Annexin A11 is one of the oldest vertebrate annexins that has a crucial role in sarcoidosis pathogenesis. The mechanism of effect in sarcoidosis granuloma cells may be due to alterations in apoptosis. Immune cells with a specific mutation at protein location 230 are resistant to apoptosis and consequently have continued effects on inflammation and progression of sarcoidosis. The mechanism of action of annexin A11 may be based upon alterations in delivering calcium to two different apoptosis pathways (caspase and P53).Annexins are Ca They are widely distributed among eukaryotes but largely absent in prokaryotes and yeasts. The first protein of this family, discovered in 1977, was called synexin. Currently known as annexin A7, it caused aggregation of chromaffin granules in the adrenal glands in the presence of free calcium . The medmeeting” . Annexinmeeting” .A11 was linked to increasing susceptibility to sarcoidosis. It was also suggested that cells with mutant annexin A11 might have altered susceptibility to apoptosis. However, the mechanism of this effect has never been discussed. We propose here a molecular mechanism for this association.There is little information about annexin A11 and many of its structural and functional characteristics are speculated based on similarity with other members of annexin family. Recently, genetic mutation in annexin annexin genes [ANX genes with mammalian ANX genes shows that three zebrafish ANX genes are homologous with human ANX1; two are homologous with human ANX2; and two are homologous with human ANX11. This information suggests that zebrafish ANX genes may have resulted from duplications after the divergence of the zebrafish and mammalian genomes [More than a 100 annexins have been identified in many different species . Twelve in genes  that are genomes .ANXA9) to 96 kb (ANXA10) and are spread throughout the genome on chromosomes 1, 2, 4, 5, 8, 9, 10, and 15 [ANXA5 and ANXA10 genes in early vertebrate evolution. The reasons for the annexin genes or their chromosomal regions to duplicate are not well understood. Their successful preservation and the extent to which they contribute to vertebrate complexity are also not well described [The 12 human annexin genes range in size from 15 kb ANXA to 96 kb6 kb ANXA and are ANXA11 gene is located on human chromosome 10q22–q23 and is composed of 15 exons and 14 introns without the 5′ flanking region [The annexins A1, A2, A4, A5, A6, A7, and A11) to selective, such as annexin A3 in neutrophils, annexin A8 in the placenta and skin, annexin A9 in the tongue, annexin A10 in the stomach and annexin A13 in the small intestine [ANXA11 has the highest gene expression in whole blood cells, particularly CD19+ cells (B-cells), CD14+ cells (monocytes) and CD33+ cells (myeloid). However, it is found in almost all tissues including lung, heart, and intestines [Annexin gene expression levels within human organs have a broad range, from universal . Some annexins, such as annexins A11 and A2, have been found in the nucleus under particular circumstances in the cell cycle , 17. It In some circumstances, annexins can be expressed at the cell surface even without a secretory signal peptide. For example, annexin A1 translocates from the cytosol to the cell surface following exposure of cells to glucocorticoids . In a stAnnexin A2 expression at surface of vascular endothelial cells has a function in the regulation of plasmin generation . It has Annexin A11 distribution changes per cell cycle. It is more prevalent in the nucleus than the cytoplasm in interphase cells and then moves to degenerating nuclear envelope. And finally during the mitotic phase, it will be concentrated within the central spindle . It has S100 proteins, which only express in vertebrates, are a well-known group of proteins that interact with the annexins. The functions of S100 proteins are diverse and include regulating actin and microtubule networks, promoting cell survival and proliferation, calcium homeostasis, and mediating muscle contraction. The complex of annexin A2-S100A10 interacts with several membrane ion channels, such as transient receptor like potential vanilloid type 5 and 6 channels (TRPV5 and TRPV6). The complex also interacts with cystic fibrosis conductance regulator protein (CFTR) and plays a role the regulation of these ion channels , 34. It 2+ to S100A6 and apoptosis-linked gene2 (ALG-2) whose protein augments apoptosis. The significance of these interactions in the pathogenesis of sarcoidosis is discussed later in this article.Annexin A11 is involved in cellular apoptotic processes. The N-terminal domain of ANXA11 contains binding sites that deliver CaANXA1 causes changes in the inflammatory response and the effects of glucocorticoids [ANXA1 null mouse line, there was altered expression of other annexins as well as cyclooxygenase-2 and cytoplasmic phospholipase A2. In addition, there was an exaggerated response to the stimuli characterized by an increase in leukocyte emigration and IL-1β generation and a partial or complete resistance to the anti-inflammatory effects of glucocorticoids [Studies on knockout mice of annexin families show a diversity of functions among these proteins. Loss of rticoids . In the rticoids .Data supports the role of annexin 2 as a regulator of cell surface plasmin generation, fibrin homeostasis, and neovascularization in laboratory mice models . HomozygANXA7 knockout mouse. The viability of the ANXA7 null mouse was compared to the heterozygous mouse [ANXA7, was viable and able to reproduce [Another study involved the formation of the us mouse . The ANXeproduce .ANXA7 null mutant mouse proposed ANXA7 function in the fusion of vesicles as a calcium channel [ANXA7 null mice were studied. When stimulated with high frequencies, the cells showed an altered cell shortening relationship. Possibly through its role in calcium regulation, this study suggested a function for annexin A7 in electromechanical coupling [Another study of  channel . Cardiomcoupling . The othANXA2 gene polymorphism was higher in the sickle cell patients compared to controls. The SNP was also present in higher frequency in sickle cell osteonecrosis patients than those without osteonecrosis [Annexins have essential roles in the pathogenesis or progression of many human diseases. Recent genetic studies discovered single nucleotide polymorphisms (SNPs) in the genomes of this group of proteins. In a study from India, annexin A2 gene SNP (rs7170178) was found to be associated with osteonecrosis in sickle cell patients . The frenecrosis .ANXA5 gene was sequenced in 243 Japanese women with recurrent pregnancy loss and 119 fertile controls [ANXA5 gene SNPs, the carrier frequency for the minor allele was significantly higher in the pregnancy loss group [In another study from Japan, the annexin A5 gene polymorphism was found to be associated with recurrent pregnancy loss. The promoter region of the controls . In a cass group . For SNPss group .Annexins are also associated with autoimmune disorders. In rheumatoid arthritis, high extracellular annexin V levels initiates the production of annexin V autoantibodies that may have a crucial role in pathogenesis of disease . SystemiDysregulation of Annexin A11 has been found in cancer, cancer treatment, and diabetes . For exaSome members of the annexin family may also be used as biomarkers and for clinical imaging. For example, ANXA1 was investigated as a potential serum biomarker for lung cancer. Lung cancer tissues exhibited higher expression of annexin A1 than normal tissues. In addition, increased serum annexin A1 was significantly associated with pathologic grade and clinical stage of lung cancer patients . QuantitSarcoidosis is a systemic immune disorder with a characteristic accumulation of epithelioid granulomas in many organs, such as the lungs, kidney, skin and eyes , 51. Chr+ and CD19+, immune cells involved in sarcoidosis, are proposed mechanisms for sarcoidosis [ANXA11 (annexin A11) gene on chromosome 10q22.3. A common nonsynonymous SNP (rs104955) was found to be strongly associated with sarcoidosis. As it is demonstrated in Fig. ANXA11 R230C SNP were more resistant to apoptosis than the wild genotype. This association was particularly prominent in subjects with the TT ANXA11 phenotype [2+ dependent. Without calcium delivery from ANXA11 to ALG-2, the apoptosis via caspase pathway would not be activated [Genetic instability and mutation in annexin A11 has been identified in single nucleotide polymorphisms in patients with sarcoidosis compared to control groups. Decreased activation of CD8coidosis . In addicoidosis . The strhenotype . The mec SNP rs10955 was fhenotype . The MAPctivated .Fig. 3AN2+ delivery from ANXA11 [The second important protein involved in apoptosis is P53 . P53 medm ANXA11 . Indeed,m ANXA11 . With a m ANXA11 . Figure In the sarcoidosis lung, granulomas develop when the immune cells attempt to wall off an unknown antigen trigger. These cells include macrophages, lymphocytes, and multinucleated giant cells. The granuloma can be effaced by apoptosis leaving only minor scar. Without apoptosis the inflammatory and granulomatous reaction continues. Patients with ANXA11 R230C cannot clear granulomas due to altered apoptosis and may have a higher risk of onset of sarcoidosis and poorer outcome Fig. .Fig. 6DeHofmann et al. were the first group of researchers to identify the association of ANXA11 with sarcoidosis . More reIn a 2013 study, the role of annexin A11 in sarcoidosis was confirmed in a US population. This association also extended to clinical manifestation of disease. The studied involved 1689 sarcoidosis cases and 1252 controls. 25 SNPs in and around ANXA11 were significantly associated with sarcoidosis risk in both African Americans and European Americans. The most significant allelic association was the minor C allele at rs1049550 among African and European Americans . This waThese recent advances of annexin A11 in the pre-clinical stage show its significant role in the pathogenesis of sarcoidosis. In the era of personalized medicine, the annexin could be a novel focus. Annexin A11 thus may be a target for clinical trials in which a patient’s genetic risk factors are used to tailor treatment .Annexins have significant effects on human health and disease. Annexin A11 affects two apoptosis related pathways (caspase and P53) and has a crucial role in sarcoidosis pathogenesis. This theory should be tested in cell culture and animal models. Recent studies show the significant role of annexin A11 in the genetic susceptibility to sarcoidosis. If the ANXA11 could increase susceptibility to sarcoiodosis via interaction with caspase and P53, a new opportunity to develop new therapeutic targets in sarcoidosis will be recognized.


A simple one-pot process to exfoliate graphite and synthesize nanographene-dimesitylporphyrin hybrids has been developed. Despite the bulky mesityl groups, which are expected to hinder the efficient π–π stacking between the porphyrin core and graphene, the liquid-phase exfoliation of graphite is significantly favored by the presence of the porphyrins. Metallation of the porphyrin further enhances this effect. The resulting graphene/porphyrin hybrids were characterized by spectroscopy  and microscopy . The −1) to NMP-exfoliated graphene (86 cm−1). Significantly, this effect is more pronounced in FLG/DMPP (78 cm−1) and even more in FLG/ZnMPP (75 cm−1). These results indicate that not only the yield, but also the efficiency of the exfoliation process is enhanced upon addition of the porphyrins. Meanwhile, the 2D band intensity diminishes due to the electron doping of graphene by porphyrin molecules, as corroborated by the significant shifts in the frequency of the G band and the decrease of the 2D/G ratio . Graphite powder  and all reagents were purchased from Aldrich . Solvents were purchased from commercial suppliers and used as received. UV-visible spectra of three different samples of each graphene solution were measured by using a Cary 50 UV-visible spectrophotometer  in quartz cuvettes with a pathlength of 1 cm. Fluorescence experiments were carried out at least three times for each sample. Emission spectra were recorded on a NanoLog 4 HORIBA . Raman spectra were acquired with a Bruker Senterra confocal Raman microscopy instrument , at excitation laser wavelength of 532 nm. The laser power at the sample was 10 mW, and the data acquisition time was 20 s. The sample preparation involved drop casting of the solutions on a Si substrate with a 300 nm oxide layer. STEM images were obtained with an Auriga Carl ZEISS operating at 25 kV . The FLG/porphyrin hybrids were deposited by drop casting of the dispersions onto holey carbon mesh grid. −1. The solutions were sonicated  for various periods from 60 to 240 min. In order to exclude the effect of temperature and to avoid the degradation of the solvent due to an increase of the bath water during sonication, a cooling system was connected to the bath sonicator performing the experiments at 20 °C. At different times during this period, aliquots of the suspensions were centrifuged at 3000 rpm for 30 min . After that, the supernatant was carefully collected for further characterization. For FLG/porphyrin hybrids, the same procedure was followed but graphite was added to solutions of 5,15-dimesityl-10,20-diphenylporphyrin (0.01 mg·mL−1 in NMP) and Zn-5,15-dimesityl-10,20-diphenylporphyrin (0.01 mg·mL−1). In this case, the supernatant of FLG/porphyrin hybrids was directly measured as obtained.Graphene dispersions were prepared via a liquid-phase exfoliation technique by adding graphite powder to 1-methyl-2-pyrrolidone (NMP) at a concentration of 1 mg·mLThe one-pot exfoliation of graphite and formation of FLG/DMPP hybrids has been demonstrated. The presence of the two bulky mesityl groups in the porphyrins does not impede their interaction with FLG. In fact, UV-vis measurements show that the presence of the porphyrins enhances the concentration of exfoliated graphene by up to 140%. We have also observed a very significant effect of the metal core of the porphyrin on the interaction with the surface of graphene. Our results show that the decrease in the efficiency of π–π interactions between the aromatic core of porphyrins and FLG can be compensated for by adequate CH–π and van der Waals interactions. This expands the range of porphyrins that can be used to synthesize FLG/porphyrin hybrids to include those with bulky substituents in the meso positions, which in turn should facilitate their use in electronic devices.


Two hundred and twenty-three CRC patient specimens were used to determine both BDNF and TrkB mRNA levels. The expression of these proteins in their primary and metastatic tumors was investigated by immunohistochemistry. CRC cell lines and recombinant BDNF and K252a  were used for in vitro cell viability, migration, invasion, anoikis resistance and in vivo peritoneal metastasis assays. Tissue BDNF mRNA was associated with liver and peritoneal metastasis. Tissue TrkB mRNA was also associated with lymph node metastasis. The co-expression of BDNF and TrkB was associated with liver and peritoneal metastasis. Patients with higher BDNF, TrkB, and co-expression of BDNF and TrkB had a significantly poor prognosis. BDNF increased tumor cell viability, migration, invasion and inhibited anoikis in the TrkB-expressing CRC cell lines. These effects were suppressed by K252a. In mice injected with DLD1 co-expressing BDNF and TrkB, and subsequently treated with K252a, peritoneal metastatic nodules was found to be reduced, as compared with control mice. BDNF/TrkB signaling may thus be a potential target for treating peritoneal carcinomatosis arising from colorectal cancer.Tropomyosin-related receptor kinase B (TrkB) signaling, stimulated by brain-derived neurotrophic factor (BDNF) ligand, promotes tumor progression, and is related to the poor prognosis of various malignancies. We sought to examine the clinical relevance of BDNF/TrkB expression in colorectal cancer (CRC) tissues, its prognostic value for CRC patients, and its therapeutic potential  The outstanding progress in the treatment of metastatic colorectal cancer (mCRC) has been founded on the recent multi-drug combination chemotherapies, which now produce median survival times exceeding 20 months NTR) with low affinity NTR which exerts diverse functions such as the regulation of cell survival and differentiation during neuronal development Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin (NT) family. BDNF plays an important role in the development and repair of the nervous system NTR involved in proliferation, differentiation, survival and apoptosis of neuronal and non-neuronal tumors NTR preferentially acts as an interacting partner of TrkB, modulates TrkB activation by BDNF, and influences prosurvival effect by BDNF/TrkB signaling Although both TrkB and p75NTR seems to have either tumor-promoting or tumor-suppressing functions according to tumor types BDNF/TrkB signaling has been reported to be associated with tumor progression, metastasis, and response to chemotherapy in several human malignancies such as neuroblastoma Previously, studies in our laboratory have revealed: an association between TrkB levels tumor progression and patient prognosis in gastric cancer In vitro studies showed that BDNF/TrkB signaling was involved in proliferative or invasive properties With regard to the BDNF/TrkB signaling in CRC, BDNF or TrkB was overexpressed in both clinical tumor samples and associated with aggressive tumor phenotypes The aim of this study was to examine: the association between BDNF/TrkB expression and clinicopathological variables in a series of human CRC tissues; the prognostic value of BDNF/TrkB signaling in CRC patients; and its therapeutic potential in vitro and in vivo.This study was reviewed and approved by the Institutional Review Board and the Local Ethics Committee of the Mie University Graduate School of Medicine (No. 2126). Written informed consent was obtained from all the patients (adults and parents of children) enrolled onto the study.The experimental protocols of in vivo studies were reviewed and approved by the Animal Care and Use Committee at the Mie University Graduate School of Medicine.A total of 223 patients with CRC, who were treated at the Department of Gastrointestinal and Pediatric Surgery in the Mie University Graduate School of Medicine from 2000 to 2008, were included in this study. Patients with incomplete clinical data, inadequate follow-up or inadequate tissue sampling were excluded from the study. All patients had histologically confirmed adenocarcinoma of the colon or rectum. The median age of the patients was 67 years (range: 12–91 years). The median follow-up time was 30.6 months (range: 1.5–107.2). A total of 49 patients died of CRC-related causes during this period.Staging was based on clinical assessment and histopathological analysis using the International Union Against Cancer (UICC) TNM staging system. Written informed consent was obtained from all the patients enrolled onto the study, according to local ethics guidelines. This study was approved by the Institutional Review Board.Cancer tissues were frozen in liquid nitrogen immediately after surgical resection and stored at −80°C until use. The diagnosis of CRC was confirmed for all 223 patients based on clinicopathologic findings.Cancer tissues were minced and homogenized with a Mixer Mill MM 300 homogenizer . Total RNA was isolated using an RNeasy mini kit (Qiagen) according to the manufacturer's instructions. cDNA was synthesized from 5 µg total RNA with a random hexamer primer and Superscript III reverse transcriptase  according to the manufacturer's instructions.Quantitative PCR (qPCR) analysis was performed using a TaqMan Universal PCR Master Mix . The expression levels of the target gene transcripts, measured using TaqMan probes for BDNF  and TrkB , were normalized to GAPDH  and evaluated using Applied Biosystems StepOne Software (v2.1).2). The level of expression of the target gene was calculated from the standard curve, and normalized against GAPDH. Finally, the target gene mRNA level was expressed as a ratio relative to the GAPDH mRNA level. Real-time quantitative PCR assays were performed in triplicate for each sample and the mean value was used in calculating mRNA expression levels.The relative BDNF and TrkB gene expression levels were determined using a standard curve. The standard curve was generated using a 5-fold serial dilution of random-primed qPCR Human Reference cDNA . All standard curves were linear within the range used for analysis, with an acceptable corresponding correlation coefficient . The cell line authentication testing has been performed for these cell lines. These cell lines were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 µg/mL streptomycin at 37 °C and 5% CO2.in vitro assays.Recombinant human BDNF was purchased from PeproTech  and prepared according to the manufacturer's instructions. Recombinant human BDNF was dissolved in PBS (10 µg/ml) for the in vitro and in vivo assays.K252a was purchased from Calbiochem  and stored at −20°C before use. K252a was dissolved in PBS (10 µg/ml) for the The CRC cell lines were washed in ice-cold PBS whilst on the dish. Cold lysis buffer  was then added directly to the dish. The cells were then scraped off the dish, collected, and homogenized using a Mixer Mill MM 300 homogenizer . The supernatants were collected and frozen at −20°C until use. The protein concentration was measured using the BCA protein assay . 20 µg of the protein lysate was mixed with an equal volume of 2× Laemmli loading buffer containing 2-mercaptoethanol, and heated at 100 °C for 5 min. Samples were electrophoretically separated on 12.5% gradient polyacrylamide gels containing 0.1% SDS, followed by semi-dry transfer to an Immun-Blot PVDF membrane . The membrane was then blocked using 5% skimmed milk in Tris-buffered saline, pH 7.5, supplemented with 0.1% Tween 20 (TBS-T).The blots were then incubated with mouse monoclonal anti-TrkB antibody  at a 1∶1000 dilution, rabbit polyclonal anti-BDNF antibody  at a 1∶100 dilution, and mouse monoclonal anti-actin (clone C4) antibody  at a 1∶400 dilution in 5% skimmed milk in TBS-T overnight at 4 °C. After washing three times in TBS-T, the blots were incubated with alkaline-phosphatase-conjugated goat anti- mouse IgG  at a 1∶200 dilution in 5% skimmed milk in TBS-T. Following treatment with an enhanced chemiluminescence detection solution, chemiluminescent signals were visualized in a CS Analyzer and AE-6962 light capture .To assess the effect of BDNF on cell viability, migration, invasion, and anoikis resistance, recombinant human BDNF and K252a, a selective pharmacological pan-Trk inhibitor, were used.Cell viability, migration, invasion, and anoikis resistance were compared among non-treated cells (control), BDNF-treated cells, K252a-treated cells, and K252a followed by BDNF-treated cells.Cytotoxicity was evaluated using a WST-8  colorimetric assay. Cancer cells (5000 cells/well) were seeded onto 96-well cell plates  in 100 µl of culture medium for 24 h. After preincubation, the cells were treated with K252a (100 nM) or serum-free medium for 2 h, and then treated with either recombinant human BDNF (100 ng/ml) or serum-free medium. 48 h later, the medium was discarded and replaced with 90 µl of fresh medium followed by the addition of 10 µl WST-8 reagent solution  and incubated for 2 h at 37 °C in an incubator.Cell viability was determined by colorimetric comparison by reading optical density (OD) values from a microplate reader  at an absorption wavelength of 450 nm. Cytotoxicity was evaluated using the Cell Counting Kit according to the manufacturer's instructions. The data were obtained from similar results of at least three independent experiments. Results are presented as mean ± standard error (SE).Confluent cancer cells were serum-deprived for 48 h. Wounds were generated using a sterile 200-µL pipette tip after preincubation with K252a (100 nM) or serum-free medium for 2 h. Recombinant human BDNF (100 ng/ml) or serum-free medium was added and the cells were incubated at 37 °C for an additional 46 h. Wound closure was assessed using an Olympus IX71 microscope  at 10× magnification. Cell migration distance was measured using Adobe Photoshop 9.0.2 software and compared with baseline measurements. Each independent experiment was performed at least three times. Results are presented as mean ±SE.Cell invasion was evaluated using Biocoat Matrigel invasion chambers and control inserts (Becton Dickinson Labware). A total of 50000 cells/well were seeded in the invasion and control chambers, and pre-incubated with 100 nM K252a or serum-free medium for 2 h. Fresh medium alone or medium containing recombinant human BDNF (100 ng/ml) was then added to the Falcon companion plates . The Matrigel invasion chambers and control inserts were incubated for 24 h at 37 °C. The incubation medium containing cells was removed from the top chamber using cotton swabs and serum-free medium. The membranes were fixed in methanol, stained with Mayer's hematoxylin, dehydrated in ethanol, and mounted on glass slides. The number of cells that invaded the underside of the membrane was then determined. Each independent experiment was performed three times. Results are presented as mean ±SE.Anoikis, detachment-induced apoptosis, is known to be induced when adherent tumor cells are forced to grow in a non-adherent fashion. Anoikis resistance was evaluated by the proportion of viable tumor cells that proliferated non-adherently in low-attachment dishes.5 cells/ml, respectively. Their cell suspensions (2 ml) were added to each well and incubated for 24 h in a humidified atmosphere (37 °C and 5% CO2).Anoikis assays were performed in six-well Costar Ultra Low Attachment Microplates . DLD1, LoVo, and SW480 cells were suspended in RPMI-1640 with BDNF (100 ng/ml), K252a (100 nM), or K252a+BDNF at a concentration of 5×103 cells/well in microtiter plates  in a final volume of 100 µL culture medium per well. To assess the viable tumor cells that proliferated non-adherently in low-attachment dishes, spectrophotometric absorbance of each well was measured using WST-8 reagent solution as described above (cell viability assay). Each independent experiment was performed six times. Results are presented as mean ±SE.After induction of anoikis, cells were seeded at 5×105 cells were washed and resuspended in 0.5 ml of 1×binding buffer, and Annexin V: fluorescein isothiocyanate/Propidium Iodide (PI) labeling performed according to the manufacturer's protocol . The analysis was performed using a FACSCalibur flow cytometer  to quantify the proportion of viable or apoptotic tumor cells under non-adherent growth condition using low-attachment dishes. Each sample contained 5×105 cells. The data were analyzed using CellQuest Pro software (BD Biosciences). Viable tumor cells that were negative for both Annexin V and PI staining (lower left quadrant) were considered as anoikis-resistant cells with non-adherent growth. Apoptotic tumor cells that were positive for Annexin V staining (lower right quadrant+upper right quadrant) were considered as anoikis-induced cells. Tumor cells treated with 2.5% formalin solution were used as a positive control for apoptosis induced cells. Flow cytometric analysis was performed to confirm the results of cell viability assay for anoikis resistance.After induction of anoikis, 5×107 cells/500 µl PBS) were injected intraperitoneally for peritoneal metastatic formation. Either K252a (500 µg/kg) or PBS (control) was injected intraperitoneally three times a week to examine the effect of K252a on the peritoneal metastatic formation. Four weeks later, the mice were sacrificed, and then the size and number of peritoneal metastatic nodules was evaluated . Results are presented as mean ±SE.Male nude mice (BALB/c) at 8 weeks of age were purchased from Japan SLC Inc. . The experimental protocols were reviewed and approved by the Animal Care and Use Committee at the Mie University Graduate School of Medicine. DLD1 cells . The results are expressed as the mean ±SE (standard error). Differences between groups were estimated using the Pearson's chi-square test, repeated-measures analysis of variance (ANOVA) analysis, or an unpaired Student's t-test. Actuarial survival curves were obtained using the Kaplan–Meier method, and comparisons were made using log-rank tests. Univariate and multivariate analyses were done using the Cox proportional hazards model to investigate the effects of the histopathological and molecular factors (BDNF and TrkB expression status) present in the primary tumor specimen on overall survival (OS). Variables associated with survival with a P-value less than 0.05 in univariate analysis were used for multivariate analysis. P-values of less than 0.05 were considered statistically significant. Two-sided P-values <0.05 were considered to be statistically significant.The BDNF mRNA expression ratio, relative to GAPDH, in CRC tissues was 0.122±0.230 (mean±SD), ranging from 0 to 1.357. For statistical analysis purposes the BDNF mRNA expression values were dichotomized as low or high. A cutoff value for the BDNF mRNA level was determined as a maximum predictive value using an OS based on receiver–operating characteristic (ROC) curve analysis. One hundred and two patients had high BDNF mRNA expression (>0.037), whereas 121 patients had low BDNF mRNA expression. As shown in According to the TaqMan Gene Expression Assay , probe of TrkB  is designed to span the exon 6/7 boundaries. Corresponding primers amplify the fragments containing exons 6–7 which encodes the extracellular portion of the TrkB receptor. Therefore, this primer and probe set detects both the full-length TrkB (TrkB.FL) and the truncated TrkB (TrkB.T1) The TrkB mRNA expression ratio, relative to GAPDH, in CRC tissues was 0.054±0.141 (mean±SD), ranging from 0 to 1.149. ROC curve analysis showed that the cutoff value for TrkB mRNA was 0.002 . One hundred and seventy-one patients had high TrkB mRNA expression, whereas 52 patients had low TrkB mRNA expression. As shown in Ninety-four patients showed both high BDNF and TrkB mRNA expression. This patient group was classified as a co-expressing both BDNF and TrkB. As shown in Immunohistochemistry showed that the BDNF protein  was expressed in the cytoplasm of human CRC cells and that the TrkB protein  was expressed in the nucleus of human CRC cells . In patiAs shown by the above results, both BDNF and TrkB expression in tumor cells seems to be involved in both primary and metastatic tumor progression in human CRC. To explore the involvement of an autocrine BDNF/TrkB signaling in CRC progression, we first examined BDNF and TrkB expression in 5 CRC cell lines including CaCO2, DLD1, HT29, LoVo, and SW480.BDNF mRNA expression was detected in all 5 cell lines by RT-PCR analysis . In contWestern blotting analysis  showed tBased on our results and evidence from previous reports, we selected 3 cell lines  which showed both BDNF and TrkB mRNA expression with either TrkB.FL or TrkB.T1 protein expression for further examination.To explore the possibility of an autocrine BDNF/TrkB signaling loop occurring in CRC, we investigated the effect of BDNF on tumor cell viability in TrkB-expressing CRC cells. As shown in These results suggest that exogenous BDNF increases tumor cell viability in TrkB-expressing CRC cells, and that TrkB receptor blockade may provide a potent means of inhibiting tumor growth.in vitro migration assays. Forty-eight hours later, BDNF significantly increased the migration of DLD1 cells, as compared with the non-treated controls. K252a decreased the migratory ability of the DLD1 cells, which was nearly identical to the non-treated controls. K252a followed by BDNF inhibited the migratory ability of DLD1 cells, as compared with BDNF treatment. The representative images of the above results were shown in To examine the effect of BDNF on tumor cell motility, DLD1 cells were used for in vitro invasion assays. Twenty-four hours later, BDNF significantly increased the invasion of SW480 cells, as compared with the non-treated controls. K252a decreased the invasive ability of SW480 cells, which was nearly identical to the non-treated controls. K252a followed by BDNF treatment inhibited the invasive ability of SW480 cells, as compared with BDNF treatment. The representative images of the above results were shown in To examine the effect of BDNF on tumor cell invasion, SW480 cells were used for in vitro experimental setting, it is induced when adherent tumor cells are forced into non-adherent growth. To examine the effect of BDNF on anoikis resistance (detachment-induced apoptosis resistance), DLD1, LoVo, and SW480 cells were used for in vitro anoikis assays. The number of anoikis resistant cells was evaluated by counting viable tumor cells that proliferated non-adherently in low-attachment dishes (cell viability assay using a WST-8 reagent solution). As shown in Anoikis is a form of detachment-induced apoptosis. In the These results suggest that exogenous BDNF enhances anoikis resistance in TrkB-expressing CRC cells, and that a TrkB receptor blockade may inhibit the anoikis resistance of these tumor cells.in vivo peritoneal metastasis assay. in vivo peritoneal metastasis assay. The representative images of PC arising from DLD1 cells and peritoneal metastases were shown in The results presented above have shown that BDNF enhances the cell viability, migration, and anoikis resistance of TrkB-expressing CRC cells, and that the TrkB inhibitor, K252a, inhibited these effects. These results suggest that an autocrine BDNF/TrkB loop may be involved in the progression of both primary and peritoneal metastatic tumors. We next investigated whether a TrkB receptor blockade suppress the peritoneal metastasis of BDNF/TrkB-co-expressing DLD1 cells using an These results suggest that a TrkB receptor blockade may inhibit either anoikis resistance, adhesion onto the peritoneal surface, or the colonization of the peritoneal cavity.Previously, we reported an inverse correlation between TrkB and E-cadherin mRNA levels in CRC patients, and an inhibition of tumor cell proliferation, migration, and invasion by TrkB knockdown in CRC cell lines The mRNA levels of BDNF, TrkB and their co-expression in CRC tissues were associated with tumor progression and poor prognosis of CRC patients. Although we couldn't show that co-expression of BDNF and TrkB had an independent prognostic significance by multivariate analysis (data not shown), BDNF/TrkB expression status of the primary CRC tissues may provide prognostic information for CRC patients.Previous reports showed that BDNF was higher in CRC tissues than adjacent normal tissues at the mRNA We found that BDNF/TrkB signaling was significantly associated with proliferation, migration, invasion, and anoikis resistance in BDNF/TrkB co-expressing CRC cells. Recent studies also demonstrated the molecular mechanisms of BDNF/TrkB signaling in tumor cell migration, invasion, and anoikis resistance. They showed the involvement of β5 integrin in BDNF/TrkB signaling mediated cell migration In this study, BDNF was detected at the mRNA and protein level in all 5 cell lines, although cell line-dependent difference was observed at the protein expression level. In contrast, TrkB was detected at the mRNA level in 3 out of 5 cell lines. Additionally, these 3 cell lines showed higher protein expression of TrkB.T1 than that of TrkB.FL.TrkB.T1, one of the truncated forms of TrkB receptor, is generated by alternative splicing during gene transcription and lacks the intracellular tyrosine kinase domain with the extracellular BDNF binding region TrkB.T1 was also frequently detected in several human malignancies rather than TrkB.FL in vitro studies.In CRC cell lines, TrkB.T1 has been also reported to be preferentially expressed at both the mRNA and protein levels, as compared with TrkB.FL Since TrkB.T1 regulates BDNF/TrkB.FL signaling in vivo. PC arising from CRC has been reported to be found in approximately 7% of CRC patients at diagnosis, or at the time of primary tumor surgery (synchronous PC) We also demonstrated that treatment of mice with the pharmacological pan-Trk inhibitor K252a suppressed the PC formation of BDNF/TrkB-co-expressing CRC cells In our series, synchronous PC was the most powerful predictive factor of overall survival, with the highest risk ratio during the last 5 years of life (data not shown). These lines of evidence indicate that PC is regarded as a primary target of CRC treatment. Although the effects of chemotherapeutic treatment on the prognosis of metastatic CRC have been well reported, it remains unclear whether these treatments provide the same benefit to the subset of CRC patients with PC.Recently, we showed that the blockade of BDNF/TrkB signaling by K252a suppressed the PC formation arising from gastric cancer cells with co-expression of BDNF and TrkB However, further studies will be needed to reveal the therapeutic potential of BDNF/TrkB signaling in CRC, especially for PC arising from CRC. To clarify the anti-tumor efficacy of the blockade of BDNF/TrkB signaling in CRC, we would first need to confirm whether a BDNF blockade, such as an anti-BDNF antibody, and a TrkB blockade, such as a pharmacological pan-Trk inhibitor or an antagonistic anti-TrkB antibody, show anti-tumor effects on CRC in patients with various BDNF/TrkB expression statuses.In conclusion, we have demonstrated that BDNF/TrkB signaling in tumor tissues is associated with tumor progression and poor prognosis in CRC patients, and that it is also associated with proliferation, migration, invasion, and anoikis resistance in BDNF/TrkB co-expressing CRC cells. The BDNF and TrkB expression status in our resected specimens may provide prognostic information for CRC patients. Finally, BDNF/TrkB signaling may be a promising potential target for PC arising from CRC.


Since graphene has been taken as the potential host material for next-generation electric devices, coexistence of high carrier mobility and an energy gap has the determining role in its real applications. However, in conventional methods of band-gap engineering, the energy gap and carrier mobility in graphene are seemed to be the two terminals of a seesaw, which limit its rapid development in electronic devices. Here we demonstrated the realization of insulating-like state in graphene without breaking Dirac cone. Using first-principles calculations, we found that ferroelectric substrate not only well reserves the Dirac fermions, but also induces pseudo-gap states in graphene. Calculated transport results clearly revealed that electrons cannot move along the ferroelectric direction. Thus, our work established a new concept of opening an energy gap in graphene without reducing the high mobility of carriers, which is a step towards manufacturing graphene-based devices. This fascinating character of extremely high carrier mobility endows graphene great advantage for electronic applications.Two-dimensional atomic materials, which are consisted of one or several atomic layers, have attracted great research interests in recent years124578101112zp-zp bonding −7 μC/cm.For the experimentally synthesized hydroxyl-functionalized BNSL (OH-BNSL), hydroxyl groups are connected with boron atoms. However, the orientation of hydroxyl groups on BNSL is not explored. As shown in Now we turn to explore the electronic properties of graphene/OH-BNSL bilayer, in which graphene is covered on the surface of ferroelectric OH-BNSL. As shown in 6 m/s).As stated in our previous reportsAlthough the character of linear band dispersion and zero-energy gap in graphene is not disturbed by the OH-BNSL, it is cursory to deduce that OH-BNSL has little effect on the electronic property of graphene. Since OH-BNSL introduces a periodic electric potential along the ferroelectric direction (the orientation of hydroxyls) in graphene, the charge density of graphene should be modulated by this periodic electric potential. As shown in Generally, the carriers transport will be modified by the charge density wave. As we know, along the direction of chain-like charge patterns, the carriers can much easier be transported because of the strong overlapping of nearest-neighbor wave function. Besides, the calculated band dispersion has demonstrated the existence of Dirac fermions, namely, such carriers have a high mobility. While for the direction perpendicular to the chain-like charge patterns, carriers are difficult to be transferred from one chain to another one because of the weaker overlapping of nearest-neighbor wave function, namely, there is a “pseudogap” along this direction although there is no real energy gap in the electronic structures.PJ and VJ, respectively. The calculated zero-bias transmission functions of two proposed graphene/OH-BNSL bilayer junctions are plotted in VJ are significant larger than that of PJ within the energy range from −0.25 to 0.12 eV. For example, at the Fermi level, the transmission coefficient of VJ is about 1.6, and the corresponding value of PJ is very small (less than 10−4), which stands for the metallic and insulating states, respectively. These results show that the transport direction of graphene/OH-BNSL bilayer system depends on the orientation of hydroxyl groups on BNSL. The metallic channel coexists with an insulating-like one in graphene/OH-BNSL bilayer system, which well confirms our above electronic structure calculations. Moreover, the metallic state can be switched into the insulating-like state by changing the orientation of hydroxyl groups on BNSL.To verify the above predictions, we perform first-principles calculations combined with non-equilibrium Green's function method to explore the transport properties of two graphene/OH-BNSL bilayer junctions, as shown in inserts of It should be noted that the “pseudogap” explored in graphene/OH-BNSL bilayer is totally different with that of previous reports181920211819202113/cm2. Therefore, in the following studies, we will limit the electron concentration below 1.0 × 1013/cm2. As shown in 13/cm2, 0.5 × 1013/cm2, 1.0 × 1013/cm2). Importantly, in all cases, the charge density around Fermi level has the chain-like patterns  code. The projected augmented wave (PAW) method25−4 eV, and 0.01 eV/Å, respectively. The accuracy of our simulation was tested through a comparison of the presented results with previous theoretical results.Our first-principles calculations were based on spin-polarized density functional theory (DFT) using the generalized gradient approximation (GGA) known as PW91LGΓRG+], in which ΓL(R) stands for the coupling matrix between the left(right) electrode and the central scattering region, and G is the retarded Green's function of the scattering region. In our calculations, we adopted Troullier-Martins nonlocal pseudopotential and double-zeta plus polarization basis sets for all elements in the junctions. The grid integration was defined by an energy cutoff of 150 Ry, while the exchange and correlation functional was treated at the level of Ceperley-Alder local-density approximation. Test calculations with more k-points and larger cutoff energy yield the similar results.The transport calculations for graphene/OH-BNSL bilayer were carried out by using the fully self-consistent non-equilibrium Green's function method combined with first-principles calculations, implemented in ATK package27E.K. conceived the project. F.W. performed the calculations of electronic properties. J.H. studied the electric conductivity and calculated the I–V curve. F.W., J.H., Q.L., K.D. and E.K. co-wrote the paper, and all authors contributed to the discussion and preparation of the manuscript.


To study the impact of soluble IL2 receptor (sIL2R), chest x-ray (CxR), and angiotensin-converting enzyme (ACE) as markers for sarcoidosis in uveitis patients.Retrospective study.Serum concentrations of sIL2R and ACE were measured in patients with active uveitis. Those with elevated sIL2R and /or ACE values were examined for suspected systemic sarcoidosis.Our main outcome parameters were the specificity and sensitivity of sIL2R, CxR and ACE in screening for ocular sarcoidosis.We measured 261 patients with uveitis for sarcoidosis using sIL2R and ACE between January 2008 and November 2011; sarcoidosis was been diagnosed using other tests  in 41 of 53 patients with elevated sIL2R values (>639 U/ml) and in one patient with normal sIL2R (582 U/ml). Their mean sIL2R value was 1310 U/ml, extending from 582 to 8659 U/ml. Only 9 patients, however, presented elevated ACE (>82 U/l). Their mean ACE value was 116.4 U/l, ranging from 84.1 to 175.5 U/l. IL2R specificity was 94% with 98% sensitivity. In contrast, ACE had a specificity of 99.5%, but a sensitivity of only 22%; the chest x-ray had a specificity of 100% with 50% sensitivity in detecting sarcoidosis. We observed the entire spectrum of uveitis: sixteen patients suffered from anterior, 8 from intermediate, 16 from posterior, and 2 from panuveitis.An elevated level of soluble IL2R suggests sarcoidosis with uveitis more convincingly than ACE, making sIL2R a more effective marker parameter for sarcoidosis than ACE or chest x-ray in uveitis patients. Sarcoidosis or Morbus Boeck is a multisystem disorder of unknown cause,2,3. TheSarcoidosis is distributed worldwide and usually starts between the ages of 20 and 40 years. Its prevalence in the USA is three times higher in the Afro-American population than in the Caucasian population,14,15,16Untreated sarcoidosis may be fatal, and intraocular involvement can lead to blindness. The ophHowever, diagnosing it can be challenging. The golIn 2009, an international group of uveitis specialists therefore defined diagnostic criteria for ocular sarcoidosis, choosing criteria enabling the ophthalmologist to make the diagnosis of ocular sarcoidosis without the invasive diagnostic procedures needed for histological proof. These criteria included signs of intraocular inflammation and laboratory investigations.Not mentioned in these criteria is the soluble interleukin-2 receptor (sIL2R), a serum marker widely used for the diagnosis of and as an activity marker in pulmonary sarcoidosis,28,29,30Although its exact pathogenesis is unknown, sarcoidosis is characterized by T-cell activation,32,33. TThe aim of this study was to evaluate the potential clinical usefulness of sIL2R as a screening parameter for sarcoidosis with uveitis in comparison to established markers .Between January 2008 and November 2011, 600 patients were seen with uveitis for the first time in the uveitis section at the Eye Center, University of Freiburg. Out of these 261 were tested for sIL2R and ACE. In 53 of those we detected elevated sIL2R. All patients suspected of having intraocular sarcoidosis were referred to a respiratory specialist (pulmonologist).Medical data were reviewed retrospectively. We analyzed age, gender, comorbidity, current medications (including ACE inhibitors) and visual acuity.Anatomical location of the uveitis and its severity were classified according to the standardisation in uveitis nomenclature (SUN) criteria. In the We also collected chest radiographs, computer tomographies, bronchoalveolar lavage results, biopsies, and laboratory data. In accordance with the European Respiratory Society patients with alveolar lymphocytes ≥ 20% were considered to present a pathology. Due to Institutional Review Board (IRB)/Ethics Committee approval was obtained (Ethik-Kommission der Albert-Ludwigs-Universität Freiburg (No.69/14)). According to the IRB requirements patient records and information were anonymized and de-identified prior to analysis.ACE levels were determined by spectrophotometric assay using the synthetic tripeptide substrate N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPGG), which is catalyzed by ACE to FAP and glycylglycine (Roche). This hydrolysis results in a decrease in absorbance at 340 nm.Soluble IL2R was measured via a solid-phase two-site chemiluminescence immunometric assay . Soluble IL2R in the sample binds to the solid-phase antibody and reagent antibody , forming a sandwich complex. A chemiluminescent signal is generated in proportion to the bound enzyme. According to the manufacturer, the threshold value for ACE is 82 U/mL and 639 U/ml for sIL2R, respectively.Contingency table to achieve specificity, sensitivity, positive predictive value and negative predictive value of ACE, chest x-ray and sIL2R were calculated using Prism from the Graph Pad. The Youden Index, a marker of the performance of a diagnostic test, was also provided. FurtherThe patients with elevated sIL2R were classified in two categories: the first included all patients with ocular sarcoidosis, including 41 patients with elevated sIL2R and one patient with normal sIL2R value (582 U/ml) and in the second, those with elevated sIL2R and no evidence of sarcoidosis.The male/female ratio did not differ between these categories, and the median age was similar . Due to Mean ACE value was 59 U/ml in the sarcoidosis patients with uveitis (n = 41) while that of the uveitis patients without sarcoidosis (n = 220) was just 35 U/ml  (p<0.001The patients with elevated sIL2R/no sarcoidosis suffered from idiopathic non-granulomatous uveitis (6), from systemic autoimmune disease (3) (undifferentiated systemic autoimmune disease (2), lupus erythematosus (1)), mycoplasma infection (1), HLA B27 associated uveitis (1) or TINU syndrome  (1).Based on results from the chest x-rays, CT scans, tissue biopsies and IWOS criteria, we detected 42 sarcoidosis patients with uveitis. A diagnosis of definite ocular sarcoidosis was made in 10 patients, presumed ocular sarcoidosis in 12 patients, and probable ocular sarcoidosis in 19 patients. One patient was diagnosed with possible ocular sarcoidosis .We classified these patients according to the SUN criteria. Sixteen suffered from anterior uveitis, 8 from intermediate uveitis, 16 from posterior uveitis and two from panuveitis .Specificity was high for the chest x-ray, sIL2R and ACE. It was lowest for sIL2R at 94%. In contrast, the sIL2R sensitivity was 98%, chest x-ray 50%, and ACE only 22%. Thus positive and negative predictive values were favorable for all factors, but Youden Index was only favorable for sIL2R  .The eye is a potential primary and/or presenting site for the manifestation of sarcoidosis. Ophthalmologists play a critical role in establishing its diagnosis,46. In 2Serum angiotensin converting enzyme (ACE) is probably the most common laboratory test for sarcoidosis, revealing an approximate sensitivity of 57–73% and a specificity of 90% in the literature, but only 22% sensitivity and 90% specificity in our patients,49. EvenACE is secreted by epithelioid cells and macrophages and is thus a marker for the general burden of granulomas. ACE is An ideal marker would be one that is easy to obtain at any time and safe for the patient. The serum sIL2R value fulfills these criteria and is already frequently used in the clinical routine for diagnosing pulmonary and systemic sarcoidosis,29,30. IIn the present retrospective study, we assessed serum sIL2R in a group of patients with ocular sarcoidosis. On the basis of our data, we can claim that soluble IL-2R is a useful screening marker for sarcoidosis with uveitis. All kinds of intraocular uveitis  were detected as being associated with subclinical pulmonary sarcoidosis. Furthermore, we measured a sensitivity of 22% and a specifity of 90% for ACE. In direct comparison, IL2R seems to be the more appropriate screening tool for the diagnosis (sensitivity 98% // specificity 94%// Youden Index 92%). Soluble IL2R is a parameter that indicates the extent of T-cell activation and has been known since the early eighties,36. ElevAn increased IL2R level has been reported to be associated with uveitis. Few studies have demonstrated a relationship between sIL2R levels in peripheral blood and in aqueous humor with uveitis,59,60,61There are some limitations in our study; it is only a retrospective, single-center study. As mentioned above, there is no real gold standard available, thus we applied the IWOS criteria. Taking this into account, you will never even in a prospective study detect all true positives and false positives. Therefore, there will be always some degree of uncertainity.In addition, the performance of a test depends tremendously on the population in which it is assessed. The specificity would be less in a population with lower incidence of sarcoidosis.Nevertheless, our results clearly indicate that sIL2R levels are superior to ACE levels and CxR for identifying patients with sarcoidosis even if the study has the aforementioned limitations.In summary: based on our data; we recommend adding the measurement of sIL2R in the routine diagnostic work-up of patients with uveitis as an additional screening parameter for intraocular sarcoidosis. Moreover, already established biomarkers such as ACE and lysozyme may be replaced by sIL2R.S1 Dataset(XLSX)Click here for additional data file.S1 Table* p<0.0001; soluble Interleukin 2 Receptor (sIL2R); Angiotensin converting enzyme (ACE).(DOC)Click here for additional data file.


Moreover, while the in-plane stiffness only increases moderately when the devices are cooled down to 10 K, it approaches 300 N m−1 when the aspect ratio of graphene membranes is increased. These results indicate that softening of graphene at temperatures <400 K is caused by static wrinkling, with only a small contribution due to flexural phonons. Together, these results explain the large variation in reported mechanical constants of graphene devices and pave the way towards controlling their mechanical properties.Free-standing graphene is inherently crumpled in the out-of-plane direction due to dynamic flexural phonons and static wrinkling. We explore the consequences of this crumpling on the effective mechanical constants of graphene. We develop a sensitive experimental approach to probe stretching of graphene membranes under low applied stress at cryogenic to room temperatures. We find that the in-plane stiffness of graphene is 20–100 N m Free-standing graphene is inherently crumpled in the out-of-plane direction due to dynamic flexural phonons and static wrinkling. Here, the authors use optical interferometry as a delicate probe to investigate the consequences of this crumpling on the effective mechanical constants of graphene. E2D=340 N m−1 and vanishing bending rigidity. This record stiffness caused by strong carbon–carbon bonds13E3D∼1 TPa∼E2D/t, where t∼0.335 nm is graphite interlayer spacing461012What is the mechanical stiffness of free-standing monolayer graphene? The answer to this question may appear trivial: graphene is universally modelled as a flat sheet with in-plane stiffness (the ratio of in-plane stress and strain) et al.At the same time, the interplay between crumpling and mechanical properties of graphene remains virtually unstudied experimentally, despite its obvious significance for fundamental understanding of mechanics of two-dimensional (2D) materials as well as applications in graphene-based Nanoelectromechanical (NEMS) devices. We believe that the likely culprit is the technique used in the majority of the experiments probing graphene mechanics—atomic force microscopy (AFM) nanoindentation3714E2D to values of as low as ∼20 N m−1. Furthermore, by performing temperature-dependent measurements and studying devices of various geometries, we separately probe the contributions of flexural phonons and static wrinkles. Static wrinkling is found to be the dominant mechanism determining the effective mechanical constants in our devices. Finally, we discover an approach to suppress static wrinkling by in situ changing the membrane's geometry.Here we quantitatively investigate the effects of crumpling on the effective mechanical stiffness of graphene. To accomplish this, we develop a non-contact approach based on interferometric profilometry. This allows us to study the mechanics of graphene at cryogenic to room temperatures in a controlled geometry with uniform loading. We confirm that out-of-plane crumpling softens 3N4) with diameter ranging between 7.5 and 30 μm form the ‘sample chip'. Pristine residue-free graphene is seen in the high-resolution scanning electron microscopy (SEM) imaging of the sample chip . The applicability of parallel-plate capacitor approximation is justified since d∼10–20 μm is much larger compared with the maximum deflection of graphene (∼600 nm). The maximum Vg that can be applied without dielectric breakdown is ∼2,000 V, which allowed us to reach maximum pressures around 30 kPa; the uncertainty in P is below 5% for all voltages.Graphene was electrostatically pressured by applying a voltage h is determined with sub-nanometre precision in the out-of-plane direction and sub-micron resolution in the in-plane direction by detecting the phase shift of light reflected from the sample surface. Large graphene–gate separation d helps to maximize the interferometric signal from nearly transparent graphene. When d is large, only the signal reflected from graphene is detected since the gating chip is out of focus. A separate measurement is performed to find d by deliberately sweeping the focus from the sample to gating chip.The deflection of the graphene membranes under applied pressure was determined via interferometric profilometery. In this technique, a profile of the surface h, and verify that the membrane is deflecting symmetrically. Third, the pressure is applied uniformly, allowing us to use simple and reliable models to extract mechanical constants. Fourth, the optical nature of the technique allows simple characterization of devices inside an optical cryostat at low temperatures. At the same time, we note that our method is not a replacement for AFM nanoindentation. Our approach operates in the regime of low applied pressure and cannot be used to determine breaking strength of 2D materials.Our method of probing the mechanical properties of graphene has several critical advantages over AFM-based nanoindentation and other techniques. First, graphene deflection is measured via a non-contact approach. This means that the membrane morphology is not disturbed with a sharp tip that applies non-uniform stress. Second, the height data from the entire membrane is recorded at the same time. This means we can find the true maximum centre-point deflection, h versus known applied pressure P σr/ɛr, where ν∼0.165 is the Poisson ratio for grapheneE2D does not deviate more than 10% from the simple analytical estimate above  data to the bulge-test equation E2D=35±29 N m−1, consistent with previous workσr(ɛr), with E2D=(1–ν)dσr/dɛr increasing from less than 10 N m−1 at low stress to 50 N m−1 at higher stress  data between loading and unloading cycles (E2D for CVD graphene (E2D∼50–80 N m−1 in two devices). This confirms that E2D in our experiments is not affected by the grain boundaries in graphene, which is consistent with conclusions from previous experimentsE2D consistent with optical profilometry measurements. It is important to note that AFM nanoindentation pushes graphene towards the substrate, while electrostatic loading pulls graphene away from it. Similarity in E2D values obtained for opposite loading directions confirms that interaction of graphene with the sidewalls does not affect the measured E2D. Finally, simple estimates show that the organic residues that may remain on graphene after the fabrication process are unlikely to affect E2D. A uniform residue layer with Young's modulus of ∼2 GPa , we would expect strong stiffening of graphene at low temperature if this were the dominant effect. We measured two different devices in the range of temperatures between 400 and 10 K . This confirms that the changes in stiffness observed in Normally, the presence of defects lowers the mechanical stiffness of any material, including grapheneE2D measured in the experiment can be loosely approximated asIt is instructive to estimate relative contributions for flexural phonons and static wrinkling to the in-plane stiffness of our devices. The in-plane stiffness of graphene Elatt∼340 N m−1, Eflex and Ewrin are contributions to stiffness from three different mechanisms—stretching of carbon–carbon bonds, flexural phonons and static wrinkles. The data in E2D from 36 to 300 N m−1. Provided that the width of graphene ribbon is much larger compared with the typical wavelength of a flexural phonons, ∼10–1,000 nm, we expect that the process of cutting does not affect the contribution due to flexural phonons. We can then use Ewrin<40 N m−1 and Eflex>2,500 N m−1 from this data. In agreement with our earlier conclusion, we see that the contribution due to wrinkles dominates E2D. A simple estimate can clarify why the contribution due to static wrinkling is larger than that of flexural phonons. The degree of crumpling of a membrane can be quantified as ΔA/A=(A′–A)/A, where A is membrane's area, and A′ is the area of membrane's projection onto a plane parallel to it flex∼0.5%. This estimate agrees with ΔA/A extracted from molecular dynamics wrin∼π2δ2/λ2>0.1–1.6%. The lower bound here is likely a very conservative estimate as it neglects wrinkles with longer wavelengths. The corresponding ɛt from this estimate is larger than the average built-in strain observed in experiment, and we, therefore, do expect softening of graphene due to static wrinkling.The degree of crumpling of graphene due to flexural phonons can be estimated using a model based on the application of the equipartition theorem to the membrane-bending modesdynamics  and moreδ=1–2 nm  we estimσ0=0.02 N m−1 fits our data well  with uniform loading and at cryogenic to room temperatures. We have confirmed that graphene is significantly softened by out-of-plane crumpling. Moreover, we developed an approach to test relative contributions of flexural phonons and static wrinkles to the in-plane stiffness of graphene, and found that the latter dominates. Our observations reinforce the idea that great care is needed when applying classical elasticity theories to atomically thick materials372 environment at 350 °C. The fabrication yield for intact suspended graphene membranes varies from 55% for our smallest (7.5 μm diameter) devices to <8% for the biggest (30 μm diameter) devices. The graphene membranes remained clamped to the sample chip via van der Waals interactions forming suspended circular graphene membranes.Silicon nitride membranes (thickness 1 μm) were fabricated by depositing low-stress silicon-rich silicon nitride on both sides of a silicon chip. An array of holes ranging between 7.5 and 30 μm were then patterned in the nitride using standard fabrication procedures. A metallic contact  was deposited onto the top surface of the nitride membrane. Monolayer graphene was then transferred onto holes in the nitride membranes. Different transfer procedures were used for CVD and exfoliated graphene. For CVD graphene, we use a high-quality atmospheric growth and wet transfer3941A Wyko 9800 interferometric profilometer equipped with a 20 × ‘through transmissive media' objective (NA=0.28) was used to perform optical measurements. In measurements of graphene deflection, phase shift interferometry mode was used with HB-LED at ∼530 nm wavelength as illumination source. To measure graphene-gate separation, vertical scanning interferometry mode was used with white light illumination source.+ ion beam .The FIB lithography was carried out using a Novalab 600 Dual-Beam (electron/ion) FEI. The system is aligned to the graphene with the electron beam  while cuts are made with GaHow to cite this article: Nicholl, R. J. T. et al. The effect of intrinsic crumpling on the mechanics of free-standing graphene. Nat. Commun. 6:8789 doi: 10.1038/ncomms9789 (2015).Supplementary Figures 1-7 and Supplementary References


Exportin-1 (XPO1) is a nuclear export protein with >220 cargo proteins, including tumor suppressors and cell cycle modulators. Selinexor is a SINE (Selective Inhibitor of Nuclear Export) compound that has been administered to >900 cancer patients in Phase I and II trials to date, with evidence of efficacy and tolerability. Selinexor blocks nuclear export of NFAT1c, STAT1 and STAT3, which are implicated in regulating the inhibitory T cell receptor PD-1 and its ligand, PD-L1. We hypothesized that selinexor would upregulate T cell checkpoint molecule expression, and that combination treatment with anti-PD-1 or anti-PD-L1 would thereby enhance the ability of selinexor to elicit antitumor activity.in vivo study.Selinexor increased PD-1 gene expression by ~2-fold in normal lymphocytes and induced PD-L1 gene expression in tumor cell lines. Mice bearing syngeneic colon tumors (colon26) treated with selinexor and anti-PD-1 for 2 weeks demonstrated a significant reduction in tumor growth rate (P < 0.05), while monotherapy with either agent had no significant effect on tumor growth. Similar results were obtained in mice bearing syngeneic B16F10 melanoma tumors, whereby combined treatment with selinexor + anti-PD-1 was superior to either single agent alone (p < 0.034). Combined therapy of mice bearing B16F10 tumors with selinexor and anti-PD-L1 was similarly effective, with significantly smaller tumors at the study endpoint (p < 0.001). No weight loss or signs of toxicity were evident in any Immunophenotypic analysis by flow cytometry revealed that selinexor alone or in combination with anti-PD-1/anti-PD-L1 significantly increased the percentage of splenic NK cells (p≤0.050), while selinexor ± anti-PD-L1 significantly increased the percentage of splenic Th1 T cells (p≤0.011), all compared to vehicle treated mice. Interestingly, combining selinexor with anti-PD-L1 significantly decreased the percentage of splenocytes that expressed PD-L1 (p < 0.001). These data indicate that the efficacy of selinexor may be enhanced by disrupting the pre-existing PD-1/PD-L1 signaling in effector cells (T and NK cells).Altogether, these data suggest that the efficacy of selinexor in combination with anti-PD-1 or anti PD L1 in mouse syngeneic tumor models may be due to both disrupting immunosuppressive PD-1/PD-L1 signaling and increasing the frequency of potentially tumor reactive NK cells and Th1 T cells. This provides a rational basis for this treatment combination as a novel therapeutic approach for advanced cancer.


This paper presents a low-cost low-power CMOS humidity sensor for passive RFID sensing applications. The humidity sensing element is implemented in standard CMOS technology without any further post-processing, which results in low fabrication costs. The interface of this humidity sensor employs a PLL-based architecture transferring sensor signal processing from the voltage domain to the frequency domain. Therefore this architecture allows the use of a fully digital circuit, which can operate on ultra-low supply voltage and thus achieves low-power consumption. The proposed humidity sensor has been fabricated in the TSMC 0.18 μm CMOS process. The measurements show this humidity sensor exhibits excellent linearity and stability within the relative humidity range. The sensor interface circuit consumes only 1.05 μW at 0.5 V supply voltage and reduces it at least by an order of magnitude compared to previous designs. Passiveetc. . The pasetc. –6. Theseetc. [Humidity measurements are essential for a wide range of applications, including environment monitoring systems, process control systems, pharmaceutical and biomedical applications, food preservation, etc. . Humiditetc.  and resietc.  are the Various materials, such as porous ceramics and hygroscopic polymers, are used as humidity-sensitive materials . Becauseetc. However, analog IC is less scalable and suffers more when going to nanometer CMOS technology. Apart from the issues of matching and noise, the threshold voltage is reduced less remarkably compared to the power supply, resulting in a reduced voltage headroom for traditional analog amplitude-based sensor interfaces [CMOS technology has been mainly developed for digital IC. Benefitting from technology scaling, digital IC achieves great improvements on speed, power dissipation, chip area, terfaces . To copeterfaces ,22. ThisThe paper aims to present a low-cost low-power humidity sensor for RFID sensing applications. The rest of the paper is organized as follows: Section 2 presents a low-cost humidity sensor which is fabricated in standard CMOS technology without any further post-processing. Section 3 introduces the Phased-Locked Loop (PLL)-based sensor interface theory first and then illustrates the implementation of the fully-digital sensor interface in detail. The measurement results are presented and compared with previous designs in Section 4. Finally, some conclusions are drawn in Section 5.2.2 layer on the Si wafer. A thick aluminum layer is then deposited and patterned with standard optical lithography and wet etching over the SiO2 in order to form the interdigitated structure. The sensing film is polyimide that is coated on the interdigitated electrodes. In order for moist air to access the sensing film, there must be a large area of the polyimide exposed to the air. As is seen from L is the length of aluminum electrodes, S is the width of each electrode, and W is the distance between adjacent electrodes. The thickness of the polyimide layer is H, which is generally larger than the metal thickness h.For a CMOS process, interdigitated top metal fingers, with polyimide filled into the finger gaps, can be utilized for capacitive humidity sensing. The line-to-line coupling capacitance of the top metal is sensitive to the dielectric constant of the filling material. Furthermore, due to the high precision of photolithography, such kinds of sensing structures are highly reproducible with less inter-die variations . Figure wetε represents the dielectric constant of the polyimide film with absorbed water, γ is the fractional volume of water absorbed in polyimide, dryε is the dry polyimide film dielectric constant, and waterε is the water dielectric constant. The relationship between γ and relative humidity can be modeled by Daubinin equation [mγ is maximum factional volume at T0=298K, ф(T) is the temperature dependence on the adsorption coefficient and β(T) is the temperature dependence of the relative dielectric constant of water and the catalytic effect. The dielectric constant of the polyimide film wetε (and in turn the capacitance of the sensor) depends on the relative humidity, which increases or decreases when moisture penetrates or leaves the film. The mechanism for moisture absorption (or desorption) depends on the film properties (not sensor geometries) and determines the sensitivity of the sensor [The dielectric constant of the sensing film can be expressed by the Looyenga empirical equation  as folloequation  as folloe sensor .N finger array sensor, the total sensor capacitance can be expressed as [For a essed as :(3)CsenH has a great influence on the sensitivity and response time of the sensor. Generally, a humidity sensor with a thicker polyimide layer has better sensitivity and longer response time. Trading off the various factors, including sensitivity, response time, hysteresis performance and chip area, this work chooses N = 40, L = 200 μm, W = 2.5 μm, S = 2.5 μm, h = 1 μm and H ≈ 2 μm. The sensor capacitance can be evaluated in accordance with wetε is given.The selection of the thickness of polyimide layer 3.3.1.sensf. The frequency-demodulating block is a digital first-order Bang-Bang-Phased-Locked-Loop (BBPLL), consisting of a single-bit phase detector and a Digitally-Controlled Oscillator (DCO). This BBPLL measures whether sensf leads or lags digf (the frequency of the DCO) and hence the DCO is only steered by a single-bit signal outb. When the entire feedback loop is locked, sensf shifts between a maximum and minimum value which corresponds to the maximal and minimal value of the sensor. The average digital frequency digf will correspond to the sensor frequency sensf. Therefore, the over-sampled output outb represents the digital value of the sensor value.The architecture of PLL-based sensor interface, developed from Danneels , is showThis architecture employs the BBPLL block, which has advantages ranging from low voltage capabilities, low power consumption, small chip area, and scalability to smaller technologies and robustness to process variation ,29. Anot3.2.sensorC acts as the variable load on a single stage of the SCO, thereby generating a sensor-controlled frequency sensf. The variable capacitive load on a single stage of the DCO consists of two capacitors, oC and mC. The capacitor oC, designed equal to the quiescent value of sensorC, is always connected to the DCO. But the capacitor mC, designed slightly larger than the maximum variation of sensorC, is swapped in or out of the DCO depending on the feedback from the single-bit phase-detector. Considering the issues of system linearity and process variation, mC is normally designed as a programmable capacitor. The dummy load capacitor dC at each stage should be carefully designed to be small enough for high resolution performance.The implementation of the proposed fully-digital capacitive sensor interface is shown in sensf and digf are equal, thus the two frequency controls of the SCO and DCO are correlated. Since both oscillators are implemented identically, the control of the SCO, the sensor value, is correlated to the control of the DCO, the single-bit output of the phase detector. Hence the value of the sensor is digitized in this single-bit DCO control signal (outb). In order to increase resolution, outb is over-sampled by taking its average duty cycle overtime [As shown in overtime .oC and mC are selected as 5 pF and 2 pF, respectively. The dummy load capacitors (oC) at each stage are chosen to be 1 pF. Due to the fully digital architecture, the power supply of the interface is selected as 0.5 V, which is close to the process threshold voltage.For this work, the humidity sensor's capacitance varies from 5–6.5 pF within the relative humidity range. Hence the capacitors 4.2 and 0.008 mm2, respectively. Two test humidity sensors, with 1 μm-thick and 2 μm-thick polyimide layers respectively, were measured in a Votsch VCL4003  temperature and climate test chamber.The proposed humidity sensor, fabricated in the Taiwan Semiconductor Manufacturing Company (TSMC) 0.18 μm 1P6M mixed-signal CMOS process, is shown in sensorC) of the humidity sensor with 2 μm-thick polyimide layer at 25 °C. The measured outputs (dotted line) and the digitized over-sampled outputs (solid line) are shown together. It is obvious that the average duty cycle of the output increases in proportion to the value of ΔsensorC.The measured sensitivity of the humidity sensor is shown in versus relative humidity on the 1st, 8th, 15th day so as to study the repeatability of the humidity sensor with time. It is obviously that the humidity sensor exhibits excellent repeatability.The stability measurement of the humidity sensor is given in 5.The paper presents a integrated capacitive humidity sensor for RFID sensing applications. The humidity sensor is implemented in a standard CMOS process without any further post-processing, which results in low fabrication costs. The sensor interface, based on PLL architecture, transfers sensor signal processing from the traditional voltage domain to the frequency domain. It employs a fully-digital architecture and therefore can operate on an ultra-low supply voltage, which results in low power dissipation. The measurement results prove its excellent performance in the areas of linearity, stability, chip area and power dissipation. In particular the proposed sensor interface can work with 0.5 V supply voltage and consumes only 1.05 μW. Therefore this humidity sensor is particularly suitable for the mass-production of RFID sensing tags.


Flavonoids are also known to exert an inhibitory effect on the vascular endothelial growth factor receptor (VEGFR) family of tyrosine kinase receptors. VEGFR2 is one of the important receptors involved in the regulation of vasculogenesis and angiogenesis and has also been implicated to exhibit various neuroprotective roles. Its upregulation and uncontrolled activity is associated with a range of pathological conditions such as age-related macular degeneration and various proliferative disorders. In this study, we investigated molecular interactions of 7,8-DHF and its derivatives with both the TrkB receptor as well as VEGFR2. Using a combination of molecular docking and computational mapping tools involving molecular dynamics approaches we have elucidated additional residues and binding energies involved in 7,8-DHF interactions with the TrkB Ig2 domain and VEGFR2. Our investigations have revealed for the first time that 7,8-DHF has dual biochemical action and its treatment may have divergent effects on the TrkB via its extracellular Ig2 domain and on the VEGFR2 receptor through the intracellular kinase domain. Contrary to its agonistic effects on the TrkB receptor, 7,8-DHF was found to downregulate VEGFR2 phosphorylation both in 661W photoreceptor cells and in retinal tissue. Recent studies have identified that a flavonoid derivative, 7,8-DHF acts as a high-affinity tropomyosin related kinase receptor B (TrkB) agonist that provokes receptor dimerization and autophosphorylation and activation of downstream signalling in vivo . This coin vivo , Parkinsin vivo , Rett syin vivo , and Hunin vivo . It can in vivo . We havein vivo . TrkB isin vivo ,7. 7,8-Din vivo ,5. Our sin vivo . The Ig2etc. [The VEGF receptor super-family is another class of tyrosine kinase receptors that play a critical role in the retina. In addition to its involvement in neovascularisation associated with several proliferative disorders, abnormal VEGF expression is implicated in several ocular disease conditions such as macular edema associated with diabetic retinopathy , choroidetc. . VEGFR2 etc. ,13. Flavetc. ,15. VEGFWe report here for the first time a dual action of compound 7,8-DHF on TrkB and the VEGFR2 receptor. Using a combination of bioinformatics and biochemical approaches we have provided critical additional insights into the molecular interactions of 7,8-DHF with both the TrkB and the VEGFR2 receptor. Structurally related derivatives of 7,8-DHF are extensively compared to determine the interactions and binding parameters with the TrkB and VEGFR2 receptors. This study also illustrates the effects of 7,8-DHF treatment on the activity of VEGFR2 in both 661W photoreceptor cells as well as in the rat retina.in silico using AutoDock4.2 to reveal the best binding modes of 7,8-DHF. Our studies revealed that the binding site of TrkB-D5 comprised of Lys312, Pro313, Ala314, Leu315, Trp317, Ile323, Leu324, Glu326, Cys331, Thr332, Lys333, Ile334 and Tyr342 residues. Hydrogen bonding with Leu315 and Ile334 indicated these to be critical residues involved in interaction with 7,8-DHF  and VEGFR2 structures were subjected to 7,8-DHF binding  7,8-DHF A,B. In t protein C,D.−1 was observed to be associated with the binding of 7,8-DHF with human TrkB-D5. An inhibitory constant (Ki) of 64.79 µM was calculated for 7,8-DHF-TrkB complex binding, which correlated well with its binding energy. In general, all 37 flavonoid derivatives could be divided into two parts: scaffold I comprised of 2,3-dihydro-4H-chromen-4-one, and scaffold II formed by a 2-phenyl group (H-chromen-4-one), seven dihydroxy flavonoid derivatives through scaffold II (phenyl ring) and the remaining 16 involved interactions through both scaffold I and scaffold II with the TrkB protein   , energy n (RMSF)  and rootn (RMSF) .315 and 236 chiral (C) atom of Lys312 respectively (313 as well as atom 21 (H) of 7,8-DHF and atom 241 (O) Pro313. In addition pi–sigma interaction was also observed between the B ring of 7,8-DHF and atom 265 (C) of Leu315. Hydrogen bonding and pi–sigma bonding with the residues Pro313 and Leu315 in MD simulation may indicate a potential conformational change in TrkB (917 and atom 746 (O) of Cys917 (915 as well as atom 18 (O) of 7,8-DHF and atom 733 (H) Phe916 which was additional to that of Cys917 hydrogen bond interaction observed in molecular docking analysis    A,B. The  < 0.05) C,D. Non-Ki 64.79 µM for TrkB. The high in silicoKi suggested that 7,8-DHF did not have a significant inhibitory effect on TrkB [This study investigated the molecular interactions underlying 7,8-DHF and various other dihydroxyflavone derivatives with the TrkB receptor using a combination of molecular docking and dynamics studies. We also examined for first time molecular interactions between various flavonoid derivatives including 7,8-DHF with the VEGFR2. 7,8-DHF is an agonist of TrkB receptor and its treatment leads to upregulation in the tyrosine phosphorylation on TrkB residues and activate its downstream signalling . Intrigu on TrkB ,21 as co on TrkB ,23.in silico docking approaches based on topological surface geometry complementarities for 7,8-DHF-TrkB and 7,8-DHF-VEGFR2 complexes indicated formation of hydrogen bond networks between surface amino acid residues [et al. showed that 7,8-DHF strongly associated with the CC2 domain and also partially interacted with the leucine-rich motif domain. This additional binding site at Ig2 may play a role in stabilising or further enhancing the 7,8-DHF binding to the TrkB. Similar involvement of the TrkB Ig2 domain in interactions with BDNF were observed with potential contributions to the TrkB receptor dimerization [N-glycosylation site in the Ig2 domain could also play a role in ligand receptor interaction, but further studies are required to establish this [The residues . The staresidues . Moleculrization . The N-gpi–sigma bonds. The 7,8-DHF-VEGFR2 complex also showed formation of two hydrogen bonds with Cys917 after molecular docking. Molecular simulations further showed additional 5 H-bonds involving Glu915, Phe916 and Cys917 residues. Detection of additional bonds and interactions following molecular simulations indicated that the protein-ligand complexes acclimatize and achieve a more stable configuration over a period of time. No significant changes were observed in the total energy of either of the protein-ligand complexes within the 10ns simulation period indicating that the complexes attained a stable conformation , serine/threonine-protein kinase 4 (STK4), phosphatidylinositol 4,5-bisphosphate 3-kinase (PK3CG) and CUB domain-containing protein 1 (CDCP1) (568 (TrkB) which corresponds to Val846 (VEGFR2) is buried in the TrkB structure and is not surface accessible for development of bond formation. These observations suggested that the absence of these structural motifs in TrkB could be the reason underlying exclusion of 7,8-DHF interactions with the ATP binding site of TrkB in contrast to that observed in VEGFR2  A. Furthen VEGFR2 . Togethein silico studies was investigated in the photoreceptor 661W cells in culture as well as in the rat retina under in vivo conditions. 7,8-DHF treatment has been shown to activate the TrkB signalling and reduce apoptosis by activating the downstream processes of Akt and Erk1/2 pathways. We chose 661W cells as these have been shown to express VEGFR2 [in silico predictions. Briefly, the fact that similar inhibitory effect was observed in the rat retinal tissues upon 7,8-DHF treatment validate our 661W results and reassure that the inhibitory effects are not an experimental artefact or not a cell specific phenomenon. The loss in VEGFR2 activity can potentially be attributed to 7,8-DHF interactions with key active site residues Glu915, Phe916, and Cys917 that may give rise to conformational changes in the geometry of the protein as observed in the molecular dynamic studies  and anti-TrkB (sc20542) antibodies were obtained from Santa Cruz Biotechnology . Anti-VEGF antibody  and VEGFR2 antibody  were also used for western blotting. β-Actin antibody was obtained from Sigma, USA. All other reagents were of analytical grade from Sigma .Animal experiments were conducted in accordance with the Australian code of practice for the care and use of animals for scientific purposes and the guidelines of the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research and were approved by the Macquarie University Animal Ethics Committee (2012/31), NSW, Australia. Male Sprague-Dawley rats were obtained from Animal Research Centre, Perth, Australia and maintained in the animal house in cyclic light , in an air-conditioned room with controlled temperature (21 ± 2 °C) and with free access to water and rodent chow.et al., San Francisco, CA, USA), implying amber parameters, followed by minimization with MMTK method in 500 steps with a step size of 0.02 Å [Crystal structure of the TrkB-D5 domain bound to Neurotrophin-4/5 (PDB id: 1HCF)  and VEGFf 0.02 Å . The actf 0.02 Å  for the The three-dimensional (3D) structures of dihydroxy flavones derivatives were collected from the pubchem database . In totahttp://www.pdb.org/). Rigid docking was performed for studying protein–ligand interactions through AutoDock tools. The atom types and bond types were assigned [Ki) were used for determination of binding energies of different docking conformations, ranking in accordance to their binding scores [Ki, binding free energy, electrostatic energy, van der Waals, hydrogen bond, desolvation energy, total intermolecular and torsional energy for 37 DHF derivatives are given in + software  [The docking of the 37 dihydroxy derivatives to the binding site of TrkB-D5 and VEGFR2 was performed using the AutoDock 4.2    were use+ and Cl− to balance the net charge of the system. In Desmond, equilibration of the whole system was carried out using default protocol made up of a series of restrained minimizations and MD simulations. During simulation, initial coordinates of the protein molecules were slowly relaxed without deviation. The minimized system was relaxed with NPT  ensemble restraining non-hydrogen solute atom for 10 ns simulation time. The full system was composed of 17,346 atoms for TrkB-D5 and 36,979 for VEGFR2 complex respectively. The temperature was maintained at 300 K and pressure at 1.01325 bars. Long-range electrostatic interactions were computed using particle-mesh Ewald method [MD simulations were performed for the complex of 7,8-DHF-TrkB and 7,8-DHF-VEGFR2 using Desmond 3.2 software  , incorpod method ,48 and v2. Approximately, 2.0 × 105 cells were seeded in each culture dish 6–12 h before treatment [in vivo experiments, 7,8-DHF (2 mg/kg) was administered intraperitoneally to the rats. The rat retinas were harvested, flash frozen and sonicated in the lysis buffer for further analysis.Photoreceptor derived 661W cells were maintained in DMEM culture media containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C at 5% COreatment ,50. Cell3, 100 mM NaF, 1 mM Na2MoO4, 10 mM Na4P2O7). The proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes as explained previously [661W cells and retinal tissues were lysed in lysis buffer  containing  and . All values with error bars are presented as mean ± SD from given n sizes and compared by Student’s in vivo study where 7,8-DHF administration led to a decrease in the activity of VEGFR2 in the retina. The combined in silico, cell culture and in vivo studies suggest emergence of 7,8-DHF as a dual action compound, which in addition to its known agonistic effects on TrkB receptor, can suppress the VEGFR2 activity.Inhibition of VEGF/VEGFR signalling may be critical in several disorders involving unregulated angiogenesis. A poorly monitored treatment, on the other hand, may give rise to unwarranted complications. In the context of retina for example, excess anti-VEGF treatment may cause onset of dry AMD leading to gradual neurodegeneration. In contrast, activation of the neurotrophic factor signalling such as TrkB may play a critical role in protecting against several neurodegenerative disorders including retinal disorders. In this study, we have evaluated the potential of flavonoid derivatives to act as VEGFR2 inhibitors and the same time evaluated their potential as an activator of neurotrophic factor signalling via activation of the TrkB receptor, with emphasis on examining additional interactions with 7,8-DHF. The interactions of 7,8-DHF and several of its derivatives with the extracellular domain of TrkB receptor using a combination of molecular docking and dynamics tools were determined and presented here. Potential interactions of 7,8-DHF and its derivatives with VEGFR2 were also evaluated. Computational studies indicated 7,8-DHF to be an inhibitor of the VEGFR2. Effects of 7,8-DHF on photoreceptor cells in culture revealed that 7,8-DHF downregulated the VEGFR2 activity. Similar results were obtained in the


A density functional theory calculation shows that covalent bonds were formed between the R6G molecules and the defect sites on the graphene nanosheets. Our study reveals the important role that defects play in tailoring the properties and sensor device applications of graphene materials.A proper understanding of the role that molecular doping plays is essential to research on the modulation of the optical and electronic properties of graphene. The adsorption of R6G molecules onto defect-rich reduced graphene oxide nanosheets results in a shift of the Fermi energy and, consequently, a variation in the optical constants. This optical variation in the graphene nanosheets is used to develop an ultrasensitive surface plasmon resonance biosensor with a detection limit of 10 The application of graphene in next-generation electronics and renewable energy devices has been hindered by the absence of a band gap and the fact that the material presents challenges to optical modulation12451114−1710−11 M and an LOD of 10 aM. The success of modulating the ERGO optical properties for use as a sensitive layer in ultra-sensitive sensing will open new pathways for designing more efficient sensors in the near future.Surface plasmon resonance (SPR) has been employed to monitor biomolecular binding events via the detection of dielectric constant changes on the surface of a thin gold chip161719205 Ha, and a maximum force of 0.002 Ha/Å and maximum displacement of 0.005 Å are adopted in the geometry optimization. Smearing techniques are used to achieve self-consistent field convergence with a smearing value of 0.005 Ha. The graphene sheet is represented using a hexagonal supercell containing 200 atoms with a p (10×10) structure in the x-y plane and a vacuum layer of 35 Å along the z direction between the sheets, which leads to negligible interaction between the periodic image2425The geometric and energetic calculations are performed using the spin unrestricted DFT, as implemented in the Dmol3 codeTwo dimensional GO nanosheets are self-assembled on a flat Au surface by utilizing the strong metal-carbon coupling between the GO and the Au surface. Cyclic voltammetry is then used to electrochemically reduce the GO nanosheets on the Au surface. The details and characterizations are described in our previous reports20SPR spectrometry was carried out using a TR2005 spectrometer . The set-up was based on the conventional Kretchmann configuration and included a He-Ne laser with a wavelength of λ = 632.8 nm coupled to the system via a high refractive index prism, LAFSN9 glass ε = 3.40. The Gold-coated sensor chip mounted was with a homemade electrochemical flow cell and attached to the prism base with high index matching oil . The reflected light was detected using a photodiode.DLD.UPS and XPS were carried out with an unfiltered He I (21.2 eV) gas discharge lamp and a hemispherical analyser using a Kratos AXIS UltraThe Raman spectra of the R6G molecules were obtained from the ERGO, Graphene, and GO substrates using an excitation wavelength of 514 nm under normal incident light with a Renishaw 1000 microspectrometer connected to a Leica microscope with an objective lens of 50 × (NA = 0.5). The typical accumulation time was 10 s.The SPR responses to R6G on different graphene materials are shown in 20−17 M to 10−11 M. It is clear that the SPR response for the R6G on the ERGO is much higher than for the other substrates. On a conventional Au film SPR chip, the changes in the SPR responses are barely detectable for the same series of concentrations, indicating the essential role that the surface modifications play. It is worth noting that the response from 10−11 M R6G on the ERGO surface is of 35 mRU, which is seven times higher than that for the GO and graphene substrates. This suggests that ERGO is a good sensitive layer for an SPR sensing platform. The ERGO sensitive layer displays the lowest LOD at 10–17 M estimated according to the IUPAC guideline of 3:1 signal to noise. However, defect-free graphene and GO exhibit poor sensing performances at low concentrations. ERGO is highly defect-rich due to the removal of oxygen functional groups in the GO by electrochemical reductionThe calibration curves for the SPR response versus the R6G concentration are shown in −6 M, a monolayer of R6G molecules was proven to adsorb onto an Au film and the ERGO after a 30 min incubation period and thorough rinsing88The enhancement of the SPR signal from the doping effect is confirmed by a control experiment using a monolayer of R6G molecules on Au and ERGO substrates. The SPR angle shift on the ERGO surface is 400 mdeg, which is more than two times greater than that observed on the Au film, as shown in A relationship between the dielectric constants and the Fermi level of the intrinsic graphene is established using the Kubo formula and the local Random Phase Approximation (RPA)31f is the Fermi-Dirac distribution function. The first term of this equation describes the intraband contribution, whereas the second term denotes the interband transition to generate the electron-hole pair. The Kubo formula has been verified to accurately describe the complex dielectric constant of graphene in the optical regime with corrections of a few percentwhere σ is the conductivity of the graphene, The effective dielectric constant −1 is usually assigned to the E2g phonon of the C sp2 atoms, the D peak at ~ 1347 cm−1 is a breathing mode of κ-point phonons of A1g symmetry, the 2D band at ~ 2700 cm−1 is due to a double resonance process, and combinational modes in the D + G band exist at 2938 cm−1 21. The D peak is visible in −1. The observed Raman bands are assigned to the R6G molecules. The Fermi energy from the doping carrier concentration is obtained from the experiential formulaThe use of Raman spectra confirms the change of the Fermi energy during doping, as shown in where n is the doping concentration, Egap is the band gap of the ERGO induced by molecular doping, and −1 is a breathing mode of κ-point phonons of A1g symmetry, and the 2D band at ∼2700 cm−1 is due to a double-resonance process. And the D-band is often used to measure the disorder of the graphite materials. The D peak is visible in where −1 observed in the Raman spectra in Ultraviolet photoelectron spectroscopy (UPS) is also used to investigate the electronic structures of the graphene-based materials before and after incubation using R6G molecules. The secondary-electron onset representing the vacuum levels of the samples is used to determine the work function obtained from the UPS38To understand the mechanism by which a R6G molecule adsorbs on defect-rich graphene, theoretical calculations have been performed using the density functional theory (DFT). We start with a hexagonal graphene supercell (10 × 10 graphene unit cell) containing a C vacancy as a simple model, as shown in Fig. 6aThe presence of R6G dopants lead to significant changes in the electronic structures of graphene with vacancies. For the density of states (DOS), as shown in 44−17−10−11 M and an LOD of 10 aM. The existence of defect in the graphene nanosheets result in abundant dangling bonds, which are beneficial for the formation of covalent bonds with R6G molecules. This works as not only a new mechanism for SPR sensing but also a new method for the modulation of the optical properties of graphene-based materials.In summary, the concept of an SPR sensor with ultra-high sensitivity, via a sensitive layer with defect-rich graphene, offers numerous opportunities to develop new biological and chemical sensors and sensing strategies. The defect-rich graphene tunable energy band is presumed to serve as a signal amplifying sensing layer on the Au surface to provide an ultralow limit of detection and a long-term stability. The results show that when small amounts of dye molecules are adsorbed on the defect-rich electrochemically reduced graphene oxide (ERGO) surface, the increase in the dielectric constant of the ERGO results in significant changes to the SPR spectrum. The detection of the target R6G molecule provides a linear dynamic range of 10How to cite this article: Xue, T. et al. R6G molecule induced modulation of the optical properties of reduced graphene oxide nanosheets for use in ultrasensitive SPR sensing. Sci. Rep.6, 21254; doi: 10.1038/srep21254 (2016).