 40 nm Au). CVD graphene purchased from Graphenea was deposited on top of prefabricated contacts using wet transfer technique. Graphene/Cu layers were covered with poly(methyl methacrylate) (PMMA) A4 495 by spin coating at 3500 rpm for 60 s and curing at room temperature overnight. Graphene layer present on the bottom side of the foils was removed using an O0 nm Au. All the measurements were performed under ambient environment and at room temperature conditions. Raman spectroscopy measurements were performed using a confocal scanning Raman microscope (WiTec Alpha300 system) in a backscattering geometry with 532 nm excitation (1 mW) and a ×100 objective (0.9 NA). Dark-field measurements were conducted in a backscattering geometry with illumination from a Nikon Intensilight C-HGFI mercury lamp, where the scattered light was collected using Nikon LU Plan ELWD ×100 (0.80 NA) objective and transmitted via a fibre to a single-photon-counting module (SPCM-AQRH) connected to a gated photon counter (SR400) or a Princeton Instruments spectrometer for microscopic and spectroscopic measurements, respectively. The transport measurements were conducted using a Keithley 2634B semiconductor parameter analyser unit with a two-point measurement for source and drain electrodes.38. However, in the plasmon-based photodetectors, cross-polarization is not applicable due to high device sensitivity to the beam polarization. To overcome this issue, a two-colour experiment configuration was selected with wavelengths of 650 and 750 nm to be on resonance with plasmonic nanostructures. It should be noted that pulsed excitation is significantly different in nature from continuous wave (CW) excitation. In particular, CW excitation leads to a steady-state temperature as the system equilibrates with time, while pulsed excitation generates a temperature spike of hot carriers that rapidly cool long before the next pulse arrives. Interestingly, at room temperature the device responsivity is the same for both types of excitation , while the open-circuit photovoltage signal was detected with a voltage pre-amplifier (SR560) and a lock-in amplifier (SR830). The back-gate bias was applied using a Keithley 2634B unit. For the time-resolved measurements, the initial pulse at 750 nm was split into two optical paths, and part of it was coupled to a Coherent Chameleon Compact OPO to produce 1300 nm signal that was introduced in second-harmonic generation system (Harmonixx-APE) to provide 650 nm excitation. The initial pulse was delayed using a retro-reflector mounted onto a motorized translation stage (Thorlabs PT1-Z8). The two pulses were combined using a beam splitter and focussed on the sample to form overlapping beams. Previously performed time-resolved photocurrent studies of graphene devices typically utilize cross-polarized beam configuration to minimize interference near zero time delay point2 to reproduce illumination conditions. The Gaussian profile has been incorporated through multiplication with xL is the position of the laser spot and RL is the laser spot size.Numerical calculations were performed using a finite-difference time-domain method . For Fig. Supplementary Information


The use of cork for a variety of applications has been gaining significance due to environmental concerns and political agendas. Consequently, its range of applications is growing rapidly. In this work, aiming to improve its mechanical response for crashworthiness applications, cork agglomerates were enriched by small quantities of graphene oxide or graphene nanoplates in order to observe a resulting improvement of the mechanical behaviour during quasi-static and dynamic compressive loading cases. To produce homogenous cork agglomerates including graphene, the material was previously dispersed into granulated cork using stirrers to achieve a good distribution. Then, the typical procedure of compression and curing was carried out. Magnified images attest a good dispersion of graphene into the cork matrix. Mechanical testing was performed for a variety of graphene concentrations , becoming clear that the beneficial effect of including graphene (either oxide or nanoplates) is related to a later densification stage while keeping the same stress plateau levels. There is increasing interest in cellular materials as a solution for many engineering applications. They have a huge potential that has been not fully exploited yet . They ofOne can state that the best material for each application depends on the application itself. Regarding crashworthiness, Expanded Polystyrene (EPS) is one of the most widespread synthetic cellular materials thanks to its excellent cost-benefits ratio. For this reason, most of protective headgear (from sports to roads) contains EPS. Nevertheless, this material is crushable, deforms plastically, hence offering minimal protection in multi-impact cases ,10,11,12As a common factor to all previously referred materials, the recyclability has been becoming a very serious issue. Oil-derived plastics are one of the major sources of world’s pollution ,14 and iQuercus Suber tree. The bark totally renews in approximately nine years. Agglomerated cork, a by-product mainly from wine-stoppers production can ally mechanical viscoelasticity to other well-known insulation properties of cork , making it a quite interesting material from the engineering point of view.Regarding crashworthiness, many researchers have been attracted by cork mainly due to its capability to absorb energy ,16,17. CAgglomerated cork is produced by compressing cork grains together (previously grinded to a specific grain size). A binder is added to keep the agglomerate structurally stable. Then, the compressed volume is cured in an oven. In the end, cork agglomerates are composite materials and their mechanical properties can be adjusted like any other composite by altering its density, percentage of binder, grain size or binder type, as demonstrated by Santos et al.  and PereSome authors have been proposing techniques and solutions to enhance the mechanical properties of cork agglomerates: Fernandes, et al.  showed tThe innovative point and main objective of this study is to test and validate the use of graphene as a tool to enhance the mechanical behaviour of agglomerated cork under compressive loading. Indeed, given its outstanding properties, graphene composites are being introduced in a wide range of fields, from electronics and mechanics to medical fields. Graphene, the thinnest existing material, provides high thermal conductivity, outstanding mechanical features , high optical transmittance and high electronic transport ,25. OtheThe authors’ study aims to add another remarkable application to graphene by dispersing graphene oxide or graphene nanoplates into the agglomerated cork matrix. Next sections will show the methods carried out and the resulting beneficial outcome in terms of mechanical properties.The cork grains used in the experiment were provided by Amorim Cork Composites (ACC), with particle size of 0.5–1 mm. The graphene nanoplates (GNP) powder was provided by Cheap Tubes Inc.  and used as given, while graphene oxide (GO) (4 mg/mL aqueous dispersion) was purchased from Graphenea . GO was dried by lyophilisation and then submitted to a ball milling process in order to obtain small sized particles and gather uniform dispersion in the final composite.The binder used in this experiment was supplied by Flexpur , and denominated as flexible due to its chemical and physical features. The binder is based on most common used isocyanates, which are the aromatic diisocyanates, toluene diisocyanate (TDI) and methylene diphenyl diisocyanate (MDI). In their work, Santos, et al.  also use3 in an analytical balance. It shall be noticed that the weight % (wt.%) is herein related to the total weight of the mixture—i.e., cork grains, binder, water and graphene. In the second step, the graphene nanoparticles or graphene oxide was added to the agglomerated cork. The components were mixed for 3 h. Hereafter, water (5 wt.%) was added to the cork part while the blend was kept homogenized using a mixer. One minute later, the binder (10 wt.%) was introduced to the mixture. In the last step, the mixture was compressed by a pressing machine until the desired density and shape was achieved. Furthermore, the samples were heated up to the temperature of 140 °C and held at that temperature for additional 2 h. The sequential production process of the cork-graphene samples is depicted in The preparation of the specimens commenced by the authors consists of four main steps. At first, it was necessary to weight the exact amount of constituents, which were necessary to obtain the assumed density of 160 kg/mWithin the production process, the authors created three samples for each kind of composite, separately for both forms of testing quasi-static and dynamic. In total, the authors performed tests on 42 samples. The description and further used nomenclature is presented in The samples were inspected by the use of a Scanning Electron Microscope VEGA3  in order to observe the distribution of the added graphene nanoplates or graphene oxide to the agglomerated cork material. Cork in its axial and tangential section is visible as brick wall and in its natural radial section mostly as heptagonal, hexagonal and pentagonal cells . ExemplaIn the experiments, it is intended to observe the effect of GNP or GO presence and concentration on the agglomerated cork mechanical behaviour. In all tests, only the percentage of the reinforcement by GNP or GO was varied to the values of 0.1, 0.5 and 1 wt.%. As it was shown in −1. The recorded load-deflection-curves were used in order to calculate the stress-strain.The experiments were carried out in two stages: quasi-static and dynamic loadings. The Shimadzu AG50 KN universal testing machine  was used for the quasi-static compression tests. Thereby, the samples were subjected during the test to compression at a strain rate of 0.1 sThe dynamic tests were performed by using the Instron Dynatup 9250hv . The dynamic tests were separated into first and second impact for each specimen. As the tests were carried out in a row on the same day, the authors ensure constant temperature conditions for all tests. The samples were impacted by a 7.05 kg flat tup with a 50 mm diameter, which is initially positioned 300 mm above the specimen top surface. Hence, the applied kinetic energy was set to 20.75 J. The tests were scheduled in the way that after a relaxation time of 10 min, the second impact was carried out for the same specimen. For the second impact, the height of the tup was adjusted for each specimen separately to 300 mm, guaranteeing the same impact energy of 20.75 J. Data acquisition for the dynamic tests was done by the use of a load cell with a sample rate of 204.8 Hz. Additionally, a high-speed camera Phantom V12  was used at a frame rate of 10,000 fps with an exposure time of 40–90 μs. The gathered video-files were processed by using TEMA Motion . The dedicated software allowed the authors to generate for each specimen accurate displacement-time data for characteristic, beforehand installed high-contrast markers. These markers were positioned on the machine’s impactor and anvil. As the sample rate was differing for load cell data and video data, the data correlation was done by the use of National Instruments LabVIEW 2014 software.The agglomerated cork samples and the cork samples enriched by Graphene Oxide exhibit firstly a comparable performance under quasi-static compression. By observing Consequently, densification establishes later. This is a desirable feature of energy absorbing materials, deforming at almost constant stress values and achieving densification for high compressive strains. Thus, a gradual enhancement of the crashworthiness properties can be recognized by increasing the wt.% of GO to the agglomerated cork specimen. According to this observation, the most significant changes majorly occur from 0 to 0.1 wt.% and from 0.5 to 1 GO wt.%.In appropriate applications of energy absorption, the plateau effect is the most relevant segment of the stress-strain curve. The longer the plateau phase, the better applicability of the solution can be stated. From this particular point of view, the GNP and GO enriched specimens are interesting, once they lasted longer showing such behaviour, with material reaching densification for more than 80% uniaxial compression. On the other hand, it needs to be noted that the plateaus stress values were not increased. Therefore, by remaining stable in terms of stress values, but with a delayed densification, it can be concluded that graphene promotes a beneficial effect in terms of mechanical properties. Consequently, the attractiveness of adding this nanomaterial is raised for any kind of composite as a reinforcement.Comparing the results of equally GNP- and GO-enriched specimens with each other, By comparing the added quantity of 1 wt.% of GO or GNP to the specimen in Adding 1 wt.% GNP has better effects on the improvement of stress-strain curves compared with GO, namely an elongated plateau stage. Such a difference is mainly related to the chemical nature of these two fillers. GO sheets owns oxygen functional groups at their surface, making it a polar nanomaterial. On the contrary, GNPs are only made by carbon . The cork grains are also apolar. Relating this to the mixing procedure within the production process, it is more likely to obtain a better dispersion of the GNP on the cork grain, yielding in better results.One must however bear in mind, that in both types of reinforcement their most significant changes in performance were due to higher quantities of added GO or GNP, higher than 0.5 wt.%. Above 1 wt.% a saturation of the material is expectable with no further improvements.The authors were especially interested in the influence of adding graphene oxide or graphene nanoparticles to agglomerated corks mechanical properties—particularly stiffness—under dynamic loading. The benefit would be seen in a material with high energy absorption capabilities, which is not about to fail suddenly by reaching higher stress levels. As cork is generally accepted as a material capable to withstand at least a second major impact, each specimen was tested for the second time in the same conditions. The behaviour of all the different samples is very similar. After analysing the results, the dynamic performance of GO- or GNP-enriched cork material can be stated as very comparable to each other, showing no significant difference, neither to the other type of enrichment nor to the untreated agglomerated cork. In this work, the authors compared the quasi-static versus dynamic response of the graphene-enriched agglomerated cork material. We noticed that unlike for the quasi-static loading, during the dynamic tests, enriching samples by graphene has no significant influence on the energy absorption capabilities compared to the cork agglomerate without added graphene. Since the data was verified, and the tests were carried out on three samples for each test, the experiment is considered to be robust. Therefore, the explanation of this interesting phenomenon is related to the binder (polyurethane glue), impact energy and the binder reinforcement i.e., GNP or GO. First of all the cork material, in comparison to the used polyurethane binder, is a very good heat insulator with small heat capacity ,33. OnceThis research was intended to create a reinforced agglomerated cork material. Therefore, graphene nanoplates or graphene oxide was added in the quantities of 0.1, 0.5 or 1 wt.%. Graphene holds promise for various material and device applications due to its exceptional mechanical properties. Nevertheless, in numerous works and research it was a struggle to blend it homogenously into cork with success. The presented work contributes to this field by creating original composites enriched by GNP or GO. The samples were observed concerning the distribution of the added GNP or GO by the usage of SEM and consecutively tested toward their mechanical response to quasi-static and dynamic loads. The authors conclude, that:▪Adding GNP or GO to agglomerated cork material extends the plateau stress and delays densification under quasi-static loads.▪The most significant changes in these effects were recognized for the change from 0 to 0.1 wt.% and 0.5 to 1 wt.% of added GO or GNP.▪In contrast to quasi-static loading, during the dynamic tests, for the particular testing conditions (impact energy and impact velocity) enriching samples with graphene has no significant influence on the energy absorption capabilities compared to the non-reinforced cork agglomerate.▪Enriching samples by GNP or GO in the tested quantities showed limited influence on the energy absorption capabilities, that is, its effect is only evident for low strain rates. Nevertheless, in the latter, it clearly delayed densification stages, promoting better crashworthiness properties. Damage resistance was also not found to be enhanced.Overall, the findings of this research contribute to possible applications of this reinforced cork material. The revealed effect of extending the plateau stress gains importance in applications exemplarily as damper of heavy-load machinery, particularly, when weight is a limitation. Thereby, the findings are opening a new material resource.


Self-healing materials have attracted much attention because that they possess the ability to increase the lifetime of materials and reduce the total cost of systems during the process of long-term use; incorporation of functional material enlarges their applications. Graphene, as a promising additive, has received great attention due to its large specific surface area, ultrahigh conductivity, strong antioxidant characteristics, thermal stability, high thermal conductivity, and good mechanical properties. In this brief review, graphene-containing polymer composites with self-healing properties are summarized including their preparations, self-healing conditions, properties, and applications. In addition, future perspectives of graphene/polymer composites are briefly discussed. Regenerative abilities allow creatures to repair damaged functions to prolong their life span. Researchers are inspired to design and prepare self-healing materials to increase the lifetime of materials and reduce the total cost of systems during the process of long-term use. Recently, great progress has been made in self-healing composite materials that possess the ability to restore their structure and functionality after damage. Early self-healing materials were focused on microcapsule or microtubule by release of healing agents to achieve repairing. However, the self-healing times of these methods are dependent on the amounts of healing agents in the microcapsule or microtubules . To addr2 carbon atoms, has attracted widespread attention in all kinds of research areas due to its large specific surface area, excellent electrical conductivity, thermal conductivity, and unique mechanical properties [Graphene, as a new type of two-dimensional planar monolayer of spoperties ,23,24,25operties ,28,29,30operties ,32,33,34In this review, the current advances in self-healing graphene/polymer composites have been summarized, including their preparation methods, self-healing conditions, properties and applications. Finally, the future prospects of the self-healing graphene/polymer composites are discussed.The simple mixing is the simplest and commonest method for preparing graphene or graphene derivative/polymer composites ,41,42,43Considering its easy agglomerate due to the strong π-π interactions, usually, graphene has been oxidized into graphene oxide (GO). Yan et al. fabricated chitosan/GO supramolecular hydrogels with self-healing properties . It was N,N-methylenebisacrylamide (MBA), and potassium persulfate, were added to the graphene dispersion in water and polymerized.In situ polymerization can be interpreted to mean that the monomer and graphene or graphene derivative were mixed firstly and subsequently polymerized by the addition of initiators ,56,57,58Taking advantage of this method, our group fabricated cationic PAM/GO self-healing hydrogels with tough, stretchable, compressive property . The GO During the in situ polymerization process, graphene can also be modified and participate in the polymerization, forming the covalent bond between graphene and polymers. For example, Karak et al. fabricated a tough self-healing elastomeric nanocomposite containing a castor oil-based hyperbranched polyurethane (PU) and an iron oxide nanoparticle decorated reduced graphene oxide (IORGO) nanohybrid . The IORSimilarly, Kim et al. synthesized a phenyl isocyanate modified GO and obtained the self-healing composites by condensation reaction of poly(tetramethylene glycol) and 4,4′-methylene diphenyl diisocyanate and phenyl isocyanate modified GO in the presence of phenylhydrazine  Figure b,c.The DA reaction and its retro-Diels-Alder (rDA) analogue is a promising route to introduce self-healing properties to polymeric systems, which can be performed under mild conditions without any catalyst or healing agent ,64,65,66The layer-by-layer (LBL) self-assembly technique is a versatile approach to fabricate multilayered nanostructural composites ,71,72,73Sun and coworkers reported an intrinsically healable, reduced graphene oxide (RGO)-reinforced polymer composite film via LBL assembly . RGO modApart from the aforementioned fabrication methods, there are other ways to fabricate self-healing graphene/polymer composites. Tang et al. reported conductive and self-healing nanocomposite hydrogels though a simple hydrothermal method . All theTo restore their original properties, graphene/polymer composites need to repair themselves autonomously or require external energy/stimuli such as mechanical force, light, heat, pH changes. In this section, different self-healing conditions of graphene/polymer composites were summarized.Heating is a common self-healing condition due to its easy operation. By heating, the mobility of polymer chains increases, thus facilitating the self-healing of graphene/polymer composites ,80,81,82Although heating is an easy way to repair damaged graphene/polymer composites, during heating other parts of devices are susceptive to heat, thus heating will cause interference of other parts of the devices and result in the deterioration of devices. Therefore, light radiation is an alternative way to repair the damaged composites. Graphene has good ability of photothermal energy transformation that makes the self-healing graphene/polymer composites usually show the same capacity . Zhang eN,N-dimethylacrylamide) hybrid hydrogel realized by NIR irradiation for only 2~3 min up to the strength recovery of ~96% [The near-infrared (NIR) irradiation has been suggested as a non-invasive, harmless and highly efficient skin-penetrating biomedical technique ,85. Tong of ~96% . GO acte of ~96% . When th of ~96% . The selN,N-dimethylacrylamide) chains on 3D graphene networks that exhibits good neural compatibility, high conductivity, low impedance and efficient NIR-triggered photothermal self-healing [Considering NIR as a non-invasive, harmless and highly efficient biomedical technique, it can be used in photothermal therapy. Wang et al. reported a hydrogel made by cross-linking poly. The polymer chains interdiffuse across the crack surface of the damaged fibrous membrane under 532 nm light irradiation, and recrystallize upon cooling after turning off the continuous-wave diode laser. The surface conductivity recovery by 91% and tensile strength of the membrane are still well maintained after multiple cutting-healing cycles .Graphene has an excellent microwaves absorption capability due to its large area conjugated π-structure ,90. UndeDeionized water or organic solvent is a practicable external stimulus to heal damage ,92,93,943+ hydrogel [3+ ions to the carboxyl groups on the PAA chains. Keeping the cut surfaces in contact and immersing them in FeCl3/HCl solution for a certain time gave rise to the self-healing process. After 15 h immersion, the healed hydrogel can bear a dumbbell of 5.5 kg. Importantly, the break position of the healed hydrogel after tensile test is not the healing position, proving the perfect connection of cut surfaces by Fe3+ ions  allow the polymer chains to diffuse and mix at room temperature, and be able to self-heal at room temperature without the need of any plasticizer, solvent, healing agents or external stimuli. Two cut pieces of samples were gently brought into contact and healed in less than 60 s. The obtained stress–strain curves were similar to that of the original uncut samples  based on GO and amine-terminated randomly branched oligomers . Both th samples . The HBN samples .3+ and catechol. No obvious cut line was seen and the healed sample can be stretched without fracturing by the tweezers.Kim et al. developed a mussel-mimetic nanocomposite hydrogel based on catechol-containing polyaspartamide and GO . Two pieApart from the aforementioned self-healing conditions, microcapsules are also utilized to heal the damaged composites, although this method cannot realize multiple healing in one damaged area. Gao et al. fabricated GO microcapsules (GOMCs) in Pickering emulsions containing linseed oil as the healing agent . The GO −3. The healed hydrogels also have favorable mechanical properties with a fracture strain of 2000% and a fracture strength of 153 kPa. GO played a role as the cross-link agent in the polyacrylamide network, which improved the tensile property . Primary GO sheets are well dispersed in the polymer matrix at low content (0.1 wt %). Sun et al. fabricated a covalent bonding GO/PU composite with significant mechanical reinforcement and thermal healable property. The Young’s modulus (21.95 ± 2.56 MPa) and fracture strain (449 ± 16%) increased twice and the fracture stress (8.01 ± 0.71 MPa) even increased nearly 4 times at a GO loading of only 0.1 wt %. The enhanced mechanical properties of the composites are ascribed to the good dispersion of GO and covalent linking based on DA chemistry . To imprco-acrylic acid) hybrid materials with shape memory behavior and self-healing ability [2/RGO nanocomposite fabricated by Karak et al. also possessed excellent shape memory behavior [Besides high mechanical properties, shape memory behavior is an attractive property for self-healing graphene/polymer composites due to their wide applications in machinery, electronic device, chemical industry, biology and other fields ,61,80,82behavior . As RGO For conductive graphene/polymer composites, the shape memory can be realized by electrical conductivity. When the content of conductive filler in a polymer matrix exceeds the percolation threshold, the conductive network can generate Joule heating. Therefore, the speed of the shape memory depends on the electrical conductivity of the filler. For instance, Mohammad and coworkers synthesized fast electroactive shape memory and self-healing PVA/graphene nanocomposites. The electrically triggered shape recovery experiments of the composite containing 3 and 4.5 wt % of graphene  were conducted under four different DC voltages  and the resulting recovery ratio as a function of recovery time is presented in +) transportation.Generally, a polymer is partially an insulator because the covalent bond of the polymer chain does not have free carries (electrons), on the other hand, as the polymer molecules pile together by van der Waals forces, the distances between the molecules are large, electrons overlap between the molecules are poor, so the free carries are very difficult to mobile in polymer . TherefoSupercapacitors are very common applications of conductive self-healing composite. For instance, Liu and Gao et al. assembled a stretchable and self-healable supercapacitor . The supFabrication of self-healing anticorrosive coatings has attracted attentions as it has the ability to extend the service life and prevent the substrate from corrosive attack. The self-healing coating on the surface of the material can repair the damaged surface automatically to protect the material ,109,110.One of the threats to human health is microbial contaminations or infections. Microbial fouling is the critical factor to degradation of polymeric self-healing materials. Therefore, antimicrobial activity is a fantastic property of self-healing composites. The nanocomposites prepared by Karak et al. exhibited good antimicrobial activity against Staphylococcus aureus, Escherichia coli and Candida albicans because sulfur-containing compounds and polysulfanes are generally considered to be antimicrobial agents .N,N-dimethylacrylamide that can achieve a self-healing ability at 37 °C [The medical field also has great interest in self-healing materials. For example, Hu et al. fabricated a double network hydrogel based on β-CD functionalized graphene and at 37 °C . CamptotDue to the biocompatibility of DNA, the hydrogels can possess a variety of biological and environmental applications. Shi et al. reported GO/DNA self-healing hydrogels with high mechanical strength, excellent environmental stability, high dye-adsorption capacity and can be applied in tissue engineering, drug delivery, and removing organic pollutant . Self-healing hydrogels were proposed to be used as biomaterials because of the capability of spontaneously healing any injury. Lu et al. prepared a mussel-inspired conductive, self-adhesive, and self-healing tough hydrogel . The hydSelf-healing graphene/polymer composites are one of the most promising intelligent materials. The latest research progress in the preparation, properties and applications of self-healing graphene/polymer composites is reviewed and some of the composites are summarized in (1) Graphene, as a component of the self-healing composites, can play an active role in the self-healing process because of the photothermal energy transformation ability. In this case, graphene acts as an energy absorber to transform irradiation or sunlight to thermal energy rapidly and efficiently to promote the mutual diffusion of the polymer chains across the damaged interface, thus facilitating the self-healing process. On the other hand, graphene in the composites is only used as a reinforcing agent to enhance the mechanical strength. In this case, the self-healing process may be impeded because some interactions between graphene and polymers will limit the movement and diffusion of the polymer chains. In addition, the glass transition temperature of the composites may be changed after the addition of graphene, thus influencing the self-healing efficiency. Therefore, graphene as a reinforcing agent can improve the mechanical performance. However, the self-healing efficiency of the composite will be reduced. The mechanical properties and self-healing efficiency are two contradictory aspects. How to balance the two aspects to achieve graphene/polymer composites with both high mechanical strength and high self-healing efficiency is a challenge.(2) For intrinsically self-healing graphene-based materials, simple contacting without any external stimuli is considered to be most potential considering practical application. Therefore, exploring this kind of self-healing graphene-based composites is desired.(3) For electrical conductive graphene/polymer composites, the mechanical strength and the conductivity are two contradictory aspects. Increasing the graphene content will increase the conductivity of the composite, but excess graphene will reduce the mechanical strength. How to obtain high mechanical strength and high conductivity of the graphene/composites with self-healing properties is a challenge.(4) A lot of polymeric materials should be developed which exhibit self-healing properties. Also, it is highly challenging to endow the combined properties in a single material. The structural incompatibility among such types of polymers is one of the crucial reasons for this difficulty.(5) To improve the compatibility of polymer and graphene, graphene usually needs to be modified. However, modification of graphene will lose some inherent properties of graphene. Therefore, how to improve the compatibility of polymer and graphene and retain the inherent properties of graphene as much as possible in the composite is a challenge.In this review, we have summarized the recent progress of graphene-containing polymer composites with self-healing capability. The preparation methods, self-healing conditions, and properties and applications of the graphene/polymer composites have been briefly discussed. Finally, the further perspectives of the composites have been proposed. Intelligent materials and self-healing materials, specifically for graphene-containing composites, are still in the initial stage. There will be a great research space in the field. The progress will guide further development of the self-healing graphene/polymer composites.


Bcl-2 pre-mRNA and consequent increase of a negative regulator of autophagy, Bcl-2, which correlate with and are caused by a progressive increase of TDP-43, one of the early events associated with ALS-TDP pathogenesis, in the spinal cord of TDP-43 (N390D/+) mice and spinal cord MN derived from their ES cells. The TDP-43 (N390D/+) knock-in mice appear to be an ideal rodent model for basic as well as translational studies of ALS- TDP.Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset degenerative disorder of motor neurons. The diseased spinal cord motor neurons of more than 95% of amyotrophic lateral sclerosis (ALS) patients are characterized by the mis-metabolism of the RNA/DNA-binding protein TDP-43 (ALS-TDP), in particular, the presence of cytosolic aggregates of the protein. Most available mouse models for the basic or translational studies of ALS-TDP are based on transgenic overexpression of the TDP-43 protein. Here, we report the generation and characterization of mouse lines bearing homologous knock-in of fALS-associated mutation A315T and sALS-associated mutation N390D, respectively. Remarkably, the heterozygous TDP-43 (N390D/+) mice but not those heterozygous for the TDP-43 (A315T/+) mice develop a full spectrum of ALS-TDP-like pathologies at the molecular, cellular and behavioral levels. Comparative analysis of the mutant mice and spinal cord motor neurons (MN) derived from their embryonic stem (ES) cells demonstrates that different ALS-associated TDP-43 mutations possess critical ALS-causing capabilities and pathogenic pathways, likely modified by their genetic background and the environmental factors. Mechanistically, we identify aberrant RNA splicing of spinal cord  SOD1 and TARDBP encoding the RNA/DNA-binding protein TDP-43, with mutations associated with ALS have been identified . The kinetics of the Suc-LLVY-AMC cleavage was measured spectrofluorometrically at 5 min intervals for 1 h under 37 °C by using an EnSpire Multiplate Reader . Negative control assay was conducted in the presence of the specific proteasomal inhibitors MG-132  and Lactacystin .For staining of the brain and spinal cord, 14 μm thick brain sections and 10 μm thick spinal cord sections were prepared as described before . AntibodFor staining of ESC-derived MN, the culture medium was removed and the cells were washed gently with PBS. MN on coverslips were fixed by freshly made and pre-colded 4% paraformaldehyde for 20 min. The samples of spinal cord sections or ESC-derived MN were then permeabilized with PBS/0.5% Triton X-100 for 7 min at room temperature. After blocking with 2% fetal bovine serum (FBS) for 1 h at room temperature, the samples were incubated overnight at 4 °C with one or more of different antibodies, including goat anti-choline acetyltransferase , rabbit anti-TDP-43 (Proteintech), and mouse anti-Tau . After washing, the samples were incubated with DAPI (Invitrogen) plus Alexa Fluor 488-conjugated, Alexa Fluor 546-conjugated, and Alexa Fluor 647-conjugated secondary antibody (Invitrogen) for 1 h at room temperature. The images were analyzed on a Zeiss LSM 780 confocal microscope.6 MNs were lysed with either RIPA buffer or urea buffer. For in vitro phosphorylation of TDP-43 as a positive control of Western blotting, the recombinant TDP-43 was expressed in E. coli. and purified through Nickel affinity column. The purified TDP-43 was then prepared in 3 μM in 10 mM Tris, pH. 8.0, 5 mM MgCl2, 200 μM ATP, and 1 μl Casein Kinase 1 (New England BioLabs) in 100 μl solution. The reaction was incubated at 30 °C for 2 h and stopped by adding SDS containing sample loading dye.The mouse tissues (200 mg /ml) were extracted with RIPA buffer  or urea buffer  containing protease and phosphatase inhibitors (Roche). After homogenization and centrifugation of the tissue at 13,000 rpm 4 °C for 30 min, the solution from the urea buffer-derived extract was defined as the “total protein extract”, and the supernatant from the RIPA buffer extract(s) was defined as the “soluble fraction”. The pellet from the RIPA buffer-derived extract was washed by RIPA buffer for 3 times, dissolved in urea buffer, and defined as the “insoluble fraction”. The cellular extracts of cultured MN were prepared in the following way. The cultured spinal MN (see below) were purified by GFP (+)-based sorting in FACSAriaII SORP. The purified GFP (+) MNs were cultures for different days and lysed with lysis buffer. 1*10The different protein extracts were separated by 12–15% SDS-PAGE and immunoblotted with the appropriate primary antibodies  and then the secondary antibodies. The bound antibodies were detected by using the chemiluminescence Western blotting detection reagent ECL . The expression levels of different proteins were compared by measuring their band intensities on the blots with Image J software (NIH).The standard process followed that by Arnold et al. (2013). Briefly, spinal cord was lysed gently with 10x (vol./weight) hypotonic buffer A  containing protease inhibitors (Roche) by homogenization. After 15 min on ice, 0.5% NP-40 was added and the samples were vortexed and centrifuged at 800 g for 5 min at 4 °C. The supernatant was defined as the cytosolic fraction. For preparation of the nuclear extract, the nuclear pellet was washed with hypotonic buffer A, added 5x (vol/wt) extraction buffer  glycerol, 1.5 mM MgCl, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM DTT) containing protease inhibitors, and then incubated at 4 °C while rotating at 60 rpm for 40 min.A modified MN induction protocol was used for this analysis. On day 5 of the differentiation phase, the EBs were directly cultured in the MN medium without dissociation by trypsin. The EBs were then examined by FACS (LSRII-12P) for 14 days in culture. The percentage of the GFP (+) MN in the EBs on the 1st day was defined as 100% and the folds of change on the following days were quantitated and compared for the WT and mutant MN.Gapdh gene. The sequences of the PCR primers are listed below:Total RNAs from the tissues or cells were isolated following the standard protocol using Trizol reagent (Thermo Fisher Scientific). cDNA synthesis was carried out using SuperScript II reverse transcriptase (Invitrogen) and subjected to PCR. Alternatively, real-time PCR (qPCR) using SYBR Green PCR Master Mix (Applied Biosystems) and ABI 7500 real-time System was carried out. All data were analyzed after normalization to the expression level of the Tardbp forward primer: 5′- GGTAATCCAGGTGCTTTG-3′Tardbp reversed primer: 5′- CCTGCATTTGATGCTGACCC-3′Bcl-2 splicing forward primer (P1):5′-TTCGGGGAAGGATGGCGCAAGC- 3′Bcl-2-201 forward primer (P2): 5′- ACGGAGGCTGGGATGCCTTTGTGG-3′Bcl-2 reversed primer (P3): 5′- TCACTTGTGGCCCAGGTATGC-3′Eif4h splicing forward primer: AAGCTAGCTATCCATGGCGGACTTCGACACCTACGACG-3′Eif4h splicing reversed primer: 5′-CCCGGGAGCTCTCATTCTTGCTCCTTTTGAACGAC-3′Mapt forward primer: 5′-AGGAAATGACGAGAAGAAAGC-3′Mapt reversed primer: 5′-GACCCTGGAGGAGTCTTAGG − 3′Rbm39 forward primer: 5′- GGCCGCTACAGAAGTCCTTACTCC - 3′Rbm39 reversed primer: 5′-ACCCAATCTTTCCTCGGATGG- 3′ANkrd12 forward primer: 5′- TTACTATTAGCCCATCAAGAAATGAAG − 3′ANkrd12 reversed primer: 5′- TATGTCCTGGATCTGAATCTGTGTC-3′cDNAs of mouse wild type TDP-43, TDP-43 (A315T) and TDP-43 (N390D) with addition of a Myc epitope tag to the 3′ -end were generated by RT-PCR of different mouse spinal cord RNAs as the templates. The PCR primers used were: forward 5′-CCG CTC GAG CGG ATG TCT GAA TAT ATT CGG GTA AC-3′; reverse 5′- TGC TCT AGA GCA CAT TCC CCA GCC AGA AGA C-3′. These cDNA fragments were first cloned into pGEM-T vector (Promega Corporation) and then subcloned into the XhoI/ XbaI sites of pEF-myc vector (Promega Corporation). The generation of expression plasmids carrying human wild-type TDP-43, TDP-43 (A315T), and TDP-43 (N390D) cDNA, respectively, was described in Wu et al. .The N2a cells were transfected with the pEF-myc vector or different mouse TDP-43 expression plasmids. HEK293T cells were transfected with the vector or different human TDP-43 expression plasmids . DNA traThe procedures followed those by Huang et al. (2014) In brief, MNs on day 2 were treated with cycloheximide (20 mg/ml) for different time periods . The levels of TDP-43 in the treated MN were analyzed by Western blotting and the relative intensities of the TDP-43 bands were quantified by AlphaEaseFC software.2 in the dark chamber and incubated at 37 °C, 5% CO2 for 45 min. The excess Fura-2-AM was washed out with HBSS (without CaCl2) and incubated for an additional 30 min in HBSS (containing 2 mM CaCl2) for recovering, and then the coverslips were transferred onto the recording chamber of an inverted fluorescence microscope (Zeiss Axiovert 200) equipped with a 20 X objective lens and MetaFluor (Molecular Devices) acquisition and analysis software. The fluorescence signals at 510 nm were acquired every 2 s in 5 min by UV excitation at wavelengths of 340 nm  and 380 nm , respectively. The formula MNs were dissociated on differentiation day 5, plated on coated 22 X 22 mm glass coverslips, and grown in MN medium until calcium imaging experiments. HEK293T cells were cultured on 0.1% gelatin-coated 22 X 22 mm glass coverslips and transfected with plasmid DNAs for 24 h before calcium imaging. Before calcium imaging, the MNs were treated with 2uM Fura-2-AM (Invitrogen) in HBSS (Invitrogen) containing 2 mM CaClStatistical differences were analyzed by Kaplan–Meier and log rank test(s) for the survival rates, two-way ANOVA and Bonferroni post hoc analysis for multiple group comparisons, and the unpaired Student’s t-test for two group comparisons . The error bars of mouse behavior were showed by SEM, and the error bars of molecule and cellular analysis were displayed by SD.The images of immunofluorescence staining were acquired with Z-stacked on a Zeiss LSM 510 META confocal microscope software; basically, one slide contain 3 spinal cord sections or 2 brain sections of one mouse at least; each sample of IF staining will duplicate; the merge images and analysis such as motor neuron number and axonal length were measured/calculated via ImageJ (NIH). The bands intensities of RT-PCR and Western blotting were also quantified through ImageJ or AlphaEaseFC 4.0 (Alpha Innotech) or ImageJ (NIH). The raw data of Calcium imaging is recorded via MetaFluor (Molecular Devices) acquisition and analysis software, and the result were calculated using Prism 7.tardbp gene . Over time, the N390D/+ male mice from either line #108 or #361 started to show abnormal hind limb-clasping and kyphosis at around 8 months of age, as exemplified in Fig. Rotarod and T-maze tests were used to assess whether the knock-in mice would develop ALS- and/or FTLD-like phenotypes. Surprisingly, while heterozygous N390D/+ male mice from either line #108 or #361 exhibited significant motor dysfunction at the ages of 6 months and older, heterozygous and homozygous A315T male mice appeared indistinguishable from +/+ littermates Fig. a. Notwittardbp mRNA , ubiquitin(+) aggregates, and depletion of nuclear TDP-43 in diseased motor neurons . The patIncreasing evidence showed that glial pathology would mediate the disease progression of ALS via reactive astrocytes and microglial activation . In partWhere ALS disease begins has remained argumentative. One of the hypotheses is “dying-back”, in which ALS begins within the lower motor neurons (LMN) and then the pathology spreads from LMN to the upper motor neurons (UMN) , 17, 66.The above data clearly revealed abnormal intracellular localization and aggregation of TDP-43 in the spinal cord of the N390D/+ mice in concert with the mis-regulation of TDP-43 metabolism. The occurrence of TDP-43 protein aggregates was known to be countered by the combined actions of autophagy and the ubiquitin proteasome system (UPS) . We therBcl-2 mRNA is one of the potential neuronal RNA targets of TDP-43 [Bcl-2 pre-mRNA splicing. Mouse Bcl-2 mRNA consists of 2 splicing variants, Bcl-2-201 and Bcl-2-202 (Ensembl), the former of which encodes the well-studied Bcl-2 protein (NP_033871 from NCBI). Indeed, there was an excellent correlation between the age-dependent increase of spinal Bcl-2 protein and that of the functional Bcl-2 mRNA, i.e. the isoform 201 in the N390D/+ mice, as shown by RT-PCR analysis  cell culture   plays a causative role in altering the alternative splicing of Bcl-2, we also checked the expression patterns of several known TDP-43 target genes. Among them, the splicing pattern of the pre-mRNA of Eif4h was altered in mutant TDP-43 transgenic mouse models [Eif4h pre-mRNA was also mis-regulated in the spinal cord of N390D/+ male mice at the age of 3 months  knock-ins on ALS pathogenesis were due, at least in part, to cell-autonomous effects on spinal cord motor neurons (MN), we generated spinal cord MN in culture. To accomplish this, the N390D/+ and A315T/+ mice were crossed with Hb9:GFP transgenic mice. Embryonic stem cells (ESCs) were derived from TDP-43 (A315T/+)/ Hb9:GFP or TDP-43 (N390D/+)/ Hb9:GFP mice, and then differentiated to GFP(+) MN in culture  MN  MN were similar compared to those of (+/+) MN on culture after day 2  MN as caused by the increased level of TDP-43. Furthermore, there is a lag between the time on-set of Bcl-2 protein increase and MN degeneration.Significantly, in parallel to the time-dependent increase of the TDP-43 level Fig. a, there  MN Fig. b and c rRNA Fig. d. On they 2 Fig. b and dayy 2 Fig. a. The da2+) [2+ release from ER. Thus, neurotoxicity indeed could be caused by up-regulation of Bcl-2 protein. The above data taken together show that elevation of the cellular level of TDP-43 (N390D) would increase the intracellular cytosolic calcium ion concentration as a result of the increase of Bcl-2 protein.Since Bcl-2 is known to increase ER calcium ion leakage resulting in overloading of the cytosolic calcium ion (Ca2+) , 46, we 2+)  also proThe availability of appropriate ALS mouse models is essential for understanding ALS disease mechanisms and the development of therapeutic drugs. Most of previously developed ALS-TDP mouse models are based on a transgenic approach, whereby wild type or mutant TDP-43 is expressed under the control of different promoters , 40, 63.A315T causes neuron degeneration [A315T under the control of endogenous mouse Tarabp promoter develop mitochondria dysfunction but without obvious others phenotypes [It is somewhat unexpected but not totally surprising that in the genetic background of C57BL/6 J mice, only N390D but not A315T mutation of TDP-43 exhibits a dominant causative role in ALS-TDP pathogenesis , Q331K , M337 V Accompanied with these phenotypes are various molecular and cellular pathologies Fig.  includinThere are also progressive changes of autophagy and UPS in the spinal cord of N390D/+ mice. Interestingly, the LC3-II/LC3-I ratio in pre-symptomatic (3-month old) N390D/+ male mice is higher than that of the wild-type male mice Fig. a, which Tardbp mRNA decay and/or processing [Tardbp mRNA level  protein on the alteration of Bcl-2 pre-mRNA splicing and consequent increase of the Bcl-2 protein was not observed for TDP-43(A315T)  mice. This leads to upregulation of the Bcl-2 protein, change of autophagy, mis-metabolism of TDP-43 and other proteins, ER stress, etc., all of which would contribute to the age-dependent cytotoxicity/ death of the spinal cord MN and other age-dependent ALS-like phenotypes TDP-43 (N390D/+) mice.Overall, our data indicate that spinal cord-specific increase of TDP-43 (N390D), due in part to its higher stability, and the associated of gain-of-toxicity in The establishment and comparative analysis of the two mouse models, TDP-43 (N390D/+) and TDP-43 (A315T/+), suggest that different human ALS-associated TDP-43 mutations display distinct pathophysiological changes in mice. The TDP-43 (N390D/+) mice should provide an excellent model to study in detail the initiation and propagation of ALS-TDP under normal physiological conditions. It would also be ideal for use in the validation of potential therapeutic approaches and reagents for the treatment of ALS-TDP.Additional file 1: Figure S1. Effects of N390D mutations on the survival of newborn pups and on motor function/ lifespan of N390D/+ female mice.Additional file 2: Figure S2. Cognition analysis of N390D/+ male mice.Additional file 3: Figure S3. Similar levels of Tardbp mRNA expression in different tissues of +/+, A315T/+ and N390D/+ male mice.Additional file 4: Figure S4. Increase of phosphorylated TDP-43 (pTDP-43) in the spinal cord, and loss of NeuN(+) neurons of primary motor cortex of the old N390D/+ male mice.Additional file 5: Figure S5. Increase of astrogliosis in the spinal cord of N390D/+ male mice at late stage of pathogenesis.Additional file 6: Figure S6. Decreased of p62 protein level and enhanced of the proteasome activities in the spinal cord, but not the forebrain, of symptomatic N390D/+ mice.Additional file 7: Figure S7. Expression change of Bcl-2 and other TDP-43 target genes in the spinal cord of N390D/+ male mice in comparison to the +/+male mice.Additional file 8: Figure S8. Scheme of MN differentiation.Additional file 9: Movie S1. 18-month old N390D/+ mouse (#108). Note the 18-month old N390D/+ mice . Note the 18-month old N390D/+ mice . Note the 18-month old N390D/+ mice (Additional file


Ultrasonication is widely used to exfoliate two dimensional (2D) van der Waals layered materials such as graphene. Its fundamental mechanism, inertial cavitation, is poorly understood and often ignored in ultrasonication strategies resulting in low exfoliation rates, low material yields and wide flake size distributions, making the graphene dispersions produced by ultrasonication less economically viable. Here we report that few-layer graphene yields of up to 18% in three hours can be achieved by optimising inertial cavitation dose during ultrasonication. We demonstrate that inertial cavitation preferentially exfoliates larger flakes and that the graphene exfoliation rate and flake dimensions are strongly correlated with, and therefore can be controlled by, inertial cavitation dose. Furthermore, inertial cavitation is shown to preferentially exfoliate larger graphene flakes which causes the exfoliation rate to decrease as a function of sonication time. This study demonstrates that measurement and control of inertial cavitation is critical in optimising the high yield sonication-assisted aqueous liquid phase exfoliation of size-selected nanomaterials. Future development of this method should lead to the development of high volume flow cell production of 2D van der Waals layered nanomaterials. The discovery of graphene has also generated significant research interest into other 2D van der Waals layered nanomaterials, such as the family of metallic and semiconducting transition metal dichalcogenides9. One of the main challenges limiting the further applications and commercialisation of graphene and other 2D layered materials is that it remains difficult to produce large quantities of high-quality flakes with application specific size distributions. Many of the useful properties of graphene are indeed dependent on the lateral size and thickness of individual flakes5; for example, graphene flakes with large lateral dimensions (>1 μm) are used in polymer composites10 and conductive graphene inks6, flakes with smaller lateral dimensions (<1 μm) are employed in ceramic composites11, and graphene quantum dots (<100 nm) are found in photovoltaics, fuel cells, and catalysis applications12.Since the discovery of graphene14, shear mixing15, wet-jet milling16, or microfluidisation17. Although some liquid phase exfoliation methods report graphene yields as high as 100%17, a recent study demonstrated that the majority of commercially available graphene had less than 10% graphene content. This was due to the flakes containing more than 10 layers, which makes them nano graphite rather than multilayer graphene according to the ISO standard on graphene nomenclature18. Due to the wide flake size distributions (nm-μm) that are characteristic of liquid phase exfoliation techniques extensive centrifugation is often required to both narrow the size distribution and remove large graphitic flakes. Although cascade centrifugation has been shown to be effective in isolating narrow size distributions, it is time intensive, lowers the bulk concentration of the dispersed graphene and can inadvertently remove thinner graphene flakes with larger lateral dimensions as it separates flakes by their length19. As liquid phase exfoliation techniques typically produce dispersions with low intrinsic graphene concentrations (~0.1 mg ml−1), centrifugation and re-dispersion is often required to produce graphene dispersions with industrially viable concentrations (≥1 mg ml−1). As such, the removal of large graphene flakes can be unavoidable when producing graphene dispersions.One of the most scalable graphene production routes is to exfoliate it from graphite in the liquid phase using ultrasonication17, ultrasonication is one of the most widely used standalone methods to produce high-quality graphene dispersions, as well as being a complementary processing technique in other graphene production methods. The main limitations of using ultrasonication are its wide flake size distributions, low exfoliation rates, and comparatively low graphene yields. This is due to a poor understanding of the fundamental mechanisms driving graphene exfoliation, and a reliance upon purely empirical parameters such as sonication time13, temperature control20 and nominal electrical input power21 to monitor and develop ultrasonication strategies. Although acoustic cavitation is often suggested as the primary mechanism driving graphene exfoliation during sonication23, this has not been experimentally validated, and there have been no efforts to directly control and optimise acoustic cavitation during sonication. Using advanced cavitation metrology tools, we demonstrate that inertial cavitation drives graphene exfoliation during sonication for the first time. Furthermore, a controlled application of inertial cavitation is critical for optimising the sonication-assisted liquid phase exfoliation of graphene, resulting in improved graphene exfoliation rates, as well as a route towards in-situ size control of the graphene flakes. These findings will be instrumental in developing advanced ultrasonication strategies that will increase the large volume production and commercialisation of a wide range of 2D nanomaterials.Despite microfluidisation, shear mixing and wet jet milling demonstrating superior exfoliation rates of pristine graphene flakes24. Both types of cavitation exhibit physicochemical effects which are strongly dependent on the properties of the liquid being sonicated  as well as the acoustic field frequency, amplitude and geometry25. Stable cavitation generates short range vortices known as microstreaming, whereas inertial cavitation collapses radiate spherical shockwaves with velocities of up to 4,000 m s−1 with peak pressures of up to 6 GPa26. Intense liquid jets (jetting) with pressures of up to 1 GPa can also be generated during inertial collapse27. In a typical sonication environment, such as a sonic bath or sonotrode, both types of cavitation can exist simultaneously.Acoustic cavitation is the stimulated expansion and collapse of microbubbles in response to an applied acoustic field Fig. . Sonic b28. The acoustic signals that cavitating bubbles emit .In this work, acoustic cavitation was generated by a multi-frequency reference cavitating vessel Fig.  that canmit Fig.  were meaEquation , paramettra Fig.  and f1 a31, where a significant volume fraction of cavitating bubbles dynamically scatter and absorb the acoustic field. As there is a significant drop in the graphene exfoliation rate at high pre-amp voltages, Fig. To study graphene exfoliation arising from the physiochemical effects of acoustic cavitation, samples were produced by sonicating graphite in 28 ml low density polyethylene vials positioned in a region where inertial cavitation activity is intense and localised Fig. S. Prelimiμm size distribution (Supplementary Information −1) was chosen to increase the dispersed graphene yield and minimise the population of the larger graphitic flakes post sonication, ensuring that the graphene size distributions are representative of the acoustic cavitation mechanisms which exfoliated them. As 34. As the value of To explore the role of inertial cavitation on the liquid phase exfoliation of graphene, graphite with a narrow 45–75 in Fig. S, absolut13. As such, the graphene exfoliation rate can be increased for a given sonication time by increasing the intensity of the inertial cavitation activity, as measured by the square root of ICD  sonication times by using an array of cooling fans to actively cool the vessel. As such, cooling strategies should be carefully considered when developing large volume sonication methodologies to ensure consistent exfoliation. Shorter sonication times, as well as pulsed ultrasonication, may help mitigate temperature increase during sonication.During exfoliation trials, it was found that sonication times beyond 120 minutes resulted in anomalously high graphene yields that appeared to deviate from the power law relationship with ICD Fig. S. This wa36. Accordingly, it can be concluded that inertial cavitation, which is characterised by stochastic and energetic bubble collapse, fractures graphite/graphene during sonication in a multiplicative stochastic process. Figure μm lateral dimensions of the flakes in the post-sonication precipitate , this indicates that the entire initial graphite population has been fractured by the accumulated inertial cavitation activity during sonication. Therefore, during sonication the mean flake size will progressively decrease until the flakes are small enough to be suspended in the solution by the electrostatic repulsion of the adsorbed sodium cholate surfactant molecules. This is demonstrated in Fig. To quantitatively investigate the evolution of the graphene size distribution as a function of ICD, the length and thickness of the graphene flakes were measured using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The graphene length Fig.  and thicte Fig. S are sign37 and carbon nanotubes38 and is demonstrated by the saturation in the graphene yield as a function of ICD despite there being an abundance of ~0.5 μm size graphite flakes in the precipitate  is much smaller than the resonant size of the cavitating bubbles present in this work (~160 μm at 21.06 kHz), jetting events within the graphene dispersion will significantly decrease in frequency as the mean flake size decreases during sonication. However, jetting may also exfoliate any graphite/graphene flakes in the vicinity of the LDPE vials’ walls. The local shear stresses generated by collapsing bubbles, known as microstreaming, are also unlikely to drive exfoliation as graphene was not observed below the inertial cavitation threshold where microstreaming still occurs. Furthermore, as the speed of microstreaming vortices is proportional to the squared frequency of cavitating bubbles39, the shear forces they generate will be more effective at megasonic frequencies40. Consequently, shockwave exfoliation, which is facilitated by a combination of fracturing41 and the high velocity interparticle collisions43, is the most probable exfoliation mechanism during sonication. However, as shockwaves lose more than 50% of their initial energy over the first 25 μm of propagation due to absorption26, and will be attenuated by dispersed graphene flakes (which will increase in density during sonication), graphene exfoliation is most likely facilitated by the shockwaves generated by immediately adjacent inertial cavitation collapse events.Graphene exfoliation is likely to be driven by a combination jetting, microstreaming and shockwaves during sonication. As jetting is facilitated by nearby extended surfaces, and the maximum size of the graphite used  of nanosheets such that the laser beam (532 nm excitation wavelength) interrogates 100 s of flakes per measurement44. The Raman spectrum of graphene, shown in Fig. 48. Specifically it has been established that the ratio of the intensity of the D peak (~1320 cm−1) to that of the G peak (~1580 cm−1) is indicative of graphene nanosheet size, and the shape and intensity of the 2D peak (~2700 cm−1) changes as function of graphene nanosheet thickness47.Spectroscopic trends in the graphene samples were explored using visible Raman spectroscopy. To ensure that the measurements were representative of the graphene dispersions, 120 spectra were collected and averaged across 20 × 20 D/IG, increases between the minima and maxima of the ICD range. As the inverse of the ID/IG ratio is proportional to the length of graphene flakes44, Fig. D/IG ratio that were observed across each two-dimensional Raman map  was sonicated in LDPE Nalgene vials  with sodium cholate surfactant . The LDPE vials were sonicated continuously at 21.06 kHz for up to 3 hours in NPL’s multi-frequency reference vessel. After sonication, the graphene dispersions were left to sediment overnight before being centrifuged at 1000 rpm (120 rcf) for 2 hours before being characterised with UV-Vis, AFM, SEM and Raman spectroscopy. The graphene yield is given by Pre-treated and sieved Asbury Carbons fine flake graphite (0.2 mg mlSupplementary Information: Controlled Sonication as a Route to in-situ Graphene Flake Size Control


This study compared the effects of pirfenidone with the pan-HDAC-inhibitor panobinostat/LBH589, a FDA-approved drug for the treatment of multiple myeloma, head-to-head on survival, fibrotic activity and proliferation of primary IPF-fibroblasts in vitro.Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with a poor prognosis. Pirfenidone is the first antifibrotic agent to be approved for IPF-treatment as it is able to slow down disease progression. However, there is no curative treatment other than lung transplantation. Because epigenetic alterations are associated with IPF, histone deacetylase (HDAC)-inhibitors have recently been proven to attenuate fibrotic remodeling Primary fibroblasts from six IPF-patients were incubated for 24h with vehicle (0.25% DMSO), panobinostat  or pirfenidone (2.7 mM), followed by assessment of proliferation and expression analyses for profibrotic and anti-apoptosis genes, as well as for ER stress and apoptosis-markers. In addition, the expression status of all HDAC enzymes was examined.BIRC5/survivin, and was associated with induction of ER stress and apoptosis in IPF-fibroblasts. In contrast, pirfenidone-treatment maintained Bcl-XL expression, and was neither associated with ER stress-induction nor any apoptotic signaling. Pirfenidone also led to increased expression of HDAC6 and sirtuin-2, and enhanced α-tubulin-deacetylation. But in line with its ability to increase histone acetylation, pirfenidone reduced the expression of HDAC enzymes HDAC1, -2 and -9.Treatment of IPF-fibroblasts with panobinostat or pirfenidone resulted in a downregulated expression of various extracellular matrix (ECM)-associated genes, as compared to vehicle-treated cells. In agreement, both drugs decreased protein level of phosphorylated (p)-STAT3, a transcription factor mediating profibrotic responses, in treated IPF-fibroblasts. Further, an increase in histone acetylation was observed in response to both treatments, but was much more pronounced and excessive in panobinostat-treated IPF-fibroblasts. Panobinostat, but not pirfenidone, led to a significant suppression of proliferation in IPF-fibroblasts, as indicated by WST1- and BrdU assay and markedly diminished levels of cyclin-D1 and p-histone H3. Furthermore, panobinostat-treatment enhanced α-tubulin-acetylation, decreased the expression of survival-related genes Bcl-XL and We conclude that, beside other antifibrotic mechanisms, pirfenidone reduces profibrotic signaling also through STAT3 inactivation and weak epigenetic alterations in IPF-fibroblasts, and permits survival of  fibroblasts. The pan-HDAC-inhibitor panobinostat reduces profibrotic phenotypes while inducing cell cycle arrest and apoptosis in IPF-fibroblasts, thus indicating more efficiency than pirfenidone in inactivating IPF-fibroblasts. We therefore believe that HDAC-inhibitors such as panobinostat can present a novel therapeutic strategy for IPF. The pan-HDACi LBH589 has an efficient inhibitory activity at nanomolar concentrations (10–200 nM) and appears to be the most potent clinically available HDAC inhibitor - 1,3-benzene disulphonate) (Roche) is a colorimetric assay that quantifies mitochondrial dehydrogenase activity and thus reflects cell viability. In brief, IPF-fibroblasts (n = 4) were seeded in 96-well plates (2×104 cells/200 μL/well) and were allowed to grow overnight in normal culture medium. Next day, the medium was replaced, and fibroblasts were incubated for 24h with vehicle, LBH589 or pirfenidone, as described above. Subsequently, fibroblastic cells were labeled with 10 μM BrdU and re-incubated for 12h, followed by fixation (30 min/RT) and incubation with HRP-coupled anti-BrdU-antibody for 90 min at RT. Thereafter, 100 μL substrate solution (tetramethyl-benzidine) per well was added, and the plate was incubated for 5–25 min at RT until color development (blue) was sufficient. The substrate reaction was then stopped by adding 25 μL 1M H2SO4 per well and thorough mixing (yellow color). Immediately, absorbance was measured at 450 nm in an ELISA microtiter plate reader. The assay was performed in triplicates.The colorimetric bromodeoxyuridine (BrdU) assay (Roche/Sigma-Aldrich) is based on incorporation of BrdU into newly synthesized DNA in proliferating cells, and quantitative binding of a monoclonal HRP-coupled anti-BrdU-antibody. In brief, IPF-fibroblasts (n = 6) were seeded in 96-well plates - and pirfenidone-treated IPF-fibroblasts, as previously described .RT-PCR for escribed . GAPDH wProtein expression of histone H3-acetyl K27, acetylated α-tubulin, phospho-histone H3 (Ser10), cyclin D1, p-STAT3 (Tyr705), α-SMA, tropomyosin, collagen-I, HDAC1, HDAC2, HDAC6, HDAC9, sirtuin-2, survivin, Bcl-XL, CHOP, p21, p53, p16, AIF , DR5, cleaved PARP1-p25 and cleaved caspase-3 in vehicle-, panobinostat (LBH589)- and pirfenidone-treated IPF-fibroblasts was analyzed by quantitative immunoblotting of cell lysates, as described before . DependeZytoChem-Plus AP Kit (Fast Red)  was used for immunohistochemical localization of research target-proteins in formalin-fixed, paraffin-embedded lung tissue sections from patients with sporadic IPF (n = 5) and organ donors (n = 5), according to the manufacturer´s instructions and previous published work . In the 4 cells/400 μL/well) and were allowed to grow overnight in normal culture medium at 37°C in 5% CO2 atmosphere. Next day, the medium was replaced, and fibroblasts were incubated for 24h with vehicle, LBH589 or pirfenidone, as described above. Cells were then fixed with freshly prepared 4% (w/v) paraformaldehyde in 1×PBS for 30 min at RT, followed by permeabilization of cells in a freshly prepared solution containing 0.2% (w/v) Triton X-100 and 0.1% (w/v) sodium citrate, on ice for 15 min. Thereafter, IPF-fibroblasts were incubated for 1h with TUNEL reaction mixture (100 μL/well) containing fluorescein (FITC)-dUTP and the enzyme terminal deoxynucleotidyl transferase (TdT), at 37°C in a humidified atmosphere in the dark. As negative control experiments, cells were incubated with label solution without the TdT enzyme. After extensive washing with 1×PBS, cells were counterstained with DAPI and mounted in Vectashield . The FITC-labeled DNA fragments in the apoptotic cells were visualised using Axio Observer.Z1 fluorescence microscope .The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-reaction is a method for detection and quantification of apoptosis at the single-cell level, based on detection of DNA fragmentation by labeling the 3′- hydroxyl termini in the double-strand DNA breaks generated during apoptosis. In brief, IPF-fibroblasts (n = 4) were seeded in 8-well chamber-slides  was performed on vehicle-, panobinostat (LBH589)-, and pirfenidone-treated IPF-fibroblasts (n = 3). Pharmacological treatments, fixation and permeabilization of cells were done in 8-well chamber slides as described above. After blocking of cells with 2% (w/v) BSA in 1×PBS, cells were incubated for 90 min with α-SMA-Cy3™ antibody, diluted 1:200 in blocking buffer.For analyses of F-actin structures, differently treated IPF-fibroblasts (n = 3) were incubated for 10 min with AlexaFluor 555-Phalloidin , diluted 1:500 in 1×PBS. Thereafter, cells were counterstained with DAPI and mounted in Vectashield.All data are presented as median ± range. Data were analyzed by GraphPad Prism 5.02 software. For the statistical comparison of differences between the vehicle- and panobinostat/LBH589 group, the vehicle- and pirfenidone group, and between the pirfenidone- and the panobinostat/LBH589 group, the non-parametric Mann Whitney test was applied. Significance level is indicated by *p<0.05, **p<0.01, ***p < 0.001.We examined the therapeutic efficacy of the HDAC inhibitor panobinostat (LBH589) and pirfenidone head-to-head in cultured primary IPF-fibroblasts (n = 4), and incubated them for 24h with vehicle [0.25% (v/v) DMSO], LBH589 (85 nM) or pirfenidone . As control experiment for effective HDAC inhibition in response to LBH589, we first examined the acetylation status of histone H3-K27 (H3K27Ac) in all treatments. As expected, LBH589-treated IPF-fibroblasts revealed a very strong acetylation of this core histone, when compared to vehicle and pirfenidone . BecauseAs LBH589 blocks as a pan-HDAC inhibitor all class-I, -II and -IV HDAC enzymes, we also assessed the acetylation status of α-tubulin, which is a substrate of the class-IIb deacetylase HDAC6 . In lineTaken together, histone- and α-tubulin-hyperacetylation in response to LBH589 confirmed the efficacy of HDAC suppression. Importantly, pirfenidone-treatment led also to a significant increase in chromatin-acetylation, but decreased α-tubulin-acetylation in cultured IPF-fibroblasts, as compared to vehicle-treatment.We next analyzed the proliferation status in vehicle-, LBH589- and pirfenidone-treated IPF-fibroblasts. As expected, LBH589-incubation resulted in significant suppressed proliferation of IPF-fibroblasts in comparison to vehicle and pirfenidone, as assessed by WST-1  and BrdUThese results were further supported by immunoblots for the proliferation markers phospho (p)-histone H3  and cyclCOL1A1-  gene expression  was not observed to be reduced in response to pirfenidone as compared to vehicle, but was significantly suppressed in LBH589-treated IPF-fibroblasts  was not decreased in response to pirfenidone, but significantly downregulated in LBH589-treated IPF-fibroblasts  in IPF-fibroblasts as compared to vehicle-treatment , which were similar to P53 found at basal levels in IPF-fibroblasts, were reduced in reponse to panobinostat-, but not pirfenidone-treatment as compared to vehicle  transcripts , a profibrotic transcription factor, was clearly observed in the nuclei of myofibroblasts in fibroblast foci  and risk factors  in common with cancer . Results in vivo . Also tr in vivo . Thus, t in vivo , and LBH in vivo –36. LBH5 in vivo ,38,39, a in vivo . In the CCND1 gene transcription versus vehicle.Surprisingly, the WST-1 and BrdU proliferation assays revealed, that pirfenidone did not greatly inhibit the proliferation of IPF-fibroblasts at 2.7 mM concentration as compared to vehicle, but which was, as expected, significantly reduced in response to the anti-cancer drug LBH589. This result was corroborated by protein expression analyses for proliferation markers p-histone H3 and cyclin D1, indicating suppression only in LBH589-incubated IPF-fibroblasts in comparison to vehicle- and pirfenidone-treatments with same expression status of both markers. In agreement, only LBH589, but not pirfendone, downregulated in vitro after 24h exposure, including mesenchymal stem cells and fibrotic fibroblasts HDAC inhibition thereby potentiating repression of HDAC activity ), while COL1A1, COL3A1 and FN was evident on the transcriptomic level in response to both drugs, whereas only the mRNA for ACTA2 was downregulated in LBH589- versus vehicle- and pirfenidone-treatment, suggesting that pirfenidone regulates ACTA2/α-SMA during translation.Furthermore, we observed that both LBH589- and pirfenidone-treatment resulted in significant suppression of STAT3-phosphorylation at Tyr705 and its activation, as well as in reduction of ECM-associated proteins α-SMA, tropomyosin and collagen-I in IPF-fibroblasts, when compared to vehicle. Notably, the inhibition of STAT3 activation in pirfenidone-treatments did not significantly differ in extent from LBH589-treated IPF-fibroblasts indicating histone-hyperacetylation. Likewise, reduction of profibrotic genes CNN1 , DES (desmin) and P4HTM (transmembrane prolyl 4-hydroxylase) as compared to LBH589- and vehicle-treatment, with CNN1 and DES being paradoxically even upregulated in IPF-fibroblasts in response to LBH589.Much to our surprise, pirfenidone only was capable in reducing transcription of profibrotic ECM-associated genes BIRC5/survivin, which is overexpressed in fibroblast foci of IPF-lungs and suspected to mediate apoptosis-resistance and persistence of myofibroblasts , panobinostat  or pirfenidone . The effects of vehicle-, LBH589- and pirfenidone-treatment were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) for indicated genes, and is depicted by representative agarose gels of RT-PCR products for (TIF)Click here for additional data file.S2 FigBIRC5 and HDAC9. GAPDH served as housekeeping gene. Data are presented as mean ± SEM of n = 4. *p<0.05 vs. vehicle; by Mann Whitney test.(A) IPF-fibroblast cell line CCL-134 (n = 4) or (B) embryonic WI-38 fibroblasts (n = 4) were incubated for 24h with vehicle , panobinostat , valproic acid , 4-phenyl-butyrate , resveratrol  or pirfenidone . Thereafter, cells were harvested and analyzed by qRT-PCR for (TIF)Click here for additional data file.S3 FigHDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, and SIRT2. Gene expression analysis for HDAC9 was performed with n = 4/6 vehicle-, LBH589- and pirfenidone-treated IPF-fibroblasts. GAPDH was used as reference gene. Results from two independent experiments are shown. -RT control = PCR of a RNA sample without reverse transcriptase.Primary IPF-fibroblasts  were incubated for 24h with vehicle , panobinostat  or pirfenidone . The effects of vehicle-, LBH589- and pirfenidone-treatment were analyzed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) for indicated HDAC genes, and is depicted by representative agarose gels of RT-PCR products for (TIF)Click here for additional data file.S4 FigRepresentative immunohistochemistry for phosphorylated (p)-STAT3 (Y705), cytokeratin-5 (KRT5) and α-SMA in  IPF- and (B) normal donor lung tissue.  In IPF, the antibody for p-STAT3 revealed nuclear staining in myofibroblasts of fibroblast foci (indicated by α-SMA staining and dashed arrows in A and C) as well as in overlying abnormal bronchiolar basal cells [indicated by KRT5 expression in (A)]. (B) Normal donor lungs indicated no or minimal staining in the interstitium as well as alveolar epithelium.(TIF)Click here for additional data file.S5 Fig Induction of p-STAT3 is observed in fibroblast foci (indicated by dashed arrows in A) and overlying abnormal bronchiolar epithelium (indicated by arrows and KRT5 expression in A), as well as in bronchioles of IPF-lungs (indicated by hashmark in B), and coincided with survivin and HDAC4 overexpression in these areas. Smooth muscle cells of IPF lungs (indicated by asterisk in B) also revealed nuclear p-STAT3 and survivin induction.(TIF)Click here for additional data file.S6 Fig(A) IPF-fibroblast cell line CCL-134 (n = 4) or (B) embryonic WI-38 fibroblasts (n = 4) were incubated for 24h with vehicle , panobinostat , valproic acid , 4-phenyl-butyrate , resveratrol  or pirfenidone . Thereafter, cells were harvested and analyzed by immunoblotting for α-SMA. GAPDH served as loading control. Data are presented as mean ± SEM of n = 4. *p<0.05 vs. vehicle; by Mann Whitney test.(TIFF)Click here for additional data file.S7 FigPrimary IPF-fibroblasts (n = 3) were incubated for 24h with vehicle , panobinostat  or pirfenidone , followed by fixation and staining with AlexaFluor 555-Phalloidin (red stain). Nuclei were counterstained with DAPI (blue stain). The cells were then analyzed by a fluorescence microscope. Vehicle-treated IPF-fibroblasts indicated beside linear F-actin structures stress fiber formation and extension of cells (left panel), which was impaired and abrogated in response to pirfenidone-treatment (right panel). In contrast to vehicle- (and pirfenidone-) treated cells, the panobinostat-treated IPF-fibroblasts revealed increased stress fiber formation in direction to a F-actin based cell expansion, resulting in a pronounced larger cell area and increased cell speading of single fibroblastic cells. Representative images for n = 3 IPF-fibroblast isolates are shown.(TIF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.


TGF beta is a multifunctional cytokine that is important in the pathogenesis of pulmonary fibrosis. The ability of TGF beta to stimulate smooth muscle actin and extracellular matrix gene expression in fibroblasts is well established. In this report, we evaluated the effect of TGF beta on the expression of HGF, FGF7 (KGF), and FGF10, important growth and survival factors for the alveolar epithelium. These growth factors are important for maintaining type II cells and for restoration of the epithelium after lung injury. Under conditions of normal serum supplementation or serum withdrawal TGF beta inhibited fibroblast expression of HGF, FGF7, and FGF10. We confirmed these observations with genome wide RNA sequencing of the response of control and IPF fibroblasts to TGF beta. In general, gene expression in IPF fibroblasts was similar to control fibroblasts. Reduced expression of HGF, FGF7, and FGF10 is another means whereby TGF beta impairs epithelial healing and promotes fibrosis after lung injury. Idiopathic pulmonary fibrosis (IPF) is a devastating disease whose pathogenesis is not fully understood and has nearly a 50% 3‐year mortality rate , fibroblast growth factor 7 , and FGF10. HGF has been shown to accelerate the closure of human alveolar epithelial cell scratch wounds in vitro  and five subjects with IPF . Total RNA was isolated using standard kits from Qiagen . The isolated total RNA was processed for next‐generation sequencing (NGS) library construction as developed in the NJH Genomics Facility for analysis with a Life Technologies  Ion Proton NGS platform. A modified Kapa Biosystems  KAPA Stranded mRNA‐Seq kit for whole transcriptome libraries was used to primarily target all poly(A) RNA. Briefly, library construction started from isolation of total RNA species, followed by mRNA (poly[A]) isolation, first and second strand cDNA synthesis, adaptor ligation, amplification, and bead templating. Once validated, the libraries were sequenced as barcoded‐pooled samples on a P1 Ion Proton chip, as routinely performed by the NJH Genomics Facility.http://feb2014.archive.ensembl.org/Homo_sapiens/). The number of reads mapping unambiguously to each gene in the Ensembl annotation was enumerated without strand specificity, using the featureCouts program from the subread package (version 1.5.0‐p1) . Reads of at least 30 nt length were mapped to the canonical chromosomes of the hg19 assembly of the human genome (UCSC) with the STAR RNAseq aligner (version 2.4.1d)  run in tris glycine buffer under reduced conditions , following the manufacturer's instructions.t tests with the assumption that the resulting values were normally distributed. In all analyses, we focused on the magnitude of possible changes in outcomes; this is in keeping with current statistical practices , FN1 (fibronectin), ACTA2 (smooth muscle actin), and LOXL2 (lysyl oxidase like 2) in cells cultured on plastic as expected , a noncollagenous extracellular matrix protein, was highly upregulated by TGFβ as has been reported by others previously but control and IPF fibroblasts responded similarly in our studies (Vuga et al. We were surprised to find limited differences in the response to TGF beta between fibroblasts isolated from IPF lungs and lungs from organ donors (Moodley et al. Although there have been numerous reports comparing fibroblasts from control and IPF lung, we could find no commonly accepted in vitro measurement to establish that the IPF fibroblasts that we isolate and study in our lab are the same phenotype as IPF fibroblasts studied in other labs. To phenotype fibroblasts from IPF patients, researchers will need better surface markers for different subpopulations of fibroblasts and direct dissociation and characterization of the cells at the time of the biopsy or lung transplantation before expansion in vitro. Our results demonstrate that TGF beta decreases expression of HGF, FGF7, and FGF10 in the presence and absence of serum in both control and IPF fibroblasts. Decrease in expression of these growth factors would be expected to impair the ability to repair the alveolar epithelium after injury.