These characteristics are compared with other technologies in this field, demonstrating that the fabrication approach described in this work is competitive and promising to fabricate large-scale flexible heaters with a very fast response and high steady-state temperatures in a cost-effective way. This technology can be easily combined with other fabrication methods, such as screen printing or spray-deposition, for the manufacturing of complete sensing systems where the temperature control is required to adjust functionalities or to tune sensitivity or selectivity. In this paper, we present a simple and inexpensive method for the fabrication of high-performance graphene-based heaters on different large-scale substrates through the laser photothermal reduction of graphene oxide . This method allows an efficient and localized high level of reduction and therefore a good electrical conductivity of the treated films. The performance of the heaters is studied in terms of steady-state temperature, power consumption, and time response for different substrates and sizes. The results show that the LrGO heaters can achieve stable steady-state temperatures higher than 200 °C when a voltage of 15 V is applied, featuring a time constant of around 4 s and a heat transfer coefficient of ~200 °C cm In recent years, heaters have attracted a growing interest because of the emergence of a broad spectrum of newly integrated sensing technologies, such as gas sensors or biosensors ,2,3, whiIn this context, graphene and graphene related materials have attracted the attention of many researchers as an alternative to the conventional expensive indium tin oxide (ITO) heaters due to their outstanding electrochemical, mechanical, and optical properties . Thus, dIn this work, we present a methodology for the fabrication of high-performance laser-reduced graphene-oxide (LrGO) heaters, which can be applied on different substrates (flexible or not). The laser reduction of GO ,13, aimiSEM, Raman spectroscopy, as well as X-ray photoelectron spectroscopy (XPS) were used to study the quality of the synthesized material, and the electrothermal experiments show promising results in terms of power consumption, response time, and steady-state temperature when compared with other existing technologies. Thus, this fabrication approach paves the way to an extremely simple, inexpensive, and eco-friendly fabrication of graphene-based heaters. The paper is structured as follows. The fabrication of the LrGO heaters is described in 2 on two different outstanding thermal insulators, Kapton® HN polyimide films with a thickness of 125 µm  and mica sheets with a thickness of ~1.5 mm . After drying the deposited GO for at least 24 h at ambient temperature (relative humidity 50%), the resulting ~50 µm layer of GO was photothermally reduced using a CO2 laser engraving machine . The laser-assisted reduction was demonstrated as a fast and scalable approach to obtain conductive patterns of laser reduced graphene-oxide (LrGO) with different lithographic patterns avoiding the use of chemicals or masks  for a 150 mm2 heater during 5 min (300 s) of bending with a bending radius and a frequency of 10 mm and 0.5 Hz , respectively. The driving voltage was set to 12.5 V to achieve a steady temperature of 145 °C, according to the electrothermal experiments shown in 2 heater. Under these conditions, the maximum changes in temperature reported were below 10 °C, resulting in a relative variation of temperature under 10%, following the periodic bending of the substrate (as shown in the inset of Further, we also analyzed the electromechanical stability of the heaters on Kapton2/W. It was also demonstrated that, while the electrical conductivity did not depend on the properties of the substrate, the heat transfer coefficient did, reporting a variation of 62 °C cm2/W from the Kapton® substrate to the mica one. The response time for the different steady-state temperatures and sizes was also analyzed, as well as its dependence on the heater area and the resistance variation. Finally, bending experiments demonstrated the stability and the consistency of the heaters when they were operating under mechanical stress. The authors believe that this technology is a big step forward in the cost-effective fabrication of high-performance heaters on large flexible substrates, which could be expanded to multiple ubiquitous applications.Laser-reduced graphene-oxide was studied as a heating element for the fabrication of flexible heaters. We presented a comprehensive study covering aspects from the production and the characterization of the raw material to the electrical and the thermal characterization of LrGO films operating as heaters for different sizes and substrates. The laser photothermal treatment was demonstrated as a fast and simple procedure to obtain conductive patterns of highly reduced graphene-oxide on flexible substrates with a competitive sheet resistance of ~200 Ω/sq. The self-heating effect of the LrGO films when voltage was applied between their terminals was studied in terms of steady-state temperature, transient response, power consumption, and size. The results illustrated that the saturation temperature depended linearly on the input power density, reporting heat transfer coefficients in the range 200–440 °C cm


We have explored the theoretical applicability of adsorption on graphene for the isotopic enrichment of aromatic compounds. Our results indicate that for nonpolar molecules, like benzene, the model compound used in these studies shows a reasonable isotopic fractionation that is obtained only for the deuterated species. For heavier elements, isotopic enrichment might be possible with more polar compounds, e.g., nitro- or chloro-substituted aromatics. For benzene, it is also not possible to use isotopic fractionation to differentiate between different orientations of the adsorbed molecule over the graphene surface. Our results also allowed for the identification of theory levels and computational procedures that can be used for the reliable prediction of the isotope effects on adsorption on graphene. In particular, the use of partial Hessian is an attractive approach that yields acceptable values at an enormous increase of speed. There are two interesting problems associated with the theoretical modeling of this process. Firstly, since no covalent changes in bonding occur during adsorption, the isotope effects on this process are expected to be very small  as in thC the ring overlays symmetrically central ring of the graphene model with all atoms (both carbons and hydrogens) occupying positions directly over the carbon atoms. Rotation of the benzene ring leads to a symmetrical structure C2 in which hydrogen atoms are placed over the centers of the ring while the midpoints of the C-C bond are placed over the carbon atoms of the central ring of the graphene model. The translation of the benzene ring along the plane parallel to that of graphene so that one of the carbon atoms of graphene is located directly under the center of the benzene ring yielded the third skewed model (S). The subsequent rotation of the benzene ring into a position in which all the hydrogen atoms of benzene overlaid the carbon atoms resulted in model S2.Benzene molecules adsorbed over a graphene surface can be oriented in several ways. We have considered the four canonical positions illustrated in 54) which is a compromise between the computational cost of Hessian calculations and the necessity to consider a sheet large enough to efficiently neglect edge effects. In fact, in the modeling of the adsorption of aromatic compounds on graphene sheets, sizes from C24 [150 [54 model of the graphene sheet. As illustrated in While a few different sizes of the graphene sheet were explored, the main model used in the present studies contains 54 carbon atoms . For example, at the M06-2X/def2-SVPP level of theory, S is more stable than S2 by 0.14 kcal/mol, but on the Gibbs free energy surface, the stability is reversed with S being more stable by 0.21 kcal/mol.In agreement with the literature data , we haveS2 structure. In all other attempts, the optimization resulted in the unrealistic structure V, presented in V structure is lower than that of S2 by 0.34 kcal/mol, while at the Gibbs free energy surface, S2 is more stable by 0.23 kcal/mol. Furthermore, we were unsuccessful in optimizing structure C to a local minimum at any considered theory level—it seems to be a transition state for the rotation of the benzene ring over the graphene plane.In all studied theory levels, the distance between the planes, the only significant geometric parameter, was about 3.3 Å in agreement with earlier reports. However, in the case of ONIOM calculations with DFTB as the lower theory level, we were able to optimize only the n is the number of vibrational degrees of freedom (of B and BG), and iu = i/(kBT)hν, where iν are isotopic frequencies, T is absolute temperature, and h and Bk are the Planck’s and Boltzmann’s constants, respectively. As already mentioned, the isotope effects on adsorption are small. Therefore, for clarity of the discussion we use isotopic fractionation, ε, which is related to the isotope effect by Equation (3) with units of parts per thousand , i.e., the isotope effect is expressed as an inverse deviation from unity (no isotope effect) multiplied by a thousand [An isotope effect on adsorption is formally an equilibrium isotope effect. We can describe the adsorption process by the following scheme:unctions . Within thousand :ε = (1/I13C6 benzene). Isotopic fractionations per position (εperD and εperC) were subsequently obtained using the rule of geometric means [Calculations of isotope effects were performed for fully isotopically substituted reactants (perdeuterated or ic means  which deic means  p. 391 f6H6 vs C6D6) at the ωB97xD/def2-TZVPP level of theory are listed in In the initial calculations, we confirmed that the expected values of IE are small, in the range of parts per thousand or less. Therefore, we studied the influence of the convergence criteria on the final value of the calculated isotope effects. Out of several cases, the most illustrative is a simple test of calculating “isotope effects” where the “substrate” is a molecule converged at a higher threshold, and the “product” is the same molecule converged with tighter convergence criteria. We have calculated two “isotope effects” using the benzene molecule optimized with the convergence criteria (RMS force 0.000300 a.u.—default in Gaussian packages), which is then increased thirty times (0.000010 a.u.), and three hundred times (0.000001 a.u.), using the tight and very-tight options, respectively. Furthermore, we have compared the results obtained using Gaussian versions 09 and 16. The results obtained for perdeuterated benzene  were identified as local minima. The results obtained with the latter parametrization suggest that isotopic fractionation can be indicative of the orientation of the benzene molecule over the graphene. This conclusion, however, does not agree with the results obtained using PM6 and since this parametrization yielded results in better agreement with the reference level, we conclude that isotopic fractionation is a rather poor indicator of the geometrical features of the benzene–graphene complex.In the quest for finding the economic theory level for reliable calculations of the isotope effects on adsorption, we have also studied the performance of the QM/QM calculations within the ONIOM scheme using the reference ωB97xd/def2-TZVPP theory level in the description of benzene and the three semiempirical parametrizations in the description of the graphene. As can be seen from the results collected in 150 model. The results are slightly smaller than those described above. This might indicate that the employed C54 model is still too small and the edge effects contribute artificially to the calculated values. It is, however, hard to test as the calculations with a full Hessian analysis for the C150 model are prohibitively time-consuming.The costliest step in the theoretical predictions of isotope effects is the calculation of Hessians. Adsorption seems to be an ideal process to test the performance of the partial Hessian analysis, as the interactions between the absorber and the adsorbed molecule are small , even more so in the studied system since the charge transfer is minimal and the dominant forces are weak van der Waals interactions. We have, therefore, calculated the isotopic fractionations using the structure optimized at the reference level using Hessian only for a part of the system (benzene molecule in our case) in the field of the point charges of the remaining part (graphene atoms). This protocol is a routine in some programs  while, B or 3nB-6 degrees of freedom, the adsorption difference between them is negligible.According to the findings of Williams and coworkers , a parti34, B97d3 [http://paneth.p.lodz.pl/en/strona-glowna/innowacje/isoeff/.The initial structures of the complexes were prepared using the HyperChem program . Subsequ4, B97d3 , B97d [44, B97d3 , M06-2X 4, B97d3 , LC-BLYP4, B97d3 , cam-B3L4, B97d3 , and B3L4, B97d3 ,54,55,564, B97d3 ,36, def24, B97d3 ,36 6-31 4, B97d3 ,58,59, a4, B97d3 ,61. The 4, B97d3 , PM7 [634, B97d3  semiempi4, B97d3 , has beeThe main questions addressed in this contribution concern the possibility of using the adsorption of aromatic compounds on graphene for aromatic enrichment and the possibility of using the values of isotope effects for distinguishing between different orientations of the adsorbed molecules. For the studied model compound (benzene), both the differentiation of the adsorbed molecule orientations over the graphene surface using isotopic fractionation, as well as the application of the adsorption on graphene for isotopic enrichment, seem realistic only for the deuterated species due to the small values and small differences of the carbon isotope effects associated with this process. Furthermore, the effects of the temperature are quite small, thus, extremely low temperatures would be needed to increase the isotopic fractionation, as illustrated in In the process, we have explored the applicability of the theoretical predictions of the isotope effects on the adsorption of aromatic compounds on graphene. Our studies indicate that in order to reliably calculate the values of these isotope effects, geometries need to be optimized with convergence limits significantly tighter than those set as default in most of the routinely used programs (Gaussian package in our case). Even with these constraints, different density functionals yield scatter results. Taking ωB97xd/def2-TZVPP as the reference level, we conclude that from among the economic theory levels, only this functional expressed in the def2-SVPP basis set gives acceptable results. In fact, a def2 family of basis sets seems better suited for calculations of isotope effects on adsorption than Pople’s type basis sets although the triple-zeta basis set gives considerably better results. The QM:QM approach within the ONIOM scheme did not yield satisfactory results with the semiempirical parametrizations tested . The use of partial Hessian, on the other hand, seems to be an attractive alternative since this approach yields acceptable values at an enormous increase of speed.


Drosophila and mice, as loss of the scaffolding factor SIN3 or the associated HDAC results in lethality. The objective of this study is to elucidate contributions of SIN3 complex components to these essential processes. We used the Drosophila model organism to carry out a systematic functional analysis of the SIN3 complex. We find that SIN3 associated proteins are essential for viability and cell proliferation during development. Additionally, tissue specific reduction of SIN3 complex components results in abnormal wing development. Interestingly, while knockdown of each factor resulted in similar phenotypes, their individual effects on recruitment of SIN3 to polytene chromosomes are distinct. Reduction of some factors leads to large changes in the morphology of the chromosome and/or greatly reduced SIN3 binding. These findings suggest that while individual SIN3 complex components work through distinct molecular mechanisms, they each make a substantial contribution to the overall function of this highly conserved histone deacetylase complex.Establishment and maintenance of histone acetylation levels are critical for metazoan development and viability. Disruption of the balance between acetylation and deacetylation by treatment with chemical histone deacetylase (HDAC) inhibitors results in loss of cell proliferation, differentiation and/or apoptosis. Histone deacetylation by the SIN3 complex is essential in  Histone acetylation is one of the earliest recognized modifications to correlate with transcriptional activity2. The enzymes that control acetylation levels, histone lysine acetyltransferases (KATs) and histone deacetylases (HDACs), typically assemble into large protein complexes3. Proteins within these enzymatic complexes serve different roles. In some cases, they are critical for recruitment to specific loci. For example, the ING2 subunit of the mammalian SIN3 complex contains a binding domain that recognizes histone H3 trimethylated at lysine 4 (H3K4me3) and is important for recruitment of HDAC1 to target genes in response to DNA damage4. In other cases, the complex subunit interacts with a transcription factor allowing for recruitment to specific gene regulatory regions. Mammalian SIN3 was first isolated as a binding partner of the DNA-binding transcriptional repressor Mad5.The template for eukaryotic transcription is chromatin, comprised of nucleosomes, DNA wrapped around a core of histone proteins. Histones are subject to post-translational chemical modifications, which impact nucleosome structure and protein accessibility to the chromatin templateDrosophila melanogaster, is found in multiple multi-subunit protein complexes. These complexes include the nucleosome remodeling (NuRD) complex, corepressor for REST (CoREST) and the SIN3 complex, named for the scaffolding factor SIN36. To add to the complexity, multiple distinct SIN3 complexes are present in a single organism7. These SIN3 HDAC complexes vary in subunit isoform specificity and accessory factor composition. Efforts to interrogate the complex components through a set of identical assays to examine the possible roles of individual factors in governing specific biological processes are limited.The histone deacetylase HDAC1, formerly named RPD3 in 5. Drosophila have a single gene, Sin3A, which has more sequence similarity to mammalian SIN3A compared to SIN3B, and from which multiple protein isoforms are produced8. Due to the presence of multiple paired amphipathic helical domains, known protein-protein interaction modules, SIN3 is believed to serve as the scaffold for assembly of the SIN3 HDAC complex9. While an HDAC was the first histone modifying enzyme found to interact with SIN310, subsequent work from multiple laboratories demonstrated that the histone demethylase KDM5 in mammals, dKDM5/LID in Drosophila, interacts with a subset of SIN3 complexes11. In studies performed by independent research groups using different organisms, a number of accessory factors including BRMS1, PF1, FAM60A, ING1/2, MRG15, RbAp46/48, RBP1, SAP180, SAP130, SAP30, SAP18 and SDS3 have been reported as members of a SIN3 complex, in addition to the two enzymatic components13. In an earlier proteomic study to identify isoform-specific SIN3 complexes, we found that the following proteins interact with each of the two major Drosophila SIN3 isoforms: HDAC1, CG14220 (SDS3), hat-trick (htk) (ARID4B), CG7379 (ING1/2), Sap130, CG3815 (PF1) and BRMS114. Additionally, we determined that Caf1-55, dKDM5/LID and CG15356 (EMSY) are factors that predominantly interact with the largest and most widely expressed SIN3 isoform, SIN3 220. Apart from the enzymatic activity of HDAC1 and dKDM5/LID, little is understood about the role of the accessory factors in the context of SIN3 complex function.SIN3 is a highly conserved protein that is expressed in organisms from yeast to mammals. Mammals have two genes encoding highly similar SIN3 isoforms, SIN3A and SIN3BDrosophila melanogaster. We sought to identify common and unique phenotypes resulting from reduced expression of complex components, either individually or in combination with reduction of the SIN3 scaffolding factor. We also set out to determine which factors participate in recruitment and/or stabilization of SIN3 onto chromatin, which is believed to be a pre-requisite for complex regulatory activity. Our data indicate that while reduced expression of the majority of the subunits results in highly similar biological phenotypes, only a subset of the factors impact chromatin recruitment. These findings highlight functional specialization of SIN3 complex components.To address this lack of comprehensive understanding, the objective of this study was to perform a systematic analysis of a defined SIN3 complex in 14 and are listed in Table 15. This dsRNA is processed by the RNAi machinery and will lead to degradation of the endogenously expressed target mRNA; ultimately resulting in reduced protein expression16. For these studies, we utilized a variety of GAL4 driver lines to express GAL4, and thus induce RNAi knock down, in different tissues. For all experiments, we compared the knockdown fly with a control that was the progeny of the same GAL4 driver crossed to either UAS-GFPRNAi or UAS-mCherryRNAi. These control flies have expression of GAL4 and induction of the RNAi pathway, but no specific Drosophila gene is targeted for knock down. As an additional control, to reduce the possibility that observed phenotypes are due to an off-target effect, when available, we utilized more than one RNAi line to target different regions of the targeted RNA. To validate the efficiency of knockdown, we performed RT-qPCR analysis  gene upstream of the GAL4 coding sequence. Ser has a complex expression pattern with prominent expression in the dorsal compartment of the wing imaginal disc18. We previously determined that this selected driver results in expression of GAL4 in the wing imaginal disc throughout larval development in a pattern that is largely consistent, however that is somewhat broader than, the reported expression of Ser19. We typically observed RNA levels reduced to about 60-70 % of the level in wild type wing discs in these heterogeneous cell populations.To examine functions of SIN3 complex subunits, we measured a number of phenotypic traits in flies with reduced levels of SIN3 complex components compared to wild type controls. The complex components investigated included factors previously determined to associate with the largest isoform of SIN3Drosophila viability. We used the Act5C-Gal4 driver line for ubiquitous expression of GAL4. Progeny of the cross of this driver to the UAS-targetRNAi line results in ubiquitous knockdown of the targeted complex component. Knockdown of each gene coding for a SIN3 complex component resulted in no adult progeny , Sap130, CG15356 (EMSY), CG3815 (PF1), CG7379 (ING1/2) and CG14220 (SDS3), are also required for viability.We first tested whether each of the complex components is essential for ny Table . Thus, eDrosophila wing is a nonessential tissue and has been used by us and others to investigate factors involved in development and cell cycle control25. We previously determined that SIN3 and dKDM5/LID are necessary for development of a normal wing21. For this study, we have repeated those experiments to allow for direct comparison with knockdown of the other complex components. As in those prior studies, here we used the Ser-Gal4 driver to induce RNAi knockdown of the SIN3 complex components in wing imaginal discs and observed the resulting adult wing morphology.Because ubiquitous RNAi knockdown resulted in loss of viability, we utilized a conditional knockdown system to further investigate SIN3 complex components. The Sin3A or lid. HDAC1 and Caf1-55 are each found in additional complexes apart from SIN3. HDAC1 is a component of other HDAC complexes including NuRD and CoREST6. Caf1-55 associates not only with HDACs, but also KATs and chromatin assembly factors26. It is possible that the loss of each of these factors impacts the function of not only the SIN3 complex but also the other chromatin organization complexes in which these factors are found. The severe disruption of wing morphology is thus likely due to effects on multiple developmental pathways. The other factor found to yield the blistered phenotype upon knockdown is CG3815 (PF1). Little is known about PF1 in flies and work in other organisms has focused on the role of this factor as a component of the SIN3 complex. In triple negative breast cancer cells, disruption of the interaction between PF1 and SIN3A altered the expression of cancer-related genes and importantly, decreased the metastatic potential of cancer cells in a mouse tumor model27. These authors additionally found that knockdown of PF1 phenocopies the effects of disruption of the PF1 and SIN3A interaction, indicating that a main role for PF1 is as part of the SIN3 complex. The data from the fly wing development assay suggests that, in flies, PF1 might function apart from SIN3. Interestingly, the blistered wings are only found in the male CG3815 (PF1) knockdown flies. The wings of the female CG3815 (PF1) knockdown flies are curved, similar to those of the Sin3A knockdown flies  and Brms1 knockdown female flies had curved wings, some had straight wings supporting the idea that females are less sensitive to reduced SIN3 complex function as compared to males. Like wild type strains, progeny of the control cross had straight wings. When multiple RNAi lines were tested for an individual gene, the resulting phenotypes were similar across the lines. These results suggest the phenotypes are not due to off-target effects but rather are due to reduced expression of the targeted gene.We first tested whether the individual complex component was necessary for wing development. Individual reduction of HDAC1, Caf1-55 or CG3815 (PF1) resulted in severely malformed wing phenotypes Fig. . The winSin3A wing specific knockdown suppresses the curved wing phenotype in female flies by not in males21. Additionally, male flies that carry a mutation in lid have shorter life spans and a higher sensitivity to the presence of paraquat-induced reactive oxygen species as compared to the females with the mutation28. Mutations in HDAC1 have also been found to yield sex specific phenotypic variation. Flies carrying certain specific alleles of HDAC1 survive at significantly lower rates as compared to their genetically identical females counterparts29. We do not have a definitive answer as to the mechanism controlling the sex specific differential sensitivity to mutation in SIN3 complex components. It is possible that the differences are related to the epigenetic variation linked to the presence of the heterochromatic Y chromosome in male flies and/or the single X chromosome, which is subject to dosage compensation controlled in part by histone acetylation30. In addition to the possible impact of dosage compensation mechanisms, male and female flies have differences in their genome-wide chromatin landscape31. It is possible that the SIN3 histone modifying complex controls some of these sex specific differences, which then differentially affect genes important for normal wing morphology.We note that sex specific phenotypic differences comparing flies with mutation or reduced expression of SIN3 complex components to wild type have been reported previously. For example, we previously observed that overexpression of dKDM5/LID in the context of GAL4 expression pattern controlled by the Ser-Gal4 driver is complex and thus the amount of knockdown is likely variable across the wing imaginal discs cells, with some cells affected very little and others a lot.An additional observation is that although we determined efficiency of knockdown in the wing imaginal discs at approximately 60 and 70% of control levels  knockdown was observed when htk (ARID4B) and Sin3A were simultaneously reduced by RNAi. These data suggest that the majority of the factors work through a similar mechanism to impact the same pathways for wing development. Reduction of HDAC1, CAF1-55 and CG3815 (PF1) leads to flies with a more severe phenotype compared to the Sin3A knockdown curved wing, which is not modified when combined with SIN3 reduction. This finding suggests that those three factors have function outside of the SIN3 complex. As noted, HDAC1 and CAF1-55 are part of multiple non-SIN3 containing chromatin modification complexes26. The number of male flies for the Caf1-55KK, Sin3A double knockdown genotype is low, possibly due to lethality in the pupal stage as we observed pupal cases from which flies did not eclose. We do not have an explanation as to reason why the wing specific knockdown would result in male lethality. Taken together, the results indicate all tested components of the SIN3 complex are required for normal wild type development of the Drosophila wing. Because wing specific RNAi of each factor results in a similar curved wing phenotype and they were found to biochemically associate in a previously conducted co-immunoprecipitation experiment14, it is possible that most of the components work as part of a single complex. Because of the distinct phenotypes observed in the wing specific RNAi knockdown flies, HDAC1, CAF1-55 and CG3815 (PF1) likely have some activity outside of their roles as members of the SIN3 complex. Whether other complex factors work outside of their association with SIN3 cannot be ruled out and future work identifying possible non-SIN3 containing additional complexes will shed light on this possibility.We next examined the effect of reducing levels of SIN3 along with a second component of the complex. We previously determined that dual knockdown of eny Fig. . The winDrosophila S2 cultured cell line32. It was subsequently determined that SIN3, HDAC1, the histone demethylase dKDM5/LID and CAF1-55 are also critical for cell proliferation and cell survival during Drosophila development35. To investigate if other components of the complex are necessary for cell proliferation during development, we performed clonal analysis in wing imaginal disc cells. For these studies, we utilized the heat shock “flip-out” GAL4 driver (Actin > GAL4) to activate expression of a UAS-linked RNAi target along with a GFP marker in random clones of cells via the heat-shock induction of FLP recombinase at a precise time point in larval development. Once activated, cell clones will continue to have reduced target gene expression and the effect of this reduced expression on cell proliferation can be measured by the size and number of the GFP positive cells. Reduction of all SIN3 complex components resulted in fewer GFP positive cells. In some instances, the clones are smaller in size and for others, the clones are fewer in number, relative to the clones produced in the mCherry RNAi controls  and Brms1 led to reduced cell proliferation in the developing wing tissue. This finding suggests that their function in these cells is similar to that of other complex components; they are important for cell proliferation rather than functioning as tumor suppressors.The human homologs of a number of SIN3 complex factors, including ARID4B, EMSY, PF1, ING1/2 and BRMS1, are known to function in cell proliferation and the level of their expression has been linked to growth of cancer cells. Elevated expression of ARID4B was found to promote metastasis in a mouse model of human breast cancer9. We asked if any of the complex components are critical for recruitment of SIN3 to chromatin. For this analysis, we used polytene chromosomes prepared from Drosophila larval salivary glands. We utilized the feb36-GAL4 driver line to induce RNAi and reduce the level of the SIN3 complex factor in salivary glands45. Chromosome preparations from third instar larval salivary glands were probed with antibody to SIN3 and also stained with DAPI to visualize the DNA.SIN3 is described as corepressor as it does not contain a DNA-binding domain for gene targeting. To recruit the complex to promoters where it functions to regulate gene expression, sequence-specific DNA-binding transcription factors interact with SIN3 and/or components of the complexHDAC1 led to disruption of polytene chromatin structure. The salivary glands were smaller and overall DAPI staining of nuclei was low compared to wild type controls. The chromosomes lack integrity; the banding pattern is disrupted and less distinct as compared to control chromosomes . cDNA was generated from total RNA using the ImProm-II Reverse Transcription System (Promega) with random hexamers. The cDNA was used as template in a quantitative real-time PCR (qPCR) assay. The analysis was performed using ABsolute SYBR Green ROX master mix (Fisher Scientific) and carried out in a Stratagene Mx3005P real-time thermocycler. Primers used for analysis are given in Supplemental Table Whole flies were imaged at 30x magnification using an Olympus DP72 camera coupled to an Olympus SZX16 microscope. Wings were imaged at 80x magnification using a SPOT RT color camera coupled to a Leica MZ125 microscope.mCherryRNAi-TRiP males or UAS-SIN3 complex component RNAi to generate random GFP positive clones via the heat shock flip-out system. Embryos were collected for four hours and then after 48–52 hours, 2nd instar larvae were subjected to heat shock at 37 °C for two hours. After returning to 27 °C, wandering 3rd instar larvae were dissected and immunostained 120 hours after egg laying19. Antibody against GFP  followed by sheep anti-mouse Alexa 488  was used for staining. Visualization and imaging was done using an Olympus BX53 compound microscope with a DP72, 12.8 megapixel camera. Images were processed using Olympus CellSens software. Clones were analyzed in a minimum of 20 discs per genotype using Photoshop CS to count the GFP positive pixels for each immunostained disc and comparing that number to the total number of pixels in the DAPI-stained disc48.hsFLP;Act5C > CD2 > Gal4,UAS-EGFP virgin females were crossed to 49. A primary antibody against SIN3 (1:1000)50 was followed by a secondary antibody Alexa Fluor 594 (1:400) (Life Technologies). Chromosome spreads were prepared from a minimum of three independent parental crosses and representative images are shown. A minimum of four slides of each control and experimental genotype were prepared at the same time and photographed using identical exposure times. Chromosomes were imaged using an Olympus BX53 compound microscope with a DP72, 12.8 megapixel camera at 400x. Five to ten chromosomal spreads were chosen from each slide for imaging, and images were processed identically using Olympus CellSens software. To quantify SIN3 immunofluorescence signals on polytene chromosomes relative to DAPI intensity, we used a program developed in Matlab 7.4.0 from the protocol outlined here51. Briefly, batches of control and experimental images, processed identically, are input into the program, which uses two fluorescent channels (DAPI and Alexa Fluor 594). The program applies a mask to each chromosome, removing non-chromosomal antibody staining from the pixel-based quantification.Polytene chromosome preparation and staining methods were modified from the protocol outlined heret test using GRAPHPAD software. http://www.graphpad.com/quickcalcs/index.cfm.All significance values were calculated by the two sample Student’s Supplementary Information


Graphene transparent conductive electrodes are highly attractive for photodetector (PD) applications due to their excellent electrical and optical properties. The emergence of graphene/semiconductor hybrid heterostructures provides a platform useful for fabricating high-performance optoelectronic devices, thereby overcoming the inherent limitations of graphene. Here, we review the studies of PDs based on graphene/semiconductor hybrid heterostructures, including device physics/design, performance, and process technologies for the optimization of PDs. In the last section, existing technologies and future challenges for PD applications of graphene/semiconductor hybrid heterostructures are discussed. A photodetector (PD) is a device for detecting light, the underlying mechanism is conversion of light to an electrical signal called a photocurrent (PC). PDs are used in a wide range of academic and industrial fields, including image sensing, optical communications, environmental monitoring, and chemical/biological sensing ,2,3. In A wide variety of optoelectronic devices based on graphene are still being studied, and some of them have already reached a level of competitiveness comparable to conventional semiconductor devices ,14. HoweThe emergence of the graphene/SiC interface in 2009  as the pChemical vapor deposition (CVD)-grown graphene was first employed in graphene/Si heterojunctions, resulting in 65% external quantum efficiency (EQE) , which sB) to prevent the electrons from flowing from the semiconductor to the graphene. Depending on the energy of the radiation (E) = hν (h = Planck’s constant and ν = photon frequency), PC is generated based on two main mechanisms. (1) When ΦB < hν < semiconductor band gap (gE), the electrons are excited from the graphene and are injected into the semiconductor; (2) When hν > gE, the electron-hole pairs are generated in the depletion layer of the semiconductor. The PDs are operated based on the photovoltaic or photogating effect depending on their device configurations.Graphene/semiconductor hybrid heterostructures can be formed by directly combining graphene with one-, two-, and three-dimensional semiconductors through relatively simple manufacturing processes. When graphene and a semiconductor material form a junction, charge transfer occurs until the Fermi energy levels of the two materials coincide because their work functions mismatch. Meanwhile, the free charge region inside the semiconductor near the interface is depleted by the charge transfer, resulting in the formation of the built-in electric field. In case of graphene/n-type semiconductor junction, the immobile positive charge formed by the depletion of electrons bends the semiconductor band near the interface upward. More details are given in previous reports ,102. In J-V) characteristics under dark. A PD operating in this mode is named as a photodiode, whose linearity, detectivity, and sensitivity are maximized due to the lowest dark current. In contrast, the responsivity (R) is usually low due to the lack of internal gain. The PD can be also operated in reverse bias (photoconductive mode), where the built-in external electromagnetic field enhances the separation efficiency of the electron-hole pairs, thereby reducing the response time due to the shortening of the passage time and the decrease of the diode capacity. The photovoltaic mode is more suitable for precise photodetection with the photoconductive mode being better suited for high-speed applications.The PD can be operated in a photovoltaic mode (=zero bias). The heterostructures generally exhibit rectifying behaviors, i.e., nonlinear current density-voltage  curve of the graphene transistor upon illumination. In addition, the direction of the curve shift represents the polarity of the captured carriers, and the photogate effect can induce positive or negative PC by gV.Photogating effects occur in the heterostructures-based phototransistors. The operating mechanism of the phototransistors is as follows. Once electron-hole pairs are generated in semiconductors, one type of carrier is transferred to the graphene, and the opposite-type carrier is trapped in the semiconductor. The trapped charges act as a local gate, thereby effectively modulating the Fermi level of graphene through capacitive coupling. This induces more carriers and consequently modulates the electrical conductance. In addition, the transferred free carriers circulate many times in the graphene channel within their lifetime, which contributes to higher photoconduction gain. The gain is expressed as gain = lifetime/transit, where transit is the transit time of the photogenerated carriers through the graphene channel. Shorter transit time and longer lifetime should be obtained to achieve higher gain. Nevertheless, the lifetime of the photocarrier is determined by the response time, which is directly related to the carrier recombination process. Thus, a high gain will extend the response time. A phototransistor can show much higher R is defined as the ratio of PC to incident light power, which indicates how efficiently a PD responds to an optical signal, as expressed by the equation R = ph/LlightI, in a unit of A/W, where phI is the PC and lightL is the incident-light power. R is proportional to EQE of a PD, meaning the conversion rate from photons to electrons/holes. Therefore, EQE is related to R based on the following equation: λ is the incident-light wavelength, q is the absolute value of electron charge, h is the Planck constant, and c is the speed of light.D* is the weakest level of light which can be detected by the device, and is determined by R and noise of a PD. D* is given by D* = (A1/2·∆f)n× R/i. Where A is the effective area of a PD, ∆f is the electrical bandwidth, and ni is the noise current. The performance of D* is limited by three kinds of noise such as dark current (DC), Johnson noise, and thermal fluctuation noise , w, w24], wR of 0.28 A/W, corresponding to an internal quantum efficiency of 10%, much higher than a typical Schottky junction (~1%). Usually, the R in the UV region is at least 1–2 times lower than that in the visible-near infrared (NIR) region due to the weak light absorption and relatively short carrier lifetime of the graphene. TiO2 [2O3 anti-reflection layers [2O3 antireflective layer has been fabricated for UV photodetection, resulting in 0.2 A/W R, 5 ns response time, 1.6 × 1013 cm·Hz1/2/W D* in wavelength range from 200 to 400 nm, comparable to those of state-of-the-art Si, GaN, and SiC Schottky PDs. The gapless and semi-metallic properties of graphene are very promising for its application for broadband photodetection from microwave to UV wavelengths. Graphene/Si Schottky PDs are also very efficient in fast carrier separation due to the presence of the built-in electric field without suffering from a sharp reduction in the lifetime of photocarriers under 1550 nm illumination , therebyne. TiO2  and Al2OR and response time of the PD at the same time, as shown in 3) has been widely used for improving the performance of the PD in organic electronics due to its high work function [2. The functionalization of the MoO3 layer on graphene led to a significant performance enhancement of the graphene/Si PDs [12 cm·Hz1/2/W D*. However, the interface between graphene and Si normally contains relatively-high density of surface states, resulting in large leakage current, which can limit the overall D* of the graphene/Si PD [2/Si PD showed 0.0078 pW·Hz−1/2 NEP at room temperature, resulting in D* up to 4.08 × 1013 cm·Hz1/2/W. Unlike traditional transparent electrodes, such as indium tin oxide or ultrathin metals, unique absorption properties of graphene in the UV range contribute to increasing the lifetime of hot electrons, thereby leading to large PC. Recently, a variety of graphene/Si-based structures have been proposed to improve the device performance. A new approach of integrating graphene/Si PD with two-dimensional (2D) Pt nanoparticles (NPs) has proven to enhance the function ,105 and e/Si PDs , based one/Si PD ,25. Recene/Si PD . The gra2/Si capacitors for high-performance optical detection [R/D* of 3 A·W−1/3.5 × 1012 cm·Hz1/2·W−1 in the visible range. Here, the PC exceeded the forward current because the photogenerated minority carriers accumulated in the Si/SiO2 interface diffused to the graphene/Si junction. According to another approach, the PC in graphene-based hybrid photodiodes was not only very efficiently generated in the graphene/Si Schottky barrier region but also in the adjacent graphene/SiO2/Si region due to the reverse bias voltage [2/Si region rose sharply by about one order of magnitude at voltages exceeding a certain threshold bias, regardless of the incident laser power. In Si under SiO2, the inversion layer not only provided a low resistance path for minority charge transport, but also acted as a passivation layer on the surface states of SiO2 to effectively collect the charge carriers, resulting in improved PC. These results provide guidelines for establishing the fundamental mechanism of PC generation in graphene-based hybrid optoelectronic devices and for designing hybrid photodetectors based on combinations of two- and three-dimensional materials. The PD was also fabricated by directly growing graphene nanowalls on a Si substrate instead of using graphene layers CVD-grown via a metal catalyst [D* was 5.88 × 1013 cm·Hz1/2/W, higher than ever reported in the planar graphene/Si based PDs, thanks to the ultra-low current noise of 3.18 fA·Hz1/2, which can be attributed to the excellent junction quality of 0.69 eV barrier height and 1.18 ideal factor. Other principal PD parameters were 107 PC/DC (light/IdarkI) ratio, 40 μs response time, 8.5 kHz cutoff frequency, and 0.52 A/W R. In addition, graphene/Si Schottky diodes were fabricated in parallel with graphene/SiOetection , thereby voltage . The PC catalyst . Resulti6light/IdarkI ratio and high response speed , better than those of pure graphene/Si NWs heterojunction [Two-dimensional Si nanostructures such as porous Si (PSi) and Si NWs have high ratio of surface to volume/light absorption, very useful for their application in graphene-based PDs. junction , as show2 matrix allow for large light absorption in the visible range based on the quantum confinement effect [3-doped graphene/SQDs: SiO2 multilayers (MLs)/Si PDs were successfully fabricated to show 0.35 A/W R and 109 cm·Hz1/2/W D* near the wavelength of 600 nm, indicating considerable performance improvement in the visible region due to the strong photoresponse of SQDs [3 doping concentration (Dn). However, the performance of the PD is limited by the surface of the AuCl3-doped graphene on which light is first irradiated due to large reduction in transmittance by the Au NPs formed by the doping. Recently, long-term stabilities have been remarkably enhanced by employing bis(trifluoromethanesulfonyl)-amide (TFSA) with low resistance and high transparency as a dopant for graphene, as shown in R and D* of the TFSA-doped graphene/SQD: SiO2 MLs/Si PDs with 0.413 A/W and 1010 Hz1/2/W at the peak wavelength of 630 nm under low voltage (−2 V), respectively, significantly better than the AuCl3-doped graphene PDs, indicating minimization of the optical loss by the use of TFSA. On the other hand, graphene/Si Schottky junction PDs with SQDs on top of the graphene, as show in −1/2 NEP, 7.4 × 109 cm·Hz1/2/W D*, and response time ≤ 25 ns, possibly resulting from significant reduction of the optical reflections, contributed by the SQDs, as shown in Si is widely used as a broadband PD, but is limited in its use for wider applications due to the inherent indirect bandgap nature. Especially, graphene/Si-based PD exhibits fast photoresponse in the UV and NIR regions, but not in the visible region. One way to overcome this drawback is to form Si quantum dots (SQDs) ,108. SQDt effect ,38. Rece of SQDs ,38. The light/IdarkI ratio (105) under UV illumination. In another approach, UV PDs were successfully fabricated by transferring large-area graphene TCE on vertically-standing GaN NW array as a light absorbing media, as shown in R of 25 A/W at 357 nm. These results represent a potential application of the graphene/GaN junctions in various optoelectronic devices operating in the UV region.GaN is a wide direct bandgap semiconductor with excellent optical/electrical properties and high chemical stabilities at high temperature, and has been employed in various PD applications. Graphene/GaN heterostructures are useful for light detection in the UV/visible region based on Schottky diode junction ,43. Grap4light/IdarkI ratio, 1.73 mA/W R, and 1011 cm·Hz1/2/W under 850 nm illumination. The PD also showed fast response speed with rise/fall times of 72/122 μs, as shown in x thin layer was employed for optimizing R of the graphene/GaAs NIR PD. Here, the AlOx serves not only as a surface protective layer for suppressing the carrier recombination, but also as a barrier for reducing the current leakage [x layer, the R and specific D* increased by 4 times to 5 mA/W and 2.8 × 1011 cm·Hz1/2/W, respectively, whilst rise/fall times sharply decreased to 320/380 ns.GaAs has long been considered as an ideal candidate material for NIR PD applications due to its striking electrical/optical properties such as high carrier mobility, direct bandgap of ~1.4 eV, and large optical absorption coefficient. As shown in  leakage , thereby7 through further device optimization, as shown in ZnO has received considerable attention over the past two decades due to its unique properties such as direct bandgap of 3.2 eV, large exciton binding energy of ~60 meV, high transmittance (>80%) in the wavelength range of 400–800 nm, and wide-variable conductivity in the metallic to insulating range ,110. To 2/Si substrate [2, thereby greatly reducing the time of the electron transfer in the channel. As a result, the photogenerated electrons transferred from the QD to the graphene are recycled several times before recombination, resulting in the increase of the photoconductivity gain. The phototransistors exhibited ~108 A/W R corresponding to ~3 × 109 gain, ~103 UV/visible rejection ratio, and ~5.1 × 1013 cm·Hz1/2/W specific D*. More recently, phototransistors consisting of graphene FET and ZnO QDs have been fabricated based on surface active process [D* increased up to 7.5 × 1014 Jones due to the efficient transfer of charge carriers from ZnO QDs to graphene. These results suggest how important the van der Waals interface is for the efficient optoelectronic processes in heterostructure PDs. On the other hand, in graphene/ZnO heterostructures photodiodes [D* [In recent years, ZnO NW or nanorod (NR) arrays have been combined with various graphene sheets on rigid or flexible substrates to form UV phototransistors ,52,53,54ubstrate . Surpris process . Resultitodiodes ,65,66,67odes [D* ,64,66. Todes [D* ,60,62,63odes [D* . R and 385 gains at −1 V [A Schottky-junction-type UV PD fabricated by depositing graphene layer on ZnO NR array was shown to be highly sensitive to UV illumination with excellent operational stability and reproducibility, resulting in 113 A/W  at −1 V . The res at −1 V  and by m at −1 V  or exter at −1 V ,62. The  at −1 V . The h-BR, measured under an external strain of 5 to 30%, showed maximum 17 μA/W and 3.8 μA/W at a strain of 30% for UV and visible light, respectively [Recently, the effect of strain modulation on PD performance was studied for a flexible graphene/ZnO nanostructure ,65. The 2 [2O3 [2 is a promising material for deep-UV detection applications. Recently, in the ZrO2 QDs-graphene heterostructure, R was shown to be as high as 22 A/W at a deep-UV wavelength of 254 nm [2 layer due to the high mobility of graphene. This explains why R is so extremely highLike graphene/ZnO hybrid structures, other semiconductor metal oxides including ZrO2 , Ga2O3 [2 [2O3 ,70 have f 254 nm . In the 2O3 junctions, as shown in R and D* of 39.3 A/W and 5.92 × 1013 Jones, respectively, comparable to, or even better than, Ga2O3 nanostructure-based PDs. The PD parameters were reproducible and stable even by repeated measurements under UV illumination, showed maxima at ~220 nm, but were almost blinded to light at wavelengths longer than 280 nm. Most recently, graphene/Ga2O3/graphene PD showed apparent rectifying characteristics and excellent solar-blind UV photoresponse [light/IdarkI ratio up to 82.88 under 254 nm illumination. As another approach, a deep UV photodiode was fabricated based on multilayer graphene/Garesponse  with fasR about 10 times larger on a piezoelectric substrate than on a SiO2 substrate [Organic semiconductors including small molecules and polymers are very attractive in practical view due to their simple solution production processes at low temperatures , low cosubstrate . The impR and specific D* being 700 A/W and of 1013 Jones, respectively, as shown in 4 s and PC being invariant for more than 200 cycles  hybrid, exhibiting the R of several hundreds of A/W and relatively-rapid response time shorter than 1 s in the UV-visible range [Perovskites have recently received strong attention as next-generation PD materials because of their high adsorption coefficient, tunable bandgap, small exciton binding energy, long carrier diffusion length, ambipolar charge transport, and high carrier mobility ,120,121.le range ,75. Espe3 hybrid phototransistors with those of graphene-based other semiconductor hybrid PDs operated based on similar mechanisms [3 stack was integrated with Au NPs having a plasmon resonance peak at ~530 nm, as shown in R and faster response speed [R was attributed to the light-harvesting effect enhanced by the near-field of the perovskite, induced by the plasmonic coupling of Au NPs. Furthermore, the time for these carriers to diffuse into graphene is shorter than that for carriers generated far from the perovskite–graphene interface, thus leading to the faster operation in the PD by the plasmonic effect.It is currently difficult to compare the performances of the graphene/MAPbIchanisms ,22,122, chanisms . The enhse speed . The hig3 films on graphene, thereby forming a compact heterostructure for efficient light harvesting and exciton separation, as shown in 7 A/W R and 2 × 1015 Jones D*, several times larger than those of PD containing a MAPbI3 film prepared by conventional spin-coating method. Very recently, a thin layer of P3HT has been inserted between MAPbI3−xClx perovskite and graphene as a hole transport layer for high-performance phototransistors [3−xClx perovskite was shown to be larger than that of the MAPbI3 perovskite, resulting in more efficient transfer of the photo-generated holes to the graphene side. In addition, the separation of the photo-generated electrons and holes was more effective due to the presence of the P3HT layer, thereby reducing the recombination of the optical carriers, resulting in the transport of high-density electrons to the perovskite layer. The gain of this PD was close to 1010, at least one order of magnitude higher than those of other PDs without the P3HT layer [As an advanced step, the sequential vapor deposition method has been developed to grow ultra-flat MAPbInsistors . In thisHT layer ,22,122. 3 NWs hybrid phototransistor was fabricated to show an R as large as 106 A/W [3−xIx nanocrystals (NCs) was done to fabricate phototransistors showing high R [D* (2.4 × 1016 Jones) thanks to the extremely-high carrier mobility of the graphene, very promising for the application of the perovskite NCs in next-generation high-performance optoelectronics.Highly-crystalline perovskite nanostructures are advantageous for the photo-detection application because the recombination rate of the photo-generated carriers is much smaller in the bulk than in the thin film. Recently, the graphene/MAPbI 106 A/W . As anotg high R . This PDLow-dimensional carbon materials such as fullerenes, carbon QDs, carbon nanotubes (CNTs), and graphene have attracted much interest over the past several decades due to their unique structural and physical properties ,125,126.2 on Si was employed to investigate the properties of the intrinsic charge transfer between graphene and graphite QDs in the graphene FET, as shown in DSI-gV characteristics of the graphene FET before and after the deposition of the graphite QDs. The Dirac points are 1.6 V and 48 V before and after doping the graphite QDs, respectively. In other words, a higher gV is required to access the Dirac point of the graphite QDs/graphene FET because the net negative charge of the graphite QDs can attract more light-induced free holes in the graphene film. Such carrier transfer is known to enable more effective charge separation in graphite QDs and consequently suppress the electron-hole recombination [DSV) under different optical powers, showing a linear dependence on bias. Here, the photoconduction gain is enhanced by the recycling of holes through the graphene channel within the lifetime of the trapped electrons. Zero-dimensional carbon materials including graphite QDs, graphene QDs, and C60 were deposited on the graphene by spin coating or thermal evaporation methods to form hybrid structures for high-performance phototransistor applications ,83,84,85bination ,127. Fig3 photoconductive gain in the UV range. Graphene/graphene QDs/graphene PD was also fabricated to form hybrid-type PD that operates over a wide spectral range from UV to NIR [R, 1011 cm·Hz1/2/W D*, ~95 dB linear dynamic range (LDR), and 100 μs response time. Recently, PDs composed of silica NPs and graphene QDs as donors and acceptors were well operated based on Förster resonance energy transfer (FRET) mechanism, as shown in R of the FRET system was more than three times greater than that of the PD containing only graphene QDs, as shown in Graphene was hybridized with graphene QDs on a rippled poly(dimethylsiloxane) substrate to fabricate stretchable PD , whose PV to NIR ,85. ThisR under different back-gate voltages. The R was shown to be ~120 A/W and ~40 A/W at 650 and 1550 nm, respectively.CNTs can be metallic or semiconducting depending on their respective chiral vector . Metallilight/IdarkI ratio, R, and specific D* of the PD are 240, 0.21 A/W, and 4.87 × 1010 Jones, respectively, much larger than other CNTs-based PDs. The PD also exhibited a fast response rate of 68/78 μs rise/fall times and 5400 Hz 3dB bandwidth. More recently, there has been a report on novel broadband graphene/CNT thin film photodiode , where t2, [2 [The van der Waals interaction between neighboring layers without dangling bonds in 2D materials allows for their integration with other materials, including graphene, to form a variety of planar or vertical functional heterostructures with fundamentally different properties. As a prototype geometry, 2D layered semiconductors such as InSe , WS2, [92, , MoTe2 [2, [2 , and gra3 A·W−1 and fast rise/fall times of 1/10 ms [R on incident light power was observed in graphene/WS2/graphene heterostructure device [R increases as the optical power increases at gV = 0 V (off state), considered to be due to an efficient photo-induced charge transfer process in which electrons reside in the graphene while the holes are located at WS2. On the other hand, at gV = 30 V (on state), the R decreases as the optical power increases. When on state in the phototransistor, the photo-gating effect can increase the Fermi level and reduce the contact resistance. As a result, the influence of illumination on the Fermi level of graphene is reduced, thereby causing different power dependence of R. Recently, the planar graphene/n-InSe/graphene van der Waals heterostructures showed high R of 4 × 10 1/10 ms . The elee device . In this2 hybrid heterostructure phototransistors exhibited a high R of 5 × 108 and 1 × 1010 A·W−1 at 130 K and room temperature, respectively [2 by local conditions, and the electrons are transferred to the graphene with the help of the gate electric field, resulting in high R. On the other hand, the trapped holes act as local gates, thereby causing a significant photogating effect on graphene through capacitive coupling. Eventually, the response time is slow because of the long life of the trapped holes. In another approach, a similar phototransistor was fabricated using CVD-induced graphene and MoS2 [7 A·W−1 due to the low carrier mobility of CVD-graphene and very slow response times of several-hundred seconds, but seemed to be better suited for practical applications by easy preparation in large areas. In device structures dominated by photogating effects, 2D semiconductor serves as a medium for harvesting light, whereas graphene serves as a conduction channel for carrier transport and circulation. Recently, mechanically exfoliated graphene-MoSectively . Here, tand MoS2 . This hy2/graphene stack heterostructures were useful for efficient light–matter interactions, leading to improved photon absorption and electron-hole generation [R increases from ~10−2 to ~10−1 A·W−1 by integrating plasmonic metal nanostructures and heterostructures to improve the light absorption. It is worth noting that these results can be used for designing the dual-gate-type graphene/WS2/graphene heterostructure to control the polarity and amplitude of the PC by adjusting the direction and intensity of the built-in electric field [2/graphene employing 2.2 nm trilayer WSe2, as shown in Recently, graphene/transition metal dichalcogenide (TMD) heterostructures have also exhibited significant functionalities in the photodiode type. Carrier generation, separation, and transport processes were shown to be adjusted by easily modulating the Fermi level of graphene and the height of the Schottky barrier in graphene/TMD/graphene vertically-stacked heterostructures. For instance, graphene/WSneration . Scanninic field . A typicR photodiodes were fabricated by integrating graphene with TMD stacked semiconductors such as MoS2, MoTe2, and WSe2 [2 Schottky junction photodiode exhibited unique optical response over a wide spectral range from 400 to 1500 nm [R values of 0.52 A·W−1 at 590 nm/1.26 A·W−1 at 1440 nm were obtained when operating in the energy gap excitation mode/when internal light emission dominated the PC generation in graphene, respectively. On the other hand, metal-insulator-semiconductor-like photodiodes with an insulator layer between graphene and MoS2 showed improved current rectification and higher current flow compared to 2D-based metal-semiconductor diodes and p-n junctions based on TMDs [2/graphene/MoS2 gate tunable rectification behavior and the broadband response ranging from 400 to 2400 nm. [R and D* of up to 105 A·W−1 and 1015 Jones in the visible range, respectively, but they sharply decrease to 10−1 A·W−1 and 109 Jones at 2400 nm IR wavelength, as shown in 2, graphene, and MoS2), resulting in a large number of photocarriers, contributing to significant increase of PC. However, when the IR light having energy smaller than the band gap of WSe2 is irradiated, only graphene can absorb light, resulting in very low PC. These results suggest that the van der Waals junctions based on 2D materials are very promising in future critical optoelectronic device applications.As another approach, relatively-low-and WSe2 ,97,98,99 1500 nm . Maximum2400 nm. . The devGraphene/semiconductor hybrid heterostructures showed their tremendous potential for PD applications. Particular attention was paid to several important issues related to device design, device performance, and processing technology to enhance figure-of-merit parameters of the PDs. By the optimization through various approaches, the PDs typically showed some new features that could open up the way to many conventional and advanced applications. The work function, surface resistance, and optical transparency of the graphene TCEs were successfully tuned by adjusting the number of layers, chemical doping, photo-induced doping, or electric field gating doping, which strongly affected the performance of the PDs. To meet the requirements for the practical applications of the PDs, some of the properties of the sensing materials should be optimized. Because the heterogeneity of the nanostructured materials can greatly affect the uniformity of the device performance, more precise control of their morphology, crystallinity, and orientation is highly needed. Some emerging methods such as plasmonic technology and integration of optical waveguides/micro cavities are also useful for improving the photo-material interactions, resulting in the increase of the light absorption. In particular, special efforts should be made for high response speed of the PDs by increasing the charge carrier mobility of the sensing materials or introducing a vertical electric field for the transfer and separation of photo-carriers. Broadband, spectrally selective, or flexible PDs should also be developed for specific applications. In addition to the improvements of the figure-of-merit parameters, long-term reliability and durability, environmentally-friendly and cost-effective machining technologies, large-scale production and consolidation are also important issues for various applications of the PDs in upcoming new situations.


Silicone rubber (SIR) is widely used as an insulation material in high voltage cable accessories. Electrical tree is a typical electrical degradation and is easily initiated because of the distorted electric field. In this study, graphene nanoplatelets at contents of 0.001–0.010 wt % (0.00044–0.00436 vol %) were added into SIR to improve the electrical tree inhibiting ability. Scanning electron microscopy, conductivity and surface potential decay tests were conducted to analyze the characteristics of graphene/SIR nanocomposites. The typical electrical treeing experiment was employed to observe the electrical tree inhibition of graphene in SIR. The results show that graphene nanoplatelets were well dispersed in SIR. The conductivity was higher after the addition of graphene nanoplatelets, and the trap distribution was affected by graphene nanoplatelets. The tree was changed from a bush-branch structure to a bush structure after the addition of graphene. Tree inception voltage improved and reached the highest mean value at 0.003 wt %. The tree length was inhibited at 0.001 to 0.007 wt % and the lowest tree length occurred at 0.005 wt %. Silicone rubber (SIR) is usually employed as insulation in both alternating current (AC) and direct current (DC) power cable accessories, forming a multilayer insulation with cross-linked polyethylene (XLPE), polypropylene (PP) or other polymers ,2,3. Giv2) and alumina (Al2O3) [Nanodielectrics with filler contents of several wt % have been proven to have a high electrical tree-inhibiting ability ,12,13,14 (Al2O3) ,16,17. W (Al2O3) ,16,17. T (Al2O3) . Accordi (Al2O3) ,20. FromGraphene is a typical 2D structure with a giant specific surface area. The effects of graphene nanoplatelets on SIR properties have been researched ,22,23,24In this work, graphene/SIR nanocomposites were prepared with filler contents of 0, 0.001, 0.003, 0.005, 0.007 and 0.010 wt %. Scanning electron microscopy (SEM), conductivity and trap distribution were tested. The electrical treeing experiment was conducted and the inhibition effect of graphene was discussed.4 g/mol, and the content of vinyl groups was 0.13–0.20 mol %. The graphene used for the sample was purchased from Tanfeng Graphene Technology Co., Ltd. . The diameter of graphene nanoplatelets was 0.5–5 μm, the thickness was 0.5–3.0 nm, with 1–2 layers and an SSA of 1000–1217 m2/g. Before mixing, a surface treatment was applied on the graphene.High temperature vulcanized methyl vinyl SIR (MVQ 110-2) was provided by Dongjue Silicone Group Co. Ltd. . Its average molecular weight was 65 × 10Zhang et al. reported that graphene modified with vinyltrimethoxysilane exhibited improved dispersing and banding properties with SIR . Thus, vThe weight content of graphene in nanocomposites was 0, 0.001, 0.003, 0.005, 0.007 and 0.010 wt % . The required graphene was weighed by a microbalance with a sensitivity of 0.01 mg . A twin-screw continuous mixer was employed to mix the graphene and SIR, with a rotation speed of 40 rpm at room temperature for 15 min. Then 2,5-dimethyl-2,5-di(tert-butylperoxy) hexane  was added into the mixture as the vulcanizing agent at 1.5 phr. After the mixing process, the compounds were placed into the moulding in the plate vulcanizing machine  at a temperature of 165 °C and pressure of 10 MPa for 10 min [A high-resolution scanning electron microscope  was employed to observe the cross-section of the graphene/SIR nanocomposites. The samples were cryo-fractured in liquid nitrogen and the surface of the cross-section was painted with platinum before scanning.DC conductivity was tested on a typical three-electrode system as in . The samI is the current in A, U is the applied voltage in V, l is the thickness of sample in m and r is the radius of sample in m.After 10,000 s, the conductivity was calculated as follows:To calculate the trap distribution in SIR, surface potential decay (SPD) measurement was used. The measurement setup was shown in our previous work . The samET is the trap energy in eV; T is the temperature in K; vATE is the attempt to escape frequency in s–1; kB is the Boltzmann’s constant in eV/K; h is the Planck constant in eV·s; NT is the trap density in eV–1m–3; ε0 and rε are the permittivity of vacuum and the relative permittivity of SIR; q is the elementary charge in C; L is the sample thickness in m; sU is the surface potential in V and t is the decay time in s.The potential decay rate is related to the trap distribution in samples. The trap energy and density can be calculated by these three equations ,30:(2)ETThe treeing test setup is shown in The graphene nanoplatelets distribution in the samples for all graphene contents were analyzed with SEM. The SEM pictures of samples with 0.001, 0.005 and 0.010 wt % are shown in −13 S/m. From 0.005 to 0.010 wt %, a sharp increase (1.55 × 10−11 S/m) in conductivity occurred, potentially caused by the shorter distance between graphene nanoplatelets at higher graphene contents.The conductivity of graphene/SIR nanocomposites is shown in The potential decay in graphene/SIR nanocomposites is related to the charge migration from SIR bulk ,32. Duri20 eV–1m–3 to lower than 2.7 × 1020 eV–1m–3 and the density of deep trap decreased from 2.2 × 1020 eV–1m–3 to lower than 1.7 × 1020 eV–1m–3 after the addition of graphene.The trap distribution in the nanocomposites can be calculated according to Equations (2)–(4). The relative permittivity used here is shown in During the experiment, two different tree structures were observed, defined as bush-branch tree and bush tree. As shown in To observe the inhibition effect of graphene, the tree inception voltage was tested. The RMS of the AC voltage was set to 5–10 kV. In the test, the voltage was increased in 0.2-kV steps and was maintained for 30 s at each step to check if the tree was triggered or not. If an electrical tree with a length of 20 μm was observed, the RMS was recorded as the tree inception voltage. Ten samples were tested for each graphene content.The average inception voltages from 10 tests and the standard deviations are shown in The treeing process was recorded using a microscope and computer. Typical tree growth processes in samples with different graphene contents are shown in As shown in As shown in As shown in The tree length of a typical tree without breakdown at different times was calculated as shown in During the experiments, nine samples of each graphene content were tested for 60 min. The tree length at 60 min was calculated to analyze the inhibition effect of graphene. If the breakdown occurred within 60 min, the length was calculated as 2 mm. In the whole experiment, breakdown only occurred in samples with 0 and 0.010 wt %. The box-plot of tree length at 60 min is shown in Under AC voltage, there is an electric field with altering direction. According to the theory proposed by Tanaka et.al., it is believed that there is an electron injection-extraction process near the needle tip . Given tIn the case of nanocomposites, the trapping-detrapping process is similar to what occurs in undoped SIR. When the electrons are injected into the sample, they will be captured by the traps around needle tip induced by the interface between SIR and graphene ,34. HoweAccording to the model proposed by Danikas et al. , the treHowever, the shorter distance between the nanoplatelets lower the dielectric strength in some regions inside the nanocomposites with higher content . BecauseGraphene nanoplatelets and SIR were employed to prepare nanocomposites with graphene content lower than 0.010 wt %. SEM, conductivity and trap distribution characteristics were tested to examine the nanocomposites.The results showed that the conductivity of SIR can be increased by the addition of graphene nanoplatelets. The trap distribution in SIR changes with the addition of graphene nanoplatelets. The shallow and deep trap levels in samples increase in nanocomposites with content ranging from 0.001 to 0.007 wt %.The electrical degradation was inhibited by the addition of graphene nanoplatelets at low contents. The average tree inception voltage increased after the addition of graphene nanoplatelets at contents of 0.001 to 0.007 wt %. The tree structure in undoped SIR was a bush-branch tree, which changed to the bush tree structure in graphene/SIR nanocomposites. The tree length in graphene/SIR nanocomposites was inhibited at contents of 0.001 to 0.007 wt % and increased at 0.010 wt %.


Sarcoidosis is a rare, chronic, multisystem disease of unknown aetiology, characterised by non-caseating epithelioid cell granulomas. Its association with internal malignancy, in particular haematological cancers has been strongly documented in the literature, while its link with solid organ malignancies is less extensively reported. We present an atypical case of cutaneous sarcoidosis occurring in association with breast cancer recurrence in a 49-year-old female. Physician recognition of this link between sarcoidosis and internal malignancy is vital because many cases of sarcoidosis in association with neoplasia present initially, or even exclusively, with cutaneous sarcoidal lesions that may precede the development of cancer by several years, or as in our case, present as a cutaneous marker of concomitant underlying malignancy. Our case highlights the importance of age-appropriate cancer screening in additional to a routine work-up for systemic sarcoidosis in a patient with cutaneous sarcoidosis. Sarcoidosis is a chronic, idiopathic, multisystem disease, characterised by non-caseating epithelioid cell granulomas. The lungs are involved in more than 90% of cases, but the lymphatic system, eyes, and skin may also be affected. Less common but usually more severe forms can involve the liver, spleen, central nervous system, heart, upper respiratory tract, and bones. Its pathogenesis appears to correspond to an aberrant immune response in a susceptible host. Sarcoidosis typically affects young adults, with a slight female general predominance , 2.Various diseases have been associated with sarcoidosis, including autoimmune disorders such as rheumatoid arthritis, psoriasis, vasculitis, thyroid disease, systemic sclerosis, and Sjogren syndrome. Haematological malignancies, in particular lymphoproliferative disorders such as Hodgkin lymphoma, are most strongly associated with sarcoidosis compared to solid organ malignancies , 3. We pA 49-year-old female presented with a 2-month history of asymptomatic lesions on the left knee found incidentally on routine full skin examination. The patient was otherwise well, with no pulmonary or systemic symptoms.She had a past history of breast cancer diagnosed 4 years ago, managed by lumpectomy and adjuvant chemoradiotherapy achieving remission. The patient had regular cancer surveillance and was currently on adjuvant tamoxifen, with a planned duration of 10 years. Her other notable medical history included lifelong asthma, gastrooesophageal reflux disease, depression, subacute thyroiditis and previous shoulder, and knee arthroscopies. Her regular medications included tamoxifen, pantoprazole, venlafaxine, budesonide/formoterol, and terbutaline. She was a lifetime non-smoker and rarely consumed alcohol. The patient had no family history of autoimmune conditions.Examination revealed numerous erythematous-to-brown, non-tender papules occurring on the anterior left knee . On the Skin biopsy showed multiple, variably sized naked sarcoidosis type granulomas scattered throughout the dermis . Chest rThe patient's management was then deferred to a medical oncologist for ongoing care of her metastatic breast cancer. She received stereotactic radiation to her spinal lesion and was commenced on a special access program with ribociclib. Following breast cancer treatment, cutaneous sarcoidal lesions completely resolved.Cutaneous involvement presents in 25% of patients with systemic sarcoidosis and may be the only manifestation . DermatoVarious diseases have been associated with sarcoidosis. Previously, an association between sarcoidosis and malignancy has been described, although no clear relationship has been identified. In most cases, sarcoidosis was diagnosed before the detection of an associated neoplasm. Haematological malignancies remain most strongly associated compared to solid tumours –3. BrincPrevious literature cases of sarcoidosis occurring with breast cancer are summarised in Our case is unique in that the cutaneous sarcoidosis most likely occurred around the same time the patient's breast cancer recurrence was diagnosed and investigation for systemic sarcoidosis revealed her metastatic disease. This may be an incidental finding or indicate that dysregulation of the immune system mediated by either the breast cancer or sarcoidosis lead to the granulomatous inflammation of sarcoidosis or neoplasm, respectively , 4, 34. Recognition by physicians of this link between sarcoidosis and internal malignancy is vital because many cases of sarcoidosis in association with neoplasia present initially, or even exclusively, with cutaneous sarcoidal lesions that may precede the development of cancer by several years or as in our case, present as a cutaneous marker of concomitant underlying malignancy. Thus, in addition to routine screening for systemic sarcoidosis, patients diagnosed with cutaneous sarcoidosis should be closely followed up, particularly including age-appropriate cancer screening to exclude the development of associated malignancy .


We numerically propose a dual-band absorber in the infrared region based on periodic elliptical graphene-black phosphorus (BP) pairs. The proposed absorber exhibits near-unity anisotropic absorption for both resonances due to the combination of graphene and BP. Each of the resonances is independently tunable via adjusting the geometric parameters. Besides, doping levels of graphene and BP can also tune resonant properties effectively. By analyzing the electric field distributions, surface plasmon resonances are observed in the graphene-BP ellipses, contributing to the strong and anisotropic plasmonic response. Moreover, the robustness for incident angles and polarization sensitivity are also illustrated. Graphene is a two-dimensional material with carbon atoms arranged in a honeycomb lattice , 2. VariIn our work, an anisotropic dual-band infrared absorber is numerically proposed using periodic elliptical graphene-BP pairs, which is ease of fabrication. The independent tunability of resonance by geometric size and doping level is demonstrated. Electric field distributions are plotted to reveal the physical mechanism. The incident angle tolerance and polarization sensitivity are also illustrated.2 layer as shown in Fig. 2 and hBN are obtained from Ref. x- and y- directions. A port with infrared wave excitation is set upon the top surface of the computational domain, while perfect electric conductor (PEC) boundary condition is set on the bottom surface. Tetrahedral meshes with user-controller mesh density are applied for the entire domain.The proposed absorber is made up of transverse and longitudinal elliptical graphene-BP pairs deposited on a SiOIn the simulation, both graphene and BP are treated as two-dimensional surface with surface conductivities instead of bulk materials with permittivity tensors. This assumption solves the problems of thickness definition for ultrathin materials and low computational efficiency .σ(ω), we use the well-known Kubo formulas as below [To describe the surface conductivity of graphene as below :1\documσ(ω) consists of the intraband and interband counterparts, namely σintra and σinter. ω is the radian frequency, μc is the chemical potential, Г is the scattering rate, and T is the Kelvin temperature. ħ, e, ξ, and kB are the reduced Planck constant, electron charge, electron energy, and Boltzmann constant, respectively.According to Eq. c ≫ kBT, Kubo formulas can be further simplified to Eq. ω, Г, and μc. Here, Г is assumed as 0.3 meV and μc is assumed to be 0.7 eV according to the previous work [In the infrared region, since the incident photon can hardly excite the interband transition, the light-graphene interaction is dominated by the intraband transition. Particularly, when μous work , 26.σj of BP with a simple semi-classical Drude model [ns is the carrier density relating with the doping level. We choose ns = 1.9 × 1013 cm−2 and ГBP = 10 meV according to the previous reference [j is the concerned direction, so σx and σy are determined by the electron mass along x- and y-direction, respectively. mx and my can be further calculated by:m0 is the standard electron mass, and Δ and a are the band gap and scale length for BP monolayer, respectively. By substituting Eqs. x-) and zigzag (y-) direction. The discrepancy between them contributes to the anisotropic surface conductivity of BP.On the other hand, we calculate the surface conductivity de model :6\documeference . j is thmentclasspt{minimaR1/R0), where R1 and R0 denote the reflectance  of different polarizations at the same wavelength, then the maximum PER of each resonance can reach up to 23 dB and 25 dB at λ = 9.5 μm and λ = 14.1 μm, respectively. Therefore, the proposed absorber can be utilized as a dual-band reflective polarizer with high performance.To illustrate the anisotropic absorption characteristic of the proposed absorber, we first simulate and compare the absorption spectra with individual graphene layer, individual BP layer, and graphene-BP pairs. As can been observed in Fig. a increases from 42 to 52 nm for both polarizations, while second resonant frequencies are almost unchanged. On the other hand, as shown in Fig. b, the second resonances are redshifted as well, while the first absorption peaks remain constant for TE and TM polarization. Therefore, the dual absorption peaks can be tuned independently by varying the corresponding axis length in the elliptical graphene-BP pairs. Moreover, the thickness of dielectric layer also plays a critical role in the performance for the proposed device, which acts as a Fabry-Perot resonator formed by the graphene-BP metasurface and the PEC substrate. Thus, the absorption spectra with different td are plotted in Fig. td increases from 0.95 to 1.75 μm, the first absorption peaks for TE and TM polarization have a dramatic drop, while the second peaks increase at first then decrease sharply. As a consequence, there is an optimal thickness td that maximizes the dual absorption peaks of the proposed absorber.We next analyze the absorption spectra with different geometric configurations to demonstrate the tunable dual-band absorption property in Fig. x-) direction. At the first peak (λ = 8.8 μm), the incident infrared light can excite electrons in graphene and BP to oscillate in the transverse direction, leading to the concentration of electric field at the short axis ends of the longitudinal ellipse as shown in Fig. λ = 14.1 μm, the localized electric field is enhanced at the long axis ends of the transverse ellipse. On the other hand, TM incidence with electric field in the zigzag (y-) direction can excite electrons to vibrate along the longitudinal direction at the absorption peak of 9.5 μm, leading to concentrated field distributions at the short axis ends of the transverse ellipse. Besides, at λ = 15.4 μm, the enhancement of electric field is focused at the long axis ends of the longitudinal ellipse. Therefore, the resonance wavelengths are directly related to the finite oscillation length of the induced dipoles in both transverse and longitudinal elliptical graphene and BP pairs.In order to elucidate the physical insight, we further reveal the electric field intensity distributions at different wavelengths in Fig. c and ns according to graphene and BP model formulas as mentioned above. μc and ns represent the doping level of graphene and BP that can be altered after geometric fabrication. Thus, performances of the proposed absorber with different μc and ns are depicted in Fig. μc is chosen between 0.4 and 0.8 eV from the previous work verified by experiments [ns of BP was demonstrated to be 2.6 × 1014 cm−2, so a moderate ns is chosen between 1013 cm−2 and 1014 cm−2 in the simulation. In Fig. μc = 0.4 eV, the first absorption peak is located at 10.9 μm and the second one is located at 17.1 μm. As μc increases to 0.8 eV, the two resonant wavelengths are blueshifted to 8.4 μm and 13.4 μm. Similarly for TM polarization, the dual absorption peaks are blueshifted from 12.4 and 19.8 μm to 8.9 and 14.4 μm, respectively, with μc increasing from 0.4 to 0.8 eV as shown in Fig. λp can be calculated as L is the effective oscillation length [L is fixed, the absorption spectra exhibit an obvious blueshift as ns increases for TE polarization as plotted in Fig. ns increases from 1013 cm−2 to 1014 cm−2 as demonstrated in Fig. One can tune the anisotropic dual-band absorption performance effectively by varying the geometric dimensions as demonstrated in Fig. eriments . In the eriments , the maxn length . Thus, iθ increases to 52°, while the second absorption peak maintains above 80% even when θ increases to 80°. When θ > 46°, the second resonant wavelength is redshifted gradually as θ becomes larger. For TM incidence, when θ is less than 62°, the absorption rate at the first peak maintains larger than 90%, while the resonant wavelength keeps constant at λ = 9.5 μm as shown in Fig. θ up to 60°, then drops slightly with the increase of θ. The excellent angular stability originates from the common feature of Fabry-Perot resonators, which are robust for oblique incident angles [In the practical applications, tolerance of wide incident angles is preferred for infrared absorbers. Therefore, absorption spectra under oblique incidences are elaborated. In Fig. t angles .Fig. 5Aφ are presented in Fig. φ increases from 0 to 90°, the absorption spectrum turns out to be the same as the TM polarization in Fig. φ < 90°, the incidence will excite electrons in BP to oscillate in both armchair and zigzag directions due to its x- and y- components of the incident electric field. Consequently, surface plasmon resonances can be induced simultaneously in armchair and zigzag directions of BP.Absorption spectra under normal incidence with different polarization angles In conclusions, we have proposed an anisotropic dual-band infrared absorber consisting of periodic transverse and longitudinal graphene-BP ellipses. The maximum PER at each resonance can reach up to 23 dB and 25 dB. The dual anisotropic resonances are attributed to the induced electric dipoles located at the ends of short and long axes. By adjusting the lengths of short axis and long axis, the first and second absorption peaks can be independently tuned, respectively. Moreover, the resonant absorption bands can also be tuned by changing the corresponding doping level of graphene and BP. Besides, high absorption rates at both peaks can be achieved under oblique incidence for any polarization. The proposed absorber can be utilized as a tunable reflective polarizer and novel infrared sensor.


Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by an exacerbated fibrotic response. Although molecular and cellular determinants involved in the onset and progression of this devastating disease are largely unknown, an aberrant remodeling of the pulmonary vasculature appears to have implications in IPF pathogenesis. Here, we demonstrated for the first time that an increase of reactive oxygen species (ROS) generation induced by sera from IPF patients drives both collagen type I deposition and proliferation of primary human pulmonary artery smooth muscle cells (HPASMCs). IPF sera-induced cellular effects were significantly blunted in cells exposed to the NADPH oxidase inhibitor diphenyleneiodonium (DPI) proving the causative role of ROS and suggesting their potential cellular source. Contrary to IPF naive patients, sera from Pirfenidone-treated IPF patients failed to significantly induce both ROS generation and collagen synthesis in HPASMCs, mechanistically implicating antioxidant properties as the basis for the in vivo effect of this drug. An aber regions .Although the fibroblast appears to be the most well established, other types of cells have been reported to be implicated in the IPF-associated fibrotic process . In addiAfter decades of having no effective medical treatment for IPF, two recent antifibrotic agents have been introduced for the management of this pathology: Nintedanib, a potent kinase inhibitor blocking the effects of growth factors implicated in the pathogenesis of IPF  , and PirWe hypothesized that prooxidant circulating factors may trigger VSMCs' phenotypic switching and induce cell proliferation and collagen I synthesis and that the antioxidant properties of Pirfenidone can counteract this phenotypic change and thus ameliorate IPF patients' conditions. To verify our two research questions, we investigated reactive oxygen species (ROS) production, cell proliferation, and collagen synthesis in primary human vascular smooth muscle cells exposed to serum obtained from naive IPF patients (IPF) or IPF patients treated with Pirfenidone (IPF + D) and healthy donors (HD).IPF was diagnosticated in accordance with evidence-based guidelines for the diagnosis and management of IPF . High-reD-penicillamine, and colchicine, or oral corticosteroids at a dose ≥ 15 mg/day or the equivalent during the preceding 3 months; and current pregnancy or breastfeeding. All enrolled patients provided written informed consent, and the protocol for our study was approved by the ethics committee of the University Hospital of the University of Sassari (2262/CE-17/11/2015). Age- and sex-matched healthy donors were recruited through posted flyers and enrolled after passing a screening questionnaire aimed at excluding the presence of any underlying vascular or autoimmune disease. Blood samples were collected at the time of the IPF diagnosis and after 24 weeks of Pirfenidone treatments, and then sera were prepared according to our previously published procedures [Patients with the following conditions were excluded from the study: currently in a period of acute exacerbation of IPF; comorbid conditions including malignancy, bleeding tendency, and severe hepatic dysfunction ; use of immunosuppressants, antifibrotic drugs including interferon, ocedures .v/v) of the subjects' sera. Sera among the different subjects were normalized based on protein content. To investigate the involvement of NADPH oxidase in the IPF sera-induced cellular effects, in selected experiments, cells were pretreated for 1 hr with 10 μM of the flavin-oxidase inhibitor diphenyleneiodonium (DPI) [In this study, pulmonary artery smooth muscle cells (HPASMCs) isolated from human pulmonary arteries of healthy donors were used . Cells were cultured in HPASMC basal medium supplemented with endothelial cell growth supplement. When confluent, HPASMCs were subcultured at a split ratio of 1 : 2 and used within three passages. Unless not specified in the text, cells were plated in 96-well black plates  and processed for experiments in basal medium containing 10% (um (DPI) , 21.2DCF-DA) (Molecular Probes) as previously described [2DCF-DA, thus trapping the reduced form of the probe (H2DCF). Intracellular ROS oxidize H2DCF, yielding the fluorescent product, DCF. For ROS measurements, cultured cells were preincubated for 30 min with PBS plus containing 1 μM H2DCFDA, then washed with PBS and treated as described. Fluorescence was measured by using a Tecan GENios Plus microplate reader  in a light-protected condition. Excitation and emission wavelengths used for fluorescence quantification were 485 nm and 535 nm, respectively. Treatment-induced variation of fluorescence was kinetically measured over a time-course of 5 hours. All fluorescence measurements were corrected for background fluorescence and protein concentration. Using untreated cells as a reference, the anti- and prooxidant outcomes were evaluated by the comparison of five measurements and expressed as a means ± SD of the relative fluorescence unit (RFU) values.Intracellular ROS levels were assessed by using the ROS molecular probe 2′,7′-dichlorodihydrofluorescein diacetate  was used to normalize the cell transduction efficiency [α-LV-FP602 lentivectors. Transduction efficiency was checked and confirmed under a fluorescence microscope. Treatment-induced variation of fluorescence was kinetically measured over a time-course of 12 hours using a Tecan GENios Plus microplate reader . Excitation wavelengths used for fluorescence quantification were 485 nm and 535 nm, while emission wavelengths were 535 nm and 590 nm for pCOL1A1-LV-tGFP and EFα-LV-FP602, respectively. Data were normalized for transduction efficiency by reporting the ratio of pCOL1A1-LV-tGFP to EFα-LV-FP602 and expressed as a means ± SD of the relative fluorescence unit (RFU) values.As previously described, collagen type-I (COLS) synthesis was investigated employing COL1A1-LV-tGFP, a GFP-based lentiviral vector (LV) driven by the human COL1A1 gene promoter , 24. A rficiency . This meficiency . HPMECs μl of peroxidase-labeled anticollagen monoclonal antibody solution was transferred into each well for an anticollagen monoclonal antibody coating. Subsequently, 20 μl of cell extract  or standard was added. Then, the solution was incubated for 3 hours at 37°C. Then, 100 μl of substrate solution was added into each well. The reaction between a peroxidase-labeled anticollagen monoclonal antibody and substrate results in color development with intensities proportional to the amount of collagen present in samples and standards. Finally, after 15 minutes of incubation at room temperature 100 μl of Stop Solution was added into each well. The amount of collagen was quantitated by measuring the absorbance (450 nm) using a Tecan GENios Plus microplate reader . Accurate concentrations of collagen in the samples were determined by comparing their specific absorbances with those obtained for the standards plotted on a standard curve. Results were expressed as a means ± SD of ng/ml collagen.Variation of collagen synthesis in response to sera treatment was quantitatively confirmed by assessing the collagen protein levels (COLP) with an enzyme immunoassay kit  following the protocol provided by the manufacturer. Briefly, after 48 h sera treatment cells were washed and recovered in the protein extraction buffer. 100 v/v) of serum from different subjects, and BrdU was added 12 hrs before the end of the experiments. After that, the culture supernatant was removed, and the cells were fixed with a Fix-Denat solution for 30 min. The Fix-Denat was discarded and cells were incubated with an anti-BrdU antibody conjugated to horseradish peroxidase (anti-BrdU-POD) for 90 min. After rinsing three times with washing buffer, the substrate solution was added and allowed to react for 3–10 min at room temperature. Within this time window, the horseradish peroxidase catalyzes the oxidation of diacyl hydrazide, where the reaction product decaying from its excited state yields light. Finally, light emission was read by using a GENios Plus microplate reader (Tecan). Results were expressed as a means ± SD of the relative light units/sec (RLU/s) values [Cell proliferation was assessed by using a chemiluminescent immunoassay method, which is based on the measurement of BrdU incorporation during DNA synthesis . When cells are pulsed with BrdU, it is incorporated into newly synthesized DNA strands of actively proliferating cells. The incorporation of BrdU into cellular DNA can be detected using anti-BrdU antibodies, allowing assessment of the population of cells synthesizing DNA. Subconfluent cells were treated for 48 with 10%  values , 26.P < 0.05. All the analyses were performed using the GraphPad Prism 6 software .Data were checked for normal distribution and processed by one-way analysis of variance (ANOVA) followed by post hoc Tukey's multiple comparison tests to determine the differences between mean values among data groups, with significance defined as P = 0.1813) and DLCO (P = 0.5770).IPF patients enrolled in the study were predominantly males (72.72%) and had an average age of 71.27 ± 5.5 years . At baseIntracellular ROS levels were kinetically determined in a 5-hour time-course  and valuExposure of HPASMCs to IPF sera also resulted in a progressive time-dependent increase of the collagen type 1 (COL1) promoter activity  with valData in β1), tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF) [β1 for instance, can exert its profibrotic effect via a NADPH oxidase-dependent increase of intracellular ROS levels [β1-induced differentiation of fibroblasts into myofibroblasts [Vascular remodeling in IPF fibrosis is a controversial area of research. It has now been reported that both areas with increased angiogenesis and fibrosis are present in the lungs of patients with this disease, clearly indicating aberrant microvascular tissue remodeling . Indeed,r (VEGF) , 3, whicr (VEGF) –32. TGF-S levels . In the S levels . ConsistS levels  and in problasts . Finallyroblasts .This preliminary pilot study provides new evidence supporting the possibility that pathological tissue restructuring in the lung of IPF patients may be driven and/or maintained by prooxidant circulating factors acting, at least in part, through the induction of HPASMC proliferation and the activation of collagen synthesis. While more studies are needed to precisely identify both the mediators and the molecular determinants of these effects, our data provide new clues concerning the mechanism of action of Pirfenidone and indicate a rationale for considering antioxidant therapies in the treatment or prevention of IPF.