Drosophila, a single Sin3A gene encodes different isoforms of SIN3, of which SIN3 187 and SIN3 220 are the major isoforms. Previous studies have demonstrated functional non-redundancy of SIN3 isoforms. The role of SIN3 isoforms in regulating distinct biological processes, however, is not well characterized.The multisubunit SIN3 complex is a global transcriptional regulator. In Drosophila S2 cell culture model system in which cells predominantly express either SIN3 187 or SIN3 220. To identify genomic targets of SIN3 isoforms, we performed chromatin immunoprecipitation followed by deep sequencing. Our data demonstrate that upon overexpression of SIN3 187, the level of SIN3 220 decreased and the large majority of genomic sites bound by SIN3 220 were instead bound by SIN3 187. We used RNA-seq to identify genes regulated by the expression of one isoform or the other. In S2 cells, which predominantly express SIN3 220, we found that SIN3 220 directly regulates genes involved in metabolism and cell proliferation. We also determined that SIN3 187 regulates a unique set of genes and likely modulates expression of many genes also regulated by SIN3 220. Interestingly, biological pathways enriched for genes specifically regulated by SIN3 187 strongly suggest that this isoform plays an important role during the transition from the embryonic to the larval stage of development.We established a These data establish the role of SIN3 isoforms in regulating distinct biological processes. This study substantially contributes to our understanding of the complexity of gene regulation by SIN3.The online version of this article (doi:10.1186/s12864-016-2428-5) contains supplementary material, which is available to authorized users. Nucleosomes are comprised of DNA wrapped around histone proteins to form a stable chromatin structure. Post translational modifications (PTMs) of histones influence chromatin structure and the transcriptional state of genes . HistoneDrosophila S2 cells compromises the G2/M phase transition during cell cycle progression [Sin3A causes curved wings in adult flies [mSin3A impacts both G1 and G2/M phases of the cell cycle [sin3 in budding yeast show an accumulation of asynchronous cell population in the G2 phase [Drosophila cell lines in which Sin3A was knocked down [. Several investigators have shown that the two highly similar mouse Sin3 proteins play distinct roles during development. For example, mSin3A is essential for early embryonic development, survival and growth of cultured cells [mSin3B plays a regulatory role during the late gestation period of embryogenesis, suggesting the functional non-redundancy of the mSIN3 genes [SIN3 activity is linked to several cellular processes throughout metazoan development. SIN3 has been shown to be involved in protein stability, oncogenic transformations, senescence and cell survival . SIN3 isgression . Additiolt flies . Rescue lt flies . In mousll cycle , 18. NulG2 phase . SIN3 haked down . In addiked down , 21. In ked down . Severaled cells , 18, whiN3 genes . DespiteDrosophila, splicing of a single gene results in the expression of multiple isoforms of SIN3 [Drosophila, Saccharomyces cerevisiae express a single Sin3 protein [The intricate mechanism of gene regulation by SIN3 is augmented by the presence of distinct SIN3 isoforms or discrete SIN3 HDAC complexes in multiple eukaryotic model systems . In Dros of SIN3 . Additio of SIN3 . Unlike  protein . Althoug protein –31. The  protein . Further protein , 33. TheDrosophila [. SIN3 187 and SIN3 220 are the most prevalent isoforms of SIN3 expressed during fly development [Drosophila S2 cells and larval imaginal disc cells, whereas SIN3 187 is the prevalent isoform during the latter stages of embryonic development and in adults [Drosophila.Previously, we demonstrated the existence of distinct SIN3 HDAC complexes in osophila . SIN3 18elopment . SIN3 22n adults . Addition adults . Differen adults , which iDrosophila cultured cells that predominantly express one or the other major isoform. To our knowledge, it is the first time the binding sites of SIN3 isoforms across the Drosophila genome have been mapped. Interestingly, we found that overexpression of SIN3 187 led to replacement of SIN3 220 at a majority of the genomic sites, indicating that the binding sites of the SIN3 isoforms overlap with each other. RNA-seq analysis, however, demonstrate that SIN3 187 plays unique gene regulatory roles, in addition to having some functions in common with SIN3 220.Genome-wide binding sites of SIN3 have been previously published by several groups , 37, 38,, we established two S2 cultured cell lines in which cells express either of the two major SIN3 isoforms with a tag for immunoprecipitation ), Cytochrome c1 (Cyt-c1), Pyruvate kinase (Pyk), twine (twe), CG9548 but not at control regions dachsous (ds) or CG31819  analysis [Through investigation of the chromosomal distribution of SIN3 isoforms, we found that they are enriched over the euchromatic regions of the analysis . Specifianalysis . We founThe enrichment patterns of SIN3 187 and SIN3 220 at genomic features  are similar to each other Fig. . AdditioDrosophila genome) and 6905 (approximately 40 % of the Drosophila genome) genes bound by SIN3 187 and SIN3 220, respectively, supporting the idea of a global transcriptional role of SIN3 isoforms. Additionally, the ChIP-qPCR data showing localization of the SIN3 isoforms at putative gene targets  and 46 % (162/349) of the genes repressed and activated by SIN3 220, respectively, are direct targets  to verify gene expression changes resulting from Sin3A knockdown. We selected S-adenosyl methionine synthetase (Sam-S) and tumbleweed (tum), which were found to be altered upon Sin3A knockdown, and bound by SIN3 220 as well  analysis on the genes that were directly repressed and activated by SIN3 220 using the DAVID gene annotation module Fig. . 52 % 1. AdditioDrosophila embryogenesis, the levels of SIN3 isoforms are comparable [Our findings demonstrate that the ectopic expression of SIN3 187 in S2 cells led to a reduction in the level of SIN3 220 Fig. . In addimparable . During mparable . Based or) or activated (Aa) by SIN3 220, B. Genes directly repressed (Br) or activated (Ba) by SIN3 220 as well as SIN3 187, C. Genes directly repressed (Cr) or activated (Ca) by SIN3 187 alone and D. Genes that are directly regulated in opposing directions by SIN3 187 and SIN3 220  and Glutamate-cysteine ligase modifier subunit (Gclm), which are shown to be repressed and activated specifically by the SIN3 187 isoform, respectively. In addition, these genes are bound by SIN3 187 at the promoter region as determined by the ChIP-seq data . Our results demonstrate that reduction of Sin3A (SIN3 220) did not impact the expression of these genes  knockdown  knockdown. For example, biological processes such as neuron development, cell morphogenesis and aging are overrepresented by both SIN3 187 regulated genes and genes that show a change in expression due to reduction of Sin3A (SIN3 220). Collectively, these data indicate that SIN3 187 specifically modulates biological processes involved in differentiation and development, in addition to regulating processes in common with the SIN3 220 isoform.Our previous research demonstrated that during the late stages of  reduced . These rIn this research, we have described the differential role of the SIN3 isoforms in regulating gene activity, summarized in Fig. Drosophila cells, ectopic expression of SIN3 187 led to a reduced level of SIN3 220  + L-glutamine supplemented with 10 % heat-inactivated fetal bovine serum (Invitrogen). Gentamycin (50 mg/ml) was added to S2 cells. S2 cell lines stably transfected with expression constructs of HA-tagged SIN3 187 and SIN3 220 were grown in the media with 0.1 mg/ml of penicillin/streptomycin and 0.1 mg/ml Geneticin. Cells were maintained at 27 °C.The 6 cells were harvested by centrifugation and lysed in Laemmli Sample buffer (Bio-Rad). Proteins (15 to 20 μg) were resolved on an 8 % SDS-polyacrylamide gel and transferred to a polyvinylidine difluoride (PDVF) membrane . Membranes were incubated with primary antibody: HA-HRP , SIN3 PAN (1:2000 [Western blot was carried out following standard protocols . To prep (1:2000 ), SIN3 2 (1:2000 ), α-Tubu7 exponentially growing S2 cells were subject to cross-linking by the addition of formaldehyde to a final concentration of 1 % for 10 min at room temperature. Excess formaldehyde was quenched by addition of glycine to a final concentration of 125 mM. The cross-linked cells were washed with 1X phosphate buffered saline and resuspended in lysis buffer ), followed by incubation on ice for 15 min. These cells were disrupted by dounce homogenizer  followed by low speed centrifugation (170 x g for 10 min). The pellet was resuspended in 200 μl MNase digest buffer  and subjected to micrococcal nuclease digestion by addition of 20 units of MNase  for 10 min at room temperature. 10 mM EDTA was added to quench the MNase activity. Following MNase digestion, samples were made up to 1.2 ml with resuspension buffer  and sonicated using a probe sonicator (Fisher Scientific) to extract cross-linked chromatin in the solution.Approximately 4x10Approximately 75 μg of crude chromatin was used to prepare input for the ChIP experiment. Briefly, samples were treated with 0.05 μg/μl of RNase A (Sigma) and incubated overnight with 200 mM NaCl at 65 °C. Samples were treated with 0.04 μM Proteinase K (Fisher Scientific), 10 μM EDTA and 20 μM Tris (pH 8) and incubated at 45 °C for 1.5 h. DNA was purified by standard phenol chloroform extraction. This method leads to the formation of mono, di, tri-nucleosomal DNA fragments as determined by agarose gel electrophoresis analysis of purified DNA.3) at 65 °C for 30 min. Elution was done twice in a total volume of 500 μl. Cross-links were reversed in the eluted samples as for the ChIP input DNA preparation. Purified DNA samples from HA-tag ChIP were quantified by real-time PCR using 2-ΔΔCT method of analysis [For HA-tag ChIP, we used monoclonal anti-HA-agarose antibody (Sigma). Anti-HA-agarose bead slurry was washed with RIPA buffer. 75 μg of chromatin was incubated with 30 μl washed anti-HA-agarose for 4 h at 4 °C. Chromatin prepared from wild type S2 cells was used as control. Chromatin bound HA-agarose was then washed, once with 1 ml of modified 2X RIPA buffer  for 8 min at room temperature, once with 1 ml of high salt buffer  at 4 °C for 10 min, once with 1 ml of low salt buffer  at 4 °C for 10 min and once with 1 ml of Tris-EDTA (pH 8). To perform SIN3 PAN ChIP, we incubated chromatin with 10 μl polyclonal SIN3 antibody  overnighanalysis . All genWe prepared sequencing libraries using the method previously described by Ford et al., 2014 . In brieDrosophila genome. Reference Drosophila melanogaster sequence (dm3) was downloaded from the UCSC table browser and raw reads were aligned to the reference genome. Only uniquely mapped tags were subsequently used for further analysis. Input DNA tags were used to normalize ChIP DNA tags. Protein bound regions were called using Model-based analysis of ChIP-seq (Macs 2.1.0) [To map the binding sites of SIN3 isoforms, we used Bowtie2  to aligns 2.1.0)  using ths 2.1.0) –59(AdditPeaks were assigned to UCSC Refseq genes (dm3). Using the bedtools  windows Sin3A knockdown was carried out based on a published protocol [6 cells were plated in 4 ml of Drosophila Schneider’s medium. After 3 h, FBS-containing media was carefully removed and supplemented with 2 ml of serum free media. 50 μg of dsRNA was added to plates and mixed by swirling. After 30 min, 4 ml of complete Drosophila media was added. Cells were incubated for 4 days. Construction of dsRNA targeting Sin3A was previously described [GFP sequence. The primer pairs to amplify GFP containing sequence are GAA TTA ATA CGA CTC ACT ATA GGG AGA TGC CAT CTT CCT TGA AGT CA (forward primer) and GAA TTA ATA CGA CTC ACT ATA GGG AGA TGA TGT TAA CGG CCA CAA GTT (reverse primer). Efficient knockdown of Sin3A compared to GFP RNAi was verified by western blot analysis.RNAi mediated protocol  with sliescribed . dsRNA a4. Ectopic expression of SIN3 187HA was verified by western blotting.S2 cells carrying stably transfected HA-tagged SIN3 187 transgene under the metallothionein promoter was induced by addition of 0.07 M CuSOSin3A was compared to GFP RNAi S2 cells. The primers used for RT-qPCR are listed in Additional file Taf1 gene was used to normalize the mRNA level. At least three independent biological replicates were performed. Results are represented as the mean of the fold difference between the experimental and control samples.Gene expression analysis by RT-qPCR was carried out as previously described . The chaSin3A knockdown versus GFP RNAi was retrieved through GEO under GSE68775 .Procedures from RNA isolation to next generation sequencing were performed at the Applied Genomics Technology Center, Wayne State University. Briefly, total RNA was extracted using the EZ1® RNA Universal Tissue Kit (Qiagen). Cell lysis and homogenization was carried out by bead-milling on the TissueLyser® II (Qiagen) in 750 μl QIAzol™ lysis reagent. Extracted RNA was purified on the EZ1® Advanced (Qiagen). Additional DNase treatment was done to remove any residual DNA. Quantification and quality assessment of purified total RNA was done using the DropSense96® Microplate Spectrophotometer (Trinean) and microfluidics using the RNA R6K assay for the Agilent 2200 TapeStation, respectively. cDNA was generated by reverse transcription of the purified mRNA. The TruSeq RNA Sample Preparation Kit (Illumina) was used to prepare sequencing libraries following the manufacturer’s protocol. Single indexed samples were multiplexed and sequenced on an Illumina HiSeq 2500 sequencing system in paired end mode with a read length of 2 x 50 bp. A total of three independent biological replicates were performed. In the current study, we report the RNA-seq analysis done on total RNA isolated from cells overexpressing SIN3 187HA. Wild type S2 cells treated in the same way as the SIN3 187HA cell line was used as control. Correlation among replicates is shown in Additional file Sin3A knockdown sample and FDR ≤ 0.001 for the SIN3 187HA overexpression sample. The R statistics environment was used to visualize the data. The volcano and scatter plots were generated using the ggplot2 package.The Tophat pipeline was utilized for the analysis . Reads oP-value <0.05 were pooled.Genes bound by SIN3 isoforms and that also showed change in expression upon alteration in the level of SIN3 isoforms were included in the GO analysis. GO analysis, performed separately on genes repressed or activated by SIN3 isoforms, was done utilizing the online tool DAVID . Utilizit-test.Statistical calculations were done using the online statistical tool Graphpad and significant values were calculated using the unpaired Student’s www.ncbi.nlm.nih.gov/geo/). ChIP-seq datasets are deposited under accession number GSE72171 and RNA-seq data for SIN3 187 overexpression are deposited under accession number GSE72172. These two datasets are included in a superseries GSE72173. RNA-seq datasets for Sin3A knockdown samples can be retrieved from GSE68775.ChIP-seq and RNA-seq data can be accessed from the GEO database (


The ability to control the charge-potential landscape at solid-liquid interfaces is pivotal to engineer novel devices for applications in sensing, catalysis and energy conversion. The isoelectric point (pI)/point of zero charge (pzc) of graphene plays a key role in a number of physico-chemical phenomena occurring at the graphene-liquid interface. Supported by theory, we present here a methodology to identify the pI/pzc of  graphene, which also allows for estimating the nature and extent of ion adsorption. The pI of bare graphene -grown) is found to be less than 3.3, which we can continuously modify up to 7.5 by non-covalent electrochemical attachment of aromatic amino groups, preserving the favorable electronic properties of graphene throughout. Modelling all the observed results with detailed theory, we also show that specific adsorption of ions and the substrate play only an ancillary role in our capability to tune the pI of graphene. Miniaturized devices with nanostructures as active elements are highly promising to realize the dream of complete chemical analysis on a single chip2467911514One of the fundamental challenges in such a scenario is the need to have control over the charge-potential landscape of the graphene-liquid interface in addition to providing for the right chemical functionalities and biological receptors for the application of interest. The charge density on graphene has been found to be largely dependent on the nature and density of chemical groups available on the surface171967σOHP) can be modelled using the Gouy-Chapman-Stern theory asFirst we present a theoretical formulation of the charge-potential relationships at the graphene-liquid interface as shown in 22ψOHP is the potential at the OHP, β = 1/kT, κ−1 is the Debye screening length given by cI is the total ionic strength of the solution, ε0 and εr are permittivity of free space and relative permittivity and e is the electronic charge. On the graphene side, we model the presence of functionalities using a layer of a definite thickness (tFL) with a dielectric constant (ε−FLr) different from that of water (εr−H2O = 80). The functionalities present ionizable groups on the graphene surface, whose charge distribution can be modeled by a Langmuir-Freundlich type of an isotherm1617Ka/Kb or pKa/pKb) of the ionizable groups dictate the magnitude and sign of the charge distribution (σ0) and the potential (ψ0) at the interface between functional layer and the Stern layer. This dissociation or protonation of the groups can be schematically generalized in a manner similar to that of amino acids aswhere An example for the former case are carboxyl groups which are dissociated at high pH, while the latter case can be thought of as hydroxyl or amine groups which are protonated at low pH. The fractions of dissociated and protonated groups are respectively given by+], which corresponds to the bulk proton concentrationσmax) at maximum dissociation or protonation, (see Notes/N1 in SI)where the square brackets indicate the surface concentration of the respective species, except for [Hi or j refers to different kinds of negatively or positively chargeable functional groups respectively each with a corresponding Ka and m specifies the degree of heterogeneity in the distribution of ionizable groups as specified by modified Langmuir-Freundlich theorywhere the superscript index σoff is a charge density offset which is constant over a range of pH and ionic strengthwhere sign[CStern is the specific capacitance of the Stern layer (thickness tStern) given by ε0εr−Stern/tStern.where VecG) applied through a reference electrode leads to an electrified interface, whose effect on the charge carrier density in graphene is given byIn order to arrive at the charge carrier distribution in graphene, we need to consider that in an experiment an external voltageψgr referring to the graphene electrostatic potential, CFL = ε0εr−FL/tFL and ψoff is a cumulative offset potential that is independent of pH or ionic strength of the solution. This includes measurement offsets, offset voltage arising due to the choice of the reference electrode and offsets due to the varying filling levels of graphene from impurities or the underlying substrate (the part which is only in contact with graphene and not with the liquid)ψOHP by ensuring charge neutrality using the transcendental equationwith ψOHP at every triple {pH, cI, VecG}, the charge-potential distribution at all planes of the graphene-liquid interface can be self-consistently computed. From the experimental perspective, we can extract the gate voltage at the Dirac point (cI of the liquid can be varied and the Dirac point recorded as a function of pH and cI. The Dirac point corresponds to the point of minimum charge carrier density10cI by solving for σgr(ψOHP) = 0. The minus sign occurs because the applied gate voltage in an experiment occurs at the reference electrode, whereas in the model it occurs at the graphene plane. Since we restrict here only to the situation at the Dirac point, the gate dependent variation in charge carrier density occurring due to the linear energy dispersion in graphene and the effect of quantum capacitanceUsing the value of Ka of 7 and hence we can talk of an isoelectric point of the surface to be 7. It is apparent that the Dirac point variation at higher pH is compressed at higher ionic strength due to the smaller thickness of the electrical double layer, consistent with previous measurements on grapheneψoff is taken as −0.1 V here (ψoff = 0), although this may vary from sample to sample due to the spatially varying chemistry of CVD-grown graphene, size of the flake and local charging effects from the underlying substrate. We can exclude this parameter by comparing the pH-Dirac point behavior at 1 M ionic strength to the behavior at the other IS values. difference Dirac curve (M-I) correlates directly with the sign of the net surface charge. We have used the data at 1 M ionic strength as the baseline due to this reason. This is due to the fact that the compressed double layer at high IS leads to an effective interface potential whose absolute magnitude is smaller than the value at low IS. Now, in order to determine the pI experimentally, we measure the Dirac point as a function of pH and IS and deduce difference Dirac curves such as the one in σ0 = 0, i.e. the net charge of the surface is found to be 02023Before going to the experimental details, it is worth clarifying the definitions of pI/pzc312 chips and patterning them using photolithography (See Methods for details)Rgr) at a frequency of 1 kHz continuously in buffer solutions of varying pH and ionic strength (see I and 1000 mM: M). From every cycle of field-effect scan, as shown in M-I difference curve representative of the sign of surface charge on graphene.Graphene devices were fabricated by transferring CVD-graphene on to Si/SiO−1L. This corresponds to an average charge density of around 1 μC/cm2 close to the values reported for homogenized alkane emulsions in water (around 5 μC/cm2)412 substrate. The capacitance due to trapped charges in SiO2 is much weaker than the double layer capacitance at the graphene-liquid interface45From 193638404-aminobenzylamine (ABA) and aniline (ANI)Ka of ABA is 8.5, in contrast to that of ANI, which is 4.253Now we turn towards the effect of attaching functional groups on the isoelectric point of graphene surface. For this purpose we choose an electrochemical route since it allows for the versatile attachment of a broad range of chemical moieties by simple variation of experimental parameters4849M-I difference Dirac curve (blue) obtained from this measurement along with the difference Dirac curve before modification (black curve). The curve before modification does not have a zero crossing consistent with the discussion that the pI/pzc of graphene is less than 3.3. However, after modification the difference curve exhibits a clear zero crossing at around a pH of 6, which is attributed to be the approximate pI of the functionalized surface. In order to obtain support for this claim, we have used the model to simulate the presence of pABA by introducing an additional type of ionizable group with a charge density that is around 1.2 times that of graphene (Ka of 8.5. The effect of the silanol groups at (15%) and that of specific ion adsorption (7%) remain the same as for bare graphene. The fitted curves are presented as solid lines in M-I difference curve occurs at 5.92. Further support for this estimation is obtained from Ka of the type of ionizable group to 4.2 (instead of 8.5 as done before). The results collected in M-I zero crossing occurs at 4.05. Further details of the model parameters are discussed in M-I difference Dirac curve directly as an approximate estimate of the surface pI.Ka (or pI) of bare (unmodified) graphene, the attached moieties and to some extent the underlying substrate. The weighting factor is then determined by the density of ionizable sites (see Ka is presented). It is worth mentioning that we are modifying the acid-base equilibrium of the graphene-liquid interface through electrochemical functionalization and hence the pI is modified in this process. The change in pzc is a consequence of this modification and sets in only when we have specific adsorption of ions. The immediate question that comes up is to see the effect of increasing thickness of the functional layers on the isoelectric point of functionalized graphene. For this purpose, we have repeated the above experiment for varying thickness of the polymer layer (see Methods) in many different devices. We first measure the pH-IS-behaviour of unmodified graphene at 4 different IS values, perform the electrochemical functionalization and repeat the pH-IS-measurement followed by an estimation of the layer thickness using AFM. The most important conclusion from the foregoing discussion is that the functionalized graphene surface assumes an effective pI that is a weighted balance between the p2 on the value of pI of the functional interface. For this purpose, we have performed a similar set of measurements on another kind of substrate (referred to as Set 2), namely an oxide prepared using a wet etching procedure (the previous devices were measured on dry oxide). The wet oxide results in a comparatively higher density of silanol groups55Another important aspect concerns the role of SiOFinally, we demonstrate that we can continuously tune the value of pI of the functionalized graphene surface by exploiting the versatility of the electrochemical modification protocol. For this purpose, we perform the characterization of pH-IS-behaviour at the initial stage and after every consecutive electrochemical modification. With every modification the density of functional groups on the graphene surface increases resulting in a shift of the isoelectric point towards higher pH values. We perform this series of experiments continuously without drying the sample in order to ensure that the functional groups remain intact during the entire measurement series. In conclusion, we have presented a strategy to estimate the isoelectric point of bare (unmodified/as-prepared) and functionalized graphene. The surface charge behavior is determined by the weighted average of acid-base properties of the ionizable groups on unmodified or functionalized graphene. While as-prepared CVD-graphene exhibits a pI below 3.3, we could tune this value to higher levels by a judicious attachment of a controlled density of ionizable groups on the graphene surface. In this manner the graphene surface can be rendered positively or negatively charged with the same type of chemical functionality at a given pH. Using a theoretical model we could also show that specific adsorption of ions indeed occurs at the graphene-liquid interface albeit with a minor effect on the capability to tune the net surface charge density of functionalized graphene. The presented theory and the methodology can be used to identify changes in pI/pzc using other chemical functionalization strategies and thereby serves as a versatile platform for engineering the interfacial properties of graphene for a targeted application.2 wafers using photolithography. Two kinds of wafers have been used – one with a dry oxide of 575 nm thickness obtained by thermal oxidation (Set 1) and the other obtained by wet chemical etching with an oxide thickness of 500 nm. CVD-graphene obtained either commercially (Graphene Supermarket Inc.) or by a peel-off process Ref. 2O2) leading to removal of copper in less than 10 minutes. Then, graphene was transferred to Si/SiO2 chips with the prepatterned Ti/Pt electrode lines and baked in the oven at 95 °C for 20 minutes before removal of PS using toluene. The patterning of the flakes (of size 2 μm × 2.5 μm) was also performed by photolithography. For this purpose, 10 nm of copper was first evaporated on graphene to be used as a sacrificial protective layer in order that the photoresist does not come in direct contact with the graphene surface. Following this, the structures are patterned using a positive process using the resist S1805 (Microposit). After exposure and development the unprotected regions are removed using mild oxygen plasma etching. After this, the resist and the remaining copper are removed in N-ethylpyrrolidone and HCl/H2O2 respectively. Subsequently we do another round of lithography to deposit SiO2 by thermal evaporation in order to passivate all electrode lines using an insulating layer. This ensures that graphene is exclusively in contact with the solution. Following this an electrochemical etching procedure was carried out in HCl in order to remove trace metal impuritiesFirst, pre-patterned electrode lines with 4 micron gaps are prepared on 4” Si/SiOThe field-effect measurements in liquid were carried out in a PDMS (poly(dimethylsiloxane)) channel placed on the graphene device to hold the liquid (volume is approx. 200 μL). The gate voltage was applied using a Ag/AgCl reference electrode (WPI Inc.). 0 mV vs Ag/AgCl corresponds to 50 ± 5 mV versus SCE measured in 0.1 M KCl. The impedance of the device was measured using an LCR Meter (Agilent) at 1 kHz. The gate voltage was continuously swept in a fixed range and the real part of the impedance recorded to obtain the data as in 4 was introduced into the microwell and the voltage at graphene was swept from −0.2 to +0.7 V against the Ag/AgCl reference for 1 cycle. For Gr/pANI, a mixture of 10 mM ANI and 100 mM LiClO4 was used, and the voltage was swept from −0.2 to +0.8 V for 4 cycles. For tuning the pI, a series of electrochemical polymerization runs were carried out, using ABA as precursor. For the first 3 modifications, 0.1 mM, 1 mM and 10 mM ABA were used at a voltage of 0.7 V for 1 cycle. The subsequent modifications were also carried out at 0.7 V and 10 mM ABA but increasing the number of cycles from 3 up to 12.The electrochemical modification (ECM) to deposit the functional layers was carried out via oxidative polymerization. The same PDMS channel was filled up with the precursor solution and an Ag/AgCl electrode was used as the reference electrode. The same electrical setup mentioned above was used to perform the modification2 chips were measured using an NT-MDT NTEGRA system or a S&I MonoVista system, with a laser excitation of 632.8 nm at a power of 2.7 mW or 1 mW respectively. This system was equipped with a 520 mm monochromator and a 600 l/mm grating. The acquisition time was 5s. AFM images were obtained using a Digital Instruments Veeco Dimension III in tapping mode. The acquired AFM images were processed using Gwyddion software.Raman spectra of graphene samples on Si/SiOAll equations are modelled symbolically and solved numerically in Mathematica 10. The fitting of model parameters to the data was performed in a semi-automatic manner by programmatically varying the parameters in a given range and minimizing the deviation between the measured and simulated curves.How to cite this article: Zuccaro, L. et al. Tuning the isoelectric point of graphene by electrochemical functionalization. Sci. Rep.5, 11794; doi: 10.1038/srep11794 (2015).


This indicates that SHI induces annealing effects at lower fluences. We also observe that the number of graphene layers is reduced at fluences higher than 1 × 1013 ions/cm2. Using inelastic thermal spike model calculations, we estimate a radius of 2.6 nm for ion track core surrounded by a halo extending up to 11.6 nm. The transient temperature above the melting point in the track core results in damage, whereas lower temperature in the track halo is responsible for annealing. The results suggest that SHI irradiation fluence may be used as one of the tools for defect annealing and manipulation of the number of graphene layers.Studies on interaction of graphene with radiation are important because of nanolithographic processes in graphene-based electronic devices and for space applications. Since the electronic properties of graphene are highly sensitive to the defects and number of layers in graphene sample, it is desirable to develop tools to engineer these two parameters. We report swift heavy ion (SHI) irradiation-induced annealing and purification effects in graphene films, similar to that observed in our studies on fullerenes and carbon nanotubes (CNTs). Raman studies after irradiation with 100-MeV Ag ions (fluences from 3 × 1060.80.x; 81.05.ue Graphene is a new member of the carbon family that has two-dimensional honeycomb lattice structure ,2. It is+ ions has shown that the disorder produced in graphene leads to two competing processes resulting in an initial increase in the formation of defects as depicted by the increase in ID/IG ratio followed by its decrease at higher fluence [+ ions [The graphene properties are sensitive to defects. For example, it has been reported that metal to insulator transition occurs with small introduction of defects in graphene perfect lattice . Hence,  fluence . The def [+ ions  and 2-Me [+ ions  show tha [+ ions . Structu [+ ions . Graphen [+ ions . Zhao et [+ ions . Yang et [+ ions .As mentioned above, most of the irradiation studies on graphene are focused on damage by electron and low-energy ions, and there are only few reports available on the ion irradiation studies of graphene at high energy . The stu2/Ni substrate from Graphene Laboratories Inc., USA were used. Though mechanically cleaved graphene samples generally provide better quality single-layer graphene, we have used commercially available CVD-grown samples which are available in larger size, with industrial applications in mind. Irradiation is carried out with 100-MeV Ag7+ ions using 15-MV Pelletron accelerator at IUAC, New Delhi, India, at fluences of 3 × 1010, 1 × 1011, 3 × 1011, 1 × 1012, 3 × 1012, 1 × 1013, 3 × 1013 and 1 × 1014 ions/cm2. The pressure in the irradiation chamber was of the order of 10−6 mbar, and the average ion current was 1 particle nanoampere . The sample was cut into pieces of 1 × 1 cm2 size for irradiation. Half of the area of each sample was masked with thin aluminium foil during irradiation to keep half the portion unirradiated for comparing the results.In the present work, graphene flakes grown with chemical vapour deposition (CVD) technique on Si/SiOThe surface topography was studied using Digital Instruments Nanoscope IIIa atomic force microscope  in the tapping mode. Raman measurements were carried out using Renishaw inVia microRaman set-up . Initially, we performed Raman measurements on pristine sample at several spots with different laser powers to check the self-heating effect. We took 10% (2 mW) of the laser power, which is below the threshold value for laser-induced heating, for measurement on all the samples. Therefore, self-annealing effect during Raman measurement is ruled out.Raman spectra of pristine (unirradiated) as well as irradiated portions of each sample were recorded at four different spots (flakes) using Ar ion laser with excitation wavelength of 514.5 nm and averaged out for further analysis. The size of the laser spot is approximately 2 μm with × 50 optical magnification of the attached optical microscope. The peak intensities have been obtained by fitting the peaks assuming Lorentzian distribution.A typical AFM topographic image of the sample is shown in Figure h ~ 0.35n + t0, where h is the measured height, t0 is the approximate distance between the first graphene layer and the substrate and n is the number of layers. Substrate to the first layer height t0 is approximately 0.6 to 0.7 nm [The film thickness shows a linear relation of −1), G peak  and 2D peak . The G peak is the characteristic peak of graphitic material, and its origin is due to Eg2 symmetry mode near the k = 0 point in the first Brillouin zone. It is the first-order process and a stretching vibrational mode of the carbon atom both in the rings and in the chain [Ag1 symmetry phonons near the K-zone boundary. In the absence of defect, these phonons are Raman inactive due to the momentum conservation in the scattering [The evolution of Raman spectra of pristine as well as irradiated part is shown in Figure he chain . Any chaattering . They beattering . So, theattering .y-axis for clarity. It has been observed that D peak areal intensity first decreases up to a fluence of 3 × 1011 ions/cm2 and then starts to increase with increasing fluences up to 1 × 1014 ions/cm2.The Raman spectra of unirradiated and irradiated graphene at different fluences with 100-MeV Ag ions are shown in Figure α defined by ID/IG ratio, where the ID and IG are the intensity of D and G peaks, respectively, in the Raman spectrum. It measures the disorder present in the sample which may be due to defects or misalignment in the graphene sample. Samples of lower disorder have low value of α, and a high value of α indicates larger amount of disorder. Since there are variations in different parts of the sample, we have normalized the value of ID/IG of irradiated part with the value of ID/IG of pristine part, and these normalized ratios have been studied for further analysis. The variation of α for different fluences is plotted in Figure The defects induced in carbon-based materials are studied using disorder parameter α is observed up to the fluence of 3 × 1011 ions/cm2, and with further increase in the fluence, there is an increase in its value. This initial decrease signifies ordering or purification of graphene at low fluences and is induced by energy dissipated by the incident energetic ion. To the best of our knowledge, this is the first kind of report on the ordering or purification of graphene. Electron beam irradiation studies [It is clear from Figure  studies ,38-40 haI2D/IG at a lower fluence also confirms the crystal quality improvement as shown in Figure 13 ions/cm2, the ratio I2D/IG decreases sharply due to higher defect density.The small increment in intensity ratio 2-bonded structure [g as compared to graphite (g = 3 × 1013 W/cm3/K) and subsequently result in a larger calculated value of core and halo radius. Figure In the present case, as the energetic ion passes through the film, a large fraction of its energy is transferred to the electronic subsystem of the target material by inelastic collisions. According to the thermal spike model, this results in high transient local energy density along the ion path. The deposited energy is transferred to the atomic system by electron-phonon coupling which generates a localized heating of the atomic system in the ion track of nanometric dimension. This results in the transient thermal spike lasting for a few picoseconds. Moreover, the temperature shows a gradual radial decay as the radial distance from the ion track increases . The spatructure . The temtructure . Hence, The temporal distribution of lattice temperature as projected from the 3D plot for radial distances of 0.0 nm (centre of the track core), 2.6 nm, 11.6 nm and 15.0 nm are plotted in Figure The temperature higher than the melting temperature is reached in a radius of 2.6 nm, thus creating a damaged molten zone along the ion trajectory, referred as the track core. Similarly, temperatures above 800 K, essential for graphene annealing , are rea12 and 3.4 × 1011 ions/cm2, respectively. Hence, we can expect that annealing will increase up to a fluence of 3.4 × 1011 ions/cm2 when halos cover the sample. Beyond this fluence, the region damaged by the track core starts to dominate. Kumar et al. have observed similar behaviour in carbon nanotubes due to ion irradiation [The damage and annealing occurs simultaneously. However, because the ratio of the core and halo radii is approximately 1:4  × 10−12 cm2, 4.3 ± 0.2 and (6.7 ± 0.8) × 10−14 cm2 respectively, with chi-square value of 0.04. The simulated curve with the above parameters is shown in Figure In Equation 1, the first term represents exponential decay due to the annealing effect of irradiation in an annular region and the second term represents exponential increase due to damage effects within the ion track core. Constants  6.7 ± 0. × 10−14 σ1 and σ2, respectively, represent the cross-sectional area of cylindrical regions which are annealed and damaged by an incident ion. The annular halo and core radii estimated from the value of σ1 and σ2 are 5.7 and 1.4 nm, respectively. The overlapping fluence calculated with this core radius is 1.6 × 1013 ions/cm2. The halo and core radii observed for graphene are found to be smaller in comparison to radii 11.6 and 2.6 nm obtained from thermal spike calculations. The smaller core and halo radii can be explained in terms of difference in the strength of electron-phonon coupling  of graphene and graphite. Larger thermal conductivity and mean free path may lead to a lower value of g in the case of graphene as compared to graphite (g = 3 × 1013 W/cm3/K) and subsequent estimated lower value for core and halo radii.−1 at fluence of 1 × 1014 ions/cm2  there is more coverage of one- and two-layer graphene instead of three to four layers observed before irradiation. The ion irradiation-induced ablation in graphene can be used for controlling the number of layers with spatial selectivity. Similar reduction in the number of graphene layers with laser ablation above a given threshold laser energy density has been demonstrated by Dhar et al. [Atomic force microscopy imaging Figure  also conr et al. . This sh11 ions/cm2 after which a rapid increase in disorder is observed. The effect is similar to that shown earlier in fullerenes and CNTs. It is concluded that graphene can be purified by the use of SHI irradiation which is of particular importance for using CVD-grown graphene in device applications. Our results suggest that swift heavy ion with selected ranges of fluence may be used as one of the tools for defect annealing and manipulation of the number of graphene layers.In summary, the annealing effect of swift heavy ion irradiation on graphene in low-fluence regime has been observed. Raman studies show that 100-MeV Ag ion irradiation induced annealing up to a fluence of 3 × 10SHI: swift heavy ion; CNTs: carbon nanotubes; EBL: electron beam lithography; GFET: graphene FET; SEM: scanning electron microscope; MWCNT: multiwalled carbon nanotubes; CVD: chemical vapour deposition; pnA: particle nanoampere; AFM: atomic force microscope; FWHM: full width at half maxima; i-TS: inelastic thermal spike.The authors declare that they have no competing interests.SK carried out the experiments and data analysis and drafted the manuscript. AT and DKA contributed in the design and discussion of this work and in the revision of the manuscript, SAK carried out the theoretical simulations and fitting of curve. FS and VB carried out the Raman characterization and helped in the analysis of data. All authors read and approved the final manuscript.


As single agents, durvalumab (MEDI4736), a human IgG1 anti-PD-L1 antibody, and tremelimumab, a human IgG2 anti-CTLA-4 antibody, have shown acceptable safety profiles and antitumor activity. Similar to other anti-PD-L1/anti-PD-1 monotherapies, durvalumab has shown greater objective tumor response rates in PD-L1-positive patients compared with PD-L1-negative patients. Anti-CTLA4 therapies activate T-cells and may increase immune infiltrate and PD-L1 expression in tumor cells and tumor infiltrating immune cells. Thus, combination therapy with durvalumab and tremelimumab could be active in NSCLC regardless of baseline PD-L1 expression.This is a phase 1, open-label, dose-escalation/expansion study (NCT02000947) of D+T in patients with Stage III/IV NSCLC . The primary endpoint is safety and tolerability; secondary endpoints include investigator-reported RECIST v1.1 response. PD-L1 expression was tested retrospectively using an immunohistochemical assay (Ventana).As of 1 June 2015, 102 patients received treatment in the dose escalation phase; combinations of durvalumab [3 mg/kg (D3) to 20 mg/kg (D20) every 2 (q2w) or 4 weeks (q4w)] and tremelimumab [1 mg/kg (T1) to 3 mg/kg (T3)] q4w, plus a D15 + T10 combination, were explored. Across all cohorts, 80% and 42% of patients had ≥1 treatment-related AE ; 28% discontinued treatment due to a related AE. A greater frequency of AEs, without a corresponding increase in tumor response, was seen with increasing T dose. In the combined T1 cohort (D10–D20), 73% and 30% of patients had ≥1 related AE ; 16% discontinued treatment due to a related AE. There were 3 treatment-related deaths .84 patients  were evaluable for response Table . The oveD+T at selected phase 3 dose D20, T1) has a manageable tolerability profile and anti-tumor activity in NSCLC. Unlike anti- PD-1/PD-L1 monotherapies, the combination of D+T appears to be active regardless of PD-L1 status, including even in patients with no tumor cell membrane PD-L1 staining, a setting where patients would not be expected to derive significant benefit from anti-PD-1/PD-L1 monotherapy over current standard of care [ has a ma


Legal problems are more numbered even in Neonatology.Hospital policy should establish strategies to limit systematic, environmental and human errors, including solutions to optimize organization and increase knowledge .Staff reSimulation works through mechanisms that are proper of our brain for decision-making (i.e. simulated mind) . The capThe aim of this lecture is to focus on methods of simulation on neonatal stabilization and transport .Simulation retraining for medical (or nurse) staff should be based on the andragogic approach: adults are conscious of their own educational needs and focus their own attention on specific interests (related to daily practice) . This meA precious additive methodological element is fun: the serious medical game is the approach to optimize technical memories trough emotions .In our experience, materials for simulation are daily clinical devices and innovative stuff developed by a multidisciplinary collaboration ;we discuAs regards neonatal stabilization and transport, we perform: i) annual retraining sessions for all the operators in our Unit, ii) low and medium fidelity simulation sessions for nearby hospitals, iii) high fidelity sessions for colleagues working at geographically uncomfortable hospitals (i.e. island) and so needing for transport by helicopter, as a kind of “full scale CRM (Crisis Resources Management)”.In our opinion the traditional approach to teaching is inadequate for retraining of adult professionals. We propose simulation as the method to deepen knowledge, strengthen abilities and so optimize performances. Recording sessions and analysing them to discuss behaviours is a main instruments for debriefing. Every kind of support  is admitted and should be creatively promoted.


Our findings suggest that TRPV4 channels regulate tumor angiogenesis by selectively inhibiting tumor endothelial cell proliferation.Endothelial cell proliferation is a critical event during angiogenesis, regulated by both soluble factors and mechanical forces. Although the proliferation of tumor cells is studied extensively, little is known about the proliferation of tumor endothelial cells (TEC) and its contribution to tumor angiogenesis. We have recently shown that reduced expression of the mechanosensitive ion channel TRPV4 in TEC causes aberrant mechanosensitivity that result in abnormal angiogenesis. Here, we show that TEC display increased proliferation compared to normal endothelial cells (NEC). Further, we found that TEC exhibit high basal ERK1/2 phosphorylation and increased expression of proliferative genes important in the G1/S phase of the cell cycle. Importantly, pharmacological activation of TRPV4, with a small molecular activator GSK1016790A (GSK), significantly inhibited TEC proliferation, but had no effect on the proliferation of NEC or the tumor cells  themselves. This reduction in TEC proliferation by TRPV4 activation was correlated with a decrease in high basal ERK1/2 phosphorylation. Finally, using a syngeneic tumor model revealed that TRPV4 activation, with GSK, significantly reduced endothelial cell proliferation  Endothelial cell (EC) proliferation is majorly regulated by soluble factors, such as vascular endothelial growth factor (VEGF), which is required for angiogenesis. However, mechanical forces, generated by pulsatile blood flow (shear and strain), also control EC proliferation, possibly via modulation of EC responses to soluble growth factors124572+ influx in EC through ion channels, including members of the transient receptor potential (TRP) family12in vitro. However, the signaling mechanism(s) upstream of this abnormal TEC phenotype are not yet known. We recently found that TEC express reduced levels of TRPV4 compared to normal endothelial cells (NEC), and exhibit abnormal Rho-dependent mechanosensing and angiogenesis, which were restored upon overexpression or pharmacological activation of TRPV4While calcium has not been widely pursued as a potential anti-angiogenic target, intracellular calcium has been demonstrated to regulate angiogenesis, including the processes of cell proliferation, survival, and migration10In the present study, we investigated the role of TRPV4 in TEC proliferation, including the potential underlying molecular mechanism(s).We investigated if TEC proliferation levels are altered compared to NEC, which may contribute to the abnormal angiogenesis and structure of tumor vessels. We found that TEC exhibited significantly (p ≤ 0.05) increased proliferation compared to NEC, as measured by XTT and cell viability assays . HoweverSince TEC express low levels of TRPV4 ;15 and pTo further confirm the specificity of GSK on TEC proliferation, we investigated if GSK treatment inhibits Lewis Lung Carcinoma (LLC) cell proliferation. While LLC cells express TRPV4 . OverallWe next investigated the molecular mechanism(s) by which TRPV4 mediates the above effects. Since members of the mitogen-activated protein kinase (MAPK/ERK) family are critical regulators of cell proliferation, and mechanical signaling is known to stimulate ERK1/216171820Activation of the ERK signaling pathway, as well as the extracellular matrix (ECM)/integrin/receptor tyrosine kinase signaling pathway, has been demonstrated to induce cyclin D1 mRNA expression, allowing entry of cells into S-phasein vivo, we induced LLC tumors in wild-type (WT) mice, as previously describedFinally, to determine the functional significance of TRPV4 in the regulation of TEC proliferation in vitro. Further, we found a correlation between TRPV4 expression, abnormal TEC proliferation, and increased ERK1/2 phosphorylation, which was attenuated by pharmacological activation or overexpression of TRPV4. Finally, we demonstrate that EC proliferation in tumors is significantly inhibited by treatment with GSK in mice subcutaneously implanted with LLC tumors.In the present study, we demonstrate that reduced expression of the mechanosensitive ion channel TRPV4 in TEC results in increased proliferation, which may contribute to abnormal tumor angiogenesis. We show that the pharmacological activation of TRPV4, with a small molecule activator GSK, decreases TEC proliferation Several studies have indicated different TRP channels to influence the angiogenic process through multiple mechanisms1223Previously, TRPV4 dysfunction has been found to increase renal cystogenesis, and pharmacological activation of TRPV4 reduces proliferation of renal duct cells as well as cholangiocytes2514in vitro, and significantly reduced TEC proliferation in vivo, revealing a correlation between TRPV4-dependent ERK1/2 activity and TEC proliferation. We have previously shown that TRPV4 expression is lower in TEC compared to NEC and treatment with TRPV4 agonist, GSK, up-regulates TRPV4 expression in TEC but not in NEC. Further, GSK treatment normalized abnormal angiogenesis exhibited by TEC in vitro. Similarly, increased expression of TRPV4 by GSK may inhibit TEC proliferation via inhibition of ERK1/2 phosphorylation. Based on these findings, we hypothesize that TRPV4 expression (and/or activity) should be at a threshold level to maintain EC mechanosensitivity and proliferation, and beyond this threshold TRPV4 expression may not have any additional effects, as seen in NEC.Indeed, our results have shown that TEC exhibit increased expression of cell cycle genes, S phase protein, PCNA, and proliferation. Importantly, in untreated animals (−GSK), we found increased co-localization of the proliferative marker ki-67 and endothelial marker CD31, confirming that there is increased proliferation of TEC in tumors. However, pharmacological activation of TRPV4 with GSK normalized ERK1/2 phosphorylation, TEC proliferation Taken together, our data supports a significant role for the mechanosensitive TRPV4 channel in regulating TEC proliferation via modulation of the ERK pathway. We have demonstrated that pharmacological activation of TRPV4 induces vascular normalization in tumors by modulating Rho activity2 incubator, split at ~90–95% confluence, and used between passages 11–22, as previously described9Normal (NEC) and tumor EC (TEC) were extensively characterized and cultured on fibronectin or gelatin coated tissue culture dishes and grown in a defined medium in a 37 °C, 5%COCells were treated with (GSK: GSK1016790A 100 nM) or vehicle (DMSO), washed with PBS, and lysed in RIPA buffer or Triton X-100 with protease and phosphatase inhibitors (Boston Bioproducts). Lysates were loaded and separated by electrophoresis on 8%, 10%, or 12% SDS acrylamide gels. Proteins were transferred onto a PVDF membrane and the membrane was blocked in 5% milk in TBS with 0.01% Tween-20 (TBS-T) for 1 h. The blots were incubated with primary antibodies: phospho-ERK1/2 (1:1000) ; total ERK1/2 (1:1000) ; phospho AKT (1:1000) , total AKT (1:1000) , anti-PCNA (1:1000) (Abcam) and tubulin (1:5000) (Abcam) overnight, rinsed three times with TBS-T, and incubated with the appropriate secondary antibody goat anti-mouse  or goat anti-rabbit  conjugated with horseradish peroxidase (Jackson Laboratories). Signals were detected using chemiluminescent substrates (Thermo Scientific) and developed with a FluorChem M Simple Imager (Protein Simple).RNA isolation of cells was performed using an RNeasy Mini Kit (Qiagen) and measured using the NanoDrop 2000 UV-Vis Spectrophotometer. cDNA synthesis was performed using qscript cDNA SuperMix (Quanta Biosciences) and qPCR analysis was performed using the Fast SYBR green master mix (Applied Biosystems) on the Fast Real-Time PCR system (Applied Biosystems). Real time PCR was performed using real time cyclin D1, cyclin A, cyclin E1, CDK1, CDK2, CDK4, CDK6, TRPV4, and GAPDH primers obtained from IDT technologies. Gene expression was first made relative to GAPDH and ΔΔCT values were expressed as fold change compared to NEC.Cells were transfected with TRPV4-EGFP or EGFP constructs using targefect (Targeting Systems)15156) were subcutaneously injected in the flank region of C57BL/6 mice. Once the tumors were palpable (after 7 days), mice either received daily intraperitoneal (i.p.) injections of TRPV4 agonist GSK1016790A (10 μg/kg) (+GSK) or saline (−GSK), until day 21, as previously describedIn vivo proliferation assays were performed using frozen sections  fixed and permeabilized in ice cold acetone (20 min), washed with Tris buffered saline (TBS), and incubated with rat-anti-CD31 (Invitrogen) (1:50) and anti-ki-67 (Abcam) (1:200) overnight. Sections were then washed with TBS (3x–5 min) and incubated for 1 h with appropriate secondary antibodies coupled to Alexa Fluor-488 or Alexa Fluor-594 (Invitrogen) and mounted with DAPI (Vector labs). Images were acquired with Olympus Epifluorescence Microscope (IX71) using QCapture Pro (QImaging) and quantified using ImageJ. Total number of endothelial cells (CD31+ stained vessels) as well as the number of EC that co-localized with ki-67 were counted and expressed them as % of proliferating EC (CD31+-ki-67+/CD31+). The rationale for this is to differentiate proliferating EC (vessels) from non-proliferating EC.All animal experiments were performed according to an approved protocol by Northeast Ohio Medical University, IACUC. Mouse Lewis Lung Carcinoma (LLC) cells  for 2 h. The cells were fixed with 4% PFA, washed with PBS containing 0.1% Triton X-100 (PBST) (3x–5 min), and incubated for 10 min with 1N HCL on ice. Fixed cells were then incubated with 2N HCL at room temperature (RT) for 10 min and moved to the incubator (37 °C) for 20 min. Borate buffer (0.1 M) was used to buffer the cells for 12 min at RT. Cells were washed with PBST (3x–5 min), blocked with PBST solution containing 1M glycine and 5% fetal bovine serum (FBS) for 1 h, and incubated with anti-BrdU primary antibody (1:100) (Abcam) overnight at RT. Following incubation, cells were washed with PBST (3x−5 min), incubated with secondary antibody, Alexa Fluor-488 (Invitrogen), and mounted with DAPI (Vector labs). Images were captured using an Olympus IX71-fluorescence microscope, and analyzed with ImageJ. Quantitative analysis was performed by measuring BrdU positive cells in NEC and TEC  and expressed as fold change over respective control conditions (NEC in the case of NEC vs TEC or TEC in the case of TEC vs TEC+GSK) (p ≤ 0.05).Cell proliferation was determined using XTT kit (Biotium) and cell viability kit (Enzo biosciences). Cultured EC were trypsinized, counted, and plated in a 96-well plate (1000–2000 cells/well). 24–48 h post plating, media was removed and cells were treated with GSK1016790A (GSK) (10–500 nM) in serum free media for overnight. Cells were washed once with PBS; and XTT or Calcein dye was added to the cells and incubated for 30 min-24 h. Wells containing no cells with the dye served as controls to determine the level of the background signal. Absorbance was read at 485–535 nm or 450–500 nm, respectively for Calcein and XTT.t test, analysis of variance (ANOVA) and Tukey’s post-hoc analysis with significance set at p ≤ 0.05.All the data shown is mean ± SEM from at least three independent experiments. Significance was determined using Student’s How to cite this article: Thoppil, R. J. et al. TRPV4 channel activation selectively inhibits tumor endothelial cell proliferation. Sci. Rep.5, 14257; doi: 10.1038/srep14257 (2015).


Background: Prior cognitive research in posttraumatic stress disorder (PTSD) has focused on automatic responses to negative affective stimuli, including attentional facilitation or disengagement and avoidance action tendencies. More recent research suggests PTSD may also relate to differences in reward processing, which has lead to theories of PTSD relating to approach-avoidance imbalances. The current pilot study assessed how combat-PTSD symptoms relate to automatic behavioral tendencies to both positive and negative affective stimuli.Method: Twenty male combat veterans completed the approach-avoidance task (AAT), Clinician Administered PTSD Scale, Beck Depression Inventory-II, and State-Trait Anger Expression Inventory-II. During the AAT, subjects pulled (approach) or pushed (avoid) a joystick in response to neutral, happy, disgust, and angry faces based on border color. Bias scores were calculated for each emotion type (avoid-approach response latency differences). Main and interaction effects for psychological symptom severity and emotion type on bias score were assessed using linear mixed models.Results: There was a significant interaction between PTSD symptoms and emotion type, driven primarily by worse symptoms relating to a greater bias to avoid happy faces. Post hoc tests revealed that veterans with worse PTSD symptoms were slower to approach as well as quicker to avoid happy faces. Neither depressive nor anger symptoms related to avoid or approach tendencies of emotional stimuli.Conclusion: Posttraumatic stress disorder severity was associated with a bias for avoiding positive affective stimuli. These results provide further evidence that PTSD may relate to aberrant processing of positively valenced, or rewarding stimuli. Implicit responses to rewarding stimuli could be an important factor in PTSD pathology and treatment. Specifically, these findings have implications for recent endeavors in using computer-based interventions to influence automatic approach-avoidance tendencies. Anhedono reward . It is so reward . For exao reward  have beeo reward . NeurocoNeurocognitive research related to PTSD has focused primarily on responses to negative or trauma-relevant affective stimuli. Results from research investigating implicit attentional processes suggest that individuals with PTSD may experience greater attentional interference by negative or trauma-relevant affective stimuli . In othebehavioral tendencies to approach or avoid affective stimuli (The approach-avoidance task (AAT) was developed to identify automatic  stimuli . Subject stimuli  based on stimuli , and spi stimuli . Results stimuli  and posi stimuli .There have been two studies utilizing the AAT to investigate behavioral tendencies for individuals with PTSD associated with trauma-related, negative stimuli . One stuInvestigations using trauma-unrelated affective stimuli  are important for understanding whether observed behavioral tendencies may generalize to other situations and contexts. The investigation of behavioral tendencies to positively valenced stimuli or situations would also be important, as this could relate not only to underlying symptoms of anhedonia or emotional numbing, but perhaps also to motivation for treatment. There have been recent endeavors to use computer-based interventions  to modifIn the current study, we examined approach and avoidance tendencies in response to trauma-unrelated affective stimuli . This choice of stimuli was to identify whether PTSD relates to differences in approach and avoidance tendencies that are pervasive in generalizing to trauma-unrelated stimuli and situations. In addition, we aimed to understand how PTSD may relate to approach and avoidance tendencies to both positive and negative affective stimuli, which would be difficult to do if focusing exclusively on trauma-related stimuli. The use of emotional faces could also have implications for understanding interpersonal deficits that are commonly associated with PTSD . We hypoSD = 7.69; mean education = 15.55, SD = 1.85) with varying PTSD symptomology  – IV; a-approacha for angry conditions. More negative values reflect greater avoidance bias whereas larger positive scores reflect greater approach tendencies.The AAT was conducted similarly to previously described tasks , and inc1. Age and education were explored as possible covariates using Spearman’s rho. Three linear mixed effects analyses (LMEs) were used to investigate main and interaction effects for each psychological measure  and emotion type  on avoid-approach bias scores. While effect sizes for LME’s are controversial in the present literature given the impact and difficulty estimating random effects, conditional effects sizes for LME models were estimated. Results were considered significant at p < 0.05, Bonferroni corrected for three LMEs .Statistical analyses were conducted using R Statistical PackagePost hoc Spearman’s rho correlations were used to clarify LME results and identify potential relationships between the avoid-approach bias scores for each specific emotion type and psychological symptom severity including PTSD, depressive, and anger symptoms. To further delineate identified effects of implicit approach and avoidance tendencies, a second bias score was calculated (a–pushn).lculated . The secSD = 19.01), depression  and anger symptoms . Neither age nor education correlated with avoid-approach bias scores .Subjects endorsed mild levels of PTSD  = 4.80, p = 0.005, conditional effect Cohen’s d = 0.518], but no main effect for either PTSD symptoms  or emotion type . The interaction between depressive symptoms and emotion type was trending with a small to medium effect , but there was no main effect for depressive symptoms or emotion type . There were no main or interaction effects with anger symptoms, yielding small effects . Figure 1 depicts relationships between PTSD and depressive symptoms and approach/avoidance tendencies by emotion type.Linear mixed effects analyses analyses revealed a significant interaction with a medium effect between PTSD symptoms and emotion type [Post hoc correlations indicated that individuals with worse PTSD symptoms exhibited a greater avoidh-approachh bias (greater avoidance) for happy faces, and demonstrated a large effect . Despite a trending interaction between depressive symptoms and emotion type, there were no significant relationships between depressive symptoms and avoid-approach bias scores .h-approachh bias score, we examined the individual bias scores for happy faces for approach and avoidance response directions separately compared to neutral faces . Large effects were found for both approach and avoidance conditions, suggesting that those with worse PTSD symptoms were slower to approach , and quicker to avoid  happy faces.To further characterize the relationship between PTSD symptoms and the avoidavoidance tendencies to positive affective stimuli . These findings are consistent with reports of decreased behavioral and neural  responsivity to reward in PTSD . Our res stimuli , the prer = 0.35; In previous research, PTSD diagnosis has been reported to relate to greater attentional biases for negative affective stimuli . The fewpositive affective stimuli may be more robust than to negative affective social stimuli in PTSD, and warrant further investigation. In particular, further research is needed to understand whether the avoidance tendencies to positive affective stimuli are specific to the social domain  or if it generalizes to other domains. Notably, one previous study reported depressive symptom severity reported by individuals with PTSD to relate to increased avoidance of more general positive affective stimuli . While our findings related specifically to PTSD symptoms and not depressive symptom severity, both of these findings suggest that implicit behavioral tendencies to positive affective stimuli play a role in trauma-related psychological symptoms.In the present study, individuals with greater PTSD symptom severity exhibited behavioral biases associated with positive affective stimuli (happy faces). Our results suggest that avoidance tendencies to Evidence-based treatments for PTSD (i.e., Prolonged Exposure therapy; The current study was limited by a small sample and the lack of a non-trauma exposed group. Furthermore, all subjects were male combat veterans. Therefore, generalizability to other populations is unknown. In addition, subjects in the present study endorsed mild to moderate levels of PTSD symptomology. This limits generalizability to more severe PTSD populations and may provide a reasonable explanation for the lack of expected results related to negatively valenced stimuli. However, this also suggests that automatic approach-avoidance imbalances may be evident even for those with only moderate symptom levels. Nevertheless, utilizing a sample with more severe PTSD symptom severity or the use of trauma-relevant stimuli might yield different results and provide additional insights into the role of approach and avoidance behavior in PTSD populations. It is important for future, larger studies to investigate the impact of a broader range of PTSD symptomology and to examine the contribution of individual PTSD symptom clusters on approach and avoidance behavioral tendencies. Despite limitations, the current results provides initial evidence that PTSD symptoms are associated with differences in implicit behavioral tendencies in response to positively valenced stimuli, characterized by both reduced approach and increased avoidance tendencies. These results highlight the potential value of targeting responses to positive affective stimuli in PTSD treatment.AC: Substantial contributions to the conception and design of the work; data acquisition, data analysis and interpretation of results. Contributions to drafting, and revising the work critically for important intellectual content. Provided final approval of the version to be published, and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. WY: Substantial contributions to data analysis. Revising the work critically for important intellectual content. Provided final approval of the version to be published, and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. J-FS: Substantial contribution to data acquisition. Revising the work critically for important intellectual content. Provided final approval of the version to be published, and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. SB: Substantial contributions to the conception and design of the work. Revising the work critically for important intellectual content. Provided final approval of the version to be published, and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. CT: Substantial contributions to data analysis and interpretation of results. Revising the work critically for important intellectual content. Provided final approval of the version to be published, and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. RA: Substantial contribution to the concept or design of the work, and interpretation of results. Revising the work critically for important intellectual content. Provided final approval of the version to be published, and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Knockdown MTDH also inhibited tumor growth in vivo. All these results indicated that MTDH protein levels in most HCC tissues are higher than non-tumor tissues, and knockdown of MTDH inhibited growth and induced apoptosis in HCC cells through the activation of PTEN. Therefore, MTDH might be an effective targeted therapy gene for HCC.The activation of oncogenes and the loss of tumor suppressor genes are believed to play critical roles in the pathogenesis of human hepatocellular carcinoma (HCC). Metaherin (MTDH), also called astrocyte elevated gene-1 (AEG-1), is frequently amplified in a variety of cancers, but the roles of MTDH with regard to growth and apoptosis in HCC have not yet been studied. In the present study, we first analyzed the expression of MTDH in HCC samples. We found that MTDH protein levels are higher in most HCC cancerous tissues compared with their matched adjacent non-tumor tissues. Additionally, the MTDH mRNA was also higher in HCC tissues compared to their matched adjacent non-tumor tissues. Knockdown of the endogenous MTDH using small interfering RNA further showed that deficiency of MTDH suppressed cell growth and caused apoptosis in HCC cells. Knockdown MTDH promoted PTEN and  Hepatocarcinogenesis is a complex multistep process in which many signalling cascades are altered, leading to a heterogeneous molecular profile. Comprehensive gene mutations and abnormal gene/protein expression are intimately correlated with hepatocellular cancer generation . SurgeryMTDH is a single-pass transmembrane protein composed of 582 amino acids with a gene located at chromosome 8q22.5 . Many siin vitro and tumor growth in vivo. Additionally, we evaluated the cell apoptosis in MTDH knockdown HepG2 cells. Finally, we tested the proliferation and apoptosis-related protein expression changes in MTDH shRNA HepG2 cells, in an attempt to explore the molecular mechanisms in MTDH mediating HCC growth.In the present study, we aimed to examine the effects of MTDH silencing on HCC cell viability To investigate the roles of MTDH in HCC progression, we first used immunohistochemistry to test MTDH in HCC tissues. The results showed that MTDH was differently expressed in HCC tissues. The positive expression of MTDH in HCC tissues was 65.22% (15/23). MTDH expression was mainly located in the cytoplasm and cell membrane, and MTDH was also found in the nucleus in HCC tissues A. The imvs. that in the LV-GFP-NC-shRNA and control cells as detected by Western blot.To further explore the molecular mechanism of MTDH-mediated pathological functions in HCC, we examined knockdown MTDH protein expression in HepG2 cells. Cells were infected by a lentivirus which contained MTDH shRNA (LV-GFP-MTDH-shRNA) or an empty vector (LV-GFP-NC-shRNA) A. UninfeTo understand whether down-regulation of MTDH inhibits HepG2 cells viability, CCK-8 assay was used to test the viability effect of shRNA silencing of MTDH expression in HepG2 cells. As shown in Cell apoptosis is an important cause of viability suppression, so we also performed a cell apoptosis assay with a flow cytometer. The percentage of apoptosis in HepG2 cells was greatly increased in the MTDH shRNA group C. Our rePTEN is tightly controlled by various non-genomic mechanisms. To further determine molecular mechanisms of MTDH in HCC growth, we tested the growth- and apoptosis-related protein PTEN expression in HepG2 stable cells with or without shRNA silencing of MTDH expression. As indicated in Nude mice was subsequently injected with LV-GFP-MTDH-shRNA or LV-GFP-NC-shRNA cells into the right axilla of BALB/c nude mice. The mice were sacrificed 6 weeks after inoculation and tumors were excised and measured A. The tuThe prognosis for HCC is still very poor due to the high frequency of late-stage cancer and metastasis at diagnosis. Despite benefits from surgical resection and a low risk of complications, only a limited proportion of HCC patients are eligible to undergo optional resection at diagnosis. Chemotherapy represents the main therapeutic option for these HCC patients, but neither single-drug nor multiple-drug treatments prolong the survival of late-stage HCC patients . Knowledin vitro and in vivo, and these results strongly suggest that MTDH could also promote HCC growth.Recently, accumulating research results have been showing that MTDH could also promote malignant tumor growth. In a MTDH transgenic mouse model with hepatocyte-specific expression of MTDH, such mice displayed profound resistance to chemotherapeutics and growth factor deprivation with activation of pro-survival signaling pathways . It is cP53−/− myoblasts transformed by K-ras overexpression resulted in impaired terminal differentiation and rapid, reproducible tumor formation following orthotopic injection into syngeneic host hindlimbs in mice [In normal tissue development, PTEN functions as part of the process of coordinating the differentiation and proliferation of cell types in time and space to form a functional organ . However in mice . However in mice . In our In vivo results also showed that MTDH shRNA could effectively inhibit HepG2 tumor growth. All these results suggested that MTDH might function as a tumor growth promoter in HCC, meaning it potentially has great value in targeted therapy in HCC treatment.In conclusion, our results showed that MTDH expression was significantly increased in HCC cancer tissues compared to matched non-cancerous liver tissues. Knockdown of MTDH expression could significantly inhibit HepG2 cell proliferation, colony formation, and induce cell apoptosis. Mechanism analysis showed that MTDH shRNA decreased phosphorylation of AKT and increased apoptosis-related protein PTEN and p53 expression in HepG2 cells. 2 incubator.HepG2 human HCC cell line was purchased from the American Type Culture Collection . Cells were cultured in Dulbecco’s modified Eagle medium  supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a 5% COAt 80% confluency, cells were dissociated with 0.25% trypsin (Invitrogen) and collected for reverse transcription polymerase chained reaction (RT-PCR). The total RNA was isolated using TRIzol reagent (Invitrogen). Primers of MTDH and GAPDH  were synthesized . The forward primer sequences for MTDH were: AAGAGGAAAACTG AGCCATCTG, and reverse: CGGCTAACATCCCACTGATAAT. The forward primer sequences for GAPDH were: AGAAGGCTGGGGCTCATTTG, and reverse: AGGGGCCATCCACAGTCTTC, respectively. PCR was performed in a DNA thermal cycler  in the following conditions: one cycle at 94 °C for 2 min; 26 cycles, at 94 °C for 30 s, at 62 °C for 30 s, and at 72 °C for 45 s; and one cycle at 72 °C for 10 min. PCR products were electrophoresed on 1.5% agarose gel containing 0.5 μg/mL ethidium bromide and visualized using an ultraviolet illuminator.HCC tissues and matched non-cancerous hepatic tissues were collected at the Department of Hepatibiliary Surgery in the Second Chongqin Medical Hospital, Chongqin, China between June 2013 and October 2014. The corresponding paraneoplastic tissues were taken at least 2 cm apart from the cancerous node. The tissues were immediately frozen in liquid nitrogen after surgical removal and stored at −80 °C until use. All the recruited patients in this study had no history of preoperative chemotherapy or radiotherapy. HCC diagnosis was based on World Health Organization criteria . Liver fv/v) NP40, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate) with proteas inhibitors. Equal amounts of proteins (50 μg) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred to a polyvinylidene difluoride membrane . After blocking with 5% non-fat milk, the membrane was incubated overnight at 4 °C with the primary MTDH antibody , GAPDH  , PCNA , p-AKT , AKT , p53 , and PTEN  then with horseradish peroxydase-coupled secondary antibody . Signal was detected with enhanced chemiluminescence . Band signals were acquired in the linear range of the scanner and analyzed using QUANTITY ONE software .HepG2 cells were collected and lysed with radio immunoprecipitation assay buffer . The MTDH RNA interference lentivirus vector named LV-GFP-MTDH-shRNA and LV-GFP-NC-shRNA (control). The shRNA targeting sequencing for MTDH was: 5′-GAGUUGAUGAUCGUAGAAG-3′ (siMTDH) and negative control siRNA was 5′-UUCUCCGAACGUGUCACGU-3′ (siNC). The HepG2 cell line was infected with LV-GFP-MTDH-shRNA or LV-GFP-NC-shRNA for 2 h and subsequently placed in fresh medium, while the uninfected cells were used as the blank control. The cells were cultured for the next 48 h and then harvested for western blot analysis or prepared for the following experiments.Cell Counting Kit-8  was used to measure the tumor cell proliferation. Cells were plated at a density of 5000 cells per well in 96-well plates with the complete medium. At the end of infection LV-GFP-MTDH-shRNA or LV-GFP-NC-shRNA for 48 h, 10 μL of the cell proliferation reagent WST-8 was added to each well and incubated for 2 h at 37 °C. The absorbance was measured at 450 nm with an ultraviolet spectrometer . The experiments were performed in quadruplicate and repeated in triplicate.2 for 10 days. The supernatants were then discarded and cells were rinsed in PBS for twice, and fixed with 70% ethanol for 15 min. The cells were stained with 0.1% crystal violet for 10 min and PBS was used to wash the rest of crystal violet twice. The plates were dried at room temperature and the colony numbers containing more than 50 cells were microscopically counted. The experiments were performed in triplicate.After the infection LV-GFP-MTDH-shRNA or LV-GFP-NC-shRNA for 48 h in HepG2 cells, 500 cells were plated into 6-well plates and continually cultured at 37 °C and at an atmosphere of 5% COHepG2 cells were infected with LV-GFP-MTDH-shRNA or LV-GFP-NC-shRNA for 48 h, and an apoptosis assay was performed with Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit (Invitrogen) in 48 h after infection according to the manufacturer’s instructions. The cell suspension (100 μL) was incubated with 5 μL of annexin-V and 1 μL of propidium iodide at room temperature for 15 min. The stained cells were analyzed with fluorescent-activated cell sorting (FACS) using BD LSR II flow cytometer . The percentage of early apoptotic cells located in the lower right quadrant (annexin V–FITC positive/PI negative cells), as well as late apoptotic cells located in the upper right quadrant (annexin V–FITC positive/PI positive cells) were determined.2O2-methanol and normal goat serum was used to close non-specific binding. An anti-human MTDH rabbit monoclonal antibodies  or PCNA  was incubated at 4 °C overnight. The next morning, the slides were washed three times with PBS and incubated with a biotin-labeled second antibody for 15 min. DAB color-substrate solution was added for half a minute. Then the slides were counterstained with hematoxylin for 1 min, dried in 60 °C for 2 h, dehydrated in 85%, 95%, and 100% gradient ethanol, and sealed with neutral gum. The slides omitted the first antibody used as negative controls, and breast cancer tissue slides which had been confirmed to overexpress the MTDH protein as positive controls.MTDH expression was detected in 4-μm thick HCC paraffin embedded slides with an immunohistochemical staining method. Briefly, the slides were first dewaxed in xylol and rehydrated in 100%, 95%, and 85% graded alcohol series, with antigen retrieval in 0.01 M sodium citrate solution 98 °C for 15 min. Endogenous peroxidase activity was blocked with 3% HA semi-quantitative assessment method which combining staining intensity and the percentage of positive cells was used to evaluate MTDH dyeing results. The staining intensity was scored as 0, negative; 1, weak; 2, moderate; and 3, strong. The percentage of positive cells was scored as . The final staining score was calculated by a staining index (SI) of MTDH ranging from 0 to 12 that was finally determined by multiplying the scores of staining intensity and proportion of immunopositive cancer cells according to previously published criteria . A total6 HepG2 cells at the end of LV-GFP-MTDH-shRNA or LV-GFP-NC-shRNA infection for 48 h was injected subcutaneously into the left posterior flank regions of each mouse. Tumor growth was examined every week. Mice were sacrificed after tumor inoculation for six weeks, and the volume of each tumor was calculated. Tumor volume was calculated using the formula LS/2, where L is the greatest tumor diameter and S is the lowest tumor diameter. All experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and approved by our institutional ethical guidelines for animal experiments.For the HCC xenograft model, 4-week-old BALB/c nude mice were housed in a pathogen-free environment and used for experimentation. Medium (0.2 mL) containing 5 × 10t-test was used to analyze the differences in MTDH expression and HepG2 cell proliferation, colony formation number, and apoptotic rate. Statistical significance was considered to be p < 0.05.All the data were analyzed with SPSS 17.0 software . Measurement data were presented as means ± SD from at least three separate experiments. A two-sided Student’s In conclusion, we found that MTDH high expression in HCC tissues and higher MTDH protein levels are found in most HCC cancerous tissues compared with their matched adjacent non-tumor tissues. Additionally, the MTDH mRNA was also higher in HCC tissues compared to their matched adjacent non-tumor tissues. Knockdown of the endogenous MTDH using small interfering RNA further showed that deficiency of MTDH suppressed cell growth and caused apoptosis in HCC cells. And mechanisms analysis showed that knockdown MTDH expression promoted PTEN and p53 expression in HCC cells. And phosphorylation of AKT was inhibited by MTDH siRNA. Knockdown MTDH also inhibited tumor growth in vivo. All these results indicated that MTDH protein levels in most HCC tissues are higher than non-tumor tissues, and knockdown of MTDH inhibited growth and induced apoptosis in HCC cells through the activation of PTEN. Therefore, MTDH might be an effective targeted therapy gene for HCC.


Thermoelectric effects allow the generation of electrical power from waste heat and the electrical control of cooling and heating. Remarkably, these effects are also highly sensitive to the asymmetry in the density of states around the Fermi energy and can therefore be exploited as probes of distortions in the electronic structure at the nanoscale. Here we consider two-dimensional graphene as an excellent nanoscale carbon material for exploring the interaction between electronic and thermal transport phenomena, by presenting a direct and quantitative measurement of the Peltier component to electronic cooling and heating in graphene. Thanks to an architecture including nanoscale thermometers, we detected Peltier component modulation of up to 15 mK for currents of 20 μA at room temperature and observed a full reversal between Peltier cooling and heating for electron and hole regimes. This fundamental thermodynamic property is a complementary tool for the study of nanoscale thermoelectric transport in two-dimensional materials. The interaction of electric and thermal transport phenomena at the nanoscale leads to Seebeck and Peltier thermoelectric effects. Here, the authors directly detect the Peltier effect in graphene, and show that it can be reversed by controlling the type and density of the majority carriers using a back gate. S=−ΔV/ΔT, used for temperature sensing in thermocouples. Graphene8911121415S up to ±100 μV K−1 at room temperature, due to its unique electronic band structure and electrostatic tunability of the density and polarity of its charge carriers. In contrast, the Peltier effect describes the heating or cooling of a junction between two different materials when an electric charge current is present. It is quantified by the Peltier coefficient Π, which can be understood as the heat transported by thermally excited charge carriers. The Peltier effect is a reversible thermodynamic phenomenon that depends linearly on the current, so it is fundamentally different from the irreversible Joule heatingΠ=ST, where T is the reference temperature, it follows that in graphene also the Peltier coefficient Π (and its associated cooling or heating action) can be controlled in both magnitude and sign. Until now, one study managed to detect Peltier heat in a graphene–metal junctionRecent advances in thermoelectrics1356This work presents a direct and quantitative electronic measurement of Peltier cooling and heating, in both single layer (SL) and bilayer (BL) graphene, demonstrating full modulation of the Peltier effect via electrostatic gating. We use nanoscale thermocouples for a sensitive and spatially resolved thermometry of the Peltier electronic heat evolved or absorbed at a graphene–metal junction. The results are consistent with the reversibility and electron-hole symmetry expected for the linear response of the Peltier effect. Furthermore, we probe both the local temperature change on the junction where the Peltier effect is induced, as well as in another junction some distance away. We successfully describe the observed temperature profile in the device using a simple one-dimensional model.I directed from graphene to Au, the evolution of the Peltier heat at the junction is then given by Πgr|⩾ΠAu ΔT=StcΔT  and higher-order contributions such as Joule heating (∝I2) by separating the first harmonic response to the heat modulation at the junction. From the second harmonic, we estimate that Joule heating at 20 μA is ∼10 mK at 300 K, similar to the Peltier cooling and heating. Our measurement scheme allows us to single out the Peltier component, excluding all other possible sources from the measured signal  and cooling (Vg>5 V). This is consistent with the location of the charge neutrality (Dirac) point =2E/π(ℏvF)2, to obtain the thermopowerwith kB the Boltzmann constant, e the electron charge, ℏ the reduced Planck's constant, vF the Fermi velocity, tox the SiO2 thickness, G the measured charge conductivity from the Dirac curve.with T estimated using Rth with a value to allow a direct comparison at large Vg. The good agreement between the two only deviates near the peak in the hole regime. This is because the Peltier effect probes the local density of states at the graphene–metal junction. Therefore, it is much more sensitive to doping from the contact than the Dirac curve (shown in the inset) of the graphene region in between the contacts, which only shows a small electron-hole asymmetryVg≈−25 V, correlates with the observed charge neutrality point at VD=−20 V in the charge transport (see inset). The nonmonotonic behaviour of the Peltier signal is visible for the electron regime, but the parabolic dispersion in BL graphene leads to a broader Peltier curve than for SL graphene. We apply a similar approach as before, to estimate the temperature at the BL graphene–metal junction. Here we use the density of states of BL graphene, ν(E)=2m/(πℏ2), together with the semiclassical Mott relation, leading to:m≈0.05me, where me is the free electron massRth. We observed an overall agreement between the data and the model, with a lower Peltier signal in the hole regime being consistent with the broader Dirac curve.with Rth. Given the geometry of the devices, this usually involves detailed numerical thermal models. To offer physical insight we use a simple one-dimensional model for the heat flow along graphene, with a non-conserved heat current as it flows away via the SiO2 insulator into the Si substrate acting as a thermal reservoir  the thermal conductivity and tgr (tox) the thickness of graphene (SiO2). Considering the thermal conductivity κgr=600 W m−1 K−1 for SL graphene supported on a Si/SiO2 substrateLtt≈320 nm. The small value indicates that the temperature modulation due to the Peltier effect diffuses laterally a short distance from the contact. With this characteristic length, we can readily estimate the thermal resistance of a heat transport channel in analogy to the study of spin resistanceRth≈1 × 105 K W−1 from the one-dimensional description serves as an upper limit for the thermal resistance, in agreement with the one order of magnitude lower scaling parameter Rth≈1 × 104 K W−1 used for fitting the modelled curves in A quantitative understanding requires estimating the magnitude of the thermal resistance eservoir . Here we channel . It is gL=280 nm. Thus, we can validate the estimated Ltt, as the new measurement should be lower by a factor Πgr∝T2. The result, a signal one order of magnitude lower  with a Seebeck coefficient of onlySAu≈2 μV K−1. All measured devices (two BLs and one SL) showed consistent results and had typical dimensions of a few micrometres. In this study, we present results for a SL with a channel width of SL and BL graphene flakes were mechanically exfoliated on a Si/SiOVtc/I≈1 mΩ. Therefore, we established a careful measurement protocol to differentiate the Peltier response from extrinsic effects.The measurement of the Peltier effect in a graphene transistor involved the challenge of a sensitive and local thermometry, for which the nanoscale thermocouples were developed. To achieve sub-mK resolution we required the measurement of thermocouple responses in the order of I≤20 μA to the graphene–metal junctions (between Au contacts 1 and 2 of I).We applied low amplitude AC currents V34=V3−V4, and then repeated for a configuration where the voltage detectors were reversed, V43. This allowed us to extract the true differential mode signal VDM=(V34−V43)/2 and to exclude common mode contributions . Therefore, there is only a correction of 30% to account for the detection efficiency of the thermocouples. We note that this model can only obtain an order of magnitude estimate. It serves as an upper limit for the actual thermal resistance, because it neglects increased lateral heat spreading near the graphene edges and the finite width of the contacts. Finally, using the Wiedemann–Franz law we calculate that electrons only contribute up to 5% to the thermal conductivity of supported graphene, validating our treatment of It is noteworthy that the distance between the NiCu/Au thermocouples and the graphene channel (≈500 nm) is smaller than the thermal transfer length of the Au leads, How to cite this article: Vera-Marun, I. J. et al. Direct electronic measurement of Peltier cooling and heating in graphene. Nat. Commun. 7:11525 doi: 10.1038/ncomms11525 (2016).Supplementary Figures 1-3, Supplementary Notes 1-3 and Supplementary References