Foxc2 is one of the earliest podocyte markers during glomerular development. To circumvent embryonic lethal effects of global deletion of Foxc2, and to specifically investigate the role of Foxc2 in podocytes, we generated mice with a podocyte‐specific Foxc2 deletion. Mice carrying the homozygous deletion developed early proteinuria which progressed rapidly into end stage kidney failure and death around postnatal day 10. Conditional loss of Foxc2 in podocytes caused typical characteristics of podocyte injury, such as podocyte foot process effacement and podocyte microvillus transformation, probably caused by disruption of the slit diaphragm. These effects were accompanied by a redistribution of several proteins known to be necessary for correct podocyte structure. One target gene that showed reduced glomerular expression was Nrp1, the gene encoding neuropilin 1, a protein that has been linked to diabetic nephropathy and proteinuria. We could show that NRP1 was regulated by Foxc2 in vitro, but podocyte‐specific ablation of Nrp1 in mice did not generate any phenotype in terms of proteinuria, suggesting that the gene might have more important roles in endothelial cells than in podocytes. Taken together, this study highlights a critical role for Foxc2 as an important gene for podocyte function. Foxc2 is a transcription factor with many vital functions during development, including skeletogenesis and patterning of the aortic branch  as well as collagen IV subunits a3 and a4 (COL4A3 and COL4A4)  and PODXL  (#5247)  and subcloned into the pPGKneobpAlox2PGKDTA vector. The 5′ loxP site was inserted into the BspEII‐site upstream of the start codon, where no known regulatory elements were identified. To avoid disruption of miRNA‐binding sites, the floxed Neo‐cassette was inserted outside of the 3′UTR. The targeting vector was linearized with NotI and electroporated into RW4 ES cells and homologous recombination was confirmed by southern blot. The Neo‐cassette was then removed by transient transfection of correctly targeted ES clones and positive clones were injected into the blastocyst obtained from C57Bl/6 mice. Chimeric mice were mated with C57Bl/6 mice to generate Foxc2fl/+ mice.Mice with a floxed Foxc2 or Nrp1 knockouts, as well as Cre reporter mice, animals with floxed alleles were crossed with Pod‐Cre transgenic mice. The strain was maintained on a mixed sv129;C57Bl/6 background.To generate podocyte‐specific All animal procedures were approved by the Ethical Committee for Animal Experiments at the University of Gothenburg, which adheres to the principles and guidelines established by the European Convention for the Protection of Laboratory Animals.Foxc2lacZ, Pod‐Cre transgene, Rosa26STOPlacZ, and floxed Nrp1 were genotyped as previously described . Expected products were 180 bp from the wild‐type allele and 220 bp from the floxed Foxc2 allele. Genotyping of the null allele of Nrp1 was performed by PCR, using sense primer 5′‐ AAGGAGTGGCACAGCATCTT‐3′ and antisense primer 5′‐ TTGGGTGAACTCAGCCACTT‐3′. Amplification from the Nrp1 null allele was estimated to generate a 350 bp PCR product, whereas wild‐type allele should generate a 739 bp band.Alleles for  Soriano . Floxed μm thickness.Embryos and kidneys for X‐gal staining and standard histology were harvested and immersed in ice‐cold 4% (w/v) paraformaldehyde (PFA)(Sigma‐Aldrich) in phosphate‐buffered saline (PBS)(Medicago) for 1 h. Fixed specimens for cryosectioning were immersed in ice‐cold 30% (w/v) sucrose in PBS until they sunk, embedded in OCT Cryomount (Histolab) and cryosectioned at 10 β‐galactosidase expression, fixed whole embryos or kidneys were incubated in 5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyranoside  staining solution (5 mmol/L K3Fe(CN)6 (ICN), 5 mmol/L K4Fe(CN)6 (ICN), 5 mmol/L EGTA (Sigma‐Aldrich), 0.01% (w/v) deoxycholate (Sigma‐Aldrich), 0.02% (v/v) NP‐40 (BDH), 2 mmol/L MgCl2 (Scharlau), and 1 mg/mL of X‐Gal (Sigma‐Aldrich) in PBS) overnight at 37°C. Here, β‐galactosidase‐expressing tissues were located and photographed under a dissecting microscope.For detection of μm. Sections were mounted on SuperFrost microscope slides and counterstained for 5 min in 0.1% (w/v) cresyl violet acetate (Sigma‐Aldrich) in distilled H2O, and washed for 15 min in 96% ethanol (Solveco) and for 5 min in PBS.Stained embryos were postfixed in 10% (v/v) formalin and embedded in a mixture of 25% (w/v) bovine serum albumin (BSA)(Sigma‐Aldrich) and 0.4% (w/v) gelatin in PBS. The solution was solidified by addition of glutaraldehyde (Sigma‐Aldrich) to a final concentration of 2.5% (v/v), and the resulting tissue blocks were serial sectioned on a vibratome at 30 Standard hematoxylin and eosin staining for histology was performed on cryosections using Mayer′s HTX (Histolab) and eosin Y with phloxine (Sigma‐Aldrich). Histochemically stained sections were mounted in Canada Balsam (Sigma‐Aldrich), and then examined and photographed using an Eclipse E800 microscope (Nikon).4) (Sigma‐Aldrich), and 1% (w/v) potassium ferrocyanide (K4Fe(CN)6) for 2 h at 4°C. Contrast was enhanced by incubation in 0.5% (w/v) uranyl acetate for 1 h in darkness at room temperature. After dehydration, the filters with attached cells were embedded in Agar 100 resin (Agar Scientific LTD). Ultrathin transverse sections (60–70 nm) of the filters with attached cells were contrasted with uranyl acetate and lead citrate before examination using a Zeiss LEO 912 Omega transmission electron microscope (Zeiss). Digital image files were captured with a MegaView III camera (Soft Imaging Systems).Kidneys for electron microscopy were fixed overnight in modified Karnovsky′s fixative (2.5% (v/v) glutaraldehyde, 2% (w/v) paraformaldehyde, 0.05 mol/L sodium cacodylate (Sigma‐Aldrich), 0.02% (w/v) sodium azide (Sigma‐Aldrich), pH 7.2) For transmission electron microscopy, kidneys were rinsed in 0.15 mol/L sodium cacodylate and postfixed in 1% (w/v) osmium tetroxide  OsO4 at 4°C, 10 min in 1% (w/v) thiocarbohydrazide (Sigma‐Aldrich) followed by 1 h incubation in 1% (w/v) OsO4 at 4°C. Sections were dehydrated in ethanol followed by immersion in hexamethyldisilazane (Sigma‐Aldrich) and evaporation in a fume hood. The dried specimens were mounted on stubs and coated with palladium and then examined in a Zeiss 982 Gemini field emission scanning electron microscope equipped with an in‐lens secondary electron detector (Zeiss).For scanning electron microscopy, 60 μm were fixed and permeabilized in ice‐cold acetone (Fisher Scientific) for 10 min or fixed in 4% (w/v) paraformaldehyde in PBS for 20 min at room temperature and permeabilized in 0.2% (v/v) Triton X‐100 in PBS for 15 min at room temperature. Sections were then blocked in 10% (v/v) fetal bovine serum in DMEM medium (Gibco) (blocking solution) for 1 h at room temperature and incubated with primary antibody diluted in blocking solution at 4°C overnight. After washing in PBS, the sections were incubated with secondary antibody and TO‐PRO®‐3 nuclear stain (Thermo Fisher Scientific 1:1000) diluted in blocking solution for 1 h at room temperature. Sections were washed again in PBS and slides were mounted in ProLong® Gold Antifade Mountant (Thermo Fisher Scientific) or ProLong® Diamond Antifade Mountant (Thermo Fisher Scientific) and photographed using an LSM 510 Meta confocal microscope (Zeiss). Antibodies used are listed in Table Kidneys were dissected and embedded in OCT Cryomount (Histolab). Cryosections at 10 TUNEL staining was performed using Click‐iT Plus TUNEL assay with Alexa488  according to manufacturer's protocol.Foxc2fl/fl or Foxc2fl/fl; Pod‐Cre+/− mice and automatically counted using ImageJ software and the built‐in function of “Analyze particles”.WT1 positive cells (according to labeling with WT1 primary antibody) were identified in glomeruli from either μL of each urine sample, and BSA standards at 0, 1, 3, and 10 μg, were separated by SDS‐PAGE on a NuPAGE Novex 4–12% Bis‐Tris gel (Thermo Fisher Scientific). After electrophoresis, the gel was immersed in fixing solution (50% (v/v) methanol (Fisher Scientific), 10% (v/v) acetic acid (Acros)) for 1 h, incubated for 20 min with agitation in staining solution (0.1% (w/v) Comassie brilliant blue R‐250 (Bio‐Rad), 50% (v/v) methanol, 10% (v/v) acetic acid), and finally incubated several times in destaining solution (40% (v/v) methanol, 10% (v/v) acetic acid) until destained.Spot urine was collected and 2 μg albumin/mL urine with mg creatinine/mL urine, getting the ratio μg albumin/mg creatinine.Urinary albumin content in spot urine was assessed using Albuwell M indirect ELISA (Exocell) and urinary creatinine content was measured, using Creatinine Companion kit (Exocell). Both measurements were performed according to manufacturer′s instructions. Albumin:creatinine ratio was calculated by dividing γ (Millipore). For differentiation, IFNγ was excluded from the medium and cells were incubated at 37°C until harvesting.An immortalized podocyte cell line E11 (Cell Lines Service GmbH) , or non‐targeting ON‐TARGETplus siRNA control (GE Dharmacon), using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer′s protocol. After transfection, cells were transferred to 37°C, in medium without IFNγ, to induce differentiation until harvesting after 48 h.For knockdown experiments, cells cultured at 33°C were transfected with either Foxc2 was achieved by retroviral transduction of undifferentiated E11 cells. Full‐length coding sequence of mouse Foxc2 (NM_013519.2) were PCR‐amplified from genomic DNA (C57Bl/6) (primers used: sense 5′‐ AAAGGATCCTCTGGGACGCAGCATGCAGG‐3′ and antisense 5′‐ AAAGATATCTCAGTATTTGGTGCAGTCGTAAGA‐3′) and the PCR product was cloned in retroviral pBabe‐puro vector (gift from Hartmut Land & Jay Morgenstern & Bob Weinberg (Addgene plasmid # 1764)) (Morgenstern and Land Overexpression of mouse 7 Dynabeads (Thermo Fisher Scientific) diluted in 10 mL of PBS. The kidneys were removed, minced, and digested in collagenase (1 mg/mL collagenase A (Roche), 100 U/mL deoxyribonuclease I (QIAGEN) in PBS) at 37°C for 15 min with gentle agitation. The collagenase‐digested tissue was gently pressed through a 100‐μm cell strainer, washed with PBS and filtered through a new cell strainer without pressing, and the cell strainer was washed with PBS. The cell suspension was then centrifuged at 200g for 5 min and the cell pellet was resuspended in 2 mL of PBS. Finally, glomeruli containing Dynabeads were gathered by a magnetic particle concentrator and washed for at least three times with PBS.Glomeruli were isolated using the magnetic bead isolation procedure essentially as described , according to the manufacturer′s instructions using random primers. Expression levels of specific mRNAs were quantified by real‐time PCR, using Power SYBR green PCR Master Mix (Thermo Fisher Scientific) on a ViiA™ 7 Real Time Detection System (Thermo Fisher Scientific) and normalized to the level of Rplp0. All samples were analyzed in quadruplicate, and mean values were calculated. Primers were designed using Primer‐BLAST software  according to the manufacturer's instructions. Reverse transcription of 1 μg of total proteins were separated by SDS‐PAGE on NuPAGE 4–12% Bis–Tris protein gels (Thermo Fisher Scientific) and transferred to Immobilon‐PSQ membrane (Millipore). The membrane was blocked by incubation in blocking solution (5% (w/v) skim milk powder (Millipore) in tris‐buffered saline (TBS)). Specific proteins were detected with indicated primary antibodies , pH 8.0, 1 mmol/L EDTA (Sigma‐Aldrich), 1% (v/v) Triton X‐100 (Sigma‐Aldrich), 0.5% (w/v) sodium deoxycholate (Sigma‐Aldrich), 0.1% (w/v) SDS (Serva), 150 mmol/L NaCl (Scharlau)), supplemented with Complete protease inhibitor cocktail (Roche Life Science) according to the manufacturer's recommendations. Protein concentrations were measured using the Pierce BCA protein assay kit (Thermo Fisher Scientific) and 20 Foxc2, we have previously generated a knocked‐in mouse model where the single exon of Foxc2 is replaced by the lacZ gene, encoding β‐galactosidase, resulting in lacZ expression under control of the endogenous Foxc2 promoter , completely lacked the Foxc2fl/fl; Pod‐Cre+/− genotype  revealed typical signs of podocyte injury. Mice lacking ent Fig. B. Occasient Fig. B. Other ent Fig. B.Foxc2 deletion. Scanning electron microscopy confirmed progression of podocyte microvillus transformation, that is, the presence of microvillus‐like structures extending from the cell body of podocytes that lack Foxc2 expression , a marker for mesangial cells, was observed, whereas the staining intensity of the endothelial marker CD31 was unaffected by the loss of Foxc2 in podocytes.Since ice Fig. A. There tes Fig. B. By labtes Fig. , we coulFoxc2 in podocytes, we examined a number of proteins that previously have been associated with podocyte damage and/or with a reported link to FOXC2. No obvious difference in expression levels could be detected for any of these , and CD2AP, was altered Fig. C. InsteaNrp1) had significantly altered expression (approx. 50% lower) in glomeruli from Foxc2fl/fl; Pod‐Cre+/− mice as compared with Foxc2fl/+ mice  was conducted. The mRNA levels in isolated glomeruli was analyzed by qPCR and, from our list of candidate genes, only neuropilin 1  and older mice (14 months old) and analyzed for microalbuminuria, an indicator of podocyte injury, using ELISA. Still, there was no difference in albumin content in the urine of the different genotypes no matter what age or gender analyzed (data not shown), suggesting a minor (if any) contribution of NRP1 to podocyte function.Although the amount of Foxc2 , and died around postnatal day 10 due to severe kidney failure. Importantly, there were no indications of kidney dysfunction in terms of albuminuria until postnatal day 5. This finding is in bright contrast to a recent work by Motojima et al. Foxc2 was conditionally deleted in podocytes, using Nephrin‐Cre. In this model, no kidney phenotype was observed unless challenged with combinatorial deletion of Foxc1. It is surprising that the phenotypes are so different. Especially, since nephrin expression appears earlier than podocin during podocyte differentiation (Putaala et al. Foxc2 mRNA in glomeruli of Foxc2fl/fl; Pod‐Cre+/− mice; but unfortunately no such data were presented by Motojima et al. Hence, it is difficult to compare data from these two models since there might be differences in knockout efficiency.To study the specific role of FOXC2 in the podocyte, we generated a floxed Foxc2fl/fl; Pod‐Cre+/− mice confirmed that podocytes lacking Foxc2 develop foot process effacement, a common, but nonspecific feature of podocyte injury, as well as extensive podocyte microvillus transformation, another manifestation of podocyte damage (Patek et al. Foxc2fl/fl; Pod‐Cre+/− mice seem to develop properly since at postnatal day 3, the presence of interdigitated foot processes and normal urinary albumin concentrations could be demonstrated. Other typical signs of glomerular injury, such as thickening of the basement membrane or loss of fenestrated endothelial cells, the latter a feature of the global Foxc2 knockout mouse (Takemoto et al. Foxc2fl/fl; Pod‐Cre+/− mice; not even at day 9 when the mice suffer from end‐stage kidney failure. Additionally, in contrast to findings in the global Foxc2 knockout mice, podocyte‐specific Foxc2 deletion did not affect kidney size.Ultrastructural analysis of kidneys from Foxc2 in podocytes does not affect podocyte number, proliferation/dedifferentiation or apoptosis, conditions known to cause foot process effacement and proteinuria (Greka and Mundel Foxc2 knockout mice is the opposite effect of what was observed in the global knockout of Foxc2, where the mesangial core failed to develop properly (Takemoto et al. Loss of Foxc2 targets, we pursued a candidate gene approach based on either phenotypic similarities and/or a reported link to Foxc2. The importance for proper glomerular function have been reported for NPHS2 (Boute et al. Foxc2 knockout mouse (Takemoto et al. Foxc2 and have an established function or expression in podocytes, that is, NPHS1 (Lenkkeri et al. To investigate potential Foxc2 knockout mice when examined by immunohistochemistry. This was particularly surprising for those candidates that were affected in the global knockout of Foxc2. However, this finding shows many similarities with studies of another transcription factor, LMX1B, where proposed downstream targets derived from a global knockout model, that is, NPHS2, COL4A3, and COL4A4, could not be confirmed when conditionally deleting Lmx1b in podocytes (Suleiman et al. Unexpectedly, none of these proteins showed detectable changes in expression levels in podocyte‐specific Foxc2 in podocytes is the change in distribution of components of the foot process. Such components normally accumulate along the capillary lining, being associated with the basement membrane, but was observed in a cytoplasmic, often granular, and rim‐like pattern in the Foxc2‐depleted podocytes. A change in distribution of podocyte markers is often observed after foot process effacement (Shirato Foxc2 global knockout mice, without any detectable changes (Takemoto et al. Foxc2 expression in podocytes is crucial to maintain the architecture of foot processes.One striking feature observed after conditional deletion of  Shirato . ProteinNrp1, a transmembrane co‐receptor that can bind either VEGF or semaphorins (Gu et al. Foxc2 target in vitro. A link between these two genes has already been proposed in endothelial cells (Hayashi and Kume Foxc2. Encouraged by these findings, and the many indications that NRP1 might have an important role in podocytes and the development of proteinuria (Bondeva et al. Nrp1 in podocytes in vivo did not cause any kidney‐related effects at all. It did not even reduce glomerular mRNA levels of Nrp1, which suggests that the reduced levels that were observed in the glomeruli from Foxc2 conditional knockouts might be derived from glomerular cells other than podocytes. Increased Nrp1 expression has also been shown to lead to increased leakage of vessels (Roth et al. Nrp1 in endothelial cells is a response to decrease the leakage in the glomeruli. A recent report also shows that expression of Nrp1 in mesangial cells is vital for proper glomerular function when deleted using Pdgfrb‐Cre (Bartlett et al. Nrp1 are more likely to play a role in mesangial cells rather than podocytes.We identified Foxc2, but not Nrp1, expression is essential for proper podocyte function in vivo. We could clearly demonstrate that podocytes that are deprived from Foxc2 lose their unique architecture and hence their ability to prevent proteins from leaking into the urine. Nevertheless, despite the dramatic phenotype, it is not associated with detectable alterations in levels of important glomerular components, and previously proposed targets, including NPHS2, COL4A3, COL4A4, or Rhpn1. Further studies will therefore be needed to identify genes regulated by Foxc2 in podocytes and how Foxc2 regulates them during normal kidney physiology to maintain proper podocyte structure and function.To conclude, we show for the first time that None.Figure S1. Strategy for generation of mice with conditional‐ready floxed Foxc2 allele.Figure S2. Analysis of urinary albumin concentration in young and old mice.Click here for additional data file.


In this study, a simple HPLC method was developed and validated to determine 2,3-DMS and 3,4-DMS in rat plasma. Chromatographic separation was obtained with a reversed-phase HPLC column through a 12.5-min gradient delivery of a mixture of acetonitrile and water at the flow rate of 1.5 mL/min at 50 °C. The lower limit of quantification was 10 ng/mL. After successful validation, the pharmacokinetic profiles of 2,3-DMS and 3,4-DMS were subsequently studied in Sprague-Dawley rats. Upon single intravenous administration (4 mg/kg), 2,3-DMS had a medium volume of distribution of the central compartment (cV = 2.71 ± 0.51 L/kg), quite rapid clearance (Cl = 52.0 ± 7.0 mL/min/kg), moderate mean transit time (0→lastMTT = 131.0 ± 4.5 min) but a fairly long terminal elimination half-life (1/2tZλ = 288.9 ± 92.9 min). Interestingly, 3,4-DMS displayed a pharmacokinetic profile apparently distinct from 2,3-DMS and it had more extensive distribution (cV = 5.58 ± 1.73 L/kg), faster clearance (Cl = 143.4 ± 40.5 mL/min/kg) and shorter residence (0→lastMTT = 61.4 ± 27.1 min). Following single oral administration (10 mg/kg), 2,3-DMS had low and erratic plasma exposure (maxC = 37.5 ± 23.7 ng/mL) and poor oral bioavailability (2.22% ± 2.13%) while the oral bioavailability of 3,4-DMS was even poorer than 2,3-DMS. Clearly, the location of the methoxy groups had a significant impact on the pharmacokinetics of resveratrol analogs. This study provided useful information for the design of resveratrol derivatives in future study. The pharmacokinetic profiles of 2,3-DMS and 3,4-DMS were subsequently assessed in Sprague-Dawley rats. The information obtained in this study can be used as reference for the design of resveratrol derivatives in future study.trans-stilbene (IS) elutes at about 7.9 min, 8.3 min and 8.5 min respectively (n = 6) or pre-dosing plasma samples (n = 22) was observed  of 2,3-DMS and 3,4-DMS, an indicator of the sensitivity of the assay was 10 ng/mL. The calibration curves were all linear with regression correlation coefficients re shown . The stare shown .A simple and sensitive HPLC-UV method was developed and validated for the quantification of 2,3-DMS and 3,4-DMS in rat plasma. To our knowledge, this is the first validated assay for the quantification of 2,3-DMS and 3,4-DMS in a biological matrix.The metabolic stabilities of 2,3-DMS and 3,4-DMS were assessed in rat liver microsomes and the results are shown in As hydroxypropyl-β-cyclodextrin (HP-β-CD) increases the aqueous solubility of resveratrol and its analogs ,10,11,12i.e., a distribution phase followed by a terminal elimination phase. Therefore, the classical two-compartment first-order open model was selected to represent the intravenous pharmacokinetic profiles of 2,3-DMS and 3,4-DMS. The plasma concentration versus time data of individual rat was fitted into the model and the fitting was excellent (2R > 0.96), indicating that an appropriate model was chosen. The major pharmacokinetic parameters of 2,3-DMS and 3,4-DMS are listed in The plasma concentration-time profiles of 2,3-DMS and 3,4-DMS are shown in cV = 2.71 ± 0.51 L/kg), quite rapid clearance (Cl = 52.0 ± 7.0 mL/min/kg), moderate mean transit time (0→lastMTT = 131.0 ± 4.5 min) but a fairly long terminal elimination half-life (1/2 λZt = 288.9 ± 92.9 min). Interestingly, 3,4-DMS displayed a pharmacokinetic profile apparently distinct from 2,3-DMS and it had more extensive distribution (cV = 5.58 ± 1.7 L/kg), faster clearance (Cl = 143.4 ± 40.5 mL/min/kg) and shorter residence (0→lastMTT = 61.4 ± 27.1 min). With a LLOQ of 10 ng/mL, the plasma level of 2,3-DMS all remained quantifiable throughout our sampling period; however, 3,4-DMS dropped to a unquantifiable level in 2 out of 5 rats at 3 h post-administration and it eventually became unmeasurable at 11 h after intravenous dosing. Clearly, 3,4-DMS had much faster elimination than 2,3-DMS.Following single bolus intravenous dosing, 2,3-DMS had a medium volume of distribution of the central compartment  varied from 15 min to 5 h. Both the maximal plasma concentration (maxC) and the plasma exposure (0→lastAUC) were very limited, leading to a poor oral bioavailability (F = 2.22% ± 2.13%). The oral bioavailability of 3,4-DMS was almost nil as 3,4-DMS could only be quantified in three post-dosing samples collected from one out of six rats. Clearly, the oral bioavailability of 2,3-DMS and 3,4-DMS was problematic.The oral pharmacokinetic profile of 2,3-DMS was highly erratic b. Only 3i.e., molecular weight, clogP  in liver microsomes in the presence of NADPH, the measured oral bioavailability of 3,4-DMS was much poorer than 2,3-DMS. This may be due to extra-hepatic metabolism and more extensive tissue distribution of 3,4-DMS. The clearance of 3,4-DMS was found to be three-fold as high as rat hepatic blood flow, indicating extensive extra-hepatic elimination of 3,4-DMS. Besides hepatic metabolism, intestinal first-pass elimination is a common cause of poor oral bioavailability as well. As 3,4-DMS had a larger apparent volume of distribution of the central compartment, it had extensive tissue distribution, leading to a lower plasma level. Therefore, more plasma samples had a 3,4-DMS concentration less than LLOQ and could not be quantified. This could lead to an underestimated oral bioavailability.Using our HPLC-UV method, we managed to measure 2,3-DMS and 3,4-DMS in rat plasma at a level as low as 10 ng/mL. The quantification sensitivity could be improved if a mass spectrometry detector were applied. In a recent study, RES and its metabolites were quantified with excellent sensitivity using UPLC-MS/MS method with electrospray ionization . Howevertrans-3,5,4'-trimethoxystilbene, trans-3,5,3',4'-tetramethoxystilbene and trans-3,5,3',4',5'-pentamethoxystilbene had excellent or quite good oral bioavailability [trans-3,5-dihydroxystilbene) was much poorer than resveratrol  [trans-stilbene skeleton. However, such hypothesis needs to be explored and confirmed in future studies.In this study, both 2,3-DMS and 3,4-DMS were found to have poor oral bioavailability. Interestingly, some polymethoxystilbenes such as lability ,12,13. Ttilbene) ,17. BaseThe intravenous pharmacokinetic profiles of 2,3-DMS and 3,4-DMS were more favorable than RES as they had longer elimination half-life, slower clearance and more abundant plasma exposure . Howevertrans-stilbene (96% purity) was obtained from Sigma–Aldrich . HPLC/Spectro-grade acetonitrile was purchased from Tedia . 2-Hydroxypropyl-β-cyclodextrin (HP-β-CD) (degree of substitution about 0.6) was from Wacker . Analytical grade dimethyl sulphoxide (DMSO) was from MP Biomedicals . Milli-Q water (18.2 MΩ·cm at 25 °C) was generated by a Millipore Direct-Q® ultrapure water system  and used to prepare mobile phase and dosing vehicle. Other reagents were at least of analytical grade. Male Sprague-Dawley rat liver microsome (RLM)  and nicotinamide adenosine dinucleotide phosphate (NADPH)  were procured from BD Biosciences . NMR spectra were run on a Varian Unity Inova spectrometer operating at 499.86 (1H) and 125.70 MHz (13C) and equipped with gradient-enhanced, reverse-detection probe. Chemical shifts (δ) are indirectly referred to tetramethylsilane (TMS) using solvent signals.White powdery trans-stilbene were carried out in dimly lit environment to avoid photo-isomerization.All laboratory procedures involving manipulations of 2,3-DMS, 3,4-DMS and Oily colourless liquid 2,3-DMS and white powdery 3,4-DMS (both with purity >97%) were synthesized employing a previously published method based on an Arbuzov rearrangement followed by the Horner-Emmons-Wadsworth reactions.trans-3,4-Dimethoxystilbene: Benzyl bromide  was stirred with triethyl phosphite (7 mL) under reflux (130 °C) for 5 h. The mixture was diluted with DMF (37 mL) and was stirred with MeONa  in an ice bath (0 °C) for 30 min. Then 3,4-dimethoxybenzaldehyde  was added to the reaction flask, and the mixture was stirred at room temperature for 1 h, at 100 °C for 1 h and finally it was kept under stirring overnight at rt. 3,4-DMS  was obtained as a pure product by crystallization from methanol of the crude precipitate obtained adding a mixture of methanol-water (1:2) to the reaction mixture. 1H-NMR  δ = 7.51 , 7.36 , 7.25 , 7.07 , 6.98 , 6.87 , 3.96 , 3.91 . 13C-NMR  δ = 149.1 , 148.9 , 137.5 , 130.5 , 128.6 , 128.4 , 127.2 # , 126.8 # , 126.2 , 119.9 , 111.2 , 108.8 , 55.9 , 55.8 . Values with identical superscript (#) may be interchanged.trans-2,3-Dimethoxystilbene: Benzyl bromide  was dissolved in triethyl phosphite (7 mL) and stirred under reflux at 130 °C for 5 h. The reaction mixture was diluted in DMF (35 mL) and the solution was stirred with MeONa  at 0 °C for about 30 min. After this time, 2,3-dimethoxybenzaldehyde  was added to this solution and the mixture was stirred at room temperature for 1 h, and then heated to 100 °C for 1 h. After cooling the reaction was kept under stirring at room temperature overnight. The reaction mixture was quenched with weakly acid solution and extracted with CH2Cl2 (200 mL × 3). Finally the combined organic layers were washed with water and dried over anhydrous sodium sulphate (Na2SO4). The recovered organic phase, evaporated to dryness in vacuo, was purified by PLC using silica gel (0.063–0.200 mm) and a gradient of diethyl ether in petroleum ether (from 0% to 2%). Purification has returned 3.8709 g of pure 2,3-DMS (yield: 64%). 1H-NMR  δ = 7.64 , 7.59 * , 7.45 , 7.35 , 7.23 * , 7.14 , 6.90 , 3.96 , 3.93 . Values with identical superscript (*) maybe interchange. 13C-NMR  δ = 152.9 , 146.8 , 137.5 , 131.3 , 129.7 , 128.5 , 127.5 # , 126.5 , 123.9 # , 122.8 , 117.7 , 111.3 , 60.8 , 55.9 . Values with identical superscript (#) may be interchanged. NMR spectra are shown in 18: 250 × 4.6 mm i.d., 5 mm), protected by a guard column , was used to quantify analytes in rat liver microsome and plasma.All high performance liquid chromatography (HPLC) analyses were carried out with a Shimadzu  2010A liquid chromatography system. This HPLC comprised a quaternary gradient low-pressure mixing pump, online degasser, auto-sampler, column oven, dual-wavelength UV–Vis detector and system controller. Class-VP Version 6.12 SP1  was used to control the HPLC and perform data analysis. A reversed-phase HPLC column  was prepared by dissolving in acetonitrile and diluted to 100 ng/mL (working solution). Equal volumes of rat liver microsome (RLM) and trans-stilbene–acetonitrile working solution were mixed to quench the reaction. After vigorous vortexing for 20 s, the samples were centrifuged at 6,000 g for for 10 min at 4 °C. Rat plasma samples were processed by adding trans-stilbene–acetonitrile working solution to rat plasma in 3:1 ratio. After vigorous vortexing for 20 s, the samples were centrifuged at 10,000 g for 4 min at 4 °C. Finally, the supernatant was transferred into a glass insert that was pre-installed in a 1.5 mL auto-sampler vial. During each assay, 75 µL supernatant was injected into the HPLC-UV system.Stock solutions of analytes were prepared in DMSO to a final concentration of 1 mg/mL, stored at room temperature (24 °C) and protected from light. The internal standard (IS)  and selectivity was further confirmed by chromatographic comparison between the pre-dosing and post-dosing rat plasma samples.Selectivity of elution conditions was examined by comparing chromatograms of blank rat plasma from six different rats and rat plasma samples containing 10 ng/mL of both 2,3-DMS and 3,4-DMS and 100 ng/mL Sensitivity was represented by lower limit of quantification (LLOQ). A signal to noise ratio of 5:1 is defined as LLOQ.trans-stilbene  was adopted as analytical response. Linear regression was analyzed using GraphPad Prism 5, version 5.03 for Windows  via least sum-of-squares method, where x was concentration of 2,3-DMS, y was analytical response and a weighting factor of 1/2x was used. Calibration standards of concentrations 10, 50, 100, 250, 500, 1000, and 1500 ng/mL were measured on 5 consecutive days to access linearity. Five replicates of calibration standards were analyzed on Day 3 while duplicates of calibration standards were analyzed on other days to determine intra- and inter-day assay respectively. Similarly, quality control (QC) samples at 25, 400, 1400 ng/mL were used for assessing intra- and inter-day precision and accuracy. Precision was defined by coefficient of variation while accuracy was determined by percentage of measured concentration against the corresponding nominal concentrations. The precision and accuracy of 3,4-DMS was assessed in the same way.The ratio between peak area of 2,3-DMS and Stabilities of 2,3-DMS in stock solution were evaluated after storage at room temperature for eight days. Stability of 2,3-DMS in rat plasma under different conditions was profiled with QC samples. Freeze-thaw stability was assessed after three freeze (−80 °C)–thaw (24 °C) cycles. Short-term room temperature stability (24 °C) of 2,3-DMS in rat plasma was examined after storing such samples at room temperature for 6 h. Long-term storage stability of 2,3-DMS in rat plasma was studied after being stored at −80 °C for 10 days. Post-preparative stability study was conducted by re-analyzing samples  24 h later. The stability of 3,4-DMS was assessed in the same way. Samples were considered stable if analytical responses were within 100% ± 15% of corresponding freshly prepared samples.B and either compounds to achieve a final compound concentration of 0.6 µg/mL. Negative control was set up, using Milli-Q water in place of NADPH. Chemical stability of compound in inactivated RLM was checked using similar system with acetonitrile/Milli-Q mixture instead of phosphate buffe.RLM metabolic stabilities of 2,3-DMS and 3,4-DMS were examined by mixing Sprague-Dawley RLM with potassium phosphate buffer (pH = 7.4), NADPH regenerating solution A was added to initiate reaction. Aliquots were drawn at 0, 5, 15, 25, 35 and 45 min and quenched using ice cold trans-stilbene–acetonitrile working solution. Chemical stability was monitored at 0, 25 and 45 min. The samples were further processed for HPLC analysis as described above.Pre-incubation was done in 37 °C water bath for 5 min before NADPH x was time in minutes, y was percentage remaining with respect to 0 min and a weighting factor of 1/2y was used.To assess extent of metabolism, percentage of compound remaining at each time point with respect to 0 min was calculated. Mean and standard deviation was obtained from column statistics using GraphPad Prism 5, version 5.03 for Windows, via raw means from the six replicates of each setup. Elimination half-lives were obtained from non-linear regression via one-phase exponential decay, where This animal model has been used to investigate the pharmacokinetics of various stilbenes ,12,13,14n = 5) received single intravenous bolus dose (4 mg/kg) of 2,3-DMS and 3,4-DMS respectively. Serial blood samples were collected before dosing (0 min) and at 5, 15, 30, 60, 90, 120, 180, 300, 480 and 660 min post-dosing. Group 3 and Group 4 (n = 6) received single oral dose (10 mg/kg) of 2,3-DMS and 3,4-DMS respectively through oral gavage and serial blood samples were collected before dosing (0 min) and at 15, 30, 45, 60, 90, 120, 180, 300, 480 and 660 min post-dosing. 0.3 mL heparin-saline (10 I.U./mL) was used to flush the cannular after each intravenous injection or blood sampling to maintain its patency. Blood samples were centrifuged at 3500 g at 4 °C for 5 min and plasma was collected and stored at −80 °C.Twenty-two rats were divided into four groups. Group 1 and Group 2 (C = −α·tA·e + −β·tB·e) using a weighting factor of 1/2y [0→lastAUC)) in rats receiving intravenous administration was calculated by the linear/log trapezoidal rule while the linear trapezoidal rule was used to calculate the plasma exposure in rats receiving oral administration [0→lastCl), mean transit time (0→lastMTT) and terminal half-life (1/2 λZt) were calculated using non-compartmental analysis. Maximal plasma concentration (maxC) and the time required to reach (maxt) were from direct experimental observations. Absolute oral bioavailability (F) of 2,3-DMS was calculated through reported method [Pharmacokinetic parameters were derived using WinNonlin standard version 1.0 . Intravenous pharmacokinetic profiles of both compounds displayed typical bi-exponential decline, thus their plasma concentration-time data were fitted into classical two-compartment first-order open model  test and Fisher’s exact test were used to compare the contingency tables for number of quantifiable intravenous plasma samples and number of quantifiable oral plasma samples of both compounds respectively. For all analysis, probability level of p ≤ 0.05 represented criterion of statistical significance.All statistical calculations were calculated using GraphPad InStat, Version 3.10, 32 bit for Windows. Two-tailed unpaired t-test was used to compare intravenous pharmacokinetic parameters of both compounds. Two-tailed Chi square (χA simple and sensitive HPLC-UV method was developed and validated for the quantification of 2,3-DMS and 3,4-DMS in rat plasma. The pharmacokinetic profiles of 2,3-DMS and 3,4-DMS were assessed in Sprague-Dawley rats. The location of the methoxy groups was found to have a significant impact on the pharmacokinetics of the resveratrol analogs. The current study also suggested that single phenyl ring substitution could be associated with poor oral bioavailability and metabolic instability. The information obtained in this study will benefit the appropriate design of resveratrol derivatives in future.


Retrotransposons are transposable elements (TEs) capable of “jumping” in germ, embryonic and tumor cells and, as is now clearly established, in the neuronal lineage. Mosaic TE insertions form part of a broader landscape of somatic genome variation and hold significant potential to generate phenotypic diversity, in the brain and elsewhere. At present, the LINE-1 (L1) retrotransposon family appears to be the most active autonomous TE in most mammals, based on experimental data obtained from disease-causing L1 mutations, engineered L1 reporter systems tested in cultured cells and transgenic rodents, and single-cell genomic analyses. However, the biological consequences of almost all somatic L1 insertions identified thus far remain unknown. In this review, we briefly summarize the current state-of-the-art in the field, including estimates of L1 retrotransposition rate in neurons. We bring forward the hypothesis that an extensive subset of retrotransposition-competent L1s may be de-repressed and mobile in the soma but largely inactive in the germline. We discuss recent reports of non-canonical L1-associated sequence variants in the brain and propose that the elevated L1 DNA content reported in several neurological disorders may predominantly comprise accumulated, unintegrated L1 nucleic acids, rather than somatic L1 insertions. Finally, we consider the main objectives and obstacles going forward in elucidating the biological impact of somatic retrotransposition. Ac/Ds loci in maize . ATREX1, SAMHD1, ADAR1, RNASEH2A, RNASEH2B, RNASEH2C and IFIH1 [SAMHD1 mutations indicated that somatic L1 insertions occurred at a rate similar to that of controls [Aicardi-Goutières syndrome (AGS) is a very rare interferonopathy that provides arguably the best developed example of a neurological phenotype potentially linked to L1-associated autoimmunity. Genetic analyses of AGS patients have revealed mutations most commonly in the genes nd IFIH1 , 243. Mond IFIH1 , 244–251nd IFIH1 , 252–254nd IFIH1 . An abunnd IFIH1 , 221, whcontrols . Whilst TREX1 mutant mice with L1 reverse transcriptase inhibitors had no impact on interferon response or the retrotransposition frequency of an engineered L1 reporter gene in vivo [Intriguingly, a recent study demonstrated that media obtained from TREX1-deficient human astrocytes was toxic to healthy neurons, whereas media from TREX1-deficient astrocytes treated with L1 reverse transcriptase inhibitors was significantly less toxic . The aut in vivo . Previou in vivo  or not r in vivo  the leth in vivo .TREX1 in controlling L1 and other endogenous retrotransposons in AGS requires further study. It should however be noted that i) the somewhat opposing results detailed above for L1 were obtained using different species and cell types, ii) assays measuring engineered and endogenous L1 activity can provide different results [At this stage, the etiological role of  results , 257, ii results , 247.Bombyx mori R2 retrotransposon previously revealed that the RNA in a hybrid DNA:RNA molecule generated during TPRT could be displaced during second strand DNA synthesis without the apparent involvement of a ribonuclease [As for TREX1, RNaseH2 has been alternatively reported as being a negative or positive regulator of L1 retrotransposition , 260. Sonuclease . Ribonucnuclease . NonetheSomatic mosaicism represents an intriguing and underexplored form of genetic and biological variation in mammals. Although L1 retrotransposon-driven mosaicism is now established to occur in brain cells, any impact of this phenomenon upon normal and abnormal neurobiological processes remains undemonstrated. Despite the recent development of tools, including single-cell genome, epigenome and transcriptome sequencing , 265–272Experiments to test the impact of individual somatic L1 insertions present a major challenge. Work in this area could be greatly accelerated through: i) the development of methods to reliably survey genome structural variation and transcription, genome-wide and from the same cell, using human brain tissue obtained post-mortem, or from tissue obtained during brain surgery , 279, or


Polypyrrole nanowires/graphene (PPyNG) nanocomposites as anticorrosive fillers were prepared by in situ polymerization in order to improve the anticorrosion performance of waterborne epoxy coatings. Field emission scanning electron microscope (FESEM) and Fourier transform infrared spectroscopy (FTIR) characterized the morphologies and structures of the synthesized PPyNG. The polypyrrole nanowires with about 50 nm in diameter were obtained. Conjugation length of PPy was increased with the addition of graphene. Open circuit potential (OCP) measurements, Tafel polarization curves, and electrochemical impedance spectroscopy (EIS) using an electrochemical workstation evaluated the anticorrosion properties of the waterborne epoxy/PPyNG coatings (EPPyNG). The studied nanocomposite coating possessed superior corrosion protection performance when the graphene content of the filler was 2 wt %. Its corrosion rate was about 100 times lower than that of neat epoxy coating. The higher barrier properties of nanocomposite coating and passivation effect of polypyrrole nanowires were beneficial in corrosion protection. Metal corrosion is a disturbing phenomenon in which chemical or electrochemical reactions damage metal materials. It would not only result in economic losses, but also threaten the safety of industrial production. Many methods for corrosion protection have been developed, such as environmental modification, anodic protection, cathodic protection, protective coatings, and corrosion inhibitors, or any combination thereof ,2,3. AmoICPs have attracted tremendous attention due to a wide range of potential applications in sensors, supercapacitor electrodes, biological industries, and corrosion protection ,11. In tDespite these advantages, the aggregation tendency of PPy particles is a major restriction of the processing and application of epoxy/PPy coating. Integration with montmorillonite and graphene turns out to be an effective method for avoiding aggregations and improve dispersion of PPy in the polymer matrix, which contributes to a great corrosion protection enhancement ,19,20. GPPy with one-dimensional nanostructure, such as nanotube , nanorod4), sulfuric acid (H2SO4), hydrogen peroxide (H2O2), N-methyl pyrrolidone (NMP), Sodium chloride (NaCl), and cetyltrimethylammonium bromide (CTAB) were obtained from Sinopharm Chemical Reagent Co., Ltd. . The flake graphite powder (325 mesh) was purchased from Sigma-Aldrich Chemicals . All of the chemicals were of analytical reagent grade and used without further purification. Hexion Inc.  provided waterborne epoxy resin (Epikote 6520-WH-53) and curing agent (Epikur 8538-Y-68). The Q235 mild steels  with area of 42 mm × 10 mm were purchased from Biuged Laboratory Instruments (Guangzhou) Co., Ltd. . The mild steels were polished while using 400 grift sand papers and then cleaned in ethanol and acetone.The pyrrole monomer was purchased from Aladdin Bio-Chem Technology Co., Ltd. . Ammonium persulfate (APS), potassium permanganate (KMnO4 (1 wt % equiv.) was added in batches into concentrated H2SO4  over a period of 45 min. in an ice-water bath. 100 g graphite  was then added in batches under stirring at 35 °C for 2 h. The black flakes were filtered through a 200-mesh sieve and poured into 2 L of ice water after reaction. Subsequently, 30 wt % H2O2 was added to decompose the insoluble manganese dioxide. The wet powders of pretreated graphite were obtained after filtering and washing. Pretreated graphite was then dispersed in the alkali water (pH = 14) under ultrasonication while using a sonic vibra-cell VC505 processor in 60% power for 1 h, resulting in black graphene slurry. The graphene slurry was centrifuged at 10,000 rpm for 10 min. and then repeatedly washed by a large amount of water (4–6 times) until pH approached 10. The prepared graphene was re-dispersed in N-methyl pyrrolidone (NMP) for further use.Graphene was prepared by following the method by Dong et al. . In brieThe PPy nanowires/graphene nanocomposites (PPyNG) were synthesized through in situ chemical oxidative polymerization. In a typical process, 0.31 mL pyrrole monomer and 0.91 g CTAB were added in a mixture of 125 mL 0.2 M hydrochloric acid and graphene-NMP dispersion and stirred for 2 h at ambient temperature, and then cooled to 0–5 °C. A precooled aqueous solution of 10 mL 0.2 M HCl containing 4 mmol APS was added into above solution in batches. The reaction was allowed to proceed under stirring for 4 h at about 0–5 °C. The resulting product was filtered and rinsed with deionized water and ethanol (4–6 times) until the filtrate was colorless. Finally, the product was dried in a vacuum oven at 40 °C for 24 h. The weight ratio of pyrrole to graphene varied as 99:1, 98:2, 97:3, and the resulting black composites were named as PPyNG1, PPyNG2, and PPyNG3, respectively. For comparison, neat PPy nanowires (PPyN) were fabricated by similar method absence of graphene dispersion.The investigated coatings in this study consisted of the epoxy without the addition of filler as well as with the incorporation of PPy nanowires and PPy nanowires/graphene as the functional additives. Three weight concentrations of PPy nanowires additives had been prepared in order to achieve the optimal level of additives, and their anticorrosion properties are shown in 2 area, and a working electrode. The bare mild steels or mild steels with coatings were sealed while using sealant (paraffin: Rosin = 1:1) and Teflon to leave 1 cm2 area opening to the electrolytic solution. The electrolyte was 3.5 wt % NaCl solution. Open-circuit potential (OCP) was recorded for the blank epoxy, EPPyN and EPPyNG coatings up to 30 days of immersion time. The potential dynamic polarization curves of blank epoxy, EPPyN, and EPPyNG coatings were performed with a sweep rate of 2 mV/s from the cathodic direction to the anodic direction. The electrochemical impedance spectroscopy (EIS) measurements were collected in the frequency range of 100 kHz to 0.01 Hz while using an alternating current signal with the amplitude of 5 mV. All of the electrochemical tests were conducted at room temperature. A ZQ-401 microscope was used to record the optical microscopic images of EPPyN coatings .The Fourier transform infrared spectrometer (FTIR) spectra of PPyNG films were collected by a Bruker-Veretex70 spectrometer  while using KBr pellets. The scanning electron microscopy (SEM) images of PPyN and PPyNG composites were obtained while using a Zeiss Sigma FE-SEM . The electrochemical measurements were carried out to characterize the anticorrosive properties of blank epoxy, EPPyN and EPPyNG coatings using a CHI 660E electrochemical workstation  that was equipped with a conventional three-electrode cell with a saturated calomel electrode (SCE) as reference, a platinum counter electrode with 1 cm−1 arose from N–H stretching vibrations [−1 were due to the asymmetric and symmetric ring-stretching modes, respectively [−1 were attributed to C–H deformation vibrations and C–N stretching vibrations, respectively [−1 were assigned to the doping states of PPy [−1, which was probably due to the π–π interactions between graphene layers and aromatic polypyrrole rings [−1 and oxidization state sensitive 1558 cm−1 band (I1478/I1558) could be used to calculate the conjugation length [brations . The chaectively . The banectively . In addis of PPy . When cole rings . Moreoven length ,36. The n length ,37.Icorr), corrosion potential (Ecorr), anodic Tafel slope (ba), and cathodic Tafel slope (bc) were estimated from the Tafel extrapolation of anodic and cathodic lines to the point of intersection. The more negative Ecorr and larger Icorr suggested a faster corrosion rate, while the more positive Ecorr and the smaller Icorr indicated a slower corrosion process [Ecorr of the neat epoxy resin coated mild steel was −784 mV. A significantly positive shift Ecorr of −565 mV was obtained for the composites coating with PPyN, confirming that EPPyN engaged in redox reactions, resulting in the formation of metal oxide passive layer [Ecorr dramatically increased to −537 mV with 1 wt % graphene and −482 mV with 2 wt % graphene. Advanced corrosion protection effect of EPPyNG coating compared to EPPyN might arose from dispersing graphene nanosheets to increase the tortuosity of the diffusion pathway of H2O, O2, and Cl− [Ecorr than EPPyNG2, probably because larger fraction of graphene affected the growth of PPy nanowires and induced the defects in PPyNG3 composites [ba/bc ratio of EPPyNG2 was observed, which revealed a reduction of the anodic dissolution [Icorr values of EPPyNG1 and EPPyNG2 coated mild steel considerably decreased when compared with EPPyN. For the EPPyNG2 coatings, the Icorr was lowest than other coatings, which indicated the best anticorrosion performance in all of the studied samples, which corresponded to results of OCP tests.The potentiodynamic polarization tests were carried out to study the anticorrosion performance of EPPyNG composites coatings.  process . The Ecove layer ,42. By a and Cl− . Howevermposites , which wsolution . The Icovcorr (mm/year) was obtained from Equation (1) [M) is 55.85 g/mol for Q235, Icorr is the corrosion current density (A/cm2), n is 2 for the oxidation of steel, and the density (ρ) is 7.85 g/cm3 for Q235, 3270 is a constant. Rp is the polarization resistance calculated by the slope of the polarization curve at the Ecorr according to the Stern–Geary Equation (2) [The quantitative analysis of Tafel curve was also investigated, and tion (1) :(1)vcorrtion (2) :(2)Rp=baba and bc are the anodic and cathodic Tafel slopes, Icorr is the corrosion current density. The protection efficiency  was calculated via Equation (3) [pR(bare) and pR(coated) denote the polarization resistance of bare and coated steel, respectively. Here, tion (3) :(3)PE=Rpvcorr value of 8.9 × 10−5 mm/year for EPPyNG2 was about 100 times lower than that for blank epoxy coating. The result of vcorr demonstrated that the EPPyNG coatings displayed higher anticorrosion performance than epoxy coating with PPyN alone, and the best performance was achieved when the graphene-doped ratio was 2 wt %. The mild steel that was coated with EPPyNG2 exhibited a Rp value of 5.6 × 106 Ωcm2, which was higher than that of bare mild steel, neat epoxy, EPPyN, EPPyNG1, and EPPyNG3 coated ones. In the case of protection efficiency, the highest PE value from EPPyNG2 (99.9%) described that PPyNG2 loading in the epoxy coating can provide superior inhibition corrosion performance.The calculated EIS is a powerful tool for investigating the corrosion protection of the coatings . Figure 0.01Hz) was used as a semi-quantitative standard of coating’s protective performance for the Bode patterns [0.01Hz of EPPyNG2 . The EPPyNG2 coating had better barrier properties when compared to neat epoxy, because the well dispersed PPyNG could fill the structural and pinhole porosity of neat epoxy and then inhibited the water penetration. The conjugation length of EPPyNG2 was longer than those of EPPyN and EPPyNG1, which resulted in electrons easier delocalization and in favor of the formation of passivation layers [6 Ωcm2) and EPPyNG1 (5.4 × 106 Ωcm2). Although EPPyNG3 possessed longer conjugation length, its excess graphene content might increase the defects due to influence of polypyrrole nanowires, and probably generated micro galvanic corrosion that showed the corrosion-promotion activity [6 Ωcm2) displayed worse anticorrosive property when compared with EPPyNG2. In addition, the impedance modulus of EPPyNG2 remained 1.2 × 105 Ωcm2 after 30 days, which was higher than other coatings.The impedance modulus at low frequency  and double-layer (steel/solution) capacitance (Cdl), respectively. Rpore modeled ionically conducting paths across the coating, which could be used to evaluate the barrier performance of the coatings [Moreover, ZSimpWin software further fitted the EIS measurements while using the equivalent electric circuits, as shown in coatings . Models Rpore and Cc on the basis of the above fitting models. Generally, a higher Rpore and lower Cc suggested that a small amount of corrosive media penetrated into the coatings [Rpore of neat epoxy coating gradually decreased from 7.3 × 105 Ωcm2 to a much lower value (1.3 × 104 Ωcm2). The Rpore of coatings with EPPyN, EPPyNG1, and EPPyNG3 decreased to some extent, while the EPPyNG2 coating always maintained much higher values. The Cc of pure epoxy coating continuously increased from 3.1 × 10−9 F/cm2 to 1.1 × 10−8 F/cm2. In contrast, the EPPyNG2 displayed a much lower Cc value when compared to those of neat epoxy and EPPyN coating. The EPPyNG3 coating also exhibited lower Rpore and higher Cc than EPPyNG2, which confirmed that excess graphene induced the defect coating and reduced corrosion protection. Consequently, it could be concluded that the EPPyNG2 composite coating showed the outstanding corrosion resistance, which was in agreement with the potentiodynamic polarization results.coatings . It coul2O, O2, and Cl-, etc.) could penetrate the coating easily due to the minute crevices of the surface. However, the well-dispersed PPyNG composite, as an anti-corrosion barrier, repaired the cracks of epoxy coating, and enhanced the tortuosity of the diffusion pathway at a great extent. Furthermore, PPyN reacted with steel to form a dense layer of passive oxide film, which resulted in substantially reduced penetration of corrosive medias. Hence, the anti-corrosion performance of the EPPyNG nanocomposite coatings was significantly improved.According to the above results, Rpore and Cc were used to study the anticorrosion performance of the coatings. The PPy nanowires/graphene nanocomposite coating exhibited improved anticorrosion performance in comparison with pure epoxy and epoxy/PPy nanowires samples. The passivation effect of the PPy nanowires contributed to the effective inhibiting corrosive effect. Meanwhile, the well-dispersed PPyNG could block the coating pores and decrease the corrosive medias diffusion toward the substrate. Therefore, PPy nanowires/graphene nanocomposite would be used as a promising anticorrosion pigment.In this study, the PPy nanowires were synthesized while using CTAB as soft template, and combined with graphene nanosheets by in situ oxidation polymerization. The incorporation of PPy nanowires/graphene into waterborne epoxy on mild steel substrate was performed. Potentiodynamic polarization plots, impedance measurements, and fitted


Recently, graphene has been extensively researched in fundamental science and engineering fields and has been developed for various electronic applications in emerging technologies owing to its outstanding material properties, including superior electronic, thermal, optical and mechanical properties. Thus, graphene has enabled substantial progress in the development of the current electronic systems. Here, we introduce the most important electronic and thermal properties of graphene, including its high conductivity, quantum Hall effect, Dirac fermions, high Seebeck coefficient and thermoelectric effects. We also present up-to-date graphene-based applications: optical devices, electronic and thermal sensors, and energy management systems. These applications pave the way for advanced biomedical engineering, reliable human therapy, and environmental protection. In this review, we show that the development of graphene suggests substantial improvements in current electronic technologies and applications in healthcare systems. Although graphene has been theoretically studied for several decades ,2,3, freD carbon −1 K−1 ,16,17. M−1 K−1 . Thus, g−1 K−1 , optical−1 K−1 , high ro−1 K−1 . Graphen−1 K−1 ,25,26,27The remarkable electronic and optical properties observed for graphene crystallites are the primary reasons for the exclusive focus of experimental and theoretical efforts on graphene while ignoring the existence of other 2D materials. The electrons in graphene have long mean free paths without disrupting the electron-electron interactions and disorder. Therefore, the properties of graphene differ from those of other common metals and semiconductors associated with the physical structures and electronic properties. K and K’ at the edge of the graphene Brillouin zone (BZ), called Dirac points, are essential for the physics of graphene. Their positions in momentum space can be expressed asThe graphene electronic structure can be predicted by graphene simulation, especially using density functional theory (DFT). The adsorption energy of graphene and other materials , density of states (DOS), geometry, work function and dipole moment of each adatom-graphene system are studied and calculated using DFT ,32,33,342 V−1 s−1 at ambient conditions and can be continually switched between electrons and holes in concentrations n of 1013 cm−2 [13 cm−2) in electrically and chemically doped devices.As shown in 013 cm−2 ,5,6,7. TN is an integer. The first plateau occurs at The QHE is another factor indicating the system’s outstanding electronic quality. Because the temperature range of the QHE for graphene is 10 times broader than that of other 2D materials, the QHE in graphene can be seen at room temperature. The unique reaction of massless fermions in a magnetic field is made more evident by their behavior at the high-field limit, where Shubnikov-de Haas oscillations (SdHOs) progress to the QHE . In Figusequence ,37,38,39sequence ,7,40. Thc is the speed of light) [T and reflectance R are also universal  . This paom thick . The unit light) ,42. AddiThe ability of photodetectors to detect broad spectral ranges of light with high responsivity and fast photodetection is critical in optoelectronic applications ranging from sensing, imaging, and spectroscopy to communications. Therefore, numerous studies have reported the use of traditional semiconductors for broader ranges of photodetection, even in the mid-infrared to far-infrared regime. As a result, narrow-bandgap semiconductor compounds such as indium antimonide (InSb), mercury cadmium telluride (HgCdTe), lead sulfide (PbS), and lead selenide (PbSe) are now widely applied in mid-infrared or far-infrared photodetectors ,44,45. H2O5/graphene heterostructure photodetector shows a remarkably high responsivity of ~1000 A W−1 under a low excitation power (~10−9 W) of 532 nm laser light at a 1 V source-drain bias voltage. Additionally, the authors fabricated a similar graphene/6-nm-thick intrinsic silicon/graphene heterostructure device with improved photoresponsivity in the infrared regime. The intrinsic-silicon-incorporated photodetector shows high responsivity in a broadband regime ranging from near-infrared (4 A W−1 at 1.3 μm light and 1.9 A W−1 at 2.1 μm light) to mid-infrared light (1.1 A W−1 at 3.2 μm). Yu et al. fabricated a mid-infrared photodetector by hybridizing graphene with an innovative narrow-bandgap semiconductor, titanium sesquioxide (Ti2O3) nanoparticles [2O3 nanoparticle photodetector, with a high responsivity of ~300 A W−1 and high detectivity of ~7 × 108 cm Hz1/2 W−1 at room temperature. Furthermore, a high responsivity of ~120 A W−1 can be achieved from the photodetector over a wide range of wavelengths from 4.5 μm to 10 μm  to near-infrared light (1550 nm) under an illuminated light intensity of 15 mW/ cm2. Fang et al. proposed a graphene photodetector with a gold snowflake-like fractal metasurface design [Vfractal and Vedge, respectively). The results showed that photovoltage enhancement factors  of 8 to 13 over the visible-light range (476 to 647 nm) can be achieved. Cakmakyapan et al. fabricated a photodetector with gold-patched graphene nanostripes showing ultrafast photodetection and high responsivity [−1 to 8 A W−1. Additionally, the photodetector shows an ultrafast photodetection speed exceeding 50 GHz. Liu et al. developed photodetectors consisting of a thin tunnel barrier sandwiched between two graphene layers . The graarticles . The schto 10 μm b. Figuree design . The autd light 10 nm undeSolid-state light-emitting diodes (LEDs) are currently widely used in the semiconductor industry  due to t−2 by transferring ultraclean and damage-free graphene supported by rosin (C19H29COOH); −1 and power efficiency of 102.6 lm W−1, exceeding the performance of ITO and poly(methyl) methacrylate (PMMA)-transferred graphene OLEDs  to red (~750 nm) composed of semireduced graphene oxide sandwiched by graphene oxide (GO) and reduced graphene oxide (RGO) [−1 and 6000 cd m−2 under a 12 V bias voltage and a 0.1 A drive current and blue light at 0.67 lm W−1 and 800 cd m−2 under a 16.5 V bias voltage and a 0.1 A drive current. This experiment enabled in situ wavelength tuning by controlling the bias voltage and doping level. Huang et al. introduced transparent LEDs based on graphene-encapsulated Cu NWs [2-day, 74% less than that of bare PET. Thus, A-2GE showed high optoelectronic properties and strong antioxidant stability and can be used for TCEs for LEDs. Zhang et al. fabricated four-inch flexible organic light-emitting diodes (OLEDs) showing a high brightness of ~10,000 cd mne OLEDs e. In addectrodes . This stde (RGO) . The LEDd Cu NWs . Cu NWs d Cu NWs ,80. Howed Cu NWs . High-quSolar cells based on organic-inorganic perovskite materials have recently been widely studied due to their high power conversion efficiencies (PCEs), long-range carrier diffusion length, intense light absorption, and facile fabrication (low cost and low temperature) ,87,88,89± 0.35% and 12.02 ± 0.32% from the graphene and fluorine-doped tin oxide (FTO) sides, respectively, with optimized conditions of double-layer graphene and an ~70 mg mL−1 2,2′,7,7′-tetrakis--9,9′-spirobifluorine (spiro-OMeTAD) solution due to the low surface roughness. Sung et al. first fabricated PSCs with over 17% efficiency by exploiting graphene as a transparent conducting anode [3) controlled the contact angle between poly:poly(styrene sulfonate) (PEDOT:PSS) and the graphene surface and helped successfully form PEDOT:PSS/MAPbI3 layers. The thickness of the MoO3 layer affected the PCE of the PSCs. In the experiment, a 1 nm-thick MoO3 layer with graphene-based devices showed a maximum PCEs of 17.1%, larger than 90% of those of ITO-based devices (18.8%). The flexibility and high PCE of this graphene-based device pave the way for the further development of flexible solar cells. Najafi et al. fabricated a PSC with a high PCE of over 20% using MoS2 QDs and a functionalized reduced graphene oxide (f-RGO) hybrid [2 QDs:f-RGO hybrids are used as both the HTL and active buffer layer (ABL), and a schematic of the PSC is shown in 2 (cTiO2)/mesoporous TiO2 (mTiO2)/MAPbI3/f-RGO:MoS2/spiro-OMeTAD/Au. The I-V characteristics of MoS2 QDs, f-RGO, and MoS2 QDs:f-RGO as ABLs are shown in 2 QDs:f-RGO showed a further improved performance, with a maximum PCE of 20.12% and a fill factor (FF) of 79.75%, compared to the reference device showing a maximum PCE of 16.85% and an FF of 76.9%. Agresti et al. fabricated a large-area PSC of 50.56 cm2 with a PCE of 12.6% by doping the mTiO2 layer with graphene flakes and inserting lithium-neutralized graphene oxide flakes (GO-Li) between the interface of the mTiO2 and perovskite [2 is improved by the GO-Li layer, and the graphene flakes dispersed in the mTiO2 layer help speed up the charge dynamic at the electrode. The interface engineering of PSCs by GRMs leads to an increased PCE, from 11.6% for the reference PSC to 12.6% for the PSC with GO-Li and mTiO2. Li et al. proposed a PSC with an MAI perovskite layer incorporating graphene nanofibers [You et al. proposed semitransparent PSCs with top graphene electrodes . The fabng anode . The prerovskite . The chanofibers . Integra−1 and mechanical durability and reliability (repeated tests of 6000 cycles). Shin et al. paved the way to use pressure sensors for diverse application areas, such as medical diagnosis, robotics and automatic electronics [Graphene can be utilized in various electronic sensors due to its unique device properties. Recently, novel integrated electronic circuitries with tactile pressure sensors based on piezoelectricity ,108, piectronics . The aut−2) than that of the CNT-Au electrode sensors. Additionally, thanks to the outstanding properties of graphene and CNT, the device shows a high optical transparency (over 80% at 550 nm) and remarkable mechanical flexibility (bending radius of 5.5 mm) with high electrical reliability. Goossens et al. reported the monolithic integration of graphene with a complementary metal–oxide–semiconductor (CMOS)-integrated circuit operating as a high-mobility phototransistor [For the true integration of woven electronics and/or optoelectronics into textiles, the direct fabrication of the device on the fiber itself with high-performance materials allowing easy unification into fabrics is required ,119. Aloansistor . BroadbaResearch on the safe and accurate monitoring of human body signals, hazardous disease detection, and stimulation systems has been highly focused in areas ranging from biological research to clinical medicine ,131,132.For early and reliable clinical cancer diagnosis and treatments, cost-efficient, dependable and sensitive monitoring and detecting systems are crucial. Wu et al. fabricated a functionalized graphene-based electrochemical sensor array system for cell sensing . The gra2), tryptophan (Trp), and uric acid (UA) [2, Trp, and UA of 1.58, 0.06, 6.45, 0.10, and 0.09 μM, respectively (S/N = 3)) with differential pulse voltammetry (DPV). As predicted by the World Health Organization (WHO), the number of diabetes patients has been gradually increasing [Detecting and monitoring various electrolytes, such as biological, organic, and inorganic electrolytes, and electroactive materials in human body fluids are important for clinical diagnosis and analytical applications ,144,145.cid (UA) . A CVD mcreasing , and succreasing ,149,150.creasing . The autThe most important consideration for systems biology and personalized and precision medicine is an acceptable affinity and binding efficiency of deoxyribonucleic acid (DNA) hybridization and the ability to distinguish DNA sequences with single-nucleotide substitutions. Xu et al. suggested next-generation multichannel graphene-based DNA sensors for a cost-effective, fast, simple and label-free biosensing system to perform kinetic studies  . ReproduA provisional WHO guideline has established a concentration limit of microcystin-LR (MC-LR) of 1 μg/L in drinking water  because 3-functionalized RGOH displays 118.6 and 58.9 times higher responses than its unmodified RGOH counterpart for NO3 and NH3, respectively. Moreover, the response increases monotonically from 6.1% at 200 ppb NO2 to 22.5% at 2 ppm NO2. Guo et al. fabricated a NO2 gas sensor from the room-temperature reduction of GO via two-beam-laser interference (TBLI) [2 and accelerated the response/recovery rates. For 20 ppm NO2, the response (Ra/Rg) of the sensor based on RGO hierarchical nanostructures is 1.27, which is higher than those of GO (1.06) and thermally reduced RGO (1.04). The response time and recovery time of the sensor based on laser-reduced RGO are 10 s and 7 s, respectively, which are much shorter than those of GO (34 s and 45 s). With fast-growing industrial development, toxic gases endangering human beings have been extensively produced and emitted ,159. Thee (TBLI) . The fab2), it can be exploited in various applications [2 concentrations is essential. Sharma et al. produced an FET hydrogen sensor integrated with a graphene-Pd-Ag-gate FET (GPA-FET) [Because of the significant properties (high heat of combustion and low minimum ignition energy) of hydrogen  . The GPAMany studies have reported the integration of graphene-based mobile gas sensors with modern technologies such as smart phones, cloud computing and the Internet of Things (IoT) with graphene nanoribbons due to the high electrical conductivity of the nanoribbons. The first monolithically integrated CMOS-monolayer graphene gas sensor was developed by Zanjani et al., with a minimal number of post-CMOS processing steps, to realize a gas sensor platform that integrates the higher gas sensitivity of monolayer graphene with the low power consumption and cost advantages of a silicon CMOS platform . FurtherC, is a distinct characteristic of a material expressing the change in the energy density U with respect to the change in temperature (1 K or 1 °C), represented as C = dU/dT, with units of joules per kelvin per unit mass, per unit volume, or per mole. The thermal time constant of a material, which indicates how quickly the material heats or cools, is determined by the specific heat. The thermal time constant is expressed as τ ≈ RCV, where V is the volume of the material and R is the thermal resistance for heat dissipation [C = Cph + Cel. However, the contribution of phonons dominates the specific heat of graphene at all temperatures [Cph ≈ 0.7 J g−1 K−1, which is approximately 30% higher than that of diamond due to the higher density of states (DOS) at low phonon frequencies caused by the weak coupling of graphite layers. For a graphene sheet at room temperature, a similar result is expected, but the specific heat can be altered when graphene interfaces with a substrate  [The specific heat, sipation . As the sipation ,170,171.eratures . In addieratures . The speulators) . K is explained by Fourier’s law, q is the heat flux per unit area; and ν is the average phonon group velocity and λ is the mean free path. Therefore, as the specific heat of graphene is dominated by phonon transport, the thermal conductivity is also dominated by phonon transport [The thermal conductivity , q=−K∇T . In thisransport .K of pyrolytic graphite is approximately 2000 W m−1 K−1 at room temperature, and the cross-plane K at room temperature is more than two orders of magnitude smaller. Because HOPG is composed of well-aligned large crystallites, the overall behavior is analogous to that of a single crystal; this fact explains the difference in K [K reaches maximum values at approximately 100 K and 70 K, respectively. In amorphous carbon, K ranges from 0.01 W m−1 K−1 to 2 W m−1 K−1 at 4 K and 500 K, respectively. Similarly, carbon allotropes have different thermal conductivities depending on the temperature. The difference is attributed to the grain size and quality of graphite and the phonon DOS, as indicated by Cph. Here, the thermal conductivity of various carbon allotropes, including two types of pyrolytic graphite (in-plane and cross-plane), diamond, and amorphous carbon, is presented in nce in K . Heat is2 substrates by reactive ion etching (RIE), and graphene was suspended over the trenches. The depth of the trenches was 300 nm, and the width varied from 2 to 5 μm [A method to measure the thermal conductivity of graphene by exploiting confocal micro-Raman spectroscopy is introduced in  to 5 μm . An opti to 5 μm . The theD ≈ −1.29 cm−1mW−1, where PD is the total dissipated power and the temperature coefficient −1 K−1 is obtained for freely suspended graphenes; this value is the highest of any known material [As shown in 2 cm−1/K . When thmaterial ,16,17. Rmaterial , detect material  and achimaterial . −1 [−1 at room temperature. It was theoretically verified that TEP behaves as n0) but is saturated at low densities. The values of the Seebeck coefficient range from ~100 to ~10 μV K−1 for temperatures ranging from ~100 to ~300 K. The Seebeck coefficient  of the efficiency of thermoelectric energy conversion is defined as ZT due to suppressed phonon scattering, which leads to decreased K (ZT for graphene suggests the possibility of its use in energy-harvesting applications. The ZT of various graphene nanostructures is very promising for the improvement of the thermoelectric energy conversion [The thermoelectric power (TEP) is the voltage induced by a temperature gradient. Experimental studies indicated that graphene has a TEP of ~ 50 to 100 μV K−1 . In additriguing . The figuctivity . GraphenKph+Kel) ,188. Thenversion . Electronic skins (E-skins) can generate electronic signals from external stimulation for use as human-machine interfaces (HMIs) and multifunctional smart skins ,193,194.2)/Si substrate, a silicon nitride (SiNx) membrane, and a Si wafer with etched rectangular boxes (10 × 20 μm2) [−4) and electron-phonon scattering. The graphene-based sensor fabricated on a SiNx membrane has a substantially faster response, better sensitivity because of the low thermal mass, and better mechanical stability than other suspended graphene sensors. As a result, a temperature sensor comprising the suggested SLG on a SiNx membrane can be exploited for various highly sensitive and fast applications. Single-layer graphene (SLG) presents impressive thermal properties, so graphene can be used for thermal management and temperature sensing applications. Davaji et al. fabricated and compared micropatterned SLGs on a silicon dioxide (SiO 20 μm2) . These s−1 (700|V|W−1) at room temperature) and low noise-equivalent power (below 1100|pW|Hz−1/2 (20|pW|Hz−1/2)) [Graphene can be utilized as not only a temperature sensor but also a sensor of other crucial parameters. Though terahertz radiation has been used in various applications ranging from security to medicine , the senHz−1/2)) . The comHz−1/2)) ,205, andHz−1/2)) ,206. EleHz−1/2)) ,208 or dHz−1/2))  through Hz−1/2)) , and theHz−1/2)) . AdditioHz−1/2)) ,213,214.Hz−1/2)) ,217,218.Hz−1/2)) ,220 was Hz−1/2)) . The detRecently, studies aiming to realize theranostic systems with simultaneous detection and therapeutic services in one single nanostructure have increased in the field of personalized nanomedicine and clinical biomedical applications ,223,224.−1 K−1, and low cost [2, respectively. The CHF of the hybrid graphene/SWCNT heater showed the greatest improvement of 18.2% compared to that of the bare ITO heater. As shown in 2 K, respectively. The HTC of the hybrid graphene/SWCNT heater improved by 55% compared to that of the bare ITO heater. These improvements are attributed to the deposition of SWCNTs on the graphene, which compensates the disconnected areas and wrinkles of the graphene. Han et al. introduced a high-performance heat spreader to manage excessive heat [−1 K−1 [−2, the temperature of the hotspot decreased by 17 °C with a GBP deposited on nonfunctionalized GO. In addition, 3-amino-propyltriethoxysilane (APTES) functionalization resulted in an additional decrease in temperature of 11 °C, or a total decrease of 28 °C. Bernal et al. proposed edge-functionalized graphene nanoplatelets (GnPs) to improve the thermal conductivity [−1 K−1, with grain sizes ranging from ~200 nm to 10 μm. The thermal conductivity of graphene was found to be significantly decreased with the decreasing grain size by a small thermal boundary conductance of ~3.8 × 109 W m−2 K−1. Ding et al. proposed graphene nanosheets (GNSs) exhibiting high dispersibility in water and thermal conductivity [−1 K−1. Efficient heat management systems have become extremely important in various fields, such as electronic, optoelectronic, and thermoelectric applications, with the fast-growing development of state-of-the-art technologies to transport heat to the field environment; a high power heating density is required, or excessive heat must be prevented from deteriorating the device reliability, lifetime, and performance ,235. In low cost ,237. Howlow cost . Therefolow cost . The autive heat . Graphen [−1 K−1 . With a uctivity . The diauctivity . Segregauctivity . Graphit2σT/(Kph + Kel); thus, enhancing the Seebeck coefficient and electrical conductivity and reducing the thermal conductivity are essential to achieving a higher efficiency. Cho et al. proposed multidimensional nanomaterials based on organic thin films exhibiting a high thermoelectric power factor at room temperature [5 S m−1 and 120 μV K−1 at 80 QLs, respectively. These results led to a high value of the thermoelectric power factor (PF) (~2710 μW m−1 K−2), where PF = S2σ. Ma et al. doped bromine (Br) onto graphene fibers to enhance the thermoelectric properties [−3 and room-temperature PF of 624 μW m−1 K−2. These values are better than those of materials composed of solely graphene and CNTs. Lin et al. observed the enhanced thermoelectric effect of lanthanum strontium titanium oxide (LSTO) upon adding graphene [Currently, with the rapid development of industry, the energy consumption of mainly fossil fuels is causing dramatic depletion worldwide . Additioperature . The thioperties ,250. Addoperties ,252. As graphene . The addIn this paper, we presented a review of the electronic and thermal properties of graphene and its up-to-date applications. Graphene has exceptional physical characteristics compared to other usual metals and semiconductors and has the potential for extensive applications. The ambipolar electric field effect in SLG proves that charge carriers have higher mobilities than those of semiconductors. Additionally, the QHE, the unique phenomenon observed with LL, is explained by graphs and unique geometrical phases. Using the experimental results of graphene crystal samples, the visual transparency of graphene was characterized by the fine-structure constant α. In addition to its electronic features, the superior thermal properties of graphene were reviewed. The specific heats of graphene and graphite were derived, and the thermal conductivity of carbon allotropes containing graphene was explained. Graphene has high thermoelectric power, which suggests its potential for use in energy-harvesting applications. These remarkable properties of graphene can be incorporated into versatile systems in various application fields: optical devices, electronic and thermal sensors and energy management systems. Here, state-of-the-art examples based on graphene were presented. Despite the development of many graphene-based electronic systems with unique properties, challenges and problems related to the development of materials for commercial electronics still exist and need to be studied further. By resolving such challenges and problems, graphene will be in the limelight as an active material for future electronics.


To investigate if animal-assisted therapy (AAT) leads to higher consciousness in patients in a minimally conscious state during a therapy session, measured via behavioral reactions, heart rate and heart rate variability.In a randomized two treatment multi-period crossover trial, 10 patients in a minimally conscious state participated in eight AAT sessions and eight paralleled conventional therapy sessions, leading to 78 AAT and 73 analyzed control sessions. Patients’ responses during sessions were assessed via behavioral video coding and the Basler Vegetative State Assessment (BAVESTA), heart rate and heart rate variability . Data were analyzed with generalized linear mixed models.p < 0.001) and active movements per tactile input during AAT compared to control sessions . No difference was found for positive emotions. With BAVESTA, patients’ overall behavioral reactions were rated higher during AAT . AAT led to significantly higher LF  and lower HF , while heart rate, SDNN, RMSSD did not differ.Patients showed more eye movements (IRR = 1.31, 95% CI: 1.23 to 1.40, Patients in a minimally conscious state showed more behavioral reactions and increased physiological arousal during AAT compared to control sessions. This might indicate increased consciousness during therapeutic sessions in the presence of an animal.NCT02629302.ClinicalTrials.gov  Acquired brain injuries can result in severe disorders of consciousness, such as minimally conscious state (MCS), with often serious lifelong consequences for patients and their families –3. EarlyM = 47.20, SD = 19.36) and diagnosed with acquired brain injury with either traumatic (N = 4) or non-traumatic causes (N = 6). Secondary diagnoses were not considered. Severity of the disorder of consciousness was assessed via the original JFK Coma Recovery Scale (CRS) [Subjects were 10 inpatients in a minimally conscious state. All patients were in stationary neurorehabilitation in a Swiss rehabilitation clinic, aged 17 to 71 years (le (CRS) ,12 that le (CRS)  and to Ble (CRS)  for the The screening process involved the family members of the patients as well as the responsible physicians and therapists. The legal representative of the patients provided written informed consent. The human-related protocols were approved by the Ethics Committee for Northwest and Central Switzerland and the animal-related protocols were approved by the Veterinary Office of the Canton Basel-Stadt, Switzerland. AAT was performed according to the guidelines of the International Association of Human Animal Interaction Organizations (IAHAIO) to ensure patient safety and animal welfare . No therN AAT = 8, N control = 8). Sessions lasted for approximately 15 minutes and were held four times a week, twice with an animal and twice without an animal. Each control session was paralleled with an AAT session such that two sessions in two consecutive weeks were as similar as possible regarding the involved therapist, day of the week, time of day and therapeutic activity. All participants were allocated randomly to start with either AAT or a control session. Allocation sequence was generated via a random number generator by the principal investigator who also enrolled and assigned participants to interventions. Some of the originally planned 160 sessions were cancelled due to illness of patient or therapist, and for some sessions data was lost due to technical problems. In total, we coded the behavior of 151 sessions  and analyzed assessment data of 136 sessions  and heart rate data of 115 sessions . All AAT and control sessions were held in a therapy room within the therapy animal facility at the rehabilitation center. The patients were transported to the therapy room by wheelchair. Patients wore a heart rate monitor belt on their chest which continuously measured heart rate and heart rate variability during the session. All sessions were videotaped and at the end of each session, the behavior of the patients was assessed via the Basler Vegetative State Assessment by the therapists. Prior to the study start, a suitable animal was selected for each patient according to preference and abilities. Included species were dogs, guinea pigs and rabbits. All animals were trained for AAT, had experience working with patients in a minimally conscious state, and were kept and handled according to the IAHAIO standards [The study was designed as a randomized two treatment multi-period crossover design to evaluate the immediate effects of the different interventions on patient reactions. Standardized therapy sessions that integrated an animal served as experimental condition and are referred to as AAT sessions. In the control condition, paralleled, comparable standardized therapy sessions without the presence of an animal  were used. Each patient participated in 16 therapy sessions over a period of 4 weeks (N = 151) were videotaped with a handheld camera (Sony HDR-CX240) and analyzed with a behavioral coding system software . Analyses were done continuously, defining each second of the video with the different variables as present or not for state behavior variables. We calculated the percentage of the duration of each state variable in relation to the observed time period of a therapy session. Count variables were coded only if they occurred, and the total occurrence within a therapy session was calculated. All videos were coded according to a strict ethogram defined by detailed descriptions of the behaviors with inclusion and exclusion examples. The coding scheme was developed for the purpose of this study. As basis, 11 existing paper-pencil behavioral assessment tools in German and English for patients with disorders of consciousness were screened. Items were pooled and reduced to behaviors that could be observed during video analysis and that occur according to a stimulus during a therapeutic situation. Our coding scheme included the dimensions “eyes open/closed”, “eye movement”, “movement”, “phonation” and “emotion” . Moreover, we coded the amount of verbal and tactile stimuli offered by the therapist as well as the amount of the patient’s physical contact with the animal. Inter-rater reliability was measured by Cohen’s kappa for all coded variables. Before coding the actual data, each rater achieved an inter-rater reliability of k > 0.80. Inter-rater reliability ranged between 0.83 and 0.99 indicating excellent agreement among coders.As primary outcome, the patient’s reactions was assessed via behavioral video coding. Therapy sessions (The Basler Vegetative State Assessment (BAVESTA) , a behav® RS800CX, Polar® Electro Oy) as further secondary outcomes. The recorded inter-beat intervals were analyzed with Kubios HRV analysis software version 3.0.2 . In each therapy session, a 5-minute recording was selected. In control sessions, the 5-minute sequence was taken from the middle of the whole session. For AAT sessions, the duration of interaction between the patient and the animal was identified via the videos and the 5-minute sequence was taken from the middle of the interaction phase. Before processing, all RR-series were visually checked and, when necessary, artifacts were corrected. If the number of corrected beats was higher than 5%, the data was excluded from analysis . We also excluded data if the total recording or the interaction between the patient and the animal was shorter than 5 minutes (N = 1). We calculated the following HRV parameters: time domain: the standard deviation of all normal-to-normal RR intervals  and root-mean square differences of successive RR intervals ; and frequency domain: relative power of the low frequency (LF) and high frequency (HF) band in normal units.Heart rate (HR) and heart rate variability (HRV) were measured using non-invasive HR monitoring belts  was used as effect size. The model holds under the assumption that there is no time effect which might be violated. Therefore, we checked the robustness of the model by fitting a second model equivalent to the previous one but including session number as a categorical fixed effect. During data inspection we noticed that the therapists behave differently in AAT and control sessions, primarily with respect to the number of tactile inputs. Because those inputs trigger most of the patients’ reactions, we fitted a third model that includes time as well as the log of tactile inputs. For descriptive statistics, the number of observed count behaviors (count variables) was transformed into rate per time ((n/time)*100 sec) and rate per tactile inputs ((n/tactile inputs)*100). To analyze the effect of AAT on BAVESTA scores and HR/HRV parameters as secondary outcomes, generalized linear mixed models with condition as fixed effect and the individual patient as random effect with the mean difference (b) as effect size was used. All variables were visually checked to detect extreme values (histogram and Q-Q-plot). Model diagnostics of linear mixed models included visual checks for normality of residuals and homogeneity of residuals. All residuals were approximately normally distributed with the exception of RMSSD, which was therefore log-normal transformed. No data were excluded except for HR/HRV data with corrected beats greater than 5% and recordings where patient and animal interacted for less than 5 minutes. Sample size was estimated based on clinical experience and on a pre-analysis of an ongoing study. The significance level was set at the 5% level and all statistical analyses were performed using SPSS, Version 24, and R, Version 3.5.1.M = 47.20, SD = 19.36), participated in this study. CRS values at study start ranged between 14 and 22. All patients were diagnosed with MCS in a clinical assessment by the responsible physician according to the Aspen diagnostic criteria [Two female and eight male participants, between age 17 to 71 with an average age of 47 years (criteria  and the p < 0.001), while verbal inputs from therapists did not differ significantly between conditions .There were more tactile inputs from therapists during control sessions than during AAT sessions . This effect was also present for the models that include time or time and tactile input. The rate of eye movement per tactile input even increased by a factor of 1.7 during AAT compared to control therapy sessions. The rate of total movements per 100 seconds was higher during control therapy sessions and decreased from 3.5 to 3 during AAT . However, this effect reversed when time and tactile inputs were added to the model  with a rate of 23 during AAT and a rate of 20 movements per 100 seconds during control therapy sessions. While there was no difference in self-initiated (active) movements in model one or two, patients showed significantly more self-initiated movements per tactile input during AAT compared to control therapy sessions . The opposite effect was found for reactive movements of the patients. The rate of reactive movements per time was lower during AAT sessions  but this difference disappeared when looking at the rate per tactile input. Patients showed a higher amount of phonation during AAT compared to control therapy sessions  but again, this effect disappeared when time and tactile inputs were included in the model. There was no difference regarding positive emotions, operationalized via positive facial expressions. Negative emotions were reduced during AAT compared to control therapy sessions but this difference was only statistically significant when the amount of tactile inputs were taken into account . While there was no difference regarding the subscales “attention”, “verbal communication” “emotional reactions” or “motor reactions”, we found significantly higher perception and information processing scores  as well as significantly more nonverbal communication  during AAT compared to standard therapy sessions  and lower HF values  during AAT compared to control sessions.Heart rate as well as heart rate variability parameters SDNN and RMSSD did not differ significantly between AAT and control sessions see . In contWe present the first randomized controlled trial of patients in a minimally conscious state assessing behavioral reactions and arousal during AAT and control therapy sessions. AAT led to significantly more eye movements, self-initiated movements as well as movements in total compared to control therapy sessions in the systematic behavior analysis. This is in line with results of Bardl and Bardl’s case-study  that docWe observed differences in the frequency domain heart rate variability parameters. During AAT sessions, HF values were significantly lower and values of LF were significantly higher compared to conventional standard therapy. The decrease in HF values reflects decreased activity of the parasympathetic nervous system, while the increase in LF values is associated with increased activity of the sympathetic nervous system ,21, so bNeither participants nor raters responsible for video coding could be blinded to the conditions. The crossover design of this study only allowed for detecting short-term effects of AAT on behavioral and heart rate measurements during therapy sessions, and the small sample size limits the study outcomes and warrants further trials with more patients. Strengths of this study are inclusion of a paralleled control condition, behavior measured with different approaches and inclusion of a physiological parameter to identify underlying mechanisms. Moreover, our results showed that the presence of an animal can also influence the behavior of the involved therapists and that patients’ reactions should be interpreted in relation to the behavior of the therapists. This is a relevant aspect that should be taken into account in further study designs.Our results indicate that AAT is a feasible approach to increase behavioral reactions and arousal in patients in a minimally conscious state. Integration of animals could be used to increase consciousness of these patients and lead to achieving a relevant therapeutic goal. Although this result is promising, the data are preliminary and it is necessary to further investigate whether AAT might be an effective approach to improve therapeutic effects of neurorehabilitation for patients in a minimally conscious state.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 File(DOC)Click here for additional data file.S2 File(PDF)Click here for additional data file.


Sarcoidosis is a non-caseating inflammatory chronic systemic disease of unknown etiology, which may affect one or more organs. Paranasal sinuses involvement occurs sporadic in sarcoidosis. We report a patient with a medical history of sarcoidosis involving her lungs, liver, and lymphatic system for four years who now presented with nasal and sinuses symptoms. The primary treatment with local cortisone showed no improvement. Computed tomography (CT) scan of the paranasal sinuses (PNS) revealed signs of chronic pansinusitis. She was successfully treated with endoscopic sinonasal surgery. Subsequent histological analysis confirmed the involvement of the PNS with sarcoidosis. Her follow-up during the last 6 months was without recurrence. Sarcoidosis is a multisystemic inflammatory disease of unknown etiology, which may involve any organ in the body -3. InvolA 31 year old woman presented with left-sided nasal obstruction and pressure pain in the left maxillary sinus and forehead for six months. She was diagnosed four years ago with sarcoidosis with involvement of the lungs and liver, hilar lymphadenopathy, as well as iritis. CT of the PNS showed typical signs of chronic left maxillary, anterior ethmoidal, and frontal sinusitis . Since cet al. in 2001 of 6 patients with pulmonary sarcoidosis showed four patients with histologically proven SNS [While sarcoidosis mainly involves the lower respiratory tract, it rarely affects the PNS. Until 2013 more than 15 studies including about 5000 sarcoidosis patients described occurrence of sinonasal sarcoidosis (SNS) in about 4.5% ,16. Bockoven SNS . Others et al. proposed clinical and radiologic criteria for the diagnosis of histologically proven SNS deduced from the study of 15 SNS patients with retrospectively involvement. The radiological criteria were presence of nodules on the septal or turbinate mucosa, nasopharyngeal or pharyngolaryngeal lesions, and complete or subtotal opacification of the sinuses and/or the nasal cavities [et al. in 2001, where only 7% of 86 patients were treated with FESS [et al. presented a retrospective analysis of 12 biopsy-proven cases, where the conservative treatment with topical and systemic corticosteroid following sinuses surgery was successful in most patients. Only 4 of them had repeated surgery because of symptoms persistence [Histologically, paranasal sarcoidosis is characterized by non-caseating granulomatous inflammation which may occur before systemic manifestation of sarcoidosis ,15,16. Rcavities . Initialith FESS . Lately,SNS was found in 4.5% of reported cases worldwide. The clinical presentation included nasal obstruction, epistaxis, rhinorrhea or facial pain. The treatment consisted of local and systemic steroid therapy and in some cases sinus surgery. Sinonasal involvement in patients with sarcoidosis is an important manifestation that can be treated successfully. Thus it should be considered in every cased since SNS may be underdiagnosed.The authors declare no competing interests.